Detection of silver nanoparticles in cells by flow cytometry using light scatter and far-red fluorescence.

The cellular uptake of different sized silver nanoparticles (AgNP) (10, 50, and 75 nm) coated with polyvinylpyrrolidone (PVP) or citrate on a human derived retinal pigment epithelial cell line (ARPE-19) was detected by flow cytometry following 24-h incubation of the cells with AgNP. A dose dependent increase of side scatter and far red fluorescence was observed with both PVP and citrate-coated 50 nm or 75 nm silver particles. Using five different flow cytometers, a far red fluorescence signal in the 700-800 nm range increased as much as 100 times background as a ratio comparing the intensity measurements of treated sample and controls. The citrate-coated silver nanoparticles (AgNP) revealed slightly more side scatter and far red fluorescence than did the PVP coated silver nanoparticles. This increased far red fluorescence signal was observed with 50 and 75 nm particles, but not with 10 nm particles. Morphological evaluation by dark field microscopy showed silver particles (50 and 75 nm) clumped and concentrated around the nucleus. One possible hypothesis to explain the emission of far red fluorescence from cells incubated with silver nanoparticles is that the silver nanoparticles inside cells agglomerate into small nano clusters that form surface plasmon resonance which interacts with laser light to emit a strong far red fluorescence signal. The results demonstrate that two different parameters (side scatter and far red fluorescence) on standard flow cytometers can be used to detect and observe metallic nanoparticles inside cells. The strength of the far red fluorescence suggests that it may be particularly useful for applications that require high sensitivity. © Published 2013 Wiley-Periodicals, Inc.

Detection of TiO2 nanoparticles in cells by flow cytometry.

Evaluation of the potential hazard of man-made nanomaterials has been hampered by a limited ability to observe and measure nanoparticles in cells. In this study, different concentrations of TiO(2) nanoparticles were suspended in cell culture medium. The suspension was then sonicated and characterized by dynamic light scattering and microscopy. Cultured human-derived retinal pigment epithelial cells (ARPE-19) were incubated with TiO(2) nanoparticles at 0, 0.1, 0.3, 1, 3, 10, and 30 microg/ml for 24 hours. Cellular reactions to nanoparticles were evaluated using flow cytometry and dark field microscopy. A FACSCalibur flow cytometer was used to measure changes in light scatter after nanoparticle incubation. Both the side scatter and forward scatter changed substantially in response to the TiO(2). From 0.1 to 30 microg/ml TiO(2), the side scatter increased sequentially while the forward scatter decreased, presumably due to substantial light reflection by the TiO(2) particles. Based on the parameters of morphology and the calcein-AM/propidium iodide viability assay, TiO(2) concentrations below 30 microg/ml TiO(2) caused minimal cytotoxicity. Microscopic analysis was done on the same cells using an E-800 Nikon microscope containing a xenon light source and special dark field objectives. At the lowest concentrations of TiO(2) (0.1-0.3 microg/ml), the flow cytometer could detect as few as 5-10 nanoparticles per cell due to intense light scattering by TiO(2). Rings of concentrated nanoparticles were observed around the nuclei in the vicinity of the endoplasmic reticulum at higher concentrations. These data suggest that the uptake of nanoparticles within cells can be monitored with flow cytometry and confirmed by dark field microscopy. This approach may help fulfill a critical need for the scientific community to assess the relationship between nanoparticle dose and cellular toxicity Such experiments could potentially be performed more quickly and easily using the flow cytometer to measure both nanoparticle uptake and cellular health.

A physiologically based pharmacokinetic model for trichloroethylene in the male long-evans rat.

A physiologically based pharmacokinetic (PBPK) model for trichloroethylene (TCE) in the male Long-Evans (LE) rat was needed to aid in evaluation of neurotoxicity data collected in this rodent stock. The purpose of this study was to develop such a model with the greatest possible specificity for the LE rat. The PBPK model consisted of 5 compartments: brain, fat, slowly perfused tissue, rapidly perfused viscera, and liver. Partition coefficients (blood, fat, muscle, brain, liver) were determined for LE rats. The volumes of the brain, liver, and fat compartments were estimated for each rat, with tissue-specific regression equations developed from measurements made in LE rats. Vapor uptake data from LE rats were used for estimation of Vmaxc. As blood flow values for LE rats were not available, values from Sprague-Dawley (SD) and Fischer-344 (F344) rats were used in separate simulations. The resulting values of Vmaxc were used to simulate tissue (blood, liver, brain, fat) TCE concentrations, which were measured during (5, 20, 60 min) and after (60 min of TCE followed by 60 min of air) flow-through inhalation exposures of LE rats to 200, 2000, or 4000 ppm TCE. Simulation of the experimental data was improved by use of F-344 blood-flow values and the corresponding Vmaxc (8.68 mg/h/kg) compared to use of SD flows and the associated Vmaxc (7.34 mg/h/kg). Sensitivity analysis was used to determine those input parameters with the greatest influence on TCE tissue concentrations. Alveolar ventilation consistently (across exposure concentration, exposure duration, and target tissue) had the greatest impact on TCE tissue concentration. The PBPK model described here is being used to explore the relationship between measures of internal dose of TCE and neurotoxic outcome.

A comparison of Haber's rule at different ages using a physiologically based pharmacokinetic (PBPK) model for chloroform in rats.

Haber's rule as commonly interpreted in inhalation toxicology, can be stated as exposure concentration times duration equals a constant biological effect, or C x t=k. In other words, identical products of concentration and duration lead to the same effect. The goals of this paper are to develop a biological and pharmacokinetic modeling approach for chloroform, and to evaluate Haber's rule for different ages by taking into account the physiological changes due to growth and aging in rats. Three-dimensional dose-response surfaces for liver toxicity were generated for each age group of interest: adolescent, adult, and senescent rats. The three-dimensional surfaces were then characterized with a generalized description of Haber's rule for each age group. The simulations suggest that adolescent rats need higher exposure levels in order to achieve similar levels of liver damage compared to adults or senescent rats, if the comparison is made using the same exposure length. In summary, a pharmacokinetic modeling approach with a biological framework including the chemical's mode of action, was used to relate concentration, exposure duration and effect. Major advantages of this approach include: the potential ability to extrapolate to humans, the inclusion of aging in the simulations, and the ability to summarize the results using a generalized form of Haber's rule.

Retinal degeneration in licensed pesticide applicators.

BACKGROUND: Retinal degeneration is the leading cause of visual impairment in older adults, but little is known about its relationship to neurotoxic exposures. METHODS: The Agricultural Health Study is a cohort study of licensed pesticide applicators from Iowa and North Carolina. We used cross-sectional data from self-administered questionnaires given at enrollment in 1994-1996 to compare pesticide use in 154 applicators who reported retinal degeneration and 17,804 controls. RESULTS: Retinal degeneration was associated with fungicide use (odds ratio = 1.8, 95% confidence interval = 1.3-2.6). This relationship was seen in subgroups defined by state, demographic characteristics, or medical history, as well as in the entire group. Risk increased with cumulative days of fungicide use (P for trend = 0.011) and was greater when application methods involving greater personal exposure were used. Retinal degeneration was also related to use of organochlorine or carbamate insecticides, but these associations were less consistent. Since nearly all applicators used organophosphate insecticides and herbicides, these exposures could not be effectively evaluated. CONCLUSIONS: These results suggest that exposure to some fungicides and insecticides may increase risk of retinal degeneration.

Neurotoxic and pharmacokinetic responses to trichloroethylene as a function of exposure scenario.

Strategies are needed for assessing the risks of exposures to airborne toxicants that vary over concentrations and durations. The goal of this project was to describe the relationship between the concentration and duration of exposure to inhaled trichloroethylene (TCE), a representative volatile organic chemical, tissue dose as predicted by a physiologically based pharmacokinetic model, and neurotoxicity. Three measures of neurotoxicity were studied: hearing loss, signal detection behavior, and visual function. The null hypothesis was that exposure scenarios having an equivalent product of concentration and duration would produce equal toxic effects, according to the classic linear form of Haber's Rule ((italic)C(/italic) times t = k), where C represents the concentration, t, the time (duration) of exposure, and k, a constant toxic effect. All experiments used adult male, Long-Evans rats. Acute and repeated exposure to TCE increased hearing thresholds, and acute exposure to TCE impaired signal detection behavior and visual function. Examination of all three measures of neurotoxicity showed that if Haber's Rule were used to predict outcomes across exposure durations, the risk would be overestimated when extrapolating from shorter to longer duration exposures, and underestimated when extrapolating from longer to shorter duration exposures. For the acute effects of TCE on behavior and visual function, the estimated concentration of TCE in blood at the time of testing correlated well with outcomes, whereas cumulative exposure, measured as the area under the blood TCE concentration curve, did not. We conclude that models incorporating dosimetry can account for differing exposure scenarios and will therefore improve risk assessments over models considering only parameters of external exposure.

Topical exposure of the eyes to the organophosphorus insecticide malathion: lack of visual effects.

Concern for toxicity following exposure to organophosphorus insecticides led us to investigate whether topical application of either malathion or malathion mixed in a protein bait as used for aerial spray applications could be toxic to the ocular/visual system. Adult male Long-Evans rats were either untreated or treated with malathion alone (two drops per day in each eye), bait alone (six drops per day in each eye) or malathion and bait (six drops per day in each eye). The dose levels of malathion alone and malathion and bait were chosen based on pilot work and provided approximately equivalent amounts of active ingredient. The rats were treated 5 days a week for 4 weeks. During the final week of treatment, the rats were implanted surgically with cranial recording electrodes overlying the visual projection area of the cerebral cortex. Visual pattern-evoked potentials (PEPs) were elicited with vertical sinusoidal gratings at three levels of stimulus spatial frequency (0. 08, 0.16 and 0.32 cycles per degree) and three levels of visual contrast (0.15, 0.30 and 0.60). After spectral analysis of the PEP waveforms, the amplitude and phase at the stimulus rate (F1) and the first harmonic (F2) were determined. Although F1 and F2 parameters were influenced significantly by manipulation of the stimulus parameters, no significant differences were observed that could be attributed to treatment with the test substances. In addition, an ophthalmological examination of the eyes and a light microscopic evaluation of ocular tissues, including retina and optic nerve, revealed no treatment-related lesions. The dose levels used in this study were high-approximately 84000 times the exposure per unit surface area expected from aerial spraying-and yet the visual function of the treated subjects was apparently normal. This study identified no significant toxicological concerns regarding direct ocular contact exposure to malathion.

A dosimetric analysis of behavioral effects of acute toluene exposure in rats and humans.

The literature on behavioral effects of exposure to toluene is difficult to assess due, in part, to a wide variety of exposure conditions employed and outcomes measured. This study investigated whether previous experiments would be more consistent with each other if toluene exposure parameters were expressed not as concentration and duration, but as estimated amount of toluene in tissues. A physiologically based pharmacokinetic (PBPK) model was used to estimate concentration of toluene in arterial blood (CaTOL) from published studies in rats and humans exposed acutely to toluene vapor. Data for rats were selected from studies of avoidance behavior using both rate of responding and measures of successful responding. Data for humans were from studies of choice reaction time (CRT). Behavioral measures were converted to proportion of baseline to place them on a common scale across experiments. A meta-analysis was done to fit dose-effect curves using CaTOL and the rescaled effects. Results demonstrated that effects were an orderly function of CaTOL and were not influenced by concentration or duration of exposure, except as exposure influenced CaTOL. In rats, response rates first increased, reached a peak, and then declined as CaTOL increased. Successful avoidance in rats and CRT in humans always declined as CaTOL increased. In rats, response rates were increased by 10% at CaTOL approximately 13 ml/L. In humans, reaction times increased by 10% at CaTOL approximately 3 ml/L. Cross-species comparisons were made with the following caveats: PBPK uncertainties, few human data, and poor task comparability.

The organophosphate pesticide chlorpyrifos affects form deprivation myopia.

PURPOSE: The effects of the anti-cholinesterase organophosphate pesticide chlorpyrifos (CPF) on the refractive development of the eye were examined. Form deprivation was used to induce eye growth to address the previously reported relationship between organophosphate pesticide use and the incidence of myopia. METHODS: Chickens, a well-established animal model for experimental myopia and organophosphate neurotoxicity, were dosed with chlorpyrifos (3 mg/kg per day, orally, from day 2 to day 9 after hatching) or corn oil vehicle (VEH) with or without monocular form deprivation (MFD) over the same period. The set of dependent measures included the refractive state of each eye measured using retinoscopy, axial dimensions determined with A-scan ultrasound, and intraocular pressure. RESULTS: Dosing with CPF yielded an inhibition of 35% butyrylcholinesterase in plasma and 45% acetylcholinesterase in brain. MFD resulted in a significant degree of myopia in form-deprived eyes resulting from significant lengthening of the vitreal chamber of the eye. CPF significantly reduced the effect of MFD, resulting in less myopic eyes (mean refraction: VEH-MFD = -16.2 +/- 2.3 diopters; CPF-MFD = -11.1 +/- 1.8 diopters) with significantly shorter vitreal chambers. Nonoccluded eyes were, on average, slightly hyperopic. Treatment with CPF for 1 week in the absence of MFD led to no significant change in ocular dimensions or refraction relative to controls. CONCLUSIONS: The use of form deprivation as a challenge suggests that CPF treatment interferes with the visual regulation of eye growth.

A comparison of the acute neuroactive effects of dichloromethane, 1,3-dichloropropane, and 1,2-dichlorobenzene on rat flash evoked potentials (FEPs).

Previous research showed that acute exposure to dichloromethane (DCM) produced a selective reduction in peak N30 of flash evoked potentials (FEPs) in rats. In contrast, acute exposures to p-xylene or toluene selectively reduced FEP peak N160. The present experiments compared the effects of DCM (log P = 1.25; oil:water partition coefficient), 1,3-dichloropropane (DCP; log P = 2.00), and 1,2-dichlorobenzene (DCB; log P = 3.38) on FEPs recorded from adult Long-Evans rats. Before administration of test compounds, FEPs were recorded for five daily sessions to develop FEP peak N160. Test compounds were dissolved in corn oil and administered i.p. at doses based on proportions of their LD50 values. The doses were: DCM, 0, 57.5, 115, 230, or 460 mg/kg; DCP, 0, 86, 172, 343, or 686 mg/kg; and DCB, 0, 53, 105, 210, or 420 mg/kg. Testing times after dosing varied among compounds and were based on pilot studies to measure both the times of peak effect and recovery. Each solvent produced significant changes in the latency and amplitude of multiple components of the FEP waveforms. However, the predominant effect of DCM was to reduce the amplitude of peak N30 (ED50 = 326.3 mg/kg), that of DCP was to reduce both peaks N30 (ED50 = 231.0 mg/kg) and N160 (ED50 = 136.8 mg/kg), and that of DCB was to reduce peak N160 (ED50 = 151.6 mg/kg). There was no consistent relationship between log P values and the potency of the compounds to alter FEP peaks N30 and N160. The results suggest that organic solvents have multiple acute effects on the function of the central nervous system, which are not predictable solely by the compound's lipid solubility.

EPA's neurotoxicity risk assessment guidelines.

The proposed Neurotoxicity Risk Assessment Guidelines (U.S. EPA, 1995c Fed. Reg. 60(192), 52032-52056) of the U.S. Environmental Protection Agency (EPA) were the subject of a workshop at the 1997 Meeting of the Society of Toxicology. The workshop considered the role of guidelines in the risk assessment process, the primary features, scientific basis, and implications of the guidelines for EPA program offices, as well as for industrial neurotoxicologists from the perspectives of both pesticides and toxic substances regulation. The U.S. National Academy of Sciences (NAS, 1983, Risk Assessment in the Federal Government: Managing the Process) established a framework for distinguishing risk management from risk assessment, the latter being the result of integrating hazard identification, hazard characterization, and exposure assessment data. The guidelines are intended to establish operating principles that will be used when examining data in a risk assessment context. The proposed neurotoxicity risk assessment guidelines provide a conceptual framework for deciding whether or not a chemically induced effect can be considered to be evidence of neurotoxicity. Topics in the proposed guidelines include structural and functional effects, dose-response and -duration considerations, and relationships between effects. Among the issues that must be considered are the multiplicity of chemical effects, the levels of biological organization in the nervous system, and the tests, measurements, and protocols used. Judgment of the adversity of an effect depends heavily on the amount and types of data available. The attribution of a chemically induced effect to an action on the nervous system depends on several factors such as the quality of the study, the nature of the outcome, dose-response and time-response relationships, and the possible involvement of nonneural factors. The guidelines will also serve as a reference for those conducting neurotoxicity testing, as well as establish a consistent approach to neurotoxicity risk assessment by regulators. Extending this approach through international harmonization would be advantageous to the development of products for a worldwide market. Thus, both risk assessors and regulated industries have a large stake in the guidelines to provide a framework that will lead to accurate risk assessment decisions.

Possible confounding effects of strobe "clicks" on flash evoked potentials in rats.

Flash evoked potentials (FEPs) undergo within- and between-session changes and are modified by auditory white noise (26). We examined whether an auditory potential produced by the "click" associated with the strobe discharge could be recorded, and if alterations in an auditory response could explain the within- and between-session changes in FEPs. We also examined differences between a frontal cortex or a nasal reference electrode location on FEPs and auditory potentials. An auditory potential associated with the strobe discharge could be clearly recorded. This response was eliminated by the presence of 80 dB SPL masking white noise. However, the within- and between-session changes in FEPs could not be explained by modifications of the auditory potential. Animals whose ear drums were ruptured did not exhibit an auditory response, and their FEPs were similar to those of controls tested in the presence of masking white noise. A nasal reference electrode decreased the impact of auditory potentials on FEPs, but allow visual potentials (electroretinogram and optic tract activity) to influence FEPs. The data show that auditory potentials associated with the strobe discharge can be recorded from the visual cortex of rats, and that these auditory responses represent a possible confounding factor in the interpretation of toxicological studies employing FEPs.

Combined effects of paired solvents on the rat's auditory system.

A number of volatile organic solvents have been shown to be ototoxic to rats, but there is little information regarding how solvents might act in this way when encountered in combination. To examine this issue, male Long Evans rats were exposed by inhalation to pairs of solvents known to be ototoxic when administered individually; those reported on here are trichloroethylene+toluene, mixed xylenes+trichloroethylene, xylenes+chlorobenzene, and chlorobenzene+toluene. Rats were exposed 8 h/day for 5 consecutive days, using complementary proportions of isoeffective concentrations of the solvents alone. Hearing was assessed by brainstem-evoked response audiometry. The effects were as predicted by a linear dose-addition model, indicating additive rather than synergistic or antagonistic interactions at the concentrations studied.

Assessment of offspring development and behavior following gestational exposure to inhaled methanol in the rat.

The prospect of widespread human exposure associated with its use as an alternative fuel has sparked concern about the toxic potential of inhaled methanol (MeOH). Previous studies have revealed congenital malformations in rats following inhaled MeOH (Nelson et al. (1985). Fundam. Appl. Toxicol. 5, 727-736) but these studies did not include postnatal behavioral assessment. In the present study, pregnant Long-Evans rats were placed in exposure chambers containing 15,000 ppm MeOH or air for 7 hr/day on Gestational Days (GD) 7-19. The total alveolar dose of methanol was estimated at about 6.1 g/kg/day, for a total dose of about 42.7 g/kg for the entire study. Maternal body weights were recorded daily and blood methanol concentrations were determined at the end of exposure on GD 7, 10, 14, and 18. Following birth (Postnatal Day 0 [PND 0]), a number of tests were performed at various points in development, including: offspring mortality and body wt (PND 1,3), motor activity (PND 13-21, 30, 60), olfactory learning (PND 18), behavioral thermoregulation (PND 20-21), T-maze learning (PND 23-24), acoustic startle response (PND 24, 60), reflex modification audiometry (PND 60), pubertal landmarks (PND 31-56), passive avoidance (PND 72), and visual-evoked potentials (PND 160). Maternal blood MeOH levels, measured from samples taken within 15 min after removal from the exposure chamber, declined from about 3.8 mg/ml on the first day of exposure to 3.1 mg/ml on the 12th day of exposure. MeOH transiently reduced maternal body wt (4-7%) on GD 8-10, and offspring BW (5%) on PND 1. No other test revealed significant effects of MeOH. Prenatal exposure to high levels of inhaled MeOH appears to have little effect on this broad battery of tests beyond PND 1 in the rat.

Work-site clinical and neurobehavioral assessment of solvent-exposed microelectronics workers.

Twenty-five workers, five currently and 20 formerly involved in the manufacture of hybrid microcircuits, underwent clinical evaluations at the request of a management-union committee concerned about chronic solvent exposures in a research and development laboratory. A battery of neurobehavioral tests was administered to compare the solvent-exposed group with 32 age-, gender-, ethnicity-, and education-matched controls. The tests included: MMPI-I, hand grip strength, tactile sensitivity, dexterity, color discrimination, visual acuity and contrast sensitivity, and tests selected from the computerized Neurobehavioral Evaluation System (NES2). Clinical narratives and retrospective exposure assessments in the study group suggested chronic low-level exposure to solvents, with intermittent acute excursions. Work-related diagnoses included upper respiratory mucosal irritation and sinusitis (44%), lower respiratory reactive airway disease (12%), and dermatitis (5%). Three workers (12%) had findings consistent with a solvent-induced encephalopathy. Significant differences (after Bonferroni correction) were found between the two groups on 5 of 11 NES subtests: symptom scale, mood scale, finger tapping, simple reaction time, and symbol-digit substitution. Differences also reached significance for overall vibration sensitivity thresholds, visual contrast sensitivity, and grip strength. The MMPI average clinical scale elevation was significantly higher in the exposed group than controls. These results support an association between chronic low-dose solvent exposure and measurable neurobehavioral changes.

Effects of organophosphates on the visual system of rats.

The possibility that exposure to organophosphate insecticides can lead to ocular damage is suggested by Japanese studies from the 1960s and 1970s indicating that exposed humans developed chronic ocular degeneration, in addition to showing more commonly accepted effects of cholinesterase-inhibiting compounds. Other papers reported ocular lesions in laboratory animals treated with organophosphates. More recently, retinal degeneration following chronic organophosphate treatment has been reported to the Environmental Protection Agency by pesticide manufacturers in studies conducted in compliance with good laboratory practice regulations. Several factors, however, have prompted scepticism regarding organophosphate-induced ocular toxicity, including the widespread use of organophosphate compounds for both agricultural and ophthalmological practices without numerous additional reports of comparable ocular toxicity. We are developing a research program to address these issues involving electrophysiological, biochemical and histological investigations of rats treated with organophosphate insecticides. The research program is young, but early results are available. Notably, retinas from rats treated with a single subcutaneous injection of 100 mg kg-1 fenthion showed decreases in carbachol-stimulated release of inositol phosphate, an indicator of cholinergically-mediated intracellular second messenger systems. These effects persisted at least 56 days after fenthion administration. This could indicate several different toxicological actions, which are currently under investigation. It is concluded that the possible association between exposure to organophosphates and ocular toxicity cannot be dismissed, and that several important research issues need to be resolved.

Rat and human sensory evoked potentials and the predictability of human neurotoxicity from rat data.

The development of comprehensive quantitative models as alternatives to risk assessment based on uncertainty factors will require many steps, among them consideration of the relationships between the health endpoints which are measured in laboratory animals and humans. Sensory evoked potentials are measures of sensory function which can be recorded from many species, including humans, and as such provide an opportunity for examining the extrapolation of neurotoxicity data from laboratory animals to humans. Our research strategy for investigating how well laboratory rat data predict human neurotoxic risk involves comparing parametric stimulus manipulations and drug treatments in both species. Finally, we are comparing results in humans with neurodegenerative conditions, including those induced by neurotoxicant exposure, with animal models. To date, we have focused on pattern-elicited visual evoked potentials (VEPs) recorded from pigmented rats and humans. Parametric manipulations of spatial frequency, temporal frequency and stimulus contrast revealed parallel functions, displaced for differences in absolute sensitivity. Additionally, diazepam produced similar effects in rats and human volunteers. A quantitative cross-species map was developed to illustrate the prediction of human effects from rat data. Exposure to carbon disulfide produced changes in rat VEP-derived contrast sensitivity functions, which resembled psychophysically-measured loss of visual contrast sensitivity in human workers exposed to organic solvents. The results of these continuing efforts should help indicate how well animal electrophysiological measures predict human neurotoxicity.

Within-session changes in peak N160 amplitude of flash evoked potentials in rats.

The negative peak occurring approximately 160 ms after stimulation (peak N160) of flash evoked potentials (FEPs) of rats changes with repeated testing. Habituation, sensitization, and arousal have all been invoked to explain these changes, but few studies have directly tested these explanations. We examined within-session changes in peak N160 amplitude with repeated testing, and the modulatory effects of stimulus intensity and auditory white noise. Peak N160 amplitude increased with daily testing (between-session changes), and was larger at greater stimulus intensities. In contrast, peak N160 amplitude underwent within-session increases on early days and within-session decreases on later days. The within-session changes were not affected by stimulus intensity. In rats previously tested in a quiet environment, exposure to acoustic white noise increased motor activity and transiently decreased peak N160 amplitude, which then increased and subsequently decreased with continued photic and acoustic stimulation. Repeated testing in the presence of noise did not alter the within-session changes in peak N160 amplitude. Heart rate showed both within- and between-session decreases, but was unaffected by noise. The data suggest that the within-session changes in peak N160 amplitude may reflect a habituation-like response to the test environment.

Prolonged neurobehavioral and visual effects of short-term exposure to 3,3'-iminodipropionitrile (IDPN) in rats.

Strategies for neurotoxicity testing often include initial screening tests, such as a functional observational battery (FOB) and motor activity assessment, followed by detailed characterization studies. In this study, a neurobehavioral screening battery (FOB and motor activity) was used to evaluate the effects of 3-day repeated exposure to 0, 100, 200, or 400 mg/kg/day IDPN. Adult Long-Evans rats (males and females) were tested before dosing and 1, 14, 28, 56, and 91 days after the third dose. IDPN initially produced generalized CNS depression, weakness, and hypothermia. Thereafter, marked hyperactivity, increased excitability, decreased reactivity to visual and auditory stimuli, neuromuscular weakness, equilibrium changes, and a "waltzing syndrome" (vertical and lateral head movements, circling, and retropulsion) emerged and persisted for 3 months. Males were more severely affected than females. Following neurobehavioral testing, the rats were examined for visual function using flash (three intensities) and pattern (three pattern sizes by three contrast levels)-elicited visual evoked potentials (VEPs). IDPN produced changes in pattern- and flash-elicited VEPs, thus verifying predictions made from the screening tests. However, the extent of the VEP changes produced by IDPN was insufficient to account for some of the deficits detected in the FOB, which are dependent on sensory, integrative, and motor functions. Thus, profound neurological effects of IDPN were detected using this screening battery and visual effects were confirmed using VEPs. These effects, following only three doses of IDPN, lasted for at least 3 months and thus may be permanent.