Page 385 - The Toxicology of Fishes

Liver Toxicity 365

Cholestasis is most commonly not fatal, although various pathologic sequelae are associated with this
condition. Chronic manifestations of cholestasis include systemic toxemia, impaired immune function,
and central nervous system and renal system stress. Hence, failure or impairment of bile excretory
mechanisms can lead to general conditions of systemic toxemia.
Bile-acid-induced toxicity, which may result in cholestasis, is often associated with perturbation of
mitochondrial respiration and depolarization (MPT) and cell death (Aguilar et al., 1996; Benz et al.,
2000; Palmeira and Rolo, 2004; Rolo et al., 2004). Increasing concentrations of the bile acids LCA,
DCA, UDCA, CDCA, glycochenodeoxycholic (GCDC), and taurochenodeoxycholic (TCDC) were
shown to decrease mitochondrial membrane potential, decrease state 3 respiration, and enhance state 4
respiration (Rolo et al., 2004). Bile salts themselves have been observed to alter energy-dependent
transport dynamics via modulation of normal cellular energy production and regulation. In short, impair-
ment of bile transport via compromise of hepatocyte or cholangiocyte function is a major source of
toxicity, from both endogenous substances and xenobiotics.
Alterations to cytoskeletal and gap- and tight-junction proteins are primary mechanisms by which
cholangiopathies may occur, and they are seen following exposure to xenobiotics. The naturally occurring
toxins microcystin-LR (MC-LR) and okadaic acid (OA), for example, inhibit cytoskeletal function via
inhibition of dephosphorylation, and microcystin, a protein phosphorylation (PP2A) inhibitor, is known to
induce cholestasis in humans via altered microtubule-dependent vesicle movement in hepatocytes (Pouria
et al., 1998). OA, which also blocks PP2A, inhibits cAMP stimulation of TC uptake, translocation, and
2+
dephosphorylation of NTCP and increases cytosolic Ca (Mukhopadhyay et al., 1998). Similarly, admin-
istration of phalloidin leads to rapid and sustained cholestasis in rats through cytoskeletal changes resulting
in impaired canalicular function. These changes were linked with a total loss of MRP2 function (possibly
due to altered translocation of the protein from the Golgi apparatus to the outer cell membrane) (Rost et
al., 1999). Phalloidin damages cytoskeletal function and arrangement and results in sustained cholestasis
in rats through impairment of canalicular function and a total loss of MRP2 expression (Rost et al., 1999).
Toxicants such as α-naphthylisothiocyanate (ANIT) are also known to alter cytoskeletal function and
induce cholestasis in rats (Lesage et al., 2001; Orsler et al., 1999; Palmeira et al., 2003), and preliminary
studies in the Hinton laboratory have shown that ANIT targets both hepatocytes and biliary epithelia in
medaka (Hinton, unpublished studies).

Related Studies of Biliary Toxicity in Livers of Fishes

A variety of diseases related to biliary dysfunction such as primary biliary cirrhosis (PBC) and primary
sclerosing cholangitis (PSC) are well recognized in mammals, but hepatobiliary-related disease states,
as well as impaired transport function, are only beginning to be understood in fish species (Cai et al.,
2001; Kirby et al., 1995; Lorent et al., 2004). Studies in cartilaginous fishes provide evidence for
cytoskeletal-related impairment to hepatobiliary transport. They have generally employed clusters of
isolated polarized skate hepatocytes, and they have provided structural and functional evidence for
microtubule-dependent transcytosis. As an example, the polarized arrangement of microtubules, presence
of cytoplasmic dynein, and inhibition of bile salt secretion by nocodozole were consistent with the
microtubule cytoskeleton playing a fundamental role in the mediation of transcytosis, endocytosis, and
bile excretory function in skate hepatocytes (Henson et al., 1995).
Published findings on xenobiotic-induced alterations of P-glycoprotein in fish are conflicting. For
example, Sturm et al. (2001a) found no induction of C219-immunoreactive Pgp protein levels after in
vivo exposure of rainbow trout to prochloraz or nonylphenol diethoxylate. Similarly, dietary exposure
of channel catfish to the xenobiotics β-naphthoflavone, benzo(a)pyrene, or 3,4,3′,4′-tetrachlorobiphenyl
did not result in elevated levels of C219-immunoreactive proteins nor in altered Pgp activity in intestine
and liver (Doi et al., 2001). In contrast, the expression of hepatic C219-immunoreactive Pgp protein was
significantly enhanced in the liver of killifish, both in vitro and in vivo, after exposure to chlorpyrifos
oxon and the carcinogen N-nitrosodiethylamine (Albertus and Laine, 2001).
Complex relations between xenobiotics and Pgp expression in fish are indicated from a study on
intertidal blennies (Anoplarchus purpurescens) (Bard et al., 2002b). Hepatic Pgp immunostaining with
C219 antibody was significantly elevated in blennies collected from field sites downstream of pulp mills,