Formation of modified DNA bases in cells exposed either to gamma radiation or to high-LET particles

The aim of the present study was to measure the formation of eight base modifications in the DNA of cells exposed to either low-LET ((60)Co gamma rays) or high-LET ((12)C(6+) particles) radiation. For this purpose, a recently optimized HPLC-MS/MS method was used subsequent to DNA extraction and hydrolysis. The background level of the measured modified bases and nucleosides was shown to vary between 0.2 and 2 lesions/10(6) bases. Interestingly, thymidine glycols constitute the main radiation-induced base modifications, with an overall yield of 0.097 and 0.062 lesion/10(6) bases per gray for gamma rays and carbon heavy ions, respectively. Both types of radiations generate four other major degradation products, in the following order of decreasing importance: FapyGua > 5-HmdUrd > 5-FordUrd > 8-oxodGuo. The yields of formation of FapyAde and 8-oxoAde are one order of magnitude lower than those of the related guanine modifications, whereas the radiation-induced generation of 5-OHdUrd was below the limit of detection of the assay. The efficiency for both types of radiation to generate base damage in cellular DNA is low because the highest yield per gray was 0.097 thymine glycols per 10(6) DNA bases. As a striking observation, the yield of formation of the measured DNA lesions was found to be, on average, twofold lower after exposure to high-LET radiation ((12)C(6+)) than after exposure to low-LET gamma radiation. These studies show that the HPLC-MS/MS assay provides an accurate, reliable and sensitive method for measuring cellular DNA base damage.     

DNA adducts of styrene-7,8-oxide in target and non-target organs for tumor induction in rat and mouse after repeated inhalation exposure to styrene

Styrene by inhalation had been shown to increase the lung tumor incidence in mice at 20 ppm and higher, but was not carcinogenic in rats at up to 1000 ppm. Styrene-7,8-oxide, the major metabolic intermediate, has weak electrophilic reactivity. Therefore, DNA adduct formation was expected at a low level and a 32P-postlabeling method for a determination of the two regioisomeric 2'-deoxyguanosyl-O6-adducts at the alpha(7)- and beta(8)-positions had been established. The first question was whether DNA adducts could be measured in the rat at the end of the 2 years exposure of a bioassay for carcinogenicity, even though tumor incidence was not increased. Liver samples of male and female CD rats were available for DNA adduct analysis. Adducts were above the limit of detection only in the highest dose group (1000 ppm), with median levels of 9 and 8 adducts per 10(7) nucleotides in males and females, respectively (sum of alpha- and beta-adducts). The result indicates that the rat liver tolerated a relatively high steady-state level of styrene-induced DNA adducts without detectable increase in tumor formation. The second question was whether different DNA adduct levels in the lung of rats and mice could account for the species difference in tumor incidence. Groups of female CD-1 mice were exposed for 2 weeks to 0, 40, and 160 ppm styrene (6h per day; 5 days per week), female CD rats were exposed to 0 and 500 ppm. In none of the lung DNA samples were adducts above a limit of detection of 1 adduct per 10(7) DNA nucleotides. The data indicate that species- and organ-specific tumor induction by styrene is not reflected by DNA adduct levels determined in tissue homogenate. The particular susceptibility of the mouse lung might have to be based on other reactive metabolites and DNA adducts, indirect DNA damage and/or cell-type specific toxicity and tumor promotion.     

Melatonin reduces the increase in 8-hydroxy-deoxyguanosine levels in the brain and liver of kainic acid-treated rats

In the present study, the effect of melatonin on oxidative DNA damage induced by kainic acid (KA) treatment was investigated. 8-hydroxy-deoxyguanosine (8-OH-dG) is a main product of oxidatively damaged DNA and was used as the endpoint in these studies. The levels of 8-OH-dG were found to be elevated in the hippocampus and frontal cortex of rats treated with KA. These elevated levels were significantly reduced in animals that were co-treated with melatonin. Thus, there was no difference in 8-OH-dG levels in the brain of control rats compared to those treated with KA (10 mg/kg) plus melatonin (10 mg/kg). The levels of 8-OH-dG also increased in the liver of rats treated with KA. This rise in oxidatively damaged DNA was also prevented by melatonin administration. Melatonin's ability to reduce KA-induced increases in neural and hepatic 8-OH-dG levels presumably relates to its direct free radical scavenging ability and possibly to other antioxidative actions of melatonin.     

Oxidative DNA damage in tissues of English sole (Parophrys vetulus) exposed to nitrofurantoin

Juvenile English sole were exposed intramuscularly to nitrofurantoin (NF) and the levels of 8-hydroxy-2′deoxyguanosine (8-OH-dG) in liver, kidney and blood were determined using reversed-phase HPLC with electrochemical detection. Identification and quantitation of the 8-OH-dG in the samples was accomplished by comparison with standard 8-OH-dG, which was characterized by UV spectroscopy and fast-atom bombardment mass spectrometry. The levels of hepatic 8-OH-dG increased (r² = 0.59, P = 0.015) with the dose of NF (0.10 – 10 mg NF/kg fish). In kidney and blood, however, the levels of 8-OH-dG were significantly higher than controls only at the highest dose tested. The level of binding in liver ranged from 0.37 to 0.76 fmol 8-OH-dG/μg DNA. The levels of hepatic 8-OH-dG reached a maximum (approx. 1 fmol 8-OH-dG/μg DNA) between 1 and 3 days after exposure, followed by a decrease to control levels (approx. 0.25 fmol 8-OH-dG/μg DNA) at 5 days post-exposure. These data demonstrate the first direct evidence for the formation of oxidized DNA bases resulting from the metabolism of a nitroaromatic compound by fish.     

Oxidative DNA damage in tissues of English sole (parophrys vetulus) exposed to nitrofurantoin

Juvenile English sole were exposed intramuscularly to nitrofurantoin (NF) and the levels of 8-hydroxy-2′deoxyguanosine (8-OH-dG) in liver, kidney and blood were determined using reversed-phase HPLC with electrochemical detection. Identification and quantitation of the 8-OH-dG in the samples was accomplished by comparison with standard 8-OH-dG, which was characterized by UV spectroscopy and fast-atom bombardment mass spectrometry. The levels of hepatic 8-OH-dG increased (r² = 0.59, P = 0.015) with the dose of NF (0.10 – 10 mg NF/kg fish). In kidney and blood, however, the levels of 8-OH-dG were significantly higher than controls only at the highest dose tested. The level of binding in liver ranged from 0.37 to 0.76 fmol 8-OH-dG/μg DNA. The levels of hepatic 8-OH-dG reached a maximum (approx. 1 fmol 8-OH-dG/μg DNA) between 1 and 3 days after exposure, followed by a decrease to control levels (approx. 0.25 fmol 8-OH-dG/μg DNA) at 5 days post-exposure. These data demonstrate the first direct evidence for the formation of oxidized DNA bases resulting from the metabolism of a nitroaromatic compound by fish.     

Age-associated accumulation of 8-hydroxydeoxyguanosine in mitochondrial DNA of human diaphragm

This is the first report that age-associated accumulation of 8-hydroxydeoxyguanosine (8-OH-dG) does occur in human mitochondrial DNA (mtDNA) in muscle of diaphragm. We extracted mtDNA from human diaphragm muscles from differing age groups, and determined the amount of 8-OH-dG by ultramicro-high performance liquid chromatography/mass-spectrometry system. With the same specimen, multiple deletions of mtDNA were detected by electrophoresis after amplification by the polymerase chain reaction method. In subjects below age 55, the level of 8-OH-dG in mtDNA was below 0.02% of the total deoxyguanosine (dG), whereas, in subjects over age 65, the level of 8-OH-dG increased with age at a rate of ca. 0.25% per 10 years, reaching 0.51% at age 85. Moreover, a concomitant increase in multiple deletions was detected with the increase in age. These results suggest that, in younger diaphragms, replication of mtDNA dilutes out 8-OH-dG being not detectable. In the elderly subjects aged over 65, the replication rate might be slowed down leading to the accumulation of 8-OH-dG in mtDNA, which would accelerate the age-associated multiple deletions of mtDNA observed among the subjects.     

The grapefruit flavanone naringin protects against the radiation-induced genomic instability in the mice bone marrow: a micronucleus study

To test the hypothesis that carcinogen exposure and oxidative stress are involved in pancreatic carcinogenesis in susceptible individuals, aromatic DNA adducts and 8-hydroxyguanosine (8-OH-dG) were measured by (32)P-postlabeling and HPLC-EC, respectively, in 31 pancreatic tumors and 13 normal tissues adjacent to the tumor from patients with pancreatic cancer. Normal pancreatic tissues from 24 organ donors, from six patients with non-pancreatic cancers, and from five patients with chronic pancreatitis served as controls. It was found that tissue samples from patients with pancreatic cancer had significantly higher levels of both aromatic DNA adducts and 8-OH-dG compared with control samples. The mean (+/-S.D.) levels of aromatic DNA adducts were 101.8+/-74.6, 26.9+/-26.6, and 11.2+/-6.6 per 10(9) nucleotides in adjacent tissues, tumors, and controls, respectively. The mean (+/-S.D.) levels of 8-OH-dG were 11.9+/-9.6, 10.8+/-10.6, and 6.7+/-4.6 per 10(5) nucleotides in adjacent tissues, tumors, and controls, respectively. Polymorphisms of the CYP1A1, CYP2E1, NAT1, NAT2, GSTM1, MnSOD, and hOGG1 genes were determined in these patients. The level of aromatic DNA adducts was significantly associated with polymorphism of the CYP1A1 gene. No significant correlation was found between the level of 8-OH-dG and the MnSOD, GSTM1, and hOGG1 polymorphisms. However, one novel polymorphism/mutation of the hOGG1 gene was found in a pancreatic tumor. Mutation at codon 12 of the K-ras gene was found in 25 (81%) of 31 pancreatic tumors, including three G-to-A transitions and 22 G-to-T transversions. Patients with the G-to-T mutation had a significantly higher level of aromatic DNA adducts than those with G-to-A or wild-type codon (P=0.02). On the other hand, the K-ras mutation profile was not related to the level of 8-OH-dG. Given the limitation of sample size, these preliminary data lend further support the hypothesis that carcinogen exposure and oxidative stress are involved in pancreatic carcinogenesis.     

Prenatal Stress Causes Oxidative Damage to Mitochondrial DNA in Hippocampus of Offspring Rats

Mitochondrion, the primary source of reactive oxygen species (ROS), is also the target of ROS. 8-Hydroxy-2′-deoxyguanosine (8-OH-dG) is the major end-product of damaged DNA caused by ROS. In our previous studies, we showed that prenatal stress (PNS) preferentially caused cognitive dysfunction and increased ROS in the hippocampus of female offspring rats. The present study aimed to determine 8-OH-dG level of mitochondria in order to elucidate the mechanism of hippocampal pyramidal neuronal damage and cognitive dysfunction induced by PNS. Pregnant rats were divided into two groups: control group (undisturbed) and PNS group (exposed to a restraint stress for 7 days at the late stage of gestation). Offspring rats were divided into four groups: female-control group, male-control group, female-stress group, male-stress group and used at 30-day-old after their birth. The content of 8-OH-dG was determined by high performance liquid chromatography-electrochemical detection (HPLC-ECD). The results showed that the contents of 8-OH-dG in female and male prenatal stressed offspring were significantly higher than that in their respective controls (P < 0.001). 8-OH-dG level was significantly higher in the female-stress group than in the male-stress group (P < 0.05), whereas there was no any gender-dependent difference in the control groups. These results suggest that accumulation of oxidative mitochondrial DNA damage may play an important role in PNS-induced cognitive dysfunction in female offspring rats. Special issue article in honor of Dr. Akitane Mori.     

Left ventricular interstitial 8-hydroxy-deoxyguanosine concentration following myocardial ischemia and reperfusion in anesthetized rats

Summary

The effect of myocardial ischemia and reperfusion on left ventricular interstitial 8-hydroxydeoxyguanosine (8-OH-dG), a possible biomarker for in vivo oxidative deoxyribonucleic acid damage, in anesthetized rats was investigated. A microdialysis probe was implanted. Levels of 8-OH-dG in microdialysates were analyzed via an on-line high performance liquid chromatography system equipped with an electrochemical detector. Myocardial ischemia for 10 or 20 min, induced by clamping of the left anterior descending coronary artery, did not affect 8-OH-dG levels. However, reperfusion following either 10-min or 20-min ischemia significantly increased 8-OH-dG levels in collected microdialysates. Reperfusion-induced increases in 8-OH-dG levels were more prominent in the 20 min ischemia group (as high as 3.5 fold relative to basal levels) than in the 10 min ischemia group as high as 2.0 fold relative to basal levels). In conclusion, we observed that left ventricular interstitial 8-OH-dG concentration increased following myocardial ischemia and reperfusion in anesthetized rats. These results suggest that 8-OH-dG might be a useful biomarker for oxidative damage following myocardial ischemia and reperfusion.     

Lipid peroxidation products mediate the formation of 8-hydroxydeoxyguanosine in DNA.

Membrane lipid peroxidation processes yield products that may react with DNA to cause oxidative modifications. We have investigated this possibility and have found that calf thymus DNA exposed to autooxidized lipids causes the formation of 8-hydroxy-2'-deoxyguanosine (8-OH-dG). 8-OH-dG formation in DNA was measured using high-pressure liquid chromatography with electrochemical detection. Methyl linolenate oxidized for different lengths of time was exposed to DNA. The amount of 8-OH-dG formed in DNA was proportional to the amount of lipid peroxidation as measured by the thiobarbituric reactive substances present. The formation of 8-OH-dG in DNA by autooxidized methyl linolenate was dependent on the presence of the transition metal ions Cu or Fe and was inhibited by various scavengers, including superoxide dismutase and catalase. This implicates the involvement of oxygen free radicals in the process. Liposomes formed from phosphatidylcholine (82%) and methyl arachidonate (18%) were peroxidized for different lengths of time and then exposed to DNA. 8-OH-dG was formed in DNA by exposure to Cu(II) and peroxidized liposomes. Under these conditions, Fe(III) was slightly less effective than Cu(II) in mediating 8-OH-dG formation. These observations clearly show that 8-OH-dG formation in DNA may result from processes that may occur during intracellular lipid peroxidation.     

Hydroxyl Free Radical Adduct of Deoxyguanosine: Sensitive Detection and Mechanisms of Formation

DNA or 2-deoxyguanosine reacts with hydroxyl free radical to form 8-hydroxy-deoxyguanosine (8-OH-dG). We found that 8-OH-dG can be effectively separated from deoxyguanosine by high pressure liquid chromatography and very sensitively detected using electrochemical detection. The sensitivity by electrochemical detection is about one-thousand fold enhanced over optical detection. Utilizing deoxyguanosine in bicarbonate buffer it was found that ferrous ion, but not ferric ion, was effective in forming 8-OH-dG. The hydroxyl free radical scavenging agents, thiourea and ethanol, were very effective in quenching Fe(11) mediated 8-OH-dG formation, but superoxide dismutase had very little effect.     

Spontaneous oxidative DNA damage in bovine retained and nonretained placental membranes

Retention of fetal membranes (RFM) is believed to be associated with conditions of oxidative stress. In this study, 8-hydroxy-2'-deoxyguanosine (8-OH-dG) was used for the determination of spontaneous oxidative DNA lesions in maternal and fetal parts of bovine retained and nonretained placentas. Placental specimens were collected directly after spontaneous delivery or during cesarean section from cows divided into 6 groups: (A) cesarean section before term without RFM, (B) with RFM, (C) cesarean section at term without RFM, (D) with RFM, (E) spontaneous delivery at term without RFM and (F) with RFM. Isolated DNA was hydrolyzed and analyzed by HPLC; native nucleosides were monitored at 254 nm and 8-OH-dG by electrochemical detection. No significant differences in 8-OH-dG levels between retained and nonretained placental tissues were found in all samples from preterm groups (mean concentrations between 13 and 42 micromol/mol deoxyguanosine (dG)). In the term cesarean section group with RFM a significant increase in 8-OH-dG concentration in DNA from maternal (8-fold) and fetal (18-fold) membranes were detected when compared to the respective nonretained tissues. Also, in the term spontaneous delivery groups maternal nonretained placental tissues showed increased levels of 8-OH-dG in comparison to the respective tissues of the retained placenta group. In placental tissues oxidative DNA lesions appear to be controlled by responsive mechanisms which, possibly following exhaustion, give rise to increased 8-OH-dG levels.     

Hydroxyl Free Radical Adduct of Deoxyguanosine: Sensitive Detection and Mechanisms of Formation

DNA or 2-deoxyguanosine reacts with hydroxyl free radical to form 8-hydroxy-deoxyguanosine (8-OH-dG). We found that 8-OH-dG can be effectively separated from deoxyguanosine by high pressure liquid chromatography and very sensitively detected using electrochemical detection. The sensitivity by electrochemical detection is about one-thousand fold enhanced over optical detection. Utilizing deoxyguanosine in bicarbonate buffer it was found that ferrous ion, but not ferric ion, was effective in forming 8-OH-dG. The hydroxyl free radical scavenging agents, thiourea and ethanol, were very effective in quenching Fe(11) mediated 8-OH-dG formation, but superoxide dismutase had very little effect.     

Electrochemical performance of 8-hydroxy-2'-deoxyguanosine and its detection at poly(3-methylthiophene) modified glassy carbon electrode

8-Hydroxy-2'-deoxyguanosine (8-OH-dG) has attracted enormous attention in recent years because it has been acknowledged as a typical biomarker of oxidative DNA damage. In this paper, the electrochemical performance of 8-OH-dG at the poly(3-methylthiophene) (P3MT) modified glassy carbon electrode (GCE) was investigated by cyclic voltammetry (CV) and linear sweep voltammetry (LSV). The conducting polymer P3MT can effectively decrease the oxidation peak potential of 8-OH-dG and greatly enhance its peak current. In 0.1 M pH 7.0 phosphate buffer solution (PBS), the anodic peak currents of cyclic voltammograms are linear with the 8-OH-dG concentration in two intervals, viz. 0.700-35.0 microM and 35.0-70.0 microM, with the correlative coefficients of 0.9992 and 0.9995, respectively. The detection limit of 8-OH-dG can be estimated to be 0.100 microM (S/N=3). This modified electrode can be used to detect the amount of 8-OH-dG in human urine. Furthermore, the effects of scan rate, pH, and interference of uric acid (UA) for the voltammetric behavior and detection of 8-OH-dG are also discussed. This proposed modified electrode also shows excellent reproducibility and stability that makes it an ideal candidate for amperometric detection of 8-OH-dG in flow injection analysis (FIA) and high performance liquid chromatography (HPLC).     

Formation of the oxidative damage marker 8-hydroxy-2'-deoxyguanosine from the nucleoside 2'-deoxyguanosine: parameter studies and evidence of Fe(II) binding

High-performance liquid chromatography (HPLC) with UV absorption detection was employed to measure the amounts of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) produced from the nucleoside 2'-deoxyguanosine (dG) under varying reaction conditions using iron and H(2)O(2). The results indicate that 8-OH-dG produced from the reaction of iron and H(2)O(2) with dG can undergo reaction with free (i.e., unchelated) Fe(III) and that adding the chelating agent ethylenediaminetetraacetic acid (EDTA) after the reaction prevents this from occurring. It also appears that the free radical species generated by iron-EDTA chelates in pH 7.4 N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (Hepes) buffer is either not formed or unstable in unbuffered aqueous solution. Finally, 8-OH-dG levels are significantly larger when Fe(II) is allowed to bind to the nucleoside dG prior to addition of H(2)O(2). However, production of 8-OH-dG from unbound Fe(II) is also relevant. The results of this work show that differing reaction conditions in vivo, especially at the cellular level, will affect significantly the measured yields of 8-OH-dG. These results also have implications for studies involving DNA and the ability to distinguish between 8-OH-dG produced from free iron and iron bound to both phosphate groups and the DNA base guanine.     

Increased cellular proliferation in adipose tissue of adult rats fed a high-fat diet.

The feeding of a high-fat diet to adult rats was shown to increase the incorporation of [3H]thymidine into DNA of the adipocyte and stromal fractions. After only 2 days on a high-fat diet there was a marked increase in the incorporation of label. When a 2-week period was interposed between [3H]thymidine administration and determination of DNA specific activity, the greatest increase in incorporation of label was found after 1 week on the diet, when incorporation increased 6-fold or more in both adipocytes and stroma and subsequently decreased to stabilize at a level two or three times that of chow-fed rats in the adipocyte fraction. Rats labeled when young and later placed on a high-fat diet showed a decrease in DNA specific activity in both adipocytes and stroma, confirming that cellular proliferation had occurred in both fractions. The specific activities of both stromal and adipocyte DNA were very similar at all time points studied. An attempt to increase the difference in specific activities by waiting many weeks after [3H]thymidine injection before isolating DNA was not successful. This may be because the total amount of DNA in the stromal and adipocyte fractions increases in parallel on the diet. The significance of these findings in terms of the normal turnover of adipose tissue DNA and the responsiveness to diet is discussed.     

Cigarette Smoking Induces Formation of 8-Hydroxydeoxyguanosine,One of the Oxidative Dna Damages in Human Peripheral Leukocytes

Active oxygen species (AOS) such as O and H₂O₂ have been shown to be generated from both gas and tar phases of cigarette smoke and it has been suggested that they are involved in carcinogenesis due to cigarette smoking. Therefore, we investigated the effect of cigarette smoking on oxidative DNA damages in human peripheral blood cells using 8-hydroxydeoxy-guanosine (8-OH-dG) as a marker.

From ten healthy male volunteers aged 20-22 years, 5 ml of blood was taken before and 10 minutes after smoking 2 cigarettes in 10 minutes. After lysis of blood cell membranes leukocyte DNA was isolated using a DNA extractor and 8-OH-dG levels were determined using high performance liquid chromatography (HPLC) with electrochemical detection.

The mean levels of 8-OH-dG increased significantly (P <0.05) from 3.3 ± 0.8/10⁶dG (mean ± SD) to 5.1 ± 2.5 after smoking.

These results indicate that cigarette smoking induces oxidative DNA damage in peripheral blood cells in a relatively short time.     

Oxidative DNA damage in cultured cells and rat lungs by carcinogenic nickel compounds.

DNA damage in cultured cells and in lungs of rats induced by nickel compounds was investigated to clarify the mechanism of nickel carcinogenesis. DNA strand breaks in cultured cells exposed to nickel compounds were measured by using a pulsed field gel electrophoresis technique. Among nickel compounds (Ni(3)S(2), NiO (black), NiO (green), and NiSO(4)), only Ni(3)S(2), which is highly carcinogenic, induced lesions of both double- and single-stranded DNA in cultured human cells (Raji and HeLa cells). Treatment of cultured HeLa cells with Ni(3)S(2) (10 microg/ml) induced a 1.5-fold increase in 8-hydroxy-2'-deoxyguanosine (8-OH-dG) compared with control, whereas NiO (black), NiO (green), and NiSO(4) did not enhance the generation of 8-OH-dG. Intratracheal instillation of Ni(3)S(2), NiO(black), and NiO(green) to Wistar rats increased 8-OH-dG in the lungs significantly. NiSO(4) induced a smaller but significant increase in 8-OH-dG. Histological studies showed that all the nickel compounds used induced inflammation in lungs of the rats. Nitric oxide (NO) generation in phagocytic cells induced by Ni(3)S(2), NiO(black), and NiO(green) was examined using macrophage cell line RAW 264.7 cells. NO generation in RAW 264.7 cells stimulated with lipopolysaccharide was enhanced by all nickel particles. Two mechanisms for nickel-induced oxidative DNA damage have been proposed as follows: all the nickel compounds used induced indirect damage through inflammation, and Ni(3)S(2) also showed direct oxidative DNA damage through H(2)O(2) formation. This double action may explain relatively high carcinogenic risk of Ni(3)S(2).     

Urinary excretion of biomarkers for radical-induced damage in rats treated with NDMA or diquat and the effects of calcium carbimide co-administration

The urinary excretion of seven aldehydes, acetone, coproporphyrin III and 8-hydroxy-2'-deoxyguanosine (8-OH-dG) as non-invasive biomarkers of oxidative damage was measured in rats treated with diquat or N-nitrosodimethylamine (NDMA), two compounds causing hepatic damage by different mechanisms. Furthermore, the effect of co-administration of the aldehyde dehydrogenase inhibitor, calcium carbimide (CC) on the urinary excretion of the aldehydes was determined. Slight hepatotoxicity was found at the end of the experiment after treatment with NDMA (0.5, 4 and 8 mg/kg at t = 0, 48 and 96 h, respectively) or diquat (6.8 and 13.6 mg/kg at t = 0 and 48 h, respectively). In diquat treated rats slight nephrotoxicity was also found. Urinary excretion of aldehydes, acetone and coproporphyrin III remained largely unchanged in rats treated with NDMA. In the rats treated with diquat, the urinary excretion of several aldehydes was several-fold increased. An increase was also found in the urinary excretion of 8-OH-dG after the second dose of diquat. Treatment of rats with CC did not significantly influence the urinary excretion of aldehydes in control and NDMA rats. However, in rats treated with diquat, CC caused a potentiating effect on the excretion of acetaldehyde, hexanal and malondialdehyde (MDA), indicating that oxidation of aldehydes to carbonylic acids by aldehyde dehydrogenases (ALDHs) might be an important route of metabolism of aldehydes. In conclusion, increased urinary excretion of various aldehydes, acetone, coproporphyrin III and 8-OH-dG was observed after administration of diquat, probably reflecting oxidative damage induced by this compound. No such increases were found after NDMA administration, which is consistent with a different toxicity mechanism for NDMA. Therefore, excretion of aldehydes, acetone, coproporphyrin III and 8-OH-dG might be used as easily accessible urinary biomarkers of free radical damage.     

Simultaneous determination of O6-methyl-2'-deoxyguanosine, 8-oxo-7,8-dihydro-2'-deoxyguanosine, and 1,N6-etheno-2'-deoxyadenosine in DNA using on-line sample preparation by HPLC column switching coupled to ESI-MS/MS

O(6)-Methyl-2'-deoxyguanosine (O(6)-mdGuo), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), and 1,N(6)-etheno-2'-deoxyadenosine (epsilondAdo) are promutagenic DNA lesions originating from both endogenous and exogenous agents and actions (methylation, hydroxylation, lipid peroxidation products). A highly sensitive quantitative method was developed to measure these DNA adducts simultaneously, using liquid chromatography tandem mass spectrometry with column switching. Deuterated O(6)-[(2)H(3)]mdGuo was synthesized and used as internal standard. The limits of quantification for O(6)-mdGuo, 8-oxodGuo, and epsilondAdo were 24, 98, and 48 fmol on column, respectively. The method showed linearity in the range 0.24-125 pmol/ml, 0.98-125 pmol/ml, and 0.49-62.5 pmol/ml for the three adducts, respectively. The inter-day precision in the linear concentration range was between 1.7 and 9.3% for O(6)-mdGuo, 10.6 and 28.7% for 8-oxodGuo, and 6.2 and 10.4%, for epsilondAdo. In DNA isolated from liver of untreated 12-week-old female F344 rats, O(6)-mdGuo was above the limit of detection (37 adducts per 10(9) normal nucleosides) but could not be quantified. 8-oxodGuo and epsilondAdo showed background levels of 500 and 130 adducts per 10(9) normal nucleosides, respectively. DNA analyzed 1h after treatment of rats with dimethylnitrosamine by oral gavage of 50 microg/kg b.wt. did not affect the levels of 8-oxodGuo and epsilondAdo but resulted in 200 O(6)-mdGuo adducts per 10(9) normal nucleosides. The method developed will be of use to study the biological significance of exogenous DNA adducts as an increment to background DNA damage and the role of modulating factors, such as DNA repair.