An efficient autometallography approach to localize lead at ultra-structural levels of cultured cells
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Biophysics Reports
METHOD
An efficient autometallography approach to localize lead at ultra-structural levels of cultured cells Han Song1,2, Gang Zheng2, Xue-Feng Shen2, Zai-Hua Zhao2, Yang Liu2, Yang Liu3, Ying-Ying Liu4, Jun-Jun Kang4, Jing-Yuan Chen1,2&, Wen-Jing Luo2 1 2
3 4
Department of Health Service, PLA General Hospital, Beijing 100853, China Department of Occupational and Environmental Health and the Ministry-of-Education’s Key Laboratory of Hazard Assessment and Control in Special Operational Environment, School of Public Health, Fourth Military Medical University, Xi’an 710032, China Department of Neurology, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, China Institute of Neurosciences, Fourth Military Medical University, Xi’an 710032, China
Received: 1 June 2020 / Accepted: 13 July 2020 / Published online: 28 September 2020
Abstract
Understanding the precise intracellular localization of lead (Pb) is a key in deciphering processes in Pbinduced toxicology. However, it is a great challenge to trace Pb in vitro, especially in cultured cells. We aimed to find an innovative and efficient approach to investigate distribution of Pb in cells and to validate it through determining the subcellular Pb content. We identified its ultra-structural distribution with autometallography under electron microscopy in a choroidal epithelial Z310 cell line. Electron microscopy in combination with energy-dispersive X-ray spectroscope (EDS) was employed to provide further evidence of Pb location. In addition, Pb content was determined in the cytosol, membrane/ organelle, nucleus and cytoskeleton fractions with atomic absorption spectroscopy. Pb was found predominantly inside the nuclear membranes and some was distributed in the cytoplasm under lowconcentration exposure. Nuclear existence of Pb was verified by EDS under electron microscopy. Once standardized for protein content, Pb percentage in the nucleus fraction reached the highest level (76%). Our results indicate that Pb is accumulated mainly in the nucleus of choroid plexus. This method is sensitive and precise in providing optimal means to study the ultra-structural localization of Pb for in vitro models. In addition, it offers the possibility of localization of other metals in cultured cells. Some procedures may also be adopted to detect target proteins via immunoreactions.
Keywords Lead, Autometallography, Subcellular fraction, Energy-dispersive X-ray spectroscope, Electron microscopy
INTRODUCTION Neurotoxicity due to lead (Pb) accumulation in specific brain areas causes a wide variety of symptoms including decreased intelligence quotient, cognitive deficits, poor attention span and increased aggression (Boucher et al. 2012; Caldero´n et al. 2001; Needleman et al. 1996; Tong & Correspondence: [email protected] (J.-Y. Chen),
Ó The Author(s) 2020
et al. 1998). The mechanism of Pb-induced disturbance of neuronal functions included interfering with neurotransmitter release, disrupting the function of GABAergic, dopaminergic, and cholinergic syste
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