Naohiro Ikeda

Genotoxicity studies on green tea catechin.

The beneficial effects of tea catechins are well documented. We evaluated the genotoxic potential of a green tea catechin preparation using established genotoxicity assays, including a bacterial reverse mutation assay (Ames test), a chromosomal aberration assay in cultured Chinese hamster lung cells (CHL/IU), a mouse lymphoma L5178Y/tk assay, and a bone marrow micronucleus (MN) assay in ICR CD mice and SD rats. No significant increases in the number of revertant colonies were observed in the Ames test, but positive responses were observed in two in vitro assays: the chromosomal aberration assay and mouse lymphoma L5178/tk assay. However, the in vivo study demonstrated no significant increase in micronucleated polychromatic erythrocytes (MNPCE) in the bone marrow of both ICR CD mice and SD rats administered a high dose of the green tea catechin preparation up to 2000mg/kg. Combined with favorable epidemiological information suggesting a chemopreventive effect of tea catechins on carcinogenesis, we conclude that green tea catechin presents no significant genotoxic concern under the anticipated conditions of use. These results are consistent with other genotoxicity studies of tea catechins, which show minimal, if any, genotoxic potential.

Measurement of glycidol hemoglobin adducts in humans who ingest edible oil containing small amounts of glycidol fatty acid esters.

Hemoglobin (Hb) adducts are frequently used to address and/or monitor exposure to reactive chemicals. Glycidol (G), a known animal carcinogen, has been reported to form Hb adducts. Here, we measure G adduct levels in humans who daily ingest DAG oil, an edible oil consisting mainly of diacylglycerol. Since DAG oil contains a small amount of glycidol fatty acid esters (GEs), possible exposure to G released from GEs has been raised as a possible concern. For measurement of Hb adducts, we employed the N-alkyl Edman method reported by Landin et al. (1996) using gas chromatography-tandem mass spectrometry with minor modifications to detect G-Hb adducts as N-(2,3-dihydroxy-propyl)valine (diHOPrVal). Blood samples were collected from 7 DAG oil users and 6 non-users, and then G-Hb adduct levels were measured. G-Hb adducts were detected in all samples. The average level of diHOPrVal was 3.5±1.9pmol/g globin in the DAG oil users and 7.1±3.1pmol/g globin in the non-users. We conclude that there is no increased exposure to G in individuals who daily ingest DAG oil.

Glycidol exposure evaluation of humans who have ingested diacylglycerol oil containing glycidol fatty acid esters using hemoglobin adducts.

Glycidol fatty acid esters (GEs) have been found as impurities in refined edible oils including diacylglycerol (DAG) oil, and concerns of possible exposure to glycidol (G), a known animal carcinogen, during digestion have been raised. We previously measured N-(2,3-dihydroxy-propyl)valine (diHOPrVal), a G hemoglobin adduct, for DAG oil exposed and non-exposed groups and showed there was no significant difference between them. In the present study, we conducted an additional analysis to verify the outcome of the previous report. The first experiment was designed as a matched case-control study to adjust variables with an increased sample size. The average levels of diHOPrVal were 6.9 pmol/g-globin (95%CI: 4.9-9.0) for 14 DAG oil exposed subjects and 7.3 pmol/g-globin (95%CI: 6.1-8.5) for 42 non-exposed volunteers, and no significant difference in levels was found between the two groups. In a second experiment, we compared the adduct levels of 12 DAG oil exposed subjects before and after discontinuing use of DAG oil, and found there was no significant change in diHOPrVal levels (from 7.1±1.1 to 7.5±1.4 pmol/g-globin). These results suggest that there was no increased exposure to G for humans who ingested DAG oil daily, although the evaluated population was limited.

The reconstructed skin micronucleus assay in EpiDerm™: reduction of false-positive results - a mechanistic study with epigallocatechin gallate.

The high rate of false-positive or misleading results in in vitro mammalian genotoxicity testing is a hurdle in the development of valuable chemicals, especially those used in cosmetics, for which in vivo testing is banned in the European Union. The reconstructed skin micronucleus (RSMN) assay in EpiDerm™ (MatTek Corporation, USA) has shown promise as a follow-up for positive in vitro mammalian genotoxicity tests. However, few studies have explored its better predictive performance compared with existing in vitro assays. In the present study, we followed the protocol of the RSMN assay and used eight chemicals to compare micronucleus (MN) induction with EpiDerm™ with that in normal human epidermal keratinocytes (NHEKs), both derived from human skin. The assessments of EpiDerm™ conformed to those of in vivo MN assay, whereas those of NHEKs did not. The effect of cell differentiation status on MN induction was further addressed using a model compound, epigallocatechin gallate (EGCG), which is a major component of green tea extract that shows positive results in in vitro mammalian genotoxicity assays via oxidative stress and negative results in in vivo MN studies. RSMN assay in an underdeveloped epidermal model, EpiDerm-201™ (MatTek Corporation), showed a negative result identical to that in EpiDerm™, indicating that the barrier function of keratinocytes has limited impact. Analysis of the gene expression profile of both EpiDerm™ and NHEKs after EGCG treatment for 12h revealed that the expression of genes related to genotoxic response was significantly induced only in NHEKs. Conversely, antioxidative enzyme activities (catalase and glutathione peroxidase) in EpiDerm™ were higher than those in NHEKs. These results indicate that EpiDerm™ has antioxidant properties similar to those of a living body and is capable of eliminating oxidative stress that may be caused by EGCG under in vitro experimental conditions.

Genotoxicity evaluation of alpha-linolenic acid-diacylglycerol oil

Highlights • We evaluated the genotoxicity of ALA-DAG oil using standard tests.• Bacterial reverse mutation and in vitro/in vivo micronucleus tests were conducted.• No genotoxicity was observed under the testing conditions.

A retrospective evaluation method for in vitro mammalian genotoxicity tests using cytotoxicity index transformation formulae

Although in vitro chromosomal aberration tests and micronucleus tests have been widely used for genotoxicity evaluation, false-positive results have been reported under strong cytotoxic conditions. To reduce false-positive results, the new Organization for Economic Co-operation and Development (OECD) test guideline (TG) recommends the use of a new cytotoxicity index, relative increase in cell count or relative population doubling (RICC/RPD), instead of the traditionally used index, relative cell count (RCC). Although the use of the RICC/RPD may result in different outcomes and require re-evaluation of tested substances, it is impractical to re-evaluate all existing data. Therefore, we established a method to estimate test results from existing RCC data. First, we developed formulae to estimate RICC/RPD from RCC without cell counts by considering cell doubling time and experiment time. Next, the accuracy of the cytotoxicity index transformation formulae was verified by comparing estimated RICC/RPD and measured RICC/RPD for 3 major chemicals associated with false-positive genotoxicity test results: ethyl acrylate, eugenol and p-nitrophenol. Moreover, 25 compounds with false-positive in vitro chromosomal aberration (CA) test results were re-evaluated to establish a retrospective evaluation method based on derived estimated RICC/RPD values. The estimated RICC/RPD values were in good agreement with the measured RICC/RPD values for every concentration and chemical, and the estimated RICC suggested the possibility that 12 chemicals (48%) with previously judged false-positive results in fact had negative results. Our method enables transformation of RCC data into RICC/RPD values with a high degree of accuracy and will facilitate comprehensive retrospective evaluation of test results.

Alpha-linolenic acid-enriched diacylglycerol oil does not promote tumor development in tongue and gastrointestinal tract tissues in a medium-term multi-organ carcinogenesis bioassay using male F344 rat

Alpha-linolenic acid (ALA)-enriched diacylglycerol (DAG) oil is an edible oil enriched with DAG (>80%) and ALA (>50%). The present study investigated whether ALA-DAG oil promotes tumorigenesis in the tongue and gastrointestinal tract, using a rat medium-term multi-organ carcinogenesis bioassay model. Rats were treated with five genotoxic carcinogens to induce multi-organ tumorigenesis until week 4, and from 1 week after withdrawal, fed a semi-synthetic diet (AIN-93G) containing ALA-DAG oil at concentrations of 0, 13,750, 27,500, and 55,000 ppm. Rats fed AIN-93G containing 55,000 ppm ALA-triacylglycerol or a standard basal diet served as reference and negative control groups, respectively. Animals were euthanized at week 30. ALA-DAG oil was shown to have no effects on survival, general condition, body weight, food consumption, or organ weight. More discolored spots were observed in the stomachs of the 13,750- and 55,000-ppm ALA-DAG groups than in those of the control groups; however, there were no differences in the frequency of histopathological findings across groups. There were no meaningful increases in the incidence of pre-neoplastic and neoplastic lesions in the tongue and gastrointestinal tract among the groups. We therefore conclude that ALA-DAG oil does not promote tumor development in the digestive system.

Repeated-dose liver and gastrointestinal tract micronucleus assays with CI Solvent Yellow 14 (Sudan I) using young adult rats.

The in vivo genotoxicity of CI Solvent Yellow 14 (Sudan I) was examined using repeated-dose liver and gastrointestinal tract micronucleus (MN) assays in young adult rats. Sudan I is a mono-azo dye based on aniline and 1-amino-2-hydroxynaphthalene. This dye was demonstrated as a rat liver carcinogen in a National Toxicology Program (NTP) bioassay, and genotoxicity was noted in a rat bone marrow micronucleus (BMMN) assay. In the present study, Sudan I was administered orally to rats for 14-days, and the MN frequency in the liver, stomach, colon, and bone marrow were analyzed. The frequency of micronucleated hepatocytes (MNHEPs) was not significantly increased by the administration of the Sudan I. Gastrointestinal tract MNs were also not induced. However, in the BMMN assay, a significant increase in micronucleated immature erythrocytes (MNIMEs) was observed in a dose-dependent manner. While Sudan I has been reported to lack hepatic genotoxicity, it has also exhibited tumor-promoting activities. These results are consistent with the lack of induction of MN in the hepatocytes. The lack of MN induction in cells of the gastrointestinal tract was also logical because azo-compounds are reported to be unlikely to induce DNA damage in the rat gut. The repeated-dose rat liver and gastrointestinal tract MN assays have the potential to be used in the evaluation of the genotoxicity of a chemical in each organ in accordance with its mode of action.

Effects of dietary alpha-linolenic acid-enriched diacylglycerol oil on embryo/fetal development in rats

Recent studies suggest that diets supplemented with alpha-linolenic acid (ALA)-enriched diacylglycerol (DAG) oil provide potential health benefits in preventing or managing obesity. However, available safety information about reproductive and developmental toxicities of ALA-DAG oil is limited. This study was conducted to clarify the effect, if any, of ALA-DAG oil on embryo-fetal development, following maternal exposure during the critical period of major organogenesis. ALA-DAG oil was administered via gavage to pre-mated female Sprague Dawley rats from gestation day 6 through 19, at dose levels of 0, 1.25, 2.5, and 5.0 mL/kg/day (equivalent to 0, 1149, 2325, and 4715 mg/kg/day, respectively), with total volume adjusted to 5 mL/kg/day with rapeseed oil. All females survived to the scheduled necropsy. There were no treatment-related changes in clinical or internal findings, maternal body weights, feed consumption, intrauterine growth, survival, and number of implantations. No ALA-DAG oil-related fetal malformations or developmental variations were noted. A maternal maximum tolerated dose for ALA-DAG oil could not be achieved in this study. Based on these results, a dose level of 5.0 mL/kg (4715 mg/kg/day), the highest dose tested, was considered as the no-observed-adverse-effect level (NOAEL) for both maternal and developmental toxicity.

A 90-day repeated-dose toxicity study of dietary alpha linolenic acid-enriched diacylglycerol oil in rats

ABSTRACT Diets supplemented with alpha‐linolenic acid (ALA)‐enriched diacylglycerol (DAG) oil—which mainly consists of oleic and linolenic, linoleic acids—have potential health benefits in terms of preventing or managing obesity. Although safety of DAG oil has been extensively investigated, toxicity of ALA‐DAG oil has not been well understood. Hence, the present study was conducted to clarify the potential adverse effects, if any, of ALA‐DAG oil in rats (10/sex/group) fed diets containing 1.375%, 2.75%, or 5.5% ALA‐DAG oil for 90 days. Compared to control rats fed rapeseed oil or ALA‐triacylglycerol oil (flaxseed oil), rats receiving ALA‐DAG oil did not reveal any toxicologically significant treatment‐related changes as evaluated by clinical signs, functional observational battery, body weight, food consumption, ophthalmology, urinalysis, hematology, clinical chemistry, organ weight, necropsy and histopathology. The no observed adverse effect levels for dietary exposure to ALA‐DAG oil for male and female rats were 2916 and 3326mg/kg body weight/day, respectively, the highest dose tested. The findings from this study suggest that consumption of ALA‐DAG oil is unlikely to cause adverse effects. HIGHLIGHTSThis study was investigated to clarify the toxicological potential of ALA‐DAG oil for 90 days.ALA‐DAG oil did not reveal any toxicologically significant treatment‐related changes.The no observed adverse effect levels for male and female rats were 2916 and 3326mg/kg body weight/day, respectively.