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Background and PurposeThe non-nucleoside analogue reverse transcriptase inhibitor efavirenz is associated with hepatic toxicity and metabolic disturbances. Although the mechanisms involved are not clear, recent evidence has pinpointed a specific mitochondrial action of efavirenz accompanied by the induction of an endoplasmic reticulum (ER) stress/unfolded protein response in human hepatic cells. The aim of this study was to further investigate the involvement of this organelle by evaluating efavirenz's effects in cells lacking functional mitochondria (rho degrees) and comparing them with those of the typical mitotoxic agent rotenone, a standard complex I inhibitor, and the ER stress inducer thapsigargin. Experimental ApproachHep3B rho(+) and rho degrees cells were treated with clinically relevant concentrations of efavirenz, then mitochondrial function and cytotoxicity were studied using standard cell biology techniques. Key ResultsEfavirenz-treated rho degrees cells exhibited a substantial reduction in parameters indicative of mitochondrial interference, such as increased superoxide production, mitochondrial mass/morphology alterations and enhanced expression of LONP, a highly conserved mitochondrial protease. In line with these results, the cytotoxic effect (cell number, chromatin condensation, cell cycle alterations and induction of apoptosis) of efavirenz was less pronounced in Hep3B respiration-depleted cells than in wild-type cells. The effect of efavirenz was both similar and different from those of two distinct mitochondrial stressors, thapsigargin and rotenone. Conclusions and ImplicationsCells lacking normal mitochondria (rho degrees) are less vulnerable to efavirenz. Our results provide further evidence that the hepatic damage induced by efavirenz involves acute interference with mitochondria and extend our knowledge of the response of mitochondria/ER to a stress stimulus. The authors would like to thank Brian Normanly for editing of the manuscript. Grants PI11/00327 and CIBER CB06/04/0071 (both from Instituto de Salud Carlos III, Ministerio de Economia y Competitividad), PROMETEOII/2014/035, ACOMP/2013/236 and GV/2014/118 (all from Generalitat Valenciana), and UV-INV-PRECOMP12-80613 (Universitat de València). M. P., H. A. F. and F. A. are recipients of Predoctoral Trainee Research Grants (ACIF/2013/136 from Generalitat Valenciana; Fundación Dr Esplugues and FI12/00198 from Instituto de Salud Carlos III, Ministerio de Economía y Competitividad respectively). A. B.-G. is recipient of a Juan de la Cierva contract (JCI-2012-15124, Ministerio de Economía y Competitividad).