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AMERICAN JOURNAL OF INDUSTRIAL MEDICINE (2007) Basal Ganglia Neurotransmitter Concentrations in Rhesus Monkeys Following Subchronic Manganese Sulfate Inhalation Melanie F. Struve, BS, Brian E. McManus, BS, Brian A. Wong, and David C. Dorman, DVM, PhD PhD, Background Manganese neurotoxicity in humans is recognized as a form of parkinsonism with lesions occurring predominantly within the globus pallidus, subthalamic nucleus, putamen, and caudate nucleus. Methods This study evaluated dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, serotonin, norepinephrine, 5-hydroxyindoleacetic acid, g-aminobutyric acid (GABA), and glutamate concentrations in the globus pallidus, caudate, and putamen of male rhesus monkeys exposed subchronically to either air or manganese sulfate (MnSO4) at 0.06, 0.3, or 1.5 mg Mn/m3. Results An approximate 1.5–6-fold increase (vs. air-exposed controls) in mean brain manganese concentration was observed following subchronic MnSO4 exposure. A marginally significant (P < 0.1) decrease in pallidal GABA and 5-hydroxyindoleacetic acid concentration and caudate norepinephrine concentration occurred in monkeys exposed to MnSO4 at 1.5 mg Mn/m3. Conclusions Despite the presence of increased tissue manganese concentrations, highdose exposure to MnSO4 was associated with relatively few changes in basal ganglial neurotransmitter concentrations. Am. J. Ind. Med. 2007. ß 2007 Wiley-Liss, Inc. KEY WORDS: parkinsonism; neurochemistry; inhalation; macaca mulatta; manganese INTRODUCTION As an essential nutrient, manganese plays an important role in blood sugar regulation, cellular energy, reproduction, digestion, bone growth, and free radical defense mechanisms. Manganese metalloenzymes include arginase, glu- CIIT Centers for Health Research, The Hamner Institutes for Health Sciences, Research Triangle Park, North Carolina 27709 Contract grant sponsor: Afton Chemical Corporation. *Correspondence to: Melanie F. Struve, CIIT Centers for Health Research, The Hamner Institutes for Health Sciences, 6 Davis Drive, P.O. Box 12137, Research Triangle Park, NC 27709. E-mail: [email protected] Accepted18 May 2007 DOI 10.1002/ajim.20489. Published online in Wiley InterScience (www.interscience.wiley.com) 2007 Wiley-Liss, Inc. tamine synthetase, phosphoenolpyruvate decarboxylase, and manganese superoxide dismutase. Human dietary manganese deficiency has not been documented [Aschner et al., 2005]. There have, however, been reports associating reduced blood or serum manganese concentrations with epilepsy [Dupont and Tanaka, 1985; Carl et al., 1986; Davidson and Ward, 1988], although reports of seizures associated with high blood manganese concentrations [Gonzalez-Reyes et al., 2007] and studies failing to support this observation have also been published [Ilhan et al., 2004]. Excessive manganese accumulation within the human caudate nucleus, putamen, and globus pallidus is observed following high-dose inhalation or oral exposure [Aschner et al., 2005]. Clinically, manganese neurotoxicity (manganism) is categorized as a form of parkinsonism because severely affected individuals demonstrate gait abnormalities, postural instability, micrographia, dystonia, rigidity, and 2 Struve et al. bradykinesia. Overt manganese neurotoxicity most often results from the chronic inhalation of very high (>1 mg/m3) concentrations of airborne manganese [Pal et al., 1999]. Pulmonary and reproductive toxicities have also been reported in occupationally exposed workers [Martin, 2006]. Individuals receiving total parenteral nutrition and patients with hepatobiliary insufficiency are also at increased risk for manganese neurotoxicity [Aschner et al., 2005]. Brain magnetic resonance imaging (MRI) studies revealing hyperintensities within the pallidum and other brain regions known to accumulate manganese have been reported for people exposed to high levels of manganese or with hepatobiliary insufficiency [reviewed in Fitsanakis et al., 2006b]. Occupational exposure to manganese occurs primarily through contact with dust or fumes generated during manganese mining operations, cutting, and welding of steel, or during the production of certain batteries and pesticides. Inhalation is the most relevant route for workplace exposure since dermal absorption is extremely limited and oral absorption of manganese is under tight physiological regulation with only a small fraction (2–5%) of the ingested dose becoming systemically absorbed [Aschner et al., 2005]. Occupational studies have found tremor, reduced hand steadiness, and slower finger-tapping speed in workers that inhale manganese at concentrations below those associated with overt manganese neurotoxicity [Olanow, 2004]. For example, a cross-sectional study conducted by Roels et al. [1992] in a Belgium dry alkaline battery plant revealed that 20–30% of the workers had subclinical neurobehavioral dysfunction associated with inhalation exposure to particulate manganese dioxide (MnO2). The mean time-weighted average (TWA) concentration of manganese in inhalable dust was 1 mg Mn/m3 with an average duration of exposure of 5.3 years. An 8-year prospective investigation of the manganese-exposed cohort identified the NOAEL and LOAEL to be approximately 120 and 400 mg Mn/m3, respectively [Roels et al., 1999]. The American Conference of Governmental and Industrial Hygienists (ACGIH) threshold limit value (TLV) expressed as a time-weighted average for manganese is 0.2 mg Mn/m3 (total dust). Neuropathology associated with manganism generally involves the globus pallidus and substantia nigra pars reticulata, with less extensive involvement of the caudate nucleus, putamen, subthalamic nucleus, and substantia nigra pars compacta [Yamada et al., 1986]. These observations suggest that the dopaminergic system is an important target for manganese. In two patients with manganese toxicity, striatal dopamine levels were reported to be markedly decreased [Mena et al., 1970; Bernheimer et al., 1973]. Unlike individuals with Parkinson’s disease, manganism patients do not respond to dopamine replacement [Koller et al., 2004; Olanow, 2004]. Likewise, functional imaging studies using fluorodopa-labeled positron emission tomography (PET) scans have failed to show reduced striatal uptake of fluorodopa in patients with manganism [Olanow, 2004]. Neurochemical changes that occur in people following high-dose manganese exposure are poorly documented. Thus, this study was conducted to improve our understanding of neurochemical changes that occur in nonhuman primates following high-dose manganese inhalation. MATERIALS AND METHODS Experimental Design Male rhesus monkeys were exposed to air (n ¼ 6) or MnSO4 at 0.06 (n ¼ 6), 0.3 (n ¼ 4), or 1.5 mg Mn/m3 (n ¼ 4) for 65 exposure days. Exposures were conducted for 6 hr/day, 5 days/week. MnSO4 aerosol concentrations of 0.18, 0.92, and 4.62 mg MnSO4/m3, corresponding to 0.06, 0.3, and 1.5 mg Mn/m3, were generated for this study. Control animals were exposed to filtered air. Our lowest exposure MnSO4 concentration (0.06 mg Mn/m3) is below the current ACGIH 8-hr TLV for inhaled manganese of 0.2 mg Mn/m3. T1weighted MRI studies of the head were performed 3–4 days prior to necropsy using the following anesthesia protocol. Animals were fasted overnight and then tranquilized with ketamine (approximately 10–15 mg/kg, IM, Fort Dodge Animal Health, Fort Dodge, IA). Animals were given atropine (approximately 0.05 mg/kg, SQ, Phoenix Scientific, Inc., St. Joseph, MO) to decrease bronchial secretions and prevent bradycardia. This was followed by an intravenous bolus of propofol (Gensia Sicor Pharmaceuticals, Inc., Irvine, CA) at an approximate dose of 1.25–2.5 mg/kg, followed by slow intravenous propofol infusion at approximately 300 mg/kg/min (range 105–470 mg/kg/min) to maintain a light plane of anesthesia for approximately 1– 2 hr. The in-life and necropsy portions of this study were performed in accordance with the U.S. Environmental Protection Agency’s Good Laboratory Practice (GLP) Standards for Inhalation Exposure Health Effects Testing (40 CFR Part 79.60). Chemicals Manganese (II) sulfate monohydrate (MnSO4H2O) was obtained from Aldrich Chemical Company, Inc. (Milwaukee, WI). Manganese sulfate is a relatively water-soluble, pale pink, crystalline powder that contains approximately 32% manganese. Unless otherwise noted, all other chemicals were purchased from Sigma–Aldrich (St. Louis, MO). Animals This study was conducted under federal guidelines for the care and use of laboratory animals [National Research Council, 1996] and was approved by the CIIT Institutional Animal Care and Use Committee. Twenty, 17–22 month Neurotransmitters in Manganese-Exposed Monkeys old, male rhesus monkeys were purchased from Covance Research Products, Inc. (Alice, TX). Monkeys were 20– 24 months old at the start of the inhalation exposure. Additional details concerning these animals, their husbandry, and assessments have been previously published [Dorman et al., 2005, 2006a,b]. Animals were observed daily by trained veterinary and animal care staff. Manganese exposures Four 8-m3 stainless steel and glass inhalation exposure chambers were used. Methods describing chamber monitoring as well as generation and characterization of the MnSO4 aerosol have been previously described [Dorman et al., 2004, 2005]. Based upon optical particle sensor measurements, the overall average concentrations (SD) for the MnSO4 atmospheres were 0.19 0.01, 0.97 0.06, and 4.55 0.33 mg/m3 for the target concentrations of 0.18, 0.92, and 4.62 mg MnSO4/m3, respectively. The geometric mean diameter, geometric standard deviation (sg), and calculated mass median aerodynamic diameters (MMAD) of the MnSO4 aerosols were determined to be 1.04 mm (sg ¼ 1.51; MMAD ¼ 1.73 mm), 1.07 mm (sg ¼ 1.54; MMAD ¼ 1.89 mm), and 1.12 mm (sg ¼ 1.58; MMAD ¼ 2.12 mm) for the target concentrations of 0.18, 0.92, and 4.62 mg MnSO4/ m3, respectively. Necropsy procedures Necropsies were performed 18–23 hr after termination of the final inhalation exposure. Food was withheld overnight prior to necropsy. Monkeys were anesthetized with ketamine (20 mg/kg, im, Fort Dodge Animal Health, Fort Dodge, IA) and blood was collected from a peripheral vein using plastic syringes with hypodermic needles. Monkeys were then euthanized with pentobarbital (80–150 mg/kg, iv, Henry Schein, Inc., Port Washington, NY) followed by exsanguination. Brains were removed, divided along the mid-sagittal plane, and dissected into regions containing the caudate, putamen, and globus pallidus. Anatomical structures were identified using a monkey brain atlas [Martin and Bowden, 2000]. All samples were stored immediately in individual plastic vials or bags, frozen in liquid nitrogen, and stored at 808C until chemical analyses were performed. Brain manganese concentrations Brain manganese concentrations were determined by graphite furnace atomic absorption spectrometry with a Perkin Elmer AAnalyst 800 atomic absorption spectrometer equipped with AA WinLab software (version 4.1 SP), as previously described [Dorman et al., 2004]. Samples (10– 30 mg) were digested in 16 M nitric acid (1 ml) prior to 3 manganese analysis using a CEM MARS5 Microwave Accelerated Reaction System (CEM, Matthews, NC). Brain neurotransmitter concentrations Monoamine neurotransmitter (dopamine, 3,4-dihydroxyphenylacetic acid [DOPAC], homovanillic acid [HVA], serotonin [5-HT], 5-hydroxyindoleacetic acid [5-HIAA], and norepinephrine) concentrations were determined using modifications of the methods described by Ali and Slikker [1994]. Approximately 10–50 mg of brain tissue were homogenized with a 20-fold volume of an aqueous solution containing 0.2 N perchloric acid (PCA) with 3,4 dihydroxybenzyl-amine (DHBA) at 1000 ng DHBA/ml and homoserine (500 mg/ml) as internal standards. Tissue samples in aqueous PCA were placed in ice and sonicated for approximately 4–8 s using a Branson 450 digital sonifier (Danbury, CT). The homogenized sample was then centrifuged for 10 min at 10,000g (12,000 rpm) at 48C. Sample supernatants were diluted as needed prior to analysis. Tissue monoamine concentrations were determined by using reverse-phase high-pressure liquid chromatography with electrochemical detection (HPLC-EC). An HPLC-EC system consisting of a Waters 717Plus refrigerated autosampler (Waters Corporation, Milford, MA), ESA 580 HPLC pump (ESA Biosciences, Inc., Chelmsford, MA), SSI pulse dampener, ESA model 5020 guard cell, and ESA Coulochem II Model 5200A electrochemical detector equipped with an ESA analytical electrochemical cell Model 5011 was used. The HPLC was equipped with a Supelco Discovery C18 analytical column (10 cm 4.6 mm, 5 mm) and a Supelguard Discovery C18 guard column (2 cm 4.0 mm, 5 mm). The mobile phase was 7 mM phosphate buffer and 11% methanol at pH 3.0. Chromatograms were analyzed using EZChrom Elite for the ESA Chromatography Data System. Representative standard curves were developed for each monoamine of interest using commercially available high-purity standards purchased from Sigma–Aldrich or a similar vendor. g-Aminobutyric acid (GABA) and glutamic acid concentrations were determined by using reverse-phase HPLCEC with pre-column derivatization of the amino acids with o-phthaldialdehyde (OPA) and 2-mercaptoethanol (b-ME) prior to detection using procedures described in ESA Application Note 70-0160 (http://www.esainc.com/ applications/application_notes.htm#GABA). An HPLC-EC system consisting of an ESA Model 540 refrigerated autosampler, ESA 580 HPLC pump, SSI pulse dampener, and ESA CoulArray Model 5600 electrochemical detector and thermal chamber equipped with twin ESA analytical electrochemical cells (Model 6210) was used. The HPLC was equipped with a Waters Xterra MS C18 2.5 mm (3.0 mm 5.0 mm) HPLC column. The mobile phase was 20% methanol and 3.5% acetonitrile in 100 mM sodium phosphate dibasic buffer at pH 6.7. Chromatograms were analyzed using ESA 4 Struve et al. CoulArray Chromatography Data Software System. Representative standard curves were developed using commercially available high-purity standards purchased from Sigma–Aldrich. controls) in mean caudate manganese concentration was observed following subchronic exposure to MnSO4 at 0.3 mg Mn/m3. Treatment-related clinical signs were not observed. Data analysis and statistics Brain Neurotransmitter Concentrations The data for quantitative, continuous variables were compared for the exposure and control groups by tests for homogeneity of variance (Levene’s test), analysis of variance (ANOVA), and Dunnett’s multiple comparison procedure for significant ANOVA. In the event the Levene’s test was significant, then the data were transformed using a natural log (ln) transformation. If the Levene’s test remained significant then the data were analyzed by nonparametric statistics (Wilcoxon/Kruskal–Wallis). Statistical analyses were performed using JMP Statistical Software (SAS Institute, Inc., Cary, NC). A probability value of <0.01 was used for Levene’s test, while unless otherwise noted, <0.05 was used as the critical level of significance for all other statistical tests. Unless otherwise noted, data presented are mean values standard error of the mean (SEM). Putamen, caudate, and globus pallidus glutamate and GABA concentrations following the end of the 13-week exposure are presented in Table I. A statistically significant decrease (vs. controls) in pallidal GABA concentration, at P < 0.1, was observed in monkeys exposed to MnSO4 at 1.5 mg Mn/m3. Putamen, caudate, and globus pallidus monoamine neurotransmitter concentrations following the end of the 13-week exposure are presented in Table II. Statistically significant decreases (vs. controls) in pallidal 5-HIAA concentration and caudate norepinephrine concentration, at P < 0.1, were observed in monkeys exposed to MnSO4 at 1.5 mg Mn/m3. Putamen neurotransmitter concentrations were unaffected by MnSO4 exposure. DISCUSSION RESULTS Brain Manganese Concentrations The results of these analyses have been previously published [Dorman et al., 2006a] and are presented in Table I. As reported, a statistically significant increase (vs. controls) in mean pallidal and putamen manganese concentration was observed following subchronic exposure to MnSO4 at 0.06 mg Mn/m3. A statistically significant increase (vs. Manganese poisoning in humans and other primates produces neuropathological changes in the basal ganglia, especially in the internal segment of the globus pallidus, caudate, and putamen [Yamada et al., 1986; Eriksson et al., 1987, 1992a,b]. In humans, these lesions are localized preand post-synaptically to the dopaminergic nigrostriatal pathway and are associated with the resultant development of dystonia [Barbeau, 1984]. This observation has led to several hypotheses concerning the mode of action of TABLE I. Mean (SEM) Regional Brain Manganese (mg Mn/g Tissue Wet Weight), and GABA and Glutamate Levels (mg/g Tissue Wet Weight) in Monkeys Exposed to MnSO4 by Inhalation for 6 hr/day, 5 days/week, for13 weeks (65 Exposure Days) Endpoint Manganese GABA Glutamate MnSO4 exposure concentration (mg/m3) Putamen Caudate Globus pallidus 0 0.06 0.3 1.5 0 0.06 0.3 1.5 0 0.06 0.3 1.5 0.36 0.01 0.58 0.04* 0.75 0.05* 1.81 0.14 * 257 18 228 10 242 19 223 10 951 80 986 19 967 62 998 10 0.34 0.02 0.47 0.04 0.69 0.03* 1.72 0.10* 249 23 227 20 222 26 201 14 1181 48 1172 23 1143 55 1214 25 0.48 0.04 0.80 0.04* 1.28 0.15 * 2.94 0.23 * 619 46 587 64 681 31 415 98{ 434 89 375 73 306 10 450 134 Group sizes were 6, 6, 4, and 4 for the 0, 0.06, 0.3, and 1.5 mg Mn/m3 exposure groups, respectively. *Significantly different from control, P < 0.05. { Significantly different from control, P < 0.1. Neurotransmitters in Manganese-Exposed Monkeys 5 TABLE II. Mean (SEM) Regional Brain Catecholamine Levels (mg/gTissue Wet Weight) in Monkeys Exposed to MnSO4 by Inhalation for 6 hr/day, 5 days/ week, for13 weeks (65 Exposure Days) Transmitter (mg/g) Dopamine DOPAC 5-HIAA 5-HT HVA Norepinephrine MnSO4 exposure concentration (mg/m3) Putamen Caudate Globus pallidus 0 0.06 0.3 1.5 0 0.06 0.3 1.5 0 0.06 0.3 1.5 0 0.06 0.3 1.5 0 0.06 0.3 1.5 0 0.06 0.3 1.5 56.46 4.51 63.56 1.92 64.37 3.24 63.80 0.76 1.96 0.45 1.85 0.30 1.71 0.30 1.54 0.13 1.06 0.10 0.85 0.07 1.01 0.17 0.70 0.13 0.27 0.04 0.27 0.02 0.29 0.08 0.29 0.04 93.8 6.4 81.6 4.0 93.7 8.7 83.8 8.4 1.42 0.09 1.47 0.08 1.45 0.03 1.53 0.12 64.72 4.31 61.48 4.25 61.49 4.10 64.82 2.39 2.19 0.36 1.89 0.17 2.22 0.37 1.63 0.09 0.59 0.06 0.49 0.06 0.82 0.13 0.39 0.02 0.52 0.10 0.38 0.07 0.45 0.09 0.49 0.15 63.0 8.5 61.6 6.9 68.0 11.0 63.6 9.0 0.88 0.04 0.73 0.08 0.92 0.10 0.65 0.05{ 2.10 0.94 0.71 0.17 1.07 0.18 1.02 0.44 0.36 0.16 0.20 0.03 0.33 0.08 0.16 0.05 2.07 0.21 2.00 0.18 2.28 0.10 1.46 0.11{ 0.56 0.07 0.55 0.11 0.80 0.11 0.50 0.12 68.2 7.6 53.2 8.5 56.5 4.4 49.1 13.7 0.56 0.16 0.58 0.17 0.77 0.26 0.59 0.22 Group sizes were 6, 6, 4, and 4 for the 0, 0.06, 0.3, and 1.5 mg Mn/m3 exposure groups, respectively. { Significantly different from control, P < 0.1. manganese. One hypothesis focuses on manganese-induced dopamine oxidation resulting in generation of reactive oxygen species, oxidative stress, and secondary cytotoxicity to dopaminergic neurons [Graham, 1984; Archibald and Tyree, 1987; Simonian and Coyle, 1996; Hussain et al., 1997]. Numerous studies conducted in rodents provide at least partial support for this hypothesis. Rodent studies have demonstrated that depletion of striatal dopamine and loss of nigro-striatal dopaminergic neurons occur following highdose manganese exposure [Chandra et al., 1978; Gianutsos and Murray, 1982; Komura and Sakamoto, 1991; Bonilla et al., 1994; Pappas et al., 1997]. Studies conducted in nonhuman primates result in similar findings. For example, Eriksson and coworkers [Eriksson et al., 1987, 1992a] reported reduced striatal and pallidal levels of dopamine and DOPAC, but not homovanillic acid concentrations, and reduced dopamine uptake sites and D1 receptors in monkeys exposed to manganese oxide subcutaneously. In our study however, we did not observe any statistically significant changes in either dopamine or DOPAC levels in the MnSO4exposed monkeys. We did however observe marginally statistically significant decreases (vs. controls, P < 0.1) in pallidal 5-HIAA concentration and caudate norepinephrine concentration in monkeys exposed to MnSO4 at 1.5 mg Mn/ m3. The toxicological significance of this observation is unclear. While research has focused on manganese-induced effects on the dopaminergic system, there is evidence that high brain manganese concentrations may also disrupt the regulation of the GABA-ergic neurons [reviewed in Gwiazda et al., 2002; Fitsanakis et al., 2006a]. Tomas-Camardiel et al. [2002] have shown that high-dose manganese exposure can decrease the levels of glutamic acid decarboxylase levels, an enzyme involved in GABA synthesis regulation. This observation has prompted the suggestion that manganese neurotoxicity progresses with increasing cumulative dose, whereby striatal GABA levels are adversely affected before changes in dopamine levels occur [Fitsanakis et al., 2006a]. Our results are consistent with this hypothesis because a marginally statistically significant decrease (vs. controls, P < 0.1) in pallidal GABA concentration was observed in monkeys exposed to MnSO4 at 1.5 mg Mn/m3. Glutamate 6 Struve et al. regulation also appears to be affected by increased brain manganese levels [reviewed in Fitsanakis et al., 2006a]. Interestingly, we did not observe any significant changes in basal ganglial glutamate levels in our monkeys. Additional work is currently underway to evaluate glutamine synthetase levels in these samples. The form of manganese used in this study (MnSO4) is more soluble than the oxide forms present in most workplaces. Increased manganese particle solubility enhances brain manganese delivery [Dorman et al., 2001]. In the present study we observed manganese accumulation within the globus pallidus, putamen, and caudate nucleus following exposure to the lowest MnSO4 exposure concentration (0.06 mg Mn/m3) used in this study. A greater than three to fivefold increase (vs. air-exposed controls) in mean tissue manganese concentration was observed in the globus pallidus, putamen, and caudate of monkeys exposed subchronically (65 exposure days) to MnSO4 at the highest exposure concentration (1.5 mg Mn/m3). Our results demonstrating manganese accumulation are consistent with previous nonhuman primate inhalation studies [Coulston and Griffin, 1977; Bird et al., 1984]. Moreover, brain MRI hyperintensities seen in our MnSO4-exposed monkeys [Dorman et al., 2006a] mimic those seen in heavily exposed people [Fitsanakis et al., 2006b]. Thus, our exposure conditions were sufficient to raise brain manganese concentrations to levels seen during manganese neurotoxicity. Overall, subchronic high-dose MnSO4 inhalation in rhesus monkeys was associated with relatively few changes in basal ganglial neurotransmitter concentrations. This lack of response may in part reflect the small sample size used, however, our reanalysis of our data using a less stringent significance level (P < 0.1) helps compensate for our experimental design. This less stringent analysis identified neurotransmitter changes in monkeys exposed to MnSO4 at 1.5 mg Mn/m3. Interestingly, however, we did not see any change in brain neurotransmitter concentrations in monkeys exposed to lower MnSO4 exposure concentrations (i.e. <1 mg Mn/m3) despite the presence of elevated brain manganese concentrations in these animals. Occupational manganese neurotoxicity often requires prolonged exposure and clinical manifestations may be delayed for up to several years [Roels et al., 1987, 1992; Lucchini et al., 1997, 1999; Martin, 2006]; thus results seen following subchronic exposure may not reflect these more chronic exposure scenarios. Our study provides additional evidence that changes in basal ganglial neurotransmitter concentrations do not occur rapidly following subchronic manganese inhalation at the current TLV. Centers for Health Research staff for their contributions. We also thank Dr. Eva Polston for her critical review of this manuscript. This publication is based on a study sponsored and funded by the Afton Chemical Corporation in satisfaction of registration requirements arising under Section 211(a) and (b) of the Clean Air Act and corresponding regulations at 40 C. F. R. Subsections 79.50 et seq. 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