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Mechanism of Inhibition of Mixed-Function Oxidation by Ethanol

  • Lester A. Reinke
  • Steven A. Belinsky
  • Frederick C. Kauffman
  • Ronald G. Thurman

Abstract

p-Nitroanisole (PNA) O-demethylation in perfused rat livers was inhibited by low concentrations of ethanol (Ki = 1 mM) and acetaldehyde (Ki = 0.5 mM). Ethanol and acetaldehyde also inhibited microsomal PNA metabolism, but only at much higher concentrations (Ki = 130 mM and 5 mM, respectively). Moreover, the dose-dependent inhibition of PNA metabolism in the perfused liver correlated strongly with changes in surface fluorescence of pyridine nucleotides and flavoproteins resulting from the metabolism of ethanol and acetaldehyde. t-Butanol (20 mM) did not cause pyridine nucleotide or flavoprotein reduction, nor did it inhibit PNA metabolism. When ethanol metabolism was blocked with 4-methylpyrazole, the inhibition of PNA metabolism by ethanol was diminished.

Ethanol infused in the presence of PNA decreased the intracellular concentrations of malate, aspartate and a-ketoglutarate. The infusion of aspartate (5 mM) partially reversed the inhibition of mixed-function oxidation by ethanol. These data indicate that changes in the oxidation-reduction state of pyridine nucleotides due to the metabolism of ethanol and acetaldehyde inhibit the transfer of mitochondrial reducing equivalents into the cytosol. Thus, inhibition of PNA metabolism by ethanol in the intact cell results from decreased availability of NADPH for NADPH-cytochrome *p < 0.005 with respect to values with no additions.
Table 1

Effect of Ethanol on p-Nitroanisole O-Demethylase Activity in Hepatic Microsomes from Fed, Phenobarbital-Treated Rats

Addition

p-Nitroanisole O-Demethylase Activity nmoles Products Formed/ Min/Mg Microsomal Protein

Percent Inhibition

None

1.21 ± 0.05

----

Ethanol, 2 mM

1.21 ± 0.02

0

Ethanol, 5 mM

1.11 ± 0.02*

8.3

Ethanol, 10 mM

1.04 ± 0.02*

14.0

Ethanol, 20 mM

0.95 ± 0.02*

21.5

Ethanol, 100 mM

0.67 ± 0.02*

44.6

Ethanol, 500 mM

0.28 ± 0.01*

76.9

Acetaldehyde, 5 mM

0.62 ± 0.04*

48.8

Methylpyrazole, 40 µM

0.94 ± 0.07

22.3

Hepatic microsomes were incubated with 0.5 mM p-nitroanisole, 10 mM nicotinamide, 5 mM Mg Cl2 and an NADPH-generating system consisting of 0.4 mM NADP+, 30 mM isocitrate and 0.2 units of isocitrate dehydrogenase. p Nitrophenol and 4-nitrocatechol formation from p-nitroanisole were determined as described in METHODS. Values are means ± SD from 3–6 individual flasks. Ki for ethanol equals 130 mM from double reciprocal plots.

Keywords

Aldehyde Dehydrogenase Perfuse Liver Pyridine Nucleotide Double Reciprocal Plot Ethanol Metabolism 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Springer Science+Business Media New York 1980

Authors and Affiliations

  • Lester A. Reinke
    • 1
  • Steven A. Belinsky
    • 1
  • Frederick C. Kauffman
    • 2
  • Ronald G. Thurman
    • 1
  1. 1.Dept. of PharmacologyUniv. Of North CarolinaChapel HillUSA
  2. 2.Dept. of PharmacologyUniv. of MarylandBaltimoreUSA

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