Although smoking cessation is the primary goal for the control of cancer and other smoking-related diseases, chemoprevention provides a complementary approach applicable to high risk individuals such as current smokers and ex-smokers. The thiol N-acetylcysteine (NAC) works per se in the extracellular environment, and is a precursor of intracellular cysteine and glutathione (GSH). Almost 40 years of experience in the prophylaxis and therapy of a variety of clinical conditions, mostly involving GSH depletion and alterations of the redox status, have established the safety of this drug, even at very high doses and for long-term treatments. A number of studies performed since 1984 have indicated that NAC has the potential to prevent cancer and other mutation-related diseases. N-Acetylcysteine has an impressive array of mechanisms and protective effects towards DNA damage and carcinogenesis, which are related to its nucleophilicity, antioxidant activity, modulation of metabolism, effects in mitochondria, decrease of the biologically effective dose of carcinogens, modulation of DNA repair, inhibition of genotoxicity and cell transformation, modulation of gene expression and signal transduction pathways, regulation of cell survival and apoptosis, anti-inflammatory activity, anti-angiogenetic activity, immunological effects, inhibition of progression to malignancy, influence on cell cycle progression, inhibition of pre-neoplastic and neoplastic lesions, inhibition of invasion and metastasis, and protection towards adverse effects of other chemopreventive agents or chemotherapeutical agents. These mechanisms are herein reviewed and commented on with special reference to smoking-related end-points, as evaluated in in vitro test systems, experimental animals and clinical trials. It is important that all protective effects of NAC were observed under a range of conditions produced by a variety of treatments or imbalances of homeostasis. However, our recent data show that, at least in mouse lung, under physiological conditions NAC does not alter per se the expression of multiple genes detected by cDNA array technology. On the whole, there is overwhelming evidence that NAC has the ability to modulate a variety of DNA damage- and cancer-related end-points.

In spite of the dominant role of cigarette smoke (CS) in cancer epidemiology, all studies performed during the past 60 years have shown that this complex mixture is either negative or weakly tumorigenic in experimental animals. We implemented studies aimed at evaluating whether exposure of mice early in life may enhance susceptibility to CS carcinogenicity. A total of 98 newborn Swiss albino mice were either untreated (controls) or received a subcutaneous injection of benzo(a)pyrene [B(a)P] (positive control) or were exposed whole-body to mainstream cigarette smoke (MCS) for 120 days, starting within 12 h after birth. Complete necropsy and histopathological analyses were performed at periodical intervals. In contrast with the lack of lung tumors in controls, MCS-exposed mice developed microscopically detectable tumors, starting only 75 days after birth and reaching an overall incidence of 78.3% after 181-230 days. The mean lung tumor multiplicities were 6.1 and 13.6 tumors per mouse in males and females, respectively, showing a significant intergender difference. Most tumors were microadenomas or adenomas, but 18.4% of the mice additionally had malignant lung cancer. MCS also induced bronchial and alveolar epithelial hyperplasia, and blood vessel proliferation. Furthermore, malignant tumors, some of which may have a metastatic origin, were detected in the urinary tract and liver of MCS-exposed mice. A somewhat different spectrum of tumors was observed in B(a)P-treated mice. In conclusion, MCS is a potent and broad spectrum carcinogen in mice when exposure starts early in life, covering stages of life corresponding to neonatal, childhood and adolescence periods in humans. This animal model will be useful to explore the mechanisms involved in CS-induced carcinogenesis and to investigate the protective effects of dietary agents and chemopreventive drugs.

The thiol N-acetylcysteine (NAC) is currently considered one of the most promising cancer chemopreventive agents by virtue of its multiple and coordinated mechanisms affecting the process of chemical carcinogenesis. Recent studies have shown that an unpaired cysteine residue in the propeptide plays a key role in inactivation of latent metastasis-associated metalloproteinases: the present study was designed to assess whether NAC could also affect tumor take, invasion and metastasis of malignant cells. As assessed by zymographic analysis, NAC completely inhibited the gelatinolytic activity of type-IV collagenases in the cells tested (gelatinases A and B). Moreover, NAC was efficient in inhibiting the chemotactic and invasive activities of tumor cells of human (A2058 melanoma) and murine origin (K1735 and B16-F10 melanoma cells as well as C87 Lewis lung carcinoma cells) in Boyden-chamber assays, which are predictive of the invasive and metastatic properties. Reduced glutathione (GSH) had a similar, although less effective activity. The number of lung metastases decreased sharply when B16-F10 murine melanoma cells, injected i.v. into nude mice, were pre-treated with NAC and resuspended in medium supplemented with 10 mM NAC. In other experiments NAC was given in drinking water, starting 48-72 hr before subcutaneous inoculation of either B16-F10 cells or of their highly metastatic variant B16-BL6, or intramuscular injection of LLC cells. In all experiments NAC treatment decreased the weight of the locally formed primary tumor and produced a dose-related delay in tumor formation. Spontaneous metastasis formation by B16-F10 and B16-BL6 tumors was slightly yet significantly reduced by oral administration of NAC. However, this was not observed for Lewis lung tumors. These data indicate that NAC affects the process of tumor-cell invasion and metastasis, probably due to inhibition of gelatinases by its sulfhydryl group, with the possible contribution of other mechanisms, including the potent antioxidant activity of this thiol.

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The thiol N-acetylcysteine (NAC), now under clinical trial for cancer chemoprevention both in Europe (project Euroscan) and in the US (National Cancer Institute), has been shown during the past decade to exert protective effects in a variety of experimental test systems. NAC inhibited spontaneous mutagenicity and that induced by a number of chemical compounds and complex mixtures. Moreover, NAC significantly decreased the incidence of neoplastic and preneoplastic lesions induced by several chemical carcinogens in rodents (mice, rats, hamsters), e.g., in lung, trachea, colon, liver, mammary gland, Zymbal gland, bladder and skin. Our studies provided evidence that multiple mechanisms contribute to NAC antimutagenicity and anticarcinogenicity. They include extracellular mechanisms, such as detoxification of reactive compounds due to the nucleophilic and antioxidant properties of NAC, inhibition of nitrosation products, and enhancement of thiol concentration in intestinal bacteria; trapping and enhanced detoxification of carcinogens in long-lived non-target cells, such as erythrocytes and bronchoalveolar lavage cells; mechanisms working in the cytoplasm of target cells, such as replenishment of GSH stores, modulation of metabolism of mutagens/carcinogens, blocking of electrophiles, and scavenging of reactive oxygen species; and nuclear effects, such as inhibition of DNA adduction by metabolites of carcinogens, inhibition of "spontaneous" mutations, attenuation of carcinogen-induced DNA damage, and protection of nuclear enzymes, such as poly(ADP-ribose) polymerase. In particular, benzo(a)pyrene diolepoxide-DNA adducts in rats exposed either to benzo(a)pyrene or cigarette smoke were prevented by NAC not only in target organs for carcinogenicity, such as lung and trachea, but also in other organs, such as heart, aorta and testis, where these molecular biomarkers have been tentatively associated with cardiomyopathies, atherosclerosis and hereditary diseases, respectively. The protective mechanisms of NAC are expected to affect not only initiation but also promotion and progression, due to the reiterate involvement of certain key mechanisms in carcinogenesis. Moreover, recent studies demonstrate that NAC can also affect the steps of invasion and metastasis, including the specific inhibition of type IV collagenases degrading basement membranes, inhibition of chemotactic and invasive activities of human and murine malignant cells, delay of primary tumor formation in mice, and inhibition of lung metastases. Evidence was also provided that administration of pharmacological doses of NAC sharply decreases urinary excretion of mutagens in smokers. Key words: N-Acetylcysteine, anticarcinogenicity, antimutagenicity, antioxidants, glutathione, molecular dosimetry, thiolsThe thiol N-acetylcysteine (NAC), an analogue and precursor of reduced glutathione (GSH), has become one of the most promising cancer chemopreventive agents [l], and is currently under clinical trial at both the National Cancer Institute (Chemoprevention Bra...

We have extensively studied the effects of N-acetylcysteine (NAC), a cytoprotective drug that can prevent in vivo carcinogenesis. Here we review our findings NAC completely inhibits gelatinolytic activity of metalloproteases and chemotactic and invasive activities of tumor cells. In addition, NAC reduces the number of lung metastases when malignant murine melanoma cells are injected into nude mice. NAC treatment decreases the weight of primary tumors and produces a dose-related increase in tumor latency. Moreover, oral administration of NAC reduces the formation of spontaneous metastases. In experimental metastasis assays, we have found a synergistic reduction in the number of lung metastases after treatment with doxorubicin (DOX) and NAC in nude mice. In tumorigenicity and spontaneous metastasis assays, the combined administration of DOX and oral NAC again has shown synergistic effects on the frequency and weight of primary tumors and local recurrences and completely prevented the formation of lung metastases. The addition of NAC to endothelial cells strongly reduces their invasive activity in response to angiogenic stimuli. NAC inhibited the degradation and release of radiolabeled type IV collagen by activated endothelial cells, indicating that NAC blocks gelatinase activity. Oral administration of NAC reduces the angiogenic response induced by KS tumor cell products, confirming the ability of NAC to inhibit the invasive activity of endothelial cells in vivo and thereby blocking angiogenesis.

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