| ObjectiveCombining with neural behavior, serum neural endocrine hormone and the concentration of metal elements in the blood and urine and the correlations among them, this cross-sectional study was to observe the Mn accumulation in the brain and Mn-induced pathological, functional and metabolic changes in the brain by applicating a non-invasive magnetic resonance imaging (MRI) and hydrogen proton magnetic resonance spectrum (’H-MRS). We aimed to explore the possibility of MRI as a noninvasive and early detection method for early diagnosis of Mn neurotoxicity and looking for central and peripheral biomarkers of subclinical manganism, providing scientific basis for screening manganism susceptible popolations and early diagnosis and prevention of chronic manganism of asymptomatic occupational Mn-exposed workers.Methods and materials(1) 19 male Mn-exposed welder workers, working in an construction machinery manufacturing company of Guangxi Province and 43 age--matched control subjects with no history of Mn exposure participated in this study. And the Mn-exposed group were divided into 3 subgroups according the lengths ofworking:<5y,5～10 y and>10 y group. Mn concentrations in air samples and red blood cells were measured by using flame atomic absorption spectrometry (AAS) and inductively coupled plasma emission mass spectrometry (ICP-MS), respectively Alteration of T1-weighted imaging (T1-WI) high signal, n-acetyl aspartate (NAA), creatine (Cr), choline (Cho), inositol(ml), glutamic acid compounds (GLX) peak height in the basal ganglia pallidum were detected by using magnetic resonance imaging (MRI) and’H-MRS.Then the pallidum index (PI), NAA/Cr, Cho/Cr, mI/Cr and that GLX/Cr ratio were calculated.(2) 9 smelter workers worked in a ferro alloy factory were selected as the high Mn-exposed group(MnH). And 14 welder workers from machine manufacturing factory were selected as the low Mn-exposed group(MnL), while 23 workers from a property management company without Mn exposure were selected as the control group. All participators are residents of Guangxi. Neurobehavioral changes of workers were measured by using the purdue pegboard test(PPT) with calculating the right hand test value (PPTR), left hand test (PPTL), both hands test value (PPTB), assembly value (PPTA) and the summation of the test (PPTS). Mn concentrations in the erythrocyte (MnE) and air were detected by using AAS and ICP-MS, respectively. Glutamate (Glu), gamma-aminobutyric acid (GABA), Cho,NAA,Cr mI, glycerol phosphate choline (GPC) and glutathione (GSH) peak height in the frontal cortex, posterior cingulate cortex, hippocampus and thalamus were detected by using’H-MRS. The ratio each of metabolite and water height of water peak were calculated. Among these metabolism, GABA need to apply a special editor MRS (MEGA-PRESS) scanning sequence scan.(3) 62 male smelter workers from a ferro alloy factory were selected as the high Mn-exposed group(MnH). And 62 welder workers from a machine manufacturing factory were selected as the low Mn-exposed group(MnL), while 43 workers from the same city without Mn exposure were selected as the control group. The groups were divided into<40 and>40 years old subgroup according to age, and<5,5-10 and>10 y subgroups according to the Mn-exposed length. The levels of Prolactin (PRL), luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone (TST) and thyroid stimulating hormone (TSH) s in the serum of workers were detected by using chemiluminescence immunoassay.(4) 80 male smelting workers were slected as high manganese exposure group(MnH),179 welder workers were slected as low manganese exposure group(MnL).48 workers without Mn exposure and contacting other harmful substances in the same city were slected as the control group. The contents of Mn, Zn, Cu, Fe, Ca, Mg and Al in the erythrocytee and urine were detected by using ICP-MS.Results1. Changes of metabolite levels in globus pallidus (basal ganglia) of welder caused by manganese exposure(1) The geometric mean concentration of MnO2 in welding work environment was 0.03 mg/m3 (0.003-0.519 mg/m3,which was Lower than the time weighted average allowable concentration of MnO2 in our current workplace air(PC-TWA, GBZ2.1-2007.0.15 mg/m3). The age of exposed and control groups workers were 32.4±8.2 and 36.7±9.5 years, respectively. There were no significant differences in age, smoking rates and alcohol consumption rates between the Mn-exposed and control group. The time of exposed group workers contact manganese was 10.5±6.6 years. Mn levels in the red blood cells of Mn-exposed group were significantly higher than those of control.(2) the results of MRI.33 of the 43 workers of Mn-exposed group showed a symmetric high signal intensity on T1-WI images in both sides of the globus pallidus. The high signal intensity rate was 76.7%.The control group showed no T1-WI hyperintensity in the globus pallidus. Compared with the control group, PI values of the Mn-exposed group were increased (P<0.01).There was also no significant difference among the PI values of working length subgroups (P>0.05)(3) the results of’H-MRS.Compared with the control group, Glx/Cr ration of the Mn-exposed group was significantly increased,however, the Cho/Cr and ml/Cr ratios were robustly decreased (P<0.05 or 0.01). There was also no significant difference among the Cho/Cr, mI/Cr and Glx/Cr rates of working length subgroup (P>0.05)(4) Correlation analysis.The levels of Mn in erythrocytee of Mn-exposed group displayed a positive correlation with NAA/Cr ratio (r=0.312, P=0.042), mI/Cr displayed a positive correlation with Cho/Cr (r=0.314,P=0.040) and Glx/Cr (r=0.515,P=0.000) ratios.2. Brain metabolite levels of workers under different occupational manganese exposure levels (1) The mean concentration of MnO2 in smelting working environments (MnH) and welding working environment were 0.597 mg/m3 (0.022-4.994 mg/m3) and 0.075 mg/m3 (0.0018-0.519 mg/m3), respectively. There were no significant differences in age, smoking rates and alcohol consumption rates among these three groups (P>0.05). The working length of Mn exposure in MnH group were significantly higher than MnL group. The Mn levels in red blood cells and urine of the MnL and MnH groups were significantly higher than those of the control group. The Mn levels in red blood cells of MnH group were significantly higher than that of MnL group(P<0.05-0.01).(2)The results of neurobehavioral test.The PPTS scores of the MnL group were significantly lower than those in the control.(3)The changes of nerve metabolite in the brain regions:Compared with the control, the levels of GSH in the frontal cortex and GPC in the posterior cingulate cortex, ml in the hippocampus, Glu and GSH in the thalamus of MnL group were robustly decreased. Forthermorethe levels of Glu in the frontal cortex and mI in the thalamus of MnL and MnH group were significantly lower than those of the control (P <0.05-0.01).(4) The results of correlation analysis.①The GAB A of MnH group displayed a negative correlation with the Purdue pegboard test scores PPTR(r=-0.721, P=0.029, PPTL(r=-0.806, P=0.009), PPTB(r=-0.816, P=0.007), PPTA(r=-0.931, P=0.000), PPTR(r=-0.922, P=0.000).②The GABA/tCr of Combined group (control+MnH group) displayed a negative correlation with the Purdue pegboard test scores PPTR(r=-0.373, P=0.036), PPTL(r=-0.388, P=0.028), PPTB(r=-0.510, P=0.003), PPTA(r=-0.392, P=0.027), PPTR(r=-0.439, P=0.012). ③The GABA/tCr of MnH group displayed a negative correlation with the Purdue pegboard test scores PPTR(r=-0.832, P=0.010), PPTL(r=-0.739, P=0.036), PPTB(r=-0.781, P=0.022), PPTA(r=-0.843, P=0.009), PPTR(r=-0.870, P=0.005). However, there were no correlations between working length of Mn-exposed, Mn levels in red blood cells and all of the Purduepegboard test scores. ④MnU of MnH group displayed a positive correlation with the GABA/tCr in thalamus. But there were no correlations between the GABA/tCr in thalamus and Mn-exposed length (r=0.505, P=0.166) and MnE(r=0.074, P=0.861).3. Neuroendocrine hormone levels in serum of workers under different occupational manganese exposure levels(1) Age, length of Mn-exposed, smoking and drinking rates of MnH group were significantly higher than those of the MnL and control groups (P<0.05-0.01). Compared with the contro group, MnU of MnL and MnH group were significantly increased. MnE of MnH group were significantly higher than those in the MnL group and control group (P<0.05-0.01).(2) The results of serum neuroendocrine hormones:①The concentration of TSH in the serum of MnL group was significantly lower than those in the control group. The concentrations of PRL, TST, TSH in the serum of MnH group were significantly lower than those of the control and MnL group. However, The concentration of LH in the serum of MnH group were significantly higher than that of the MnL group (P<0.01, table3).②The age stratified analysis:The concentration of TSH in the<40a-MnL group was significantly lower than that in<40a～control group. The concentrations of PRL, TST and TSH of<40a-MnH group were significantly lower than those of the<40a～control and<40a-MnL group, but the concentration of LH was higher than that in the<40a-MnL group. LH concentration of the 40a-MnL group was significantly lower than that in 40a～control group. PRL and TSH concentrations of 40a-MnH group was significantly lower than those in the 40a～control group, while the LH concentration was significantly higher than that of the<40a-MnL group(P<0.05～0.01).③The length of working stratified analysis:Compared with the control group, LH and TST concentration of 5～10 y MnL subgroup were significantly decreased, while the TST concentration of<5y～MnL subgroup was increased. LH concentration of 5-10y and>10y～MnL subgroup was lower than<5y～MnL subgroup, while the LH concentration of>10y～MnL subgroup were significantly higher than that of 5-10y MnL subgroup. With the Mn exposed length increasing, TST concentration showed first increased and then decreased (P<0.05-0.01).(3) Correlation analysis:Age was positively correlated with MnE, serum LH and FSH, and negative correlated with the serum PRL and TST. Mn-exposed length was positively correlated with MnE, LH,FSH,but negatively correlated with PRL. Furthermore, MnE was positively correlated with MnU, LH,but negatively correlated with PRL,TST. MnU was positively correlated with LH,but negatively correlated with PRL. The LH in the serum was positively correlated with the serum FSH (P<0.05-0.01).4. The levels of metal elements in blood and urine of workers under different occupational manganese exposure levels(1) There are no statistical difference on age, smoking and drinking rates between MnL group and the control group. The age of MnH group was significantly higher than those in control and MnL group. Mn-exposed length of MnH group was significantly higher than MnL group, smoking rate of MnH group was significantly higher than that of control group, drinking rate of MnH group was significantly higher than the control and MnL group (P<0.05-0.01).(2) The contents of Ca and Al in the erythrocyte of MnL group was obviously lower than the control group. Compared with control group and MnL group, The contents of Mn, Fe, Ca, Mg and Zn the erythrocyte of MnH were significantly increased, while Cu and Al was significantly decreased (P<0.05-0.01).(3) The contents of Mn, Ca and Mg levels in urine of MnL group were higher than those of the control group. In addition, the contents of Mn, Fe, Ca and Zn in urine of MnH group were significantly higher than those in the control and MnL groups. Compared with the control group, the contents of Cu and Mg in urine of MnH group were obviously higher(P<0.05-0.01).(4) Correlation analysis:①There was positive correlation between MnE and age,length of Mn-exposue,Al, Ca, Fe, Zn in the erythrocyte of Mn-exposued group(P<0.01); There was positive correlation between age, length of Mn-exposue and Al, Ca, Fe, Zn in the erythrocyte of Mn-exposued group(P <0.01).②There was positive correlation between MnU and age,length of Mn-exposue, Fe,Zn in the urine of Mn-exposued group(P<0.01). There was inverse correlation between age,length of Mn-exposue and Al in the urine of Mn-exposued group; There was positive correlation between age,length of Mn-exposue and Fe, Zn in the urine of Mn-exposued group; There was inverse correlation between length of Mn-exposue and Mg in the urine of Mn-exposued group(P<0.05 or P<0.01).Conclusions1. Long-term,low levels of Mn exposure can lead to the accumulation of Mn in the globus pallidus, including the increasing of MnE, the high symmetry T1-WI signal appeared in the basal ganglia pallidum, and the increasing of PI values. These results indicates that high symmetry T1-WI signal and PI value of globus pallidus may be a sensitive index in evaluating the accumulation of Mn in the brain.2. Different levels of Mn exposure may produce changes of metabolites(including Cho, ml,d GLX, Glu, GPC, GSH) in different brain regions(globus pallidus, frontal cortex, thalamus, posterior cingulate cortex, hippocampus). These suggests that excessive Mn exposure may cause changes of brain function and metabolic.’H-MRS is an effective methods to assess brain function and metabolism changes in the asymptomatic occupational Mn-exposed workers.3. There is a negative correlation between thalamic GABA levels and purdue roof test scores. This indicates that GABA may be a subclinical biomarker of Mn-induced movement disorder.4. The Mn concentration of smelting workplace was higher than welding workplace, while the changes of brain metabolites of welders was more serious than the smelters. This suggests that the Mn neurotoxicity may be related to the features of Mn dust/fume, such as particle size, sedimentation rate and solubility. However, more research is needed to confirm this phenominon.5. Different levels of Mn exposure may affect on the levels of serum PRL, LH, FSH, TST and TSH, and it is more obvious in high levels of Mn exposure group. And this suggestes that PRL, TST, TSH in the serum may be the peripheral biomarkers of Mn neurotoxicity.6. Different levels of Mn exposure may cause changes of the levels of Mn, Zn, Cu, Fe, Ca, Mg and Al in red blood cells and urine, and it is more obvious in high levels of Mn exposure group. This suggests that excessive Mn exposure may disturb the homeostasis of metal elements and they may be the peripheral biomarkers of Mn neurotoxicity. |
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