R. Arden James

Olfactory Neuron Loss in Adult Male CD Rats Following Subchronic Inhalation Exposure to Hydrogen Sulfide

Dysosmia and anosmia are reported to occur following human exposure to hydrogen sulfide (H2S) gas. The clinical association between H2S exposure and olfactory dysfunction in humans necessitates evaluation of the nasal cavity and olfactory system in experimental animals used to study H2S toxicity. The purpose of this study was to subchronically expose 10-week-old male CD rats to relatively low concentrations of H2S and to histologically evaluate the nasal cavity for exposure-related lesions. Rats (n = 12/ group) were exposed via inhalation to 0, 10, 30, or 80 ppm H2S 6 h/d and 7 d/wk for 10 weeks. Following exposure to 30 and 80 ppm H2S, a significant increase in nasal lesions limited to the olfactory mucosa was observed. The lesions, which consisted of olfactory neuron loss and basal cell hyperplasia, were multifocal, bilaterally symmetrical, and had a characteristic rostrocaudal distribution pattern. Regions of the nasal cavity affected included the dorsal medial meatus and the dorsal and medial portions of the ethmoid recess. The no observed adverse effect level for olfactory lesions in this study was 10 ppm. For perspective, the American Conference of Governmental Industrial Hygienists threshold limit value (TLV) recommendation for H 2S is currently 10 ppm (proposed revision: 5 ppm), so the concentrations employed in the present study were 3 and 8 times the TLV. These findings suggest that subchronic inhalation exposure to a relatively low level of H2 S (30 ppm) can result in olfactory toxicity in rats. However, because of differences in the breathing style and nasal anatomy of rats and humans, additional research is required to determine the significance of these results for human health risk assessment.

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.

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.

Design and evaluation of an olfactometer for the assessment of 3-methylindole-induced hyposmia.

Few studies objectively evaluate olfactory function in animals following exposure to chemicals that induce nasal toxicity. An olfactometer capable of generating a reproducible olfactory stimulus and measuring an odorant-cued behavioral response was developed for rats from a commercially available two-way shuttle box. The box was modified to deliver the test odorant, acetaldehyde, to either of two chambers separated by a physical barrier consisting of a downward-directed airwall sandwiched between two exhaust panels. Male Fisher 344 rats were trained with either a coupled odorant- or tone-cued active avoidance paradigm in order to compare auditory-cued versus olfactory-cued learning and memory. Odorant-cued animals had faster acquisition and longer retention of the avoidance behavior than tone-cued animals. Animals given the model olfactory toxicant 3-methylindole (3-MI, 400 mg/kg, ip) had reduced odorant-cued avoidance, while no effect on tone-cued behavior was observed. In a follow-up study, additional odorant-trained rats were dosed with 0, 100, 200, or 300 mg/kg of 3-MI ip and olfactory function reassessed 6 days later. Histopathologic evidence of moderate to severe olfactory epithelial damage was observed in all rats 7 days after 3-MI administration. Only the highest 3-MI dose (300 mg/kg) was associated with a significant reduction in odor-cued avoidance behavior as compared to that seen in control. These results indicate that use of this olfactometer can provide a functional assessment of chemically induced olfactory toxicity and complements more routine nasal pathology.

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...

Chloroform-induced olfactory mucosal degeneration and osseous ethmoid hyperplasia are not associated with olfactory deficits in Fischer 344 rats

Adult female F-344 rats were trained (avoidance rate > 70%) over four days with a coupled tone- (n = 10 rats/dose) or 2 ppm acetaldehyde-cued (n = 6 rats/dose) foot shock paradigm. Rats were gavaged with chloroform dissolved in corn oil for 5 days/week for 3 week at 0 or 400 (tone-cued) or 0, 34, 100, or 400 (odor-cued) mg/kg body weight/day. Tone-cued response was reevaluated 6, 16, and 38 days after the first chloroform dose (day 1). Olfaction was assessed on days 6-7, 20-21, and 41-42 using 2 or 0.0002 ppm acetaldehyde. Nasal histopathology (n = 4-5 rats/dose) was assessed on days 6, 20, and 42. Significantly decreased body weights were observed following a single 100 or 400 mg/kg chloroform dose. Body weights in the 400 mg/kg/day chloroform group remained depressed for 17 days. Histopathology revealed degenerative changes in olfactory mucosa and underlying ethmoid turbinate bones that were essentially identical in nature and severity, including dose-response and progression, to those reported previously for chloroform gavage (Larson et al., Food Chem. Toxicol., 1995;33:443 456). At all dose level and sacrifice timepoints, however, regions of morphologically normal olfactory mucosa were present, especially in dorsal medial and ventral lateral regions of the nose. Neither odor- nor tone-cued avoidance behaviors were affected, indicating that even fairly severe and extensive chloroform-induced olfactory mucosal degeneration is not associated with a detectable olfactory deficit in rats.

Reciprocal translocations in somatic and germ cells of mice chronically exposed by inhalation to ethylene oxide: implications for risk assessment

Groups of male B6C3F1 mice were exposed by inhalation to 0, 25, 50, 100 or 200 p.p.m. ethylene oxide (EO) for up to 48 weeks (6 hours/day, 5 days/week). Animals were sacrificed at 6, 12, 24 and 48 weeks after the start of the exposure for analyses of reciprocal translocations in peripheral blood lymphocytes and germ cells. The frequency of the total chromosomal aberrations in the peripheral blood lymphocytes was significantly increased at the 100 and 200 p.p.m. exposure concentrations at the 12-week time point, at 50, 100 and 200 p.p.m. at the 24-week time point and at all EO concentrations at the 48-week time point. The frequency of stable reciprocal translocations, which can be used as biomarkers, was increased (P < 0.05) at 100 and 200 p.p.m. at the 12-week time point, at 100 and 200 p.p.m. at the 24-week time point and at 50, 100 and 200 p.p.m. at the 48-week time point. No statistically significant increase could be observed in translocation frequencies at the 6-week time point in the peripheral blood lymphocytes. The exposure-response curves were non-linear when the frequencies of translocations were plotted against EO exposure durations or against EO exposure concentrations. There was no effect of exposure concentration rate on reciprocal translocation frequency. Reciprocal translocations induced in spermatogonial stem cells (observed at the sprematocyte stage) showed significant increases in translocation frequencies over controls at all EO concentrations at 48 weeks. However, increases were small and they did not occur in a dose-responsive manner. The statistically significant increase observed at 12 weeks in the spermatocytes was equivocal. This study provides low-level chronic exposure somatic cytogenetic data generated in mice that can be used to support the shape of the tumour dose-response in rodents and humans The germ cell cytogenetic data are discussed in terms of its relevance for a threshold response for genetic effects at low exposures.

Respiration in Sprague-Dawley Rats During Pregnancy

Minute ventilation and tidal volume increase in humans during pregnancy. Little data exists, however, on the respiration in pregnant rats, despite their widespread use as an animal model. Since respiration will affect the pharmacokinetics of volatile compounds and ultimately the dose to the fetus, we conducted a study to evaluate respiration in rats during pregnancy. Whole-body plethysmography was used to measure the breathing frequency and tidal volume approximately every other day from gestation day (GD) 1 to 21 in 16 timed pregnant and 16 nonpregnant, female, Sprague-Dawley rats. Minute ventilation was calculated as a product of the breathing frequency and tidal volume, and the body weight of each rat was used to determine the scaled ventilation. Multivariate analysis of variance methods for a repeated-measures design were used to analyze the respiratory data. Breathing frequency was not affected by pregnancy; however, tidal volume was somewhat greater in pregnant versus nonpregnant rats. The increase in tidal volume resulted in significantly increased minute ventilation in pregnant rats compared to nonpregnant rats during the latter period of gestation. Due to the increased body weight of the pregnant rats, the scaled ventilation at the end of gestation was significantly lower in pregnant rats compared to nonpregnant rats. This study provides important reference values that can be used in pharmacokinetic models during pregnancy.

Development and use of a single-animal whole-body system for inhalation exposure.

Inhalation exposure studies, in which test subjects are fully or partially immersed in an atmosphere containing a compound of interest, are usually carried out using one of two possible exposure systems: large whole-body chambers or systems that expose only the animals nose or head. Whole-body chambers may require large quantities of test compound, which can pose a problem if the chemical is expensive or available in limited quantities. Nose- or head-only systems can help conserve test compound but may cause stress or injury to animals. To address these concerns, the authors developed an exposure system consisting of small single-animal whole-body chambers. They exposed 80 mice and 80 rats to five test compounds at various concentrations. Though the system was labor-intensive for animal care technicians, it effectively exposed animals to precise chemical doses without causing adverse effects, using less test compound than would have been required in a conventional whole-body chamber.