Preclinical safety and biodistribution assessment of Ad-KCNH2-G628S administered via atrial painting in New Zealand white rabbits.

Post-operative atrial fibrillation (POAF) is the most common complication after cardiac surgery. Despite implementation of several pharmacological strategies, incidence of POAF remains at approximately 30%. An adenovirus vector encoding KCNH2-G628S has proven efficacious in a porcine model of AF. In this preclinical study, 1.5 × 1010 or 1.5 × 1012 Ad-KCNH2-G628S vector particles (vp) were applied to the atrial epicardium or 1.5 × 1012 vp were applied to the whole epicardial surface of New Zealand White rabbits. Saline and vector vehicle served as procedure controls. Animals were followed for up to 42 days. Vector genomes persisted in the atria up to 42 days, with no distribution to extra-thoracic organs. There were no adverse effects attributable to test article on standard toxicological endpoints or on blood pressure, left atrial or ventricular ejection fractions, electrocardiographic parameters, or serum IL-6 or troponin concentrations. Mononuclear infiltration of the myocardium of the atrial free walls of low-dose, but not high-dose animals was observed at 7 and 21 days, but these changes did not persist or affect cardiac function. After scaling for heart size, results indicate the test article is safe at doses up to 25 times the maximum proposed for the human clinical trial.

Comparative pulmonary and genotoxic responses to inhaled nickel subsulfide and nickel sulfate in F344 rats.

Inhalation studies with nickel (Ni) subsulfide (Ni3 S2 ) and Ni sulfate hexahydrate (NiSO4 ·6H2 O) investigated differences in mode of action that could explain why the former induced lung tumors in rats and the latter did not. Male rats were exposed to ≤0.22 mg Ni/m3 NiSO4 ·6H2 O or 0.44 mg Ni/m3 Ni3 S2 , 6 h/day, 5 days/week for 3 and 13 weeks; subsets of the rats exposed for 13 weeks were held for an additional 13 weeks. Analyses of bronchoalveolar lavage fluid, isolated cells, and whole lung tissue were conducted to compare the extent and persistence of any induced lung effects. Histological findings were qualitatively identical for both compounds and consistent with lesions reported in earlier studies. After 13 weeks of exposure, the incidence and severity of pulmonary inflammation and epithelial cell hyperplasia were greater among Ni3 S2 -exposed rats, whereas the reverse response was seen for apoptosis. Only Ni3 S2 exposure significantly increased epithelial and non-epithelial cell proliferation after 13 weeks of exposure. Both compounds induced DNA damage in isolated lung cells and DNA hypermethylation of whole lung tissue after 13 weeks of exposure at the highest exposure concentrations. Increases in cell proliferation, DNA damage, and tissue DNA hypermethylation did not persist during the 13-week recovery period. In summary, the highest concentrations of each compound produced marked pulmonary toxicity, but the lowest concentrations produced minimal or no effects. Differences in the proliferative and apoptotic responses between the two compounds may help explain differences in carcinogenicity, whereas the identification of no observed adverse effect concentrations (NOAECs) contributes to the risk characterization for inhalation exposure to nickel compounds.

Bridging from Intramuscular to Limb Perfusion Delivery of rAAV: Optimization in a Non-human Primate Study.

Phase 1 and phase 2 gene therapy trials using intramuscular (IM) administration of a recombinant adeno-associated virus serotype 1 (rAAV1) for replacement of serum alpha-1 antitrypsin (AAT) deficiency have shown long-term (5-year) stable transgene expression at approximately 2% to 3% of therapeutic levels, arguing for the long-term viability of this approach to gene replacement of secreted serum protein deficiencies. However, achieving these levels required 100 IM injections to deliver 135 mL of vector, and further dose escalation is limited by the scalability of direct IM injection. To further advance the dose escalation, we sought to bridge the rAAV-AAT clinical development program to regional limb perfusion, comparing two methods previously established for gene therapy, peripheral venous limb perfusion (VLP) and an intra-arterial push and dwell (IAPD) using rAAV1 and rAAV8 in a non-human primate (rhesus macaque) study. The rhesus AAT transgene was used with a c-myc tag to enable quantification of transgene expression. 5 cohorts of animals were treated with rAAV1-IM, rAAV1-VLP, rAAV1-IAPD, rAAV8-VLP, and rAAV8-IAPD (n = 2-3), with a dose of 6 × 1012 vg/kg. All methods were well tolerated clinically. Potency, as determined by serum levels of AAT, of rAAV1 by the VLP method was twice that observed with direct IM injection; 90 µg/mL with VLP versus 38 µg/mL with direct IM injection. There was an approximately 25-fold advantage in estimated vector genomes retained within the muscle tissue with VLP and a 5-fold improvement in the ratio of total vector genomes retained within muscle as compared with liver. The other methods were intermediate in the potency and retention of vector genomes. Examination of muscle enzyme (CK) levels indicated rAAV1-VLP to be equally safe as compared with IM injection, while the IAPD method showed significant CK elevation. Overall, rAAV1-VLP demonstrates higher potency per vector genome injected and a greater total vector retention within the muscle, as compared to IM injection, while enabling a much greater total dose to be delivered, with equivalent safety. These data provide the basis for continuation of the dose escalation of the rAAV1-AAT program in patients and bode well for rAAV-VLP as a platform for replacement of secreted proteins.

The Gene Therapy Resource Program: A Decade of Dedication to Translational Research by the National Heart, Lung, and Blood Institute.

Over a 10-year period, the Gene Therapy Resource Program (GTRP) of the National Heart Lung and Blood Institute has provided a set of core services to investigators to facilitate the clinical translation of gene therapy. These services have included a preclinical (research-grade) vector production core; current Good Manufacturing Practice clinical-grade vector cores for recombinant adeno-associated virus and lentivirus vectors; a pharmacology and toxicology core; and a coordinating center to manage program logistics and to provide regulatory and financial support to early-phase clinical trials. In addition, the GTRP has utilized a Steering Committee and a Scientific Review Board to guide overall progress and effectiveness and to evaluate individual proposals. These resources have been deployed to assist 82 investigators with 172 approved service proposals. These efforts have assisted in clinical trial implementation across a wide range of genetic, cardiac, pulmonary, and blood diseases. Program outcomes and potential future directions of the program are discussed.

A Preclinical Study in Rhesus Macaques for Cystic Fibrosis to Assess Gene Transfer and Transduction by AAV1 and AAV5 with a Dual-Luciferase Reporter System.

Cystic fibrosis (CF) is an autosomal recessive disease that is potentially treatable by gene therapy. Since the identification of the gene encoding CF transmembrane conductance regulator, a number of preclinical and clinical trials have been conducted using the first generation of adeno-associated virus, AAV2. All these studies showed that AAV gene therapy for CF is safe, but clinical benefit was not clearly demonstrated. Thus, a new generation of AAV vectors based on other serotypes is needed to move the field forward. This study tested two AAV serotypes (AAV1 and AAV5) using a dual-luciferase reporter system with firefly and Renilla luciferase genes packaged into AAV1 or AAV5, respectively. Two male and two female Rhesus macaques were each instilled in their lungs with both serotypes using a Penn-Century microsprayer. Both AAV1 and AAV5 vector genomes were detected in all the lung samples when measured at the time of necropsy, 45 days after instillation. However, the vector genome number for AAV1 was at least 10-fold higher than for AAV5. Likewise, luciferase activity was also detected in the same samples at 45 days. AAV1-derived activity was not statistically greater than that derived from AAV5. These data suggest that gene transfer is greater for AAV1 than for AAV5 in macaque lungs. Serum neutralizing antibodies were increased dramatically against both serotypes but were less abundant with AAV1 than with AAV5. No adverse events were noted, again indicating that AAV gene therapy is safe. These results suggest that with more lung-tropic serotypes such as AAV1, new clinical studies of gene therapy using AAV are warranted.

Safety and biodistribution assessment of sc-rAAV2.5IL-1Ra administered via intra-articular injection in a mono-iodoacetate-induced osteoarthritis rat model.

Interleukin-1 (IL-1) plays an important role in the pathophysiology of osteoarthritis (OA), and gene transfer of IL-1 receptor antagonist (IL-1Ra) holds promise for OA treatment. A preclinical safety and biodistribution study evaluated a self-complementary adeno-associated viral vector carrying rat IL-1Ra transgene (sc-rAAV2.5rIL-1Ra) at 5 × 10(8), 5 × 10(9), or 5 × 10(10) vg/knee, or human IL-1Ra transgene (sc-rAAV2.5hIL-1Ra) at 5 × 10(10) vg/knee, in Wistar rats with mono-iodoacetate (MIA)-induced OA at days 7, 26, 91, 180, and 364 following intra-articular injection. The MIA-induced OA lesions were consistent with the published data on this model. The vector genomes persisted in the injected knees for up to a year with only limited vector leakage to systemic circulation and uptake in tissues outside the knee. Low levels of IL-1Ra expression and mitigation of OA lesions were observed in the vector-injected knees, albeit inconsistently. Neutralizing antibodies against the vector capsid developed in a dose-dependent manner, but only the human vector induced a small splenic T-cell immune response to the vector capsid. No local or systemic toxicity attributable to vector administration was identified in the rats as indicated by clinical signs, body weight, feed consumption, clinical pathology, and gross and microscopic pathology through day 364. Taken together, the gene therapy vector demonstrated a favorable safety profile.

Evaluation of Readministration of a Recombinant Adeno-Associated Virus Vector Expressing Acid Alpha-Glucosidase in Pompe Disease: Preclinical to Clinical Planning.

A recombinant serotype 9 adeno-associated virus (rAAV9) vector carrying a transgene that expresses codon-optimized human acid alpha-glucosidase (hGAA, or GAA) driven by a human desmin (DES) promoter (i.e., rAAV9-DES-hGAA) has been generated as a clinical candidate vector for Pompe disease. The rAAV9-DES-hGAA vector is being developed as a treatment for both early- and late-onset Pompe disease, in which patients lack sufficient lysosomal alpha-glucosidase leading to glycogen accumulation. In young patients, the therapy may need to be readministered after a period of time to maintain therapeutic levels of GAA. Administration of AAV-based gene therapies is commonly associated with the production of neutralizing antibodies that may reduce the effectiveness of the vector, especially if readministration is required. Previous studies have demonstrated the ability of rAAV9-DES-hGAA to correct cardiac and skeletal muscle pathology in Gaa(-/-) mice, an animal model of Pompe disease. This article describes the IND-enabling preclinical studies supporting the program for a phase I/II clinical trial in adult patients with Pompe. These studies were designed to evaluate the toxicology, biodistribution, and potential for readministration of rAAV9-DES-hGAA injected intramuscularly into the tibialis anterior muscle using an immune modulation strategy developed for this study. In the proposed clinical study, six adult participants with late-onset Pompe disease will be enrolled. The goal of the immune modulation strategy is to ablate B-cells before the initial exposure of the study agent in one leg and the subsequent exposure of the same vector to the contralateral leg four months after initial dosing. The dosing of the active agent is accompanied by a control injection of excipient dosing in the contralateral leg to allow for blinding and randomization of dosing, which may also strengthen the evidence generated from gene therapy studies in the future. Patients will act as their own controls. Repeated measures, at baseline and during the three months following each dosing will assess the safety, biochemical, and functional impact of the vector.

Assessment of toxicity and biodistribution of recombinant AAV8 vector-mediated immunomodulatory gene therapy in mice with Pompe disease.

A preclinical safety study was conducted to evaluate the short- and long-term toxicity of a recombinant adeno-associated virus serotype 8 (AAV2/8) vector that has been developed as an immune-modulatory adjunctive therapy to recombinant human acid α-glucosidase (rhGAA, Myozyme) enzyme replacement treatment (ERT) for patients with Pompe disease (AAV2/8-LSPhGAApA). The AAV2/8-LSPhGAApA vector at 1.6 × 10(13) vector particles/kg, after intravenous injection, did not cause significant short- or long-term toxicity. Recruitment of CD4(+) (but not CD8(+)) lymphocytes to the liver was elevated in the vector-dosed male animals at study day (SD) 15, and in group 8 animals at SD 113, in comparison to their respective control animals. Administration of the vector, either prior to or after the one ERT injection, uniformly prevented the hypersensitivity induced by subsequent ERT in males, but not always in female animals. The vector genome was sustained in all tissues through 16-week postdosing, except for in blood with a similar tissue tropism between males and females. Administration of the vector alone, or combined with the ERT, was effective in producing significantly increased GAA activity and consequently decreased glycogen accumulation in multiple tissues, and the urine biomarker, Glc4, was significantly reduced. The efficacy of the vector (or with ERT) was better in males than in females, as demonstrated both by the number of tissues showing significantly effective responses and the extent of response in a given tissue. Given the lack of toxicity for AAV2/8LSPhGAApA, further consideration of clinical translation is warranted in Pompe disease.

Intrapleural administration of an AAVrh.10 vector coding for human α1-antitrypsin for the treatment of α1-antitrypsin deficiency.

Alpha-1 antitrypsin (α1AT) deficiency is a common autosomal recessive disorder characterized by a marked reduction in serum α1AT levels, lung and liver disease. α1AT is mainly produced and secreted by hepatocytes, with its primary function to protect the lung against the proteolytic activity of neutrophil elastase. Serum α1AT levels <11 µM are associated with progressive destruction of lung parenchyma and early-onset of panacinar emphysema in the age range 35-45. The current approved treatment for α1AT deficiency is a costly protein augmentation therapy requiring weekly intravenous infusion of purified α1AT from pooled human plasma. Gene therapy offers the advantage of a single vector administration, eliminating the burden of the repeated purified protein infusions, with the consequent reduced overall drug cost and improved compliance. We have developed a novel, highly efficient gene therapy approach for α1AT deficiency based on the administration of AAVrh.10hα1AT, an adeno-associated viral vector serotype rh.10 coding for normal M-type human α1AT via the intrapleural route. On the basis of prior murine studies, this approach provides sustained α1AT proximal to the lung with a highly efficient vector. In support of a clinical trial for this approach, we carried out a study to assess the safety of intrapleural administration of AAVrh.10hα1AT to 280 mice and 36 nonhuman primates. The data demonstrate that this approach is safe, with no toxicity issues. Importantly, there was persistent expression of the human α1AT mRNA in chest cavity cells for the duration of the study (6 months in mice and 1 year in nonhuman primates). Together, these data support the initiation of a clinical trial of intrapleural human AAVrh.10hα1AT for the treatment of α1AT deficiency.

Advancing translational research through the NHLBI Gene Therapy Resource Program (GTRP).

Abstract Translational research is a lengthy, complex, and necessary endeavor in order to bring basic science discoveries to clinical fruition. The NIH offers several programs to support translational research including an important resource established specifically for gene therapy researchers-the National Heart, Lung, and Blood Institute (NHLBI) Gene Therapy Resource Program (GTRP). This paper reviews the core components of the GTRP and describes how the GTRP provides researchers with resources that are critical to advancing investigational gene therapy products into clinical testing.

The acute toxicity, tissue distribution, and histopathology of inhaled ricin in Sprague Dawley rats and BALB/c mice.

Ricin is a highly toxic ribosome-inactivating protein derived from the castor bean (Ricinus communis). Due to the relative ease of producing ricin, it is characterized as a category B priority pathogen by the Center for Disease Control and Prevention. The purpose of this study was to compare the acute toxicity, associated histopathology, as well as the regional respiratory tract deposition and clearance kinetics of inhaled ricin in rats and mice using a single pure preparation. Acute toxicity was evaluated in five groups of six animals per species exposed nose-only to ricin aerosols and followed up to 7 days post-exposure. Tissues were collected for histopathology. The calculated median lethal doses (LD50s) were 0.24 µg/kg (rats) and 0.58 µg/kg (mice). Histological changes were noted in nose, larynges, trachea, lung, thymus, and spleen of both species. Pulmonary deposition in rats inhaling 94-99 ng/L ricin for 20 min (low dose) or 40 min (high dose) were 45.9 and 96 ng/g lung, respectively. Clearance was best described by a single-component negative exponential function. Estimated lung doses were 0.38 and 1.43 µg/g·h among the low and high dose rats, respectively. In mice inhaling 94 ng/L ricin for 20 min, pulmonary deposition was 91.1 ng/g lung and the estimated tissue dose was 1.72 µg/g·h. No ricin was detected in extra-respiratory tract tissue or in excreta. Results of this study demonstrate differences exist in pulmonary deposition, clearance rates, and tissue dose and histopathological changes between rats and mice inhaling ricin.

Chronic carcinogenicity study of gasoline vapor condensate (GVC) and GVC containing methyl tertiary-butyl ether in F344 rats.

Chronic inhalation studies were conducted to compare the toxicity and potential carcinogenicity of evaporative emissions from unleaded gasoline (GVC) and gasoline containing the oxygenate methyl tertiary-butyl ether (MTBE; GMVC). The test materials were manufactured to mimic vapors people would be exposed to during refueling at gas stations. Fifty F344 rats per gender per exposure level per test article were exposed 6 h/d, 5 d/wk for 104 wk in whole body chambers. Target total vapor concentrations were 0, 2, 10, or 20 g/m³ for the control, low-, mid-, and high-level exposures, respectively. Endpoints included survival, body weights, clinical observations, organs weights, and histopathology. GVC and GMVC exerted no marked effects on survival or clinical observations and few effects on organ weights. Terminal body weights were reduced in all mid- and high-level GVC groups and high-level GMVC groups. The major proliferative lesions attributable to gasoline exposure with or without MTBE were renal tubule adenomas and carcinomas in male rats. GMV exposure led to elevated testicular mesothelioma incidence and an increased trend for thyroid carcinomas in males. GVMC inhalation caused an increased trend for testicular tumors with exposure concentration. Mid- and high-level exposures of GVC and GMVC led to elevated incidences of nasal respiratory epithelial degeneration. Overall, in these chronic studies conducted under identical conditions, the health effects in F344 rats following 2 yr of GVC or GMVC exposure were comparable in the production of renal adenomas and carcinomas in male rats and similar in other endpoints.

Preclinical evaluation of a recombinant adeno-associated virus vector expressing human alpha-1 antitrypsin made using a recombinant herpes simplex virus production method.

Recombinant adeno-associated virus (rAAV) vectors offer promise for gene therapy of alpha-1 antitrypsin (AAT) deficiency. A toxicology study in mice evaluated intramuscular injection of an rAAV vector expressing human AAT (rAAV-CB-hAAT) produced using a herpes simplex virus (HSV) complementation system or a plasmid transfection (TFX) method at doses of 3 × 10(11) vg (1.2 × 10(13) vg/kg) for both vectors and 2 × 10(12) vg (8 × 10(13) vg/kg) for the HSV-produced vector. The HSV-produced vector had favorable in vitro characteristics in terms of purity, efficiency of transduction, and hAAT expression. There were no significant differences in clinical findings or hematology and clinical chemistry values between test article and control groups and no gross pathology findings. Histopathological examination demonstrated minimal to mild changes in skeletal muscle at the injection site, consisting of focal chronic interstitial inflammation and muscle degeneration, regeneration, and vacuolization, in vector-injected animals. At the 3 × 10(11) vg dose, serum hAAT levels were higher with the HSV-produced vector than with the TFX-produced vector. With the higher dose of HSV-produced vector, the increase in serum hAAT levels was dose-proportional in females and greater than dose-proportional in males. Vector copy numbers in blood were highest 24 hr after dosing and declined thereafter, with no detectable copies present 90 days after dosing. Antibodies to hAAT were detected in almost all vector-treated animals, and antibodies to HSV were detected in most animals that received the highest vector dose. These results support continued development of rAAV-CB-hAAT for treatment of AAT deficiency.

Effects of LTB4 receptor antagonism on pulmonary inflammation in rodents and non-human primates.

Asthma, chronic obstructive pulmonary disease (COPD) and acute lung injury/acute respiratory distress syndrome (ALI/ARDS) are characterized by neutrophilic inflammation and elevated levels of leukotriene B4 (LTB4). However, the exact role of LTB4 pathways in mediating pulmonary neutrophilia and the potential therapeutic application of LTB4 receptor antagonists in these diseases remains controversial. Here we show that a novel dual BLT1 and BLT2 receptor antagonist, RO5101576, potently inhibited LTB4-evoked calcium mobilization in HL-60 cells and chemotaxis of human neutrophils. RO5101576 significantly attenuated LTB4-evoked pulmonary eosinophilia in guinea pigs. In non-human primates, RO5101576 inhibited allergen and ozone-evoked pulmonary neutrophilia, with comparable efficacy to budesonide (allergic responses). RO5101576 had no effects on LPS-evoked neutrophilia in guinea pigs and cigarette smoke-evoked neutrophilia in mice and rats. In toxicology studies RO5101576 was well-tolerated. Theses studies show differential effects of LTB4 receptor antagonism on neutrophil responses in vivo and suggest RO5101576 may represent a potential new treatment for pulmonary neutrophilia in asthma.

A review of whole animal bioassays of the carcinogenic potential of naphthalene.

This report provides a summary of deliberations conducted under the charge for members of Module A participating in the Naphthalene State-of-the-Science Symposium (NS3), Monterey, CA, October 9-12, 2006. Whole animal bioassays have been performed by the National Toxicology Program in mice and rats to ascertain the carcinogenic potential of naphthalene by inhalation exposure. A statistically significant increased incidence of pulmonary alveolar/bronchiolar adenoma (a benign lesion), was observed among female mice; an observed increase among the males did not reach statistical significance. No nasal tumors were observed in either sex. A tumorigenic response was observed in both sexes of rats, in males an increased incidence of nasal respiratory epithelium adenoma (a benign rather than malignant lesion) and in females, olfactory epithelial neuroblastoma. Interpretations of these studies vary. On the one hand, evidence of extensive non-neoplastic response in both sexes of both species indicates cytotoxicity occurred at all doses, and strongly suggests that cytotoxicity played a significant role in the tumor responses observed in the target tissues. On the other hand, olfactory epithelial neuroblastoma has rarely been observed in NTP bioassays. This review seeks to develop a consensus understanding of the scientific evidence provided by these studies, taking into account that they have been used as the basis for quantitative human cancer risk assessment, and suggests scientific studies that, if performed, could resolve scientific uncertainties.

Naphthalene metabolism in relation to target tissue anatomy, physiology, cytotoxicity and tumorigenic mechanism of action.

This report provides a summary of deliberations conducted under the charge for members of Module C Panel participating in the Naphthalene State-of-the-Science Symposium (NS(3)), Monterey, CA, October 9-12, 2006. The panel was charged with reviewing the current state of knowledge and uncertainty about naphthalene metabolism in relation to anatomy, physiology and cytotoxicity in tissues observed to have elevated tumor incidence in these rodent bioassays. Major conclusions reached concerning scientific claims of high confidence were that: (1) rat nasal tumor occurrence was greatly enhanced, if not enabled, by adjacent, histologically related focal cellular proliferation; (2) elevated incidence of mouse lung tumors occurred at a concentration (30 ppm) cytotoxic to the same lung region at which tumors occurred, but not at a lower and less cytotoxic concentration (tumorigenesis NOAEL=10 ppm); (3) naphthalene cytotoxicity requires metabolic activation (unmetabolized naphthalene is not a proximate cause of observed toxicity or tumors); (4) there are clear regional and species differences in naphthalene bioactivation; and (5) target tissue anatomy and physiology is sufficiently well understood for rodents, non-human primates and humans to parameterize species-specific physiologically based pharmacokinetic (PBPK) models for nasal and lung effects. Critical areas of uncertainty requiring resolution to enable improved human cancer risk assessment were considered to be that: (1) cytotoxic naphthalene metabolites, their modes of cytotoxic action, and detailed low-dose dose-response need to be clarified, including in primate and human tissues, and neonatal tissues; (2) mouse, rat, and monkey inhalation studies are needed to better define in vivo naphthalene uptake and metabolism in the upper respiratory tract; (3) in vivo validation studies are needed for a PBPK model for monkeys exposed to naphthalene by inhalation, coupled to cytotoxicity studies referred to above; and (4) in vivo studies are needed to validate a human PBPK model for naphthalene. To address these uncertainties, the Panel proposed specific research studies that should be feasible to complete relatively promptly. Concerning residual uncertainty far less easy to resolve, the Panel concluded that environmental, non-cytotoxic exposure levels of naphthalene do not induce tumors at rates that can be predicted meaningfully by simple linear extrapolation from those observed in rodents chronically exposed to far greater, cytotoxic naphthalene concentrations.

The toxicity of microcystin LR in mice following 7 days of inhalation exposure.

Microcystins, a family of cyclic heptapeptides produced by the cyanobacteria, Microcystis aeruginosa, have documented hepatotoxic and tumor promoting activities. The purpose of this study was to evaluate the toxicity of inhaled microcystin LR (microcystin). Male BALB/c mice were exposed by nose-only inhalation to 260-265 microg microcystin/m(3) for 7 days. The low-, mid- and high-dose groups were exposed for 0.5, 1, and 2h, respectively. Control animals were sham exposed to aerosolized vehicle. Treatment-related microscopic lesions were observed only in the nasal cavity of the mid- and high-dose groups. These lesions consisted of minimal to moderate multifocal degeneration and necrosis of the respiratory epithelium, with variable neutrophilic inflammation and minimal to marked degeneration, necrosis, and atrophy of the olfactory epithelium. The no-adverse-effect dose for the nasal lesions was approximately 3 microg/kg body weight, or 20 ng/cm(2) of nasal epithelium. In serum, only two protein peaks, occurring at m/zs of 11,688 and 11,829 Da, exhibited decreases in intensity that were microcystin dose-dependent. While these proteins have not been positively identified, they may be useful in the future as biomarkers of microcystin exposure in humans.

A biphasic response to silica: I. Immunostimulation is restricted to the early stage of silicosis in lewis rats.

Inhalation of crystalline silica may lead to acute or chronic silicosis. Although chronic silicosis is associated with increased incidence/exacerbation of autoimmune disorders, the immunologic effects of chronic silicosis are not completely understood. In an animal model of chronic silicosis, Lewis rats were exposed to filtered air or silica (1.75 microm average particle size) at an exposure concentration of 6.2 mg/m(3), 6 h/d, 5 d/wk for 6 wk, and observed up to 27 wk after the exposure. Based on silica burden, lung histopathology, and immunologic changes, two distinct stages were identified in the development of chronic silicosis. Stage 1 (4-28 d after exposure) was characterized by silica deposition in various tissues, and augmented antibody and cellular immunity. Although bronchoalveolar lavage contained an increased number of activated macrophages, protein and lactate dehydrogenase levels were comparable to controls. In Stage 2 (>/= 10 wk), silica was localized in epithelioid macrophages, and T cell immunity had returned to normal, but the lavage fluids contained increased protein concentration and lactate dehydrogenase activity. Moreover, lungs from silica-treated animals contained neutrophils and lymphocytes, and exhibited granulomatous changes around the silica-containing epithelioid macrophages. Thus, in the early stages of silicosis, silica activates the immune system; however, the progression of lung granulomas does not depend on a continually activated adaptive immune system.

The uptake, distribution, metabolism, and excretion of methyl tertiary-butyl ether inhaled alone and in combination with gasoline vapor.

The purpose of these studies was to evaluate the tissue uptake, distribution, metabolism, and excretion of methyl tertiary-butyl ether (MTBE) in rats and to determine the effects of coinhalation of the volatile fraction of unleaded gasoline on these parameters. Male F344 rats were exposed nose-only once for 4 h to 4, 40, or 400 ppm 14C-MTBE and to 20 and 200 ppm of the light fraction of unleaded gasoline (LFG) containing 4 and 40 ppm 14C-MTBE, respectively. To evaluate the effects of repeated inhalation of LFG on the fate of inhaled MTBE, rats were exposed for 7 consecutive days to 20 and 200 ppm LFG followed on d 8 by exposure to LFG containing 14C-MTBE. Three subgroups of rats were included for evaluation of respiratory parameters, rates and routes of excretion, and tissue distribution and elimination. MTBE and its chief metabolite, tertiary-butyl alcohol, were quantitated in blood and kidney (immediately after exposure), and the major urinary metabolites, 2-hydroxyisobutyric acid and 2-methyl-1,2- propanediol, were identified and quantified in urine. Inhalation of MTBE alone or as a component of LFG had no concentration-dependent effect on respiratory minute volume. The initial body burdens (IBBs) of MTBE equivalents achieved after 4 h of exposure to MTBE did not increase linearly with exposure concentration. MTBE equivalents rapidly distributed to all tissues examined, with the largest percentages distributed to liver. Between 40 and 400 ppm, there was a significant reduction in percentage of the IBB present in the major organs examined, both immediately and 72 h after exposure. At 400 ppm, the elimination rates of MTBE equivalents from tissues changed significantly. Furthermore, at 400 ppm there was a significant decrease in the elimination half-time of volatile organic compounds (VOCs) in breath and a significant increase in the percentage of the IBB of MTBE equivalents eliminated as VOCs in breath. LFG coexposure significantly decreased the percentage of the MTBE equivalent IBBs in tissues and increased rates of elimination of MTBE equivalents. The study results indicate that the uptake and fate of inhaled MTBE are altered upon increasing exposure levels from 4 to 400 ppm, suggesting that toxic effects observed previously upon repeated inhalation of concentrations of 400 ppm or greater may not necessarily be linearly extrapolated to effects that might occur at lower concentrations. Furthermore, coexposure to LFG, whether acute or repeated, decreases tissue burdens of MTBE equivalents and enhances the elimination rate of MTBE and its metabolites, thereby potentially reducing the toxic effects of the MTBE compared to when it is inhaled alone.