Menadione-induced DNA fragmentation without 8-oxo-2'-deoxyguanosine formation in isolated rat hepatocytes.

Menadione (2-methyl-1,4-naphthoquinone) induces oxidative stress in cells causing perturbations in the cytoplasm as well as nicking of DNA. The mechanisms by which DNA damage occurs are still unclear, but a widely discussed issue is whether menadione-generated reactive oxygen species (ROS) directly damage DNA. In the present study, we measured the effect of menadione on formation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG), an index of oxidative DNA base modifications, and on DNA fragmentation. Isolated hepatocytes from phenobarbital-pretreated rats were exposed to menadione, 25-400 microM, for 15, 90 or 180 min with or without prior depletion of reduced glutathione (GSH) by diethyl maleate. Menadione caused profound GSH depletion and internucleosomal DNA fragmentation, which was demonstrated by a prominent fragmentation ladder on agarose gel electrophoresis. We found no oxidative modification of DNA in terms of increased 8-oxodG formation. In contrast, the positive control of sunlamp light increased 8-oxodG 5-fold in rat hepatocytes. We conclude that oxidative modification of DNA bases is unlikely to be important in menadione-induced DNA damage.

Oxidative DNA damage after transplantation of the liver and small intestine in pigs.

Oxidative damage is thought to play an important role in ischemia/reperfusion injury, including the outcome of transplantation of the liver and intestine. We have investigated oxidative DNA damage after combined transplantation of the liver and small intestine in 5 pigs. DNA damage was estimated from the urinary excretion of the repair product 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG). In the first 1-3 hr after reperfusion of the grafts, 8-oxodG excretion was increased 2.9-fold (1.7-4.1; 95% confidence intervals; P < 0.05). A control experiment included sham surgery with clamping of the suprarenal inferior caval vein in 2 pigs during steady state infusion of 8-oxodG. While the caval vein was clamped, the urinary excretion of 8-oxodG was almost blocked, whereas after removal of the clamp, the excretion returned to and did not exceed the preclamp levels. In a separate experiment with 2 pigs, the elimination of injected 8-oxodG was shown to adhere to first-order kinetics with a clearance and a terminal elimination half-life of approximately 4 ml min-1 kg-1 and 2 1/2 hr, respectively. The injected dose was completely excreted into the urine within 4 hr. It is concluded that substantial oxidative damage to DNA results from reperfusion of transplanted small intestine and liver in pigs, as estimated from the readily excreted repair product 8-oxodG.

Radiation-induced formation of 8-hydroxy-2'-deoxyguanosine and its prevention by scavengers.

Reactive oxygen species (ROS) induce 8-hydroxy-2'-deoxyguanosine (8-OHdG) formation, which has been proposed as a key biomarker relevant to carcinogenesis. 8-OHdG has been induced in a number of different ways, most often without knowledge of the specific type and amount of ROS generated. We have measured 8-OHdG formation in calf thymus DNA exposed to ionizing radiation under conditions generating either hydroxyl radicals (OH.), superoxide anions (O2-) or both. Additionally, we investigated the relationship between the scavenger effect of the drug 5-aminosalicylic acid (5-ASA) and increasing OH. exposure toward 8-OHdG formation. The effect of this drug was compared to those of the physiological scavengers ascorbate and reduced glutathione (GSH). We found that OH. generated 8-OHdG in a dose-dependent manner, whereas O2- did not cause 8-OHdG formation. 5-ASA, ascorbate and GSH all acted as hydroxyl radical scavengers, although with different concentration-effect curves, emphasizing the importance of using relevant pharmaco-/physiological concentrations in studies focusing on therapeutic applications of scavengers. The scavenger effect of 5-ASA at concentrations > or = 0.1 mM was similar at 30 and 100 Gy radiation, i.e. within a wide range of OH. exposure, which is useful information considering clinical applications where the exact amount of ROS formed is unknown. Both 5-ASA and ascorbate at low concentrations (< or = 0.1 mM) were less efficient in preventing 8-OHdG formation from X-ray generated OH. than was shown in a previous comparable study using light as the source of ROS. This differentiation probably reflects variations in both number and type of ROS formed in the two systems.

Effect of ascorbate and 5-aminosalicylic acid on light-induced 8-hydroxydeoxyguanosine formation in V79 Chinese hamster cells.

Recently we showed that ascorbate and 5-aminosalicylic acid (5-ASA) prevented 8-hydroxydeoxyguanosine (8-OHdG) formation in calf thymus DNA exposed to UV-visible light. However, the ultimate defense against oxidative DNA damage depends on an intracellular/intranuclear effect of the compounds. In the present study we investigated the effect of ascorbate and 5-ASA on 8-OHdG formation in V79 Chinese hamster cells exposed to light from a sun-lamp. Exposure for 1 min (4560 mJ/cm2) increased 8-OHdG formation in cellular DNA to 30-40 times background level. Preincubation of the cells with ascorbate or 5-ASA at concentrations of 0.1, 1 and 10 mM diminished the 8-OHdG formation to 0.67, 0.74 and 0.49 times controls (P < 0.05) for ascorbate respectively, and to 0.82, 0.66 and 0.33 times controls (P < 0.05), for 5-ASA. These findings demonstrate that both ascorbate and 5-ASA prevent oxidative DNA damage in cells by acting as intracellular/intranuclear antioxidants.

8-Hydroxydeoxyguanosine as a urinary biomarker of oxidative DNA damage.

Living organisms are continuously exposed to reactive oxygen species as a consequence of biochemical reactions as well as external factors. Oxidative DNA damage has been implicated in aging, carcinogenesis and other degenerative diseases. The urinary excretion of the DNA repair product 8-hydroxydeoxyguanosine (8OHdG) has been proposed as a noninvasive biomarker of oxidative DNA damage in humans in vivo. We have developed a three-dimensional HPLC analysis with electrochemical detection for the analysis of 8OHdG in urine and studied factors affecting the excretion of this biomarker in 83 healthy humans and in various laboratory animals, including dog, pig, and rat. Previously, other groups have used comparable HPLC methods or gas chromatography-mass spectrometry with selective ion monitoring for measuring the excretion of 8OHdG in humans, rats, mice, and monkeys. In the 169 humans studied so far, the average 8OHdG excretion was 200-300 pmol/kg per 24 h with a sevenfold range, and the coefficient of variation was 30-40%. This excretion corresponds 140-200 oxidative modification of guanine bases per cell per day. Thirty-two smokers from our study population excreted 50% (31-69%; 95% confidence interval) more 8OHdG than 53 nonsmokers. This indicates a 50% increased rate of oxidative DNA damage from smoking, adding to the other well-known health hazards of smoking. The biochemical-physiological basis is unknown but may be related to smoke constituents including or generating reactive oxygen species and/or consuming antioxidants and/or the well-known enhancing effect of smoking on the metabolic rate. In our 83 healthy subjects the 8OHdG excretion correlated with body composition. Thus, lean and/or male subjects excreted more than obese and/or female subjects, possibly related to differences in metabolic rate. In accordance, the excretion of 8OHdG decreased after calorie restriction, which will cause a decline in the metabolic rate. Across the investigated species, humans, dogs, pigs, and rats, the excretion of 8OHdG correlated with the specific metabolic rate, confirming data from other groups on humans, monkeys, rats, and mice. The excretion of 8OHdG decreased with age in rats in parallel with the decline in metabolic rate with advancing age. The excretion of 8OHdG reflects the formation and repair of only one out of approximately 20 described oxidative DNA modifications. So far, methods are not available for the determination of the corresponding repair products, except 8OHdG and thymidine glycol, in urine. Moreover, the importance in terms of mutagenicity, particularly regarding tumour suppressor genes and oncogenes, is mainly documented for 8OHdG in DNA.(ABSTRACT TRUNCATED AT 400 WORDS)

8-Hydroxydeoxyguanosine in vitro: effects of glutathione, ascorbate, and 5-aminosalicylic acid.

Oxidative DNA damage, as expressed by 8-hydroxydeoxyguanosine (8-OHdG), was investigated in calf thymus DNA exposed to either ultraviolet radiation or to FeCl2/H2O2 in a Fenton-like reaction. The influence of iron (absent in the UV system and present in the FeCl2/H2O2 system) and pH (7.4 and 4.0) on the effect of glutathione (GSH), ascorbate, and 5-aminosalicylic acid (5-ASA, a drug used in the treatment of chronic inflammatory bowel diseases) was examined in these systems. Without iron, all three compounds considerably reduced 8-OHdG formation (i.e., acted as scavengers), while in the presence of iron salts, 8-OHdG formation was accelerated (except for GSH at pH 7.4), i.e., the compounds acted as prooxidants. This effect was augmented at low pH. The prooxidant property of 5-ASA may have implications for its clinical use. Maximum scavenging effect for all the compounds investigated was obtained at much lower doses than the maximum enhancing effect. This demonstrates that to the end of oxy-radical scavenging, the concentration of the GSH, ascorbate, and 5-ASA, respectively, should be chosen to obtain maximum antioxidant effect and minimum prooxidant effects. The significance of this finding for the selection of antioxidant dose is important but remains to be investigated further.

CCl4 cirrhosis in rats: irreversible histological changes and differentiated functional impairment.

Cirrhosis of the rat liver was induced by a 12 week individualized CCl4/phenobarbital treatment. After treatment, all surviving animals (81%) showed cirrhosis of the liver. The cirrhosis induced was irreversible when evaluated 24 weeks after cessation of treatment. Quantitative liver function measurements were reduced in a differentiated manner. Ranked according to the most pronounced changes they are: capacity of urea-N synthesis (CUNS), galactose elimination capacity (GEC) and antipyrine clearance (APC). Hepatic glutathione concentrations were only slightly decreased after the CCl4 treatment. It is possible to produce a high incidence of irreversible cirrhosis with differentiated functional impairment in the rat.

Age dependence of rat liver function measurements.

Changes in the galactose elimination capacity, the capacity of urea-N synthesis and antipyrine clearance were studied in male Wistar rats at the age of 8, 20 and 44 weeks. Further, liver tissue concentrations of microsomal cytochrome P-450, microsomal protein and glutathione were measured. All liver function measurements increased from the age of 8 to 44 weeks when expressed in absolute values. In relation to body weight, these function measurements were unchanged or reduced from week 8 to week 20. At week 44, galactose elimination capacity and capacity of urea-N synthesis related to body weight were increased by 10% and 36%, respectively, and antipyrine plasma clearance was reduced to 50%. Liver tissue concentrations of microsomal cytochrome P-450 and microsomal protein increased with age when expressed in absolute values, but were unchanged per g liver, i.e., closely related to liver weight in the age range studied. Glutathione showed an increase of 35% from 8 to 44 weeks of age expressed per g liver. Careful age matching of control animals is important for experimental rat studies.