Effects of dietary transition metals on oxidative DNA lesions in neonatal rats

Bulky endogenous oxidative lesions (type II I-compounds) reflect DNA damage associated with oxidative stress. As shown by 32P-postlabeling, their levels are enhanced by pro-oxidant genotoxins and also shortly after normal birth in several rat tissues as a function of time and the maternal diet. In order to elucidate which dietary components contribute to postnatal DNA damage, we have focused, herein, on the possible role of transition metals (iron, copper, and nickel). Pregnant Fischer 344 (F344) rats were fed AIN-93G purified diet containing different amounts of iron, copper, and nickel, or Purina-5001 natural-ingredient diet (which contains relatively high concentrations of these metals). Type II I-compounds were estimated by nuclease P1-enhanced 32P-postlabeling in liver and lung DNA of fetuses and at 24h and day 9 post-partum. Increased postnatal oxidative damage was detected in liver but not lung DNA of neonates exposed to higher amounts of dietary transition metals. There were significant positive linear correlations between maternal transition metal intake and neonatal, but not fetal and maternal type II I-compound levels. The results show that transition metals in the maternal diet affect perinatal oxidative DNA damage, presumably via a Fenton-type reaction. They also provide evidence for optimal levels in the maternal diet of transition metals, which on one hand, are essential for life, but on the other, can cause potentially deleterious DNA alterations in the offspring.     

Protection of DNA damage by dietary restriction.

Dietary restriction is known to retard the aging processes and delay the onset of age-related neoplastic diseases. The mechanisms underlying these remarkable actions of nutritional intervention are not known in spite of recently intensified research efforts. However, the last couple of years' research on dietary restriction produced strong evidence indicating that its effective antiaging actions might be related to its ability to modulate free radical damage. In the present study, DNA damage and attenuation of the damage by dietary restriction were assessed by measuring 8-hydroxydeoxyguanosine 8-OH dG) in both nuclear DNA (nuDNA) and mitochondrial DNA (mitDNA) fractions. The data show that substantially more damage (approximately 15 times) occurred in mitDNA compared to nuDNA. More interestingly, the DNA damage was significantly attenuated in dietarily restricted rats.     

Effect of dietary taurine on endogenous hypercholesterolemia in rats fed on phenobarbital-containing diets.

The effect of dietary taurine on endogenous hypercholesterolemia induced by a phenobarbital-containing diet was investigated. Supplemented taurine did not affect the concentrations of serum cholesterol, but further potentiated the accumulation of hepatic cholesterol in the hypercholesterolemic state induced by phenobarbital. It is suggested that taurine might amplify the hepatic cholesterogenesis in phenobarbital-induced hypercholesterolemia.     

Effects of environmental air pollution on endogenous oxidative DNA damage in humans

Epidemiological studies conducted in metropolitan areas have demonstrated that exposure to environmental air pollution is associated with increases in mortality. Carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) are the major source of genotoxic activities of organic mixtures associated with respirable particulate matter, which is a constituent of environmental air pollution. In this study,we wanted to evaluate the relationship between exposure to these genotoxic compounds present in the air and endogenous oxidative DNA damage in three different human populations exposed to varying levels of environmental air pollution. As measures of oxidative DNA damage we have determined 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) by liquid chromatography–tandem mass spectrometry (LC–MS/MS) and cyclic pyrimidopurinone N-1,N² malondialdehyde-2′-deoxyguanosine (M₁dG) by the immunoslot blot assay from lymphocyte DNA of participating individuals. The level of endogenous oxidative DNA damage was significantly increased in individuals exposed to environmental air pollution compared to unexposed individuals from Kosice (8-oxodG adducts) and Sofia (M₁dG adducts). However, there was no significant difference in the level of endogenous oxidative DNA and exposure to environmental air pollution in individuals from Prague (8-oxodG and M₁dG adducts) and Kosice (M₁dG adducts). The average level of M₁dG adducts was significantly lower in unexposed and exposed individuals from Kosice compared to those from Prague and Sofia. The average level of 8-oxodG adducts was significantly higher in unexposed and exposed individuals from Kosice compared to those from Prague. A significant increasing trend according to the interaction of c-PAHs exposure and smoking status was observed in levels of 8-oxodG adducts in individuals from Kosice. However, no other relationship was observed for M₁dG and 8-oxodG adduct levels with regard to the smoking status and c-PAH exposure status of the individuals. The conclusion that can be made from this study is that environmental air pollution may alter the endogenous oxidative DNA damage levels in humans but the effect appears to be related to the country where the individuals reside. Genetic polymorphisms of the genes involved in metabolism and detoxification and also differences in the DNA repair capacity and antioxidant status of the individuals could be possible explanations for the variation observed in the level of endogenous oxidative DNA damage for the different populations.     

The effect of aging and dietary restriction on DNA repair

DNA repair was studied as a function of age in cells isolated from both the liver and the kidney of male Fischer F344 rats. DNA repair was measured by quantifying unscheduled DNA synthesis induced by UV irradiation. Unscheduled DNA synthesis decreased approximately 50% between the ages of 5 and 30 months in both hepatocytes and kidney cells. The age-related decline in unscheduled DNA synthesis in cells isolated from the liver and kidney was compared in rats fed ad libitum and rats fed a calorie-restricted diet; calorie restriction has been shown to increase the survival of rodents. The level of unscheduled DNA synthesis was significantly higher in hepatocytes and kidney cells isolated from the rats fed the restricted diet. Thus, calorie restriction appears to retard the age-related decline in DNA repair.     

Arsenite induces oxidative DNA adducts and DNA-protein cross-links in mammalian cells.

Arsenic is generally recognized as a nonmutagenic carcinogen because sodium arsenite induces DNA damage only at very high concentrations. In this study we demonstrate that arsenite concentrations above 0.25 microM induce DNA strand breaks in both human leukemia cells and Chinese hamster ovary cells. Therefore, DNA damage may be involved in arsenic-induced carcinogenesis. Formamidopyrimidine-DNA glycosylase and proteinase K greatly increased DNA strand breaks in arsenite-treated cells, providing evidence that a large portion of arsenite-induced DNA strand breaks come from excision of oxidative DNA adducts and DNA-protein cross-links. Because DNA strand breaks appear only temporarily during excision repair, the level of detectable DNA strand breaks will be low at any given time point. For this reason many previous studies have only detected low levels of DNA strand breaks. We also show that catalase, and inhibitors of calcium, nitric oxide synthase, superoxide dismutase, and myeloperoxidase, could modulate arsenite-induced DNA damage. We conclude that arsenite induces DNA adducts through calcium-mediated production of peroxynitrite, hypochlorous acid, and hydroxyl radicals.     

Effects of dietary calcium restriction and acute exercise on the antioxidant enzyme system and oxidative stress in rat diaphragm

Calcium deficiency is considered to increase intracellular calcium level; thus the aim of the current study was to elucidate whether dietary calcium restriction enhanced exercise-induced oxidative stress in rat diaphragm. Twenty male Wistar rats were randomly assigned to either a control group or a group subjected to 1 mo of calcium restriction. In addition, each group was subsequently subdivided into rested or acutely exercised group. Dietary calcium restriction significantly (P < 0.05) upregulated the activities of manganese-superoxide dismutase (Mn-SOD), copper-zinc-superoxide dismutase (Cu-Zn-SOD), and glutathione peroxidase (Gpx) but not catalase. Acute exercise, in addition to calcium restriction, decreased both SOD isoenzymes in the diaphragm of calcium-restricted rats (P < 0.05). On the other hand, calcium restriction resulted in increased Gpx mRNA expression (P < 0.05). In control rats, acute exercise significantly (P < 0.05) increased the expressions of both SOD mRNAs, whereas in the calcium-restricted rats, it increased that of Mn-SOD mRNA (P < 0.05) but decreased that of Gpx mRNA (P < 0.05). Furthermore, reactive carbonyl derivative, a marker of protein oxidation, was significantly greater in the calcium-restricted rats than in the control rats after acute exercise (P < 0.05). The results suggest that antioxidant enzymes in rat diaphragm were upregulated in response to an increased oxidative stress by dietary calcium restriction but that upregulation is not enough to cope with exercise-induced further increase of oxidative stress.     

DNA polymerase mutagenic bypass and proofreading of endogenous DNA lesions

DNA polymerases differentiate between correct and incorrect substrates during synthesis on undamaged DNA templates through the biochemical steps of base incorporation, primer-template extension and proofreading excision. Recent research examining DNA polymerase processing of abasic, alkylation and oxidative lesions is reviewed in light of these discrimination mechanisms. Inhibition of DNA synthesis results from correct polymerase discrimination against utilization of geometrically incorrect template bases or 3' terminal basepairs. The efficiency of translesion synthesis is thus related to the physical structure of the lesion containing DNA. However, variations in enzyme structure and kinetics result in translesion synthesis efficiencies that are also dependent upon the DNA polymerase. With a low probability, polymerase misinsertion events create a 3' lesion terminus which is geometrically favored over the correct lesion basepair, resulting in mutagenic translesion synthesis. For example, both polymerase alpha and polymerase beta appear to require the formation of a stable 3' primer-template structure for efficient abasic site translesion synthesis. However, the enzymes differ as to the precise molecular make-up of the stable DNA structure, resulting in different mutational specificities. Similar mechanisms may be applicable to oxidative damage, where mutational specificities dependent upon the DNA polymerase also have been observed. In vitro reaction conditions also influence DNA polymerase processing of lesions. Using an in vitro herpes simplex virus thymidine kinase (HSV-tk) gene forward mutation assay, we demonstrate that high dNTP substrate concentrations affect the mutagenic specificity of translesion synthesis using alkylated templates. The exonuclease-deficient Klenow polymerase error frequency for G-->A transition mutations using templates modified by N-ethyl-N-nitrosourea (ENU) was four-fold higher at 1000 microM [dNTP], relative to 50 microM [dNTP], consistent with an increased efficiency of extension of the etO6G.T mispair. Moreover, the frequency of other ENU-induced polymerase errors was suppressed when polymerase reactions contained 50 microM dNTP, relative to 1000 microM dNTP. The efficiency of proofreading as a polymerase error discrimination mechanism reflects a balance between the competing processes of 3'-->5' exonuclease removal of mispairs and polymerization of the next correct nucleotide. Polymerases that are devoid of a proofreading exonuclease generally display enhanced abasic site translesion synthesis relative to proofreading-proficient enzymes. In addition, the proofreading exonucleases of Escherichia coli Pol I and T4 DNA polymerases have been found to remove mispairs caused by abasic sites and oxidative lesions, respectively, resulting in lowered polymerase error rates. However, the magnitude of the exonuclease effect is small (less than 10-fold), and highly dependent upon the DNA polymerase-exonuclease. We have studied proofreading exonuclease removal of alkylation damage in the HSV-tk forward assay. We observed no significant reduction in the magnitude of the mutant frequency vs. dose-response curves when N-methyl-N-nitrosourea or ENU-treated templates were used in exonuclease-proficient Klenow polymerase reactions, as compared to the exonuclease-deficient polymerase reactions. Thus, available data suggest that proofreading excision of endogenous lesion mispairs does occur, but the efficiency is dependent upon the lesion and the DNA polymerase-exonuclease studied.     

Effects of dietary restriction on physical performance in mice

Dietary restriction is known to prolong life in laboratory animals. However, little is known about the effects of dietary restriction on physical performance. To evaluate physical performance, we measured four item indices: time to climb out of obstacles, time to escape restraint by gummed tape, time hanging from a bar, and ability to resist slipping every week. The diets of ICR mice were restricted from the age of 7 weeks through 24 weeks. Body weight of the diet-restricted mice decreased during the 7th to 9th weeks of age. After the 10th week, weight gain resumed. In response to assigned tasks, the diet-restricted mice performed better in all activities: they climbed out of obstacles faster, freed themselves sooner from restraint by gummed tape, hung from a bar longer, and better resisted slipping down a slope. These results suggest that diet-restricted mice have superior physical abilities, such as those required to overcome or avoid risks to life, than do ad-libitum-fed mice.     

Effects of dietary caloric restriction and aging on thyroid hormones of rhesus monkeys.

Plasma levels of thyroid hormones - triiodothyronine (T 3 ), thyroxin (T 4 ), and thyroid-stimulating hormone (TSH) were measured in male and female rhesus monkeys (Macaca mulatta) fed either ad libitum or a 30 % calorie-restricted (CR) diet (males for 11 years; females for 6 years). The same hormones were measured in another group of young male rhesus monkeys during adaptation to the 30 % CR regimen. Both long- and shorter-term CR diet lowered total T 3 in plasma of the monkeys. The effect appeared to be greater in younger monkeys than in older counterparts. No effects of CR diet were detected for either free or total T 4, although unlike T 3, levels of this hormone decreased with age. TSH levels also decreased with age, and were increased by long-term CR diet in older monkeys only. No consistent effects of shorter-term CR diet were observed for TSH. In the light of the effects of the thyroid axis on overall metabolism, these results suggest a possible mechanism by which CR diets may elicit their well-known beneficial 'anti-aging' effects in mammals.     

MutS recognition of exocyclic DNA adducts that are endogenous products of lipid oxidation.

The ability of the methyl-directed mismatch repair system to recognize and repair the exocyclic adducts propanodeoxyguanosine (PdG) and pyrimido[1,2-alpha]purin-10(3H)-one (M(1)G), the major adduct derived from the endogenous mutagen malondialdehyde, has been assessed both in vivo and in vitro. Both adducts were site-specifically incorporated into M13MB102 DNA, and the adducted genomes were electroporated into wild-type or mutS-deficient Escherichia coli strains. A decrease in mutations caused by both adducts was observed in mutS-deficient strains, suggesting that MutS was binding to the adducts and blocking repair by nucleotide excision repair. This hypothesis was supported by the differences in mutation frequency observed when hemimethylated genomes containing PdG on the (-)-strand were electroporated into a uvrA(-) strain. The ability of purified MutS to bind to PdG- or M(1)G-containing 31-mer duplexes in vitro was assessed using both surface plasmon resonance and gel shift assays. MutS bound to M(1)G:T-containing duplexes with similar affinity to a G:T mismatch but less strongly to M(1)G:C- and PdG-containing duplexes. Dissociation from each of the adduct-containing duplexes occurred at a faster rate than from a G:T mismatch. The present results indicate that MutS can bind to exocyclic adducts resulting from endogenous DNA damage and trigger their removal by mismatch repair or protect them from removal by nucleotide excision repair.     

Exocyclic DNA adducts as oxidative stress markers in colon carcinogenesis: potential role of lipid peroxidation,dietary fat and antioxidants

Molecular pathways to colorectal cancer involve multiple genetic changes, whereby extensive oxyradical damage causes mutations in cancer-related genes and leads to a cycle of cell death and regeneration. Besides direct oxidative DNA-damage, reactive oxygen and nitrogen species can induce etheno (epsilon)-DNA adducts mainly via trans-4-hydroxy-2-nonenal, generated as the major aldehyde by lipid peroxidation (LPO) of omega-6 PUFAs. Patients with familial adenomatous polyposis (FAP) develop multiple colorectal adenomas. In affected tissues increased LPO could be triggered due to increased arachidonic acid metabolism as a result of elevated cyclooxygenases. Our studies demonstrated an increased epsilon-DNA adduct level in affected colon epithelia of FAP patients. Epsilon-DNA adducts are promutagenic and can cause genomic instability that drives colorectal adenoma to malignancy. We have further investigated the potential chemopreventive properties of olive oil and its polyphenolic components. 'Mediterranean diet', of which olive oil is a major fatty acid source, has protective effects against human breast and colorectal cancers. Olive oil extracts and the newly identified lignan fractions showed high antioxidant capacity in vitro. As epsilon-DNA adducts are biomarkers for oxidative stress and LPO induced DNA damage, they can verify the efficacy of newly identified antioxidants, e.g. from olive oil, as chemopreventive agents against colon carcinogenesis.     

Effects of benzo[a]pyrene-deoxyguanosine lesions on DNA methylation catalyzed by EcoRII DNA methyltransferase and on DNA cleavage effected by EcoRII restriction endonuclease

DNA methylation is an important cellular mechanism for controlling gene expression. Whereas the mutagenic properties of many DNA adducts, e.g., those arising from polycyclic aromatic hydrocarbons, have been widely studied, little is known about their influence on DNA methylation. We have constructed site-specifically modified 18-mer oligodeoxynucleotide duplexes containing a pair of stereoisomeric adducts derived from a benzo[a]pyrene-derived diol epoxide [(+)- and (-)-r7,t8-dihydroxy-t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, or B[a]PDE] bound to the exocyclic amino group of guanine. The adducts, either (+)- or (-)-trans-anti-B[a]P-N(2)-dG (G*), positioned either at the 5'-side or the 3'-side deoxyguanosine residue in the recognition sequence of EcoRII restriction-modification enzymes (5'-...CCA/TGG...) were incorporated into 18-mer oligodeoxynucleotide duplexes. The effects of these lesions on complex formation and the catalytic activity of the EcoRII DNA methyltransferase (M.EcoRII) and EcoRII restriction endonuclease (R.EcoRII) were investigated. The M.EcoRII catalyzes the transfer of a methyl group to the C5 position of the 3'-side cytosine of each strand of the recognition sequence, whereas R.EcoRII catalyzes cleavage of both strands. The binding of R.EcoRII to the oligodeoxynucleotide duplexes and the catalytic cleavage were completely abolished when G was positioned at the 3'-side dG position (5'-...CCTGG*...). When G* was at the 5'-side dG position, binding was moderately diminished, but cleavage was completely blocked. In the case of M.EcoRII, binding is diminished by factors of 5-30 but the catalytic activity was either abolished or reduced 4-80-fold when the adducts were located at either position. Somewhat smaller effects were observed with hemimethylated oligodeoxynucleotide duplexes. These findings suggest that epigenetic effects, in addition to genotoxic effects, need to be considered in chemical carcinogenesis initiated by B[a]PDE, since the inhibition of methylation may allow the expression of genes that promote tumor development.     

Effects of dietary protein restriction on nephron number in the mouse

In rats, maternal protein restriction reduces nephron endowment and often leads to adult hypertension. Sex differences in these responses have been identified. The molecular and genetic bases of these phenomena can best be identified in a mouse model, but effects of maternal protein restriction on kidney development have not been examined in mice. Therefore, we determined how combined prenatal and postnatal protein restriction in mice affects organ weight, glomerular number and dimensions, and renal expression of angiotensin receptor mRNA, in both male and female offspring. C57/BL6/129sv mice received either a normal (20% wt/wt; NP) or low (9% wt/wt; LP) protein diet during gestation and postnatal life. Offspring were examined at postnatal day 30. Protein restriction retarded growth of the kidney, liver, spleen, heart, and brain. All organs except the brain weighed less in female than male offspring. Protein restriction increased normalized (to body weight) brain weight, with females having relatively heavier brains than males. The effects of protein restriction were not sex dependent, except that normalized liver weight was reduced in males but increased in females. Glomerular volume, but not number, was greater in female than in male mice. Maternal protein restriction reduced nephron endowment similarly in male and female mice. Renal expression of AT(1A) receptor mRNA was approximately sixfold greater in female than male NP mice, but similar in male LP and female LP mice. We conclude that maternal protein restriction reduces nephron endowment in mice. This effect provides a basis for future studies of developmental programming in the mouse.     

DNA abasic lesions in a different light: solution structure of an endogenous topoisomerase II poison

Topoisomerase II is the target for several anticancer drugs that "poison" the enzyme and convert it to a cellular toxin by increasing topoisomerase II-mediated DNA cleavage. In addition to these "exogenous topoisomerase II poisons," DNA lesions such as abasic sites act as "endogenous poisons" of the enzyme. Drugs and lesions are believed to stimulate DNA scission by altering the structure of the double helix within the cleavage site of the enzyme. However, the structural alterations that enhance cleavage are unknown. Since abasic sites are an intrinsic part of the genetic material, they represent an attractive model to assess DNA distortions that lead to altered topoisomerase II function. Therefore, the structure of a double-stranded dodecamer containing a tetrahydrofuran apurinic lesion at the +2 position of a topoisomerase II DNA cleavage site was determined by NMR spectroscopy. Three major features distinguished the apurinic structure ( = 0.095) from that of wild-type ( = 0.077). First, loss of base stacking at the lesion collapsed the major groove and reduced the distance between the two scissile phosphodiester bonds. Second, the apurinic lesion induced a bend that was centered about the topoisomerase II cleavage site. Third, the base immediately opposite the lesion was extrahelical and relocated to the minor groove. All of these structural alterations have the potential to influence interactions between topoisomerase II and its DNA substrate.     

Effects of dietary fat content on adiposity during energy restriction in genetically obese rats.

The effects of the amount of fat provided in a restricted diet on weight loss and body composition were studied in this work. Lean male (Fa/?) Zucker rats were fed a control diet ad libitum. Obese (fa/fa) Zucker rats were divided into three groups: one group was fed a control diet ad libitum and the other two groups were fed 75% energy-restricted diets, which provided 10 or 50% of calories as fat. After 4 weeks, energy restriction normalized body weight but not body composition in the genetically obese rats. Reductions in adipose tissue weights and adipocyte size, without changes in the cellularity, were observed. Differences only reached statistical significance in subcutaneous adipose tissue. A standard fat content in the diet induced the same fat-free mass reduction as a higher amount of this macronutrient, but a greater body fat reduction. This suggests that the restriction of dietary fat, as well as energy, is necessary to achieve dietary management in obesity.     

Metal ion dependence of DNA cleavage by SepMI and EhoI restriction endonucleases

Most of type II restriction endonucleases show an absolute requirement for divalent metal ions as cofactors for DNA cleavage. While Mg²⁺ is the natural cofactor other metal ions can substitute it and mediate the catalysis, however Ca²⁺ (alone) only supports DNA binding. To investigate the role of Mg²⁺ in DNA cleavage by restriction endonucleases, we have studied the Mg²⁺ and Mn²⁺ concentration dependence of DNA cleavage by SepMI and EhoI. Digestion reactions were carried out at different Mg²⁺ and Mn²⁺ concentrations at constant ionic strength. These enzymes showed different behavior regarding the ions requirement, SepMI reached near maximal level of activity between 10 and 20 mM while no activity was detected in the presence of Mn²⁺ and in the presence of Ca²⁺ cleavage activity was significantly decreased. However, EhoI was more highly active in the presence of Mn²⁺ than in the presence of Mg²⁺ and can be activated by Ca²⁺. Our results propose the two-metal ion mechanism for EhoI and the one-metal ion mechanism for SepMI restriction endonuclease. The analysis of the kinetic parameters under steady state conditions showed that SepMI had a K_m value for pTrcHisB DNA of 6.15 nM and a V_max of 1.79 × 10^?2 nM min^?1, while EhoI had a K_m for pUC19 plasmid of 8.66 nM and a V_max of 2 × 10^?2 nM min^?1.     

Influence of controlled dietary restriction on immunologic function and aging.

The underfeeding regimens tested in rodents for life span prolongation and/or immunologic effects result in a complex blend of protein and energy restriction while offering at least adequate amounts of all other essential nutrients. Underfeeding started at weaning and continued throughout life represents the only proved way of slowing the rate of aging in homeotherms. Mounting evidence indicates that underfeeding initiated later in life may also influence life span favorably. Old mice after lifelong restriction, or moderately aged mice (16.5 months) after 4.5 months of restriction, display "younger" immune systems than do age-matched, normally fed controls, as judged by response to mitogens, the mixed lymphocyte reaction, and other determinants. The immunologic effects of underfeeding, when measured quite early in life, are strain-dependent in the mouse. Diets designed to restrict the intake per week of energy (but not protein) produce the same effects on immune response capacity in very young mice as do energy restricted, protein unbalanced regimens. Underfeeding early in life drastically dampens thymic growth and alters the timing of involution. Early restriction also produces a profound lowering of body temperature in young (C57BL/10Sn x C3H/HeDiSn)F1 females. On the other hand, temperature lowering was not observed in males of this hybrid fed normally for the first year of life and restricted for 3 months prior to measurement. The mechanisms behind these various effects of controlled dietary restriction (i.e., undernutrition without malnutrition) are poorly understood at present.     

Chemical and biochemical postlabeling methods for singling out specific oxidative DNA lesions.

A survey of the main available chemical and biochemical postlabeling assays for measuring oxidative DNA damage is reported. Two main approaches, radio and fluorescent postlabeling, have been used in order to reach a high level of sensitivity of detection. This is required for the measurement of DNA damage within cells and tissues upon exposure to agents of oxidative stress. Most of the methods are based on liquid chromatographic separation of defined DNA modifications following either acidic hydrolysis or enzymic digestion of DNA. In a subsequent step, the isolated base or sugar damages are either radiolabeled or made fluorescent by chemical or enzymatic reactions. Emphasis is placed on the recently developed high performance liquid chromatographic 32P-postlabeling assay, which allows the specific and sensitive measurement of various base damages including adenine N-1 oxide and 5-hydroxymethyluracil at the level of one modification per 10(7) normal bases in a sample size of 1 microgram of DNA. Examples of application of radioactive postlabeling to the measurement of DNA base damage following exposure of human cells to oxidizing agents including hydrogen peroxide and UVA radiation are provided.