Adenine and pyridine nucleotide concentrations and relationships to 2,6-dinitrotoluene metabolism in cultured rat liver slices.
The relationships between nucleotide (ATP, ADP, AMP, NADPH, NADP(+), NADH and NAD(+)) concentrations and the metabolism of the hepatocarcinogen 2,6-dinitrotoluene (2,6-DNT) in cultured slices of rat liver were investigated. ATP, NADPH and NADH concentrations in freshly prepared rat liver slices at the beginning of culture were 48-67% lower than those measured in freeze-clamped rat liver (in vivo values). ATP concentrations in cultured liver slices increased with time of incubation, and after 4 hr ATP concentrations in liver slices were 25% greater than in vivo values. In contrast, NADPH and NADH concentrations did not recover with time of culture, and after 4 hr NADPH and NADH concentrations in liver slices were 50 and 24% of in vivo values, respectively. The addition of ethanol (50 mm) to cultured liver slices increased NADH concentrations by 132%, relative to untreated liver slices. Treatment of liver slices with ammonium chloride (10 mm), 6-aminonicotinamide (1 mm), or the substitution of fructose for glucose in the incubation medium significantly decreased NADPH concentrations by 64, 44 and 63%, respectively. All treatments significantly decreased ATP concentrations; fructose was the most effective agent tested and decreased liver slice ATP concentrations by 69%. These results indicate that the changes in liver slice nucleotide concentrations that occur during culture are not irreversible, and we suggest that these changes are a homoeostatic response to culture conditions and not a reflection of tissue damage or cytotoxicity. Rates of 2,6-dinitrotoluene metabolism by rat liver slices were unaffected by fructose or ammonium chloride, despite the decrease in NADPH concentrations produced by these agents. These results suggest that NADPH concentrations are not rate-limiting to 2,6-DNT metabolism by rat liver slices.
Chapman
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Division of Pharmacokinetics and Drug Metabolism, Wellcome Research Laboratories, Burroughs Wellcome Co., Research Triangle Park, NC 27709, USA.
Michener
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Powis
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