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In order to evaluate the optimal experimental conditions and to identify the best growth phase for yeast genotoxicity studies, comparative experiments were performed with stationary and growing cells. Methyl methanesulfonate (MMS) and cyclophosphamide (CP) were used as chemical mutagens and strain D7 of Saccharomyces cerevisiae as detector of induced mitotic gene conversion (trp+ convertants) and point reverse mutation (ilv+ revertants) in log or stationary phase cells after either 4 or 16 h of treatment. The highest MMS-induced toxicity and genotoxicity were observed after 16 h of exposure in a suspension test with log phase cells, which is consistent with the greater permeability and sensitivity of growing yeast cells. The maximal induction of genetic effects and toxicity by CP was conversely obtained after 16 h of treatment in stationary phase cells. This may be ascribed to the greater ability of detoxication of growing cells as compared to resting cells. Our results suggest that in evaluating the mutagenicity of chemicals in yeast systems it is important to consider factors such as growth phase and exposure time.

In recent years there has been increasing interest in antimutagenesis, and studies have been done using both prokaryotic and eukaryotic systems. In eukaryotic systems the first studies were performed with different strains of Schizosaccharomyces pombe. In particular, caffeine and L-methionine were investigated. Different strains of Saccharomyces cerevisiae were employed in studies of a wide variety of compounds, including acridine, saccharin, salts, tumor promoters and co-carcinogens. Strain D7 was widely employed and antimutagenic activity of spermine, chlorophyllin, cobaltous chloride and fermented milk is reported.

Chloral hydrate (CH), a metabolite of trichloroethylene (TCE), was studied in vitro using the D7 diploid strain of Saccharomyces cerevisiae, with and without a mammalian microsomal activation system (S9 fraction), and in vivo by intrasanguineous host-mediated assay (HMA). The in vivo effects on the hepatic microsomal monooxygenase induced by CH in mice pretreated with β-naphthoflavone (β-NF) and Naphenobarbital (PB) were also investigated. Chloral hydrate induced a significant increase of mitotic gene conversion in D7 strain both in vivo and in vitro. The enzymatic determinations in mice showed a decrease in aminopyrine N-demethylase (APD) and p-nitroanisole O-demethylase (p-NAD) activities (about 37% and 29% respectively) after one acute dose of CH. Moreover, stability experiments, carried out in the conditions of the liver microsomal assay (LMA), showed an increase of residual activity, after 1 h of preincubation with respect to the control (about 22% and 9% for APD and p-NAD respectively).

Styrene and its presumed metabolite, styrene oxide, were tested for their mutagenic effect on a forward mutation system of yeast and of Chinese hamster cells, and on a gene-conversion system of yeast. Experiments with liver microsomal preparations and host-mediated assay with yeast were also carried out. Styrene oxide was mutagenic in all test systems. Styrene was mutagenic only in the host-mediated assay.

The effect of NADPH supply on enzymatic activity and its stability were investigated with respect to the mono-oxygenase activities of 7-ethoxyresorufin O-deethylase (ERD), dinemorphan N-demethylase (DND), aminopyrine N-demethylase (APD), 7-ethoxycoumarin O-deethylase (ECD) and p-nitroanisole O-demethylase (p-NAD) under incubation conditions for the liver microsomal assay (LMA). Experiments with S9 liver fractions of mouse (induced with Na-phenobarbital and beta-naphthoflavone) and rat (induced with Aroclor 1254) were set out at different pre-incubation times with and without exogenous isocitrate dehydrogenase (IC-DH) in the LMA. Such LMA mixtures contain Mn2+, NADP+, DL-isocitrate (IC) and endogenous IC-DH as NADPH-generating machinery. No changes in mono-oxygenase stability and lipid peroxidation (LP) were observed in the presence of exogenous IC-DH. The metabolizing capability at the considered times was the maximal one, as shown by no stability changes after the direct addition of IC-DH to the enzymatic assays. Exogenous IC-DH in the incubation for LMA did not alter the mitotic crossing-over and the mitotic gene conversion of dimethylnitrosamine (DMNA) and AR2MNFN (a nitroimidazo[2,1-b]thiazole) in the tester D7 strain of Saccharomyces cerevisiae. It was concluded that endogenous IC-DH seems to be sufficient to provide a saturating level of NADPH for mono-oxygenase activities during incubations for LMA without additional external NADPH-generating enzyme activity.

3 structurally related epoxides, 3,4-epoxycyclohexene, trans -1,2,3,4-diepoxycyclohexane and trans -3,4-epoxycyclohexane- r -1, trans -2-diol ( anti isomer) were tested for their ability to induce both point mutation, mitotic gene conversion and recombination in a diploid strain (D7) of the yeast Saccharomyces cerevisiae , with and without a mammalian microsomal activation system, and the formation of 6-thioguanine-resistant mutants in V79 hamster cells. Genetic effects were related to the alkylating properties of the epoxides, as measured by alkylation of4-( p -nitrobenzyl) pyridine (NBP). Of the 3 epoxides, only 3,4-epoxycyclohexene, characterized by the highest reactivity towards NBP, induced all genetic effects in both test systems. A marginal activity was shown by trans -1,2,3,4-diepoxycyclohexane only in the yeast. The lack of genetic activity of the anti isomer of 3,4-epoxycyclohexane-1,2-diol, in spite of the formal similarity of its functional groups with those present in mutagenic polycyclic arene epoxydiols, was attributed to the dramatic reduction of lipophilicity of the molecule.

Certain aspects of cytochrome P-450 induction were studied in a diploid strain (D7) of the yeast Saccharomyces cerevisiae in order to obtain cells containing a high level of metabolizing enzymes. The highest level of cytochrome P-450 was reached during the logarithmic growth phase in a 20%-glucose liquid medium. Yeast cells harvested in these conditions were used in the mutagenesis test with dimethyl nitrosamine (DMNA) as a positive control and with styrene (Sty). Both substances gave positive results, whereas Sty never showed any mutagenic activity in the conventional test with stationary growth phase cells and external metabolic activation. The test with cells from the logarithmic growth phase is proposed as a possible alternative to the liver-microsome assay, and its reliability is discussed.

At high survival levels (85%), point mutation and gene conversion frequencies were determined in strain D7 of Saccharomyces cerevisiae after treatment with methyl methanesulfonate (MMS) either after cells were incubated in complete medium before plating or following a split-dose protocol. It is shown that induction of gene conversion by MMS post-incubation leads to an additional enhancement in frequency. This increase is not observed for point mutation. By fractionation of the MMS dose (1 mM + 1 mM) with incubation in complete medium between the 2 doses the frequency of gene conversion is twice as high as with a single equal total dose (2 mM). This treatment does not modify the frequencies of point mutation. These data support the notion that an inducible recombinogenic function exists in wild-type yeast.

Activity levels of 7-ethoxycoumarin O-deethylase (ED), aminopyrine N-demethylase (APD), p-nitroanisole O-demethylase (p-NAD) and glucose-6-phosphate dehydrogenase (G-6-PDH) were determined in incubation mixtures for the liver-microsomal assay (LMA) at time 0 and after 1 and 2 h incubation under conditions for mutagenic assay. The experiments were performed with S9 liver fractions from mice (induced with Na-phenobarbital and beta-naphthoflavone) and rats (induced with Aroclor 1254) with and without G-6-PDH in the incubation mixtures. In the absence of G-6-PDH the activities were significantly lower at time 0 in the mouse. The pattern of stability, however, was similar for the activities, with an increase of stability after 1 and 2 h of pre-incubation (an exception for p-NAD). Only ED activity showed a similar behaviour in the rat. No differences were present for APD and p-NAD activities at time 0 in the rat, but the enzyme stabilities were significantly decreased after 2 h of incubation (about 15% and 10% for APD and p-NAD respectively) in the absence of G-6-PDH. At time 0, the amounts of G-6-PDH differed between mouse and rat fractions; however, during the incubations for LMA they decreased by about 57% and 53% for the two species, respectively. In addition to the above biochemical results, the presence of exogenous G-6-PDH in the incubations for the mutagenic assay, significantly increased the mitotic gene conversion and mitotic crossing-over of dimethylnitrosamine (DMN) and AR2MNFN (a nitroimidazo[2,1-b]thiazole) in the D7 strain of Saccharomyces cerevisiae.