Berberrubine attenuates potassium oxonate- and hypoxanthine-induced hyperuricemia by regulating urate transporters and JAK2/STAT3 signaling pathway.

Phellodendri Chinensis Cortex (PC) is a traditional medicinal material used to treat gout and hyperuricemia (HUA) in China. Berberine (BBR), the main component of PC, possesses anti-hyperuricemic and anti-gout effects. However, BBR exhibits low bioavailability due to its extensive metabolism and limited absorption. Thus, the metabolites of BBR are believed to be the potential active forms responsible for its in vivo biological activities. Berberrubine (BRB), one of the major metabolites of BBR, exhibits appreciable biological activities even superior to BBR. In this work, the anti-hyperuricemic efficacy of BRB was investigated in HUA model mice induced by co-administration with intraperitoneal potassium oxonate (PO) and oral hypoxanthine (HX) for 7 days. Results showed that administration with BRB (6.25, 12.5, and 25.0 mg/kg) significantly decreased the serum levels of uric acid (UA) by 49.70%, 75.35%, and 75.96% respectively, when compared to the HUA group. In addition, BRB sharply decreased the levels of blood urea nitrogen (BUN) (by 19.62%, 28.98%, and 38.72%, respectively) and serum creatinine (CRE) (by 16.19%, 25.07%, and 52.08%, respectively) and reversed the PO/HX-induced renal histopathological damage dose-dependently. Additionally, BRB lowered the hepatic XOD activity, downregulated the expressions of glucose transporter 9 (GLUT9) and urate transporter 1 (URAT1), upregulated expressions of organic anion transporter 1/3 (OAT1/3) and ATP-binding cassette transporter subfamily G member 2 (ABCG2) at both protein and mRNA levels, and suppressed the activation of the JAK2/STAT3 signaling pathway. In addition, BRB significantly decreased the levels of inflammatory mediators (IL-1ß, IL-6, and TNF-α). In conclusion, our study indicated that BRB exerted anti-hyperuricemic effect, at least in part, via regulating the urate transporter expressions and suppressing the JAK2/STAT3 signaling pathway. BRB was believed to be promising for further development into a potential therapeutic agent for HUA treatment.

Mutagenic potential of hypoxanthine in live human cells.

Hypoxanthine (Hx) is a major DNA lesion generated by deamination of adenine during chronic inflammatory conditions, which is an underlying cause of various diseases including cancer of colon, liver, pancreas, bladder and stomach. There is evidence that deamination of DNA bases induces mutations, but no study has directly linked Hx accumulation to mutagenesis and strand-specific mutations yet in human cells. Using a site-specific mutagenesis approach, we report the first direct evidence of mutation potential and pattern of Hx in live human cells. We investigated Hx-induced mutations in human nonmalignant HEK293 and cancer HCT116 cell lines and found that Hx is mutagenic in both HEK293 and HCT116 cell lines. There is a strand bias for Hx-mediated mutations in both the cell lines; the Hx in lagging strand is more mutagenic than in leading strand. There is also some difference in cell types regarding the strand bias for mutation types; HEK293 cells showed largely deletion (>80%) mutations in both leading and lagging strand and the rest were insertions and A:T→G:C transition mutations in leading and lagging strands, respectively, whereas in HCT116 cells we observed 60% A:T→G:C transition mutations in the leading strand and 100% deletions in the lagging strand. Overall, Hx is a highly mutagenic lesion capable of generating A:T→G:C transitions and large deletions with a significant variation in leading and lagging strands in human cells. In recent meta-analysis study A→G (T→C) mutations were found to be a prominent signature in a variety of cancers, including a majority types that are induced by inflammation. The deletions are known to be a major cause of copy-number variations or CNVs, which is a major underlying cause of many human diseases including mental illness, developmental disorders and cancer. Thus, Hx, a major DNA lesion induced by different deamination mechanisms, has potential to initiate inflammation-driven carcinogenesis in addition to various human pathophysiological consequences.

Aag Hypoxanthine-DNA Glycosylase Is Synthesized in the Forespore Compartment and Involved in Counteracting the Genotoxic and Mutagenic Effects of Hypoxanthine and Alkylated Bases in DNA during Bacillus subtilis Sporulation.

Aag from Bacillus subtilis has been implicated in in vitro removal of hypoxanthine and alkylated bases from DNA. The regulation of expression of aag in B. subtilis and the resistance to genotoxic agents and mutagenic properties of an Aag-deficient strain were studied here. A strain with a transcriptional aag-lacZ fusion expressed low levels of ß-galactosidase during growth and early sporulation but exhibited increased transcription during late stages of this developmental process. Notably, aag-lacZ expression was higher inside the forespore than in the mother cell compartment, and this expression was abolished in a sigG-deficient background, suggesting a forespore-specific mechanism of aag transcription. Two additional findings supported this suggestion: (i) expression of an aag-yfp fusion was observed in the forespore, and (ii) in vivo mapping of the aag transcription start site revealed the existence of upstream regulatory sequences possessing homology to σG-dependent promoters. In comparison with the wild-type strain, disruption of aag significantly reduced survival of sporulating B. subtilis cells following nitrous acid or methyl methanesulfonate treatments, and the Rifr mutation frequency was significantly increased in an aag strain. These results suggest that Aag protects the genome of developing B. subtilis sporangia from the cytotoxic and genotoxic effects of base deamination and alkylation. IMPORTANCE: In this study, evidence is presented revealing that aag, encoding a DNA glycosylase implicated in processing of hypoxanthine and alkylated DNA bases, exhibits a forespore-specific pattern of gene expression during B. subtilis sporulation. Consistent with this spatiotemporal mode of expression, Aag was found to protect the sporulating cells of this microorganism from the noxious and mutagenic effects of base deamination and alkylation.

Gold(I)-triphenylphosphine complexes with hypoxanthine-derived ligands: in vitro evaluations of anticancer and anti-inflammatory activities.

A series of gold(I) complexes involving triphenylphosphine (PPh3) and one N-donor ligand derived from deprotonated mono- or disubstituted hypoxanthine (HLn) of the general composition [Au(Ln)(PPh3)] (1-9) is reported. The complexes were thoroughly characterized, including multinuclear high resolution NMR spectroscopy as well as single crystal X-ray analysis (for complexes 1 and 3). The complexes were screened for their in vitro cytotoxicity against human cancer cell lines MCF7 (breast carcinoma), HOS (osteosarcoma) and THP-1 (monocytic leukaemia), which identified the complexes 4-6 as the most promising representatives, who antiproliferative activity was further tested against A549 (lung adenocarcinoma), G-361 (melanoma), HeLa (cervical cancer), A2780 (ovarian carcinoma), A2780R (ovarian carcinoma resistant to cisplatin), 22Rv1 (prostate cancer) cell lines. Complexes 4-6 showed a significantly higher in vitro anticancer effect against the employed cancer cells, except for G-361, as compared with the commercially used anticancer drug cisplatin, with IC50 ≈ 1-30 µM. Anti-inflammatory activity was evaluated in vitro by the assessment of the ability of the complexes to modulate secretion of the pro-inflammatory cytokines, i.e. tumour necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß), in the lipopolysaccharide-activated macrophage-like THP-1 cell model. The results of this study identified the complexes as auspicious anti-inflammatory agents with similar or better activity as compared with the clinically applied gold-based antiarthritic drug Auranofin. In an effort to explore the possible mechanisms responsible for the biological effect, the products of interactions of selected complexes with sulfur-containing biomolecules (L-cysteine and reduced glutathione) were studied by means of the mass-spectrometry study.

Attenuating effect of daidzein on polychlorinated biphenyls-induced oxidative toxicity in mouse testicular cells.

The attenuating effect of daidzein (DAI) on oxidative toxicity induced by Aroclor 1254 (A1254) was investigated in mouse testicular cells. Cells were exposed to A1254 alone or with DAI. The oxidative damage was estimated by measuring malondialdehyde (MDA) formation, superoxide dismutase (SOD) activity and glutathione (GSH) content. Results show that A1254 induced a decrease of germ cell number, an elevation in thiobarbituric acid reactive substances (TBARS) but a decrease in SOD activity and GSH content. However, simultaneous supplementation with DAI decreased TBARS level and increased SOD activity and GSH content. Consequently, dietary DAI may restore the intracellular antioxidant system to attenuate the oxidative toxicity of A1254 in testicular cells.

Biochemical effects of pretreatment with vitamins E and C in rats submitted to intrastriatal hypoxanthine administration.

We previously demonstrated that intrastriatal injection of hypoxanthine, the major metabolite accumulating in Lesch-Nyhan disease, inhibited Na+,K+-ATPase activity and induced oxidative stress in rat striatum. In the present study, we evaluated the action of vitamins E and C on the biochemical alteration induced by hypoxanthine administration on Na+,K+-ATPase, TBARS, TRAP, as well as on superoxide dismutase (SOD), catalase (CAT) and glutathione-peroxidase (GPx) activities in striatum of adult rats. Animals received pretreatment with vitamins E and C or saline during 7 days. Twelve hours after the last injection of vitamins or saline, animals were divided into two groups: (1) vehicle-injected group and (2) hypoxanthine-injected group. For all parameters investigated in this research, animals were sacrificed 30 min after drug infusion. Results showed that pretreatment with vitamins E and C prevented hypoxanthine-mediated effects on Na+,K+-ATPase, TBARS and antioxidant enzymes (SOD, CAT and GPx) activities; however the reduction on TRAP was not prevented by these vitamins. Although extrapolation of findings from animal experiments to humans is difficult, it is conceivable that these vitamins might serve as an adjuvant therapy in order to avoid progression of striatal damage in patients affected by Lesch-Nyhan disease.

Intrastriatal injection of hypoxanthine reduces striatal serotonin content and impairs spatial memory performance in rats.

The aim of this study was to investigate the effects of intrastriatal injection of hypoxanthine, a metabolite accumulated in Lesch-Nyhan disease, on rats' performance in the Morris water maze tasks, along with the monoamine content in striatum of rats. Male adult Wistar rats were divided in two groups: (1) saline-injected and (2) hypoxanthine-injected group. Seven days after solutions infusion, animals were trained in the Morris Water Maze or were sacrificed for evaluation of the striatal monoamine content. Results show that hypoxanthine administration caused impairment on spatial navigation in the acquisition phase in reference memory task in the Morris Water Maze, as well as in the latency to cross over the platform location in probe trial, when compared to the saline group (control). Hypoxanthine also altered rat performance in the working memory. Although striatal dopamine metabolites content did not change, treated animals showed a reduction of tissue levels of serotonin (5-HT) and 5- hydroxyl-indoleacetic acid (5-HIAA). These results show that intra-striatal hypoxanthine administration provoked impairment of spatial learning/memory in rats without affecting striatal dopaminergic system, although serotonergic pathways seem to have been affected.

Intrastriatal hypoxanthine reduces Na(+),K (+)-ATPase activity and induces oxidative stress in the rats.

The main objective of this study was to investigate the effects of a single intrastriatal injection of hypoxanthine, a metabolite accumulated in Lesch Nyhan disease and possibly involved in its neuropathology, on Na(+),K(+)-ATPase activity, as well as on some parameters of oxidative stress, namely chemiluminescence (an index of lipid peroxidation), total radical-trapping antioxidant parameter--TRAP (an index of total antioxidant capacity of the tissue) and total thiol protein membrane content, in striatum, cerebral cortex and hippocampus of rats. Results show that hypoxanthine significantly decreased Na(+),K(+)-ATPase activity and TRAP while increased chemiluminescence in all ipsislateral structures tested. However, no effect on total thiol protein membrane content was detected. We suggest that hypoxanthine induces oxidative stress in all cerebral structures studied (striatum, hippocampus and cerebral cortex) and that the reduction of Na(+),K(+)-ATPase activity was probably mediated by reactive oxygen species.

Cancer chemopreventive activities of S-3-1, a synthetic derivative of danshinone.

Salvia miltiorrhiza is a traditional Chinese medicine which has been well documented for its anti-cancer effects. Based on the structure of danshinone, one of the active compounds derived from Salvia miltiorrhiza, we synthesized a simplified phenolic analog, S-3-1, and tried to explore its possible actions in preventing the development of cancer. With the Ames test, S-3-1 was found to efficiently suppress the mutagenicity of benzo[alpha]pyrene. This result is consistent with the inhibitory effect of S-3-1 on the activation of benzo[alpha]pyrene by hepatic microsomal enzymes. Besides the anti-initiation effects, S-3-1 could significantly inhibit the croton oil-induced increase of mouse skin epithermal ornithine decarboxylase activity. Moreover, S-3-1 quenched both superoxide and hydroxyl free radicals whereas it inhibited lipid peroxidation in the in vitro model. These results suggest that S-3-1 might act as anti-initiation and anti-promotion agents through reversing the biochemical alterations induced by carcinogen during carcinogenesis. Therefore, we further investigated the effects of S-3-1 on carcinogenesis. In vitro, S-3-1 inhibited the benzo[alpha]pyrene-induced transformation of V79 Chinese hamster lung fibroblasts. At 10-40 mg/kg, S-3-1 was found to inhibit the development of DMBA/croton oil-induced skin papilloma in mice through decreasing the incidence of papilloma, prolonging the latent period of tumor occurrence and reducing tumor number per mouse in a dose-dependent manner. We concluded from this study that S-3-1 might be developed as a new chemopreventive drug.

Carvedilol reduces mitochondrial damage induced by hypoxanthine/xanthine oxidase: relevance to hypoxia/reoxygenation injury.

The cardioprotective properties of new pharmaceuticals such as carvedilol might be explained by enhanced mitochondrial protection. The aim of this work was to determine the role of carvedilol in the protection of heart mitochondria from oxidative damage induced by hypoxanthine/xanthine oxidase, a known source of oxidative stress in the vascular system. Carvedilol reduced oxidative-stress-induced mitochondrial injury, as seen by the delay in the loss of the mitochondrial transmembranar potential (Delta Psi), the decrease in mitochondrial swelling, and the increase in mitochondrial calcium uptake. Carvedilol improved the mitochondrial respiratory activity in state III and offered an overall protection in the respiratory control and in the P/O ratios in mitochondria under oxidative stress. The data indicated that carvedilol was able to partly protect heart mitochondria from oxidative stress-induced damage. Our results suggest that mitochondria can be important targets for some cardioprotective pharmaceuticals.

Iron, alpha-tocopherol, oxidative damage and micronucleus formation in rat splenocytes.

The influence of low and high alpha-tocopherol diets in concert with a high polyunsaturated fat content and a modest increase in dietary iron has been studied. Iron supplementation at five times the recommended dietary level was not associated with any increased sensitivity of the splenocytes to any of the oxidative challenges. Despite the significantly higher alpha-tocopherol concentrations in the plasma and liver of animals supplemented with this vitamin, there was no apparent protection against oxidative genotoxicity, as judged by the formation of micronuclei in splenocytes subjected to oxidative stress ex vivo. These results add to the evidence that vitamin E supplementation has little effect against oxidative genomic damage, at least as demonstrated by an increase in micronucleus frequency.

Influence of melatonin on free radical-induced changes in rat pancreatic beta-cells in vitro.

Free radicals may produce cytotoxicity to pancreatic islets under pathophysiological conditions. The aim of our in vitro investigations was to compare functional and morphological changes in pancreatic beta-cells induced by reactive oxygen species (ROS) generated by alloxan or xanthine oxidase/hypoxanthine (XO/HX), respectively. We demonstrate that short-term exposure to alloxan or to XO/HX leads to a temporarily elevated insulin release from isolated pancreatic islets. On application of alloxan, this effect is caused by beta-cell necrosis and can be prevented by administration of melatonin, while in contrast, XO/HX did not lead to long-term morphological changes in the majority of the cells. Among the cells destroyed by alloxan, only necrosis could be detected, while in contrast, some apoptotic cells were identified by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) reaction and electron microscopic examinations of cells treated with XO/HX. Melatonin was able to prevent the changes caused by alloxan, but failed to influence the alterations caused by XO/HX. Using electron spin resonance and lipid peroxidation assay, respectively, it was confirmed that melatonin effectively detoxifies hydroxyl radicals. Therefore, we believe that hydroxyl radicals are the toxic principle of alloxan, but not of XO/HX toxicity.

The role of polymorphonuclear leukocytes and oxygen-derived free radicals in experimental acute pancreatitis: mediators of local destruction and activators of inflammation.

Using a retrograde infusion sodium taurocholate pancreatitis model in the rat treatment with oxygen radical scavengers or monoclonal anti-ICAM-1 antibody decreased tissue damage and polymorphonuclear leukocytes (PMN) infiltration. Scavengers or anti-ICAM-1 treatment attenuated the activating capacity of blood PMNs following zymosan stimulation. The local production of oxygen free radicals in the pancreas by systemic infusion of hypoxanthine and regional infusion of xanthine oxidase did not induce acute pancreatitis, although an increase of infiltrating PMNs was observed. Our data suggest that oxygen free radicals and infiltrating PMNs aggravate acute pancreatitis and that both are important mediators of local destruction and systemic activation of PMNs.

Protection against the co-operative toxicity of nitric oxide and oxygen free radicals by overexpression of antioxidant enzymes in bioengineered insulin-producing RINm5F cells.

AIMS/HYPOTHESIS: The importance of different antioxidative enzymes for the defence of insulin-producing cells against the toxicity of nitric oxide (NO) was characterised in bioengineered RINm5F cells. METHODS: RINm5F insulin-producing cells stably overexpressing glutathione peroxidase (GPX), catalase (CAT) or Cu/Zn superoxide dismutase (SOD) were exposed to S-nitroso-N-acetyl-D,L-penicillamine (SNAP), sodium nitroprusside (SNP) and 3 morpholinosydnonimine (SIN-1), which generate both NO and reactive oxygen species, and to the polyamine/ NO, complex DETA/NO which generates NO alone. The viability of the cells was tested by the MTT assay. RESULTS: Overexpression of antioxidant enzymes provided significant protection against the toxicity of SNAP, SNP and SIN-1, with an individual specificity related to their chemical characteristics, but was without effect upon the toxicity of DETA/NO. Cells overexpressing GPX were well protected against SNP and SNAP, while CAT was most effective against SIN-1. SOD overexpression provided less protection against the toxicity of SNAP and SNP than overexpression of GPX but was more effective in protecting against SIN-1. Co-incubation of cells with NO donors and hydrogen peroxide or hypoxanthine and xanthine oxidase showed an overadditive synergism of toxicity. CONCLUSION/INTERPRETATION: The results emphasise the importance of a synergism between NO and reactive oxygen species for pancreatic beta-cell death. Such a synergism has also been observed after exposure of beta cells to cytokines. The component of the toxicity that is mediated by oxygen radicals can be suppressed effectively through overexpression of CAT, GPX or SOD or both.

Morin hydrate: a better protector than purpurogallin of corneal endothelial cell damage induced by xanthine oxidase and SIN-1.

PURPOSE: Free radicals are responsible for tissue injury in corneal preservation and transplantation. Morin hydrate, a flavonoid from Brazil wood, has been shown to be cytoprotective in several types of cells. The aim of this study was to investigate the effectiveness of morin hydrate on rabbit corneal endothelial cells against damage induced by oxyradicals and nitric oxide. METHODS: Corneal endothelial cell cultures were prepared from New Zealand white rabbits, using standard microcarrier technique. Two free-radical-generating systems were used-17 IU/L xanthine oxidase/1 mM hypoxanthine and 5 mM 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1, a nitric oxide-donating agent). RESULTS: Over 95% of cultured corneal endothelial cells necrosed within 3.6 +/- 1.5 min after exposure to xanthine oxidase/hypoxanthine. Adding morin hydrate delayed cell necrosis to 5.8 +/- 0.3 min (0.25 mM morin hydrate), 13.3 +/- 5.0 min (0.5 mM), and 41.5 +/- 8.6 min (1.0 mM). Exposed to nitric oxide generated by SIN-1, cells necrosed by 9.5 +/- 2.5 min, versus 14.1 +/- 1.3 min (0.25 mM morin hydrate), 27.2 +/- 2.0 min (0.5 mM), and 43.3 +/- 5.4 min (1.0 mM). Morin hydrate significantly prolonged survival of cells compared to equimolar concentrations of purpurogallin, Trolox, or ascorbate (P < 0.01). CONCLUSION: This study demonstrates that morin hydrate behaves as a broad-spectrum antioxidant: it scavenges not only xanthine oxidase/hypoxanthine-generated oxyradicals, but also nonenzymatic, nitrogen-derived radicals, better than those above mentioned antioxidants. This property of morin hydrate may help prevent free radical damage in corneal preservation solutions.

Role of rebamipide on induction of heat-shock proteins and protection against reactive oxygen metabolite-mediated cell damage in cultured gastric mucosal cells.

Reactive oxygen metabolites (ROM) have been reported to be important in the pathogenesis of ischemia/ reperfusion-, ethanol-, nonsteroidal antiinflammatory drug-, or Helicobacter pylori-induced gastric mucosal injury. Rebamipide, a novel antiulcer agent, has been reported either to prevent various acute experimental gastric mucosal lesions or to accelerate the healing of chronic gastric ulcers. The underlying mechanism by which rebamipide exerts its cytoprotective effect in the damaged stomach is not fully determined. We investigated the role of rebamipide in protecting against ROM-mediated cell damage in gastric mucosal cells and in inducing cytoprotective proteins. Cells were exposed to ROM enzymatically generated by hypoxanthine-xanthine oxidase. Cytotoxicity was quantified by measuring specific 51Cr release from prelabeled cells. ROM caused dose-dependent increase in cytotoxicity and amount of thiobarbituric acid-reactive substances (TBA-RS). ROM-induced cytotoxicity and TBA-RS were dose-dependently decreased by the addition of rebamipide and/or catalase, but not by superoxide dismutase alone. The effects of rebamipide on electric spin resonance signal were investigated. We found that the DMPO spin adduct ESR signal of hydroxyl radicals (DMPO-OH) was significantly attenuated by rebamipide. Western blot analysis showed that induction of heat-shock protein (HSP70) was significantly increased following rebamipide administration in a dose-dependent manner. Based on these results, it is concluded that rebamipide exerted a protective effect on HX-XO-induced gastric mucosal cell cytotoxicity through one or more of the following mechanism(s): (1) inhibition of lipid peroxidation of the cell membrane; (2) hydroxyl radical scavenging activity; and (3) induction of cellular cytoprotective protein such as HSP70.

Role of lipid peroxidation and antioxidants in gastric mucosal injury induced by the hypoxanthine-xanthine oxidase system in rats.

Free radical-induced gastric mucosal injury was caused by severe depletion of glutathione and alpha-tocopherol. Intravenous infusion of hypoxanthine (HX) via the jugular vein and local intra-arterial infusion of xanthine oxidase (XO) via the left gastric artery caused marked gastric mucosal injury in the antrum and the corpus. This study was performed to determine whether antioxidants in the gastric mucosa are mobilized during oxidative stress in the rat stomach. The level of thiobarbituric acid (TBA) reactive substance in the gastric mucosa was not significantly changed. The levels of total glutathione and alpha-tocopherol in the gastric mucosa significantly decreased. Total superoxide dismutase (Cu/Zn-and Mn-SOD) and glutathione peroxidase activities were not significantly changed. Administration of SOD reversed the glutathione level but not the alpha-tocopherol level in the gastric mucosa. To determine the role of glutathione and alpha-tocopherol in oxidative stress, the stomach was removed from a normal, alpha-tocopherol supplemented, and glutathione-depleted rat and used for experimentation. Frozen slices of the rat stomach were infused with HX-XO then examined histochemically using cold Schiff's reagent for signs of lipid peroxidation. It was found that the alpha-tocopherol supplemented stomach inhibited lipid peroxidation induced by HX-XO. Biochemical measurements and histochemical examination showed that the glutathione-depleted frozen tissue section and the homogenate had increased by lipid peroxidation induced by HX-XO. These findings suggested that alpha-tocopherol and glutathione may play a role in protecting the gastric mucosa against oxygen free radicals.

Effects of allopurinol on free-radical-induced reduction of the proliferation of retinal pigment epithelial cells.

PURPOSE: To investigate the effects of allopurinol on free-radical-induced reduction of growth of retinal pigment epithelial (RPE) cells. METHODS: Bovine RPE cells were seeded at a density of 1 x 10(5) cells/Petri dish (60 x 15 mm) containing 4 ml MEM-Earle plus 15% FCS plus antibiotics. Twenty-four hours after seeding, cultures were exposed to a free radical generating system hypoxanthine(HX)/xanthine oxidase (XO) (HX: 0.1 micromol/l; XO: 700 microU/ml) for 10 or 60 min. Thereafter, the cells were washed. After washing the cells, allopurinol was added at 500 or 1000 micromol. To evaluate the effect of the radical generating system one group was not washed after treatment ('no wash'). The cultures were divided into eight groups: (1) no treatment (control); (2) HX/XO, no wash; (3,4) HX/XO, washed after 10 or 60 min; (5,6) HX/XO, washed after 10 or 60 min, application of 500 micromol of allopurinol; (7,8) HX/XO, washed after 10 or 60 min, application of 1000 micromol of allopurinol. Cell counts were carried out 96 h post-seeding. The values are expressed as means +/- SE. RESULTS: After 72 h (HX/XO, no wash), the radical generating system resulted in a significant decrease in cell growth as compared to controls. When being eliminated after 10 or 60 min, the radical generating system also led to significant decrease in the values as compared to controls. Allopurinol treatment: All therapy (scavenger) groups (4,5) were significantly different from the respective control group; following exposition to the radical generating system for 60 min, allopurinol showed significantly higher values when given at 1000 micromol as compared to 500 micromol. CONCLUSIONS: The results demonstrate that allopurinol, when given at a scavenger dose (500-1000 micromol), can prevent free-radical-induced cell damage and stop chain reactions. Thus, the possible beneficial value of allopurinol on diseases with involvement of oxidative tissue damage, such as age-related macular degeneration, should be investigated further.