In vitro proteomic analysis of methapyrilene toxicity in rat hepatocytes reveals effects on intermediary metabolism.

The antihistaminic drug methapyrilene was withdrawn from the market in 1979 because of hepatocarcinogenicity in rats. Since then, the drug has been used as a model hepatotoxin especially for transcriptomic analyses using material from in vivo studies. Much less transcriptomics data are available from in vitro studies, and no studies have investigated proteomic effects of methapyrilene in vitro. Thus, the present study was aimed to characterize the proteomic response of primary rat hepatocytes to methapyrilene, to broaden our knowledge on the molecular mechanisms of methapyrilene toxicity, and to compare the results of collagen sandwich-cultured hepatocytes to in vivo data. In vitro methapyrilene concentrations (0.39 µM, 6.25 µM, and 100 µM) were chosen to cover an in vivo-relevant range. Based on published pharmacokinetic data they correspond to concentrations in portal vein blood for previously in vivo-tested doses of methapyrilene, up to a concentration showing slight cytotoxicity. Analysis of proteomic alterations by two-dimensional gel electrophoresis and mass-spectrometric protein identification demonstrated consistent and concentration-dependent effects of methapyrilene, in particular on mitochondrial proteins. Data suggest substantial deregulation of amino acid and ammonia metabolism and effects on mitochondrial energy supply pathways. The effects identified in vitro concur well with into previous in vivo observations. Several effects, for example, the influence of methapyrilene on S-adenosylmethionine metabolism, have not been described previously. The data suggest that already non-toxic concentrations of methapyrilene alter components of the intermediary metabolism, such as branched-chain amino acid metabolism, as well as urea and tricarboxylic cycle enzymes. In summary, data substantially add to our knowledge on molecular mechanisms of methapyrilene hepatotoxicity at the protein level.

Effect of aflatoxin B1, benzo[a]pyrene, and methapyrilene on transcriptomic and epigenetic alterations in human liver HepaRG cells.

The increasing number of man-made chemicals in the environment that may pose a carcinogenic risk highlights the need for developing reliable time- and cost-effective approaches for carcinogen detection and identification. To address this issue, we investigated the utility of high-throughput microarray gene expression and next-generation genome-wide DNA methylation sequencing for the in vitro identification of genotoxic and non-genotoxic carcinogens. Terminally differentiated and metabolically competent human liver HepaRG cells were treated at minimally cytotoxic concentrations of (i) the genotoxic human liver carcinogen aflatoxin B1 (AFB1) and its structural non-carcinogenic analog aflatoxin B2 (AFB2); (ii) the genotoxic human lung carcinogen benzo[a]pyrene (B[a]P) and its non-carcinogenic isomer benzo[e]pyrene (B[e]P); and (iii) the non-genotoxic liver carcinogen methapyrilene for 72 h and transcriptomic and DNA methylation profiles were examined. Treatment of HepaRG cells with the liver carcinogens AFB1 and methapyrilene generated distinct gene-expression profiles, whereas B[a]P had only a slight effect on gene expression. In contrast to transcriptomic alterations, treatment of HepaRG cells with the carcinogenic and non-carcinogenic chemicals resulted in profound changes in the DNA methylation footprint; however, the correlation between gene-specific DNA methylation and gene expression changes was minimal. Among the carcinogen-altered genes, transferrin (TF) emerged as sensitive marker for an initial screening of chemicals for their potential liver carcinogenicity. Potential liver carcinogens (i.e., chemicals causing altered TF gene expression) could then be subjected to gene-expression analyses to differentiate genotoxic from non-genotoxic liver carcinogens. This approach may substantially enhance the identification and assessment of potential liver carcinogens.

Effect of methapyrilene hydrochloride on hepatic intracellular iron metabolism in vivo and in vitro.

The liver, a central detoxification organ and main regulator of systemic iron homeostasis, is prone to damage by xenobiotics. In the present study, we investigated the effect of the hepatotoxicant and hepatocarcinogen methapyrilene hydrochloride on iron metabolism in rat liver in a repeat-dose in vivo toxicity study and in human HepaRG cells in vitro. Treatment of male Fischer 344 (F344) rats with methapyrilene at doses 40 and 80mg/kg body weight (bw)/day by gavage for 6 weeks resulted in changes in the expression of classic hepatotoxicity-related marker genes and iron homeostasis-related genes, especially a prominent, dose-dependent down-regulation of the transferrin (Tf) gene and an up-regulation of the ferritin, light chain (Ftl) gene. A decrease in the level of TF and an increase in the level of FTL also occurred in methapyrilene-treated differentiated HepaRG cells, indicating the existence of interspecies and in vitro-in vivo similarities in the disturbance of cellular iron homeostasis upon liver injury. In contrast, there was minimal overlap in the expression of liver toxicity-marker genes in the livers of rats and in HepaRG cells treated with methapyrilene. Importantly, the decrease of transferrin at mRNA and protein levels occurred after the treatment with a low dose of methapyrilene that exhibited minimal cytotoxicity. These results demonstrate the significance of the dysregulation of hepatic iron metabolism in the pathogenesis and mechanism of chemical-induced liver toxicity and suggest that these changes may be sensitive and useful indicators of potentially hepatotoxic chemicals.

Editor's Highlight: Organ-Specific Epigenetic Changes Induced by the Nongenotoxic Liver Carcinogen Methapyrilene in Fischer 344 Rats.

Continuous lifetime exposure to certain natural and man-made chemicals is a major cause of cancers in humans; therefore, evaluating the carcinogenic risks of chemicals remains important. Currently, substantial progress has been made in identification of genotoxic carcinogens; in contrast, predicting the carcinogenic potential of nongenotoxic compounds is a challenge due to many different modes of action that may lead to tumorigenesis. In the present study, we investigated the effects of the nongenotoxic liver carcinogen methapyrilene and the nongenotoxic noncarcinogen usnic acid, at doses that do not exhibit organ cytotoxicity, on epigenomic alterations in the livers and kidneys of Fischer 344 (F344) rats. We demonstrate that a repeat-dose oral treatment of male F344 rats with methapyrilene for 6 weeks caused target organ-specific epigenetic alterations in the livers. In contrast, only very slight epigenetic changes were found in the livers of F344 rats treated with hepatotoxicant, but noncarcinogen, usnic acid. The methapyrilene-induced epigenetic changes consisted of changes in histone lysine acetylation and methylation, with the greatest increase occurring in global and gene-specific histone H3 lysine 9 (H3K9) deacetylation. Importantly, the results of the present study show an association between gene-specific histone H3K9 deacetylation and a reduced expression of critical cancer-related genes, including prospero homeobox 1 (Prox1), HNF1 homebox A (Hnf1a), and peroxisome proliferator activated receptor alpha (Ppara), which provides a mechanistic link between methapyrilene-induced epigenetic aberrations and liver carcinogenesis.

Disruption of spindle checkpoint function ahead of facilitation of cell proliferation by repeated administration of hepatocarcinogens in rats.

We aimed to clarify the hepatocarcinogen-specific disruption of cell cycle checkpoint functions and its time course after repeated administration of hepatocarcinogens. Thus, rats were repeatedly administered with hepatocarcinogens (methapyrilene, carbadox and thioacetamide), a marginal hepatocarcinogen (leucomalachite green), hepatocarcinogenic promoters (oxfendazole and ß-naphthoflavone) or non-carcinogenic hepatotoxicants (promethazine and acetaminophen) for 7, 28 or 90 days, and the temporal changes in cell proliferation, expression of G1/S and spindle checkpoint-related molecules, and apoptosis were examined using immunohistochemistry and/or real-time RT-PCR analysis. Hepatocarcinogens facilitating cell proliferation at day 28 of administration also facilitated cell proliferation and apoptosis at day 90. Hepatocarcinogen- or hepatocarcinogenic promoter-specific cellular responses were not detected by immunohistochemical single molecule analysis even after 90 days. Expression of Cdkn1a, Mad2l1, Chek1 and Rbl2 mRNA also lacked specificity to hepatocarcinogens or hepatocarcinogenic promoters. In contrast, all hepatocarcinogens and the marginally hepatocarcinogenic leucomalachite green induced Mdm2 upregulation or increase in the number of phosphorylated MDM2(+) cells from day 28, irrespective of the lack of cell proliferation facilitation by some compounds. However, different Tp53 expression levels suggest different mechanisms of induction or activation of MDM2 among hepatocarcinogens. On the other hand, hepatocarcinogenic methapyrilene and carbadox downregulated the number of both ubiquitin D(+) cells and proliferating cells remaining in M phase at day 28 and/or day 90, irrespective of the lack of cell proliferation facilitation in the latter. These results suggest that hepatocarcinogens disrupt spindle checkpoint function after 28 or 90 days of administration, which may be induced ahead of cell proliferation facilitation.

Detection of initiating potential of non-genotoxic carcinogens in a two-stage hepatocarcinogenesis study in rats.

We previously reported a toxicogenomics-based prediction model for hepatocarcinogens in which the expression patterns of signature genes following repeated doses of either genotoxic or non genotoxic compounds were similar. Based on the results of our prediction model, we hypothesized that repeated doses of non-genotoxic carcinogens might have initiating potential. Here, we conducted a two stage hepatocarcinogenesis study in rats exposed to the initiating agent nitrosodiethylamine (DEN), and hepatotoxic compounds thioacetamide (TAA), methapyrilene (MP) and acetaminophen (APAP) for 1-2 weeks followed by the liver tumor promoter phenobarbital (PB). The duration of initial treatment was determined based on positive results from our prediction model. Combined treatment of 3 or 30 mg/kg of genotoxic DEN and PB induced marked increases in altered hepatocellular foci and a DEN dose-dependent increase in the number and area of glutathione S-transferase-placental form (GST-P)-positive foci. A low number of altered hepatocellular foci were also observed in rats treated with TAA at a dose of 45 mg/kg.MP at a dose of 100 mg/kg induced a very low number of foci, but APAP did not. Hierarchical clustering analysis using gene expression data revealed that 2-week treatment with TAA at a dose of 30 mg/kg and MP at 45 mg/kg induced specific expression of DNA damage-related genes, similar to 1-week treatment with DEN at a dose of 30 mg/kg. These results suggest that TAA and MP induce DNA damage, which partially supports our hypothesis. Although this study does not indicate whether tumor growth in response to these compounds can be assessed in this model, our results suggest that cumulative treatment with non genotoxic TAA might have initiating potential in the liver.

Pharmacokinetics explain in vivo/in vitro discrepancies of carcinogen-induced gene expression alterations in rat liver and cultivated hepatocytes.

Cultivated hepatocytes represent a well-established in vitro system. However, the applicability of hepatocytes in toxicogenomics is still controversially discussed. Recently, an in vivo/in vitro discrepancy has been described, whereby the non-genotoxic rat liver carcinogen methapyrilene alters the expression of the metabolizing genes SULT1A1 and ABAT, as well as the DNA damage response gene GADD34 in vitro, but not in vivo. If the collagen sandwich cultures of hepatocytes really produce false-positive data, this would compromise its application in toxicogenomics. To revisit the putative in vivo/in vitro discrepancy, we first analyzed and modeled methapyrilene concentrations in the portal vein of rats. The relatively short half-life of 2.8 h implies a rapid decrease in orally administered methapyrilene in vivo below concentrations that can cause gene expression alterations. This corresponded to the time-dependent alteration levels of GADD34, ABAT and SULT1A1 RNA in the liver: RNA levels are altered 1, 6 and 12 h after methapyrilene administration, but return to control levels after 24 and 72 h. In contrast, methapyrilene concentrations in the culture medium supernatant of primary rat hepatocyte cultures decreased slowly. This explains why GADD34, ABAT and SULT1A1 were still deregulated after 24 h exposure in vitro, but not in vivo. It should also be considered that the earliest analyzed time point in the previous in vivo studies was 24 h after methapyrilene administration. In conclusion, previously observed in vitro/in vivo discrepancy can be explained by different pharmacokinetics present in vitro and in vivo. When the in vivo half-life is short, levels of some initially altered genes may have returned to control levels already 24 h after administration.

The genotoxic potential of methapyrilene using the alkaline Comet assay in vitro and in vivo.

The genotoxicity of methapyrilne (MP) has been evaluated in a number of assays since it was found to be a rat hepatocarcinogen with subsequent withdrawal as an over-the-counter antihistamine. Whilst it has not been classified as a genotoxin, there are reports of positive findings from mammalian cell gene mutation and transformation assays. To investigate further the genotoxic potential of MP, the alkaline Comet assay was used to evaluate DNA damage both in primary hepatocytes in culture and in vivo in the rat. To confirm bioactivation was required to induce the hepatotoxic mechanism, aminobenzotriazole, a broad spectrum cytochrome P450 enzyme inhibitor was used as a pre-treatment. The levels of glutathione and glutathione disulfide were determined in both hepatocytes in culture and in the liver following in vivo exposure. MP showed significant increases in DNA damage in freshly isolated male rat hepatocyte suspensions that could be significantly reduced by pre-incubation of aminobenzotriazole (ABT). DNA damage showed a marked sex difference, with male hepatocytes being more susceptible, and showing a concurrent depletion of glutathione (GSH) compared with female hepatocytes. Modulation of the GSH levels by diethylmaleate and γ-glutamylcysteinylethyl ester, elevated and reduced the levels of DNA damage, respectively. In the in vivo Comet assay, there was no evidence of DNA damage following MP (150mg/kg p.o) treatment for three consecutive days, although histological and liver enzyme changes were seen. Total protein GSH content was elevated in MP-treated animals and superoxide dismutase levels were increased specifically in periportal regions. Taken together, these data support the potential for MP to induce oxidative stress. The differences in DNA damage detected by the Comet assay in vitro, and in rat liver in vivo, could be attributed to differences in metabolism and response to oxidant insult or the inability of the assay to discriminate damage in a small number of individual cells in the whole liver.

HPLC-MS/MS methods for the quantitative analysis of ophthalmic acid in rodent plasma and hepatic cell line culture medium.

Ophthalmic acid (OA), an endogenous tripeptide analogue of glutathione, has been suggested as a potential biomarker for paracetamol/acetaminophen hepatotoxicity. Here HPLC-MS/MS methods have been developed for the precise, sensitive and specific detection and quantification of OA in in vitro cell culture medium and plasma. For the cell culture medium the LLOQ was found to be 1 ng/ml, with less than 1% between sample carry over at all concentrations and precision below 15% for within day and below 9% for between day analyses. For rat plasma the presence of endogenous OA resulted in the LLOQ being 25 ng/ml (defined as the lowest concentration on the calibration curve where the base peak was less than 20% of the LLOQ). For the plasma assay the percentage carry over was less than 1% for all concentrations and within and between batch precision was below 21%. The methods were linear for both sample types from the LLOQ up to 5 µg/ml. The method was successfully applied to the determination of OA in samples obtained following the chronic administration of the rat hepatotoxin methapyrilene, where plasma OA concentrations were observed to show a weak negative correlation with those of established liver injury biomarkers such as aspartate aminotransferase (AST).

Toxicogenomics and cancer risk assessment: a framework for key event analysis and dose-response assessment for nongenotoxic carcinogens.

In order to determine a threshold for nongenotoxic carcinogens, the traditional risk assessment approach has been to identify a mode of action (MOA) with a nonlinear dose-response. The dose-response for one or more key event(s) linked to the MOA for carcinogenicity allows a point of departure (POD) to be selected from the most sensitive effect dose or no-effect dose. However, this can be challenging because multiple MOAs and key events may exist for carcinogenicity and oftentimes extensive research is required to elucidate the MOA. In the present study, a microarray analysis was conducted to determine if a POD could be identified following short-term oral rat exposure with two nongenotoxic rodent carcinogens, fenofibrate and methapyrilene, using a benchmark dose analysis of genes aggregated in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO) biological processes, which likely encompass key event(s) for carcinogenicity. The gene expression response for fenofibrate given to rats for 2days was consistent with its MOA and known key events linked to PPARα activation. The temporal response from daily dosing with methapyrilene demonstrated biological complexity with waves of pathways/biological processes occurring over 1, 3, and 7days; nonetheless, the benchmark dose values were consistent over time. When comparing the dose-response of toxicogenomic data to tumorigenesis or precursor events, the toxicogenomics POD was slightly below any effect level. Our results suggest that toxicogenomic analysis using short-term studies can be used to identify a threshold for nongenotoxic carcinogens based on evaluation of potential key event(s) which then can be used within a risk assessment framework.

Functional and toxicological consequences of metabolic bioactivation of methapyrilene via thiophene S-oxidation: Induction of cell defence, apoptosis and hepatic necrosis.

UNLABELLED: Methapyrilene, [N,N-dimethyl-N'-pyridyl-N'(2-thienylmethyl)-1,2-ethanediamine] (MP) was withdrawn from, clinical use due to reported periportal hepatic necrosis and hepatocarcinogenicity in the rat, via S-oxidation of the thiophene group. In this study MP is used as a model hepatotoxin to further characterise the functional consequences of S-oxidation of the thiophene group in vivo, in rat models and in vitro, in freshly isolated rat hepatocyte suspensions. In vivo histological studies revealed the early depletion of glutathione (GSH), which was confined to the damaged periportal area, in contrast to an increase in GSH levels in the centrilobular region. Additionally, the induction of cell defence was demonstrated by an increase in the protein levels of heme-oxygenase 1 (HO-1) and glutamate cysteine ligase, catalytic subunit (GCLC) in vivo. Histological examination demonstrated that cytotoxicity progresses initially via apoptosis before an increase in necrosis over the 3-day administration. An apoptotic-like mechanism was observed in vitro via the measurement of cytochrome c release and caspase activation. CONCLUSION: This study provides evidence for a complex pathway of MP-induced hepatotoxicity which progresses through early adaptation, apoptosis, necrosis and inflammation, all underpinned by the zonal induction and depletion of GSH within the liver.

Primary rat hepatocytes as in vitro system for gene expression studies: comparison of sandwich, Matrigel and 2D cultures.

Recent studies have presented evidence that in vivo obtained gene expression data can be used for carcinogen classification, for instance to differentiate between genotoxic and non-genotoxic carcinogens. However, although primary rat hepatocytes represent a well-established in vitro system for drug metabolism and enzyme induction, they have not yet been systematically optimized for toxicogenomic studies. The latter may be confounded by the fact that cultured hepatocytes show strong spontaneous alterations in gene expression patterns. Therefore, we addressed the following questions: (1) which culture system is optimal, comparing sandwich, Matrigel and 2D cultures, (2) how critical is the impact of culture period on substance-induced alterations in gene expression and (3) do these substance-induced alterations in cultured hepatocytes occur already at in vivo relevant concentrations? For this purpose we analyzed the expression of four genes, namely Abat, Gsk3beta, Myd116 and Sult1a1 that recently have been reported to be influenced by the antihistamine and non-genotoxic carcinogen methapyrilene (MPy). The most reproducible effects of MPy were observed in sandwich cultures. Induction factors of Gsk3beta and Myd116 at 100 microM MPy were 2 and 4 (medians), respectively, whereas expression of Abat and Sult1a1 were inhibited by factors of 7 and 5, respectively. Similar results were observed in hepatocytes maintained for 24 h or 3 weeks in sandwich culture with respect to the influence of MPy on the expression of Abat, Gsk3beta, Myd116 and Sult1a1. To determine whether MPy influences gene expression at in vivo relevant concentrations, 3.5 mg/kg MPy were administered to male Wistar rats intraperitoneally, resulting in plasma concentrations ranging between 1.72 and 0.32 microM 5 and 80 min after injection. Inhibition of Abat and Sult1a1 expression in vitro already occurred at in vivo relevant concentrations of 0.39 microM MPy. Induction of Myd116 was observed at 6.25 microM which is higher but in the same order of magnitude as in vivo relevant concentrations. In conclusion, the presented data strongly suggest that sandwich cultures are most adequate for detection of MPy-induced gene expression alterations and the effect of MPy was detected at in vivo relevant concentrations.

A toxicogenomics approach for early assessment of potential non-genotoxic hepatocarcinogenicity of chemicals in rats.

For assessing carcinogenicity in animals, it is difficult and costly, an alternative strategy has been desired. We explored the possibility of applying a toxicogenomics approach by using comprehensive gene expression data in rat liver treated with various compounds. As prototypic non-genotoxic hepatocarcinogens, thioacetamide (TAA) and methapyrilene (MP) were selected and 349 commonly changed genes were extracted by statistical analysis. Taking both compounds as positive with six compounds, acetaminophen, aspirin, phenylbutazone, rifampicin, alpha-naphthylisothiocyanate, and amiodarone as negative, prediction analysis of microarray (PAM) was performed. By training and 10-fold cross validation, a classifier containing 112 probe sets that gave an overall success rate of 95% was obtained. The validity of the present discriminator was checked for 30 chemicals. The PAM score showed characteristic time-dependent increases by treatment with several non-genotoxic hepatocarcinogens, including TAA, MP, coumarin, ethionine and WY-14643, while almost all of the non-carcinogenic samples were correctly predicted. Measurement of hepatic glutathione content suggested that MP and TAA cause glutathione depletion followed by a protective increase, but the protective response is exhausted during repeated administration. Therefore, the presently obtained PAM classifier could predict potential non-genotoxic hepatocarcinogenesis within 24 h after single dose and the inevitable pseudo-positives could be eliminated by checking data of repeated administrations up to 28 days. Tests for carcinogenicity using rats takes at least 2 years, while the present work suggests the possibility of lowering the time to 28 days with high precision, at least for a category of non-genotoxic hepatocarcinogens causing oxidative stress.

Identification of the thiophene ring of methapyrilene as a novel bioactivation-dependent hepatic toxicophore.

Methapyrilene (MP), a 2-thiophene H(1)-receptor antagonist, is a model toxicant in the genomic and proteomic analyses of hepatotoxicity. In rats, it causes an unusual periportal necrosis that is hypothetically attributed to chemically reactive and cytotoxic metabolites. We have characterized the bioactivation of MP by hepatic microsomes and primary rat hepatocytes, and we established a possible causal linkage with cytotoxicity. Methapyrilene tritiated at C-2 of the diaminoethane moiety ([3H]MP) was metabolized via an NADPH-dependent pathway to intermediates that combined irreversibly with microsomes (rat > mouse approximately human). This binding was attenuated by the cytochrome P450 (P450) inhibitor 1-aminobenzotriazole and thiols but not by trapping agents for iminium ions and aldehydes. Reactive intermediates were trapped as thioether adducts of monooxygenated MP. Mass spectrometric and hydrogen/deuterium exchange analysis of the glutathione adduct produced by rat liver microsomes indicated that the metabolite was most probably a thioether of MP S-oxide substituted in the thiophene ring. The glutathione adduct was formed by rat hepatocytes and eliminated in bile by rats administered [3H]MP intravenously. MP produced concentration- and time-dependent cytotoxicity, depleted glutathione, and underwent irreversible binding to the hepatocytes before a significant increase in cell damage was observed. P450 inhibitors reduced turnover of the drug, production of the glutathione adduct, irreversible binding, and cytotoxicity but inhibited glutathione depletion selectively. MP underwent lesser turnover and bioactivation in mouse hepatocytes and was not cytotoxic. Analogs with phenyl and p-methoxyphenyl rings were much less hepatocytotoxic than MP. Hepatotoxicity in rats was diminished by predosing with 1-aminobenzotriazole. For the first time, a thiophene ring substituent is identified as a bioactivation-dependent toxicophore in hepatocytes.

Gene expression profiling of methapyrilene-induced hepatotoxicity in rat.

The present study was conducted as a model case of the toxicogenomics approach for analyzing toxicological mechanisms and toxicity assessments in the early stage of drug development by comparing with classical toxicology data. Methapyrilene (MP) 100 mg/kg produced obvious histopathological changes in liver of rats by single or repeated dose up to 28 days with significant elevation of ALT and AST. In the middle dose groups (30 mg/kg MP), no apparent changes were noted in blood biochemical data by single dosing or repeated dosing up to one week, and no obvious histopathological changes were observed except a slight hypertrophy in the hepatocytes. Comprehensive gene expression changes were analyzed using Affymetrix GeneChip and differentially expressed probe sets were statistically extracted. These contained many genes related to "glutathione metabolism", "apoptosis", "MAPK signaling pathway" and "regulation of cell cycle", which were all thought to be involved in the development of presently observed phenotypes. In the high dose groups, TGP1 scores (developed in our system in order to overview the responsiveness of drugs to multiple marker gene lists) for these categories were markedly increased from the early time point after single dose and kept their high expression throughout the repeated dose period. In the middle dose groups, the increment of the scores were noted not only at the time points when apparent pathological changes emerged, but also at the earlier stage of repeated dosing and even after single dosing. We conclude that toxicogenomics would enable a more sensitive assessment at the earlier time point than classical toxicology evaluation.

Methapyrilene toxicity: anchorage of pathologic observations to gene expression alterations.

Methapyrilene (MP) exposure of animals can result in an array of adverse pathological responses including hepatotoxicity. This study investigates gene expression and histopathological alterations in response to MP treatment in order to 1) utilize computational approaches to classify samples derived from livers of MP treated rats based on severity of toxicity incurred in the corresponding tissue, 2) to phenotypically anchor gene expression pattems, and 3) to gain insight into mechanism(s) of methapyrilene hepatotoxicity. Large-scale differential gene expression levels associated with the exposure of male Sprague-Dawley rats to the rodent hepatic carcinogen MP for 1, 3, or 7 days after daily dosage with 10 or 100 mg/kg/day were monitored. Hierarchical clustering and principal component analysis were successful in classifying samples in agreement with microscopic observations and revealed low-dose effects that were not observed histopathologically. Data from cDNA microarray analysis corroborated observed histopathological alterations such as hepatocellular necrosis, bile duct hyperplasia, microvesicular vacuolization, and portal inflammation observed in the livers of MP exposed rats and provided insight into the role of specific genes in the studied toxicological processes.

Principal component analysis of mass spectra of peptides generated from the tryptic digestion of protein mixtures.

Principal component analysis (PCA) has been used to analyse mass spectral peptide profiles obtained from the enzymatic digestion of standard protein mixtures. Scores and loadings plots clearly revealed peptide fragments that differentiated one protein mixture from another. Peptide map search results identified with a high degree of certainty any additional proteins in these mixtures. As a proof-of-concept this methodology was applied to hepatic protein mixtures obtained from rats treated with two hepatotoxic compounds: methapyriline and SB-219994. Liver proteins were extracted, pre-separated by one-dimensional polyacrylamide gel electrophoresis, subjected to tryptic digestion and analysed by mass spectrometry. Two up-regulated proteins, glutathione S-transferase with methapyrilene and peroxisomal bifunctional enzyme with SB-219994, were identified in this manner.

Methapyrilene hepatotoxicity is associated with increased hepatic glutathione, the formation of glucuronide conjugates, and enterohepatic recirculation.

The mechanisms by which acute administration of methapyrilene, an H(1)-receptor antihistamine causes periportal necrosis to rats are unknown. This study investigated the role of the hepato-biliary system in methapyrilene hepatotoxicity following daily administration of 150 mg/kg per day over 3 consecutive days. Biliary metabolites of methapyrilene were tentatively identified. In male Han Wistar rats administration of methapyrilene significantly increased hepatic reduced glutathione (GSH) to 140% of control levels 24 h following the last dose. There were no significant changes in the activities of glutathione-related enzymes, glutathione peroxidase (GPx) and reductase (GSH), glutathione S-transferase (GST), and gamma-glutamyl cysteine synthetase (gamma-GCS) over 3 days of methapyrilene administration. Methapyrilene treatment resulted in no significant increase in excretion of biliary oxidized glutathione (GSSG), a sensitive marker of oxidative stress in vivo, following the third dose. [3H]Methapyrilene-derived radioactivity was detected in bile, to a greater extent than in feces, indicating that methapyrilene and/or metabolites underwent enterohepatic recirculation. Cannulation and exteriorization of the bile duct (to interrupt enterohepatic recirculation) afforded some protection against the hepatotoxicity, assessed by clinical chemistry and histopathology. Liquid chromatography-mass spectrometry (LC-MS) analysis of bile indicated the presence of unmetabolized methapyrilene, methapyrilene O-glucuronide and desmethyl methapyrilene O-glucuronide. These data demonstrate that acute methapyrilene hepatotoxicity in vivo is not a consequence of GSH depletion, or oxidative stress, but that enterohepatic recirculation of biliary metabolites may be important. Progressive exposure to non-oxidizing, reactive metabolic intermediates may be responsible for hepatotoxicity.

Methapyrilene hepatotoxicity is associated with oxidative stress, mitochondrial disfunction and is prevented by the Ca2+ channel blocker verapamil.

Methapyrilene (MP) is an unusual hepatotoxin in that it causes periportal necrosis in rats. The mechanism of acute methapyrilene hepatotoxicity has, therefore, been investigated in cultured male rat hepatocytes. Addition of methapyrilene to rat hepatocytes resulted in a time- and dose-dependent loss in cell viability between 4 and 8 h of incubation as judged by cellular enzyme leakage. The cytochrome P450 (CYP) inhibitor metyrapone protected against methapyrilene-mediated toxicity suggesting that MP is metabolised by CYP for toxicity. The concentration-dependent protection from methapyrilene toxicity afforded by metyrapone correlated with an inhibition of microsomal CYP2C11-associated androstenedione 16alpha hydroxylase activity, and hepatocytes prepared from hypophysectomised rats (containing reduced levels of microsomal immunodetectable CYP2C11 and associated androstenedione 16alpha hydroxylase activity) showed resistance to the toxic effects of methapyrilene. These data suggest that the toxicity of methapyrilene is predominantly dependent on the CYP2C11 isoform. Treatment of hepatocytes with a toxic concentration of MP caused oxidative stress as indicated by increases in NADP+ levels within 2 h and cellular thiol oxidation as evidenced by a reduction--but not complete loss--in glutathione levels. Methapyrilene hepatotoxicity was associated with an early loss in mitochondrial function, as indicated by mitochondrial swelling and significant losses in cellular ATP within 2 h. Co-incubation of methapyrilene-treated hepatocytes with inhibitors of inner mitochondrial transition permeability pore opening--cyclosporin A or the thiol reductant dithiothreitol--abrogated cell death suggesting that pore opening and loss of mitochondrial Ca2+ homeostasis play a significant role in methapyrilene-mediated cell death. Co-incubation of methapyrilene-treated hepatocytes with the phenylalkylamine calcium channel blocker verapamil--but not by treating cells in a nominally calcium-free medium--also abrogated cell death, suggesting that if Ca2+ is involved in cell killing then it is dependent on an intracellular Ca2+ pool. Pre-treatment of hepatocytes for 1 h with verapamil--to inhibit intracellular Ca2+ pool filling--increased the potency of verapamil protection against methapyrilene toxicity by approximately 100-fold. Taken together, these data indicate that methapyrilene intoxication leads to mitochondrial disfunction and suggest a critical role for a loss of mitochondrial Ca2+ homeostasis in this model of hepatocyte death.

Synergistic/comutagenic action in the Ames test as an indication of irrelevant positive findings.

A number of structurally very diverse compounds which cause weak positive effects in the Ames test by evident or suspect irrelevant mechanisms is discussed. As a unifying observation we describe synergistic effects in combination with known mutagens in the responsive strains and comutagenic effects in initially unresponsive strains. We argue that the compounds enhance the formation of spontaneous (or mutagen-induced) revertant colonies by test-specific mechanisms likely to be of no relevance to multicellular eukaryotic organisms rather than possessing intrinsic genotoxic (i.e. DNa-damaging) properties in the Ames test.