Differential role of hydrogen peroxide and organic peroxides in augmenting asbestos-mediated DNA damage: implications for asbestos induced carcinogenesis

The incubation of asbestos with DNA in presence of peroxides augmented DNA damage several fold as compared to the damage caused by individual treatments. Asbestos in presence of hydrogen peroxide causes DNA double strand breaks, damage to its deoxyribose sugar moiety and enhanced DNA fidelity. However, only DNA double strand breaks and enhanced DNA fidelity could be recorded in presence of organic hydroperoxide/peroxide but no DNA sugar damage could be observed. Further, the extent of DNA damage could be correlated to the carcinogenic potential of asbestos fibre. Crocidolite, the most carcinogenic variety of asbestos, produces maximum damage to DNA in presence of both hydrogen peroxide and organic hydroperoxide/peroxide while chrysolite which is only a co-carcinogen produces significantly less DNA damage. The observed differences in DNA damage by hydrogen peroxide and organic hydroperoxide/peroxide have been ascribed to the differential reactivity of DNA with hydroxyl and alkoxy/aryloxy free radicals produced respectively from these inorganic and organic peroxides.     

Indole-3-propionate: a potent hydroxyl radical scavenger in rat brain

The hydroxyl radical scavenging activity of indole-3-propionate was evaluated by kinetic competition studies with the hydroxyl radical trapping reagent 2,2'-azino-bis-(3-ethyl-benz-thiazoline-6-sulfonic acid) (ABTS) and by measuring hydroxyl radical-initiated lipid peroxidation in the rat striatum. Using ABTS, the indole was shown to act as a potent hydroxyl radical scavenger with a rate constant of 7.8x1010 mol l-1 s-1. Hydroxyl radical-initiated lipid peroxidation, determined by measuring tissue malondialdehyde formation, was inhibited dose-dependently both in vitro and in vivo. Indole-3-propionate reacts with hydroxyl radicals at a diffusion controlled rate and can thereby provide on-site protection against the oxidative damage of biomolecules induced by these highly reactive and toxic oxygen intermediates. While it remains to be established if endogenous brain tissue levels of indole-3-propionate are sufficiently high to have a significant impact on total antioxidative capacity, the compound itself or a structurally related agent may be useful as an antioxidant adjuvant to combat hydroxyl radical-mediated oxidative stress.     

The role of ferredoxin-NADP+ reductase in the concerted cell defense against oxidative damage -- studies using Escherichia coli mutants and cloned plant genes

Ferredoxin-NADP+ reductases (FNR) participate in cellular defense against oxidative damage. Escherichia coli mutants deficient in FNR are abnormally sensitive to methyl viologen and hydrogen peroxide. Tolerance to these oxidants was regained by expression of plant FNR, superoxide dismutase, or catalase genes in the mutant cells. FNR contribution to the concerted defense against viologen toxicity under redox-cycling conditions was similar to that of the two major E. coli superoxide dismutases together, as judged by the phenotypes displayed by relevant mutant strains. However, FNR expression in sodA sodB strains failed to increase their tolerance to viologens, indicating that the FNR target is not the superoxide radical. Sensitivity of FNR-deficient cells to oxidants is related to extensive DNA damage. Incubation of the mutant bacteria with iron chelators or hydroxyl radical scavengers provided significant protection against viologens or peroxide, suggesting that oxidative injury in FNR-deficient cells was mediated by intracellular iron through the formation of hydroxyl radicals in situ. The NADP(H)-dependent activities of the reductase were necessary and sufficient for detoxification, without participation of either ferredoxin or flavodoxin in the process. Possible mechanisms by which FNR may exert its protective role are discussed.     

Artificial background and induced levels of oxidative base damage in DNA from human cells

The pre-mutagenic oxidative DNA base damage of 8-hydroxy-guanine is present in DNA isolated from cells and the amount present increases with exposure of cells to oxidative stress. The oxidative DNA base damage may be present before isolation of DNA or it may be produced during isolation and processing of DNA. We have found that the amount of oxidative base damage measured in DNA can be reduced to a stable lower level by adding increasing concentrations of the antioxidants desferrioxamine, histidine and reduced glutathione immediately before cell lysis. Inclusion of these antioxidants after cell lysis did not affect the level of DNA damage. Oxidative DNA base damage produced by ultraviolet A irradiation of human cells was also reduced by adding antioxidants after irradiation and before cell lysis. Thus, unidentified oxidants induced by ultraviolet A irradiation may damage DNA significantly during extractions of DNA from cells subsequent to ultraviolet A irradiation.     

Copper ion-mediated modification of bases in DNA in vitro by benzoyl peroxide

The mouse skin tumor promoter benzoyl peroxide (BzPO), in conjunction with Cu(I), causes promutagenic damage in DNA. Because free radical intermediates are produced by the reaction of BzPO with Cu(I), we sought to determine whether BzPO plus Cu(I) caused DNA base damage typical of that caused by the hydroxyl radical. A broad range of modified DNA bases were measured by GC-MS with selected-ion monitoring after exposure of purified plasmid pCMV beta gal DNA to BzPO +/- Cu(I). Exposure to BzPO/Cu(I) caused up to 20-fold increases in the levels of adenine-derived modified bases, up to 4-fold increases in guanine- and cytosine-derived modified bases, and only a < 2-fold increase in thymine-derived modified bases. The guanine-derived modified base 8-hydroxyguanine was elevated to the highest net amount, approximately 160 molecules/10(5) DNA bases. Exposure to BzPO alone or Cu(I) alone induced only minor (< < 2-fold) DNA base modification. Also, benzoic acid, the major non-radical metabolite of BzPO, or BzPO plus Fe(II) were ineffective at inducing DNA base modification. The hydroxyl radical scavenger dimethyl sulfoxide inhibited BzPO/Cu(I)-induced base modification by 10-50%. These data suggest that the reaction of BzPO with Cu(I) generates hydroxyl radical or a similarly reactive intermediate which causes DNA base damage. This damage may be responsible for BzPO/Cu(I)-mediated mutagenesis.     

Relationship between the intracellular reactive oxygen species and the induction of oxidative DNA damage in human neutrophil-like cells

To clarify the mechanisms of intracellular induction of oxidative DNA damage, we have investigated the concentrations of intracellular reactive oxygen species and the amounts of 8-hydroxydeoxyguanosine (8OHdG), a mutagenic oxidative DNA damage, in human neutrophil-like cells, dimethylsulfoxide-differentiated HL60 (DMSO-HL60). We determined intracellular concentrations of hydrogen peroxide and superoxide by flow cytometry with dichlorofluorescein diacetate and hydroethidine, respectively. We determined the 8OHdG amounts with an electrochemical detector connected to HPLC after anaerobic sample processing. DMSO-HL60 releases superoxide upon stimulation with phorbol myristate acetate, and the released superoxide dismutates to hydrogen peroxide. Stimulation of DMSO-HL60 with 100 nM phorbol myristate acetate increased intracellular hydrogen peroxide, superoxide and 8OHdG (control). Addition of 1000 U/ml catalase decreased hydrogen peroxide (31.3% of control) and 8OHdG (20.3%). Addition of 100 U/ml SOD decreased superoxide (18.7%) and 8OHdG (41.6%). Addition of 1 mM deferoxamine decreased 8OHdG (30.4%), but increased hydrogen peroxide (129.6%). Addition of 200 microM 4-acetamido-4'- isothiocyanostilbene-2,2'-disulfonic acid decreased superoxide (59.9%) and 8OHdG (42.0%). Addition of 0.4% ethanol had no effect on superoxide concentration (102.2%), but tended to decrease hydrogen peroxide (83.5%) and 8OHdG (84.3%). Pretreatment of DMSO-HL60 with 0.1 mM FeSO4 increased 8OHdG (117.3%), but decreased hydrogen peroxide (75.8%). These findings indicate that the extracellularly released superoxide and hydrogen peroxide diffuse into the cell, but that such reactive oxygen species are not the direct molecules to induce 8OHdG. Our results suggest that 8OHdG is induced by the hydroxyl radical which is generated from intracellular hydrogen peroxide and superoxide-reduced Fe.     

Thyrotropin receptor expression in adrenal, kidney, and thymus

Inflammatory conditions characterized by neutrophil activation are associated with a variety of chronic diseases. Reactive oxygen species are produced by activated neutrophils and produce DNA damage which may lead to tissue damage. Previous studies have shown that activated murine neutrophils induce DNA strand breaks in a target plasmacytoma cell, RIMPC 2394. We studied the effect of a water soluble nitroxide anti-oxidant, Tempol, on murine neutrophil induction of DNA strand breaks in this system. Murine neutrophils were isolated from the peritoneal cavity of BALB/cAn mice after an i.p. injection of pristane oil. Neutrophils were activated by the phorbol ester PMA and co-incubated with RIMPC 2394 cells. Control alkaline elution studies revealed progressive DNA strand breaks in RIMPC cells with time. The addition of Tempol to the incubation mixture prevented DNA damage in a dose dependent fashion. Five mM Tempol provided complete protection. Tempol protection against DNA strand breaks was similar for both stimulated neutrophils and exogenously added hydrogen peroxide. Measurement of hydrogen peroxide produced by stimulated neutrophils demonstrated that Tempol did not decrease hydrogen peroxide concentration. Oxidation of reduced metals, thereby interfering with the production of hydroxyl radical, is the most likely mechanism of nitroxide protection, although superoxide dismutase (SOD) like activity and scavenging of carbon-based free radicals may also account for a portion of the observed protection. The anti-oxidant activity of Tempol inhibited DNA damage by activated neutrophils. The nitroxides as a class of compounds may have a role in the investigation and modification of inflammatory conditions.     

Hypervalent chromium mimics reactive oxygen species as measured by the oxidant-sensitive dyes 2',7'-dichlorofluorescin and dihydrorhodamine

Intracellular metabolism of the carcinogen chromate [Cr(VI)] produces the oxidative stress and oxidative DNA damage associated with its genotoxicity. Such oxidative stress has previously been measured by fluorescence using oxidant-sensitive dyes and attributed to the formation of reactive oxygen species (ROS). However, metabolism of Cr(VI) also produces Cr(IV) and Cr(V) which can directly damage biological macromolecules without forming ROS. We used the high-valence chromium species, bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) [Cr(V)-EHBA], to test whether high-valence chromium would also react with the oxidant-sensitive dyes 2',7'-dichlorofluorescin (DCFH) and dihydrorhodamine (DHR). Cr(V)-EHBA caused both dyes to fluoresce over a wide dynamic range and under conditions which indicated that Cr(V) had reacted directly with both dyes without first forming a diffusible radical species. Dimethylthiourea (DMTU) and ethanol did not affect Cr(V)-induced fluorescence in vitro or Cr(VI)-induced fluorescence in A549 cells. Under the same conditions, ethanol and DMTU increased the extent of hydrogen peroxide-induced fluorescence. As chromium-induced fluorescence was unaffected by radical scavengers and was qualitatively different from hydrogen peroxide-induced fluorescence, we conclude that DCF and R123 fluorescence in chromate-treated A549 cells is a qualitative and cumulative measure of intracellular Cr(V) formation and not ROS.     

Scavenging of reactive oxygen species by letosteine, a molecule with two blocked-SH groups. Comparison with free-SH drugs

Acute production of reactive oxygen species by polymorphonuclear neutrophils during the respiratory burst may induce tissue injuries. In this in vitro study, it was demonstrated that letosteine, a mucolytic agent containing two blocked thiol groups, had antioxidant activity, but only when it was first submitted to alkaline hydrolysis. In a cell-free system, hydrogen peroxide, hypochlorous acid and hydroxyl radical concentrations were reduced by half by letosteine concentrations of 200, 15 and 350 mumol/l, respectively. The mechanism of letosteine action may be related to the -SH group liberated in vitro by hydrolysis, which seemed to react by scavenging the reactive oxygen species in the same way as acetylcysteine and MESNA, free-thiol drugs known for their antioxidant properties. So, letosteine, a compound with blocked -SH groups which in vivo can metabolically become free, may have a therapeutic application in preventing oxidative tissue injury damage induced by the respiratory burst.     

Oxidative damage does not alter membrane phospholipid asymmetry in human erythrocytes

Oxidant-induced damage has been proposed to be the underlying mechanism for loss of membrane phospholipid asymmetry in the erythrocyte membrane. In sickle cell disease, thalassemia, and diabetes as well as in senescent erythrocytes, an apparent correlation between oxidative damage and loss of phosphatidylserine asymmetry has been reported. In the present study, erythrocytes were subjected to various levels of oxidative stress and/or sulfhydryl modifying agents. The transmembrane location of phosphatidylserine (PS) was assessed by FITC-conjugated annexin V labeling and the PS-dependent prothrombinase assay. Transbilayer movement of spin-labeled PS was used to determine aminophospholipid translocase activity. Our data show that cells did not expose PS as the result of oxidative stress induced by phenylhydrazine, hydrogen peroxide, tert-butyl hydroperoxide, cumene hydroperoxide, or sulfhydryl modification by N-ethylmaleimide (NEM) and diamide, even under conditions that led to severe cellular damage and impairment of aminophospholipid translocase activity. In contrast, the increase of intracellular calcium induced by treatment with calcium and ionophore A23187 leads to a rapid scrambling of the lipid bilayer and the exposure of PS, which can be exacerbated by the inhibition of aminophospholipid translocase activity. Oxidation of the cells with hydrogen peroxide or phenylhydrazine did not affect A23187-induced uptake of calcium, but partly inhibited calcium-induced membrane scrambling. In conclusion, oxidative damage of erythrocytes does not induce exposure of phosphatidylserine on the membrane surface, but can interfere with both aminophospholipid translocase activity and calcium-induced randomization of membrane phospholipids.     

A Saccharomyces cerevisiae mutant defines a new locus essential for resistance to the antitumour drug bleomycin

The antitumor drug bleomycin can produce a variety of lesions in the cellular DNA by a free radical dependent mechanism. To understand how these DNA lesions are repaired, bleomycin-hypersensitive mutants were isolated from the yeast Saccharomyces cerevisiae. We report here the analysis of one mutant, DRY25, that showed extreme sensitivity to bleomycin. This mutant also exhibited hypersensitivity to hydrogen peroxide and t-butyl hydroperoxide, but showed no sensitivity to other DNA-damaging agents, including gamma-rays, ultraviolet light, and methyl methanesulfonate. Subsequent analysis revealed that strain DRY25 was severely deficient in the repair of bleomycin-induced DNA lesions. Under normal growth conditions, DRY25 displayed a 3-fold increase in the frequency of chromosomal translocation that was further stimulated by 5- to 15-fold when the cells were treated with either bleomycin or hydrogen peroxide, but not by methyl methanesulfonate, as compared with the wild type. Genetic analysis indicated that the mutant defect was independent of the nucleotide excision, postreplication, or recombinational DNA-repair pathways. These data suggest that one conceivable defect of DRY25 is that it lacks a protein that protects the cell against oxidative damage to DNA. A clone that fully complemented DRY25 defect was isolated and the possible roles of the complementing gene are discussed.     

Increased 8-hydroxyguanine levels in DNA and its repair activity in rat kidney after administration of a renal carcinogen, ferric nitrilotriacetate

The renal carcinogen, ferric nitrilotriacetate (Fe-NTA), is known to induce oxidative stress and the subsequent formation of a type of oxidative DNA damage, 8-hydroxyguanine (8-OH-Gua), in the rat kidney (Umemura et al., 1990). Using an improved DNA isolation method (Nakae et al., 1995), which reduces the background level of 8-OH-Gua, we found a five-fold increase in the 8-OH-Gua level in kidney DNA after a single i.p. injection of Fe-NTA. On the basis of the report that 8-OH-Gua repair activity is enhanced after cells are exposed to oxidative stress due to ionizing radiation (Bases et al., 1992), the measurement of 8-OH-Gua repair activity will also be useful to assess cellular oxidative stress. The 8-OH-Gua repair enzyme activity was determined with an endonuclease assay using a 22 mer DNA that contains 8-OH-Gua at a specific position. A five-fold increase in the 8-OH-Gua repair activity as compared with the control, was observed in the target organ, the rat kidney, 120 h after Fe-NTA administration. In the non-target organ, the liver, the increase was not as large (two-fold). This simple assay of oxidative DNA damage repair will be useful for evaluating the carcinogenicity of oxygen radical forming chemicals, in addition to chemical analyses of oxidative DNA damage.     

Heat killing of Bacillus subtilis spores in water is not due to oxidative damage

The heat resistance of wild-type spores of Bacillus subtilis or spores (termed alpha-beta-) lacking DNA protective alpha/beta-type small, acid-soluble spore proteins was not altered by anaerobiosis or high concentrations of the free radical scavenging agents ethanethiol and ethanedithiol. Heat-killed wild-type and alpha-beta- spores exhibited no increase in either protein carbonyl content or oxidized bases in DNA. These data strongly suggest that oxidative damage to spore macromolecules does not contribute significantly to spore killing by heat.     

Enhanced oxidative damage in cystic fibrosis patients

Antioxidant depletion and increased free radical production by inflammatory cells have been described in cystic fibrosis (CF) patients. To evaluate oxidative damage intensity, we measured plasma concentrations of malondialdehyde, hydroperoxides and protein carbon groups as markers of oxidative injury to lipids and proteins in a group of 101 CF patients free of acute exacerbation, and in 43-112 controls. Moreover, we estimated antioxidant function by measuring activities of erythrocyte superoxide dismutase, glutathione reductase and vitamin E concentrations. In CF patients, malondialdehyde and hydroperoxide plasma levels were significantly higher than in controls (p < 0.001). Increased lipid peroxidation was documented by these two markers. Parallel rises in protein carbonyls in plasma of CF patients were observed (p < 0.0001). These patients presented biochemical but not clinical vitamin E deficiency. Glutathione reductase and superoxide dismutase activities were significantly higher than in controls. These results show a serious imbalance of CF patients between oxidant-antioxidant status leading to oxidative stress.     

Effect of antithyroid drugs on hydroxyl radical formation and alpha-1-proteinase inhibitor inactivation by neutrophils: therapeutic implications

The release of proteolytic enzymes and generation of strong oxidants such as the hydroxyl radical by activated neutrophils has been proposed to play an important role in mediating toxin-induced liver injury. The antithyroid drug propylthiouracil protects against liver injury induced by many hepatotoxic agents and markedly reduces mortality in patients with alcoholic liver disease. However, the mechanism(s) by which propylthiouracil protects against liver injury is not well understood. The present studies investigate the effect of antithyroid drugs on proteolytic enzyme activity and on hydroxyl radical generation from activated neutrophils. In the presence of hydrogen peroxide and chloride, neutrophil myeloperoxidase, an enzyme from the same gene superfamily as thyroid peroxidase, generates hypochlorous acid which inactivates alpha-1-proteinase inhibitor (A1PI) present in serum. This inactivation allows neutrophil-released proteolytic enzymes to attack cells. In the present study myeloperoxidase activity was inhibited fully at therapeutic concentrations by antithyroid drugs (propylthiouracil and methimazole). Antithyroid drugs fully prevented hypochlorous acid formation, and prevented neutrophil-mediated inactivation of A1PI, with concomitant blockage of proteolytic activity. Conversely, generation of both superoxide and hydroxyl radicals by activated neutrophils was unaffected by propylthiouracil. The production of these oxygen radicals was fully inhibited by the NADPH oxidase inhibitor diphenylene iodonium chloride, however. These studies indicate that antithyroid drugs are unlikely to prevent cell injury by inhibiting hydroxyl radical generation or by scavenging hydroxyl radicals, but are likely to exert their hepatoprotective anti-inflammatory action by inhibiting neutrophil myeloperoxidase, an enzyme akin to thyroid peroxidase.     

Use of casual plasma glucose levels in community screening for diabetes mellitus

The purpose of this study was to evaluate the hydroxyl radical scavenging activities of hydroxybenzoic acids and their esters from both chemical and biological aspects. These activities of hydroxybenzoic acids and their related compounds were estimated by ESR-spin trapping method, in which 3,4,5-trihydroxybenzoic acid and its ethyl and propyl esters showed the highest activities as estimated by IC50 value (50% inhibition concentration of hydroxyl radicals generated in the system): 78.04 +/- 11.23, 95.95 +/- 2.64, and 86.46 +/- 2.31 microM, respectively. In addition, 3,4,5-trihydroxybenzoic acid (gallic acid) at a concentration of 25 microM, protected against dermal fibroblast cell damage induced by H2O2, and enhanced the survival to 83.8 +/- 3.1%, in which the survival of control was 44.2 +/- 1.0%. Based on these results, the pretreatment effects of 3,4,5-trihydroxybenzoic acid n-alkyl esters on cell damage induced by H2O2 were examined. The survival of fibroblasts pretreated with the esters increased depending on the alkyl chain-length. Both C12 and C16 alkyl esters gave almost complete cell survival of 89.5 +/- 2.0% and 91.3 +/- 1.0%, respectively. The order of the protective effects of the compounds was in good agreement with that of their partition coefficients, suggesting that 3,4,5-trihydroxybenzoic acid alkyl esters are incorporated into fibroblasts, and thus prevent the cells from the toxicity caused by H2O2. In addition, an increase of intracellular peroxide formation in fibroblasts induced by UVA-irradiation, was suppressed to 2.27 +/- 0.41 nmol/10(4) cells by pretreatment with C16 alkyl ester at a concentration of 25 microM. Since 3,4,5-trihydroxybenzoic group has been demonstrated to possess a potent scavenging activity of hydroxyl radicals, this moiety was indicated to be important in preventing cell damage induced by UVA or H2O2: in turn, these produce hydroxyl radicals in the presence of trace metal ions such as iron and copper in cells.     

Inhibitory effect of atractylon on tert-butyl hydroperoxide induced DNA damage and hepatic toxicity in rat hepatocytes

Atractylon, a main sesquiterpenic constituent of Atractylodes rhizomes, was studied for the mechanism of its inhibitory effects on the tert-butyl hydroperoxide (t-BHP)-induced cytotoxicity and lipid peroxidation in primary culture of rat hepatocytes. In the preliminary study, atractylon showed an effective antioxidant property tested by its capacity for quenching 1,1-diphenyl-2-picrylhydrazyl radical (DPPH). Further investigations showed that atractylon at the concentrations of 0.01, 0.1 and 1.0 mg/ml decreased the formation of malondialdehyde (MDA), leakage of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) and repair synthesis of DNA induced by 30-min treatment of t-BHP (1.5 mM) in primary cultured rat hepatocytes. Addition of atractylon also attenuated the genotoxicity of t-BHP evaluated by unscheduled DNA synthesis. The sum of the results suggested that the protective effect of atractylon against oxidative stress induced by t-BHP is via its ability to quench free radicals.     

Protection of U937 cells against oxidative injury by a novel series of iron chelators

A new series of iron chelators designed to protect tissues against iron-catalysed oxidative damage is described. These compounds are aminocarboxylate derivatives bearing pendant aromatic groups. They were designed to have a relatively low affinity for both ferrous and ferric iron and to be site-specifically oxidizable by hydrogen peroxide through intramolecular aromatic hydroxylation into species with strong iron binding capacity which do not catalyse hydroxyl radical formation. Thus, at the cellular level, oxidative injury is used to convert weak iron chelators into strong iron chelators in order to promote cell survival. The purpose of this local activation process is to minimise toxicity compared to strong iron chelators which may interfere with normal iron metabolism. Compounds within this series were evaluated in vitro in view of their capacity to undergo intramolecular hydroxylation and to protect cultured cells against oxidative injury. Results show that the intramolecular aromatic hydroxylation capacity is critically dependent upon the amino carboxylate chelating moieties and the substituents of the aromatic rings. Cell protection against oxidative injury is only observed with compounds possessing sufficient lipophilicity. The monohydroxylation product of N,N'-dibenzylethylenediamine N,N'-diacetic acid, protects cells against both H2O2 and tBuOOH toxicity with IC50's of 12 and 60 microM, respectively, in agreement with the oxidative activation concept. These results represent the first step toward the development of a new strategy to safe iron chelation for the prevention of oxidative damage.     

Aromatic hydroxylation in animal models of diabetes mellitus

Although the involvement of oxidative stress is well documented in the diabetic state, the individual active oxygen species generated have not been demonstrated in animal models of diabetes currently used. Since streptozotocin-induced diabetes mellitus in animals still serves as an animal model of diabetes mellitus, but streptozotocin induces diabetes and generates oxidative stress per se, we decided to study whether aromatic hydroxylation reflecting hydroxyl radical attack was found in three animal models of diabetes mellitus without streptozotocin induction or in streptozotocin-induced diabetes only. For this purpose, we compared lipid peroxidation, aromatic hydroxylation of phenylalanine, glycoxidation in genetically determined diabetic mouse strains db/db and kk, and the diabetic BB rat to these parameters in the streptozotocin-treated rat. Kidney malondialdehyde concentrations, reflecting lipid peroxidation, pentosidine, and Nepsilon-caboxymethyllysine concentrations, reflecting glycoxidation, were significantly elevated in all diabetic groups as compared to their nondiabetic mates. Aromatic hydroxylation was significantly elevated in the streptozotocin-induced diabetic state exclusively. We conclude that biochemical, pathophysiological, and treatment studies in the streptozotocin model of diabetes mellitus may be confounded by the presence of products, reactions, and tissue damage generated by aromatic hydroxylation reflecting hydroxyl radical attack. We suggest it is not the diabetic state but streptozotocin that generates the hydroxyl radical, as reflected by aromatic hydroxylation in this model.