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.     

Induction of 8-hydroxydeoxyguanosine but not initiation of carcinogenesis by redox enzyme modulations with or without menadione in rat liver.

Inducibility of oxidative stress in rat liver in vivo by menadione-associated redox cycling activation under redox enzyme modulating conditions was examined by monitoring hepatocyte injury and 8-hydroxydeoxyguanosine (8-OHdG) levels of liver DNA. In addition, the treatment-associated liver tumor initiating activity was assessed in terms of development of gamma-glutamyl-transpeptidase (GGT)- and glutathione S-transferase placental form (GST-P)-positive foci and hyperplastic nodules. With or without following menadione treatment (50 mg/kg, i.g.), redox enzyme modulations of increased cytochrome P450 reductase activity induced by phenobarbital (PB)-Na (100 mg/kg, i.p. for 5 days), inhibition of DT-diaphorase by dicumarol (25 mg/kg, i.p.) and depletion of glutathione by phorone (200 mg/kg, i.p.), with or without further supplement of iron EDTA-Na-Fe(III) (70 mg/kg, i.p.), caused both substantial hepatocyte necrosis and 8-OHdG production in Fischer 344 male rats. Subsequent feeding with a 0.05% PB diet for 64 weeks resulted in slightly increased development of GGT-positive foci but not GST-P positive lesions or hyperplastic nodules, suggesting a lack of tumor-initiating activity of the oxidative DNA damage associated with redox enzyme modulations with or without menadione.     

Search for Ha-ras codon 61 mutations in liver tumours caused by hexachlorobenzene and Aroclor 1254 in C57BL/10ScSn mice with iron overload.

C57BL/10ScSn mice administered iron--dextran and fed the environmental pollutants hexachlorobenzene (HCB) and polychlorinated biphenyls (PCBs) develop hepatic nodules and carcinomas within 18 months. A range of lesions from the livers were analysed for the presence of mutations in the Ha-ras proto-oncogene at codon 61 using the polymerase chain reaction to amplify DNA from formalin-fixed sections, followed by oligonucleotide hybridization. Only two mutations from 23 preneoplastic and neoplastic lesions induced by HCB were detected (a focus of altered cells and a trabecular cell carcinoma). With Aroclor 1254 no mutations were detected in 28 areas at various stages of carcinogenesis analysed. Sequencing of the two mutations generated by HCB showed a C-->T transversion at the first base of codon 61 (carcinoma) and an A-->T transversion at the second base (proliferative focus). Thus, in marked contrast to some other systems of mouse liver tumour induction, hepatocarcinogenesis caused by HCB and PCBs in C57BL/10ScSn mice is an example of carcinogenesis which does not involve a high frequency of Ha-ras gene mutation at codon 61.     

Promoting effects of polychlorinated biphenyls (Aroclor 1254) and polychlorinated dibenzofuran-free Aroclor 1254 on diethylnitrosamine-induced tumorigenesis in the rat

The hepatic tumor-promoting activity of a commercial polychlorinated biphenyl mixture, Aroclor 1254 (AR 1254), with and without its intrinsic polychlorinated dibenzofuran (PCDF) impurities, was investigated. Male Sprague-Dawley non-inbred albino rats were treated with 66 microgram diethylnitrosamine (DENA)/ml drinking water for 5 weeks and subsequently given a control diet or a diet supplemented (100 ppm for 18 wk) with either AR 1254 or AR 1254 from which the PCDF moieties were removed (AR 1254-PCDF). Of those animals receiving DENA alone, 16% exhibited hepatocellular carcinomas. Of those rats treated with DENA followed by administration of AR 1254 or AR 1254-PCDF, 64 or 84%, respectively, developed hepatocellular carcinomas. Thus promotion with either AR 1254 or AR 1254-PCDF significantly (P less than 0.05) increased the incidence of DENA-initiated hepatocellular carcinomas. Administration of AR 1254 or AR 1254-PCDF alone did not induce hepatic tumors. Therefore, PCDF impurities were not necessary for the promoting activity of AR 1254.     

Generation of 8-hydroxydeoxyguanosine from DNA using rat liver homogenates

In relation to carcinogenesis, aging and other pathologic conditions, urinary 8-hydroxydeoxyguanosine (8OHdG) is widely used as a marker for evaluating the effect of oxidative stress on DNA. Because no reports have described how 8OHdG is generated from DNA in vivo or by biological materials, and how it is excreted into urine, the authors investigated the generation of 8OHdG from DNA, using rat liver homogenate. Oxidatively damaged DNA samples containing different levels of 8OHdG were prepared using ultraviolet irradiation with three different concentrations of riboflavin. Following incubation of damaged DNA samples with rat liver homogenates, the generation of 8OHdG from the DNA was determined using high-performance liquid chromatography with electrochemical detection after ultrafiltration of the incubation mixtures. The generation of 8OHdG was also tested with an anti-8OHdG antibody. The quantity of 8OHdG generated from the DNA by rat liver homogenates was dependent on the 8OHdG levels in the DNA: almost all 8OHdG in the DNA was released as 8OHdG by rat liver homogenates. Generation of 8OHdG correlated with the degradation of DNA. Interestingly, the generated 8OHdG was stable in the presence of rat liver homogenates, whereas deoxyguanosine (dG) rapidly disappeared in the same conditions. Less than 1/10,000 of dG was converted to 8OHdG when dG was incubated with rat liver homogenate. Incubation of 8-hydroxyguanine with rat liver homogenates did not generate 8OHdG. These findings suggest that most of the 8OHdG in DNA is released as 8OHdG during DNA degradation and that, because of its stability, 8OHdG is excreted into urine, thus providing a convenient measure of oxidative damage to DNA.     

Induction of 8-hydroxydeoxyguanosine in DNA by chromium(III) plus hydrogen peroxide and its prevention by scavengers

The capability of Cr(III) to induce DNA lesions generated byoxidative damage was investigated in this study by examiningthe formation of 8-hydroxydeoxyguanosine (8-OHdG) in calf thymusDNA by CrCl₃ and/or H₂O₂ in 10 mM phosphate buffer. In the presenceof 0.5 mM H₂O₂, the formation of 8-OHdG markedly increased withincreasing CrCl₃ concentration. In contrast, H₂O₂ or CrCl₃ alonedid not cause any increase in 8-OHdG level above background.The amount of 8-OHdG induced by CrCl₃ plus H₂O₂ was time dependent;its generation increased linearly over an incubation periodof 90 min. The formation of 8-OHdG was unfavorable in an acidicsolution (pH < 6); the highest level of 8-OHdG was observedat pH 7–8. Scavengers of reactive oxygen species markedlyinhibited the formation of 8-OHdG by CrCl₃ plus H₂O₂; the inhibitioneffect was sodium azide > D-mannitol > Tris-HCI at anequal concentration. The induction of 8-OHdG by CrCl₃ plus H₂O₂remained unchanged in D₂O. Moreover, an addition of catalase(2.2 U/ml) to the reaction mixture completely inhibited theformation of 8-OHdG by CrCl₃/H₂O₂, whereas only 22% of thatformation was inhibited by superoxide dismutase (11 U/ml). Alarge amount of bovine serum albumin (1.1 mg/ml) could reducethe formation of 8-OHdG by CrCl₃ plus H₂O₂, thereby implyingthat Cr(III)-mediated DNA-protein crosslinks are unfavorablefor 8-OHdG formation. Furthermore, ascorbate could prevent theformation of 8-OHdG by CrCl₃ plus H₂O₂; the extent of preventionincreased with increasing ascorbate concentration (10 µM-3mM). Thus, ascorbate acts as a free radical scavenger in theCrCl₃/H₂O₂ system. The above findings suggest that Cr(III)/H₂O₂could generate oxidative damage to DNA, possibly through a Fenton-likereaction, i.e. Cr(III) + H₂O₂     

Induction of liver tumors in male Wistar rats by feeding polychlorinated biphenyls (Aroclor 1260)

Two groups of 32 male Wistar rats, each 5 weeks of age, were fed on protein diet containing polychlorinated biphenyl (Aroclor 1260), at 50 ppm and 100 ppm levels, respectively, for 120 days. This not only brought about gross hepatic changes but induced neoplastic nodules with adenofibrosis in 75% and 50% of the rats of the respective groups. None of the control animals showed such changes. The study revealed that feeding of the PCBs can not only induce liver adenofibrosis in young male Wistar rats in a short duration of time, but also showed that the carcinogenic potentiality in male rats fed Aroclor 1260 is greater when fed at a lower dose.     

Is 8-hydroxydeoxyguanosine a mediator of carcinogenesis by a choline-devoid diet in the rat liver?

The mechanism(s) by which a diet devoid of choline (CD) induceshepatocellular carcinomas in rats remains unknown. Althoughanimals fed this diet develop nuclear lipid peroxidation, suggestingoxidative DNA damage, there is no direct evidence that thisoccurs. In this study, 8-hydroxy-deoxyguanosine (8-OHdG), aDNA adduct generated by reactive oxygen species, was analyzedin the liver of rats fed a CD diet and in controls receivinga choline-sufficient (CS) diet. After partial hepatectomy, theanimals were injected with diethylnitrosamine (DEN, 50 mg/kgbody wt) or with saline and fed a CD or CS diet for 24 weeks.While liver DNA from rats injected either with DEN or salineand fed a CS diet did not show detectable amounts of the nucleotide,those who were fed DEN/CD and saline/CD demonstrated similar,easily measurable levels of 8-OHdG. These results indicate thatthere is a positive association between the continuous administrationof a CD diet and the production of 8-OHdG in liver DNA, andsupport the idea that oxidative DNA damage is involved in carcinogenesisby a CD diet.     

Induction of 8-hydroxydeoxyguanosine in isolated DNA and HeLa cells exposed to fecapentaene-12: evidence for the involvement of prostaglandin H synthase and iron

The genotoxic/mutagenic mechanism(s) of action of fecapentaene-12(fec-12) is complex but there is evidence to suggest that thegeneration of active oxygen species (AOS) may be involved. Thishas been assessed by measuring the formation of 8-hydroxydeoxyguanosine(8-OHdG) in isolated DNA and HeLa cells exposed in vitro tofec-12. The possibility that fec-12 may form AOS via peroxidative‘activation’ by prostaglandin H synthase (PHS) hasbeen investigated by measuring 8-OHdG in HeLa cells exposedto fec-12 in the absence or presence of PHS inhibitors. Therole of iron as a catalyst in this pathway has also been investigated.A 4-fold increase in the level of 8-OHdG in isolated DNA wasseen after exposure to fec-12 (1 mM) alone. This increase wasenhanced synergistically by ferrous iron. Fec-12 exposure ofHeLa cells at 50 and 100 /µM induced 2- and 3-fold increases(P < 0.001) respectively in the level of 8-OHdG in cellularDNA. No increase was seen at 10 /µM fec-12. The PHS inhibitorsindomethacin and acetylsalicylate blocked the formation of 8-OHdGinduced by fec-12 (50 /µM) but did not inhibit the formationof 8-OHdG in these cells after exposure to H₂O₂ and Fe²⁺. Additionof the iron chelating agent o-phenanthroline to cells priorto fec-12 exposure blocked the increase in 8-OHdG induced byfec-12 (50 /µM). Addition of the radical scavenging agentDMSO (10%) to cells prior to fec-12 exposure reduced the levelof 8-OHdG to within 10% of control. Specific inhibition of fec-12induced 8-OHdG formation in HeLa cells by PHS inhibitors suggeststhat this enzyme may be involved in ‘activating’fec-12 to form AOS in cells. Inhibition of fec-12 induced 8-OHdGformation in cells by o-phenanthroline suggests a role for intracellulariron as a catalyst in this process.     

Formation of 8-hydroxydeoxyguanosine within DNA of mouse keratinocytes exposed in culture to UVB and H2O2.

Exposure to UV light contributes to the development of skin cancer. The importance of reactive oxygen species in UV-radiation carcinogenesis has been recognized for some time and several associated DNA base modifications have been identified. In particular, 8-hydroxydeoxyguanosine (8-OHdG) has been well studied as an indicator of oxidative damage to calf thymus DNA exposed to a variety of oxygen-generating systems, including UV light. However, to date, few studies of 8-OHdG have been conducted in cell or animal systems and those in vitro investigations that studied UV exposure have used UVC (< 290 nm), not the UVB (290-320 nm) or UVA (320-400 nm) ranges to which organisms are exposed through sunlight. The objective of this study was to measure 8-OHdG formation in the DNA of cultured mouse keratinocytes exposed to UVB. Using HPLC with electrochemical detection, background levels of 8-OHdG were approximately 6 fmol/micrograms DNA in DNA isolated and digested to the nucleoside level. UVB induced 8-OHdG up to 100% above that for mock-treated cells at a dose of 630 mJ/cm2 (dose-response range: 210-630 mJ/cm2). UVB exposure at 630 mJ/cm2 combined with 5 mM H2O2 elevated 8-OHdG formation up to 280% above that in control cells, whereas H2O2 alone had no effect. These results suggest that factors which increase the generation of reactive oxygen species by UV light may be potent cofactors of UV-radiation carcinogenesis.     

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.     

Induction of minisatellite DNA rearrrangements by genotoxic carcinogens in mouse liver tumors

We Investigated whether somatic rearrangements in minisatelliteDNA are more frequent in chemically induced mouse liver tumorsthan they are in spontaneous tumors. CD-1 mouse liver tumorswere induced by either a single dose or 15 consecutive dailydoses of 7,12-dimethylbenz[a]anthracene, 4-aminoazobenzene,N-hydroxy-2-acetyl-aminofluorene or diethylnitrosamine (DEN).Using DNA fingerprinting analysis, we found that the single-and multiple-dose carcinogen treatments caused a 2- to 5-foldhigher frequency of minisatellite DNA rearrangements comparedwith that found in spontaneous tumors-with the exception ofsingle-dose DEN tumors, which showed no increase in rearrangements.Our results suggest that DNA fingerprinting may be a valuahleassay for differentiating certain chemically induced tumorsfrom spontaneous tumors.     

Mechanisms of anti-carcinogenesis: the distribution and metabolism of aflatoxin B1 in rainbow trout fed Aroclor 1254

Polychlorinated biphenyls (PCBs) have previously been shownto be inhibitors of carcinogenesis in trout. The mechanism ofthis inhibition was investigated by studying the effects ofPCBs on aflatoxin B₁ (AFB₁) distribution, metabolism and DNAadduct formation, both in vivo and in vitro. A 24 h distributionstudy of injected tritiated AFB₁ showed more radioactivity inblood, liver and bile in fish fed PCBs, but less in residualcarcass. The metabolites of AFB₁ found in vivo in blood plasmaand liver homogenates were shifted by PCB pre-treatment towardsgreater production of the polar metabolite aflatoxin M₁ (AFL-M₁)and glucuronide conjugates. The major metabolite in bile ofPCBs fish was the glucuronide of aflatoxi-col M₁ (AFL-M₁), whichwas enhanced 15-fold over controls. Levels of aflatoxicol (AFL)glucuronide, the major conjugate in controls, were unalteredby PCBs. The pattern of AFB1 metabolism in isolated bepatocytesfrom PCB-prefed fish was consistent with in vivo metabolism.AFB₁-DNA adduct formation in a 1 h assay was similar in hepatocytesfrom PCB-fed and control fish. However, the total rate of AFB₁metabolism was significantly elevated in hepatocytes from PCB-fedfish such that the degree of AFB₁-DNA adduct formed per unitAFB₁ metabolized was 42% lower than control. Similarly, adductformation in vivo during the first 24 h post-AFB₁ injectionin PCB fish was not significantly different from controls. However,over a longer 21 day period, adduct levels in PCB fish wereonly 48–69% of controls (P < 0.005, analysis of variance),once peak adduct formation was reached. Thus, initial ratesof adduct formation may be misleading in the absence of furtherinformation on rates of carcinogen metabolism in vitro and/orpharmacokinetics of peak adduct formation in vivo. These resultsindicate that PCB inhibition of AFB₁ carcinogenesis in troutinvolves dramatic initial changes in carcinogen distribution,metabolism and elimination which, over time, results in a netreduction of DNA adduct formation.     

Induction of lung and liver tumors by flouranthene in a preweanling CD-1 mouse bioassay

Fluoranthene (FA), a major enviromental pollutant, induced lungand liver tumors 6–9 months after intraperitoneal injectionof 0.7, 1.75 and 3.5 mg FA into preweanling CD-1 mice. Therewas a dose-dependent increase in lung tumors with a maximumtumor incidence of nearly 45% and a maximum tumor multiplicityof 0.6–0.7 lung tumors/mouse. No significant differencewas noted in lung tumors in the 6 and 9 month bioassays, althoughfewer tumors were consistently noted in mice treated with thetwo lowest doses of FA. Indices of lung tumor incidence (ED₅₀)and multiplicity (TM_1.0) were similar for the two bioassaysand ranged from 18.9–19.5 and 26.2–27.2 µmolrespectively. Male mice had up to two times more lung tumorsthan females but these results were not statistically significant.Liver tumors (nodular hyperplasia) appeared only in FA-treatedmales but no dose-response relationship was evident. However,liver tumors were observed in only 0–10% of the male micein the 6 month treatment groups, but in 20–60% of themales in the 9 month groups. Because the CD-1 preweanling mouseresponded to the weak lung tumorigen FA, it is a viable, limited-termbioassay for measuring tumorigenicity of PAH and combustionemissions.     

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).