Abstract
Evolution of reactive oxygen species (ROS), generated during the patho-physiological stress of nervous tissue, has been implicated in the etiology of several progressive human neurological disorders including Alzheimer’s disease (AD) and amylotrophic lateral sclerosis (ALS). In this brief communication we used mixed isomers of 5-(and-6)-carboxy-2′,7′-dichlorofluorescein diacetate (carboxy-DCFDA; C25H14Cl2O9; MW 529.3), a novel fluorescent indicator, to assess ROS generation within human neuronal-glial (HNG) cells in primary co-culture. We introduced pathological stress using the sulfates of 12 environmentally-, industrially- and agriculturally-relevant divalent and trivalent metals including Al, Cd, Cu, Fe, Hg, Ga, Mg, Mn, Ni, Pb, Sn and Zn. In this experimental test system, of all the metal sulfates analyzed, aluminum sulfate showed by far the greatest ability to induce intracellular ROS. These studies indicate the utility of using isomeric mixtures of carboxy-H2DCFDA diacetates as novel and highly sensitive, long-lasting, cell-permeant, fluorescein-based tracers for quantifying ROS generation in intact, metabolizing human brain cells, and in analyzing the potential epigenetic contribution of different metal sulfates to ROS-generation and ROS-mediated neurological dysfunction.
Highlights
The evolution of reactive oxygen species (ROS) is a persistent and ongoing metabolic process during the course of normal human aging
As an initial step to quantify and understand ROS-inducibility by relatively common metal sulfates, in these experiments we studied the ROS-inducing capabilities of
Fluorescent signals from stressed human neuronal-glial (HNG) cells were quantified using digital electronic imaging photography under ultraviolet (UV) light (Ex 502 nm; Em 530 nm) employing an Axioskop/Zeiss MC63 photo control unit and a Nikon Optiphot-2 microscope equipped with an additional differential-Interference Contrast/Nikon UFX–DX photo control unit
Summary
The evolution of reactive oxygen species (ROS) is a persistent and ongoing metabolic process during the course of normal human aging. Excessive ROS generation that overwhelms cellular anti-oxidant defenses, and ensuing free-radical damage to cellular lipids, proteins, and nucleic acids, lies at the core of the widely accepted free radical theory of aging [1,2,3,4]. Carboxy-DCFDA has exceedingly low sensitivity of intrinsic fluorescence yield at intracellular pH, and the 5′-and 6′-carboxy-ligands significantly further stabilize the internalized fluorescent signal enabling longer data-collection times. Using this novel and highly sensitive analytical tracer in this study we have quantified the ROS-producing capability of 12 environmentally- and industrially-relevant metal sulfates in human neuronal-glial (HNG) cells in primary culture. CH3COO-R-groups facilitate cellular entry; intracellular esterases cleave these to “trap” the molecule within the cell; (B) peak excitation (λEx 502 nm; shown in green) and peak emission (λEm 530 nm; shown in red) for 5-(and-6)-carboxy-2′,7′-dichlorofluorescein diacetate after removal of acetyl groups by cellular esterases; the dicarboxyl groups at positions 5 and 6 appear to stabilize the carboxy-DCFDA flurophor to prolong intracellular fluorescence yield
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