Biomedical subjects

S Grilli

Publications and source records attributed to S Grilli.

At least 19 recordsLinked to original sources

PubMed1992

Initiating activity of 1,1,2,2-tetrachloroethane in two-stage BALB/c 3T3 cell transformation.

By using in vitro two-stage BALB/c 3T3 cell transformation assay, we have tested the effect of promoting treatment with tetradecanoylphorbol acetate (TPA) on transformation induced by 1,1,2,2-tetrachloroethane (1,1,2,2-TTCE). Cells were treated with subeffective or transforming concentrations of 1,1,2,2-TTCE in the presence of an S9-mix activating system, followed by TPA promoting treatment. The transforming activity of 1,1,2,2-TTCE is evident only by reseeding confluent cells and allowing additional rounds of cell replications in the amplification test. Treatment with TPA leads to a marked transformation yield in all plates scored even at the lowest assayed dosage of 1,1,2,2-TTCE, without performing amplification of transformation.

3T3 Cells↗

PubMed1992

Strategies for advancement of short-term mutagenicity tests: on the optimal ionic strength for the liver microsomal assay.

The aim of this work was to optimize the ionic strength (tau) in the liver microsomal assay (LMA) in performing short-term genotoxicity tests. tau optimization would increase the sensitivity (i.e. decrease false negatives) and at the same time increase the specificity (decrease false positives). Such optimization depends upon the relative activities and stabilities of the liver polysubstrate cytochrome P450- and FAD-containing monooxygenase-dependent metabolizing enzymes present in the incubation mixtures. With regard to phase-I pathway, the expression of various P450-like activities (IA1, IA2, IIB1, IIE1, IIIA P450 classes) and thiobenzamide s-oxidase (as FAD-MFO marker), were examined in terms of their exact incubation conditions for the LMA during a period of preincubation (1 h) over the tau range 0.06-1.40. As a comparison with the phase-II pathway, the behaviour of glutathione S-transferases (total and pi class), glutathione S-epoxide transferase, epoxide hydrolase and UDP-glucuronosyl transferase were studied. Lipid peroxidation (LP) was also determined. Experiments were performed on S9 fractions derived from sodium phenobarbital, beta-naphthoflavone, isosafrol, ethanol and pregnenolone 16-alpha carbonitrile super-induced mouse liver. The maximal value of the mean specific activity (Asp), up to a 46% increase, was found at tau = 0.864 for oxidative reactions considered. On the contrary, a slight modulation of Asp for post-oxidative reactions was seen. LP was not changed appreciably by varying tau. In vitro DNA binding of the well-known premutagenic agent [14C]dimethylnitrosamine ([14C]DMNA), mediated by mouse hepatic microsomal enzymes, showed a significant increase of specific activity at tau = 0.864 (2.25-fold) compared to the usual tau (0.06) used. Additional confirmation of these results stems from mutagenesis experiments using DMNA on the diploid D7 strain of Saccharomyces cerevisiae as a biological test system. Indeed, a significant enhancement of mitotic gene conversion (up to 1.8-fold), mitotic crossing-over (2.6-fold) and reverse point mutation (2.6-fold) frequencies was achieved at tau = 0.86 compared to tau = 0.06 (traditional). These data show that tau = 0.86 can provide more convenient conditions for in vitro bioactivation (as exemplified by an increased Asp phase-I/Asp phase-II ratio), as well as DNA binding and genotoxic response.

Animals↗

PubMed1992

In vivo and in vitro interaction of trichloroethylene with macromolecules from various organs of rat and mouse.

Trichloroethylene was covalently bound in vivo to DNA, RNA and proteins of rat and mouse organs 22 hr after ip injection. The covalent binding index values of rat and mouse liver DNA classify trichloroethylene as a weak initiator. Labeling of RNA and proteins from various organs of both species was higher than that of DNA. In vitro, trichloroethylene was bioactivated by microsomal fractions dependent on cytochrome P450, mainly from liver of both species, to intermediate(s) capable of binding to exogenous DNA. No particular species-specific difference was evident except for mouse lung microsomes which were more efficient than rat lung microsomes. GSH-transferases capable of bioactivating P450-dependent were present in mouse lung microsomes and in liver microsomes of both species. These data, along those previously reported, provide sufficient evidence for a weak ability of TCY to interact covalently with DNA.

Animals↗

PubMed1992

Induction of invasive and experimental metastasis potential in BALB/c 3T3 cells by benzo(a)pyrene transformation.

A clone of BALB/c 3T3 cells (A-31), which is highly resistant to spontaneous in vitro transformation, was treated with the carcinogen benzo(a)pyrene [B(a)P]. This agent was capable of inducing in vitro transformation in the presence of S9 activating system and 6 weeks after treatment large foci were detected. Transformation frequency in solvent control groups was very low. Three foci from a single plate of two different experiments were pooled and the cells tested for their in vitro invasive properties and in vivo tumorigenic and metastatic potential. B(a)P-transformed 3T3 cells grew in soft agar and were highly tumorigenic when injected s.c. in nude mice (75% incidence within 7 weeks). Untreated cells were poorly tumorigenic (0/4 mice had tumors within 7 weeks), though they also gave rise to neoplasms after a longer latency. Spontaneous metastasis incidence was low for both controls and treated cells; however, almost all animals (15/16) injected i.v. with B(a)P-transformed cells had pulmonary nodules in the experimental metastasis assay. A few nodules in some of the animals in the control group were detected (4/16). B(a)P-transformed cells were able to invade a thin coating of matrigel in the chemoinvasion assay and also grew in matrigel showing an invasive, branching morphology. Untreated cells did not grow or invade. Our data suggest that a single treatment with a chemical carcinogen can increase tumorigenicity as well as confer invasive and experimental metastasis potential in BALB/c 3T3 cells. This work provides evidence for a role of chemical carcinogens in tumor progression.

3T3 Cells↗

PubMed1991

Chloroform bioactivation leading to nucleic acids binding.

Chloroform was bound covalently to DNA, RNA and proteins of rat and mouse organs in vivo after i.p. injection. Covalent Binding Index values of rat and mouse liver DNA classify chloroform as a weak initiator. Labelings of RNA and proteins from various organs of both species were higher than that of DNA. In an in vitro cell-free system, chloroform was bioactivated by cytochrome P450-dependent microsomal fractions, by cytosolic GSH-transferases from rat and mouse liver, and particularly by the latter enzymes from mouse lung. This observation suggests that GSH plays a role in the binding of chloroform metabolites to DNA. The presence of both microsomal and cytosolic enzymatic systems in the standard incubation mixture generally led to an additive or synergistic bioactivating effect for rat and mouse, respectively.

Animals↗

PubMed1991

In vivo unwinding fluorimetric assay as evidence of the damage induced by fenarimol and DNOC in rat liver DNA.

Five pesticides [amitraz, cyanazine, cyhexatin, dinitro-o-cresol (DNOC), and fenarimol] were tested as pure active ingredients for in vivo induction of DNA strand breaks on rat hepatocytes after intraperitoneal (ip) treatment. Two pesticides, fenarimol and DNOC, were capable of inducing DNA damage because they significantly increased the DNA unwinding rate. On the contrary, amitraz, cyanazine, and cyhexatin were not DNA-damaging agents.

Animals↗

PubMed1991

DNA damaging activity of methyl parathion.

14C-methyl parathion was covalently bound to DNA, RNA and proteins of various rat and mouse organs 22 hr after i.p. injection. Covalent binding index (CBI) to liver DNA was low in both species and typical of weak initiators. The labelings of RNA and proteins from different organs of both species was slightly higher than DNA binding. No interaction with brain nucleic acids was observed (CBI detection limit: 2.8). The in vitro enzyme-mediated interaction of methyl parathion with calf thymus DNA was mainly performed by rodent liver microsomes and, to a lesser extent, by microsomes from mouse kidney and lung whereas brain microsomes were inefficient. Activation of methyl parathion by cytosolic fractions from different organs of both species to form(s) capable of binding to DNA was negligible. When microsomes and cytosolic fractions from rodent liver and lung or mouse kidney were simultaneously present in the incubation mixture, a synergistic effect in catalyzing DNA binding was observed. The extent of DNA binding was reduced by adding SKF 525-A to the microsomal standard incubation mixture, whereas it was enhanced by adding GSH to liver or lung murine microsomes or to mouse kidney microsomes. These results suggest that methyl parathion is bioactivated by P450-dependent microsomal mixed function oxidase system and by microsomal GSH-transferases. By contrast, cytosolic GSH- transferases play a detoxificant role in the metabolism of this compound.

Animals↗

PubMed1991

Transforming activity of ethylene dibromide in BALB/c 3T3 cells.

Ethylene dibromide was capable of inducing in vitro transformation of BALB/c 3T3 cells either in the presence or in the absence of exogenous metabolic activation (S9-mix). This transforming effect was evidenced by the induction of a higher number of transformed foci as compared to the controls performed with untreated cells or solvent vehicle-treated cells. In the absence of exogenous activation, all assayed doses (ranging from 23.4 micrograms/ml to 187.9 micrograms/ml) exerted transforming activity. Number of foci obtained in EDB-treated plates antransformation frequency of the target cells were higher than those detected in the transformation test performed in the presence of S9-mix.

3T3 Cells↗

PubMed1991

Lack of correlation between alkaline DNA fragmentation and DNA covalent binding induced by polychloroethanes after in vivo administration. Problems related to the assessment of a carcinogenic hazard.

The DNA-damaging activity of polychloroethanes was tested in mouse liver by the fluorometric assay of DNA unwinding. With the exception of 1,2-dichloroethane, all components of this chemical class had negative results. The failure of the parameter alkaline "DNA fragmentation" to detect the DNA-damaging activity of polychloroethanes is in sharp contrast with the measurement of DNA covalent binding, another short-term parameter of genotoxicity. Since covalent DNA adducts appear to be quantitatively well correlated with the oncogenic potencies of chloroethanes in liver, the negative results obtained with the present method can perhaps be explained in terms of quality of DNA adducts; these may be incapable of producing DNA breaks or alkali-labile sites detectable as alkaline DNA fragmentation. It is however worth noting that carcinogenicity of chloroethanes appears to depend not only on DNA damaging capability, but also on promoting activity during the carcinogenic process.

Alkalies↗

PubMed1990

In vivo and in vitro interaction of 1,2-dichlorobenzene with nucleic acids and proteins of mice and rats.

Twenty-two hours after i.p. injection into male Wistar rats and BALB/c mice, 1,2-dichlorobenzene (1,2-DCB) was covalently bound to DNA, RNA, and proteins of liver, kidney, lung and stomach. The covalent binding index to liver DNA was typical of carcinogens classified as weak initiators. The enzyme-mediated in vitro interaction of 1,2-DCB with calf thymus DNA of synthetic polyribonucleotides was carried out by a microsomal mixed-function oxidase system and microsomal GSH-transferases, which seemed to be effective only in liver and lung of rat and mouse. Cytosolic GSH-transferases played a minor role in 1,2-DCB bioactivation. The latter finding provides the first evidence of 1,2-DCB genotoxicity in mammalian cells. The type of halide, the number of halosubstituents and their spatial disposition on the benzene ring are the major determinants of halobenzenes activability to intermediate(s) capable of interacting covalently with DNA and other macromolecules in biologic systems.

Animals↗

PubMed1990

Evaluation of genotoxic effects of the herbicide dicamba using in vivo and in vitro test systems.

The genotoxic effects of the herbicide dicamba have been studied by measuring 1) the unwinding rate of liver DNA from intraperitoneally (i.p.) treated rats (fluorimetric assay); 2) DNA repair as unscheduled DNA synthesis (UDS) induced in cultured human peripheral blood lymphocytes (HPBL); and 3) sister chromatid exchanges (SCE) in HPBL. Results show that dicamba is capable of inducing DNA damage since it significantly increases the unwinding rate of rat liver DNA in vivo and also induces UDS in HPBL in vitro in the presence of exogenous metabolic activation (S-9 mix). Furthermore, dicamba causes a very slight increase in SCE frequency in HPBL in vitro.

Animals↗

PubMed1990

Mutagenic and carcinogenic potency indices and their correlation.

We have analyzed a significant number of studies existing in the literature, in which the ability of different short-term tests for predicting carcinogenicity in rodents was investigated. We have separated these studies into two groups. In the better known group of studies, qualitative predictivity was investigated (sensitivity and specificity). In the second group of studies (analyzed in greater detail), positive results were examined for the correlation between carcinogenic potency and potency of response in a given short-term test. There is substantial agreement between qualitative and quantitative predictivity; both appear to be situated between a low and moderate level. We have analyzed the interesting possibility of using the quantitative approach not only for positive data but for combined positive and negative data as well. We have stressed that short-term tests of genotoxicity should be asked to predict only initiation and irreversible alterations in the genome and not to predict a combination of these events, including promotion and modulation of differentiation. Even with regard to only initiation, genotoxicity data should be related to comparative metabolism, as well as to considerations of the significance of different end points and structure-activity relationship data. In conclusion, the information coming from short-term tests of genotoxicity is probably useful but should be used in conjunction with other types of information and only for predicting one particular class of events in the entire process of carcinogenesis.

Carcinogenicity Tests↗

PubMed1990

The covalent interaction of 1,4-dibromobenzene with rat and mouse nucleic acids: in vivo and in vitro studies.

1,4-Dibromobenzene (1,4-DBB) was covalently bound to DNA from liver, kidney, lung and stomach of mice after intraperitoneal administration. The covalent binding index (CBI) value (23 in mouse liver) was typical of weak initiators. On the contrary, no interaction with DNA from rat organs was observed (CBI detection limit: 1.3-2.6). The in vitro interaction of 1,4-DBB with calf thymus DNA was mediated mainly by microsomes, especially those from liver of both species and from mouse lung. Mouse subcellular fractions were more active then rat subcellular fractions. Unlike liver cytosol, subcellular cytosolic fractions from lung, kidney and stomach were capable of bioactivating 1,4-DBB, although to a lesser extent than liver microsomes. Both cytochrome P-450 and GSH-transferases are involved in 1,4-DBB bioactivation.

Animals↗

PubMed1990

Strategies for optimization of short-term genotoxicity tests: the synergistic effect of NADPH and NADH on P450 function in processing pre-mutagens.

The synergistic effect of NADPH and NADH on P450 functions upon pre-mutagens requiring metabolism during the incubation conditions used in the liver microsomal assay (LMA) was studied. The mean specific activity (Asp) during 1 h of pre-incubation (LMA) of some microsomal mono-oxygenases (i.e. ethylmorphine N-demethylase, p-nitroanisole O-demethylase and aminopyrine N-demethylase) examined with S9 fractions from sodium phenobarbital and beta-naphthoflavone pre-treated mice, was doubled when both NADPH and NADH were present. In contrast, when lipid peroxidation was used as the main enzymatic inactivation index, there was no appreciable change. In agreement with biochemical data, in vitro DNA binding of the pre-mutagenic agent [14C]-1,1,1,2-tetrachloroethane ([14C]TTCE), mediated by mouse hepatic enzymes, showed a significant enhancement (4.4-fold) of specific activity in the presence of both pyridine nucleotides. Mutagenesis experiments using TTCE in the diploid D7 strain of Saccharomyces cerevisiae (from stationary growth phase) as a biological test system, showed a significant enhancement of mitotic gene conversion and reverse point mutation frequencies when using NADPH plus NADH in the medium. Conversely, no positive results without NADH were seen. These findings lead us to suggest the routine use of both NADPH and NADH in order to increase the 'sensitivity' of in vitro mutagenicity screens.

Animals↗

PubMed1990

In vitro transformation of BALB/c 3T3 cells by 1,1,2,2-tetrachloroethane.

1,1,2,2-Tetrachloroethane (1,1,2,2-TTCE) was shown to be capable of inducing in vitro transformation of BALB/c 3T3 cells (clone A-31) either in the presence or in the absence of S9 activating system using an amplification-transformation (level-II) assay by reseeding confluent cells from each treatment and allowing additional rounds of cell replication. In the absence of metabolic activation, the highest assayed dose (1000 micrograms/ml), exerting the highest toxicity, was the only transforming dose. Lower doses of 1,1,2,2-TTCE were capable of transforming BALB/c cells in the presence of S9 activating system, the dose of 500 micrograms/ml exerting the highest transforming activity. The number and size of transformed foci recognized in the level-II plates were a function of the number of cells reseeded in the amplification assay. Foci obtained in the presence of S9 activating systems were larger in size, more deeply basophilic, and exhibited denser multilayering of constituent cells than foci recognized in the absence of exogenous metabolic activation.

Animals↗

PubMed1990

Quantitative predictability of carcinogenicity of the covalent binding index of chemicals to DNA: comparison of the in vivo and in vitro assays.

The capability of covalent binding to DNA to predict the initiating potential of chemical carcinogens was compared for the assays performed in vivo (rodent liver DNA) and in vitro (purified DNA incubated in the presence of mouse and rat liver microsomes). A quantitative correlation between DNA adducts and carcinogenic potency was investigated. The in vivo assay appeared slightly, but not significantly, more predictive than the in vitro assay. Also predictivity was slightly higher both in vivo and in vitro when we referred to liver carcinogenicity instead of overall carcinogenicity. The predictive ability found for DNA covalent binding (both in vivo and in vitro) was similar to that of many short-term tests (such as mutagenicity, DNA damage/repair, SCEs, and cell transformation tests). The covalent DNA binding, measured after incubation with DNA in vitro in the presence of liver microsomes, could therefore be a reasonable short-term test offering greater rapidity of execution and requiring the sacrifice of fewer animals than the corresponding in vivo test.

Animals↗

PubMed1989

The different genotoxicity of p-dichlorobenzene in mouse and rat: measurement of the in vivo and in vitro covalent interaction with nucleic acids.

Twenty-two hours after i.p. injection to male Wistar rats and BALB/c mice para-dichlorobenzene (p-DCB) is bound covalently to DNA from liver, kidney, lung and stomach of mice but not of rats. DNA adducts in mouse liver are repaired in seventy-two hours. The covalent binding index value, calculated on the labelling of mouse liver DNA, classifies p-DCB as a weak initiator with an oncogenic activity lower than that of chlorobenzene. The labelling of RNA and proteins from the different organs of both species is, however, low. In vitro interaction with calf thymus DNA mediated by mouse and rat microsomes from liver and lung did occur. Binding extent was strongly reduced by addition of 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride (SKF 525-A) to the microsomal standard incubation mixture, whereas it was enhanced by adding GSH. Cytosolic fractions from kidney and lung were able to induce binding of p-DCB to DNA to a lower extent with respect to microsome-mediated binding. These results indicate that microsomal mixed function oxidase system and microsomal GSH-transferases can be involved in overall activating metabolism whereas cytosolic GSH-transferases play a minor role. This study, which is a part of a structure-activity relationship approach on benzene and its haloderivatives, provides the first evidence of genotoxicity of p-DCB in mammalian cell. It allows to partly explain variations of susceptibility of different species to hepatocarcinogenesis and of hepatotoxicity of different isomers.

Animals↗

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↗