Environmental Health Perspectives Volume
103, Supplement 4, May 1995
[Citation
in PubMed] [Related
Articles]
Morphometric Abnormalities in Brains of Great Blue Heron Hatchlings
Exposed in the Wild to PCDDs
Diane S. Henshel,1,2,5 J. William Martin,1 Ross
Norstrom,3 Phillip Whitehead,4 John D. Steeves,2
and Kimberly M. Cheng5
1School of Public and Environmental Affairs, Indiana University,
Bloomington, Indiana; 2Department of Zoology, University of British
Columbia, Vancouver, British Columbia, Canada; 3National Wildlife
Research Center, Canadian Wildlife Service, Hull, Quebec, Canada; 4Canadian
Wildlife Service, Pacific and Yukon Region, Delta, British Columbia, Canada;
5Department of Animal Science, University of British Columbia,
Vancouver, British Columbia, Canada
Abstract
Great blue heron hatchlings from colonies in the Strait of Georgia, British
Columbia, Canada are being monitored for environmental contaminant exposure
and effects by the Canadian Wildlife Service. The contaminants of concern
are polychlorinated dibenzodioxins (PCDDs) and polychlorinated dibenzofurans
(PCDFs), primarily derived from kraft pulp mill effluent. The levels of
PCDDs and PCDFs in eggs from the most contaminated colonies peaked in 1988
and 1989 and dropped dramatically through 1990 to 1992. Brains of heron
hatchlings (taken as eggs from the wild and hatched in the laboratory) were
analyzed for gross morphological abnormalities. Brains from highly contaminated
colonies (Crofton, British Columbia and University of British Columbia Endowment
Lands) in 1988 exhibited a high frequency of intercerebral asymmetry. The
frequency of this abnormality decreased in subsequent years as the levels
of TCDD and TCDD-TEQs (toxic equivalence factors) decreased. The asymmetry
was significantly correlated with the level of TCDD and TCDD-TEQs in eggs
taken from the same nest. Yolk-free body weight negatively correlated and
the brain somatic index positively correlated with the TCDD level in such
pair-matched eggs. These results indicate that gross brain morphology, and
specifically intercerebral asymmetry, may be useful as a biomarker for the
developmental neurotoxic effects of PCDDs and related chemicals.
-- Environ Health Perspect 103(Suppl 4):00-00 (1995)
Key words: polychlorinated dibenzodioxin, polychlorinated dibenzofuran,
development, birds, asymmetry, TCDD, morphology, central nervous system,
brain, kraft pulp mill effluent
This paper was presented at the Conference on Environmentally
Induced Alterations in Development: A Focus on Wildlife held 10-12 December
1993 in Racine, Wisconsin.
This work was supported by grants from the Wildlife Toxicology
Fund of the World Wildlife Fund Canada, the British Columbia Environment
Ministry, Canfor Corporation Inc., Fletcher-Challenge Canada, MacMillan
Bloedel Ltd. Inc., the National Science and Engineering Research Council
(NSERC, Canada), the British Columbia Wildlife Federation, the Northwest
Territories Wildlife Federation, and the Arde Bulova Foundation. J.W.M.
was supported by a research assistantship from the School of Public and
Environmental Affairs, Indiana University.
Address correspondence to Dr. Diane S. Henshel, School
of Public and Environmental Affairs, Indiana University, Bloomington, IN
47405. Telephone (812) 855-4556. Fax (812) 855-7802.
Introduction
The province of British Columbia in Canada has a resource-based economy.
Forests are one of the main resources. Thus, the forestry and pulp and paper
industries are the largest industrial base for the province. Most of the
pulp and paper companies of British Columbia use the bleached kraft process,
which ultimately releases polychlorinated dibenzodioxins (PCDDs; including
2,3,7,8-tetrachloro- dibenzodioxin [TCDD], 1,2,3,7,8-pentachlorodibenzodioxin,
and 1,2,3,6,7, 8-hexachlorodibenzodioxin) and polychlorinated dibenzofurans
(PCDFs; including 2,3,7,8-tetrachlorodibenzofuran) in the effluent. As a
result of the persistent halogenated aromatic hydrocarbon (HAH)-related
industrial contaminants in the Strait of Georgia (off the west coast of
the mainland of British Columbia), the most significant are the PCDDs and
PCDFs, with only a minor contribution of polychlorinated biphenyl (PCB)
from other industries. Analysis of great blue heron (Ardea herodias)
eggs from a colony near a pulp mill in British Columbia in 1988 showed that
PCBs contributed less than half of the total TCDD-like activity as measured
by toxic equivalents [TCDD-TEQs; (1,2)]. This situation contrasts
strongly with that in the Great Lakes, where PCBs contribute most of the
TCDD-like activity in piscivorous birds such as the double-crested cormorant
(Phalocrocorax auritus) (3). The Strait of Georgia, British
Columbia is thus an ideal location to study the effects of PCDDs and PCDFs
on wildlife.
Since 1982, the Canadian Wildlife Service (CWS) has been monitoring the
levels of PCDDs and PCDFs in the Strait of Georgia, in part by evaluating
the levels of these compounds in the eggs of great blue herons. Since 1983
they have also been monitoring the reproductive success of the heron colonies
(4). In 1987 and 1988 one colony failed to produce any fledglings.
This colony is located near a pulp and paper mill in Crofton on Vancouver
Island. The Crofton colony also had the highest levels of PCDDs in the eggs.
Between 1988 and 1990, the Crofton pulp mill instituted major changes in
their pulp processing that has led to a decrease in the level of PCDDs and
PCDFs in the effluent released into the Strait of Georgia (5).
Evaluations of the various heron colonies and of hatchlings taken (as
eggs) from the colonies in 1988 indicated that a) there were significant
colony differences in yolk-free body weight, kidney weight, stomach weight,
tibia dry and ash weight, tibia and beak length, and down follicle density
(as examined on the head) (6); b) all of the above differences
also correlated negatively with log TCDD concentrations in eggs taken from
the same nests as the evaluated hatchlings; c) analysis of P450 enzyme
activity indicated that ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin
O-deethylase (PROD) activity also varied by colony, being highly
induced in the more contaminated colonies (7); and d) the
behavior of the parent herons in the most contaminated colony (Crofton)
differed from the behavior of the herons in the control colony; nesting
was less synchronized from nest to nest, and nest attentiveness was lower
and more variable at the Crofton colony compared to Sydney Island (a control
colony) (8).
Since the nervous system interacts with the endocrine system to control
reproduction, growth, and behavior, we initiated studies to evaluate whether
the brain was affected in the herons from the more contaminated colonies.
Preliminary observations (9,10) on brains from heron hatchlings taken
in 1988 and 1989 (as eggs) from colonies with low, moderate, and high levels
of PCDD and PCDF contamination indicated that a) many of the hatchling
brains developed a gross cerebral asymmetry that varied in frequency by
colony and that increased in frequency and degree with increasing levels
of TCDD in eggs taken from the same nests as the evaluated hatchlings; b)
there was a significant increase in the cell density and width of the pyriform
cortex of the forebrains of heron hatchlings taken from the most contaminated
colony (Crofton) compared to a relatively uncontaminated "control"
colony. Many of the cells that were increased in number were immunoreactive
for taurine, a putative inhibitory neurotransmitter); and c) brain
weight decreased with increasing levels of TCDD. At this time it is unclear
what relationship these changes have to the growth, behavioral, and reproductive
changes seen in the contaminated heron hatchlings. No one has fully identified
how the pyriform cortex, for example, is controlled or how it is linked
into many of the other structures in the avian brain. However, the cerebral
hemispheres contain the integrative part of the brain. The pyriform cortex
(on the lateral aspects of the cerebral hemispheres) is an accessory structure
for the limbic system, which controls, among other things, emotions and
instinctive behavior. In addition, the pyriform cortex is the second structure
to receive olfactory input, which in many animals plays a role in instinctive
behavior (11).
The present experiments extend these preliminary observations to the
subsequent years when, due to improvements in the kraft process by the local
pulp mills, the overall contamination level of PCDDs and PCDFs in the Strait
of Georgia decreased to moderately low "background" levels, as
indicated by the amount of contamination of heron eggs (2).
Materials and Methods
Methods were as described in detail in Henshel et al. (9). Briefly,
two eggs from each nest were collected by the Canadian Wildlife Service.
One egg was taken for analysis (individual or pooled as indicated) and one
egg was incubated until hatching in the laboratory. These eggs were termed
pair-matched eggs. All birds were euthanized within 24 hr of hatching. The
brains were removed and preserved in 100 ml 4% paraformaldehyde (4°C),
and subsequently transferred to phosphate-buffered saline containing azide
for storage. Brains were weighed postfixation to reduce risk of damage to
the brains during the weighing process.
Eggs were taken from a total of five colonies: Nicomekl, University of
British Columbia Endowment Lands (UBC), and Crofton in 1988, Chilliwack
and UBC in 1990, Nicomekl, UBC, Crofton, Chilliwack, and Tillicum in 1991,
and UBC in 1992. The Nicomekl, Chilliwack, and Tillicum colonies were considered
to be our reference, i.e., less contaminated, colonies.
Brains were measured by eye, using an engineering ruler (with 0.5 mm
gradations) held parallel to the plane of measurement. Measurements that
fell between the gradations were recorded as 0.25 mm. All difference measurements
used reflect the right brain measurement subtracted from the left brain
measurement. Care was taken to have the brains measured and weighed by the
same person to reduce inconsistencies between observations. Brains were
coded when measured. The measurements were subsequently decoded and analyzed
by a different person.
PROC GLM (General Linear Model) and PROC REG (Linear regression) (SAS
6.04; SAS Institute; Cary, North Carolina) were used to analyze the data.
Measurements were compared to the source colony (nested in year) and TCDD
and TCDD-TEQ values from eggs taken from the same nest. TCDD-TEQs were calculated
based on PCDD and PCDF measured values using Safe's toxic equivalence factors
(1). Significance was determined using an a of 0.05 and is
expressed as the probability of occurrence of the null hypothesis. Mean
values ±standard error of the mean (SEM) were calculated and are
listed in Table 1 along with sample sizes. Some chemical analyses were made
on pooled samples. These are indicated in Table 1.
Results
Cerebral Asymmetry
Normal avian brains typically are grossly symmetrical. These brains appear
asymmetrical in that there is a greater convexity in the left, compared
to the right, cerebral hemisphere. To quantify the observable brain asymmetry
(Figure 1), we made four measurements of the forebrain region (Figure 2).
The width, height, and angle measurements are only affected by brain nuclei
(functional aggregates of neurons) in the cerebral hemispheres. The depth
measurement, however, is also affected by the rostral thalamic and hypothalamic
nuclei, especially the preoptic area (POA).
Figure 1. Photograph
of the dorsal aspect of brains taken from the UBC colony (left) and the
Chilliwack colony (right) in 1990. The arrow points to the right hemisphere
of the brain of a heron from the UBC colony; it has a flatter arc compared
to the right hemisphere of the brain from a heron from the Chilliwack colony.
The three-dimensional increased convexity in the left hemisphere does not
visualize well from this angle.
Figure 2. Diagram
of the four measurements made. The width, height, and angle measurements
(drawn in dorsal view) were all made with the brain lying parallel to the
desktop. The depth measurement (drawn from the lateral view) was made with
the brain lying coronally perpendicular to the desktop.
Of the four measurements made, only the width, angle, and depth measurements
varied significantly with colony nested in year (p<0.0001 for
all three measurements; r2=0.60, 0.69, and 0.41, respectively;
Figure 3). For all three measurements, the asymmetry was significantly greater
in the brains from herons from the UBC and Crofton colonies in 1988 compared
to either the Nicomekl colony in 1988, or any of the colonies in subsequent
years (p<0.001-0.0001, depending on the measurement). Figure 4
illustrates the mean TCDD (A) and TCDD-TEQ (B) values in the
pair-matched eggs for each colony by year. Analysis of variance indicated
a significant relationship between the TCDD and TEQ values and the colony
nested in year (r2=0.80, 0.74, respectively; p<0.0001).
Note that as the TCDD and TCDD-TEQ levels decreased in the most contaminated
colonies (Crofton and UBC), the degree of cerebral asymmetry correspondingly
decreased. This relationship is emphasized in the scattergrams plotting
TCDD against the width and angle difference measurements and TCDD-TEQ against
the depth difference measurement (Figure 5A,B,C, respectively).
As the TCDD (or TCDD-TEQ) levels increased in eggs taken from the same nest,
the likelihood and degree of observed asymmetry also increased. (Log TCDD
and log TCDD-TEQ are used for the X axes because, when the measurements
are regressed against the dose or log dose alone, the log dose correlation
coefficient is higher. The correlation coefficients (r2)
for each of these relationships are: width: 0.57 (TCDD), 0.42 (TCDD-TEQ);
angle: 0.61 (TCDD), 0.46 (TCDD-TEQ); and depth: 0.40 (TCDD), 0.44 (TCDD-TEQ).
Figure 3. Histograms
illustrating the mean values for the differences in width (A), angle
(B) and depth (C) of the heron brains analyzed by colony and
year. Of the colonies, N is Nicomekl, UBC is the colony near the University
of British Columbia, Cr is Crofton, Ch is Chilliwack, T is Tillicum. SEMs
and sample size for each measurement are listed in Table 1.
A |
B |
Figure 4. Histograms
illustrating the mean TCDD and TCDD-TEQ levels in each colony in each year
as determined in an egg taken from the same nest as each egg/hatchling used
in these measurements. SEMs and sample sizes are listed in Table 1. SEMs
of 0 indicate pooled samples were used for the measurements of those colonies
in those years.
Figure 5. Scattergrams
illustrating the relationship between TCDD (A, width; B, angle)
and TCDD-TEQ (C, depth).
The regression equations with the highest r2 value
that best describes the interactions of the factors believed to be affecting
the brain asymmetries are
width difference= 0.002 x TCDD+13.897
x brain wt/body wt-0.353
r2=0.61, p<0.0001
angle difference=0.459 x log TCDD-0.456
r2=0.69, p<0.0001;
depth difference=0.001 x TCDD-TEQ
+13.619 x brain wt/body wt+0.010
r2=0.49, p<0.0001.
Brain and Body Weights
As seen previously (9), the body weight and yolk-free body weight
decreased with increasing TCDD concentrations in the pair-matched egg (Figure
6).
Figure 6. Scattergram
depicting the relationship between yolk-free body weight and TCDD levels.
The correlation coefficient for this relationship is -0.34.
The regression equation for this relationship is
yolk-free body weight= -3.853 x log TCDD
+51.797
r2=0.19, p<0.001.
Brain weight and the brain somatic index (BSI, brain weight/body weight),
both tend to increase with increasing TCDD and TCDD-TEQ levels (Figure 7A,B;
p<0.0001 for log TCDD vs BSI). This was not clear from the 1988
results in which there was a significant decrease in brain weight in the
brains of herons from the Crofton colony (the colony in which there were
the highest contamination levels) compared to brains of herons from both
the Nicomekl (low contamination) and UBC (moderate contamination) colonies
(Figure 8). However, as the TCDD levels decreased in subsequent years, there
was a decrease in the brain weight and the brain somatic index. Unlike the
clear relationship between the BSI and log TCDD, the relationship between
brain weight and log TCDD is not so clear. Log TCDD only varies significantly
with brain weight (p<0.0001) when the colony-related factors are
also included as covariates in the analysis of (co)variance. One contribution
to the change in brain weight may be the change in body weight. In general,
when bodies decrease in size, the organs decrease correspondingly. However,
the brain weight has a tendency to increase with increasing TCDD, a factor
that decreases body weight. The majority of the variation in brain weight
is due to the colony-related factors (evaluated as colony nested within
year; r2=0.97; p<0.0001). When considering the
contaminant-related increase in the brain somatic index, the decrease in
body weight is also a significant factor (r2=0.85, p<0.0001).
Since body weight is one of two factors in the BSI, it was not included
in the regression equation to avoid a problem with collinearity.
Figure 7. Scattergrams
relating brain weight (A) and Brain somatic index (BSI, B)
to log TCDD levels. The correlation coefficients for these relationships
are 0.05 (brain weight) and 0.31 (BSI).
Figure 8. Histogram
of the mean brain somatic index in the heron brains averaged by colony and
year. SEMs and sample size for each mean are listed in Table 1.
The regression equation relating BSI to log TCDD and colony nested within
year ("colony by year") is
Brain somatic index=0.003248
x log TCDD-0.0000011
x (Colony by year)+0.02754
r2=0.49 p>0.0001
Discussion
We have shown that heron embryos exposed in ovo to a mixture of
PCDDs and PCDFs hatch with asymmetric brains. This effect has appeared not
only in comparison of colonies with different degrees of exposure in the
same year, but also in the same colony as the level of contamination decreased
over time. This phenomenon, which is easily quantifiable, could be used
as a morphometric biomarker for TCDD-related exposure.
The brain asymmetry also provides an indication of how the PCDDs and
PCDFs interact with the brain. The width and angle measurements, which are
only affected by the cerebral hemispheres, correlate best with the TCDD
(or log TCDD) levels, while the depth measurement (which is affected by
the steroid-sensitive anterior hypothalamus) correlates best with the TCDD-TEQ
levels. Thus, it is possible that different PCDD and PCDF congeners have
differing effects or different potencies in the individual brain regions
potentially related to the steroid sensitivity of the region.
In addition to the TCDD-related brain asymmetry, the BSI tended to increase
with increasing TCDD concentrations in the pair-matched egg. The corresponding
decrease in body weight probably affected this relationship. The BSI also
tended to vary among colonies, indicating that some other factor besides
simply TCDD levels might be influencing brain weight. These factors might
include genetic differences, gender (which was not determined, as the hatchling
gonads are undifferentiated), hormonal status, nutrition, or other unmeasured
contaminants.
As expected, the yolk-free body weight also decreased with increasing
TCDD and TEQ values. Hoffman et al. (12) first suggested that yolk-free
body weight could be used as a biomarker for the effects of PCBs on the
growth of heron embryos. They noted that yolk-free body weight decreases
with increasing PCB concentration, as noted in this dataset as well.
TCDD is arguably the most toxicologically potent of the group of related
halogenated aromatic hydrocarbons (HAHs), which includes PCDDs, PCDFs, and
PCBs. Most biomarkers of toxicity that are now in use to assess the effects
of TCDD (and related chemicals) on developmental processes evaluate teratogenic,
cellular, or biochemical changes in the palate and beak, kidney, liver,
spleen, bursa, thymus, thyroid, gonads, white blood cells, and blood hormone
and vitamin levels (estrogen, androgens, thyroid hormone [T3,T4],
and vitamin A) (13-17). Other previously noted effects include pericardial
and abdominal edema and (as mentioned above) decreased body weight and growth
(18,19). Several studies have noted that exposure to TCDD-related
chemicals is correlated with changes in behavior, especially reproductive
and parenting behaviors (16,20). As behavior is largely controlled
by the central nervous system, it is important to find and develop biomarkers
for central nervous system toxicity that may represent the anatomical correlates
of the behavioral changes. Such a nervous system biomarker will both validate
the behavioral changes and potentially provide an indication for where in
the nervous system the chemical is acting.
Recent studies by Seegal et al. (21) have indicated that HAH-correlated
changes in nigro-striatal dopamine levels are most potently induced by the
ortho-substituted, noncoplanar PCBs (2,2´-dichlorophenyl and
2,2´,4-trichlorophenyl). These PCBs are toxicologically dissimilar
to the meta and para (laterally)-substituted, coplanar analogs of TCDD when
evaluated using a number of different assays (1). In this study,
the forebrain asymmetry (width and angle measurements) correlates best with
the TCDD levels in the pair-matched egg. Yet the variation in the depth
measurement and its higher correlation with the TCDD-TEQ values may reflect
the contribution of other, nonlaterally substituted PCDDs and PCDFs. It
is important to recognize, however, that even though a number of PCDDs and
PCDFs are contributing to the determined TEQs (1), all PCDDs and
PCDFs are inherently coplanar. The response of the central nervous system
to the PCDDs, PCDFs, and PCBs may include more than one response potentially
mediated by a variety of the coplanar and noncoplanar halogen-substituted
congeners. It becomes therefore even more important to understand the multiplicity
of mechanisms by which the HAHs interact with the central nervous system.
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