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.

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)

Non-induction of extrahepatic antipyrine and metronidazole metabolism evaluated from partially hepatectomized rats.

1. The effect of beta-naphthoflavone (BNF), given i.p. (n = 9) and orally (n = 9), on the metabolism of antipyrine and metronidazole was investigated in rats. 2. The clearances of antipyrine and metronidazole were determined on a single saliva sample. The rates of formation of antipyrine and metronidazole metabolites were determined from a 20 h urine sample and saliva clearance. 3. Administration of beta-naphthoflavone i.p. was significantly more effective than oral dosage on the induction of antipyrine and metronidazole metabolism (p < 0.05). 4. The capacity of extrahepatic tissues to metabolize antipyrine and metronidazole was quantitatively assessed in rats with and without pretreatment with beta-naphthoflavone immediately after sham operation or 70% partial hepatectomy (n = 40). 5. Antipyrine and metronidazole clearances correlated with liver weight in induced and non-induced rats. Linear regression of antipyrine and metronidazole clearances did show a non-significant Y-intercept (p > 0.05), indicating a negligible extrahepatic metabolism in both induced and in non-induced rats. 6. From a quantitative point of view this study indicates that induction of extrahepatic cytochrome P450 metabolism of antipyrine and metronidazole is negligible.