In vitro neurotoxic hazard characterisation of dinitrophenolic herbicides.
Dinitrophenolic compounds are powerful toxicants with a long history of use in agriculture and industry. While (high) human exposure levels are not uncommon, in particular for agricultural workers during the spraying season, the neurotoxic mechanism(s) that underlie the human health effects are largely unknown. We therefore investigated the in vitro effects of two dinitrophenolic herbicides (DNOC and dinoseb) on a battery of neurotoxicity endpoints in (dopaminergic) rat PC12 cells. Cell viability, mitochondrial activity, oxidative stress and caspase activation were assessed using fluorescence-based bioassays (CFDA, alamar Blue, H2DCFDA and Ac-DEVD-AMC, respectively), whereas changes in intracellular [Ca(2+)]i were assessed using single-cell fluorescence microscopy with Fura-2AM. The combined results demonstrate that exposure to both DNOC and dinoseb is linked to calcium release from the endoplasmic reticulum and activation of caspase-mediated apoptotic pathways. In subsequent experiments, immunofluorescent labelling with specific antibodies was used to determine changes in intracellular α-synuclein levels, demonstrating that both DNOC and dinoseb increase levels of intracellular α-synuclein. The combined results indicate that in vitro exposure to DNOC and dinoseb activates pathways that are not only involved in acute neurotoxicity but also in long-term effects as seen in neurodegeneration.
Neurotoxicology Research Group, Division of Toxicology, Institute for Risk Assessment Sciences, Faculty of Veterinary Medicine, Utrecht University, 3508 TD Utrecht, The Netherlands; IUF-Leibniz Research Institute for Environmental Medicine, Auf'm Hennekamp 50, 40225 Düsseldorf, Germany; Centre for Sustainability, Environmental & Health, National Institute for Public Health and the Environment, P.O. Box 1, 3720 BA, Bilthoven, The Netherlands. Electronic address: Harm.Heusinkveld@IUF-Duesseldorf.de.
Harm J
HJ
Heusinkveld
Neurotoxicology Research Group, Division of Toxicology, Institute for Risk Assessment Sciences, Faculty of Veterinary Medicine, Utrecht University, 3508 TD Utrecht, The Netherlands.
Arie C
AC
van Vliet
Department of Neurology, St. Elisabeth Hospital, P.O. Box 90151, NL-5000 LC Tilburg, The Netherlands.
Peter C G
PC
Nijssen
Neurotoxicology Research Group, Division of Toxicology, Institute for Risk Assessment Sciences, Faculty of Veterinary Medicine, Utrecht University, 3508 TD Utrecht, The Netherlands.
Remco H S
RH
Westerink
D019297:2,4-Dinitrophenol / Q000031:analogs & derivatives* / Q000633:toxicity; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D004138:Dinitrocresols / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D006540:Herbicides / Q000633:toxicity*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D020258:Neurotoxicity Syndromes / Q000209:etiology* / Q000378:metabolism / Q000473:pathology; D018384:Oxidative Stress / Q000187:drug effects; D016716:PC12 Cells; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D018570:Risk Assessment; D013997:Time Factors; D015854:Up-Regulation; D051844:alpha-Synuclein / Q000378:metabolism
D004138:Dinitrocresols; D006540:Herbicides; D017382:Reactive Oxygen Species; D051844:alpha-Synuclein; C024132:4,6-dinitro-o-cresol; D020169:Caspases; D019297:2,4-Dinitrophenol; D002118:Calcium; C018527:dinoseb
Caspase-mediated apoptosis; Dinitrophenolic herbicides; Endoplasmic reticulum; In vitro neurotoxicology; Single cell Fura-2 Ca(2+)-imaging; α-Synuclein