HELGOI_~NDER MEERESUNTERSUCHUNGEN
Helgol~inder Meeresunters. 37, 493-504 (1984)
Alterations in patterns of excretion and other metabolic
functions in d e v e l o p i n g fish embryos e x p o s e d to
benzo(a)pyrene
R. M. K o c a n & M. L. L a n d o l t
University of Washington, School of Fisheries, WH-I O; Seattle, Washington 98195, USA
ABSTRACT: Steelhead trout (Salmo gairdneri) embryos were used as a model to study some of the
physiological and biochemical changes which occur following exposure to sublethal levels of
benzo(a)pyrene B(a)P, a k n o w n environmental contaminant. Embryos were e x p o s e d to B(a)P on day
1, 15, or 25 post fertilization, corresponding to the pre-blastula, eyed stage and late organogenesis
stage of development. These times were chosen to d e t e r m i n e w h e t h e r the age of the embryo at the
time of exposure influenced their response to this model compound. The c h a n g e s which were
observed were related to the age of the embryo at the time of exposure but not to the concentration
of the B(a)P to which they were exposed. It was found that the longer the embryo developed, the
more p e r m e a b l e the eggs became, taking up twice to 10 times as much B(a)P on day 25 as they did
on day 1. Embryos exposed later in d e v e l o p m e n t were also able to excrete the metabolites of B(a)P
through the egg m e m b r a n e 10 times more rapidly than their counterparts which were exposed early
during development. Following hatching, the larvae from those groups exposed later in development contained 2 to 5 times as much B(a)P b o u n d to or dissolved in the tissues than did those
exposed on day 1. Hatching time was also modified by the times of exposure, with those exposed on
days 15 and 25 hatching later and over a longer period of time than either the untreated or DMSO
controls. Although physical abnormalities were rare, there a p p e a r e d to be a consistent increase in
the level of teratogenesis in those groups which were exposed early during development.
INTRODUCTION
O u r i n a b i l i t y to d o c u m e n t a c c u r a t e l y t h e c a u s e of p o p u l a t i o n d e c l i n e s i n a q u a t i c
s p e c i e s is l a r g e l y t h e r e s u l t of t h e i n a c c e s s a b i l i t y of m o r i b u n d i n d i v i d u a l s . M a s s i v e ,
s u d d e n m o r t a l i t y is a n e x c e p t i o n i n w h i c h w e c a n f r e q u e n t l y d e t e r m i n e b o t h t h e c a u s e of
t h e d i e - o f f a s w e l l as t h e s o u r c e of t h e c a u s a t i v e a g e n t ( s ) . M o r e f r e q u e n t l y , h o w e v e r , a
species gradually declines in abundance and finally disappears entirely from an area in
w h i c h it o n c e f l o u r i s h e d . T h e r e is u s u a l l y n o s h o r t a g e of s p e c u l a t i o n to e x p l a i n t h e s e
d e c l i n e s , s u c h as o v e r e x p l o i t a t i o n , h a b i t a t d e g r a d a t i o n , c o m p e t i t i o n w i t h i n t r o d u c e d
s p e c i e s a n d r e c e n t l y e v e n t h e E1 Nifio. H a r d s c i e n t i f i c e v i d e n c e h o w e v e r , is u s u a l l y
l a c k i n g . T h e r e a s o n for t h i s is t h a t t h e c a u s e of s u c h i n s i d i o u s m o r t a l i t y is g r e a t l y
s e p a r a t e d i n t i m e f r o m t h e f i n a l r e c o g n i z a b l e e f f e c t - t h e l o s s of a s p e c i e s f r o m its
habitat.
Our interest in this s u b j e c t has c e n t r e d l a r g e l y on the n o n l e t h a l d a m a g e p r o d u c e d in
d e v e l o p i n g f i s h e m b r y o s as a r e s u l t of e x p o s u r e s to t o x i c a g e n t s d u r i n g v a r i o u s p e r i o d s of
their e m b r y o n i c d e v e l o p m e n t . F r o m t h e s e s t u d i e s w e h a v e s h o w n that fish cells in vitro
© Biologische Anstalt Helgoland, Hamburg
494
R . M . Kocan & M. L. Landolt
e x h i b i t c h r o m o s o m a l d a m a g e (Kocan et al., 1982), as well as d e v e l o p i n g mutations
(Kocan et al., 1981) m u c h as m a m m a l i a n cells do. T h e chromosome d a m a g e seen in vitro
has also b e e n verified i n vivo i n our laboratory b y Liguori (Ph.d. Thesis, 1984) as well as
b e i n g d e s c r i b e d u n d e r n a t u r a l conditions (Longwell & Hughes, 1980). K l i g e r m a n &
Bloom (1976) a n d K l i g e r m a n (1979) d e m o n s t r a t e d chromosomal d a m a g e of a different
type (SCE) a n d i n a different species, i n d i c a t i n g that such s u b l e t h a l DNA d a m a g e is not
uncommon.
In addition to physical defects observed in D N A , w e have also seen changes in the
capacity of trout embryos and fry to excrete xenobiotics, such as polycyclic aromatic
hydrocarbons, following embryonic exposure. Biochemical alterations in enzyme function have also been reported by several investigators working with embryonic fish of
different species (Binder & Stegeman, 1980, 1983).
All of these observations suggest that sublethal exposure of fish embryos to toxic
substances m a y result in their inability to successfully compete with normal individuals
at some later period in life due to persistent sublethal d a m a g e which occurred earlier. In
order to clarify some of the causes and relationships of these early biochemical alterations, w e examined the dynamics of embryo development and some of the critical
processes associated with early life, after the embryos were exposed to a k n o w n toxic
substance. The concentrations of toxin and methods of exposure were chosen to insure
experimental uniformity and consistent uptake by the eggs, and in no w a y were
intended to duplicate natural exposures.
MATERIALS AND M E T H O D S
Experimental embryos
S t e e l h e a d trout (Salmo gairdneri) e m b r y o s were o b t a i n e d from a single female
w h i c h had r e t u r n e d to the U n i v e r s i t y of W a s h i n g t o n e x p e r i m e n t a l hatchery to spawn.
F o l l o w i n g fertilization by a s i n g l e m a l e steelhead, the eggs were m a i n t a i n e d in lots of
100 i n n e t e g g c h a m b e r s s u s p e n d e d i n a flow-through system of d e c h l o r i n a t e d city water
at 10 °C. T e m p e r a t u r e , w a t e r flow a n d chlorine were m o n i t o r e d continuously.
Test compound
14C-benzo(a)pyrene (14C-B(a)P) (Sp. Act. 58.5 m C i / m M o l - Amersham) was diluted
with u n l a b e l l e d B(a)P to m a k e a stock solution of 10btg/~tl i n spectrophotometric grade
d i m e t h y l s u l p h o x i d e (DMSO). This solution was u s e d to m a k e final exposure concentrations of 2~tg a n d 20btg/ml of w a t e r (0.5% DMSO). The final activity of 14C-B(a)P was
0.23 t~Ci/tlg.
Exposure of eggs consisted of a s i n g l e 24-h p u l s e at one of the above concentrations
i n 0.5 ml of w a t e r p e r egg. I m m e d i a t e l y following exposure each group of eggs was
e x t e n s i v e l y w a s h e d in f l o w i n g w a t e r to r e m o v e a n y a d h e r i n g B(a)P. A s u b s a m p l e of 10
14C-B(a)P l a b e l l e d eggs was r e m o v e d following exposure to m e a s u r e the a m o u n t of B(a)P
w h i c h was t a k e n up. Radioactivity was d e t e r m i n e d by h o m o g e n i z i n g the entire egg a n d
larva i n a s c i n t i l l a t i o n vial, a d d i n g s c i n t i l l a t i o n cocktail a n d c o u n t i n g the entire a m o u n t
of activity i n the egg or larva on a Packard Tri Carb Liquid scintillation counter, Model
D e v e l o p i n g fish e m b r y o s e x p o s e d to b e n z o ( a ) p y r e n e
495
300. W h e n comparisons b e t w e e n eggs or larvae c o n t a i n i n g different a m o u n t s of p i g m e n t
were b e i n g made, q u e n c h i n g for each system was t a k e n into account, thus e l i m i n a t i n g
any possibility of error w h e n c o m p a r i n g different types of tissue.
Exposures
Three groups of 400 eggs each were exposed to u n l a b e l l e d B(a)P (Sigma, Gold
Lable) on day 1, day 15, a n d day 25 post fertilization. Controls consisted of 600 u n t r e a t e d
a n d 200 DI~SO treated eggs. The three exposure periods (early, m i d a n d late d e v e l o p ment) correspond to p r e - b l a s t u l a (day 1), eyed e m b r y o (day 15), a n d late o r g a n o g e n e s i s
(day 25), a n d serve to d e m o n s t r a t e w h e t h e r the stage of d e v e l o p m e n t of the e m b r y o
i n f l u e n c e s the outcome of exposure to B(a)P (Fig. 1). A p a r a l l e l set of 50 eggs was
exposed to 14C-B(a)P i n order to d e t e r m i n e the rate of u p t a k e of the test c o m p o u n d at
each exposure period.
1
1
yolk
~-hotching Jf- resor Pti on --i
i//
55
Days post fertilization
i
45
~
55
i
65
Fig. 1. Trout embryo development and benzo(a)pyrene exposure schedule. Following fertilization,
eggs were allowed to water harden for 24 h, after which they were exposed to B(a)P in water (0.5 %
DMSO) for 24 h. On days 15 and 25 post fertilization, two additional groups of eggs were similarly
exposed. Eggs were sampled following each exposure period and again just prior to hatching. After
hatching, sac fry (larva and yolk) were also collected for analysis and for collection of excreted
water soluble metabolites of B(a)P
Hatching
The effect of B(a)P on h a t c h i n g was d e t e r m i n e d b y r e c o r d i n g the n u m b e r of eggs
which hatched each day until all eggs were hatched. Each of the t r e a t m e n t groups a n d
controls consisted of four replicates of 100 eggs each. The four t r e a t m e n t groups were: (1)
u n t r e a t e d controls, (2) DMSO treated controls, (3) eggs exposed one day post fertilization
(pre-blastula), (4) eggs e x p o s e d 15 days post fertilization (eyed stage), a n d (5) eggs
exposed 25 days post fertilization (late organogenesis).
Metabolism/excretion
Excretion of B(a)P m e t a b o l i t e s w a s m e a s u r e d b y two methods. O n e m e t h o d determ i n e d the total radioactivity in each of 10 eggs s a m p l e d following e x p o s u r e a n d
w a s h i n g (day 2), a n o t h e r 10 eggs s a m p l e d just prior to h a t c h i n g (day 35), a set of 10 sac
fry s a m p l e d i m m e d i a t e l y after h a t c h i n g (day 37), a n d 10 f i n g e r l i n g s s a m p l e d following
total yolk resorption (day 70). By c o m p a r i n g the levels of 14C-B(a)P i n e a c h of these
groups we could calculate the a m o u n t lost (excreted) d u r i n g i n c u b a t i o n , the a m o u n t lost
496
R . M . Kocan & M. L. Landolt
at h a t c h i n g (perivitelline fluid) a n d the a m o u n t p r e s e n t i n the larva a n d yolk after
hatching. By d i s s e c t i n g the larva free from the yolk we could also d e t e r m i n e the a m o u n t
p r e s e n t i n the yolk a n d larva separately.
A second m e t h o d of d e t e r m i n i n g the a m o u n t of l a b e l l e d test c o m p o u n d excreted b y
the d e v e l o p i n g eggs was to place i n d i v i d u a l eggs i n small vials a n d cover t h e m with a
small a m o u n t of sterile water. By r e m o v i n g the entire v o l u m e of water three times per
w e e k a n d r e p l a c i n g it with fresh sterile water, we could d e t e r m i n e the a m o u n t of
l a b e l l e d c o m p o u n d lost into the w a t e r every three days. The a c c u m u l a t e d counts
t h r o u g h o u t i n c u b a t i o n w o u l d t h e n r e p r e s e n t the total a m o u n t of l a b e l l e d B(a)P lost from
the time of exposure u n t i l hatching. In this w a y it was also possible to m e a s u r e the
a m o u n t of radioactivity r e l e a s e d with the p e r i v i t e l l i n e fluids at the time of hatching.
This same t e c h n i q u e of i n c u b a t i n g i n a closed system was also u s e d to collect the
excreted m e t a b o l i t e s of sac fry d u r i n g the period of yolk resorption. D u r i n g yolk
resorption fry were kept i n a closed system w h e r e the w a t e r was collected three to four
times per w e e k a n d r e p l a c e d with fresh sterile water. This water c o n t a i n e d all of the
excreted m a t e r i a l from the d e v e l o p i n g larvae a n d was used to d e t e r m i n e if a n y differe n c e s i n m e t a b o l i c c a p a b i l i t y had r e s u l t e d from exposure of the eggs at different
d e v e l o p m e n t a l periods.
D u r i n g the collection period, w a t e r from each t r e a t m e n t group was p l a c e d into a
dark bottle, g a s s e d with n i t r o g e n a n d frozen at -- 20 °C u n t i l it was extracted. Extractions
consisted of a s i n g l e h e x a n e extraction (4 h e x a n e : 1 water) followed by centrifugation at
2000 r p m for 5 m i n to e n s u r e that the two layers were fully separated. Following the
h e x a n e extraction the w a t e r layer was a g a i n extracted twice with ethylacetate (4:1). The
h e x a n e extraction was i n t e n d e d to r e m o v e a n y n o n p o l a r m a t e r i a l which m i g h t have
b e e n p r e s e n t a n d the e t h y l a c e t a t e to r e m o v e a n y slightly polar m e t a b o l i t e s (Phase I
reactants).
A n a l i q u o t of each w a t e r s a m p l e was also i n c u b a t e d with [3-glucuronidase (1,000
units/ml) a n d s u l p h a t a s e (45 units/ml) for 60 m i n to d e c o n j u g a t e a n y sulphate or
g l u c u r o n i c acid c o n j u g a t e s (Phase II reactants) w h i c h m i g h t b e present. In this w a y we
were a b l e to d e t e r m i n e the relative a m o u n t of s o l u b l e u n c o n j u g a t e d a n d c o n j u g a t e d
m e t a b o l i t e s excreted b y the various t r e a t m e n t groups.
Teratogenesis
From the time of h a t c h i n g u n t i l the yolk was fully resorbed, the four groups of sac fry
were e x a m i n e d three times per w e e k u n t i l they were 60 days old. D u r i n g this period all
a n i m a l s w h i c h a p p e a r e d to b e p h y s i c a l l y or b e h a v i o u r a l l y a b n o r m a l were r e m o v e d a n d
fixed i n 10 % formalin for future classification.
RESULTS
U p t a k e of B ( a ) P
By u s i n g 14C-B(a)P it was p o s s i b l e to m e a s u r e the a m o u n t of B(a)P e n t e r i n g the trout
eggs at each exposure period as well as m o n i t o r for the c o n c e n t r a t i o n of B(a)P in the
exposure water, T a b l e 1 s u m m a r i z e s the c o n c e n t r a t i o n s of B(a)P found in the eggs
497
D e v e l o p i n g fish e m b r y o s e x p o s e d to b e n z o ( a ) p y r e n e
Table 1. Concentration of 14C-B(a)P in trout eggs following 24 h exposure
Exposure concentration (~g/ml)
N
2
20
10
10
Exposure day (post-fertilization)
1
24 ± 2"
83 ± 11
15
54 ± 10
729 ± 123
25
59 ± 10
664 ± 87
* Values represent nanograrns (ng) of B(a)P for entire egg (i.e. yolk, embryo, chorion and
perivitelline fluid ± S.D.)
i m m e d i a t e l y after the 24-h e x p o s u r e period. T h e s e v a l u e s w e r e u s e d to c a l c u l a t e the
p e r c e n t loss a n d r e t e n t i o n p r e s e n t e d l a t e r in this section u n d e r " E x c r e t i o n " . It is e v i d e n t
that the n e w l y fertilized e g g is not as p e r m e a b l e to B(a)P in t h e form w e u s e d for
exposure, as are the m o r e d e v e l o p e d eggs.
The actual m e a s u r e d c o n c e n t r a t i o n s of B(a)P in the w a t e r at the start of e x p o s u r e
w e r e 1.6-1.8 ~tg/ml for the 2 ~tg/ml e x p o s u r e water, and 16.1-19.8 ~ g / m l for the 20 ~tg/ml
exposure water. F o l l o w i n g the 24-h exposure, t h e s e c o n c e n t r a t i o n s w e r e r e d u c e d to
1.4-1.6~tg/ml a n d 12.8-15.1~tg/ml: a 12% a n d 2 0 % loss r e s p e c t i v e l y .
Excretion
A distinct d i f f e r e n c e w a s o b s e r v e d in the rate of e x c r e t i o n of w a t e r s o l u b l e m e t a b o lites of B(a)P from e m b r y o s e x p o s e d e a r l y vs those e x p o s e d late in d e v e l o p m e n t (Fig. 2).
T h o s e e x p o s e d prior to b l a s t u l a f o r m a t i o n e x c r e t e only 2 to 3 % of t h e total a m o u n t of
B(a)P w h i c h was in t h e e g g f o l l o w i n g e x p o s u r e , w h i l e those e x p o s e d late in i n c u b a t i o n
lost as m u c h as 20 % of the total a m o u n t o r i g i n a l l y t a k e n up d u r i n g e x p o s u r e . T h e g r o u p
e x p o s e d 15 days f o l l o w i n g fertilization (eyed stage), e x h i b i t e d an e x c r e t i o n rate m i d w a y
b e t w e e n the early and late e x p o s u r e groups. T h e loss of l a b e l l e d B(a)P w h i c h o c c u r r e d in
the day 1 e x p o s u r e g r o u p took p l a c e d u r i n g the entire i n c u b a t i o n p e r i o d (30+ days),
w h i l e those e x p o s e d late in d e v e l o p m e n t h a d only 10 to 15 days to e x c r e t e the B(a)P prior
to hatching. C o n s i d e r i n g this, t h e p e r d a y loss of B(a)P w a s e v e n m o r e s p e c t a c u l a r in the
late e x p o s u r e group. This e x p e r i m e n t w a s r e p e a t e d w i t h t h r e e s e p a r a t e e g g lots from
three different females, a n d the results w e r e e s s e n t i a l l y i d e n t i c a l e a c h time. Likewise,
the loss of s o l u b l e B(a)P from the e g g w a s the s a m e w h e t h e r m e a s u r e d d i r e c t l y by
s a m p l i n g the i n c u b a t i o n w a t e r or by m e a s u r i n g the d i f f e r e n c e in r a d i o a c t i v i t y i n s i d e the
e g g from the t i m e of e x p o s u r e until just prior to h a t c h i n g . In a s u b s e q u e n t e x p e r i m e n t a
s i n g l e b a t c h of e g g s w a s split into two e q u a l lots w i t h only o n e b e i n g fertilized. Both
groups w e r e e x p o s e d to 14C-B(a)P a n d a l l o w e d to d e v e l o p for 20 days, d u r i n g w h i c h e g g s
w e r e s a m p l e d t w i c e p e r w e e k . At the e n d of t h e 20 days the r a d i a t i o n l e v e l s in the e g g s
from both groups w a s c o m p a r e d a n d found to b e identical. This i n d i c a t e d that t h e s m a l l
a m o u n t of c o m p o u n d lost from the e g g s e x p o s e d e a r l y in d e v e l o p m e n t w a s not the result
of e m b r y o m e t a b o l i s m a n d excretion, since e v e n e g g s w h i c h c o n t a i n e d no e m b r y o lost
the s a m e small a m o u n t d u r i n g the s a m e p e r i o d (i.e. < 2 %).
T h e a m o u n t of B(a)P in the p e r i v i t e l l i n e fluid at t h e t i m e of h a t c h i n g also a p p e a r e d
different in the e a r l y a n d later e x p o s u r e groups. T h o s e e x p o s e d later d u r i n g d e v e l o p -
498
R . M . Kocan & M, L. Landolt
fluid
DAY 1 EXPOSURE
15%
~ p e r i v i t e i l i n e
(pre-blostulo) ~ socf r y ~
excreted2%
.p.erivitelline fluid 13%
DAY
EXPOSURE [[
DAY15
15EXPOSURE
(eyed embryo)
i f / / ~ " ~ @ . . ~ e x c r e t e d 9%
perivitetline fluid 21%
~ e x c r e t e d
18%
DAY25 EXPOSURE ~ x ' x,~,,,....,...............
~ ~ i ,
(orgon0genesis)
Fig, 2. Distribution of benzo(a)pyrene in trout eggs from exposure to hatching, B(a)P was identified
in three components of the egg/embryo complex: (1) excretions out of the egg during embryo
development up to the time of hatching; (2) losses along with perivitelline fluid at the time of
hatching; (3) a component bound to or dissolved in the embryo/yolk, Embryos exposed later in
development excreted considerably more of their B(a)P content than did those exposed early.
Conversely, the amount present in the embryo/yolk was about 25 % greater in the early exposure
group than in the late exposure group. Embryos exposed midway through development exhibited
intermediate values in all three categories
m e n t a g a i n r e l e a s e d more l a b e l l e d c o m p o u n d at the time of h a t c h i n g t h a n did those
exposed early,
Sac fry c o n t i n u e d to excrete l a b e l l e d s o l u b l e c o m p o u n d s into the water throughout
yolk resorption. O n c e the yolk was totally resorbed a n d the a b d o m i n a l suture line closed
(30 to 45 days post hatching), no l a b e l could be d e t e c t e d in the water in w h i c h the fry
were kept.
Hatching
Both day 15 a n d day 25 exposures of trout eggs to B(a)P r e s u l t e d i n differences in the
h a t c h i n g d y n a m i c s w h e n c o m p a r e d to the u n t r e a t e d a n d DMSO controls, T a b l e 2
s u m m a r i z e s the h a t c h i n g data from the 6 t r e a t m e n t groups a n d the controls. Eggs which
were exposed early in d e v e l o p m e n t e x h i b i t e d no statistical difference in h a t c h i n g times
w h e n c o m p a r e d to the controls. Those eggs e x p o s e d later i n d e v e l o p m e n t b e g a n
h a t c h i n g later t h a n either the early exposure group or the controls, a n d took a b o u t 3 days
l o n g e r to hatch t h a n either of the other groups. No difference in h a t c h i n g success was
D e v e l o p i n g f i s h e m b r y o s e x p o s e d to b e n z o ( a ) p y r e n e
499
Table 2. M e a n hatching day of s t e e l h e a d trout eggs treated with benzo(a)pyrene on days 1, 15 and
25 post fertilization
Exposure day
(postfertilization)
Exposure
concentration
(ptg/ml)
p<*
M e a n day to
50 % hatch
( ± S,D.)
P <: *
M e a n day to
90 % hatch
( ± S.D.)
39.8 ± 1,47
-
43.0 ± 2.37
(0.5 %)
39.4 -- 1.14
NS
41.6 + 0.89
NS
2
20
39.8 ± 2.98
39.0 ± 2.16
NS
NS
43.3 ± 4.72
42.5 ± 3.11
NS
NS
15
2
20
43.0 ± 1.41
43.0 ± 2.31
.01
.01
47.5 ± 2.38
47.3 ± 3.30
.01
.05
25
2
20
43.5 ± 3.32
41.8 ± 0.96
.05
.05
47.3 ± 3.30
45.5 ± 1.73
.05
.05
Untreated
control
DMSO
control
* ANOVA: 100 eggs/replicate; controls = 6 replicates; treated = 4 replicates; DMSO = 2 replicates; NS = not significant (P >0.25)
n o t e d as a r e s u l t of e x p o s u r e to B(a)P, e v e n t h o u g h s o m e e g g s w e r e e x p o s e d to
c o n c e n t r a t i o n s a s h i g h a s 20 btg/ml, a c o n c e n t r a t i o n a b o u t 1000 t i m e s g r e a t e r t h a n w o u l d
n a t u r a l l y b e e n c o u n t e r e d . B o t h B(a)P c o n c e n t r a t i o n s p r o d u c e d t h e s a m e h a t c h i n g p a t t e r n
in their r e s p e c t i v e t r e a t m e n t group.
Yolk and larval tissue
C o m p a r i s o n of t h e a m o u n t of l a b e l l e d c o m p o u n d p r e s e n t i n y o l k a n d l a r v a l t i s s u e at
t h e t i m e of h a t c h i n g s h o w e d t h a t t h e y o l k c o n t a i n e d t h e m a j o r i t y of t h e o r i g i n a l e g g
c o n t e n t of l a b e l l e d B(a)P. T a b l e 3 s u m m a r i z e s t h e p e r c e n t d i s t r i b u t i o n of r a d i o a c t i v i t y
Table 3. Concentration of B(a)P in embryo and yolk immediately post hatching
Exposure
concentration
(~g/ml)
N
Exposure day (post-fertilization)
1
*
2
Embryo
Yolk
5
5
1.3 _±_0.2" (6%)**
19 _ 2,1 (94%)
20
Embryo
Yolk
5
5
5.0 ± 1.3(10%)
44 ± 3.9 (90%)
15
3,7 ± 1.4 (9%)
38 +_ 9.9(91%)
18
183
± 3.2 (9%)
± 33 (91%)
25
5.7 +
31 ±
26
93
3.0(16%)
2.5(84%)
-- 4.5(22%)
- 30 (78%)
* Values represent nanograms (ng) of B(a)P bound to or dissolved in embryonic tissues or yolk
( ± S,D,}
Values in ( ) represent % of B(a)P in larvae or yolk relative to the total amount of B(a)P in
larva + yolk (sac fry) at hatching
*
500
R . M . Kocan & M. L. Landolt
levels detected i n yolk a n d larval tissue at the time of hatching. It is a p p a r e n t from these
data that those e m b r y o s w h i c h were exposed earlier d u r i n g i n c u b a t i o n a n d excreted less
B(a)P t h a n did those exposed later, r e t a i n e d the extra l a b e l l e d m a t e r i a l in the yolk (90 to
94%) as c o m p a r e d to those exposed on day 25 w h i c h r e t a i n e d m u c h less (78 to 84%).
From this it is a p p a r e n t that the larvae w h i c h were exposed early a c c u m u l a t e more B(a)P
i n their tissues at the time of h a t c h i n g t h a n those exposed later in d e v e l o p m e n t . By the
time the yolk h a d b e e n fully resorbed the fry from each exposure group c o n t a i n e d the
same level of b o u n d 14C, regardless of the time of their exposure.
Metabolism/excretion
T a b l e 4 s u m m a r i z e s the results of o r g a n i c extractions of waters i n w h i c h sac fry from
the various exposure groups were h e l d u n t i l they h a d fully resorbed their yolk material.
It is a p p a r e n t that both slightly polar a n d c o n j u g a t e d forms of B(a)P metabolites were
excreted into the water. The large a m o u n t of l a b e l l e d m a t e r i a l w h i c h r e m a i n e d in the
Table 4. Percent water soluble 14C-B(a)P excreted by sac fry
Extraction procedure*
Exposure day (post-fertilization)
1
Hexane (4 : 1)
Ethylacetate (4 : 1)
Ethylacetate (~-Glucuronidase/sulphatase)
90
71
52
15
93
63
43
25
97
55
36
* Extractions done on water collected daily from 10 sac fry from each treatment group over a
5-day period
water following o r g a n i c extraction a n d ~ - g l u c u r o n i d a s e / s u l p h a t a s e d e c o n j u g a t i o n m a y
r e p r e s e n t g l u t a t h i o n e c o n j u g a t e s of B(a)P metabolites.
There a p p e a r s to b e a r e l a t i o n s h i p b e t w e e n the time of exposure d u r i n g developm e n t a n d the a m o u n t of s o l u b l e metabolites s u b s e q u e n t l y excreted by the larva d u r i n g
yolk resorption, with those exposed later excreting larger q u a n t i t i e s of soluble a n d
c o n j u g a t e d material. By the time the yolk is fully resorbed, however, all of these
differences disappear. Both exposure groups (2 ~g a n d 20 ~g) e x h i b i t e d the same patterns
of solubility.
Teratogenesis
A m o n g the control fish ( u n e x p o s e d a n d DMSO treated), less t h a n 1% of the 600
a n i m a l s e x h i b i t e d a n y type of physical defect b y the time they had totally resorbed their
yolk. T h e t r e a t e d groups, however, all s h o w e d i n c r e a s e d physical defects by this time
with the majority a p p e a r i n g in those a n i m a l s treated early d u r i n g their d e v e l o p m e n t .
T a b l e 5 s u m m a r i z e s the data from the t e r a t o g e n e s i s evaluation.
The majority of defects r e p r e s e n t e d in this e x p e r i m e n t were ocular abnormalities,
cephalic deformity, s p i n a l deformity a n d fin r e d u c t i o n s or e l i m i n a t i o n s .
D e v e l o p i n g fish e m b r y o s e x p o s e d to b e n z o ( a ) p y r e n e
501
Table 5. Teratogenic effects on fish embryos following exposure to benzo(a)pyrene.
B(a)P
concentration
No. exposed
Post-fertilization
Exposure time
% abnormal at
hatching
2 ~g/ml
2 ~g/ml
100
100
day 1
day 25
6
1
2 ~tg/ml
2 ~g/ml
230
340
day 1
day 15
5
3
20 ~tg/ml
20 ~g/ml
20 ~g/ml
290
280
280
day 1
day 15
day 25
13
I1
4
normal/DMSO
normal/DMSO
300
300
day I
day 25
< 1
< 1
Day 1 = pre-blastula; day 15 ~ eyed stage (1st eye pigment); day 25 = late organogenesis; day
35 = hatching
DISCUSSION
U n l i k e infectious diseases, g e n e t i c a n d m e t a b o l i c d i s e a s e s are u s u a l l y d e t e c t a b l e
only w h e n c o m p a r a t i v e m e a s u r e m e n t s c a n b e m a d e from a s i n g l e i n d i v i d u a l , a n d a w e l l
e s t a b l i s h e d n o r m a l b a s e l i n e for those m e a s u r e m e n t s is a v a i l a b l e for c o m p a r i s o n .
T h e data w e h a v e p r e s e n t e d h e r e d e m o n s t r a t e that d r a m a t i c n o n l e t h a l d e v i a t i o n s
from the n o r m c a n occur in e m b r y o s f o l l o w i n g e x p o s u r e to a k n o w n e n v i r o n m e n t a l
contaminant. Since no s i g n i f i c a n t m o r t a l i t y o c c u r r e d in t h e test animals, a n d p r e c i s e
m e a s u r e m e n t s w e r e n e e d e d on l a r g e n u m b e r s of i n d i v i d u a l s w i t h s i m i l a r g e n e t i c
b a c k g r o u n d , it is u n l i k e l y that t h e s e s a m e c h a n g e s w o u l d h a v e b e e n d e t e c t a b l e u n d e r
natural conditions. It is not c l e a r w h a t effect e a c h of t h e a l t e r a t i o n s w e o b s e r v e d w o u l d
h a v e on the survival a n d r e p r o d u c t i v e p o t e n t i a l of the e x p o s e d p o p u l a t i o n . Based on
w h a t is k n o w n of some of the e n z y m e systems w h i c h w e r e altered, t h e r e p r e s u m a b l y
could b e d r a m a t i c effects at s o m e later p e r i o d in t h e o r g a n i s m ' s d e v e l o p m e n t .
It has b e e n w e l l e s t a b l i s h e d that trout as w e l l as o t h e r fish s p e c i e s h a v e a c t i v e m i x e d
function o x y g e n a s e (MFO) a n d aryl h y d r o c a r b o n h y d r o x y l a s e (AHH) systems (Bend &
J a m e s , 1978; I w a o k a et al., 1979; P e d e r s o n et al., 1976), a l t h o u g h the l e v e l of activity
varies by species. This s u b j e c t is e x t e n s i v e l y r e v i e w e d by S t e g e m a n (1981). S i n c e t h e s e
e n z y m e s function both to e l i m i n a t e c e r t a i n e n v i r o n m e n t a l c o n t a m i n a n t s as w e l l as
e n d o g e n o u s steroids, t h e i r a l t e r a t i o n c o u l d p o s e serious p r o b l e m s to t h e o r g a n i s m . T h e
mechanism(s) u n d e r l y i n g t h e a l t e r e d e x c r e t o r y p a t t e r n s w e h a v e o b s e r v e d h a v e not b e e n
w o r k e d out for fish, a l t h o u g h t h e y h a v e b e e n e x a m i n e d in s e v e r a l m a m m a l i a n systems
(Lucier, 1981). It is not u n l i k e l y that t h e r e are s i m i l a r c o u n t e r p a r t s in fish. O n e possibility
is that e x p o s u r e of the e m b r y o in the p r e - b l a s t u l a s t a g e results in an i n h i b i t i o n or
r e p r e s s i o n of the M F O or c o n j u g a t i o n systems, t h e r e b y r e d u c i n g t h e c a p a c i t y of t h e
e m b r y o a n d fry to q u i c k l y m e t a b o l i z e a n d e x c r e t e m a n y xenobiotics. A l t e r n a t i v e l y , t h e
p r e s e n c e of a functional e m b r y o w i t h most of its organs a n d e n z y m e systems intact at the
time of e x p o s u r e (organogenesis) could result in the i n d u c t i o n of t h e s e systems (Binder &
502
R . M . Kocan & M. L. Landolt
S t e g e m a n , 1983), t h e r e b y i n c r e a s i n g the capacity of the o r g a n i s m to excrete the foreign
c o m p o u n d . Those e m b r y o s w h i c h were exposed as a p r e - b l a s t u l a w o u l d e n c o u n t e r B(a)P
o n l y via the yolk, w h i c h is b e i n g resorbed, a n d p o s s i b l y not r e s p o n d the same as those
w h i c h r e c e i v e d total b o d y exposure. A n o t h e r possibility is that different responses result
from early a n d late exposures, thus c a u s i n g each to r e s p o n d differently t h a n w o u l d a n
u n e x p o s e d embryo. W h i c h e v e r the case, if these differences i n m e t a b o l i c processing a n d
excretion of B(a)P persist into later life, they could alter the o r g a n i s m ' s ability to deal
with s u b s e q u e n t exposure to similar c o m p o u n d s a n d its ability to process e n d o g e n o u s
c o m p o u n d s such as steroids.
As we have p o i n t e d out, the age of a n e m b r y o at the time of exposure to a xenobiotic
i n f l u e n c e s the u l t i m a t e capacity of the later larva to excrete that c o m p o u n d d u r i n g yolk
resorption. Those exposed early excrete the c o m p o u n d more slowly t h a n those exposed
later i n d e v e l o p m e n t . W h e t h e r rapid e l i m i n a t i o n of xenobiotics is desirable or not
d e p e n d s on w h e t h e r the r e d u c t i o n in b o d y b u r d e n of the chemical outweighs the rapid
formation of activated i n t e r m e d i a t e c o m p o u n d s w h i c h have the capacity to b i n d to
p r o t e i n a n d n u c l e i c acid a n d t h e r e b y to alter e n z y m e function, DNA stability, a n d
initiate neoplasia.
S i n n h u b e r & Wales (1974) a n d Wales et al. (1978) observed that the e m b r y o ' s age at
the time of exposure to aflatoxin i n f l u e n c e d the d e v e l o p m e n t of tumors later i n life. This
could b e e x p l a i n e d b y different rates of m e t a b o l i s m a n d excretion by the different ages
of the e m b r y o s at the time of exposure.
It is clear that differences in b o d y b u r d e n , excretory rate a n d the form of the
c o n j u g a t e d c o m p o u n d s e x t e n d s b e y o n d h a t c h i n g a n d t h r o u g h yolk resorption. It has yet
to be d e m o n s t r a t e d , however, w h e t h e r these differences persist w h e n the o r g a n i s m is
exposed to similar c o m p o u n d s as it matures, or w h e t h e r these e n z y m a t i c alterations
u l t i m a t e l y affect the i n d i v i d u a l t h r o u g h b e h a v i o u r a l modification or h o r m o n a l imbal a n c e s (Lorz et al., 1979).
T h e c h a n g e s we o b s e r v e d i n h a t c h i n g d y n a m i c s m a y l i k e w i s e b e related to alterations i n e n z y m e systems w h i c h partially control hatching. It has b e e n well established
(Blaxter, 1969) that h a t c h i n g e n z y m e s are r e s p o n s i b l e for the onset of h a t c h i n g a n d egg
m e m b r a n e disruption. It is also k n o w n that a variety of e n v i r o n m e n t a l factors, i n c l u d i n g
pollution, can alter these e n z y m e s a n d affect the h a t c h i n g d y n a m i c s of a species.
Therefore, if polycyclic aromatics such as B(a)P c a n alter h a t c h i n g g l a n d enzymes, it
could e x p l a i n the c h a n g e s w e observed.
T e r a t o g e n i c a b n o r m a l i t i e s are r e l a t i v e l y rare i n n o n - i n b r e d trout, b u t a p p e a r to
i n c r e a s e as a result of exposure to B(a)P. T h e more o b v i o u s t e r a t o g e n i c effects which we
o b s e r v e d i n the treated groups, w o u l d p r o b a b l y l e a d to the death of the affected
i n d i v i d u a l s i n their n a t u r a l h a b i t a t t h r o u g h starvation a n d / o r predation. It is likely that a
n u m b e r of i n d i v i d u a l s with only m i n o r defects escape detection, b u t w o u l d u l t i m a t e l y be
lost from the p o p u l a t i o n d u e to b e h a v i o u r a l a n o m a l i e s a n d associated physical defects.
As Laale points out (1981), the d e v e l o p i n g fish e m b r y o is a sensitive o r g a n i s m which
r e s p o n d s to c h a n g e s i n its e n v i r o n m e n t , i n c l u d i n g c h e m i c a l pollution. Skeletal a n d
c e p h a l i c a b n o r m a l i t i e s of n e w l y h a t c h e d fish, w h i c h w e e n c o u n t e r e d most frequently,
are b e l i e v e d b y some to result from g e n e t i c alterations (SchrSder, 1969, 1973; a n d
Nelson, 1977), w h i c h c a n result from exposure to B(a)P a n d other polycyclic aromatic
hydrocarbons. Hose et al. (1981, 1982) showed that d e v e l o p i n g flatfish eggs exposed to
D e v e l o p i n g f i s h e m b r y o s e x p o s e d to b e n z o ( a ) p y r e n e
503
B(a)P o f t e n s u f f e r e d f r o m d e v e l o p m e n t a l a b n o r m a l i t i e s a s d i d t h o s e e m b r y o s p r o d u c e d
b y f e m a l e s w h i c h h a d b e e n e x p o s e d to B(a)P p r i o r to e g g l a y i n g .
Although infectious diseases are a significant factor influencing the health and
s u r v i v a l of a q u a t i c o r g a n i s m s , t h e r e is a m o r e i n s i d i o u s f o r m of d i s e a s e w h i c h r e s u l t s
f r o m e x p o s u r e to s u b l e t h a l t o x i c s u b s t a n c e s i n t h e e n v i r o n m e n t . T h o s e o r g a n i s m s w h i c h
a r e e x p o s e d , a n y t i m e f r o m f e r t i l i z a t i o n to s e x u a l m a t u r i t y , r u n t h e r i s k of b e c o m i n g
g e n e t i c a l l y d a m a g e d , a n d t h e r e b y b e i n g l e s s fit to r e s p o n d to o t h e r f o r m s of e n v i r o n m e n tal i n s u l t s , a n d m o r e s e r i o u s l y , h a v i n g a r e d u c e d c a p a c i t y to p r o d u c e n o r m a l h e a l t h y
o f f s p r i n g . E f f e c t s s u c h a s i n d u c t i o n a n d / o r s u p p r e s s i o n of e x i s t i n g e n z y m e s y s t e m s
d u r i n g e m b r y o n i c d e v e l o p m e n t a l s o p o s e t h e t h r e a t of c o m p r o m i s i n g a n i n d i v i d u a l ' s
a b i l i t y to c o p e w i t h l a t e r e x p o s u r e to e n v i r o n m e n t a l c o n t a m i n a n t s .
Acknowledgements: This work was supported in part by grants from the NIEHS (ES-02190-02), EPA
(R-810057-1), NOAA (OMPA-NA 80 RAD 00053) and NOAA (MESA-04-78-B01-13). The authors
also acknowledge the assistance of K. Sabo, V. Liguori and L. Ginno.
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