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Racial Differences in Paraoxonase-1 (PON1): A Factor in the Health of Southerners?

Abstract

Background

The southern United States (excluding Florida) has the highest age-adjusted rate of cardiovascular disease (CVD) in the country, with African Americans having a higher prevalence of CVD than Caucasians. Paraoxonase-1 (PON1), an enzyme associated with high-density lipoprotein particles, participates both in the hydrolysis of oxidized lipids (thus protecting against atherosclerosis) and in the hydrolysis of organophosphates. Higher paraoxonase activity has been associated with lower risk of atherosclerosis.

Objectives

In this study we characterized the distribution of the functional PON1Q192R polymorphisms (PON status as assessed by diazoxonase to paraoxonase ratios) and the PON1 activity levels in 200 adult males and females of both races (50 in each race/sex class) from the southern United States from commercially obtained blood bank serum samples.

Methods

We used spectrophotometric methods with serum to determine PON1 status, arylesterase activities (phenyl acetate hydrolysis), and levels of cotinine and C-reactive protein (CRP).

Results

African Americans had higher paraoxonase activities but lower diazoxonase activities than did Caucasians, consistent with African Americans having a lower proportion of the functional genotype QQ (QQ 15%, QR 34%, RR 44%, 7% indeterminate), than did Caucasians (QQ 60%, QR 31%, RR 7%, 2% indeterminate). Cotinine levels indicated that all samples came from non-smokers and that CRP levels were higher in African Americans than in Caucasians and higher in females than in males. CRP levels showed no association with paraoxonase activities.

Conclusions

These data present initial observations for use in characterizing the poorer cardiovascular health status of the population in the southern United States and more specifically southern African Americans.

Keywords: atherosclerosis, cardiovascular disease, C-reactive protein, health disparities

Paraoxonase-1 (PON1; EC 3.1.8.1) is an enzyme that has three important known functions: an antioxidative function in preventing the formation of oxidized lipoproteins (Shih et al. 1998), a hydrolytic function on the active metabolites of some organophosphate insecticides (e.g., paraoxon and diazoxon) and other xenobiotic substrates (van Himbergen et al. 2006), and a hydrolytic function that degrades quorum sensing factors of Pseudomonas aeruginosa, thereby increasing host resistance to the bacteria (Ozer et al. 2005; Stoltz et al. 2008). PON1 is synthesized primarily in the liver and is secreted into the blood, where it is associated with high-density lipoproteins (HDLs) (Costa et al. 2005; Deakin et al. 2002; James and Deakin 2004; Sorenson et al. 1999). PON1 belongs to a family of calcium-dependent lactonases/hydrolases that also include PON2 and PON3 (Gaidukov et al. 2006). The three enzymes hydrolyze aromatic and long-chain aliphatic lactones, but PON2 and PON3 lack paraoxonase (POase; phosphotriesterase) and arylesterase (phenyl acetate hydrolysis) activities (Costa et al. 2005). Lactones, such as those formed from the enzymatic and nonenzymatic oxidation of arachidonic acid and docosa-hexaenoic acid, are the endogenous substrates of PON1 (Draganov et al. 2005). PON1’s physiologic activity is thought to be that of a lipolactonase whose activity results in the prevention of atherosclerosis (Gaidukov et al. 2006). PON1 prevents the formation of oxidized low-density lipoproteins (LDLs) and protects phospholipids in HDLs from oxidation (Costa et al. 2005).

The PON1 gene contains a variety of single-nucleotide polymorphisms in the promoter and the coding sequences (Chen et al. 2005). Previously, the PON1Q192R polymorphism in the coding sequence was thought to influence susceptibility to cardiovascular disease (CVD) (Costa et al. 2005). The PON1192 polymorphism affects PON1’s hydro lytic activity toward several nonphysiologic substrates and thus was thought to influence its ability to protect against LDL oxidation. The Q alloform was believed to be more protective of cardiovascular health than the R alloform because it had a greater capacity to metabolize oxidized lipids (Costa et al. 2005). However, a meta-analysis of 43 published studies revealed only a very weak association of the PON1192 polymorphism with coronary heart disease (Wheeler et al. 2004) and concluded the association was of uncertain significance. Gaidukov et al. (2006) reported that the R alloform of PON1 bound HDL with a higher affinity than did the Q alloform and thus exhibited increased stability and lipolactonase activity. Bhattacharyya et al. (2008) found patients with the QQ genotype had an increased incidence of major cardiac events. Others have reported no association of PON1 genotype with CVD (Gardemann et al. 2000; Ombres et al. 1998). Thus, the association, if any, of the PON1192 polymorphism with atherosclerosis remains uncertain. Other studies have shown an association of lower POase activity with atherosclerotic diseases (Ayub et al. 1999; Jarvik et al. 2000; Mackness et al. 2001), thus demonstrating the importance of determining the phenotype (i.e., actual enzyme activity or enzyme concentration) and not just the genotype when studying atherosclerosis. Therefore the continued investigation of both genotype and phenotype in more populations at risk of CVD is warranted.

Plotting the rates of diazoxon hydrolysis [diazoxonase (DZOase)] versus paraoxon hydrolysis (POase) (i.e., DZOase/POase ratio) separates individuals into three functional genotypes of PON1 activity: PON1192QQ, PON1192QR, and PON1192RR (Richter and Furlong 1999). However, both genotype and phenotype (i.e., overall PON1 activity) are important for determining the relationship of PON1 polymorphisms with susceptibilities to disease, sensitivity to organophosphate insecticides, and pharmacokinetic status of drug metabolism (Richter et al. 1999). Because arylesterase activity, using phenyl acetate as the substrate instead of the more toxic organophosphates as substrates, is frequently monitored as an index of PON1 activity, we also determined arylesterase activity in this study.

The significance of PON1 to cardiovascular health and sensitivity to organophosphate insecticides has been addressed experimentally through the use of PON1 knockout mice, which were more susceptible to atherosclerosis, had HDL and LDL particles that were more susceptible to in vitro oxidation, and had HDL particles that were inefficient for hydrolysis of oxidized LDL particles in vitro (Shih et al. 1998). HDL particles isolated from transgenic mice with human PON1 had an enhanced ability to protect LDL particles from oxidation (Tward et al. 2002).

The southern United States (except Florida) has higher annual age-adjusted mortality rates from CVD (e.g., coronary heart disease and stroke) than do other regions of the country (American Heart Association 2005), and African Americans have higher annual age-adjusted mortality rates of CVD than do Caucasians. The well-characterized critical risk factors for CVD are hypertension, dyslipidemias (e.g., high LDL cholesterol and low HDL cholesterol), smoking, diabetes mellitus, a family history of disease, and obesity. Several theories have been advanced to explain the higher stroke mortality rate in the South, including low socioeconomic status, an increased prevalence of severity of hypertension, and the presence of environmental toxicants (Perry and Rocella 1998). Data on the frequency distribution of PON1192 genotypes have been reported on numerous populations, but not specifically the American southern populations, which have the worst American health statistics. Previous studies reported that African Americans have a weighted PON1Q192 allele frequency of 0.37 and a weighted PON1R192 allele frequency of 0.63, whereas Caucasians have a weighted PON1Q192 allele frequency of 0.73 and a weighted PON1R192 allele frequency of 0.27 (Chen et al. 2003; Scacchi et al. 2003). Knowledge of PON1 enzymatic activities as related to genotype, phenotype, race, sex, and age within a population may provide a useful explanation for some of the disparities in CVD among demographic groups.

To characterize the functional genotype distribution (as assessed by DZOase/POase ratios) and activity levels of PON1 as contributors to the higher CVD in southern populations, in the present study we investigated PON1 activity levels with three substrates (paraoxon, diazoxon, and phenyl acetate) in the serum of African-American and Caucasian southerners within race, sex, and age groups. In addition, we also investigated the levels of the nicotine metabolite cotinine (as a possible influence of cigarette smoking on PON1 activity and concentrations) (James et al. 2000; Nishio and Watanabe 1997) and the inflammatory marker C-reactive protein (CRP), a biomarker for increased risk of coronary heart disease (reviewed by Casas et al. 2008). Our study samples came from a commercial vendor that had obtained serum samples from blood banks in Alabama and Tennessee. We obtained a total of 200 serum samples from adult men and women in equally distributed race and sex classes who self-identified as being Caucasian or African American. With the data obtained, we observed that African Americans in the South have lower DZOase activities, higher POase activities, and lower DZOase/POase ratios than Caucasians, reflecting the significantly different distribution of QQ, QR, and RR functional genotypes observed in African Americans than that found in Caucasians. Our laboratory is expanding these studies at present with samples from individuals whose cardiovascular health status is known to determine what role the difference in frequency distribution of QQ, QR, and RR functional genotypes and differences in activity levels between the racial groups may play in the CVD health disparities observed and to investigate the associations that might exist with exposure to environmental chemicals.

Materials and Methods

Chemicals and samples

We purchased all biochemicals from Sigma Chemical Company (St. Louis, MO). We synthesized paraoxon as described previously by Chambers et al. (1990). Diazoxon was purchased from ChemService (West Chester, PA). Serum cotinine was determined using the Cotinine Direct ELISA (Calbiotech, Inc., Spring Valley, CA). Serum CRP was determined using a high-sensitivity CRP ELISA (Calbiotech, Inc.). We purchased serum samples from Integrated Laboratory Services-Biotech (Chestertown, MD), which had obtained serum samples from blood banks in Alabama and Tennessee. We excluded from the study individuals who were < 25 years of age or > 65 years of age, who were known or suspected to be infected with HIV (human immunodeficiency virus) or hepatitis, or who did not self-declare as Caucasian or African American; no demographic information was available on any of the samples other than age, sex, and self-declared race. The mean ages (± SD) were 33.5 ± 6.9 years for African-American females, 40.4 ± 8.5 years for African-American males, 39.3 ± 9.2 years for Caucasian females, and 36.4 ± 8.5 years for Caucasian males. The Institutional Review Board for the Protection of Human Subjects in Research at Mississippi State University approved the study protocol.

POase assay

We measured paraoxon hydrolysis spectrophotometrically in micro-titer plates according to a method described by Richter and Furlong (1999). We incubated paraoxon (1.2 mM final concentration; stock solution in dry ethanol) in paired serum samples of 1 μL serum in a reaction volume of 200 μL. A calcium buffer solution of Tris-HCl (0.1 M, pH 8.0), 2 mM CaCl2, and 2 M NaCl was used to activate PON1 in one set of triplicate samples made from the same three independent dilutions of serum. We used an EDTA buffer solution with 1 mM EDTA instead of CaCl2 to eliminate PON1 activity in the paired set of triplicate samples made from three independent dilutions of serum. After mixing and incubation at 37°C for 5 min, paraoxon was added, mixed, and incubated at 37°C for 20 min with shaking. After incubation, the enzyme reactions were terminated by adding a solution of 50 μL of 20 mM EDTA plus 2% Tris base solution in deionized water. The 4-nitrophenol released was quantified at 405 nm. We subtracted the mean of the EDTA triplicates from the mean of the CaCl2 triplicates for each individual to correct for non-PON1-mediated hydrolysis. Data were expressed as micromoles of paraoxon hydrolyzed per minute per liter of serum. The reaction rate was linear during the 20-min incubation time.

DZOase assay

We measured the 2-isopropyl- 4-methyl-6-hydroxypyrimidine (IMHP) released from diazoxon hydrolysis spectrophotometrically in a continuous assay according to the method described by Richter and Furlong (1999). We incubated diazoxon (2.0 mM final concentration; solution in dry ethanol) in each sample. Diluted serum (20 μL serum plus 1,950 μL of the calcium buffer described above) was pre incubated at 37°C for 5 min, and then 20 μL diazoxon (2.0 mM final concentration) was added. The solution was mixed and immediately placed into a cuvette. We quantified the IMHP continuously for 2 min at 270 nm. Samples were run in quadruplicate and the values averaged as micromoles of diazoxon hydrolyzed per minute per liter of serum.

Arylesterase assay

We assessed arylesterase activity using phenyl acetate as the substrate. We diluted serum to 5 μL/mL in 0.05 M Tris-HCl plus 2 M NaCl buffer (pH 8.0) containing either 2 mM CaCl2 to activate PON1 or 1 mM EDTA to serve as a blank. These dilutions (2.5 mL) were warmed to 37°C, and then 25 μL of 50 mM phenyl acetate in ethanol was added. Absorbance was measured at 270 nm for 3 min, and the slopes were calculated. PON1 activity was expressed as micromoles of phenyl acetate hydrolyzed per minute per liter of serum.

C-reactive protein

Serum CRP was quantified spectrophotometrically at 450 nm using a high sensitivity CRP ELISA kit (solid-phase direct sandwich method) following the manufacturer’s directions and interpolating the values from a standard curve. Data were expressed as milligrams of CRP per liter of serum. We did not include CRP values > 10 mg/L in these analyses because these values are not used in assessing cardiovascular risks (American Heart Association 2005).

Cotinine assay

We quantified serum cotinine spectrophotometrically at 450 nm using a cotinine ELISA kit following manufacturer’s directions. Data were expressed as nanograms per milliliter of serum.

Statistical analysis

All data were analyzed using the SAS System for Windows (version 9.1; SAS Institute Inc., Cary, NC). We performed efficacy analyses using a linear mixed model (PROC MIXED), and we used the least square means when we found statistical significance to determine the differences between groups (race, age groups, and sex) with respect to the mean DZOase and POase activities. In the mixed models, the fixed effects were race, age group, and sex. All statistical comparisons were two-sided using a 0.05 significance level.

Results

DZOase and POase activities, and DZOase/POase ratios

The overall DZOase and POase activities for all subjects and for both races are shown in Table 1, and the DZOase versus POase plots are shown in Figure 1. Similar to results of Richter and Furlong (1999), plotting hydrolytic rates separated individuals into one of three functional PON1192 genotypes for determining PON1 status. The frequency distribution of the functional genotypes for the entire population was QQ (0.375), QR (0.325), and RR (0.255). Nine data points (frequency of 0.045) were indeterminate for apparent QR and RR genotypes, seven of which were from African Americans (three females, four males) and two from Caucasians (one female, one male); these were not included in Figure 1. These nine indeterminate individuals all fell in the region between the QR and RR functional genotype. These individuals may have nonsense or missense mutations as previously described by Jarvik et al. (2002).

Figure 1
DZOase activities versus POase activities in sera of male and female Caucasian and African-American southerners, as well as the distribution of individuals into three PON1192 functional genotypes (QQ, QR, and RR). Abbreviations: AAF, African-American ...
Table 1
POase and DZOase activities and DZOase/POase ratios in the serum of African-American and Caucasian southerners (mean ± SD).

Differences in race

The frequency distribution of QQ (0.15), QR (0.34), and RR (0.44) functional genotypes within the African-American population was substantially different from the distribution in the Caucasian population [QQ (0.60), QR (0.31), and RR (0.07); Figure 2]. Caucasians had a 4-fold higher proportion (± 95% confidence interval) for the QQ genotype (0.60 ± 0.15) than did African Americans (0.15 ± 0.11), and this difference was significant. In our study population, Caucasians had a PON1192Q allele frequency of 0.77 and a PON1192R allele frequency of 0.23. African Americans had a PON1192Q allele frequency of 0.34 and a PON1192R allele frequency of 0.66.

Figure 2
DZOase activities versus POase activities in sera of male and female Caucasian southerners (A) and African-American southerners (B). Abbreviations: AAF, African-American female; AAM, African-American male; CF, Caucasian female; CM, Caucasian male.

The mean DZOase activities of African Americans were significantly lower than those of Caucasians (p = 3.25−08), whereas the mean POase activities of African Americans were significantly higher than those of Caucasians (p = 6.52−09; Table 1). The mean DZOase/POase ratio of African Americans was significantly lower (p < 0.05) than the ratio of Caucasians (Table 1). The racial differences in the DZOase activity, the POase activity, and the DZOase/POase ratio are all to be expected given the differences in the functional genotype distributions and the differences in the PON1192 allele frequencies observed between Caucasians and African Americans in our study population. When we compared African Americans and Caucasians within the same functional genotype, we found no statistically significant differences in mean POase and DZOase activities. However, within the QQ genotype, we found a nonsignificant trend, with Caucasians having a higher mean DZOase activity than African Americans.

Differences in sex and PON1192

The mean POase activities of all males and all females and the mean DZOase activities of all males and all females were not significantly different (Table 2). The mean DZOase/POase ratio of all males and all females was also not significantly different. In addition, we observed no differences between sexes in activities and the DZOase/POase ratios within each race. However, the mean POase activities, the mean DZOase activities, and the mean DZOase/POase ratios were significantly different in African-American females compared with Caucasian females and in African-American males compared with Caucasian males (Table 2), again likely reflecting the difference in functional genotype distribution between the racial groups.

Table 2
POase and DZOase activities and DZOase/POase ratios in the serum of African-American and Caucasian southerners by sex (mean ± SD).

Differences in age and PON1192

We separated individuals into three age groups (25–30 years, 31–40 years, and > 40 years) for statistical analysis. In females, POase activities appeared to be higher (p = 0.0366) in the youngest group (n = 32) compared with the oldest group (n = 32) (Figure 3). The reverse appeared to be true in males, with POase activities lower (p = 0.0209) in the youngest group (n = 28) compared with the oldest group (n = 45). However, these differences are probably the result of uneven sampling of functional genotypes among these arbitrary age ranges. The females had QQ distributions of 27%, 47%, and 63% in the youngest, middle, and oldest age groups, respectively. Conversely, the males had QQ distributions of 46%, 32%, and 25% in the youngest, middle, and oldest age groups, respectively. We found no significant differences in DZOase activities among the three age groups.

Figure 3
DZOase (A) and POase (B) activities in three age classes of male and female southerners. Female age groups: 25–30 years (n = 32), 31–40 years (n = 36), > 40 years (n = 32); male age groups: 25–30 years (n = 28), 31–40 ...

Arylesterase activity

Results plotting phenyl acetate hydrolysis (arylesterase activity) versus POase activity showed a trend toward separating individuals into the three functional genotypes (Figure 4), but did not yield as clear-cut a distinction among the three functional genotypes as did the plot of POase versus DZOase. Caucasians had slightly higher activities (16,407 ± 341 U/L) than African Americans (14,276 ± 327 U/L), but this was not statistically significant.

Figure 4
Arylesterase activity as monitored with phenyl acetate versus POase activities in sera of male and female African-American and Caucasian southerners. Abbreviations: AAF, African-American female; AAM, African-American male; CF, Caucasian female; CM, Caucasian ...

Cotinine levels

Serum cotinine levels were less than the levels indicative of active smokers (> 78 ng/mL; Wall et al. 1988) for all samples.

CRP levels

CRP levels were significantly higher in African Americans (4.70 ± 3.50 mg/L) than in Caucasians (3.71 ± 2.60 mg/L; p < 0.05) and higher in females (4.93 ± 3.20 mg/L) than in males (3.53 ± 2.80 mg/L; p < 0.05), similar to previously published data (Lakoski et al. 2006). We found no significant association between POase and CRP levels or DZOase and CRP levels. Individuals with CRP levels > 10 mg/L were not included in this analysis because CRP levels > 10 mg/L are not considered reliable for indicating increased risk of CVD.

Discussion

PON1 plays an important role in hydrolyzing many substrates, including oxidized lipids and active metabolites of organophosphate insecticides (Chambers 2008). The single-nucleotide polymorphism at position 192 has been proposed to have a significant effect on PON1 hydrolytic activity, xenobiotic metabolism, and the onset of CVD, with the R alloform earlier considered to be associated with vascular disease (Harel et al. 2004). Despite the fact that southerners (except Floridians) in general and African Americans in particular have higher annual age-adjusted mortality rates from CVD than do people in other regions of the United States, data on the distribution of PON1Q192R in the serum of Caucasians and African Americans in the South have not been documented.

In the present study we determined the PON1192 functional genotype distribution (as assessed by DZOase/POase ratios) and PON1 enzymatic activities (paraoxon and diazoxon hydrolysis, phenyl acetate hydrolysis) in the serum of 200 African-American and Caucasian southerners. We assumed that the individuals sampled from Alabama and Tennessee blood banks were residents of the South and therefore representative of southerners in general. This study revealed significant differences in the functional PON1192 genotype distribution between Caucasians and African Americans. Caucasians had an overwhelmingly higher distribution of the functional QQ genotype (60%) than did African Americans (15%). Our study agrees with results of Chen et al. (2003) and Scacchi et al. (2003), who reported that African Americans have a higher R allele frequency (0.63) and Caucasians have a higher Q allele frequency (0.73). However these two studies did not report the activity level (phenotype), which is ultimately more important in functional capacity to hydrolyze oxidized lipids.

The arylesterase activity plot, where phenyl acetate was substituted for diazoxon, did not yield as straightforward a separation of functional genotypes as did the paraoxon versus diazoxon plot. Most laboratories report on aryl esterase activity using phenyl acetate, probably because of the high cost, toxicity, and instability of diazoxon. However, phenyl acetate is not as useful as the toxic organophosphate substrate for characterization of PON1 status. Although the African Americans had a significantly lower mean DZOase activity than did Caucasians, the lower activity of African Americans with phenyl acetate was not statistically significant. Probably because of the low numbers after the population was divided into functional genotypes, we found a trend within the QQ functional genotype (not statistically significant) that Caucasians had higher DZOase activity; this suggests that phenotype is important in characterizing risk factors and indicates that more research with larger groups of known cardiovascular health status would be useful.

Much of the difference in DZOase and POase activities between the races probably resulted from the difference in the PON1192 genotype distribution; however, racial differences in other genotypic influences (e.g., promoter polymorphisms that influence expression) and in environmental and lifestyle influences, (e.g., diet, statins and other drugs, exposure to environmental factors) also probably contribute to the difference. However, the cotinine levels indicated that none of the serum samples came from smokers, so smoking was not an influence on the PON1 levels measured.

Differences in PON1 activities between the sexes have not been shown to be significant in other studies (Mueller et al. 1983). Results from our study also do not indicate a significant difference when comparing DZOase and POase activities of females and males separately or within each race.

In the present study we found that with age, POase activity appeared to decrease in females but increase in males, whereas DZOase activity did not change significantly with age in either sex. The difference in POase activity just reaches significance but is likely the result of uneven sampling of functional genotypes within the various age ranges, as mentioned in the “Results.” This interpretation emphasizes the need to consider potential confounders (in this case, functional genotype sampling disparities among age classes) when drawing conclusions about differences in activity levels among groups of individuals. Other studies have shown that serum PON1 activity is lower during development and at birth but increases over time (Chen et al. 2003). PON1 activities have also been reported to remain constant during adulthood or decrease in elderly subjects (Costa et al. 2005; Jarvik et al. 2002; Milochevitch and Khalil 2001).

The individuals sampled here did not appear to be smokers, as indicated by the low cotinine levels. Although we do not know whether the blood banks screened out smokers, it would be highly unlikely to obtain 200 random samples with no smokers among them by chance. Because smoking can affect PON1 levels, we can assume that this potential factor did not influence the PON1 activity levels measured here. Certainly other xenobiotics may have influenced the activity levels, but we had no knowledge of drugs (e.g., statins) or environmental chemicals to which these individuals may have been exposed.

Although we did not know the health status of the individuals in this study, we did determine CRP concentrations as a biomarker of inflammation and CVD. We did not include individuals with CRP levels > 10 mg/L in these calculations because recommendations of the American Heart Association and the Centers for Disease Control and Prevention suggest excluding these CRP concentrations from clinical decisions (Pearson et al. 2003). We found that females had higher CRP concentrations than did males (p < 0.05) and that African Americans had higher CRP concentrations than did Caucasians (p < 0.05). These results on sex and race are consistent with literature reports (Kelley-Hedgepeth et al. 2008; Lakoski et al. 2006). CRP is influenced by modifiable risk factors such as body mass index, exogenous estrogen, diabetes, hypertension, smoking, alcohol use, HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase inhibitors, aspirin use, physical activity, LDL, and HDL (Cushman et al. 1999; Ridker et al. 1999). However, we have no information regarding these factors in our study population.

One potential confounder worth noting is the potential inaccuracy of the self-identification of race. Although individuals may be primarily one race, in many cases there has been racial mixing in the past that may or may not be known to the individuals, so the racial designation may not have been totally accurate.

Conclusions

In this article we report the PON1192 functional genotype distribution, PON1 enzymatic activities (POase, DZOase, and arylesterase), and mean DZOase/POase ratios in the serum of African-American and Caucasian southerners, along with the CRP and cotinine levels of these individuals. Most important, we found significant racial differences in the PON1192 functional genotype distribution, with African Americans—the race with higher CVD—displaying a greater proportion of the functional RR genotype. Although, at present, the relationship of the functional genotypes to CVD is equivocal, our data support the hypothesis that the functional RR genotype is less protective of cardiovascular health. These data suggest that further study using samples of southerners from both races—where cardio vascular health status, lifestyle, and environmental influences are known—would be of value in characterizing risk of both CVD and susceptibility to organophosphate insecticide toxicity. In addition, determining the complete PON1 genotype (i.e., determining the promoter and protein polymorphisms) and quantitation of PON1 expression would allow the evaluation of what factors other than genotype contribute to the differences in PON1 activity observed between Caucasians and African Americans. Our laboratory is currently engaged in such a study with southerners of both races and both sexes where the presence of CVD and any therapeutic interventions are known.

Footnotes

We thank S. Givaruangsawat for the statistical analyses.

This research was supported in part by National Institutes of Health grant R21 ES015107 and by the Center for Environmental Health Sciences and the College of Veterinary Medicine, Mississippi State University. This is Center for Environmental Health Sciences publication 123 and Mississippi Agricultural and Forestry Experiment Station publication J-11545.

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4-Vinylcyclohexene Diepoxide (VCD) Inhibits Mammary Epithelial Differentiation and Induces Fibroadenoma Formation in Female Sprague Dawley Rats

Abstract

4-Vinylcyclohexene diepoxide (VCD), an occupational chemical that targets ovarian follicles and accelerates ovarian failure in rodents, was used to test the effect of early-onset reproductive senescence on mammary fibroadenoma formation. One-month female Sprague Dawley rats were dosed with VCD (80 mg/kg or 160 mg/kg) and monitored for 22 months for persistent estrus and tumor development. Only high-dose VCD treatment accelerated the onset of persistent estrus relative to controls. However, both doses of VCD accelerated mammary tumor onset by 5 months, increasing incidence to 84% (vs. 38% in controls). Tumor development was independent of time in persistent estrus, 17β-estradiol, androstenedione and prolactin. Delay in VCD administration until after completion of mammary epithelial differentiation (3 months) did not alter tumor formation despite acceleration of ovarian senescence. VCD administration to 1-month rats acutely decreased mammary alveolar bud number and expression of β-casein, suggesting that VCD’s tumorigenic effect requires exposure during mammary epithelial differentiation.

Keywords: Fibroadenoma, VCD, Mammary epithelium, β-casein, c-KIT

1. Introduction

Fibroadenomas are the most common form of benign breast disease, occurring in 25% of women [1]. In addition to being associated with an increased risk of breast cancer, mammary fibroadenomas are also of clinical importance as these benign tumors account for up to 50% of all breast biopsies [1]. However, little is known about the precise pathogenesis of mammary fibroadenomas; thus further exploration of their etiology is warranted.

Spontaneous mammary tumors occur with varying incidence in most strains of rats, including Sprague Dawley (SD) rats. The majority are benign fibroadenomas, with only 18% of these being malignant [2,3]. Although 70% of female SD rats ultimately develop mammary tumors over their ~24 month life span [4], tumor onset does not typically occur before post-natal day (PND) 400 [5,6]. The initiation and/or progression of mammary tumors in SD rats is hormone-dependent as ovariectomy with or without adrenalectomy between PND 77–102 almost completely prevents mammary tumor development, reducing the overall incidence from 70% to 4% [4,7]. Tumor onset in rats typically occurs concurrently with the onset of acyclicity, which in rats, unlike in women, is marked by a transient phase characterized by persistent secretion of 17β-estradiol from existing follicles (persistent estrus) [8,9]. Because of this temporal association, a causal role for 17β-estradiol’s direct or indirect effects (e.g. inducing lactrotrope hypertrophy and prolactin secretion) on mammary tumor development during this transitional period has been proposed, but not proven [10]. However, no additional information regarding the hormone-responsiveness of these spontaneous tumors is available.

In order to elucidate specific elements of the hormonal milieu that impact fibroadenoma formation in female SD rats, we chose to examine the effect of an ovary-intact, chemically-induced model of premature ovarian failure on mammary tumor occurrence. The occupational chemical 4-vinylcyclohexene diepoxide (VCD), produced in the plastics industry and studied by the National Toxicology Program in the 1980’s [1116], has been exploited in translational research after its effects on the female reproductive system were discovered [1014]. In this chemical model of pre-mature ovarian failure, repeated administration of VCD to one-month rodents can accelerate the natural process of pre-antral folliclular atresia within the ovary by inhibition of c-KIT survival signaling [13]. In the absence of a pool of immature follicles, the onset of ovarian failure is accelerated [1216]. Thus, one might initially postulate that this chemically-accelerated form of ovarian failure could limit mammary tumor development, analogous to the protective effects of surgically–induced ovarian failure (ovariectomy). However, a careful comparison of the hormonal changes occurring in each of these ovarian failure models suggests alternative hypotheses. Firstly, because androgens suppress mammary tumor formation [17], the persistence of ovarian androgen production in ovary-intact, VCD-induced ovarian failure (vs. complete ablation of the ovary with ovariectomy) may prevent the occurrence of spontaneous mammary tumors. Alternatively, by accelerating the onset and/or prolonging the duration of persistent estrus during the transition to ovarian failure, VCD-treatment could actually speed mammary tumor development. To assess the relative importance of these alterations in ovarian hormone production on fibroadenoma development, experiments were undertaken to assess the dose-dependent effects of VCD on spontaneous mammary tumor development during persistent estrus and the onset of ovarian senescence in SD rats.

2. Materials and methods

2.1 Animals procedures

Young (1-month) female Sprague Dawley rats (Harlan Laboratories) were housed in plastic cages and maintained on a 12L/12D schedule at 22 ±2 °C, with food (Teklad Global Diet 2018S) and water available ad libitum. After one week of acclimation to the animal facility, rats were randomly assigned to treatment groups. 4-Vinylcyclohexene diepoxide (VCD; ≥ 96% purity) was obtained from Sigma and stored at −20 °C. Following established protocols [16], young SD rats were administered 25 intraperitoneal (ip) doses of VCD between post-natal days (PND) 35–68 (80 mg/kg or 160 mg/kg; n = 12 and 21, respectively), or vehicle (1.25 µL/g/d DMSO; n = 17). VCD dosages were selected based on their known ovotoxic effects in the Fischer F344 rat strain [16]. A subset of animals from vehicle (n = 7) and high dose VCD (160 mg/kg; n = 10) treatment groups was sacrificed following dosing on PND 53 in order to assess direct effects of VCD on the developing breast. The remaining animals were monitored visually and by palpation for mammary tumor onset and incidence by individuals blinded to treatment group for 570 days post-treatment, which was equivalent to PND 604, or 22 months of age, where 1 month = 28 days). In order to determine whether the stage of mammary development at time of VCD exposure affected outcome, a separate study was conducted in which mature (3-month) SD rats were treated with the same VCD doses (80 mg/kg or 160 mg/kg × 25 d; n = 7 or n = 12, respectively) or vehicle (n = 17) between PND 94–119 and monitored for mammary tumor onset and incidence for 261 days post-treatment, which was equivalent to PND 355, or 13 months of age. Morning blood serum and plasma samples were collected periodically throughout the experiment from the tail vein in anesthetized rats (8 mL xylazine, 5 mL ketamine, 2 mL acepromazine; 1 µL/g), and stored at −80 °C for subsequent hormone assays. All experiments were approved by The University of Arizona IACUC and conformed to the Guide for the Care and Use of Experimental Animals.

2.2 Mammary tumor histopathology

Mammary tumors were removed at the termination of the experiment or were surgically excised during the experimental period from anesthetized animals if tumors reached a size that restricted the animal’s mobility (~3 cm diameter). Animals with excised tumors remained in the study and were monitored for tumor formation at new sites. Mammary tumors were fixed in phosphate buffered formalin, and transferred to 70% ethanol after 48 hours. Tumors were then paraffin embedded, and 5 µm sections were stained with hematoxylin and eosin for histologic evaluation. Tumor histopathology was determined by an ACVP board-certified veterinary pathologist (DGB) who was blinded to treatment groups.

2.3 Hormone assays

Circulating 17β-estradiol and androstenedione were measured using commercially available radioimmunoassays (Siemens) as per manufacturer’s protocol with sensitivities of 2.5 pg/mL and 0.03 ng/mL, respectively. Plasma prolactin levels were measured by enzyme-linked immunosorbent assay (ELISA, Calbiotech) with a sensitivity of 0.2 ng/ml.

2.4 Evaluation of estrous cyclicity

Temporal changes in reproductive status were monitored each month for 10 day intervals by assessment of vaginal cytology based on standard techniques [8,9]. Briefly, vaginal smears were considered indicative of proestrus if round, nucleated epithelial cells were observed, estrus if large cornified cells were observed, metestrus if a combination of leukocytes, cornified and round epithelial cells were observed, and diestrus if round epithelial cells and leukocytes were observed. As previously described [8], the reproductive stage of each animal was further classified as “epithelial phase” when only nucleated or cornified cells were present in the vaginal smear, and the beginning of ovarian senescence was considered to be ≥ 75% of days in the epithelial phase, which is indicative of persistent estrus and precedes ovarian failure, defined here as the end of persistent estrus [8,9].

2.5 Mammary whole mounts analysis

The fourth mammary gland, harvested on PND 53 after administration of 15 doses of 160 mg/kg/d VCD or vehicle to 1-month rats (n = 7 per group) was mounted on slides and fixed overnight in 1:3 acetic acid:ethanol, followed by de-fatting in acetone for 10 days. Acetone was refreshed every 48 hours. Tissues were then stained overnight with carmine alum, dehydrated in a series of graded ethanol, and stored in glycerol. The distal 5 mm margin of the mammary whole mount was examined for terminal end buds (TEB), terminal ducts (TD) and alveolar buds (AB; 3–5 buds), by light microscopy. Epithelial structures were determined using the criteria of Russo and Russo [18] by an individual blinded to treatment groups. Briefly, a bulb-shaped terminal ductal structure > 100 µm in diameter with three to six epithelial cell layers in the perimeter of the bulb was designated a terminal end bud (TEB). A terminal duct (TD) was classified as a terminal structure < 100 µm in size with one to three epithelial layers between the ductal lumen and the outer edge of the structure. Alveolar buds (ABs) were lobular structures composed of three to five buds in one cluster. Alveolar bud lobules (> 5 buds) were not detected in the distal 5 mm margin at the time point examined.

2.6 Real-time quantitative RT-PCR

RNA was isolated from mammary tissue of vehicle-treated or VCD-treated rats (160 mg/kg/d; n = 7 and n = 10, respectively) on PND 53 after 15 doses. Mammary tissue was harvested, flash-frozen in liquid nitrogen and stored at −80 °C. Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA) and followed by 2.5M lithium chloride precipitation, as previously described [19]. RNA (250 µg) was reverse transcribed (iScript; BioRad) and changes in expression of c-KIT (Rn00573942_m1), KIT-ligand (Rn01502851_m1), cyclin D1 (Rn00432359_m1) and β-casein (Rn01524626_m1) were determined by TaqMan real-time RT-PCR analysis in using rat-specific primers obtained from Applied Biosystems. Data were analyzed using the comparative cycle threshold (C1) method as a means of relative quantitation of gene expression, normalized to an endogenous reference (18s RNA), as previously described [19].

2.7 Immunofluorescence and confocal microscopy

The fourth mammary glands, isolated from 53 day-old rats treated with 15 daily doses of vehicle vs. 160 mg/kg/d VCD, were fixed in phosphate buffered formalin, paraffin embedded, sectioned (5 µm), deparaffinized, and prepared for confocal microscopy, as previously described [13]. Briefly, sections were treated for antigen retrieval by immersion in citrate buffer (10 mM sodium citrate; pH 6.0) and microwave heating for 2.5 minutes at boiling temperature, followed by 7 minutes at sub-boiling temperature. Tissue sections were blocked with 5% BSA/PBS, and polyclonal rabbit antihuman c-KIT (1:400 dilution; A4502, Dako), which is known to react with rat c-KIT [20], was applied for 18 h, followed by biotinylated goat anti-rabbit secondary antibody (Vector) at a 1:75 dilution for 1 h (4 °C) with further streptavidin/biotin amplification. Sections were treated with Cy5-streptavidin (Jackson ImmunoResearch Labs), followed by the nuclear stain YOYO-1 (Molecular Probes). Slides were washed with PBS, coverslipped with aqueous mounting medium, and stored in the dark at 4 °C until viewed on Zeiss (LSM 510 NLOMeta) confocal microscope with an argon and helium-neon laser projected though the tissue into a photomultiplier at λ = 633 and 488 nm for Cy5 and YOYO-1, respectively. Specificity of primary antibody for detection of c-KIT was determined by verification of the absence of staining when primary antibody was omitted (data not shown).

2.8 Statistical methods

Kaplan Meier survival plots were analyzed by Logrank Test with post-hoc Logrank Test for trend to confirm dose-dependency of tumor development. Chi-square analysis was performed to compare tumor incidence between VCD- and vehicle-treated groups at individual time points. One-way analysis of variance (ANOVA) with Bonferroni post-hoc testing, repeated measures one-way ANOVA for serial hormone analysis, or student unpaired t-test was performed on parametric data, as appropriate. All analyses were performed with InStat software (Graphpad, San Diego, CA).

3. Results

3.1 Ovarian Function Following PND 35–68 VCD Administration

The onset of persistent estrus in low dose VCD (80 mg/kg)-treated animals was no different than vehicle-treated rats (PND 329 ±32 vs PND 314 ±19, p > 0.05; Table 1). In contrast, administration of high dose VCD significantly accelerated the onset of persistent estrus (PND 167 ±19, p < 0.001 vs. vehicle; Table 1). Similarly, the transition from persistent estrus to ovarian failure was unchanged in rats treated with low dose VCD and was accelerated in high dose VCD-treated rats (PND 317 ±27 vs. 445 ±21 in vehicle, p < 0.001; Table 1). Circulating levels of 17β-estradiol and androstenedione, assayed at 90 day intervals, were not different in VCD-treated animals as compared to controls, including at the end of the experiment (Table 2).

TABLE I
Effect of VCD on reproductive cyclycity of female Sprague Dawley rats a
TABLE II
Effect of VCD on sex steroids of female Sprague Dawley rats a

3.2 Mammary Tumor Onset and Incidence Following PND 35–68 VCD Administration

In control (vehicle-treated) animals, as anticipated from prior reports, mammary tumors were not clinically evident until PND 394 (Figure 1A). At this time point, all control rats had been in a state of persistent estrus for approximately 2.5 months (Table 1; Figure 1C). In contrast, mammary tumors were evident as early as PND 219 in both high and low dose VCD-treated animals, preceding tumor onset in vehicle-treated controls by 6 months (175 days; Figure 1A). In rats treated with high dose VCD, tumor onset occurred less than 2-months after the accelerated onset of persistent estrus, while tumor onset in low dose VCD rats actually preceded persistent estrus by 4 months (110 days; Figure 1A,C). Mammary tumor incidence over time was higher in VCD-treated rats vs. vehicle-treated controls (p = 0.03) and was dose-dependent (p < 0.01; Figure 1A). Final tumor incidence in both low and high dose VCD animals significantly exceeded that of controls (83–85% vs. 38%; Figure 1A). Additionally, VCD had a dose-dependent effect on tumor burden, as the average number of tumors per affected rat was double and triple that of controls with 80 and 160 mg/kg VCD, respectively (Figure 1B).

Fig. 1Fig. 1Fig. 1
Effect of VCD on fibroadenoma formation in female SD rats. (A) Tumor incidence and onset were assessed over time with 80 and 160 mg/kg VCD relative to vehicle-treated controls, expressed in a Kaplan Meier plot and analyzed by Logrank Test for treatment ...

3.3 Effect of Delayed (PND 94–119) VCD Administration on Ovarian Function and Mammary Tumors

As initial data suggested that the accelerated onset and higher incidence of mammary tumors in VCD-treated rats were not closely related to the onset or duration of persistent estrus, mature 3-month SD rats were dosed with VCD to assess the importance of mammary developmental stage at time of VCD exposure. Effects of VCD on ovarian function in 3-month animals recapitulated those demonstrated in 1-month rats, as low dose VCD did not alter the onset of persistent estrus, while the onset of persistent estrus in high dose VCD-treated animals was accelerated as compared to controls (PND 290 ±8.2 vs. PND 341 ±12, p < 0.01). However, treatment with either dose of VCD in mature animals did not increase mammary tumor incidence relative to control animals (tumor incidence at PND 355 was 0% in all 3 groups). When normalized to time post-VCD treatment (261 days), tumor incidence in young VCD-treated animals was significantly increased (38%; PND 295) relative to VCD-treated mature animals (0%, p < 0.01; PND 355), suggesting that tumor onset was dependent on age at time of VCD exposure.

3.4 Mammary Gland Histopathology

On gross inspection and palpation, all mammary tumors in vehicle and VCD-treated animals were subcutaneous, moderately firm and freely movable. A subset of tumors excised from the animals was examined histologically (27%, n = 14 of 52). All tumors were benign, with histopathology consistent with fibroadenoma for all specimens (Figure 2A), with the exception of one papillary cystadenoma (Figure 2B). The fibroadenomas (Figure 2A) were characterized as non-encapsulated, well-demarcated masses composed of multiple lobules of dense fibrous connective tissue separating numerous small ductules lined by low cuboidal epithelium. Cells within the connective tissue were spindle shaped with plump round to ovoid vesiculate nuclei, while ductular epithelial cells had minimal cytoplasm and round nuclei with finely stippled chromatin and occasional multiple nucleoli. The mitotic index was 0–2/HPF. The papillary cystadenoma (Figure 2B) was a partially encapsulated, well-demarcated mass composed of multiple lobules composed of cysts and intercalated papillary projections lined by a single layer of cuboidal to low columnar epithelium. Scant fibrous stroma was present, although there were a few small foci of dense fibrous tissue randomly interspersed in the mass. Epithelial cells had minimal cytoplasm and round to oval nuclei with finely stippled chromatin and occasional multiple nucleoli. Mitotic figures were rare. The cysts often contained hemorrhage with occasional hemoglobin crystals and clusters of hemosiderin-laden macrophages.

Fig. 2
VCD-induced mammary tumor histology. (A) Mammary fibroadenomas were characterized by multiple lobules of dense fibrous connective tissue separating numerous small ductules lined by low cuboidal epithelium that are occasionally filled with proteinaceous ...

3.5 Prolactin Assay

Because spontaneous fibroadenomas in SD rats have been postulated to occur in response to elevated prolactin during persistant estrus [21], serum prolactin levels were compared between groups at multiple times throughout the experimental period. Prolactin levels, while tending to increase with age in all groups, were not different between VCD-treated and control animals at any time point during the study (Figure 3).

Fig. 3
Effect of VCD treatment on plasma prolactin. Following dosing with 4-vinylcyclohexene diepoxide (VCD, 80 or 160 mg/kg) or vehicle control, plasma prolactin levels were assessed by enzyme-linked immunosorbent assay (ELISA, Calbiotech) on PND 59, 219, 282 ...

3.6 Mammary Whole Mount Analysis During VCD Exposure

As VCD only stimulated fibroadenoma formation when exposure occurred during a time of rapid mammary gland proliferation and differentiation, direct effects of VCD (160 mg/kg/d, PND 28–53) vs. vehicle on mammary ductal epithelial structures were assessed (Figure 4). The number of terminal end buds (TEB) and terminal ducts (TD) were not significantly different between vehicle- and VCD-treated animals (Figure 4A,B); however, the number of alveolar buds (AB) was significantly decreased (−36%) with VCD dosing (p = 0.02; Figure 4A–D).

Fig. 4Fig. 4
Direct effect of VCD on mammary epithelium. The number of terminal end buds (TEB), alveolar buds (AB) and terminal ducts (TD) per mm2 were assessed in whole mount preparations of the fourth mammary gland of 53 d female Sprague Dawley rats immediately ...

3.7 Direct Effect of VCD on Gene Expression of Markers of Mammary Gland Proliferation and Differentiation

Expression of cyclin D1, a proliferation marker for the mammary epithelium [20], was not changed with VCD treatment (160 mg/kg/d, PND 28–53; Figure 5A), whereas, gene expression of β-casein, a milk protein and biomarker for differentiation and maturation of the mammary epithelium [22], was down-regulated with VCD exposure (Figure 5A). Expression of c-KIT and KIT-ligand, genes associated with cell growth and differentiation in the mammary gland [23], was next assessed in mammary ductal epithelium since VCD is known to target c-KIT signaling in ovarian follicles [13]. c-KIT and KIT-ligand genes were expressed in control mammary tissue, but their expression levels were not significantly changed following 15-days of VCD treatment (160 mg/kg/d) in PND 53 rats (Figure 5A). Consistent with this finding, immunofluorescent c-KIT protein, which localized to mammary ductal epithelial structures in control tissue (PND 53, Figure 5B,D), was not noticeably different with respect to expression pattern or intensity in VCD-treated vs. control animals (Figure 5C,E).

Fig. 5Fig. 5Fig. 5Fig. 5Fig. 5
Effect of VCD on mammary gene expression. (A) Cyclin D1, β-casein, c-KIT and KIT-ligand mRNA levels were determined by real-time RT-PCR analysis in mammary tissue isolated from 53 d female Sprague Dawley rats immediately following 15 doses of ...

4. Discussion

Benign mammary fibroadenomas have been linked to increased risk of breast cancer, indicating that, while not necessarily causative, benign and malignant breast lesions may share a common underlying mechanism or risk factor. Pre-malignant stages of mammary tumor development can best be manipulated and studied in rodent models, and these models may ultimately shed light on the etiology of human disease. Mammary tumor development has been linked to a number of risk factors, both exogenous (e.g. environmental exposure) and endogenous (e.g. sex hormones) [24,25]. The ovotoxic chemical 4-vinylcyclohexene diepoxide (VCD) was selected in this study due to its ability to manipulate the time of onset of ovarian failure by accelerating the normal process of follicular atresia in rats [16]. The initial aim of this study was to elucidate the specific effect of accelerated onset of persistent estrus, as well as ovary-intact, androgen-replete ovarian failure on spontaneous mammary tumor development.

VCD treatment, when begun at 1 month of age, did in fact alter mammary tumor development, accelerating the onset and increasing the incidence of mammary tumors, with a latency period of 6 months (185 days) relative to onset of exposure. Observed differences in mammary tumor onset and incidence, however, could not be attributed to VCD-induced changes in ovarian function, as mammary tumors appeared in 1-month VCD-treated animals even before changes in ovarian function and achieved a higher incidence despite a similar duration of persistent estrus in VCD-treated vs. control animals. Moreover, high dose VCD treatment of 3-month old rats, while accelerating the onset of persistent estrus, did not alter the onset or incidence of mammary tumor development. Thus, these results suggest that, contrary to our initial hypotheses, VCD’s tumorigenic effect may be mediated via direct effects on the mammary gland, and that early onset of ovarian senescence was not a factor in stimulating tumor onset or incidence in this model.

The absence of a role for ovarian hormones in mediating VCD-induced mammary tumor formation was further confirmed by assessment of ambient serum levels of 17β-estradiol and androstenedione in VCD-treated rats, which were no different than controls at any time. Further, because rising prolactin (PRL) levels in a setting of elevated follicle stimulating hormone have also been postulated to drive mammary tumor formation in normal rats [21], serum PRL levels were compared between groups. While tending to increase with age in all animals, PRL levels were not different between VCD and control animals at any time point during the study, suggesting that prolactin was not a causative factor driving the high tumor incidence seen in VCD-treated animals.

While finding no evidence for a hormonal cause of increased tumor formation in VCD-treated rats, the evidence instead pointed to a possible direct tumorigenic effect of VCD on the mammary epithelium, as tumor incidence only increased when VCD exposure occurred during the period from PND 35–68 when terminal end buds proliferate and differentiate into alveolar buds [18]. It would thus appear that VCD can be included in the growing list of polycyclic hydrocarbons, such as 7,12 dimethylbenz [α] anthracene (DMBA), that induce mammary neoplasia by targeting the developing mammary ductal system during this developmental stage, classically known as “the window of susceptibility” [18,26,27]. DMBA, which stimulates proliferation of undifferentiated mammary epithelium during TEB development, is frequently used experimentally to induce mammary carcinoma formation [22,2631]. In contrast to DMBA, VCD appeared to target mammary epithelial differentiation, reducing mammary alveolar bud number and expression of β-casein milk protein, a biomarker for mature mammary epithelium [22], while having no effect on proliferation (i.e. TEB number and cyclin D1 expression). Thus, en toto, these data suggest that blockade of mammary epithelial differentiation may induce fibroadenoma formation, while enhanced proliferation of undifferentiated epithelium drives carcinoma formation. Chemical disregulation of epithelial proliferation vs. differentiation may thus have an impact on the type of epithelial transformation (malignant vs. benign) that develops during the lifespan of an animal. Accelerated tumor formation with VCD treatment could also be a species- and strain-specific effect, as Sprague Dawley rats are highly prone to spontaneous fibroadenoma development with age [46].

The c-KIT signaling pathway is a key regulator of cell differentiation and survival in a wide range of tissues [3234]. While its role in mammary epithelium has not been a focus of study, it is known that loss of c-KIT expression is associated with malignant transformation of mammary epithelia in both rodents and humans [3537]. Because abrogation of c-KIT signaling by VCD and decreased c-KIT gene expression is the primary driver of VCD-induced ovotoxicity [13], we investigated the possibility that disruption of the c-KIT pathway in mammary tissue by VCD during epithelial differentiation could be permissive for later tumor development. However, neither c-KIT nor its ligand was significantly altered by VCD treatment on PND 53 after 15 days of VCD treatment. Although it remains possible that c-KIT expression was altered at other time points in the study, mammary c-KIT expression may be more highly correlated with malignant than benign transformation [20], potentially explaining why changes in c-KIT were not detected. It should be noted that these data are the first, to our knowledge, to document expression and localization of c-KIT, an understudied mammary signaling pathway, in the mammary ductal epithelium of female Sprague Dawley rats.

In conclusion, VCD-induced mammary tumorigenesis may be a useful tool for examining perturbations in the physiology of branching morphogenesis in the breast, as well as the pathophysiology of fibroadenomas, a common disorder in women that is associated with increased breast cancer risk and is responsible for the majority of mammary biopsies [1]. Because human mammary fibroadenoma is an understudied disease, this novel model may provide new insights into the underlying cellular and molecular mechanisms responsible for mammary neoplasia.

Acknowledgements

This work was supported by the National Institutes of Health [grant number 5R21AT003614].

Footnotes

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Arsenic exposure through drinking water increases the risk of liver and cardiovascular diseases in the population of West Bengal, India

Abstract

Background

Arsenic is a natural drinking water contaminant affecting 26 million people in West Bengal, India. Chronic arsenic exposure causes cancer, cardiovascular disease, liver disease, neuropathies and ocular diseases. The aims of the present study were to assess bioindicators of hepatocellular injury as indicated by the levels of liver enzymes, to determine the auto immune status, as indicated by the amounts of anti-nuclear antibodies (ANA) and anti-dsDNA antibodies in their serum, and to predict cardiovascular risk in the arsenic exposed population.

Methods

Effect of chronic arsenic exposure on liver was determined by liver function tests. Autoimmune status was measured by measuring ANA and anti-dsDNA in serum. Inflammatory cytokines associated with increased cardiovascular disease risk, IL6, IL8 and MCP-1 were determined.

Results

Our results indicated that serum levels of bilirubin, alanine transaminase, aspartate transaminase, alkaline phosphatase and ANA were increased in the arsenic exposed population. Serum levels of IL6 and IL8 also increased in the arsenic exposed group.

Conclusions

Chronic arsenic exposure causes liver injury, increases the serum levels of autoimmune markers and imparts increased cardiovascular risk.

Keywords: Arsenic, Antinuclear antibody, Liver function tests, Cytokines

Background

Arsenic is a paradoxical human carcinogen affecting millions of people around the world. At present, people in more than 35 countries across the globe are affected by drinking arsenic-contaminated ground water. In India, West Bengal is the worst affected state where more than 26 million people in 9 out of 18 districts are drinking heavily contaminated ground water through hand-pumped tube-wells [1]. The arsenic concentrations in these districts are far above the current maximum contaminant level (MCL) established by WHO and US EPA i.e. 10 μg/l [2,3]. This is regarded as the greatest arsenic calamity in the world [4]. Long term exposure to arsenic-contaminated water causes a wide range of adverse health effects, including cancer [5], cardiovascular disease [6], diabetes mellitus [7], neuropathies [8], liver disease [9], ocular diseases [8], and skin lesions [5,10]. The skin lesions include rain drop pigmentation, hypopigmentation, hyperpigmentation, keratoses and skin cancers including Bowen’s disease, basal cell carcinoma and squamous cell carcinoma [11,12]. More than 300,000 people in West Bengal have these skin lesions that are hallmarks of chronic arsenic toxicity. Prolonged arsenic ingestion leads to its accumulation in the liver, kidneys, heart and lungs and in smaller amounts in the muscles, nervous system, gastrointestinal tract and spleen [13]. Respiratory disease is also common in arsenic toxicity [8,11,14].

Among the various internal organs affected by chronic exposure to arsenic in humans, liver is one of the important targets. Epidemiological studies have shown association between chronic arsenic exposure and liver disease including hepatomegaly, hepatoportal sclerosis, liver fibrosis and cirrhosis of liver [9,15-17]. Abnormal liver functions as manifested by severe gastrointestinal problems and clinical elevations of liver enzymes in plasma including alanine amino transferase (ALT), asparatate amino transferase (AST), alkaline phosphatase (ALP) also are associated with chronic arsenic exposure [9,15,18]. Exposure of mice to arsenic in drinking water causes elevation of liver enzymes in plasma [19] and capilarization of liver sinusoidal endothelium [20]. Infact, liver is the major site of arsenic metabolism [21] and hence arsenic exposure causes liver disease in exposed humans [9].

Antinuclear antibodies (ANA) are a diverse group of autoantibodies that are found in autoimmune disorders like systemic lupus erythomatosus, Rheumatoid arthritis, Sjorgen’s syndrome, systemic sclerosis and inflammatory myositis. Arsenic exposure increases the incidence of auto-immune diseases like diabetes mellitus [7]. ANA are present in lower titers in liver diseases, leprosy, multiple sclerosis and juvenile rheumatoid arthritis [22].The association of chronic arsenic exposure and autoimmune disorders has received only minimal attention [23,24].

Inflammation has been found to be one of the most important factors that contribute to cardiovascular disease. Elevated levels of monocyte chemoattractant protein (MCP-1), interleukin 6 (IL6), and tumor necrosis factor alpha (TNFα), produced by the immune system play important roles in increasing the risk of cardiovascular disease. Both IL6 and TNFα play important roles in the regulation of the synthesis of other acute phase proteins which are established risk factors for atherosclerosis [25]. Exposure of mice to arsenic in drinking water causes the induction of inflammatory cytokines associated with increased atherosclerosis [26].

We have been studying health effects, cytogenetic damage, genetic variants, and immunological changes in the population exposed to arsenic through drinking water in West Bengal [5,10,12,27-32]. During our epidemiological survey, we found that the arsenic exposed population complains of muscular cramps and joint pains, which are characteristics symptoms of rheumatoid arthritis. In the present study we assessed bioindicators of hepatocellular injury as indicated by the serum levels of liver enzymes, determined the autoimmune status as indicated by the amounts of serum ANA and anti-dsDNA and measured circulating inflammatory cytokines related to cardiovascular diseases in the population exposed to very high arsenic content in their drinking water. The results have been compared with a group of arsenic unexposed individuals (controls). We show that the arsenic exposed population exhibits increased liver enzymes, increased serum ANA and inflammatory cytokines indicating liver injury, heightened autoimmunity, and increased likelihood of cardiovascular disease.

Methods

Chemicals

Ascorbic acid, acetone, nitric acid (69% GR), hydrochloric acid (35%), potassium iodide, sodium hydroxide, and sodium borohydride (96% pure) were obtained from Merck (Hohenbrunn, Germany). Arsenic (III) and arsenic (V) AAS (Atomic Absorption Spectrometry) standards were obtained from Qualigens (Shanon Co. Clare, Ireland). ELISA kits for ANA and anti-ds DNA were obtained from Calbiotech Inc. (Spring valley, CA, USA). Kits for estimation of ALT, AST,ALP, bilirubin, total protein and GGT were obtained from Randox laboratories limited (Antrim, United Kingdom). ELISA kits for IL6, IL8, and MCP-1 were obtained from Thermo Scientific (Pierce Biotechnologies, Rockford, USA).

Study site and study population

Murshidabad district is a highly contaminated district in West Bengal India. Here people are exposed to arsenic by drinking heavily contaminated ground water, which is much above the permissible limits laid by WHO [2]. In our epidemiological survey, we initially recruited a large number of participants, from which we selected those with the highest current exposure (as measured by arsenic concentration in water and urine) in Murshidabad district for the current study. Thus, 103 arsenic exposed individuals (both with and without skin lesions) were chosen while 107 unexposed individuals were chosen from arsenic unexposed East Midnapur district, of the same state, where arsenic in drinking water ranges between 3-10 μg/L. Individuals ranging from 15 to 78 years of age with at least 10 years of exposure were included as exposed study participants. Initially, four trained volunteers were sent to the villages for door-to-door survey to identify individuals with skin lesions among the villagers. All the villagers were requested to attend the medical camp irrespective of the presence of arsenic-induced skin lesions. An interview was performed using a structured questionnaire that elicited information about demographic factors, life-style, occupation, diet, tobacco usage, medical, and residential histories [8,31]. Detailed information on current and life time tobacco usage were obtained. Of the study population 10% were randomly re-interviewed in the field to verify the accuracy of the information provided. A team of physicians consisting of specialists in the fields of dermatology, neurology, ophthalmology and respiratory diseases, each with 15 years of experience examined the study participants. Samples were collected only from those subjects who provided informed consent to participate and fulfilled the inclusion criteria. Arsenic-exposed individuals and unexposed individuals were matched with respect to age, sex, and socio-economic status. Occupationally, the majority of the study participants were farmers and household workers. In general these population are from the rural Bengal and belongs to low income group. The arsenic exposed individuals show various non cancerous, precancerous and cancerous skin lesions which are hallmarks of chronic arsenic toxicity. The non-dermatological health outcomes include conjunctivitis, peripheral neuropathy and respiratory problems [8]. In the course of our study, we have found that the arsenic-exposed individuals are more susceptible to opportunistic infections, respiratory problems, fungal and parasitic infections. Our earlier studies revealed that these individuals had impaired macrophage functions and increased death of immune cells by apoptosis [10,28].

Since arsenic containing pesticides were not very common in these areas and arsenic mining was not done in this region, occupational exposure to arsenic was ruled out. Therefore, drinking water is the principle source of arsenic in this region. Water and urine samples were collected from the subjects on the same day, which carried code numbers. Information from questionnaire-sourced data on the subjects was not revealed before arsenic analyses were completed. This study was conducted in accord with the Helsinki II Declaration (https://www.wma.net/e/policy/b3.htm) and approved by the Institutional Ethics Committee named “The Ethical Committee on Human Subjects, Indian Institute of Chemical Biology” dated April 26, 2010.

Physical examination of skin

A careful examination of skin was conducted under natural daylight to reveal the presence of typical arsenic-induced skin lesions including raindrop, hypo- and hyper-pigmentation, palmo- and planter-hyperkeratosis. Visible or palpable dermal lesions were documented. Physical examination of skin identified three types of cancerous lesions: squamous cell carcinoma, Bowen’s disease and basal cell carcinoma. Two dermatologists with more than 15 years of experience in the relevant field, jointly diagnosed any “probable or doubtful” skin lesion. The cases that still remained doubtful were not considered. Arsenic-induced skin lesions served as a biological marker of arsenicosis. The dermatologists confirmed that the individuals without skin lesions did not develop arsenic-induced skin lesions even after prolonged arsenic exposure.

Analysis of arsenic in water and urine samples

All the study participants were provided with acid-washed [nitric acid–water (1:1)] polypropylene bottles for collection of drinking water (approximately 100 ml) into which nitric acid (1.0 ml/L) was added later as a preservative [32]. First morning voids (approx. 100 ml) were collected in precoded polypropylene bottles for arsenic estimation as these give the best measure of the recent arsenic exposure [33]. Immediately after collection, the samples were stored in salt–ice mixture and brought to the laboratory where they were kept at –20°C until estimation of arsenic was carried out. Flow injection–hydride generation–atomic absorption spectrometry was used for the determination of total arsenic in the collected samples. A Perkin-Elmer Model-Analyst 700 (Boston, MA, USA) spectrometer equipped with a Hewlett-Packard (Houston,TX, USA) Vectra computer with GEM software, Perkin-Elmer EDL System-2, arsenic lamp (lamp current 380 mA) was utilized for the purpose [28].

Isolation of serum from blood

Blood samples were collected by veinpuncture method. About 2 ml blood was stored aseptically in sterile 10 ml polypropylene tubes without any anti coagulant for serum collection. Care was taken to prevent any mechanical damage which might cause haemolysis of the blood. The tubes were allowed to stand in room temperature for few minutes and then put on ice and transferred to laboratory for further processing. Coagulated blood was centrifuged at 3000 rpm for 10 minutes and the clear serum was collected and stored at -80°C till analyzed.

Assay of bilirubin, liver enzymes and autoimmune markers

Total serum protein, bilirubin and AST, ALT, ALP and GGT activities were determined in serum samples of both the exposed and unexposed individuals using a Randox Daytona autoanalyser (Randox laboratories Ltd., Crumlin, Co. Antrim, UK). Serum ANA and anti-dsDNA were determined using ELISA kit from Calbiotech Inc. (Spring valley, CA,USA) according to manufacturer’s instructions.

Cytokine quantification

Serum levels of cytokines IL6, IL8, and MCP-1 were measured in a subset of the total population under study. A total of 65 (32 arsenic exposed and 33 arsenic unexposed) individuals were randomly chosen for measurement of serum IL6, IL8 and MCP1 levels .The individuals were matched with respect to age-sex-tobacco usage status. Serum samples were collected from coagulated blood. IL6, IL8, and MCP1 concentrations were measured by ELISA using IL6, IL8, and MCP1 ELISA kits from Thermo Scientific (Pierce Biotechnologies), following manufacturer’s instructions.

Statistical analysis

Mann Whitney test was performed to test for significant differences in all parameters, including arsenic contents in urine, water, liver function tests, ANA, anti ds-DNA, IL6, IL8, and MCP-1 between the unexposed and exposed groups. Data are expressed as mean ± SD. One way ANOVA with Tukey-Kramar Multiple Comparisons Post-Test (for parametric data) and Kruskal-Wallis Test with Dunn’s Post test (for non parametric data) were used to compare differences in the central tendencies of different liver function and autoimmune marker parameters in more than two groups. Microsoft Excel and Graph Pad Instat (San Diego, CA, USA) software were used for the purpose.

Results

Demographic characteristics of study participants

Descriptive characteristics of the exposed individuals and unexposed individuals are summarized in Table 1. The average ages of unexposed and exposed individuals 40.0 ± 13.0 and 40.1 ± 13.4 years respectively. Age distribution pattern of the study participants in both the groups are shown in Table 1. Total study population is divided into six subgroups of 10 years interval. Occupationally, majority of the male individuals were farmers and females were housewives in both categories. There was no significant difference in the age or sex distribution patterns, tobacco usage and socio-economic status between the unexposed and exposed groups (Table 1).

Table 1
Demographic characteristic of the arsenic unexposed and exposed study participants

Arsenic content in urine and water samples of the study populations

Arsenic concentration was measured in water and urine samples in the study populations and the results are summarized in Table 1. Arsenic content in water of the exposed group was significantly higher than the recommended MCL of 10 μg/L established by WHO and USEPA. In the unexposed control group, the arsenic content in water was always less than 10 μg/L. Results show that the concentration of arsenic in urine of arsenic exposed individuals also was significantly higher (p < 0.001) compared to the unexposed individuals.

Arsenic induced effects on enzyme markers of liver injury

We determined the status of liver injury in our study population by determining serum protein, bilirubin, and ALT, AST, ALP and GGT activities (Table 2). Bilirubin, AST, ALT, and ALP were increased in the exposed individuals, indicating chronic arsenic exposure had caused liver injury and malfunction in these people.

Table 2
Serum levels of the liver function test parameters in arsenic exposed and unexposed population

Arsenic induced effects on autoimmunity markers

The arsenic exposed population very often complains of muscular cramps and joint pain, which are typical symptoms of rheumatoid arthritis (an autoimmune disorder). Therefore, we predicted that autoimmunity markers might be elevated in the arsenic exposed population. Thus, we determined the serum levels of ANA and anti-dsDNA in our study population. We found that ANA levels were increased in the arsenic exposed population (Table 3). Interestingly, we did not find any difference in serum levels of anti-dsDNA in the unexposed and exposed groups.

Table 3
Serum levels of ANA and Anti-dsDNA in the arsenic exposed and unexposed population

Dose response relationship with urine arsenic concentration

We have grouped our exposed study population into four sub-groups depending upon the arsenic concentration in their urine. Group A consists of individuals having arsenic content in urine below 300 ppb, group B, C and D consists of individuals having urinary arsenic concentration of 301-600 ppb, 601-900 ppb and more than 900 ppb respectively. The results are shown in Table 4 which indicates that there is a significant increase in both liver function test parameters (AST and ALT) and autoimmune markers (ANA and anti-dsDNA) with increase in arsenic content in urine.

Table 4
Dose response measurement of liver function test parameters and autoimmune markers with urinary arsenic concentration

Arsenic induced secretion of cardiovascular markers

IL6, IL8 and MCP-1 are inflammatory cytokines associated with cardiovascular disease. Thus, we determined the circulating levels of these cytokines as indicators of cardiovascular disease associated with arsenic exposure (Table 5). We have found that IL6 and IL8 were increased significantly in the exposed group. The apparent increase in MCP-1 was not statistically significant.

Table 5
Comparison of three cytokine levels in serum of arsenic exposed and unexposed individuals

Discussion

Chronic arsenic exposure is well established as carcinogenic but interest in the non-cancer disease endpoints of arsenic exposure is of great interest. The non-cancer disease endpoints include cardiovascular disease [6] and immune dysfunction [34], as well as neuropathies and ocular diseases [8,35,36]. In the present study, we focused on biological markers of liver disease, autoimmunity and cardiovascular disease in a population in West Bengal exposed to high levels of arsenic in drinking water. Increased bilirubin and liver enzyme levels in serum indicated the presence of liver injury in the arsenic exposed individuals. Increased serum ANA indicated increased likelihood of autoimmunity, and increased serum inflammatory cytokine levels indicated increased systemic inflammation and risk of cardiovascular diseases in the arsenic exposed individuals.

Liver function tests are a helpful screening tool to detect hepatic dysfunction. Liver has to perform different kinds of biochemical, synthetic and excretory functions, so no single biochemical test can detect the global functions of liver. To detect the proper functioning of the liver, various tests are performed to detect specific liver activities. Among various parameters, serum bilirubin is the marker the liver’s capacity to transport organic anions and to metabolize drugs. Aminotransferases (ALT, AST), alkaline phosphatase (ALP), aminopeptidase are the tests to detect injury to hepatocytes. Total protein is the marker for liver’s biosynthetic capacity [37]. Bilirubin is an endogenous anion derived from hemoglobin degradation. Underlying liver disease is indicated when the liver function tests are abnormal and serum bilirubin levels are elevated. Hyperbilirubinemia seen in acute viral hepatitis is directly proportional to the degree of histological injury of hepatocytes and the longer course of the disease [38]. The aminotransferases, AST and ALT are the most frequently utilized indicators of hepatocellular necrosis. ALT is primarily localized to the liver but the AST is present in a wide variety of tissues like the heart, skeletal muscle, kidney, and brain as well as liver. The AST and ALT levels are increased to some extent in almost all liver diseases. The highest elevations occur in severe viral hepatitis, drug or toxin induced hepatic necrosis and circulatory shock [38]. Higher levels of alkaline phosphatase occur in cholestatic disorders. Elevations occur as a result of both intrahepatic and extrahepatic obstruction to bile flow and the degree of elevation does not help to distinguish between the two. The mechanism by which alkaline phosphatase reaches the circulation may be due to leakage from the bile canaliculi into hepatic sinusoids via leaky tight junctions [39]. In healthy people most circulating alkaline phosphatase originates from liver or bone [40]. In liver disease, glutamyl transpeptidase activity correlates well with alkaline phosphatase levels [41]. So, clinicians are often confused when they see elevated alkaline phosphatase levels and are unable to differentiate between liver diseases and bony disorders and in such situations measurement of glutamyl transferase helps as it is raised only in cholestatic disorders and not in bone diseases. Elevated levels of these liver function enzymes have been associated with hepatic dysfunctions in lead [42,43] and cadmium [44] exposure. In our study, we have found that serum levels of bilirubin, ALT, AST, and ALP have increased significantly in the arsenic exposed population when compared to the unexposed group with similar socio-economic status. These increases clearly indicate that chronic arsenic exposure causes injury to hepatocytes with damage to the liver’s capacity to transport organic anions and to metabolize drugs, cholestatic injury and impairment of the liver’s biosynthetic capacity. Our results are consistent with many findings of chronic exposure to arsenic associated with hepatomelagy, hepatoportal sclerosis, liver fibrosis and cirrhosis with concomitant increase in serum bilirubin, ALT, AST, and ALP [9,45,46].

ANA and anti-ds DNA are frequently found in serum of patients with different types of autoimmune disorders and are biomarkers of autoimmune disorders [47-50]. Rheumatic diseases that affect connective tissue, including the joints, bone, and muscle are associated with these antibodies. Autoimmune and rheumatic diseases can be difficult to diagnose. People with the same disease can have very different symptoms. A helpful strategy in the diagnosis of these diseases is to find and identify an autoantibody in the person's blood. ANA bind to several nuclear antigens. It is useful as a screen for many autoantibodies associated with systemic diseases. Presence of anti-dsDNA is one of the diagnostic criteria for systemic lupus erythomatosus, which is an autoimmune disorder. IgG class antibodies, antibodies to single stranded DNA (ssDNA) and IgM antibodies to DNA are found in number of connective diseases as well as in rheumatoid disorders [51,52]. Increased ANA titres have also been reported in arsenic exposed population earlier [23]. Thus, ANA can be utilized as a biomarker in early risk assessment of arsenic induced autoimmune disease in high risk arsenic zones, since most of the individuals did not show any symptoms of rheumatoid arthritis although they were ANA positive. We have also found a significant increase in the serum levels of ANA and anti ds DNA in arsenic exposed population compared to the unexposed population in West Bengal. Therefore ANA appears to be a useful biomarker in early risk assessment of arsenic exposure induced autoimmune disease, which might lead to rheumatoid arthritis or other types of autoimmune diseases.

In order to find out the dose response of the LFT parameters and autoimmune markers with the increase of arsenic concentration in urine, we have stratified the data of the exposed population into four sub-groups depending upon the arsenic content in urine as mentioned in the results section. Our findings show that an increasing trend is observed in both LFT parameters and autoimmune markers with increasing urinary arsenic concentration. The dose response increase is significant in case of AST, ALT, ANA and anti-dsDNA indicating that with increase in arsenic exposure (as urine is the best current exposure marker), the levels of hepatic damage and autoimmune response increases in the exposed individuals. Although no dose response is observed in bilirubin and alkaline phosphatase levels with urinary arsenic content compared to unexposed group , the significant increase in levels of other hepatic parameter indicates that increase in arsenic exposure causes hepatic injury since all the parameters together contribute to hepatic damage.

Cytokines play pivotal roles in systemic inflammation and thus in cardiovascular diseases [52]. In atherosclerotic plaque, inflammatory cytokines (IL6, IL8, MCP-1, TNFα, IL1b) secreted by macrophages, dendritic cells, T cells and smooth muscle cells, aggravate plaque instability by inhibiting extracellular matrix synthesis and promoting smooth muscle cell apoptosis. Elevated levels of IL8 found in atherosclerotic plaques suggest that it acts as important mediator of angiogenesis in this tissue contributing to plaque formation [53]. Elevated plasma levels of IL6 and TNFα were detected in patients with stable or unstable angina and myocardial infarction [54,55]. It was also found that increased plasma levels of IL6, IL8 and MCP-1 were associated with cardiovascular risk in patients with systemic lupus erythematosus [56]. Arsenic exposure has been connected with a host of cardiovascular risk factors and endpoints including peripheral arterial disease (blackfoot disease), atherosclerosis, coronary heart disease, stroke, and hypertension [57]. It has been found that the expression of several cytokine related genes is increased in human subjects with increase in arsenic exposure, including heme oxygenase-1 (HO-1), MCP-1 and IL6 [58]. The secretion of theses cytokines are regulated by reactive oxygen species (ROS) and they are involved in modulating the biological functions of vascular smooth muscle cells are thus are involved in atherosclerosis [59]. In our previous study we have found increased generation of ROS [10] in the arsenic exposed population of West Bengal. The increased oxidative stress due to arsenic exposure may influence inflammatory responses in the vascular cells. We can conclude that increased serum concentrations of these cytokines and chemokines (IL6, IL8 and MCP-1) may act as early biomarkers of increased cardiovascular risk in the arsenic exposed subjects. In this context it is worthwhile to discuss that Kupffer cells in the liver are a major source of the inflammatory cytokines upon hepatotoxic insult [60]. Our results show that liver injury by arsenic exposure. Thus, the injured hepatic cells may induce increased secretion of the inflammatory cytokines (IL6, IL8, TNFα) in the arsenic exposed individuals which in turn contributes to cardiovascular risk.

Conclusions

Chronic arsenic exposure causes a wide variety of human diseases. Exposed individuals are at higher risk of developing liver and cardiovascular disease, as indicated by elevated serum levels of liver injury biomarkers and inflammatory cytokines. Increase of autoimmune markers in the serum suggests that arsenic exposure also induces autoimmune diseases such as rheumatoid arthritis. Both rheumatoid arthritis and liver disease are risk factors for cardiovascular disease. Thus, the arsenic exposure induced systemic inflammatory disease and the hepatic injury likely contributes to increased cardiovascular risk observed in arsenic exposed populations.

Abbreviations

ALP, Alkaline phosphatase; ALT, Alanine transaminase; ANA, Anti-nuclear antibody; AST, Aspartate transaminase; GGT, γ-Glutamyl transpeptidase; IL6, Interleukin 6; IL8, Interleukin 8; MCL, Maximum contaminant level; MCP-1, Monocyte chemoattractant protein-1; TNFα, Tumor necrosis factor alpha.

Competing interests

The authors declare that there exists no competing financial interest among any of the authors.

Authors’ contributions

AKG and JCS planned, designed and wrote the final manuscript, ND and SP collected samples, performed experimental works and prepared manuscript draft, NB performed experimental works and wrote manuscript draft, DC and NSM collected samples and performed field works, NS, TJS, SB, PM, AKB performed epidemiological survey. All authors read and approved the final manuscript.

Authors’ information

Dr. Ashok K. Giri is a Chief Scientist at the CSIR-Indian Institute of Chemical Biology. His work includes various epidemiological and molecular biological aspects of the highly arsenic exposed population of West Bengal. He has more than 20 peer reviewed publications projecting research work of more than 10 years in this area.

Pre-publication history

The pre-publication history for this paper can be accessed here:

https://www.biomedcentral.com/1471-2458/12/639/prepub

Acknowledgements

Authors are grateful to Fogarty International Training Program (2D43TW000815-11) jointly with University of California, Berkeley, for providing training to S.P., N.B. N. D., N.S.M. and S.B. for research on molecular epidemiology and environmental health. This study was funded by Council of Scientific and Industrial Research, Government of India (NWP-0004 and NWP-0052); U.S. Public Health Service (ES011314, ES014443) to J.C.S.

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Perfluorinated compounds are related to breast cancer risk in greenlandic inuit: A case control study

Abstract

Background

Breast cancer (BC) is the most common cancer for women in the western world. From very few cases an extraordinary increase in BC was observed in the Inuit population of Greenland and Canada although still lower than in western populations. Previous data suggest that exposure to persistent organic pollutants (POPs) might contribute to the risk of BC. Rat studies showed that perfluorinated compounds (PFCs) cause significantly increase in mammary fibroadenomas. This study aimed at evaluating the association between serum levels of POPs/PFCs in Greenlandic Inuit BC cases and their controls, and whether the combined POP related effect on nuclear hormone receptors affect BC risk.

Methods

Thirty-one BC cases and 115 controls were sampled during 2000-2003 from various Greenlandic districts. The serum levels of POPs, PFCs, some metals and the combined serum POP related effect on estrogen- (ER), androgen- (AR) and Ah-receptor (AhR) transactivity were determined. Independent student t-test was used to compare the differences and the odds ratios were estimated by unconditional logistic regression models.

Results

We observed for the very first time a significant association between serum PFC levels and the risk of BC. The BC cases also showed a significantly higher concentration of polychlorinated biphenyls at the highest quartile. Also for the combined serum POP induced agonistic AR transactivity significant association to BC risk was found, and cases elicited a higher frequency of samples with significant POP related hormone-like agonistic ER transactivity. The AhR toxic equivalent was lowest in cases.

Conclusions

The level of serum POPs, particularly PFCs, might be risk factors in the development of BC in Inuit. Hormone disruption by the combined serum POP related xenoestrogenic and xenoandrogenic activities may contribute to the risk of developing breast cancer in Inuit. Further investigations are needed to document these study conclusions.

Keywords: PFCs, POPs, combined serum xenohormone and dioxin-like activities, n-3 fatty acids

Background

Breast cancer (BC) is the most common cancer for women in the western world and the incidence has been increasing since 1940. The highest incidence rates are observed in North America, and the lowest risk is found in Asia and Africa [1]. Breast cancer is also the most common cancer in females in Europe with the highest incidence in The Netherlands and Denmark and lowest in the eastern part of Europe [2]. From very few cases in the 1970's an extraordinary increase in BC has been observed in the Inuit population of Greenland and Canada today [3,4]. Known established breast cancer risk factors include genetic inheritance e.g. mutations in the BRCA-1 and BRCA-2 genes [5], lifelong exposure to estrogens (early menarche and late menopause increases the risk), obesity after menopause, alcohol, smoking and high intake of fat [2,6,7]. Some factors seem to reduce the risk such as low age at first birth, large number of full term pregnancies and long duration of breastfeeding [6,7]. Thus, BC risk is influenced by genetics and reproductive history, but the known risk factors only explain less than a third of all cases and more than 70% of women diagnosed with BC have no inherited or sporadic cancer. The risk of BC is thought to be modified by lifestyle and environmental exposures. Results from many studies have confirmed that BC is not a single disease with variable morphologic features and biomarkers, but rather a group of molecularly distinct neoplastic disorders [8].

The susceptibility to BC upon environmental exposures might not only be as child, in puberty or young but can be pre-determined in utero through alterations of the hormonal environment caused by either maternal diet and/or exposure to environmental chemicals with endocrine activities that can modify the epigenome. These epigenetic modifications, inherited by somatic daughter cells, lead to changes in mammary gland development and increase the vulnerability for malignant transformation [9]. An array of legacy persistent organic pollutants (POPs) including polychlorinated dibenzodioxins and polychlorinated dibenzofurans (PCDD/PCDF), polychlorinated biphenyls (PCBs), and organochlorine pesticides (OCPs) are potential endocrine disrupters and can play an important role in the risk of BC. These persistent and lipophilic compounds do biomagnify through the food web and bioaccumulate age dependently in human and animals. PCBs have been associated with effects being relevant for development of BC such as estrogenic, tumour promoting, and immunosuppressive activities [10]. Evidence regarding organochlorine exposure and BC risk is controversial. A case-control study of Danish women showed a significant doses relation between the risk of BC and the serum level of the pesticide dieldrin, and a non-significant relation of BC risk at the highest level of dieldrin and PCBs among BC women with a tumour with mutant p53 [11,12]. In contrast, Raaschou et al. 2005 found no association between persistent organochlorines (14 pesticides and 18 PCBs) levels in adipose tissues and breast cancer in postmenopausal Danish women [13]. Thus, no strong evidence for the role of organochlorines, including PCBs, in BC development was found. However, two studies found a 3-fold risk in postmenopausal breast cancer with a A2455G mutation in the P450 polymorphic CYP1A1 gene and high PCB levels compared with wild-type alleles and low PCBs [14]. These associations between PCB exposure and BC risk among genetically susceptible and racially subgroups warrant further studies.

Perfluorinated compounds (PFCs) are a large group of chemicals used since the 1950's in different industrial and commercial applications (e.g. Teflon, carpets, furniture, food stuff packing etc.). For a long time, these fluorinated chemicals were considered metabolically inert and nontoxic [15] and the carbon-fluorine bond renders theses chemicals very resistant to biodegradation and therefore persistent in the environment [15]. Available evidence suggest the transformation or biodegradation of precursor perfluorinated chemicals occurs by both abiotic and biotic degradation pathways where perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) are typical final degradation products [16,17]. In 2001, it was discovered that the PFCs were accumulating in the environment and human tissues with a global distribution [16-18]. Unlike the legacy POPs (e.g. PCBs, OCPs) - which accumulate in lipid rich tissues - PFCs bind to blood proteins and accumulate mainly in liver, kidney and bile secrets [19]. Humans are exposed to PFCs through occupational settings, environmental exposures and/or through contact with consumer goods (diet, air, water, food and household dust) where PFCs have been found.

The perfluoroalkyl acids (PFAAs) include the perfluorocarboxylated acids (PFCAs) and perfluorosulfonated acids (PFSAs), and the PFAAs include the two most studied PFCs: PFOS and PFOA. These two compounds are the most studied because existing laboratory procedures in the past did not allow analyses of other PFCs that in general exist in lower concentrations. PFOA and PFOS are persistent in the environment and found in human blood, breast milk and liver with half lives of 4 to 10 years [20]. The PFCs are found globally and governmental regulations in USA and Europe on use and production of specific compounds such as PFOS and PFOA have been made. Recently, PFOS has been added to Annex B of the Stockholm Convention on POPs [21].

Biomonitoring studies have been carried out in almost all part of the world in order to assess PFAAs levels and temporal trends in the general population [22,23]. European studies observed serum and plasma concentrations range from 1 to 116 ng/ml for PFOS and from 0.5 to 40 ng/ml for PFOA. The average plasma levels of PFOS and PFOA in Danish pregnant women was 35.3 and 5.6 ng/ml, respectively, similar to most levels reported for western country populations during the same decade [24]. For middle aged women in Norway slightly lower levels were reported (medians: PFOS; 20 ng/ml; PFOA; 4.4 ng/ml). Mean and median concentrations from North American populations appear to be slightly higher than European, Asian and Australian populations [22]. Recently we performed a survey of serum PFCs in Greenlandic Inuit [25] showing similar PFOS levels in Inuit women as found for Danish and European women but lower PFOA levels.

The maternal level of PFOA or PFOS was found associated with various reproductive and child health outcomes [24,26-29], and seems to impact maternal fecundity [27].

The biological effects of PFCs have been studied in more detail mainly in rodents; little data are available for other species and humans [19,30]. Studies in animals have documented an array of toxicological outcomes including liver hypertrophy and tumours [31], thyroid hormone alterations, decreased serum cholesterol and glucose, developmental toxicity, immunotoxicity, and carcinogenic potency [32,33]. Animal and in vitro studies have also suggested that PFAAs may have potential geno- and neurotoxic effects [34-36]. U.S. EPA has proposed PFOA to be deemed as a rodent carcinogen with relevance to humans [37].

A 2-year study in rats [38] reported a statistically significant increase in mammary fibroadenomas and Leydig cell adenomas suggesting impact of PFOA on reproductive tissues. Because of these data the U.S. EPA Science Advisory Board recommended to reconsider the possible impact of PFOA on mammary tissues. In 2007 White and coworkers reported [39] that gestational exposure to PFOA in mice compared to non exposed controls was associated with altered mammary gland development in dams and female offspring. A significant reduction in mammary differentiation among exposed dams was evident, and also affected the epithelial involution and altered milk protein gene expression [39,40].

Recently, estrogen-like properties of PFCs were reported in human MCF-7 breast cancer cells suggesting endocrine potentials [41].

The objective of this BC case-control epidemiologic study in Greenlandic Inuit women was to evaluate the serum level of legacy POPs and PFCs, blood metals, and the combined xenobiotic serum POP related effect on the functions of the estrogen- (ER) and androgen receptors (AR) and the aryl hydrocarbon receptor (AhR) functions.

Methods

Study population and Data collection

The subjects of breast cancer cases were taken from Inuit women at the "Dronning Ingrids Hospital" in Nuuk, where all breast cancer cases in Greenland are registered. Approximately 80% of all BC cases were included in the sampling period 2000-2003. Subjects of controls were selected by frequency matching age and districts from a cross sectional study of POP concentrations and bone ultrasound measurement [42] and the Arctic Monitoring and Assessment Programme (AMAP) study [43]. The controls were matched with the cases having similar frequency of age ≤ 50, 51-55, 56-59, ≥ 60yrs (see table table1)1) and then frequency matched with the cases of the districts (see table of Figure Figure1).1). All subjects, 31 cases and 115 controls, were of Greenland Inuit decent, defined as having more than two grandparents born in Greenland. The sampling period was 2000-2003 with subjects from Nuuk, Upernavik, Qeqertarsuaq, Narsaq, Tasiilaq, Qaqortoq, Sisimiut, Assiaat, and Nanortalik. Figure Figure11 shows a map of Greenland, with the collection sites.

Table 1
Demographic, lifestyle and reproductive characteristics of breast cancer patients and controls
Figure 1
Geographical distribution of the breast cancer cases and controls.

All subjects completed the validated Danish standard questionnaire. It included questions about demographic and lifestyle parameters and allowed to document the following risk factors for breast cancer: age, breastfeeding, Body Mass Index (BMI), smoking, menopause status.

Blood sample collection and analyses

Blood samples were taken when the breast cancer was diagnosed and for controls when enrolled in the study following the standard procedure and stored at -80°C until analyses.

Concerning the chemical analyses and bio-activity measurements both cases and controls were batch together in the run of assays and the persons running the assays were blinded to the samples of cases and controls

Plasma legacy POPs and plasma lipid

The lipophilic plasma legacy POPs and lipids were analysed at the Centre de Toxicologie of the Institut National de Sante Publique du Quebec (Quebec, Canada) (CTQ), a certificated laboratory by Canadian Association for Environmental Analytical Laboratories. Plasma was extracted by 1:1:3 mixture of ammonium sulphate: ethanol: hexane and then concentrated and purified on two florosil columns. Twelve PCB congeners [International Union for Pure and Applied Chemistry (IUPAC) no. 99, 101, 105, 118, 128, 138, 153, 156, 170, 180, 183, 187], and eight OCPs [p,p'-DDT(dichlorodiphenyltrichloroethane) and its major metabolite p,p'-DDE (p,p'-dichlorodiphenyl-dichloroethylene), β-hexachlorocyclohexane (β-HCH), aldrin, hexachlorobenze (HCB), oxychlorodane, cis-nonachlor and trans-nonachlor] were analysed in purified extracts by high-resolution gas chromatography with electron capture detection. The detection limit was 0.08 μg/L for p, p'-DDE, p, p'-DDT and β-HCH, and 0.04 μg/L for other pesticides and PCBs [42]. The measured PCBs and OCPs were grouped as sum PCB and sum OCPs.

Plasma lipids were measured at CTQ using standard enzymatic procedure. The total plasma lipid concentration was obtained from cholesterol esters, free cholesterol, triglycerides and phospholipids as described previously [44]. To obtain the lipid adjusted POPs data (μg/kg lipid), the measured PCBs and pesticides (μg/L) were divided by total plasma lipid (g/L) and multiplied by 1000.

Serum perfluorinated compounds (PFCs)

Serum PFCs were measured at the National Environmental Research Institute, Aarhus University, Denmark. Perfluoroheptanoic acid (PFHpA), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluoroundecanoic acid (PFUnA), perfluorododecanoic acid (PFDoA), perfluorotridecanoic acid (PFTrA), perfluorohexane sulfonate (PFHxS), perfluorooctane sulfonate (PFOS) and perfluorooctane sulfonamide (PFOSA) were measured in serum extracts. The extraction method was based on ion pairing as described previously [45]. Instrumental analysis was performed by liquid chromatography-tandem mass spectrometry (LC-MS-MS) with electrospray ionization (ESI). The analytes were separated on a C18 Betasil column (2.1 × 50 mm, Thermo Hypesil-Keystone, Bellafonte, PA, USA) using an Agilent 1100 Series HPLC (Agilent Technologies, Palo Alto, CA). The HPLC was interfaced to a triple quadruple API 2000 (Sciex, Concorde, Ontario, Canada) equipped with a TurboIon Spray™ source operating in negative ion mode. Detection of the analytes was based on retention time and the most abundant mass transition corresponding to an authentic standard. Confirmation of analyte identity was based on the relative response of the secondary mass transition to the primary mass transition. Quantification of the analytes was done using response factors calculated from a four point calibration curve consisting of blank samples (rabbit serum) spiked with the analytes in the concentration range 1-50 ng/ml and extracted following the same procedure as samples.

The samples were extracted and analyzed in batches together with a procedural blank. The target compounds were not detected in any of the blank samples. The detection limit of the analytical method (MDL) was defined as those concentrations of the analytes needed to produce a signal-to-noise ratio (S/N) of 3:1. Detection limits ranged from 0.1 to 0.4 ng/ml. Method performance is currently tested by participating in the AMAP Ring Test for Organic Pollutants in Human Serum organized by Institute Nationale de santé publique du Québec [46]. Satisfactory Z-scores were obtained by our laboratory in the tests for PFCs.

The analysed PFCs were grouped into sumPFSA (sum of PFOS, PFHxS and PFOSA) and sumPFCA (sum of PFHpA, PFOA, PFNA, PFDA, PFUnA, PFDoA and PFTrA).

Blood metals

Whole blood metals including selenium (Se), zinc (Zn) and the heavy metals lead (Pb), mercury (Hg) and cadmium (Cd) were measured using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) after digesting blood with nitric acid in the microwave at the National Environmental Research Institute, Aarhus University, Denmark.

Plasma fatty acids

Plasma fatty acids were determined by capillary gas-liquid chromatography at the Biology Department, University of Guelph, Canada [47]. The fatty acids composition of plasma phospholipids was expressed as a percentage of the total area of all fatty acid peaks from 14:0 to 24:0. Plasma phospholipids of fatty acids correspond to relative percentages of total fatty acids by weight. The n-3 polyunsaturated fatty acids were reported on the sum of C18:3, n-3; C18:4, n-3; C20:3, n-3; C20:4, n-3; C20:5, n-3; C22:5, n-3; C22:6, n-3, and the n-6 fatty acid acids was the sum of C18:2, n-6; C18:3, n-6; C20:2, n-6; C20:3, n-6; C20:4, n-6; C22:2, n-6; C22:4, n-6 and C22:5, n-6. The ratio between n-3 and n-6 is known to be a strong indicator of marine food intake and thus a good indicator of the relative consumption of traditional versus imported food [48,49].

Serum cotinine determination

Cotinine is a metabolite of nicotine. The level of cotinine in the blood is proportionate to the amount of exposure to tobacco smoke and thus it is a valuable indicator of tobacco smoke exposure. The Calbiotech Cotinine Direct ELISA Kit was used to measure the serum cotinine (Calbiotech Inc., CA, USA) at Centre of Arctic Environmental Medicine & Cellular & Molecular Toxicology,, Department of Public Health, Aarhus University, Denmark. This assay is a solid phase competitive ELISA and the absorbance was read on ELISA Reader at 450 nm. The serum cotinine concentration was expressed as ng/ml and the limit detection was 1 ng/ml.

Measurement of serum estradiol

Solid phase fluoronimmunoassay was employed to determine serum 17 β-Estradiol using DELFIA® Estradiol kit (PerkinElmer Life and Analytical Sciences, Wallac Oy, Turku, Finland) at Centre of Arctic Environmental Medicine & Cellular & Molecular Toxicology, Department of Public Health, University of Aarhus, Denmark. The principle of this assay is based on competition between europium-labelled estradiol and sample estradiol for polyclonal anti-estradiol antibodies. The fluorescence of the samples were measured on a fluorometer (Wallac 1420 Multilabel Counter, Perkin Elmer life science, FIN) with protocol "56 E2" for automatic measurement and result calculation. The serum E2 concentration was expressed as nmol/L. The sensitivity of the assay was 0.05 nmol/L, and the intra- and inter-assay coefficient of variation were less than 10%.

Serum POP related xenobiotic induced receptor transactivities

SPE-HPLC fractionation of the serum samples for determination of ER and AR transactivity.

For determination of the combined serum POP related xenoestrogen (XER) and xenoandrogen (XAR) receptor transactivity a Solid Phase Extraction (SPE) and a High-Performance Liquid Chromatography (HPLC) fractionation was performed on 3.6 ml serum to obtain the serum fraction (F1) containing the actual mixture of bio-accumulated lipophilic POPs separated from endogenous hormones. The SPE-HPLC F1 extracts were analyzed for the actual combined xenohormone activity in the ER and AR transactivation assays [50-52].

Measurement of XER-transactivity and XAR-transactivity

Determination of the ER transactivation was carried out in the stable transfected MVLN cells, carrying the ERE-luciferase reporter vector (kindly provided by M. Pons, France) [51,53].

The AR transactivation was determined in the Chinese Hamster Ovary cells (CHO-K1) by transient co-transfection with the MMTV-LUC reporter vector (kindly provided by Dr. Ronald M. Evans, Howard Huges Medical institute, CA) and the AR expression plasmid pSVAR0 (kindly provided by Dr. A.O. Brinkmann, Erasmus University, Rotterdam) [52]. The luciferase activity was measured in a LUMIstar luminometer (BMG Lumistar, RAMCON, Denmark) and corrected for cell protein by fluorometric measurements in the WALLAC VICTOR2 (PerkinElmer, USA) at 355/460 nm wavelength as described [53-55].

In each assay all samples were tested in triplicate in two sets of tests: 1) the effect of the serum F1 POP extract only (termed XER/XAR) to test primarily for agonistic effect. The response of the serum F1 POP extract was compared to the reference solvent control of the analyses. When the F1 POP extract was significantly higher compared to that of solvent control (p <0.05), it was termed as significantly agonistic XER/XAR (i.e. significantly increased xenoestrogenic/xenoandrogenic transactivity); 2) the competitive xenohormone transactivity was determined upon co-treatment with 17 β-estradiol (E2) at EC40-E2 or the synthetic testosterone R1881 at EC50-R1881 and the serum F1 POP extract (termed XERcomp/XARcomp) to test primarily for antagonistic effects on ligand induced receptor transactivity (i.e. response of serum F1 POP extract plus E2 or R1881 were significantly lower than the response of E2 or R1881 alone), but if the response was significantly higher than the ligand reference values an additive or synergistic effect is indicated [51,52]. Measurements of the serum xeno-estrogen and xeno-androgen receptor transactivity were conducted at Centre of Arctic Environmental Medicine & Cellular & Molecular Toxicology, Department of Public Health, Aarhus University, Denmark.

Serum extraction of POPs for AhR-transactivity determination

A semi-automated solid-phase extraction method was used to prepare purified extracts for AhR-mediated transcriptional activity analyses from a single 5-ml plasma sample as described in Medehouenou et al. [54]. In short, the plasma sample was mixed with equal parts of formic acid and deionised water and extracted using a methanol: dichloromethane mixture (1/9). Then the extract was cleaned up on activated silica/acidic silica column and the compounds were eluted with dichloromethane. The solvent was evaporated to dryness and the resulting fraction reconstituted in 5 μl of dimethylsulfoxide for the measurement of AhR-mediated transcriptional activity [54].

Measurement of AhR-mediated transactivity

Detail procedure of AhR-mediated transactivity measurement was described elsewhere [54,55]. In short, the bioassay used to measure the AhR mediated transactivity was based on the expression of the firefly luciferase in H4IIE.Luc cells resulting from the activation of the AhR pathway by AhR activating compounds. H4IIE.Luc cells (kindly donated by A. Brouwer, BioDetection Systems B.V., Amsterdam, The Netherlands) were obtained by transfecting rat hepatoma H4IIE cells with the luciferase reporter gene plasmid pGudLuc1.1 [56,57]. After H4IIE.Luc cells were exposed to TCDD standards and the cleaned plasma extracts for 24 h, the luciferase activity was measured. AhR-mediated transcriptional activity of cleaned plasma extracts was interpolated onto the TCDD dose-response curve of AhR mediated luciferase activity and expressed as TCDD equivalents (AhR-TEQ), which represents the total TCDD-toxic potency of a mixture of dioxin-like compounds. The limit of detection was 30 pg TEQ/L, corresponding to approximately 5 pg TEQ/g lipids [54]. Measurement of AhR-mediated transactivity was performed at the Centre de Toxicologie (CTQ) of the Institut National de Sante Publique du Quebec (Quebec, Canada).

Statistical analysis

The distribution of data was checked by Q-Q plots. The natural logarithmic transformed variables improved the normality and homogeneity of variance and thus the comparison analysis was performed on the ln-transformed data. Independent student t-test was used to compare the graphical variables (age, BMI, fatty acids), chemical variables (PFCs, POPs, metals), xenobiotic induced transactivities (XER/XERcomp, XAR/XARcomp, AhR-TEQ) between cases and controls. Comparison of chemical variables and xenobiotic induced receptor transctivities was also performed by adjusting for age, BMI, number of pregnancies and smoking (serum cotinine) using ANCOVA analysis. Pearson's chi-square test was used to check the difference between cases and controls for frequency of breastfeeding, menopausal status and agonistic XER/XAR.

Unconditional logistic regression models were used to estimate the odds ratios (ORs) and 95% confidence intervals (95% CI) under controlling for potential confounders. Potential confounders considered for this analysis included age, BMI, total number of full-term pregnancies, breastfeeding, menopausal status and serum cotinine based on a priori consideration of the research design and well-established breast cancer risk factors. Each potential confounder was included in the model one by one with the chemical variables or xenobiotic induced receptor transactivities and compared to the model only with the chemical measurements or xenobiotic induced receptor transactivities. The confounder was identified when the difference of beta coefficients was more than 15%.

All statistical analysis was performed using SPSS version 17.0 (SPSS Inc. Chicago, IL, USA) conducted at Centre of Arctic Environmental Medicine & Cellular & Molecular Toxicology,, Department of Public Health, Aarhus University, Denmark. The statistical significant level was set to p ≤ 0.05.

Results

Demographic, lifestyle and reproductive characteristics of the study population

Table Table11 summarize the demographic, lifestyle and reproductive characteristics of the breast cancer (BC) cases and controls. Median age of the BC patients and controls was 50 and 54 years, respectively, and no significant age difference was observed. The age distributions at ≤ 55, 56-59 and ≥ 60 of cases and controls were similar. Cases and controls had similar BMI. The seafood intake, represented by the ratio of n-3 fatty acid to n-6 fatty acid (n-3/n-6), did not differ between cases and controls. To evaluate the current smoking status of the participants, serum cotinine levels of participants were measured, and borderline lower level was found for BC cases compared to their controls, indicating less current smoking among the cases than control participants. For the smoking behaviour obtained from the questionnaire, no significant difference between cases and controls was observed (Table (Table11).

As shown in Table Table1,1, the BC cases had lower full pregnancies numbers compared to the controls. However, this difference might be suggestive since only 52% of cases and 77% of controls had information on pregnancy; the proportion of breast feeding of BC patients was similar to that of the control participants. Similar percent of information on menopausal status for cases and controls (65% and 77%, respectively) was given. A substantial proportion of breast cancer patients were premenopausal (55%) while most of controls were postmenopausal (82%). The serum level of estradiol (E2) was non-significantly higher in BC cases compared with controls. In both cases and controls the premenopausal women had higher E2 level (Table (Table11).

Serum levels of POPs, metals and xenobiotic induced receptor transactivities POPs

The serum level of perfluorinated compounds (PFCs) was clearly significant higher in BC cases than controls (Table (Table2),2), and this significant difference persisted upon adjustment for age, BMI, pregnancy and cotinine. For PFOS and the sum of perfluorsulfonated acids (sumPFSA), breast cancer patients had double median level compared to the controls (p < 0.0001, Table Table2),2), and for PFOA and sum PFCA the significant difference between cases and controls was given by p = 0.04 and p = 0.001, respectively. No significant difference was observed between cases and controls for the legacy POPs such as PCBs and OCPs (Table (Table2).2). However, when the PCBs were subdivided into quartiles, the highest PCBs quartile (PCBs > 2645 ug/kg lipid) was significantly higher for the cases compared with the controls (p = 0.02, Table Table2).2). Also upon pooling the data for legacy POPs and PFCs, the cases had significantly higher level than that of controls (p = 0.007, Table Table2).2). Similar tendency was observed when data was stratified by menopausal status (see additional file 1).

Table 2
Serum levels of POPs and blood metals in breast cancer patients and controls

Metals

Levels of selected trace elements and heavy metals in the study participants are given in Table Table2.2. The level of Zn was significantly higher in cases, and a borderline higher level of Cd was found for controls. Although not significant the selenium (Se) level in BC cases tended to be lower than that of controls. But for the heavy metals Pb and Hg, no significant difference was observed between cases and controls.

POP related receptor transactivities

The xenobiotic potential of the extracted serum POP mixture was analyzed for effects on nuclear receptor transactivities such as ER, AR and AhR ex vivo upon exposure of the respective cell cultures as shown in Table Table3.3. Serum xenoestrogenic transactivities (XER, XERcomp) did not differ significantly between the two groups, but BC cases elicited a higher subject sample frequency of significant agonistic xenoestrogenic transactivity compared to controls (38.7% vs. 32.7%; Table Table33 and Figure Figure2).2). The agonistic xenoandrogenic transactivity (XAR) of cases was significantly higher than that of controls (p = 0.01, Table Table3),3), and the cases also elicited higher subject frequency of significantly increased agonistic XAR (18.5% vs. 5.2%; Table Table33 and Figure Figure2)2) compared to the controls. For the XER and XAR data no differences was found before and upon adjustment for the confounder's age, BMI, pregnancy and cotinine. Moreover, the serum AhR toxic equivalent (AhR-TEQ) of cases was lower than controls (p = 0.009, Table Table33 and Figure Figure2);2); however, upon adjustment for age, BMI, pregnancy and cotinine the significance disappeared (data not shown).

Table 3
Serum POP related xenobiotic induced receptor transactivities in breast cancer patients and controls
Figure 2
Levels of serum POP related xenobiotic induced transactivities of breast cancer cases and controls. A) serum xenobiotic agonistic induced ER and AR receptor transactivity; B) serum xenobiotic induced AhR transactivity (see legend to Table 3). % agonistic ...

Odd Ratios of correlation of serum POPs, metals and xenobiotic transactivities with the risk of breast cancer

Both before and after adjustment for the corresponding confounders, BC risk was associated with serum levels of PFOS (adjusted OR = 1.03, p = 0.05) and the sum of perfluorsulfonated acids (sumPFSA) (adjusted OR = 1.03, p = 0.02) (Table (Table4).4). Breast cancer risk was not associated with serum legacy POPs neither before nor after adjustment for the corresponding confounders (Table (Table4).4). However, the sum of legacy POPs and PFCs was significantly associated with the risk of BC (adjusted OR = 1.02, p = 0.01, Table Table44).

Table 4
Odds ratios of breast cancer and 95% confidence intervals associated with PFCs and POPs among breast cancer patients and controls

The blood level of the trace element Se was not associated with BC risk (Table (Table4).4). None of the heavy metals were found to associate to the risk of breast cancer (data not shown). The integrated xenoestrogenic transactivity (XER) in serum was not associated with the risk of BC either before or after adjustment for confounders, whereas the integrated agonistic xenoandrogenic transactivity (XAR) in serum was significantly associated with the BC risk (adjusted OR = 44.1, p = 0.016, Table Table4)4) both before and after adjusting for confounders. However, the AhR-TEQ was not found associated with the BC risk (Table (Table44).

Discussion

This study aimed at evaluating the association between serum levels of legacy POPs and PFCs in Greenlandic Inuit breast cancer cases and their controls. The present data shows for the very first time a relation between the serum level of PFCs and the risk of breast cancer in Greenlandic Inuit. Moreover, the breast cancer cases also had a significant higher serum PCB level at the highest quartile. In addition, the cases elicited a higher frequency of subjects samples inducing POP related hormone-like agonistic xeno-ER (XER) and xeno-AR (XAR) transactivity being significantly different for XAR. In contrast, the serum POP related dioxin-like AhR-TEQ was lowest in BC cases, although the significance disappeared for the adjusted data.

Until now very few reports are published concerning the incidence of breast cancer in the Arctic population and to our knowledge none have ever evaluated the factors affecting the risk of breast cancer in Greenlandic Inuit. In the western world BC is the most common cancer for women [1,2], where established risk factors can explain less than 30% of the cases. The incidence of BC has traditionally been low among the Inuit, but since the 1970's a considerable increase has been observed [3,4] although still at a level being approximately 60% of the incidence in e.g. Denmark [58]. In the Arctic, before 1966 BC was reported absent from the western and central Canadian Arctic and from 1967 to 1980 only 2 BCs out of 107 cancers were found [59]. Breast cancer was studied over a 20-year period in Inuit populations in the circumpolar region. A total of 193 BC's were observed in women with an incidence increase from 28.2 per 100.000 in 1969-1973 to 34.3 per 100.000 in 1984-1988 [60]. In Greenland, the age adjusted incidence in women increased from 35 to 46.4/100000 in the period 1973-87 and 1988-97, respectively [58]. Among Greenlandic women, an increase is particular observed among older women e.g. at the age from 40 to 70 the increase was (~65/100,000) in 1973 - 87 compared to (~170/100,000) the period 1988-97. [58,4]. There is a pattern shift typically seen in low-risk countries with stagnation or falling rates after menopause to increased rates after menopause as observed in the Western countries. This pattern shift does not support a general improved diagnosis that would be expected to be reflected across all age groups. Ethnic and international differences in BC risk can mostly be explained by differences in environmental exposures and lifestyle, particularly reproductive and hormonal factors [6-8]. The increasing prevalence of obesity and type-2 diabetes [61], and change in breastfeeding from prolonged feeding continuing through childbearing years to a pattern of breastfeeding now more similar to the western world [4] could be consisting with the increase in Arctic BC rates. However, low parity, late age at first birth and obesity is also risk factors for hormone-related cancers such as uterine and ovarian cancer that have not elicited a parallel increase in incidence in the Arctic and thus other factors may be important for the increase in BC incidence [4].

In this study the median age and BMI were similar for BC cases and the controls and no difference in breastfeeding was found. The only established reproductive BC risk factor found significantly different from the controls was, that BC cases had a lower number of full term pregnancies, although this information was only obtained for approximately 50% and 77% of the cases and control participants, respectively. In contrast to the expected menopausal related BC risk a higher frequency of cases were premenopausal (55% vs. 18%), and for the controls a higher percent of the subjects were postmenopausal. We found that postmenopausal women had higher burden of PFCs and legacy POPs than premenopausal women both in cases and controls, supporting the known age-dependent bioaccumulations of these compounds [25,43]. It is expected that premenopausal women are at general lower risk for BC than postmenopausal women. However, we observed a higher frequency of premenopausal women in the case group during the sampling period 2000-2003. We suggest that the combined endogenous and xenobiotic related hormone disruption might be higher for cases compared to controls as hypothesized in the following: Premenopausal cases, although relatively shorter lifetime exposure to endogenous estrogens but higher actual estrogen levels, the higher legacy POPs and PFC serum levels contributes to a higher POP related xenobiotic estrogenic disruptions. For the postmenopausal cases, although lower actual level of endogenous estrogen, the BC risk is influenced by a longer lifetime estrogenic exposure and the higher legacy POPs and PFC serum level contributes to a higher POP related xenobiotic estrogenic disruptions. This is supported by our data analysis stratifying the menopause status showing that cases had higher levels of PFCs and legacy POPs than controls both for the premenopausal women as well as postmenopausal women. In addition, postmenopausal women might be at higher risk because of significant higher xenoandrogenic bioactivity in accordance to the hypothesis of Adams, J.B. [62].

Result from the questionnaire showed similar proportion of reported current smoking for cases and controls. However, since questionnaire is subjective, the measurement of serum cotinine is more precise to reflect the current smoking status. The lower level of serum cotinine for cases indicates that the cases smoked less but it can be a result of their disease although the non significant lower level of serum Cd in cases supports different smoking habits for cases.

The Arctic Inuit population have one of the highest burden of POPs globally, particularly in some districts in Greenland, and the hormone disrupting potential of the actual mixture of serum POPs have been reported [63]. The recent increase in BC incidence might be explained by the high burden of legacy POPs and increased exposure to new emerging POPs such as PFCs together with the recent transition in the Inuit diet from the traditional marine food to more western food and lifestyle factors such as smoking and alcohol intake.

The ubiquitous presence of PFAAs globally including the Arctic regions has been documented [64,25], and unlike legacy POPs the level of PFAAs in marine mammals from Greenland still show an increasing trend in the past years [65,66].

In the present study we measured 3 PFSA isomers and 7 PFCA isomers and the most abundant isomers were PFOS and PFOA, and to a less extend PFHxS. The statistic data for PFHxS was similar to that of PFOS (data not shown). Since PFOS and PFOA were the most abundant we use these two compounds as the representative isomer of PFSAs and PFCAs. For BC cases we observed a significant higher serum level of PFCs and for PCBs in the highest quartile, and the adjusted odds ratios (ORs) indicated a significant risk for BC in relation to the level of serum PFOS and sumPFSA and total sum of legacy POPs plus PFCs.

Our data shows to our knowledge for the very first time an association between the serum level of PFCs and the risk of breast cancer. The higher levels of PCBs in BC cases in the present study are supported by previous reported data [10-12,14].

In general, there have been reported non consistent association between serum fluorochemical levels and adverse health effects in human but elevated bladder cancer mortality among male workers exposed to PFOS for a minimum of one year [33]. In rats [38] and mice [39] studies upon gestational exposures to PFOA significant increase in mammary fibroadenomas and Leydig cell adenomas and significant reduction in mammary differentiation and gland development in dams and female offspring were reported, respectively. In addition, PFOA-exposed female pups showed stunted mammary gland epithelial branching and growth [39]. However, the hypothesis that the PPAR-α agonistic potential of PFOA was involved was not supported since PPAR-α null mice exhibited normal mammary gland development [39]. In contrast, over expression of PPAR-α during pregnancy in mice impair normal differentiation of the mammary gland [33]. Thus far, data suggest that PPAR-α is the most likely target of PFOA and PFOS with the former having highest affinity in both human and mouse isoforms [33]. Further research is needed to elucidate the role of the PPAR pathway on the mammary gland. Moreover, comparing animal (rodent) data with human health risk must always be done with some caution because the exposure profile and often the body elimination is very different: in animals the exposure is often with single compounds, higher concentration but relatively short time, whereas in human the exposure is lifelong with low doses and complex mixtures.

We found that the serum level of both PFAAs, PFOS and PFOA, as well as the legacy POPs were significant higher in the BC cases compared to the controls but the adjusted OR only indicated significant BC risk for PFOS and PFSA and upon pooling all the chemical groups. However, we found in both cases and controls a high inter-correlation between serum PFSA and PFCA (r = 0.85-0.96, p <0.05) and between these two PFC groups and the legacy POPs (r = 0.42-0.55, p <0.05). Taken that into consideration it cannot be predicted which single compounds or chemical group that play the main role in the observed increase in BC risk.

Preliminary data suggest that some PFCs might be weak xenoestrogens [41]. In adult rats it was found that PFOA led to a decrease in serum testosterone and increase in serum estradiol levels [33]. Future studies are needed to understand the possible roles of PFCs as endocrine disrupters on sex hormone homoeostasis and function.

In our study the total combined serum POP related xenobiotic bio-activities reflects the lipophilic legacy POPs only and does not include the more hydrophilic PFCs. In cases we found that the POP related serum agonistic xenoandrogenic transactivity was significantly associated with the risk of BC as well as a higher number of subjects with significant increased serum agonistic xenoestrogenic transactivity.

With reference to the induced XAR a significant association to BC risk was found both before and after adjustment for related confounders. Using a similar analytical system the biological effects of the total combined xenoestrogens have been set in relation to the risk of BC by N. Olea and his co-workers [67,68]. It is well established that exposure to estrogens can increase the risk of breast cancer mainly attributed via their potential to increase the receptor induced cell proliferation and thereby expand possible gene damage and mutations in e.g. either tumour suppressing genes or proto-oncogenes causing inactivation or activation of the gene expressions of growth factors that act on mammary epithelial cells in an autocrine or paracrine loop [69]. Thus the body burden of exogenous estrogens such as POP related xenostrogens may further increase the BC risk.

Adams, J.B. [62] proposed a role of adrenal androgens (AA) in the aetiology of breast cancer. Premenopausal women that develop BC tend to have subnormal serum levels of AA and thus lower androgens acting via the AR opposing the estrogenic stimulated cell growth during this life period. In contrast, subjects developing the disease postmenopausal have supranormal levels of AA, and the elevated AA levels stimulate cell growth by the action of 5-androsten-3β, 17β-diol, termed hermaphrodiol, via its combination with the ER in a milieu having low concentrations of the classical E2. It might be hypothesized that the significantly higher combined serum POP related xenoAR activity level in cases has contributed to the BC development predominantly in the postmenopausal cases whereas the higher frequency of cases eliciting significant agonistic POP related xenoestrogenic transactivity might affect the BC risk in both pre- and postmenopausal cases. However, further research is needed before any conclusion can be taken.

In the present study we observed a significant lower level of AhR-TEQ in BC cases compared to their controls although the significance disappeared upon adjustment for the corresponding confounders (age, BMI, pregnancy, breastfeeding and cotinine). The higher serum sum POP/sumPCB levels in cases may explain the observed difference between cases and controls. Non-dioxin-like PCBs are shown to have the potential to antagonize the AhR pathway [70,71], supporting our unpublished in vitro studies (manuscript in preparation). Moreover, we found in an earlier study that Inuit with high serum levels of PCB had lower AhR-TEQ compared to Europeans with lower PCB levels [55]. Dioxins are reported to have antiestrogenic potentials [72-74]. It can be speculated whether the lower dioxin-like AhR-TEQ level in cases could play a role in BC risk via its lower antiestrogenic potential. Alternatively, it may be explained by differences between cases and controls regarding metabolic pathways involved in the biotransformation of both mono-ortho PCBs and estrogens as suggested by Demers et al. 2002 [75].

In spite of the POPs the traditional diet might also offer some protection against breast cancer because the marine diet is rich in 22:6, n-3 fatty acids and the antioxidant selenium (Se) [76], both factors suggested to have inhibitory effect on breast cancer [77,78] and chemically induced carcinogenesis in animals [79]. In addition, the intake of n-3 fatty acids is associated to the level of Vitamin A and D. An inverse relation between Zn and breast cancer was reported [80], and solar radiation and thus Vitamin D was suggested to reduce BC risk [81]. The intake of Zn has decreased since 1976 [48,82]. Thus, by changing to the western-lifestyle food intake the Greenlandic Inuit have a decreased intake of food factors, which might be protective against the risk of BC, including n-3 fatty acids, Zn, Vitamin D and Se.

In this study we found a trend to lower n-3/n-6 fatty acid ratio and lower Se but significantly higher levels of serum Zn in BC cases. To evaluate whether these factors contributed to the development of BC needs further research. However, a recent physiologically based pharmacokinetic (PBPK) model accounting for any given physiologic lifetime history using data on pregnancies, height, weight, and age, estimated the values of physiologic parameters (e.g., organ volume, composition, and blood flow) throughout a woman's entire life was developed. This PBPK model showed the limitations of using a single sample value obtained around the time of diagnosis for lifetime exposure assessment and point out the need to estimate the past POP exposure during time windows that are hypothesized to be mechanistically critical in carcinogenesis [83]. These factors may to some degree explain the controversial reports on POP exposure and breast cancer risk.

In the present study the metals were measured in the whole blood which primarily reflects the actual metal exposure. The limitation in reflecting the body burden prior to the disease will be further discussed in a manuscript particularly focusing on the effects of metals on the risk of breast cancer from the same study population (manuscript is in preparation).

There are some weaknesses in the presented study. Firstly, the few subjects involved, 31 cases and 115 controls, gives a poor statistically power. However, the highly related serum PFC levels with the risk of BC cancer did persist in all our effort to make up a better case-control frequency match. The number of controls in the West region (especially in Nuuk) was higher than other regions and may have impact on the final results. Therefore we also evaluated the data by reducing the number of Nuuk control subjects by matching the age and BMI with the cases in Nuuk to obtain similar cases/control ratio as for the other regions. Similar data was found between the data presented and upon including the reduced and matched data of Nuuk controls (not shown). However, a trend of higher level of organochlorine pesticides (OCPs) and competitive xenoestrogenic transactivity (XERcomp) for cases was observed being significant by including the reduced and matched data of Nuuk controls, whereas the observed significant difference between cases and controls at the highest PCB quartile and XAR transactivity disappeared probably due to reduced statistical power (data not shown).

Conclusions

The presented data show a higher level of PFCs and legacy POP in BC cases indicating that the level of serum POPs in particularly PFCs might be risk factors in the development of breast cancer in Greenlandic Inuit. Furthermore, our data suggest that the higher level of legacy POP related xenoestrogenic and xenoandrogenic agonistic activities in cases compared to controls can contribute to the development of BC in Inuit. Further investigations are needed to document these study conclusions.

Abbreviations

AA: adrenal androgens; AR: androgen receptor; AhR: aryl hydrocarbon receptor; AMAP: Arctic Monitoring and Assessment Programme; BC: Breast cancer; BMI: Body Mass Index; p,p'-DDT: dichlorodiphenyltrichloroethane; p,p'-DDE: p,p'-dichlorodiphenyl-dichloroethylene; E2:estradiol; ER: estrogen receptor; ESI: electrospray ionization; HCB: hexachlorobenze; β-HCH: β-hexachlorocyclohexane; LC-MS-MS: liquid chromatography-tandem mass spectrometry; OCPs: organochlorine pesticides; ORs: odds ratios; PBPK: physiologically based pharmacokinetic; PCBs: polychlorinated biphenyls; PCDD/PCDF: Polychlorinated Dibenzodioxins and Polychlorinated Dibenzofurans; PFAAs: perfluoroalkyl acids; PFCs: Perfluorinated contaminants; PFCAs: perfluorocarboxylated acids; PFDA: perfluorodecanoic acid; PFDoA: perfluorododecanoic acid; PFHpA: Perfluoroheptanoic acid; PFHxS: perfluorohexane sulfonate; PFNA: perfluorononanoic acid; PFOA: perfluorooctanoic acid; PFOS: perfluorooctane sulfonate; PFOSA: perfluorooctane sulfonamide;PFSAs: perfluorosulfonated acids;PFTrA: perfluorotridecanoic acid; PFUnA: perfluoroundecanoic acid;POPs: persistent organic pollutants;SPE-HPLC: Solid Phase Extraction - High-Performance Liquid Chromatography;TEQ: TCDD equivalents;XAR: xeno-androgen receptor transactivity of serum alone;XER: xeno-estrogen receptor transactivity of serum alone;XARcomp: competitive xenoandrogenic transactivity;XERcomp: competitive xenoestrogenic transactivity.

Conflict of interest statement

The authors declare that they have no competing interests.

Authors' contributions

ECB-J conceived the study and made the overall study design, participated in data analyses and drafted the manuscript; ML participated in the study design, performed the statistical analyses, participated in drafting the manuscript and reviewed the manuscript drafts; RB were responsible for the PFC analyses and reviewed the manuscript drafts, PA were responsible for the AhR-CALUX analyses and reviewed the manuscript drafts; GA performed the trace element analyses and reviewed the manuscript drafts, TK performed the AR-transactivity analyses, guided the ER- transactivity analyses and reviewed the manuscript drafts, MG performed the SPE-HPLC analyses and reviewed the manuscript drafts, GM was the responsible person for cases and control sampling in Greenland, PK and PN took the samples from cases and reviewed the manuscript drafts, ED participated in the study design, handed over the control samples from Nuuk and reviewed the manuscript drafts. All authors read and approved the final manuscript.

Supplementary Material

Additional file 1:

Results after being stratified by menopausal status. Levels of POPs, metals and POP related xenobiotic induced receptor transactivities of BC cases and controls within the premenopausal and postmenopausal women are given.

Acknowledgements

We thank all the member of the Centre of arctic Environmental Medicine for critically scientific and technical support, and particularly research assistant Cao Yi for technical support doing the XER, cotinine and estradiol measurements and the technical staff at NERI for performing the chemical analyses. We thank the International Polar Year Committee, the Commission for Scientific Research in Greenland and the Danish Environmental Agency for economically support.

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Use of cotinine urinalysis to verify self-reported tobacco use among male psychiatric out-patients

Abstract

Context:

There is a complex and significant correlation between respiratory disorders and psychiatric conditions. Reliability of self-reported tobacco use has been questioned in recent times.

Aims:

The current study aims at assessment of accuracy of self-reported tobacco use (both smoked and smokeless) among psychiatric out-patients.

Settings and Design:

We recruited 131 consecutive subjects from the out-patient psychiatry department of a tertiary care hospital.

Materials and Methods:

Male patients meeting the study criteria were approached for participation in the study. They were asked about their recent tobacco use history. Those reporting recent use were assessed for severity of dependence using Fagerstrom Test for Nicotine Dependence (FTND)-smoking and FTND-smokeless scales. Quantitative urine cotinine analysis was performed using the Enzyme Linked Immunesorbant Assay (ELISA) method. Based on this method, a (50 ng/ml) cut off score for urinary cotinine level for tobacco use was set.

Statistical Analysis Used:

Concordance between the self-report of tobacco use and urinary cotinine level was assessed using the Cohen's kappa. Additionally, Pearson's correlation coefficient was used to examine the correlation between the FTND-smoking and FTND-smokeless scales and the urinary cotinine levels.

Results:

The values of Cohen's kappa suggest no significant concordance between the self-reported recent tobacco use and urinary cotinine levels for both smoking and smokeless tobacco forms. The discordance was present irrespective of a higher (550 ng/ml) or a lower (50 ng/ml) cut off score for a urinary cotinine level. Pearson's correlation coefficient failed to reveal any significant direct correlation between the FTND scores and urinary cotinine levels.

Conclusions:

It is recommended to use biological markers such as urinary cotinine levels to corroborates the information provided by the patients.

KEY WORDS: Psychiatric illness, tobacco use, urinalysis

INTRODUCTION

There is a complex and significant correlation between respirator disorders and psychiatric conditions. An increased prevalence of respiratory disorders has been reported among patients with psychiatric illnesses.[13] It has been observed that psychiatric symptoms might be the first presenting symptoms for small cell lung cancer.[4] However, the physical illnesses including respiratory illness largely remain undetected and untreated. Alarmingly, low rates of 13% (psychiatric inpatients) and 8% (psychiatric outpatients) for physical examination have been reported among psychiatric patients.[5,6] Consequently, most of these conditions are likely to get undetected. Similarly, the rate of psychiatric morbidity is high among patients with lung cancer.[7] Mood and anxiety disorders are also prevalent among both adult and pediatric patients with asthma and severe lung diseases.[8,9]

Smoking rates have been found to be high among the patients with psychiatric illness. Epidemiological studies have found smoking rates among psychiatric patients to be twice that of general population.[10] Thus, a high rate of tobacco use has been implicated as a possible risk factor for respiratory disease in psychiatric patients.[11,12] Most of the literature on tobacco and psychiatric disorders is limited to cigarette smoking, and information on other forms of smoking is limited. Smokeless tobacco use remains understudied and under-researched in the association of respiratory disorders with psychiatric conditions, and it is tempting to speculate that this is due to the potential of oral/smokeless tobacco to adversely impact on respiratory disease in the first place. Nevertheless, tobacco use, smoking as well as smokeless, is the single largest preventable cause of death globally.[13]

The literature on accuracy of self-reported tobacco use among psychiatry patients is limited. Takeuchi et al. (2010) conducted a study on validity of self-report of tobacco use among patients with schizophrenia. In the absence of accurate and precise information on tobacco use, it is likely to get unnoticed and hence unmanaged among patients with psychiatric illness.[14]

The current study aims at assessment of accuracy of self-reported tobacco use (both smoked and smokeless) among male psychiatric out-patients.

MATERIALS AND METHODS

All the male subjects attending the psychiatry out-patient department of the tertiary care multispecialty teaching hospital constituted the sample frame for the current study. The subjects were recruited from the follow up OPD in order to ensure adequate participation of the subjects with regards to self-report. All male patients aged 18 or more coming for follow-up visit were approached for participation in the study. Those willing to participate and giving informed consent were included in the study. Subjects with significant cognitive impairment (MMSE score <24) or having a significant medical co-morbidity interfering with participation in the study were excluded from the study. A total of 175 consecutive male patients were approached for participation in the study. Five subjects were excluded as they were having significant cognitive deficit or significant medical co-morbidity. Finally, 131 consecutive male subjects (75% of all patients approached) were included in the study. Since this is the first reported study of its kind, it was left to the study group to arrive at the sample size. Each potential subject recruited in the study was to serve its own control using the urinalysis report. So it was decided to collect the study sample over a 2-month period. This was done with an aim to conduct a pilot study on this non-researched issue.

The study subjects were asked about their recent tobacco use during the past week. Active tobacco use was defined as daily use of tobacco products. Information was gathered for both smoking and smokeless forms of tobacco. Those reporting recent tobacco use were asked “How many cigarettes/biri/gutkha/etc do you use per day?”; “Which is your most preferred tobacco product?” Additionally, they were assessed for severity of dependence using the Fagerstrom Test for Nicotine Dependence (FTND) (smoking as well as smokeless). FTND-smoking is a widely used six-item questionnaire used to screen for severity of dependence on smoked tobacco.[15] FTND-smokeless (FTND-ST) is a nine-item instrument used to evaluate the level of nicotine dependence for smokeless tobacco.[16]

Subsequently 50 ml of urine samples were collected from each subject under close supervision and were submitted for laboratory analysis. Quantitative urinary cotinine was done by using Enzyme Linked Immunesorbant Assay (ELISA) kits of Calbiotech Inc., USA, which uses solid phase competitive ELISA. The assay was carried out as directed by the manufacturers. The detection limit of the cotinine assay was 2 ng/ml.

Data was analyzed using SPSS ver. 17. The concordance between the self-report of tobacco use and urinary cotinine level was assessed using the Cohen's kappa. Additionally, Pearson's correlation coefficient was used to find the correlation between the FTND-smoking and FTND-smokeless scales and the urinary cotinine levels.

There is no unanimity of appropriate cut off value of urine cotinine levels to screen for recent tobacco use. The recommended cut off values range from 50 to 550 ng/ml in different studies.[17,18] The Calbiotech ELISA method recommends a cut off value of >50 ng/ml.

The ethical standards of the responsible committee on human experimentation (institutional or regional) and with the Helsinki Declaration of 1975 as revised in 1983 were followed. Conditions of anonymity and confidentiality were ensured throughout the conduct of the study.

RESULTS

A total of 131 male subjects attending the psychiatry out-patient department were recruited in the study. The mean age of study subjects was 31.05 (SD±11.66) years. Eighty-six (65%) of the subjects were from urban background and 70% were married. Self-reported recent use of cigarettes and biri was reported by 8 (6.1%) and 16 (12.1%) of the subjects, respectively. Self-reported use of smokeless tobacco products was by 13 (9.8%) for gutkha, 8 (6.1%) for tobacco powder, and 2 (1.5%) for khaini, respectively. The findings have been summarized in Figure 1. The mean FTND scores were 2.59 (SD±1.37) and 3.35 (SD±1.72) for smoking and smokeless forms.

Figure 1
Self-reported use of different tobacco products by study subjects (smoking and smokeless)

In order to assess the accuracy of self-reported recent tobacco use, an ELISA method for quantitative urinary cotinine level was used as biomarker. The values of Cohen's kappa suggest no significant concordance between the self-reported recent tobacco use and urinary cotinine levels for both smoking and smokeless tobacco forms [Table 1]. The discordance was present at the recommended cut off value of >50 ng/ml as recommended for the Calbiotech ELISA method.

Table 1
Concordance between self.report of recent tobacco product use and urine cotinine levels (N=131)

There was a statistically significant negative correlation between the FTND-smoking form and urine cotinine levels (correlation coefficient=–0.59, P=0.02). However, no such correlation was observed between FTND-ST scores and urine cotinine levels (correlation coefficient=–0.08, P=0.75).

DISCUSSION

We recruited the subjects from follow-up psychiatry out-patient so that they could provide information about their tobacco use. The findings of the current study raise concerns about the reliance on the self-report for tobacco use among psychiatric out-patients.

There is a complex and significant correlation between respiratory disorders and psychiatric conditions. An increased prevalence of respiratory disorders has been reported among patients with psychiatric illnesses.[13] It has been observed that psychiatric symptoms might be the first presenting symptoms for small cell lung cancer.[4] A higher prevalence of paraneoplastic syndrome in this cancer type and higher metastasis rates to brain might be possible explanations for this association. A high rate of tobacco use has been implicated as a possible contributor to this association.[10,11] Cigarette smoking has been postulated to a common underlying factors for both respiratory illness and panic attacks among patients with these co-morbidities.[19]

However, the physical illness including respiratory illness largely remains undetected and untreated. Alarmingly low rates of 13% (psychiatric inpatients) and 8% (psychiatric outpatients) of physical examination have been reported among psychiatric patients.[5,6] Consequently, most of these conditions are likely to get undetected. An out-patient study from India reported the rate of undetected respiratory illness to be 15%, second only to hypertension.[20]

Similarly, the rates of psychiatric morbidity is high among patients with lung cancer.[7] Mood and anxiety disorders are also prevalent among both adult and pediatric patients with asthma and severe lung diseases.[8,9] Anxiety and depressive symptoms are predictive of poorer asthma management, associated functional impairment, and inferior treatment outcomes among asthma patients.[21,22]

The accuracy of self-report of tobacco use was found to be low in the current study when it was cross checked with urinary cotinine levels for both smoking and smokeless forms. Even among those reporting recent use of tobacco products, the FTND scores were not find to be directly correlated with the urinary cotinine levels. In fact, there was a negative correlation between the FTND-smoking scale scores and urine cotinine levels. Both the smoking and smokeless versions of FTND have been shown to be valid and reliable instruments for assessing tobacco dependence.[15,16] These findings suggest that psychiatric out-patients tend to under report recent use of tobacco products. Additionally, the severity of tobacco dependence (as estimates by FTND) does not correlate with the amount of tobacco consumed by these individuals.

The issue of reliability of self-report about tobacco use among psychiatric patients has not been studied adequately. Takeuchi et al. (2010) reported the first study of reliability of self-report of smoking among patients with schizophrenia.[14] The correlation between the self-reported smoking and breath CO levels was lost with an increase in duration of psychiatric illness. We could not come across any other study on this issue. We made use of urinary cotinine levels as a biomarker for recent tobacco use. Urine cotinine level has been recognized as a useful biomarker of recent tobacco use. Use of urinary cotinine levels (>50 ng/ml) to validate the self-report of tobacco use has been recommended in medical settings.[23]

Use of tobacco products by psychiatric patients is associated with a poor treatment response and worsening of the long-term course.[24] Additionally, it exposes these individuals to the harmful effects of tobacco. Tobacco use continues to be the single largest preventable cause of death globally. Tobacco use is a likely contributor to a relatively higher mortality rate seen among patients with psychiatric disorders.[25] Smoking has also shown to increase the requirement of neuroleptics.[26]

Co-morbid use of tobacco products by psychiatric patients is likely to get unnoticed if not assessed properly. Mentally ill receive tobacco treatment on only 12% of their visits to a psychiatrist and 38% of their visits to a primary care physician.[27]

A focus on the presenting axis I psychiatric illness could overshadow the tobacco use problem among these individuals. Use of biomarkers such as urinary cotinine level can help improve the recognition rate of recent tobacco use by these patients. This information would be of help while planning an appropriate management for them.

The current study has certain strengths. We used an objective biomarker in urine cotinine levels to corroborate the self-report. Additionally, we carried out a quantitative analysis of the urine cotinine levels. We analyzed the data using the two extreme cut off values of urinary cotinine levels. This was done keeping in mind use of different threshold for this value across studies.[17,18] However, the concordance rates were poor with both these cut-off values.

However, it is a pilot study with a relatively small sample size. Use of the study subjects as self-controls partly takes care of the issue of sample size. A conclusive sample size could not be estimated due to absence of prior work. The current study sample comprised of a heterogeneous group of different psychiatric disorders as we recruited a consecutive sample presenting to the out-patient psychiatry department. The issue needs to be studied among specific psychiatric illness groups. Also the findings need to be replicated in larger samples from different centers and settings. Additionally, we recruited only male subjects in the current study from a follow up out-patient setting. It would be interesting to compare the findings from female psychiatric patients.

Footnotes

Source of Support: Nil

Conflict of Interest: None declared.

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