Modulation of apoptosis by cancer chemopreventive agents.

A review of almost 2000 studies showed that the large majority of 39 putative cancer chemopreventive agents induced "spontaneous" apoptosis. Inhibition of the programmed cell death triggered by a variety of stimuli was consistently reported only with ascorbic acid, alpha-tocopherol, and N-acetylcysteine (NAC). We performed experimental studies in rodents exposed to cigarette smoke, either mainstream (MCS) or environmental (ECS), and UV-A/B-containing light. The nonsteroidal anti-inflammatory drug sulindac did not affect the apoptotic process in the skin of light-exposed mice and in the lungs of ECS-exposed mice. Likewise, 5,6-benzoflavone, indole-3-carbinol, 1,2-dithiole-3-thione and oltipraz failed to modulate apoptosis in the respiratory tract of ECS-exposed rats. Phenethyl isothiocyanate further enhanced the frequency of apoptosis in pulmonary alveolar macrophages and bronchial epithelial cells, and upregulated several genes in the lung of ECS-exposed rats. Both individually and in combination with oltipraz, NAC inhibited apoptosis in the respiratory tract of rats exposed either to MCS or ECS. Moreover, NAC attenuated the ECS-related overexpression of proapoptotic genes and normalized the levels of proapoptotic proteins in rat lung. The transplacental administration of NAC to mice considerably attenuated gene overexpression in the liver of fetuses exposed to ECS throughout pregnancy. Inhibition of apoptosis by chemopreventive agents reflects their ability to counteract certain upstream signals, such as genotoxic damage, redox imbalances, and other forms of cellular stress that trigger apoptosis. On the other hand, enhancement of apoptosis is a double-edged sword, since it represents a protective mechanism in carcinogenesis but may contribute to the pathogenesis of other degenerative diseases. We suggest that stimulation of apoptosis by so many chemopreventive agents, as reported in the literature, may often reflect the occurrence of toxic effects at high doses.

The epidemiological revolution of the 20th century.

Until 100 years ago the epidemiological scenario of human diseases had substantially remained unchanged. The 20th century has been characterized by a fantastic advance in life expectancy and by a shift from infectious to chronic degenerative diseases as prevailing causes of death. As an example of the epidemiological revolution in a developed country, we reconstructed, year by year from 1901 to 2000, the situation in Italy. Reference to the situation in other countries is also made. Both crude and age-adjusted mortality data were made available for males and females. A new turning point became evident in the second half of the 20th century with the decline of mortality for cardiovascular diseases and, more recently, for tumors. This review discusses the roots and rationale for these epidemiological changes. The discoveries made in the area of biomedical sciences, the progress in preventive and curative medicine, and the improvement of hygienic conditions have been so spectacular that 1 million lives are saved every year in Italy as compared with the late 19th century.

Induction and modulation of lung tumors: genomic and transcriptional alterations in cigarette smoke-exposed mice.

Cigarette smoke plays a major role in the epidemiology of lung cancer, and smoke components have extensively been investigated in carcinogenicity and chemoprevention studies in experimental animals. However, it is much more difficult to reproduce the tumorigenicity of the whole complex mixture in preclinical models. The authors review here some results obtained in their laboratories, dealing with the induction of lung tumors, and genomic and transciptional alterations in smoke-exposed mice. The authors were successful in inducing lung tumors in 4 strains of mice exposed whole-body to environmental cigarette smoke, including Swiss albino, A/J, SKH-1 hairless, and p53 mutant (UL533 x A/J)F1 mice. However, the tumorigenic response was rather weak in all strains. Much more intense were the smoke-induced alterations of a variety of intermediate biomarkers, such as cytogenetic end points in pulmonary alveolar macrophages, bone marrow and peripheral blood erythrocytes; apoptosis, p53 oncoprotein, and proliferating cell nuclear antigen in the bronchial epithelium; bulky DNA adducts, 8-hydroxy-2-deoxyguanosine; multigene expression, and thiobarbituric acid-reactive aldehydes in whole lung and several other organs. Smoke-induced genomic and transcriptional alterations were suitable for evaluating their modulation by chemopreventive agent, as shown in studies using the thiol N-acetylcysteine and the nonsteroidal anti-inflammatory drug sulindac.

Modulation of cigarette smoke-related end-points in mutagenesis and carcinogenesis.

The epidemic of lung cancer and the increase of other tumours and chronic degenerative diseases associated with tobacco smoking have represented one of the most dramatic catastrophes of the 20th century. The control of this plague is one of the major challenges of preventive medicine for the next decades. The imperative goal is to refrain from smoking. However, chemoprevention by dietary and/or pharmacological agents provides a complementary strategy, which can be targeted not only to current smokers but also to former smokers and passive smokers. This article summarises the results of studies performed in our laboratories during the last 10 years, and provides new data generated in vitro, in experimental animals and in humans. We compared the ability of 63 putative chemopreventive agents to inhibit the bacterial mutagenicity of mainstream cigarette smoke. Modulation by ethanol and the mechanisms involved were also investigated both in vitro and in vivo. Several studies evaluated the effects of dietary chemopreventive agents towards smoke-related intermediate biomarkers in various cells, tissues and organs of rodents. The investigated end-points included metabolic parameters, adducts to haemoglobin, bulky adducts to nuclear DNA, oxidative DNA damage, adducts to mitochondrial DNA, apoptosis, cytogenetic damage in alveolar macrophages, bone marrow and peripheral blood erytrocytes, proliferation markers, and histopathological alterations. The agents tested in vivo included N-acetyl-L-cysteine, 1,2-dithiole-3-thione, oltipraz, phenethyl isothiocyanate, 5,6-benzoflavone, and sulindac. We started applying multigene expression analysis to chemoprevention research, and postulated that an optimal agent should not excessively alter per se the physiological background of gene expression but should be able to attenuate the alterations produced by cigarette smoke or other carcinogens. We are working to develop an animal model for the induction of lung tumours following exposure to cigarette smoke. The most encouraging results were so far obtained in models using A/J mice and Swiss albino mice. The same smoke-related biomarkers used in animal studies can conveniently be applied to human chemoprevention studies. We participated in trials evaluating the effects of N-acetyl-L-cysteine and oltipraz in smokers from Italy, The Netherlands, and the People's Republic of China. We are trying to develop a pharmacogenomic approach, e.g. based on genetic metabolic polymorphisms, aimed at predicting not only the risk of developing cancer but also the individual responsiveness to chemopreventive agents.

Molecular alterations and lung tumors in p53 mutant mice exposed to cigarette smoke.

Mutations and deletions in p53 are the most common genetic lesions in human cancer,and an extraordinarily high incidence of lung cancer occurs in smokers suffering from Li-Fraumeni syndrome, which is characterized by germ-line inactivation of one p53 allele. In contrast, p53 mutations are infrequent in lung tumors formed in A/J mice. Moreover, despite the dominant role of cigarette smoke in the epidemiology of human lung cancer, it is very difficult to reproduce the lung tumorigenicity of this complex mixture in animal models. We used a transgenic mouse with a dominant-negative p53 mutation to examine the effects of a mutant p53 on smoke-induced lung carcinogenesis in mice. p53 mutant (UL53-3 x A/J)F(1) mice of both genders and their wild-type (wt) littermate controls were exposed whole-body to environmental cigarette smoke (ECS) for up to 9.5 months. Untreated mutant mice of both genders underwent an early stimulus of bronchial cell proliferation, and an age-related formation of DNA adducts in lung and heart. In males, there was an age-related increase of micronucleated normochromatic erythrocytes in peripheral blood and an impairment of body weight gain. These findings underscore a physiological protective role of p53 in wt A/J mice. The response of wt and mutant mice to ECS was similar in terms of oxidative DNA damage in lung and heart, proliferation of the bronchial epithelium, and levels of p53 oncoprotein, as assessed after exposure for 28 days. In contrast, ECS-exposed mutant mice underwent a lower induction of apoptosis in bronchial epithelium, a greater formation of DNA adducts in lung and heart, and a more intense cytogenetic damage, shown by a higher frequency of micronuclei in pulmonary alveolar macrophages and in peripheral blood normochromatic erythrocytes. Interestingly, at the end of the experiment, DNA adducts were not repaired in either wt or mutant mice after discontinuing exposure to ECS for 1 week. A weak but significant increase of lung tumor incidence and multiplicity was induced in p53 mutant (UL53-3 x A/J)F(1) mice after exposure to ECS for either 5 months, followed by recovery in air for 4.5 months, or 9.5 continuative months. Conversely, no tumorigenic effect was observed in their wt littermate controls, carrying a 99.9% A/J background and 5% FVB genome. This contrasts with the weakly positive results obtained in previous studies using wt A/J mice. Thus, in agreement with the results of previous lung tumorigenicity studies performed with the smoke carcinogens benzo(a)pyrene and 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone, (UL53-3 x A/J)F(1) mice carrying a mutant p53 transgene appear to be more sensitive to ECS than the corresponding wt littermate controls. These findings provide evidence that p53 mutations play a role in smoke-related carcinogenesis not only in humans but also in A/J mice.

Induction of apoptosis in the lung but not in the liver of rats receiving intra-tracheal instillations of chromium(VI).

Several studies have shown that hexavalent chromium [Cr(VI)] induces apoptosis in a variety of in vitro test systems. We instilled intra-tracheally either saline or sodium dichromate (0.25 mg/kg body weight), for three consecutive days, to Sprague-Dawley rats. TUNEL analyses showed a marked increase of the apoptotic index in both bronchial epithelium and lung parenchyma of Cr(VI)-treated rats, but no effect was detected in their liver. In parallel, the expression of 13 out of 18 apoptosis-related genes, evaluated by cDNA array analysis, was significantly enhanced in rat lung. The overexpressed genes included c-Jun N-terminal kinases 1, 2 and 3, bcl-x, bcl-2-associated death promoter and bcl-2-related ovarian killer protein, caspases 1, 3 and 6, DNase I precursor, DNA topoisomerases I and II alpha, and poly(ADP-ribose) polymerase. The enhancement of p53 expression in the lung was borderline to statistical significance. Expressions of bcl-2, bax-alpha, mdm2 and DNA topoisomerase IIB were not enhanced to a significant extent in lung. No induction of gene expression was observed in rat liver. RT-PCR analyses confirmed that Cr(VI) enhances the expression of c-Jun N-terminal kinase 1, caspase 6, and DNase I precursor but not that of bcl-2 in lung, while none of these genes was overexpressed in the liver of Cr(VI)-treated rats. The lack of stimulation of apoptosis in the liver parallels the failure of Cr(VI) to produce genotoxic damage, as we previously observed under identical experimental conditions. These negative findings may be ascribed to reduction of Cr(VI) to Cr(III) when traveling from the respiratory tract to the liver. On the other hand, induction of apoptosis in the respiratory tract parallels the occurrence of genotoxic effects and oxidative DNA damage produced by Cr(VI) in the same tissue. As previously shown in another laboratory, Cr(VI) did not induce lung tumors after 30 months of administration of the same daily dose. Therefore, apoptosis is likely to provide a protective mechanism at a post-genotoxic stage of Cr(VI) carcinogenesis.