Nanomolar aluminum induces pro-inflammatory and pro-apoptotic gene expression in human brain cells in primary culture

Generation of Reactive Oxygen Species (Ros) and Pro-Inflammatory Signaling in Human Brain Cells in Primary Culture

“…HNG cell lines, derived from normal human neural progenitor cells (PT-2599; Lonza Clonetics Cell Systems, Walkersville, MD) were cultured in 6-well (3.5 cm diameter) plates (Costar 3506, Corning Life Sciences, Acton, MA) at 5% CO 2 , 20% O 2 and 37ºC in an HNG cell maintenance medium (HNGMM) supplemented with 2.5% serum containing hFGF (human fibroblast growth factor), NSF-1 (neuronal survival factor 1), hEGF (human epidermal growth factor) and GA-1000 (gentamicin-amphotericin B G/A 1000) as previously described [9,[12][13][14][15][16][17][18][19]. HNGMM was completely changed every 3.5 days.…”

“…Coded isolation reagent and media samples were analyzed for potential trace metal contamination using a Perkin Elmer 5000PC Zeeman-type electro thermal atomic absorbance (EAA) spectrophotometer equipped with an automated sampler and IBM/AT-supported analysis package for trace metal analysis [9,18,19]. Wherever possible, ultrapure HNO 3 washed polysulfone plasticware was used according to the URI-GSO protocols to stringently eliminate trace metal contamination [17][18][19].…”

“…Based on a previously verified and highly sensitive assay in vitro stress-test system for the effects of ROS on human brain cell pathogenic gene expression [9,[12][13][14][15][16][17][18], in these experiments we tested the ROSinducing effects of the two most widely used non-prescription drugs -simvastatin and acetylsalicylic acid (aspirin); the pro-inflammatory cytokines -interleukin-6 (IL-6) and interleukin-1beta (IL-1β); tissue necrosis factor alpha (TNFα); amyloid precursor protein (βAPP) and the AD-associated neurotoxic peptides -Aβ40 and Aβ42; the neurotoxic metals -Hg, Cu, Zn, Mn, Fe and Al; and hydrogen peroxide at 50 nM or 100 nM ambient concentrations in the growth medium of human neuronal-glial (HNG) cells in primary culture. Several of these factors were tested in combination.…”

“…Hoechst 33258 bis-benzimide (H-1398), 2' ,7'-dichlorofluorescein diacetate (H 2 DCFDA; D399) was obtained from Molecular Probes-Invitrogen (Eugene, OR) and used according to the manufacturer's instructions. All other reagents were of the highest ultrapure grades commercially available and were used without further purification [18][19][20].…”

“…While cellular systems have evolved elaborate anti-oxidant systems to neutralize the effects of ROS, degenerative disease processes such as those associated with AD and PD may overwhelm these neuroprotective anti-oxidant defenses [5][6][7][8][9][10][11]. The progressive generation of ROS during the course of human aging lies at the core of the free radical theory of aging originally proposed by Harman 55 years ago [10]; this theory implies that aging is associated with increased ambient levels of ROS, ROS-oxidized biomolecules and their deleterious biological effects [8][9][10][11].Based on a previously verified and highly sensitive assay in vitro stress-test system for the effects of ROS on human brain cell pathogenic gene expression [9,[12][13][14][15][16][17][18], in these experiments we tested the ROSinducing effects of the two most widely used non-prescription drugs -simvastatin and acetylsalicylic acid (aspirin); the pro-inflammatory cytokines -interleukin-6 (IL-6) and interleukin-1beta (IL-1β); tissue necrosis factor alpha (TNFα); amyloid precursor protein (βAPP) and the AD-associated neurotoxic peptides -Aβ40 and Aβ42; the neurotoxic metals -Hg, Cu, Zn, Mn, Fe and Al; and hydrogen peroxide at 50 nM or 100 nM ambient concentrations in the growth medium of human neuronal-glial (HNG) cells in primary culture. Several of these factors were tested in combination.…”