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Reproductive endocrine disruption in the freshwater catfish, Heteropneustes fossilis, in response to the pesticide γ-hexachlorocyclohexane
ARTICLE IN PRESS
Ecotoxicology and Environmental Safety 58 (2004) 77–83
Reproductive endocrine disruption in the freshwater catfish, Heteropneustes fossilis, in response to the pesticide g-hexachlorocyclohexane Pratap B. Singha, and Adelino V.M. Canariob b
a Department of Zoology, Tilak Dhari College, Jaunpur 222002, India Centre of Marine Sciences, University of Algarve, Campus of Gambelas, Faro 8000-810, Portugal
Received 1 November 2002; received in revised form 17 July 2003; accepted 19 July 2003
Abstract Both male and female freshwater catfish, Heteropneustes fossilis, were exposed to safe (SC; 0.1 and 1.0 mg/L) and sublethal (SL; 10 mg/L) concentrations of an agricultural pesticide, g-hexachlorocyclohexane (g-HCH) for 4 weeks during the active pre-spawning (vitellogenic) phase of their annual reproductive cycle. On the last day of exposure, 18 h before killing, fish were treated intramuscularly (i.m.) with [I-14C]acetic acid (74 kBq per fish). After 4 weeks of exposure, we monitored the effects of g-HCH on gonadosomatic index (GSI); on plasma concentrations of gonadotropin (GtH), testostosterone (T), 11-ketotestosterone (11-KT), 17b-estradiol (E2); and on hepatic incorporation of [I-14C]acetic acid into total phospholipids (TP) and the fractions thereof: phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI), and phosphatidylethanolamine (PE). In both the sexes, GSI and plasma GtH were decreased significantly in response to g-HCH exposure. Plasma T and 11-KT in males, and plasma T and E2 in females declined significantly in response to g-HCH exposure. In both the sexes, hepatic incorporation of [I-14C]acetic acid into PS and PI increased significantly, whereas incorporation into TP, PC, and PE was significantly reduced after g-HCH exposure. Our findings demonstrated that g-HCH exposure depressed GSI, plasma GtH, sex steroids, and [I-14C]acetic acid incorporation into hepatic TP, and had very selective and specific effects on various classes of TP, resulting either from the hypothalamo-hypophyseal-gonadal axis or from direct action on hepatic and steroidogenic enzymes during the pre-spawning phase, causing reproductive endocrine disruptions. r 2003 Elsevier Inc. All rights reserved. Keywords: g-HCH; Gonadotropin; Sex steroids; Phospholipids; Endocrine disruptions; Fish reproduction
1. Introduction g-Hexachlorocyclohexane (g-HCH) is widely used to kill pests for crop protection. Paddy-cum-fish culture is very common in India, where fish are directly exposed to g-HCH as nontarget species. Fish are a major source of protein for human societies, and fisheries are a major commercial activity. Fish accumulate g-HCH preferentially in their fatty tissues, such as liver and gonads, but the effects may become apparent only when concentration in such tissues passes a certain threshold. These effects can include damaged immune response, tumors, or respiratory problems, all of which can shorten the life span and decrease the population through both pre
Corresponding author. Fax: +91-5452-263338. E-mail address: pratap p [email protected] (P.B. Singh).
0147-6513/$ - see front matter r 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2003.07.014
mature mortality and a decreased number of fish surviving to enter the spawning season (Macek, 1968; Anderson, 1996). Toxic substances, even in very low concentration, have been reported to suppress reproduction (Hirose, 1975; Sivarajah et al., 1978a, b; Singh et al., 1994; Ankley et al., 2001), steroidogenesis (Wester et al., 1990; Van Der Kraak et al., 1992; Singh et al., 1994; Kime and Singh, 1996), lipid metabolism (Singh, 1992; Singh and Kime, 1994), GtH concentrations (Thomas, 1990; Singh et al., 1994), vitellogenin production (Chen et al., 1986; Pereira et al., 1992), and reproductive physiology (Santos and Pacheco, 1996; Hontela et al., 1997; Pacheco and Santos, 2001). Additional reports of Wiegand and Idler (1982) have indicated that [I-14C]acetic acid was incorporated primarily into polar lipids in fish undergoing ovarian recrudescence. Hansen
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(1987) had demonstrated that when [I-14C] or [32P]phosphate was injected directly into the bloodstream of the eel, the resulting phospholipid patterns were the same in both gill and liver dominated not by PE but by PC. Most previous studies on pollution effects have been restricted to fatty acids, cholesterol, or TP, but there are few reports on simultaneous studies on GSI, plasma GtH, sex steroids production, and various classes of phospholipids of either sex after long-term g-HCH exposure in the Indian freshwater catfish, H. fossilis. Therefore, the present study was conducted during the pre-spawning phase (May–June), when the parameters of interest showed maximum activity and involvement in reproduction, as reported in this teleost (Singh and Singh, 1990). The aim of this experiment was to assess the endocrine disruption resulting from g-HCH exposure by taking into consideration the parameters of GSI; plasma GtH, the sex steroid hormones T, E2, and 11-KT; and hepatic incorporation of [I-14C]acetic acid into various classes of phospholipids: PS, PI, PE, and PC—the latter of which is a major constituent of phospholipid for vitellogenin synthesis in liver by ovarian E2 under the control of GtH during gonadal recrudescence.
2. Materials and methods 2.1. Experimental fish The original research reported in this monograph was conducted under ethical guidelines (Anonymous, 1998) established for animal use by Tilak Dhari College, Jaunpur (UP), India. Male and female H. fossilis (65–75 g body wt, 21–22 cm length) were collected on 2 May 2001 from a local pond of the Pratapgarh district (Lat. 25 340 S; Long. 61 590 E) during the pre-spawning phase and maintained in a tank (3500 L) supplied with a constant flow of dechlorinated tap water (temperature 2071 C, pH 7.2, hardness 154 mg/L (as CaCO3), alkalinity 68 mg/L (as CaCO3), dissolved oxygen (7.2 mg/L), and conductivity 0.56 mO: The fish were exposed to a natural photoperiod (13 h light/11 h dark) and a temperature of 3072 C, having a GSI (total gonadal wt/total body wt 100) of 5.1270.25 for males and 7.1170.35 for females. They were fed ad libitum with minced goat liver comprising 20% protein, 5% lipid, and 15% carbohydrate, with the remaining 60% composed of water, minerals, and vitamins. After 10 days of acclimation, experiments were begun. 2.2. Chemicals Analytical-grade chemicals were obtained from BDH (India). Solvents were redistilled before use. Thinlayer chromatography (TLC) pre-coated plastic sheets
(E. Merck Silica gel G60 F254; 20 cm 20 cm 0.20 mm) were obtained from BDH. Lipid standards were obtained from Sigma Chemical Co. (UK): lecithin for TP, l-a-phosphatidylcholine for PC, l-a-phosphatidyl-lserine for PS, l-a-phosphatidylinositol for PI, l-aphosphatidylethanolamine for PE. g-HCH (g-BHC; 1a; 2a; 3b; 4a; 5a; 6b-hexachlorocyclohexane, g-isomer, 99% pure) was also purchased from Sigma. [I-14C]acetic acid sodium salt (2.15 GBq/mmol) was obtained from Amersham International (UK). Labeled hormones [1,2,6,7-3H]testosterone (sp. act. 103 Ci/mmol), [2,4,6,7-3H]17b-estradiol (sp. act. 89 Ci/mmol), and [3H]ketotestosterone were obtained from Radiochemical Centre Amersham (UK). Authentic steroids were purchased from Sigma (USA). Lyophilized aliquots of antibodies against T, 11-KT, and E2 were provided by G.D. Niswender, Colorado State University, Colorado. 2.3. Exposure studies To test the pesticide concentration to be used, a single fish was exposed (according to the method described by Singh et al., 1994) briefly to concentrations of 20, 10, 1, and 0.1 mg/L of g-HCH (dissolved in 1 mL acetone). Fish exposed at 20 mg/L showed extreme hyperactivity and were immediately transferred to uncontaminated water. Fish at 10, 1, and 0.1 mg/L showed no apparent distress even after 24 h. Pesticide concentrations for the experiments were therefore chosen as 10, 1, and 0.1 mg/L. After 4 weeks, the fish exposed to 0.1 and 1 mg/L showed no behavioral differences from the controls, but those exposed to 10 mg/L showed increased mucus and locomotor activity indicative of mild stress. We therefore classed the two pesticide levels as safe (SC) and sublethal (SL) concentrations, respectively. In our earlier experiments, paddy-cum-fish culture in which the g-HCH exposure was intended to kill the pests, the g-HCH concentrations in the water used for rearing was recorded as 10 mg/L (unpublished data). After acclimation, the fish were categorized by sex and were divided into eight batches, each comprising six fish in a plastic aquarium (46 25 30 cm3) containing 20 L of water at 20 C. All eight aquaria were surrounded by a water jacket at 20 C. The g-HCH was dissolved in acetone and diluted with water to the required concentrations. Both male and female fish were exposed to g-HCH at the selected safe (SC; 0.1 and 1.0 mg/L) and sublethal (SL; 10 mg/L) concentrations for 4 weeks during the pre-spawning phase. Control fish were maintained in plain dechlorinated tap water containing acetone at the same concentration as the treated groups (20 mL per tank). A separate true control was also kept without any treatment. During the experiment, fish were fed every fourth day, when the aquarium water was exchanged for freshwater containing the appropriate pesticide concentrations. On day 29
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2.4. Plasma gonadotropin and sex steroid hormones measurements Duplicate samples were run for measurements of reproductive hormones. Plasma GtH was measured using the enzyme-linked immunosorbent assay method of Kah et al. (1989). Sex steroids were measured by radioimmunoassay using the methods of Singh et al. (1994). 2.5. Effect of g-hexachlorocyclohexane on conversion of different phospholipids by [I-14C]acetic acid in exposed fish To observe whether g-HCH had an effect on PC and its conversion by radiolabeled acetic acid to other phospholipids, pollutant-exposed and control fish were sampled 18 h after injection. Duplicate samples were prepared for the study of the incorporation of [I-14C]acetic acid into different phospholipids. Liver lipids were extracted following the method of Folch et al. (1957), so as to extract TPs. TPs were separated on TLC using the double-solvent system (system I, diethylether:benzene:ethanol:acetic acid, 40:50:2:0.2; system II, hexane:diethylether, 94:6) of Freeman and West (1966). The Rf value of TP was 00. Authentic lipids were visualized by exposing the plates to iodine vapor, and radioactive areas were detected with a TLC radiochromatogram scanner. The TP residue was scraped from the TLC plate and eluted with chloroform:methanol (2:1) and the solvent evaporated. Samples were rechromatographed on TLC, and the various phospholipid classes (Rf values for PC, PS, PI, and PE were 0.12, 0.20, 0.27, and 0.41, respectively) were separated using methyl acetate:isopropanol:chloroform:methanol:0.25% aqueous KCl (25:25:25:10:9) by volume (Vitiello and Zanetta, 1978). The different phospholipids were visualized by brief exposure to iodine vapor and the individual classes scraped into scintillation minivials. After adding 3 mL Packard Scintillator 299, the minivials were vortexed and radioactivity was determined.
2.6. Statistical analysis The contents of the different phospholipids were expressed as mean7SEM in dpm/g of individual liver samples. GtH and sex steroids contents were expressed as ng/mL plasma, and values were expressed as mean7SEM ðn ¼ 5Þ: The data were analyzed by Student’s t-test followed by Newman–Keuls’ multiplerange test at the level of 0.05 (Bruning and Kintz, 1977).
3. Results There was no difference in the values between the controls (i.e., vehicle-treated and nontreated proper control); hence, vehicle-treated control values were taken into consideration. 3.1. Effect of g-hexachlorocyclohexane exposure on gonadosomatic index GSI was significantly decreased in both the sexes during the reproductively active pre-spawning phase after 4 weeks of g-HCH exposure at 1 and 10 mg/L dose levels as compared with the controls. However, g-HCH at 0.1 mg/L exposure did not decrease GSI significantly in either sex (Fig. 1). 3.2. Effect of g-hexachlorocyclohexane exposure on plasma gonadotropin Plasma GtH was significantly reduced in both the sexes after exposure to 1 and 10 mg/L concentrations of g-HCH, whereas the 0.1 mg/L exposure failed to show any such effect when compared to the controls (Fig. 2). 8 NS
7 6 5 GSI
of exposure, 18 h before killing, all fish were injected i.m. with 25 mL aqueous solution of [I-14C]acetic acid sodium salt at a dose rate of 74 kBq per fish. All fish were bled by caudal incision, and blood samples were collected in heparinized glass culture tubes. Plasma was separated by centrifugation at 17,000g at 4 C for 15 min and stored frozen at 20 C until assayed for GtH, male sex steroids (T and 11-KT), and female sex steroids (T and E2). Individual gonad weights were recorded for calculation of GSI. Individual livers were extirpated, washed in 0.6% saline, blotted, weighed, and kept frozen at 20 C for further analysis of hepatic incorporation of [I-14C]acetic acid into phospholipids after g-HCH exposure.
79
**
NS
**
4 3
*
*
2 1 0 Male
Female
Fig. 1. Gonadosomatic index (GSI) of male and female H. fossilis (Bloch) held in freshwater [ ], 0.1 ppm [ ], 1.0 ppm [ ] and 10 ppm [ ] g-HCH treated fish. Bar indicates mean7SEM ðn ¼ 5Þ: Asterisks denotes a significant difference ð Po0:001; Po0:005Þ from control. NS—not significant ðP40:05Þ:
ARTICLE IN PRESS P.B. Singh, A.V.M. Canario / Ecotoxicology and Environmental Safety 58 (2004) 77–83
80
25.0
14 12
20.0
15.0 NS
*
**
10.0
*
ng ml-1 Plasma
GtH ng ml-1
NS
*
10
*
*
8 6
* *
4
*
*
2
5.0
0 T
0.0 Male
E
2
Female
Female
Fig. 2. Plasma gonadotropin (GtH) concentrations in male and female H. fossilis held in freshwater [ ],0.1 ppm [ ],1.0 ppm [ ] and 10 ppm [ ] g-HCH treated fish. Bars indicates mean7SEM ðn ¼ 5Þ: Asterisks denotes a significant difference ð Po0:001; Po0:01Þ from control, NS—not significants ðP40:05Þ:
Fig. 4. Plasma T (testosterone) and E2 (estradiol-17b) concentrations in female H. fossilis held in freshwater [ ], 0.1 ppm [ ], 1.0 ppm [ ] and 10 ppm [ ] g-HCH treated fish. Bars indicates mean7SEM ðn ¼ 5Þ Asterisks denotes a significance difference ð Po0:001Þ from control.
10000
12
8000
NS
dpm/g of liver
-1
ng ml Plasma
10
9000
8 ***
**
6
7000 6000 5000
**
*
4000 *
3000
4
** *
2
*
NS
**
2000
*
** *
NS
NS
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NS
* * *
1000 0
0
TP
T
PC
PS
PI
PE
11 KT
Fig. 3. Plasma T (testosterone) and 11-K T (11-ketotestosterone) concentrations in male H. fossilis held in freshwater [ ], 0.1 ppm [ ], 1.0 ppm [ ] and 10 ppm [ ] g-HCH treated fish. Bars indicates mean7SEM ðn ¼ 5Þ: Asterisks denotes a significance difference ð Po0:001; Po0:005; Po0:02Þ from control, NS—not significant ðP40:05Þ:
Fig. 5. Effect of g-HCH exposure on hepatic incorporation of [I-14C]acetic acid into TP (total phospholipid), PC (phosphatidylcholine), PS (phosphatidylserine), PI (phosphatidylinositol) and PE (phosphatidylethanolamine) during the prespawning phase of male H. fossilis held in freshwater [ ], 0.1 ppm [ ],1.0 ppm [ ] and 10.0 ppm [ ] g-HCH treated fish. Asterisks denotes a significant difference Po0:001; Po0:005; Po0:01 from control. NS—not significant ðP40:05Þ:
3.3. Effect of g-hexachlorocyclohexane exposure on plasma sex steroids
3.4. Effect of g-hexachlorocyclohexane exposure on hepatic incorporation of [I-14C]acetic acid into total phospholipids and various fractions
In males, plasma T concentration significantly declined at the 1 and 10 mg/L g-HCH exposures, whereas the 0.1 mg/L dose was not effective in inducing any significant change when compared to the control. However, 11-KT values were significantly lowered at all doses of g-HCH exposure carried out in the present study. In females, plasma T and E2 were recorded to be decreased significantly at all tested concentrations of g-HCH when compared with the controls (Figs. 3,4).
Total phospholipids were significantly decreased in both sexes at all the concentrations of g-HCH exposure when compared with controls. In males, PE showed a significant decrease at all doses, whereas PC reflected such an effect only at the 1 and 10 mg/L g-HCH exposures when compared with the controls. PS increased significantly at the 0.1 and 1 mg/L g-HCH doses, and PI increased only at the 1 mg/L dose as compared with controls (Fig. 5).
Male
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dpm/g liver
10000 8000
** * *
6000 4000 2000
***
** **
NS
***
* * *
NS
* * *
0 TP
PC
PS
PI
PE
Fig. 6. Effect of g-HCH exposure on hepatic incorporation of [I-14C]acetic acid into TP (total phospholipid), PC (phosphatidylcholine), PS (phosphatidylserine), PI (phosphatidylinositol) and PE (phosphatidylethanolamine) during the prespawning phase of female H. fossilis held in freshwater [ ], 0.1 ppm [ ],1.0 ppm [ ] and 10.0 ppm [ ] g-HCH treated fish. Asterisks denotes a significant difference Po0:001; Po0:005; Po0:01 from control. NS—not significant ðP40:05Þ:
In females, when compared with the controls, PE and PC decreased at all doses of g-HCH exposure, whereas both PS and PI increased. However, PS showed an increase at all doses tested, whereas PI increased only at the 1 mg/L g-HCH dose (Fig. 6).
4. Discussion This is the first comprehensive study on the effect of g-HCH exposure on GSI, GtH, sex steroid hormones, and hepatic incorporation of [I-14C]acetic acid into various phospholipids during the reproductively active pre-spawning phase in H. fossilis. The decline in GSI in H. fossilis subsequent to g-HCH exposure indicates the inhibition of gonadal growth during the reproductive phase studied. A similar decrease in GSI in response to pesticide exposure has been recorded in several teleosts (Thomas, 1989; Mukherjee et al., 1991; Singh et al., 1994). According to our results, g-HCH exposure at SC and SL concentrations significantly decreased the plasma concentrations of GtH required for ovarian steroidogenesis during the pre-spawning phase of H. fossilis. Our findings are supported by the earlier reports on carp, Carassius auratus, after g-HCH exposure (Singh et al., 1994). The decreased spontaneous pituitary secretion of GtH in vitro resulting from polychlorinated biphenyl treatment in Micropogonias undulatus L. (Thomas, 1989) and the disruption of the pituitary-gonadal axis in the teleost white sucker, Catostomus commersoni L. by bleached kraft mill effluent (Van Der Kraak et al., 1992) have also been reported as endocrine disruptors. Inhibition of steroidogenesis, microsomal enzymes, and
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histopathological changes in response to toxicants have been reported in some teleosts (Sivarajah et al., 1978a, b; Singh et al., 1994). Singh and Singh (1991) have demonstrated that injection of Mystus sp. GtH elevated the sex steroids (T and E2) and phospholipids during the pre-spawning phase in H. fossilis. In another experiment, Mercure et al. (2001) have indicated that E2 treatment of immature rainbow trout Oncorhynchus mykiss increases PC, PI, PE, and plasma vitellogenin. Among the polar lipids, PC content was higher in this species. Fremont and Riazi (1988) have indicated that fish vitellogenin comprises 18% of total lipids, of which approximately two-thirds is phospholipids and the remaining fraction is largely triacylglycerides, sterols, and sterol esters. On the basis of results obtained here, it can be argued that the secretion of E2 by the ovary (under maturational GtH control), which stimulates the liver to secrete phospholipid components and vitellogenin—ultimately deposited in growing oocytes under the influence of vitellogenic GtH during the reproductively active pre-spawning phase—is disrupted by gHCH. Suneja et al. (1984) found reduced activity of PC and PE on [I-14C]acetate incorporation in male rats after T-2 toxin administration. Therefore, it is concluded that g-HCH exposure inhibited production of plasma GtH, steroidogenesis, lipogenesis, and vitellogenesis in this species via the hypothalamo-hypophyseal-gonadal axis. Thus it is possible that decreased plasma sex steroids in both the sexes might be indicative of a change in the biosynthetic capacity of the gonad, either directly by the inhibition of aromatization activity, which is responsible for estrogen synthesis, or indirectly by suppressing the secretion of GtH. Considering the results obtained, it is reasonable to assume that the inhibition of hepatic lipogenesis and translocation of hepatic lipids to the ovary is brought about by the impairment of steroid metabolism. This contention is supported by a number of earlier reports in which it was well established that GtH and the gonadal steroids are critically involved in the regulation of lipid metabolism in teleosts in relation to reproduction (Wiegand and Peter, 1980a, b; Singh and Singh, 1991; Schulz et al., 2001; Devlin and Nagahama, 2002). In a separate report, Singh (1992) demonstrated decreased lipogenesis in the liver and its mobilization to the gonads in H. fossilis after g-HCH treatment for 28 days during preparatory and pre-spawning phases. They have further attributed these decreases to impaired steroidogenesis as well as synthesis and secretion of GtH. In this species, decreases in plasma GtH after g-HCH exposure indicate that GtH has direct control over steroidogenesis as well as phospholipid biosynthesis. Decreases in GtH and sex steroids have also been reported in C. auratus (Singh et al. 1994) after g-HCH exposure. Leslie and Buckley (1976) have reported that in the goldfish liver PC was the major component of TP, with
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the rest being PE, PI, and PS in decreasing order. They have also shown that liver microsomal choline phosphotransferase enzyme activity decreased as the temperature increased from 10 C to 30 C. It has also been reported that pulp mill effluent has inhibited UDPglucuronyl transferase (UDP-GT) activity in trout (Castren and Oikari, 1987). They have further proposed that pollutants that affect hepatic UDP-GT activity may therefore also affect the gonadal enzyme and possibly interfere with chemical signaling between the sexes or balances of free and conjugated steroids within the gonad, where the enzyme may provide protection. Recently, Ibanez et al. (2003) have suggested that lipoprotein lipase is probably involved in the incorporation of neutral lipids into the oocytes of sea bass, and the follicular cells not only participate in steroidogenesis but may also be important for the accumulation of nutrients into oocytes. Further studies on the individual enzyme involved in the synthesis of different phospholipids and sex steroids are necessary to determine the role of g-HCH exposure in this species during reproductive growth.
5. Conclusions Our findings clearly demonstrated that g-HCH significantly depressed the GSI, plasma GtH, sex steroids, and [I-14C]acetic acid incorporation into hepatic phospholipid components, and had very selective and specific effects on various classes of phospholipid, resulting from either decreased GtH levels or direct action on the enzymes involved in steroidogenesis and lipogenesis during the reproductively active prespawning phase in H. fossilis. PC, a major constituent of phospholipids and the predominant lipid of vitellogenin, and reproductive hormones are affected by SC as well as SL exposure to g-HCH, causing reproductive endocrine disruptions acting via the hypothalamo-pituitary-gonadal axis.
Acknowledgments A grant-in-aid from UGC MRP (F.8.4(55) 1999-2000/ MRP/NR dated 31.03.2000) to P.B.S. is greatly appreciated. The authors are grateful to R.B. Raizada, Industrial Toxicology Research Centre, Lucknow, for laboratory facilities, and to S.S. Srivastava and G.N. Shukla for reading of this manuscript.
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Immune System. Organism, Pathogen and Environment. Academic Press, San Diego, CA, pp. 289–310. Ankley, G.T., Jensen, K.M., Kahl, M.D., Makynen, E.A., 2001. Description and evaluation of short term reproduction test with fathead minnow (Pimephales promelas). Environ. Toxicol. Chem. 20, 1276–1290. Anonymous, 1998. Guidelines for the treatment of animals in behavioral research and teaching. Anim. Behav. 55, 251–257. Bruning, J.L., Kintz, B.L., 1977. Computational Handbook of Statistics 2nd Edition. Scott, Foresman, Glenview, IL. Castren, M., Oikari, A., 1987. Changes of the liver UDP-glucuronosyltransferase activity in trout (Salmo gairdneri Rich.) acutely exposed to selected aquatic toxicants. Comp. Biochem. Physiol. 86C, 357–360. Chen, T.T., Reid, P.C., Van Beneden, R., Sonstegard, R.A., 1986. Effect of aroclor 1254 and mirex on estradiol-induced vitellogenin production in juvenile rainbow trout (Salmo gairdneri). Can. J. Fish. Aquat. Sci. 43, 169–173. Devlin, R.H., Nagahama, Y., 2002. Sex determination and sex differentiation in fish: an overview of genetic, physiological and environmental influences. Aquaculture 208, 191–364. Folch, J., Lees, M., Sloane-Stanley, G.H., 1957. A simple method for isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 497–509. Freeman, C.P., West, D., 1966. Complete separation of lipid classes on a single thin layer plate. J. Lipid Res. 7, 324–327. Fremont, L., Riazi, A., 1988. Biochemical analysis of vitellogenin from rainbow trout (Salmo gairdneri). Fatty acid composition of phospholipids. Reprod. Nutr. Dev. 28, 939–952. Hansen, H.J.M., 1987. Comparative studies on lipid metabolism in various salt transporting organs of the European eel (Anguilla anguilla). Mono-unsaturated phosphatidylethanolamine as a key substance. Comp. Biochem. Physiol. 88B, 323–332. Hirose, K., 1975. Reproduction in medaka, Oryzias latipes, exposed to sublethal concentration of g-benzenehexachloride (BHC). Bull. T. Reg. Fish. Res. Lab. 81, 139–149. Hontela, A., Daniel, C., Rasmussen, J.B., 1997. Structural and functional impairment of the hypothalamo-pituitary-interrenal axis in fish exposed to bleached kraft mill effluent in the St. Maurice River. Que. Ecotoxicol. 6, 1–12. Ibanez, A.J., Peinada-Onsurbe, J., Sanchez, E., Prat, F., 2003. The role of lipoprotein Lipase (LPL) in the incorporation of neutral lipids into the oocytes of the European Sea Bass (Dicentrarchus labrax L.) during gonadal development. Proceedings of the Seventh International Symposium on Reproductive Physiology of Fish, Mie, 18–23 May; Abstract O-VI-4, p. 76. Kah, O., Pontet, A., Rodriguez, J.N., Calas, A., Breton, B., 1989. Development of an enzyme-linked immunoassay for goldfish gonadotropin. Biol. Reprod. 40, 68–73. Kime, D.E., Singh, P.B., 1996. In vitro effects of g-hexachlorocyclohexane on in vitro biosynthesis and metabolism of steroids in goldfish Carassius auratus. Ecotoxicol. Environ. Saf. 34, 165–171. Leslie, J.M., Buckley, J.T., 1976. Phospholipid composition of goldfish (Carassius auratus L.). Liver and brain and temperature dependence of phosphatidylcholine synthesis. Comp. Biochem. Physiol. 53B, 335–337. Macek, K.J., 1968. Reproduction in brook trout (Salvelinus fontinalis) fed sublethal concentration of DDT. J. Fish. Res. Bd. Can. 25, 1787–1796. Mercure, F., Holloway, A.C., Tocher, D.R., Sheridan, M.A., Van Der Kraak, G.J., Letherland, J.F., 2001. Influence of plasma lipid changes in response to 17b-oestradiol stimulation of plasma growth hormone, somatostatin and thyroid hormone levels in immature rainbow trout. J. Fish Biol. 59, 605–615. Mukherjee, D., Guha, D., Kumar, V., Chakrabarty, S., 1991. Impairment of steroidogenesis and reproduction in sexually mature
ARTICLE IN PRESS P.B. Singh, A.V.M. Canario / Ecotoxicology and Environmental Safety 58 (2004) 77–83 Cyprinus carpio by phenol and sulfide under laboratory conditions. Aquat. Toxicol. 21, 29–40. Pacheco, M., Santos, M.A., 2001. Biotransformation, endocrine and genetic responses of Anguilla anguilla L. to petroleum distillate products and environmentally contaminated waters. Ecotoxicol. Environ. Saf. 49, 64–75. Periera, J.J., Ziskowski, J., Mercaldo-Allen, R., Luedke, D., Gould, E., 1992. Vitellogenin studies in winterflounder (Pleuronectus americanus) from Long Island sound and Boston Harbor. Estauries 15, 289–297. Santos, M.A., Pacheco, M.G., 1996. Anguilla anguilla L. Stress biomarker recovery in clean water and secondary treated pulp mill effluent. Ecotoxicol. Environ. Saf. 35, 96–100. Schulz, R.W., Vischer, H.F., Cavaco, J.E.B., Santos, E.M., Tyler, C.R., Goos, H.J.Th., Bogerd, J., 2001. Gonadotropins, their receptors, and the regulation of testicular functions in fish. Comp. Biochem. Physiol. (Part B), 129, 407–417. Singh, P.B., 1992. Impact of malathion and g-BHC on lipid metabolism in the freshwater female catfish, Heteropneustes fossilis. Ecotoxicol. Environ. Saf. 23, 22–32. Singh, P.B., Singh, T.P., 1990. Seasonal correlative changes between sex steroids and lipid levels in the freshwater female catfish, Heteropneustes fossils (Bloch). J. Fish Biol. 37, 793–802. Singh, P.B., Singh, T.P., 1991. Impact of oLH-RH and mGTH on lipid metabolism and steroidogenesis in the freshwater catfish, Heteropneustes fossilis. Zool. Jb. Physiol. 95, 221–232. Singh, P.B., Kime, D.E., 1994. In vivo incorporation of [I-14C]acetic acid into liver lipids of goldfish, Carassius auratus, during g-hexachlorocyclohexane exposure. Aquat. Toxicol. 30, 237–248. Singh, P.B., Kime, D.E., Epler, P., Chyb, J., 1994. Impact of g-hexachlorocyclohexane exposure on plasma gonadotropin levels and in vitro stimulation of gonadal steroid production by carp hypophyseal homogenate in Carassius auratus. J. Fish Biol. 44, 195–204.
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Sivarajah, K., Franklin, C.S., William, W.P., 1978a. The effect of polychlorinated biphenyls on plasma steroid levels and hepatic microsomal enzymes in fish. J. Fish Biol. 13, 401–409. Sivarajah, K., Franklin, C.S., William, W.P., 1978b. Some histopathological effect of Aroclor 1254 on the liver and gonads of rainbow trout, salmo gairdneri and carp, Cyprinus carpio. J. Fish Biol. 13, 411–414. Suneja, S.K., Ram, G.C., Wagle, D.S., 1984. Effect of T-2 toxin administration to rats on lipid metabolism in liver. Toxicol. Lett. 22, 113–118. Thomas, P., 1989. Effect of Arclor 1254 and cadmium on reproductive endocrine function and ovarian growth in Atlantic croaker. Mar. Environ. Res. 28, 499–503. Thomas, P., 1990. Teleost model for studying the effect of chemicals on female reproductive endocrine function. J. Exp. Zool. 4 (Suppl.), 126–128. Van Der Kraak, G.J., Munkittrick, K.R., McMaster, M.E., Portt, C.B., Chang, J.P., 1992. Exposure to bleeched kraft pulp mill effluent disrupts pituitary gonadal axis of white sucker at multiple sites. Toxicol. App. Pharmacol. 115, 224–233. Vitiello, F., Zanetta, J.P., 1978. Thin layer chromatography of phospholipids. J. Chromatogr. 166, 637–640. Wester, P.W., Canton, J.H., Van Iersel, A.A.J., Krajnc, E.I., Vaessen, H.A.M.G., 1990. The toxicity of bis(tri-n-butyltin)oxide (TBTO) and di-n-butyltindichloride (DBTC) in the small fish species Oryzias latipes (medaka) and Poecilia reticulata (guppy). Aquat. Toxicol. 16, 53–72. Wiegand, M.D., Peter, R.E., 1980a. Effect of salmon gonadotropin (SG-100) on plasma lipids in the goldfish, Carassius auratus. Can. J. Zool. 58, 957–966. Wiegand, M.D., Peter, R.E., 1980b. Effect of sex steroids on plasma lipids in the goldfish, Carassius auratus. Can. J. Zool. 58, 967–979. Wiegand, M.D., Idler, D.R., 1982. Synthesis of lipids by the rainbow trout (Salmo gairdneri) ovary in vitro. Can. J. Zool. 60, 2683–2693.
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Reproductive endocrine disruption in the freshwater catfish, Heteropneustes fossilis, in response to the pesticide g-hexachlorocyclohexane Pratap B. Singha, and Adelino V.M. Canariob b
a Department of Zoology, Tilak Dhari College, Jaunpur 222002, India Centre of Marine Sciences, University of Algarve, Campus of Gambelas, Faro 8000-810, Portugal
Received 1 November 2002; received in revised form 17 July 2003; accepted 19 July 2003
Abstract Both male and female freshwater catfish, Heteropneustes fossilis, were exposed to safe (SC; 0.1 and 1.0 mg/L) and sublethal (SL; 10 mg/L) concentrations of an agricultural pesticide, g-hexachlorocyclohexane (g-HCH) for 4 weeks during the active pre-spawning (vitellogenic) phase of their annual reproductive cycle. On the last day of exposure, 18 h before killing, fish were treated intramuscularly (i.m.) with [I-14C]acetic acid (74 kBq per fish). After 4 weeks of exposure, we monitored the effects of g-HCH on gonadosomatic index (GSI); on plasma concentrations of gonadotropin (GtH), testostosterone (T), 11-ketotestosterone (11-KT), 17b-estradiol (E2); and on hepatic incorporation of [I-14C]acetic acid into total phospholipids (TP) and the fractions thereof: phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI), and phosphatidylethanolamine (PE). In both the sexes, GSI and plasma GtH were decreased significantly in response to g-HCH exposure. Plasma T and 11-KT in males, and plasma T and E2 in females declined significantly in response to g-HCH exposure. In both the sexes, hepatic incorporation of [I-14C]acetic acid into PS and PI increased significantly, whereas incorporation into TP, PC, and PE was significantly reduced after g-HCH exposure. Our findings demonstrated that g-HCH exposure depressed GSI, plasma GtH, sex steroids, and [I-14C]acetic acid incorporation into hepatic TP, and had very selective and specific effects on various classes of TP, resulting either from the hypothalamo-hypophyseal-gonadal axis or from direct action on hepatic and steroidogenic enzymes during the pre-spawning phase, causing reproductive endocrine disruptions. r 2003 Elsevier Inc. All rights reserved. Keywords: g-HCH; Gonadotropin; Sex steroids; Phospholipids; Endocrine disruptions; Fish reproduction
1. Introduction g-Hexachlorocyclohexane (g-HCH) is widely used to kill pests for crop protection. Paddy-cum-fish culture is very common in India, where fish are directly exposed to g-HCH as nontarget species. Fish are a major source of protein for human societies, and fisheries are a major commercial activity. Fish accumulate g-HCH preferentially in their fatty tissues, such as liver and gonads, but the effects may become apparent only when concentration in such tissues passes a certain threshold. These effects can include damaged immune response, tumors, or respiratory problems, all of which can shorten the life span and decrease the population through both pre
Corresponding author. Fax: +91-5452-263338. E-mail address: pratap p [email protected] (P.B. Singh).
0147-6513/$ - see front matter r 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2003.07.014
mature mortality and a decreased number of fish surviving to enter the spawning season (Macek, 1968; Anderson, 1996). Toxic substances, even in very low concentration, have been reported to suppress reproduction (Hirose, 1975; Sivarajah et al., 1978a, b; Singh et al., 1994; Ankley et al., 2001), steroidogenesis (Wester et al., 1990; Van Der Kraak et al., 1992; Singh et al., 1994; Kime and Singh, 1996), lipid metabolism (Singh, 1992; Singh and Kime, 1994), GtH concentrations (Thomas, 1990; Singh et al., 1994), vitellogenin production (Chen et al., 1986; Pereira et al., 1992), and reproductive physiology (Santos and Pacheco, 1996; Hontela et al., 1997; Pacheco and Santos, 2001). Additional reports of Wiegand and Idler (1982) have indicated that [I-14C]acetic acid was incorporated primarily into polar lipids in fish undergoing ovarian recrudescence. Hansen
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(1987) had demonstrated that when [I-14C] or [32P]phosphate was injected directly into the bloodstream of the eel, the resulting phospholipid patterns were the same in both gill and liver dominated not by PE but by PC. Most previous studies on pollution effects have been restricted to fatty acids, cholesterol, or TP, but there are few reports on simultaneous studies on GSI, plasma GtH, sex steroids production, and various classes of phospholipids of either sex after long-term g-HCH exposure in the Indian freshwater catfish, H. fossilis. Therefore, the present study was conducted during the pre-spawning phase (May–June), when the parameters of interest showed maximum activity and involvement in reproduction, as reported in this teleost (Singh and Singh, 1990). The aim of this experiment was to assess the endocrine disruption resulting from g-HCH exposure by taking into consideration the parameters of GSI; plasma GtH, the sex steroid hormones T, E2, and 11-KT; and hepatic incorporation of [I-14C]acetic acid into various classes of phospholipids: PS, PI, PE, and PC—the latter of which is a major constituent of phospholipid for vitellogenin synthesis in liver by ovarian E2 under the control of GtH during gonadal recrudescence.
2. Materials and methods 2.1. Experimental fish The original research reported in this monograph was conducted under ethical guidelines (Anonymous, 1998) established for animal use by Tilak Dhari College, Jaunpur (UP), India. Male and female H. fossilis (65–75 g body wt, 21–22 cm length) were collected on 2 May 2001 from a local pond of the Pratapgarh district (Lat. 25 340 S; Long. 61 590 E) during the pre-spawning phase and maintained in a tank (3500 L) supplied with a constant flow of dechlorinated tap water (temperature 2071 C, pH 7.2, hardness 154 mg/L (as CaCO3), alkalinity 68 mg/L (as CaCO3), dissolved oxygen (7.2 mg/L), and conductivity 0.56 mO: The fish were exposed to a natural photoperiod (13 h light/11 h dark) and a temperature of 3072 C, having a GSI (total gonadal wt/total body wt 100) of 5.1270.25 for males and 7.1170.35 for females. They were fed ad libitum with minced goat liver comprising 20% protein, 5% lipid, and 15% carbohydrate, with the remaining 60% composed of water, minerals, and vitamins. After 10 days of acclimation, experiments were begun. 2.2. Chemicals Analytical-grade chemicals were obtained from BDH (India). Solvents were redistilled before use. Thinlayer chromatography (TLC) pre-coated plastic sheets
(E. Merck Silica gel G60 F254; 20 cm 20 cm 0.20 mm) were obtained from BDH. Lipid standards were obtained from Sigma Chemical Co. (UK): lecithin for TP, l-a-phosphatidylcholine for PC, l-a-phosphatidyl-lserine for PS, l-a-phosphatidylinositol for PI, l-aphosphatidylethanolamine for PE. g-HCH (g-BHC; 1a; 2a; 3b; 4a; 5a; 6b-hexachlorocyclohexane, g-isomer, 99% pure) was also purchased from Sigma. [I-14C]acetic acid sodium salt (2.15 GBq/mmol) was obtained from Amersham International (UK). Labeled hormones [1,2,6,7-3H]testosterone (sp. act. 103 Ci/mmol), [2,4,6,7-3H]17b-estradiol (sp. act. 89 Ci/mmol), and [3H]ketotestosterone were obtained from Radiochemical Centre Amersham (UK). Authentic steroids were purchased from Sigma (USA). Lyophilized aliquots of antibodies against T, 11-KT, and E2 were provided by G.D. Niswender, Colorado State University, Colorado. 2.3. Exposure studies To test the pesticide concentration to be used, a single fish was exposed (according to the method described by Singh et al., 1994) briefly to concentrations of 20, 10, 1, and 0.1 mg/L of g-HCH (dissolved in 1 mL acetone). Fish exposed at 20 mg/L showed extreme hyperactivity and were immediately transferred to uncontaminated water. Fish at 10, 1, and 0.1 mg/L showed no apparent distress even after 24 h. Pesticide concentrations for the experiments were therefore chosen as 10, 1, and 0.1 mg/L. After 4 weeks, the fish exposed to 0.1 and 1 mg/L showed no behavioral differences from the controls, but those exposed to 10 mg/L showed increased mucus and locomotor activity indicative of mild stress. We therefore classed the two pesticide levels as safe (SC) and sublethal (SL) concentrations, respectively. In our earlier experiments, paddy-cum-fish culture in which the g-HCH exposure was intended to kill the pests, the g-HCH concentrations in the water used for rearing was recorded as 10 mg/L (unpublished data). After acclimation, the fish were categorized by sex and were divided into eight batches, each comprising six fish in a plastic aquarium (46 25 30 cm3) containing 20 L of water at 20 C. All eight aquaria were surrounded by a water jacket at 20 C. The g-HCH was dissolved in acetone and diluted with water to the required concentrations. Both male and female fish were exposed to g-HCH at the selected safe (SC; 0.1 and 1.0 mg/L) and sublethal (SL; 10 mg/L) concentrations for 4 weeks during the pre-spawning phase. Control fish were maintained in plain dechlorinated tap water containing acetone at the same concentration as the treated groups (20 mL per tank). A separate true control was also kept without any treatment. During the experiment, fish were fed every fourth day, when the aquarium water was exchanged for freshwater containing the appropriate pesticide concentrations. On day 29
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2.4. Plasma gonadotropin and sex steroid hormones measurements Duplicate samples were run for measurements of reproductive hormones. Plasma GtH was measured using the enzyme-linked immunosorbent assay method of Kah et al. (1989). Sex steroids were measured by radioimmunoassay using the methods of Singh et al. (1994). 2.5. Effect of g-hexachlorocyclohexane on conversion of different phospholipids by [I-14C]acetic acid in exposed fish To observe whether g-HCH had an effect on PC and its conversion by radiolabeled acetic acid to other phospholipids, pollutant-exposed and control fish were sampled 18 h after injection. Duplicate samples were prepared for the study of the incorporation of [I-14C]acetic acid into different phospholipids. Liver lipids were extracted following the method of Folch et al. (1957), so as to extract TPs. TPs were separated on TLC using the double-solvent system (system I, diethylether:benzene:ethanol:acetic acid, 40:50:2:0.2; system II, hexane:diethylether, 94:6) of Freeman and West (1966). The Rf value of TP was 00. Authentic lipids were visualized by exposing the plates to iodine vapor, and radioactive areas were detected with a TLC radiochromatogram scanner. The TP residue was scraped from the TLC plate and eluted with chloroform:methanol (2:1) and the solvent evaporated. Samples were rechromatographed on TLC, and the various phospholipid classes (Rf values for PC, PS, PI, and PE were 0.12, 0.20, 0.27, and 0.41, respectively) were separated using methyl acetate:isopropanol:chloroform:methanol:0.25% aqueous KCl (25:25:25:10:9) by volume (Vitiello and Zanetta, 1978). The different phospholipids were visualized by brief exposure to iodine vapor and the individual classes scraped into scintillation minivials. After adding 3 mL Packard Scintillator 299, the minivials were vortexed and radioactivity was determined.
2.6. Statistical analysis The contents of the different phospholipids were expressed as mean7SEM in dpm/g of individual liver samples. GtH and sex steroids contents were expressed as ng/mL plasma, and values were expressed as mean7SEM ðn ¼ 5Þ: The data were analyzed by Student’s t-test followed by Newman–Keuls’ multiplerange test at the level of 0.05 (Bruning and Kintz, 1977).
3. Results There was no difference in the values between the controls (i.e., vehicle-treated and nontreated proper control); hence, vehicle-treated control values were taken into consideration. 3.1. Effect of g-hexachlorocyclohexane exposure on gonadosomatic index GSI was significantly decreased in both the sexes during the reproductively active pre-spawning phase after 4 weeks of g-HCH exposure at 1 and 10 mg/L dose levels as compared with the controls. However, g-HCH at 0.1 mg/L exposure did not decrease GSI significantly in either sex (Fig. 1). 3.2. Effect of g-hexachlorocyclohexane exposure on plasma gonadotropin Plasma GtH was significantly reduced in both the sexes after exposure to 1 and 10 mg/L concentrations of g-HCH, whereas the 0.1 mg/L exposure failed to show any such effect when compared to the controls (Fig. 2). 8 NS
7 6 5 GSI
of exposure, 18 h before killing, all fish were injected i.m. with 25 mL aqueous solution of [I-14C]acetic acid sodium salt at a dose rate of 74 kBq per fish. All fish were bled by caudal incision, and blood samples were collected in heparinized glass culture tubes. Plasma was separated by centrifugation at 17,000g at 4 C for 15 min and stored frozen at 20 C until assayed for GtH, male sex steroids (T and 11-KT), and female sex steroids (T and E2). Individual gonad weights were recorded for calculation of GSI. Individual livers were extirpated, washed in 0.6% saline, blotted, weighed, and kept frozen at 20 C for further analysis of hepatic incorporation of [I-14C]acetic acid into phospholipids after g-HCH exposure.
79
**
NS
**
4 3
*
*
2 1 0 Male
Female
Fig. 1. Gonadosomatic index (GSI) of male and female H. fossilis (Bloch) held in freshwater [ ], 0.1 ppm [ ], 1.0 ppm [ ] and 10 ppm [ ] g-HCH treated fish. Bar indicates mean7SEM ðn ¼ 5Þ: Asterisks denotes a significant difference ð Po0:001; Po0:005Þ from control. NS—not significant ðP40:05Þ:
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80
25.0
14 12
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GtH ng ml-1
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*
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*
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* *
4
*
*
2
5.0
0 T
0.0 Male
E
2
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Female
Fig. 2. Plasma gonadotropin (GtH) concentrations in male and female H. fossilis held in freshwater [ ],0.1 ppm [ ],1.0 ppm [ ] and 10 ppm [ ] g-HCH treated fish. Bars indicates mean7SEM ðn ¼ 5Þ: Asterisks denotes a significant difference ð Po0:001; Po0:01Þ from control, NS—not significants ðP40:05Þ:
Fig. 4. Plasma T (testosterone) and E2 (estradiol-17b) concentrations in female H. fossilis held in freshwater [ ], 0.1 ppm [ ], 1.0 ppm [ ] and 10 ppm [ ] g-HCH treated fish. Bars indicates mean7SEM ðn ¼ 5Þ Asterisks denotes a significance difference ð Po0:001Þ from control.
10000
12
8000
NS
dpm/g of liver
-1
ng ml Plasma
10
9000
8 ***
**
6
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*
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4
** *
2
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2000
*
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1000 0
0
TP
T
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11 KT
Fig. 3. Plasma T (testosterone) and 11-K T (11-ketotestosterone) concentrations in male H. fossilis held in freshwater [ ], 0.1 ppm [ ], 1.0 ppm [ ] and 10 ppm [ ] g-HCH treated fish. Bars indicates mean7SEM ðn ¼ 5Þ: Asterisks denotes a significance difference ð Po0:001; Po0:005; Po0:02Þ from control, NS—not significant ðP40:05Þ:
Fig. 5. Effect of g-HCH exposure on hepatic incorporation of [I-14C]acetic acid into TP (total phospholipid), PC (phosphatidylcholine), PS (phosphatidylserine), PI (phosphatidylinositol) and PE (phosphatidylethanolamine) during the prespawning phase of male H. fossilis held in freshwater [ ], 0.1 ppm [ ],1.0 ppm [ ] and 10.0 ppm [ ] g-HCH treated fish. Asterisks denotes a significant difference Po0:001; Po0:005; Po0:01 from control. NS—not significant ðP40:05Þ:
3.3. Effect of g-hexachlorocyclohexane exposure on plasma sex steroids
3.4. Effect of g-hexachlorocyclohexane exposure on hepatic incorporation of [I-14C]acetic acid into total phospholipids and various fractions
In males, plasma T concentration significantly declined at the 1 and 10 mg/L g-HCH exposures, whereas the 0.1 mg/L dose was not effective in inducing any significant change when compared to the control. However, 11-KT values were significantly lowered at all doses of g-HCH exposure carried out in the present study. In females, plasma T and E2 were recorded to be decreased significantly at all tested concentrations of g-HCH when compared with the controls (Figs. 3,4).
Total phospholipids were significantly decreased in both sexes at all the concentrations of g-HCH exposure when compared with controls. In males, PE showed a significant decrease at all doses, whereas PC reflected such an effect only at the 1 and 10 mg/L g-HCH exposures when compared with the controls. PS increased significantly at the 0.1 and 1 mg/L g-HCH doses, and PI increased only at the 1 mg/L dose as compared with controls (Fig. 5).
Male
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dpm/g liver
10000 8000
** * *
6000 4000 2000
***
** **
NS
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* * *
NS
* * *
0 TP
PC
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Fig. 6. Effect of g-HCH exposure on hepatic incorporation of [I-14C]acetic acid into TP (total phospholipid), PC (phosphatidylcholine), PS (phosphatidylserine), PI (phosphatidylinositol) and PE (phosphatidylethanolamine) during the prespawning phase of female H. fossilis held in freshwater [ ], 0.1 ppm [ ],1.0 ppm [ ] and 10.0 ppm [ ] g-HCH treated fish. Asterisks denotes a significant difference Po0:001; Po0:005; Po0:01 from control. NS—not significant ðP40:05Þ:
In females, when compared with the controls, PE and PC decreased at all doses of g-HCH exposure, whereas both PS and PI increased. However, PS showed an increase at all doses tested, whereas PI increased only at the 1 mg/L g-HCH dose (Fig. 6).
4. Discussion This is the first comprehensive study on the effect of g-HCH exposure on GSI, GtH, sex steroid hormones, and hepatic incorporation of [I-14C]acetic acid into various phospholipids during the reproductively active pre-spawning phase in H. fossilis. The decline in GSI in H. fossilis subsequent to g-HCH exposure indicates the inhibition of gonadal growth during the reproductive phase studied. A similar decrease in GSI in response to pesticide exposure has been recorded in several teleosts (Thomas, 1989; Mukherjee et al., 1991; Singh et al., 1994). According to our results, g-HCH exposure at SC and SL concentrations significantly decreased the plasma concentrations of GtH required for ovarian steroidogenesis during the pre-spawning phase of H. fossilis. Our findings are supported by the earlier reports on carp, Carassius auratus, after g-HCH exposure (Singh et al., 1994). The decreased spontaneous pituitary secretion of GtH in vitro resulting from polychlorinated biphenyl treatment in Micropogonias undulatus L. (Thomas, 1989) and the disruption of the pituitary-gonadal axis in the teleost white sucker, Catostomus commersoni L. by bleached kraft mill effluent (Van Der Kraak et al., 1992) have also been reported as endocrine disruptors. Inhibition of steroidogenesis, microsomal enzymes, and
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histopathological changes in response to toxicants have been reported in some teleosts (Sivarajah et al., 1978a, b; Singh et al., 1994). Singh and Singh (1991) have demonstrated that injection of Mystus sp. GtH elevated the sex steroids (T and E2) and phospholipids during the pre-spawning phase in H. fossilis. In another experiment, Mercure et al. (2001) have indicated that E2 treatment of immature rainbow trout Oncorhynchus mykiss increases PC, PI, PE, and plasma vitellogenin. Among the polar lipids, PC content was higher in this species. Fremont and Riazi (1988) have indicated that fish vitellogenin comprises 18% of total lipids, of which approximately two-thirds is phospholipids and the remaining fraction is largely triacylglycerides, sterols, and sterol esters. On the basis of results obtained here, it can be argued that the secretion of E2 by the ovary (under maturational GtH control), which stimulates the liver to secrete phospholipid components and vitellogenin—ultimately deposited in growing oocytes under the influence of vitellogenic GtH during the reproductively active pre-spawning phase—is disrupted by gHCH. Suneja et al. (1984) found reduced activity of PC and PE on [I-14C]acetate incorporation in male rats after T-2 toxin administration. Therefore, it is concluded that g-HCH exposure inhibited production of plasma GtH, steroidogenesis, lipogenesis, and vitellogenesis in this species via the hypothalamo-hypophyseal-gonadal axis. Thus it is possible that decreased plasma sex steroids in both the sexes might be indicative of a change in the biosynthetic capacity of the gonad, either directly by the inhibition of aromatization activity, which is responsible for estrogen synthesis, or indirectly by suppressing the secretion of GtH. Considering the results obtained, it is reasonable to assume that the inhibition of hepatic lipogenesis and translocation of hepatic lipids to the ovary is brought about by the impairment of steroid metabolism. This contention is supported by a number of earlier reports in which it was well established that GtH and the gonadal steroids are critically involved in the regulation of lipid metabolism in teleosts in relation to reproduction (Wiegand and Peter, 1980a, b; Singh and Singh, 1991; Schulz et al., 2001; Devlin and Nagahama, 2002). In a separate report, Singh (1992) demonstrated decreased lipogenesis in the liver and its mobilization to the gonads in H. fossilis after g-HCH treatment for 28 days during preparatory and pre-spawning phases. They have further attributed these decreases to impaired steroidogenesis as well as synthesis and secretion of GtH. In this species, decreases in plasma GtH after g-HCH exposure indicate that GtH has direct control over steroidogenesis as well as phospholipid biosynthesis. Decreases in GtH and sex steroids have also been reported in C. auratus (Singh et al. 1994) after g-HCH exposure. Leslie and Buckley (1976) have reported that in the goldfish liver PC was the major component of TP, with
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the rest being PE, PI, and PS in decreasing order. They have also shown that liver microsomal choline phosphotransferase enzyme activity decreased as the temperature increased from 10 C to 30 C. It has also been reported that pulp mill effluent has inhibited UDPglucuronyl transferase (UDP-GT) activity in trout (Castren and Oikari, 1987). They have further proposed that pollutants that affect hepatic UDP-GT activity may therefore also affect the gonadal enzyme and possibly interfere with chemical signaling between the sexes or balances of free and conjugated steroids within the gonad, where the enzyme may provide protection. Recently, Ibanez et al. (2003) have suggested that lipoprotein lipase is probably involved in the incorporation of neutral lipids into the oocytes of sea bass, and the follicular cells not only participate in steroidogenesis but may also be important for the accumulation of nutrients into oocytes. Further studies on the individual enzyme involved in the synthesis of different phospholipids and sex steroids are necessary to determine the role of g-HCH exposure in this species during reproductive growth.
5. Conclusions Our findings clearly demonstrated that g-HCH significantly depressed the GSI, plasma GtH, sex steroids, and [I-14C]acetic acid incorporation into hepatic phospholipid components, and had very selective and specific effects on various classes of phospholipid, resulting from either decreased GtH levels or direct action on the enzymes involved in steroidogenesis and lipogenesis during the reproductively active prespawning phase in H. fossilis. PC, a major constituent of phospholipids and the predominant lipid of vitellogenin, and reproductive hormones are affected by SC as well as SL exposure to g-HCH, causing reproductive endocrine disruptions acting via the hypothalamo-pituitary-gonadal axis.
Acknowledgments A grant-in-aid from UGC MRP (F.8.4(55) 1999-2000/ MRP/NR dated 31.03.2000) to P.B.S. is greatly appreciated. The authors are grateful to R.B. Raizada, Industrial Toxicology Research Centre, Lucknow, for laboratory facilities, and to S.S. Srivastava and G.N. Shukla for reading of this manuscript.
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