Page 180 - The Toxicology of Fishes

160 The Toxicology of Fishes

CYP1
Phylogeny — CYP1 split from the CYP2 family approximately 450 million years ago (MYA) (Nelson
et al., 1993) (Figure 4.4). The CYP1 gene family represents one of the most studied families of CYP
in fish, primarily with regard to the role that CYP1A subfamily members (see below) play in the
biotransformation of environmentally persistent aromatic hydrocarbons (e.g., TCDD, PAHs) and their
relationship with disease processes resulting from exposure to compounds of this nature (Stegman and
Hahn, 1994).

Regulation—Expression of CYP genes are regulated by diverse mechanisms. Basal levels of individual
CYP mRNAs and proteins are regulated via transcriptional and post-transcriptional processes, including
mRNA and protein stabilization or degradation. CYP1 gene expression is induced by structurally diverse
aromatic hydrocarbons wherein the induction response requires initial binding to the aryl hydrocarbon
receptor (AhR). The AhR is a basic helix–loop–helix DNA-binding protein that has been extensively
characterized in laboratory animals and human cell lines. In mammals, the AhR controls the transcription
of the genes CYP1A1, CYP1A2, CYP1B1, as well as phase II enzymes such as glutathione S-transferase,
uridine diphosphate (UDP)-glucuronosyltransferase, and aldehyde-3-dehydrogenase (Safe, 1995). Ago-
nists for the AhR include both synthetic and naturally occurring compounds. The best characterized
ligands include certain PAHs and planar halogenated aromatic hydrocarbons (HAHs), including poly-
chlorinated dibenzo-p-dioxins (PCDDs) and -furans (PCDFs), as well as polychlorinated biphenyls
(PCBs). Planar HAHs bind to the AhR in predictable structure–activity relationships. 2,3,7,8-Tetrachlo-
rodibenzo-p-dioxin (TCDD) has the highest AhR binding affinity among the HAHs and is also correlated
with the high toxic potency of this congener (Safe, 1990). Structure–activity relationships are not as
clear-cut for the PAHs because the toxic and genotoxic responses induced by these compounds are not
all AhR mediated; however, Billiard and coworkers (2002) found a positive relationship between PAH
binding to the teleost AhR and PAH potency for CYP1A induction. More recently, numerous naturally
occurring dietary AhR ligands have been identified, including indole-3-carbinol, indolo-(3,2-b)-carba-
zole, dibenzoylmethanes, curcumin, and carotinoids (Denison and Nagy, 2003; Jeuken et al., 2003).
Studies with the AhR knockout mouse have shown that this protein plays an important role in normal
–/–
embryonic development (Abbott et al., 1995) and development of the liver and immune system (Fernan-
dez-Salguero et al., 1995). The AhR was first identified in 1976 in hepatic cytosol from C57BL/6 mice
(Poland et al., 1976). Apparent molecular masses of the photoaffinity-labeled cytosolic AhR are highly
species dependent, ranging from 95 kDa for mouse to 124 kDa for hamster (Safe, 1995). The AhR has
been identified by photoaffinity labeling in several species of teleosts and elasmobranchs (Hahn et al.,
1994), as well as in the fish cell lines PLHC-1 and RTG-2 (Hahn et al., 1993). Nuclear AhR levels found
in the killifish (Fundulus heteroclitus) were 203 fmol/mg, which was relatively high compared to most
rodent species (Willett et al., 1995). Whereas mammals have a single AhR gene, two genes (AhR1 and
AhR2) have been cloned in killifish (Karchner et al., 1999), zebrafish (Andreasen et al., 2002), and
rainbow trout (Oncorhynchus mykiss) (Abnet et al., 1999). Four forms of the AhR (AhR1α, β; AhR2α,
β) are found in Atlantic salmon (Salmo salar L.) (Hansson et al., 2004). The presence of AhR in fish
indicates that this protein has been conserved for at least 450 million years. For more on AhR-mediated
toxicities, refer to Chapter 5.
A simplified mechanism of AhR-mediated induction of CYP1A involves a ligand entering the cell
where it associates with the cytosolic AhR, which exists as a multiprotein complex with two molecules
of heat shock protein 90 (Hsp90), the X-associated protein 2 (XAP2), and a 23-kDa co-chaperone protein
(p23) (Denison and Nagy, 2003). Binding of ligand causes a conformational change that facilitates nuclear
translocation and association with the AhR nuclear translocator (ARNT). The AhR/ARNT heterodimer,
in turn, associates with dioxin response elements (DREs; sometimes called AhR response elements, or
AhREs) and with various coregulators (Carlson and Perdew, 2002) to initiate transcription and translation
of AhR-responsive genes, including CYP1s. Induction of CYP1 protein can be determined at the tran-
scriptional (mRNA) level using northern blot or quantitative reverse transcription polymerase chain
reaction (RT-PCR) and at the protein level by using western blot, enzyme-linked immunosorbent assays
(ELISAs), or immunohistochemistry. Finally, CYP1-dependent catalytic activities such as aryl hydrocar-
bon hydroxylase (AHH) or ethoxyresorufin-O-deethylase (EROD) can also be used to measure induction.