Phosphorylase-mediated mobilization of the amino group of adenine in Bacillus cereus.

Mobilization of the ribose moiety of purine nucleosides as well as of the amino group of adenine may be realized in Bacillus cereus by the concerted action of three enzymes: adenosine phosphorylase, adenosine deaminase, and purine nucleoside phosphorylase. In this pathway, ribose-1-phosphate and inorganic phosphate act catalytically, being continuously regenerated by purine nucleoside phosphorylase and adenosine phosphorylase, respectively. As a result of such a metabolic pathway, adenine is quantitatively converted into hypoxanthine, thus overcoming the lack of adenase in B. cereus.

Nitrous oxide production by nitrogen-fixing, fast-growing Rhizobia.

Rhizobium trifolii, R. leguminosarum, andR. "hedysarum", grownex planta under anoxic conditions in a chemically defined medium, evolve N2O from NO3 (-), NO2 (-), and (NH4)2NO3. The amount of nitrous oxide formed after 96 hours is about 0.2µM×mg(-1) cells d.w. Large availability of organic matter enhances the production of N2O from nitrate by free-livingR. trifolii in peat/sand mixtures. Denitrification of the above species andR. meliloti was detected also in planta. Nitrous oxide production increases almost linearly from 10-45µM×mg(-1) nodules d.w. when nitrogen-fixing plants are exposed to increasing concentrations of nitrate (1-12µM).

Studies on microsomal metabolizing systems for mutagenesis tests. Stability of fortified fractions.

Rat liver S-9 microsomal fractions were fortified with NADP+, G-6-P, and MgCl2 and stored at -80 degrees C and -20 degrees C. At various times aminopyrine demethylase and lipid peroxidation were determined and compared with that of non-fortified fractions. No difference in stability was noted between fortified and not fortified fractions. The known stability pattern of non fortified fractions was confirmed. Lipid peroxidation was found blocked in the fractions stored at -80 degrees C whereas a small release of malonyl dialdehyde proceeded in the frozen state at -20 degrees C, contributing to the loss of activity at this temperature. The use of pre-fortified fractions stored at -80 degrees C is justified.

Genetic activity of vinylidene chloride in yeast.

Vinylidene chloride (VDC) was tested for its ability to induce both point mutation and mitotic gene conversion in a diploid strain (D7) of the yeast Saccharomyces cerevisiae in a suspension test with and without a mammalian microsomal activation system, and in the intrasanguineous host-mediated assay in mice. In suspension tests with D7, VCD was toxic but not genetically active without microsomal activation. When a mouse liver 10 000 X g supernatant was included in the suspension tests, dose-related increases in both point mutation and mitotic gene conversion were seen at survival levels greater than 50%, at doses of VCD above 20 mM. In the host-mediated assay, VDC induced both point mutation and mitotic gene conversion when recovered from the liver and kidneys after both acute and sub-acute dosing. Yeasts recovered from the lungs showed little, if any, increase in either point mutation or mitotic gene conversion.

Studies on the incubation mixtures for the in vitro mutagenesis test with metabolic activation (microsomal assay) = 2. Behaviour of cytochromes P-450, P-420, b5, of NADPH-CYT. C-reductases, and p-nitroanisole demethylase.

Cytochromes P-450, P-420, b5, NADPH-cytochrome c-reductase, aminopyrine and p-nitroanisole demethylase and lipid peroxidation were determined at various times in the incubation mixtures for the in vitro microsomal assay for the mutagenic activity of xenobiotics. No effect was observed on cytochromes b5 and P-420. A decrease in cytochrome P-450 (about 50% in 2 hrs.) and a much faster decrease of aminopyrine demethylase and NADPH cytochrome c-reductase (about 50% in 30 min) was noted with mice microsomes. With S9 liver fraction of rat, p-nitroanisole demethylase activity was much more stable than aminopyrine demethylase activity in the presence of lipid peroxidation, but the decrease was faster and at comparable rates for both activities in the presence of 50 mM styrene. The use of simple colorimetric assay as proves of microsomal monooxygenase activity and the importance of this kind of enzyme studies for a better understanding of the in vitro mutagenesis results are discussed.

[Role of cytochrome P-450 in strain D7 of the yeast Saccharomyces cerevisiae]. / Ruolo del citocromo P-450 nel ceppo D7 lievito Saccharomyces cerevisiae.

Usual "in vitro" mutagenesis tests combine microbial test systems with mammalian metabolism (microsomal assay). Some cases are known in which expected positive results are not obtained due to many factors operating between the microsomes in the incubation mixtures and the nucleus of the test microbial cells (detoxifying mechanisms, permeability effects, unstable intermediate, alternative targets in the pathway, etc.). In these cases, useful additional information could be obtained using microbial test systems with incorporated metabolism. Microbial systems and in particular yeasts in Cytochrome P-450 dependent metabolizing activity are known since many years. We studied under this aspect the strain D7 of Saccharomyces cerevisiae, used in our laboratory, in order to standardize the conditions for optimal metabolizing activity. The composition of the culture medium, the growth phase, and the effect of the amount of the initial inoculum, were studied. Cytochrome P-450 content was determined by the technique of difference spectra (reduced cytochrome +/- CO). Results on the effect of glucose and sodium phenobarbital concentration in the culture medium were in agreement with the literature. Moreover we found that the cytochrome P-450 contents was markedly dependent on the size of the initial inoculum, being higher and the smaller was the initial inoculum.

The problem of negative results for styrene in the in vitro mutagenesis test with metabolic activation (microsomal assay). - 2. Behaviour of epoxide hydrolase in the incubation mixtures.

The behaviour of epoxide hydrolase and monooxygenase was studied in incubation mixtures for the in vitro mutagenesis test with metabolic activation (microsomal assay) both in the presence and in absence of styrene 50mM. Epoxide hydrolase activity was much more stable than monooxygenase in all tested systems (S-9 fractions or microsomes of rats or mice, males or females) both in the presence and in absence of styrene. Membrane bound epoxide hydrolase was slightly less stable than the soluble one. These results further substantiate the explanation for the negative results of in vitro mutagenesis tests with styrene.

The use of organic solvents in mutagenicity testing.

13 organic substances (dimethylsulfoxide, methanol, ethanol, n-propyl alcohol, sec-butyl alcohol, tert-butyl alcohol, dl-sec-amyl alcohol, ethylene glycol, ethylene glycol monomethyl ether, 1,4-diethylene dioxide, acetone, methyl acetate and formamide) were considered from the standpoint of their use as solvents for water-insoluble chemicals to be tested for mutagenicity. First, the effect of these solvents on cell survival was studied in the yeast Schizosaccharomyces pombe and in V79 Chinese hamster cells. 8 solvents showing relatively low toxicity on either cell system (dimethylsulfoxide, ethanol, ethylene glycol, ethylene glycol monomethyl ether, 1,4-diethylene dioxide, acetone, methyl acetate and formamide) were tested for their effect on aminopyrine demethylase. 4 solvents (ethanol, 1,4-diethylene dioxide, methyl acetate and formamide) showed a more or less pronounced adverse effect on the microsomal enzymic activity. The remaining 4 and methanol (whose effect on aminopyrine demethylase was not testable) were assayed for mutagenicity in S. pombe. They all gave negative results both with and without the post-mitochondrial fraction from mouse liver.

Genetic effects of vinylcyclohexene diepoxide in yeast.

Using D7 strain of S. cerevisiae where we can consider three genetic effects such as mitotic gene conversion, mitotic cross over and reverse mutation we tested vinylcyclohexene diepoxide "in vitro" without metabolic activation. In this condition VCD is very toxic and induces both three genetic effects namely mitotic gene conversion, mitotic cross over and reverse mutation.

The problem of negative results for styrene in the in vitro mutagenesis test with metabolic activation (microsomal assay):explanation by gas chromatographic analysis.

Mutagenic experiments in vitro were performed on yeast (S. pombe) with styrene with metabolic activation by adding at fixed times fresh mouse microsomes to the incubation mixture. Preceding studies with a single initial addition of microsomes were negative up to the maximal dose of 100 mM styrene for 60 min (5). The addition of fresh microsomes at the times 12, 24, 36 and 48 min in the presence of EDTA 40 mM resulted in an increased toxicity of styrene in vitro, but not in increased mutagenicity. Gas chromatographic determination of styrene oxide in the incubation mixture with 50 mM styrene revealed a concentration of active metabolite not capable of acting mutagenically.

Studies on the incubation mixtures for the in vitro mutagenesis test with metabolic activation (microsomal assay). Behaviour of mixed function oxidase and lipid peroxidation in the presence of some xenobiotics.

The effect of some xenobiotics on microsomal mixed function oxidase and lipid peroxidation, in mice, in incubation mixtures for the in vitro mutagenesis test with metabolic activation was studied. Aniline 1 or 2 mM and aminopyrine 0.38 or 8.33 mM completely inhibited the lipid peroxidation with small protection of the monooxygenase. Styrene 50 or 100 mM inhibited to a lesser extent the lipid peroxidation with marked increase in the inactivation of the monooxygenase. By a technique based on successive additions of fresh microsomes it was possible to evaluate the part of the inactivation due to enzyme denaturation and that due to inhibition. EDTA 40 mM was not able to protect from inactivation in the presence of aniline 1mM. Data of this type could be utilized to obtain more reliable results of in vitro mutagenesis tests with metabolic activation by suitably managing the enzyme activity in the incubation mixtures in order to keep it as constant as possible.

[Role of NADPH-dependent lipid peroxidation in the oxidase inactivation in mixed hepatic microsomal function]. / Ruolo della perossidazione lipidica NADPH-dipendente nella inattivazione dell'ossidasi a funzione mista microsomiale epatica.

When mouse liver microsomes were preincubated at 37 degrees with buffer, the monooxygenase activity was nearly constant and lipid peroxidation very low over 15 min. When preincubated with NADPH, there was an increase in lipid peroxidation accompanied by loss in enzyme activity (about 40%). The addition of EDTA 5 or 40 mM depressed lipid peroxidation and protected the monooxygenase activity with a rate increasing with concentration. The addition of FeSO4 0.5 mM had only little effect one enzyme activity whereas stimulated the response of the 2-thiobarbituric acid test.

Induction of gene mutations and gene conversions by vinyl chloride metabolites in yeast.

Chloroethylene oxide and 2-chloroacetaldehyde, two metabolites of vinyl chloride, and 2-chloroethanol, a putative metabolic intermediate, were assayed for their genetic activity in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae. Chloroethylene oxide was found to be the most effective in inducing forward mutations in Sch. pombe and gene conversions in S. cerevisiae, increasing the mutation and conversion frequencies 340 and 50 times, respectively, over those of the controls. In either the presence or the absence of mouse liver microsomes, 2-chloroacetaldehyde showed only feeble genetic activity, and 2-chloroethanol was completely inactive in both yeast strains. In contrast to vinyl chloride, 2-chloroacetaldehyde did not induce forward mutations in Sch. pombe inthe host-mediated assay in mice. The results strongly support the hypothesis that chloroethylene oxide is one of the principal mutagenic agents formed from vinyl chloride in the presence of mouse liver enzymes.

Mutagenicity of industrial compounds: styrene and its possible metabolite styrene oxide.

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.

Evaluation of the genetic effects induced by vinyl chloride monomer (VCM) under mammalian metabolic activation: studies in vitro and in vivo.

As part of a programme of investigations on the biological effects of the industrial compound vinyl chloride monomer (VCM), the raw material for the production of polyvinyl chloride (PVC), analyses on the genetic effects by this compound have been done by experiments (in vitro) which have taken mammalian metabolism into account. Vinyl chloride in the presence of purified microsomes (sedimented at 105,000 g) obtained from mouse liver was converted into an active metabolite(s) which produced gene mutations in the yeast Schizosaccharomyces pombe (forward mutation) and gene conversions in two loci of a diploid Saccharomyces cerevisiae. Moreover, the compound was active in the host-mediated assay, when mice were treated with an oral dose of 700 mg/kg. The role is discussed of mutagenicity tests for the prediction of both genetic and carcinogenic risks of chemical compounds in industrial use.