1 Genetic Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, Nih, Bethesda, MD 20892-7236, USA
2 EPOCA, Epidemiology Research Center, University of Milan, Milan, Italy
3 Environmental Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA
4 Department of Laboratory Medicine, University of Milanbicocca, Hospital of Desio, Desio, Milan, Italy
5 Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA
6 Health and Nutrition Sciences, Brooklyn CollegeCUNY, Brooklyn, NY, USA
7 To whom correspondence should be addressed Email: landim{at}mail.nih.gov
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Abstract |
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Abbreviations: AhR, aryl hydrocarbon receptor; ARNT, aryl hydrocarbon receptor nuclear translocator; BMI, body mass index; CI, confidence interval; EROD, 7-ethoxyresorufin O-deethylase; FBS, fetal bovine serum; HBSS, Hank's balanced salt solution; IS, internal standard; PBS, phosphate-buffered saline; p.p.t., parts per trillion; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TEF, toxic equivalency factor; TEQ, toxic equivalent.
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Introduction |
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In 1976, an industrial accident exposed several thousand people to substantial quantities of TCDD in Seveso, Italy. The exposed population consisted of both genders, a wide distribution of ages and a broad range of exposures and therefore provides a unique opportunity to study the effect of dioxin in a non-occupational setting. Three contamination zones (A, B and R) were delimited. A zone-based cohort including nearly 300 000 subjects living in these zones as well as in the surrounding non-contaminated area has been followed for mortality and cancer incidence studies (2,3). An increase in cancer incidence of (3,4) and mortality from (2) lymphohemopoietic neoplasms has been reported.
Approximately 20 years after the exposure, we designed a population-based study to evaluate the impact of TCDD exposure upon mechanistically based biomarkers of dioxin response in humans. We randomly selected the study's subjects from the most exposed zones (A and B) and from the surrounding non-contaminated zone in order to estimate TCDD plasma levels and gene expression in the general population of the entire area. In individuals from zones A and B, elevated plasma TCDD levels [ranging from background values to 90 ng/kg lipid, or parts per trillion (p.p.t.)] were still present after a period of time roughly equivalent to two biological half-lives, with significantly higher levels in women (5,6). In contrast, other dioxin-like congeners were at background levels in both TCDD-exposed and non-exposed areas. TCDD levels in study subjects were within the range of body burdens associated with sensitive dioxin-dependent responses in animal studies, such as induction of CYP1A1 (7).
Genetic and biochemical studies indicate that the AhR is necessary for most of the toxic effects of TCDD, such as tumor promotion, thymic involution, craniofacial anomalies, skin disorders and alterations in the endocrine, immunological and reproductive systems (8,9). TCDD-activated AhR can induce changes in growth factor receptor signaling, cytosolic signaling proteins, calcium mobilization, tumor suppressor proteins and oncogene or cell cycle proteins (1012) and can form a complex with the retinoblastoma protein (9,13) or the RelA NF-B subunit (14). AhR has been known for a long time as a ligand-activated receptor and transcription factor that forms an active heterodimer with the aromatic hydrocarbon nuclear translocator (ARNT/HIF-1ß) and activates the transcription of xenobiotic metabolizing enzymes, such as cytochrome P4501A1 (CYP1A1) and P4501B1 (CYP1B1) as well as other genes (15). Prolonged expression of CYP1A1 may increase the likelihood of deleterious DNA lesions, due to an increase in the generation of genotoxic metabolites and reactive oxygen species (18). Polymorphisms of the CYP1A1 gene and the magnitude of induction of CYP1A1 gene expression by AhR agonists in mitogen-activated human lymphocytes have been correlated with an increased risk of lung cancer in some studies (19), but not in others (20). Similarly, CYP1B1 may be involved in the mechanism of carcinogenesis through its metabolism of 17ß-estradiol and bioactivation of polycyclic aromatic hydrocarbons and arylamines (21,22). Both CYP1A1 (23) and CYP1B1 (24) mRNA levels, measured by quantitative RTPCR in peripheral lymphocytes, have been proposed as biomarkers of TCDD biological effective dose in humans.
We report here results on the measurement of expression of several genes involved in the AhR pathway, specifically AhR, ARNT, CYP1A1 and CYP1B1 and CYP1A1-associated 7-ethoxyresorufin O-deethylase (EROD) activity in study subjects' peripheral blood lymphocytes. Some AhR-dependent markers, such as CYP1A1 expression and EROD activity, are known to be only barely detectable in uncultured lymphocytes (25,26). Therefore, we measured the AhR-related markers in both unstimulated cells and in lymphocytes treated with mitogen and in vitro TCDD. The main objective of the study was to verify whether plasma levels of dioxin, measured approximately two decades after the accident, were associated with this pattern of AhR-dependent gene expression and activity and to identify environmental or host factors which could modify such an association.
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Materials and methods |
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Measurement of TCDD in plasma
The dioxin measurements in human plasma were performed at the CDC using a high resolution gas chromatography/high resolution mass spectrometry analysis (27). Specifically, TCDD and 21 other dioxin or dioxin-like congeners were measured, including 10 dibenzofurans, four co-planar polychlorinated biphenyls and seven additional dibenzo-p-dioxins. Results are reported in p.p.t., lipid adjusted. Of the 121 subjects, 11 samples (four from zone B and seven from zone non-ABR) were inadequate and were excluded from the analyses based on plasma TCDD. In another 23 subjects (nine from zone B and 14 from zone non-ABR) levels were determined to be below the detection threshold and so values were estimated by dividing the lipid-adjusted detection limit by 2 (28). Excluding or assigning 0 values for these samples did not substantially change the reported findings. The toxic equivalent (TEQ) for a mixture of dioxin-like compounds is defined as the product of the concentration of each congener multiplied by its specific toxic equivalency factor (TEF). The TEF of polychlorinated dibenzo-p-dioxins and dibenzofurans (2931) was defined as the toxic potency of the individual congener relative to TCDD, which is assigned a TEF of 1.0.
Biological sample acquisition and storage
Donors provided 550 ml of whole blood, which was collected into tubes treated with sodium heparin. The blood was diluted with Hank's balanced salt solution (HBSS) (Life Technologies, Gaithersburg, MD) at a proportion of 17 ml blood per 13 ml salt solution. Mononuclear cells were separated by Ficoll Hypaque density gradient centrifugation at 1000 g (Histopaque 1077; Sigma Chemical Co., St Louis, MO). The buffy coat containing the mononuclear cells was washed with 25 ml basal lymphocyte culture medium (basal medium) consisting of RPMI 1640 (Life Technologies) with 10% sterile filtered fetal bovine serum (FBS) (Hyclone, Logan, UT), 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin and 0.25 µg/ml amphotericin (Life Technologies). After centrifugation at 250 g for 10 min, the pellet was washed in 45 ml HBSS. The cell concentration was adjusted to 2 x 107 cells/ml and an equal volume of freeze medium (RPMI 1640; Life Technologies) with 7.5% cell culture grade DMSO (American Type Culture Collection, Rockville, MD), 20% FBS, 2 mM glutamine, 100 U/ml penicillin and 100 µg/ml amphotericin (Life Technologies) was added. A 1 ml aliquot of cells was frozen at a rate of 1°C/min and then stored in the vapor phase of liquid nitrogen.
Peripheral blood mononuclear cell culture
Mononuclear cells were thawed in a 37°C water bath and washed in 40 ml of basal medium at 37°C. After centrifugation at 200 g for 10 min, the cells were resuspended in 1 ml fresh stimulation medium consisting of basal medium supplemented with 1.25 µg/ml phytohemagglutinin (Murex Diagnostics, Norcross, GA), 0.15% (v/v) pokeweed mitogen (Life Technologies) and 50 µM 2-mercaptoethanol (Sigma). Treated cells received stimulation medium containing 20 nM TCDDtreated FBS. The method for incorporating and verifying the concentration of TCDD is detailed in Spencer et al. (24) and Tucker et al. (32). Stimulation medium was added until the cells were at a density of 2 x 106/ml. Half of each cell suspension was transferred to each of two culture flasks. One flask was treated by adding an equal volume of stimulation medium containing 20 nM TCDD for a final concentration of 10 nM TCDD. The control culture flask received an equal volume of stimulation medium without the TCDD.
Cells were cultured in a 37°C incubator at 95% relative humidity and 5% CO2. After 72 h (± 2 h), the cells were removed and resuspended in stimulation medium. Fourteen millilitres of the resuspension solution was used for subsequent RNA analysis. The remaining cells were pelleted by centrifugation at 300 g for 10 min at room temperature and resuspended in 3 ml phosphate-buffered saline (PBS) containing 1 mM EGTA. Cell concentrations were adjusted to 20 x 106 cells/ml in PBS/EGTA. Of this, 200 µl (2 x 106 cells) was removed for the EROD assay.
The cell suspension intended for RNA analysis was centrifuged at 300 g for 10 min and the pellet was resuspended in 1 ml Tri-reagent (Sigma) at room temperature. The Tri-reagent containing the mononuclear cell lysate was stored at -70°C until removal for total RNA isolation.
RNA isolation
Total RNA was isolated from mononuclear cells before and after incubation at 37°C. Tri-reagent, which employs an acidphenolguanidine thiocyanate procedure, was used to isolate total RNA as per the manufacturer's instruction. Briefly, samples were centrifuged, extracted with chloroform and washed with isopropanol and 75% cold ethanol. After the addition of 100 µl DEPC-treated water, the RNA concentration was measured at 260 nM, diluted to 40 ng/µl and stored at -70°C.
Quantitative competitive reverse transcription PCR (RTPCR)
RTPCR was accomplished by titrating 100 ng test RNA against varying but known concentrations of a heterologous recombinant internal standard (IS) as previously described (23,24). The recombinant IS RNA consisted of a spacer sequence derived from the human GSTM1 gene. The GSTM1 spacer was flanked by target RNA-specific forward and reverse primer sites and a reverse transcriptase primer site. A unique IS was constructed for each target RNA. Table I shows the primers used to detect the genes and their associated GenBank listings. Details of the recombinant IS construction, amplification and purification are described in Spencer et al. (24).
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After reverse transcription, 30 µl of PCR product was added to each tube with final concentrations of 0.50.6 µM for each of the forward and reverse primers (Bioserve), NEB buffer and 1.25 U Taq DNA polymerase (Promega) per reaction tube. The reactions were heated to 94°C for 4 min and then cycled at 94°C for 30 s, from 54 to 65°C for 30 s (the temperature varied according to the specific gene) and 75°C for 30 s. The number of cycles varied by gene transcript. The optimized conditions for each of the RTPCR assays are summarized in Table I.
Initially, a series of six 10-fold IS dilutions was performed for each sample in order to determine the approximate range of expression. The quantitation was then repeated using a series of six to seven 2-fold IS dilutions for each RNA. After amplification by PCR, the test and IS cDNAs were separated by electrophoresis (100 V for 3 h) on a 2% NuSieve 3:1 agarose (FMCBioProducts, Rockland, ME) gel in 40 mM Trisacetate, 1 mM EDTA buffer. Gels also contained 0.5 µg/ml ethidium bromide for subsequent detection. A digitized image of the cDNA was obtained using a Molecular Dynamics fluorimager (Amersham, Piscataway, NJ). The digitized images were analyzed by measuring the intensity of the DNA bands with NIH Image software (NIH Image v.1.61). Quantitation was based upon the series of six or seven IS standard concentrations run for each sample by plotting log10 ratio of band intensity of the test RNA and IS RNA against log10 copies of IS RNA. Linear regression was used to interpolate the number of RNA copies present in each 100 ng of test RNA by calculating the x-intercept, which corresponds to the equivalent band intensity and, hence, copy number of the test and IS RNAs.
Ethoxyresorufin deethylase (EROD) assay
An aliquot of 500 µl of EROD buffer [5.0 mM MgSO4 in 0.1 M KPO4 with 2.0 mg/ml bovine serum albumin (Sigma)], 20 µg NADPH and 50 pmol ethoxyresorufin in 10 µl DMSO were added to 106 mononuclear cells in 100 µl PBS/EGTA buffer and incubated for 30 min at 37°C. The reactions were stopped by the addition of a volume of methanol equivalent to the total reaction volume (620 µl). The reactions were centrifuged at room temperature for 3 min and transferred to a 96-well plate in triplicate. Each plate also included a negative control (prepared from heat-inactivated mononuclear cells), a positive control (prepared from phenobarbitol-induced murine microsomes) and a standard curve of 0.19100 pmol resorufin/well. Fluorescence was measured on a Perkin Elmer LS-50B fluorescence plate reader with excitation at 550 nM and emission at 585 nm. Activity was expressed as activity per 106 lymphocytes or per mg protein. Results are reported as pmol/min/mg protein throughout the paper. Protein was measured using a bicinchoninic acid assay kit, which includes a BSA standard (Pierce BCA protein assay kit).
Statistical analysis
Differences in gene expression and EROD activity between experiments performed in different cell culture conditions were evaluated using the paired t-test. The unpaired Student's t-test was used for group comparisons. A Bonferroni correction was used for multiple comparisons. Two-sided P values are reported. Logarithmic transformations of all variables were used to improve the fit to a normal distribution. Geometric means and 95% confidence intervals (CI) are reported throughout the manuscript. Body mass index (BMI) was defined as weight in kg/height in m2 (kg/m2). Percentage body fat was defined as in Knapik et al. (33). Pearson's correlation coefficients (r) and Wald test t values for simple and multiple regression analyses, respectively, are reported throughout the manuscript. We performed simple and multiple linear regression analyses to assess associations between variables. Independent variables for the regression models in the analyses of uncultured cells included: plasma TCDD, related AhR pathway markers, age, gender, actin expression and date of culture assay. For the cultured cells, the independent variables included: actin expression, post-culture viability, batch of experiment (17 batches were categorized in four consecutive groups for the analyses), percentage cell growth, plasma TCDD and related AhR pathway markers. All uncultured cells with viability <75% were excluded from the analyses. Results in cultured cells indicate results from lymphocytes cultured with mitogen and 10 nM TCDD, unless otherwise specified. All analyses were performed with the use of the Stata statistical package Release 7.0 (Stata Corp., College Station, TX).
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Results |
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Plasma TCDD and TEQ by zone
In subjects with detectable results in uncultured lymphocytes, mean plasma TCDD levels were 54.6 p.p.t. in subjects from zone A (n = 6, 95%CI = 22.5132.9), 11.3 p.p.t. in subjects from zone B (n = 35, 95% CI = 8.016.1) and 4.2 p.p.t. in subjects from the surrounding non-contaminated area (n = 35, 95% CI = 3.45.4). There was a strong correlation between TCDD and TEQ levels overall (n = 76, r = 0.86, P < 0.0001) and within zones (n = 41, r = 0.88, P < 0.0001 and n = 35, r = 0.73, P < 0.0001 in zones A + B and non-ABR, respectively). The percentage of TEQ due to TCDD was 26% overall (range 1084%) and varied by zone: it was 35% in zone A + B (range 1284%) and 18% in zone non-ABR (range 1041%).
Association between dioxin exposure and AhR-dependent biomarkers
Uncultured cells
In the univariate analysis, AhR (in all cell conditions) and ARNT (in uncultured cells) were lower in subjects with higher exposure (higher TCDD or TEQ plasma levels or resident in zones A + B) (Table II). In mitogen-stimulated cells, CYP1A1 expression was higher in subjects with higher TCDD levels.
In the multivariate model, the negative association between TCDD plasma levels and AhR was statistically significant in uncultured lymphocytes (t = -2.28, P = 0.026, model adjusted for age, gender, actin and date of assay) (Table III). The association between TEQ levels and AhR was also negative, but not significant in the multivariate model (t = -0.73, P = 0.467). TCDD was not significantly associated with ARNT (P = 0.21) and CYP1B1 (P = 0.60) expression.
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Cultured cells
In cells cultured with mitogen and in vitro TCDD, the association between TCDD or TEQ plasma levels and AhR, CYP1A1 or CYP1B1 expression was not significant (Table III), after adjustment for actin expression, post-culture viability, experiment group and cell growth. Although based on a small number of subjects, there was a positive association between plasma TCDD and CYP1A1 expression in zones A + B (n = 33, t = 2.06, P = 0.05). Since CYP1A1 expression was correlated with BMI (r = -0.24, P = 0.03) and with percentage body fat (r = -0.23, P = 0.03), we added these variables to the regression model, with no substantial change in the results. In subjects from zones A + B there was also a significant positive association between TEQ and AhR expression (t = 2.53, P = 0.02).
Overall, plasma TCDD and TEQ levels (Table III) were negatively and significantly associated with EROD activity (t = 0.33, P = 0.01 and t = -2.20, P = 0.03 for TCDD and TEQ levels, respectively).
Association among markers within the AhR-dependent pathway
Uncultured cells
There was a strong correlation between AhR and ARNT gene expression (t = 4.20, P < 0.001), AhR and CYP1B1 expression (t = 4.50, P < 0.001) and ARNT and CYP1B1 expression (t = 2.26, P = 0.028) in the multivariate model adjusted for age, gender, actin and date of assay (Table IV). When AhR and ARNT were fitted in one singular regression model, AhR (t = 3.30, P = 0.002), but not ARNT (t = 0.43, P = 0.67), significantly predicted CYP1B1 levels. In addition, there was no significant interaction between AhR and ARNT in this model. The median ratio between AhR and ARNT was 2.3, with an interquartile range of 1.54.0.
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Discussion |
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TCDD plasma levels were significantly associated with lower levels of AhR expression in uncultured cells. Females, who had higher levels of dioxin in our study (5,6), showed lower AhR transcripts. Although not significantly, TEQ levels were also negatively associated with AhR expression. The negative association between TCDD or TEQ levels and AhR was still evident when we considered subjects within their respective zones of residence, even though the association was not significant, possibly because of the low number of subjects in each category and/or the possible misclassification of the true biological exposure when zone is used as a surrogate for measured TCDD values.
Our findings may suggest that, at least in lymphocytes, long-term presence of dioxin in the human body does not result in an increase in AhR pathway responsiveness or that responsiveness is eventually lost or reduced decades after the initial acute exposure. Both exposed and unexposed study subjects donated their blood at approximately the same time in the morning, thus it is unlikely that a circadian variation of AhR (36) could have affected the results. However, transcript levels may have been influenced by other factors that intervened between the exposure to TCDD and our measurement. Most importantly, in the two decades since the initial exposure, responsiveness may have been lost due to adaptation, i.e. death or alteration of the responsive cell populations. Future studies may evaluate the receptor protein levels in order to determine whether the AhR protein is also reduced.
We did not find a similar negative association between TCDD plasma levels and ARNT expression. This is consistent with animal models, which indicate a stronger TCDD-dependent down-regulation of AhR protein in comparison with that of ARNT, a difference that may be due to the need to conserve ARNT for other signaling pathways (35,37,38).
As previously reported (23,24,39,40), addition of mitogen to the cell culture and in vitro TCDD resulted in a significant increase in AhR-dependent gene expression. Measured markers within the AhR pathway were highly correlated with each other. Overall, all gene expression, including that of the AhR gene, was not correlated with plasma TCDD levels. The variation in mRNA levels following stimulation with mitogen and, to a lesser extent, with TCDD in culture may have masked the effect of the accident-related TCDD present in blood. Also, after two half-lives, TCDD levels may have been too low to elicit a strong induction of the AhR pathway.
Plasma TCDD and TEQ levels were associated with decreased EROD activity in cultured cells. Cigarette constituents, such as benzo[a]pyrene and nicotine, are known to induce CYP1A1 expression (41) and EROD activity has been shown to be correlated with daily cigarette consumption in surgical lung samples (42). However, recent use of tobacco products was not significantly associated with CYP1A1 mRNA expression in cultured cells, so this variable did not account for the findings. In a previous study (43) we found that EROD activity was significantly higher in subjects carrying a polymorphic variant of the CYP1A1 gene, while expression of the CYP1A1 mRNA did not vary across genotypes. Further studies including genotype data on CYP1A1 and other related genes would be required to clarify this issue.
The observation that overall gene expression in uncultured cells were lower in subjects from zones A + B in comparison with the subjects from the non-contaminated area suggests that the zone classification may reflect the effect of acute exposure to TCDD and the presence of other unknown factors at the moment of the accident. In addition, the percentage of TEQ due to TCDD in zones A + B was almost 2-fold that in zone non-ABR. TCDD may elicit a different effect on gene expression in comparison to the effect due to other congeners. The acute exposure to TCDD may have resulted in a down-regulation of the AhR pathway, death or unresponsiveness of key cells or in a differential co-induction of a repressor molecule (44) or down-regulation of the Ah receptor-interacting protein (45) in the exposed individuals. Moreover, alternative pathways, such as the retinoic acid receptors signal transduction pathway, may have interfered with AhR signaling (46) or with the protein kinase C-mediated events required for the AhR signaling pathway.
We measured all biomarkers in peripheral blood lymphocytes. While TCDD has effects on diverse cell types, lymphocytes are readily available from blood. Mitogen-stimulated lymphocytes express the AhR, CYP1A1 and CYP1B1 genes (23,24,47,48) and similarities in the regulation of lymphocyte CYP1A1 with the liver isoenzyme have been found (49), suggesting that CYP1A1 expression in peripheral blood lymphocytes can be used to monitor hepatic enzyme activity (49). In addition, an excess of lymphoproliferative cancers is a postulated dioxin consequence (2,3), particularly in this population, and peripheral lymphocytes may provide the best feasible surrogate for lymphatic cell populations for epidemiological investigations.
Consistent with effects observed in endometrial cells (50), AhR, together with ARNT and CYP1B1, increased with age in uncultured cells and CYP1A1 increased with BMI and percentage body fat in our study. All the other factors identified in previous studies (5,6) as important determinants of TCDD levels in the population, such as distance from the accident site, consumption of domestic livestock and poultry and smoking, were not associated with AhR-dependent markers. In contrast, many laboratory-related factors, such as pre- and post-culture cell viability, storage and shipment conditions, cell growth, day of experiment and actin expression, were strongly associated with gene expression and EROD activity. We have adjusted the regression models for the possible confounders, controlled the analyses for multiple comparisons and excluded results obtained in uncultured cells with low viability. The present study highlights the need to experimentally and statistically assess these types of factors for their impact on gene expression results in molecular epidemiology studies.
In conclusion, TCDD plasma levels were associated with a reduction in AhR expression in unstimulated cells. If substantiated, this finding may suggest that long-term exposure to TCDD perturbs AhR pathway regulation. Precisely how this modulation of AhR alters the potency of dioxin as a carcinogen is not known, and will need to be explored. In mitogen-stimulated cells cultured in vitro with TCDD, plasma dioxin levels showed a lack of association with AhR-dependent gene expression and a negative association with EROD activity. Gene message levels within the AhR signaling pathway were highly correlated. Larger studies investigating gene and protein interactions in subjects with high TCDD exposure levels are needed to further elucidate TCDD action and possible carcinogenic effects in humans as well as the influence of individual susceptibility on TCDD-related adverse health outcomes.
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Acknowledgments |
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References |
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