Interactions of paraquat and triadimefon: behavioral and neurochemical effects.

Triadimefon (TDF), a triazole fungicide, and paraquat (PQ), a non-selective herbicide/dessicant, are both known to adversely impact brain dopaminergic function and are used in overlapping geographical areas of the US. Since "real world" situations indicate humans are exposed to a diverse mixture of chemicals, this study hypothesized that combined exposures to PQ+TDF could produce interactive effects by simultaneously attacking multiple target sites of dopamine systems. Thus, 10 mg/kg PQ (PQ10) and 25 or 50 mg/kg TDF (TDF25 and 50, respectively) were administered i.p. to male C57BL/6 mice, 2x per week for 12 weeks, either alone or in combination. Acutely, TDF50 increased horizontal and vertical activity with increased vertical activity still occurring 24h later, indicative of sustained behavioral sensitization. Acutely, PQ decreased horizontal but not vertical activity with a lack of residual effects at 24h. PQ prevented the increased levels of activity associated with TDF50. These interactions differed for horizontal and vertical activity, indicating their differential neurochemical mediation, and suggesting that they did not arise from simple additivity of PQ and TDF effects. Nor could the interactive effects be readily ascribed to corresponding neurochemical interactions, since all treatments generally increased levels of DA and metabolites acutely in striatum and were associated with general reductions in levels of DA and metabolites and turnover in striatum and frontal cortex 7 days after the final treatment. Thus, TDF and PQ both separately and through interactions may serve as environmental risk factors through different mechanisms for dopaminergically-mediated behavioral dysfunctions.

Antitumor activity and toxicity of novel nitroheterocyclic phosphoramidates.

A series of novel nitroheterocyclic phosphoramidates has been evaluated for antitumor activity in murine and xenograft tumor models and for toxicity in mice. Significant increases in lifespan and long-term survivors were noted in L1210 leukemia and B16 melanoma models, and both complete and partial tumor regressions were observed in the MX-1 breast cancer xenograft model. All compounds exhibited some degree of toxicity to granulocyte/macrophage progenitors in the bone marrow of mice. Two drugs were selected for further toxicologic, histopathologic, and pharmacokinetic evaluations. Toxicity of potential clinical significance was observed only in the bone marrow at the highest drug dose; otherwise no significant abnormalities in blood chemistries or organ histopathology were noted. The bone marrow lesions consisted of reduced numbers of progenitor cells in the myeloid and erythroid series; platelets were not affected. The compounds were eliminated rapidly by first-order kinetics, with half-lives in the 4-12 min range. The best of these compounds exhibits excellent antitumor activity and minimal toxicity at therapeutically effective doses in mice.

Potentiated and preferential effects of combined paraquat and maneb on nigrostriatal dopamine systems: environmental risk factors for Parkinson's disease?

The absence of any compelling basis for a heritable basis of idiopathic Parkinson's disease (PD) has focused attention on environmental exposures as causative agents. While the herbicide paraquat has repeatedly been implicated, its impact on dopamine systems following systemic exposures is equivocal. The restricted focus on paraquat also ignores the extensive geographical overlap of its use with other agrichemicals known to adversely impact dopamine systems, including ethylenebisdithiocarbamate fungicides such as maneb. The present study sought to determine whether combined exposures to paraquat and maneb would produce additive effects and support a multiple-hit environmental contribution to PD. C57BL/6 mice were exposed to either paraquat (5-10 mg/kg) or maneb (15-30 mg/kg) i.p. alone or in combination once a week for 4 weeks. Sustained decreases in motor activity immediately following injections were consistently observed only with combined exposures, with activity levels returning to control values 24 h later. Concurrently, levels of dopamine and metabolites and dopamine turnover were increased immediately post-injection only by combined exposures, and returned to control levels or below within 48 h. Reductions in tyrosine hydroxylase immunoreactivity, measured 3 days after the last injection, resulted only from combined exposure and were detected in dorsal striatum, but not in the nucleus accumbens. The fact that combined exposures resulted in potentiated effects that appear to target nigrostriatal dopamine systems suggests that these combinations may be important environmental risk factors for Parkinsonism. These findings also raise questions about the adequacy of current risk assessment guidelines for these chemicals which are based on effect levels derived from exposures to single agents.

Pulmonary chemokine and mutagenic responses in rats after subchronic inhalation of amorphous and crystalline silica.

Chronic inhalation of crystalline silica can produce lung tumors in rats whereas this has not been shown for amorphous silica. At present the mechanisms underlying this rat lung tumor response are unknown, although a significant role for chronic inflammation and cell proliferation has been postulated. To examine the processes that may contribute to the development of rat lung tumors after silica exposure, we characterized the effects of subchronic inhalation of amorphous and crystalline silica in rats. Rats were exposed for 6 h/day, on 5 days/week, for up to 13 weeks to 3 mg/m(3) crystalline or 50 mg/m(3) amorphous silica. The effects on the lung were characterized after 6.5 and 13 weeks of exposure as well as after 3 and 8 months of recovery. Exposure concentrations were selected to induce high pulmonary inflammatory-cell responses by both compounds. Endpoints characterized after silica exposure included mutation in the HPRT gene of isolated alveolar cells in an ex vivo assay, changes in bronchoalveolar lavage fluid markers of cellular and biochemical lung injury and inflammation, expression of mRNA for the chemokine MIP-2, and detection of oxidative DNA damage. Lung burdens of silica were also determined. After 13 weeks of exposure, lavage neutrophils were increased from 0.26% (controls) to 47 and 55% of total lavaged cells for crystalline and amorphous silica, with significantly greater lavage neutrophil numbers after amorphous silica (9.3 x 10(7) PMNs) compared to crystalline silica (6.5 x 10(7) PMNs). Lung burdens were 819 and 882 microg for crystalline and amorphous silica, respectively. BAL fluid levels of LDH as an indicator of cytotoxicity were twice as high for amorphous silica compared to those of crystalline silica, at the end of exposure. All parameters remained increased for crystalline silica and decreased rapidly for amorphous silica in the 8-month recovery period. Increased MIP-2 expression was observed at the end of the exposure period for both amorphous and crystalline silica. After 8 months of recovery, those markers remained elevated in crystalline silica-exposed rats, whereas amorphous silica-exposed rats were not significantly different from controls. A significant increase in HPRT mutation frequency in alveolar epithelial cells was detected immediately after 13 weeks of exposure to crystalline, but not to amorphous silica. A significant increase in TUNEL staining was detected in macrophages and terminal bronchiolar epithelial cells of amorphous silica-exposed rats at the end of the exposure period; however, crystalline silica produced far less staining. The observation that genotoxic effects in alveolar epithelial cells occurred only after crystalline but not amorphous silica exposure, despite a high degree of inflammatory-cell response after subchronic exposure to both types of silica, suggests that in addition to an inflammatory response, particle biopersistence, solubility, and direct or indirect epithelial cell cytotoxicity may be key factors for the induction of either mutagenic events or target cell death.

Immunohistochemical localization of the acylases that catalyze the deacetylation of N-acetyl-L-cysteine and haloalkene-derived mercapturates.

Acylases catalyze the hydrolysis of a range of S-substituted N-acetyl-L-cysteines. The hydrolysis of N-acetyl-L-cysteine is catalyzed by cytosolic acylase I, and activity is present in human endothelial cells and rat lung, intestinal, and liver homogenates. Many haloalkenes are metabolized to mercapturates, which also undergo acylase-catalyzed hydrolysis. The acylases that catalyze the deacetylation of N-acetyl-L-cysteine and several haloalkene-derived mercapturates have been recently identified: acylase I catalyzes the deacetylation of N-acetyl-L-cysteine and some haloalkene-derived mercapturates whereas an acylase purified from rat kidney cytosol catalyzes the deacetylation of a distinct set of substrates, including several haloalkene-derived mercapturates. The objective of these studies was to examine the tissue and subcellular localization of acylase I and purified rat kidney acylase. Immunoblotting showed the presence of immunoreactive acylase I and purified rat kidney acylase in rat kidney, liver, lung, and brain. Both acylases were identified by immunohistochemistry in several rat organs, including kidney, liver, lung, brain, stomach, intestines, adrenals, pancreas, and testis, indicating that acylase activity is widespread in rat tissues.

Effect of 40-kHz ultrasound on acute thrombotic ischemia in a rabbit femoral artery thrombosis model: enhancement of thrombolysis and improvement in capillary muscle perfusion.

BACKGROUND: We have shown previously that 40-kHz ultrasound (US) at low intensity accelerates fibrinolysis in vitro with little heating and good tissue penetration. These studies have now been extended to examine the effects of 40-kHz US on thrombolysis and tissue perfusion in a rabbit model. METHODS AND RESULTS: Treatment was administered with either US alone at 0.75 W/cm(2), streptokinase alone, or the combination of US and streptokinase. US or streptokinase resulted in minimal thrombolysis, but reperfusion was nearly complete with the combination after 120 minutes. US also reversed the ischemia in nonperfused muscle in the absence of arterial flow. Tissue perfusion decreased after thrombosis from 13. 7+/-0.2 to 6.6+/-0.8 U and then declined further to 4.5+/-0.4 U after 240 minutes. US improved perfusion to 10.6+/-0.5 and 12.1+/-0. 5 U after 30 and 60 minutes, respectively. This effect was reversible and declined to pretreatment values after US was discontinued. Similarly, tissue pH declined from normal to 7.05+/-0. 02 after thrombosis, but US improved pH to 7.34+/-0.03 after 60 minutes. US-induced improvement in tissue perfusion and pH also occurred after femoral artery ligation, indicating that thrombolysis did not cause these effects. CONCLUSIONS: 40-kHz US at low intensity markedly accelerates fibrinolysis and also improves tissue perfusion and reverses acidosis, effects that would be beneficial in treatment of acute thrombosis.

Ablation of tumor necrosis factor receptor type I (p55) alters oxygen-induced lung injury.

Hyperoxic lung injury, believed to be mediated by reactive oxygen species, inflammatory cell activation, and release of cytotoxic cytokines, complicates the care of many critically ill patients. The cytokine tumor necrosis factor (TNF)-alpha is induced in lungs exposed to high concentrations of oxygen; however, its contribution to hyperoxia-induced lung injury remains unclear. Both TNF-alpha treatment and blockade with anti-TNF antibodies increased survival in mice exposed to hyperoxia. In the current study, to determine if pulmonary oxygen toxicity is dependent on either of the TNF receptors, type I (TNFR-I) or type II (TNFR-II), TNFR-I or TNFR-II gene-ablated [(-/-)] mice and wild-type control mice (WT; C57BL/6) were studied in >95% oxygen. There was no difference in average length of survival, although early survival was better for TNFR-I(-/-) mice than for either TNFR-II(-/-) or WT mice. At 48 h of hyperoxia, slightly more alveolar septal thickening and peribronchiolar and periarteriolar edema were detected in WT than in TNFR-I(-/-) lungs. By 84 h of oxygen exposure, TNFR-I(-/-) mice demonstrated greater alveolar debris, inflammation, and edema than WT mice. TNFR-I was necessary for induction of cytokine interleukin (IL)-1beta, IL-1 receptor antagonist, chemokine macrophage inflammatory protein (MIP)-1beta, MIP-2, interferon-gamma-induced protein-10 (IP-10), and monocyte chemoattractant protein (MCP)-1 mRNA in response to intratracheal administration of recombinant murine TNF-alpha. However, IL-1beta, IL-6, macrophage migration inhibitory factor, MIP-1alpha, MIP-2, and MCP-1 mRNAs were comparably induced by hyperoxia in TNFR-I(-/-) and WT lungs. In contrast, mRNA for manganese superoxide dismutase and intercellular adhesion molecule-1 were induced by hyperoxia only in WT mice. Differences in early survival and toxicity suggest that pulmonary oxygen toxicity is in part mediated by TNFR-I. However, induction of specific cytokine and chemokine mRNA and lethality in response to severe hyperoxia was independent of TNFR-I expression. The current study supports the prediction that therapeutic efforts to block TNF-alpha receptor function will not protect against pulmonary oxygen toxicity.

Evaluation of the safety of fenbendazole in cats.

OBJECTIVE: To evaluate the safety of fenbendazole in domestic cats. ANIMALS: 28 six- to seven-month old domestic short-hair cats. PROCEDURE: Cats were randomly assigned to 1 of 3 treatment groups or a control group (n = 7/group). Cats in the treatment groups were given fenbendazole at a dosage of 50, 150, or 250 mg/kg, PO, every 24 hours for 9 days; control cats were given a placebo. A fecal examination, coagulation tests, serum biochemical analyses, CBC, and urinalyses were performed before and 5, 9, and 21 days after initiation of treatment; cats were closely monitored for adverse reactions. After the last dose of fenbendazole was given, 4 control cats and 4 cats given fenbendazole at the highest dosage were euthanatized, and necropsies were performed. RESULTS: None of the cats developed any adverse reactions. For cats in the control and all treated groups, laboratory test results were within reference limits, and there were no significant differences in results of laboratory tests among groups. No gross or histologic lesions were identified in the control or treated cats that were euthanatized. CONCLUSIONS AND CLINICAL RELEVANCE: Fenbendazole administered to healthy cats at a dosage 5 times the dosage and 3 times the duration approved for use in dogs and wild felids did not cause any acute or subacute adverse reactions or pathologic changes. Results suggest that cats may be safely treated with fenbendazole.

The nigrostriatal dopaminergic system as a preferential target of repeated exposures to combined paraquat and maneb: implications for Parkinson's disease.

Experimental evidence supporting 1,1'-dimethyl-4,4'-bipyridinium [paraquat (PQ)] as a risk factor for Parkinson's disease (PD) is equivocal. Other agricultural chemicals, including dithiocarbamate fungicides such as manganese ethylenebisdithiocarbamate [maneb (MB)], are widely used in the same geographical regions as paraquat and also impact dopamine systems, suggesting that mixtures may be more relevant etiological models. This study therefore proposed that combined PQ and MB exposures would produce greater effects on dopamine (DA) systems than would either compound administered alone. Male C57BL/6 mice were treated twice a week for 6 weeks with intraperitoneal saline, 10 mg/kg paraquat, 30 mg/kg maneb, or their combination (PQ + MB). MB, but not PQ, reduced motor activity immediately after treatment, and this effect was potentiated by combined PQ + MB treatment. As treatments progressed, only the combined PQ + MB group evidenced a failure of motor activity levels to recover within 24 hr. Striatal DA and dihydroxyphenylacetic acid increased 1-3 d and decreased 7 d after injections. Only PQ + MB reduced tyrosine hydroxylase (TH) and DA transporter immunoreactivity and did so in dorsal striatum but not nucleus accumbens. Correspondingly, striatal TH protein levels were decreased only by combined PQ + MB 5 d after injection. Reactive gliosis occurred only in response to combined PQ + MB in dorsal-medial but not ventral striatum. TH immunoreactivity and cell counts were reduced only by PQ + MB and in the substantia nigra but not ventral tegmental area. These synergistic effects of combined PQ + MB, preferentially expressed in the nigrostriatal DA system, suggest that such mixtures could play a role in the etiology of PD.

Lack of cyclooxygenase-2 inhibits growth of teratocarcinomas in mice.

Two isoforms of cyclooxygenase (COX-1 or COX-2) have been identified in the prostanoid biosynthetic pathway. The constitutive form, COX-1, is thought to maintain cellular homeostasis and the inducible form, COX-2, is recognized as a primary response gene thought to be involved in modulating cell proliferation and differentiation. To further characterize the role of the cyclooxygenases in cell proliferation, differentiation, and tumorigenicity we developed embryonic stem (ES) cell lines which contain homozygous disruptions in either the COX-1 or the COX-2 gene. These lines were then examined in terms of their viability, proliferation, and in vitro differentiation potential. Our results demonstrate that the wild-type ES cells do not express either COX-1 or COX-2 until the cells undergo differentiation. And the lack of either cyclooxygenase has no apparent effect on ES cell proliferation in vitro. However, the absence of a functional COX-2 gene leads to a dramatic reduction in the formation and growth of teratocarcinomas that appear when ES cells are injected into syngeneic mice. Histological microscopy shows that the few very small tumors that were generated from ES cells lacking COX-2 appear more differentiated than tumors emerging from COX-1 -/- or wild-type cells by exhibiting greater keratinization in the areas of squamous epithelium and the ossification of bone-forming cartilage. We conclude that the presence of a functional COX-2 enzyme is necessary for the efficient growth of these teratocarcinomas in animals.

Acute pulmonary effects of ultrafine particles in rats and mice.

Ambient fine particles consist of ultrafine particles (< 100 nm) and accumulation-mode particles (approximately 100 to 1,000 nm). Our hypothesis that ultrafine particles can have adverse effects in humans is based on results of our earlier studies with particles of both sizes and on the finding that urban ultrafine particles can reach mass concentrations of 40 to 50 micrograms/m3, equivalent to number concentrations of 3 to 4 x 10(5) particles/cm3. The objectives of the exploratory studies reported here were to (1) evaluate pulmonary effects induced in rats and mice by ultrafine particles of known high toxicity (although not occurring in the ambient atmosphere) in order to obtain information on principles of ultrafine particle toxicology; (2) characterize the generation and coagulation behavior of ultrafine particles that are relevant for urban air; (3) study the influence of animals' age and disease status; and (4) evaluate copollutants as modifying factors. We used ultrafine Teflon (polytetrafluoroethylene [PTFE]*) fumes (count median diameter [CMD] approximately 18 nm) generated by heating Teflon in a tube furnace to 486 degrees C to evaluate principles of ultrafine particle toxicity that might be helpful in understanding potential effects of ambient ultrafine particles. Teflon fumes at ultrafine particle concentrations of approximately 50 micrograms/m3 are extremely toxic to rats when inhaled for only 15 minutes. We found that neither the ultrafine Teflon particles alone when generated in argon nor the Teflon fume gas-phase constituents when generated in air were toxic after 25 minutes of exposure. Only the combination of both phases when generated in air caused high toxicity, suggesting the existence of either radicals on the particle surface or a carrier mechanism of the ultrafine particles for adsorbed gas-phase compounds. We also found rapid translocation of the ultrafine Teflon particles across the epithelium after their deposition, which appears to be an important difference from the behavior of larger particles. Furthermore, the pulmonary toxicity of the ultrafine Teflon fumes could be prevented by adapting the animals with short 5-minute exposures on 3 days prior to a 15-minute exposure. This shows the importance of preexposure history in susceptibility to acute effects of ultrafine particles. Aging of the fresh Teflon fumes for 3.5 minutes led to a predicted coagulation resulting in particles greater than 100 nm that no longer caused toxicity in exposed animals. This result is consistent with greater toxicity of ultrafine particles compared with accumulation-mode particles. When establishing dose-response relationships for intratracheally instilled titanium dioxide (TiO2) particles of the size of the urban ultrafine particles (20 nm) and of the urban accumulation-mode particles (250 nm), we observed significantly greater pulmonary inflammatory response to ultrafine TiO2 in rats and mice. The greater toxicity of the ultrafine TiO2 particles correlated well with their greater surface area per mass. Ultrafine particles of carbon, platinum, iron, iron oxide, vanadium, and vanadium oxide were generated by electric spark discharge and characterized to obtain particles of environmental relevance for study. The CMD of the ultrafine carbon particles was approximately 26 nm, and that of the metal particles was 15 to 20 nm, with geometric standard deviations (GSDs) of 1.4 to 1.7. For ultrafine carbon particles, approximately 100 micrograms/m3 is equivalent to 12 x 10(6) particles/cm3. Homogeneous coagulation of these ultrafine particles in an animal exposure chamber occurred rapidly at 1 x 10(7) particles/cm3, so that particles quickly grew to sizes greater than 100 nm. Thus, controlled aging of ultrafine carbon particles allowed the generation of accumulation-mode carbon particles (due to coagulation growth) for use in comparative toxicity studies. We also developed a technique to generate ultrafine particles consisting of the stable isotope 13C by using 13C-graphite electrodes made in our laboratory from amorphous 13C powder. These particles are particularly useful tools for determining deposition efficiencies of ultrafine carbon particles in the respiratory tracts of laboratory animals and the translocation of particles to extrapulmonary sites. For compromised animals, we used acute and chronic pulmonary emphysema; a low-dose endotoxin inhalation aimed at priming target cells in the lung was also developed. Other modifying factors were age and copollutant (ozone) exposure. Exposure concentrations of the generated ultrafine particles for acute rodent inhalation studies were selected on the basis of lung doses predicted to occur in people inhaling approximately 50 micrograms/m3 urban ultrafine particles. Concentrations that achieved the same predicted lung burden per unit alveolar surface were used in rodents. (ABSTRACT TRUNCATED)

Bcl-2 family gene expression during severe hyperoxia induced lung injury.

Exposure of the lung to severe hyperoxia induces terminal transferase dUTP end-labeling (TUNEL) indicative of DNA damage or apoptosis and increases expression of the tumor suppressor p53 and of members of the Bcl-2 gene family. Because cell survival and apoptosis are regulated, in part, by the relative abundance of proteins of the Bcl-2 family, we hypothesized that lung cells dying during exposure would show increased expression of pro-apoptotic members, such as Bax, whereas surviving cells would have increased expression of anti-apoptotic members, such as Bcl-X(L). The hypothesis is tested in the current study by determining which Bcl-2 genes are regulated by hyperoxia, with specific focus on correlating expression of Bax and Bcl-X(L) with morphologic evidence of apoptosis or necrosis. Adult mice exposed to greater than 95% oxygen concentrations for 48 to 88 hours had increased whole-lung mRNA levels of Bax and Bcl-X(L), no change in Bak, Bad, or Bcl-2, and decreased levels of Bcl-w and Bfl-1. In situ hybridization revealed that hyperoxia induced Bax and Bcl-X(L) mRNA in uniform and overlapping patterns of expression throughout terminal bronchioles and parenchyma, coinciding with TUNEL staining. Electron microscopy and DNA electrophoresis, however, suggested relatively little classical apoptosis. Unexpectedly, Western analysis demonstrated increased Bcl-X(L), but not Bax, protein in response to hyperoxia. Bax and Bfl-1 were not altered by hyperoxia in p53 null mice; however, oxygen toxicity was not lessened by p53 deficiency. These findings suggest that oxygen-induced lung injury does not depend on the relative expression of these Bcl-2 members.

A chimeric aryl hydrocarbon receptor knockout mouse model indicates that aryl hydrocarbon receptor activation in hematopoietic cells contributes to the hepatic lesions induced by 2,3,7, 8-tetrachlorodibenzo-p-dioxin.

Pathologic changes associated with 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) exposure have been reported in the livers of a wide range of species. While these changes have been extensively described, the mechanisms of toxic interaction(s) that produce these lesions remain unclear. Using an aryl hydrocarbon receptor (Ahr) knockout male mouse chimeric model, we investigated whether the presence of this receptor in hematopoietic and/or parenchymal cells affects TCDD-induced hepatotoxicity. Bone marrow chimeras were produced by hematopoietic reconstitution of irradiated mice. Specifically, chimeras were generated with aryl hydrocarbon receptor (AHR) positive hematopoietic and parenchymal cells (Ahr+/+ animal bone marrow cells into irradiated Ahr+/+ animals), AHR positive hematopoietic and negative parenchymal cells (Ahr+/+ into Ahr-/-), AHR negative hematopoietic and positive parenchymal cells (Ahr-/- into Ahr+/+), and AHR negative hematopoietic and parenchymal cells (Ahr-/- into Ahr-/-). Male wild-type (Ahr+/+) and knockout (Ahr-/-) animals were used as nonchimeric controls. Following TCDD treatment (30 microg/kg body wt), liver sections from mice in each control and chimeric group were histologically evaluated for necrotic and inflammatory changes. TCDD treatment produced moderate inflammation in Ahr+/+ controls and Ahr+/+ into Ahr+/+ chimeras. This response was mild in TCDD-treated Ahr-/-, Ahr-/- into Ahr-/-, Ahr+/+ into Ahr-/-, and Ahr-/- into Ahr+/+ animals and was not different from the corresponding vehicle-treated groups. Moderate necrosis was observed in all TCDD-treated controls or chimeras with AHR-positive parenchyma. No or mild necrosis was observed in TCDD- and vehicle-treated animals containing AHR-negative parenchyma. These data indicate that the presence of AHR in hepatic parenchyma alone is sufficient for TCDD induction of hepatic necrosis, and its presence in hematopoietic cells is necessary for the inflammatory response to TCDD-induced hepatic lesions.

Blockade of CD40-CD40 ligand interactions protects against radiation-induced pulmonary inflammation and fibrosis.

This study investigated whether CD40-CD40 ligand (L) interactions are important in mediating ionizing radiation-induced lung toxicity. Radiotherapy is a key component in the management of malignant diseases and is a conditioning regimen for bone marrow transplantation. Unfortunately, radiation therapy is particularly toxic to the lung, potentially inducing a fatal pneumonitis and fibrosis, thus limiting its effectiveness. There are no therapies that protect against the development of radiation-induced lung toxicity. Using a mouse model of radiation-induced lung toxicity, a monoclonal anti-CD40L antibody (MR1) that disrupts CD40-CD40L interactions was tested for the ability to reduce lung injury. C57BL/6 mice were pretreated with either nothing, MR1, or hamster IgG 24 h prior to a single dose of 15 Gray ionizing radiation to the thorax. During the following 26 weeks, mice continued to receive MR1 or hamster IgG twice per week. MR1 protected against death from radiation pneumonitis and fibrosis and dramatically reduced lung pathology as evidenced by a limited influx of inflammatory cells, minimal collagen deposition, and septal thickening. MR1 also prevented radiation-induced pulmonary mastocytosis and blunted expression of cyclooxygenase-2, a proinflammatory enzyme responsible for prostaglandin synthesis. Disruption of CD40-CD40L interactions may offer a new mode of intervention to protect against radiation-induced pulmonary toxicity.

Prenatal and lactational exposure to methylmercury affects select parameters of mouse cerebellar development.

Animal studies of the neuropathological effects of prenatal methylmercury (MeHg) seldom use regimens that represent environmental exposures. While acute administration of high doses of MeHg to developing rodents can model some of the outcomes MeHg produces in the human cerebellum, their long-term relevance to cerebellar development is unknown. The present study was undertaken to determine the effect of chronic dietary exposure to MeHg. Pregnant mice were exposed throughout gestation to 0.0 or 4.0 ppm methylmercury in their drinking water. Postpartum exposure of pups and lactating dams continued to postnatal day (PND) 30. On PND7, 14, 21, and 30, several morphometric indices of cerebellar cortex development, as well as blood and brain levels of total Hg, were measured in pairs of male and female littermates. No signs of overt toxicity were observed in the dams or offspring. Blood and brain levels of total Hg were highest in the exposed PND7 offspring and fell throughout the sampling period despite continued exposure. In a region of molecular layer in the anterodorsal lobe, MeHg exposure reduced the density of migrating cells in PND7 offspring. Molecular layer widths were reduced in PND30 offspring. In a region of the inferior lobe of PND7 offspring, MeHg exposure reduced external granular layer widths and decreased the density of migrating cells in the molecular layer. However, MeHg did not affect cerebellar cortex development in the central lobe, suggesting a regional sensitivity to chronic, low-level MeHg exposure during development.

Fate and toxicity of 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (compound A)-derived mercapturates in male, Fischer 344 rats.

BACKGROUND: 2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (compound A) is formed in the anesthesia circuit by the degradation of sevoflurane. Compound A is nephrotoxic in rats and undergoes metabolism by the mercapturic acid pathway in rats and humans to yield the mercapturates S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-N-acetyl-L -cysteine (compound 3) and S-[2(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cys teine (compound 5). These experiments were designed to examine the fate and nephrotoxicity of compound A-derived mercapturates in rats. METHODS: The deacetylation of compounds 3 and 5 by human and rat kidney cytosol and with purified acylases I and III was measured, and their nephrotoxicity was studied in male Fischer 344 rats. The metabolism of the deuterated analogs of compounds 3 and 5, [acetyl-2H3]S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl ]-N-acetyl-L-cysteine (compound 3-d3) and [acetyl-2H3]S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N -acetyl-L-cysteine (compound 5-d3), respectively, was measured. RESULTS: Compound 5, but not compound 3, was hydrolyzed by human and rat kidney cytosols and by acylases I and III. 19F nuclear magnetic resonance spectroscopic analysis showed no urinary metabolites of compound 3, but unchanged compound 5 and its metabolites 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid and 2-[1-(fluoromethoxy)-2,2,2-trifluoroethyl]-4,5-dihydro-1,3-thiazol e-4-carboxylic acid were detected in urine. Compound 5 (250 microM/kg) produced clinical chemical and morphologic evidence of renal injury in two of three animals studied. CONCLUSIONS: Compounds 3 and 5 underwent little metabolism. Compound 5, but not compound 3, was mildly nephrotoxic. These results indicate that compound A-derived mercapturate formation constitutes a detoxication pathway for compound A.

Aryl hydrocarbon receptor activation in genital tubercle, palate, and other embryonic tissues in 2,3,7, 8-tetrachlorodibenzo-p-dioxin-responsive lacZ mice.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates the toxicity of 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) and related halogenated aromatic hydrocarbons. Although the normal function and endogenous ligand for this receptor are not known, it is thought to have a role in growth regulation processes. The AhR has been found in both adult and certain developing tissues, and AhR agonists like the environmental contaminant TCDD cause a number of developmental anomalies. We sought to determine whether the AhR is directly activated to a transcriptionally functional form in tissues known to be adversely affected by AhR agonist exposure. To this end, a transgenic mouse model was developed that could be used to indicate the temporal and spatial context of transcriptionally active AhR following agonist exposure in vivo. A synthetic promoter containing two dioxin-responsive elements (DREs) and a minimal TATA box was strongly induced by TCDD in transfected cells when linked to the lacZ or luciferase reporter gene. Transgenic mice harboring the lacZ construct had TCDD-inducible beta-galactosidase activity in tissues following adult and in utero exposure. Embryonic lacZ expression was induced in hard and soft palates, genital tubercle, certain facial regions, shoulder, as well as other tissues by in utero exposure to 30 microg TCDD/kg at Gestational Day 13. The most intense reporter response was observed in the genital tubercle. Histopathology of the palate and tubercle demonstrated the reporter gene activity to be both cell- and region-specific. This is the first publication to correlate reported TCDD-elicited toxicity (e.g., cleft palate in mice) with TCDD-dependent AhR activation. These data indicate the ability of TCDD to initiate a signal transduction process leading to a transcriptionally active AhR in these tissues, thereby identifying potential targets of dioxin-induced toxicity during development. Weak activation of the reporter gene was consistently observed only in the genital tubercle in the absence of exogenous inducer. This indicates minimal or no endogenous AhR activators at the developmental stage examined. This mouse model will prove useful for both the examination of the endogenous role of the AhR in proliferation or differentiation and of the developmental targets of dioxin-like compounds.

Acoustic backscatter properties of the particle/bubble ultrasound contrast agent.

Bubble-based suspensions with diameters in the 1-5 microns range have been developed for use as ultrasound contrast agents. Bubbles of these dimensions have resonance frequencies in the diagnostic ultrasonic range, thus improving their backscatter enhancement capabilities. The durability of these bubbles in the blood stream has been found to be limited, providing impetus for a number of approaches to further stabilize them. One of the approaches has been the development of micrometer-size porous particles or 'nano-sponges' with properties suitable for the entrapment and stabilization of gas bubbles. However, the complex morphology and surface chemistry involved in the production of this type of agent makes it unfeasible to directly measure the volume of the entrained gas. A model based on acoustic scattering principles is proposed which indicates that only a small volume fraction of gas should be necessary to significantly enhance the echogenicity of this type of particle-based contrast agent. In the model, the effective scattering cross-section is evaluated as a function of the volume fraction of gas contained in the overall scatterer and the overall scatterer diameter. Initially, the volume fraction of gas is considered as a discrete entity of single bubble. Using common mixture rules, it is then shown that the gas can be considered to be distributed throughout the particle and still arrive at a result that is similar to that for a single, discrete volume of gas. The main contribution to the increased scattering cross-section is due to the compressibility difference between gas and water. The backscatter coefficient is computed as the product of the resulting differential scattering cross-section and the scatterer number density. This approach facilitates comparison with known backscatter coefficients of biological targets such as liver and blood. Simple experimental results are presented for comparison with the model, and the implications relevant to clinical use are suggested.

NF-kappa B-dependent inhibition of apoptosis is essential for host cellsurvival during Rickettsia rickettsii infection.

The possibility that bacteria may have evolved strategies to overcome host cell apoptosis was explored by using Rickettsia rickettsii, an obligate intracellular Gram-negative bacteria that is the etiologic agent of Rocky Mountain spotted fever. The vascular endothelial cell, the primary target cell during in vivo infection, exhibits no evidence of apoptosis during natural infection and is maintained for a sufficient time to allow replication and cell-to-cell spread prior to eventual death due to necrotic damage. Prior work in our laboratory demonstrated that R. rickettsii infection activates the transcription factor NF-kappa B and alters expression of several genes under its control. However, when R. rickettsii-induced activation of NF-kappa B was inhibited, apoptosis of infected but not uninfected endothelial cells rapidly ensued. In addition, human embryonic fibroblasts stably transfected with a superrepressor mutant inhibitory subunit Ikappa B that rendered NF-kappa B inactivatable also underwent apoptosis when infected, whereas infected wild-type human embryonic fibroblasts survived. R. rickettsii, therefore, appeared to inhibit host cell apoptosis via a mechanism dependent on NF-kappa B activation. Apoptotic nuclear changes correlated with presence of intracellular organisms and thus this previously unrecognized proapoptotic signal, masked by concomitant NF-kappa B activation, likely required intracellular infection. Our studies demonstrate that a bacterial organism can exert an antiapoptotic effect, thus modulating the host cell's apoptotic response to its own advantage by potentially allowing the host cell to remain as a site of infection.

Disruption of the CD40-CD40 ligand system prevents an oxygen-induced respiratory distress syndrome.

Oxygen therapy is a mainstay treatment for infants and adults with poor lung function. Unfortunately, oxygen itself is toxic and incites respiratory cell damage and inflammation. Therapies for oxygen-induced lung damage are nonexistent. Employing a mouse model of hyperoxic lung injury, a monoclonal anti-CD40 ligand (L) antibody (MR1), which disrupts CD40-CD40L interactions, was tested for the ability to reduce pulmonary injury. Intraperitoneal administration of MR1, either before or after oxygen exposure, was remarkably effective in reducing and in many cases preventing lung injury. The pro-inflammatory enzyme cyclooxygenase-2 (Cox-2), responsible for prostaglandin production, is massively up-regulated in the lungs after hyperoxic exposure. Immunohistochemical staining for Cox-2 revealed that MR1 greatly reduces the oxygen-induced induction of Cox-2. The remarkable effectiveness of MR1 in blunting hyperoxic lung injury in this preclinical model may be relevant to the hundreds of thousands of patients who require treatment with high oxygen and who are at risk for developing severe pulmonary inflammation and consequent fibrosis. Strategies to disrupt CD40-CD40L interactions may offer a new mode of intervention for oxygen-induced acute respiratory distress syndrome and other inflammatory lung disorders.