A formamidopyrimidine derivative from the deoxyguanosine adduct produced by food contaminant acrylamide induces DNA replication block and mutagenesis.

Acrylamide, a common food contaminant, is metabolically activated to glycidamide, which reacts with DNA at the N7 position of dG, forming N7-(2-carbamoyl-2-hydroxyethyl)-dG (GA7dG). Owing to its chemical lability, the mutagenic potency of GA7dG has not yet been clarified. We found that GA7dG undergoes ring-opening hydrolysis to form N6-(2-deoxy-d-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-[N-(2-carbamoyl-2-hydroxyethyl)formamido]pyrimidine (GA-FAPy-dG), even at neutral pH. Therefore, we aimed to examine the effects of GA-FAPy-dG on the efficiency and fidelity of DNA replication using an oligonucleotide carrying GA-FAPy-9-(2-deoxy-2-fluoro-ß-d-arabinofuranosyl)guanine (dfG), a 2'-fluorine substituted analog of GA-FAPy-dG. GA-FAPy-dfG inhibited primer extension by both human replicative DNA polymerase ε and the translesion DNA synthesis polymerases (Polη, Polι, Polκ, and Polζ) and reduced the replication efficiency by less than half in human cells, with single base substitution at the site of GA-FAPy-dfG. Unlike other formamidopyrimidine derivatives, the most abundant mutation was G:C > A:T transition, which was decreased in Polκ- or REV1-KO cells. Molecular modeling suggested that a 2-carbamoyl-2-hydroxyethyl group at the N5 position of GA-FAPy-dfG can form an additional H-bond with thymidine, thereby contributing to the mutation. Collectively, our results provide further insight into the mechanisms underlying the mutagenic effects of acrylamide.

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.

Histopathological and immunohistochemical evaluation for detecting changes in blood hormone levels caused by endocrine disruptors in a 28-day repeated-dose study in rats.

Although measurements of blood hormone levels in rodent toxicological studies can provide important information on the mechanisms of toxicity and extrapolation to humans, there are several difficulties such as large individual differences and limited sample volume. To develop a more simplified method that does not depend solely on blood samples, we examined the possible application of immunohistochemistry for detecting endocrine disruptors in short-term studies. Aminotriazole (AMT), propylthiouracil (PTU), phenobarbital, aminoglutethimide (AGT), estradiol, and vitamin D3 were administered orally to 6-week-old male and female SD rats (five/group) for 28 days. Measurements of serum hormone levels revealed decreases in triiodothyronine (T3) and thyroxine (T4) in the AMT and PTU groups, an increase in thyroid stimulating hormone (TSH) in the AMT, PTU, and AGT groups, and an increase in adrenocorticotrophic hormone in the AGT group. Increased thyroid, pituitary, and adrenal gland weights; histopathological lesions, including follicular hypertrophy/hyperplasia, hypertrophy/vacuolation of anterior pituitary cells, and increased adrenocortical vacuolation were observed in association with the hormone level changes. Immunohistochemical analysis revealed a decreased T4 level in the thyroid gland of the AMT and PTU groups and an increased area of TSH positive immunostaining in the pituitary gland of the AMT, PTU, and AGT groups, consistent with the changes in serum T4 and TSH levels, respectively. These results suggest that histopathological analysis and immunohistochemistry for T4 and TSH might be useful and sensitive methods of detecting thyroid dysfunction, and that combining organ weight measurements is a reliable parameter of detecting endocrine disruptors.

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.

Spontaneous adenolipoma of the mammary gland in the male F344 rat.

A 110-week-old male F344 rat from the high-dose group of a 104-week carcinogenicity study, exhibited a spontaneously occurring subcutaneous mass in the left axilla extending to the chest. Histologically, the mass was well-demarcated from the adjacent mammary tissue and slightly encapsulated without evidence of infiltration into the surrounding tissues. The mass contained both epithelial and adipose components. The epithelial component consisted of ductal structures of various sizes lined by a single layer of flattened to cuboidal epithelial cells with relatively clear or vacuolated cytoplasm. These ductal structures were well-intermingled with an adipose component that consisted of a uniform monomorphic cell population of mature adipocytes. Both cell types were well-differentiated and did not exhibit cellular atypia. Within the mass, fibrous connective tissue was found in the stroma with infiltration of numerous mast cells. Based on these findings, the mass was diagnosed as an adenolipoma of the mammary gland.

A 90-day repeated oral dose toxicity study of four stereoisomers of 2,4-dimethyl-4-phenyltetrahydrofuran, a synthetic flavoring substance, in F344 rats.

2,4-Dimethyl-4-phenyltetrahydrofuran (CAS no. 82461-14-1) is a food additive used as a synthetic flavoring substance. To investigate the toxicological properties and determine the no-observed-adverse-effect level (NOAEL), a 90-day repeated oral dose toxicity study of 2,4-dimethyl-4-phenyltetrahydrofuran containing four stereoisomers was conducted in F344 rats at doses of 0, 6, 24, and 96 mg/kg body weight (BW)/day. No mortality or abnormal clinical signs related to treatment in any group was observed. At a dose of 96 mg/kg BW, fluctuated serum total protein and total cholesterol and increased absolute and relative liver weights and relative kidney weights were observed in both sexes. Increased serum albumin in males and decreased Na and Cl in females were also observed. On histopathological assessment, at a dose of 96 mg/kg BW, diffuse hepatocellular hypertrophy in the liver in both sexes and tubular regeneration with scattered proximal tubular degeneration and/or necrosis throughout the cortex in the kidney in males were detected. Based on these findings, the NOAEL for 2,4-dimethyl-4-phenyltetrahydrofuran used in the current study was found to be 24 mg/kg BW/day for both sexes.

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.

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.

A 13-week subchronic toxicity study of hexyl acetate in SD rats.

Hexyl acetate (CAS No. 142-92-7) is a naturally occurring ester compound that has a fruity odor. Despite its frequent use as a nature-identical flavoring agent, there are limited repeated dose toxicity data for hexyl acetate. Here we performed a 13-week subchronic toxicity study of hexyl acetate in male and female Crl:CD(SD) rats under GLP regulations. Hexyl acetate was given orally by gavage at doses of 0, 100, 300, or 1,000 mg/kg/day using corn oil as the vehicle. No significant toxicological changes in general condition, body weights, food intake, ophthalmology, hematology, organ weights, and histopathological findings were observed in any groups. Urinalysis revealed occult blood in two male animals treated with 1,000 mg/kg/day hexyl acetate, and one showed red blood cells in the urine sediment. Furthermore, blood biochemistry showed a significant increase in inorganic phosphorus levels in males treated with 1,000 mg/kg/day hexyl acetate. These results indicated that the no-observed-adverse-effect level (NOAEL) of hexyl acetate was 300 mg/kg/day for males and more than 1,000 mg/kg/day for females.

Twist1 was detected in mesenchymal cells of mammary fibroadenoma and invasive components of breast carcinoma in rats.

Fibroadenoma (FA) is a common mammary fibroepithelial tumor. The tumor size of the FA is increased by estrogen, progesterone, prolactin, and pregnancy, whereas it decreases after menopause. These observations in humans indicate that FA is hormone dependent. In rats, the most common mammary neoplasm is also FA. Expression levels of Twist1, a transcriptional regulator of epithelial-mesenchymal transition, were examined in paraffin-embedded tissue sections of an experimental rat breast model to find physiological alternations coincident with reproductive hormonal changes. Twenty-three Fischer 344/Brown Norway F1 hybrid rats were used as 14- to 16-week-old adolescent rats (n=3), pregnant rats (n=4), and lactating rats (n=6) in addition to rats over 100-weeks-old that exhibited aging (n=3) and FA (n=7). Seventy-six cases of chemically induced breast carcinoma and two cases of FA in Sprague Dawley rats were also examined. Using tissue sections, we observed that Twist1-positive mesenchymal cells were predominantly located in the periductal region in adolescent and pregnant rats and in the terminal duct lobular unit in pregnant and elderly rats. Twist1 was also expressed diffusely in the mesenchymal cells of FA rats. Twist1-positive cancer-associated mesenchymal cells were found more frequently in the invasive components of breast carcinomas than in intraductal components. The expressions of Twist1 in mesenchymal cells were induced by physiological and pathological stimuli, suggesting the biological role of Twist1 in tissue structure. Further study may reveal the role of Twist1 in mesenchymal cells of mammary glands in rats.

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.

Size-dependent acute toxicity of silver nanoparticles in mice.

In this study, we aimed to evaluate changes in the acute toxicity of intraperitoneally administered silver nanoparticles (AgNPs) of varying sizes in BALB/c mice. Seven-week-old female BALB/c mice were intraperitoneally administered AgNPs measuring 10, 60, or 100 nm in diameter (0.2 mg/mouse) and then sacrificed 1, 3, or 6 h after treatment. In mice administered 10 nm AgNPs, reduced activity and piloerection were observed at 5 h post administration, and lowered body temperature was observed at 6 h post administration, with histopathological changes of congestion, vacuolation, single cell necrosis, and focal necrosis in the liver; congestion in the spleen; and apoptosis in the thymus cortex. These histopathological changes were not evident following administration of either 60 or 100 nm AgNPs. These results suggested that smaller AgNPs, e.g., those measuring 10 nm in diameter, had higher acute toxicity in mice.

Horseradish extract promotes urinary bladder carcinogenesis when administered to F344 rats in drinking water.

Horseradish extract (HRE), consisting mainly of a mixture of allyl isothiocyanate and other isothiocyanates, has been used as a food additive. To evaluate the potential hazards of HRE, a 104-week chronic study, a 2-week analysis of cell proliferation in the urinary bladder and a medium-term promotion bioassay of HRE were conducted with administration at concentrations of up to 0.04% HRE in the drinking water to male F344 rats. In the 104-week chronic study with 32 male rats per group, no treatment-related increases in the incidences of neoplastic lesions in any organ, including urinary bladder, were observed, except for simple hyperplasia in the urinary bladder in rats treated with HRE at concentrations of more than 0.01% (5.0 mg kg-1 body weight day-1 ). In the promotion study, HRE treatment after N-butyl-N-(4-hydroxybutyl)nitrosamine initiation caused a clear increase in papillary or nodular hyperplasia, papilloma, and urothelial carcinoma of the urinary bladder in the groups given HRE for 13 weeks at doses higher than 0.005%, 0.01%, and 0.04% (2.7, 5.4 and 20.5 mg kg-1 body weight day-1 ), respectively. In the 2-week cell proliferation analysis, treatment with HRE at concentrations greater than 0.005% (3.9 mg kg-1 body weight day-1 ) caused transient increases in 5-bromo-2'-deoxyuridine labeling indices in the urothelium. Although clear tumor induction was not observed, administration of relatively low-dose HRE increased cell proliferation in the urothelium and exerted obvious promoting effects on rat urinary bladder carcinogenesis. Further studies are needed to elucidate the mode of action of HRE in the rat urinary bladder to facilitate data extrapolation from the present study and provide insights into risk assessment. Copyright © 2017 John Wiley & Sons, Ltd.

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.

Anti-Inflammatory Effects of Capsaicin and Piperine on Helicobacter pylori-Induced Chronic Gastritis in Mongolian Gerbils.

BACKGROUND: Spices have been used for thousands of years, and recent studies suggest that certain spices confer beneficial effects on gastric disorders. The purpose of this study was to evaluate possible chemopreventive effects of spice-derived compounds on Helicobacter pylori (H. pylori)-induced gastritis. METHODS: We examined the inhibitory effects of curcumin, capsaicin, and piperine on H. pylori in vitro by determining the colony-forming units and real-time RT-PCR in H. pylori stimulated AGS gastric cancer cells. For in vivo analysis, 6-week-old SPF male Mongolian gerbils were infected with H. pylori, fed diets containing 5000 ppm curcumin, 100 ppm capsaicin, or 100 ppm piperine, and sacrificed after 13 weeks. RESULTS: All three compounds inhibited in vitro proliferation of H. pylori, with curcumin being the most effective. Infiltration of neutrophils and mononuclear cells was suppressed by piperine both in the antrum and corpus of H. pylori-infected gerbils. Capsaicin also decreased neutrophils in the antrum and corpus and mononuclear cell infiltration and heterotopic proliferative glands in the corpus. mRNA expression of Tnf-α and formation of phospho-IκB-α in the antrum were reduced by both capsaicin and piperine. In addition, piperine suppressed expression of Il-1ß, Ifn-γ, Il-6, and iNos, while H. pylori UreA and other virulence factors were not significantly attenuated by any compounds. CONCLUSION: These results suggest that capsaicin and piperine have anti-inflammatory effects on H. pylori-induced gastritis in gerbils independent of direct antibacterial effects and may thus have potential for use in the chemoprevention of H. pylori-associated gastric carcinogenesis.

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.

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.

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.

Absence of in vivo genotoxicity of 3-monochloropropane-1,2-diol and associated fatty acid esters in a 4-week comprehensive toxicity study using F344 gpt delta rats.

3-Monochloropropane-1,2-diol (3-MCPD) is regarded as a rat renal and testicular carcinogen and has been classified as a possible human carcinogen (group 2B) by International Agency for Research on Cancer. This is potentially of great importance given that esters of this compound have recently found to be generated in many foods and food ingredients as a result of food processing. There have been a few reports about their toxicity, although we have recently found that the toxicity profile of 3-MCPD esters was similar to that of 3-MCPD in a rat 13-week repeated dose study, except for the acute renal toxicity seen in 3-MCPD-treated females. In the present study, to examine in vivo genotoxicity we administered equimolar doses of 3-MCPD or 3-MCPD fatty acid esters (palmitate diester, palmitate monoester and oleate diester) to 6-week-old male F344 gpt delta rats carrying a reporter transgene for 4 weeks by intragastric administration. In vivo micronucleus, Pig-a mutation and gpt assays were performed, as well as investigations of major toxicological parameters including histopathological features. As one result, the relative kidney weights of the 3-MCPD and all three ester groups were significantly increased compared with the vehicle control group. However, the frequency of micronucleated reticulocytes and Pig-a mutant red blood cells did not differ among groups. Moreover, no changes were observed in mutant frequencies of gpt and red/gam (Spi(-)) genes in the kidney and the testis of 3-MCPD and 3-MCPD-fatty-acid-esters-treated rats. In histopathological analyses, no treatment related changes were observed, except for decrease of eosinophilic bodies in the kidneys of all treated groups. These results suggest that 3-MCPD and its fatty acid esters are not in vivo genotoxins, although they may exert renal toxicity.