J Toxicol Pathol 2012; 25: 27–35 Original Article N-Ethyl-N-Nitrosourea Induces Retinal Photoreceptor Damage in Adult Rats Katsuhiko Yoshizawa1, Tomo Sasaki1,2, Norihisa Uehara1, Maki Kuro3, Ayako Kimura1, Yuichi Kinoshita1,4, Hisanori Miki1, Takashi Yuri1, and Airo Tsubura1 1 Department of Pathology II, Kansai Medical University, Moriguchi, Osaka 570-8506, Japan 2 Kyoto R & D Center, Maruho Co., Ltd., Kyoto, Kyoto 600-8815, Japan 3 Department of Ophthalmology, Kansai Medical University, Moriguchi, Osaka 570-8507, Japan 4 Department of Cytopathology, Kansai Medical University Takii Hospital, Moriguchi, Osaka 570-8507, Japan Abstract: Seven-week-old male Lewis rats received a single intraperitoneal injection of N-ethyl-N-nitrosourea (ENU) (100, 200, 400 or 600 mg/kg), and retinal damage was evaluated 7 days after the treatment. Sequential morphological features of the retina and retinal DNA damage, as determined by a TUNEL assay and phospho-histone H2A.X (γ-H2AX), were analyzed 3, 6, 12, 24 and 72 hr, 7 days, and/or 30 days after 400 mg/kg ENU treatment. Activation of the nuclear enzyme poly (ADP-ribose) polymerase (PARP) was analyzed immunohistochemically by poly (ADP-ribose) (PAR) expression in response to DNA damage of the retina. All rats that received ≥ 400 mg/kg of ENU developed retinal degeneration characterized by the loss of photoreceptor cells in both the central and peripheral retina within 7 days. In the 400 mg/kg ENU-treated rats, TUNEL-positive signals were only located in the photoreceptor cells and peaked 24 hr after ENU treatment. The γ-H2AX signals in inner retinal cells appeared at 24 hr and peaked at 72 hr after ENU treatment, and the PAR signals selectively located in the photoreceptor cell nuclei appeared at 12 hr and peaked at 24 hr after ENU treatment. However, degeneration was restricted to photoreceptor cells, and no degenerative changes in inner retinal cells were seen at any time points. Retinal thickness and the photoreceptor cell ratio in the central and peripheral retina were significantly decreased, and the retinal damage ratio was significantly increased 7 days after ENU treatment. In conclusion, ENU induced retinal degeneration in adult rats that was characterized by photoreceptor cell apoptosis through PARP activity. (DOI: 10.1293/tox.25.27; J Toxicol Pathol 2012; 25: 27–35) Key words: N-ethyl-N-nitrosourea, γ-H2A.X, PAR, PARP, photoreceptor cell, retinal degeneration, rat Introduction damage in adult rodents is unclear. Double-strand breaks (DSBs), the most deleterious N-Ethyl-N-nitrosourea (ENU) is an alkylating agent kind of alteration to DNA, are produced by exogenous that is particularly effective in inducing congenital malfor- agents. One of the earliest events in the DNA damage re- mations1,2 and tumors of systemic organs including the ner- sponse is the Ser139 phosphorylation of histone H2A.X vous tissue in rodents3. ENU mutagenesis has been used to (γ-H2AX), which is a critical factor for cellular protection create a large number of random point mutations in the ge- against DNA alkylating agents13–16. DNA alkylating agents nomic DNA of mice and zebrafish and is a powerful tool for also activate poly (ADP-ribose) polymerase (PARP)17. In creating animal models of disease4,5, such as morphological several retinal damage models, such as NMDA-induced defects in the eyes6,7 and retinal degeneration in the prog- ganglion cell loss18, retinal ischemia/reperfusion injury19, eny of treated animals8–10. A single intraperitoneal injection retinal degeneration 1 (rd1) mice carrying the Pde 6b gene20, of ENU also induces retinal and corneal damage in mature and N-methyl-N-nitrosourea (MNU)-induced photoreceptor mice11,12; however, the mechanism of ENU-induced retinal apoptosis21, cell death is linked to PARP hyperactivation. The purpose of this research is to evaluate the effect of ENU on the retinal cells of adult rats and to focus on the Received: 1 September 2011, Accepted: 31 October 2011 Mailing address: Katsuhiko Yoshizawa, Department of Pathology activation of γ-H2AX and PARP as DNA damage responses II, Kansai Medical University, 10-15 Fumizono, Moriguchi, Osaka in the retina after ENU exposure. 570-8506, Japan TEL: 81-6-6993-9431 FAX: 81-6-6992-5023 Materials and Methods E-mail: [email protected] ©2012 The Japanese Society of Toxicologic Pathology Animals This is an open-access article distributed under the terms of the Cre- Fifty-two male Lewis rats [LEW/CrlCrlj] obtained ative Commons Attribution Non-Commercial No Derivatives (by-nc- nd) License <http://creativecommons.org/licenses/by-nc-nd/3.0/>. from Charles River Laboratories Japan (Osaka, Japan) were 28 ENU-induced Retinal Damage in Rats used at 7 weeks of age. Rats were maintained in specific tal Pathology, Hamamatsu Photonics, Hamamatsu, Japan) to pathogen-free conditions and received free access to a com- prepare digital images. The ndpi image files were opened mercial diet (CMF 30Gy; Oriental Yeast, Chiba, Japan) and in color mode with the NDP.view software (Hamamatsu tap water. The animals were housed in plastic cages with Photonics). The total retinal thickness (from the internal paper-chip bedding (Paper Clean, SLC, Hamamatsu, Japan) limiting membrane to the pigment epithelium), inner reti- in an air-conditioned room at 22 ± 2 °C and 60 ± 10% rela- nal thickness (from the internal limiting membrane to the tive humidity with a 12-hr light/dark cycle. The illumination outer plexiform layer) and outer retinal thickness (from the intensity was less than 60 lux in the cages. All procedures outer nuclear layer to the pigment epithelial cell layer) were were in accordance with the guidelines for animal experi- individually measured from methacarn-fixed HE slides us- mentation at Kansai Medical University. ing NDP.view, as described in our previous reports11,22. The measurements were conducted at the central retina (approx- Chemical and dose formulation imately 400 μm from the optic nerve) and peripheral retina ENU (N-Nitroso-N-ethylurea bulk package®; chemi- (approximately 400 μm from both sides of the ciliary bod- cal formula, C H N O ) was purchased from Sigma-Aldrich ies). To further evaluate the photoreceptor cell loss, the pho- 3 7 3 2 Japan (Tokyo, Japan) and kept at −80 °C in the dark. It was toreceptor ratio [(outer retinal thickness / total retinal thick- dissolved in physiologic saline just prior to use. ness) × 100] was calculated. To determine the area of retinal damage, the entire length of the retina and the length of the Experimental procedures damaged area in HE preparations were measured. Damaged The experiments consisted of 2 independent studies, the retina was designated as the presence of less than four rows purpose being to confirm the dose dependency of the retinal of photoreceptor nuclei in the outer nuclear layer11,22, and the lesions in the 1st study and to confirm the sequential changes retinal damage ratio was calculated as: (length of damaged in the 2nd study. Sixteen rats received an intraperitoneal (ip) retina / whole retinal length) × 100. injection of ENU at a dose of 100, 200, 400 or 600 mg/kg (4 rats/group). Seven days after injection, rats were anesthe- TUNEL, phospho-histone H2A.X, and poly (ADP- tized with isoflurane (Forane®, Abbott Japan, Tokyo, Japan) ribose) immunohistochemistry and sacrificed by exsanguination from abdominal aortic Formalin-fixed eye sections at 6 time points (3, 6, 12, 24 transection. In another experiment, 28 rats received a single and 72 hr and 7 days) after treatment with 400 mg/kg ENU ip injection of 400 mg/kg of ENU, and 4 randomly selected and at 7 days after vehicle treatment were used for the analy- mice were sacrificed at 7 time points (3, 6, 12, 24 and 72 hr ses of cell death and DNA damage responses. Cell death was and 7 and 30 days) after treatment. In each experiment, the observed by terminal deoxynucleotidyl transferase (TdT)- control groups consisted of 4 rats that were treated with ve- mediated dUTP digoxigenin nick end-labeling (TUNEL) us- hicle (physiological saline) only. All rats were observed daily ing an in situ apoptosis detection kit (ApopTag; Millipore, for clinical signs of toxicity and were weighed at the time of Bellerica, MA, USA) according to previous reports11,22. ENU treatment and on the day of sacrifice. Both eyes were Sequential sections were immunohistochemically stained quickly removed at the time of sacrifice, and complete nec- with anti-phospho-histone H2A.X (γ-H2AX) monoclonal ropsies were conducted on all animals. antibody (Ser139, 1:200 in dilution; Cell Signaling Technol- ogy, Danvers, MA, USA), an immunomarker of the DNA Tissue fixation and processing damage response14,16,23 and anti-poly (ADP-ribose) (PAR) One eye from each rat was fixed overnight in 10% monoclonal antibody (10H, 1:200 in dilution; Enzo Life Sci- neutral buffered formalin, and the other eye was fixed over- ences International, Plymouth Meeting, PA, USA), an immu- night in methacarn (60% methanol, 30% chloroform and nomarker of the activation of poly (ADP-ribose) polymerase 10% acetic acid). Subsequently, the eyes were embedded (PARP)19–21. PAR is synthesized after the activation of the in paraffin, sectioned at a thickness of 4 μm, and stained nuclear DNA repair enzyme PARP. Antigen retrieval was with hematoxylin and eosin (HE). Ocular sections were cut necessary for γ-H2AX visualization and was conducted by along a line parallel to the optic axis and nerve (including pressure-cooker heating (Pascal, Dako, Carpinteria, CA, the ora serrata). Histologic and morphometric evaluations USA). Each primary antibody was reacted overnight under were performed by a toxicologic pathologist certified by the 4 °C, and the antigen–antibody complexes were identified Japanese Society of Toxicologic Pathology and the Inter- using an LSAB staining kit (Dako) according to the manu- national Academy of Toxicologic Pathology (K.Y.) and an facturer’s instructions. The reaction products were visual- ophthalmologist certified by the Japanese Ophthalmological ized with 3–3′-diaminobenzidine tetrahydrochloride. Society (M.K.), according to the previously defined histo- pathological terminology and diagnostic criteria11,22. Apoptotic and γ-H2AX cell ratio and PAR immunore- activity Morphometric analysis of retinal thickness, photore- TUNEL- and γ-H2AX-stained retinal sections were ceptor cell ratio and retinal damage ratio scanned with a high-resolution digital slide scanner to pre- HE-stained sections of the retina were scanned with a pare digital images. The images were captured at 40 × mag- high-resolution digital slide scanner (NanoZoomer 2.2 Digi- nification. The apoptotic cell ratio was calculated from TU- Yoshizawa, Sasaki, Uehara et al. 29 NEL-stained slides by determining the number of apoptotic significantly decreased in both the central and peripheral nuclei of photoreceptor cells per microscopic field at the retina, as compared with the control rats (Fig. 1), whereas central retina and peripheral retina. The γ-H2AX-positive 400 mg/kg ENU caused a decrease in thickness in only the cells were counted in three microscopic fields per whole central retina with statistical significance (Fig. 2a). Changes retina in a random fashion. The intensity grade of immune in the inner retinal thickness were not observed in any of the staining and expression for PAR in the retina was scored as ENU-treated groups (data not shown). To further evaluate negative, weak, moderate or strong. the ENU-induced effects on retinal thickness, the photore- ceptor cell ratio was calculated (Fig. 2a). The photoreceptor Statistical analysis cell ratios at the central and peripheral retinas were 43% All discrete values, expressed as means ± standard er- and 51% for control rats, but only 10% and 11% (P<0.01) in ror (SE), were analyzed using the two-tailed independent the 600 mg/kg ENU-treated rats and 9% (P<0.01) and 43% Student’s t-test for unpaired samples after confirming the (not significant) in the 400 mg/kg ENU-treated rats. The homogeneity of variances. The results presented below in- photoreceptor cell ratios at the central and peripheral retina clude comparisons between ENU-treated mice and vehicle- were not significantly different in rats treated with 200 mg/ treated mice. P values < 0.05 were considered to show sta- kg ENU or less (46% and 50% in the 200 mg/kg group and tistical significance. 46% and 50% in the 100 mg/kg group, respectively). To evaluate the degree of disease progression, the retinal dam- Results age ratio was compared among the groups (Fig. 2b). The 600 and 400 mg/kg ENU-induced damages were 100% and General remarks 56%, respectively, while the damage was 0% in the 200 and Mortality and body weight of ENU-treated rats were 100 mg/kg ENU-treated groups and vehicle-treated rats. affected in a dose-dependent manner. Rats treated with 600 mg/kg ENU exhibited decreased locomotor activity, Morphological and morphometric analyses of sequen- and 2 of the 4 rats in this group died on day 7. There was a tial changes in retinal damage statistically significant decrease in the growth rates (weight Six hours after treatment with 400 mg/kg ENU, con- gain) in all ENU-treated groups; the growth rates were densation and fragmentation of photoreceptor cell nuclei 61%, 92%, 115%, 120% and 127% in the 600, 400, 200 and and vacuolar change in the photoreceptor layer occurred. 100 mg/kg groups and the control group, respectively. The The disruption and loss of the photoreceptor cell nuclei pro- growth rate of the 400 mg/kg ENU-treated group decreased gressed at 24 hr, and widespread destruction and disappear- at 24 hr, peaked at day 7, and recovered at day 30 (data not ance of photoreceptor segments occurred at 72 hr (Fig. 3). shown). A majority of the photoreceptor cell nuclei were lost in the central retina at day 7. As a result, the thickness of the total Morphological and morphometric analysis of dose- retina and the outer retina of ENU-treated rats decreased dependent retinal damage progressively, and cystoid degeneration was seen in the in- The results of histological evaluation of the retinas of ner retinal layer 30 days after treatment (Fig. 3). The pho- ENU- and vehicle-treated rats were compared 7 days after toreceptor cell ratio at the central and peripheral retina was ENU treatment. The retinas of control rats contained more decreased significantly from 24 hr after ENU treatment; the than 10 layers of photoreceptor nuclei in the central retina photoreceptor cell ratios in the central and peripheral retina and more than 8 layers of cells in the peripheral retina (Fig. were decreased by 39% and 45% at 24 hr, 28% and 40% at 1). All rats treated with 600 mg/kg ENU had either no photo- 72 hr, and 9% and 44% at day 7, respectively, and by 43% receptor cell nuclei or only a few rows of photoreceptor cell and 51% in control rats (Fig. 4). The retinal thickness on day nuclei, such that the outer nuclear layer and photoreceptor 30 was similar to the thickness 7 days after treatment. Dur- layer almost disappeared in both the central and peripheral ing the disease course, no inflammatory cell infiltration was retina (Fig. 1). In 400 mg/kg ENU-treated rats, the central seen in the retina. retina contained no photoreceptor cell nuclei or only a few rows of photoreceptor cell nuclei (Fig. 1), and the peripheral Apoptotic cell ratio retina contained 5 or fewer rows of photoreceptor cell nuclei. In both the central and peripheral retina of the 400 mg/ The remaining photoreceptor nuclei were densely stained kg ENU-treated rats, the TUNEL signal appeared selective- and contained clumped chromatin, suggesting that they were ly in the photoreceptor cell nuclei, and some pyknotic photo- degenerating. The ENU-induced changes were restricted to receptor cell nuclei at 6 and 12 hr showed TUNEL positivity photoreceptor cells; all other layers of the retina remained without obvious nuclear destruction. Many photoreceptor intact 7 days after ENU treatment. No other ENU-induced cell nuclei showed TUNEL positivity at 24 hr after treat- changes to the components of the eye (including the cornea) ment (Fig. 5a). The sequential changes in the apoptotic cell were observed. The retinas of all rats that received 200 mg/ ratio at 0, 3, 6, 12, 24 and 72 hr and 7 days were 0.6, 0.5, 2.8, kg ENU or less were histopathologically intact (Fig. 1). 13.4, 44.5, 19.8 and 1.4, respectively, in the central retina Seven days after treatment with 600 mg/kg ENU, and 0.1, 0.1, 2.4, 15.5, 46.3, 21.1 and 0.6, respectively, in the the total retinal thickness and outer retinal thickness were peripheral retina (Fig. 5b). 30 ENU-induced Retinal Damage in Rats Fig. 1. Retinal change in adult rats 7 days after a single ip injection of ENU. The outer nuclear layer and photoreceptor layer degenerated and/ or disappeared in both the peripheral and central retina of rats who received ≥ 400 mg/kg ENU. No change was seen in the retinas of rats treated with 200 or 100 mg/kg ENU. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PRL, photoreceptor layer; and PEL, pigment epithelial layer. HE staining, ×400. Fig. 3. Sequential retinal changes in adult rats after a single ip injection of 400 mg/kg ENU. Retinal cross sections were collected 3, 6, 12, 24 and 72 hr and 7 and 30 days after ENU treatment. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PRL, photoreceptor layer; and PEL, pigment epithelial layer. HE staining, ×400. Yoshizawa, Sasaki, Uehara et al. 31 Fig. 4. Sequential changes in the photoreceptor cell ratios of the cen- tral and peripheral retina after a single ip injection of 400 mg/ kg ENU into adult rats. The index was calculated as [(outer retinal thickness/total retinal thickness) × 100]. The mean ± SE of the four rats in each treatment group is shown. * and **: P<0.05 and P<0.01, respectively. the cell nuclei in the inner nuclear layer and was not accom- panied by any obvious morphological nuclear changes. The signal appeared 24 hr after ENU treatment, and the index peaked 72 hr after ENU treatment (Fig. 6a). The sequential changes in the γ-H2AX cell ratio were 0, 0, 0, 0, 3, 16.3 and 1.8 at 0, 3, 6, 12, 24 and 72 hr and 7 days (Fig. 6b). PAR Fig. 2. Photoreceptor cell ratios in the central retina and peripheral signals were not detected in the control retina. At 12 hr after retina 7 days after a single ip injection of ENU into adult rats ENU treatment, the signals were localized to the photore- (a). Rats treated with 600 and 400 mg/kg ENU had a statisti- ceptor cell nuclei at the central and peripheral retina. The cally significant decrease in photoreceptor ratio at the central expression peaked at 24 hr after treatment, and the signals and/or peripheral retina, as compared with saline-treated con- disappeared 7 days after treatment. trols. The index was calculated as [(outer retinal thickness/ total retinal thickness) × 100]. Retinal damage ratios in ENU- Discussion treated adult rats (b). The index was evaluated as [(length of retina composed of less than four rows of photoreceptor cells/ whole retinal length) × 100]. The mean ± SE of the four rats in The present study provides new evidence of ENU-in- each treatment group is shown. **: P<0.01. duced retinal changes. Retinal degeneration characterized by the loss of the outer nuclear layer and photoreceptor layer in the central and peripheral retina was detected in ENU- γ-H2AX cell ratio and PAR immunoreactivity treated adult rats after a single ip injection of ≥ 400 mg/kg In the retinas of the 400 mg/kg ENU-treated rats, the ENU. The outer nuclear layer and photoreceptor layer of γ-H2AX-positive cells were scattered almost evenly in the ENU-treated rats decreased progressively due to photore- the whole retina. The γ-H2AX signal was localized to the ceptor cell apoptosis, which was characterized by the pres- 32 ENU-induced Retinal Damage in Rats Fig. 5. TUNEL-positive photoreceptor cells in the outer nuclear layer 24 hr after a single ip injection of 400 mg/kg ENU into Fig. 6. γ-H2AX-positive cells per field in the inner nuclear layer 72 adult rats (a). The signals are seen in many photoreceptor hr after a single ip injection of 400 mg/kg ENU into adult rats cell nuclei, although no or few signals are seen in the con- (a). The signals are seen in several inner retinal cell nuclei, trol retina (b). GCL, ganglion cell layer; IPL, inner plexiform although no signals are seen in the control retina (b). GCL, layer; INL, inner nuclear layer; OPL, outer plexiform layer; ganglion cell layer; IPL, inner plexiform layer; INL, inner ONL, outer nuclear layer; PRL, photoreceptor layer; and PEL, nuclear layer; OPL, outer plexiform layer; and ONL, outer pigment epithelial layer. TUNEL staining, ×400. Sequential nuclear layer. γ-H2AX immunohistochemical staining, ×800. changes in the apoptotic cells per field were evaluated in the Sequential changes in the γ-H2AX-positive cells of the inner central and peripheral retina, respectively (c). Mean ± SE; retina (c). Mean ± SE, each bar represents four rats. * and **: each bar represents four rats. * and **: P<0.05 and P<0.01, P<0.05 and P<0.01, respectively. respectively. ence of TUNEL-positive nuclei restricted to photoreceptor cells. Photoreceptor cell apoptosis was evident at 6 hr and peaked at 72 hr after ENU treatment, followed by extensive photoreceptor cell loss at day 7. A morphometric analysis showed that photoreceptor cell damage occurred similarly in both the central and peripheral retina in rats treated with 600 mg/kg ENU, while 400 mg/kg ENU caused loss of outer retinal layers located mainly in the central retina at 7 days after exposure. Therefore, the central retina may be more sensitive to ENU-induced damage. ENU is a stem-cell mutagen that also affects hematopoietic stem cells and im- mune cells, and animals are highly susceptible to infection after injection of ENU24. In the present study, the deaths in the 600 mg/kg dose group may have been caused by hema- topoietic toxicity and immunotoxicity. Fig. 7. Sequential PAR expression in the retina after a single ip injec- tion of 400 mg/kg ENU into adult rats. PAR immunoreactivi- ties located in the photoreceptor cell nuclei were seen at 12 hr and peaked at 24 hr after ENU treatment. INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; and PRL, photoreceptor layer. PAR immunohistochemical staining, ×600. Yoshizawa, Sasaki, Uehara et al. 33 ENU is the most potent mutagen in rodents4,5,25, and sion involves the formation of foci by more persistent dou- it can be used to generate point mutations throughout the ble-strand breaks that tend to accumulate and remain unre- mouse genome25. Various mouse models for human diseases paired in cells14. In ENU-induced retinal damage, γ-H2AX have been generated by a systematic, large-scale, genome- signals appeared in the inner retinal cells 24 hr after ENU wide phenotyping screen in the last decade5. The eyes of exposure, peaked at 72 hr and then disappeared. No cell ENU-treated animals exhibit morphological defects, such death in the inner retina could be seen at any point, sug- as lens opacity, microphthalmia, corneal adhesion, corneal gesting that the cells recovered from the DNA damage. The opacity, pink eye, iris anomalies, coloboma, and preretinal identity of the γ-H2AX-positive cells located in the inner blood vessels5–7. The progeny of ENU-treated animals ex- nuclear layer (bipolar cells, horizontal cells, amacrine cells hibit retinal degeneration8–10. In addition, a single ip injec- or Muller cells) was not determined. Moreover, γ-H2AX tion of 100 mg/kg ENU on day 14 of gestation or of 200 mg/ expression was not detected in the outer retina where cell kg ENU on day 3 after birth induces rosette-like structures death occurred. Further studies concerning the DNA dam- (dysplasia) in the retinas of fetal and neonatal rats, respec- age cascade and cell type in the inner retina are necessary tively2,26. Although ENU has been used as a carcinogen and to understand the detailed pathogenesis of ENU-induced mutagen in many animal toxicity and carcinogenesis stud- γ-H2AX expression in the inner retina. ies, there has been only one report concerning the effect of Hypofluorescence in indocyanine green angiography is ENU on the retinas of adult mice11, and this previous study seen in the chorioretinal atrophic area in MNU-treated reti- was conducted by our group. To the best of our knowledge, nal degeneration34. Also, the vascular sequential changes in the present study is the first report on ENU-induced retinal MNU-treated retinal degeneration have been reported35; the injury in adult rats. numbers of acellular capillaries, ghost cells and vessels ex- Inherited night blindness is a fairly widespread dis- hibiting narrowing in trypsin-digested retinal vessels were ease in humans, and it affects approximately one in 5,000 increased after MNU exposure. These data may suggest individuals worldwide. A common form of inherited blind- ischemic or hypoxic conditions in the retina after MNU ex- ness is retinitis pigmentosa, which is a degenerative pig- posure. Under ischemic or hypoxic conditions, target cells mentary retinopathy that is noninflammatory, bilateral and express γ-H2AX as a DNA damage response and progress progressive27,28. Animal models of retinal degeneration are through the DNA repair pathway36. In our study, although important for elucidating the mechanism of human retinitis angiographic analysis was not conducted, the inner retina pigmentosa28–30 and exploring potential treatments28,31. The may have been under ischemic or hypoxic conditions after retinal degeneration (photoreceptor cells loss) induced by ENU exposure, and this may have been followed by progres- N-methyl-N-nitrosourea (MNU), which is an ENU-related sion through the DNA repair pathway. Therefore, γ-H2AX compound25, has been well studied as one of the rodent expression in the inner retina may be a secondary effect due models of retinitis pigmentosa22,31. MNU causes DNA ad- to the ischemic or hypoxic conditions that developed after duct formation in photoreceptor nuclei, followed by apop- the outer retinal damage was induced by ENU exposure. tosis via the downregulation of Bcl-2, upregulation of Bax The survival of neurons from the potentially damag- and activation of the PARP and caspase families in rats. In ing events caused by unregulated cell-protective signaling contrast, few studies have focused on ENU-induced retinal and DNA repair is a complex process that requires multiple damage in adult rodents. steps and enzymes. Exposure to ENU causes the forma- In the present study, ENU induced an increased number tion of DNA adducts, such as O6-alkylguanine, in the target of TUNEL-positive photoreceptor cells in parallel with the cells37–39. The adduct is removed by repair enzymes such as expression of PAR signals. The PAR signals were expressed O6-methylguanine-DNA methyltransferase (MGMT)38,40. selectively in the photoreceptor cell nuclei. The signals ap- Additionally, DNA polymerase β is essential for the repair peared 12 hr after ENU treatment, and the immunoreactiv- of DNA lesions damaged by exogenous genotoxin41. DNA ity peaked 72 hr after ENU treatment. PAR is synthesized polymerase β is responsible for 99% of the polymerase ac- after activation of the nuclear DNA repair enzyme PARP. tivity in neurons42 and is upregulated during light-induced However, under conditions of severe DNA damage, exces- retinal degeneration43. Further studies of the DNA repair sive activation of PARP results in depletion of the cellular cascade in the inner retina are necessary to understand the pool of nicotinamide adenine dinucleotide and ATP, which detailed pathogenesis of ENU-induced retinal degeneration. is followed by cell death21. In conclusion, retinal degeneration occurred in adult γ-H2AX is a critical factor for cellular protection rats after a single intraperitoneal injection of 400 mg/ against DNA alkylating agents15, and it can mediate DNA kg ENU. ENU-induced retinal degeneration was related repair13,14,16,32. Methylating agents such as MNU can cause to photoreceptor cell apoptosis via PARP activation. The cell-cycle arrest, γ-H2AX expression and cell death, either γ-H2AX signals were expressed only in inner retinal cells via caspase-dependent apoptosis or via a PARP-dependent and disappeared promptly, suggesting that the inner retinal form of apoptosis33. Two forms of γ-H2AX expression have cells recovered from DNA damage. Investigations of the de- been elucidated. The transient form of γ-H2AX expression tailed mechanisms of ENU-induced retinal degeneration are is associated with the repair and removal of double-strand necessary for a better understanding of the pathogenesis of breaks from the nucleus. The other form of γ-H2AX expres- chemical-induced retinal degeneration. 34 ENU-induced Retinal Damage in Rats Acknowledgements: The authors thank Ms. T. Akamatsu 13. Mah LJ, El-Osta A, and Karagiannis TC. γ-H2AX: a sensi- for her technical assistance and Ms. A. Shudo for prepar- tive molecular marker of DNA damage and repair. Leuke- mia. 24: 679–686. 2010. 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