Naomichi Inui

Transplacental action of sodium nitrite on embryonic cells of Syrian golden hamster

Hamster embryos were treated with various doses of NaNO2 in utero, by its oral administration to the mothers, and then the embryonic cells were examined for micronucleus formation, chromosomal aberrations, morphological or malignant transformation and drug-resistant mutations. For induction of resistant mutations, the cells were cultured in normal medium for 72 h, and then selected in media containing 8-azaguanine (10 or 20 microgram/ml) or 1 mM ouabain. This treatment with NaNO2 caused marked dose-dependent induction of 8-azaguanine- and ouabain-resistant mutations. Cultured embryonic fibroblasts in the resting state also showed a marked dose-dependent increase in micronucleus formation but not an increase in chromosomal aberrations. This treatment also caused morphological and neoplastic transformation of the cells. Transplacental oral treatment with DMN, as a positive control, caused changes of similar extent in biological effects of embryonic fibroblasts, and in addition it caused chromosomal aberrations in metaphase plates. On the contrary, transplacental oral application of NaNO2 did not induce any biological change in cultured embryonic fibroblasts.

Mouse strain differences in the induction of micronuclei by polycyclic aromatic hydrocarbons

The frequency of micronucleated erythrocytes (MNE) in 3 inbred mouse strains and 2 of their hybrids (C57BL/6, BALB/c, DBA/2, BDF1 and CDF1) were examined after polycyclic aromatic hydrocarbons (PAHs; 7,12-dimethylbenz[a]anthracene (DMBA), 3-methylcholanthrene (3-MC), benzo[a]pyrene (BaP), benzo[e]pyrene (BeP) and anthracene (ANT] were injected i.p. PAHs are thought to form active metabolites after being administered to mammals. In mouse strains with inducible PAH activating enzymes, such as C57BL/6 or BALB/c, MNE were significantly induced, as compared to control mice, 48 h after DMBA, BaP, or 3-MC was injected. No increase in the frequency of MNE occurred in the DBA/2 strain which cannot induce the activating enzymes. BeP and ANT did not increase the frequency of MNE in any mouse used. The levels of MNE induction in BDF1 or CDF1 hybrids were similar to those in C57BL/6 or BALB/c. These results support the view that the genetic capacity to metabolize PAHs is strongly associated with micronucleus induction as in the case of PAH carcinogenesis.

Comparative studies on chromosome aberrations induced by polycyclic hydrocarbons in cell-mediated and microsome-mediated assay

Studies were made on chromosome changes in Chinese hamster V79 cells induced by the polycyclic hydrocarbons, benzo[alpha]pyrene (BP), 3-methylchol-anthrene (MC) and 7,12-dimethylbenz[alpha]anthracene (DMBA) in the presence and absence of cell-mediated and microsome-mediated activation systems. In cell-mediated assay, BP, MC and DMBA induced concentration-dependent chromosome aberrations in V79 cells, at concentrations of 0.5--1.0 to 10.0 micrograms/ml in the presence of lethally irradiated Syrian hamster embryonal feeder cells at 2.0 X 10(6) cells/60-mm dish. The highest incidences of cells with aberrant chromosomes, observed at concentrations of 20 micrograms/ml, were 24.0% (BP), 23.0% (MC) and 80.0% (DMBA). In the absence of feeder cells, these chemicals did not induce chromosome aberrations in V79 cells, even at the maximum concentrations tested, only background levels being observed. In microsome-mediated assay, BP, MC and DMBA at 20 micrograms/ml also induced chromosome aberrations when combined with microsomes. However, the highest incidences of aberrations were observed with lower amounts of microsomes than usual, the optimal amount of microsomes ranging from 1 to 10%. In this range, the highest incidences of cells with aberrant chromosomes were 10.0% (BP), 9.0% (MC) and 28.0% (DMBA), that is about half to one-third those observed in cell-mediated assay.

Inhibition of 12-O-tetradecanoylphorbol-13-acetate-enhanced transformation in vitro by radical scavengers.

The inhibitory effects of some scavengers of oxygen radicals were studied, using a two-stage transformation assay system in vitro 3-methylcholanthrene (3-MC)-initiation and 12-O-tetradecanoylphorbol-13-acetate-(TPA)-promotion in Balb 3T3 cells. Mannitol, a scavenger for hydroxyl radicals, strongly inhibited the TPA-enhanced transformation in a dose-dependent manner. Superoxide dismutase (SOD) and catalase, which are specific scavengers for O2-. and H2O2, respectively, also inhibited the transformation. These results suggest that oxygen radicals may play an important role in the TPA-enhanced transformation in Balb 3T3 cells.

A comparison of intraperitoneal injection and oral gavage in the micronucleus test with mitomycin C in mice.

Intraperitoneal (i.p.) injection and oral (p.o.) gavage were evaluated in the mouse micronucleus test with mitomycin C (MMC). The tests were carried out in 2 laboratories with the MS/Ae and CD-1 mouse strains. On the basis of a small-scale acute toxicity study and a pilot experiment, the full-scale micronucleus test was performed with a 24-h sampling time at doses of 1, 2, 4, and 8 mg/kg for both treatment routes. In both strains, a clear positive dose-response relation was shown by both routes. Although the frequency of micronucleated polychromatic erythrocytes (MNPCEs) was higher with i.p. on a mg/kg basis, this tendency was reversed when dose was expressed as a percentage of the LD50.

Retinoic acid and butylated hydroxyanisole inhibit promoter-enhanced transformation in vitro

The inhibitory effects of some antipromoters were studied using a two-stage transformation assay system in vitro with 3-methylcholanthrene (3-MC)-initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promotion in BALB 3T3 cells. Butylated hydroxyanisole (BHA), a phenolic antioxidant, inhibited TPA-enhanced transformation in a dose-dependent manner, but butylated hydroxytoluene (BHT) did not. Among the three antipromoters tested, retinoic acid (RA) was the most effective inhibitor.

A rapid simple screening method for skin tumor promoters using mouse peritoneal macrophages in vitro

The enhancing effects in vitro of a potent skin-tumor promoter, phorbol myristate acetate (PMA), and its derivatives on nitroblue tetrazolium (NBT) reduction, phagocytosis and cell spreading of mouse peritoneal macrophages were compared. The enhancing effects on these markers of macrophage function, especially NBT reduction, were found to correlate well with the skin-tumor promoting activities of the phorbol esters. Another potent promoter, teleocidin, strongly enhanced NBT reduction, although teleocidin is structurally different from phorbol esters. Other tumor promoters, namely saccharin, phenobarbital, cantharidin and Tween 60 did not enhance NBT reduction by macrophages. These data indicate that this test system is appropriate for screening of PMA-type tumor promoters such as phorbol esters and teleocidin.

Effects of diesel exhaust particles on chromosome aberration, sister chromatid exchange and morphological transformation in cultured mammalian cells

Abstract The ability of diesel exhaust particles (diesel tar) from light-duty (LD) and heavy-duty (HD) engines to induce chromosome aberrations, sister chromatid exchanges (SCEs) and morphological transformations is examined. Chinese hamster V79 cells were treated with LD and HD diesel tar for 3 h in order to analyze chromosome aberrations and SCEs. 3T3 cells were used for the morphological transformation test. LD tar induced significant numbers of chromosome aberrations, whereas HD did not. Both LD and HD samples increased the number of SCEs in a dose-dependent fashion, with LD being more potent. In the transformation test, LD tar also induced a significant number of Type III foci, while HD was only weakly active. The transformed cells isolated from these Type III foci produced tumors when injected into nude mice. These results show that LD possesses clear clastogenic and transforming capabilities but that HD is weaker in this regard.

Mouse strain differences in induction of micronuclei by base analogues and nucleosides.

The frequency of induced micronucleated polychromatic erythrocytes (MNPCEs) was compared in BALB/c, C57BL/6, and DBA/2 mice after intraperitoneal (i.p.) injection of 5-bromodeoxyuridine (BUdR), 5-fluorodeoxyuridine (FUdR), cytosine arabinoside (Ara-C), 6-mercaptopurine (6-MP), 5-bromouracil (5-BU), thymidine (TdR), uridine (UdR), adenosine (AdR) and guanosine (GdR). The experimental procedure was a single i.p. injection followed by harvest at 30 h. The frequency of MNPCEs was significantly increased in all strains by treatment with BUdR, FUdR, Ara-C and 6-MP compared to vehicle control. TdR and UdR induced MNPCEs slightly in BALB/c mice but showed no effect on C57BL/6 and DBA/2 mice. 5-BU, AdR, and GdR did not increase the frequency of MNPCEs in any mouse strain used. These results suggest that BALB/c mice are more susceptible to induction of MNPCEs by clastogenic base analogues and nucleosides than are C57BL/6 or DBA/2 mice.

Changes DNA polymerases α, β and γ in mouse liver as a function of age

Abstract The activities of DNA polymerases α, β, γ were determined in mouse liver as a function of age by a combination of glycerol density gradient centrifugation with polymerase specific assays. Although α polymerase was preserved througout the life span, the activity dropped sharply from a high level at the fetal and neonatal stages to a level one order lower after maturation through adjustment of the amount of protein administered. β polymerase showed similar but less drastic changes than α. DNA polymerase γ activity increased about two-fold in going from newborn to adult stages and remained constant after maturation. According to the amount of DNA, DNA polymerase α decreased after birth, but the change was less drastic compared to that through adjustment of the amount of protein. DNA polymerase β increased the activity 2–3-fold within a period of 3 months following birth. γ polymerase underwent more than a 10-fold increase in activity through adjustment of the amount of DNA within the same period.