EcOTOXICOLOGYANDENVIRONMENTALSAFETYW,
22-32(1992)
Impact of Malathion and y-BHC on Lipid Metabolism in the Freshwater Female Catfish, Heteropneustes fossilis P. B. SINGH Reproductive Toxicology Laboratory, Postgraduate Department of Zoology, T. D. College, Jaunpur (U.P.), India Received September 25, 1990 Female Heteropneustes fossilis were exposed to sublethal concentrations of malathion (5 and 20 ~1 liter-‘) and r-BHC (4 and 16 I.cgliter-‘) for 4 weeks during different phases of their annual reproductive cycle. The impact of these pesticides on free fatty acids (FFA), monoglycerides (MG), diglycerides (DG), triglycerides (TG), phospholipids (PL), free cholesterol (CF), and ester&d cholesterol (CE) in the liver, plasma, and ovary was asses&. During the preparatory phase both the pesticides reduced the levels of all the hepatic and ovarian lipids with elevated hepatic CE. During the prespawning phase these pesticides decreased all the lipids in the liver, plasma, and ovary with the elevation of hepatic FFA and CE. During the spawning phase a reduction of hepatic MG and CF with a decreased level of plasma and ovarian levels of FFA and PL was recorded, whereas ovarian levels of TG and CE were elevated in response to both the pesticides. During postspawning and resting phases all the hepatic lipids were reduced with the elevation of CE in response to the exposure. These pesticides also restricted their mobilization to the ovary. Cholesterol biosynthesis seemed unaffected but the hydrolysis of CE to CF was adversely affected during preparatory and prespawning phases which is a period of sex steroid hormone biosynthesis. 0 1992 Academic
Press, Inc.
INTRODUCTION Toxic substances are known to suppress gonadal growth (Freeman and Idler, 1975; Kocan and Landolt, 1989; Dey and Bhattacharya, 1989; Gill et al., 1990), ovarian 3@-hydroxysteroid dehydrogenase activity (Kapoor et al., 1978), steroidogenesis (Sir@ and Singh, 1987; Singh and Singh, 199 la), and lipid metabolism (Lal and Sir@, 1987; Singh and Singh, 199 lb). In earlier reports (Singh and Singh, 1980; Murty and Devi, 1982) the authors have measured total lipids gravimetrically to observe the effect of pesticides, but these studies do not provide a clear picture of lipid metabolism, except for the report of Lal and Singh (1987), who have observed different lipid fractions in Clurius butruchusduring different phases of its annual reproductive cycle. These reports are restricted only to a single teleost. The aim of this experiment is to ascertain whether or not the pattern of lipid mobilization in response to pesticide exposure is like that of C. butruchus. Therefore, in the present study the effects of an organophosphate, malathion (S( 1,2dicarbethoxyethyl) O,O-dimethyldithio phosphate), and an organochlorine, yBHC (1,2,3,4,5,6-hexachlorocyclohexane), on lipid metabolism in the female catfish, Heteropneustesfossilis were studied by estimating free fatty acids, mono-, di-, and triglycerides (an energy source), phospholipids (a constituent of vitellogenin), free cholesterol (a steroid precursor), and esterified cholesterol in the liver, plasma, and ovary during different phases of its annual reproductive cycle, using chromatographic and spectrophotometric techniques. 0147-6513192 $3.00 Copyright 0 I992 by Academic Press, Inc. All rights of reproduction in any form nserved.
22
LIPID
METABOLISM
MATERIALS Experimental
IN RESPONSE
AND
TO
23
PESTICIDES
METHODS
Fish
Live female H. fossilis weighing 60-65 g and measuring 2 1-23 cm were collected from the ponds around Jaunpur (U.P.), India, during the first week of each reproductive phase (Table 1). They were brought to the laboratory for acclimation, kept in circulating dechlorinated tap water for 10 days prior to experimentation in each phase under a natural photoperiod and temperature, and fed standardized rations containing 2OY0 protein, 5% lipids, and 15% carbohydrates, the rest being 60% water, minerals, and vitamins, etc., on alternate days. During each phase, after acclimation, fish were divided into five batches, each having 10 individuals, in an aquarium of 90 liters capacity. Fish were exposed to sublethal concentrations (SL) of malathion (5 and 20 ~1 liter-‘) and y-BHC (4 and 16 pg liter-‘) for 4 weeks under static condition following procedures which had been reported (Singh and Singh, 1986). Malathion was added directly to the water but y-BHC was first dissolved in 1 ml of acetone and then added to the water. Control fish for malathion were kept in plain water, but for r-BHC, 1 ml of acetone was added in the aquarium water. During the experiment fish were fed on every 4th day, when the aquarium water was changed with freshwater having the same concentration of pesticides. Chemicals Lipid standards-palmitic acid, monopalmitin, dipalmitin, tripalmitin, lecithin, cholesterol, and cholesterol oleate-were purchased from Sigma (St. Louis, MO). Silica gel GhO was purchased from E. Merck (Darmstadt, Germany) and AR grade solvents (BDH, India) were distilled and used. y-BHC and malathion were obtained from Bharat Pulversing Mill Private Ltd., India, and Cynamid India Ltd., respectively. Lipid Extraction,
Separation, and Quantification
by Thin Layer Chromatography
At the end of the experiment, on the 29th day, five fish from each batch were bled by caudal puncture separately into heparinized tubes, blood was centrifuged at 4000 rpm for 15 min in a refrigerated centrifuge (IEC-B20A) at 4°C and plasma was then frozen at -20°C for further analysis. Individual livers and ovaries were extirpated, washed in 0.6% saline, blotted, and kept with plasma samples. Tissue and plasma TABLE
1
GONAMX~MATIC INDEX(GSI)OFFEMALEH.~O&~S BEFORE PESTICIDEEXPOSURE ANDPHOTOPERIODANDTEMPERATIJREDURINGEXPERIMENTATION
Reproductive phase
Date of collection of fish
GSI before pesticide exposure
Photoperiod
(“C)
Preparatory Prespawning Spawning Postspawning Resting
March 3 May 6 July 7 September 4
0.49 + 0.05 7.01 + 0.35
11.5L112.5D 13.OL:l l.OD
28 + 2 30 + 2
10.90 + 0.63 0.41 + 0.03
13.3L: 10.7D
31 t2
12.1L:11.9D 10.3L: 13.7D
26 -t 2 25 + 2
December
2
0.26 + 0.04
Temperature
24
P. B. SINGH
lipids were extracted in chlorofotmmethanol (2: 1) following the method of Folch et al. (1957). Triplicate samples of each tissue from a single specimen were taken for analysis. Each samples consisted of 50 mg of liver and ovary, and 100 ~1 of plasma. Different fractions of lipids were separated by thin layer chromatography (&-values for FFA, MG, DG, TG, PL, CF, and CE were 0.47, 0.25, 0.73, 0.78, 0.00, 0.62, and 0.94, respectively) using the double solvent system (System I, diethyletherbenzene: ethanol:acetic acid, 40:50:2:0.2; and System II, hexanediethylether, 94:6) of Freeman and West (1966). Spots of various lipids were made visible by exposing the plates to iodine vapor. Spots of different lipid fractions from the samples and standards, and corresponding areas of the silica gel from the blanks were scraped and transferred to separate test tubes. Quantitative estimation of various lipids was made spectrophotometrically on a Spectronic-2000 at 375 nm by the method of Matzo et al. ( 197 1). Data were expressed in mg/g of tissue or mg/ml plasma (mean f SEM). For statistical analysis of the data, analysis of variance and Newman-Keuls’ multiple range t test were employed, at the probability level of 0.05 (Bruning and Kintz, 1977). RESULTS The responses of different lipids to malathion and T-BHC caused appreciable change in various classes of lipids of the liver, plasma, and ovary. Results are shown in Figs. 1-7. Preparatory Phase During this phase, both pesticides suppressed the levels of FFA, MG, IX, TG, PL, and CF, and elevated CE was noticed in the liver. Plasma levels of FFA, MG, DG, TG, CF, and CE remained unchanged while decreased levels of PL were recorded after exposure to the pesticides. Ovarian levels of FFA, MG, DG, and CE were unaf fected while those of TG, PL, and CF were reduced. Prespawning Phase During this phase, both pesticides decreased the levels of MG, IX, TG, PL, and CF, while elevated levels of FFA and CE were recorded in the liver. In the plasma and ovary similarly low levels of TG, PL, and CF and elevated levels of FFA and CE were noticed, whereas MG and DG remained unaffected after pesticide exposure. Spawning Phase During this phase, these pesticides elevated the levels of FFA, MG, DG, TG, and PL, with a reduction in CF by malathion, while T-BHC could not alter the CF in the liver. In the plasma reduced levels of FFA, TG, and PL were noticed, while MG, DG, and CF remained unaffected by these pesticides. The levels of CE were enhanced by exposure to 4 and 16 pg liter-’ of y-BHC and 20 ~1 liter-’ of malathion. In the ovary, decreased levels of FFA and PL were recorded while TG and CE were elevated after pesticide exposure. No alteration in ovarian MG, DG, and CF was recorded after pesticide exposure.
LIPID METABOLISM
25
IN RESPONSE TO PESTICIDES I a •Kl QJ 01
CONTROL MALATHION 5ppm MALATHION 20ppm $-BHC 4 ppm $-BHC 16ppm
fl 3.00u al z 5 2.00ak w ‘W
iW
2 1.00 -
O.OOPREP
PRES
SPAW
POSTS
REST
FIG. 1. Changes in free fatty acids in the liver, plasma, and ovary in response to malathion and -y-BHC in female H. fossitis. PREP, preparatory phase; PRES, prespawning phase; SPAW, spawning phase; POSTS. postspawning phase; REST, resting phase. (X) Not significant at the level of 0.05.
Postspawning Phase During this phase, decreased levels of FFA, MG, DG, and TG and elevated levels of CF and CE were recorded in the liver after pesticide exposure, except for PL which was unaffected. Except for the elevated levels of plasma TG, ah the lipids remained unaltered in the plasma and ovary after exposure to both concentrations of pesticides. Resting Phase During this phase, after the exposure to both pesticides decreased levels of FFA, MG, TG, PL, and CF and elevated levels of CE were recorded in the liver. Except for the increased levels of ovarian FFA, all the studied lipids remained unchanged in the plasma and ovary after pesticide exposure.
26
P. B. SINGH lllIl0 CONTROL 6Y MALATHION •l MALATHION
r
5ppm ZOppm
5 0.60g d0 (x60aFi ?r i 0.30-
o.ooPREP
PRES
SPAW
POSTS
REST
FIG.2. Changes in monoglycerides in the liver, plasma, and ovary in response to malathion and y-BHC in female H. fossilis. PREP, preparatory phase; PRES, prespawning phase; SPAW, spawning phase; POSTS, postspawning phase; REST, resting phase. (X) Not significant at the level of 0.05.
DISCUSSION During the preparatory phase of normal female H. fossilis, the liver shows major lipogenic activity, with little or no mobilization of hepatic lipids to the ovary (Singh and Singh, 1990). A reduction in hepatic FFA, MG, DG, TG, PL, and CF with an elevation in CE after exposure to the pesticide suggests an inhibition of lipogenic activity in this species. Further a drop in hepatic levels of CF with the elevation of CE suggests that the hydrolysis of CE to CF is prevented, concomitantly hepatic CF is esterified into CE which is evident from the decrease in the levels of hepatic and ovarian CF by these pesticides which is required for steroidogenesis. Lal and Singh (1987) have observed similar trends in lipid mobilization from the liver to the ovary via the plasma in a similar catfish, C. batrachus, but they could not find any change
LIPID METABOLISM
27
IN RESPONSE TO PESTICIDES P
CONTROL
k3 MALATHION c87 MALATHION
0.80
5ppm 20ppm
;i 0.60 E s x 0.40 z T
0.20 I
5as? 0.00
.I: 5
r o&o-
F g
0.40-
F 4 E
0.20-
i.5 : s
o.oo-
ii ;
2.00
W v 2 (3 0
1.50-
-
1.00
-
0.50
-
O.OOL PREP
PRES
SPAW
POSTS
REST
FIG. 3. Changes in diglycerides in the liver, plasma, and ovary in response to malathion and y-BHC in female H. fossilis. PREP, preparatory phase; PRES, prespawning phase; SPAW. spawning phase; POSTS, postspawning phase; REST, resting phase. (X) Not significant at the level of 0.05.
in ovarian CF. Kling et al. (1978) have reported that the conversion of acetate to fatty acids in the rat liver was slowed by the PCB Aroclor 1254. Similarly Dzogbefia et al. ( 1978) have also demonstrated decreased glyceride biosynthesis in the livers of PCBtreated rats in vitro. During the prespawning phase, increased hepatic FFA with a reduction in MG, DG, and TG in H. fossilis suggests that the biosynthesis of FFA is not impaired; only the conversion of FFA to MG, DG, and TG is arrested by exposure to these pesticides. Elevated levels of plasma and ovarian FFA suggest that FFA is transferred to the ovary via the plasma, hence biosynthesis is not impaired. Our findings are somewhat different from those reported by Lal and Singh (1987) for the same pesticide in C. batruchus, namely, decreased levels of ovarian FFA. The TG concentration in H. fossilis after pesticide exposure decreased in all the studied tissue, whereas increased ovarian TG was recorded in C. batrachus by La1 and Singh (1987). Decreased levels of hepatic
28
P. B. SINGH IllllO CONTROL EZi MALATHION 5ppm •B] MALATHION 20ppm 0 ?-BHC lppm q 3-BHC 16ppm
9. % 4’ z-
n 5E 3.00
& 3
6. :
PREP
PRES
SPAW
POSTS
REST
FIG. 4. Changes in triglycerides in the liver, plasma, and ovary in response to malathion and -y-BHC in female H. fossilis. PREP, preparatory phase; PRES, prespawning phase; SPAW, spawning phase; POSTS, postspawning phase; REST, resting phase. (X) Not significant at the level of 0.05.
DG and PL in response to these pesticides suggest that the conversion of DG into PL is inhibited. Dzogbefia et al. (1978) have also reported decreased PL biosynthesis in the liver of PCB-treated rat. Earlier reports by Wiegand and Peter (1980a, 1980b) indicated that the teleost ovary imports various lipids from the liver during the vitellogenic phase, involving the sex hormones and gonadotropins. Hence it is likely that inhibited mobilization of these various lipids from the liver to the ovary is caused by these pesticides, by decreasing the sex hormones and gonadotropin. This suggestion gets support from the work of Kapoor et al. (1978), Singh and Singh (1980), and Singh and Singh ( 1987), who have found reduced 3/3-HSD in Cyprzhus carpio, gonadotrophic potency in H. fossilis, and decreased sex hormones in C. batrachus, respectively. Further, high levels of CE, along with a reduced level of CF in the liver, plasma, and ovary of pesticide-treated fish, suggest that these pesticides might have checked the conversion of CE to its free form, which probably leads to decreased steroid production in this species (Sir@ and Sir@, 199 la) and in C. batrach (Singh and Singh, 1987).
LIPID METABOLISM
29
IN RESPONSE TO PESTICIDES CONTROL MALATHION
5ppm
MALATHION
20ppm
q-BHC ?-BHC
.” *
CIppm 16ppm
r
PREP
PRES
SPAW
POSTS
REST
FIG. 5. Changes in phospholipids in the liver, plasma, and ovary in response to malathion and YBHC in female H. fossilis. PREP, preparatory phase; PRES, prespawning phase; SPAW, spawning phase; POSTS, postspawning phase; REST, resting phase. (x) Not significant at the level of 0.05.
During the spawning phase, the elevation in hepatic TG after malathion and yBHC exposure can be attributed to a decrease in their use. Under natural conditions, H. fossilis reduces its food intake during the prespawning phase and depends mainly on stored lipids for the energy required for spawning. A decreased level of FFA in the ovary, with an increased level of TG, also suggests a decrease in the hydrolysis of TG to FFA and ultimately in the &oxidation of the latter to yield metabolic water, as well as ATP. A failure of ovulation in response to exposure to these pesticides may also result from inadequate ovarian hydration. Hydration of the ovary normally precedes ovulation (Hirose, 1976; Greeley et al., 1986). The effect of these pesticide on PL, CF, and CE metabolism appears to be similar to that found during the prespawning phase. During the postspawning and resting phases the impact of these pesticides on lipid metabolism seems to be restricted to the liver, where they arrest hepatic lipogenesis.
30
P. B. SINGH IIll CONTROL kXi7 MALATHION CxI MALATHION fiB 3-BHC q 3-BHC
aooPREP
PRES
SPAW
POSTS
5ppm 20ppm
Ippm 16ppm
REST
FIG. 6. Changes in free cholesterol in the liver, plasma, and ovary in response to malathion and -y-BHC in female H. fossilis. PREP, preparatory phase; PRES, prespawning phase; SPAW, spawning phase; POSTS, postspawning phase; REST, resting phase. (X) Not significant at the level of 0.05.
CONCLUSION The present findings clearly demonstrate that malathion and y-BHC both significantly alter lipid metabolism and have very selective and specific effects on various classes of lipids in H. fossilis during different phases of the annual reproductive cycle. Cholesterol biosynthesis is not impaired by these pesticides but the conversion.of CE to CF is checked. The mobilization of various hepatic lipids to the ovary is also restricted by both pesticides. The impact of these pesticides in H: fossilis followed a pattern of lipid mobilization similar to that in C. batruchus with some differences in one or two specific classes of lipids. Thus it appears that these pesticides interfere with the production of lipid-deprived energy, vitellogenin, and sex hormone precursor, which ultimately results in decreased breeding potential and prevention of spawning in the female H. fossilis.
LIPID METABOLISM
31
IN RESPONSE TO PESTICIDES q CONTROL q MALATHION a
MALATHION
fZ?d ?-BHC •l %BHC
Sppm 20ppm
lppm 16ppm
z e 1.00 I .g 0.00
P z! 1.205 ij 0.80n 2 g OAO0
v i
o.oo-
0.00 -
PREP
PRES
SPAW
POSTS
REST
FIG. 7. Changes in esterified cholesterol in the liver, plasma, and ovary in response to malathion and yBHC in female H. fossilis. PREP, preparatory phase; PRES, prespawning phase; SPAW, spawning phase; POSTS, postspawning phase; REST, resting phase. (X) Not significant at the level of 0.05.
ACKNOWLEDGMENTS We are grateful to Dr. P. K. Singh, Principal, T. D. College, Jaunpur (U.P.), for providing laboratory facilities, and financial assistance from a UGC minor research project (26-1(743)/89(SR-IV)) dated August 26, 1989, to P.B.S. is gratefully acknowledged.
REFERENCES BRUNING, J. L., AND K~NTZ, B. L. (1977). Computational Handbook of Statistics, 2nd ed. Scott, Foresman, Glenview, IL. DEY, S., AND BHATTACHARYA, S. (1989). Ovarian damage to Channa punctatus after chronic exposure to low concentrations of elsan, mercury, and ammonia. Ecotoxicol. Environ. Saf 17,247-257. DZOGBEFIA, V. P., KLING, D., AND GAMBLE, W. (1978). Polychlorinated biphenyk In vitro and in vitro modifications of phospholipid and glycerides biosynthesis. J. Environ. Pathol. Toxicol. 1, 841-856. FOLCH, J., LEES, M., AND STANLEY, G. H. S. (1957). A simple method for isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226,497-507.
32
P. B. SINGH
C. P., AND WEST, D. (1966). Complete separation of lipid classes on a single thin layer plates. J. Lipid Res. 7, 324-327. FREEMAN, H. C., AND IDLER, D. R (1975). The effect of polychlorinated biphenyl on steroidogenesis and reproduction in the brook trout Salvelinus fontinalis. Can. J. B&hem. 53,666-670. GILL, T. S., PANDE, J., AND TEWARI, H. (1990). Sub-lethal effects of an organophosphorus insecticide on certain metabolite levels in a freshwater fish, Puntius conchonius. Pestic. B&hem. Physiol. 36(3), 290299. GREELEY, M. S., CALDER, D. R., AND WALLACE, R. A. (1986). Changes in teleost yolk protein during oocyte maturation: Correlation of yolk proteolysis with oocyte hydration. Comp. Biochem. Physiol. B 84, 1-11. HIROSE, K. (1976). Endocrine control of ovulation in Medaka (Oryzias latipes) and Ayu (Plecoglossus altivelis). J. Fish. Res. Board Can. 33, 989-994. KAPOOR, K., KAMALDEEP, K., AND TOOR, H. S. (1978). The effect of fenitrothion on reproduction of a teleost fish Cyprinus carpio communis Linn: A biochemical study. Bull. Environ. Contam. Toxicol. 20, 438-442. KLING, D., KUNKLE, J., ROLLER, A. S., AND GAMBLE, W. (1978). Polychlorinated biphenyl: In vivo and in vitro modifications of cholesterol and fatty acid biosynthesis. J. Environ. Pathol. Toxicol. 1, 8 13-828. KOCAN, R. M., AND LANDOLT, M. L. (1989). Survival and growth to reproductive maturity of coho salmon following embryonic exposure to a model toxicant. Mar. Environ. Res. 27, 177-194. LAL, B., AND SINGH, T. P. (1987). The effect of malathion and y-BHC on the lipid metabolism in relation to reproduction in the tropical teleost, Clarias batrachus. Environ. Pollut. 48, 37-47. MARZO, A., GHRARCH, P., SERDINI, D., AND MJZRONI,D. (197 I). Simplified measurement of monoglycerides, diglycerides, triglycerides and fatty acids in biological samples. Clin. Chem. 17(3), 145-147. MURTY, A. S., AND DEVI, A. P. (1982). The effect of endosulfan and its isomers on tissue protein glycogen and lipids in the fish, Channa punctatus. Pestic. B&hem. Physiol 17,280-286. SINGH, H., AND SINGH, T. P. (1980). Effect of two pesticides on total lipid and cholesterol content of ovary, liver and blood serum during different phases of the annual reproductive cycle in the freshwater teleost, Heteropneustes fossilis (Bloch). Environ. Pollut. 23, 9- 17. SINGH, P. B., AND SINGH, T. P. (1986). Pesticide induced impairment of lipid metabolism, steroidogenesis and gonadal recrudescence during preparatory phase of its annual reproductive cycle in the freshwater female catfish, Heteropneustes fossilis. Proceedings, Sixth All India Seminar on Ichthyology, December 21-24, 1986. Manipur University, India. SINGH, S., AND SINGH, T. P. (1987). Impact of malathion and hexachlorocyclohexane on plasma profiles of three sex hormones during different phases of the reproductive cycle in Clarias batrachus. Pestic. Biochem. Physiol. 27,301-308. SINGH, P. B., AND SINGH, T. P. (1990). Seasonal correlative changes between sex steroids and lipid levels in the freshwater female catfish, Heteropneustes fossilis. J Fish Biol. 37, 793-802. SINGH, P. B., AND SINGH, T. P. (1991a). Impact of r-BHC on sex steroids level and their modulation by ovine luteinizing hormone-releasing hormone and Mystus gonadotropin in the freshwater catfish, Heteropneustes fossilis. Aquat. Toxicol., in press. SINGH, P. B., AND SINGH, T. P. (1991b). Impact of r-BHC on the lipid class levels and their modulation by reproductive hormones in the freshwater cat&h, Heteropne-ustesfossilis. Bull. Environ. Contam. Toxicol., in press. WIEGAND, M. D., AND PETER, R. E. (1980a). Effect of salmon gonadotropin (SG-100) on plasma lipids in the goldfish, Carassius auratus. Can. J. Zool. B&957-966. WIEGAND, M. D., AND PETER, R. E. ( 1980b). Effect of sex steroids on plasma lipids in the goldfish, Carassius auratus. Can. J. Zool. 58,967-979. FREEMAN,
22-32(1992)
Impact of Malathion and y-BHC on Lipid Metabolism in the Freshwater Female Catfish, Heteropneustes fossilis P. B. SINGH Reproductive Toxicology Laboratory, Postgraduate Department of Zoology, T. D. College, Jaunpur (U.P.), India Received September 25, 1990 Female Heteropneustes fossilis were exposed to sublethal concentrations of malathion (5 and 20 ~1 liter-‘) and r-BHC (4 and 16 I.cgliter-‘) for 4 weeks during different phases of their annual reproductive cycle. The impact of these pesticides on free fatty acids (FFA), monoglycerides (MG), diglycerides (DG), triglycerides (TG), phospholipids (PL), free cholesterol (CF), and ester&d cholesterol (CE) in the liver, plasma, and ovary was asses&. During the preparatory phase both the pesticides reduced the levels of all the hepatic and ovarian lipids with elevated hepatic CE. During the prespawning phase these pesticides decreased all the lipids in the liver, plasma, and ovary with the elevation of hepatic FFA and CE. During the spawning phase a reduction of hepatic MG and CF with a decreased level of plasma and ovarian levels of FFA and PL was recorded, whereas ovarian levels of TG and CE were elevated in response to both the pesticides. During postspawning and resting phases all the hepatic lipids were reduced with the elevation of CE in response to the exposure. These pesticides also restricted their mobilization to the ovary. Cholesterol biosynthesis seemed unaffected but the hydrolysis of CE to CF was adversely affected during preparatory and prespawning phases which is a period of sex steroid hormone biosynthesis. 0 1992 Academic
Press, Inc.
INTRODUCTION Toxic substances are known to suppress gonadal growth (Freeman and Idler, 1975; Kocan and Landolt, 1989; Dey and Bhattacharya, 1989; Gill et al., 1990), ovarian 3@-hydroxysteroid dehydrogenase activity (Kapoor et al., 1978), steroidogenesis (Sir@ and Singh, 1987; Singh and Singh, 199 la), and lipid metabolism (Lal and Sir@, 1987; Singh and Singh, 199 lb). In earlier reports (Singh and Singh, 1980; Murty and Devi, 1982) the authors have measured total lipids gravimetrically to observe the effect of pesticides, but these studies do not provide a clear picture of lipid metabolism, except for the report of Lal and Singh (1987), who have observed different lipid fractions in Clurius butruchusduring different phases of its annual reproductive cycle. These reports are restricted only to a single teleost. The aim of this experiment is to ascertain whether or not the pattern of lipid mobilization in response to pesticide exposure is like that of C. butruchus. Therefore, in the present study the effects of an organophosphate, malathion (S( 1,2dicarbethoxyethyl) O,O-dimethyldithio phosphate), and an organochlorine, yBHC (1,2,3,4,5,6-hexachlorocyclohexane), on lipid metabolism in the female catfish, Heteropneustesfossilis were studied by estimating free fatty acids, mono-, di-, and triglycerides (an energy source), phospholipids (a constituent of vitellogenin), free cholesterol (a steroid precursor), and esterified cholesterol in the liver, plasma, and ovary during different phases of its annual reproductive cycle, using chromatographic and spectrophotometric techniques. 0147-6513192 $3.00 Copyright 0 I992 by Academic Press, Inc. All rights of reproduction in any form nserved.
22
LIPID
METABOLISM
MATERIALS Experimental
IN RESPONSE
AND
TO
23
PESTICIDES
METHODS
Fish
Live female H. fossilis weighing 60-65 g and measuring 2 1-23 cm were collected from the ponds around Jaunpur (U.P.), India, during the first week of each reproductive phase (Table 1). They were brought to the laboratory for acclimation, kept in circulating dechlorinated tap water for 10 days prior to experimentation in each phase under a natural photoperiod and temperature, and fed standardized rations containing 2OY0 protein, 5% lipids, and 15% carbohydrates, the rest being 60% water, minerals, and vitamins, etc., on alternate days. During each phase, after acclimation, fish were divided into five batches, each having 10 individuals, in an aquarium of 90 liters capacity. Fish were exposed to sublethal concentrations (SL) of malathion (5 and 20 ~1 liter-‘) and y-BHC (4 and 16 pg liter-‘) for 4 weeks under static condition following procedures which had been reported (Singh and Singh, 1986). Malathion was added directly to the water but y-BHC was first dissolved in 1 ml of acetone and then added to the water. Control fish for malathion were kept in plain water, but for r-BHC, 1 ml of acetone was added in the aquarium water. During the experiment fish were fed on every 4th day, when the aquarium water was changed with freshwater having the same concentration of pesticides. Chemicals Lipid standards-palmitic acid, monopalmitin, dipalmitin, tripalmitin, lecithin, cholesterol, and cholesterol oleate-were purchased from Sigma (St. Louis, MO). Silica gel GhO was purchased from E. Merck (Darmstadt, Germany) and AR grade solvents (BDH, India) were distilled and used. y-BHC and malathion were obtained from Bharat Pulversing Mill Private Ltd., India, and Cynamid India Ltd., respectively. Lipid Extraction,
Separation, and Quantification
by Thin Layer Chromatography
At the end of the experiment, on the 29th day, five fish from each batch were bled by caudal puncture separately into heparinized tubes, blood was centrifuged at 4000 rpm for 15 min in a refrigerated centrifuge (IEC-B20A) at 4°C and plasma was then frozen at -20°C for further analysis. Individual livers and ovaries were extirpated, washed in 0.6% saline, blotted, and kept with plasma samples. Tissue and plasma TABLE
1
GONAMX~MATIC INDEX(GSI)OFFEMALEH.~O&~S BEFORE PESTICIDEEXPOSURE ANDPHOTOPERIODANDTEMPERATIJREDURINGEXPERIMENTATION
Reproductive phase
Date of collection of fish
GSI before pesticide exposure
Photoperiod
(“C)
Preparatory Prespawning Spawning Postspawning Resting
March 3 May 6 July 7 September 4
0.49 + 0.05 7.01 + 0.35
11.5L112.5D 13.OL:l l.OD
28 + 2 30 + 2
10.90 + 0.63 0.41 + 0.03
13.3L: 10.7D
31 t2
12.1L:11.9D 10.3L: 13.7D
26 -t 2 25 + 2
December
2
0.26 + 0.04
Temperature
24
P. B. SINGH
lipids were extracted in chlorofotmmethanol (2: 1) following the method of Folch et al. (1957). Triplicate samples of each tissue from a single specimen were taken for analysis. Each samples consisted of 50 mg of liver and ovary, and 100 ~1 of plasma. Different fractions of lipids were separated by thin layer chromatography (&-values for FFA, MG, DG, TG, PL, CF, and CE were 0.47, 0.25, 0.73, 0.78, 0.00, 0.62, and 0.94, respectively) using the double solvent system (System I, diethyletherbenzene: ethanol:acetic acid, 40:50:2:0.2; and System II, hexanediethylether, 94:6) of Freeman and West (1966). Spots of various lipids were made visible by exposing the plates to iodine vapor. Spots of different lipid fractions from the samples and standards, and corresponding areas of the silica gel from the blanks were scraped and transferred to separate test tubes. Quantitative estimation of various lipids was made spectrophotometrically on a Spectronic-2000 at 375 nm by the method of Matzo et al. ( 197 1). Data were expressed in mg/g of tissue or mg/ml plasma (mean f SEM). For statistical analysis of the data, analysis of variance and Newman-Keuls’ multiple range t test were employed, at the probability level of 0.05 (Bruning and Kintz, 1977). RESULTS The responses of different lipids to malathion and T-BHC caused appreciable change in various classes of lipids of the liver, plasma, and ovary. Results are shown in Figs. 1-7. Preparatory Phase During this phase, both pesticides suppressed the levels of FFA, MG, IX, TG, PL, and CF, and elevated CE was noticed in the liver. Plasma levels of FFA, MG, DG, TG, CF, and CE remained unchanged while decreased levels of PL were recorded after exposure to the pesticides. Ovarian levels of FFA, MG, DG, and CE were unaf fected while those of TG, PL, and CF were reduced. Prespawning Phase During this phase, both pesticides decreased the levels of MG, IX, TG, PL, and CF, while elevated levels of FFA and CE were recorded in the liver. In the plasma and ovary similarly low levels of TG, PL, and CF and elevated levels of FFA and CE were noticed, whereas MG and DG remained unaffected after pesticide exposure. Spawning Phase During this phase, these pesticides elevated the levels of FFA, MG, DG, TG, and PL, with a reduction in CF by malathion, while T-BHC could not alter the CF in the liver. In the plasma reduced levels of FFA, TG, and PL were noticed, while MG, DG, and CF remained unaffected by these pesticides. The levels of CE were enhanced by exposure to 4 and 16 pg liter-’ of y-BHC and 20 ~1 liter-’ of malathion. In the ovary, decreased levels of FFA and PL were recorded while TG and CE were elevated after pesticide exposure. No alteration in ovarian MG, DG, and CF was recorded after pesticide exposure.
LIPID METABOLISM
25
IN RESPONSE TO PESTICIDES I a •Kl QJ 01
CONTROL MALATHION 5ppm MALATHION 20ppm $-BHC 4 ppm $-BHC 16ppm
fl 3.00u al z 5 2.00ak w ‘W
iW
2 1.00 -
O.OOPREP
PRES
SPAW
POSTS
REST
FIG. 1. Changes in free fatty acids in the liver, plasma, and ovary in response to malathion and -y-BHC in female H. fossitis. PREP, preparatory phase; PRES, prespawning phase; SPAW, spawning phase; POSTS. postspawning phase; REST, resting phase. (X) Not significant at the level of 0.05.
Postspawning Phase During this phase, decreased levels of FFA, MG, DG, and TG and elevated levels of CF and CE were recorded in the liver after pesticide exposure, except for PL which was unaffected. Except for the elevated levels of plasma TG, ah the lipids remained unaltered in the plasma and ovary after exposure to both concentrations of pesticides. Resting Phase During this phase, after the exposure to both pesticides decreased levels of FFA, MG, TG, PL, and CF and elevated levels of CE were recorded in the liver. Except for the increased levels of ovarian FFA, all the studied lipids remained unchanged in the plasma and ovary after pesticide exposure.
26
P. B. SINGH lllIl0 CONTROL 6Y MALATHION •l MALATHION
r
5ppm ZOppm
5 0.60g d0 (x60aFi ?r i 0.30-
o.ooPREP
PRES
SPAW
POSTS
REST
FIG.2. Changes in monoglycerides in the liver, plasma, and ovary in response to malathion and y-BHC in female H. fossilis. PREP, preparatory phase; PRES, prespawning phase; SPAW, spawning phase; POSTS, postspawning phase; REST, resting phase. (X) Not significant at the level of 0.05.
DISCUSSION During the preparatory phase of normal female H. fossilis, the liver shows major lipogenic activity, with little or no mobilization of hepatic lipids to the ovary (Singh and Singh, 1990). A reduction in hepatic FFA, MG, DG, TG, PL, and CF with an elevation in CE after exposure to the pesticide suggests an inhibition of lipogenic activity in this species. Further a drop in hepatic levels of CF with the elevation of CE suggests that the hydrolysis of CE to CF is prevented, concomitantly hepatic CF is esterified into CE which is evident from the decrease in the levels of hepatic and ovarian CF by these pesticides which is required for steroidogenesis. Lal and Singh (1987) have observed similar trends in lipid mobilization from the liver to the ovary via the plasma in a similar catfish, C. batrachus, but they could not find any change
LIPID METABOLISM
27
IN RESPONSE TO PESTICIDES P
CONTROL
k3 MALATHION c87 MALATHION
0.80
5ppm 20ppm
;i 0.60 E s x 0.40 z T
0.20 I
5as? 0.00
.I: 5
r o&o-
F g
0.40-
F 4 E
0.20-
i.5 : s
o.oo-
ii ;
2.00
W v 2 (3 0
1.50-
-
1.00
-
0.50
-
O.OOL PREP
PRES
SPAW
POSTS
REST
FIG. 3. Changes in diglycerides in the liver, plasma, and ovary in response to malathion and y-BHC in female H. fossilis. PREP, preparatory phase; PRES, prespawning phase; SPAW. spawning phase; POSTS, postspawning phase; REST, resting phase. (X) Not significant at the level of 0.05.
in ovarian CF. Kling et al. (1978) have reported that the conversion of acetate to fatty acids in the rat liver was slowed by the PCB Aroclor 1254. Similarly Dzogbefia et al. ( 1978) have also demonstrated decreased glyceride biosynthesis in the livers of PCBtreated rats in vitro. During the prespawning phase, increased hepatic FFA with a reduction in MG, DG, and TG in H. fossilis suggests that the biosynthesis of FFA is not impaired; only the conversion of FFA to MG, DG, and TG is arrested by exposure to these pesticides. Elevated levels of plasma and ovarian FFA suggest that FFA is transferred to the ovary via the plasma, hence biosynthesis is not impaired. Our findings are somewhat different from those reported by Lal and Singh (1987) for the same pesticide in C. batruchus, namely, decreased levels of ovarian FFA. The TG concentration in H. fossilis after pesticide exposure decreased in all the studied tissue, whereas increased ovarian TG was recorded in C. batrachus by La1 and Singh (1987). Decreased levels of hepatic
28
P. B. SINGH IllllO CONTROL EZi MALATHION 5ppm •B] MALATHION 20ppm 0 ?-BHC lppm q 3-BHC 16ppm
9. % 4’ z-
n 5E 3.00
& 3
6. :
PREP
PRES
SPAW
POSTS
REST
FIG. 4. Changes in triglycerides in the liver, plasma, and ovary in response to malathion and -y-BHC in female H. fossilis. PREP, preparatory phase; PRES, prespawning phase; SPAW, spawning phase; POSTS, postspawning phase; REST, resting phase. (X) Not significant at the level of 0.05.
DG and PL in response to these pesticides suggest that the conversion of DG into PL is inhibited. Dzogbefia et al. (1978) have also reported decreased PL biosynthesis in the liver of PCB-treated rat. Earlier reports by Wiegand and Peter (1980a, 1980b) indicated that the teleost ovary imports various lipids from the liver during the vitellogenic phase, involving the sex hormones and gonadotropins. Hence it is likely that inhibited mobilization of these various lipids from the liver to the ovary is caused by these pesticides, by decreasing the sex hormones and gonadotropin. This suggestion gets support from the work of Kapoor et al. (1978), Singh and Singh (1980), and Singh and Singh ( 1987), who have found reduced 3/3-HSD in Cyprzhus carpio, gonadotrophic potency in H. fossilis, and decreased sex hormones in C. batrachus, respectively. Further, high levels of CE, along with a reduced level of CF in the liver, plasma, and ovary of pesticide-treated fish, suggest that these pesticides might have checked the conversion of CE to its free form, which probably leads to decreased steroid production in this species (Sir@ and Sir@, 199 la) and in C. batrach (Singh and Singh, 1987).
LIPID METABOLISM
29
IN RESPONSE TO PESTICIDES CONTROL MALATHION
5ppm
MALATHION
20ppm
q-BHC ?-BHC
.” *
CIppm 16ppm
r
PREP
PRES
SPAW
POSTS
REST
FIG. 5. Changes in phospholipids in the liver, plasma, and ovary in response to malathion and YBHC in female H. fossilis. PREP, preparatory phase; PRES, prespawning phase; SPAW, spawning phase; POSTS, postspawning phase; REST, resting phase. (x) Not significant at the level of 0.05.
During the spawning phase, the elevation in hepatic TG after malathion and yBHC exposure can be attributed to a decrease in their use. Under natural conditions, H. fossilis reduces its food intake during the prespawning phase and depends mainly on stored lipids for the energy required for spawning. A decreased level of FFA in the ovary, with an increased level of TG, also suggests a decrease in the hydrolysis of TG to FFA and ultimately in the &oxidation of the latter to yield metabolic water, as well as ATP. A failure of ovulation in response to exposure to these pesticides may also result from inadequate ovarian hydration. Hydration of the ovary normally precedes ovulation (Hirose, 1976; Greeley et al., 1986). The effect of these pesticide on PL, CF, and CE metabolism appears to be similar to that found during the prespawning phase. During the postspawning and resting phases the impact of these pesticides on lipid metabolism seems to be restricted to the liver, where they arrest hepatic lipogenesis.
30
P. B. SINGH IIll CONTROL kXi7 MALATHION CxI MALATHION fiB 3-BHC q 3-BHC
aooPREP
PRES
SPAW
POSTS
5ppm 20ppm
Ippm 16ppm
REST
FIG. 6. Changes in free cholesterol in the liver, plasma, and ovary in response to malathion and -y-BHC in female H. fossilis. PREP, preparatory phase; PRES, prespawning phase; SPAW, spawning phase; POSTS, postspawning phase; REST, resting phase. (X) Not significant at the level of 0.05.
CONCLUSION The present findings clearly demonstrate that malathion and y-BHC both significantly alter lipid metabolism and have very selective and specific effects on various classes of lipids in H. fossilis during different phases of the annual reproductive cycle. Cholesterol biosynthesis is not impaired by these pesticides but the conversion.of CE to CF is checked. The mobilization of various hepatic lipids to the ovary is also restricted by both pesticides. The impact of these pesticides in H: fossilis followed a pattern of lipid mobilization similar to that in C. batruchus with some differences in one or two specific classes of lipids. Thus it appears that these pesticides interfere with the production of lipid-deprived energy, vitellogenin, and sex hormone precursor, which ultimately results in decreased breeding potential and prevention of spawning in the female H. fossilis.
LIPID METABOLISM
31
IN RESPONSE TO PESTICIDES q CONTROL q MALATHION a
MALATHION
fZ?d ?-BHC •l %BHC
Sppm 20ppm
lppm 16ppm
z e 1.00 I .g 0.00
P z! 1.205 ij 0.80n 2 g OAO0
v i
o.oo-
0.00 -
PREP
PRES
SPAW
POSTS
REST
FIG. 7. Changes in esterified cholesterol in the liver, plasma, and ovary in response to malathion and yBHC in female H. fossilis. PREP, preparatory phase; PRES, prespawning phase; SPAW, spawning phase; POSTS, postspawning phase; REST, resting phase. (X) Not significant at the level of 0.05.
ACKNOWLEDGMENTS We are grateful to Dr. P. K. Singh, Principal, T. D. College, Jaunpur (U.P.), for providing laboratory facilities, and financial assistance from a UGC minor research project (26-1(743)/89(SR-IV)) dated August 26, 1989, to P.B.S. is gratefully acknowledged.
REFERENCES BRUNING, J. L., AND K~NTZ, B. L. (1977). Computational Handbook of Statistics, 2nd ed. Scott, Foresman, Glenview, IL. DEY, S., AND BHATTACHARYA, S. (1989). Ovarian damage to Channa punctatus after chronic exposure to low concentrations of elsan, mercury, and ammonia. Ecotoxicol. Environ. Saf 17,247-257. DZOGBEFIA, V. P., KLING, D., AND GAMBLE, W. (1978). Polychlorinated biphenyk In vitro and in vitro modifications of phospholipid and glycerides biosynthesis. J. Environ. Pathol. Toxicol. 1, 841-856. FOLCH, J., LEES, M., AND STANLEY, G. H. S. (1957). A simple method for isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226,497-507.
32
P. B. SINGH
C. P., AND WEST, D. (1966). Complete separation of lipid classes on a single thin layer plates. J. Lipid Res. 7, 324-327. FREEMAN, H. C., AND IDLER, D. R (1975). The effect of polychlorinated biphenyl on steroidogenesis and reproduction in the brook trout Salvelinus fontinalis. Can. J. B&hem. 53,666-670. GILL, T. S., PANDE, J., AND TEWARI, H. (1990). Sub-lethal effects of an organophosphorus insecticide on certain metabolite levels in a freshwater fish, Puntius conchonius. Pestic. B&hem. Physiol. 36(3), 290299. GREELEY, M. S., CALDER, D. R., AND WALLACE, R. A. (1986). Changes in teleost yolk protein during oocyte maturation: Correlation of yolk proteolysis with oocyte hydration. Comp. Biochem. Physiol. B 84, 1-11. HIROSE, K. (1976). Endocrine control of ovulation in Medaka (Oryzias latipes) and Ayu (Plecoglossus altivelis). J. Fish. Res. Board Can. 33, 989-994. KAPOOR, K., KAMALDEEP, K., AND TOOR, H. S. (1978). The effect of fenitrothion on reproduction of a teleost fish Cyprinus carpio communis Linn: A biochemical study. Bull. Environ. Contam. Toxicol. 20, 438-442. KLING, D., KUNKLE, J., ROLLER, A. S., AND GAMBLE, W. (1978). Polychlorinated biphenyl: In vivo and in vitro modifications of cholesterol and fatty acid biosynthesis. J. Environ. Pathol. Toxicol. 1, 8 13-828. KOCAN, R. M., AND LANDOLT, M. L. (1989). Survival and growth to reproductive maturity of coho salmon following embryonic exposure to a model toxicant. Mar. Environ. Res. 27, 177-194. LAL, B., AND SINGH, T. P. (1987). The effect of malathion and y-BHC on the lipid metabolism in relation to reproduction in the tropical teleost, Clarias batrachus. Environ. Pollut. 48, 37-47. MARZO, A., GHRARCH, P., SERDINI, D., AND MJZRONI,D. (197 I). Simplified measurement of monoglycerides, diglycerides, triglycerides and fatty acids in biological samples. Clin. Chem. 17(3), 145-147. MURTY, A. S., AND DEVI, A. P. (1982). The effect of endosulfan and its isomers on tissue protein glycogen and lipids in the fish, Channa punctatus. Pestic. B&hem. Physiol 17,280-286. SINGH, H., AND SINGH, T. P. (1980). Effect of two pesticides on total lipid and cholesterol content of ovary, liver and blood serum during different phases of the annual reproductive cycle in the freshwater teleost, Heteropneustes fossilis (Bloch). Environ. Pollut. 23, 9- 17. SINGH, P. B., AND SINGH, T. P. (1986). Pesticide induced impairment of lipid metabolism, steroidogenesis and gonadal recrudescence during preparatory phase of its annual reproductive cycle in the freshwater female catfish, Heteropneustes fossilis. Proceedings, Sixth All India Seminar on Ichthyology, December 21-24, 1986. Manipur University, India. SINGH, S., AND SINGH, T. P. (1987). Impact of malathion and hexachlorocyclohexane on plasma profiles of three sex hormones during different phases of the reproductive cycle in Clarias batrachus. Pestic. Biochem. Physiol. 27,301-308. SINGH, P. B., AND SINGH, T. P. (1990). Seasonal correlative changes between sex steroids and lipid levels in the freshwater female catfish, Heteropneustes fossilis. J Fish Biol. 37, 793-802. SINGH, P. B., AND SINGH, T. P. (1991a). Impact of r-BHC on sex steroids level and their modulation by ovine luteinizing hormone-releasing hormone and Mystus gonadotropin in the freshwater catfish, Heteropneustes fossilis. Aquat. Toxicol., in press. SINGH, P. B., AND SINGH, T. P. (1991b). Impact of r-BHC on the lipid class levels and their modulation by reproductive hormones in the freshwater cat&h, Heteropne-ustesfossilis. Bull. Environ. Contam. Toxicol., in press. WIEGAND, M. D., AND PETER, R. E. (1980a). Effect of salmon gonadotropin (SG-100) on plasma lipids in the goldfish, Carassius auratus. Can. J. Zool. B&957-966. WIEGAND, M. D., AND PETER, R. E. ( 1980b). Effect of sex steroids on plasma lipids in the goldfish, Carassius auratus. Can. J. Zool. 58,967-979. FREEMAN,
