Cellular damage to human hepatocytes through repeated application of 5-aminolevulinic acid Thomas S. Weiss 1,2, * , Sascha Pahernik 3 , Irmgard Scheruebl 2 , Karl-Walter Jauch 4 , Wolfgang E. Thasler 1,2 1 Center for Liver Cell Research, University of Regensburg Hospital, F.-J.-S.-Allee 11, D-93042 Regensburg, Germany 2 Department of Surgery, University of Regensburg Hospital, Regensburg, Germany 3 Department of Urology, Johannes Gutenberg University Mainz, Mainz, Germany 4 Department of Surgery, LM University Munich, Hospital Grosshadern, Germany Background/Aims: 5-Aminolevulinic acid (ALA), a precursor of porphyrins is used for photodynamic diagnosis and therapy within topical or systemic applications. A potential toxic effect on the human liver is of major interest and therefore we investigated the impact of a repeated application of ALA without illumination on cultures of human hepatocytes. Methods: After ALA treatment of hepatocytes in vitro the porphyrin synthesis, albumin secretion, liver-specific enzyme release, and malondialdehyde levels were determined. In order to reduce levels of reactive oxygen substances, mannitol and the antioxidant enzymes superoxide dismutase and catalase were supplemented. Results: Porphyrin biosynthesis by human hepatocytes in vitro was repeatedly stimulated by ALA (0.001–1.0 mM), which was indicated by an accumulation of protoporphyrin IX. A repetitive treatment (up to four times) of hepatocytes with ALA resulted in an impairment of the hepatic function and viability, depending on the ALA concentration (0.1–1.0 mM) and frequency of application (2–3 times). This was also accompanied by increased malondialdehyde levels indicating enhanced lipid peroxidation. Only superoxide dismutase was able to reduce cellular damage and prevent specific function. Conclusions: Repeated, not single, ALA treatment without illumination may cause deleterious effects to the liver, which are mediated by oxygen radicals and inhibited by an antioxidant. q 2003 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. Keywords: 5-Aminolevulinic acid; Porphyrin; Photodynamic therapy/diagnosis; Human hepatocyte; Reactive oxygen substance; Lipid peroxidation; Antioxidant 1. Introduction Porphyrins play an important role not only as a prerequi- site for heme synthesis, but also in the approach to the treatment or detection of cancer where they are used as photosensitizers in photodynamic therapy (PDT) or as a fluorescent dye in photodynamic diagnosis (PDD). After the exogenous administration of 5-aminolevulinic acid (ALA), tumors selectively accumulate porphyrins, which under suitable irradiation generate either cytotoxic species leading to cellular damage or fluorescent light indicating the tumor spread within the tissue [1–3]. Therefore, ALA is widely used as a topical drug in PDT [1,4–7], but also studies with a systemic application of ALA aimed at basal cell carcinomas [8] or gastrointestinal tumors [3] are currently under investigation. Even though ALA has a relatively rapid clearance from the body compared to many other agents [1], photosensitiv- ity due to elevated porphyrin levels after systemic ALA treatment has been reported [9]. Photosensitization and toxic effects of non-tumor affected tissue after the applica- tion of ALA or porphyrin were evaluated using not only models for investigating the side effects of PDT [10,11], but also experimental designs elucidating the aspects of porphyria [12–16]. Toxic effects to the liver (toxic dark Journal of Hepatology 38 (2003) 476–482 0168-8278/03/$30.00 q 2003 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0168-8278(02)00454-3 www.elsevier.com/locate/jhep Received 28 August 2002; received in revised form 29 November 2002; accepted 17 December 2002 * Corresponding author. Tel.: 149-941-944-6837; fax: 149-941-944- 6838. E-mail address: thomas.weiss@klinik.uni-regensburg.de (T.S. Weiss).