Ken-ichi Fujita

Inhibition by green tea catechins of metabolic activation of procarcinogens by human cytochrome P450.

Catechins, major polyphenol constituents of green tea, are potent chemopreventive agents against cancers caused by chemical carcinogens in rodents. The effects of four epicatechin derivatives, epigallocatechin gallate (EGCG), epicatechin gallate (ECG), epigallocatechin (EGC) and epicatechin (EC), on the metabolic activation of benzo[a]pyrene (B[a]P), 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine (PhIP) and aflatoxin B(1) (AFB(1)) by human cytochrome P450 (CYP) were examined. B[a]P, PhIP and AFB(1) were activated by respective human CYP1A1, CYP1A2 and CYP3A4 expressed in the membrane fraction of genetically engineered Salmonella typhimurium (S. typhimurium) TA1538 cells harboring the human CYP and human NADPH-CYP reductase (OR), when the membrane fraction was added to S. typhimurium TA98. Galloylated catechins, ECG and EGCG inhibited the mutagenic activation potently, while EGC and EC showed relatively weak inhibitory effects. Catechins also inhibited the oxidations of typical substrates catalyzed by human CYPs, namely ethoxycoumarin O-deethylation by CYP1A1, ethoxyresorufin O-deethylation by CYP1A2 and midazolam 1-hydroxylation by CYP3A4. The IC(50) values of catechins for the inhibition of human CYP were roughly the same as those seen in the mutagenic activation. EGCG inhibited other forms of human CYP such as CYP2A6, CYP2C19 and CYP2E1, indicating the non-specific inhibitory effects of EGCG toward human CYPs. Furthermore, EGCG inhibited human NADPH-cytochrome CYP reductase (OR) with a K(i) value of 2.5 microM. These results suggest that the inhibition of the enzyme activity of CYP is accounted for partially by the inhibition of OR.

High Catalytic Activity of Human Cytochrome P450 Co-expressed with Human NADPH-Cytochrome P450 Reductase in Escherichia coli

Forms of human cytochrome P450 (P450 or CYP), such as CYP1A1, CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4, were expressed or co-expressed together with human NADPH-P450 reductase in Escherichia coli. When P450 was expressed alone in E. coli, the expression level of holo-P450 ranged from 310 to 1620 nmol/L of culture. The expression level of holo-P450 decreased by co-expression with the reductase, and the level ranged from 66 to 381 nmol/L of culture. The expression level of the reductase varied depending on the forms of P450 co-expressed, and ranged from 204 to 937 U/L of culture. We assayed the catalytic activity of P450 using E. coli cells disrupted by freeze-thaw. When co-expressed with the reductase, human P450 catalyzed the oxidation of representative substrates at efficient rates. The rates appeared comparable to the reported activities of P450 in a reconstituted system containing purified preparations of P450 and the reductase.

Genetic polymorphism of CYP2A6 in relation to cancer

To clarify the roles of human cytochrome P450 (P450 or CYP) 2A6 and 2E1 on the metabolic activation of N-nitrosamines, we established genetically engineered Salmonella typhimurium strains harboring human CYP2A6 or CYP2E1 together with NADPH-P450 reductase (OR). The 5-terminus of CYP cDNA was modified to achieve a high-level expression in S. typhimurium. Modified CYP2A6 or CYP2E1 cDNA and native OR cDNA were introduced into a pCW vector. S. typhimurium YG7108 cells were transformed with this vector. The mutagen producing ability of these enzymes for some N-nitrosamines were evaluated using the established S. typhimurium cells. We found that the substrate specificity of CYP2A6 and CYP2E1 was different among mutagens. CYP2A6 was responsible for the metabolic activation of N-nitrosamines possessing relatively long alkyl chains, whereas CYP2E1 was responsible for the metabolic activation of N-nitrosamines with relatively short alkyl chains. It is likely that CYP2A6 gene polymorphism is responsible for the interindividual variability on the cancer susceptibility. We found the whole deletion of CYP2A6 gene as a type of genetic polymorphism in Japanese. Thus, we developed a gene diagnosis method to detect the variant. We evaluated the relationship between the CYP2A6 gene whole deletion and the susceptibility to the lung cancer. The frequency of CYP2A6 gene whole deletion was significantly lower in the lung cancer patients than that of healthy volunteers.

Role of human cytochrome P450 (CYP) in the metabolic activation of N-alkylnitrosamines : application of genetically engineered Salmonella typhimurium YG7108 expressing each form of CYP together with human NADPH-cytochrome P450 reductase

The role of human cytochrome P450 (CYP) in the metabolic activation of N-alkylnitrosamines was examined by Ames test using genetically engineered Salmonella typhimurium (S. typhimurium)YG7108 cells expressing each form of human CYP together with human NADPH-cytochrome P450 reductase (OR). The relationship between the structure of N-alkylnitrosamines and CYP form(s) involved in the activation was evaluated. Eleven strains of S. typhimurium YG7108 cells expressing each form of CYP (CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 or CYP3A5) were employed. Eight N-alkylnitrosamines including N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodipropylamine (NDPA), N-nitrosodibutylamine (NDBA), N-nitrosomethylethylamine (NMEA), N-nitrosomethylpropylamine (NMPA), N-nitrosomethylbutylamine (NMBA) and N-nitrosoethylbutylamine (NEBA) were examined. Minimal concentration (MC) value of a promutagen was defined as the concentration of a chemical giving a positive result. Mutagen-producing capacity of CYP, as indicated by induced revertants/nmol promutagen/pmol CYP, for an N-alkylnitrosamine was determined for all forms of CYP. These N-alkylnitrosamines were mainly activated by CYP2E1, CYP2A6 and CYP1A1. N-alkylnitrosamines with relatively short alkyl chains such as NDMA and NMEA were primarily activated by CYP2E1 as judged by mutagen-producing capacity. With the increase of the number of the carbon atoms of the alkyl chains, the contribution of CYP2A6 increased. CYP2A6 played major roles in the activation of NDEA, NDPA, NMPA, NMBA and NEBA. Interestingly, CYP1A1 became a molecular form of CYP playing a major role in the metabolic activation of NDBA.

Down-regulation of 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx)-induced CYP1A2 Expression Is Associated with Bovine Lactoferrin Inhibition of MeIQx-induced Liver and Colon Carcinogenesis in Rats

The inhibitory influence of bovine lactoferrin (bLF) on induction of preneoplastic hepatic glutathione S‐transferase placental form‐positive (GST‐P+) cell foci and colon aberrant crypt foci (ACF) by diethylnitrosamine (DEN) and 2‐amino‐3,8‐dimethylimidazo[4,5‐f]quinoxaline (MeIQx) was investigated in F344 rats. Rats were initially treated with DEN, then placed on basal diet containing MeIQx (200 ppm) alone, MeIQx plus 2% bLF, or MeIQx plus 0.2% bLF from week 2 to week 8, with partial hepatectomy performed at week 3. Concomitant administration of 2% or 0.2% bLF with MeIQx caused significant dose‐dependent decreases in both number and unit area of GST‐P+ cell foci (2% bLF, P <0.001; 0.2% bLF, P <0.01). Similar results were observed for MeIQx‐induced colon ACF in the groups without DEN treatment (2% and 0.2% bLF, P <0.05). To investigate the underlying mechanisms, we analyzed the influence of bLF on levels of cytochrome P4501A2 (CYP1A2), a metabolically activating enzyme of MeIQx in the liver. The results demonstrated that combined administration of 2% bLF significantly reduced levels of MeIQx‐induced CYP1A2 mRNA (P <0.05) and protein (P <0.05) to the normal levels, in association with reduced values for MeIQx‐DNA adducts (P <0.05), liver GST‐P+ cell foci and colon ACF. These results suggest that bLF is a chemopreventive agent for DEN alone or DEN plus MeIQx‐induced liver, and MeIQx‐induced colon carcinogenesis in rats. One possible mechanism is a normalizing down‐regulation of CYP1A2 expression by bLF, with consequent reduction of carcinogen activation and adduct formation.

Development of a Salmonella tester strain sensitive to promutagenic N-nitrosamines: expression of recombinant CYP2A6 and human NADPH-cytochrome P450 reductase in S. typhimurium YG7108.

We developed a new Salmonella tester strain highly sensitive to promutagenic N-nitrosamines by introducing a plasmid carrying human cytochrome P450 2A6 (CYP2A6) and NADPH-cytochrome P450 reductase (OR) cDNA into the ada- and ogt-deficient strain YG7108. The YG7108 2A6/OR cells expressed high levels of CYP2A6 (77+/-8nmol/l) and OR (470+/-20 micromol cytochrome c reduced/min/l). The expressed CYP2A6 efficiently catalyzed coumarin 7-hydroxylation. N-Nitrosodiethylamine (NDEA), N-nitrosomethylphenylamine (NMPhA), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were mutagenic in the new strain in the absence of any exogenous activation system. The concentrations of promutagen that caused a two-fold increase in revertants were 7.1, 0.14, and 1.4 microM for NDEA, NMPhA, and NNK, respectively. YG7108 2A6/OR cells showed about 10- and 100-fold higher sensitivity to NDEA and NNK, respectively, than parental YG7108 cells assayed in the presence of rat liver S9 (final concentration, 21% (v/v)). Parental YG7108 cells did not detect NMPhA mutagenicity even in the presence of rat liver S9. We believe that this is the first demonstration that CYP2A6 is responsible for the metabolic activation of NMPhA. The established tester strain may be useful to predict human activation of N-nitrosamine promutagens.

Establishment of a Salmonella tester strain highly sensitive to mutagenic heterocyclic amines

Heterocyclic amines (HCAs) that are present in cooked foods require metabolic activation to exert their genotoxicity. They undergo activation via N-hydroxylation by cytochrome P450 1A2 (CYP1A2), followed by O-esterification by O-acetyltransferase (OAT). To develop a Salmonella tester strain that is highly sensitive to mutagenic HCAs, we introduced a coexpression plasmid (p1A2OR) carrying human CYP1A2 and NADPH-CYP reductase cDNAs and an expression plasmid (pOAT) carrying Salmonella OAT to Salmonella typhimurium TA1538 to yield a TA1538/ARO strain. The TA1538/ARO strain was proven to express the enzymes, as indicated by high activities of 7-ethoxyresorufin O-deethylase and isoniazid N-acetylase. The TA1538/ARO strain exhibited very high sensitivity to mutagenic HCAs 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and a somewhat higher sensitivity to 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine compared with the parent Ames tester strain TA1538. The minimum concentrations of 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, IQ, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine giving positive results were defined by evidence that the number of colonies increased in a dose-dependent manner and reached a number two times higher than that obtained by vehicle alone as a control in the TA1538/ARO strain at concentrations of 0.3, 3, 30, and 1000 pM, respectively. When the membrane and cytosol fractions prepared from TA1538/ARO were added to a mixture containing the parental TA1538, the sensitivity of TA1538 to IQ was much lower than that seen with TA1538/ARO. These results indicate that the intracellular expression of drug-metabolizing enzymes makes the established strain of Salmonella highly sensitive to mutagenic HCAs.

Establishment of Salmonella strain expressing catalytically active human UDP-glucuronosyltransferase 1A1 (UGT1A1)

Human uridinediphosphate-glucuronosyltransferase 1A1 (UGT1A1) was expressed in Salmonella typhimurium TA1535 cells by transfection of the cells with plasmids carrying the UGT1A1 cDNA. UGT1A1 cDNA was isolated by a polymerase chain reaction from human liver total RNA and was inserted into the pSE420 plasmid, linked to the trc promoter and terminator. The plasmid thus constructed was introduced into Salmonella TA1535 cells. The expression of human UGT1A1 protein was confirmed by Western blot analysis. The maximal expression was observed at 24 h after the addition of isopropyl-beta-D-thiogalactopyranoside, an inducer. However, the bilirubin conjugation activity of the membrane fraction from the Salmonella cells was not detectable. When a beta-glucuronidase inhibitor such as saccharic acid 1,4-lactone, glycyrrhizin or 1-naphtyl-beta-D-glucuronide was added to the reaction mixture, the bilirubin conjugation activity of the human UGT1A1 was detected. When geniposide was added to the reaction mixture, the bilirubin conjugation activity of UGT1A1 was not seen. Taking these results into account, the established Salmonella strain possesses the beta-glucuronidase activity. Since the beta-glucuronidase activity of the Salmonella was lower than that of E. coli, it was concluded that Salmonella seemed to be a good host to express UGT protein. This is the first study to demonstrate the establishment of a bacterial strain expressing native human UGT protein showing catalytic activity.