MALDI-TOF MSI and electrochemical detection of metallothionein in chicken liver after cadmium exposure

Metallothioneins (MTs) are intracellular, low-molecular weight, proteins where cysteines form at least one third of all protein amino acids and thiol groups serve as pockets for coordination with divalent metal ions, especially Zn and Cu [1]. Metallothioneins are connected with cancer development, protection of organism against environmental pollution effects and also with chemoresistance of cells. Their main functions include detoxification of heavy metals, maintenance of ion homeostasis and protection against oxidative stress. MTs exist in all kind of mammalian cells. Four isoforms of human MT (MT-1, MT-2, MT-3, MT 4) were found so far [2] and according to UniProt database two chicken MTs (MT1 and MT3) have been reported. The expression of MTs in the liver can be induced by several metals and drugs. MTs serve as cellular regulators by coordination of essential metals (Zn, Cu) and reducing the toxicity of heavy metals (Cd, Pb) [3].
MTs can be detected electrochemically in Brdicka solution. Here, three characteristic signals are measured for construction of electrochemical fingerprint of MT: RS2Co (about -1.3 V), Cat1 (-1.4 V) and Cat2 (-1.5 V). The obtained voltammograms are then analyzed by mathematical model [4,5].
Cadmium (Cd), a non-essential heavy metal and a selective toxicant, is accumulated mainly in the kidneys where it causes cellular injury [6]. Cd ions stimulate expression of MTs and toxicity occurs when the concentration of Cd ions exceeds the buffering capacity of intracellular MTs [7]. Cd naturally occurs in the environment as a pollutant from agriculture and industry [8-10]. Cadmium is a known teratogen and can cause damage to chicken embryos [11]. Cadmium levels can be determined by different methods. In this study, atomic absorption spectroscopy (AAS) method was used as described in our previous work [12]. Matrix assisted laser desorption/ionization time-of-flight mass spectrometry imaging (MALDI TOF MSI) is an imaging technique used for determining spatial distribution of various molecules (biomarkers) in a variety of tissue samples. MALDI is “soft” ionization technique, which does not completely fragment the molecules of analytes and therefore a molecular weight of whole analyte can be measured [13]. MALDI-TOF has been used for detection of cancer biomarkers of different tumors, namely; gastrointestinal tumors, tumors of respiratory system, renal and bladder tumors, prostate, breast and ovarian tumors [14]. In this study we have used MALDI-TOF MSI for imaging MT in chicken liver for the first time.
The purpose of this study was the detection of MT in liver from chicken embryos exposed to different concentrations of cadmium by electrochemistry and MALDI-TOF MSI. Also, we attempted to find some correlation between measured MT concentrations and administered concentrations of cadmium.

Figure 1: (A) Determination of concentration of Cd relative to the weight of liver from chicken embryos. Cd was measured by AAS (paragraph 3.6.). (B) Determination of concentration of metallothionein (MT) relative to the total protein amount measured in liver from chicken embryos. MT was measured by differential pulse voltammetry (DPV) using Brdicka solution (paragraph 3.4.). (C) Kaplan-Meier survival analysis of chicken embryos after the application of 0, 3.75, 7.5, 15, 30 and 60 µg/egg of cadmium in the form of Cd2+ ions – these concentrations are shown as groups with different color according to the legend.

Figure 2. MALDI mass spectrometry images of spatial distribution of chicken metallothionein MT1 (6277 Da) in liver from chicken embryo after exposure to Cd: (A) control sample (0 µg of Cd), (B) 7.5 µg of Cd and (C) 15 µg of Cd. Pictures on the left represent scanned images of liver slices, pictures on the right represent results from MSI, and pictures in the middle represent scanned images of liver slices merged with results from MSI and.

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