Multiple-endpoint genotoxicity assay for colon carcinogen 1,2-dimethylhydrazine.

Human risk assessment of the toxic potency of chemicals typically includes genotoxicity assays for predicting carcinogenicity. Gene mutation frequency and chromosomal aberration are two major genotoxicity endpoints in standardized in vitro and in vivo assays. The weight-of-evidence approach in risk assessment is more focused on in vivo assay results; however, animal welfare considerations are aimed at the reduction, replacement, and refinement (3R's) of animal experiments, including a reduction in the number of experimental animals. Proposals to reduce experimental animals in genotoxicity testing include the incorporation of genotoxicity endpoint(s) into other toxicological studies and the combination of two or more assays detecting different genotoxicity endpoints in the same animals. In this study, we used 1,2-dimethylhydrazine as a model chemical of colon carcinogen to assess gene mutation frequency and chromosomal aberration in vivo simultaneously. Specifically, a gene mutation frequency assay was combined with a multiple-organ micronucleus test (peripheral blood, bone marrow, liver, and colon) in F344 gpt delta transgenic rats. Both gpt mutant frequency and micronucleated cell frequency significantly increased in colon and liver but not in bone marrow. Interestingly, we found that the colon carcinogen induced both gene mutations and micronuclei in the targeted colon tissue. Thus, we demonstrated that the mechanism of a carcinogen could be derived from an animal experiment using a lower number of experimental animals as currently recommended. Moreover, a significant increase in mutant frequency in colon and liver was already observed on the first day after treatment completion, as well as on the third day, which is the guideline-recommended period. Thus, this endpoint is compatible with other genotoxicity assays. We confirmed that performing the micronucleus assay in combination with a gene mutation assay in F344 gpt delta transgenic rats is useful to evaluate different genotoxic endpoints simultaneously in the same animals, which reduces the number of experimental animals.

Target-specific micronucleus induction by colon carcinogens: 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 1,2-dimethylhydrazine.

Genotoxicity occurring at the target organs of carcinogenesis is important for understanding the mechanisms of chemical carcinogenicity and also for setting of threshold estimation. In vivo gene mutations have been evaluated by transgenic animal models in which any organ can be targeted; however, the methodologies that have been applied to assess chromosomal aberrations including micronucleus induction, are organ restricted, (often to bone marrow hematopoietic cells, as a common example). For food and food-related chemicals, the digestive tract is the important target organ as it is the organ of first contact. In the present study, we used 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 1,2-dimethylhydrazine (DMH) as model chemicals of carcinogens primarily targeting the colon. We evaluated the applicability of colon cells and hepatocytes, together with bone marrow cells, in the micronucleus assay. Both model chemicals induced micronuclei in the colon, which is the target organ of these carcinogens, after short- and long-term treatment(s). The results demonstrate the target specificity of micronucleus induction and the assay using organs other than bone marrow will play an important role in understanding the mechanism of carcinogenicity and predicting new carcinogenic agents.

Sesame Lignans Suppress Age-Related Cognitive Decline in Senescence-Accelerated Mice.

Sesame lignans, which are biologically active compounds present in sesame seeds and oil, are known to have neuroprotective effects in several models of brain dysfunction. However, the effects of sesame lignans on age-related brain dysfunction are not clear and were thus investigated in the present study using a senescence-accelerated mouse (SAMP10). Two-month-old male SAMP10 mice were administrated a basal diet with 0% or 0.05% sesame lignans for two months, or with 0%, 0.02%, or 0.05% sesame lignans for 10 months and subjected to step-through passive avoidance tasks and forced swim tests. Reactive carbonyl species (RCs) were evaluated as markers of oxidative stress using a recently developed comprehensive analytical method. Both learning time in passive avoidance tasks and immobile time in forced swim tests became longer with aging (p < 0.05). However, the administration of sesame lignans significantly ameliorated age-related effects in both tests (p < 0.05). Age-related increases in RCs such as 4-hydroxy-2-nonenal in the cerebral cortex and liver were reduced in mice fed sesame lignans. These results suggest that sesame lignans can prevent age-related brain dysfunction via anti-oxidative activity.

Integration of micronucleus tests with a gene mutation assay in F344 gpt delta transgenic rats using benzo[a]pyrene.

Reduction of the number of animals used in in vivo genotoxicity tests is encouraged. For this purpose, we conducted integrated toxicity tests combining gene mutation assays with multiple-organ micronucleus (MN) tests (peripheral blood, bone marrow, liver, and colon) in F344 gpt delta transgenic (Tg) rats. Seven-week-old male F344 gpt delta rats were orally administered 62.5 or 125 mg/kg/day benzo[a]pyrene (B[a]P) for 28 days. One day after the final day of treatment (day 29) and three days after the final treatment (day 31), bone marrow, liver, and colon samples were collected, and mutation assays and MN tests were performed. The gpt mutant frequency (MF) significantly increased in bone marrow, liver and colon but MN induction was only significant in bone marrow but not in liver and colon. Similarly MN induction was only observed in bone marrow in non-Tg F344 rats. In peripheral blood obtained on day 4, 15, 29, 31, a time-dependent increase was observed in reticulocyte MN frequency during the treatment. Thus, our integrated method successfully detected both gene mutations and MN induction caused by B[a]P. In addition, no significant differences were observed between sampling times (day 29 versus 31), suggesting that sampling on day 29 is also valid to evaluate gene mutations. On the other hand, MN results in bone marrow and peripheral blood were different depending on the sampling day. An appropriate sampling day should be designated according to which assays are integrated. We confirmed that integration of the MN test with a gene mutation assay using F344 gpt delta Tg rats is useful to evaluate different endpoints related to genotoxicity using the same animals and to reduce animal use.

Genotoxicity evaluation of sesamin and episesamin.

Sesamin is a major lignan that is present in sesame seeds and oil. Sesamin is partially converted to its stereoisomer, episesamin, during the refining process of non-roasted sesame seed oil. We evaluated the genotoxicity of these substances through the following tests: a bacterial reverse mutation assay (Ames test), a chromosomal aberration test in cultured Chinese hamster lung cells (CHL/IU), a bone marrow micronucleus (MN) test in Crlj:CD1 (ICR) mice, and a comet assay using the liver of Sprague-Dawley (SD) rats. Episesamin showed negative results in the Ames test with and without S9 mix, in the in vitro chromosomal aberration test with and without S9 mix, and in the in vivo comet assay. Sesamin showed negative results in the Ames test with and without S9 mix. In the in vitro chromosomal aberration test, sesamin did not induce chromosomal aberrations in the absence of S9 mix, but induced structural abnormalities at cytotoxic concentrations in the presence of S9 mix. Oral administration of sesamin at doses up to 2.0g/kg did not cause a significant increase in either the percentage of micronucleated polychromatic erythrocytes in the in vivo bone marrow MN test or in the % DNA in the comet tails in the in vivo comet assay of liver cells. These findings indicate that sesamin does not damage DNA in vivo and that sesamin and episesamin have no genotoxic activity.

A novel mouse model for type 2 diabetes and non-alcoholic fatty liver disease: spontaneous amelioration of diabetes by augmented beta cell mass.

Given the potential for beta-cells to increase their mass, glucose intolerance might be ameliorated by a compensatory increase in beta-cell mass. However, it remains uncertain whether such amelioration is feasible in vivo. In this study, we investigated glucose tolerance, islet morphology, and islet gene expression of Fatty Liver Shionogi (FLS) mice, a model for non-alcoholic fatty liver disease (NAFLD). Relative to control mice, FLS mice showed an age-dependent increase in glucose intolerance up to the age of 24 weeks, leading to the development of diabetes. After this time, glucose tolerance ameliorated spontaneously and diabetes resolved by 48 week of age, associated with marked hyperinsulinemia. Islets of the FLS mice demonstrated a marked increase in beta-cell mass with an increase in beta-cell numbers. Islet gene expression analysis in FLS mice demonstrated no changes in gene expression of glucokinase or insulin receptor substrate 2. These data demonstrated that the 24-week-old FLS mouse is a model for type 2 diabetes with NAFLD and that the 48-week-old FLS mouse exhibits spontaneous amelioration of type 2 diabetes associated with augmented beta-cell number/mass.