William K. Boyes

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 TiO2 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 TiO2 nanoparticles at 0, 0.1, 0.3, 1, 3, 10, and 30 μg/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 TiO2. From 0.1 to 30 μg/ml TiO2, the side scatter increased sequentially while the forward scatter decreased, presumably due to substantial light reflection by the TiO2 particles. Based on the parameters of morphology and the calcein‐AM/propidium iodide viability assay, TiO2 concentrations below 30 μg/ml TiO2 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 TiO2 (0.1–0.3 μg/ml), the flow cytometer could detect as few as 5–10 nanoparticles per cell due to intense light scattering by TiO2. 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. Published 2010 Wiley‐Liss, Inc.

In Vitro Phototoxicity and Hazard Identification of Nano-scale Titanium Dioxide

Titanium dioxide nanoparticles (nano-TiO(2)) catalyze reactions under UV radiation and are hypothesized to cause phototoxicity. A human-derived line of retinal pigment epithelial cells (ARPE-19) was treated with six samples of nano-TiO(2) and exposed to UVA radiation. The TiO(2) nanoparticles were independently characterized to have mean primary particle sizes and crystal structures of 22nm anatase/rutile, 25nm anatase, 31nm anatase/rutile, 59nm anatase/rutile, 142nm anatase, and 214nm rutile. Particles were suspended in cell culture media, sonicated, and assessed for stability and aggregation by dynamic light scattering. Cells were treated with 0, 0.3, 1, 3, 10, 30, or 100μg/ml nano-TiO(2) in media for 24hrs and then exposed to UVA (2hrs, 7.53J/cm(2)) or kept in the dark. Viability was assessed 24hrs after the end of UVA exposure by microscopy with a live/dead assay (calcein-AM/propidium iodide). Exposure to higher concentrations of nano-TiO(2) with UVA lowered cell viability. The 25nm anatase and 31nm anatase/rutile were the most phototoxic (LC(50) with UVA<5μg/ml), while the 142nm anatase and 214nm rutile were the least phototoxic. An acellular assay ranked TiO(2) nanoparticles for their UVA photocatalytic reactivities. The particles were found to be capable of generating thiobarbituric acid reactive substances (TBARS) under UVA. Flow cytometry showed that nano-TiO(2) combined with UVA decreased cell viability and increased the generation of reactive oxygen species (ROS, measured by Mitosox). LC(50) values under UVA were correlated with TBARS reactivity, particle size, and surface area.

Combined effects of solvents on the rat's auditory system: styrene and trichloroethylene

Because exposures to toxic agents typically involve more than one substance, it is necessary to know if combined exposures pose different risks than those to single agents. Many solvents have been implicated in central nervous disorders and some of them are known to produce hearing loss, probably mediated by damage to cochlear hair cells. Hearing loss was studied by recording the brainstem auditory evoked response (BAER) in male Long Evans rats exposed 8 h/day for 5 days to mixtures of styrene (STY) and trichloroethylene (TCE). Dose groups included air or solvent pairs (STY/TCE) in the following concentrations (ppm): (0:3000), (250:2250), (500:1500), (750:750) and (1000:0). Decreased BAER amplitude, indicative of hearing loss, was correlated with blood levels of total solvent. The effects were as predicted by a linear dose-addition model, indicating neither synergistic nor antagonistic interactions at the concentrations studied.

Focal lesions of visual cortex—Effects on visual evoked potentials in rats

Focal lesions were placed in the visual cortex of Long-Evans hooded rats, immediately below skull screw recording electrodes. Lesions were produced by heat, and extended an average depth of about 0.9 mm below the cortical surface. Evoked potentials recorded from the electrode overlying the cortical lesion were compared with simultaneously recorded potentials from a contralateral homotopic site. The effects of the lesion were selective. Flash-evoked potential peaks P1, P2, and N2 were depressed by the lesion, and peaks N1 and P3 were augmented; peak N3 was unaffected. Pattern reversal evoked potential peak N3 was depressed by the lesion, and peaks N1 and P2 were made more distinct. The results emphasized that different peaks have different generators, and suggest in particular that flash-evoked potential peaks P1 and N2, and peak N3 of the pattern reversal-evoked potential require the superficial layers of the cortex.

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.

Electrophysiological Analysis of Complex Brain Systems: Sensory-Evoked Potentials and Their Generators

Publisher Summary This chapter focuses on neural sources of selected sensory-evoked potentials for electrophysiological analysis of complex brain systems. Sensory-evoked potentials represent a class of electrophysiological procedures that involve stimulation of sensory receptors or afferent nerves, and recording the evoked electrical activity from some portion or portions of the neural pathways. Typical stimuli might be a flash of light or a changing visual pattern for eliciting visual-evoked potentials, a click or tone of a specific frequency for auditory-evoked potentials, or an electrical shock to a peripheral nerve for somatosensory-evoked potentials. It is highly important that the recording of sensory-evoked potentials overcome signal-to-noise problems because of the small voltage of most of the evoked responses relative to other ongoing neural and non-neural electrical activity. This is typically accomplished through a combination of optimizing electrode placements, amplification and filtering of signals, and signal averaging or processing in phase with repetitive stimulation. This chapter provides basic concepts about localization of evoked potential generators. The chapter also elaborates electroretinograms, flash-evoked potentials, and pattern-evoked potentials. Brain stem auditory-evoked responses are also explained in detail.

Chlordimeform produces profound, selective, and transient changes in visual evoked potentials of hooded rats

Rat visual function was tested after acute exposure to chlordimeform (CDM), a formamidine insecticide/acaricide. Adult male Long-Evans rats were surgically implanted with epidural recording electrodes overlying visual cortex and tested 1 week later. Pattern reversal-evoked potentials (PREPs), flash-evoked potentials (FEPs), and FEP recovery ratios were measured after acute CDM administration. Averaged recordings obtained during 200 reversals of a black-and-white square wave grating comprised the PREPs, and those obtained during 128 paired strobe lamp flashes comprised the FEPs. In the first study, which examined dose-response relationships, i.p. injections of 0 (saline), 5, 15, or 40 mg/kg CDM-HCl were administered 30 min prior to testing. The PREP amplitudes showed large dose-related changes in the CDM-treated rats. PREP N1P1 and P1N3 peak-to-peak amplitudes increased more than 200% in the 40 mg/kg group. In contrast, FEP amplitudes and FEP recovery ratios were unchanged by CDM. Both PREP and FEP peak latencies were increased by CDM in dose-related fashions. In the second study, which examined the time course of CDM action, PREPs and paired-pulse FEPs were recorded 3, 6, and 24 h after dosage with either 0 or 40 mg/kg CDM. All evoked potential changes were large at 3 and 6 h, but had returned to control values by 24 h. In summary, acute exposure to CDM temporarily increased both the amplitude and latency of PREPs, but only the latency of FEPs.

Moving From External Exposure Concentration to Internal Dose: Duration Extrapolation Based on Physiologically Based Pharmacokinetic Derived Estimates of Internal Dose

The potential human health risk(s) from chemical exposure must frequently be assessed under conditions for which adequate human or animal data are not available. The default method for exposure-duration adjustment, based on Habers rule, C (external exposure concentration) or C n (the ten Berge modification) × t (exposure duration) = K (a constant toxic effect), has been criticized for prediction errors. A promising alternative approach to duration adjustment is based on equivalence of internal dose, that is, target-tissue dose levels, across different exposure durations. A proposed methodology for dose-duration adjustments for acute exposure guideline levels (AEGLs) based on physiologically based pharmacokinetic (PBPK) estimates of dose is illustrated with trichloroethylene (TCE). Steps in this methodology include: (1) selection and evaluation, or development and evaluation, of an appropriate PBPK model; (2) determination of an appropriate measure of internal dose; (3) estimation with the PBPK model of the tissue dose (the target tissue dose) resulting from the external exposure conditions (concentration, duration) of the critical effect; (4) estimation of the external exposure concentrations required to achieve tissue doses equivalent to the target tissue dose at exposure durations of interest; and (5) evaluation of sources of variability and uncertainty. For TCE, this PBPK modeling approach has allowed determination of dose metrics predictive of the acute neurotoxic effects of TCE and dose-duration adjustments based on estimates of internal dose.

Essentiality, Toxicity, and Uncertainty in the Risk Assessment of Manganese

Risk assessments of manganese by inhalation or oral routes of exposure typically acknowledge the duality of manganese as an essential element at low doses and a toxic metal at high doses. Previously, however, risk assessors were unable to describe manganese pharmacokinetics quantitatively across dose levels and routes of exposure, to account for mass balance, and to incorporate this information into a quantitative risk assessment. In addition, the prior risk assessment of inhaled manganese conducted by the U.S. Environmental Protection Agency (EPA) identified a number of specific factors that contributed to uncertainty in the risk assessment. In response to a petition regarding the use of a fuel additive containing manganese, methylcyclopentadienyl manganese tricarbonyl (MMT), the U.S. EPA developed a test rule under the U.S. Clean Air Act that required, among other things, the generation of pharmacokinetic information. This information was intended not only to aid in the design of health outcome studies, but also to help address uncertainties in the risk assessment of manganese. To date, the work conducted in response to the test rule has yielded substantial pharmacokinetic data. This information will enable the generation of physiologically based pharmacokinetic (PBPK) models capable of making quantitative predictions of tissue manganese concentrations following inhalation and oral exposure, across dose levels, and accounting for factors such as duration of exposure, different species of manganese, and changes of age, gender, and reproductive status. The work accomplished in response to the test rule, in combination with other scientific evidence, will enable future manganese risk assessments to consider tissue dosimetry more comprehensively than was previously possible.

Investigations of Amitraz Neurotoxicity in Rats I. Effects on Operant Performance

Amitraz (AMZ) is a formamidine pesticide which is often compared to chlordimeform (CDM). The effects of AMZ (6.25-75 mg/kg) and CDM (5-20 mg/kg) on the schedule-controlled performance of rats were examined using a multiple fixed-ratio (FR) 10 fixed-interval (FI) 300-sec schedule of milk reinforcement. Following dose-effect determinations, rats received three daily doses of AMZ (50 mg/kg). Low to intermediate doses of AMZ (6.25-25 mg/kg), administered 20 min presession, significantly decreased FI responding but not FR responding. In contrast, CDM appeared to decrease responding similarly under both components. Both compounds disrupted the temporal pattern of responding within the interval; AMZ had only a moderate influence at all effective doses but CDM produced a marked dose-dependent disruption of temporal response patterning. The effects of the high doses of AMZ (50-75 mg/kg) were more pronounced 24 hr after dosing, whereas the rats had recovered from lower doses at this time. Performance was progressively disrupted and the rats health rapidly deteriorated during the course of three daily injections of AMZ (50 mg/kg). Operant performance recovered more quickly than did the general health of the rats. Thus AMZ produced effects of multiple-schedule performance that were distinct from the effects of CDM. Moreover, the signs of intoxication and the effects on schedule-controlled behavior following a high dose (50 mg/kg) were augmented and persistent with short-term repeated administration.