The aim of this in vitro study was to determine the effect of TCDD and luteinizing hormone (LH) on mRNA expression of aryl hydrocarbon receptor 1 (AHR1), AHR1 nuclear translocator 1 (ARNT1), and the CYP1 family monooxygenases (CYP1A4, CYP1A5, CYP1B1), and to assess the basal and TCDD-induced activity of these enzymes in chicken ovarian follicles. White (WF) and yellowish (YF) prehierarchical follicles and fragments of the theca (TL) and granulosa (GL) layers of the 3 largest preovulatory follicles (F3-F1) were exposed to TCDD (10nM), ovine LH (oLH; 10ng/mL) or a combination of TCDD (10nM) and oLH (10ng/mL), and increasing doses of TCDD (0.01-100nM). AHR1 and ARNT1 mRNA transcripts were found in all examined follicles. The effect of TCDD and oLH on AHR1 and ARNT1 mRNA expression depended on the maturational state of the follicle. CYP1A4 was predominantly expressed in the GL of the F3-F1 follicles; in comparison with the WF, a higher level of CYP1A5 mRNA was found both in the GL and TL of F3-F1 follicles. Alternatively, the highest level of CYP1B1 mRNA was noticed in the WF follicles. In different developmental stages of the follicle TCDD and oLH induced a different CYP1 isoform. TCDD increased EROD and MROD activities in all the investigated ovarian follicles. In conclusion, AHR1 and ARNT1 mRNA expression indicate that the chicken ovary is a target tissue for dioxin and dioxin-like compounds. The expression of CYP1-family genes and TCDD-inducible EROD and MROD activities in ovarian follicles suggest the possibility of xenobiotic detoxification in the chicken ovary.

We examined the effects of amiloride derivatives, especially 5-(N-ethyl-N-isopropyl)amiloride (EIPA), on the activity of cytochrome P450 (CYP) 1 isoforms, known to metabolize carcinogenic polycyclic aromatic hydrocarbons (PAHs), such as benzo(a)pyrene (BP), into mutagenic metabolites and whose cellular expression can be induced through interaction of PAHs with the arylhydrocarbon receptor. EIPA was found to cause a potent and dose-dependent inhibition of CYP1-related ethoxyresorufine O-deethylase (EROD) activity in both liver cells and microsomes. It also markedly reduced activity of human recombinant CYP1A1 enzyme through a competitive mechanism; activities of other human CYP1 isoforms, i.e. CYP1A2 and CYP1B1, were also decreased. However, EIPA did not affect BP-mediated induction of CYP1A1 mRNA and protein levels in rat liver cells, likely indicating that EIPA does not block activation of the arylhydrocarbon receptor by PAHs. Inhibition of CYP1 activity by EIPA was associated with a decreased metabolism of BP, a reduced formation of BP-derived DNA adducts and a diminished BP-induced apoptosis in liver cells. The present data suggest that amiloride derivatives, such as EIPA, may be useful for preventing toxicity of chemical carcinogens, such as PAHs, through inhibition of CYP1 enzyme activity.

CYP1B1 is implicated to have a role in the development of breast, ovarian, renal, skin and lung carcinomas. It has been suggested that identification of potent and specific CYP1B1 inhibitors can lead to a novel treatment of cancer. Flavonoids have a compact rigid skeleton which fit precisely within the binding cavity of CYP1B1. Systematic isosteric replacement of flavonoid 'O' atom with 'N' atom led to the prediction that a 'quinazoline' scaffold could be the basis for designing potential CYP1B1 inhibitors. A total of 20 quinazoline analogs were synthesized and screened for CYP1B1 and CYP1A1 inhibition in Sacchrosomes™. IC50 determinations of six compounds with capability of inhibiting CYP1B1 identified quinazolines 5c and 5h as the best candidates for CYP1B1 inhibition, with IC50 values in the nM range. Further selectivity studies with homologous CYPs, belonging to the CYP1, CYP2 and CYP3 family of enzymes, showed that the compounds are likely to be free from critical drug-drug interaction liability. Molecular modelling studies were performed to rationalize the observed enzymatic inhibitions. Further biological studies in live yeast and human cells, harboring CYP1A1 and CYP1B1 enzymes, have illustrated the most potent compounds' cellular permeability and capability of potently inhibiting CYP1B1 enzyme expressed within live cells.

Selenium (Se), an essential trace element in human nutrition, is thought to have an important role in the prevention of oxygen damage by organic hydroperoxides generated by oxidative metabolism. Epidemiological studies have shown an association between placental cytochrome P450-1A1 (CYP1) activity and threatened preterm delivery (TPD), and other experimental studies have shown alterations in fetal development with CYP1 activity or toxicity. The present study examined the possible protective effect of selenium on the potential toxicity of maternal exposure to polycyclic aromatic hydrocarbons (PAHs) on the normal course of pregnancy. Placental CYP1 activity was used as a risk factor resulting from maternal exposure to PAHs. TPD occurrence was used as a general indicator of troubles in the normal course of pregnancy. A group of TPD patients and a group of controls were selected from 178 pregnant women attending obstetrical care in a maternity hospital. Selenium concentrations in maternal plasma were lower in the TPD group: 63.7 ng/ml (CI 95% confidence bounds = 43.6-82.2) vs 69.2 ng/ml (CI 95% confidence bounds = 49. 3-96.3) (t test, P<0.01). When placental CYP1 was induced, an association between TPD and selenium was found, with an increase of 10 ng/ml for the latter. An adjusted odds ratio of 0.55 (CI 95% confidence bounds = 0.34-0.88; chi(2), P<0.01) was estimated. When placental CYP1 was not activated, the odds ratio was estimated at 0.99 (CI 95% confidence bounds=0.95-1.03; NS). This epidemiologic finding suggests that antioxidant Se status may be a protective factor against the potential toxic effect of PAHs on the normal course of pregnancy. The downward trend that we observed supports the hypothesis that the one-electron pathway metabolism of PAHs may explain a large fraction of TPD and some preterm deliveries.

Epidemiological studies on 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD) exposure indicated high incidences of pulmonary dysfunctions and lung cancer. Animal studies also demonstrated lung cancer development in female, but not in male, rats exposed to TCDD. Such effects, however, have not been reported in polychlorinated biphenyls (PCB) exposure. In our present study, we have investigated the effects of TCDD and PCB126, with or without cotreatment with 17 beta-estradiol (E2), on a human bronchial epithelial cell line BEAS-2B. We found that treatment with either TCDD or PCB126 alone reduced cell numbers as well as thymidine incorporation. Cell death, however, was only detected in PCB126-, but not TCDD-, treated cultures. The TCDD-induced cell reduction, therefore, could not be contributed to cell death. Meanwhile, because TCDD- and PCB126-enhanced CYP1A1 and CYP1B1 expressions were significantly reduced by the AhR antagonist and CYP1 inhibitor alpha-naphthoflavone (ANF), this indicated that the effects of TCDD and PCB126 were AhR and cytochrome p450 1 dependent. We also found that while E2 itself did not alter CYP1A1 and CYP1B1 expressions, cotreatment of E2 with TCDD or PCB126 would significantly enhance TCDD-, but not PCB126-, induced toxicity. We further demonstrated that in the presence of E2, 1 nM TCDD increased the production of E2 metabolites, 2-methoxyestradiol (2-MeOE2) and 4-methoxyestradiol (4-MeOE2). PCB126, however, only increased 2-MeOE2 formation without significant induction of 4-MeOE2. We believe that these metabolites, especially 4-MeOE2, interacted with TCDD to further suppress cell growth. Our data provided the first demonstration on the enhancement of TCDD-induced toxicity in human lung cells via interaction with estrogen.

The purpose of the study was to determine how multi-vitamin deficiency affects xenobiotic-metabolizing enzyme (XME) activities in the rat liver. Vitamin levels and XME activities were studied in the livers of male Wistar rats who were fed for 4 weeks with semi-synthetic diets containing either adequate (100 % of recommended vitamin intake) levels of vitamins (control), or decreased vitamin levels (50 % or 20 % of recommended vitamin intake). The study results have shown that moderate vitamin deficiency (50 %) leads to a decrease of vitamin A levels only, and to a slight increase, as compared with the control, in the following enzyme activities: methoxyresorufin O-dealkylase (MROD) activity of CYP1 A2 - by 34 % (p < 0.05), UDP-glucuronosyl transferase - by 26 % (p < 0.05), and quinone reductase - by 55 % (p < 0.05). Profound vitamin deficiency (20 %) led to a decrease of vitamins A, E, B1, B2, and C, and enzyme activities in the liver: MROD - to 78 % of the control level (p < 0.05), 4-nitrophenol hydroxylase - to 74 % (p < 0.05), heme oxygenase-1 - to 83 % (p < 0.05), and quinone reductase - to 60 % (p < 0.05). At the same time, the UDP-glucuronosyl transferase activity and ethoxyresorufin O-dealkylase activity of CYP1A1, pentoxyresorufin O-dealkylase activity of CYP2B1/2 and 6β-testosterone hydroxylase, as well as the total activity of glutathione transferase did not differ from the control levels. The study has demonstrated that profound multi-vitamin deficiency is associated with a decrease in the expression of CYP1A2 and CYP3A1 mRNAs to 62 % and 79 %, respectively. These data indicated that a short-term but profound multi-vitamin deficiency in rats leads to a decrease in the activities and expression of the some XME that play an important role in detoxification of xenobiotics and metabolism of drugs and antioxidant protection.

Recently, CYP1 enzymes are documented for selective metabolism of anticancer leads in cancer prevention and/or progression. Elucidation of specificity of substrates/inhibitors of CYP1 isoforms plays a vital role in design of more selective and potent anticancer leads. However, an area of concern is the broad range of substrate specificities and planar nature of substrates with limited dataset which makes it difficult to predict their site of metabolism (SOM) accurately. In the present study, various models for prediction of site of metabolism in case of CYP1A1, CYP1A2, and CYP1B1 substrates were developed using MetaSite, molecular docking, and quantum chemical descriptors. The predictive accuracy of MetaSite, molecular docking, and quantum chemical descriptors in identifying experimental site of metabolism was analyzed at three levels; top rank, top three ranks, and top five ranks. Two quantum chemical descriptors, chemical hardness and local nucleophilicity are proposed for the prediction of CYP-mediated SOM for the first time. The predictive accuracy shown by chemical hardness at top three ranks was 83.3, 85.7, and 84.6 % for CYP1A1, CYP1A2 and CYP1B1, respectively, whereas local nucleophilicity gave poor predictions of 50, 42.8, and 46.2 %, respectively. The predictability of chemical hardness descriptor outperformed at all three levels of ranks for CYP1A1, CYP1A2, and CYP1B1. Hence, we propose chemical hardness as an useful quantum chemical descriptor for prediction of metabolically vulnerable prints in CYP1A1, CYP1A2, and CYP1B1 mediated metabolism and support the optimization efforts in drug discovery and development programs.

The number of fatalities in Japan attributable to lung cancer exceeded 50000 in 2001. It is socially desirable that various markers, which can be utilized for the prevention of lung cancer, be established. We believe that smoking or exposure to carcinogens in air induces mutations in bronchial and alveolar epithelia, leading to the development of lung cancer. It would be useful to have markers of individual differences in susceptibility to chemical carcinogen-induced lung cancer 1) to identify genetic polymorphisms of enzymes metabolizing chemical carcinogens and 2) to investigate the expression of enzymes metabolizing chemical carcinogens. In this paper, we review CYP expression in the bronchial epithelium. CYP1, CYP2 and CYP3 are expressed in the bronchial epithelium. We also show the relationship between the genetic polymorphisms of cytochrome P450 (CYP) and a person's susceptibility to chemical carcinogen-induced lung cancer. We demonstrate the relationship between cigarette consumption and the CYP expression profile in the bronchial epithelium. To maintain and promote public health, we must apply evidence, such as CYP polymorphisms and CYP profiles to disease prevention and also to aggressively advance evidence-based prevention (EBP) of lung cancer.

The inhibition activity of 36 flavonoids against CYP1A2 was determined by our previously developed in vitro method. The Comparative Molecular Similarity Indexes Analysis (CoMSJA) approach was used to probe the quantitative relationships between the flavonoids' molecular structural descriptors and their inhibitory activities. A reliable CoMSIA model with the combined electrostatic and hydrophobic fields was derived with the regression coefficient R2 of 0.948 and the cross-validation regression coefficient q2 of 0.630, separately, which is capable of elucidating the quantitative relationship between the 3D structural descriptors of the flavones and their bioactivities. Comparing with flavone, the larger pi-pi conjugated system of alpha-naphthoflavone significantly improved the biologically inhibitory ability. Based on the core structure of the latter, either electropositive substituents or hydrophobic groups at the 6, 3', and 4' ring positions or electronegative counterparts at the 5 ring position, can enhance the inhibitory potency against CYP1 A2 according to the CoMSIA contour maps.

Pesticide formulations typically contain adjuvants added to enhance the performance of the active ingredient. Adjuvants may modify the bioavailability and toxicity of pesticides. In this study, the aim was to examine to which degree nonylphenol (NP) may interfere with the toxicity of two organophosphorus pesticides found in aquafeeds, chlorpyrifos-methyl (CPM) and pirimiphos-methyl (PPM). Atlantic salmon liver cells were exposed to these compounds singly or in combinations for 48 h using 3D cell cultures. Cytotoxicity, gene expression (RT-qPCR), and lipidomics endpoints were used to assess toxicity. The dose-response assessment showed that NP was the most toxic compound at equimolar concentrations (100 μM). Shotgun lipidomics pointed to a general pattern of elevated levels of saturated 18:0 fatty acids and declined levels of 18:1 monounsaturated fatty acids by the combined treatment. All three compounds had a distinct effect on membrane phospholipids, in particular on phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Lipid species patterns predicted inhibited stearoyl CoA desaturase (SCD) activity and increased Δ6 desaturase (D6D) activity in co-treated cells. While all three compounds alone mitigated increased triacylglycerol (TAG) accumulation, combined treatment resulted in lower total TAG in the cells. Multivariate analysis with PLS regression showed significant combined effects for nine genes (d5d, d6d, scd, srebf2, vtg, esr1, cyp1, ugt1a, and cat) and four lipid species (FFA 22:5, LPC 18:0, TAG52:1-FA16:0, and TAG52:1-FA18:0). In summary, this study demonstrates that the adjuvant can be the main contributor to the toxicity of a mixture of two organophosphorus pesticides with relatively low toxicity in fish cells.

OBJECTIVE

To explore the mechanism of nourishing kidney and reducing urine effect of suoquan capsule by discussing the adjusting action on ALD synthase.

METHODS

Built the model of deficiency of the kidney and diuresis by adenine,inspectd the contents of Cort, ALD in blood and the mRNA expression of <em>CYP1</em>1B2 with ELISA and RT-PCR technology of the mice.

RESULTS

Compared with model group, Suoquan capsule could remarkedly increase the contents of Cort, ALD in blood and the mRNA expression of CYP1 1 B2 of model rats.

CONCLUSIONS

Suoquan capsule can increase the contents of Cort, ALD in blood and the mRNA expression of <em>CYP1</em>1B2 in deficiency of the kidney and diuresis rats. Promote the combining of ALD by adjusting the ALD synthase may be the mechanism of nourishing the kidney and reducing urine of Souquan capsule from ALD synthase approach.

OBJECTIVE

Glucosinolates/isothiocyanates are an established class of naturally occurring chemopreventive agents, a principal mechanism of action being to limit the generation of genotoxic metabolites of chemical carcinogens, as a result of modulation of cytochrome P450 and phase II detoxification enzymes. The objective of this study was to assess whether a glucosinolate-rich extract from Daikon sprouts, containing glucroraphasatin and glucoraphenin, is a potential chemopreventive agent by modulating such enzymes in the liver and lung of rats.

METHODS

Rats were exposed to the glucosinolate-rich Daikon extract through the diet, at three dose levels, for 14 days, so that the low dose simulates dietary intake.

RESULTS

At the low dose only, a modest increase was noted in the hepatic dealkylations of methoxy-, ethoxy-, pentoxyresorufin and benzyloxyquinoline that was accompanied by elevated expression of CYP1 and CYP3A2 apoprotein levels. In lung, only a modest increase in the dealkylation of pentoxyresorufin was observed. At higher doses, in both tissues, these increases were abolished. At the same low dietary dose, the Daikon extract elevated markedly glutathione S-transferase activity paralleled by rises in GSTα, GSTμ and GSTπ protein expression. An increase was also noted in quinone reductase activity and expression. Finally, glucuronosyl transferase and epoxide hydrolase activities and expression were also up-regulated, but necessitated higher doses.

CONCLUSIONS

Considering the ability of Daikon glucosinolates to effectively enhance detoxification enzymes, in particular glutathione S-transferase, it may be inferred that consumption of this vegetable may possess significant chemopreventive activity and warrants further evaluation through epidemiology and studies in animal models of cancer.

Cytochrome P450 (CYP) genes, which make up a large gene superfamily, are known to play an important role in drug metabolism. The CYP1 family, one of the gene families of the CYP superfamily, has three subfamilies of genes whose sequences have been deposited in the GenBank/EMBL thus far: CYP1A, CYP1B, and CYP1C. Mammals as well as fish confront numerous foreign chemicals in the environment that may accumulate to toxic levels unless they are metabolized and eliminated by processes largely mediated by CYP enzymes. A new complementary DNA of the CYP1C subfamily encoding CYP1C2 was isolated from the carp liver after a single intraperitoneal injection of beta-napthoflavone (BNF). The full-length cDNA obtained contained a 5' noncoding region of 198 bp, an open reading frame of 1575 bp coding for 524 amino acids and a stop codon, and a 3' noncoding region of 531 bp. The predicted molecular weight of the protein was approximately 59.3 kDa. The amino acid sequence deduced from the carp CYP1C2 sequence showed a similarity of 76.6% with that deduced from our previously reported carp CYP1C1. It exhibited similarities of 77.3, 73.7, and 76.4% with those deduced from scup CYP1C2, scup CYP1C1, and Japanese eel CYP1C1 sequences, respectively. Carp CYP1C2 cDNA showed similarities with reported CYP1Bs of teleosts and mammals, namely, 47.6, 45.3, 45.7, 44.0, and 44.6% for carp, plaice, human, rat, and mouse CYP1B1s, respectively, while it exhibited a similarity of 49.0% with carp CYP1B2. The carp CYP1C2 sequence was aligned with the CYP1 sequences and has been deposited in the GenBank/EMBL data bank with the accession number AY437777. The phylogenetic tree constructed using fish and mammalian CYP1 sequences suggested a closer relationship of CYP1C with CYP1B than with CYP1A. The tree showed possibile existence of CYP1C subfamily genes in mammalian species. Northern blot analysis of the liver, intestines, kidneys, and gills revealed a distinct induced expression only in the kidneys, with no detectable constitutive expression in the other organs studied.

OBJECTIVE

To characterize the distribution of mathematically derived human hepatic CYP1A1 activity using differential inhibition of ethoxyresorufin O-deethylation (EROD) by fluvoxamine.

METHODS

Quantitative CYP1A1- and CYP1A2-mediated EROD activities were determined in 42 human livers using differential inhibition of EROD by fluvoxamine. CYP1A2-specific activity was also measured by phenacetin O-deethylation and caffeine 3-demethylation. Distributions of CYP1A1-mediated EROD and CYP1-A2 probe activities were analyzed using cumulative distribution (probit) plots and the Kolgomorov-Smirnov test. Age effect on CYP1A1- and CYP1A2-mediated EROD activities was evaluated using descriptive statistics and analysis of variance.

RESULTS

The derived CYP1A1 protein concentration of 0.58 +/- 1.04 pmol/mg was only 4% of the derived CYP1A2. Since CYP1A1 is intrinsically far more active than CYP1A2 in mediating EROD, contribution of CYP1A1 to EROD represented approximately 25-40% of CYP1A2 contribution. Three of the 42 livers exhibited no CYP1A1-mediated EROD. Approximately 8% of the individuals showed high CYP1A1 activity phenotype based on cumulative distribution curve analysis. Hepatic CYP1A1 activity was more variable than that of CYP1A2. The variance of CYP1A1-mediated EROD was significantly different from that of CYP1A2, using the Kolgomorov-Smirnov statistical test. Even though not statistically significant, an age-related pattern in CYP1A1-mediated activity was identified: activity was high in the pre-puberty group, then decreased in the young/mature adult group and, finally, a slight increase was observed in old age.

CONCLUSIONS

Distribution pattern in CYP1A1-mediated EROD suggests that the low derived CYP1A1 expression is most likely induced rather than constitutive. CYP1A1 activity deviates from log-normal distribution; the variations in hepatic CYP1A1 activity may affect the conversion of procarcinogens to carcinogens. The age-related trend in CYP1A1-mediated EROD activity hints that CYP1A1 responsiveness to inducers may change with age as well as with exposure to environmental inducers. These findings prompt (1) future genotyping studies to determine whether increased CYP1A1 inducibility is a result of genetic factors and (2) studies to address whether CYP1A1 inducibility changes with age.

Several epidemiological studies have suggested that the incidence of male reproductive organ malformations, including hypospadias or cryptorchidism, has increased due to fetal-stage exposure to environmental pollutants. However, the association of chemical exposure with the expression of target regulatory genes in the tissues of patients has not yet been reported. Because experimental approaches or clinical trials in human studies are limited, especially those using fetal and/or infants, it is difficult to obtain clear physiological evidence of mechanisms underlying male reproductive malformations. Thus, the lack of physiological evidence makes this issue controversial. We analyzed preputial tissues from patients with hypospadias (n = 23) and phimosis (n = 16). The atypical CYP1 family genes, CYP1A1 and CYP1B1, are potential biomarkers of environmental chemical exposure. We then compared the expression levels of CYP1A1 and CYP1B1 between hypospadias and phimosis samples by quantitative RT-PCR analysis. The mRNA expression levels of SRD5A2 and AR also were measured, because the androgen-related genes involved in the onset of disorders of male reproductive system. A significantly higher CYP1B1 expression level and a lower AR expression level were observed in the hypospadias groups than in the phimosis group. Positive correlations (P < 0.001) between the mRNA expression levels of the CYP1 family and SRD5A2 were found in patients with hypospadias but not in those with phimosis. Moreover, the methylation levels of the four genes were determined by bisulfite genomic sequencing. Although the SRD5A2 promoter region showed moderate methylation, no methylation was detected in CYP1A1, CYP1B1, or AR. There was no significant difference in SRD5A2 promoter methylation level between hypospadias and phimosis patients. Negative correlations were found between the methylation level of SRD5A2, especially at the - 221 Sp1 site, and the CYP1 family mRNA expression levels (CYP1A1, p = 0.002; CYP1B1, p = 0.007) in hypospadias patients, but not in phimosis patients. The significant positive association of mRNA expression level and the negative association of methylation level of the SRD5A2 gene with the mRNA expression levels of CYP1 family genes in the preputial tissue seem to indicate the chemical exposure of patients with hypospadias.

The methylated benzo[a]pyrenes (MeBaPs) are present at significant levels in the environment, especially in the sediments contaminated by petrogenic PAHs. However, the existing data on their toxic effects in vitro and/or in vivo are still largely incomplete. Transcription factor AhR plays a key role in the metabolic activation of PAHs to genotoxic metabolites, but the AhR activation may also contribute to the tumor promoting effects of PAHs. In this study, the AhR-mediated activity of five selected MeBaP isomers was estimated in the DR-CALUX reporter gene assay performed in rat hepatoma cells. Detection of other effects, including induction of CYP1A1, CYP1B1, and AKR1C9 mRNAs, DNA adduct formation, production of reactive oxygen species, oxidation of deoxyguanosine, and cell cycle modulation and apoptosis, was performed in the rat liver epithelial WB-F344 cell line, a model of liver progenitor cells. We identified 1-MeBaP as the most potent inducer of AhR activation, stable DNA adduct formation, checkpoint kinase 1 and p53 phosphorylation, and apoptosis. These effects suggest that 1-MeBaP is a potent genotoxin eliciting a typical sequence of events ascribed to carcinogenic PAHs: induction of CYP1 enzymes, formation of high levels of DNA adducts, activation of DNA damage responses (including p53 phosphorylation), and cell death. In contrast, 10-MeBaP, representing BaP isomers substituted with the methyl group in the angular ring, elicited only low levels DNA adduct formation and apoptosis. Other MeBaPs under study also elicited strong apoptotic responses associated with DNA adduct formation as the prevalent mode of toxic action of these compounds in liver cells. MeBaPs induced a weak production of ROS, which did not lead to significant oxidative DNA damage. Importantly, 1-MeBaP and 3-MeBaP were found to be potent AhR agonists, one order of magnitude more potent than BaP, thus suggesting that the AhR-dependent modulations of gene expression, deregulation of cell survival mechanisms, and further nongenotoxic effects associated with AhR activation may further contribute to their tumor promotion and carcinogenicity.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a prominent heterocyclic aromatic amine (HAA) found in meat and fish cooked at moderate to high temperature. It is considered as a potent dietary factor promoting colon carcinogenesis. However, the role of intestinal cells in PhIP bioactivation has not been fully explained, particularly when cells are pre-malignant. Loss of function of the adenomatous polyposis coli (APC) gene product is an early and frequent event in human colorectal carcinogenesis. Normal (Apc(+/+)) and pre-malignant (Apc(Min/+), where Min=multiple intestinal neoplasia) colonic epithelial cells of mice can be used to study promotion of carcinogenesis, but these cells have not been characterized for bio-activation of HAA. We investigated the metabolism of (14)C-PhIP in these two murine cell lines. Cells induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) metabolized PhIP into 4'-OH-PhIP as the main metabolite in PhiP detoxification. Besides, 5-OH-PhIP was identified, revealing the formation of intermediary reactive metabolites, since it results from a degradation of conjugates of N-acetoxy-PhIP. Apc(Min/+) cells produce significantly higher amounts of these metabolites. Demethylated metabolites are also observed, indicating that the colon contains a significant CYP1 family dependent metabolic activity. A minor hydroxy-glucuronide-PhIP metabolite is observed in Apc(Min/+) cells, the glucuronidation being known as an important step in the detoxification pathway. Quantitative real-time reverse transcription polymerase chain reaction experiments demonstrate that induction by TCDD has prevailing effects in gene expression of CYP1A1, CYP1A2 and CYP1B1 in Apc(Min/+) cells. In these cells, N-acetyltransferase-2 is also expressed at higher levels. So, the more important potency to metabolically bio-activate PhIP, as measured in Apc(Min/+) cells, can be linked to a higher probability to generate new in situ mutations.

Multiple forms of CYP exist in humans. P450s comprise a superfamily of enzymes. These P450s play important roles in the oxidation of structurally diverse endobiotics and xenobiotic chemicals including anticancer drugs and carcinogens. The genes encoding most of the CYP1, CYP2, and CYP3 families show genetic polymorphism. In this article, we describe recent findings that these polymorphic enzymes can abolish or quantitatively or qualitatively alter drug metabolism in humans.

The cytochrome P450 (CYP) 1-3 families are involved in xenobiotic metabolism, and are expressed primarily in the liver. Ostriches (Struthio camelus) are members of Palaeognathae with the earliest divergence from other bird lineages. An understanding of genes coding for ostrich xenobiotic metabolizing enzyme contributes to knowledge regarding the xenobiotic metabolisms of other Palaeognathae birds. We investigated CYP1-3 genes expressed in female ostrich liver using a next-generation sequencer. We detected 10 CYP genes: CYP1A5, CYP2C23, CYP2C45, CYP2D49, CYP2G19, CYP2W2, CYP2AC1, CYP2AC2, CYP2AF1, and CYP3A37. We compared the gene expression levels of CYP1A5, CYP2C23, CYP2C45, CYP2D49, CYP2G19, CYP2AF1, and CYP3A37 in ostrich liver and determined that CYP2G19 exhibited the highest expression level. The mRNA expression level of CYP2G19 was approximately 2-10 times higher than those of other CYP genes. The other CYP genes displayed similar expression levels. Our results suggest that CYP2G19, which has not been a focus of previous bird studies, has an important role in ostrich xenobiotic metabolism.

The results of quantitative structure-activity relationship (QSAR) studies on series of P450 inhibitors are reported. Cytochrome P450 families CYP1, CYP2 and CYP51 have been investigated for QSAR analysis, including those of CYP2 subfamilies: CYP2A, CYP2B, CYP2C, CYP2D and CYP2E. The accumulated evidence indicates different structural descriptors being involved, depending on the P450 enzyme concerned, although compound lipophilicity in the form of either logP or logD(7.4) appears to represent a common factor in some cases. This is thought to represent desolvation of the P450 active site, although quadratic expressions in lipophilicity tend to suggest that membrane transport is important, especially for CYP2B and CYP2E isoforms. In general, there is close agreement (R = 0.95-0.99) between experimental pKi values and those calculated via QSAR analysis.

The cytochrome P450 CYP1A1 and CYP1B1 enzymes are phase I extrahepatic enzymes involved in the activation of pro-carcinogenic compounds to carcinogenic metabolites. Although differential overexpression of CYP1A1 and CYP1B1 has been documented at the messenger RNA (mRNA) and protein level, studies that have examined CYP1 expression by enzyme activity assays are limited. In the current study, the expression of CYP1A1 and CYP1B1 was investigated in a panel of human tumors of endometrial origin by quantitative reverse transcriptase PCR (qRT-PCR), Western blotting, and enzyme activity assays. The data revealed that approximately 36 % (5/14) and 43 % (6/14) of the endometrial tumors overexpressed CYP1A1 and CYP1B1 mRNA, whereas in 57 % of the endometrial tumors, CYP1 mRNA levels were downregulated. The mean mRNA levels of CYP1B1 and CYP1A1 in endometrial tumors did not show a significant difference compared to normal tissues (p>> 0.05). Western blotting confirmed the qRT-PCR results and CYP1A1 and CYP1B1 proteins were shown to be downregulated in 7/14 (50 %) of the tumors and overexpressed in 4/14 (29 %) of the tumors. As regards to enzyme activity, 21 % (3/14) of the endometrial samples revealed elevated CYP1 activity levels across the tumor counterparts. Overall, the data suggest a putative downregulation of CYP1A1 and CYP1B1 expression in endometrial tumors, whereas overexpression of active CYP1 enzymes in 21 % of the tumors highlights the potential use of the latter enzymes as chemotherapeutic targets in endometrial cancer.

In its classical genomic mode of action, the aryl hydrocarbon receptor (AhR) acts as a ligand activated transcription factor regulating expression of target genes such as CYP1A1 and CYP1B1. Some ligands may also trigger more rapid nongenomic responses through AhR, including calcium signaling (Ca2+). In the present study we observed that pyrene induced a relatively rapid increase in intracellular Ca2+-concentrations ([Ca2+]i) in human microvascular endothelial cells (HMEC-1) and human embryonic kidney cells (HEK293) that was attenuated by AhR-inhibitor treatment and/or transient AhR knockdown by RNAi. In silico molecular docking based on homology models, suggested that pyrene is not able to bind to the human AhR in the agonist conformation. Instead, pyrene docked in the antagonist conformation of the AhR PAS-B binding pocket, although the interaction differed from antagonists such as GNF-351 and CH223191. Accordingly, pyrene did not induce CYP1A1 or CYP1B1, but suppressed CYP1-expression by benzo[a]pyrene (B[a]P) in HMEC-1 cells, confirming that pyrene act as an antagonist of AhR-induced gene expression. Use of pharmacological inhibitors and Ca2+-free medium indicated that the pyrene-induced AhR nongenomic [Ca2+]i increase was initiated by Ca2+-release from intracellular stores followed by a later phase of extracellular Ca2+-influx, consistent with store operated calcium entry (SOCE). These effects was accompanied by an AhR-dependent reduction in ordered membrane lipid domains, as determined by di-4-ANEPPDHQ staining. Addition of cholesterol inhibited both the pyrene-induced [Ca2+]i-increase and alterations in membrane lipid order. In conclusion, we propose that pyrene binds to AhR, act as an antagonist of the canonical genomic AhR/Arnt/CYP1-pathway, reduces ordered membrane lipid domains, and activates AhR nongenomic Ca2+-signaling from intracellular stores.

In order to elucidate the mechanism for diastereoisomer-specific toxicity and metabolism of hexabromocyclododecanes (HBCDs) in biota, zebrafish (Danio rerio) were exposed to different concentrations of individual HBCD diastereoisomers (α-, β- and γ-HBCD) in water for 7 and 21d. We examined the gene expression of aryl hydrocarbon receptor (AHR) and cytochrome P450 (CYP), as well as ethoxyresorufin-O-deethylase (EROD) activity in zebrafish livers. Exposure to different HBCD diastereoisomers caused different expression of AHRs in zebrafish livers. For instance, 10 and 100μgL(-1) of α- and β-HBCD up-regulated the expressions of ahr1a and ahr1b in zebrafish liver, whereas 10 and 100μgL(-)(1) of γ-HBCD down-regulated them after 7d exposure. α-HBCD showed the most significant up-regulation of ahr1a and ahr1b expression, whereas γ-HBCD showed the most significant down-regulation of their expression among three HBCD diastereoisomers. Moreover, HBCDs could affect the expression of CYP1s as well as EROD activity in a gene-specific and diastereoisomer-specific manner. α-, β- and γ-HBCD inhibited cyp1a expression but enhanced the expression of cyp1b1 and cyp1c1. α-, β- and γ-HBCD showed different degrees of effect on the same CYP1 gene in a concentration-dependent way. The different effects of HBCD diastereoisomers on these genes we examined and EROD activity not only indicate diastereoisomer-specific toxic effect, but also in turn explain diastereoisomer-specific accumulation of HBCDs in zebrafish.

The objective of the current study was to investigate the effect of vanadium (V(5+)) on Cyp1 expression and activity in C57BL/6 mice liver and isolated hepatocytes. For this purpose, C57BL6 mice were injected intraperitoneally with V(5+) (5 mg/kg) in the absence and presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (15 μg/kg) for 6 and 24 h. Furthermore, isolated hepatocytes from C57BL6 mice were treated with V(5+) (5, 10, and 20 μM) in the absence and presence of TCDD (1 nM) for 3, 6, 12, and 24 h. In vivo, V(5+) alone did not significantly alter Cyp1a1, Cyp1a2, or Cyp1b1 mRNA, protein, or catalytic activity levels. Upon co-exposure to V(5+) and TCDD, V(5+) significantly potentiated the TCDD-mediated induction of the Cyp1a1, Cyp1a2, and Cyp1b1 mRNA, protein, and catalytic activity levels at 24 h. In vitro, V(5+) decreased the TCDD-mediated induction of Cyp1a1 mRNA, protein, and catalytic activity levels. Furthermore, V(5+) significantly inhibited the TCDD-induced AhR-dependent luciferase activity. V(5+) also increased serum hemoglobin (Hb) levels in animals treated for 24 h. Upon treatment of isolated hepatocytes with Hb alone or in the presence of TCDD, there was an increase in the AhR-dependent luciferase activity. When isolated hepatocytes were treated for 2 h with V(5+) in the presence of TCDD, followed by replacement of the medium with new medium containing Hb, there was further potentiation to the TCDD-mediated effect. The present study demonstrates that there is a differential modulation of Cyp1a1 by V(5+) in C57BL/6 mice livers and isolated hepatocytes and demonstrates Hb as an in vivo specific modulator.