Hajime Sui

Evaluation of the repeated-dose liver and gastrointestinal tract micronucleus assays with 22 chemicals using young adult rats: summary of the collaborative study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/The Japanese Environmental Mutagen Society (JEMS) - Mammalian Mutagenicity Study Group (MMS).

The repeated-dose liver micronucleus (RDLMN) assay using young adult rats has the potential to detect hepatocarcinogens. We conducted a collaborative study to assess the performance of this assay and to evaluate the possibility of integrating it into general toxicological studies. Twenty-four testing laboratories belonging to the Mammalian Mutagenicity Study Group, a subgroup of the Japanese Environmental Mutagen Society, participated in this trial. Twenty-two model chemicals, including some hepatocarcinogens, were tested in 14- and/or 28-day RDLMN assays. As a result, 14 out of the 16 hepatocarcinogens were positive, including 9 genotoxic hepatocarcinogens, which were reported negative in the bone marrow/peripheral blood micronucleus (MN) assay by a single treatment. These outcomes show the high sensitivity of the RDLMN assay to hepatocarcinogens. Regarding the specificity, 4 out of the 6 non-liver targeted genotoxic carcinogens gave negative responses. This shows the high organ specificity of the RDLMN assay. In addition to the RDLMN assay, we simultaneously conducted gastrointestinal tract MN assays using 6 of the above carcinogens as an optional trial of the collaborative study. The MN assay using the glandular stomach, which is the first contact site of the test chemical when administered by oral gavage, was able to detect chromosomal aberrations with 3 test chemicals including a stomach-targeted carcinogen. The treatment regime was the 14- and/or 28-day repeated-dose, and the regime is sufficiently promising to incorporate these methods into repeated-dose toxicological studies. The outcomes of our collaborative study indicated that the new techniques to detect chromosomal aberrations in vivo in several tissues worked successfully.

Partial hepatectomy strongly increased the mutagenicity of N-ethyl-N-nitrosourea in Muta™Mouse liver

The relationship between cell proliferation and mutagenesis can be investigated in vivo due to the advent of transgenic animal mutation assays. In these assays, slowly proliferating tissues, such as mammary gland and liver, that are exposed to mutagens generally have longer manifestation times for mutations and lower mutant frequencies. This may be because the cells have enough time prior to cell division to repair DNA damage. We carried out this study using the Muta™Mouse positive selection system to investigate the effect of a high rate of cell proliferation induced by partial hepatectomy (PH) on mutation induction. We used a 2 × 2 experimental design for PH (or no PH) and 50 mg/kg N‐ethyl‐N‐nitrosourea (ENU) (or phosphate buffer), with the chemical injected 16–19 hr after PH. In the non‐ENU groups, the mean MF was slightly but not significantly higher in the PH group than in the non‐PH group. In the ENU non‐PH group, the MF was also slightly but not significantly increased. In the ENU PH group, in contrast, the mean MF was 7 times the mean MF of the group that received either treatment alone. These results strongly support the hypothesis that ENU induced pre‐mutational DNA lesions in liver are completely repaired prior to cell division, and PH increases the mutagen‐induced MF by reducing the amount of time available for such repair. Environ. Mol. Mutagen. 34:121–123, 1999

Mutation induction by N-propyl-N-nitrosourea in eight Muta™Mouse organs

As a part of the 2nd Collaborative Study for the Transgenic Mouse Mutation Assay, we studied the organ specificity and the temporal changes in mutant frequency (MF) of the lacZ gene following intraperitoneal injection of 250 mg/kg N-propyl-N-nitrosourea into male MutaMouse. We used a positive selection system and examined eight organs, i.e., bone marrow, liver, kidney, lung, spleen, brain, heart, and testis. The chemical caused a significant increase in MF in all organs except for brain, and the bone marrow was the most sensitive organ, exhibiting a MF on day 7 that was 10 times that of the control. The MF increased from day 7 to day 28 in liver, kidney, and testis, while it decreased in bone marrow. The relationship between the results of this study and the target organs of carcinogenesis, and the cause of the temporal changes in MF, are discussed.

Effects of O (6)-alkylguanine-DNA alkyltransferase deficiency in Escherichia coli as the host for the detection of mutations in lacI transgenic mice.

Transgenic mice are widely used to detect gene mutations in vivo induced by a variety of chemicals. It is known, however, that no mutagenicity of methyl methanesulfonate (MMS) is detected in epididymal sperm in various transgenic mice assays, although MMS induces the dominant lethal and specific locus mutations in male mice. To investigate the issue of whether unrepaired lesions in DNA of mature sperm can be transformed into mutations during replication of the lambda phage in Escherichia coli cells, we developed an E. coli strain YG5152, which is a derivative of strain SCS‐8 but is deficient in the genes encoding O 6‐alkylguanine‐DNA alkyltransferases. When lambda LIZα phages were treated with MMS or N‐ethyl‐N‐nitrosourea (ENU) in vitro and infected to the E. coli strains, the mutant frequencies of lacI were markedly higher in strain YG5152 than in strain SCS‐8. When Big Blue™ mice were treated with MMS (160 mg/kg) or ENU (125 or 250 mg/kg) and the phages rescued from mature sperm were infected to the strains, the mutation frequency (MF) of phages from ENU‐treated mice at a dose of 250 mg/kg in strain YG5152 was about two times higher than that in strain SCS‐8. However, no increase in the MF was observed in the MMS‐treated mice even in strain YG5152. These results suggest that, although strain YG5152 efficiently detects ex vivo mutations caused by mutagenic alkyl adducts formed by MMS in lambda phage DNA, no detectable levels of mutagenic methyl adducts are present in mature sperm of MMS‐treated mice. Possible reasons for this lack of mutagenicity of MMS in mature sperm using transgenic mice assays are discussed. Environ. Mol. Mutagen. 34:221–226, 1999

In vivo comet assay of acrylonitrile, 9-aminoacridine hydrochloride monohydrate and ethanol in rats

As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the in vivo rat alkaline comet assay, we examined the ability of acrylonitrile, 9-aminoacridine hydrochloride monohydrate (9-AA), and ethanol to induce DNA damage in the liver and glandular stomach of male rats. Acrylonitrile is a genotoxic carcinogen, 9-AA is a genotoxic non-carcinogen, and ethanol is a non-genotoxic carcinogen. Positive results were obtained in the liver cells of male rats treated with known genotoxic compounds, acrylonitrile and 9-AA.

Genotoxic potential and in vitro tumour-promoting potential of 2-dodecylcyclobutanone and 2-tetradecylcyclobutanone, two radiolytic products of fatty acids.

The DNA-damaging and tumour-promoting effects of two 2-alkylcyclobutanones (2-ACBs), which are found in irradiated fat-containing foods, were investigated by use of the comet assay and in an azoxymethane (AOM)-induced colon-carcinogenesis study in rats, respectively. We conducted genotoxicity tests of 2-dodecylcyclobutanone (2-dDCB) and 2-tetradecylcyclobutanone (2-tDCB) according to the test guidelines for chemicals or drugs. In addition, a cell-transformation assay with Bhas 42 cells was performed to investigate their promoting potential in vitro. The Salmonella typhimurium mutagenicity assay (Ames test), conducted with five tester strains, revealed that neither 2-dDCB nor 2-tDCB possessed mutagenic activity. Moreover, both in the in vitro chromosomal aberration test on CHL/IU cells and the in vivo bone-marrow micronucleus test where mice were given 2-dDCB and 2-tDCB (orally, up to 2000 mg/kg bw/day), we did not detect any clastogenic effects. Furthermore, DNA strand-breaks were not detected in the in vitro comet assay with CHL/IU cells, and DNA adducts derived from 2-dDCB and 2-tDCB were not detected in the colon tissues of the mice used for the micronucleus tests, in rats from a repeated dose 90-day oral toxicity test (0.03% 2-tDCB in the diet), or in rats from the AOM-induced carcinogenesis study (0.025% 2-tDCB in the diet). An in vitro tumour-promotion assay with Bhas 42 cells revealed that the number of transformed foci increased significantly following treatment of cells in the stationary phase with 2-dDCB or 2-tDCB for 10 days. Our results indicate that neither 2-dDCB nor 2-tDCB were genotoxic chemicals. However, they exhibited promoting activity, at least in vitro, when Bhas 42 cells were continuously exposed to these chemicals at toxic doses.

Evaluation of in vivo genotoxicity by thioacetamide in a 28-day repeated-dose liver micronucleus assay using male young adult rats

The repeated-dose liver micronucleus (RDLMN) assay has the potential to detect liver carcinogens and can be integrated into general toxicological studies. In this study, thioacetamide (TAA) was tested in 14- and 28-day RDLMN assays to assess the performance of the assay. The test substance, TAA, was administered orally to 6-week-old male Crl:CD (SD) rats once daily for 14 or 28 days at a dosage of 5, 10 or 20mg/kg/day. Hepatocytes were collected approximately 24h after the last TAA administration, and the incidence of micronuclei was assessed. In this study, bone marrow micronucleus assays were also conducted in the same animals. The 14- and 28-day RDLMN assays indicated that none of the TAA dosages significantly increased the proportion of micronucleated hepatocytes. Bone marrow micronucleus assays with TAA also provided negative results. It is known that TAA is a liver carcinogen in mice and rats. In the previous genotoxic studies, the Ames test and the chromosomal aberration test using CHL/IU cells have yielded negative results [1-4]. The liver micronucleus assay using young adult rats singly dosed with TAA (75 and 150mg/kg) also produced negative results [5]. TAA gave positive results only in the mouse bone marrow micronucleus assays [6,7].

Evaluation of the repeated-dose liver micronucleus assay with p-dimethylaminoazobenzene.

The micronucleus induction by p-dimethylaminoazobenzene (DAB), a genotoxic rat liver carcinogen, was assessed in the liver and bone marrow of young adult rats after the repeated administration of DAB for 14 (Lab. 1) and 28 (Lab. 2) days. Three dose levels, 25, 50 and 100mg/kg/day, were used for the investigations in both labs. The frequency of micronucleated hepatocytes was significantly increased in a dose-dependent manner after the repeated administration of DAB at 50mg/kg/day or more for 14 and 28 days. Similarly, the frequency of micronucleated immature erythrocytes in the bone marrow was increased after the repeated administration of DAB at 100mg/kg/day for 14 and 28 days. These results indicate that the repeated-dose liver micronucleus assay allowed for the detection of micronucleus induction by DAB, and that the lowest detectable dose for micronucleus induction in the liver was lower than in the bone marrow. Thus, the repeated-dose liver micronucleus assay using young adult rats is considered suitable for the detection of micronucleus induction by liver carcinogens, such as DAB.