Forms Of Cytochrome P-450 Research Articles

Development of UV- Spectrophotometric Method for Simultaneous Estimation of Metformin and Piperine in Combined Tablet Dosage Form

Piperine is an alkaloid obtained from Piper nigrum Linn., is known to modify the biotransformation of the drugs by inhibiting Cytochrome P450 forms. Piperine inhibits the bio-transformation and metabolism of metformin leading to higher levels of drug in systemic circulation, thus increases the bioavailabilty when co-administered with metformin. In the present study a simple, accurate, precise, economical procedures, entailing neither irksome sample treatment nor tedious extraction process a combined tablet dosage form of metformin and piperine have been developed for the simultaneous estimation of piperine and metformin using UV- Spectrophotometry method. The developed method was based on employing simultaneous equation method for analysis of both the drugs. Metformin and piperine have shown absorbance maxima at 237nm and 342nm respectively, in methanol and the linearity was obeyed over the concentration range of 2-10μg/ml and 2-10 μg/ml respectively for metformin and piperine. The validation parameters like precision, accuracy, limit of quantitation (LOQ), limit of detection (LOD), were determined according to ICH guidelines. All the results were found to be within the limits.

Proteomic and Bioinformatics Analysis of Membrane Lipid Domains after Brij 98 Solubilization of Uninduced and Phenobarbital-Induced Rat Liver Microsomes: Defining the Membrane Localization of the P450 Enzyme System.

The proteomes of ordered and disordered lipid microdomains in rat liver microsomes from control and phenobarbital (PB)-treated rats were determined after solubilization with Brij 98 and analyzed by tandem mass tag (TMT)-liquid chromatography-mass spectrometry (LC-MS). This allowed characterization of the liver microsomal proteome and the effects of phenobarbital-mediated induction, focusing on quantification of the relative levels of the drug-metabolizing enzymes._The microsomal proteome from control rats was represented by 333 (23%) proteins from ordered lipid microdomains, 517 (36%) proteins from disordered lipid domains, and 587 (41%) proteins that uniformly distributed between lipid microdomains. Most enzymes related to drug metabolism were mainly localized in disordered lipid microdomains. However, cytochrome P450 (CYP) 1A2, multiple forms of CYP2D, and several forms of UDP glucuronosyltransferases (UGT) 1A1 and 1A6) localized to ordered lipid microdomains. Other drug-metabolizing enzymes, including several forms of cytochromes P450, were uniformly distributed between the ordered and disordered regions. The redox partners, NADPH-cytochrome P450 reductase and cytochrome b5, localized to disordered microdomains. PB induction resulted in only modest changes in protein localization. Less than five proteins were variably associated with the ordered and disordered membrane microdomains in PB and control microsomes. PB induction was associated with fewer proteins localizing in the disordered membranes and more being uniformly distributed or localized to ordered domains. Ingenuity Pathway Analysis (IPA) was used to ascertain the effect of PB on cellular pathways, resulting in attenuation of pathways related to energy storage/utilization and overall cellular signaling and an increase in those related to degradative pathways. SIGNIFICANCE STATEMENT: This work identifies the lipid microdomain localization of the proteome from control and phenobarbital-induced rat liver microsomes. Thus, it provides an initial framework to understand how lipid/protein segregation influences protein-protein interactions in a tissue extract commonly used for studies in drug metabolism and uses bioinformatics to elucidate the effects of phenobarbital induction on cellular pathways.

Drug Interactions Involving 17α-Ethinylestradiol: Considerations Beyond Cytochrome P450 3A Induction and Inhibition.

It is widely acknowledged that drug-drug interactions (DDIs) involving estrogen (17α-ethinylestradiol (EE))-containing oral contraceptives (OCs) are important. Consequently, sponsors of new molecular entities (NMEs) often conduct clinical studies with priority given to OCs as victims of cytochrome P450 (CYP) 3A (CYP3A) induction and inhibition. Such scenarios are reflected in the US Food and Drug Administration-issued guidance documentation related to OC DDI studies. Although CYP3A is important, OCs such as EE are metabolized by sulfotransferase 1E1 and UDP-glucuronosyltransferase (UGT) 1A1, expressed in the gut and liver, and so both can also serve as loci of victim OC DDI. Therefore, for any NME, one should carefully consider its induction and inhibition profile involving CYP3A4/5, UGT1A1, and SULT1E1. As DDI perpetrators, available clinical DDI data indicate that EE-containing OCs can induce (e.g., UGT1A4 and CYP2A6) and inhibit (CYP1A2 ≥ CYP2C19 > CYP3A4/5 > CYP2C8, CYP2B6, CYP2D6, and CYP2C9) various CYP forms. Although available in vitro CYP inhibition data do not explain such a graded inhibitory effect in vivo, it is hypothesized that EE differentially modulates CYP expression via potent agonism of the estrogen receptor expressed in the gut and liver. From the standpoint of the NME as potential OC DDI victim, therefore, it is important to assess its projected (pre-phase I) or known therapeutic index and pharmacokinetic profile (fraction absorbed, absolute oral bioavailability, clearance/extraction class, fraction metabolized by CYP1A2, CYP2C19, CYP2A6, and UGT1A4). Such information can enable the prioritization, design, and interpretation of NME-OC DDI studies.

Roles of cytochrome P450 2A6 in the oxidation of flavone, 4′-hydroxyflavone, and 4′-, 3′-, and 2′-methoxyflavones by human liver microsomes

Nine forms of recombinant cytochrome P450 (P450 or CYP) enzymes were used to study roles of individual P450 enzymes in the oxidation of flavone and some other flavonoids, 4′-hydroxyflavone and 4′-, 3′-, and 2′-methoxyflavones, by human liver microsomes using LC-MS/MS analysis. As has been reported previously , 4′-, 3′-, and 2′-methoxyflavones were preferentially O-demethylated by human liver P450 enzymes to form 4′-, 3′-, and 2′-hydroxylated flavones and also 3′,4′-dihydroxyflavone from the former two substrates. In comparisons of product formation by oxidation of these methoxylated flavones, CYP2A6 was found to be a major enzyme catalysing flavone 4′- and 3′-hydroxylations by human liver microsomes but did not play significant roles in 2′-hydroxylation of flavone, O-demethylations of three methoxylated flavones, and the oxidation of 4′-hydroxyflavone to 3′,4′-dihydroxyflavone. The effects of anti-CYP2A6 IgG and chemical P450 inhibitors suggested that different P450 enzymes, as well as CYP2A6, catalysed oxidation of these flavonoids at different positions by liver microsomes. These studies suggest that CYP2A6 catalyses flavone 4′- and 3′-hydroxylations in human liver microsomes and that other P450 enzymes have different roles in oxidizing these flavonoids.

Enantioselective inhibition of human CYP2C19 by the chiral pesticide ethofumesate: Prediction of pesticide-drug interactions in humans

Ethofumesate is a chiral herbicide that may display enantioselective behavior in humans. For this reason, the enantioselective potential of ethofumesate and its main metabolite ethofumesate-2-hydroxy to cause pesticide-drug interactions on cytochrome P450 forms (CYPs) has been evaluated by using human liver microsomes. Among the evaluated CYPs, CYP2C19 had its activity decreased by the ethofumesate racemic mixture (rac-ETO), (+)-ethofumesate ((+)-ETO), and (−)-ethofumesate ((−)-ETO). CYP2C19 inhibition was not time-dependent, but a strong inhibition potential was observed for rac-ETO (IC50 = 5 ± 1 μmol L−1), (+)-ETO (IC50 = 1.6 ± 0.4 μmol L−1), and (−)-ETO (IC50 = 1.8 ± 0.4 μmol L−1). The reversible inhibition mechanism was competitive, and the inhibition constant (Ki) values for rac-ETO (2.6 ± 0.4 μmol L−1), (+)-ETO (1.5 ± 0.2 μmol L−1), and (−)-ETO (0.7 ± 0.1 μmol L−1) were comparable to the Ki values of strong CYP2C19 inhibitors. Inhibition of CYP2C19 by ethofumesate was enantioselective, being almost twice higher for (−)-ETO than for (+)-ETO, which indicates that this enantiomer may be a more potent inhibitor of this CYP form. For an in vitro-in vivo correlation, the Food and Drug Administration's (FDA) guideline on the assessment of drug-drug interactions used in the early stages of drug development was used. The FDA's R1 values were estimated on the basis of the obtained ethofumesate Ki and distribution volume, metabolism, unbound plasma fraction, gastrointestinal and dermal absorption data available in the literature. The correlation revealed that ethofumesate probably inhibits CYP2C19 in vivo for both chronic (oral) and occupational (dermal) exposure scenarios.

Cytochrome P450 content in primary tumors and liver metastases of patients with metastatic colorectal cancer.

To determine the content of low-spin form of cytochrome P450in primary tumors and liver metastases of the patients with metastatic colorectal cancer (mCRC) and and assess its prognostic significance. The levels of the oxidized and low-spin forms of cytochrome P450in the tissues of patients with mCRC were studied by electron paramagnetic resonance. To detect CYP1A2and CYP1B1isoforms, Western blot analysis was used. The activity of metalloprotreinases was studied by gelatine zymography. In the liver metastases and tissues adjacent to metastasis, the levels of low-spin forms of cytochrome P450 are lower than in the samples of conventionally normal liver tissue. Western blot analysis revealed that low-spin form of cytochrome P450in primary tumors and liver metastases detected by electron paramagnetic resonance is attributed largely to CYP1B1isoform. The content of low-spin form of cytochrome P450inversely correlated with the the activity of gelatinases (matrix metalloproteinase-2 and -9). The survival of patients with high levels of the low-spin form of cytochrome P450in tumor tissues was higher than that of patients with low levels of the enzyme (105months vs 61months). In the primary tumors and liver metastases of patients with mCRC, the content of the low-spin form of cytochrome P450decreased, which correlated inversely with patient's survival.

Identification of suberosin metabolites in human liver microsomes by high-performance liquid chromatography combined with high-resolution quadrupole-orbitrap mass spectrometer.

Suberosin is a natural prenylated coumarin derivative isolated from Citropsis articulata. It has various pharmacological properties, especially as an anticoagulant, for which it has been used since antiquity. However, its metabolic pathway and metabolites have not yet been studied. Therefore, this study characterizes its metabolic pathway and metabolites in human liver microsomes (HLMs) using high-resolution quadrupole-orbitrap mass spectrometry (HRMS/MS). Eight metabolites (M1-M8) were found, including three monohydroxylated (M1-M3), one hydrated (M4), three dihydroxylated (M5-M7), and one glucuronide conjugate (M8). Furthermore, forms of cytochrome P450 (CYPs) responsible for suberosin metabolism in HLMs were characterized. CYP1A2 was identified as a major enzyme for the production of M1 and M5 metabolites. The M2, M3, and M7 metabolites were predominantly generated by CYP2B6. M8 was the only phase II metabolite, identified as a glucuronide conjugate from either M1 or M2. This glucuronide conjugate may be the only promising metabolite from phase II metabolism. Phase I metabolism, especially hydroxylation, was found to provide a predominant metabolic pathway of suberosin in HLMs. Further studies should be conducted to explore the metabolites, examining their efficacy and their toxicity in an in vivo system.

Heterologous Expression and Functional Characterization of Novel CYP2C9 Variants Identified in the Alaska Native People.

CYP2C9 is a major form of human liver cytochrome P450 that is responsible for the oxidative metabolism of several widely used low-therapeutic index drugs, including (S)-warfarin and phenytoin. In a cohort of Alaska Native people, ultrarare or novel CYP2C9 protein variants, M1L (rs114071557), N218I (rs780801862), and P279T (rs182132442, CYP2C9*29), are expressed with higher frequencies than the well characterized CYP2C9*2 and CYP2C9*3 alleles. We report here on their relative expression in lentivirus-infected HepG2 cells and the functional characterization of purified reconstituted enzyme variants expressed in Escherichia coli toward (S)-warfarin, phenytoin, flurbiprofen, and (S)-naproxen. In the infected HepG2 cells, robust mRNA and protein expression were obtained for wild-type, N218I, and P279T variants, but as expected, the M1L variant protein was not translated in this liver-derived cell line. His-tagged wild-type protein and the N218I and P279T variants, but not M1L, expressed well in E. coli and were highly purified after affinity chromatography. Upon reconstitution with cytochrome P450 oxidoreductase and cytochrome b5, the N218I and P279T protein variants metabolized (S)-warfarin, phenytoin, flurbiprofen, and (S)-naproxen to the expected monohydroxylated or O-demethylated metabolites. Steady-state kinetic analyses revealed that the relative catalytic efficiency ratios of (S)-warfarin metabolism by the P279T and N218I variants were 87% and 24%, respectively, of wild-type CYP2C9 protein. A similar rank ordering was observed for metabolism of phenytoin, flurbiprofen, and (S)-naproxen. We conclude that carriers of the variant N218I and, especially, the M1L alleles would be at risk of exacerbated therapeutic effects from drugs that rely on CYP2C9 for their metabolic clearance. SIGNIFICANCE STATEMENT: Novel gene variants of CYP2C9-M1L, and N218I, along with P279T (CYP2C9*29)-are expressed in Alaska Native people at relatively high frequencies. In vitro characterization of their functional effects revealed that each variant confers reduced catalytic efficiency toward several substrates, including the low-therapeutic index drugs (S)-warfarin and phenytoin. These data provide the first functional information for new, common CYP2C9 variants in this understudied population. The data may help guide dose adjustments in allele carriers, thus mitigating potential healthcare disparities.

Preference for O-demethylation reactions in the oxidation of 2′-, 3′-, and 4′-methoxyflavones by human cytochrome P450 enzymes

2′-, 3′-, and 4′-Methoxyflavones (MeFs) were incubated with nine forms of recombinant human cytochrome P450 (P450 or CYP) enzymes in the presence of an NADPH-generating system and the products formed were analyzed with LC-MS/MS methods.CYP1B1.1 and 1B1.3 were highly active in demethylating 4′MeF to form 4′-hydroxyflavone (rate of 5.0 nmol/min/nmol P450) and further to 3′,4′-dihydroxyflavone (rates of 2.1 and 0.66 nmol/min/nmol P450, respectively). 3′MeF was found to be oxidized by P450s to m/z 239 (M-14) products (presumably 3′-hydroxyflavone) and then to 3′,4′-dihydroxyflavone. P450s also catalyzed oxidation of 2′MeF to m/z 239 (M-14) and m/z 255 (M-14, M-14 + 16) products, presumably mono- and di-hydroxylated products, respectively.At least two types of ring oxidation products having m/z 269 fragments were formed, although at slower rates than the formation of mono- and di-hydroxylated products, on incubation of these MeFs with P450s; one type was products oxidized at the C-ring, having m/z 121 fragments, and the other one was the products oxidized at the A-ring (having m/z 137 fragments).Molecular docking analysis indicated the preference of interaction of O-methoxy moiety of methoxyflavones in the active site of CYP1A2.These results suggest that 2′-, 3′-, and 4′-methoxyflavones are principally demethylated by human P450s to form mono- and di-hydroxyflavones and that direct oxidation occurs in these MeFs to form mono-hydroxylated products, oxidized at the A- or B-ring of MeF.

Inhibitory and inductive effects of 4- or 5-methyl-2-mercaptobenzimidazole, thyrotoxic and hepatotoxic rubber antioxidants, on several forms of cytochrome P450 in primary cultured rat and human hepatocytes

Effects of 4-methyl-2-mercaptobenzimidazole (4-MeMBI) and 5-methyl-2- mercaptobenzimidazole (5-MeMBI) on cytochrome P450 (CYP) activity were examined in primary cultured rat hepatocytes. Hepatocytes from male Wistar rats were cultured in the presence of 4-MeMBI or 5-MeMBI (0−400 μM), and the activity of CYPs 3A2/4 (48 and 96 h) and 1A1/2 (48 h) was determined by measuring the activity of testosterone 6β-hydroxylation and 7-ethoxyresorufin O-deethylation, respectively. As a result, 4-MeMBI and 5-MeMBI (≥12.5 μM) inhibited CYP3A2 activity. On the other hand, 4-MeMBI (≥25 μM) and 5-MeMBI (≥100 μM) induced CYP1A1/2 activity, being consistent with the previous in vivo results. In a comparative metabolism study using primary cultured human hepatocytes from two Caucasian donors, 4-MeMBI and 5-MeMBI induced the activity of CYPs 3A4 and 1A1/2 with individual variability. It was concluded from these results that 4-MeMBI, 5-MeMBI and MBI caused inhibition of CYP3A2 activity in primary cultured rat hepatocytes, suggesting their potential for metabolic drug-drug interactions. Primary cultured rat and human hepatocytes were considered to be useful for the evaluation of effects of the benzimidazole compounds on their inducibility and inhibitory activities of cytochrome P450 forms.

Development of uv-spectrophotometric method for the simultaneous estimation of nateglinide and piperine in combined tablet dosage form

Simple, accurate, precise, economical procedure entailing neither irksome sample treatment nor tedious extraction process have been developed for the simultaneous estimation of piperine and nateglinide in a combined tablet dosage form of nateglinide and piperine using UV-Spectrophotometry method. Piperine is an alkaloid obtained from Piper nigrum Linn., is known to modify the biotransformation of the drugs by inhibiting Cytochrome P450 forms. Piperine inhibits the bio-transformation and metabolism of nateglinide leading to higher levels of drug in systemic circulation, thus increases the bioavailabilty when co-administered with nateglinde. Nateglinide and piperine have shown absorbance maxima at 258nm and 244nm respectively, in methanol and the linearity was obeyed over the concentration range of 50–250μg/ml and 5–25μg/ml respectively for nateglinide and piperine. The validation parameters like precision, accuracy, limit of quantitation (LOQ), limit of detection (LOD), were determined according to ICH guidelines. All the results were found to be within the limits.

Development of new Coumarin-based profluorescent substrates for human cytochrome P450 enzymes

Cytochrome P450 (CYP) enzymes constitute an essential xenobiotic metabolizing system that regulates the elimination of lipophilic compounds from the body. Convenient and affordable assays for CYP enzymes are important for assessing these metabolic pathways.In this study, 10 novel profluorescent coumarin derivatives with various substitutions at carbons 3, 6 and 7 were developed. Molecular modeling indicated that 3-phenylcoumarin offers an excellent scaffold for the development of selective substrate compounds for various human CYP forms, as they could be metabolized to fluorescent 7-hydroxycoumarin derivatives. Oxidation of profluorescent coumarin derivatives to fluorescent metabolites by 13 important human liver xenobiotic-metabolizing CYP forms was determined by enzyme kinetic assays.Four of the coumarin derivatives were converted to fluorescent metabolites by CYP1 family enzymes, with 6-methoxy-3-(4-trifluoromethylphenyl)coumarin being oxidized selectively by CYP1A2 in human liver microsomes. Another set of four compounds were metabolized by CYP2A6 and CYP1 enzymes. 7-Methoxy-3-(3-methoxyphenyl)coumarin was oxidized efficiently by CYP2C19 and CYP2D6 in a non-selective fashion.The advantages of the novel substrates were (1) an excellent signal-to-background ratio, (2) selectivity for CYP1 forms, and (3) convenient multiwell plate measurement, allowing for precise determination of potential inhibitors of important human hepatic forms CYP1A2, CYP2C19 and CYP2D6.

1-Aminobenzotriazole: A Mechanism-Based Cytochrome P450 Inhibitor and Probe of Cytochrome P450 Biology.

1-Aminobenzotriazole (1-ABT) is a pan-specific, mechanism-based inactivator of the xenobiotic metabolizing forms of cytochrome P450 in animals, plants, insects, and microorganisms. It has been widely used to investigate the biological roles of cytochrome P450 enzymes, their participation in the metabolism of both endobiotics and xenobiotics, and their contributions to the metabolism-dependent toxicity of drugs and chemicals. This review is a comprehensive evaluation of the chemistry, discovery, and use of 1-aminobenzotriazole in these contexts from its introduction in 1981 to the present.

Metabolic changes from liver regeneration after metastatic colorectal cancer surgery

Background. The pathological effect of the surgical ischemia-reperfusion technique is a cause for the development of liver damage and occurrence of acute liver failure during the postoperative period in cancer patients after major resection as well as after transplantation. Despite the urgency of the problem, most of the pathological processes that accompany the above-mentioned conditions are still only incompletely studied. Objective. To investigate the mechanisms of the damaging effects of stress reaction after liver resection, to evaluate the functional state of residual liver tissue and to find ways to influence the regeneration and apoptosis of the resected liver, which in the future would make the liver surgery safer. Methods. The results of treatment of 15 patients with colorectal cancer (mCRC) and liver metastases, who had undergone surgery in the period from December 2015 to November 2016 at the National Cancer Institute. Determination of the state of the detoxifying function of hepatocytes and the coupling of biological oxidation and phosphorylation in the mitochondria of hepatocytes were performed by electron paramagnetic resonance at the temperature of liquid nitrogen. Results. It has been found that the levels of the oxidized and low-spin forms of cytochrome P-450 in the catalytic cycle of the detoxification system were 0.33 ± 0.08 relative units and 1.11 ± 0.13 relative units, respectively (the normal levels are 0.59 ± 0.03 relative units and 2.56 ± 0.02 relative units). Also, an increase registered in the levels of formation of complexes of NO-FeS-proteins up to the values of 0.33 ± 0.08 relative units (the normal level is 0.14 ± 0.07). We believe that such changes are the cause of disturbances in the energy function of mitochondria and the formation of cell hypoxia, which correlates with an increase of lactate levels in tissue of the future liver remnant. The liver remnant levels are > 2.00 mM/g·tissue, while the normal is 1.80 ± 0.26 mm/g·tissue. This confirms the functioning of hepatocytes under hypoxic conditions. Conclusions. In the residual liver tissue, we have found a decrease in detoxification system deficiency and the energy supply of hepatocytes. In hepatocytes of the future liver remnant, reprogramming of the metabolism of mitochondria from oxidative phosphorylation in glycolysis is registered, resulting in the formation of cell hypoxia and an increase in levels of lactate and superoxide radicals. To assess the degree of ALF in the postoperative period, we suggest use of the levels of lactate, the activity of cytochrome P-450, the levels of formation of complexes of NO and FeS-proteins in mitochondria ETC and the generation rate of SR as the markers.

Imidacloprid is hydroxylated by Laodelphax striatellus CYP6AY3v2.

Laodelphax striatellus (Fallén) is one of the most destructive pests of rice, and has developed high resistance to imidacloprid. Our previous work indicated a strong association between imidacloprid resistance and the overexpression of a cytochrome P450 gene CYP6AY3v2 in a L.striatellus imidacloprid resistant strain (Imid-R). In this study, a transgenic Drosophila melanogaster line that overexpressed the L.striatellus CYP6AY3v2 gene was established and was found to confer increased levels of imidacloprid resistance. Furthermore, CYP6AY3v2 was co-expressed with D.melanogaster cytochrome P450 reductase (CPR) in Spodoptera frugiperda 9 (SF9) cells. A carbon monoxide difference spectra analysis indicated that CYP6AY3v2 was expressed predominately in its cytochrome P450 (P450) form, which is indicative of a good-quality functional enzyme. The recombinant CYP6AY3v2 protein efficiently catalysed the model substrate P-nitroanisole to p-nitrophenol with a maximum velocity (Vmax ) of 60.78 ± 3.93 optical density (mOD)/min/mg protein. In addition, imidacloprid itself was metabolized by the recombinant CYP6AY3v2/nicotinamide adenine dinucleotide 2'-phosphate reduced tetrasodium salt (NADPH) CPR microsomes in in vitro assays (catalytic constant (Kcat ) = 0.34 pmol/min/pmol P450, michaelis constant (Km ) = 41.98 μM), and imidacloprid depletion and metabolite peak formation were with a time dependence. The data provided direct evidence that CYP6AY3v2 is capable of hydroxylation of imidacloprid and conferring metabolic resistance in L.striatellus.

CYP4F2 repression and a modified alpha-tocopherol (vitamin E) metabolism are two independent consequences of ethanol toxicity in human hepatocytes

The expression of CYP4F2, a form of cytochrome P-450 with proposed role in α-tocopherol and long-chain fatty acid metabolism, was explored in HepG2 and HepaRG human hepatocytes during ethanol toxicity.Cytotoxicity, ROS production, and JNK and ERK1/2 kinase signaling increased in a dose and time-dependent manner during ethanol treatments; CYP4F2 gene expression decreased, while other CYP4F forms, namely 4F11 and 12, increased along with 3A4 and 2E1 isoforms. α-Tocopherol antagonized the cytotoxicity and CYP4F2 gene repression effect of ethanol in HepG2 cells. Ethanol stimulated the tocopherol-ω-hydroxylase activity and the other steps of vitamin E metabolism, which points to a minor role of CYP4F2 in this metabolism of human hepatocytes. PPAR-γ and SREBP-1c followed the same expression pattern of CYP4F2 in response to ethanol and α-tocopherol treatments. Moreover, the pharmacological inhibition of PPAR-γ synergized with ethanol in decreasing CYP4F2 protein expression, which suggests a role of this nuclear receptor in CYP4F2 transcriptional regulation.In conclusion, ethanol toxicity modifies the CYP expression pattern of human hepatic cells impairing CYP4F2 transcription and protein expression. These changes were associated with a lowered expression of the fatty acid biosynthesis regulators PPAR-γ and SREBP-1c, and with an increased enzymatic catabolism of vitamin E. CYP4F2 gene repression and a sustained vitamin E metabolism appear to be independent effects of ethanol toxicity in human hepatocytes.

Cytochrome P450 expression system for high-throughput real-time detection of genotoxicity: Application to the study of human CYP1A2 variants

Individual variations in cytochrome P450-mediated metabolism are believed to contribute to individual susceptibility to chemical carcinogenesis. CYP1A2 is one of the major forms of cytochrome P450 involved in drug metabolism and bioactivation of carcinogens. We have applied a recently developed high-throughput Salmonella typhimurium TA1535 system for detection of DNA damaging agents to the study of CYP1A2 polymorphisms. Non-synonymous variants T83M [CYP1A2*9], S212C [CYP1A2*12], S298R [part of CYP1A2*21], G299S [CYP1A2*13], I314V [no allele designation], I386F [CYP1A2*4], C406Y [CYP1A2*5] and R456H [CYP1A2*8] were examined. The cDNAs for each of these variants and the wild-type were co-expressed with human NADPH cytochrome P450 oxidoreductase in the TA1535-based system. The bioactivation capacity of these CYP1A2 variants was investigated using three CYP1A2-dependent pro-mutagens, 1-aminopyrene (1AP), 2-aminoanthracene (2AA), and 2-amino-3-methylimidazo(4,5-f)quinoline (IQ). All CYP1A2 variants except R456H, T83M, and I386F gave positive responses with all three compounds. Variant R456H generated no detectable holoenzyme and no detectable response for any of the compounds; I386F did not bioactivate IQ; T83M did not bioactivate 1AP. Multivariate analysis indicated variant T83M to be substantially altered in catalytic properties when compared with wild-type CYP1A2; variants G299S and I386F are slightly but significantly different. These results corroborate our previous studies, indicating the effectiveness of this new high-throughput system, not only for examining the effect of CYP1A2 polymorphisms on pro-mutagen bioactivation, but also for obtaining insights on CYP1A2 function at the mechanistic level.

Interaction of isoflavonoids with human liver microsomal cytochromes P450: inhibition of CYP enzyme activities

1. The possibility of interaction of isoflavonoids with concomitantly taken drugs to determined isoflavonoids safety was studied. Inhibition of nine forms of cytochrome P450 (CYP3A4, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2C9, CYP2D6 and CYP2E1) by 12 isoflavonoids (daidzein, genistein, biochanin A, formononetin, glycitein, equol and six glucosides, daidzin, puerarin, genistin, sissotrin, ononin and glycitin) was studied systematically.2. The most potent inhibitors were genistein and daidzein inhibiting noncompetitively the CYP2C9 with Ki of 35.95 ± 6.96 and 60.56 ± 3.53 μmol/l and CYP3A4 (inhibited by genistein with Ki of 23.25 ± 5.85 μmol/l also by a noncompetitive mechanism). Potent inhibition of CYP3A4 was observed also with biochanin A (Ki of 57.69 ± 2.36 μmol/l) and equol (Ki of 38.47 ± 2.32 μmol/l).3. Genistein and daidzein inhibit noncompetitively CYP3A4 and CYP2C9. With plasma levels in micromolar range, a clinically important interaction with concomitantly taken drugs does not seem to be probable.

Alteration of cytochrome P450 1 regulation and HSP 70 level in brain of juvenile common carp (Cyprinus carpio) after chronic exposure to tributyltin.

Tributyltin (TBT), a toxic contaminant in aquatic environments, has bio-accumulated in aquatic food webs throughout the world and can be found at toxic levels in some biota. However, the molecular mechanisms and effects of TBT are not fully understood. The aim of the present study was to investigate the effect of long-term exposure of TBT on cytochrome P450 (CYP450) 1 regulation and heat-shock proteins (HSPs) profiling in brain of freshwater teleost. The effects of long-term exposure to TBT on mRNA expression of cytochrome P450 (CYP450) 1 family genes and ethoxyresorufin O-deethylase (EROD) activity in the brain of common carp were evaluated, as well as HSP 70 level. Fish were exposed to sublethal concentrations of TBT (75 ng/L, 0.75 μg/L and 7.5 μg/L) for 15, 30, and 60 days. Based on the results, long-term exposure (more than 15 days) to TBT could lead to obvious physiological-biochemical responses (based on EROD activity, HSP 70 level and CYP450 1 family genes expression). The mRNA expression of CYP450 1 family genes (CYP1A, CYP1B, CYP1C1 and CYP1C2) suggested that CYP1A was to accommodate most EROD activity in fish, but other CYP450 forms also involved in this proceeding. Thus, the measured physiological responses in fish brain could provide useful information to better understand the mechanisms of TBT-induced bio-toxicity and could be used as potential biomarkers for monitoring the TBT pollution in the field.

How to spot heritable breast cancer: A primary care physician’s guide

Because breast cancer is common, primary care physicians will encounter many patients who have a personal or family history of it. Many patients may benefit from referral to a cancer genetics program for assessment, genetic counseling, and consideration of genetic testing. This article discusses the complexities of risk assessment in cancer genetics (focusing on breast cancer), and highlights the primary care physician's role in identifying and caring for patients at risk.