Hepatotoxicity of di-(2-ethylhexyl)phthalate is attributed to calcium aggravation, ROS-mediated mitochondrial depolarization, and ERK/NF-κB pathway activation.
Di-(2-ethylhexyl)phthalate (DEHP) is a widely used plasticizer found in a variety of polyvinyl chloride medical products. Although DEHP-induced cytotoxicity and apoptosis are well studied in various cell types, the precise mechanisms are not well understood so far. This study, aimed at going beyond the toxicology approach, focuses on the molecular mechanisms through which DEHP causes hepatotoxicity. We show that DEHP induces apoptotic cell death in a dose-dependent manner, as proven by an increase in annexin V-positively stained cells, DAPI/PI staining, and immunofluorescence studies. The DEHP-induced decrease in cell viability was significantly inhibited by adding catalase (CAT), but CAT treatment did not suppress the DEHP-stimulated calcium flux in the hepatocytes, whereas BAPTA-AM significantly reduced the DEHP-stimulated DCF intensity. These results demonstrate that DEHP increases the intracellular calcium level, which mediates the generation of H(2)O(2) in hepatocytes. Investigating cell-signaling mechanisms, we found that DEHP induced apoptotic cell death by mitochondrial-dependent caspase-3 activation and PARP cleavage. These changes due to DEHP exposure were associated with increased IKK and NF-κB phosphorylation. Preexposure of hepatocytes to an IKK inhibitor (PS-1145) prevented DEHP-induced caspase-3 and PARP cleavage. DEHP also markedly increased the activity of ERK1/2 MAPK. Pretreatment with the ERK inhibitor PD98059 attenuated NF-κB and IKK phosphorylation, indicating that ERK MAPK is mainly involved in DEHP-induced NF-κB activation. These results, for the first time, reveal that DEHP induces apoptosis in hepatocytes via the activation of the ERK/NF-κB signaling pathway, in which calcium ions and hydrogen peroxide act as the pivotal mediators of the apoptotic signaling.
Ghosh
Jyotirmoy
J
Division of Molecular Medicine, Bose Institute, Kolkata, India.
Das
Joydeep
J
Manna
Prasenjit
P
Sil
Parames C
PC
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004051:Diethylhexyl Phthalate / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D008099:Liver / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects*; D016328:NF-kappa B / Q000378:metabolism*; D010968:Plasticizers / Q000633:toxicity; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects
D016328:NF-kappa B; D010968:Plasticizers; D017382:Reactive Oxygen Species; D004051:Diethylhexyl Phthalate; D048049:Extracellular Signal-Regulated MAP Kinases; D002118:Calcium