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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.

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.

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.

In the last few years there has been much concern about the problem connected to the exposure to mutagens present in the environment of industrialized countries. Particularly, the mutagenic activity of airborne particulate matter has been studied by many investigators and correlated with elevated lung cancer mortality rates. In most cases the Salmonella typhimurium/microsome test has been used for these studies. This short-term test, which is the most validated among the short-term genotoxicity tests, provides an important indication on the carcinogenic potential of environmental pollutants. That are complex mixtures containing a wide variety of compounds potentially capable of causing additive, antagonistic or synergistic genotoxic response in living organisms. Several studies have suggested that diverse factors, such as traffic and meteorological conditions, could affect the levels of pollutants in the air. In our work, we have investigated three different areas in Pisa, where the intensity and the kind of the road traffic were different. Airborne particles have been collected during a year and the genotoxic activity has been studied using TA98 and TA100 strains of Salmonella typhimurium. 20 refs., 1 fig., 3 tabs.

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.

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.

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.

Vinylidene chloride (VDC) was tested for its ability to induce both point mutation and mitotic gene conversion in a diploid strain (D7) of the yeast Saccharomyces cerevisiae in a suspension test with and without a mammalian microsomal activation system, and in the intrasanguineous host-mediated assay in mice. In suspension tests with D7, VCD was toxic but not genetically active without microsomal activation. When a mouse liver 10000Xg supernatant was included in the suspension tests, dose-related increases in both point mutation and mitotic gene conversion were seen at survival levels greater than 50%, at doses of VCD above 20 mM. In the host-mediated assay, VDC induced both point mutation and mitotic gene conversion when recovered from the liver and kidneys after both acute and sub-acute dosing. Yeasts recovered from the lungs showed little, if any, increase in either point mutation or mitotic gene conversion.

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.

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.

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

Styrene and its presumed metabolite, styrene oxide, were tested for their mutagenic effect on a forward mutation system of yeast and of Chinese hamster cells, and on a gene-conversion system of yeast. Experiments with liver microsomal preparations and host-mediated assay with yeast were also carried out. Styrene oxide was mutagenic in all test systems. Styrene was mutagenic only in the host-mediated assay.

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.

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 ρ-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 β-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.

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.

3 structurally related epoxides, 3,4-epoxycyclohexene, trans -1,2,3,4-diepoxycyclohexane and trans -3,4-epoxycyclohexane- r -1, trans -2-diol ( anti isomer) were tested for their ability to induce both point mutation, mitotic gene conversion and recombination in a diploid strain (D7) of the yeast Saccharomyces cerevisiae , with and without a mammalian microsomal activation system, and the formation of 6-thioguanine-resistant mutants in V79 hamster cells. Genetic effects were related to the alkylating properties of the epoxides, as measured by alkylation of4-( p -nitrobenzyl) pyridine (NBP). Of the 3 epoxides, only 3,4-epoxycyclohexene, characterized by the highest reactivity towards NBP, induced all genetic effects in both test systems. A marginal activity was shown by trans -1,2,3,4-diepoxycyclohexane only in the yeast. The lack of genetic activity of the anti isomer of 3,4-epoxycyclohexane-1,2-diol, in spite of the formal similarity of its functional groups with those present in mutagenic polycyclic arene epoxydiols, was attributed to the dramatic reduction of lipophilicity of the molecule.

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.

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.

This study was designed to investigate the possible genotoxic and teratogenic actions of diphenyl (DP), diphenyl ether (DPE), and their eutectic mixture, in a comparative approach including different test systems. Two microbial systems and a metazoan model were used: (1) diploid D7 strain of Saccharomyces cerevisiae; (2) Salmonella typhimurium strains TA100, TA98, TA1535, TA1537, TA1538, TA1532, TA2636; and (3) sea urchins (Paracentrotus lividus and Sphearechinus granularis). Both compounds resulted in severe toxicity in all of test organisms at levels greater than or equal to 10(-5) M (approximately 2 ppm). DP caused genetic effects in yeast with and without activating system, while the two chemicals appeared to be ineffective in Salmonella up to toxic levels. The action of DP and DPE on sea urchins resulted in developmental defects and mitotic abnormalities, following exposure of embryos or by pretreatment of sperm or eggs. In this system DPE appeared to be more effective than DP by about one order of magnitude (minimal active concentrations: 10(-5) M vs 10(-4) M). The eutectic mixture, industrially used as a heat transfer medium, was tested in its virgin and used form, for genotoxicity and embryotoxicity. The latter appeared to be more effective than the virgin eutectic. This increase in the embryo- and genotoxicity of the used eutectic may be related to the appearance of newly formed compounds in the heat transfer process. These compounds have been separated by high-pressure liquid chromatography and detected by fluorimetry.

The use of sodium phenobarbital (PB, CYP2B1 inducer) combined with β-naphthoflavone (β-NF, 1A1) to induce certain Phase I reactions in S9 liver fractions is a standard method for conducting short-term bioassays for genotoxicity. However, because post-oxidative enzymes are also able to activate many precarcinogens, we tested the possibility of adapting S9 liver fractions derived from Phase II-induced rodents to the field of genetic toxicology. In this study, S9 liver fractions derived from Swiss albino CD1 mice fed 7.5g/kg 2-(3)-tert-butyl-4-hydroxyanisole (BHA; a monofunctional Phase II-inducer) for 3 weeks, show a clear pattern of induction with an approximately 3.5–9.5-fold increase in glutathione S-transferase activity. In vitro DNA binding of the promutagenic agents, [14C]-l,4-dichlorobenzene (DCB) and [14C]-1,4-dibromobenzene (DBB), is mediated by such metabolic liver preparations and showed a significant increase in covalent binding capability. In some instances, enzyme activity was more elevated when compared to that obtained with traditional (Phase I-induced) S9. Together with DNA binding, the genetic response of these chemicals in the diploid D7strain of Saccharomyces cerevisiae used as a biological test system, revealed the ability of the BHA-derived preparations to activate the promutagenic agents, as exemplified by the significant enhancement of mitotic gene-conversion (up to 5.2-fold for DCB and 3.4-fold for DBB) and reverse point mutation (up to 3.6-fold for DCB and 2.5-fold for DBB) at a 4mM concentration. This novel metabolising biosystem, with enhanced Phase II activity, is recommended together with a traditional S9, for detecting unknown promutagens in genotoxicity studies. The routine use of either oxidative or post-oxidative S9 increases the responsiveness of the test and can contribute to the identification of promutagens not detected when using traditional protocols.