impact of detergents on the protein histochemistry of various cell types of the gill epithelium of...

ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY 1$206-2 11 ( 1988)

Impact of Detergents on the Protein Histochemistry of Various Cell Types of the Gill Epithelium of Rita rita

DEBASISH ROY’

Department of Zoology, Banaras Hindu University, Varanasi-221005, India and Department of Zoology, Utkal University, Bhubaneswar, India

Received September 23, I98 7

Fish, Rita rita, were exposed to an anionic detergent, dodecylbenzene sodium sulfonate, 6.9 mg per litre of tap water (96~hr LCso of the detergent). A gradual decrease in the protein constituents of the major cell types, viz, the epithelial cells and the goblet mucous cells in the epithelium lining the gill arch, gill filament, and club cells present only in the gill arch epithelium has been observed by using a series of h&chemical techniques. B 1988 Academic FWZ, IDC.

INTRODUCTION

The widespread use of synthetic detergents, due to their lower cost and better wash- ing capability, has considerably added to the pollution of cultivable waters and has posed a great threat to the whole of the aquatic environment. Abel ( 1974) studied in detail earlier reports of the effects induced by synthetic detergents on various systems of aquatic organisms, including fish and microinvertebrates. This review infers that a considerable amount of literature exists on the histomorphological changes influ- enced by detergents in fish gill. Subsequently, Abel and Skidmore (1975), Abel (1976), Fukuda (1983), and Misra et al. (1985) shed light on the histopathological changes in gills caused by detergents at both light and electron microscopic levels. However, a detailed examination of histochemical alterations in the nature of different cell types, present in fish gills, has altogether been neglected. In this communica- tion, the changes effected by an anionic detergent, dodecylbenzene sodium sulfonate, in the protein constituents of various cell types of the gills of a freshwater teleost, Rita rita, have been incorporated.

MATERIALS AND METHODS

Live specimens of R. rita ( 12- 16 cm in length) were collected from the riversides of the Ganges at Varanasi, U.P., and were acclimated to optimum laboratory condi- tions for about 30 days before experimentation. Fish during acclimatization were fed with minced goat liver on alternate days. Fecal material and uneaten food were siphoned out of the aquaria every day. Static bioassay tests were performed as de- scribed in the Standard methods (APHA, AWWA, & WPCF, 15th edition) for evalu- ating the 96-hr LC5,, of an anionic detergent, dodecylbenzene sodium sulphonate. All the subsequent experiments were set at 6.9 mg litre-’ (96~hr LCX, of the detergent). Calculated amounts of detergent for 30 litres of water in aquaria of 2 X 1 X 1.5 ft were dissolved in a small amount of water and then thoroughly mixed into the aquaria water. Fish from both control and experimental aquaria were removed at

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206

DETERGENT-INDUCED CHANGES IN GILL EPITHELIUM 207

various time intervals (i.e., 15, 30, and 45 min, 1,2,3,6, and 12 hr, 1,2, 3,4, 5,6,7, and 8 days) and first the right gills were dissected and fixed in 10% neutral formalin. Tissue pieces (8 mm in length) were processed and sectioned at 5 pm. Sections were then stained with a series of histochemical techniques for the demonstration of different protein constituents in various cell types, present in gills.

Sections were also subjected to reactions for blocking protein end groups following Pearse (1968).

RESULTS

Epithelial Cells

Moderate reaction in the cytoplasmic material is observed with mercury bromphe- no1 blue for general proteins, with acid solochrome cyanine R for basic proteins, and with Millon’s reactions for tyrosine. The same areas of outer and middle layer epithelial cells of the gill arch epithelium as well as gill filament epithelium stain weakly with ninhydrin-SchifFs method for protein-bound -NH2 groups and with the Bis- mark brown Y technique for mucoproteins. The cell peripheries, with the DMAB nitrite technique, show a weak reaction for tryptophan. Due to the absence of cystein (-SH)-bound groups and cystine (-SS)-bound groups, the epithelial cells remain unstained with the DDD technique (Table 1). The basal cells of the gill arch epithelium showed more or less similar but slightly stronger reactions.

No change in the intensity of reactions with bromphenol blue for general proteins or with Ninhydrin-Schiffs method for protein-bound -NH2 groups could be observed in the outer and middle layer epithelial cells of the epithelium lining the gill arch up to the end of the experiment. The reaction for basic proteins with acid solochrome cyanine R remained the same even after 6 days of detergent treatment and no reaction for basic proteins could be located in the cells after this period. With the DMAB-nitrite technique for tryptophan, a positive reaction for epithelial cells in any layer could not be observed at any stage of experimentation.

The intensity of staining in the nuclei of the epithelial cells is reduced after 5 days and a weak reaction for the general proteins and basic nucleoproteins with the mercury bromphenol blue and Biebrich scarlet methods, respectively, persists until the end of the experiment (Table 1).

Goblet Mucous Cells

With the Bismark brown Y technique, goblet mucous cells of the epithelial lining of the gill arch weakly show the presence of small amounts of mucoproteins in their secretory contents. With various other techniques for proteins (Table I), the contents of the goblet mucous cells remain unstained (See Table 2.)

Transferring the fish into the detergent medium did not induce any signifi- cant change in the histoehemical property of the protein contents of the goblet mucous cells.

Club Cells

While the cytoplasmic contents of club cells of the epithelium lining the gill arch have a moderate reaction with the mercury bromphenol blue method for genera1 proteins, with Ninhydrin-Schiff s method for protein-bound -NH* groups, and with the acid solochrome cyanine R method for basic proteins, the nuclei and the cell

TABL

E 1

A SU

MM

ARY

OF

HIS

T~C

HEM

ICAL

C

HAN

GES

IN

F~O

TEIN

CO

NSTI

TUEN

TS

OF

EPIT

HEL

IAL

CELL

S IN

THE

GIL

L EP

ITH

ELIIJ

M

OF

Ritu

rit

a AT

VAR

IOU

S IN

TER

VALS

O

F DE

TERG

ENT

EX

PO

SU

RE

Epith

elial

ceUs

Outer

lay

er Mi

ddle

layer

Basa

l lay

er

Chem

ical

ccm

stitu

ents

Histo

chem

ical

techn

ique

Color

C

I 2

3 4

5 6

1 8

C 1

2 3

4 5

6 7

8 C

1 2

3 4

5 6

7 8

Gene

ral

pmtei

n

Basic

pro

teins

Ba

sic

0ucIe

opm

tein.s

Ty

msin

e

Tlypto

phan

Qstei

n bo

und

~ulry

dryl

(-SH)

gr

oups

Cysti

ne

boun

d dis

ulfide

(-S

S)

grou

ps

Muc

apm

teins

Hg-b

mm

phen

ol blu

e BI

Ninh

ydrin

/Sch

iff P

Deam

inatio

n/ninh

ydrin

/ -

Schil

T Ac

id so

lochr

ome

cyan

ine

R BI

Bie

brich

sc

arlet

S

Millio

n rea

ction

P

kdina

tion/M

illion

reacti

on

- DM

AEnit

rite

BI

Perfo

rmic

acid/

oxida

tion/

- DM

AB

nitrite

an

d iod

inatio

n/DM

AB

nitrite

Di

bydm

xydin

aphth

yl-

- dis

ultide

(D

DD)

Male

imide

ox

idatio

n/DDD

-

and

icdina

tion/D

DD

Petfo

rmic

acid/

AkGn

blu

e -

Perfo

rmic

acid/

oxida

tion/

- pe

rform

ic ac

id/Al&

n blu

e an

d thi

c&co

late

redU

CtiO

~lpn

form

iC

acid/

Alcian

blu

e Bi

smar

k br

own

Y

Br

r

__-

- -

- -

- --

:r +.

+*

f.

+.

- -

+. p

;:

+b +

b fb

*b

_+b

p +’

+b

+*

+a

+. +

. +.

+.

- -

+. +

. __

_-

- -

----

- -

- -

- -

- -

+*

- -

- _

- -

- -

- -

-

1:

;I +.

-

- 1:

;.I

*t

J *b

*b

+.

+.

+.

+

* +.

-

- -

- -

- -

- -

_ _

- -

- -

- -

- -

- -

- -

___-

_-

- -

---

----

--

- *.

-

- -

- -

- -

-

t+’ +a

t+’ +b

t+’ f=

*’

- -

- -

- _

- _

1:

;: +*

+.

-

- 1:

+b

+b

;I

*b

+b

!z

g p

+a

+.

+a

+.

+.

- 1

E -

- -

- -

- -

_ iz

-

- -

- -

_ -

_ -

- -

- -

- -

- 5

TABL

E 2

A SU

MM

ARYO

FHIS

TOC

HEM

ICAL

CH

ANG

ESIN

PRO

TEIN

CO

NST

ITU

ENTS

OFG

LAN

DC

ELLS

INTH

EGIL

LEPI

THEL

IUM

OF

Ritu

rita

ATV

AR

IOU

SIN

TER

VA

LSO

FDE

TER

GE

NTE

XP

~SU

RE

Club

cells

Goble

t HU

MUS

cells

outer

lay

er M

iddl

elay

eran

dbas

alla

yn

CO

kX

Chem

ical

cons

tituen

t Hi

stach

emica

itech

nique

sy

mbo

l C

1 2

3 4

5 67

8C

1234

5678

C I23

4567

8

Gene

ral

protei

ns

Hg-br

omph

enol

blue

F’m

tein

boun

d-NH1

Ni

nhyd

rin/S

cbiff

grou

ps

Deam

inatio

n/ninh

ydrin

/ sC

hi5

Basic

pro

teins

Ac

id so

lochr

ome

cyan

ine

R

Basic

nu

cleop

rotein

s Bie

brich

sc

arlet

Ty

msin

e

Tryp

topha

n

Cyste

in bo

und

sWdry

1 (-S

H)

groJ

Jps

Cysti

ne

boun

d dis

ulphid

e (-S

S)

grou

ps

Mien

re

actio

n Io

dina

tion/

Mill

onre

atiio

n D

MA

B-n

itrite

P

erfo

rmic

ac

id ox

idatio

n/ DM

AB

nitrite

an

d iad

inatio

n/DM

A&oit

rite

Diby

droxy

dinap

hthyl-

dis

ulphid

e (D

DD)

Male

imide

ox

idatio

n/DDD

an

d lod

inatio

n/DDD

Pe

rform

ic ac

id/Al&

n blu

e

Per

form

icac

idox

idal

ion/

pe

rfotic

ac

id/Alc

ian

blue

and

thiqt

lywlat

e re

ducti

on/

perfo

rmic

acid/

Al&n

blue

Bi.&

ark

brow

n Y

Bl

P - BI

s P - BI

- - - - BI

- -

-__

----

----

- -

- _

__

- -

--_

- P

tbd

;b - - - - - - -

- -

- -

- i’

9 +’

210 DEBASISH ROY

boundaries have a strong reaction with all these techniques. With Millon’s reaction for tyrosine, with Bismark brown Y for mucoproteins, and with DMAB-nitrite for tryptophan, the cytoplasmic contents of these cells show a weak reaction. However, only the nuclei of these cells exhibit a moderate reaction with the Biebrich scarlet method for basic nucleoproteins.

The contents and the nuclei of club cells in the deeper layer of the epithelium lining the gill arch stain comparatively less darkly with the mercury bromphenol blue and acid solochrome cyanine R methods.

Histochemical analysis does not indicate any substantial change in the staining behavior of the club cells of the epithelium lining the gill arch up to 2 days of detergent treatment. Subsequently, the intensity of the staining reaction decreases and a weak reaction for general proteins, basic proteins, and protein-bound -NH2 groups in the club cells of all the strata is left, after the third day onward, until the end of the experiment. Positive reaction for tyrosine with Millon’s reaction, for mucoproteins with Bismark brown Y, and for tryptophan with the DMAB-nitrite technique disappears after the fourth day.

The staining intensity with Biebrich scarlet for basic nucleoproteins becomes weak alter 2 days and continues to remain weak until the end of the experiment.

DISCUSSION

The present investigation observes a gradual decrease in the intensity ofthe staining reaction in different protein constituents of various cell types, present in the gill arch and gill filament epithelium, indicating the occurrence of certain changes in the chemistry of the protein moieties induced by the detergent.

The series of events and the mechanism of detergent poisoning leading to death of the fish are not yet known. The immediate cause ofdeath for acute detergent intoxica- tion where extensive gill damage occurs is likely to be either asphyxiation or loss of osmotic or ionic stability. But asphyxiation alone is never the only cause of fish death, rather, it is difficult to say whether asphyxiation is the primary cause associated with various other secondary factors or whether these secondary factors together give rise to the primary one.

Much research has been carried out in the past on the interaction of proteins and synthetic detergents Putnam (1948). It is established that detergents in low concentra- tion can alter proteins reversibly or irreversibly. At higher concentrations, when the detergent is largely in the form of micelles, detergents have the property of solubilizing organic material. Hotchkiss (1946), while discussing the bactericidal effects of synthetic detergents, suggested that a succession of events takes place, the first of which involves combination with oppositely charged ions on the bacterial sites. He believes this to be succeeded by membrane injury causing cytolysis, autolysis, possibly selec- tive inactivation of metabolic systems and eventual death. The possibility of a similar mechanism in fish gill epithelium may not be ignored. Helenius and Simon (1975) explained that the presence of highly charged proteins or glycoproteins, or a dense protein network on the surface of the biological membrane, may affect the penetra- tion, thereby facilitating the effects of detergents on the membrane. Few authors in the 1940s considered that synthetic detergents could produce their diverse effects by a combination with proteins primarily through the action of electrostatic forces.

DETERGENT-INDUCED CHANGES IN GILL EPITHELIUM 211

CONCLUSIONS

Synthetic detergents are known to exert disruptive effect on the gills of fish. This report, for the first time, aims at the study of change in the different protein moieties in various cell types of gill arch epithelium. The typical manner of decrease of staining reaction of protein constituents indicate the detergent action on the gill epithelium to be instant and the detergents may not only be exerting their effects through contact, but also may be penetrating through the membrane.

ACKNOWLEDGMENTS

Thanks are due to the University Grants Commission, New Delhi, and the concerned Principal Investi- gator for necessary facilities and financial assistance.

REFERENCES

ABEL, P. D. (1974). Toxicity of synthetic detergents to fish and aquatic invertebrates. J. Fish Biol. 6,279- 298.

ABEL, P. D. ( 1976). Toxic action of several lethal concentrations of an anionic detergent on the gills of the brown trout (Salmo trutta. L) J. Fish Biol. 9,441~446.

ABEL, P. D., AND SKIDMORE, J. F. (1975). Toxic effects of an anionic detergent on the gills of rainbow trout. Water Res. 9,759-765.

APHA, AWWA, AND WPCF (1980). Standard methods for the examination of water and waste water, 15th ed., Washington.

FUKUDA, Y. (1983). Specific reactions of gold fish gills exposed to linear alkylbenzene sulfonate. Japan. .I. Ichthyol. 30(3), 268-274.

HELENIUS, A., AND SIMON, K. ( 1975). Solubilization of membranes by detergents. B&him. Biophys. Acta 41529-79.

HOTCHKISS, R. D. (1946). The nature of the bacteriocidal action of surface active agents. Ann. N. Y. Acad. Sci. 46,479-493.

MISRA, V., LAL, H., CHAWLA, G., AND VISWANATHAN, P. N. (1985). Pathomorphological changes in the gills of fish fingerlings (Cirrhina mrigala) by linear alkylbenzene sulfonate. Ecotoxicol. Environ. Sat 10, 302-308.

PEAR%, A. G. E. (1968). Histochemistry: Theoretical and Applied, Vol. 1,3rd ed. Churchill, London. PUTNAM, F. W. (1948). The interactions of proteins and synthetic detergents. Adv. Protein Chem. 4, 79-

122.

impact of detergents on the protein histochemistry of various cell types of the gill epithelium of...

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