Effect of aflatoxin on mitochondrial transcription and translation

Abstract

The effect of aflatoxin B₁ on RNA synthesis in phosphate-swollen rat liver mitochondria was examined. In the assay system used the incorporation of ³H-UTP into acid-insoluble product was inhibited 52% by actinomycin D (50µg/ml) and the labeled product was sensitive to pancreatic RNase. Aflatoxin B₁ inhibited in vitro mitochondrial ³H-UTP incorporation only 12% at high concentrations (0.4 mM or 130 nmol/mg protein). On the other hand, 25% inhibition was observed with mitochondria isolated 4 hr after animals had been treated with aflatoxin B₁ (5.3 mg/kg body weight by i.p. injection). This suggested that a metabolite of aflatoxin B₁ may inhibit mitochondrial RNA synthesis.

The effect of aflatoxin B₁ on mitochondrial protein synthesis was examined using sterile mitochondrial preparations and two assay systems. In the first assay mitochondrial respiration served as an energy source, and in the second an ATP-generating system was used. As expected for mitochondrial protein synthesis, the incorporation of ¹⁴C-leucine into hot acid-insoluble product was inhibited 86% by chloramphenicol but was not affected by cycloheximide.

Aflatoxin B₁ inhibited in vitro mitochondrial protein synthesis only at very high concentrations (13-25% inhibition at 0.4 mM or 135 nmol/mg protein). Treatment of animals with aflatoxin B₁ (5.0 mg/kg body weight), which is expected to result in relatively low concentrations of aflatoxin in the mitochondrial fraction (0.1 nmol/mg protein), produced a 23% inhibition of protein synthesis in mitochondria isolated 4 hr after injection. These data suggest that a metabolite of aflatoxin B₁ may inhibit mitochondrial protein synthesis.

Using an in vitro system for the metabolism of aflatoxin B₁ by the mixed-function oxidase system in the presence of mitochondria, it was found that a metabolite(s) of aflatoxin B₁ inhibited mitochondrial protein synthesis by 40% at a concentration of 0.5 nmol/mg mitochondrial protein. This inhibition was not due to an alteration of mitochondrial respiration, as inhibition was observed in both the respiration and ATP supported assays. In addition, mitochondrial respiration, respiratory control ratios and P:O ratios were not affected. Formation of the inhibitory metabolite(s) required NADPH and 2, 3-unsaturation of the aflatoxin molecule.