Page 183 - The Toxicology of Fishes

Biotransformation in Fishes 163

of BNF-induced EROD activity in channel catfish (Ictalurus punctatus) (Watson et al., 1995). In both
in vitro and in vivo experiments, 2-AA was a mechanism-based inhibitor of CYP1A. Similarly, 3,3′,4,4′-
tetrachlorinated biphenyl (PCB 77) at high doses causes competitive inhibition of CYP1A enzyme
activity and decreased induction of CYP1A protein (Gooch et al., 1989; White et al., 1997b). White and
coworkers’ (1997a) data suggested that, in scup, tetrachlorinated biphenyl (TCB) decreased CYP1A
protein by enhancing protein degradation. TCB also initiated redox cycling through an uncoupling of
CYP1A (Schlezinger and Stegeman, 2001). Finally, in killifish cotreated with BaP and the four-ring
PAH fluoranthene (FL), both hepatic EROD activity and CYP1A immunoreactive protein levels were
significantly inhibited (Willett et al., 2001). Although a covalent interaction between FL and CYP1A
was not detected, the relative composition of DNA adducts changed in cotreated fish, suggesting that
BaP metabolism is significantly affected when fish are co-exposed to PAH mixtures. Antifungal imidazoles
(clotrimazole and ketoconazole) inhibit CYP1A-mediated EROD activities in gizzard shad (Dorosoma
cepedianum), rainbow trout, and Atlantic cod (Hasselberg et al., 2004; Hegelund et al., 2004; Levine et
al., 1997). Acrylamide, an environmental contaminant, is also an inhibitor of CYP1A and suggests
feedback regulation on CYP1A mRNA transcription (Haasch et al., 1992; Petersen and Lech, 1987).

CYP1B—Until the mid-1990s, the CYP1 gene family was believed to contain a single subfamily with
the well-known members CYP1A1 and CYP1A2; however, in 1994, the cDNA for CYP1B1 was isolated
from TCDD-induced human keratinocyte cells (Sutter et al., 1994). Human CYP1B1 is a single-copy
gene that is located on chromosome 2. It contains three exons and two introns and generates a 5.2-kb
mRNA (Murray et al., 2001). In mammals, CYP1B1 expression is high in vascular endothelial cells,
breast, prostate, uterus, epithelial lining of the head and neck, and the adrenal cortex (Nebert et al.,
2004). CYP1B1 protein has also been reported in human breast, kidney, lung, brain, and testis tumors
(McFadyen et al., 2001; Murray et al., 2001). Recombinant human CYP1B1 is highly active in oxidizing
the potent PAHs BaP and dimethylbenzanthracene (DMBA) to their respective carcinogenic metabolites.
Shimada and coworkers (1999) found that CYP1B1 was more active than CYP1A1 in metabolizing BaP
to the proximate toxicant BaP-7,8-diol. Their study suggests that species or tissues with less CYP1B1
may be less likely to form DNA-reactive PAH metabolites and thereby may be more resistant to
carcinogenesis. This finding is supported by studies with CYP1B1-null mice. Seventy percent of DMBA-
treated wild-type mice developed highly malignant lymphomas, whereas the CYP1B1-null mice only
had 7.5% cancer incidence (Buters et al., 1999). Likewise, metabolism of DMBA to toxic intermediates
in MCF7 and T47D breast cancer cells is blocked by CYP1B1 antibodies (Angus et al., 1999; Christou
et al., 1994).
A significant void in CYP1B1 research exists in nonmammalian species such as fish. There are only
two published studies where CYP1B was studied in four fish species: scup, killifish, zebrafish (Danio
rerio) (Godard et al., 2000), and plaice (Leaver and George, 2000). The plaice CYP1B has two protein-
coding exons with similar exon–intron boundaries compared to the human CYP1B1. The amino acid
sequence of the plaice has 54% identity with the human CYP1B1 sequence but only 39% identity with
the plaice CYP1A (Leaver and George, 2000). CYP1B was detected in plaice gill by northern blot but
did not appear to be induced by BNF. For carp (Cyprinus carpio), two partial CYP1B and two CYP1C
sequences have been submitted to GenBank. Similarly, scup genes and the killifish partial sequence
recently have been reclassified CYP1C1 and CYP1C2, suggesting that the genes have less than 55%
amino acid identity with either the plaice or mammalian CYP1Bs. The cloned channel catfish and brown
bullhead (Ameiurus nebulosus) CYP1B genes are 71, 61, and 55% similar, respectively, to carp, plaice,
and human CYP1B1 (accession number DQ088663). Currently, none of the CYP1B-dependent metabolic
studies has been done in fish or fish cells, yet fish are commonly used in toxicology and carcinogenicity
testing. In vivo and in vitro data in catfish do indicate that CYP1B mRNA is induced in gill (but not
liver) primary cultured cells and gill, kidney, blood, gonad, and liver following BaP exposure (Butala,
unpublished data; Metzger, unpublished data).
In addition to its involvement in PAH metabolism, human CYP1B1 is an estradiol hydroxylase
primarily at the C-4 position, whereas CYP1A1 and CYP1A2 have activity at the C-2, C-6α, and C-15α
positions of estradiol (Hayes et al., 1996). Tumors have been reported in tissues where estradiol was
converted to the 4-hydroxyestradiol metabolite, but tumors did not form where there was primarily