Page 708 - The Toxicology of Fishes

688 The Toxicology of Fishes

Schlenk and Di Giulio (2002) and Van der Oost et al. (2003). This portion of the chapter focuses on
inducible proteins, metabolites, and genotoxic endpoints, but it should be noted that other candidate
responses for biomarkers include the inhibition (i.e., via cholinesterases) or repression of specific proteins.

Inducible Proteins
Most biochemical defenses respond to cellular injury by increasing levels of defenses through self-reg-
ulating signal transduction mechanisms. These defenses are usually proteins that serve numerous cellular
functions, many still unknown. Depending on the dose (or concentration) of the toxicant, these systems
are often adaptive; however, when the dose exceeds the capacity of such systems to function properly,
irreversible cell injury and toxicity may result. Thus, measuring these systems may provide early warning
of danger to the cell as well as help elucidate potential mechanisms of cellular injury.
These particular markers are proteins primarily regulated at the transcriptional level. The protein itself
can be measured by enzyme-linked immunosorbent assay (ELISA) or western blot using protein-specific
antibodies, many of which are commercially available, or, alternatively, the transcript can be measured
by (1) northern blot or ribonuclease protection assay (RPA) using respective cDNA and cRNA probes
or (2) quantitative reverse-transcriptase polymerase chain reaction (qPCR) using conserved nucleotide
sequences found in several species. If the protein has enzymatic activity, then an additional measure of
catalytic activity might be added to one or two of the above. Of the three methods, measurement of
protein content is typically the most robust, as activity and transcript levels can be easily degraded by
excessive temperature or by nonspecific ribonucleases, respectively. Although -omic studies have already
led to the discovery of several other transcriptional biomarkers of toxicity, the discussion here focuses
specifically on a few endpoints that have been more well-characterized in fish. (Note: Vitellogenin is
not discussed in this chapter, as it is described in other chapters within this text.)

CYP1A
Cytochrome P450 monooxygenases (CYPs) are a multi-gene family of enzymes that occur in nearly all
plants and animals. These enzymes carry out an array of reactions, and before the recent identification
of individual isoforms and their substrate specificities P450s were known collectively as the mixed-
function oxidases. Because of the plethora of genes that encode these proteins, a nomenclature committee
has been appointed to classify each protein into specific gene families (http://drnelson.utmem.edu/nel-
sonhomepage.html). One of the most common and highly conserved is the CYP1A subfamily. Excellent
reviews have been published on the natural history, function, and regulation of this subfamily of enzymes
and their potential use in biomonitoring (Bucheli and Fent, 1995; Stegeman, 1993; Stegeman and Hahn,
1994; Stegeman and Lech, 1991; Van der Oost et al., 2003). CYP1A expression is increased primarily
through activation of a cytosolic receptor known as the aryl hydrocarbon (Ah) receptor, which eventually
serves as its own transcription factor initiating CYP1A mRNA expression. Ah receptors have a relatively
selective binding region and prefer planar aromatic hydrocarbons as agonists (Hahn et al., 2005); thus,
expression of CYP1A, which has a typically low basal expression rate, can be used as a biomarker of
exposure to various planar aromatic hydrocarbons ranging from polychlorinated biphenyls (PCBs) and
dioxins to numerous polycyclic aromatic hydrocarbons (PAHs).
Although most of the metabolites resulting from CYP1A-catalyzed reactions are hydroxylated deriv-
atives of parent compounds, the reaction often involves the formation of reactive electrophilic interme-
diates that may undergo nucleophilic attack by critical macromolecules such as sulfhydral groups of
proteins and amine moieties of nucleic acids. Because of the enhanced possibility of protein or DNA
adduction, which may result in cellular dysfunction, induction of CYP1A has been proposed to be used
as a biomarker of effect. Studies have shown relationships between CYP1A expression and reproductive
alterations following exposure to PCBs or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Cook et al.,
1997; Teraoka et al., 2003). CYP1A catalytic activity in embryos and eggs has been used to determine
toxicity equivalency factors (TEFs), which allow calculations of threshold values for dioxin-like com-
pounds to adversely affect development in eggs of salmonids (Cook et al., 1997). These data suggest
that CYP1A expression may be used as biomarker not only of effect but also of susceptibility.