Effect of microsampling (50 µL) on toxicological evaluation of methapyrilene, a hepatotoxic substance, in a collaborative 28-day study in female rats.

Although microsampling of blood is recommended to promote the 3Rs in toxicokinetic (TK) evaluation, there are few reports applying microsampling in actual toxicity evaluation. Here, we assessed the effects of microsampling on toxicological evaluation of methapyrilene hydrochloride, a hepatotoxic substance. Female SD rats received methapyrilene hydrochloride orally at dose levels of 0 (vehicle), 10, and 30 mg/kg BW, once daily for 4 weeks. Each dose level included a microsampling group and a non-microsampling group (n = 5). In the microsampling groups, blood sampling (50 µL/time point) was performed at 6 time points on day 1 of administration and 7 time points on day 27-28; all the animals underwent necropsy on day 29. Toxicity studies and TK analysis were performed, and through these studies in 2 organizations, cross-organization validation of the effect on toxicity evaluation was conducted. In one organization, microsampling obscured changes in some parameters in hematology due to the administration of methapyrilene hydrochloride. In the other organization, although the relationship between the developing pattern of histopathological findings in the liver and the blood sampling was suspected, it was associated with poor reproducibility; this was considered as a change within a variation range of biological reactions. Each of these phenomena was observed in only one organization without consistency. In both organizations, no effect of blood microsampling was observed in other endpoints. In conclusion, microsampling is considered to be a technique applicable to safety studies of drugs showing hepatotoxicity, as it did not show a marked influence on the toxicological evaluation of methapyrilene hydrochloride.

In vitro - in vivo correlation of gene expression alterations induced by liver carcinogens.

Although cultivated hepatocytes are widely used in the studies of drug metabolism, their application in toxicogenomics is considered as problematic, because previous studies have reported only little overlap between chemically induced gene expression alterations in liver in vivo and in cultivated hepatocytes. Here, we identified 22 genes that were altered in livers of rats after oral administration of the liver carcinogens aflatoxin B1 (AB1), 2-nitrofluorene (2-NF), methapyrilene (MP) or piperonyl-butoxide (PBO). The functions of the 22 genes have been classified into two groups. Genes related to stress response, DNA repair or metabolism and genes associated with cell proliferation, respectively. Next, rat hepatocyte sandwich cultures were exposed to AB1, 2-NF, MP or PBO for 24h and expression of the above mentioned genes was determined by RT-qPCR. Significant correlations between the degree of gene expression alterations in vivo and in vitro were obtained for the stress, DNA repair and metabolism associated genes at concentrations covering a range from cytotoxic concentrations to non-toxic/in vivo relevant concentrations. In contrast to the stress associated genes, no significant in vivo/in vitro correlation was obtained for the genes associated with cell proliferation. To understand the reason of this discrepancy, we compared replacement proliferation in vivo and in vitro. While hepatocytes in vivo, killed after administration of hepatotoxic compounds, are rapidly replaced by proliferating surviving cells, in vitro no replacement proliferation as evidenced by BrdU incorporation was observed after washing out hepatotoxic concentrations of MP. In conclusion, there is a good correlation between gene expression alterations induced by liver carcinogens in vivo and in cultivated hepatocytes. However, it should be considered that cultivated primary hepatocytes do not show replacement proliferation explaining the in vivo/in vitro discrepancy concerning proliferation associated genes.

Non-genotoxic carcinogens: early effects on gap junctions, cell proliferation and apoptosis in the rat.

Non-genotoxic carcinogens are thought to induce tumour formation by disturbing the balance between cell growth and cell death. Gap junctions (GJ) contribute to the maintenance of tissue homeostasis by allowing the intercellular exchange of growth regulatory signals and potential inhibition of GJ intercellular communication through loss of connexin (Cx) plaques has been shown to be involved in the cancer process. We have investigated the time- and dose-dependent effects of the non-genotoxic hepatocarcinogens Wy-14,643, 2,3,7,8-tetrachlorodibenzo-p-dioxin, methapyrilene and hexachlorobenzene and the male rat kidney carcinogens chloroform, p-dichlorobenzene and d-limonene on gap junction plaque expression in relation to proliferation and apoptosis. With the exception of limonene, all non-genotoxic carcinogens significantly reduced the expression of GJ plaques containing Cx32 in their respective target tissue. No dose-dependent, significant effects were seen in non-target organs. Although alteration of Cx32 expression did not appear to correlate with induction of cell proliferation, out data suggest that the interaction of both processes-interference of GJ coupled with a proliferative stimulus (at the carcinogenic dose)-may be important in non-genotoxic carcinogenesis and provide a potential alert for non-genotoxic carcinogens in short-term toxicity tests.

Gliotoxin stimulates the apoptosis of human and rat hepatic stellate cells and enhances the resolution of liver fibrosis in rats.

BACKGROUND & AIMS: Hepatic stellate cells (HSCs) play a pivotal role in liver fibrosis and stimulating their apoptosis could be an effective treatment for liver fibrosis. METHODS: Activated HSCs, hepatocytes, and rats with liver fibrosis were treated with gliotoxin. RESULTS: Addition of gliotoxin to activated (alpha-smooth muscle actin positive) rat and human HSCs resulted in morphologic alterations typical of apoptosis. Within 2-3 hours of incubation, caspase 3 activity was markedly induced and caspase inhibitor 1 (Z-VAD-FMK)-sensitive oligonucleosome-length DNA fragments were detectable by gel electrophoresis of low molecular weight DNA. Apoptosis was widespread as judged by fluorescence-activated cell sorter analysis and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining in both rat and human HSCs at concentrations that had no effect on the viability of rat hepatocytes. Gliotoxin treatment significantly reduced the number of activated stellate cells and mean thickness of bridging fibrotic septae in livers from rats treated with carbon tetrachloride. CONCLUSIONS: These data demonstrate proof-of-concept that by up-regulating HSC apoptosis, the extent of fibrosis can be decreased in inflammatory liver injury.

Comparisons of protein changes in human and rodent hepatocytes induced by the rat-specific carcinogen, methapyrilene.

There is a growing concern that the rodent bioassay may not always serve as an appropriate model to assess the carcinogenic risk for humans exposed to certain compounds. Mechanistic research that examines the effects of a compound in rodent and man could help in the interpretation of bioassay results. This paper reports a novel use of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) technology to assess similarities and differences in the response of rodents and humans to the rat-specific hepatocarcinogen, methapyrilene (MP). A sequential examination of rodent and human hepatic proteins was conducted following in vivo exposure of rats and mice and in vitro exposure of rat, mouse, and human hepatocytes to MP. Results showed that covalent modifications observed in rats and mice in vivo were duplicated both qualitatively and quantitatively in the corresponding in vitro systems and that these modifications correlated with carcinogenic susceptibility. Covalent modifications in human hepatocytes were minimal despite exposure to concentrations of MP that were 6-fold higher than those used in rodent hepatocytes. These studies suggest that in the case of MP the rat is not the most appropriate model for assessing the human situation. Furthermore, these data show that in vitro-in vivo comparisons based on 2-D PAGE may provide adjunctive information for extrapolating rodent toxicity/bioassay results to human risk assessment.

Effects of methapyrilene measured in mitochondria isolated from naive and methapyrilene-treated rat and mouse hepatocytes.

Methapyrilene (MP) is a rat-specific liver carcinogen that alters mitochondrial number and morphology both in vivo and in vitro. This biological phenomenon may be due to the effects of MP on mitochondrial function. To test this hypothesis, studies were conducted to examine the effects of MP on DNA and protein synthesis and respiration in isolated mitochondria. DNA and protein synthesis activities were measured using [3H]thymidine and [3H]leucine incorporation. Mouse liver mitochondria were also examined for comparison since no tumor formation or alterations in mitochondrial morphology have been associated with MP treatment in mice. A significant decrease in basal DNA and protein synthesis levels was observed in mitochondria isolated from rats and mice following in vivo MP treatment. This effect could not be reproduced when mitochondria were exposed to 0 or 100 microM MP following isolation, despite the presence of an S9 activation system. Electron microscopic examinations were performed on isolated rat mitochondria and revealed morphologic differences between mitochondria from naive and MP-treated rats. Although significant differences in State 3 and State 4 respiratory rates were noted, the respiratory control ratio, ADP/O ratio, and uncoupler-stimulated respiratory rates were unaffected. Results demonstrate that: (1) MP irreversibly depresses DNA and protein synthesis in a majority of mitochondria, despite only localized morphologic changes; (2) these changes are not reflected by a decrease in respiratory function; and (3) depression of DNA and protein synthesis does not correlate with carcinogenic susceptibility.

Modulation of rat hepatic cytochromes P450 by chronic methapyrilene treatment.

The antihistaminic compound methapyrilene (MP) when chronically administered has been shown to be a rat-specific hepatocarcinogen. To examine the effects of chronic MP treatment on the hepatic microsomal cytochromes P450. Fischer 344 rats were gavaged for 10 weeks (5 days on, 2 days off) with either vehicle or 50, 100, or 150 mg MP/kg body weight. Chronic MP treatment was found to have a significant effect on several microsomal enzymatic activities. Small (17-28%) but significant (P less than 0.05) decreases were observed for total P450 levels and the activities of erythromycin N-demethylase (catalyzed by P450IIIA), N-nitrosodimethylamine demethylase (catalyzed by P450IIE1) and pentoxyresorufin O-dealkylase (catalyzed by P450IIB1). In addition, a relatively large decrease (approximately 80%) was observed for the activity of benzphetamine N-demethylase (representative of P450IIC11) and an induction of about 40% was observed for ethoxyresorufin O-dealkylase (catalyzed by P450IA). The metabolism of testosterone by microsomes isolated from the rats chronically treated with MP indicated that several reactions were compromized. Specifically, testosterone 2 alpha-hydroxylase, indicative of P450IIC11, was reduced greatly (86%), whereas testosterone 6 beta-hydroxylase, reflecting P450IIIA, and testosterone 7 alpha-hydroxylase, indicative of P450IIIA1, were affected only slightly by MP treatment (approximately 25%). Immunoblot analyses of the various microsomal samples were performed to determine if chronic MP treatment had direct effects on the level of expression of the cytochromes P450. Decreases in the levels of P450IIIA, IIE1, and IIC11, determined by immunoblot analyses, closely paralleled those observed for their marker catalytic activities. Further studies will be required to determine the mechanism by which MP affects the levels of the cytochromes P450 (i.e. increased degradation or decreased synthesis).

Glutathione and lipid peroxide levels in rat liver following administration of methapyrilene and analogs.

The possibility was examined that the induction of tumors in rat liver by feeding methapyrilene, which is not mutagenic, is related to effects on glutathione levels and lipid peroxidation. Fischer 344 rats were given single-dose and multiple-dose treatments with the anti-histamine methapyrilene (MP), which is carcinogenic in rats, and with two non-carcinogenic analogs, methafurylene (MF) and thenyldiamine (TD) and the effects on malonaldehyde (MDA) formation and glutathione (GSH) levels in the liver were investigated. After a single dose, MDA levels were increased at 6 h by MF and TD and at 24 h by MP. MDA levels returned to normal after 30 h with MP and MF, but not with TD. Levels of MDA (and other TBA-reactive products) after four daily treatments were most elevated by TD, less elevated by MP, and were lowered by MF. Forty-two hours following treatment with both MP and MF, MDA levels had returned to normal, but in TD-treated animals MDA remained high. GSH levels were highest after MF and MP, and remained high at 42 h, but TD induced only a small increase. There appears to be increased lipid peroxidation in the liver as a result of treatment of rats with MP, MF and TD. The greater response induced by TD, as well as the increased liver GSH levels after repeated administration of all three drugs indicate that lipid peroxidation in rat liver is not a particular effect related to the liver carcinogen methapyrilene.

The potent hepatocarcinogen methapyrilene does not form DNA adducts in livers of Fischer 344 rats.

The antihistamine methapyrilene hydrochloride has been shown to be a potent hepatocarcinogen in Fischer 344 rats. It has also been evaluated in a number of short-term in vitro genotoxicity assays resulting in conflicting reports. Short-term in vivo assays suggest that it may act as a promoter. We studied its ability to form DNA adducts in the target organ using the highly sensitive 32P-postlabeling technique. Methapyrilene failed to induce formation of DNA adducts in hepatocellular DNA at doses which induced S-phase DNA synthesis. These data suggest that methapyrilene does not induce the carcinogenesis process through a direct genotoxic mechanism.

Choline deficient diet enhances the initiating and promoting effects of methapyrilene hydrochloride in rat liver as assayed by the induction of gamma-glutamyltranspeptidase-positive hepatocyte foci.

Earlier we demonstrated that short-term feeding of methapyrilene hydrochloride (MPH) and of a choline deficient (CD) diet to rats induced peroxidative damage of microsomal membrane lipids of liver cells. In the present study, we investigated whether a CD diet modifies the extent of MPH-induced lipid peroxidation and whether the modifications lead to changes in the initiating and promoting action of these agents using assays of the induction of gamma-glutamyltranspeptidase (GGT)-positive hepatocyte foci. Addition of 0.1% MPH to a CD diet enhanced the extent of microsomal lipid peroxidation induced by a CD diet alone. Feeding a choline supplemented (CS) or a CD diet containing 0.1% MPH for 2 weeks followed by 7 weeks promotion by a CD diet plus phenobarbital was ineffective in inducing GGT-positive foci. Feeding MPH in a CS or a CD diet for 4 weeks, however, resulted in the development of substantial numbers of GGT-positive foci. There was a 3 fold increase in the number of foci in rats initiated with a CD + MPH diet over that in rats initiated with a CS + MPH diet. 0.1% MPH in a CS diet or a CD diet exerted significant promotional effects on the induction of GGT-positive foci in rats initiated with a single injection of diethylnitrosamine. Addition of MPH to a CD diet was additive in inducing GGT-positive foci. The results suggest that lipid peroxidation of the liver may be involved in the carcinogenic and/or promoting effects of MPH and a CD diet.

Methapyrilene is a genotoxic carcinogen: studies on methapyrilene and pyrilamine in the L5178Y/TK +/- mouse lymphoma assay.

Methapyrilene (MP), a sedating antihistamine, is a potent rat hepatocarcinogen which has been thought to be non-genotoxic on the basis of the negative results in a small number of short-term mutagenicity tests. The present studies show that MP is a moderately active mutagen in the L5178Y/TK +/-----TK-/- mouse lymphoma assay (MLA) in the presence of aroclor-induced rat-liver S9, and that it induces predominantly small-colony thymidine kinase-deficient (TK-/-) mutants of demonstrated chromosomal origin. 10 of 12 small colony TK-/- mutants analyzed by banded karyotype (230-band level of resolution) show aberrations to chromosome 11b, the known location of the single functional TK gene in these cells. The observed aberrations from nine of the mutants included insertions, deletions and translocations while the tenth mutant had highly rearranged, multiple copies of chromosome 11 segments. By varying the concentrations of the S9 protein and cofactors it was shown that our standard S9 composition was close to optimum for activating MP to a mutagen. The activity and stability of various lots of S9 prepared in-house or purchased from a contract laboratory revealed significant differences. The ability of 2 lots of in-house S9 to activate a standard concentration of MP increased rapidly over the first 4 weeks of liquid nitrogen storage then declined slowly over the next 16 weeks. Three separate lots of purchased S9 were essentially inactive for the first 2 weeks of liquid nitrogen storage then increased in activity thereafter; these were the only occasions in which MP was not mutagenic in our hands. The mutagenic activity of pyrilamine (PYR), a structurally related antihistamine which is far less carcinogenic in rats, but easily detected in short-term tests as being genotoxic, was also investigated in the MLA. PYR was slightly less mutagenic than MP over a comparable range of concentrations, and also induced predominantly small-colony mutants. These studies fail to adequately explain the great carcinogenic differences between these two compounds, but are consistent with the potent hepatocarcinogenicity of MP resulting through a mutagenic mechanism.

The effect of chronic methapyrilene treatment on methapyrilene metabolism in vitro.

Rats were fed 100 or 1000 p.p.m. methapyrilene (MPH) in their diet for 1, 2, 4, 8 or 16 weeks. Liver microsomes were prepared from both control and treated rats. After incubation with 1 mM MPH, eight metabolites were detected and six were quantitated. Five of the metabolites have been previously identified as 2-hydroxymethyl-thiophene (ThM), thiophene-2-carboxylic acid (ThCA), N-(2-thienylmethyl)-2-aminopyridine (TMAP), N-2-pyridyl-N',N'-dimethylethylenediamine (PMED), and 2-aminopyridine (AP). Three other metabolites have been tentatively identified based on their mass spectral fragmentation patterns as normethapyrilene (N-MPH), (5-hydroxypyridyl)methapyrilene (HP-MPH), and methapyrileneamide (MPH-A). The same metabolites were found in both control and treated animals, the most abundant being N-MPH and PMED. Pretreatment with MPH resulted in inhibition of both consumption of MPH and formation of some metabolites. However increases in the formation of all of the metabolites also occurred under different treatment conditions. In both control and treated tissue, the preliminary mass balance was less than 55%, except in incubations with tissue from rats treated with 1000 p.p.m. for 8 or 16 weeks where it was 92 and 89%, respectively. Dramatic increases in the fraction of TMAP, MPH-A, N-MPH, and HP-MPH relative to MPH consumed account for the increase in the mass balance after 8 weeks pretreatment with 1000 p.p.m. MPH, and increases in the amounts of PMED, HP-MPH and ThCA account for the higher mass balance after 16 weeks. The toxicological consequences of these complex metabolic changes may be important in the induction of cancer by MPH.

Influence of ovarian steroids on prostaglandin- and leukotriene-induced uterine contractions.

The mammalian uterus is capable of metabolizing arachidonic acid via the lipoxygenase pathway, and the uterus responds to lipoxygenase products. We postulated that progesterone influences the production of leukotrienes in the uterus in a way similar to that in which estradiol influences prostaglandin production. Uterine contractions were measured in actively sensitized guinea pigs throughout the estrous cycle and in ovariectomized, hormonally primed, sensitized guinea pigs. Antigen challenge stimulated uterine contractions (caused by prostaglandins) that increased throughout the estrous cycle to a maximum in day 15, when estradiol is at its peak. Pretreatment with indomethacin abolished uterine contractions except on day 9 of the cycle, when progesterone levels are at their highest. Day 9 contractions were blocked by FPL 55712, a selective receptor antagonist of leukotrienes. These findings were confirmed in ovariectomized/sensitized guinea pigs. Our data suggest that endogenous synthesis of leukotrienes in the uterus may be directly related to the rise of progesterone.

Methapyrilene induces membrane lipid peroxidation of rat liver cells.

Male Sprague Dawley rats were fed either a basal diet or the same diet containing 0.1% methapyrilene hydrochloride for 1, 2 and 3 days and 1 and 2 weeks. Peroxidative degradation of membrane lipids from liver cell mitochondria and microsomes was evaluated by determining the extent of diene conjugate (u.v. absorption 233 nm) formation. Feeding methapyrilene for 1, 2 and 3 days resulted in lipid peroxidation of both mitochondrial and microsomal membranes. After 1 and 2 weeks feeding, peroxidation was no longer evident in lipids isolated from mitochondria, while the microsomal membrane lipids showed persistent conjugated dienes. The results suggest that membrane lipid peroxidation of the liver may play a role in the promoting and/or carcinogenic effects of methapyrilene hydrochloride.

Genetic activity in yeast assays of reputed nonmutagenic, carcinogenic N-nitroso compounds and methapyrilene hydrochloride.

Methapyrilene hydrochloride (MPHC), N-nitrosomethylaniline (NMA), N-nitrosomethyl-3-carboxypropylamine (NMCP) and N-nitrosodiethanolamine (NDELA) are reputed to be nonmutagenic carcinogens because they are genetically inactive in Salmonella mutagenesis tests but produce cancer in rats. We have assayed these compounds for their genetic activity with diploid strains D7, D7-144, and RMO52 of Saccharomyces cerevisiae. The compounds MPHC and NMA were highly toxic to the cells and induced gene conversion and reverse mutations in strains D7, D7-144 and RMO52. Metabolic activation was not required for this activity. However, in acidic (pH 5) medium, the genetic activity and cell toxicity of MPHC and NMA were markedly reduced. Ascorbic acid suppressed the mutagenicity and toxic effects of MPHC. Mutagenicity of NDELA was enhanced in strain D7-144 when cells were treated in acidic medium. At pH 7, NDELA was not mutagenic. NMCP induced reversed mutations in strains D7-144 and RMO52 in the absence of metabolic activation. Our results indicate that the four carcinogens, MPHC, NMA, NMCP and NDELA, require different physiological conditions for the expression of their genetic activity.

Inversion of the metabolic zonation of rat liver parenchyma by methapyrilene treatment.

Treatment with the antihistaminic agent methapyrilene led to a decrease of glucose-6-phosphatase activity and to an increase of glucose phosphorylating activity in the periportal zone of the liver acinus. However, the glucogenic capacity was maintained by a compensatory elevation of glucose-6-phosphatase and simultaneous reduction of hexokinase and glucokinase in the perivenous zone. The normal metabolic zonation with a glucogenic periportal and a glycolytic perivenous zone was not abolished but inverted by these alterations.

Inability of methapyrilene to induce sister chromatid exchanges in vitro and in vivo.

The induction of sister chromatid exchanges (SCE) by the hepatocarcinogen methapyrilene hydrochloride was investigated using appropriate in vitro and in vivo mammalian cell systems. Methapyrilene, even at the maximum tolerated dose, did not induce SCE in Chinese hamster ovary cells (CHO) or when CHO cells or hamster lung fibroblasts, V-79, were cocultivated with early cultures of rat liver epithelial cells, which are known to metabolize different classes of chemical carcinogens to active forms. Moreover, a hybrid clone of cells (formed by fusion of CHO cells with rat liver epithelial cells), which is highly sensitive to SCE formation by a number of xenobiotics, failed to produce SCE after treatment with methapyrilene. Experiments in vivo with bone marrow cells and in vitro with CHO cells cocultivated with primary hepatocytes from rats also confirmed the inability of methapyrilene to induce SCE in the indicator cells. Since aflatoxin B1 induced SCE in the in vitro and in vivo models, it may be concluded that methapyrilene does not induce SCE at a concentration which is not cytotoxic to the indicator cells in the different systems described. Autoradiographic studies in cultured rat liver cells with tritiated methapyrilene showed that the label was localized in the cytoplasm but not in the interphase nuclei or in the metaphase chromosomes, indicating a lack of interaction of methapyrilene with the nuclear macromolecules of the putative target cells for methapyrilene.

Cytotoxicity of xenobiotics in adult rat hepatocytes in primary culture.

Primary cultures of hepatocytes isolated by collangenase perfusion of adult rats were used as an in vitro system for assessing cytotoxicity of xenobiotics. The leakage of two intracellular enzymes, lactate dehydrogenase (LDH) and glutamic oxaloacetic transaminase (GOT) were compared as indicators of cell damage. Although some differences in sensitivity were detected, either enzyme could be used to evaluate the cytotoxic potential of a chemical. Release of LDH was also compared with determination of cell viability by trypan blue uptake. For cultures exposed to 2-aminofluorene or alpha-naphthylisothiocyanate both endpoints gave comparable results. The cytotoxicities of benzo(a)pyrene, 2-aminofluorene, 3,2'-dimethyl-4-aminobiphenyl, methylene-bis-2-chloroaniline, aflatoxin B1, pyrene, aflatoxin G2, fluorene, methapyrilene, and alpha-naphthylisothiocyanate were monitored by the release of LDH and were found to be related to the length of exposure and concentration of the chemical. For chemicals that elicited DNA repair, little or no cytoxicity was observed at genotoxic concentrations in the hepatocyte primary culture/DNA repair test. Thus, measurement of enzyme release provides a means of quantifying cytotoxicity. Moreover, the use of hepatocyte primary cultures permits identification of genotoxic effects versus general cytotoxic effects of chemicals.