Induction of unscheduled DNA synthesis in primary cultures of rat, mouse, hamster, monkey, and human hepatocytes

Variation in hepatic metabolism between species may be an important factor in the differences observed in chemical carcinogenesis. We examined 6 chemicals representative of 4 chemical classes in the in vitro hepatocyte DNA repair assay using cells isolated from the Fischer-344 rat, B6C3F1 mouse, Syrian golden hamster, cynomolgus monkey and from human liver. Hepatocytes were isolated by in situ or biopsy liver perfusion and incubated with [3H]-thymidine and the test chemical. Unscheduled DNA synthesis (UDS) was measured as net grains/nucleus (NG) by quantitative autoradiography. Qualitative and quantitative differences in UDS responses were observed for every chemical. Liver cultures isolated from the rat, mouse, hamster, human, and monkey and treated with aflatoxin B1 or dimethylnitrosamine all yielded dose-related increases in NG. Human, rat, and hamster hepatocyte cultures yielded positive responses following exposure to the aromatic amines 2-acetylaminofluorene, 4-aminobiphenyl, and benzidine, whereas cultures isolated from the monkey and mouse yielded less than 0 NG. Treatment with benzo[a]pyrene (BAP) produced strong positive responses in monkey and human hepatocyte cultures, weak positive responses in hamster cultures, and equivocal or negative responses in rat and mouse hepatocyte cultures. Hepatocyte function was assessed by measurement of DNA content, glutathione content, BAP hydroxylase activity, p-nitroanisole-O-demethylase activity, p-nitrophenol conjugation, and urea synthesis rates. The functional capabilities of isolated hamster, monkey, and human hepatocyte cultures do not appear to correlate with UDS responses observed for any compound; however, they indicate that the cultures were metabolically competent at the time of chemical exposure. These studies suggest that rat hepatocytes are a suitable model for human hepatocytes, whereas mouse and male monkey hepatocytes may be insensitive to aromatic amines.     

Induction of unscheduled DNA synthesis in primary rat hepatocytes by benzidine-congener-derived azo dyes in the in vitro and in vivo/in vitro assays

The genotoxicity of the benzidine-congener-derived azo dyes. Direct Blue 1 ( DB1 ), Direct Blue 14 ( DB14 ), Direct Brown 95 ( DB95 ), and Direct Red 46 ( DR46 ) was studied in the in vitro and in vivo/in vitro unscheduled DNA synthesis (UDS) assays in primary rat hepatocytes to determine if in vivo metabolism of these compounds was required for induction of UDS. Hepatocytes were isolated, cultured, and treated with the azo dyes and [3H]thymidine (in vitro assay); alternatively, in the in vivo/in vitro assay, rats were intubated with the azo dyes, the hepatocytes isolated at 17 h after dosing and incubated in a medium containing [3H]thymidine. UDS was quantified by an autoradiographic method. None of the azo dyes induced UDS in the in vitro assay. However, DR46 did induce marginal, but significant UDS in 1 experiment (1.2 net grains at 500 micrograms/ml media). No significant UDS was observed when DR46 was tested in a subsequent in vitro assay. In the in vivo/in vitro assay, DB95 (100 mg/kg), DB14 (125 mg/kg), and DR46 (100 mg/kg) induced significant UDS (12, 2.1, and 3.5 net grains, respectively). None of the azo dyes tested was mutagenic in the Salmonella/microsome assay in the presence and absence of rat liver enzymes. Therefore, in vivo reduction of azo dyes, presumably by the gut microflora, is a requirement for the genotoxicity of these azo dyes in the primary rat hepatocyte UDS assay.     

Unscheduled DNA synthesis in human hair follicles after in vitro exposure to 11 chemicals: comparison with unscheduled DNA synthesis in rat hepatocytes.

A new method is described to investigate unscheduled DNA synthesis (UDS) in human tissue after exposure in vitro: the human hair follicle. A histological technique was applied to assess cytotoxicity and UDS in the same hair follicle cells. UDS induction was examined for 11 chemicals and the results were compared with literature findings for UDS in rat hepatocytes. Most chemicals inducing UDS in rat hepatocytes raised DNA repair at comparable concentrations in the hair follicle. However, 1 of 9 chemicals that gave a positive response in the rat hepatocyte UDS test, 2-acetylaminofluorene, failed to induce DNA repair in the hair follicle. Metabolizing potential of hair follicle cells was shown in experiments with indirectly acting compounds, i.e., benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene and dimethylnitrosamine. The results support the conclusion that the test in its present state is valuable as a screening assay for the detection of unscheduled DNA synthesis. Moreover, the use of human tissues may result in a better extrapolation to man.     

Unscheduled DNA synthesis in human hair follicles after in vitro exposure to 11 chemicals: comparison with unscheduled DNA synthesis in rat hepatocytes

A new method is described to investigate unscheduled DNA synthesis (UDS) in human tissue after exposure in vitro: the human hair follicle. A histological technique was applied to assess cytotoxicity and UDS in the same hair follicle cells.UDS induction was examined for 11 chemicals and the results were compared with literature findings for UDS in rat hepatocytes. Most chemicals inducing UDS in rat hepatocytes raised DNA repair at comparable concentrations in the hair follicle. However, 1 of 9 chemicals that gave a positive response in the rat hepatocyte UDS test, 2-acetylaminofluorene, failed to induce DNA repair in the hair follicle.Metabolizing potential of hair follicle cells was shown in experiments with indirectly acting compounds, i.e., benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene and dimethylnitrosamine.The results support the conclusion that the test in its present state is valuable as a screening assay for the detection of unscheduled DNA synthesis. Moreover, the use of human tissues may result in a better extrapolation to man.     

Hepatocyte ploidy in normal young rat

The aim of the present study was to examine the relation between hepatocyte size and ploidy in Sprague-Dawley rat liver. Therefore, subpopulations of hepatocytes of various sizes were separated from the isolated crude hepatocyte population either mechanically or by using centrifugal elutriation. Hepatocyte size was determined on scanning electron microscopy photographs. Ploidy of hepatocytes was assessed by flow cytometry. The crude hepatocyte population was very heterogeneous in sizes, with diameters ranging from 8 to 39 microm. Hepatocyte ultrastructure was well preserved as demonstrated by transmission electron microscopy. The distribution of hepatocytes within the ploidy classes was the following: 19.6+/-3.6% diploid, 56.2+/-3.2% tetraploid and 3.4+/-0.6% octoploid mononucleated cells. Thus approximately 79% of hepatocytes appeared mononucleated. The binucleated hepatocytes (21%) had two diploid nuclei (18.7+/-2.9%) or two tetraploid nuclei (2.1+/-0.6%). A similar distribution of hepatocytes into ploidy classes was obtained in subpopulations of hepatocytes of various sizes. Our findings suggest that distribution into ploidy classes is not strictly correlated with hepatocyte size. In accordance with previous observations, our results on hepatocyte ploidy from periportal or perivenous origin using digitonin perfusion, is in favour of the existence of ploidy zonation within the rat hepatic lobule.     

Characterization of the effects of human placental HGF on rat hepatocytes.

Hepatocyte growth factor (HGF) also known as hepatopoietin A (HPTA) (Michalopoulos, FASEB J., 4:176-187, 1990) is a heparin-binding growth factor whose characterization and tissue distribution have been reported elsewhere. This growth factor was recently cloned and its amino acid sequence determined under the name of hepatocyte growth factor (HGF) (Miyazawa et al., Biochem. Biophys. Res. Commun., 163:967-973, 1989; Zarnegar et al., Biochem. Biophys. Res. Commun., 163:1370-1376, 1989; Nakamura et al., Nature, 342:440-443, 1989). Human placenta is one of the tissues that contains significant amounts of HGF. We isolated HGF from human placenta and characterized its biologic effect on rat hepatocytes. Human placenta HGF was isolated in high purity as a single chain molecule. Single chain HGF stimulated DNA synthesis in primary rat hepatocyte cultures in serum-free medium. The maximal effect was seen at 5-10 ng/ml. The maximal response occurred at 25-48 hours after plating of the hepatocytes. Protein synthesis was also stimulated by HGF in primary rat hepatocyte cultures. There were peak responses at 19-24 and 37-42 hours after plating of the hepatocytes. TGF beta 1 inhibited more than 95% of HGF-induced DNA synthesis but only 25% of HGF-induced protein synthesis. HGF interacted in an additive manner with EGF, a well-known hepatocyte mitogen. There was not an additive interaction between HGF and aFGF. Regenerating liver hepatocytes obtained from rats which underwent two-thirds partial hepatectomies (PHX) also responded to HGF in a dose-dependent manner as the hepatocytes from normal liver.     

Species differences in cytotoxic and genotoxic effects of phenacetin and paracetamol in primary monolayer cultures of hepatocytes

The cytotoxic and genotoxic effects of phenacetin and paracetamol were examined in monolayer cultures of hepatocytes isolated from the mouse, hamster, rat and guinea pig. No marked increase in unscheduled DNA synthesis (UDS) after exposing hepatocytes from any of the species to phenacetin was observed. At cytotoxic concentrations of paracetamol, an increased UDS in mouse hepatocytes in vitro was observed. Pretreatment of the mice by inducers of drug-metabolizing enzymes, such as 3-methylcholanthrene and Aroclor 1254, lowered the concentration threshold for the toxic responses. With rat hepatocytes only a minor increase in UDS was noted, while with hepatocytes from hamsters and guinea pigs in fact a decrease was seen. The narrow range observed between the cytotoxic and genotoxic effects of paracetamol makes it difficult to predict whether the initial DNA damage could lead to a mutation or whether the cells will die before the mutation is expressed. With respect to the cytotoxic effects, hamster hepatocytes were found to be most susceptible to paracetamol, followed by mouse, while rat and guinea pig were less affected. These data were in accordance with in vivo findings (Davis et al., 1974), indicating the potential value of hepatocyte culture when screening for possible liver toxic substances.     

Application of the neutral red assay (NR assay) to monolayer cultures of primary hepatocytes: rapid colorimetric viability determination for the unscheduled DNA synthesis test (UDS)

The neutral red (NR) absorption method was adapted for the determination of cell viability in the UDS assay with primary hepatocyte cultures of the rat. The NR method is rapid, easy to perform, and suitable for handling of large numbers of cultures simultaneously. It can be used for concentration range-finding pre-experiments. In addition, it can easily be integrated into a UDS test protocol for documentation of toxic effects if supplementary cultures for each concentration are established. The time schedule required for the NR assay makes it possible for one person to process the hepatocytes for autoradiography and at the same time determine the toxicity.     

Evaluation of calcium channel blockers as potential hepatoprotective agents in oxidative stress injury of perfused hepatocytes

The aim of this study was to investigate the effects of calcium channel blockers on tertbutyl hydroperoxide (TBH) induced liver injury using isolated perfused rat hepatocytes. Rat hepatocytes were immobilized in agarose threads and perfused with Williams E medium. Hepatocyte injury was induced by the addition of tertbutyl hydroperoxide (1 mM) to the perfusion medium 30 min after the addition of either verapamil or diltiazim. Hepatocyte injury was observed by monitoring the functional and metabolic competence of hepatocytes or by ultrastructural morphological examination of hepatocytes. Verapamil (0.5 mM) reduced lactate dehydrogenase leakage in TBH-injured hepatocytes as compared to the controls (154+/-11% vs. 247+/-30%). Lipid peroxides production was reduced after verapamil pretreatment as compared to the controls and oxygen consumption was increased by pretreatment of hepatocytes with verapamil. Verapamil pretreatment increased the protein synthesis activity at both levels of granular endoplasmic reticulum and free polysomes in cytoplasm and decreased ATPase activity. Diltiazem was qualitatively effective as verapamil. It is concluded that in hepatocyte oxidative injury, calcium channel blockers exhibited hepatoprotective properties. The hepatoprotective effect of calcium channel blockers was accompanied by a decrease in ATPase activity, which may implicate a normalization of Ca2+i after TBH intoxication.     

Contribution of serum protein association to discrepancy between the in vivo and in vitro UDS results for 6,7-dimethyl-2,4-di-1-pyrrolidinyl-7H-pyrrolo[2,3-d]pyrimidine (U-89843)

U-89843 has been shown to undergo biotransformation, both in vitro and in vivo, to form U-97924 as a major primary metabolite. U-89843 was found to be positive in an in vitro UDS mutagenesis screen conducted with primary rat hepatocytes in serum-free media. In contrast to in vitro results, no evidence of genetic toxicity of U-89843 was observed in rats in the in vivo/in vitro version of the UDS test with single oral doses up to 1400 mg/kg. The negative results may be related to more robust in vivo detoxification mechanisms or relatively lower exposure to reactive metabolites formed by bioactivation of U-89843 as compared to that observed in the serum-free in vitro hepatocyte test system. Further studies showed rat serum suppressed the in vitro metabolism of U-89843 as well as the formation of the corresponding hydroxylated metabolite, U-97924, the putative precursor of proposed reactive electrophilic metabolite. The measured in vivo systemic clearance of U-89843 (0.53 l/h/kg) in rats was about 1000-fold slower than the in vitro intrinsic clearance (606 l/h/kg) estimated by measuring the formation of U-97924 in rat liver microsomal incubations. Since U-89843 is extensively associated with serum proteins a poor extraction ratio into the liver may account for the slower biotransformation of U-89843 in vivo as compared to that exhibited in in vitro serum-free hepatocyte incubations. Addition of bovine serum albumin (1–40 mg/ml) to the in vitro UDS assay medium decreased the UDS mean net grains per nucleus response of U-89843. These results suggest that the effect of serum protein should be considered when comparing serum-free in vitro UDS and in vivo UDS results for highly serum protein bound compounds.     

DNA-repair studies with sodium fluoride: comparative evaluation using density gradient ultracentrifugation and autoradiography

Sodium fluoride (NaF) was assayed for the induction of DNA-repair synthesis in WI-38 human diploid fibroblasts and in primary cultures of rat hepatocytes. DNA-repair synthesis in non-replicating DNA was measured by ultracentrifugation of density-labeled DNA in CsCl gradients. When this method was used, NaF did not induce DNA-repair synthesis in either of these cell types. However, when NaF was assayed for induction of unscheduled DNA synthesis (UDS) in rat hepatocytes by autoradiography, an increased net nuclear grain count was observed. Because the autoradiographic results were not confirmed by density-gradient ultracentrifugation of hepatocyte DNA, which is a more definitive technique, it is doubtful whether the autoradiographic results actually represent DNA-repair synthesis. Modifications of the UDS/autoradiography protocol to include more extensive washing resulted in no UDS response. Published reports (Hellung-Larsen and Klenow, 1969; Srivastava et al., 1981) describe the formation of precipitable complexes of Mg2+, F-, and [3H]thymidine triphosphate which suggests that autoradiographic measurement of UDS may lead to artifacts when testing NaF unless extensive washing of the cultures is employed.     

Effects of medium composition on the morphology and function of rat hepatocytes cultured as spheroids and monolayers

Primary hepatocytes cultured as monolayers or as spheroids were studied to compare the effects of four different culture media (Williams' E, Chee's, Sigma Hepatocyte, and HepatoZYME medium). Rat hepatocytes were cultured as conventional monolayers for 3 d or as spheroids for 2 wk. For spheroid formation a method was emplOyed that combined the use of a nonadherent substratum with rotation of cultures. Hepatocyte integrity and morphology were assessed by light and electron microscopy and by reduced glutathione content. Hepatocyte function was measured by albumin secretion and 7-ethoxycoumarin metabolism. Chee's medium was found to be optimal for maintenance of hepatocyte viability and function in monolayers, but it failed to support spheroid formation. For spheroid formation and for the maintenance of spheroid morphology and function, Sigma HM was found to be optimal. These results demonstrate that the medium requirements of hepatocytes differ markedly depending on the culture model employed. Spheroid culture allowed better preservation of morphology and function of hepatocytes compared with conventional monolayer culture. Hepatocytes in spheroids formed bile canaliculi. and expressed an actin distribution resembling that found in hepatocytes in vivo. Albumin secretion was maintained at the same level as that found during the first d in primary culture, and 7-ethoxycoumarin metabolism was maintained over 2 wk in culture at approximately 30% of the levels found in freshly isolated hepatocytes. The improved morphology and function of hepatocyte cultures as spheroids may provide a more appropriate in vitro model for certain applications where the maintenance of liver-specific functions in long-term culture is crucial.     

Evaluation of four methods for scoring cytoplasmic grains in the in vitro unscheduled DNA synthesis (UDS) assay

The in vitro unscheduled DNA synthesis (UDS) assay measures DNA repair (incorporation of [³H]thymidine) following in vitro treatment of rat primary hepatocytes. The autoradiographic method was used to detect UDS by counting developed silver grains in the photographic emulsion overlaying nuclei and cytoplasmic areas of the hepatocytes. In this communication we report results using 4 scoring methods: (1) the most heavily labeled cytoplasmic areas adjacent to the nucleus (our standard method), (2) the cytoplasmic area left of the nucleus, (3) the cytoplasmic areas left and right of the nucleus, and (4) 2 cytoplasmic areas whose positions were selected at random. Rat primary hepatocyte cultures treated with a medium control, a solvent control (dimethyl sulfoxide) and 5 known genotoxic chemicals (2-acetylamino-fluorene, dimethylnitrosamine, diethylnitrosamine, methyl methanesulfonate and ethyl methanesulfonate) were scored using these 4 methods. The average or maximum cytoplasmic grain count was subtracted from the nuclear grain count to yield net grains/nucleus (NG). In general, NG counts for Methods 2,3 and 4 were similar, although shifted about 3–10 grains higher than Method 1 for controls and most treated groups. Methods 2, 3 and 4 showed more experiment-to-experiment variability in sensitivity for detecting statistically significant increases in treated groups than did our standard method. Thus, the alternative methods afforded no consistent improvements in sensitivity or reduction of variability for this assay. Subtraction of the average or the highest cytoplasmic count had virtually no effect on the sensitivity of the assay, but simply requires an appropriate adjustment of the criteria for a positive response.     

Induction of DNA-repair synthesis in primary rat hepatocytes by epoxides

The genotoxicity of 10 epoxides was investigated in the UDS test with primary rat hepatocytes. The sensitivity of the assay was demonstrated using 2-acetylaminofluorence. The epoxides 1,2-epoxyoctane, 1,2-epoxydecane, epoxycyclooctane, epoxycyclododecane, (+)-limoneoxide, alpha-pinaneoxide, transstilbeneoxide, and cis-2,3-epoxysuccinic acid, which are known to be non-mutagenic in the Ames test, as well as the bacterial mutagen, 1,2-epoxyphenoxypropane did not induce UDS in primary hepatocytes of the rat. However, a positive UDS response obtained with glycidyltrimethylammonium chloride showed that metabolic inactivation of the oxirane ring in hepatocytes is influenced by further structural substituents.     

DNA synthesis in rat hepatocytes: Inhibition by a platelet factor and stimulation by an endogenous factor

Primary monolyer cultures of adult rat hepatocytes can be induced to undergo DNA synthesis in serum-free medium in the presence of insulin, glucagon, and epidermal growth factor (three factors). We have found that hepatocyte DNA synthesis is affected not only by an endogenous stimulant produced by the hepatocytes and released into the culture medium. Serum has a strong inhibitory effect on hepatocyte DNA synthesis. Partially purified human platelet extract (“platelet inhibitor”) inhibits the three-factor-induced DNA synthesis in a concenration-dependent manner. Pure βTGF at 0.5 ng/ml as well as HPLC-purified PDGF at 10 ng/ml completely inhibit the three-factor-induced DNA synthesis. Determination of the time required for the presence of the three factors and the platelet inhibitor to exert their effects indicated that the inhibition of DNA synthesis is caused not by competition of the platelet inhibitor with any of the three factors but through an independent pathway. Hepatocyte DNA synthesis is density-dependent and is greater if medium is not changed during the course of an experiment than if medium is changed daily. Hepatocyte-conditioned medium is also affective in stimulating DNA synthesis beyond the level induced by the three factors. These results suggest that an endogenous stimulant for hepatocyte DNA synthesis is produced by the hepatocytes themselves. Our studies demonstrate that hepatocyte DNA synthesis is subject to both stimulatory and inhibitory controls. Unlike the three factors, the endogenous stimulant can overcome the inhibition by the platelet inhibitor, suggesting the importance of these factors in the physiological control of hepatocyte DNA synthesis.     

Functional role of RhoA in growth regulation of primary hepatocytes

The expression, activation and involvement in growth regulation of a small GTPase, RhoA, were examined in rat primary hepatocyte cultures. Hepatocytes freshly isolated from liver expressed RhoA protein at high levels. The total level of RhoA protein in the cells decreased markedly within a day in monolayer cultures. Thereafter, RhoA expression recovered as cell-cell attachment occurred during the culture. On the other hand, the level of the active form of RhoA decreased as the culture proceeded. Ca(2+) depletion in the medium to disrupt cadherin engagement triggered RhoA activation without de novo protein synthesis, indicating cadherin engagement regulates RhoA activation in hepatocytes. Hepatocyte growth stimulation by HGF was enhanced by Ca(2+) depletion or introduction of a constitutively active form of RhoA. The Clostridium botulinum C3 enzyme inhibited hepatocyte growth with stimulation by HGF. These results suggest that RhoA has a crucial role in hepatocyte growth control.     

Genotoxicity in the hepatocyte/DNA repair test and toxicity to liver mitochondria of 1-hydroxyanthraquinone and several dihydroxyanthraquinones

1-Hydroxyanthraquinone and dihydroxyanthraquinones (alizarin, quinizarin, anthrarufin and chrysazin) were examined for genotoxicity in the hepatocyte/DNA repair test and for effects on oxidative phosphorylation in isolated rat liver mitochondria. Of the anthraquinone compounds tested, 1-hydroxyanthraquinone and 1,8-dihydroxyanthraquinone (chrysazin) induced DNA repair synthesis in rat hepatocytes, indicating their genotoxic activity. Only 1,2-dihydroxyanthraquinone (alizarin) exerted an uncoupling and inhibitory effect on mitochondrial respiration. The possible relationships of the genotoxic potencies and the uncoupling activities of these anthraquinones to their chemical structures are discussed.Abbreviations ADP adenosine-5-diphosphate - ETP electron transport particles - RC respiratory control - TdR thymidine deoxyribonucleotide - UDS unscheduled DNA synthesis     

Induction of DNA repair in human and rat hepatocytes by 1,6-dinitropyrene

1,6-Dinitropyrene (DNP) was found to be an extremely potent genotoxicant in metabolically competent primary cultures of human and rat hepatocytes. Dose-dependent increases in DNA repair as measured by unscheduled DNA synthesis (UDS) were observed in the range from 0.05 to 5 microM 1,6-DNP for both species, indicating that the rat-hepatocyte assay is an appropriate model for assessing genotoxic potential in human hepatocytes for this class of compound. Unlike some nitroaromatic compounds, 1,6-DNP did not require gut flora for metabolic activation. No DNA repair was observed in hepatocytes isolated from rats treated with 50 mg/kg 1,6-DNP in corn oil by gavage 2, 12 or 24 h previously. The reason for the lack of a response in vivo is not known, but may relate to detoxification or distribution of the compound in the animal.     

Improved maintenance of adult rat hepatocytes in a new serum-free medium in the presence or absence of barbiturates

Summary For serum-free primary culture of adult rat hepatocytes, a synthetic medium DM-160 and rat-tail collagen were selected for the basal medium and for the culture substratum, respectively. Barbiturates, such as phenobarbital and 1-ethyl-5-isobutylbarbiturate, efficiently supported survival of hepatocytes and maintained their morphologic features at lower concentrations under the serum-free conditions than under the serum-supplemented conditions. However, the hepatocyte survival rates under the serum-free conditions were lower than those under the serum-supplemented conditions in the presence or absence of barbiturates. Supplementation of the basal medium with a combination of five groups of factors (5Fs), such as eight amino acids (Ala, Arg, Gly, Ile, Met, Phe, Pro, and Trp), two unsaturated fatty acids (linoleate and oleate), a protease inhibitor (aprotinin), three vitamins (A, C, and E), and five trace elements (Mn, Fe, Cu, Zn, and Se), improved the hepatocyte survival under the serum-free conditions in the presence or absence of barbiturates. In other words, the serum could be completely substituted by the 5Fs. Hepatocyte cultures maintained in the 5Fs-suppelemented basal medium showed excellent induction of tyrosine aminotransferase activity in response to dexamethasone in the presence or absence of barbiturates. The efficiency of the 5Fs-supplemented basal medium for maintaining hepatocytes was not inferior to those of other media in common use with hepatocytes, such as Williams' medium E and Waymouth's medium MB-752/1. In conclusion, maintenance of functional hepatocytes in serum-free primary culture could be improved by use of the new medium preparation (the 5Fs-supplemented DM-160) in the presence of barbiturates. This work was supported by a grant no. 61771923 from the Ministry of Education, Science and Culture of Japan.