We studied comparative expression and activity of cytochrome P450 family 1 (CYP1) isoforms in rat embryo cells, both primary and immortalized by Rausher leukemia virus (RLV). In RLV-infected embryonal cells compared with the initial ones the expression levels of CYP1A1 and 1B1 mRNAs and benzo[a]pyrene (BP) hydroxylase activity were higher, regardless of their treatment with the CYP1 inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin. The sensitivity to BP and 7,12-dimethylbenzo[a]anthracene was higher in the cells immortalized with RLV. The expression level of mRNAs of induction-mediating proteins aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator was the same in both cell cultures tested. Higher sensitivity of cells immortalized with RLV compared with the initial embryo cells to transforming effect of BP, which was described previously, is possibly associated with elevated expression of CYP1 isoforms.

Environmental carcinogen benzo(a)pyrene (BaP) is a representative compound of polycyclic aromatic hydrocarbons (PAHs). BaP is strongly associated with prostate carcinogenesis. However, the molecular mechanism of BaP in development of prostate carcinoma remains largely unknown. The aim of this study was to investigate the effect and mechanism of BaP on the development in prostate cancer. PC-3 cells were exposed to different concentrations of BaP for 24, 48, 72 h, respectively. We analyzed the effect of BaP on PC-3 cell viability, cell cycle, DNA strand breaks, mutagenic activity, and migration. The expression of associated regulatory genes and the effect of JAK2/STAT3 signaling were also measured to explore the relationships among BaP metabolism, the JAK2/STAT3 pathway and proliferative activity in PC-3 cells. We observed significant effects on proliferation, DNA strand breaks and mutagenic activity after BaP exposure in PC-3 cells, and inhibitors of CYP1 and the AhR transcription factor α -naphthoflavone (ANF) and CH223191 treatment clearly reduced both cell survival and mutagenesis associated with BaP exposure. Reduction in G0-G1 phase population and elevation in S phase were observed after BaP exposure. Migratory cells for PC-3 were significantly increased. The results were further confirmed by the expression of mRNA levels in the significant increments of Snail, Slug, MMP-9, CYP1A1, CYP1B1, CycilnD1, CDK4 and significant reduction of E-cadherin. Significant enhancements were found in the expression of JAK2, STAT3 after BaP treatment. Additionally, activator IL-6 significantly enhanced the effect of BaP on cell survival, mutagenic activity, Cyclin D1, CDK4, Snail, and JAK2/STAT3 expression in PC-3 cells. Significant reductions in cell survival, mutagenic activity, Cyclin D1, CDK4, Snail, and JAK2/STAT3 expression were found after inhibitor AG490, ANF and CHJ223191 treatment. These findings reveal that BaP enhances the proliferative and mutagenic activity via JAK2-STAT3 pathway in PC-3 cells, and provide the additional evidence to understand the crucial role of BaP in prostate cancer carcinogenesis.

Keywords: Benzo(a)pyrene; JAK2/STAT3 pathway; Mutagenic activity; PC-3 cell; Proliferative activity.

ATP-binding cassette (ABC) transporters together with phase I and II detoxification enzymes have been considered as included in a cellular detoxification system. Previous studies have highlighted the involvement of aryl hydrocarbon receptor (AHR) and Cyp1a in PAH-induced embryo toxicity. However, the response of other xenobiotic enzymes/transporters in PAH-mediated embryo toxicity is not fully characterized. In the present study, rare minnow embryos were exposed to 10 and 100µg/L BaP within 4h post-fertilization (hpf) up to 168 hpf. RNA was extracted at 24, 48, 96, and 168 hpf. The basal and BaP-induced expression of phase I enzyme genes (cyp1a, 1b1, and 1c1), phase II enzyme gene (gstm and ugt1a), and ABC transporter genes (abcb1, abcc1, abcc2, and abcg2) mRNA was determined using real-time PCR. Severe developmental defects (e.g., spinal deformities, pericardial and yolk-sac edema) were observed in the BaP treated groups. The basal expression showed that gstm was most strongly expressed, followed by abcb1, ugt1a, and abcc2, whereas cyp1a, 1b1, 1c1, and abcg2 showed weak expression. BaP significantly induced the mRNA expression of three CYP1s (cyp1a, 1b1, and 1c1) (p<0.05) and the ABC transporters (abcc1, abcc2, and abcg2) in a dose-dependent manner. However, the mRNA expression of Phase II enzymes (gstm, ugt1a) for the BaP treatments showed no significant difference with that of the controls. Furthermore, distinct induced patterns of these genes were observed during different exposure periods. Simultaneous up-regulation of the cyp and ABC transporter gene transcripts suggests that a possible involvement and cooperation in the detoxification process could provide protection against the BaP toxicity of rare minnows at the early life stage.

Polybrominated diphenyl ethers (PBDEs) were once widely used as flame retardants in furniture and electronic products, and contamination persists in developing countries due to the dismantling of electronic waste. Our previous study confirmed that 2,2',4,4',5-pentabromodiphenyl ether (BDE-99) induced cytochrome P450 1A (Cyp1a) via aryl hydrocarbon receptor (Ahr)-mediated signaling in the zebrafish liver cell line (ZFL) in vitro. In this study, the toxicities of BDE-47 and BDE-99 at environmentally relevant concentrations (50 and 500 nM) were evaluated in newly hatched zebrafish (Danio rerio) larvae in vivo. A time-course study (8, 24, 48, and 96 h) was performed. BDE-99 was observed to cause yolk sac edema and pericardial edema after 72 h of exposure. Real-time polymerase chain reaction assay and whole-mount in situ hybridization assay confirmed cyp1a induction by BDE-99 in the liver and intestine. Continuous down-regulation of trβ by as much as 2.1-fold after 96 h and transient down-regulation of ttr by 7.1-fold after 24 h indicated the interference of BDE-99 in the thyroid hormone system. cyp1a induction was also observed in BDE-47-treated larvae, but cellular localization of cyp1a was not confirmed by whole-mount in situ hybridization. The induction of four cyp1 genes (cyp1a, cyp1b1, cyp1c1 and cyp1c2) by both BDE congeners warrants further study to understand the in vivo metabolism of BDE-47 and BDE-99 and the dioxin-like toxicity potencies of the OH-/MeO-PBDEs. The data obtained in this study will aid the characterization of molecular disorders caused by PBDEs in fish and help to delineate better models for toxicity assessment of environmental pollutants in ecological systems and in other vertebrates such as humans.

Polyhalogenated carbazoles (PHCZs), which have the similar structure of dioxin, have been reported ubiquitous in the environments and drawn wide concerns. However, their potential ecological and health risks are still poorly understood. Here, wildtype zebrafish embryos were used to evaluate the environmental risks of 2,7-dibromocarbazole (2,7-DBCZ), 3,6-dibromocarbazole (3,6-DBCZ), and 3,6-dichlorocarbazole (3,6-DCCZ). 2,7-DBCZ was the most toxic compound with the 96-h LC₅₀ value of 581.8 ± 29.3 μg·L^-1 and the EC₅₀ value of 201.5 ± 6.5 μg·L^-1 for pericardial edema. The teratogenic effects of 2,7-DBCZ were tested using transgenic zebrafish larvae. The transcriptomic analysis revealed that 90 genes in zebrafish expressed differently after exposure to 2,7-DBCZ, and many pathways were related to aryl hydrocarbon receptor (AhR) activation. The qRT-PCR also showed that expression levels of AhR1 and CYP1 A in zebrafish were significantly up-regulated after exposure to 2,7-DBCZ. In conclusion, 2,7-DBCZ exhibited more potent toxicity and cardiac teratogenic effects, and presented developmental toxicity partially consistent with AhR activation. Our results will be of great help to the risk assessment and regulation-making of PHCZs. Meanwhile, further studies should be promoted to illustrate the potential mechanism between PHCZs and AhR in the near future.

7,12-Dimethylbenzanthracene (DMBA), a polycyclic aromatic hydrocarbon, exhibits mutagenic, carcinogenic, immunosuppressive, and apoptogenic properties in various cell types. To achieve these functions effectively, DMBA is modified to its active form by cytochrome P450 1 (CYP1). Exposure to DMBA causes cytotoxicitymediated apoptosis in bone marrow B cells and ovarian cells. Although uterine endometrium constitutively expresses CYP1A1 and CYP1B1, their apoptotic role after exposure to DMBA remains to be elucidated. Therefore, we chose RL95-2 endometrial cancer cells as a model system for studying DMBA-induced cytotoxicity and cell death and hypothesized that exposure to DMBA causes apoptosis in this cell type following CYP1A1 and/or CYP1B1 activation. We showed that DMBA-induced apoptosis in RL95-2 cells is associated with activation of caspases. In addition, mitochondrial changes, including decrease in mitochondrial potential and release of mitochondrial cytochrome c into the cytosol, support the hypothesis that a mitochondrial pathway is involved in DMBA-induced apoptosis. Exposure to DMBA upregulated the expression of AhR, Arnt, CYP1A1, and CYP1B1 significantly; this may be necessary for the conversion of DMBA to DMBA-3,4-diol-1,2-epoxide (DMBA-DE). Although both CYP1A1 and CYP1B1 were significantly upregulated by DMBA, only CYP1B1 exhibited activity. Moreover, knockdown of CYP1B1 abolished DMBA-induced apoptosis in RL95-2 cells. Our data show that RL95-2 cells are susceptible to apoptosis by exposure to DMBA and that CYP1B1 plays a pivotal role in DMBA-induced apoptosis in this system.

The expression and inducibility of cytochrome P4501A (CYP1A) were investigated in hepatic microsomes of ringed seals (Phoca hispida) and grey seals (Halichoerus grypus) from polluted (Baltic Sea) and less polluted (Svalbard and Sable Island) areas. Liver CYP1A protein levels and activities were assessed by immunoblot analysis and determining catalytic activities towards ethoxyresorufin (EROD) and pentoxyresorufin (PROD). The amount of CYP1A apoprotein and its catalytic activity were increased up to 5-fold in Baltic ringed and grey seal populations in comparison with ringed seals from Svalbard and grey seals from Sable Island. EROD and PROD activities correlated in all seal groups, indicating catalysis by the same CYP form(s). Enzyme kinetic studies suggested that PROD activity is catalysed by CYP1A enzymes in both ringed and grey seals. In immunoblot analysis, a protein was revealed in liver with an antibody against human CYP1B1, indicating that a CYP1B1 like protein could be present in ringed and grey seals. In conclusion, these data strengthen the concept that CYP1A is markedly induced in seals living in polluted waters and that both EROD and PROD activities are mediated by CYP1A forms in the seal liver. In addition, this study provides the first evidence for the presence of a CYP1B like protein in seal liver.

A straightforward methodology, based on first principles, for the estimation of human cytochrome P450-substrate binding energies is outlined, and the system has then been applied successfully to a relatively large dataset of P450 substrates totalling 90 compounds. The results of Quantitative Structure-Activity Relationship (QSAR) analysis on the same dataset of cytochrome P450 (CYP) substrates from the CYP1, CYP2, and CYP3 families, involving a total of 90 compounds, agree favourably with the original analysis based on first principles, thus confirming the use of average values for hydrogen bond and pi-pi stacking energies, together with utilizing log P values as an estimation of desolvation energies. This method is based on a linear summation of the various contributary factors to the process, including: desolvation, hydrogen bonding, pi-pi stacking, restricted bond rotation and other energies relating to loss in translational and rotational energy. It is found that, for the majority of P450 substrates investigated, the first four terms are required for a relatively good estimation (R = 0.98) of the substrate binding affinity (deltaGbind) towards CYP1 and CYP2 enzymes. Consequently, it would appear that the loss in rotational and translational energy, which is thought to occur on substrate binding, apparently has little effect in most cases, possibly due to some degree of residual motion of the enzyme-substrate complex within the endoplasmic reticulum membrane. However, the appearance of a small constant term in the QSAR equation could possibly relate to an average loss in translational and rotational energy for the 90 compounds studied in this investigation.

1. Primates exhibit a high degree of among-species dietary diversity, which likely exposes them to varying levels of xenobiotic compounds. Here, we examined the evolution of primate CYP1-3 gene families, and we classified the 15 CYP1-3 gene subfamilies as either xenobiotic-metabolizing (XM) or endogenous-metabolizing (EM) based on sources in the P450 literature. 2. We predicted that XM P450s would show (1) greater variability in gene-copy number and (2) more evidence of diversifying selection and, especially on codons that encode the substrate-recognition sites (SRSs) for the final enzymes.3. Counter to our first prediction, EM and XM P450s showed similar levels of variation in gene-copy number. We did find, however, that four XM P450 subfamilies (CYP2C, CYP2D, CYP2E, and CYP3A) showed evidence of diversifying selection while no EM subfamilies demonstrated any consistent signal of diversifying selection. Of these four, CYP2C, CYP2D, and CYP3A showed significant links between SRSs and diversifying selection.4. These results reveal an amount of evolutionary dynamism that would not be expected when viewing P450 subfamilies along a simple binary EM/XM spectrum. We recommend that comparative studies of cytochrome P450 evolution should focus on the CYP2C, CYP2D, CYP2E, and CYP3A subfamilies.

Keywords: Primates; cytochrome P450; diversifying selection; positive selection.

Several cytochrome P450s have been identified in guinea pig adrenal microsomes which are distinct from the known steroidogenic P450s, c17 and c21, and are immunochemically related to cytochrome P450s found in liver. One, a 52 K protein related to P450 I (CYP1), occurs almost exclusively in males, is localized to the inner zone, and is suppressed by ACTH. Its levels correlate with microsomal capacity for xenobiotic metabolism. The others, related to P450s II and III (CYP2 and 3), are more predominant in males, but not exclusive to them, are found in both the inner and outer zones, and are not suppressed by ACTH. Their functions remain to be elucidated. The male predominance of the CYP1-related protein has recently been shown to be due to suppression of the protein in females by estrogen. To determine if estrogen is also involved in the regulation of the CYP2-related proteins, ovariectomized and sham-operated animals were treated with a long-acting estrogen, estradiol valerate, or with the vehicle alone. These P450s reached male levels in ovariectomized females treated only with the vehicle. Their enhanced levels were suppressed by treatment with estrogen. Estrogen treatment also suppressed the levels of the P450s seen in sham-operated females. Endogenous estrogen produced similar effects. In hemi-ovariectomized females the contralateral ovary hypertrophied, a state in which estrogen levels would be maintained or increased. In these females no increase occurred in the immunodetectable P450s. In normal females, estrogen levels are low in prepubertal animals, rise at the time of puberty and drop again after ovarian cycling is completed. The CYP2-related proteins were present in adrenal microsomes of prepubertal females, but were suppressed after puberty. On the other hand, post-estrous females, in whom estrogen levels would be low, acquired male levels of these proteins in their adrenal microsomes. P450c17 and P450c21, as well as 3β-hydroxysteroid dehydrogenase, were not affected by surgery or estrogen. Taken together, these experiments indicate that suppression by estrogen in females can account, in large part, for the predominance of several immunochemical homologs of liver P450s in adult male guinea pig adrenals.

Drosophila cyclophilin 1 (Cyp1) is a structural and functional homolog of mammalian cyclophilin D (CypD), a unique mitochondrial cyclophilin (Cyp) that regulates the inner mitochondrial membrane permeability transition and cell survival under cellular stresses such as oxidative damage. In this study, we generated and characterized a Drosophila Cyp1 mutant. Cyp1 mutant flies successfully developed into adults and showed no significant defects in mitochondrial morphology, function, and content. However, oxidative damage significantly decreased in Cyp1 mutant flies, and inhibition of Cyp1 expression substantially increased the survival under various oxidative stress paradigms. Moreover, Cyp1 mutation successfully ameliorated survival rates, locomotor activity, and dopaminergic neuron quantity in a Drosophila DJ-1 mutant under oxidative stress, further confirming the protective role of Cyp1 mutation against oxidative stress. In conclusion, these results suggest Cyp1 and its human homolog CypD as putative molecular targets for the treatment of DJ-1 deficiency-associated diseases, including Parkinson's disease.

The principal objectives of this study were to evaluate whether exposure of rats to low doses of isothiocyanates modulates the overall metabolism of heterocyclic amine 2-amino-3-methylimidazo-[4,5-f]quinoline (IQ), as exemplified by urinary mutagenicity, a food carcinogen, and to relate any modifications in metabolism to changes in CYP1 and glutathione S-transferase activities. Animals were exposed to isothiocyanates either for 2 wk (long-term) or 1 day (short-term), and all animals were then treated with a single oral dose of IQ, and urine was collected daily for 3 days; animals continued to receive the isothiocyanates during this period. Urinary mutagenic activity was determined using the Ames mutagenicity assay in the presence of an activation system from Aroclor 1254-treated rats. At the end of the study, animals were killed and hepatic methoxy- and ethoxyresorufin dealkylations were determined as well as glutathione S-transferase activity. All isothiocyanates studied, namely sulforaphane, erucin, and phenethyl isothiocyanate, decreased urinary mutagenic activity, implying enhanced IQ metabolism, but only after long-term intake. Changes in mutagenic activity were not related to changes of any of the enzyme activities determined. It is concluded that long-term intake of isothiocyanates may stimulate the metabolism of IQ, but this effect is not linked to changes in hepatic CYP1A2 and glutathione S-transferase activities.

The cytotoxicity of the carbamate insecticide carbaryl, the organophosphate insecticide quinalphos, the benzimidazole fungicide benomyl, and its debutylcarbamoylated derivative carbendazim was investigated in rat (Fa32) and human (Hep G2) hepatoma-derived established cell lines. Benomyl was the most toxic of the four pesticides, followed by quinalphos, carbaryl and the least toxic carbendazim, suggesting that the butylcarbamoyl group plays an important role in the toxicity of benomyl. EROD (7-ethoxyresorufin-O-deethylase) and PROD (7-pentoxyresorufin-O-depentylase) activity were moderately activated in Fa32 cells. In Hep G2 cells no PROD was measured, but EROD was activated 2.5 to 28 times the control values. Piperonyl butoxide and 1-aminobenzotriazole did not influence the cytotoxicity of the pesticides. However, when the endogenous glutathione content was reduced by pretreatment of the cells with l-buthionine-S,R-sulfoximine, the cytotoxicity of the pesticides strongly increased in both cell lines. In conclusion, the same cytotoxicity was observed for the four pesticides in both the animal and the human cell line. CYP1/2-dependent enzymes were activated to different degrees. No evidence was obtained for a cytochrome P450-dependent cytotoxicity, but glutathione showed a protective effect against the four pesticides in both cell lines.

The protoberberine alkaloid berberine carries methylenedioxy moiety and exerts a variety of pharmacological effects, such as anti-inflammation and lipid-lowering effects. Berberine causes potent CYP1B1 inhibition, whereas CYP1A2 shows resistance to the inhibition. To reveal the influence of oxidative metabolism on CYP1 inhibition by berberine, berberine oxidation and the metabolite-mediated inhibition were determined. After NADPH-fortified preincubation of berberine with P450, the inhibition of CYP1A1 and CYP1B1 variants (CYP1B1.1, CYP1B1.3, and CYP1B1.4) by berberine was not enhanced, and CYP1A2 remained resistant. Demethyleneberberine was identified as the most abundant metabolite of CYP1A1- and CYP1B1-catalyzed oxidations, and thalifendine was generated at a relatively low rate. CYP1A1-catalyzed berberine oxidation had the highest maximal velocity (V max) and exhibited positive cooperativity, suggesting the assistance of substrate binding when the first substrate was present. In contrast, the demethylenation by CYP1B1 showed the property of substrate inhibition. CYP1B1-catalyzed berberine oxidation had low K m values, but it had V max values less than 8% of those of CYP1A1. The dissociation constants generated from the binding spectrum and fluorescence quenching suggested that the low K m values of CYP1B1-catalyzed oxidation might include more than the rate constants describing berberine binding. The natural protoberberine/berberine fmetabolites with methylenedioxy ring-opening (palmatine, jatrorrhizine, and demethyleneberberine) and the demethylation (thalifendine and berberrubine) caused weak CYP1 inhibition. These results demonstrated that berberine was not efficiently oxidized by CYP1B1, and metabolism-dependent irreversible inactivation was minimal. Metabolites of berberine caused a relatively weak inhibition of CYP1.

Methylation of the non-carcinogen 15,16-dihydrocyclopenta[a]phenanthren-17-one (CPP-17-one) at the bay region to form 11-CH3-CPP-17-one confers carcinogenic potential. In the present study we have investigated the in vitro metabolism and mutagenicity of the methylated compound by hepatic microsomal preparations from rats pretreated with various prototype inducers of cytochrome P450 proteins in order to provide a rationale for this marked difference in carcinogenic activity. The most effective metabolism of 11-CH3-CPP-17-one occurred in the presence of Aroclor 1254-induced microsomes, the principal metabolites being oxidative products of the A- and D-rings and of the methyl substituent. When benzo[a]pyrene-induced microsomes served as the metabolising system, the major A-ring metabolite was the 3,4-diol. A similar metabolic pattern was seen with microsomes from rats treated with 11-CH3-CPP-one itself, but the overall effect of metabolism was lower than that observed with benzo[a]pyrene-treated microsomes but higher than that of control animals. In contrast, microsomes from rats treated with clofibrate, dexamethasone, isoniazid and phenobarbitone failed to enhance the metabolism of 11-CH3-CPP-17-one when compared with control microsomes and the metabolites reflected primarily oxidation of the D-ring. When 11-CH3-CPP-17-one was employed as a promutagen in the Ames test, a mutagenic response was evident only in the presence of microsomes from benzo[a]pyrene-induced rats, but induction with phenobarbitone, isoniazid, dexamethasone, clofibrate and the compound itself, failed to elicit a positive mutagenic response. When 3,4-dihydroxy-11-CH3-CPP-17-one served as the promutagen, a mutagenic response was observed in the presence of benzo[a]pyrene-induced and, to a lesser extent, 11-CH3-CPP-17-one-induced microsomes. Treatment of rats with 11-CH3-CPP-17-one caused a marked increase in the O-deethylation of ethoxyresorufin and, to a much lesser extent in epoxide hydrolase activity. It is concluded that (i) 11-CH3CPP-17-one is an inducer of the CYP1 family; (ii) under the present experimental conditions only the CYP1 family can oxidise the A-ring to form the 3,4-dihydroxy-11-CH3-CPP-17-one, the precursor of the ultimate carcinogen and (iii) only the CYP1 family oxidizes the diol to generate the ultimate carcinogen.(ABSTRACT TRUNCATED AT 400 WORDS)

A novel actinomycete strain CYP1-5^T was isolated from the mountain soil sample collected from Chaiyaphum province, Thailand and its taxonomic position was clarified by using a polyphasic taxonomic approach. The chemotaxonomic properties of strain CYP1-5^T were consistent within the genus Actinomadura. Cell-wall peptidoglycan of this strain contained meso-diaminopimelic acid. Galactose, madurose, and ribose were presented as the diagnostic sugars in whole-cell hydrolysates. The major menaquinone was MK-9(H₆). Major cellular fatty acids were iso-C_16:0 and C_16:0. Phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, and phosphatidylinositol mannoside were observed as predominant phospholipids. Based on the results of phylogenetic analyses of 16S rRNA gene sequence, strain CYP1-5^T was constituent with the genus Actinomadura and was closely related to Actinomadura syzygii GKU157^T (99.5%) and Actinomadura chibensis IFM 10266^T (= JCM 14158^T) (98.2%). The draft genome size of strain CYP1-5^T was 9.30 Mb with 72.2 mol% of G + C content. Strain CYP1-5^T showed ANIb values of 94.9% with A. syzygii GKU157^T and 93.2% with A. chibensis JCM 14158^T. Phenotypic characteristics, phylogenetic analysis and genome data support that strain CYP1-5^T could be discriminated from its closest relatives, representing a novel species of the genus Actinomadura, for which the name Actinomadura decatromicini sp. nov. is proposed. The type strain is CYP1-5^T (= JCM 16996^T = KCTC 19916^T = TISTR 2901^T).

The taxonomic position of the mountain soil actinomycete, strain CYP1-1BT, was clarified by a polyphasic study. The strain produced a single spore, or occasionally a chain of spores, on aerial mycelium. Chemotaxonomic data supported the classification of CYP1-1BT as representing a member of the genus Actinomadura on the basis of the presence of meso-diaminopimelic acid in the peptidoglycan; galactose, glucose, madurose and ribose as whole cell sugars; MK-9(H6), MK-9(H8) and MK-9(H4) as dominant menaquinones; C16 : 0, 10-methylated C18 : 0 and C18 : 1ω9c as the major cellular fatty acids; and diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositol manosides as the predominant phospholipids. The DNA G+C content was 74.3 mol%. On the basis of the combination of morphological and chemotaxonomic characteristics, CYP1-1BT was identified as representing a member of the genus Actinomadura. On the basis of the results of 16S rRNA gene analysis, CYP1-1BT, was shown to be closely related to Actinomadura nitritigenes DSM 44137T (98.9 %). Phenotypic, genotypic and DNA-DNA hybridization data supported the hypothesis that CYP1-1BT represents a novel species of the genus Actinomadura for which the name Actinomadura montaniterrae sp. nov. is proposed. The type strain is CYP1-1BT (=JCM 16995T=KCTC 39784T=PCU 349T=TISTR 2400T).

Oral treatment with alpha-tocopherol for 4 days dose-dependently increased the content of cytochrome P450 (CYP), catalytic activities of CYP1A1, CYP1A2, CYP2B1, CYP2C, and activity of NADPH-cytochrome-P450 reductase in the liver of male rats, but did not change activity of glutathione S-transferase. These results suggest that alpha-tocopherol induced the enzymes of phase I of xenobiotic metabolism, including CYP1 and CYP2 families involved in the metabolism of drugs and procarcinogenes.

Frequent recreational use of Anabolic Androgenic Steroids (AAS) is an instance of substance abuse which mimics the status of a natural hormone and upon prolonged exposure may lead to adverse drug reactions. These adverse drug reactions proceed in a manner so as to alter the normal metabolism of an enzyme mediated pathway such as the Cytochrome P450 (CYP) family of enzymes.

The present study was conducted to investigate the impact of overuse of Nandrolone Decanoate (ND), an AAS, upon CYP enzyme activity and a CYP gene, belonging to CYP1 family.

The study was carried out using normal and ND treated male albino mice. Genetic analysis was conducted using normalized and treated cDNA and reverse transcriptase polymerase chain reaction based assays. For enzyme assay, 0.1ml of 25 mg ND was administered to the animals twice a week for a period of 90 days. Genetic analysis was carried out with the same dose but administered for a period of 360 days.

CYP enzyme activity increased significantly (p<0.01) in the ND treated group of animals compared to that in the normal group. However, no noticeable alteration was observed at the molecular level.

From the present study it could be inferred that, at elevated doses, ND has the potential to alter hepatic CYP enzyme activity without any modification in the CYP gene. This could be due to a possible adaptive response of the living system to such drugs.

Polycyclic aromatic hydrocarbons (PAHs) are carcinogenic compounds which are emitted through incomplete combustion of organic materials, fossil fuels, consumption of processed meat, smoked food, and from various industrial activities. High molecular mass and mobility make PAHs widespread and lethal for human health. A cellular system in human detoxifies these toxicants through specialized enzymatic machinery called xenobiotic-metabolizing (CYP450) and phase-II (GSTs) enzymes (XMEs). These metabolizing enzymes include cytochromes P450 family (CYP1, CYP2), glutathione s-transferases, and ALDHs. Gene polymorphisms in XMEs encoding genes can compromise their metabolizing capacity to detoxify ingested carcinogens (PAHs etc.) that may lead to prolong and elevated exposure to ingested toxicants and may consequently lead to cancer. Moreover, PAHs can induce cancer through reprograming XMEs' gene functions by altering their epigenetic markers. This review article discusses possible interplay between individual's gene polymorphism in XMEs' genes, their altered epigenetic markers, and exposure to PAHs in cancer susceptibility in Pakistan.

Keywords: ALDH; CYPP450; Carcinogenic; GSTM 1; PAHs; Polymorphism.

Consumption of cooked/processed meat and ethanol are lifestyle risk factors in the aetiology of breast cancer. Cooking meat generates heterocyclic amines such as 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Epidemiology, mechanistic and animal studies indicate that PhIP is a mammary carcinogen that could be causally linked to breast cancer incidence; PhIP is DNA damaging, mutagenic and oestrogenic. PhIP toxicity involves cytochrome P450 (CYP1 family)-mediated metabolic activation to DNA-damaging species, and transcriptional responses through Aryl hydrocarbon receptor (AhR) and estrogen-receptor-α (ER-α). Ethanol consumption is a modifiable lifestyle factor strongly associated with breast cancer risk. Ethanol toxicity involves alcohol dehydrogenase metabolism to reactive acetaldehyde, and is also a substrate for CYP2E1, which when uncoupled generates reactive oxygen species (ROS) and DNA damage. Here, using human mammary cells that differ in estrogen-receptor status, we explore genotoxicity of PhIP and ethanol and mechanisms behind this toxicity. Treatment with PhIP (10-7-10-4 M) significantly induced genotoxicity (micronuclei formation) preferentially in ER-α positive human mammary cell lines (MCF-7, ER-α+) compared to MDA-MB-231 (ER-α-) cells. PhIP-induced CYP1A2 in both cell lines but CYP1B1 was selectively induced in ER-α(+) cells. ER-α inhibition in MCF-7 cells attenuated PhIP-mediated micronuclei formation and CYP1B1 induction. PhIP-induced CYP2E1 and ROS via ER-α-STAT-3 pathway, but only in ER-α (+) MCF-7 cells. Importantly, simultaneous treatments of physiological concentrations ethanol (10-3-10-1 M) with PhIP (10-7-10-4 M) increased oxidative stress and genotoxicity in MCF-7 cells, compared to the individual chemicals. Collectively, these data offer a mechanistic basis for the increased risk of breast cancer associated with dietary cooked meat and ethanol lifestyle choices.

OBJECTIVE

The effects of miroestrol (MR), an active phytoestrogen from Pueraria candollei var. mirifica, on expression of cancer-related genes were determined.

METHODS

Seven-week-old female ICR mice (n = 5 each) were subcutaneously administered estradiol (E2, 0.5 mg/kg/day) or MR (0.5 or 5 mg/kg/day) daily for 7 days. Some were given ER or MR in combination with β-naphthoflavone (BNF, 30 mg/kg/day) for the last 3 days. The expression of cancer-related genes including cytochrome P450 1A (Cyp1a), cytochrome P450 1B1 (Cyp1b1), aromatase P450 (Cyp19), NAD(P)H: quinone oxidoreductase 1 (Nqo1) and catechol-O-methyltransferase (Comt) were evaluated.

RESULTS

In the presence of BNF, MR suppressed hepatic <em>CYP1</em>A1 activity and <em>CYP1</em>A2 activity, expression of <em>CYP1</em>B1 mRNA and expression of <em>CYP1</em>A1/2 and <em>CYP1</em>B1 protein. E2, by contrast, did not. MR restored expression levels of hepatic NQO1 and uterine COMT in BNF-treated mice. Furthermore, MR increased expression of uterine <em>CYP1</em>9 to the same extent as E2.

CONCLUSIONS

MR may be superior to E2 as it downregulates expression of CYP1. Moreover, MR normalized expression of both NQO1 and COMT, the protective enzymes, in murine liver and uteri. These results support the use of MR as an alternative supplement for menopausal women, MR having the extra benefit of reducing cancer risk.

CYP1A1 is thought to mediate carcinogenesis in oral, lung and epithelial cancers. In order to identify a CYP1A1 inhibitor from an edible plant, 394 natural products in the IIIM's natural product repository were screened, at 10 μM concentration, using CYP1A1-Sacchrosomes™ (i.e. microsomal enzyme isolated from recombinant baker's yeast). Twenty-seven natural products were identified that inhibited 40-97% of CYP1A1's 7-ethoxyresorufin-O-deethylase activity. The IC50 values of the 'hits', belonging to different chemical scaffolds, were determined. Their selectivity was studied against a panel of 8 CYP-Sacchrosomes™. In order to assess cellular efficacy, the 'hits' were screened for their capability to inhibit CYP enzymes expressed within live recombinant human embryonic kidney (HEK293) cells from plasmids encoding specific CYP genes (1A2, 1B1, 2C9, 2C19, 2D6, 3A4). Isopimpinellin (IN-475; IC50, 20 nM) and karanjin (IN-195; IC50, 30 nM) showed the most potent inhibition of CYP1A1 in human cells. Isopimpinellin is found in celery, parsnip, fruits and in the rind and pulp of limes whereas different parts of the Indian beech tree, which contain karanjin, have been used in traditional medicine. Both isopimpinellin and karanjin negate the cellular toxicity of CYP1A1-mediated benzo[a]pyrene. Molecular docking and molecular dynamic simulations with CYP isoforms rationalize the observed trends in the potency and selectivity of isopimpinellin and karanjin.