Toxicological Sciences, Vol 47, 118-125, Copyright © 1999 by Society of Toxicology
DA Hill, PA Jean and RA Roth
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.
ARTICLES
Bile duct epithelial cells exposed to alpha-naphthylisothiocyanate produce a factor that causes neutrophil-dependent hepatocellular injury in vitro
Department of Pharmacology and Toxicology, Michigan State University, East Lansing 48824, USA.
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