Biomolecules 2021, 11, 1497. https://doi.org/10.3390/biom11101497 www.mdpi.com/journal/biomolecules Article Alcohol-Induced Lysosomal Damage and Suppression of Lysosome Biogenesis Contribute to Hepatotoxicity in HIV-Exposed Liver Cells Moses New-Aaron 1,2, *, Paul G. Thomes 2,3 , Murali Ganesan 2,3 , Raghubendra Singh Dagur 2,3 , Terrence M. Donohue, Jr. 2,3 , Kharbanda K. Kusum 2,3 , Larisa Y. Poluektova 4 and Natalia A. Osna 1,2,3,4, * 1 Department of Environmental Health, Occupational Health, and Toxicology, College of Public Health, University of Nebraska Medical Center, Omaha, NE 68198, USA 2 Research Service, Veterans Affairs Nebraska-Western Iowa Health Care System, Omaha, NE 68105, USA; paul.thomes@unmc.edu (P.G.T.); murali.ganesan@unmc.edu (M.G.); raghu.dagur82@gmail.com (R.S.D.); tdonohue@unmc.edu (T.M.D.J.); kkharbanda@unmc.edu (K.K.K.) 3 Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68105, USA 4 Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68105, USA; lpoluekt@unmc.edu * Correspondence: moses.newaaron@unmc.edu (M.N.-A.); nosna@unmc.edu (N.A.O.) Abstract: Although the causes of hepatotoxicity among alcohol-abusing HIV patients are multifac- torial, alcohol remains the least explored “second hit” for HIV-related hepatotoxicity. Here, we in- vestigated whether metabolically derived acetaldehyde impairs lysosomes to enhance HIV-induced hepatotoxicity. We exposed Cytochrome P450 2E1 (CYP2E1)-expressing Huh 7.5 (also known as RLW) cells to an acetaldehyde-generating system (AGS) for 24 h. We then infected (or not) the cells with HIV-1ADA then exposed them again to AGS for another 48 h. Lysosome damage was assessed by galectin 3/LAMP1 co-localization and cathepsin leakage. Expression of lysosome biogenesis– transcription factor, TFEB, was measured by its protein levels and by in situ immunofluorescence. Exposure of cells to both AGS + HIV caused the greatest amount of lysosome leakage and its im- paired lysosomal biogenesis, leading to intrinsic apoptosis. Furthermore, the movement of TFEB from cytosol to the nucleus via microtubules was impaired by AGS exposure. The latter impairment appeared to occur by acetylation of α-tubulin. Moreover, ZKSCAN3, a repressor of lysosome gene activation by TFEB, was amplified by AGS. Both these changes contributed to AGS-elicited disrup- tion of lysosome biogenesis. Our findings indicate that metabolically generated acetaldehyde dam- ages lysosomes and likely prevents their repair and restoration, thereby exacerbating HIV-induced hepatotoxicity. Keywords: lysosome damage; lysosome biogenesis; HIV; ethanol metabolites; hepatotoxicity 1. Introduction Since the emergence of antiretroviral therapy, a significant decline in AIDS-related mortality among people living with HIV (PLWH) precipitated an upsurge in non-AIDS- related mortality (NARM). Comorbidities implicated in NARM among PLWH are cardi- ometabolic disorders, renal disease, mental illness, and liver disease [1]. While cardiomet- abolic disease ranks as the leading comorbidity among PLWH [2], liver disease remains the leading injury for NARM, accounting for approximately one-fifth of all cases [3–5]. This makes the management of liver disease a priority for HIV care delivery. Pathogenesis of HIV-related liver comorbidities is exacerbated by “second hits”, such as alcohol or co- infection with hepatotropic viruses [6]. However, the mechanism(s) by which alcohol in- duces liver injury during HIV infection is/are not entirely clear, which prevents improving Citation: New-Aaron, M.; Thomes, P.G.; Ganesan, M.; Dagur, R.S.; Donohue, T.M.; Kusum, K.K.; Poluektova, L.Y.; Osna, N.A. Alcohol-Induced Lysosomal Damage and Suppression of Lysosome Biogenesis Contribute to Hepatotoxicity in HIV-Exposed Liver Cells. Biomolecules 2021, 11, 1497. https://doi.org/10.3390/biom11101497 Academic Editor: Enrico Moro Received: 28 September 2021 Accepted: 6 October 2021 Published: 11 October 2021 Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional claims in published maps and insti- tutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses /by/4.0/).