Suppression of apoptosis and induction of DNA synthesis in vitro by the phthalate plasticizers monoethylhexylphthalate (MEHP) and diisononylphthalate (DINP): a comparison of rat and human hepatocytes in vitro

Abstract

Diethylhexylphthalate (DEHP) and diisononylphthalate (DINP) are plasticizers with many important commercial, industrial and medical applications. However, both DEHP and DINP are rodent peroxisome proliferators (PPs), a class of compounds that cause rodent liver tumours associated with peroxisome proliferation, induction of hepatic DNA synthesis and the suppression of apoptosis. Despite these effects in the rodent, humans appear to be nonresponsive to the adverse effects of PPs. Previously, we have shown that the fibrate hypolipidaemic peroxisome proliferator, nafenopin, induced DNA synthesis and suppressed apoptosis in rat but not in human hepatocytes. In this work, we have examined species differences in the response of rat and human hepatocytes to DEHP and DINP in vitro. In rat hepatocytes in vitro, both DINP and MEHP (a principle metabolite of DEHP and the proximal peroxisome proliferator) caused a concentration-dependent induction of DNA synthesis and suppression of both spontaneous and transforming growth factor beta1 (TGFbeta1)-induced apoptosis. Similarly, both MEHP and DINP caused a concentration-dependent induction of peroxisomal beta-oxidation although the response to DINP was less robust. In contrast to the pleiotropic response noted in rat hepatocytes, neither DINP nor MEHP caused an induction of beta-oxidation, stimulation of DNA synthesis and suppression of apoptosis in human hepatocytes cultured from three separate donors. These data provide evidence for species differences in the hepatic response to the phthalates DEHP and DINP, confirming that human hepatocytes appear to be refractory to the hepatocarcinogenic effects of PPs first noted in rodents.

Modes of Action and Species-Specific Effects of Di-(2-ethylhexyl)Phthalate in the Liver

“…While the induction of peroxisomal genes in vitro in rodent hepatocytes is robust and comparable to that in whole liver [43,44], the increase in DNA synthesis, a marker of cell proliferation in vitro has been reported to be only 120-200%, which is markedly less than that observed in vivo [43,44]. Suppression of apoptosis studies in cultured rat hepatocytes were conducted under both "spontaneous" and growth factor (e.g., TGFβ1)-induced conditions [43,44,31].…”

“…It was concluded that hepatocytes from all species included in the study were responsive to MEHP by inducing peroxisomal palmitoyl-CoA oxidase, but rat hepatocytes were about 30-fold more sensitive based on the dose-response studies. The effects of MEHP on human hepatocytes have also been studied in vitro by several groups [43,44,31]. A general conclusion from these experiments is that human hepatocytes are largely non-responsive to MEHP when DNA synthesis, apoptosis, or peroxisomal gene induction are evaluated.…”

“…In cultured rat hepatocytes, MEHP shows a modest (up to 2-fold) yet significant effect on replicative DNA synthesis [43,44,100]. It should be noted, however, that the magnitude of response is much lower than that seen in rat liver in vivo and it has been hypothesized that other cells in liver (e.g., Kupffer cells) may play an important role by potentiating the proliferative response of the hepatocytes by producing mitogenic cytokines (see below).…”

Epigenetic Effects of Endocrine Disrupting Chemicals: Phthalates: Review

“…Ayrıca, MEHP'nin transforme edici büyüme faktörü [transforming growth factor (TGFβ1)] ve DNA'da hasar oluşturucu ajanlar olan etoposid, hidroksiüre ve anti-Fas antikorlarıyla indüklenen karaciğer apoptozunu da baskıladığı gösterilmiştir. 34,[39][40][41] Sıçan hepatositleri üzerinde yapılan canlılık deneylerinde, peroksizom proliferatörlerine maruz kalan hepatositlerin en az dört hafta canlı kaldığı; ancak kontrol hayvanlardan elde edilen hücrelerin ortalama sekiz gün canlı kalabildiği; peroksizom proliferatörlerine maruz kalan kültürlerde apoptoz belirtilerinin (kondense ve fragmante DNA) daha az sıklıkla görüldüğü belirtilmiştir. 42 PPAR-α'nın apoptozun inhbisyonu için gerekli olduğu düşünülmektedir ve PPAR-α-farelerin nafenopin gibi peroksizom proliferatörleri-nin etkilerine yanıt vermediği, spontan ve TGFβ1 ile indüklenen apoptoza açık olduğu belirtilmiştir.…”

“…42 PPAR-α'nın apoptozun inhbisyonu için gerekli olduğu düşünülmektedir ve PPAR-α-farelerin nafenopin gibi peroksizom proliferatörleri-nin etkilerine yanıt vermediği, spontan ve TGFβ1 ile indüklenen apoptoza açık olduğu belirtilmiştir. 34,40 PPAR-α aracılıklı apoptoz inhibisyonu bu reseptö-rün negatif efektör regülatör etkisine bağlıdır. 42 …”