Roles of Cytochrome P450 in Metabolism of Ethanol and Carcinogens

Abstract

Cytochrome P450 (P450) enzymes are involved in the metabolism of carcinogens, as well as drugs, steroids, vitamins, and other classes of chemicals. P450s also oxidize ethanol, in particular P450 2E1. P450 2E1 oxidizes ethanol to acetaldehyde and then to acetic acid, roles also played by alcohol and aldehyde dehydrogenases. The role of P450 2E1 in cancer is complex in that P450 2E1 is also induced by ethanol, P450 2E1 is involved in the bioactivation and detoxication of a number of chemical carcinogens, and ethanol is an inhibitor of P450 2E1. Contrary to some literature, P450 2E1 expression and induction itself does not cause global oxidative stress in vivo, as demonstrated in studies using isoniazid treatment and gene deletion studies with rats and mice. However, a major fraction of P450 2E1 is localized in liver mitochondria instead of the endoplasmic reticulum, and studies with site-directed rat P450 2E1 mutants and natural human P450 2E1 N-terminal variants have shown that P450 2E1 localized in mitochondria is catalytically active and more proficient in producing reactive oxygen species and damage. The role of the mitochondrial oxidative stress in ethanol toxicity is still under investigation, as is the mechanism of altered electron transport to P450s that localize inside mitochondria instead of their typical endoplasmic reticulum environment.

Keywords: Chemical carcinogens; Cytochrome P450 2E1; Endoplasmic reticulum, and cytochrome P450; Enzyme kinetics; Ethanol; Mitochondria, and cytochrome P450; Mitochondrial toxicity; Oxidation by cytochrome P450, in ethanol oxidation; P450 2E1; Reactive oxygen species.

Fig. 2.1

Human enzymes involved in bioactivation of carcinogens [63]. (a) fractions of enzyme classes involved in bioactivation; (b) individual human P450 contributions to carcinogen activation. FMO microsomal flavin-containing monooxygenase, NAT N-acetyltransferase, SULT sulfotransferase, AKR aldo-keto reductase, COX cyclooxygenase (prostaglandin synthase)

Fig. 2.2

Possible mechanisms of oxidation of ethanol to acetaldehyde and acetic acid [33]. (a) oxidation of ethanol to acetaldehyde. (b) oxidation of acetaldehyde by a perferryloxo mechanism (Compund I). C, oxidation of acetaldehyde by a ferric peroxide mechanism

Fig. 2.3

Kinetic deuterium isotope effects with human P450 2E1 [11, 12]. The results are presented in Hanes-Wolff plots ([S]/v vs. [S]). (a) ethanol to acetaldehyde. CH₃CH₂OH as substrate (k_cat, 2.7 (± 0.2) min⁻¹, K_m 11 (± 2) mM); CH₃CD₂OH as substrate (■, k_cat 2.4 (± 0.1) min⁻¹, K_m 53 (± 6) mM). (b) acetaldehyde to acetic acid. CH₃CHO as substrate (□, k_cat 7.5 (± 0.5) min⁻¹, K_m 0.50 (± 0.2) mM); CH₃CDO as substrate (◆, k_cat 5.0 (± 0.4) min⁻¹, K_m 1.5 (± 0.3) mM). This research was originally published in Bell, L. C., and Guengerich, F. P. [11] Oxidation kinetics of ethanol by human cytochrome P450 2E1. Rate-limiting product release accounts for effects of isotopic hydrogen substitution and cytochrome b₅ on steady-state kinetics. The Journal of Biological Chemistry 272, 29,643–29,651 and Bell-Parikh, L. C., and Guengerich, F. P. [12] Kinetics of cytochrome P450 2E1-catalyzed oxidation of ethanol to acetic acid via acetaldehyde. The Journal of Biological Chemistry 274, 23,833–23,840. © The American Society for Biochemistry and Molecular Biology

Fig. 2.4

A mutational approach for altering the bimodal targeting efficiency of rat P450 2E1 [7]. (a) the WOLFPSORT program was utilized to alter the SRP binding and mitochondria-targeting efficiencies of the N-terminal signal regions. (b) predicted targeting efficiencies of WT and mutant P450 2E1 proteins. This research was originally published in Bansal, S., Liu, C. P., Sepuri, N. B., Anandatheerthavarada, H. K., Selvaraj, V., Hoek, J., Milne, G. L., Guengerich, F. P., and Avadhani, N. G. [7] Mitochondria-targeted cytochrome P450 2E1 induces oxidative damage and augments alcohol-mediated oxidative stress. The Journal of Biological Chemistry 285, 24,609–24,619. © The American Society for Biochemistry and Molecular Biology

Fig. 2.5

Ethanol-induced F₂-isoprostanes in P450 2E1-expressing cells and in liver fractions from ethanol-treated rats [7]. (a) F₂-isoprostanes were assayed using gas chromatography-mass spectrometry. Asterisks represent significant increase in F₂ -isoprostanes in ER⁺, Mt+, and Mt⁺⁺ cells after ethanol treatment (p < 0.05). Values represent the means ± SD of three assays. B, F₂- isopreostanes were measured in mitochondria and microsomes isolated from the livers of rats fed with alcohol for 2–8 weeks (W) and pair-fed controls. In each case 100 μg of protein was used. The means ± SD in the 8-week-fed rats were based on assays carried out in three rats each in control and fed groups. Asterisks represent significant difference (p < 0.05) from pair-fed controls. The values presented in boxes below the graph indicate the ratios of P450 (CYP) 2E1 contents between pair-fed controls and alcohol-fed rat livers. This research was originally published in Bansal, S., Liu, C. P., Sepuri, N. B., Anandatheerthavarada, H. K., Selvaraj, V., Hoek, J., Milne, G. L., Guengerich, F. P., and Avadhani, N. G. [7] Mitochondria-targeted cytochrome P450 2E1 induces oxidative damage and augments alcohol-mediated oxidative stress. The Journal of Biological Chemistry 285, 24,609–24,619. © The American Society for Biochemistry and Molecular Biology

Fig. 2.6

Mitochondrial P450 2E1-induced respiratory deficiency in yeast cells [7]. (a) mitochondrial and microsomal CYP2E1 contents in yeast cells stably expressing WT and mutant rat P450 (CYP) 2E1 cDNA constructs. The mitochondrial and microsomal proteins (50 μg each) were analyzed using immunoblotting with anti-P450 2E1. Two identically run (parallel) blots were probed with antibody to the mitochondria-specific marker Tim23 and the microsome-specific marker dolicholphosphate mannose synthase (DPMS). (b), yeast cells expressing ER⁺, WT, and Mt⁺⁺ rat P450 2E1 were grown in yeast extract/peptone/dextrose medium supplemented with appropriate amino acids. Cells (~ 2.0 OD₆₀₀ units) were pelleted and resuspended in 1 ml of sterile water. The culture was serially diluted 10 times, and 10 μl of each dilution was spotted onto plates containing 2% glucose (w/v) (left panel) and 2% lactate (w/v) (right panel), which were incubated at 30 °C for 4 days. This research was originally published in Bansal, S., Liu, C. P., Sepuri, N. B., Anandatheerthavarada, H. K., Selvaraj, V., Hoek, J., Milne, G. L., Guengerich, F. P., and Avadhani, N. G. [7] Mitochondria-targeted cytochrome P450 2E1 induces oxidative damage and augments alcohol-mediated oxidative stress. The Journal of Biological Chemistry 285, 24,609–24,619. © The American Society for Biochemistry and Molecular Biology

Fig. 2.7

Interindividual variations in P450 2E1 content of human liver samples [8]. (a) immunoblotting analysis of mitoplast (Mt) and microsomal (Mc) fractions isolated from human liver samples (50 μg of protein each) using polyclonal antibodies to human P450 (CYP) 2E1 (1:1000, v/v) and the mitochondrial marker protein mtTFA (1:3000, v/v) and a monoclonal antibody to microsomal NADPH-P450 reductase (1:1500, v/v). (b) densitometric analysis was performed to determine the distribution of P450 2E1 in human mitochondria and microsomes. (c) immunoblot analysis (with anti-human P450 2E1) of human liver mitochondrial and microsomal proteins from liver samples (HL, ‘human liver’) HL114 and HL134, subjected to limited trypsin digestion (150 μg/mg protein, 20 min on ice). (d) N-terminal amino acid sequence of human P450 2E1 protein indicating the ER targeting domain, mitochondrial targeting domain, and proline-rich domain. Variations within the putative signal sequence region are shown by the arrows. This research was originally published in Bansal, S., Anandatheerthavarada, H. K., Prabu, G. K., Milne, G. L., Martin, M. V., Guengerich, F. P., and Avadhani, N. G. [8] Human cytochrome P450 2E1 mutations that alter mitochondrial targeting efficiency and susceptibility to ethanol-induced toxicity in cellular models. The Journal of Biological Chemistry 288, 12,627–12,644. © The American Society for Biochemistry and Molecular Biology

Fig. 2.8

Effects of ethanol on cellular toxicity in cells expressing WT and mutant human P450 2E1 constructs [8] (a) cellular GSH levels (nmol/mg protein). (b) ethanol-induced F₂-isoprostanes in human P450 2E1-expressing cells, assayed using gas chromatography-mass spectrometry (expressed as ng/2 × 10⁶ total cells). (c) mitochondrial membrane potential (ΔΨ_m) measured spec-trophotometrically in stable cells using a fluorescent dye, tetramethylrhodamine methyl ester (TMRM). Briefly, cells were incubated with and without ethanol and also treated with disulfiram (25 μM) overnight. Cells were washed and loaded with 150 nM tetramethylrhodamine methyl ester in an assay involving a Chameleon microplate reader (excitation wavelength 535 nm, emission wavelength 590 nm). For a control, 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used in each series. *, p <0.05; **, p <0.001. Values represent means ± SE from three independent measurements. A.U., arbitrary units. This research was originally published in Bansal, S., Anandatheerthavarada, H. K., Prabu, G. K., Milne, G. L., Martin, M. V., Guengerich, F. P., and Avadhani, N. G. [8] Human cytochrome P450 2E1 mutations that alter mitochondrial targeting efficiency and susceptibility to ethanol-induced toxicity in cellular models. The Journal of Biological Chemistry 288, 12,627–12,644. © The American Society for Biochemistry and Molecular Biology

Fig. 2.9

Subcellular distribution and ethanol-mediated ROS in HepG2 cells stably expressing WT or mutant human P450 2E1. [8] (a) mitochondria and microsomes from HepG2 cells stably expressing human P450 2E1 cDNAs. Mitochondrial and microsomal fractions (50 μg of protein) were subjected to immunoblotting analysis (anti- human P450 2E1). The blot was also co- developed with anti-NADPH-P450 reductase (a microsomal marker) and porin (a mitochondrial marker) to assess relative cross-contamination. (b) percentage subcellular distribution was calculated based on band intensity. (c) ROS levels in whole cells grown with or without ethanol (300 mM) were estimated using the dye dichlorofluorescin (DCF) as the substrate. Cells were treated with the mitochondria-targeted antioxidant Mito-CP (2.5 μM)and the P450 2E1 inhibitor disulfiram (25 μM) (cell-permeable superoxide dismutase and catalase were used as controls). Values represent means ± SD (error bars) from three independent experiments. *, p <0.05; **, p <0.001. This research was originally published in Bansal, S., Anandatheerthavarada, H. K., Prabu, G. K., Milne, G. L., Martin, M. V., Guengerich, F. P., and Avadhani, N. G. [8] Human cytochrome P450 2E1 mutations that alter mitochondrial targeting efficiency and susceptibility to ethanol-induced toxicity in cellular models. The Journal of Biological Chemistry 288, 12,627–12,644. © the American Society for Biochemistry and Molecular Biology