Metabolic activation of o-phenylphenol to a major cytotoxic metabolite, phenylhydroquinone: role of human CYP1A2 and rat CYP2C11/CYP2E1.

1. The in vitro metabolic activation of o-phenylphenol has been evaluated as yielding a toxic metabolite, 2,5-dihydroxybiphenyl (phenylhydroquinone), by p-hydroxylation in liver microsomes of rat and human. The involvement of rat CYP2C11, CYP2E1 and human CYP1A2 in the p-hydroxylation of o-phenylphenol is suggested. 2. 2,3- and phenylhydroquinone, which induced DNA single-strand scission in the presence of 1 microM CuCl2, were the most cytotoxic chemicals examined to cultured mammalian cell lines among o-phenylphenol, m-phenylphenol, p-phenylphenol, 2,2'-, 4,4'-, 2,3- and phenylhydroquinone. 3. Rat and human liver microsomes catalysed the formation of phenylhydroquinone, but not 2,3-dihydroxybiphenyl, using o-phenylphenol as a substrate. A higher rate of metabolic activation of o-phenylphenol was observed with livers of the male than the female rats by 5.6- and 2.6-fold respectively. 4. Inhibitory antibodies against the male-specific CYP2C11 inhibited hepatic o-phenylphenol p-hydroxylation in the male F344 and Sprague-Dawley rat by > 70%. Liver microsomes from the isoniazid-treated rats produced 1.8- and 3-fold induction of o-phenylphenol p-hydroxylation and chlorzoxazone 6-hydroxylation (a CYP2E1-dependent activity) respectively. 5. Human CYP1A2, expressed by baculovirus-mediated cDNA expression systems, exhibited a remarkably higher capacity for o-phenylphenol p-hydroxylation at concentrations of 5 (> 5-fold), 50 (> 2-fold) and 500 microM (> 2-fold) than CYP2A, CYP2B, CYP2Cs, CYP2D6, CYP2E1 and CYP3A4 on the basis of pmol P450. 6. Among various CYP inhibitors tested here, 7,8-benzoflavone and furafylline, typical human CYP1A2 inhibitors, inhibited the microsomal p-hydroxylation of o-phenylphenol in human livers most potently by 70 and 50% respectively. 7. The results thus indicate the involvement of rat CYP2C11/CYP2E1 and human CYP1A2 in the hepatic p-hydroxylation of o-phenylphenol.

Interlaboratory validation of the In Vitro eye irritation tests for cosmetic ingredients (7) evaluation of cytotoxicity test by CornePack((R)).

The cytotoxicity test of neutral red (NR) uptake in normal rabbit corneal epithelial cells (CornePack((R))) was validated as an alternative method to the Draize rabbit eye irritation test (Draize test). We tested 38 cosmetic ingredients as well as isotonic sodium chloride solution in three phases of the validation study. The test procedures were controlled among participating laboratories under a common standard operating procedure (SOP). The concentration of test substances that showed a 50% reduction in NR uptake relative compared with controls (median NR uptake concentration: NR(50), mug/ml) was determined and compared with in vivo Draize scores. Six laboratories participated in the first phase of the validation study, seven in the second, and five in the third. The average interlaboratory coefficient of variation (CV) was 32.9%. The correlation and rank correlation coefficients between the maximal average Draize total scores (MAS) and NR(50) were -0.583 and 0.587, respectively. When the anionic detergents were excluded from analysis, the correlation coefficient increased to -0.738. When the cut-off point for positive and negative irritation was set at MAS of 15 and the predictability of this method was assessed by liner regression line, six substances (two acids, two alkanolamines and two alcohols) were false negative. Through this project, it appeared that CornePack, supplied in kit form with frozen secondary cultured cell in serum-free medium, could provide an effective, highly sensitive and simple preliminary screen for determining the cytotoxicity of substances. These results suggested that CornePack might have the potential to predict the MAS if definite criteria can be established for the compounds to be applicable. However, it is important to understand the nature of CornePack responses since its NR(50) profile was quite different from other cytotoxicity assays.

Interlaboratory validation of the in vitro eye irritation tests for cosmetic ingredients (9). Evaluation of cytotoxicity test on HeLa cells.

The cytotoxicity test using MTT on HeLa cells (HeLa-MTT) was evaluated as an alternative method to the Draize eye irritation test (Draize test) by six to eight laboratories. The 50% inhibition concentration (EC(50)) for MTT reduction was calculated. A total of thirty-nine test chemicals were examined. The average interlaboratory coefficient of variation (CV) of the present assay was 25%. Comparison of the in vitro test results with those of the Draize test revealed a correlation coefficient of -0.799 between the log(EC(50)) value and the maximum average total score (MAS) for a 10% solution or suspension. The following characteristics of HeLa-MTT have become apparent through this validation: (1) HeLa-MTT could be applied to all substances including water-insoluble substances, esters, a colour additive, and a substance which is known to directly reduce MTT; (2) there is good interlaboratory reproducibility and strong correlation between HeLa-MTT and Draize MAS; (3) results for strong acids, alkanolamines and alcohols (lower mono-ol) clearly deviate from other samples with respect to the correlation between HeLa-MTT and Draize MAS. These results suggest that HeLa-MTT may be useful for predicting the Draize MAS if definite criteria can be established for applicable compounds.

Interlaboratory validation of the in vitro eye irritation tests for cosmetic ingredients. (10) Evaluation of cytotoxicity test on CHL cells.

The present interlaboratory validation study was performed in order to evaluate the use of Chinese hamster lung cell lines that employs crystal violet staining (CHL-CVS) as an alternative cytotoxicity test to the Draize eye irritation test (Draize test) for cosmetic ingredients. Ten substances, nine of which were surfactants, were evaluated at seven laboratories in the first phase of the validation study; 15 substances including dyes and lipids were evaluated at seven laboratories in the second phase of the validation study; 14 substances including acids and alkalis were evaluated at four laboratories in the third phase of the validation study. The logEC(50) values obtained for CHL-CVS were compared with the maximal average Draize total score (MAS) for a 10% (w/v) solution of 38 cosmetic ingredients as well as isotonic sodium chloride solution. The interlaboratory coefficient of variation (CV) for EC(50)s was 35.6%, which was considered to be within a tolerable range. The correlation coefficient and the Spearman's rank correlation coefficient between the in vitro and in vivo tests were -0.729 and 0.709, respectively. The prediction ability of the proposed method was assessed from the linear regression line for a MAS cut-off point of 15. According to this analysis, four substances (two alcohols and two acids) were determined to be false negative. The present study revealed the following characteristic factors of this method: (1) CHL-CVS could be applied to all the test substances including dyes and lipids in this study; (2) The results for medium-insoluble substances varied according to the laboratory; (3) The correlation between the in vivo and in vitro data for acids and alcohols (lower mono-ol) differed from that of the other substances. These results suggested that the CHL-CVS might have a potential to predict the Draize MAS if definite criteria can be established for the compounds to be applicable.

Alternative methods for mechanistic studies in toxicology. Screening of hepatotoxicity of pesticides using freshly isolated and primary cultured hepatocytes and non-liver-derived cells, SIRC cells.

We constructed a screening battery for the evaluation of hepatotoxicity using freshly isolated and primary cultured rat hepatocytes (abbreviated to FIH and PCH, respectively) and rabbit eye derived cell line, SIRC cells. Effects on cell viability and drug metabolizing enzyme activities were examined by several pesticides and compared to those of in vivo. Among the pesticides studied, prometryn and ametryn showed cytotoxicity on PCH at lower concentration than on SIRC cells. Cytotoxicities of these chemicals on FIH were inhibited by metyrapone. They also increased rat serum AST in vivo. On the other hand, cytotoxicities of IBP, erusan, alanicarb, benfuracarb, and swep in PCH were observed at similar concentration to those in SIRC cells. Linuron, nitrofen, and chlomethoxifen increased ethoxycoumarin O-deethylation activities by almost similar concentration to those of benzo[a]pyrene. Linuron also induced ethoxycoumarin O-deethylation activity in vivo. These findings indicated that a battery of in vitro tests consisting of FIH, PCH and SIRC cells was useful to screen the hepatotoxicity of pesticides.

Oxysterols inhibit gap junctional communication between rat hepatocytes in primary culture.

Several oxysterols were examined for their effect on gap junctional communication between rat hepatocytes in primary culture. 25-Hydroxycholesterol, 22(S)-hydroxycholesterol and 7 beta-hydroxycholesterol, in decreasing order of potency, markedly inhibited gap junctional communication. In contrast, 7-ketocholesterol showed no inhibitory effect. The inhibition of gap junctional communication by oxysterols was not a consequence of changes in cell viability, as measured by lactate dehydrogenase leakage and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction activity. The addition of exogenous cholesterol to the culture medium did not abolish the effect of 25-hydroxycholesterol, suggesting that the capacity of oxysterols to inhibit gap junctional communication is independent of their inhibitory effect on cholesterol synthesis. We suppose that inhibition of gap junctional communication may be an early sign of oxysterols-induced toxicity on hepatocytes.

Indium inhibits gap junctional communication between rat hepatocytes in primary culture.

The effect of indium on gap junctional communication was investigated in primary cultured rat hepatocytes. Treatment of hepatocytes with indium chloride at concentrations of 100 microM to 1 mM for 2 h resulted in dose-dependent inhibition of gap junctional communication between hepatocytes. The effect of indium on hepatocytes was also evaluated using two indices for cell viability: lactate dehydrogenase (LDH) leakage and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. Indium did not cause any increase in LDH leakage from hepatocytes at the above concentrations, but inhibition of MTT reduction was observed at concentrations above 500 microM. These results suggest that the gap junctions between hepatocytes may be vulnerable sites to indium toxicity.

Induction of cytochrome P-450c and P-450d by metyrapone in the primary culture of rat hepatocytes.

The effects of metyrapone on qualitative changes in cytochrome P-450-dependent drug metabolizing activities in primary cultures of rat hepatocytes were investigated. Metyrapone apparently increased benzo(a)pyrene hydroxylation and maintained both ethoxycoumarin-O-deethylation and propoxycoumarin-O-depropylation, whereas it had little effect on methoxycoumarin-O-demethylation. Furthermore, P-450d (high spin type of P-448) as well as P-450c (low spin type of P-448) were induced by metyrapone, while P-450b and P-450e were not. In conclusion, metyrapone act as a 3-methylcholanthrene-like inducer in the primary cultures of rat hepatocytes.

Characterization of three forms of cytochrome P-450 inducible by 3-methylcholanthrene in Golden hamster livers with special reference to aflatoxin B1 activation.

Three forms of cytochrome P-450 of liver microsomes of 3-methylcholanthrene-treated Golden hamsters were purified and characterized as regards their catalytic activity toward aflatoxin B1-related hepatocarcinogenic mycotoxins. These include two major forms, designated as cytochrome P-450-AFB (P-450-I) and P-450-II, and one minor form, P-450-III. Cytochromes P-450-AFB, P-450-II, and P-450-III have their absorption maximum in the carbon monoxide-complex of the reduced form at 448.5, 447.0, and 448.0 nm, have apparent molecular weights of 56,000, 58,000, and 59,500, and are in the low spin, high spin, and low spin state, respectively. Of these, cytochrome P-450-AFB was shown to be highly active in the mutagenic activation of aflatoxin B1-related hepatocarcinogens such as sterigmatocystin and O-methylsterigmatocystin. Activation of aflatoxin B1 by hepatic microsomes of 3-methylcholanthrene-treated hamsters was inhibited almost completely by the antibody against P-450-AFB but not by the antibody against P-450-II, indicating that P-450-AFB is the major component responsible for the activation of aflatoxin B1 by hamster liver. Western blot analysis demonstrated that no protein cross-reacted with the antibody to P-450-AFB in the liver microsomes from guinea pig, rat, mouse, and house musk shrew (Suncus murinus) treated with 3-methylcholanthrene, while one or two proteins cross-reacted with the antibody to P-450-II in the liver microsomes of these animals.

Effects of various inducers on the activities of cytochrome P-450-mixed function oxidases and aflatoxin B1 activation in microsomes of hamster livers.

The effects of various inducers on the activities of drug-metabolizing enzymes including aflatoxin B1 activation were studied in Syrian golden hamsters. Activity for aflatoxin B1 was determined by aflatoxin B1-DNA adducts formation. The treatments of hamsters with 3-methylcholanthrene, alpha-naphthoflavone and benzo(a)pyrene elevated markedly the activity for aflatoxin B1 by 2460, 1380 and 450%, respectively. Phenobarbital induced slightly and isosafrole and ethanol did not induce the activity for aflatoxin B1. Pregnenolone-16 alpha-carbonitrile decreased aflatoxin B1 activation to 51% of that of the non-treated animals. These results were in good accordance with the induction rate of a form of cytochrome P-450 (P-450AFB) which has potent activity to aflatoxin B1. Characteristics in the induction of mixed function oxidases of hamsters by these inducers, especially in respect to benzo(a) pyrene metabolizing enzyme, seemed to differ from those of rats. These results suggest that the activity for aflatoxin B1 in hamster is inducible by 3-methylcholanthrene-type inducers and that hamster is a suitable animals model to study the mechanism of aflatoxin B1-hepatocarcinogenesis.

Preparation and characterization of monoclonal antibodies against a form of hamster liver cytochrome P-450 highly specific to aflatoxin B1.

Monoclonal antibodies were prepared against a form of cytochrome P-450 (designated as cytochrome P-450-I) purified from 3-methylcholanthrene-treated hamster livers which is highly specific to aflatoxin B1. The cytochrome P-450-I was detected in ELISA and Western blots in liver microsomes from 3-methylcholanthrene-treated hamsters and also from non-treated and phenobarbital-treated hamsters in smaller amounts. However, none of the liver microsomes from 3-methylcholanthrene-treated rat, rabbit, guinea pig and Suncus murinus contained the cytochrome P-450-I. These results suggest that cytochrome P-450-I is specific to hamster and is induced mainly by 3-methylcholanthrene.

Characterization of mixed-function oxidases and purified cytochrome P-450 of livers of house musk shrew, Suncus murinus.

Characteristics of mixed-function oxidases in the liver of house musk shrew, Suncus murinus, were studied. The basal level of cytochrome P-450 and the activity of drug-metabolizing enzyme in hepatic microsomes of Suncus murinus is relatively lower than that of rats, while the level of cytochrome b5 and NADPH-cytochrome c reductase is approximately the same as that of rats. The treatment of Suncus murinus with phenobarbital and 3-methylcholanthrene elevated the level of cytochrome P-450 and the activity of drug-metabolizing enzymes, but not the level of cytochrome b5. Two distinct forms of cytochrome P-450 have been purified from hepatic microsomes of 3-methylcholanthrene-treated Suncus murinus. These forms have their absorption maximum at 448.0 nm and 448.5 nm in CO-bound reduced form, and one is in the high-spin state and the other is in the low-spin state. They are different in their molecular weights (53,500 and 55,000) and in their spectral and catalytic properties. Characteristics of these forms were compared with those of the major forms of cytochrome P-450 purified from livers of rats treated with 3-methylcholanthrene.

Effects of oral contraceptive steroids (norethisterone/mestranol) on the activities of hepatic drug-metabolizing enzymes in iron-deficient anemic rats.

Either oral contraceptive steroid (norethisterone/mestranol; N/M) treatment or iron-deficiency (Fe(-] anemia alone caused an increase in NADPH cytochrome c reductase and in three hepatic microsomal mixed-function oxidase activities in female rats. When N/M treatment and the Fe(-) diet are combined, no further change in hepatic enzyme activity is seen compared with that with either treatment alone.

Comparison of hepatic drug-metabolizing enzymes induced by 3-methylcholanthrene and phenobarbital between pre- and postnatal rats.

Effects of 3-methylcholanthrene (3MC) and phenobarbital (PB) on the hepatic drug-metabolizing enzyme system in fetal liver of rats were investigated. Intraperitoneal administration of 3MC (25 mg/kg, 72 and 48 hr before death) to pregnant rats significantly increased hexobarbital (HB) and aminopyrine (AM)-metabolizing activities in fetuses on the 21st day of gestation to 148.0 and 150.6% of control fetuses, respectively. In contrast, HB and AM-metabolizing activities in 4-day-old neonates and mothers were decreased by administration of 3MC on the 21st day of gestation. Benzo[a]pyrene (BP)-metabolizing activity, NADPH-cytochrome c reductase activity, and cytochrome P-450 content in 3MC-treated fetuses were significantly increased to 2143.6, 137.6, and 323.8% of the control, respectively. Following 3MC administration, the maximum absorption of the cytochrome P-450-CO difference spectra in liver microsomes of fetuses was observed at 449-450 nm. The induction profile following 3MC administration in the fetal livers was different from that in the neonatal and the maternal livers. On the other hand, intraperitoneal administration of PB (60 mg/kg, 72, 48, and 24 hr before death) significantly increased HB, AM, and BP-metabolizing activities in fetal livers to 263.7, 231.0, and 151.2% of the respective controls. The profile induced by PB in the fetal livers was similar to that in maternal livers. These results suggest that HB and AM-metabolizing enzymes in fetal livers treated with 3MC or PB possess the capacity to be induced, and the responsiveness of the drug-metabolizing enzyme system to 3MC during the prenatal stage may differ from the postnatal stage.

Simultaneous determination of aminopyrine hydroxylation and aminopyrine N-demethylation in liver microsomes by high-performance liquid chromatography.

Aminopyrine and its metabolites, including 3-hydroxymethyl-2-methyl-4-dimethylamino-1-phenyl-3-pyrazoline-5-one which is a hydroxylated metabolite of aminopyrine, were separated on a reversed-phase (C8) Radial-Pak column using a mobile phase of methanol-triethylamine-water (30:1:69) adjusted to pH 5.40 with acetic acid. Detection of the peak was performed by an ultraviolet detector at 254 nm. By the rapid and simple method, aminopyrine hydroxylation as well as aminopyrine N-demethylation in liver microsomes can be examined simultaneously.

The heterogeneity of arylhydrocarbon hydroxylase in fetal liver microsomes of rats.

The characteristic of arylhydrocarbon hydroxylase system in fetal liver microsomes of rat was investigated. NADH-synergistic effect on NADPH-dependent arylhydrocarbon hydroxylase was observed in fetal liver microsomes of rat but not in maternal liver microsomes. NADH-synergistic effect decreased in parallel with the decrease of the ratio of cytochrome b5/cytochrome P-450 in liver microsomes. The cytochrome P-450 in arylhydrocarbon hydroxylase system in fetal liver microsomes of rat seemed to be different from that in offspring liver microsomes in respect of its dependency on cytochrome b5 system for its maximum activity.

Cytotoxic effects of methylnitrosourea on developing brain.

The effect of methylnitrosourea (MNU) on cerebellar and cerebral DNA, RNA, protein, lysosomal enzymes (acid DNase, RNase, phosphatase, and beta-glucuronidase), and 2',3'-cyclic nucleotide 3'-phosphohydrolase (2',3'-CNPase) activities was studied in rats from birth through 12 days of age. Subcutaneous injection of MNU in a dose of 0.625 mmol/kg caused a suppression of increase in weights and content of DNA, RNA, and protein of cerebellum, but no changes in those of the cerebrum or in body weight. Ratios of protein and RNA to DNA were substantially elevated by MNU in the cerebellum but not in the cerebrum. Acid DNase and acid RNase activities of MNU-treated rats were significantly elevated beyond the increase of these activities in controls in the cerebellum, but no change in these activities by MNU was observed in the cerebrum. A slight elevation in acid phosphatase activity was observed in the cerebellum but not in the cerebrum after MNU pretreatment. Beta-glucuronidase and 2',3'-CNPase activities were not changed in the cerebellum or in the cerebrum. These results suggest that in the developing brain, especially in the cerebellum at the mitotic stage, MNU caused cell damage and inhibited cell mitosis.

Studies on nitrosamine formation by the interaction between drugs and nitrite. II. Hepatotoxicity by the simultaneous administration of several drugs and nitrite.

The alterations of biochemical parameters, namely, glutamic oxaloacetic transaminase and glutamic pyruvic transaminase in serum, hepatic microsomal drug oxidation systems, glucose-6-phosphate dehydrogenase and lysozomal enzymes in hepatic soluble fraction were investigated for the purpose of semiquantitatively estimating the hepatotoxicity caused by the interaction of several drugs and sodium nitrite in rats. The simultaneous administration of aminopyrine and sodium nitrite induced the alterations of these parameters. However, these alterations were not induced by the administration of antipyrine and sodium nitrite. Therefore the alterations were thought to be mainly due to N-nitrosodimethylamine formed. The administration of 0.4 mmole/kg of aminopyrine and 1.0 mmole/kg of sodium nitrite was thought to induce the alterations to almost the same extent as induced by 0.15 mmole/kg of N-nitrosodimethylamine. The simultaneous administrations of sodium nitrite and several other drugs with tertiary amino groups, namely oxytetracycline, diphenhydramine, oleandomycin, erythromycin and minocycline induced small alterations of these parameters. However, these alterations were not thought to relate to the hepatic injury induced by N-nitrosodimethylamine. Therefore the amount of N-nitrosodimethylamine formed from these drugs in rats was thought to be rather small. We also investigated the effect of several antioxidants on the alterations of biochemical parameters induced by aminopyrine and sodium nitrite. Ascorbic acid, sodium erythorbate and propyl gallate inhibited these alterations. On the other hand, sorbic acid did not.