Metabolism of 5-methoxypsoralen by Saccharomyces cerevisiae.

Incubation of methoxypsoralen (5-MOP) in the presence of diploid yeast cells (Saccharomyces cerevisiae) before UV-A exposure leads to an incubation-time dependent decrease of photoinduced genotoxic effects. The reduction in photoinduced genotoxicity is stronger in cells grown in the presence of 20% glucose and containing high levels of cytochrome P-450 than in cells grown in the presence of 0.5% glucose and containing undetectable levels of cytochrome P-450. Inhibition of P-450 activity by specific inhibitors, such as tetrahydrofuran and metyrapone, strongly affects the observed decrease in 5-MOP genotoxicity, indicating the involvement of P-450 in 5-MOP metabolism. As demonstrated by spectrophotometric and chromatographic (HPLC) analysis during incubation of 5-MOP with P-450 containing yeast cells, 5-MOP gradually disappears from the cell supernatant of the incubation mixture. The reduction in the chromatographic peak corresponding to 5-MOP is accompanied by the appearance of a new peak that probably corresponds to a metabolite. As shown by the use of P-450 specific inhibitors, the metabolite appears to be due to P-450 mediated 5-MOP metabolisation. Its UV absorption spectrum suggests an alteration of the pyrone moiety of the 5-MOP molecule.

Inhibition of yeast cytochrome P-450 by ammonium metavanadate.

Incubation of diploid D7 strain cells of Saccharomyces cerevisiae (grown in 20% glucose) in the presence of ammonium metavanadate (AMV) led to a decrease in the cytochrome P-450-dependent monooxygenase system (cytochrome P-450 level and 7-ethoxycoumarin O-deethylase). The electrophoretic analysis of microsomal fractions of yeast cells treated with metavanadate revealed a decrease in the intensity of the bands corresponding to a M(r) in the range of 51,000-58,000 Da compared with those observed in controls, i.e., cells grown in 20% glucose. Analysis of the cytochrome P-450 transcript showed that AMV treatment reduced the mRNA level. Our results suggest that AMV inhibits the yeast cytochrome P-450 system by acting at both the pre- and post-transcriptional levels.

Mutagenicity and toxicity of electromagnetic fields.

Humans are exposed daily to electromagnetic fields (EMFs) originating from a variety of devices and systems. During the 1980s many reports of potential mutagenic, teratogenic, and carcinogenic effects of EMFs were published, sometimes with contrasting results. To date, no study has established unequivocally a causal relationship between EMFs and cancer. Cell cultures can provide a simple and inexpensive tool for the study of the effects of EMFs. We have used the Chinese hamster V79 cell line to evaluate the influence of a sinusoidal EMF at 50-Hz with a constant flow of 2 G on the induction of HGPRT- mutants and on survival. Our results showed that the EMF employed did not induce any modification of mutation frequency, but the results on survival were contrasting. When only 10(2) cells were plated, a reduction in the number of colonies, reaching about 50% after 10 days of treatment, was observed; however, when 2 x 10(5) cells or more were seeded, no reduction in viability was recorded. An intercellular metabolic interaction may explain these results.

Induction of cytochrome P-450 by 5-methoxypsoralen in the yeast Saccharomyces cerevisiae.

Yeast cells (D7 strain) incubated in the presence of 5-methoxypsoralen (5-MOP) increase the activity of the monooxygenase system cytochrome P-450 dependent (cytochrome P-450 level and 7-ethoxycoumarin-O-diethylase activity). Northern analysis of cytochrome P-450 specific RNA shows that 5-MOP treatments induce an increase in mRNA. The induction of cytochrome P-450 appears to occur at the transcriptional level. The capacity of 5-MOP to induce the cytochrome P-450 system in eukaryotic cells, in which it is known to be involved in the metabolism of the psoralen, may decrease the availability of the compound for photo-induced genotoxic reactions, which may explain the good tolerance in patients.

Mutagenicity and chemical analysis of airborne particulate matter collected in Pisa.

The genotoxic effects of airborne particulate samples collected from two urban areas during various traffic intensity levels were evaluated using the yeast Saccharomyces cerevisiae (D7 strain) and Salmonella typhimurium (TA98 and TA100 strains). Standard protocols were used, in compliance with recent legislation. Airborne particulate data reveal that genotoxic effects were more evident in the samples collected in the area with intense moving traffic than in the area with limited traffic. Particulate, benzene, toluene, NO2, CO, and heavy metal determinations in these two areas show that their concentrations are related to the intensity of traffic.

Biochemical and genetic interactions of two commercial pesticides with the monooxygenase system and chlorophyllin.

Two commercial preparations of atrazine and zineb were tested on a diploid D7 strain of the yeast Saccharomyces cerevisiae using cells from logarithmic growth phase (with a high level of cytochrome P-450) and from stationary growth phase. The compounds induced marked increases of both gene conversion and point mutation frequencies in the logarithmic phase cells, while in the stationary phase no genotoxic effect was observed. The results obtained employing TA98 and TA100 strains of Salmonella typhimurium (Ames test) confirmed that neither atrazine nor zineb were mutagenic. The interaction between zineb and chlorophyllin, a known antimutagen in several biological systems, has been evaluated in yeast cells from logarithmic growth phase. The results showed that chlorophyllin seems to have a protective role against the genotoxic effects of zineb. The in vivo effects on cytochrome P-450 content (Cyt. P-450) and on monooxygenase activities were examined in hepatic microsomes of induced animals (rat, pig, and rabbit) 24 hrs after acute treatment. The results obtained with atrazine showed that it caused different effects in the three animal species. Preliminary data with zineb indicated that it can act both as an inducer or as an inhibitor of the monooxygenase system, depending on the dose used.

Induction of cytochrome P-450 and catalase activity in Saccharomyces cerevisiae by UV and X-ray irradiation. Possible role for cytochrome P-450 in cell protection against oxidative damage.

Cytochrome P-450 was induced both in the diploid wild-type D7 strain and in two isogenic DNA-repair-deficient strains (rad3 and rad56) of Saccharomyces cerevisiae following UV- and X-irradiation. The induction occurred only in logarithmic growth phase cells and it was transient showing a peak 3 h after irradiation. The maximal amount of cytochrome P-450 was directly proportional to the radiation dose applied. Under the same experimental conditions an increase of the catalase activity was also observed, suggesting that activated oxygen species produced by irradiation might be implicated in the induction of both enzymes. The sensitivity to H2O2 of cells containing high cytochrome P-450 levels was enhanced when this enzyme was specifically inhibited by tetrahydrofuran and metyrapone. This supports the hypothesis that cytochrome P-450, as well as catalase, might be involved in cell protection against oxidative damage.

Vanadium: genetical and biochemical investigations.

Ammonium metavanadate was studied for its ability to induce mitotic gene conversion and reverse point mutation in the D7 strain of Saccharomyces cerevisiae. Metavanadate increased the convertant and revertant frequencies; the highest activity was observed without metabolic activation. This indicated that the S9 hepatic fraction and yeast cells in logarithmic phase (and containing a high level of cytochrome P450) biotransform vanadate, probably reducing it to vanadyl. In addition, the effect of ammonium metavanadate on the hepatic monooxygenase system was studied in mice by measuring the level of cytochrome P450 and determining the activities of aminopyrine N-demethylase, p-nitroanisole O-demethylase and 7-ethoxycoumarin O-deethylase in mouse liver microsomal fraction. The results indicated that this compound reduced mono-oxygenase activity and also the level of cytochrome P450.

Expression of cytochrome P450 in yeast after different chemical treatments.

In Saccharomyces cerevisiae a number of chemical agents induce synthesis of cytochrome P450. A cytochrome P450 gene has been well characterized in this yeast: CYP51, which codes for a constitutive enzyme involved in the 14 alpha-demethylation of lanosterol, a key step in the biosynthesis of ergosterol. In this work, we have analysed the level of transcription of the CYP51 gene in correlation with cytochrome P450 enzymatic activity after treatment with several chemical agents known to interact with cytochrome P450. Using as a probe a DNA fragment whose identity to the CYP51 gene was established by sequence analysis and mapping on chromosome VIII, a unique RNA species was observed in all treatment samples. The increased level found for this transcript in cells treated with ethanol, 20% glucose, phenobarbital or 5-methoxypsoralen correlates with the levels of induction in cytochrome P450 enzymatic activity measured in cells grown under the same conditions, indicating that induction of cytochrome P450 by these treatments is regulated at the transcriptional level.

Effect of magnetic fields on rodent monooxygenase enzymes.

The effects of 50 Hz, 1.2 mT magnetic fields (MFs) were tested on hepatic monooxygenase enzymes of basal and beta-naphthoflavone-phenobarbital-preinduced rats and mice. An inductive effect on cytochrome P-450 level and on some enzymatic cytochrome P-450-dependent activities was observed in basal mice after MF exposure. Enzymatic activities in preinduced mice and rats were reduced by MFs, the degree of reduction depending on the enzyme. A specific inhibitory effect was determined in some of the assayed activities and in the relative peculiar P-450 isoforms detected by Western blot analysis.

Tetrachloroethane, pentachloroethane, and hexachloroethane: genetic and biochemical studies.

Tetrachloroethane (TTCE), pentachloroethane (PCE), and hexachloroethane (HCE) were tested in diploid strain (D7) of the yeast Saccharomyces cerevisiae in suspension test with and without mammalian metabolic activation (S9). TTCE, PCE, and HCE gave positive results on cells harvested from logarithmic growth phase; only PCE induced a significant increase (P less than or equal to .01) of mitotic gene conversion and point reverse mutation on cells from stationary growth phase with metabolic activation (S9). The in vivo effects on cytochrome P450 content (cyt. P450), pentoxyresorufin O-dealkylase (P450-like, class IIB, PROD), and ethoxy-resorufin O-deethylase (P448-like, class IA, EROD) activities were examined in hepatic microsomes from mice 24 h after acute intoxication. All the halogenated hydrocarbons displayed a marked toxic effect as shown by the significant decrease in cyt. P450 levels (maximum of 76% decrease, with TTCE 753.2 mg/kg) and EROD (maximum of 69% decrease, with PCE 925.4 mg/kg), and to a lesser extent in PROD (maximum of 52.4% decrease, with HCE 3150 mg/kg). Although a general decrease of P450 functions was observed, the toxic effects of TTCE and PCE seem to be preferentially related to P448 forms.

Genotoxicity of vanadium compounds in yeast and cultured mammalian cells.

The ability of vanadium compounds to induce genetic activity was investigated in D7 and D61M strains of Saccharomyces cerevisiae and in Chinese hamster V79 cell line. In our previous work, ammonium metavanadate (pentavalent form, V5) induced mitotic gene conversion and point reverse mutation in the D7 strain of yeast. The genotoxicity was reduced by the presence of S9 fraction, which probably reduced pentavalent vanadium to the tetravalent form. In the present study, vanadyl sulfate (tetravalent form, V4) induced no convertants and revertants in yeast cells harvested from stationary growth phase. With yeast cells from logarithmic growth phase, which contain high levels of cytochrome P-450, a significant increase in genetic effects was observed. Further experiments, performed by treating cells harvested from logarithmic growth phase in the presence of cytochrome P-450 inhibitors, indicated that the monooxygenase system influenced the genotoxicity of metavanadate while the genetic activity of vanadyl remained unaffected. Aneuploidy effect in the D61M strain of Saccharomyces cerevisiae was induced by either V5 or V4, confirming that vanadium compounds are potentially antitubulin agents in eukaryotic cells. Although these compounds are very toxic in V79 cells, no mutagenic effect was observed in the presence or in the absence of S9 fraction.