- Email: [email protected]
γ-BHC- and Cythion-induced alterations in lipid metabolism in a freshwater catfish, Clarias batrachus, during different phases of its annual reproductive cycle
ECOTOXICOLOGY
AND ENVIRONMENTAL
SAFETY
14,38-47 (1987)
r-BHC- and Cythion-Induced Alterations in Lipid Metabolism in a Freshwater Catfish, C/arias batrachus, during Different Phases of Its Annual Reproductive Cycle B. LAL AND T. P. SINGH* Fish Endocrinology Laboratory, Department of Zoology, Banaras Hindu University, Varanasi- 221005, India Received December 24, 1986 Specimens of either sex of the freshwater catfish Clark batrachus were exposed to safe and sublethal concentrations of r-BHC (2 and 8 ppm) and Cythion ( 1 and 4 ppm) for 4 weeks during diierent phases of the annual reproductive cycle. Their effects on various lipid fractions, viz., triglycerides (TG), phospholipids, free cholesterol, and ester&A cholesterol, were studied in liver, plasma, gonads, and muscle. These pesticides suppressed the level of hepatic TG in females during the preparatory, prespawning, and postspawning phases, while during the spawning phase they stimulated an increase in its level. However, in males, these pesticides were ineffective in having any effect on liver TG during the spawning and postspawning phases, but in the preparatory phase, as with the female, they increased its levels, while in the prespawning phase they decreased liver TG levels. Hepatic phospholipid biosynthesis was impaired by -y-BHC but Cythion had no effect on it. Cholesterol biosynthesis as such appeared to be unaffected by these pesticides but the dynamics of free and esterified cholesterol levels were disturbed in response to Cythion and y-BHC. These pesticides greatly reduced the mobilization of these hepatic lipids to gonads. Muscle lipids were least a&ted by these pesticides. Q 1987 Academic RW, I~C.
INTRODUCTION In recent years, there has been increasing concern and potential threat to fish culture programs as a result of indiscriminate and wide use of pesticides. Pesticide actions have been correlated with reproductive dysfunction in a variety of teleosts (for review, see, Singh and Sir&, 1983; Sir@ et al., 1985; Safe, 1984). Inspite of the established fact that in the teleost gonadal growth and development are greatly associated with sequential changes in physiological and biochemical events, most of the studies on pesticide actions in fishes are restricted to the toxicity test, growth rate assessment, fecundity, and reproductive activity (Wildish et al., 197 1; Burdick et al., 1972; Jackson, 1976; Anees, 1978). Very little attention has been paid to the effects of pesticides on the biochemistry of reproduction, particularly on lipid metabolism as lipids serve as an energy source, a major constituent of vitellogenin (de Vlaming et al., 1984) and a processor of sex hormones. In earlier reports, Singh and Singh (1980b,c,d) and Murty and Devi ( 1982) have measured gravimetrically total lipids to observe the effects of pesticides but these studies do not provide a clear picture of lipid metabolism as lipids undergo rapid breakdown, resynthesis, and interconversion with a slight change in extero- and interoceptic stimuli. Thus, the effects of pesticides on lipid metabolism in fish remain obscure. Therefore, in the present study effects of pesticides (r-BHC, an organochlorine, 1,2,3,4,5,6-hexachlorocyclohexane), and Cythion (malathion), an organophospho* To whom requests for reprints should be addressed. 0147-6513187 $3.00 Copyrisbt 0 1987 by Academic Press, Inc. All rinhts ofremoduction in any form resmed.
38
LIPID METABOLISM
39
AND PESTICIDES
TABLE
1
GONADOSOMATICINDEX (GSI) OF C. batrachus BEFORE PESTICIDE EXPOSURE AND PHOTOPERIOD AND TEMPERATUREDURING EXPERIMENTATION GSI before pesticide exposure
Reproductive phases
Date of fish collection
Female
Preparatory Prespawning Spawning Postspawning
March 5 May 7 July 3 Sept. 8
1.0 6.2 8.5 0.4
Male
Photoperiod
Temperature (“Cl
0.05
12L12D 13.5L10.5D 13L:llD 11.5L12.5D
28+2 33 f 2 31+2 26 + 2
0.15
0.20 0.06
rus, S( 1,2-dicarbethoxyethyl) ester of O-O-dimethylphosphorodithioate) were studied on lipid metabolism in either sex of a freshwater catfish, Clurias batrachus, by using chromatographic and spectrophotometric techniques to estimate triglycerides (energy source), phospholipids (vitellogenin constituent), and free and esterified cholesterol (sex hormone precursor) in the liver, plasma, gonads, and muscle during different phases of its annual gonadal cycle. MATERIALS
AND
METHODS
Fish Adult specimens of either sex of C. batruchus (weight between 60 and 70 g and size between 18 and 19 cm) were collected the first week of each reproductive phase from ponds around Varanasi, India (Table 1). Fish were acclimatized to laboratory conditions for 2 weeks prior to experimentation in each phase and were fed standardized rations (20% protein, 5% lipid, and 15% carbohydrate) on alternate days. They were maintained under natural photoperiod and temperature. Chemicals Lipid standards. Tripalmitin for triglycerides (TG), lecithin for phospholipids (PL), cholesterol for free cholesterol (CF), and cholesterol oleate for esterified cholesterol (CE) were obtained from Sigma (U.S.A.). Silica gel Gso was purchased from E. Merck (Germany) and AR grade solvents (BDH, India) were distilled and used. r-BHC and Cythion were obtained from Bharat Pulverising Mill Private Ltd., India, and Cynamid India Ltd., India, respectively. Pesticide Exposure During each phase, after acclimatization fish were sexed and specimens of each sex were divided into six batches, each having 10 fish, in an aquarium of 90-liter capacity. Fish were exposed to 96 hr safe concentrations (SC) and sublethal concentrations (SL) of Cythion (1 and 4 ppm) and r-BHC (2 and 8 ppm) for 4 weeks, a procedure which had already been standardized and reported (Lal and Singh, 1984). During experimentation fish were fed every 4th day when aquarium water was changed with fresh water having similar concentrations of the above pesticides.
40
LAL AND SINGH
Lipid Extraction and Separation On termination of the experiments on Day 28 blood was collected separately from each fish of each group in heparinized tubes by caudal puncture and centrifuged at 2000g at 4°C and plasma was frozen at -20°C for further analysis. Individual liver, ovary, testis, and muscle were extirpated, washed in 0.6% saline, blotted, and kept frozen at -20°C until analysis. Tissues and plasma lipids were extracted in chloroforrnmethanol(2: 1) following the method of Folch et al. (1957). 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 the volume obtained for plasma was 100 ~1. Different fractions of lipids were separated by thin-layer chromatography (&values for TG, PL, CF, and CE were 0.80,0.00, 0.44, and 0.90, respectively) by using the double system (System I, diethylether:benzene:ethanol:acetic acid, 40:50:2:0.2, and Solvent system II, hexane:diethylether, 94.6) of Freeman and West (1966). Spots of various lipids were visualized by exposing the plates to the iodine vapor. Spots of different lipid fractions of samples, standards, and the corresponding area of silica gel from blanks were scraped and transferred to separate test tubes. Quantitation Quantitative estimation of various lipids was made spectrophotometrically on Spectronic-2000 (Bausch & Lomb, U.S.A.) at 375 nm by the method of Marzo et al. (197 1) with one modification in which incubation time was reduced from 45 to 30 min. Data were expressed in milligrams per gram tissue or milligrams per milliliter plasma. For statistical analysis of data, analysis of variance along with supplemented Newman-Keuls multiple range t test were employed at the cxlevel of 0.05. RESULTS Analysis of variance clearly indicated that the responses of different lipids varied with phase, tissue, nature, and concentrations of pesticides. Changes in TG, PL, CF, and CE are shown in Figs. l-4. Preparatory Phase Triglycerides. Hepatic level of TG was suppressed by both pesticides in either sex of C. batrachus but their levels in the plasma and muscle were unresponsive to pesticide exposure. Ovarian TG was also unchanged; however, in testes its level was decreased. Phospholipids. In both sexes, a significant reduction in hepatic as well as plasma levels of PL was recorded in response to y-BHC. Similarly, Cythion also caused a decrease in their levels, but only in females. In the ovary, PL was unaltered, while in the testes it was depressed by both pesticides. These pesticides had no effect on muscular PL levels. Free and esterijied cholesterol. Significantly reduced levels of CF were registered in the liver in the female, but in the male, its levels were unchanged with respect to these pesticides. Its levels in plasma, gonads, and muscle in either sex remained unaltered after pesticide exposure. Unlike free cholesterol, CE was raised by Cythion and yBHC in the liver, while plasma levels were unresponsive. Testicular CE was also elevated by both pesticides but in the ovary only Cythion could raise its levels. In muscle, however, it was increased in females and remained unchanged in males.
LIPID METABOLISM
41
AND PESTICIDES MUSCLE
2.30
d
t
GONAD
p 3
CONTROL CYTHION CYTNION
-
SC -SL
PLASMA
0 0
,.
. .
t 400 0 5
13* 0 t
il.ml
12.0
z I5.0
. a’ 0
m PREP
PRESPO
SPO
POSTSPO
PREP
PMSPB
SPO
POBTEPO
FIG. 1. Level of triglycerides in response to Cythion and r-BHC in C. batrachus. x, not significant.
Prespawning Phase Triglycerides. These pesticides at either concentration decreased TG in the liver, plasma, and muscle in female C. batrachus. Ovarian TG levels were enhanced at safe concentrations and remained unchanged at sublethal concentrations of both pesticides. However, in male C. batrachus, these pesticides provoked an increase in TG levels in the liver and testes, whereas in the plasma, either pesticide reduced TG levels. Muscular TG was also reduced but only in response to Cythion. Phospholipids. In either sex of C. batrachus, Cythion and y-BHC at both concentrations caused a decrease in the plasma and gonadal PL but hepatic levels were raised by Cythion and reduced by r-BHC. Muscular PL, however, remained unchanged in males and was elevated in females by both pesticides. Free and ester$ed cholesterol. These pesticides at either concentration evoked a significant reduction in CF levels in the gonads and muscle in both sexes. While its
42
LAL AND SINGH 3.0-
MUSCLE
17.or
o-
GONAO
w
PLASMA
2Q)
14.0
LIVER
0 2 7.0
0i
FIG.
PRSP
WISP0
SPO
POSTSPO
PREP
2. Level of phospholipids in response to Cythion and yBHC in C. butruchux x, not significant.
hepatic levels remained unaltered in females and were reduced in males by Cythion, T-BHC induced a decrease and increase in its hepatic levels in females and males, respectively. Moreover, plasma CF was decreased by both pesticides in females, but in males only T-BHC reduced its level while Cythion increased it. Contrary to these data, CE levels were elevated by both pesticides in all the tissues studied in either sex of C. batrachus except in muscle where it was decreased. Spawning Phase Triglycerides. Both pesticides at either concentration elevated the levels of TG in the liver and ovary and decreased it in the plasma without affecting it’s level in the muscle in female C. batrachus. In males, TG levels of liver, plasma, and muscle were unresponsive but in testes they were reduced in response to these pesticides. Phospholipids. As in the preparatory phase, Cythion provoked an increase in PL levels in the liver with marked decrease in the plasma and gonads in both the sexes
LIPID 1 .o
METABOLISM
43
AND PESTICIDES MUSCLE
OT
0.6
2
FIG.
4.Or
6
GONAD
3. Level of free cholesterol in response to Cythion and yBHC in C. batrach.
x, not significant.
of C. batrachus. y-BHC also behaved similarly except that it caused a significant reduction in the hepatic level in females. Free and esteriJied cholesterol. In both sexes, Cythion and r-BHC failed to induce any appreciable change in CF levels in any of the studied tissues, whereas CE in the liver and gonads were increased. Plasma CE was also increased in females but only at sublethal and safe concentrations of Cythion and -r-BHC, respectively. In males, Cythion could not produce any change and r-BHC raised CE levels in the plasma. Muscular CE, however, remained unaffected. Postspawning Phase During this stage, very little effects could be noticed in either sex of C. batrachus. Lipid levels studied in males remained totally unaffected by these pesticide exposures. However, in females, a decreased level of TG and increased levels of free and esterified cholesterol in the liver were registered after pesticide exposure. Plasma TG was also increased.
44
LAL AND SINGH MUSCLE
@&;i
& PLASMA
PliESPO FIG.
SPO
4. Level of esterified cholesterol in response to Cythion and y-BHC in C. butruchus. x, not signiikant.
DISCUSSION In retrospect of the present findings it can be suggested that the effects of pesticides (Cythion and r-BHC) on lipid metabolism in C. batruchus vary with the change in the nature of lipid class, tissue, period of annual reproductive cycle, sex, as well as nature and concentration of pesticides. Decreased hepatic triglycerides in female C. batrachus during preparatory, prespawning, and postspawning phases suggested that these pesticides inhibited TG biosynthesis in liver. Singh and Singh ( 198Oc) have also reported decreased levels of hepatic total lipids in Cythion- and hexadrin-exposed Heterbpneustes fossilis during the preparatory phase. Murty and Devi (1982) have shown in Channapunctutusdecreased liver lipids after endosulfan treatment. Dzogbefia et al. (1978) have observed suppressed glyceride biosynthesis in FCB-treated rat liver in vitro. Though the comparison of these results with our findings may be misleading due to variations in experimental design and species, they partially support our results indirectly. Further, pesticide-induced increase in hepatic TG levels during the spawning phase in females and during the prespawning in males can be attributed to their decreased utilization. Because under natural conditions, normal
LIPID
METABOLISM
AND PESTICIDES
45
female C. batrachus during the spawning phase diminish food intake and show prominent lipolytic activity to meet the caloric demand of spawning behavior and activity (Lal and Sir&, 1984). Moreover, these pesticides appeared not to interfere with triglycerides of the plasma, ovary, and muscle during preparatory and postspawning phases, whereas in the prespawning phase decreased triglycerides might have resulted from restricted mobilization of hepatic triglycerides to the ovary. de Vlaming et al. (1977a,b), Upadhyay (1977), Wiegand and Peter (1980a,b), and Lal and Singh (1983) have demonstrated increased lipogenic activity as well as transfer of lipids to ovary during the prespawning phase under the influence of sex hormones and gonadotropins under natural conditions. Moreover, Singh and Singh (1980a) in H. fossilis, Kapoor et al., ( 1978) in Cyprinus carpio, and Sir@ (1985) in C. batrachus have found decreased gonadotropic potency, 3&hydroxysteroid dehydrogenase, and sex steroids (esterone, estradiol- 178, and testosterone), respectively. Thus, in view of these results it can be suggested that these pesticides inhibited hepatic lipogenesis and lipid deportation from liver to gonads in C. batrachus by decreasing the level of sex steroids and gonadotropins. However, increased ovarian TG during spawning and testicular TG in the prespawning phase have again suggested the inhibited utilization and hydrolysis of TG to free fatty acids and ultimately ,&oxidation of the latter. That is perhaps why sufficient metabolic water is also not produced which is required for hydration of the ovary preceding ovulation. Free fatty acids, upon oxidation, produce enough metabolic water and ATP. Cythion and r-BHC also seemed to inhibit hepatic biosynthesis of phospholipids in females during the preparatory phase, while during the prespawning phase only r-BHC was effective in reducing phospholipid hepatic biosynthesis. In males, a similar mechanism appeared to be operative. Dzogbefia et al. ( 1978) have also reported decreased hepatic phospholipid biosynthesis in PCB-treated rat liver. These pesticides also impaired the mobilization of phospholipids to gonads in C. batrachus. In C. batrachus these pesticides seemed to be totally ineffective in altering total cholesterol content or biosynthesis as a whole, except for inhibiting the hydrolysis of esterified cholesterol to free cholesterol in liver and gonads during the period of gonadal growth and maturation. This was perhaps one of the reasons that Singh and Singh (1987) found decreased estradiol, estrone, and testosterone in Cythion- and y-BHC-exposed C. batrachus. The possibility of reduced hydrolysis of ester&d cholesterol to the free form is further supported indirectly by the fact that gonadotropin, required for this hydrolysis (ZaIkroni et al., 1958), is reduced in response to Cythion and hexadrin in the Indian catfish H. fossilis (Singh and Singh, 1980a). Thus, probably in C. batrachus also these pesticides exert their impact on cholesterol metabolism by reducing gonadotropic potency. CONCLUSION The present study has clearly demonstrated that Cythion and y-BHC both have altered lipid metabolism significantly and have very selective and specific effects on various lipids in C. batrachus, probably by acting at several sites in metabolic processes. Changes in extero- and interoceptic conditions may also have some impact on actions of these pesticides on lipids as was evident from their varied effects during
46
LAL AND SINGH
different phases of the annual reproductive cycle. Their actions on apolar and less polar lipids were more or less similar and were inhibitory to lipogenesis except during the spawning and prespawning periods in females and males, respectively, when these pesticides probably inhibited the use of lipids. Cholesterol biosynthesis as such was not impaired by these pesticides but the conversion of CE to CF was adversely affected. In general, r-BHC inhibited the phospholipid biosynthesis but Cythion did not. Mobilization of various hepatic lipids to the ovary was also restricted by Cythion and y-BHC. Muscle was the least affected tissue throughout the annual reproductive cycle in C. batrachus. Thus, these pesticides possibly derange the production in lipidderived energy, vitellogenin, and sex hormone precursor which ultimately results in decreased breeding potentials and in cessation of spawning. ACKNOWLEDGMENTS Grant-in-Aid for research from HCS/DST/928/80 and ICAR-PG480 Project No. IN-AR!&21 3, 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 of Cynamid Ltd. for Cythion.
REFERENCES ANEES, M. A. (1978). Hepatic pathology in a freshwater teleost Channa punctatus (Bloch) exposed to sublethal and chronic levels of three organophosphorus insecticides. Bull. Environ. Contum. Toxicol. 19,524-527. BIJRDICK, 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 GameJ. 19,97-l 15. DE VLAMING, V. L., FITZGERALD, R., DELAHUNTY, G., CEEH, J., SILMAN, K., AND BARKLEY, M. (1984). Dynamics of oocyte development and related changes in serum estradiol- 178, yolk precursor and lipid levels in the teleostean fish, Leptocottus armatus. Comp. B&hem. Physiol. 7lA 599-610. DE VLAMING, V. L., SHING, J., PAQUETTE, G., AND VUCHS, R. (1977a). In viva and in vitro effects of oestradiol 17 on lipid metabolism in Notemigenus crysoleucas. J. Fish Biol. 10,273-285. DE VLAMING, V. L., VODICNIK, M. J., BAUER, G., MURPHY, T., AND EVAN, D. (1977b). Estradiol-17 effect on lipid and carbohydrate metabolism and on the induction in yolk precursor in goldfish, Curassium auratus. Life Sci. 12, 1945-1952. DZOGBEFIA, V. P., KLING, D., AND GAMBLE, W. (1978). Polychlorinated biphenyl: In vivo and In vitro modifications of phospholipid and glycerides biosynthesis. J. Environ. Pathol. Toxicol. 1,84 l-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. FREEMAN, C. P., AND WEST, D. (1966). Complete separation of lipid classeson a single thin layer plates. J. Lipid Res. 7,324-327. JACKSON, G. A. (1976). Biological halflife of Eldrin in Channel catfish tissues. Bull. Environ. Contam. Toxicol. 16,505-507. KAPOOR, K., KAMALDEEP, K., AND TOOR, H. S. (1978). The effect of fenitrotbion on reproduction of a teleost fish, Cyprinus carpio communis Linn. A biochemical study. Bull. Environ. Contam. Toxicol. 20, 438-442. LAL, B., AND SINGH, T. P. (1983). Effect of ovariectomy and sex steroid administration on lipid metabolism in freshwater teleost, Clarias batrachus. In Proceedings of the First Znt. Symp. on Recent Advances in Life Sciences, Bodhgaya, India, 29-3 1 December, pp. 136. LAL, B., AND SINGH, T. P. (1984). Pesticides induced alterations in lipid metabolism during prespawning phase of reproductive cycle in freshwater teleost; Clark batrachus. In Proceedings of the Symposium on Comparative Endocrinology Varanasi, India, 29-3 1 December. LAL, B., SINGH, A., KUMARI, A., AND SINHA, N. (1986). Biochemical and haematological changes following malathion treatment in the freshwater catfish, Heteropneustes fossilis (Bloch). Environ. Pollut. A42, 151-156. LAL, B., YADAV, A. K., AND SINGH, T. P. (1985). Lipid programme and reproduction in catfish, Cfarias batrachus. In Second International Conference on Life Science, Shillong, Meghalaya, India, 14-16 November.
LIPID METABOLISM
AND PESTICIDES
47
MARZO, A., GHIRARCH, P., SERIDINI, D., AND MARONI, D. (197 1). Simplified measurement of monglyo erides, diglycerides, triglycerides and fattyacids in biological samples. Clin. Chem. 17, 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. SAFE, S. (1984). Polychlorinated (PCBs) and polybrominated biphenyls (PBBs): Biochemistry, toxicology, mechanism of action. CRC Crit. Rev. Toxicol. 13,3 19-393. SINGH, S. (1985). Steroidogenesis in Female Cathsh, Clarias batrachus. Ph.D. thesis, Department of Zoology, B.H.U., Varanasi, India. 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 freshwater catfish, Heteropneustesfossilis (Bloch). Environ. Res. 22, 190-200. SINGH, H., AND SINGH, T. P. (1980b). Effect of two pesticides on testicular 32P uptake, gonadotrophic potency, lipid and cholesterol content of testis, liver and blood serum during spawning phase in Heteropneustes fossilis (Bloch). EndokrinoIogie 76,288-296. SINGH, H., AND SINGH, T. P. (198Oc). Effect of two pesticides on total lipid and cholesterol content of overy, liver and blood serum during different phases of the annual reproductive cycle in the freshwater teleost, Heteropneustesfossiiis (Bloch). Environ. Pollut. A23,9-17. 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. A22,85-90. SINGH, H., AND SINGH, T. P. ( 1983). Effect of pesticides on fish reproduction. Ichthyologia 15,7 l-8 1. SINGH, S., AND SINGH, T. P. (1987). Impact of malathion and hexachlorocyclohexane on plasma profile of 3 sex steroids during different phases of the reproductive cycle in Clarias batrachus. Pestic. Biochem. Physiol. 22,301-308. SINGH, T. P., YADAV, A. K., LAL, B., AND SINGH, S. (1985). Impact of pesticides on fish reproduction, In Proceedings of International Conference on Pesticides: Toxicity, Safety and Risk, Assessment, Lucknow, India, in press. UPADHYAY, S. N. (1977). Morphologie des gonads immatures eletude experimental del’ induction de la gametogenese chez la truite arc-en-ciel juvenile Salmo gairdneri (R.). These de doctorate d’ Elat es Science Naturelles, L’ Universite Pierre et Marie Curie, Paris, France. WIEGAND, M. D., AND PETER, R. E. (1980a). Effect ofsalmon gonadotropin @G-100) on plasma lipids in the goldfish, Carassius auratus. Canad. J. Zool. S&957-966. WIEGAND, M. D., AND PETER, R. E. (1980b). Effect of sex steroids on plasma lipids in the goldfish, Carassius auratus. Canad. J. Zool. S&967-972. WILDISH, D. J., CARSON, W. G., CUNNINGHAM, T., ANLI LISTER, N. J. (197 1). Toxicological effects of some organophosphate insecticides to atlantic salmon. Fish. Res. Board Canad. Manscr. Rep. Ser. 1157, l-22.
ZAF’FARONI, A., HECHTER, O., AND PINCUS, G. (1958). (As cited by Hetchler, O., 1958.) Conversion of cholesterol to steroid hormone. In Cholesterol (R. P. Cook, Ed.), Academic Press, New York.
AND ENVIRONMENTAL
SAFETY
14,38-47 (1987)
r-BHC- and Cythion-Induced Alterations in Lipid Metabolism in a Freshwater Catfish, C/arias batrachus, during Different Phases of Its Annual Reproductive Cycle B. LAL AND T. P. SINGH* Fish Endocrinology Laboratory, Department of Zoology, Banaras Hindu University, Varanasi- 221005, India Received December 24, 1986 Specimens of either sex of the freshwater catfish Clark batrachus were exposed to safe and sublethal concentrations of r-BHC (2 and 8 ppm) and Cythion ( 1 and 4 ppm) for 4 weeks during diierent phases of the annual reproductive cycle. Their effects on various lipid fractions, viz., triglycerides (TG), phospholipids, free cholesterol, and ester&A cholesterol, were studied in liver, plasma, gonads, and muscle. These pesticides suppressed the level of hepatic TG in females during the preparatory, prespawning, and postspawning phases, while during the spawning phase they stimulated an increase in its level. However, in males, these pesticides were ineffective in having any effect on liver TG during the spawning and postspawning phases, but in the preparatory phase, as with the female, they increased its levels, while in the prespawning phase they decreased liver TG levels. Hepatic phospholipid biosynthesis was impaired by -y-BHC but Cythion had no effect on it. Cholesterol biosynthesis as such appeared to be unaffected by these pesticides but the dynamics of free and esterified cholesterol levels were disturbed in response to Cythion and y-BHC. These pesticides greatly reduced the mobilization of these hepatic lipids to gonads. Muscle lipids were least a&ted by these pesticides. Q 1987 Academic RW, I~C.
INTRODUCTION In recent years, there has been increasing concern and potential threat to fish culture programs as a result of indiscriminate and wide use of pesticides. Pesticide actions have been correlated with reproductive dysfunction in a variety of teleosts (for review, see, Singh and Sir&, 1983; Sir@ et al., 1985; Safe, 1984). Inspite of the established fact that in the teleost gonadal growth and development are greatly associated with sequential changes in physiological and biochemical events, most of the studies on pesticide actions in fishes are restricted to the toxicity test, growth rate assessment, fecundity, and reproductive activity (Wildish et al., 197 1; Burdick et al., 1972; Jackson, 1976; Anees, 1978). Very little attention has been paid to the effects of pesticides on the biochemistry of reproduction, particularly on lipid metabolism as lipids serve as an energy source, a major constituent of vitellogenin (de Vlaming et al., 1984) and a processor of sex hormones. In earlier reports, Singh and Singh (1980b,c,d) and Murty and Devi ( 1982) have measured gravimetrically total lipids to observe the effects of pesticides but these studies do not provide a clear picture of lipid metabolism as lipids undergo rapid breakdown, resynthesis, and interconversion with a slight change in extero- and interoceptic stimuli. Thus, the effects of pesticides on lipid metabolism in fish remain obscure. Therefore, in the present study effects of pesticides (r-BHC, an organochlorine, 1,2,3,4,5,6-hexachlorocyclohexane), and Cythion (malathion), an organophospho* To whom requests for reprints should be addressed. 0147-6513187 $3.00 Copyrisbt 0 1987 by Academic Press, Inc. All rinhts ofremoduction in any form resmed.
38
LIPID METABOLISM
39
AND PESTICIDES
TABLE
1
GONADOSOMATICINDEX (GSI) OF C. batrachus BEFORE PESTICIDE EXPOSURE AND PHOTOPERIOD AND TEMPERATUREDURING EXPERIMENTATION GSI before pesticide exposure
Reproductive phases
Date of fish collection
Female
Preparatory Prespawning Spawning Postspawning
March 5 May 7 July 3 Sept. 8
1.0 6.2 8.5 0.4
Male
Photoperiod
Temperature (“Cl
0.05
12L12D 13.5L10.5D 13L:llD 11.5L12.5D
28+2 33 f 2 31+2 26 + 2
0.15
0.20 0.06
rus, S( 1,2-dicarbethoxyethyl) ester of O-O-dimethylphosphorodithioate) were studied on lipid metabolism in either sex of a freshwater catfish, Clurias batrachus, by using chromatographic and spectrophotometric techniques to estimate triglycerides (energy source), phospholipids (vitellogenin constituent), and free and esterified cholesterol (sex hormone precursor) in the liver, plasma, gonads, and muscle during different phases of its annual gonadal cycle. MATERIALS
AND
METHODS
Fish Adult specimens of either sex of C. batruchus (weight between 60 and 70 g and size between 18 and 19 cm) were collected the first week of each reproductive phase from ponds around Varanasi, India (Table 1). Fish were acclimatized to laboratory conditions for 2 weeks prior to experimentation in each phase and were fed standardized rations (20% protein, 5% lipid, and 15% carbohydrate) on alternate days. They were maintained under natural photoperiod and temperature. Chemicals Lipid standards. Tripalmitin for triglycerides (TG), lecithin for phospholipids (PL), cholesterol for free cholesterol (CF), and cholesterol oleate for esterified cholesterol (CE) were obtained from Sigma (U.S.A.). Silica gel Gso was purchased from E. Merck (Germany) and AR grade solvents (BDH, India) were distilled and used. r-BHC and Cythion were obtained from Bharat Pulverising Mill Private Ltd., India, and Cynamid India Ltd., India, respectively. Pesticide Exposure During each phase, after acclimatization fish were sexed and specimens of each sex were divided into six batches, each having 10 fish, in an aquarium of 90-liter capacity. Fish were exposed to 96 hr safe concentrations (SC) and sublethal concentrations (SL) of Cythion (1 and 4 ppm) and r-BHC (2 and 8 ppm) for 4 weeks, a procedure which had already been standardized and reported (Lal and Singh, 1984). During experimentation fish were fed every 4th day when aquarium water was changed with fresh water having similar concentrations of the above pesticides.
40
LAL AND SINGH
Lipid Extraction and Separation On termination of the experiments on Day 28 blood was collected separately from each fish of each group in heparinized tubes by caudal puncture and centrifuged at 2000g at 4°C and plasma was frozen at -20°C for further analysis. Individual liver, ovary, testis, and muscle were extirpated, washed in 0.6% saline, blotted, and kept frozen at -20°C until analysis. Tissues and plasma lipids were extracted in chloroforrnmethanol(2: 1) following the method of Folch et al. (1957). 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 the volume obtained for plasma was 100 ~1. Different fractions of lipids were separated by thin-layer chromatography (&values for TG, PL, CF, and CE were 0.80,0.00, 0.44, and 0.90, respectively) by using the double system (System I, diethylether:benzene:ethanol:acetic acid, 40:50:2:0.2, and Solvent system II, hexane:diethylether, 94.6) of Freeman and West (1966). Spots of various lipids were visualized by exposing the plates to the iodine vapor. Spots of different lipid fractions of samples, standards, and the corresponding area of silica gel from blanks were scraped and transferred to separate test tubes. Quantitation Quantitative estimation of various lipids was made spectrophotometrically on Spectronic-2000 (Bausch & Lomb, U.S.A.) at 375 nm by the method of Marzo et al. (197 1) with one modification in which incubation time was reduced from 45 to 30 min. Data were expressed in milligrams per gram tissue or milligrams per milliliter plasma. For statistical analysis of data, analysis of variance along with supplemented Newman-Keuls multiple range t test were employed at the cxlevel of 0.05. RESULTS Analysis of variance clearly indicated that the responses of different lipids varied with phase, tissue, nature, and concentrations of pesticides. Changes in TG, PL, CF, and CE are shown in Figs. l-4. Preparatory Phase Triglycerides. Hepatic level of TG was suppressed by both pesticides in either sex of C. batrachus but their levels in the plasma and muscle were unresponsive to pesticide exposure. Ovarian TG was also unchanged; however, in testes its level was decreased. Phospholipids. In both sexes, a significant reduction in hepatic as well as plasma levels of PL was recorded in response to y-BHC. Similarly, Cythion also caused a decrease in their levels, but only in females. In the ovary, PL was unaltered, while in the testes it was depressed by both pesticides. These pesticides had no effect on muscular PL levels. Free and esterijied cholesterol. Significantly reduced levels of CF were registered in the liver in the female, but in the male, its levels were unchanged with respect to these pesticides. Its levels in plasma, gonads, and muscle in either sex remained unaltered after pesticide exposure. Unlike free cholesterol, CE was raised by Cythion and yBHC in the liver, while plasma levels were unresponsive. Testicular CE was also elevated by both pesticides but in the ovary only Cythion could raise its levels. In muscle, however, it was increased in females and remained unchanged in males.
LIPID METABOLISM
41
AND PESTICIDES MUSCLE
2.30
d
t
GONAD
p 3
CONTROL CYTHION CYTNION
-
SC -SL
PLASMA
0 0
,.
. .
t 400 0 5
13* 0 t
il.ml
12.0
z I5.0
. a’ 0
m PREP
PRESPO
SPO
POSTSPO
PREP
PMSPB
SPO
POBTEPO
FIG. 1. Level of triglycerides in response to Cythion and r-BHC in C. batrachus. x, not significant.
Prespawning Phase Triglycerides. These pesticides at either concentration decreased TG in the liver, plasma, and muscle in female C. batrachus. Ovarian TG levels were enhanced at safe concentrations and remained unchanged at sublethal concentrations of both pesticides. However, in male C. batrachus, these pesticides provoked an increase in TG levels in the liver and testes, whereas in the plasma, either pesticide reduced TG levels. Muscular TG was also reduced but only in response to Cythion. Phospholipids. In either sex of C. batrachus, Cythion and y-BHC at both concentrations caused a decrease in the plasma and gonadal PL but hepatic levels were raised by Cythion and reduced by r-BHC. Muscular PL, however, remained unchanged in males and was elevated in females by both pesticides. Free and ester$ed cholesterol. These pesticides at either concentration evoked a significant reduction in CF levels in the gonads and muscle in both sexes. While its
42
LAL AND SINGH 3.0-
MUSCLE
17.or
o-
GONAO
w
PLASMA
2Q)
14.0
LIVER
0 2 7.0
0i
FIG.
PRSP
WISP0
SPO
POSTSPO
PREP
2. Level of phospholipids in response to Cythion and yBHC in C. butruchux x, not significant.
hepatic levels remained unaltered in females and were reduced in males by Cythion, T-BHC induced a decrease and increase in its hepatic levels in females and males, respectively. Moreover, plasma CF was decreased by both pesticides in females, but in males only T-BHC reduced its level while Cythion increased it. Contrary to these data, CE levels were elevated by both pesticides in all the tissues studied in either sex of C. batrachus except in muscle where it was decreased. Spawning Phase Triglycerides. Both pesticides at either concentration elevated the levels of TG in the liver and ovary and decreased it in the plasma without affecting it’s level in the muscle in female C. batrachus. In males, TG levels of liver, plasma, and muscle were unresponsive but in testes they were reduced in response to these pesticides. Phospholipids. As in the preparatory phase, Cythion provoked an increase in PL levels in the liver with marked decrease in the plasma and gonads in both the sexes
LIPID 1 .o
METABOLISM
43
AND PESTICIDES MUSCLE
OT
0.6
2
FIG.
4.Or
6
GONAD
3. Level of free cholesterol in response to Cythion and yBHC in C. batrach.
x, not significant.
of C. batrachus. y-BHC also behaved similarly except that it caused a significant reduction in the hepatic level in females. Free and esteriJied cholesterol. In both sexes, Cythion and r-BHC failed to induce any appreciable change in CF levels in any of the studied tissues, whereas CE in the liver and gonads were increased. Plasma CE was also increased in females but only at sublethal and safe concentrations of Cythion and -r-BHC, respectively. In males, Cythion could not produce any change and r-BHC raised CE levels in the plasma. Muscular CE, however, remained unaffected. Postspawning Phase During this stage, very little effects could be noticed in either sex of C. batrachus. Lipid levels studied in males remained totally unaffected by these pesticide exposures. However, in females, a decreased level of TG and increased levels of free and esterified cholesterol in the liver were registered after pesticide exposure. Plasma TG was also increased.
44
LAL AND SINGH MUSCLE
@&;i
& PLASMA
PliESPO FIG.
SPO
4. Level of esterified cholesterol in response to Cythion and y-BHC in C. butruchus. x, not signiikant.
DISCUSSION In retrospect of the present findings it can be suggested that the effects of pesticides (Cythion and r-BHC) on lipid metabolism in C. batruchus vary with the change in the nature of lipid class, tissue, period of annual reproductive cycle, sex, as well as nature and concentration of pesticides. Decreased hepatic triglycerides in female C. batrachus during preparatory, prespawning, and postspawning phases suggested that these pesticides inhibited TG biosynthesis in liver. Singh and Singh ( 198Oc) have also reported decreased levels of hepatic total lipids in Cythion- and hexadrin-exposed Heterbpneustes fossilis during the preparatory phase. Murty and Devi (1982) have shown in Channapunctutusdecreased liver lipids after endosulfan treatment. Dzogbefia et al. (1978) have observed suppressed glyceride biosynthesis in FCB-treated rat liver in vitro. Though the comparison of these results with our findings may be misleading due to variations in experimental design and species, they partially support our results indirectly. Further, pesticide-induced increase in hepatic TG levels during the spawning phase in females and during the prespawning in males can be attributed to their decreased utilization. Because under natural conditions, normal
LIPID
METABOLISM
AND PESTICIDES
45
female C. batrachus during the spawning phase diminish food intake and show prominent lipolytic activity to meet the caloric demand of spawning behavior and activity (Lal and Sir&, 1984). Moreover, these pesticides appeared not to interfere with triglycerides of the plasma, ovary, and muscle during preparatory and postspawning phases, whereas in the prespawning phase decreased triglycerides might have resulted from restricted mobilization of hepatic triglycerides to the ovary. de Vlaming et al. (1977a,b), Upadhyay (1977), Wiegand and Peter (1980a,b), and Lal and Singh (1983) have demonstrated increased lipogenic activity as well as transfer of lipids to ovary during the prespawning phase under the influence of sex hormones and gonadotropins under natural conditions. Moreover, Singh and Singh (1980a) in H. fossilis, Kapoor et al., ( 1978) in Cyprinus carpio, and Sir@ (1985) in C. batrachus have found decreased gonadotropic potency, 3&hydroxysteroid dehydrogenase, and sex steroids (esterone, estradiol- 178, and testosterone), respectively. Thus, in view of these results it can be suggested that these pesticides inhibited hepatic lipogenesis and lipid deportation from liver to gonads in C. batrachus by decreasing the level of sex steroids and gonadotropins. However, increased ovarian TG during spawning and testicular TG in the prespawning phase have again suggested the inhibited utilization and hydrolysis of TG to free fatty acids and ultimately ,&oxidation of the latter. That is perhaps why sufficient metabolic water is also not produced which is required for hydration of the ovary preceding ovulation. Free fatty acids, upon oxidation, produce enough metabolic water and ATP. Cythion and r-BHC also seemed to inhibit hepatic biosynthesis of phospholipids in females during the preparatory phase, while during the prespawning phase only r-BHC was effective in reducing phospholipid hepatic biosynthesis. In males, a similar mechanism appeared to be operative. Dzogbefia et al. ( 1978) have also reported decreased hepatic phospholipid biosynthesis in PCB-treated rat liver. These pesticides also impaired the mobilization of phospholipids to gonads in C. batrachus. In C. batrachus these pesticides seemed to be totally ineffective in altering total cholesterol content or biosynthesis as a whole, except for inhibiting the hydrolysis of esterified cholesterol to free cholesterol in liver and gonads during the period of gonadal growth and maturation. This was perhaps one of the reasons that Singh and Singh (1987) found decreased estradiol, estrone, and testosterone in Cythion- and y-BHC-exposed C. batrachus. The possibility of reduced hydrolysis of ester&d cholesterol to the free form is further supported indirectly by the fact that gonadotropin, required for this hydrolysis (ZaIkroni et al., 1958), is reduced in response to Cythion and hexadrin in the Indian catfish H. fossilis (Singh and Singh, 1980a). Thus, probably in C. batrachus also these pesticides exert their impact on cholesterol metabolism by reducing gonadotropic potency. CONCLUSION The present study has clearly demonstrated that Cythion and y-BHC both have altered lipid metabolism significantly and have very selective and specific effects on various lipids in C. batrachus, probably by acting at several sites in metabolic processes. Changes in extero- and interoceptic conditions may also have some impact on actions of these pesticides on lipids as was evident from their varied effects during
46
LAL AND SINGH
different phases of the annual reproductive cycle. Their actions on apolar and less polar lipids were more or less similar and were inhibitory to lipogenesis except during the spawning and prespawning periods in females and males, respectively, when these pesticides probably inhibited the use of lipids. Cholesterol biosynthesis as such was not impaired by these pesticides but the conversion of CE to CF was adversely affected. In general, r-BHC inhibited the phospholipid biosynthesis but Cythion did not. Mobilization of various hepatic lipids to the ovary was also restricted by Cythion and y-BHC. Muscle was the least affected tissue throughout the annual reproductive cycle in C. batrachus. Thus, these pesticides possibly derange the production in lipidderived energy, vitellogenin, and sex hormone precursor which ultimately results in decreased breeding potentials and in cessation of spawning. ACKNOWLEDGMENTS Grant-in-Aid for research from HCS/DST/928/80 and ICAR-PG480 Project No. IN-AR!&21 3, 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 of Cynamid Ltd. for Cythion.
REFERENCES ANEES, M. A. (1978). Hepatic pathology in a freshwater teleost Channa punctatus (Bloch) exposed to sublethal and chronic levels of three organophosphorus insecticides. Bull. Environ. Contum. Toxicol. 19,524-527. BIJRDICK, 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 GameJ. 19,97-l 15. DE VLAMING, V. L., FITZGERALD, R., DELAHUNTY, G., CEEH, J., SILMAN, K., AND BARKLEY, M. (1984). Dynamics of oocyte development and related changes in serum estradiol- 178, yolk precursor and lipid levels in the teleostean fish, Leptocottus armatus. Comp. B&hem. Physiol. 7lA 599-610. DE VLAMING, V. L., SHING, J., PAQUETTE, G., AND VUCHS, R. (1977a). In viva and in vitro effects of oestradiol 17 on lipid metabolism in Notemigenus crysoleucas. J. Fish Biol. 10,273-285. DE VLAMING, V. L., VODICNIK, M. J., BAUER, G., MURPHY, T., AND EVAN, D. (1977b). Estradiol-17 effect on lipid and carbohydrate metabolism and on the induction in yolk precursor in goldfish, Curassium auratus. Life Sci. 12, 1945-1952. DZOGBEFIA, V. P., KLING, D., AND GAMBLE, W. (1978). Polychlorinated biphenyl: In vivo and In vitro modifications of phospholipid and glycerides biosynthesis. J. Environ. Pathol. Toxicol. 1,84 l-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. FREEMAN, C. P., AND WEST, D. (1966). Complete separation of lipid classeson a single thin layer plates. J. Lipid Res. 7,324-327. JACKSON, G. A. (1976). Biological halflife of Eldrin in Channel catfish tissues. Bull. Environ. Contam. Toxicol. 16,505-507. KAPOOR, K., KAMALDEEP, K., AND TOOR, H. S. (1978). The effect of fenitrotbion on reproduction of a teleost fish, Cyprinus carpio communis Linn. A biochemical study. Bull. Environ. Contam. Toxicol. 20, 438-442. LAL, B., AND SINGH, T. P. (1983). Effect of ovariectomy and sex steroid administration on lipid metabolism in freshwater teleost, Clarias batrachus. In Proceedings of the First Znt. Symp. on Recent Advances in Life Sciences, Bodhgaya, India, 29-3 1 December, pp. 136. LAL, B., AND SINGH, T. P. (1984). Pesticides induced alterations in lipid metabolism during prespawning phase of reproductive cycle in freshwater teleost; Clark batrachus. In Proceedings of the Symposium on Comparative Endocrinology Varanasi, India, 29-3 1 December. LAL, B., SINGH, A., KUMARI, A., AND SINHA, N. (1986). Biochemical and haematological changes following malathion treatment in the freshwater catfish, Heteropneustes fossilis (Bloch). Environ. Pollut. A42, 151-156. LAL, B., YADAV, A. K., AND SINGH, T. P. (1985). Lipid programme and reproduction in catfish, Cfarias batrachus. In Second International Conference on Life Science, Shillong, Meghalaya, India, 14-16 November.
LIPID METABOLISM
AND PESTICIDES
47
MARZO, A., GHIRARCH, P., SERIDINI, D., AND MARONI, D. (197 1). Simplified measurement of monglyo erides, diglycerides, triglycerides and fattyacids in biological samples. Clin. Chem. 17, 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. SAFE, S. (1984). Polychlorinated (PCBs) and polybrominated biphenyls (PBBs): Biochemistry, toxicology, mechanism of action. CRC Crit. Rev. Toxicol. 13,3 19-393. SINGH, S. (1985). Steroidogenesis in Female Cathsh, Clarias batrachus. Ph.D. thesis, Department of Zoology, B.H.U., Varanasi, India. 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 freshwater catfish, Heteropneustesfossilis (Bloch). Environ. Res. 22, 190-200. SINGH, H., AND SINGH, T. P. (1980b). Effect of two pesticides on testicular 32P uptake, gonadotrophic potency, lipid and cholesterol content of testis, liver and blood serum during spawning phase in Heteropneustes fossilis (Bloch). EndokrinoIogie 76,288-296. SINGH, H., AND SINGH, T. P. (198Oc). Effect of two pesticides on total lipid and cholesterol content of overy, liver and blood serum during different phases of the annual reproductive cycle in the freshwater teleost, Heteropneustesfossiiis (Bloch). Environ. Pollut. A23,9-17. 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. A22,85-90. SINGH, H., AND SINGH, T. P. ( 1983). Effect of pesticides on fish reproduction. Ichthyologia 15,7 l-8 1. SINGH, S., AND SINGH, T. P. (1987). Impact of malathion and hexachlorocyclohexane on plasma profile of 3 sex steroids during different phases of the reproductive cycle in Clarias batrachus. Pestic. Biochem. Physiol. 22,301-308. SINGH, T. P., YADAV, A. K., LAL, B., AND SINGH, S. (1985). Impact of pesticides on fish reproduction, In Proceedings of International Conference on Pesticides: Toxicity, Safety and Risk, Assessment, Lucknow, India, in press. UPADHYAY, S. N. (1977). Morphologie des gonads immatures eletude experimental del’ induction de la gametogenese chez la truite arc-en-ciel juvenile Salmo gairdneri (R.). These de doctorate d’ Elat es Science Naturelles, L’ Universite Pierre et Marie Curie, Paris, France. WIEGAND, M. D., AND PETER, R. E. (1980a). Effect ofsalmon gonadotropin @G-100) on plasma lipids in the goldfish, Carassius auratus. Canad. J. Zool. S&957-966. WIEGAND, M. D., AND PETER, R. E. (1980b). Effect of sex steroids on plasma lipids in the goldfish, Carassius auratus. Canad. J. Zool. S&967-972. WILDISH, D. J., CARSON, W. G., CUNNINGHAM, T., ANLI LISTER, N. J. (197 1). Toxicological effects of some organophosphate insecticides to atlantic salmon. Fish. Res. Board Canad. Manscr. Rep. Ser. 1157, l-22.
ZAF’FARONI, A., HECHTER, O., AND PINCUS, G. (1958). (As cited by Hetchler, O., 1958.) Conversion of cholesterol to steroid hormone. In Cholesterol (R. P. Cook, Ed.), Academic Press, New York.