Biomedical subjects

S Grilli

Publications and source records attributed to S Grilli.

At least 37 records · Page 2Linked to original sources

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↗

PubMed1989

Covalent binding of 1,1,1,2-tetrachloroethane to nucleic acids as evidence of genotoxic activity.

Twenty-two hours after ip administration to male Wistar rats and BALB/c mice, 1,1,1,2-tetrachloroethane (1,1,1,2-TTCE) is bound covalently to DNA, RNA, and proteins of liver, lung, kidney, and stomach. The in vivo reactivity leads to binding values to DNA generally higher in mouse organs than in rat organs. The covalent binding index (CBI) values (82 in mouse liver DNA and 40 in rat liver DNA) classify 1,1,1,2-TTCE as a weak to moderate initiator. Both microsomal and cytosolic enzymatic systems from rat and mouse organs are capable of bioactivating 1,1,1,2-TTCE in vitro. Liver fractions are the most effective. When the activating systems are simultaneously present in the incubation mixture a synergistic effect is observed. Unlike the related chemical 1,1,2,2-tetrachloroethane (1,1,2,2-TTCE), which is bioactivated only through an oxidative route, 1,1,1,2-TTCE metabolism is carried on by oxidative and reductive pathways, both dependent on cytochrome P-450. 1,1,1,2-TTCE is also bioactivated by microsomal GSH-transferases from liver and lung. These data further confirm that correlations exist between structure and genotoxic activity of halocompounds.

Animals↗

PubMed1989

Results of animal studies suggest a nonlinear dose-response relationship for benzene effects.

Considering the very large industrial usage of benzene, studies in risk assessment aimed at the evaluation of carcinogenic risk at low levels of exposure are important. Animal data can offer indications about what could happen in humans and provide more diverse information than epidemiological data with respect to dose-response consideration. We have considered experiments investigating metabolism, short-term genotoxicity tests, DNA adduct formation, and carcinogenicity long-term tests. According to the different experiments, a saturation of benzene metabolism and benzene effects in terms of genotoxicity seems evident above 30 to 100 ppm. Below 30 to 60 ppm the initiating effect of benzene seems to be linear for a large interval of dosages, at least judging from DNA adduct formation. Potential lack of a promoting effect of benzene (below 10 ppm) could generate a sublinear response at nontoxic levels of exposure. This possibility was suggested by epidemiological data in humans and is not confirmed or excluded by our observations with animals.

Animals↗

PubMed1989

Benzene adducts with rat nucleic acids and proteins: dose-response relationship after treatment in vivo.

The dose-response relationship of the benzene covalent interaction with biological macromolecules from rat organs was studied. The administered dose range was 3.6 x 10(7) starting from the highest dosage employed, 486 mg/kg, which is oncogenic for rodents, and included low and very low dosages. The present study was initially performed with tritium-labeled benzene, administered by IP injection. In order to exclude the possibility that part of the detected radioactivity was due to tritium incorporated into DNA from metabolic processes, 14C-benzene was then also used following a similar experimental design. By HPLC analysis, a single adduct from benzene-treated DNA was detected; adduct identification will be attempted in the near future. Linear dose-response relationship was observed within most of the range of explored doses. Linearity was particularly evident within low and very low dosages. Saturation of benzene metabolism did occur at the highest dosages for most of the assayed macromolecules and organs, especially in rat liver. This finding could be considered as indicative of the dose-response relationship of tumor induction and could be used in risk assessment.

Animals↗

PubMed1989

Metabolic activation and covalent binding to nucleic acids of pentachloroethane as short-term test of genotoxicity.

The in vivo covalent binding of 14C-pentachloroethane to DNA, RNA and proteins of rats and mouse organs was detected 22 hr after i.p. injection. The covalent binding index, calculated on the liver labeling was comparable to those of compounds considered as weak-moderate initiators. Like other haloalkanes, 14C-pentachloroethane was bioactivated in in vitro cell-free system by both microsomal and cytosolic enzymatic fractions from mouse and rat organs to react covalently with DNA and other macromolecules. The binding extents obtained from in vitro incubation and the binding values detected after in vivo administration of labeled pentachloroethane were comparable each other and showed a high correlation with oncogenic potency index of this compound. This result confirms the efficiency of in vitro binding as short-term test of genotoxicity prediction.

Animals↗

PubMed1988

Binding of hexachloroethane to biological macromolecules from rat and mouse organs.

Hexachloroethane (HCE) binds to macromolecules of rat and mouse both in vivo and in vitro after metabolic activation. The covalent binding index (CBI) to liver DNA in vivo is comparable to that of compounds classified as weak-moderate initiators and is of approximately the same order of magnitude as those of other halocompounds such as 1,2-dichloroethane. HCE is bioactivated in vitro by microsomal enzymatic systems from murine liver and kidney and, to a greater extent, by cytosolic fractions from all assayed organs. HCE is less reactive than 1,1,2,2-tetrachloroethane, which is more toxic and oncogenic. The ability of hexachloroethane and five other chloroethanes to react covalently with mouse liver DNA both in vivo and in vitro parallels the relative oncogenic potency of these hepatocarcinogenic chemicals in mouse liver.

Animals↗

PubMed1988

Improvement of short-term tests for mutagenicity: on the optimal pH for the liver microsomal assay.

The aim of this study was to optimize the pH in the liver microsomal assay (LMA) in processing short-term mutagenicity tests. pH optimization would increase the sensitivity (i.e. decrease the presence of false negatives) and increase the specificity (decrease false positives). Such optimization is a function of the relative activities and stabilities of the liver microsomal cytochrome P-450- and FAD-containing monooxygenase-dependent biotransformation enzymes present in the incubation mixtures used. The enzyme activities ethoxyresorufin O-deethylase, dinemorphan N-demethylase, aminopyrine N-demethylase, p-nitroanisole O-demethylase and thiobenzamide S-oxidase (as phase-I markers), were examined in terms of their exact incubation conditions for the LMA during a period of pre-incubation (1 h) over the pH range 6-9. As a comparison, the behaviours of glutathione S-transferase and epoxide hydrase activities (as phase-II markers) were also studied. Lipid peroxidation was also determined. Experiments were carried out on S9 fractions derived from Na-phenobarbital and beta-naphthoflavone induced mouse liver. The maximal value of the mean specific activity (Asp) was found at pH 7.8 for the phase-I drug metabolizing enzymes considered (30-45% increase). On the contrary, a lower increase of Asp for epoxide hydrase and glutathione S-transferase (approximately 14%), was observed between pH 7.4 and 7.8. Lipid peroxidation was not changed appreciably by varying pH. In vitro DNA binding of the well-known pre-mutagenic agent [14C]dimethylnitrosamine ([14C]DMNA), mediated by mouse hepatic microsomal enzymes, showed a significant increase of specific activity at pH 7.8 (2.8-fold) compared to the usual pH (7.4) employed. Additional support for the above results has come from mutagenesis experiments using DMNA on the diploid D7 strain of Saccharomyces cerevisiae as a biological test system. In fact, a significant enhancement of mitotic gene conversion (1.7-fold), mitotic cross-over (2.6-fold) and reverse point mutation (2.3-fold) frequencies were observed at pH 7.8 compared to pH 7.4. These data indicate that pH 7.8 provides a more favourable condition for in vitro mutagenesis tests resulting in greater rates of biotransformation (as measured by an increased Asp phase-I/Asp phase-II ratio), DNA binding and genotoxic response.

Animals↗

PubMed1988

Isolation of S9 fractions from mouse and rat with increased enzyme activities after repeated administration of cytochrome P-450 and P-448 inducers.

Cytochrome P-450 (cyt P-450), NADPH cytochrome P-450 reductase and various microsomal monooxygenase activities [e.g. aminopyrine N-demethylase, p-nitroanisole O-demethylase, dinemorphan N-demethylase, ethoxycoumarin O-deethylase and ethoxyresorufin O-deethylase (ERD)], were determined in hepatic post-mitochondrial supernatant from mice and rats. Experiments were performed on male and female animals treated with a combination of sodium phenobarbital and beta-naphthoflavone according to the standard protocol schedule for short-term genotoxicity testing. A second inductive treatment after 2, 3, 4 or 5 weeks was provided. The increase in cyt P-450 and in all enzymatic activities measured was enhanced in both species by a second induction treatment, particularly when given after 4 weeks. ERD activity was the only monooxygenase activity which was sex-dependent, being more active in female than in male animals. To extend the biochemical data, experiments were performed with the proposed S9 fractions on styrene, which previously has proved difficult to detect in short-term in vitro mutagenicity tests. Using the new induction conditions positive results were obtained with the D7 strain of Saccharomyces cerevisiae. It was concluded that a simple pre-induction of the animals 3-4 weeks before the main induction treatment leads to a more active S9 fraction for in vitro genotoxicity studies.

Animals↗

PubMed1987

The covalent binding of 1,1,2,2-tetrachloroethane to macromolecules of rat and mouse organs.

The in vivo interaction of the hepatocarcinogen 1,1,2,2-tetrachloroethane (1,1,2,2-TTCE) with DNA, RNA, and proteins of male Wistar rats and BALB/c mice was measured 22 hr after i.p. injection. Covalent binding index (CBI) to liver DNA was about 500 and was comparable to those of carcinogens classified as moderate initiators. It was higher than those of other chloroethanes, even than that of 1,2-dichloroethane (1,2-DCE), a symmetrically substituted haloethane whose genotoxicity has been widely demonstrated. In in vitro cell-free systems, 1,1,2,2-tetrachloroethane was bioactivated by mixed-function oxidase(s) and glutathione-S-transferase(s) (GSH-T) from microsomal and cytosolic fractions of rat and mouse liver and, to a lesser extent, of mouse lung. The in vitro activation led to formation of reactive species capable of binding to exogenous DNA and to the subcellular constituents of enzymatic fractions. These data, along with previous literature reports, provide sufficient evidence of 1,1,2,2-TTCE genotoxicity.

Animals↗

PubMed1987

Possible implications from results of animal studies in human risk estimations for benzene: nonlinear dose-response relationship due to saturation of metabolism.

To date, all risk assessment studies on benzene have been based almost exclusively on epidemiological data. We have attempted a more integrated and quantitative evaluation of carcinogenic risk for humans, trying to utilize, in addition to the epidemiological data, all data available, specifically data on metabolism, genotoxicity, and carcinogenicity in small rodents. An integrated evaluation of the globality of the available data seems to suggest a progressive saturation of metabolic capacity both for man and rodents between 10 and 100 ppm. The most susceptible target cells seem to be different in humans (predominant induction of myelogenous leukemia) and small rodents (induction of a wide variety of tumors). Nevertheless, both epidemiological and experimental carcinogenicity data tend to indicate a flattening of the response for the highest dosages, again suggesting a general saturation of mechanisms of metabolic activation, extended to different target tissues. From a quantitative point of view, the data suggest a carcinogenic potency at 10 ppm two to three times higher than that computable by a linear extrapolation from data in the 100 ppm range. These observations are in accord with the recent proposal of the European Economic Community of reducing benzene time-weighted average occupational levels from 10 to 5 ppm.

Animals↗

PubMed1987

Evidence of DNA binding activity of perchloroethylene.

14C-Perchloroethylene is covalently bound to DNA, RNA and proteins of rat and mouse organs in vivo after ip injection. Covalent Binding Index values are typical of weak-moderate and weak initiators, for mouse and rat liver, respectively. The greater amounts of labelings detected in mouse liver and in rat kidney macromolecules are consistent with the known toxic and carcinogenic actions of this compound. In vitro binding of perchloroethylene to nucleic acids and proteins proceeds through the involvement of the P-450-dependent mixed function oxidase system from liver microsomes. Kidney, lung and stomach microsomal fractions are uneffective. Cytosolic enzymes from all assayed organs are much more efficient than liver microsomes in bioactivating the compound. GSH addition to liver microsomal system greatly enhances binding extent. This observation suggests that GSH plays a role in the binding of perchloroethylene metabolites as for symmetrically substituted haloethanes.

Animals↗

PubMed1986

Interaction of halocompounds with nucleic acids.

The binding of epichlorohydrin, 1,2-dichloroethane, 1,2-dibromoethane, chlorobenzene, bromobenzene, and benzene to nucleic acids and proteins of different murine organs was studied in in vivo and in vitro systems. The extent of in vivo enzymatic activation of brominated compounds was higher than that of chlorinated chemicals. Aryl halides were bound mainly to liver DNA whereas interaction of alkyl halides with DNA of liver, kidney, and lung gave rise to similar binding extent. In vitro activation of all chemicals was mediated by microsomal P-450-dependent mixed function oxidase system which is present in rat and mouse liver and, in smaller amount, in mouse lung. Activation of alkyl halides by liver cytosolic GSH-transferases even occurred. The relative reactivity of chemicals in vivo, expressed as Covalent Binding Index (CBI) to rat liver DNA, was: 1,2-dibromoethane greater than bromobenzene greater than 1,2-dichloroethane greater than chlorobenzene greater than epichlorohydrin greater than benzene. On the whole, it agreed with in vitro activation of chemicals, with genotoxicity data from other short-term assays and also with oncogenicity of benzene, epichlorohydrin, 1,2-dichloroethane, and 1,2-dibromoethane. CBI values of chlorobenzene and bromobenzene gave the first clear evidence of genotoxicity and of possible carcinogenicity of these two chemicals.

Animals↗

PubMed1986

1,1,2-Trichloroethane: evidence of genotoxicity from short-term tests.

At 22 hr after ip injection into adult male Wistar rats, 1,1,2-trichloroethane was covalently bound to DNA, RNA and proteins of the liver, kidney, lung and stomach, as has been found with various weakly carcinogenic halo compounds. The extent of interaction of 1,1,2-trichloroethane with mouse liver DNA was much higher than that with rat liver DNA. This result provides evidence of a correlation between adducts formation and species susceptibility to hepatocarcinogenesis (only the mouse is sensitive to the oncogenic effect of this compound). Interaction with DNA mediated by murine liver microsomes occurred in vitro. No particular differences between the two species were found. In vitro binding was enhanced (approximately 5-fold) by pretreatment in vivo with phenobarbitone but was suppressed by addition of 2-diethylamino-ethyl-2,2-diphenylvalerate X HCl in vitro. Cytochrome P-450 was, therefore, involved in the interaction process. Glutathione suppressed the microsome-mediated interaction, acting as a "scavenger" of reactive intermediate(s). Murine lung microsomes were less effective bioactivators than liver microsomes for the interaction with DNA and microsomal RNA, but not microsomal protein. Kidney and stomach microsomes were ineffective, as were cytosolic fractions from all of the assayed organs of the two species. The extent of in vitro interaction of 1,1,2-trichloroethane with synthetic polyribonucleotides was of the same order of magnitude as that with DNA. The results represent further clear evidence for genotoxicity of 1,1,2-trichloroethane.

Animals↗