Cardiac changes following overdose Faa A et al. CLINICS 2012;67(7):821-826 Figure 4 - S100B positivity and type of substance. requires induction of an appropriate factor to counter the action of some negative regulatory elements (47). In this study, the S100B protein was also detectable in extracellular spaces, in which it might exert multiple important effects. Previous research has shown that astro- cytes may be stimulated to excrete S100B by relatively high levels of cytosolic Ca (48). A similar enhancing action of calcium ions could also be hypothesized in cardiomyocytes under hypoxic stress. A secondary hypothesis to explain the presence of S100B outside of cells is leakage from damaged cells (49). Our observation of coagulative myocytolysis in all cases of drug abuse might confirm this hypothesis. Whatever the source of extracellular S100B and the mechanism underlying its release, extracellular S100B might attain local concentrations sufficient to affect cellular activities by paracrine, autocrine and endocrine manners. The extracellular effects of S100B protein on cardiomyo- cytes and inflammatory cells could be mediated by the receptor for advanced glycation end-products (RAGE), a member of the immunoglobulin superfamily that is gen- erally considered a central mediator of ischemia/reperfu- sion injury in the heart (50-53). Dissecting the complex pattern of intracellular signaling resulting from hypoxic insults to ischemia and reperfusion (I/R) may be a considerable challenge. In the complex environmental interactions in the injured heart, several ‘danger signals’ are commonly liberated from ischemic cells (52). These signals signal the ischemic insult to other cells and mediate an inflammatory response but also promote tissue growth and remodeling that is partially Figure 5 - Oedema, wavy arrangement of cardiomyocytes. 824 mediated via RAGE activation. The S100B protein could participate in this process. Our data confirm the hypothesized existence of a common pathway through which multiple drugs (heroin and cocaine) might cause S100B protein cardiac overload and release. The reported increase in S100B urine levels in newborns at higher risk of neonatal death (33) due to hypoxic-ischemic encephalopathy or asphyxia (34) suggest that severe hypoxia is the common mechanism of drug- induced S100B protein accumulation at the cardiac level. Finally, our results, though limited by the small number of cases available and the numerous uncontrolled variables involved (e.g., doses, survival time, individual tolerance, post-mortem redistribution, drug stability, etc.), support the hypothesis that the immunohistochemical detection of sig- nificant S100B protein in the heart supports a suspicion of drug addiction and could distinguish overdose-related death from traumatic or ischemia-related death. Based on our preliminary data, we suggest that the immunodetection of S100B protein in the heart could be useful to not only help determine the cause of death in complicated or atypical cases but also provide evidence in individual cases to be shown in court. Further studies are needed to clarify the metabolic link between overdose and S100B protein overload in the heart of drug abuse-related sudden deaths. ACKNOWLEDGMENTS The authors wish to thank Mr. Ignazio Ferru for secretarial assistance and Mrs. Sandra Serra for technical work. AUTHOR CONTRIBUTIONS Faa A analyzed and interpreted the data and prepared the manuscript. Senes G processed the heart tissues (hematoxylin–eosin and S100B immunostaining). Locci A processed the heart tissues (hematoxylin–eosin and S100B immunostaining). Pampaloni P provided assistance to the manuscript preparation and assembled images. Pais ME performed the experiments. Piras B performed the experiments and critically revised the manuscript. D’Aloja E critically revised the final version of the manuscript. Faa G contributed to the conception and design of the study. REFERENCES 1. Hollander JE, Hollander RS. In. Goldfrank LR, ed. Goldfrank’s Toxicologic Emergencies. New York: McGraw-Hill Medical Pub Division. 2002;1004-19. 2. Hale SL, Lehmann MH, Kloner RA. Electrocardiographic abnormalities after acute administration of cocaine in the rat. Am J Cardiol. 1989;63(20):1529-30, http://dx.doi.org/10.1016/0002-9149(89)90024-6.