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Effects of combined exposure to lead and cadmium on the hypothalamic–pituitary axis function in proestrous rats
Food and Chemical Toxicology 41 (2003) 379–384 www.elsevier.com/locate/foodchemtox
Effects of combined exposure to lead and cadmium on the hypothalamic–pituitary axis function in proestrous rats A. Pillai, L. Priya, S. Gupta* Department of Biochemistry, Faculty of Science, M.S. University of Baroda, Vadodara, Gujarat 390002, India Accepted 30 August 2002
Abstract The effects of lead and cadmium on the hypothalamic–pituitary axis were studied in proestrous rats. Adult female rats were treated intraperitonially with either lead acetate and cadmium acetate alone or in combination at a dose of 0.05 mg/kg daily for 15 days. Serotonin (5-HT) and norepinephrine (NE) levels decreased in individually and combined metal treated groups whereas dopamine (DA) levels were decreased only in the cadmium-exposed group. The pituitary levels of luteinizing hormone (LH) and follicle stimulating hormone (FSH) were decreased significantly in cadmium and combined treatment groups. In contrast, lead exposure failed to cause any change in serum LH and FSH levels, whereas cadmium and combined treatments showed significant decrease in serum LH and FSH levels as compared with control. The accumulation of both metals increased in the hypothalamus and pituitary after treatment. These data suggest that the metal accumulation disrupts the regulatory mechanisms of the hypothalamic–pituitary axis where the effects produced by the combined treatment of metals are not additive. # 2002 Elsevier Science Ltd. All rights reserved.
1. Introduction Lead and cadmium are known endocrine disrupters present in the environment. Exposure to these metals is associated with changes in the activity of endocrine system in male and female animals. It was shown that metals can affect the activity of the hypothalamus–pituitary–ovarian axis by acting at the hypothalamus (Das et al., 1993; Andersson et al., 1997; Antonio, et al., 1999), the pituitary (Lorenson et al., 1983; Ronis et al., 1998; Lafuente et al., 1999a), the ovary (Paksy et al., 1990) and/or the accessory organs (Klinefelter and Hiss, 1998). A number of different studies have shown that both these metals modify plasma levels of luteinizing hormone (LH) and follicle stimulating hormone (FSH) (Zylber-Haran et al., 1982; Lorenson et al., 1983; Paksy et al., 1989; Lafuente et al., 1997, 1999a). It is known that catecholamines and indoleamines as well as other neurotransmitters of the Abbreviations: ANOVA, analysis of variance; DA, dopamine; FSH, follicle stimulating hormone; 5-HT, serotonin; LH, luteinizing hormone; NE, norepinephrine. * Corresponding author. Tel.: +91-265-795594. E-mail address: [email protected] (S. Gupta).
central nervous system modulate pituitary hormone secretion (Drouva and Gallo, 1976; Lopez et al., 1989). Thus the metal accumulated at this level after the exposure may change the regulatory mechanism. Various studies have shown that lead exposure can cause changes in catecholaminergic functions (Shih and Hanin, 1978; Cooper and Manalis, 1983; Winder and Kitchen, 1984; Nation et al., 1989). Low-level Cd exposure results in increased catecholamine neurotransmission (Rastogi et al., 1977; Williams et al., 1978; Arito et al., 1981; Cooper and Manalis, 1983; Nation et al., 1989). Most of the above-cited studies were carried out with a single metal. Available data on combined exposure of lead and cadmium shows that these metals show additive effects on acetyl choline release at the frog neuromuscular junction (Cooper and Manalis, 1984) whereas Nation et al. (1989) observed lesser effects of combined exposure than in isolation. As the hypothalamus is an important tissue which modulates pituitary hormone secretion it is of interest to know whether these alterations in pituitary hormones are mediated by changes in neurotransmitter content at the hypothalamic level. According to WHO (1993) the permitted dose of cadmium for humans is 70 mg/person/day and that of
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lead is 245 mg/person/day. In the present study the administered dose is 10 mg/rat/day for both lead and cadmium, which is lower than the permitted dose for humans. However, it is the final accumulation of metal in the tissues that plays major role in metal toxicity (Paksy et al., 1990; Marquez et al., 1998). So it is very interesting to study the effects produced by the low concentration of metals when administered together. Hence, the present study involves the effect of simultaneous exposure of lead and cadmium via intraperitoneal injection on the hypothalamic neurotransmitter content and plasma and pituitary levels of LH and FSH. This route of administration results in higher internal doses than when orally administered and competition in absorption of other elements via the intestinal barrier does not occur. We also estimated the metal content both in hypothalamus and pituitary to study whether the changes in the hypothalamic–pituitary unit are related to metal accumulation.
2. Materials and methods 2.1. Animals and treatment Adult virgin female rats weighing 180–220 g were maintained under controlled conditions of light and temperature and having free access to diet and tap water. Ovarian cycle was checked daily by vaginal cytology. Animals displaying at least three 4-day cycles were selected for the experiment. There were four groups of 10 animals each in the study. Group 1 animals were given sodium acetate as control, group 2 lead acetate, group 3 cadmium acetate and group 4 received lead acetate and cadmium acetate in combination. The animals were treated ip with 0.05 mg/kg body weight dose per day for 15 days. The dose was selected on the basis of our previous studies on the effect of simultaneous exposure of lead and cadmium on hepatic estradiol metabolism (Pillai et al., 2002). The animals were killed by decapitation on the proestrous stage at 10.00 and the decapitation procedure was completed within 30 s. Proestrous is the period before ovulation, when maximal ovarian hormone production occurs. Blood was collected and serum samples obtained after centrifugation at 2500 g for 10 min were stored at 20 C until LH and FSH were measured. The hypothalamus and pituitary were immediately removed and processed for further assays. The pituitary gland of each rat was homogenized separately in cold 0.9% NaCl (1 ml/10 mg wet weight), centrifuged at 500 g for 15 min at 4 C and the supernatant fraction was used for hormone analysis. Also the body weight gain of the animals of the four groups was recorded.
2.2. Amine measurements Amines were estimated by the flourimetric method of Shellenberger and Gordon (1971). The hypothalamus was dissected out immediately, weighed and homogenized in chilled 0.4 m perchloric acid solution containing 0.1% (w/v) Na2S2O5 and 0.025% Na2EDTA at 4 C in a motor driven glass homogenizer. The homogenates were centrifuged at 4000 g at 4 C for 15 min. The supernatant was taken and mixed with 25 mg alumina. The solution was shaken well, centrifuged at 500 g for 5 min and the supernatant was used for 5-HT estimation. Alumina was further washed four times with 5 ml of H2O, and extracted with 150 ml 0.2 m HClO4 solution. The supernatant was used for the estimation of norepinephrine and dopamine. 2.3. Estimation of 5-HT The fluorescence was developed by reaction with ninhydrin as utilized by Synder et al. (1965). An aliquot (4 ml) was taken and the pH was adjusted to 10 with 0.8 ml 10 m NaOH. It was transferred to a 30-ml stoppered glass tube containing 0.5 ml 0.5 m borate buffer (pH 10), 1.5 ml saturated NaCl solution (approx. 1.5 g) and 15 ml butanol, and vortexed for 10 min. After removing the aqueous phase, the organic phase was then vortexed for 3 min with 2 ml 0.1 m borate buffer (pH 10), previously saturated with NaCl. The butanol layer (10 ml) was transferred to another tube containing 4 ml phosphate buffer (0.05 ng, pH 7) and 15 ml n-heptane. After vortexing for 2 min, 3.6 ml phosphate layer was carefully aspirated into a 15-ml glass tube containing 0.3 ml 0.1 m freshly prepared ninhydrin solution. The tubes were then heated for exactly 30 min at 75 C in a waterbath. One hour later, the solution was transferred to a quartz cuvette and fluorescence was estimated at 385/ 490 nm. 2.4. Estimation of norepinephrine and dopamine A 1-ml aliquot of the perchloric acid eluate was made to pH 6.5 0.2 with 1.5 ml of 0.1 m phosphate buffer– EDTA solution. Iodine reagent (0.2 ml) was added (shaking immediately to mix) kept for exactly 2 min, after which 0.5 ml of the alkaline sodium sulfite solution was added. After 2 min, 0.4 ml of glacial acetic acid was added to bring the pH to 4.4–4.8 and kept in a hot-air oven at 100 C for 3–4 min. Tubes were placed in an icebath for cooling and the NE fluorescence was read at 380/495 nm. The samples were returned to the oven and heated at 100 C for an additional 40 min to develop DA fluorescence. After cooling, the DA fluorescence of the tubes was read at 325/380 nm. The accuracy and precision of the assay were checked by running, in parallel, different concentrations of external and internal
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standards and blanks with tissue samples. The standard curves were linear over the range of concentrations obtained from the tissue samples. 2.5. Hormone measurement Both LH and FSH levels were measured by double antibody radioimmunoassay, using 125I-labeled radioligands. r-LH antiserum (AFP115368) and r-FSH antiserum (AFP19896) were obtained from National Hormone and Pituitary Program (NHPP, MD, USA). Hormones were labeled with 125I (Taramani Institute, Chennai, India) according to the modified procedure of Greenwood et al. (1963). The intra-assay coefficients of variation were 9.5 and 7.3% for LH and FSH, respectively. All samples were measured in the same assay to avoid inter-assay variations. Sensitivities of the assays were 0.065 and 0.08 ng/ml for LH and FSH, respectively. 2.6. Metal analysis Lead and cadmium concentrations were determined in hypothalamus and pituitary samples. The samples were digested in reagent grade nitric acid–perchloric acid (2:1) mixture. The digestion was continued until samples became colorless. Then the acid mixture was evaporated and the precipitate thus obtained was dissolved in a few drops of concentrated HCl. The sample was diluted to 1 ml with distilled water and the readings were taken in GBC 902 double beam atomic absorption spectrophotometer. Sensitivities of the assays were 0.06 and 0.009 mg/ml for lead and cadmium, respectively.
Fig. 1. Serotonin, dopamine and norepinephrine levels in hypothalamus of female rats treated with lead acetate and cadmium acetate alone and in combination for 15 days (0.05 mg/kg body weight). The values are expressed as meanS.E.M. (n=5 in each group). *P40.05 vs control group. **P40.05 vs lead group. #P40.05 vs cadmium group. ## P40.05 vs lead+cadmium group.
treatment groups did not show any significant change in the dopamine level. The animals in the cadmium and combined groups showed a significant decrease in serum LH (P40.05, Fig. 2) and FSH (P40.05, Fig. 3) levels as compared to the control group, whereas the lead-treated group did not show any significant change in the LH and FSH levels as compared to control. The cadmium-exposed animals showed a significant decrease in pituitary LH (P40.05, Fig. 2) and FSH content (P40.05, Fig. 3) as compared to control and lead-exposed groups. The
2.7. Statistical analysis Comparison of values was done by analysis of variance (ANOVA) followed by Student’s t-test. P40.05 were considered statistically significant. All values represent the mean S.E.M.
3. Results Fig. 1 shows the estimated concentration of hypothalamic serotonin, norepinephrine and dopamine in various groups. The serotonin content was decreased in all metal-treated groups with cadmium showing maximum reduction. The combined treatment with lead and cadmium was associated with changes in serotonin relatively similar to those induced by either metal administered alone. The norepinephrine content was decreased in both isolated and combined treatment groups (P40.05). Among the three groups cadmium exposure was showing maximum reduction in NE content. The dopamine content was decreased in the cadmium exposed animals (P40.05), whereas other
Fig. 2. Serum and pituitary levels of LH in female rats treated with lead acetate and cadmium acetate alone and in combination for 15 days (0.05 mg/kg body weight). The values are expressed as meanS.E.M. (n=5 in each group). *P40.05 vs control group. **P40.05 vs lead group. #P40.05 vs cadmium group. ## P40.05 vs lead+cadmium group.
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Fig. 3. Serum and pituitary levels of FSH in female rats treated with lead acetate and cadmium acetate alone and in combination for 15 days (0.05 mg/kg body weight). The values are expressed as meanS.E.M. (n=5 in each group). *P40.05 vs control group. **P40.05 vs lead group. #P40.05 vs cadmium group. ##P40.05 vs lead+cadmium group.
simultaneous treatment with lead and cadmium induced similar changes in the LH and FSH content as with cadmium alone (P40.05). The lead exposure failed to cause any change in both LH and FSH levels. The analysis of metal concentrations showed that the tissues of cadmium and combined metal exposed groups contained a greater concentration of cadmium residues than the tissues of control and lead-exposed groups (Table 1). Similarly in the case of lead and combined metal exposed groups the lead concentration was significantly higher than that in control and cadmium exposed groups (Table 1).
4. Discussion The present study shows the effects of lead and cadmium on the hypothalamic neurotransmitter content as well as serum and pituitary levels of LH and FSH in the
proestrous stage of rats. The decrease in 5-HT content due to the inhibitory effect of cadmium agrees with the results found by Lafuente et al. (2000) where they have shown the effect in different hypothalamic regions. Das et al. (1993) observed a decrease in 5-HT level in the whole hypothalamus after cadmium exposure. Our results indicate that the decrease in serotonin content was greater in the cadmium-exposed group than that in animals receiving cadmium in combination with lead. Norepinephrine plays an important role in modulating luteinizing hormone releasing hormone (LHRH) neurons that are involved in the regulation of gonadotropin secretion by the anterior pituitary. It has been reported that lead can act at the hypothalamic level to alter LHRH secretion in the rat (Bratton, Hiney, & Dees, 1994). Lead blocks the NE-induced release of PGE2 resulting in diminished LHRH secretion. This decrease could be due to the altered Ca2+ mobilisation, a limiting step in PGE2 formation and subsequent LHRH release. In the present study we have observed decrease in NE in both isolated and combined metal treated groups. Dopamine content was not affected by lead or simultaneous exposure with lead and cadmium, whereas cadmium alone was showing significant decrease in the DA content. Various rodent studies on the subchronic exposure of low levels of lead resulted in significant reduction in DA and its metabolites 3,4 dihydroxyphenylacetic acid and homovanillic acid in the nucleus accumbens of rats (Jadhav and Kala, 1994; Kala et al., 1994; Kala and Jadhav, 1995). The discrepancies with other studies could be due to differences in the age of animals, mode of treatment, duration and brain areas analyzed. Lead has been reported to alter calcium homeostasis by affecting both voltage-dependent and receptor-operated calcium channels (Bressler and Goldstein, 1991; Audesirk, 1993; Oortgiesen et al., 1993), whereas the in vitro studies have shown that lead enhances calcium-activated release in brain transmitters (Minnema et al., 1988). Cadmium is shown to inhibit calcium entry and the attendent release of peripheral catecholamines (Hirning et al., 1988). Therefore the
Table 1 Lead and cadmium levels at the hypothalamus and pituitary of female rats exposed to lead acetate and cadmium acetate alone and in combination for 15 days (0.05 mg/kg body weight)a Group
Hypothalamus
Pituitary
Lead
Cadmium
(mg/g) Control Pb Cd Pb+Cd
2.52 0.095 4.1 0.27*** 2.8 0.021# 3.02 0.125**
Lead
Cadmium
(mg/g)
#
0.5250.032 0.5370.062 1.0350.056***## 0.840.021*** # ###
5.05 0.032 9.55 0.68*** 5.7 0.251## 6.25 0.263**
#
*P40.05; **P40.01; ***P40.001 vs control group. #P40.01; ##P40.001 vs lead group. ###P40.05 vs cadmium group. a The values are expressed as meanS.E.M. (n=5 in each group).
0.7450.365 0.82 0.096 1.47 0.102* 1.2820.375
#
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changes observed in the co-treatment group might be due to the fact that lead and cadmium compete with calcium at the channel site and compete with each other for entry through terminal membrane channels (Cooper and Manalis, 1984). The studies on pituitary hormones show that lead and cadmium affect the gonadotropin secretion differently. It is known that serotonin increases both LH and FSH secretion (Wuttke et al., 1978; Ellis et al., 1983). A maximum reduction in both pituitary and serum gonadotropin levels was found in cadmium-treated group. Lead does not produce any change in the gonadotropin levels compared with control. Significant changes observed in the combined treatment group can be due to the inhibitory effects produced by cadmium. The hormonal disturbances following combined treatment with lead and cadmium more often paralleled with the effects of cadmium alone than the changes seen in lead alone, suggesting antagonism between lead and cadmium. Decreased plasma levels of LH and FSH have been reported after administration of higher doses of cadmium to female rats (Paksy et al., 1989; Varga and Paksy, 1991). The decrease in LH and FSH levels after cadmium exposure could also be due to the accumulation of cadmium at the pituitary causing a direct effect on the hypophyseal level since there are some literature available indicating this mechanism (Lorenson et al., 1983; Cooper et al., 1987). Several studies have shown that cadmium can compete with calcium at the pituitary level (Waalkies and Poirier, 1984; Milos et al., 1989), which results in altered calcium regulation. Thus it either interferes with calcium influx through the membrane channel (Poirier et al., 1983; Kasprzak and Poirier, 1985; Cooper et al., 1987) or alters intracellular calcium mobilization. The results of our study agree with this hypothesis as both LH and FSH levels were decreased on cadmium accumulation. However, it is interesting to note that the decrease is observed more in the case of FSH than LH, suggesting additional mechanism for such differential effect. Lafuente et al. (2000) correlated the decease in FSH levels found in postpubertal rats after cadmium exposure to inhibin levels more than to cadmium accumulation at the pituitary. Dopamine is a known inhibitor of gonadotropin secretion and this fact cannot be correlated with the decreased levels of LH and FSH observed in the present study. Since gonadotropin secretion is under the control of multiple regulators it must be the net effect of all these regulators which has resulted in decreased levels of gonadotropins. The present investigation found that combined exposure to lead and cadmium showed less concentrations of both metals in the hypothalamus and pituitary relative to the metal retention in individual metal-treated groups. Nation et al. (1990) found a similar change in blood lead concentration in their studies on the effects of combined exposure of lead and cadmium on behavioral changes.
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The lesser accumulation of metals after combined exposure could be a reason for the observed antagonistic effects. From the above results it is clear that cadmium exerts more disruptions in the hypothalamic– pituitary axis function compared with lead, and the combined treatment effects are not additive.
Acknowledgements We are thankful to NIDDKs National Hormone and Pituitary Program (NHPP, USA) and to Dr. A. Parlow for the gift of LH and FSH. The work was supported by grants from UGC, New Delhi, India.
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Effects of combined exposure to lead and cadmium on the hypothalamic–pituitary axis function in proestrous rats A. Pillai, L. Priya, S. Gupta* Department of Biochemistry, Faculty of Science, M.S. University of Baroda, Vadodara, Gujarat 390002, India Accepted 30 August 2002
Abstract The effects of lead and cadmium on the hypothalamic–pituitary axis were studied in proestrous rats. Adult female rats were treated intraperitonially with either lead acetate and cadmium acetate alone or in combination at a dose of 0.05 mg/kg daily for 15 days. Serotonin (5-HT) and norepinephrine (NE) levels decreased in individually and combined metal treated groups whereas dopamine (DA) levels were decreased only in the cadmium-exposed group. The pituitary levels of luteinizing hormone (LH) and follicle stimulating hormone (FSH) were decreased significantly in cadmium and combined treatment groups. In contrast, lead exposure failed to cause any change in serum LH and FSH levels, whereas cadmium and combined treatments showed significant decrease in serum LH and FSH levels as compared with control. The accumulation of both metals increased in the hypothalamus and pituitary after treatment. These data suggest that the metal accumulation disrupts the regulatory mechanisms of the hypothalamic–pituitary axis where the effects produced by the combined treatment of metals are not additive. # 2002 Elsevier Science Ltd. All rights reserved.
1. Introduction Lead and cadmium are known endocrine disrupters present in the environment. Exposure to these metals is associated with changes in the activity of endocrine system in male and female animals. It was shown that metals can affect the activity of the hypothalamus–pituitary–ovarian axis by acting at the hypothalamus (Das et al., 1993; Andersson et al., 1997; Antonio, et al., 1999), the pituitary (Lorenson et al., 1983; Ronis et al., 1998; Lafuente et al., 1999a), the ovary (Paksy et al., 1990) and/or the accessory organs (Klinefelter and Hiss, 1998). A number of different studies have shown that both these metals modify plasma levels of luteinizing hormone (LH) and follicle stimulating hormone (FSH) (Zylber-Haran et al., 1982; Lorenson et al., 1983; Paksy et al., 1989; Lafuente et al., 1997, 1999a). It is known that catecholamines and indoleamines as well as other neurotransmitters of the Abbreviations: ANOVA, analysis of variance; DA, dopamine; FSH, follicle stimulating hormone; 5-HT, serotonin; LH, luteinizing hormone; NE, norepinephrine. * Corresponding author. Tel.: +91-265-795594. E-mail address: [email protected] (S. Gupta).
central nervous system modulate pituitary hormone secretion (Drouva and Gallo, 1976; Lopez et al., 1989). Thus the metal accumulated at this level after the exposure may change the regulatory mechanism. Various studies have shown that lead exposure can cause changes in catecholaminergic functions (Shih and Hanin, 1978; Cooper and Manalis, 1983; Winder and Kitchen, 1984; Nation et al., 1989). Low-level Cd exposure results in increased catecholamine neurotransmission (Rastogi et al., 1977; Williams et al., 1978; Arito et al., 1981; Cooper and Manalis, 1983; Nation et al., 1989). Most of the above-cited studies were carried out with a single metal. Available data on combined exposure of lead and cadmium shows that these metals show additive effects on acetyl choline release at the frog neuromuscular junction (Cooper and Manalis, 1984) whereas Nation et al. (1989) observed lesser effects of combined exposure than in isolation. As the hypothalamus is an important tissue which modulates pituitary hormone secretion it is of interest to know whether these alterations in pituitary hormones are mediated by changes in neurotransmitter content at the hypothalamic level. According to WHO (1993) the permitted dose of cadmium for humans is 70 mg/person/day and that of
0278-6915/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0278-6915(02)00247-8
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lead is 245 mg/person/day. In the present study the administered dose is 10 mg/rat/day for both lead and cadmium, which is lower than the permitted dose for humans. However, it is the final accumulation of metal in the tissues that plays major role in metal toxicity (Paksy et al., 1990; Marquez et al., 1998). So it is very interesting to study the effects produced by the low concentration of metals when administered together. Hence, the present study involves the effect of simultaneous exposure of lead and cadmium via intraperitoneal injection on the hypothalamic neurotransmitter content and plasma and pituitary levels of LH and FSH. This route of administration results in higher internal doses than when orally administered and competition in absorption of other elements via the intestinal barrier does not occur. We also estimated the metal content both in hypothalamus and pituitary to study whether the changes in the hypothalamic–pituitary unit are related to metal accumulation.
2. Materials and methods 2.1. Animals and treatment Adult virgin female rats weighing 180–220 g were maintained under controlled conditions of light and temperature and having free access to diet and tap water. Ovarian cycle was checked daily by vaginal cytology. Animals displaying at least three 4-day cycles were selected for the experiment. There were four groups of 10 animals each in the study. Group 1 animals were given sodium acetate as control, group 2 lead acetate, group 3 cadmium acetate and group 4 received lead acetate and cadmium acetate in combination. The animals were treated ip with 0.05 mg/kg body weight dose per day for 15 days. The dose was selected on the basis of our previous studies on the effect of simultaneous exposure of lead and cadmium on hepatic estradiol metabolism (Pillai et al., 2002). The animals were killed by decapitation on the proestrous stage at 10.00 and the decapitation procedure was completed within 30 s. Proestrous is the period before ovulation, when maximal ovarian hormone production occurs. Blood was collected and serum samples obtained after centrifugation at 2500 g for 10 min were stored at 20 C until LH and FSH were measured. The hypothalamus and pituitary were immediately removed and processed for further assays. The pituitary gland of each rat was homogenized separately in cold 0.9% NaCl (1 ml/10 mg wet weight), centrifuged at 500 g for 15 min at 4 C and the supernatant fraction was used for hormone analysis. Also the body weight gain of the animals of the four groups was recorded.
2.2. Amine measurements Amines were estimated by the flourimetric method of Shellenberger and Gordon (1971). The hypothalamus was dissected out immediately, weighed and homogenized in chilled 0.4 m perchloric acid solution containing 0.1% (w/v) Na2S2O5 and 0.025% Na2EDTA at 4 C in a motor driven glass homogenizer. The homogenates were centrifuged at 4000 g at 4 C for 15 min. The supernatant was taken and mixed with 25 mg alumina. The solution was shaken well, centrifuged at 500 g for 5 min and the supernatant was used for 5-HT estimation. Alumina was further washed four times with 5 ml of H2O, and extracted with 150 ml 0.2 m HClO4 solution. The supernatant was used for the estimation of norepinephrine and dopamine. 2.3. Estimation of 5-HT The fluorescence was developed by reaction with ninhydrin as utilized by Synder et al. (1965). An aliquot (4 ml) was taken and the pH was adjusted to 10 with 0.8 ml 10 m NaOH. It was transferred to a 30-ml stoppered glass tube containing 0.5 ml 0.5 m borate buffer (pH 10), 1.5 ml saturated NaCl solution (approx. 1.5 g) and 15 ml butanol, and vortexed for 10 min. After removing the aqueous phase, the organic phase was then vortexed for 3 min with 2 ml 0.1 m borate buffer (pH 10), previously saturated with NaCl. The butanol layer (10 ml) was transferred to another tube containing 4 ml phosphate buffer (0.05 ng, pH 7) and 15 ml n-heptane. After vortexing for 2 min, 3.6 ml phosphate layer was carefully aspirated into a 15-ml glass tube containing 0.3 ml 0.1 m freshly prepared ninhydrin solution. The tubes were then heated for exactly 30 min at 75 C in a waterbath. One hour later, the solution was transferred to a quartz cuvette and fluorescence was estimated at 385/ 490 nm. 2.4. Estimation of norepinephrine and dopamine A 1-ml aliquot of the perchloric acid eluate was made to pH 6.5 0.2 with 1.5 ml of 0.1 m phosphate buffer– EDTA solution. Iodine reagent (0.2 ml) was added (shaking immediately to mix) kept for exactly 2 min, after which 0.5 ml of the alkaline sodium sulfite solution was added. After 2 min, 0.4 ml of glacial acetic acid was added to bring the pH to 4.4–4.8 and kept in a hot-air oven at 100 C for 3–4 min. Tubes were placed in an icebath for cooling and the NE fluorescence was read at 380/495 nm. The samples were returned to the oven and heated at 100 C for an additional 40 min to develop DA fluorescence. After cooling, the DA fluorescence of the tubes was read at 325/380 nm. The accuracy and precision of the assay were checked by running, in parallel, different concentrations of external and internal
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standards and blanks with tissue samples. The standard curves were linear over the range of concentrations obtained from the tissue samples. 2.5. Hormone measurement Both LH and FSH levels were measured by double antibody radioimmunoassay, using 125I-labeled radioligands. r-LH antiserum (AFP115368) and r-FSH antiserum (AFP19896) were obtained from National Hormone and Pituitary Program (NHPP, MD, USA). Hormones were labeled with 125I (Taramani Institute, Chennai, India) according to the modified procedure of Greenwood et al. (1963). The intra-assay coefficients of variation were 9.5 and 7.3% for LH and FSH, respectively. All samples were measured in the same assay to avoid inter-assay variations. Sensitivities of the assays were 0.065 and 0.08 ng/ml for LH and FSH, respectively. 2.6. Metal analysis Lead and cadmium concentrations were determined in hypothalamus and pituitary samples. The samples were digested in reagent grade nitric acid–perchloric acid (2:1) mixture. The digestion was continued until samples became colorless. Then the acid mixture was evaporated and the precipitate thus obtained was dissolved in a few drops of concentrated HCl. The sample was diluted to 1 ml with distilled water and the readings were taken in GBC 902 double beam atomic absorption spectrophotometer. Sensitivities of the assays were 0.06 and 0.009 mg/ml for lead and cadmium, respectively.
Fig. 1. Serotonin, dopamine and norepinephrine levels in hypothalamus of female rats treated with lead acetate and cadmium acetate alone and in combination for 15 days (0.05 mg/kg body weight). The values are expressed as meanS.E.M. (n=5 in each group). *P40.05 vs control group. **P40.05 vs lead group. #P40.05 vs cadmium group. ## P40.05 vs lead+cadmium group.
treatment groups did not show any significant change in the dopamine level. The animals in the cadmium and combined groups showed a significant decrease in serum LH (P40.05, Fig. 2) and FSH (P40.05, Fig. 3) levels as compared to the control group, whereas the lead-treated group did not show any significant change in the LH and FSH levels as compared to control. The cadmium-exposed animals showed a significant decrease in pituitary LH (P40.05, Fig. 2) and FSH content (P40.05, Fig. 3) as compared to control and lead-exposed groups. The
2.7. Statistical analysis Comparison of values was done by analysis of variance (ANOVA) followed by Student’s t-test. P40.05 were considered statistically significant. All values represent the mean S.E.M.
3. Results Fig. 1 shows the estimated concentration of hypothalamic serotonin, norepinephrine and dopamine in various groups. The serotonin content was decreased in all metal-treated groups with cadmium showing maximum reduction. The combined treatment with lead and cadmium was associated with changes in serotonin relatively similar to those induced by either metal administered alone. The norepinephrine content was decreased in both isolated and combined treatment groups (P40.05). Among the three groups cadmium exposure was showing maximum reduction in NE content. The dopamine content was decreased in the cadmium exposed animals (P40.05), whereas other
Fig. 2. Serum and pituitary levels of LH in female rats treated with lead acetate and cadmium acetate alone and in combination for 15 days (0.05 mg/kg body weight). The values are expressed as meanS.E.M. (n=5 in each group). *P40.05 vs control group. **P40.05 vs lead group. #P40.05 vs cadmium group. ## P40.05 vs lead+cadmium group.
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Fig. 3. Serum and pituitary levels of FSH in female rats treated with lead acetate and cadmium acetate alone and in combination for 15 days (0.05 mg/kg body weight). The values are expressed as meanS.E.M. (n=5 in each group). *P40.05 vs control group. **P40.05 vs lead group. #P40.05 vs cadmium group. ##P40.05 vs lead+cadmium group.
simultaneous treatment with lead and cadmium induced similar changes in the LH and FSH content as with cadmium alone (P40.05). The lead exposure failed to cause any change in both LH and FSH levels. The analysis of metal concentrations showed that the tissues of cadmium and combined metal exposed groups contained a greater concentration of cadmium residues than the tissues of control and lead-exposed groups (Table 1). Similarly in the case of lead and combined metal exposed groups the lead concentration was significantly higher than that in control and cadmium exposed groups (Table 1).
4. Discussion The present study shows the effects of lead and cadmium on the hypothalamic neurotransmitter content as well as serum and pituitary levels of LH and FSH in the
proestrous stage of rats. The decrease in 5-HT content due to the inhibitory effect of cadmium agrees with the results found by Lafuente et al. (2000) where they have shown the effect in different hypothalamic regions. Das et al. (1993) observed a decrease in 5-HT level in the whole hypothalamus after cadmium exposure. Our results indicate that the decrease in serotonin content was greater in the cadmium-exposed group than that in animals receiving cadmium in combination with lead. Norepinephrine plays an important role in modulating luteinizing hormone releasing hormone (LHRH) neurons that are involved in the regulation of gonadotropin secretion by the anterior pituitary. It has been reported that lead can act at the hypothalamic level to alter LHRH secretion in the rat (Bratton, Hiney, & Dees, 1994). Lead blocks the NE-induced release of PGE2 resulting in diminished LHRH secretion. This decrease could be due to the altered Ca2+ mobilisation, a limiting step in PGE2 formation and subsequent LHRH release. In the present study we have observed decrease in NE in both isolated and combined metal treated groups. Dopamine content was not affected by lead or simultaneous exposure with lead and cadmium, whereas cadmium alone was showing significant decrease in the DA content. Various rodent studies on the subchronic exposure of low levels of lead resulted in significant reduction in DA and its metabolites 3,4 dihydroxyphenylacetic acid and homovanillic acid in the nucleus accumbens of rats (Jadhav and Kala, 1994; Kala et al., 1994; Kala and Jadhav, 1995). The discrepancies with other studies could be due to differences in the age of animals, mode of treatment, duration and brain areas analyzed. Lead has been reported to alter calcium homeostasis by affecting both voltage-dependent and receptor-operated calcium channels (Bressler and Goldstein, 1991; Audesirk, 1993; Oortgiesen et al., 1993), whereas the in vitro studies have shown that lead enhances calcium-activated release in brain transmitters (Minnema et al., 1988). Cadmium is shown to inhibit calcium entry and the attendent release of peripheral catecholamines (Hirning et al., 1988). Therefore the
Table 1 Lead and cadmium levels at the hypothalamus and pituitary of female rats exposed to lead acetate and cadmium acetate alone and in combination for 15 days (0.05 mg/kg body weight)a Group
Hypothalamus
Pituitary
Lead
Cadmium
(mg/g) Control Pb Cd Pb+Cd
2.52 0.095 4.1 0.27*** 2.8 0.021# 3.02 0.125**
Lead
Cadmium
(mg/g)
#
0.5250.032 0.5370.062 1.0350.056***## 0.840.021*** # ###
5.05 0.032 9.55 0.68*** 5.7 0.251## 6.25 0.263**
#
*P40.05; **P40.01; ***P40.001 vs control group. #P40.01; ##P40.001 vs lead group. ###P40.05 vs cadmium group. a The values are expressed as meanS.E.M. (n=5 in each group).
0.7450.365 0.82 0.096 1.47 0.102* 1.2820.375
#
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changes observed in the co-treatment group might be due to the fact that lead and cadmium compete with calcium at the channel site and compete with each other for entry through terminal membrane channels (Cooper and Manalis, 1984). The studies on pituitary hormones show that lead and cadmium affect the gonadotropin secretion differently. It is known that serotonin increases both LH and FSH secretion (Wuttke et al., 1978; Ellis et al., 1983). A maximum reduction in both pituitary and serum gonadotropin levels was found in cadmium-treated group. Lead does not produce any change in the gonadotropin levels compared with control. Significant changes observed in the combined treatment group can be due to the inhibitory effects produced by cadmium. The hormonal disturbances following combined treatment with lead and cadmium more often paralleled with the effects of cadmium alone than the changes seen in lead alone, suggesting antagonism between lead and cadmium. Decreased plasma levels of LH and FSH have been reported after administration of higher doses of cadmium to female rats (Paksy et al., 1989; Varga and Paksy, 1991). The decrease in LH and FSH levels after cadmium exposure could also be due to the accumulation of cadmium at the pituitary causing a direct effect on the hypophyseal level since there are some literature available indicating this mechanism (Lorenson et al., 1983; Cooper et al., 1987). Several studies have shown that cadmium can compete with calcium at the pituitary level (Waalkies and Poirier, 1984; Milos et al., 1989), which results in altered calcium regulation. Thus it either interferes with calcium influx through the membrane channel (Poirier et al., 1983; Kasprzak and Poirier, 1985; Cooper et al., 1987) or alters intracellular calcium mobilization. The results of our study agree with this hypothesis as both LH and FSH levels were decreased on cadmium accumulation. However, it is interesting to note that the decrease is observed more in the case of FSH than LH, suggesting additional mechanism for such differential effect. Lafuente et al. (2000) correlated the decease in FSH levels found in postpubertal rats after cadmium exposure to inhibin levels more than to cadmium accumulation at the pituitary. Dopamine is a known inhibitor of gonadotropin secretion and this fact cannot be correlated with the decreased levels of LH and FSH observed in the present study. Since gonadotropin secretion is under the control of multiple regulators it must be the net effect of all these regulators which has resulted in decreased levels of gonadotropins. The present investigation found that combined exposure to lead and cadmium showed less concentrations of both metals in the hypothalamus and pituitary relative to the metal retention in individual metal-treated groups. Nation et al. (1990) found a similar change in blood lead concentration in their studies on the effects of combined exposure of lead and cadmium on behavioral changes.
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The lesser accumulation of metals after combined exposure could be a reason for the observed antagonistic effects. From the above results it is clear that cadmium exerts more disruptions in the hypothalamic– pituitary axis function compared with lead, and the combined treatment effects are not additive.
Acknowledgements We are thankful to NIDDKs National Hormone and Pituitary Program (NHPP, USA) and to Dr. A. Parlow for the gift of LH and FSH. The work was supported by grants from UGC, New Delhi, India.
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