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.

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.

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.

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.

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.

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.

Regression of pulmonary lesions produced by inhaled titanium dioxide in rats.

Inhaled ultrafine particles of TiO2 (TiO2-D, 20 nm particle size) lead to a greater pulmonary inflammatory response than larger pigment-grade particles (TiO2-F, 250 nm). Male Fisher 344 rats were exposed for 6 hours a day, 5 days a week, for 3 months to 1) filtered air (control); 2) TiO2-F, 22.3 mg/m3; 3) TiO2-D, 23.5 mg/m3; or 4) crystalline SiO2, a positive control particle (approximately 800 nm particle size, 1.3 mg/m3). Groups of 3-4 animals were sacrificed at 6 and 12 months following the completion of exposure. Pulmonary effects of exposure were evaluated using standard hematoxylin and eosin-stain sections, histochemical stains for collagen, and immunohistochemical assays for cell turnover. Six months after animals were exposed to SiO2, they had moderate focal interstitial fibrosis and moderately severe focal alveolitis. Animals exposed to TiO2-D had slightly less fibrosis. The least fibrosis was seen in the TiO2-F group. At 1 year after exposure, fibrosis was still present but decreased in the SiO2 group. The amount of interstitial fibrosis in the TiO2-D- and TiO2-F-treated animals had largely returned to untreated control levels, although an increased number of alveolar macrophages persisted, usually with retained particles. There was discordance between bromodeoxyuridine and proliferating cell nuclear antigen indices, most probably due to cytokine elaboration in the areas of inflammation, which may have altered the expression of proliferating cell nuclear antigens. There was no detectable fibroblast labeling at the 6-month observation and only very low levels at 12 months. Thus, although initially irritant, TiO2-induced lesions regressed during a 1-year period following cessation of exposure.

Intratracheal instillation versus intratracheal inhalation: influence of cytokines on inflammatory response.

Our laboratory has developed a method of particle exposure whereby anesthetized rats intratracheally inhale, at a regulated breathing rate and pressure, an aerosolized test material. This method is capable of delivering considerable doses in a short time period and, unlike the commonly used method of intratracheal instillation, does so with an even particle distribution throughout the lung. Early studies comparing the response of male Fischer 344 rats exposed to TiO2 particles of two differing primary particle sizes showed that at similar particle doses animals exposed by the two methods showed differences in response, as measured by bronchoalveolar lavage (BAL) parameters. Building on this, we sought to study the roles that macrophage inflammatory protein-2 (MIP-2) and tumor necrosis factor alpha (TNF-alpha), two cytokines thought to have proinflammatory roles in the lung, may play in the differences observed. Increases in MIP-2 protein levels in the lavaged cells, but not the supernatant, were observed in those groups where increased polymorphonuclear cells (PMN) in the lung lavage were found, but not in those where no increase in PMN levels was observed. BAL TNF-alpha levels, measured by enzyme-linked immunosorbent assay, showed no apparent correlation with cellular or biochemical BAL parameters for either particle size or dosing method. Increases in immunocytochemical staining for TNF-alpha, compared to unexposed controls, were observed in several particle-exposed groups. Thus, it appears that increased BAL MIP-2 protein levels, but not TNF-alpha, correlate well with the inflammatory response, as measured by PMN numbers in lavaged cells, for both exposure systems.

Teratogenicity of methanol following a single oral dose in Long-Evans rats.

The teratogenicity of methanol was investigated following a single oral exposure preceded by an equal volume of mineral oil to guard against local gastric irritation. Four groups of pregnant Long-Evans rats were gavaged on day 10 of gestation with the following solutions: 0.0 (n = 13), 1.3 (n = 12), 2.6 (n = 11), and 5.2 (n = 10) mL MeOH/kg. Wilson sectioning (head only), gross necropsy, and Alizarin red skeletal examinations were performed on day 20 of gestation. At 5.2 mL/kg, the dams demonstrated > 20% decrease in weight gain in comparison to the control, which was the only clinical toxic manifestation or histopathologic change noted for the dams. Methanol at all doses failed to produce any significant change in standard reproductive indices (e.g., postimplantation loss). A significant decrease in fetal body weight (11 to 19.5%), however, was associated with prenatal oral ingestion of methanol. Both internal and external examination of the fetuses demonstrated a dose-dependent increase in anomalies [0 = 0.6%, 1.3 mL/kg = 3.7%, 2.6 mL/kg = 7%, 5.2 mL/kg = 16.5% (litter percents)]. The dose-related anomalies were undescended testes, exophthalmia, and anophthalmia. Thus, acute methanol given orally produces anomalies, even when there is no apparent maternal toxic response.

Changes in surfactant protein gene expression in a neonatal rabbit model of hyperoxia-induced fibrosis.

Lung injuries, including bronchopulmonary dysplasia, alter the surfactant system. We developed a newborn rabbit model of acute, followed by chronic, hyperoxic injury to study surfactant protein (SP) gene expression. Initial litters were exposed to >95% O2 until 50% died (LD50; 7-11 days old). Subsequent litters were exposed to >95% O2 for 8 days, followed by 60% O2 until 22-36 days. Controls were exposed to room air. LD50 animals displayed acute pulmonary inflammation, edema, protein leak, and surfactant dysfunction. These changes resolved, and fibrosis developed by 22 days. Whole lung SP-A mRNA expression (measured by membrane hybridization) was twice control levels at 4 days of >95% O2, with specific elevations in terminal bronchioles and type II cells at 4 days and the LD50 by in situ hybridization. Whole lung SP-B and SP-C mRNA were unchanged from control throughout exposure. However, in situ hybridization showed elevations in SP-B and SP-C mRNA in type II cells in inflamed areas at the LD50. SP mRNA alterations resolved by 22-36 days. The surfactant system recovers from acute hyperoxic injury, despite continued 60% O2 exposure.

Immunolocalization of microsomal glutathione S-transferase in rat tissues.

Distribution of microsomal glutathione transferase (mGST) protein in rat tissues was investigated by immunohistochemistry. Studies on the localization of mGST are of interest because of its involvement in the detoxication and bioactivation of xenobiotics. mGST antigen was detected in the cytoplasm of some hepatocytes and in bile ducts. In kidney, focal staining of mGST was observed in distal tubules and collecting ducts. Cerebral cortical and cerebellar Purkinje neurons showed good immunoreactivity, and nuclear staining was observed in the choroid plexus. The antigen was detected in epithelial cells of respiratory bronchioles and in the crypt cells of the duodenum. Exocrine cells of the pancreas stained for mGST. Nuclear immunostaining for this protein was observed in primary spermatocytes. mGST antigen was detected in the cytoplasm of the adrenal medulla as a granular stain. Leydig and Sertoli cells in testis also stained for the antigen. Distribution of mGST protein differs from that observed with cytosolic transferases and may be important in determining cell-selective susceptibility to xenobiotics.

Nephrotoxicity of the glutathione and cysteine S-conjugates of the sevoflurane degradation product 2-(fluoromethoxy)-1,1,3,3, 3-pentafluoro-1-propene (Compound A) in male Fischer 344 rats.

2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (Compound A) is a halogenated alkene that is nephrotoxic in rats when administered by inhalation or intraperitoneally. Compound A undergoes glutathione-dependent metabolism: Compound A-derived glutathione S-conjugates and mercapturates are excreted in the bile and urine, respectively, of rats given Compound A. The present experiments were designed to study the nephrotoxicity of the Compound A-derived glutathione and cysteine S-conjugates, S-[2-(fluoromethoxy)-1,1,3,3, 3-pentafluoropropyl]glutathione , S-[2-(fluoromethoxy)-1,3,3, 3-tetrafluoro-1-propenyl]glutathione , S-[2-(fluoromethoxy)-1,1,3,3, 3-pentafluoropropyl]-L-cysteine and S-[2-(fluoromethoxy)-1,3,3, 3-tetrafluoro-1-propenyl]-L-cysteine . Conjugates , and given intraperitoneally produced dose-dependent nephrotoxicity that was characterized by diuresis, increased excretion of glucose and protein, elevated blood urea nitrogen concentrations and severe morphological changes in the kidneys, particularly in the proximal tubules. Glutathione S-conjugate , at a dose of 500 micromol/kg, was hepatotoxic. Cysteine S-conjugate was not nephrotoxic, apparently because of its facile cyclization to the thiazoline 2-[1-(fluoromethoxy)-2,2,2-trifluoroethyl]-4,5-dihydro-1, 3-thiazole-4-carboxylic acid, which is not a beta-lyase substrate. Also, the alpha-methyl analog of cysteine S-conjugate S-[2-(fluoromethoxy)-1,1,3,3, 3-pentafluoropropyl]-DL-alpha-methylcysteine, which cannot undergo beta-lyase-dependent bioactivation, was not nephrotoxic. These in vivo data show that Compound A-derived S-conjugates are nephrotoxic and that the toxicity is associated with beta-lyase-dependent bioactivation.