Cross-sectional study of expression of divalent metal transporter-1, transferrin, and hepcidin in blood of smelters who are occupationally exposed to manganese

Study population

This cross-sectional human study was approved by the Office of Clinical Investigation at the ZMC and the Institutional Review Board for human study at Purdue University. Subjects were recruited from a Mn–Fe ferroalloy smelting factory for the Mn-exposed workers or from an Fe only smelting factory as the control subjects; both factories are located in Zunyi city of Guizhou province in China. Workers in both smelting factories usually operated the smelting process within 2–5 m of the smelting furnaces. The workers worked 7–8 h per day with the average employment history of 5.2 years. Both groups were also matched for socioeconomic status (salary, education, etc.) and background environmental factors (place of residence, etc.). Subjects in these two groups at the time of interview had no reported exposure to other toxic substances, radiation therapy or substance abuse. Subjects who had taken medications which could interfere with Fe metabolism, such as vitamin D, aspirin or herbal supplements, were excluded from the study.

Determination of airborne Mn levels in work sites

Airborne Mn level were evaluated by individual sampler with the SKC pumps (model 224-44XR, calibrated at 2 L/min), tygon tubing and a sampling cassette with a close-face MCE filter (37 mm, pore size: 0.8 µm) within the worker’s breathing zone. A time-weighted average concentration was calculated following each 8 h work shift. Mn and Fe were determined by atomic absorption spectrophotometry (AAS) as briefly described below and in our published papers (Cowan et al., 2009a; Cowan et al., 2009b).

Physical examination and blood sample collection

Clinical examination took place in the Zunyi Medical College, Zunyi, China. The written informed consent forms were obtained from all subjects prior to interview and physical examination. The study protocol received official approvals from the Office of Clinical Investigation at the ZMC and the human study Institutional Review Board at Purdue University. Participants agreed to use their blood samples for this biological monitoring research. The participants were asked to fast overnight prior to the study. Blood samples were collected in a heparinized tube and stood at room temperature for 30 min. After centrifuging at 600 g/min for 5 min, the supernatant and pellet were collected as the plasma fraction and the erythrocytes fraction, respectively for Mn and Fe analysis (Cowan et al., 2009a; Cowan et al., 2009b). For gene expression analysis, blood samples (0.5 ml of venous blood) were collected into autoclaved tubes (pre-loaded with 0.7 ml of Trizol). All samples were stored at −20 °C prior to laboratory analyses.

Determination of Mn and Fe concentrations in air or blood samples

For analysis of Mn in air samples, the sampled filters were completely digested with 5 mL of HClO₄–HNO₃mixture (1:9 vol/vol) at 200 °C until dry, and the residues were dissolved in 10 mL of 1% HNO₃ and diluted for AAS analysis.

For analysis of Mn and Fe in plasma, an aliquot (0.5 mL) of plasma was mixed with 5 mL of digestion solution (HClO₄:HNO₃; 3:7), stood at room temperature for 4–6 hr, and then heated until dry. The dried samples were then re-dissolved in 5 mL of 1% HNO₃. For analysis of metals in erythrocytes, approximately 1.5 g of the pellet blood cells was mixed with saline to a volume of 4 mL. An aliquot (2 mL) of this fraction was mixed with 10 mL of the digestion solution and heated until dry; 5 mL of 1% HNO₃ was added to dissolve the residue. The solution was diluted appropriately prior to atomic absorption spectrophotometry (AAS).

Mn and Fe concentrations were determined by AA240FS Varian AAS (Australia Pty Ltd.) using a China National Standard Operation Protocol (GB/T16018-1995) for occupational safety surveillance. The detection limits of graphite furnace AAS for Mn and Fe were 0.1 ng/mL and 0.09 ng/mL, respectively.

Real-time RT-PCR analysis

The total RNA was extracted with TRIzol (Invitrogen, Carlsbad, CA), according to the Manufacturer’s recommendations, followed by purification process (Qiagen, Valencia, CA). The purified RNA was conveyed to cDNA with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). The forward and reverse primer sequences for selected genes were designed with the ABI Primer Express software (Foster City, CA) and listed in Table 1. Power SYBR Green Master Mix (Applied Biosystems, Cheshire, UK) was used for real-time PCR analysis. The relative differences in expression between groups were expressed using cycle time (Ct) values as follows: the Ct values of the interested genes were first normalized with β-actin of the same sample, and then the relative differences between control and treatment groups were calculated and expressed as relative increases, setting control as 100%.

Data calculation and statistical analysis

Records of interviews and other reports were reviewed and abstracted for demographic data. All data are expressed as the mean ± SD unless otherwise stated. The cumulative Mn exposure for each worker was calculated by multiplying airborne Mn concentrations with years of employment, assuming 8 h per working day and 5 days per week. Linear regression models and Spearman correlation coefficients were used to evaluate the associations between mRNA expressions and Mn/Fe concentrations in plasma and erythrocytes, and between mRNA expressions and the accumulative Mn exposure, following the data transformation to logarithm. This transformation is valid with regards to the symmetric distribution and the linearity of variables after the logarithm transformation. Differences between two means were considered significant when p value was less than 0.05. Data were analyzed using SPSS13.0 software.

Materials

Chemicals were obtained from the following sources: AAS standards of all metals from Alfa Products (Danvers, MA, USA). All reagents were of analytical grade, HPLC grade or the highest available pharmaceutical grade.

Demographic and Mn exposure in the study population

The information on the study population, age and gender differences, Mn exposure and accumulated Mn exposures are summarized in Table 2. The subjects in Mn-exposed group were about 3 years younger than those of controls (p < 0.05). The ratio of male vs. female subjects in controls (5:1) is higher than that in the Mn-exposed group (3.7:1); however, this difference is not statistically significant. Daily monitoring of air samples using the stationary monitors indicated the mean airborne Mn concentrations of 0.002 mg/m³ and 0.18 mg/m³ in control and Mn-exposed workers, respectively. While the airborne Mn level was closed to the maximum allowable concentration (MAC) of 0.2 Mn mg/m³ in the work place by the standards of Chinese Ministry of Public Health (TJ36-79) and American Conference of Governmental Industrial Hygienists, the mean accumulative Mn exposure was about 214 fold higher in Mn-exposed workers than in the control group (Table 2).

Mn and Fe concentrations in the plasma and erythrocytes

Mn concentrations in plasma and erythrocytes were about 3.3 and 2.6 fold higher, respectively, in Mn-exposed workers than in controls (p < 0.05; Table 3). The Fe concentrations in Mn-exposed workers, on the other hand, were significantly decreased by 33% and 36% in plasma and erythrocytes, respectively, as compared to controls (Table 3). The results indicate that Mn exposure increased Mn concentrations while it decreased Fe concentrations in the blood compartment; the observation is consistent with our previous reports (Cowan et al., 2009a; Cowan et al., 2009b).

Expression of DMT1, Tf, TfR and hepcidin in blood cells

Among the genes involved in Fe metabolism, Tf molecules bind Fe and transport Fe in extracellular space; DMT1 and TfR function to take Fe into the cells; and hepcidin can increase intracellular Fe storage. Data by real-time RT-PCR showed that the relative transcript levels of DMT1,Tf and hepcidin in Mn-exposed workers were significantly decreased by 58%, 68% and 61%, respectively, as compared to controls (Table 4). However, the expression of TfR was not altered.

Changes of mRNA levels of DMT1, Tf and hepcidin as the function of blood Mn and Fe levels

By linear regression analysis, the levels of mRNAs encoding DMT1, Tf and hepcidin appeared to show the tendency to decline as the Mn concentrations in erythrocytes increased (Figs. 1A, 1C and 1E); however, the correlations between mRNA levels and erythrocytic Mn levels approached to, but did not achieve, the statistical significance, except for Tf mRNA (p = 0.05; Fig. 1C). For plasma samples, the mRNA expression levels of all 3 Fe regulatory transport proteins were inversely correlated with the Mn-concentrations in plasma (p < 0.01; Figs. 1B, 1D and 1F).

The changes of DMT1, Tf and hepcidin mRNA expressions as the function of Fe concentrations in erythrocytes and plasma were also analyzed; the results are summarized in Table 5. There was a general positive correlation between mRNA expressions and Fe concentrations in plasma and erythrocytes; however, none of these correlations reached the statistical significance (Table 5).

Changes of biological parameters as the function of cumulative Mn exposure

Mn concentrations in erythrocytes were significantly correlated with the cumulative Mn exposure among the study subjects (r = 0.46, p < 0.01) (Fig. 2A), so were the Mn concentrations in plasma (r=0.59, p < 0.01) (Fig. 2B). These positive correlations reflect a job-related Mn exposure. Importantly, the liner regression analysis revealed that the mRNA levels of all 3 Fe transport proteins, i.e., DMT1, Tf and Hepcidin, were significantly decreased as the cumulative Mn exposure increased (p < 0.01; Fig. 3), suggesting a Mn exposure-associated alteration in Fe regulatory proteins.