Differential transcriptomic alterations in nasal versus lung tissue of acrolein-exposed rats.

Introduction: Acrolein is a significant component of anthropogenic and wildfire emissions, as well as cigarette smoke. Although acrolein primarily deposits in the upper respiratory tract upon inhalation, patterns of site-specific injury in nasal versus pulmonary tissues are not well characterized. This assessment is critical in the design of in vitro and in vivo studies performed for assessing health risk of irritant air pollutants. Methods: In this study, male and female Wistar-Kyoto rats were exposed nose-only to air or acrolein. Rats in the acrolein exposure group were exposed to incremental concentrations of acrolein (0, 0.1, 0.316, 1 ppm) for the first 30 min, followed by a 3.5 h exposure at 3.16 ppm. In the first cohort of male and female rats, nasal and bronchoalveolar lavage fluids were analyzed for markers of inflammation, and in a second cohort of males, nasal airway and left lung tissues were used for mRNA sequencing. Results: Protein leakage in nasal airways of acrolein-exposed rats was similar in both sexes; however, inflammatory cells and cytokine increases were more pronounced in males when compared to females. No consistent changes were noted in bronchoalveolar lavage fluid of males or females except for increases in total cells and IL-6. Acrolein-exposed male rats had 452 differentially expressed genes (DEGs) in nasal tissue versus only 95 in the lung. Pathway analysis of DEGs in the nose indicated acute phase response signaling, Nrf2-mediated oxidative stress, unfolded protein response, and other inflammatory pathways, whereas in the lung, xenobiotic metabolism pathways were changed. Genes associated with glucocorticoid and GPCR signaling were also changed in the nose but not in the lung. Discussion: These data provide insights into inhaled acrolein-mediated sex-specific injury/inflammation in the nasal and pulmonary airways. The transcriptional response in the nose reflects acrolein-induced acute oxidative and cytokine signaling changes, which might have implications for upper airway inflammatory disease susceptibility.

Serum metabolome and liver transcriptome reveal acrolein inhalation-induced sex-specific homeostatic dysfunction.

Acrolein, a respiratory irritant, induces systemic neuroendocrine stress. However, peripheral metabolic effects have not been examined. Male and female WKY rats were exposed to air (0 ppm) or acrolein (3.16 ppm) for 4 h, followed by immediate serum and liver tissue collection. Serum metabolomics in both sexes and liver transcriptomics in males were evaluated to characterize the systemic metabolic response. Of 887 identified metabolites, > 400 differed between sexes at baseline. An acrolein biomarker, 3-hydroxypropyl mercapturic acid, increased 18-fold in males and 33-fold in females, indicating greater metabolic detoxification in females than males. Acrolein exposure changed 174 metabolites in males but only 50 in females. Metabolic process assessment identified higher circulating free-fatty acids, glycerols, and other lipids in male but not female rats exposed to acrolein. In males, acrolein also increased branched-chain amino acids, which was linked with metabolites of nitrogen imbalance within the gut microbiome. The contribution of neuroendocrine stress was evident by increased corticosterone in males but not females. Male liver transcriptomics revealed acrolein-induced over-representation of lipid and protein metabolic processes, and pathway alterations including Sirtuin, insulin-receptor, acute-phase, and glucocorticoid signaling. In sum, acute acrolein inhalation resulted in sex-specific serum metabolomic and liver transcriptomic derangement, which may have connections to chronic metabolic-related diseases.

Sex-specific respiratory and systemic endocrine effects of acute acrolein and trichloroethylene inhalation.

Acrolein and trichloroethylene (TCE) are priority hazardous air pollutants due to environmental prevalence and adverse health effects; however, neuroendocrine stress-related systemic effects are not characterized. Comparing acrolein, an airway irritant, and TCE with low irritancy, we hypothesized that airway injury would be linked to neuroendocrine-mediated systemic alterations. Male and female Wistar-Kyoto rats were exposed nose-only to air, acrolein or TCE in incremental concentrations over 30 min, followed by 3.5-hr exposure to the highest concentration (acrolein - 0.0, 0.1, 0.316, 1, 3.16 ppm; TCE - 0.0, 3.16, 10, 31.6, 100 ppm). Real-time head-out plethysmography revealed acrolein decreased minute volume and increased inspiratory-time (males>females), while TCE reduced tidal-volume. Acrolein, but not TCE, inhalation increased nasal-lavage-fluid protein, lactate-dehydrogenase activity, and inflammatory cell influx (males>females). Neither acrolein nor TCE increased bronchoalveolar-lavage-fluid injury markers, although macrophages and neutrophils increased in acrolein-exposed males and females. Systemic neuroendocrine stress response assessment indicated acrolein, but not TCE, increased circulating adrenocorticotrophic hormone, and consequently corticosterone, and caused lymphopenia, but only in males. Acrolein also reduced circulating thyroid-stimulating hormone, prolactin, and testosterone in males. In conclusion, acute acrolein inhalation resulted in sex-specific upper respiratory irritation/inflammation and systemic neuroendocrine alterations linked to hypothalamic-pituitary-adrenal axes activation, which is critical in mediating extra-respiratory effects.

Toluene inhalation exposure for 13 weeks causes persistent changes in electroretinograms of Long-Evans rats.

Studies of humans chronically exposed to volatile organic solvents have reported impaired visual functions, including low contrast sensitivity and reduced color discrimination. These reports, however, lacked confirmation from controlled laboratory experiments. To address this question experimentally, we examined visual function by recording visual evoked potentials (VEP) and/or electroretinograms (ERG) from four sets of rats exposed repeatedly to toluene. In addition, eyes of the rats were examined with an ophthalmoscope and some of the retinal tissues were evaluated for rod and M-cone photoreceptor immunohistochemistry. The first study examined rats following exposure to 0, 10, 100 or 1000ppm toluene by inhalation (6hr/d, 5d/wk) for 13 weeks. One week after the termination of exposure, the rats were implanted with chronically indwelling electrodes and the following week pattern-elicited VEPs were recorded. VEP amplitudes were not significantly changed by toluene exposure. Four to five weeks after completion of exposure, rats were dark-adapted overnight, anesthetized, and several sets of electroretinograms (ERG) were recorded. In dark-adapted ERGs recorded over a 5-log (cd-s/m(2)) range of flash luminance, b-wave amplitudes were significantly reduced at high stimulus luminance values in rats previously exposed to 1000ppm toluene. A second set of rats, exposed concurrently with the first set, was tested approximately one year after the termination of 13 weeks of exposure to toluene. Again, dark-adapted ERG b-wave amplitudes were reduced at high stimulus luminance values in rats previously exposed to 1000ppm toluene. A third set of rats was exposed to the same concentrations of toluene for only 4 weeks, and a fourth set of rats exposed to 0 or 1000ppm toluene for 4 weeks were tested approximately 1year after the completion of exposure. No statistically significant reductions of ERG b-wave amplitude were observed in either set of rats exposed for 4 weeks. No significant changes were observed in ERG a-wave amplitude or latency, b-wave latency, UV- or green-flicker ERGs, or in photopic flash ERGs. There were no changes in the density of rod or M-cone photoreceptors. The ERG b-wave reflects the firing patterns of on-bipolar cells. The reductions of b-wave amplitude after 13 weeks of exposure and persisting for 1year suggest that alterations may have occurred in the inner nuclear layer of the retina, where the bipolar cells reside, or the outer or inner plexiform layers where the bipolar cells make synaptic connections. These data provide experimental evidence that repeated exposure to toluene may lead to subtle persistent changes in visual function. The fact that toluene affected ERGs, but not VEPs, suggests that elements in the rat retina may be more sensitive to organic solvent exposure than the rat visual cortex.

Toxicological assessments of rats exposed prenatally to inhaled vapors of gasoline and gasoline-ethanol blends.

The primary alternative to petroleum-based fuels is ethanol, which may be blended with gasoline in the United States at concentrations up to 15% for most automobiles. Efforts to increase the amount of ethanol in gasoline have prompted concerns about the potential toxicity of inhaled ethanol vapors from these fuels. The well-known sensitivity of the developing nervous and immune systems to ingested ethanol and the lack of information about the neurodevelopmental toxicity of ethanol-blended fuels prompted the present work. Pregnant Long-Evans rats were exposed for 6.5h/day on days 9-20 of gestation to clean air or vapors of gasoline containing no ethanol (E0) or gasoline blended with 15% ethanol (E15) or 85% ethanol (E85) at nominal concentrations of 3000, 6000, or 9000 ppm. Estimated maternal peak blood ethanol concentrations were less than 5mg/dL for all exposures. No overt toxicity in the dams was observed, although pregnant dams exposed to 9000 ppm of E0 or E85 gained more weight per gram of food consumed during the 12 days of exposure than did controls. Fuel vapors did not affect litter size or weight, or postnatal weight gain in the offspring. Tests of motor activity and a functional observational battery (FOB) administered to the offspring between post-natal day (PND) 27-29 and PND 56-63 revealed an increase in vertical activity counts in the 3000- and 9000-ppm groups in the E85 experiment on PND 63 and a few small changes in sensorimotor responses in the FOB that were not monotonically related to exposure concentration in any experiment. Neither cell-mediated nor humoral immunity were affected in a concentration-related manner by exposure to any of the vapors in 6-week-old male or female offspring. Systematic concentration-related differences in systolic blood pressure were not observed in rats tested at 3 and 6 months of age in any experiment. No systematic differences were observed in serum glucose or glycated hemoglobin A1c (a marker of long-term glucose homeostasis). These observations suggest a LOEL of 3000 ppm of E85 for vertical activity, LOELs of 9000 ppm of E0 and E85 for maternal food consumption, and NOELs of 9000 ppm for the other endpoints reported here. The ethanol content of the vapors did not consistently alter the pattern of behavioral, immunological, or physiological responses to the fuel vapors. The concentrations of the vapors used here exceed by 4-6 orders of magnitude typical exposure levels encountered by the public.

Toxicological outcomes in rats exposed to inhaled ethanol during gestation.

Recent legislation has encouraged replacing petroleum-based fuels with renewable alternatives including ethanol, which is typically blended with gasoline in the United States at concentrations up to 10%, with allowances for concentrations up to 85% for some vehicles. Efforts to increase the amount of ethanol in gasoline have prompted concerns about the potential toxicity of inhaled ethanol vapors from these fuels. The well-known sensitivity of the developing nervous and immune systems to ingested ethanol, and the lack of information about its toxicity by inhalation prompted the present work on its potential developmental effects in a rat model. Pregnant Long-Evans rats were exposed for 6.5h/day on days 9-20 of gestation to clean air or ethanol vapor at concentrations of 5000, 10,000, or 21,000 ppm, which resulted in estimated peak blood ethanol concentrations (BECs) of 2.3, 6.7, and 192 mg/dL, respectively. No overt toxicity in the dams was observed. Ethanol did not affect litter size or weight, or postnatal weight gain in the pups. Motor activity was normal in offspring through postnatal day (PND) 29. On PND 62, the 5000 and 21,000 ppm groups were more active than controls. On PND 29 and 62, offspring were tested with a functional observational battery, which revealed small changes in the neuromuscular and sensorimotor domains that were not systematically related to dose. Cell-mediated and humoral immunity were not affected by ethanol exposure in 6-week-old offspring. Systolic blood pressure was increased by 10,000 ppm ethanol in males at PND 90 but not at PND 180. No differences in lipoprotein profile, liver function, or kidney function were observed. In summary, prenatal exposure to inhaled ethanol caused some mild changes in physiological and behavioral development in offspring that were not clearly related to inhaled concentration or BEC, and did not produce detectable changes in immune function. This low toxicity of inhaled ethanol may result from the slow rise in BEC by the inhalation route.

Use of novel inhalation kinetic studies to refine physiologically-based pharmacokinetic models for ethanol in non-pregnant and pregnant rats.

Ethanol (EtOH) exposure induces a variety of concentration-dependent neurological and developmental effects in the rat. Physiologically-based pharmacokinetic (PBPK) models have been used to predict the inhalation exposure concentrations necessary to produce blood EtOH concentrations (BEC) in the range associated with these effects. Previous laboratory reports often lacked sufficient detail to adequately simulate reported exposure scenarios associated with BECs in this range, or lacked data on the time-course of EtOH in target tissues (e.g. brain, liver, eye, fetus). To address these data gaps, inhalation studies were performed at 5000, 10 000, and 21 000 ppm (6 h/d) in non-pregnant female Long-Evans (LE) rats and at 21 000 ppm (6.33 h/d) for 12 d of gestation in pregnant LE rats to evaluate our previously published PBPK models at toxicologically-relevant blood and tissue concentrations. Additionally, nose-only and whole-body plethysmography studies were conducted to refine model descriptions of respiration and uptake within the respiratory tract. The resulting time-course and plethysmography data from these in vivo studies were compared to simulations from our previously published models, after which the models were recalibrated to improve descriptions of tissue dosimetry by accounting for dose-dependencies in pharmacokinetic behavior. Simulations using the recalibrated models reproduced these data from non-pregnant, pregnant, and fetal rats to within a factor of 2 or better across datasets, resulting in a suite of model structures suitable for simulation of a broad range of EtOH exposure scenarios.

Behavioral effects of sub-acute inhalation of toluene in adult rats.

Reports of behavioral effects of repeated inhalation of toluene in rats have yielded inconsistent findings. A recent study from this laboratory (Beasley et al., 2010) observed that after 13 weeks of inhaled toluene ("subchronic" exposure scenario), rats showed mild but persistent changes in behavior, primarily involving acquisition of an autoshaped lever-press response. The present experiment sought to systematically replicate these findings, using a 4-week "sub-acute" exposure scenario. Adult male Long-Evans rats inhaled toluene vapor (0, 10, 100, or 1000 ppm) for 6h/day, 5 days/week for 4 weeks. As in the subchronic study, toluene had no effect on motor activity, anxiety-related behavior in the elevated plus-maze, or acquisition of the visual discrimination. However, sub-acute toluene did not affect appetitively-motivated acquisition of the lever-press response, but did reduce accuracy of signal detection at the end of training. Analysis of the deficit in accuracy in the 1000 ppm group by means of manipulations of different task parameters suggested a greater influence of attentional impairment than visual or motor dysfunction as a source for the deficit. These results confirm a pattern of subtle and inconsistent long-term effects of repeated daily exposure to concentrations of toluene vapor of 1000 ppm and below, in contrast to robust and reliable effects of acute inhalation of the solvent at concentrations above 1000 ppm.

Behavioral effects of subchronic inhalation of toluene in adult rats.

Whereas the acute neurobehavioral effects of toluene are robust and well characterized, evidence for persistent effects of repeated exposure to this industrial solvent is less compelling. The present experiment sought to determine whether subchronic inhalation of toluene caused persistent behavioral changes in rats. Adult male Long-Evans rats inhaled toluene vapor (0, 10, 100, or 1000 ppm) for 6h/day, 5 days/week for 13 weeks and were evaluated on a series of behavioral tests beginning 3 days after the end of exposure. Toluene delayed appetitively-motivated acquisition of a lever-press response, but did not affect motor activity, anxiety-related behavior in the elevated plus maze, trace fear conditioning, acquisition of an appetitively-motivated visual discrimination, or performance of a visual signal detection task. Challenges with acute inhalation of toluene vapor (1200-2400 ppm for 1 h) and injections of quinpirole (0.01-0.03 mg/kg) and raclopride (0.03-0.10 mg/kg) revealed no toluene-induced latent impairments in visual signal detection. These results are consistent with a pattern of subtle and inconsistent long-term effects of daily exposure to toluene vapor, in contrast to robust and reliable effects of acute inhalation of the solvent.

Neurotrophin mediation of allergic airways responses to inhaled diesel particles in mice.

Neurotrophins, including nerve growth factor (NGF), partially mediate many features of allergic airways disease including airway hyperresponsiveness. Diesel exhaust particulates (DEP) associated with the combustion of diesel fuel exacerbate many of these allergic airways responses in humans. We tested the hypothesis that DEP-induced enhancement of allergic airways disease in a murine model is dependent on normal function of the low affinity pan-neurotrophin receptor p75(NTR), or tyrosine kinase A (trkA), the primary receptor for NGF. Ovalbumin (OVA)-sensitized and nonallergic BALB/c mice were intranasally instilled with anti-p75(NTR), anti-trkA, or vehicle, 1 h before OVA aerosol challenge, and then exposed nose-only to the particulate matter fraction that was less than 2.5 microns in aerodynamic diameter fraction of Standard Reference Material 2975 DEP (2.0 mg/m(3)) or filtered air for 5 h. One day later, DEP-exposed OVA-allergic mice had significantly greater increases in ventilatory responses to methacholine (Mch), but not increased lung resistance, suggesting that the airflow changes may have originated in the nasal passages. DEP-exposed OVA-allergic mice also had increased lung IL-4 levels relative to all other groups. The instillation of anti-p75(NTR) or anti-trkA completely reversed the DEP-induced increases in ventilatory responses and lung IL-4 protein to levels similar to control mice. OVA-allergic DEP-exposed mice treated with anti-p75(NTR) had significantly less lung resistance in response to Mch relative to OVA-allergic DEP-exposed mice treated with anti-trkA. The results of this study demonstrate that the enhancement of allergic airways responses by DEP exposure is partly dependent on neurotrophins in mice. In addition, neurotrophins that bind p75(NTR), but not trkA, may mediate pulmonary central airways and tissue resistance responses to allergen and DEP exposure.

Inhibition of pan neurotrophin receptor p75 attenuates diesel particulate-induced enhancement of allergic airway responses in C57/B16J mice.

Recent investigations have linked neurotrophins, including nerve growth factor (NGF), neurotrophin-3 (NT-3), and brain-derived neurotrophic factor (BDNF), to allergic airways diseases. Antibody blockade of NGF attenuates airway resistance in allergic mice. Diesel exhaust particle (DEP) exposure has been linked to asthma exacerbation in many cities with vehicular traffic congestion. We tested the hypothesis that DEP-induced enhancement of the hallmark features of allergic airway disease in a murine model is dependent on the function of the pan neurotrophin receptor p75. Ovalbumin (OVA)-sensitized C57B1/6J mice were intranasally instilled with an antibody against the p75 receptor or saline alone 1 h before OVA challenge. The mice were then exposed nose-only to the PM2.5 fraction of SRM2975 DEP or air alone for 5 h beginning 1 h after OVA challenge. Two days later, air-exposed OVA-allergic mice developed a small but insignificant increase in methacholine-induced airflow obstruction relative to air-exposed, vehicle-sensitized mice. DEP-exposed OVA-allergic mice had a significantly greater degree of airway obstruction than all other groups. Instillation of anti-p75 significantly attenuated the DEP-induced increase in airway obstruction in OVA-allergic mice to levels similar to non-sensitized mice. The DEP-induced exacerbation of allergic airway responses may, in part, be mediated by neurotrophins.