Introduction
Manganese (Mn) is an enzymatic cofactor that plays an important role in a number of physiologic processes. However, when present at high tissue concentration, Mn produces cellular toxicity, including neurotoxicity in brain. Although the mechanisms by which Mn induces neuronal damage are not well defined, Mn neurotoxicity appears to be regulated by a number of factors, including oxidative injury, mitochondrial dysfunction, and neuroinflammation (Milatovic et al.
2007,
2009; Zhang et al.
2013). Occupational Mn exposure results in an imbalance between dopamine (DA) and γ-aminobutyric acid (GABA) in the basal ganglia, eliciting the syndrome called manganism, which shares multiple features with Parkinson’s disease (Huang
2007). The strongest correlation between any type of environmental exposure and increased susceptibility to Parkinsonism is observed in the Mn-exposed population (Gorell et al.
1999; Racette et al.
2001).
Occupational exposure (miners, smelters, welders, and workers in dry-cell battery factories; also inhalation of Mn in aerosols/dusts) accounts for the major source of Mn intoxication in humans. An important but non-occupational source of Mn is methylcyclopentadienyl manganese tricarbonyl (MMT), the antiknock agent in gasoline. Another source is potassium permanganate, a powerful oxidizing agent for purifying drinking water, treating waste water, and as an agricultural fungicidal and bactericidal agent (Huang
2007). Manganism has been observed recently in intravenous methcathinone abusers because this substance is illicitly produced by a potassium permanganate oxidation process (Stepens et al.
2008). Additionally, Mn is a natural component of many foods, particularly of nuts, grains, and tea, and is an essential trace element used by humans in enzymatic processes. There are numerous reports of Mn intoxication related to long-term total parenteral nutrition (Reynolds et al.
1994; Reimund et al.
2000).
The effects of Mn on the adult mammalian brain have been studied for decades in connection with manganism. However, the risk of Mn-induced neurotoxicity during brain development, both pre- and postnatally, has received little attention. Several studies report the aftereffects (e.g., reduced intellectual function) of Mn exposure on children at various developmental stages (Woolf et al.
2002; Wasserman et al.
2006). Mn crosses the placenta to impair embryonic development (Spencer
1999) and produce behavioral abnormalities through childhood and into adulthood (Kwieciński and Nowak
2009; Nowak et al.
2010,
2011; Brus et al.
2012; Szkilnik et al.
2014). It is evident that Mn in excess is toxic to human embryos and fetuses (Colomina et al.
1996). The progressive and latent nature of some neurodegenerative disorders (e.g., Parkinson’s disease) suggest that the triggering event for these disorders occurs much sooner than the appearance of visible symptoms. In humans, only after the loss of about 80 % of pars compacta substantia nigra DA neurons do symptoms (e.g., Parkinsonian tremor) arise. Therefore, it is important to identify possible environmental trigger(s), to pinpoint the period during which such factors pose the greatest risk, and to determine the mechanism(s) involved.
The current study was conducted in order to assess the effects of paired gestational Mn exposure with overt dopaminergic neurotoxicity (i.e., 6-hydroxydopamine, 6-OHDA) on hydroxyl radical (HO
•) production in rat brain. A better understanding of the causes of HO
• production in brain and the effects of on neurodegenerative processes are especially important because the brain is at high risk for oxidative injury because of (1) high levels of oxygen consumption combined with (2) low catalase, superoxide dismutase, and glutathione peroxidase activity, (3) high iron concentration, and (4) elevated polysaturated fatty acid content of neuronal membranes (Halliwell
2006).
Discussion
In the present study, we demonstrated that Mn content in the brain (frontal cortex, hippocampus, and neostriatum), kidney, liver, and bone was significantly elevated in rat dams exposed to this metal during pregnancy. Conversely, in neonates (P14) whose mothers were exposed to Mn during the prenatal and perinatal periods, Mn primarily accumulated in the femoral bone and liver. In P14 pups, no significant differences in Mn content in other tissues (brain, kidney, femoral muscle, and heart) were noted in comparison to control. Interestingly, Mn accumulation in the femoral bone and liver was not observed in 8-week-old Mn-exposed rats. This is in agreement with other reports indicating that Mn can be transiently deposited in various rat tissues (mostly in the liver), both after parenteral and oral administration (Roels et al.
1997). Dorman et al. (
2005) showed that newborn rats exposed to Mn via aerosol inhalation during lactation had a 2–3-fold increase in Mn content in brain and in other tissues (stomach, blood, liver, and skull cap); however, following cessation of Mn administration, tissue Mn concentrations returned to control values by P45 in all offspring tissues. Others have demonstrated that the level of Mn in perinatally exposed pups was 6–8 times higher than in controls. When Mn intoxication was discontinued, the Mn contents decreased nearly to control levels (Kostial et al.
2005). The data cited above largely corroborate the present findings. It is notable that Siddappa et al. (
2002) and Garcia et al. (
2006) observed that the expressions of proteins involved in Mn transport, i.e., divalent metal transporter-1 (DMT-1) or transferrin receptors in the central nervous system (cerebral cortex, hippocampus, and neostriatum), appear in rats at P5. This may, at least in part, explain the results of our study concerning the lack of accumulation of this metal in the brains of neonates (14-day-old rats). These studies, as well as our own observations, indicate that Mn crosses the placenta. Although, in contrast to the other heavy metals (cadmium, lead) (Brus et al.
1995; Nowak et al.
2006; Szczerbak et al.
2007), Mn is not deposited in the tissue for long periods of time. Following cessation of Mn exposure, Mn is very quickly and efficiently eliminated by the body.
To determine the possible role of HO
• generation in Mn-induced neurotoxicity, we estimated the content of 2.3- and 2.5-DHBA (spin trap products of salicylate; HO
• provides an index of in vivo reactive oxygen species generation). We demonstrated that DA denervation resulted in enhancement of HO
• formation in the brains of newborn rats. Similar data were obtained from adult animals (8-week-old rats lesioned with 6-OHDA at a dose of 30, 60, or 134 µg at P3). In the current study, 2.3-DHBA and 2.5-DHBA contents were significantly increased in the frontal cortex, hippocampus, neostriatum, thalamus with hypothalamus, and pons of 6-OHDA-lesioned rats. Additionally, there was no association between the extent of DA denervation and increased HO
• formation. These data are in agreement with the results published by Kostrzewa et al. (
2000) who found that in the DA-denervated neostriatum (6-OHDA, 134 µg), 2.3-DHBA was increased more than 4-fold, and 2.5-DHBA was increased 2.5-fold in comparison to fully DA-innervated rats.
An important novelty of our work is the demonstration that HO
• overproduction was observed not only in rats with profound dopaminergic system damage (6-OHDA, 134 μg), but also in animals with intermediate (6-OHDA, 60 μg) and minor (6-OHDA, 30 μg) DA depletion. This indicates that even modest injury to the dopaminergic system acts as a “trigger mechanism,” initiating a cascade of adverse signaling events that lead to a protracted increase in HO
• generation. Concurrently, we showed that if this process is launched in early postnatal life (14-day-old rats), then it persists throughout adult life. One hypothesis that could explain this phenomenon is that 6-OHDA administered to laboratory animals induces up-regulation of DMT-1 localized on DA neurons, with subsequent increases in iron overload—a possible substrate for HO
• production (Fenton reaction) (Song et al.
2007; Jiang et al.
2010). Furthermore, others found reduced expression of ferroportin 1 and hephaestin in the substantia nigra of 6-OHDA-lesioned rats. These two iron export proteins are involved in removal of this metal from neurons in the substantia nigra (Wang et al.
2007). This hypothesis could also serve as an explanation for the absence of changes in DHBA in the cerebellum, since the cerebellum has sparse dopaminergic innervation, and abnormal iron homeostasis seems to be of marginal relevance to this part of the brain.
In the present work, we also showed that perinatal Mn exposure increases the generation of HO• in the brains of newborn rats; in the frontal cortex, hippocampus, thalamus with hypothalamus, and partly in the pons at 8-weeks. Moreover, 6-OHDA-induced DA denervation enhanced this effect in the neostriatum at P14 and through 8 weeks.
Because of the latter effect, it was of interest to assess neostriatal extraneuronal HO
• levels. We found that microdialysate levels of both 2.3- and 2.5-DHBA were significantly elevated in DA-denervated neostriatum. These results were counter to our expectation because the extraneuronal compartment is effectively protected by various antioxidants (e.g., ascorbic acid, uric acid, etc.) prior to HO
• formation. Additionally, endogenous melatonin may play an active role in maintaining oxidative homeostasis in the extracellular compartment of the neostriatum (Rocchitta et al.
2005). Extracellular ascorbic acid concentrations in the neostriatum range between 350 and 500 µM, similarly to uric acid; and increases by ~50 % following an increase in evoked DA release (e.g., after systemic amphetamine injection) (Miele and Fillenz
1996).
Interestingly, no differences were found in microdialysate contents of 2.3- and 2.5-DHBA in the neostriatum between Mn-exposed and control rats. These results are counter to findings on isolated neostriatal tissues. However, in isolated neostriatal tissues, the measurement of the intensity in HO
• generation reflects an intracellular compartment, which represents more than 90 % of its weight, as well as the extraneuronal compartment. Furthermore, extracellular HO
• is dwarfed by the intraneuronal environment wherein the majority of metabolic processes occur (mitochondria, lysosomes, etc.). These considerations find confirmation in the work by Milatovic et al. (
2007,
2009) who showed that Mn induces neuronal damage through oxidative injury and mitochondrial dysfunction (intracellular compartment). Conversely, it is known that DA is a source of free radicals, which are formed during its enzymatic metabolism (monoamine oxidase), or by non-enzymatic autoxidation that leads to production of highly reactive DA quinones and DA semiquinones (Halliwell
2006). Under conditions of a marked DA deficit, glutamate could initiate free radicals formation through stimulation of intracellular signaling, in the process termed excitotoxicity. This hypothesis is supported by the study of Golembiowska and Dziubina (
2012) who showed that a marked increase in striatal extracellular glutamate level in rats with 6-OHDA-induced DA depletion could account for enhanced extraneuronal formation of hydroxyl radicals. Conversely, restoration of the striatal DA-glutamate balance suppressed 6-OHDA-induced overproduction of hydroxyl radical. In our recently published study in which the same experimental model was engaged (as in the present work), we found no difference in extraneuronal DA in control versus Mn-exposed rats (Szkilnik et al.
2014). Under the assumption that Mn exposure minimally affected the extraneuronal milieu (i.e., DA release), we cannot expect overt changes in extraneuronal hydroxyl radical formation.
DHBA studies coincide with antioxidant enzyme activity deterioration that we found in the brains of rats lesioned with 6-OHDA and/or intoxicated with Mn. The most prominent impairments were observed in the prefrontal cortex, striatum, and brain stem i.e., significant decrease in activity of SOD isoenzymes and GST was noted in 6-OHDA-, Mn-, and Mn + 6-OHDA groups in comparison to control. In mammals, SODs represent the major antioxidant defense system against superoxide anion (O
2
•−
). We hypothesize that ineffectiveness of SOD activity may result in accumulation of O
2
•−
which, in turn, in the presence of hydrogen peroxide (H
2O
2) (Haber–Weiss reaction) brings about enhancement of HO
• generation, the effect demonstrated in the current study. Furthermore, from our work, we have also learned that H
2O
2 cleavage mechanisms are nearly unaffected (in exception with prefrontal cortex and hippocampus) because negligible changes in catalase and glutathione-associated enzymes activity were found. Our data are in agreement with others who also demonstrated that 6-OHDA treatment resulted in impairment (decrease) in antioxidant enzymes activity in rats and mice (Ahmad et al.
2012; Haleagrahara et al.
2013). Also, Santos et al. (
2012) found that in rats injected ip for 4 or 8 days with 25 mg MnCl
2/kg/day, a significant increase in Mn-SOD protein expression was noted in brains after 4 Mn doses, but the expressions of these proteins were decreased after 8 Mn doses. Chtourou et al. (
2010) demonstrated that Mn intoxication (20 mg/ml manganese chloride in drinking water for 30 days) was accompanied by a decrease of enzymatic (SOD, CAT, and GPx) and non-enzymatic (glutathione and ascorbic acid) antioxidants in the rat’s cerebral cortex.
In summary, the current study demonstrates HO• overproduction in brain tissue (prefrontal cortex, hippocampus, thalamus/hypothalamus, striatum) and in striatal in vivo (extraneuronal) microdialysates of adulthood rats, despite lack of a measurable residual of Mn consequent to perinatal Mn exposure (i.e., Mn addition to the drinking water of mother rats during gestation and through the 21 day suckling period). Moreover, HO• elevation was enhanced in perinatal Mn-exposed rats by mild-, moderate-, or extensive-postnatal 6-OHDA lesioning of dopaminergic neurons, which alone resulted in increased tissue- and microdialysate levels of HO•. On the basis of this study, it appears that perinatal Mn exposure may represent a life-long risk toward the incidence or severity of neurodegenerative disorders, such as Parkinson’s disease, in humans.