Application of duplex sequencing to evaluate mutagenicity of aristolochic acid and methapyrilene in Fisher 344 rats.

Duplex sequencing (DS) is an error-corrected next-generation sequencing (NGS) method that can overcome notorious high error rate from the process of NGS and detect ultralow-frequency mutations. In this study, we evaluated the mutagenicity of aristolochic acid, a known genotoxic carcinogen, and methapyrilene, a known nongenotoxic carcinogen using DS. Four male Fisher 344 rats were treated with aristolochic acid, methapyrilene, or the vehicle control for 6 weeks, liver tissues were collected one day after the treatment, and the DNA was isolated for analysis. The mutation frequency for the aristolochic acid-treated group was significantly increased over the vehicle control (44-fold), whereas no significant difference in the mutation frequency was observed between the methapyrilene-treated and the control groups. The primary type of mutation induced by aristolochic acid was A:T > T:A transversion, which occurred frequently at ApT sites, whereas the major type of mutation in the control and methapyrilene-treated groups was G:C > A:T transition, which occurred frequently at CpG sites. These findings are consistent with previously published data obtained with other in vivo mutation assays. Thus, our results suggest that the DS mutation assay is a promising technology for assessing mutagenicity of chemicals in vivo.

Development of a single mobile phase for LC-IM-MS-based discovery lipidomics and metabolic phenotyping: Application to methapyrilene hepatotoxicity in the rat.

The untargeted global profiling of endogenous metabolites and lipids has the potential to increase knowledge and understanding in many areas of biology. LC-MS/MS is a key technology for such analyses however, several different LC methodologies, using different mobile phase compositions, are required to cover the diversity in polarity and analyte structure encountered in biological samples. Most notably many lipid screening methods make use of isopropanol (IPA) as a major component of mobile phases employed for comprehensive lipidomic profiling. In order to increase laboratory efficiency, and minimize opportunities for errors, a suite of methods, based on a single acetonitrile (ACN)-aqueous buffer mobile phase combination, has been developed. This mobile phase can be used for hydrophobic interaction liquid chromatography on an amide stationary phase (for polar analytes), reversed-phase (RP) LC analysis on a C8 stationary phase (for moderately polar-non-polar compounds) and RPLC using a CSH phenyl-hexyl bonded column (for lipids). All of these sub 10 minute separations had good throughput and reproducibility with CV's of analyte response <25 % whilst eliminating the need for complex mobile phase preparation and the use of IPA as an organic modifier for lipidomics. Advantages of removing IPA and replacing it with the ACN-based method were a 58 % increase in peak capacity for lipids, with improved resolution for the di- and triglycerides and cholesterol esters compared to current methods. Compared to the IPA-containing solvent system the ACN-based mobile phase also resulted in a 61 % increase in lipid feature detection. The utility of this "universal" mobile phase approach was demonstrated by its application to a rat toxicology study investigating the consequences of methapyrilene administration through on the endogenous metabolite profiles of plasma and urine. Methapyrilene and its metabolites were also profiled in these samples.

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.

Downregulation of low-density lipoprotein receptor class A domain-containing protein 4 (Ldlrad4) in the liver of rats treated with nongenotoxic hepatocarcinogen to induce transforming growth factor ß signaling promoting cell proliferation and suppressing apoptosis in early hepatocarcinogenesis.

We previously found downregulation of low-density lipoprotein receptor class A domain-containing protein 4 (LDLRAD4), a negative regulator of transforming growth factor (TGF)-ß signaling, in glutathione S-transferase placental form (GST-P) expressing (+ ) pre-neoplastic lesions produced by treatment with nongenotoxic hepatocarcinogens for up to 90 days in rats. Here, we investigated the relationship between LDLRAD4 downregulation and TGFß signaling in nongenotoxic hepatocarcinogenesis. The transcripts of Tgfb and Hb-egf increased after ≥28 days of treatment. After 84 or 90 days, Snai1 increased transcripts and the subpopulation of GST-P+ foci downregulating LDLRAD4 co-expressed TGFß1, phosphorylated EGFR, or phosphorylated AKT2, and downregulated PTEN, showing higher incidences than those in GST-P+ foci expressing LDLRAD4. The subpopulation of GST-P+ foci downregulating LDLRAD4 also co-expressed caveolin-1 or TACE/ADAM17, suggesting that disruptive activation of TGFß signaling through a loss of LDLRAD4 enhances EGFR and PTEN/AKT-dependent pathways via caveolin-1-dependent activation of TACE/ADAM17 during nongenotoxic hepatocarcinogenesis. The numbers of c-MYC+ cells and PCNA+ cells were higher in LDLRAD4-downregulated GST-P+ foci than in LDLRAD4-expressing GST-P+ foci, suggesting a preferential proliferation of pre-neoplastic cells by LDLRAD4 downregulation. Nongenotoxic hepatocarcinogens markedly downregulated Nox4 after 28 days and later decreased cleaved caspase 3+ cells in LDLRAD4-downregulated GST-P+ foci, suggesting an attenuation of apoptosis by LDLRAD4 downregulation through activation of the EGFR pathway. At the late hepatocarcinogenesis stage in a two-stage model, LDLRAD4 downregulation was higher in adenoma and carcinoma than in pre-neoplastic cell foci, suggesting a role of LDLRAD4 downregulation in tumor development. Our results suggest that nongenotoxic hepatocarcinogens cause disruptive activation of TGFß signaling through downregulating LDLRAD4 toward carcinogenesis in the rat liver.

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.

Quantitative targeted bile acid profiling as new markers for DILI in a model of methapyrilene-induced liver injury in rats.

Recently, bile acids (BAs) were reported as promising markers for drug-induced liver injury (DILI). BAs have been suggested to correlate with hepatocellular and hepatobiliary damage; however a clear connection of BA patterns with different types of DILI remains to be established. To investigate if BAs can improve the assessment of liver injury, 20 specific BAs were quantitatively profiled via LC-MS/MS in plasma and liver tissue in a model of methapyrilene-induced liver injury in rats. Methapyrilene, a known hepatotoxin was dosed daily over 14-days at doses of 30 and 80mg/kg, followed by a recovery phase of 10days. Conventional preclinical safety endpoints were related to BA perturbations and to hepatic gene expression profiling for a mechanistic interpretation of effects. Histopathological signs of hepatocellular and hepatobiliary damage with significant changes of clinical chemistry markers were accompanied by significantly increased levels of indivdual BAs in plasma and liver tissue. BA perturbations were already evident at the earliest time point after 30mg/kg treatment, and thereby indicating better sensitivity than clinical chemistry parameters. Furthermore, the latter markers suggested recovery of liver injury, whereas BA levels in plasma and liver remained significantly elevated during the recovery phase, in line with persistent histopathological findings of bile duct hyperplasia (BDH) and bile pigment deposition. Gene expression profiling revealed downregulation of genes involved in BA synthesis (AMACR, BAAT, ACOX2) and hepatocellular uptake (NTCP, OATs), and upregulation for efflux transporters (MRP2, MRP4), suggesting an adaptive hepatocellular protection mechanism against cytotoxic bile acid accumulation. In summary, our data suggests that specific BAs with high reliability such as cholic acid (CA) and chenodeoxycholic acid (CDCA) followed by glycocholic acid (GCA), taurocholic acid (TCA) and deoxycholic acid (DCA) can serve as additional biomarkers for hepatocellular/hepatobiliary damage in the liver in rat toxicity studies.

B-13 progenitor-derived hepatocytes (B-13/H cells) model lipid dysregulation in response to drugs and chemicals.

Lipid dysregulation is a common hepatic adverse outcome after exposure to toxic drugs and chemicals. A donor-free rat hepatocyte-like (B-13/H) cell was therefore examined as an in vitro model for investigating mechanisms. The B-13/H cell irreversibly accumulated triglycerides (steatosis) in a time- and dose-dependent manner when exposed to fatty acids, an effect that was potentiated by the combined addition of hyperglycaemic levels of glucose and insulin. B-13/H cells also expressed the LXR nuclear receptors and exposure to their activators - T0901317 or GW3965 - induced luciferase expression from a transfected LXR-regulated reporter gene construct and steatosis in a dose-dependent manner with T0901317. Exposing B-13/H cells to a variety of cationic amphiphilic drugs - but not other hepatotoxins - also resulted in a time- and dose-dependent accumulation of phospholipids (phospholipidosis), an effect that was reduced by over-expression of lysosomal phospholipase A2. Through application of this model, hepatotoxin methapyrilene exposure was shown to induce phospholipidosis in both B-13 and B-13/H cells in a time- and dose-dependent manner. However, methapyrilene was only toxic to B-13/H cells and inhibitors of hepatotoxicity enhanced phospholipidosis, suggesting phospholipidosis is not a pathway in toxicity for this withdrawn drug. In contrast, pre-existing steatosis had minimal effect on methapyrilene hepatotoxicity in B-13/H cells. These data demonstrate that the donor free B-13 cell system for generating hepatocyte-like cells may be employed in studies of fatty acid- and LXR activator-induced steatosis and phospholipidosis and in the dissection of pathways leading to adverse outcomes such as hepatotoxicity.

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.

Phenotypic and biomarker evaluation of zebrafish larvae as an alternative model to predict mammalian hepatotoxicity.

Zebrafish phenotypic assays have shown promise to assess human hepatotoxicity, though scoring of liver morphology remains subjective and difficult to standardize. Liver toxicity in zebrafish larvae at 5 days was assessed using gene expression as the biomarker approach, complementary to phenotypic analysis and analytical data on compound uptake. This approach aimed to contribute to improved hepatotoxicity prediction, with the goal of identifying biomarker(s) as a step towards the development of transgenic models for prioritization. Morphological effects of hepatotoxic compounds (acetaminophen, amiodarone, coumarin, methapyrilene and myclobutanil) and saccharin as the negative control were assessed after exposure in zebrafish larvae. The hepatotoxic compounds induced the expected zebrafish liver degeneration or changes in size, whereas saccharin did not have any phenotypic adverse effect. Analytical methods based on liquid chromatography-mass spectrometry were optimized to measure stability of selected compounds in exposure medium and internal concentration in larvae. All compounds were stable, except amiodarone for which precipitation was observed. There was a wide variation between the levels of compound in the zebrafish larvae with a higher uptake of amiodarone, methapyrilene and myclobutanil. Detection of hepatocyte markers (CP, CYP3A65, GC and TF) was accomplished by in situ hybridization of larvae to coumarin and myclobutanil and confirmed by real-time reverse transcription-quantitative polymerase chain reaction. Experiments showed decreased expression of all markers. Next, other liver-specific biomarkers (i.e. FABP10a and NR1H4) and apoptosis (i.e. CASP-3 A and TP53) or cytochrome P450-related (CYP2K19) and oxidoreductase activity-related (ZGC163022) genes, were screened. Links between basic mechanisms of liver injury and results of biomarker responses are described. Copyright © 2016 John Wiley & Sons, Ltd.

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.

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.

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.