567 RADIATION RESEARCH 168, 567–573 (2007) 0033-7587/ 07 $15.00  2007 by Radiation Research Society. All rights of reproduction in any form reserved. Mrad9 and Atm Haploinsufficiency Enhance Spontaneous and X-Ray-Induced Cataractogenesis in Mice Norman J. Kleiman, a,b Janice David, b Carl D. Elliston, c Kevin M. Hopkins, c Lubomir B. Smilenov, c David J. Brenner, a,c Basil V. Worgul, b,c,1 Eric J. Hall c and Howard B. Lieberman a,c,2 a Department of Environmental Health Sciences, Columbia University, Mailman School of Public Health, New York, New York 10032; b Eye Radiation and Environmental Research Laboratory, Columbia University, College of Physicians and Surgeons, New York, New York 10032; and c Center for Radiological Research, Columbia University, College of Physicians and Surgeons, New York, New York 10032 Kleiman, N. J., David, J., Elliston, C. D., Hopkins, K. M., Smilenov, L. B., Brenner, D. J., Worgul, B. V., Hall, E. J. and Lieberman, H. B. Mrad9 and Atm Haploinsufficiency En- hance Spontaneous and X-Ray-Induced Cataractogenesis in Mice. Radiat. Res. 168, 567–573 (2007). Rad9 and Atm regulate multiple cellular responses to DNA damage, including cell cycle checkpoints, DNA repair and ap- optosis. However, the impact of dual heterozygosity for Atm and Rad9 is unknown. Using 50 cGy of X rays as an environ- mental insult and cataractogenesis as an end point, this study examined the effect of heterozygosity for one or both genes in mice. Posterior subcapsular cataracts, characteristic of radi- ation exposure, developed earlier in X-irradiated double het- erozygotes than in single heterozygotes, which were more prone to cataractogenesis than wild-type controls. Cataract onset time and progression in single or double heterozygotes were accelerated even in unirradiated eyes. These findings in- dicate that the cataractogenic effect of combined heterozygos- ity is greater than for each gene alone and are the first to demonstrate the impact of multiple haploinsufficiency on ra- diation effects in an intact mammal. These observations may help explain observed interindividual differential radiosensi- tivity in human populations and have important implications for those undergoing radiotherapy or exposed to elevated lev- els of cosmic radiation, such as the astronaut corps. These findings demonstrate that Mrad9 and Atm are important de- terminants of lens opacification and, given the roles of Atm and Rad9 in maintaining genomic stability, are consistent with a genotoxic basis for radiation cataractogenesis.  2007 by Radi- ation Research Society INTRODUCTION The cellular response to DNA damage caused by normal metabolic processes or exposure to exogenous insults is 1 During the final stages of this study and the preparation of this article, Dr. Basil V. Worgul passed away unexpectedly. We miss him as a good friend, long-time collaborator and valued colleague and dedicate this pa- per to his memory and to the continuation of the research he held so dear. 2 Address for correspondence: Center for Radiological Research, Co- lumbia University College of Physicians and Surgeons: 630 W. 168th St., New York, NY 10032; e-mail: lieberman@cancercenter.columbia.edu. critical for determining whether deleterious effects will oc- cur. It is well established that complete loss of gene func- tion (homozygous defects) for cellular components in- volved in DNA damage recognition, DNA repair or cell cycle checkpoint control might lead to genomic instability, mutation, carcinogenesis or lethality (1–3). There is also compelling evidence that heterozygous gene defects that lead to haploinsufficiency can have negative biological ef- fects [for reviews, see refs. (4, 5)]. For example, haploin- sufficiency for pTEN, Blm, Rb, Atm or Plk4 enhances car- cinogen-induced tumor formation (6–11). In mice, hetero- zygosity for Atm causes early onset of cataracts induced by X rays or heavy ions (12, 13). In humans, ATM heterozy- gosity might predispose individuals to deleterious late ef- fects of radiotherapy (14). Women heterozygous for BRCA1 or BRCA2 might have increased risk for breast cancer after diagnostic chest X rays (15), and individuals with hetero- zygous defects in the melancortin 4 receptor might be ab- normally susceptible to obesity (16). In contrast to these studies of single-gene heterozygous mutations, no studies have examined the specific pathological effects of multiple gene heterozygosity in complex mammalian systems and, in particular, for genes regulating the cellular response to DNA damage. Atm and Mrad9 play critical roles in the cellular response to DNA damage (17, 18) and are important for maintaining genomic integrity at least in part by regulating cell cycle checkpoints induced by radiation. Furthermore, Rad9 is phosphorylated by the ATM kinase activity after cells are irradiated (19). Recently, mouse embryo fibroblasts hap- loinsufficient for both Atm and Mrad9 were shown to be more sensitive to radiation-induced transformation than ei- ther wild-type controls or those heterozygous for just one gene (20). On the basis of this and related observations, our group and others have suggested that differential radio- sensitivity of some individuals might be explained by mul- tiple heterozygosity for genes involved in cell cycle check- point control, DNA damage recognition, or DNA repair (21–23). In contrast to these in vitro investigations, studies with