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Query: UNIPROT:P06889 (Mol)
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Carcinogenic polycyclic aromatic hydrocarbons (PAHs) form DNA adducts via a complex metabolic activation pathway that includes cytochrome P450 (CYP) 1A1, whereas intermediate metabolites can be detoxified by conjugation through pathways including glutathione s-transferase M1 (GSTM1). PAH-DNA adducts can be measured in peripheral white blood cells (WBCs) and should reflect the net effect of competing activation and detoxification pathways and DNA repair as well as exposure. We have previously shown that WBC PAH-DNA adducts measured by an enzyme-linked immunosorbent assay (ELISA) were associated with recent, frequent consumption of charbroiled food among 47 nonsmoking wildland fire-fighters who provided two blood samples 8 wk apart. In the investigation reported here, which was performed in the same population, we measured the association between the GSTM1 null genotype, which results in loss of enzyme activity, and PAH-DNA adduct levels, hypothesizing that subjects with this genotype would have higher levels of DNA adducts because of their decreased ability to detoxify PAH metabolites. However, PAH-DNA adduct levels were nonsignificantly lower in subjects with the GSTM1 null genotype (n = 28) compared with other subjects (n = 19) (median 0.04 fmol/microgram DNA vs 0.07 fmol/microgram DNA, respectively, P = 0.45, Wilcoxon rank-sum test). Adduct levels were also lower in the nine subjects heterozygous or homozygous for the CYP1A1 exon 7 polymorphism (which codes for a valine rather than isoleucine and is thought to be associated with greater CYP1A1 activity) compared with the 38 wild-type subjects (P = 0.12). In the entire group, there was a positive association between consuming charbroiled food and PAH-DNA adduct formation (r = 0.24, P = 0.02, Spearman rank-order correlation). This association was weaker in the subgroup of subjects with the GSTM1 null genotype (r = 0.03, P = 0.84) and stronger among the remaining subjects (r = 0.57, P = 0.0002). These results suggest that the GSTM1 null genotype and CYP1A1 exon 7 polymorphism are not associated with increased susceptibility for PAH-DNA adduct formation in peripheral WBCs measured by ELISA in nonsmoking populations.
Mol Carcinog 1995 Sep
PMID:The impact of glutathione s-transferase M1 and cytochrome P450 1A1 genotypes on white-blood-cell polycyclic aromatic hydrocarbon-DNA adduct levels in humans. 754 26

Many natural environments are contaminated with carcinogenic polycyclic aromatic hydrocarbons (PAHs) and N-heterocyclic aromatic hydrocarbons (NHAs) as complex mixtures of coal tar, petroleum, and shale oil. These potentially hazardous substances are prevalent at many former tar production and coal gasification sites. Three polycyclic [benzo(a)pyrene (BaP), benz(a)anthracene (BAA), and 7,12-dimethylbenz(a)anthracene (DMBA)] and two N-heterocyclic [7H-dibenzo(c,g)carbazole (DBC), and dibenz(a,j)acridine (DBA)] aromatic hydrocarbons were analyzed for cytotoxic and genotoxic effects on human lymphocytes. All of these polyaromatic compounds are normally present in the environment, except for DMBA. Lymphocytes from healthy donors were isolated from whole blood. The 5-ring polycyclic aromatic BaP consistently induced micronuclei in a linear dose-dependent manner with doses from 0.1-10.0 micrograms/ml, whereas the 4-ring compounds (BAA and DMBA) had no effect on the induction of micronuclei above controls except at 5 and 10 micrograms/ml. Of the two N-heterocyclic compounds, DBC produced a significant increase in micronuclei in lymphocytes, but the dose response tended to plateau above 0.1 microgram/ml. DBA showed an effect on the frequency of micronuclei above controls only at high doses of 5 and 10 micrograms/ml. The average background frequency of micronuclei for 7 lymphocyte donors averaged 3.1 per 1,000 stimulated cells, whereas the average frequency of micronuclei at 10 micrograms/ml BaP was 36.8 per 1,000 stimulated cells. The lowest effective dose in 2 donors for BaP occurred at 0.1 microgram/ml. At a challenge dose of 1 microgram/ml (4 microM) of BaP, considerable variation in micronuclei induction between 7 individuals was observed, ranging from 2-6-fold increases above spontaneous frequency. Over a dose range of 1-10.0 micrograms/ml (4-40 microM), BaP also induced sister chromatid exchanges (SCEs) in lymphocytes, whereas BAA had no effect above controls. Parallel studies of both cytogenetic endpoints showed that the micronucleus assay is a more sensitive indicator of BaP exposure at equivalent doses. Mitotic and replication indices of BaP-exposed lymphocytes showed that cell proliferation is only moderately inhibited even at the highest dose; this shows that bulky DNA-adducts are generally compatible with cell survival. The cytogenetic data are consistent, first-off, with reports that individuals in the population vary widely with respect to the inducibility of the CYP1A1 gene, which is known to be involved in polycyclic aromatic hydrocarbon metabolism, in particular, in BaP. Secondly, the data support the fact that polyaromatic compounds differ with regard to micronucleus induction within the same sample(s) of human lymphocytes, indicating selective metabolism of polyaromatic compounds that may reflect carcinogen sensitivity of the individual.(ABSTRACT TRUNCATED AT 400 WORDS)
Environ Mol Mutagen 1995
PMID:Induction of micronuclei and sister chromatid exchanges by polycyclic and N-heterocyclic aromatic hydrocarbons in cultured human lymphocytes. 755 7

Previous studies in our laboratory identified a negative regulatory domain in the 5'-flanking region of the human CYP1A1 gene containing two negative regulatory elements (NRE). Characterization of one of these elements revealed three nuclear protein binding regions: a 21-bp palindrome with a point of symmetry at -784 and two guanine- and cytosine-rich elements that flank the palindrome. Functional studies suggested the palindrome is critical for transcriptional repression, whereas the guanine- and cytosine-rich sequences play a secondary role. In this study, the interaction between nuclear proteins and the CYP1A1 NRE was further defined. Electrophoretic mobility shift assays (EMSA) indicated that the NRE -784 palindrome alone, but not the guanine- and cytosine-rich sequences minus the palindrome, was capable of specific nuclear protein binding. Competitive cotransfection experiments confirmed this observation in intact cells. Specific residues important for DNA-protein interactions were identified by site-directed mutagenesis and competitive EMSA. The loss of specific protein binding was also correlated with the loss of negative regulatory activity in a transient-expression assay. Finally, competitive EMSA was performed with consensus oligonucleotides for known transcription factors. An NF-Y consensus sequence efficiently competed with the NRE probe for specific nuclear protein binding. EMSA supershift analyses indicate that a protein immunologically related to NF-YB is part of the specific nuclear protein complex binding the human CYP1A1 NRE. These studies have refined our understanding of the sequences critical for the transcriptional repression of human CYP1A1. To our knowledge, this is also the first report implicating a member of the NF-Y transcription factor family in negative gene regulation.
Mol Cell Biol 1995 Sep
PMID:Partial characterization of the human CYP1A1 negatively acting transcription factor and mutational analysis of its cognate DNA recognition sequence. 765 31

To determine whether human liver responds to treatment with aromatic hydrocarbons (AHs) with induction of the multidrug resistance (mdr) gene product P-glycoprotein and whether AH induction of mdr involves the Ah receptor, we compared induction of mdr mRNA with induction of cytochrome P450 (CYP)1A1 mRNA in AH-treated cultures of primary human hepatocytes. Hepatocytes from all 15 individuals tested responded to treatment with 3-methylcholanthrene (MC) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with induction of CYP1A1 mRNA. However, only 62% and 55% of the preparations responded to treatment with MC and TCDD, respectively, with induction of mdr mRNA. Indeed, in some individuals mdr mRNA was suppressed by MC and TCDD despite robust CYP1A1 induction. These studies provide the first evidence that not only does individual variation in mdr induction by AH exist but that AHs regulate mdr in humans by a novel mechanism distinguishable from the classical Ah receptor pathway. The dramatic variability in AH induction of mdr may be a predictive risk factor that will help to identify an individual's risk of AH-associated toxicities.
Mol Carcinog 1995 Feb
PMID:Phenotypic variability in induction of P-glycoprotein mRNA by aromatic hydrocarbons in primary human hepatocytes. 766 17

DNA repair-deficient (xeroderma pigmentosum group A (XPA)) and DNA repair-proficient (normal) human skin fibroblasts were genetically engineered by transformation with a controllable human cytochrome P450 (CYP)1A1 expression vector. Induction of CYP1A1 enabled these cells to metabolize (+/-)-benzo[a]pyrene-trans-7,8-dihydrodiol (BPD) into a potent cytotoxicant and mutagen. The XPA cells were more susceptible than the normal cells to the cytotoxic effects of both CYP1A1-metabolized BPD and exogenously supplied (+/-)-anti-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10- epoxide (BPDE). Furthermore, the differential cytotoxicity between XPA and normal cells induced by CYP1A1-metabolized BPD was 8.4-fold greater than that induced by exogenously supplied BPDE. The two cell lines had similar CYP1A1 activities, suggesting that a difference in metabolic potential was not the cause of the differential response to BPD. At comparable cytotoxicity in both XPA and normal cells, BPD treatment induced more mutants and more DNA adducts than BPDE treatment did. At similar levels of DNA adducts in XPA cells, the levels of cytotoxicity induced by CYP1A1-metabolized BPD and exogenously supplied BPDE were similar, but CYP1A1-metabolized BPD induced a threefold higher hypoxanthine phosphoribosyltransferase mutation frequency. In contrast, at similar levels of adducts in CYP1A1-expressing normal cells, BPD induced less cytotoxicity and a lower mutation frequency. DNA adducts were identified and quantified by 32P-postlabeling analyses. The principal adduct formed by both CYP1A1-metabolized BPD and exogenously supplied BPDE was 10-beta-(deoxyguanosin-N2-yl)-7 beta,8 alpha,9 alpha-trihydroxy-7,8,9,10- tetrahydrobenzo[a]pyrene, indicating that the differential effects of BPD- and BPDE-induced adducts were not due to a difference in the types of adducts formed. The results of these studies suggest that CYP1A1-metabolized BPD may form adducts preferentially in transcriptionally active genes or that the intracellular concentration of BPDE may influence the balance between cytotoxicity and mutagenicity (or both).
Mol Carcinog 1995 Feb
PMID:Differential mutagenicity and cytotoxicity of (+/-)-benzo[a]pyrene-trans-7,8-dihydrodiol and (+/-)-anti-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide in genetically engineered human fibroblasts. 766 21

The 2,3,7,8-tetrachlorodibenzo-p-dioxin-transformed aryl hydrocarbon receptor (AHR) complex binds to xenobiotic-responsive element (XRE) sequences in the 5' flanking region of the CYP1A1 gene, resulting in initiation of transcription. Both components of the transformed AHR complex [the ligand-binding AHR monomer and the AHR nuclear translocator (ARNT)] directly contact the XRE. These proteins belong to a novel subclass of basic helix-loop-helix transcription factors. The binding sites of AHR and ARNT on the asymmetric XRE were determined using nuclear extracts of 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated Hepa-1c1c7 cells and a panel of double-stranded oligonucleotides containing XRE1 of the CYP1A1 gene (5'-TTGCGTGAGAA-3'), in which all combinations of three, two, or one of the thymines indicated were substituted by the photoreactive thymine analog 5-bromodeoxyuracil. Covalent cross-linking analysis and immunoprecipitation with antibodies specific for AHR or ARNT demonstrated that ARNT directly contacts the 3'-most thymine position, that AHR directly contacts the second thymine position, and that neither protein contacts the 5'-most thymine position. The thymine position contacted by ARNT lies within a three-nucleotide sequence (5'-GTG-3') identical to a half-site of an E-box element (5'-CACGTG-3') that is recognized by a number of other basic helix-loop-helix transcription factors. AHR binds to a portion of the XRE that does not resemble an E-box. Additional experiments demonstrated that neither protein loops over to contact residues located beyond the other's binding site.
Mol Pharmacol 1995 Mar
PMID:Orientation of the heterodimeric aryl hydrocarbon (dioxin) receptor complex on its asymmetric DNA recognition sequence. 770 Feb 40

The aryl hydrocarbon receptor (AhR) is a transcriptional enhancer that is activated by the binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related toxic xenobiotics, as well as some naturally occurring compounds. Ligand binding initiates 1) dissociation of the ligand-bound monomeric AhR from the ligand-unoccupied multimeric complex and 2) biochemical and/or conformational changes that enable association of the ligand-bound monomer with other proteins. This heteromeric complex has high affinity for specific elements [dioxin-responsive elements (DREs)] in the regulatory regions of a number of structural genes, the induction and/or repression of which may be a mechanism of toxicity of TCDD. We have developed a relatively simple and rapid procedure that enables purification to homogeneity of a TCDD-bound receptor complex. The final step of purification is based on binding to an oligonucleotide containing the specific DRE sequence that is found in the upstream region of the CYP1A1 structural gene. The purified complex retains in vitro DRE-binding function. Silver staining and Western blot analyses demonstrate that the complex consists of the AhR ligand-binding monomer of approximately 104 kDa, plus two proteins (94 and 96 kDa) that are recognized by antibodies prepared against the AhR nuclear translocator protein. Previous attempts to purify a DRE-binding form of the AhR were unsuccessful because of dissociation of the complex during chromatography; this is the first report of an isolated functional complex. The purified preparation will be valuable in further studies of receptor regulation and function.
Mol Pharmacol 1994 Dec
PMID:Purification to homogeneity of the heteromeric DNA-binding form of the aryl hydrocarbon receptor from rat liver. 780 20

The effects of interleukin (IL)-1 beta, IL-4, IL-6, tumor necrosis factor (TNF)-alpha, interferon (IFN)-alpha, IFN-gamma, and transforming growth factor (TGF)-beta 1 on cytochrome P-450 (CYP) 1A expression and polycyclic aromatic hydrocarbon (PAH)-mediated induction in primary human hepatocyte cultures were determined. Most cytokines that were previously found to decrease basal CYP expression could counteract PAH induction of CYP1A mRNA and its associated ethoxyresorufin-O-deethylation (EROD) activity. IL-1 beta and TNF-alpha blocked 3-methylcholanthrene (3-MC)-induced EROD activity by up to 25 and 44%, respectively. IFN-alpha and IFN-gamma antagonized EROD induction by up to 61 and 70%, respectively. TGF-beta 1 proved to be the most effective cytokine, because 72 hr of treatment with 2 ng/ml TGF-beta 1 produced nearly 100% inhibition of 3-MC- and benzo(a)pyrene-induced CYP1A1 and CYP1A2 mRNAs and EROD activity. Treatment with cycloheximide in combination with 3-MC led to superinduction of CYP1A mRNA, under which conditions TGF-beta 1 did not block induction, suggesting the requirement for protein synthesis for the suppressive effect of the cytokine. In addition, TGF-beta 1 augmented AP-1-binding activity, suggesting that fos and/or jun protooncogene products could be implicated in the response. Our results demonstrate that IL-1 beta, TNF-alpha, and IFNs antagonized PAH-mediated induction of CYP1A gene expression in human hepatocytes. In addition, we report the finding of a novel effect of TGF-beta 1, which was able to prevent CYP1A1 and -1A2 induction by two different PAHs.
Mol Pharmacol 1994 Dec
PMID:Transforming growth factor-beta 1 down-regulates basal and polycyclic aromatic hydrocarbon-induced cytochromes P-450 1A1 and 1A2 in adult human hepatocytes in primary culture. 780 30

The ability of indole-3-carbinol (IC), an anticarcinogen present in cruciferous vegetables, to induce CYP1A1, CYP1A2, CYP2B1/2, CYP2E1 and CYP3A1/2 in female rat liver was determined by Western analysis using monoclonal antibodies and compared to effects produced by pregnenolone carbonitrile in animals of both sexes. The ontogeny of induction of these cytochrome P450 isozymes in response to oral administration of IC was also investigated. An inverse correlation was observed between the 6 beta-hydroxylation of androsterone (A) and the induction by IC of CYP3A1/2, the P450 isozyme responsible for the bulk of hepatic 6 beta-hydroxylation of 4-androstenedione (AD). The effect of inhibitors on the formation of 6 beta-OHA from A or AD was also determined and shown to differ from their action on the P450 isozymes involved in the formation of the 6 beta-hydroxylated derivatives of AD or lithocholic acid. The results indicate that the enzyme induced by IC is distinct from the CYP3A1/2 which catalyzes hydroxylations at position 6 beta, allylic in AD but not in the fully saturated ring system of A. The increased hepatic conversion of A to its biologically less active 6 beta-OHA metabolite after treatment of female rats with IC could possibly contribute to the anticarcinogenic action of indole carbinols. It is also proposed that the action of multiple inducers present in cruciferous and other vegetables might produce androgen metabolic profiles very different from those produced by individual components isolated from them.
J Steroid Biochem Mol Biol 1994 Nov
PMID:Distinct forms of hepatic androgen 6 beta-hydroxylase induced in the rat by indole-3-carbinol and pregnenolone carbonitrile. 798 Nov 31

Mutations in tumor suppressor genes are intricately associated with the etiology of neoplasia. Often, such mutations are followed by the loss of the second, functional alleles of tumor suppressor genes, a phenomenon known as loss of heterozygosity. Loss of heterozygosity may occur by different molecular mechanisms, including mitotic recombination, and it is conceivable that these molecular events are influenced by endogenous as well as exogenous factors. To test whether mitotic recombination is induced by certain carcinogens, we genetically engineered a Saccharomyces cerevisiae tester strain so that it metabolizes two important classes of carcinogens, polycyclic aromatic hydrocarbons and heterocyclic arylamines. This was accomplished by expressing human cDNA's coding for the cytochrome P450 (CYP) enzymes CYP1A1 or CYP1A2 in combination with NADPH-CYP oxidoreductase in a strain heterozygous for two mutations in the trp5 gene. Microsomes isolated from the transformed yeast strains activated various xenobiotics to powerful mutagens that were detected in the Ames test. Of these, the mycotoxin aflatoxin B1, when activated intracellularly in the strains containing either human CYP enzyme, significantly induced mitotic recombination. These results are discussed in light of possible mechanisms that are involved in aflatoxin B1-mediated hepatocarcinogenesis. Similarly, benzo[a]pyrene-trans-7,8-dihydrodiol and 3-amino-1-methyl-5H-pyrido[4,3-b]indole were activated to recombinagenic products, whereas benzo[a]pyrene and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline were negative in this assay. Our results argue that the constructed yeast strains may be a valuable tool for the investigation of drug-induced mitotic recombination.
Mol Carcinog 1994 Dec
PMID:DNA recombination induced by aflatoxin B1 activated by cytochrome P450 1A enzymes. 799 64


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