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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 effect of prolactin action on nuclear polyphosphoinositide synthesis was investigated in isolated rat liver nuclei. An increased uptake of phosphate from [gamma 32P] adenosinetriphosphate was observed in both phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate with a maximum response at 10(-12) M concentration of hormone. Pulse-chase experiments in isolated nuclei following prolactin treatment indicate that the observed increase in accumulation of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate is mainly due to a decrease in their rate of turnover possibly induced by a change in activity of polyphosphoinositide-specific monoesterases. In vitro prolactin also reduces the activity of nuclear phospholipase C specific for phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. Moreover, this feature is strongly supported by the concomitant decrease in nuclear diacylglycerol mass. Thus these data suggest that once prolactin reaches the nucleus an intranuclear signalling is evoked through inositol lipid metabolism.

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.

In order to investigate the kinetics of vocal fold epithelium a bromodeoxyuridine-anti bromodeoxyuridine method has been applied in vivo at both light and electron microscopy level. This method is able to define the length of both epithelium turnover and cell-cycle in basal elements, as well as the existence of a higher proliferation rate during night time in comparison with day time. Moreover distinct labeling patterns observed in incorporating cells allow us to define the precise localization in S-phase of cycling elements.

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.

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.

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.

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.

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.

Covalent binding of 14C-1,1,1-trichloroethane to macromolecules from rat and mouse liver, kidney, lung and stomach was analyzed under the same experimental conditions previously utilized in studying 1,1-dichloroethane and 1,1,2-trichloroethane. Labeling of DNA, RNA and proteins was very low both in in vivo interaction and in in vitro microsome-mediated binding. Interaction proceeded through the involvement of the P-450-dependent mixed function oxidase system from liver microsomes and, to a lesser extent, from lung microsomes. Covalent Binding Index of 1,1,1-trichloroethane in liver DNA was typical of very weak initiators. However, overall evaluation of the short-term assays available for 1,1,1-trichloroethane leads to limited evidence of genotoxicity. On the other hand, the evidence of 1,1,1-trichloroethane carcinogenicity in animals is still inadequate.

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.

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.

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.

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.

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.