Endocrine Disruptors

Early Cerebral Activities of the Environmental Estrogen Bisphenol A Appear to Act via the Somatostatin Receptor Subtype sst2 Rosa Maria Facciolo,1 Raffaella Alò,1 Maria Madeo,1 Marcello Canonaco,1 and Francesco Dessì-Fulgheri 2 1Comparative Anatomy Laboratory of the Ecology Department, University of Calabria, Cosenza, Italy; 2Animal Biology Department, University of Florence, Florence, Italy

subtypes, designated as sst1–5, have been Recently, considerable interest has been aroused by the specific actions of bisphenol A (BPA). identified as a family of G protein–coupled The present investigation represents a first study dealing with the interaction of BPA with the receptors (11). Of these five receptor sub- biologically more active somatostatin receptor subtype (sst2) in the rat limbic circuit. After treat- types, sst2, which is densely distributed in ing pregnant female Sprague-Dawley rats with two doses (400 µg/kg/day; 40 µg/kg/day) of BPA, the various brain regions, is considered to be, the binding activity of the above receptor subtype was evaluated in some limbic regions of the from a functional point of view, one of the offspring. The higher dose proved to be the more effective one, as demonstrated by the elevated most important cerebral subtypes (12,13). 125 0 affinity of sst2 with its specific radioligand, [ I]-Tyr somatostatin-14. The most dramatic This subtype also promotes neurotransmis- effects of BPA on sst2 levels occurred at the low-affinity states of such a subtype in some telen- sion actions, probably through the involve- cephalic limbic areas of postnatal rats (10 days of age; postnatal day [PND] 10). These included ment of both the high and low receptor- lower (p < 0.05) sst2 levels in the gyrus dentate of the hippocampus and basomedial nucleus of binding states (14), as revealed by the suc- the amygdala; significantly higher (p < 0.01) levels were observed only for the high-affinity states cessful neurobiological functions of sst2, of the periventricular nucleus of the hypothalamus. A similar trend was maintained in PND 23 especially in the presence of E2 (15,16). rats with the exception of much lower levels of the high-affinity sst2 receptor subtype in the Recently, the subtype sst2 has provided a amygdala nucleus and ventromedial hypothalamic nucleus. However, greater changes produced specific analgesic type of behavioral response by this environmental estrogen were reported when the binding activity of sst2 was checked in similar to that noted for another major neu- the presence of the two more important selective agonists (zolpidem and Ro 15-4513) specific for ronal inhibitory system, the hetero- the α-containing γ-aminobutyric acid (GABA) type A receptor complex. In this case, an even oligomeric γ-aminobutyric acid (GABA) greater potentiating effect (p < 0.001) was mainly obtained for the low-affinity sst2 receptor sub- type A receptor (17). This receptor system, type in PND 10 animals, with the exception of the high-affinity type in the ventromedial hypo- which constitutes nearly 60% of neuronal thalamic nucleus and gyrus dentate. These results support the contention that an sst2 subtype synapses, consists of at least seven classes of α–containing GABA type A receptor system might represent an important neuromediating sta- genes that encode for the following subunits: tion capable of promoting estrogenlike mechanisms of BPA, especially during the early develop- α, β, γ, δ, ε, π, ρ. Among this long list of mental phases. Key words: autoradiography, brain, development, GABA, rats, somatostatin subunits, the first, which is involved in the receptors, xenoestrogen. Environ Health Perspect 110(suppl 3):397–402 (2002). assembly of the other sequences and also http://ehpnet1.niehs.nih.gov/docs/2002/suppl-3/397-402facciolo/abstract.html determines the overall biophysical and phar- macological properties of the GABA type A receptor (18), has been chosen for this study. It has been widely accepted that estrogen contact with food products, the residual On the basis of the specific interactions activities are mediated either genomically monomer solvents and/or additives in the between sex steroids and sst2 receptor sub- through binding of specific intracellular polymer may migrate to the nourishing type, as well as the colocalization of estrogen receptors located within target cells or component (3,5). Thus, major attention has receptors and GABA type A neurons (19), through local membrane types of mecha- been directed toward the potentially toxic we investigated whether the binding activity α nisms at the cell surface (1,2). A number of steroidal actions of this environmental estro- of sst2 alone or in the presence of some environmental agents recognized as environ- gen as displayed by some reproductive GABA type A receptor subunit isoforms mental estrogens or simply xenoestrogens malformations of offspring after maternal occurs in an estrogenic fashion in the pres- interact at the estrogen receptor level. As a exposure to BPA (6,7). ence of BPA. These relationships may bring result, public and scientific interests regard- Despite the 1,000-fold less potent activ- us closer to identifying molecular receptor ing estrogenic functions have focused their ity of BPA with respect to that of estradiol activities operating at the brain level after attention on both the toxic and biologically (E2), it is still able to mimic E2 biological exposure to BPA. beneficial actions of some classes of environ- actions such as vaginal cornification (7) and mental chemicals. A member of this class of growth and differentiation of the mammary This article is part of the monograph Impact of environmental estrogen is the industrial phe- gland (8). Consequently, it appears that not Endocrine Disruptors on Brain Development and nolic chemical bisphenol A (BPA). Such a only estrogens but also xenoestrogen deriva- Behavior. xenoestrogen is widely used in the manufac- tives have important and diverse pleiotropic Address correspondence to M. Canonaco, ture of polycarbonate plastics, epoxy resins actions in both reproductive and nonrepro- Comparative Anatomy Laboratory, Ecology Dept., for lining food cans, and dental sealants, and ductive tissues. In this context, the growth University of Calabria, 87036 Rende, Cosenza, Italy. Telephone: 0039 984 492974. Fax: 0039 as a stabilizing agent in plastics such as hormone system, and above all somatostatin 984 492986. E-mail: [email protected] polyvinyl chloride (3,4). These man-made (SRIF) play a crucial role in neurosecretory We are grateful to Synthélabo (Bagneux, France) chemicals, because of their leaching from function (9) at the encephalic level, espe- and Hoffmann-La Roche (Basel, Switzerland) for numerous reservoirs, can enter the body by cially in relation to tissue content (10), having provided zolpidem and Ro 15-4513, ingestion or adsorption and mimic the which is still an unresolved facet of BPA respectively. This study was supported in part by actions of estrogens. Earlier studies revealed effects. SRIF is widely distributed in the the contract grant sponsor COFIN (F.D.F.) and MEMO-BIOMAR (M.C.) Project of MIUR that when derivatives of BPA, such as the mammalian brain and is contained in short (Italy). well-known diglycidyl ether of BPA, com- interneurons plus projecting neuronal path- Received 8 January 2002; accepted 26 February monly used in food packaging, come into ways. To date, five distinct receptor 2002.

Environmental Health Perspectives • VOLUME 110 | SUPPLEMENT 3 | JUNE 2002 397 Endocrine Disruptors • Facciolo et al.

Materials and Methods BPA-treated rats at PND 10 (hBPA, n = 5; A lBPA, n = 6) and PND 23 (hBPA, n = 6; 500 Animals ▲ ▲ ▲ ▲ lBPA, n = 4), along with their respective ● ● ● 400 ▲ ● ● ■ Sexually mature Sprague-Dawley female rats controls (n = 4; n=5), were used. The ■ ■ ■ ▲● ■ (200–250 g) were purchased from Charles autoradiographic analyses of the SRIF sub- ● ■ 300 ▲ ■ River (Como, Italy), caged individually, type sst2 were conducted on posterior lim- ● ■ ▲ housed in the Cellular Biology Department bic brain sections (12 mm) according to 200 ● ■ ▲ (University of Calabria, Cosenza, Italy) stab- previously described methods (23), plus ● ▲ ■ OIL (fmol/mg protein) ▲ ularium, and maintained on a 12-hr modifications. In such a trial it was impor- 100 ● ● 1BPA dark/12-hr light schedule (lights on 14:00– tant that brain tissue was prewashed 3 Specific binding level ■▲ ■ hBPA 0▲●■ 2:00 hr). Animal maintenance and experi- times at room temperature in 50 mM Tris- 0 100 200 300 400 500 mental procedures were in accordance with HCl buffer, pH 7.4, with one of these [125I]-Tyr0-SRIF14 (pM) the Guide for Care and Use of Laboratory washes (30 min) performed in the presence B Animals (20). Efforts were made to minimize of 10–5 M guanosine 5´-triphosphate 3 animal suffering and reduce the number of (GTP) (Sigma Chemical) to allow the dis- ▲ OIL ● ▲ 1BPA specimens used. sociation of endogenous ligand as well as ■ ● hBPA ■ the binding specificity of this class of 2 ▲ -SRIF14 (pM)

Bisphenol A Administration 0 ● G-coupled receptor subtype (sst2) with ■ ▲

● ▲

Thirty-two 60-day-old female rats were sub- respect to those considered the deglycosy- I]-Tyr ■ 1 divided into three experimental groups in lated type (24). Subsequently, slices were 125 ● ▲ ■ ● ▲ which four females were housed per cage incubated for 1 hr at the same temperature ● ▲ ■ ● ▲ ▲ ■ ● ● with stainless steel wire lids for 3 days for and in the same buffer containing 0.5% ■ ■ ■ 0 protein) [ acclimation purposes. Afterward these rats bovine serum albumin and varying concen- Specific binding level (fmol/mg 0 100 200 300 400 500 received either 40 µg/kg/day BPA (lBPA; trations (5–500 pM) of [125I]-Tyr0- Specific binding level (fmol/mg protein) Sigma Chemical, Milan, Italy) po or 400 SRIF14 (81.4 TBq/mmole; New England Figure 1. Ligand-binding assay. (A) Saturation µg/kg/day BPA (hBPA) po dissolved in Nuclear Division, Milan, Italy) ± 1 mM curves of [125I]-Tyr0-SRIF14 (pM) versus specific arachis oil and were compared with controls cold SRIF14 (nonspecific binding). The binding level (fmol/mg protein) ± SEM in posterior that consisted of only arachis oil (OIL)- selection of this radioligand was based on brain regions of rats treated with lBPA (n = 6) and treated rats for 10 days. BPA was given its preferential affinity toward sst receptor hBPA (n = 5) compared with those treated with OIL 2 (n = 5) were obtained by wipe assay procedures orally because this route of exposure is more subtypes (13). After an exposure period of using different concentrations (5–500 pM) of the relevant to human exposure. These two 18 days at room temperature, autoradi- radioligand, as reported in “Materials and doses of BPA were chosen on the basis of ographic films (Hyperfilm; Amersham, Methods.” (B) The linear Scatchard plots of spe- their capability to promote evident morpho- Milan, Italy) of dried sections plus relative cific binding level (fmol/mg protein) versus specific metrical changes in offspring (21,22), as well standards were evaluated with a Zeiss binding levels/[125I]-Tyr0-SRIF14 (fmol/mg as to represent the concentrations that may VIDAS image analyzer (Zeiss, Milan, protein/pM) ± SEM enabled us to calculate the negative slope that provided the mean dissociation be released when resins for lining food cans Italy). Labeled sections were stained with constant, while the intercept of the curve at the and dental sealants come into contact with cresyl violet acetate to identify the different abscissa provided the maximal number of binding food products. During this treatment period, posterior limbic areas. sites. Evaluation of saturation-binding study sup- a sexually mature Sprague-Dawley male was To evaluate interaction of the sst2 recep- plied similar results in five separate tests. introduced into each cage and left there for 5 tor subtypes with certain α GABA type A days. Pregnant females were isolated in sin- receptor subunits, adjacent brain slices were the absence of the selective agonists. The gle cages; at birth, litters were culled to eight, incubated in a fashion similar to that used in choice of hBPA treatment group and the and pups (one per litter) were randomly the saturation study. This time incubation concentration of [125I]-Tyr0-SRIF14 to apply assigned to dams of the same treatment. For was handled, as determined in a previous in the in vitro autoradiographic evaluation the next 23 days, while the dam reared her study (14), in the presence of the best [125I]- were determined by both saturation binding foster pups within the same cage, she contin- Tyr0-SRIF14 affinity state (25 pM) and dif- and wipe assay evaluations (26) of posterior ued to receive the same treatment until the ferent concentrations (5 nM–500 µM) of the brain regions. weaning of the pups at postnatal day (PND) selective agonists (the imidazopyridine zolpi- 23, to assure exposure both via the placenta dem [Synthelabo Recherche, Paris, France] Statistical Analysis and lactation. During this period PND 10 or the imidazobenzodiazepinone Ro 15-4513 Results were reported as means ± SEM for rats from the treatment groups lBPA (n = 6), [Hoffmann-LaRoche, Basel, Switzerland]), or all trials. For the receptor binding study, hBPA (n = 7), and OIL (n = 4) were decapi- a selective agonist of the GABA type A com- Scatchard analysis of saturation binding tated and their brains quickly removed for plex, isoguvacine (ICN Biomedicals, Milan, data, which were fitted by a one-site and/or sst2 receptor autoradiography. Others were Italy). The former two drugs, specific for the two-site model [based on the significance of decapitated at PND 23 (lBPA, n = 7; hBPA, a subunit, are noted for their strong analgesic extra-sum square using LIGAND program n = 7; OIL, n = 5). actions, whereas the latter, which is specific (27)], supplied relative affinity states and for the β subunit, was included as a means of maximal receptor binding densities. A two- Effects of Bisphenol A on Interaction α receptor subunit specificity (25). The levels tailed Student’s t-test was applied for BPA between sst2 and -Containing of sst2 induced by the higher dose of BPA effects on sst2 receptor activity. One-way GABA Type A Receptor with respect to arachis-treated animals in rats analysis of variance (ANOVA) was also used Receptor autoradiography approaches were of both 10 (n = 12) and 23 days of age (n = for a GABA type A-sst2 receptor differences, used to identify the relationship between 13) in the presence of the GABA type A followed where necessary by Newman-Keuls BPA, sst2, and GABA type A receptor selective agonists were expressed as a ratio multiple range test. Significance was checked activity. For this part, the brain of with respect to the same treatment groups in when the p value was less than 0.05.

398 VOLUME 110 | SUPPLEMENT 3 | JUNE 2002 • Environmental Health Perspectives Endocrine Disruptors • Bisphenol A and sst2 receptor subtype in the rat limbic system

A 400 400 ** 350 A OIL 350 A OIL hBPA hBPA iii300 300 ** * 250 250 200 200 ** 150 150 100 100 (fmol/mg protein) (fmol/mg protein) 50 50 Specific binding level Specific binding level 0 0 VMN Pe GD RAD Co-Me Bm VMN Pe GD RAD Co-Me Bm

250 350 B OIL * B OIL 200 hBPA 300 hBPA *** 250 150 * 200 * B * 100 150 iii 100

(fmol/mg protein) 50 (fmol/mg protein) 50 Specific binding level Specific binding level 0 0 VMN Pe GD RAD Co-Me Bm VMN Pe GD RAD Co-Me Bm Figure 3. BPA-dependent binding activity varia- Figure 4. BPA-dependent binding activity varia- tions of the sst2 receptor subtype in PND-10 rats. tions of the sst2 receptor subtype in PND-23 rats. [125I]-Tyr0-SRIF14 binding levels (fmol/mg pro- [125I]-Tyr0-SRIF14 binding (fmol/mg protein; χ– + tein; χ– ± SEM) to the high (A) and low (B) affinity SEM) to the (A) high-affinity and (B) low-affinity state of sst2 receptor subtypes were evaluated state of sst2 receptor subtypes were evaluated in in some limbic areas of PND-10 rats (n = 10) some limbic areas of PND-23 rats (n = 11) treated 4 mm treated with either hBPA or OIL, as described in with either hBPA or OIL, as described in “Materials and Methods.” Mean values in each “Materials and Methods.” Mean values in each Figure 2. Representative binding autoradiograms brain region of the same treatment group were brain region of the same treatment group were of posterior brain regions. Abbreviations: HP, analyzed using a two-tailed Student’s t -test. analyzed using a two-tailed Student’s t-test. *p < hippocampus; VL, ventral lateral posterior thala- *p< 0.05; **p < 0.01. 0.05; **p < 0.01; ***p < 0.001. mic nucleus. (i) A heterogeneous binding activity of the sst2 receptor subtype was obtained in some labeling of this receptor subtype with its registered in the cortico-medial (Co-Me) limbic areas of PND-23 rats when the posterior specific radioligand provided a heteroge- nucleus of the amygdala and RAD, respec- brain regions of (A) OIL-treated and (B) hBPA- neous and uniform type of binding in the tively (Figure 4B). In the high-affinity type, treated animals were incubated in the presence different posterior limbic areas, as shown in significantly diminished levels were found in of 25 pM [125I]-Tyr0-SRIF14, as described in “Materials and Methods.” This binding pattern the representative autoradiograms (Figure 2). Bm and the ventromedial hypothalamic was compared with that of their respective (ii) When the binding parameters of the sst2 nucleus (VMN), whereas higher levels were nonspecific binding autoradiograms. Bar = 4 mm. subtype were identified by Scatchard analy- observed only in the GD (Figure 4A) of the sis, it was possible to observe two different same animals. binding affinities under the influence of Next, the influence of BPA on cerebral ≤ ≥ Results hBPA (high affinity 75 pM; low affinity sst2 binding activity in the presence of the ≤ α Effects of BPA on Interaction 75 500 pM), as reported in previous work selective agonists of the -containing GABA α for another rodent study (14). type A receptor system was examined to between sst2 and -Containing The variations of sst2 binding parameters determine the role of this other major neu- GABA Type A Receptor after treatment with this xenoestrogen ronal system on BPA-dependent effects. This study dealing with early cerebral activi- proved to be primarily of a mixed nature in Displacement activities of [125I]-Tyr0- ties of the environmental estrogen BPA via the telencephalic regions of both PND 10 SRIF14 binding by these two agonists the sst2 receptor subtypes in some limbic and PND 23 rats. In the former animals, (zolpidem and Ro 15-4513) resulted in the areas of the rat displayed a fairly specific and hBPA was responsible for the diminished shifting of the curve to the left, which corre- stable binding activity, as shown by the rep- levels of the low-affinity sst2 receptors (p < sponds to a greater affinity for sst2 receptor resentative saturation curve of posterior lim- 0.05) in the gyrus dentate (GD) of the hip- subtype compared with the other GABA bic regions of rats 23 days of age (Figure 1). pocampus and basomedial nucleus of the type A agonist (Figure 5). Consequently, Additionally, comparison of the two BPA amygdala (Bm) (Figure 3B). A similar varia- incubation of the same brain regions with doses (40 and 400 µg/kg/day) permitted us tion, this time of the enhanced type, was these two selective agonists accounted for to ameliorate the heterogeneous and stable obtained for the stratum radiatum lacuno- even greater potentiating activities of BPA 125 0 binding activity of [ I]-Tyr -SRIF14 to its sum CA1 layer of the hippocampus (RAD). on the two sst2 affinity states, especially the preferred receptor subtype (sst2), especially As far as the high-affinity type of sst2 recep- low-affinity type in postnatal animals. When in the case of the latter dose, which is tors were concerned, BPA significantly the effects of BPA in the presence of the two regarded as the most effective dose capable enhanced (p < 0.01) the levels of this affinity α-containing GABA type A agonists were of disrupting any endocrine function (28). type in the hypothalamus and specifically the compared with those in the absence of the For this purpose, and because of the weaker periventricular nucleus (Pe) (Figure 3A). A two agonists (Figures 6, 7), higher levels of 125 0 affinity of the radioligand [ I]-Tyr - comparable trend was reported for mainly the low-affinity sst2 receptor occurred in the SRIF14 toward sst2 subtype in the presence the same limbic areas of PND 23 animals Pe and GD of PND 10 animals, whereas sig- of lBPA, only hBPA was tested for binding (Figure 4). Diminished (p < 0.05) and even nificantly higher Ro 15-4513–induced levels activities with sst2 in the posterior limbic significantly (p < 0.001) lower levels of the were reported for both the Bm and VMN areas of animals 10 and 23 days of age. The low-affinity type of sst2 receptor were (Figure 6B). In the same biological stage, a

Environmental Health Perspectives • VOLUME 110 | SUPPLEMENT 3 | JUNE 2002 399 Endocrine Disruptors • Facciolo et al.

A 1.0 A 120 * ● Control ■ 100● Isoguvacine ■ 0.5 ● ▲ Zolpidem ▲ ■ ● ■ 80 ▲ ● ■ ● ▲ ■ ● 0 60 ▲ ■ ● ▲ ■ ▲ ● 40 ■

-SRIF14 (% of total binding) –0.5

0 ● ▲ ■ 20 ▲ * ▲ ●

I]-Tyr ■ ▲ ● ▲ ■ 125 ▲ ■● [ 0 –10 –9 –8 –7 –6 –5 –1.0 **

Log [drug] M plus GABA type A agonists hBPA/OIL B without GABA type A agonists hBPA/OIL Zolpidem 120 –1.5 Ro 15-4513 ● Control Ratio = *** ● ■ 100■ Isoguvacine ● ▲ Zolpidem ▲ ■ ● –2.0 ▲ ■ ● 80 ■ ● VMN Pe GD RAD Co-Me Bm ▲ ■ ● 60 ▲ ■ ● 5.0 ▲ ■ B 40 ▲ ● *** Zolpidem ■ 4.5

-SRIF14 (% of total binding) *** 0 ▲ ● ■ Ro 15-4513 20 ▲ *** ▲ ● 4.0 ■ I]-Tyr ▲ ● ▲ ■

125 ▲ ■● 3.5 [ 0 –10 –9 –8 –7 –6 –5 Log [drug] M 3.0 Figure 5. Competition assay. Displacement curves 2.5 of [125I]-Tyr0-SRIF14 (% of total binding) in some 2.0 limbic areas of animals treated with (A) OIL (n = 5) *** *** and (B) hBPA (n = 5) were generated in the pres- 1.5 ence of different concentrations (5 nM–500 µM) ** of selective benzodiazepine (zolpidem) and GABA 1.0 type A (isoguvacine) agonists, as described in hBPA/OIL plus GABA type A agonists hBPA/OIL 0.5 “Materials and Methods.” Each point represents without GABA type A agonists hBPA/OIL the mean of five separate tests. 0

Ratio = –0.5 notable BPA influence was registered, this –1.0 time for the high-affinity type of sst2 recep- VMN Pe GD RAD Co-Me Bm tor in the presence of Ro 15-4513 (Figure 6A). When the effects of GABA type A ago- Figure 6. BPA-dependent changes of sst2 in presence of GABA type A selective agonists in PND-10 rats. Effects of 20 nM of the selective benzodiazepine agonists, the imidazopyridine zolpidem, or the imida- nists were checked in animals 23 days of age, zobenzodiazepinone Ro 15-4513 on [125I]-Tyr0-SRIF14 binding to both (A) high high-affinity and (B) low- variations of low-affinity receptors were sim- affinity sst2 receptor binding states were tested in some limbic areas of PND 10 rats (n = 12) treated with ilar to those of PND 10 animals (Figure 7B), hBPA (400 µg/kg/day) compared with OIL-treated rats, as described in “Materials and Methods.” Values whereas a marked reduction of the changes of sst2 induced by the hBPA dose compared with OIL-treated animals in the presence of the GABA type A in the high-affinity type was observed again, selective agonists were expressed as a ratio to the same treatment groups in the absence of the selec- χ– mostly in the presence of Ro 15-4513 tive agonists. The ratios ( ± SEM) were compared using ANOVA, and differences, where necessary, were obtained by Newman-Keuls multiple range test. *p < 0.05; **p < 0.01; ***p < 0.001. (Figure 7B).

Discussion morpho-functional aspects have led This distinction was made possible by the Investigation of the specific SRIF receptor researchers to consider doses greater than binding conditions adopted in our study, subtype (sst2) under the influence of the envi- 200 µg as a selective developmental toxicant specifically a radioligand exhibiting a strong ronmental estrogen BPA made it feasible to (16,28). At the brain level, the actions of both affinity for the sst2 subtype along with an ele- discern, for the first time, the importance of BPA doses appeared to behave in an estro- vated concentration of GTP. This concentra- this xenoestrogen on cerebral-dependent genic fashion, as demonstrated by the similar tion, higher than that used in the past (22), mechanisms that might be considered critical Scatchard curves of [125I]-Tyr0-SRIF14 bind- allowed us to explore the more extreme satu- β for morphological aspects and neuroen- ing in the presence of 17 -estradiol (16,31). ration ranges of the sst2 receptor subtype. The docrine activities in the developing rat. In this However, it was the high dose that was associ- identification of both high- and low-affinity study, the binding activity of the sst2 subtype ated with the shifting of the curve toward a states as probable targets of xenoestrogen was compared with a somewhat low dose and greater affinity of the sst2 receptor subtype. interactions, as shown in a previous work a sufficiently high BPA dose that accounted Indeed, application of the higher BPA dose (14), was also facilitated by the greater prefer- for, in the case of the latter dose, the alter- was responsible for the evident binding capac- ence of the [125I]-Tyr0-SRIF14 toward the ation of both body weight and reproductive ities of both affinity states of the sst2 subtype deglycosylated form of sst receptors, a condi- tract morphology in rodents (29,30). These in some areas of the posterior limbic regions. tion typical for the sst2 subtype (13,32).

400 VOLUME 110 | SUPPLEMENT 3 | JUNE 2002 • Environmental Health Perspectives Endocrine Disruptors • Bisphenol A and sst2 receptor subtype in the rat limbic system

1.5 A release of SRIF by GABA agonists, with the consequent alteration of the growth hor- mone secretion levels (38) in areas that lack ** 1.0 E2 receptors. Moreover, the prevalence of the low-affinity type of sst2 receptor as a target * of the xenoestrogen, plus the major changes occurring in the PND 10 stage, tend to 0.5 point to either a predominance of this type of affinity state at the early developmental stages responsible for neuronal communicat- 0 ing functions (39) or simply that this repre- sents the preferred targets of BPA in an age-independent manner. hBPA/OIL plus GABA type A agonists hBPA/OIL

hBPA/OIL without GABA type A agonists hBPA/OIL Zolpidem The differences of BPA-induced actions –0.5 on the sst subtype were even greater in the * 2 Ro 15-4513 presence of major agonists zolpidem and Ratio = α Ro 15-4513, which are specific for 1 and α –1.0 4 GABA type A receptor subunits (17), fur- VMN Pe GD RAD Co-Me Bm ther emphasizing the cruciality of such receptor binding conditions for the success of this environmental E2. That GABAergic components are involved in the enhance- 4.0 B *** Zolpidem ment of BPA-dependent sst2 levels, with the 3.5 Ro 15-4513 exception of the VMN and GD, is not sur- 3.0 prising if we consider that these two receptor 2.5 systems are both widely distributed. *** 2.0 Moreover, the two receptor systems are not only colocalized functionally (40,41) but 1.5 ** * also structurally, as revealed by the identifi- 1.0 cation of a novel site for sst2 on GABA type 0.5 A complex (42). However, it is worth noting α 0 that although the two isoforms are impli-

–0.5 cated, in a similar fashion, on steroidal regu- * latory interactions (2,43), it is the latter hBPA/OIL plus GABA type A agonists hBPA/OIL –1.0 * hBPA/OIL without GABA type A agonists hBPA/OIL isoform that exerts greater BPA-induced sst2 –1.5 receptor subtype changes. The lack of a con- *** –2.0 sistent zolpidem-dependent modulatory Ratio = –2.5 action could be due to the GABA type A VMN Pe GD RAD Co-Me Bm receptor complex not being assembled by Figure 7. BPA-dependent changes of sst in presence of GABA type A selective agonists in PND-23 rats. this subunit isoform, as some hypothalamic 2 α Effects of 20 nM of the selective benzodiazepine agonists, the imidazopyridine zolpidem or the imida- stations do not contain the 1 subunit (44). 125 0 α zobenzodiazepinone Ro 15-4513 on [ I]-Tyr -SRIF14 binding to both (A) high-affinity and (B) low-affinity On the other hand, it is specifically the 4 sst2 receptor binding states were tested in some limbic areas of PND-23 rats (n = 13) treated with hBPA isoform that seems to be the preferred target compared with OIL-treated rats, as described in “Materials and Methods.” Values of sst2 induced by the of steroids involved in the restoration of anx- hBPA dose compared with OIL-treated animals in the presence of the GABA type A selective agonists were expressed as a ratio to the same treatment groups in the absence of the selective agonists. The iolytic and analgesic states (45,46). Thus, in ratios (χ– ± SEM) were compared using ANOVA, and differences, where necessary, were obtained by line with these observations, our results seem Newman-Keuls multiple range test. *p < 0.05; **p < 0.01; ***p < 0.001. to further extend the participation of α 4-dependent GABAergic functions. Taken together, these results provide for The evaluation of the two affinity states whereas an inverse trend was provided by the first time direct evidence of BPA activi- of the sst2 subtype in some posterior limbic the VMN, Bm, and Co-Me of PND 23 ani- ties being regulated in a heterogeneous fash- areas of postnatal rats treated with a high mals. Whereas the major effects seemed to ion at the cerebral level through the BPA dose showed that mainly the low-affin- occur in steroid-enriched brain regions, interaction of the sst2 receptor subtype at ity state was the preferential target of this studies have demonstrated that this female the early developmental phases. In most environmental estrogen. In fact, the greater sex steroid does not always require the colo- cases, BPA was responsible for greater changes occurred in hypothalamic, hip- calization of their specific steroids as in the diminished levels of the sst2 receptor sub- pocampal, and amygdalar areas that are not case of the Pe (37), indicating that perhaps type. This accounts for the lower inhibitory only noted for a marked binding density of the estrogenic effects in similar areas are activities of this subtype, with the exception the sst2 receptor subtype (22,33,34) but also accomplished by indirect means such as a of Pe, in which case the higher quantity of for the E2-dependent regulation of this local membrane type of mechanism (2) or sst2 is probably linked to the hypothalamic receptor subtype (35,36), though at different through the sharing of colocalized E2 recep- area being the major site of SRIF mRNA concentrations. This was particularly evident tors on GABAergic terminals of neighboring expression (36). by the higher levels of the sst2 receptor areas (19). This relationship appears to sup- However, the sensitivity of this inter- subtype in the Pe and RAD of PND 10 rats, port the strong E2-dependent modulatory action appears to rely heavily on participation

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of some α GABA type A receptor subunits, Neuroscience 65:551–561 (1995). N, Theveniau M, Nahmias C, Vaysse N, Susini C. The in particular the α isoform. Observations of 13. Rohrer SP, Birzin ET, Mosley RT, Berk SC, Hutchins SM, tyrosine phosphatase SHP-1 associates with the sst2 4 Shen DM, Xiong Y, Hayes EC, Parmar RM, Foor F, et al. somatostatin receptor and is an essential component of the present study could provide further Rapid identification of subtype-selective agonists of the sst2-mediated inhibitory growth signaling. J Biol Chem insights into phenomena such as the accelera- somatostatin receptor through combinatorial chemistry. 272: 24448–24454 (1997). tion of puberty after treatment with this envi- Science 282:737–740 (1998). 33. Martin J-L, Chesselet M-F, Raynor K, Gonzales C, Reisine 14. Facciolo RM, Alò R, Pappaianni F, Madeo M, Carelli A, T. Differential distribution of somatostatin receptor sub- ronmental estrogen (47), as well as contribute Canonaco M. Estrogenic influence on SST2 receptors-α types in rat brain revealed by newly developed somato- to the understanding of the steroidal influ- GABA type A receptor subunit interaction in the hamster statin analogs. Neuroscience 41:581–593 (1991). ence on hypothalamic circadian pacemakers limbic areas during hibernation. In: Perspective in 34. Mathé AA, Nomikos GG, Svensson TH. In vivo release of Comparative Endocrinology (Goos HJT, Rastogi RK, somatostatin from rat hippocampal and striatum. under stress and estrous cycle (48). In the lat- Vaudry H, Pierantoni R, eds). Bologna:Monduzzi Editore, Neurosci Lett 149:201–204 (1993). ter case, BPA could assume a prominent role, 2001;555–564. 35. Slama A, Videau C, Kordon C, Epelbaum J. Estradiol reg- especially because of its more recently recog- 15. Herbison AE. Somatostatin-immunoreactive neurones in ulation of somatostatin receptors in the arcuate nucleus the hypothalamic ventromedial nucleus possess oestro- of the female rat. Neuroendocrinology 56:240–245 (1992). nized feature: the capability of promoting gen receptors in the male and female rat. J 36. Simonian SX, Murray HE, Gillies GE, Herbison AE. gene transcriptional activities necessary for the Neuroendocrinology 6:323–328 (1994). Estrogen-dependent ontogeny of sex differences in synthesis of progesterone receptors in hypo- 16. Visser-Wisselaar HA, Van Uffelen CJC, Van Koetsveld somatostatin neurons of the hypothalamic periventricu- PM, Lichtenauer-Kaligis EGR, Waaijers AM, Uitterlinder lar nucleus. Endocrinology 139:1420–1428 (1998). thalamic neurons of ovariectomized animals P, Mooy DM, Lamberts SW, Hofland LJ. 17-β-Estradiol- 37. Herbison AE, Theodosis DT. Absence of estrogen (49). Indeed it is obvious that we are still at dependent regulation of somatostatin receptor subtype immunoreactivity in somatostatin (SRIF) neurons of the the beginning, but interests concerning the expression in the 7315b prolactin secreting rat pituitary periventricular nucleus but sexually dimorphic colocal- type of models for studying this endocrine tumor in vitro and in vivo. Endocrinology 138:1180–1189 ization of estrogen receptor and SRIF immunoreactivities (1997). in neurons of the bed nucleus of the stria terminalis. disruptor are rapidly emerging. Perhaps the 17. Ács Z, Zsom L, Mergl Z, Makara. Significance of chloride Endocrinology 132:1707–1714 (1993). exploitation of its estrogenic-like activity channel activation in the gamma-aminobutyric acid 38. Gillies G, Davidson K. GABAergic influences on somato- might represent a potential value for the induced growth hormone secretion in the neonatal rat statin secretion from hypothalamic neurons cultured in pituitary. Life Sci 52:1733–1739 (1993). defined medium. Neuroendocrinology 56:248–256 (1992). screening of environmental E2 as agents of 18. Barnard EA, Skolnick P, Olsen, RW, Möhler H, Sieghart 39. Schumacher M, McEwen BS. Steroid and barbiturate congenital neural problems and memory loss W, Braestrup C, Bateson AN, Langer S. International modulation of the receptor GABAA. Mol Neurobiol that are linked to glutamate-induced Union of Pharmacology. XV. Subtypes of γ-aminobutyric 3:275–304 (1989). acidA receptors: classification on the basis of subunit 40. Esclapez M, Houser CR. Somatostatin neurons are a sub- neuronal cell death (50). structure and function. Pharmacol Rev 50:291–310 (1998). population of GABA neurons in the rat dentate gyrus: 19. Flugge G, Oertel WH, Wuttke W. Evidence for estrogen evidence from colocalization of pre-prosomatostatin and REFERENCES AND NOTES receptive GABAergic neurons in the preoptic anterior glutamate decarboxylase messenger RNAs. hypothalamic area of rat brain. Neuroendocrinology Neuroscience 64:339–355 (1995). 43:1–5 (1986). 41. Leresche N, Asprodini E, Emry Z, Cope DW, Crunelli V. 1. Green PS, Bishop J, Simpkins JW. 17α-estradiol exerts 20. European Communities Council Directive. Guide for use Somatostatin inhibits GABAergic transmission in the neuroprotective effects on SK-N-SH cells. J Neurosci 17:511–515 (1997). and care of laboratory animals. Off J Eur Commun sensory thalamus via presynaptic receptors. 2. Canonaco M, Facciolo RM, Alò R. Neuroactive steroid L358:1–29 (1986). Neuroscience 98:513–522 (2000). mechanisms and GABA type A receptor subunit assem- 21. Vom Saal FS, Cooke PS, Buchanan DL, Palanza P, Thayer 42. Vincens M, Mauvais-Jarvis F, Behar S. A novel recogni- bly in hypothalamic and extrahypothalamic regions. Int KA, Nagel SC, Parmigiani S, Welshons WV. A physiologi- tion site for somatostatin-14 on the GABAA receptor Cytol Rev 214:69–112 (2002). cally based approach to the study of bisphenol A and complex. Eur J Pharmacol 344:R1–R2 (1998) 3. Biedermann M, Grob K, Bronz M, Curcio R, Huber M, other estrogenic chemicals on the size of reproductive 43. Gulinello M, Gong QH, Li X, Smith SS. Short-term expo- α Lopez-Fabal F. Bisphenol A-diglycidyl ester (BADGE) in organs, daily sperm production, and behavior. Toxicol sure to a neuroactive steroid increases 4 GABAA edible-oil-containing canned foods: determination by LC- Ind Health 14:239–260 (1998). receptor subunit levels in association with increased LC-fluorescence detection. Mitt Geb Lebensmittelunters 22. Canonaco M, Facciolo RM, Alò R, Madeo M, Franzoni anxiety in the female rat. Brain Res 910:55–66 (2001). Hyg 87:547–558 (1998). MF. Unpublished data. 44. Davies AM, McCarthy MM. Developmental increase in 3 γ 4. Olea N, Pulgar R, Perez P, Olea-Serrano F, Rivas A, 23. Leroux P, Weissmann D, Pujol J-F, Vaudry H. H-muscimol binding to the -aminobutyric acidA recep- Novillo-Fertrell A, Pedraza V, Soto AM, Sonnenschein C. Quantitative autoradiography of somatostatin receptors tor in hypothalamic and limbic areas of the rat: why is Estrogenicity of resin-based composites and sealants in the rat limbic system. J Comp Neurol 331:389–401 the ventromedial nucleus of the hypothalamus an excep- used in dentistry. Environ Health Perspect 104:298–305 (1993). tion? Neurosci Lett 288:223–227 (2000). (1996). 24. Hervieu G, Emson PC. Visualisation of somatostatin 45. Smith SS, Gong QH, Li X, Moran MH, Bitran D, Frye CA, α α 5. Korach KS. Surprising places of estrogenic activity receptor sst3 in the rat central nervous system. Mol Hsu F-C. Withdrawal from 3 -OH-5 -pregnan-20-one [Editorial]. Endocrinology 132:2277–2278 (1993). Brain Res 71:290–303 (1999). using a pseudopregnancy model alters the kinetics of 6. Ramos JG, Varayoud J, Sonnenschein C, Soto AM, 25. Siegel E, Buhr A. The benzodiazepine binding site of hippocampal GABAA-gated current and increases α Munoz De Toro M, Luque EH. Prenatal exposure to low receptors Trends Pharmacol Sci 18:425–429 (1997). GABAA receptor 4 subunit in association with doses of bisphenol A alters the periductal stroma and 26. Canonaco M, O’Connor LH, Pfaff DW, McEwen BS. increased anxiety. J Neurosci 18:5275–5284 (1998). glandular cell function in the rat ventral prostate. Biol GABAA receptor level changes in female hamster fore- 46. Follesa P, Cagetti E, Mancuso L, Biggio F, Manca A, Reprod 65:1271–1277 (2001). brain following in vivo estrogen progesterone and benzo- Maciocco E, Massa F, Desole MS, Carta M, Busonero F, 7. Gupta C. Reproductive malformation of the male off- diazepine treatment: a quantitative autoradiography et al. Increase in expression of the GABAA receptor ?4 spring following maternal exposure to estrogenic chemi- analysis. Exp Brain Res 75:644–652 (1989). subunit gene induced by withdrawal of, but not by long- cals. Proc Soc Exp Biol 224:61–68 (2000). 27. Munson PJ, Rodbard D. LIGAND: a versatile computerize term treatment with, benzodiazepine full or partial ago- 8. Colerangle JB, Roy D. Profound effects of the weak envi- approach for characterization of ligand-binding systems. nists. Brain Res Mol Brain Res 92:138–148 (2001). ronmental estrogen-like chemical bisphenol A on the Anal Biochem 107:220–239 (1980). 47. Howdeshell KL, Hotchkiss AK, Thayer KA, Vandenbergh growth of the mammary gland of Noble rats. J Steroid 28. Laws SC, Carey SA, Ferrell JM, Bodman GJ, Cooper RL. JG, vom Saal FS. Exposure to bisphenol A advances Biochem Mol Biol 60:153–160 (1997). Estrogenic activity of octylphenol, nonylphenol, bisphe- puberty. Nature 401:763–764 (1999). 9. Tannenbaum GS, Ling N. The interrelationship of growth nol A and methoxychlor in rats. Toxicol Sci 54:154–167 48. Arancibia S, Estupina C, Pesco J, Belmar J, Tapia- hormone (GH)-releasing factor and somatostatin in gen- (2000). Arancibia L. Responsiveness to depolarization of hypo- eration of the ultradian rhythm of GH secretion. 29. Steinmetz R, Brown NG, Allen DL, Bigsby RM, Ben- thalamic neurons secreting somatostatin under stress Endocrinology 115:1952–1957 (1984). Jonathan N. The environmental estrogen bisphenol A and estrous cycle conditions: involvement of GABAergic 10. Quintela M, Senaris R, Heiman ML, Casanueva FF, stimulates prolactin release in vitro and in vivo. and steroidal interactions. J Neurosci Res 50:575–584 Dieguez C. Leptin inhibits in vitro hypothalamic somato- Endocrinology 138:1780–1786 (1997). (1997). statin secretion and somatostatin mRNA levels. 30. Papaconstantinou AD, Umbreit TH, Fisher BR, Goering 49. Funabashi T, Kawaguchi M, Kimura F. The endocrine dis- Endocrinology 138:5641–5644 (1997). PL, Lappas NT, Brown KM. Bisphenol A-induced ruptors butyl benzyl phthalate and bisphenol A increase 11. Burns C, Weckbecker G, Raulf F, Kaupmann K, increases in uterine weight and alterations in uterine the expression of progesterone receptor messenger Schoeffter P, Hoyer D, Lubbert H. Molecular pharmacol- morphology in ovariectomized B6C3F1 mice: role of the ribonucleic acid in the preoptic area of adult ovariec- ogy of somatostatin receptor subtypes. Ann NY Acad Sci estrogen receptor. Toxicol Sci 56:332–339 (2000). tomized rats. Neuroendocrinology 74:77–81 (2001). 733:138–146 (1994). 31. Kimura N, Hayafuji C, Kimura N. Characterization of 17-β- 50. Behl C, Widman M, Trapp T, Holsboer F. 17β-estradiol 12. Beaudet A, Greenspun D, Raelson J, Tannenbaum GS. estradiol–dependent and –independent somatostatin protects neurons from oxidative stress-induced cell Patterns of expression of SSTR1 and SSTR2 somato- receptor subtypes in rat anterior pituitary. J Biol Chem death in vitro. Biochem Biophy Res Commun 216:473–482 statin receptor subtype in the hypothalamus of the adult 264:7033–7044 (1989). (1995). rat: relationship to neuroendocrine functions. 32. Lopez F, Esteve JP, Buscail L, Delesque N, Saint-Laurent

402 VOLUME 110 | SUPPLEMENT 3 | JUNE 2002 • Environmental Health Perspectives