Age-dependent effects of Aroclor 1254R on calcium uptake by subcellular organelles in selected brain regions of rats

Toxicology 156 (2000) 13–25

13

Age-dependent effects of Aroclor 1254R on calcium uptakeby subcellular organelles in selected brain regions of rats�

Rashmi Sharma a,b, Ethel C. Derr-Yellin b, Dennis E. House c,Prasada Rao S. Kodavanti b,*

a National Research Council, National Academy of Sciences, Washington, DC, USAb Cellular and Molecular Toxicology Branch, Neurotoxicology Di6ision, MD74B,

National Health and En6ironmental Effects Research Laboratory, US En6ironmental Protection Agency, Research Triangle Park,NC 27711, USA

c Biostatistics and Research Support Di6ision, National Health and En6ironmental Effects Research Laboratory,US En6ironmental Protection Agency, Research Triangle Park, NC 27711, USA

Received 4 May 2000; accepted 21 August 2000

Abstract

Earlier reports from our laboratory have indicated that polychlorinated biphenyls (PCBs) affect signal transductionmechanisms in brain, including Ca2+ homeostasis, phosphoinositol hydrolysis, and protein kinase C (PKC)translocation in mature neurons and adult brain homogenate preparations. Present studies were designed toinvestigate whether there were any brain region-, gender-, or age-dependent effects of PCBs on 45Ca2+-uptake by twosubcellular organelles, microsomes and mitochondria. We have studied in vitro effects of a widely studied commercialPCB mixture, Aroclor 1254R, on 45Ca2+-uptake by microsomes and mitochondria in cerebellum, frontal cortex andhippocampus of postnatal day (PND) 7, 21, and 90–120 (adult) male and female Long–Evans (LE)-rats. In general,microsomal and mitochondrial 45Ca2+-uptake in selected brain regions increased with age; PND 7BPND 215adults. Among three brain regions, hippocampus had relatively lower microsomal 45Ca2+-uptake than cerebellum andfrontal cortex throughout the development. Mitochondrial 45Ca2+-uptake was comparable in three brain regions ofPND 7 and adult animals, but in PND 21 rats, the cerebellum had much higher activity than frontal cortex andhippocampus. No gender-related differences were seen in 45Ca2+-uptake by either microsomes or mitochondria in

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Abbre6iations: ANOVA, analysis of variance; DMSO, dimethyl sulfoxide; EGTA, ethylene glycol-bis(b-aminoethylether)N,N,N %,N %-tetraacetic acid; ER, endoplasmic reticulum; GABA, g-aminobutyric acid; GD, gestational day; HEPES, (N [2-hy-droxyethyl]piperazine-N %[2-ethanesulfonic acid]); IC50, concentration that inhibits control activity by 50%; IP, inositol phosphate;LE, Long–Evans; LSD, least significant difference; PCBs, polychlorinated biphenyls; PKC, protein kinase C; PND, postnatal day.� The research described in this article has been reviewed by the National Health and Environmental Effects Research

Laboratory, US Environmental Protection Agency, and approved for publication. The approval of this work does not necessarilyreflect upon the views and policies of the Agency. Mentioned trade names or commercial products have neither been endorsed norrecommended for use.

* Corresponding author. Tel.: +1-919-5417584; fax: +1-919-5414849.E-mail address: [email protected] (P.R.S. Kodavanti).

0300-483X/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.

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R. Sharma et al. / Toxicology 156 (2000) 13–2514

selected brain regions throughout development. Inhibition of 45Ca2+-uptake by Aroclor 1254 in a concentration-de-pendent manner was observed throughout the study. However, the degree of inhibition of microsomal 45Ca2+-uptakein these brain regions by Aroclor 1254 increased with age, PND 7BPND 215adults (IC50s=21–34, 8–20 and10–14 mM, respectively). Brain region-specific differential sensitivity to Aroclor 1254 on the inhibition of microsomal45Ca2+-uptake was not seen in PND 7 and adult animals but in PND 21 rats, hippocampus was more sensitive thanthe other selected brain regions. There were no age-, gender- or brain region-specific differential effects of Aroclor1254 on mitochondrial 45Ca2+-uptake. These results indicate that a commercial PCB mixture, Aroclor 1254, inhibited45Ca2+-uptake by both microsomes and mitochondria uniformly in selected brain regions of males and femalesduring development. However, the inhibition of microsomal 45Ca2+-uptake by Aroclor 1254 increased with age. Theage- and gender-related differential sensitivity to Aroclor 1254 may be attributed to the changes in calciumhomeostasis in various brain regions during development. © 2156 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Aroclor 1254R; Brain regions; Calcium uptake; Development; Gender; Microsomes; Mitochondria; Polychlorinatedbiphenyls; Neurotoxicity

1. Introduction

Polychlorinated biphenyls (PCBs) are the mem-bers of halogenated hydrocarbon class of environ-mental chemicals introduced more than 70 yearsago for use in the electrical industry, specificallyfor capacitors, cables and transformers. PCBs,also classified as ‘persistent bioaccumulative toxi-cants’ (Vallack et al., 1998; Fisher, 1999) weresold commercially as mixtures under the tradenames of Aroclor, Chlophen, Kanechlor andFenchlor with different weight percentage of chlo-rine contents (Erickson, 1986). These chemicalsare not biodegradable and in spite of the ban oftheir use, they are still a significant threat tohuman health (Kodavanti and Tilson, 1997;Tilson and Kodavanti, 1997). Two major acci-dents of PCB poisoning ‘Yusho’ (Kuratsune et al.,1972) and ‘YuCheng’ (Rogan et al., 1988) andseveral epidemiological studies of environmen-tally-induced PCB contamination in humans(Michigan cohort, North Carolina cohort, andDutch cohort) have been reported (Jacobson etal., 1990; Rogan and Gladen, 1992; Winnekee etal., 1998; Patandin et al., 1999).

Due to various degrees of chlorination on theten positions of biphenyl rings, 209 congeners ofPCBs are possible, out of which, 135 congenershave been detected in environmental samples.Among ortho-, meta- and para-substituted con-geners, the ortho-substituted non-coplanar PCBsconstitute the major portion of total PCB residuesin environmental samples (World Health Organi-

zation, 1993) and reported to have the highestpotency in nervous system preparations (Koda-vanti and Tilson, 1997; Tilson and Kodavanti,1997). In addition, both experimental and epi-demiological evidences suggest that PCBs causedevelopmental neurotoxicity (Seegal, 1996). Inutero exposure to PCBs has been known to causedelayed neuronal development, cognitive deficitsand motor dysfunction in both the animals andhumans (Kuratsune et al., 1972; Tilson et al.,1979, 1990; Pantaleoni et al., 1988; Schantz et al.,1989; Jacobson et al., 1990; Ahlborg et al., 1992;Rogan and Gladen, 1992; Rogan et al., 1988;Winnekee et al., 1998; Patandin et al., 1999).

The mechanism by which PCBs cause thesedevelopmental neurotoxic effects is still not com-pletely understood. Also, there is little informa-tion whether PCB effects are age-, gender-, orbrain region-dependent. Earlier reports from ourlaboratory have shown that non-coplanar PCBcongeners and three commercial mixtures at lowmicromolar concentrations have a significant ef-fect on intracellular second messenger systemsincluding Ca2+-homeostasis, enzymes involved inATP synthesis, inositol phosphate hydrolysis, andprotein kinase C (PKC) activation/translocationin mature neurons and homogenate preparationsof adult brain (Kodavanti et al., 1993a, 1994,1996; Maier et al., 1994; Shafer et al., 1996;Mundy et al., 1999). These second messengers notonly have a significant role in the development ofthe nervous system (Kater and Mills, 1991), butalso play a major role in several neurophysiologi-

R. Sharma et al. / Toxicology 156 (2000) 13–25 15

cal functions such as long-term potentiation, andlearning and memory (Gibson and Peterson, 1987;Vander Zee and Douma, 1997). It has been pro-posed that perturbations in second messenger sys-tems including Ca2+ homeostasis might be themode of action for PCB effects on the nervoussystem (Kodavanti and Tilson, 1997; Tilson andKodavanti, 1997). Calcium is an important com-ponent of the second messenger system and playsa significant role as a universal messenger ofextracellular signals in a variety of cells (Car-valho, 1982; McGraw et al., 1982; Rasmussen,1986a,b). Several cellular mechanisms, which reg-ulate calcium homeostasis play a crucial role inneurophysiological functions and the developmentof brain. The expression of plasma membranecalcium-pumping ATPases has been known tovary with age in different brain regions of devel-oping and adult brains (Paul and Neve, 1992;Singh, 1999) and differential expression of theisoforms of Ca2+-ATPases may reflect cellulardifferences in calcium handling properties (Zac-charias et al., 1997).

In light of the above information that PCBs aredevelopmental neurotoxicants and second messen-gers may be targets for PCBs, present studies werefocused on whether Ca2+ uptake by microsomesand mitochondria was age-, brain region- andgender-dependent and also if the effects of PCBson the calcium uptake process depended on thesefactors. We have studied the in vitro effects of awidely studied PCB mixture, Aroclor 1254, onCa2+-uptake by microsomes (endoplasmic reticu-lum, ER) and mitochondria in cerebellum, frontalcortex and hippocampus of postnatal day (PND)7, 21, and adult (PND 90–120) male and femaleLong–Evans (LE)-rats. Aroclor 1254 is a com-mercial mixture and contains \99% ortho-substi-tuted PCBs by weight (Frame et al., 1996;Kodavanti et al., 1998). In addition, the referencedoses (RFDs) derived from animal studies withAroclor 1254 and other commercial mixtures havebeen used in the assessments of risks to humans(Cogliano, 1998). Results from this study indi-cated that the rate of Ca2+-uptake by microsomesand mitochondria varied with age in three brainregions. Some gender-related differential effectswere also found on microsomal 45Ca2+-uptake in

PND 21 rats. Aroclor 1254 inhibited 45Ca2+-up-take in both microsomes and mitochondria; how-ever, only microsomal 45Ca2+-uptake wasinhibited differentially by Aroclor 1254 as a func-tion of age.

2. Materials and methods

2.1. Animals

Timed pregnant dams (gestational day (GD 13)and adult male and female (90–120 days old,300–400 g) LE rats were obtained from CharlesRiver Laboratories (Portage, OR and Raleigh,NC, respectively). Pregnant dams were housedindividually and adult rats housed two per cage inAmerican Association for Accreditation of Labo-ratory Animal Care (AAALAC)-approved animalfacilities. Food (NIH diet no. 31) and water wereprovided ad libitum. Temperature was maintainedat 2192°C and relative humidity at 50910%with a 12-h light/dark cycle. Beginning on GD 22,rats were checked twice daily (a.m. and p.m.) forbirths, and the date that birth was first discoveredwas assigned PND 0. The litter size varied be-tween 10 and 14 pups and left alone untilsacrifice.

2.2. Chemicals

Aroclor 1254R (purity, \99%) was purchasedfrom Ultra Scientific (LotcNTO1022; NorthKingstown, RI). 45CaCl2 (34.12 mCi/mg; purity,\99%) was purchased from Dupont New Eng-land Nuclear (Boston, MA). All other chemicalsused in the study were obtained from well-knowncommercial sources. Aroclor 1254 was dissolvedin dimethyl sulfoxide (DMSO) and various stockconcentrations of 0.5, 1.5, 5, 15, and 25 mM wereprepared. A 3-ml aliquot of these stock solutionswas added to a 1.5-ml incubation buffer toachieve the final concentrations of 1, 3, 10, 30 and50 mM in Ca2+-uptake studies and the samevolume of DMSO used in controls. DMSO at theconcentrations used did not affect either microso-mal or mitochondrial Ca2+-uptake.


2.3. Isolation of microsomes and mitochondria

Animals from three age-groups and both gen-ders were sacrificed on different days of experi-ment by decapitation. The cerebella, frontalcortices, and hippocampi were quickly isolated onice. To obtain sufficient amount of tissues, brainregions were pooled from 15 males or 15 femalesin PND 7 group, six males or six females in PND21 group, and five males or five females in thePND 90–120 (adults) group. There was no crossover of the pups from each litter as the subcellularfractionation for each age was done on threedifferent weeks to achieve an n of 3. The brainregions were homogenized in 20-ml cold homoge-nizing buffer containing 250 mM sucrose, 5 mMHEPES, 5 mM KCl, 10 mM dithiothreitol, 1 mMMgCl2; pH 7.05.The isolation of microsomes andmitochondria was done by the method describedby Gray and Whittaker (1962) and Dodd et al.(1981) with slight modifications (Kodavanti et al.,1993a,b). The homogenate was centrifuged at1000×g at 4°C for 10 min to remove cell debrisand nuclei, and the resulting supernatant cen-trifuged at 9000×g at 4°C for 20 min. The super-natant was further centrifuged at 105 000×g at4°C for 1 h to obtain microsomes and suspendedin homogenizing buffer. The pellet obtained aftercentrifugation at 9000×g was suspended in 0.32M sucrose (10 ml) and layered over 1.2 M sucrose(10 ml). After high-speed centrifugation(105 000×g for 30 min; Beckman model L8-70,rotor Ti 70 type), the resulting mitochondrialpellet was resuspended in the homogenizingbuffer. Protein contents in microsomal and mito-chondrial fractions were determined by Lowry’smethod (Lowry et al., 1951). Freshly isolatedmicrosomes and mitochondria were used forCa2+-uptake studies.

2.4. Measurement of 45Ca2+-uptake

With slight modifications (Kodavanti et al.,1993a,b), uptake of 45Ca was measured by themethod described earlier by Moore et al. (1975).Briefly, the assay mixture (1.5 ml) contained 30mM histidine–imidazole buffer (pH 6.8), 100 mMKCl, 5 mM MgCl2, with or without 5 mM

sodium azide (for microsomes or mitochondria,respectively), 5 mM ammonium oxalate, microso-mal/mitochondrial protein (80–120 mg), and 0.1mCi 45CaCl2 containing 5 mM free Ca2+ in cal-cium-ethylene glycol-bis (b-aminoethyl ether)N,N,N %N %-tetraacetic acid (Ca2+-EGTA) bufferedmedium. Various concentrations of Aroclor 1254(0–50 mM in DMSO; final concentration) alongwith protein were preincubated at 37°C for 5 min.The reaction was initiated by the addition of ATP(5 mM final concentration) and carried on for 20min at 37°C in shaking water bath. The reactionwas terminated by the addition of 5 ml 10 mMTris–HCl buffer (pH 7.4) and the samples filteredthrough 0.45 mm Millipore® filters. The filterswere then collected in scintillation vials containing10 ml of Ultima Gold scintillation fluid and theamount of radioactivity measured by liquid scin-tillation spectroscopy. Non-specific 45Ca2+-up-take in the absence of ATP was subtracted fromthe total binding to get the specific 45Ca2+-uptakeand calculated as pmol/mg protein per minute.

2.5. Statistical analysis

Multi-factors analysis of variance (ANOVA) ina split–split-plot design was used with three repli-cations. Split–split–split plot analysis was doneusing five factors such as age, gender, brain re-gion, fraction and dose with three replications ineach experiment. Three brain regions (subplots)were further split into the two fractions (sub-sub-plots) which are further split into six samples(sub-sub-subplots) for dose–response curve of theAroclor 1254. ANOVA was used with only thefirst order interactions of these effects, i.e. interac-tion of only two factors at one time. In addition,the slopes of dose–response curves for three age-groups in each fraction of the brain region andeach gender were compared. To determine theIC50 values, the regression line was fitted to thelinear portion of the dose–response curves usingGraphPad INSTAT software. Also, the averageIC50 values were used for comparisons amongfour factors including age, gender, brain region,and fraction in a split–split-plot design. In addi-tion, the rate of calcium uptake was also com-


pared using factors such as age, brain region,gender and fraction. Comparisons between threeage groups in each brain region of male andfemale rats were made using Tukey’s multiplecomparison (SAS, 1990).

3. Results

3.1. Basal le6els of microsomal calcium uptake

3.1.1. Brain-region specific differencesIn PND 7 rats, Tukey’s multiple comparison

showed that the microsomal 45Ca2+-uptake wascomparable in cerebellum and frontal cortex butthe levels were significantly lower in hippocam-pus. However, the uptake differed significantlyamong three brain regions in PND 21 animals;the highest being in cerebellum and lowest inhippocampus. Further, the uptake in cerebellumand frontal cortex of adults were not differentfrom each other, but were significantly greaterthan hippocampus (Fig. 1).

3.1.2. Age- and gender-related differencesTukey’s multiple comparison showed that the

microsomal 45Ca2+-uptake in cerebellum washighest in PND 21 and lowest in PND 7 rats; infrontal cortex, highest in adult animals and lowestin PND 7 animals. In hippocampus, the uptakewas clearly age-dependent (adults\PND 21\PND 7). There were no gender-related differencesin calcium uptake by microsomes isolated fromthe cerebella and hippocampi in all the age-groups, except in PND21 animals where the mi-crosomal 45Ca2+-uptake in frontal cortex wasslightly lower in females compared with the males(Fig. 1). Overall, results indicate that microsomalcalcium uptake in three brain regions was age-de-pendent (PND 7BPND 215adults).

3.2. Basal le6els of mitochondrial calcium uptake

3.2.1. Brain-region specific differencesIn PND 7 rats, 45Ca2+-uptake by mitochondria

was comparable in cerebellum and frontal cortexbut relatively low in hippocampus. Tukey’s multi-ple comparison showed that in PND 21 animals,

the mitochondrial 45Ca2+-uptake was significantlyhigher in cerebellum compared with frontal cortexand hippocampus. In adult animals, no significant

Fig. 1. Age-dependent changes in the basal 45Ca2+-uptake bymicrosomes isolated from cerebellum (A), frontal cortex (B),and hippocampus (C) of male () and female (a) rats. The45Ca2+-uptake was measured as described in material andmethods and expressed as pmols 45Ca2+ per mg protein permin. Values represent mean9S.E. obtained from three sepa-rate experiments, assayed in triplicate samples.


Fig. 2. Age-dependent changes in the basal 45Ca2+-uptake bymitochondria isolated from cerebellum (A), frontal cortex (B),and hippocampus (C) of male () and female (a) rats. The45Ca2+-uptake was measured as described in material andmethods and expressed as pmols 45Ca2+ per mg protein permin. Values represent mean9S.E. obtained from three sepa-rate experiments, assayed in triplicate samples.

chondrial 45Ca2+-uptake in cerebellum was signifi-cantly higher than frontal cortex andhippocampus. Overall, PND 21 animals hadhigher mitochondrial calcium uptake than PND 7and adult animals. There were no gender-relateddifferences in calcium uptake by mitochondriaisolated from the selected brain regions in any ofthe age-groups (Fig. 2).

Taken together, microsomal and mitochondrial45Ca2+-uptake in PND 7 rats were similar inrespective brain regions, but at PND 21 and adultage, the rate of microsomal 45Ca2+-uptake washigher than mitochondrial 45Ca2+-uptake in allthe selected brain regions.

3.3. Inhibition of microsomal 45Ca2+-uptake byAroclor 1254

3.3.1. Age-related inhibitory effectIn general, Aroclor 1254 inhibited microsomal

45Ca2+-uptake to a lesser extent in PND 7 animalsthan the adult animals (Fig. 3, Table 1). Tukey’smultiple comparison showed that the inhibition ofcerebellar microsomal 45Ca2+-uptake in PND 7and PND 21 animals was not significantly differ-ent, but they were significantly less sensitive thanadult animals. In both frontal cortex andhippocampus, the inhibition of microsomal45Ca2+-uptake in PND 21 and adult animals wasnot significantly different, but significantly moresensitive than that of PND 7 animals (Table 1).

3.3.2. Brain region-specific and gender-related in-hibitory effect

In PND 7 and adult animals, the inhibition ofmicrosomal 45Ca2+-uptake by Aroclor 1254 wassimilar in cerebellum, frontal cortex, andhippocampus. However, there were brain-regionspecific effects in PND 21 animals; the inhibitionof microsomal 45Ca2+-uptake was greater inhippocampus than cerebellum. Based on IC50 val-ues, there were no gender-related effects of Aro-clor 1254 on microsomal 45Ca2+-uptake incerebellum and hippocampus in all age groups(Table 1). However, inhibition of microsomal45Ca2+-uptake by Aroclor 1254 in frontal cortexof PND 21 females was 2-fold more sensitive thanmales (Table 1).

differences were seen among three brain regions(Fig. 2).

3.2.2. Age and gender-related differencesTukey’s multiple comparison showed that mito-


Fig. 3. Concentration–response curves representing the in vitro effect of Aroclor 1254 on 45Ca2+ uptake by microsomes isolatedfrom male cerebellum (A), male frontal cortex (B), male hippocampus (C), female cerebellum (D), female frontal cortex (E), andfemale hippocampus (F) of PND 7 (), PND 21 (�), and adult (PND 90–120; �) rats. 45Ca2+-uptake was measured as describedin Section 2. Data were converted to percent of control. Values represent mean9S.E. obtained from three separate experiments,assayed in triplicate samples.


Table 1IC50 values (mM) of Aroclor 1254 representing the inhibition of 45Ca2+-uptake by microsomes and mitochondria of cerebellum,frontal cortex and hippocampus of LE ratsa

MaleBrain region Female

PND 21 PND 90–120 PND 7 PND 21PND 7 PND 90–120

MicrosomesCerebellum 17.3691.5422.4993.30 12.0192.58 23.6392.80 19.7091.32 12.0794.11

18.3293.45 12.0193.94 29.1792.5524.3295.09 8.9591.42Frontal cortex 13.9193.639.0990.64 10.8592.95 21.2790.68 8.2791.05Hippocampus 10.9693.2233.6492.12

Mitochondria18.7491.56Cerebellum 19.1493.1511.9093.44 20.1194.61 16.4691.01 13.1493.2416.3192.19 19.5597.43 19.8093.24 25.5092.38 13.2493.39Frontal cortex 17.0891.0315.2993.16 12.6594.99 17.3691.25 18.5590.2318.2291.79 10.9693.17Hippocampus

a Values represent mean9S.E. obtained from three separate dose–response curves obtained from three separate experimentsassayed in triplicate samples. IC50, concentration that inhibits control activity by 50%.

3.4. Inhibition of mitochondrial 45Ca2+-uptake byAroclor 1254

3.4.1. Age-related, brain region-specific, and gen-der-related inhibitory effect

Aroclor 1254 inhibited mitochondrial 45Ca2+-uptake to a similar extent in all three age groupsof male and female rats in selected three brainregions. Aroclor 1254 also inhibited mitochon-drial 45Ca2+-uptake uniformly in three brain re-gions in both male and female rats (Fig. 4; Table1). Although IC50 value for mitochondrial 45Ca2+

-uptake in frontal cortex of female PND 21 ratswas 1.5-fold higher than the corresponding males,these differences were not statistically significant(Table 1).

4. Discussion

Calcium is an important component of thesecond messenger system, plays a significant roleas a universal messenger of extracellular signals,and regulates diverse neuronal processes includingdevelopment and maturation, gene expression,synaptic plasticity, and long-term memory process(Morris et al., 1988; Malenka et al., 1989; Ginty,1997). Several cellular mechanisms, which regu-late the Ca2+-homeostasis, play a crucial role inthe neurophysiological functions and the develop-

ment of brain. Altered Ca2+-homeostasis hasbeen proposed to be a fundamental mediator ofage-related changes in the nervous system (Gibsonand Peterson, 1987; Khachaturian, 1994). One ofthe mechanisms by which neurons maintain nor-mal calcium homeostasis is by the effective opera-tion of intracellular Ca2+-pumps located insubcellular organelles, namely ER and mitochon-dria. In ER, Ca2+-uptake is an active high-affinity process involving ATP hydrolysis byCa2+-ATPase, whereas mitochondrial Ca2+-up-take is a low-affinity electrophoretic uniport pro-cess driven by the potential difference establishedacross the mitochondrial inner membrane by aproton pump energized by ATP hydrolysis (Borle,1981; Berridge, 1987; Carafoli, 1987).

Present studies showed a significant age-relateddifferences in microsomal Ca2+-uptake in threeselected brain regions and some gender-relateddifferential expression in frontal cortex of PND21 rats. Although the mechanisms were different,Ca2+-uptake by microsomes and mitochondria inbrain regions of PND 7 pups did not differ fromeach other significantly. However, differences be-tween these two Ca2+-uptake processes becameevident in three brain regions of PND 21 andadult animals suggesting that during development,the role of ER might have become prominent inCa2+ homeostasis, which is in agreement withprevious reports (Hanahisa and Yamaguchi,


1998). Among three brain regions, the cerebellumhad highest microsomal and mitochondrial cal-cium uptake in PND 21 and adult rats, which is

also in agreement with previously published re-ports (Verma et al., 1990). Under physiologicalconditions, mitochondria are known to store

Fig. 4. Concentration–response curves representing the in vitro effect of Aroclor 1254 on 45Ca2+ uptake by mitochondria isolatedfrom male cerebellum (A), male frontal cortex (B), male hippocampus (C), female cerebellum (D), female frontal cortex (E), andfemale hippocampus (F) of PND 7 (), PND 21 (�), and adult (PND 90–120; �) rats. 45Ca2+ uptake was measured as describedin Section 2. Data were converted to percent of control. Values represent mean9S.E. obtained from three separate experiments,assayed in triplicate samples.


more calcium than ER; however, ER is moreefficient in sequestering cytosolic calcium thanmitochondria (Berridge, 1987; Carafoli, 1987), asalso seen in our studies and the contribution ofmitochondrial Ca2+ uptake in calcium homeosta-sis seemed to be less significant during aging.

During perturbation of Ca2+ homeostasis, cy-tosolic [Ca2+]i levels may increase within the celleither through increased Ca2+ influx (Carafoli,1987) or by Ca2+-release from intracellular stores(Berridge, 1987). Increased [Ca2+]i may activateseveral intracellular Ca2+-dependent processes(Drapeau and Blaustein, 1983; Carafoli, 1987;Kostyuk and Verkhratsky, 1994). Perturbations inCa2+-homeostasis have been proposed duringneurotoxicity by a number of chemicals (Koda-vanti et al., 1993b). PCBs are known to increaseintracellular free Ca2+ levels in a number of cellsystems (Kodavanti et al., 1993a; Carpenter et al.,1997; Voie and Fonnum, 1998), mainly by amechanism that requires extracellular calcium(Mundy et al., 1999) and inhibition of microsomaland mitochondrial Ca2+ buffering (Kodavanti etal., 1993a, 1996). We have reported previouslythat a widely studied commercial PCB mixture,Aroclor 1254R, inhibited Ca2+-uptake by micro-somes and mitochondria in cerebellum of adultrats at physiological concentrations with IC50 val-ues of 6–9 mM (Kodavanti et al., 1996). SuchPCB concentrations have been detected in severalsamples from human breast milk (3.5–36 mM)(Greizerstein et al., 1999), human blood (7–12mM) (Bush et al., 1985), human brain (\6 mM,Dewailly et al., 1995), and rat brain (40–50 mM)(Shain et al., 1986; Kodavanti et al., 1998). Theresults of the present study confirm these findingsand the IC50 values ranged from 10 to 13 mM incerebellum of adult rats (Table 1). In addition,current results showed that the inhibition of mi-crosomal and mitochondrial Ca2+-uptake byAroclor 1254 was similar in cerebellum, frontalcortex, and hippocampus of PND 7 and adult ratsindicating that the sensitivity of intracellularCa2+ buffering is similar among the selectedbrain regions in these age groups. However,hippocampus was more sensitive than the otherselected brain regions in PND 21 rats. The micro-somal Ca2+-uptake in frontal cortex of PND 21

males was about 2-fold less sensitive than thecorresponding females. Interestingly, current re-sults indicate an age-related and gender-relateddifferential inhibition of microsomal Ca2+-uptakeby Aroclor 1254 in selected brain regions. Mito-chondrial Ca2+ uptake was also inhibited by Aro-clor 1254 in a dose-dependent manner, however,no differential inhibition was observed. In addi-tion, results showed that the inhibition of micro-somal 45Ca2+-uptake was less than mitochondrial45Ca2+-uptake in cerebellum, frontal cortex, andhippocampus of PND 7 rats suggesting highersensitivity of low affinity calcium uptake systemto Aroclor 1254 exposure during early develop-ment. The Ca2+ buffering mechanisms regulatedby microsomes and mitochondria were not inhib-ited by Aroclor 1254 in a gender-specific manner.

It is likely that age-dependent effects of Aroclor1254 on microsomal 45Ca2+-uptake could be dueto differential expression of Ca2+-ATPase in dif-ferent brain regions during development. Somerecent studies have also shown that high-affinityCa2+-extrusion system provided by the isoformsof Ca2+-ATPases may play an essential role inthe dynamic processing of short- and long-termchanges in intracellular Ca2+. Distinct differencesin the amount and/or isoform expression patternmay critically influence the susceptibility of agroup of neurons toward Ca2+-mediated excito-toxicity (Zaccharias et al., 1997). The differentialsensitivity of specific isoform may be an impor-tant factor in brain-region and age-related differ-ential sensitivity to PCBs. Significant differencesin microsomal and mitochondrial calcium uptakein cerebellum and frontal cortex at PND 21 agealso indicate the role of hormones in regulation ofcalcium homeostasis and the expression of Ca2+-ATPase in the brain of LE-rats, since the involve-ment of calcitonin and pituitary functions oncalcium homeostasis has been known (Borle,1981).

In conclusion, current results indicate that (a)the basal 45Ca2+-uptake in microsomes and mito-chondria was age-dependent and brain-region spe-cific, but not gender-specific; (b) Aroclor 1254inhibited the 45Ca2+-uptake by both microsomesand mitochondria at low micromolar concentra-tions, and the inhibition was age-dependent only


in microsomes, suggesting that microsomal Ca2+

buffering was more sensitive during development;(c) brain region-specific differential sensitivity toAroclor 1254 on the inhibition of microsomal45Ca2+-uptake was not seen in PND 7 and adultanimals but in PND 21 rats, hippocampus wasmore sensitive than the other selected brain re-gions; (d) there were some gender-related differ-ences in the inhibition of intracellularCa2+-buffering by Aroclor 1254. The age-relateddifferential sensitivity may be attributed to differ-ential expression of ER Ca2+-ATPase(s) in vari-ous brain regions during development and theirsensitivity to Aroclor 1254.

Acknowledgements

The authors thank Dr Joe A. Elder, Dr WilliamR. Mundy, and Dr Hugh A. Tilson for theirvaluable comments on the earlier version of thismanuscript. Thanks to Tiffany Redmond (sum-mer trainee) for technical assistance. RashmiSharma is a recipient of NRC Senior ResearchAssociate award.

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Age-dependent effects of Aroclor 1254R on calcium uptake by subcellular organelles in selected brain regions of rats