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M Paolini

Publications and source records attributed to M Paolini.

At least 109 records · Page 6Linked to original sources
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↗
PubMed1987

Inducibility of NADPH cytochrome c (P-450) reductase in yeast: role in the bioactivation of nitroimidazo(2,1-b)thiazoles.

Nine nitroimidazo(2,1-b)thiazoles were tested for their ability to induce convertants, revertants and mitotic recombinants on growing and stationary phase cells of Saccharomyces cerevisiae D7 strain. All compounds were genetically active only on cells from the logarithmic growth phase (from a 20% glucose medium), inducing dose-related increases in conversion and reversion frequencies. The addition of S9 microsomal fraction to stationary phase cells gave positive results at levels of doses 10-100 times lower than that used with growing cells. NADPH cytochrome c (P-450) reductase activity was determined in order to compare the capability of hepatic and yeast microsomes in bioactivating the test compounds. Results showed that nitroreductive enzymes are inducible by conditions supporting growth. The greater sensitivity of the treatment with external metabolic activation could be due to the greater content (about 14-fold) of NADPH cytochrome c-reductase activity in hepatic microsomes.

Animals↗
PubMed1987

In vivo protective role of antioxidants against genotoxicity of metronidazole and azanidazole.

The mutagenicity of metronidazole and azanidazole has been extensively reported. Previous experiments demonstrated, by means of the intrasanguineous host-mediated assay, that they significantly induced mutagenicity in liver, kidney and lung of mice. The treatment of mice with butylated hydroxyanisole (BHA) or butylated hydroxytoluene (BHT) by two different routes of administration (i.p. injection and oral intubation) significantly reduced liver- and kidney-mediated mutagenicity of azanidazole and metronidazole. No significant differences were observed between the routes of treatment in terms of protective effect on genotoxicity of azanidazole in the considered organs, whereas i.p. administration was the most suppressive on the mutagenicity of metronidazole. Even if BHT was the most effective agent in preventing mutation induction in mice, a detectable toxicity, in terms of increased mutagenicity, was evaluated in the liver. Evidence of lung abnormalities was also seen. The results suggest that the possible adverse effects on biological systems limit the prophylactic use of BHA and BHT in preventing the action of chemical carcinogens in man.

Administration, Oral↗
PubMed1986

The direct-acting mutagenicity of nitroimidazo[2,1-b]thiazoles in Salmonella typhimurium.

A series of nitroimidazo[2,1-b]thiazole derivatives was investigated for direct-acting mutagenic potency with the Salmonella assay. All of the nine derivatives tested were mutagenic. The compounds induced predominantly base displacements resulting in frame-shift mutations. The mutagenic activity did not require the S9 fraction but was largely dependent on "classical" bacterial nitroreductase. The primary basis of the mutagenic activity of nitroimidazo[2,1-b]thiazoles appears to be a reduction of the nitro-function to the corresponding hydroxylamine. Mutagenicity seems to be paralleled by an increase of the nitro groups: dinitroderivatives were more active than nitroderivatives. Other electrophiles and sterically constrained nitro groups could account for differences in genotoxicity.

Mutagenicity Tests↗
PubMed1986

Structure-activity relationship of nitroimidazo (2,1-b) thiazoles in the Salmonella mutagenicity assay.

The mutagenic activity of a series of nitroimidazo (2,1-b) thiazoles was determined in Salmonella typhimurium TA-100 strain by means of the Ames test. A multiple regression analysis showed a highly significant parabolic relationship between mutagenic activity and the Rm values as an expression of the lipophilic character of molecules. The lipophilic character should be important in determining an optimal cell permeation. However when the lipophilic character was expressed by means of the sigma pi values the equation was much less satisfactory. This could be due to the fact that the Rm values of nitroheterocyclic compounds actually represent a measure both of lipophilic and polar character.

Mutagenicity Tests↗
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↗
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

The organospecific activity of metronidazole and azanidazole in the intrasanguineous host-mediated assay.

The genotoxicity of nitroimidazoles and, in particular, their potential carcinogenicity has been demonstrated. In order to investigate the specific target organ(s) for these drugs or their metabolites, a method for measuring mutations in microorganisms, with reference to the metabolism of mammals, was used in mice. Metronidazole and azanidazole were tested for their ability to induce genetic effects in a diploid strain (D7) of Saccharomyces cerevisiae in the Intrasanguineous Host-Mediated Assay. The test compounds showed dose-related increases of point mutation and mitotic gene conversion frequencies in liver, kidney and lung. Azanidazole seemed to favour the kidney and the liver, although increases in genotoxicity were observed also in the lung. Metronidazole was toxic and induced both point mutation and mitotic gene conversion when recovered from the liver. Yeast recovered from the kidney and the lung showed an increase especially in point mutation. This work provides more information about the mechanisms involved in the mutagenicity of nitroimidazoles at the site of action.

Animals↗
PubMed1984

Comparative genetic activity of cis- and trans-1,2-dichloroethylene in yeast.

The cis and trans isomers of 1,2-dichloroethylene were tested for mutagenic effects in a diploid strain (D7) of the yeast Saccharomyces cerevisiae in suspension tests with and without a mammalian microsomal activation system, an S9 mouse liver fraction, and by an in vivo intrasanguineous host mediated assay. The effects of the same agents on aminopyrine N-demethylase activity and cytochrome P-450 level in liver were studied in nonpretreated and in phenobarbital + beta-naphtoflavone-pretreated mice. In the suspension test, both isomers exhibited dose dependent toxicity, and survival was lower with metabolic activation than without. In this test also, both isomers exhibited genetic activity as measured by increases in recombinants at the ade 2 locus in experiments with metabolic activation. In the host-mediated assay, only the cis isomer showed evidence of mutagenic activity with significant increases in convertants at the trp locus and revertants at the ilv locus. Such mutagenic activity was found both after acute and chronic doses and in liver, kidney, and lung tissue. The two isomers exhibited different effects with respect to aminopyrine N-demethylase activity and cytochrome P-450 level. In general, the trans isomer appeared to emphasize induction of enzyme activity or level while the cis isomer more frequently tended to inhibit activity or destroy the enzyme.

Aminopyrine N-Demethylase↗
PubMed1984

Effect of repeated ether anesthesias on the mono-oxygenase system of rat liver S-9 fraction.

This study was designed to investigate the effect of ether anesthesia in rats, before i.p. injections to induce the mono-oxygenase enzyme system, on biochemical properties of liver S9 fractions. Aminopyrine N-demethylase and rho-nitroanisole O-demethylase activity levels, their stability, and lipid peroxidation were determined in S9 fractions after etherization (about 1 min in ether vapor chamber daily for 3 consecutive days, before i.p. injections of Na-phenobarbital and beta-naphthoflavone) and compared with controls receiving the same injections without etherization. The activities were slightly (but not significatively) enhanced after this treatment, but stability was markedly and significatively greater after 1 h of incubation in the conditions of the liver microsomal assay (+ 14.8% and + 74.7%, respectively); lipid peroxidation was strongly and significatively depressed (-76.0%). Etherization sufficient to kill the animals on the 4th day resulted in equally active but less stable S9 fraction enzymes. Dimethylnitrosamine (as a standard premutagen) was assayed with the D7 strain of Saccharomyces cerevisiae using S9 fractions obtained from both anesthetized and nonanesthetized rats. According to biochemical data, results obtained with S9 from partially anesthetized rats were comparable with the conventional ones (S9 from nonanesthetized rats). On the contrary, the use of more prolonged ether anesthesia, including one on the day the animals are killed, gives S9 fraction significantly less effective. We conclude that if brief etherizations are used, for i.p. injections only, the S9 fractions obtained are entirely satisfactory and the procedures involved in production are simplified; the additional animal treatment (etherization) must be specified.

Aminopyrine N-Demethylase↗
PubMed1984

NADPH-generating system: influence on microsomal mono-oxygenase stability during incubation for the liver-microsomal assay with rat and mouse S9 fractions.

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.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1984

Genetic and biochemical investigation on chloral hydrate in vitro and in vivo.

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 beta-naphthoflavone (beta-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).

Aminopyrine N-Demethylase↗
PubMed1984

Studies of genetic effects in the D7 strain of Saccharomyces cerevisiae under different conditions of pH.

The genetic effects of variation in pH in culture media and in suspension tests were examined in a diploid strain (D7) of the yeast, Saccharomyces cerevisiae. Deviation from the normal pH of 6.24 in the liquid culture medium, has a significant effect on cellular growth and on mitotic gene conversion at the trp5 locus. Frequencies of reversion at the ilv I-92 locus and of mitotic crossing-over at the ade2 locus are not significantly influenced. Suspension tests, performed using phosphate buffer (pH 5.8), strongly confirm the original results. Our data suggest that the increase in mitotic gene conversion under various conditions of pH is due to a specific effect of pH itself on the cells of S. cerevisiae. In fact, increases were obtained using the same pH in both cellular growth and non-growth conditions. The maximum effect detected with both procedures was obtained at pH 5.8; in the growth test, at this pH, gene conversion frequency appeared to be most pronounced, being about 10 times higher than that of the control. These results suggest that pH exerts its specific action both on growing and non-growing yeast cells, and the difference in induction of genetic effect between these two conditions is probably due to a time factor.

Cell Division↗
PubMed1983

Mutagenicity of styrene on metabolizing D7 strain of saccharomyces cerevisiae.

The level of cyt.p-450 in the D7 strain of the yeast S.cerevisiae depended on the substrate supporting the growth, on its concentration, on the starting inoculated number of cells. (1) In the yeast grown on D-mannose where fermentation and respiration occurred concomitantly, cytochrome P-450 was also formed. It was detected a maximal concentration during the logarithmic phase when in the cultures there are about 50 . 10(6) cell/ml. We use cells harvested at this moment of the growth for mutagenesis tests. The tested substances were dimethylnitrosamine and styrene. DMNA to probe the sensibility of our cells and styrene that has always given contrasting results but from which the formation is known of genetically active metabolite: styrene oxide(6-7). Styrene gave positive results with our metabolizing yeast cells.

Culture Media↗