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IS J-llct
H isto c h e m ic a l Jo u rn a l 1 4 , 7 1 3 - 7 1 8 ( 1 9 8 2 )
Use of microstereology and quantitative
cytochemistry to determ ine the effects of crude
oil-derived aromatic hydrocarbons on lysosomal
structure and function in a marine bivalve mollusc,
M y t i l u s edulis
M I C H A E L N. M O O R E an d K. R O B E R T C L A R K E
N atural Environm ent Research Council, In stitu te for M arine Environm ental Research, Prospect Place, The Hoe,
P lym outh P L I 3DH, Devon, U.K.
R eceiv ed 29 S e p te m b e r 198 1 a n d in re v ise d fo rm 7 D e c e m b e r 1981
Summary
T h e m a rin e bivalve m o llu sc, M ytilu s edulis (b lu e m u sse l), is a n o te d a c c u m u la to r o f m a n y e n v ir o n
m e n ta l p o llu ta n ts a n d is in c re a sin g ly u s e d fo r th e ch e m ic a l a n d biolo g ical a s s e s s m e n t o f e n v ir o n m e n
ta l im p a c t. T h e toxic effe cts o f c ru d e o il-d e riv e d a ro m a tic h y d ro c a rb o n s (30 p g /l to ta l h y d ro c a rb o n s )
o n th e ly s o s o m a l-v a c u o la r sv s te m o f th e d ig e stiv e cells h av e b e e n in v e s tig a te d in c ry o s ta t se c tio n s o f
h e x a n e -fro z e n d ig estiv e g la n d s . E x p o su re to aro m atic h y d ro c a rb o n s re d u c e d th e cy to ch e m ically
d e te r m in e d laten c y o f ly so so m a l ß -N -a c e ty lh e x o sa m in id a se ; ly so so m a l v o lu m e d e n s ity a n d su rface
d e n s ity in c re a se d w h ile th e n u m e ric a l d e n s ity d e c re a s e d . E x p e rim e n ta l e x p o su re re s u lte d in th e
fo rm a tio n o f v e ry large ly s o so m e s w h ic h a re b e lie v e d to be larg ely a u to p h a g ic in fu n c tio n a n d th e s e
re s u lts in d ic a te a sig n ific a n t stru c tu ra l a n d fu n c tio n a l d is tu rb a n c e o f d ig e stiv e cell ly s o so m e s in
re s p o n s e to h y d ro c a rb o n s.
Introduction
T h e m a r i n e b iv a lv e m o l l u s c , M y t il u s e d u lis ( b l u e m u s s e l ) , is a n o t e d a c c u m u l a t o r o f m a n y
e n v i r o n m e n t a l p o l l u t a n t s a n d is i n c r e a s i n g l y u s e d in t h e c h e m i c a l a n d b io lo g ic a l a s s e s s
m e n t o f e n v i r o n m e n t a l i m p a c t ( B a y n e , 1 9 7 6 ) . T h e d ig e s ti v e c e lls o f t h e m u s s e l h a v e a
h i g h l y d e v e l o p e d l y s o s o m a l - v a c u o l a r s y s t e m w i t h la r g e s e c o n d a r y ly s o s o m e s w h i c h a re
i n v o l v e d in t h e i n t r a c e l l u l a r d i g e s t i o n o f i n g e s t e d f o o d ( S u m n e r , 1 9 6 9 ; O w e n , 1 9 7 2 ;
M o o r e , 1 9 7 6 , 1 9 8 0 a ). T h e s e c e lls a r e o f v it a l i m p o r t a n c e to th e 'n o r m a l ' f u n c t i o n i n g o f t h e
a n im a l. P r e v i o u s i n v e s t i g a t i o n s h a v e s h o w n t h a t t h e l y s o s o m a l - v a c u o l a r s y s t e m i n th e
d i g e s t i v e c e lls o f th e d i g e s t i v e d i v e r t i c u l a is h i g h l y r e s p o n s i v e to e n v i r o n m e n t a l f a c to r s
( M o o r e , 1 9 8 0 a ). T h is r e s p o n s i v e n e s s o f t h e l y s o s o m a l s y s t e m h a s i n v o l v e d c h a n g e s in
0 0 1 8 -2 2 1 4 /8 2 /0 5 0 7 1 3 -0 6 S 0 3 .2 2 /0
© 198 2 C h a p m a n a n d H aii Ltd.
714
M O O R E and CLARKE
th e c y to c h e m ic a l ly d e t e r m i n e d la t e n c y ( l a t e n t a c ti v it y = t o t a l a c ti v it y -
f r e e a c tiv ity ) o f
/L N -a c e ty lh e x o s a m in id a s e , ß - g lu c u ro n id a s e a n d a r y ls u lp h a ta s e (M o o re , 1 9 7 6 , 1 9 8 0 b ;
M o o r e e t a l., 1 9 7 8 a , b , 1 9 8 0 a ; B a y n e e t a l., 1 9 8 1 ) a n d h a s b e e n u s e d to d e v e l o p a n in d e x
o f c e l l u l a r ( ly s o s o m a l) r e s p o n s e to e n v i r o n m e n t a l s t r e s s o r s (B a y n e et a l., 1 9 7 6 ; M o o r e
1 9 8 0 a ) . L a te n c y o f /L N - a c e t y l h e x o s a m i n i d a s e d e t e r m i n e d c y to c h e m ic a l ly in s e c t io n s
u s i n g n a p h t h o l A S -B I N - a c e ty l - / ? - D - g lu c o s a m in i d e h a s a ls o s h o w n g o o d a g r e e m e n t
w i t h la t e n c y d e t e r m i n e d in c e ll- f r e e l y s o s o m a l p r e p a r a t i o n s f r o m t h e d i g e s t i v e g la n d
u s i n g p - n i t r o p h e n y l - / V - a c e t y l - ß - D - g l u c o s a m i n i d e ( B a y n e e t a l., 1 9 8 1 ).
A d d itio n a lly , s tru c tu r a l a lte ra tio n s h a v e
b e e n o b s e r v e d in t h e ly s o s o m e s o f th e
d i g e s t i v e c e lls f o l lo w in g a v a r i e t y o f e x p e r i m e n t a l t r e a t m e n t s i n c l u d i n g s e x s t e r o i d s
( M o o r e e t a l ., 1 9 7 8 a ) , a n t h r a c e n e ( M o o r e e t a l., 1 9 7 8 b ) , t e m p e r a t u r e , s t a r v a t i o n a n d
s a l i n i t y (B a y n e e t a l., 1 9 7 8 ). T h e s e a l t e r a t i o n s , h o w e v e r ,
w e r e n o t d e s c r i b e d in a
q u a n tita tiv e m a n n e r.
T h e p r e s e n t i n v e s t i g a t i o n w a s d e s i g n e d to e x a m i n e t h e to x ic e f f e c ts o f t h e a r o m a ti c
h y d r o c a r b o n r ic h w a t e r - a c c o m m o d a t e d f r a c ti o n (W A F ) o f N o r t h S e a ( A u k F ie ld ) c r u d e
o il o n th e l a t e n c y o f l y s o s o m a l / L N - a c e t y l h e x o s a m i n i d a s e a n d l y s o s o m a l s t r u c t u r a l
p a r a m e t e r s i n t h e d ig e s ti v e c e lls .
Materials and methods
T h e c o n c e n tra tio n o f to tal h y d ro c a rb o n s in th e e x p e rim e n ta l s e a w a te r w a s 30 n g/1 a n d sa m p le s w e re
ta k e n a fte r 3 4 a n d 103 d a y s o f e x p o su re to W A F . C o n tro l s a m p le s w e re also ta k e n a t th e s e tim es.
D e ta ils o f p re p a r a tio n a n d d o s in g o f W A F h a v e b e e n d e s c rib e d p re v io u sly (M o o re et a l., 1980b).
T h e la te n c y o f ly so so m a l ß -N - a c e ty lh e x o sa m in id ase w a s d e te r m in e d in 10 ^ m c ry o s ta t se c tio n s of
h e x a n e c h ille d ( - 7 0 ° C) d ig e stiv e g la n d slices as d e s c rib e d b y M o o re (1976). T h is in v o lv e d p re
tr e a tm e n t o f th e se c tio n s in 0.1 M citra te b u ffer, p H 4 .5 , c o n ta in in g 2.5% N aC l a t 37° C a t in te rv a ls
fro m 2 to 25 m in in o rd e r to a ctiv ate la te n t e n z y m e a ctiv ity . T h is w a s fo llo w ed b y in c u b a tio n (20 m in)
fo r /1 -N -acety lh e x o sam in id a se u sin g n a p h th o l A S -B I-N -acety l-/?-D -g lu co sam in id e (0 .4 m g /m l) d is
so lv e d in 2 -m e th o x y e th a n o i a t p H 4 .5 a n d 37° C in 0.1 M c itra te b u ffe r c o n ta in in g 2.5% N aC l a n d 7%
p o ly p e p tid e (P o ly ep P 5 1 1 5 ) as a s ta b iliz e r (B iten sk y et a l., 1 9 7 3 ; M oo re, 1976). T h e se c tio n s w e re
rin s e d in 3% salin e a n d p o st-c o u p le d u sin g F a st V io let B (1 m g /m l) in 0.1 M p h o s p h a te , p H 7.4.
S ta in in g in te n s ity w a s m e a s u re d in th e d ig e stiv e cells u s in g a V ickers M 85 s c a n n in g m ic ro d e n s i
to m e te r a t 560 n m a s d e sc rib e d b y M o o re et al. (1 9 7 8 a). R elativ e a b s o rb a n c e m e a s u re d a fte r 2 m in
p r e - tr e a tm e n t w as ta k e n as free a ctiv ity a s th e re is o fte n d iffu sib le a ctiv ity in se c tio n s s ta in e d w ith o u t
a n y p re - tre a tm e n t. T h is d iffu sib le activ ity m a y re s u lt fro m d a m a g e to so m e ly o so m e s d u rin g
se c tio n in g a s w e ll a s from n o n -ly so so m a l e n z y m e . T o tal a c tiv ity w a s ta k e n a s th e m a x im u m relativ e
a b s o rb a n c e m e a s u re d in se c tio n s fro m th e p re - tre a tm e n t se ries. L a te n t activ ity w a s e x p re s se d as a
p e rc e n ta g e o f to ta l activ ity a s s u m m a riz e d b e lo w :
T o tal a ctiv ity — F re e activ ity
P e rc e n ta g e la te n t a ctiv ity = ----------------- 1------------------------- x 10 0
T o ta l activ ity
Free a ctiv ity = s ta in in g in te n s ity a fte r 2 m in p re - tre a tm e n t
T o tal activ ity = m ax im al s ta in in g in te n s ity o b ta in e d fro m m e a s u re m e n ts o n p re
tr e a te d se rie s o f se c tio n s (2 - 2 5 m in )
H yd ro carb o n s and m ollusc lysosom es
715
M icro stereo lo g ical a n a ly s e s o f s e c o n d a ry ly s o so m a l stru c tu re w ith in th e d ig e stiv e cells w e re p e r
fo rm e d o n 10 /im c ry o sta t se c tio n s s ta in e d fo r ß -N - a c e ty lh e x o sa m in id a se in w h ic h th e la te n t e n z y m e
h a d b e e n a c tiv a te d . T h e m e th o d s fo r th e m ic ro ste re o lo g ic a l a n a ly s e s h a v e b e e n fully d e s c rib e d b y
L o w e et al. (1981). P a ra m e te r e s tim a to rs w e re d e te rm in e d for v o lu m e d e n s ity (to tal v o lu m e of
ly s o s o m e s /to ta l v o lu m e o f c y to p la sm in th e d ig e s tiv e cells s a m p le d ; V J V C), su rface d e n s ity (to tal
ly so so m a l su rface a re a /to ta l v o lu m e o f c y to p la s m in th e d ig e stiv e c e lls sa m p le s; S J V C), n u m e ric a l
d e n s ity (total n u m b e r o f ly s o so m e s /to ta l v o lu m e o f c y to p la sm in th e d ig e stiv e cells sam p led ;.V L/ V C)
a n d th e s ta n d a rd d e v ia tio n s (e stim a te d ) o f th e s e p a ra m e te rs.
R esults and discussion
T h e r e s u lts s h o w th a t th e p e rc e n ta g e la te n c y o f ß -N - a c e ty lh e x o s a m in id a s e w a s s ig n ifi
c a n tly r e d u c e d fro m th e c o n tr o l v a lu e s b y tr e a tm e n t w ith W A F a t b o th s a m p lin g tim e s
( T a b le 1 ). T h e d e c lin e i n p e r c e n t a g e la t e n c y o f ß - N - a c e t y l h e x o s a m i n i d a s e o b s e r v e d f r o m
3 4 d a y s to 1 0 3 d a y s ( T a b le 1 ) r e f l e c t e d a g r a d u a l d e c li n e in b o t h p h y s i o lo g i c a l a n d c e ll u la r
c o n d i t i o n o f t h e c o n t r o l a n i m a l s d u r i n g t h e c o u r s e o f t h i s e x p e r i m e n t ( W i d d o w s e t a l.,
1 9 8 2 ) . T h is d e t e r i o r a t i o n w a s p r o b a b l y a l o n g - t e r m e f f e c t o f m a i n t a i n i n g t h e m u s s e l s i n a n
a q u a r i u m s y s te m .
T h e s t r u c t u r a l c h a n g e s i n t h e s e c o n d a r y l y s o s o m e s i n d u c e d b y e x p o s u r e to W A F c a n b e
c le a r ly s e e n i n F ig s . 1 a n d 2 ; m a n y o f t h e s e c o n d a r y l y s o s o m e s h a v e a g r e a t l y e n l a r g e d
a p p e a r a n c e a n d t h e r e is a t h i n n i n g o f t h e d i g e s t i v e t u b u l e e p i t h e l i u m . T h e m i c r o s t e r e o l o g i
c a l r e s u l t s s h o w t h a t t h e r e a r e s i g n if i c a n t i n c r e a s e s i n l y s o s o m a l v o l u m e p e r u n i t v o l u m e o f
d i g e s t i v e c e ll c y t o p l a s m ( V J V C) a t b o t h s a m p l i n g t i m e s ( T a b le 1 ). T h is c le a r ly s u p p o r t s
t h e v i s u a l e v i d e n c e in F i g s . l a n d 2.
I n a d d i t i o n to t h e v o l u m e in c r e a s e t h e r e w e r e s i g n if i c a n t i n c r e a s e s i n l y s o s o m a l
s u r f a c e d e n s i t y a t b o t h s a m p l i n g t i m e s ( T a b le 1 ) a n d a s i g n if i c a n t d e c r e a s e in ly s o s o m a l
n u m e r i c a l d e n s i t y a f t e r 1 0 3 d a y s o f e x p o s u r e to W A F ( T a b le 1).
T a b le 1. T h e effects o f a ro m a tic h y d ro c a r b o n s in th e w a te r a c c o m m o d a te d frac tio n o f N o rth S ea
c ru d e oil (30 p g /\) o n la te n cy o f ly so so m a l ß - N -h e x o s a m in id a se a n d ly so so m a l stru c tu ra l c h a ra c te r
istics in th e d ig e stiv e cells.
Experim ental
treatm ent
% latency o f ß - N acetylhexosaminidase
Volum e density
Surface density
Nutnerical densih/
3 4 d a v s co n tro l
3 4 d a y s e x p o se d
3 4 .1 6 ± 8.79
8 .7 0 ± 1 0 .2 9
P = 0 .0 0 8 , L i-test
0 .0 6 4 ± 0 .0 3 0
0 .1 2 7 ± 0 .0 5 8
P < 0 .0 5 , f-te st
0 .1 4 7 ± 0 .0 2 6
0 .2 0 9 ± 0 .0 1 9
P < 0 .0 5 , f-te s t
0 .0 1 7 ± 0 .0 0 6
0 .0 1 5 ± 0 .0 0 4
N .S .t, f-te st
1 03 d a y s c o n tro l
1 0 3 d a y s e x p o se d
1 6 .7 0 ± 1 4 .8 0
0
P = 0 .0 4 8 , L i-test
0 .0 6 1 ± 0 .0 2 4
0 .1 9 2 ± 0 .0 6 2
P < 0 .0 5 , f-te st
0 .1 4 8 ± 0 .0 2 4
0 .1 9 0 ± 0 .0 1 7
P < 0 .0 5 , t-te s t
0 .0 2 0 ± 0 .0 1 4
0 .0 0 5 ± 0 .0 0 3
P < 0 .0 5 , f- te s t
Five m u ssels p e r sam ple.
t N .S ., n o t significant.
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Fig. 1. A c ry o s ta t s e c tio n th r o u g h a d ig e stiv e tu b u le fro m a c o n tro l m u s se l s a m p le d a fte r 1 0 3 d a y s
s h o w in g ly so so m e s (a rro w e d ) re a c te d fo r ß -N - h e x o sa m in id a se a c tiv ity in th e d ig e s tiv e cells. Scale
b ar: 1 0 ¿¿m.
Fig. 2. A se c tio n a s in Fig. 1 fro m a m u s se l e x p o se d to a ro m a tic h y d ro c a rb o n s (3 0 /¿g/1) in W A F
for 103 d a y s. T h e s e c o n d a ry ly s o so m e s are g re a tly e n la rg e d (a rro w e d ) a n d th e re is a n a p p a re n t
re d u c tio n in n u m b e r . T h e d ig e s tiv e tu b u le e p ith e liu m d is p la y s a re d u c tio n in h e ig h t a n d th e
tu b u le lu m e n (L) is e n la rg e d in c o m p a riso n w ith th e c o n tro l. S cale b a r: 10 u m .
H y d ro carb o n s an d m ollusc lysosom es
717
E x p o su re to a ro m a tic h y d ro c a rb o n s in W A F r e d u c e d th e latency of ly so so m al
ß - N - a c e ty lh e x o sa m in id a s e a n d re s u lte d in th e fo rm a tio n of large a n d p re s u m a b ly
u n s ta b le ly so so m e s. T h e se larg e ly so so m e s (Fig. 2) are b e lie v e d to be largely a u to p h a g ic
in fu n c tio n a n d h av e b e e n o b s e rv e d p re v io u sly in sta rv e d m u s s e ls (B ayne et al., 1978).
T h a t a u to p h a g y is o c c u rrin g is fu rth e r s u p p o rte d b y th e fac t th a t th e d ig estiv e cells fro m
th e sa m e sa m p le s w e re sig n ifican tly re d u c e d in h e ig h t b y W A F as d e te rm in e d b y im age
a n a ly s is (L o w e et al., 1981). T h e m e c h a n is m b y w h ic h th e s e large ly so so m es are fo rm ed
m a y b e in d ic a te d b y th e d ec re a se iii n u m e ric a l d e n sity o f th e ly so so m es, w h ic h c o u ld
s u g g e s t a fu sio n o f sm a lle r ly so so m e s to fo rm th e large u n s ta b le ty p e (b a se d o n laten c y
d a ta ).
T h is a lte ra tio n in ly so so m a l s tru c tu re w ith in th e d ig e stiv e cells re p re s e n ts a
c o n s id e ra b le d is tu rb a n c e o f th e n o rm a l (control) s tru c tu re a n d can p ro b a b ly be
in te r p r e te d as e n h a n c e d c a ta b o lism o f c y to p lasm ic c o m p o n e n ts by a ly so so m al
a u to p h a g ic m e c h a n ism . T h e re s u ltin g in c rease in tissu e c a ta b o lism is fu rth e r s u p p o rte d
b y p h y sio lo g ic a l d a ta w h ic h s h o w s a sig n ific a n t d e c rea se in sco p e for g ro w th (W id d o w s
et al., 1982).
A cknow ledgem ents
T h is w o rk fo rm s p a rt o f th e e x p e rim e n ta l e co lo g y p ro g ra m m e o f th e In stitu te for M arine
E n v iro n m e n ta l R esearch , a c o m p o n e n t o f th e N a tu ra l E n v iro n m e n t R e sea rc h C ouncil. It
w a s c o m m issio n e d in p a rt b y th e D e p a rm e n t o f th e E n v iro n m e n t (C o n tract N o. D G R
4 8 0 /2 8 8 ). N o rth S ea c ru d e oil w a s k in d ly su p p lie d to u s b y S hell (U .K .) L td.
References
B. L. (1976) W a tc h o n m u s se ls . M ar. Pollut. Bull. 7, 2 1 7 -8 .
& W I D D O W S , J. ( 1 9 7 8 ) F u rth e r
s tu d ie s o n th e effe c ts o f s tre s s in th e a d u lt o n th e e g g s o f M ytilu s edulis. ]. mar. biol. Ass. U.K. 58,
BAYNE,
B A Y N E , B. L . , H O L L A N D , D . L . , M O O R E , M . N . , L O W E , D . M.
825-41.
& w i d d o w s , J. ( 1 9 7 6 ) A c y t o c h e m i c a l a n d
M ytilu s edulis L. Mar. Pollut. Bull. 7, 2 2 1 - 4 .
B A Y N E , B. L . , M O O R E , M . N . & K O E H N , R. K. (1981) L y so so m e s a n d th e re s p o n s e b y M ytilu s
edulis L. to a n in c re a se in salin ity . Mar. Biol. Lett. 2, 1 9 3 -2 0 4 .
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