Metallomics of the human lens: a focus on the zinc-metallothionein system

Abstract

Purpose: Metallothioneins (MTs) are zinc-ion-binding proteins involved in defense mechanism against oxidative damage and inflammatory stress. The purpose of the present work was: (i) to determine the total amount and the quantitative speciation of trace elements (i.e., Zn, Fe, Cu and Se) in human lenses and capsules; and (ii) to study the effect of metals (i.e., ZnSO₄) and inflammatory stress (i.e., IL1a) on MT protein levels and zinc-MT redox system in a human lens epithelial cell line (HLEsv).

Methods: A total of 20 human lenses and their corresponding capsules were used for total analysis of Zn, Fe, Cu and Se, and their quantitative speciation by Inductively Coupled Plasma Mass Spectrometry (ICPMS), and HPLC-ICP-MS, respectively. HLEsv cells were treated, in a dose-dependent manner with the enriched-stable isotope ⁶⁸ZnSO₄ alone (i.e., 25mM, 50mM, and 100 mM), with IL1a alone (100U/mL), or in combination ⁶⁸ZnSO₄ + IL1a, during 24h. The concentration of zinc and zinc-binding proteins, the absolute quantification and the stoichiometry of intracellular MTs, were determined by Isotope Pattern Deconvolution (IPD) and HPLC-ICP-MS.

Results: Total multielemental determinations: Zn was the element in the highest concentration, in both, the capsule (9.7±2.5 mg/g tissue) and the lens (9.3±1.1 mg/g tissue). When comparing the distribution of the analyzed elements between lens and capsule, Fe was the sole element showing significant differences (1.2±0.4 mg/g capsule; 0.3±0.1 mg/g lens). Quantitative speciation: Zinc in lens is mainly associated to high molecular weight (HMW) proteins, whereas in the capsule is mostly bound to low and medium molecular weigh (LMW and MMW) proteins. HLEsv in vitro experiments: ⁶⁸ZnSO₄, when added alone exerted an increased MT protein synthesis, and elicited a synergistic effect when added in combination with IL1a. Under control conditions intracellular MTs contained 3 zinc atoms (Zn₃MTs), and upon zinc treatment alone or in combination with IL1a, it favored the formation of Zn₇MT species.

Conclusions: Zinc is highly concentrated in human lens and capsule. It is mostly associated to HMW proteins in the lens, and to LMW and MMW proteins in the capsule. The LMW proteins are most likely MTs. Zn is capable to induce a stoichiometry change in MTs from Zn₃-MT to Zn₇-MT, rendering lens cells in culture potentially more resistant to oxidative stress insults.