~ Pergamon 0887-2333(94)E0006-F Toxic'. in Vitro Vol. 8, No. 3, pp. 317-328, 1994 Copyright © 1994 ElsevierScienceLtd Printed in Great Britain. All rights reserved 0887-2333/94 $7.00 + 0.00 EFFECTS OF AFLATOXIN B~ IN A LIVER CELL LINE FROM RAINBOW TROUT (ONCORHYNCHUS MYKISS) D. G. BECHTEL and L. E. J. LEE* Department of Veterinary Anatomy, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, Canada S7N 0W0 (Received 30 September 1993; revisions received 18 November 1993) Abstract--The cytotoxic response to ariatoxin B 1 (AFB) was investigated in RTL-W1, a cell line derived from the normal liver of a mature rainbow trout (Oncorhynchus mykiss). AFB altered RTL-Wl morphology and ultrastructure and inhibited DNA synthesis. The effective concentration required for 50% inhibition (ECs0) of DNA synthesis, after 2 days of treatment was 0.04 #g/ml. This response was compared with that of two other salmonid, but non-liver, cell lines [rainbow trout gonad (RTG-2) and Chinook salmon embryo (CHSE-214)]. Although RTG-2 cells were as sensitive as RTL-W1 cells (ECs0 for inhibition of DNA synthesis was 0.05 pg/ml), CHSE-214 cells were unresponsive to AFB at concentrations as high as 2 # g/ml. After a single AFB exposure, RTL-W1 sublines were isolated that had phenotypic changes typical of malignant transformation. These were increased growth rate, reduced contact inhibition of growth, altered cellular morphology and growth in soft agar. In addition, RTL-Wl metabolized AFB: the major metabolites, aflatoxin (AFL) and aflatoxin MI (AFM), were detected by thin-layer chromatography and HPLC. The relative amounts of these metabolites, unlike those observed with RTG-2 cells, were in close agreement with those produced by trout liver in vivo. Thus, RTL-W 1 could provide a sensitive in vitro model system for studying the action of biotransformation requiring xenobiotics. Overall, the observed responses were similar to those reported for liver ceils in AFB-exposed trout, suggesting that RTL-Wl cells are suitable for studying cytotoxic effects and malignant transform- ation in vitro. INTRODUCTION Cell cultures provide useful means for studying cyto- toxicity without involvement of the complex control processes of the whole organism (Frazier, 1992; Paganuzzi-Stammati et al., 1981; Watson, 1992). Sentinel cells that could be readily and consistently used for monitoring environmental toxicants, and that maintain xenobiotic-metabolizing activity have been sought for their applicability in toxicology (Glatt et al., 1987). Usually, mammalian cell lines have been used for monitoring the effects that toxicants might have in those species and ultimately these are correlated with effects in humans. In con- trast, relatively few efforts have been aimed at inves- tigating the effect toxicants that are particularly abundant in aquatic environments have in the lower vertebrates such as fish, and few fish cell lines have *To whom correspondence should be addressed. Abbreviations: AFB=aflatoxin B6 AFB2=aflatoxin B2; AFB2~ = ariatoxin B2~; AFL = ariatoxicol; AFL-M = aflatoxicol M 6 AFM = aflatoxin M~; AFP = aria- toxin P6 AFQ = aflatoxin Q6 B[a]P = benzo[a]pyrene; CHSE-214 = Chinook salmon embryo ceils; DMSO = dimethyl sulfoxide; ECs0= effective concentration required for 50% inhibition; FBS = foetal bovine serum; SPE = solid phase extraction; TEM = transmission electron microscopy; TLC = thin-layer chromatog- raphy; RTG-2 = rainbow trout gonad cell line; RTL- W1 = rainbow trout liver-Waterloo 1 cell line. been developed or used for ecotoxicity testing in these organisms (Babich and Borenfreund, 1987 and 1991). Established cell lines are advantageous for routine use in toxicology because of their immortality and phenotype stability, but they have limited appli- cations because of their poor capacity to metabolize chemicals. Much effort has been spent in developing culture systems with metabolically competent cells such as hepatocytes (Kremers et al., 1990). Most toxicology studies performed with fish cells also use primary cultures of hepatocytes (Baksi and Frazier, 1990), as their functioning have been found to be consistent with the role of the liver in situ (Moon et al., 1985). But in general, primary cultures are cumbersome to prepare, are short lived and produce inconsistent results. Thus liver cell lines that maintain metabolic capability have been sought. Fish liver cell lines are few (Bols and Lee, 1991) and most have been derived from malignant tumours, which renders them unsuitable for correlating the effects toxicants might have in normal tissues as opposed to altered ceils. The RTL-Wl cell line derived from rainbow trout liver, expressed cytochrome P4501A1 activity, was sensitive to benzo[a]pyrene, and possessed some attributes of the normal organ of origin (Lee et al., 1993). Thus the present study investigated whether the RTL-WI cell line could be useful as a model fish sentinel cell line 317