Regioselective Glucuronidation of Flavones at C5, C7, and C4' Positions in Human Liver and Intestinal Microsomes: Comparison among Apigenin, Acacetin, and Genkwanin.

Flavones, which are distributed in a variety of plants and foods in nature, possess significant biological activities, including antitumor and anti-inflammatory effects, and are metabolized into glucuronides by uridine 5'-diphosphate (UDP)-glucuronosyltransferase (UGT) enzymes in humans. In this study, apigenin, acacetin, and genkwanin, flavones having hydroxyl groups at C5, C7, and/or C4'positions were focused on, and the regioselective glucuronidation in human liver and intestinal microsomes was examined. Two glucuronides (namely, AP-7G and AP-4'G for apigenin, AC-5G and AC-7G for acacetin, and GE-5G and GE-4'G for genkwanin) were formed from each flavone by liver and intestinal microsomes, except for only GE-4'G formation from genkwanin by intestinal microsomes. The order of total glucuronidation activities was liver microsomes > intestinal microsomes for apigenin and acacetin, and liver microsomes < intestinal microsomes for genkwanin. The order of CLint values (x-intercept) based on v versus V/[S] plots for apigenin glucuronidation was AP-7G > AP-4'G in liver microsomes and AP-7G < AP-4'G in intestinal microsomes. The order of CLint values was AC-5G < AC-7G for acacetin and GE-5G < GE-4'G genkwanin glucuronidation in both liver and intestinal microsomes. This suggests that the abilities and roles of UGT enzymes in the glucuronidation of apigenin, acacetin, and genkwanin in humans differ depending on the chemical structure of flavones.

Less Carcinogenic Chlorinated Estrogens Applicable to Hormone Replacement Therapy.

Human estrogens prescribed for hormone replacement therapy (HRT) are known to be potent carcinogens. To find safer estrogens, several chlorinated estrogens were synthesized and their carcinogenic potential were determined. A pellet containing either 2-chloro-17ß-estradiol (2-ClE2) or 4-chloro-17ß-estradiol (4-ClE2) was implanted subcutaneously for 52 weeks into August Copenhagen Irish (ACI) rats, a preferred animal model for human breast cancer. 17ß-Estradiol (E2) frequently induced mammary tumors while both 2-ClE2 and 4-ClE2 did not. Their 17α-ethinyl forms, thought to be orally active estrogens, were also synthesized. Neither 2-chloro-17α-ethinylestradiol (2-ClEE2) nor 4-chloro-17α-ethinylestradiol (4-ClEE2) induced tumors. The less carcinogenic effects were supported by histological examination of mammary glands of ACI rats treated with the chlorinated estrogens. A chlorine atom positioned at the 2- or 4-position of E2 may prevent the metabolic activation, resulting in reducing the carcinogenicity. 2-ClE2 and 4-ClE2 administered subcutaneously and 2-ClEE2 and 4-ClEE2 given orally to ovariectomized rats all showed uterotrophic potency, albeit slightly weaker than that of E2. Our results indicate that less carcinogenic chlorinated estrogens retaining estrogenic potential could be safer alternatives to the carcinogenic estrogens now in use for HRT.

Evaluation of the sensitization potential of volatile and semi-volatile organic compounds using the direct peptide reactivity assay.

The purpose of this study was to evaluate the sensitization potential of 82 compounds classified as volatile and/or semi-volatile organic compounds using the direct peptide reactivity assay (DPRA), given that these chemical compounds have been detected frequently and at high concentrations in a national survey of Japanese indoor air pollution and other studies. The skin sensitization potential of 81 of these compounds was evaluable in our study; one compound co-eluted with cysteine peptide and was therefore not evaluable. Twenty-five of the evaluated compounds were classified as positive. Although all glycols and plasticizers detected frequently and at high concentrations in a national survey of Japanese indoor air pollution were negative, hexanal and nonanal, which are found in fragrances and building materials, tested positive. Monoethanolamine and 1,3-butanediol, which cause clinical contact dermatitis, and several compounds reported to have weak sensitization potential in animal studies, were classified as negative. Thus, it was considered that compounds with weak sensitization potential were evaluated as negative in the DPRA. Although the sensitization potential of the formaldehyde-releasing preservative bronopol has been attributed to the release of formaldehyde (a well-known contact allergen) by its degradation, its degradation products-bromonitromethane and 2-bromoethanol-were classified as positive, indicating that these degradation products also exhibit sensitization potential. The compounds that tested positive in this study should be comprehensively assessed through multiple toxicity and epidemiological studies.

Catecholamine oxidation-mediated transcriptional inhibition in Mn neurotoxicity.

Manganese (Mn) poisoning may result in a neurological disorder called manganism. Although the neurotoxic mechanism of Mn is unclear, oxidative stress may be involved based on the interactions between neurotransmitter catecholamines and metals such as iron. Here, we propose a novel mechanism in which Mn oxidizes catecholamines and inhibits cellular transcription. Mn accelerated the oxidation of adrenaline (Ad) and produced adrenochrome (AdC) more effectively than iron. Furthermore, the oxidation of DNA bases increased when Ad, Mn, and iron were present. However, despite the absence of iron, cell viability decreased in the presence of AdC or Ad with Mn, which suggests there is another mechanism independent of oxidative DNA damage. AdC or preincubated Ad with Mn reduced mRNA synthesis in T7 RNA polymerase-driven transcription. RNA synthesis decreased in AdC-treated cells dose-dependently. These results show that Mn disrupts neuronal function via catecholamine oxidation-mediated transcriptional inhibition.

Carcinogenic potential of fluorinated estrogens in mammary tumorigenesis.

Fluorination preventing metabolic hydroxylation of 17ß-estradiol (E2) was applied to investigate the mechanisms underlying estrogen-induced carcinogenesis. Either 2-fluoro-17ß-estradiol (2-FE2) or 4-fluoro-17ß-estradiol (4-FE2) was administered subcutaneously for 52 weeks to August Copenhagen Irish (ACI) rats, the preferred animal model for human breast cancer. 4-FE2 induced frequent mammary tumors whereas 2-FE2 did not. The cumulative incidence of mammary tumors in rats treated with 4-FE2 was comparable to that observed with E2. The carcinogenic results were supported by histological examination of mammary glands of fluorinated estrogen-treated ACI rats. To evaluate the estrogenic potential of the fluorinated estrogens, 2-FE2 or 4-FE2 was administrated subcutaneously to ovariectomized rats. Both 4-FE2 and 2-FE2 showed high uterotrophic potency. Our results indicate that estrogenic potential may not be the sole factor driving mammary tumorigenesis. Since fluorination inhibits metabolic hydroxylation of E2 at the substituted position, the carcinogenic effect may occur through the metabolic activation of 2-hydroxylated E2, in combination with the compound's estrogenic potency.

[Ensuring Traceability Using qNMR in the Quantitative Analysis of Formaldehyde and Acetaldehyde].

　Currently, indoor air quality guidelines for formaldehyde and acetaldehyde are set by the Ministry of Health, Labour and Welfare of Japan. Aldehydes are widely used in adhesives and preservatives, and exposure to these compounds via indoor air is a matter of concern. Considering that contact with indoor air is part of daily life, evaluation of indoor air quality is extremely important. 2,4-Dinitrophenylhydrazine (DNPH) derivatization is widely used for quantitative analysis of aldehydes. A certified reference material with traceability to the International System of Units (SI) is required for this method. However, currently, there are no certified reference materials available for aldehyde-DNPH derivatives, which means that the quantified values obtained by this method are not sufficiently reliable. In this study, we determined the actual content and purity of commercially available aldehyde-DNPH derivatives using 1H-quantitative NMR (qNMR), which can be measured with SI-traceability. Although the commercial DNPH derivatives of formaldehyde and acetaldehyde were low concentration solutions, we were able to determine their purities using 1H-qNMR. Furthermore, we were able to separate and quantify the acetaldehyde isomers generated by the derivatization reaction. In conclusion, it is possible to obtain highly accurate results using 1H-qNMR with commercially available reagents that are not certified metrologically.

Metabolomic analysis uncovered an association of serum phospholipid levels with estrogen-induced mammary tumors in female ACI/Seg rats.

Estrogen is reported to be involved in mammary tumorigenesis. To unveil metabolic signatures for estrogen-induced mammary tumorigenesis, we carried out serum metabolomic analysis in an estrogen-induced mammary tumor model, female August Copenhagen-Irish/Segaloff (ACI/Seg) rats, using liquid chromatography-mass spectrometry. In contrast to the control group, all rats with an implanted 17ß-estradiol (E2) pellet developed mammary tumors during this experiment. E2 treatment significantly suppressed body weight gain. But no significant differences in food consumption were observed between the two groups, suggesting that metabolic alteration depended on E2 treatment. Serum metabolomic analysis detected 116 features that were statistically different (p < 0.01) between the groups. Quantitation analysis revealed that several phospholipids such as phosphatidylcholines and lysophosphatidylcholines (LPCs) were identified as significantly different metabolites. E2-treated rat serum stimulated the proliferation of human breast cancer MDA-MB-231 cells. In addition, the proliferation effect was diminished by pretreating cells with either autotaxin inhibitor or antagonist for lysophosphatidic acid receptor whose ligands are metabolites of LPCs via autotaxin-mediated hydrolysis. In summary, our results suggest that not only are phospholipids potential biomarkers for mammary tumors but importantly, LPCs themselves could be associated with E2-induced mammary tumorigenesis in female ACI/Seg rats.

Hepatic glucuronidation of 4-tert-octylphenol in humans: inter-individual variability and responsible UDP-glucuronosyltransferase isoforms.

4-tert-Octylphenol (4-tOP) is an endocrine-disrupting chemical. It is mainly metabolized into glucuronide by UDP-glucuronosyltransferase (UGT) enzymes in humans. The purpose of this study was to assess inter-individual variability in and the possible roles of UGT isoforms in hepatic 4-tOP glucuronidation in the humans. 4-tOP glucuronidation activities in the liver microsomes and recombinant UGTs of humans were assessed at broad substrate concentrations, and kinetics were analyzed. Correlation analyses between 4-tOP and diclofenac or 4-hydroxybiphenyl activities in pooled and individual human liver microsomes were also performed. Typical CLint values were 17.8 mL/min/mg protein for the low type, 25.2 mL/min/mg protein for the medium type, and 47.7 mL/min/mg protein for the high type. Among the recombinant UGTs (13 isoforms) examined, UGT2B7 and UGT2B15 were the most active of catalyzing 4-tOP glucuronidation. Although the K m values of UGT2B7 and UGT2B15 were similar (0.36 and 0.42 µM, respectively), the CLint value of UGT2B7 (6.83 mL/min/mg protein) >UGT2B15 (2.35 mL/min/mg protein). Strong correlations were observed between the glucuronidation activities of 4-tOP and diclofenac (a probe for UGT2B7) or 4-hydroxybiphenyl (a probe for UGT2B15) with 0.79-0.88 of Spearman correlation coefficient (r s) values. These findings demonstrate that 4-tOP glucuronidation in humans is mainly catalyzed by hepatic UGT2B7 and UGT2B15, and suggest that these UGT isoforms play important and characteristic roles in the detoxification of 4-tOP.

Selenium uptake through cystine transporter mediated by glutathione conjugation.

Selenium (Se) is an essential trace element and is regarded as a protective agent against cancer. In particular, antioxidant effects of selenoenzymes contribute to cancer prevention. Se can also produce reactive oxygen species and, thereby, exert cancer-selective cytotoxicity. Selenodiglutathione (SDG) is a primary Se metabolite conjugated to two glutathione (GSH) moieties. SDG increases intracellular Se accumulation and is more toxic than selenous acid (H2SeO3), but the mechanisms for importing Se compounds into cells are not fully understood. Here, we propose a novel mechanism for importing Se, in the form of SDG. Cellular intake of Se compounds was assessed based on Se accumulation, as detected by ICP-MS. SDG incorporation was decreased in the presence of thiols (GSH, cysteine or their oxidized forms, GSSG and cystine), whereas H2SeO3 uptake was increased by addition of GSH or cysteine. Cellular SDG uptake was decreased by pretreatment with specific inhibitors against gamma-glutamyl transpeptidase (GGT) or the cystine/glutamate antiporter (system xc-). Furthermore, siRNA against xCT, which is the light chain component of system xc-, significantly decreased SDG incorporation. These data suggest an involvement of SDG in Se incorporation, with SDG processed at the cell surface by GGT, leading to formation of selenodicysteine which, in turn, is likely to be imported via xCT. Because GGT and xCT are highly expressed in cancer cells, these mechanisms mediated by the cystine transporter might underlie the cancer-selective toxicity of Se. In addition, the system described in our study appears to represent a physiological transport mechanism for the essential element Se.

Glucuronidation of 4-tert-octylphenol in humans, monkeys, rats, and mice: an in vitro analysis using liver and intestine microsomes.

4-tert-Octylphenol (4-tOP) is an endocrine-disrupting chemical. It is mainly metabolized into glucuronide by UDP-glucuronosyltransferase (UGT) enzymes in mammals. In the present study, the glucuronidation of 4-tOP in humans, monkeys, rats, and mice was examined in an in vitro system using microsomal fractions. The kinetics of 4-tOP glucuronidation by liver microsomes followed the Michaelis-Menten model for humans and monkeys, and the biphasic model for rats and mice. The K m, V max, and CL int values of human liver microsomes were 0.343 µM, 11.6 nmol/min/mg protein, and 33.8 mL/min/mg protein, respectively. The kinetics of intestine microsomes followed the Michaelis-Menten model for humans, monkeys, and rats, and the biphasic model for mice. The K m, V max, and CL int values of human intestine microsomes were 0.743 µM, 0.571 nmol/min/mg protein, and 0.770 mL/min/mg protein, respectively. The CL int values estimated by Eadie-Hofstee plots were in the order of mice (high-affinity phase) (3.0) > humans (1.0) ≥ monkeys (0.9) > rats (high-affinity phase) (0.4) for liver microsomes, and monkeys (10) > mice (high-affinity phase) (5.6) > rats (1.4) > humans (1.0) for intestine microsomes. The percentages of the CL int values of intestine microsomes to liver microsomes were in the order of monkeys (27 %) > rats (high-affinity phase in liver microsomes) (7.9 %) > mice (high-affinity phase in liver and intestine microsomes) (4.2 %) > humans (2.3 %). These results suggest that the metabolic abilities of UGT enzymes expressed in the liver and intestine toward 4-tOP markedly differ among species and imply that species differences are strongly associated with the toxicities of alkylphenols.

Peroxiredoxin 6 is a molecular target for 1,2-naphthoquinone, an atmospheric electrophile, in human pulmonary epithelial A549 cells.

1,2-Naphthoquinone (1,2-NQ) is an electrophile found in the atmosphere, which reacts readily with protein nucleophiles to form a stable protein adduct. Peroxiredoxin 6 (Prdx6) is predominantly expressed in lung tissue and functions in antioxidant defense by facilitating the repair of damaged cell membranes via reduction of peroxidized phospholipids. In the present study, human A549 pulmonary epithelial cells were exposed to 1,2-NQ to explore whether 1,2-NQ can bind covalently to Prdx6, thereby disrupting its catalytic activity. Two-dimensional SDS/PAGE followed by western blot analysis with a specific antibody against 1,2-NQ showed that Prdx6 was covalently modified by 1,2-NQ. Using purified human Prdx6, it was found that 1,2-NQ bound covalently to Prdx6 through Cys47, Lys144 and Cys91, resulting in a significant reduction in phospholipase A(2) activity. These results suggest that arylation of Prdx6 by 1,2-NQ may, at least in part, be involved in the cellular toxicity induced by 1,2-NQ.

Initial response and cellular protection through the Keap1/Nrf2 system during the exposure of primary mouse hepatocytes to 1,2-naphthoquinone.

Quinones are reactive chemical species that cause cellular damage by modifying protein thiols and/or catalyzing the reduction of oxygen to reactive oxygen species, thereby promoting oxidative stress. Transcription factor Nrf2 plays a crucial role in cellular defense against electrophilic modification and oxidative stress. In studies using 1,2-naphthoquinone (1,2-NQ) as a model quinone, we found that Keap1, the negative regulator of Nrf2, was readily arylated at its reactive thiols by 1,2-NQ. Exposure of primary mouse hepatocytes to 1,2-NQ resulted in the activation of Nrf2 and the upregulation of some of Nrf2's downstream genes. This interaction was further investigated in hepatocytes from Nrf2 knockout mice in which the proteins responsible for the metabolism and excretion of 1,2-NQ are minimally expressed. The chemical modification of cellular proteins by 1,2-NQ was enhanced by Nrf2 deletion, resulting in increased toxicity. However, deletion of the negative regulatory protein, Keap1, drastically reduced the covalent binding by 1,2-NQ and its cellular toxicity. Experiments with chemicals that inhibit the biotransformation and extracellular excretion of 1,2-NQ suggest that 1,2-NQ undergoes detoxification and excretion into the extracellular space predominantly by two-electron reduction and subsequent glucuronidation by NAD(P)H:quinone oxidoreductase 1 and uridine 5'-diphosphate-glucuronosyltransferases, followed by multidrug resistance-associated protein-dependent excretion. These findings suggest that the Keap1/Nrf2 system is essential for the prevention of cell damage resulting from exposure to 1,2-NQ.

Functional characterization of human and cynomolgus monkey UDP-glucuronosyltransferase 1A1 enzymes.

AIMS: UDP-glucuronosyltransferase 1A1 (UGT1A1) plays important roles in the glucuronidation of various drugs and endogenous substances. Cynomolgus monkeys are regarded as experimental animals closer to humans in studies on safety evaluation and biotransformation for drug development. In this study, the similarities and differences in the enzymatic properties of UGT1A1 between humans and cynomolgus monkeys were precisely identified. MAIN METHODS: Human and cynomolgus monkey UGT1A1s (humUGT1A1 and monUGT1A1, respectively) were cloned, and the corresponding proteins were heterologously expressed in insect cells. The enzymatic properties of UGT1A1 proteins were characterized by kinetic analysis of 7-hydroxy-4-trifluoromethylcoumarin (7-HFC), estradiol at 3-hydroxy position (E-3OH) and 7-ethyl-10-hydroxycamptothecin (SN-38) glucuronidation. KEY FINDINGS: There were no significant differences in the levels of kinetic parameters for 7-HFC, E-3OH and SN-38 glucuronidation between humans and cynomolgus monkeys in both enzyme sources of liver microsomes and recombinant UGT1A1s. 7-HFC and E-3OH glucuronidation by human liver microsomes exhibited biphasic and sigmoidal kinetics, respectively, whereas the kinetics by cynomolgus monkey liver microsomes fitted the typical Michaelis-Menten model. SN-38 glucuronidation by human and cynomolgus monkey liver microsomes exhibited autoactivation kinetics. In recombinant UGT1A1 enzymes expressed in insect cells, the kinetics of 7-HFC, E-3OH and SN-38 glucuronidation fitted the substrate inhibition (7-HFC glucuronidation) or Hill equation (E-3OH and SN-38 glucuronidation), and each glucuronidation showed the same kinetic profile between humans and cynomolgus monkeys. SIGNIFICANCE: These findings suggest that the enzymatic properties of human and cynomolgus monkey UGT1A1 enzymes are very similar.

Functional characterization of human cytochrome P4502E1 allelic variants: in vitro metabolism of benzene and toluene by recombinant enzymes expressed in yeast cells.

Benzene and toluene are common organic solvents currently in worldwide industrial usage, which are metabolized mainly by hepatic cytochrome P450 2E1 (CYP2E1) in humans. Genetic polymorphism of CYP2E1 in 5'-flanking and coding regions has been found previously in Caucasian and Chinese populations. In this study, the effects of CYP2E1 alleles causing amino acid substitutions (CYP2E1*2, CYP2E1*3 and CYP2E1*4; wild-type, CYP2E1.1A) on benzene hydroxylation and toluene methylhydroxylation were studied using recombinant CYP2E1 enzymes of wild-type (CYP2E1.1) and variants (CYP2E1.2 having Arg76His, CYP2E1.3 having Val389Ile and CYP2E1.4 having Val179Ile) expressed in yeast cells. The K (m), V (max) and CL (int) values of CYP2E1.1 were 10.1 mM, 9.38 pmol/min/pmol CYP and 0.99 nL/min/pmol CYP for benzene hydroxylation, and 3.97 mM, 19.9 pmol/min/pmol CYP and 5.26 nL/min/pmol CYP for toluene methylhydroxylation, respectively. The K (m), V (max) and CL (int) values for benzene and toluene metabolism of CYP2E1.2, CYP2E1.3 and CYP2E1.4 were comparable to those of wild-type CYP2E1. These findings may mean that the polymorphic alleles of CYP2E1 causing amino acid substitutions are not directly associated with the metabolic activation of benzene and toluene. The information gained in this study should help to identify the variations in the toxicity of environmental pollutants.

Haplotypes and a novel defective allele of CES2 found in a Japanese population.

Human carboxylesterase 2 (hCE-2) is a member of the serine esterase superfamily and is responsible for hydrolysis of a wide variety of xenobiotic and endogenous esters. hCE-2 also activates an anticancer drug, irinotecan (7-ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxycamptothecin, CPT-11), into its active metabolite, 7-ethyl-10-hydroxycamptothecin (SN-38). In this study, a comprehensive haplotype analysis of the CES2 gene, which encodes hCE-2, in a Japanese population was conducted. Using 21 single nucleotide polymorphisms (SNPs), including 4 nonsynonymous SNPs, 100C>T (Arg(34)Trp, *2), 424G>A (Val(142)Met, *3), 1A>T (Met(1)Leu, *5), and 617G>A (Arg(206)His, *6), and a SNP at the splice acceptor site of intron 8 (IVS8-2A>G, *4), 20 haplotypes were identified in 262 Japanese subjects. In 176 Japanese cancer patients who received irinotecan, associations of CES2 haplotypes and changes in a pharmacokinetic parameter, (SN-38 + SN-38G)/CPT-11 area under the plasma concentration curve (AUC) ratio, were analyzed. No significant association was found among the major haplotypes of the *1 group lacking nonsynonymous or defective SNPs. However, patients with nonsynonymous SNPs, 100C>T (Arg(34)Trp) or 1A>T (Met(1)Leu), showed substantially reduced AUC ratios. In vitro functional characterization of the SNPs was conducted and showed that the 1A>T SNP affected translational but not transcriptional efficiency. These findings are useful for further pharmacogenetic studies on CES2-activated prodrugs.

Inducibility of UDP-glucuronosyltransferase 1As by beta-naphthoflavone in HepG2 cells.

UDP-glucuronosyltransferases (UGTs) are conjugation enzymes, which are regulated in a tissue-specific manner by endogenous and environmental factors. In this study, we focused on UGT1A isoforms (UGT1A1, UGT1A6 and UGT1A9), mainly expressed in the human liver, and examined the inducibility of UGT1As by beta-naphthoflavone (BNF) in human hepatoma HepG2 cells. The cells were pretreated for 72 h with BNF at concentrations of 25, 50 and 100 microM. 7-Ethyl-10-hydroxycamptothecin (SN-38) glucuronidation, used as a probe for UGT1A1, showed sigmoidal kinetics with a Hill coefficient (n) of 1.2-1.3 in control and BNF-pretreated HepG2 cells. The Vmax values were significantly increased 3.6- to 4.3-fold by BNF, whereas there was no significant change in the S50 values by BNF at any concentration examined. On the other hand, 4-methylumbelliferone (4-MU) glucuronidation as a probe for UGT1A6 and UGT1A9 in the control and BNF-pretreated HepG2 cells exhibited a biphasic kinetic pattern. Although Km1 values for the low-Km phase were similar between the control and BNF-pretreated HepG2 cells, Km2 values for the high-Km phase of BNF-pretreated HepG2 cells were reduced to 54-69% of control HepG2 cells. The values of Vmax1 and Vmax2 for the low- and high-Km phases, respectively, were significantly increased 1.9- to 2.6-fold by BNF at 25 and/or 50 microM but not 100 microM. With respect to Vmax (Vmax1 and Vmax2) and Vmax/Km (Vmax1/Km1 and Vmax2/Km2), the values were significantly increased 2.0- to 3.2-fold by BNF at all concentrations examined. Furthermore, real-time reverse transcription polymerase chain reaction using TaqMan probes demonstrated that BNF concentration-dependently induced mRNA levels of UGT1A1 but not UGT1A6 or UGT1A9 in HepG2 cells (1.3- to 6.0-fold). These results suggest that the inducibility of UGT1A isoforms in HepG2 cells by BNF is different from other aryl hydrocarbon receptor agonists previously reported, and should provide useful information for the prediction of drug-drug interactions and toxicological assessment of environmental chemicals.

Transfection assays with allele-specific constructs: functional analysis of UDP-glucuronosyltransferase variants.

Adverse drug reactions (ADRs) are a major clinical problem. A rapidly growing body of evidence suggests that genetic factors, at least in part, determine individual susceptibility to ADRs. A large number of pharmacogenetic studies have identified a number of polymorphisms as predictors of drug efficacy and/or adverse events. These candidate markers should be investigated further to ascertain the underlying mechanism of action, for example, changes in the kinetic parameters of an enzyme, or transcriptional activity of a promoter region. In this chapter, we describe a transient transfection assay for the functional characterization of naturally occurring variants of UDP-glucuronosyltransferase (UGT) 1A1. This phase II drug metabolizing enzyme is involved in the glucuronidation of SN-38, an active metabolite of the anti-cancer drug irinotecan. Single-nucleotide polymorphisms of the UGT1A1 gene have been correlated to irinotecan-induced ADRs. Variant UGT1A1s are heterologously expressed in COS-1 cells and characterized in terms of the level of protein expression and enzyme kinetics.

Genetic variations and haplotypes of UGT1A4 in a Japanese population.

Nineteen genetic variations, including 11 novel ones, were found in exon 1 and its flanking region of the UDP-glucuronosyltransferase (UGT) 1A4 gene from 256 Japanese subjects, consisting of 60 healthy volunteers, 88 cancer patients and 108 arrhythmic patients. These variations include -217T>G and -36G>A in the 5'-flanking region, 30G>A (P10P), 127delA (43fsX22; frame-shift from codon 43 resulting in the termination at the 22nd codon, codon 65), 175delG (59fsX6), 271C>T (R91C), 325A>G (R109G), and 357T>C (N119N) in exon 1, and IVS1+1G>T, IVS1+98A>G and IVS1+101G>T in the following intron. Among them, 127delA and 175delG can confer early termination of translation, resulting in an immature protein that probably lacks enzymatic activity. Variation IVS1+1G>T is located at a splice donor site and thus may lead to aberrant splicing. Since we did not find any significant differences in the frequencies of all the variations among the three subject groups, the data were analyzed as one group. The allele frequencies of the novel variations were 0.006 for IVS1+101G>T, 0.004 for 30G>A (P10P) and 357T>C (N119N), and 0.002 for the 8 other variations. In addition, the two known nonsynonymous single nucleotide polymorphisms (SNPs), 31C>T (R11W) and 142T>G (L48V), were found at 0.012 and 0.129 frequencies, respectively. The SNP 70C>A (P24T), mostly linked with 142T>G (L48V) in German Caucasians, was not detected in this study. Sixteen haplotypes were identified or inferred, and some haplotypes were confirmed by cloning and sequencing. It was shown that most of 142T>G (L48V) was linked with -219C>T, -163G>A, 448T>C (L150L), 804G>A (P268P), and IVS1+43C>T, comprising haplotype *3a; haplotype *4a harbors 31C>T (R11W); 127delA (43fsX22) and 142T>G (L48V) were linked (haplotype *5a); 175delG (59fsX6) was linked with 325A>G (R109G) (*6a haplotype); and -219C>T, -163G>A, 142T>G (L48V), 271C>T (R91C), 448T>C (L150L), 804G>A (P268P), and IVS1+43C>T comprised haplotype *7a. Our results provide fundamental and useful information for genotyping UGT1A4 in the Japanese and probably Asian populations.

Functional characterization of five novel CYP2C8 variants, G171S, R186X, R186G, K247R, and K383N, found in a Japanese population.

Cytochrome P450 2C8 is one of the primary enzymes responsible for the metabolism of a wide range of drugs such as paclitaxel, cerivastatin, and amiodarone. We have sequenced the CYP2C8 gene from 201 Japanese subjects and found five novel nonsynonymous single nucleotide polymorphisms (SNPs): 511G>A (G171S), 556C>T (R186X; X represents the translational stop codon), 556C>G (R186G), 740A>G (K247R), and 1149G>T (K383N), with the allele frequency of 0.0025. The CYP2C8 variants were heterologously expressed in COS-1 cells and functionally characterized in terms of expression level, paclitaxel 6alpha-hydroxylase activity, and intracellular localization. The prematurely terminated R186X variant was undetectable by Western blotting and inactive toward paclitaxel 6alpha-hydroxylation. The G171S, K247R, and K383N variants exhibited properties similar to those of the wild-type CYP2C8. Paclitaxel 6alpha-hydroxylase activity of the R186G transfectant was only 10 to 20% that of wild-type CYP2C8. Furthermore, the R186G variant displayed a lower level of protein expression in comparison to the wild type, which was restored by the addition of a proteasome inhibitor (MG-132; Z-Leu-Leu-Leu-aldehyde). The reduced CO-difference spectral analysis using recombinant proteins from an insect cell/baculovirus system revealed that the R186G variant has a minor peak at 420 nm in addition to the characteristic Soret peak at 450 nm, suggesting the existence of improperly folded protein. These results indicate that the novel CYP2C8 SNPs, 556C>T (R186X) and 556C>G (R186G), could influence the metabolism of CYP2C8 substrates such as paclitaxel and cerivastatin.

Inorganic arsenic compounds and methylated metabolites induce morphological transformation in two-stage BALB/c 3T3 cell assay and inhibit metabolic cooperation in V79 cell assay.

We have performed two-stage transformation assay using BALB/c 3T3 cells to determine initiating and promoting activities of disodium arsenate, sodium arsenite, monomethylarsonic acid (MMAA) and dimethylarsinic acid (DMAA). Treatment with these arsenic compounds at the initiating stage induced significant numbers of transformed foci when cells were post-treated with 12-O-tetradecanoylphorbol-13-acetate (TPA). Disodium arsenate was active at the concentrations of 15-30 microM, sodium arsenite 5-20 microM, and DMAA 1-2 mM. MMAA required 10 mM to induce cell transformation. The concentrations of these compounds (except DMAA) that induced transformation were highly growth-inhibitory (more than 50%). DMAA induced transformation foci at growth inhibition levels of 66 to 84%. In experiments on promoting activity, cells pretreated with a sub-threshold dose of 20-methylcholanthrene (MCA, 0.2 microg/ml) or sodium arsenite (10 microM) were used. Transformation was enhanced by post-treatment with disodium arsenate (1-10 microM), sodium arsenite (0.5-2 microM), and MMAA (200-1000 microM), but not with DMAA. Studies of gap junctional intercellular communication using the V79 cell metabolic cooperation assay showed that the arsenic compounds (except DMAA) exhibited inhibitory activity. Thus, most arsenicals were shown to have not only initiating activity, but also promoting activity. In addition, inorganic arsenicals, especially trivalent sodium arsenite, were more active than organic ones and exhibited promoting activity at one-order of magnitude lower than initiating activity. These results suggest that from the viewpoint of human hazard, more attention should be paid to the tumor promoting activity of inorganic arsenic compounds.