Apoptosis as a mechanism of tributyltin cytotoxicity to thymocytes: relationship of apoptotic markers to biochemical and cellular effects

Abstract

Recent in vitro studies have suggested that activation of apoptosis could account for the profound depletion of cortical thymocytes, which characterizes tributyltin (TBT) immunotoxicity. However, it has also been shown that TBT disrupts macromolecular synthesis and cellular energetics to an extent that might be expected to interfere with the initiation of apoptosis. The purpose of these studies was to further evaluate the morphological and biochemical characteristics of thymocyte killing by TBT and to relate this to key cellular processes. Ex vivo thymocyte cultures from immature rats were treated with bis(tri-n-butyltin) oxide (TBTO) at concentrations ranging from those which rapidly produced necrosis (5-10 microM), down to cytotoxic but subnecrotic concentrations (0.1-2 microM). In cells exposed to TBTO concentrations that caused a rapid and near maximal inhibition of protein synthesis, it remained possible to demonstrate the stereotypic internucleosomal DNA cleavage and morphological changes indicative of apoptosis. Further confirmation that apoptosis was occurring independently from protein synthesis was provided by the absence of a protective effect following cycloheximide pretreatment. Apoptosis still occurred in TBTO-treated thymocytes although intracellular ATP levels were depressed to 20% or less of control values. Cytoprotective effects were noted with the intracellular Ca2+ chelators BAPTA-AM and Quin-2 AM, and also with zinc. Cell killing by TBTO occurred without overt disturbance of thymocyte cell cycle parameters. These results indicate that thymocyte apoptosis stimulated by TBT exposure occurs independently of a requirement for protein synthesis and does not require fully conserved cellular energetics.