Research ReportProlactin is a peripheral marker of manganese neurotoxicity☆
Research Highlights
►Mn decreases spontaneous motor activity, both ambulation and rearing. ►Mn reduces brain glutathione levels, suggesting Mn-induced production of ROS. ►Mn increases serum prolactin levels, indicating changes in dopaminergic function. ►A good correlation exists between brain Mn content and prolactin serum levels. ►Prolactin may serve as a predictive biomarker of Mn-induced neurotoxic effects.
Introduction
Manganese (Mn) is an abundant and essential metal acquired naturally through regular dietary intake (Aschner et al., 2002). Accordingly, Mn levels in organs are usually kept at optimal concentrations (Michalke et al., 2007, Au et al., 2008). Blood Mn levels are maintained by both gastrointestinal (GI) absorption and efficient biliary excretion, the latter representing the main route for Mn elimination from the body (Dorman et al., 2006, Greenberg and Campbell, 1940). In general, humans are exposed to low levels of Mn in water, air and food. Nevertheless, Mn can accumulate in certain brain regions following elevated exposures, and Mn-induced neurotoxicity may ensue. Overexposure to Mn can also occur in occupational environments, and cases of Mn neurotoxicity (manganism) have been reported particularly in miners, smelters and workers in the alloy industry where exposures occur predominantly via the inhalation of Mn fumes or Mn-containing dusts (ATSDR (Agency for Toxic Substances and Diseases), 2000, Aschner et al., 2007). The gasoline additive, methylcyclopentadyenyl manganese tricarbonyl (MMT), is another source of airborne Mn (Kaiser, 2003).
The early onset of Mn intoxication is typically subtle, but once established, usually becomes progressive and irreversible, leading to permanent neurological damage (Jiang et al., 2006, Aschner et al., 2007). Thus, early diagnosis is crucial for preventing Mn neurotoxicity in settings of occupational and environmental exposure. Once absorbed, Mn is rapidly distributed throughout the body, concentrating primarily in the bones and in metabolically active organs. However, in all studied mammalian species, including humans, the brain represents the primary target tissue in which Mn persists for the longest duration (Maeda et al., 1997, Lucchini et al., 2000, Crossgrove and Zheng, 2004, Dorman et al., 2006, Kim et al., 2007). T1-weighted magnetic resonance imaging (MRI) corroborates the accumulation of Mn predominantly in the basal ganglia, namely in the globus pallidus, striatum and substantia nigra pars reticulata (Cersosimo and Koller, 2006, Reaney et al., 2006, Roth, 2006).
The precise mechanisms by which Mn enters the brain are not yet fully understood. As an essential metal, it readily crosses the blood-brain-barrier (BBB) in both the adult and the developing fetus (Aschner and Aschner, 1991). Recently, Aschner et al., 2007, Au et al., 2008 documented that facilitated diffusion, active transport, divalent metal transport 1 (DMT-1; also known as DCT-1/NRAMP2)-mediated transport and transferrin (Tf)-dependent transport mechanisms are involved in shuttling Mn across the BBB. Mn accumulates in brain regions that are normally rich in iron, most likely due to the fact that the two metals share common transporters (Tf and DMT-1). Studies in Fe-deficient rats have shown Mn accumulation in the globus pallidus, hippocampus and substantia nigra (ATSDR, 2000), corroborating a close relationship between low Fe levels and increased Mn uptake into the brain.
Although Mn is a component of several critical enzymes that prevent cellular oxidative stress, such as mitochondrial superoxide dismutase (Aschner et al., 2002, Crossgrove and Zheng, 2004), it can also interact negatively with cellular dopamine, promoting dopamine autoxidation and causing dopaminergic cell death. Hypo- or hyper-activity in dopaminergic neurotransmission leads to a number of disorders, such as Parkinson's disease and schizophrenia (Ben-Jonathan and Hnasko, 2001). Dopamine also acts by binding to D2-receptors on specific pituitary cells (lactrophs) which are responsible for the secretion of prolactin, thereby inhibiting both the release and the synthesis of this neurohormone (Ellingsen et al., 2003, Takser et al., 2004, Ellingsen et al., 2007). In addition, hypothalamic dopaminergic neurons, which provide dopaminergic efferents to the anterior pituitary gland are themselves regulated by feedback from prolactin through a short-loop feedback mechanism. A variety of other modulators of prolactin secretion (e.g., serotonin, GABA, estrogens and opioids) act at the hypothalamic level by inhibiting or reinforcing the dopaminergic tone (e.g., substance P) (Fitzgerald and Dinan, 2008). Evidence proving that dopamine regulates prolactin release in humans has also been corroborated by observations that drugs which interfere with its release affect circulating prolactin levels (Ben-Jonathan and Hnasko, 2001).
Since Mn stimulates dopamine autoxidation in dopaminergic neurons, it indirectly modulates prolactin secretion, thereby leading to an increase in circulating prolactin levels. Recently, Kim et al. (2009) studied mechanisms that govern changes in rat brain dopamine levels and prolactin production by evaluating the transacting factor of the prolactin gene (PIT-1). The results of these studies showed a increases in PIT-1 and prolactin, as well as a significant decrease in dopamine levels in rats exposed to Mn. These results are consistent with the role of PIT-1 as a regulator of both dopamine and prolactin levels.
Occupational biomonitoring in populations at risk for Mn exposure has attempted to address the utility of prolactin as a biological index of exposure (Sloot et al., 1996, Smargiassi and Mutti, 1999). Indeed, a strong relationship between Mn exposure and serum prolactin levels has been established in multiple studies (Aschner, 2006, Kim et al., 2007). Mn was positively associated with prolactin in human neonates (Takser et al., 2004) and occupationally exposed men (Mutti et al., 1996, Smargiassi and Mutti, 1999, Ellingsen et al., 2007). However, negative relationships between Mn and prolactin have also been reported (Roels et al., 1992, Popek et al., 2006). For example, Roels et al. (1992) found that serum prolactin as a predictive measure of effect was unrelated to atmospheric Mn exposure. Such differences in outcomes may be attributable to different exposure times and differences in accumulated brain Mn levels.
In addition to its effect on the dopaminergic system, Mn also inhibits mitochondrial respiration and antioxidant systems. Mn readily induces free radical formation and oxidative stress (Chen and Liao, 2002, Hazell and Normandin, 2002; Erikson et al., 2004, Marreilha dos Santos et al., 2010) leading to the excessive production of reactive oxygen species (ROS). ROS generation depletes GSH, perturbing the intracellular redox balance as well as the conjugation and excretion of toxic molecules (Meister, 1988, Meister, 1991, Marreilha dos Santos et al., 2008). In vitro studies have shown that the inhibition of GSH synthesis and the consequent impairment of neuronal antioxidant system activity play a detrimental role in oxidative stress-mediated Mn neurotoxicity (Desole et al., 1997, Marreilha dos Santos et al., 2010). Dorman et al. (2000) detected significantly decreased striatal GSH levels following subchronic Mn sulfate (MnSO4) inhalation. A similar mechanism for Mn neurotoxicity was proposed by Erikson et al. (2004) using lipid peroxides, GSH and metallothionein as biomarkers of free radical production.
The identification and validation of biomarkers is crucial to human toxicology (Smith et al., 2007). To prevent the neurotoxic effects resulting from chronic exposure to Mn, at-risk populations must receive initial and subsequent examinations and necessary follow-up treatment using specific, sensitive and predictive biomarkers. Occupational exposure to Mn has been largely monitored by the determination of Mn levels in blood and urine. Lucchini et al. (1999) observed an association between blood Mn and exposure levels. Apostoli et al. (2000) investigated the suitability of blood and urine Mn levels for exposure assessment, concluding that the two measures could discriminate only between groups of occupationally exposed and control subjects; however, within the exposed groups, there was no association between blood and urinary Mn levels and Mn exposure. Furthermore, blood and urine Mn analyses did not provide information about previous exposures, due to Mn's relatively short blood half-life (t1/2 < 2 h) (Li et al., 2006). Accordingly, published data question the utility of Mn blood and urine levels as biomarkers of chronic exposure, as they reveal substantial variability in Mn levels in exposed subjects. Since clinical diagnostic medicine relies on effect-related biomarkers (Liu et al., 2008) and the currently available tests (Mn in blood and urine) fail to establish this relationship, attention must be focused on alternative indicators (Smargiassi and Mutti, 1999, Apostoli et al., 2000, Montes et al., 2008).
Given these deficiencies in biomonitoring, it is prudent to perform in vivo assays to evaluate the relationship between biomarkers of exposure (blood and brain Mn levels) and effect (prolactin, GSH and behavioral assays). Accordingly, the present study was designed to address the consequences of repeated exposures to MnCl2 and to correlate its neurotoxic sequalae with reliable and predictive biomarkers of exposure (prolactin), which, in the future, could serve to validate risk in Mn-exposed human populations.
Section snippets
Animal weight
The rats' weights were recorded on the first day of treatment and after the 4 and 8 doses of Mn, before the sacrifice. As shown in Table 1, on comparing the rats' weights after the 4 and 8 doses of Mn with the respective controls, there was a reduction in the body weight gain in both treated groups but that reduction was only significant for the 8 doses group (p < 0,001).
Behavioral assays
Behavioral parameters were studied in control and Mn-treated rats (8 doses) at the beginning of the experiment and 24 h after
Discussion
The control and prevention of chronic Mn-induced neurotoxicity has particular clinical relevance in occupational, clinical and environmental toxicology as overexposure to this metal can lead to progressive and permanent neurodegenerative damage, resulting in a syndrome analogous to idiopathic Parkinson's disease (Aschner et al., 2002, Crossgrove and Zheng, 2004, Aschner et al., 2007). The need to biomonitor populations at risk has become increasingly important in preventing early and
Chemicals
Manganese chloride tetrahydrate (MnCl2∙ 4H2O, 99.99%: Sigma-Aldrich), nitric acid (HNO3, 65% suprapure: Merck), hydrogen peroxide (H2O2, 30%: Aldrich), sterile saline solution.
Animals
Experiments were performed according to the Guidelines for Animal Experimentation as set forth by the NIH. Male Wistar rats (Charles River Laboratories®, Barcelona, Spain) weighing 150–171 g were used for the experiments. Animals were housed in an isolated room and adapted to controlled environmental conditions for 10 days
Acknowledgments
The authors wish to thank Doctor Rita Castro for her support with the HPLC analysis and Dr. Virginia Carvalho for assisting in the GFAAS analysis.
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