Peroxidative in vitro metabolism of diethylstilbestrol induces formation of 8-hydroxy-2'-deoxyguanosine

The generation of superoxide and hydroxyl radicals is knownto be implicated in the hydroxylation of 2'-deoxyguanosine (dG)at the C-8 position and of guanine base residues in DNA. Itwas also shown previously that in the presence of horseradishperoxidase, hydrogen peroxide and Fe³⁺-EDTA complex, diethylstilbestrol(DES) induces single strand breaks in DNA, caused by the productionof superoxide anion (O^–) and hydroxyl (OH) radicals. Bymeans of high-pressure liquid chromatography and electrochemicaldetection a strong indication is adduced that dG is oxidizedto 8-hydroxy- 2'-deoxyguanosine during peroxidative in vitrometabolism of DES, which might be at the basis of DES inducedcell transformation.     

Dimethylarsinic acid induces 8-hydroxy-2'-deoxyguanosine formation in the kidney of NCI-Black-Reiter rats

Dirnethylarsenic peroxyl radical [(CH(3))(2)AsOO] has been postulated to be responsible for DNA damage induced by dimethylarsinic acid (DMA). In an effort to elucidate the possible mechanism of tumor-inducing potential of DMA, an experiment was designed to investigate the formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a specific marker of oxidative base damage in the kidney tissues of NCI-Black Reiter (NBR) rats. Animals were divided into four groups and administered the vehicle - saline, 5, 10 and 20 mg/kg body weight respectively of DMA by gavage, once a day, 5 days a week, for a period of 4 weeks. DMA induced increase of 8-OHdG levels in the kidney of the rats treated, with the highest level at the dose of 10 mg/kg body weight. Analysis of the kidney for cell proliferation employing PCNA-positive index showed greater proliferation in the tissues of treated rats. However, DMA did not have any influence on apoptosis in this regimen. Histopathological examination of the kidney selections revealed the presence of vacuolated degeneration and dilation of the proximal tubule cells in two groups (10 and 20 mg/kg body weight). This study provides evidence to substantiate the role of DMA in inducing oxidative DNA damage in the kidney.     

5-Aminolevulinic acid mediates the in vivo and in vitro formation of 8-hydroxy-2'-deoxyguanosine in DNA

5-Aminolevulinic acid (ALA), a heme precursor accumulated inchemical and inborn porphyrias, may behave as an endogenouspro-oxidant In chronically treated rats (40 mg ALA/kg body wtevery 2 days for 15 days) the steady-state level of 8-hydroxy-2'-deoxyguanosine(8-OHdG) in liver DNA (94.5 ± 233 residues/106 dG) was4.5 times higher than in non-treated rats (21 ± 7.5 residues/10⁶ dG). In vitro exposure of calf thymus DNA to ALA (0.05-5mM) in the presence of 10 uM Fe²+ caused the formation of 8-OHdG.The amount of 8-OHdG rose from 135 ± 15 residues/10⁶dG in the control system to 1140 ± 150 residues/106 dGafter incubation with 5 mM ALA and 10 µM Fe²1. Diethylenetriaminepentaaceticacid (5 mM) or mannitol (100 mM) inhibited the formation of8-OHdG by 63 and 69% respectively, evidencing the involvementof both H2O2 and HO in this process. Hydrogen peroxide (100µM) or Fe²+ alone did not cause DNA oxidation. The presentdata support the hypothesis that ALA-generated reactive oxygenspecies can oxidize DNA and may be involved in the developmentof primary liver cell carcinoma in individuals with symptomaticacute intermittent porphyria.     

Quantitative immunoanalysis of promutagenic 8-hydroxy-2'-deoxyguanosine in oxidized DNA

The modified DNA base 8-hydroxyguanine has been implicated inspontaneous mutagenesis, carcinogenesis and cellular aging.Polyclonal antibodies specific for the 8-hydroxy-2'-deoxyguanosinemoiety in oxidized DNA were used for sensitive detection andquantitation of this biomarker of oxidative damage to cellularDNA. The analysis was performed with immunoslot blot assay (ISB)of oxidized DNA modified in vitro with methylene blue plus lightand upon H₂O₂ treatment of cultured human cells. The level of8-OHdG in DNA exposed to 90 and 120 min light in the presenceof 100 µM methylene blue showed 15.96 ± 2.4 and22.65 ± 3.65 pmol/µg DNA compared to 0.107 ±0.024 pmol/µg in commercial calf thymus DNA control. Inherentdamage, due to cellular endogenous oxidation of DNA, increasedsignificantly upon inhibition of catalase activity in humancells with 10 mM azide. The damage increased further on exposureof azide-treated cells to H₂O₂. The amounts of 8-OHdG followingtreatment of cells with 10 and 100 µM H₂O₂ were determinedto be 205 ± 42 and 333 ± 17.5 pmol/µg DNArespectively. Very low but quantifiable antibody binding wasseen with the ‘control unoxidized’ human nuclearDNA. This DNA, obtained under controlled conditions to restrictthe induction of 8-OHdG during isolation, provides a backgroundlevel of 0.022 ± 0.005 pmol 8-OHdG/µg DNA. Thequantitative assessment of 8-OHdG by ISB assay, with fmol sensitivityand direct analysis using unhydrolyzed DNA, should prove a highlyvaluable alternative to currently used approaches to detecting8-OHdG in enzymatic DNA hydrolysates.     

Radiation-induced formation of 8-hydroxy-2'-deoxyguanosine and its prevention by scavengers

Reactive oxygen species (ROS) induce 8–hydroxy–2'-deoxyguanosine(8-OHdG) formation, which has been proposed as a key biomarkerrelevant to carcinogenesis. 8-OHdG has been induced in a numberof different ways, most often without knowledge of the specifictype and amount of ROS generated. We have measured 8-OHdG formationin calf thymus DNA exposed to ionizing radiation under conditionsgenerating either hydroxyl radicals (OH·), superoxideanions (O2^–) or both. Additionally, we investigated therelationship between the scavenger effect of the drug 5-aminosalicylicacid (5-ASA) and increasing OH· exposure toward 8-OHdGformation. The effect of this drug was compared to those ofthe physiological scavengers ascorbate and reduced glutathione(GSH). We found that OH· generated 8-OHdG in a dose-dependentmanner, whereas O₂^– did not cause 8-OHdG formation. 5-ASA,ascorbate and GSH all acted as hydroxyl radical scavengers,although with different concentration-effect curves, emphasizingthe importance of using relevant pharmaco-/physiological concentrationsin studies focusing on therapeutic applications of scavengers.The scavenger effect of 5-ASA at concentrations     

Formation of DNA adduct 8-hydroxy-2'-deoxyguanosine induced by man-made mineral fibres

Two man-made mineral fibres, rockwool and glasswool, were found to mediate hydroxylation of deoxyguanosine and calf thymus DNA to form the DNA adduct 8-hydroxy-2'-deoxyguanosine. The modification of the nucleoside is probably mediated by hydroxyl radicals and may play a role in fibre-induced carcinogenesis.     

In vivo accumulation of 8-hydroxy-2'-deoxyguanosine in DNA correlates with release of reactive oxygen species in Fanconi's anaemia families

The present study was aimed at verifying the occurrence, ifany, of in vivo oxidative DNA damage in FA homozygotes, theirparents and siblings. 8-Hydroxy-2'-deoxyguanosine (8-OHdG) wasmeasured, by HPLC/EC, in DNA from circulating blood leucocytesfrom FA homozygotes and their relatives and compared with agroup of paediatric and adult healthy subjects. The populationstudied consisted of: (i) 15 FA homozygotes; (ii) 24 FA heterozygotes;(iii) 11 siblings. The 8-OHdG level in FA homozygotes was significantlyhigher with respect to age-matched controls, with a mean levelof 33.3 ± 6.8 (mean ± SE) and 3.9 ± 0.268-OHdG/10⁵ dG respectively. The FA parents (heterozygotes) alsodisplayed higher 8-OHdG levels relative to controls. The releaseof hydroxyl ('OH) and 'OH-like radicals from leucocytes wasdetermined by luminol-dependent chemiluminescence (LDCL) ina subgroup of FA homo- and heterozygotes, showing a very largein vivo formation of non-superoxide radicals. Chromosomal instabilitywas also measured in the FA population. When relating either8-OHdG or LDCL levels to spontaneous or diepoxybutane-inducedchromosomal instability (S-CI and DEB-CI respectively), a significantcorrelation was observed between the 8-OHdG, LDCL and S-CI data.Within families a positive association was found between 8-OHdGlevels in homozygotes and their related heterozygotes, suggestingsegregation of the genetic defect(s) underlying the abnormaloxidative metabolism. The present study provides evidence foran in vivo pro-oxidant state in FA, in terms of excess formationof 'OH and 'OH-like radicals, and of DNA hydroxyl adducts. Thisfinding appears to be shared by homozygotes and, to a lesserextent, by heterozygotes.     

Sensitive detection of 8-hydroxy-2'-deoxyguanosine in DNA by 32P-postlabeling assay and the basal levels in rat tissues

Oxidative damage from reactive oxygen species including freeradicals has been considered to play a vital role in many degenerativediseases and measurement of 8-hydroxy-2'-deoxyguanosine (Oh⁸dG)in tissue DNA has been used as a benchmark for oxidative DNAdamage. We report here an ultrasensitive ³²P-postlabeling methodto detect and quantitate Oh⁸dG in DNA and have determined basallevels of Oh⁸dG in rat tissues. The method is comprised of DNAdigestion to 3'-monophosphates, 5'-³²P-labeling, conversionto 5'-monophosphates and separation by a 2-directional PEI-cellulose TLC (Dl = 1.5 M formic acid; and D2 = 0.6 M ammoniumformate, pH 6.0). Under these conditions, all radioactive contaminantswere either removed from the chromatogram (normal nucleotidesand ³²Pi) or remained at the origin (ATP and other contaminants),while Oh⁸dG migrated in the middle of the chromatogram. Calfthymus DNA incubated with ascorbic acid and H₂O₂ produced predominantlyone spot under the chromatography conditions used; a chromatographicallyidentical spot was also detected in untreated DNA, but at amuch lower level (125 ± 40 Oh⁸dG/10⁶ nucleotides). Achromatographically identical spot was also found in dGp incubatedwith ascorbic acid and H₂O₂, but not with dAp, dCp or dTp. Whenapplied to rat tissue DNA, the assay readily permitted detectionof Oh⁸dG in the liver, lung, kidney, heart, brain, spleen, intestinesand mammary epithelial cells of 3-month old female Sprague-Dawleyrats. The tissue Oh⁸dG levels were found in the range of 87± 29 to 133 ± 49 per 10⁶ nucleotides, with liverand heart being the highest and kidney and brain the lowestThese values are in the vicinity to those found by gas chromatography/massspectrometry but 10–50 times higher than those reportedby HPLC-electrochemical detection. Because of its high sensitivity(<1 Oh⁸dG per 10^5–6 nucleotides) to detect Oh⁸dG usingnanogram quantity of DNA digest, the ³²P-postlabeling methodis likely to be valuable in quantitating Oh⁸dG in human tissuebiopsies.     

Formation and persistence of DNA adducts from the carcinogen N-hydroxy-2-acetylaminofluorene in rat mammary gland in vivo

The rat mammary carcinogen, N-hydroxy-2-acetylamino-fluorene(N-hydroxy-2-AAF), has been proposed to be metabolically activatedby mammary cytosolic N,O-acetyltransferase to a DNA bindingspecies. To test this hypothesis, adult female Sprague-Dawleyderived CD rats were treated, l.p., with 4.0 mg/kg [ring-³H]N-hydroxy-2-AAF.After 4 h, 1, 3, 14, and 28 days, the animals were killed, themammary epithelium DNA was isolated and the carcinogen-deoxyribonucleosideadducts present were analyzed by high pressure liquid chromatography.At each time, only one adduct was detected and it was chromatographicallyidentical to N-(deoxyguanosin-8-yl)-2-aminofluorene. The levelof the adduct was maximal at 4 h (1.5 adducts/10⁶ nucleotides)and then decreased, following first order kinetics with a t_1/2of 14.2 days. The detection of a single non-acetylated aminofluoreneadduct is consistent with N,O-acyltransferase being involvedin the metabolic activation of N-hydroxy-2-AAF in the rat mammarygland.     

Formation of reactive oxygen species and of 8-hydroxy-2'-deoxyguanosine in DNA in vitro with betel-quid ingredients

Using a chemiluminescence technique, superoxide anion (O2-.) and H2O2 were shown to be formed in vitro, above pH 9.5, from betel-quid (BQ) ingredients, such as areca-nut extract and catechu. The formation of O2-. was enhanced by Fe2+, Fe3+ and Cu2+ and inhibited by Mn2+. Saliva was found to inhibit both O2-. and H2O2 formation from BQ ingredients. Upon incubation of DNA at alkaline pH with areca-nut extract or catechu, in the presence or absence of Fe3+, 8-hydroxy-2'-deoxyguanosine was formed, as quantified by high-performance liquid chromatography. The data suggest a possible role of reactive oxygen species (ROS) in the etiology of oral cancer in betel quid chewers.     

Dietary hydrogen peroxide enhances hepatocarcinogenesis in trout: correlation with 8-hydroxy-2'-deoxyguanosine levels in liver DNA.

The tumor-enhancing effect of hydrogen peroxide (H2O2) in N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-initiated rainbow trout hepatocarcinogenesis was investigated and correlated with the levels of the mutagenic DNA adduct 8-hydroxy-2'-deoxyguanosine (oh8dG). In addition, the protective role of vitamin E was examined in relation to tumor enhancement and oh8dG levels in liver DNA. Trout were fed diets containing two levels of vitamin E (1000 or 20 mg/kg wet wt), each of which were made up to contain three levels of H2O2 (0, 600 or 3000 p.p.m.). Dietary vitamin E levels had no significant effect on tumor incidence or levels of oh8dG in liver DNA. On the other hand, dietary H2O2 enhanced liver tumors in a dose-dependent manner. Liver tumor incidence correlated significantly with the mean level of liver DNA oh8dG content (r = 0.87). We conclude that the H2O2 tumor-enhancing effect coincides with higher levels of oh8dG in the trout liver genome. Thus, rainbow trout may be a useful model for the study of the relationship of oh8dG levels in vivo to enhancement or promotion of carcinogenesis and its modulation by dietary enhancers and inhibitors of oxidative stress.     

Spontaneous and 2-nitropropane induced levels of 8-hydroxy-2'-deoxyguanosine in liver DNA of rats fed iron-deficient or manganese- and copper-deficient diets

Rats (Wistar, female, 4 weeks old) were fed iron-deficient (Fe^–;2.2 µg Fe/g) or manganese- and copper-deficient (Mn.Cu^–;0.3 µg Mn/g, 0.4 ug Cu/g) diets for 8 weeks to determinethe oxidative damage of DNA by element deficiency. After feedingof the diets, 2-nitropropane (2-NP, 80 mg/kg body weight) wasadministered i. p. as an inducer of 8-hydroxy-2'-deoxyguanosine(8-OH-dG) to the element-deficient rats. The hemoglobin concentrationof rats in the Fe- group showed an induction of severe anemia(8.4 g/100 ml whole blood). In the Mn.Cu^– group, Mn-super-oxidedismutase (SOD) activities of plasma and Cu.Zn-SOD activitieswere significantly lower than that of the normal diet group.However, total SOD activities of plasma were not depressed severelyin contrast to that of the liver in the Mn.Cu^– group.Background (spontaneous) levels of 8-OH-dG in normal diet groupwere 0.96 + 0.37/105 deoxyguanosine (dG), however, significantlyhigher levels were detected in the Fe^– group (1.56±0.19,P < 0.01). Conversely, a lower (but not significant) levelof 8-OH-dG than the normal diet group were detected in the Mn.Cu^–group (0.78 ±0.08). Six hours after 2-NP treatment,8-OH-dG levels in liver DNA were significantly induced to 1.44+ 0.24 in the normal diet fed group 1.89±0.22 in theFe^– and 1.08±0.12 in the Mn.Cu^– groups. Comparedto the normal diet group, these induced levels of 8-OH-dG inthe Fe-group were significantly higher (P < 0.05), and thatin Mn. Cu^– group were significantly lower (P < 0.05).The high levels of 8-OH-dG in severe iron deficiency might bethe results of (i) an increase of hydroxyl radical generationby accumulated copper in hepatocytes; or (ii) a depression ofenzymatic activity for removing 8-hydroxy-2' -deoxyguanosinein DNA, which is dependent on divalent cations. On the otherhand, the low level of 8-OH-dG in manganese and copper deficiencymight be the result of a decrease of lipid peroxidation whichhas been suggested to be an intermediator from active oxygenspecies to hydroxyl radical.     

DNA adduct 8-hydroxyl-2'-deoxyguanosine (8-hydroxyguanine) affects function of human DNA methyltransferase

8-Hydroxyl-2'-deoxyguanosine (also referred to as 8- hydroxyguanine[8-OH-dG] or 7,8-dihydro-8-oxoguanine), a common DNA adductresulting from injury to DNA via reactive oxygen species, affectsthe in vitro methylation of nearby cytosine moieties by thehuman DNA methyltransferase. The exact position of 8-OH-deoxyguanosinerelative to a CpG dinucleotide appears important to this effect.Our data indicate that 8-OH-deoxyguanosine diminishes the abilityof the methyltransferase to methylate a target cytosine whenthe 8-OH-deoxyguanosine is one or two nucleotides 3' from thecytosine, on the same strand On the other hand 8-OH-deoxyguanosinedoes not diminish the ability of the enzyme to respond to amethyl director (5-methylcytosine) when the 8-OH-deoxyguanosineis on the same strand but one or two nucleotides 3' from themethyl director. Differences in methylation rates as great as13-fold have been detected using various 8-OH-deoxyguanosine-containingoligonucleotides as substrates in methylation assays. Our findingssuggest that oxidative damage of parental strand guanines wouldpermit normal copying of methylation patterns through maintenancemethylation, while oxidative damage of guanines in the nascentstrand DNA would inhibit such methylation.     

Urinary 8-hydroxy-2'-deoxyguanosine excretion as a biomarker for estimating DNA oxidation in patients undergoing external radiotherapy and/or brachytherapy

Urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) is considered to be a biomarker of cellular oxidative stress and to be associated with carcinogenesis. To estimate the oxidative stress caused by radiotherapy, we used the enzyme-linked immunosorbent assay (ELISA) to monitor urinary 8-OHdG levels in 72 patients undergoing radiotherapy. Subjects consisted of 23 breast cancer patients undergoing post-operative external radiotherapy for breast conserving therapy, 12 tongue and seven prostate cancer patients undergoing interstitial radiotherapy, 19 esophageal cancer patients undergoing external radiotherapy and chemotherapy, and 11 cervical cancer patients undergoing external radiotherapy and brachytherapy with or without chemotherapy. Before radiotherapy, cervical cancer patients showed a higher urinary 8-OHdG level (16.0+/-8.8 ng/mg) than breast cancer patients (5.3+/-5.5 ng/mg, p=0.001). In the other three groups, urinary 8-OHdG levels were in the medium range (prostate cancer patients: 6.6+/-6.3 ng/mg; esophageal cancer patients: 8.8+/-11.1 ng/mg; tongue cancer patients: 10.2+/-6.7 ng/mg). Radiotherapy did not cause changes in the excretion level of urinary 8-OHdG in patients with breast, esophageal and tongue cancer. However, radiotherapy reduced 8-OHdG excretion levels in patients with cervical cancer, whereas interstitial radiotherapy transiently increased these levels in patients with prostate cancer.     

2-Nitropropane induces DNA repair synthesis in rat hepatocytes in vitro and in vivo

The genotoxicity of 2-nitropropane (2-NP) and 1-nitropropane(1-NP) was investigated by measuring the induction of DNA repairsynthesis in rat liver cells in vitro and in vivo. 2-NP stronglyinduced DNA repair synthesis in both cases. When applied invivo, 2-NP was considerably more effective in hepatocytes frommales than in those from females. 1-NP was not active in vitroor in vivo. 2-NP and 1-NP did not induce repair in cell linesof extrahepatic origin derived from rat, mouse, hamster andman. The results are consistent with the reported carcinogenicityof 2-NP in rat liver and suggest that the formation of hepatocarcinomasby 2-NP is due to the generation of a genotoxic metabolite from2-NP by liver-specific metabolism.     

[32P]ATP mediates formation of 8-hydroxy-2'-deoxyguanosine from 2'- deoxyguanosine, a possible problem in the 32P-postlabeling assay

8-Hydroxy-2'-deoxyguanosine (8-OH-dG) is a biomarker for oxidative stress on DNA, a common lesion in mammalian cells. A correlation between increased levels of 8-OH-dG and diseases like diabetes, infections and cystic fibrosis has been found in humans. 8-OH-dG levels have been shown to be decreased by antioxidants, an indication of the importance of dietary habits. 8-OH-dG is used as a biomarker for oxidative stress in vivo as well as in vitro and is suggested to be a mutagenic DNA lesion. Different methods are used for the analyses of 8- OH-dG, i.e. GC-MS, HPLC-EC and 32P-postlabeling. The most commonly used method is HPLC-EC. In the analysis of 8-OH-dG, the work-up procedure for DNA, as well as the preparation for analysis, are of critical importance as there is a risk for auto-oxidation of deoxyguanosine (dG), which would result in false high background levels and low sensitivity in analysis. 32P-Postlabeling has recently been applied to the analysis of 8-OH-dG and has shown to be a very sensitive method for the detection of DNA adducts. It is shown here that after extrapolation to normal 32P-postlabeling conditions, [32P]ATP generated 8-OH-dG to levels of 25 8-OH-dG/10(5) dG. [32P]ATP mediated the formation of 8-OH- dG from dG in a dose-dependent manner at all dose levels (0.13-12 microCi). The reaction occurred immediately and increased with time in a linear dose-response fashion. At high doses (6.0 and 12 microCi) the dose-response declined after 24 h, which indicates a possible decomposition or rearrangement of 8-OH-dG. A repeated experiment with 5 microCi [32P]ATP during 2 h resulted in a linear formation of 8-OH-dG and a level of 19 8-OH-dG/10(5) dG. The results indicated that awareness of the auto-oxidation generated by 32P[ATP] in the postlabeling assay is of utmost importance and that dG must be separated before 32P-postlabeling of 8-OH-dG.      

Estrous cycle modification of rat mammary gland DNA alkylation by N-methyl-N-nitrosourea

Virgin Sprague-Dawley rats exhibiting regular estrous cycles were used as a model system to determine whether the level of circulating estrogen modifies the alkylation pattern of mammary gland DNA by a direct-acting carcinogen, N-methyl-N-nitrosourea (NMU). The concentration of 7-methylguanine and O6-methylguanine were similar in mammary epithelial DNA 0.25, 0.50, and 1.0 h after i.v. injection of 50 mg/kg body weight NMU on different days of the rat estrous cycle. However, O6-methylguanine was significantly higher in mammary gland DNA 8 and 24 h after a single i.v. dose of carcinogen on proestrus or estrus, compared to rats receiving carcinogen on diestrus. There was no difference in the 7-methylguanine levels at 8 h in any group, but this adduct was higher in estrous-treated rats at 24 h. The ratio of O6-methylguanine to 7-methylguanine was significantly lower at 8 h in mammary gland DNA from diestrous-injected rats, and this difference reflected the lower level of O6-methylguanine adducts in this group. In contrast, O6-methylguanine concentrations in DNA extracted from the liver of the same animals were virtually identical at all time periods examined. 7-Methylguanine levels were higher in the liver at 0.5, 1, 8, and 24 h post-NMU in proestrus as compared with diestrous-injected rats. The observed adduct clearance suggests that rat mammary epithelium may contain repair systems capable of removing O6-methylguanine. These results also suggest that the initial removal of the O6-methylguanine lesions in mammary epithelial DNA (rather than the initial rate of alkylation) is affected by the hormonal environment during carcinogen exposure. This effect may be tissue specific since removal of O6-methylguanine from liver DNA is apparently not altered by the stage of the estrous cycle at which NMU is administered.     

beta-Ionone suppresses mammary carcinogenesis, proliferative activity and induces apoptosis in the mammary gland of the Sprague-Dawley rat

beta-Ionone demonstrates potent anticancer activity both in vitro and in vivo. We determined tumor incidence and the number of rats bearing tumors as well as cell proliferation and apoptosis in a rat mammary cancer model induced by 7, 12-dimethylbenz[a]anthracene (DMBA). Rats were fed an AIN-76A diet containing beta-ionone (0, 9, 18 or 36 mmol/kg), starting 2 weeks before DMBA administration and continuing for 24 weeks. A dose-dependent inhibition of mammary carcinogenesis by dietary beta-ionone was observed. Corresponding tumor incidence values were 82.1, 53.3, 25.9 and 10.0% (p < 0.01 or 0.05). Time to tumor appearance increased and tumor multiplicity decreased with increasing dietary beta-ionone. Histopathological and immunohistochemical evaluations of tumors were performed on the 64, 31, 15 and 3 tumors, respectively, identified in rats from the respective groups of 30. The proportions of adenocarcinomas, adenomas and benign masses were equally distributed in the latter group. In proportions within the other groups, the proportions of adenocarcinomas and benign masses decreased and increased with increasing dietary beta-ionone. Proliferating cell nuclear antigen (PCNA), cyclin D1 and Bcl-2 expression decreased, and Bax expression and nuclear fragmentation increased with increasing dietary beta-ionone. These results demonstrate the potent capacity of dietary beta-ionone to suppress DMBA-initiated mammary cancer in rats.     

8-hydroxy-2'-deoxyguanosine in cervical cells: correlation with grade of dysplasia and human papillomavirus infection

In this study, the 8-hydroxy-2'-deoxyguanosine (8-OHdG) level was assessed in human cervical cells by an immunoperoxidase method and was related to the presence of human papillomavirus (HPV) infection and precancerous lesions. After optimizing the immunohistochemical method of detecting oxidative DNA damage in whole cells, we have used this technique to estimate the oxidative damage in cervical cells collected during a routine PAP test. The analysis of variance (ANOVA) of the data from human samples showed significant differences in the 8-OHdG content among normal, low-grade and high-grade squamous intraepithelial lesion (SIL, HGSIL and LGSIL, respectively; P < 0.001). In the comparison of the three groups, statistically significant differences were detected between normal SIL and HGSIL (P < 0.001) and between LGSIL and HGSIL (P = 0.003), whereas no statistically significant difference was found between normal SIL and LGSIL (P = 0.1). Grouping observations by HPV status, no significant difference was detected in 8-OHdG levels between HPV(+) and HPV(-) subjects (P = 0.8). The polytomous and proportional odds models, extensions of the logistic regression analysis, showed that the effect of 8-OHdG levels in rising the risk of dysplasia was roughly constant through SIL grades. In conclusion, the immunoperoxidase method, applied to single human cervical cells, provides clear evidence that significant differences exist in 8-OHdG content between normal and dysplastic cells and that oxidative DNA damage might play an important role in cervical carcinogenesis.     

Identification and in vivo formation of 32P-postlabeled rat mammary DMBA-DNA adducts

The in vivo formation of 32P-postlabeled mammary 7,12-dimethylbenz[a]anthracene (DMBA)-DNA adducts was evaluated for female Sprague-Dawley rats following administration of DMBA (i.g.) at 1, 3, 5, 10 and 20 mg/rat. Adduct formation was also measured as a function of time following DMBA intubation. At least eight adducts were formed in vivo in mammary epithelial cells. The identities of four of these nucleoside bisphosphate adduct spots were determined by cross-referencing with previously characterized 3H-labeled nucleoside DMBA adducts. These identified adducts constitute four of the five major adducts formed in vivo. Two adducts were identified as the anti-dihydrodiolepoxide of DMBA reacted with deoxyguanosine (dGuo). Two other major adducts were derived from the syn-dihydrodiolepoxide and bound to dGuo and to deoxyadenosine (dAdo). Total DMBA-DNA binding increased at all DMBA doses investigated (r = 0.94). Total binding values were (mean +/- SEM) 39.3 +/- 6.1, 158.0 +/- 16.9, 194.7 +/- 9.9, 326.9 +/- 21.5 and 443.2 +/- 20.8 nmol DMBA/mol DNA for rats administered DMBA at 1, 3, 5, 10 and 20 mg/rat respectively. The anti-dGuo adduct predominated at all doses and times evaluated, contributing to approximately 52% of total binding. The occurrence of anti-derived adducts was greater than that of syn-derived adducts. Binding of DMBA to dGuo substantially exceeded binding to dAdo. The 32P-postlabeling procedure represents a sensitive technique for detecting specific DMBA-DNA adducts formed in vivo in the rat mammary gland.