Liver metal levels and expression of genes related to iron homeostasis in rhesus monkeys after inhalational manganese exposure

Here we present data on liver metal levels and expression of genes related to iron homeostasis in rhesus monkeys after inhalational manganese exposure. Archived liver samples from rhesus monkeys exposed to 0 (n=6), 0.06 (n=6), 0.3 (n=4) and 1.5 (n=4) mg/m3 manganese inhalation for 65 days were obtained from a published study (“Tissue manganese concentrations in young male rhesus monkeys following subchronic manganese sulfate inhalation” [1]). Samples were analyzed by spectroscopy, immunoblotting and quantitative PCR to assess metal levels and gene expression. Liver manganese and iron levels were linearly correlated although only the intermediate manganese exposure level (0.3 mg Mn/m3) led to a statistically significant increase in liver iron levels.


a b s t r a c t
Here we present data on liver metal levels and expression of genes related to iron homeostasis in rhesus monkeys after inhalational manganese exposure. Archived liver samples from rhesus monkeys exposed to 0 (n ¼6), 0.06 (n ¼6), 0.3 (n ¼ 4) and 1.5 (n ¼4) mg/m 3 manganese inhalation for 65 days were obtained from a published study ("Tissue manganese concentrations in young male rhesus monkeys following subchronic manganese sulfate inhalation" [1]). Samples were analyzed by spectroscopy, immunoblotting and quantitative PCR to assess metal levels and gene expression. Liver manganese and iron levels were linearly correlated although only the intermediate manganese exposure level (0.3 mg Mn/m 3 ) led to a statistically significant increase in liver iron levels. &

Value of the data
Inhalational manganese exposure perturbed liver iron levels and expression of BMP6, a known regulator of systemic iron homeostasis.
While sample size in this study is small, the potential value of this data is that it includes an analysis of factors involved in both cellular and systemic iron homeostasis.
Our analysis may be useful as a reference for future, larger studies.

Data
We analyzed levels of manganese, iron, copper and zinc and expression of genes involved in iron homeostasis in archived liver samples from rhesus monkeys after inhalational manganese exposure [1]. Fig. 1   factors involved in iron storage, export, import and regulation of gene expression. Specifically, Fig. 2 depicts levels of ferritin, an intracellular iron storage protein abundantly expressed in conditions of iron excess. Fig. 3 depicts levels of factors that regulate cellular iron excess: BMP6, a protein abundantly expressed in conditions of iron excess; HAMP, a liver-derived hormone also known as hepcidin that negatively regulates iron excess and is stimulated by BMP6; SLC40A1, a cellular iron export protein also known as ferroportin that is negatively regulated by HAMP. Fig. 4   transferrin receptor, an iron import protein that mediates cellular uptake of iron bound to the serum protein transferrin. Fig. 5 depicts levels of iron regulatory protein 2, an RNA-binding protein that regulates expression of genes such as ferritin and transferrin receptor in response to changes in cellular iron levels. Table 1 presents analysis of correlations between all measured parameters in Figs 1-5. Table 2 presents hematologic data previously referenced as data not shown in the original study [1].

Animals
Archived samples of rhesus monkey liver were obtained from a previously published study [1]. Details concerning the animals, their husbandry and manganese inhalation have been published [1]. Male rhesus monkeys purchased from Covance Research Products, Inc. (Alice, TX) were used.  Monkeys were approximately 20-24 months old at the start of the inhalation exposure. Monkeys were exposed to MnSO 4 for 6 h/day, 5 days/week, for 13 weeks (65 exposure days). These monkeys were allocated as follows: 0.0 (n ¼ 6), 0.06 (n ¼6), 0.3 (n ¼4), and 1.5 (n ¼4) mg Mn/m 3 . Food was withheld overnight prior to necropsy. Monkeys were anesthetized with ketamine (20 mg/kg, i.m., Fort Dodge Animal Health, Fort Dodge, IA), blood collected, and then euthanized with pentobarbital (80-150 mg/kg, i.v., Henry Schein, Inc., Port Washington, NY) followed by exsanguination. Liver samples were stored in individual plastic vials or bags, frozen in liquid nitrogen, and stored at approximately À80°C until analyses were performed. atomic emission spectrometry (ICP-AES) using a JY2000 Ultrace spectrometer (Horiba) or by graphite furnace atomic absorption spectrometry (GF-AAS) using an AAnalyst 600 spectrometer (Perkin Elmer) in the Environmental Chemistry Facility at Brown University using previously described protocols [2]. Reference standards were analyzed repeatedly during each run to ensure run consistency. Additional samples of liver were used to assess biochemical endpoints. RNA was extracted from thawed liver tissue and analyzed by quantitative polymerase chain reaction (QPCR) using Taqman assays (Life Technologies) Rh02839540_m1 (BMP6), Rh02819165_m1 (HAMP), Rh02913303_m1 (ID1), Rh00-998191_m1 (SMAD7), Rh02621719_u1 (IL6), Rh02621758_m1 (TFRC) and Rh02621745_g1 (GAPDH) as previously described [2]. Proteins were extracted from liver tissue and immunoblotted as previously described [2] using antibodies against ferritin heavy chain (FTH1) (Cell Signaling), ferritin light chain Table 1 Spearman's rank correlations between parameters of iron homeostasis in monkeys exposed to inhalation of 0-1.5 mg Mn/m 3 over 65 days.

Statistics
Following an assessment of normality by Shapiro-Wilk test, the data for continuous variables were inter-compared for the exposure groups by analysis of variance (ANOVA). If the exposure`s main effect was significant, a Dunnett's test was used to compare the three MnSO 4 exposure levels to the airexposed controls. Unless otherwise noted, data presented are for the mean values 7 standard deviation. Correlations between measured parameters were assessed by Spearman Rank Order Correlation test. A probability value of p¼ 0.05 was used as the critical level of significance for all statistical tests. Statistical analysis was performed using Sigmaplot (Systat Software). Analysis did not take into account effect of multiple comparisons on calculation of statistical significance.