Cytogenetic studies on 1,1-dichloroethylene and its two isomers in mammalian cells in vitro and in vivo

Chromosomal aberration and sister-chromatid exchange (SCE) tests in vitro on 1,1-dichloroethylene (1,1-DCE), its two isomers, cis- and trans-1,2-DCE, and two possible metabolites of 1,1-DCE, chloroacetyl chloride and chloroacetic acid, were carried out using a Chinese hamster cell line, CHL. 1,1-DCE induced chromosomal aberrations in the presence of S9 mix prepared from the rat liver, but not in the absence of S9 mix. SCEs were also slightly induced by 1,1-DCE only in the presence of S9 mix. On the other hand, two isomers and two metabolites of 1,1-DCE induced neither chromosomal aberrations nor SCEs with and without S9 mix. 1,1-DCE, however, was negative even at a sublethal dose in the micronucleus test using mouse bone marrow, fetal liver and blood.     

Negative micronucleus tests on caprolactam and benzoin in ICR/JCL male mice

Male ICR/JCL mice were given a single intraperitoneal injection of 125, 250, and 500 mg/kg of caprolactam (CAP), or 250, 500, and 1000 mg/kg of benzoin (ZOIN). Bone marrow preparations were made 24, 30, and 48 h after treatment with the maximum dose, and 30 h after treatment with the other doses. The slides were coded before microscopic examination. No significant increase was found in the incidence of micronucleated polychromatic erythrocytes after treatment with either CAP or ZOIN.     

A sensitive method for the detection of mutagenic nitroarenes: construction of nitroreductase-overproducing derivatives of Salmonella typhimurium strains TA98 and TA100

'Classical nitroreductase' is an enzyme involved in the intracellular metabolic activation of mutagenic nitroarenes. The nitroreductase gene of Salmonella typhimurium TA1538 was cloned into pBR322 and the plasmids harboring the gene were introduced into TA98 and TA100. The resulting strains (YG1021 and YG1026) had more than 50 times higher nitrofurazone-reductase activity than TA1538 containing pBR322, and were extremely sensitive to the mutagenic action of 2-nitrofluorene, 1-nitropyrene and 2-nitronaphthalene. These results indicate that the new strains permit the efficient detection of mutagenic nitroarenes.     

Localization and possible function of 20 kDa actin-modulating protein (actolinkin) in the sea urchin egg

We have previously described a novel actin-capping protein, a 20,000-molecular weight protein (20K protein)-actin complex (20K-A) isolated from sea urchin eggs. In the present study, the localization and possible function of this 20K protein were investigated. The 20K protein was localized in the sea urchin egg cortex. Its distribution in the cortex as revealed by immunofluorescence microscopy did not change during or after fertilization up to the first mitosis, but it was concentrated to some extent in the cleavage furrow region. Exogenously added actin polymerized on the cortex isolated from unfertilized egg; however, actin did not polymerize on the cortex extracted with 0.6 M KCl, that is, the cell membrane, which lost the 20K protein. The cell membrane preincubated with 20K-A restored the activity to grow actin filaments. When decorated with myosin subfragment 1, almost all the actin filaments showed the arrowhead configuration pointing away from the membrane, indicating that they were connected to the membrane at their barbed ends. These results strongly suggest that the 20K protein connects actin filaments to the plasma membrane of sea urchin eggs. Because of this property we call this protein "actolinkin".     

Decreased clastogenicity of dinitropyrenes in Chinese hamster lung (CHL) subclone cells with low NADPH-cytochrome P-450 reductase activity

Clastogenic potentials of 1,3-, 1,6- and 1,8-dinitropyrenes (DNPs) were compared between Chinese hamster lung (CHL) cells and its subclone MM1 cells, which were recently isolated as menadione-resistant cells after treatment with MNNG. NADPH-cytochrome P-450 reductase activity of the MM1 cells decreased to 50% of that in the parental CHL cells. All 3 DNPs induced chromosomal aberrations without exogenous metabolic activation systems in the CHL cells. 1,6- and 1,8-DNP showed equivalent clastogenic potency: the maximum frequency of cells with chromosomal aberrations was about 50% for both chemicals. The clastogenic potential of 1,3-DNP was lower than that of 1,6- and 1,8-DNP: the maximum frequency of aberrant cells was 10%. In the MM1 cells, in contrast, the frequencies of aberrant cells decreased to about 30% of those observed for the parental CHL cells after treatment with 1,6- and 1,8-DNP, and to the same level as that of the concurrent control after treatment with 1,3-DNP. These results suggest a possibility that the reduced clastogenic effect of 3 DNPs in MM1 cells may correlate with the reduced activity of NADPH-cytochrome P-450 reductase which is thought to contribute to the metabolic conversion of these DNPs to their clastogenic forms in the CHL cells.     

Mutagenicity of 30 chemicals in Salmonella typhimurium strains possessing different nitroreductase or O-acetyltransferase activities

Salmonella typhimurium YG1021, YG1024, YG1026 and YG1029 are new derivatives of the Ames tester strains TA98 and TA100, with elevated 'classical' nitroreductase or acetyl-CoA:N-hydroxyarylamine O-acetyltransferase level. Thirty mutagens with different structures were tested using these strains and the sensitivities were compared with those of the conventional strains and of the enzyme-deficient strains. Elevated O-acetyltransferase activity of the indicator strains specifically increased their ability to detect the mutagenicity of aromatic nitro, amino and hydroxylamino compounds, whereas the strains with high nitroreductase activity were very sensitive to some nitroaromatics. The combined use of the isogenic tester strains with different metabolic capacities was quite useful to assess the intracellular metabolic activation and detoxification mechanisms of chemical mutagens.     

Kinetics of micronucleus formation in relation to chromosomal aberrations in mouse bone marrow

A simulation analysis of the kinetics of micronucleus formation in polychromatic erythrocytes in mouse bone marrow was performed after a single administration of 3 chemicals--mitomycin C (MMC), 6-mercaptopurine (6-MP) and 1-beta-D-arabinofuranosylcytosine (Ara-C)--with different modes of action. The time-response patterns in the incidence of chromosomal aberrations and micronuclei after treatment with each chemical were compared and subjected to the simulation study with 3 parameters. Two of them, the time between the final mitotic metaphase of the erythroid series and nucleus expulsion (T1), and the duration of the polychromatic erythrocyte (PCE) stage in the bone marrow (T2), were almost identical for the 3 chemicals. However, the coefficients of formation rate of micronucleated cells resulting from cells with chromosomal aberration(s) (k) differed: Ara-C differed from the other two. These results indicate that chromosomal aberrations, especially chromatid breaks and probably gaps, induced by this chemical, effectively contribute to micronucleus formation. The DNA content of micronuclei was also compared to the length of acentric fragments induced by Ara-C and it was found that their distributions were comparable. These findings strongly suggest that chromosomal aberrations induced by chemicals are essential events for the induction of micronuclei in the PCE of bone marrow.