Mouse Cytochrome P450 Enzymes Research Articles

Effects of Polygonum hydropiper on the expressions of inflammatory cytokines and cytochrome P450 enzymes in mice with E. coli-induced diarrhea

Effects of Polygonum hydropiper on the expressions of inflammatory cytokines and cytochrome P450 enzymes in mice with E. coli-induced diarrhea

Exposure to an Environmental Mixture of Polycyclic Aromatic Hydrocarbons Induces Hepatic Cytochrome P450 Enzymes in Mice.

Cytochrome P450 enzymes (CYPs) play an important role in bioactivating or detoxifying polycyclic aromatic hydrocarbons (PAHs), common environmental contaminants. While it is widely accepted that exposure to PAHs induces CYPs, effectively increasing rates of xenobiotic metabolism, dose- and time-response patterns of CYP induction are not well-known. In order to better understand dose- and time-response relationships of individual CYPs following induction, we exposed B6129SF1/J mice to single or repeated doses (2-180 μmol/kg/d) of benzo[a]pyrene (BaP) or Supermix-10, a mixture of the top 10 most abundant PAHs found at the Portland Harbor Superfund Site. In hepatic microsomes from exposed mice, we measured amounts of active CYPs using activity-based protein profiling and total CYP expression using global proteomics. We observed rapid Cyp1a1 induction after 6 h at the lowest PAH exposures and broad induction of many CYPs after 3 daily PAH doses at 72 h following the first dose. Using samples displaying Cyp1a1 induction, we observed significantly higher metabolic affinity for BaP metabolism (Km reduced 3-fold), 3-fold higher intrinsic clearance, but no changes to the Vmax. Mice dosed with the highest PAH exposures exhibited 1.7-5-fold higher intrinsic clearance rates for BaP compared to controls and higher Vmax values indicating greater amounts of enzymes capable of metabolizing BaP. This study demonstrates exposure to PAHs found at superfund sites induces enzymes in dose- and time-dependent patterns in mice. Accounting for specific changes in enzyme profiles, relative rates of PAH bioactivation and detoxification, and resulting risk will help translate internal dosimetry of animal models to humans and improve risk assessments of PAHs at superfund sites.

Novel Therapeutic Approaches Against Acetaminophen-induced Liver Injury and Acute Liver Failure.

Liver injury and acute liver failure caused by acetaminophen (APAP, N-acetyl-p-aminophenol, paracetamol) overdose is a significant clinical problem in most western countries. The only clinically approved antidote is N-acetylcysteine (NAC), which promotes the recovery of hepatic GSH. If administered during the metabolism phase, GSH scavenges the reactive metabolite N-acetyl-p-benzoquinone imine. More recently, it was shown that NAC can also reconstitute mitochondrial GSH levels and scavenge reactive oxygen/peroxynitrite and can support mitochondrial bioenergetics. However, NAC has side effects and may not be efficacious after high overdoses. Repurposing of additional drugs based on their alternate mechanisms of action could be a promising approach. 4-Methylpyrazole (4MP) was shown to be highly effective against APAP toxicity by inhibiting cytochrome P450 enzymes in mice and humans. In addition, 4MP is a potent c-Jun N-terminal kinase inhibitor expanding its therapeutic window. Calmangafodipir (CMFP) is a SOD mimetic, which is well tolerated in patients and has the potential to be effective after severe overdoses. Other drugs approved for humans such as metformin and methylene blue were shown to be protective in mice at high doses or at human therapeutic doses, respectively. Additional protective strategies such as enhancing antioxidant activities, Nrf2-dependent gene induction and autophagy activation by herbal medicine components are being evaluated. However, at this point, their mechanistic insight is limited, and the doses used are high. More rigorous mechanistic studies are needed to advance these herbal compounds. Nevertheless, based on recent studies, 4-methylpyrazole and calmangafodipir have realistic prospects to become complimentary or even alternative antidotes to NAC for APAP overdose.

Role of Mouse Cytochrome P450 Enzymes of the Cyp2abfgs Subfamilies in the Induction of Lung Inflammation by Cigarette Smoke Exposure.

Many constituents of tobacco smoke (TS) require bioactivation to exert toxic effects; however, few studies have examined the role of bioactivation enzymes in the adverse effects of TS exposure. This knowledge gap is a major source of uncertainty for risk assessment and chemoprevention efforts. Our aim is to test the hypothesis that cytochrome P450 (P450) enzyme-mediated bioactivation is essential to the development of TS exposure-induced lung toxicity, by determining the contributions of P450 enzymes in the mouse Cyp2abfgs gene subfamilies to environmental tobacco smoke (ETS)-induced lung inflammation. Adult female wildtype (WT) and Cyp2abfgs-null mice (both on C57BL/6J background) were exposed to filtered air or ETS, intermittently, for 1 or 2 weeks. Lung inflammation was assessed by quantification of inflammatory cells, cytokines, chemokines, and proteins in bronchoalveolar lavage fluid (BALF) and histopathological analysis. Glutathione (GSH) conjugates of 2 ETS constituents, naphthalene (NA), and 3-methylindole (3MI), were measured in mice exposed to ETS for 4 h. Persistent macrophagic and neutrophilic lung inflammation was observed in ETS-exposed WT mice; the extent of which was significantly reduced in ETS-exposed Cyp2abfgs-null mice. Levels of proinflammatory cytokines and chemokines, along with the total protein concentration, were increased in cell-free BALF from ETS-exposed WT mice, but not Cyp2abfgs-null mice. Additionally, GSH conjugates of NA and 3MI were detected in the lungs of WT, but not Cyp2abfgs-null, mice following ETS exposure. These results provide the first in vivo evidence that the mouse Cyp2abfgs gene cluster plays an important role in ETS-induced lung inflammation.

Benzyl alcohol protects against acetaminophen hepatotoxicity by inhibiting cytochrome P450 enzymes but causes mitochondrial dysfunction and cell death at higher doses

Acetaminophen (APAP) hepatotoxicity is a serious public health problem in western countries. Current treatment options for APAP poisoning are limited and novel therapeutic intervention strategies are needed. A recent publication suggested that benzyl alcohol (BA) protects against APAP hepatotoxicity and could serve as a promising antidote for APAP poisoning. To assess the protective mechanisms of BA, C56Bl/6J mice were treated with 400 mg/kg APAP and/or 270 mg/kg BA. APAP alone caused extensive liver injury at 6 h and 24 h post-APAP. This injury was attenuated by BA co-treatment. Assessment of protein adduct formation demonstrated that BA inhibits APAP metabolic activation. In support of this, in vitro experiments also showed that BA dose-dependently inhibits cytochrome P450 activities. Correlating with the hepatoprotection of BA, APAP-induced oxidant stress and mitochondrial dysfunction were reduced. Similar results were obtained in primary mouse hepatocytes. Interestingly, BA alone caused mitochondrial membrane potential loss and cell toxicity at high doses, and its protective effect could not be reproduced in primary human hepatocytes (PHH). We conclude that BA protects against APAP hepatotoxicity mainly by inhibiting cytochrome P450 enzymes in mice. Considering its toxic effect and the loss of protection in PHH, BA is not a clinically useful treatment option for APAP overdose patient.

Characterization of Four New Mouse Cytochrome P450 Enzymes of the CYP2J Subfamily

The cytochrome P450 superfamily encompasses a diverse group of enzymes that catalyze the oxidation of various substrates. The mouse CYP2J subfamily includes members that have wide tissue distribution and are active in the metabolism of arachidonic acid (AA), linoleic acid (LA), and other lipids and xenobiotics. The mouse Cyp2j locus contains seven genes and three pseudogenes located in a contiguous 0.62 megabase cluster on chromosome 4. We describe four new mouse CYP2J isoforms (designated CYP2J8, CYP2J11, CYP2J12, and CYP2J13). The four cDNAs contain open reading frames that encode polypeptides with 62-84% identity with the three previously identified mouse CYP2Js. All four new CYP2J proteins were expressed in Sf21 insect cells. Each recombinant protein metabolized AA and LA to epoxides and hydroxy derivatives. Specific antibodies, mRNA probes, and polymerase chain reaction primer sets were developed for each mouse CYP2J to examine their tissue distribution. CYP2J8 transcripts were found in the kidney, liver, and brain, and protein expression was confirmed in the kidney and brain (neuropil). CYP2J11 transcripts were most abundant in the kidney and heart, with protein detected primarily in the kidney (proximal convoluted tubules), liver, and heart (cardiomyocytes). CYP2J12 transcripts were prominently present in the brain, and CYP2J13 transcripts were detected in multiple tissues, with the highest expression in the kidney. CYP2J12 and CYP2J13 protein expression could not be determined because the antibodies developed were not immunospecific. We conclude that the four new CYP2J isoforms might be involved in the metabolism of AA and LA to bioactive lipids in mouse hepatic and extrahepatic tissues.

Addendum

This addendum was compiled by the authors to clarify some points presented in the review article titled “Expression, Function and Regulation of Mouse Cytochrome P450 Enzymes: Comparison With Human Cytochrome P450 Enzymes”, which appeared in Current Drug Metabolism (2009) 10 (10), 1151-1183. Table 1 lists Cyp2c22, Cyp2c23, Cyp2c51 and Cyp2b20 as mouse P450 genes. Mouse Cyp2c22 and Cyp2c23 genes (ref. 440) should not be included in Table 1 because they are believed to be the same as Cyp2c70 and Cyp2c44, respectively (see mouse Master Table in Dr. Nelsons P450 homepage, http://drnelson.uthsc.edu/mouse.table.html). The mouse Cyp2c51 gene (ref. 119) is probably a combination of mouse Cyp2c52-ps and Cyp2c69 genes and may not be a separate gene (see Dr. Nelsons P450 homepage). The mouse Cyp2b20 gene (Swiss-Prot accession number Q62397) may be identical with Cyp2b10, although recent studies (refs. 9, 60 and 180) have suggested that Cyp2b20 and Cyp2b10 may be different genes. This matter remains to be resolved. If the following mouse P450 genes, Cyp2c22, Cyp2c23, Cyp2c51 and Cyp2b20, are removed from Table 1 and the Cyp2ab1 gene (which is not a pseudogene, ref. 8) is added, then the total mouse P450 gene count would be 102 genes, as originally reported in ref. 8, and not 105 genes as stated on page 1151. With regard to the human cytochrome P450 data presented in Table 2, CYP4F3A and CYP4F3B enzymes were generated by alternative splicing of the CYP4F3 gene that encodes the CYP4F3 enzyme (ref. 174). Thus, CYP4F3A and CYP4F3B enzymes are not encoded by separate cytochrome P450 genes. It thus appears that there are 57 human P450 genes, as originally reported in ref. 8, and not 58 genes as stated on page 1151. In addition to the human P450 enzymes listed in Table 2, additional human P450 enzymes have been discovered as a result of gene splicing. The authors are indebted to Drs. Daniel Nebert and David Nelson for their expert advice on the subject matter presented in this addendum.

Expression, Function and Regulation of Mouse Cytochrome P450 Enzymes: Comparison With Human Cytochrome P450 Enzymes

The present review focuses on the expression, function and regulation of mouse cytochrome P450 (Cyp) enzymes. Information compiled for mouse Cyp enzymes is compared with data collected for human CYP enzymes. To date, approximately 40 pairs of orthologous mouse-human CYP genes have been identified that encode enzymes performing similar metabolic functions. Recent knowledge concerning the tissue expression of mouse Cyp enzymes from families 1 to 51 is summarized. The catalytic activities of microsomal, mitochondrial and recombinant mouse Cyp enzymes are discussed and their involvement in the metabolism of exogenous and endogenous compounds is highlighted. The role of nuclear receptors, such as the aryl hydrocarbon receptor, constitutive androstane receptor and pregnane X receptor, in regulating the expression of mouse Cyp enzymes is examined. Targeted disruption of selected Cyp genes has generated numerous Cyp null mouse lines used to decipher the role of Cyp enzymes in metabolic, toxicological and biological processes. In conclusion, the laboratory mouse is an indispensable model for exploring human CYP-mediated activities.

Cloning, expression, and characterization of three new mouse cytochrome p450 enzymes and partial characterization of their fatty acid oxidation activities.

The mammalian CYP2C subfamily is one of the largest and most complicated in the cytochrome P450 superfamily. In this report, we describe the organization of the mouse Cyp2c locus, which contains 15 genes and four pseudogenes, all of which are located in a 5.5-megabase region on chromosome 19. We cloned three novel mouse CYP2C cDNAs (designated CYP2C50, CYP2C54, and CYP2C55) from mouse heart, liver, and colon, respectively. All three cDNAs contain open reading frames that encode 490 amino acid polypeptides that are 57 to 95% identical to other CYP2Cs. The recombinant CYP2C proteins were expressed in Escherichia coli after N-terminal modification, partially purified, and shown to be active in the metabolism of both arachidonic acid (AA) and linoleic acid, albeit with different catalytic efficiencies and profiles. CYP2C50 and CYP2C54 metabolize AA to epoxyeicosatrienoic acids (EETs) primarily, and linoleic acid to epoxyoctadecenoic acids (EOAs) primarily, whereas CYP2C55 metabolizes AA to EETs and hydroxyeicosatetraenoic acids and linoleic acid to EOAs and hydroxyoctadecadienoic acids. Northern blotting and reverse transcription-polymerase chain reaction analysis reveal that CYP2C50 transcripts are abundant in liver and heart; CYP2C54 transcripts are present in liver, kidney, and stomach; and CYP2C55 transcripts are abundant in liver, colon, and kidney. Immunoblotting studies demonstrate that CYP2C50 protein is expressed in liver and heart, CYP2C54 protein is detected primarily in liver, and CYP2C55 protein is present primarily in colon. Immunohistochemistry reveals that CYP2C55 is most abundant in surface columnar epithelium in the cecum. We conclude that these new CYP2C enzymes are probably involved in AA and linoleic acid metabolism in mouse hepatic and extrahepatic tissues.

Effects of musk xylene and musk ketone on rat hepatic cytochrome P450 enzymes

The purpose of the present work was to characterize the effect of musk xylene (MX) and musk ketone (MK) treatment on rat hepatic cytochrome P450 enzymes. Male F344 rats were dosed orally with MX (10, 50 or 200 mg/kg) or MK (20, 100 or 200 mg/kg) for 7 days, after which CYP1A, 2B and 3A enzyme activities and protein levels were determined. MX treatment resulted in a two- to four-fold increase in the activity of CYP1A, 2B and 3A enzymes. For CYP1A and 3A, these changes were consistent with small increases in immunoreactive proteins. However, for CYP2B, despite only a three-fold increase in enzyme activity, protein levels were increased nearly 50-fold relative to control. This induction occurred by transcriptional activation of the CYP2B1 gene as evidenced by increased steady state CYP2B1 mRNA levels. In contrast to MX, MK treatment increased CYP2B activity, protein and mRNA levels. However MK treatment also increased CYP1A enzyme activity nearly 30-fold higher than control rats, a profile that was markedly different from MX, and very different from its effects in mice (Stuard, S.B., Caudill, D., Lehman-McKeeman, L.D., 1997. Characterization of the effects of musk ketone on mouse cytochrome P450 enzymes. Fund. Appl. Toxicol. 40, 264–271). These results indicate that in rats, MX is an inducer of CYP2B enzymes, but these enzymes are not functionally active. In contrast, MK also induces CYP2B enzymes, with no concurrent inactivation. MK also exhibits a unique pattern of cytochrome P450 induction by increasing both CYP1A and CYP2B in rats.

Initial characterization of the major mouse cytochrome p450 enzymes involved in the reductive metabolism of the hypoxic cytotoxin 3-Amino-1,2,4-Benzotriazine-1,4- di- N-Oxide (Tirapazamine, SR 4233, Win 59075)

The benzotriazine di- N-oxide SR4233 (tirapazamine, WIN 59075) is currently in phase I clinical trials as the lead compound in a series of novel and highly selective antitumour hypoxic cytotoxins. Reductive bioactivation is thought to proceed via a one-electron reduced, oxidizing nitroxide radical and also forms the inactive single N-oxide SR4317 via radical disproportionation or a second oneelectron reduction. In mouse liver microsomes reductive metabolism is catalysed predominantly by cytochrome P450 (70%) and cytochrome P450 reductase (30%). The aim of the present study was to examine which cytochrome P450 isozymes may be involved. Reduction of SR4233 to SR4317 was monitored by HPLC analysis. Metabolism by microsomes from both control and dexamethasone-induced BALB/c male mice was 70% inhibited by carbon monoxide. The cytochrome P450 inhibitor SKF 525A, following aerobic preincubation, also inhibited SR4233 reduction by 58%. Reduction was induced 2–3-fold by dexamethasone and was not accountable by increases in cytochrome P450 reductase or DT-diaphorase. The induction data and the greater degree of inhibition of SR 4233 reduction by metyrapone compared to α-naphthoflavone suggested a possible involvement of Cyp2b, Cyp2c and Cyp3a cytochrome P450 subfamilies. Both Cyp3a (7.4-fold) and Cyp2b (1.8-fold) type enzymes were shown by western immunoblot analysis to be induced by dexamethasone, the latter correlating more closely with increased SR 4233 reductase activity and also with the 2-fold induction of benzphetamine TV-demethylase, a Cyp2b-type enzyme. No inhibition of SR4233 reduction was seen with erythromycin or cyclosporin A which act as substrates/inhibitors for Cyp3a-type enzymes, but inhibition was seen with p-nitrophenol and tolbutamide which are substrates for Cyp2el- and Cyp2c-type enzymes, respectively (11% and 25% inhibition in induced microsomes). SR4233 itself inhibited benzphetamine N-demethylase, which is catalysed by Cyp2b-type enzymes but not erythromycin N-demethylase which is catalysed by Cyp3a-type isoforms. Immunoinhibition studies with epitope specific monoclonal antibodies were consistent with the major involvement of phenobarbitone- and steroid-inducible products of the Cyp2b and Cyp2c subfamilies. These forms contributed at least 53% and 26%, respectively, of the cytochrome P450-associated SR4233 reductase activity in the induced microsomes. The findings support our earlier conclusion that cytochrome P450 is the major SR 4233 reductase in mouse liver and provides leads as to the possible involvement of specific isoforms in human tumours and normal tissues.