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.     

The effects of vitamin C and vitamin E on oxidative DNA damage: results from a randomized controlled trial.

Oxidative DNA damage may be important in mutagenic, carcinogenic, and aging processes. Although it is plausible that antioxidant vitamins may reduce oxidative DNA damage, evidence from human studies has been sparse and inconsistent. We determined the short-term effects of vitamin C (500 mg/day) and vitamin E (400 IU d-alpha-tocopheryl acetate/day) supplements on oxidative DNA damage in a double-masked, placebo-controlled, 2x2 factorial trial in 184 nonsmoking adults. Mean duration of supplementation was 2 months. Oxidative DNA damage was measured by 24-h urinary excretion of 8-hydroxy-2'-deoxyguanosine (8-OHdG). At baseline, urinary 8-OHdG (mean +/- SE; ng/mg creatinine) was associated with race (15.6 +/- 0.8 in African Americans versus 20.3 +/- 1.2 in Caucasians, P = 0.001), prior antioxidant supplement use (18.6 +/- 0.8 in users versus 13.8 +/- 1.5 in non-users, P = 0.007), and regular exercise (19.2 +/- 1.1 in exercisers versus 16.6 +/- 0.9 in non-exercisers, P = 0.04). Fruit and vegetable intake and serum ascorbic acid were inversely associated with urinary 8-OHdG (P-trend = 0.02 and 0.016, respectively). The benefits of fruit and vegetable intake became evident with the consumption being at least three servings/day. At the end of supplementation, change from baseline in urinary 8-OHdG (mean +/- SE; ng/mg creatinine) was -0.6 +/- 1.4 (P = 0.61), 0.6 +/- 1.1 (P = 0.59), 0.5 +/- 1.0 (P = 0.61), and 1.6 +/- 1.4 (P = 0.27) in the placebo, vitamin C alone, vitamin E alone, and combined vitamins C and E groups, respectively. In overall and subgroup analyses, there was no significant main effect or interaction effect of the supplements on urinary 8-OHdG. In conclusion, supplementation of diet with vitamin C (500 mg/day) and vitamin E (400 IU d-alpha-tocopheryl acetate/day) had no significant main effect or interaction effect on oxidative DNA damage as measured by urinary 8-OHdG in nonsmoking adults. However, several aspects of a healthy lifestyle were associated with lower oxidative DNA damage.     

Increased 8-oxodeoxyguanosine levels in lung DNA of A/J mice and F344 rats treated with the tobacco-specific nitrosamine 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone.

Evidence for the involvement of free radicals in nitrosamine carcinogenesis comes mainly from increased lipid peroxidation as a result of nitrosamine treatment. More direct evidence for nitrosamine-induced oxidative DNA damage has been lacking. In this study we examined the levels of 8-oxodeoxyguanosine or 8-hydroxydeoxyguanosine (8-OH-dG) in tissue DNA of mice and rats treated with the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Multiple doses of NNK (0.25 or 0.50 mg/mouse, 3 times weekly for 3 weeks) administered by gavage resulted in a significant elevation of 8-OH-dG in lung DNA, from 2.1 to 3.8 adducts/10(5) dG for the lower dose or to 6.6 adducts/10(5) dG for the higher dose, 2 h after the last NNK administration. A single dose treatment of NNK by gavage (4 mg/mouse) also resulted in an increase of this lesion in the lung DNA, however, the increase was not statistically significant. In liver, however, the increase was only significant by multiple doses at the higher dose, from 2.3 to 3.4 adducts/10(5) dG. This lesion appeared to be repaired efficiently. At 4 and 24 h after NNK treatment, the 8-OH-dG levels declined to the basal levels in both liver and lung. A single dose of NNK (20 mg/rat) also caused a significant increase of 8-OH-dG from 3.0 to 5.1 adducts/10(5) dG in rat lung DNA. An increase of 8-OH-dG in liver DNA was also seen, however, it was not statistically significant. Unlike the liver and the lung, the 8-OH-dG levels in rat kidney, a non-target tissue, were inert to NNK treatment. These results provide for the first time direct evidence supporting the role of oxidative DNA damage in NNK lung tumorigenesis.     

Inhibition of UVB induced DNA photodamage in mouse epidermis by topically applied alpha-tocopherol

Ultraviolet B (UVB, 290-320 nm) exposure results in a variety of cellular insults including induction of cyclobutane pyrimidine dimers in DNA. Accumulation of these lesions can lead to mutations in critical genes and contribute to the development of nonmelanoma skin cancer. Topically applied alpha-tocopherol (vitamin E) has previously been shown to prevent the induction of skin tumors in UVB irradiated female C3H/HeNTac mice. We hypothesized that alpha-tocopherol, which absorbs strongly in the UVB, may act as a sunscreen to prevent photodamage. To explore possible mechanisms of photoprotection, we topically applied alpha-tocopherol dispersed in a neutral cream vehicle to the dorsal epidermis of female C3H/HeNTac mice and exposed them to 2.5 J/m2/s of UVB for 60 min. Immediately after exposure, we analyzed thymine dimer levels in DNA by capillary gas chromatography with electron capture detection. Epidermal DNA from mice receiving this UVB dose contained 247 +/- 42 pmol thymine dimers/micromol thymine. Topical application of alpha-tocopherol inhibited dimer formation in a dose-dependent manner. A 1% alpha-tocopherol dispersion inhibited the formation of thymine dimers to 43% of levels in vehicle controls. Several vitamin E compounds, including alpha-tocopherol acetate, alpha-tocopherol methyl ether, gamma-tocopherol, and delta-tocopherol also inhibited thymine dimer formation, but were five- to ten-fold less potent than alpha- tocopherol. A variety of commercially available sunscreens were also less potent than alpha-tocopherol in their ability to reduce dimer formation. These results suggest that DNA photoprotection is an important mechanism by which topically applied alpha-tocopherol can inhibit UVB induced skin cancer. Alpha-Tocopherol acetate, the most common form of vitamin E in commercial skin care products, conferred less protection, perhaps due to its lower absorptivity in the UVB. Our results further underscore the importance of determining which forms of vitamin E can inhibit specific lesions involved in photocarcinogenesis.      

Radioprotection by vitamin E: injectable vitamin E administered alone or with WR-3689 enhances survival of irradiated mice.

Radioprotection by injectable vitamin E (alpha-tocopherol) was investigated in mice exposed to 60Co radiation (0.2 Gy/min). Vitamin E injected subcutaneously either 1 hr before or within 15 min after irradiation significantly increased 30-day postirradiation survival in CD2F1 male mice. A dose reduction factor (DRF) of 1.11 (95% confidence interval [1.08, 1.14]) was observed for vitamin E at a dose of 100 IU/kg body weight administered within 15 min after irradiation. Combination studies with the phosphorothioate WR-3689 (S-2([3-methylaminopropyl]amino)ethylphosphorothioic acid) were undertaken to determine whether radioprotection by WR-3689 could be enhanced by vitamin E. Mice were given WR-3689 (150-225 mg/kg, intraperitoneally) 30 min before irradiation and were given vitamin E (100 IU/kg) either 1 hr before or within 15 min after irradiation. Survival was significantly increased in mice given vitamin E and WR-3689 before irradiation as compared to mice given WR-3689 alone: the DRF for WR-3689 (150 mg) was 1.35 [1.32, 1.38]; for WR-3689 combined with vitamin E (100 IU), the DRF was 1.49 [1.45, 1.53].     

Effects of vitamin E and selenium supplementation on esophageal adenocarcinogenesis in a surgical model with rats

Two well-known antioxidative nutrients, vitamin E and selenium, were used in this study to investigate possible inhibitory action against the formation of esophageal adenocarcinoma (EAC) in rats. In this model, carcinogenesis is believed to be driven by oxidative stress. Male Sprague-Dawley rats (8 weeks old) were divided into four groups and received esophagoduodenal anastomosis (EDA) surgery plus iron supplementation (12 mg/kg/week). Vitamin E and selenium were supplemented in the diet in the forms of alpha-tocopheryl acetate (750 IU/kg) and sodium selenate (1.7 mg Se/kg), which were 10 times the regular amounts in the basic AIN93M diet. At 40 weeks after surgery, all the EDA groups had lower body weights than the non-operated control group. Iron nutrition (hemoglobin, total serum iron and transferrin saturation) was normal as a result of iron supplementation after EDA. Vitamin E supplementation maintained the normal plasma level of alpha-tocopherol in EDA rats, but not those of gamma-tocopherol and retinol. Selenium supplementation increased the serum and liver selenium contents of the EDA rats. Histopathological analysis showed that selenium supplementation increased the incidence of EAC and the tumor volume. The selenium level in the tumor is higher than that in the duodenum of the same animal.Vitamin E supplementation, however, inhibited carcinogenesis, especially in the selenium-supplemented group. We believe that vitamin E exerts its effect through its antioxidative properties, and a high dose of inorganic selenium may promote carcinogenesis by enhancing oxidative stress.     

Protective effects of vitamin A against the genotoxicity of NNK, a nicotine-derived N-nitrosamine

In this investigation, the effect of vitamin A on 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induced genotoxic damage in rat liver was studied and correlated with NNK metabolism and DNA single strand breaks (DNA-SSBs). Male adult Fischer rats were fed a vitamin A deficient diet for 8 weeks and then divided into two sub-groups. Rats in group Ia and group Ib were fed vitamin A supplemented and vitamin A deficient diets respectively for an additional 4 weeks. Rats were then treated with a single i.p. injection of NNK (25-100 mg/kg) and subjected to partial hepatectomy 1 week later. After 72 h, liver perfusions were performed for cell preparation. Non-hepatectomized rats were used as controls (group IIa and IIb, consisting of rats fed vitamin A supplemented and deficient diets respectively). In non-hepatectomized rats (groups IIa and IIb), micronucleus (MN) frequencies were not significantly increased. This confirms the importance of cell proliferation for MN formation. In rats fed a vitamin A deficient diet and subjected to partial hepatectomy (group Ib), a significant increase of MN was observed (2.2 +/- 0.3, 5.7 +/- 0.9, 12.8 +/- 3.8 and 21.2 +/- 4.3% for control, 25, 50 and 100 mg NNK/kg body wt respectively). In rats fed a vitamin A supplemented diet (group Ia) we have observed a significant decrease of MN induction, as compared to rats fed a vitamin A deficient diet (group Ib) (2.7 +/- 1.0, 2.9 +/- 0.9, 9.7 +/- 2.7, 14.8 +/- 3.2% for control, 25, 50 and 100 mg NNK/kg body wt respectively). Freshly isolated hepatocytes from rats fed vitamin A deficient and supplemented diets but without NNK treatment in vivo were used to study NNK metabolism and DNA-SSBs. Results showed that hepatocytes isolated from rats fed a vitamin A supplemented diet (group C) metabolized NNK less effectively than hepatocytes isolated from rats fed a vitamin A deficient diet (group B) (NNK metabolism was decreased by 0.5-fold in group C as compared to group B). This inhibition was observed only with the NNK activation pathway, alpha-carbon hydroxylation, but not with a deactivation pathway: pyridine-N-oxidation. DNA-SSBs induced by NNK were also reduced in hepatocytes isolated from rats fed a vitamin A supplemented diet as compared to hepatocytes isolated from rats fed a deficient diet. These reductions were 7.8, 12.4 and 4.5% after 6 h of elution and 9.1, 8.5 and 3.0% after 9 h of elution for 1, 5 and 10 mM NNK respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Elevated 8-hydroxy-2'-deoxyguanosine levels in lung DNA of A/J mice and F344 rats treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and inhibition by dietary 1,4-phenylenebis(methylene)selenocyanate

1,4-Phenylenebis(methylene)selenocyanate (p-XSC) is an effective chemopreventive agent against 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK)-induced lung adenoma in female A/J mice. While p-XSC can effectively inhibit NNK-induced DNA methylation in female A/J mice and in male F344 rats, its effect on NNK-induced oxidative DNA damage had not been determined. Thus, the effect of p-XSC on the levels of 8- hydroxy-2'-deoxyguanosine (8-OH-dG) in lung DNA from A/J mice and F344 rats treated with NNK was examined. Mice were given NNK by gavage (0.5 mg/mouse in 0.2 ml corn oil, three times per week for 3 weeks) or by a single i.p. injection (2 mg/mouse in 0.1 ml saline) while maintained on a control diet (AIN-76A) or control diet containing p-XSC at 10 or 15 p.p.m. (as Se) starting 1 week before NNK administration and continuing until termination. Mice were killed 2 h after the last NNK gavage in the multiple administration protocol or 2 h after the single i.p. injection. Treatment with NNK by gavage significantly elevated the levels of 8-OH-dG in lung DNA of A/J mice from 0.7 +/- 0.1 to 1.6 +/- 0.2 adducts/10(5) 2'-deoxyguanosine (dG) (P < 0.001), while dietary p- XSC (at 10 p.p.m. Se) prevented significant elevation of the levels of this lesion caused by NNK, keeping them at 0.9 +/- 0.1 adducts/10(5) dG (P < 0.003). Injection of NNK in saline also significantly increased the levels of 8-OH-dG in lung DNA of A/J mice from 1.2 +/- 0.6 to 3.6 +/- 0.8/10(5) dG adducts (P < 0.01), while dietary p-XSC (at 15 p.p.m. Se) kept these levels at 1.9 +/- 0.5 adducts/10(5) dG (P < 0.03). Rats were given a single i.p. injection of NNK (100 mg/kg body wt) in saline while being maintained on control diet (AIN-76A) or control diet containing p-XSC (15 p.p.m. as Se) starting 1 week before NNK administration and continuing until termination. The rats were killed 2 h after injection. Treatment with NNK using this protocol significantly elevated the levels of 8-OH-dG in lung DNA of F344 rats from 2.6 +/- 0.5 to 3.5 +/- 0.5 adducts/10(5) dG (P < 0.03), while dietary p-XSC (at 15 p.p.m. Se) kept the levels of this lesion at 2.2 +/- 0.6 adducts/10(5) dG (P < 0.01). Our findings suggest that the chemopreventive efficacy of p-XSC against NNK-induced lung tumorigenesis in A/J mice and F344 rats may be due in part to inhibition of oxidative DNA damage.      

Dose-dependent effects of dietary alpha- and gamma-tocopherols on genetic instability in mouse Mutatect tumors

Vitamin E in foodstuffs is a mixture of tocopherols. In mouse Mutatect tumors, a model designed to detect DNA mutations, the hypoxanthine phosphoribosyltransferase (Hprt) gene mutation frequency is associated with the number of tumor-infiltrating neutrophils and both are markedly decreased in mice fed high levels of alpha-tocopherol. Dietary alpha-tocopherol is also associated with a decrease in neutrophil-associated loss of an interleukin 8 (IL-8)-expressing transgene in this tumor model. We examined Hprt gene mutation frequency (expressed as the number of 6-thioguanine-resistant colonies per 10(5) clonable tumor cells), IL-8 transgene loss, and myeloperoxidase activity (an indirect measure of neutrophil number) in tumors from Mutatect mice fed diets supplemented with various concentrations of D-alpha-tocopherol acetate and/or D-gamma-tocopherol acetate or neither tocopherol for 4 weeks. Hprt gene mutation frequency and myeloperoxidase activity were statistically significantly lower in tumor cells from mice fed alpha-tocopherol at 50 or 100 mg/kg body weight per day than in tumor cells from mice fed 0 mg/kg body weight per day alpha-tocopherol (P<.001 for each comparison). IL-8 transgene loss occurred in 28 of 28 tumors (100%; 95% confidence interval [CI] = 86% to 100%) from mice fed alpha-tocopherol at 50 mg or less/kg body weight per day and seven of 18 tumors (39%; 95% CI = 24% to 54%) from mice fed 100 mg/kg body weight per day (P<.001, Fisher's exact test, referent groups [pooled] 0, 25, and 50 mg/kg). gamma-Tocopherol had no detectable effect on any of the three endpoints. Thus, dietary alpha-tocopherol decreases two forms of genetic instability in a dose-dependent manner in this experimental tumor model.     

Oxidative DNA damage estimated by 8-hydroxydeoxyguanosine excretion in humans: influence of smoking, gender and body mass index.

Oxidative DNA damage may be implicated in ageing, carcinogenesis and other degenerative diseases. Oxidative DNA damage can be assessed in humans in vivo from the urinary excretion of the DNA-repair product 8-hydroxydeoxyguanosine (8OHdG). We investigated factors influencing the excretion of 8OHdG in 24 h urine from 83 randomly selected healthy subjects (52 women) aged 40-64 years. For 2 weeks prior to urine collection the subjects kept a weighed diet record. 8OHdG was quantified by an automatic three-dimensional HPLC analysis with electrochemical detection. The 8OHdG excretion was 252 +/- 103 (mean +/- SD) pmol kg body weight/24 h with a range from 78 to 527. Multiple regression analysis identified three factors, smoking, body mass index (BMI) and gender, as significant predictors of the 8OHdG excretion. In 30 smokers the 8OHdG excretion was 320 +/- 99 pmol/kg/24 h opposed to 213 +/- 84 pmol/kg/24 h in 53 non-smokers. According to multiple regression analysis smokers excreted 50% (31-69%; 95% confidence interval) more 8OHdG than non-smokers. In 52 women the 8OHdG excretion was 240 +/- 106 pmol/kg/24 h opposed to 271 +/- 96 pmol/kg/24 h in 31 men. According to the multiple regression analysis men excreted 29% (10-48%) more 8OHdG than women. According to multiple regression analysis the 8OHdG excretion decreased with 4% (2-6%) per increment in BMI measured in kg/m2. The dietary distribution of energy demonstrated no important predictive value with respect to 8OHdG excretion. The intake of the antioxidant vitamins C and E and of vitamin A equivalents, including beta-carotene, was not associated with 8OHdG excretion. The results suggest that smoking increases oxidative DNA damage by approximately 50%. This effect implies potential serious health effects adding to the other well-known health hazards of smoking. The higher 8OHdG excretion in men and lean subjects may be related to a higher rate of metabolism with increased availability of reactive oxygen species. The apparent 7-fold individual variation in oxidative DNA damage carries implications regarding the rate of ageing and the risk of cancer and other degenerative diseases. The excretion of 8OHdG into urine offers a valuable tool for testing such hypotheses in humans.     

The Protective Role of Glutathione, Cysteine and Vitamin C against Oxidative DNA Damage Induced in Rat Kidney by Potassium Bromate

The roles of glutathione (GSH), cysteine, vitamin C., liposome-encapsulated superoxide dismutase (L-SOD) and vitamin E in preventing oxidative DNA damage and cytotoxicity in the rat kidney after administration of potassium bromate (KBrO₃) to male F344 rats were investigated by measuring 8-hydroxydeoxyguanosine (8-OH-dG), an oxidative DNA product, lipid peroxidation (LPO) levels and relative kidney weight (RKW). Combined pre- and posttreatment of animals with 2 × 800 mg/kg GSH i.p. inhibited the increase of 8-OH-dG, LPO levels and RKW caused by 80 mg/kg KBrO₃ i.p. administration. In contrast, pretreatment with 0.3 ml/kg diethylmaleate (DEM) i.p., a depletor of tissue GSH, was associated with elevation of 8-OH-dG, LPO levels and RKW after a 20 mg/kg KBrO₃ i.p. treatment, which itself caused no change. Administration of KBrO₃ itself reduced renal non-protein thiol levels, but this was inhibited by the two doses of exogenous GSH. Combined treatment with DEM and KBrO₃ lowered the non-protein thiol level in the kidney more than did DEM treatment alone. Protective effects against the oxidative damage caused by KBrO₃ were also observed for pre- and posttreatment with 400 mg/kg cysteine i.p., another sulfhydryl compound, and daily i.g. application of 200 mg/kg vitamin C for 5 days. However, no influence was evident after pre- and posttreatment with 18,000 U/kg L-SOD i.p. or daily i.g. 100 mg/kg of vitamin E for 5 days. The results suggest that intracellular GSH plays an essential protective role against renal oxidative DNA damage and nephrotoxicity caused by KBrO₃.     

The protective role of glutathione, cysteine and vitamin C against oxidative DNA damage induced in rat kidney by potassium bromate.

The roles of glutathione (GSH), cysteine, vitamin C, liposome-encapsulated superoxide dismutase (L-SOD) and vitamin E in preventing oxidative DNA damage and cytotoxicity in the rat kidney after administration of potassium bromate (KBrO3) to male F344 rats were investigated by measuring 8-hydroxydeoxyguanosine (8-OH-dG), an oxidative DNA product, lipid peroxidation (LPO) levels and relative kidney weight (RKW). Combined pre- and posttreatment of animals with 2 x 800 mg/kg GSH i.p. inhibited the increase of 8-OH-dG, LPO levels and RKW caused by 80 mg/kg KBrO3 i.p. administration. In contrast, pretreatment with 0.3 ml/kg diethylmaleate (DEM) i.p., a depletor of tissue GSH, was associated with elevation of 8-OH-dG, LPO levels and RKW after a 20 mg/kg KBrO3 i.p. treatment, which itself caused no change. Administration of KBrO3 itself reduced renal non-protein thiol levels, but this was inhibited by the two doses of exogenous GSH. Combined treatment with DEM and KBrO3 lowered the non-protein thiol level in the kidney more than did DEM treatment alone. Protective effects against the oxidative damage caused by KBrO3 were also observed for pre- and posttreatment with 400 mg/kg cysteine i.p., another sulfhydryl compound, and daily i.g. application of 200 mg/kg vitamin C for 5 days. However, no influence was evident after pre- and posttreatment with 18,000 U/kg L-SOD i.p. or daily i.g. 100 mg/kg of vitamin E for 5 days. The results suggest that intracellular GSH plays an essential protective role against renal oxidative DNA damage and nephrotoxicity caused by KBrO3.     

Effect of two years' supplementation with natural antioxidants on vitamin and trace element status biomarkers: preliminary data of the SU.VI.MAX study.

The "SUpplementation en VItamines et Minéraux AntioXidants" (SU.VI.MAX) study is a randomized double-blind, placebo controlled, primary-prevention trial designed to test the efficacy of a daily supplementation with antioxidant vitamins (vitamin C, 120 mg; vitamin E, 30 mg; and beta-carotene, 6 mg) and minerals (selenium, 100 microg; and zinc, 20 mg) at nutritional doses (one to three times the daily recommended dietary allowances), in reducing the frequency of cancers and cardiovascular diseases. The study involves 12,735 eligible subjects (women aged 35-60 years, men aged 45-60 years) included in 1994 in France. They will be followed up for 8 years. The targeted population is the general population. The aim of this specific analysis is to assess the effect of 2 years of supplementation on biochemical indicators of vitamin and trace element on a subsample of 1000 subjects. The mean (+/- standard deviation) concentrations of plasma beta-carotene, alpha-tocopherol, vitamin C, selenium and zinc among participants who were randomly assigned to receive a daily supplementation with beta-carotene, vitamin E, vitamin C, selenium and zinc for 2 years were significantly higher than those who were assigned to receive placebo. Specifically, the mean concentrations among men in the intervention group were 0.86 +/- 0.70 micromol/L for beta-carotene, 35.3 +/- 9.3 micromol/L for alpha-tocopherol, 11.5 +/- 4.7 microg/ mL for vitamin C, 1.65 +/- 0.33 micromol/L for selenium, and 16.2 +/- 3.9 micromol/L for zinc. The mean concentrations among women in the intervention were 1.25 +/- 0.90 micromol/L for beta-carotene, 34.9 +/- 8.4 micromol/L for alpha-tocopherol, 12.6 +/- 4.0 microg/mL for vitamin C, 1.68 +/- 0.37 micromol/L for selenium, and 15.3 +/- 3.9 micromol/L for zinc. The values observed for beta-carotene and vitamin E in the supplementation group after 2 years of intervention are those that have been associated with the lowest risk of cancer in observational studies. They are definitely lower than concentrations reported in intervention studies showing an apparent negative effect of high levels of beta-carotene supplementation on the lung cancer incidence rate in high-risk subjects (initial level multiplied by 12-18). Data from the follow-up will ascertain if any plausible reduction in the incidence rate of cancers may be associated with such amounts of antioxidant agents.     

Formation of 8-hydroxy-2'-deoxyguanosine in the DNA of cultured human keratinocytes by clinically used doses of narrowband and broadband ultraviolet B and psoralen plus ultraviolet A

Psoralen plus ultraviolet A (PUVA) and narrowband ultraviolet B (UVB) are widely used in skin disease phototherapy. Recently, the efficacy of UVB therapy has been greatly improved by narrowband UVB, compared to conventional broadband UVB. The objectives of the current study were to evaluate the influence of UVB-induced and PUVA-induced oxidative stress on cultured keratinocytes. We analyzed 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in human keratinocytes (HaCaT cell line) using a high-performance liquid chromatography system equipped with an electrochemical detector. Non-irradiated human keratinocytes contained a baseline of 1.48 +/- 0.22 (mean +/- SD) 8-OH-dG per 10(6) deoxyguanosine (dG) residues in cellular DNA, which increased linearly with higher doses of UVB. When their abilities to induce 8-OH-dG were compared to each other, based on the minimal erythemal and therapeutically used doses, by irradiating them with broadband UVB at 100 mJ/cm(2), the amount of 8-OH-dG increased to 3.42 +/- 0.46 residues per 10(6) dG, while a narrowband UVB treatment at 1000 mJ/cm(2), with biological effects comparable to those elicited by 100 mJ/cm(2) broadband UVB, increased it to 2.06 +/- 0.31 residues per 10(6) dG. PUVA treatment, with 100 ng/mL 8-methoxypsoralen and 5000 mJ/cm(2) UVA, increased the 8-OH-dG level to 4.52 +/- 0.42 residues per 10(6) dG. When HaCaT cells treated with 2000 mJ/cm(2) narrowband UVB were cultured and the amount of 8-OH-dG was monitored in the living cells, 65.6% of the residues were repaired 24 h after treatment. Our study provides a warning that widely used narrowband UVB and PUVA induce cellular oxidative DNA damage at the therapeutically used doses, although to a lesser degree than broadband UVB with the same clinically effective dose.     

The effect of hOGG1 and glutathione peroxidase I genotypes and 3p chromosomal loss on 8-hydroxydeoxyguanosine levels in lung cancer

Polymorphic genes for the peroxide scavenger glutathione peroxidase I (GPX1) and 8-hydroxydeoxyguanosine (8-OHdG) DNA glycosylase/apurinic (AP) lyase (hOGG1) map to loci on chromosome 3p which are subject to frequent loss of heterozygosity (LOH) in lung tumours. Levels of the pro-mutagenic, oxidative DNA lesion 8-OHdG, were measured in 37 paired normal and tumorous lung specimens using HPLC with electrochemical detection. Lung tumours were also analysed for 3p LOH by fluorescent PCR with Genescan analysis. No significant difference was observed between 8-OHdG levels in tumour [7.7 +/- 6.7 (mean +/- SE) 8-OHdG/10(6) 2'-deoxyguanosine (dG)] and normal (8.1 +/- 8.8 8-OHdG/10(6) dG) lung tissue. Adduct levels in normal lung tissue DNA were not associated with constitutive hOGG1 genotype although there was a trend towards lower 8-OHdG levels in individuals possessing the ALA6 GPX1 polymorphism. Lung tumours exhibiting 3p LOH (40%) contained higher levels of 8-OHdG adducts (10.9 +/- 2.6 8-OHdG/10(6) dG) (P = 0.05) and lower GPX1 enzyme activity [45.5 nmol glutathione (GSH)/min/mg] (P = 0.09) when compared with tumours without LOH at these sites (5.55 +/- 0.87 8-OHdG/10(6) dG and 63.6 nmol GSH/min/mg, respectively). In conclusion, tumours with 3p LOH at loci associated with hOGG1 and GPX1 appear to have compromised oxidative defence mechanisms as measured by reduced GPX1 enzyme activity and elevated 8-OHdG levels and this may affect the prognosis of lung cancer patients.     

Products of oxidative DNA damage and repair as possible biomarkers of susceptibility to lung cancer

The broad spectrum of oxidative DNA damage biomarkers [urinary excretion of 8-hydroxy-2'-deoxyguanosine (8-OH-dGuo) and 8-hydroxyguanine (8-OH-Gua)] and the level of oxidative DNA damage and repair in leukocytes DNA were analyzed in three groups of subjects: (a) lung cancer patients [all smokers (n = 51)]; (b) healthy smokers with comparable smoking status (n = 26); and (c) healthy nonsmokers (n = 38). The mean level of 8-OH-Gua in urine samples of 38 healthy nonsmokers reached a value of 1.783 +/- 0.785 nmol/day/kg. This level was significantly lower than that in the urine of the two smoker groups (cancer patients and healthy smokers), in whom the levels reached values of 2.319 +/- 1.271 and 2.824 +/- 0.892 nmol/day/kg, respectively. Urinary excretion of 8-OH-dGuo was similar in all groups of subjects. The level of 8-OH-dGuo in DNA isolated from leukocytes of cancer patients was significantly higher than that in DNA isolated from the group of healthy smokers and nonsmokers (9.44 +/- 4.77 versus 7.20 +/- 2.83 and 5.88 +/- 2.47 molecules/10(6) deoxyguanosine, respectively). Repair activity of 8-OH-Gua, as estimated by the nicking assay, was significantly higher in blood leukocytes of healthy volunteers (44.6 +/- 20.21 and 37.54 +/- 13.43 pmol/h/mg protein for smokers and nonsmokers, respectively) than in the leukocytes of lung cancer patients (24.56 +/- 11.28 pmol/h/mg protein). Because oxidative DNA insult represented by urinary excretion of oxidative DNA lesions was similar in both groups of subjects with similar smoking status, it appears likely that a higher rate of generation of oxidative damage in cellular DNA of lung cancer patients is a result of deficiency of the repair mechanism(s) in this group.     

The DNA damaging drug cyproterone acetate causes gene mutations and induces glutathione-S-transferase P in the liver of female Big Blue transgenic F344 rats [published erratum appears in Carcinogenesis 1998 Apr;19(4):707]

The gestagenic and antiandrogenic drug cyproterone acetate (CPA) is mitogenic, tumorigenic and induces DNA-adducts and DNA-repair synthesis in rat liver. Thus CPA is expected to be mutagenic. However in vitro mutagenicity test systems were negative. To examine whether CPA induces mutations in rat liver, the in vivo mutation assay based on Big Blue transgenic F344 rats was employed. Single oral doses of 25, 50, 75, 100 and 200 mg CPA/kg b.w. respectively were administered to female Big Blue rats. Six weeks after treatment, liver DNA was assayed for mutations. At the highest dose, 200 mg CPA/kg b.w., the frequency of (17 +/- 4) x 10(-6) spontaneous mutations was increased to a maximum of (80 +/- 8) x 10(-6) mutations. One-hundred and 75 mg CPA/kg b.w. resulted in mutation frequencies of (35 +/- 5) and (27 +/- 5) x 10(-6), respectively. The mutation frequency at doses of 50 and 25 mg CPA/kg b.w. was similar to that of vehicle treated controls. Statistical analysis of the dose-effect relationship revealed that it was not possible to decide whether a threshold dose exists or not. DNA adducts were analyzed by the 32P-postlabelling technique. The total level of the major and the two minor adducts observed in the autoradiograms increased between doses of 25 to 75 mg CPA/kg b.w. to a maximum of approximately 12,000 +/- 3000 adducts per 10(9) nucleotides. The level did not further increase significantly with 100 and 200 mg CPA/kg b.w. After CPA treatment no preneoplastic liver foci were observed. However, single glutathione-S-transferase placental form (GST-P) positive hepatocytes were observed and the frequency was dependent on the dose. These cells are not supposed to represent initiated cells, since they occurred only transiently after 6 weeks and disappeared thereafter completely. In conclusion, our results demonstrate that CPA is mutagenic in vivo. The mutation frequency increased at high CPA doses, when the increase of the DNA adduct formation had already ceased. This suggests that the mitogenic activity of CPA is required to express the mutations.      

Contribution of antioxidants to preventive effect of mesna in cyclophosphamide-induced hemorrhagic cystitis in rats

Purpose The aim of this study was to evaluate whether combination of antioxidants and mesna may prevent cystitis induced by cyclophosphamide better than mesna alone.Materials and methods A total of 46 male Spraque-Dawley rats were divided into six groups. Five groups received single dose of cyclophosphamide (CP, 100 mg/kg) intraperitoneally with the same time intervals: group 2 received CP only, group 3 received mesna (21.5 mg/kg for three times), group 4 beta-carotene (20 mg/kg for two times) and mesna, group 5 received alpha-tocopherol (20 mg/kg for two times) and mesna, and group 6 received melatonin (5 mg/kg for two times) and mesna on the day of CP injection. Group 1 served as control.Results CP injection resulted in severe cystitis. Mesna has showed meaningful but not full protection against CP toxicity. Although beta-carotene did not show any additional beneficial effect when combined with mesna, alpha-tocopherol and especially melatonin with mesna resulted full protection that the pathologist, blinded to the slides, could not differ from sham control.Conclusion Oxidants may be important in the pathogenesis of CP-induced cystitis. Melatonin and alpha-tocopherol may help to ameliorate bladder damage along with other drugs such as mesna and diuretics.     

Preclinical pharmacologic evaluation of bleomycin A6

Bleomycin A6 is an antineoplastic antibiotic. Toxicologic study showed that the LD50 values of bleomycin A6 in mice were 92 +/- 7.4 mg/kg (IV), 72 +/- 5.6 mg/kg (IM), 82 +/- 4.5 mg/kg (SC), 102 +/- 0.6 mg/kg (IP) and 520 +/- 0.1 mg/kg (PO), respectively. Wistar rats were divided into 6 groups. Doses of 20, 10, 5, 2 and 0.5 mg/kg/day were given intraperitoneally every other day for 60 days. Renal damage to various degrees was found in rats with doses higher than 2 mg/kg. However, no pathologic changes were observed in the liver, heart, lungs, spleen, stomach, intestines, pancreas or brain. Three groups of dogs (3 in each group) were injected i.v. every other day for 60 days with a dose of 1.0, 0.5 and 0.1 mg/kg, respectively. The three dogs receiving 1.0 mg/kg died between the 4th and 7th week. Dogs treated with 1.0 and 0.5 mg/kg had renal damage and skin ulceration. With a dose of 0.1 mg/kg, dogs showed good tolerance and no renal damage was found. Multiple doses of bleomycin A6 injected into rabbit muscles caused no obvious local changes. After IV injection into rabbits, the maximal serum concentration of bleomycin A6 was 18.5 micrograms/ml, having a half life of 2.8 hours and 7.7% of the drug were excreted in the urine within 12 hours.     

Promutagenic methylation damage in bladder DNA from patients with bladder cancer associated with schistosomiasis and from normal individuals.

Radioimmunoassays (RIAs) have been used to detect the promutagenic lesion O6-methyldeoxyguanosine (O6-MedG) in DNA isolated from the bladder tissues of Egyptian patients presenting with bladder carcinoma and concomitant schistosomiasis (bilharziasis), a parasitic disease known to be associated with the presence of N-nitrosamines in the urine. Alkylation damage was present in the DNA of the majority of the samples (44/46, 96%); 38 samples were of tumour tissue and 8 from uninvolved bladder mucosa. Levels of O6-MedG ranged from undetectable (ND; i.e. less than 0.01 mumol O6-MedG/mol dG) to 0.485 mumol/mol dG with an overall mean of 0.134 +/- 0.10 mumol/mol dG, including the two samples that were below the limit of detection. In contrast, methylation damage was detected in only 4/12 (33%) of the DNA samples from normal bladder tissue of European origin. In these samples levels of O6-MedG ranged from ND to 0.225 mumol/mol dG with an overall mean of 0.046 +/- 0.082 mumol O6-MedG/mol dG. These results confirm that alkylation events can be detected in the DNA of schistosome-infected human bladder tissue. The methylation of uninvolved and tumour tissue DNA to similar extents suggests that the alkylating intermediate may have been present in the urine. These results indicate the need for further investigation to determine whether relationships exist between levels of DNA damage and the prevalence of schistosome infection and/or the extent and type of bacterial infection that frequently accompanies this disease.