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Dietary modulation of carcinogenesis-related pathwaysDietary item or component studied: hioglucosidic precursor glucoraphanin (GRP).(Brasicaceae)Pathways studied: up-regulation of the xenobiotic-detoxifying Phase-II enzymes and Down regulation of the xenobiotic- activating Phase-I enzymesStudy type (in vitro, animals, humans): Sprague-Dawley ratsMode of exposure (if in vivo) (acute, chronic, root of exposure): administration through dietImpact on pathway (including dose-response): CYP2E1 activity (P < 0.01) increased in rats treated with 240 mg/kg b.w. GRP (up to 1.8- and 2-fold, in males and females, respectively).CYP1A1-linked EROD monooxygenase (P < 0.01) induced (up to 4.6- and 3.4-fold, in males and females, respectively) at the higher dose.CYP2B1/2-supported PROD monooxygenase increasedup to ?5.5-fold following repeated administration of 120 mg/kg b.w. GRP, and the 'mixed' ECOD activity as well, which was (P < 0.01) induced up to ?1.8-fold, at the higher dose tested.Repeated administration of GRP: the CYP1A1/2 (7_ position), was the most affected one, reaching an 8.8-fold increase (P < 0.01) in males treated with 240 mg/kg b.w. GRPTestosterone 2_-(CYP3A1/2, CYP1A1) hydroxylase in female rats, with an 8.4-fold increase at the higher dose compared with a 4.4-fold (P < 0.01) increase recorded in malesThe CYP2B1/2-supported 16_- (CYP2B1) testosterone hydroxylase was significantly (P < 0.01) affected by the lower GRP dosage, with a 2.1- and 2.2-fold increase. KEYWORDS CLASSIFICATION: analogs & derivatives;Animals;Anticarcinogenic Agents;Biological Markers;Blotting,Northern;Blotting,Western;Brassica;chemistry;Chromatography,High Pressure Liquid;Cytochrome P-450 Enzyme System;drug effects;dietary modulation of carcinogenesis-related pathways;Dietary Supplements;enzymology;Electron Spin Resonance Spectroscopy;Fluorometry;Free Radicals;Glucose;Imidoesters;Italy;Liver;metabolism;Metabolic Detoxication,Phase I;Metabolic Detoxication,Phase II;Molecular Probes;pharmacology;Rats;Rats,Sprague-Dawley;Research;Thiocyanates;Xenobiotics. Epidemiological and animal studies linking high fruit and vegetable consumption to lower cancer risk have strengthened the belief that long-term administration of isolated naturally occurring dietary constituents could reduce the risk of cancer. In recent years, metabolites derived from phytoalexins, such as glucoraphanin found in broccoli and other cruciferous vegetables (Brassicaceae), have gained much attention as potential cancer chemopreventive agents. The protective effect of these micronutrients is assumed to be due to the inhibition of Phase-I carcinogen-bioactivating enzymes and/or induction of Phase-II detoxifying enzymes, an assumption that still remains uncertain. The protective effect of glucoraphanin is thought to be due to sulforaphane, an isothiocyanate metabolite produced from glucoraphanin by myrosinase. Here we show, in rat liver, that while glucoraphanin slightly induces Phase-II enzymes, it powerfully boosts Phase-I enzymes, including activators of polycyclic aromatic hydrocarbons (PAHs), nitrosamines and olefins. Induction of the cytochrome P450 (CYP) isoforms CYP1A1/2, CYP3A1/2 and CYP2E1 was confirmed by Western immunoblotting. CYP induction was paralleled by an increase in the corresponding mRNA levels. Concomitant with this Phase-I induction, we also found that glucoraphanin generated large amount of various reactive radical species, as determined by electron paramagnetic resonance (EPR) spectrometry coupled to a radical-probe technique. This suggests that long-term uncontrolled administration of glucoraphanin could actually pose a potential health hazard.

This work aimed to investigate whether the insecticide acephate (125 or 250 mg/kg b.w.) or diflubenzuron (752 or 1075 mg/kg b.w.), two of the most widely used pesticides worldwide, impairs CYP-linked murine metabolism in liver, kidney and lung microsomes after repeated (daily, for three consecutive days) i.p. administration. The regio- and stereo-selective hydroxylation of testosterone was used as multibiomarker of different CYP isoforms. Both gender and tissue specific effects were observed. Lung was the most responsive tissue to induction by lower diflubenzuron dose, as exemplified by the marked increase of testosterone 7alpha-hydroxylation (CYP2A) (up to 13-fold) in males. Higher dose produced a generalized inactivation. At the lower dose acephate induced 6beta- (CYP3A1/2, liver) as well as 2beta- (CYP2B1/2, kidney) hydroxylase activities ( approximately 5 and approximately 4-fold increase, respectively) in males. In females, a marked suppression of the various hydroxylations was observed. At 250 mg/kg of acephate, animals did not survive. Induction of the most affected isoforms was sustained by immunoblotting analysis. Corresponding human CYP modulations might disrupt normal physiological functions related to these enzymes. Furthermore, the co-mutagenic and promoting potential of these pesticides, phenomena linked to CYP upregulation (e.g. increased bioactivation of ubiquitous pollutants and generation of oxygen free radicals) are of concern for a more complete definition of their overall toxicological potential.

An elevated level of total plasma homocysteine (tHcy) is considered to be a predictor of the mortality risk for all diseases. A high consumption of vegetables and citrus fruit, both good sources of folate, decreases the concentration of tHcy. We investigated if supplementation of concentrated fruit and vegetables is able to decrease tHcy concentrations. For this purpose, we used a product that is presently on the market, made up of fruit and vegetable powders. 26 subjects participated in a cross-over design intervention trial. At the end of the study, each participant received 2 capsules of fruit and 2 capsules of vegetables a day for 4 weeks and then acted his/her own control for another 4 weeks. Daily extract intake decreased plasma tHcy from an expected mean ± SD level of 12.71 ± 3.23 umol/L observed after the 28-day control period to 7.98 ± 1.70 umol/L, a difference of 4.73 ± 1.153 (p Elevated tHcy is a risk factor causally linked to chronic disease. The plasma tHcy concentration was decreased as a result of taking a powdered fruit and vegetable extract on a daily basis.

In the last 10 years, there is an increasing interest in selenium (Se) because of its environmental, biological, and toxicological importance, and in particular, because of its antioxidant properties. However, inspite of extensive studies, the optimal concentration of Se to be used for its beneficial effects in not yet known. In addition, the mechanisms of Se antioxidant property require further study. We report on the effects of various mutagens/carcinogens such as azoxymethane, methylmethanesulphonate, and hydrogen peroxide on Chinese V79 hamster cells, in presence of sodium selenite in the concentration of 0.5 μM. We found that Se reduced the genotoxic effect of these mutagens/carcinogens. We also investigated enzymatic activities of glutathione peroxidase, catalase, superoxide dismutase, and glutathione S-transferase, in order to understand the Se involvement in the detoxification of free radicals. Sodium selenite increased the activities of glutathione peroxidase and catalase.

Antimutagens and anticarcinogens are known to play an important role in decreasing damages induced by oxidants. In this study, we investigated the genotoxic and antimutagenic potential of two selenium compounds (sodium selenite: Na(2)SeO(3); seleno-DL-methionine: C(5)H(11)NO(2)Se) and Vitamins A and E in yeast cells of Saccharomyces cerevisiae. An oxidative mutagen (hydrogen peroxide (H(2)O(2)), HP) was chosen as positive control. We determined the enzymatic activities involved in the protection against oxidative damages (catalase: CAT; superoxide dismutase: SOD; glutathione peroxidase: GPx) in the cytosolic extract of yeast cells. The results demonstrated that selenium compounds exerted both mutagenic and antimutagenic effect at different concentrations. Antimutagenesis was evident both in stationary and in logarithmic phase cells. Catalase, SOD, and GPx were significantly increased in the presence of all the compounds assayed. Vitamins A (retinol) and E (alpha-tocopherol) did not have toxic or mutagenic action.

Murine S9 liver fractions isolated from mice fed 7.5 g kg-1 2(3)-tert-Butyl-4-hydroxyanisole (BHA) for 3 weeks were tested to determine: (a) the profile of both phase-I and phase-II xenobiotic metabolizing enzymes; (b) their ability to induce in vitro covalent binding of some precarcinogens to calf thymus DNA; and (c) their activation in a standard genetic toxicology assay. With regard to phase-I pathway, the S9 fraction expressed various cytochrome P-450-(CYP) (classes 1A1, 1A2, 2B1, 2E1, and 3A)-dependent biotransformation enzymes at levels comparable with those present in murine control liver. For post-oxidative enzymes, the S9 expressed high levels of glutathione S-transferases (up to 12-fold increase), glutathione S-epoxide-transferase (up to 2.6-fold), UDP-glucuronosyl transferase (up to 5.3-fold) and epoxide hydrolase (up to 2.6-fold) activities, as compared to untreated mice. The in vitro DNA binding of the precarcinogenic agents [14C]-1,4-dichlorobenzene, [14C]-1,2-dichlorobenzene and [14C]-1,4-dibromobenzene, mediated by BHA-induced cytosol and/or microsomal preparation, showed an increase in specific activity comparable to that observed with phase-I (PB/beta NF) induced S9. In some instances, covalent binding was even more elevated using the BHA-induced systems as compared with traditional S9 fractions. For example, cytosol derived from BHA-administered mice was able to induce a significant binding to calf thymus DNA up to 26.2-fold increase for [14C]-1,4-dichlorobenzene, while cytosol from PB/beta NF was not. A high mutagenic response on diploid D7 strain of Saccharomyces cerevisiae as exemplified by a marked induction of mitotic gene conversion and point (reverse) mutation confirmed that BHA-derived S9 fractions activate precarcinogens to final genotoxins. Because a number of chemicals are activated by either oxidative or post-oxidative enzymes, the use of metabolizing biosystems, with an enhanced phase-II pathway, together with classical S9 fractions, can improve the sensitivity of the assay in detecting unknown genotoxins.

“In vivo” effects of a commercial preparation of zineb on monooxygenase activities were examined on hepatic microsomes of induced and not induced (basal) rats. Moreover additional “in vitro” experiments with the D7 strain of yeast Saccharomyces cerevisiae and Salmonella typhimurium were performed. Our results show that zineb caused a decrease in cytochrome P‐450 (cyt P‐450) content and in some enzymatic activities such as aminopyrine‐N‐demethylase (APD), p‐nitroanisole‐O‐demethylase (p‐NAD) and aniline hydroxylase (AnH). Immunoblotting analysis, using anti P‐450 2E1 antibodies, confirmed data obtained in AnH activity. Experiments on yeast Saccharomyces cerevisiae showed that zineb induced marked increase of gene conversion and point reverse mutation in logarithmic growth phase cells. Finally, in experiments with Salmonella typhimurium, zineb had not mutagenic effects. It can be concluded from “in vivo” and “in vitro” experiments that zineb has toxic action both in animals, expecially in basal rats, and in...

The effect of the antimutagenic flavoring cinnamaldehyde on the induction of HGPRT− mutants by methyl methanesulfonate (MMS), N-nitroso-N-methylurea (MNU), ethyl methanesulfonate (EMS) and (UV) light was investigated in the Chinese hamster V79 cell line. Cinnamaldehyde did not show any mutagenic or toxic effects in this experimental system by itself and did not modify mutation frequency when given to cells simultaneously with chemical mutagens. Under these conditions, the cytotoxicity of MMS, but not that of MNU and EMS, was increased. Cell viability was also reduced in MNU-, EMS-, or UV light-pretreated cells when they were postincubated in the presence of cinnamaldehyde. Moreover, cinnamaldehyde reduced the frequency of mutations induced by MMS but not by the other mutagens. The results obtained suggest that cinnamaldehyde inhibits some cellular function(s) promoting the repair of a variety of different cytotoxic lesions. At the same time, stimulation by cinnamaldehyde of an error-free DNA repair mechanism acting on MMS-induced mutagenic damage was indicated.

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 Mr 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.

The genotoxicity of dimethylamine (DMA) was investigated in the D7 strain of Saccharomyces cerevisiae. DMA was able to induce mitotic gene conversion and point reverse mutation in the presence of metabolic activation (S9 fraction). To study the co-mutagenicity/co-carcinogenicity or toxicity of DMA, changes in xenobiotic metabolizing enzymes were studied in purified microsomes from DMA-induced mice or rats receiving a single or three consecutive doses (25 or 50 mg/kg body wt). Pentoxyresorufin O-dealkylase (class IIB1 P450, PROD), ethoxyresorufin O-deethylase (IA1, EROD) and p-nitrophenol hydroxylase (IIE1, pNPH) were all affected by DMA treatment with a loss of activity of 62, 55 and 54% in the mouse, or 64, 25 and 56% in the rat for PROD, EROD and pNPH activities, respectively, at the highest tested dose. No significant alteration of P450 IIIA-like activity was seen. Results indicated that the metabolites of DMA induced genetic activity in yeast. In addition, a clear non-specific hepatotoxic effect was recorded as a significant reduction in activity of the selected monooxygenases.

A commercial preparation of atrazine was tested in diploid strain (D7) of yeast Saccharomyces cerevisiae in suspension test using cells from logarithmic growth phase (with a high level of cytochrome P‐450) and stationary growth phase. This compound induced a marked increase of both gene conversion and point mutation in logarithmic phase cells, while in stationary phase cells only an increase of gene conversion was observed. Results obtained employing TA98 and TA100 strains of Salmonella typhimurium (Ames test) confirm that atrazine has no mutagenic effect. The in vivo effects on cytochrome P‐450 content (Cyt.P‐450), 7‐ethoxycoumarin‐O‐deethylase (ECD), aminopyrine‐N‐demethylase (APD), p‐nitroanisole‐O‐demethylase (p‐NAD) activities were examined in hepatic microsomes from Naphenobarbital and β‐naphthoflavone induced pigs 24 h after acute intoxication with atrazine. Our results indicated a marked decrease of cytochrome P‐450 level and of microsomal protein content, suggesting a hepatotoxic effect of atrazi...

The in vivo effects of a commercial preparation of maneb on mono-oxygenase activities of hepatic microsomes of basal and induced rats were examined. In vitro experiments with the D7 strain of yeast Saccharomyces cerevisiae were also performed. In both basal and induced rats maneb caused a decrease in cytochrome P-450 content and aniline hydroxylase. Immunoblotting analysis using anti-P-450 IIE1 antibodies confirmed the data obtained for aniline hydroxylase activity. Maneb was toxic in cells of S. cerevisiae. On the basis of in vivo and in vitro experiments it can be concluded that maneb possesses a toxic activity attributable to its main metabolite ethylene thiourea. Immunoblotting analysis indicates that maneb biotransformation influences the IIE1 P-450 isoform.

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 baal 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 inhibitor effect was determined in some of the assayed activities and in the relative peculiar P-450 isoforms detected by Western blot analysis.

Spermine is a polyamine found in bacteria, animal, and plant tissues. It is involved in a variety of biological processes, and its interaction with DNA stabilizes the secondary structure of the double helix. Spermine is one of the first reported antimutagens, reducing the mutation rate in several prokaryotic test systems, while in eukaryotic organisms conflicting results have been obtained. In light of the significant antimutagenic effect of spermine, it is important to evaluate its activity in mammalian cells in culture. The present study was undertaken to evaluate the ability of spermine to suppress the level of HGPRT- mutants induced by ethylmethanesulfonate, methylmethanesulfonate, and mitomycin C. Spermine reduced the mutation frequency induced by ethylmethanesulfonate and methylmethanesulfonate but did not affect survival; with mitomycin C survival was reduced but mutation rate was not influenced.

The role of diet in the etiology of cancer has received increasing attention in the last ten years (1-5). Consumption of milk reduced the incidence of human stomach cancer induced by alkylating agents (6). Human subjects (7) and experimental animals (8) receiving dietary supplements of Lactobacillus acidophilus exhibited significantly lower levels of fecal enzymes associated with colon carcinogenesis. In addition, extracts prepared from L. bulgaricus, L. casei, and L. acidophilus spent media were shown to inhibit the growth of Sarcoma and Ehrlich tumors, suggesting that lactobacillic cultures have an antitumorigenic effect (9,10).

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.

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.

Chlorinated ethanes as 1,1,1,2-tetrachloroethane (TTCE), hexachloroethane (HCE) and pentachloroethane (PCE) have a wide application in different fields and their use increases every yearl. They are used as accelerators in rubber, retardants in fermentation processes, degreasers and solvents in extraction processes2; moreover, they may occur as intermediates in the production of chlorinated ethylenes. These compounds are diffused in the environment; they have been detected in trace quantities in air, river and drinking-water1,3. Their extensive diffusion and widespread use indicate that human exposure occurs.

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.

It has been suggested that the majority of human cancers are caused by environmental exposures, but little is known about the effective environmental and occupational risks of cancer. It is likely that the process of mutagenesis and the intricate balance between mutagenesis and antimutagenesis are involved in aging, evolution, and other fundamental life processes (15).

Perchloroethylene (PCE) was tested in a diploid strain (D7) of the yeast Saccharomyces cerevisiae in suspension tests with and without a mammalian microsomal activation system (S9) and 'in vivo' by the intrasanguineous host-mediated assay. In addition, enzyme alteration studies were performed in mice non-pretreated or pretreated with phenobarbital + beta-naphthoflavone. PCE did not induce any genetic effect either 'in vitro' or 'in vivo'. In the suspension test, PCE was more toxic without metabolic activation and less toxic with mammalian microsomal activation. The enzymatic determinations showed an increase of the aminopyrine demethylase activity and of the level of cytochrome P-450.

The influence of NTA on chromium genetic activity was studied in the D7 strain of Saccharomyces cerevisiae. At low dose (subgenotoxic) of sodium chromate (CrVI) (5mM), NTA increased the point mutation while at higher dose (25 mM) of chromate in the presence of NTA a decrease of point reverse mutation was observed. Probably NTA affected either the uptake of Cr(VI) favouring the intracellular reduction to Cr(III), or the recombinational repair of DNA breaks induced by chromate oxiding activity. An increase of point (reverse) mutation was obtained in the experiments with NTA and chromium chloride, suggesting the hypothesis that NTA might interact with Cr(III) forming NTA‐Cr(III) complexes able to permeate cellular membranes and bind to DNA. In addition NTA genetic inactivity was confirmed.

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.

The influence of nitrilotriacetic acid (NTA), a substitute of poliphosphates in laundry detergents, on mitotic gene conversion and point reverse mutation induced by sodium chromate and chromium chloride was analyzed on D7 strain of Saccharomyces cerevisiae. NTA determined a significant decrease of chromate convertogenic activity while the induced point reverse mutation was not affected. This can be due to a direct interaction between chromate and NTA or to a possible NTA effect on recombinational repair of DNA‐breaks induced by the chromate oxidizing activity. Both chromium chloride and NTA dissolved into liquid growth medium inhibited cell growth but the simultaneous presence of both compounds allowed cells to grow. In this condition the cell population, growing up to the stationary phase, accumulated a significant number of convertants and revertants. Our results indicate that NTA, interacting with Cr3+ , probably forms NTA‐Cr3+ complexes able to cross the membranes and to react with DNA. In addition th...

Mytilus galloprovincialis has a hepatopancreatic monooxygenase activity cytochrome P450-dependent. The present paper studies the induction of this activity in the hepatopancreatic microsomes after treatment of M. galloprovincialis with Na-phenobarbital. Mussels were collected from the Gulf of La Spezia in 1985. We measured the increase in levels of cytochrome P450, b5, and P420 and the 7-ethoxycoumarin deethylase activity. Furthermore, the fraction of mouse liver was replaced by the correspondent mussels' hepatopancreatic fraction in the yeast genotoxicity test to determine the ability of mussels to biotransform cyclophosphamide and styrene oxide. The results suggest that edible mussels are capable of detoxicating styrene oxide but not of bioactivating cyclophosphamide.

The genotoxicity of chromium chloride was investigated in cells of D7 strain of Saccharomyces cerevisiae harvested either from logarithmic or stationary growth phase. A weak induction of mifotic gene conversion and point reverse mutation was obtained when the incubations were performed using phosphate buffer. No genetic effect was observed when the incubations were performed using Tris‐HCl buffer. The experiments with 51Cr radiotracer demonstrated that Cr3+ ion enters the yeast cells and binds to DNA even if the incubation mixture was performed with Tris HCl buffer. This behaviour could be due to the highest concentration of CrCl3 that cause some damages to cytoplasmatic membrane.

Aminopyrine- N -demethylase and p -nitroanisole- O -demethylase activities were determined in incubation mixtures for the liver microsomal assay at time zero and after 1 h of incubation in the conditions for the mutagenic assay. The experiments were performed with the S9 liver fraction of mice in the basal state and induced with sodium phenobarbital, β-naphthoflavone or both. Lipid peroxidation was also determined. The experiments were repeated with female mice and also in the presence of styrene, as an example of a xenobiotic substance. The activity of pNAD was much more stable than that of APD in all the conditions tested. The pattern of stability, however, was similar for the two activities: more stable than controls with S9 fractions from β-NF-induced mice, less stable than controls in PB-induced mice, intermediate between controls and PB-induced mice in those with combined induction by PB + βNF. Styrene 50 mM in the incubation mixtures led to a marked inactivation of enzymic activity, similar in all cases and reaching about 90% in 1 h. S9 fractions from female mice gave enzymes slightly more stable in almost all cases. Lipid peroxidation was appreciably more elevated in basal than in induced animals. It was concluded that, for a mutagenesis test on an unknown xenobiotic, S9 fractions from mice following PB and β-NF induction are to be preferred from the point of view of activation.

Incineration of industrial and urban waste materials is an important problem for the environmental contamination and therefore for human health. Environmental contaminants spread by urban incinerators can contain a complex mixture of toxic compounds such as dioxin, benzofurans, alogenate acids. It is important to evaluate the genetic damage induced by complex mixtures widespread in the environment. In the present work, the genotoxic activity of samples obtained from the urban incinerator of Florence was analyzed. The results were compared with those obtained with samples drawn from the urban Snamprogetti incinerator of Schio (Vicenza), where halogenated acids contained in the smoke are neutralized with the lime wash in a salification column. Samples were tested using prokaryotic (Salmonella typhimurium TA98, TA100 and TA102 strains) and eukaryotic (Saccharomyces cerevisiae, D7 strain) microorganisms. These systems permit one to obtain rapid, reproducible and reliable results in order to evaluate the genotoxic activity of substances present in the environment.

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-nitroanisoleO-demethylase (p-NAD) activities as well as lipid peroxidation development (LP) in S9 liver fractions from β-naphthoflavone and sodium phenobarbital (β-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.

The wide variety of genetic alterations that can be induced in human populations when exposed to chemical genotoxic substances present in our environment may be predictable using laboratory organisms such as yeasts. In the present paper methodologies are described for analysing the genetic effects induced by a well known chemical mutagen, ethyl methanesulfonate (EMS). Haploid or diploid yeast cells have been treated in vitro , in buffer or in the presence of mouse liver microsomes, and in vivo , in the peritoneum of the mouse (host-mediated assay). With these different methods of assaying the genetic activity of a compound, its metabolic activation occuring in the mammalian body is taken into account: this might lead to a more reliable extrapolation of data from laboratory experiments to man. The relationships between doses and frequencies of the induced genetic effects are described by equations obtained after regression analysis of the data, thus allowing a quantitative comparison among different methodologies and different genetic systems. One genetic system analyzed is represented by forward-mutations scored phenotypically on a non selective medium. Mutations induced in five loci with different sensitivity and average data of mutation-induction per locus have been derived. The second genetic system was provided by scoring on a selective medium mitotic gene-conversions induced in two loci with different kinetics. Haploid cells of Schizosaccharomyces pombe and diploid cells of Saccharomyces cerevisiae were submitted to analysis for the evaluation of gene-mutations and gene-conversions respectively.

Potassium dichromate and chromium chloride were analyzed for their ability to induce mitotic gene conversion and point reverse mutation in D 7 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 K 2 Cr 2 O 7 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 CrCl 3 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.

Commercial compounds used in the leather and tannery industries near Florence were tested in Salmonella‐mammalian microsomes mutagenicity test and in yeast suspension test with and without metabolic activation. Eight compounds of the 28 tested increased the number of his+ revertants in Salmonella typhimurium and five of them increased the recombinants in yeast.

TCDD (2,3,7,8-Tetrachlorodibenzo-p-dioxin) induces genetic effects both “in vitro” with and without metabolic activation and “in vivo” Intrasanguineous host mediated assay using D7 strain of S.cerevisiae . Ashes from urban incenerator of S.Donnino (Florence, Italy) by thenselves and its acqueuos extract induced strong genetic effect and alteration of enzyme levels.

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.