Behavioral and neurotoxicological effects of subchronic manganese exposure in rats
Introduction
Manganese (Mn) is, in small amounts, an essential micronutrient (WHO, 1981), being cofactor in metallo-enzymes. In prolonged exposure to higher doses it is, however, a potential environmental neurotoxicant. In the general population, excess dietary intake (beyond the estimated safe and adequate daily amount) is more typical, as in case of babies fed by cow milk or soybean-based formulas (Marlowe and Bliss, 1993), or in the population having environmental exposure via the drinking water. In Greece, 50 mg/l Mn in the drinking water was associated with neurological effects (Kondakis et al., 1989). Mn as man-made environmental contaminant originates from Mn-containing waste (e.g. batteries), methylcyclopentadienyl manganese tricarbonyl (MMT) used as anti-knock petrol additive, and organo-Mn agricultural fungicides (ATSDR, 2000).
Following ingestion, inorganic Mn is absorbed in the intestine mostly in trivalent form (Cotzias et al., 1971) in a homeostatic self-limiting process. The main sites of deposition are the mitochondria-rich tissues, e.g. liver, pancreas, pituitary gland and the muscles (Barceloux, 1999). Brain is among the primary target organs in chronic Mn exposure (Roels et al., 1987), and the turnover of Mn there is much slower than in other parts of the body (Feldman, 1992).
In subchronic dosage, Mn appears in the cerebral and cerebellar cortex (Dorman et al., 2000). In case of chronic intake, Mn is found in motor control centers such as the basal ganglia, particularly the globus pallidus, striatum and substantia nigra where it causes degeneration (Yamada et al., 1986). Mn accumulation was also described in the pons and medulla (Chan et al., 1992), and in the hippocampus (Takeda et al., 1998).
The high sensitivity of different behavior types and the integration of different (motor, sensory, attentional, motivational) behavioral functions is especially important in assessment neurotoxic risk due, e.g. to Mn. Excess Mn was shown to cause several neurotoxic effects such as childhood hyperactivity disorder (Barlow, 1983), manganese psychosis, extrapyramidal dysfunction (Quaghebeur et al., 1996), motor deficit (above 7.5 μg/l blood Mn; Mergler et al., 1999); as well as altered childhood psychomotor development (Takser et al., 2003) and impairments of several memory process (list learning, visual recognition, digit span). Lucchini et al. (1999) found irritability and motor functional damage in exposed workers having ca. 10 μg/l blood Mn level. A child developed severe epilepsy following exposure to welding fumes resulting in 15–20 μg/l blood Mn (Hernandez et al., 2003).
Similar deficits were also induced by Mn in animals. Öner and Sentürk (1992) saw impaired T-maze learning in rats receiving 375 μg/kg b.w. Mn for 30 days. Ingersoll et al. (1995) elicited spontaneous hypoactivity by intrathecal administration of MnCl2. Dorman et al. (2000) described altered acoustic startle responses (ASR) in neonatal rats receiving 25 and 50 mg/kg b.w. MnCl2.
Significant relationship between neurotoxic alterations, neurotransmitter or modulator metabolism (first of all catecholamines), and Mn exposure have been described (Neff et al., 1969). Mn, in case of prolonged low level exposure, accumulates in the nigrostrial dopaminergic pathway (Subhash and Padmashree, 1991). The resulting dopaminergic–glutamatergic interactions are involved in the Mn effects on extrapyramidal motor functions and, indirectly, in the sensorimotor integration (Calabresi et al., 1997).
Direct toxicity of Mn to dopaminergic neurons was described by Parenti et al. (1987). The involvement of other transmitters can result from dopaminergic control upon these (Takeda et al., 2002) or by direct effect of Mn. The latter is know to exist in the GABAergic (Trepper et al., 1998) and glutamatergic system. Reduced glial glutamate uptake (Aschner et al., 1999) leads, as a direct result of impaired astrocytic-neuronal interactions (Hazell, 2002), to increased extracellular glutamate concentration, the excitotoxic effect of which could play a key role in manganese-induced neuronal cell death. Marked reactive astrocytosis, with significant hypertrophy of glial fibrillary acid protein-immunoreactive—GFAP-IR—astrocytes, was seen on Mn exposure in the globus pallidus (Baeck et al., 2003). In the hippocampal dentate gyrus, Mn exposure caused a decrease of GFAP-IR area in juvenile, but an increase in adult, rats (Pappas et al., 1997). This, together with the numerous effects of Mn exposure on the behavior, raised the possibility that these functional and morphological changes induced by Mn develop in parallel and are in detectable causal relationship.
There has been ample literature on the negative effects of MnCl2 on behavioral outcomes in rats. Several mechanisms have been purposed, such as influence on transmitter systems (Tran et al., 2002, Gwiazda et al., 2002), or directly on structural elements like hippocampal astrocytes (Aschner, 1996) and the midbrain and basal ganglia (Erikson and Aschner, 2002). The dentate gyrus (primarily the granule cells) plays an important role in the acquisition of new information (Ogura et al., 2002) and is possibly neural substrate of spatial reference and working memory, and of synaptic plasticity (Ikegaya et al., 1995). Agents acting on the dentate gyrus possibly affect learning and memory performance, locomotor activity, radial maze performance and spontaneous motor activity, and psychomotor performance.
In the works cited above, however, behavioral effects were not supported by histochemical or electrophysiological findings, and no follow-up in the after-exposure period was included. The electrophysiological effect of Mn exposure is in itself an open question: some authors found disturbances of spontaneous or stimulus-evoked cortical activity in workers exposed to comparable airborne Mn levels (Sinczuk-Walczak et al., 2001) while others did not (Deschamps et al., 2001).
Hence, our experiment involved a complex behavioral test battery (8-arm radial maze, open field activity—OF, acoustic startle response—ASR, and pre-pulse inhibition—PPI). Our behavioral investigations were supplemented by cortical electrophysiology (electrocorticogram—ECoG, evoked potentials—EP), immunhistochemistry (GFAP in various parts of the hippocampus), and by Mn level determination in blood, cortex and hippocampus. All investigations were performed both during and after the period of Mn administration. It was also attempted to prove the involvement of the dopamine (DA) system by applying a dopaminergic agonist in the elimination period.
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Animals, housing, treatment
Young adult male Wistar rats (220–250 g body weight at start), obtained at the University's Breeding Center, were used. The animals were housed under controlled conditions of temperature (22–24 °C) and photoperiod (12-h light:12-h dark cycle with light starting at 06:00 h), with free access to drinking water. Three weeks before starting the treatment, the animals (up to 4 rats per cage) were put in cages of 28 cm × 40 cm × 20 cm. The memory test used required that during the 10 weeks of treatment the
Manganese levels
Levels of Mn in various tissues, at the end of the 5th and 10th week of MnCl2 application and the 12th post-treatment week, are given in Table 2. Compared to the 0th week (pre-administration) value of 0.0181 ± 0.003 μg/g, blood Mn levels increased in a dose- and time-dependent way. In the high dose group, the increase was significant both in the 5th treatment week (F2,6 = 12.16; high dose versus control, p < 0.01; high versus low dose, p < 0.01) and in the 10th treatment week (F2,12 = 9.51; high dose
Discussion
Subchronic administration of MnCl2 to the rats in this study resulted in marked internal exposure (shown by the Mn levels in the cortex and hippocampus; Table 2). Mn levels in the control rats’ brains, originating from the background Mn present in standard food and drinking water, were similar to that described earlier by Rehnberg et al. (1982).
From the gastrointestinal tract, absorption of ionic Mn is low (<10%) and self-limited in the rats (Davis et al., 1992). Once absorbed, Mn is bound to
Conclusion
The main points of conclusion drawn from the above results are as given below.
Oral MnCl2 treatment, 14.84 and 59.36 mg/kg b.w., for 10 weeks brought about, primarily in the high-dose group, an increase in first the blood, then cortex and hippocampus Mn levels.
During the treatment period, altered PPI and cortical spontaneous activity in the treated rats suggested the involvement of brainstem cholinergic systems (pre-pulse inhibition pathway and ascending cortical activation) due to Mn-induced
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