Bile duct epithelial cells exposed to alpha-naphthylisothiocyanate produce a factor that causes neutrophil-dependent hepatocellular injury in vitro

Abstract

The acute hepatotoxicity induced by alpha-naphthylisothiocyanate (ANIT) in rats is manifested as neutrophil-dependent necrosis of bile duct epithelial cells (BDECs) and hepatic parenchymal cells. This hepatotoxicity mirrors that of drug-induced cholangiolitic hepatitis in humans. Since BDECs are primary targets of ANIT-induced toxicity, we hypothesized that after exposure to ANIT, BDECs produce a factor(s) that causes neutrophil chemotaxis and neutrophil-dependent hepatocellular injury. To test this hypothesis BDECs were isolated from male Sprague Dawley rats and incubated with ANIT (6.25, 12.5, 25, or 50 microM) or vehicle for 24 h. The conditioned medium (CM) was collected and placed in the bottom chamber of a two-chambered chemotaxis system, while isolated neutrophils were placed in the top chamber. Chemotaxis was indicated by neutrophil migration through a membrane to the bottom chamber. CM from BDECs exposed to each concentration of ANIT was chemotactic, whereas CM from vehicle-treated BDECs was not. ANIT alone caused a modest degree of chemotaxis at 50 microM. The conditioned media were added to isolated hepatocytes or to hepatocyte-neutrophil cocultures and incubated for 24 h. Hepatocyte toxicity was indicated by alanine aminotransferase release into the culture medium. CM from vehicle-treated BDECs did not cause hepatocyte killing in either hepatocyte-neutrophil cocultures or hepatocyte cultures. In contrast, the addition of CM from ANIT-treated BDECs (CM-BDEC-A) to hepatocyte-neutrophil cocultures resulted in hepatocyte killing. The same CM was not cytotoxic to hepatocyte cultures devoid of neutrophils. The hepatocyte killing could not be explained by residual ANIT in the CM, which was below the limit of detection (< or = 0.5 microM). The addition of antiproteases afforded protection against neutrophil-dependent hepatocellular injury induced by CM-BDEC-A. These results indicate that ANIT causes BDECs to release a factor(s) that attracts neutrophils and stimulates them to injure hepatocytes in vitro.

Non invasive high resolution in vivo imaging of α-naphthylisothiocyanate (ANIT) induced hepatobiliary toxicity in STII medaka

“…Results of this study suggest cells of medaka intrahepatic biliary system respond to ANIT, and that responses, both cellular and system level, are similar to those described in rodents Carpenter-Deyo et al 1991;Connolly et al 1988;Hill et al 1999;Kossor et al 1993;Lesage et al 2001;Orsler et al 1999;Waters et al 2001). Changes observed in vivo, and confirmed with ex-vivo analyses (histological, immunohistochemical, ultrastructural), were; attenuation and dilation of bile canaliculi, bile preductular lesions, hydropic vacuolation of hepatocytes and BPDECs, BPDEC cytomegaly, and hyperplasia of BECs in the hilar and peri-hilar region of the liver.…”

Characterization of the protective effects of melatonin and related indoles against ?-naphthylisothiocyanate-induced liver injury in rats

“…In fact, BECs are the primary targets of ANIT-induced toxicity and injury to these cells occurs prior to hepatocellular damage [Connolly et al, 1988]. Furthermore, it has been demonstrated that after exposure to ANIT, BECs produce a factor(s) that causes neutrophil chemotaxis and neutrophil-dependent hepatocellular injury [Hill et al, 1999]. A recent study has demonstrated that human BECs exposed to certain proin¯ammatory cytokines (IL-1 and TNFa) rapidly express IL-8 and monocyte chemotactic protein-1 (MCP-1), potent chemotactic agents for neutrophils and monocytes or T cells, respectively [Morland et al, 1997].…”

Resveratrol effectively attenuates α-naphthyl-isothiocyanate-induced acute cholestasis and liver injury through choleretic and anti-inflammatory mechanisms

“…Taken together, our results indicate that the impairment of mitochondrial biogenesis, rather than oxidative injury and mitochondrial oxidative phosphorylation, is a critical event at this stage of ANIT-induced cholestasis. The activity of antioxidant enzymes, ATP synthesis and mtDNA copy number remained unchanged after resveratrol treatment in ANIT-intoxicated rats, implying that the antioxidant properties and mitochondrial protection of resveratrol The release of cytotoxic and inflammatory mediators, including those that attract neutrophils, has been suggested to contribute to the hepatic injury induced by ANIT; however, the exact mechanisms are not well understood [27][28][29] . In the present study, an enhancement in the hepatic activity of MPO, an index of tissue neutrophil infiltration, occurred after ANITinduced liver injury.…”