Page 560 - The Toxicology of Fishes

540 The Toxicology of Fishes

Wales, 1970). The high sensitivity of trout to AFB , relative to rodents, is illustrated by the findings that
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a diet of 4 ppb AFB induced ~60% tumors in trout livers (Sinnhuber et al., 1974), while a 5-ppb AFB 1
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diet fed to rats for a comparable time period elicited less than 1/10 the incidence of tumors (Wogan et
al., 1974).
Aflatoxin B is metabolized by the CYP system to various metabolites, including hydroxylation to
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AFM and AFB -8,9-epoxide. The epoxide is thought to lead to carcinogenesis due to its high reactivity
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with DNA (Eaton and Gallagher, 1994; Essigmann et al., 1982, Swenson et al., 1977). Various human
CYPs (1A1, 1A2, 2A6, 2B6, 3A4, and 3A5) are capable of metabolizing AFB to the 8,9-epoxide;
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however, mammalian CYP1A2 and CYP3A4 appear to be especially involved in AFB activation to the
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carcinogenic epoxide (Gallagher et al., 1996; Hengstler et al., 1999). In trout, CYP2K1 appears to be
primarily involved in forming the AFB -epoxide (Williams and Buhler, 1983; Yang et al., 2000). Dif-
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ferences in both the formation of AFB -epoxide and in detoxification reactions appear to contribute to
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the different sensitivities various species exhibit to AFB -induced carcinogenesis. The reactive AFB -
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epoxide can be detoxified by conjugation with glutathione via glutathione S-transferases in mammalians.
In rodents, the lower sensitivity of mice to AFB -induced carcinogenesis, relative to rats, appears to be
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largely attributed to more efficient glutathione conjugation of the epoxide in mice than in rats (Degen
and Neumann, 1981; Raney et al., 1992). In rodents, a high level of glutathione conjugation is associated
with a low level of AFB –DNA binding (Raj et al., 1984). In fishes, the major excreted AFB metabolites
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are AFL and AFL–glucuronide conjugates, while glutathione conjugates represent minor metabolites
(Dashwood et al., 1992; Gallagher and Eaton, 1995; Toledo et al., 1987; Troxel et al., 1997a). The high
sensitivity of trout to AFB -induced carcinogenesis is thought to be at least partially attributed to high
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bioactivation to AFB -epoxide by CYP2K1 and to low glutathione conjugation of this reactive metabolite
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(Dashwood et al., 1992).
DMBA
7,12-Dimethylbenzanthracene is one of the most carcinogenic PAHs in mammals. In fish, DMBA is also
a potent carcinogen that induces tumors in liver, stomach, and swim bladder (Aoki et al., 1993; Harrtig
et al., 1996; Weimer et al., 2000). Although DMBA is not considered an environmental pollutant, it is
modeled after polyaromatic hydrocarbon environmental contaminants. In mammalian cells, DMBA can
be bioactivated by cytochrome P450s to a reactive “bay region” diol-epoxide metabolite that subsequently
adducts to adenine and guanine residues in DNA (Baird and Dipple, 1977; Diamond et al., 1972; Dipple
et al., 1983). The bay region diol-epoxide DMBA-3,4-diol-1,2-oxide is thought to be the ultimate
carcinogenic metabolite (Vigny et al., 1985) in mammalian tissues. In addition, DMBA can be activated
by one-electron oxidation, as demonstrated by the depurinating DNA adducts observed in mouse skin
(Devanesan et al., 1993). Phase I DMBA metabolites can be conjugated via phase II reactions (detoxi-
fication) to water-soluble glucuronide, sulfate, and glutathione metabolites.
In rainbow trout, DMBA was shown (Schnitz and O’Connor, 1992; Schnitz et al., 1987) to be
metabolized to water-soluble compounds. Initially, most of the identified phase II conjugates were
sulfates, especially with low doses of DMBA. As the concentration of DMBA increased, there was a
shift to more glucuronide conjugates (Schnitz et al., 1993), consistent with the idea that sulfation has a
high affinity with a low capacity, whereas glucuronidation has a low affinity with a high capacity (Pang
et al., 1981). In cultured primary trout liver cells, DMBA was also rapidly converted to water-soluble
metabolites (Weimer et al., 2000). Enzymatic digestion indicated that ≤5% of these metabolites were
conjugated with sulfate, 10 to 35% were glucuronide conjugates, and the majority of these metabolites
remained unidentified. Depleting cellular glutathione did not significantly alter the metabolism of DMBA
to water-soluble metabolites or the proportion of sulfate or glutathione conjugates. Schnitz et al. (1993)
also reported a large fraction of DMBA metabolites that were not identified. Although the rate of
metabolism of DMBA to water-soluble metabolites was similar between mouse embryo fibroblasts and
cultured trout liver cells, the level of unconjugated, nonpolar (phase I) metabolites was much lower in
trout liver cells than in mammalian fibroblasts (Weimer et al., 2000). This finding suggests that phase
II metabolism (detoxification) of DMBA primary (phase I) metabolites is more efficient in trout liver
cells than in mammalian fibroblasts.