The environmental pollutant 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD) is the prototype of a large number of non-genotoxic carcinogens, dietary phytochemicals and endogenous metabolites that act via binding the aryl hydrocarbon receptor (AHR). The TCDD-liganded AHR massively upregulates CYP1A1, CYP1A2 and CYP1B1 in many mammalian organs. We demonstrated that TCDD treatment markedly increases the levels of several epoxides and diol metabolites of the epoxides of both ω-6 and ω-3 polyunsaturated fatty acids (PUFA) in the liver and lungs of mice, in an aryl hydrocarbon receptor-dependent fashion, and most likely via the activities of the CYP1 family members. ω-6 Epoxides are known to stimulate tumor growth, angiogenesis, and metastasis in mice. Interestingly, ω-3 epoxides have the opposite effect on these parameters. TCDD and other AHR agonists may, therefore, impact angiogenesis, growth and metastasis of tumors in either a positive or negative way, depending on the relative levels of ω -6 epoxides and ω-3 epoxides generated in the host and/or tumor cells. This is of potential relevance to carcinogenesis by AHR agonists in the human, since the human population is exposed to widely varying ω-6: ω-3 PUFA ratios in the diet.

Benzo[a]pyrene (BaP), a polyaromatic hydrocarbon produced by the combustion of cigarettes and coke ovens, is a known procarcinogen. BaP activates the aryl hydrocarbon receptor (AhR) and induces the expression of a battery of genes, including CYP1A1, which metabolize BaP to toxic compounds. The possible role of CYP1 enzymes in mediating BaP detoxification or metabolic activation remains to be elucidated. In this study, we assessed the effects of CYP1 enzymes (CYP1A1, CYP1A2 and CYP1B1) on BaP-induced AhR transactivation and DNA adduct formation in HEK293 cells and HepG2 cells. Transfection of CYP1A1 and CYP1B1, but not CYP1A2, suppressed BaP-induced activation of AhR. Expression of CYP1A1 and CYP1A2, but not CYP1B1, inhibited DNA adduct formation in BaP-treated HepG2 cells. These results indicate that CYP1A1 and CYP1B1 play a role in deactivation of BaP on AhR and that CYP1A1 and CYP1A2 are involved in BaP detoxification by suppressing DNA adduct formation. BaP treatment did not induce DNA adduct formation in HEK293 cells, even after transfection of CYP1 enzymes, suggesting that expression of CYP1 enzymes is not sufficient for DNA adduct formation. Lower expression of epoxide hydrolase and higher expression of glutathione S-transferase P1 (GSTP1) and GSTM1/M2 were observed in HEK293 cells compared with HepG2 cells. Dynamic expression of CYP1A1, CYP1A2 and CYP1B1 along with expression of other enzymes such as epoxide hydrolase and phase II enzymes may determine the detoxification or metabolic activation of BaP.

Little is known about the impact of genetic variation on the genetic damage induced by urban air pollution or environmental tobacco smoke (ETS) in exposed populations. The levels of bulky DNA adducts ( 32P-postlabelling, nuclease P1 enrichment) and chromosomal aberrations were measured in lymphocytes of 194 non-smoking students living in the city of Athens, and the rural region of Halkida, Greece. In these individuals personal exposure to PAH was also measured. Furthermore, genetic polymorphisms were examined in cytochromes P450 1A1, 1B1, in the GSTM1, GSTP1 and GSTT1 as well as in microsomal epoxy hydrolase (EPHX) genes. Subjects with the CYP1*2A mutant genotype also suffering significant ETS exposure tended to exhibit higher adduct levels and % aberrant cells. In contrast, CYP1B1 polymorphisms seemed to have an impact on the DNA adduct levels only among individual with negligible ETS exposure. Subjects carrying both the CYP1*2A mutant genotype and the GSTM1 null genotype tended to have higher DNA adduct levels. A similar effect was also observed with the combined CYP1A1*2A/GSTP1 (Ile/Val) and the CYP1A1*2A/mEH "slow" polymorphisms. In both cases, the effect was more pronounced among subjects with higher levels of ETS exposure. Stepwise restriction of the observations to subjects characterised by (a) GSTP1 mutant, (b) GSTM1 null, (c) mEH "slow" (His139His) genotypes and (d) ETS exposure resulted in a significant trend of increasing DNA adduct levels only among individuals with at least one CYP1A1*2A mutated allele, illustrating the importance and complexity of gene-exposure and gene-gene interactions in determining the level of genotoxic damage on an individual levels.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates many of the toxic effects of dioxin-like compounds (DLCs) and some polycyclic aromatic hydrocarbons (PAHs). Strong AHR agonists, such as certain polychlorinated biphenyls and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), cause severe cardiac teratogenesis in fish embryos. Moderately strong AHR agonists, such as benzo[a]pyrene and β-naphthoflavone, have been shown to cause similar cardiotoxic effects when coupled with a cytochrome P450 1A (CYP1A) inhibitor, such as fluoranthene (FL). We sought to determine if weak AHR agonists, when combined with a CYP1A inhibitor (FL) or CYP1A morpholino gene knockdown, are capable of causing cardiac deformities similar to moderately strong AHR agonists (Wassenberg and Di Giulio Environ Health Perspect 112(17):1658-1664, 2004a; Wassenberg and Di Giulio Res 58(2-5):163-168, 2004b; Billiard et al. Toxicol Sci 92(2):526-536, 2006; Van Tiem and Di Giulio Toxicol Appl Pharmacol 254(3):280-287, 2011). The weak AHR agonists included the following: carbaryl, phenanthrene, 2-methylindole, 3-methylindole, indigo, and indirubin. Danio rerio (zebrafish) embryos were first exposed to weak AHR agonists at equimolar concentrations. The agonists were assessed for their relative potency as inducers of CYP1 enzyme activity, measured by the ethoxyresorufin-O-deethylase (EROD) assay, and cardiac deformities. Carbaryl, 2-methylindole, and 3-methylindole induced the highest CYP1A activity in zebrafish. Experiments were then conducted to determine the individual cardiotoxicity of each compound. Next, zebrafish were coexposed to each agonist (at concentrations below those determined to be cardiotoxic) and FL in combination to assess if CYP1A inhibition could induce cardiac deformities. Carbaryl, 2-methylindole, 3-methylindole, and phenanthrene significantly increased pericardial edema relative to controls when combined with FL. To further evaluate the interaction of the weak AHR agonists and CYP1A inhibition, a morpholino was used to knockdown CYP1A expression, and embryos were then exposed to each agonist individually. In embryos exposed to 2-methylindole, CYP1A knockdown caused a similar level of pericardial edema to that caused by exposure to 2-methylindole and FL. The results showed a complex pattern of cardiotoxic response to weak agonist inhibitor exposure and morpholino-knockdown. However, CYP1A knockdown in phenanthrene and 3-methylindole only moderately increased pericardial edema relative to coexposure to FL. AHR2 expression was also knocked down using a morpholino to determine its role in mediating the observed cardiac teratogenesis. Knockdown of AHR2 did not rescue the pericardial edema as previously observed with strong AHR agonists. While some of the cardiotoxicity observed may be attributed to the combination of weak AHR agonism and CYP1A inhibition, other weak AHR agonists appear to be causing cardiotoxicity through an AHR2-independent mechanism. The data show that CYP1A is protective of the cardiac toxicity associated with weak AHR agonists and that knockdown can generate pericardial edema, but these findings are also suggestive of differing mechanisms of cardiac toxicity among known AHR agonists.

Metabolic activation and DNA adduct formation of the carcinogenic aromatic hydrocarbon dibenzo[a,l]pyrene (DBP) was investigated in human mammary carcinoma MCF-7 cells and human cytochrome P450 (CYP) 1B1-expressing Chinese hamster V79 cells in culture. It has been shown that DBP is metabolically activated to DNA-binding diol epoxides both in vitro and in vivo. To further establish the role of human CYP1B1 in the activation of DBP, both cell lines were cotreated with DBP and a selective chemical inhibitor of CYP1B1, 2,4,3' ,5'-tetramethoxy-stilbene (TMS). Results from DBP-DNA adduct analyses revealed the complete inhibition of DNA binding when cells were cotreated with DBP and TMS in comparison to DBP alone. Inactivation of CYP1B1 by TMS was also demonstrated through a decrease in the 7-ethoxyresorufin O-deethylase (EROD) activity in microsomes isolated from these cells. Emodin, 3-methyl-1,6,8-trihydroxyanthraquinone, an active ingredient of an herb, has been recently shown of being able to induce CYP1 gene expression. Examination of human CYP1B1 induction and EROD activity confirmed an increase in protein levels upon cotreatment with emodin and DBP. Despite increases in protein levels and enzyme activity, there was no significant change in DBP-DNA binding levels at very low substrate concentrations (17 nM). The data obtained in this study emphasize the central role of CYP1B1 in the activation of DBP in human cells in culture.

In the present study, we investigated the effect of Ginkgo biloba extracts and some of its individual constituents on the catalytic activity of human cytochrome P450 enzymes CYP1B1, CYP1A1, and CYP1A2. G. biloba extract of known abundance of terpene trilactones and flavonol glycosides inhibited 7-ethoxyresorufin O-dealkylation catalyzed by human recombinant CYP1B1, CYP1A1, and CYP1A2, and human liver microsomes, with apparent Ki values of 2 +/- 0.3, 5 +/- 0.5, 16 +/- 1.4, and 39 +/- 1.2 microg/ml (mean +/- SE), respectively. In each case, the mode of inhibition was of the mixed type. Bilobalide, ginkgolides A, B, C, and J, quercetin 3-O-rutinoside, kaempferol 3-O-rutinoside, and isorhamentin 3-O-rutinoside were not responsible for the inhibition of CYP1 enzymes by G. biloba extract, as determined by experiments with these individual chemicals at the levels present in the extract. In contrast, the aglycones of quercetin, kaempferol, and isorhamentin inhibited CYP1B1, CYP1A1, and CYP1A2. Among the three flavonol aglycones, isorhamentin was the most potent in inhibiting CYP1B1 (apparent Ki = 3 +/- 0.1 nM), whereas quercetin was the least potent in inhibiting CYP1A2 (apparent Ki = 418 +/- 50 nM). The mode of inhibition was competitive, noncompetitive, or mixed, depending on the enzyme and the flavonol. G. biloba extract also reduced benzo[a]pyrene hydroxylation, and the effect was greater with CYP1B1 than with CYP1A1 as the catalyst. Overall, our novel findings indicate that G. biloba extract and the flavonol aglycones isorhamnetin, kaempferol, and quercetin preferentially inhibit the in vitro catalytic activity of human CYP1B1.

Ginseng extract has been reported to decrease the incidence of 7,12-dimethylbenz[a]anthracene (DMBA)-initiated tumorigenesis in mice. A potential mechanism for this effect by ginseng is inhibition of DMBA-bioactivating cytochrome P450 (P450) enzymes. In the present in vitro study, we examined the effect of a standardized Panax ginseng (or Asian ginseng) extract (G115), a standardized Panax quinquefolius (or North American ginseng) extract (NAGE), and individual ginsenosides (Rb1, Rb2, Rc, Rd, Re, Rf, and Rg1) on CYP1 catalytic activities, as assessed by 7-ethoxyresorufin O-dealkylation. G115 and NAGE decreased human recombinant CYP1A1, CYP1A2, and CYP1B1 activities in a concentration-dependent manner. Except for the competitive inhibition of CYP1A1 by G115, the mode of inhibition was the mixed-type in the other cases. A striking finding was that NAGE was 45-fold more potent than G115 in inhibiting CYP1A2. Compared with G115, NAGE also preferentially inhibited 7-ethoxyresorufin O-dealkylation activity in human liver microsomes. Rb1, Rb2, Rc, Rd, Re, Rf, and Rg1, either individually or as a mixture and at the levels reflecting those found in an inhibitory concentration (100 microg/ml) of NAGE or G115, did not influence CYP1 activities. However, at a higher ginsenoside concentration (50 microg/ml), Rb1, Rb2, Rc, Rd, and Rf inhibited these activities. Overall, our in vitro findings indicate that standardized NAGE and G115 extracts, which were not treated with calf serum or subjected to acid hydrolysis, inhibited CYP1 catalytic activity in an enzyme-selective and extract-specific manner, but the effects were not due to Rb1, Rb2, Rc, Rd, Re, Rf, or Rg1.

Cyclophilins are peptidyl-prolyl cis-trans isomerases that are highly conserved throughout eukaryotes and are the cellular target of the immunosuppressive drug cyclosporin A (CsA). We cloned cyp1, a cyclophilin A-encoding gene in the phytopathogenic fungus Cryphonectria parasitica, and showed that this gene was downregulated following infection by a virulence-attenuating hypovirus. The function of cyp1 was further investigated by construction of a cyp1 deletion mutant. Although the wild-type C. parasitica strain EP155 was sensitive to CsA, the Δcyp1 strain was highly tolerant to CsA, indicating that CYP1 was the target of CsA. Deletion of cyp1 resulted in reduced virulence when inoculated to chestnut stems. Transcriptional analysis revealed that deletion of cyp1 also reduced transcript levels for genes encoding key components of the heterotrimeric guanosine triphosphate-binding protein signalling pathway that are essential for sensing environmental cues and are involved in C. parasitica development and virulence.

Injection of microparticle-encapsulated DNA elicits immune responses to plasmid-encoded antigens in mice and humans. Cytochrome P450 CYP1B1 (CYP1B1) is a member of the CYP1 P450 enzyme family that is overexpressed in a variety of solid tumors. The work described herein was performed to study the kinetics of stimulating T cell responsiveness with an encapsulated DNA encoding CYP1B1 and provides support for the clinical development of this formulation. Immunization of HLA-A2/Kb transgenic mice with human CYP1B1 encoding plasmid DNA formulated in poly(lactide-co-glycolide) (PLG) microparticles elicits CD8+ T cells that respond to human CYP1B1-positive target cells. The duration of the immune response, the effect on the immune response of multiple injections, and the safety of repeated injections were studied. These results show that the PLG-encapsulated DNA therapeutic elicits durable immune responses to CYP1B1, the responses are dependent on repeat immunization, and that the formulation is well tolerated.

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcriptional regulator of several genes including the cytochrome P4501 (CYP1) family as well as genes encoding factors involved in cell growth and differentiation. In mice, several polymorphic forms of the AHR are known, some of which have altered affinity for toxic and carcinogenic ligands. Remarkably little genetic variation has been detected in the human AHR gene. In studies on human AHR, Kawajiri et al. (Pharmacogenetics 1995; 5:151-158) reported a variation at codon 554 that results in an amino acid change from arginine to lysine; the frequency of the variant allele in a Japanese population (n = 277) was 0.43. We investigated the Lys554 allele in 386 individuals of various ethnic origins and found the frequency to be: 0.58 in Ivory Coast Africans (n = 58); 0.53 in a mixed African group (n = 20); 0.39 in Caribbean-Africans (n = 55); 0.32 in Canadian Chinese (n = 41); 0.14 in North American Indians (n = 47); 0.12 in French Canadian Caucasians (n = 20); 0.11 in a mixed ethnicity North American group (n = 45); 0.09 in Canadian Inuits (n = 22); and 0.07 in German Caucasians (n = 78). We expressed the human Lys554 allele in an in-vitro transcription-translation system and found that the receptor bearing the R554L substitution had an equivalent ability to that of the wild-type receptor to bind to a dioxin-responsive element following treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The Lys554 allele also was equivalent to the wild-type receptor at stimulating CYP1A1 mRNA expression when transfected into TCDD-treated receptor-deficient mouse Hepa-1 cells. It is not yet known if any of the wide variations in allele frequency at codon 554 are related to ethnic differences in susceptibility to adverse effects of environmental chemicals.

Cytochrome P450 (CYP) enzymes constitute a multigene family of many endogenous and xenobiotic substances. The CYP1 family is of particular interest in environmental toxicology because its members are dominant in the metabolism of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and aryl amines. A new complementary DNA of the CYP1C subfamily encoding CYP1C1 was isolated from carp liver after intraperitoneal injection of beta-napthoflavone (BNF). The full-length cDNA obtained contained a 5' noncoding region of 244 bp, an open reading frame of 1572 bp coding for 524 amino acids, a stop codon, and a 3' noncoding region of 965 bp. The predicted molecular weight of the protein was approximately 59.3 kDa. The deduced amino acid sequence of this cDNA was 82.1% and 80.2% similar to Japanese eel and scup CYP1C1 sequences, respectively, while it exhibited a similarity of 74.9% with the scup CYP1C2 sequence. The deduced amino acid sequence of carp CYP1C1 showed similarities with those of the reported CYP1B1s of teleosts and mammals of 48.4, 48.8, 48.2, 48.6, 45.3, and 45.5% for carp CYP1B1, carp CYP1B2, plaice CYP1B1, and human, rat, and mouse CYP1B1, respectively. The phylogenetic tree constructed using fish and mammalian CYP1 sequences suggested a closer relationship of the CYP1C subfamily to CYP1B than to CYP1A. The tree showed the possibility of the existence of CYP1C subfamily genes in mammalian species. Northern blot analysis for the liver, intestine, gills, and kidney showed no detectable induced expression but constitutive expression in the gill organs.

We were aimed at investigating the activation of the carcinogenic polycyclic aromatic hydrocarbon (PAH) dibenzo[a,l]pyrene (DB[a,l]P) in Chinese hamster V79 cells that express single human, rat or fish cytochrome P450 (CYP) enzymes. DB[a,l]P is detectable in environmental samples and has been characterized as the most potent carcinogenic species among all PAHs as yet tested in rodent bioassays. Metabolite profiles and metabolite-dependent cytotoxic and clastogenic activities were monitored. The total turnover of CYP-mediated transformation of DB[a,l]P was as follows: human CYP1B1>fish CYP1A1 approximately human CYP1A1>)rat CYP1A2>rat CYP1A1. By contrast, enzyme forms that are not classified as being members of family CYP1, such as CYP2A6, 2E1, 2B1, and 3A4, failed to catalyze any detectable conversion of this substrate. All CYP1A1 enzymes tested formed both the K-region trans-8,9- and the trans-11,12-dihydrodiol, whereas human CYP1B1 failed to catalyze K-region activation. In cells expressing human or fish CYP1A1, human CYP1B1, and rat CYP1A2, the (-)-trans-11,12-dihydrodiol was formed enantiospecifically. DB[a,l]P-dependent cytotoxicities (EC(50)) were found in the following order: human CYP1A1 (12 nM>>fish CYP1A1 (30 nM>>human CYP1B1 (45 nM>>)other forms. In addition, an appreciable micronuclei formation was detected in human CYP1A1- and 1B1-expressing cells during exposure to DB[a,l]P. Our study demonstrates that human CYP1A1, 1B1 and fish CYP1A1 are able to transform DB[a,l]P into genotoxic derivatives in appreciable amounts. In contrast, CYP enzymes from rat predominantly target the K-region of DB[a,l]P and thus are serving more a rather protective route of biotransformation. Together our data suggest that humans might be more susceptible to DB[a,l]P-induced carcinogenicity than rats.

Chronic inhalation of cigarette smoke is a major risk factor for the development of lung cancer. It has been suggested that genetic susceptibility may contribute to the risk, because only a small portion of smokers develops the disease. Several polymorphisms that involve the metabolic activation or detoxification of carcinogens derived from cigarette smoke have been found to be associated with lung cancer risk. Many studies have focused on the relation between the distribution of polymorphic variants of different forms of the metabolic enzymes and lung cancer susceptibility. In this respect two groups of genetic polymorphisms of enzymes involved in xenobiotic metabolism, cytochrome P450 (CYP) and glutathione S-transferases (GSTs), have been discussed.CYP multigene superfamily consists of 10 subfamilies (CYP1-CYP1CYP1A1 gene has been reported in several Japanese populations but such an association has not been observed in either Caucasians or African-Americans. The relation betweenCYP2D6 and lung cancer remains conflicting and inconclusive. Several polymorphisms have been identified at theCYP2E1 locus. No definitive link between the polymorphisms ofCYP2E1 and the risk of lung cancer has, however, been identified. The role of otherCYP2 isoforms in lung carcinogenesis has not been sufficiently investigated.GSTs form a superfamily of genes consisting of five distinct families, namedGSTA, GSTM, GSTP, GSTT andGSTS. The role ofGSTM, GSTT1 orGSTP1 polymorphism in modifying the lung cancer risk may be more limited than has been so far anticipated.Although some genetic polymorphisms discussed here have not shown significant increases/decreases in risk, individuals with differing genotypes may have different susceptibilities to lung cancer. Hopefully, in future studies it will be possible to screen for lung cancer using specific biomarkers.

In Saccharomyces cerevisiae, the two genes, CYC1 and CYC7, that encode the isoforms of cytochrome c are expressed at different levels. Oxygen regulation is mediated by the expression of the CYP1 gene, and the CYP1 protein interacts with both CYC1 upstream activation sequence 1 (UAS1) and CYC7 UASo. In this study, the homology between the CYP1-binding sites of both genes was investigated. The most noticeable difference between the CYC1 and CYC7 UASs is the presence of GC base pairs at the same positions in a repeated sequence in CYC7 compared with CG base pairs in CYC1. Directed mutagenesis changing these GC residues to CG residues in CYC7 led to CYC1-like expression of CYC7 both in a CYP1 wild-type strain and in a strain carrying the semidominant mutation CYP1-16 which reverses the oxygen-dependent expression of the two genes. Our results strongly support the hypothesis that the CYP1-binding sites in CYC1 and CYC7 are related forms of the same sequence and that the CYP1-16 protein has altered specificity for the variant forms of the consensus sequences in both genes.

Administration of melatonin to rodents decreases the incidence of tumorigenesis initiated by benzo[a]pyrene or 7,12-dimethylbenz[a]anthracene, which requires bioactivation by cytochrome P450 enzymes, such as CYP1A1, CYP1A2 and CYP1B1, to produce carcinogenic metabolites. The present study tested the hypothesis that melatonin is a modulator of human CYP1 catalytic activity and gene expression. As a comparison, we also investigated the effect of melatonin on the catalytic activity of CYP2A6, which is also a procarcinogen-bioactivating enzyme. Melatonin (3-300 microm) decreased 7-ethoxyresorufin O-dealkylation catalyzed by human hepatic microsomes and recombinant CYP1A1, CYP1A2 and CYP1B1, whereas it did not affect coumarin 7-hydroxylation catalyzed by hepatic microsomes or recombinant CYP2A6. Melatonin inhibited CYP1 enzymes by mixed inhibition, with apparent K(i) values (mean +/- S.E.M.) of 59 +/- 1 (CYP1A1), 12 +/- 1 (CYP1A2), 14 +/- 2 (CYP1B1) and 46 +/- 8 microm (hepatic microsomes). Additional experiments indicated that melatonin decreased benzo[a]pyrene hydroxylation catalyzed by hepatic microsomes and CYP1A2 but not by CYP1A1 or CYP1B1. Treatment of MCF-10A human mammary epithelial cells with melatonin (up to 300 microm) did not affect basal or benzo[a]pyrene-inducible CYP1A1 or CYP1B1 gene expression. Consistent with this finding, melatonin did not influence reporter activity in aryl hydrocarbon receptor-dependent pGudluc6.1-transfected MCF-10A cells treated with or without benzo[a]pyrene, as assessed in an in vitro cell-based luciferase reporter gene assay. Overall, melatonin is an in vitro inhibitor of human CYP1 catalytic activity, and it may be useful to develop potent analogues of melatonin as potential cancer chemopreventive agents that block CYP1-mediated chemical carcinogenesis.

Ultraviolet (UV) radiation damages cell molecules, and has been suggested to up-regulate mammalian cytochrome P4501 (CYP1) genes through an aryl hydrocarbon receptor (AHR) mediated mechanism. In this study, embryos and larvae of zebrafish (Danio rerio) were exposed to UV to determine the effects on expression of CYP1 and stress response genes in vivo in these fish. Zebrafish embryos were exposed for varying times to UV on two consecutive days, with exposure beginning at 24 and 48h post-fertilization (hpf). Embryos exposed for 2, 4 or 6h twice over 2 days to UVB (0.62 W/m(2); 8.9-26.7 kJ/m(2)) plus UVA (2.05 W/m(2); 29.5-144.6 kJ/m(2)) had moderately (2.4+/-0.8-fold) but significantly up-regulated levels of CYP1A. UVA alone had no effect on CYP1A expression. Proliferating cellular nuclear antigen (PCNA) and Cu-Zn superoxide dismutase (SOD1) transcript levels were induced (2.1+/-0.2 and 2.3+/-0.5-fold, respectively) in embryos exposed to two 6-h pulses of 0.62 W/m(2) UVB (26.8 kJ/m(2)). CYP1A was induced also in embryos exposed to higher intensity UVB (0.93 W/m(2)) for two 3-h or two 4-h pulses (20.1 or 26.8 kJ/m(2)). CYP1B1, SOD1 and PCNA expression was induced by the two 3-h pulses of the higher intensity UVB, but not after two 4-h pulses of the higher intensity UVB, possibly due to impaired condition of surviving embryos, reflected in a mortality of 34% at that UVB dose. A single 8-h long exposure of zebrafish larvae (8dpf) to UVB at 0.93 W/m(2) (26.8 kJ/m(2)) significantly induced CYP1A and CYP1B1 expression, but other CYP1 genes (CYP1C1, CYP1C2 and CYP1D1) showed no significant increase. The results show that UVB can induce expression of CYP1 genes as well stress response genes in developing zebrafish, and that UVB intensity and duration influence the responses.

Several flavonoids have shown their anti-carcinogenic effects in various models. The soyabean isoflavone genistein was demonstrated earlier in our laboratory to be an effective inhibitor of dimethylbenz[a]anthracene (DMBA)-induced DNA damage in MCF-7 cells by curbing cytochrome P450 (CYP) 1 enzymes. The red clover (Trifolium pratense) isoflavone biochanin A is a methylated derivative of genistein, and its anti-mutagenic effect in bacterial cells has been shown previously. Because of its protection against chemical carcinogenesis in an animal model, biochanin A was selected for testing in our established MCF-7 cell system. From the results obtained in the semi-quantitative reverse transcription-polymerase chain reaction and xenobiotic response element (XRE)-luciferase reporter assays, biochanin A could reduce xenobiotic-induced CYP1A1 and -1B1 mRNA abundances through the interference of XRE-dependent transactivation. Enzyme kinetic studies also indicated that biochanin A inhibited both CYP1A1 and -1B1 enzymes with inhibition constant (Ki) values 4.00 and 0.59 microm respectively. Since the biotransformation of DMBA was dependent on CYP1 enzyme activities, biochanin A was able to decrease the DMBA-DNA lesions. The present study illustrated that the red clover isoflavone could protect against polycylic aromatic hydrocarbon-induced DNA damage.

Since Human CYP1 enzymes catalyze the metabolic activation of procarcinogens and deactivation of certain anticancer drugs, the inhibition of these enzymes has been considered as an effective approach for chemoprevention and treatment of CYP1-mediated drug resistance. Recent knowledge relating to the enhanced expression of CYP1B1 in tumors also provided certain advantages in cancer therapy by the activation of prodrugs only in tumor cells. This review concentrates on the characterized CYP1 inhibitors and CYP1-activatied anticancer prodrugs. The mechanism for enzyme inhibition and activation of prodrugs, the cancer preventive/therapeutic potential of these chemicals and their related SARs are highlighted. According to their structural features, CYP1 inhibitors are divided into the following categories: flavonoids, trans-stilbenes, coumarins, terpenoids, alkaloids, quinones, isothiocyanates and synthetic aromatics. In the same way, CYP1-activatied prodrugs are categorized into four groups: benzothiazoles, flavonoids, stilbenes and alkylating agents. Almost all of these inhibitors and prodrugs are planar molecules with one aromatic ring and some have similarity with identified CYP1 substrates. CYP1 inhibitors could effectively block the procarcinogen-induced tumor initiation in animal models and benefit us with chemoprevention. The advent of Phortress and aminoflavone as clinical candidates shows promising perspectives in developing CYP1-mediated prodrugs as chemotherapeutic drugs that are specifically activated in tumors. All of these preclinical and clinical studies indicate that inhibitors and prodrugs target CYP1 are promising anticancer strategies.

As the incidence of skin tumors has been steadily growing, there is an urgent need for the preventive measures as well as the improved therapeutic approaches. In the last two decades, natural plant derived polyphenols (PPs, resveratrol, silibinin, green tea polyphenols, flavonoids, anthocyanins, etc.) have been drawing particular interest as emerging active substances in dermatological/cosmeceutical compositions for the prevention, slowing, or reversion of skin tumorigenesis (chemoprevention). When chronically applied to the skin, they supposedly would not damage normal skin cells or negatively affect their functions while they would suppress tumorigenic cell transformation, inhibit tumor cell proliferation, and activate tumor cell apoptosis. PPs are also reported to synergize with conventional anti-cancer therapies. The major aim of this critical review is to provide recent updates on the molecular and cellular targets for the prevention and therapy of skin tumors with a special focus on the crossroad between inflammation and carcinogenesis as the most promising approach to chemoprevention. Novel therapeutic targets as different as epidermal stem cells, cellular senescence, epigenetic enzymes involved in carcinogenesis, epidermal growth factor and aryl hydrocarbon receptors, and metabolic CYP1 subfamily enzymes are highlighted. The mechanisms of PPs interaction with these molecular and cellular targets are reviewed. The feasibility of PPs to prevent/ cure specific cutaneous toxicity connected to anti-EGFR therapy and to reduce multidrug resistance of skin tumors is also discussed.

The dioxin receptor, also known as arylhydrocarbon receptor (AhR), is a ligand-activated transcription factor that mediates the toxicity of dioxins and related environmental contaminants. In addition, there is a growing list of natural compounds, mainly plant polyphenols that can modulate AhR function and downstream signaling with quite unknown consequences for cellular function. We investigate the potential of four different beta-carboline alkaloids to stimulate AhR signaling in human hepatoma cells and keratinocytes. Three test substances, namely rutaecarpine, annomontine and xestomanzamine A, increase AhR-driven reporter gene activity as well as expression of two AhR target genes in a dose-dependent and time-dependent manner. Additionally, the three test alkaloids stimulate cytochrome P450 (CYP) 1 enzyme activity without showing any antagonistic effects regarding benzo(a)pyrene-stimulated CYP1 activation. The AhR-activating property of the beta-carbolines is completely abrogated in AhR-deficient cells providing evidence that rutaecarpine, annomontine and xestomanzamine A are natural stimulators of the human AhR. The toxicological relevance of beta-carboline-mediated AhR activation is discussed.

Breast cancer is a major cause of death worldwide. Amongst the various forms of treatment chemoprevention is favoured and natural products such as the dietary flavonoids have been examined for their cancer preventative activity. In this study we investigated the anticancer activity of the flavonoid diosmetin, as a result of cytochrome P450 CYP1 metabolism. Diosmetin was metabolized to luteolin via an aromatic demethylation reaction on the B-ring from CYP1A1, CYP1B1 and the hepatic isozyme CYP1A2. CYP1A1 and CYP1A2 also produced additional unidentified metabolites. CYP1B1 showed the lowest apparent KM and CYP1A1 the highest apparent Kcat. Diosmetin was also metabolized to luteolin in estrogen receptor positive breast cell-line (MCF-7 cells) preinduced for 24 h with the potent CYP1 inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Treatment of MCF-7 cells with TCDD caused bioactivation of diosmetin enhancing its cytotoxicity. Taken together these data suggest that the flavonoid diosmetin is metabolised to the more active molecule luteolin by CYP1 family enzymes.

Human epidermal keratinocytes express subsets of cytochromes P450 (P450) (CYP gene products) that are strongly up-regulated, not regulated, or down-regulated by differentiation-specific factors. We investigated how drug exposure affects epidermal expression of <em>CYP1</em>-4 genes, which encode many drug-metabolizing P450s. Real-time polymerase chain reaction (PCR) assays measured <em>CYP1</em>-4 mRNA levels in epidermal keratinocytes differentiated in vitro in the presence of drug or vehicle for 6 days. We confirmed the spinous phenotype at day 6 by changes in cellular morphology and upregulation of cytokeratin 10 and transglutaminase (TGM)1 mRNA in the differentiating keratinocytes. Effects of drug exposure depended on the influence of differentiation-specific factors in controlling epidermal <em>CYP1</em>-4 expression. CYP2C18, 2C19, 2C9, 2W1, 3A4, and 4B1 are up-regulated by cellular differentiation; mRNA levels for these CYP genes were inhibited in differentiating keratinocytes exposed to retinoic acid and aryl hydrocarbon receptor (AhR) ligands. These same drugs effected <or=2-fold change or even augmented mRNA levels for CYP genes that are not regulated by differentiation (CYP2S1, 2J2, 1B1, 1A1, 1A2, 2E1, and 2D6) and for CYP2U1, which is expressed at highest levels in undifferentiated keratinocytes. The clinically relevant drugs miconazole, dexamethasone, rifampicin, and dapsone had little effect on <em>CYP1</em>-4 mRNA levels under assay conditions. The AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin also up-regulated keratinocyte TGM1 mRNA in a concentration- and time-dependent manner. This effect was blocked by the AhR antagonist resveratrol. These findings implicate AhR-dependent up-regulation of TGM1 mRNA in differentiating keratinocytes as one mechanism contributing toward chloracne in humans exposed to toxic levels of dioxin.

An inhibitory effect on both constitutive and inducible expression of cytochrome P450 isoenzymes has been shown for different cytokines and growth factors. We previously described an inhibition of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced CYP1A1 mRNA and enzyme activity by transforming growth factor-beta1 (TGF-beta1) in human lung cancer A549 cells. In the present study, we report that not only TCDD-induced expression of CYP1A1 but also basal mRNA expression of CYP1A1, CYP1B1, and aryl hydrocarbon receptor (AHR) was down-regulated by TGF-beta1 in cells not treated with TCDD. In contrast, mRNA expression of the AHR partner protein Arnt (aryl hydrocarbon receptor nuclear translocator) was not influenced. Furthermore, TCDD-induced expression of CYP1B1 and NMO-1 was inhibited, and the IC50 values of 5-10 pM TGF-beta1 were in the same range as observed for inhibition of CYP1A1 and AHR mRNA expression. Transfection studies with a plasmid containing a luciferase reporter gene under control of two dioxin-responsive elements indicate an effect on AHR protein expression. Results of time-course studies revealed a parallel inhibition of AHR and CYP1 mRNA expression, indicating that TGF-beta1 is a direct negative regulator of transcription of these genes. The treatment of cells with cycloheximide led to a superinduction of TCDD-induced CYP1A1 and CYP1B1 mRNA expression and abolished the inhibitory effect of TGF-beta1 on basal as well as TCDD-induced CYP1 and AHR mRNA expression. TGF-beta1 seems not to influence the stability of AHR mRNA. The results suggest that TGF-beta1 induces rapid transcription and translation of an as-yet-unknown negative regulatory factor or factors that may directly regulate expression of AHR and genes of Ah gene battery.

We identify cytochrome P450 1A1 (CYP1A1) as a target for tumor-selective drug development in bladder cancer and describe the characterization of ICT2700, designed to be metabolized from a prodrug to a potent cytotoxin selectively by CYP1A1. Elevated CYP1A1 expression was shown in human bladder cancer relative to normal human tissues. RT112 bladder cancer cells, endogenously expressing CYP1A1, were selectively chemosensitive to ICT2700, whereas EJ138 bladder cells that do not express CYP1A1 were significantly less responsive. Introduction of CYP1A1 into EJ138 cells resulted in 75-fold increased chemosensitivity to ICT2700 relative to wild-type EJ138. Negligible chemosensitivity was observed in ICT2700 in EJ138 cells expressing CYP1A2 or with exposure of EJ138 cells to CYP1B1- or CYP3A4-generated metabolites of ICT2700. Chemosensitivity to ICT2700 was also negated in EJ138-CYP1A1 cells by the CYP1 inhibitor α-naphthoflavone. Furthermore, ICT2700 did not induce expression of the AhR-regulated CYP1 family, indicating that constitutive CYP1A1 expression is sufficient for activation of ICT2700. Consistent with the selective activity by CYP1A1 was a time and concentration-dependent increase in γ-H2AX protein expression, indicative of DNA damage, associated with the activation of ICT2700 in RT112 but not EJ138 cells. In mice-bearing CYP1A1-positive and negative isogenic tumors, ICT2700 administration resulted in an antitumor response only in the CYP1A1-expressing tumor model. This antitumor response was associated with detection of the CYP1A1-activated metabolite in tumors but not in the liver. Our findings support the further development of ICT2700 as a tumor-selective treatment for human bladder cancers.