Aquatic Toxicology 57 (2002) 203– 215 www.elsevier.

com/locate/aquatox

Effects of benzo[a]pyrene exposure on a fish population

resistant to the toxic effects of dioxin-like compounds
Diane E. Nacci a,*, Michael Kohan b, Marguerite Pelletier a, Elizabeth George b
a
US En6ironmental Protection Agency, Office of Research and De6elopment, Atlantic Ecology Di6ision, 27 Tarzwell Dri6e,
Narragansett, RI 02882, USA
b
National Health En6ironmental research Laboratory, En6ironmental Carcinogenesis Di6ision, 86 T.W. Alexander Dri6e,
Research Triangle Park, NC 27711, USA
Received 1 December 2000; received in revised form 10 April 2001; accepted 10 April 2001

Abstract

Effects of a model polycyclic aromatic hydrocarbon (PAH) were compared in populations of the estuarine fish
Fundulus heteroclitus indigenous to a reference site and one highly contaminated with polychlorinated biphenyls
(PCBs) and other compounds. The fish population resident to the PCB-contaminated site is genetically resistant to
those PCB congeners categorized as dioxin-like compounds (DLCs) that act through the aryl hydrocarbon receptor
(AHR). In response to DLC exposures, these DLC-resistant fish showed poor inducibility for enzymes known to be
regulated by the AHR pathway and important for the metabolism of xenobiotics including some PAHs that also act
as AHR agonists. Therefore, a laboratory study using the model PAH, benzo[a]pyrene (BaP), was conducted to
evaluate how PAHs might affect these wild fish populations that differed in their inherent sensitivities to DLCs and
in their tissue concentrations of contaminants. Following BaP treatment, the activities of two xenobiotic metabolizing
enzymes and the concentrations of BaP-DNA adducts, as measured using the 32P-postlabeling method, were lower in
the livers of DLC-resistant than reference fish. These results suggest that DLC-resistance could provide protection
following chronic exposures to PAHs from the long-term consequences of DNA adduct formation, such as cancer.
Alternatively, reduced metabolism and elimination of toxic or photo-activated PAHs could have acute consequences
to the health and reproduction of these DLC-resistant fish and their progeny. These fish populations provide useful
models to evaluate the potential costs and benefits of genetic adaptation in wildlife populations subject to
anthropogenic stress. Published by Elsevier Science B.V.

Keywords: Xenobiotic resistance; Benzo[a]pyrene; DNA adducts; Aryl hydrocarbon receptor


This is contribution number AED-00-081 of the EPA ORD NHEERL Atlantic Ecology Division. This manuscript has been
reviewed and approved for publication by the U.S. EPA. Approval does not signify that the contents necessarily reflect the views
and policies of the U.S. EPA. Mention of trade names, products, or services does not convey, and should not be interpreted as
conveying, official U.S. EPA approval, endorsement, or recommendation.
* Corresponding author. Tel.: + 1-401-7823143; fax: + 1-401-7823030.
E-mail address: nacci.diane@epa.gov (D.E. Nacci).

0166-445X/02/$ - see front matter. Published by Elsevier Science B.V.

PII: S 0 1 6 6 - 4 4 5 X ( 0 1 ) 0 0 1 9 6 - 5
204 D.E. Nacci et al. / Aquatic Toxicology 57 (2002) 203–215
1. Introduction
Fundulus heteroclitus (‘mummichog’) is a non-
migratory fish species indigenous to estuaries
along the East Coast of the U.S. and Canada (e.g.
Abraham, 1985). Mummichogs are a predominant
fish species in many urban harbors, including
New Bedford Harbor (NBH), MA, USA (Nelson
et al., 1996; Black et al., 1998), where the concen-
trations of certain contaminants in sediment and
biota are extremely high. The northernmost por-
tion of NBH, designated a Superfund site, has
sediment concentrations of polychlorinated
biphenyls (PCBs) as high as 2100 mg/g dry weight,
but other contaminants, including polycyclic aro-
matic hydrocarbons (PAHs), are also relatively
high (e.g. 170 mg summed PAHs /g dry weight,
Pruell et al., 1990). Livers of mummichogs col-
lected from the Superfund area of NBH in 1996
contained an average of 324 mg total PCBs /g dry
weight which was 135-fold more than those from
a local, relatively uncontaminated reference site at
West Island (WI, MA, Nacci et al., 1999). Despite
high tissue concentrations of contaminants, NBH
mummichogs are apparently healthy (review see
Nacci et al., 2001a), as indicated by high fecundity
rates (Gleason and DellaVecchia, 2001), high con-
dition indices (M. Huber, personal communica-
tion), and nearly normal levels of stored vitamin
A (Nacci et al., 2001b).
One explanation for the persistence of a fish
population indigenous to this potentially toxic
environment is that NBH mummichogs are rela-
tively unresponsive to specific classes of local or-
ganic contaminants (Bello, 1999; Nacci et al.,
1999; Bello et al., 2001). These contaminants,
which include non- and mono-ortho PCB con-
geners, act partially or wholly through the aryl
hydrocarbon receptor (AHR), and are categorized
toxicologically as dioxin-like compounds (DLCs)
(e.g. Safe, 1990). While DLCs are extremely toxic
to the early life stages of fish species (e.g. Walker
and Peterson, 1991; Peterson et al., 1993; Eisler
and Belisle, 1996), including F. heteroclitus
(Toomey et al., 2001), NBH mummichogs are
relatively unresponsive to their effects. In NBH
fish, non-responsiveness to DLCs is demonstrated
as resistance to lethal effects (Nacci et al., 1999)
but also as minimal increases in the activities
cytochrome P450 enzymes regulated by the AHR
(Nacci et al., 1999; Bello et al., 2001). Increased
cytochrome P450 1A (CYP1A) activity, whether
measured as mRNA induction or ethoxyresorufin-
o-deethylase (EROD) activity, is considered a
hallmark of AHR-signal transduction pathway
activation (e.g. Stegeman and Hahn, 1994; Whyte
et al., 2000). This evidence suggests that NBH
mummichogs demonstrate altered responsiveness
of the AHR pathway (also, Hahn, 1998; Bello et
al., 2001; Powell et al., 2000). Consistent with
these findings, NBH fish also demonstrate low
induction or enzyme activities in response to some
PAHs which act as AHR agonists (Nacci et al.,
1999; Bello et al., 2001). Because some AHR-reg-
ulated enzymes are important for the metabolism
and elimination of some PAHs (Baird and Ral-
ston 1997), the biological fate and effects of these
PAHs might differ between NBH and reference
fish.
In this study, an experiment was conducted to
address the hypothesis that wild populations of F.
heteroclitus that have adapted genetically to the
toxic effects of DLCs (Nacci et al., 1999) respond
differently to PAH exposure than non-adapted
fish. To test this hypothesis, field-collected fish,
known to contain varying tissue concentrations of
DLCs (Nacci et al., 1999) were challenged in the
laboratory with the prevalent environmental con-
taminant, carcinogen and model PAH, ben-
zo[a]pyrene (BaP). Responses to BaP were
compared between a contaminated, DLC-adapted
and an uncontaminated, DLC-sensitive fish popu-
lation. Specifically, activities were compared for
two enzymes that are believed to be regulated by
the AHR pathway in fish and are of potential
significance to the rates of metabolism and elimi-
nation of PAHs, including BaP. In addition, the
quality and quantity of DNA adducts produced
by these fish were assessed to infer differences in
the biological activity of BaP between popula-
tions. Results of this study will be used to develop
testable hypotheses concerning specific costs and
benefits associated with toxic exposures and ge-
netic adaptation to anthropogenic stressors by
wild fish populations.
 D.E. Nacci et al. / Aquatic Toxicology 57 (2002) 203–215
2. Materials and methods
2.1. BaP challenge experiments
In July of 1998, fish were collected from WI
and NBH and maintained in the laboratory for a
period of approximately two weeks before use in
experimentation. The experimental design was
based on that of Willett et al. (1995). The average
weights (sd) of female fish used in this experiment
were 5.4 (1.7) and 4.4 (1.7) g wet weight for WI
and NBH, respectively. The average weights (sd)
of male fish used in this experiment were 4.9 (1.7)
and 3.7 (1.6) g wet weight for WI and NBH,
respectively. In this species with a life span of
about three years (Valiela et al., 1977), length
data (not shown) suggested that these fish were
two years old.
Fish were maintained in flowing sea water
aquaria at 20°C and fed TetraMin® (TetraWerke,
Germany) ad libidum before and during experi-
mentation. There were three treatments for each
of two fish populations. For each treatment, 12
male and female fish from one population were
anesthetized with three-amino benzoic acid ethyl
ester (also known as MS-222, Sigma Chemical
Company, St. Louis, MO) and injected with 10
ml/g of treatment solution. Injections were made
into the peritoneal cavity using a 16 gauge needle.
The injection site was sealed with a small amount
of rapid-cure glue (Duro® superglue, Loctite
Corp., Hartford CT) before the fish were returned
to a recovery tank. Treatment solutions consisted
of corn oil solvent, low dose BaP (nominally, 5
mg/kg) and high dose BaP (50 mg/kg). Each
treatment group was divided into two replicate
holding tanks so that there were 12 fish (six males
and females) from a single population in each of
two 80 l aquaria. Therefore, for each population,
there were 72 fish divided into six aquaria. After
10 days, fish were sacrificed using an overdose of
MS-222. Fish were held on ice while livers were
dissected and split in an anterior-posterior fash-
ion. These replicate liver samples from each fish
were frozen and stored at − 80°C for up to 3
months prior to analysis. Livers from male fish
were used to measure enzyme activities while the
livers of female fish were used to meaure the
205
activity of a single enzyme (EROD), only. A
subset of livers from male fish were also analyzed
for DNA adduct concentrations.
2.2. Enzyme acti6ities
Fish liver microsomes were prepared as de-
scribed by Willett et al. (1995), using reagents
supplied by Sigma Chemical Company, unless
otherwise specified. Briefly, livers were homoge-
nized in 0.1 M Tris, pH 7.4 buffer containing 0.25
M sucrose, centrifuged at 600× g for 10 min to
remove nuclear debris, then at 5000× g for 10
min and 13,000×g for 10 min to remove mito-
chondrial material. The post-mitochondrial super-
natant was then centrifuged at 100 000× g for 60
min. The microsomal pellet was resuspended in 50
mM Tris, pH 7.5 buffer containing 1 mM EDTA
in 20% glycerol.
EROD and total protein were measured using a
96-multiwell plate method adapted from Kennedy
and Jones (1994). Sodium phosphate buffer (pH
8.0), microsomal suspension and ethoxyresorufin
were added to sample wells of Co-Star® (Corning,
NY) white wall low fluorescence plates. The plate
was warmed to 25°C, and reactions were initiated
with the addition of NADPH (1 mM final concen-
tration) then incubated for 10 min. Reactions
were terminated by the addition of acetonitrile
that contained fluorescamine (300 mg/ml). A plate
reader was used to measure fluorescence indica-
tive of EROD activity (resorufin production) and
protein concentration (fluorescamine concentra-
tion). Resorufin fluorescence was measured using
a 530-nm excitation filter (25-nm bandwidth) and
a 590-nm emission filter (35-nm bandwidth).
Protein fluorescence was measured using a 400-
nm excitation filter (35-nm bandwidth) and emis-
sion 460-nm excitation filter (40-nm bandwidth).
Bovine serum albumin and resorufin were used to
generate standard curves for each plate.
Uridine diphosphate glucuronyl transferase
(UGT) was measured using 4-nitrophenol sub-
strate as described in Palace et al. (1997) for use
with lake trout microsomes. Briefly, microsomal
suspensions were mixed with an incubation mix-
ture containing Tris–maleate buffer, (250 mM,
pH 7.4), MgCl2 (5 mM), and 4-nitrophenol (0.5
206 D.E. Nacci et al. / Aquatic Toxicology 57 (2002) 203–215
mM), then warmed to 25°C. The reaction was
initiated by the addition of UDP-glucuronic acid
(5 mM) and incubated for 30 min. The reaction
was stopped with the addition of two volumes of
ice – cold 0.5 M trichloroacetic acid. The superna-
tant solution was neutralized with an equal vol-
ume of 2 M sodium hydroxide and diluted with
three volumes of water. Absorbance was mea-
sured at 405 nm to assess the reduction in color
caused by the formation of 4-nitrophenyl b-glu-
curonide. The extinction coefficient of 4-nitrophe-
nol (18.1×103 cm2/mol, Burchell and Weatherill,
1981) was used to calculate transferase activity as
the formation of 4-nitrophenyl b-glucuronide per
min.
2.3. DNA preparation and adduct Quantification
2.3.1. Reagents
[g-32P]Adenosine triphosphate (3000 Ci/mmol)
was purchased from Amersham Pharmacia Bio-
tech (Piscataway, NJ). RNAse A and T1, a-amy-
lase, calf thymus DNA, and micrococcal nuclease
were obtained from Sigma Chemical Company
(St. Louis, MO). Calf spleen phosphodiesterase
and nuclease P1 were commercially available from
Calbiochem (La Jolla, CA). Polynucleotide kinase
(3% phosphatase free) was obtained from
Boehringer Mannheim (Indianapolis, IN). Poly-
ethyleneimine (PEI)-cellulose thin layer chro-
matography (TLC) plates were purchased from
Alltech Associates, Inc. (Deerfield, IL). Opti-
fluor® was supplied by Packard Instrument Co.
(Meriden, CT).
2.4. DNA Extraction
Frozen liver tissues were homogenized (c2
setting, Omni, 2000, Waterbury, CT) for 2– 3 s in
5 × volumes of buffer (10 mM Tris– HCl,10 mM
EDTA, pH 7.4). The homogenates were cen-
trifuged at 4000× g for 10 min to pellet the
nuclear fraction and resuspended in 1 ml of 1%
SDS, 20 mM EDTA, pH 7.4. An RNase mixture
that included RNase A (80 mg), RNAse T1 (80
units), and a-amylase (20 mg) was added and
incubated at 37°C for 1 h. Proteinase K (0.4 mg in
80 ml of 50 mM Tris– HCl, pH 7.4) was added
and incubated at 37°C for 1 h. Protein was re-
moved by successive extractions with phe-
nol:chloroform:isoamyl alcohol (25:24:1)
saturated with 50 mM Tris–HCl, pH 8.0, and
chloroform:isoamyl alcohol (24:1). DNA was pre-
cipitated in absolute ethanol, washed twice with
70% ethanol, and resuspended in 10 mM Tris–
HCl, 1 mM EDTA, pH 7.4. DNA concentrations
were determined spectrophotometrically, using
absorbance at 260 nm. The ratio of absorbance at
260:280 generally ranged from 1.75–1.85.
32
2.5. P-postlabeling analysis
To measure BaP-DNA adducts, 32P-postlabel-
ing procedures were conducted on DNA extracts
of individual fish livers. For this purpose, fish
liver DNA (6 mg) was digested to 3%-mononucle-
otides at 37°C for 3.5 h with micrococcal nuclease
and calf spleen phosphodiesterase as described by
Gupta (1985). DNA digests were treated with
nuclease P1 as reported by Reddy and Randerath
(1986) with the following modifications. Aliquots
of 13.75 ml of undiluted DNA digest (5.5 mg), 1.3
ml nuclease P1 (6.5 Units), 2.0 ml zinc chloride (1
mM), and 3.3 ml sodium acetate buffer (0.4 M, pH
5.0) were mixed and incubated at 37°C for 45
min. After incubation, 2.4 ml of Tris –HCl (0.5 M,
pH 7.5) was added to the nuclease P1 mix and
evaporated to dryness. HPLC-quality water (10
ml) was added to the digest and incubated at 37°C
for 30 min with 7.5 ml of a radioactive mixture
containing 50 mCi [g-32P] ATP, 1.5 ml kinase
buffer (100 mM MgCl, 100 mM dithiothreitol, 10
mM spermidine, 300 mM Tris–HCl, pH 7.5), and
3% phosphatase free, polynucleotide kinase (5.4
Units). The total labeled mix was spotted onto the
origin of a 10× 10 cm PEI-cellulose TLC plate.
The adducts were resolved according to the
method of Ploch et al. (1998). Labeled DNA
adducts were detected on the TLC plate by au-
toradiography after exposing Kodak Biomax®
MS X-ray film for 16 h at −80°C. The spots were
excised and placed into 5 ml of Opti-fluor® and
counted.
To measure the total number of nucleotides, an
aliquot containing 0.5 mg of the digested DNA
sample was diluted 1000×. An aliquot (2.5 ml)
 D.E. Nacci et al. / Aquatic Toxicology 57 (2002) 203–215
was labeled as above with an equal volume of
radioactive mix. The labeled nucleotides were di-
luted 80× with 10 mM Tris base, 5 mM EDTA,
pH 9.5 and 5 ml were applied to a 20× 20 cm
PEI-cellulose TLC plate. Chromatography was in
one direction with 0.3 M ammonium sulfate, 10
mM sodium phosphate, pH 7.4. After a 30 min
exposure at room temperature using Kodak
Biomax MR X-ray film, the labeled bases were
excised and counted as above. Relative adduct
labeling (RAL) values were calculated by dividing
adduct counts per minute (cpm) by the total
nucleotide cpm after correcting for the back-
ground activity, dilution factors, and volumes
spotted. Because values for control livers are used
to calculate background activity for the RAL,
these relative adduct labeling values cannot be
calculated for the experimental controls.
2.6. Statistical analyses
Percent survival data were arc sine square root
transformed to normalize variance across the re-
sponse range before one- and two-way analysis of
variance followed by a test of least significant
difference. EROD activity data were ordered by
rank then analyzed using one- and two-way vari-
ance procedures to evaluate effects of factors in-
cluding sex, population, and treatment. This
procedure was equivalent to Kruskal– Wallis k-
testing (SAS Institute, 1990). Differences were
considered significant when P B0.05. All analyses
except those on DNA adducts were conducted
using SAS® (SAS Institute, 1990). To compare
DNA adduct concentrations, data were analyzed
using a t-test or, when unequal variance was
determined, the Mann-Whitney Rank Sum Test,
using SigmaStat® software (Jandel Scientific, San
Rafael, CA).
3. Results
During laboratory exposure to BaP, fish sur-
vival ranged from 67 to 100% and did not differ
significantly between populations (P =0.122) or
BaP treatments (P= 0.317) (Fig. 1). However,
population (P B0.001) and treatment (P B0.001)
207
had significant effects on EROD activity. While
BaP treatment tended to increase EROD activity,
this effect was lower in NBH than WI fish (PB
0.001) (Fig. 2). Sex affected EROD response, also
(PB 0.001). BaP-induced EROD activity was
lower in female than male fish, and not signifi-
cantly increased in NBH female fish, relative to
their respective controls. Although fish from both
sites used in laboratory experiments were gener-
ally similar in size, fish from WI were slightly
larger than those from NBH (P= 0.004). How-
ever, the weights of males did not differ between
sites (P= 0.178). When male fish only were com-
pared, BaP treatment increased EROD response
by 14-fold over control in WI fish but not signifi-
cantly in NBH fish (P= 0.071) (Fig. 3a). Simi-
larly, BaP increased UGT activity in males from
WI by 2.8-fold over control, while there was no
increase demonstrated in NBH males (P= 0.407)
(Fig. 3b).
Chromatograms of 32P-postlabeled hepatic
DNA from male fish showed dose-related in-
creases in the magnitude of DNA adduct spots
(Fig. 4), the chemical identities of which have not
yet been determined. Up to six unique spots were
characterized by position, measured, and ranked
by order of magnitude. The largest spots, referred
to as one and two, were evident in BaP-dosed fish
Fig. 1. Survival (mean 9sd, n =2 replicates of 12 fish each) of
adult F. heteroclitus following injection with corn oil carrier
(white bars), 5 mg/kg benzo[a]pyrene (BaP, gray bars), or 50
mg/kg BaP (black bars). Fish were collected from West Island
(WI), or New Bedford Harbor (NBH), MA, USA. Trans-
formed values were not significantly different by Duncan’s
multiple range test.
208 D.E. Nacci et al. / Aquatic Toxicology 57 (2002) 203–215

Fig. 2. Hepatic microsomal activity for ethoxyresorufin-o-deethylase (EROD, mean 9 sd, n = 5 – 8 fish) measured in adult male (m)
and female (f) F. heteroclitus following injection with corn oil carrier (white bars), 5 mg/kg benzo[a]pyrene (BaP, gray bars), or 50
mg/kg BaP (black bars). Fish were collected from West Island (WI), or from New Bedford Harbor (NBH), MA, USA. Letters
indicate significant differences among data that were rank transformed prior to one-way analysis of variance procedure, equivalent
to Kruskal – Wallis k-testing.

from both populations. While the ranking pat-
terns among spots were similar between popula-
tions, there were differences between populations
in relative magnitudes for similar spots and for
total radioactivity in all spots (Table 1). Follow-
ing exposure to five or 50 mg/kg BaP, spot one
and total spots were significantly elevated in WI
over NBH fish. The magnitude of spot 2 in WI
fish was also increased above that measured in
NBH fish.
4. Discussion
Effects of a model PAH and important envi-
ronmental pollutant, BaP, were compared in pop-
ulations of F. heteroclitus indigenous to a highly
PCB-contaminated site and a local reference site.
Fish from these sites vary widely in their tissue
concentrations of contaminants, including those
that act through the AHR (Nacci et al., 1999). In
addition, F. heteroclitus from these sites have been
shown to differ genetically in their responsiveness
to dioxin-like contaminants that act through the
AHR (Bello, 1999; Nacci et al., 1999; Bello et al.,
2001). In this study, responses to laboratory BaP
treatments were compared between contaminated,
DLC-adapted fish collected from NBH and rela-
tively uncontaminated, DLC-sensitive fish from
WI. Consistent with prior studies of NBH fish
and some AHR agonists (Nacci et al., 1999; Bello
et al., 2001), activities of two xenobiotic metabo-
lizing enzymes believed to be regulated by the
AHR pathway in fish were affected minimally by
BaP treatment. These differences between WI and
NBH fish suggest potential differences in the
metabolism, activation and/or elimination of BaP
that could be reflected in differences in the con-
centration of total BaP-DNA adducts produced
by BaP treatment. In fact, BaP-DNA adduct con-
centrations were relatively low in NBH fish. How-
ever, the types and relative quantities of DNA
adducts produced by BaP treatment were appar-
ently similar between populations.
In response to BaP exposure, F. heteroclitus
from a reference population demonstrated dra-
matically increased EROD activity. EROD re-
sponses following BaP exposure were similar for
 D.E. Nacci et al. / Aquatic Toxicology 57 (2002) 203–215
F. heteroclitus from this reference site and related
fish species (Fundulus grandis and F. simulus, Wil-
lett et al., 1995), also presumably from relatively
uncontaminated sites. Since EROD activity can
be indicative of AHR-regulated induction of
phase I-type enzymes that can use BaP as a
substrate (Stegeman and Hahn 1994), these find-
ings suggest that BaP exposure can increase the
rate of BaP activation in reference Fundulus.
However, consistent with other studies using this
fish species and others, CYP1A expression and
Fig. 3. (a) Hepatic microsomal activity for ethoxyresorufin-o-
deethylase (EROD mean 9 SD, n= 5–8 fish) or (b) uridine
diphosphate glucuronyl transferase (UGT, mean 9 SD, n=5 –
8 fish) measured in adult male F. heteroclitus following injec-
tion with corn oil carrier (white bars), 5 mg/kg benzo[a]pyrene
(BaP, gray bars), or 50 mg/kg BaP (black bars). Fish were
collected from West Island (WI), or from New Bedford Har-
bor (NBH), MA, USA. Letters indicate significant differences
among data that were rank transformed prior to one-way
analysis of variance procedure, equivalent to Kruskal –Wallis
k-testing.
209
Fig. 4. Typical chromatograms of hepatic DNA adducts from
fish from New Bedford Harbor (NBH, left panels), and a
reference site, West Island, (WI, right panels), following expo-
sure to corn oil control (0), 5 mg/kg benzo[a]pyrene (BaP) or
50 mg/kg BaP. Six unique spots, whose chemical identities
have not yet been established, were characterized by position,
measured for total radioactivity and ranked by order of mag-
nitude (1 – 6).
EROD activity can be suppressed in reproduc-
tively active females (e.g. Elskus 1992) like those
from WI and NBH that were used in this study.
Male and female mummichogs from NBH
demonstrated minimal increases in the activities
of phase I-type xenobiotic metabolizing enzymes
following BaP exposure. These results are consis-
tent with published studies which demonstrated
poor responsiveness of NBH mummichogs when
challenged with some other contaminants that act
through the AHR (Nacci et al., 1999; Bello et al.,
2001). Nacci et al. (1999) showed that embryonic
mummichogs from NBH showed increased
EROD activity only at the highest tested concen-
trations of the dioxin-like PCB congener
3,3%,4,4%,5-pentachlorobiphenyl (PCB126), and the
210 D.E. Nacci et al. / Aquatic Toxicology 57 (2002) 203–215

PAH, 3-methyl cholanthrene (3MC). Similarly, and xenobiotics (Poland and Knutson, 1982), in-
Bello et al. (2001) showed that while the DLC, cluding CYP1A, and the conjugation enzymes,
tetrachlorodibenzofuran, was a potent inducer of UGT and glutathione-s-transferase (GST). How-
CYP1A1 in reference fish, it was not effective in ever, differences in the magnitude and temporal
NBH fish or their progeny. However, the PAH, patterns of inducibility by AHR agonists suggest
b-napthaflavone, was a relatively effective inducer that these enzymes may not be coordinately regu-
of CYP1A1 in fish raised in the laboratory from lated by the AHR pathway in all fish species,
NBH parents (F1). Therefore, although NBH fish during all developmental stages (Andersson et al.,
have displayed some variation in responsiveness 1985). In several fish species some isoforms of
following exposure to different AHR agonists, UGT, characterized by their substrate specificity,
they have shown generally poor increases in have been shown to be inducible by AHR ago-
AHR-regulated phase I-type activities. Disparities nists (Clarke et al., 1992; George, 1994). For
between field-collected fish and their progeny sug- example, exposure to DLCs that induced EROD
gest that responses of NBH fish may reflect ge- also induced glucuronidation of the substrate p-
netic differences between fish populations but also nitro phenol in immature rainbow trout
may reflect inhibitory effects produced by high (Oncorhynchus mykiss, Huuskonen et al., 1996).
tissue concentrations of DLCs and other contami- Conversely, Palace et al. (1996) demonstrated that
nants (e.g. Besselink et al., 1998; Schlezinger et in lake sturgeon (Acipenser ful6escens) exposed to
DLCs that elevated EROD minimally, UGT did
al., 1999; Whyte et al., 2000).
not differ significantly among treatments. Consis-
Reference mummichogs also responded to BaP
tent with the findings of this study, xenobiotic-as-
exposure with increased activity of a phase II- or
sociated increases in the activity of phase II-type
conjugation-type enzyme. The activities of these
enzymes is typically low (e.g. two- to three-fold)
enzymes are generally interpreted as affecting
relative to the high levels of induction often
xenobiotic elimination (George, 1994). In mam-
demonstrated for phase I-type enzymes (i.e.
mals, AHR pathway activation coordinately regu-
CYP1A, Stegeman and Hahn, 1994). UGT activ-
lates a bank of enzymes that metabolize endo- ity that was measured in this study using p-nitro-
phenol substrate may be indicative of
Table 1
Differences in hepatic benzo[a]pyrene-DNA adduct formation glucuronidation activity towards BaP metabolites
in livers of male F. heteroclitus from contaminated and refer- (George, 1994). This interpretation suggests that
ence sites the rate of glucuronidation and subsequent excre-
tion of BaP may be moderately increased follow-
Dose (mg/kg) Site Spot 1 Spot 2 Totala
ing BaP exposure to mummichogs from reference
5 NBH 49 2b 39 2 79 4 (8) populations. While in this study UGT activity in
4c 3 6 NBH fish was not elevated by laboratory expo-
WI 149 8 69 3 21912 (12) sure to BaP, Bello (1999) showed that UGT activ-
12* 5 17* ity was high in the livers of field-collected NBH
50 NBH 509 49 129 8 669 60 (8) mummichogs. Results from this study, also sug-
30 10 36 gestive of modest elevation in the chronic or
WI 385 9 264 108 974 510 9347 (7) constitutive levels of UGT, are consistent with a
318* 89* 425*
potentially important role for conjugation in
Values are reported as nmole adduct/mole nucleotide, with the xenobiotic elimination.
number of fish per treatment indicated in parentheses. While some BaP metabolism results in elimina-
a
Total adducts in all spots measured (up to 6). tion, metabolic activation can also produce inter-
b
Mean9 standard deviation of duplicate analyses from two mediates that interact directly with biological
or three separate 32P-postlabeling procedures.
c
Median values. Asterisks indicate adduct levels signifi-
targets, producing adverse effects such as DNA
cantly different (PB0.05) from those of fish from the NBH adduct formation. As indicated by the quantity of
site using the Mann–Whitney Rank Sum Test. total DNA adducts produced by BaP exposure,
 D.E. Nacci et al. / Aquatic Toxicology 57 (2002) 203–215
the biological activity of BaP was higher in the
reference than the NBH fish population. As in
this study, Willett et al. (1995) showed that un-
contaminated Fundulus sp. exposed to BaP pro-
duced dose-responsive increases in hepatic DNA
adduct concentrations. NBH fish demonstrated
dose responsive DNA adduct formation following
BaP exposure, also. However, significantly lower
DNA adduct concentrations were measured in
NBH than reference fish livers. Interestingly,
lifelong exposure to high concentrations of PAHs,
including BaP, in NBH sediment (Pruell et al.,
1990), did not produce high concentrations of
DNA adducts in NBH controls. It is not known
to what extent the relatively low concentrations of
adducts in field-collected fish from NBH reflects
the limited bioavailability of PAHs in NBH sedi-
ment or the limited biological activity of BaP in
NBH fish.
Low DNA adduct concentrations in NBH fish
livers may reflect low rates of metabolic activation
consistent with low AHR-regulated enzyme activi-
ties but could also reflect high rates of DNA
adduct loss via DNA repair. However, DNA re-
pair activities have been reported as generally low
in fish species (Maccubbin, 1994). For example,
the major BaP adduct formed in English sole and
brown bullhead persisted for up to 60 days (Mac-
cubbin, 1994). Willett et al. (1995) also showed
that in Fundulus species exposed to BaP, DNA
adduct levels were stable from 2 to 14 days post-
injection, suggesting that DNA repair may not be
an important factor in the experiment described
here. However, further experiments will be neces-
sary to test directly whether low production or
high losses contribute to the relatively low DNA
adduct concentrations measured in NBH fish.
Other researchers have compared fish species
for their ability to metabolize BaP and produce
BaP-DNA adducts to explain their relative sensi-
tivity or resistance to known health effects of
PAH exposure, i.e. cancer. In fish species, BaP
responses were compared between the cancer-
prone brown bullhead (Americanus nebulosos) and
the cancer-resistant channel catfish (Ictalurus
punctatus). Consistent with cancer resistance,
BaP-treated channel catfish showed lower DNA
adduct concentrations than brown bullhead
211
(Ploch et al., 1998). Channel catfish also demon-
strated higher EROD and GST activities (Has-
spieler et al., 1994; Gallagher et al., 1996),
indicative of faster rates of BaP metabolism. Sim-
ilarly, BaP exposure produced lower DNA adduct
concentrations in the cancer-resistant starry
flounder than the cancer-prone English sole
(Varanasi et al., 1986). Although the rate of phase
I-type BaP activation was similar between these
fish species, Stein et al. (1990) suggested that
higher phase II-type conjugation activity might
explain lower DNA adduct formation in the
starry flounder. This observation may be impor-
tant in reconciling the correlation between higher
rates of enzyme activities and higher concentra-
tions of DNA adducts in AHR-responsive, refer-
ence versus poorly responsive, NBH
mummichogs. Specifically, the relative activities or
ratios of phase I-type (i.e. activation) and phase
II-type (i.e. elimination) activities, rather than the
rates for any one enzyme, may dictate the out-
come of PAH metabolism, biological availability
and health effects.
The finding that similar PAH exposure pro-
duces fewer DNA adducts in NBH fish suggests
that this fish population may be relatively resis-
tant to PAH-mediated cancer formation. There-
fore, the adaptation demonstrated by NBH fish
may be beneficial for population persistence in an
environment polluted with co-occurring AHR-ag-
onists. The physiological adaptation that reduces
the toxicity of DLCs to early life stages also
reduces the rates of PAH-associated cancer for-
mation, a compensatory strategy that protects
reproductively mature life stages. However, evolu-
tionary theory predicts that inherited traits that
are adaptive for specific environmental conditions
may present fitness disadvantages under alternate
conditions and that these specific fitness disadvan-
tages or ‘costs’ can be predicted based upon an
understanding of the mechanisms associated with
toxicity and resistance (review Coustau et al.,
2000). In this case, it was hypothesized that while
the long-term risk of cancer from PAH exposure
may be relatively reduced in fish that are poorly
AHR responsive, there may be other health risks
associated with the retention of high tissue con-
centrations of contaminants due to low rates of
metabolism and elimination.
212 D.E. Nacci et al. / Aquatic Toxicology 57 (2002) 203–215
Data from field-collected NBH fish are consis-
tent with the hypothesis that poor AHR respon-
siveness may be correlated with low rates for
xenobiotic activation that may limit elimination.
Specifically, certain PCB congeners that can be
metabolized by AHR-regulated enzymes and
that are usually depleted selectively in PCB-ex-
posed fish tissues (i.e. 3,3%,4,4% tetrachloro-
biphenyl, PCB77, Gutjahr-Gobell et al., 1999)
are present in relatively high concentrations in
NBH mummichogs (Lake et al., 1995). Consis-
tently, PAH exposures to NBH fish could result
in high, retained PAH concentrations in tissues
of exposed fish, and enhanced maternal transfer
of these compounds to their young. Toxic conse-
quences could result following exposure to sun-
light that non-enzymatically activates certain
PAHs, like BaP, into toxic intermediates. Exper-
iments are underway to determine whether under
certain environmental conditions, PAHs are
more acutely toxic to NBH than reference fish.
In addition to these theoretical predictions
concerning how high exposures to PAHs could
affect DLC-adapted NBH mummichogs, it may
be useful to review evidence concerning another
population of mummichogs indigenous to a site
highly contaminated with PAHs. The Atlantic
Wood Superfund site (Elizabeth River, VA) is
contaminated with creosote, and has sediment
PAH concentrations 13-fold higher than those
measured at NBH (2200 mg PAH/g, Vogelbein et
al., 1990). Like NBH mummichogs, Atlantic
Wood mummichogs are resistant to the acutely
toxic effects of local contaminants (in this case,
whole sediment, Vogelbein et al., 1996), are rela-
tively insensitive to CYP1A inducers (Van Veld
and Westbrook, 1995) and demonstrate elevated
activities for a phase-II type enzyme, GST (Van
Veld et al., 1991; Armknecht et al., 1998). How-
ever, unlike predictions for NBH fish, Atlantic
Wood mummichogs exhibit a high prevalence of
hepatocellular carcinoma and other lesions
(Vogelbein et al., 1990; Fournie and Vogelbein,
1994). Whether these findings contradict the pre-
dictions for the long-term effects of PAH expo-
sures on fish that are poorly AHR responsive
will not be understood until the effects of simi-
lar high dose, long-term exposures to AHR re-
sponsive fish can be compared.
This initial experiment using field-collected fish
has confirmed that PAH exposures produce
smaller changes in the activities of certain xeno-
biotic metabolizing or detoxifying enzymes and
lower BaP-DNA adduct concentrations in NBH
versus reference fish. It is not possible to tell
from these results to what extent the differences
between these two fish populations is related to
genetic versus environmental differences. In or-
der to distinguish the contributions of these fac-
tors, BaP responses will be compared in the
laboratory-reared (uncontaminated) progeny of
field-collected fish. Such experiments will con-
tribute to an increased understanding of the di-
rect and indirect costs and the benefits of
compensatory strategies demonstrated by popu-
lations exposed to persistent, bioaccumulative
and toxic contaminants. Research underway to
characterize the quantity and identity of metabo-
lites and DNA adducts produced by BaP-ex-
posed mummichogs from these populations may
provide further insight into how metabolic route
may contribute to the biological activity of
xenobiotic compounds and their potential health
risks to exposed fish populations. Additional ex-
periments to characterize similarities and differ-
ences between the mechanisms by which NBH
and Atlantic Wood fish respond to PAHs and
DLCs will provide further insight into toxic ef-
fects and compensatory mechanisms elicited by
anthropogenic stressors on wildlife populations.
Acknowledgements
The authors appreciate the helpful advice pro-
vided by two anonymous reviewers as well as
reviewers and technical consultants at the U.S.
Environmental Protection Agency (EPA), Na-
tional Health and Environmental Effects Re-
search Laboratory, Atlantic Ecology Division,
Narragansett, RI: Dr W. Boothman, Dr R.
Burgess, L. Mills. Valuable assistance was pro-
vided by Laura Coiro, Denise Champlin, Mark
Tagliabue, Jonathan Serbst, Adam Swank and
Lance Brooks. All experiments conducted for
this report comply with current laws of the
USA.
 D.E. Nacci et al. / Aquatic Toxicology 57 (2002) 203–215
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