Benzo[a]pyrene (BaP) is a human carcinogen that covalently binds to DNA after metabolic activation by cytochrome P450 (CYP) enzymes. In this study human recombinant CYPs (CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C19, 2E1, 3A4, and 3A5) were expressed in Supersomes™ together with their reductases, NADPH:CYP oxidoreductase, epoxide hydrolase and cytochrome b5 , to investigate BaP metabolism. Human CYPs produced up to eight BaP metabolites. Among these, BaP-7,8-dihydrodiol and BaP-9-ol, which are intermediates in BaP-derived DNA adduct formation, were mainly formed by CYP1A1 and 1B1, and to a lesser extent by CYP2C19 and 3A4. BaP-3-ol, a metabolite that is a 'detoxified' product of BaP, was formed by most human CYPs tested, although CYP1A1 and 1B1 produced it the most efficiently. Based on the amounts of the individual BaP metabolites formed by these CYPs and their expression levels in human liver, we determined their contributions to BaP metabolite formation in this organ. Our results indicate that hepatic CYP1A1 and CYP2C19 are most important in the activation of BaP to BaP-7,8-dihydrodiol, whereas CYP2C19, 3A4, and 1A1 are the major enzymes contributing to the formation of BaP-9-ol. BaP-3-ol is predominantly formed by hepatic CYP3A4, while CYP1A1 and 2C19 are less active.

• Keywords
• benzo[a]pyrene, cytochrome P450, human liver, metabolism,
• MeSH
• DNA Adducts metabolism   MeSH
• Benzo(a)pyrene metabolism   pharmacokinetics   MeSH
• Microsomes, Liver metabolism   MeSH
• Liver enzymology   metabolism   MeSH
• Rabbits MeSH
• Humans MeSH
• Inactivation, Metabolic MeSH
• Oxidation-Reduction MeSH
• Cytochrome P-450 Enzyme System genetics   metabolism   MeSH
• Chromatography, High Pressure Liquid MeSH
• Animals MeSH
• Check Tag
• Rabbits MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA Adducts MeSH
• benzo(a)pyrene-DNA adduct MeSH Browser
• Benzo(a)pyrene MeSH
• Cytochrome P-450 Enzyme System MeSH

ABSTRACT: The microsomal protein cytochrome b5 , which is located in the membrane of the endoplasmic reticulum, has been shown to modulate many reactions catalyzed by cytochrome P450 (CYP) enzymes. We investigated the influence of exposure to the anticancer drug ellipticine and to two environmental carcinogens, benzo[a]pyrene (BaP) and 1-phenylazo-2-naphthol (Sudan I), on the expression of cytochrome b5 in livers of rats, both at the mRNA and protein levels. We also studied the effects of these compounds on their own metabolism and the formation of DNA adducts generated by their activation metabolite(s) in vitro. The relative amounts of cytochrome b5 mRNA, measured by real-time polymerase chain reaction analysis, were induced by the test compounds up to 11.7-fold in rat livers. Western blotting using antibodies raised against cytochrome b5 showed that protein expression was induced by up to sevenfold in livers of treated rats. Microsomes isolated from livers of exposed rats catalyzed the oxidation of ellipticine, BaP, and Sudan I and the formation of DNA adducts generated by their reactive metabolite(s) more effectively than hepatic microsomes isolated from control rats. All test compounds are known to induce CYP1A1. This induction is one of the reasons responsible for increased oxidation of these xenobiotics by microsomes. However, induction of cytochrome b5 can also contribute to their enhanced metabolism.

• Keywords
• DNA, Drug research, Enzymes, High pressure liquid chromatography,
• Publication type
• Journal Article MeSH

Aristolochic acid I (AAI) is a plant alkaloid causing aristolochic acid nephropathy, Balkan endemic nephropathy and their associated urothelial malignancies. AAI is detoxified by cytochrome P450 (CYP)-mediated O-demethylation to 8-hydroxyaristolochic acid I (aristolochic acid Ia, AAIa). We previously investigated the efficiencies of human and rat CYPs in the presence of two other components of the mixed-functions-oxidase system, NADPH:CYP oxidoreductase and cytochrome b₅, to oxidize AAI. Human and rat CYP1A are the major enzymes oxidizing AAI. Other CYPs such as CYP2C, 3A4, 2D6, 2E1, and 1B1, also form AAIa, but with much lower efficiency than CYP1A. Based on velocities of AAIa formation by examined CYPs and their expression levels in human and rat livers, here we determined the contributions of individual CYPs to AAI oxidation in these organs. Human CYP1A2 followed by CYP2C9, 3A4 and 1A1 were the major enzymes contributing to AAI oxidation in human liver, while CYP2C and 1A were most important in rat liver. We employed flexible in silico docking methods to explain the differences in AAI oxidation in the liver by human CYP1A1, 1A2, 2C9, and 3A4, the enzymes that all O-demethylate AAI, but with different effectiveness. We found that the binding orientations of the methoxy group of AAI in binding centers of the CYP enzymes and the energies of AAI binding to the CYP active sites dictate the efficiency of AAI oxidation. Our results indicate that utilization of experimental and theoretical methods is an appropriate study design to examine the CYP-catalyzed reaction mechanisms of AAI oxidation and contributions of human hepatic CYPs to this metabolism.

• Keywords
• contribution of cytochromes P450 in detoxification of aristolochic acid I in human and rat livers, cytochrome P450-mediated detoxification of aristolochic acid I, molecular modeling, plant nephrotoxin and carcinogen aristolochic acid I,
• MeSH
• Cytochrome P-450 Enzyme Inhibitors pharmacology   MeSH
• Microsomes, Liver drug effects   metabolism   MeSH
• Liver drug effects   metabolism   MeSH
• Catalytic Domain MeSH
• Catalysis MeSH
• Rats MeSH
• Aristolochic Acids adverse effects   chemistry   metabolism   MeSH
• Humans MeSH
• Activation, Metabolic MeSH
• Inactivation, Metabolic MeSH
• Methylation drug effects   MeSH
• Molecular Conformation MeSH
• Models, Molecular MeSH
• Kidney Diseases etiology   metabolism   MeSH
• Oxidation-Reduction drug effects   MeSH
• Cytochrome P-450 Enzyme System chemistry   metabolism   MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• aristolochic acid I MeSH Browser
• Cytochrome P-450 Enzyme Inhibitors MeSH
• Aristolochic Acids MeSH
• Cytochrome P-450 Enzyme System MeSH

Sudan I [1-(phenylazo)-2-hydroxynaphthalene, C.I. Solvent Yellow 14, CAS No: 842-07-9] is used as the compound employed in chemical industry and to color materials such as hydrocarbon solvents, oils, fats, waxes, plastics, printing inks, shoe and floor polishes and gasoline. Such a wide used could result in a considerable human exposure. Sudan I is known to cause developments of tumors in the liver or urinary bladder in rats, mice, and rabbits, and is considered a possible weak human carcinogen and mutagen. This carcinogen is also a potent contact allergen and sensitizer. Here, we compare the data concerning the Sudan I oxidative metabolism catalyzed by cytochrome P450 (CYP) and peroxidase enzymes, which has been investigated in our laboratory during the last two decades. These two types of enzymes are responsible both for Sudan I detoxication and activation. Among the Sudan I metabolites, C-hydroxylated derivatives and a dimer of Sudan I are suggested to be the detoxication metabolites formed by CYPs and peroxidases, respectively. Metabolic activation of Sudan I by both types of enzymes leads to formation of reactive species (the benzenediazonium ion by CYP and Sudan I radicals by peroxidase) that bind to DNA and RNA, generating covalent adducts in vitro and in vivo. Whereas the structure of the major adduct formed by the benzenediazonium ion in DNA has already been identified to be the 8-(phenylazo)guanine adduct, the structures of adducts formed by peroxidase, have not been characterized as yet. Biological significance of the DNA adducts of Sudan I activated with CYP and peroxidase enzymes and further aims of investigations in this field are discussed in this study.

• Keywords
• Sudan I, carcinogenic azo dye, cytochrome P450, oxidative activation, peroxidase,
• Publication type
• Journal Article MeSH

Cytochrome b(5) (cyt b(5)), a component of endoplasmic reticulum membrane, plays a role in modulation of enzymatic activity of some cytochrome P450 (CYP) enzymes. The effect of apo-cytochrome b(5) on this enzymatic system has not been investigated in details, because preparation of cyt b(5) as a pure protein failed in many laboratories. In order to prepare the native apo-cytochrome b(5) in a large scale we utilized a protein with higher affinity toward the heme; the apo-myoglobin from the equine skeletal muscle. In the first step, we extracted heme moiety from the native myoglobin by butanone extraction. Than the effect of pH on spontaneous heme release from both proteins was investigated: purified rabbit cyt b(5) as well as equine skeletal muscle myoglobin. The prepared apo-myoglobin was incubated with the cyt b(5) and heme transfer was monitored as a shift of absorption maximum from 413 to 409 nm in pH varying between 3-6 (10 mM KH(2)PO(4), pH 3-6). Here, we obtained 43 mg of the equine skeletal muscle apo-myoglobin (43% yield). The optimal pH range for heme transfer from cyt b(5) into apo-myoglobin was between 4.2 and 5. Native apo-cytochrome b(5) was successfully prepared using procedure described here.

• Keywords
• apo-myoglobin, butanon extraction, cytochrome b5, pH,
• Publication type
• Journal Article MeSH

2-Nitroanisole (2-NA) is an important industrial pollutant and a potent carcinogen for rodents. Understanding which cytochrome P450 (CYP) enzymes are involved in its metabolism are important to assess an individual's susceptibility to this environmental carcinogen. The aim of this study was to evaluate the efficiency of rat hepatic CYPs to oxidize 2-NA, to examine the metabolites formed during such an oxidation, and to compare such efficiencies of rat CYPs with those of human. 2-NA is oxidized by rat hepatic microsomes to 2-nitrophenol (2-NP) as the major metabolite, and to 2,6-dihydroxynitrobenzene (2,6-DNB) and 2,5-dihydroxynitrobenzene (2,5-DNB) as the minor products. All these metabolites are suggested as detoxication products. Using hepatic microsomes of rats pre-treated with specific CYP inducers and microsomes from Baculovirus transfected insect cells expressing recombinant rat and human CYP enzymes we found that rat recombinant CYP2E1, 2D2, 2B2, 2C6 and 1A1, as well as orthologous human CYP enzymes are the most efficient enzymes metabolizing 2-NA. However, human CYP1A1 oxidize 2-NA with a higher efficiency than the enzyme of rats. The results show the participation of orthologous CYPs in 2-NA oxidation by both species and underline the suitability of rat species as a model to evaluate human susceptibility to 2-NA.

• Keywords
• 2-nitroanisole, 2-nitrophenol, cytochrome P450, detoxication, metabolism, oxidation,
• Publication type
• Journal Article MeSH