Carmelo F. Cesarone

Antioxidant Activity and Other Mechanisms of Thiols Involved in Chemoprevention of Mutation and Cancer

Our studies provide evidence that thiols, such as N-acetyl-L-cysteine, inhibit both spontaneous mutations and induced mutations in bacteria, prevent the in vivo formation of carcinogen-DNA adducts, and suppress or delay the development of tumors or preneoplastic lesions in rodents. N-Acetylcysteine and other thiols exert antioxidant activity toward superoxide anion, hydrogen peroxide, and singlet oxygen, assessed in bacterial genotoxicity models. In addition, several other mechanisms were shown to contribute to their antimutagenic and anticarcinogenic activities, in the extracellular environment and in nontarget or target cells. These mechanisms include blocking of electrophilic metabolites and of direct-acting compounds, either of endogenous or exogenous source, modulation of several xenobiotic-metabolizing pathways, and protection of DNA-dependent nuclear enzymes. Chemoprevention of mutation and cancer by thiols is particularly useful under conditions of reduced glutathione (GSH) depletion due to toxic agents or to cancer-associated viral diseases, such as acquired immunodeficiency syndrome (AIDS) or viral hepatitis B.

Changes in activity and mRNA levels of poly(ADP-ribose) polymerase during rat liver regeneration

ADP-ribosylation of nuclear proteins, catalysed by the enzyme poly(ADP-ribose) polymerase, is involved in the regulation of different cellular processes of DNA metabolism. To further clarify the role of the enzyme during proliferating activity of mammalian cells, we have studied the control of gene expression in regenerating rat liver. The changes in activity and mRNA levels were analysed during the early and late phases of the compensatory model. When enzyme activity was measured in isolated liver nuclei obtained at different times after hepatectomy, two different phases were observed: an early wave occurring before the onset of DNA synthesis, and a second one, starting several hours after the onset of DNA synthesis and returning to control values at later times. The evaluation of the enzymatic level in nuclear extracts and by activity gel analysis showed a more gradual increase starting 1 day after hepatectomy, in concomitance with the peak of DNA synthesis. By using a specific murine cDNA probe, a significant enhancement of mRNA levels for poly(ADP-ribose) polymerase was observed during liver regeneration, slightly preceding the onset of DNA synthesis. The results obtained show that changes in poly(ADP-ribose) polymerase activity, during liver regeneration, are associated both to early events preceding the increase in DNA synthesis and to later phases of the cell proliferation process.

Antimutagenic and Anticarcinogenic Mechanisms of Aminothiols

Glutathione (GSH) and synthetic aminothiols, such as N-acetylcysteine (NAC), play an important role as inhibitors of mutagenesis and carcinogenesis (1). The present paper will update the studies carried out in our laboratories, which will be discussed according to a mechanistic approach. In Table 1, reporting the classification of inhibitors of mutagenesis and carcinogenesis proposed by De Flora and Ramel (2), the arrows indicate the mechanisms that we have investigated so far in the case of aminothiols.

Relationship between poly(ADP-ribose) polymerase activity and DNA synthesis in cultured hepatocytes

Previous studies have demonstrated that an increase in poly(ADP-ribose) polymerase activity could be closely related to DNA replication during liver regeneration and to DNA repair synthesis in different experimental systems. This relationship was further investigated by studying the time course of endogenous and total poly(ADP-ribose) polymerase activity in cultured rat hepatocytes stimulated by epidermal growth factor. This mitogen has been shown to stimulate DNA synthesis in liver cells both in vivo and in vitro. A 6-fold increase in endogenous activity was observed early after epidermal growth factor addition, just before DNA synthesis. A subsequent 4-fold increment in total enzyme activity, concomitant with DNA synthesis, was detected. Orotic acid, which has recently shown mitoinhibitory effect, abolished the epidermal-growth-factor-induced increase in endogenous and total poly(ADP-ribose) polymerase activity, as well as DNA synthesis. On the contrary, 3-aminobenzamide inhibitor of poly(ADP-ribose) polymerase completely suppressed the endogenous activity but only partially modified the increase in total catalytic level and the overall pattern of thymidine incorporation. Taken together, these data indicate that, in cultured hepatocytes, the induction of DNA synthesis is supported by an increased poly(ADP-ribose) polymerase activity.

Relationship between poly(ADP-ribose) polymerase activity and DNA damage induced by zinc dithiocarbamates in mouse and rat liver

The genotoxic effects due to in vivo treatment with zinc dithiocarbamates were evaluated in rat and mouse liver. The two pesticides Zineb and Ziram, belonging to this chemical class, induced an increase in single-strand DNA breaks, as measured by the alkaline elution technique. The nuclear enzyme poly(ADP-ribose) polymerase (pADPRP), a chromatin-bound catalytic protein, utilizing NAD+ as a substrate, was tested by a radiometric procedure. A close relationship between the increased extent of DNA damage and the enhanced level of endogenous pADPRP activity was obtained in rat liver, whereas both parameters remained unchanged in mouse liver.

Differential assay and biological significance of poly(ADP-ribose) polymerase activity in isolated liver nuclei.

Poly(ADP-ribosyl)ation of nuclear proteins is catalyzed by poly(ADP-ribose) polymerase. This enzyme is involved in the regulation of basic cellular functions of DNA metabolism. DNA breaks induced by DNA-damaging agents trigger the activation of poly(ADP-ribose) polymerase increasing its endogenous level. This increase modifies the pattern of poly(ADP-ribosyl)ated chromatin proteins. In this paper we describe a procedure for the isolation of intact nuclei from rat liver to be used for the endogenous activity assay. Artifactual activation of the enzyme was avoided since a very low level of DNA-strand breaks occurs during the isolation of nuclei. We present a series of experiments which prove the ability of this procedure to detect increases in endogenous liver activity without modification of the total level. The application of this technique can be useful for a better understanding of the role of early changes in poly(ADP-ribose) polymerase level in physiological conditions and during exposure to DNA-damaging agents.

Protection of Nuclear Enzymes by Aminothiols

The maintenance of an intact DNA repair capacity is a necessary requirement for cell physiology and for survival. On the other hand, long-lasting DNA lesions, induced by physical or chemical agents, represent the first step in aging (1), mutagenesis (2,3) or in the multistage carcinogenic process (4-6). Impaired DNA repair synthesis has been shown to be responsible for increased neoplastic development in human subjects affected by repairdeficient syndromes such as: Xeroderma pigmentosum, Ataxia telangiectasia, Bloom’s syndrome, Fanconi’s anemia (7,8).

Antigenotoxic and Anticarcinogenic Effects of Thiols. In Vitro Inhibition of the Mutagenicity of Drug Nitrosation Products and Protection of Rat Liver ADP-Ribosyl Transferase Activity

Reduced glutathione (GSH) is well-known to play a fundamental role in the protection of the organism against toxic, mutagenic and/or carcinogenic agents (see e.g. refs. 1–3 for reviews). Among synthetic aminothiols, acting as analogs and precursors of GSH, N-acetyl-L-cysteine (NAC) is of particular interest, because this molecule is already extensively used in the treatment of chronic respiratory diseases, and is extremely well tolerated in humans4. In addition, NAC is known to possess various antitoxic and antioxidant properties3–5.

Inhibition of Mutagenesis and Carcinogenesis by N-Acetylcysteine

N-acetylcysteine (NAC), a precursor of intracellular cysteine and glutathione (GSH)) (1), is extensively used in the treatment of patients suffering from respiratory diseases. Moreover, this synthetic aminothiol has been shown to possess a variety of antitoxic properties in humans, animals and in vitro test systems (2).

Effect of Glutathione and N-Acetylcysteine on Hepatocellular Modifications Induced by 2-Acetylaminofluorene

The exposure of rats to a dietary regimen containing 2-acetylaminofluorene induces a sequence of hepatocellular alterations leading to the development of preneoplastic nodules. Groups of 2-acetylaminofluorenetreated rats were given glutathione or N-acetylcysteine to evaluate the effects of these different thiols on the sequence of events that originate transformed cells. It is well known that intracellular thiols protect biological macromolecules from scavenging free radicals and electrophilic compounds produced by the metabolism of chemical agents. Male Wistar rats were maintained on a feeding regimen containing 0.05% 2-acetylaminofluorene. The diet of 2 groups of 2-acetylaminofluorene-treated animals was supplemented with either 0.1% glutathione or N-acetylcysteine. The effects in the liver of the exogenously supplied thiols during 2-acety-laminofluorene treatment were assessed evaluating DNA damage, glutathione levels, activity of marker enzymes glucose-6-phosphatase, γ-glutamyltranspeptidase, and glutathione-S-transferase, survival rates, and development of salivary gland tumors. Our results demonstrate that the mortality due to 2-acetylaminofluorene exposure was reduced or completely abolished by thiols and that the development of salivary gland tumors was inhibited. Exogenously supplied thiols significantly reduced DNA damage as assessed by alkaline elution. At the doses employed, glutathione and N-acetylcysteine induce early stimulation of glutathione-S-transferase, had little effect on the loss of glucose-6-phosphatase activity and scanty influence on the net increase in γ-glutamyltranspeptidase activity.