Inhibition of neurite outgrowth and enhanced effects compared to baseline toxicity in SH-SY5Y cells

Abstract

Early life exposure to environmental chemicals can cause developmental neurotoxicity (DNT). The impairment of key neurodevelopmental processes such as neurite outgrowth inhibition can be used as endpoints for screening of DNT effects. We quantified neurite-specific effects using the ratio of effect concentrations for cytotoxicity and neurite outgrowth inhibition (SRcytotoxicity). Baseline cytotoxicity, the minimal toxicity of any chemical, was used to quantify enhanced cytotoxicity (toxic ratio, TR) and neuronal-specific toxicity (SRbaseline) by comparing baseline cytotoxicity with the effects on cell viability and neurite outgrowth, respectively. The effects on cell viability and neurite length were measured based on image analysis in human neuroblastoma SH-SY5Y cells. Baseline cytotoxicity was predicted from hydrophobicity descriptors using a previously published model for SH-SY5Y cells. Enhanced cytotoxicity and neuronal-specific toxicity were more often observed for hydrophilic chemicals, which indicates that they are more likely to act through specific modes of action (MOA) on cell viability and neurite outgrowth. Hydrophobic chemicals showed a tendency to act through baseline toxicity without showing specific or enhanced toxicity, but were highly potent considering their low effect concentrations for both cytotoxicity and neurite outgrowth inhibition. The endpoint-specific controls (narciclasine, colchicine, cycloheximide, and rotenone), two carbamates (3-hydroxycarbofuran and carbaryl), and two redox cyclers (diquat and paraquat) showed distinct neurite-specific effects (SRcytotoxicity > 4). By comparing neurite-specific effects with enhanced cytotoxicity, one can explain whether the observed effects involve specific inhibition of neurite outgrowth, other specific MOAs, or merely baseline toxicity arising from hydrophobicity.

Monitoring Mixture Effects of Neurotoxicants in Surface Water and Wastewater Treatment Plant Effluents with Neurite Outgrowth Inhibition in SH-SY5Y Cells

“…A total of 381 chemicals were analyzed for surface water samples according to the analytical method described by Neale et al 23 For the WWTP effluent, 499 chemicals were analyzed, and the details of the analytical method and results of chemical analysis were published by Finckh et al 26 As has been previously shown, chemical analysis by direct injection of water samples can be compared with bioassay results from SPE extracts due to the high chemical recovery of the applied SPE method. 24 Bioanalysis SH-SY5Y cells were maintained and differentiated prior to the assay as described by Lee et al 22 The methanol extracts of surface water and WWTP effluent were blown down under a stream of nitrogen gas and reconstituted with the assay medium. The cells were exposed with 11 different concentrations in serial dilution with a relative enrichment factor (REF, L water /L bioassay ) of 100 as the maximum concentration.…”

“…For chemicals or samples where a plateau was observed in their CRC with poor linearity, a log-logistic model was applied for CRC modeling. The details of image and data analysis and the equations for CRC models including the linear regression and loglogistic model were described by Lee et al 22 For the reporter gene assays, data were analyzed as described by Neale et al, 23 and a 30% effect cutoff was applied for the linear CRC without extrapolation for both chemicals and samples. For sensitivity distribution, water samples were ranked based on their IC 10 and EC 10 values, and their cumulative probability distributions were derived.…”

“…The CRCs of the tested chemicals are given in Figure S7 with their IC 10 and EC 10 in Table S8. Effect concentrations for clothianidin and fipronil were already available for SH-SY5Y cells together with a number of other chemicals that were tested in a previous study, 22 not all of which were active and/or cytotoxic (Table S2, total number of tested chemicals 97, active 59). Among the newly prioritized 41 chemicals, the specificity ratios, SR cytotoxicity and SR baseline , could be determined only for 21 chemicals.…”

“…Mebendazole is an antihelmintic human and veterinary drug that inhibits polymerization of microtubule by binding to tubulin, which corresponds to the MOA of a known neurite-specific toxicant, colchicine. 13,15,22 Mebendazole was more frequently detected in the WWTP effluent and at higher concentrations than in surface water. The second most neurite-specific toxicant, BIT, is used as antimicrobial and is an electrophile that can react with functional groups in enzymes or other proteins.…”

Mixture Risk Assessment of Complex Real-Life Mixtures—The PANORAMIX Project

“…For this purpose, each extract will be assessed in a panel of up to 20 well-established and carefully selected cell-based or cell-free in vitro bioassays with endpoints related to reproductive toxicity and neuro(developmental)toxicity ( Figure 2 ). This panel includes measurement of neurotoxicity in human neuronal cells, including neurite outgrowth inhibition [ 26 ], acetylcholinesterase inhibition [ 27 ] and mitochondrial toxicity [ 28 ], neurodevelopmental assessment in zebrafish embryos [ 29 ], developmental toxicity in embryoid bodies derived from human-induced pluripotent stem cells [ 30 ], thyroid hormone disruption, such as binding to the transport protein transthyretin [ 31 ], inhibition of uptake and efflux of thyroid hormones [ 32 ], thyroid hormone receptor activity [ 33 ], activation/inactivation of thyroid hormones, iodide utilisation and recycling [ 32 , 34 , 35 ], oestrogen, androgen and aryl hydrocarbon receptor activities [ 36 ], genotoxicity, including identification of potential aneugenic and clastogenic activity [ 37 ], and finally measurement of oxidative stress [ 38 ].…”