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The use of gasolines blended with a range of ethanol concentrations may result in inhalation of vapors containing a variable combination of ethanol with other volatile gasoline constituents. The possibility of exposure and potential interactions between vapor constituents suggests the need to evaluate the possible risks of this complex mixture. Previously we evaluated the effects of developmental exposure to ethanol vapors on neurophysiological measures of sensory function as a component of a larger project evaluating developmental ethanol toxicity. Here we report an evaluation using the same battery of sensory function testing in offspring of pregnant dams exposed during gestation to condensed vapors of gasoline (E0), gasoline blended with 15% ethanol (E15) or gasoline blended with 85% ethanol (E85). Pregnant Long-Evans rats were exposed to target concentrations 0, 3000, 6000, or 9000 ppm total hydrocarbon vapors for 6.5h/day over GD9 - GD20. Sensory evaluations of male offspring began as adults. The electrophysiological testing battery included tests of: peripheral nerve (compound action potentials, nerve conduction velocity [NCV]), somatosensory (cortical and cerebellar evoked potentials), auditory (brainstem auditory evoked responses), and visual functions. Visual function assessment included pattern elicited visual evoked potentials (VEP), VEP contrast sensitivity, dark-adapted (scotopic) electroretinograms (ERGs), light-adapted (photopic) ERGs, and green flicker ERGs. The results included sporadic statistically significant effects, but the observations were not consistently concentration-related and appeared to be statistical Type 1 errors related to multiple dependent measures evaluated. The exposure concentrations were much higher than can be reasonably expected from typical exposures to the general population during refueling or other common exposure situations. Overall the results indicate that gestational exposure of male rats to ethanol/gasoline vapor combinations did not cause detectable changes in peripheral nerve, somatosensory, auditory, or visual function when the offspring were assessed as adults., (Published by Elsevier Inc.)

Cytokines, low-molecular-weight messenger proteins that act as intercellular immunomodulatory signals, have become a mainstream preclinical marker for assessing the systemic inflammatory response to external stressors. The challenge is to quantitate from healthy subjects cytokine levels that are below or at baseline and relate those dynamic and complex cytokine signatures of exposures with the inflammatory and repair pathways. Thus, highly sensitive, specific, and precise analytical and statistical methods are critically important. Investigators at the U.S. Environmental Protection Agency (EPA) have implemented advanced technologies and developed statistics for evaluating panels of inflammatory cytokines in human blood, exhaled breath condensate, urine samples, and murine biological media. Advanced multiplex, bead-based, and automated analytical platforms provided sufficient sensitivity, precision, and accuracy over the traditional enzyme-linked immunosorbent assay (ELISA). Thus, baseline cytokine levels can be quantified from healthy human subjects and animals and compared to an in vivo exposure response from an environmental chemical. Specifically, patterns of cytokine responses in humans exposed to environmental levels of ozone and diesel exhaust, and in rodents exposed to selected pesticides (such as fipronil and carbaryl), were used as case studies to generally assess the taxonomic applicability of cytokine responses. The findings in this study may aid in the application of measureable cytokine markers in future adverse outcome pathway (AOP)-based toxicity testing. Data from human and animal studies were coalesced and the possibility of using cytokines as key events (KE) to bridge species responses to external stressors in an AOP-based framework was explored.

Exposure of pregnant Long-Evans rats to elemental mercury (Hg0) vapor resulted in a significant accumulation of Hg in tissues of neonates. Because elevated Hg in neonatal tissues may adversely affect growth and development, we were interested in how rapidly Hg was eliminated from neonatal tissues. Pregnant rats were exposed to 1, 2, or 4 mg Hg0 vapor/m3 or air (controls) for 2 hr/day from gestation day 6 (GD6) through GD15. Neonatal brain, liver, and kidney were analyzed for total Hg at various times between birth and postnatal day 90 (PND90). Milk was analyzed for Hg between birth and weaning (PND21). Before weaning, the Hg levels in neonatal tissues were proportional to maternal exposure concentrations and were highest in kidney followed by liver and then brain. There was no elimination of Hg between birth and weaning, indicating that neonates were exposed continuously to elevated levels of Hg during postpartum growth and development. Consumption of milk from exposed dams resulted in a slight increase in kidney Hg concentration during this period. Unexpectedly, neonatal Hg accumulation increased rapidly after weaning. Increased Hg was measured in both control and exposed neonates and was attributed to consumption of NIH-07 diet containing trace levels of Hg. By PND90, tissue Hg levels equilibrated at concentrations similar to those in unexposed adult Long-Evans rats fed the same diet. These data indicate that dietary exposure to trace amounts of Hg can result in a significantly greater accumulation of Hg in neonates than gestational exposure to high concentrations of Hg0 vapor.

Carbonyl sulfide (COS), a high-priority Clean Air Act chemical, was evaluated for neurotoxicity in short-term studies. F344 rats were exposed to 75-600 ppm COS 6 h per day, 5 days per week for up to 12 weeks. In rats exposed to 500 or 600 ppm for up to 4 days, malacia and microgliosis were detected in numerous neuroanatomical regions of the brain by conventional optical microscopy and magnetic resonance microscopy (MRM). After a 2-week exposure to 400 ppm, rats were evaluated using a functional observational battery. Slight gait abnormality was detected in 50% of the rats and hypotonia was present in all rats exposed to COS. Decreases in motor activity, and forelimb and hindlimb grip strength were also detected. In rats exposed to 400 ppm for 12 weeks, predominant lesions were in the parietal cortex area 1 (necrosis) and posterior colliculus (neuronal loss, microgliosis, hemorrhage), and occasional necrosis was present in the putamen, thalamus, and anterior olivary nucleus. Carbonyl sulfide specifically targeted the auditory system including the olivary nucleus, nucleus of the lateral lemniscus, and posterior colliculus. Consistent with these findings were alterations in the amplitude of the brainstem auditory evoked responses (BAER) for peaks N3, P4, N4, and N5 that represented changes in auditory transmission between the anterior olivary nucleus to the medial geniculate nucleus in animals after exposure for 2 weeks to 400 ppm COS. A concentration-related decrease in cytochrome oxidase activity was detected in the posterior colliculus and parietal cortex of exposed rats as early as 3 weeks. Cytochrome oxidase activity was significantly decreased at COS concentrations that did not cause detectable lesions, suggesting that disruption of the mitochondrial respiratory chain may precede these brain lesions. Our studies demonstrate that this environmental air contaminant has the potential to cause a wide spectrum of brain lesions that are dependent on the degree and duration of exposure.

In this carbonyl sulfide (COS) study, magnetic resonance microscopy (MRM) and detailed light microscopic evaluation effectively functioned in parallel to assure that the distribution and degree of pathology in the brain was accurately represented. MRM is a powerful imaging modality that allows for excellent identification of neuroanatomical structures coupled with the ability to acquire 200 or more cross-sectional images of the brain, and the ability to display them in multiple planes. F344 rats were exposed to 200-600 ppm COS for up to 12 weeks. Prior to MRM, rats were anesthetized and cardiac perfused with McDowell Trump's fixative containing a gadolinium MR contrast medium. Fixed specimens were scanned at the Duke Center for In Vivo Microscopy on a 9.4 Tesla magnetic resonance system adapted explicitly for microscopic imaging. An advantage of MRM in this study was the ability to identify lesions in rats that appeared clinically normal prior to sacrifice and the opportunity to identify lesions in areas of the brain which would not be included in conventional studies. Other advantages include the ability to examine the brain in multiple planes (transverse, dorsal, sagittal) and obtain and save the MRM images in a digital format that allows for postexperimental data processing and manipulation. MRM images were correlated with neuroanatomical and neuropathological findings. All suspected MRM images were compared to corresponding H&E slides. An important aspect of this study was that MRM was critical in defining our strategy for sectioning the brain, and for designing mechanistic studies (cytochrome oxidase evaluations) and functional assessments (electrophysiology studies) on specifically targeted anatomical sites following COS exposure.