Suppression of tumor promoter-induced oxidative events and DNA damage in vivo by sarcophytol A: a possible mechanism of antipromotion.

Sarcophytol A (Sarp A), a nontoxic compound isolated from marine soft coral, inhibits the in vivo effects of tumor promoters. However, the mechanism of its action is unknown. Our studies show that Sarp A suppresses oxidant formation and DNA oxidation in the epidermis of SENCAR mice exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA). In the short-term experiments, mice were topically pretreated with different doses of Sarp A before 6.5 nmol TPA, and the same treatment was repeated 20 h later. Sarp A significantly decreased the TPA-induced infiltration of neutrophils, the levels of myeloperoxidase in the dermis, and the formation of H2O2, cis-thymidine glycol, 8-hydroxyl-2'-deoxyguanosine, and 5-hydroxymethyl-2'-deoxyuridine in the epidermis. In the long-term studies, repeated TPA applications (3.2 nmol twice a week for 16 weeks) increased cis-thymidine glycol 2.7-fold, 5-hydroxymethyl-2'-deoxyuridine 3.4-fold, and 8-hydroxyl-2'-deoxyguanosine 3.3-fold in epidermal DNA over the basal levels. Application of 350 nmol Sarp A before each TPA treatment significantly decreased the formation of oxidized DNA bases even below those present in the control mouse skin. Histological examination showed that Sarp A also alleviated the TPA-induced inflammatory response and infiltration of phagocytes. Thus, it is possible that suppression of tumor promotion by Sarp A is due (at least in part) to its inhibitory effects on tumor promoter-mediated migration and activation of phagocytes, oxidant formation, and DNA base oxidation.

In vivo formation of oxidized DNA bases in tumor promoter-treated mouse skin.

There has been a paucity of evidence showing that 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a potent tumor promoter, causes DNA damage in vivo. We show that oxidized DNA bases are formed in the epidermis of TPA-treated SENCAR mice in a dose- and time-dependent manner. As measured by high-performance liquid chromatography and acetylation of nucleosides with [3H]acetic anhydride, these oxidized DNA derivatives include cis-thymidine glycol, 5-hydroxymethyl-2'-deoxyuridine, and 8-hydroxyl-2'-deoxyguanosine. Their maximal formation induced by a single TPA dose occurred within 6-8 h (a 2-5-fold increase). The level of 8-hydroxyl-2'-deoxyguanosine was the lowest (3.2/10(5) bases) and remained almost unchanged for 18 h; thymidine glycol (29.1/10(4) bases) and 5-hydroxymethyl-2'-deoxyuridine (17.3/10(4) bases) declined gradually but were still above controls at 24 h. Reapplication of TPA 20 h after the first dose (time of the maximal polymorphonuclear leukocyte infiltration) enhanced the net formation of 8-hydroxyl-2'-deoxyguanosine by 3.8-fold (P less than 0.05), of cis-thymidine glycol by 1.9-fold (P less than 0.001), and of 5-hydroxymethyl-2'-deoxyuridine by 2.0-fold (P less than 0.01), as compared to those maximally produced by a single TPA dose. Thus, the infiltration of polymorphonuclear leukocytes into TPA-treated mouse skin, which was corroborated by histological examination and the presence of polymorphonuclear leukocyte-specific myeloperoxidase, might play an important role in TPA-induced DNA oxidation in vivo. Our findings provide proof that tumor promoters can induce genetic modification in vivo that is oxidative in nature. Hence, formation of oxidized DNA bases may be responsible for the genetic effects of tumor promoters in carcinogenesis.

Tamoxifen suppresses tumor promoter-induced hydrogen peroxide formation by human neutrophils.

Trans-tamoxifen (TAM) has been used successfully in therapy for estrogen-dependent human breast tumors and prevention of their recurrence. The mechanism of this prevention was thought to be due to the interference of TAM with estrogen promotion. TAM has a wider anticarcinogenic action that is similar to other chemopreventive agents in that it suppresses tumor promotion in 2-stage carcinogenesis by interfering with the action of protein kinase C. We report that TAM (5 microM) totally inhibits hydrogen peroxide (H2O2) formation by 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-treated human neutrophils. Interestingly, beta-estradiol (10 microM) also slightly inhibits the oxidative burst of neutrophils. Pretreatment of neutrophils with varying amounts of TAM and beta-estradiol caused additive inhibition of H2O2 formation by the 2 agents. 4-Hydroxy-tamoxifen, a metabolite with the highest affinity for the estrogen receptor, was only as inhibitory as beta-estradiol. Other derivatives (cis-, N-desmethyl-, and N-desdimethyl-tamoxifen) with low biological activities had a smaller effect on H2O2 formation. TPA-treated neutrophils were shown to contain 5-hydroxymethyl uracil (HMU). TAM prevented the TPA-induced formation of HMU in other cells. Like TPA, dietary fat, which is a risk factor for breast cancer, induces formation of HMU in the DNA of human white blood cells. TAM may suppress the dietary fat-induced HMU in the same manner at it does in TPA-induced neutrophils.

Inhibition of oxidative stress in HeLa cells by chemopreventive agents.

12-O-Tetradecanoylphorbol-13-acetate (TPA)-mediated oxidative stress in HeLa cells and its inhibition were studied by fluorometric measurement of H2O2 and by 3H-postlabeling of the oxidized bases 8-hydroxyl-2'-deoxyguanosine (8-OHdG) and 5-hydroxymethyl-2'-deoxyuridine (HMdU). TPA treatment (10 fmol/cell) caused approximately 7-fold increase in H2O2 levels (0.1 nmol TPA/ml), and 5-10-fold increase in 8-OHdG and HMdU (10 nmol TPA/ml). Naturally occurring compounds [caffeic acid phenethyl ester (CAPE), (-).epigallocatechin gallate (EGCG), penta-O-galloyl-beta-D-glucose (PGG) and sarcophytol A (Sarp A)] and the anticancer drug tamoxifen (TAM) were tested as potential chemopreventive agents. These agents dose-dependently inhibited TPA-induced H2O2, 8-OHdG and HMdU. The doses required for a 50% decrease in H2O2 were approximately 2.5 microM for TAM; 5 microM for CAPE, EGCG and PGG; and 75 microM for Sarp A. TAM and PGG (10 microM), EGCG (25 microM), and CAPE (50 microM) abolished TPA-mediated H2O2 production, even below the normal cellular levels. TAM (2.5-20 microM) decreased TPA-mediated HMdU and 8-OHdG formation 2-29 times. Maximum inhibition occurred at 20 microM TAM, which caused an approximately 95% decline in HMdU and 8-OHdG. CAPE was effective at 0.5-50 microM. CAPE (25 microM) decreased 8-OHdG 95%, and HMdU 58%, while Sarp A (250 microM) reduced 8-OHdG by 93% and HMdU by 78%. EGCG (1-25 microM) and PGG (1-10 microM) inhibited of 8-OHdG and HMdU dose-dependently. However, higher doses (50 and 100 microM) decreased the efficacy of that inhibition. Of those agents tested, TAM appears to be the most and Sarp A the least effective. Our results point to these 5 compounds as being potential chemopreventive agents, which at very low doses decrease the tumor promoter-mediated oxidative processes.

Carcinogenic sulfide salts of nickel and cadmium induce H2O2 formation by human polymorphonuclear leukocytes.

Some derivatives of nickel, cadmium, and cobalt are carcinogenic in humans and/or animals but their mechanisms of action are not known. We show that they are capable of stimulating human polymorphonuclear leukocytes (PMNs), as measured by H2O2 formation, a known tumor promoter. Most effective were the carcinogens nickel subsulfide, which caused a 550% net increase in H2O2 over that formed by resting PMNs, followed by cadmium sulfide, 400%, and nickel disulfide, 200%. Nickel sulfide and cobalt sulfide caused statistically nonsignificant increases of 45 and 20%, respectively. Noncarcinogenic barium and manganese sulfides, and sulfates of nickel, cadmium, and cobalt were inactive. The enhancement of H2O2 formation by CdS and Ni3S2 (1 mumol/2.5 x 10(5) PMNs) was comparable to that mediated by the potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate, used at 0.5 and 1 nM, respectively. Concurrent treatment of 12-O-tetradecanoylphorbol-13-acetate-stimulated PMNs with Ni3S2 or NiS caused a decrease in H2O2 accumulation from that expected if the effects were additive. Including catalase in the reaction mixture proved that the oxidant formed by stimulated PMNs was H2O2, whereas adding superoxide dismutase showed that superoxide was also present in PMN samples treated with NiS but not with Ni3S2. Since nickel- and cadmium-containing particulates are deposited in the lungs and cause infiltration of PMNs, the ability to activate those cells and induce H2O2 formation may contribute to their carcinogenicity.

Radiation-like modification of bases in DNA exposed to tumor promoter-activated polymorphonuclear leukocytes.

Oxygen species generated by human polymorphonuclear leukocytes (PMNs) activated by 12-O-tetradecanoylphorbol-13-acetate (TPA) caused the formation of 5-hydroxymethyl-2'-deoxyuridine (HMdUrd), and (+) and (-) diastereoisomers of cis-thymidine glycol (dTG) in DNA that was exposed to them. There were 9 HMdUrds and 31 dTGs formed per 1 X 10(6) thymidine residues. When Fe(II)/ethylenediaminetetraacetic acid was added to TPA-activated PMNs at 0, 10, 15, and 20 min after TPA, HMdUrd formation increased 5-, 13-, 30-, and 35-fold. Although dTG was initially formed in larger amounts than HMdUrd, it eventually decreased but was still 5-, 6-, 5.5-, and 3-5-fold, respectively, higher than in the absence of iron. From 65 to 1800 times more HMdUrd was formed in DNA when autologous plasma was present during incubation of DNA with TPA-activated PMNs than in its absence. The levels of dTG also varied from about the same as HMdUrd to the nondetectable. Reconstituted human serum transferrin used instead of plasma or Fe(II) also supported the formation of HMdUrd and dTG. When DNA was treated with Fe(II)-reduced H2O2 in the absence of PMNs and TPA, both derivatives were formed. However, the same treatment of marker dTG of dTG-containing polydeoxyadenylic-thymidylic acid caused the decomposition of dTG. Thus, the reduction of hydrogen peroxide by Fe(II) complexed to either ethylenediaminetetraacetic acid or amino acids amy be responsible for the formation of HMdUrd and dTG and for subsequent decomposition of dTG in DNA exposed to the TPA-activated PMNs.

Inhibition of tumor promoter-mediated processes in mouse skin and bovine lens by caffeic acid phenethyl ester.

Caffeic acid phenethyl ester (CAPE) was isolated from propolis (a product of honeybee hives) that has been used in folk medicine as a potent antiinflammatory agent. CAPE is cytotoxic to tumor and virally transformed but not to normal cells. Our main goal was to establish whether CAPE inhibits the tumor promoter (12-O-tetradecanoylphorbol-13-acetate)-induced processes associated with carcinogenesis. Topical treatment of SENCAR mice with very low doses (0.1-6.5 nmol/topical treatment) of CAPE strongly inhibits the following 12-O-tetradecanoylphorbol-13-acetate-mediated oxidative processes that are considered essential for tumor promotion: (a) polymorphonuclear leukocyte infiltration into mouse skin and ears, as quantified by myeloperoxidase activity; (b) hydrogen peroxide (H2O2) production; and (c) formation of oxidized bases in epidermal DNA, as measured by 5-hydroxymethyluracil and 8-hydroxylguanine. A 0.5-nmol dose of CAPE suppresses the oxidative burst of human polymorphonuclear leukocytes by 50%. At higher doses (1-10 mumol), CAPE inhibits edema and ornithine decarboxylase induction in CD-1 and SENCAR mice. Interestingly, we discovered that 12-O-tetradecanoylphorbol-13-acetate-induced H2O2 production in bovine lenses also is inhibited by CAPE. Cumulatively, these findings point to CAPE as being a potent chemopreventive agent, which may be useful in combating diseases with strong inflammatory and/or oxidative stress components, i.e., various types of cancer and possibly cataract development.

Anticarcinogenic action of protease inhibitors.

Protease inhibitors are synthesized in biological systems and play a critical role in controlling a number of diverse physiological functions. They participate in blood clotting and lysis of clots, in growth processes by modulation of proteolytic digestion of proteins and thus availability of amino acids, and in the induction of selective DNA amplification. When incorporated into the diet, protease inhibitors appear to suppress many types of cancer. In vitro, they suppress neoplastic transformation caused by chemical carcinogens, ionizing radiation, and oncogenes. These observations offer the hope that judiciously applied protease inhibitors in small concentrations may prevent a wide range of human cancers. This hope is further supported by epidemiological studies which show that populations consuming relatively large amounts of protease inhibitors have a lower occurrence of cancer. The tasks remaining are to determine the kind and the level of protease inhibitors that are most effective in preventing cancer without also having toxic side effects and to incorporate them into our diet. Perhaps the most encouraging investigations are those using small nontoxic protease inhibitors available in pure form (epsilon-aminocaproic acid, a trypsin plasminogen activator inhibitor, and nicotinamide, a chymotrypsin inhibitor and known vitamin). Both agents have been shown to be preventive agents of cancer in animals and in vitro models. Further studies with natural protease inhibitors may yield even more effective agents which when incorporated into our diet will prevent the development of many types of cancer.

Carcinogen-mediated oxidant formation and oxidative DNA damage.

This article reviews the experimental data that points to formation of reactive oxygen species (ROS) and oxidative DNA base damage as being important contributors to cancer development. Particular emphasis is placed on the role they play in genetic changes occurring during tumor promotion. A number of structurally different anticarcinogenic agents inhibit ROS production and oxidative DNA damage as they inhibit inflammation and tumor promotion. This underlines the importance of ROS and oxidative genetic damage to the carcinogenic process. It also points to the possibility that some types of cancer may be preventable if the cycles of tumor promotion can be interrupted.

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.

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.

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.

Effect of metals on nucleoside hydroperoxide, a product of ionizing radiation in DNA.

Ionizing radiation causes formation of thymine hydroperoxides in DNA. Their decomposition generates more stable products and active oxygen species which may oxidize other DNA bases. We have determined the effects of free and chelated metal ions on the degradation of 5-hydroperoxymethyl-2'-deoxyuridine (HPMdU). Two products were formed as analyzed by HPLC: 5-hydroxymethyl-2'-deoxyuridine (HMdU) and 5-formyl-2'-deoxyuridine (FdU). Sn(II) and Fe(II) caused instantaneous HPMdU degradation; Sn(II) generated only HMdU, whereas Fe(II) formed about equal amounts of both. Sn(IV) and Fe(III) were inactive. Cu(I), Cu(II), and Co(II) caused a time-dependent formation of both products, with FdU predominating. In the presence of Cu(I), Cu(II), and Fe(II), formate inhibited formation of HMdU but enhanced that of FdU. EDTA abolished Cu(I)-induced decomposition of HPMdU but only decreased that which was mediated by Cu(II). In contrast, EDTA enhanced the activity of Fe(III) with a time-dependent formation of FdU. EDTA and diethylenetriaminepentaacetic acid (DTPA) caused an instantaneous Fe(II)-mediated decomposition of HPMdU to FdU. Only desferal partially inhibited the activity of Fe(II), whereas the activities of Cu(I), Cu(II), and Fe(III) were blocked by desferal and DTPA. Possible mechanisms of HPMdU degradation by metal ions in the absence or presence of formate or chelators as well as formation of the .OH are discussed.

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.

Recognition of oxidized DNA bases by sera of patients with inflammatory diseases.

Chronic inflammatory conditions result from or contribute to many diseases. Prominent among them is systemic lupus erythematosus (SLE). Sera of SLE patients contain an array of various auto-antibodies (Ab), including antinuclear Ab of unknown etiologies. The most puzzling is formation of Ab directed against autologous DNA. Our hypothesis was that increased oxidant production causes oxidation of DNA bases, which provide antigenic determinants that elicit antioxidized DNA Ab. To test this hypothesis, we used oxidized DNA nucleoside (5-hydroxymethyl-2'-deoxyuridine [HMdU]) conjugated to bovine serum albumin (HMdU-BSA) as the antigen. The results of the enzyme-linked immunosorbent assay showed that these Abs are sensitively detectable in SLE sera and sera of various other inflammatory autoimmune diseases. The titers of anti-HMdU Ab were significantly higher (p < .01) than those present in the control sera. Anti-HMdU Ab were predominantly of the IgM isotype, with low levels of IgG and no IgA. Anti-HMdU Ab bound to the HMdU-BSA-coated wells in a concentration- and time-dependent manner. That binding was inhibited by HMdU-BSA and to a lesser extent by thymidine-BSA, a normal nucleoside conjugate. The specific binding appears to be inversely related to the age of the patients, but no significant differences were observed between the sexes of the same age.

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.

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.

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

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.