Cellular and molecular responses to ethyl-parathion in undifferentiated SH-SY5Y cells provide neurotoxicity pathway indicators for organophosphorus impacts.

High-fidelity nonanimal screening methods are needed that can rapidly and accurately characterize organophosphorus compound (OP)-induced neurotoxicity. Herein, the efficacy of human neuroblastoma cell line (SH-SY5Y) to provide molecular and cellular responses characteristic of the OP neurotoxicity pathway was investigated in response to the OP-model compound, ethyl-parathion. Undifferentiated SH-SY5Y cells were exposed to ethyl-parathion for 30 min at 0 (control), 0.5, 2.5, 5, 10, and 25 µg/ml. Dose-responsive reductions in cell viability were observed with significant reductions at ≥10 µg/ml. From these results, ethyl-parathion exposures of 0 (control), 5, and 10 µg/ml were selected to examine bioindicators underlying the OP neurotoxicity pathway including: reactive oxygen species (ROS), cell membrane peroxidation, mitochondrial membrane potential (MMP), and apoptosis. Ethyl-parathion elicited highly significant increases in ROS relative to controls (p < .01) at both exposure concentrations, confirmed using N-acetyl cysteine (NAC) as a ROS quencher which alleviated ROS increases. A response characteristic of increased ROS exposure, cell membrane-lipid peroxidation, significantly increased (p < .05) at the highest ethyl-parathion exposure (10 µg/ml). As a likely consequence of membrane-lipid peroxidation, ethyl-parathion-induced reductions in MMP were observed with significant effects at 10 µg/ml, reducing MMP by 58.2%. As a culmination of these cellular-damage indicators, apoptosis progression was investigated by phosphatidylserine translocation where ethyl-parathion-induced dose-responsive, highly significant (p < .01) increases at both 5 and 10 µg/ml. Overall, the mechanistic responses observed in undifferentiated SH-SY5Y cells corresponded with in vivo mammalian results demonstrating potential for this nonanimal model to provide accurate OP neurotoxicology screening.

Triplet-Energy Quenching Functions of Antioxidant Molecules.

UV-like DNA damage is created in the dark by chemiexcitation, in which UV-activated enzymes generate reactive oxygen and nitrogen species that create a dioxetane on melanin. Thermal cleavage creates an electronically excited triplet-state carbonyl whose high energy transfers to DNA. Screening natural compounds for the ability to quench this energy identified polyenes, polyphenols, mycosporine-like amino acids, and related compounds better known as antioxidants. To eliminate false positives such as ROS and RNS scavengers, we then used the generator of triplet-state acetone, tetramethyl-1,2-dioxetane (TMD), to excite the triplet-energy reporter 9,10-dibromoanthracene-2-sulfonate (DBAS). Quenching measured as reduction in DBAS luminescence revealed three clusters of 50% inhibitory concentration, ~50 µM, 200-500 µM, and >600 µM, with the former including sorbate, ferulic acid, and resveratrol. Representative triplet-state quenchers prevented chemiexcitation-induced "dark" cyclobutane pyrimidine dimers (dCPD) in DNA and in UVA-irradiated melanocytes. We conclude that (i) the delocalized pi electron cloud that stabilizes the electron-donating activity of many common antioxidants allows the same molecule to prevent an electronically excited species from transferring its triplet-state energy to targets such as DNA and (ii) the most effective class of triplet-state quenchers appear to operate by energy diversion instead of electron donation and dissipate that energy by isomerization.

Photosensitized rose Bengal-induced phototoxicity on human melanoma cell line under natural sunlight exposure.

Rose Bengal (RB) is an anionic water-soluble xanthene dye, which used for many years to assess eye cornea and conjunctiva damage. RB showed strong absorption maxima (λmax) under visible light followed by UV-B and UV-A. RB under sunlight exposure showed a time-dependent photodegradation. Our results show that photosensitized RB generates (1)O2 via Type-II photodynamic pathway and induced DNA damage under sunlight/UV-R exposure. 2'dGuO degradation, micronuclei formation, and single- and double-strand breakage were the outcome of photogenotoxicity caused by RB. Quenching studies with NaN3 advocate the involvement of (1)O2 in RB photogenotoxicity. RB induced linoleic acid photoperoxidation, which was parallel to (1)O2-mediated DNA damage. Oxidative stress in A375 cell line (human melanoma cell line) was detected through DCF-DA assay. Photosensitized RB decreased maximum cellular viability under sunlight followed by UV-B and UV-A exposures. Apoptosis was detected as a pattern of cell death through the increased of caspase-3 activity, decreased mitochondrial membrane potential, and PS translocation through inner to outer plasma membrane. Increased cytosolic levels of Bax also advocate the apoptotic cell death. We propose a p53-mediated apoptosis via increased expression of Bax gene and protein. Thus, the exact mechanism behind RB phototoxicity was the involvement of (1)O2, which induced oxidative stress-mediated DNA and membrane damage, finally apoptotic cell death under natural sunlight exposure. The study suggests that after the use of RB, sunlight exposure may avoid to prevent from its harmful effects.