Effects of p-Aminosalicylic acid on the Neurotoxicity of Manganese and Levels of Dopamine and Serotonin in the Nervous System and Innervated Organs of Crassostrea virginica

Abstract

Manganese is a neurotoxin causing Manganism in individuals chronically exposed to elevated levels in their environment. Toxic manganese exposure causes mental and emotional disturbances, and a movement disorder similar to Idiopathic Parkinsons Disease. Manganese interferes with dopamine neurons involved in control of body movements. Recently, p-aminosalicylic acid (PAS) is being used to alleviate symptoms of Manganism, but its mechanism of action is unknown. The eastern oyster, Crassostrea virginica, possesses a dopaminergic innervation of its gill. Oysters exposed to manganese have reduced levels of dopamine in the cerebral ganglia, visceral ganglia and gill, but not of norepinephrine, octopamine or serotonin. Those results are consistent with reported mechanisms of action of manganese in human and mammalian systems. In this study we determined the effects of PAS treatments on dopamine and serotonin levels in oysters exposed to manganese. Adult C. virginica were exposed to 500 µM and 1 mM of manganese with and without 500 µM and 1 mM of PAS by removing one shell and maintaining the animals in individual containers of aerated artificial sea water at 18° C for 3 days. Control animals were similarly treated without manganese or PAS. Dopamine and serotonin levels were measured by HPLC with fluorescence detection. PAS protected the ganglia and gill against the effects of 500 µM manganese, but not against the 1 mM manganese treatments. Serotonin levels were not affected by the treatments. The study demonstrates PAS can protect against reductions in dopamine levels caused by neurotoxic manganese exposure, but is concentration dependent. These findings may provide insights into the actions of PAS in therapeutic treatments of Manganism.

Introduction

Manganese is present in animal tissues and is required as an enzyme cofactor or activator for numerous reactions of metabolism¹. While essential in trace amounts, excessive manganese exposure can result in toxic accumulations in human brain causing extrapyramidal symptoms similar to those seen in patients with Idiopathic Parkinson’s disease^2–5, a dopaminergic cell disorder. This Parkinson-like neurological condition was first described in 1837 in two manganese ore-crushing mill workers⁶ and has since been referred to as Manganism^7–10. Symptoms common to both disorders include gait imbalance, rigidity, tremors and bradykinesia^7,11–13, suggesting a similar etiology of neuronal damage in the substantia nigra with a resulting deficiency of the neurotransmitter dopamine for the striatum. However, compared to Parkinsons disease, there are some differentiating features seen with Manganism including symmetry of effects, more prominent dystonia, a characteristic “cock walk,” an intention rather than resting tremor, earlier behavioral and cognitive dysfunction, difficulty turning, and a poor response to Levodopa^2,14–20, suggesting different or more extensive damage in the basal ganglia or to the dopaminergic system.

The primary cause of manganese toxicity is believed to be by inhalation of manganese from the atmosphere²¹. In addition to mining and manganese ore processing, high levels of airborne manganese are possible in a number of other occupational settings, including welding, dry battery manufacture and use of certain organochemical fungicides like Maneb^{14, 22–25}.

Although manganese toxicity has been recognized for some time, the primary mechanism underlying its neurotoxic effects remains elusive. Human and animal studies have shown that toxic exposure to manganese results in metal accumulations in various areas of the basal ganglia and dysfunction of cells of both the striatum and the globus pallidus^2,26–32. Other studies have shown that manganese selectively targets dopaminergic neurons in the human basal ganglia^14,31 and decreases dopamine levels in the striatum^{11,27,33–35}. Considering the clinical similarities between Manganism and Parkinson’s Disease, and the fact that manganese accumulates in brain regions rich in dopaminergic neurons, it has long been suggested that the neurotoxicity caused by manganese involves a disruption in dopaminergic neurotransmission^36–38.

Until recently, clinical interventions for Manganism have not been successful³⁹. However in 2006, Jaing et al.³⁹ reported the first effective treatment of Manganism. Treatments with the drug p-aminosalicylic acid (PAS) reversed many of the symptoms of severe manganese intoxication in the patient. PAS is an anti-inflammatory drug which has been used to treat tuberculosis. They indicate that the exact mechanism of the drug action is unknown and that further studies of PAS in the treatment of manganese intoxication is necessary.

Bivalves and other marine invertebrates are often used in metal environmental toxicology studies because their tissues readily accumulate trace metals to concentrations that are usually much higher on a wet weight basis than what is present in the surrounding seawater^40–42. Numerous reports have been made on the bioaccumulation of various heavy metals in the eastern oyster, Crassostrea virginica, and other oyster species^43–49. Dopamine, serotonin and other biogenic amines are present in the nervous tissue and gill of C. virginica⁵⁰. The animal has a reciprocal dopaminergic and serotonergic innervation of the lateral ciliated cells of the gill, originating in the cerebral and visceral ganglia, which slow down and speed up the beating rates of the cilia, respectively⁵¹.

The animal is a simple system with which to study the relationships among biogenic amines, neurotoxins and other chemicals which may interact with them. Our lab reported that C. virginica incubated in MnCl₂, readily accumulated manganese into its ganglia and tissues⁵² and caused a reduction in the levels of dopamine in the oyster’s cerebral ganglia, visceral ganglia and gill, while having no effects on levels of other biogenic amines, including serotonin, epinephrine and octopamine⁵³. We also showed that manganese treatments impaired the animals dopaminergic innervation of the lateral ciliated cells of the gill, but did not impair the serotonin innervation⁵⁴.

In the present study we sought to use C. virginica as a model to study the effects of PAS treatments on the effects of manganese on a known dopaminergically innervated system.

Materials and Methods

Oysters were maintained in Instant Ocean® artificial seawater (ASW) obtained from Aquarium Systems Inc. (Mentor, OH). Dopamine, serotonin, norepinephrine, epinephrine, octopamine and 1-octanesulfonic acid (sodium salt, SigmaUltra) were obtained from Sigma-Aldrich (St. Louis, MO). All other reagents including manganese chloride (MnCl₂·4H₂O, ASC grade) were obtained from Fisher Scientific (Pittsburgh, PA).

Adult C. virginica of approximately 80 mm shell length were obtained from Frank M. Flower and Sons Oyster Farm in Oyster Bay, NY. They were maintained in the lab for up to two weeks in temperature-regulated aquaria in ASW at 16 – 18° C, specific gravity of 1.024 ± 0.001, salinity of 31.9 ppt, and pH of 7.2 ± 0.2. Each animal was tested for health prior to experimentation by the resistance it offered to being opened. Animals that fully closed in response to tactile stimulation and required at least moderate hand pressure to being opened were used for the experiments. In order to ensure that each oyster would receive equal exposure to manganese during the experiment and not just close up, healthy specimens were shucked by removing their right shell before being placed into individual temperature-controlled aerated containers of ASW for 3 days in the presence of up to 1.0 mM of manganese and PAS. Control animals were similarly prepared without exposure to added manganese and PAS. Both control and experimentally treated animals tolerated the 3-day treatment well. Survival was excellent, there were no fatalities, and only animals with visible signs of heart pumping were used in subsequent experiments.

Endogenous serotonin and dopamine levels of the cerebral and visceral ganglia, as well as the gill were measured using high performance liquid chromatography (HPLC) with fluorescence detection based on the method of Fotopoulou and Ioannou⁵⁵. Animals were treated for three days with or without manganese and PAS after which time each animal’s cerebral ganglia, visceral ganglia and gill were excised and prepared for HPLC measurements of amine levels. The dissected tissues were homogenized with a Brinkman Polytron homogenizer with Omni International disposable probe tips in 0.4 M HCl. They were centrifuged at 12,000 × g for 20 minutes and then vacuum filtered through a 0.24 micron filter. Aliquots (20 µl) of the samples were injected into a Beckman System Gold 126/168 HPLC system fitted with a Phenomenex-Gemini (Torrance, CA) 5µ C18 reverse phase, ion pairing column with a guard column. The mobile phase was 50 mM acetate buffer (pH 4.7) containing 1-octanesulfonic acid (1.1 mM) and EDTA (0.11 mM), mixed with methanol (85:15 v/v). All reagents were HPLC grade. The flow rate was 2 ml/min in isocratic mode. A Jasco FP 2020 Plus Spectrofluorometer was used for detection of native fluorescence (280 nm excitation, 320 nm emission) and was fitted with a 16 µl flow cell. HPLC results are reported as ng/g wet weight for gill and ng/ganglion for the ganglia. Statistical analysis comparing dopamine and serotonin levels in gill and ganglia of treated animals to the controls was determined by a Two-way ANOVA.

Results

Serotonin and dopamine are able to be separated and quantified using HPLC with fluorescence detection. Chromatographs of the amine peaks for standards and a cerebral ganglion are shown in Figures 1a,b. The sensitivity is in the high picogram levels (Fig. 2).

Figure 1.

a. Chromatogram of amine standards.

b. Chromatogram of cerebral ganglia

Figure 2.

Graph showing relative fluorescence of amine standards at excitation wavelength of 280 nm and emission wavelength of 320 nm.

Endogenous dopamine levels in the cerebral and visceral ganglia of untreated animals are in the range of 20 ng/ganglion and 200 ng/g wet weight for the gill. Treating animals for 3 days with 500 µM and 1 mM of manganese significantly reduced dopamine levels in the ganglia and gills (Fig. 3). Serotonin levels were not significantly changed in any of the tissues as a result of either treatment (Fig. 4). The dopamine levels in the ganglia and gill of animals which were co-treated with manganese and PAS were not significantly reduced when 500 µM of PAS was administered with the 500 µM of manganese. However, when 1 mM of PAS was administered with 1 mM of manganese, the dopamine levels in the ganglia and gill were significantly reduced as compared to the untreated controls (Fig. 3). The treatment with manganese and PAS caused no changes in serotonin levels in the ganglia and gill (Fig. 4).

Figure 3.

a. dopamine levels (ng/ganglion ± sem) in the cerebral (CG) and visceral ganglia (VG) of animals treated with 500 µM of manganese (Mn), and 500 µM of Mn and PAS (p-aminosalicylic acid). Figure 3b. dopamine levels CG and VG of animals treated with 1 mM of Mn, and 1 mM of Mn and PAS. Figure 3c. dopamine levels (ng/g wet weight ± sem) in the gill of animals treated with 500 µM of Mn, and 500 µM of Mn and PAS. Figure 3d. dopamine levels in the gill of animals treated with 1 mM of Mn, and 1 mM of Mn and PAS. Statistical significance comparing treated animals to untreated controls was determined by a Two way ANOVA.

Figure 4.

a. serotonin levels in the CG and VG of animals treated with 500 µM of manganese (Mn), and 500 µM of Mn and PAS (p-aminosalicylic acid). Figure 4b. serotonin levels CG and VG of animals treated with 1 mM of Mn, and 1 mM of Mn and PAS. Figure 4c. serotonin levels (ng/g wet weight ± sem) in the gill of animals treated with 500 µM of Mn, and 500 µM of Mn and PAS. Figure 4d. serotonin levels in the gill of animals treated with 1 mM of Mn, and 1 mM of Mn and PAS. Statistical significance comparing treated animals to untreated controls was determined by a Two way ANOVA.

Discussion

An earlier study showed that a 3-day treatment of C. virginica with manganese disrupted the oyster’s dopaminergic, cilio-inhibitory mechanism, while not impairing the serotonergic cilio-excitatory mechanism⁵⁴. The study also showed that manganese treatments lowered endogenous dopamine, but not serotonin levels, in the gill, cerebral ganglia and visceral ganglia of manganese-exposed animals compared to controls. Taken together, the results indicate the specificity of manganese toxicity on the animal’s dopaminergic system. The results of that study suggest that the observed physiological deficits resulting from manganese treatments could be due to several contributing factors, most likely a combination of effects involving reduced levels of neuronal dopamine, possible destruction or damage of dopamine neurons, reduction of terminally released dopamine, and impaired post-synaptic responses to terminally released of dopamine.

It the present study, manganese treatments decreased dopamine levels in the ganglia and gill, but not serotonin levels and PAS protected against the effects of the low dose of manganese on reducing dopamine levels in ganglia and gill.

Manganese is a trace element in animal systems required for normal carbohydrate, lipid, amino acid and protein metabolism, as well as a required cofactor for various antioxidant enzymes such as mitochondrial superoxide dismutase^1,56. However when in excess, manganese is cytotoxic and has been shown to raise levels of reactive oxygen species^57,58, deplete glutathione⁵⁹, impair energy metabolism^60,61, and cause oxidation of catecholamine and other biological chemicals⁶². The prooxidant character of excess manganese and the fact that metal accumulates in dopamine-rich areas of the brain strongly suggests that manganese toxicity is causing further oxidative stress on an already stressed dopaminergic system^56,63–66.

While the cellular and molecular mechanism of manganese toxicity remains unclear, several lines of evidence suggest that exposure to manganese or manganese containing compounds induces oxidative stress-mediated dopaminergic cell death^67–69 which is in agreement with current theories on oxidative stress as a mediator of neuronal death in Parkinson’s disease and other neurodegenerative diseases^70–74.

Dopaminergic neurons and dopamine-rich areas of the brain are particularly vulnerable to oxidative stress, because the enzymatic and non-enzymatic metabolism of dopamine can generate reactive oxygen species and various neurotoxic catecholamine metabolites such as 6-hydroxydopamine^73,75–78. In a related bivalve, Mytilus edulis, a previous study showed that treatments with 6-hydroxydopamine caused a reduction in the animal’s ganglionic levels of dopamine⁷⁹. A recent study using transgenic mice provided in vivo evidence that chronic exposure to unregulated cytosolic dopamine alone was sufficient to cause neurodegeneration in striatal neurons and resulting motor dysfunction⁸⁰.

Jiang et al.³⁹ speculated that the mechanism of action of PAS in alleviating Manganism may be due to a chelating ability of PAS or that the salicylic acid moiety in PAS, which possesses an antiinflammatory effect, may contribute to therapeutic effectiveness of PAS in treatment of neurodegenerative Manganism. Other studies have suggested that nonsteroidal anti-inflammatory drugs, including sodium salicylic acid, may have neuroprotective benefit, because the inflammatory processes have been shown to play a role in the pathogenesis of neurodegenerative diseases such as Alzheimer disease, Parkinson’s disease, and amyotrophic lateral sclerosis^81,82.

The present study demonstrates that the gill and ganglia preparations of C. virginica can be used to investigate mechanisms that underlie manganese neurotoxcity, and may also serve as a model in the pharmacological study of drugs to treat or prevent Manganism and perhaps other dopaminergic cell disorders.