Research Article Relative Mutagenic Potencies of Several Nucleoside Analogs, Alone or in Drug Pairs, at the HPRT and TK Loci of Human TK6 Lymphoblastoid Cells Meghan M.Carter, Salina M.Torres, Dennis L. Cook Jr., Consuelo L. McCash, Mia Yu, Vernon E.Walker, * and Dale M.Walker Lovelace Respiratory Research Institute, Albuquerque, New Mexico Experiments were performed to investigate the impact of didanosine (ddI), lamivudine (3TC), and stavudine (d4T) on cell survival and mutagenicity in two reporter genes, hypoxanthine-guanine phosphor- ibosyltransferase (HPRT) and thymidine kinase (TK), using a cell cloning assay for assessing the effects of individual nucleoside analogs (NRTIs)/drug combi- nations in human TK6 B-lymphoblastoid cells. Three- day treatments with 0, 33, 100, or 300 lM ddI, 3TC, or ddI-3TC produced positive trends for increased HPRT and TK mutant frequencies. While dose-related trends were too small to reach signifi- cance after treatments with d4T or d4T-3TC, pairwise comparisons with control cells indicated that expo- sure to 100 lM d4T or d4T-3TC caused significant elevations in HPRT mutants. Measurements of muta- genicity in cells exposed to d4T (or d4T-3TC) were complicated by the cytotoxicity of this NRTI. Enhanced increases in mutagenic responses to com- bined NRTI treatments, compared with single drug treatments, occurred as additive to synergistic effects in the HPRT gene of cells exposed to 100 lM ddI- 3TC or 100 lM d4T-3TC, and in the TK gene of cells exposed to 100 or 300 lM ddI-3TC. Comparisons of these data to mutagenicity studies of other NRTIs in the same system (Meng Q et al. [2000c]: Proc Natl Acad Sci USA 97:12667–126671; Torres SM et al. [2007]: Environ Mol Mutagen 48:224–238) indi- cate that the relative mutagenic potencies for all drugs tested to date are: AZT-ddI > ddI-3TC > AZT- 3TC % AZT-3TC-ABC (abacavir) > AZT ddI > d4T- 3TC > 3TC > d4T  ABC. These collective data sug- gest that all NRTIs with antiviral activity against HIV-1 may cause host cell DNA damage and mutations, and impose a cancer risk. Environ. Mol. Mutagen. 48:239–247, 2007. V V C 2007 Wiley-Liss, Inc. Key words: didanosine; HPRT , lamivudine; nucleoside analogs; stavudine; TK INTRODUCTION Highly active antiretroviral therapy (HAART), which typically includes one or more nucleoside reverse tran- scriptase inhibitors (NRTIs) along with a protease inhibi- tor or a non-NRTI, has been successful in controlling viral burden, in prolonging the lifespan of HIV-infected persons, and in reducing viral transmission of HIV from mother-to-infant to <2% [Moodley et al., 1998; Capparelli et al., 2005]. However, exposures to NRTIs are genotoxic and mutagenic, leading to increased risk for cancer [Olivero et al., 1999; O’Neill et al., 2001; Bonnet et al., 2004; Brock et al., 2006; Escobar et al., 2007; Meng et al., 2007; Witt et al., 2007]. Research conducted to determine the safety of NRTIs for use in HIV prophy- laxis, especially for the prevention of vertical transmis- sion, and in the treatment of HIV infection indicates that these compounds function as chemical mutagens and mi- tochondrial toxins [Olivero et al., 1997; Blanche et al., Abbreviations: ABC, abacavir or (1S, 4R)-4-[2-amino-6-(cyclopropyla- mino)-9H-purin-9-yl]-2cyclopentene-1-methanol; AZT, zidovudine or 3 0 - azido-2 0 ,3 0 -dideoxythymidine; CHAT, cytidine (C), hypoxanthine (H), aminopterin (A), and thymidine (T); d4T, stavudine or 2 0 ,3 0 -didehydro- 3 0 -deoxythymidine; ddI, didanosine or 2 0 ,3 0 -dideoxyinosine; HAART, highly active antiretroviral therapy; HPRT, hypoxanthine-guanine phos- phoribosyltransferase; NRTIs, nucleoside reverse transcriptase inhibitors; TK, thymidine kinase; 3TC, lamivudine or 2 0 ,3 0 -dideoxy-3 0 -thiacytidine. The contents of this communication are the sole responsibility of the authors and do not necessarily represent the official views of the NCI, NHLBI, or NIH. Grant sponsor: The National Cancer Institute; Grant Number 1 R01 CA95741 The National Heart, Lung, and Blood Institute; Grant Number 1 R01 HL72727. *Correspondence to: Vernon E. Walker, Lovelace Respiratory Research Institute, 2425 Ridgecrest Drive SE, Albuquerque, NM 87108, USA. E-mail: vwalker@lrri.org Invited article on the genotoxicity of perinatal NRTI therapy. DOI 10.1002/em.20282 Published online 14 March 2007 in Wiley InterScience (www.interscience. wiley.com). V V C 2007 Wiley-Liss, Inc. Environmental and Molecular Mutagenesis 48:239^247 (2007)