Genetically Engineered Bacterial Cells Expressing Human Cytochrome P450 as an Alternative Tool in the Prediction of Metabolism and Toxicity of Chemicals in Humans

Ken-ichi Fujita, Hirotaka Kushida, Hiroshi Iwata, Akihiro Suzuki,Yoshiyuki Yamazaki, Kazuo Nakayama and Tetsuya Kamataki

Laboratory of Drug Metabolism, Division of Pharmacobio-dynamics, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-ku N12 W6, Sapporo, 060-0812, Japan

Correspondence: Ken-ichi Fujita
Laboratory of Drug Metabolism, Division of Pharmacobio-dynamics, Graduate School of Pharmaceutical Sciences, Hokkaido University, N12W6 Kita-ku, Sapporo, 060-0812 JAPAN
Tel.+81-11-706-3234 Fax.+81-11-716-5324

AATEX 6(2,3):97-127,2000

Genetically engineered bacterial cells expressing human cytochrome P450 (P450 or CYP) have provided new tools, as an alternative method for experimental animals and human specimens. Using the bacterial system, it is possible to investigate the functions of human P450 in the detoxification or the metabolic activation of various xenobiotics and the metabolism of endogenous compounds. This review focused on the development of bacterial cells expressing human P450, and the application of this system to drug metabolism and toxicological studies. There are many kinds of host cells for the heterologous expression of a form of P450. Among them, bacterial cells including Escherichia coli (E. coli) and Salmonella have advantages with regard to ease of use and the high yield of protein. The modification of the N-terminal amino acid sequence of P450 allowed it to express P450 protein in bacterial cells (Barnes et al., 1991). It was an excellent breakthrough for the establishment of an expression system of P450 using bacterial cells. Since then, many isoforms of human P450 have been successfully expressed in bacterial cells. Many reports that appeared so far have indicated that the P450 enzyme expressed in E. coli after modification of the N-terminus showed considerable catalytic activities in systems reconstituted with NADPH-P450 reductase purified from liver microsomes from appropriate animals. Bacterial cells do not possess endogeneous electron transport systems to support the full catalytic activity of P450 expressed in bacterial cells. Thus, systems co-expressing both P450 and other electron transport enzymes from NADPH or NADH to P450s have been established. The catalytic activities were detected even if the whole cells of bacteria co-expressing P450 with the reductase were used. Recently, these strains of bacteria were applied to analyze the toxicological and pharmacological roles of P450 in humans. For example, we established Salmonella strains harboring human CYP2A6 or CYP2E1 together with the reductase, and clarified that CYP2A6 was responsible for the activation of N-nitrosamines with relatively long alkyl chain(s) such as NNK, NNN and NMPhA, whereas CYP2E1 was involved in the activation of N-nitrosamines with relatively short alkyl chain(s) such as NDMA and NDEA. These strains of bacteria may be useful to study drug metabolism and toxicology in humans, and may be an alternative method to those using experimental animals.

Key words: E.coli, Salmonella Typhimurium, Catalytic Activity, Substrate Specificity, Drug-drug Interraction, Mutation Assay

Abbreviations: 2-AA, 2-aminoanthracene; 2-AAF, 2-acetylaminofluorene; AFB1, aflatoxin B1; B[a]P, benzo[a]pyrene; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; IPTG, isopropyl-b-D-thiogalactopyranoside; IQ, 2-amino-3-methylimidazo[4,5-f]quinoline; MeIQ, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline; MeIQx, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline; NADH, nicotinamide adenine dinucleotide reduced form; NADPH, nicotinamide adenine dinucleotide phosphate-reduced form; NDEA, N-nitrosodiethylamine; NDMA, N-nitrosodimethylamine; NMPhA, N-nitrosomethyphenylamine; NNK, 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone; NNN, N-nitrosonornicotine; PAH, polycyclic aromatic hydrocarbon; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; P450 or CYP, cytochrome P450; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin