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Impact of pesticides on lipid metabolism in the freshwater catfish, Clarias batrachus, during the vitellogenic phase of its annual reproductive cycle
13,13-23(
ECOTOXIC~LOGYANDENVIRONMENTALSAFETY
1987)
Impact of Pesticides on Lipid Metabolism in the Freshwater C/arias batrachus, during the Vitellogenic Phase of Its Annual Reproductive Cycle BECHAN Fish Endocrinology
Laboratory,
Catfish,
LAL AND T. P. SINGH’
Department Varanasi-221005, Received
of Zoology, India
March
Banaras
Hindu
University,
7, 1986
Specimens of either sex of the freshwater catfish Clarias batrachus were exposed to safe and sublethal concentrations ofan organochlorine, -/-BHC (2 and 8 ppm). and an organophosphorus compound, malathion (I and 4 ppm), for 4 weeks during the vitellogenic phase of their annual reproductive cycle. The effectson total lipid and its various fractions, viz.. free fattyacids, monoglycerides, diglycerides, triglycerides. phospholipids. free cholesterol, and esterified cholesterol, were studied in the liver. plasma. gonads, and muscle. Except for elevated liver lipid in the male in response to malathion, no significant change in total lipid could be observed following pesticide exposure. However, various lipid fractions responded differently to two concentrations of the pesticides. Both pesticides affected the metabolism of nonpolar and less polar lipids alike. Malathion inhibited only mobilization of hepatic phospholipid to gonads but not its hepatic biosynthesis, whereas -r-BHC reduced its synthesis in the liver as well. These pesticides seemed to restrict the conversion of esterified cholesterol into free cholesterol without affecting the biosynthesis of cholesterol as such. In both sexes,ester&cation of free fatty acids to acyl glycerides and their mobilization from liver to gonads seemed to be restricted as a result of pesticides aCtiOfl.
B 1987 Academic
Press, Inc.
INTRODUCTION
Pesticides, even in very low concentration, have been reported to interfere with basal metabolism (Litterst and Vanloon, 1972; Sanders et al., 1974; Write et al., 1977). K.ollar and Zinkle (1973) have shown that PCB increased serum cholesterol in the rabbit. Total lipid and glycerides of liver were increased by oral administration of PCB in the rabbit (Ito et al., 1971). In clinical cases of PCB poisoning, serum triglyceride levels were found to have increased, but post-heparin plasma lipoprotein lipases and serum lecithin-cholesterol acyltransferase activity were found to have decreased (Uzawa et al., 197 1). Kling et al. (1978) have noted that cholesterol and fatty acid biosyntheses were decreased in response to Aroclor 1254 in the rat. Dzogbefia et al. ( 1978) have reported decreased levels of phospholipid and glyceride synthesis after PCB exposure. In contrast to mammal studies, little attention has been paid to similar studies in fish. Much information regarding the effects of pesticides on fish describers the survival, growth rate, fecundity, or reproductive activity (Wildish et al., 197 1; Burdick et al., 1972; Jackson, 1976; Anees, 1978), but information dealing with pesticide effects on biochemical parameters is meager. Organochlorine and organophosphorus compounds are known to suppress gonadal growth (Freeman and Idler, 1975) and ovarian 3&hydroxysteroid dehydrogenase activity in Cyprinus carpio (Kapoor et al., 1978). Except for the reports of Singh and Singh (1980a,b,c,d) on ’ To whom reprint requests should be addressed. 13
Ol47-6513/87 $3.00 Copyright 0 1987 by Academic Press. Inc. All rights of reproduction in any form reserved
14
LAL
AND
SINGH
Heteropneustes fossilis and that of Murty and Devi ( 1982) in Channa punctatus, no comprehensive study of lipid metabolism in response to pesticides is available. Lipids undergo rapid breakdown resynthesis and interconversion with the changes in exteroand interoceptic stimuli. Hence, unless various classes of lipid in different tissues are analyzed simultaneously, a clear picture of lipid metabolism in response to pesticides cannot emerge. Therefore, in the present investigation an attempt had been made to assess the impact of an organochlorine compound, -/-BHC (1,2,3,4,5,6-hexachlorocyclohexane), and organophosphorus compound, malathion (S-( 1,2-dicarbethoxyethyl) ester of O,O-dimethyl phosphorothioate), on lipid metabolism by estimating the levels of seven classes of lipid in the liver, plasma, gonads, and muscle during the vitellogenic phase of the annual reproductive cycle in both sexes of Clarias batrachus. MATERIALS
AND
METHODS
Adult and healthy specimens of both sexes of C. batrachus with an average weight of 65 g (60-70 g) and size of 18.5 cm (18-19 cm) were collected in the first week of May from the ponds around Varanasi, India. Fish were acclimatized to laboratory conditions for 2 weeks and were fed minced goat liver containing 20% protein, 5% lipid, and 15% carbohydrate every fourth day. The fish were maintained under natural photoperiod and temperature. After acclimatization fish were sexed and specimens of each sex were divided into six batches, each comprising 10 fish, in an aquarium of 90-liter capacity. Fish were exposed to 96-hr safe concentrations (SC) and sublethal concentrations (SL) of malathion (1 and 4 ppm) and BHC (2 and 8 ppm) which have been standardized and reported (Lal and Singh, 1984). Malathion was added directly to water, but 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 BHC 1 ml of acetone was also added to the aquarium water. Experiments were conducted for 4 weeks (3rd week of May-2nd week of June) during the vitellogenic phase at natural photoperiod ( 13L: 11 D) and temperature (30 & 2°C). During experimentation, fish were fed every 4th day when aquarium water was changed with fresh water having similar concentrations of the above pesticides. Chemicals Lipid standards. Monopalmitin for monoglycerides (MG), dipalmitin for diglycerides (DG), tripalmitin for triglycerides (TG), palmitic acid for free fatty acids (FFA), lecithin for phospholipids (PL), cholesterol for free cholesterol (CF), and cholesterol oleate for esterified cholesterol (CE) were purchased from Sigma (U.S.A.). Silica gel GhO was purchased from E. Merck (Germany) and AR grade solvents (BDH, India) were distilled and used. Tricane methane sulfonate (MS 222) for anesthesia was a gift from Sandoz Ltd. (Switzerland). y-BHC and malathion were obtained from Bharat Pulverising Mill Private Ltd., India, and Cynamid India Ltd., India, respectively. Lipid Extraction
and Separation
When experiments were terminated, blood was collected separately by caudal puncture from individual fish of each group, in heparinized tubes, and centrifuged at 2000g at 4°C; plasma was frozen at -20°C for further analysis. Individual liver, ovary,
IMPACT
OF
PESTICIDES
ON
LIPID
METABOLISM
15
testis, and muscle were extirpated, washed in 0.6% saline, blotted, and kept frozen at -20°C until further analysis. Tissues and plasma were extracted in chloroform: methanol (2: l), following the method of Folch et al. (1957) for total lipid concentration, which was measured gravimetrically. Triplicate samples of each tissue from a single specimen were taken for analysis. Each sample weighed 30-40 mg for liver, gonads, and muscle and 100 ~1 for plasma. Further, different fractions of lipid were separated by thin-layer chromatography (&values for MG, DG, TG, FFA, PL, CF, and CE were 0.15,0.64,0.80,0.27,0.00,0.44, and 0.90, respectively) by the double solvent system of Freeman and West (1966) (system I-diethylether:benzene:ethanol:acetic iacid, 40:50:2:0.2, and solvent system II-hexane:diethylether, 94:6). Spots of various lipids were visualized by exposing the plates to iodine vapor. Spots of different lipid fractions of samples, standards, and corresponding areas of silica gel from blanks were scraped and transferred to separate test tubes. Quantitation Quantitative estimation of various lipids were made spectrophotometrically on Spectronic-2000 (Bausch & Lomb, U.S.A.) at 375 nm by the method of Matzo et al. (197 1). D,ata were expressed in milligrams per gram of tissue or per milliliter of plasma (mlean -t SE), and Student’s t test was employed for statistical analysis. RESULTS Female Four weeks’ exposure of this fish to malathion and -/-BHC at SC and SL caused appreciable changes in various classes of lipids without affecting total lipid concentration of the liver, plasma, ovary, and muscle. Results are summarized in Tables 1 and 2. Efects on Free Fatty Acids (FFA) Malathion at safe and sublethal concentrations increased FFA levels in the liver and plasma but reduced their levels in the ovary and muscle. Similarly, y-BHC also increased FFA of the liver and plasma at both concentrations. However, ovarian FFA was decre,ased by safe concentration of -y-BHC, but its sublethal concentration could not produce any change, whereas the muscular FFA was reduced by sublethal concentration of -/-BHC and remained unchanged in response to its safe concentration. l$ects on Acyl Glycerides SublethLal concentration of both pesticides decreased mono- and triglycerides in the liver, plasma, ovary, and muscle. Moreover, diglycerides were also decreased in the liver, plasma, and muscle, but in the ovary they were elevated in response to malathion and remained unchanged after y-BHC treatment. These pesticides at a safe concentration also caused a decrease in mono-, di-, and triglycerides of all the tissues studied except ovarian triglycerides, which were elevated.
1
Level of
51.17 f
53.10 f 4.89”
SC
10.95 k
10.07 f
SL
SC
34.35 IL 4.17
37.71 * 3.03”
36.01 k 2.81”
SC
5 I .03 k 4.09”
SC
SL
56.18 + 5.61”
SL
Control
65.79 +- 5.79
Control
1.87”
1.01 a
10.6 I z!z 0.96
Control
5.76“
46.79 + 3.69
SL
TL
Control
malathion
Nore. Values are expressed as mg/g tissue or m&l except those marked ‘I”‘.
Muscle
Ovary
Plasma
Liver
Tissue
0.15 f0.01
0.21 f 0.01
0.45 f 0.0 1
0.12 * 0.01
0.35 + 0.02
0.40 * 0.03
0.5 I + 0.02
0.08 f 0.0 1
0.54 +- 0.02
0.56 +- 0.02
I. I5 2 0.05
7.45 l!z 0.37
2.83 t- 0.18
5.54 + 0.89
0.69 k 0.03
0.59 + 0.06
0.96 1: 0.02
4.13 +-0.10
4.31 IO.21
10.94 $0.21
TG
2.61 f 0.12
0.19+0.01
1.16 -to.05 0.17~0.01
1.26 +- 0.07
2.38 f 0.12
2.06 + 0.2 I
3.98 + 0.17
0.14f0.01
2.41 + 0.07
9.39 + 0.27
10.13 + 0.32
13.89 -t 0.42
1.69 + 0.35
0.17 kO.01
0.74 f 0.03
2.80 + 0.23 1.53 * 0.33
0.5 1 f 0.07”
0.45 f 0.04”
0.46 f 0.03
CF
9.36 f 0.32
9.04 + 0.29
6.31 + 0.12
PL
0.36 z!z 0.03
0.39 Ik 0.03
3.14 + 0.43
5.07 + 0.46
5.74 + 0.48
2.07 f0.17
0.64 f 0.04
0.36 k 0.01 0.84 AI 0.03
8.01 I? 0.15
8.13 to.18
0.68 t 0.06
CE
plasma. and are means + SE; n = 5. P values for SL and SC were compared against their respective controls and were found significant
5.41 + 0.36
4.04 f 0.36
8.42 k 0.46
9.38 2 0.86
1.88 + 0.05
1.01 + 0.12
0.19 -t 0.01
0.38 k 0.02
8.91 rt 0.23
14.93 Ik 1.37
0.29 f 0.02
0.07 f 0.0 1
0.37 t- 0.03
0.21 f 0.01
0.05 + 0.0 I
0.42 f 0.02
1.12 -t 0.23
0.92 50.13
2.52 -I- 0.43
DG
0.40 * 0.04
0.08 + 0.0 1
1.15 ItO.
16.58 + 0.89
0.29 + 0.01
1.55 f0.15 0.64 IL 0.02
17.21 + 0.99
MG
13.66 + 0.27
FFA
EFFECT OF MALATHION ON TOTAL LIPID (TL), FREE FATTY ACIDS (FFA), MONOGLYCERIDES (MG), DIGLYCERIDES (DG), TRIGLYCERIDES (TG), PHOSPHOLIPIDS (PL), FREE CHOLESTEROL (CF), AND ESTERIFIED CHOLESTEROL (CE) LEVELS IN LIVER, PLASMA, OVARY, AND MUSCLE IN FEMALE Clarias batrachus
TABLE
c
$
z g
r
IMPACT
OF
PESTICIDES
ON
LIPID
METABOLISM
17
18
LAL
AND
SINGH
Efects on Phospholipids Malathion and y-BHC at both concentrations elevated the phospholipid levels in the muscle and decreased them in the plasma and ovary. But, hepatic levels were increased in response to malathion and were decreased in r-BHC-exposed fish. Efects on Cholesterol Both pesticides, at either concentration, caused reduction in free cholesterol levels of the plasma, ovary, and muscle, whereas hepatic levels remained unchanged by malathion and were decreased in y-BHC-treated fish. Contrary to free cholesterol, esterified cholesterol was raised in all the tissues except in muscle, where it was reduced by both pesticides. Male Malathion evoked an increase in total lipid level of the liver in male C. batrachus in contrast to that of the female, whereas in other tissues it failed to induce any change in total lipid levels. -/-BHC also could not produce any obvious change in total lipid of the tissues studied. However, varied responses were noted in various classes of lipids in different tissues after pesticide exposure. Results are summarized in Tables 3 and 4. Effects on Free Fatty Acids and AcyI Glycerides Free fatty acid levels along with mono-, di-, and triglycerides in the liver and monoand triglycerides in testes were enhanced by these pesticides at either concentration, but these lipids were suppressed in the plasma. However, in muscle their levels remained unchanged in response to y-BHC and were reduced in malathion-exposed fish. Efects on Phospholipids
and Free and EsteriJied Cholesterol
High and low hepatic phospholipids were noticed in malathion- and -y-BHCtreated fish, respectively. Phospholipid levels in the plasma and testes were suppressed but raised in muscle. Malathion exposure caused a decrease in concentration of free cholesterol in the liver, testes, and muscle but elevate levels in the plasma, whereas r-BHC evoked an increase in hepatic free cholesterol but a decrease in the plasma, testes, and muscle. Both pesticides increased the esterified cholesterol levels in all the tissues studied. DISCUSSION y-BHC and malathion caused appreciable changes in various classes of lipid of different tissues in C. batrachus but the spectrum of total lipid apparently remained unchanged. A similar observation for total lipid in the liver and ovary in H. fossilis in response to malathion and hexadrin during the prespawning phase has been recorded by Singh and Singh (1980d). However, Murty and Devi ( 1982) have reported decreased liver lipids after technical endosulfan treatment in C. punctatus. In female C. batrachus increased hepatic free fatty acids accompanied by low mono-, di-, and
30.54 3I 5.05”
SC
31.07zk4.13a
SC
k 3.06”
23.12
SL
+ 2.17
27.54
Control
k 4.81”
31.23
SL
+- 3.79
26.51
10.50 _+ 2.10”
SC
Control
7.21 + 1.89”
SL 0.03
0.85kO.21
0.77 f 0.1 1
3.00 310.48
12.44 + 0.46
13.49 -t 0.85
4.61 f 0.06
0.51 f
0.59 -t 0.03
1.63 + 0.33
19.67 + 0.64 20.72 f 0.63
7.11 -to.01
FFA
0.06 2 0.0 I
0.06 310.01
1.58 k 0.09 0.20 IL 0.02
1.41 20.08
0.25 f 0.01
0.31 -to.01
0.27 ?I 0.01
0.17 f 0.04 1.16 -to.05
0.15 f 0.01
0.28 -t 0.02
0.22 + 0.0 1
0.16 +O.Ol
0.07 + 0.0 1 0.07 f 0.01
0.34 AI 0.02
4.61 f 0.07 4.76 It_ 0.32
3.67 -t 0.10
DG
0.09 + 0.0 1
1.87 k 0.11 2.47 k 0.14
1.22 f 0.04
MG
0.85 + 0.03
0.80 k 0.03
2.1 I + 0.23 1.41 f 0.03
2.21 + 0.23
1.90 + 0.08
1.08 -t 0.08
1.43 -t 0.08
1.84 k 0.08
9.72 +_ 0.82 10.72 * 0.93
2.71 f0.13
TG
0.55 f 0.08 0.12 f 0.01
0.56 kO.16
1.22 +_ 0.05
1.35 rt 0.05
0.02
2.83 k 0.12
against their respective controls and were found significant
0.13f0.01
0.37 f
0.1 I fO.O1
8.2 1 f 0.82 0.29 f 0.04
0.83 f 0.17
2.92kO.13
0.14 f 0.01
7.2 1 + 0.79
1.03 + 0.03
0.87 + 0.03
12.09 f 0.3 1
0.87 IL 0.03
0.67 + 0.02
1.07 +- 0.04
0.28 + 0.02
7.47 + 0.89 7.74 + 0.46
1.52 ?c 0.03
CE
1.79 31 0.06
0.71 * 0.02
0.32 + 0.03
2.01 f 0.05 1.81 +- 0.05
0.98 f 0.02 0.88 f 0.02
2.73 f 0.17
7.23 f 0.13 14.05 f 0.84 13.54 f 0.95
CF
PL
LIPID(TL),FREEFATTY ACIDS(FFA),MONOGLYCERIDE~(MG),DIGLYCERIDES(DG),TRIGL~~ERIDES(TG), (PL), FREE CHOLESTEROL (CF), AND ESTERIFIED CHOLESTEROL (CE) LEVELS IN LIVER, PLASMA, TESTES,ANDMUSCLEOFMALE Clariasbatrachus
3
NW,. Values are expressed as mg/g tissue or mg/ml plasma, and are means 2 SE: n = 5. P values for SL and SC were compared except those marked ““‘.
Muscle
Testes
8.08 2 1.14
Control
50.09 IL 4.02 53.28 Ik 4. I I
SL SC
Plasma
36.17 -t 3.04
Control
Liver
TL
malathion
Level of
ONTOTAL PHOSPHOLIPIDS
MALATHION
Tissue
EFFECTOF
TABLE
5
W
i5
$
r z u g
z
E
2 E E;
41 3
2 3
4
SC
32.92 f 4.07”
33.61 + 3.20”
30.70 k 4.12”
SL
SC
26.07 f 3.54
Control
34.26 f 3.48“
4.18 + 0.66
12.17f2.51”
SC
27.18 -t 2.72
0.30 Ik 0.02
11.81 + 1.68”
SL
SL
1.40 + 0.03
10.13 _+ 1.72
Control
Control
14.71 -t 0.92
45.89 f 6.15”
SC
0.25 zk 0.02”
1.04 r+ 0.05
0.93 + 0.06”
0.96 iz 0.04”
1.39 f 0.02
0.40 Ik 0.03”
0.51 f 0.02
0.49 _+ 0.02
0.59 +_ 0.04
0.65 Z!I 0.03
0.41 f 0.01
0.81 + 0.04
0.15 zk 0.01
O.l8t-0.01
0.30 -t 0.01
14.87 k 0.86
3.42 zk 0.09
2.20 -t 0.10
CF
0.39 * 0.03
0.3 1 xk 0.02”
0.34 t 0.0 1
I .86 * 0.01
1.52 f 0.04
1.10f0.04
1.31 * 0.03
1.43 -t 0.03
0.35 * 0.03
5.69 -+ 1.17
18.54 k 2.39
1.50 f 0.06
CE
(TG),
against their respective controls and were found significant
2.42 f 0.12
1.09 + 0.04
1.57 It_ 0.40
3.41 f 0.09
0.42 + 0.02”
0.49 zk 0.0 1
1.78 f 0.01
1.98 k 0.02
11.89 zk 0.27
0.73 f 0.01
0.57 -t 0.03 1.5 1 t- 0.07
0.45 * 0.03
0.63 + 0.01
2.01 + 0.36
0.83 f 0.04
6.48 f 0.10 1.68 f 0.06
4.57 -t 0.17
7.76 + 0.57
PL
6.28 t- 0.17
2.86 f 0.13
TG
plasma. and are means ?z SE; n = 5. P values for SL and SC were compared
4.01 x!I 0.55”
0.27 + 0.02”
0.17 f 0.02
0.24 f 0.0 1
0.12 + 0.01
2.34 + 0.03
0.31 f 0.03
12.3 1 t- 0.46
3.90 -t 0.38
1.27
1.52
0.23 f 0.01
0.21 * 0.02
0.25 + 0.01
0.08 f 0.01’
4.07 z!I 0.19”
18.00 k
23.18 k
0.49 _t 0.02
0.35 ?I 0.02
0.03 f 0.01
5.53 f 0.24
5.17 +_ 0.36
3.87 + 0.13
DG
0.08 f 0.0 1
11.37 + 0.96
1.10~0.04 2.18 + 0.26
1.87
7.00 t 0.29
31.50 k
3.17
38.73 t- 4.50”
MG
34.84?
FFA
SL
TL
ON TOTAL LIPID (TL),FREE FATTY ACIDS (WA), MONOGLYCERIDES (MG), DIGLYCERIDES (DG), TRIGLYCERIDES PHOSPHOLIPIDS (PL). FREE CHOLESTEROL (CF), AND ESTERIFIED CHOLESTEROL (CE) LEVELS IN LIVER, PLASMA, TESTES, AND MUSCLE OF MALE Clarias batrachus
Control
BHC
Level of
OF BHC
Note. Values are expressed as mg/g tissue or mg/ml except those marked ‘Ia”.
Muscle
Testes
Plasma
Liver
Tissue
EFFECT
TABLE
IMPACT
OF
PESTICIDES
ON
LIPID
METABOLISM
21
triglyceridles after pesticide exposure indicated that the biosynthesis of fatty acids was probably not affected, but their conversion to acyl glycerides was impaired. Increased fatty acid content in liver has been reported in PCB-treated rats (Kling et al., 1978). However, elevated plasma free fatty acids in the pesticide-exposed fish might be the consequence of either their release from liver being greater than their uptake by other tissues, or as a byproduct of complete hydrolysis of circulating dietary triglycerides, which was evident by decreased plasma triglycerides. Decreased levels of free fatty acids in ovary and muscle appeared to be due either to their restricted transportation from liver to ovary or to faster utilization. In earlier reports, it has been shown that the teleostean ovary imports lipids and phospholipids from liver during the vitellogenie phase under the influence of sex steroids and gonadotropin (Wiegand and Peter, 1980; Upadhyay, 1977). Thus, it appeared that mobilization of these lipids was probably inhibited by these pesticides in C. batruchus by decreasing the levels of sex steroids and gonadotropin. Singh and Singh (1980a,b) and Kapoor et al. (1978) have recorded reduced gonadotropic potency in malathion-treated H. ,fossilis and 3-P-hydroxysteroid dehydrogenase activity in the ovary of fenitrothion-exposed C. carpio, respectively, findings which support our above views. However, increased levels of triglycerides in the ovary in response to malathion and r-BHC at the safe concentration were interesting and unusual, but cannot be interpreted at this stage. In malIe C. batruchus, unlike in the female, these pesticides appeared to stimulate lipogenic activity in liver by increasing fatty acid synthesis and its conversion to acyl glycerideis. This increase might also be due to inhibited P-oxidation of free fatty acids as well as their utilization, whereas elevated levels of free fatty acids and triglycerides in testes s,uggested that these pesticides probably reduced their utilization by suppressing testicular activity. In normal male C. batrachus, La1 and Singh (unpublished) have found decreased free fatty acids and triglycerides in testes during the vitellogenic phase under natural conditions. They have attributed these decreases to increased consumption (as fuel) to sustain enhanced testicular activity during this phase. However, reduced levels of these lipids in plasma might be due to their reduced release from liver to plasma. Hepatic phospholipids were raised in malathion-treated C. batrachus of either sex but were reduced in r-BHC-exposed fish, indicating that y-BHC probably interfered with their biosynthesis in liver, whereas both pesticides apparently inhibited their mobilization from liver to gonads, as was evident by their low levels in plasma and gonads. Here again it might be due to decreased sex steroids and gonadotropin, which are required for phospholipid uptake by ovary (Upadhyay, 1977; La1 and Singh, 1983). Singh and Singh ( 1980a,b) have also reported decreased 32P uptake by ovary and testes in H. fossilis after malathion and hexadrin exposure. Decreased free cholesterol and increased esterified cholesterol levels were recorded in all the tissues studied in pesticide-exposed C. batrachus, suggesting that conversion of esterified to free cholesterol was inhibited without affecting the cholesterol synthesis as such. Decreased levels of gonadotropin, as a consequence of pesticide action, might possibly be responsible for this inhibited conversion. CONCLUSIONS From the data it can be concluded that the actions of both pesticides on the metabolism of nonpolar lipids were alike and sex related. However, hepatic biosynthesis of
22
LAL
AND
SINGH
polar lipids, like phospholipids, seemed to be affected differently by these two pesticides. ,&Oxidation of free fatty acids, as well as mobilization of various lipids from liver to ovary, was inhibited by these pesticides in C. batrachus. ACKNOWLEDGMENTS Grant-in-Aid HCS/DST/928/80 for research and ICAR-PL-480 Project IN-ARS-213 New Delhi to T.P.S. are gratefully acknowledged. We are grateful to Mr. S. W. Vaidya of Bharat Pulverishing Mill Ltd., India, for y-BHC and to Mr. S. R. Maley ofcynamid India Ltd. for malathion.
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teleost Chunnn punctatus (Bloch) exposed to insecticides. Bull. Environ. Contam. Toxicol.
19,524-527. BURDICK, G. E., DEAN, H. J., HARRIS. E. J., SKEA, J., KARCHER, R.. AND FRISA, C. ( 1972). Effect of rate and duration of feeding DDT on the reproduction of salmonid fish reared and held under controlled conditions. N. Y. Fish Game J. 19,97-I 15. DZOGBEF~A, V. P., KLING, D., AND GAMBLE, W. (1978). Polychlorinated biphenyl: In vivo and in vitro modifications of phospholipid and glycerides biosynthesis. J. Environ. Pathol. Tu~icol. 1,84 l-856. FOLCH. J., LEES, M., AND STANLEY, S. (1957). A simple method for isolation and purification of total lipids from animal tissues. J. Biochem. 226,497-507. FREEMAN, 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. (I 975). The effect of polychlorinated biphenyl on steroidogenesis and reproduction in the brook trout Salvelinus fontinalis. Canad. J. Biochem. 53,666-670. ITO, Y. H.. UZAWA, H., AND NOTOMI, A. (1971). Lipid composition of the rabbit liver poisoned with chlorobiphenyl. Fukuoka Igaku Zasshi 62,48-50. JACKSON, G. A. (1976). Biological halflife of Eldrin in Channel catfish tissues. BUN. Environ. Contam. Tax-icol. 16, 505-507. KAPOOR, K., KAMALDEEP, K., AND TOOR. H. S. (I 978). The effect of fenitrothion on reproduction of a teleost fish Cyprinus carpio mmmunis Linn. A biochemical study. Bull. Environ. Contam. To,xicol. 20. 438-442. KLING, D., KLJNKLE, J., ROLLER, A. S., AND GAMBLE, W. (1978). Polychlorinated biphenyl: In vivo and In vitro modifications of cholesterol and fatty acid biosynthesis. J. Environ. Putho/. Toxicol. 1,8 13-828. KOLLAR, L. D., AND ZINKLE, J. G. (1973). Pathology of polychlorinated biphenyl in rabbits. Amer. J.
Pathol. 70,263-278. LAL, B.. AND SINGH, T. P. (1983). Effect of ovariectomy and sex steroids administration on lipid metabolism in freshwater teleost, Clarias batrachus. In Proceedings ofFirst InternationalSymposium on Recent Advances in Lt@ Sciences, Bodhgaya, India, 29-3 1 Dec., p. 136. LAL, B., AND SINGH, T. P. (1984). Pesticides induced alterations in the lipid metabolism during prespawning phase of reproductive cycle in freshwater teleost, Clarias batrachus. In Proceedings of Symposium on Comparative Endocrinology. Varanasi. India, 29-3 1 Dec., p. 85. LITTERST, C. L., AND VANLOON, E. J. (1972). Enzyme induction by polychlorinated biphenyls relative to known inducing agents (36867). Proc. Sot. Exp. Biol. Med. 141,765-768. MARZO, A., GHIRARCH, P., SERDINI, D.. AND MERONI, D. (1971). 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. Biochem. Physiol. 17,280-286. SANDERS, O.T., ZEPP, R. L.. AND KIRKPATRICK, R. L. ( 1974). Effect of PCB ingestion on sleeping times, organ weight, food consumption. serum corticosterone and survival of albino mice. Bull. Environ. Contam. Toxicol. 12,394-399. SINGH, H., AND SINGH, T. P. (1980a). Effect of two pesticides on ovarian ‘*P uptake and gonadotropin concentration during different phases of annual reproductive cycle in the fresh water catfish Heteropneustes/ossilis (Bloch). Environ. Res. 22, 190-200.
IMPACT
OF PESTICIDES
ON LIPID METABOLISM
23
SINGH, H.. AND SINGH, T. P. (1980b). Effect of two pesticides on testicular 32P uptake, gonadotrophic potency, lipid and cholesterol content oftestis, liver and blood Serum during spawning phase in Heferopneustes/izssilis (Bloch). Endokrinologie 76(3), 288-296. 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,fossili.s (Bloch). Environ. Pollut. Ser. .4 23,9-17. SINGH, H., AND SINGH, T. P. (1980d). Short-term effect of two pesticides on lipid and cholesterol content of liver. ovary and blood serum during the prespawning phase in the freshwater teleost, Heteropneustes fossilis (Bloch). Environ. Pollut. Ser. A 22, 85-90. UPADHYAY, S. N. (1977). Morphologic des gonads immatures etude experimental de l’induction de la gametogenese chew la truite arc-en-ciel juvenileSalmogairdneri (R). Thbe de doctorate d’ Elates Science Naturelles. L. Universite Pierre et Marie Curie, Paris, France. UZAWA, H.. ITO. Y.. NOTOMI. A., HORI, S., IKEURA. Y., AND KATSUKI, S. ( 197 1). Clinical and experimental studies on hyperglyceridemia induced by oral ingestion of chlorinated biphenyl. Fukuoka Igaku Zasshi62.66-73. WIEGAND, M. D.. AND PETER, R. E. ( 1980). Effect of sex steroids on plasma lipids in the goldfish. Carassius auratus. Canad. J. Zool. 58,967-972. WILDISH, D. J., CARSON, W. G.. CUNNINGHAM, T., AND LISTER, N. J. (197 1). Toxicological effects of some organophosphate insecticides to atlantic salmon. Fish. Res. Board Canad. Manscr. Rep. Ser. 1157, 1-22.
WRITE, A. !j.\ AKINTONWA, D. A. A., AND WOOTER, M. F. (1977). Studies on the interaction of Dieldrin with mammalian liver cells at the subcellular level. Ecofoxicol. Environ. Saf 1,7- 16.
ECOTOXIC~LOGYANDENVIRONMENTALSAFETY
1987)
Impact of Pesticides on Lipid Metabolism in the Freshwater C/arias batrachus, during the Vitellogenic Phase of Its Annual Reproductive Cycle BECHAN Fish Endocrinology
Laboratory,
Catfish,
LAL AND T. P. SINGH’
Department Varanasi-221005, Received
of Zoology, India
March
Banaras
Hindu
University,
7, 1986
Specimens of either sex of the freshwater catfish Clarias batrachus were exposed to safe and sublethal concentrations ofan organochlorine, -/-BHC (2 and 8 ppm). and an organophosphorus compound, malathion (I and 4 ppm), for 4 weeks during the vitellogenic phase of their annual reproductive cycle. The effectson total lipid and its various fractions, viz.. free fattyacids, monoglycerides, diglycerides, triglycerides. phospholipids. free cholesterol, and esterified cholesterol, were studied in the liver. plasma. gonads, and muscle. Except for elevated liver lipid in the male in response to malathion, no significant change in total lipid could be observed following pesticide exposure. However, various lipid fractions responded differently to two concentrations of the pesticides. Both pesticides affected the metabolism of nonpolar and less polar lipids alike. Malathion inhibited only mobilization of hepatic phospholipid to gonads but not its hepatic biosynthesis, whereas -r-BHC reduced its synthesis in the liver as well. These pesticides seemed to restrict the conversion of esterified cholesterol into free cholesterol without affecting the biosynthesis of cholesterol as such. In both sexes,ester&cation of free fatty acids to acyl glycerides and their mobilization from liver to gonads seemed to be restricted as a result of pesticides aCtiOfl.
B 1987 Academic
Press, Inc.
INTRODUCTION
Pesticides, even in very low concentration, have been reported to interfere with basal metabolism (Litterst and Vanloon, 1972; Sanders et al., 1974; Write et al., 1977). K.ollar and Zinkle (1973) have shown that PCB increased serum cholesterol in the rabbit. Total lipid and glycerides of liver were increased by oral administration of PCB in the rabbit (Ito et al., 1971). In clinical cases of PCB poisoning, serum triglyceride levels were found to have increased, but post-heparin plasma lipoprotein lipases and serum lecithin-cholesterol acyltransferase activity were found to have decreased (Uzawa et al., 197 1). Kling et al. (1978) have noted that cholesterol and fatty acid biosyntheses were decreased in response to Aroclor 1254 in the rat. Dzogbefia et al. ( 1978) have reported decreased levels of phospholipid and glyceride synthesis after PCB exposure. In contrast to mammal studies, little attention has been paid to similar studies in fish. Much information regarding the effects of pesticides on fish describers the survival, growth rate, fecundity, or reproductive activity (Wildish et al., 197 1; Burdick et al., 1972; Jackson, 1976; Anees, 1978), but information dealing with pesticide effects on biochemical parameters is meager. Organochlorine and organophosphorus compounds are known to suppress gonadal growth (Freeman and Idler, 1975) and ovarian 3&hydroxysteroid dehydrogenase activity in Cyprinus carpio (Kapoor et al., 1978). Except for the reports of Singh and Singh (1980a,b,c,d) on ’ To whom reprint requests should be addressed. 13
Ol47-6513/87 $3.00 Copyright 0 1987 by Academic Press. Inc. All rights of reproduction in any form reserved
14
LAL
AND
SINGH
Heteropneustes fossilis and that of Murty and Devi ( 1982) in Channa punctatus, no comprehensive study of lipid metabolism in response to pesticides is available. Lipids undergo rapid breakdown resynthesis and interconversion with the changes in exteroand interoceptic stimuli. Hence, unless various classes of lipid in different tissues are analyzed simultaneously, a clear picture of lipid metabolism in response to pesticides cannot emerge. Therefore, in the present investigation an attempt had been made to assess the impact of an organochlorine compound, -/-BHC (1,2,3,4,5,6-hexachlorocyclohexane), and organophosphorus compound, malathion (S-( 1,2-dicarbethoxyethyl) ester of O,O-dimethyl phosphorothioate), on lipid metabolism by estimating the levels of seven classes of lipid in the liver, plasma, gonads, and muscle during the vitellogenic phase of the annual reproductive cycle in both sexes of Clarias batrachus. MATERIALS
AND
METHODS
Adult and healthy specimens of both sexes of C. batrachus with an average weight of 65 g (60-70 g) and size of 18.5 cm (18-19 cm) were collected in the first week of May from the ponds around Varanasi, India. Fish were acclimatized to laboratory conditions for 2 weeks and were fed minced goat liver containing 20% protein, 5% lipid, and 15% carbohydrate every fourth day. The fish were maintained under natural photoperiod and temperature. After acclimatization fish were sexed and specimens of each sex were divided into six batches, each comprising 10 fish, in an aquarium of 90-liter capacity. Fish were exposed to 96-hr safe concentrations (SC) and sublethal concentrations (SL) of malathion (1 and 4 ppm) and BHC (2 and 8 ppm) which have been standardized and reported (Lal and Singh, 1984). Malathion was added directly to water, but 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 BHC 1 ml of acetone was also added to the aquarium water. Experiments were conducted for 4 weeks (3rd week of May-2nd week of June) during the vitellogenic phase at natural photoperiod ( 13L: 11 D) and temperature (30 & 2°C). During experimentation, fish were fed every 4th day when aquarium water was changed with fresh water having similar concentrations of the above pesticides. Chemicals Lipid standards. Monopalmitin for monoglycerides (MG), dipalmitin for diglycerides (DG), tripalmitin for triglycerides (TG), palmitic acid for free fatty acids (FFA), lecithin for phospholipids (PL), cholesterol for free cholesterol (CF), and cholesterol oleate for esterified cholesterol (CE) were purchased from Sigma (U.S.A.). Silica gel GhO was purchased from E. Merck (Germany) and AR grade solvents (BDH, India) were distilled and used. Tricane methane sulfonate (MS 222) for anesthesia was a gift from Sandoz Ltd. (Switzerland). y-BHC and malathion were obtained from Bharat Pulverising Mill Private Ltd., India, and Cynamid India Ltd., India, respectively. Lipid Extraction
and Separation
When experiments were terminated, blood was collected separately by caudal puncture from individual fish of each group, in heparinized tubes, and centrifuged at 2000g at 4°C; plasma was frozen at -20°C for further analysis. Individual liver, ovary,
IMPACT
OF
PESTICIDES
ON
LIPID
METABOLISM
15
testis, and muscle were extirpated, washed in 0.6% saline, blotted, and kept frozen at -20°C until further analysis. Tissues and plasma were extracted in chloroform: methanol (2: l), following the method of Folch et al. (1957) for total lipid concentration, which was measured gravimetrically. Triplicate samples of each tissue from a single specimen were taken for analysis. Each sample weighed 30-40 mg for liver, gonads, and muscle and 100 ~1 for plasma. Further, different fractions of lipid were separated by thin-layer chromatography (&values for MG, DG, TG, FFA, PL, CF, and CE were 0.15,0.64,0.80,0.27,0.00,0.44, and 0.90, respectively) by the double solvent system of Freeman and West (1966) (system I-diethylether:benzene:ethanol:acetic iacid, 40:50:2:0.2, and solvent system II-hexane:diethylether, 94:6). Spots of various lipids were visualized by exposing the plates to iodine vapor. Spots of different lipid fractions of samples, standards, and corresponding areas of silica gel from blanks were scraped and transferred to separate test tubes. Quantitation Quantitative estimation of various lipids were made spectrophotometrically on Spectronic-2000 (Bausch & Lomb, U.S.A.) at 375 nm by the method of Matzo et al. (197 1). D,ata were expressed in milligrams per gram of tissue or per milliliter of plasma (mlean -t SE), and Student’s t test was employed for statistical analysis. RESULTS Female Four weeks’ exposure of this fish to malathion and -/-BHC at SC and SL caused appreciable changes in various classes of lipids without affecting total lipid concentration of the liver, plasma, ovary, and muscle. Results are summarized in Tables 1 and 2. Efects on Free Fatty Acids (FFA) Malathion at safe and sublethal concentrations increased FFA levels in the liver and plasma but reduced their levels in the ovary and muscle. Similarly, y-BHC also increased FFA of the liver and plasma at both concentrations. However, ovarian FFA was decre,ased by safe concentration of -y-BHC, but its sublethal concentration could not produce any change, whereas the muscular FFA was reduced by sublethal concentration of -/-BHC and remained unchanged in response to its safe concentration. l$ects on Acyl Glycerides SublethLal concentration of both pesticides decreased mono- and triglycerides in the liver, plasma, ovary, and muscle. Moreover, diglycerides were also decreased in the liver, plasma, and muscle, but in the ovary they were elevated in response to malathion and remained unchanged after y-BHC treatment. These pesticides at a safe concentration also caused a decrease in mono-, di-, and triglycerides of all the tissues studied except ovarian triglycerides, which were elevated.
1
Level of
51.17 f
53.10 f 4.89”
SC
10.95 k
10.07 f
SL
SC
34.35 IL 4.17
37.71 * 3.03”
36.01 k 2.81”
SC
5 I .03 k 4.09”
SC
SL
56.18 + 5.61”
SL
Control
65.79 +- 5.79
Control
1.87”
1.01 a
10.6 I z!z 0.96
Control
5.76“
46.79 + 3.69
SL
TL
Control
malathion
Nore. Values are expressed as mg/g tissue or m&l except those marked ‘I”‘.
Muscle
Ovary
Plasma
Liver
Tissue
0.15 f0.01
0.21 f 0.01
0.45 f 0.0 1
0.12 * 0.01
0.35 + 0.02
0.40 * 0.03
0.5 I + 0.02
0.08 f 0.0 1
0.54 +- 0.02
0.56 +- 0.02
I. I5 2 0.05
7.45 l!z 0.37
2.83 t- 0.18
5.54 + 0.89
0.69 k 0.03
0.59 + 0.06
0.96 1: 0.02
4.13 +-0.10
4.31 IO.21
10.94 $0.21
TG
2.61 f 0.12
0.19+0.01
1.16 -to.05 0.17~0.01
1.26 +- 0.07
2.38 f 0.12
2.06 + 0.2 I
3.98 + 0.17
0.14f0.01
2.41 + 0.07
9.39 + 0.27
10.13 + 0.32
13.89 -t 0.42
1.69 + 0.35
0.17 kO.01
0.74 f 0.03
2.80 + 0.23 1.53 * 0.33
0.5 1 f 0.07”
0.45 f 0.04”
0.46 f 0.03
CF
9.36 f 0.32
9.04 + 0.29
6.31 + 0.12
PL
0.36 z!z 0.03
0.39 Ik 0.03
3.14 + 0.43
5.07 + 0.46
5.74 + 0.48
2.07 f0.17
0.64 f 0.04
0.36 k 0.01 0.84 AI 0.03
8.01 I? 0.15
8.13 to.18
0.68 t 0.06
CE
plasma. and are means + SE; n = 5. P values for SL and SC were compared against their respective controls and were found significant
5.41 + 0.36
4.04 f 0.36
8.42 k 0.46
9.38 2 0.86
1.88 + 0.05
1.01 + 0.12
0.19 -t 0.01
0.38 k 0.02
8.91 rt 0.23
14.93 Ik 1.37
0.29 f 0.02
0.07 f 0.0 1
0.37 t- 0.03
0.21 f 0.01
0.05 + 0.0 I
0.42 f 0.02
1.12 -t 0.23
0.92 50.13
2.52 -I- 0.43
DG
0.40 * 0.04
0.08 + 0.0 1
1.15 ItO.
16.58 + 0.89
0.29 + 0.01
1.55 f0.15 0.64 IL 0.02
17.21 + 0.99
MG
13.66 + 0.27
FFA
EFFECT OF MALATHION ON TOTAL LIPID (TL), FREE FATTY ACIDS (FFA), MONOGLYCERIDES (MG), DIGLYCERIDES (DG), TRIGLYCERIDES (TG), PHOSPHOLIPIDS (PL), FREE CHOLESTEROL (CF), AND ESTERIFIED CHOLESTEROL (CE) LEVELS IN LIVER, PLASMA, OVARY, AND MUSCLE IN FEMALE Clarias batrachus
TABLE
c
$
z g
r
IMPACT
OF
PESTICIDES
ON
LIPID
METABOLISM
17
18
LAL
AND
SINGH
Efects on Phospholipids Malathion and y-BHC at both concentrations elevated the phospholipid levels in the muscle and decreased them in the plasma and ovary. But, hepatic levels were increased in response to malathion and were decreased in r-BHC-exposed fish. Efects on Cholesterol Both pesticides, at either concentration, caused reduction in free cholesterol levels of the plasma, ovary, and muscle, whereas hepatic levels remained unchanged by malathion and were decreased in y-BHC-treated fish. Contrary to free cholesterol, esterified cholesterol was raised in all the tissues except in muscle, where it was reduced by both pesticides. Male Malathion evoked an increase in total lipid level of the liver in male C. batrachus in contrast to that of the female, whereas in other tissues it failed to induce any change in total lipid levels. -/-BHC also could not produce any obvious change in total lipid of the tissues studied. However, varied responses were noted in various classes of lipids in different tissues after pesticide exposure. Results are summarized in Tables 3 and 4. Effects on Free Fatty Acids and AcyI Glycerides Free fatty acid levels along with mono-, di-, and triglycerides in the liver and monoand triglycerides in testes were enhanced by these pesticides at either concentration, but these lipids were suppressed in the plasma. However, in muscle their levels remained unchanged in response to y-BHC and were reduced in malathion-exposed fish. Efects on Phospholipids
and Free and EsteriJied Cholesterol
High and low hepatic phospholipids were noticed in malathion- and -y-BHCtreated fish, respectively. Phospholipid levels in the plasma and testes were suppressed but raised in muscle. Malathion exposure caused a decrease in concentration of free cholesterol in the liver, testes, and muscle but elevate levels in the plasma, whereas r-BHC evoked an increase in hepatic free cholesterol but a decrease in the plasma, testes, and muscle. Both pesticides increased the esterified cholesterol levels in all the tissues studied. DISCUSSION y-BHC and malathion caused appreciable changes in various classes of lipid of different tissues in C. batrachus but the spectrum of total lipid apparently remained unchanged. A similar observation for total lipid in the liver and ovary in H. fossilis in response to malathion and hexadrin during the prespawning phase has been recorded by Singh and Singh (1980d). However, Murty and Devi ( 1982) have reported decreased liver lipids after technical endosulfan treatment in C. punctatus. In female C. batrachus increased hepatic free fatty acids accompanied by low mono-, di-, and
30.54 3I 5.05”
SC
31.07zk4.13a
SC
k 3.06”
23.12
SL
+ 2.17
27.54
Control
k 4.81”
31.23
SL
+- 3.79
26.51
10.50 _+ 2.10”
SC
Control
7.21 + 1.89”
SL 0.03
0.85kO.21
0.77 f 0.1 1
3.00 310.48
12.44 + 0.46
13.49 -t 0.85
4.61 f 0.06
0.51 f
0.59 -t 0.03
1.63 + 0.33
19.67 + 0.64 20.72 f 0.63
7.11 -to.01
FFA
0.06 2 0.0 I
0.06 310.01
1.58 k 0.09 0.20 IL 0.02
1.41 20.08
0.25 f 0.01
0.31 -to.01
0.27 ?I 0.01
0.17 f 0.04 1.16 -to.05
0.15 f 0.01
0.28 -t 0.02
0.22 + 0.0 1
0.16 +O.Ol
0.07 + 0.0 1 0.07 f 0.01
0.34 AI 0.02
4.61 f 0.07 4.76 It_ 0.32
3.67 -t 0.10
DG
0.09 + 0.0 1
1.87 k 0.11 2.47 k 0.14
1.22 f 0.04
MG
0.85 + 0.03
0.80 k 0.03
2.1 I + 0.23 1.41 f 0.03
2.21 + 0.23
1.90 + 0.08
1.08 -t 0.08
1.43 -t 0.08
1.84 k 0.08
9.72 +_ 0.82 10.72 * 0.93
2.71 f0.13
TG
0.55 f 0.08 0.12 f 0.01
0.56 kO.16
1.22 +_ 0.05
1.35 rt 0.05
0.02
2.83 k 0.12
against their respective controls and were found significant
0.13f0.01
0.37 f
0.1 I fO.O1
8.2 1 f 0.82 0.29 f 0.04
0.83 f 0.17
2.92kO.13
0.14 f 0.01
7.2 1 + 0.79
1.03 + 0.03
0.87 + 0.03
12.09 f 0.3 1
0.87 IL 0.03
0.67 + 0.02
1.07 +- 0.04
0.28 + 0.02
7.47 + 0.89 7.74 + 0.46
1.52 ?c 0.03
CE
1.79 31 0.06
0.71 * 0.02
0.32 + 0.03
2.01 f 0.05 1.81 +- 0.05
0.98 f 0.02 0.88 f 0.02
2.73 f 0.17
7.23 f 0.13 14.05 f 0.84 13.54 f 0.95
CF
PL
LIPID(TL),FREEFATTY ACIDS(FFA),MONOGLYCERIDE~(MG),DIGLYCERIDES(DG),TRIGL~~ERIDES(TG), (PL), FREE CHOLESTEROL (CF), AND ESTERIFIED CHOLESTEROL (CE) LEVELS IN LIVER, PLASMA, TESTES,ANDMUSCLEOFMALE Clariasbatrachus
3
NW,. Values are expressed as mg/g tissue or mg/ml plasma, and are means 2 SE: n = 5. P values for SL and SC were compared except those marked ““‘.
Muscle
Testes
8.08 2 1.14
Control
50.09 IL 4.02 53.28 Ik 4. I I
SL SC
Plasma
36.17 -t 3.04
Control
Liver
TL
malathion
Level of
ONTOTAL PHOSPHOLIPIDS
MALATHION
Tissue
EFFECTOF
TABLE
5
W
i5
$
r z u g
z
E
2 E E;
41 3
2 3
4
SC
32.92 f 4.07”
33.61 + 3.20”
30.70 k 4.12”
SL
SC
26.07 f 3.54
Control
34.26 f 3.48“
4.18 + 0.66
12.17f2.51”
SC
27.18 -t 2.72
0.30 Ik 0.02
11.81 + 1.68”
SL
SL
1.40 + 0.03
10.13 _+ 1.72
Control
Control
14.71 -t 0.92
45.89 f 6.15”
SC
0.25 zk 0.02”
1.04 r+ 0.05
0.93 + 0.06”
0.96 iz 0.04”
1.39 f 0.02
0.40 Ik 0.03”
0.51 f 0.02
0.49 _+ 0.02
0.59 +_ 0.04
0.65 Z!I 0.03
0.41 f 0.01
0.81 + 0.04
0.15 zk 0.01
O.l8t-0.01
0.30 -t 0.01
14.87 k 0.86
3.42 zk 0.09
2.20 -t 0.10
CF
0.39 * 0.03
0.3 1 xk 0.02”
0.34 t 0.0 1
I .86 * 0.01
1.52 f 0.04
1.10f0.04
1.31 * 0.03
1.43 -t 0.03
0.35 * 0.03
5.69 -+ 1.17
18.54 k 2.39
1.50 f 0.06
CE
(TG),
against their respective controls and were found significant
2.42 f 0.12
1.09 + 0.04
1.57 It_ 0.40
3.41 f 0.09
0.42 + 0.02”
0.49 zk 0.0 1
1.78 f 0.01
1.98 k 0.02
11.89 zk 0.27
0.73 f 0.01
0.57 -t 0.03 1.5 1 t- 0.07
0.45 * 0.03
0.63 + 0.01
2.01 + 0.36
0.83 f 0.04
6.48 f 0.10 1.68 f 0.06
4.57 -t 0.17
7.76 + 0.57
PL
6.28 t- 0.17
2.86 f 0.13
TG
plasma. and are means ?z SE; n = 5. P values for SL and SC were compared
4.01 x!I 0.55”
0.27 + 0.02”
0.17 f 0.02
0.24 f 0.0 1
0.12 + 0.01
2.34 + 0.03
0.31 f 0.03
12.3 1 t- 0.46
3.90 -t 0.38
1.27
1.52
0.23 f 0.01
0.21 * 0.02
0.25 + 0.01
0.08 f 0.01’
4.07 z!I 0.19”
18.00 k
23.18 k
0.49 _t 0.02
0.35 ?I 0.02
0.03 f 0.01
5.53 f 0.24
5.17 +_ 0.36
3.87 + 0.13
DG
0.08 f 0.0 1
11.37 + 0.96
1.10~0.04 2.18 + 0.26
1.87
7.00 t 0.29
31.50 k
3.17
38.73 t- 4.50”
MG
34.84?
FFA
SL
TL
ON TOTAL LIPID (TL),FREE FATTY ACIDS (WA), MONOGLYCERIDES (MG), DIGLYCERIDES (DG), TRIGLYCERIDES PHOSPHOLIPIDS (PL). FREE CHOLESTEROL (CF), AND ESTERIFIED CHOLESTEROL (CE) LEVELS IN LIVER, PLASMA, TESTES, AND MUSCLE OF MALE Clarias batrachus
Control
BHC
Level of
OF BHC
Note. Values are expressed as mg/g tissue or mg/ml except those marked ‘Ia”.
Muscle
Testes
Plasma
Liver
Tissue
EFFECT
TABLE
IMPACT
OF
PESTICIDES
ON
LIPID
METABOLISM
21
triglyceridles after pesticide exposure indicated that the biosynthesis of fatty acids was probably not affected, but their conversion to acyl glycerides was impaired. Increased fatty acid content in liver has been reported in PCB-treated rats (Kling et al., 1978). However, elevated plasma free fatty acids in the pesticide-exposed fish might be the consequence of either their release from liver being greater than their uptake by other tissues, or as a byproduct of complete hydrolysis of circulating dietary triglycerides, which was evident by decreased plasma triglycerides. Decreased levels of free fatty acids in ovary and muscle appeared to be due either to their restricted transportation from liver to ovary or to faster utilization. In earlier reports, it has been shown that the teleostean ovary imports lipids and phospholipids from liver during the vitellogenie phase under the influence of sex steroids and gonadotropin (Wiegand and Peter, 1980; Upadhyay, 1977). Thus, it appeared that mobilization of these lipids was probably inhibited by these pesticides in C. batruchus by decreasing the levels of sex steroids and gonadotropin. Singh and Singh (1980a,b) and Kapoor et al. (1978) have recorded reduced gonadotropic potency in malathion-treated H. ,fossilis and 3-P-hydroxysteroid dehydrogenase activity in the ovary of fenitrothion-exposed C. carpio, respectively, findings which support our above views. However, increased levels of triglycerides in the ovary in response to malathion and r-BHC at the safe concentration were interesting and unusual, but cannot be interpreted at this stage. In malIe C. batruchus, unlike in the female, these pesticides appeared to stimulate lipogenic activity in liver by increasing fatty acid synthesis and its conversion to acyl glycerideis. This increase might also be due to inhibited P-oxidation of free fatty acids as well as their utilization, whereas elevated levels of free fatty acids and triglycerides in testes s,uggested that these pesticides probably reduced their utilization by suppressing testicular activity. In normal male C. batrachus, La1 and Singh (unpublished) have found decreased free fatty acids and triglycerides in testes during the vitellogenic phase under natural conditions. They have attributed these decreases to increased consumption (as fuel) to sustain enhanced testicular activity during this phase. However, reduced levels of these lipids in plasma might be due to their reduced release from liver to plasma. Hepatic phospholipids were raised in malathion-treated C. batrachus of either sex but were reduced in r-BHC-exposed fish, indicating that y-BHC probably interfered with their biosynthesis in liver, whereas both pesticides apparently inhibited their mobilization from liver to gonads, as was evident by their low levels in plasma and gonads. Here again it might be due to decreased sex steroids and gonadotropin, which are required for phospholipid uptake by ovary (Upadhyay, 1977; La1 and Singh, 1983). Singh and Singh ( 1980a,b) have also reported decreased 32P uptake by ovary and testes in H. fossilis after malathion and hexadrin exposure. Decreased free cholesterol and increased esterified cholesterol levels were recorded in all the tissues studied in pesticide-exposed C. batrachus, suggesting that conversion of esterified to free cholesterol was inhibited without affecting the cholesterol synthesis as such. Decreased levels of gonadotropin, as a consequence of pesticide action, might possibly be responsible for this inhibited conversion. CONCLUSIONS From the data it can be concluded that the actions of both pesticides on the metabolism of nonpolar lipids were alike and sex related. However, hepatic biosynthesis of
22
LAL
AND
SINGH
polar lipids, like phospholipids, seemed to be affected differently by these two pesticides. ,&Oxidation of free fatty acids, as well as mobilization of various lipids from liver to ovary, was inhibited by these pesticides in C. batrachus. ACKNOWLEDGMENTS Grant-in-Aid HCS/DST/928/80 for research and ICAR-PL-480 Project IN-ARS-213 New Delhi to T.P.S. are gratefully acknowledged. We are grateful to Mr. S. W. Vaidya of Bharat Pulverishing Mill Ltd., India, for y-BHC and to Mr. S. R. Maley ofcynamid India Ltd. for malathion.
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IMPACT
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