Dietary exposure to diesel exhaust particles and oxidatively damaged DNA in young oxoguanine DNA glycosylase 1 deficient mice

Pulmonary exposure to diesel exhaust particles (DEP) has been associated with high levels of oxidized DNA in lung cells, whereas long-term oral DEP exposure appears to induce the DNA repair system with concomitant unaltered levels of oxidized DNA in the colon and liver of rats. Here we studied the generation of oxidatively damaged DNA in young wild type (WT) and oxoguanine DNA glycosylase 1 (OGG1) deficient mice after dietary exposure to 0mg/kg, 0.8 mg/kg, or 8 mg/kg Standard Reference Material 1650 in the feed for 21 days. The ingestion of DEP did not increase the levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine and comet assay endpoints in terms of strand break, endonuclease III, and formamidopyrimidine glycosylase (FPG) in the colon, liver, and lung tissue of WT or Ogg1(-/-) mice. The level of OGG1 mRNA could only be measured in WT mice and it was not increased by DEP feeding. On the contrary, the level of FPG sites was twofold higher in the liver and lung of Ogg1(-/-) mice compared to the levels in the WT mice tissues. In conclusion, although Ogg1(-/-) mice have high levels of oxidized guanine lesions, they do not appear to be markedly vulnerable to the genotoxicity by oral administration of DEP.     

Oxidative DNA damage in vitamin C-supplemented guinea pigs after intratracheal instillation of diesel exhaust particles

The health effects of diesel exhaust particles (DEP) are thought to involve oxidative damage. We have investigated the effect of intratracheal DEP instillation to guinea pigs in three groups of 12 animals each given 0, 0.7, or 2.1 mg. Five days later guinea pigs exposed to DEP had increased levels of oxidized amino acids (gamma-glutamyl semialdehyde), DNA strand breaks, and 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG) in the lung. Bulky DNA ad- ducts were not significantly elevated in the lung. The antioxidant enzyme activity of glutathione reductase was increased in the lung of DEP-exposed guinea pigs, whereas glutathione peroxidase and superoxide dismutase enzyme activities were unaltered. There was no difference in DNA strand breaks in lymphocytes or urinary excretion of 8-oxodG at the two doses tested. Protein oxidations in plasma and in erythrocytes were not altered by DEP exposure. The concentrations of ascorbate in liver, lung, and plasma were unaltered by the DEP exposure. The results indicate that in guinea pigs DEP causes oxidative DNA damage rather than bulky DNA adducts in the lung. Guinea pigs, which are similar to humans with respect to vitamin C metabolism, may serve as a new model for the study of oxidative damage induced by particulate matter.     

Sucrose, glucose and fructose have similar genotoxicity in the rat colon and affect the metabolome.

We have shown previously that a high sucrose intake increases the background level of somatic mutations and the level of bulky DNA adducts in the colon epithelium of rats. The mechanism may involve either glucose or fructose formed by hydrolysis of sucrose. Male Big Blue rats were fed 30% sucrose, glucose, fructose or potato starch as part of the diet. Mutation rates and bulky DNA adduct levels were determined in colon and liver. The concentration of short-chain fatty acids and pH were determined in caecum, C-peptide was determined in plasma, biomarkers for oxidative damage and proliferation were determined in colon, and a metabonomic analysis was performed in plasma and urine. The sugars increased the mutation rates in colon and the bulky adduct levels in colon and liver to a similar extent. All sugars decrease the caecal concentration of acetic acid and propionic acid. The metabonomic studies indicated disturbed amino acid metabolism and decrease in plasma and urinary acetate as a common feature for all sugars and confirmed triglyceridemic effects of fructose. In conclusion, the genotoxicity may be related to the altered chemical environment in the caecum and thereby also in the colon but we found no related changes in insulin resistance or oxidative stress.     

Oxidatively damaged DNA and inflammation in the liver of dyslipidemic ApoE-/- mice exposed to diesel exhaust particles

Epidemiological studies have shown that exposure to air pollution particles is associated with cardiovascular diseases, whereas the role in the initiation of atherosclerosis is unresolved. Atherosclerosis is considered to be an inflammatory disease that also involves oxidative stress. Here we investigated effects of oxidative stress elicited by diesel exhaust particles (DEP) in the aorta, liver, and lung of dyslipidemic ApoE(-/-) mice at the age when visual plaques appear in the aorta (11-13 weeks). DEP was administrated by intraperitoneal injection (0, 50, 500 and 5,000 microg DEP/kg bodyweight) in order to omit vascular effects secondary to pulmonary inflammation. The mice were killed either 6 or 24h after the administration. Inflammation was measured as the expression of inducible nitric oxide synthase (iNOS) and serum nitric oxide and DNA damage was measured by the comet assay. The expression of iNOS mRNA was increased in the liver 6h after the administration. The level of oxidized purine bases, determined as formamidopyrimidine DNA glycosylase sites was increased by 67% (95% CI: 11-124%) in the liver after 24h in the mice administrated with only 50 microg/kg bodyweight. However, there was no indication of systemic inflammation determined as the serum concentration of nitric oxide and iNOS expression, and DNA damage was not increased in the aorta. These observations indicate that intraperitoneal DEP injection does not induce inflammation or oxidatively damaged DNA in the lung and aorta, whereas a direct effect in terms of inflammation and oxidized DNA was observed in the liver of dyslipidemic ApoE(-/-) mice.     

Microcystin-LR induces oxidative DNA damage in human hepatoma cell line HepG2.

Microcystins are naturally occurring hepatotoxins produced by strains of Microcystis aeruginosa. They are involved in promoting primary liver tumours and a previous study showed that they might also be tumour initiators. In this study we demonstrate that microcystin-LR (MCLR) at doses that were not cytotoxic (0.01-1 microg/ml), induced dose and time dependent DNA strand breaks in human hepatoma cell line HepG2. These DNA strand breaks were transient, reaching a maximum level after 4h of exposure and declining with further exposure. In the presence of the DNA repair inhibitors cytosine arabinoside (AraC) and hydroxyurea (HU), together with MCLR, DNA strand breaks accumulated after prolonged exposure. These results suggest that DNA strand breaks are intermediates, produced during the cellular repair of MCLR induced DNA damage. Digestion of DNA with purified, oxidative DNA damage specific enyzmes, endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg) markedly increased DNA strand breaks in MCLR treated cells, providing evidence that a substantial portion of the MCLR induced DNA strand breaks originate from excision of oxidative DNA adducts. A hydroxyl radical scavenger (DMSO) significantly reduced MCLR induced DNA damage. From these results we conclude that MCLR induces formation of reactive oxygen species that cause DNA damage, and that MCLR may act as an initiator of liver cancer.     

Inhibition of oxidative DNA repair in cadmium-adapted alveolar epithelial cells and the potential involvement of metallothionein

This study evaluated the effects of cadmium (Cd) adaptation in cultured alveolar epithelial cells on oxidant-induced DNA damage and its subsequent repair. Using the comet assay, we determined that lower levels of DNA damage occurred in Cd-adapted cells compared with non-adapted cells following treatment of cells with hydrogen peroxide (H(2)O(2)). This may be a consequence of increased thiol-containing antioxidants that were observed in adapted cells, including metallothionein and glutathione. Cd-adapted cells were, however, less efficient at repairing total oxidative DNA damage compared with non-adapted cells. Subsequently, we investigated the effect of Cd adaptation on the repair of particular oxidized DNA lesions by employing lesion-specific enzymes in the comet assay, namely formamidopyrimidine DNA glycosylase (Fpg), an enzyme that predominantly repairs 8-oxoguanine (8-oxoG), and endonuclease III, that is capable of repairing oxidized pyrimidines. The data demonstrated that adaptation to Cd results in significantly impaired repair of both Fpg- and endonuclease III-sensitive lesions. In addition, in situ detection of 8-oxoG using a recombinant monoclonal antibody showed that Cd-adaptation reduces the repair of this oxidative lesion after exposure of cells to H(2)O(2). Activities of 8-oxoG-DNA glycosylase and endonuclease III were determined in whole cell extracts using 32P-labeled synthetic oligonucleotides containing 8-oxoG and dihydrouracil sites, respectively. Cd adaptation was associated with an inhibition of 8-oxoG-DNA glycosylase and endonuclease III enzyme activity compared with non-adapted cells. In summary, this study has shown that Cd adaptation: (1) reduces oxidant-induced DNA damage; (2) increases the levels of key intracellular antioxidants; (3) inhibits the repair of oxidative DNA damage.     

Commonly consumed and naturally occurring dietary substances affect biomarkers of oxidative stress and DNA damage in healthy rats.

The influence of black currant juice, Bowman-Birk protease inhibitor (BBI), kolaviron (a biflavonoid fraction of Garcinia kola seed), sugars, vitamin C and tert-butyl hydroperoxide on a wide range of biomarkers for oxidative stress, DNA damage and sugar or lipid metabolism has been investigated in male F 344 rats. The selected pro-oxidant control, tert-butyl hydroperoxide, significantly increased plasma and liver 2-amino-adipic semialdehyde (AAS), a marker of protein oxidation (p <0.05) whereas lipid oxidation assessed as malon dialdehyde (MDA) and DNA oxidation were not significantly increased. Feeding BBI also increased the level of oxidized protein in plasma and liver at the higher dose level (0.5%). No effect was observed at the lower dose level (0.25%), which even decreased lipid oxidation in plasma. BBI did not affect background levels of DNA strand breaks or oxidation (comets). In rats exposed to black currant juice, a statistically significant decrease in liver AAS and MDA was observed. This effect could not be explained by its content of sugars or of the known redox active constituent, vitamin C. The lowering effect of black currant juice on protein and lipid oxidation was similar in magnitude to that of the known liver protectant, kolaviron. In rats treated with kolaviron (200 mg/kg body weight), background AAS levels were significantly reduced in both plasma and liver whereas the effect on MDA only reached statistical significance in plasma. Kolaviron was the only extract tested which decreased oxidative damage to DNA in the liver. The erythrocyte antioxidant enzyme activities, catalase and glutathione peroxidase were decreased in rats treated with tert-butyl hydroperoxide (p <0.05) but were not affected by the other treatments. Black currant juice and sugars increased plasma triglyceride levels and black currant juice increased plasma cholesterol but neither of them nor any other treatment affected blood glucose, erythrocyte HbA1c or fructosamine. We conclude that markers of oxidative stress may be modified by several mechanisms after feeding rats with complex dietary factors and that both pro- and antioxidant effects may consequently be observed simultaneously after short-term feeding of antioxidant-rich foods, herb medicines, or known pro- and antioxidants.     

Oxidative DNA damage and its repair in rat spleen following subchronic exposure to aniline

The mechanisms by which aniline exposure elicits splenotoxic response, especially the tumorigenic response, are not well-understood. Splenotoxicity of aniline is associated with iron overload and generation of reactive oxygen species (ROS) which can cause oxidative damage to DNA, proteins and lipids (oxidative stress). 8-Hydroxy-2′-deoxyguanosine (8-OHdG) is one of the most abundant oxidative DNA lesions resulting from ROS, and 8-oxoguanine glycosylase 1 (OGG1), a specific DNA glycosylase/lyase enzyme, plays a key role in the removal of 8-OHdG adducts. This study focused on examining DNA damage (8-OHdG) and repair (OGG1) in the spleen in an experimental condition preceding a tumorigenic response. To achieve that, male Sprague-Dawley rats were subchronically exposed to aniline (0.5 mmol/kg/day via drinking water for 30 days), while controls received drinking water only. Aniline treatment led to a significant increase in splenic oxidative DNA damage, manifested as a 2.8-fold increase in 8-OHdG levels. DNA repair activity, measured as OGG1 base excision repair (BER) activity, increased by ∼ 1.3 fold in the nuclear protein extracts (NE) and ∼ 1.2 fold in the mitochondrial protein extracts (ME) of spleens from aniline-treated rats as compared to the controls. Real-time PCR analysis for OGG1 mRNA expression in the spleen revealed a 2-fold increase in expression in aniline-treated rats than the controls. Likewise, OGG1 protein expression in the NEs of spleens from aniline-treated rats was ∼ 1.5 fold higher, whereas in the MEs it was ∼ 1.3 fold higher than the controls. Aniline treatment also led to stronger immunostaining for both 8-OHdG and OGG1 in the spleens, confined to the red pulp areas. It is thus evident from our studies that aniline-induced oxidative stress is associated with increased oxidative DNA damage. The BER pathway was also activated, but not enough to prevent the accumulation of oxidative DNA damage (8-OHdG). Accumulation of mutagenic oxidative DNA lesions in the spleen following exposure to aniline could play a critical role in the tumorigenic process.     

8-Oxoguanine DNA glycosylase and MutY homolog are involved in the incision of arsenite-induced DNA adducts.

Since arsenite is known to induce oxidative DNA damage in human cells, we asked if it induces other types of DNA damage and how the DNA damage is repaired. Treatment of human promyelocytic leukemia NB4 cells with 0.5muM As(2)O(3) for 30 min induced no DNA breaks, as analyzed by a standard comet assay. However, breaks were detected if these cells were then digested with endonuclease III (EnIII), formamidopyrimidine-DNA glycosylase (Fpg), or a nuclear extract (NE) of NB4 cells. Using either H(2)O(2)-Fe-treated nuclei or As(2)O(3)-treated cells, digestion with either NE or EnIII + Fpg generated the same amount of breaks, and subsequent treatment with EnIII + Fpg resulted in no increase in breaks in NE-digested cells and vice versa. The human cell lines, defective in nucleotide excision protein, such as xeroderma pigmentosum (XP) A, XPD, and XPG, excised Ultraviolet C-induced adducts less rapidly than normal fibroblasts, but excised As(2)O(3) adducts at the same rate as the normal cells. Immunodepletion of the NE with antibody against 8-oxoguanine DNA glycosylase (OGG1) or MutY homolog (MYH) decreased the incision of As(2)O(3)-induced adducts, while antibodies against XPA, XPB, XPD, XPF, or XPG, did not. These results suggest that As(2)O(3) induces the formation of only oxidative DNA adducts and that OGG1 and MYH are involved in this incision process.     

Ochratoxin a causes DNA damage and cytogenetic effects but no DNA adducts in rats

Ochratoxin A (OTA) is a potent nephrotoxin and renal carcinogen in rats, but the mechanism of OTA tumorigenicity is unknown. Ochratoxin A has been shown to be negative in many genetic toxicology test in vitro. However, the potential of OTA to induce genotoxic effects has not been investigated in male rats, the most sensitive species for OTA-induced tumor formation. In this study, male F344 rats were repeatedly administered OTA (0, 250, 500, 1000, and 2000 microg/kg of body wt) or the non-chlorinated analogue ochratoxin B (OTB; 2000 microg/kg of body wt) for 2 weeks (5 days/week), and DNA breakage was analyzed in target and nontarget tissues using the comet assay both in the absence and presence of formamidopyrimidine-DNA (Fpg) glycosylase. Potential DNA-adduct formation was also analyzed in the target organ kidney by 32P-postlabeling using two different solvent systems. DNA-strand breaks were evident in liver, kidney, and spleen of animals treated with OTA, and a similar degree of DNA damage was observed in rats treated with OTB, despite the lower toxicity of OTB. Moreover, the presence of DNA damage did not correlate with histopathological alterations, which were evident in the kidney but not in the liver. In liver and kidney, the extent of DNA damage was further enhanced in the presence of Fpg glycosylase, which is known to convert oxidative DNA damage into strand breaks, suggesting the presence of oxidative DNA damage. Oxidative DNA damage as a mechanism of OTA-dependent DNA damage is consistent with the absence of lipophilic DNA adducts as assessed by 32P-postlabeling analysis. No spots indicative of OTA-related DNA adducts were observed in kidney DNA extracted from OTA-treated animals by 32P-postlabeling analysis, despite the use of synthetic standard for postulated adducts. A small, but not significant, increase in the incidence of chromosomal aberrations (essentially chromatid and chromosome-type deletions) was observed in splenocytes from rats treated with OTA in vivo and subsequently cultured in vitro to express chromosomal damage. These aberrations are also compatible with oxidative DNA lesions since they are not typically caused by chemical carcinogens which form covalent DNA adducts. Together, with the lack of evidence for formation of lipophilic DNA adducts as assessed by postlabeling, these data suggest that OTA may cause genetic damage in both target and nontarget tissues independent of direct covalent binding to DNA.     

Rapid detection of the hOGG1 Ser326Cys polymorphism using LightCycler technology

Human 8-oxoguanine DNA glycosylase 1 (hOGG1) plays an important role in the repair of 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodGuo), one of the major constituents in DNA damage. A recent in vitro study showed that the hOGG1 326Cys polymorphism (rs1052133) exhibits reduced 8-oxodGuo repair activity. This study aimed to develop a LightCycler (LC) assay to analyze the C>G polymorphism (Ser326Cys) in exon 7 of the hOGG1 gene followed by validation of the method using DNA samples from 260 polycyclic aromatic hydrocarbons(PAH)-exposed workers with known 8-oxodGuo DNA-adduct values measured by HPLC. Twenty DNA samples were analyzed by a PCR-RFLP analysis with Fnu4H I to generate control DNA. LC melting curve analyses of the hOGG1 exon 7 PCR product were characteristic of the probes hybridized to the non-mutated Ser-type (CC) at 65 degrees C, or to the Cys mutant (GG) at 59 degrees C. The distribution in the population of PAH-exposed workers (N=260) was 58% (CC), 38%(CG), and 4% (GG). The minor G allele displayed a frequency of 23 %. The distribution of 8-oxodGuo adducts for the Ser326Cys variants of hOGG1 revealed geometric means (GM) of 5.83 (CC), 5.27 (CG), and 6.53 (GG) 8-oxodGuo adducts/10(6)dGuo. Corresponding GM values of current smokers were 5.7 (CC), 5.6 (CG) and 7.0 (GG) 8-oxodGuo adducts/10(6) dGuo. The analysis of the Ser326Cys polymorphism in 260 DNA samples with this new LC assay revealed that this method is reliable for high throughput analysis of this key polymorphism in the hOGG1 gene.     

Reduced cytochrome P4501A activity and recovery from oxidative stress during subchronic benzo[a]pyrene and benzo[e]pyrene treatment of rainbow trout

The mechanisms by which aniline exposure elicits splenotoxic response, especially the tumorigenic response, are not well-understood. Earlier, we have shown that aniline-induced oxidative stress is associated with increased oxidative DNA damage in rat spleen. The base excision repair (BER) pathway is the major mechanism for the repair of oxidative DNA base lesions, and we have shown an up-regulation of 8-oxoguanine glycosylase 1 (OGG1), a specific DNA glycosylase involved in the removal of 8-hydroxy-2′-deoxyguanosine (8-OHdG) adducts, following aniline exposure. Nei-like DNA glycosylases (NEIL1/2) belong to a family of BER proteins that are distinct from other DNA glycosylases, including OGG1. However, contribution of NEIL1/2 in the repair of aniline-induced oxidative DNA damage in the spleen is not known. This study was, therefore, focused on evaluating if NEILs also contribute to the repair of oxidative DNA lesions in the spleen following aniline exposure. To achieve that, male SD rats were subchronically exposed to aniline (0.5 mmol/kg/day via drinking water for 30 days), while controls received drinking water only. The BER activity of NEIL1/2 was assayed using a bubble structure substrate containing 5-OHU (preferred substrates for NEIL1 and NEIL2) and by quantitating the cleavage products. Aniline treatment led to a 1.25-fold increase in the NEIL1/2-associated BER activity in the nuclear extracts of spleen compared to the controls. Real-time PCR analysis for NEIL1 and NEIL2 mRNA expression in the spleen revealed 2.7- and 3.9-fold increases, respectively, in aniline-treated rats compared to controls. Likewise, Western blot analysis showed that protein expression of NEIL1 and NEIL2 in the nuclear extract of spleens from aniline-treated rats was 2.0- and 3.8-fold higher than controls, respectively. Aniline treatment also led to stronger immunoreactivity for NEIL1 and NEIL2 in the spleens, confined to the red pulp areas. These studies, thus, show that aniline-induced oxidative stress is associated with an induction of NEIL1/2. The increased NIEL-mediated BER activity is another indication of aniline-induced oxidative damage in the spleen and could constitute another important mechanism of removal of oxidative DNA lesions, especially in transcribed DNA following aniline insult.     

Cell-specific oxidative DNA damage induced by estrogen in rat testicular cells in vitro

17 alpha-Ethinylestradiol (EE) can induce oxidative DNA damage in terms of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in rat testicular cells by an apparent estrogen receptor-mediated mechanism. We investigated differential susceptibility to EE in cell sub-populations from rat testes and the role of rat 8-oxo-guanine DNA glycosylase (rOGG1). Isolated rat testicular cells were incubated with EE concentrations ranging from 0.1 to 1000 nM. Single strand DNA breaks and oxidised purines as fapyguanine glycosylase (FPG) sensitive sites were assessed by the comet assay. In the total cell population and in round haploid cells, oxidised purines showed a bell-shaped concentration-response relationship with a maximally increased levels at 10 nM EE, whereas, no significant effects were seen in diploid, S-phase or tetraploid cells. The mRNA level of rOGG1 in testes cells was unaffected by EE, whereas, baseline levels were higher than in liver tissue and similar to colon tissue.     

Potential Application of Benzo(a)Pyrene-Associated Adducts (Globin or Lipid) as Blood Biomarkers for Target Organ Exposure and Human Risk Assessment

In order to investigate the potential application of blood biomarkers as surrogate indicators of carcinogen–adduct formation in target-specific tissues, temporal formation of benzo[a]pyrene (BaP)-associated DNA adducts, protein adducts, or lipid damage in target tissues such as lung, liver, and kidney was compared with globin adduct formation or plasma lipid damage in blood after continuous intraperitoneal (ip) injection of [³H]BaP into female ICR mice for 7 d. Following treatment with [³H]BaP, formation of [³H]BaP–DNA or –protein adducts in lung, liver, and kidney increased linearly, and persisted thereafter. This finding was similar to the observed effects on globin adduct formation and plasma lipid damage in blood. The lungs contained a higher level of DNA adducts than liver or kidneys during the treatment period. Further, the rate of cumulative adduct formation in lung was markedly greater than that in liver. Treatment with a single dose of [³H]BaP indicated that BaP–globin adduct formation and BaP–lipid damage in blood reached a peak 48 h after treatment. Overall, globin adduct formation and lipid damage in blood were significantly correlated with DNA adduct formation in the target tissues. These data suggest that peripheral blood biomarkers, such as BaP–globin adduct formation or BaP–lipid damage, may be useful for prediction of target tissue-specific DNA adduct formation, and for risk assessment after exposure.     

Higher DNA adduct levels in urinary bladder and prostate of slow acetylator inbred rats administered 3,2'-dimethyl-4-aminobiphenyl

Human epidemiological studies suggest associations between acetylator phenotype and aromatic amine-induced tumors. The aromatic amine carcinogen 3,2'-dimethyl-4-aminobiphenyl (DMABP) induces colon, prostate, and urinary bladder tumors in the rat, and a rapid and slow acetylator rat model has been characterized. The formation of DNA adducts has been used as a valuable biomarker in tumorigenesis. In order to examine the relationship between the acetylation polymorphism and aromatic amine-induced cancer, DNA adducts were measured in three target organs (colon, prostate, and urinary bladder) and two nontarget organs (liver and heart) of male rapid (F344) and slow (WKY) acetylator inbred rats administered DMABP. Two DMABP-DNA adducts, N-(deoxyguanosin-8-yl)-DMABP (C8-DMABP) and 5-(deoxyguanosin-N2-yl)-DMABP (N2-DMABP), were identified in each target and nontarget organ examined. C8-DMABP-DNA adduct levels were highest in liver and were dose related in liver, colon, urinary bladder, and prostate. DMABP-DNA adduct levels were significantly higher in the prostate and urinary bladder of slow acetylator vs rapid acetylator rats. These studies suggest that DMABP-induced DNA damage is acetylator phenotype-dependent in urinary bladder and prostate, two target organs for DMABP-induced tumorigenesis in the rat.     

Repair of rat liver DNA in vivo damaged by ethylene dibromide.

Tube feeding of [¹⁴C]Jethylene dibromide (EDB) to non-fasted rats resulted in the incorporation of the radioactivity into liver DNA, RNA and protein. Using alkaline and neutral sucrose gradients, it was observed that administration of the pesticide to non-fasted rat caused slower sedimentation of liver DNA in alkaline and not in neutral sucrose gradients. Slower sedimentation of liver DNA in alkaline sucrose gradients was apparent within 2 hr after the administration of a dose of 22 mg/100 g or 4 hr after a dose of 7.5 mg/100 g of body weight. Using a dose of 7.5 mg/100 g, the EDB-induced liver DNA damage was repaired significantly by 17.5 hr and almost completely by 96 hr. Administration of diethyldithiocarbamate, a free radical scavenger, did not inhibit liver DNA damage caused by EDB.     

Effects of a Brussels sprouts extract on oxidative DNA damage and metabolising enzymes in rat liver.

The apparent anticarcinogenic effect of cruciferous vegetables found in numerous epidemiological and experimental studies has been associated with their influence on phase I and phase II metabolising enzymes as well as on the antioxidant status. In the present study we investigated the effect of administration of a Brussels sprouts extract on the expression at the mRNA level and/or catalytic activity in rat liver of three phase I enzymes [cytochrome P450-1A2 (CYP1A2),-2B1/2 (CYP2B1/2) and-2E1 (CYP2E1)] and two phase II enzyme [NADPH:quinone reductase (QR) and glutathione S-transferase pi 7 (GSTpi)], all previously suggested to be induced by vegetables. We also examined the activity and/or expression of several important antioxidant enzymes: glutathione peroxidase (GPx), catalase and gamma-glutamyl-cysteine synthetase (GCS) and the activity of the repair enzyme 8-oxoguanine DNA glycosylase (OGG1). QR, GPx and catalase activity was also assessed in the kidneys. In order to examine a possible effect of the Brussels sprouts related to oxidative stress, we measured oxidative DNA damage in terms of 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG) and lipid peroxidation in terms of malondialdehyde (MDA) formation in the liver. Oral administration of an aqueous Brussels sprouts extract for 4 days was found to induce the expression of GST 1.3-fold (P < 0.05) and the activity of QR 2.6-fold in rat liver (P < 0.05). No significant differences were seen in the expression of the phase I enzymes. No differences in antioxidant enzyme activity/expression or OGG1 activity were observed. In a second experiment, administration of the Brussels sprouts extract for 3 or 7 days was found to increase the level of 8-oxodG in rat liver from 0.75 to 0.97 per 10(5) dG and from 0.81 to 0.97 per 10(5) dG, respectively (P < 0.05). No effects on MDA levels were found. The present results support the data obtained in several studies that consumption of cruciferous vegetables is capable of inducing various phase II enzyme systems. However, the observed increase in oxidative DNA damage raises the question of whether greatly increased ingestion of cruciferous vegetables is beneficial.     

Genotoxicity of environmental agents assessed by the alkaline comet assay

Generation of DNA damage is considered to be an important initial event in carcinogenesis. A considerable battery of assays exists for the detection of different genotoxic effects of compounds in experimental systems, or for investigations of exposure to genotoxic agents in environmental or occupational settings. Some of the tests may have limited use because of complicated technical setup or because they only are applicable to a few cell types. The single cell gel electrophoresis (comet) assay is technically simple, relatively fast, cheap, and DNA damage can be investigated in virtually all mammalian cell types without requirement for cell culture. The aim of this thesis was to evaluate the comet assay as a genotoxicity test in genetic toxicology of environmental agents, encompassing both experimental animal models and biomonitoring. The comet assay detects strand breaks (SB). The cells are embedded in agarose and lysed, generating nucleus-like structures in the gel (referred to as nucleoids). Following alkaline electrophoresis, the DNA strands migrate toward the anode, and the extent of migration depends on the number of SB in the nucleoid. The migration is visualized and scored in a fluorescence microscope after staining. Broad classes of oxidative DNA damage can be detected as additional SB if nucleoids are incubated with bacterial DNA glycosylase/endonuclease enzymes. Oxidized pyrimidines and purines can be detected by incubation with endonuclease III and formamidopyrimidine DNA glycosylase, respectively. The animal experimental studies indicated that the comet assay was able to detect genotoxic effects of diesel exhaust particles in lung tissue, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-induced DNA damage in colon epithelial cells and liver tissue, and benzene-induced damage in bone marrow and liver cells. The strength of the comet assay was further outlined by application of repair enzymes, indicating no oxidative DNA base damage following IQ treatment. High levels of oxidative DNA lesions were detected after exposure to benzene or X-ray irradiation. The comet assay did not detect DNA damage in colon or liver following ingestion of diets containing of high contents of animal fat or sucrose, although other indices of DNA damage were found. Determined from the results of a large Japanese study, the discrimination between carcinogens and non-carcinogens appears to be similar between the comet assay and alkaline elution, which also detects SB. This suggests that the comet assay is a reliable genotoxicity test in animal experimental systems. In the biomonitoring studies, we investigated the effect of common exposures and lifestyle factors (rather than effects of known carcinogens) on the level of oxidative DNA damage in mononuclear blood cells of humans. In the first study, based on repeated measurements, it was shown that interindividual variation and seasonal variation were major determinants for the basal level of SB, whereas no effect of age, exercise, or antioxidant intake could be detected. The effect of exercise was further investigated under both normoxic and hypoxic circumstances, showing a strong effect of hypoxia, and only effect of exercise in terms of SB in hypoxia. In a placebo-controlled parallel dietary fruit and vegetable (or the corresponding amount of antioxidants) intervention study, no effects of the level of oxidative DNA damage or sensitivity to hydrogen peroxide were observed. Although this may seem in contrast to other antioxidant intervention studies, a critical literature survey of antioxidant intervention studies on oxidative DNA damage suggested that well-controlled studies tended to show no effect of antioxidant supplementation. In summary, the aggregated data from the publications included in this thesis, and other publications encompassing the comet assay, indicate that the comet assay is a reliable method for detection of DNA damage in tissues of experimental animals. Although not all types of genotoxic exposures should be expected to result in DNA damage in mononuclear blood cells, the comet assay seems to be a valuable tool for detection of genotoxic exposure in humans. The comet assay indicates that DNA damage is abundant in mammalian cells and affected by lifestyle and many environmental exposures, including diet, exercise, hypoxia, and sunlight.     

Ultrasensitive and specific detection methods for exocylic DNA adducts: markers for lipid peroxidation and oxidative stress

Among exocyclic DNA adducts, etheno (epsilon) bases (epsilond A, epsilond C, N(2),3-epsilond G) are generated by reactions of DNA bases with lipid peroxidation (LPO) products derived from endogenous sources and from the carcinogens vinyl chloride or urethane. The recent development of ultrasensitive methods has made it possible to detect these epsilon-adducts in vivo and to study their formation and role in experimental and human carcinogenesis. The promutagenic epsilon-DNA modifications can be detected by immunoaffinity/32P-postlabelling or by immunohistochemistry. When epsilon-adducts are excised from tissue DNA, the modified nucleosides can be quantified in urine by an immunoaffinity-HPLC-fluorescence method. Highly variable background levels of epsilon-adducts were detected in tissues from unexposed humans and rodents, suggesting an endogenous pathway of formation from reaction of trans-4-hydroxy-2-nonenal (via its 2,3-epoxide) with DNA bases. Several known cancer risk factors increased the level of these DNA lesions: Elevated epsilon-adducts were found in hepatic DNA from patients with excess metal storage (haemochromatosis, Wilson's disease), resulting in oxidative stress and high risk of liver cancer. Reactive O/N-intermediates generated during inflammatory processes, for example in patients with inflammatory bowel disease (IBD) and familial adenomatous polyposis (FAP) led to the formation of epsilon-adducts likely through peroxynitrite-mediated LPO and/or increased oxidative arachidonic acid metabolism. A high omega-6-polyunsaturated fatty acid (PUFA) diet increased epsilon-DNA adducts in white blood cells (WBC), particularly in female subjects (about 40-fold), while the level of adducted malondialdehyde in deoxyguanosine of WBC-DNA was only moderately elevated. In conclusion, there is now growing evidence that epsilon-adducts were elevated in cancer-prone patients and in rodents (liver, pancreas, colon, skin), suggesting that promutagenic epsilon-adducts, when formed as a consequence of persistent oxidative stress, can drive cells to malignancy. Therefore, biomonitoring of exocyclic DNA adducts offers useful tools: (i) to evaluate the etiological contributions of dietary fats, oxidative stress, and chronic inflammatory/infectious processes; (ii) to verify the efficacy of chemopreventive agents on endogenous DNA damage and cancer risk; and (iii) to gain mechanistic insights into the role of oxidative stress/LPO-derived lesions in the initiation and progression of human cancer.