Marianne W. Marshall

Toxic Interactions in the Rat Nose: Pollutants from Soiled Bedding and Methyl Bromide

Interactions between test chemicals and pollutants can confound toxicology studies. To test the sensitivity of the regenerating olfactory epithelium to additional challenge with the olfactory epithelial toxicant methyl bromide (MeBr), Fischer 344 (F344) rats received 2 6-hr inhalation exposures (separated by a 28-day recovery period) to either 0 or 175 ppm MeBr. The regenerating epithelium was resistant to the second MeBr exposure. In addition, histopathologic examination revealed squamous epithelial hyperplasia in the vestibule; inflammation, epithelial necrosis, mucosal erosions, and squamous metaplasia of the respiratory epithelium in the anterior nose; and olfactory sensory cell loss in the dorsal medial meatus. These changes could not be attributed to MeBr, but they were correlated with housing in filter-capped cages between MeBr exposures and were presumably caused by volatile pollutants from soiled bedding. Moreover, olfactory sensory cell loss in the dorsal medial meatus was associated with local resistance to MeBr-induced damage in rats with pollutant-induced changes. Analysis of cage air revealed a progressive increase in ammonia levels between bedding changes (up to 50 ppm), but exposure to 300 ppm ammonia in an additional experiment reproduced only the anterior nasal lesions and not olfactory sensory cell loss. This study demonstrates that 1) regenerating olfactory epithelium is refractory to further MeBr toxicity; 2) pollutants from soiled bedding (in addition to ammonia) produce nasal lesions; and 3) pollutant-induced changes modify the nasal response to inhaled MeBr.

Fertility and developmental neurotoxicity effects of inhaled hydrogen sulfide in Sprague–Dawley rats

In this study, we examined whether perinatal exposure by inhalation to hydrogen sulfide (H2S) had an adverse impact on pregnancy outcomes, offspring prenatal and postnatal development, or offspring behavior. Virgin male and female Sprague-Dawley rats (12 rats/sex/concentration) were exposed (0, 10, 30, or 80 ppm H2S; 6 h/day, 7 days/week) for 2 weeks prior to breeding. Exposures continued during a 2-week mating period (evidence of copulation = gestation day 0 = GD 0) and then from GD 0 through GD 19. Exposure of dams and their pups (eight rats/litter after culling) resumed between postnatal day (PND) 5 and 18. Adult male rats were exposed for 70 consecutive days. Offspring were evaluated using motor activity (PND 13, 17, 21, and 60+/-2), passive avoidance (PND 22+/-1 and 62+/-3), functional observation battery (PND 60+/-2), acoustic startle response (PND 21 and 62+/-3), and neuropathology (PND 23+/-2 and 61+/-2). There were no deaths and no adverse physical signs observed in F0 male or female rats during the study. A statistically significant decrease in feed consumption was observed in F0 male rats from the 80-ppm H2S exposure group during the first week of exposure. There were no statistically significant effects on the reproductive performance of the F0 rats as assessed by the number of females with live pups, litter size, average length of gestation, and the average number of implants per pregnant female. Exposure to H2S did not affect pup growth, development, or performance on any of the behavioral tests. The results of our study suggest that H2S is neither a reproductive toxicant nor a behavioral developmental neurotoxicant in the rat at occupationally relevant exposure concentrations (< or =10 ppm).

Olfactory Mucosal Necrosis in Male CD Rats Following Acute Inhalation Exposure to Hydrogen Sulfide: Reversibility and the Possible Role of Regional Metabolism

Hydrogen sulfide (H 2S) is a potent inhibitor of cytochrome oxidase (CO) and is associated with dysosmia and anosmia in humans and nasal lesions in exposed rodents. An improved understanding of the pathogenesi s of these lesions is needed to determine their toxicological relevance. We exposed 10-week-old male CD rats to 0, 30, 80, 200, or 400 ppm H2S for 3 hours/day for 1 or 5 days consecutively. The nose was histologically examined 24 hours after H2S exposure, and lesion recovery was assessed at 2 and 6 weeks following the 5-day exposure. A single 3-hour exposure to ≥80 ppm H 2S resulted in regeneration of the respiratory mucosa and full thickness necrosis of the olfactory mucosa localized to the ventral and dorsal meatus, respectively. Repeated exposure to the same concentrations caused necrosis of the olfactory mucosa with early mucosal regeneration that extended from the dorsal medial meatus to the caudal regions of the ethmoid recess. Acute exposure to 400 ppm H2S induced severe mitochondrial swelling in sustentacular cells and olfactory neurons, which progressed to olfactory epithelial necrosis and sloughing. CO immunoreactive cells were more frequently observed in regions of the olfactory mucosa commonly affected by H2S than in regions that were not. These findings demonstrate that acute exposure to ≥80 ppm H2S resulted in reversible lesions in the respiratory and olfactory mucosae of the CD rat and that CO immunoreactivity may be a susceptibility factor for H2S-induced olfactory toxicity in the rat.

Respiratory Tract Responses in Male Rats Following Subchronic Acrolein Inhalation

The goal of this study was to characterize the respiratory tract toxicity of acrolein, including nasal and pulmonary effects, in adult male F344 rats. Animals underwent whole-body exposure to 0, 0.02, 0.06, 0.2, 0.6, or 1.8 ppm acrolein for 6 hr/day, five days/week for up to 65 exposure days (13 exposure weeks). Respiratory tract histopathology was evaluated after 4, 14, 30, and 65 exposure days, as well as 60 days after the end of the 13 week exposure. Acrolein exposure was associated with reduced body weight gain. Rats exposed to ≥ 0.06 ppm acrolein had depressed terminal body weights when compared with air-exposed controls. Histologic evaluation of the nasal cavity showed olfactory epithelial inflammation and olfactory neuronal loss (ONL) following exposure to 1.8 ppm acrolein. Moderately severe ONL in the dorsal meatus and ethmoid turbinates occurred within four days while septal involvement developed with ongoing exposure. A rostral-caudal gradient in lesion severity was noted, with the anterior portion of the nasal cavity being more severely affected. Acrolein exposure was associated with inflammation, hyperplasia, and squamous metaplasia of the respiratory epithelium. The lateral wall was amongst the most sensitive locations for these responses and increased respiratory epithelial cell proliferation occurred at this site following 4 to 30 days of exposure to ≥ 0.6 ppm acrolein. The NOAEL for nasal pathology seen in this study was 0.2 ppm acrolein.

Neurotoxicological effects associated with short-term exposure of Sprague-Dawley rats to hydrogen sulfide.

Although hydrogen sulfide (H2S) is a known neurotoxic hazard, only a limited number of experimental animal studies have examined its neurochemical or behavioral effects. Our aim was to determine if short-term inhalation exposure of rats to H2S would result in altered brain catecholamnine levels or impaired learning and memory. Three groups of adult male CD rats were tested; two groups were exposed by nose-only inhalation (0, 30, 80, 200, or 400 ppm H2S) and one group was exposed by whole-body inhalation (0, 10, 30, or 80 ppm H2S) for 3 h per day forfive consecutive days. The first group (n = 10 rats per concentration) was tested immediately following each daily nose-only H2S exposure for spatial learning with a Morris water maze. Core body temperatures were also monitored in these animals during and after the last H2S exposure. The second group of rats (n = 10 rats per concentration) was tested for spontaneous motor activity immediately following the fifth exposure. These rats were then euthanized and striatal, hippocampal, and hindbrain catecholamnine levels determined. A third group of rats (n = 5-7 rats per concentration) was pretrained on a multiple fixed- interval (FI) schedule and exposed whole-body. Daily performance on the FI schedule was compared for the week pre-exposure, for the exposure week immediately following daily exposures, and for the week postexposure. We observed significant reductions in motor activity, water maze performance, and body temperature following exposure only to high concentrations (> or = 80 ppm) of H2S. Exposure to H2S did not affect regional brain catecholamine concentrations or performance on the FI schedule. Additional studies using other measures of behavior and longer-term exposure to H2S may be required to more definitively address conditions under which H2S exposure results in behavioral toxicity.

Nasal uptake of inhaled acrolein in rats.

An improved understanding of the relationship between inspired concentration of the potent nasal toxicant acrolein and delivered dose is needed to support quantitative risk assessments. The uptake efficiency (UE) of 0.6, 1.8, or 3.6 ppm acrolein was measured in the isolated upper respiratory tract (URT) of anesthetized naive rats under constant-velocity unidirectional inspiratory flow rates of 100 or 300 ml/min for up to 80 min. An additional group of animals was exposed to 0.6 or 1.8 ppm acrolein, 6 h/day, 5 days/wk, for 14 days prior to performing nasal uptake studies (with 1.8 or 3.6 ppm acrolein) at a 100 ml/min airflow rate. Olfactory and respiratory glutathione (GSH) concentrations were also evaluated in naive and acrolein-preexposed rats. Acrolein UE in naive animals was dependent on the concentration of inspired acrolein, airflow rate, and duration of exposure, with increased UE occurring with lower acrolein exposure concentrations. A statistically significant decline in UE occurred during the exposures. Exposure to acrolein vapor resulted in reduced respiratory epithelial GSH concentrations. In acrolein-preexposed animals, URT acrolein UE was also dependent on the acrolein concentration used prior to the uptake exposure, with preexposed rats having higher UE than their naive counterparts. Despite having increased acrolein UE, GSH concentrations in the respiratory epithelium of acrolein preexposed rats were higher at the end of the 80 min acrolein uptake experiment than their in naive rat counterparts, suggesting that an adaptive response in GSH metabolism occurred following acrolein preexposure.

Changes in intracellular pH play a secondary role in hydrogen sulfide-induced nasal cytotoxicity.

Hydrogen sulfide (H2S) is a naturally occurring gas that is also associated with several industries. The potential for widespread human inhalation exposure to this toxic gas is recognized as a public health concern. The nasal epithelium is particularly susceptible to H2S-induced pathology. Cytochrome oxidase inhibition is postulated as one mechanism of H2S toxicity. Another mechanism by which the weak acid H2S could cause nasal injury is intracellular acidification and cytotoxicity. To further understand the mechanism by which H2S damages the nasal epithelium, nasal respiratory and olfactory epithelial cell isolates and explants from naive rats were loaded with the pH-sensitive intracellular chromophore SNARF-1 and exposed to air or 10, 80, 200, or 400 ppm H2S for 90 min. Intracellular pH was measured using flow cytometry or confocal microscopy. Cell lysates were used to quantify total protein and cytochrome oxidase activity. A modest but statistically significant decrease in intracellular pH occurred following exposure of respiratory and olfactory epithelium to 400 ppm H2S. Decreased cytochrome oxidase activity was observed following exposure to >10 ppm H2S in both respiratory and olfactory epithelia. None of the treatments resulted in cytotoxicity. The intracellular acidification of nasal epithelial cells by high-dose H2S exposure and the inhibition of cytochrome oxidase at much lower H2S concentrations suggest that changes in intracellular pH play a secondary role in H2S-induced nasal injury.

Development of a Mouse Whole-Body Exposure System from a Directed-Flow, Rat Nose-Only System

AbstractIn many inhalation exposure experiments, such as pharmacokinetic studies in unrestrained pregnant animals, it is desirable to expose unrestrained animals and to remove animals from the exposure system at intermediate time points. A two-tiered, 32-port, directed-flow, nose-only exposure system was modified with extended 0.635-mm stainless steel inlet tubes to create a whole-body, modified nose-only (WB-MNO) exposure system. Individual pregnant CD-1 mice held within a rat nose-only tube were exposed to a well-mixed methanol (MeOH) atmosphere. The volume of an individual mouse (∼30 ml) constituted approximately 5% of the 600-ml tube volume. Maternal MeOH pharmacokinetics were obtained on gestational day 8 following 6-h WB-MNO MeOH exposures at either 10,000 or 15,000 ppm. Results from these WB-MNO exposures were compared with a 6-h, 15,000 ppm MeOH exposure using a Hinners-type 1-m3 whole-body inhalation chamber (WB-H). The WB-MNO exposure atmosphere was produced using a wick generator heated to 29-3...