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The role of biomarkers in risk

assessment
a
Daniel Schlenk
a
Department of Pharmacology, Environmental
Toxicology Program , University of Mississippi ,
University, MS, 38677 Phone: (601) 232–5150 Fax:
(601) 232–5150
Published online: 02 Dec 2008.

To cite this article: Daniel Schlenk (1996) The role of biomarkers in risk
assessment, Human and Ecological Risk Assessment: An International Journal, 2:2,
251-256, DOI: 10.1080/10807039609383604

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 Human and Ecological Risk Assessment: Vol. 2, No. 2, pp. 251-256,1996

The Role of Biomarkers in

Risk Assessment

Daniel Schlenk
Department of Pharmacology, Environmental Toxicology Program, University of
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Mississippi, University, MS*

Biomarkers have been defined as biochemical, physiological, or pathological

responses measured in individual organisms which provide information concerning
exposures to environmental contaminants and/or sublethal effects arising from such
exposures (Benson and Di Giulio, 1992). Exposure (exposure assessment) and
sublethal effects (hazard assessment) can be theoretically used to perform risk
assessments. While exposure assessments are based on dosimetry in both human
health and ecological risk assessments (ERA), exposure-response assessments are
considerably more difficult to carry out with ERAs because the endpoints examined
vary widely between assessments. In addition, ERAs have historically been
constructed by measuring the lethality of an indicator organism as the "measurable"
response endpoint with subsequent extrapolation to the expected environmental
concentration of the Stressor. This may or may not include the use of a safety factor.
The problem with this type of assessment is that most environmental exposures
from anthropogenic sources occur at chronic sublethal levels. Thus, it is difficult to
accurately evaluate the risk of a chronic Stressor with the use of endpoints developed
to assess acute exposures. Consequently, the use oí adverse sublethal responses, such
as those measured by biomarkers, has been proposed to assess the risk of
environmental Stressors.
Although physiological and pathological biomarkers have merit, this discussion
will focus primarily on two hepatic biochemical indicators, the cytochrome P450 1A
monooxygenase (CYP1A) and the metal-binding protein metallothionein (MT). In
order for a biomarker to be useful in a risk assessment, it must indicate exposure
and/or a hazardous effect. CYP1A is an enzyme which monooxygenates planar
aromatic hydrocarbons and is also induced by exposure to its substrates. Although
CYP1A protein and mRNA can be measured with appropriate probes, the most
common measurement of CYP1A is through the deethylation of substrate
ethoxyresorufin to resorufin (EROD). Since no endogenous substrate has been

* Department of Pharmacology, Environmental Toxicology Program, University of Mississippi,

University, MS 38677; Tel: (601) 232-5150; Fax: (601) 232-5148

251
 Schlenk

identified for CYP1A, induction of CYP1A expression in tissues of animals is

indicative of exposure to exogenous planar aromatic chemicals. CYP1A and exposure
to planar aromatic compounds, such as polynuclear aromatic hydrocarbons, have
been observed to be directly related in several field studies ( Varanasi et al, 1986;
Collier et al, 1992a; 1995). Likewise, studies examining residue levels of chlorinated
dibenzodioxins and furans have shown a direct correlation with CYP1A activity
(Hodson et ai, 1992; Gagnon, Dodson, and Hodson, 1994a; Kloepper-Sams et al,
1994). Although several studies have documented relationships between CYP1A
activity and reproductive disorders (Andersson et al., 1988; Norrgren et al, 1993;
Gagnon et al., 1994b) as well as hepatic neoplasms (Fabacher and Baumann, 1985;
Collier et al, 1992b), other studies have found little or no correlation with whole
animal effects (Kloepper-Sams et al, 1994). CYP1A catalyzes the bioactivation of
PAHs to more reactive intermediates which can bind critical macromolecules, such
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as DNA and, depending on the dose, eventually lead to toxicity. However, the
majority of CYPlA-derived metabolites (86%), at least in rainbow trout, are
generally water soluble and nonmutagenic (Williams and Buhler, 1984). Thus, the
overall role of CYP1A is to help cell eliminate these compounds. In addition,
metabolites generated by CYP1A do not necessarily result in greater toxicity. For
example, CYP1A1 in trout detoxifies aflatoxin Bi to the less reactive metabolite,
alfatoxin Mi, whereas CYP2K1 catalyzes the formation of the 8,9 epoxide which
binds DNA (Loveland et al, 1983). Consequently, although CYP1A induction can
be used as a biomarker of exposure, its induction does not exclusively mean that the
animal is experiencing an adverse effect.
MTs are inducible metal-binding proteins characterized by having a low
molecular weight ranging from 6,000 to 10,000, a high content of cysteine (about
30%), a lack of aromatic amino acid residues and a wide distribution in various
organisms (Kagi and Schaffer, 1988). Recent studies with transgenic mice have
shown that the primary function of MT appears to be that of metal detoxification
(Masters et al, 1994). It has been established that numerous physiological stresses,
organic and inorganic chemicals, especially heavy metals such as cadmium, copper,
and mercury are particularly powerful in the transcription^ induction of MTs
(Hamer, 1986; Andrews, 1990). Several field studies have demonstrated a correlation
of M T expression with heavy metal exposure (Klaverkamp etal, 1984; Hamilton and
Mehrle, 1986; George et al, 1990; George, 1990; Hogstrand, Lithner, and Haux,
1991; Roesijadi, 1992). In recent years, the development of molecular nucleotide and
antibody probes have greatly enhanced the ease and sensitivity of measuring MT in
biological samples (Hogstrand and Haux, 1990; Kille et al, 1992). Since MTs carry
out endogenous functions in essential metal homeostasis and are induced by a
number of acute endogenous stress responses (such as infection or inflamation), their
presence is not exclusively indicative of exposure to exogenous, metals. For example,
when marine arthropods molt, massive amounts of copper have been shown to be
released from the copper-containing protein, hemocyanin, which subsequently
induced MT (Engel and Brouwer, 1987). Consequently, if M T measurements were
made on feral organisms that had recently molted, then M T levels would be elevated
which might be misinterpreted as an exposure to exogenous heavy metals. A similar

252 Hum. Ecol. Risk Assess. Vol. 2, No. 2,1996

 Invited Debate/Commentary

relationship occurs in spawning female trout where the catabolism of large amounts
of the zinc-containing protein, vitellogenin, leads to MT induction (Olsson et al,
1995). Regarding its association with adverse effects, few studies have been carried
out comparing animal health with M T induction.
CYP1A and M T have evolved as defense mechanisms. Since most defense
mechanisms help the organism to cope with endogenous stress, animals tend to use
such processes not only to counter anthropogenic-induced changes, but also to adapt
to natural changes in their environment. Determining when an animal is simply
adapting or undergoing stress that is adversely affecting that organism is a challenge
that can severely limit the use of biomarkers as consistent endpoints for exposure-
response models, such as is necessary in risk assessments. Just as it is extremely
difficult to extrapolate (top)down from an acute response resulting from a lethal
concentration of a Stressor to low-level effects occurring over longer periods, it is
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also difficult to extrapolate (bottom)up from a cellular response caused by chronic

exposure, and assess its effect on a whole organism or an ecosystem.
In an attempt to remedy this problem and determine whether biochemical
responses can be used in hazard assessments or whether expression of these defense
mechanisms have any relationship to adverse whole animal or population effects, we
have been exploring the use of M T and CYP1A as bioindicators of exposure and
sublethal effects in feral fish collected from various waterways that have been
exposed to a host of compounds. For example, on the Ouachita River in
Southwestern Arkansas, large predatory fish, such as largemouth bass (Micropertus
salmoides) were found to have residual mercury in excess of the 1.0 ug/g limit for the
United States Food and Drug Administration. Examining nonspawning female
bass, we showed that hepatic MT levels correlated with muscle residues of mercury.
Thus, we were able to demonstrate the use of M T as a biomarker of exposure
through an understanding of its function {i.e., regulation during sexual
development). Similar studies have shown relationships between M T expression
and other metals (Klaverkamp et al., 1984; Hamilton and Mehrle, 1986; George et
al., 1990; Hogstrand et al., 1991; Roesijadi, 1992). Consequently, M T maybe used
as a biomarker of exposure, but only if the biological condition of the collected
sample is well characterized.
Since we were able to show relationships between M T and exposure, we then
wanted to explore whether there was any correlation to animal health (i.e., could
MT be used in a hazard assessment). During our study comparing M T and mercury,
it was our opinion (not documented) that fish which contained large body burdens
of mercury (up to 1.5 [ig/g) did not appear to be significantly different externally
compared to those which did not contain appreciable mercury. Consequently, to
determine how well biochemical or cellular endpoints correlate with whole animal
or population responses in feral fish, we subsequently collected 134 animals of 44
different species from 13 equidistant sites from another 150 mile-long waterway in
Southeastern Arkansas, Bayou Bartholomew (Schlenk et al, in press). The
expression of three hepatic biomarkers (CYP1A, MT, and HSP30) were examined
in four of the most commonly collected species: largemouth bass (Micropterus
salmoides), white crappie (Pomoxis annularus), blue gill (Lepomis macrochirus), and

Hum. Ecol. Risk Assess. Vol. 2, No. 2,1996 253

 Schlenk

common carp {Cyprinus carpió). Animals were differentiated according to sex and
development, with only the carp having enough animals to separate into both sexes.
Of the remaining species, only female bass, male crappie, and male bluegill were
found in enough numbers to be examined.
Linear regression analysis was performed relating each biochemical response to a
fish health index containing 14 morphological and physiological parameters
previously described in centrarchids (Adams, Brown, and Goede, 1993) including
such items as tissue appearance, color, presence of parasites, hematocrit, etc.
Interestingly, a direct correlation was observed between hepatic CYP1A protein and
the index of animal health with an lvalue of 0.656. Supporting the relationship with
CYP1A, a negative correlation of hepatic HSP30 and fish health (r2 = 0.589) was
observed. This is due to the fact that HSP30 is the enzyme heme oxygenase which is
induced by acute cellular stress and catabolizes heme to protective antioxidants
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(Pimestone et al, 1971). The inverse relationship of HSP30 and CYP1A indicates
that heme oxygenase may be catabolizing the heme-containing CYPlA as well as
other heme-proteins in response to some form of acute oxidative stress. Although
M T has also been shown to be induced in response to acute stress in fish (Baer and
Thomas, 1990), expression of hepatic M T failed to significantly correlate with the
fish health assessment index. The lack of a correlation between hepatic M T
expression and feral animal health indicates that either these animals are not affected
by metal-related toxicity at the time of sampling, or some other endogenous process
is confounding the comparison. In contrast, a recently completed laboratory study
(Schlenk, unpublished) comparing hepatic M T and body weight and growth in male
channel catfish exposed to copper sulfate for 10 weeks showed an inverse relationship
indicating adverse sublethal toxicity. No relationship was observed in females which
were significantly more resistant to copper toxicity.
It is important to note that each of these biomarkers are proteins. Consequently,
processes that impair protein synthesis, such as acute cellular stress, will significantly
influence the induction or reduction of any biomarker protein. Thus, if acute stress
is present in a sampled tissue, a direct correlation with toxicity may not be observed.
However, utilizing a suite of endpoints, such as acute stress response proteins, allows
a better evaluation of the hazard within the sampled tissue.
In summary, although fraught with uncertainties, biomarkers can be useful in
ERAs under very specific assumptions and conditions. For example, when induction
is present (Type I error) biomarkers can be useful as a cost-effective method for
determination of xenobiotic exposure in animals, if the biomarker is calibrated
against the natural history of the organism. However, if induction is absent (Type II
error), exposure to xenobiotics cannot be ruled out because of other confounding
factors such as inhibitors of specific protein activities {i.e., metals and CYPlA) or
acute cellular stress which may reduce overall protein synthesis. Currently, the
association between biochemical and whole animal/population responses is not well
established. More mechanistic research is needed to determine the differences
between cellular adaptation and stress. With recent advances in recombinant DNA
technologies and cell biology, it may eventually be possible to develop a cost-
effective and predictive measurement that will provide an accurate measurement of
adverse sublethal effects in feral organisms which could be used in risk assessments.

254 Hum. Ecol. Risk Assess. Vol. 2, No. 2,1996

 Invited Debate/Commentary

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