Metal-sulfate induced generation of ROS in human brain cells: detection using an isomeric mixture of 5- and 6-carboxy-2',7'-dichlorofluorescein diacetate (carboxy-DCFDA) as a cell permeant tracer

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; C(25)H(14)C(l2)O(9); 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-H(2)DCFDA 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.

Keywords: 5-carboxy-2′,7′-dichlorofluorescein diacetate; 6-carboxy-2′,7′-dichlorofluorescein diacetate (5- and 6-carboxy-DCFDA; carboxy-DCFDA); Alzheimer’s disease; Parkinson’s disease; aluminum; amyotrophic lateral sclerosis; carboxy-DCFDA; epigenetic human neural cells; inflammation; metal sulfates; prion disease; synergistic effects.

Figure 1

(A) Molecular structure of the mixed isomer, cell permeant 5-(and-6)-carboxy-2′,7′-dichlorofluorescein diacetate [carboxy-DCFDA; C₂₅H₁₄C_l2O₉; MW 529.3; Molecular Probes C-369; CAS name = 3′,6′-bis(acetyloxy)-2′,7′-dichloro-3-oxo-spiro-[iso-benzofuran-1(3H),9′-(9H)xanthene-ar-carboxylic acid; CAS number 127770-45-0]; the twin CH₃COO-R-groups facilitate cellular entry; intracellular esterases cleave these to “trap” the molecule within the cell; (B) peak excitation (λE_x 502 nm; shown in green) and peak emission (λE_m 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.

Figure 2

(A) Human neuronal-glial (HNG) cells after 2.5 weeks in primary co-culture; the cell density is approximately 35% neurons and 65% astroglia at 60% confluency; human primary neuronal and glial “support” cell co-cultures are used as human neuronal cells do not culture well by themselves [19,22]; neuronal cells are stained with neuron-specific β-tubulin (red; λ_max = 690 nm), glial cells are stained with glial-specific glial fibrillary acidic protein (GFAP; green; λ_max = 525 nm), and nuclei are stained with Hoechst 33258 (blue; λ_max = 470 nm); photo magnification 20×; (B) co-incubation with 5-(and-6)-carboxy-2′,7′-dichloro-fluorescein diacetate (C₂₅H₁₄C_l2O₉; carboxy-DCFDA) indicates appreciable ROS generation throughout the entire neuronal-glial cell soma and neurite extensions in all cell types; 100% of the cells are stained and exhibit varying degrees of ROS generation depending on anatomical location; treatment shown after 3 h with 50 nM Al₂(SO₄)₃ displays significant ROS signal yield with a green fluorescence emission λ_max 530 nm (Figure 1); photo magnification 30×.