North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition Annual Meeting, October 19-22, 2006, Orlando, Florida: Abstracts: ORAL ABSTRACT PRESENTATIONS: HEPATOLOGY/IBD/CELIAC DISEASE SATURDAY, OCTOBER 21, 2006, 10 a.m.- 12 Noon: 181
Several genetic metabolic liver diseases are characterized by hepatocellular steatosis and cholestasis, a combined lesion we refer to as steatocholestasis. In in vitro and in vivo models of steatocholestasis in leptin receptor-deficient Zucker rats, we demonstrated that glycochenodeoxycholic acid (GCDC) induces more severe injury in the obese liver and that antioxidants protect against this injury (Gastroenterology 2005;128:S1512). The objective of this study was to further characterize the pathways of hepatocellular injury in steatocholestasis in an in vitro primary culture model.
Sprague-Dawley rats were fed a high-carbohydrate diet to generate hepatic steatosis or a control diet (J Hepatol 2003;39:978). Lean and fat-laden hepatocytes were isolated and cultured overnight in the presence of 250μM trolox (TRX), 20mM n-acetylcysteine (NAC), 100 μM tauroursodeoxycholic acid (TUDC), or vehicle (control). The following morning, 100 μM GCDC, 500 μM tert-butylhydroperoxide (tBOOH), or vehicle (control) was added. Hepatocyte necrosis was measured by LDH release, apoptosis by caspase-3,7 activity, and hydroperoxide generation by dichlorofluorescin (DCF) fluorescence.
After 4 hr in both lean and fat-laden hepatocytes, GCDC caused significant elevation in LDH release, caspase-3,7 activation, and DCF fluorescence vs control (P ≤ 0.001). Apoptosis was significantly reduced in fat-laden vs lean hepatocytes (P < 0.05), but there was no difference in necrosis or ROS generation. TUDC protected against GCDC-induced apoptosis and necrosis (P < 0.05) in both obese and lean hepatocytes, but it did not affect ROS generation. Neither TRX nor NAC reduced hepatocyte apoptosis, necrosis, or ROS generation following GCDC, although they did significantly reduce tBOOH-induced ROS generation and LDH release (P < 0.05).
These surprising results suggest that alternate, ROS-independent pathways of hepatocellular injury may be operative in this primary hepatocyte model of steatocholestasis in contrast to findings in Zucker rat hepatocytes.