Oltipraz-induced phase 2 enzyme response conserved in cells lacking mitochondrial DNA

Oltipraz, a member of a class of 1,2-dithiolethiones, is a potent phase 2 enzyme inducing agent used as a cancer chemopreventive. In this study, we investigated regulation of the phase 2 enzyme response and protection against endogenous oxidative stress in lymphoblastic leukemic parental CEM cells and cells lacking mitochondrial DNA (mtDNA) (rho0) by oltipraz. Glutathione (GSH) levels (total and mitochondrial) and glutathione S-transferase (GST) activity were significantly increased after pretreatment with oltipraz in both parental (rho+) and rho0 cells, and both cell lines were resistant to mitochondrial oxidation, loss of mitochondrial membrane potential, and cell death in response to the GSH depleting agent diethylmaleate. These results show that the phase 2 enzyme response, by enhancing GSH-dependent systems involved in xenobiotic metabolism, blocks endogenous oxidative stress and cell death, and that this response is intact in cells lacking mtDNA.     

Effect of oltipraz [5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3-thione] on azoxymethane-induced biochemical changes related to early colon carcinogenesis in male F344 rats

Epidemiologic studies suggest that the consumption of cruciferous vegetables is associated with a reduced risk for several types of cancer including cancer of colon. Experimental studies indicate that dithiolthiones, naturally occurring substances in cruciferous vegetables, possess anticarcinogenic properties. 5-(2-Pyrazinyl)-4-methyl-1,2-dithiol-3-thione (oltipraz), a substituted dithiolthione, has been tested for its chemopreventive activity. We studied the effect of dietary oltipraz on liver and colonic mucosal enzymes and DNA adducts to evaluate the modulating role of this agent during the early period of azoxymethane (AOM)-induced carcinogenesis. At 6 weeks of age, groups of animals were fed the AIN-76A diet containing 0 and 300 ppm oltipraz. At 8 weeks of age, all of the animals except vehicle-treated animals were administered a subcutaneous injection of AOM (15 mg/kg body wt/week for 2 weeks). Animals intended for vehicle treatment were administered normal saline subcutaneously. Fifteen hours after the second AOM injection, six animals each from control oltipraz diet groups were sacrificed and liver and colonic mucosa from each animal were used for DNA adduct analysis. Animals intended for liver and colonic mucosal glutathione S-transferase, tyrosine specific protein kinase (TPK), and ornithine decarboxylase (ODC) enzyme assays were killed 5 days after the second AOM or saline injection. The results of this study indicated that dietary oltipraz significantly increased liver (P less than 0.001) and colonic mucosal (P greater than 0.05) weights, but had no effect on body weights (P greater than 0.05). In saline-treated animals, feeding of oltipraz significantly increased the cytosolic glutathione S-transferase (P less than 0.001) and ODC (P less than 0.05) activities in the liver and colon when compared with those fed the control diet. Although our unpublished results indicate an inhibitory role of oltipraz when fed during the initiation and postinitiation phases of intestinal carcinogenesis, the increased ODC activity may indicate a possible role of oltipraz in colon tumor promotion. Additional studies are indicated to test the antitumor properties of oltipraz administered during the postinitiation phases. AOM treatment significantly increased the TPK (P less than 0.0001) and ODC (P less than 0.01) activities in the liver and colon of animals fed the control diet. Dietary oltipraz significantly suppressed the AOM-induced TPK (P less than 0.001) activities in liver and colon and ODC (P less than 0.01) activity of colon. Analysis of nucleic acid bases, O6-methylguanine, and 7-methylguanine revealed that dietary oltipraz significantly (P less than 0.05) inhibited the AOM-induced adduct species. These results suggest that dietary oltipraz enhances the colonic and liver glutathione S-transferase activity and reduced the formation of DNA adducts. In addition, dietary oltipraz modulates liver and colonic ODC and TPK activities that have been shown to play a role in tumor promotion.     

Redox regulation of apurinic/apyrimidinic endonuclease 1 activity in Long-Evans Cinnamon rats during spontaneous hepatitis

The Long-Evans Cinnamon (LEC) rat is an animal model for Wilson’s disease. This animal is genetically predisposed to copper accumulation in the liver, increased oxidative stress, accumulation of DNA damage, and the spontaneous development of hepatocellular carcinoma. Thus, this animal model is useful for studying the relationship of endogenous DNA damage to spontaneous carcinogenesis. In this study, we have investigated the apurinic/apyrimidinic endonuclease 1 (APE1)-mediated excision repair of endogenous DNA damage, apurinic/apyrimidinic (AP)-sites, which is highly mutagenic and implicated in human cancer. We found that the activity was reduced in the liver extracts from the acute hepatitis period of LEC rats as compared with extracts from the age-matched Long-Evans Agouti rats. The acute hepatitis period had also a heightened oxidative stress condition as assessed by an increase in oxidized glutathione level and loss of enzyme activity of glyceraldehyde 3-phosphate dehydrogenase, a key redox-sensitive protein in cells. Interestingly, the activity reduction was not due to changes in protein expression but apparently by reversible protein oxidation as the addition of reducing agents to extracts of the liver from acute hepatitis period reactivated APE1 activity and thus, confirmed the oxidation-mediated loss of APE1 activity under increased oxidative stress. These findings show for the first time in an animal model that the repair mechanism of AP-sites is impaired by increased oxidative stress in acute hepatitis via redox regulation which contributed to the increased accumulation of mutagenic AP-sites in liver DNA.     

Polymorphisms of the CHRNA4 gene encoding the alpha4 subunit of nicotinic acetylcholine receptor as related to the oxidative DNA damage and the level of apoptotic proteins in lymphocytes of the patients with Alzheimer's disease

The study aimed at the analysis of polymorphisms in the gene coding for the nicotinic acetylcholine receptor alpha4 subunit (CHRNA4) and the evaluation of the extent of the oxidative damage to DNA (8-oxo2dG), as well as the level of proteins participating in DNA repair (p53, PARP) and DNA degradation (Bax:Bcl-2, 85-kDa fragment) in the peripheral blood lymphocytes of the patients suffering from Alzheimer's disease (AD) and in the healthy individuals of the control group. In the AD patients the increased levels of oxidized guanine were demonstrated in DNA, accompanied by the elevated expression of p53, Bax, PARP, and of a 85-kDa protein subunit as well as an augmented ratio of Bax:Bcl-2. Also, the level of Bcl-2 protein was decreased. In the AD patients with the CHRNA4 polymorphisms the highest level of 8-oxo2dG and of proteins involved in DNA repair were documented in patients with polymorphisms in exon 5, in contrast to the patients with polymorphisms in intron 5. In the former patients, levels of pro- and antiapoptotic proteins remained at the same level. Both CHRNA4 polymorphisms and the extent of dementia seem to affect the levels of DNA oxidative damage as well as to activate factors that participate in the DNA degradation and its repair.     

Exogenous 8-oxo-7,8-dihydro-2′-deoxyguanosine: Biomedical properties,mechanisms of action,and therapeutic potential

Abstract—8-Oxo-7,8-dihydroguanine (8-oxo-G) is a key biomarker of oxidative damage to DNA in cells, and its genotox- icity is well-studied. In recent years, it has been confirmed experimentally that free 8-oxo-G and molecules containing it are not merely inert products of DNA repair or degradation, but they are actively involved in intracellular signaling. In this review, data are systematized indicating that free 8-oxo-G and oxidized (containing 8-oxo-G) extracellular DNA function in the body as mediators of stress signaling and initiate inflammatory and immune responses to maintain homeostasis under the action of external pathogens, whereas exogenous 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGuo) exhibits pro- nounced antiinflammatory and antioxidant properties. This review describes known action mechanisms of oxidized guanine and 8-oxo-G-containing molecules. Prospects for their use as a therapeutic target are considered, as well as a pharmaceu- tical agent for treatment of a wide range of diseases whose pathogenesis is significantly contributed to by inflammation and oxidative stress.     

Is 5-aminolevulinic acid involved in the hepatocellular carcinogenesis of acute intermittent porphyria?

5-Aminolevulinic acid (ALA) is a heme precursor that accumulates in acute intermittent porphyria (AIP) due to enzymatic deficiencies in the heme biosynthetic pathway Its accumulation has been associated with several symptoms, such as abdominal pain attacks, neuromuscular weaknesses, neuropsychiatric alterations and increased hepatocellular carcinoma (HCC) incidence. The use of exogenous ALA to elevate porphyrin levels in tumor photodynamic therapy, adds further significance to ALA toxicology. Under ferritin mediated and metal catalyzed oxidation, ALA produces reactive oxygen species that can damage plasmid and isolated DNA in vitro, and increases the steady-state level of 8-oxo-7,8-dihydro-2'-deoxyguanosine in liver, spleen and kidney DNA and 5-hydroxy-2'-deoxycytidine in liver DNA of ALA-treated rats. The in vitro DNA damage could be partially inhibited by SOD, catalase, DTPA, mannitol and melatonin. ALA also promotes the formation of radical-induced base degradation products in isolated DNA. 4,5-Dioxovaleric acid, the final oxidation product of ALA, alkylates guanine moieties within both nucleoside and isolated DNA, producing two diastereoisomeric adducts. Dihydropyrazine derivatives of ALA generated by its dimerization, promote DNA strand-breaks and 8-oxodGuo formation in the presence of Cu2+. Together these results reinforce the hypothesis that the DNA damage induced by ALA may be associated with the development of HCC in individuals suffering from AIP.     

Use of intermediate endpoints in quantitating the response of precancerous lesions to chemopreventive agents

A current area of emphasis in cancer research is the determination of whether cancer can be prevented through the use of naturally occurring chemopreventive agents such as beta-carotene. A major area of concern in the design of long-term, large-scale population studies to ascertain the efficacy of such chemopreventive agents lies in the paucity of biological data on the activity of these agents in man. The studies described in this paper were performed to determine whether a series of short-term markers could be used in chemopreventive trials as indicators of the possible success of a chemopreventive regime. Three such markers are described. The first involves the measurement of genotoxic damage in the target tissues of carcinogen-exposed individuals by using the micronucleus test on exfoliated cells. This end point has been successfully used to demonstrate a reduction in carcinogen damage (micronuclei production) in the oral cavity of individuals in population groups at elevated risk for oral cancer (tobacco and betel quid users in the Philippines, snuff users in the Northwest Territories). The second marker involves the determination of DNA adducts in exfoliated cells of carcinogen-exposed individuals by the use of DNA postlabelling procedure. The final marker discussed involves a chemical determination of the levels of a chemopreventive agent in target tissues of individuals receiving a supplement in the diet. In this case, the example described is beta-carotene in exfoliated cells of carcinogen-exposed individuals. These three markers may be combined to determine whether a chemopreventive agent reaches a target tissue, affects DNA adduct formation, and prevents genotoxic damage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fruit juice consumption modulates antioxidative status,immune status and DNA damage

Polyphenolic compounds exert a variety of physiological effects in vitro including antioxidative, immunomodulatory and antigenotoxic effects. In a randomized crossover study in healthy men on a low-polyphenol diet, we determined the effects of 2 polyphenol-rich juices (330 ml/d) supplemented for 2 weeks on bioavailability of polyphenols, markers of antioxidative and immune status, and reduction of DNA damage. Juices provided 236 mg (A) and 226 mg (B) polyphenols with cyanidin glycosides (A) and epigallocatechin gallate (B) as major polyphenolic ingredients. There was no accumulation of plasma polyphenols after two weeks of juice supplementation. In contrast, plasma malondialdehyde decreased with time during juice interventions. Moreover, juice consumption also increased lymphocyte proliferative responsiveness, with no difference between the two juices. Interleukin-2 secretion by activated lymphocytes and the lytic activity of natural killer cells were significantly increased by both juices. Juice intervention had no effect on single DNA strand breaks, but significantly reduced oxidative DNA damage in lymphocytes. A time-delay was observed between the intake of fruit juice and the reduction of oxidative DNA damage and the increase in interleukin-2 secretion. We conclude that consumption of either juice enhanced antioxidant status, reduced oxidative DNA damage and stimulated immune cell functions. However, fruit juice consumption for 2 weeks did not result in elevated plasma polyphenols in subjects after overnight fasting. Further studies should focus on the time-delay between juice intake and changes in measured physiological functions, as well as on active polyphenolic metabolites mediating the observed effects.     

Repair of oxidative DNA damage by amino acids

Guanyl radicals, the product of the removal of a single electron from guanine, are produced in DNA by the direct effect of ionizing radiation. We have produced guanyl radicals in DNA by using the single electron oxidizing agent (SCN)₂^–, itself derived from the indirect effect of ionizing radiation via thiocyanate scavenging of OH. We have examined the reactivity of guanyl radicals in plasmid DNA with the six most easily oxidized amino acids cysteine, cystine, histidine, methionine, tryptophan and tyrosine and also simple ester and amide derivatives of them. Cystine and histidine derivatives are unreactive. Cysteine, methionine, tyrosine and particularly tryptophan derivatives react to repair guanyl radicals in plasmid DNA with rate constants in the region of ~10⁵, 10⁵, 10⁶ and 10⁷ dm³ mol^–1 s^–1, respectively. The implication is that amino acid residues in DNA binding proteins such as histones might be able to repair by an electron transfer reaction the DNA damage produced by the direct effect of ionizing radiation or by other oxidative insults.     

Exopolysaccharide-peptide complex from oyster mushroom (Pleurotus ostreatus) protects against hepatotoxicity in rats

Liver damage involves oxidative stress and a progression from chronic hepatitis to hepatocellular carcinoma (HCC). The increased incidence of liver disease in Egypt and other countries in the last decade, coupled with poor prognosis, justify the critical need to introduce alternative chemopreventive agents that may protect against liver damage. The aim of this study was to evaluate the efficacy of exopolysaccharide-peptide (PSP) complex extracted from Pleurotus ostreatus as a hepatoprotective agent against diethylnitrosamine (DEN)/carbon tetrachloride (CCL₄)-induced hepatocellular damage in rats. The levels of liver injury markers (ALT, AST and ALP) were substantially increased following DEN/CCl₄ treatment. DEN/CCl₄ - induced oxidative stress was confirmed by elevated levels of lipid peroxidation and decreased levels of superoxide dismutase, glutathione-S-transferase, and reduced glutathione. PSP reversed these alterations in the liver and serum, and provided protection evidenced by reversal of histopathological changes in the liver. The present study demonstrated that PSP extract from P. ostreatus exhibited hepatoprotective and antioxidant effects against DEN/CCl₄-induced hepatocellular damage in rats. Given the high prevalence of HCV-related liver damage in Egypt, our results suggest further clinical evaluation of P. ostreatus extracts and their potential hepatoprotective effects in patients with liver disease.     

Effect ofmutY andmutM/fpg-1 mutations on starvation-associated mutation inEscherichia coli: implications for the role of 7,8-dihydro-8-oxoguanine

MutY specifies a DNA glycosylase that removes adenines unnaturally paired with various bases including oxidized derivatives of guanine, such as 7,8-dihydro-8-oxoguanine (8-oxoG). The rate of mutation in starvedEscherichia coli cells is markedly raised inmutY mutants defective in this glycosylase. As predicted, the mutations produced include G to T transversions. Bacteria carryingmutM orfpg-1 mutations (defective in Fapy glycosylase, which removes oxidized guanine residues such as 8-oxoG) show little or no enhancement of mutation under starvation conditions. When present together withmutY, however,mutM clearly further enhances the rate of mutation in starved cells. Plasmids resulting in overproduction of MutY or Fapy glycosylases reduce the rate of mutation in starved cells. We conclude that, in non-growing bacteria, oxidized guanine residues, including 8-oxoG, constitute an important component of spontaneous mutation. Addition of catalase to the plates did not reduce the mutant yield, indicating that extracellular hydrogen peroxide is not involved in the production of the premutational damage. Singlet oxygen, known to give rise to 8-oxoG, may be the ultimate oxidative species.     

Effect ofmutY andmutM/fpg-1 mutations on starvation-associated mutation inEscherichia coli: implications for the role of 7,8-dihydro-8-oxoguanine

MutY specifies a DNA glycosylase that removes adenines unnaturally paired with various bases including oxidized derivatives of guanine, such as 7,8-dihydro-8-oxoguanine (8-oxoG). The rate of mutation in starvedEscherichia coli cells is markedly raised inmutY mutants defective in this glycosylase. As predicted, the mutations produced include G to T transversions. Bacteria carryingmutM orfpg-1 mutations (defective in Fapy glycosylase, which removes oxidized guanine residues such as 8-oxoG) show little or no enhancement of mutation under starvation conditions. When present together withmutY, however,mutM clearly further enhances the rate of mutation in starved cells. Plasmids resulting in overproduction of MutY or Fapy glycosylases reduce the rate of mutation in starved cells. We conclude that, in non-growing bacteria, oxidized guanine residues, including 8-oxoG, constitute an important component of spontaneous mutation. Addition of catalase to the plates did not reduce the mutant yield, indicating that extracellular hydrogen peroxide is not involved in the production of the premutational damage. Singlet oxygen, known to give rise to 8-oxoG, may be the ultimate oxidative species.     

Iron supplementation and oxidative damage to DNA in healthy individuals with high plasma ascorbate

Previously, we have investigated the potential for a pro-oxidant interaction of iron and ascorbate in vivo in iron and ascorbate cosupplementation or ascorbate supplementation studies. In this study, for the first time, the effects of iron supplementation on oxidative damage to DNA in healthy individuals with plasma ascorbate levels at the upper end of the normal range were examined. Forty female and male volunteers (mean plasma ascorbate approximately equal to 70 micromol/L) were supplemented with a daily dose of syrup (ferrous glycine sulphate equivalent to 12.5 mg iron) for 6 weeks. Serum ferritin, transferrin bound iron, % saturation of transferrin and plasma ascorbate were assessed and the mean dietary intakes of all subjects were estimated through food frequency questionnaires. Oxidative damage to DNA bases from white blood cells was measured by gas chromatography/mass spectrometry with selected-ion monitoring (GC/MS-SIM), using isotope-labelled standards for quantification. Iron supplementation did not affect any of the iron status parameters. There were also no detrimental effects, over the period under investigation, in terms of oxidative damage to DNA. However, the effects of larger doses or of longer supplementation periods should also be investigated.     

Formation of the carboxamidine precursor of cyanuric acid from guanine oxidative lesion dehydro-guanidinohydantoin

DNA damage under oxidative stress leads to oxidation of guanine base. The identification of the resulting guanine lesions in cellular DNA is difficult due to the sensitivity of the primary oxidation products to hydrolysis and/or further oxidation. We isolated dehydroguanidino-hydantoin (DGh) (or oxidized guanidinohydantoin), a secondary oxidation product of guanine, and showed that this lesion reacts readily with nucleophiles such as asymmetric peroxides and transforms to 2,4,6-trioxo-1,3,5-triazinane-1-carboxamidine residue. Further hydrolysis of this intermediate leads to cyanuric acid and finally to urea residue. This work demonstrates a new possible pathway for the formation of the well-known carboxamidine precursor of cyanuric acid lesion.     

Gender differences in steady-state levels of oxidative damage to DNA in healthy individuals

Oxidative damage to DNA has often been used as a biomarker for oxidative stress and more specifically for cancer risk. Indeed, the measurement of oxidative damage to DNA, particularly of 8-hydroxyguanine (8OHG) and 8-hydroxy-2'-deoxyguanosine (8OHdG), has been adopted as a method for establishing the effects of antioxidant supplementation towards protection from certain cancers, cardiovascular and neuro-degenerative diseases, both in patients and healthy individuals. However, reported levels of 8OHdG or 8OHG vary considerably, possibly due to the different methodologies used, and only few data are available for the non-smoking and the female population. In this paper, steady-state levels of oxidative damage to DNA measured in a group of 20 males and 19 females are reported. Significant gender differences in levels of modified DNA bases such as 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FAPy guanine), 8-hydroxyadenine (8OHA) and 5-hydroxycytosine (5OHC), measured by gas chromatography-mass spectrometry (GC/MS), were observed. The results are discussed in relation to the Vitamin C and iron status of the subjects and to the existing, yet limited, literature data. The role of gender in predisposition to oxidative damage to DNA needs to be addressed in future studies.     

Deletion of Ogg1 DNA glycosylase results in telomere base damage and length alteration in yeast

Telomeres consist of short guanine‐rich repeats. Guanine can be oxidized to 8‐oxo‐7,8‐dihydroguanine (8‐oxoG) and 2,6‐diamino‐4‐hydroxy‐5‐formamidopyrimidine (FapyG). 8‐oxoguanine DNA glycosylase (Ogg1) repairs these oxidative guanine lesions through the base excision repair (BER) pathway. Here we show that in Saccharomyces cerevisiae ablation of Ogg1p leads to an increase in oxidized guanine level in telomeric DNA. The ogg1 deletion (ogg1Δ) strain shows telomere lengthening that is dependent on telomerase and/or Rad52p‐mediated homologous recombination. 8‐oxoG in telomeric repeats attenuates the binding of the telomere binding protein, Rap1p, to telomeric DNA in vitro. Moreover, the amount of telomere‐bound Rap1p and Rif2p is reduced in ogg1Δ strain. These results suggest that oxidized guanines may perturb telomere length equilibrium by attenuating telomere protein complex to function in telomeres, which in turn impedes their regulation of pathways engaged in telomere length maintenance. We propose that Ogg1p is critical in maintaining telomere length homoeostasis through telomere guanine damage repair, and that interfering with telomere length homoeostasis may be one of the mechanism(s) by which oxidative DNA damage inflicts the genome.     

Mitochondrial DNA repair of oxidative damage in mammalian cells

Nuclear and mitochondrial DNA are constantly being exposed to damaging agents, from endogenous and exogenous sources. In particular, reactive oxygen species (ROS) are formed at high levels as by-products of the normal metabolism. Upon oxidative attack of DNA many DNA lesions are formed and oxidized bases are generated with high frequency. Mitochondrial DNA has been shown to accumulate high levels of 8-hydroxy-2'-deoxyguanosine, the product of hydroxylation of guanine at carbon 8, which is a mutagenic lesion. Most of these small base modifications are repaired by the base excision repair (BER) pathway. Despite the initial concept that mitochondria lack DNA repair, experimental evidences now show that mitochondria are very proficient in BER of oxidative DNA damage, and proteins necessary for this pathway have been isolated from mammalian mitochondria. Here, we examine the BER pathway with an emphasis on mtDNA repair. The molecular mechanisms involved in the formation and removal of oxidative damage from mitochondria are discussed. The pivotal role of the OGG1 glycosylase in removal of oxidized guanines from mtDNA will also be examined. Lastly, changes in mtDNA repair during the aging process and possible biological implications are discussed.     

Oxidative DNA damage in circulating leukocytes occurs as an early event in chronic HCV infection

Chronic hepatitis C virus (HCV) infection is associated with an increased production of reactive oxygen species within the liver that are responsible for the oxidation of intracellular macromolecules. To ascertain whether the increased risk of hepatocellular carcinoma in individuals with chronic HCV infection is related to an accumulation of oxidative DNA damage, the 8-hydroxydeoxyguanosine (8-OHdG) content in the DNA of liver tissue and leukocytes of 87 individuals with HCV- or HBV-related liver disease and of 10 healthy controls was measured. Serum levels of thiobarbituric acid reactive substances (TBARS) were also assessed as an index of lipid peroxidation. RESULTS: The 8-OHdG content in the circulating leukocytes correlated with that of liver tissue (r = 0.618, p < .0004). HCV patients had the highest median 8-OHdG levels (p < .0004). 8-OHdG leukocyte levels in HCV patients were higher than in HBV patients (p < .04) and they significantly correlated with the clinical diagnosis (p < .025), the serum ferritin levels (p < .05), and the amount of liver steatosis (p < .001). No correlation was found with age, gender, history of drinking or smoking, ALT or GGT levels, ESR, alpha-1, or gamma-globulin level and Ishak score. TBARS levels were significantly higher in cirrhotics than in noncirrhotics (p < .01). CONCLUSIONS: The 8-OHdG level in circulating leukocytes is a reliable marker of oxidative stress occurring in the liver of individuals with chronic HCV infection. DNA oxidative damage appears to be an early and unique event in the natural history of HCV-related hepatitis. This injury increases the risk of genomic damage and may be one of the important factors involved in the carcinogenic process in cases of HCV-related chronic liver disease.