Correlation between the results of in vitro and in vivo chromosomal damage tests in consideration of exposure levels of test chemicals.

INTRODUCTION: We examined the correlation between the results of in vitro and in vivo chromosomal damage tests by using in-house data of 18 pharmaceutical candidates that showed positive results in the in vitro chromosomal aberration or micronucleus test using CHL/IU cells, and quantitatively analyzed them especially in regard to exposure levels of the compounds. FINDINGS: Eight compounds showed that the exposure levels [maximum plasma concentration (Cmax) and AUC0-24h] were comparable with or higher than the in vitro exposure levels [the lowest effective (positive) concentration (LEC) and AUCvitro = LEC (µg/mL) × treatment time (h)]. Among them, 3 compounds were positive in the in vivo rodent micronucleus assays using bone marrow cells. For 2 compounds, cytotoxicity might produce false-positive results in the in vitro tests. One compound showed in vitro positive results only in the condition with S9 mix which indicated sufficient concentration of unidentified active metabolite(s) might not reach the bone marrow to induce micronuclei. CONCLUSION: These facts suggested that the in vivo exposure levels being equal to or higher than the in vitro exposure levels might be an important factor to detect in vivo chromosomal damage induced by test chemicals.

The suitability of rat hepatoma cell line H4IIE for evaluating the potentials of compounds to induce CYP3A23 expression.

To investigate the suitability of H4IIE cells for detecting cytochrome P450 (CYP) induction in vitro, we compared CYP induction by typical CYP inducers in H4IIE cells and rat primary hepatocytes by examining gene expression and enzyme activity, and by immunocytochemistry. The cells were preincubated with 0.1 µM of dexamethasone (DEX) for 24 h, followed by 48 h of exposure to 10 µM of beta-naphthoflavone (bNF), 100 µM of phenobarbital (PB) and 10 µM of DEX. Cyp1a1, Cyp2b1/2 and Cyp3a23/3a1 (Cyp3a23) expressions in H4IIE cells were up-regulated 280-, 1.5- and 65-fold relative to those in vehicle-treated cells, respectively. The fold inductions of those expressions in rat primary hepatocytes were 80-, 33- and 152-fold, respectively. Comprehensive gene expression analysis using DNA microarrays showed that Cyp3a23, Gsta2, Ugt2b12, Udpgt and Sult2a1 expressions were up-regulated in H4IIE cells exposed to 10 µM of DEX. CYP3A activity was not increased, but some H4IIE cells exposed to DEX were stained strongly with anti-CYP3A antibody. We cloned these cells and obtained cloned H4IIE (cH4IIE) cells with expression level of Cyp3a23 higher than those of vehicle-treated cells. It was confirmed that preincubation with 0.1 µM of DEX increased pregnane X receptor (Pxr) expression level and enhanced the Cyp3a23 induction effects of test compounds significantly. Retrospective examination of in vitro CYP induction assay using cH4IIE cells resulted in 80% correlation with the data from in vivo rat toxicity studies. These results suggested that cH4IIE cells are suitable for evaluating the potentials of a compound to induce CYP3A23 expression.

Evaluation of a liver micronucleus assay in young rats (III): a study using nine hepatotoxicants by the Collaborative Study Group for the Micronucleus Test (CSGMT)/Japanese Environmental Mutagen Society (JEMS)-Mammalian Mutagenicity Study Group (MMS).

We have been investigating a liver micronucleus assay to detect genotoxic chemicals using young rats for several years, and had established its advantages with respect to using autonomous proliferation of young rat hepatocytes. Nine chemicals known to induce hepatotoxic effects such as necrosis (2,6-dinitrotolune, bromobenzene, isoniazid, phenacetin, allyl alcohol and thioacetamide), cholestasis (chlorpromazine hydrochloride and alpha-naphthyl isothiocyanate) and oxidative stress (clofibrate) were selected for this study. A liver micronucleus assay was conducted in 4-week-old male F344 rats using two or three dose levels of test chemicals given orally by gavage to evaluate the compound's ability to induce micronucleated hepatocytes. Several of these test chemicals were additionally examined in a peripheral blood micronucleus assay conducted concurrently and in the same animals. The genotoxic rodent hepatocarcinogen, 2,6-dinitrotoluene showed a positive result in the liver micronucleus assay, but the nongenotoxic hepatocarcinogens, clofibrate and thioacetamide gave negative responses. Bromobenzene, known to produce DNA adducts but is noncarcinogenic in rodent liver, was judged equivocal in this assay. alpha-Naphthyl isothiocyanate is noncarcinogenic and showed negative response in the liver. The other four chemicals, known to be either noncarcinogenic or carcinogenic in other non-liver target organs, showed negative results in the liver micronucleus assay. Based on the results in the present study and previous report described above, it was concluded that this technique is able to effectively predict genotoxic rodent hepatocarcinogenicity, and does not give false positives due to hepatotoxicity.

Characterization and interlaboratory comparison of a gene expression signature for differentiating genotoxic mechanisms.

The genotoxicity testing battery is highly sensitive for detection of chemical carcinogens. However, it features a low specificity and provides only limited mechanistic information required for risk assessment of positive findings. This is especially important in case of positive findings in the in vitro chromosome damage assays, because chromosome damage may be also induced secondarily to cell death. An increasing body of evidence indicates that toxicogenomic analysis of cellular stress responses provides an insight into mechanisms of action of genotoxicants. To evaluate the utility of such a toxicogenomic analysis we evaluated gene expression profiles of TK6 cells treated with four model genotoxic agents using a targeted high density real-time PCR approach in a multilaboratory project coordinated by the Health and Environmental Sciences Institute Committee on the Application of Genomics in Mechanism-based Risk Assessment. We show that this gene profiling technology produced reproducible data across laboratories allowing us to conclude that expression analysis of a relevant gene set is capable of distinguishing compounds that cause DNA adducts or double strand breaks from those that interfere with mitotic spindle function or that cause chromosome damage as a consequence of cytotoxicity. Furthermore, our data suggest that the gene expression profiles at early time points are most likely to provide information relevant to mechanisms of genotoxic damage and that larger gene expression arrays will likely provide richer information for differentiating molecular mechanisms of action of genotoxicants. Although more compounds need to be tested to identify a robust molecular signature, this study confirms the potential of toxicogenomic analysis for investigation of genotoxic mechanisms.

Gene expression profiling in streptozotocin treated mouse liver using DNA microarray.

Streptozotocin (SZ) is known to exert toxic effects not only on pancreatic islet beta cells but also on other organs including liver. For analyzing changes in genes expression associated with SZ toxicity, we performed DNA microarray analyses on the liver obtained from SZ-treated mice. Eight-week-old male ICR mice were treated i.p. with 200 mg/kg of SZ, and the blood and liver were taken at 6, 24 and 48 h after the treatment. Labeled cRNA prepared from total RNA of the liver was hybridized to the GeneChip Murine Genome U74A V.2 (Affymetrix). The number of the probe sets, which were clearly up-regulated or down-regulated, were over 100 at 6 and 24h after the SZ-treatment, and it decreased at 48 h after the treatment. Many of the up-regulated genes were categorized into cell cycle/apoptosis related genes, immune/allergy related genes and stress response/xenobiotic metabolism related genes. On the other hand, genes related to glucose, lipid and protein metabolisms were down-regulated. These changes started prior to the elevation of the serum glucose levels, indicating the direct action of SZ on the liver rather than the secondary effect of diabetes. This may be related with the previously reported hepatic changes such as lipid peroxidation, mitochondrial swelling and inhibition of hepatocyte proliferation observed before the development of hyperglycemia.

Morphological and gene expression analysis in mouse primary cultured hepatocytes exposed to streptozotocin.

Streptozotocin (SZ) is known to exert toxic effects not only on pancreatic islet beta cells but also on other organs including the liver. For analyzing direct effects of SZ on hepatocytes, we performed morphological analysis and DNA microarray analysis on mouse primary cultured hepatocytes. Hepatocytes were taken from non-treated Crj:CD-1(ICR) mice. The primary cultured hepatocytes were treated with SZ at concentrations of 0, 1, 3, 10, 30 and 100 mM. After the treatment for about 6 or 24h, cell survival assay using tetrazolium salt (WST-1), light microscopic/electron microscopic analysis and gene expression analysis were performed. For the gene expression analysis, target (labeled cRNA) prepared from total RNA of the hepatocytes was hybridized to the GeneChip Murine Genome U74A V.2 (Affymetrix). The signal intensity calculation and scaling were performed using Microarray Suite Software Ver 5.0. IC50 of the cell survival assay was around 62 mM at 6 h exposure and 7 mM at 24 h exposure. Marked chromatin margination was observed in nuclei of the hepatocytes treated with SZ at concentrations of 3 or 10mM. Gene expression analysis revealed similar expression changes to those of in vivo, i.e. up-regulation in cell proliferation/ apoptosis related genes, and down-regulation of lipid metabolism related genes. These results potently supported the hypothesis that many of the hepatic alteration including histopathological and gene expression changes are induced by direct effect of SZ rather than by the secondary effect of the hyperglycemia or hypoinsulinemia.

Hepatic changes in the acute phase of streptozotocin (SZ)-induced diabetes in mice.

We have reported the streptozotocin (SZ)-induced hepatic lesions in the subacute phase (4 to 12 weeks after the treatment), which are characterized by appearance of oncocytic hepatocytes, cytomegalic hepatocytes and bile duct hyperplasia. In this study, we focused on the acute phase (6 to 48 hours after the treatment) of the SZ-induced hepatic lesions of mice to clarify the onset of the hepatic alterations, especially before the induction of hyperglycemia. Livers were taken from 8-week-old Crj:CD-1 (ICR) male mice at 6, 12, 24, 36 and 48 hours after the 200 mg/kg b.w. of SZ-injection. SZ-induced hyperglycemia was noted at 36 and 48 hours after the treatment, but the hepatic changes including lipid peroxidation, mitochondrial swelling, peroxisome proliferation and inhibition of hepatocyte proliferation occurred before the elevation of the serum glucose levels. The present findings indicate the direct effects of SZ on hepatocytes rather than the secondary effects of diabetes, and certain correlations between the hepatocytic changes in the acute phase and those in the subacute one. In addition, ulcer and submucosal edema of the gallbladder were observed at 36 or 48 hours after the SZ-treatment, which can be a novel finding in SZ-treated animal.