Mutagenesis vol. 18 no. 5 pp. 477-483,
September 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press
Meeting Report |
Prevention of degenerative diseases; clues from studies investigating oxidative stress, Brussels, 13 November 2002
Division of Toxicology and Cancer Risk Factors, German Cancer Research Center (DKFZ), 280 Im Neuenheimer Feld, 69120-Heidelberg, Germany
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The European Commission held a major conference from 11 to 13 November 2002 in the Palais du Heysel in Brussels to mark the launch of the European Union’s Sixth Framework Programme (FP-6) for research, which covers the period 2002–2006.
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The conference was a major forum to present the objectives and priorities of the FP-6 and to explain rules for participation. The Commission dedicated one part of the conference programme to the participants themselves. The resulting participants’ forum was organized by scientists, industrial researchers, research users, organizations and associations and others working in or affected by research and technology on topical cutting-edge subjects relevant to research and society. Following a call for proposals in March 2002, 250 proposals were received, of which 80 events were selected based on the following criteria: potential interest to the scientific community; a European and/or international dimension; relevance to European society; the participation of young scientists. Here we summarize event 61 entitled ‘Prevention of degenerative diseases: clues from studies investigating oxidative stress’, coordinated by J.Nair, German Cancer Research Centre (DKFZ), Germany. The forum event was structured to cover different aspects of the importance of oxidative stress in degenerative diseases with the ultimate goal of harnessing the knowledge gained in preventive and therapeutic strategies.
Oxidative processes are involved in ageing as well as in the pathogenesis of different degenerative diseases. Oxidative stress enhances lipid peroxidation (LPO), implicated in the promotion and progression stages of carcinogenesis and development of atherosclerosis, in particular under conditions of chronic inflammation and infections. Besides impaired quality of life and the loss of human resources, the cost of medical care in combating the above degenerative diseases within Europe in future will become enormous, as it is estimated that more than 2 000 000 new cases of degenerative diseases will be detected each year. Understanding the common underlying genetic disturbances that occur in pathways of disease progression will facilitate mechanism-based preventive strategies for lowering degenerative disease prevalence within European countries and world wide.
The event was chaired by H.Bartsch (German Cancer Research Centre, Heidelberg, Germany), who set the scene of the forum event by providing an overview on the role of oxidative stress in disease. Common pathways of chronic degenerative diseases (CDD) were through biologically relevant reactive oxygen species (ROS) and reactive nitrogen species (RNS), which can be generated by biochemical redox reactions, phagocytes and up-regulation of emergency enzymes like cyclooxygenase-2 (COX-2), lipoxygenase (LOX) and inducible nitric oxide synthase (iNOS). The resulting oxidative stress is currently implicated in over 100 human and animal diseases, including cancer, inflammatory, infectious, cardiovascular and neurological diseases. Etheno-DNA adducts, which are formed by LPO products resulting from increased oxidative stress, are promutagenic DNA lesions. The development of ultra-sensitive detection methods for etheno-DNA adducts (Nair et al., 1995
) has led to growing evidence that these adducts are elevated in cancer-prone patients and in rodents (liver, pancreas, colon and skin), indicating that etheno-adducts, when formed as a consequence of persistent oxidative stress, can drive cells to malignancy. Endogenous sources of DNA damage include known cancer risk factors such as metal storage, chronic inflammatory processes and a high 
-6 polyunsaturated fatty acid (PUFA) diet, which significantly increase the background level of these miscoding DNA adducts. Enhanced levels of etheno-DNA base adducts were detected in (i) colon tissue of ulcerative colitis and Crohn’s disease patients (both prone to colon cancer), (ii) urine of HBV-associated chronic hepatitis, liver cirrhosis and hepatocellular carcinoma patients from Thailand, (iii) abdominal aorta from patients with cardiovascular disease and (iv) human Alzheimer brain hippocampus. High dietary intake of -6 PUFA markedly increased etheno adduct levels in white blood cells of female volunteers on a controlled diet (Nair et al., 1997) while a protective effect of dietary vitamin E and vegetable intake against LPO-induced DNA damage was demonstrated in women on a free diet (Hagenlocher et al., 2001). These studies indicate that biomonitoring of exocyclic DNA adducts appears to be a promising tool to understand disease aetiopathogenesis of human CDD, including cancer and neurodegenerative diseases. These biomarkers should allow monitoring of the progression of disease, identifying targets for interventive therapy and implementing effective chemopreventive measures.
C.P.Wild (Molecular Epidemiology Unit, University of Leeds, Leeds, UK) further outlined common pathways in degenerative diseases due to oxidative stress. While a vast range of radicals can contribute to oxidative stress, a major question is whether oxidative stress is a cause or consequence of the disease. A range of events, including infection, radiation, toxins, ischaemia, exercise and temperature extremes, provoke a response to injury. Tissue injury subsequently triggers a whole variety of responses that can generate radicals. For example, about 15–20% of cancers develop via infectious agents. Major cancers associated with infections include those of liver, cervix, stomach, Kaposi’s sarcoma and cholangiocarcinoma. The 8-oxo-deoxyguanosine adduct was consistently increased in, for example, infection with HBV (liver), HCV (lymphocytes) and Helicobacter pylori (stomach) (Shimoda et al., 1994; Baik et al., 1996; Cardin et al., 2001). Barrett’s oesophagus is associated with at least an order of magnitude increased risk of developing adenocarcinoma of the oesophagus. This is probably due to chronic reflux, which is associated with inflammation and increased oxidative DNA damage. An understanding of underlying disease processes in each of these instances is crucial to developing a rationale for prevention and treatment. LPO may also be a key event in neurodegenerative diseases and vascular diseases. High levels of isoprostane 8–12 iso-isoprostane F2
-VI, a specific marker of LPO, were observed in patients with Alzheimer’s disease compared with cognitively normal elderly patients (Pratico and Delanty, 2000; Pratico and Trojanowski, 2000). A polymorphism in human glutathione peroxidase (hGPX1 Ala6 allele), which has been reported to be associated with increased lung cancer risk (Ratnasinghe et al., 2000), was also found to be associated with increased risk for coronary artery disease (Winter et al., 2003). Thus, understanding the common insult of oxidative stress to the different target molecules of DNA, lipids and proteins in different tissues in the context of different diseases illustrates that there are common pathways in the natural history of a number of CDD. Understanding these underlying pathways and having appropriate biomarkers, including markers of genetic susceptibility, would provide a rationale for developing preventive measures.
F.-J.van Schooten (Department of Health Risk Analysis and Toxicology, Maastricht University, Maastricht, The Netherlands) reviewed the role of detoxifying enzymes in oxidative stress and CDD. Under normal conditions, the body is well equipped with a variety of defence mechanisms that serve to inactivate the ROS generated. If these mechanisms are saturated or faulty, for instance by improper functioning of detoxifying enzymes, by low endogenous levels of antioxidants, following excess exposure to environmental factors (e.g. irradiation, iron loading or tobacco smoke) or following inflammation, a condition occurs that leads to oxidative stress (Sies, 1997). Important detoxifying enzymes of ROS are dismutase, peroxidase and catalase enzymes. Superoxide dismutase is responsible for decreasing superoxide levels by conversion to hydroperoxide and is present in the cytosol and the mitochondria. The cytosolic form is dependent upon zinc and copper cofactors while the mitochondrial form requires manganese. Catalase is a highly reactive enzyme found in peroxisomes which breaks down hydrogen peroxide to oxygen and water. Glutathione peroxidase is a selenium-requiring enzyme found in the cytoplasm and mitochondria which reduces hydroperoxide and hydrogen peroxide in reactions requiring glutathione. Myeloperoxidase (MPO) is involved in DNA damage via generation of peroxyl free radicals, hypochloric acid and activation of certain carcinogens (Petruska et al., 1992). A frequent –463 G
A polymorphism in the MPO gene diminishes MPO activity and is associated with lower DNA damage in cigarette smokers or coal tar-treated eczema patients (Rojas et al., 2001). Cytochrome P450 enzymes in animals and humans constitute one of the primary defence systems against toxic chemicals in food or the environment. The induction of these enzymes prevents acute toxic effects due to foreign chemicals but also results in oxidant by-products. Therefore, enzymes responsible for detoxification of xenobiotics, for instance glutathione S-transferases, are additionally important to control cellular oxidative stress. People differ in their potential to efficiently control ROS-induced damage, to eliminate hydrogen peroxide, to protect and repair oxidized phospholipids and to detoxify harmful environmental compounds. One of the explanations for the differences in handling oxidative stress and xenobiotic exposures can be found in inherited differences in enzyme activities. Consequently, genetic polymorphisms in oxidative stress genes have been associated with amyotrophic lateral sclerosis, heart disease, cancer and neurological degeneration (Forsberg et al., 2001).
G.Fürstenberger (German Cancer Research Center, Heidelberg, Germany) elaborated on oxidative stress response signals. Transient fluctuations of ROS serve as important intracellular signals, unlike exposure to high or sustained levels that cause damage to cells and tissues. Consistently associated with these processes is stimulation of the arachidonic acid cascade. Arachidonic acid (-6 PUFA) released from phospholipids by phospholipase A2 (PLA2) is oxygenated through two major routes, the COX and LOX pathways. The COX isozymes (COX-1 or inducible COX-2) catalyse the formation of endohydroperoxide PGG2 that, via reduction to PGH2, is converted to prostaglandins and thromboxanes. The LOX pathway oxidises arachidonic acid to hydroperoxy derivatives, which on conversion to hydroxy derivatives (HETE) leads to the formation of leukotrienes and lipoxins. PLA2, COX and LOX enzymes, the rate-limiting components of the arachidonic acid cascade, are targeted by ROS along various pathways, e.g. by initiating the COX and LOX reaction cycle, by activating signalling cascades leading to an increase in cPLA2 activity and by inducing the expression of COX and LOX via NF-
B- and/or AP1-dependent pathways. In addition, ROS are generated in the course of COX- and LOX-catalysed arachidonic acid metabolism, amplifying the oxidative stress response.
Aberrant expression and activity of inducible COX-2 and individual LOX isozymes is a consistent feature of epithelial tumours in humans and experimental animals. In the initiation–promotion model of multistage mouse skin carcinogenesis, the LOX enzymes are differentially up- or down-regulated in the course of tumour development in a stage-dependent manner (Furstenberger et al., 2002; Muller et al., 2002). In addition, inhibitors of LOX have been shown to suppress tumour formation in various epithelial animal tissues. COX-2, but not COX-1, is aberrantly expressed in papillomas and squamous cell carcinomas, leading to a massive accumulation of PGE2 and PGF2 in the tumour tissue (Muller-Decker et al., 1995). Selective COX-2 inhibitors strongly reduce tumour development in mouse skin (Muller-Decker et al., 1998). Transgenic mice overexpressing COX-2 under the control of the keratin 5 promoter in basal keratinocytes develop skin hyperplasia and dysplasia and a sebaceous gland hyperplasia accompanied by neo-angiogenesis (Neufang et al., 2001). Moreover, COX-2 overexpression induces autopromotion in mouse skin allowing tumour development in skin upon initiation by DMBA alone, indicating that COX-2 overexpression is causally related to the promotion stage of mouse carcinogenesis (Muller-Decker et al., 2002). Most importantly, most epithelial tumours of humans show very similar expression profiles of COX-2 and LOX, although data for LOX is still limited. Thus, COX-2 and specific LOX appear to be appropriate molecular targets for chemopreventive measures (Marks and Furstenberger, 2000).
J.Nair (German Cancer Research Center, Heidelberg, Germany) continued to elaborate on oxidative stress response signals and their role in cellular damage. He referred to oxidative stress as a double-edged sword. While transient oxidative stress activates defence mechanisms in the cell, other enzymes induced by oxidative stress (COX-2, LOX and iNOS) can generate reactive intermediates that can damage cellular macromolecules. Survival of cells depends on their ability to resist stress, to perform repair and to remove damaged molecules or cells. The -6 PUFAs, arachidonic acid and linoleic acid are metabolized by enzymes overexpressed during chronic inflammation to form the reactive aldehydes, such as malondialdehyde and trans-4-hydroxy-2-nonenal (HNE), which can attack DNA to form adducts. Familial adenomatous polyposis is a high penetrance autosomal dominant disease that predisposes to colorectal adenoma and carcinoma. Significantly higher levels of etheno-DNA adducts (etheno-dA and etheno-dC) were detected in polyps of patients with familial adenomatous polyposis (Schmid et al., 2000). In the multistage mouse skin carcinogenesis model, a strong positive correlation was observed between the formation of etheno-DNA adducts and the LOX-catalysed arachidonic acid metabolite HETE (Nair et al., 2000). In APC min mice, deletion of cPLA2 (which decreases arachidonic acid supply to COX-2) results in a significant decrease in the number of intestinal polyps (Hong et al., 2001). Pre-B lymphoma cells (RcsX) when injected into SJL mice were shown to increase NO production 50-fold (Tamir et al., 1995). Increased levels of etheno adducts were observed in spleen DNA upon this treatment and the adduct levels could be significantly reduced by simultaneous treatment with the iNOS inhibitor N6-methyl-L-arginine (Nair et al., 1998). NO and derived species modulate COX activity and eicosanoid production as shown in the iNOS knockout mice (Marnett et al., 2000). In patients with Alzheimer’s disease, PGH2 accelerated the formation of amyloid ß1–42 oligomers, as observed by immunohistochemistry (Boutaud et al., 2002), and plasma HNE concentrations were also significantly enhanced (McGrath et al., 2001). Increased etheno-dA levels could be visualized by immunohistochemistry in the brain of OXYS rats, a strain that mimics human degenerative diseases and develops cataract, scoliosis and cancer (Nair et al., in preparation). In cardiovascular diseases the importance of oxidative stress in the causal path is also indicated by increased levels of COX-2 in arterial plaques (McGeer et al., 2002), the presence of malondialdehyde-modified low density lipoproteins and proteins in atherosclerotic human vascular tissue and the protective effects of selective COX-2 inhibitors on the adverse outcome in acute coronary syndrome (Altman et al., 2002). These studies substantiate the important role of ROS in intricate signal transduction involved in cell survival and death.
A.Barbin (International Agency for Research on Cancer, Lyon, France) deliberated upon DNA damage induced by LPO. Aldehydes derived from LPO are mutagenic and clastogenic in different test systems (Esterbauer et al., 1991). Malondialdehyde forms a pyrimidopurinone adduct (M1G) with guanine moieties in DNA (Marnett, 1999). Enals derived by LPO from -6 PUFA react with nucleobases, yielding propano adducts (Pan and Chung, 2002). Alternatively, they can be further oxidized into epoxides that form etheno adducts with DNA bases (Chung et al., 1996). Exocyclic DNA adducts exhibit promutagenic properties in site-directed mutagenesis assays, inducing mainly base pair substitution mutations (Marnett, 1999; Barbin, 2000; Yang et al., 2002). Identical or structurally related exocyclic adducts are also formed by various mutagenic and carcinogenic xenobiotics (Bartsch et al., 1994). The mutations observed in the ras and p53 genes in tumours induced by vinyl chloride or by urethane in rodents and in humans are compatible with the promutagenic properties of etheno-bases (Barbin, 2000). In rodents exposed to these carcinogens or their metabolites, linear correlations have been established between the levels of etheno DNA adducts and the incidence of tumours (Swenberg et al., 1999). Pilot studies have demonstrated elevated levels of etheno-DNA or M1G adducts in target tissues from humans suffering from genetic or acquired diseases associated with increased LPO (Bartsch and Nair, 2000; Leuratti et al., 2002). This has been confirmed in animal models of these human diseases (Nair et al., 1996). Levels of exocyclic adducts are increased by certain lifestyle habits, such as a diet rich in -6 PUFA, high consumption of red meat, alcohol drinking and smoking, which are considered as risk factors for cancer and cardiovascular diseases (Nair et al., 1997; Leuratti et al., 2002; Zhang et al., 2002). Conversely, lower levels of exocyclic DNA adducts have been associated with consumption of vegetables, fruit, whole meal bread and vitamin E intake (Hagenlocher et al., 2001; Leuratti et al., 2002). Modulating effects of diet and alcohol on endogenous levels of etheno-DNA adducts have also been observed in rodents (Navasumrit et al., 2001). A background level of exocyclic DNA adducts has been detected in all human and animal tissues examined so far, showing a high inter-individual variability in different tissues. Compared with other oxidative DNA lesions, exocyclic adducts are not subject to artifactual degradation or formation during the analysis.
H.Ohshima (International Agency for Research on Cancer, Lyon, France) focused on DNA damage induced by inflammatory oxidants. Chronic infection and inflammation activate a variety of inflammatory cells and the diverse free radicals and oxidants are produced in high concentrations. They react with each other to generate new ROS and RNS such as peroxynitrite and nitryl chloride (NO2Cl), which damage DNA, RNA, lipids and proteins by nitration, oxidation and chlorination reactions. Peroxynitrite was shown to react with guanine and deoxynucleosides to form 8-nitroguanine and cytotoxic base propenals, respectively, and cause DNA strand breakage (Szabo and Ohshima, 1997). 8-Nitroguanine in DNA is easily depurinated to yield an apurinic site and thus it can potentially induce G:CT:A transversion mutations (Yermilov et al., 1995). 8-Nitroguanine in DNA could not be used as a biomarker for DNA damage caused by RNS as it is not stable. However, recently it was found that 8-nitroguanine formed in RNA was relatively stable and can be used as an exposure marker for RNS (Masuda et al., 2001). Formation of 8-nitroguanine in cellular RNA has recently been shown using polyclonal and monoclonal antibodies against 8-nitroguanine (Akaike et al., 2003). NO2Cl and HOCl also react with nucleosides, DNA and RNA to form various chlorinated bases, including 8-chloroguanine, 5-chlorocytosine and 8-chloroadenine (Masuda et al., 2002). Human MPO and activated neutrophils catalyse the formation of chlorinated bases in the presence of H2O2 and Cl–. Tertiary amines such as nicotine dramatically enhance the chlorination reaction mediated by HOCl and MPO (Masuda et al., 2002). Genetic polymorphism of MPO has been associated with a modified lung cancer risk, suggesting that chlorination damage mediated by MPO, HOCl and nicotine could play an important role in lung cancer development in smokers. In collaboration, both polyclonal and monoclonal antibodies against 8-chloroguanine have been successfully produced, which could be useful to detect this adduct in cellular DNA/RNA or in urine. 8-Oxoguanine, which is being measured as a marker of oxidative DNA damage in many laboratories, is further oxidized by oxidants to yield spiroiminodihydantoin deoxyribonucleoside and other products (Suzuki et al., 2001, 2002). As these further oxidized products of 8-oxoguanine could be better and more stable markers for oxidative DNA damage, methods are currently being developed to detect such modified bases in DNA. Thus base modifications caused by inflammatory oxidants in tissues could be measured as biomarkers for the development of infection- or inflammation-associated human cancers and, equally important, to study the efficacy of chemoprevention trials using anti-inflammatory or antioxidative agents.
M.Saparbaev (Institute Gustave Roussy, Villejuif, France) addressed repair mechanisms of oxidative stress-induced DNA damage, pointing out that about 100 different base- and sugar-damaged DNA lesions have been identified. To immediately counteract the deleterious effects leading to genomic disturbance, cells have evolved a number of extremely well conserved DNA repair mechanisms. ROS generate mostly non-bulky DNA lesions; most of them are substrates for the base excision repair (BER) pathway, although recently it was shown that other pathways would also be efficient in the removal of oxidized bases. Key enzymes involved in the excision of oxidized bases in the BER pathway are DNA glycosylases (Gros et al., 2002), which cleave the N-glycosylic bond between the abnormal base and deoxyribose, leaving an abasic site in DNA. However, the BER pathway, requiring the sequential action of two enzymes for incision of DNA (Laval, 1977), raises theoretical problems for the efficient repair of oxidative DNA damage because it generates genotoxic intermediates such as abasic (AP) sites and/or blocking 3'-terminal groups that must be eliminated by additional steps before initiating DNA repair synthesis. In addition, biological evidence hints at the existence of an alternative repair pathway. Escherischia coli and mice mutants deficient in the DNA glycosylases that remove oxidized bases are not sensitive to ROS (Klungland et al., 1999b; Blaisdell and Wallace, 2001; Takao et al., 2002). Recent findings indicate that E.coli Nfo and Nfo-like endonucleases of yeast and human origin nick DNA on the 5'-side of various oxidative damaged bases, generating 3'-hydroxyl and 5'-phosphate termini. This provides an alternative, DNA glycosylase-independent nucleotide incision repair (NIR) pathway (Ischenko and Saparbaev, 2002). This alternative repair pathway is evolutionarily conserved from E.coli to humans. The NIR pathway directly generates proper ends for DNA replication on one side and on the other side for elimination of the lesion by specific nucleases. This mechanistic characteristic creates an advantage as compared with BER.
In most tumours, genome disturbances are seen at the chromosomal level, with frequent gains and losses of part or whole chromosomes. In a small subset of tumours, the instability is at the nucleotide level. Evidence has accumulated that gross structural alterations in chromosomes are due to double-strand breaks, whereas subtle sequence changes could be due to the non-efficient repair of various types of base damages in DNA (Weeda et al., 1998). Several lines of evidence suggest that the excision/incision repair of damaged DNA can generate single- and double-strand breaks. Moreover, the single-strand breaks could be converted to double-strand breaks upon replication (Blaisdell and Wallace, 2001; Kuzminov, 2001). Therefore, in vivo the repair of oxidative DNA damage should be highly regulated and avoid the persistence of genotoxic intermediates such as AP and/or single-strand breaks with blocking 3'-terminal groups. Indeed, it has been shown that certain repair proteins can modulate the activity of the DNA glycosylases (Klungland et al., 1999a; Privezentzev et al., 2001; Hardeland et al., 2002). These proteins could orchestrate excision repair. Altering the fine balance of such proteins could have profound implications on the genome.
E.Dogliotti (Istituto Superiore di Sanità, Rome, Italy) gave an example of how cells have evolved multiple protective strategies to prevent the deleterious effects of DNA oxidation. There are multiple layers of control of DNA 8-oxo-2'-deoxyguanosine (8oxoG), which is the most stable product of oxidative DNA damage. A specific DNA glycosylase, OGG1, corrects 8oxoG:C pairs, MYH glycosylase excises A residues misincorporated opposite unrepaired 8oxoG during replication and MTH, an 8oxodGTPase, prevents the incorporation of 8oxodGMP into nascent DNA. The BER pathway is the main repair mechanism for either 8oxoG:C pairs in resting DNA (Laval et al., 1998; Boiteux and Radicella, 1999) or 8oxoG:A mismatches produced during replication (Parlanti et al., 2002). The mismatch repair (MMR) system also plays an important role by contributing to the excision of 8oxodGMP incorporated during replication. A large part of the mutator phenotype of MMR-defective cells is indeed a consequence of defective processing of oxidative DNA damage (Colussi et al., 2002). Although the basic BER of 8oxoG could be reconstituted in vitro with only four proteins (Pascucci et al., 2002), several lines of evidence indicate that the repair of oxidative damage in mammalian cell nuclei is more complex. For instance, the preferential repair of 8oxoG located in the transcribed sequences involves DNA glycosylases and mechanisms other than OGG1-initiated BER (Le Page et al., 2000a,b) and the replication-associated repair of oxidized bases incorporated into the nascent strand from the deoxynucleotide pool has yet to be clarified (reviewed in Mitra et al., 2002). It is believed that DNA polymerase-mediated BER, which shares several components with DNA replication, is a good candidate for replication-associated repair of oxidative DNA damage (Parlanti et al., 2002). If distinct complexes are involved in repair of transcriptionally active regions and in repair of quiescent versus dividing cells, the challenge for the future is to identify and characterize these complexes.
Up to now mouse models have provided limited clues to the function of the genes involved in repair of oxidative damage. OGG1-deficient mice, similar to other DNA glycosylase-defective mice, do not exhibit an excess of tumours (Klungland et al., 1999b). However, if the role of these repair genes is to protect the long-term integrity of the genome, a knockout animal might be a poor model for screening for reduction in fitness. Interestingly, MTH1-deficient mice display greater numbers of tumours in the lungs, liver and stomach compared with their wild-type littermates (Tsuzuki et al., 2001). Until recently, inherited deficiencies in the repair of oxidative DNA damage had not been causally linked with any human genetic disorder, but it has now been demonstrated that inherited defects of MYH predispose to multiple colorectal adenomas and carcinoma (Al Tassan et al., 2002; Jones et al., 2002). Moreover, polymorphic variants of some BER partners (e.g. XRCC1) have been shown to be associated with increased gastric cancer risk, and microsatellite instability, secondary to deficient function of MMR, is present in 27–40% of gastric cancer cases (reviewed in Gonzalez et al., 2002).
A.Collins (Rowett Research Institute, Aberdeen, UK) addressed the unresolved problem of whether antioxidants are the only good reason to eat fruits and vegetables. There is convincing epidemiological evidence that fruit and vegetables in the diet can help to prevent certain kinds of cancer. The idea arose, at a time when high levels of oxidized bases were being found in DNA of human cells, that this oxidative damage might be a significant cause of cancer and that antioxidants might prevent it and in turn protect against cancer. Several large-scale human supplementation trials were set up to test the efficacy of ß-carotene, believed to be a likely candidate for antioxidant activity in vivo. After several years of supplementation, two trials (The Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study Group, 1994; Omenn et al., 1996), involving smokers and/or asbestos workers as subjects, reported an increase in lung cancer incidence in the groups taking ß-carotene compared with those taking a placebo. Another trial, with mainly non-smokers, reported no effect of ß-carotene (Hennekens et al., 1996). Clearly, not enough is known about how plant micronutrients might affect human health.
Regarding the level of oxidative DNA damage actually present in the cell, after correcting for methodological problems, it now seems likely that the actual level of oxidative damage is around 1–5 8-oxoguanines per 106 guanines (ESCODD, 2002). Determining whether this low level can be further decreased by antioxidant supplementation requires the use of sensitive techniques that are not subject to adventitious oxidation. There are numerous reports that mixtures of dietary antioxidants or foods rich in antioxidants increase the ability of lymphocytes to withstand DNA oxidation in vitro and decrease the level of endogenous DNA oxidation (Duthie et al., 1996; Pool-Zobel et al., 1997; Mitchell and Collins, 1999; Porrini and Riso, 2000; Collins et al., 2003). However, DNA oxidation is a measure of exposure to DNA-damaging agents, rather than of cancer risk. Another biomarker, chromosome aberrations, is known to be associated with elevated cancer incidence, and it is therefore noteworthy that chromosome aberrations are decreased by supplementation with antioxidants (Du
inská et al., in preparation). The emphasis on antioxidants has diverted attention from other effects that phytochemicals might have. The decrease in DNA damage could be caused by a stimulation of DNA repair, as addition of kiwi fruit to the normal diet does indeed lead to a substantial enhancement of repair of oxidized guanine (Collins et al., 2003). Polymorphisms in genes coding for antioxidant enzymes or DNA repair pathways are likely to affect individual responses to oxidative stress. Polymorphisms in glutathione S-transferase genes influence whether smokers have low vitamin C levels in the blood and elevated DNA damage in the lymphocytes relative to non-smokers (Duinská et al., 2001). The role of fruits and vegetables in preventing cancer and other chronic degenerative diseases is more complex than that of simply providing dietary antioxidants.
In conclusion, the meeting covered in depth and has up-dated the concept that various chronic degenerative diseases with different clinical appearances share common biochemical, genetic and cellular alterations that appear to be related to disease pathogenesis. The presentations also revealed important lacunae in knowledge and pointed to principal research areas to be addressed. Altogether this platform event provided an excellent opportunity for interdisciplinary scientific exchange and initiation of a network of integrated collaborative projects ranging from molecular biology to clinical and molecular epidemiological investigations. Finally, a concerted effort to apply the knowledge gained in implementing common mechanism-based preventive strategies for lowering degenerative disease prevalence in Europe and world wide was proposed.
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We wish to thank Helmholtz Gemeinschaft (Bonn, Germany) for financial support for the forum event.
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1To whom correspondence should be addressed. Tel: +49 (0)6221 423306; Fax: +49 (0)6221 423359; Email: j.nair{at}dkfz.de
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Received on March 27, 2003; revised on April 3, 2003; accepted on April 7, 2003.
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