ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY 17,59-66 (1989)

Detergent-Induced Changes in the Mapping of Certain Enzymes in Various Cell Types of Rita rita

I. Opercular Epidermis

DEBASISH ROY’

Department of Zoology, Banaras Hindu University, Varanasi-221005, India, and Central Marine Fisheries Research Institute, Post Box No. 2704, Ernakulam, Cochin, India

Received March 16, I988

The different cell types in the opercuiar epidermis of Riia rifa when exposed to a synthetic detergent, dodecylbenzene sodium sulfonate in the concentration of 6.9 mg liter-’ (96-hr LCsO of the detergent), had a gradual decrease in the activity of various enzymes responsible for different metabolic processes occurring inside the cell. After a certain period of detergent exposure, the activities of all the enzymes in these cells are completely inhibited. o 1989 Academic press, h.

INTRODUCTION

Detergent intoxication is one severe hazard to the aquatic environment caused by modern human activities. Abel ( 1974) reviewed various aspects of the influence of detergents on both freshwater and marine fishes. These works mainly focus on the histopathology of gill under the influence of detergent treatment. On the other hand, the literature regarding the harmful aspects of detergents on fish skin are insignificant (Bardach et al., 1965; Pohla-Gubo and Adam, 1982; Zaccone et al., 1985; Roy, 1988). Recently, Zaccone et al. ( 1985) discussed the histological and histochemical changes in the pattern ofthe enzymes malate: NAD-oxidoreductase, isocitrate: NAD-oxidore- ductase, lactate dehydrogenase, glucosed-phosphate dehydrogenase on the epider- mis of Heteropneustes fossilis induced by an anionic detergent, sodium alkyl benzene sulfonate. In the present investigation, an attempt has been made to examine the changes in the activities of several important enzymes, namely acid phosphatase, alkaline phosphatase, nonspecific esterase, Ca*+- and Mg*+-dependent ATPases, 5’- nucleotidase, glucose-6-phosphatase, succinate dehydrogenase, glucose-g-phosphate dehydrogenase, and monoamine oxidase engaged in various metabolic processes in the epidermis of a catfish, Rita rita, under the influence of an anionic detergent, dodecylbenzene sodium sulfonate.

MATERIALS AND METHODS

Fish R. rita ( 12- 16 cm in length) were bought from fishermen, who caught them using hooks and lines, in the month of January from the river Ganges in Varanasi, India. The fish then were acclimated to normal laboratory conditions for nearly 30 days prior to experimentation. They were fed minced goat liver on alternate days.

’ Present address for reprint requests and all communication: Debasish Roy, c/o Mr. R. N. Roy, 464, Saheednagar, Bhubaneswar-75 1 007, Orissa, India.

59 0147-6513/89$3.00 Copyright 0 1989 by Academic Press. Inc. All rights of reproduction in any form resewed.

60 DEBASISH ROY

Left over food and fecal material were siphoned out of the aquaria every morning. Only healthy fish with clean skin surfaces were used. A static bioassay test was per- formed to determine the 96-hr LC5,, of the anionic detergent to the fish, R. rita, fol- lowing the methods of APHA et al. (1980). Fish under control as well as experimental conditions kept in 6.9 mg liter-’ detergent (96-hr L&J and aquaria 2 X 1 X 1.5 ft were sacrificed at varying 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. Right opercula of the fish were dissected out and small epidermal layers were separated manually with needles, forceps, and scissors for frozen sectioning. For the demonstration of alkaline phosphatase, acid phospha- tase, and nonspecific esterase activities, the tissues were fixed for l-2 hr in cold (4°C) 10% neutral formalin and formal calcium. For other enzymes, however, fresh unfixed tissue blocks were subjected to sectioning. Frozen sections of freeze-dried (both fixed and unfixed) tissues were cut at lo-15 pm using an A0 (Model 849C) cryostat at -30°C.

Histochemical Procedure

The activities of alkaline phosphatases (ALP, EC 3.1.3.1) and acid phosphatase (ACP, EC 3.1.3.2) were demonstrated following Pearse (1968) and succinate dehy- drogenase (SDH, EC 1.3.99.1) and glucose-6-phosphate dehydrogenase (G-6-PDH, EC 1.1.1.49) following Pearse (1972). The a-naphthylacetate method of Gomori (1952) for nonspecific esterase (NSE, EC 3.1.1.1, 3.1.1.2, and 3.1.1.6) and lead method of Wachstein and Meisel(1957) for 5’nucleotidase (EC 3.1.3.5) and magne- sium-dependent adenosine triphosphatase (Mg*+ ATPase, EC 3.1.3.9) were em- ployed. For the demonstration of calcium-dependent adenosine triphosphatase (Ca*’ ATPase, EC 3.6.1.3) and monoamine oxidase (MAO, EC 1.4.3.4) the techniques of Padykula and Herman (1955) and Glenner et al. (1957), respectively, were applied.

Control reactions for all these enzymes were also performed by omitting the sub- strate from the incubating medium or subjecting the tissue sections to prior hot water vapor treatment for lo- 15 min or both.

RESULTS

Epithelial Cells

The peripheral zones of the epithelial cells of the outer and middle layers of the opercular epidermis exhibit strong activity for acid phosphatase and nonspecific es- terases. The basal cells of stratum germinativum, however, give a negative reaction for these enzymes. Moderate reaction for alkaline phosphatase could be observed only in the epithelial cells of the basal layer, whereas the outer and middle layer cells remain unstained. The nuclei of these cells at all the layers in the epidermis exhibit moderate activity for 5’-nucleotidase. The epithelial cells show a weak reaction for glucose-6-phosphatase, glucose-6-phosphate dehydrogenase, and Ca*+- and Mg*+-de- pendent ATPases and negative reaction for monoamine oxidase. A weak activity of succinate dehydrogenase is seen in the epithelial cells of the outer and the middle layer. The activity for this enzyme is comparatively stronger, being moderate in the basal layer (Table 1).

Twenty-four hours of detergent treatment induces a decrease in acid phosphatase, alkaline phosphatase, and 5’-nucleotidase activity while no reaction for these en-

DETERGENT-INDUCED CHANGES IN OPERCULAR EPIDERMIS 61

TABLE 1

A SUMMARY OF HISTOCHEMICAL REACTIONS SHOWING THE ACTIVITIES OF VARIOUS ENZYMES IN THE EPITHELIAL CELLS OF THE OPERCULAR EPIDERMIS OF Rim rita, AT

DIFFERENT TIME INTERVALS OF DETERGENT TREATMENT

Epithelial cells

Outer layer Middle layer Basal layer Histochemical Color

Enzymes techniques symbol C I 2 3 4to8 C 1 2 3 4to8 C I 2 3 4to8

Alkaline Azodye coupling phosphataae method

Control Acid Azodye coupling

phosphatase method Control

Nonspecific esterases

a-naphthylaeetate method

Control 5’-Nucleotidase Lead method

Control

Glucosed- Lead method phosphatase Control

Calcium- Calcium method dependent Control ATPase

Magnesium- Lead method dependent Control ATPase

Succinate Nitro-BT method dehydrogenase Control

Glucose-6- Nitro-BT method phosphate Control dehydrogenase

Monoamine Tetrazolium method oxidase Control

Br - --- - ++-- -

-

RBr

-

RBr

- BrB

-

BrB -

BrB

BrB -

Bl -

Bl -

++b +b - - -

++b +b *b - ~

+= *a-- -

* --- -

+a --- -

fa - -- -

+ -

f --- -

- --~ _ --~- -

++b +b - - _ - - - - -

+= *a - - ~ +a *a - - -

- --- - ---- _

2 - f--- -

+a - - - - 2’ - - - -

- --- - ---- -

fa --- - &a--- -

- --- - ---- -

I ! I - f--- -

2 - +--- - - --- _ --__ _

- --- - --__ _

Note. Symbols: a, cell nuclei; b, cell boundaries; Bl, blue; Br, brown; BrB, brownish black; RBr, reddish brown; -, negative: f, weak; +, moderate; ++, strong reactions; C, control conditions; 1 to 8, days of detergent treatment.

zymes, after 2 days, could be observed in the epithelial cells. However, the reaction for nonspecific esterases persists up to 2 days of detergent exposure. The effect of the detergent medium on the activity of glucose-6-phosphatase, succinate dehydroge- nase, and glucosed-phosphate dehydrogenase is rapid and so, within 24 hr of treat- ment the staining disappears completely from the epithelial cells. For monoamine oxidase, the epithelial cells remain unstained as do those of the control condition.

Goblet Mucus Cells

The peripheral areas of the goblet mucus cells in the opercular epidermis exhibit strong reaction for acid phosphatase and nonspecific esterases. A weak activity in the peripheral region is observed for alkaline phosphatase, calcium-dependent and

62 DEBASISH ROY

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64 DEBASISH ROY

magnesium-dependent ATPases, succinate dehydrogenase, and glucose-6-phosphate dehydrogenase. The secretory products demonstrate strong activity for acid phospha- tase and weak activity for glucose-6-phosphatase, whereas for alkaline phosphatase, nonspecific esterases, calcium- and magnesium-dependent ATPases, succinate dehy- drogenase, and glucosed-phosphate dehydrogenase, the bulk contents remain un- stained. The nuclei of the goblet mucus cells give moderate reaction for 5’-nucleo- tidase (Table 2).

The detergent treatment causes quick inhibition of the activity of alkaline phospha- tase, glucose-6-phosphatase, succinate dehydrogenase, and glucosed-phosphate de- hydrogenase. No immediate change in the activity of acid phosphatase and nonspe- cific esterases could be observed. The decrease in the acid phosphatase is well noted, being moderate in the secretory contents and slightly stronger in the peripheral cyto- plasm after 24 hr, whereas the peripheral region shows a decreased nonspecific ester- ases activity, i.e., moderate enzyme activity, after 48 hr. At 72 hr, the activities for both these enzymes become significantly weak and finally after 96 hr, no enzyme activity could be localized in any area of the goblet mucus cells. The decline in the activity of 5’-nucleotidase in the nuclei and calcium-dependent and magnesium-de- pendent ATPases in the periphery starts 24 hr after transferring the fish to the deter- gent medium and after, 48 hr onward, no activity for these enzymes was observed.

Club Cells

The cytoplasmic contents of the club cells exhibit moderate reaction for glucose- 6-phosphatase, glucosed-phosphate dehydrogenase, and alkaline phosphatase. Only the boundaries of the club cells of the opercular epidermis give weak reaction for calcium- and magnesium-dependent ATPases, whereas the nuclei of the cells have weak activity for 5’-nucleotidase. Reactions for acid phosphatase, nonspecific ester- ases, and monoamine oxidase were not observed in any part of the club cells of the opercular epidermis.

After the fish are transferred to the detergent medium, moderate glucosed-phos- phatase reaction in the contents of the club cells gradually decreases and becomes weak after 24 hr in the lower layer, whereas the activity of the enzyme remains the same in the outer layers up to 48 hr. In 72-hr treated tissues, the reaction becomes weak in all the layers and in subsequent stages of experimentation, the club cells in all the layers of opercular epidermis remain altogether unstained for glucose-6- phosphate dehydrogenase. The boundaries of the club cells give positive, although weak, reactions for both calcium- and magnesium-dependent ATPases even after 24 hr of detergent treatment and after this period, no activity for these enzymes could be localized throughout the duration of the experiment. The reactions for acid phos- phatase, succinate dehydrogenase, and monoamine oxidase in the club cells of the opercular epidermis could not be demonstrated at any stage after the start of the experiment.

DISCUSSION

Normal epithelial, club, and goblet mucus cells have enzyme activity for both cal- cium- and magnesium-dependent ATPases. The primary function of this enzyme is to convert ATP to ADP, releasing energy for metabolic processes. Zaccone and Licata ( 1982) correlated the presence of sodium-activated ATPase with its significant role

DETERGENT-INDUCED CHANGES IN OPERCULAR EPIDERMIS 65

in the process of maintenance of electrolyte balance in the milieu interior. Action of the detergent on the ATPase enzyme is so quick that the activity of the enzyme in all the epithelial cells throughout the epidermis stops instantly.

The presence of 5’-nucleotidase activity in the nuclei of various cell types in the opercular epidermis may relate to the dephosphorylation of nucleotides during the metabolism of nucleic acids. Due to the severe toxic action of the detergent, the en- zyme activity in the nuclei also deactivates. This also supports the view that the action of the detergent is through contact. Both acid phosphatase and nonspecific esterases, being hydrolytic in nature, actively take part in various catabolic processes. The pres- ence of these enzymes in the outer layer epithelial cells relates to their autolytic func- tion by which the older cells after completion of their cycle are exfoliated to make room for the new cells coming up from below to replace them. The present investiga- tion shows strong activity for acid phosphatase and nonspecific esterases in the pe- riphery of the goblet mucus cells of opercular epidermis. The activity of these en- zymes starts diminishing from the very first day due to the direct and immediate penetration of the detergent in the outer layer of the epidermis where the goblet mu- cus cells are mainly concentrated.

The presence of glucose-6-phosphatase in the epidermal cells indicates its role in releasing glucose for the routine metabolic processes including the synthesis of mu- tins. A lethal dose of detergent treatment completely deactivates the enzyme within 24 hr. Due to the cessation of this enzyme activity, the production of free glucose is considerably reduced or totally stopped, causing short supply of the chief energy source which, in turn, results in the total disorder and breakdown of the normal or routine physiological activities of these cells. Consequently, the cells not only lose the capability to sustain the toxic effect of the medium but also degenerate and die.

CONCLUSION

The present study observes either a gradual or a sudden decrease in the intensity of reaction for certain metabolically important enzymes in various cell types of the epidermis. There may be one or many probable reasons for such a change and hence confirmed inference on the mechanism of detergent action cannot be drawn unless similar studies in various other organs are conducted.

ACKNOWLEDGMENTS

Thanks are due to the University Grants Commission, New Delhi, for financial assistance and to the

Head of the Department of Zoology, Banaras Hindu University, for providing necessary laboratory facilities during the tenure of this work. Encouragement given by the Principal Investigator is also acknowledged.

REFERENCES

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

298. APHA, AWWA, AND WPCF (1980). Standard Methodsfor the Examination of Water and Waste Water,

15th ed. Washington, DC. BARDACH, J. E., FUJIYA, M., AND HOLL, A. (1965). Detergents: Effects on the chemical senses of the fish

Ictalurus natalis (le sueur). Science 148, 1605-1607. GLENNER, G. G., BURTNER, H. J., AND BROWN, G. W. (1957). Histochemical demonstration of mono-

amine oxidase activity by tetrazolium salt. J. Histochem. Cytochem. 5,59 l-600.

66 DEBASISH ROY

GOMORI, G. ( 1952). Microscopic Histochemistry: Principles and Practice. Univ. of Chicago Press, Chicago, IL.

PADYKULA, H. A., AND HERMAN, E. (1955). The specificity of the histochemical method for adenosine triphosphatase. J. Histochem. Cytochem. 3,170-195.

PEARSE, A. G. E. (1968). Histochemistry: Theoretical andApplied, 3rd ed., Vol. 1. Churchill, London. PEARSE, A. G. E. (1972). Histochemistry: Theoretical and Applied, Vol. 2. Churchill and Livingstone,

London. POHLA-GUBO, G., AND ADAM, H. (1982). Influence of the anionactive detergent Na-alkyl-benzenesulpho-

nate (LAS) on the head-epidermis of juvenile rainbow trout (Salmo gairdneri Richardson). Zoo/. Anz. 209,97-l 10.

ROY, D. (1988). Statistical analysis of anionic detergent-induced changes in the goblet mucous cells of opercular epidermis and gill epithelium of Rita rita (Ham.) (Bagridae: Pisces). Ecotoxicol. Environ. Saf 15,260-27 1.

WACHSTEIN, M., AND MEISEL, E. (1957). Histochemical localization of 5’-nucleotidase. J. Histochem. Cytochem. 5,200-220.

ZACCONE, G., AND LICATA, A. (1982). Histochemistry and fine structure of the flame cone cells in the skin epidermis of the sea horse fish Hippocampus ramulosus Leach 18 14 (Teleostei: Syngnathidae). Arch.

Biol. (Bruxelles) 93,249-266. ZACCONE, G., Lo CASCIO, P., FASULO, S., AND LICATA, A. (1985). The effect of an anionic detergent on

complex carbohydrates and enzyme activities in the epidermis of the catfish Heteropneustes fossilis

(Bloch). Histochem. J. 17,453-466.