Treatment of fetal rat hepatocytes with transforming growth factor beta (TGF‐ß) is followed by apoptotic cell death. Analysis of radical oxygen species (ROS) content and mitochondrial transmembrane potential (Δψ _m ), using specific fluorescent probes in FACScan and confocal microscopy, showed that TGF‐ß mediates ROS production that precedes the loss of Δψ _m , the release of cytochrome c, and the activation of caspase 3. TGF‐ß induces a decrease in the protein and mRNA levels of bcl‐x _L , an antiapoptotic member of the Bcl‐2 family. In contrast, there is no change in the expression and/or translocation of Bax, a proapoptotic member of the same family. EGF maintains Bcl‐x _L , preventing Δψ _m collapse and release of cytochrome c. The presence of radical scavengers blocks the decrease in bcl‐x _L levels, Δψ _m collapse, cytochrome c release, and activation of caspase 3; in contrast, the presence of glutathione synthesis inhibitors such as BSO accentuated the effect. The incubation of fetal hepatocytes in the presence of ter‐butyl‐hydroperoxide alone produces a decrease in bcl‐x _L . These results indicate that during the apoptosis mediated by TGF‐ß in fetal hepatocytes, ROS may be responsible for the decrease in bcl‐x _L mRNA levels that precedes the loss of Δψ _m , the release of cytochrome c , and the activation of caspase 3, culminating in cell death.—Herrera, B., Alvarez, A. M., Sanchez, A., Fernandez, M., Roncero, C., Benito, M., Fabregat, I. Reactive oxygen species (ROS) mediates the mitochondrial‐dependent apoptosis induced by transforming growth factor ß in fetal hepatocytes. FASEB J. 15, 741‐751 (2001)

Most of the morphologic changes that are observed in apoptotic cells are caused by a set of cysteine proteases (caspases) that are activated during this process. In previous works from our group we found that treatment of rat fetal hepatocytes with transforming growth factor β1 (TGF-β1) is followed by apoptotic cell death. TGF-β1 mediates radical oxygen species (ROS) production that precedes bcl-x_L down-regulation, loss of mitochondrial transmembrane potential, release of cytochrome c, and activation of caspase-3 (Herrera et al., FASEB J 2001;15:741-751). In this work, we have analyzed how TGF-β1 activates the caspase cascade and whether or not caspase activation precedes the oxidative stress induced by this factor. Our results show that TGF-β1 activates at least caspase-3, -8, and -9 in rat fetal hepatocytes, which are not required for ROS production, glutathione depletion, bcl-x_L down-regulation, and initial cytochrome c release. However, caspase activation mediates cleavage of Bid and Bcl-x_L that could originate an amplification loop on the mitochondrial events. An interesting result is that transmembrane potential disruption occurs later than the initial cytochrome c release and is mostly blocked by the pan-caspase inhibitor Z-VAD.fmk, indicating that the decrease in mitochondrial transmembrane potential (ΔΨm) may be a consequence of caspase activity rather than the mechanism by which TGF-β induces cytochrome c efflux. Finally, although Z-VAD.fmk completely blocks nucleosomal DNA fragmentation, it only delays cell death, which suggests that activation of the apoptotic program by TGF-β in fetal hepatocytes inevitably leads to death, with or without caspases. (Hepatology 2001;34:548-556.)