Pancreatic Islets Are Very Poor in Rectifying Oxidative DNA Damage Manisha A. Modak, MSc,* Pradeep Bhaskar Parab, PhD,Þ and Saroj S. Ghaskadbi, PhD* Objective: Free radicals that escape scavenging by antioxidant defense damage lipids, proteins, and DNA. Damage to DNA can be repaired. Therefore, both cells’ antioxidant defense and their ability to repair oxidatively damaged DNA decide its fate to survive oxidative stress. Pancreatic islets cells with poor antioxidant defense were checked for their ability to remove oxidative damage form DNA. Methods: For ex vivo DNA repair, assay-cultured pancreatic islets and liver slices were treated with 1 and 10 mM H 2 O 2 , respectively, for 30 minutes. After incubation for different time intervals, 8-hydroxy-2¶- deoxyguanosine (8-OHdG) in DNA of these cells was estimated using monoclonal antibody raised against 8-OHdG by competitive enzyme- linked immunosorbent assay. For invitro DNA repair assay, oxidatively damaged pBR322 was incubated with nuclear extracts of islet and liver cells, and 8-OHdG retained in the plasmid was quantitated. Results: Oxidative damage induced by H 2 O 2 was removed quickly and efficiently from DNA by liver cells compared with islet cells. The repair of oxidatively damaged plasmid DNA in vitro was also performed more efficiently (P G 0.05) by nuclear extracts from liver cells compared with islet cell. Conclusions: We clearly demonstrate that in addition to their low antioxidant defense, islets are very poor in rectifying the oxidative DNA damage. Key Words: oxidative DNA damage, 8-OHdG, DNA repair (Pancreas 2009;38: 23Y29) T he pancreatic A-cell toxin streptozotocin (STZ) is routinely used as diabetogen in experimental diabetes. The cytotoxic action of this diabetogen is mediated by reactive oxygen species (ROS). Streptozotocin enters A cells via a glucose transporter GLUT2, 1,2 causes alkylation of DNA, 3,4 and induces generation of free radicals that damage DNA by inducing single-strand breaks. This DNA damage leads to activation of poly adenosine diphosphate ribose polymerase, which in turn brings about depletion of nicotinamide adenine dinucleotide + and adenosine- 5¶-triphosphate (ATP). Enhanced ATP dephosporylation is a substrate for xanthine oxidase resulting in the formation of superoxide radical that leads to formation of hydrogen peroxide and hydroxyl radical formation. 5 Damage due to ROS is determined primarily by the amount of generated radicals and the antioxidant defense status of the cell. Therefore, different organs can exhibit considerable dif- ferences in their susceptibility toward cytotoxic damage. 6 The primary defense includes antioxidant enzymes, namely, super- oxide dismutase (SOD), catalase, and peroxidases, whereas sec- ondary defense is offered by small antioxidant molecules such as reduced glutathione, uric acid, and others. In addition, cells also have systems to repair the damage induced by these ROS. It is well established that the activities of antioxidant en- zymes are very low in pancreatic islets. 6Y10 Low levels of SOD, catalase, and glutathione peroxidase enzymes are responsible for the extraordinary sensitivity of pancreatic A cells toward oxi- dative stress in diabetes. 6 Overexpression of cellular enzymes like SOD in vitro leads to protection of A cells against oxidative stressYinduced A-cell damage/death. 11 Overexpression of mito- chondrial Mn-SOD is also shown to have protective effect on isolated islets against oxidative damage in vitro. 12 Adenoviral vectorYmediated overexpression of glutamyl cysteine ligase cat- alytic subunit, a primary regulator of de novo synthesis of glu- tathione in mammalian cells, protects pancreatic islets against oxidative stress in vitro. 13 Although there are many reports 6Y10 describing low anti- oxidant defense of pancreatic islets, very little is known about the repair efficiencies of pancreatic islet cells. Streptozotocin treatment leads to increase in the expression of gadd 153 and gadd 45 genes in rat pancreatic islets, which are relevant in repair of islet DNA damage. 14 In this study, we have quantitated oxi- dative DNA repair in pancreatic islet cells and compared it with that of liver cells using liver slice culture. The major advantage of liver slice culture is that slices contain all the cell types present in liver and retain tissue-specific microarchitecture with main- tained cell diversity, identity, and functional heterogeneity. Liver slice culture has already been proven to be a good model for studying xenobiotics 15 and its kinetics, 16 effect of hepatoto- xins, 17 hormone-regulated glucose metabolism, 18 phase I and II hepatic drug metabolism, 19 and drug-induced apoptosis 20 and in genotoxicity studies. 21 The concentration of STZ, which is lethal to pancreatic islets, is not cytotoxic to liver cells. Therefore, we have used hydrogen peroxide as damaging agent. DNA repair capacity of both islet and liver cells was quantitated by esti- mating removal of 8-hydroxy-2¶-deoxyguanosine (8-OHdG), a standard marker for oxidative DNA damage using monoclonal antibody raised in our laboratory against 8-OHdG by compe- titive enzyme-linked immunosorbent assay (ELISA) following a protocol of Chiou et al. 22 In addition, cell-free extracts from these cells were also tested for their ability to remove 8-OHdG from oxidatively damaged plasmid pBR 322 in vitro. MATERIALS AND METHODS Isolation of Mouse Islets Pancreatic islets were isolated from Swiss albino mice of 8 to 10 weeks old of either sex (18Y20 g body weight) as described by Lacy and Kostianovsky 23 and Shewade et al. 24 Briefly, pan- creatic tissue was dissected out under sterile conditions, washed in Hanks balanced salt solution and digested using 1 mg/mL collagenase Type V (Sigma, USA) for 10 to 15 minutes at 37-C. ORIGINAL ARTICLE Pancreas & Volume 38, Number 1, January 2009 23 From the *Department of Zoology, University of Pune, Ganeshkhind; and †National Centre for Cell Science, Pune University Campus, Ganeshkhind, Pune (M.S.), India. Received January 8, 2008. Accepted for publication May 29, 2008. Present address of P.B.P.: Yashraj BiotechnologyLtd, C-232,TTC Industrial Area, Navi, Mumbai 4000705, India. Reprints: Saroj S. Ghaskadbi, PhD, Department of Zoology, University of Pune, Ganeshkhind, Pune 411 007, India (e-mail: ssg@unipune.ernet.in). This research was supported by the financial assistance from UGC, New Delhi. Copyright * 2008 by Lippincott Williams & Wilkins ISSN: 0885-3177 DOI: 10.1097/MPA.0b013e318181da4e Copyright @ 2009 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.