[CHARACTERISTICS OF THE RETINA IN CHRONIC STRESS IN LABORATORY RATS OF DIFFERENT AGE GROUPS].

The retina was studied in albino laboratory male rats of two age groups (12 and 24 months), 10 animals in each subjected to chronic combined stress. The stress was caused in animals by simultaneous exposure to pulsed light, loud sound, swinging and restriction of mobility for 7 days, 30 mm daily. The retina of intact rats of the corresponding age groups (n = 20) served as control. Enucleated eyes of stressed and control animals were processed with standard histological technique and stained with Nissl's method and hematoxylin-eosin. The retina of the stressed animals of both age groups showed the decrease in the number of cells and the disarrangement of its layers, most pronounced in the layers of photoreceptor neurons and ganglion cells. The comparative morphometric analysis demonstrated a reduction of the layer thickness and cell numerical density in the retina of stressed animals, both young (12 months) and old (24 months), as compared to that of control animals.

Modulation of catalase peroxidatic and catalatic activity by nitric oxide.

Previously, we found that catalase enhanced the protection afforded by superoxide dismutase to Escherichia coli against the simultaneous generation of superoxide and nitric oxide (Brunelli et al., Arch. Biochem. Biophys. 316:327-334, 1995). Hydrogen peroxide itself was not toxic in this system in the presence or absence of superoxide dismutase. We therefore investigated whether catalase might consume nitric oxide in addition to hydrogen peroxide. Catalase rapidly formed a reversible complex stoichiometrically with nitric oxide with the Soret band shifting from 406 to 426 nm and two new peaks appeared at 540 and at 575 nm, consistent with the formation of a ferrous-nitrosyl complex. Catalase consumed more nitric oxide upon the addition of hydrogen peroxide. Conversely, micromolar concentrations of nitric oxide slowed the catalase-mediated decomposition of hydrogen peroxide. Catalase pretreated with nitric oxide and hydrogen peroxide regained full activity after dialysis. Our results suggest that catalase can slowly consume nitric oxide while nitric oxide modestly inhibits catalase-dependent scavenging of hydrogen peroxide. The protective effects of catalase in combination with superoxide dismutase may result from two actions; reducing peroxynitrite formation by scavenging nitric oxide and by scavenging hydrogen peroxide before it reacts with superoxide dismutase to form additional superoxide.

Effects of carbon dioxide/bicarbonate on induction of DNA single-strand breaks and formation of 8-nitroguanine, 8-oxoguanine and base-propenal mediated by peroxynitrite.

Carbon dioxide has been reported to react with peroxynitrite (ONOO-), a strong oxidant and nitrating agent, to form an ONO2CO2- adduct, altering the reactivity characteristic of peroxynitrite. We found that bicarbonate (0-10 mM) caused a dose-dependent increase of up to 6-fold in the formation of 8-nitroguanine in calf-thymus DNA incubated with 0.1 mM peroxynitrite, whereas it produced no apparent effect on 8-oxoguanine formation. In contrast, bicarbonate inhibited peroxynitrite-induced strand breakage in plasmid pBR322 DNA and thymine-propenal formation from thymidine. We conclude that C02/HCO3- reacts with peroxynitrite to form a potent nitrating agent, but also to inactivate hydroxyl-radical-like activity of peroxynitrous acid.

Formation of 8-nitroguanine in DNA treated with peroxynitrite in vitro and its rapid removal from DNA by depurination.

Peroxynitrite is a strong oxidant formed by reaction of nitric oxide with superoxide in inflamed tissues. We have demonstrated that 8-nitroguanine is formed dose-dependently in calf thymus DNA incubated with low concentrations of peroxynitrite in vitro. 8-Nitroguanine in acid-hydrolyzed DNA was chemically reduced into 8-aminoguanine, which was analyzed using high performance liquid chromatography with electrochemical detection. Only peroxynitrite, but not nitrite, tetranitromethane nor NO-releasing compounds, formed 8-nitroguanine. Antioxidants and desferrioxamine inhibited the reaction. 8-Nitroguanine was depurinated from DNA incubated at pH 7.4, 37 degrees C (t1/2 = approximately 4 h). Peroxynitrite did not increase 8-oxoguanine levels in DNA.

Formation of 8-nitroguanine by the reaction of guanine with peroxynitrite in vitro.

Nitric oxide and superoxide anion, both formed in inflamed tissues, react rapidly to form the peroxynitrite anion (ONOO-), a strong oxidant which can initiate reactions characteristic of hydroxyl radical (HO.), nitronium ion (NO2+) and nitrogen dioxide radical (NO2.). Peroxynitrite, therefore, may cause DNA or tissue damage, contributing to the multistage carcinogenesis process. We have studied reactions of various bases, nucleosides or deoxynucleosides with peroxynitrite in vitro. Guanine reacted rapidly with peroxynitrite under physiological conditions and formed several substances, two of which were yellow, a characteristic of nitro and nitroso compounds. On the basis of chromatographic and spectral evidence we identified the major compound (which accounts for approximately 80% of all compounds formed) as 8-nitroguanine. Its formation was maximal at approximately pH 8 and increased dose-dependently with peroxynitrite concentration, but was not dependent on guanine concentration. The presence of ferric ions, which has been shown to catalyse nitration of tyrosine, did not affect nitration of guanine. 8-Nitroguanine could act as a specific marker for DNA damage induced by peroxynitrite in inflamed tissues.

Individual values of excretion of benzo[a]pyrene metabolites and susceptibility to its carcinogenic effect in rats.

In white outbred LIO rats exposed to multiple intraperitoneal (i.p.) doses (10 mg/kg) of benzo[a]pyrene (BP) in the form of a water-lipid emulsion, individual peculiarities of the excretion of its metabolites, BP-7,8-diol and 3-hydroxy-BP (3-OH-BP) in urine and feces were detected and compared with the carcinogenic effect. Parameters of BP metabolite excretion differed from those found in our previous experiments with rats exposed to single high i.p. doses of BP (100 and 200 mg/kg), dissolved in sunflower oil [11,12]. In comparison with our previous observation, in the present study, the carcinogenic effect was considerably weaker (5/22 versus 10/19). The rats that developed tumours of internal tissues (four peritoneal malignant histiocytomas and one lung lymphosarcoma), excreted higher quantities of BP-7,8-diol in the urine than other rats. The possible implication of monitoring excretion of BP metabolites for predicting individual susceptibility to its carcinogenic effect is discussed.

Biomarkers of individual susceptibility to carcinogens: application for biological monitoring.

In order to develop new markers of individual susceptibility to various human carcinogens, we studied some parameters of formation and metabolism of carcinogens, as well as DNA adducts formation and DNA repair in animals and humans. Following an i.p. administration of benzo(a)pyrene (BP) to the rats, levels of urinary excretion of BP-7,8-diol correlated with tumour latency. A high correlation was found between excretion of this metabolite and BP-DNA adducts level in the liver. Healthy smokers excreted higher quantities of BP-7,8-diol, than smoking lung cancer patients, thus confirming the suggestion on existence of cancer-prone phenotype. N-nitroso compounds formed most efficiently in stomach juice of children with superficial gastritis who therefore could be at high risk of stomach cancer. N-ethyl-N'-nitro-N-nitrosoguanidine induced stomach cancer earlier in monkeys with a low level of DNA repair enzyme, O6-alkylguanine-DNA alkyltransferase (AGT) in gastric mucosa. Overall, these markers can be helpful in predicting individual susceptibility to carcinogens.

Biomarkers for individual susceptibility to carcinogenic agents: excretion and carcinogenic risk of benzo[a]pyrene metabolites.

In rats exposed to a single intraperitoneal dose of 200 mg/kg of the environmental carcinogen benzo[a]pyrene (BP) in sunflower oil, significant individual variations in excretion of the BP activation (BP-7,8-diol) and deactivation (3-OH-BP) derivatives were found. Most rats developed peritoneal sarcomas. Only the levels of BP-7,8-diol excreted in the urine correlated directly with the latency of tumor formation. After a similar exposure to a dose of 100 mg/kg BP, Macaca fascicularis monkeys excreted smaller quantities than rats of both metabolites. After rats were given 10 intraperitoneal injections each of 10 mg/kg of BP in a water-lipid emulsion, the excreted levels of both metabolites after the first, fifth, and tenth injection were lower than those of the rats that received 200 mg/kg. BP metabolites were also detected in the urine of lung cancer patients who were heavy smokers. The applicability of monitoring the excretion of the BP metabolites to predicting individual cancer risk is discussed.