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Changes in the function of the inhibitory neurotransmitter system in the rat brain following subchronic inhalation exposure to 1-bromopropane
NeuroToxicology 28 (2007) 415–420
Changes in the function of the inhibitory neurotransmitter system in the rat brain following subchronic inhalation exposure to 1-bromopropane Susumu Ueno a,*, Yasuhiro Yoshida b, Yukiko Fueta c, Toru Ishidao c, Jiqin Liu b, Naoki Kunugita d, Nobuyuki Yanagihara a, Hajime Hori c a
Department of Pharmacology, School of Medicine, University of Occupational and Environmental Health, Japan Department of Immunology, School of Medicine, University of Occupational and Environmental Health, Japan c Department of Environmental Management I, School of Health Sciences, University of Occupational and Environmental Health, Japan d Department of Health Information Science, School of Health Sciences, University of Occupational and Environmental Health, Japan b
Received 18 October 2005; accepted 10 March 2006 Available online 24 March 2006
Abstract 1-Bromopropane (1-BP) has been widely used as a cleaning agent and a solvent in industries, but the central neurotoxicity of 1-BP remains to be clarified. In the present study, we investigated the effects of subchronic inhalation exposure to 1-BP vapor on the function of the inhibitory neurotransmitter system mediated by g-aminobutyric acid (GABA) in the rat brain. Male Wistar rats were exposed to 1-BP vapor for 12 weeks (6 h/ day, 5 days/week) at a concentration of 400 ppm, and, in order to investigate the expression and function of brain GABA type A (GABAA) receptors, total/messenger RNA was prepared from the neocortex, hippocampus, and cerebellum of the control and 1-BP-exposed rats. Moreover, hippocampal slices were prepared, and the population spike (PS) amplitude and the slope of the field excitatory postsynaptic potential (fEPSP) were investigated in the paired-pulse configuration of the extracellular recording technique. Using the Xenopus oocyte expression system, we compared GABA concentration–response curves obtained from oocytes injected with brain subregional mRNAs of control and 1-BP exposed rats, and observed no significant differences in apparent GABA affinity. On the other hand, paired-pulse inhibition of PS amplitude was significantly decreased in the hippocampal dentate gyrus (DG) by exposure to 1-BP, without any effect on the paired-pulse ratio of the fEPSP slopes, suggesting neuronal disinhibition in the DG. Moreover, RT-PCR analysis indicated decreased levels of GABAA receptor b3 and d subunit mRNAs in the hippocampus of 1-BP-exposed rats. These results demonstrate that subchronic inhalation exposure to 1-BP vapor reduces the function of the hippocampal GABAergic system, which could be due to changes in the expression and function of GABAA receptors, especially the d subunitcontaining GABAA receptors. # 2006 Elsevier Inc. All rights reserved. Keywords: 1-Bromopropane; GABAA receptor; Xenopus oocytes; d Subunit; Disinhibition
1. Introduction Since chlorofluorocarbons have been identified as ozonedepleting solvents, several new compounds have been introduced as alternatives in the workplace. 1-Bromopropane (1-BP) is one of the most important substitutions used in Japan and the United States as a cleaning agent for metals, electronics, ceramics, etc. However, recent studies have demonstrated that
* Corresponding author at: Department of Pharmacology, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishiku, Kitakyushu, Fukuoka 807-8555, Japan. Tel.: +81 93 691 7424; fax: +81 93 601 6264. E-mail address: [email protected] (S. Ueno). 0161-813X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2006.03.006
exposure to 1-BP induces not only reproductive toxicity (Ichihara et al., 2000; Yamada et al., 2003) but also neurotoxicity in the peripheral nervous system (Yu et al., 1998; Zao et al., 1999). The number of workers at risk for exposure to 1-BP vapor is increasing, so that the toxicities of this alternative compound to other organs and/or tissues need to be clarified. The central nervous system (CNS) neurotoxicity caused by inhalation exposure to 1-BP vapor is a topic of great interest, and it has recently been demonstrated that inhalation exposure to 1-BP causes neuronal dysfunction in the rat hippocampus, that is, disinhibition (Fueta et al., 2000, 2002a,b, 2004). In their studies, such disinhibition was observed in the hippocampal CA1 region and the dentate
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gyrus (DG) after repeated exposure to 1-BP vapor at 1500 or 700 ppm, which appeared at the end of the 1st week (1500 ppm) or the 4th week (700 ppm) before convulsion occured. These observations suggest that, even before behavioral abnormalities become apparent, subchronic exposure to 1-BP could alter neuronal excitability in the CNS. Accordingly, it is urgently necessary to elucidate the mechanism of neuronal disinhibition induced by 1-BP exposure. In the CNS, neuronal excitability is regulated by ion channels, including voltage-gated and transmitter-gated ion channels. Among the latter, the g-aminobutyric acid (GABA) type A (GABAA) receptor/chloride ion channel complex is the major inhibitory neurotransmitter receptor in the mammalian brain. Seven classes of GABAA receptor subunits (a1–6, b1–3, g1–3, d, e, p, u) have been cloned to date (Barnard et al., 1998; Whiting et al., 1999), and the GABAergic system, together with the glutamatergic system, has been demonstrated to be critical for overall control of hippocampal excitability and seizures. Moreover, several lines of evidence have suggested that alterations in the expression and function of GABAA receptors in the hippocampus are associated with epilepsy (Coulter, 2001). Therefore, disinhibition in the hippocampus caused by 1-BP exposure could be associated with alterations in the function of the GABAergic system, especially GABAA receptormediated inhibition. In the present study, we describe the effects of subchronic inhalation exposure to 1-BP vapor at 400 ppm, a lower concentration than that tested in previous reports (Fueta et al., 2000, 2002a), primarily on the function of the inhibitory GABAergic system in the rat brain. We assessed the function of brain regional GABAA receptors using a Xenopus oocyte expression system with two-electrode voltage-clamp recordings and hippocampal excitability using slice preparations with extracellular recording techniques. We also examined the expression of GABAA receptor subunit mRNAs in the hippocampus using RT-PCR analysis. 2. Materials and methods 2.1. Animals and inhalation exposure to 1-BP vapor Male Wistar rats (8 weeks of age) were purchased from Kyudo Co. Ltd. (Kumamoto, Japan). Breeding conditions of rats and the inhalation system apparatus were described previously (Hori et al., 1999; Ishidao et al., 2002). The rats were placed in an inhalation chamber containing 1-BP vapor at a concentration (exposure group) or only fresh air (control group). This treatment was performed 6 h a day (mainly from 9 a.m. to 3 p.m.), 5 days a week for 12 weeks. The experiments were carried out under the control of the Ethics Committee of Animal Care and Experimentation in accordance with The Guiding Principle for Animal Care Experimentation, the University of Occupational and Environmental Health, Japan and the Japanese Law for Animal Welfare and Care (No. 221).
2.2. Messenger RNA preparation and expression of brain receptors in Xenopus oocytes After 12 weeks of exposure to 1-BP vapor, rats were deeply anesthetized with diethyl ether and then decapitated, and the neocortex, hippocampus, and cerebellum were dissected. Total RNA was isolated using TRIzol (Life Technologies, Rockville, MD), and mRNA was prepared by using the PolyATtract1 mRNA Isolation System (Promega, Madison, MI) according to the manufacturer’s protocol. The isolation of Stage V and VI oocytes from adult female Xenopus laevis, the injection of mRNA (50 ng per oocyte) into oocytes, and two-electrode voltage-clamp recordings were performed as described previously (Beckstead et al., 2000; Ueno et al., 2004). Either GABA or kainate was applied for 30– 60 s, in increasing concentrations (as listed in Fig. 1) to each oocyte, allowing 5–15 min between applications. 2.3. Hippocampal slice preparation and electrophysiological recording The procedures for hippocampal slice preparation and measurements of the population spike (PS) amplitudes and the slope of the field excitatory postsynaptic potential (fEPSP) from the DG were described previously in detail (Fueta et al., 2004). To analyze paired-pulse responses, calculation of the paired-pulse ratio (PPR) was performed as follows: PPR of fEPSP ¼
2nd fEPSP slope ; 1st fEPSP slope
PPR of PS ¼
2nd PS 1st PS
The conventional measurements of the slope of the fEPSP and the PS amplitude in the paired-pulse configuration are also shown in Fig. 2. 2.4. RT-PCR analysis Total RNA (0.25 mg) was reverse-transcribed in a total volume of 20 ml, containing 1 ml (five units) Superscript II RNase H-reverse transcriptase (Fermentas, Hanover, MD) and 0.25 mg of random primer (Life Technologies). One-twentieth of the synthesized cDNA was amplified by PCR with specific primers representing the rat GABAA receptor subunit. The primer sequences were as follows: a1 (580): [forward] 50 -ATCTTTGGGCCTGGACCCTCATTCT-30 [reverse] 50 -CGGGCTGGCTCCCTTGTCCACTC-30 ; a4 (387): [forward] 50 -TTTAAACGAATCCCCAGGACAGAA-30 , [reverse] 50 -TGCCATTTCTCATAATTCTAA-30 ; b3 (419): [forward] 50 -TGGAGCACCGTCTGGTCTCCAGGA-30 , [reverse] 50 -TCGATCATTCTTGGCCTTGGCTGT-30 ; g2 (335): [forward] 50 -GTGGAGTATGGTACCCTGCACTATTTTGTG-30 , [reverse] 50 -CAGAAGGCGGTAGGGAAGAAGATCCGAGCA-30 ; d (398): [forward] 50 -GACTACGTGGGCTCCAACCTGGA30 , [reverse] 50 -ACTGTGGAGGTGATGCGGATGCT-30 .
S. Ueno et al. / NeuroToxicology 28 (2007) 415–420
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Fig. 1. (A) Sample tracings from an oocyte injected with hippocampal mRNA obtained from the control rats demonstrate GABA-evoked inward currents with increasing doses of GABA. (B) GABA dose–response curves obtained from Xenopus oocytes injected with mRNAs of hippocampus, neocortex, and cerebellum from the control (open circles) and 1-BP-exposed (closed squares) rats. The EC50 values are summarized in Table 1. All data are represented as mean S.E.M. from 3 to 6 oocytes.
The numbers in parentheses represent the predicted size of each amplification product, in base pairs. The primer sequences for the amplification of b-actin mRNA (516 bp) were 50 -CCCTAAGGCCAACCGTGAAAAGATG-30 [forward] and 50 -GAACCGCTCATTGCCGATAGTGATG-3 0 [reverse]. PCR amplification was performed in a 50-ml reaction volume containing 1 mM MgCl2, 200 mM dNTPs, 100 pmol of each primer, and 1 unit of Taq DNA polymerase (Fermentas). Following an initial denaturation at 94 8C for 2 min, amplification was performed at 94 8C for 1 min and 72 8C for 2 min for 24 cycles, with a final extension at 72 8C for 7 min. The annealing temperature was 55 8C for all reactions. The PCR products, 5 ml/well, were separated on 1% agarose gels. The optical densities of the products were measured using an ATTO Densitograph (Atto Corporation, Tokyo, Japan). 2.5. Data analysis All values are presented throughout as mean S.E.M. Nonlinear regression analysis and statistical analysis were carried out by analysis of variance, paired/unpaired, or Welch’s/ Student’s t-tests using GraphPad Prism software (San Diego, CA).
3. Results Fig. 1 shows GABA concentration–response curves obtained from Xenopus oocytes injected with mRNAs of hippocampus, neocortex, and cerebellum, respectively, which were mainly mediated by the activation of GABAA receptors. Non-linear regression analysis indicated that there were no significant differences in the GABA EC50 between the control and 1-BP-exposed rats (Table 1). Moreover, we investigated kainate-evoked currents with increasing concentrations of kainate (Fig. 2). These were thought to be mediated by the a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) type of glutamate receptor, because the EC50 values were similar to that obtained from Xenopus oocytes expressing recombinant rat AMPA (GluR1/GluR2) receptors (Yamakura and Harris, 2000). No significant changes in kainate EC50 were found for all three subregions of the brain between the two groups of rats (Table 1). Next, we examined the effects of 1-BP exposure on neural excitability using hippocampal slices, since it has been reported that inhalation exposure to 1-BP at 1500 and 700 ppm causes hyperexcitability in the CA1 region and the DG (Fueta et al., 2000, 2002b). The slope of the fEPSP (Fig. 3(A)) evoked by the
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Fig. 2. (A) Sample tracings from an oocyte injected with hippocampal mRNA obtained from the control rats demonstrate kainate-evoked inward currents with increasing doses of kainate. (B) Kainate dose–response curves obtained from Xenopus oocytes injected with mRNAs of hippocampus, neocortex, and cerebellum from the control (open circles) and 1-BP-exposed (closed squares) rats. The EC50 values are also summarized in Table 1. All data are represented as mean S.E.M. from 3 to 6 oocytes.
first electrical stimulation was measured in the DG in increasing stimulus intensity, and no significant differences in stimulation–response relationships were observed between the control and 1-BP-exposed rats (Fig. 3(B)). In paired-pulse profiles of PS amplitudes, however, a significant increase in paired-pulse ratios (PPRs) was observed at 5–20-ms interpulse intervals (IPIs), without a change in the PPRs of the fEPSP slopes during the corresponding IPIs (Fig. 4). Since no change in potency for GABA at hippocampal GABAA receptors expressed in oocytes was detected for 1-BPexposed rats compared with controls, the RT-PCR technique Table 1 Mean EC50 values of GABA and kainate obtained from oocytes injected with mRNAs of rat brain subregions Hippocampus
Neocortex
Cerebellum
Control GABA (mM) Kainate (mM)
95.7 (79.3–116) 79.2 (74.1–84.7)
136 (117–159) 61.6 (58.7–64.6)
89.7 (76.6–105) 79.8 (76.3–83.4)
1-BP-exposed GABA (mM) Kainate (mM)
128 (96.4–171) 71.9 (66.7–77.6)
184 (146–231) 66.8 (63.3–70.6)
97.4 (86.1–110) 79.3 (74.6–84.4)
Values were obtained from 3 to 6 oocytes. The 95% confidence intervals are shown in parentheses.
was used to investigate hippocampal GABAA receptor subunit expression. We analyzed the levels of GABAA receptor a1, a4, b3, g2 and d subunit mRNAs, which have been demonstrated to be expressed predominantly in the hippocampus (Wisden et al., 1992), by using optical density measurements, and found that mRNAs of two subunits, b3 and d, were expressed at lower levels in rats exposed to 1-BP compared with controls (Fig. 5). 4. Discussion In the present study, we investigated the effects of subchronic inhalation exposure to 1-BP vapor on CNS function, with particular focus on the GABAergic system. The results obtained from oocytes injected with brain mRNAs of the control and 1-BP-exposed rats suggest that subchronic inhalation exposure to 1-BP at 400 ppm produces no significant effects on the potency of GABA and kainate at brain regional – not only hippocampal, but also neocortical or cerebellar – GABAA and AMPA receptors, respectively (Figs. 1 and 2). In the electrophysiological experiments using hippocampal slices, we did not find any significant change in stimulation– response relationships of the fEPSP slopes in the DG between the control and 1-BP-exposed rats, indicating that inhalation
S. Ueno et al. / NeuroToxicology 28 (2007) 415–420
Fig. 3. (A) Conventional measurements of the slope of the fEPSP and the PS amplitude in the paired-pulse configuration for a typical current recorded from the DG. (B) Stimulation–response curves of the slope 1 of the fEPSP in the DG obtained from control (open circles) and 1-BP-exposed (closed squares) rat hippocampal slices. All values are presented as mean S.E.M.
exposure to 400 ppm of 1-BP vapor does not affect excitatory synaptic drive in the DG (Fig. 3(B)). These fEPSP slopes are thought to reflect the function of the AMPA type of glutamate receptor. Therefore, the data demonstrating no alterations in apparent affinity to kainate at hippocampal AMPA receptors expressed in oocytes after exposure to 1-BP seems to be consistent with the results of the fEPSP slopes. However, paired-pulse profiles of PS in the DG demonstrate that, like exposure at concentrations of 700 and 1500 ppm, even inhalation exposure to 400 ppm of 1-BP for 12 weeks induces disinhibition in the DG (Fig. 4(B)). On the other hand, no increase in PPRs was obtained from the recording in the CA1 (data not shown), suggesting that there could be differences in sensitivity to 1-BP between the CA1 and the DG, detailed studies for which are in progress in our laboratory. Since neuronal hyperexcitability observed in the DG was not associated with a change in PPRs of the fEPSP slopes (Fig. 4(B), inset), it is likely that this hyperexcitability may not be due to a change in feed-forward inhibition of synaptic input, but instead to a reduction in feedback (recurrent) inhibition, which is mediated mainly by GABA and GABAA receptors. Although no alteration was observed in apparent affinity to GABA in the control and 1-BP-exposed hippocampal GABAA receptors expressed in oocytes, we found a decrease in the expression level of the GABAA receptor b3 and d subunit mRNAs by RT-PCR analysis (Fig. 5). It should be noted that,
419
Fig. 4. (A) Sample tracings of paired-pulse PS responses in the DG obtained from control and 1-BP-exposed rat hippocampal slices with a 10-ms interpulse interval. (B) Paired-pulse profiles of the PS in the DG obtained from 1-BPexposed rats (closed squares) in comparison to those from controls (open circles). yp < 0.05 and *p < 0.05, vs. control using Welch’s t-test and Student’s t-test, respectively. Inset, paired-pulse profiles of the fEPSP in the DG obtained from control (open circles) and 1-BP-exposed (closed squares) rats with 5–20ms interpulse intervals.
Fig. 5. (Top) mRNA expression levels of the GABAA receptor a1, a4, b3, g2, and d subunits in the hippocampus, together with the b-actin mRNA level, were compared between the control (1-BP[]) and 1-BP-exposed (1-BP[+]) rats by RT-PCR. (Bottom) histogram showing the relative levels of expression of each subunit in the hippocampus of 1-BP-exposed rats. Expression levels were normalized to the b-actin band and represented as relative ratio to the control.
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while most GABAA receptors in the brain appear to be composed of a, b, and g subunits (Whiting et al., 1999), d subunit-containing GABAA receptors in the DG, in combination with a4 and b2/3 subunits, are thought to be located outside the synapse and to play an important role to inhibit cell excitability via a continuously active mode of inhibition (Nusser and Mody, 2002; Semyanov et al., 2004). Moreover, in animal models of epilepsy, the expression of the GABAA receptor d subunit has been demonstrated recently to be altered (Peng et al., 2004). Taken together, these findings suggest that the changes in the expression and function of d-containing GABAA receptors may be related to symptoms and/or diseases caused by neuronal hyperexcitability in the hippocampus. In this study, we were not able to detect any change in GABA potency from whole hippocampal GABAA receptors expressed in oocytes, and have not yet examined in more detail the function of d subunit-containing GABAA receptors after subchronic exposure to 1-BP. Our results, however, raise the possibility that changes in the expression of the GABAA b3 and d subunits by 1-BP exposure could alter the function of extrasynaptic GABAA receptors, resulting in a reduction of the recurrent inhibition, which leads to convulsions. The interesting observations that rats manifest convulsions after 4 weeks of inhalation exposure to 1-BP vapor at 1500 ppm (Ishidao et al., 2002) and after 14 weeks of exposure at 700 ppm (Ishidao, Unpublished data) would support this idea. In conclusion, we demonstrated that subchronic inhalation exposure to 1-BP vapor at 400 ppm produces hyperexcitability in the DG, which could be related to alterations in the expression and function of specific neurotransmitter receptors; the d subunit-containing GABAA receptors could be one of the specific targets of 1-BP. Our observations should help to enhance the understanding of the mechanism of CNS neurotoxicity caused by 1-BP inhalation exposure, which requires further investigation to be clarified. Acknowledgements We thank Ms. Kayoko Yahata for technical assistance. This study was supported by a Grant-in-Aid for Scientific Research (C) (no. 16590214) from the Japan Society for the Promotion of Science (JSPS) to S.U., Y.Y., Y.F., and T.I., and a University of Occupational and Environmental Health (UOEH) Research Grant for Promotion of Occupational Health to S.U., Y.Y., T.I., and N.K. References Barnard EA, Skolnick P, Olsen RW, Mo¨hler H, Sieghart W, Biggio G, et al. International union of pharmacology. XV. Subtypes of g-aminobutyric
acidA receptors: classification on the basis of subunit structure and receptor function. Pharmacol Rev 1998;50:291–313. Beckstead MJ, Weiner JL, Eger EI II, Gong DH, Mihic SJ. Glycine and gaminobutyric acidA receptor function is enhanced by inhaled drugs of abuse. Mol Pharmacol 2000;57:1199–205. Coulter DA. Epilepsy-associated plasticity in g-aminobutyric acid receptor expression, function, and inhibitory synaptic properties. Int Rev Neurobiol 2001;45:237–52. Fueta Y, Fukuda T, Ishidao T, Hori H. Electrophysiology and immunohistochemistry in the hippocampal CA1 and the dentate gyrus of rats chronically exposed to 1-bromopropane, a substitute for specific chlorofluorocarbons. Neuroscience 2004;124:593–603. Fueta Y, Fukunaga K, Ishidao T, Hori H. Hyperexcitability and changes in activities of Ca2+/calmodulin-dependent kinase II and mitogen-activated protein kinase in the hippocampus of rats exposed to 1-bromopropane. Life Sci 2002a;72:521–9. Fueta Y, Ishidao T, Arashidani K, Endo Y, Hori H. Hyperexcitability of the hippocampal CA1 and the dentate gyrus in rats subchronically exposed to a substitute for chlorofluorocarbons, 1-bromopropane vapor. J Occup Health 2002b;44:156–65. Fueta Y, Ishidao T, Kasai T, Hori H, Arashidani K. Decreased paired-pulse inhibition in the dentate gyrus of the brain in rats exposed to 1-bromopropane vapor. J Occup Health 2000;42:149–51. Hori H, Ishidao T, Oyabu T, Yamato H, Morimoto Y, Tanaka I. Effect of simultaneous exposure to methanol and toluene vapor on their metabolites in rats. J Occup Health 1999;41:149–53. Ichihara G, Yu X, Kitoh J, Asaeda N, Kumazawa T, Iwai H, et al. Reproductive toxicity of 1-bromopropane, a newly introduced alternative to ozone layer depleting solvents, in male rats. Toxicol Sci 2000;54:416–23. Ishidao T, Kunugita N, Fueta Y, Arashidani K, Hori H. Effects of inhaled 1bromopropane vapor on rat metabolism. Toxicol Lett 2002;134:237–43. Nusser Z, Mody I. Selective modulation of tonic and phasic inhibitions in dentate gyrus granule cells. J Neurophysiol 2002;87:2624–8. Peng Z, Huang CS, Stell BM, Mody I, Houser CR. Altered expression of the d subunit of the GABAA receptor in a mouse model of temporal lobe epilepsy. J Neurosci 2004;24:8629–39. Semyanov A, Walker MC, Kullmann DM, Silver RA. Tonically active GABAA receptors: modulating gain and maintaining the tone. Trends Neurosci 2004; 27:262–9. Ueno S, Tsutsui M, Toyohira Y, Minami K, Yanagihara N. Sites of positive allosteric modulation by neurosteroids on ionotropic g-aminobutyric acid receptor subunits. FEBS Lett 2004;566:213–7. Whiting PJ, Bonnert TP, McKernan RM, Farrar S, Le Bourdelles B, Heavens RP, et al. Molecular and functional diversity of the expanding GABA-A receptor gene family. Ann N Y Acad Sci 1999;868:645–53. Wisden W, Laurie DJ, Monyer H, Seeburg PH. The distribution of 13 GABAA receptor subunit mRNAs in the rat brain. I. Telencephalon, diencephalon, mesencephalon. J Neurosci 1992;12:1040–62. Yamada T, Ichihara G, Wang H, Yu X, Maeda K, Tsukamura H, et al. Exposure to 1-bromopropane causes ovarian dysfunction in rats. Toxicol Sci 2003; 71:96–103. Yamakura T, Harris RA. Effects of gaseous anesthetics nitrous oxide and xenon on ligand-gated ion channels. Comparison with isoflurane and ethanol. Anesthesiology 2000;93:1095–101. Yu X, Ichihara G, Kitoh J, Xie Z, Shibata E, Kamijima M, et al. Preliminary report on the neurotoxicity of 1-bromopropane, an alternative solvent for chlorofluorocarbons. J Occup Health 1998;40:234–5. Zao W, Aoki K, Xie T, Misumi J. Electrophysiological changes induced by different doses of 1-bromopropane and 2-bromopropane. J Occup Health 1999;41:1–7.
Changes in the function of the inhibitory neurotransmitter system in the rat brain following subchronic inhalation exposure to 1-bromopropane Susumu Ueno a,*, Yasuhiro Yoshida b, Yukiko Fueta c, Toru Ishidao c, Jiqin Liu b, Naoki Kunugita d, Nobuyuki Yanagihara a, Hajime Hori c a
Department of Pharmacology, School of Medicine, University of Occupational and Environmental Health, Japan Department of Immunology, School of Medicine, University of Occupational and Environmental Health, Japan c Department of Environmental Management I, School of Health Sciences, University of Occupational and Environmental Health, Japan d Department of Health Information Science, School of Health Sciences, University of Occupational and Environmental Health, Japan b
Received 18 October 2005; accepted 10 March 2006 Available online 24 March 2006
Abstract 1-Bromopropane (1-BP) has been widely used as a cleaning agent and a solvent in industries, but the central neurotoxicity of 1-BP remains to be clarified. In the present study, we investigated the effects of subchronic inhalation exposure to 1-BP vapor on the function of the inhibitory neurotransmitter system mediated by g-aminobutyric acid (GABA) in the rat brain. Male Wistar rats were exposed to 1-BP vapor for 12 weeks (6 h/ day, 5 days/week) at a concentration of 400 ppm, and, in order to investigate the expression and function of brain GABA type A (GABAA) receptors, total/messenger RNA was prepared from the neocortex, hippocampus, and cerebellum of the control and 1-BP-exposed rats. Moreover, hippocampal slices were prepared, and the population spike (PS) amplitude and the slope of the field excitatory postsynaptic potential (fEPSP) were investigated in the paired-pulse configuration of the extracellular recording technique. Using the Xenopus oocyte expression system, we compared GABA concentration–response curves obtained from oocytes injected with brain subregional mRNAs of control and 1-BP exposed rats, and observed no significant differences in apparent GABA affinity. On the other hand, paired-pulse inhibition of PS amplitude was significantly decreased in the hippocampal dentate gyrus (DG) by exposure to 1-BP, without any effect on the paired-pulse ratio of the fEPSP slopes, suggesting neuronal disinhibition in the DG. Moreover, RT-PCR analysis indicated decreased levels of GABAA receptor b3 and d subunit mRNAs in the hippocampus of 1-BP-exposed rats. These results demonstrate that subchronic inhalation exposure to 1-BP vapor reduces the function of the hippocampal GABAergic system, which could be due to changes in the expression and function of GABAA receptors, especially the d subunitcontaining GABAA receptors. # 2006 Elsevier Inc. All rights reserved. Keywords: 1-Bromopropane; GABAA receptor; Xenopus oocytes; d Subunit; Disinhibition
1. Introduction Since chlorofluorocarbons have been identified as ozonedepleting solvents, several new compounds have been introduced as alternatives in the workplace. 1-Bromopropane (1-BP) is one of the most important substitutions used in Japan and the United States as a cleaning agent for metals, electronics, ceramics, etc. However, recent studies have demonstrated that
* Corresponding author at: Department of Pharmacology, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishiku, Kitakyushu, Fukuoka 807-8555, Japan. Tel.: +81 93 691 7424; fax: +81 93 601 6264. E-mail address: [email protected] (S. Ueno). 0161-813X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2006.03.006
exposure to 1-BP induces not only reproductive toxicity (Ichihara et al., 2000; Yamada et al., 2003) but also neurotoxicity in the peripheral nervous system (Yu et al., 1998; Zao et al., 1999). The number of workers at risk for exposure to 1-BP vapor is increasing, so that the toxicities of this alternative compound to other organs and/or tissues need to be clarified. The central nervous system (CNS) neurotoxicity caused by inhalation exposure to 1-BP vapor is a topic of great interest, and it has recently been demonstrated that inhalation exposure to 1-BP causes neuronal dysfunction in the rat hippocampus, that is, disinhibition (Fueta et al., 2000, 2002a,b, 2004). In their studies, such disinhibition was observed in the hippocampal CA1 region and the dentate
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gyrus (DG) after repeated exposure to 1-BP vapor at 1500 or 700 ppm, which appeared at the end of the 1st week (1500 ppm) or the 4th week (700 ppm) before convulsion occured. These observations suggest that, even before behavioral abnormalities become apparent, subchronic exposure to 1-BP could alter neuronal excitability in the CNS. Accordingly, it is urgently necessary to elucidate the mechanism of neuronal disinhibition induced by 1-BP exposure. In the CNS, neuronal excitability is regulated by ion channels, including voltage-gated and transmitter-gated ion channels. Among the latter, the g-aminobutyric acid (GABA) type A (GABAA) receptor/chloride ion channel complex is the major inhibitory neurotransmitter receptor in the mammalian brain. Seven classes of GABAA receptor subunits (a1–6, b1–3, g1–3, d, e, p, u) have been cloned to date (Barnard et al., 1998; Whiting et al., 1999), and the GABAergic system, together with the glutamatergic system, has been demonstrated to be critical for overall control of hippocampal excitability and seizures. Moreover, several lines of evidence have suggested that alterations in the expression and function of GABAA receptors in the hippocampus are associated with epilepsy (Coulter, 2001). Therefore, disinhibition in the hippocampus caused by 1-BP exposure could be associated with alterations in the function of the GABAergic system, especially GABAA receptormediated inhibition. In the present study, we describe the effects of subchronic inhalation exposure to 1-BP vapor at 400 ppm, a lower concentration than that tested in previous reports (Fueta et al., 2000, 2002a), primarily on the function of the inhibitory GABAergic system in the rat brain. We assessed the function of brain regional GABAA receptors using a Xenopus oocyte expression system with two-electrode voltage-clamp recordings and hippocampal excitability using slice preparations with extracellular recording techniques. We also examined the expression of GABAA receptor subunit mRNAs in the hippocampus using RT-PCR analysis. 2. Materials and methods 2.1. Animals and inhalation exposure to 1-BP vapor Male Wistar rats (8 weeks of age) were purchased from Kyudo Co. Ltd. (Kumamoto, Japan). Breeding conditions of rats and the inhalation system apparatus were described previously (Hori et al., 1999; Ishidao et al., 2002). The rats were placed in an inhalation chamber containing 1-BP vapor at a concentration (exposure group) or only fresh air (control group). This treatment was performed 6 h a day (mainly from 9 a.m. to 3 p.m.), 5 days a week for 12 weeks. The experiments were carried out under the control of the Ethics Committee of Animal Care and Experimentation in accordance with The Guiding Principle for Animal Care Experimentation, the University of Occupational and Environmental Health, Japan and the Japanese Law for Animal Welfare and Care (No. 221).
2.2. Messenger RNA preparation and expression of brain receptors in Xenopus oocytes After 12 weeks of exposure to 1-BP vapor, rats were deeply anesthetized with diethyl ether and then decapitated, and the neocortex, hippocampus, and cerebellum were dissected. Total RNA was isolated using TRIzol (Life Technologies, Rockville, MD), and mRNA was prepared by using the PolyATtract1 mRNA Isolation System (Promega, Madison, MI) according to the manufacturer’s protocol. The isolation of Stage V and VI oocytes from adult female Xenopus laevis, the injection of mRNA (50 ng per oocyte) into oocytes, and two-electrode voltage-clamp recordings were performed as described previously (Beckstead et al., 2000; Ueno et al., 2004). Either GABA or kainate was applied for 30– 60 s, in increasing concentrations (as listed in Fig. 1) to each oocyte, allowing 5–15 min between applications. 2.3. Hippocampal slice preparation and electrophysiological recording The procedures for hippocampal slice preparation and measurements of the population spike (PS) amplitudes and the slope of the field excitatory postsynaptic potential (fEPSP) from the DG were described previously in detail (Fueta et al., 2004). To analyze paired-pulse responses, calculation of the paired-pulse ratio (PPR) was performed as follows: PPR of fEPSP ¼
2nd fEPSP slope ; 1st fEPSP slope
PPR of PS ¼
2nd PS 1st PS
The conventional measurements of the slope of the fEPSP and the PS amplitude in the paired-pulse configuration are also shown in Fig. 2. 2.4. RT-PCR analysis Total RNA (0.25 mg) was reverse-transcribed in a total volume of 20 ml, containing 1 ml (five units) Superscript II RNase H-reverse transcriptase (Fermentas, Hanover, MD) and 0.25 mg of random primer (Life Technologies). One-twentieth of the synthesized cDNA was amplified by PCR with specific primers representing the rat GABAA receptor subunit. The primer sequences were as follows: a1 (580): [forward] 50 -ATCTTTGGGCCTGGACCCTCATTCT-30 [reverse] 50 -CGGGCTGGCTCCCTTGTCCACTC-30 ; a4 (387): [forward] 50 -TTTAAACGAATCCCCAGGACAGAA-30 , [reverse] 50 -TGCCATTTCTCATAATTCTAA-30 ; b3 (419): [forward] 50 -TGGAGCACCGTCTGGTCTCCAGGA-30 , [reverse] 50 -TCGATCATTCTTGGCCTTGGCTGT-30 ; g2 (335): [forward] 50 -GTGGAGTATGGTACCCTGCACTATTTTGTG-30 , [reverse] 50 -CAGAAGGCGGTAGGGAAGAAGATCCGAGCA-30 ; d (398): [forward] 50 -GACTACGTGGGCTCCAACCTGGA30 , [reverse] 50 -ACTGTGGAGGTGATGCGGATGCT-30 .
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Fig. 1. (A) Sample tracings from an oocyte injected with hippocampal mRNA obtained from the control rats demonstrate GABA-evoked inward currents with increasing doses of GABA. (B) GABA dose–response curves obtained from Xenopus oocytes injected with mRNAs of hippocampus, neocortex, and cerebellum from the control (open circles) and 1-BP-exposed (closed squares) rats. The EC50 values are summarized in Table 1. All data are represented as mean S.E.M. from 3 to 6 oocytes.
The numbers in parentheses represent the predicted size of each amplification product, in base pairs. The primer sequences for the amplification of b-actin mRNA (516 bp) were 50 -CCCTAAGGCCAACCGTGAAAAGATG-30 [forward] and 50 -GAACCGCTCATTGCCGATAGTGATG-3 0 [reverse]. PCR amplification was performed in a 50-ml reaction volume containing 1 mM MgCl2, 200 mM dNTPs, 100 pmol of each primer, and 1 unit of Taq DNA polymerase (Fermentas). Following an initial denaturation at 94 8C for 2 min, amplification was performed at 94 8C for 1 min and 72 8C for 2 min for 24 cycles, with a final extension at 72 8C for 7 min. The annealing temperature was 55 8C for all reactions. The PCR products, 5 ml/well, were separated on 1% agarose gels. The optical densities of the products were measured using an ATTO Densitograph (Atto Corporation, Tokyo, Japan). 2.5. Data analysis All values are presented throughout as mean S.E.M. Nonlinear regression analysis and statistical analysis were carried out by analysis of variance, paired/unpaired, or Welch’s/ Student’s t-tests using GraphPad Prism software (San Diego, CA).
3. Results Fig. 1 shows GABA concentration–response curves obtained from Xenopus oocytes injected with mRNAs of hippocampus, neocortex, and cerebellum, respectively, which were mainly mediated by the activation of GABAA receptors. Non-linear regression analysis indicated that there were no significant differences in the GABA EC50 between the control and 1-BP-exposed rats (Table 1). Moreover, we investigated kainate-evoked currents with increasing concentrations of kainate (Fig. 2). These were thought to be mediated by the a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) type of glutamate receptor, because the EC50 values were similar to that obtained from Xenopus oocytes expressing recombinant rat AMPA (GluR1/GluR2) receptors (Yamakura and Harris, 2000). No significant changes in kainate EC50 were found for all three subregions of the brain between the two groups of rats (Table 1). Next, we examined the effects of 1-BP exposure on neural excitability using hippocampal slices, since it has been reported that inhalation exposure to 1-BP at 1500 and 700 ppm causes hyperexcitability in the CA1 region and the DG (Fueta et al., 2000, 2002b). The slope of the fEPSP (Fig. 3(A)) evoked by the
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Fig. 2. (A) Sample tracings from an oocyte injected with hippocampal mRNA obtained from the control rats demonstrate kainate-evoked inward currents with increasing doses of kainate. (B) Kainate dose–response curves obtained from Xenopus oocytes injected with mRNAs of hippocampus, neocortex, and cerebellum from the control (open circles) and 1-BP-exposed (closed squares) rats. The EC50 values are also summarized in Table 1. All data are represented as mean S.E.M. from 3 to 6 oocytes.
first electrical stimulation was measured in the DG in increasing stimulus intensity, and no significant differences in stimulation–response relationships were observed between the control and 1-BP-exposed rats (Fig. 3(B)). In paired-pulse profiles of PS amplitudes, however, a significant increase in paired-pulse ratios (PPRs) was observed at 5–20-ms interpulse intervals (IPIs), without a change in the PPRs of the fEPSP slopes during the corresponding IPIs (Fig. 4). Since no change in potency for GABA at hippocampal GABAA receptors expressed in oocytes was detected for 1-BPexposed rats compared with controls, the RT-PCR technique Table 1 Mean EC50 values of GABA and kainate obtained from oocytes injected with mRNAs of rat brain subregions Hippocampus
Neocortex
Cerebellum
Control GABA (mM) Kainate (mM)
95.7 (79.3–116) 79.2 (74.1–84.7)
136 (117–159) 61.6 (58.7–64.6)
89.7 (76.6–105) 79.8 (76.3–83.4)
1-BP-exposed GABA (mM) Kainate (mM)
128 (96.4–171) 71.9 (66.7–77.6)
184 (146–231) 66.8 (63.3–70.6)
97.4 (86.1–110) 79.3 (74.6–84.4)
Values were obtained from 3 to 6 oocytes. The 95% confidence intervals are shown in parentheses.
was used to investigate hippocampal GABAA receptor subunit expression. We analyzed the levels of GABAA receptor a1, a4, b3, g2 and d subunit mRNAs, which have been demonstrated to be expressed predominantly in the hippocampus (Wisden et al., 1992), by using optical density measurements, and found that mRNAs of two subunits, b3 and d, were expressed at lower levels in rats exposed to 1-BP compared with controls (Fig. 5). 4. Discussion In the present study, we investigated the effects of subchronic inhalation exposure to 1-BP vapor on CNS function, with particular focus on the GABAergic system. The results obtained from oocytes injected with brain mRNAs of the control and 1-BP-exposed rats suggest that subchronic inhalation exposure to 1-BP at 400 ppm produces no significant effects on the potency of GABA and kainate at brain regional – not only hippocampal, but also neocortical or cerebellar – GABAA and AMPA receptors, respectively (Figs. 1 and 2). In the electrophysiological experiments using hippocampal slices, we did not find any significant change in stimulation– response relationships of the fEPSP slopes in the DG between the control and 1-BP-exposed rats, indicating that inhalation
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Fig. 3. (A) Conventional measurements of the slope of the fEPSP and the PS amplitude in the paired-pulse configuration for a typical current recorded from the DG. (B) Stimulation–response curves of the slope 1 of the fEPSP in the DG obtained from control (open circles) and 1-BP-exposed (closed squares) rat hippocampal slices. All values are presented as mean S.E.M.
exposure to 400 ppm of 1-BP vapor does not affect excitatory synaptic drive in the DG (Fig. 3(B)). These fEPSP slopes are thought to reflect the function of the AMPA type of glutamate receptor. Therefore, the data demonstrating no alterations in apparent affinity to kainate at hippocampal AMPA receptors expressed in oocytes after exposure to 1-BP seems to be consistent with the results of the fEPSP slopes. However, paired-pulse profiles of PS in the DG demonstrate that, like exposure at concentrations of 700 and 1500 ppm, even inhalation exposure to 400 ppm of 1-BP for 12 weeks induces disinhibition in the DG (Fig. 4(B)). On the other hand, no increase in PPRs was obtained from the recording in the CA1 (data not shown), suggesting that there could be differences in sensitivity to 1-BP between the CA1 and the DG, detailed studies for which are in progress in our laboratory. Since neuronal hyperexcitability observed in the DG was not associated with a change in PPRs of the fEPSP slopes (Fig. 4(B), inset), it is likely that this hyperexcitability may not be due to a change in feed-forward inhibition of synaptic input, but instead to a reduction in feedback (recurrent) inhibition, which is mediated mainly by GABA and GABAA receptors. Although no alteration was observed in apparent affinity to GABA in the control and 1-BP-exposed hippocampal GABAA receptors expressed in oocytes, we found a decrease in the expression level of the GABAA receptor b3 and d subunit mRNAs by RT-PCR analysis (Fig. 5). It should be noted that,
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Fig. 4. (A) Sample tracings of paired-pulse PS responses in the DG obtained from control and 1-BP-exposed rat hippocampal slices with a 10-ms interpulse interval. (B) Paired-pulse profiles of the PS in the DG obtained from 1-BPexposed rats (closed squares) in comparison to those from controls (open circles). yp < 0.05 and *p < 0.05, vs. control using Welch’s t-test and Student’s t-test, respectively. Inset, paired-pulse profiles of the fEPSP in the DG obtained from control (open circles) and 1-BP-exposed (closed squares) rats with 5–20ms interpulse intervals.
Fig. 5. (Top) mRNA expression levels of the GABAA receptor a1, a4, b3, g2, and d subunits in the hippocampus, together with the b-actin mRNA level, were compared between the control (1-BP[]) and 1-BP-exposed (1-BP[+]) rats by RT-PCR. (Bottom) histogram showing the relative levels of expression of each subunit in the hippocampus of 1-BP-exposed rats. Expression levels were normalized to the b-actin band and represented as relative ratio to the control.
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while most GABAA receptors in the brain appear to be composed of a, b, and g subunits (Whiting et al., 1999), d subunit-containing GABAA receptors in the DG, in combination with a4 and b2/3 subunits, are thought to be located outside the synapse and to play an important role to inhibit cell excitability via a continuously active mode of inhibition (Nusser and Mody, 2002; Semyanov et al., 2004). Moreover, in animal models of epilepsy, the expression of the GABAA receptor d subunit has been demonstrated recently to be altered (Peng et al., 2004). Taken together, these findings suggest that the changes in the expression and function of d-containing GABAA receptors may be related to symptoms and/or diseases caused by neuronal hyperexcitability in the hippocampus. In this study, we were not able to detect any change in GABA potency from whole hippocampal GABAA receptors expressed in oocytes, and have not yet examined in more detail the function of d subunit-containing GABAA receptors after subchronic exposure to 1-BP. Our results, however, raise the possibility that changes in the expression of the GABAA b3 and d subunits by 1-BP exposure could alter the function of extrasynaptic GABAA receptors, resulting in a reduction of the recurrent inhibition, which leads to convulsions. The interesting observations that rats manifest convulsions after 4 weeks of inhalation exposure to 1-BP vapor at 1500 ppm (Ishidao et al., 2002) and after 14 weeks of exposure at 700 ppm (Ishidao, Unpublished data) would support this idea. In conclusion, we demonstrated that subchronic inhalation exposure to 1-BP vapor at 400 ppm produces hyperexcitability in the DG, which could be related to alterations in the expression and function of specific neurotransmitter receptors; the d subunit-containing GABAA receptors could be one of the specific targets of 1-BP. Our observations should help to enhance the understanding of the mechanism of CNS neurotoxicity caused by 1-BP inhalation exposure, which requires further investigation to be clarified. Acknowledgements We thank Ms. Kayoko Yahata for technical assistance. This study was supported by a Grant-in-Aid for Scientific Research (C) (no. 16590214) from the Japan Society for the Promotion of Science (JSPS) to S.U., Y.Y., Y.F., and T.I., and a University of Occupational and Environmental Health (UOEH) Research Grant for Promotion of Occupational Health to S.U., Y.Y., T.I., and N.K. References Barnard EA, Skolnick P, Olsen RW, Mo¨hler H, Sieghart W, Biggio G, et al. International union of pharmacology. XV. Subtypes of g-aminobutyric
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