Myocardial oxidative damage is induced by cardiac Fas-dependent and mitochondria-dependent apoptotic pathways in human cocaine-related overdose

Abstract

The aim of this study is to analyse cardiac specimens from human cocaine-related overdose, to verify the hypothesis that cardiac toxicity by acute exposure to high dosage of cocaine could be mediated by unbalanced myocardial oxidative stress, and to evaluate the apoptotic response. To address these issues, biochemical and immunohistological markers of oxidative/nitrosative stress were evaluated. We found that i-NOS, NOX2 and nitrotyrosine expression were significantly higher in the hearts of subjects who had died from high doses of cocaine, compared to the control group. Increase of these markers was associated with a dramatic increase in 8-OHdG, another marker of oxidative stress. A high number of TUNEL-positive apoptotic myocells was observed in the study group compared to the control group. The immunoexpression of TNF-α was significantly higher in the cocaine group compared to the control group. Furthermore, we detected a significantly stronger immunoresponse to anti-SMAC/DIABLO in our study group compared to control cases. Both cardiac Fas-dependent and mitochondria-dependent apoptotic pathways appeared to be activated to a greater extent in the cocaine group than in the control group. Our results highlight the central role of oxidative stress in cocaine toxicity. High levels of NOS can promote the oxidation process and lead to apoptosis.

Figure 1. The level of MDA was significantly elevated (p < 0.05) in study group cardiac specimens compared to the control group.

Cardiac cytosolic levels of AA were significantly lower (p < 0.05) and the GSH/GSSG ratio was dramatically lower (p < 0.01) in the study group cases compared to controls. Results were expressed as mean ± SD. The comparison between groups was conducted using the comparison Student’s t test. A value of p < 0.05 was considered statistically significant. *p < 0.05; **p < 0.01 study group vs control group.

Figure 2

(A) Hypereosinophilic, hypercontracted myocardial cells with rhexis of the myofibrillar apparatus into cross-fibre, anomalous, and irregular bands (insert: normal aspect of myocytes in control group specimens) (haematoxylin and eosin, original magnification 100x). (B,C) Increase of apoptotic cells was measured by TUNEL assay: values of myocyte cell apoptosis in the cocaine group were significantly higher than control cases. The percentage of apoptotic myocyte nuclei was determined. Apoptosis was measured by the determination of the fraction of myocyte nuclei labelled by TUNEL: 60 ± 13% and 3 ± 1% apoptotic cells were observed in cocaine group and control group (D) (original magnification for B-C-D 63x), respectively. (E) Quantification of apoptosis positive-stained nuclei between groups: the percentage of apoptotic myocyte nuclei was significantly elevated (p < 0.001) in study group specimens compared to the control group. Values are presented as mean ±SD (standard deviation). The unpaired two-way Student’s t-test was used to compare the results obtained for cocaine group with the control group. p < 0.05 was accepted as indicative of significant difference among groups.

Figure 3

Intense and massive positive (brown in A and in B) myocyte immunoreaction of NOX2 (A,B) in the cocaine group, with representative images of NOX2 immunostaining demonstrated by brown reaction in the cardiac cells compared by the negativity in the control group (C) (original magnification for A-B-C 40x).

Figure 4

(A,B) Immunohistochemistry analysis of i-NOS showing positive staining (yellow reaction) in cocaine group heart tissue and compared with the control group (original magnification for A-B-C 40x) (C).

Figure 5

Intense nitrotyrosine reaction (darker reaction in cardiac cell) in cocaine group study (A,B), compared with control group (original magnification for A-B-C 40x) (C).

Figure 6

8-OHdG positive staining in about 70% of cardiomyocytes nuclei (nuclear brown reaction) from study group specimens (A,B), while they were detected in less than 5% in control group cardiac specimens (C) (original magnification for A-B-C 80x). Values are presented as mean ±SD (standard deviation). The unpaired two-way Student’s t-test was used to compare the results obtained for cocaine group with the control group. p < 0.05 was accepted as indicative of significant difference among groups.

Figure 7

SMAC Diablo immunostaining in cardiomyocytes nuclei (nuclear brown reaction) from cocaine group specimens (A,B), while they were detected in less than 5% in control group cardiac specimens (C) (original magnification for A-B-C 100x).

Figure 8

(A,B) Increase of Bcl-2 positive (nuclear brown reaction) immunostaining in the cells’nuclei of the cocaine group; positive cardiomyocytes nuclei were significantly higher than control case group (C) (original magnification for A-B-C 80x).

Figure 9. Mechanisms of the pathological effects of cocaine: apoptotic myocells process is due both to cardiac Fas-dependent and mitochondria-dependent apoptotic pathways.

Evaluation of myocardial oxidative damage is the hypothesis that cardiac toxicity caused in humans by acute exposure to high dosage of cocaine could be mediated, at least in part, by unbalanced myocardial oxidative stress.