No association between alcohol supplementation and autoantibodies to DNA damage in postmenopausal women in a controlled feeding study.

Alcohol consumption is linked to increased breast cancer risk. Since oestrogens increase breast cancer risk, possibly through oxidative damage, and we have shown that alcohol consumption increases serum oestrogens, we tested whether moderate alcohol supplementation increased oxidative DNA damage among healthy postmenopausal women not on hormone replacement therapy in a randomized controlled crossover study. We used serum 5-hydroxymethyl-2-deoxyuridine (5-HMdU) autoantibodies (aAbs) as a marker of oxidative DNA damage. The results showed no evidence for increased or decreased levels of oxidative DNA damage among women who consumed 15 g or 30 g alcohol per day for 8 weeks compared with women in the 0 g alcohol group. We conclude that among healthy women, it is possible that an 8-week trial of moderate alcohol supplementation might be too short to make enough 5-HMdU aAbs to compare differences by alcohol dose. In future studies, a panel of biomarkers for DNA damage should be used.

Influence of postmenopausal hormone replacement therapy on an estrogen metabolite biomarker of risk for breast cancer.

Whether postmenopausal hormone-replacement therapy (HRT) increases the risk of breast cancer remains controversial, despite numerous epidemiological studies. We approached the question from a biochemical rather than an epidemiological direction - we hypothesized that if estrogen administration increases the risk of breast cancer, it should also alter a known estrogen biomarker of risk towards what has been observed in patients who already have breast cancer. The specific biomarker we studied was the ratio of the urinary excretion of two principal estradiol metabolites, 2-hydroxyestrone and 16 alpha-hydroxyestrone, which is markedly decreased in women with breast cancer and women with familial risk for breast cancer. We studied 34 healthy postmenopausal women not on HRT and 19 women on HRT (Premarin 0.625 mg daily plus Provera, 2.5 mg daily, in women with a uterus and Premarin alone in women without a uterus); treatment duration ranged from 3 months to 15 years. We also studied four women with recently diagnosed, untreated breast cancer. The women with breast cancer showed a significantly lower 2-hydroxyestrone to 16 alpha-hydroxyestrone ratio than control women on HRT (1.35 +/- 0.13 vs. 2.71 +/- 0.84; p < 0.0001). There was no significant difference in the metabolite ratio between healthy women on HRT and women not on HRT (2.82 +/- 0.92 vs. 2.71 +/- 0.84). There was no significant difference between women receiving Premarin alone and women receiving Premarin plus Provera (2.46 +/- 0.84 vs. 3.13 +/- 0.90), and neither differed significantly from women not on HRT (2.71 +/- 0.84). The finding that the ratio of women on HRT was not decreased to or toward the ratio in women with breast cancer can be interpreted, we believe, as a suggestive item of biochemical evidence that HRT is not a risk for breast cancer.

Importance of complete DNA digestion in minimizing variability of 8-oxo-dG analyses.

Estimates of 8-oxo-2'-deoxyguanosine (8-oxo-dG) in DNA vary at least one order of magnitude using different quantitative methods or even the same method. Our hypothesis is that an incomplete DNA hydrolysis to nucleosides by the conventional nuclease P1 (NP1) and alkaline phosphatase (AP) digestion system plays an important role in contributing to the variability of measurements using HPLC coupled with UV and electrochemical (EC) detection. We show here that factors, such as the amount of DNA, choice of enzymes, their activities, and incubation time, can affect DNA digestion and, thus, cause variability in 8-oxo-dG levels. The addition of DNase I and phosphodiesterases I and II to the NP1 + AP system improves the DNA digestion by completely releasing normal nucleosides and 8-oxo-dG, thereby reducing the interday variations of 8-oxo-dG levels. Diethylenetriamine pentaacetic acid (DTPA), an iron chelator, prevented background increases of 8-oxo-dG during DNA digestion, as well as during the waiting period in the autosampler when a batch of DNA samples is analyzed by HPLC. After optimization of the DNA digestion conditions, the interday variability of 8-oxo-dG measurements using commercially available salmon testes DNA (ST DNA) were 26% over a period of 2 years. Under these optimal conditions, our laboratory variability may contribute as little as 13% to the overall variability as shown by assessment of oxidative DNA damage in a population of smokers. Based on our results, we believe that the modified DNA digestion conditions will provide much more accurate 8-oxo-dG determinations and, thus, more reliable estimates of cancer risk.

Association of tocopherols with circulating autoantibody levels against an oxidized DNA nucleoside in humans.

Autoantibodies against oxidized DNA bases are found in vivo and have been used as an indicator of oxidative damage, yet little is known concerning their individual variation and relation to serum micronutrients. Human plasma anti-5-hydroxymethyl-2'-deoxyuridine (HMdU) autoantibody (aAb) levels were repeatedly determined in 41 women and 11 men, and found to have small within-individual variation over time, but large between-individual differences. A positive association in both women (r = .5762, p = .0001) and men (r = .415, p = .2) between plasma total tocopherols and antibody levels was observed. Autoantibody levels were lower in postmenopausal women (8.37 +/- 1.61 vs. 17.18 +/- 2.85 in premenopausal women, p < .01), independently of plasma tocopherol. However, aAb titers in postmenopausal women were still significantly associated with plasma tocopherol levels and adjustment for menopausal status in women yielded a highly significant correlation between HMdU aAb levels and total tocopherol (r = .7342, p = .0001). Plasma malondialdehyde equivalents (MDA), a measure of lipid peroxidation, were also higher in individuals with either high plasma alpha-tocopherol or high beta+gamma-tocopherol levels. The positive association of tocopherols with markers of oxidative damage may reflect a response to the generation of endogenous oxidants associated with enhanced immune function. The decrease in aAb level in postmenopausal women may similarly reflect decreased immune function associated with decreased estrogen levels.

Gender differences in autoantibodies to oxidative DNA base damage in cigarette smokers.

Oxidative DNA damage and antibodies to that damage have been implicated in lung, breast, and colorectal cancer. In this observational validation study, the relationship between anti-5-hydroxymethyl-2'-deoxyuridine (HMdU) autoantibody (aAb) and plasma micronutrients was assessed in 140 heavy smokers by ELISA. Anti-HMdU aAbs were 50% higher in women after adjustment for cigarettes/day (CPD; P = 0.002), although men smoked more and had higher plasma cotinine levels. The women reported taking more vitamin C (P < 0.005) and had higher plasma levels of alpha-carotene and beta-carotene (P < 0.001) and cryptoxanthin (P < 0.01) than men. Neither CPD nor cotinine was associated with aAb titers. Anti-HMdU aAbs were associated inversely with alpha-tocopherol (P = 0.10), retinol (P = 0.06), and age (P = 0.04) in women but not in men. In contrast to the men, women 50 years of age (P = 0.05). Given the same duration of exposure, women had higher anti-HMdU aAbs and also reached peak levels at a lower cumulative smoking exposure (30 years) compared with male smokers (40 years). Subjects smoked an average of 28.9 +/- 0.81 CPD and initiated smoking at 17.2 +/- 0.33 (SE) years of age. Therefore, smokers who reported smoking for 30 years were typically <50 years old. Women

Estrogens as endogenous genotoxic agents--DNA adducts and mutations.

Estrogens induce tumors in laboratory animals and have been associated with breast and uterine cancers in humans. In relation to the role of estrogens in the induction of cancer, we examine formation of DNA adducts by reactive electrophilic estrogen metabolites, formation of reactive oxygen species by estrogens and the resulting indirect DNA damage by these oxidants, and, finally, genomic and gene mutations induced by estrogens. Quinone intermediates derived by oxidation of the catechol estrogens 4-hydroxyestradiol or 4-hydroxyestrone may react with purine bases of DNA to form depurinating adducts that generate highly mutagenic apurinic sites. In contrast, quinones of 2-hydroxylated estrogens produce less harmful, stable DNA adducts. The catechol estrogen metabolites may also generate potentially mutagenic oxygen radicals by metabolic redox cycling or other mechanisms. Several types of indirect DNA damage are caused by estrogen-induced oxidants, such as oxidized DNA bases, DNA strand breakage, and adduct formation by reactive aldehydes derived from lipid hydroperoxides. Estradiol and the synthetic estrogen diethylstilbestrol also induce numerical and structural chromosomal aberrations and several types of gene mutations in cells in culture and in vivo. In conclusion, estrogens, including the natural hormones estradiol and estrone, must be considered genotoxic carcinogens on the basis of the evidence outlined in this chapter.

The decomposition of peroxynitrite to nitroxyl anion (NO-) and singlet oxygen in aqueous solution.

The mechanism of decomposition of peroxynitrite (OONO(-)) in aqueous sodium phosphate buffer solution at neutral pH was investigated. The OONO(-) was synthesized by directly reacting nitric oxide with superoxide anion at pH 13. The hypothesis was explored that OONO(-), after protonation at pH 7.0 to HOONO, decomposes into (1)O(2) and HNO according to a spin-conserved unimolecular mechanism. Small aliquots of the concentrated alkaline OONO(-) solution were added to a buffer solution (final pH 7.0-7.2), and the formation of (1)O(2) and NO(-) in high yields was observed. The (1)O(2) generated was trapped as the transannular peroxide (DPAO(2)) of 9, 10-diphenylanthracene (DPA) dissolved in carbon tetrachloride. The nitroxyl anion (NO-) formed from HNO (pKa 4.5) was trapped as nitrosylhemoglobin (HbNO) in an aqueous methemoglobin (MetHb) solution. In the presence of 25 mM sodium bicarbonate, which is known to accelerate the rate of decomposition of OONO(-), the amount of singlet oxygen trapped was reduced by a factor of approximately 2 whereas the yield of trapping of NO(-) by methemoglobin remained unaffected. Because NO(3)(-) is known to be the ultimate decomposition product of OONO(-), these results suggest that the nitrate anion is not formed by a direct isomerization of OONO(-), but by an indirect route originating from NO(-).

Alpha-tocopherol dietary supplement decreases titers of antibody against 5-hydroxymethyl-2'-deoxyuridine (HMdU).

This study evaluated the effects of vitamin E (alpha-tocopherol) on oxidative DNA damage in a randomized double-blind Phase II chemoprevention trial. Oxidative DNA damage was measured by the level of auto-antibody (Ab) against 5-hydroxymethyl-2'-deoxyuridine (HMdU) in plasma. After the baseline screening, eligible subjects (n = 31; plasma samples from 28 subjects were available for this study) were randomized to receive 15, 60, or 200 mg of alpha-tocopherol per day for 28 days. Biomarkers were measured twice at baseline--on day 1 (visit 1) and day 3 (visit 2)--and twice after intervention--on day 17 (visit 3) and day 31 (visit 4). At baseline, there was a highly significant inverse correlation between anti-HMdU Ab titer and plasma vitamin E level (r = -0.53; P = 0.004; n = 28). Smoking did not affect baseline anti-HMdU Ab titer; however, anti-HMdU Ab titer levels at baseline were significantly lower in subjects with above-median (0.75 ounce/day) alcohol consumption (P = 0.008). No significant change in anti-HMdU Ab level occurred at either visit 3 or visit 4 for subjects on the lowest dose, 15 mg alpha-tocopherol per day. Subjects receiving 60 mg of alpha-tocopherol per day had a significant decrease in anti-HMdU Ab level at visits 3 and 4 compared with baseline (P = 0.049 and P = 0.02, respectively). However, subjects receiving the highest dose, 200 mg/day, had less consistent results: a significant decrease in anti-HMdU Ab level was seen at visit 4 (P = 0.04) but not at visit 3. Our results demonstrate an inverse relationship between alpha-tocopherol and anti-HMdU Abs in plasma; oxidative DNA damage can be modulated by short-term dietary supplementation of alpha-tocopherol in some subjects.

Effect of supplementation with chromium picolinate on antibody titers to 5-hydroxymethyl uracil.

Recent in vitro studies have shown that chromium (III) compounds such as chromium picolinate, a popular dietary supplement among people trying to lose weight, produce chromosome damage. We monitored levels of DNA damage in a chromium picolinate supplement trial by measuring antibodies titers to an oxidized DNA base, 5-hydroxymethyl-2'-deoxyuridine (HMdU), by enzyme-linked immunosorbent assays. Ten obese volunteer women completed a 8-week course of 400 micrograms chromium picolinate per day. In either absolute titers or percent of the baseline value, there were no changes in antibody titers at 4 or 8 weeks. The titers were very stable within individuals and those of one individual rarely crossed over others, which was reflected in an intraclass correlation coefficient of 0.99 (95% confidence interval: 0.96-1.00). There were no effects on glucose and lipid metabolism in this period. The results of this trial suggest that chromium (III) picolinate in a dose typically used for nutrient supplementation dose not increase oxidative DNA damage, as measured by anti-HMdU antibody levels.

Serum autoantibodies recognizing 5-hydroxymethyl-2'-deoxyuridine, an oxidized DNA base, as biomarkers of cancer risk in women.

Human sera contain anti-5-hydroxymethyl-2'-deoxyuridine (HMdU; an oxidized thymidine) autoantibodies (aAbs), which are significantly higher in chronic inflammatory diseases. The intent of this study was to establish whether anti-HMdU aAbs can serve as predictors of breast and colorectal cancer risk. Sera of 169 women were analyzed by ELISA. Women healthy at blood donation but who were diagnosed 0.5-6 years later with breast or colorectal cancer exhibited significantly increased anti-HMdU aAbs over the age-matched controls (P = 0.028 and P < 0.001, respectively). Subjects diagnosed with rectal cancer had the highest levels of anti-HMdU aAbs (44.80 +/- 11.50; n = 6) in comparison to colon (29.03 +/- 2.49; n = 33) and breast (35.86 +/- 8.55; n = 9) cancers. Individuals with benign breast disease also had elevated anti-HMdU aAb (35.12 +/- 8.77; n = 10), with a borderline statistical significance (P = 0.095), whereas those with benign gastrointestinal tract diseases had those titers (30.95 +/- 3.64; n = 8) significantly increased (P < 0.02). Anti-HMdU aAb levels in subjects with a family history of any cancer (23.57 +/- 2.86; n = 55) did not significantly differ from those of the controls (19.41 +/- 2.90; n = 48), but women with a family history of breast cancer (two primary relatives or one with a bilateral disease) showed increased levels (34.48 +/- 8.16; n = 8; P = 0.024). Ps for linear trend of age-adjusted odds ratios were 0.049 for breast and < 0.001 for colorectal cancers. Anti-HMdU aAb titers showed a remarkable stability over a period of 6 years, with a low (14%) intraindividual variance. Thus, elevated anti-HMdU aAb titers may be an early signal of cancer risk, because they were significantly increased in otherwise healthy women who had a family history of breast cancer; in those who had benign breast disease or benign gastrointestinal tract diseases; and, most importantly, in those who at 0.5-6 years after the initial blood donation developed breast or colorectal cancer.

Molecular markers of exposure to cadmium and nickel among alkaline battery workers.

T he goal of the study wasto evaluate the usefulness of metallothionein mRNA, anti-5- hydroxymethyl-2'-deoxyuridine antibodies titres (anti-HMdU Ab), and 8-hydroxydeoxyguanosine (8OHdG) in urine as markers of the biologically active dose after exposure to airborne cadmium and nickel in human studies. Exposed persons (n = 38) were chosen from workers involved in the production and assembly, chemistry, and maintenance departments of a nickel-cadmium battery factory in Poland. Controls (n = 52) were chosen from administration personnel at the factory. Biological samples from workers were collected twice: once in the summer, after a month of vacation, and again in the winter, after 3 months of regular working activity within the plant. Controls were recruited during the second phase of the study. When exposure groups were defined on the basis of ambient air cadmium measurements, we found a two-fold increase in mean metallothionein mRNA values in the highest exposure group (air cadmium above 1000 g m-3) and a positive correlation of metallothionein mRNA with blood cadmium levels (r = 0 46, p < 0 008). Future studies can be designed to investigate further the interand intra-subject component of the variability and the possibility of the existence of M T gene polymorphisms, determining different responses and susceptibilities to cadmium exposure. We did not find any difference in the mean values of anti-HMdU Ab titres and 8OHdG in urine in any of the exposure groups analysed. Nickel exposure appeared to have greater impact on anti-HMdU Ab titres than cadmium.

Low serum thiol levels predict shorter times-to-death among HIV-infected injecting drug users.

OBJECTIVES: To investigate whether serum thiol levels are altered by HIV disease, and whether low serum thiols predict time to death among HIV-infected injecting drug users (IDU). DESIGN: A cross-sectional study of serum thiol levels among 13 HIV-seronegative IDU, 116 HIV-seropositive IDU, and 17 HIV-seropositive IDU with a history of AIDS, and a cohort study of the 133 HIV-infected IDU who took part in the cross-sectional study. METHODS: Subjects were recruited from a methadone-maintenance treatment program during 1990-1991. Total serum thiols were determined spectrophotometrically at enrolment; low serum thiols were defined as those with an absorbance at 412 nm < or = 0.46. Deaths through 31 December 1993 were determined from the National Death Index (NDI). Twenty-six HIV-seropositive subjects died during follow up; death certificates, which were obtained for 23 subjects, indicated AIDS or HIV infection for 20. Product-limit estimation was used to calculate survival. Multivariate analyses employed Cox proportional-hazards regression. RESULTS: Analysis of cross-sectional data showed that serum thiols did not differ significantly among HIV-free subjects, HIV-infected subjects, and HIV-infected subjects with a history of AIDS. Cohort analysis, adjusted for age, revealed that persons with those with high serum thiols (relative hazard = 2.83; 95% confidence interval (CI), 1.15, 6.97); a significant interaction between low serum thiols and a history of AIDS was associated with a relative hazard of 5.65 (95% CI, 1.22-2.61). CONCLUSIONS: Among HIV-infected persons, low serum thiols, especially in concert with a history of AIDS, predict mortality risk. These findings support the hypothesis that oxidative stress is critical to the pathogenesis of HIV infection.

Inhibitory effects of topical application of low doses of curcumin on 12-O-tetradecanoylphorbol-13-acetate-induced tumor promotion and oxidized DNA bases in mouse epidermis.

The effects of topical applications of very low doses of curcumin (the major yellow pigment in turmeric and the Indian food curry) on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced oxidation of DNA bases in the epidermis and on tumor promotion in mouse skin were investigated. CD-1 mice were treated topically with 200 nmol of 7,12-dimethylbenz[a]anthracene followed one week later by 5 nmol of TPA alone or together with 1, 10, 100 or 3000 nmol of curcumin twice a week for 20 weeks. Curcumin-mediated effects on TPA-induced formation of the oxidized DNA base 5-hydroxymethyl-2'-deoxyuridine (HMdU) and tumor formation were determined. All dose levels of curcumin inhibited the mean values of TPA-induced HMdU formation in epidermal DNA (62-77% inhibition), but only the two highest doses of curcumin strongly inhibited TPA-induced tumor promotion (62-79% inhibition of tumors per mouse and tumor volume per mouse). In a second experiment, topical application of 20 or 100 nmol (but not 10 nmol) of curcumin together with 5 nmol TPA twice a week for 18 weeks markedly inhibited TPA-induced tumor promotion. Curcumin had a strong inhibitory effect on DNA and RNA synthesis (IC50 = 0.5-1 microM) in cultured HeLa cells, but there was little or no effect on protein synthesis.

Inhibitory effects of curcumin on tumorigenesis in mice.

Curcumin (diferuloylmethane), the naturally occurring yellow pigment in turmeric and curry, is isolated from the rhizomes of the plant Curcuma longa Linn. Curcumin inhibits tumorigenesis during both initiation and promotion (post-initiation) periods in several experimental animal models. Topical application of curcumin inhibits benzo[a]pyrene (B[a]P)-mediated formation of DNA-B[a]P adducts in the epidermis. It also reduces 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced increases in skin inflammation, epidermal DNA synthesis, ornithine decarboxylase (ODC) mRNA level, ODC activity, hyperplasia, formation of c-Fos, and c-Jun proteins, hydrogen peroxide, and the oxidized DNA base 5-hydroxymethyl-2'-deoxyuridine (HmdU). Topical application of curcumin inhibits TPA-induced increases in the percent of epidermal cells in synthetic (S) phase of the cell cycle. Curcumin is a strong inhibitor of arachidonic acid-induced edema of mouse ears in vivo and epidermal cyclooxygenase and lipoxygenase activities in vitro. Commercial curcumin isolated from the rhizome of the plant Curcuma longa Linn contains 3 major curcuminoids (approximately 77% curcumin, 17% demethoxycurcumin, and 3% bisdemethoxycurcumin). Commercial curcumin, pure curcumin, and demethoxycurcumin are about equipotent as inhibitors of TPA-induced tumor promotion in mouse skin, whereas bisdemethoxycurcumin is somewhat less active. Topical application of curcumin inhibits tumor initiation by B[a]P and tumor promotion by TPA in mouse skin. Dietary curcumin (commercial grade) inhibits B[a]P-induced forestomach carcinogenesis, N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG)-induced duodenal carcinogenesis, and azoxymethane (AOM)-induced colon carcinogenesis. Dietary curcumin had little or no effect on 4-(methylnitosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung carcinogenesis and 7,12-dimethylbenz[a]anthracene (DMBA)-induced breast carcinogenesis in mice. Poor circulating bioavailability of curcumin may account for the lack of lung and breast carcinogenesis inhibition.

Inhibitory effects of caffeic acid phenethyl ester (CAPE) on 12-O-tetradecanoylphorbol-13-acetate-induced tumor promotion in mouse skin and the synthesis of DNA, RNA and protein in HeLa cells.

Topical application of caffeic acid phenethyl ester (CAPE), a constituent of the propolis of honeybee hives, to the backs of CD-1 mice previously initiated with 7,12-dimethylbenz[a]anthracene (DMBA) inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumor promotion and the formation of 5-hydroxymethyl-2'-deoxyuridine (HMdU) in epidermal DNA. Topical application of 5 nmol TPA twice weekly for 20 weeks to mice previously initiated with 200 nmol of DMBA resulted in 18.8 skin papillomas per mouse. Topical application of 1, 10, 100 or 3000 nmol of CAPE together with 5 nmol of TPA twice a week for 20 weeks inhibited the number of skin papillomas per mouse by 24, 30, 45 or 70%, respectively, and tumor size per mouse was decreased by 42, 66, 53 or 74%, respectively. Topical application of 5 nmol of TPA twice weekly for 20 weeks to mice previously initiated with DMBA produced an average of 12.6 HMdU residues per 10(4) normal bases in epidermal DNA. Topical application of 1, 10, 100 or 3000 nmol of CAPE with 5 nmol of TPA twice weekly for 20 weeks to DMBA-initiated mice decreased the level of HMdU in epidermal DNA by 40-93%. The in vitro addition of 1.25, 2.5, 5, 10 or 20 microM CAPE to cultured HeLa cells inhibited the synthesis of DNA by 32, 44, 66, 79 or 95%, respectively, the synthesis of RNA was inhibited by 39, 43, 58, 64 or 75%, respectively, and the synthesis of protein was inhibited by 29, 30, 37, 32 or 47%, respectively. The results indicate a potent inhibitory effect of CAPE on TPA-induced tumor promotion and TPA-induced formation of HMdU in DNA of mouse skin as well as an inhibitory effect of CAPE on the synthesis of DNA, RNA and protein in culture HeLa cells.

7,12-dimethylbenz[a]anthracene induces oxidative DNA modification in vivo.

Initiation and promotion are major stages in the multistage carcinogenesis process. Formation of initiating carcinogen-DNA base adducts leads to heritable genetic changes, but the tumor-promoting events induced by complete carcinogens have not, as yet, been elucidated. Oxidant production and oxidative DNA damage induced by phorbol esters (i.e., 12-O-tetradecanoyl-phorbol-13-acetate) are associated with tumor promotion, while antioxidants and inhibitors of oxidative DNA damage suppress promotion and carcinogenesis. Our goal was to establish whether a carcinogen that requires oxidative metabolism for its activity can also induce oxidant production and DNA base oxidation. We found that topical treatment of SENCAR mice with 7,12-dimethylbenz[a]anthracene, which induces tumors in 40-50% of the mice, also causes hydrogen peroxide production and formation of oxidized bases (i.e., 8-hydroxyl-2'-deoxyguanosine and 5-hydroxymethyl-2'-deoxyuridine) in epidermal DNA. The levels of oxidized bases were of comparable magnitude to those mediated by the potent tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate. The oxidized bases persisted over several weeks in epidermal DNA. These oxidative events appear to be temporally associated with inflammatory responses that include edema and polymorphonuclear leukocyte infiltration, which remained elevated over longer periods of time and at higher levels than those induced by phorbol ester. Because these processes are usually associated with tumor promotion, our results support the conjecture that oxidative events may be involved in what is operationally referred to as the tumor promotion process by 7,12-dimethylbenz[a]anthracene.

Immunotoxicity of low level cadmium exposure in fish: an alternative animal model for immunotoxicological studies.

Cadmium represents a major aquatic pollutant in many parts of the world. Yet, despite the fact that cadmium accumulates in high concentrations in fish tissues, is found in polluted aquatic environments, and is carcinogenic and immunotoxic in a variety of mammalian species, the effects of cadmium on the immune responses of directly exposed aquatic species have not been clearly defined. This study was designed to assess the effects of in vivo cadmium exposure, at a concentration found in contaminated aquatic environments, on the immune defense mechanisms of fish. In this study, no effects were observed upon body weight, lysozyme activity, of cell viability, despite the high concentration of accumulated cadmium in the gills and liver. Furthermore, in the absence of any clinical manifestations or overt toxicity, exposure of rainbow trout to waterborne cadmium at 2 ppb altered macrophage-mediated immune functions, including phagocytosis and free radical production, in a time-dependent manner. Similar immunotoxic effects of cadmium have also been observed in mammals. Although interspecies comparisons between mammalian and fish immune responses are extremely complicated and need to be approached with caution, results from this study suggest the applicability of fish as an additional/alternative animal model for immunotoxicological studies.

Human promyelocytic leukemia cells (HL-60): a new model to study the effects of chemopreventive agents on H2O2 production.

Tumor promoter-stimulated polymorphonuclear leukocytes (PMNs) produce excessive H2O2, which contributes to inflammation and carcinogenesis. A new model to study 12-O-tetradecanoylphorbol-13-acetate (TPA)-mediated H2O2 formation and its suppression by chemopreventive agents was developed using human promyelocytic leukemic HL-60 cells and validated by comparing results with those obtained using human PMNs. Equal H2O2 (20 to 25 nmol/ml) was generated by TPA-activated PMNs (2.5 x 10(5)/ml) and TPA-treated dimethylsulfoxide (DMSO)-differentiated HL-60 cells (5 x 10(5)/ml). The chemopreventive agent-mediated inhibition of TPA-induced H2O2 formation was also comparable in both cell types. These results suggest that HL-60 cells can become a useful in vitro system to screen rapidly for chemopreventive agents and to study their properties.

Occupational exposures to Cd, Ni, and Cr modulate titers of antioxidized DNA base autoantibodies.

This study was undertaken to establish whether occupational exposures to derivatives of carcinogenic metals evoke inflammatory immune responses, as determined by the presence of elevated titers of antibodies (Ab) that recognize oxidized DNA bases. Sera obtained from the blood of steel welders (Delaware) and from workers of the Centra Ni-Cd Battery Factory (Poznan, Poland) were analyzed by the enzyme-linked immunosorbent assay. To determine specific and nonspecific binding, an oxidized thymidine [5-hydroxymethyl-2'-deoxyuridine (HMdU)] coupled to bovine serum albumin (HMdU-BSA) as well as mock-coupled BSA (M-BSA) were used as antigens for coating the wells of microtiter plates. Titers of anti-HMdU Ab were significantly elevated in the high Cd and Ni exposure groups (18.3 +/- 3.2 vs 10.8 +/- 2.1 A492/microliters; p < 0.05). The sera of the groups with low exposures to Cd and Ni also had enhanced titers of those Ab but those increases were not statistically significant. Interestingly, the Ab titers present in the sera of controls for Cd and Ni exposures appear to be constant regardless of the protein content. In contrast, both lightly and heavily exposed subjects exhibited Ab titers that increased with increasing protein content. When 12 randomly selected workers (4 from each of the control, lightly, and heavily exposed groups) were outfitted with personal monitors, anti-HMdU Ab titers of those workers showed a significant difference between the groups with light (< 100 micrograms/m3) and heavy (> 200 micrograms/m3) exposures to Cd (9.8 +/- 3.7 vs 22.1 +/- 3.7 A492/microliters; p < 0.01) and Ni (11.7 +/- 1.4 vs 31.0 +/- 1.8; p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

The role of nickel and nickel-mediated reactive oxygen species in the mechanism of nickel carcinogenesis.

Increasing evidence demonstrates the reactive oxygen species (ROS) are implicated in metal carcinogenesis. Exposure of cultured Chinese hamster ovary (CHO) cells to several nickel compounds, i.e. NiS, Ni3S2, NiO (black and green), and NiCl2 has been shown to increase oxidation of 2',7-dichlorofluorescein to the fluorescent 2',7-dichlorofluorescein (DCF), suggesting that nickel compounds increased the concentration of oxidants in CHO cells. This fluorescence can be attenuated by addition of exogenous catalase to the extracellular media, indicating that H2O2 is one of the formed oxidants in this system. Fluorimetric measurements of chromogens following thiobarbituric acid reaction showed that nickel compounds also induce lipid peroxidation with a decreasing potency NiS, Ni3S2 > black NiO > green NiO > NiCl2. These results suggest that lipid hydroperoxides may also be produced through the action of nickel in intact cells. MgCl2, an antagonist of Ni-induced DNA strand breaks and cell transformation, has no effect on the formation of DCF fluorescence induced in CHO cells by nickel. The results suggest that nickel is an active inducer of ROS in intact mammalian cells and that the molecular mechanism of nickel carcinogenesis may involve multiple steps of nickel-mediated ROS.