Page 372 - The Toxicology of Fishes

352 The Toxicology of Fishes

Hepatic Phase II Metabolism
During phase II reactions, xenobiotic metabolites produced from the first phase are conjugated to polar
endogenous substrates (e.g., sulfate, glucuronide), facilitating their subsequent elimination from the body
via bile or urine (Stegeman, 1989). Biliary excretion is often chemical specific (see detailed coverage
below). Low-molecular-weight compounds are poorly excreted into bile, but high-molecular-weight xeno-
biotic compounds conjugated with sulfate and glucuronide are readily excreted into the bile (Klaassen and
Watkins, 1984). Several types of petroleum hydrocarbon metabolites have specific fluorescence properties,
and exposure of fish (e.g., Atlantic cod, Atlantic salmon, English sole) to crude oil results in increases in
bile fluorescent aromatic compounds (FACs) (Aas et al., 2000; Gagnon and Holdway 2002; Krahn et al.,
1986). Significant correlation has been established between the prevalence of hepatic lesions and mean
biliary metabolite concentration in fish, and analysis of FAC in fish bile is recommended as a sensitive
method for detection of PAH contamination from both pyrolytic and petroleum origin (Krahn et al., 1986).
In general, conjugation reactions, such as glucuronidation or glutathione conjugation, have not received
sufficient attention, and further studies must be conducted in the future. Just as was shown for phase I
metabolism, the liver is quantitatively the most important site for phase II metabolism in fish (Clarke et
al., 1991). In mammals, phase II enzymes such as glutathione S-transferases and UDP-glucuronyltrans-
ferases represent multigene families, and there is evidence that this applies for fish as well, although the
characterization of the various isoenzymes requires more attention (Leaver et al., 1992, 1993). In field
studies of fish from contaminated sites, phase II conjugates of xenobiotics were detected in bile. Examples
include fuel-oil PAHs (Collier and Varanasi, 1991), phthalate ester plasticizers (Melancon and Lech, 1983),
and the pulp mill constituents chlorophenols and resin acids (Hardig et al., 1988; Oikari et al., 1985;
Stuthridge et al., 1997). The importance of phase II metabolism for the bioconcentration and toxicity of
xenobiotics in fish may be exemplified by an organophosphorous compound. In several fish species,
dimethylphosphorothioates are rapidly metabolized in the liver and eliminated via bile as glutathione
conjugates. In addition, this rapid phase II metabolism is associated with lower bioconcentration and
lower toxicity than observed for related organophosphorous compounds that are metabolized by the
oxidative system (Debruijn et al., 1993). Another example is provided by the lampricide 3-trifluoromethyl-
4-nitrophenol. The selective toxicity of this substance to sea lampreys was found to be related to a greatly
reduced capacity of this species to glucuronidate the compound (Lech and Statham, 1975).
To study liver xenobiotic metabolism, in vitro preparations of fish liver cells have proven to be a valuable
experimental model. Isolated hepatocytes can be kept as fresh isolates (Moon et al., 1985), as longer term
monolayers (Pesonen and Andersson, 1997; Segner 1998), or as aggregate cultures (Cravedi et al., 1996).
These preparations have the advantage of making it possible to analyze the fate of xenobiotics specifically
in the liver, in the absence of systemic influences. Further, they offer the possibility for interspecies com-
parison of chemical metabolic conversion under fairly comparable conditions (Coulombe et al., 1984;
Cravedi and Baradat, 1991; Murk et al., 1994). Fish hepatocytes in vitro are valuable tools for study of
induction of biotransformation enzymes as well as the formation of xenobiotic metabolites (as reviewed in
Segner and Cravedi, 2001). Generally, the metabolite pattern produced by fish liver cells in vitro agrees well
with the in vivo metabolism of the substances, although possible differences should not be overlooked when
extrapolating from in vitro to in vivo hepatic metabolism (Cravedi et al., 1999; Morrison et al., 1985;
Nishimoto et al., 1992). An interesting example of such an in vitro and in vivo differences was provided by
Cravedi et al. (2001), who found the toxicologically relevant hydroxylamine metabolite of 2,4-dichloroaniline
in the in vitro hepatocyte preparations but not in vivo in the bile fluid. The likely explanation for this difference
is that the hydroxylamine metabolite is unstable; therefore, it can be detected only in the culture media,
which are processed after a 1-hour incubation period, while the bile is sampled only at 24-hour intervals.

Nuclear Receptors in Liver Biology
Major functions of livers—the uptake, metabolism, storage, and redistribution of nutrients and endoge-
nous molecules; metabolism of xenobiotics; and formation and excretion of bile—are regulated by
nuclear receptors (NRs). Mammalian liver NRs are endogenous sensors, regulating many of the above