Atypical cytochrome P450 induction profiles in glomerular mesangial cells at the mRNA and enzyme level. Evidence for CYP1A1 and CYP1B1 expression and their involvement in benzo[a]pyrene metabolism.

Recent studies in this laboratory have shown that benzo[a]pyrene (BaP) modulates growth factor-related gene expression and proliferation of renal glomerular mesangial cells (GMCs) in vitro. Because many of the toxic and biochemical effects of this polycyclic aromatic hydrocarbon are mediated through oxidative metabolism, the present studies were conducted to examine the patterns of cytochrome P450IA1 (CYP1A1) and P4501B1 (CYP1B1) inducibility in mesangial cells and the molecular consequences of this response. Exposure of cultured GMCs to BaP (30 microM) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 10 nM) for 24 hr induced CYP1A1 mRNA levels, a response abolished by cotreatment with 10 microM cycloheximide. The pattern of hydrocarbon inducibility was atypical in that BaP was a more effective inducer of CYP1A1 gene expression than TCDD, and both hydrocarbons induced aryl hydrocarbon hydroxylase (AHH) activity, but not ethoxyresorufin-O-deethylase activity. Cotreatment with alpha-naphthoflavone (alpha NF, 1 microM) or ellipticine (ELLIP, 0.1 nM) only partially inhibited the induction of AHH activity by BaP (30 microM). BaP and TCDD also induced expression of the CYP1B1 protein and the pattern of induction was comparable to that observed for CYP1A1. Treatment of GMCs with 30 microM BaP was associated with the formation of eight DNA adducts, and their occurrence could be inhibited by pretreatment with alpha NF (1 microM), but not ELLIP (0.1 nM). These results demonstrate that CYP1A1 and CYP1B1-related activities are induced in GMCs by BaP and TCDD and this induction is associated with metabolism of BaP to reactive intermediates that bind covalently to DNA.

Cytochromes CYP1A1 and CYP1B1 in the rat mammary gland: cell-specific expression and regulation by polycyclic aromatic hydrocarbons and hormones.

Cultured rat mammary cells express both CYP1A1 and CYP1B1 in response to polycyclic aromatic hydrocarbons (PAH) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in a cell type-specific manner. The expression of each P450 was determined functionally (regioselective PAH metabolism), as apoprotein (immunoblots) and as mRNA (Northern hybridization). The epithelial rat mammary cells (RMEC) expressed CYP1A1, however only after PAH or TCDD treatment. CYP1B1 protein was scarcely detected in these induced RMEC but was surprisingly active as a participant in 7,12-dimethylbenz[a]anthracene (DMBA) metabolism shown through selective antibody inhibition (40% of total activity). CYP1B1 was selectively expressed in the stromal fibroblast population of rat mammary cells to the exclusion of CYP1A1. In the rat mammary fibroblasts (RMF), CYP1B1 protein and associated activity were each present at low levels constitutively and were highly induced by benz[a]anthracene (BA) to a greater extent than by TCDD (12- versus 6-fold). However, BA (10 microM) and TCDD (10 nM) stimulated the 5.2-kb CYP1B1-specific mRNA equally. These increases are consistent with the involvement of the aryl hydrocarbon (Ah) receptor in the transcription of the CYP1B1 gene and with the additional stabilization of CYP1B1 protein by BA, previously observed in embryo fibroblasts. Exactly this regulation of CYP1B1-dependent activity was seen in RMEC suggesting that this arises from exceptionally active CYP1B1 in a small proportion (5%) of residual RMF. The constitutive expression and PAH inducibility of CYP1B1 and CYP1A1 proteins in RMF and RMEC, respectively, were each substantially decreased (approximately 75%) by a hormonal mixture (17 beta-estradiol (0.2 microM) progesterone (1.5 microM) cortisol (1.5 microM) and prolactin (5 micrograms/ml)). Progesterone and cortisol, added singly to RMF suppressed CYP1B1 protein expression (approximately 80%) in both untreated and BA-induced cells, while cortisol also suppressed the 5.2-kb CYP1B1 mRNA. In contrast, 17 beta-estradiol stimulated constitutive expression of CYP1B1 protein (50-75%) and mRNA level (2- to 3-fold), but did not affect CYP1B1 expression in BA-treated RMF. The expression of CYP1A1 and CYP1B1 is therefore highly cell specific even though each is regulated through the Ah receptor. Each P450 exhibits a surprisingly similar pattern of hormonal regulation even though expressed in different cell types.

Dual regulation of cytochrome P450EF expression via the aryl hydrocarbon receptor and protein stabilization in C3H/10T1/2 cells.

The major cytochrome P450 (P450EF) in the mouse embryo fibroblast C3H/10T1/2CL8 (10T1/2) cell line, which is very active in polycyclic aromatic hydrocarbon metabolism, is immunologically distinct from known P450 families but shares homology with an adrenocorticotropin hormone-regulated P450 from rat adrenal glands (P450RAP). P450EF is more effectively induced by benz[a]anthracene (BA) than by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which is anomalous for aryl hydrocarbon receptor (AhR)-mediated transcriptional activation. Evidence is presented here that induction of P450EF is consistent with mediation by the AhR but also involves an additional selective stabilization of P450EF by BA. P450EF-specific mRNA was measured by in vitro translation of 10T1/2 mRNA and subsequent immunoprecipitation with antibodies that recognize P450EF. P450EF mRNA was equally stimulated (> 10-fold) by BA (10 microM) and TCDD (10 nM) after 6 hr of induction in 10T1/2 cells. This equal stimulation of P450EF by BA and TCDD is consistent with transcriptional activation of the gene by the AhR. BA induction of mRNA declined 3-fold between 6 and 18 hr, due to metabolism of BA. Steady state P450EF mRNA levels declined quickly once this stimulation was removed, whereas total P450EF protein levels, measured by immunoblotting, continued to increase. During a 6-hr inhibition of protein synthesis with cycloheximide, both total P450EF and functional cytochrome, measured by polycyclic aromatic hydrocarbon metabolism, decreased by 60% in uninduced and TCDD-induced transformed 10T1/2 cells. This is consistent with relatively rapid degradation of P450EF (t1/2 = 4 hr). No such decline was seen when BA was present, indicating a stabilization of P450EF, which can explain the additional effectiveness of BA in enhancing the level of P450EF.

Mouse endometrium stromal cells express a polycyclic aromatic hydrocarbon-inducible cytochrome P450 that closely resembles the novel P450 in mouse embryo fibroblasts (P450EF).

Stromal cells from mouse endometrium, E041 cells, at passages 21, 23 and 26 were metabolically active towards 7,12-dimethylbenz[a]anthracene (DMBA). The total DMBA-metabolizing activity of E041 cells was preferentially increased by benz[a]anthracene (BA) relative to 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) treatment (7-fold by BA and 4-fold by TCDD), but the relative proportions of DMBA metabolites formed remained unchanged. Profiles of DMBA metabolites generated from E041 cell microsomes were very different from that of mouse P4501A1 but exhibited similarities to that of P450EF, the polycyclic aromatic hydrocarbon (PAH)-inducible cytochrome P450 in the mouse embryo fibroblast cell line C3H10T1/2 (10T1/2). Notably, both induced and uninduced E041 cell microsomes were very effective in the formation of the proximate carcinogen DMBA-3,4-dihydrodiol (15-20% of total) and DMBA-10,11-dihydrodiol (13-18% of total) but ineffective in forming the 7-hydroxymethyl derivative of DMBA (< 1% of total). Antibodies to P450EF completely inhibited the DMBA-metabolizing activities of both induced and uninduced E041 cell microsomes, with an effectiveness similar to that in microsomes prepared from identically treated 10T1/2 cells. Anti-P4501A1 had no inhibitory effect on DMBA metabolism by either induced or uninduced E041 cell microsomes. Total DMBA-metabolizing activities in BA- and TCDD-induced E041 cells were consistently 2-fold lower compared to those in similarly treated 10T1/2 cells. In addition, both induced and uninduced E041 cell microsomes formed a lower proportion of DMBA dihydrodiols relative to phenols in comparison to identically treated 10T1/2 cell microsomes (0.5 versus 1). Addition of exogenous epoxide hydrolase to E041 cell microsomes resulted in a product distribution indistinguishable from that in 10T1/2 cells. Immunoblots demonstrated 5-fold lower levels of epoxide hydrolase in E041 cell microsomes compared to 10T1/2 cell microsomes. Anti-P450EF immunoblotted a 55 kd protein in E041 cell microsomes that was induced 14-fold by BA and 6-fold by TCDD, thus paralleling the increases in the respective DMBA metabolism. It is therefore concluded that following PAH exposure endometrial stromal cells express the novel PAH-inducible mouse embryo fibroblast P450 and fail to express P4501A1.

Biological oxidations and P450 reactions. Recombinant mouse CYP1B1 expressed in Escherichia coli exhibits selective binding by polycyclic hydrocarbons and metabolism which parallels C3H10T1/2 cell microsomes, but differs from human recombinant CYP1B1.

Orthologs of a previously identified CYP1B subfamily designated CYP1B1, which are constitutively expressed in mammary, uterine, and embryonic cells, have previously been functionally linked to 7,12-dimethylbenz-a-anthracene (DMBA) metabolism. A chimeric construct of mouse CYP1B1 in which the 20 NH2-terminal amino acids have been replaced by eight residues from human CYP17 has been expressed in Escherichia coli. This recombinant mouse CYP1B1 (recCYP1B1m) exhibited DMBA metabolism accurately reproducing the characteristic product distribution and specific activity of 3.4 nmol/nmol P450/min seen in C3H10T1/2 cells from which this cDNA has been cloned. The high proportion of 10,11- and 3,4-dihydrodiols and near absence of 5,6-dihyrodiol- and 7-hydroxy-DMBA metabolites are seen only in rodent microsomes where CYP1B1 is highly expressed. This distribution of products from recCYP1B1m was highly dependent on addition of epoxide hydrolase, particularly the ratio of 3,4-dihydrodiol to 4-phenol metabolites. These characteristics in addition to inhibition by antibodies raised to recCYP1B1m establish that the CYP1B1 cDNA indeed encodes the P450 responsible for polycyclic aromatic hydrocarbon (PAH) metabolism from C3H10T1/2 cells. DMBA metabolites from cDNA-expressed human CYP1B1 (recCYP1B1h) however, exhibited a different regioselectivity toward DMBA resembling human CYP1A1 catalyzed DMBA metabolism. Reconstitution of recCYP1B1m with different concentrations of NADPH-P450 reductase indicated a high affinity interaction with an apparent Km of 3 nM. Large PAH such as benz[a]pyrene, benz[e]pyrene, benz[a]anthracene, DMBA, 3-methylcholanthrene, and 1-ethynylpyrene bound to recCYP1B1m with high affinity (Kd 0.08 to 0.22 microM) concomitant with substantial spectral shifts (40% low to high spin state change). Smaller PAHs like pyrene, phenanthrene, and naphthalene neither produced spectral changes nor inhibited the spectral change caused by benz[a]pyrene. Among tested steroids, progesterone bound weakly to recCYP1B1m (Kd > 20 microM) with a comparable spectral shift and was a weak inhibitor of DMBA metabolism, but was not metabolized. While 17beta-estradiol is a substrate for human CYP1B1 we have found no evidence for binding to mouse CYP1B1. This data establishes CYP1B1 as an important contributor to activation of PAHs, particularly in extra hepatic tissues that are susceptible to cancer where CYP1B1 in contrast to CYP1A1 is constitutively expressed.

Identification of peroxisome proliferator-responsive human genes by elevated expression of the peroxisome proliferator-activated receptor alpha in HepG2 cells.

In mice and other sensitive species, PPARalpha mediates the induction of mitochondrial, microsomal, and peroxisomal fatty acid oxidation, peroxisome proliferation, liver enlargement, and tumors by peroxisome proliferators. In order to identify PPARalpha-responsive human genes, HepG2 cells were engineered to express PPARalpha at concentrations similar to mouse liver. This resulted in the dramatic induction of mRNAs encoding the mitochondrial HMG-CoA synthase and increases in fatty acyl-CoA synthetase (3-8-fold) and carnitine palmitoyl-CoA transferase IA (2-4-fold) mRNAs that were dependent on PPARalpha expression and enhanced by exposure to the PPARalpha agonist Wy14643. A PPAR response element was identified in the proximal promoter of the human HMG-CoA synthase gene that is functional in its native context. These data suggest that humans retain a capacity for PPARalpha regulation of mitochondrial fatty acid oxidation and ketogenesis. Human liver is refractory to peroxisome proliferation, and increased expression of mRNAs for the peroxisomal fatty acyl-CoA oxidase, bifunctional enzyme, or thiolase, which accompanies peroxisome proliferation in responsive species, was not evident following Wy14643 treatment of cells expressing elevated levels of PPARalpha. Additionally, no significant differences were seen for the expression of apolipoprotein AI, AII, or CIII; medium chain acyl-CoA dehydrogenase; or stearoyl-CoA desaturase mRNAs.

Differential time-course and dose-response relationships of TCDD-induced CYP1B1, CYP1A1, and CYP1A2 proteins in rats.

This study examined the relationship between dose- and time-dependent hepatic localization of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and expression of CYP1B1, CYP1A1 and CYP1A2 proteins. A dose-dependent increase in hepatic TCDD in female Sprague-Dawley rats treated with 0.01-30.0 microg TCDD/kg was observed. TCDD induced CYP1A1 protein in rats treated with 0.3 microg TCDD/kg or higher. TCDD induced CYP1A2 and CYP1B1 proteins in rats treated with 1.0 microg TCDD/kg or higher. The in vivo ED50 (microg TCDD/kg) for TCDD-induced CYP1A1, CYP1A2 and CYP1B1 proteins were 0.22, 0.40 and 5.19, respectively. Hepatic accumulation of TCDD reached a maximum at 8 hours post dosing with a t1/2 of approximately 10 days. TCDD-induced CYP1A1/CYP1A2 protein expression was increased time-dependently, reaching a maximum at 3 days after dosing and remaining elevated for 35 days. In contrast, TCDD-induced CYP1B1 protein showed significant expression at 3 days after dosing and decreased to basal concentrations by 35 days. This study demonstrates that TCDD exhibits differential dose-response and time-course relationships on hepatic localization and cytochrome P-450 protein expression.

Co-expression of human CYP1A1 and a human analog of cytochrome P450-EF in response to 2,3,7,8-tetrachloro-dibenzo-p-dioxin in the human mammary carcinoma-derived MCF-7 cells.

Cultured human mammary carcinoma (MCF-7) cells exhibited constitutive cytochrome P450-dependent metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) (45-75 pmol/mg microsomal protein). Exposure of the cells to 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD), which is known to induce CYP1A1, not only resulted in a 30-fold increase in the total microsomal metabolism of DMBA but produced substantial differences in the distribution of DMBA metabolites formed. This suggested that different cytochrome P450 (P450) forms predominated in untreated and induced cells. Comparative studies with TCDD-induced human hepatoblastoma (HepG2) and skin cell carcinoma (SCC-13) cells and also recombinantly expressed human CYP1A1, confirmed that the DMBA-metabolite profile in TCDD-induced MCF-7 cells was that of human CYP1A1. This distribution, however, differed substantially from the regioselectivity of rat CYP1A1 and mouse Cyp1a-1. Rabbit antibodies to rat CYP1A1 completely inhibited the DMBA-metabolizing activity of TCDD-induced MCF-7 cells but had no inhibitory effect on constitutive DMBA metabolism which was, however, completely inhibited by chicken antibodies to the novel P450 in mouse embryo fibroblasts (P450-EF). Anti-P450-EF inhibited only 10% of the DMBA-metabolizing activity in the TCDD-induced MCF-7 cell microsomes. Microsomes from untreated MCF-7 cells expressed a 52 kDa protein that was immunodetectable by rabbit anti-P450-EF and failed to express immunodetectable levels of human CYP1A1. DMBA metabolism, therefore, switches from P450-EF in uninduced microsomes to CYP1A1 in TCDD-induced microsomes. The mobility of the P450-EF-like protein in MCF-7 cells was higher than that of P450-EF from C3H/10T1/2CL8 (10T1/2) cells (55 kDa). The 52 kDa protein from MCF-7 cells was induced approximately 8-fold by TCDD while CYP1A1 immunodetectable protein was increased to much higher levels. The SCC-13 cell line exhibited a similar pattern of expression of a 52 kDa P450-EF-like protein and CYP1A1. HepG2 cells expressed the highest levels of CYP1A1 in response to TCDD without expression of the 52 kDa protein.

Mouse cytochrome P-450EF, representative of a new 1B subfamily of cytochrome P-450s. Cloning, sequence determination, and tissue expression.

A novel benz[a]anthracene and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible cytochrome P-450 (P450EF), which is very active in polycyclic aromatic hydrocarbon metabolism, has been purified from C3H10T1/2 mouse embryo fibroblasts (Pottenger, L. H., Christou, M., and Jefcoate, C. R. (1991) Arch. Biochem. Biophys. 286, 488-497). P450EF was shown to be immunologically unrelated to the major known P-450 families. A 4.9-kilobase (kb) cDNA encoding P450EF has been isolated from a lambda ZAP cDNA expression library generated from mRNA of TCDD-induced C3H10T1/2 cells. This cDNA comprises 175-base pair (bp) 5'-noncoding, 1629-bp open reading, and about 3100-bp 3'-noncoding sequence. A SmaI fragment of the 4.9-kb cDNA hybridized to a 5.2-kb mRNA species equally induced by benz[a]anthracene (10 microM) and TCDD (10 nM) in C3H10T1/2 cells, consistent with the involvement of the Ah receptor in this induction process. The deduced amino acid sequence (543 amino acids), the longest of any known cytochrome P-450, exhibits 41 and 38% identity to mouse CYP1A1 and CYP1A2, respectively, and less but substantial similarity (30-33% identity) to many members of the CYP2 family. There are five extended regions of > or = 50% identity to CYP1A1 as follows: (a) 51-118; (b) 199-222; (c) 326-343 (I-helix, O2-binding threonine); (d) 357-430; and (e) 460-487 (heme-binding cysteine). These sequence relationships suggest that P450EF is a member of a new CYP1B subfamily (mouse CYP1B1). Hybridization of mRNA and immunoblot analyses of microsomes both demonstrated beta-naphthoflavone (beta-NF) inducibility of Cyp1b-1 expression in C3H mouse lung, liver, and uterus although at lower levels relative to Cyp1a-1. The mobility of the beta-NF-inducible immunoreactive liver protein was significantly higher than that of the CYP1B1 protein detected in mouse lung, uterus, and C3H10T1/2 cells. Compared with the beta-NF-induced uterus, polycyclic aromatic hydrocarbon-induced uterine fibroblasts exhibited 10-20-fold higher levels of CYP1B1, suggesting that stromal fibroblasts are a major source of the protein.

Rabbit pregnane X receptor is activated by rifampicin.

Reverse transcriptase-polymerase chain reaction was used to amplify a partial cDNA from rabbit lung mRNA that shared 77% protein sequence identity with the mouse pregnane X receptor (PXR). Rapid amplification of cDNA ends from a rabbit kidney lambdaZAP expression library resulted in the isolation of overlapping cDNAs spanning the complete coding sequence. The deduced amino acid sequence of 411 residues exhibited 79% overall amino acid identity with human PXR and 77% identity with mouse PXR. Based on this protein sequence relationship and a similar degree of conservation exhibited by the mouse and human PXR orthologs, the cDNA appears to encode the rabbit PXR ortholog. 5'-rapid amplification of cDNA ends performed on an adaptor-ligated cDNA library from rabbit liver revealed the presence of an alternate mRNA, which differed at the 5'-terminus. RNase protection assays indicated that the alternate mRNA was expressed at >50-fold lower levels in rabbit kidney and liver. Rifampicin treatment of CV-1 cells cotransfected with a rabbit PXR expression plasmid and a luciferase reporter construct containing two copies of the DR3 enhancer from CYP3A23 produced a 6-fold induction of luciferase activity. In contrast, rat PXR was not responsive to this antibiotic under the same conditions. Pregnenolone 16alpha-carbonitrile was an efficacious activator of rat PXR, but failed to significantly activate rabbit PXR at equivalent concentrations. These results indicate that the ligand activation profile of rabbit PXR is distinct from rat PXR and more closely resembles that of human PXR. The rabbit PXR activation profile is consistent with the cytochrome P450 (P450) 3A6 induction profile in rabbits.

Characterization of the mouse Cyp1B1 gene. Identification of an enhancer region that directs aryl hydrocarbon receptor-mediated constitutive and induced expression.

Transcriptional activation of the Cyp1B1 gene in rodents is stimulated by both polycyclic hydrocarbons and cAMP. The mouse Cyp1B1 gene structure contains three exons, of which the second nucleotide of exon 2 is the translation start site. Primer extension analysis identified a transcription start domain defining an exon 1 of 371 base pairs. The sequence 1.075 kilobases upstream of the transcription start site showed 11 xenobiotic-responsive elements (XRE) (TnGCGTG or GCGTG) that are putative aryl hydrocarbon receptor (AhR)-binding sites and three steroidogenic factor-1 motifs that are associated with cAMP-mediated transcriptional activation of genes. A transiently transfected Cyp1B1-luciferase construct, composed of exon 1 and 1.075 kilobases of 5'-flanking region, was induced by 2,3, 7,8-tetrachlorodibenzo-p-dioxin (TCDD; 10.0 +/- 3.0-fold, n = 6) in C3H10T1/2 cells, which exclusively express Cyp1B1. The 90-base pair basal promoter contains two SP-1 sites, one SF-1 site, and a TATA-like box. TCDD induction and basal expression were dependent on positive regulatory elements present between -1075 and -810. Five XRE motifs localized in the enhancer region were completely conserved between mouse and human CYP1B1 sequences. Similar inductions were seen in Hepa-1 cells, which express Cyp1A1 but not Cyp1B1. However, basal Cyp1B1 promoter activities were 4-10-fold higher in C3H10T1/2 cells providing the enhancer region was present, partially reproducing the in vivo cell-specific expression of Cyp1B1. Gel shift experiments established that TCDD stimulates AhR binding to the downstream XRE in the enhancer region. However, oligonucleotides that encompass two other XREs show a high affinity complex of similar size that is evident even without TCDD treatment and that does not contain either the AhR or AhR nuclear translocator. The fourth XRE is immediately adjacent to an E-box, and this oligonucleotide formed a smaller complex that was dependent on this E-box sequence. Negative regulatory sequences have been located between the promoter and TCDD-responsive enhancer regions. Constitutive expression of the Cyp1B1 gene was lost in AhR-deficient cells and was restored by transfected AhR cDNA. Reporter constructs function in a parallel manner, demonstrating the key role of the AhR in constitutive as well as TCDD-induced expression of Cyp1B1 in mouse embryo fibroblasts.

Cytochrome P450 CYP1B1 determines susceptibility to 7, 12-dimethylbenz[a]anthracene-induced lymphomas.

CYP1B1-null mice, created by targeted gene disruption in embryonic stem cells, were born at the expected frequency from heterozygous matings with no observable phenotype, thus establishing that CYP1B1 is not required for mouse development. CYP1B1 was not detectable in cultured embryonic fibroblast (EF) or in different tissues, such as lung, of the CYP1B1-null mouse treated with the aryl hydrocarbon receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin whereas the equivalent wild-type EF cells express basal and substantial inducible CYP1B1 and lung expresses inducible CYP1B1. CYP1A1 is induced to far higher levels than CYP1B1 in liver, kidney, and lung in wild-type mice and is induced to a similar extent in CYP1B1-null mice. 7,12-dimethylbenz[a]anthracene (DMBA) was toxic in wild-type EFs that express CYP1B1 but not CYP1A1. These cells effectively metabolized DMBA, consistent with CYP1B1 involvement in producing the procarcinogenic 3,4-dihydrodiol as a major metabolite, whereas CYP1B1-null EF showed no significant metabolism and were resistant to DMBA-mediated toxicity. When wild-type mice were administered high levels of DMBA intragastrically, 70% developed highly malignant lymphomas whereas only 7.5% of CYP1B1-null mice had lymphomas. Skin hyperplasia and tumors were also more frequent in wild-type mice. These results establish that CYP1B1, located exclusively at extrahepatic sites, mediates the carcinogenicity of DMBA. Surprisingly, CYP1A1, which has a high rate of DMBA metabolism in vitro, is not sufficient for this carcinogenesis, which demonstrates the importance of extrahepatic P450s in determining susceptibility to chemical carcinogens and validates the search for associations between P450 expression and cancer risk in humans.