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Effect of colchicine on P-glycoprotein expression and activity in Caco2 cells
S108
Abstracts / Toxicology Letters 196S (2010) S37–S351
P107-008 Multidrug resistance-associated proteins (MRPS) are involved in the detoxification of benzo[a]pyrene in the intestinal barrier S. Hessel 1 , A. John 2 , A. Seidel 2 , A. Lampen 1 1
Federal Institute For Risk Assessment, Berlin, Germany, Biochemical Institute for Environmental Carcinogens, Grosshansdorf, Germany 2
The human gastrointestinal tract represents the main portal for the entry of xenobiotics. The epithelium of the small intestine plays an important role in the detoxification of xenobiotics due to the expression of a number of phase I and phase II xenobioticmetabolizing enzymes (XMEs) as well as several transport proteins of the ATP-binding cassette (ABC) superfamily. In the present study, differentiated human intestinal Caco2 cells were used as a model for the human small intestine to investigate the metabolism and transport of the procarcinogenic food-contaminant benzo[a]pyrene (B[a]P). Previous studies revealed that B[a]P phenols are excreted as phase II metabolites such as B[a]P-glucuronides and B[a]P-sulfates. Excretion of these metabolites is mediated by breast cancer resistance protein (BCRP) (Ebert et al., 2005). In contrast, detoxification of the ultimate carcinogenic phase I B[a]P metabolite anti-B[a]P-7,8-diol9,10-epoxide (BPDE) occurs as glutathione conjugates formed by glutathione-S-transferases. This study demonstrates that these BPDE glutathione conjugates are excreted overall to the basolateral side of a polarized Caco-2 monolayer. Furthermore, it was shown by induction and inhibition studies that the multidrug resistance-associated proteins (MRPs), but not BCRP, are involved in the transport of the BPDE glutathione conjugates. The unspecific MRP inhibitor MK571 completely blocked transport of BPDE glutathione conjugates. For the identification of the specific MRP proteins mediating the apical and basolateral efflux of BPDE glutathione conjugates, respectively, stable MRP1, MRP2 and MRP3 knock down clones were generated by using siRNA technique. The treatment of the knock down clones supported the hypothesis that MRP1 mediates the basolateral and MRP2 the apical excretion in the Caco-2 cell line. Finally it was shown that the transport rates are increased by pre-treatment of the cells with the flavonoid quercetin, which stimulates expression of various phase I and transport proteins. doi:10.1016/j.toxlet.2010.03.383
P107-009 Association of CYP2E1 gene polymorphisms with atherosclerosis: A preliminary study L. S¸ahiner 1 , Z. Kayaaltı 2 , T. Soylemezoglu 2 Ankara Oncology Hospital, Ankara, Turkey, 2 Ankara University Institute of Forensic Medicine, Turkey
1
Atherosclerotic cardiovascular diseases form the leading cause of death in industrialized countries and multiple genetic factors are suggested to play a role in the pathogenesis of atherosclerosis. On the basis of epidemiological evidence, it has been suggested that several factors associated with an increased risk of cancer are also associated with an increased risk of atherosclerosis. Cytochrome P4502E1 (CYP2E1) polymorphism have been found to be associated with smoking related cancers and may therefore also be associated with vascular disease. CYP2E1 shows genetic polymorphisms that vary markedly in frequency among different ethnic groups.
CYP2E1 has very broad substrate specificity, these substrates are small hydrophobic substances and many of them exert a high affinity to the enzyme and most of them are also inducers of the enzyme. Among the substrates are alcohols, ketones, aldehydes, aromatic compounds, halogenated alkanes or alkanes, anaesthetics, drugs and carcinogens. In this study we investigated the risk of coronary atherosclerosis associated with polymorphisms of CYP2E1. For this purpose, 100 atherosclerotic patients (60 males, 40 females) and 100 healthy controls (60 males, 40 females) were studied by PCRRFLP technique. The allele frequencies for CYP2E1*6 and CYP2E1*7 were consistent with Hardy-Weinberg equilibrium in patients and control groups (p > 0.05). CYP2E1*6 A allele (atypical allele) frequency was found as 4.5% and 9% respectively in the patient and control groups. CYP2E1*7 T allele (atypical allele) frequency was 5.5% in patient group and 6.5% in control group. Although the frequencies of atypical alleles were apparently higher in both male and female patients compared to healthy controls; the difference could not reach statistical significance because the study groups were relatively small in number. We suggest that with the inclusion of more patients and healthy volunteers to the study, the differences in frequencies of atypical alleles would be statistically significant. doi:10.1016/j.toxlet.2010.03.384
P107-010 Effect of colchicine on P-glycoprotein expression and activity in Caco2 cells T. Houdkova 1 , R. Silva 1 , A. Cordeiro-Da-Silva 2 , H. Carmo 1 , F. Remião 1 Faculty of Pharmacy, University of Porto, Portugal, 2 IBMC – Institute of Molecular and Cellular Biology, University of Porto, Portugal
1
The Caco2 cell line is a widely used human cell culture model, derived from human colorectal adenocarcinoma, and accepted as a reliable in vitro model for intestinal drug excretion mediated by P-glycoprotein (P-gp). P-gp is an ATP-dependent efflux pump encoded by the MDR1 gene in humans that is highly expressed in several cancer cells conferring a multidrug resistance phenotype. P-gp is inducible by many drugs including dexamethasone, rifampicin, the herbal antidepressant St. Johns wort (hyperforin and hypericin) and chemotherapeutic agents including doxorubicin, daunorubicin and vinblastine. The sensitivity of Caco2 cells to different P-gp inducers is yet to be established. Colchicine is a toxic natural product originally extracted from plants of the genus Colchicum (Colchicum autumnale). It is used as an anticancer drug and is a known P-gp inducer. The aim of the present work was to evaluate the changes in P-gp expression and activity in Caco2 cells upon colchicine exposure. Caco2 cells were incubated with a range of colchicine concentrations (0.1–100 m) up to 96 h. Colchicine cytotoxity was evaluated by the MTT assay. P-gp expression and transport activity were evaluated by flow cytometry, using a fluorescein isothiocyanate conjugated antibody (CD243) and the P-gp fluorescent subtract rhodamine 123, respectively. The results show that colchicine is cytotoxic at all tested concentrations when cells are exposed for more than 24 h. Therefore, P-gp expression and transport activity were evaluated after 24 h of incubation with colchicine. Exposure to colchicine for 24 h resulted in a small but significant increase in P-gp expression levels, although no significant changes were observed in P-gp transport activity. These results support further studies aiming at the characterization of the P-gp induction mechanism
Abstracts / Toxicology Letters 196S (2010) S37–S351
for cell protection against toxic compounds, including therapeutic drugs. Acknowledgements: Fundac¸ão para a Ciência e Tecnologia is acknowledged for financial support (PTDC/SAU-OSM/ 101437/2008). doi:10.1016/j.toxlet.2010.03.385
P107-011 In vitro metabolism of M1 vinclozolin metabolite by rat liver microsomes A. Sierra-Santoyo, M.D.L. Lopez-Gonzalez, F.G. García-Montes De Oca Departamento de Toxicologia, Cinvestav-IPN, Mexico Vinclozolin (V) is a fungicide widely used worldwide for agricultural settings. V is non-enzimatically hydrolyzed to 2-[[(3 ,5 dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid (M1) and 3 ,5 -dichloro-2-hydroxy-2-methylbut-3-enanilide (M2). Classified as an endocrine-disrupting chemical, V and its metabolites M1 and M2 competitively inhibit androgen receptor. M1 is the main metabolite to explain anti-androgenic effects attributed to V. The information about M1 biotransformation is limited. The objective of this study was to determine the M1 biotransformation by rat liver microsomes. M1 (50–700 M) was incubated in phosphate buffer 100 mM pH 7.4, MgCl2 5 mM, 0.5 mg of rat liver microsomal protein and 1 mM NADPH at 37 ◦ C for 30 min. M1 metabolites from incubation media were extracted in acetonitrile and analyzed by HPLC/DAD. M1 was metabolized to 2 products, called A and B. The kinetics of formation for both metabolites was Michaelis-Menten type. KM values for A and B metabolites were 249.1 and 197.6 M, respectively, while VMax values were 0.172 and 0.094 nmol/min/mg protein, respectively. CLint values were 0.689 and 0.476 l/min/mg protein, respectively. The addition of anti-rat cytochrome P450 (CYP) antibodies to the incubation media permitted to determine that the formation of metabolite A is catalyzed mainly by CYP3A1/2 and 2B1 by 26 and 15%, respectively, while CYP1A1/2, 2E1 and 3A1/2 are involved in the formation of metabolite B by about 12% each one. These results indicate that M1 biotransformation is CYP-dependent, several CYP isoforms are involved in its biotransformation and the metabolite A is the main product of M1 biotransformation, which corresponds to the dihydroxylated derivated. Metabolic characterization of M1 is needed for a better understanding of the adverse effects associated with the exposure to V. Funded by Conacyt 45688-Q. doi:10.1016/j.toxlet.2010.03.386
P107-012 Influence of aging and prostatic disease in the expression and activity of P-glycoprotein in human lymphocytes V. Vilas boas 1 , A.M. Martins 1 , R. Silva 1 , S.A. Lima 2 , R. Medeiros 3 , A. Cordeiro-Da-Silva 2 , F. Remião 1 1
REQUIMTE, Portugal, 2 IBMC, Portugal, 3 Molecular Oncology Unit, Portugal P-glycoprotein (P-gp) is a transmembrane protein, which mediates the efflux of numerous drugs, being related with multidrug resistance phenomenon of tumour cells. In this work the expression and activity of P-gp were evaluated in human male whole blood isolated
S109
lymphocytes in order to study the influence of aging and prostatic disease in the functionality of this protein. Three donor groups were formed, distinguished by age [groups 1 (<30 years, n = 34) and 2 (>50 years, n = 31)] and absence/presence of a prostatic disease [groups 2 and 3 (n = 19), respectively]. P-gp expression was evaluated using the monoclonal antibody UIC2 and P-gp activity was determined by UIC2 shift assay and by measuring the efflux of P-gp fluorescent substrate (rhodamine 123). Flow cytometric analysis was used in all these proceedings. P-gp expression in the lymphocytes significantly increased with aging. On the other hand, a significant decrease in P-gp expression in the lymphocytes of the patients with of prostatic disease was observed. In addition, the lymphocyte’s Pg-p activity diminished significantly with aging and in the presence of prostatic disease. A strong correlation between expression and activity of P-gp in group 1 was found, whereas in groups 2 and 3 this correlation diminished or disappeared. Both methods used to determine the activity of P-gp led to similar results. In conclusion, these results suggest that when implementing a new therapeutic regimen, the evaluation of P-gp expression should not be the only parameter to be considered. Therefore, the observed decrease in the activity of lymphocytic P-gp with both aging and in the presence of a prostatic disease, in addition to the poor correlation between expression and activity in the older groups, are important data to consider when initiating a systemic therapy with P-gp substrates. Acknowledgment: This work received financial support from the Portuguese State through “Fundac¸ão para a Ciência e Tecnologia” (PTDC/SAU-OSM/101437/2008). doi:10.1016/j.toxlet.2010.03.387
P107-013 Characterization of human aryl hydrocarbon receptor polymorphisms in MCF-7 breast cancer cells T. Celius 1 , C. Rowlands 2 , J. Matthews 1 1
University of Toronto, Canada, 2 Dow Chemical Company, Canada
The aryl hydrocarbon receptor (AHR) mediates the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Although the mechanism of action of the AHR is conserved across mammalian species, numerous molecular differences in receptor activity and sensitivity to TCDD have been reported. A number of SNPs in the human AHR loci have been identified some of which have been associated with reduced receptor function. The most widely studied polymorphism in the human AHR is the R554K; however, the interpretation of the phenotypic effects of this polymorphism is unclear. This SNP has be reported to segregate with V570I and in vitro experiments suggest that the R554K;V570I variant of AHR exhibits reduced transactivation ability in mouse Hepa-1 cells. However, these studies have not been confirmed in a human cell lines. In this study we sequenced 108 human AHR genes in an effort to further identify single SNPs within the open reading frames (ORFs) of the AHR locus. We identified eight SNPs; six have been described previously and two are novel. Five of the SNPs lead to amino acid changes in the AHR protein (I277V, P517S, R554K, V570I, Q666K). To determine the ability of AHR and the single AHR variants and the combined variant R554K;V570I to regulate CYP1A1- and CYP1B1-transcription, we performed luciferase reporter gene assays in MCF-7 AHR100 cells; a human breast cancer cell line that expresses very low endogenous levels of AHR. Western blots revealed all receptors were expressed to equivalent levels. No significant differences in CYP1A1 or CYP1B1-regulated reporter gene activity or mRNA expression
Abstracts / Toxicology Letters 196S (2010) S37–S351
P107-008 Multidrug resistance-associated proteins (MRPS) are involved in the detoxification of benzo[a]pyrene in the intestinal barrier S. Hessel 1 , A. John 2 , A. Seidel 2 , A. Lampen 1 1
Federal Institute For Risk Assessment, Berlin, Germany, Biochemical Institute for Environmental Carcinogens, Grosshansdorf, Germany 2
The human gastrointestinal tract represents the main portal for the entry of xenobiotics. The epithelium of the small intestine plays an important role in the detoxification of xenobiotics due to the expression of a number of phase I and phase II xenobioticmetabolizing enzymes (XMEs) as well as several transport proteins of the ATP-binding cassette (ABC) superfamily. In the present study, differentiated human intestinal Caco2 cells were used as a model for the human small intestine to investigate the metabolism and transport of the procarcinogenic food-contaminant benzo[a]pyrene (B[a]P). Previous studies revealed that B[a]P phenols are excreted as phase II metabolites such as B[a]P-glucuronides and B[a]P-sulfates. Excretion of these metabolites is mediated by breast cancer resistance protein (BCRP) (Ebert et al., 2005). In contrast, detoxification of the ultimate carcinogenic phase I B[a]P metabolite anti-B[a]P-7,8-diol9,10-epoxide (BPDE) occurs as glutathione conjugates formed by glutathione-S-transferases. This study demonstrates that these BPDE glutathione conjugates are excreted overall to the basolateral side of a polarized Caco-2 monolayer. Furthermore, it was shown by induction and inhibition studies that the multidrug resistance-associated proteins (MRPs), but not BCRP, are involved in the transport of the BPDE glutathione conjugates. The unspecific MRP inhibitor MK571 completely blocked transport of BPDE glutathione conjugates. For the identification of the specific MRP proteins mediating the apical and basolateral efflux of BPDE glutathione conjugates, respectively, stable MRP1, MRP2 and MRP3 knock down clones were generated by using siRNA technique. The treatment of the knock down clones supported the hypothesis that MRP1 mediates the basolateral and MRP2 the apical excretion in the Caco-2 cell line. Finally it was shown that the transport rates are increased by pre-treatment of the cells with the flavonoid quercetin, which stimulates expression of various phase I and transport proteins. doi:10.1016/j.toxlet.2010.03.383
P107-009 Association of CYP2E1 gene polymorphisms with atherosclerosis: A preliminary study L. S¸ahiner 1 , Z. Kayaaltı 2 , T. Soylemezoglu 2 Ankara Oncology Hospital, Ankara, Turkey, 2 Ankara University Institute of Forensic Medicine, Turkey
1
Atherosclerotic cardiovascular diseases form the leading cause of death in industrialized countries and multiple genetic factors are suggested to play a role in the pathogenesis of atherosclerosis. On the basis of epidemiological evidence, it has been suggested that several factors associated with an increased risk of cancer are also associated with an increased risk of atherosclerosis. Cytochrome P4502E1 (CYP2E1) polymorphism have been found to be associated with smoking related cancers and may therefore also be associated with vascular disease. CYP2E1 shows genetic polymorphisms that vary markedly in frequency among different ethnic groups.
CYP2E1 has very broad substrate specificity, these substrates are small hydrophobic substances and many of them exert a high affinity to the enzyme and most of them are also inducers of the enzyme. Among the substrates are alcohols, ketones, aldehydes, aromatic compounds, halogenated alkanes or alkanes, anaesthetics, drugs and carcinogens. In this study we investigated the risk of coronary atherosclerosis associated with polymorphisms of CYP2E1. For this purpose, 100 atherosclerotic patients (60 males, 40 females) and 100 healthy controls (60 males, 40 females) were studied by PCRRFLP technique. The allele frequencies for CYP2E1*6 and CYP2E1*7 were consistent with Hardy-Weinberg equilibrium in patients and control groups (p > 0.05). CYP2E1*6 A allele (atypical allele) frequency was found as 4.5% and 9% respectively in the patient and control groups. CYP2E1*7 T allele (atypical allele) frequency was 5.5% in patient group and 6.5% in control group. Although the frequencies of atypical alleles were apparently higher in both male and female patients compared to healthy controls; the difference could not reach statistical significance because the study groups were relatively small in number. We suggest that with the inclusion of more patients and healthy volunteers to the study, the differences in frequencies of atypical alleles would be statistically significant. doi:10.1016/j.toxlet.2010.03.384
P107-010 Effect of colchicine on P-glycoprotein expression and activity in Caco2 cells T. Houdkova 1 , R. Silva 1 , A. Cordeiro-Da-Silva 2 , H. Carmo 1 , F. Remião 1 Faculty of Pharmacy, University of Porto, Portugal, 2 IBMC – Institute of Molecular and Cellular Biology, University of Porto, Portugal
1
The Caco2 cell line is a widely used human cell culture model, derived from human colorectal adenocarcinoma, and accepted as a reliable in vitro model for intestinal drug excretion mediated by P-glycoprotein (P-gp). P-gp is an ATP-dependent efflux pump encoded by the MDR1 gene in humans that is highly expressed in several cancer cells conferring a multidrug resistance phenotype. P-gp is inducible by many drugs including dexamethasone, rifampicin, the herbal antidepressant St. Johns wort (hyperforin and hypericin) and chemotherapeutic agents including doxorubicin, daunorubicin and vinblastine. The sensitivity of Caco2 cells to different P-gp inducers is yet to be established. Colchicine is a toxic natural product originally extracted from plants of the genus Colchicum (Colchicum autumnale). It is used as an anticancer drug and is a known P-gp inducer. The aim of the present work was to evaluate the changes in P-gp expression and activity in Caco2 cells upon colchicine exposure. Caco2 cells were incubated with a range of colchicine concentrations (0.1–100 m) up to 96 h. Colchicine cytotoxity was evaluated by the MTT assay. P-gp expression and transport activity were evaluated by flow cytometry, using a fluorescein isothiocyanate conjugated antibody (CD243) and the P-gp fluorescent subtract rhodamine 123, respectively. The results show that colchicine is cytotoxic at all tested concentrations when cells are exposed for more than 24 h. Therefore, P-gp expression and transport activity were evaluated after 24 h of incubation with colchicine. Exposure to colchicine for 24 h resulted in a small but significant increase in P-gp expression levels, although no significant changes were observed in P-gp transport activity. These results support further studies aiming at the characterization of the P-gp induction mechanism
Abstracts / Toxicology Letters 196S (2010) S37–S351
for cell protection against toxic compounds, including therapeutic drugs. Acknowledgements: Fundac¸ão para a Ciência e Tecnologia is acknowledged for financial support (PTDC/SAU-OSM/ 101437/2008). doi:10.1016/j.toxlet.2010.03.385
P107-011 In vitro metabolism of M1 vinclozolin metabolite by rat liver microsomes A. Sierra-Santoyo, M.D.L. Lopez-Gonzalez, F.G. García-Montes De Oca Departamento de Toxicologia, Cinvestav-IPN, Mexico Vinclozolin (V) is a fungicide widely used worldwide for agricultural settings. V is non-enzimatically hydrolyzed to 2-[[(3 ,5 dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid (M1) and 3 ,5 -dichloro-2-hydroxy-2-methylbut-3-enanilide (M2). Classified as an endocrine-disrupting chemical, V and its metabolites M1 and M2 competitively inhibit androgen receptor. M1 is the main metabolite to explain anti-androgenic effects attributed to V. The information about M1 biotransformation is limited. The objective of this study was to determine the M1 biotransformation by rat liver microsomes. M1 (50–700 M) was incubated in phosphate buffer 100 mM pH 7.4, MgCl2 5 mM, 0.5 mg of rat liver microsomal protein and 1 mM NADPH at 37 ◦ C for 30 min. M1 metabolites from incubation media were extracted in acetonitrile and analyzed by HPLC/DAD. M1 was metabolized to 2 products, called A and B. The kinetics of formation for both metabolites was Michaelis-Menten type. KM values for A and B metabolites were 249.1 and 197.6 M, respectively, while VMax values were 0.172 and 0.094 nmol/min/mg protein, respectively. CLint values were 0.689 and 0.476 l/min/mg protein, respectively. The addition of anti-rat cytochrome P450 (CYP) antibodies to the incubation media permitted to determine that the formation of metabolite A is catalyzed mainly by CYP3A1/2 and 2B1 by 26 and 15%, respectively, while CYP1A1/2, 2E1 and 3A1/2 are involved in the formation of metabolite B by about 12% each one. These results indicate that M1 biotransformation is CYP-dependent, several CYP isoforms are involved in its biotransformation and the metabolite A is the main product of M1 biotransformation, which corresponds to the dihydroxylated derivated. Metabolic characterization of M1 is needed for a better understanding of the adverse effects associated with the exposure to V. Funded by Conacyt 45688-Q. doi:10.1016/j.toxlet.2010.03.386
P107-012 Influence of aging and prostatic disease in the expression and activity of P-glycoprotein in human lymphocytes V. Vilas boas 1 , A.M. Martins 1 , R. Silva 1 , S.A. Lima 2 , R. Medeiros 3 , A. Cordeiro-Da-Silva 2 , F. Remião 1 1
REQUIMTE, Portugal, 2 IBMC, Portugal, 3 Molecular Oncology Unit, Portugal P-glycoprotein (P-gp) is a transmembrane protein, which mediates the efflux of numerous drugs, being related with multidrug resistance phenomenon of tumour cells. In this work the expression and activity of P-gp were evaluated in human male whole blood isolated
S109
lymphocytes in order to study the influence of aging and prostatic disease in the functionality of this protein. Three donor groups were formed, distinguished by age [groups 1 (<30 years, n = 34) and 2 (>50 years, n = 31)] and absence/presence of a prostatic disease [groups 2 and 3 (n = 19), respectively]. P-gp expression was evaluated using the monoclonal antibody UIC2 and P-gp activity was determined by UIC2 shift assay and by measuring the efflux of P-gp fluorescent substrate (rhodamine 123). Flow cytometric analysis was used in all these proceedings. P-gp expression in the lymphocytes significantly increased with aging. On the other hand, a significant decrease in P-gp expression in the lymphocytes of the patients with of prostatic disease was observed. In addition, the lymphocyte’s Pg-p activity diminished significantly with aging and in the presence of prostatic disease. A strong correlation between expression and activity of P-gp in group 1 was found, whereas in groups 2 and 3 this correlation diminished or disappeared. Both methods used to determine the activity of P-gp led to similar results. In conclusion, these results suggest that when implementing a new therapeutic regimen, the evaluation of P-gp expression should not be the only parameter to be considered. Therefore, the observed decrease in the activity of lymphocytic P-gp with both aging and in the presence of a prostatic disease, in addition to the poor correlation between expression and activity in the older groups, are important data to consider when initiating a systemic therapy with P-gp substrates. Acknowledgment: This work received financial support from the Portuguese State through “Fundac¸ão para a Ciência e Tecnologia” (PTDC/SAU-OSM/101437/2008). doi:10.1016/j.toxlet.2010.03.387
P107-013 Characterization of human aryl hydrocarbon receptor polymorphisms in MCF-7 breast cancer cells T. Celius 1 , C. Rowlands 2 , J. Matthews 1 1
University of Toronto, Canada, 2 Dow Chemical Company, Canada
The aryl hydrocarbon receptor (AHR) mediates the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Although the mechanism of action of the AHR is conserved across mammalian species, numerous molecular differences in receptor activity and sensitivity to TCDD have been reported. A number of SNPs in the human AHR loci have been identified some of which have been associated with reduced receptor function. The most widely studied polymorphism in the human AHR is the R554K; however, the interpretation of the phenotypic effects of this polymorphism is unclear. This SNP has be reported to segregate with V570I and in vitro experiments suggest that the R554K;V570I variant of AHR exhibits reduced transactivation ability in mouse Hepa-1 cells. However, these studies have not been confirmed in a human cell lines. In this study we sequenced 108 human AHR genes in an effort to further identify single SNPs within the open reading frames (ORFs) of the AHR locus. We identified eight SNPs; six have been described previously and two are novel. Five of the SNPs lead to amino acid changes in the AHR protein (I277V, P517S, R554K, V570I, Q666K). To determine the ability of AHR and the single AHR variants and the combined variant R554K;V570I to regulate CYP1A1- and CYP1B1-transcription, we performed luciferase reporter gene assays in MCF-7 AHR100 cells; a human breast cancer cell line that expresses very low endogenous levels of AHR. Western blots revealed all receptors were expressed to equivalent levels. No significant differences in CYP1A1 or CYP1B1-regulated reporter gene activity or mRNA expression