Showing papers in "Mutation Research in 1999"

Lipid peroxidation—DNA damage by malondialdehyde

Abstract: Malondialdehyde is a naturally occurring product of lipid peroxidation and prostaglandin biosynthesis that is mutagenic and carcinogenic. It reacts with DNA to form adducts to deoxyguanosine and deoxyadenosine. The major adduct to DNA is a pyrimidopurinone called M1G. Site-specific mutagenesis experiments indicate that M1G is mutagenic in bacteria and is repaired by the nucleotide excision repair pathway. M1G has been detected in liver, white blood cells, pancreas, and breast from healthy human beings at levels ranging from 1–120 per 108 nucleotides. Several different assays for M1G have been described that are based on mass spectrometry, 32 P -postlabeling, or immunochemical techniques. Each technique offers advantages and disadvantages based on a combination of sensitivity and specificity. Application of each of these techniques to the analysis of M1G is reviewed and future needs for improvements are identified. M1G appears to be a major endogenous DNA adduct in human beings that may contribute significantly to cancer linked to lifestyle and dietary factors. High throughput methods for its detection and quantitation will be extremely useful for screening large populations.

Molecular mechanisms of chemopreventive effects of selected dietary and medicinal phenolic substances.

Abstract: Recently, considerable attention has been focused on identifying naturally occurring chemopreventive substances capable of inhibiting, retarding, or reversing the multi-stage carcinogenesis. A wide array of phenolic substances, particularly those present in dietary and medicinal plants, have been reported to possess substantial anticarcinogenic and antimutagenic activities. The majority of these naturally occurring phenolics retain antioxidative and anti-inflammatory properties which appear to contribute to their chemopreventive or chemoprotective activity. Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide), a pungent ingredient of hot chili pepper, protects against experimentally-induced mutagenesis and tumorigenesis. It also induces apoptosis in various immortalized or malignant cell lines. Plants of ginger family (Zingiberaceae) have been frequently and widely used as spices and also, in traditional oriental medicine. Curcumin, a yellow ingredient from turmeric (Curcuma longa L., Zingiberaceae), has been extensively investigated for its cancer chemopreventive potential. Yakuchinone A [1-(4'-hydroxy-3'-methoxyphenyl)-7-phenyl-3-heptanone] and yakuchinone B [1-(4'-hydroxy-3'-methoxyphenyl)-7-phenylhept-1-en-3-one] present in Alpinia oxyphylla Miquel (Zingiberaceae) have inhibitory effects on phorbol ester-induced inflammation and skin carcinogenesis in mice, and oxidative stress in vitro. These diarylheptanoids suppress phorbol ester-induced activation of ornithine decarboxylase and production of tumor necrosis factor-alpha or interleukin-1alpha and their mRNA expression. They also nullified the phorbol ester-stimulated induction of activator protein 1 (AP-1) in cultured human promyelocytic leukemia (HL-60) cells. In addition, both yakuchinone A and B induced apoptotic death in HL-60 cells. Ginger (Zingiber officinale Roscoe, Zingiberaceae) contains such pungent ingredients as [6]-gingerol and [6]-paradol, which also have anti-tumor promotional and antiproliferative effects. Resveratrol (3, 5,4'-trihydroxy-trans-stilbene), a phytoalexin found in grapes and other dietary and medicinal plants, and (-)-epigallocatechin gallate, a major antioxidative green tea polyphenol, exert striking inhibitory effects on diverse cellular events associated with multi-stage carcinogenesis. In addition, these compounds have ability to suppress proliferation of human cancer cells via induction of apoptosis.

Hydroxyl radicals and DNA base damage

Abstract: Modified purine and pyrimidine bases constitute one of the major classes of hydroxyl-radical-mediated DNA damage together with oligonucleotide strand breaks, DNA-protein cross-links and abasic sites. A comprehensive survey of the main available data on both structural and mechanistic aspects of.OH-induced decomposition pathways of both purine and pyrimidine bases of isolated DNA and model compounds is presented. In this respect, detailed information is provided on both thymine and guanine whereas data are not as complete for adenine and cytosine. The second part of the overview is dedicated to the formation of.OH-induced base lesions within cellular DNA and in vivo situations. Before addressing this major point, the main available methods aimed at singling out.OH-mediated base modifications are critically reviewed. Unfortunately, it is clear that the bulk of the chemical and biochemical assays with the exception of the high performance liquid chromatographic-electrochemical detection (HPLC/ECD) method have suffered from major drawbacks. This explains why there are only a few available accurate data concerning both the qualitative and quantitative aspects of the.OH-induced formation of base damage within cellular DNA. Therefore, major efforts should be devoted to the reassessment of the level of oxidative base damage in cellular DNA using appropriate assays including suitable conditions of DNA extraction.

The HUman MicroNucleus Project--An international collaborative study on the use of the micronucleus technique for measuring DNA damage in humans.

Abstract: The International Collaborative Project on Micronucleus Frequency in Human Populations (HUMN) was organized to collect data on micronucleus (MN) frequencies in different human populations and different cell types. The test procedures considered by this project are assays using human lymphocytes (cytokinesis-block method), exfoliated epithelial cells, and other cell types. Data (including descriptions of the populations monitored, detailed test protocols, and test results) are being obtained from a large number of laboratories throughout the world and are being entered into a unified database. The information will be used to: (1) determine the extent of variation of 'normal' values for different laboratories and the influence of other factors potentially affecting baseline MN frequency, e.g., age, gender and life-style; (2) provide information on the effect of experimental protocol variations on MN frequency measurements; (3) design and test optimal protocols for the different cell types; and (4) determine the extent to which MN frequency is a valid biomarker of ageing and risk for diseases such as cancer.

The chemistry of DNA damage from nitric oxide and peroxynitrite

Abstract: Nitric oxide is a key participant in many physiological pathways; however, its reactivity gives it the potential to cause considerable damage to cells and tissues in its vicinity. Nitric oxide can react with DNA via multiple pathways. Once produced, subsequent conversion of nitric oxide to nitrous anhydride and/or peroxynitrite can lead to the nitrosative deamination of DNA bases such as guanine and cytosine. Complex oxidation chemistry can also occur causing DNA base and sugar oxidative modifications. This review describes the different mechanisms by which nitric oxide can damage DNA. First, the physiological significance of nitric oxide is discussed. Details of nitric oxide and peroxynitrite chemistry are then given. The final two sections outline the mechanisms underlying DNA damage induced by nitric oxide and peroxynitrite.

DNA adduct formation from tobacco-specific N-nitrosamines

Abstract: Tobacco-specific N-nitrosamines are a group of carcinogens derived from the tobacco alkaloids. They are likely causative factors for cancers of the lung, esophagus, pancreas, and oral cavity in people who use tobacco products. The most carcinogenic tobacco-specific nitrosamines in laboratory animals are 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and N'-nitrosonornicotine (NNN). DNA adduct formation from NNK and NNN has been studied extensively and is reviewed here. NNK is metabolically activated by cytochromes P450 to intermediates which methylate and pyridyloxobutylate DNA. The resulting adducts have been detected in cells and tissues susceptible to NNK carcinogenesis in rodents. The methylation and pyridyloxobutylation pathways are both important in carcinogenesis by NNK. NNK also induces single strand breaks and increases levels of 8-oxodeoxyguanosine in DNA of treated animals. NNAL, which like NNK is a potent pulmonary carcinogen, is also metabolically activated to methylating and pyridyloxobutylating intermediates. NNN pyridyloxobutylates DNA in its rat target tissues, esophagus and nasal mucosa. Methyl and pyridyloxobutyl DNA adducts are detected in human tissues. The methyl adducts most likely result in part from exposure of smokers to NNK, but these adducts are also detected in non-smokers. Some of the methyl adducts detected in non-smokers may be due to environmental tobacco smoke exposure. There are also potential dietary and endogenous sources of these adducts. Pyridyloxobutyl DNA adducts in human tissues result mainly from exposure to tobacco-specific N-nitrosamines. In laboratory animals, DNA adduct formation and carcinogenicity of tobacco-specific N-nitrosamines are closely correlated in many instances, and it is likely that similar relationships will hold in humans.

DNA damage by mycotoxins.

Abstract: Mycotoxins are toxic fungal metabolites which are structurally diverse, common contaminants of the ingredients of animal feed and human food. To date, mycotoxins with carcinogenic potency in experimental animal models include aflatoxins, sterigmatocystin, ochratoxin, fumonisins, zearalenone, and some Penicillium toxins. Most of these carcinogenic mycotoxins are genotoxic agents with the exception of fumonisins, which is currently believed to act by disrupting the signal transduction pathways of the target cells. Aflatoxin B1 (AFB1), a category I known human carcinogen and the most potent genotoxic agent, is mutagenic in many model systems and produces chromosomal aberrations, micronuclei, sister chromatid exchange, unscheduled DNA synthesis, and chromosomal strand breaks, as well as forms adducts in rodent and human cells. The predominant AFB1-DNA adduct was identified as 8, 9-dihydro-8-(N7-guanyl)-9-hydroxy-AFB1 (AFB1-N7-Gua), which derives from covalent bond formation between C8 of AFB1-8,9-epoxides and N7 of guanine bases in DNA. Initial AFB1-N7-guanine adduct can convert to a ring-opened formamidopyrimidine derivative, AFB1-FAPY. The formation of AFB1-N7-guanine adduct was linear over the low-dose range in all species examined, and liver, the primary target organ, had the highest level of the adduct. Formation of initial AFB1-N7-guanine adduct was correlated with the incidence of hepatic tumor in trout and rats. The AFB1-N7-guanine adduct was removed from DNA rapidly and was excreted exclusively in urine of exposed rats. Several human studies have validated the similar correlation between dietary exposure to AFB1 and excretion of AFB1-N7-guanine in urine. Replication of DNA containing AFB1-N7-guanine adduct-induced G-->T mutations in an experimental model. Activation of ras protooncogene has been found in AFB1-induced tumors in mouse, rat, and fish. More strikingly, the relationship between aflatoxin exposure and development of human hepatocellular carcinoma (HHC) was demonstrated by the studies on the p53 tumor suppressor gene. High frequency of p53 mutations (G-->T transversion at codon 249) was found to occur in HHC collected from populations exposed to high levels of dietary aflatoxin in China and Southern Africa. Furthermore, AFB1-induced DNA damage and hepatocarcinogenesis in experimental models can be modulated by a variety of factors including nutrients, chemopreventive agents, and other factors such as food restriction and viral infection, as well as genetic polymorphisms.

Oxidative DNA damage mediated by copper(II), iron(II) and nickel(II) Fenton reactions: evidence for site-specific mechanisms in the formation of double-strand breaks, 8-hydroxydeoxyguanosine and putative intrastrand cross-links

Abstract: The role of metal ion-DNA interactions in the Fenton reaction-mediated formation of putative intrastrand cross-links, 8-hydroxydeoxyguanosine (8-OHdG) and single- and double-strand breaks was investigated. Salmon sperm DNA and pBluescript K+ plasmid were incubated with hydrogen peroxide and either copper(II), iron(II), or nickel(II), which differ in both their affinity for DNA and in the spectrum of oxidative DNA damage they induce in Fenton reactions. EDTA was included in these incubations according to two different strategies; the first (strategy 1) in which DNA and metal ions were mixed prior to the addition of EDTA, the second (strategy 2) in which EDTA and metal ions were mixed prior to the addition of DNA. The formation of the putative intrastrand cross-links, monitored by 32P-postlabelling, was not affected by the addition of between 10 microM and 5 mM EDTA to the copper(II) Fenton reaction according to strategy 1. In contrast, the level of cross-links declined significantly upon inclusion of 20 microM EDTA and above when added according to strategy 2. Similarly, formation of these lesions declined in the iron(II) Fenton reaction more dramatically upon addition of 5 mM EDTA when added according to strategy 2 compared to strategy 1, while the yield of cross-links formed in the nickel(II) Fenton reaction declined equally with both strategies with up to 25 mM EDTA. The formation of single- and double-strand breaks was investigated in plasmid DNA by agarose gel electrophoresis and subsequent densitometry. The formation of linear DNA in the iron(II) Fenton reaction decreased dramatically upon inclusion of EDTA according to strategy 2, while no such decline was observed using strategy 1. In contrast, the formation of linear DNA in the copper(II) Fenton reaction decreased upon inclusion of EDTA according to both strategies. A decrease in the formation of open-circular DNA was also observed upon inclusion of EDTA according to both strategies; however this decrease occurred at a lower EDTA concentration in strategy 2 (100 microM) compared to strategy 1 (200 microM), and the level of open-circular DNA reached a lower level (8. 5% compared to 24.2%). The nickel(II) Fenton reaction generated only open-circular DNA, and this was completely inhibited upon addition of 25 microM EDTA according to both strategies. There was less formation of 8-OHdG in the copper(II) and iron(II) Fenton reactions when EDTA was added according to strategy 2 than according to strategy 1. These results suggest that a site-specific mechanism is involved in the formation of double-strand breaks and, to a lesser extent, 8-OHdG and the putative intrastrand cross-links, while the formation of single-strand breaks is more likely to involve generation of hydroxyl radicals in solution.

Sperm chromatin damage associated with male smoking.

Abstract: Cigarette smoke is a rich source of mutagens and carcinogens; thus, we have investigated the effects of male smoking on the DNA of human sperm. This was performed using the sperm chromatin structure assay (SCSA), which measures the sensitivity of sperm DNA to acid induced denaturation, and the terminal deoxynucleotidyl transferase assay (TdTA), which measures DNA strand breaks by addition of the biotinylated nucleotide dUTP to 3'-OH ends of DNA, sites of DNA breakage, using the enzyme terminal deoxynucleotidyl transferase. Sperm from subjects who smoked were significantly more sensitive to acid induced denaturation than non-smokers (P<0.02) and possessed higher levels of DNA strand breaks (P<0.05). We hypothesise that smoking damages the chromatin structure and produces endogenous DNA strand breaks in human sperm. These changes may result in sperm DNA mutations, that predispose offspring to greater risk of malformations, cancer and genetic diseases.

Adverse reproductive outcomes from exposure to environmental mutagens.

Abstract: The effect of environmental pollution on reproductive outcomes has been studied in the research project 'Teplice Program' analyzing the impact of air pollution on human health. Genotoxicity of urban air particles <10 microm (PM10) in in vitro system was determined by the analysis of DNA adducts. The highest DNA binding activity was observed in aromatic fraction, identifying DNA adducts of carcinogenic polycyclic aromatic hydrocarbons (PAHs) presumably diolepoxide-derived from: 9-hydroxybenzo[a]pyrene (9-OH-B[a]P), benzo[a]pyrene-r-7,-dihydrodiol-t-9,10-epoxide[+] (anti-BPDE), benzo[b]fluoranthene (B[b]F), chrysene (CHRY), benz[a]antracene (B[a]A), indeno[1,2,3-cd]pyrene (I[cd]P). Reproductive studies were conducted in both females and males. A study of the effects of PM10 exposure on pregnancy outcomes found the relationship between the intrauterine growth retardation (IUGR) and PM10 levels over 40 microg/m(3) in the first gestational month (Odds Ratio for 40-50 microg/m(3)50 microg/m(3)=1.9). Selected biomarkers were analyzed in venous blood, cord blood (chromosomal aberrations, comet assay) and placenta (DNA adducts, genetic polymorphisms of GSTM1 and NAT2 genotypes) of women enrolled in a nested case-control study. DNA adduct levels were higher in polluted vs. control districts, in smoking vs. nonsmoking mothers, and in GSTM1 null genotype, which was more pronounced in polluted district. No effect of air pollution was observed by cytogenetic analysis of chromosomal aberrations or by comet assay. The reproductive development of young men was followed by measures of semen quality, adjusted for ambient SO(2) exposure. The analysis identified significant associations with air pollution for <13% morphologically normal sperm, <29% sperm with normal head shape, <24% motile sperm. Analysis of aneuploidy in human sperm by FISH showed, aneuploidy YY8 was associated with season of heaviest air pollution. These findings are suggestive for an influence of air pollution on YY8 disomy. All these results indicate that air pollution may increase DNA damage in human population, which may be even higher for susceptible groups. Biomarkers of exposure (DNA adducts) and susceptibility (GSTM1 and NAT2) may indicate the risk of presumable low environmental exposure. Pregnancy outcome and semen studies imply that relatively low air pollution (higher than 40 microg PM10/m(3)) can significantly increase the adverse reproductive outcomes affecting both genders.

Green tea and cancer chemoprevention.

Abstract: Worldwide interest in green tea as a cancer preventive agent for humans has increased, because it is non-toxic and it is effective in a wide range of organs. (-)-Epigallocatechin gallate (EGCG) is the main constituent of green tea; the others are (-)-epicatechin gallate, (-)-epigallocatechin and (-)-epicatechin (EC). This paper reports the results of our latest pharmacological and biochemical studies with 3H-EGCG, along with studies on human subjects. The study on bioavailability of 3H-EGCG in mice revealed the wide distribution of radioactivity in multiple organs. Specifically, radioactivity was found in all reported target organs of EGCG and green tea extract (digestive tract, liver, lung, pancreas, mammary gland and skin) as well as other organs (brain, kidney, uterus and ovary or testes) in mice. Recently, we demonstrated that EC enhanced incorporation of 3H-EGCG into human lung cancer cell line PC-9 cells. EC along with another cancer preventive agent sulindac also synergistically enhanced apoptosis in PC-9 cells induced by EGCG. Moreover, a case-control study on breast cancer patients revealed that high daily consumption of green tea was associated with a lower recurrence rate among Stages I and II patients. All the results suggest that consumption of green tea is a practical and effective cancer preventive both before cancer onset and after cancer treatment.

Estrogen, DNA damage and mutations.

Abstract: Estrogen administration to rodents results in various types of DNA damage and ultimately leads to tumors in estrogen-responsive tissues. Yet these hormones have been classified as nonmutagenic, because they did not induce mutations in classical bacterial and mammalian mutation assays. In this review, we have discussed the induction by estrogens of DNA and chromosomal damage and of gene mutations, because the classical assays were designed to uncover mutations only at one specific locus and could not have detected other types of mutations or changes in other genes. Various types of estrogen-induced DNA damage include: (a) direct covalent binding of estrogen quinone metabolites to DNA; (b) enhancement of endogenous DNA adducts by chronic estrogen exposure of rodents; (c) free radical generation by metabolic redox cycling between quinone and hydroquinone forms of estrogens and free radical damage to DNA such as strand breakage, 8-hydroxylation of purine bases of DNA and lipid hydroperoxide-mediated DNA modification. Two different types of chromosomal damage have also been induced by estrogen in vivo and in cells in culture such as numerical chromosomal changes and also structural chromosomal aberrations. Gene mutations have been induced in several cell types in culture either by the parent estrogen or by reactive estrogen quinone metabolites. Furthermore, in estrogen-induced kidney tumors in hamsters, several mutations have been observed in the DNA polymerase beta gene mRNA. Estradiol also induces microsatellite instability in these kidney tumors and in premalignant kidney exposed to estradiol. Although this work is still ongoing, it can be concluded that estrogens are complete carcinogens capable of tumor initiation by mutation potentially in critical genes. The hormonal effects of estrogens may complete the development of tumors.

Etheno DNA-base adducts from endogenous reactive species

Abstract: Promutagenic etheno (epsilon) adducts in DNA are generated through reactions of DNA bases with LPO products derived from endogenous sources or from exposure to several xenobiotics. The availability of sensitive methods has made it possible to detect three epsilon-adducts in vivo, namely epsilon dA, epsilon dC and N2,3-epsilon dG. One probable endogenous source for the formation of these adducts arises from LPO products such as trans-4-hydroxy-2-nonenal (HNE), resulting in highly variable background epsilon-adduct levels in tissues from unexposed humans and rodents. The range of background levels of epsilon dAx10-8dA detected inhuman tissues was <0.05 to 25 and in rodent tissues 0.02 to 10; the corresponding values for epsilon dCx10-8dC were 0.01 to 11 and 0.03 to 24, respectively. Part of this variability may be associated with different dietary intake of antioxidants and/or omega-6 PUFAs which oxidize readily to form 4-hydroxyalkenals, as epsilon dA and epsilon dC levels in WBC-DNA of female volunteers on a high omega-6 PUFA diet were drastically elevated. Increased levels of etheno adducts were also found in the liver of cancer-prone patients suffering from hereditary metal storage diseases, i.e., Wilson's disease (WD) and primary hemochromatosis (PH) as well as in Long-Evans Cinnamon rats, an animal model for WD. Increased metal-induced oxidative stress and LPO-derive epsilon-adducts, along with other oxidative damage, may trigger this hereditary liver cancer. Epsilon-Adducts could hence be explored as biomarkers (i) to ascertain the role of LPO mediated DNA damage in human cancers associated with oxidative stress imposed by certain lifestyle patterns, chronic infections and inflammations, and (ii) to verify the reduction of these epsilon-adducts by cancer chemopreventive agents. This article summarizes recent results on the formation, occurrence and possible role of epsilon-DNA adducts in carcinogenesis and mutagenesis.

The molecular pathways of ultraviolet-induced carcinogenesis.

Abstract: Cancer development requires the accumulation of numerous genetic changes which are usually believed to occur through the presence of unrepaired DNA lesions. Exogenous or endogenous DNA-damaging agents can lead to mutations in the absence of efficient error-free repair, via replication of DNA damage. Several DNA repair pathways are present in living cells and well-conserved from bacteria to human cells. The nucleotide excision repair (NER), the most versatile of these DNA repair systems, recognizes and eliminates a wide variety of DNA lesions and particularly those induced by ultraviolet (UV) light. The phenotypic consequences of a NER defect in humans are apparent in rare but dramatic diseases characterized by hypersensitivity to UV and a striking clinical and genetic heterogeneity. The xeroderma pigmentosum (XP) syndrome is a human disorder inherited as an autosomal recessive trait. Persistence of unrepaired DNA damage produced by exposure to UV light is associated, in the XP syndrome, with an extremely high level of skin tumors in sun-exposed sites. Several key genes are mutagenized by UV-light and are responsible for skin cancer development. Mutations are found on ras oncogenes, p53 and PTCH tumour suppressor genes in skin cancers from DNA repair proficient as well as XP patients. The typical signature of UV-induced mutations found on these genes allows one to conclude that the uvB part of sunlight is responsible for the initiation of the carcinogenesis process.

Endogenous oxidative damage of mtDNA.

Abstract: Almost a decade ago, based on analytical measurements of the oxidative DNA adduct 8-oxo-deoxyguanosine (oxo8dG), it was reported that mitochondrial DNA suffers greater endogenous oxidative damage than nuclear DNA. The subsequent discovery that somatic deletions of mitochondrial DNA occur in humans, and that they do so to the greatest extent in metabolically active tissues, strengthened the hypothesis that mitochondrial DNA is particularly susceptible to endogenous oxidative attack. This hypothesis was (and is) appealing for a number of reasons. Nevertheless, solid direct support for the hypothesis is lacking. Since the initial measurements, attempts to repeat the observation of greater oxidation of mitochondrial DNA have resulted in a range of measurements that spans over four orders of magnitude. Moreover, this range includes values that are as low as published values for nuclear DNA. In the last 2 years or so, it has become apparent that the quantification of oxidative DNA adducts is prone to artifactual oxidation. We have reported that the analysis of small quantities of DNA may be particularly susceptible to such interference. Because yields of mitochondrial DNA are generally low, a systematic artifact associated with low quantities of DNA may have elevated the apparent level of adduct oxo8dG in mitochondrial DNA relative to nuclear DNA in some studies. Whatever the cause for the experimental variation, the huge disparity between published measurements of oxidative damage makes it impossible to conclude that mitochondrial DNA suffers greater oxidation than nuclear DNA. Despite the present confusion, however, there are reasons to hypothesize that this is indeed the case. We briefly describe methods being developed by a number of workers that are likely to surmount current obstacles and allow the hypothesis to be tested definitively.

New approaches to understanding p53 gene tumor mutation spectra

Abstract: The first p53 gene mutation arising in a human tumor was described a decade ago by Baker et al. [S.J. Baker, E.R. Fearon, J.M. Nigro, S.R. Hamilton, A.C. Preisinger, J.M. Jessup, P. van Tuinen, D.H. Ledbetter, D.F. Barker, Y. Nakamura, R. White, B. Vogelstein, Chromosome 17 deletions and p53 gene mutations in colorectal carcinomas, Science 244 (1989) 217-221]. There are now over 10,000 mutations extracted from the published literature in the IARC database of human p53 tumor mutations [P. Hainaut, T. Hernandez, A. Robinson, P. Rodriguez-Tome, T. Flores, M. Hollstein, C.C. Harris, R. Montesano, IARC database of p53 gene mutations in human tumors and cell lines: updated compilation, revised formats and new visualization tools, Nucleic Acids Res. 26 (1998) 205-213; Version R3, January 1999]. A large and diverse collection of tumor mutations in cancer patients provides important information on the nature of environmental factors or biological processes that are important causes of human gene mutation, since xenobiotic mutagens as well as endogenous mechanisms of genetic change produce characteristic types of patterns in target DNA [J.H. Miller, Mutational specificity in bacteria, Annu. Rev. Genet. 17 (1983) 215-238; T. Lindahl, Instability and decay of the primary structure of DNA, Nature 362 (1993) 709-715; S.P. Hussain, C.C. Harris, Molecular epidemiology of human cancer: contribution of mutation spectra studies of tumor suppressor genes, Cancer Res. 58 (1998) 4023-4037; P. Hainaut, M. Hollstein, p53 and human cancer: the first ten thousand mutations, Adv. Cancer Res. 2000]. P53 gene mutations in cancers can be compared to point mutation spectra at the HPRT locus of human lymphocytes from patients or healthy individuals with known exposure histories, and accumulated data indicate that mutation patterns at the two loci share certain general features. Hypotheses regarding specific cancer risk factors can be tested by comparing p53 tumor mutations typical of a defined patient group against mutations generated experimentally in rodents or in prokaryotic and eukaryotic cells in vitro. Refinements of this approach to hypothesis testing are being explored that employ human p53 sequences introduced artificially into experimental organisms used in laboratory mutagenesis assays. P53-specific laboratory models, combined with DNA microchips designed for high through-put mutation screening promise to unmask information currently hidden in the compilation of human tumor p53 mutations.

Higher plant assays for the detection of chromosomal aberrations and gene mutations-a brief historical background on their use for screening and monitoring environmental chemicals.

Abstract: Higher plants are recognized as excellent indicators of cytogenetic and mutagenic effects of environmental chemicals and are applicable for the detection of environmental mutagens both indoor and outdoor. They are highly reliable bioassays with a high sensitivity for monitoring and testing for genotoxins. A brief review of major steps in the development of higher plant genotoxic assays is given.

Genotoxicity of wastewater samples from sewage and industrial effluent detected by the Allium root anaphase aberration and micronucleus assays.

Abstract: The genotoxicity of wastewater samples from sewage, and industrial effluent from the Amritsar, India, area were investigated using the Allium micronucleus and anaphase aberration assays. Raw sewage samples and acetone extracts of the dehydrated sewage were use for treatment of the Allium roots. Industrial effluents were collected and stored in the form of sludge (semi-dried matter). The acetone extracts of the sludge samples were also used for treatment of the Allium roots. From the Allium root micronuclei tests on the sewage extracts, no significant increase in the number of micronuclei was found in comparison with negative controls. All the other extracts from industrial effluent showed positive responses both in the micronucleus and anaphase aberration assays.

Oxidative damage elicited by imbalance of free radical scavenging enzymes is associated with large-scale mtDNA deletions in aging human skin.

Abstract: Mitochondrial DNA (mtDNA) mutations and impaired respiratory function have been demonstrated in various tissues of aged individuals. We hypothesized that age-dependent increase of ROS and free radicals production in mitochondria is associated with the accumulation of large-scale mtDNA deletions. In this study, we first confirmed that the proportion of mtDNA with the 4977 bp deletion in human skin tissues increases with age. We then investigated the 8-hydroxy-2'-deoxyguanosine (8-OH-dG) content in skin tissues and lipid peroxides content of the skin fibroblasts from subjects of different ages. The results showed an age-dependent increase of 8-OH-dG level in the total DNA of skin tissues of the subjects above the age of 60 years. The specific content of malondialdehyde, an end product of lipid peroxidation, was also found to increase with age. On the other hand, we examined the enzyme activities of Cu, Zn-superoxide dismutase (Cu,Zn-SOD), Mn-superoxide dismutase (Mn-SOD), catalase, and glutathione peroxidase (GPx) in the skin fibroblasts. The activities of Cu,Zn-SOD, catalase and glutathione peroxidase were found to decrease with age. However, the activity of Mn-SOD was increased with age before 60 years but was decreased thereafter. Moreover, the activity ratios of Mn-SOD/catalase and Mn-SOD/GPx exhibited the same pattern of change with age. This indicates that free radical scavenging enzymes can effectively dispose of ROS and free radicals before 60 years of age. However, elevated oxidative stress caused by an imbalance between the production and removal of ROS and free radicals occurred in skin fibroblasts after 60 years of age. Taken together, we suggest that the functional decline of free radical scavenging enzymes and the elevation of oxidative stress may play an important role in eliciting oxidative damage and mutation of mtDNA during the human aging process.

Reactive oxygen species: the unavoidable environmental insult?

Abstract: Reactive oxygen species (ROS) are generated by a variety of sources from the environment (e.g., photo-oxidations and emissions) and normal cellular functions (e.g., mitochondrial metabolism and neutrophil activation). ROS include free radicals (e.g., superoxide and hydroxyl radicals), nonradical oxygen species (e.g., hydrogen peroxide and peroxynitrite) and reactive lipids and carbohydrates (e. g., ketoaldehydes, hydroxynonenal). Oxidative damage to DNA can occur by many routes including the oxidative modification of the nucleotide bases, sugars, or by forming crosslinks. Such modifications can lead to mutations, pathologies, cellular aging and death. Oxidation of proteins appears to play a causative role in many chronic diseases of aging including cataractogenesis, rheumatoid arthritis, and various neurodegenerative diseases including Alzheimer's Disease (AD). Our goal is to elucidate the mechanism(s) by which oxidative modification results in the disease. These studies have shown that (a) cells from old individuals are more susceptible to oxidative damage than cells from young donors; (b) oxidative protein modification is not random; (c) some of the damage can be prevented by antioxidants, but there is an age-dependent difference; and (d) an age-related impairment of recognition and destruction of modified proteins exists. It is believed that mechanistic insight into oxidative damage will allow prevention or intervention such that these insults are not inevitable. Our studies are also designed to identify the proteins which are most susceptible to ROS damage and to use these as potential biomarkers for the early diagnosis of diseases such as AD. For example, separation of proteins from cells or tissues on one- and two-dimensional gels followed by staining for both total protein and specifically oxidized residues (e.g., nitrotyrosine) may allow identification of biomarkers for AD.

Satellite DNA hypomethylation vs. overall genomic hypomethylation in ovarian epithelial tumors of different malignant potential.

Abstract: Rearrangements in heterochromatin in the vicinity of the centromeres of chromosomes 1 and 16 are frequent in many types of cancer, including ovarian epithelial carcinomas. Satellite 2 DNA is the main sequence in the unusually long heterochromatin region adjacent to the centromere of each of these chromosomes. Rearrangements in these regions and hypomethylation of satellite 2 DNA are a characteristic feature of patients with a rare recessive genetic disease, ICF (immunodeficiency, centromeric region instability, and facial anomalies). In all normal tissues of postnatal somatic origin, satellite 2 DNA is highly methylated. We examined satellite 2 DNA methylation in ovarian tumors of different malignant potential, namely, ovarian cystadenomas, low malignant potential (LMP) tumors, and epithelial carcinomas. Most of the carcinomas and LMP tumors exhibited hypomethylation in satellite 2 DNA of both chromosomes 1 and 16. A comparison of methylation of these sequences in the three types of ovarian neoplasms demonstrated that there was a statistically significant correlation between the extent of this satellite DNA hypomethylation and the degree of malignancy (P<0.01). Also, there was a statistically significant association (P<0.005) between genome-wide hypomethylation and undermethylation of satellite 2 DNA among these 17 tumors. In addition, we found abnormal hypomethylation of satellite α DNA in the centromere of chromosome 1 in many of these tumors. Our findings are consistent with the hypothesis that one of the ways that genome-wide hypomethylation facilitates tumor development is that it often includes satellite hypomethylation which might predispose cells to structural and numerical chromosomal aberrations. Several of the proteins that bind to pericentromeric heterochromatin are known to be sensitive to the methylation status of their target sequences and so could be among the sensors for detecting abnormal demethylation and mediating effects on chromosome structure and stability.

p53 mutation spectrum and load: the generation of hypotheses linking the exposure of endogenous or exogenous carcinogens to human cancer.

Abstract: The activation of protooncogenes and inactivation of tumor suppressor genes in affected cells are considered as the core events that provide a selective growth advantage and clonal expansion during the multistep process of carcinogenesis. Somatic mutations, induced by exogenous or endogenous mechanisms, were found to alter the normal functions of the p53 tumor suppressor gene. p53 is the most prominent example of tumor suppressor genes because it is mutated in about half of all human cancer. In contrast to other tumor suppressor genes (like APC and RB ), about 80% of p53 mutations are missense mutations that lead to amino acid substitutions in proteins and can alter the protein conformation and increase the stability of p53 . These changes can also alter the sequence-specific DNA binding and transcription factor activity of p53 . These abnormalities can abrogate p53 dependent pathways involved in important cellular functions like cell-cycle control, DNA repair, differentiation, genomic plasticity and programmed cell death. A number of different carcinogens have been found to cause different characteristic mutations in the p53 gene. For example, exposure to ultraviolet light is correlated with transition mutations at dipyrimidine sites; aflatoxin B 1 exposure is correlated with a G:C to T:A transversion that leads to a serine substitution at residue 249 of p53 in hepatocellular carcinoma; and exposure to cigarette smoke is correlated with G:C to T:A transversions in lung carcinoma. Therefore, measuring the characteristic p53 mutation load or frequency of mutated alleles in nontumorous tissue (before the clonal expansion of mutated cells), can generate hypotheses, e.g., providing a molecular linkage between exposure to a particular carcinogen and cancer, and identifying individuals at increased cancer risk.

Mechanism of oxidative DNA damage induced by quercetin in the presence of Cu(II).

Abstract: Quercetin, one of flavonoids, has been reported to be carcinogenic. There have been no report concerning carcinogenicity of kaempferol and luteolin which have structure similar to quercetin. DNA damage was examined by using DNA fragments obtained from the human p53 tumor suppressor gene. Quercetin induced extensive DNA damage via reacting with Cu(II), but kaempferol and luteolin induced little DNA damage even in the presence of Cu(II). Excessive quercetin inhibited copper-dependent DNA damage induced by quercetin. Bathocuproine, a Cu(I)-specific chelator, catalase and methional inhibited the DNA damage by quercetin, whereas free hydroxyl radical scavengers did not. Site specificity of the DNA damage was thymine and cytosine residues. The site specificity and the inhibitory effects suggested that DNA-copper-oxygen complex rather than free hydroxyl radical induced the DNA damage. Formation of 8-oxodG by quercetin increased extensively in the presence of Cu(II), whereas 8-oxodG formation by kaempferol or luteolin increased only slightly. This study suggests a good relationship between carcinogenicity and oxidative DNA damage of three flavonoids. The mechanism of DNA damage by quercetin was discussed in relation to the safety in cancer chemoprevention by flavonoids.

Assessment of the genotoxicity of contaminated soil with the Allium/Vicia-micronucleus and the Tradescantia-micronucleus assays.

Abstract: The present study concerns the genotoxicity of contaminated soil near Metz, France. Three plant bioassays, the Vicia faba (broad bean), the Allium cepa (white onion) and the Tradescantia (spiderwort) micronuclei tests were used to evaluate for genotoxicity. Two soil samples were tested: soil sample A (from an industrial waste site) and soil sample B (from a cokeworks waste site). Maleic hydrazide was used as the positive control. Aqueous extracts of the soil samples were used to treat the roots of Vicia and Allium, and plant cuttings of Tradescantia according to the standard protocol for these plant assays established by the International Program on Chemical Safety and the World Health Organization. The results of these tests showed differential sensitivity in the three different bioassays. Soil sample A was more toxic than soil sample B.

Dietary fat and carcinogenesis.

Abstract: Epidemiologic investigations have suggested a relationship between dietary fat intake and various types of cancer incidences. Furthermore, epidemiologic studies as well as studies with animal models have demonstrated that not only the amount but also the type of fat consumed is important. At present, the mechanism by which dietary fat modulates carcinogenesis has not been elucidated. The effects of dietary fat on the development of tumours have been summarized in the present review with emphasis on colorectal, pancreas, breast and prostate cancer. It is concluded that influence on synthesis of prostaglandins and leukotrienes may be the universal mechanism by which dietary fats modulate carcinogenesis.

Correlation of DNA adduct levels with tumor incidence: carcinogenic potency of DNA adducts.

Abstract: The quantitative relationship between DNA adducts and tumor incidence is evaluated in this review. All available data on DNA adduct levels determined after repeated administration of a carcinogen to rats or mice have been compiled. The list comprised 27 chemicals, of all major structural classes of carcinogens. For the correlation with tumor incidence, the DNA adduct levels measured at the given dose were normalized to the dose which resulted in a 50% tumor incidence under the conditions of a 2-year bioassay (TD 50 dose). In rat liver, the calculated adduct concentration `responsible' for a 50% hepatocellular tumor incidence spanned from 53 to 2083 adducts per 10 8 nucleotides, for aflatoxin B1, tamoxifen, IQ, MeIQx, 2,4-diaminotoluene, and dimethylnitrosamine (in this order). In mouse liver, the respective figures were 812 to 5543 adducts per 10 8 nucleotides, for ethylene oxide, dimethylnitrosamine, 4-aminobiphenyl, and 2-acetylaminofluorene. The observed span (40-fold in rats, 7-fold in mice) reflects differences between the various DNA adducts to lead to critical mutations. If additional carcinogens fit in with this astonishingly narrow range, the measurement of DNA adduct levels in target tissue has the potential to be not only an exposure marker but an individual cancer risk marker. For toremifen and styrene, low levels of DNA adducts were detected in rat liver at the end of a negative long-term bioassay. This shows that the limit of detection of DNA adducts can be well below the limit of detection of an increased tumor incidence. For a cancer risk assessment at low levels of DNA damage, treatment-related adducts must be discussed in relation to the background DNA damage and its inter- and intraindividual variability.

Endogenous formation and significance of 1,N2-propanodeoxyguanosine adducts.

Abstract: The detection of 1,N2-propanodeoxyguanosine adducts in the DNA of rodent and human tissues as endogenous lesions has raised important questions regarding the source of their formation and their roles in carcinogenesis. Both in vitro and in vivo studies have generated substantial evidence which supports the involvement of short- and long-chain enals derived from oxidized polyunsaturated fatty acids (PUFAs) in their formation. These studies show that: (1) the cyclic propano adducts are common products from reactions of enals with DNA bases; (2) they are formed specifically from linoleic acid (LA; ω-6) and docosahexaenoic acid (ω-3) under in vitro stimulated lipid peroxidation conditions; (3) the levels of propano adducts are dramatically increased in rat liver DNA upon depletion of glutathione; (4) the adduct levels are increased in the liver DNA of the CCl4-treated rats and the mutant strain of Long Evans rats which are genetically predisposed to increased lipid peroxidation; and (5) adduct levels are significantly higher in older rats than in newborn rats. These studies collectively demonstrate that tissue lipid peroxidation is a main endogenous pathway leading to propano adduction in DNA. The possible contribution from environmental sources, however, cannot be completely excluded. The mutagenicity of enals and the mutations observed in site-specific mutagenesis studies using a model 1,N2-propanodeoxyguanosine adduct suggest that these adducts are potential promutagenic lesions. The increased levels of the propano adducts in the tissue of carcinogen-treated animals also provide suggestive evidence for their roles in carcinogenesis. The involvement of these adducts in tumor promotion is speculated on the basis that oxidative condition in tissues is believed to be associated with this process.

Antioxidant status in humans after exposure to hyperbaric oxygen.

Abstract: Hyperbaric oxygen (HBO) treatment (i.e., exposure to 100% oxygen at a pressure of 2.5 atmosphere absolute (ATA) for a total of 3×20 min periods) of human subjects induced DNA damage in the alkaline comet assay with leukocytes and protected against the DNA damaging effects of subsequent in vivo HBO exposures. Furthermore, blood taken 24 h after the first HBO was well protected against the in vitro induction of genotoxic effects by hydrogen peroxide. To investigate the mechanisms which led to this apparent adaptive response, we determined the antioxidant status of blood from subjects before and after HBO. We did not find differences in the plasma concentrations of the antioxidant vitamins A, C and E after HBO treatment. HBO had also no effect on the `antioxidant power' of the plasma as measured with the FRAP-assay or on the concentration of reduced glutathione determined in the plasma or in lymphocytes. Red cell concentrate activities of superoxide dismutase, catalase, glutathione peroxidase were not influenced by HBO. In contrast, synthesis of the heat shock protein HSP70 which has been implicated to play an important role in cellular protection against oxidative stress, was significantly induced in lymphocytes after a single HBO treatment. To investigate whether intake of antioxidants may protect against HBO-induced DNA damage, we supplemented subjects with vitamin E (800 mg for 7 days) or with N -acetylcysteine (400 mg, 1 h before the HBO treatment). However, these supplementations did not influence the induction of DNA damage by HBO.

Melatonin and protection from whole-body irradiation: survival studies in mice

Abstract: The radioprotective ability of melatonin was investigated in mice exposed to an acute whole-body gamma radiation dose of 815 cGy (estimated LD50/30 dose). The animals were observed for mortality over a period of 30 days following irradiation. The results indicated 100% survival for unirradiated and untreated control mice, and for mice treated with melatonin or solvent alone. Forty-five percent of mice exposed to 815 cGy radiation alone, and 50% of mice pretreated with solvent and irradiated with 815 cGy were alive at the end of 30 days. Irradiated mice which were pretreated with 125 mg/kg melatonin exhibited a slight increase in their survival (60%) (p=0.3421). In contrast, 85% of irradiated mice which were pretreated with 250 mg/kg melatonin were alive at the end of 30 days (p=0.0080). These results indicate that melatonin (at a dose as high as 250 mg/kg) is non-toxic, and that high doses of melatonin are effective in protecting mice from lethal effects of acute whole-body irradiation.