INORGANIC COMPOUNDS

Anil Pillai Æ P.N. Laxmi Priya Æ Sarita Gupta

Effects of combined exposure to lead and cadmiumon pituitary membrane of female rats

Received: 27 June 2002 /Accepted: 12 July 2002 / Published online: 24 September 2002� Springer-Verlag 2002

Abstract The effects of combined exposure to lead andcadmium on pituitary membrane were studied. Adultfemale rats were treated intraperitoneally with eitherlead acetate and cadmium acetate alone or in combi-nation at a dose of 0.05 mg/kg daily for 15 days. Bothmetals accumulated in the pituitary after the exposure.The membrane fluidity was decreased after the heavy-metal treatment. Among the three groups cadmiumtreatment showed more effect than did other treatmentsand the combined treatment showed intermediate val-ues. Na+K+ATPase activity was decreased significantlyby cadmium and combined treatments. The Schiff’s baseand inorganic peroxide levels were increased after themetal exposure. In conclusion, exposure to lead andcadmium caused accumulation of the metals in the pi-tuitary and lowered the membrane fluidity, which mayaffect membrane function and cause alterations in re-ceptor binding and secretory mechanism(s) of pituitaryhormones. The combined treatment with metals pro-duced intermediate results.

Keywords Cadmium Æ Fluidity Æ Lead ÆNa+K+ATPase

Introduction

Lead and cadmium are major environmental contami-nants that have been shown to cause disruptions in theendocrine system. These metals can affect the activity ofhypothalamus–pituitary–ovary axes by acting at thehypothalamus (Das et al. 1993; Antonio et al. 1998), thepituitary (Lorenson et al. 1983; Ronis et al. 1998;Lafuente et al. 1999), the ovary (Paksy et al. 1990) and/

or the accessory organs (Klinefelter and Hess 1998).Different studies have shown that both these metalsmodify plasma levels of luteinizing hormone (LH) andfollicle stimulating hormone (FSH) (Zylber-Haran et al.1982; Lorenson et al. 1983; Paksy et al. 1989; Lafuenteet al. 1997, 1999); however the exact mechanism fordecreased gonadotropin levels is not fully understood.There are reports that both lead and cadmium cangenerate free radicals (Hassoun and Stohs 1996; Sarkaret al. 1998; Daggett et al. 1998; Adonaylo and Oteiza1999). Toxic effects of free radicals on membranestructure and function have been reported, such aschanges in permeability, activity of enzymes, channels,transport proteins, membrane fluidity and receptors(Amoruso et al. 1987; Nivsarkar et al. 1998; Thevenodand Friedmann 1999; Jarrar and Mahamoud 2000).However, there are no reports on the toxic effects of leadand cadmium on pituitary membrane.

The pituitary gland plays an important role in theregulation of reproduction. Pituitary function is regu-lated by three intermating elements – hypothalamicneurosecretions (the so-called releasing factors or hypo-physiotropic hormones), feedback effects of circulatinghormones, and paracrine and autocrine secretions of thepituitary itself. Hypothalamic gonadotropin regulatinghormone (GnRH) action is initiated by binding to spe-cific cell surface receptors in the pituitary, leading toincreased synthesis and release of gonadotropins fromthe pituitary which in turn regulate gonadal function.Thus, pituitary membrane integrity is very important fornormal endocrine functioning.

Pituitary membrane interaction with toxic metalsmay be linked to a variety of toxicological effects. Mostresearch on these two metals has dealt with each metal inisolation. On the other hand, populations in real lifealways have simultaneous multiple exposures, indicatingthe need for experimental work with combinations ofsubstances. Therefore, the purpose of the present studywas to examine the potential alterations in pituitarymembrane by the simultaneous exposure to lead andcadmium.

Arch Toxicol (2002) 76: 671–675DOI 10.1007/s00204-002-0399-6

A. Pillai Æ P.N. Laxmi Priya Æ S. Gupta (&)Department of Biochemistry,Faculty of Science, M.S. University of Baroda,Vadodara, Gujarat, 390 002, IndiaE-mail: saritagupta9@yahoo.comTel.: +91-265-795594

Materials and methods

Animals and treatment

Adult virgin female rats of the Charles-Foster strain weighing 180–220 g, kept under controlled conditions of light (lights on from0700 to 2000) and temperature (24±2 �C), and having access tofood and water, were used. Their ovarian cycles were checked dailyby vaginal cytology. Animals displaying at least three 4-day cycleswere selected for the experiment. There were four groups of animalsin the study. Group 1 animals were given sodium acetate as control,group 2 lead acetate, group 3 cadmium acetate and group 4 re-ceived lead acetate and cadmium acetate in combination. The metalsolutions were prepared in distilled water. The animals were treatedintraperitoneally with a dose of 0.05 mg/kg body weight per dayfor 15 days. The combined-treated group was also exposed to thesame dose by taking half concentrations (0.025 mg/kg body weight)of each metal. The dose was selected on the basis of our earlierreports on minimum inhibition of d-ALAD, which is the marker oflead toxicity (Gupta et al. 1994), and also on hepatic estradiolmetabolism (Pillai et al. 2002). In the course of the study, theanimals were monitored continuously for their health status.

Tissue preparation

Animals of all groups were killed after 15 days of metal exposure inthe pro-estrous stage (checked by vaginal cytology), and the pitu-itaries quickly removed. Pituitary membrane was prepared ac-cording to the modified method of Poirier et al. (1974). Tissue wasweighed, homogenized in 0.25 M sucrose buffer, and centrifuged at600 g for 5 min at 4 �C. The pellet obtained was re-homogenized in0.25 M sucrose buffer and re-centrifuged at 600 g for 5 min at4 �C. The pooled supernatant was centrifuged at 10,000 g for30 min at 4 �C, the pellet collected and dissolved in 10 mmol/l Trisbuffer, pH 7.4, centrifuged at 10,000 g for 30 min at 4 �C and thepellet was finally dissolved in Tris buffer.

Measurement of membrane fluidity

Fluorescence polarization studies were performed as indicated byShinitzky and Barenholz (1978). A suspension of pituitary mem-brane was incubated for 60 min with 0.6 lmol/l 1,6-diphenyl-1,3,5-hexatriene (DPH) in 0.32 M sucrose buffer containing 10 mmol/lTris HCl, pH 7.4. The excitation and emission wavelengths were360 and 430 nm, with bandwidths 5 nm and 10 nm, respectively.Polarization values for parallel (vertical) and perpendicular (hori-zontal) were taken with a Shimadzu RF-540 spectrofluorometer.The polarization value (P) was calculated from the equation

P ¼ IVV �GIVHIVV þGIVH

where IVV and IVH are vertical and horizontal components ofemitted light, respectively, when emitted with vertically polarizedlight, and G is the correction factor for the emission monochro-mator.

Na+K+ATPase assay

ATPase was measured by the release of inorganic phosphorus fromATP (Floreani et al. 1981). The Pi was assayed according to Fiskeand Subbarow (1925). The incubation mixture contained 50 mmol/l Tris HCl (pH 7.4), 100 mmol/l NaCl, 20 mmol/l KCl, 3 mmol/lMgCl2, 3 mmol/l ATP, and 20–100 lg membrane protein, in a finalvolume of 1 ml. The assay was initiated with ATP. Enzyme incu-bation lasted for 20 min at 37 �C in a water bath, and the reactionwas stopped by the addition of cold trichloro-acetic acid to 10% (v/v). To determine the basal Mg2+ATPase activity, we added0.2 mmol/l ouabain to the incubation medium; the Na+K+AT-

Pase activity was calculated as the difference between total ATPaseand Mg2+ATPase activity.

Lipid peroxidation

We measured the formation of Schiff’s base (R1–N=CH–CH=CH–NH–R2) to assess the lipid peroxidation (Tappel 1975).Lipids were extracted from pituitary membrane samples withchloroform–methanol (2:1 v/v), dried and re-dissolved in chloro-form. Fluorescence emission was determined at 420 nm (excitationat 360 nm) on a spectrofluorometer.

Inorganic peroxides

We measured pituitary membrane samples for inorganic peroxidesusing a method similar to that of Bernt and Bergmeyer (1965) andMiettini (1985). This procedure measures the oxidation of o-dian-isidine after treatment of samples with peroxidase to liberate mo-lecular oxygen. The optical density (436 nm) of the samples wascompared with that of H2O2 as standard.

Metal analysis

Lead and cadmium concentrations were determined in the pituitarysamples. The samples were digested in reagent-grade nitric–per-chloric acid (2:1) mixture. The digestion was continued till samplesbecame colorless, then the acid mixture was evaporated and theprecipitate dissolved in a few drops of concentrated HCl and fur-ther diluted to 1 ml with distilled water. Readings were taken in aGBC 902 double-beam atomic absorption spectrophotometer. Theminimum detection limit was 0.06 lg/ml and 0.009 lg/ml for leadand cadmium, respectively.

Statistical analysis

Statistical analysis of results was performed by ANOVA followedby Students t-test. The level of significance was P £ 0.05 for eachanalysis.

Results

There were no significant differences in body weight intreated animals compared with control animals. Bothmetals were accumulated in the pituitary in amountssignificantly higher than in the control group after ex-posure (P £ 0.05; Table 1). The fluidity study shows anincrease in the fluorescence polarization ratio indicativeof a decrease in pituitary membrane fluidity, with theprobe DPH in the metal-exposed groups (P £ 0.005;

Table 1 Lead and cadmium levels in the pituitary of female ratsexposed to lead acetate and cadmium acetate alone and in com-bination for 15 days (0.05 mg/kg body weight per day). Values areexpressed as mean ± SEM (n=5 in each group)

Group Lead (lg/g) Cadmium (lg/g)

Control 5.05±0.032 0.745±0.365Lead 9.55±0.68*** 0.82±0.096Cadmium 5.7±0.251# 1.47±0.102*##

Lead + cadmium 6.25±0.263**## 1.282±0.375

*P £ 0.05, **P £ 0.01, ***P £ 0.001, compared with controlgroup; #P £ 0.001, ##P £ 0.01, compared with lead group

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Table 2). The results show that all the treated groupshad polarization values (i.e., decreased fluidity) signifi-cantly higher than that of controls (Table 2). Among thethree groups, cadmium treatment showed more effectthan did other treatments, and the combined treatmentshowed intermediate values. The formation of Schiff’sbase was also measured in this study to assess lipidperoxidation. The fluorescence intensity increased ap-proximately 25%, 23% and 19% in the cadmium, lead+ cadmium, and lead groups, respectively (P £ 0.01;Table 2) compared with controls. The metal treatmentcaused an increase in inorganic peroxide level in thepituitary membrane. Cadmium exposure resulted in amaximum increase in the inorganic peroxide level com-pared with lead and lead + cadmium (P £ 0.001;Fig. 1). The Na+K+ATPase activity was decreasedsignificantly in the cadmium (P £ 0.001; Fig. 2) andcombined-metal treated groups (P £ 0.05; Fig. 2) com-pared with the control, i.e., among the three groupscadmium treatment showed a more inhibitory effectthan did other treatments.

Discussion

To our knowledge, this is the first report showing theeffects of both lead and cadmium on pituitary membrane

integrity. The data reported here demonstrate that theplasma membrane of the pituitary is very sensitive toexposure to lead and cadmium. It is known that bio-logical membranes, besides their function as selectiveboundaries, play a fundamental role in the regulation ofmany biochemical processes. Modulation of membraneactivity may depend on membrane physical properties,in particular fluidity (Chautan et al. 1990). The results ofthe present study show that both lead and cadmium,individually and in combination, cause changes in thebiophysical properties of the pituitary membrane, i.e.,membrane fluidity is decreased by these metals.

Numerous factors have been shown to alter mem-brane fluidity in various cell systems. Hannan et al.(1989) suggested that changes in membrane fluidity byheavy metals could result from oxidation of doublebonds of the membrane fatty acids. The double bonds inthe cis configuration bring about the formation of kinksin the acyl chains and prevent them from packingtightly. But oxidation of the double bond leads to tighterpacking. Lipid peroxides and other phospholipid ca-tabolites disrupt membrane bi-layer organization(Sevanian et al. 1988), promote non-bi-layer organiza-tion (Thompson et al. 1987) and affect the packing ofphospholipids, thus increasing membrane viscosity(Sevanian et al. 1988). This indicates that an increase inlipid peroxide level due to metal exposure causes ap-pearance of Schiff’s products followed by decrease in theplasma membrane fluidity. The increased levels ofSchiff’s products and inorganic peroxides found after thetreatment with metals in our study agrees with the re-sults observed by others (Ribarov et al. 1983; Berndt andAnsari 1990).

Gwozdzinski (1991) showed that mercury and copperincreased the rigidity of the RBC membrane and at-tributed this change to the conformational changes inthe membrane proteins brought about by these metallic

Table 2 Schiff’s base and fluidity values in pituitary membranesamples of female rats exposed to lead acetate and cadmium acetatealone and in combination for 15 days (0.05 mg/kg body weight perday). Values are expressed as mean ± SEM (n=5 in each group)

Group Schiff’s base(fluorescence intensity)

Polarization

Control 29.95±1.98 0.88±0.007Lead 38.85±1.57* 0.120±0.003**Cadmium 40.17±1.73* 0.138±0.008**Lead + cadmium 36.78±0.80* 0.126±0.006**

*P £ 0.01; **P £ 0.005, compared with control group

Fig. 1 Inorganic peroxide levels in pituitary membrane samples offemale rats exposed to lead acetate and cadmium acetate alone andin combination for 15 days (0.05 mg/kg body weight per day).Values are expressed as mean ± SEM (n=5 in each group).*P £ 0.01; **P £ 0.001 compared with control group

Fig. 2 Effect of lead acetate and cadmium acetate alone and incombination on pituitary Na+K+ATPase activity of female rats(0.05 mg/kg body weight per day for 15 days). Values are expressedas mean ± SEM (n=5 in each group). *P £ 0.05, **P £ 0.001compared with control group; #P £ 0.001 compared with leadgroup, ##P £ 0.01 compared with cadmium group

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ions. It is known that most membrane-bound enzymesrequire membrane lipids to be in a ‘‘fluid’’ state foroptimum activity. Changes in membrane fluidity as aresult of lipid peroxidation can affect membrane pro-teins, since permeability and function of membrane-bound protein is known to be intimately associated withthe dynamic state of the membrane lipids (Kaplan et al.1995). The correlation between lipid peroxidation, flu-idity and membrane function has been documented inseveral studies (Dinis et al. 1993; Kaplan et al. 1995).The present study also demonstrate such correlationbetween lipid peroxidation and membrane fluidity, to-gether with significant reduction in the activity of theimportant membrane-bound enzyme Na+K+ATPaseby metal exposure. The relationship between membranelipids and enzyme activity was explored for ATPases(Moller et al. 1982; Mishra et al. 1989) and adenylatecyclase (Moller et al. 1982; Schachter 1984). Variousother studies have shown the inhibition of Na+K+

ATPase enzyme by lead and cadmium (Thevenod andFriedmann 1999; Jarrar and Mahamoud 2000) andother metals (Rohn et al. 1996; Stanimirovic et al. 1995)due to oxidative stress and increased lipid peroxidation.

We carried out an ‘‘in-vitro’’ study to understandwhether the inhibitory effect observed in the presentstudy is a direct or secondary effect (through changes inmembrane lipid composition) of the metals. The pitu-itary membrane samples were incubated with the sameconcentration of metals as their concentration in thepituitary after the ‘‘in-vivo’’ exposure. The resultsshowed that Na+K+ATPase activity was affected by themetal exposure, whereas other parameters remainedunchanged (data not included). Also the percentage ofinhibition in the ‘‘in-vitro’’ study was less than that inthe ‘‘in-vivo’’ experiment, suggesting both primary andsecondary effects of metal in ‘‘in-vivo’’ treated groups ofanimals.

In the present investigation, combined exposure tolead and cadmium showed intermediate results in vari-ous parameters studied. In most of the studies reportingon combined exposure to metals, researchers have usedthe same concentrations of the metal both in individualand combined treatment (Nation et al. 1990; Zikic et al.1998). The results obtained from such studies showedeither additive effects in the combined exposure groupas the concentration of the metals are increased, orantagonistic effects depending on the nature of themetals used, whereas in the present study, the totalconcentration of metals in the combined-exposuregroup is the same as that in the individual-metaltreatment group. This pattern avoids multiple stress inthe combined-treatment group. It is interesting to notethat the changes following combined treatment withlead and cadmium in the present study more oftenparalleled the effects of cadmium alone than thechanges seen with lead alone. This suggests that whenlead and cadmium are present together in similar con-centrations, cadmium mediates major effects due to itsmore reactive nature.

These results demonstrate that both lead and cad-mium, individually and in combination, have a pro-nounced effect on pituitary plasma membrane. It seemsplausible to speculate that the marked decrease inmembrane fluidity by metals in this investigation couldaccount, at least in part, for the neuroendocrine changesby these metals reported earlier by several workers.

Acknowledgements This work was supported by a grant fromUGC, New Delhi, India (F.12-42/98, SR-1).

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