Michael P. Moorman

Gender Differences in Murine Airway Responsiveness and Lipopolysaccharide-Induced Inflammation

The roles of gender and sex hormones in lung function and disease are complex and not completely understood. The present study examined the influence of gender on lung function and respiratory mechanics in naive mice and on acute airway inflammation and hyperresponsiveness induced by intratracheal LPS administration. Basal lung function characteristics did not differ between naive males and females, but males demonstrated significantly greater airway responsiveness than females following aerosolized methacholine challenge as evidenced by increased respiratory system resistance and elastance (p < 0.05). Following LPS administration, males developed more severe hypothermia and greater airway hyperresponsiveness than females (p < 0.05). Inflammatory indices including bronchoalveolar lavage fluid total cells, neutrophils, and TNF-α content were greater in males than in females 6 h following LPS administration (p < 0.05), whereas whole-lung TLR-4 protein levels did not differ among treatment groups, suggesting that differential expression of TLR-4 before or after LPS exposure did not underlie the observed inflammatory outcomes. Gonadectomy decreased airway inflammation in males but did not alter inflammation in females, whereas administration of exogenous testosterone to intact females increased their inflammatory responses to levels observed in intact males. LPS-induced airway hyperresponsiveness was also decreased in castrated males and was increased in females administered exogenous testosterone. Collectively, these data indicate that airway responsiveness in naive mice is influenced by gender, and that male mice have exaggerated airway inflammatory and functional responses to LPS compared with females. These gender differences are mediated, at least in part, by effects of androgens.

Evidence for oxidative damage to red blood cells in mice induced by arsine gas

In animals and human beings exposed to arsine gas (AsH3) a severe and fulminant lysis of erythrocytes occurs. Little is known about the effects of subchronic exposure on the hematopoietic system or about the mechanism of hemolysis produced by arsine gas. To examine these, we exposed male and female mice to 0.000, 0.025, 0.500 and 2.500 ppm arsine gas for 6 h a day, 5 days a week during a 90-day period. After 5, 15, and 90 days of exposure, blood was collected and routine hematologic profiles were performed to document the effects of arsine gas on peripheral blood. A moderate hemolytic anemia, indicated by decreases in erythrocyte counts, hematocrits, hemoglobin concentrations and increases in mean corpuscular hemoglobins and mean corpuscular hemoglobin concentrations, was seen in blood samples collected after 5 days of exposure. In blood collected after 15 and 90 days of exposure, the anemia was less severe but a greater increase in mean corpuscular volumes and absolute reticulocyte counts revealed an active regenerative response. Higher concentrations of methemoglobin in animals in the 2.500 ppm exposure group (measured after 90 days of exposure) indicated that the rate of oxidation of heme (ferrous to ferric) increased due to exposure to arsine gas. Additionally, the presence of Heinz bodies in blood smears stained with brilliant cresyl blue and decreases in reduced glutathione concentrations in red blood cells exposed to arsine gas in vitro provide evidence that the mechanism of hemolysis involves depletion of intracellular reduced glutathione resulting in an oxidation of sulfhydryl groups in hemoglobin and possibly red cell membranes.

Comparative toxicity of arsine gas in B6C3F1 mice, Fischer 344 rats, and Syrian Golden hamsters: System organ studies and comparison of clinical indices of exposure

In order to examine possible species differences in response to arsine exposure, multiple inhalation studies consisting of acute (1-day), subacute (14- and 28-day), and subchronic (90-day) exposures to this agent were conducted using three different species of rodents. Evaluations of hematopoietic organs and alterations in the heme biosynthetic pathway were the focus of these studies. Species used were B6C3F1 mice (exposed 1, 14, or 90 days), Fischer 344 rats (exposed 14, 28, or 90 days), and Syrian Golden hamsters (exposed 28 days). All arsine exposures were at concentrations of 0.5, 2.5, or 5.0 ppm except for 90-day studies, in which concentrations were lowered to 0.025, 0.5, or 2.5 ppm. No changes in body weight gain were observed in either sex of mice or hamsters. The only decrease in body weight gain occurred in male rats exposed to 5.0 ppm arsine for 28 days. Significant exposure-related increases in relative spleen weights occurred in both sexes of mice and rats in the 0.5 (except 14-day female rats), 2.5, and 5.0 ppm exposure groups from all studies and in hamsters in the 2.5 and 5.0 ppm exposure groups. Generally, increases in relative liver weight occurred in fewer exposure groups and were of a lesser magnitude than increases in spleen weight. Other parameters affected included decreased packed cell volumes (mice, rats, and hamsters), hematology profiles (rats), and an increase in delta-aminolevulinic acid dehydratase activity in all species. Arsenic content was measured in livers of rats after 90 days of exposure. Concentrations increased in relation to atmospheric concentrations of arsine. Histopathologic changes included increased hemosiderosis and extramedullary hematopoiesis in spleen and intracanalicular bile stasis (mice only) in liver. Additionally, bone marrow hyperplasia was observed in rats. Effects on other organs were not observed, suggesting that the hematopoietic system is the primary target for arsine. In conclusion, we have determined that the effects of arsine exposure upon mice, rats, and hamsters are similar. Most importantly, even though no effects on the hematopoietic system were observed following a single exposure to 0.5 ppm arsine which is 10 times the Threshold Limit Value (TLV) set by the American Conference of Governmental Industrial Hygienists, repeated exposure to 0.025 ppm (one-half the TLV) caused a significant anemia in rats.

Cyclooxygenase-1 Overexpression Decreases Basal Airway Responsiveness but Not Allergic Inflammation

Pharmacological inhibition or genetic disruption of cyclooxygenase (COX)-1 or COX-2 exacerbates the inflammatory and functional responses of the lung to environmentally relevant stimuli. To further examine the contribution of COX-derived eicosanoids to basal lung function and to allergic lung inflammation, transgenic (Tr) mice were generated in which overexpression of human COX-1 was targeted to airway epithelium. Although no differences in basal respiratory or lung mechanical parameters were observed, COX-1 Tr mice had increased bronchoalveolar lavage fluid PGE2 content compared with wild-type littermates (23.0 ± 3.6 vs 8.4 ± 1.4 pg/ml; p < 0.05) and exhibited decreased airway responsiveness to inhaled methacholine. In an OVA-induced allergic airway inflammation model, comparable up-regulation of COX-2 protein was observed in the lungs of allergic wild-type and COX-1 Tr mice. Furthermore, no genotype differences were observed in allergic mice in total cell number, eosinophil content (70 vs 76% of total cells, respectively), and inflammatory cytokine content of bronchoalveolar lavage fluid, or in airway responsiveness to inhaled methacholine (p > 0.05). To eliminate the presumed confounding effects of COX-2 up-regulation, COX-1 Tr mice were bred into a COX-2 null background. In these mice, the presence of the COX-1 transgene did not alter allergen-induced inflammation but significantly attenuated allergen-induced airway hyperresponsiveness, coincident with reduced airway leukotriene levels. Collectively, these data indicate that COX-1 overexpression attenuates airway responsiveness under basal conditions but does not influence allergic airway inflammation.

Time‐varying concentration profile as a determinant of the inhalation toxicity of carbon tetrachloride

Certain limitations on the flexibility of small-animal inhalation exposure systems are overcome by the machine control and monitoring of the concentration of the gas or vapor of interest. Computer assistance of chamber operation allows the user to simulate time-varying concentration profiles accurately and repeatedly. We exposed rats to seven different profiles in which the maximum concentration of carbon tetrachloride (CCl4) was 1500 ppm and the product of concentration times time (C X T) was 4500 ppm X h. The purpose was to determine the effects of systematically varying the shape of the concentration profile on the expression of hepatotoxicity of a chemical about which much was already known. All of the exposures were conducted within a span of 6 h. Examination of the severity of vacuolation and pattern of necrosis could be used to distinguish some of the exposure profiles from others. For example, vacuolation was less severe when two equal pulses were presented with an interval of 60 min, rather than 180-240 min. The indexes of necrosis varied in a more complex way, and the differences among the profiles that accounted for the differences in the patterns of the histopathological changes were not immediately apparent. We concluded that the characteristic of a time-related variation in concentration is one of the determinants of the inhalation hepatotoxicity of CCl4 and that the simple, time-weighted, average concentration may not always fairly represent the best model for the study of problems in inhalation toxicology.

Inhalation Toxicity of Phosphine for Fischer 344 Rats and B6C3F1 Mice

AbstractBecause of the potential increased use of phosphine (PH3) as a fumigant and the lack of adequate toxicity data, short-term inhalation studies were conducted to characterize the toxicity of PH3 for Fischer 344 (F344) rats and B6C3F1 mice. Male rats and mice were exposed to 0, 1, 5, or 10 ppm PH3 for up to 4 days, and males and females to 0, 1.25, 2.5, or 5 ppm for 2 wk. In the 4-day study, all rats died by the end of the third exposure to 10 ppm, and all mice were euthanized in moribund condition after the fourth exposure to 10 ppm. Clinical pathology data were obtained only for mice, due to early mortality of rats. There were no significant treatment-related effects in hematological indices in mice exposed to 1 or 5 ppm; at 10 ppm there was a moderate anemia, and leukocyte counts were significantly decreased. There were significant biologically relevant increases in serum activity of alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) and in the concentration of urine nitrogen (UN) at ...

INHALATION TOXICITY STUDIES OF THE a ,b -UNSATURATED KETONES: 2-Cyclohexene-1-One

2-Cyclohexene-1-one (CHX) is a cyclic alpha,beta-unsaturated ketone with broad human exposure. CHX is an environmental pollutant and is present in tobacco smoke and in soft drinks sweetened with cyclamate. Interest in the toxicity of this class of compounds is due to their structural similarity to the cytotoxin acrolein. In a pilot study, rats and mice were exposed to 0, 20, 40, or 80 ppm CHX for 6 h/day. The study was terminated after 4 days due to acute toxicity in the high-dose groups. In a subsequent 14-day study, mice and rats were exposed to 0, 2.5, 5, or 10 ppm CHX for 6 h/day. All animals survived exposure until terminal sacrifice. Body weights were not significantly different from controls after 14 days of exposure. Liver/body weights were increased in male and female mice exposed to 5 and 10 ppm, and in male and female rats exposed to 10 ppm CHX. Ninety-day toxicity studies were conducted to provide data required to design chronic toxicity and carcinogenicity studies of CHX if it is determined such studies are necessary. Groups of 10 male and female F-344 rats and B6C3F1 mice were exposed to 0, 2.5, 5, or 10 ppm CHX for 6 h/day for 13 wk. All animals survived until sacrifice. Body weights were not significantly different from controls after 13 wk of exposure. Liver weights were increased in male and female mice exposed to 5 and 10 ppm and in male and female rats exposed to 10 ppm CHX. No adverse effects on bone-marrow micronuclei, sperm motility, or vaginal cytology were observed. Microscopic lesions included hyperplasia, and squamous metaplasia in the nasal cavity in rats and mice of both sexes at all doses. Nasal-cavity erosion and suppurative inflammation also occurred in high-dose mice. Larynx and lung were not affected in either sex or species. Dose-related hepatic centrilobular cytoplasmic vacuolation was seen in male rats only. These data suggest that CHX acts as an alkylating agent primarily producing toxicity at the exposure site.

Distribution and disposition of morpholine in the rabbit

The results of a variety of in vivo and in vitro experiments suggested that the kidneys were the primary route of elimination of morpholine in the rabbit. Morpholine was not bound to serum proteins and was excreted 90% unchanged. The concentration of [14C]morpholine in the renal cortex was 6.6 times the concentration in the blood, and in the renal medulla was 15.3 times the concentration in the blood 30 min after the intravenous injection of single boluses of labelled compound. The chemical appeared to be distributed mainly to the extracellular space, and its rate of elimination was enhanced by acidification of the urine.

Effects of various pretreatments on the hepatotoxicity of inhaled styrene in the B6C3F1 mouse.

1. The roles of cytochrome P450 monooxygenases (P450) and glutathione (GSH) in styrene hepatotoxicity were investigated in mice by pretreating with either phenobarbital (PB; P450 inducer), SKF 525A (P450 inhibitor), N-acetylcysteine (NAC; GSH precursor), or saline (vehicle control) prior to a 6-h exposure to either 500 ppm styrene on air. 2. Styrene caused hepatocellular degeneration or necrosis in all groups; these changes were more extensive and severe in mice pretreated with PB. Styrene significantly increased relative liver weights and serum ALT and SDH levels only in mice pretreated with PB. NAC did not prevent GSH depletion or hepatotoxicity. 3. In the fat of SKF 525A-pretreated mice a slight but statistically significant increase in styrene levels was observed, suggesting that metabolism was decreased; the SO/styrene ratio in the fat of PB-pretreated mice showed a slight, but statistically significant, increase indicating a slight increase in styrene metabolism. Neither SKF 525A nor PB caused changes in microsomal enzyme activity in vitro. 4. These results suggest that styrene may be activated by a pathway not totally dependent upon P450 enzyme activity, or more likely that PB and SKF 525A are not specific for the P450 enzymes involved in activation and detoxification of styrene.

Failure of carbon disulfide and levothyroxine to modify the cardiovascular response of rabbits to a high-cholesterol diet

Exposure of rabbits for 12 weeks to 300 ppm carbon disulfide (CS2) for 6 h/day, 5 days/week, or to 25 mg/day of thiourea or 2% cholesterol in the diet, or to any combination thereof caused a significant reduction in the concentration of serum thyroxine (T4). The reduction of the concentration of serum T4 in rabbits by the treatments was completely offset by the inclusion of 0.1 mg/day of sodium levothyroxine in the diet. Ingestion of feed containing 2% cholesterol significantly increased the degree of atherosclerosis present in the aortic arch and significantly increased the oil red O positive lipid present in the heart and the aorta, with the aortic arch being the most severely affected. The response of the aorta and the heart to the 2% cholesterol diet was not significantly modified by concurrent exposure to CS2 by inhalation or by treatment with thiourea, a metabolite of CS2. We found no evidence that the development of cardiovascular lesions induced by a 2% cholesterol diet in rabbits was mediated by a mechanism involving a component of hypothyroidism.