The processes of cell injury and cell death are important in many disease states as well as chemical injury, so understanding the stages of cellular response to an injury stimulus is of practical as well as theoretical importance.
In some instances cell death is rapid, arising as a consequence of loss of a major function, as when complement injury makes large holes in the cell membrane in minutes or seconds (Lachmann 1992). Far more often injury starts as a change of environment leading to activation of a process that ends in cell death many hours or days later (CCI4 1-2 days (Christie and Judah 1954); paracetamol 3-4 days (Prescott et. al. 1976)).
Many of the model systems used to study injury have not distinguished between the stages of initiation of cell damage and the later progression of the process leading to loss of function and death. Many of the model systems have used exposures that lead to rapid cell death within an hour or two, often with continuous exposure to the injury stimulus throughout the experiment. As a result of such a compressed system, it becomes difficult to distinguish the events of exposure from the events of progression, and to distinguish the key events of progression from the incidental side effects of injury.
Paracetamol induced cell injury and death have been intensively studied both in vitro and in vivo. However, the in vitro model systems do not always reflect events which occur in drug induced injury of the liver in patients. In the in vitro models using paracetamol as the toxin, all too many agents prevent cell death;
ethanol, DPPD, fructose, glycerol, glutamate, propylene glycol and prostaglandin’s (table 3.3, 3.4).
In the whole animal, none of these agents have more than marginal protective activity once the injury process has been initiated, that is after covalent binding is complete. We can prevent paracetamol metabolites ifom reaching intracellular targets by giving methionine or N-acetyl cysteine, only if given early enough (Prescott 1984). It is claimed by Harrison et. al. (1990) that NAC even if given later offers protection, (probably due to delayed metabolism of a large dose of paracetamol). But once injury has been initiated we have no effective therapy. This is true even when agents are given many hours before the expression of failure of function or cell death. Our picture of the course of cell injury is built up largely on the basis of in vitro models, where metabolism to the reactive metabolite NAPQl which covalently binds cellular proteins, is extensive by 1 hour (Devalia et. al. 1982). ATP levels are depleted by 30 minutes (Martin and McLean 1995), mitochondrial effects are seen as early as 30 minutes and enzyme leakage is significant by 4 hours (Nazareth et. al. 1991). The in vivo findings suggest that this picture is incomplete or perhaps incorrect. In vivo the time course is very different. In mice covalent binding takes place after GSH has been depleted at around 2 hours (follow et. al. 1973). ATP levels start to fall at 2 hours and enzyme leakage is not seen until 12 hours later (Martin and McLean
1995).
Initially an early attempt to resolve some of the difficulties employed a “two stage in vitro system” (McLean and Nuttall 1978), liver slices were incubated firstly
for up to 2 hours with paracetamol to initiate injury, then transferred to fresh medium without paracetamol for a further 4 hours to follow the progression stages of injury. During this stage the protective agents were added. This system was designed to try and separate initiation events from the progression of injury. However, this is still a compressed system, showing signs of injury 4-6 hours after exposure. Although many compounds prevented injury at 4-6 hours, when the system is extended to 18 hours all protection is lost (see table 3.3 and 3.4). I have developed an in vivo / in vitro system, with initiation of injury in vivo and subsequent progression of injury followed in vitro, this seems to provide a model which more closely reflects the in vivo situation.
I have examined paracetamol hepatotoxicity and shown that in this in vivo / in vitro model a combination of compounds is required to give cell protection suggesting that more than one point of injury is critical, and that each of these points needs to be targeted for cell survival. My results indicate that cyclosporin A, trifluoperazine and fructose (FCAT) when used in combination can protect against paracetamol toxicity to some extent. All appear to interact closely with aspects of mitochondrial function. It would appear that mitochondrial function is one of the key factors in cellular injury since loss of ATP is an early event in paracetamol injury and seems independent of paracetamol metabolism (Martin and McLean 1996; Nazareth et. al. 1991).
Mitochondria are important organelles and complete loss of function can lead to the cells’ death. Mitochondrial dysfunction with disruption of ATP production by dinitrophenol (DNP) can lead to cell death (Martin and McLean 1997). DNP is a
proton translocator and uncoupler of oxidative phosphorylation. At low concentrations of DNP (5pM) there is considerable ATP loss (> 5 0 % ) without injury to the cell. With higher doses ( > lOpM) there is a very rapid drop in ATP to less than 5nmoles/mg protein which precedes cell death. Inherited disorders of mitochondrial function may not lead to such low ATP levels and so may not be life threatening (Scholte et. al. 1987).
If we consider mitochondrial structure and function we observe that the outer membrane is permeable to most metabolites but the inner membrane is selectively permeable. The mitochondria possess many transporter systems for facilitating the transport of metabolites and nucleotides across the inner membrane. The adenine nucleotide transporter system allows the exchange of ATP and ADP but not AMP. The respiratory chain, an integral part of the inner mitochondrial membrane, acts as a proton pump. The transfer of electrons through the respiratory chain leads to the pumping of protons (H^) from the matrix to the other side of the inner mitochondrial membrane. The inner membrane is practically impermeable to protons, so the protons accumulate on the outside of the inner mitochondrial membrane creating an electrochemical potential difference across the membrane. This drives the ATP synthase which in the presence of Pi + ADP forms ATP,
Collapse of the electrochemical potential will therefore result in loss of ATP production and if membrane damage is severe, ATP is utilised by the reversal of the synthetase (e.g. DNP), leading to loss of even the glycolytic ATP.