Martin, Francis L; (1994) Energy status of hepatic cells during progression and prevention of cell injury in vitro. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Cell injury takes place in two phases, initial interaction with an adverse chemical environment, and subsequent stages of progression of injury to cell death, or else adaptation and survival. In this study paracetamol, 2,4-dinitrophenol (DNP), antimycin A, and ethionine injury in hand cut rat liver slices were used to investigate the relationship between ATP levels and progression of cell injury. Slices were exposed to 10mM paracetamol for 120min and, then, incubated without paracetamol for up to 240min to investigate paracetamol-induced injury. Other slices were exposed to various concentrations of DNP, antimycin A, and ethionine to investigate the injury induced by these other compounds. Introduction of 10mM or 20mM fructose in the second phase of incubation prevented paracetamol-induced injury. 100µM adenine introduced at the start of incubation reversed ATP depletion induced by ethionine. Adenine did not reverse paracetamol-induced injury. Injury was quantified by measuring leakage of lactate dehydrogenase (LDH) and potassium (K+). ATP levels were measured using the luciferin-luciferase bioluminescent assay. ATP levels were markedly depleted after exposure to toxic concentrations of paracetamol, DNP, antimycin A and ethionine. This depletion of ATP was found to precede the onset of cell injury as quantified by significant loss of K+ and LDH. However, protection against paracetamol-induced injury using 10mM or 20mM fructose was not dependent on ATP levels. Histological examination of paracetamol-treated slices showed extensive centrilobular injury correlating well with the in vivo situation. Indices of viability i.e. ATP and K+ levels in control slices, were found to compare favourably with levels reported in precision cut liver slices and isolated hepatocytes. ATP levels were maintained at in vivo levels for six hours following a pre-incubation step. The effect of 800mg paracetamol/kg in vivo was examined. Depletion of ATP and total adenine nucleotide levels was observed. However, ATP/ADP and ATP/AMP ratios and energy charge levels were not markedly altered. ATP depletion preceded cell injury as measured by increased enzyme activity in the plasma, visible evidence of cell injury and increase in water content of liver tissue. The metabolic status of the livers was also examined using 31P-nmr. However, no gross alterations were observed using this technique. This study shows that ATP levels are crucial to the maintenance of ionic balance and membrane integrity. However, it also shows that cells can survive with depleted ATP levels. It is probable that ATP levels need to be depleted to a certain threshold level before a loss of homeostatic control becomes apparent. Liver slices cut by hand were found to be a good and reproducible model for investigating the underlying mechanisms leading to injury.

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