Dysfunction of cortical synapse-specific mitochondria in developing rats exposed to lead and its amelioration by ascorbate supplementation

Figures & data

Figure 1 Isolation of purified synapse-specific mitochondrial fraction from rat brain cortices.

Notes: Enrichment in activity of mitochondrial marker SDH was observed for SM fraction isolated by discontinuous Ficoll medium-based gradient ultracentrifugation method. SDH activity levels in SM fraction were increased by 7.2±0.4 (mean ± SEM) times compared to homogenate. Negative control comprising SM fraction preincubated with malonate was used to confirm validity of the assay. Data are represented as mean ± SEM (n=4 samples from 4 rats).
Abbreviations: SDH, succinate dehydrogenase; SM, synapse-specific mitochondria; malo, malonate; PNS, post-nuclear supernatant; PMS, post-mitochondrial supernatant; CMP, crude mitochondrial pellet.

Figure 2 SM of rats developmentally exposed to Pb have increased levels of ROS and RNS and the increase is ameliorated by ascorbate.

Notes: (A) DCFH-DA-based fluorometric analysis of ROS species show elevated ROS levels in SM of Pb-exposed rats. The increase is attenuated by ascorbic supplementation. (B) Increase in levels of RNS was observed in SM from brain cortices of rats chronically exposed to Pb. This Pb-induced increase in RNS levels was not observed in rats supplemented with ascorbic acid. Data are represented as mean ± SEM (n=5 pups from 3 litters) and * and # represent statistical significance (p<0.05) when compared to Ctrl and Pb+Asc groups, respectively (ANOVA with Newman-Keuls correction).
Abbreviations: SM, synapse-specific mitochondria; Pb, lead; ROS, reactive oxygen species; RNS, reactive nitrogen species; DCFH-DA, 2′,7′-dichlorofluorescein diacetate; Ctrl, control; Asc, ascorbic acid; SEM, standard error of the mean; NOx, total nitrites and nitrates.

Figure 3 SM from cerebral cortices of rat pups exposed to Pb show increased oxidative damage to lipids and proteins and have reduced antioxidant capacity and their redox status is rescued by ascorbic acid supplementation.

Notes: Increased oxidative damage to lipids and proteins was observed in SM fractions of Pb-exposed rats as evidenced from elevated TBARS (A), increased protein carbonylation (B) and increased protein thiol oxidation (C). Moreover, ascorbic acid supplementation could rescue the oxidative damage in SM fraction induced by Pb exposure. (D) Antioxidant capacity of SM fraction, as measured by ABTS radical decolorization assay, was found to be significantly lowered upon chronic Pb exposure but was recovered to control levels upon ascorbate supplementation. Data are represented as mean ± SEM (n=5 pups from 3 litters) and * and # represent statistical significance (p<0.05) when compared to Ctrl and Pb+Asc groups, respectively (ANOVA with Newman-Keuls correction).
Abbreviations: SM, synapse-specific mitochondria; Pb, lead; TBARS, thiobarbituric acid reactive substances; ABTS, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); Ctrl, control; Asc, ascorbic acid; SEM, standard error of the mean.

Figure 4 SM from cerebral cortices of rat pups exposed to Pb show alterations in basal mitochondrial membrane potential as well as energization-induced generation of mitochondrial membrane potential and ascorbic acid abolishes these alterations.

Notes: MMP at both basal states (A) and in presence of substrates (B) was reduced in SM fraction isolated from Pb-exposed rats. Pb-induced alterations in MMP and substrates-induced MMP generation were rescued upon ascorbate supplementation. Data are represented as mean ± SEM (n=5 pups from 3 litters) and * and # represent statistical significance (p<0.05) when compared to Ctrl and Pb+Asc groups, respectively (ANOVA with Newman-Keuls correction).
Abbreviations: SM, synapse-specific mitochondria; Pb, lead; MMP, mitochondrial membrane potential; Ctrl, control; Asc, ascorbic acid.

Figure 5 Pb induced alternations in the bioenergetic properties of cortical SM are mitigated by ascorbate supplementation.

Notes: While activities of complex I (A) and IV (D) of the ETC were unaltered in SM of Pb-exposed rats when compared to aged matched controls, activities of complex II (B) and III (C) were found to be significantly reduced in SM fraction isolated from Pb-exposed rats. However, ascorbate supplementation rescued the loss in activity of complex II alone without affecting loss in activity of complex III. (E) Significant decrease in the rate of ATP synthesis was observed for SM of Pb-exposed rats when compared to aged matched controls and the reduction was ameliorated by ascorbic acid supplementation. Data are represented as mean ± SEM (n=5 pups from 3 litters) and * and # represent statistical significance (p<0.05) when compared to Ctrl and Pb+Asc groups, respectively (ANOVA with Newman-Keuls correction).
Abbreviations: SM, synapse-specific mitochondria; Pb, lead; ETC, electron transport chain; Ctrl, control; Asc, ascorbic acid.

Figure S1 Supplementation of ascorbic acid alone does not affect the bioenergetics of cortical SM.

Notes: MMP at both basal states (A) and in presence of substrates (B) was not altered in SM fraction isolated from rat pups supplemented with ascorbic acid when compared to aged matched controls. Ascorbic acid supplementation did not affect other mitochondrial functions, including ATP synthesis (C) and activities of complexes I–IV (D–G). Data are represented as mean ± SEM (n=5 pups from 2 litters). Difference between the groups were assessed statistically using unpaired two-tailed Student’s t-test.

Abbreviations: SM, synapse-specific mitochondria; MMP, mitochondrial membrane potential; Ctrl, control; Asc, ascorbic acid; SEM, standard error of the mean.