The Effects of Pyrene on Complex I and Complex II‐mediated Respiration in Isolated Skeletal Muscle Mitochondria in the Leopard frog, Rana pipiens

Abstract

We have previously shown that in‐water exposure of the Northern Leopard frog, Rana pipiens, to pyrene significantly reduces many ATP‐dependent processes including hepatocyte cell volume regulation, skin Na+ transport, and skeletal muscle contractility. Furthermore, skin, liver, and muscle mitochondrial O2 consumption is significantly less in pyrene‐exposed frogs when compared to frogs under control conditions. One plausible explanation for the pyrene‐induced reduction in mitochondrial performance is through inhibition of mitochondrial complex activity. Therefore, the purpose of the present study was to isolate skeletal muscle mitochondria from Northern Leopard frogs and to examine Complex I and Complex II‐mediated respiration following direct exposure to pyrene or control solutions. After isolation of skeletal muscle mitochondria, Complex I and Complex II‐mediated respiration was examined using glutamate/malate and succinate, respectively, in an Oroboros O2k‐fluorespirometer equipped with a polarographic O2 sensor. To verify that decreased mitochondrial functionality could be detected, Complex I enzyme activity was inhibited with rotenone, whereas Complex II enzyme activity was inhibited with malonic acid. In order to assess the direct effects of exposure on mitochondrial function, 1.48 mM pyrene was added to extraction and suspension media. Isolated mitochondria were then incubated in suspension media with 1.48 mM pyrene for 1‐1.5 h. The Complex I and II enzyme activity was assessed and compared with mitochondria exposed to control solutions. There were no significant differences in mitochondrial Complex I and II‐mediated respiration following incubation with pyrene. These data suggest that the previously measured decrease in skeletal muscle mitochondrial performance following in‐water exposure of Northern Leopard frogs to pyrene is the result of effects on the proteins downstream of Complex I and II. Deficiencies in CoQ, Complex III, CytoC, and Complex IV activities could lead to the observed reduced mitochondrial respiration.