RESEARCH ARTICLE Resistance to DNA damage and enhanced DNA repair capacity in the hypoxia-tolerant blind mole rat Spalax carmeli Vered Domankevich, Hossam Eddini, Amani Odeh and Imad Shams* ABSTRACT Blind mole rats of the genus Spalax are the only mammalian species to date for which spontaneous cancer has never been reported and resistance to carcinogen-induced cancers has been demonstrated. However, the underlying mechanisms are still poorly understood. The fact that Spalax spp. are also hypoxia-tolerant and long-lived species implies the presence of molecular adaptations to prevent genomic instability, which underlies both cancer and aging. We previously demonstrated the upregulation of transcripts related to DNA replication and repair pathways in Spalax. Yet, to date, no direct experimental evidence for improved genomic maintenance has been demonstrated for this genus. Here, we show that compared with skin fibroblasts of the above-ground rat, Spalax carmeli skin fibroblasts in culture resist several types of genotoxic insult, accumulate fewer genotoxic lesions and exhibit an enhanced DNA repair capacity. Our results strongly support that this species has evolved efficient mechanisms to maintain DNA integrity as an adaptation to the stressful conditions in the subterranean habitat. KEY WORDS: DNA repair, Hypoxia, Longevity, Cell cycle, Genotoxic stress, Cancer INTRODUCTION The blind mole rats, Spalax, endure extreme and abrupt fluctuations in O 2 and CO 2 levels and survive very low oxygen content (down to 3% O 2 ) (Shams et al., 2005; Avivi et al., 1999). Under hypoxic conditions, deoxynucleotide triphosphates (dNTPs) are depleted and DNA repair pathways are repressed. The subsequent re- oxygenation leads to replication restart in the presence of oxidative DNA damage even though DNA repair mechanisms have not yet been recovered (Pires et al., 2010; Klein and Glazer, 2010). In other mammalian species, cycles of acute oxygen fluctuations act as a driving force of genomic instability (Klein and Glazer, 2010). However, Spalax spp. are long-lived (Tacutu et al., 2013) and, to date, no single case of spontaneous cancer has been reported for these species, during decades of research. In a previous study, we attempted to induce cellular transformation in Spalax, in vivo, using two types of carcinogens (Manov et al., 2013): (1) DMBA/TPA – a skin carcinogenesis protocol was used to treat eight Spalax and six mice individuals; and (2) 3MCA – a local fibrosarcoma induction protocol was used to treat 12 Spalax, six mouse and six rat individuals. Whereas all mice and rats developed the expected tumors within 2–6 months, no tumors were observed among Spalax individuals treated with DMBA/TPA. 3MCA treatmentinduced benign fibroblastic proliferation in only two Spalax individuals, and malignancies in another two individuals within 18 months (Manov et al., 2013) and 30 months (I.S., unpublished data). The naked mole rat (Heterocephalus glaber) is another subterranean rodent that is phylogenetically distant from Spalax (estimated divergence time from Spalax is 73 million years ago, whereas that of the rat and mouse is 45 million years ago) (Hedges et al., 2006). Remarkably, in addition to their hypoxia tolerance, three very exceptional traits have evolved in these species: (1) an ability to secrete anti-cancer substances in conditioned media (Manov et al., 2013); (2) a very low frequency of spontaneous cancers (Manov et al., 2013; Delaney et al., 2016; Buffenstein, 2005); and (3) an increased lifespan, relative to body mass (Tacutu et al., 2013; Gorbunova et al., 2014). This suggests that inhabiting hypoxic environments may require the ability to overcome a greater risk for cancer and aging, possibly because of threats placed by the conditions in these environments on the integrity of genome. In previous studies, Spalax and H. glaber genomes have been sequenced, genomic and transcriptomic data have been analyzed (Fang et al., 2014; Kim et al., 2011; Macrae et al., 2015b; Malik et al., 2011; Vinogradov, 2015), cross-species comparative studies have been carried out (Gorbunova et al., 2014; Macrae et al., 2015a,b; Malik et al., 2016; Ma et al., 2016) and reviewed (Gorbunova et al., 2014; Ma and Gladyshev, 2017; Lewis et al., 2016), and several specific candidate mechanisms have been investigated (Tian et al., 2013; Gorbunova et al., 2012; Nasser et al., 2009; Ellis et al., 2016; Zhao et al., 2014; Salmon et al., 2008; Lewis et al., 2012, 2015; Shams et al., 2013; Domankevich et al., 2016; Avivi et al., 2007; Miyawaki et al., 2016). These studies showed that several factors may act in concert to reduce cancer and aging in these species, and that these two subterranean taxa have evolved distinct mechanisms, which allow each to survive its unique physical and social environments. Nevertheless, it was clearly identified that these hypoxia-tolerant mammals express higher transcript levels of DNA repair factors (Macrae et al., 2015a; Malik et al., 2016; Shams et al., 2013), strengthening the assumption that these mammals have evolved common adaptations to hypoxia that include efficient DNA repair mechanisms. Yet, to date, no direct experimental evidence has been provided for this assumption. Here, we investigated the response to genotoxic stress and the DNA repair capacity in Spalax carmeli (Nevo et al., 2001), compared with the above-ground murine Rattus norvegicus (hereafter, rat). Our main hypothesis was that S. carmeli survives better genotoxic stress and repairs DNA more efficiently. The results presented here provide the first evidence for genotoxic stress resistance and enhanced repair capacity in this species. MATERIALS AND METHODS Animals All experimental protocols were approved by the Institutional Ethics Committee (Institutional Review Board to Evaluate Animal Subject Received 22 November 2017; Accepted 2 March 2018 The Institute of Evolution and Department of Evolutionary and Environmental Biology, University of Haifa, Haifa 3498838, Israel. *Author for correspondence (imad.shams@univ.haifa.ac.il) V.D., 0000-0003-3579-4616; I.S., 0000-0001-6496-2928 1 © 2018. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2018) 221, jeb174540. doi:10.1242/jeb.174540 Journal of Experimental Biology