TCDD-mediated alterations in the AhR-dependent pathway in Seveso, Italy, 20 years after the accident

Maria Teresa Landi1,7, Pier Alberto Bertazzi2, Andrea Baccarelli1, Dario Consonni2, Scott Masten3, George Lucier3, Paolo Mocarelli4, Larry Needham5, Neil Caporaso1 and Jean Grassman3,6

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 Milan–bicocca, 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 College–CUNY, Brooklyn, NY, USA

7 To whom correspondence should be addressed Email: landim{at}mail.nih.gov


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Approximately 20 years after the Seveso, Italy, accident we conducted a population-based study to evaluate the impact of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure on cancer using mechanistically based biomarkers of dioxin response in humans. TCDD toxic effects are mediated by the aryl hydrocarbon receptor (AhR). We studied the AhR-dependent pathway in lymphocytes from 62 subjects randomly sampled from the highest exposed zones and 59 subjects from the surrounding non-contaminated area, frequency matched for age, gender and smoking. To our knowledge, this is the most comprehensive investigation to date designed to evaluate the key genes in the pathway, including AhR, aryl hydrocarbon receptor nuclear translocator, CYP1A1 and CYP1B1 transcripts and CYP1A1-associated 7-ethoxyresorufin O-deethylase (EROD) activity in a population heavily exposed to dioxin. Current lipid-adjusted plasma TCDD concentrations in these subjects ranged from 3.5 to 90 ng/kg (or p.p.t.) and were negatively associated with AhR mRNA in unstimulated peripheral blood mononuclear cells (P = 0.03). When mitogen-induced lymphocytes were cultured with 10 nM TCDD, all AhR-dependent genes were induced 1.2- to 13-fold. In these cells, plasma TCDD was associated with decreased EROD activity. In addition, there was a strong positive correlation between AhR and CYP1A1 expression (P = 0.001) and between AhR and CYP1B1 expression (P = 0.006). CYP1A1 expression was also strongly correlated with EROD activity (P = 0.001). The analysis of the expression of dioxin-inducible genes involved in carcinogenesis may help in determining dose–response relationships for human exposure to dioxin in vivo and in assessing the variability of human response, which may indicate the presence of subjects more susceptible to disease as a result of such exposures.

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.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In 1997, the International Agency for Research on Cancer established that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a human carcinogen (1) based on a plausible mechanism involving the highly conserved aryl hydrocarbon receptor (AhR), animal models and human data from industrial exposures and accidents. However, the ability to assess risk to the public has been hampered because most of the existing human cohorts are primarily males with mixed exposures in occupational settings.

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-{kappa}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 RT–PCR 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study population
The study subjects were recruited between December 1992 and March 1994. Sixty-two subjects were randomly sampled from the highest exposed zones (A and B) and 59 subjects from the surrounding non-contaminated area (non-ABR), frequency matched for age, gender and smoking, as previously described (6). Informed consent was obtained from participants and the study was reviewed and approved by the local Institutional Review Board. Residence in the specific zone (A, B or non-ABR) was established by determining address and verified by establishing actual domicile and presence in the specified area at the time of the accident. A questionnaire including data on demographics, smoking status and number of cigarettes smoked the day before the study, foods consumed at the time of the accident, residential history, occupation and reproductive and medical history was administered by trained interviewers. Subjects with severe medical illness (liver, kidney, cardiac, immune, neoplastic or major psychiatric disease) were excluded through telephone calls assisted by a physician. Exclusion rates were low and similar across the zones (five from zone non-ABR and four from zone B).

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 {surd}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 5–50 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 TCDD–treated 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 acid–phenol–guanidine 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 (RT–PCR)
RT–PCR 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).


View this table:
[in this window]
[in a new window]
 
Table I. Quantitative RT–PCR assay primers and conditions

 
Depending on the gene, each reverse transcription reaction contained the following concentrations and quantities: 100 ng test RNA, a quantity of IS RNA in a range appropriate for the anticipated expression level, 2–7.5 mM MgCl2 (Promega, Madison, WI), NEB buffer [16.6 mM NH4SO4, 5 mM 2-mercaptoethanol, 6.8 µM EDTA, 67 mM Tris–HCl, pH 8.8, 0.1 mg/ml bovine serum albumin (BSA) (Sigma)], 1 mM deoxyribonucleoside triphosphate (Promega), 15–20 U recombinant RNasin RNase inhibitor (RNase), 80 U Moloney murine Leukemia virus reverse transcriptase and 1–1.25 µM RT primer (Bioserve Biotechnologies, Laurel, MD) in a final volume of 20 µl. Reverse transcription was performed in a Perkin Elmer 9700 thermocycler. Samples were heated to 37°C for 15 min followed by 5 min at 99°C.

After reverse transcription, 30 µl of PCR product was added to each tube with final concentrations of 0.5–0.6 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 RT–PCR 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 Tris–acetate, 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 0.19–100 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).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
AhR-dependent markers in different cell conditions
Uncultured cells
In uncultured cells, only AhR, CYP1B1, ARNT and actin expression could be detected. Mean mRNA levels per µg total RNA were 11.9 x 100 000 copies for AhR, 4.7 x 100 000 copies for ARNT and 1.1 x 100 000 copies for CYP1B1 (Table II). Women had lower levels of gene expression, particularly of the AhR gene (Table II), even after adjustment for age, actin and date of assay (t = 2.68, P = 0.009). However, when plasma TCDD was added to the model, the association between gender and AhR was no longer significant (t = 1.52, P = 0.13). Biomarker mRNA levels increased with age (Table II), with a statistically significant association in the univariate analysis (r = 0.33, P = 0.002, r = 0.26, P = 0.03 and r = 0.33, P = 0.003 for AhR, ARNT and CYP1B1, respectively) and for AhR and CYP1B1 in the multivariate models (t = 2.27, P = 0.027 and t = 2.51, P = 0.015 for AhR and CYP1B1, respectively, models adjusted for plasma TCDD levels, gender, actin, smoking and date of assay). AhR expression was higher in zone non-ABR than in zones A + B (Table II). This association was significant in the multivariate analysis (t = 0.16, P = 0.03).


View this table:
[in this window]
[in a new window]
 
Table II. AhR-dependent gene expression and EROD activity (geometric means and 95% confidence intervals) by subject characteristics and dioxin exposure

 
Cultured cells
All markers, including AhR, CYP1A1 and CYP1B1 expression and EROD activity, were highly induced when cells were cultured with mitogen and with mitogen + 10 nM TCDD (Table II). We did not measure ARNT expression in cultured cells because too few cells were available for the assay. In addition, ARNT has been shown to be poorly inducible in culture (34,35). For AhR and CYP1B1, the ratio of the expression in mitogen-stimulated cells compared with expression in uncultured cells (3.0- and 16.6-fold for AhR and CYP1B1, respectively) was larger than the ratio of the expression in cells cultured with mitogen + TCDD compared with mitogen-cultured cells (1.2- and 3.8-fold for AhR and CYP1B1, respectively) (P < 0.001). As expected, CYP1A1 mRNA levels and EROD activity were strongly induced (8.8- and 13.1-fold, respectively, P < 0.001) when 10 nM TCDD was added to the mitogen-stimulated cells, particularly in subjects with low TCDD exposure or resident in zone non-ABR, in the youngest and in current smokers (Table II).

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 = 22.5–132.9), 11.3 p.p.t. in subjects from zone B (n = 35, 95% CI = 8.0–16.1) and 4.2 p.p.t. in subjects from the surrounding non-contaminated area (n = 35, 95% CI = 3.4–5.4). 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 10–84%) and varied by zone: it was 35% in zone A + B (range 12–84%) and 18% in zone non-ABR (range 10–41%).

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.


View this table:
[in this window]
[in a new window]
 
Table III. Association between plasma TCDD or TEQ and AhR-dependent markers in uncultured cells and in cells cultured with mitogen and 10 nM TCDD

 
When we divided the subjects by zone of residence, the association between TCDD and AhR expression (t = -0.89, P = 0.38 and t = -1.50, P = 0.15 in zones A + B and non-ABR, respectively) and between TEQ and AhR expression (t = -0.34, P = 0.74 and t = -0.81, P = 0.43 in zones A + B and non-ABR, respectively) were still negative, but did not reach statistical significance.

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 1.5–4.0.


View this table:
[in this window]
[in a new window]
 
Table IV. Association among markers within the AhR pathway in uncultured cells and in cells cultured with mitogen and 10 nM TCDD

 
Cultured cells
Markers measured in mitogen-treated cells and markers measured in cells cultured with mitogen + TCDD were strongly correlated (P < 0.0001). AhR and CYP1B1 expression levels measured in uncultured cells were not significantly associated with the corresponding AhR and CYP1B1 expression levels in TCDD-stimulated cultured cells (r = 0.01, P = 0.93 for AhR and r = 0.14, P = 0.22 for CYP1B1). Within cells cultured with mitogen + TCDD, AhR expression was highly and significantly associated with that of the CYP1A1 (P = 0.001) and CYP1B1 (P = 0.006) genes in the multivariate model adjusted for actin expression, post-culture viability, batch of experiment and cell growth (Table IV). In addition, a borderline significant association between AhR expression and EROD activity (P = 0.06) was observed. As expected, CYP1A1 expression was highly correlated with the corresponding EROD activity (P = 0.001).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
TCDD is considered a potent carcinogen based on in vitro experiments and animal studies (1) and exerts most of its toxic effects through the AhR. To our knowledge, this is the first population-based study that has investigated multiple components of the AhR pathway in lymphocytes from individuals accidentally exposed or not exposed to higher than background levels of TCDD.

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.


    Acknowledgments
 
The technical assistance of Diane Spencer, Monica Ter-Minassian, Chris Miller, Theresa Cambre and Vicki Walker is gratefully acknowledged. Thanks are due to the study subjects for their invaluable participation in the study and to Nigel Walker PhD for his helpful comments.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. IARC (1997) Polychlorinated Dibenzo-para-Dioxins and Polychlorinated Dibenzofurans. IARC Monographs on the Evaluation of the Carcinogenic Risks to Humans, no. 69, IARC, Lyon.
  2. Bertazzi,P.A., Consonni,D., Bachetti,S., Rubagotti,M., Baccarelli,A., Zocchetti,C. and Pesatori,A.C. (2001) Health effects of dioxin exposure: a 20-year mortality study. Am. J. Epidemiol., 153, 1031–1044.[Abstract/Free Full Text]
  3. Bertazzi,A., Pesatori,A.C., Consonni,D., Tironi,A., Landi,M.T. and Zocchetti,C. (1993) Cancer incidence in a population accidentally exposed to 2,3,7,8-tetrachlorodibenzo-para-dioxin. Epidemiology, 4, 398–406.[ISI][Medline]
  4. Pesatori,A.C., Tironi,A., Consonni,D., Baccarelli,A., Rubagotti,M., Bachetti,S., Bernucci,I., Landi,M.T., Zocchetti,C. and Bertazzi,P.A. (1999) Cancer incidence in the Seveso population, 1977–1991. Organohalogen Compounds, 44, 411–412.
  5. Landi,M.T., Needham,L.L., Lucier,G., Mocarelli,P., Bertazzi,P.A. and Caporaso,N. (1997) Concentrations of dioxin 20 years after Seveso. Lancet, 349, 1811.[CrossRef]
  6. Landi,M.T., Consonni,D., Patterson,D.G.,Jr et al. (1998) 2,3,7,8-Tetrachlorodibenzo-p-dioxin plasma levels in Seveso 20 years after the accident. Environ. Health Perspect., 106, 273–277.[ISI][Medline]
  7. DeVito,M.J., Birnbaum,L.S., Farland,W.H. and Gasiewicz,T.A. (1995) Comparisons of estimated human body burdens of dioxin like chemicals and TCDD body burdens in experimentally exposed animals. Environ. Health Perspect., 103, 820–831.[ISI][Medline]
  8. Safe,S.H. (1986) Comparative toxicology and mechanism of action of polychlorinated dibenzo-p-dioxins and dibenzofurans. Annu. Rev. Pharmacol. Toxicol., 26, 371–399.[CrossRef][ISI][Medline]
  9. Puga,A., Barnes,S.J., Dalton,T.P., Chang,C., Knudsen,E.S. and Maier,M.A. (2000) Aromatic hydrocarbon receptor interaction with the retinoblastoma protein potentiates repression of E2F-dependent transcription and cell cycle arrest. J. Biol. Chem., 275, 2943–2950.[Abstract/Free Full Text]
  10. Enan,E., El Sabeawy,F., Scott,M., Overstreet,J. and Lasley,B. (1998) Alterations in the growth factor signal transduction pathways and modulators of the cell cycle in endocervical cells from macaques exposed to TCDD. Toxicol. Appl. Pharmacol., 151, 283–293.[CrossRef][ISI][Medline]
  11. Gierthy,J.F., Silkworth,J.B., Tassinari,M., Stein,G.S. and Lian,J.B. (1994) 2,3,7,8-Tetrachlorodibenzo-p-dioxin inhibits differentiation of normal diploid rat osteoblasts in vitro. J. Cell Biochem., 54, 231–238.[ISI][Medline]
  12. Puga,A., Xia,Y. and Elferink,C. (2002) Role of the aryl hydrocarbon receptor in cell cycle regulation. Chem.-Biol. Interact., 141, 117–130.[CrossRef][ISI][Medline]
  13. Elferink,C.J., Ge,N.L. and Levine,A. (2001) Maximal aryl hydrocarbon receptor activity depends on an interaction with the retinoblastoma protein. Mol. Pharmacol., 59, 664–673.[Abstract/Free Full Text]
  14. Kim,D.W., Gazourian,L., Quadri,S.A., Romieu-Mourez,R., Sherr,D.H. and Sonenshein,G.E. (2000) The RelA NF-kappaB subunit and the aryl hydrocarbon receptor (AhR) cooperate to transactivate the c-myc promoter in mammary cells. Oncogene, 19, 5498–5506.[CrossRef][ISI][Medline]
  15. Whitlock,J.P.,Jr (1999) Induction of cytochrome P4501A1. Annu. Rev. Pharmacol. Toxicol., 39, 103–125.[CrossRef][ISI][Medline]
  16. Hoffman,E.C., Reyes,H., Chu,F.F., Sander,F., Conley,L.H., Brooks,B.A. and Hankinson,O. (1991) Cloning of a factor required for activity of the Ah (dioxin) receptor. Science, 252, 954–958.[ISI][Medline]
  17. Jiang,B.H., Rue,E., Wang,G.L., Roe,R. and Semenza,G.L. (1996) Dimerization, DNA binding and transactivation properties of hypoxia-inducible factor 1. J. Biol. Chem., 271, 17771–17778.[Abstract/Free Full Text]
  18. Kurachi,M., Hashimoto,S., Obata,A. et al. (2002) Identification of 2,3,7,8-tetrachlorodibenzo-p-dioxin-responsive genes in mouse liver by serial analysis of gene expression. Biochem. Biophys. Res. Commun., 292, 368–377.[CrossRef][ISI][Medline]
  19. Kiyohara,C., Nakanishi,Y., Inutsuka,S., Takayama,K., Hara,N., Motohiro,A., Tanaka,K., Kono,S. and Hirohata,T. (1998) The relationship between CYP1A1 aryl hydrocarbon hydroxylase activity and lung cancer in a Japanese population. Pharmacogenetics, 8, 315–323.[ISI][Medline]
  20. Houlston,R.S. (2000) CYP1A1 polymorphisms and lung cancer risk: a meta-analysis. Pharmacogenetics, 10, 105–114.[CrossRef][ISI][Medline]
  21. Hayes,C.L., Spink,D.C., Spink,B.C., Cao,J.Q., Walker,N.J. and Sutter,T.R. (1996) 17 Beta-estradiol hydroxylation catalyzed by human cytochrome P450 1B1. Proc. Natl Acad. Sci. USA, 93, 9776–9781.[Abstract/Free Full Text]
  22. Watanabe,J., Shimada,T., Gillam,E.M., Ikuta,T., Suemasu,K., Higashi,Y., Gotoh,O. and Kawajiri,K. (2000) Association of CYP1B1 genetic polymorphism with incidence to breast and lung cancer. Pharmacogenetics, 10, 25–33.[CrossRef][ISI][Medline]
  23. Vanden Heuvel,J.P., Clark,G.C., Thompson,C.L., McCoy,Z., Miller,C.R., Lucier,G.W. and Bell,D.A. (1993) CYP1A1 mRNA levels as a human exposure biomarker: use of quantitative polymerase chain reaction to measure CYP1A1 expression in human peripheral blood lymphocytes. Carcinogenesis, 14, 2003–2006.[Abstract]
  24. Spencer,D.L., Masten,S.A., Lanier,K.M., Yang,X., Grassman,J.A., Miller,C.R., Sutter,T.R., Lucier,G.W. and Walker,N.J. (1999) Quantitative analysis of constitutive and 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced cytochrome P450 1B1 expression in human lymphocytes. Cancer Epidemiol. Biomarkers Prev., 8, 139–146.[Abstract/Free Full Text]
  25. Grassman,J., Landi,M.T., Masten,S. et al. (1999) Determinants of ethoxyresorufin-O-deethylase (EROD) activity in human peripheral blood lymphocytes challenged in vitro with dioxin. Organohalogen Compounds, 44, 375–379.
  26. Masten,S.A., Grassman,J.A., Miller,C.R., Spencer,D.L., Walker,N.J., Jung,D., Edler,L., Patterson,D.G.,Jr, Needham,L.L. and Lucier,G.W. (1998) Population-based studies of dioxin responsiveness: individual variation in CYP1A1 levels and relationship to dioxin body burden. Organohalogen Compounds, 37, 13–16.
  27. Patterson,D.G.,Jr, Hampton,L., Lapeza,C.R.,Jr, Belser,W.T., Green,V., Alexander,L. and Needham,L.L. (1987) High-resolution gas chromatographic/high-resolution mass spectrometric analysis of human serum on a whole-weight and lipid basis for 2,3,7,8-tetrachlorodibenzo-p-dioxin. Anal. Chem., 59, 2000–2005.[ISI][Medline]
  28. Hornung,R.W. and Reed,L.D. (1990) Estimation of average concentration in the presence of non-detectable values. Appl. Occup. Environ. Hyg., 5, 48–51.
  29. North Atlantic Treaty Organization Committee on the Challenges of Modern Society (1988) TEFs for Dioxins and Furans, Report no. 176. US Environmental Protection Agency in Washington, DC.
  30. US Environmental Protection Agency (1989) Update EPA/625/3-89/016. US Environmental Protection Agency in Washington, DC.
  31. WHO European Center for Environment and Health and International Program on Chemical Safety (1994) Analytical intercalibration study. Chemosphere, 28, 1049–1067.[CrossRef][ISI]
  32. Tucker,A.N., Vore,S.J. and Luster,M.I. (1986) Suppression of B cell differentiation by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Mol. Pharmacol., 29, 372–377.[Abstract]
  33. Knapik,J.J., Burse,R.L. and Vogel,J.A. (1983) Height, weight, percent body fat and indices of adiposity for young men and women entering the U.S. Army. Aviat. Space Environ. Med., 54, 223–231.[ISI][Medline]
  34. Sommer,R.J., Sojka,K.M., Pollenz,R.S., Cooke,P.S. and Peterson,R.E. (1999) Ah receptor and ARNT protein and mRNA concentrations in rat prostate: effects of stage of development and 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment. Toxicol. Appl. Pharmacol., 155, 177–189.[CrossRef][ISI][Medline]
  35. Song,Z. and Pollenz,R.S. (2002) Ligand-dependent and independent modulation of aryl hydrocarbon receptor localization, degradation and gene regulation. Mol. Pharmacol., 62, 806–816.[Abstract/Free Full Text]
  36. Richardson,V.M., Santostefano,M.J. and Birnbaum,L.S. (1998) Daily cycle of bHLH-PAS proteins, Ah receptor and Arnt, in multiple tissues of female Sprague–Dawley rats. Biochem. Biophys. Res. Commun., 252, 225–231.[CrossRef][ISI][Medline]
  37. Pollenz,R.S., Santostefano,M.J., Klett,E., Richardson,V.M., Necela,B. and Birnbaum,L.S. (1998) Female Sprague–Dawley rats exposed to a single oral dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin exhibit sustained depletion of aryl hydrocarbon receptor protein in liver, spleen, thymus and lung. Toxicol. Sci., 42, 117–128.[Abstract]
  38. Gradin,K., McGuire,J., Wenger,R.H., Kvietikova,I., Whitelaw,M.L., Toftgard,R., Tora,L., Gassmann,M. and Poellinger,L. (1996) Functional interference between hypoxia and dioxin signal transduction pathways: competition for recruitment of the Arnt transcription factor. Mol. Cell. Biol., 16, 5221–5231.[Abstract]
  39. de Morais,S.M., Giannone,J.V. and Okey,A.B. (1994) Photoaffinity labeling of the Ah receptor with 3-[3H]methylcholanthrene and formation of a 165-kDa complex between the ligand-binding subunit and a novel cytosolic protein. J. Biol. Chem., 269, 12129–12136.[Abstract/Free Full Text]
  40. Bryant,P.L., Clark,G.C., Probst,M.R. and Abbott,B.D. (1997) Effects of TCDD on Ah receptor, ARNT, EGF and TGF-alpha expression in embryonic mouse urinary tract. Teratology, 55, 326–337.[CrossRef][ISI][Medline]
  41. Wei,C., Caccavale,R.J., Weyand,E.H., Chen,S. and Iba,M.M. (2002) Induction of CYP1A1 and CYP1A2 expressions by prototypic and atypical inducers in the human lung. Cancer Lett., 178, 25–36.[CrossRef][ISI][Medline]
  42. Anttila,S., Tuominen,P., Hirvonen,A., Nurminen,M., Karjalainen,A., Hankinson,O. and Elovaara,E. (2001) CYP1A1 levels in lung tissue of tobacco smokers and polymorphisms of CYP1A1 and aromatic hydrocarbon receptor. Pharmacogenetics, 11, 501–509.[CrossRef][ISI][Medline]
  43. Landi,M.T., Bertazzi,P.A., Shields,P.G., Clark,G., Lucier,G.W., Garte,S.J., Cosma,G. and Caporaso,N.E. (1994) Association between CYP1A1 genotype, mRNA expression and enzymatic activity in humans. Pharmacogenetics, 4, 242–246.[ISI][Medline]
  44. Baba,T., Mimura,J., Gradin,K., Kuroiwa,A., Watanabe,T., Matsuda,Y., Inazawa,J., Sogawa,K. and Fujii-Kuriyama,Y. (2001) Structure and expression of the Ah receptor repressor gene. J. Biol. Chem., 276, 33101–33110.[Abstract/Free Full Text]
  45. Ma,Q. and Whitlock,J.P.,Jr (1997) A novel cytoplasmic protein that interacts with the Ah receptor, contains tetratricopeptide repeat motifs and augments the transcriptional response to 2,3,7,8-tetrachlorodibenzo-p-dioxin. J. Biol. Chem., 272, 8878–8884.[Abstract/Free Full Text]
  46. Lorick,K.L., Toscano,D.L. and Toscano,W.A.,Jr (1998) 2,3,7,8-Tetrachlorodibenzo-p-dioxin alters retinoic acid receptor function in human keratinocytes. Biochem. Biophys. Res. Commun., 243, 749–752.[CrossRef][ISI][Medline]
  47. Masten,S.A. and Shiverick,K.T. (1995) The Ah receptor recognizes DNA binding sites for the B cell transcription factor, BSAP: a possible mechanism for dioxin-mediated alteration of CD19 gene expression in human B lymphocytes. Biochem. Biophys. Res. Commun., 212, 27–34.[CrossRef][ISI][Medline]
  48. Spatzenegger,M., Horsmans,Y. and Verbeeck,R.K. (2000) CYP1A1 but not CYP1A2 proteins are expressed in human lymphocytes. Pharmacol. Toxicol., 86, 242–244.[CrossRef][ISI][Medline]
  49. Dey,A., Parmar,D., Dayal,M., Dhawan,A. and Seth,P.K. (2001) Cytochrome P450 1A1 (CYP1A1) in blood lymphocytes evidence for catalytic activity and mRNA expression. Life Sci., 69, 383–393.[CrossRef][ISI][Medline]
  50. Pitt,J.A., Feng,L., Abbott,B.D., Schmid,J., Batt,R.E., Costich,T.G., Koury,S.T. and Bofinger,D.P. (2001) Expression of AhR and ARNT mRNA in cultured human endometrial explants exposed to TCDD. Toxicol. Sci., 62, 289–298.[Abstract/Free Full Text]
Received December 22, 2002; accepted January 2, 2003.