Beales, Denise; (1997) Mechanisms of cell injury and protection in the rat after exposure to hepatotoxins. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

It is of importance when studying cell injury that the models used give information that is applicable to in vivo use. Paracetamol has been used as a model compound for the study of cell injury by many researchers (Jollow et. al. 1974, McLean and Nuttall 1978, Beales et. al. 1985, Shen et. al. 1992). The initial phase of toxicity with absorption and activation of paracetamol to a reactive metabolite is well understood and therapies for treating overdose during this phase are well established. The later progression of cell injury has yet to be fully elucidated and therefore no successful treatment is available. Many models for examining cell injury and protection use in vitro models that produce cell death in a short time and do not correlate well with events found in vivo. Many compounds have been shown to protect in the in vitro system after the initiation stage, but when tried in vivo do not offer any protection. Using an in vitro liver slice model I have shown that many compounds e.g. glycerol, fructose, diphenyl para phenylene diamine (DPPD) and ethanol, will prevent paracetamol injury in the short term i.e. up to 6 hours. But when the incubation time is extended to 18 hours we do not see any protection. The concentration of paracetamol used is 10mM, this is in the concentration range found in the plasma following an i.p. dose of 1g/Kg in vivo. So although, the concentration of paracetamol is similar in each model the time course of injury in the in vitro system is much shorter with significant leakage of enzymes by 6 hours, where as in vivo enzyme leakage is not apparent until around 12 hours. I have therefore developed a model which is comparable to the time course of injury in the in vivo situation. Paracetamol 1g/Kg i.p. is given to initiate injury in vivo, then several hours later liver slices are taken and incubated in an in vitro system for up to 18 hours. In this system the compounds that protected against paracetamol injury in the 6 hour in vitro model, showed no protective effects. Cell injury from paracetamol cannot be prevented by a single compound as in the short term in vitro model. But using paracetamol and carbon tetrachloride as models for liver cell injury, I have shown that in my new model of in vivo initiation and in vitro progression, the combination of fructose, cyclosporin A and trifluoperazine (FCAT) does offer protection in the late stage of injury, as measured by lactate dehydrogenase leakage and potassium content of the slice. This combination also maintains the mitochondrial membrane potential. Ultimately one would hope that the results found in any in vitro model would help in devising a system of protection in vivo. I have found that in vivo, glycerol does not protect against paracetamol injury. Neither does ibuprofen or dexamethasone which was shown by Horton and Wood (1989) to protect against late paracetamol injury in vivo. Protection was found in vivo in one experiment using rats dosed with paracetamol 1g/Kg i.p. then given a combination of fructose (5g/Kg), cyclosporin A (20mg/Kg) and trifluoperazine (80mg/Kg) (FCAT) orally either 3 hours or 3 and 8 hours post II the paracetamol dose. A reduction in the alanine transaminase levels in the plasma at 24 hours was found, although in a second experiment there was no significant difference between the groups. To try and establish the mechanisms by which these agents are working, I have looked at other compounds which either have been shown to act in the same way or do not act at a particular point. I have replaced fructose with glycerol which can also enter glycolysis as a potential source of ATP. Cyclosporin A was replaced with FK506 another immunosuppressant which lacks the mitochondrial effect on the pore of the inner membrane. Trifluoperazine was replaced with a variety of phenothiazines to see if there were similar effects. My results would indicate that protection from cell injury by xenobiotics such as paracetamol will require a combination of compounds targeting different sites of injury. No one compound is likely to prevent the progression of cell injury. My in vivo / in vitro model may help in devising such a combination, this model shows a progression of injury from paracetamol, similar to the time course of injury in vivo. By using a model that shows a similar response in vivo, one can improve the protection combination without using large numbers of animals.

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