In vitro assessment of inhibition by natural polyphenols of metabolic activation of procarcinogens by human CYP1A1

Previously, inhibitors of CYP1A1 were rated as candidate chemopreventive agents against cancer mainly according to their effects on the 7-ethoxyresorufin O-deethylation (EROD) of diagnostic probe substrates. Surprisingly, several polyphenols including resveratrol, formerly identified as potent inhibitors by the EROD assay, exhibited no or weak inhibition of procarcinogen activation. We compared the effects of 11 representative natural polyphenols, which normally occur in food, on different activities of CYP1A1, namely epoxidation of 7,8-dihydrodiol-benzo[a]pyrene, the terminal step in the activation leading to the ultimate carcinogenic diolepoxides, hydroxylation of benzo[a]pyrene, and EROD. For the first time, a reconstituted system was used for the determination of IC(50) values, consisting of purified enzymes (human CYP1A1 and human NADPH-cytochrome P450 reductase) and dilaurylphosphatidylcholine. The results demonstrate that the inhibitory effects of dietary polyphenols on CYP1A1 activity depend on both the structure of the inhibitor and the type of the reaction and substrate used in the assay. Consequently, a potent EROD inhibition alone is insufficient to count a substance among the chemoprotective agents.     

Constitutive and inducible expression of cytochromes P4501A (CYP1A1 and CYP1A2) in normal prostate and prostate cancer cells

Constitutive and benzo[a]pyrene (B[a]P) inducible expression of CYP1A1 and CYP1A2 in prostate cancer and normal prostate epithelial cells were examined by immunoblotting. Androgen independent prostate cancer cell lines DU145 and PC3 have constitutive expression of CYP1A and CYP1A1 and CYP1A2, respectively. Four micromolar B[a]P did not appear to induce CYP1A1 or CYP1A2 expression in DU145 or PC3 cells. The androgen dependent prostate cancer cell line, LnCap, also has constitutive expression of CYP1A1 and CYP1A2. However, both CYP1A1 and CYP1A2 are induced by treatment of LnCap cells with 4 microM B[a]P. Untreated normal prostate and primary prostate tumor cells have no detectable CYP1A1 expression. Treatment with 4 microM B[a]P induced CYP1A1 expression in both normal and primary tumor prostate cells. Constitutive CYP1A2 expression was detected in normal prostate cells with little or no induction by exposure to 4 microM B[a]P. Primary prostate tumor cells did not show constitutive expression of CYP1A2. However, CYP1A2 was induced by 4 microM B[a]P in primary prostate tumor cells. These observations indicate that hormonal and cancer specific factors affect the expression and induction of the phase I metabolic enzymes, CYP1A1 and CYP1A2 in prostate cells. These observations may be related to the potential smoking-linked higher risk of prostate cancer development and morbidity of prostate cancer patients who smoke.     

Potency of various polycyclic aromatic hydrocarbons as inducers of CYP1A1 in rat hepatocyte cultures

A number of highly toxic environmental pollutants including certain polychlorinated dibenzo-p-dioxins (PCDD), polychlorinated dibenzofurans (PCDF), and 'dioxin-like' polychlorinated biphenyls (PCB) are among the most potent agonists of the aryl hydrocarbon receptor (AHR). Induction of cytochrome P4501A1 (CYP1A1) in mammalian cell culture is widely used as a functional parameter for AHR activation providing an estimate for 'dioxin-like' inducing equivalents in extracts from environmental samples. Since a number of polycyclic aromatic hydrocarbons (PAHs) also act as AHR-agonists, the CYP1A1-inducing potencies, measured as induction of 7-ethoxyresorufin O-deethylase (EROD) activity in rat hepatocyte cultures were analyzed for 16 PAHs frequently present in environmental samples. Among these, seven PAHs including benzo[a]pyrene were relatively potent inducers allowing the determination of Induction Equivalency Factors (IEF). For three PAHs including benzo[k]fluoranthene which acted as weak inducers, IEFs were estimated, while six PAHs including acenaphthylene were classified as inactive. Based on different efficacies the concentration-response characteristics of CYP1A1 induction were analyzed in more detail for benzo[a]pyrene, benzo[k]fluoranthene, and acenaphthylene. Benzo[k]fluoranthene was markedly less effective than benzo[a]pyrene as inducer of EROD activity but even more effective than benzo[a]pyrene as inducer of CYP1A1 protein and mRNA. Acenaphthylene was highly more effective on the level of mRNA than on the levels of protein or EROD activity. Further analysis revealed that the low efficacy of acenaphthylene as inducer of CYP1A1 protein and EROD activity is due to its marked cytotoxicity while no clear-cut explanation was found for the differences in efficacy between benzo[k]fluoranthene and benzo[a]pyrene. The EROD-inducing potency of a mixture of 16 PAH was about 2-fold higher than that calculated on the basis of IEFs of the individual constituents of the mixture.     

CYP330A1 and CYP4C39 enzymes in the shore crab Carcinus maenas: sequence and expression regulation by ecdysteroids and xenobiotics

Cytochrome P450 enzymes (CYP enzymes) catalyse important metabolic reactions of exogenous and endogenous substrates, including steroid hormones. Here, we report the first two CYP sequences from the shore crab, Carcinus maenas. Two complete cDNAs isolated from crab hepatopancreas encode CYP enzymes named CYP330A1, the first member of a new family, and CYP4C39. CYP330A1 is closest related to members of the CYP2 family (37.3% identical to mouse CYP2J6) and CYP4C39 is most identical to crayfish CYP4C15 (59.5%). CYP330A1 gene expression was induced in hepatopancreas of male green intermoult crabs by ecdysone and ponasterone A, but also by benzo(a)pyrene and phenobarbital. CYP330A1 induction was not observed in red crabs. The present results indicate that the CYP330A1 enzyme may be involved in ecdysteroid metabolism, presumably catabolism, and in the detoxification of environmental pollutants. Ecdysteroids or xenobiotics did not affect CYP4C39 gene expression. The fact that both ecdysteroids and xenobiotics affect CYP330A1 gene expression indicates that mutual interactions between chemical exposures and endocrine functions may exist in the shore crab.     

Modulation of rat pulmonary carcinogen-metabolising enzyme systems by the isothiocyanates erucin and sulforaphane

The objective of this study was to evaluate the potential of the structurally related aliphatic isothiocyanates erucin and sulforaphane to modulate the pulmonary carcinogen-metabolising enzyme systems in rat lung, a target organ of their chemopreventive activity. Precision-cut rat lung slices were prepared and incubated for 24 h with a range of concentrations of either erucin or sulforaphane, up to 50 μM. Neither compound modulated the O-deethylation of ethoxyresorufin whereas they elevated markedly CYP1A1 and, to a lesser extent, CYP1B1 apoprotein levels. Neither compound influenced the O-depentylation of pentoxyresorufin or CYP2B apoprotein levels, but sulforaphane caused a modest increase in CYP3A2 apoprotein levels. Pulmonary quinone reductase activity, monitored using 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide as substrate, was markedly up-regulated by both compounds and was paralleled by a similar rise in protein levels. Both compounds increased cytosolic glutathione S-transferase activity, measured using 1-chloro-2,4-dinitrobenzene as the accepting substrate; a modest rise was seen in GSTα protein levels, determined immunologically, whereas GSTπ levels were un-affected by the same treatment. Finally, both erucin and sulforaphane increased total glutathione concentration in lung cytosol. It is concluded that these aliphatic isothiocyanates have the potential to antagonise the carcinogenicity of pulmonary carcinogens by stimulating the in situ detoxication of their DNA-binding genotoxic metabolites.     

Characterisation of two novel CYP4 genes from the marine polychaete Nereis virens and their involvement in pyrene hydroxylase activity

Cytochrome P450 enzymes (CYP enzymes) catalyse the initial step in biotransformation of xenobiotics like polycyclic aromatic hydrocarbons (PAHs). The marine polychaete Nereis virens has a high capacity for biotransformation of PAHs. In the present study, the complete cDNA sequences of two novel CYP genes isolated from N. virens gut tissue are reported. One named CYP342A1, the first member of a new family and the other named CYP4BB1, the first member of a new subfamily. This is the first investigation of specific CYP enzymes from marine polychaetes in which catalytic activity has been determined. Both CYP enzymes had monooxygenase activity and catalysed hydroxylation of pyrene to 1-hydroxypyrene. Based on the present results it is likely that both CYP4BB1 and CYP342A1 are involved in xenobiotic biotransformation. Furthermore, site-directed mutagenesis of the conserved cysteine residue of the heme binding domain resulted in complete loss of monooxygenase activity of both CYP enzymes, indicating that this cysteine residue is indispensable for monooxygenase activity of invertebrate CYP enzymes, as has been well documented in vertebrates. Considering the important role of CYP enzymes in biotransformation of xenobiotics and the presence of N. virens in estuarine environments that accumulates organic xenobiotics, our results are important in understanding the molecular mechanism of biotransformation in marine polychaetes.     

Furanoflavones pongapin and lanceolatin B blocks the cell cycle and induce senescence in CYP1A1-overexpressing breast cancer cells

Expression of cytochrome P450-1A1 (CYP1A1) is suppressed under physiologic conditions but is induced (a) by polycyclic aromatic hydrocarbons (PAHs) which can be metabolized by CYP1A1 to carcinogens, and (b) in majority of breast cancers. Hence, phytochemicals or dietary flavonoids, if identified as CYP1A1 inhibitors, may help in preventing PAH-mediated carcinogenesis and breast cancer. Herein, we have investigated the cancer chemopreventive potential of a flavonoid-rich Indian medicinal plant, Pongamia pinnata (L.) Pierre. Methanolic extract of its seeds inhibits CYP1A1 in CYP1A1-overexpressing normal human HEK293 cells, with IC₅₀ of 0.6?µg/mL. Its secondary metabolites, the furanoflavonoids pongapin/lanceolatin B, inhibit CYP1A1 with IC₅₀ of 20?nM. Although the furanochalcone pongamol inhibits CYP1A1 with IC₅₀ of only 4.4?µM, a semisynthetic pyrazole-derivative P5b, has ～10-fold improved potency (IC₅₀, 0.49?μM). Pongapin/lanceolatin B and the methanolic extract of P. pinnata seeds protect CYP1A1-overexpressing HEK293 cells from B[a]P-mediated toxicity. Remarkably, they also block the cell cycle of CYP1A1-overexpressing MCF-7 breast cancer cells, at the G₀-G₁ phase, repress cyclin D1 levels and induce cellular-senescence. Molecular modeling studies demonstrate the interaction pattern of pongapin/lanceolatin B with CYP1A1. The results strongly indicate the potential of methanolic seed-extract and pongapin/lanceolatin B for further development as cancer chemopreventive agents.     

Aldrin Epoxidation in Flathead Mullet (Mugil cephalus): Possible Involvement of CYP1A and CYP3A

The primary objective of this study was to determine specific cytochrome P450 isozyme(s) involved in the metabolism of aldrin to its toxic metabolite dieldrin in flathead mullet (Mugil cephalus) liver microsomes. To identify the cytochrome P450 isozyme responsible for the aldrin metabolism in mullet liver, the effects of mammalian‐specific cytochrome P450 inhibitors and substrates were determined in the epoxidation reaction of aldrin. CYP3A‐related inhibitors, ketoconazole, SKF‐525A, and cimetidine, inhibited the metabolism of aldrin. The contribution of CYP1A to the aldrin metabolism was shown by the inhibition of 7‐ethoxyresorufin‐O‐deethylase activity in the presence of aldrin. The results indicate that CY1A and CYP3A are the cytochrome P450s involved in aldrin epoxidase activity in mullet. In addition, the suitability of aldrin epoxidase activity for monitoring of environmental pollution was also assessed in the fish samples caught from four different locations of the West Black Sea coast of Turkey.     

CYP2D6 and CYP1A1 mutations in the Turkish population

Drugs and carcinogens are substrates of a group of metabolic enzymes including cytochrome p450 enzymes and gluthatione S-transferases. Many of the genes encoding these enzymes exhibit functional polymorphisms that contribute individual cancer susceptibility and drug response. Molecular studies based on these polymorphic enzymes also explain the aetiology of cancer and therapeutic management in clinics. We analysed the cytochrome p4501A1 (CYP1A1) and 2D6 (CYP2D6) variant genotype and allele frequencies by PCR-RFLP in Turkish individuals (n=140). The frequency of the CYP1A1*2A mutant allele was found to be 15.4%, and the CYP2D6*3 and *4 mutant allele (poor metabolizer) frequencies were 2.5% and 13.9%, respectively. This study presents the first results of CYP1A1 and CYP2D6 mutant allele distributions in the Turkish population and these data provide an understanding of epidemiological studies that correlate therapeutic approaches and aetiology of several types of malignancy in Turkish patients.     

cDNA cloning and characterization of feline CYP1A1 and CYP1A2

Deficiency of drug glucuronidation in the cat is one of the major reasons why this animal is highly sensitive to the side effects of drugs. The characterization of cytochrome P450 isoforms belonging to the CYP1A subfamily, which exhibit important drug oxidation activities such as activation of pro-carcinogens, was investigated. Two cDNAs, designated CYP1A-a and CYP1A-b, corresponding to the CYP1A subfamily were obtained from feline liver. CYP1A-a and CYP1A-b cDNAs comprise coding regions of 1554 bp and 1539 bp, and encode predicted amino acid sequences of 517 and 512 residues, respectively. These amino acid sequences contain a heme-binding cysteine and a conserved threonine. The cDNA identities, as well as the predicted amino acid sequences containing six substrate recognition sites, suggest that CYP1A-a and CYP1A-b correspond to CYP1A1 and CYP1A2, respectively. This was confirmed by the kinetic parameters of the arylhydrocarbon hydroxylase and 7-ethoxyresorufin O-deethylase activities of expressed CYPs in yeast AH22 cells and by the tissue distribution of each mRNA. However, theophylline 3-demethylation is believed to be catalyzed by CYP1A1 in cats, based on the high V(max) and low K(m) seen, in contrast to other animals. Because feline CYP1A2 had a higher K(m) for phenacetin O-deethylase activity with acetaminophen, which cannot be conjugated with glucuronic acid due to UDP-glucuronosyltransferase deficiency, it is supposed that the side effects of phenacetin as a result of toxic intermediates are severe and prolonged in cats.     

Inhibition of CYP450 1A and 3A by berberine in crucian carp Carassius auratus gibelio

Berberine has long been considered as an antibiotic candidate in aquaculture. However, studies regarding its effects on drug-metabolizing enzymes in fish are still limited. In the present study, the effects of berberine on cytochrome P4501A (CYP1A) and CYP3A in crucian carp were investigated. Injection of different concentrations of berberine (0, 5, 25, 50, and 100 mg/kg) inhibited the CYP1A mRNA expression, thereby inhibiting further the catalytic activity of CYP1A-related ethoxyresorufin-O-deethylase (EROD). Furthermore, both CYP1A expression and EROD activity were further inhibited with increasing berberine concentrations. In addition, the CYP3A expressions at both the mRNA and the protein levels were downregulated by higher berberine concentrations. The catalytic activity of CYP3A-related erythromycin N-demethylase (ERND) was also inhibited by berberine at a dose of no less than 25 mg/kg. Moreover, at the berberine concentration exceeding 25 mg/kg, the inhibition of CYP3A expression and ERND activity increased with increasing berberine concentrations. In vitro experiments were also performed. When berberine was pre-incubated with the crucian carp liver microsomes, it competitively inhibited the corresponding EROD activity with the IC₅₀ of 11.7 μM. However, the ERND activity was slightly inhibited by berberine with the IC₅₀ of 206.4 μM. These results suggest that, in crucian carp, berberine may be a potent inhibitor to CYP1A, whereas the CYP3A inhibition needs a higher concentration of berberine.     

(E)-3-(3,4,5-Trimethoxyphenyl)-1-(pyridin-4-yl)prop-2-en-1-one,a heterocyclic chalcone is a potent and selective CYP1A1 inhibitor and cancer chemopreventive agent

The overexpression of CYP1 family of enzymes is reported to be associated with development of human carcinomas. It has been well reported that CYP1A1 specific inhibitors prevents carcinogenesis. Herein, thirteen pyridine-4-yl series of chalcones were synthesized and screened for inhibition of CYP1 isoforms 1A1, 1B1 and 1A2 in Sacchrosomes? and live human HEK293 cells. The structure-activity relationship analysis indicated that chalcones bearing tri-alkoxy groups (8a and 8k) on non-heterocyclic ring displayed selective inhibition of CYP1A1 enzyme, with IC₅₀ values of 58 and 65?nM, respectively. The 3,4,5-trimethoxy substituted derivative 8a have shown >10-fold selectivity towards CYP1A1 with respect to other enzymes of the CYP1 sub-family and >100-fold selectivity with respect to CYP2 and CYP3 family of enzymes. The potent and selective CYP1A1 inhibitor 8a displayed antagonism of B[a]P mediated activation of aromatic hydrocarbon receptor (AhR) in yeast cells, and also protected human cells from CYP1A1-mediated B[a]P toxicity in human cells. This potent and selective inhibitor of CYP1A1 enzyme have a potential for development as cancer chemopreventive agent.     

氯吡格雷对大鼠肝药物酶CYP3A4和CYP1A2表达的影响

目的：氯吡格雷主要由CYP3A4催化使其激活,CYPlA2也参与氯吡格雷活化。关于氯吡格雷对肝微粒体酶的影响国内外文献报道不多,因此本实验通过检测肝细胞色素氧化酶CYP3A4和CYPlA2的表达,探讨氯吡格雷对大鼠肝药物酶的影响。方法：生理盐水为对照组,氯吡格雷设高、中、低三个剂量组（27,13．5,6．75mg／kg／d）,雄性健康大鼠连续灌胃给药7天,脱臼处死,取肝组织,通过westernblot法检测大鼠肝脏CYP3A4和CYPlA2蛋白表达情况。结果：1）、氯吡格雷抑制大鼠CYP3A4蛋白表达,氯吡格雷高中低剂量组分别比生理盐水组大鼠CYP3A4蛋白表达量降低（P〈0．05）;氯吡格雷低中高剂量组间进行比较,大鼠CYP3A4蛋白表达量呈梯度减少（P〈0．05）;2）、氯吡格雷抑制大鼠CYPlA2蛋白表达,氯吡格雷高中低剂量组分别比生理盐水组大鼠CYPlA2蛋白表达量降低（P〈0．05）,氯吡格雷低中高剂量组间进行比较,大鼠CYPlA2蛋白表达量呈梯度减少（P〈0．05）。结论：氯吡格雷使肝细胞色素氧化酶CYP3A4和CYPlA2的表达量减少,因此氯吡格雷高、中、低3个剂量组均不同程度的抑制大鼠肝脏CYP3A4和CYPlA2的表达,提示当氯吡格雷与某些主要经CYP3A4和CYPlA2代谢的药物合用时,发生代谢性相关作用的可能性大。     

Silybin and dehydrosilybin inhibit cytochrome P450 1A1 catalytic activity: A study in human keratinocytes and human hepatoma cells

The flavonolignan silybin and its derivative dehydrosilybin have been proposed as candidate UV-protective agents in skin care products. This study addressed the effect of silybin and dehydrosilybin on the activity of cytochrome P450 isoform CYP1A1 in human keratinocytes (HaCaT) and human hepatoma cells (HepG2). CYP1A1 catalytic activity was assessed as O-deethylation of 7-ethoxyresorufin using fluorescence detection. Silybin and dehydrosylibin inhibited basal and dioxin-inducible CYP1A1 catalytic activity in both cell lines used. The inhibitory effect of tested compounds was more pronounced in HaCaT cells than in HepG2 cells, and dehydrosilybin was a much stronger inhibitor than silybin. Analyses on CYP1A1 human recombinant protein yielded IC₅₀ values of 22.9 ± 4.7 μmol/L and 0.43 ± 0.04 μmol/L for silybin and dehydrosilybin, respectively. Since CYP1A enzymes are some of the most prominent actors in the process of chemically induced carcinogenesis, the inhibitory activity of the flavonolignans tested against CYP1A1 favors their use as cytoprotective agents in terms of skin and hepatic metabolism. In addition, the capability of dehydrosilybin to inhibit CYP1A1 in submicromolar concentrations makes this compound a potential biological probe in CYP1A1 analyses.     

CYP1A1 and CYP2D6 polymorphism and risk of lung cancer in a North Indian population

This case–control study was conducted to examine the association between the CYP1A1 and CYP2D6 genotypes and lung cancer risk among North Indians. The estimated relative risk for lung cancer associated with the CYP1A1 Val/Val allele was 2.68, and was four-fold when cases with small cell lung cancer (SCLC) were considered alone. With regard to the metabolism of debrisoquine, no poor metabolizers were found amongst the subjects. The odds ratio of risk with the heterozygous extensive metabolizer (HEM) genotype was 1.5. However, in the presence of at least a single copy of the variant CYP1A1 MspI allele and the CYP2D6 HEM genotype, the risk was two-fold for squamous cell carcinoma (SQCC). When the CYP1A1 Val/Val and CYP2D6 HEM genotypes were taken together, the risk for SCLC was four-fold. Stratified analysis indicated an interaction between bidi smoking and variant CYP1A1 genotypes on the risk for SQCC and SCLC. Heavy smokers (Brinkman index>400) with Val/Val genotypes were at a very high risk of developing lung cancer (odds ratio 29.30, 95% confidence interval 2.42–355, p=0.008). Heavy smokers with CYP1A1 MspI (CYP1A1*1/2A or CYP1A1*2A/*2A) genotype had a seven-fold risk for SCLC compared with non-smokers. This study is the first to be carried out on a North Indian population, and, although small, suggests that CYP1A1 and CYP2D6 polymorphisms might have a role in determining the risk for lung cancer and should be investigated further.     

Benzo(a)pyrene exposure induces CYP1A1 activity and expression in human endometrial cells

Estrogen is a major risk factor for endometrial cancer and it has been well-established that smokers have a significantly reduced risk of endometrial cancer. Localized levels of estrogen within the uterus may determine the estrogenic response. The objective of this research was to investigate effects of cigarette smoke related hydrocarbons (benzo(a)pyrene, BP) on uterine CYP1A1/2 and 1B1, enzymes involved in estrogen metabolism. Human endometrium epithelial cells (RL95-2) were incubated with various concentrations (0.05, 0.1, 0.5, 1, and 10mM) of BP for 48h. CYP1 catalytic activity, protein and mRNA levels were determined. Selective chemical and immuno-inhibitors were used to determine the contribution of individual CYP1 isoenzymes. Cells expressing CYP1A1, CYP1A2 and CYP1B1 were used for comparisons. CYP1A1/2 protein and mRNA levels were significantly elevated by BP. Low level of constitutive CYP1 activity was observed in RL95-2 cells, which was significantly induced by BP exposure (12-fold at 1mM). CYP1 activity in BP-induced cells was significantly inhibited by specific anti-CYP1A1 and high concentration of alpha-naphthoflavone (ANF, 100nM), but not by selective CYP1A2 (furafylline) and CYP1B1 (homoeriodictoyl) inhibitors and low concentration of ANF (5nM). These studies suggest that CYP1A2 and CYP1B1 are not induced by BP in the endometrial cells. It also appears that CYP1A1 is one of the major CYP450 enzymes induced by BP.