Page 380 - The Toxicology of Fishes

360 The Toxicology of Fishes

occurs. In the mammalian liver, altered bile transport (cholestasis) is a common response of the liver to
xenobiotic exposure and a key mechanism of toxicity for many drugs. Changes to cytoskeletal function,
for example, are associated with a loss of canalicular microvilli and diminished canalicular contractility
(Phillips et al., 1986; Song et al., 1998; Watanabe et al., 1983).
In the intrahepatic biliary system of mammals, biliary epithelial cells (BECs), like hepatocytes, are
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key regulators of bile transport, responsible for regulating: bile fluid alkalinity (through the Na -depen-
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dent Na /HCO and CL /HCO exchange symporters), electrolyte content (via ion channels), water
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composition (a major components of bile, via active transport of aquaporins), and bile salts. Bile salt
uptake into cholangiocytes and gallbladder epithelial cells may also serve an important role via cell
signaling for regulation of secretory and proliferative processes (in response to injury) within the biliary
tree (Alpini et al., 2001, 2002).
Biliary epithelial cells (BECs) are known target cells in a number of pathologic conditions (cholan-
giopathies) of mammalian liver, including primary biliary cirrhosis (PBC) and primary sclerosing cho-
langitis (PSC), as well as diseases associated with BEC proliferation or loss. For example, in rodents,
cholangiopathies that result in impaired bile synthesis or flow can be induced by several experimental
conditions, such as chronic administration of CCl or the reference toxicant α-naphthylisothiocyanate
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(ANIT) (Kanno et al., 2001). Because of the critical role of BECs in bile homeostasis, impairment of
BEC function can be a primary source of hepatotoxicity through altered bile synthesis and transport.
Although BECs have been identified in several fish species and their morphology characterized, their
complex transport and bile regulatory mechanisms remain poorly understood. It is likely, however, that
their functional characteristics are equally important in the hepatobiliary toxicity of fishes.

Bile Synthesis
Hepatocytes and biliary epithelial cells coregulate bile synthesis, volume, composition, and transport in
response to changing physiologic needs. In all vertebrates, bile synthesis is part of the mechanism of
cholesterol elimination, accomplished by conversion of cholesterol to water-soluble amphipathic bile
salts (Moschetta et al., 2005). In higher vertebrates, metabolic conversion of cholesterol to the primary
bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA) occurs in hepatocytes via two pathways.
The classic (neutral) synthetic pathway involves modification of the sterol nucleus (e.g., 7α-hydroxylation
and β-oxidations of the side chain of either cholesterol or one of three oxysterols containing a hydroxyl
group at the C24, C25, or C27 position of the side chain) by 7α-hydroxylase (CYP7A1), leading to
formation of CA and CDCA. The acidic, or alternative, pathway is catalyzed by sterol 27-hydroxylase
(CYP27A1) located on inner mitochondrial membranes, leading to the generation of CDCA. In both
processes, the end products CA and CDCA are subsequently amidated to anionic bile salts (bile acids
exist as bile salts at physiological pH) via glycine or taurine conjugation, rendering them impermeable
to cell membranes and hydrophilic. During enterohepatic transport, CA and CDCA are metabolized by
intestinal flora (bacteria) to the secondary bile acids deoxycholic acid (DCA; from CA) and lithocholic
acid (LCA; from CDCA).
In primitive vertebrates, including fish, bile synthesis pathways consist of different means of hydrox-
ylating cholesterol, followed by conjugation with a strong anion (usually sulfate). The most common
products in lower vertebrates are bile alcohol sulfates with four, five, or six hydroxyl groups per sterol
nucleus. It is presumed that, through evolution, bile salts gradually assumed more diverse biological
roles, reflected in structural variations (lipid solubilization, bactericidal). One of the more significant
evolutionary advancements in bile synthesis was the shift from nonrecycling C27 bile alcohol sulfates
(e.g., fish and reptiles) to enterohepatically cycling C24 bile acids. C24 bile acid structures have been
achieved in virtually all vertebrate groups through convergence, with each group achieving the C24 bile
acid structure independently. A survey of the transition in bile synthesis in various species found three
primary evolutionary transitions: (1) C27 alcohol sulfates to C24 taurine-conjugated acids, (2) C27
alcohol sulfates to C27 taurine-conjugated acids to C24 taurine-conjugated acids, and (3) C27 alcohol
sulfates to C24 glycine-conjugated acids to C24 taurine-conjugated acids. The first pathway is considered
quite rare and has been observed only in fish. The second pathway is the classical, most commonly used
route in lower vertebrates, and the third pathway is the one utilized in mammals. Vertebrate groups in