Mutation Research 519 (2002) 163–170 The Liverbeads ® as a tool for the comet assay Laurence Vian a,∗ , Ayda Trismawaty Yusuf a , Claire Guyomard b , Jean-Paul Cano a a Laboratoire de Toxicologie, Faculté de Pharmacie, BP 14491, 34093 Montpellier Cedex 5, France b Biopredic International, rue du Professeur J. Pecker, F-35000 Rennes, France Received 7 September 2001; received in revised form 10 May 2002; accepted 29 May 2002 Abstract Liverbeads ® , cryopreserved hepatocytes entrapped within an alginate matrix, were examined for their relevance in the comet assay. It was estimated by their capacity to activate the indirectly acting mutagens, cyclophosphamide (CP), benzo[a]pyrene (BP), dimethylbenzanthracene (DMBA) and 2-acetylaminofluorene (2-AAF), into DNA reactive metabolites. The comet assay performed in alkaline condition is a sensitive method for detecting strand breaks at the level of individual cells and allows use of quiescent cells. Experimental conditions as treatment time, cell density, beads dissociation and viability were investigated. Significant statistical positive results assessed by the tail extent moment (TEM) were observed with both human and rat Liverbeads ® after 12 h duration incubation compared to metabolic non-competent cells, HeLa S3. Due to the maintenance of specific functions assessed by the observed capacity to metabolize xenobiotics, Liverbeads ® represent a suitable tool system, easy to handle, for the detection of promutagens using the comet assay. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Hepatocytes; Rat; Human; Comet assay; Promutagen 1. Introduction The assessment of metabolism activation is of par- ticular importance in interpretation of the results of mutagenicity studies as many carcinogens are not active per se but require metabolic activation to form ultimate carcinogens. The liver is the major organ involved in carcinogen activation and isolated hepa- tocytes represent the most appropriate in vitro model to mimic the in vivo activation profile. However, in suspension, they do not survive more than 3–4 h and in standard culture they exhibit phenotype alterations within a few hours. Their entrapment in an algi- nate gel is an interesting alternative and this system, ∗ Corresponding author. Tel.: +33-4-67-54-80-74; fax: +33-4-67-41-08-32. E-mail address: lvian@pharma.univ-montp1.fr (L. Vian). named Liverbeads ® , makes hepatocytes available to laboratories without cell isolation process. It al- lows maintenance of hepatocyte shape resulting in an enhanced recovery of hepatic function even when the immobilized cells have been previously cryop- reserved [1,2]. Liverbeads ® can be produced from several animal species, i.e. rat, mouse, dog, monkey, and from human beings. When compared to hepato- cytes monolayers, rat Liverbeads ® expressed similar levels of total cytochrome P450s content, and remain functional for several days even after cryoconserva- tion [1,3]. In the same way, immobilization does not markedly affect human CYP1A1, 1A2, 2C9, 2C19, 2D6 and 3A4 [2]. So, Liverbeads ® may represent a useful toxicological tool for in vitro mutagenesis stud- ies for which metabolism capacity is a pre-requisite to assess indirect-acting mutagens. 1383-5718/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII:S1383-5718(02)00137-7