Ultrastructural variability of mitochondrial cristae induced in vitro by bee (Apis mellifera) venom and its derivatives, melittin and phospholipase A2, in isolated rat adrenocortical mitochondria

Abstract

We tested the ability of bee venom (BV), melittin (Mlt), and phospholipase A2 (PLA) – used in 5 concentrations each (5, 10, 15, 20 and 40 μg/100 μl) – to promote ultrastructural changes and reorganization of cristae in vitro in mitochondria isolated from rat adrenal cortex after a protocol optimized by us. Thus, apart from two control grups (CI and CS), in which the mitochondria were suspended into saline buffer and isolation medium respectively, 15 more groups of mitochondria were constituted, corresponding to the five different doses of the three substance tested (BV5 to M40; M5 to M40 and P5 to P40). The ultrastructural effects were quantified on transmission electron micrographs using a morphometry software. Values of 84.49 nm and 95.45 nm were calculated for median diameters of mitochondrial cristae in two control groups. Large and very large vesicular cristae, many with 2 or 3 membranes, were generated depending on dose among normal cristae in all treated groups. In the BV and Mlt treated groups, after an initial increase (up to 127.27 nm in V15 group and 151.2 nm in M10 group) due to stimulation of cristae fusion, the cristae diameter diminished as the doses increased, mainly by the collapse of the cristae. In the PLA treated groups, the cristae diameter increased continuously from 83.84 nm to 136.01 nm, by stimulated fusion of cristae, only the two largest doses promoting the collapse of cristae in some mitochondria. The highest percentage of abnormal cristae was found in the Mlt treated groups and next in BV treated groups. All substances tested produced pronounced ultrastructural variability of mitochondrial cristae in vitro: they also changed (depending on dose) mitochondrial shapes, generated matrix debris and the highest concentrations of BV and Mlt were responsible for mitochondrial breakdown. These ultrastructural alterations of mitochondrial criste in the presence of the BV molecules suggest a reduced capacity of adrenocortical mitochondria to synthetize steroid hormones consequently to BV envenomations and partially explain the toxic effects of the BV.