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G Bronzetti

Publications and source records attributed to G Bronzetti.

At least 55 records · Page 3Linked to original sources
PubMed1991

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.

5-Methoxypsoralen↗
PubMed1990

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.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1989

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.

Animals↗
PubMed1988

EDTA affects cytochrome P450-dependent biotransformation reactions during incubations for the liver microsomal assay.

In order to optimize the condition of the liver microsomal assay (LMA), studies were carried out to determine the effects of EDTA on mixed-function oxidase activity and its stability under the exact incubation conditions for the LMA. Aminopyrine N-demethylase (APD) and p-nitroanisole O-demethylase (p-NAD) activities as well as lipid peroxidation development (LP) in S9 liver fractions from beta-naphthoflavone and sodium phenobarbital (beta-NF + PB)- or Aroclor 1254 (AC)-treated mice were examined during a period of preincubation with EDTA ranging from 1 to 40 mM. At 5 mM EDTA, we obtained a strong inhibition of the microsomal LP as well as the greatest value of the mean specific activity (Asp) for both APD and pNAD activities. In agreement with the biochemical data, the presence of 5 mM EDTA in the incubation mixtures for the LMA significantly increased the mitotic gene conversion, mitotic crossing-over and point-reverse mutation of the well-known premutagen cyclophosphamide (30 mM) on the diploid D7 strain of Saccharomyces cerevisiae as the outcome of a greater metabolic activity. We concluded that the systematic use of 5 mM EDTA in LMA mixtures could improve the reliability and sensitivity of such a test.

Aminopyrine N-Demethylase↗
PubMed1987

NADPH as rate-limiting factor for microsomal metabolism. An alternative and economic NADPH-generating system for microsomal mono-oxygenase in in vitro genotoxicity studies.

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.

Animals↗
PubMed1987

Stability of drug metabolizing enzymes during the incubation conditions of the liver microsomal assay with non-induced and induced mouse liver S-9 fractions.

The purpose of this work was to study the relative activities and stabilities of phase-I and phase-II drug metabolizing enzymes in incubation mixtures used in vitro genotoxicity testing in order to optimize the conditions of the assay, increase sensitivity and eliminate false negative results. Cytochrome P-450, NADPH-cytochrome P-450 (cytochrome c) reductase activity and various phase-I and phase-II enzyme activities of the drug-metabolizing system were determined in incubation mixtures used in liver microsomal assays. The behaviour of aminopyrine N-demethylase and p-nitroanisole O-demethylase activities as phase-I markers have been reported previously. Other activities measured were glutathione S-transferase, glutathione S-epoxide transferase and epoxide hydrase, and lipid peroxidation (LP) was determined. The experiments were carried out on liver S9 fractions derived from non-induced mice or mice induced with sodium phenobarbital (PB), and/or beta-naphthoflavone (beta-NF). The phase-II enzymes were much more stable (70-90% residual activity) than phase-I enzyme activities (35-60%) in all conditions tested. The residual cytochrome P-450 was approximately 70% stable and the remaining activity of NADPH-cytochrome c-reductase about 80%, indicating that this latter enzyme does not limit the rate of the monoxygenase system in these conditions. Phase-II enzymes were induced to a smaller extent (about 2 times) than in phase-I enzymes (5-6 times) by beta-NF + PB. NADPH-cytochrome c-reductase behaved as phase-II enzymes in this respect as well as for stability. LP was appreciably higher in non-induced than in induced animals. Treatment with the beta-NF + PB mixture, however, showed that induced enzymes were more stable than those obtained by simple induction with either beta-NF or PB alone. These results lead to the conclusion that prolonged incubation times in mutagenicity assays are unnecessary when considering the relative stabilities of the various phase-I and phase-II enzyme activities in the drug-metabolizing system.

Animals↗
PubMed1987

Methylglyoxal: genotoxicity studies and its effect in vivo on the hepatic microsomal mono-oxygenase system of the mouse.

The genotoxic potential of methylglyoxal (MG) was studied in Saccharomyces cerevisiae D7 and in Salmonella typhimurium TA97 and TA102 in the presence and in the absence of metabolic activation system (S9 fraction) prepared from mouse liver induced with beta-naphthoflavone (beta-NF) and sodium phenobarbital (PB). The in vivo effects on the hepatic microsomal mixed function mono-oxygenase system induced by MG were studied in untreated, beta-NF or PB pre-treated mice. MG was a direct-acting mutagen in S. typhimurium TA97 and TA102 when tested up to a maximum concentration of 0.47 mg/plate. Mitotic gene conversion was also induced by MG in the yeast S. cerevisiae D7. A weak but significant effect on reverse point mutation was also found in S. cerevisiae. Genetic activity was lower in the presence of S9 fraction in yeast test. In the in vivo studies, MG (at the total dose of 600 mg/kg) was shown to increase the aminopyrine N-demethylase (APD) and p-nitroanisole O-demethylase (p-NAD) activities in uninduced mice. Cytochrome P-450 content (cyt P-450) and ethoxycoumarin O-deethylase activity (ECD) were also weakly enhanced by MG treatment. In contrast, no significant changes in mono-oxygenase activities were seen in beta-NF- or PB-treated mice after MG injection.

Aldehydes↗
PubMed1986

Multiple inducible cytochromes P-450 in yeast.

A form of cytochrome P-450 is produced in Saccharomyces cerevisiae during the logarithmic growth phase. This form is inhibited by metyrapone, tetrahydrofuran, and carbon oxide which are inhibitors of ethanol-inducible cytochromes P-450 of mammals. Addition of Na-phenobarbital to the 0.5% glucose liquid medium induced a form of cytochrome P-450 inhibited only by metyrapone. Thus, yeast possess multiple forms of cytochrome P-450 that are individually inducible.

4-Nitroquinoline-1-oxide↗
PubMed1986

Inducibility of gene conversion in Saccharomyces cerevisiae treated with MMS.

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.

DNA Repair↗
PubMed1986

Genetic activity of 2-aminofluorene in the salmonella/erythrocyte mutagenicity assay.

In a previous study, we demonstrated the activation of cyclophosphamide by mouse erythrocytes in a yeast test using the D7 strain of Saccharomyces cerevisiae. The present study provides further information on the ability of washed red blood cells from mice to activate 2-aminofluorene (2-AF) detected as an increase in mutation frequency of the tester strain, TA1538 (frameshift mutation) of Salmonella typhimurium. The 2-AF was tested at different concentrations (1-8 micrograms/plate) using both the liquid-suspension test and the agar-plate test. For comparison, the bioactivation of 2-AF by the hepatic postmitochondrial supernatant (S9 fraction) from Aroclor-1254-induced rats was studied. 2-AF was only found to be clearly mutagenic in the agar-plate test with both activation systems. The genetic response obtained with the erythrocytes appeared to be related to the number of cells/plate. At the lowest dose, slight differences are observed when genotoxic effects were compared to those with the S9 fraction.

Animals↗
PubMed1986

Specific inhibitors of the monooxygenase system of Saccharomyces cerevisiae modified the mutagenic effect of 4-nitroquinoline 1-oxide and the deethylation activity of the yeast.

A form of cytochrome P-450 is produced in Saccharomyces cerevisiae strain D7 during the logarithmic growth phase in 20% glucose liquid medium. This form was inhibited by metyrapone, tetrahydrofuran and by carbon monoxide, specific inhibitors of cytochrome P-450 in mammals. The inhibition was observed by means of the increase of the genetic activity of 4-nitroquinoline 1-oxide (4-NQO) on logarithmic growth phase cells of D7 strain, adding the inhibitors to the incubation mixture. 4-NQO is a strong direct mutagen on stationary phase cells that is detoxified by the monooxygenase system. The inhibition was measured by determining the decrease of the O-deethylation of 7-ethoxycoumarin in whole cells of yeast depending on different concentrations of inhibitors. A decrease of O-deethylation activity was found in the presence of tetrahydrofuran and metyrapone and this behaviour is typical of the cytochrome P-450 species induced by ethanol, as in mammals. Adding sodium phenobarbital to 0.5% glucose liquid medium, a form inhibited only by metyrapone was obtained. The presence of different inducible forms of cytochrome P-450 is evident.

4-Nitroquinoline-1-oxide↗
PubMed1985

Erythrocytes-mediated metabolic activation of cyclophosphamide in yeast mutagenicity test.

The degree of conversion of cyclophosphamide (CP) into mutagenic intermediates was studied using mouse erythrocytes as the metabolic activation system. The amount of mutagenic intermediates produced was measured indirectly in terms of induced frequencies of mitotic recombination, mitotic gene conversion, and reverse mutation in the diploid D7 strain of Saccharomyces cerevisiae. In the absence of S9 microsomal fraction or erythrocytes, CP did not induce any genetic response. In the presence of erythrocytes, on the other hand, CP clearly induced increases in the three genetic endpoints. The responses, however, were lower than those observed with the S9 activation system. The activating principle seems to be the oxyhemoglobin. In fact, neither p-nitroanisole O-demethylase activity nor genotoxic responses performed with red-blood cells from uninduced and PB-induced mice indicate that (possible) water-soluble forms of cytochrome P-450 were responsible for the activation of CP by erythrocytes.

Alanine Transaminase↗
PubMed1985

Study of the optimal temperature for the liver microsomal assay with mice S9 fractions.

The effect of temperature on enzymatic activity and stability was studied with respect to the monooxygenase activities of aminopyrine-N-demethylase (APD) and p-nitroanisole O-demethylase (pNAD) under incubation conditions for the liver microsomal assay. The activities of S9 liver fractions of mice induced with sodium phenobarbital and beta-naphthoflavone were determined during a period of preincubation in a range of temperatures from 30 to 44 degrees C. The greatest value of the mean specific activity was found at 40-42 degrees C for both APD and pNAD. The rapid increase of lipid peroxidation after 1 h of incubation at temperatures higher than 42 degrees C can provide an explanation of the enhancement of the rate of inactivation. In order to determine whether biological response is affected by the modifications induced by temperature in the metabolic activating system, tester strain D7 of Saccharomyces cerevisiae was used to assay the genetic activity of the well known premutagenic agent cyclophosphamide by incubating the mixtures both at the traditional temperature of 37 degrees C and at 42 degrees C. We suggest that the use of more favourable conditions for LMA with respect to enzymatic activity, than the traditional ones could improve the reliability and the sensitivity of such tests.

Aminopyrine N-Demethylase↗
PubMed1985

Conditions that influence the genetic activity of potassium dichromate and chromium chloride in Saccharomyces cerevisiae.

Potassium dichromate and chromium chloride were analyzed for their ability to induce mitotic gene conversion and point reverse mutation in D7 diploid strain of S. cerevisiae. We used cells from the stationary phase of growth with and without metabolic activation (S9 hepatic fraction) and cells from the logarithmic phase, that contain a high level of cytochrome P-450 and have a greater permeability. In the present work we confirmed the genetic activity of K2Cr2O7 in cells from the stationary phase, with and without S9 fraction and in cells from the logarithmic growth phase. A slight increase in genetic activity was observed in experiments with CrCl3 using phosphate buffer, but no genetic effects were noted in Tris-HCl buffer. Our studies suggest that phosphate ion may be the carrier responsible of the entrance of trivalent chromium in the cells. The higher cellular permeability may account for the different results obtained with both compounds in cells from the stationary and logarithmic phases of growth.

Biotransformation↗