Environmental Toxicology and Chemistry, Vol. 17, No. 10, pp. 2006–2018, 1998
q 1998 SETAC
Printed in the USA
0730-7268/98 $6.00 1 .00
IN VITRO INDUCTION OF ETHOXYRESORUFIN-O-DEETHYLASE AND PORPHYRINS
BY HALOGENATED AROMATIC HYDROCARBONS IN AVIAN PRIMARY
HEPATOCYTES
J. THOMAS SANDERSON,*† SEAN W. KENNEDY,‡ and JOHN P. GIESY§
†Department of Fisheries and Wildlife, Pesticide Research Center, and Institute for Environmental Toxicology,
Michigan State University, 13 Natural Resources Building, East Lansing, Michigan 48824-1222, USA
‡National Wildlife Research Centre, Canadian Wildlife Service, Environment Canada, 100 Gamelin Boulevard,
Hull, Quebec K1A 0H3, Canada
§Department of Zoology, National Food Safety and Toxicology Center, and Institute for Environmental Toxicology,
Michigan State University, 13 Natural Resources Building, East Lansing, Michigan 48824-1222, USA
(Received 1 September 1997; Accepted 10 February 1998)
Abstract—Ethoxyresorufin-O-deethylase (EROD) and porphyrin induction responses of primary hepatocytes to halogenated aromatic hydrocarbons (HAHs) were examined in newly hatched domestic chickens, herring gulls, ring-billed gulls, double-crested
cormorants, and Forster’s terns. Concentration–response relationships were determined for both biochemical responses in hepatocyte
preparations derived from individual avian livers (except for the tern). The choice of vehicle used to dose chicken hepatocytes
greatly affected the potencies and efficacies of HAHs. Dimethyl sulfoxide resulted in median effective concentration (EC50) values
for EROD induction that were between 10 and 15 times less than isooctane (isooctane was used throughout the study). Neither
vehicle induced EROD activity by itself. Concentration-dependent increases in EROD activity were observed with several HAHs,
and their potencies (EC50 values) were compared to that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) within each hepatocyte
preparation to determine relative potency factors (RPFs). Differences in sensitivity to these responses were observed among
individuals within each of the species and among species. Median EC50 values (nM) for EROD induction by TCDD were 0.72,
13, 20, 25, and 150 for the chicken, cormorant, ring-billed gull, herring gull, and tern hatchling, respectively. Relative potency
factors for several HAHs were different, in both ranking and potency, from those generally derived in mammalian hepatocytes.
Porphyrin accumulation was observed occasionally with the most potent aryl hydrocarbon receptor agonists, but most HAHs were
not tested at concentrations sufficiently high to observe a consistent response. This study provides information on interindividual
and interspecies differences in responsiveness to TCDD-like compounds and provides species-specific RPFs that may prove useful
for the purpose of hazard and risk assessment for fish-eating birds.
Keywords—Avian hepatocytes
2,3,7,8-Tetrachlorodibenzo-p-dioxin
Ethoxyresorufin O-deethylase
Halogenated aromatic hydrocarbons
Porphyrins
their toxic potencies in vivo [8]. 2,3,7,8-Tetrachlorodibenzop-dioxin (TCDD), for example, has a high affinity for the
cytosolic Ah receptor, is a potent inducer of CYP1A1 in vitro
and in vivo, and can cause toxicities in vivo at environmentally
relevant concentrations. Therefore, the ability of a persistent
chemical to induce CYP1A1 and its associated EROD activity
in an in vitro system is considered to be a reasonable measure
of its toxic potential in vivo.
Investigators have examined the EROD induction potential
of HAHs in primary hepatocytes of a number of avian species
[11–13]. Differences among species were observed in the sensitivity and magnitude of their response to HAHs, with domestic chicken embryos consistently the most sensitive. These
observations are consistent with reported differences among
bird species in EROD induction potency and toxic potency of
HAHs observed in embryos in vivo [14–18]. Chicken embryo
hepatocytes have also been observed to accumulate porphyrins
upon exposure to TCDD and certain other HAHs [19–21]. To
further characterize the sensitivity of these responses to HAHs
in birds, we determined potencies of HAHs to induce EROD
activity and porphyrin accumulation in primary hepatocytes
derived from hatchlings of the domestic chicken (Gallus gallus), herring gull (Larus argentatus), ring-billed gull (Larus
delawarensis), double-crested cormorant (Phalacrocorax au-
INTRODUCTION
Halogenated aromatic hydrocarbons (HAHs) such as polychlorinated dibenzo-p -dioxins (PCDDs), dibenzofurans
(PCDFs), and biphenyls (PCBs) are persistent environmental
contaminants that can bioaccumulate to toxic concentrations
in species at higher trophic levels. Elevated concentrations of
these chemicals in fish-eating birds in the Great Lakes [1,2]
and elsewhere [3–6] have been associated with numerous adverse effects, including reproductive toxicities, embryolethality, deformities, subcutaneous edema, enzyme induction, and
porphyrin accumulation. Laboratory studies have shown that
certain of these HAHs have a common mechanism of action
that involves initial binding to the aryl hydrocarbon (Ah) receptor [7–9]. A sensitive response directly mediated by the Ah
receptor is the induction of hepatic cytochrome P4501A1
(CYP1A1) and its associated ethoxyresorufin-O-deethylase
(EROD) activity [10]. Aryl hydrocarbon receptor binding affinities of HAHs correlate well with their EROD induction
potencies in vitro and, depending on the endpoint measured,
* To whom correspondence may be addressed
(t.sanderson@ritox.vet.uu.nl). The current address of J. T. Sanderson
is Research Institute for Toxicology, University of Utrecht, Yalelaan
2, P.O. Box 80176, 3508 TD, Utrecht, The Netherlands.
2006
Environ. Toxicol. Chem. 17, 1998
EROD and porphyrin induction by HAHs in avian hepatocytes
2007
Table 1. Effect of dimethyl sulfoxide (DMSO) or isooctane as a vehicle on the effective concentration (EC50) (nM) of 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD) and polychlorinated biphenyl (PCB) 77 for ethoxyresorufin-O-deethylase (EROD) induction (nmol/min·mg21 protein) in 19-dold chicken embryo primary hepatocytesa
DMSO
Isooctane
24 h
TCDD
PCB 77
a
48 h
24 h
48 h
EC50
ERODmax
EC50
ERODmax
EC50
ERODmax
EC50
ERODmax
0.0064
0.0088
0.26
0.25
88
97
97
98
0.011
0.014
2.4
4.2
131
195
32
69
0.096
0.11
2.4
2.9
115
147
107
106
0.093
0.10
2.7
3.9
140
155
79
85
The observed decrease in potency when using isooctane instead of DMSO after a 24-h exposure was 15- and 12.5-fold for TCDD and 9.2and 11.6-fold for PCB 77. A correction factor of 12 was applied when comparing results of the present study to those reported by investigators
using DMSO.
ritus), and Forster’s tern (Sterna forsteri). The objectives were
(1) to examine species differences in sensitivity to these biochemical responses, (2) to determine interindividual variabilities in sensitivity, and (3) to determine structure–activity relationships for several HAHs in each species.
METHODS
Chemicals
All HAHs used were verified to be more than 99% pure by
gas chromatography, using mass-specific (GC–MS) and electron capture detectors (GC–ECD), and did not contain Ahactive impurities at biologically significant concentrations.
Birds
Herring gull eggs were collected from a colony on Kidney
Island, Saginaw Bay, Michigan, USA. Ring-billed gull eggs
were collected from Sulfur Island, Thunder Bay, Lake Huron,
Michigan, USA. Double-crested cormorant eggs were collected from a relatively uncontaminated colony in Lake Winnipegosis, Manitoba, Canada [22–24], and from a more polluted site on Gull Island in Thunder Bay [23,24]. Forster’s tern
eggs were collected from a breeding colony in Lake St. Clair,
Michigan, USA. Domestic white leghorn chicken eggs were
obtained from the poultry farm at Michigan State University,
East Lansing, Michigan, USA. All collections took place in
June and July 1995. Fertile eggs were incubated artificially in
the laboratory at 378C and 55% relative humidity, and the birds
were killed within 24 h after hatching.
Hepatocyte preparation and treatment
Hepatocytes were prepared from individual livers (except
in the case of the Forster’s tern, for which pools of three livers
were used) under sterile conditions and cultured in 48-well
plates in serum-free medium according to the methods of Fisher and Marks [25] and Kennedy et al. [20,26]. After 24 h in
culture, the cells were dosed with HAHs in 2.5 ml of isooctane
and exposed for 24 h. Each concentration was tested in triplicate. Concentration–response curves for TCDD were generated in each individual hepatocyte preparation. When sufficient material was available, concentration–response experiments for other HAHs were also conducted in each preparation. The choice of HAH was made on the basis of a
combination of environmental relevance, availability to our
laboratory, and use in other investigations (to make comparisons).
Biochemical assays
The EROD activity and total protein and porphyrin concentrations were measured fluorometrically [27]. Standard
curves for resorufin, bovine serum albumin, and uroporphyrin
III (Porphyrin Products, Logan, UT, USA) were prepared on
the same 48-well plates used for the concentration–response
experiments. Protein concentrations were 20 to 40 mg/well for
the domestic chicken, 10 to 25 mg/well for the ring-billed gull,
20 to 50 mg/well for the herring gull, 15 to 40 mg/well for the
cormorant, and 30 to 60 mg/well for the tern. Effective concentration (EC50) values (expressed in nM) derived from concentration–response curves for EROD induction (expressed in
pmol resorufin/min·mg21 protein) were used as measures of
the Ah receptor-mediated potency of a compound, and the
maximal level of EROD induction was used as a measure of
its efficacy. Both parameters were determined by Woolf plot
analysis and visually. Relative potency factors (RPFs) were
determined on the basis of concentration–response data generated in the same hepatocyte preparation by dividing the EC50
value of TCDD by that of the test compound. Porphyrin concentrations were expressed in picomoles of uroporphyrin III
per milligram of protein.
RESULTS
Effect of vehicle and time on EROD induction potencies
and efficacies of HAHs
All HAH stock solutions and dilutions in the laboratory at
Michigan State University are dissolved in isooctane, because
this solvent is compatible with both analytical and bioassay
techniques used to determine TCDD equivalents in environmental extracts. However, dimethyl sulfoxide (DMSO) has
been used as a vehicle to administer HAHs to avian cells by
other investigators [11,12]. Therefore, we compared the influence of these two vehicles on the EROD induction potencies
and efficacies of two HAHs in 19-d-old chicken embryo hepatocytes (Table 1). After a 24-h exposure, TCDD and
3,39,4,49-tetrachlorobiphenyl (PCB 77) were about 12-fold less
potent when administered to the cells in isooctane than in
DMSO. This factor was taken into consideration when comparing our EC50 values with those of investigators using
DMSO. The relative potency and efficacy of PCB 77 declined
between 24 and 48 h of exposure in the hepatocytes, when the
compound was administered in DMSO (Table 1). The decrease
in potency was 10 to 20-fold; the decrease in efficacy was 1.5to 3-fold. On the other hand, TCDD administered in DMSO
showed a lesser decrease in potency (2-fold) and an increase
2008
Environ. Toxicol. Chem. 17, 1998
Fig. 1. Concentration–response curves for ethoxyresorufin-O-deethylase (EROD) induction by 2,3,7,8-tetrachlorodibenzo-p -dioxin
(TCDD) in a hepatocyte preparation from a single liver of a 1-d-old
domestic chicken hatchling. Each concentration was tested in triplicate. Each concentration–response curve was measured on a separate
48-well plate using the same hepatocyte preparation. EC50 5 effective
concentration.
of about 2-fold in efficacy over time. When administered in
isooctane, TCDD showed no marked differences in potency
or efficacy between 24 and 48 h. The effect of time on the
potency and efficacy of PCB 77 was also much less when
isooctane was used as vehicle.
Ethoxyresorufin-O-deethylase induction by TCDD in avian
hepatocytes
The EC50 values of TCDD (administered in isooctane) for
EROD induction were determined in hepatocyte preparations
of individual 1-d-old livers of the domestic chicken, ring-billed
gull, herring gull, and double-crested cormorant hatchling. In
Forster’s tern hatchlings, which are smaller than those of the
other species, determinations were performed in hepatocyte
preparations of pools of three livers per pool. Initial experiments in chicken hatchling hepatocytes demonstrated that concentration–response curves for EROD induction by TCDD
were reproducible within a single hepatocyte preparation (Fig.
1), with similar values for EC50 and fold induction. However,
EC50 values were found to vary among different hepatocyte
preparations within a given species, with an apparently skewed
distribution around a mean (Fig. 2). When individual hepatocyte preparations were pooled, this variability among EC50
values disappeared (not shown). Several concentration–response characteristics of EROD induction by TCDD in avian
hepatocytes are summarized in Table 2. Basal EROD activities
in the hepatocyte preparations differed considerably among
individuals within and among bird species, although maximal
EROD activities were not significantly different (Table 2).
When induction was expressed as fold induction above control,
the wild bird species showed similar levels of induction, and
the chicken hepatocytes appeared exceptionally responsive
(15-fold induction) compared with those of the wild birds (2.5to 3-fold). Median EC50 values (nM) of TCDD for the induction of EROD activity in hepatocyte preparations from different bird species were determined from the histograms in
Figure 2 and are rank-ordered from low to high as follows:
J.T. Sanderson et al.
0.72 for the domestic chicken (n 5 19), 13 for the cormorant
(n 5 23), 20 for the ring-billed gull (n 5 15), and 25 for the
herring gull (n 5 12) (Table 2). In the Forster’s tern, the median
EC50 value was 150 nM of TCDD, based on eight determinations in hepatocyte preparations of three livers per preparation. In the domestic chicken and the wild species (except
for the tern), two hepatocyte preparations were found to be
nonresponsive to TCDD (Fig. 2) or any other HAH. The distributions of the EC50 values for TCDD (Fig. 2) were further
analyzed using a logit regression model to determine several
distribution parameters useful for conducting probabilistic risk
assessments [28]. In these analyses, the nonresponsive hepatocyte preparations were omitted. Geometric means, standard
deviations, and 95% confidence intervals of the EC50 values,
together with the parameters of the regression equations, are
listed in Table 3.
The mean EC50 concentration of TCDD was significantly
lower in the chicken and significantly higher in the Forster’s
tern than in the other bird species (Table 3). Double-crested
cormorant eggs were collected from two different colonies,
Lake Winnipegosis, Manitoba, Canada, and Gull Island, Thunder Bay, Lake Huron, Michigan, USA. Historically, Lake Winnipegosis has had significantly (about 10-fold) lower levels of
HAH contamination than Gull Island [20–22]. Basal EROD
activities in the hepatocytes of cormorant chicks from Gull
Island were significantly higher than those from Lake Winnipegosis (Table 2). Maximal EROD activities and fold induction, as well as median and mean EC50s for EROD induction by TCDD, were not different between the relatively
clean and contaminated colonies. Therefore, the EC50 determinations from each colony were taken together when comparisons were made with the other bird species.
Ethoxyresorufin-O-deethylase induction by HAHs in avian
hepatocytes
Typical concentration–response curves for EROD induction
by several HAHs in a hepatocyte preparation from a single
bird liver are shown in Figure 3 for the domestic chicken, ringbilled gull, herring gull, and double-crested cormorant. Concentration–response curves generated in hepatocytes prepared
from pools of Forster’s tern livers are also shown. The EC50
values of these curves were used to determine the potency of
each HAH relative to that of TCDD and to calculate RPFs for
each bird species (Tables 4 to 8). The EC50 values for EROD
induction by TCDD were compared to results of other investigators (Table 9), and the RPFs derived in the present study
were compared to those determined by others (Table 10).
Porphyrin induction by HAHs in avian hepatocytes
Typically in avian hepatocytes, concentration–response
curves for the induction of EROD activity by the most potent
HAHs declined at the highest tested concentrations. In some
cases, the decline in EROD activity coincided with the first
measurable increase in porphyrins (Fig. 4). This phenomenon
was most consistent with TCDD, 1,2,3,7,8-PCDD, 2,3,4,7,8PCDF, and PCB 126 in hepatocytes of the domestic chickens
and wild birds, except the Forster’s tern, in which no porphyrin
accumulation was observed. The magnitude of the porphyrin
accumulation response was variable among hepatocyte preparations, HAHs, and species. Lowest-observed-effect concentrations for this response are summarized in Tables 4 to 8.
EROD and porphyrin induction by HAHs in avian hepatocytes
Environ. Toxicol. Chem. 17, 1998
2009
Fig. 2. Distribution of effective concentration (EC50) values of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for ethoxyresorufin-O-deethylase
induction in hepatocyte preparations from single livers among individuals of five different avian species, with the exception of the Forster’s tern
where 8 pools of 3 livers were used.
2010
Environ. Toxicol. Chem. 17, 1998
J.T. Sanderson et al.
Table 2. Concentration–response characteristics of 2,3,7,8-tetrachlorodibenzo-p-dioxin administered in isooctane for ethoxyresorufin-O-deethylase
(EROD) induction in primary hepatocytes of 1-d-old hatchlings of several avian species
EROD activity (mean 6 SEM) (nmol/min·mg21 protein)
Avian species
na
Median EC50b
(nM)
Domestic chicken
Herring gull
Ring-billed gull
Double-crested cormorant
19
12
15
23
0.72
25
20
13
Individual colonies
Lake Winnipegosis
Gull Island
13
10
13
13
8
150
Forster’s tern
Basalc
2.0
7
7
14
6
6
6
6
Maximal
0.4A
1B
1B
2C
20
22
17
26
962
20 6 4d
4.1 6 0.3D
6
6
6
6
Fold induction
2
4
2
4
15
2.8
2.7
2.4
6
6
6
6
3e
1.1
0.3
0.3
20 6 3
35 6 6
2.5 6 0.2
2.4 6 0.6
12 6 0.8
3.0 6 0.2
a
Sample sizes represent determinations in hepatocyte preparations from individual livers, except for the Forster’s tern, in which it represents
determinations in hepatocytes from eight individual pools of three livers per pool.
b EC50 5 effective concentration.
c Basal EROD activities with different uppercase letters were significantly different from one another (one-way analysis of variance followed by
Tukey’s test, p , 0.05).
d Significantly higher in Gull Island than Lake Winnipegosis (Student’s t test, p , 0.05).
e Significantly higher in the domestic chicken than in the other bird species (one-way analysis of variance, p , 0.05).
DISCUSSION
Effect of vehicle and time on EROD induction potency and
efficacy of HAHs
Induction experiments in 19-d-old chicken embryo hepatocytes demonstrated that the choice of vehicle for delivery
of HAHs to cells influenced the potency and in some cases
efficacy of these compounds (Table 1). Dimethyl sulfoxide
mixes readily with the cell culture medium and is known to
increase cell membrane permeability [29,30], whereas isooctane remains on top of the medium, allowing the HAHs to
diffuse slowly into the cell. It is therefore likely that DMSO
is more efficient in delivering HAHs to the cell than isooctane,
explaining the observed differences in potency of TCDD and
PCB 77. It is unclear what the reasons were for the changes
in efficacy of TCDD (increased) and PCB 77 (decreased) between 24 and 48 h when administered in DMSO, but pharmacokinetic aspects related to rates of cellular uptake and
metabolism may be responsible. In the case of PCB 77, metabolism to more polar, inactive metabolites is a likely explanation [31]; metabolism of PCB 77 to products with greater
water solubility was also observed by Lambrecht et al. [19]
in cultured chicken embryo hepatocytes. In any case, no major
changes in potency or efficacy were observed over time when
the HAHs were administered in isooctane, the vehicle used
throughout the present study. The observed difference in potency (for a 24-h exposure) by using isooctane instead of
DMSO was taken into account by applying a correction factor
of 12 (Table 1) when comparing our concentration data to those
of investigators using DMSO (Table 9).
Interindividual differences in sensitivity to EROD induction
by TCDD
One major difference between the present study and those
reported previously is that concentration–response experiments were performed in hepatocyte preparations derived from
individual livers (with the exception of the Forster’s tern),
whereas other studies have generally used pooled samples. An
exception is a recent study that examined the response of hepatocytes from individual 17-d-old double-crested cormorant
embryos to b-naphthoflavone (BNF) [32]. In our study, TCDD
concentration–response curves for EROD and porphyrin induction were generated in every hepatocyte preparation as
positive controls, as well as several HAHs, depending on the
amount of material available. This design was intended to
Table 3. Probability distributions of the effective concentration (EC50) values of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for ethoxyresorufinO-deethylase (EROD) induction in avian primary hepatocyte preparations derived from individual livers (except for the Forster’s tern, in which
pools of three livers per pool were used) using a logistic regression modela
Regression equation logit(p) 5 a 1 b 3 log(mean EC50) 1 e
n
Chicken
Herring gull
Ring-billed gull
Double-crested cormorant
Forster’s tern
17c
10c
13c
21c
8
a 6 SE
0.63
27.9
13
23.7
215
6
6
6
6
6
0.10
1.2
0.3
0.2
3.1
b 6 SE
SMSEb
6
6
6
6
6
0.34
0.43
0.38
0.28
0.61
4.2
5.5
2.6
3.5
7.1
0.26
0.88
0.22
0.1
1.4
Mean EC50 6 SE
(nM)
95%
Confidence
interval
6
6
6
6
6
0.4–1.0
15–42
3.6–32
7.1–16
64–240
0.72
28
18
11
152
0.15d
7
7
2
45e
a and b are curve-fitting parameters, and EC50 is the concentration of TCDD (nM) required to cause half-maximal induction of EROD activity
(used as a measure of sensitivity).
SMSE 5 square root of the mean squared error.
c Hepatocyte preparations nonresponsive to TCDD were omitted from the logistic regression analyses.
d Significantly lower in the domestic chicken than in the other avian species (one-way analysis of variance, p , 0.05).
e Significantly higher in the Forster’s tern than in the other avian species (one-way analysis of variance, p , 0.05).
a
b
EROD and porphyrin induction by HAHs in avian hepatocytes
Environ. Toxicol. Chem. 17, 1998
2011
Fig. 3. Concentration–response curves for ethoxyresorufin-O-deethylase (EROD) induction by several halogenated aromatic hydrocarbons in a
hepatocyte preparation from a single liver: examples of the response in the domestic chicken, herring gull, ring-billed gull and double-crested
cormorant. For the Forster’s tern, concentration–response curves were determined in a pool of three livers per pool. PCB 5 polychlorinated
biphenyl; PCDD 5 1,2,3,7,8-pentachlorodibenzo-p-dioxin; PCDF 5 2,3,4,7,8-pentachlorodibenzofuran; and TCDD 5 2,3,7,8-tetrachlorodibenzop-dioxin.
2012
Environ. Toxicol. Chem. 17, 1998
J.T. Sanderson et al.
Table 4. Domestic chicken: Effective concentration (EC50) values (nM) for ethoxyresorufin-Odeethylase (EROD) induction of several halogenated aromatic hydrocarbons (HAHs) and their potencies
relative to that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (relative potency factors [RPFs]) and
lowest-observed-effect concentrations (LOECs) for porphyrin accumulation determined in primary
hepatocyte preparations from livers of individual domestic chicken hatchlings
EROD inductionb,c
HAHa
n
EC50s (nM)
RPFs
Porphyrin inductionc
LOECs (nM)
TCDD
1,2,3,7,8-PCDD
2,3,4,7,8-PCDF
PCB 126
PCB 77
PCB 81
PCB 169
PCB 105
PCB 118
HCB
17
2
2
3
2
2
2
2
2
2
0.1–2
2, 1
1, 2
30, 5, 10
25, 10
10, 10
150, 40
NR, NR
1,000, NR
NR, NR
1
0.7, 1
0.5, 0.2
0.05, 0.08, 0.1
0.01, 0.1
0.03, 0.1
0.007, 0.01
NR, NR
0.001, NR
NR, NR
3–300
150, 100
100, 200
NR, 500, NR
NR, 300
500, 400
NR, NR
NR, NR
NR, NR
NR, NR
HCB 5 hexachlorobenzene; PCB 5 polychorinated biphenyl; PCDD 5 1,2,3,7,8-pentachlorodibenzop-dioxin; and PCDF 5 2,3,4,7,8-pentachlorodibenzofuran.
b Order of EC50 values within each row corresponds with order of RPFs and LOECs for porphyrin
induction (e.g., PCB 126 has an EC50 of 30 nM, and its RPF is equal to 0.05; therefore, the corresponding EC50 of TCDD is 1.5 nM; porphyrins were not induced by PCB 126 in this case).
c NR 5 no response within the concentration range tested.
a
characterize the interindividual variability in sensitivity of
these responses to the prototype and environmentally relevant
inducer TCDD and to determine the variability in the RPFs
for selected HAHs among individuals of each bird species.
We have observed that individual sensitivity to TCDD is
variable, with EC50 values for EROD induction differing from
3- to 80-fold within a given species (Tables 4 to 7 and Fig.
2). In contrast, TCDD concentration–response curves produced in the same hepatocyte preparation varied less than 2fold in EC50 (Fig. 1), and pooling two or three livers before
hepatocyte preparation considerably reduced the variability in
EC50s. This indicates that the observed variability is a reflection of differences in individual sensitivity of the EROD
induction response. We also observed that two hepatocyte
preparations per species were not responsive to TCDD (Fig.
2) or any other HAH tested. These nonresponsive hepatocytes
were found only among individual preparations and not among
pools (i.e., Forster’s terns), most likely because pooling would
mask any individual variability in such a low frequency re-
sponse. This occasional nonresponsiveness was also observed
by Davis et al. [32] in individual double-crested cormorant
hepatocyte preparations exposed to BNF with a similar rate
of incidence. No explanation for this phenomenon could be
offered.
Ethoxyresorufin-O-deethylase induction response in two
populations of double-crested cormorants
We prepared hepatocytes from double-crested cormorants
collected from two colonies differing in levels of contamination with HAHs. We hypothesized that cormorants from the
more polluted site (Gull Island) may have developed greater
resistance to the biological responses of TCDD than birds from
a historically less polluted location (Lake Winnipegosis). This
postulated greater resistance would possibly be reflected in a
higher EC50 of TCDD for EROD induction, because of selective survival in a contaminated area of only those individuals that have a lower-affinity Ah receptor and consequently
lower sensitivity to Ah-active chemicals. We also wished to
Table 5. Herring gull: Effective concentration (EC50) values (nM) for ethoxyresorufin-O-deethylase
(EROD) induction of several halogenated aromatic hydrocarbons (HAHs) and their potencies relative
to that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (relative potency factors [RPFs]) and lowestobserved-effect concentrations (LOECs) for porphyrin accumulation determined in primary hepatocyte
preparations from livers of individual herring gull hatchlings
EROD inductionb,c
HAHa
n
EC50s (nM)
RPFs
Porphyrin inductionc
LOECs (nM)
TCDD
1,2,3,7,8-PCDD
2,3,4,7,8-PCDF
PCB 126
PCB 77
PCB 81
HCB
10
2
2
3
2
2
2
15–50
3, 3
12, 5
150, 150, 100
200, NR
25, 25
NR, NR
1
6.7, 10
1.4, 4
0.2, 0.2, 0.5
0.15, NR
0.8, 1.2
NR
50–500
100, 500
400, 150
2,000, 2,000, 500
2,000, NR
500, NR
NR, NR
HCB 5 hexachlorobenzene; PCB 5 polychlorinated biphenyl; PCDD 5 1,2,3,7,8-pentachlorodibenzop-dioxin; and PCDF 5 2,3,4,7,8-pentachlorodibenzofuran.
b Order of EC50 values within each row corresponds with order of RPF values and LOECs for porphyrin
induction.
c NR 5 no response within the concentration range tested.
a
Environ. Toxicol. Chem. 17, 1998
EROD and porphyrin induction by HAHs in avian hepatocytes
2013
Table 6. Ring-billed gull: Effective concentration (EC50) values (nM) for ethoxyresorufin-O-deethylase (EROD) induction of several halogenated
aromatic hydrocarbons (HAHs) and their potencies relative to that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (relative potency factors [RPFs])
and lowest-observed-effect concentrations (LOECs) for porphyrin accumulation determined in primary hepatocyte preparations from livers of
individual ring-billed gull hatchlings
EROD inductionb,c
HAHa
n
EC50s (nM)
RPFs
Porphyrin inductionc
LOECs (nM)
TCDD
1,2,3,7,8-PCDD
2,3,4,7,8-PCDF
PCB 126
PCB 77
PCB 81
PCB 169
PCB 105
HCB
13
2
2
4
3
3
2
2
2
2–80
20, 30
200, 6
160, 2,000, 1,000, 150
300, 300, NR
2,000, NR, NR
300, 1,000
3,000, NR
NR, NR
1
1.0, 2.7
0.1, 6.7
0.01, 0.01, 0.04, 0.07
0.07, 0.13, NR
0.002, NR, NR
0.01, 0.02
0.001, NR
NR, NR
50–1,500
1,000, 50
1,500, 20
NR, 6,000, NR, 2,000
8,000, 3,000, NR
NR, NR, NR
500, NR
NR, NR
NR, NR
HCB 5 hexachlorobenzene; PCB 5 polychlorinated biphenyl; PCDD 5 1,2,3,7,8-pentachlorodibenzo-p-dioxin; and PCDF 5 2,3,4,7,8-pentachlorodibenzofuran.
b Order of EC50 values within each row corresponds with order of RPF values and LOECs for porphyrin induction.
c NR 5 no response within the concentration range tested.
a
examine whether differences in background contamination
with HAHs in ovo in the study would affect the response of
the hepatocytes to HAH administration in vitro. Hepatocyte
preparations from the more polluted site had basal EROD activities that were significantly (twofold) higher than those from
the relatively uncontaminated location (Table 2). Whether the
difference in basal EROD activity is a direct result of the
difference in contamination level with HAHs is not known. It
is possible that hepatocytes induced in vivo, once isolated and
cultured, rapidly lose the induced enzyme activity because of
the (partial) removal of the original inducing agent. Such a
loss was seen in cultured hepatocytes after isolation from
chicken embryos induced in ovo with BNF [32]. The EROD
activity decreased by half about 24 h after isolation and had
reached basal activity after about 60 h. However, it is important
to note that BNF is generally metabolized much faster than
TCDD, so pharmacokinetic factors are likely to play a greater
role when using BNF as the ‘‘prototype’’ inducer. Inducibility
of individually derived cormorant hepatocytes in our study
was similar to that observed by Davis et al. [32], with about
a 2- to 2.5-fold maximal increase of EROD activity above
basal levels. We did not observe a significant difference among
the two different populations of cormorants in EC50 values
for EROD induction or maximal fold induction after exposure
to TCDD (Table 2). This suggests that despite the differences
in contamination levels between the two cormorant populations, no obvious differences in sensitivity of the cultured hepatocytes to these Ah receptor-mediated biological responses
of HAHs exist. Davis et al. [32] examined the responsiveness
of cultured hepatocytes from two different populations of cormorants to BNF and could not find a conclusive difference in
median EC50 values between the two colonies.
Interspecies differences in sensitivity to EROD induction
by HAHs
To obtain estimates of the median and mean population
sensitivities of each avian species, histograms were generated
and logit regression analyses were performed to determine the
distribution characteristics of the EC50s of TCDD for EROD
induction (Fig. 2 and Table 3). These analyses demonstrated
that the mean EC50 value was significantly lower in the domestic chicken and significantly higher in the Forster’s tern
than in the other avian species. They also indicated that dif-
ferences in sensitivity among individuals can be as great as
the average sensitivity is among species. The interindividual
and interspecies differences in sensitivity observed in the present study and by others are at least partly due to differences
in affinity for the Ah receptor [18], possibly in combination
with differences in pharmacokinetic behavior of TCDD, and
other genetic factors.
The particularly sensitive response of the domestic chicken
is consistent with its known great sensitivity to the in vivo
toxicities of HAHs that are Ah receptor agonists [7]. The median EC50 value of TCDD for EROD induction in primary
hepatocytes of domestic chicken hatchlings was 0.72 nM in
our study, which, after dividing by a correction factor of 12
because of differences in vehicle (see Table 1), was comparable
to the value of 0.043 nM determined by Bosveld et al. [33]
in hatchlings of the same age. The increase we observed in
the EC50 between embryonic day 19 (about 0.1 nM; Table 1)
and 1 d after hatching (median, 0.72 nM) was also observed
by Bosveld et al. [33], although in their study the increase was
only about 2.3 fold.
Consistent with other reports [11–13], the present study
demonstrates that relative to the chicken, the EROD induction
response is considerably less sensitive to HAHs in certain other
avian species, such as fish-eating birds. In relation to the chicken hatchling, the cormorant was, on average, about 20-fold
less sensitive to TCDD; the two gull species, about 30- to 35fold less sensitive; and the Forster’s tern, more than 200-fold
less sensitive (Tables 2 and 3). In another study using primary
hepatocytes, the herring gull was found to be about 50 times
less sensitive to EROD induction by TCDD than the domestic
chicken [12]. An EC50 value for TCDD of 0.88 nM (n 5 2)
was found for EROD induction in 19-d-old embryo hepatocytes using DMSO as vehicle [12] (see Table 9). Another study
reported an EC50 value of 2.6 nM in hepatocytes from herring
gulls of the same age as those in the present study [11]. Using
a larger sample size, we determined a median EC50 value of
25 nM, which, after correction for the effect of vehicle (Table
9), is similar to this latter report. The relative sensitivity of
herring gull hepatocytes compared to those of domestic chickens based on EROD induction potency (EC50 chicken/EC50
gull) is about 0.03 in the present study. This value is comparable to that of 0.02 found by Kennedy et al. [12]. Using
EC50 values derived from Bosveld et al. [11,33] (Table 9), we
2014
Environ. Toxicol. Chem. 17, 1998
J.T. Sanderson et al.
Table 7. Double-crested cormorant: Effective concentration (EC50) values (nM) for ethoxyresorufin-Odeethylase (EROD) induction of several halogenated aromatic hydrocarbons (HAHs) and their potencies
relative to that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (relative potency factors [RPFs]) and
lowest-observed-effect concentrations (LOECs) for porphyrin accumulation determined in primary
hepatocyte preparations from individual livers of double-crested cormorant hatchlings
EROD inductionb,c
HAHa,b
n
EC50s (nM)
RPFs
Prophyrin inductionc
LOECs (nM)
TCDD
1,2,3,7,8-PCDD
2,3,4,7,8-PCDF
PCB 126
PCB 77
PCB 81
PCB 169
PCB 105
PCB 118
HCB
21
3
2
4
3
3
2
2
2
2
1.0–80
0.1, 1, 2
1, 2
90, 30, 100, 100
300, NR, NR
30, 50, 30
400, NR
NR, NR
NR, NR
NR, NR
1
10, 10, 13
10, 20
0.2, 0.3, 0.4, 0.8
0.13, NR, NR
0.1, 0.1, 0.3
0.2, NR
NR, NR
NR, NR
NR, NR
50–450
60, 15, 70
NR, NR
800, 300, NR, NR
NR, NR, NR
3,000, NR, 4,000
7,000, NR
NR, NR
NR, NR
NR, NR
HCB 5 hexachlorobenzene; PCB 5 polychlorinated biphenyl; PCDD 5 1,2,3,7,8-pentachlorodibenzop-dioxin; and PCDF 5 2,3,4,7,8-pentachlorodibenzofuran.
b Other than PCB 105, PCB 118, and HCB, Aroclorst 1242, 1254, and 1260 were also unable to induce
EROD activity in double-crested cormorant hepatocytes within the concentration range examined (10–
10.103 mg/L).
c Order of EC50 values within each row corresponds with order of RPFs and LOECs for porphyrin
induction.
d NR 5 no response within the concentration range tested.
a
determined a relative sensitivity of about 0.02 as well. These
values are similar, considering the differences in experimental
design, such as the age of the birds, the dosing vehicle, the
sample size, and the fact that in the present study hepatocytes
were prepared from individual livers instead of pools.
Limited in vivo or in ovo studies of the effects of TCDD
in fish-eating birds exist. Sanderson and Bellward [18] found
that EROD induction potencies of TCDD administered in ovo
in two fish-eating birds were between 30- and 100-fold less
in hatchlings of both the great blue heron and double-crested
cormorant than in those of the domestic chicken (Table 9).
This difference could not be explained by tissue concentrations
of TCDD, which were similar among bird species, but could
be explained partly by a more than 10-fold lower affinity of
TCDD for the hepatic Ah receptor in the heron and cormorant
relative to that in the chicken. Consistent with these observations, Powell et al. [34] found that the double-crested cormorant embryo was about 69-fold less sensitive to mortality
by PCB 126 than the domestic chicken. The difference in
sensitivity is somewhat greater than that observed for the cormorant in the present in vitro experiments (about 18-fold).
However, it should be noted that a direct comparison to the
in vivo situation is difficult to make; for instance, the in ovo
exposures to TCDD were longer and occurred during embryonic development [18,34], when hepatocytes are more sensitive to TCDD than after hatching [33].
Relative potency factors
Relative potency factors for HAHs based on EROD induction in avian primary hepatocytes of different species were
compared (Tables 4 to 8). Relative potency factors derived in
this study were determined in hepatocyte preparations from
individual avian livers (except for the Forster’s tern). As observed for the EC50 values of TCDD, the RPFs were also
variable among individuals within a given species. In the tern
the variability of the RPF determinations for a particular HAH
was less, probably as a consequence of pooling (Table 8). The
RPFs determined in chicken hepatocytes were similar to those
Table 8. Forster’s tern: Effective concentration (EC50) values (nM) for ethoxyresorufin-O-deethylase
(EROD) induction of several halogenated aromatic hydrocarbons (HAHs) and their potencies relative
to that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (relative potency factors [RPFs]) and lowestobserved-effect concentrations (LOECs) for porphyrin accumulation determined in primary hepatocyte
preparations from individual pools of three livers per pool of Forster’s tern hatchlings
EROD inductionb,c
HAHa
n
EC50s (nM)
RPFs
Porphyrin inductionc
LOECs (nM)
TCDD
1,2,3,7,8-PCDD
2,3,4,7,8-PCDF
PCB 126
PCB 77
PCB 169
8
2
2
2
2
2
75–200
15, 15
10, 10
500, 500
NR, NR
NR, NR
1
10–15
10–18
0.3, 0.4
NR, NR
NR, NR
NR
NR, NR
NR, NR
NR, NR
NR, NR
NR, NR
PCB 5 polychlorinated biphenyl; PCDD 5 1,2,3,7,8-pentachlorodibenzo-p-dioxin; and PCDF 5
2,3,4,7,8-pentachlorodibenzofuran.
b Order of EC50 values within each row corresponds with order of RPFs and LOECs for porphyrin
induction.
c NR 5 no response within the concentration range tested.
a
Environ. Toxicol. Chem. 17, 1998
EROD and porphyrin induction by HAHs in avian hepatocytes
2015
Table 9. Comparison of ethoxyresorufin-O-deethylase (EROD) induction potencies of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in vitro in
avian primary hepatocytes with in ovo induction and toxic potencies in several bird speciesa
EC50 in vitro for EROD induction (nM)
ED50 in ovo
for EROD induction
(pmole/g egg)
Sanderson et al.b
Bosveld et al.c
Kennedy et al.d
0.72b
0.060e
(1.0)
25b
2.1e
(0.03)
20b
1.6e
(0.04)
13b
1.1e
(0.05)
150b
13e
(0.005)
ND
0.043
(1.0)
2.6
(0.02)
ND
0.004–0.016
(1.0)
0.88
(0.02)
ND
0.3f
(1.0)
ND
ND
ND
ND
ND
10–30 f
(0.01–0.03)
ND
ND
ND
Common tern
ND
Black-headed gull
ND
0.32
(0.01)
ND
Ring-necked pheasant
ND
0.12
(0.36)
0.71
(0.06)
ND
Avian species
Domestic chicken
Herring gull
Ring-billed gull
Double-crested cormorant
Forster’s tern
Great blue heron
0.14
(0.11)
ND
10–30f
(0.01–0.03)
ND
ND
1.0g
(0.3)
Values in parentheses indicate the relative sensitivity compared with that of the domestic chicken (EC50 chicken/EC50 species). EC50 5 median
or mean effective concentration; ED50 5 median or mean effective dose; and ND 5 not determined.
b Present study.
c [11,33].
d [12,13].
e Correction factor of 12 applied.
f [18].
g [17].
a
reported by Kennedy et al. [12] (Table 10) and Bosveld et al.
[11,33] (not shown). Our RPFs for the chicken were also in
good agreement with RPFs for hepatic EROD induction in ovo
[11,35]. Comparison of the avian RPFs with those derived for
HAHs in mammalian systems, such as H4IIE rat hepatoma
cells [36,37] or the toxic equivalency factors proposed by Safe
in 1990 [38] and 1994 [39], indicates a number of differences
(Tables 4 to 8 and 10). The main observation was that for
avian species other than the chicken, RPFs for 1,2,3,7,8-PCDD
and 2,3,4,7,8-PCDF were generally greater (.1.0) than those
Table 10. Relative potency factors (RPFs) of halogenated aromatic hydrocarbons (HAHs) derived in avian hepatocytes in the present study
compared to other published avian RPFs or RPFs determined in mammalian H4IIE cells
RPFs of HAHs based on ERODa induction in vitro or in ovo
HAHb
2,3,7,8-TCDD
1,2,3,7,8-PCDD
2,3,7,8-TCDF
2,3,4,7,8-PCDF
PCB 126
PCB 77
PCB 81
PCB 169
PCB 105
PCB 118
Chickenc
Chickend
1.0
0.7, 1.0
ND
0.2, 0.5
0.05, 0.08, 0.1
0.01, 0.1
0.03, 0.1
0.007, 0.01
NR
NR, 0.001
1.0
ND
(1.0),j 1.1
ND
(0.3),j 0.3
(0.02),j 0.03
(0.2),j 0.2
(0.005),j 0.02
(0.00004),j 0.005
(0.00007),j 0.00008
Chickene Herring gullc
Herring gulld
Common
ternf
H4IIE cellsg
TEFsh,i
1.0
6.7, 10
ND
1.4, 4
0.2, 0.2, 0.5
NR, 0.15
0.8, 1.2
ND
ND
ND
1.0
ND
0.7, 0.9
ND
0.02, 0.06
,0.0003
ND
0.0002, 0.07
,9 3 1025
,9 3 1025
1.0
ND
0.4
ND
0.03
,0.0003
ND
0.02
ND
ND
1.0
0.3
0.09i
0.3
0.047
0.00034
0.0069
0.0015
,1.5 3 1026
, ,1.5 3 1026
1.0h
0.5h
0.1h
0.5h
0.1h
0.01h
ND
0.05h
0.001h
0.0001h
1.0
0.9
1.2
1.1
0.06
0.02
ND
0.001
ND
0.002
EROD 5 ethoxyresorufin-O-deethylase; ND 5 not determined; NR 5 no response within the concentration ranges tested.
PCB 5 polychlorinated biphenyl; PCDD 5 1,2,3,7,8-pentachlorodibenzo-p-dioxin; PCDF 5 2,3,4,7,8-pentachlorodibenzofuran; TCDD 5
tetrachlorodibenzo-p-dioxin; and TCDF 5 tetrachlorodibenzofuran.
c Relative potency factors were derived in the present study on the basis of effective concentrations (EC50) of HAHs for EROD induction in
primary hepatocytes derived from individual livers of 1-d-old chicken or herring gulls.
d Relative potency factors were determined in hepatocytes from pools of livers from 19-d-old chicken or herring gull embryos [12].
e Relative potency factors were determined on the basis of EROD induction in ovo in 17-d-old chicken embryos [11,35].
f Relative potency factors were determined in hepatocyte preparations from 18-d-old common tern embryos [13].
g Relative potency factors based on EROD induction in H4IIE cells [36].
h Toxic equivalency factors based on numerous endpoints derived from mammalian studies [39].
i Relative potency factor of 2,3,7,8-TCDF based on EROD induction in H4IIE cells [37].
j Relative potency factors were based on 10% effective concentration (EC10) values (concentration of HAH at which response is equal to that
of the EC10 of TCDD) [12].
a
b
2016
Environ. Toxicol. Chem. 17, 1998
Fig. 4. Concentration–response curves for the induction of ethoxyresorufin-O-deethylase (EROD) activity and porphyrins by 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,4,7,8-pentachlorodibenzofuran (PCDF) in a hepatocyte preparation derived from a single
liver of a 1-d-old herring gull hatchling.
in mammalian systems (0.1–1.0). The RPFs for most of the
PCBs examined in the bird species appeared to be greater than
those determined in H4IIE cells, although not that different
from the toxic equivalency factors proposed by Safe [39].
Porphyrin induction by HAHs in avian primary
hepatocytes
Porphyrin accumulation was observed infrequently in the
present study. Typically in cultured avian primary hepatocytes,
porphyrin accumulation is observed after exposure to relatively high concentrations of potent Ah receptor agonists and
after a postmaximal decline in EROD activity is observed [19–
21,40]. In our study, many of the HAHs, including TCDD,
were not tested at concentrations sufficiently high to observe
a definite postmaximal decline in EROD activity and concomitant increase in porphyrin contents in all cases. Also, the
sensitivity of this biochemical response to HAHs in avian hepatocytes appears to vary among individuals and among species (Tables 4 to 8). Consistent with other reports, domestic
chicken hatchlings displayed the greatest sensitivity (i.e., at
the lowest concentrations of HAHs) to the porphyrin accumulation response, whereas Forster’s terns were nonresponsive, similar to common tern embryos [13]. Large differences
in responsiveness have been observed in adult birds, with Japanese quail (Coturnix japonica) being relatively sensitive [41]
and American kestrels (Falco sparverius) appearing nonresponsive [42] to porphyrin accumulation induced by PCB 126.
Porphyrin accumulation is a complex response that can be
caused by a number of mechanisms [40] and is not specific
to Ah receptor agonists. Typically, TCDD and related HAHs
increase the concentrations of uroporphyrin and heptacarboxylic acid porphyrin [20,21,40]. However, the underlying mechanism by which TCDD and other Ah receptor agonists can
cause porphyrin accumulation in cultured hepatocytes is not
well understood. It is also not clear whether the response is
directly, indirectly, or not at all mediated by the Ah receptor.
Hexachlorobenzene (HCB), a fungicide with porphyrinogenic
properties, results in a porphyrin accumulation pattern in vivo
J.T. Sanderson et al.
that is similar to that of TCDD and related chemicals [40].
The mechanism by which HCB causes porphyria appears to
involve inhibition of uroporphyrinogen decarboxylase [40],
although one report indicates that HCB may also have a weak
affinity for the Ah receptor [43]. In the present study, HCB
up to a concentration of 170 mM was not able to induce porphyrins in hepatocytes of any the avian species studied. It is
possible that, under our experimental conditions, either concentrations of HCB at the biological target were not sufficiently
high (due to poor solubility) to elicit this response or that the
hepatocytes, once isolated and cultured, were no longer able
to accumulate porphyrins via the specific mechanism required
for the porphyrinogenic action of HCB. Another possibility is
that the TCDD-like porphyrin accumulation response observed
in some reports may be due to small quantities of TCDD-like
impurities in the HCB preparations, which generally elicit their
responses at micromolar concentrations [43,44]. The importance of verifying purity was recently exemplified by the observation that the ability of polychlorinated diphenyl ether
preparations to induce EROD activity in H4IIE rat hepatoma
cells was due to low concentrations of PCDF impurities, which
were well below 1% [45]. Aryl hydrocarbon-active impurities,
such as PCDDs, PCDFs, and non-ortho-PCBs were not detected by GC–MS and GC–ECD in the HCB preparation used
in the present study.
Implications for hazard assessment of HAH exposure in
avian species
The limited data available on toxic potencies of dioxin-like
HAHs in avian species indicate that EROD induction potencies
of HAHs in avian hepatocytes correspond reasonably well to
in vivo and in ovo responses such as EROD induction (Table
9) or mortality [12]. Therefore, it can be assumed that relative
EROD induction potencies in vitro are reasonable measures
of the Ah receptor-mediated toxic potencies in vivo, both for
different HAHs within a given species and for a given HAH
among different species. Consequently, on the basis of the
results of the present study, in combination with those from
other recent reports, we can formulate a number of implications
for the hazard assessment of HAH exposure in avian species.
Domestic chickens and their embryos are considerably more
sensitive to Ah receptor-mediated responses than many other
studied avian species [7,11,12,14,16–18]. When findings of
the present study (Tables 2 and 9) are considered together with
those of Kennedy et al. [12] and Lorenzen et al. [13], a rank
order of species sensitivity can be made: domestic chicken .
ring-necked pheasant . turkey ø double-crested cormorant ø
great blue heron ø ring-billed gull ø herring gull ø common
tern . Forster’s tern. From an ecotoxicological point of view,
the comparison of fish-eating bird species with domestic chickens or turkeys and pheasants is not very relevant, because the
latter species are not at great risk to accumulate toxic concentrations of dioxin-like HAHs through the food chain. It
would further appear that the fish-eating bird species examined
so far are at least one order of magnitude less sensitive than
a laboratory species such as the domestic chicken, again making the chicken a less appropriate choice for risk assessments
of avian wildlife species.
The relatively great interindividual variability within a given species indicates that the mean population sensitivity of a
given species does not provide adequate information on the
sensitivity of individual birds. In other words, there are probably herring gulls that are as sensitive as the average chicken
EROD and porphyrin induction by HAHs in avian hepatocytes
and chickens that are as sensitive or insensitive as the average
herring gull to the toxic effects of TCDD and related HAHs.
However, the relative proportions of the populations that would
be expected to exhibit extreme sensitivities would be small.
In environmental risk assessments of wildlife populations, adverse effects are usually documented on a whole-population
basis by examining population parameters such as reproduction, incidence of birth defects, and rate of mortality. The
sensitivity of any specific individual member of that population
is not considered important as long a stable viable population
is maintained. This situation is different from risk assessment
for human populations, in which knowledge of risks to individuals is considered essential for the protection of human
health [46]. However, it becomes important to consider the
sensitivity of individual members of a wildlife species when
investigators or conservation managers are dealing with severely depleted populations of rare or endangered species. In
risk assessments, safety factors are often applied to correct for
possible interindividual (10-fold) and interspecies (10- or 100fold) differences in sensitivity to an adverse effect. We suggest
that probabilistic risk assessments be applied instead of worstcase scenarios. The ranges of intra- and interspecies variation
presented here would be useful in conducting such assessments
instead of arbitrary safety factors, particularly since the present
study indicates that in some instances intra- and interspecies
differences can be greater than the applied safety factors. Although the interindividual variabilities described in the present
study were determined from distinct populations of birds from
single sites per species (except for the cormorant) and may
not necessarily reflect the interindividual variability at another
location, we point out that no difference in interindividual
variability in sensitivity (based on EC50 values for EROD
induction) was observed between the two distinct populations
of cormorants. This indicates that potential bias due to choice
of single sites is probably small.
Species-specific differences in RPFs for certain HAHs may
also have an effect on the hazard these compounds pose to a
particular wildlife species. Results from the present study indicate that 1,2,3,7,8-PCDD and 2,3,4,7,8-PCDF may be greater
relative contributors to the Ah receptor-mediated toxicity of
environmental mixtures of HAHs in wild birds than they would
be in chickens or mammals (Tables 4 to 8 and 10). Also, certain
non-ortho-substituted PCBs may be more potent in wild birds
and chickens than they would be in mammals.
Certain wild fish-eating bird populations have recovered in
the last decade with declines in environmental concentrations
of HAHs. However, populations of common terns and Forster’s
terns continue to decline [1]. The limited data available on the
potential sensitivity of these two species to TCDD-like chemicals appears to suggest that their intrinsic sensitivity is relatively low (Table 9). Factors such as habitat disturbance and
destruction or high sensitivity to other types of chemicals (e.g.,
certain pesticides) may currently be playing a more direct role
in their low reproductive success.
CONCLUSIONS
Ethoxyresorufin-O-deethylase and porphyrin induction responses of avian primary hepatocytes derived from individual
livers of 1-d-old hatchlings of the domestic chicken, herring
gull, ring-billed gull, and double-crested cormorant to TCDD
and other environmentally relevant contaminants were examined in detail. The same experiments were performed in the
Forster’s tern, using hepatocytes derived from pools of three
Environ. Toxicol. Chem. 17, 1998
2017
livers per pool. The EROD induction potencies of TCDD varied among individuals within a given species and among species. A strong effect of vehicle was also observed: EROD
induction potencies of TCDD and PCB 77 were about 12-fold
less in chicken hepatocytes when isooctane was used as vehicle
instead of DMSO. Median EC50 values for EROD induction
by TCDD could be rank-ordered as follows: white leghorn
chicken , double-crested cormorant ø ring-billed gull ø herring gull , Forster’s tern. Other investigators have reported
EC50 values for EROD induction by TCDD in hepatocytes of
the chicken and the herring gull that (after correction for potency differences due to differences in vehicle) were in good
agreement with the median EC50 values found in the present
study. Relative potency factors of several HAHs were determined in each of the species and differed from those derived
in mammalian systems. Both 1,2,3,7,8-PCDD and 2,3,4,7,8PCDF had considerably greater RPFs (and were more potent
than TCDD) in the wild bird species than in the chicken, H4IIE
cells, or the RPFs proposed by Safe [38,39]. The non-orthosubstituted PCBs (PCBs 126, 77, 81, and 169) had generally
greater RPFs in the five bird species than in H4IIE cells, although the values were not that different from those proposed
by Safe. Porphyrin accumulation was observed infrequently,
with the most potent HAHs in every species except for the
Forster’s tern. Considering that this response occurs after exposure to relatively high concentrations of Ah agonists, usually
after a postmaximal decline in EROD activity is observed, it
is probable that the HAHs were not tested at concentrations
high enough to elicit this biochemical response in a consistent
manner. Taken together, the results of the present study are
useful for the refinement of hazard assessments of HAH exposure in fish-eating birds because they provide data necessary
to conduct probabilistic risk assessments that consider the relative sensitivity of avian species and species-specific RPFs for
several environmentally relevant HAHs instead of arbitrary
safety factors.
Acknowledgement—We are very grateful to Stephanie Jones for her
technical assistance. We are indebted to Dave Best for supervising
the collection of the wild bird eggs. We thank Angelo Napolitano for
use of his incubator and for supplying the chicken eggs. This study
was supported by National Institutes of Health grant ES-04911.
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