Takashi Mikami

Comparison between in vivo mutagenicity and carcinogenicity in multiple organs by benzo[a]pyrene in the lacZ transgenic mouse (Muta Mouse).

To evaluate whether the in vivo mutagenicity test system using the lacZ transgenic mice (Muta Mouse) may be applied to carcinogenesis studies, both the in vivo mutagenicity and carcinogenicity of benzo[a]pyrene (BP) was tested in mice under the same administration conditions. The eleven organs of the mice on the 14th day after the final oral administration of BP at a dose of 125 mg kg(-1) day(-1) or corn oil for 5 consecutive days were tested for in vivo mutation by the positive-selection method. The data show that the colon had the highest lacZ mutant frequency (37-fold increase over the spontaneous frequency), followed by the ileum > forestomach > bone marrow, spleen > glandular stomach > liver, lung > kidney and heart. No significant mutations were found in the brain. These results may suggest that, in general, the organs with rapidly proliferative tissues have a marked increase in vivo mutant frequencies under the conditions of this experimental design. The forestomach and lymphatic organs including the spleen (malignant lymphoma) were the main target organs for BP carcinogenesis by 5 daily oral doses of 75 and 125 mg kg(-1) day(-1). These results suggest that the mutation results from the transgenic assay with BP reflect the carcinogenicity of BP in the mouse. They also indicate, however, that the magnitude of the in vivo lacZ mutant frequencies induced by BP in different organs did not fully correlate with the target organs for carcinogenicity.

Comparison of the mutational spectra of the lacZ transgene in four organs of the Muta™Mouse treated with benzo[a]pyrene: target organ specificity

We recently demonstrated that not all organs with a high rate of induction of mutation in the lacZ transgene develop tumors in the lambdalacZ transgenic mice (MutaMouse) used for a long-term carcinogenicity study with benzo[a]pyrene (BP). To better understand the role of chemical-induced in vivo mutations in carcinogenesis, we compared the mutational spectra of the lacZ transgene in four organs of the MutaMouse obtained 2 weeks after five daily consecutive oral treatments with 125 mg/kg/day BP. lacZ transgenes were analyzed in two target organs (forestomach and spleen) and two non-target organs (colon and glandular stomach) for BP-induced carcinogenesis in MutaMouse, and all of these organs were highly mutated in the lacZ transgene. The sequence data showed similar mutational spectra of the lacZ transgene between the two target organs; the predominant mutations were G:C-->T:A transversions (55% and 50% for forestomach and spleen, respectively), followed by deletions (20% and 21% for forestomach and spleen, respectively) mainly at G:C site. The frequent G:C-->T:A transversions are consistent with reports of the mutational spectra produced in the p53 gene in tumors generated in rats and mice exposed to BP. In contrast, the mutational spectra of the lacZ transgene in the two non-target organs are different from those in the target organs, and are also suggested to differ from one another. These findings suggest an organ/tissue-specific mechanism of mutagenesis.

Multiple organ mutation in the lacZ transgenic mouse (Muta mouse) 6 months after oral treatment (5 days) with benzo[a]pyrene.

We have recently demonstrated that not all organs with high rates of mutation in the lacZ transgene develop tumors using the Muta Mouse. To better understand the role of in vivo mutation in carcinogenesis, we examined the mutant frequencies (MF) of the lacZ transgene in tumor-bearing and non tumor-bearing organs. MF, recovered after 2 weeks (the data taken from our previous study) and after 26 weeks following oral doses of 125 mg kg-1 day-1 benzo[a]pyrene (BP) for five days were compared. The organs examined included the target organs (forestomach, spleen, and lung) and non-target organs (colon, glandular stomach, and liver) for BP carcinogenesis. The data indicated that lacZ MF were markedly increased over spontaneous frequencies in the organs examined and that the organ which showed the highest MF was the colon, followed by the forestomach>spleen>glandular stomach, liver, and lung in that order. These findings indicate that the MF of the lacZ transgene in each organ, even 26 weeks after the start of the treatment does not fully correlate with the known target organs of BP. Furthermore, the lacZ MF in a non-papilloma region of a forestomach with a papilloma was equivalent to the two highest MF observed in the healthy colon (non-target organ) of mice at 26 weeks. These observations also indicate that the generation of tumors requires the induction of mutations as well as other factor(s) specific to the target organs. These results clearly suggest that highly mutated organs do not always progress to tumors in the transgenic mouse.

Mutagenicity of dihydroxybenzenes and dihydroxynaphthalenes for Ames Salmonella tester strains.

The mutagenicity of 3 dihydroxybenzene (DHB) and 9 dihydroxynaphthalene (DHN) isomers was examined by using 5 different Ames Salmonella mutagenicity tester strains in the presence and absence of phenobarbital and 5,6-benzoflavone-treated rat liver S9-mix. Of the 3 DHB isomers, 1,4-DHB (hydroquinone) was mutagenic, and of the 9 DHN isomers, 1,3-DHN (naphthoresorcinol), 1,4-DHN (hydronaphthoquinone), 1,6-DHN and 1,7-DHN were mutagenic. Mutagenicity of all the compounds tested was observed in the absence of S9-mix, while 1,4-DHN and 1,6-DHN were also mutagenic in the presence of S9-mix. The mutagenicity of 1,4-DHB and 1,4-DHN for TA104, which is a strain sensitive to oxidative mutagens, was almost completely or partially inhibited by superoxide dismutase (SOD) and/or catalase, indicating the involvement of activated oxygen species in mutagenesis. Furthermore, from the finding that the 4 DHNs were mutagenic for TA2637, the strain sensitive to frameshift mutagens, it is possible that the mutagenicity of DHNs for S. typhimurium was also attributable to DNA adducts that form with quinones and/or semiquinones through oxidation of DHNs. The mutagenicity of 1,3-DHN, which showed the largest number of revertants in strains TA100, TA98, TA2637 and TA104, was greatly decreased, when their pKM101 plasmid-deficient strains, TA1535, TA1538, TA1537 and TA2659 were used. This observation suggests that an SOS repair system was involved in the mutagenesis of 1,3-DHN for S. typhimurium.

Morphogenetic process of dysraphic malformations induced by thalidomide in JW‐NIBS rabbits

Abstract: Our previous study demonstrated thalidomide‐induced anencephaly and holoprocencephaly at a high frequency in JW‐NIBS rabbits. In order to know the early morphogenetic process of these two malformations, thalidomide was orally given to pregnant JW‐NIBS rabbits on day 7 or on days 6–9 of gestation, and the embryos were examined on days 9–13 in histological sections and scanning electronmicroscopic specimens.