Enhanced allergic airway disease in old mice is associated with a Th17 response.

BACKGROUND: The prevalence of asthma in the elderly is increasing and associated with higher mortality than in children or young adults. However, the effects of age on the development and character of allergic asthma have been understudied. It has been suggested that mixed Th2/Th17 responses cause more severe forms of asthma, but the role of Th17 response in allergic airway disease and aging is not well understood. OBJECTIVE: To investigate age-dependent characteristics and Th17 immune response in allergic airway disease in a murine house dust mite (HDM)-allergen model. METHODS: Twelve-week-old and 15-month-old male BALB/c mice were sensitized and challenged with HDM. Bronchoalveolar lavage fluid (BALF), airway inflammation and hyperresponsiveness (AHR), serum immunoglobulin and splenic T cells were assessed. Age-related T cell activation was analyzed in a co-culture with bone marrow-derived dendritic cells (BMDC) and splenic CD4(+) T cells from young and old mice. RESULTS: Features of allergic airway disease such as mucous cell hyperplasia, infiltration of airway eosinophils and lymphocytes, Th2 cytokine expression and serum IgG1 levels were greater in old compared to young mice. In contrast to the more marked inflammatory/immune responses to HDM in old mice, AHR was greater in young HDM-treated mice. Only the old mice developed airway neutrophil infiltration and a Th17 immune response upon HDM exposure, with increases in BALF cytokines IL-17A and KC, and Th17 cytokine producing T cells in the spleen. Stimulation of CD4(+) T cells and BMDC co-cultures with HDM, resulted in an enhanced Th17 cytokine response in cells isolated from old mice. CONCLUSIONS AND CLINICAL RELEVANCE: Our findings in mice suggest that the severity and character of allergic airway disease are age dependent, with a bias towards a Th17 immune response with aging. Elderly, asthmatics may be prone to develop severe allergic airway inflammation with a mixed Th2/Th17 immune response.

CYP2F2-generated metabolites, not styrene oxide, are a key event mediating the mode of action of styrene-induced mouse lung tumors.

Styrene induces lung tumors in mice but not in rats. Although metabolism of styrene to 7,8-styrene oxide (SO) by CYP2E1 has been suggested as a mediator of styrene toxicity, lung toxicity is not attenuated in CYP2E1 knockout mice. However, styrene and/or SO metabolism by mouse lung Clara cell-localized CYP2F2 to ring-oxidized cytotoxic metabolite(s) has been postulated as a key metabolic gateway responsible for both lung toxicity and possible tumorigenicity. To test this hypothesis, the lung toxicity of styrene and SO was evaluated in C57BL/6 (WT) and CYP2F2⁻/⁻ knockout mice treated with styrene (400 mg/kg/day, gavage, or 200 or 400 mg/kg/day, ip) or S- or R-SO (200 mg/kg/day, ip) for 5 days. Styrene treated WT mice displayed significant necrosis and exfoliation of Clara cells, and cumulative BrdU-labeling index of S-phase cells was markedly increased in terminal bronchioles of WT mice exposed to styrene or S- or RSO. In contrast, Clara and terminal bronchiole cell toxicity was not observed in CYP2F2⁻/⁻ mice exposed to either styrene or SO. This study clearly demonstrates that the mouse lung toxicity of both styrene and SO is critically dependent on metabolism by CYP2F2. Importantly, the human isoform of CYP2F, CYP2F1, is expressed at much lower levels and likely does not catalyze significant styrene metabolism, supporting the hypothesis that styrene-induced mouse lung tumors may not quantitatively, or possibly qualitatively, predict lung tumor potential in humans.

Magnetic resonance imaging and computational fluid dynamics (CFD) simulations of rabbit nasal airflows for the development of hybrid CFD/PBPK models.

The percentages of total airflows over the nasal respiratory and olfactory epithelium of female rabbits were calculated from computational fluid dynamics (CFD) simulations of steady-state inhalation. These airflow calculations, along with nasal airway geometry determinations, are critical parameters for hybrid CFD/physiologically based pharmacokinetic models that describe the nasal dosimetry of water-soluble or reactive gases and vapors in rabbits. CFD simulations were based upon three-dimensional computational meshes derived from magnetic resonance images of three adult female New Zealand White (NZW) rabbits. In the anterior portion of the nose, the maxillary turbinates of rabbits are considerably more complex than comparable regions in rats, mice, monkeys, or humans. This leads to a greater surface area to volume ratio in this region and thus the potential for increased extraction of water soluble or reactive gases and vapors in the anterior portion of the nose compared to many other species. Although there was considerable interanimal variability in the fine structures of the nasal turbinates and airflows in the anterior portions of the nose, there was remarkable consistency between rabbits in the percentage of total inspired airflows that reached the ethmoid turbinate region (approximately 50%) that is presumably lined with olfactory epithelium. These latter results (airflows reaching the ethmoid turbinate region) were higher than previous published estimates for the male F344 rat (19%) and human (7%). These differences in regional airflows can have significant implications in interspecies extrapolations of nasal dosimetry.

Differential association of MUC5AC and CLCA1 expression in small cartilaginous airways of RAO-affected and control horses.

REASONS FOR PERFORMING STUDY: Airway mucus accumulation is associated with indoor irritant and allergen exposure in horses with recurrent airway obstruction (RAO). Epidermal growth factor receptor (EGFR) and a chloride channel (calcium activated, family member 1; CLCA1) are key signalling molecules involved in mucin gene expression. OBJECTIVES: We hypothesised that exposure to irritants and aeroallergens would lead to increased expression of the mucin gene eqMUC5AC and increased stored mucosubstance in the airways of RAO-affected horses, associated with increased neutrophils and CLCA1 and EGFR mRNA levels. METHODS: We performed quantitative RT-PCR of eqMUC5AC, CLCA1 and EGFR; volume density measurements of intraepithelial mucosubstances; and cytological differentiation of intraluminal inflammatory cells in small cartilaginous airways from cranial left and right and caudal left and right lung lobes of 5 clinically healthy and 5 RAO-affected horses that had been exposed to indoor stable environment for 5 days before euthanasia. RESULTS: Neutrophils were increased in RAO-affected horses compared to clinically healthy controls. EqMUC5AC mRNA levels were positively correlated with both CLCA1 and EGFR mRNA levels in RAO-affected horses but only with CLCA1 in controls. The relationship between eqMUC5AC and CLCA1 differed in the 2 groups of horses with RAO-affected animals overexpressing CLCA1 in relation to eqMUC5AC. CONCLUSIONS: These data implicate CLCA1 as a signalling molecule in the expression of eqMUC5AC in horses but also suggest differential regulation by CLCA1 and EGFR between horses with RAO and those with milder degrees of airway inflammation.

Gamma-tocopherol prevents airway eosinophilia and mucous cell hyperplasia in experimentally induced allergic rhinitis and asthma.

BACKGROUND: Traditional therapies for asthma and allergic rhinitis (AR) such as corticosteroids and antihistamines are not without limitations and side effects. The use of complementary and alternative approaches to treat allergic airways disease, including the use of herbal and dietary supplements, is increasing but their efficacy and safety are relatively understudied. Previously, we have demonstrated that gamma-tocopherol (gammaT), the primary form of dietary vitamin E, is more effective than alpha-tocopherol, the primary form found in supplements and tissue, in reducing systemic inflammation induced by non-immunogenic stimuli. OBJECTIVE: We used allergic Brown Norway rats to test the hypothesis that a dietary supplement with gammaT would protect from adverse nasal and pulmonary responses to airway allergen provocation. METHODS: Ovalbumin (OVA)-sensitized Brown Norway rats were treated orally with gammaT before intranasal provocation with OVA. Twenty-four hours after two challenges, histopathological changes in the nose, sinus and pulmonary airways were compared with gene expression and cytokine production in bronchoalveolar lavage fluid and plasma. RESULTS: We found that acute dosing for 4 days with gammaT was sufficient to provide broad protection from inflammatory cell recruitment and epithelial cell alterations induced by allergen challenge. Eosinophil infiltration into airspaces and tissues of the lung, nose, sinus and nasolacrimal duct was blocked in allergic rats treated with gammaT. Pulmonary production of soluble mediators PGE(2), LTB(4) and cysteinyl leukotrienes, and nasal expression of IL-4, -5, -13 and IFN-gamma were also inhibited by gammaT. Mucous cell metaplasia, the increase in the number of goblet cells and amounts of intraepithelial mucus storage, was induced by allergen in both pulmonary and nasal airways and decreased by treatment with gammaT. CONCLUSIONS: Acute treatment with gammaT inhibits important inflammatory pathways that underlie the pathogenesis of both AR and asthma. Supplementation with gammaT may be a novel complementary therapy for allergic airways disease.

Effect of different stages of the follicular wave on in vitro developmental competence of bovine oocytes.

This study aimed to investigate the developmental competence of ovum pick-up collected oocytes on three stages of the follicular wave: Days 2, 5 and 8. A group of 11 cows was used in successive cycles to perform ovum pick-up on either Day 2, 5 or 8 of an induced follicular wave (three sessions per stage). Follicular waves were initiated by puncturing the dominant follicle and all other follicles sized > or = 5 mm at Days 5-7 of the cycle. The plasma progesterone concentrations did not differ between the days of ovum pick-up: 4.0 +/- 1.8, 5.1 +/- 1.6 and 5.2 +/- 1.7 ng/ml for Days 2, 5 and 8, respectively. The proportion of oocytes with three or more layers of non-expanded cumulus cells was higher for Day 5 than Day 8, while Days 2 and 5 did not significantly differ from each other (85, 96 and 68% of 113, 60 and 101 oocytes for Days 2, 5 and 8, respectively). The proportion of oocytes competent to develop a blastocyst in an in vitro production system was higher for Days 2 and 5 than for Day 8: 27, 29 and 15% for the oocytes with fair to good cumulus investment and 23, 27 and 11%, respectively, when all oocytes were taken in account. This indicates that the dominant follicle reduces the developmental competence of oocytes from subordinate follicles at a relatively late stage of dominance. This finding has practical consequences for the handling of cows that undergo ovum pick-up only once or very irregularly. The embryo yield can then be improved by performing the ovum pick-up at Days 2-5 of the cycle or 2-5 days after ablation of the large follicles.

Effects of ozone and endotoxin coexposure on rat airway epithelium: potentiation of toxicant-induced alterations.

Tropospheric ozone is the major oxidizing component in photochemical smog and is one of the most pervasive problems to human health of the criteria air pollutants for which the National Ambient Air Quality Standards have been designated by the Clean Air Act. Although many adverse health effects of ozone exposure have been documented in both humans and laboratory animals, controversy surrounds the establishment and implementation of ozone standards set forth by the U.S. Environmental Protection Agency. Because people are commonly exposed to more than one air pollutant at a time, studies that examine coexposures to airborne materials may be more relevant for assessing their risks to human health. Airborne biogenic substances such as pollens, spores, and bacterial products are ubiquitous in the environment, and when inhaled can cause adverse respiratory symptoms. One such biogenic agent, bacterial endotoxin, is a potent stimulus of airway inflammation and is a ubiquitous airborne contaminant commonly found in domestic, agricultural, and industrial settings. Little is known about the interaction of exposures to biogenic substances and criteria air pollutants such as ozone. In the last few years we have performed a series of studies in rodents that examined the biologic responses of the respiratory epithelium after airway exposures to both endotoxin and ozone. When exposed to ozone (0.5 ppm 8 hr/day for 3 days), Fischer rats develop lesions in the nasal transitional epithelium, whereas intranasal instillation of endotoxin (20 microg) elicits epithelial lesions in the respiratory epithelium of the nose and conducting airways. Our studies were designed to examine how exposure to one toxicant may affect the airway epithelial lesions induced by the other toxicant. We investigated the potential role of acute inflammation in the enhancement of airway epithelial lesions after exposure of these two toxicants in neutrophil-sufficient and neutrophil-deficient rodents. A summary of these results indicates that epithelial and inflammatory responses to coexposure of these two pollutants are greater than those elicited by either agent alone. Interestingly, each toxicant enhances the epithelial alterations induced by the other. Furthermore, the synergistic effects elicited by coexposure to ozone and endotoxin are mediated partly by neutrophils. These studies provided some new insights into how inhaled co-pollutants interact to initiate and promote alterations of airway epithelium. Further studies with these and other air pollutants will help define their true risk to human health.

Hepatic ischemia/reperfusion injury in P-selectin and intercellular adhesion molecule-1 double-mutant mice.

Neutrophil adhesion and recruitment represents one of the early cellular events that occur during hepatic ischemia/reperfusion (IR) injury and plays a critical role in determining the extent of tissue damage. The adhesion molecules, such as selectins and intercellular adhesion molecules (ICAM), are important in mediating neutrophil-endothelial cell interactions and neutrophil emigration. The goal of this study was to evaluate the role of P-selectin and ICAM-1 in hepatic IR injury. Male wild-type and P-selectin/ICAM-1-deficient (P/I null) mice underwent 90 minutes of partial hepatic ischemia followed by reperfusion at various time points (0, 1.5, 3, and 6 hours). Reperfusion caused a time-dependent hepatocellular injury in both wild-type and P/I null mice as judged by plasma alanine aminotransferase (ALT) levels and liver histopathology examination. Although ALT levels were slightly lower in the P/I null mice compared with the wild-type mice the differences were not statistically significant. Neutrophil infiltration to the ischemic liver was observed in both mouse groups after 6 hours of reperfusion; however, the infiltration to the midzonal region of the ischemic liver was more pronounced in the wild-type group. This study suggests that hepatocellular injury induced after hepatic IR was independent of P-selectin and ICAM-1 in this model of acute inflammatory tissue injury.

Persistent mucin glycoprotein alterations in equine recurrent airway obstruction.

Horses with the episodic asthmalike condition of recurrent airway obstruction (RAO) have bouts of inflammation and bronchoconstriction associated with indoor housing. To assess the potential differences in airway secretions between RAO-affected and control horses, methods to quantify mucus secretions were developed and applied to bronchoalveolar lavage fluid. The relative difference in the amount of mucin glycoproteins between control and RAO-affected horses was assessed with a carbohydrate side chain-specific monoclonal antibody (4E4) in an enzyme-linked immunosorbent assay and by carbohydrate-specific enzyme-linked lectin assays. Significantly increased levels of 4E4-immunoreactive glycoprotein and the mucin-associated carbohydrates fucose (alpha-1,2 linkage) and N-acetylglucosamine were detected in RAO-affected horses in acute disease. RAO-affected horses in remission maintained significantly elevated levels of alpha-1,2-fucose and N-acetylglucosamine, whereas the 4E4-immunoreactive glycoprotein levels displayed a trend toward an increase over control levels. These results indicated that persistent changes in the quantity and/or quality of mucus glycoproteins occurred in the RAO-affected horses.

Respiratory tract toxicity in rats exposed to Mexico City air.

The rat has been used extensively as a health sentinel, indicator, or monitor of environmental health hazards, but this model has not been directly validated against human exposures. Humans in Mexico City show upper respiratory tract lesions and evidence of pulmonary damage related to their environmental inhalation exposure. In this study, male and female F344 rats were exposed (23 hr/day) in Mexico City to local Mexico City air (MCA)* for up to seven weeks. Controls were maintained at the same location under filtered air. Prior to these exposures, several steps were taken. First, the nasal passages of normal male rats shipped from the United States and housed in Mexico City were examined for mycoplasma infection; no evidence of infection was found. In addition, a mobile exposure and monitoring system was assembled and, with an ozone (O3) exposure atmosphere, was tested along with supporting histopathology techniques and analysis of rat nasal and lung tissues. Last, the entire exposure model (equipment and animals) was transported to Mexico City and validated for a three-week period. During the seven-week study there were 18 one-hour intervals during which the average O3 concentration of MCA in the exposure chamber exceeded the US National Ambient Air Quality Standard (NAAQS) of 0.120 ppm 03 (hourly average, not to be exceeded more than once per year). This prolonged exposure of healthy F344 rats to MCA containing episodically low to moderate concentrations of 03 (as well as other urban air pollutants) did not induce inflammatory or epithelial lesions in the nasal airways or lung as measured by qualitative histologic techniques or quantitative morphometric techniques. These findings agree with those of previous controlled O3 inhalation studies, but they are in contrast to reports indicating that O3-polluted MCA causes significant nasal mucosal injury in adults and children living in southwestern Mexico City. Taken together, these findings may suggest that human airways are markedly more susceptible to the toxic effects of MCA than are the airways of the F344 rat.

Identification of mucosal injury in the murine nasal airways by magnetic resonance imaging: site-specific lesions induced by 3-methylindole.

A magnetic resonance imaging (MRI) technique was developed to identify mucosal damage to the nasal passages of mice resulting from exposure to respiratory toxicants. 3-Methylindole (3-MI) was chosen as a model nasal toxicant because systemic administration of this compound in mice results in a well-characterized necrotizing nasal lesion that is restricted to the olfactory mucosa. MRI technology allows imaging of the same mice before and at time points after injection. In addition, morphological alterations and increases in the area of sinus cavity airspace can be followed as a function of dose and time following exposure. For 3-MI, the cross-sectional area of the sinus airspaces increased by 1.7-fold in mice injected with 200 mg/kg and 2.6-fold in mice injected with 300 mg/kg at 3 days after injection. Alterations in the nasal turbinates lined by olfactory mucosa were identified 1, 3, and 6 days postadministration of 3-MI using MRI. Postmortem histological examination of the nasal tissue confirmed the intranasal location and distribution of the 3-MI-induced lesions observed by MRI. MRI can be a useful technique to identify toxicant-induced mucosal injury in the nasal passages at an in-plane resolution less than 60 microm.

Inhibition of Kupffer cells reduced CXC chemokine production and liver injury.

BACKGROUND: Cytokine production is a critical component of ischemia/reperfusion (IR) injury. In the liver, Kupffer cells produce cytokines and chemokines (i.e., cytokines with chemoattractant properties) that are important mediators in neutrophil recruitment and subsequent hepatocellular injury. Therefore, the role of Kupffer cells in chemokine production in hepatic IR injury was investigated. METHODS: Adult male C57BL/6 mice underwent 90 min of partial hepatic ischemia followed by various reperfusion times (i.e., 0, 1.5, 3, and 6 h). Gadolinium chloride (GC), which inhibits Kupffer cell activity, was administered to mice 48 and 24 h prior to ischemia. The control group received a corresponding volume of normal saline. Plasma levels of the cytokine macrophage inflammatory protein-2 (MIP-2), KC, and tumor necrosis factor (TNF)-alpha and liver mRNA were measured. Liver injury was assessed by plasma level of alanine transaminase (ALT) and histopathology. RESULTS: A reperfusion time-dependent liver injury occurred as indicated by increased levels of plasma ALT and histopathology. The injury was associated with increased plasma TNF-alpha, MIP-2, and KC and their hepatic mRNA expression and neutrophil infiltration into ischemic lobes of the liver. GC treatment significantly reduced the number of Kupffer cells as determined by the immunostained liver tissue sections. The extent of liver injury significantly decreased in GC-treated mice that were associated with decreased levels of plasma ALT, TNF-alpha, MIP-2, and KC and neutrophil infiltration. CONCLUSIONS: This study suggests that Kupffer cells are major contributors to cytokine production in hepatic IR and their modulation may serve as a potential target for therapeutic intervention.

L-selectin blockade and liver function in rats after uncontrolled hemorrhagic shock.

Hemorrhagic shock (HS) and resuscitation can be seen as a global body ischemia-reperfusion (I/R) injury characterized by neutrophil infiltration and organ damage. Liver dysfunction occurs early after HS. Adhesion molecules are needed for the first steps ofneutrophil migration. Thus, the purpose of this study was to investigate the role of L-selectin in the liver after uncontrolled HS and resuscitation. Forty-eight Sprague Dawley rats were subjected to uncontrolled HS and resuscitation. Animals were divided into three groups: sham, uncontrolled HS and resuscitation, and uncontrolled HS and resuscitation with anti-L-selectin treatment. At 6 we evaluated liver injury tests, liver tissue myeloperoxidase (MPO), and liver histology. Survival was followed for 3 days and compared between groups. Statistical analysis included Fisher's exact test and one-way analysis of variance. Survival significantly increased from 30% in the control group to 60% in the treated group (p < .05). Hepatocellular and structural injury as well as neutrophil infiltration was significantly decreased in treated animals (p < .05). Thus, blockade of L-selectin resulted in decreased hepatocellular injury and increased survival in our model of uncontrolled HS. Selectins may be important therapeutic targets for blockade in the treatment of HS.

Endotoxin enhancement of ozone-induced mucous cell metaplasia is neutrophil-dependent in rat nasal epithelium.

Ozone, the primary oxidant gas in photochemical smog, causes neutrophilic inflammation and mucous cell metaplasia (MCM) in the nasal transitional epithelium (NTE) of rats and monkeys. Bacterial endotoxin is another common airborne agent that induces acute neutrophilic inflammation, but not MCM, in NTE. It does, however, enhance ozone-induced MCM in rat nasal airways (Fanucchi et al., 1998, Toxicol. Appl. Pharmacol. 152, 1-9). In the present study, F344 rats exposed to filtered air or 0.5 ppm ozone (8 h/day for 3 days) were intranasally instilled with sterile saline or 100 microg endotoxin 24 h and 48 h after the third ozone exposure. To determine the role of neutrophilic inflammation in endotoxin-induced potentiation of the MCM caused by ozone, half of the rats were depleted of circulating neutrophils prior to saline or endotoxin instillations. Rats were killed 6 h or 3 days after the last intranasal instillation, and nasal tissues were processed for (1) light microscopy and morphometric analysis to determine the number of infiltrating neutrophils and the volume amount (density) of stored mucosubstances in the NTE, and (2) quantitative RT-PCR analysis of steady-state mucin gene (rMuc-5AC) mRNA levels in the NTE. Endotoxin induced a transient influx of neutrophils in both air- and ozone-exposed rats that was completely blocked by neutrophil depletion. Endotoxin increased rMuc-5AC mRNA levels in the NTE of ozone-exposed rats. Neutrophil depletion, however, had no effect on endotoxin-induced upregulation of mucin gene mRNA levels. Endotoxin enhanced the ozone-induced increase in stored mucosubstances (4-fold increase), but only in neutrophil-sufficient rats. These data indicate that endotoxin enhancement of ozone-induced upregulation of rMuc-5AC mRNA levels is neutrophil-independent, while its effects on intraepithelial production and storage of mucus glycoproteins is dependent on the presence of neutrophils.

Production of interferon-gamma by influenza hemagglutinin-specific CD8 effector T cells influences the development of pulmonary immunopathology.

This study examined the inflammation, lung function impairment, and immune protection associated with either wild-type or interferon (IFN)-gamma-deficient Tc1- or Tc2-CD8 effector cells responding to influenza pneumonia. The adoptive transfer of influenza hemagglutinin-specific Tc1 effectors afforded protection and elicited only minimal impairment of lung function. IFN-gamma-deficient Tc1 effector cells were equally protective, but were associated with an eosinophil influx and slightly more lung function impairment early in the response. Relative to Tc1, Tc2 effector cells were less protective, elicited an eosinophil influx and a greater impairment of lung functions. IFN-gamma-deficient Tc2 effector cells were not protective and were associated with the severest impairment of lung function throughout the response, an accumulation of neutrophils, and extensive pulmonary vasculitis and alveolar hemorrhaging. Deletion of IFN-gamma was associated with a delay in effector cell recruitment and the elicitation of a more intense inflammatory response that resulted in more severe lung function impairment in the recipients of either Tc1 or Tc2 IFN-gamma-deficient effector cells. Thus, during influenza infections, IFN-gamma production by the responding CD8 T cells is associated with effector cell recruitment and mitigation of the associated inflammation and of the resulting impairment in lung functions but is not necessary for optimal protection.

Nitric oxide diminishes apoptosis and p53 gene expression after renal ischemia and reperfusion injury.

BACKGROUND: The role of nitric oxide in the ischemic injury of the kidney is still controversial. The aim of this study was to reevaluate the beneficial effect of exogenous nitric oxide and define its effects as regulator of gene p53 expression and apoptosis in the ischemic renal injury. METHODS: Sprague-Dawley rats were subjected to 75 min of renal warm ischemia and contralateral nephrectomy. The animals were divided into six groups (n=6 per group): Two sham groups at 4 and 24 hr, two ischemic control (IC) at same times and two treated groups (Na-NP), studied at same intervals, where sodium nitroprusside (5 mg/kg) was given 15 min before reperfusion. The parameters evaluated included: serum creatinine, blood urea nitrogen, neutrophil infiltration determined by myeloperoxidase, gene p53 expression determined by reverse transcriptase polymerase chain reaction, apoptosis determined by peroxidase in situ technique and light histology. RESULTS: There were significant improvements in serum creatinine and blood urea nitrogen at 24 hr in the NA-NP group when compared with the IC group (P<0.05). Myeloperoxidase levels were higher in the IC when evaluated against the Na-NP groups. Na-NP exhibited a downregulating effect in the expression of gene p53 when compared to the IC group. Apoptosis was more evident in the IC group and had moderately increased histological damage when compared to the Na-NP group. CONCLUSIONS: Nitric oxide demonstrated a protective effect in the ischemic injury of the kidney and exerted an antiapoptotic action dowregulating the expression of gene p53.

Neutrophil-dependent and neutrophil-independent alterations in the nasal epithelium of ozone-exposed rats.

Ozone induces epithelial hyperplasia and mucous cell metaplasia (MCM) in nasal transitional epithelium (NTE) of rats. A transient neutrophil influx accompanies upregulation of mucin messenger RNA (mRNA) before the onset of MCM. The present study was designed to examine the role of neutrophils in ozone-induced epithelial changes in the NTE of rats. Fourteen hours before inhalation exposure, male F344/N rats were injected intraperitoneally with antirat neutrophil antiserum to deplete circulating neutrophils, or were injected with normal (control) serum. Rats were then exposed to 0 ppm (filtered air) or 0.5 ppm ozone (8 h/d) for 1 or 3 d. Maxilloturbinates lined with NTE were analyzed to determine the epithelial labeling index; numeric densities of neutrophils, total epithelial cells, and mucous secretory cells; amount of stored intraepithelial mucosubstances; and steady-state ratMUC-5AC (mucin) mRNA levels. At 2 h after 3 d of exposure, rats treated with antiserum had 90% fewer circulating neutrophils than did rats treated with control serum. Antiserum-treated, ozone-exposed rats had 87% fewer infiltrating neutrophils than did control serum-treated, ozone-exposed rats. At 4 d after 3 d of exposure, antiserum-treated, ozone-exposed rats had 66% less stored intraepithelial mucosubstances and 58% fewer mucous cells in their NTE than did control serum-treated, ozone-exposed rats. Antiserum treatment had no effects on ozone-induced epithelial cell proliferation or mucin mRNA upregulation. The results of this study indicated that ozone-induced MCM was neutrophil-dependent, whereas ozone-induced epithelial cell proliferation and mucin gene upregulation were neutrophil-independent.

Synergistic hepatotoxicity from coexposure to bacterial endotoxin and the pyrrolizidine alkaloid monocrotaline.

Individuals are commonly exposed to bacterial endotoxin (lipopolysaccharide [LPS]) through gram-negative bacterial infection and from its translocation from the gastrointestinal lumen into the circulation. Inasmuch as noninjurious doses of LPS augment the hepatotoxicity of certain xenobiotic agents, exposure to small amounts of LPS may be an important determinant of susceptibility to chemical intoxication. Monocrotaline (MCT) is a pyrrolizidine alkaloid phytotoxin that at large doses produces centrilobular liver lesions in rats. In the present study, MCT was coadministered with LPS to determine whether LPS would enhance its hepatotoxicity. Doses of MCT (100 mg/kg, ip) and LPS (7.4 x 10(6) EU/kg, iv), which were nonhepatotoxic when administered separately, produced significant liver injury in male, Sprague-Dawley rats when given in combination. Within 18 h after MCT administration, this cotreatment resulted in enhanced plasma alanine aminotransferase and aspartate aminotransferase activities, two markers of liver injury. Histologically, overt hemorrhage and necrosis appeared between 12 and 18 h. The lesions were centrilobular and midzonal and exhibited characteristics similar to lesions associated with larger doses of MCT and LPS, respectively. In the presence of LPS, the threshold for MCT toxicity was reduced to 13-33% of the dose required for toxicity with MCT alone. A study in isolated, hepatic parenchymal cells revealed no interaction between MCT and LPS in producing cytotoxicity. In summary, coexposure of rats to noninjurious doses of MCT and LPS resulted in pronounced liver injury. Results in vitro suggest that the enhanced toxicity does not result from a direct interaction of MCT and LPS with hepatic parenchymal cells. These results provide additional evidence that exposure to small amounts of LPS may be a determinant of susceptibility to food-borne hepatotoxins.

Lipopolysaccharide and the trichothecene vomitoxin (deoxynivalenol) synergistically induce apoptosis in murine lymphoid organs.

Human exposure to Gram-negative bacterial lipopolysaccharide (LPS) is common and may have an important influence on chemical toxicity. LPS has been shown previously to enhance synergistically the toxicity of trichothecene mycotoxins. Because either of these toxin groups alone characteristically target lymphoid organs at high doses, we evaluated the effects of coexposure to subthreshold doses of Salmonella typhimurium LPS and vomitoxin (VT) administered by intraperitoneal injection and oral gavage of B6C3F1 mice, respectively, on apoptosis in lymphoid tissues after 12-h exposure. The capacity of LPS (0.5 mg/kg body weight) and VT (25 mg/kg body weight) to act synergistically in causing apoptosis in thymus, spleen, and Peyer's patches was suggested by increased internucleosomal DNA fragmentation in whole cell lysates as determined by gel electrophoresis. Following terminal deoxynucleotidyl transferase (TdT)-mediated fluorescein-dUTP nick end-labeling (TUNEL) of tissue sections, a dramatic enhancement of fluorescence intensity indicative of apoptosis was observed in thymus, spleen, Peyer's patches, and bone marrow from coexposed animals as compared to those given the agents alone. Evaluation of hematoxylin and eosin-stained tissue sections of treatment mice revealed the characteristic features of lymphocyte apoptosis, including marked condensation of nuclear chromatin, fragmentation of nuclei, and formation of apoptotic bodies in tissues from mice. Combined treatment with VT (25 mg/kg body weight) and LPS (0.5 mg/kg body weight) significantly increased (p<0.05) the amount of apoptotic thymic and splenic tissue as compared to the expected additive responses of mice receiving either toxin alone. When apoptosis was examined in cell suspensions of thymus, spleen, Peyer's patches, and bone marrow by flow cytometry in conjunction with propidium iodide staining, the percentage of apoptotic cells was significantly increased (p<0.05) in cotreatment groups as compared to the additive responses to LPS and VT given alone. The results provide qualitative and quantitative evidence for the hypothesis that LPS exposure markedly amplifies the toxicity of trichothecenes and that the immune system is a primary target for these interactive effects.

Histopathology of nasal olfactory mucosa from selected inhalation toxicity studies conducted with volatile chemicals.

In recent years, histopathologic changes have been reported in the olfactory mucosa of rodents exposed, by inhalation, to a variety of volatile chemicals. In order to better characterize these lesions, a panel of experienced pathologists reviewed microscopic lesions of the olfactory epithelium of rats reported in 10 inhalation studies conducted with 8 different chemicals. The objectives were to determine if the olfactory epithelial lesions are morphologically similar or different for the chemicals of interest, to develop and recommend appropriate diagnostic criteria and nomenclature to characterize the morphology of these olfactory lesions, and to provide specific criteria for judging the degree of severity of the olfactory changes in these studies. The results indicated that the distribution and nature of the lesions were similar in all the examined studies in which olfactory changes were observed. Recommended standardized nomenclature and diagnostic criteria and a uniform method for scoring lesion severity based on the extent of distribution and severity of tissue damage are presented.