Francesco D'Agostini

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.

Protection by N-acetylcysteine of the histopathological and cytogenetical damage produced by exposure of rats to cigarette smoke

Adult male Sprague-Dawley rats were exposed whole-body to mainstream cigarette smoke (CS) once daily for 40 consecutive days. Such a treatment resulted in a significant decrease of body weight growth and in intense histopathological changes of terminal airways, including a severe inflammation of bronchial and bronchiolar mucosae, with multiple hyperplastic and metaplastic lesions and foci of micropapillomatous growth as well as emphysema, with extensive disruption of alveolar walls. All histopathological changes were efficiently prevented by the daily administration of the thiol N-acetyl-L-cysteine (NAC) by gavage. Cytological and cytogenetical changes were monitored in bronchoalveolar lavage (BAL) fluid and bone marrow cells of groups of rats killed after 1, 3, 8, 28, or 40 days of treatment. From the first day of exposure, CS significantly enhanced the proportion of polymorphonucleates among BAL cells and the frequency of micronucleated (MN) bone marrow polychromatic erythrocytes. After 8 days, a reduction was observed in the polychromatic/normochromatic erythrocytes ratio and an increase in the frequency of MN pulmonary alveolar macrophages (PAM) was also recorded, followed, after 28 days, by an increase of binucleated PAM. All these alterations immediately reached a plateau and persisted unchanged until the end of the experiment. NAC administration exhibited a significant and considerable protective effect towards the CS-induced alterations of BAL cellularity, the increase of MN PAM and bone marrow cytotoxicity.

Modulation of the mutagenic response in prokaryotes

Short-term tests investigating genetic end-points in prokaryotes have been extensively used worldwide not only for risk assessment purposes but also for evaluating the modulation of the mutagenic response. In spite of some intrinsic limitations, such as the lack of cell compartmentalization or the need for an exogenous metabolic system working extracellularly, experimental systems in bacteria can provide useful preliminary indications and some information on the mechanisms involved. In the large majority of studies the putative modulator is mixed with a known mutagen and then assayed in target bacteria, with suitable controls. However, under natural conditions exposure of target cells to modulators may either precede, co-exist with, or follow exposure to mutagens. Therefore, a variety of methodological variations, involving pre-treatment, co-treatment, or post-treatment of bacteria with the putative modulator, have been designed. Application of these procedures showed that the effects of modulators can be completely upset, from inhibition to enhancement, or vice versa, by changing the experimental conditions. Use of methodological variations may provide more complete information on the spectrum of possible effects in bacteria as well as a better insight into modulation mechanisms. Several examples illustrating the flexibility of the Salmonella test in this field of research are available. On the other hand, the widespread use of these relatively simple techniques, yet requiring skillfulness and experience, may lead to some misuse or oversimplifications. A rather common inadequacy is to use excessive amounts of test mutagens, or to express the results in terms of revertants/survivors, rather than revertants/plate. In fact, in the Salmonella test the number of revertants is rather unrelated to the initial number of plated bacteria, provided a normal background lawn of bacterial growth is formed. Thus, a 50% killing of bacteria will not appreciably influence the number of revertants/plate, but expressed as revertants/survivors the effect will look twice as large.

Mutagenicity of polycyclic aromatic hydrocarbon fractions extracted from urban air particulates

Polycyclic aromatic hydrocarbon (PAH) fractions, purified from extracts of airborne particles collected in the area of Genoa municipality, were assayed for mutagenicity in the Salmonella/microsome test. PAH fractions accounted for only a portion of the total mutagenic activity and also displayed a different specificity of genetic activity, as compared to unfractionated material. The analysis of 224 samples collected from January 1986 to November 1987 in 10 different localities led to a large number of positive results in strain TA100 with S9 mix and, less frequently, also in TA98 without metabolic activation. Mutagenicity was related to the intensity of anthropogenic atmospheric pollution, and showed some seasonal variations, although it was not possible to discriminate particular sources of pollution on the basis of mutagenicity patterns. The mutagenic potency in TA100 (S9+) of airborne PAH fractions was significantly correlated with the concentration of individual PAHs in most of the monitored localities. The spectrum of mutagenicity of monthly samples pooled from several localities in S. typhimurium strains, with and without S9 mix, provided evidence for some contribution of nitro derivatives of PAHs or possibly also of other compounds present in the same fractions. The results obtained are discussed in view of their predictive value as indicators of potential health hazards, and of the reliability of this biological tool as a complement to chemical analyses in the evaluation of ambient air pollution.

Mutagenicity of active oxygen species in bacteria and its enzymatic or chemical inhibition

The mono-electronic reduction of oxygen in the hypoxanthine-xanthine oxidase system led to the formation of active species eliciting an evident and highly reproducible mutagenic response in strain TA104 of S. typhimurium. Similar effects were observed by generating oxy radicals either extracellularly or inside bacterial cells. Mutagenicity was selectively detected in TA104 and not in other Salmonella strains, which points out the importance of the hisG428 mutation and of the deletion excising the uvrB gene, as far as sensitivity to oxy radicals is concerned. The mutagenicity of the system was further enhanced in the presence of superoxide dismutase. Catalase did not affect the mutagenicity of hypoxanthine plus xanthine oxidase, whereas it inhibited the mutagenicity induced by the mixture of hypoxanthine with xanthine oxidase and superoxide dismutase. This demonstrates that not only hydrogen peroxide but also the superoxide radical anion is positive in this system. Glutathione and 2 synthetic thiols, i.e., N-acetylcysteine and alpha-mercaptopropionylglycine, besides decreasing the high spontaneous mutagenicity of TA104, efficiently prevented the mutagenicity of active oxygen species.

Smoke-induced microRNA and related proteome alterations. Modulation by chemopreventive agents

Dysregulation of microRNAs (miRNAs) has important consequences on gene and protein expression since a single miRNA targets a number of genes simultaneously. This article provides a review of published data and ongoing studies regarding the effects of cigarette smoke (CS), either mainstream (MCS) or environmental (ECS), on the expression of miRNAs and related proteins. The results generated in mice, rats, and humans provided evidence that exposure to CS results in an intense dysregulation of miRNA expression in the respiratory tract, which is mainly oriented in the sense of downregulation. In parallel, there was an upregulation of proteins targeted by the downregulated miRNAs. These trends reflect an attempt to defend the respiratory tract by means of antioxidant mechanisms, detoxification of carcinogens, DNA repair, anti‐inflammatory pathways, apoptosis, etc. However, a long‐lasting exposure to CS causes irreversible miRNA alterations that activate carcinogenic mechanisms, such as modulation of oncogenes and oncosuppressor genes, cell proliferation, recruitment of undifferentiated stem cells, inflammation, inhibition of intercellular communications, angiogenesis, invasion, and metastasis. The miRNA alterations induced by CS in the lung of mice and rats are similar to those observed in the human respiratory tract. Since a number of miRNAs that are modulated by CS and/or chemopreventive agents are subjected to single nucleotide polymorphisms in humans, they can be evaluated according to toxicogenomic/pharmacogenomics approaches. A variety of cancer chemopreventive agents tested in our laboratory modulated both baseline and CS‐related miRNA and proteome alterations, thus contributing to evaluate both safety and efficacy of dietary and pharmacological agents.

Prevention by N-acetylcysteine of benzo[a]pyrene clastogenicity and DNA adducts in rats

The daily i.t. administration of benzo[a]pyrene (BP) to Sprague-Dawley rats, for 3 consecutive days, did not cause any toxicity or clastogenicity in bone marrow cells, as evaluated by monitoring the ratio of polychromatic to normochromatic erythrocytes and the frequency of micronucleated polychromatic erythrocytes. However, BP produced a considerable enhancement of binucleated and micronucleated pulmonary alveolar macrophages, as well as a significant increase in polymorphonucleates recovered by bronchoalveolar lavage. These effects were prevented by administering the thiol N-acetylcysteine (NAC) by gavage 5 h before each BP instillation. In addition, the i.t. treatment with BP resulted in the formation of BP diolepoxide (BPDE)-DNA adducts in lungs and liver, as assessed by synchronous fluorescence spectrophotometry, with fluorescence peaks of similar magnitude in the 2 tissues. Pretreatment with NAC by gavage completely prevented BPDE adducts to liver DNA and significantly decreased those to lung DNA.

Experimental databases on inhibition of the bacterial mutagenicity of 4-nitroquinoline 1-oxide and cigarette smoke

Two antimutagenicity databases were prepared by applying a co-treatment procedure to the Salmonella reversion assay. Ninety compounds belonging to various chemical classes were quantitatively tested for antimutagenicity towards the direct-acting mutagen 4-nitroquinoline 1-oxide (4NQO) in strain TA100 of S. typhimurium and 63 of them were additionally tested for antimutagenicity towards unfractionated mainstream cigarette smoke (CS) in strain TA98, in the presence of S9 mix. Twelve compounds (13.3%) inhibited 4NQO mutagenicity by at least 50%, with a MID50 (dose inhibiting 50% of mutagenicity) varying over a 1226-fold range. Twenty-six compounds (41.3%) inhibited CS mutagenicity, with a MID50 varying over a 520-fold range. Three compounds only, i.e., bilirubin, curcumin and myricetin, were capable of inhibiting the mutagenicities of both 4NQO and CS. However, myricetin and the other flavonoid rutin were at the same time mutagenic by inducing frameshift mutations following metabolic activation. There was a rather rigorous selectivity of antimutagenicity data depending on the chemical class of inhibitors and it was possible to discriminate protective effects within several pairs or series of structurally related compounds. For instance, all eight thiols and aminothiols inhibited 4NQO mutagenicity, which contrasted with the inactivity of the remaining 17 sulfur compounds tested, all of them lacking a free sulfhydryl group. The mutagenicity of CS was consistently inhibited by the majority of phenols (eight out of 10 tested) and by all two isothiocyanates, two dithiocarbamates, three indole derivatives, three tetrapyrrole compounds and three flavonoids tested. Although the results obtained cannot be extrapolated to other mutagens or test systems, they may provide a useful source of information for research in the area of antimutagenesis and for the development of chemopreventive agents.

Selective induction of gene expression in rat lung by hexavalent chromium

Multigene‐expression analysis provides a formidable tool for evaluating cellular functions, under either physiological or pathological conditions, and for assessing their modulation by exogenous agents. We investigated multigene expression in the liver and lung of rats receiving intratracheal instillations of sodium dichromate for 3 consecutive days. Nylon membrane cDNA arrays were hybridized with standardized amounts of 32P‐labeled probes, and the results were normalized by making reference to housekeeping genes. The basal expression of 52 of 216 tested genes was 2.1–11.1 times higher in the liver than in the lung of control rats. No alteration of gene expression occurred in the liver of chromium(VI)‐treated rats, consistent with the fact that this metal species, being reduced upstream, can exert effects only locally but not at a distance from the respiratory tract. In contrast, the expression of 56 genes was increased 2.1 to 3.0 times in the lung as an early response to chromium(VI) administration. The altered genes are involved in the metabolic reduction of chromium(VI) and in a variety of interconnected functions, such as multidrug resistance and stress response, protein and DNA repair mechanisms, signal transduction pathways, apoptosis, and cell‐cycle modulation. Thus, short‐term treatment with chromium(VI) by intratracheal administration triggered a variety of defense processes in the lung. Although the use of selected genes does not provide an exhaustive picture of overall gene expression, these findings contribute to our understanding of chromium toxicology and provide a further mechanistic support to the involvement of thresholds in chromium(VI) carcinogenesis.

Interactions between N‐acetylcysteine and ascorbic acid in modulating mutagenesis and carcinogenesis

Both ascorbic acid (AsA, vitamin C) and N‐acetylcysteine (NAC), a precursor and analogue of glutathione, possess a broad array of biological properties underlying their protective role in a variety of pathophysiological conditions. However, under certain circumstances, AsA behaves as a pro‐oxidant rather than an anti‐oxidant and produces adverse effects. This prompted us to evaluate whether NAC could interact with AsA in preventing mutation and cancer. AsA significantly increased spontaneous revertants in the Salmonella typhimurium strains TA102 and TA104, which are sensitive to oxidative mutagens. In contrast, NAC lowered the spontaneous background in TA104 and neutralized the negative effects of AsA. Moreover, NAC and AsA showed additive effects in reducing chromium(VI) and in reverting its mutagenicity. A single i.p. injection of urethane (1 g/kg body weight) to 120 A/J mice resulted, after 4 months, in the formation of a total of 1,532 lung tumors, 425 in the 30 mice treated with the carcinogen only, 404 in those treated with urethane plus AsA, 365 in those treated with urethane plus NAC and 338 in those treated with urethane plus the combination of AsA and NAC (both given daily with drinking water at the dose of 1 g/kg body weight). Compared to positive controls, tumor multiplicity was poorly affected by AsA, whereas it was significantly decreased by NAC and even more so by its combination with AsA. The overall volumes of lung tumors in the 4 groups were 107.5, 89.3, 61.3 and 49.7 mm3, respectively. Tumor sizes were slightly but significantly decreased in mice treated with AsA and more so in those treated with NAC and NAC plus AsA, their combination being significantly more effective than each individually. All protective effects elicited by combining the 2 drugs were additive. Therefore, NAC prevents the adverse effects of AsA on spontaneous mutagenicity; at the same time, this thiol behaves in an additive fashion with AsA, inhibiting the mutagenicity of chromium(VI) and the lung tumorigenicity of urethane in mice. These findings suggest that NAC and AsA could conveniently be combined in cancer chemoprevention and other pharmacological interventions. Int. J. Cancer 88:702–707, 2000.