statistical analysis of anionic detergent-induced changes in the goblet mucous cells of opercular...
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ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY 15,260-27 l(l988)
Statistical Analysis of Anionic Detergent-Induced Changes in the Goblet Mucous Cells of Opercular Epidermis and Gill Epithelium
of Rita rifa (Ham.) (Bagridae: Pisces)
DEBASISH ROY*
Department ofZoology. Banaras Hindu University, Varanasi- 221005, India
ReceivedMay 14.1987
Rita rita exposed to 96-hr LCsO (6.9 mg/liter) of an anionic detergent, dodecylbenzene sodium sulfonate, show significant changes in the number and size ofgoblet mucous cells in the opercu- lar epidermis as well as in the lining epithelium of the gill arch and the gill filament at different time intervals oftreatment. A shift in the staining nature ofthese cells from acidic glycoprotein to neutral glycoprotein, acidic glycosaminoglycans in the opercular epidermis and acidic glyco- protein to neutral glycoprotein and then again to acidic glycoprotein + acidic glycosaminogly- cans in the gill filament epithelium reflects a change in the physiological status of fish. o 1988 Academic Press, Inc.
INTRODUCTION
A substantial amount of work has been done on the effects of synthetic detergents on the gills of fishes at both light and electron microscopical levels. The ultimate pathological effects reported are gross destruction in the gill filaments with resultant death due to asphyxia (Schmid and Mann, 1961; Lemke and Mount, 1963; Lang, 1967; Brown et al., 1968; Abel, 1976; Abel and Skidmore, 1975; Umezawa et al., 1979; Misra et a/., 1985; Zaccone et al., 1985a). The earliest and most prominent effects in the gills are reflected by the changes in the goblet mucous cell activities. However, there is a lack of statistical analysis of the goblet mucous cells in relation to their number, size, and staining behavior induced by the detergent treatment. Also the deleterious effects of this highly toxic substance on fish skin which comes into direct contact with the environment remain relatively unknown (Pohla-Gubo and Adam, 1982; Zaccone et al., 1985b). Therefore, this paper deals with statistical change in the number, size, and staining behavior of the goblet mucous cells of skin and gill under the influence of a commonly used detergent, dodecylbenzene sodium sulfonate.
MATERIALS AND METHODS
Live specimens of Rita rita (approximately 12- 16 cm in length) were caught from the River Ganges at Varanasi in January and acclimated to laboratory conditions for 4 weeks before the start of the experiment. Injured fish were not used. The 96-hr LCsO value of an anionic detergent, dodecylbenzene sodium sulfonate (&-LAS) obtained from Koch Light Laboratories, Colnbrook, England, was determined following Stan- dard methods (APHA, AWWA, WPCF, 15th ed.). Static experiments of the 96-hr LCsO (6.9 mg/liter) value of the detergent were performed for this study. The experi- ments consisted of eight batches in order to avoid overcrowding. Each batch con- tained 10 healthy fish kept in glass aquaria (2 X 1 X 1.5 ft). The first aquarium served
* Current address for reprint request: 464 Sabeednagar, Bhubaneswar-75 1007, Orissa, India.
0147-6513/88 $3.00 Copyright 0 I988 by Academic Prey. Inc. A,, rigbt.9 orreprcduction in any form reserved.
260
DETERGENT-INDUCED CHANGES IN Ritu MUCOUS CELLS 261
as control and contained only tap water and no detergent. The rest of the aquaria were used for detergent treatment and were numbered 2 to 8 (experimental). Each experimental aquarium contained 30 liters of detergent medium having a concentra- tion of 6.9 mg/liter. All the aquaria were kept under constant observation and any dead fish, if detected, were immediately removed and discarded. Fish from experi- mental as well as control aquaria were sacrificed at different time intervals: 15, 30, and 45 min; I, 2, 3,6, and 12 hr; and I, 2, 3,4, 5, 6, 7, and 8 days. Operculum and first let? gill were dissected out and fixed in aqueous Bouin’s fluid for a short duration (6 hr) before they were washed thoroughly in 70% alcohol. The epithelium from both operculum and gill was separated mechanically from the underlying tissues under a dissection microscope. The tissues pieces (2 X 2 mm, approximately) were then stained with PAS (McManus, 1948), AB (pH 2,5)/PAS (Mowry, 1963), and AB pH 2.5 (Spicer et al., 1967) techniques to visualize and differentiate the goblet mucous cells and to elaborate various types of glycoproteins. They were subsequently dehy- drated and cleared and whole mount preparations were made. The number and per- centage of goblet mucous cells containing acidic glycoproteins, neutral glycoproteins, and acidic glycosaminoglycans, and the total number of cells/mm’ area, were calcu- lated using the standard statistical procedure based on random sampling of 10 differ- ent sites on each tissue sample, For each estimation, samples of three fish from each experimental condition as well as from the control were taken into consideration. Analysis of variance and Duncan’s multiple range test were performed to find out whether the number of goblet mucous cells was significantly different with increase in exposure time during the course of experimentation.
RESULTS
Operculum
Under the influence of the detergent, the number of the goblet mucous cells in the opercular epidermis rises to 24,065 (-t866.3)/mm2 after 45 min, nearly doubling the number of goblet mucous cells observed in the control (12,783.3 (246 1.4)). Subse- quently, the number of goblet mucous cells decreases. Similar increases followed by decreases continue in a cyclic manner up to the end of the experiment (Fig. 1).
Gill Arch In the gill arch epithelium, the number of goblet mucous cells gradually decreases
up to 30 min to 11,410.2 (t-125.3)/mm2 from 13,973.l (k340.9) in normal fish, fol- lowed by an increase to 13,719.5 (+-161.13)/mm2 after 45 min. After this period a cyclic decrease followed by an increase in the number of goblet mucous cells of gill arch epithelium also continues up to 5 days of detergent exposure. Thereafter, the number remains almost unchanged (Fig. 2).
Gill Filament
One hour of detergent treatment decreases the number ofgoblet mucous cells in the gill filament epithelium to 3448.4 (-+52.81)/mm2 from 7599.0 (4483.1) in control. Subsequently, a continuous increase followed by a decrease in the number of goblet mucous cells persists until the end of the experiment.
Analysis of variance (ANOVA) indicates that the number of goblet mucous cells of the opercular epidermis, gill arch, and gill filament epithelium under the influence of the detergent changes significantly (P < 0.001) with respect to time intervals (Ta-
262 DEBASISH ROY
a
I 0 1 2 3 6tz24 68 72 96 120 144 168 192
TlMEINHOURS
1 2 3 61224 48 72 96 120 144 168 19i TIME IN HOURS
FIG. 1. (a) Variations in the number of goblet mucous c&/mm2 of the opercular epidermis of Mu ritu at different time intervals of exposure to detergent medium. (b) Variations in the number ofgoblet mucous cells/mm* of the gill arch epithelium of Ritu ritu at different time intervals of exposure to detergent me- dium. (c) Variations in the number of goblet mucous cells/mm2 of the gill filament epithelium of Rita rita
at different time intervals of exposure to detergent medium.
DETERGENT-INDUCED CHANGES IN Ritu MUCOUS CELLS 263
I I
1 2 3 612 x &a 72 96 120 tU 168 192
TINE IN HOURS
FIG. l-Continued.
bles 1 a, 1 b, ic). Duncan’s multiple range tests performed to compare variations in the number of goblet mucous cells at two specific time intervals also show that, except very few cases (Tables 2a, 2b, 2c), the change in the number of goblet mucous cells at two different time intervals is statistically significant.
Change in CeZl Size
Marked change is observed in the average diameter of the goblet mucous cells of the opercular epidermis, gill arch, and gill filament epithelium at different time inter- vals of exposure of fish to detergent medium (Table 3).
Change in the Staining Properties
Operculurn. Under normal conditions the goblet mucous cells contain acidic glyco- protein. The nature of the contents under the influence of detergent medium remains the same up to 2 hr of treatment, after which the contents of some of the mucous cells (14.73 ?I 1.03%) begin staining for neutral glycoproteins also. Gradually, the percentage of neutral glycoprotein-containing cells increases and that of acidic glyco- protein-containing cells decreases with occasional fluctuations in their number. At the end of 8 days of detergent exposure, the percentage of acidic glycoprotein-con- taining cells becomes 16.63 (+_O. 15) and neutral glycoprotein-containing cells, 7 1.6 1 (22.15). A small percentage of cells (11.76 5 0.22) also stains only for acidic glyco- saminoglycans (Fig. 2a).
Gilljilument. Similarly, in the gill filament epithelium after 2 hr of detergent treat- ment, contents of most of the goblet mucous cells (98.40 & 0.38%) show the presence
264 DEBASISH ROY
a loo- =
90-
80 - In im Y
i?m 8 2 50-
= 40- $ ts _ L 3o 0 3 20 -
10 -
0 1 2 3 61224 48 72 96 l&J 144 166 192 TIME IN HOURS
b 100 b
90-
80 - ul -1 d70 - " y60 - 8 z50 -
t d40 -
$ % 30 -
s20 -
10 -
4- 0 1 2 3 612 24 48 72 96 120 144 168 192
TIME IN HOURS
FIG. 2. (a) Percentage of goblet mucous cells containing acidic glycoprotein (O), neutral glycoprotein (0), and acidic glycosaminoglycans (A) in the opercular epidermis of Rifa rita at different time intervals of exposure to detergent medium. (b) Percentage of goblet mucous cells containing acidic glycoprotein (O), neutral glycoprotein (0), and acidic glycosaminoglycans (A) in the gill arch epithelium of Rita rita at different time intervals of exposure to detergent medium. (c) Percentage of goblet mucous cells containing acidic glycoprotein (a), neutral glycoprotein (0), and acidic glycosaminoglycans (A) in the gill filament epithelium of Rita rita at different time intervals of exposure to detergent medium.
of acidic glycoprotein, while a few (1.60 rt 0.12%) stain for neutral glycoproteins (Fig. 2b). Subsequently, up to 5 days of detergent exposure, the number of neutral glycoprotein-containing cells increases (38.07 + 0.89%) with the simultaneous de- crease in the number of acidic glycoprotein containing cells (6 1.93 + 1 S%). During the last 3 days of detergent exposure, again a large number of cells (70.69 k 2.05 to
DETERGENT-INDUCED CHANGES IN Rita MUCOUS CELLS 265
m 3 70 - 3 '460 -
8 $J50 -
10
1 %-u 3 612 24 48 72 96 120 144 168 192
I - 0 1 2
TIME IN HOURS
FIG. 2-Continued.
72.5 + 4.03%) contain acidic glycoprotein, (9.45 & 0.35 to 24.7 4 1.65%) neutral glycoprotein, and (19.86 + 0.2 to 2.7 1 -t 0.79%) acidic glycosaminoglycans.
Gill arch. No marked change in the goblet mucous cells of epithelium lining the gill arch is observed, except for the appearance of a very small number of neutral glycoprotein-containing ( 1.88%) and acidic glycosaminoglycan-secreting (0.66%) mucous cells in 24-hr detergent-exposed tissues.
TABLE 1
ANALYSISOFVARIANCESHOW~NGSIGNIL~CANTCHANGESINTHENUMBEROFGOBLET MUCOUSCELLS OFRita rita ATDIFFERENTTIMEINTERVALSOF
EXP~SURETODETERGENTMEDIUM
Source Sum square 4 Mean square F P
Total Treatment ElT0r
Total Treatment Error
Total Treatment Error
85265.76 84287.69
978.07
a. Opercular epidermis
50 16 5267.90 34 28.76 183.16 <o.oo I
35213.62 34761.34 16 2172.50
452.28 34 13.30
b. Gill arch epithelium
50
163.35 to.001
c. Gill filament epithelium
26735.52 50 26357.40 16 1647.30
378.12 34 11.12 148.10 <O.ool
TABL
E 2a
1 2
3 4
5 6
7 8
9 10
11
12
13
14
I5
16
17
(3
hr)
(6 h
r) (6
day
s)
(7 d
ays)
(5
day
s)
(4 d
ays)
(C
ontro
l) (1
5 m
in)
(2 d
ays)
(1
2 hr
) (1
day
) (8
day
s)
(1 h
r) (3
day
s)
(30
min)
(2
hr)
(45
min)
71
.89
85.5
4 88
.58
loo.
17
105.
16
126.
38
127.
82
135.
25
139.
92
140.
31
142.
73
144.
09
145.
57
146.
82
168.
18
202.
7 24
0.64
71.8
9 85
.54
88.5
8 10
0.17
10
5.16
12
6.38
12
1.82
13
5.25
13
9.92
14
0.31
14
2.73
14
4.09
14
5.57
14
6.82
16
8.18
20
2.70
24
0.64
13.6
5**
16.6
9**
28.2
8”
37.2
7**
54.4
9”
55.9
3’:
63.3
6**
68.0
3*+
3.04
14
.63*
* 19
.62*
* 40
.84*
* 42
.28*
+ 49
.71*
* 54
.38*
* 11
.59;
16
.58”
37
.80*
* 39
.24”
46
.67+
* 5
1.34
*’ 4.
99
26.2
1’”
27.6
5**
35.0
8**
39.7
5+*
21.2
2**
22.6
6**
30.0
9**
34.7
6**
1.44
8.
87
13.5
4*
7.43
12
.10’
4.
67
68.4
2**
54.7
7.”
51.3
7**
40.1
4**
35.1
5**
13.9
3**
12.4
9*
5.06
0.
39
70.8
4**
57.1
9*+
53.7
9”
42.5
6,;
37.5
7**
16.3
5**
14.9
1**
7.48
2.
8 I
2.42
72.2
0**
58.5
5**
55.5
1+*
43.9
2**
38.9
3**
17.7
1**
16.2
7**
8.84
4.
17
3.78
1.
36
73.6
8*’
60.0
3,’
56.9
9”
45.4
0**
40.4
1**
19.1
9**
17.7
5**
10.3
2 5.
65
5.26
2.
84
1.48
74.9
3”
6 1.
28**
58
.24*
* 46
.65*
* 4
I .66
**
20.4
4**
19.0
0**
11.5
7*
6.90
6.
51
4.09
2.
73
1.25
96.2
9”
82.6
4..
79.6
0**
68.0
1**
63.0
2-
4 1.
80**
40
.36*
* 32
.93*
* 28
.26*
* 27
.87”
25
.45*
* 24
.09”
22
.61*
* 21
.36’
;
130.
81**
11
7.16
**
lJ4.12
+*
102.5
3**
97.5
4"
76.32
** 74
.88**
67.45
** 62
.78*'*
62.3
9**
59.9
7"
58.61
" 57
.13+*
55
.88**
34.52
+*
168.7
5**
155.1
0**
152.0
6**
140.4
7**
135.4
8**
114.
26**
i2
112.8
2**
$
105.3
9**
g 10
0.72*
* x
100.3
3*+
97.91
** 96
.55"
95.07
** 93
.82**
72.46
** 37
.94**
LSD
= M
ean
squa
reer
ror
of A
NOVA
= ng
roup
s LS
DX
Dun
can'
stab
leva
lue
= va
lue f
or
com
paris
on
0.05
8.94
9.4
1 9.
66
9.90
10
.06
10.1
8 10
.27
10.37
10
.43
10.43
10
.52
10.52
10
.62
10.62
10
.65
10.65
0.
01
12.04
12
.57
12.88
13
.06
13.37
13
.49
13.65
13
.77
13.87
13
.87
14.05
14
.05
14.18
14
.18
14.27
14
.27
Not
e. Nu
mbe
rs
1 thr
ough
17
indi
cate
th
e m
ean
value
of
the
gobl
et
muc
ous
cells
of
thre
e fis
h at
the
exp
osur
e tim
e as
indi
cate
d.
*p<o
.05.
+*
p<0.
01.
TABL
E 2b
DU
NC
AN’S
M
ULTI
PLE
RANG
E TE
ST
APPL
IED
TO E
XAM
INE
SIG
NIF
ICAN
T C
HAN
GE
IN T
HE
DEN
SITY
O
F G
OBL
ET
Muc
ous
CELL
S IN
THE
GIL
L AR
CH
EPIT
HEL
IUM
UN
DER
THEI
NFL
UEN
CEO
F TH
E DE
TERG
ENT
I 2
3 4
5 6
7 8
9 10
11
12
13
14
15
16
17
(1
day
) (1
2 hr
) (5
day
s)
(4 d
ays)
(2
hr)
(6 h
r) (1
5 m
in)
(30
min)
(3
days
) (Z
days
) (1
hr)
63.9
1 64
.83
83.9
0 (7
day
s)
(8 d
ays)
(4
5 m
in)
(6da
ys)
(Con
trol)
(3 h
r) 86
.71
89.7
1
95.9
6 11
2.57
11
4.09
11
7.45
12
3.54
13
2.31
13
4.19
13
4.50
13
7.19
13
7.54
13
9.73
14
7.21
63.9
1 0.
92
19.9
9”
22.8
0**
25.8
0**
32.0
5**
48.6
6**
50.1
8**
53.5
4**
59.6
3**
68&l
**
70.2
8**
70.5
9**
73.2
8**
73.6
3**
75.8
2**
83.3
0**
64.8
3 19
.07*
* 21
.88*
* 24
.88*
* 31
.13*
* 47
.74*
* 49
.26*
* 52
.62’+
58
.71*
* 67
.48*
* 69
.36*
+ 69
.67*
* 72
.36*
* 72
.7 I
**
74.9
0**
82.3
8**
83.9
0 2.
8 I
5.81
12
.06*
* 28
.67*
* 30
.19*
’ 33
.55*
* 39
.64*
* 48
.41*
* 50
.29*
* 50
.60*
’ 53
.29*
’ 53
.64*
* 55
.83*
* 63
.31*
* 86
.71
3.00
9.
25”
25.8
6’;
27.3
8**
30.7
4**
36.8
3**
45.6
0**
47.4
8**
47.8
0**
50.4
8**
50.8
3’;
53.0
2”
60.5
0**
89.7
1
6.25
22
.86”
24
.38*
* 27
.74’
* 33
.83*
’ 42
.60.
. 44
.48*
* 44
.79*
’ 47
.48*
* 47
.83;
’ 50
.02*
* 57
.50*
* 95
.96
16.6
1**
18.1
3**
21.4
9**
27.5
8**
36.3
5**
38.2
3**
38.5
4**
41.2
3**
41.5
8**
43.7
7**
5 1.
25**
11
2.57
1.
52
4.88
10
.97*
* 19
.74*
* 21
.62*
* 21
.93*
* 24
.62.
; 24
.97*
’ 27
.16’
* 34
.64’
* 11
4.09
3.
36
9.45
8’
18.2
2”*
20.1
0**
20.4
1**
23.1
0**
23.4
5.’
25.6
4.;
33.
It**
117.
45
6.09
14
.86*
* 16
.74*
* 17
.05*
* 19
.74*
* 20
.098
’ 22
.2s*
* 29
.76”
12
3.54
8.
77*
10.6
5”
10.9
6*’
13.6
5’;
14.0
0**
16.1
9**
23.6
7+”
132.
3 1
1.88
2.
19
4.88
5.
23
7.42
* 14
.90*
* 13
4.19
0.
31
3.00
3.
35
5.54
13
.02*
* 13
4.50
2.
69
3.04
5.
23
12.7
1**
137.
19
0.54
2.
54
10.0
2”
137.
54
2.19
9.
67*
139.
73
7.48
14
7.2
1
LSD
= M
eans
quar
e er
roro
fANO
VA=
n gr
oups
LS
D X
Dunc
an’s
tabl
e va
lue
= va
lue
for
com
paris
on
0.05
6.
083
6.40
6.
56
6.74
6.
84
6.92
6.
98
7.05
7.
09
7.09
7.
16
7.16
7.
22
7.22
7.
24
7.24
0.
01
8.19
8.
54
8.75
8.
88
9.09
9.
18
9.28
9.
37
9.43
9.
43
9.56
9.
56
9.64
9.
64
9.70
9.
70
No@
. Nu
mbe
rs
I th
roug
h 17
indi
cate
th
e m
ean
value
of
the
gob
let
muc
ous
cells
of
thre
e fis
h at
the
exp
osur
e tim
e as
indi
cate
d.
* p <
0.0
5.
+*p<
o.o1
.
TABL
E 2c
DU
NC
AN'S
MU
LTIP
LE
RANG
ETES
T AP
PLIE
D T
O
EXAM
INES
IGN
IL~C
ANTC
HAN
GEI
NTH
EDEN
SITY
OFG
OBL
ETM
UC
OU
SCEL
LS
INTH
EEPI
THEL
IUM
LININ
G TH
EG~L
LRLA
MEN
TUN
DER
THEI
NFL
UEN
CEO
FTH
EDET
ERG
ENT
I 2
3 4
5 (66
hr)
7 8
12
13
14
15
16
17
(I hr
) (15
m
in)
(45
min)
(30
m
in)
(2 hr
) ( I
2 hr
) (6
days
) (A
) (7C
ys)
dk,
(Con
trol)
(1 da
y) (2
days
) (4
days
) (3
days
) (8
days
) 34
.40
35.41
39
.19
39.47
51
.60
65.29
65
.84
67.67
70
.21
70.52
73
.68
75.98
79
.38
82.70
86
.36
96.07
12
3.65
34.40
35
.41
39.19
39
.47
51.60
65
.29
65.84
67
.67
70.2
1 70
.52
73.68
75
.98
79.38
82
.70
86.36
96
.07
123.6
5
1.01
4.7
9 5.0
7 17
.20**
30.89
** 31
.44*+
33
.27”
35.84
” 3.7
8 4.0
6 16
. I9*
* 29
.88’.
30.43
” 32
.26**
34
80**
0.28
12.41
** 26
.10**
26.65
.; 28
.48**
31:02
** 12
.13**
25.82
** 26
.37;:
28.20
*’ 30
.74**
13.69
** 14
.24**
16.07
** 18
.61**
0.55
2.38
4.92
1.83
4.37
2.54
36.12
** 35
.11**
31.33
** 31
.05**
18.92
** 5.2
3 4.6
8 2.8
5 0.3
1
39.28
** 38
.27++
34
.49**
34.21
** 22
.08**
8.39’
7.84;
6.01
3.47
3.16
41.58
** 40
.57**
36.79
+*
36.51
** 24
.38**
10.69
** 10
.14**
8.31*
5.7
7 5.4
6 2.3
0
44.98
** 43
.97.’
40.19
** 39
.91**
27.78
** 14
.09**
13
54-
,1:71
** 9.1
7” 8.8
6#
5.70
3.40
48.30
** 47
.29*+
43
.51.;
43.23
*+
31
lo**
17:41
** 16
.86’;
15.03
+*
12.49
** 12
.18**
9.02*
*
6.72’
3.32
LSD
= Me
an
squa
re
erro
r of
ANOV
A =
n gr
oups
LS
D X
Dunc
an’s
table
value
=
value
for
co
mpa
rison
5 1.9
6**
50.9s
. 47
.17**
46.89
** 34
.76”
21.07
” 20
.52,’
18.69
** 16
.15,.
15.84
** 12
.68**
10.38
** 6.9
8+
3.66
61.67
** 60
.66**
56.88
** 56
.60,;
44.47
;. 30
.78**
30.23
** 28
.40**
25.86
** 25
.55**
22.39
*+
20.09
.8 16
.69**
13.37
” 9.7
1**
89.25
’* 88
.24*
84.46
** 84
.18**
72.05
*’ 58
.36.’
si
57.81
,’ $
55.98
” =
53.44
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53.13
** 49
.97**
47.67
** 44
.27”
40.95
** 37
.29**
27.58
**
0.05
5.56
5.85
6.00
6.16
6.26
6.33
6.39
6.45
6.49
6.49
6.55
6.55
6.60
6.60
6.62
6.62
0.01
7.49
7.8 1
8.0
1 8.1
2 8.3
2 8.3
9 8.4
9 8.5
7 8.6
2 8.6
2 8.7
4 8.7
4 8.8
1 8.8
1 8.8
7 8.8
7
No&.
Nu
mbe
rs I
throu
gh
I7 ind
icate
the
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.
DETERGENT-INDUCED CHANGES IN Rita MUCOUS CELLS 269
TABLE 3
AVERAGE DIAMETER + SD (in pm) OF GOBLET Mucous CELLS OF DIFFERENT TIZXJES UNDER THE INFLUENCE OF THE DETERGENT AT VARIOUS TIME INTERVALS
Average diameter of goblet cells f SD (in pm)
Time intervals
Length of fish in mm.
Opercular epidermis
Gill arch epithelium
Gill filament epithelium
Control 15min 30 min 45 min
1 hr 2 hr 3 hr 6 hr
12hr 1 day 2 days 3 days 4 days 5 days 6 days 7 days 8 days
125 11.0 Ik 1.56 130 10.0 k 1.92 122 8.3 + 1.66 140 6.66+ 1.92 145 11.0 + 1.56 120 8.3 + 1.3 150 13.3 Ik 3.3 153 8.3 + 3.6 144 10.6 k2.16 143 8.3 t 1.6 127 8.6 * 1.1 137 8.99kO.79 124 8.6 Ik 1.1 133 8.6 + 1.7 139 7.66 + 1.8 141 5.99-t 1.1 147 7.66 zk 1.56
8.3 + 1.03 8.0 -+ 1.03 8.3 -+ 1.33
10.2 kO.6 10.9 -+ 1.5 10.9 +0.8 14.43 f 2.7 10.43 -t 0.73 10.43 XL 0.73 8.33 rt 1.53 9.26 + 1.0 8.73? 1.8
10.0 t 0.7 8.33 + 1.43 8.93 +- 1.4
10.2 kO.96 9.16 +- 1.16
6.36+ 1.13 8.6 f 1.3 9.43 kO.76 9.14 kO.56 9.43 k 2.06 9.39 zk 0.56
10.9 t 1.2 7.13 kO.73 7.13 to.73 6.4 zk 1.63 6.16 -to.73 6.03 + 1.2 6.16 kO.73 7.36 t- 1.2 9.5 -t 1.46 4.5 ?I 1.13 5.0 -+ 1.23
DISCUSSION
The immediate reaction in the epidermis, consequent to detergent treatment, is manifested by the sudden increase doubling the number of goblet mucous cells within 45 min. This increase is also associated with the release of their secretory contents onto the surface, forming a thick slimy protective coating in an attempt to prevent deleterious effects of the detergent medium. Emptying of the goblet mucous cells results in their loss and consequent decrease in the density of the cells. It appears that new goblet cells are transformed from the existing epithelial cells in the outermost layer, as few mucous goblet cells could be observed developing in the basal or middle layers. The rate of loss of goblet mucous cells due to emptying and exhaustion is much faster than the rate of regeneration of new cells and hence, the number of goblet mucous cells at many stages drops down very low. Further, the goblet mucous cells develop in groups. Therefore, at any occasion, the number of goblet mucous cells increases suddenly and, in subsequent stages, these cells empty their contents and disappear in groups. Thus, there seems to be no continuity of goblet mucous cell regeneration as has been described by Agrawal et al. ( 1979) in Heteropneustes fossilis, where the hyperosmotic stress of 1% saline caused continual regeneration of cells in the lower layers, replacing those which are lost at the surface due to exhaustion.
The slow pace of increase and decrease in the number of goblet mucous cells in the later stages of the experiment suggests the disturbances in normal metabolic processes of mucous secretion and gross damage upsetting the normal physiology of the epider- mis prior to the death of the fish. Zaccone et a2. (1985a) has also marked an increase in the number of goblet mucous cells caused by a sublethal dose of an anionic deter-
270 DEBASISH ROY
gent (LAS). Many other external stresses, e.g., handling (Pickering and Macey, 1977) heavy metals (Westfall, 1945; McKone et al., 1971; Olson et a/., 1973; Varanasi et al., 1975; Lock and Van Overbeeke, 198 I), hormones (Mattheij and Stroband, 197 1; Marshall, 1979), salinity (Agrawal et al., 1979), and ionic calcium (Wendelaar-Bonga, 1978), also cause an increase in the density of the goblet mucous cells’in the gill and skin epithelium. However, there has been no previous research that includes detailed observations of the goblet mucous cells at different time intervals of detergent treatment.
Regarding the changes in the staining property of the contents ofthe goblet mucous cells under the influence of detergent, a large percentage of cells in the opercular epidermis and in the gill filament epithelium show a marked shift from acidic glyco- protein to neutral glycoprotein. However, at a later stage, many of the goblet mucous cells in the gill filament epithelium again stain for acidic glycoprotein and a few goblet cells stain for acidic glycosaminoglycans. The changes in staining behavior of the goblet mucous cells is not understood. It is probable that under stress conditions, the mucous cells may secrete acidic glycoprotein and acidic glycosaminoglycans which represents a pathological condition ofthe skin, such as is observed in mammals under stress conditions (Mawdesley-Thomas et al., 1971; Jones et al., 1973) and in fish (Gona, 1979).
CONCLUSION
The goblet mucous cells in the opercular epidermis and in the epithelium lining the gill arch and gill filament react suddenly to changed conditions of the external medium by introduction of detergent as is evidenced by profuse secretion by the cells. Statistically, the number per square millimeter of area and size of the cells decreases as a result of the secretion and subsequently empty and increase as a result of the synthesis and regeneration of new batches of cells. The goblet mucous cells develop in groups; therefore, the number at certain time intervals increases, whereas in the subsequent stage it decreases abruptly.
ACKNOWLEDGMENTS
Thanks are due to the Head of the Zoology Department, Bananas Hindu University, and the Principal Investigator, U.G.C. research project for necessary laboratory facilities and financial assistance in the form of Junior Research Fellowship.
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DETERGENT-INDUCED CHANGES IN Rita MUCOUS CELLS 271
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