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of hepatotoxicity enhanced phospholipidosis, suggesting phospholipidosis is not a pathway in toxicity for this withdrawn drug.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.

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.

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.

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.

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.

Systems toxicology: integrated genomic, proteomic and metabonomic analysis of methapyrilene induced hepatotoxicity in the rat.

Administration of high doses of the histamine antagonist methapyrilene to rats causes periportal liver necrosis. The mechanism of toxicity is ill-defined and here we have utilized an integrated systems approach to understanding the toxic mechanisms by combining proteomics, metabonomics by 1H NMR spectroscopy and genomics by microarray gene expression profiling. Male rats were dosed with methapyrilene for 3 days at 150 mg/kg/day, which was sufficient to induce liver necrosis, or a subtoxic dose of 50 mg/kg/day. Urine was collected over 24 h each day, while blood and liver tissues were obtained at 2 h after the final dose. The resulting data further define the changes that occur in signal transduction and metabolic pathways during methapyrilene hepatotoxicity, revealing modification of expression levels of genes and proteins associated with oxidative stress and a change in energy usage that is reflected in both gene/protein expression patterns and metabolites. The difficulties of combining and interpreting multiomic data are considered.

Dose-dependent induction of GST-P+ staining foci by the rat hepatocarcinogen methapyrilene in the medium-term bioassay.

Previous studies have demonstrated that methapyrilene hydrochloride (MP) is a rat-specific nongenotoxic carcinogen which induces liver tumors in a dose-dependent manner following chronic exposure in the diet. This study was conducted to determine the dose response of MP in the medium-term bioassay and to compare the response to tumor incidence. Two weeks following a single initiating dose of diethylnitrosamine (DEN), male F344 rats were administered MP at doses of 0, 62.5, 125, 250, or 1000 ppm in the diet for 6 weeks. A 2/3 partial hepatectomy was performed 3 weeks post-DEN. At termination, sections from the remaining three lobes were stained with GST-P antibody. Number and size of foci were measured using an image analysis system with a digitizing board. MP induced a dose-dependent increase in the number of GST-P+ foci/cm2 (0 ppm = 0.85 foci/cm2; 62.5 ppm = 1.29 foci/cm2; 125 ppm = 1.59 foci/cm2; 250 ppm = 6.55 foci/cm2; 1000 ppm = 28.23 foci/cm2). A significantly greater number of foci were observed in the caudate lobe than in the anterior and posterior lobes. The size of individual foci was largely unaffected. This study demonstrates a strong correlation between foci induction and tumor incidence and suggests that this assay may have utility in predicting dose responses for the chronic bioassay.

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).

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.

In vitro release of therapeutically active ingredients from polymer matrixes.

Several therapeutically active ingredients including benzocaine, cyclomethycaine, and methapyrilene hydrochloride were incorporated into ethylcellulose and polyamide films. The effect of cetyl alcohol and tributyl citrate upon the release of these ingredients was studied. The films containing the active ingredient and plasticizer were cast upon a mercury substrate, and the in vitro release of these drugs from each film into a desorbing medium of distilled water was measured. The results indicated that the film-forming agent and plasticizer affected the drug release rate and that the release followed first-order kinetics. Benzocaine was slowly released from polyamide-cetyl alcohol films and polyamide-cetyl alcohol-tributyl citrate films. Polyamide-tributyl citrate films showed enhanced release of benzoacaine and cyclomethycaine. Ethylcellulose films plasticized with tributyl citrate produced a fast drug release. Based upon these results, a water-soluble, highly polar, noncomplexing additive would tend to increase the drug release from the film. When the amount of benzocaine released from ethylcellulose was plotted as a function of the square root of time, a linear plot was obtained. Since this linear plot passed through the origin, ethylcellulose should be an ideal matrix for benzocaine according to the Higuchi diffusion-controlled model. These studies demonstrated the in vitro release of thf the solubility of the active agent in both the polymer film as a function of the solubility of the active agent in both the polymer matrix and the desorbing medium.