COMPARATIVE EFFECTS OF THE AFLATOXINS ON SOME ENERGY-LINKED FUNCTIONS OF RAT LIVER MITOCHONDRIA IN VITRO

Abstract

Aflatoxins B1, B2, M1, G1 and G2 were isolated and purified. By chemical, spectrophotometric and polarographic techniques, the effects of these compounds on some energy-linked functions of rat liver mitochondria were investigated by seven criteria; (a) osmotic response, (b) dehydrogenase activities. (c) adenosine triphosphatase activity. (d) activity of electron transfer complexes. (e) reversed electron transfer. (f) calcium uptake. (g) substrate-dependent oxygen uptake of various metabolic states. Crude aflatoxin causes a significant enhancement of mitochondrial swelling in vivo. However, the present results indicate that the aflatoxins affect the osmotic behaviour of rat liver mitochondria maximally at concentrations of 2 - 3 x 10-5M in vitro. In this respect, the degree of mitochondrial swelling was of the order of only 5% over that of the control. When studied by the redox-dye technique, the activities of NAD specific dehydrogenase of glutamate, β-hydroxy-butyrate, malate, isocitrate and succinate dehydrogenase were markedly stimulated by crude aflatoxin, aflatoxins B1, B2, M1 and G1 and only slightly by G2. On pre-incubation of these toxic substances with mitochondria, these enzymes have been shown to be activated by at least a factor of 2. Aflatoxin G2 was significantly inhibitory under this condition. No consistent or significant effect of these substances on adenosine triphosphatase activity at the concentration of 2 - 3 x 10-5M were observed. However, a maximum stimulation of 10% over that of the control was observed in states l and 4. Spectrophotometric measurements reveal that these aflatoxins affect electron transfer and phosphorylation processes depending on the metabolic state of the mitochondria, concentration of the toxins and whether the toxins were pre-incubated with the mitochondria. The present results suggest that inhibition is mainly localised between the second and third phosphorylation coupling sites. Polarographic studies confirm the inhibitory effects of these aflatoxins in the region of cytochrome C. When reversed electron transfer was studied spectrophotometrically, it was further observed that these aflatoxins have no effect on the first phosphorylation coupling site and electron transfer between substrate and cytochrome b when energy was derived from ATP. However, there was partial inhibition when the reaction depended on oxidative generation of high energy intermediates. Calcium uptake was slightly enhanced by these aflatoxins. Classical respiratory inhibitors and uncouplers like rotenone, antimycin, amytal, oligomycin, cyanide, malonate, dicoumarol and 2, 4-dinitrophenol were used as markers in these investigation. In general, these substances are more potent respiratory inhibitors and uncouplers than the aflatoxins.