detergent-induced changes in the mapping of certain enzymes in various cell types of rita rita: ii....
TRANSCRIPT
ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY 17,67-74 (1989)
Detergent-Induced Changes in the Mapping of Certain Enzymes in Various Cell Types of Rita rita
II. Gill Epithelium
DEBASISH ROY’
CentralMarine Fisheries Research Institute, Post Box No. 2704, Ernakulam, Cochin, India, and
Department of Zoology, Utkal University, Bhubaneswar- 751004, India
Received March 16, 1988
Rita rita were exposed to 6.9 mg/liter (96-hr LC50 of an anionic detergent, dodecylbenzene sodium sulfonate). Loss of activity of certain enzymes responsible for various metabolic pro- cesses in fish gill epithelium was observed using a battery of histochemical techniques. The information of this investigation may possibly add to an understanding of the as yet unknown mechanism of detergent action. 0 1989 Academic Press, ILK.
INTRODUCTION
The era of synthetic detergents began in 1930 and, due to their extensive use as cleansing agents in household activities as well as in industry, are involved in almost all segments of the ecosystem today. Consequently, numerous hazards to several non- target species like fish have been reported (Abel, 1974). Prior to that, Schmid and Mann (196 l), Cairns and Scheier (1962), Lemke and Mount (1963), Lang (1967), and Brown et al. ( 1968) demonstrated the deleterious effects caused by detergents on fish gill. Later, Abel and Skidmore (1975), Abel (1976), Fukuda (1982), Misra et al. ( 1985), and Roy ( 1988a) examined the histopathological changes induced by deter- gents in gills at a light or electron microscopical level. Roy (1988b) attempted to demonstrate the changes due to detergent toxicity in the lipid constituents of various cell types present in fish gill epithelium. But very little attention has been given to the study of alterations in the activity of different enzymes present in various cell types of this tissue epithelium (Zaccone et al., 1985). In his previous paper, Roy (1989) showed changes in the activity of certain metabolically important enzymes in various cell types of the opercular epidermis of Rita rita under the influence of a commonly used detergent, dodecylbenzene sodium sulfonate. In this paper, the effects of this detergent on the activity of those enzymes in various cell types of the gill epithelium have been incorporated.
MATERIALS AND METHODS
Live specimens of R. rita ( 12- 16 cm in length) were collected from a local supplier and acclimated to laboratory conditions for 4 weeks before the start of the experi-
’ Present address for reprint requests and all communication: Debasish Roy, c/o Mr. R. N. Roy, 464, Saheednagar, Bhubaneswar - 75 1007, Orissa, India.
67 0147-6513189 $3.00 Copyright 0 1989 by Academic Press. Inc. All rights of reproduction in any form reserved.
TABL
E 1
A SU
MM
ARY
OF
HIST
~CHE
MIC
AL
REAC
XONS
SH
OW
ING
TH
E AC
TIVI
TIES
O
F VA
RIO
US
ENZY
MES
IN
THE
EPI
THEL
IAL
CELL
S O
F TH
E G
ILL
EPIT
HELI
UM
OF
Rita
rit
a,
AT D
IFFE
REN
T TI
ME
INTE
RVAL
S O
F DE
TERG
ENT
TREA
TMEN
T
Epi
thel
ial c
ells
Out
er la
yer
Mid
dle
laye
r B
asal
laye
r H
isto
chem
ical
C
olor
E
nzym
es
tech
niqu
es
sym
bol
C
1 2
3 4t
o8
C
1 2
3 4t
o8
C
1 2
3 4t
o8
Alk
alin
e ph
osph
atas
e
Aci
d ph
osph
atas
e
Non
spec
ific
este
rase
s
S-N
ucle
otid
ase
Azo
dye
cou
plin
g
met
hod
Con
trol
Azo
dye
cou
plin
g
met
hod
Con
trol
cy-n
apht
hyla
ceta
te
met
hod
Con
trol
Lead
met
hod
Con
trol
Br
-
- -
RB
r +S
b
- -
RB
r ++
b -
BrB
+n
-
-
- - +b
- Sb
- *a
-
- - -tb
- +b
- *a
-
- - - - kb
- - -
- - ++b - ++b - ka
-
- - +b
- +b
- fa -
- - + 6
- +b - ka -
- - - - + b
zk
- - - - - kn
-
Glu
cose
d-
phos
phat
ase
Cal
cium
-
depe
nden
t
ATPa
se
Mag
nesi
um-
depe
nden
t AT
Pase
Succ
inat
e de
hydr
ogen
ase
Glu
cose
-6-
phos
phat
e de
hydr
ogen
ase
Mon
oam
ine
oxid
ase
Lead
met
hod
BrB
Con
trol
-
Cal
cium
m
etho
d Br
B
Con
trol
-
Lead
met
hod
Con
trol
BrB -
Nitr
o-BT
m
etho
d C
ontro
l
Nitr
o-BT
m
etho
d C
ontro
l
Bl - Bl -
Tetra
zoliu
m
met
hod
Con
trol
- -
+ - IL
- k - + - k - - -
- - - - - - - - - - - -
- - - - - - - - - - - -
- - - - - - - - - - - -
- - - - - - - - - - - -
-+
- f - + - + - + - - -
- - - - - - - - - - - -
- - - - - - - - - - - -
- - - - - - - - - -
- - - - - - - - - - - -
* - + - + - + - f - - -
- -
-
8 r
-
Not
e. S
ymbo
ls: a
, cel
l nuc
lei; b
, cel
l bou
ndar
ies;
Bl,
blue
; Br
, bro
wn;
BrB,
bro
wnish
bl
ack;
RBr
, re
ddish
br
owh;
-,
nega
tive;
+, w
eak;
+,
mod
erat
e;
++,
stro
ng r
eact
ion;
C
, con
trol
cond
itions
; 1-
8, d
ays
of d
eter
gent
tre
atm
ent.
TABL
E 2
A SU
MM
ARY
OF
HIST
OCH
EMIC
AL
REA
CTI
ON
S SH
OW
ING
TH
E AC
TIVI
TIES
O
F VA
RIO
US
ENZY
MES
IN
THE
GLA
ND
CE
LLS
OF
THE
GIL
L EP
ITHE
LIUM
O
F R
ita
rita,
AT
DIF
FER
ENT
TIM
E IN
TERV
ALS
OF
DETE
RGEN
T TR
EATM
ENT
Gla
nd c
ells
Clu
b ce
lls
Enz
ymes
Out
er la
yer
Dee
per la
yers
G
oble
t muc
us ce
lls
His
toch
emic
al
Col
or
tech
niqu
es
sym
bol
C
1 2
3 4t
o8
C
1 2
3 4t
o8
C
1 2
3 4t
o8
0 ml
Alk
alin
e A
zo d
ye c
oupl
ing
phos
phat
ase
met
hod
Con
trol
Aci
d ph
osph
atas
e A
zo d
ye c
oupl
ing
met
hod
Con
trol
Non
spec
ific
cu-n
apht
hyl ac
etat
e es
tera
ses
met
hod
Con
trol
S-N
ucle
otid
ase
Lead
met
hod
Con
trol
Br
+
-
RB
r ++
c
+ - ++c
- ++c
- fU
-
- - +c
- +=
- - -
- -
+ +-
- -
--
- -
---
- __
-
-
- -
_ +b
+b
+
b
d +
- -
- - -
-
RB
r ++
’
- -
-
*c
- -
- +c
- -
- -
-
- -
+”
- -
-
- -
_ -
- -
---
- ++
b +b
- -
f b
* b
-
BrB
&
a -
---
- __
+a
- -
- fa
&a
---
-
__
- - -
- - -
.~.
z Fi
z -
5 - - - -
Glu
cose
-6-
phos
phat
ase
Cal
cium
-
depe
nden
t AT
Pase
Mag
nesi
um-
depe
nden
t
ATPa
se
Succ
inat
e
dehy
drog
enas
e
Glu
cose
-6-
phos
phat
e de
hydr
ogen
ase
Mon
oam
ine
oxid
ase
Lead
met
hod
Con
trol
Cal
cium
met
hod
Con
trol
Lead
met
hod
Con
trol
Nitr
o-BT
m
etho
d
Con
trol
Nitr
o-BT
m
etho
d
Con
trol
Tetra
zoliu
m
met
hod
Con
trol
BrB -
BrB -
BrB -
Bl - Bl - - -
SC
- +c
- -tc
- SC
- +c
- - -
+c
kc
kc
kc
fC
- - -
kc
- - - - - fC
- kc
- -
kc
- - - - - -tc
- kc
- - -
- - - - - - - - - - - -
+c
- +c
- SC
- +c
- SC
- - -
SC
- kc
- kc
- fC
- +c
- - -
kc
- - - - - kc
- kc
- - -
kc
- - - - - tC
- kc
- - -
- - - - - - - - - - - -
kd
- +b
- +b
- + b
- f b
- -
- -
-t b
-
- -
-t b
-
- -
- -
- -
- -
- -
- -
- -
- -
- -
- -
- -
- -
- -
- -
- -
- -
- -
Note.
Sy
mbo
ls: a
, ce
ll nu
clei
; b.
narro
w pe
riphe
ry
ofgo
blet
m
ucus
cel
ls: c
. cel
l bou
ndar
ies;
d,
secr
etor
y co
nten
ts;
Br, b
rown
; Br
B, b
rown
ish
blac
k; R
Br,
redd
ish
brow
n;
-3 n
egat
ive;
f, w
eak;
f,
mod
erat
e;
++,
stro
ng r
eact
ions
; C
, con
trol
cond
itions
; 1-
8, d
ays
ofde
terg
ent
treat
men
t.
72 DEBASISH ROY
ment. The technical details of the methods and procedures applied in the study of various enzymes have been outlined in the previous paper by Roy ( 1989).
RESULTS
Epithelial Cells
The peripheral areas of the epithelial cells of outer and middle layers of the epithe- lium lining the gill arch as well as gill filament demonstrate a strong reaction for acid phosphatase and nonspecific esterases, while the basal layer epithelial cells remain unstained. A moderate activity for alkaline phosphatase and succinate dehydroge- nase is seen in the epithelial cells of the basal layer while the reaction for the former is negative and for the latter is weaker in the outer and middle layers. The epithelial cells of all the sites show a weak reaction for glucose-6-phosphatase, glucosed-phos- phate dehydrogenase, and calcium-dependent and magnesium-dependent ATPases. The nuclei ofthese cells in the gill epithelium show a weak reaction for S-nucleotidase (see Table 1).
The influence of the detergent is so sudden and fast that the activity for glucosed- phosphatase, succinate dehydrogenase, and glucosed-phosphate dehydrogenase could not be observed even after 24 hr of detergent treatment. A slight decrease was noted in the staining intensity for acid phosphatase, alkaline phosphatase, and S- nucleotidase, 24 hr after transfer of the fish into the detergent medium, whereas, after 3 days, positive reaction for any of these enzymes could not be localized in the epithelial cells of the gill epithelium. The reaction for nonspecific esterases also lasts up to 3 days, and from the fourth day onward, it was negative. The activity for mono- amine oxidase could not be demonstrated throughout the duration ofthe experiment.
Goblet Mucus Cells
The peripheral zones of the goblet mucus cells of the gill filament epithelium show moderate reaction for acid phosphatase, nonspecific esterases, and calcium- and mag- nesium-dependent ATPases while the cells of the gill arch epithelium exhibit strong reaction for acid phosphatase and nonspecific esterases. These zones of the goblet mucus cells of both gill arch and gill filament epithelium give weak activity for succi- nate dehydrogenase and glucosed-phosphate dehydrogenase. The secretory contents and nuclei of these cells exhibit weak enzyme activity for glucose-6-phosphatase and 5’-nucleotidase, respectively (see Table 2).
The activity for acid phosphatase in the peripheral area of the goblet mucus cells of gill filament and gill arch epithelium decreases after 24 and 48 hr of detergent treatment, respectively, as evidenced by weak positive reaction. Reaction could not be visualized after another 24 hr of detergent treatment. The reaction for nonspecific esterases in both sites becomes moderate after 24 hr of detergent exposure and the reaction in the goblet mucus cells of the gill arch and gill filament epithelium is weak- ened after 72 and 48 hr of treatment, respectively, after which, no reaction for this enzyme could be seen in any subsequent stage. The enzyme activity for calcium- and magnesium-dependent ATPases and S-nucleotidase in these cells of the epithelium lining the gill arch and the gill filament becomes weak, 24 hr after transfer of the fish into the detergent medium, and in subsequent stages, positive reaction for these enzymes could not be observed. There was no activity for glucose-6-phosphatase,
DETERGENT-INDUCED CHANGES IN GILL EPITHELIUM 73
succinate dehydrogenase, and glucosed-phosphate dehydrogenase even after 24 hr of detergent treatment. Positive reaction for alkaline phosphatase and monoamine oxidase could not be observed in the control or under the experimental conditions.
Club Cells
The peripheral zones of the club cells of the gill arch epithelium exhibit strong activity for acid phosphatase and nonspecific esterases while these zones give moder- ate activity for alkaline phosphatase, glucose-6-phosphatase, succinate dehydroge- nase, and glucose-6-phosphate dehydrogenase. The boundaries and the nuclei of these cells of the gill arch epithelium give moderate reaction for calcium- and magnes- ium-dependent ATPases and S-nucleotidase, respectively.
Detergent treatment does not induce any sudden change in the activity of acid phosphatase and nonspecific esterases. After 2 days of detergent treatment, the en- zyme activity decreases considerably and becomes moderate. Such a condition pre- vails up to 3 days, and in no subsequent stage could positive reaction for these en- zymes be observed. Activity for glucose-6-phosphatase, succinate dehydrogenase, and glucose-6-phosphate dehydrogenase in the club cells of gill arch epithelium is weakened gradually, the enzyme activity remains up to 3 days, and no reaction for the above enzymes could be observed after 3 days of exposure. Within 24 hr of transfer of the fish into the detergent medium, the activity for alkaline phosphatase and calcium- and magnesium-dependent ATPases weakens considerably and no enzyme activity could be observed in the club cells in subsequent stages. The pattern of change in staining intensity was almost the same in nuclei for S-nucleotidase.
DISCUSSION
The present investigation observes a decrease in the activity of many important metabolic enzymes responsible for different physiological processes in various cell types, especially goblet mucus cells. It is known that low surfactant concentrations affect most membrane-bound enzymatic activities in one way or another (Coleman, 1973). These may be activated and inhibited or modified. It is difficult to ascertain whether the changes in the activity results from the direct action of the surfactant around the enzyme (Helenius and Simon, 1975). In some cases, the surfactant may also affect the state of the substrate (Gatt, 1973). The effects of the detergent on mem- brane enzymes are sometimes biphasic; activation is observed at low detergent con- centrations and inhibition at higher concentrations (Zaccone et al., 1985). Optimal activation occurs usually when the enzyme is still membrane bound. When higher concentrations of the detergent are added to the membranes, separation of lipids from the proteins takes place (Helenius and Simon, 1975).
On the basis of the present studies, it can be assumed that this inhibition may be due to the higher dose used, causing rapid and unbearable destruction leading to the death of the fish. The reasons may be that either the conditions inside the cells are made noncongenial for the enzymes to act by the heavy dose of the detergent or the configuration of the enzyme structure is destroyed. This appears to be true because Zaccone et al. (1985) have observed an increase in the enzyme activity caused by a sublethal dose of an anionic detergent, perhaps engaged in mucus production. The other reason for the disappearance of the enzyme activity may be due to their exhaus- tion caused by a sudden load of activity for excessive mucus secretion.
74 DEBASISH ROY
CONCLUSIONS
The activity for different enzymes under the influence of the detergent in the vari- ous cell types of the gill epithelium of R. rita was found to be inhibited, the manner being either gradual or instant. The higher dose used in this study has either rendered the enzyme inactive by making the inside of the cell improper for the enzyme to act or totally changed the configuration of the enzyme molecule.
ACKNOWLEDGMENT
Thanks are due to Professor A. G. Sathyanesan, Emeritus Scientist, CSIR, Central Marine Fisheries Research Institute, Cochin, for encouragement and necessary assistance.
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