In vivo neurophysiological assessment of in silico predictions of neurotoxicity: Citronellal, 3,4-dichloro-1-butene, and benzyl bromoacetate

Abstract

Neurotoxicants may be widespread in the environment and can produce serious health impacts in the human population. Screening programs that use in vitro methods have generated data for thousands of chemicals. However, these methods often do not evaluate repeated or prolonged exposures, which are required for many neurotoxic outcomes. Additionally, the data produced by such screening methods may not include mechanisms which play critical biological roles necessary for in vivo neurotoxicity. The Hard and Soft Acids and Bases (HSAB) in silico model focuses on chemical structure and electrophilic properties which are important to the formation of protein adducts. A group of structurally diverse chemicals have been evaluated with an in silico screening approach incorporating HSAB parameters. However, the predictions from the expanded chemical space have not been evaluated using in vivo methods. Three chemicals predicted to be cumulative toxicants were selected for in vivo neurotoxicological testing. Adult male Long-Evans rats were treated orally with citronellal (CIT), 3,4-dichloro-1-butene (DCB), or benzyl bromoacetate (BBA) for 8 weeks. Behavioral observations were recorded weekly to assess motor function. Peripheral neurophysiological measurements were derived from nerve excitability (NE) tests which involved compound muscle action potentials (CMAPs) in the tail and foot, and mixed nerve action potentials (MNAPs) in the tail. Compound nerve action potentials (CNAPs) and nerve conduction velocity (NCV) in the tail were also quantified. Peripheral inputs into the central nervous system were examined using somatosensory evoked potentials recorded from the cortex (SEP_CTX) and cerebellum (SEP_CEREB). CIT or BBA did not result in significant alterations to peripheral nerve or somatosensory function. DCB reduced grip-strength and altered peripheral nerve function. The MNAPs required less current to reach 50% amplitude and had a lower calculated rheobase, suggesting increased excitability. Increased CNAP amplitudes and greater NCV were also observed. Novel changes were found in the SEP_CTX with an abnormal peak forming in the early portion of the waveforms of treated rats, and decreased latencies and increased amplitudes were observed in SEP_CEREB recordings. These data contribute to testing an expanded chemical space from an in silico HSAB model for predicting cumulative neurotoxicity and may assist with prioritizing chemicals to protect human health.

Keywords: 3,4-Dichloro-1-butene; Benzyl bromoacetate; Citronellal; Hard and soft acids and bases; Nerve excitability; Neurophysiology.

Figure 1.

Chemical structures of the three compounds selected for in vivo testing. A.) Citronellal is an aldehyde. B.) 3,4-Dichlorobutene is an alkene. C.) Benzyl bromoacetate is an ester.

Figure 2.

Mean body weight calculated as percent of pre-dosing day 1 weight. Bars represent standard error. Missing error bars are contained within the symbols. A.) Citronellal (CIT): (n = 14–20 rats/treatment) animals treated with 0, 79, 104, or 116 mg/kg/day of CIT. No significant treatment-related changes in body weight were observed. B.) 3,4-Dichloro-1-butene (DCB): (n = 16–20 rats/treatment) for animals treated with 0, 100, 175, or 225 mg/kg/day of DCB. Groups treated with DCB gained significantly less weight than control animals starting at week 1, and throughout the 8-week dosing period. C.) Benzyl bromoacetate (BBA): (n = 10–20 rats/treatment) for animals treated with 0, 65, 75, and 90 mg/kg/day of BBA. Groups treated with BBA gained less weight than controls. **High- and middle-dose significant from controls; ***High-, middle-, and low-dose significant from controls; ^#High-dose significant from low-dose; ^{# #}High- and middle-dose significant from low-dose; ⁺High-dose significant from middle-dose.

Figure 3.

Effect of 3,4-Dichloro-1-butene (DCB) on grip-strength. A.) Forelimb grip strength: Treatment decreased forelimb grip strength when averaged over weeks at 100 and 225 mg/kg/day. B.) Hindlimb grip-strength: Treatment decreased hindlimb grip strength when averaged over weeks, similarly to forelimb grip strength. * = significant from controls; # = significant from 175 mg/kg/day.

Figure 4.

Mean nerve excitability data (± SE) derived from tail mixed-nerve action potentials (MNAPs) using a 40% target threshold to tail caudal mixed-nerves after treatment with 3,4-dichloro-1-butene (DCB) (n = 15–19 rats/curve). A.) Stimulus-Response (SR): All treatment levels had a reduction in the current required to induce 50% of maximal action potential. B.) Strength-Duration Time Constant (SDTC): Treatment of 100 and 225 mg/kg/day had a lower SDTC than controls. C.) Threshold Electrotonus (TE): Data showed no changes to threshold in response to depolarizing or hyperpolarizing currents. D.) Current-Voltage Relationship (I/V): Data showed no changes to depolarizing or hyperpolarizing stimuli. E.) Recovery Cycle (RC): Less subexcitability was found in the 175 mg/kg/day group compared to the other treatments.

Figure 5.

Average compound nerve action potential (CNAP) waveforms (shaded regions represent the 95% confidence intervals) following evoked stimulation to tail caudal nerves after treatment with 3,4-dichloro-1-butene (DCB) (n = 12–20 rats/waveform). A.) CNAP1: Peak averages, with peaks P₁ and N₁ labeled, following a 1, 2, or 3 mA stimulus. As expected, an increase in stimulus intensity increased the peak latencies and decreased the peak-to-peak amplitudes. B.) Nerve Conduction Velocity (NCV) at 3mA: NCV measurements were based on latency difference of P₁ peaks in CNAP1 and CNAP2. C.) P₁N₁ Peak Amplitude: Treatment of 225 mg/kg/day increased the peak-to-peak amplitude of peak P₁N₁ when averaged over stimulus intensities. D.) NCV: Treatment of 225 mg/kg/day increased NCV. * = significant from controls.

Figure 6.

Average somatosensory evoked potential waveforms (n = 11–19 rats/waveform) recorded from the cortex (SEP_cortex) following tail stimulation after treatment with 3,4-dichloro-1-butene (DCB). A.) SEP_cortex waveforms: Average waveforms with a 1, 2, or 3 mA stimulus. Shaded regions represent the 95% confidence intervals. B.) Peak N₁P₁: Treatment produced novel changes in the SEP_cortex. A dose-related increase in occurrence of a newly formed peak (labeled N₁ and P₁) between peaks P₁₄ and N₂₇ of the SEP_CTX waveform was indicated. Single animal waveforms from each treatment which represent the group median P₁₄N₁ amplitude are presented for ease of viewing. C.) Peak P₁₄N₁ amplitude: Stimulus-intensity related increase in the amplitude of peak P₁₄N₁ for the different DCB treatment groups. * = significant from controls; # = significant from 100 mg/kg/day DCB.