Transgene mutagenesis in the testicular cells of Muta™Mouse treated transplacentally with N-ethyl-N-nitrosourea at the primordial germ cell stages: Comparisons with the specific-locus test and the intragenic gene-recombination assay.

N-Ethyl-N-nitrosourea (ENU) induces recessive mutations (RM) at a high frequency in male mouse primordial germ cells (PGCs)　in a dose-dependent and stage-specific manner when administered during embryonic development as confirmed by a specific locus test (SLT) (Shibuya et al., 1993, 1996 [1,2]). ENU also induces intragenic recombination (IGR) in the pun allele at E10.5 in PGCs of male mice (Shibuya et al., 2022 [3]). In this study, the induced mutant frequencies (MF) in testicular cells of male Muta™Mousetreated at the same developmental stages of PGCs were determined with a positive selection system (MM/PS). Although the mutant frequencies　of MM/PS were consistently lower than for the SLT/RM, they showed similar stage-specificity and dose-dependency. Expressed as a linear equation, the correlation coefficient on the MF from SLT and MM/PS was extremely high (r2 = 0.920).

Intragenic recombination within the pun allele of the pink-eyed dilution locus in pre-melanocytes and primordial germ cells of embryonic mice treated with N-ethyl-N-nitrosourea.

The forward or reverse processes of intragenic recombination (IGR), which occur through the addition or deletion of duplicated homologous exons of the pun allele in Pun mice, was observed in vivo, after introducing an homozygous pun allele in a C57BL/6 background. We assessed the frequency of IGR upon N-ethyl-N-nitrosourea (ENU) treatment of pre-melanocytes (PMCs: somatic cells) and primordial germ cells (PGCs: germ cells) of embryonic mice at 10.5 days of development (E10.5). We simultaneously examined IGR and other mutations at the p locus of PMCs responsible for coat color in the offspring obtained by crossing pun/pun with pun/P mice. The frequencies of both spontaneous and ENU-induced IGR were markedly higher than that of the recessive mutation (RM) in PMCs obtained from crossing C57BL/6 and PW strains (Shibuya et al., 1982). ENU also induces IGR at a higher frequency in PGCs at E10.5, which was observed in the next generation. These results indicate that ENU, which preferentially induces gene mutations through base substitution, also induces IGR at a high frequency in the pun allele in both somatic and germ cells of embryonic mice at the E10.5 developmental stage.

Chemical category approach of genotoxicity studies for branched alkylphenols.

A chemical category is a group of chemicals whose toxicological properties are expected to be similar or follow a regular pattern as a result of structural similarity. The category approach is beneficial for decreasing in the resource of risk assessment for huge amount of unevaluated existing chemicals, and also in the use of all kinds of animal tests including even in vivo genotoxicity tests from a point of view of the animal welfare. The present paper reports the results of in vivo micronucleus tests of o-sec-butylphenol (CAS:89-72-5) and 2-isopropyl-5-methylphenol (CAS:89-83-8) and discusses genotoxic potential of seven alkylphenols, o-sec-butylphenol, 2-isopropyl-5-methylphenol, p-sec-butylphenol (CAS:99-71-8), 2-tert-butylphenol (CAS:88-18-6), 2, 4-di-tert-butylphenol (CAS:96-76-4), 4-tert-butylphenol (CAS:98-54-4) and 6-tert-butyl-m-cresole (CAS:88-60-8) by the category approach. Based on the negative results of in vivo micronucleus tests, it can be concluded that these category chemicals are not likely clastogenic in vivo. Further in vivo micronucleus assays on untested substances may not be required by using the category approach, but further supporting information such as physicochemical profiles and (Q) SAR predictions may be necessary to strengthen the rationale for the category approach.

Photo-reactive polyvinylalcohol for photo-immobilized microarray.

A new photo-reactive polymer, polyvinylalcohol modified with phenylazido groups, was synthesized as a microarray matrix. The polymer is soluble in water and spin-coated onto glass plate. Aqueous solutions of proteins were micro-spotted onto the coated glass and were fixed by ultraviolet light irradiation. Subsequently, cell adhesion on the photo-immobilized protein microarray was investigated. Non-specific adhesion of cells onto non-protein-spotted regions was reduced in comparison with the previously prepared microarray chip (Biomaterials 24 (2003) 3021). The adhesion behavior of cells depended on the kind of immobilized proteins and the type of cells. The microarray will be useful for cell diagnosis and for the selection of biomaterials to regulate cell behavior.