Detection of 8-oxo-2'-deoxyguanosine, a marker of oxidative DNA damage, in culture medium from human mesothelial cells exposed to crocidolite asbestos

Crocidolite asbestos is associated with the development of mesothelioma.Although chromosomal changes have been documented in mesothelialcells, the mechanisms of interaction of crocidolite with DNAremain obscure. Since human mesothelial cells are exquisitelysensitive to asbestos, oxidative DNA damage was measured inan asbestos-exposed human mesothelial cell line (MET5A) by assayingoxidized guanine bases [8-oxo-2'-deoxyguanosine (oxo⁸dG), 8-oxoguanine(oxo⁸G), and 8-oxoguanosine (oxo⁸Gua)] excreted into the spentculture medium after DNA repair or turnover. At growth inhibitory,but not cytolytic concentrations, asbestos caused significantelevation of all bases in the spent medium over a 48-h period.In contrast, riebeckite, a chemically similar, nonfibrous analogof crocidolite did not cause increased adduct release. Resultsshow that oxidative RNA and DNA bases are produced in responseto asbestos in target cells of asbestos-induced cancers.     

Iron-dependent formation of 8-hydroxydeoxyguanosine in isolated DNA and mutagenicity in Salmonella typhimurium TA102 induced by crocidolite

Treatment of isolated DNA with crocidolite asbestos significantlyincreased the concentration of 8-hydroxydeoxyguanosine (8-OHdG)above background. Furthermore, incubating DNA with H₂O₂ andcrocidolite potentiated the formation of 8-OHdG above levelsobserved with crocidolite alone. In the presence of desferrioxamine,desferrioxamine and ferrozine, dimethylsulphoxide (DMSO) oro-phenanthroline, crocidolite-induced DNA oxidation was reducedby 36, 73, 74 and 70% respectively. Crocidolite, but not chrysotileasbestos, enhanced background revertants in Salmonella typhimuriumTA102, at sub-cytotoxic concentrations in a dose-dependent manner.The mutagenic effects of crocidolite were quite small and thisindicates that crocidolite was a weak mutagen in this study.The number of revertants was reduced to the spontaneous ratefor this strain after the fibres had been pretreated with desferrioxaminebefore assaying for genotoxicity in this oxygen radical-sensitivestrain. These results help to explain a mechanistic role foriron in crocidolite-induced DNA oxidation and mutagenicity inTA102.     

Aflatoxin B1-induced 8-hydroxydeoxyguanosine formation in rat hepatic DNA

A time- and dose-dependent increase in 8-hydroxydeoxy-guanosine(8-OHdG) was observed in rat hepatic DNA after a single i.p.injection of aflatoxin B₁ (AFB₁). It was also found that pre-treatmentwith selenium or deferoxamine significantly reduced 8-OHdG levelin AFB₁-administered rats. In contrast, no reduction in 8-OHdGconcentration was found in vitamin E-pre-treated rats. Theseresults provide evidence that AFB₁ causes oxidative DNA damagein rat liver, which may involve hydroxyl radicals as the initiatingspecies. It is postulated that AFB₁-induced oxidative DNA damage(8-OHdG formation) may constitute an important pathway in AFB₁hepatocarcinogenesis.     

Role of oxygen derivatives in the cytotoxicity and DNA damage produced by asbestos on rat pleural mesothelial cells in vitro

The role of reactive oxygen metabolites in the toxic effectsof asbestos on pleural mesothelial cells is not well defined.We exposed rat pleural mesothelial cells (RPMC) to chrysotileand crocidolite fibers (0–40 µg/cm²) in the presenceor absence of catalase and superoxide dismutase (SOD). Cellinjury was measured using the colorimetric 3–4 (5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay and DNA damage was evaluated in terms of unscheduledDNA synthesis (UDS). Catalase (100 U/ml) and SOD (250 U/ml)protected RPMC against asbestos-induced cytotoxicity and DNAdamage. However, the inactivated enzymes and bovine serum albuminalso showed some protection, suggesting that the effect of antioxidantenzymes may be partly related to their protein nature. Theseresults suggest that oxygen derivatives are partly involvedin the toxic effects of asbestos on cultures of RPMC. The presenceof extracellular proteins may also decrease asbestos-producedtoxicity by reducing the degree of RPMC–fiber interaction.     

Increased 8-hydroxyguanine in DNA and its repair activity in hamster and rat lung after intratracheal instillation of crocidolite asbestos.

Asbestos and man-made-mineral fibers are known to increase one type of oxidative DNA damage, 8-hydroxyguanine (8-OH-(Gua), in vitro. In this study, we analyzed the 8-OH-Gua level in DNA and its repair activity after a single intratracheal instillation of fibers (crocidolite or glass) or saline to Syrian hamsters or Wistar rats. The 8-OH-Gua level was measured with a high-performance liquid chromatography-electrochemical detector (HPLC-ECD) system. The 8-OH-Gua repair enzyme activity was determined with an endonuclease nicking assay using a 32P-labeled or fluorescently labeled 22mer DNA that contains 8-OH-Gua at a specific position. A significant increase in the 8-OH-Gua level in the lung DNA was observed 1 day after the exposure to crocidolite, as compared to the saline control. The repair activity was increased significantly at 7 days. On the other hand, after exposure to glass fibers, little or no increase of these carcinogenicity indicators was detected. These assays of 8-OH-Gua and its repair activity in short-term animal experiments will be useful for evaluating the carcinogenicity of fibers. This is the first report of the increase of 8-OH-Gua and its repair activity in the animal lung after the instillation of asbestos fibers.     

Evaluation of 8-hydroxydeoxyguanosine, a typical oxidative DNA damage, in human leukocytes

8-Hydroxydeoxyguanosine (8-OHdG) is a typical form of oxidativeDNA damage which causes mutation in vitro and in vivo. We investigatedpotential factors confounding 8-OHdG determination and, basedon the results, then determined the 8-OHdG levels in human peripheralblood leukocytes. 8–OHdG was detected electrochemicallyafter, extraction of DNA from the cells without the use of phenolby a DNA extractor under helium. In the preliminary experiments,the mononuclear leukocytes (MN) in blood samples obtained from19 laboratory workers and students were separated from the polymorphonuclearleukocytes (PMN) with Mono-Poly resolving medium. The 8-OHdGin the MN (1.157 ± 0.414 molecules per 10⁵ deoxyguanosine)did not differ significantly from that in PMN (1.131 ±0.418). The effect of red blood cells (RBC) on 8-OHdG formationduring DNA extraction was then examined by adding RBC to thehuman lymphoblastoid cell line FA72. Addition of RBC at ratiosof up to 4 RBC per FA72 cell did not increase 8-OHdG levels,while addition at a RBC/FA72 cell ratio of 20 increased the8-OHdG level 1.43-fold over that without RBC. The potentialeffect of histidine, a scavenger of both hydroxyl radicals andsinglet oxygen, on reduction of artificial 8-OHdG formationduring DNA extraction was examined during DNA extraction inthe human promyelocytic leukemia cell line HL60. Addition ofHis decreased the 8-OHdG level dose-dependently (30% reductionat 30 mM His concentration). Based on these results, we determinedthe 8-OHdG levels in human leukocyte samples obtained from 79healthy male factory workers aged 24–59 years. The leukocytefraction containing both MN and PMN was separated from RBC withMono-Poly resolving medium and DNA was extracted from the leukocytesin the presence of 30 mM His. The mean 8-OHdG level in thesesamples was 1.072 ± 0.230. To evaluate the reliabilityof the assay, FA72 was used as a standard sample in all assaydeterminations and the 8-OHdG levels of both the leukocyte samplesand the FA72 sample(s) were measured in each determination.The inter- and intra-assay coefficients of variation (CV) werecalculated to be 14.4% (n = 14) and 3.9–13.5% (n = 3–5per assay) respectively. The 8-OHdG level was measured twicein 19 leukocyte samples; the value at the first determinationwas not correlated with that at the second determination. Therange of 8-OHdG levels in the samples was relatively small comparedwith the CV of the assay. The 8–OHdG level was correlatedwith neither the age nor the smoking status of the donors.     

Erionite and asbestos differently cause transformation of human mesothelial cells

Malignant mesothelioma (MM) is an aggressive tumor associated with environmental or occupational exposure to asbestos fibers. Erionite is a fibrous zeolite, morphologically similar to asbestos and it is assumed to be even more carcinogenic. Onset and progression of MM has been suggested as the result of the cooperation between asbestos and other cofactors, such as SV40 virus infection. Nevertheless, several cases of MM were associated with environmental exposure to erionite in Turkey, where SV40 was never isolated in MM specimens. We show here that erionite is poorly cytotoxic, induces proliferating signals and high growth rate in human mesothelial cells (HMC). Long term exposure to erionite, but not to asbestos fibers, transforms HMC in vitro, regardless of the presence of SV40 sequences, leading to foci formation in cultured monolayers. Cells derived from foci display constitutive activation of Akt, NF-kappaB and Erk1/2, show prolonged survival and a deregulated cell cycle, involving cyclin D1 and E overexpression. Our results reveal that erionite is able per se to turn HMC into transformed highly proliferating cells and disclose the carcinogenic properties of erionite, prompting for a careful evaluation of environmental exposure to these fibers. The genetic predisposition to the effect of erionite is a separate subject for investigation.     

Induction of DNA strand breaks in cultured rat embryo cells by crocidolite asbestos as assessed by nick translation

Asbestos, a proven carcinogen, is reported to have no genotoxic effects. We hypothesized, however, in light of its clastogenic effects that one mechanism by which asbestos induces cell transformation and tumorigenesis involves the induction of DNA strand scission. Cultured rat embryo cells were exposed to low concentrations of International Union Against Cancer crocidolite and examined at intervals ranging from 2 to 48 h. The induction of DNA strand breaks was examined using the technique of nick translation followed by autoradiography or scintillation counting. Our results indicate that cells exposed to crocidolite have a higher incidence of DNA breaks and that this effect becomes apparent within 2-6 hours of exposure. Ball-milled crocidolite as well as riebeckite have a significantly lower effect while glass fibers induce a more pronounced DNA strand damage. These observations support the role fiber length plays in carcinogenesis and suggest that the classification of asbestos as a nongenotoxic carcinogen be reconsidered.     

Long-term mortality from pleural and peritoneal cancer after exposure to asbestos: Possible role of asbestos clearance

Models based on the multistage theory of carcinogenesis predict that the rate of mesothelioma increases monotonically as a function of time since first exposure (TSFE) to asbestos. Predictions of long-term mortality (TSFE >or= 40 years) are, however, still untested, because of the limited follow-up of most epidemiological studies. Some authors have suggested that the increase in mesothelioma rate with TSFE might be attenuated by clearance of asbestos from the lungs. We estimated mortality time trends from pleural and peritoneal cancer in a cohort of 3,443 asbestos-cement workers, followed for more than 50 years. The functional relation between mesothelioma rate and TSFE was evaluated with various regression models. The role of asbestos clearance was explored using the traditional mesothelioma multistage model, generalized to include a term representing elimination over time. We observed 139 deaths from pleural and 56 from peritoneal cancer during the period 1950-2003. The rate of pleural cancer increased during the first 40 years of TSFE and reached a plateau thereafter. In contrast, the rate of peritoneal cancer increased monotonically with TSFE. The model allowing for asbestos elimination fitted the data better than the traditional model for pleural (p = 0.02) but not for peritoneal cancer (p = 0.22). The risk for pleural cancer, rather than showing an indefinite increase, might reach a plateau when a sufficiently long time has elapsed since exposure. The different trends for pleural and peritoneal cancer might be related to clearance of the asbestos from the workers' lungs.     

Role of oxyradicals in mutagenicity and DNA damage induced by crocidolite asbestos in mammalian cells

Crocidolite, one of the most carcinogenic forms of asbestos, is mutagenic in cultured mammalian cells when assayed using a system that can detect multilocus deletions. In the present study, we examined the effect of buthionine sulfoximine (BSO) on mutation frequency and the formation of 8-hydroxydeoxyguanosine (8-OHdG) in human-hamster hybrid (A(L)) cells induced by crocidolite fibers in an attempt to determine the role of oxyradicals in mediating fiber mutagenesis. BSO, a competitive inhibitor of the enzyme gamma-glutamyl cysteine synthetase, depleted nonprotein sulfhydryls to <5% of control within 24 h at a nonmutagenic dose of 25 microM. In cells pretreated with BSO for 24 h, the mutation yield at the CD59 locus induced by a 4 microg/cm2 dose of crocidolite fibers was increased by more than 3-fold (P < 0.05). Using immunoperoxidase staining with a monoclonal antibody specific for 8-OHdG, we demonstrated that crocidolite fibers induced a dose-dependent increase in oxidative DNA damage in A(L) cells. Furthermore, addition of DMSO, a well-established hydroxyl radical (OH*) scavenger, dramatically suppressed 8-OHdG induction (P < 0.005). Our results definitely demonstrate that reactive oxygen species mediate fiber-induced DNA damage mutagenesis in A(L) cells in a concentration-dependent manner.     

DNA adduct formation and oxidative stress in colon and liver of Big Blue rats after dietary exposure to diesel particles

Exposure to diesel exhaust particles (DEP) via the gastrointestinal route may impose risk of cancer in the colon and liver. We investigated the effects of DEP given in the diet to Big Blue rats by quantifying a panel of markers of DNA damage and repair, mutation, oxidative damage to proteins and lipids, and antioxidative defence mechanisms in colon mucosa cells, liver tissue and the blood compartment. Seven groups of rats were fed a diet with 0, 0.2, 0.8, 2, 8, 20 or 80 mg DEP/kg feed for 21 days. DEP induced a significant increase in DNA strand breaks in colon and liver. There was no effect on oxidative DNA damage (8-oxodG) in colon or liver DNA or in the urine. However, the mRNA expression of OGG1, encoding an enzyme involved in repair of 8-oxodG, was increased by DEP in both liver and colon. DNA adduct levels measured by 32P-post-labelling were elevated in colon and liver, and the expression of ERCC1 gene was affected in liver, but not in colon. In addition to these effects, DEP exposure induced apoptosis in liver. There was no significant change in mutation frequency in colon or liver. The levels of oxidative protein modifications (oxidized arginine and proline residues) were increased in liver accompanied by enhanced vitamin C levels. In plasma, we found no significant effects on oxidative damage to proteins and lipids, antioxidant enzymes or vitamin C levels. Our data indicate that gastrointestinal exposure to DEP induces DNA adducts and oxidative stress resulting in DNA strand breaks, enhanced repair capacity of oxidative base damage, apoptosis and protein oxidation in colon mucosa cells and liver.     

Crocidolite asbestos increased 8-hydroxydeoxyguanosine levels in cellular DNA of a human promyelocytic leukemia cell line, HL60

Crocidolite, one of the most carcinogenic asbestos fibers, inducesthe release of reactive oxygen species (ROS) from neutrophilsand macrophages. Using HPLC combined with electrochemical detection,we determined that 8-hydroxy-deoxyguanosine (8OHdG), a moleculetypical of mutagenic oxidative DNA damage, was induced in thecellular DNA of a human promyelocytic leukemia cell line, HL60,incubated with crocidolite. Crocidolite increased 8OHdG in thecellular DNA of phorbol myristate acetate (PMA)-differentiatedHL60, which phagocytosed crocidolite. PMA-differentiated HL60released ROS spontaneously, as determined by ESR with 5,5-dimethylpyrrolone-N-oxideas a spin trap. However, the release of ROS from the cell linedid not increase after the addition of crocidolite. The additionof superoxide dismutase at a sufficient concentration to scavengeROS released from the cell did not inhibit the 8OHdG increaseinduced by crocidolite. Cytochalasin B, which inhibited phagocytosis,did not inhibit the release of ROS. However, it inhibited thecrocidolite-induced 8OHdG increase by 48.3%. Contrary to PMA-differentiatedHL60, undifferentiated HL60 neither phagocytosed crocidolitenor showed a crocidolite-induced increase in 8OHdG formation.The 8OHdG increase induced by crocidolite was not correlatedwith ROS release, but with the internalization of crocidolite,suggesting that the increase was not due to an increase in ROSrelease from the cell but was due to the conversion of relativelyinert ROS to highly reactive ROS, such as hydroxyl radicals,by crocidolite that was internalized and close to DNA.     

Inhibition of 8-hydroxydeoxyguanosine formation by curcumin in mouse fibroblast cells

Curcumin, a pigment responsible for the yellow color of curry,has been shown to be an anti-inflammation agent, an antioxidantand an antipromoter. 8-Hydroxydeoxyguanosine (8-OH-dG), an oxidizednucleoside, may be responsible for a genetic event of tumorpromotion in carcinogenesis. 8-OH-dG can be detected selectivelyand sensitively at the fmol level by HPLC–electrochemicaldetection at an applied potential of+0.8 V versus Ag/AgCl. Phorbol-12-myristate13-acetate (PMA), a potent tumor promoter, induces lipid peroxidationand 8-OH-dG formation. Curcumin can strongly scavenge the hydroxylradical (OH.) to prevent 8-OH-dG formation from dG (deoxyguanosine)in vitro and reduce the production of PMA-induced lipid peroxidationand 8-OH-dG in mouse fibroblast cells. These results suggestthat curcumin inhibits the PMA-induced tumor promotion by functioningas an OH. radical scavenger to prevent 8-OH-dG formation withinthe DNA molecule.     

Formation of 8-hydroxydeoxyguanosine in liver DNA of rats following long-term exposure to a peroxisome proliferator

The mechanism by which nongenotoxic peroxisome proliferators induce hepatocellular carcinomas in rats and mice remains intriguing. The available experimental evidence suggests that the proliferation of peroxisomes and induction of peroxisome-associated enzymes results in oxidative stress which then leads to tumorigenesis. However, so far no direct evidence for oxidative DNA damage in livers of peroxisome proliferator-treated animals has been established. In the present study we have examined the DNA obtained from the livers of rats treated with ciprofibrate, a potent peroxisome proliferator, for variable periods of time for 8-hydroxydeoxyguanosine (8-OH-dG), an adduct that results from the damage of DNA caused by hydroxyl radical. Administration of ciprofibrate in diet at a concentration of 0.025% for 16, 28, 36, or 40 weeks resulted in progressive increases in the levels of 8-OH-dG. At 16, 28, and 40 weeks of ciprofibrate treatment, the 8-OH-dG in the liver DNA was significantly increased as compared to controls. This increase in 8-OH-dG levels is attributed to persistent peroxisome proliferation resulting from chronic ciprofibrate treatment as no increase in 8-OH-dG was found in liver DNA of rats that received a single large dose of ciprofibrate. The results of this study clearly demonstrate, for the first time, that persistent proliferation of peroxisomes leads to specific oxidative DNA damage.     

Exposure of mammalian cell cultures to benzo[a] and light results in oxidative DNA damage as measured by 8-hydroxydeoxyguanosine formation

Carcinogenic polycyclic aromatic hydrocarbons induce DNA damagethrough direct covalent interactions with nucleotides of theDNA in cells in which they are activated to ‘ultimatecarcinogenic metabolites’. To determine whether they alsoinduce oxidative damage to DNA under the same circumstances,early passage Syrian hamster embryo and human mammary carcinomacell line MCF-7 cultures were treated for 24 h with 0–5µg/ml benzo[a]pyrene (BaP) or for 1 h with 0–100µM methylene blue (as a positive control for oxidativedamage). The cells were then exposed to fluorescent light for1 or 4 h or retained in darkness. After cell harvest, DNA isolationand enzymatic digestion of the DNA to deoxyribonucleosides,the amounts of 8-hydroxy- 2'-deoxyguanosine (8-OH-dGuo) andunmodified deoxy guanosine present were determined by reverse-phaseHPLC with electrochemical and UV detection respectively. Culturestreated with methylene blue for 1 h followed by light exposurefor 1 h contained 5-fold (10 µM) and 8- to 28-fold (100µM) higher 8-OH-dGuo levels than cells treated with methyleneblue not exposed to light or untreated cells exposed to light.There was no significant change in 8-OH-dGuo levels in culturestreated with 1–5 µg/ml BaP for 24 h in the absenceof light. However, both the human and hamster cell culturestreated with BaP and then exposed to fluorescent light for 4h contained 3-fold (1 µg/ml) and 8- to 10-fold (5 µg/ml)higher 8-OH-dGuo levels than those not exposed to light or nottreated with BaP. These results indicate that BaP treatmentdoes not cause 8-OH-dGuo formation in DNA of cells maintainedin darkness. Exposure of BaP-treated cells to fluorescent lightcauses formation of significant amounts of oxidative DNA damageas measured by 8-OH-dGuo formation. These findings suggest thatoxidative damage of DNA could be involved in tumor inductionby BaP in tissues, such as skin, in which exposure to BaP canoccur In the presence of light.     

Increased formation of 8-hydroxydeoxyguanosine, an oxidative DNA damage, in lymphoblasts from Fanconi's anemia patients due to possible catalase deficiency

Fanconi's anemia (FA) cells are highly susceptible to both reactiveoxygen species and mitomycin C (MMC), a DNA cross-linking agent.In this study we have determined the amounts of 8-hydroxydeoxyguanosine(8OHdG), typical of oxidative DNA damage, in Epstein Barr virustransformed lymphoblasts from FA patients and normal controlsby the use of HPLC combined with electrochemical detection.FA cells (HSC72 and 99 cells being assigned to FA complementationgroup A) formed 2–3 times more 8OHdG than control cellsafter incubation with 20 mM H₂O₂ at 37°C for 30 min. FAcells also formed more 8-hydroxyguanosine, typical of oxidativeRNA damage, than control cells. FA cells showed decreased activityto decompose H₂O₂. Although the activity in FA cells was only20–30% less than control cells, the remaining, undecomposedH₂O₂ concentration was almost twice as much in FA cells as incontrol cells, and the remaining H₂O₂ concentration correlatedwell with the amounts of 8OHdG formation. The H₂O₂ decomposingactivity was almost completely inhibited by sodium azide (NaN₃)or aminotriazole, both catalase inhibitors. With these inhibitorsthe amounts of 8OHdG formation were much higher than in thosecells without inhibitors, and were almost the same in controlcells as in FA cells. Catalase activity in FA cell lysates was70–80% of controls. MMC also increased 8OHdG formationin FA cells only at ED₁₀₀ but not at ED₅₀. These results indicatethat FA cells, at least FA complementation group A cells, haveincreased susceptibility to oxidative DNA damage, and that thisincreased susceptibility is possibly due to decreased catalaseactivity. These results also suggest that catalase plays animportant role in protecting DNA from oxidative damage. However,this increased susceptibility to oxidative DNA damage is considerednot to be the major cause of the increased susceptibility toMMC.     

Determination of 8-hydroxydeoxyguanosine in human cells under oxygen-free conditions

To establish an accurate 8-hydroxydeoxyguanosine (8OHdG) determinationsystem, we examined two potential factors causing experimentalerror in 8OHdG determination. First, we examined the efficiencyof the enzymatic digestion of DNA, that could cause misestimationof 8OHdG. Second, since we considered that the oxygen moleculesin atmosphere and in reagents were the main factor contributingto the experimental errors, we carried out the 8OHdG determinationunder oxygen-free conditions and compared the 8OHdG value withthat determined by the methods under ambient atmosphere. Thecalf thymus DNA was sufficiently digested in the condition weused and the yields of dG were constant, even when the DNA wasdamaged with H₂O₂ (80mM) and UV irradiation. By carrying outthe DNA extraction manually, instead of using the DNA extractor,we could reduce the additional 8OHdG formation during sampleprocessing. No trend was found in the difference betweenthe8OHdG values determined under oxygen-free conditions and underambient atmosphere. However, when the 8OHdG values werecomparedin samples with asbestos, the value determined under oxygen-freeconditions was significantly lower than that determined underambient atmosphere. These findings suggest that the removalof oxygen molecules was effective in reducing accidental ROSgeneration by impurities in the sample, which could cause theadditional 8OHdG formation, and that the oxygen-free systemmade the determination of 8OHdG reproducible and more accuratethan before. When the oxygen-free system was applied to humanleukocytes, the system showed good reproducibility (r = 0.535,P < 0.001), even though the 8OHdG level was low. With thesystem, we could detect a significant difference between 8OHdGin polymorphonuclear leukocytes(0.241± 0.129) and mononuclearleukocytes (0.188 ± 0.126, P < 0.01).     

Iron mobilization from crocidolite asbestos greatly enhances crocidolite-dependent formation of DNA single-strand breaks in phi X174 RFI DNA.

The ability of the iron associated with asbestos to catalyze damage to phi X174 RFI DNA was determined and compared with iron mobilized from asbestos. Asbestos (1 mg/ml) suspended for 30 min in 50 mM NaCl containing 0.5 micrograms phi X174 RFI DNA, pH 7.5, did not catalyze detectable amounts of DNA single-strand breaks (SSB). However, addition of ascorbate (1 mM) resulted in 19, 26, 7 or 8% DNA with SSB for crocidolite, amosite, chrysotile or tremolite respectively. The percentage of DNA with SSB induced by each form of asbestos was directly related to its iron content. Inclusion of desferrioxamine B, which binds Fe(III) rendering it redox inactive, completely inhibited asbestos-dependent formation of DNA SSB, suggesting that iron was responsible for catalyzing the formation of DNA SSB. Mobilization of Fe(II) from crocidolite by citrate, EDTA or nitrilotriacetate (1 mM) in the absence of ascorbate resulted in 15, 33 or 63% DNA with SSB respectively. This activity was completely inhibited by compounds considered to be .OH scavengers, i.e. mannitol, 5,5-dimethyl-1-pyrroline N-oxide or salicylate (100 mM). Preincubation of crocidolite with citrate (1 mM) for 24 h resulted in mobilization of 52 microM iron and increased ascorbate-dependent induction of DNA SSB compared with crocidolite that was preincubated without citrate. Iron mobilized by citrate was entirely responsible for crocidolite-dependent formation of DNA SSB as evidenced by complete inhibition with desferrioxamine B. Therefore, the results of the present study strongly suggest that iron was responsible for asbestos-dependent generation of oxygen radicals, which resulted in the formation of DNA SSB. Mobilization of iron by chelators, followed by redox cycling, greatly enhanced crocidolite-dependent formation of DNA SSB. Thus, mobilization of iron in vivo by low mol. wt chelators may lead to the increased production of reactive oxygen species resulting in damage to biomolecules, such as DNA.     

Elevated 8-hydroxydeoxyguanosine in hepatic DNA of rats following exposure to peroxisome proliferators: relationship to mitochondrial alterations

Recent studies have indicated a lack of correlation betweenhepatic 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels and thecarcinogenicity of peroxisome proliferators (PP) and suggestedthat DNA in intact hepatic nuclei may be insensitive to increasesin 8-OHdG resulting from PP exposure. The possibility that PP-inducedelevations in acyl CoA oxidase (ACO) activity might result inoxidative damage to mitochondrial DNA (mtDNA) was thereforeinvestigated by feeding male F344 rats the hepatocarcinogenicPP Wy-14,643 (Wy, 0.1% in the diet) for 3, 6, 11, or 22 weeks,or clofibric acid (CA, 0.5% in the diet) for 22 weeks. Followingthe respective PP exposures, hepatic peroxisomal acyl CoA oxidaseactivity was determined and DNA isolated from either mitochondriaor unfractionated liver homogenates and analysed for the presenceof 8-OHdG. PP treatment caused an increase in ACO activity (10-to 15-fold) at all time points examined and an increase of 8-OHdG(1.5- to 2-fold) in DNA isolated from unfractionated liver homogenatesfollowing PP treatment for 11 or 22 weeks. No increase of 8-OHdGin mtDNA was detected. However, quantitation of a PCR amplifiedregion from the D-loop of mtDNA demonstrated a 2- to 3-foldincrease in the relative amount of mtDNA in DNA isolated fromunfractionated liver homogenates following 3, 11, and 22 weeksexposure to Wy or CA (22 weeks only). In addition, a slightincrease in the mitochondrial volume density (1.4-fold) wasobserved in electron micrographs of liver samples from ratsexposed to Wy for 22 weeks. These results (i) demonstrate thatPP treatment, at levels which cause an increase in ACO activity,does not cause oxidative damage to mtDNA, and (ii) suggest thatone reason for the observed increase of 8-OHdG in DNA from unfractionatedliver homogenates may be an increase in the amount of mtDNApresent in these samples. Furthermore, these studies provideadditional evidence against a role of oxidative DNA damage,measured as 8-OHdG, in PP-induced rodent hepatocarcinogenesisand suggest that alterations in mitochondria or other effectsmay be more pertinent to PP-related carcinogenesis.