Protective effects of selenium against DNA adduct formation in Inuit environmentally exposed to PCBs

Environment International 36 (2010) 980–986

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Environment International j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e n v i n t

Protective effects of selenium against DNA adduct formation in Inuit environmentally exposed to PCBs Srivani Ravoori a, Cidambi Srinivasan b, Daria Pereg c, Larry W. Robertson d, Pierre Ayotte c, Ramesh C. Gupta a,e,⁎ a

James Graham Brown Cancer Center, University of Louisville, Louisville, KY 40202 USA Department of Statistics, University of Kentucky, Lexington, KY 40506, USA Laval University Medical Research Center, Quebec, QC, Canada G1V 5B3 d University of Iowa, Iowa City, IA 52242, USA e Department of Pharmacology and Toxicology, University of Louisville, Louisville, KY 40202, USA b c

a r t i c l e

i n f o

Available online 6 September 2009 Keywords: Selenium PCBs Inuit 8-oxodG DNA adducts 32 P-postlabeling

a b s t r a c t Dietary habits that expose populations to potential toxicants as well as protective agents simultaneously are a realistic scenario where a meaningful assessment of the interactions and net benefit or damage can be made. A group of Inuit from Salluit, Northern Canada are exposed to high levels of PCBs and selenium, both present in the Inuit traditional foods such as blubber from sea mammals and fatty fish. Blood samples were collected from 83 Inuit, 22–70 years old. Blood selenium and PCB levels were determined previously and ranged from 227 to 2069 µg/L and 1.7 to 143 µg/L, respectively. DNA isolated from white blood cells were analyzed by modified 32P-postlabeling adductomics technology that detects a multitude of highly polar to lipophilic adducts. The levels of 8-oxodG adducts ranged from 470 to 7400 adducts/109 nucleotides. Other as yet unidentified polar adducts showed a 30 to 800-fold inter-individual variability. Adduct levels were negatively associated with PCB and selenium levels. The subjects were classified into high and low ratio groups, with respect to selenium/PCB. In the high ratio group, the coefficient of selenium is significantly negatively correlated with 8-oxodG (r = − 0.38, p = 0.014) and total adducts (r = − 0.41, p = 0.009) while there was no correlation within the low selenium/PCB group. This study suggests that increasing selenium has mitigating effect in reducing DNA adducts and therefore, possible negative effects of PCB were not seen. A protective effect of selenium is highlighted. © 2009 Elsevier Ltd. All rights reserved.

1. Introduction Investigating the effects of dietary constituents on the overall health has gained attention in the past several years, as it helps in the evaluation of a particular nutrient or a combination that would have major health benefits, and pre-empt large-scale clinical trials with dietary supplements. The American Institute for Cancer Research and the World Cancer Research Fund have predicted about 30–40% prevention of cancers by appropriate diets and fitness (WCRF/AICR, 2007). Information on the benefits of certain nutrients can be obtained by research on ethnic groups/populations that feed on traditional diets and have benefited/harmed by it. Inuit is one such Aboriginal population from Northern Canada, where food is perceived as a major component of a healthy life. Traditional country food of Inuit includes large sea mammals such as white whales and seals (skin, meat and blubber), fatty fish, besides plants. The dietary habits and health risks/benefits contributed by their traditional diet have ⁎ Corresponding author. 304E Baxter II Research Building, 580 S. Preston St., Louisville, KY 40202, USA. Tel.: +1 502 852 3682; fax: +1 502 852 3662. E-mail address: [email protected] (R.C. Gupta). 0160-4120/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.envint.2009.08.001

been extensively reviewed, and good health attributed to their food has been recognized (Van Oostdam et al., 2005). The Inuit, by virtue of their traditional diet, are exposed to several contaminants including polychlorinated biphenyls (PCBs). PCBs constitute a family of 209 congeners used extensively in industrial and commercial products until mid 1970s. These organochlorine compounds are persistent organic pollutants (POPs), as reflected in their ubiquitous prevalence and lipid solubility. Due to their resistance to degradation, PCBs accumulate in fatty tissues and are biomagnified through food chains, including the marine Arctic food chain. They therefore accumulate in varying high concentrations in populations consuming the fat (blubber) from sea mammals which are positioned at the highest trophic level. Potential negative health effects of PCBs such as immunological disturbances, neurological defects and implications in cardiovascular and liver diseases have been discussed (Carpenter, 2006). Studies have also shown that long term low dose exposure and higher burden of PCBs increase the risk of prostate cancer (Ritchie et al., 2003, 2005). Although hormonallyactive organochlorines did not show any such associations with breast cancer risk (Demers et al., 2000), case control studies on a similar population from southern Quebec have shown an association of

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increased levels of PCBs, particularly congeners 105, 118 and 156 with breast cancer (Demers et al., 2002). Several other health issues, including visual evoked potentials and acute respiratory infections were found to be associated with the organochlorine levels in the Inuit (Dallaire et al., 2004, 2006; Saint-Amour et al., 2006). The traditional marine diet of Inuit also comprises large quantities of selenium, a dietary essential trace element. Selenium is incorporated in amino acids as selenocysteines or replaces sulfur in methionine forming selenomethionine. Selenocysteines are antioxidants preventing lipid peroxidation and cellular damage. The antioxidant properties and protective effects of selenium have been widely studied (El-Bayoumy, 2001; Patrick, 2004). However, the selenium and vitamin E cancer prevention trial (SELECT) sponsored by the NCI showed no protection against prostate cancers (Klein et al., 2001). This large study was based on the promising findings from earlier studies (1994; Clark et al., 1996; Duffield-Lillico et al., 2002; Heinonen et al., 1998). Limitations of this study such as not testing different formulations and doses of selenium, the baseline selenium levels of the study population, not having identified a suitable study population with advanced prostate cancer, and assessment of the outcome in specific subgroups who might have responded differently, have been recognized (Lippman et al., 2009). The whole blood selenium levels in the Inuit are about 8- to 15-fold higher than those reported for reference white populations consuming little fish (Belanger et al., 2006). DNA adducts are intermediate biomarkers of exposure to agents that are capable of producing electrophilic species that interact directly and/or indirectly with DNA. PCBs can produce such intermediate electrophiles as semiquinones and quinones that covalently bind to DNA as detected by 32P-postlabeling (Arif et al., 2003; McLean et al., 1996; Oakley et al., 1996b; Pereg et al., 2002) and liver cytosolic proteins (Lin et al., 2000). Furthermore, these electrophiles can also produce reactive oxygen species such as hydroxyl radicals and singlet oxygen producing a host of oxidative DNA adducts, including 8-oxodG (Marnett, 2000; Oakley et al., 1996a). Blubbers of marine mammals caught along the Taiwan coast showed higher levels of 8-oxodG, and in mature female cetaceans, the 8-oxodG levels correlated with their PCB levels (Li et al., 2005). Coadministration of equal amounts of PCB 126 and PCB 153 in rats resulted in a dose-dependent increase in 3-(2′-deoxy-[β]-D-erythropentofuranosyl)-pyrimido[1,2-a]-purin-10(3H)-one (M1dG) adducts in liver, and a constant amount of PCB 126 combined with increasing amounts of PCB 153 resulted in more accumulation of these adducts, suggesting their potential role in carcinogenicity (Jeong et al., 2008). A recent study found an inverse relationship between methylcytosine levels and persistent organic pollutants in Greenlandic Inuit, indicating PCB's ability to affect chromosomal stability and gene expression (Rusiecki et al., 2008). The 32P-postlabeling/TLC adductomics systems, developed recently in this laboratory, have been employed to measure collectively a host of DNA adducts encompassing high polarity to high lipophilicity (Ravoori et al., 2006). In this study, we investigated the effects of varying levels of selenium and PCB exposure occurring via the traditional diet of sea mammals on DNA adduct formation in a group of Inuit from Salluit, Northern Canada.

2. Materials and methods 2.1. Enzymes and chemicals 32

Enzymes, chemicals, [γ- P]ATP and TLC solvents used for DNA isolation and 32P-postlabeling were as described elsewhere (Gupta, 1996; Ravoori et al., 2006).

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2.2. Specimens Blood samples were collected from 83 Inuit subjects, 56 women and 27 men. The age ranged from 17 to 77 with a median of 42. Plasma PCB levels were determined by high-resolution gas chromatography with electron-capture detection as described previously (Demers et al., 2000). The congeners that are found abundantly in this population, as reported previously, are 138, 153 and 180 (Dewailly et al., 1993). Smoking status of these subjects was determined by a questionnaire. Blood selenium levels were determined by inductivelycoupled plasma-mass spectrometry after nitric acid digestion (Labat et al., 2003). Demographic and clinical data are summarized in Table 1. 2.3. DNA isolation DNA was isolated from the white blood cells (WBCs) by a solvent extraction procedure (Gupta, 1996) involving removal of RNA and proteins by digestion of isolated crude nuclei with RNases (sigma Chemical Co., St. Louis, MO) and proteinase K (Roche Diagnostics, Indianapolis, IN,), respectively, followed by extractions with phenol (Amresco, Solon, OH), phenol:Sevag and Sevag (Choloroform: isoamyl alcohol) (Fisher Scientific, Pittsburgh, PA). DNA was recovered by precipitation with ethanol, washed, dissolved in water and its concentration and purity was estimated by spectrophotometry. 2.4. Analysis of adducts DNA adduct profiles were determined by 32P-postlabeling/TLC systems (Ravoori et al., 2006) to assess global DNA damage comprising of polar adducts, including 8-oxodG and lipophilic adducts. Briefly, following enzymatic digestion of DNA (15–20 µg), adducts were enriched by treatment with nuclease P1 (Calbiochem, Gibbstown, NJ), 32P-labeled and resolved by 2-D PEI-cellulose TLC by development with 1 M formic acid in the presence of increasing concentration of sodium phosphate (50 mM–1000 mM) (D1) and isopropanol: 4 M ammonium hydroxide, 1:1 (D2). 8-OxodGp was enriched by PEI-cellulose TLC, 32P-labeled and resolved by 2directional TLC as described (Gupta and Arif, 2001). Normal nucleotides were labeled in parallel with adducts and separated by 1-D PEIcellulose TLC. Adduct and normal nucleotide radioactivity was measured by Packard InstantImager. Adduct levels were calculated as relative adduct labeling (RAL) and expressed as adduct(s) per 109 nucleotides. 2.5. Statistical analysis The effect of age, gender, smoking status, PCB levels and selenium levels on DNA adduct accumulation was performed individually using analysis of variance. A p-value of less than 0.05 was considered significant. Analysis of the effect of PCB and selenium on DNA adducts

Table 1 Characteristics of participants, smoking status, plasma selenium and PCB concentrations. Variable

Range

Mean

Median

Std. dev.

Age Gender Females Males Smoking status Current smoker Ex-smoker Never smoker Selenium (µg/L) PCB (µg/L)

22–70

44.67

41.5

14.5

569.3 20

397.7 26.7

56 (67%) 27 (33%)

227–2069 1.7–143

57 (68.7%) 25 (30.1%) 1 (1.2%) 673.9 29.1

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was carried out in the context of generalized linear models (McCullagh and Nelder, 1989), using negative binomial regression analysis with log-link function. The data was segmented into two groups on the basis of selenium/PCB. Since the PCBs studied here are a mixture of several congeners, a molar ratio could not be obtained. Therefore, a unit-free ratio of selenium to PCB as absolute weights was considered. The segmentation cut-off ratio was determined using non-linear segmented regression analysis (SAS program “PROC NLIN”) of 8-oxodG (in log scale) and total adducts on selenium. Subsequent to the segmentation, a full-fledged negative binomial regression analysis with log-link was performed on each group, regressing (a) 8-oxodG counts and (b) the adduct counts on the variables selenium, PCB, age, smoking status, and gender to detect the effects of the variables on the adduct burden. The negative binomial regression was carried out using SAS Genmod procedure (version 9.1) (McCullagh and Nelder, 1989; Wiencke et al., 1999). 3. Results A wide array of DNA adducts, ranging from highly polar to highly lipophilic, were detected in the blood samples from the Inuit (Fig. 1). They were qualitatively similar to those found in human uterine cervical (Ravoori et al., 2006) and other tissues (unpublished data). The unidentified adducts, based on their chromatographic properties, were grouped into P-1, P-2, PL-1, PL-2 and L-1 subgroups, where P stands for polarity, L for lipophilicity, and PL is designated for adducts that had both polar and lipophilic properties based on salt concentration requirement during the chromatography. There was no qualitative variation in adducts among the samples. However, there were large quantitative differences. The adduct levels varied by 31, 58, 165, 89 and 805-fold for P-1, P-2, PL-1, PL-2 and L-1 subgroups, respectively. The known oxidative lesion, 8-oxodG, which constitutes part of the P-2 subgroup, showed 87-fold inter-subject variability. Fig. 2 shows mean adduct levels of the different adduct categories and inter-subject variability. Highly lipophilic adducts (L-2) that resolve with high-salt, high-urea chromatography (Ravoori et al., 2006) were below detection limits in this study. A preliminary analysis of the data showed significant positive correlations between age and PCB (Spearman's r = 0.82, p < 0.0001), between age and selenium (r = 0.48, p < 0.0001), and between selenium and PCB (r = 0.65, p < 0.0001) (Fig. 3a–c). No direct association between adduct levels and gender or smoking status was found (not shown). A negative correlation of 8-oxodG (r = − 0.23; p = 0.038) and total adducts (r = − 0.31; p = 0.004) with age was observed (Fig. 3d, e). When stratified by age after controlling for the effects of smoking and gender, the levels of 8-oxodG showed a negative correlation with PCB levels among subjects 60 years or older (Table 2a), and the total adducts were negatively correlated with PCB among subjects under 30 years of age. (Table 2b). When analyzed for a correlation with factors PCB and selenium, the adduct levels seemed to be negatively associated, although it was significant only with PCB concentration (Fig. 4). The effect of selenium on the adduct burden, however, appeared to vary with selenium/PCB. To understand this phenomenon, the population was divided into two groups with high ratio and low ratio on the basis of selenium/PCB values in order to carry out more focused analysis. While there is not much experimental evidence as to the effect of PCB on adduct levels, it is reasonable to postulate that PCB can produce DNA damage. However, in this population the adduct level significantly decreased with increase in PCB levels. A plausible explanation for this peculiarity is the presence of selenium which is known to elicit antioxidant activity. Since selenium and PCB have counteracting effects on DNA adducts, the selenium/PCB ratio was considered a logical criterion to stratify the subjects. Individuals with selenium/PCB > 33 (mean ratio 75.5± 7.9; n = 41) were grouped as high- and those with ≤33 (mean ratio 18± 1.1; n = 42) as low ratio group. The cut-off value 33, which happens to be close to the median 33.4 of selenium/PCB ratio was determined using non-linear segmented regression analysis of 8-oxodG and total adducts on selenium. A comparison of the two groups (Table 3) indicates that there is a statistically significant difference with respect to all the variables, except selenium. The selenium/PCB ratio negatively correlated with age (r = − 0.745, p < 0.0001). It is worth noting that the 8-oxodG and total adduct levels were lower for the low selenium/PCB group (2250 ± 358 and 3429 ± 375/109 nucleotides, respectively) compared with the high ratio group (2722 ± 263 and 5085 ± 395/109 nucleotides, respectively). The differences were significant for both 8-oxodG (p = 0.0316) and total adducts (p = 0.0014). In contrast, the average age for the low ratio group (mean = 53.7, median = 55) is much higher than the average age for the high ratio group (mean = 35.6, median = 33). In other words, the low ratio group comprises largely of older subjects while the high ratio group is mostly younger subjects. Also, there is nearly a four-fold increase in PCB levels for the low selenium/PCB group (mean = 45.1 ± 4.2) compared to the high ratio group (mean = 12.8 ± 2.0). Subsequently, a negative binomial regression analysis was carried out separately for each group to assess the effects of selenium, smoking status, and gender

Fig. 1. Representative maps of DNA adducts from WBCs analyzed by 32P-postlabeling in conjunction with PEI-cellulose TLC. Following enzymatic digestion of DNA, adducted nucleotides were enriched by treatment with nuclease P1, except for 8-oxodG which was enriched by PEI-cellulose TLC. Adducts were 32P-labeled and chromatographed by 2- or 3-directional PEI-cellulose TLC. Chromatography conditions are as described under Materials and methods. Adducts resolved are grouped as P-1 (panel A); P-2 (B); PL-1 (C—lower), L-1 (C—upper); PL-2 (D) and 8-oxodG (E) adducts. P stands for polarity and L for lipophilicity. Adduct polarity increases with P-1 > P-2 > PL-1 > PL2 > L-1; 8-oxodG falls under the P-2 group.

on 8-oxodG and total adducts. The variable, age was not included in the model due to its correlation with selenium (r = 0.48, p < 0.0001). Also, PCB was not included directly in the model as an independent variable because it is also highly correlated with selenium (r = 0.65, p < 0.0001), leading to confounding of the PCB and selenium effects. The smoking status and gender had no significant effect for either group on the levels of 8-oxodG and total adducts (not shown). However, selenium had significant negative effect on 8-oxodG and total adducts in the high selenium/PCB group, indicating that increase in selenium reduces the adduct burden in the high ratio group, whereas no significant effect was detectable for the low ratio group. More precisely, in the high ratio group, for 8-oxodG and total adducts in log scale, the coefficient of selenium is significantly negative (r = −0.38, p = 0.014 and r = −0.41, p = 0.009), respectively (Fig. 5).

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Table 2a Estimates of the coefficients from regression of 8-oxodG adducts on PCB, gender and smoking status.

Fig. 2. Box plots highlighting the distribution and inter-individual variability of different subgroups of adducts. Total adducts represent the sum of all the subgroups of adducts.

4. Discussion This study is unique in that we have determined both DNA adduct profiles as well as the mitigating effects of selenium, in a population that is known to have high exposure to both oganochlorines as well as selenium via the traditional diet comprising of sea mammals. Smoking status is also reported to be high in this population compared to others. Information on all the variables including age, gender and smoking status provides a unique opportunity to understand the interaction between the dietary elements that have biological significance. Preliminary analyses showed a significant decrease in the total DNA adduct levels with increasing PCBs. When stratified for age, the 8-oxodG lesions showed no association with the PCB levels for subjects aged 59 years or below, and a negative correlation for subjects 60 years and older. However, we have demonstrated previously that the human hepatic enzymes metabolize lower chlorinated biphenyl congeners to reactive intermediates that form DNA adducts (Arif et al., 2003; Pereg et al., 2002). In addition to direct binding, the semiquinone intermediates formed can also produce reactive oxygen species, capable of damaging the DNA. The subjects had a mixture of PCB congeners; however, the effect of individual congeners of DNA adduct formation could not be assessed. Previous studies to assess the effect of PCBs stratified by abundance, on DNA adducts, did not show any association (Ravoori et al., 2008). The protective effects of selenium against carcinogen-induced DNA adduct formation is substantiated in several organs with animal

Age group

Intercept

PCB

Gender

Smoking Status

< 30 years (n = 13) 30–39 years (n = 27) 40–49 years (n = 12) 50–59 years (n = 12) 60 years and above (n = 19)

9 (< 0.001) 8.07 (< 0.001) 8.65 (< 0.001) 6.92 (< 0.001) 8.19 (< 0.001)

− 0.12 ( 0.4154) − 0.02 ( 0.4785) − 0.02 ( 0.506) 0.03 ( 0.1321) − 0.02 (0.0326)

0.11 ( 0.8782) − 0.01 ( 0.982) − 0.54 ( 0.3954) 0.36 ( 0.7552) 0.24 ( 0.6033)

− 0.47 ( 0.2933) 0.05 ( 0.9135) 0.06 ( 0.9452) − 0.71 ( 0.3655) 0.16 ( 0.6481)

Numbers in parentheses are p values of the estimates; numbers in bold - significant values.

studies (El-Bayoumy, 2001). While the adduct levels tend to decrease with increasing selenium levels, the effect was not significant. A population that is exposed only to PCBs or a group of people with high levels of selenium, not accompanied by increased PCB levels is not available to study a direct cause–effect relationship. A plausible explanation for the decrease in adduct levels with increasing PCBs is therefore the presence of selenium, which increases with increasing levels of PCBs, in the same diet. We postulated that selenium and PCBs have counteracting effects on DNA adducts, and a ratio of selenium to PCB was determined. At ratios of selenium/PCB below 33, determined by statistical evaluation, DNA damage could occur either due to high levels of PCBs or low selenium. However, a comparison between the high and low selenium/PCB ratio groups suggests that the selenium levels are similar in both groups. Therefore, the DNA damaging effects could be caused primarily by the PCBs. Studies on the effect of different congeners of PCBs on glutathione enzymes and selenium status have shown suppression of glutathione peroxidase activities by lower halogenated PCBs by depleting selenium (Twaroski et al., 2001). A recent study to assess the countering effects of dietary mercury and selenium has shown that rats fed low selenium diets showed greatest toxicity effects from high mercury exposures due to diminution of selenium bioavailability by methylmercury (Ralston et al., 2008). Nevertheless, the damage to DNA could still be countered by the low levels of selenium, which is probably why an association of the adduct levels with PCBs could not be made in our study. Extensive studies

Fig. 3. Correlation of A) PCB with age, B) selenium with age, C) PCB with selenium, D) 8-oxodG with age and E) total adducts with age.

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Table 2b Estimates of the coefficients from regression of total adducts on PCB, gender and smoking status. Age group

Intercept

PCB

Gender

Smoking Status

< 30 years

9.04 (< 0.0001) 8.82 (< 0.001) 8.46 (< 0.001) 7.72 (< 0.001) 8.56 (< 0.001)

− 0.1 (0.0124) − 0.03 (0.1346) − 0.01 (0.4255) 0.01 (0.3482) − 0.01 (0.1324)

− 0.18 (0.4109) − 0.05 (0.8865) 0.56 (0.1464) − 0.42 (0.5348) 0.15 (0.6185)

0.23 ( 0.3723) − 0.05 ( 0.8686) − 0.31 ( 0.5295) 0.27 ( 0.7216) − 0.02 ( 0.9442)

30–39 years 40–49 years 50–59 years 60 years and above

Numbers in parentheses are p values of the estimates; numbers in bold - significant values.

were carried out previously by Belanger and colleagues to understand the effect of PCBs and other contaminants in this population on oxidative stress, by measuring the redox states of α-tocopherol and Co-Q10 (Belanger et al., 2006, 2008). These studies failed to make any definitive associations of PCB to oxidative stress, suggesting a possible protective effect from selenium. Further Ralston et al. (2008) have also shown that presence of a potential toxic agent such as mercury and a protective agent such as selenium in the same diet confounds the assessment of the toxic agent as an independent risk factor. Studies have shown that biomarkers of hepatic enzyme induction such as D-glucaric acid, uroporphyrins and coproporphyrins showed no relation with high levels of organochlorine exposure in another Canadian fish-eating population (Ayotte et al., 2005). Several studies in the past have suggested the accumulation of DNA adducts with age (Randerath et al., 1990; Singh, 2006; Ward et al., 2005). However, in this study, the 8-oxodG and total adduct levels are significantly higher in the high ratio group comprising younger subjects compared to the low ratio group comprising older subjects. The adduct levels decline with increasing age in this population, when the study group is considered as a whole. There is also a decrease in selenium/PCB with increasing age. This highlights the ability of the PCBs to accumulate at a higher rate while the selenium is used up. Other factors such as a possible change in the dietary habits also need consideration. For example, although both PCBs and selenium showed significant increase with age, the correlation coefficient of selenium is much lower than that of PCBs. With the younger population inclined towards a more Western diet than the traditional one (Kuhnlein et al., 2004), the intake of PCBs and selenium decrease. PCBs, albeit ingested in smaller quantities, bioaccumulate, while selenium is homeostatically controlled. Over the past 30 years, a significant decrease in the intake of proteins and an increase in total lipid intake have been observed in this population (Van Oostdam et al., 2005). This increase has been attributed to increased consumption of prepared meals and meats as against the traditional Inuit diet, as a result of urbanization. When stratified by age, subjects younger than 30 years showed a negative correlation of total adducts levels with their PCB levels, or in other words, though their PCB levels are low,

Fig. 4. Correlation of total adducts with A) PCB and B) selenium.

Table 3 Comparison of various parameters between selenium/PCB high and low groups. Variable

High selenium/PCB group (n = 41) (Mean ± std. dev)

Low selenium/PCB group (n = 42) (Mean ± std. dev)

p-value$

Age (years) Selenium (µg/L) PCB (µg/L) Selenium/PCB

35.6 ± 1.5 652.4 ± 67.5 12.8 ± 2.0 75.5 ± 7.9

53.7 ± 2.0 694.8 ± 56.3 45.1 ± 4.2 18.0 ± 1.1

<0.0001 0.2156 <0.0001 <0.0001

$

p-values are based on the non-parametric Wilcoxon (Kruskal–Wallis) tests.

their adduct levels are high. Therefore, it is possible that agents other than PCBs, such as heterocyclic amines, nitrosamines and polycyclic aromatic hydrocarbons (PAHs) from fried foods in Western diet, that cause damage to DNA (Jagerstad and Skog, 2005), are responsible for the increase in adduct levels in the younger generation. Increased intake of ω-6 polyunsaturated fatty acids, implicated in several cancers including colon and breast, have been shown to increase DNA adducts in white blood cells (Nair et al., 1997). However, the adduct levels decrease in this study population, which can partially be explained by the presence of high levels of selenium. One study assessing the association between 2, 2′, 4, 4′, 5, 5′-hexachlorobiphenyl (CB-153) and alteration in sperm DNA integrity has shown that while the European population showed increased DNA fragmentation and Bcl-xL expression, no such correlations could be found among the Inuit (Stronati et al., 2006). Compared to the whole blood selenium levels of North American population, Inuit have levels that are at least 8–15 times higher and have been shown to be protective against prostate cancers (Dewailly et al., 2003). In our study, the specific form of selenium that may afford protection against DNA damage was not determined. However, total selenium levels seem to increase with age, suggesting accumulation. The organic form of selenium, selenomethionine, is stored in tissues at higher levels and is also the most bioavailable form compared to selenocysteine, selenate and selenite, which are converted to either selenocysteine or to methylated selenoforms. Several animal studies have shown selenomethionine as a potent chemopreventive agent (Behne and Kyriakopoulos, 2001). The overall cancer incidence and heart diseases are at lower rates in this population, after adjusting for age (Dewailly et al., 2001; Friborg and Melbye, 2008). Other factors such as effect of long chain polyunsaturated fatty acids

Fig. 5. Correlation analysis of DNA adducts with selenium in the selenium/PCB A) low and B) high groups.

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and consumption of wild berries by this population not assessed in this study also need consideration. The Inuit of Greenland, for example, have a very low prevalence of heart diseases, and a study has shown that their coenzyme Q10 levels are high, and correlate positively with their selenium levels (Pedersen et al., 1999). Although the protective effects of selenium have been assessed extensively in this population, its potential mitigating effects on DNA adduct formation which may be related to PCB exposure are explored in this study for the first time. Compounds that may have a major benefit in humans may sometimes not show promising results in animal studies due to inherent shortcomings in the models, including the differences in the way the compounds are metabolized. Thus, the importance and implications of such studies on populations with specific dietary habits are far reaching. In conclusion, a direct demonstration of DNA damaging effects of PCBs was not recognized in this population, suggesting that a consistent intake of selenium was overwhelmingly protective. While the Inuit may be protected from potential harmful effects of environmental pollutants due to selenium intake, it is noteworthy that exposures to environmental pollutants in several other regions such as the Detroit River–Western Lake Erie basin and San Francisco Bay in the United States and several other parts of the world may have adverse health implications in those populations. The benefit of protective agents such as selenium is highlighted. Correlation of DNA adduct inhibition in various tissues with reduction in tumor formation when intervened with selenium (El-Bayoumy, 2001) reinstates the applicability of DNA adducts as biomarkers in chemoprevention studies. Acknowledgements This work was supported by USPHS grants ES 07380, CA-77114, P30ES 014443 ES 013661 and, in part, from the Agnes Brown Duggan Endowment Funds. References The effect of vitamin E and beta carotene on the incidence of lung cancer and other cancers in male smokers. The Alpha-Tocopherol, Beta Carotene Cancer Prevention Study Group. N Engl J Med 1994;330:1029–35. Arif JM, Lehmler HJ, Robertson LW, Gupta RC. Interaction of benzoquinone- and hydroquinone-derivatives of lower chlorinated biphenyls with DNA and nucleotides in vitro. Chem Biol Interact 2003;142:307–16. Ayotte P, Dewailly E, Lambert GH, Perkins SL, Poon R, Feeley M, et al. Biomarker measurements in a coastal fish-eating population environmentally exposed to organochlorines. Environ Health Perspect 2005;113:1318–24. Behne D, Kyriakopoulos A. Mammalian selenium-containing proteins. Annu Rev Nutr 2001;21:453–73. Belanger MC, Dewailly E, Berthiaume L, Noel M, Bergeron J, Mirault ME, et al. Dietary contaminants and oxidative stress in Inuit of Nunavik. Metab Clin Exp 2006;55:989–95. Belanger MC, Mirault ME, Dewailly E, Berthiaume L, Julien P. Environmental contaminants and redox status of coenzyme Q10 and vitamin E in Inuit from Nunavik. Metab Clin Exp 2008;57:927–33. Carpenter DO. Polychlorinated biphenyls (PCBs): routes of exposure and effects on human health. Rev Environ Health 2006;21:1-23. Clark LC, Combs Jr GF, Turnbull BW, Slate EH, Chalker DK, Chow J, et al. Effects of selenium supplementation for cancer prevention in patients with carcinoma of the skin. A randomized controlled trial. Nutritional Prevention of Cancer Study Group. JAMA 1996;276:1957–63. Dallaire F, Dewailly E, Muckle G, Vezina C, Jacobson SW, Jacobson JL, et al. Acute infections and environmental exposure to organochlorines in Inuit infants from Nunavik. Environ Health Perspect 2004;112:1359–65. Dallaire F, Dewailly E, Vezina C, Muckle G, Weber JP, Bruneau S, et al. Effect of prenatal exposure to polychlorinated biphenyls on incidence of acute respiratory infections in preschool Inuit children. Environ Health Perspect 2006;114:1301–5. Demers A, Ayotte P, Brisson J, Dodin S, Robert J, Dewailly E. Risk and aggressiveness of breast cancer in relation to plasma organochlorine concentrations. Cancer Epidemiol Biomarkers Prev 2000;9:161–6. Demers A, Ayotte P, Brisson J, Dodin S, Robert J, Dewailly E. Plasma concentrations of polychlorinated biphenyls and the risk of breast cancer: a congener-specific analysis. Am J Epidemiol 2002;155:629–35. Dewailly E, Ayotte P, Bruneau S, Laliberte C, Muir DC, Norstrom RJ. Inuit exposure to organochlorines through the aquatic food chain in arctic quebec. Environ Health Perspect 1993;101:618–20.

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