EpCAM and APN expression in combination with γ-H2AX as biomarkers for detecting hepatocarcinogens in rats.

The phosphorylated form of histone H2AX (γ-H2AX) serves as a commonly utilized biomarker for DNA damage. Based on our previous findings, which demonstrated the formation of γ-H2AX foci as a reliable biomarker for detecting bladder carcinogens in repeated dose 28-day study in rats, we hypothesized that γ-H2AX could also function as a biomarker for detecting hepatocarcinogens. However, we found that γ-H2AX foci formation was not effectively induced by hepatocarcinogens that did not stimulate hepatocyte proliferation. Therefore, we explored alternative biomarkers to detect chemical hepatocarcinogenicity and discovered increased expressions of epithelial cell adhesion molecule (EpCAM/CD326)- and aminopeptidase N (APN/CD13) in the hepatocytes of rats administered various hepatocarcinogens. Significant increases in EpCAM- and APN-positive hepatocytes were observed for eight and five of the 10 hepatocarcinogens, respectively. Notably, five and two of them, respectively, were negative for γ-H2AX foci. These results highlight the potential of EpCAM and APN as useful biomarkers in combination with γ-H2AX for the detection of chemical hepatocarcinogenicity.

Oral toxicological study of titanium dioxide nanoparticles with a crystallite diameter of 6 nm in rats.

BACKGROUND: Though titanium dioxide (TiO2) is generally considered to have a low impact in the human body, the safety of TiO2 containing nanosized particles (NPs) has attracted attention. We found that the toxicity of silver NPs markedly varied depending on their particle size, as silver NPs with a diameter of 10 nm exhibited fatal toxicity in female BALB/c mice, unlike those with diameters of 60 and 100 nm. Therefore, the toxicological effects of the smallest available TiO2 NPs with a crystallite size of 6 nm were examined in male and female F344/DuCrlCrlj rats by repeated oral administration of 10, 100, and 1000 mg/kg bw/day (5/sex/group) for 28 days and of 100, 300, and 1000 mg/kg bw/day (10/sex/group) for 90 days. RESULTS: In both 28- and 90-day studies, no mortality was observed in any group, and no treatment-related adverse effects were observed in body weight, urinalysis, hematology, serum biochemistry, or organ weight. Histopathological examination revealed TiO2 particles as depositions of yellowish-brown material. The particles observed in the gastrointestinal lumen were also found in the nasal cavity, epithelium, and stromal tissue in the 28-day study. In addition, they were observed in Peyer's patches in the ileum, cervical lymph nodes, mediastinal lymph nodes, bronchus-associated lymphoid tissue, and trachea in the 90-day study. Notably, no adverse biological responses, such as inflammation or tissue injury, were observed around the deposits. Titanium concentration analysis in the liver, kidneys, and spleen revealed that TiO2 NPs were barely absorbed and accumulated in these tissues. Immunohistochemical analysis of colonic crypts showed no extension of the proliferative cell zone or preneoplastic cytoplasmic/nuclear translocation of ß-catenin either in the male or female 1000 mg/kg bw/day group. Regarding genotoxicity, no significant increase in micronucleated or γ-H2AX positive hepatocytes was observed. Additionally, the induction of γ-H2AX was not observed at the deposition sites of yellowish-brown materials. CONCLUSIONS: No effects were observed after repeated oral administration of TiO2 with a crystallite size of 6 nm at up to 1000 mg/kg bw/day regarding general toxicity, accumulation of titanium in the liver, kidneys, and spleen, abnormality of colonic crypts, and induction of DNA strand breaks and chromosomal aberrations.

Early detection of hepatocarcinogens in rats by immunohistochemistry of γ-H2AX.

We have developed an early detection method for bladder carcinogens with high sensitivity and specificity using immunohistochemistry of γ-H2AX, a well-known marker of DNA damage. To investigate the potential application of γ-H2AX as a biomarker for early detection of hepatocarcinogens, we examined γ-H2AX formation in the liver of rats treated with several different chemicals for 28 days. Six-week-old male F344 rats were orally treated for 28 days with five hepatocarcinogens: N-nitrosodiethylamine (DEN), di(2-ethylhexyl) phthalate, 1,4-dioxane (DO), 3,3'-dimethylbenzidine dihydrochloride, or thioacetamide (TAA), or with two non-hepatocarcinogens: 4-chloro-o-phenylenediamine and N-ethyl-N-nitrosourea. At the end of the treatment period, immunohistochemistry for γ-H2AX and Ki67 and expression analysis of DNA repair-related genes were performed. Significant increases in γ-H2AX-positive hepatocytes with upregulation of Rad51 mRNA expression were induced by three of five hepatocarcinogens (DEN, DO, and TAA), whereas no changes were seen for the other two hepatocarcinogens and the two non-hepatocarcinogens. Significant increases in Ki67 expression with upregulation of Brip1, Xrcc5, and Lig4 were observed in rats treated with TAA, a nongenotoxic hepatocarcinogen, suggesting that both direct DNA damage and secondary DNA damage due to cell replication stress may be associated with γ-H2AX formation. These results suggest that γ-H2AX immunostaining has potential value for early detection of hepatocarcinogens, but examination of the effects of more chemicals is needed, as is whether γ-H2AX immunostaining should be combined with other markers to increase sensitivity. γ-H2AX immunostaining using formalin-fixed paraffin-embedded specimens can be easily incorporated into existing 28-day repeated-dose toxicity studies, and further improvements in this method are expected.

Expression of stem cell markers as useful complementary factors in the early detection of urinary bladder carcinogens by immunohistochemistry for γ-H2AX.

We previously demonstrated that immunohistochemistry for γ-H2AX, a biomarker of DNA damage, is useful for early detection of urinary bladder carcinogens in rats. In a 28-day repeated-dose study, γ-H2AX was shown to have high sensitivity for detection of bladder carcinogens. However, no reports have evaluated whether a combination of multiple biomarkers may further improve sensitivity. Accordingly, in this study, we aimed to evaluate the applicability of bladder tissue and cancer stem cell markers, including cytokeratin 14 (KRT14), aldehyde dehydrogenase 1A1 (ALDH1A1), and cluster of differentiation 44 (CD44), as complementary markers for early detection of bladder carcinogens. Bladder samples obtained from male F344 rats orally treated with 14 bladder carcinogens and five nonbladder carcinogens for 28 days were used for immunohistochemical analysis of stem cell markers. In the bladder carcinogen-treated rats, increases in KRT14, ALDH1A1, and CD44 expression were observed in 9, 10, and 10 out of 14 groups, respectively, whereas the five nonbladder carcinogens did not cause upregulation of these markers. Although most epithelial cells with KRT14 or ALDH1A1 expression were also positive for CD44, KRT14 and ALDH1A1 expression were mutually exclusive. Twelve bladder carcinogens showed increases in at least one of the three markers, indicating that the combined evaluation showed higher sensitivity than the use of individual markers alone. Importantly, two of three bladder carcinogens that did not induce γ-H2AX immunostaining showed stem cell marker expression. Our results demonstrated that these stem cell markers may be useful as complementary markers for γ-H2AX in evaluation of bladder carcinogens.

Dimethylarsinic acid may promote prostate carcinogenesis in rats.

Arsenic is a known human carcinogen, inducing tumors of the lung, urinary bladder, skin, liver and prostate. However, there are no reports of prostate tumors induced by arsenicals in in vivo animal models. In a previous study, we found that HMGB2 expression was a predictive marker for prostate carcinogens in the rat 4-week repeated dose test. In this study, six-week-old male F344 rats were orally treated with a total of six chemicals (2-acetylaminofluorene (2-AAF), p-cresidine, dimethylarsinic acid (DMA), glycidol, N-nitrosodiethylamine and acrylamide) for four weeks. Animals were sacrificed at the end of the study, and HMGB2 and Ki-67 immunohistochemistry was performed. The numbers of HMGB2- and Ki-67- positive cells in all prostate lobes were significantly increased by DMA, one of the arsenicals, compared with the controls. Meanwhile, the number of Ki-67-positive cells in lateral and dorsal prostate lobes was significantly decreased by 2-AAF with the reduction of body weight, but HMGB2 expression was not. The other chemicals did not change HMGB2 and Ki-67 expression. These data indicate that DMA may have an ability to enhance prostate carcinogenesis.

4-Methylthio-3-butenyl isothiocyanate (MTBITC) induced apoptotic cell death and G2/M cell cycle arrest via ROS production in human esophageal epithelial cancer cells.

To investigate the chemopreventive mechanisms of 4-Methylthio-3-butenyl isothiocyanate (MTBITC), we analyzed cell viability, cell cycle distribution, and expression levels for cell cycle and apoptosis-related proteins in MTBITC-treated malignant esophageal KYSE510 cells, with and without the reactive oxygen species (ROS) scavenger N-acethyl-L-Cysteine (NAC). MTBITC dose-dependently reduced cell viability and Bcl2 protein expression, while it induced cleavages of caspase-3, caspase-9, and PARP-1, suggesting that reduced cell viability occurred through the mitochondrial apoptotic pathway in KYSE510 cells. In cell cycle distribution analysis, MTBITC (20-40 µM) induced cell cycle arrest at G2/M phase. Furthermore, MTBITC induced Chk1 and Akt phosphorylations and decreased p27 protein expression. Both apoptotic- and cell cycle-related changes induced by MTBITC treatment were abolished by NAC. These results suggest that MTBITC has chemopreventive potential for esophageal carcinogenesis by elimination of cancer cells via induction of mitochondrial apoptotic cell death, G2/M cell cycle arrest, and ROS production.

Immunohistochemistry of γ-H2AX as a method of early detection of urinary bladder carcinogenicity in mice.

Phosphorylated histone H2AX (γ-H2AX) has been demonstrated as a DNA damage marker both in vitro and in vivo. We previously reported the effects of genotoxic carcinogens in the urinary bladder of rats by immunohistochemical analysis of γ-H2AX using samples from 28-day repeated-dose tests. To evaluate the application of γ-H2AX as a biomarker of carcinogenicity in the bladder, we examined species differences in γ-H2AX formation in the urinary bladder of mice. Six-week-old male B6C3F1 mice were treated orally with 12 chemicals for 4 weeks. Immunohistochemical analysis demonstrated that N-butyl-N-(4-hydroxybutyl)nitrosamine, p-cresidine and 2-acetylaminofluorene (2-AAF), classified as genotoxic bladder carcinogens, induced significant increases in γ-H2AX levels in the bladder urothelium. In contrast, genotoxic (2-nitroanisole, glycidol, N-nitrosodiethylamine and acrylamide) and non-genotoxic (dimethylarsinic acid and melamine) non-bladder carcinogens did not upregulate γ-H2AX. Importantly, 2-nitroanisole, a potent genotoxic bladder carcinogen in rats, significantly increased the proportion of γ-H2AX-positive cells in rats only, reflecting differences in carcinogenicity in the urinary bladder between rats and mice. Significant upregulation of γ-H2AX was also induced by uracil, a non-genotoxic bladder carcinogen that may be associated with cell proliferation, as demonstrated by increased Ki67 expression. 2-AAF caused γ-H2AX formation mainly in the superficial layer, together with reduced and disorganized expression of uroplakin III, unlike in rats, suggesting the mouse-specific cytotoxicity of 2-AAF in umbrella cells. These results suggest γ-H2AX is a useful biomarker reflecting species differences in carcinogenicity in the urinary bladder.

Subchronic toxicity evaluation of isoeugenyl methyl ether in F344/DuCrj rats by 13-week oral administration.

Isoeugenyl methyl ether (CAS No. 93-16-3) is a food additive used as a nature identical flavoring agent. To determine the toxicity profile and the no-observed-adverse-effect level (NOAEL), we performed a subchronic toxicity test in male and female F344/DuCrj rats by intragastric administration of isoeugenyl methyl ether at doses of 8, 40, and 200 mg/kg body weight (BW)/day for 13 weeks. In this study, BW gain in the male 200 mg/kg BW/day group was decreased from week 9. In serum biochemistry, decreased triglycerides were observed in the male 200 mg/kg BW/day group. In organ weights, increases in both absolute and relative liver weights were observed in both sexes in the 200 mg/kg BW/day group. In histopathological examination, hepatocyte hypertrophy was observed in the male 200 mg/kg BW/day group. Based on these results, we concluded that the main target organ of isoeugenyl methyl ether was the liver and that the NOAEL of isoeugenyl methyl ether for both male and female F344/DuCrj rats was estimated to be 40 mg/kg BW/day.

4-Methylthio-3-butenyl isothiocyanate mediates nuclear factor (erythroid-derived 2)-like 2 activation by regulating reactive oxygen species production in human esophageal epithelial cells.

4-Methylthio-3-butenyl isothiocyanate (MTBITC) extracted from daikon (Raphanus sativus), which shows antimutagenicity, may have applications as an effective chemopreventive agent in several cancers; however, few reports have described the associated mechanisms. We investigated whether MTBITC induced cytoprotective genes, including phase II enzymes, in Het-1A human esophageal epithelial cells. HMOX1, NQO1, and GCLC mRNA levels and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) protein levels were increased in Het-1A cells treated with 10 µM MTBITC. Reactive oxygen species (ROS) tended to increase when Het-1A cells were treated with MTBITC, and the increases in ROS and Nrf2 expression in the cells treated with MTBITC were completely abolished by treatment with N-acetyl-l-cysteine. We also examined the relationships between Nrf2 activation and mitogen-activated protein kinase (MAPK) signaling by western blot analysis. MTBITC induced extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 phosphorylation in Het-1A cells; however, MTBITC did not affect the relationship between Nrf2 activation and MAPK responses. In the present study, we found that MTBITC induced Nrf2 activation and cytoprotective genes via ROS production in Het-1A cells. These results suggest that MTBITC may have the potential for preventing esophageal carcinogenesis through modification of carcinogen metabolism by phase II enzyme induction via ROS production.

Altered susceptibility of an obese rat model to 13-week subchronic toxicity induced by 3-monochloropropane-1,2-diol.

3-Monochloropropane-1,2-diol (3-MCPD) is a heat-induced food contaminant that has been shown to be a nongenotoxic renal carcinogen. Although the toxicity of 3-MCPD has been widely investigated for decades, there is a further concern that 3-MCPD might exert more potent toxicity in high-risk population with underlying diseases such as hyperlipidemia associated with obesity. In the present study, we performed a 13-week subchronic toxicity study for 3-MCPD using an obesity rat model to investigate the differences in susceptibility between obese and normal individuals. Male F344 and obese Zucker (lean and fatty) rats were administered 0, 9, 28.5, 90, 285, or 900 ppm 3-MCPD in drinking water for 13 weeks. 3-MCPD treatment decreased body weight gain, increased relative kidney weights, induced anemia, and induced epithelial cell necrosis in epididymal ducts in all 3 strains. The degrees of epididymal damage were higher in F344 and lean rats than in fatty rats, while renal toxicity was most potent in F344 rats and comparable in lean and fatty rats. In contrast, the hematology data indicated that anemia was worse in fatty rats than in F344 and lean rats, and a significant decrease in hematopoietic cells in the bone marrow was observed only in fatty rats. The no-observed-adverse-effect level was estimated to be 28.5 ppm in all 3 strains for 3-MCPD. These results suggested that obese Zucker rats may be more susceptible to 3-MCPD-dependent toxicity in the hematopoietic tissues than their lean counterparts.

Lack of in vivo mutagenicity of 1,2-dichloropropane and dichloromethane in the livers of gpt delta rats administered singly or in combination.

1,2-Dichloropropane (1,2-DCP) and dichloromethane (DCM) are possible causative agents associated with the development of cholangiocarcinoma in employees working in printing plant in Osaka, Japan. However, few reports have demonstrated an association between these agents and cholangiocarcinoma in rodent carcinogenicity studies. Moreover, the combined effects of these compounds have not been fully elucidated. In the present study, we evaluated the in vivo mutagenicity of 1,2-DCP and DCM, alone or combined, in the livers of gpt delta rats. Six-week-old male F344 gpt delta rats were treated with 1,2-DCP, DCM or 1,2-DCP + DCM by oral administration for 4 weeks at the dose (200 mg kg-1 body weight 1,2-DCP and 500 mg kg-1 body weight DCM) used in the carcinogenesis study performed by the National Toxicology Program. In vivo mutagenicity was analyzed by gpt mutation/Spi- assays in the livers of rats. In addition, gene and protein expression of CYP2E1 and GSTT1, the major enzymes responsible for the genotoxic effects of 1,2-DCP and DCM, were analyzed by quantitative polymerase chain reaction and western blotting. Gpt and Spi- mutation frequencies were not increased by 1,2-DCP and/or DCM in any group. Additionally, there were no significant changes in the gene and protein expression of CYP2E1 and GSTT1 in any group. These results indicated that 1,2-DCP, DCM and 1,2-DCP + DCM had no significant impact on mutagenicity in the livers of gpt delta rats under our experimental conditions. Copyright © 2016 John Wiley & Sons, Ltd.

Toxic effects of 4-methylthio-3-butenyl isothiocyanate (Raphasatin) in the rat urinary bladder without genotoxicity.

We recently reported that 4-methylthio-3-butenyl isothiocyanate (MTBITC) exerts chemopreventive effects on the rat esophageal carcinogenesis model at a low dose of 80 ppm in a diet. In contrast, some isothiocyanates (ITCs) have been reported to cause toxic effects, promotion activity, and/or carcinogenic potential in the urinary bladder of rats. In the present study, we investigated whether MTBITC had toxic effects in the urinary bladder similar to other ITCs, such as phenethyl ITC (PEITC). First, to examine the early toxicity of MTBITC, rats were fed a diet supplemented with 100, 300 or 1000 ppm MTBITC for 14 days. Treatment with 1000 ppm MTBITC caused increased organ weights and histopathological changes in the urinary bladder, producing lesions similar to those of 1000 ppm PEITC. In contrast, rats treated with 100 or 300 ppm MTBITC showed no signs of toxicity. Additionally, we performed in vivo genotoxicity studies to clarify whether MTBITC may exhibit a carcinogenic potential through a genotoxic mechanism in rats. Rats were treated with MTBITC for 3 days at doses of 10, 30 or 90 mg kg-1 body weight by gavage, and comet assays in the urinary bladder and micronucleus assays in the bone marrow were performed. No genotoxic changes were observed after treatment with MTBITC at all doses. Overall, these results suggested that the effects of MTBITC in the rat urinary bladder are less than those of PEITC, but that MTBITC could have toxic effects through a nongenotoxic mechanism in the urinary bladder of rats at high doses. Copyright © 2016 John Wiley & Sons, Ltd.

4-Methylthio-3-butenyl isothiocyanate (raphasatin) exerts chemopreventive effects against esophageal carcinogenesis in rats.

To examine the effects of 4-methylthio-3-butenyl isothiocyanate on esophageal carcinogenesis, male 6-week-old F344 rats were subcutaneously injected with 0.5 mg/kg body weight N-nitrosomethylbenzylamine three times per week for 5 weeks and fed a diet supplemented with 80 ppm 4-methylthio-3-butenyl isothiocyanate, equivalent to 6.05 mg/kg body weight/day for the initiation stage, 4.03 mg/kg body weight/day for the promotion stage, or 4.79 mg/kg body weight/day for all stages. Although the incidence of lesions was not affected by 4-methylthio-3-butenyl isothiocyanate treatment, the multiplicity of squamous cell papilloma in the esophagus was significantly decreased in rats in the 4-methylthio-3-butenyl isothiocyanate initiation stage group (1.13 ± 0.74), 4-methylthio-3-butenyl isothiocyanate promotion stage group (1.47 ± 0.99), and 4-methylthio-3-butenyl isothiocyanate all stage group (1.47 ± 1.13) as compared with rats treated with N-nitrosomethylbenzylamine alone (3.00 ± 1.46). Immunohistochemical analysis revealed that 4-methylthio-3-butenyl isothiocyanate induced apoptosis, suppressed cell proliferation, and increased p21 expression when administered in the promotion phase. These modifying effects were not observed in the rats treated with 4-methylthio-3-butenyl isothiocyanate alone. Our results indicated that 4-methylthio-3-butenyl isothiocyanate may exert chemopreventive effects against N-nitrosomethylbenzylamine-induced esophageal carcinogenesis in rats.

Orally administered glycidol and its fatty acid esters as well as 3-MCPD fatty acid esters are metabolized to 3-MCPD in the F344 rat.

IARC has classified glycidol and 3-monochloropropane-1,2-diol (3-MCPD) as group 2A and 2B, respectively. Their esters are generated in foodstuffs during processing and there are concerns that they may be hydrolyzed to the carcinogenic forms in vivo. Thus, we conducted two studies. In the first, we administered glycidol and 3-MCPD and associated esters (glycidol oleate: GO, glycidol linoleate: GL, 3-MCPD dipalmitate: CDP, 3-MCPD monopalmitate: CMP, 3-MCPD dioleate: CDO) to male F344 rats by single oral gavage. After 30 min, 3-MCPD was detected in serum from all groups. Glycidol was detected in serum from the rats given glycidol or GL and CDP and CDO in serum from rats given these compounds. In the second, we examined if metabolism occurs on simple reaction with rat intestinal contents (gastric, duodenal and cecal contents) from male F344 gpt delta rats. Newly produced 3-MCPD was detected in all gut contents incubated with the three 3-MCPD fatty acid esters and in gastric and duodenal contents incubated with glycidol and in duodenal and cecal contents incubated with GO. Although our observation was performed at 1 time point, the results showed that not only 3-MCPD esters but also glycidol and glycidol esters are metabolized into 3-MCPD in the rat.

Early Detection of Genotoxic Urinary Bladder Carcinogens by Immunohistochemistry for γ-H2AX.

DNA double-strand breaks (DSBs) induced by exposure to genotoxic agents are known to cause genome instability and cancer development. To evaluate the applicability of γ-H2AX, a sensitive marker of DSBs, in the early detection of genotoxicity and carcinogenicity of chemicals using animal models, we examined γ-H2AX expression in urinary bladders of rats. Six-week-old male F344 rats were orally treated for 4 weeks with a total of 12 chemicals divided into 4 categories based on genotoxicity and carcinogenicity in the urinary bladder. Animals were sacrificed at the end of administration or after 2 weeks of recovery, and immunohistochemistry for γ-H2AX was performed. At week 4, γ-H2AX expression in bladder epithelial cells was significantly increased by all 4 genotoxic bladder carcinogens as compared with the controls, whereas the 3 chemicals that were genotoxic but not carcinogenic in the bladders did not cause upregulation of γ-H2AX. After the recovery period, γ-H2AX expression was markedly reduced in all groups but remained significantly elevated in rats treated with 3 of the 4 genotoxic bladder carcinogens. Although slight increases in γ-H2AX expression were induced by a weak bladder carcinogen with equivocal genotoxicity (phenethyl isothiocyanate) and 2 nongenotoxic bladder carcinogens (melamine and uracil) at week 4, these differences were not significant and were thought to be associated with activated proliferation by urothelial hyperplasia, as demonstrated by increased Ki67-positive cells. These results suggested that γ-H2AX may be a potential biomarker for the early detection of genotoxic bladder carcinogens.

Excision of translesion synthesis errors orchestrates responses to helix-distorting DNA lesions.

In addition to correcting mispaired nucleotides, DNA mismatch repair (MMR) proteins have been implicated in mutagenic, cell cycle, and apoptotic responses to agents that induce structurally aberrant nucleotide lesions. Here, we investigated the mechanistic basis for these responses by exposing cell lines with single or combined genetic defects in nucleotide excision repair (NER), postreplicative translesion synthesis (TLS), and MMR to low-dose ultraviolet light during S phase. Our data reveal that the MMR heterodimer Msh2/Msh6 mediates the excision of incorrect nucleotides that are incorporated by TLS opposite helix-distorting, noninstructive DNA photolesions. The resulting single-stranded DNA patches induce canonical Rpa-Atr-Chk1-mediated checkpoints and, in the next cell cycle, collapse to double-stranded DNA breaks that trigger apoptosis. In conclusion, a novel MMR-related DNA excision repair pathway controls TLS a posteriori, while initiating cellular responses to environmentally relevant densities of genotoxic lesions. These results may provide a rationale for the colorectal cancer tropism in Lynch syndrome, which is caused by inherited MMR gene defects.

Validation study of the combined repeated-dose toxicity and genotoxicity assay using gpt delta rats.

Transgenic rodents carrying reporter genes to detect organ-specific in vivo genetic alterations are useful for risk assessment of genotoxicity that causes cancer. Thus, the Organization for Economic Co-operation and Development has established the guideline for genotoxicity tests using transgenic animals, which may be combined with repeated-dose toxicity studies. Here, we provide evidence to support equivalence of gpt delta and wild type (WT) rats in terms of toxicological responses to a genotoxic hepatocarcinogen, N-nitrosodiethylamine (DEN), and a non-genotoxic hepatocarcinogen, di(2-ethylhexyl)phthalate (DEHP). gpt delta rats treated with DEHP showed similar increases in liver and kidney weights, serum albumin, albumin/globulin ratios, and incidence of diffuse hepatocyte hypertrophy compared to WT F344 and Sprague-Dawley (SD) rats. DEN-treated gpt delta rats showed equivalent increases in the number and area of precancerous GST-P-positive foci in the liver compared to WT rats. The livers of DEN-treated gpt delta rats also showed increased frequencies of gpt and Spi(-) mutations; such changes were not observed in DEHP-treated gpt delta rats. These results indicated that gpt delta rats (both F344 and SD backgrounds) showed comparable DEHP-induced toxicity and DEN-induced genotoxicity to those observed in WT rats. With regard to the administration period, the general toxicity of 1.2% DEHP was evident throughout the experimental period, and the genotoxicity of 10 p.p.m. DEN could be detected after 2 weeks of administration and further increased at 4 weeks. These results suggested that combined assays using gpt delta rats could detect both general toxicity and genotoxicity by the canonical 4-week administration protocol. Therefore, this assay using gpt delta rats would be applicable for risk assessment including early detection of genotoxic carcinogens and ultimately serve to reduce cancer risks in humans from environmental chemicals.

A 13-week subchronic toxicity study of sodium iron chlorophyllin in F344 rats.

Sodium iron chlorophyllin (SIC), a water-soluble chlorophyll derivative, has been used as a food additive for green coloration. In the present study, a subchronic toxicity study of SIC was performed in male and female F344 rats with oral administration in diet at concentrations of 0%, 0.2%, 1.0%, and 5.0% for 13 weeks. No mortalities, abnormal clinical signs, and hematological changes were observed in any of the groups during the experiment. Significant reduction of body weight gain was noted in 5.0% males. In serum biochemistry, serum transferrin levels were significantly increased in 5.0% males and females. Relative spleen weights of both sexes were markedly reduced with 5.0% SIC as compared to the controls, and absolute weights of spleen were also significantly decreased in males. On histopathological assessment, diffuse hypertrophy of acinar cells in the parotid gland was observed in all examined 5.0% males and females, but not in the other groups. Based on the histopathology of the parotid glands, the no-observed-adverse-effect level (NOAEL) of SIC in the present study was estimated to be 1.0% (609 mg/kg bw/day for males and 678 mg/kg bw/day for females).

Detection of γ-H2AX, a Biomarker for DNA Double-strand Breaks, in Urinary Bladders of N -Butyl- N -(4-Hydroxybutyl)-Nitrosamine-Treated Rats.

To evaluate the potential role of DNA repair in bladder carcinogenesis, we performed an immunohistochemical analysis of expression of various DNA repair enzymes and γ-H2AX, a high-sensitivity marker of DNA double-strand breaks, in the urothelium of male F344 rats treated with N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN), a bladder-specific carcinogen. Our results clearly demonstrated that γ-H2AX aggregation was specifically generated in nuclei of bladder epithelial cells of BBN-treated rats, which was not found in untreated controls or mesenchymal cells. γ-H2AX-positive cells were detected not only in hyperplastic and neoplastic areas but also in the normal-like urothelium after BBN treatment. These data indicate that γ-H2AX has potential as a useful biomarker for early detection of genotoxicity in the rat urinary bladder. To the best of our knowledge, this is the first report demonstrating expression of γ-H2AX during bladder carcinogenesis.

Stalled Polη at its cognate substrate initiates an alternative translesion synthesis pathway via interaction with REV1.

DNA polymerase η (Polη), whose gene mutation is responsible for the inherited disorder xeroderma pigmentosum variant (XP-V), carries out accurate and efficient translesion synthesis (TLS) across cyclobutane pyrimidine dimer (CPD). As Polη interacts with REV1, and REV1 interacts with other TLS polymerases including Polι, Polκ and Polζ, Polη may play a role in recruitment of these TLS polymerases at lesion site. But it is unclear whether UV sensitivity of XP-V patients is caused not only by defect of Polη activity but also by dysfunction of network between Polη and other TLS polymerases. Here, we examined whether the TLS polymerase network via Polη is important for replicative bypass of CPDs and DNA damage tolerance induced by UV in mouse cells. We observed that UV sensitivity of Polη-deficient mouse cells was moderately rescued by the expression of a catalytically inactive Polη. Moreover, this recovery of cellular UV sensitivity was mediated by the interaction between Polη and REV1. However, expression of the inactive mutant Polη was not able to suppress the incidence of UV-induced mutation observed in Polη-deficient cells. We propose the model that REV1 and Polκ are involved in DNA damage tolerance via Polη-REV1 interaction when Polη fails to bypass its cognate substrates.