Radiotherapy and radiosensitivity syndromes in DNA repair gene mutations.

BACKGROUND: Ionizing radiation DNA damage is the main mechanism of radiotherapy (RT) action and the outcome of treatment and healthy tissue toxicity is influenced by a number of external and internal factors, including mutations in DNA damage recognition and repair. Disorders of DNA repair may result in increased sensitivity to cancer treatment. PURPOSE: The mechanism of DNA repair and an overview of genetic syndromes with mutations in genes involved in DNA repair clarify the accelerated carcinogenesis and increased radiosensitivity in RT cancers. Most radiosensitivity syndromes are autosomal recessively inherited; examples are ataxia teleangiectasia, Nijmegen breakage syndrome, xeroderma pigmentosum, Cockayne syndrome, Bloom syndrome and Werner syndrome. CONCLUSION: Radiotherapy is contraindicated in most homozygous patients with recessive radiosensitivity syndromes. Asymptomatic heterozygotes may have an increased risk of tumor incidence and a small part of them slightly increased risk of RT intolerance; however, this does not limit RT treatment. The high risk of secondary malignancies after radiotherapy is a contraindication to adjuvant RT in Li-Fraumeni syndrome.

Changes of corneal optical properties after UVB irradiation investigated spectrophotometrically.

Ozone depletion leads to an increase in UV rays of solar radiation reaching the surface of the Earth which is harmful to biological systems. Of the eye, the cornea is directly open to increased amount of UV rays of which mainly UVB rays are capable to induce reactive oxygen species damaging the cells. Previous studies showed that the irradiation of the cornea with UVB rays leads to morphological as well as metabolic disturbances of the cornea. Also, corneal hydration and corneal light absorption are increased after UVB rays. These changes were observed after five days of repeated irradiation of the cornea with UVB rays. The aim of the present paper was to examine how early the changes of corneal hydration and light absorption occur after UVB irradiation. The rabbit corneas were irradiated with UVB rays for one, two, three or four days. Corneal light absorption was examined spectrophotometrically and corneal hydration measured by pachymeter (as corneal thickness). Results show that changes of corneal hydration and light absorption appear early after UVB irradiation and increase along with the number of irradiations. In conclusion, irradiation of the rabbit cornea with UVB rays leads to harmful changes of its optical properties.

Differences in activities of antioxidant superoxide dismutase, glutathione peroxidase and prooxidant xanthine oxidoreductase/xanthine oxidase in the normal corneal epithelium of various mammals.

Under normal conditions, antioxidants at the corneal surface are balanced with the production of reactive oxygen species without any toxic effects. Danger from oxidative stress appears when natural antioxidants are overwhelmed leading to antioxidant/prooxidant imbalance. The aim of the present study was to examine the activities of enzymes contributing to the antioxidant/prooxidant balance in normal corneal epithelium of various mammals. The enzyme activities of antioxidant superoxide dismutase and glutathione peroxidase, as well as prooxidant xanthine oxidoreductase/xanthine oxidase were examined using biochemical methods. Results show that superoxide dismutase activity is high in rabbits and guinea pigs, whereas in pigs the activity is low and in cows it is nearly absent. In contrast, glutathione peroxidase activity is high in cows, pigs and rabbits, whereas in guinea pigs the activity is low. As far as prooxidant enzymes are concerned, elevated xanthine oxidoreductase/xanthine oxidase activities were found in rabbits, lower activities in guinea pigs, very low activity in cows and no activity in pigs. In conclusion, the above results demonstrate inter-species variations in activities of enzymes participating in antioxidant/prooxidant balance in the corneal epithelium. It is suggested that the levels of antioxidant and prooxidant enzymes studied in the corneal epithelium might be associated with the diurnal or nocturnal activity of animals. UV rays decompose hydrogen peroxide to damaging hydroxyl radicals and perhaps for this reason large animals with diurnal activity (cow, pig) require more effective peroxide removal (high glutathione peroxidase activity) together with the suppression of peroxide production (low superoxide dismutase activity, low xanthine oxidoreductase activity).

Irradiation of the rabbit cornea with UVB rays stimulates the expression of nitric oxide synthases-generated nitric oxide and the formation of cytotoxic nitrogen-related oxidants.

Until now, the role of nitric oxide (NO) in cornea irradiated with UVB rays remains unknown. Therefore, we investigated nitric oxide synthase isomers (NOS), enzymes that generate NO, nitrotyrosine (NT), a cytotoxic byproduct of NO, and malondialdehyde (MDA), a byproduct of lipid peroxidation, in rabbit corneas repeatedly irradiated with UVB rays (312 nm, 1x daily for 6 days, the dose per day 1.01 J/cm2) using immunohistochemical methods. The biochemical measurement of nitrite and nitrate has been used for the indirect investigation of NO concentration in the aqueous humor. Results show that in contrast to normal corneas, where of the NOS isomers only endothelial nitric oxide synthase (NOS3) was expressed in a significant amount (in the epithelium and endothelium), in irradiated corneas all NOS isomers (also brain nitric oxide synthase, NOS1, and inducible nitric oxide synthase, NOS2) as well as an indirect measure of ONOO-formation and MDA were gradually expressed, first in the epithelium, the endothelium and the keratocytes beneath the epithelium and finally in the cells of all corneal layers and the inflammatory cells that invaded the corneal stroma. This was accompanied by an elevated concentration of NO in the aqueous humor. In conclusion, repeated irradiation with UVB rays evoked the stimulation of NO production, peroxynitrite formation (demonstrated by NT residues) and lipid peroxidation (evaluated by MDA staining).

Effects of inhibition of urokinase-type plasminogen activator (u-PA) by amiloride in the cornea and tear fluid of eyes irradiated with UVB.

The purpose of the present study was to test our hypothesis that amiloride, a specific u-PA inhibitor, effectively decreases u-PA activity in cornea as well as in tear fluid and favourably affects corneal healing. Therefore, comparative histochemical and biochemical studies of u-PA and the effects of amiloride were performed on rabbit corneas and tear fluid using the sensitive fluorogenic substrate Z-Gly-Gly-Arg-7-amino-4-trifluoromethylcoumarin. Rabbit eyes were repeatedly irradiated with UVB for 9 days and during the irradiation topically treated with amiloride (1 mg/ml saline) or placebo (saline) (dropwise, 5 times daily). Results show that in placebo-treated eyes, UVB evoked the appearance of u-PA activity in cornea and tear fluid in early stages of irradiation, and u-PA levels increased during irradiation. Corneal epithelium was gradually lost and remnants of the epithelium as well as keratocytes in the upper part of corneal stroma showed high u-PA activity. Finally, corneas lost their epithelium completely. In corneal stroma, numerous u-PA-containing inflammatory cells were present. Corneas were vascularized. When amiloride was dropped on the eye surface on the first day of irradiation and subsequently daily until the end of the experiment, u-PA activity in both cornea and tear fluid was strongly inhibited. Corneas were covered with a continuous epithelium until the end of the experiment. The number of inflammatory cells was significantly decreased. Corneal vascularization was reduced by 50%. In conclusion, early application of amiloride inhibited u-PA activity in UVB-irradiated corneas as well as in tear fluid and diminished the development of corneal pathology.

Enzyme histochemistry of corneal wound healing.

The usefulness of enzyme histochemical methods for the localization of enzymes as catalysts of molecular interactions in the cells and tissues of healing corneal wounds is shown in rabbits. The current data on corneal wound healing in humans as well as in rabbits with particular reference to serine proteases are reviewed. Some inflammatory mediators are also discussed. Plasmin is a serine protease which is absent (or present only in very low concentration) in the tear fluid, and its activity appears under various pathological conditions in humans or following experimental injuries in rabbits. The role of increasing plasmin activity in the tear fluid in the depending upon the severity of corneal injury is evaluated. Great attention is devoted to conditions leading to long-lasting elevated levels of plasmin activity in the tear fluid correlated with corneal ulceration. The differences between the histochemical pattern of untreated corneas or corneas treated with some serine protease inhibitors are shown, and the efficacy of these drugs is discussed in light of present knowledge.

Dipeptidyl peptidase IV (DPPIV) activity in the tear fluid as an indicator of the severity of corneal injury: a histochemical and biochemical study.

Comparative histochemical and biochemical studies on the catalytically active protease Dipeptidyl peptidase IV (DPPIV), have been performed in the rabbit cornea and the tear fluid using a sensitive fluorogenic substrate, Gly-Pro-7-amino-4-Trifluoromethyl Coumarine (AFC). In both normal and experimentally injured corneas, DPPIV activity was detected histochemically and in the tear fluid biochemically. In contrast to the normal cornea where DPPIV activity was absent and in the tear fluid where it was low, during continuous wearing of contact lenses or repeated irradiation of the cornea with UVB rays, slight DPPIV activity appeared first in the superficial layers of the corneal epithelium, while later increased activity was present in the whole epithelium. This paralleled elevated DPPIV activity in the tear fluid. Moreover, during continuous contact lens wear, the increased DPPIV activity in the tear fluid was, in many cases, coincidental with the presence of capillaries in the limbal part of the corneal stroma. After severe alkali burns when corneal ulcers appeared, collagen fragments were active for DPPIV, which was associated with high DPPIV activity in the tear fluid. In conclusion, Gly-Pro-AFC was found to be useful for comparative histochemical and biochemical studies on DPPIV activity in the experimentally injured rabbit eye. Using the method of the tear film collection by a short touch of substrate punches to the respective site of the cornea or conjunctiva we can show that in experimental injuries (wearing of contact lenses, irradiation of the cornea with UVB rays), the damaged corneal cells were the main source for DPPIV activity in the tear fluid. It is suggested that the activity of DPPIV measured in the tear fluid might serve as an indicator of early corneal disorders, e.g. corneal vascularization related to contact lens wear.

Changes of superoxide dismutase, catalase and glutathione peroxidase in the corneal epithelium after UVB rays. Histochemical and biochemical study.

In this study, the effects of UVA and UVB rays on antioxidant enzymes (superoxide dismutase, glutathione peroxidase, catalase) were examined in the corneal epithelium. The corneas of albino rabbits were irradiated with a UV lamp generating UVA (365 nm wavelength) or UVB rays (312 nm wavelength), 1 x daily for 5 min, from a distance of 0.03 m, over 4 days (shorter procedure) or 8 days (longer procedure). In contrast to UVA rays, which did not evoke significant disturbances, UVB rays changed the activities of antioxidant enzymes. The longer repeated irradiation with UVB rays was performed, the deeper the observed decrease in antioxidant enzymes. The shorter procedure evoked a more profound decrease of glutathione peroxidase and catalase (the enzymes cleaving hydrogen peroxide) than of superoxide dismutase, an enzyme scavenging superoxide radical and producing hydrogen peroxide during the dismutation reaction of a superoxide free radical. This may contribute to an insufficient hydrogen peroxide cleavage at the corneal surface and danger to the cornea from oxidative damage. After the longer procedure (UVB rays), the activities of all antioxidant enzymes were very low or completely absent. In conclusion, repeated irradiation of the cornea with UVB rays evokes a deficiency in antioxidant enzymes in the corneal epithelium, which very probably contributes to the damage of the cornea (and possibly also deeper parts of the eye) from UVB rays and the reactive oxygen products generated by them.

Xanthine oxidoreductase and xanthine oxidase in human cornea.

Xanthine oxidoreductase (xanthine dehydrogenase + xanthine oxidase) is a complex enzyme that catalyzes the oxidation of hypoxanthine to xanthine, subsequently producing uric acid. The enzyme complex exists in separate but interconvertible forms, xanthine dehydrogenase and xanthine oxidase, which generate reactive oxygen species (ROS), a well known causative factor in ischemia/reperfusion injury and also in some other pathological states and diseases. Because the enzymes had not been localized in human corneas until now, the aim of this study was to detect xanthine oxidoreductase and xanthine oxidase in the corneas of normal post-mortem human eyes using histochemical and immunohistochemical methods. Xanthine oxidoreductase activity was demonstrated by the tetrazolium salt reduction method and xanthine oxidase activity was detected by methods based on cerium ion capture of hydrogen peroxide. For immunohistochemical studies. we used rabbit antibovine xanthine oxidase antibody, rabbit antihuman xanthine oxidase antibody and monoclonal mouse antihuman xanthine oxidase/xanthine dehydrogenase/aldehyde oxidase antibody. The results show that the enzymes are present in the corneal epithelium and endothelium. The activity of xanthine oxidoreductase is higher than that of xanthine oxidase, as clearly seen in the epithelium. Further studies are necessary to elucidate the role of these enzymes in the diseased human cornea. Based on the findings obtained in this study (xanthine oxidoreductase/xanthine oxidase activities are present in normal human corneas), we hypothesize that during various pathological states, xanthine oxidase-generated ROS might be involved in oxidative eye injury.

Comparative histochemical and biochemical studies on acid beta-galactosidase activity in the experimentally injured rabbit cornea and tear fluid using the sensitive substrate beta-galactoside-4-trifluoromethylumbelliferyl (HFC).

Comparative histochemical and biochemical studies on acid beta-galactosidase activity in the rabbit eye after various experimental injuries were performed using the same sensitive fluorogenic substrate beta-galactoside-4-trifluoromethylumbelliferyl (HFC). The aim of the study was to examine whether the severity of corneal damage corresponds with the level of the enzyme activity in the tear fluid. As until recently the substrate beta-galactoside-4-HFC had not been used for the histochemical detection of acid beta-galactosidase in the cornea, results obtained with this substrate in a fluorescent method were compared in parallel cryostat sections with results obtained using the substrate 5-bromo-4-chloro-3-indoxyl beta-galactoside in the indigogenic method (previously shown to be very sensitive for the detection of acid beta-galactosidase activity in the cornea). Both methods revealed similar localization and changes in enzyme activity; using beta-galactoside-4-HFC an acceptable cellular localization was achieved. For the measurement of acid beta-galactosidase activity in the tear fluid a semiquantitative biochemical method was elaborated using filter paper punches with the substrate (beta-galactoside-4-HFC) soaked with tears and incubated at 37 degrees C. The time of the first appearance of a greenish-yellow fluorescence (enzyme positivity) was recorded by UV lamp and compared with the appearance of fluorescence in calibrated punches containing known acid beta-galactosidase activities. The results show that beta-galactoside-4-HFC is useful for the biochemical assessment of acid beta-galactosidase activity in the tear fluid. Comparing histochemical and biochemical results, it can be concluded that increased enzymatic activity in tears parallels the severity of corneal damage. Further studies are necessary to evaluate whether the detection of acid beta-galactosidase activity in tears might be useful for diagnostic purposes in humans.

Reactive oxygen species (ROS)-generating oxidases in the normal rabbit cornea and their involvement in the corneal damage evoked by UVB rays.

The corneas of albino rabbits were irradiated (5 min exposure once a day) with UVB rays (312 nm) for 4 days (shorter procedure) or 8 days (longer procedure). The eyes were examined microbiologically and only the corneas of sterile eyes or eyes with non-pathogenic microbes were employed. Histochemically, the activities of reactive oxygen species (ROS)-generating oxidases (xanthine oxidase, D-amino acid oxidase and alpha-hydroxy acid oxidase) were examined in cryostat sections of the whole corneas. Biochemically, the activity of xanthine oxidoreductase/xanthine oxidase was investigated in the scraped corneal epithelium. UVB rays significantly changed enzyme activities in the corneas. In comparison to the normal cornea, where of ROS-generating oxidases only xanthine oxidase showed significant activity in the corneal epithelium and endothelium, D-amino acid oxidase was very low and alpha-hydroxy acid oxidase could not be detected at all, in the cornea repeatedly irradiated with UVB rays, increased activities of xanthine oxidase and D-amino acid oxidase were observed in all corneal layers. Only after the longer procedure the xanthine oxidase and D-amino acid oxidase activities were decreased in the thinned epithelium in parallel with its morphological disturbances. Further results show that the xanthine oxidase/xanthine oxidoreductase ratio increased in the epithelium together with the repeated irradiation with UVB rays. This might suggest that xanthine dehydrogenase is converted to xanthine oxidase. However, in comparison to the normal corneal epithelium, the total amount of xanthine oxidoredutase was decreased in the irradiated epithelium. It is presumed that xanthine oxidoreductase might be released extracellularly (into tears) or the enzyme molecules were denatured due to UVB rays (particulary after the longer procedure). Comparative histochemical and biochemical findings suggest that reactive oxygen species-generating oxidases (xanthine oxidase, D-amino acid oxidase) contribute to the corneal damage evoked by UVB rays.

UV Rays, the prooxidant/antioxidant imbalance in the cornea and oxidative eye damage.

In this minireview, the factors involved in the development of corneal injury due to an increased amount of UVB rays are summarized. Experimental studies have shown that an increased number of UVB rays leads to a profound decrease in corneal antioxidants (high molecular weight, antioxidant enzymes as well as low molecular weight, mainly ascorbic acid) so that a prooxidant/antioxidant imbalance appears. The decrease of corneal antioxidant protective mechanisms results in oxidative injury of the cornea and causes damage of the inner parts of the eye by UVB rays and by reactive oxygen species generated by them.

The damaging effect of UV rays (with the wavelength shorter than 320 nm) on the rabbit anterior eye segment. I. Early changes and their prevention by catalase-aprotinin application.

In experiments carried out in rabbit eyes, UV rays of 254 or 312 nm wavelength damaged the anterior eye segment, whereas those of 365 nm wavelength did not. Two min irradiation with 254 nm UV rays led to a decrease of catalase activity in the corneal epithelium. After 5 min irradiation the catalase activity in the epithelium was not detectable at all. Catalase activity was also diminished in the corneal endothelium and lens epithelium. In this stage the changes were accompanied by decreased activities of Na(+)--K(+)-dependent adenosine triphosphatase, gamma-glutamyl transpeptidase and increased activities of lysosomal enzymes in the corneal and lens epithelium as well as in the corneal endothelium. The transparency of the cornea and lens was decreased. Plasmin activity appeared in the tear fluid. The irradiation with UV rays of 312 nm caused similar disturbances, however, a longer exposure was necessary. In contrast, irradiation with UV rays of 365 nm did not produce any changes. The described corneal disturbances were prevented by dropping of catalase solution on the eye surface during the irradiation or shortly after it. However, after a protracted irradiation aprotinin had to be added to catalase to achieve the healing. The decrease of catalase activity and its prevention by a local application of catalase suggests a key role of oxyradicals in the damage of the eye by UV rays.

Biochemical and histochemical studies of plasminogen activator of urokinase type (u-PA) activity. I. A simple rapid semiquantitative fluorescent method for its detection in the tear fluid.

A simple rapid fluorescent method for the detection of plasminogen activator activity of urokinase type (u-PA) in the tear fluid is described. Small filter paper punches were soaked in the substrate solution (Z-Gly-Gly-Arg-trifluoromethylcoumarinyl-7-amide, 1 mg/1 ml) and aprotinin 100 micrograms/1 ml) dissolved in 0.1 M Tris-HCl buffer, pH 7.2 and dried. The dried punches were soaked with tears (by direct contact of the punch with the site where the activity should be assessed or by dropping of 3-5 microliters of tears collected by a glass micropipette). The punches were incubated in a thermostat (37 degrees C) together with punches containing a known u-PA activity (calibrated punches) in preheated (37 degrees C) Petri dishes. In 1 min intervals (during the first 15 min) and in 5 min intervals thereafter the probes were exposed to UV light, and the time of the first appearance of a bright yellow fluorescence was recorded. In punches containing 5 IU u-PA activity fluorescence appeared after 2 min incubation; 2.5 IU were detected after 5 min, 1.25 IU after 15 min, 0.625 IU after 30 min, 0.313 IU after 60 min, 0.156 IU after 90 min, and 0.078 IU after 120 min incubation. This simple method is recommended for use particularly in clinical laboratories. It enables e.g. to obtain a rather quick information about the urokinase activity in the tear fluid and to start the treatment with an appropriate inhibitor, if necessary.

Histochemical study on xanthine oxidase activity in the normal rabbit cornea and lens and after repeated irradiation of the eye with UVB rays.

In the normal rabbit cornea and lens the activity of xanthine oxidase, an enzyme belonging to oxidases generating reactive oxygen species (ROS), is present in the corneal epithelium as well as endothelium and lens epithelium. Repeated irradiation of the eyes with UVB rays (5 min 1 x daily, for 1 to 4 days) caused a gradual increase of xanthine oxidase activity, particularly in the corneal epithelium. Application of catalase, a scavenger of hydrogen peroxide, to the eye surface during the irradiation diminished the increase of xanthine oxidase activity. On the contrary, the pretreatment of the rabbit eyes with 3-aminotriazole, an inhibitor of catalase, for 3 days before the irradiation enhanced the increase of xanthine oxidase activity. In comparison to untreated eyes, protracted irradiation of the eyes with UVB rays (up to 10 days) caused a decrease of xanthine oxidase activity in the same cell layers of the cornea and lens. It is suggested that xanthine oxidase is involved in the generation of ROS in the anterior eye segment during early irradiation of the eyes with UVB rays and participates in its damage. Prolonged repeated irradiation of the eye (5 min 1 x daily for 5 to 10 days) caused a decrease of xanthine oxidase activity in the cornea and lens which is attributed to profound damage of the whole anterior eye segment.

The damaging effect of UV rays below 320 nm on the rabbit anterior eye segment. II. Enzyme histochemical changes and plasmin activity after prolonged irradiation.

Prolonged irradiation of the rabbit eyes with UVB rays (312 nm) caused serious enzymatic disturbances in the cornea and lens and the development of an inflammatory reaction in the whole anterior eye segment, particularly in the cornea. In the corneal stroma many inflammatory cells with high activities of acid glycosidases and lysosomal proteases were present. This was accompanied with significantly elevated plasmin activity in the tear fluid (1.6 IU/ml). Plasmin appeared also in the aqueous humour (0.8 IU/ml). For the treatment of these changes catalase (1 mg/1 ml saline), aprotinin (100 micrograms/1 ml saline) and catalase-aprotinin mixture (1:1) were applied on the eye surface during irradiation. The catalase-aprotinin mixture was most efficient and decreased plasmin activity in the tear fluid and diminished disturbances of the anterior eye segment. Obviously both, active oxygen species and elevated plasmin activity in the tear fluid contribute to the damage of the anterior eye segment and development of intracorneal inflammation after irradiation of the eye with UVB rays.

The appearance of active plasminogen activator of urokinase type (u-PA) in the rabbit anterior eye segment irradiated by UVB rays. A histochemical and biochemical study.

Repeated irradiation of the rabbit eye with UV rays of 312 nm wavelength (UVB) evoked the appearance of active plasminogen activator of urokinase type (u-PA) in the anterior eye segment. Using histochemistry, active u-PA appeared first in the corneal epithelium followed by the corneal endothelium, inflammatory cells in the corneal stroma and the lens epithelium. With a semiquantitative fluorescent method active u-PA was also found in the tear fluid and aqueous humour. UV rays of 365 nm wavelength (UVA) under the same conditions did not cause the appearance of active u-PA in the anterior eye segment.

Reactive oxygen species (ROS) generated by xanthine oxidase in the corneal epithelium and their potential participation in the damage of the corneal epithelium after prolonged use of contact lenses in rabbits.

Prolonged use of contact lenses (for 14 days) evoked an imbalance between the activity of xanthine oxidase (an enzyme belonging to reactive oxygen species-generating oxidases) and catalase (an enzyme belonging to reactive oxygen species-scavenging oxidases) in the corneal epithelium of rabbits. The activity of catalase decreased, while xanthine oxidase activity was very high. Of other enzymes studied in the corneal epithelium, the activities of xanthine oxidoreductase, glucoso-6-phosphate dehydrogenase and succinate dehydrogenase were decreased. In contrast, the activities of lactate dehydrogenase and lysosomal hydrolases (acid beta-galactosidase, dipeptidyl peptidase II) were increased and appeared in animals sacrificed immediately after contact lens removal. In rabbits sacrificed later (after 1 h), an additional increase of lactate dehydrogenase and lysosomal hydrolase activities developed in the superficial layers of the corneal epithelium. Catalase supplementation during use of contact lenses prevented both the significant decrease of catalase activity in the corneal epithelium and the development of additional epithelial damage. In contrast, topical treatment with 3-aminotriazole (an inhibitor of catalase) resulted in the nearly complete loss of catalase activity in the corneal epithelium and the appearance of more serious epithelial damage. We conclude that ROS generated by xanthine oxidase induce additional damage of the corneal epithelium related to the use of contact lenses.

Central corneal thickness considered an index of corneal hydration of the UVB irradiated rabbit cornea as influenced by UVB absorber.

UVB radiation from sunlight induces an acute corneal inflammation, photokeratitis, accompanied by changes in corneal hydration. We employed a method of ultrasonic pachymetry for daily examination of central corneal thickness as an index of corneal hydration of the rabbit cornea repeatedly irradiated by UVB radiation (312 nm, daily dose of 0.25 J/cm(2) during three or four days) as influenced by UVB absorber (actinoquinol combined with hyaluronic acid) dropped on the ocular surface during irradiation. One day after the third irradiation procedure the animals were sacrificed and corneas examined immuno-histochemically for peroxynitrite formation, a marker of oxidative damage, the antioxidant aldehyde dehydrogenase 3A1 and endothelial nitric oxide synthase, an enzyme generated nitric oxide. Results show that UV absorber combined with hyaluronic acid protected the cornea against UVB-induced changes in corneal thickness and microscopical disturbances to the cornea (both seen after buffered saline application) until the fourth experimental day. These UVB doses are equivalent to a daily exposure of 2.5 hrs of the human cornea to solar UVB radiation for three consecutive days. It is suggested that actinoquinol/ hyaluronic acid drops might be helpful for the human eye in the defence against photooxidative and other oxidative processes.