Dean P. Jones

Cytochrome c release and caspase activation in hydrogen peroxide‐ and tributyltin‐induced apoptosis

The ability of H2O2 and tributyltin (TBT) to trigger pro‐caspase activation via export of cytochrome c from mitochondria to the cytoplasm was investigated. Treatment of Jurkat T lymphocytes with H2O2 resulted in the appearance of cytochrome c in the cytosol within 2 h. This was at least 1 h before caspase activation was observed. TBT caused cytochrome c release already after 5 min, followed by caspase activation within 1 h. Measurement of mitochondrial membrane potential (ΔΨm) showed that both H2O2 and TBT dissipated ΔΨm, but with different time courses. TBT caused a concomitant loss of ΔΨm and release of cytochrome c, whereas cytochrome c release and caspase activation preceded any apparent ΔΨm loss in H2O2‐treated cells. Thus, our results suggest that different mechanisms are involved in triggering cytochrome c release with these apoptosis‐inducing agents.

Apoptosis: cell death defined by caspase activation.

In 1972, Kerr et al recognized that developmental, physiologic and some pathologic forms of cell death shared common features that were distinct from necrosis. The authors proposed the term `apoptosis to refer to this morphologic form of cell death. During the intervening period, there has been an explosive growth of research in this area, with over 8000 papers published in the past year alone. These studies have addressed cell death in a very broad spectrum of cell types and physiologic and pathologic conditions. The accumulation of data shows that the constellation of morphologic features identified as apoptosis are common but not uniform. Each of the characteristics, i.e. formation of nuclear and plasma membrane blebs, condensation of chromatin, loss of cell volume, detachment of cells from basement membranes, when considered alone, does not provide an unambiguous definition of apoptosis. There are time-dependent changes, cell-specific characteristics, and normal physiologic processes that introduce sufficient variation in morphology so that no single morphologic trait can define this type of cell death. Accordingly, it is highly desirable to have a definition of apoptosis based upon the underlying mechanism. Efforts to understand the mechanistic basis of apoptosis have yielded compelling evidence that activation of a caspase cascade provides a common biochemical pathway that explains both the morphologic features and the irreversible commitment point for this type of cell death. The purpose of this communication is to propose that apoptosis be redefined in biochemical terms as a caspase-mediated cell death with associated apoptotic morphology, i.e. cytoplasmic and nuclear condensation, chromatin cleavage and exposure of surface markers targeting cells for phagocytosis. Multicellular organisms (animals) share a common mechanism to control cell populations that has been conserved during evolution. The genetic basis of programmed cell death in Caenorhabditis elegans, was elucidated over a decade ago. This involves a cell death protease (CED-3) which is associated with activating (CED4) and inhibiting (CED-9) proteins. Homologous proteins have been found in organisms throughout the animal kingdom, including insects, amphibians, rodents and primates. The homologues of CED-3 are termed caspases, or cysteine-dependent aspartate-specific proteases. Overexpression of these proteins can result in cell death with apoptotic morphology. However, not all of these caspases normally function in cell death. A recent classification divided caspases into two groups, one involved in cytokine processing (caspase-1-like proteases) and the other playing a role in cell death. This latter group can be further subdivided according to function as activators or executioners of cell death and represent an evolution of this common mechanism for control of cell populations. Although full details and variations of the pathway have not been completely delineated, cell death in a variety of models involves activation of caspase(s). In surfacereceptor-mediated killing, activation of caspase-8 is an early event that initiates a caspase cascade and results in cleavage of different proteins located throughout the cell. In cytotoxic T lymphocyte-mediated killing, granzyme B activates caspases in target cells which results in cleavage of both nuclear and cytoplasmic proteins. In cells exposed to ionizing radiation or other DNA-damaging agents where p53 is activated, cell death is mediated by caspases. In stress-activated cell death induced by heat shock, cold shock, viral infection, bacterial infection, or growth factor withdrawal, caspase activation is a common feature of cell death (for review see reference 5). In steroidmediated cell killing, caspases are also activated. In mitochondria-mediated cell death, cytochrome c release leads to a caspase activity which is dependent upon a CED-4 homologue, Apaf-1. In all of these cases, small peptide-based caspase inhibitors delay or block appearance of apoptotic morphology and cell death. Moreover, the recent development of several caspase or Apaf-1 knockout animals, has provided more definitive support for the importance of these proteases in apoptosis. Thus, abundant evidence leads to the conclusion that caspase activation is a common pathway for the execution of this type of cell death. The proteolytic activity of caspases provides a molecular basis for apoptotic morphology. Structural components such as nuclear lamins and cytoskeletal proteins that bind to the plasma membrane are cleaved by caspases, preceding nuclear condensation and membrane blebbing. Caspase-dependent cleavage of ICAD (inhibitor of the caspase-activated DNase) allows activation of CAD and cleavage of DNA into characteristic oligonucleosomallength fragments. Exposure of phosphatidylserine on the cell surface, which is a marker for phagocytosis, is blocked by caspase inhibitors. Similarly, functional changes associated with controlled cell deletion are also linked to the cleavage of protein substrates for caspases (for review see reference 16). In Cell Death and Differentiation (1999) 6, 495 ± 496 ã 1999 Stockton Press All rights reserved 13509047/99

Use of isolated kidney cells for study of drug metabolism

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Formation and metabolism of a glutathione-S-conjugate in isolated rat liver and kidney cells

Abstract Isolated kidney cells were prepared from rat kidneys using a recirculating perfusion system with collagenase. The preparation was rapid and provided a high yield of intact metabolically active kidney cells predominantly of tubular origin. The respiration rate was 2.7 μmol O 2 /hr per 10 6 cells and was not stimulated by ADP. GSH content was 28.9 nmol/10 6 cells and did not decline during 2 hr of incubation. Cytochrome P450 content was 0.064 nmol/10 6 cells. The cells were characterized for their drug metabolizing activity using paracetamol as substrate. The rate of formation of the glucuronide and sulfate derivatives was linear for 2 hr, but slower than previously reported for rat liver cells. In contrast to incubations with liver cells, no glutathione conjugate was detected. However, formation of both cysteine and N -acetylcysteine derivatives was observed. The rate of formation of total sulfhydryl conjugates was about 50 per cent of that reported for liver cells when expressed on a cytochrome P450 basis. These studies establish the reliability and utility of this cell preparation as a model system for the study of drug metabolism by the kidney.

Calcium-activated DNA fragmentation in rat liver nuclei.

Abstract The reaction sequence involved in drug metabolism, which includes initial oxidation by cytochrome P-450, conjugation with GSH, conversion of the glutathione-S-conjugate to the cysteine-S-conjugate, and N-acetylation of the cysteine-S-conjugate to form the mercapturic acid derivative, was reconstituted in vitro using isolated rat liver and kidney cells with paracetamol as substrate. Oxidation and GSH conjugation were catalyzed primarily by the liver cells, while conversion of the GSH conjugate to the mercapturic acid derivative was catalyzed primarily by the kidney cells. With kidney cells, but not with liver cells, added GSH was oxidized to GSSG. Subsequently, GSSG concentration decreased with stoichiometric increase in glutamate concentration. Addition of GSSG to kidney cells inhibited metabolism of the GSH conjugate of paracetamol to the mercapturic acid derivative. These data indicate a relationship between metabolism of GSH conjugates and of GSSG by kidney cells and demonstrate the overall conversion in vitro of a drug to urinary products.