Absorption, distribution, metabolism and excretion (ADME) study with 2,2',4,4',5,6'-hexabromodiphenyl ether (BDE-154) in male Sprague-Dawley rats.

A metabolism study of orally administered 2,2',4,4',5,6'-hexabromodiphenyl ether (BDE-154; 11.3 micromoles kg(-1)) was conducted in conventional and bile duct-cannulated male Sprague-Dawley rats. In conventional rats, approximately 31% of the radiolabelled dose was retained at 72 h, and lipophilic tissues were the preferred sites for disposition. Urinary excretion of BDE-154 was very low (1.0%), and parent compound was detected. Cumulative biliary excretion was 1.3%, and glutathione conjugates were suggested. Over 62% of the dose in conventional male rats was excreted in faeces, and was composed of parent compound (7.3%), free metabolites (13.1%), and covalently bound residues (41.4%). Faecal metabolites characterized by gas chromatography/mass spectrometry included multiple isomers of monohydroxylated hexa-/penta-/tetrabromodiphenyl ethers, and di-hydroxylated hexa/pentabromodiphenyl ethers. The adipose tissue 14C was extractable BDE-154, but 40% of liver 14C was bound to macromolecules. The study demonstrated the importance of performing individual polybrominated diphenyl ether (PBDE) metabolism studies to understand fully PBDE pharmacokinetics.

Mechanisms of cholinergic dysfunction in rabbits following recurrent aspiration of cow's milk.

Recurrent aspiration of cow's milk has been shown to alter neural control of airways in young rabbits (Gelfand et al., 1997). The purpose of this study was to define the mechanisms responsible for in vitro cholinergic hyperresponsiveness in this model. Beginning at 1 week of age, rabbits received either 0.5 mL/kg whole cow's milk or sterile saline intranasally while under light anesthesia. This was repeated each weekday for 2 weeks. At 8 weeks of age, rabbits were sacrificed. Portions of lungs underwent lavage with sterile saline. Tracheal smooth muscle (TSM) segments were also removed. Segments were assessed for acetylcholine (ACh) release by high-performance liquid chromatography ( HPLC) with electrochemical detection or acetylcholinesterase (AChE) kinetic activity by spectrophotometry. Substance P (SP), a neuropeptide that can increase ACh release from nerves, was also assessed using an enzyme immunoassay to define the content in lavage and TSM segments. Immunohistochemistry for SP within airways was also assessed. We found that recurrent aspiration of milk led to statistically significant alterations in many parameters. Acetylcholine release was significantly greater in segments of airways from rabbits that had aspirated cow's milk (27.5 +/- 1.7 vs. 20.1 +/- 1.6 pmol/min/g tissue) than saline. At the same time, AChE activity was less in the group that aspirated milk (8.7 +/- 0.4 vs. 10.2 +/- 0.5 nmol/min/mg protein) compared to saline. The amount of SP within both lavage as well as tissue homogenates was greater in the group that had aspirated the foreign protein (159.1 +/- 28.9 vs. 41.9 +/- 5.2 pmol/mg protein in lavage; 158.7 +/- 31.9 vs. 80.5 +/- 7.8 pmol/mg protein in tissues) than saline controls. While total cholinergic nerve density as assessed by choline acetyltransferase was not significantly different between groups, SP-positive immunoreactive nerves were easily identified in the group that aspirated cow's milk. This study suggests that cholinergic hyperresponsiveness caused by repeated aspiration of milk is due to several abnormalities, including prejunctional (increase in ACh release) as well as junctional (decrease in AChE) mechanisms within the airways. In addition, an upregulation of SP within airways is part of this process.

Metabolism of [14C]1,2,7,8-tetrachlorodibenzo-p-dioxin in a ruminating Holstein calf.

1. [UL-7,8-ring 14C]-1,2,7,8-tetrachlorodibenzo-p-dioxin (1278-TCDD) was administered orally to a ruminating Holstein bull calf (43.6 kg; 1.2 mg kg(-1) body weight). Urine and faeces were collected daily for 96 h, while blood was sampled at multiple time points. Tissues were removed for combustion analysis. 2. Each tissue contained < 0.65 of the dose at 96h. Tissues with highest levels of 1278-TCDD, as a percentage of administered dose, were the large and small intestine, rumen, liver and carcass. 3. Urinary excretion accounted for 10.6% of the dose, and faecal excretion accounted for 81.6% of the administered dose. The major urinary and faecal metabolites were isolated and characterized by mass spectrometry and 1H-NMR. 4. Plasma levels of 14C peaked at 24h, and decreased to near background at 96 h. Detectable plasmal levels of 1278-TCDD were observed by 2 h. 5. A hydroxylated metabolite of 1278-TCDD was detected in calf plasma, which has the potential to interfere with thyroid hormone homeostasis.

Differences between adult and childhood asthma.

During the last 2 decades, we have gained new insights into the pathogenesis of asthma; consequently, new therapeutic agents and approaches to therapy have emerged. Nevertheless, significant gaps remain in our understanding of this disease. Important new treatment issues affect childhood (the usual time of asthma onset), and researchers have recently described increases in asthma incidence in children. Yet, most clinical studies have been performed with adults, and our knowledge about major determinants of childhood asthma remains incomplete. Major challenges in pediatric asthma include methods of easily assessing lung function and noninvasive methods of assessing asthma's inflammatory nature. Research that addresses the mechanisms responsible for disease onset is also critical to decrease the prevalence of asthma. What we know about adult asthma cannot be used in the treatment of children without further study, but it is now clear that effective treatment should begin during childhood. (J Allergy Clin Immunol 2000;106:S153-7.)

Induction and regulation of nitric oxide synthase in airway epithelial cells by respiratory syncytial virus.

In this study, we evaluated the effects of respiratory syncytial virus (RSV) infection on nitric oxide (NO) production in human airway epithelial cells. In addition, we evaluated whether T-helper type 1 (Th1)- and Th2-type cytokines modulate the release of NO in response to RSV infection. To do this, we infected monolayers of A549 cells with RSV and determined nitrite levels in the supernatant fluids. We also measured nitrite levels in human small-airway epithelial cells (SAEC) in primary culture and in the bronchoalveolar lavage fluid (BALF) obtained from Balb/c mice after RSV infection. To further support our observations in these analyses, we performed immunocytochemistry and Western blot analysis for inducible nitric oxide synthase (iNOS) in A549 cells. To evaluate the regulation of NO production in response to RSV, we performed experiments in the absence and presence of the Th1 and Th2 type cytokines: interferon (IFN)-gamma, interleukin (IL)-4, and IL-13. In addition, we assessed the inhibitory effect of dexamethasone on iNOS in RSV infected A549 cells. Results were expressed in terms of nmol/mg protein and shown as percents of control values (mean +/- SE). RSV increased the release of nitrites in A549 cells, SAEC, and BALF. The increase in nitrite levels was supported by immunocytochemistry and Western blot analysis for iNOS protein in A549 cells, indicating activation of iNOS in response to RSV infection. IFN-gamma and IL-13 did not affect the RSV-induced increase in NO production. By contrast, IL-4 and dexamethasone suppressed the release of NO in response to RSV infection. These observations show that RSV infection leads to activation of iNOS within the airway epithelium and that IL-4 and dexamethasone inhibit the production of NO in response to RSV infection.

Recurrent milk aspiration produces changes in airway mechanics, lung eosinophilia, and goblet cell hyperplasia in a murine model.

Recurrent aspiration of milk into the respiratory tract has been implicated in the pathogenesis of a variety of inflammatory lung disorders including asthma. However, the lack of animal models of aspiration-induced lung injury has limited our knowledge of the pathophysiological characteristics of this disorder. This study was designed to evaluate the effects of recurrent milk aspiration on airway mechanics and lung cells in a murine model. Under light anesthesia, BALB/c mice received daily intranasal instillations of whole cow's milk (n = 7) or sterile physiologic saline (n = 9) for 10 d. Respiratory system resistance (Rrs) and dynamic elastance (Edyn,rs) were measured in anesthetized, tracheotomized, paralyzed and mechanically ventilated mice 24 h after the last aspiration of milk. Rrs and Edyn,rs were derived from transrespiratory and plethysmographic pressure signals. In addition, airway responses to increasing concentrations of i.v. methacholine (Mch) were determined. Airway responses were measured in terms of PD(100) (dose of Mch causing 100% increase from baseline Rrs) and Rrs,max (% increase from baseline at the maximal plateau response) and expressed as % control (mean +/- SE). We found recurrent milk aspiration did not affect Edyn and baseline Rrs values. However, airway responses to Mch were increased after milk aspiration when compared with control mice. These changes in airway mechanics were associated with an increased percentage of lymphocytes and eosinophils in the bronchoalveolar lavage, mucus production, and lung inflammation. Our findings suggest that recurrent milk aspiration leads to alterations in airway function, lung eosinophilia, and goblet cell hyperplasia in a murine model.

Differences between adult and childhood asthma.

During the last 2 decades, we have gained new insights into the pathogenesis of asthma; consequently, new therapeutic agents and approaches to therapy have emerged. Nevertheless, significant gaps remain in our understanding of this disease. Important new treatment issues affect childhood (the usual time of asthma onset), and researchers have recently described increases in asthma incidence in children. Yet, most clinical studies have been performed with adults, and our knowledge about major determinants of childhood asthma remains incomplete. Major challenges in pediatric asthma include methods of easily assessing lung function and noninvasive methods of assessing asthma's inflammatory nature. Research that addresses the mechanisms responsible for disease onset is also critical to decrease the prevalence of asthma. What we know about adult asthma cannot be used in the treatment of children without further study, but it is now clear that effective treatment should begin during childhood.

Mice that overexpress Cu/Zn superoxide dismutase are resistant to allergen-induced changes in airway control.

Within the respiratory epithelium of asthmatic patients, copper/zinc-containing superoxide dismutase (Cu/Zn SOD) is decreased. To address the hypothesis that lung Cu/Zn SOD protects against allergen-induced injury, wild-type and transgenic mice that overexpress human Cu/Zn SOD were either passively sensitized to ovalbumin (OVA) or actively sensitized by repeated airway exposure to OVA. Controls included nonsensitized wild-type and transgenic mice given intravenous saline or airway exposure to saline. After aerosol challenge to saline or OVA, segments of tracheal smooth muscle were obtained for in vitro analysis of neural control. In response to electrical field stimulation, wild-type sensitized mice challenged with OVA had significant increases in cholinergic reactivity. Conversely, sensitized transgenic mice challenged with OVA were resistant to changes in neural control. Stimulation of tracheal smooth muscle to elicit acetylcholine release showed that passively sensitized wild-type but not transgenic mice released more acetylcholine after OVA challenge. Function of the M(2) muscarinic autoreceptor was preserved in transgenic mice. These results demonstrate that murine airways with elevated Cu/Zn SOD were resistant to allergen-induced changes in neural control.

IL-10 is necessary for the expression of airway hyperresponsiveness but not pulmonary inflammation after allergic sensitization.

Cytokines play an important role in modulating inflammatory responses and, as a result, airway tone. IL-10 is a regulatory cytokine that has been suggested for treatment of asthma because of its immunosuppressive and anti-inflammatory properties. In contrast to these suggestions, we demonstrate in a model of allergic sensitization that mice deficient in IL-10 (IL-10-/-) develop a pulmonary inflammatory response but fail to exhibit airway hyperresponsiveness in both in vitro and in vivo assessments of lung function. Reconstitution of these deficient mice with the IL-10 gene fully restores development of airway hyperresponsiveness comparable to control mice. These results identify an important role of IL-10, downstream of the inflammatory cascade, in regulating the tone of the airways after allergic sensitization and challenge.

Response of C2C12 mouse and turkey skeletal muscle cells to the beta-adrenergic agonist ractopamine.

The effects of ractopamine (RAC) and ractopamine stereoisomers (RR, RS, SR, and SS) on cyclic AMP (cAMP) production, total protein, and DNA concentrations in mouse skeletal muscle cells (C2C12) were evaluated. The RAC (10 microM) caused an approximately 30% increase in cell number, protein, and DNA concentrations in myoblasts after 48 h; no differences were found in myotubes. The RAC-stimulated increase of these variables in myoblasts was blocked by the presence of equimolar concentrations of propranolol. At a later passage, myoblasts failed to exhibit an increase in cell number, protein, or DNA upon exposure to RAC. Both myoblasts and myotubes increased cAMP production in response to 10 microM RAC. The RAC isomers ranked RR >> SR > RS approximately SS in ability to stimulate cAMP production, with essentially no response to SS. The SR produced about 50% of the RR response. Coincubation of propranolol (10 microM) and RAC (10 microM) prevented RAC-stimulated cAMP production in myotubes but not in myoblasts (approximately 35% of cAMP produced by RAC alone). Turkey satellite cells (derived from biceps femoris of 12-wk-old toms) produced essentially no increased cAMP when exposed to 10 microM RAC stereoisomers. Stability of RAC was evaluated under laboratory storage and culture conditions. The RAC was stable for more than 4 mo when stored in deuterated DMSO (>98% purity) at room temperature or in aqueous solutions at -80 degrees C, as determined from sequential nuclear magnetic resonance studies. Radiolabeled RAC was incubated for 72 h in the presence of serum-containing medium, with or without C2C12 cells. Ninety-eight percent of the parent compound found in the medium at time zero was present in the medium as parent at the end of 72 h. The cellular cAMP response to RAC through beta-adrenergic receptors seems to be stereospecific. If the state of myoblasts and myotubes in vitro reflects the in vivo state, then the ractopamine effect in vivo on cellular processes (including cell division and protein and DNA accumulation) may be independent of beta-adrenergic receptors in muscle.

Pulmonary infiltrates with eosinophilia syndromes in children.

Pulmonary infiltrates with eosinophilia (PIE) are a group of heterogeneous disorders having the common findings of lung disease and eosinophilia in the peripheral blood, bronchoalveolar lavage fluid, or pulmonary interstitium. Eleven cases of PIE syndromes were identified through a retrospective and prospective chart review: drug-induced (2), acute eosinophilic pneumonia (3), infant pulmonary eosinophilia (2), parasite-induced (2), Churg-Strauss syndrome (1), and atypical chronic PIE (1). Patient demographics, clinical presentation, and disease severity varied considerably among groups. Therapeutic interventions included bronchodilators (10), oxygen (7), corticosteroids (9), and mechanical ventilation (3). A single patient with acute eosinophilic pneumonia died. Our experience suggests that PIE syndromes are rare in childhood and that clinical presentation can vary widely. Because of the potential for significant morbidity and mortality, aggressive diagnostic evaluations are warranted, particularly in children with respiratory failure of unknown etiology.

Metabolism of N-isopropylacetanilide in rat.

1. Radioactivity from oral doses of N-isopropyl[1-14C]acetanilide was excreted in urine (53.5%), faeces (8.1%) and expired air (17.0%) of rat. 2. Enterohepatic circulation occurred during formation of approximately 34% of the metabolites. N-isopropylacetanilide was metabolized by oxidation in all moieties of the molecule with subsequent conjugation with glucuronic and sulphuric acids. 3. The sulphate ester of 4'-hydroxyacetanilide (acetaminophen) was the major metabolite (28 % of the dose).

Neural control mechanisms within airways: disruption by respiratory syncytial virus.

UNLABELLED: Among the factors influencing airway function are neural control mechanisms, including adrenergic, cholinergic, nonadrenergic noncholinergic inhibitory, and nonadrenergic noncholinergic excitatory pathways. Respiratory infections affect these pathways in ways that are not entirely clear. OBJECTIVE: To determine acute and chronic effects of respiratory syncytial virus infection on airway neural control mechanisms. STUDY DESIGN: Acute effects were studied in cotton rats, which received human respiratory syncytial virus or uninfected cell culture medium intranasally at 5 weeks of age. Chronic effects were studied in ferrets, which received human respiratory syncytial virus or uninfected cell culture medium intranasally during the first 10 days of life. The responsiveness of tracheal smooth muscle segments was studied in vitro 4 days after infection of cotton rats and when ferrets were 4, 8, and 24 weeks of age. RESULTS: Tracheal smooth muscle segments from cotton rats demonstrated significant increases in contractile responses to nerve stimulation (cholinergic responses). In the presence of neurokinin A, contractile responses increased (enhanced nonadrenergic noncholinergic excitatory response), and relaxation of airways by nerve stimulation (nonadrenergic noncholinergic inhibitory response) was severely impaired. Airway epithelium was also disrupted. These alterations favor airway obstruction and a hyper-responsive state. Contractile responses to nerve stimulation were increased in 4- and 8-week-old ferrets infected with human respiratory syncytial virus compared with ferrets in a control group, a difference that resolved by 24 weeks. Nonadrenergic noncholinergic inhibitory responses were absent in all 4-week-old ferrets and significantly decreased in 8-week-old ferrets infected with human respiratory syncytial virus. A significant difference persisted at 24 weeks of age. CONCLUSION: Human respiratory syncytial virus causes acute and chronic changes in neural control of airways in animal models. When infection occurs early in life, the alterations persist for long periods.

Effects of ractopamine HCl stereoisomers on growth, nitrogen retention, and carcass composition in rats.

The objectives of this study were to determine the effects of ractopamine HCl (RAC) stereoisomers (RR, RS, SR, and SS) on performance, carcass composition, and nitrogen retention in growing female rats. Forty-eight rats (eight rats/treatment) were treated with 0 or 320 microg/d of RAC or with 80 microg/d of the RR, RS, SR, or SS stereoisomers of ractopamine. Rats had free access to feed and water before and during the experiment. Ractopamine and stereoisomers were delivered via i.p. implanted osmotic pumps for 14 d, and rats were then slaughtered. Control rats were fitted with osmotic pumps containing saline. Ractopamine increased (P < .05) feed intake (d 1 to 6); body weight; carcass CP; and intake, apparent absorption, retention, and retained:intake ratio of CP on d 1 to 6 of the study. Ractopamine decreased (P < .05) carcass lipid and visceral lipid. Rats dosed with the RR stereoisomer responded similarly to rats dosed with RAC, except for carcass lipid. Carcass lipid was decreased (P < .01) by RAC relative to controls, but it was not different from controls in rats treated with the RR isomer. Compared with controls, BW, carcass CP, and CP retention were increased by the RR stereoisomer, and visceral lipid was decreased. The RS isomer also decreased visceral lipid (P < .10), but variables measured in rats dosed with the RS, SR, and SS isomers generally did not differ from controls. Results of this study indicate that the RR isomer of RAC is responsible for a majority of the leanness-enhancing effects of RAC in rats.

Identification of the major urinary and fecal metabolites of 3,5-dinitrobenzamide in chickens and rats.

Colostomized chickens given oral doses of 3,5-dinitrobenzamide (nitromide) cleared nitromide predominantly through the urine (58% of dose) and feces (21% of dose). Rats cleared 52% of nitromide via urinary excretion and 44% via feces. Major urinary metabolites for both chickens and rats include: 3-amino-5-nitrobenzamide, 3-acetamido-5-nitrobenzamide, 3-acetamide-5-aminobenzamide, and 3,5-diacetamidobenzamide. The major fecal metabolite in chickens was 3-acetamido-5-nitrobenzamide (67% of fecal 14C) and 3-acetamido-5-aminobenzamide in rats (approximately 50%).

Intermediary metabolism of pentachloronitrobenzene in the control and germ-free rat and rat with cannulated bile ducts.

1. Nearly 70% of single oral doses of 14C-labelled pentachloronitrobenzene (PCNB) was excreted in bile within 24 h. 2. The characterized biliary metabolites of PCNB were either mercapturic acid pathway metabolites or catabolites thereof (thiols, methylthiols, S-glucuronides). 3. A major biliary metabolite was S-(aminotetrachlorophenyl)glutathione. 4. Conjugation with glutathione with subsequent catabolism to bis-methylthiotetrachlorobenzene was the major pathway in the control rat. 5. Germ-free experiments showed that only nitro- group displacement occurred, and no nitro- group reduction was detected.

Metabolism and disposition of 1,4,7,8-tetrachlorodibenzo-p-dioxin in rats.

Metabolism studies of 1,4,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a relatively nontoxic dioxin congener, were undertaken to gain a better understanding of mammalian metabolism of dioxins without the problems associated with the use of the most toxic congener, 2,3,7,8-TCDD. 14C-1,4,7,8-TCDD was dosed to conventional and bile-cannulated rats at a level of 8 mg/kg. The 14C was excreted almost entirely in 72 hours with the major routes of excretion feces and bile. Metabolites were identified from the feces, bile, and urine by GC-MS or negative ion FAB MS and 1H NMR. The two major fecal metabolites were hydroxylated tetra- and triCDDs. Glucuronide and sulfate conjugates of these hydroxyl metabolites were found in the urine and bile. Minor metabolites included dichlorocatechol, dihydroxylated tetra- and triCDDs, and conjugates of these compounds.