Mechanistic toxicogenomic analysis of WAY-144122 administration in Sprague-Dawley rats.

Application of global gene expression analysis in the study of mechanisms of toxicity could provide a more comprehensive interpretation of the molecular basis of drug action. WAY-144122 has pharmacological activity against several targets improving insulin responsiveness and favorably altering lipid profiles. Normal rats treated with suprapharmacological doses of WAY-144122 for 28 days exhibited drug-related effects in the liver and ovary. To determine the molecular mechanism underlying these effects, we employed global gene expression profiling to measure RNA levels in these target organs obtained from WAY-144122-treated rats administered test article for 1, 3, 7, and 14 days. Genes altered in expression by WAY-144122 were functionally categorized and related to their biological activity. In the liver, WAY-144122 caused a widespread up-regulation of genes involved in lipid mobilization, peroxisomal proliferation, and fatty acid beta-oxidation. In the ovary, we observed reduced expression of genes encoding luteinizing hormone receptor, follistatin, and enzymes in the estradiol synthesis pathway. Transcriptional changes in both organs precede histopathological effects. Profiling analysis allowed us to formulate hypotheses for molecular mechanisms underlying the physiological observations. In the liver, transcriptional changes suggest that WAY-144122 induced increased metabolic activity and peroxisomal proliferation resulting in increased liver weight and hepatocellular hypertrophy. We propose decreased estradiol synthesis as the underlying mechanism for the observed follicular atrophy in the ovary. Importantly, in this study, we have identified potential molecular mechanisms of drug effect in expression profiles before observation of physiological changes.

Efficacy and mechanisms of action of rmB7.2-Ig as an antitumor agent in combination with Adriamycin and Cytoxan chemotherapy.

The efficacy of chemotherapy for cancer is often limited by toxicity. Immune approaches to cancer immunotherapy, while promising for specificity and long-term protection, have not typically proven potent enough to generate significant therapeutic responses. We have shown therapeutic benefit using recombinant murine B7.2-Ig (rmB7.2-Ig) in murine tumor models. Efficacy was dependent on immune activity and was not associated with toxicity. Recently, the efficacy of rmB7.2-Ig was demonstrated in leukemia tumor models in combination with chemotherapeutic agents. To further explore the potential of this approach, we evaluated the efficacy in solid tumor models of rmB7.2-Ig given in combination with chemotherapeutics commonly used in clinical practice, testing the effects of dose and schedule. RmB7.2-Ig in combination with some chemotherapeutics enhances the activity and efficacy of reduced chemotherapeutic doses. However, the relative timing of chemotherapy and rmB7.2-Ig dosing can be important. Investigation of mechanisms of action based on histological studies suggests that inflammatory as well as T cell mechanisms comprise the response. Additional studies of mice deleted of B7.1, B7.2, and CTLA-4 suggest that the enhanced response induced by rmB7.2-Ig may not be mediated through CD28 ligation alone. The efficacy suggests potential for recombinant human B7.2-Ig as an adjuvant to chemotherapy in promoting immune-mediated mechanisms to augment the activity of chemotherapy.

Hepatic toxicity and recovery of Fischer 344 rats following exposure to 2-aminoanthracene by intraperitoneal injection.

Humans may be exposed to 2-aminoanthracene (2-AA), a substituted polycyclic aromatic hydrocarbon, and a recognized mutagen and carcinogen, through oral and respiratory routes from contact with a variety of environmental sources. For the present study, we sought to evaluate hepatic damage and recovery in Fischer 344 rats following multiple i.p. injections of 5 mg of 2-AA. Rats were injected weekly for up to 5 weeks. Subgroups were then allowed to recover for 1, 5, or 9 weeks, and biochemical and pathologic changes were evaluated. We observed that weight gains were reduced relative to controls for all groups receiving > or = 2 injections. Serum enzyme levels indicative of liver damage were evident and included alterations in serum aspartate aminotransferase, alkaline phosphatase, total protein, albumin, and globulin. These alterations usually returned to normal by 5 weeks following cessation of 2-AA administration. In contrast, histologic liver changes, including hepatocyte hypertrophy, biliary hyperplasia with oval cell proliferation, altered foci, nodular hyperplasia, and one hepatocellular adenoma became more severe with time. This experiment demonstrates patterns of hepatic damage and recovery in rats exposed to 2-AA.

Establishment and validation of an isolated rat lung model for pulmonary metabolism studies.

An isolated rat lung model was established and validated for use in pulmonary metabolism studies. During the establishment phase of the study, several problems were encountered and overcome in order to maintain the lungs in physiological condition. In the validation phase of the study, the lungs were removed, ventilated and perfused from 34 male Fischer 344 rats. After an equilibration period, lungs were ventilated and perfused for up to 4 h. Morphological, biochemical and functional parameters were evaluated to validate the physiological condition of the lungs. Morphological parameters included wet/dry lung weight ratios and gross and histological scoring for edema. Biochemical parameters included assays for tissue ATP and reduced glutathione content, glutathione reductase activity and glucose utilization. Functional parameters included changes in lung tidal volume, dynamic compliance and airway resistance. Results indicated that edema formation was only detected histologically, that lungs remained nearly biochemically normal for 210 min and that pulmonary function declined to about 80-90% of normal. Overall, these findings indicated that the isolated, perfused rat lung remained in acceptable physiological condition for ca. 210 min. This period of time should be adequate for conducting pulmonary metabolism studies with a variety of exogenous compounds.

Resorbable calcium phosphate bone substitute.

The in vitro and in vivo properties of a novel, fully resorbable, apatitic calcium phosphate bone substitute (ABS) are described. The ABS was prepared from calcium phosphate precursors that were hydrated to form an injectable paste that hardens endothermically at 37 degrees C to form a poorly crystalline apatitic calcium phosphate (PCA). The PCA reaction product is stable in vivo as determined by FTIR and XRD analysis of rabbit intramuscular implants of ABS retrieved 4, 7, and 14 days postimplantation. Bone formation and resorption characteristics of the ABS material were characterized in a canine femoral slot defect model. Femoral slot defects in dogs were filled with either autologous bone implants or the ABS material. Sections of femoral bone defect site from animals sacrificed at 3, 4, 12, 26, and 52 weeks demonstrated that new bone formation proceeded similarly in both autograft and ABS filled slots. Defects receiving either material were filled with trabecular bone in the first 3 to 4 weeks after implantation; lamellar or cortical bone formation was well established by week 12. New bone formation in ABS filled defects followed a time course comparable to autologous bone graft filled defects. Histomorphometric evaluation of ABS resorption and new bone formation indicated that the ABS material was greater than 99% resorbed within 26 weeks; residual ABS occupied 0.36+/-0.36% (SEM, n = 4) of the original defect area at 26 weeks. Quantitatively and qualitatively, the autograft and ABS were associated with similar new bone growth and defect filling characteristics.

In vitro release of colchicine using poly(phosphazenes): the development of delivery systems for musculoskeletal use.

A colchicine release system utilizing biodegradable poly(phosphazenes) was investigated in vitro for intra-articular administration. Polymer degradation and drug release studies were performed on colchicine-loaded poly(phosphazenes) containing either imidazolyl (I-PPHOS) or ethyl glycinato (EG-PPHOS) side chain substituents over a 21-day period. To study the effects of an implantable colchicine-PPHOS delivery system on local musculoskeletal tissue in vitro, osteoblast-like cells were grown on the matrices. Colchicine release was 20% for I-PPHOS and 60% for EG-PPHOS over the 21-day period. Release appeared to proceed through a combination of diffusional and degradative mechanisms. Environmental scanning electron microscopy (ESEM) studies revealed large pores in the drug-depleted devices in contrast to the control matrices without drug, which may have contributed to the release seen, especially with ethyl glycinato-containing matrices. Cell growth on matrices containing colchicine was significantly (p < 0.05) inhibited in contrast to growth on tissue culture polystyrene (TCPS) and EG-PPHOS matrices without drug. The in vitro cell kinetic data suggest that designs for in vivo studies must take into account possible toxicity of colchicine and the polymer matrix on local tissue. Biodegradable PPHOS systems are promising candidates for use as intra-articular delivery vehicles for drugs with potential for systemic toxicity.

Tissue specific toxicities of the anticancer drug 6-thioguanine is dependent on the Hprt status in transgenic mice.

6-Thioguanine (6TG) a cytostatic antimetabolite is currently used to treat patients with cancer, in particular leukemias. However, one drawback of such use is the development of 6TG resistance. Hypoxanthine-guanine phosphoribosyl transferase (Hprt) plays a crucial role in the bioactivation of 6TG. Loss of Hprt has been associated with the resistance of leukemias to 6TG chemotherapy, however, nothing has been known about the effect of Hprt status on tissue specific toxicity of 6TG in vivo. We determined the effect of Hprt status on the tissue-specific toxicity of 6TG in vivo in transgenic Hprt-deficient mice. The approximate lethal dose for Hprt-deficient mice was 23-fold higher than for the wild-type. Serum biochemical analyses of 6TG-treated wild-type mice showed elevated serum enzyme levels characteristic of liver damage whereas the levels in Hprt-deficient 6TG-treated mice were within normal physiological limits. Histopathological examination of tissues from wild-type and from Hprt-deficient mice showed contrasting spectrums of microscopic lesions. Wild-type mice had loss of hematopoietic cells from bone marrow starting at the lowest dose of 25 mg/kg 6TG whereas Hprt-deficient mice had normal bone marrow and spleen even at doses of 720 mg/kg 6TG. Wild-type mice also experienced severe loss of epithelial cells from the gastrointestinal tract starting at 50 mg/kg; however, the gastrointestinal tract of Hprt -/- mice remained unaffected. Wild-type livers revealed atrophy and necrosis at doses of 25 mg/kg 6TG although Hprt -/- livers displayed no effect until 507 mg/kg. In this study we show that Hprt-deficient mice had 6TG-resistant bone marrow and there are several other factors contributing to 6TG resistance in patients. Because variations among people exist in terms of their 6TG sensitivity, determining 6TG sensitivity of lymphocytes prior to 6TG chemotherapy and restricting treatment to 6TG-sensitive patients may improve the efficacy.

Expression of hygR in transgenic mice causes resistance to toxic effects of hygromycin B in vivo.

Aminoglycoside antibiotics are indispensable for treatment of serious bacterial infections, and despite careful attention to dosage regimens, nephrotoxicity and ototoxicity still cause concern. In the present study, we tested whether side effects of aminoglycoside therapy could be limited by expression of prokaryotic genes of antibiotic resistance in vivo. We characterized the acute and tissue-specific toxicity of hygromycin B in transgenic mice bearing the hygromycin B phosphotransferase (hygR) gene under control of a constitutive promoter. We characterized the tissue-specific expression of hygR mRNA and also investigated the acute toxicity of hygromycin B in hygR and wild-type mice. The hygR mRNA reached its highest levels in brain and reached intermediate levels in spleen, muscle, kidney, liver and testis. The lowest levels were detected in heart and lungs. The hygR expression in transgenic animals caused an 89-fold increase in the approximate lethal dose of hygromycin B compared with wild-type mice. Serum biochemical analysis of hygR and wild-type mice treated with lethal doses of hygromycin B indicated liver and kidney damage measured as ALT, AST and BUN. On the morphological level, these changes led to acute tubular nephrosis in wild-type mice and acute liver damage in hygR mice. Our results show that constitutive expression of the bacterial hygR gene in transgenic mice in vivo confers resistance to hygromycin B.

An experimental model of acute and subacute viral myocarditis in the pig.

Twenty-six young pigs were infected with encephalomyocarditis virus, observed clinically, studied at intervals by noninvasive and invasive methods to assess cardiac function and eventually examined pathologically. All infected animals appeared ill, usually manifesting diminished appetite, lethargy and fever. Spontaneous mortality occurred either 1 to 4 or 20 to 21 days after infection. Electrocardiographic abnormalities, seen in the majority of animals, comprised ST-T wave changes, conduction disturbances or ventricular ectopic rhythm. The majority of animals manifested echocardiographic evidence of left ventricular dilation and decreased systolic function, which improved with time in some animals. Hemodynamic studies revealed elevation of biventricular filling pressures in 3 of 10 animals; as a group, infected animals manifested significantly elevated right ventricular filling pressures. In selected animals, the feasibility of gallium scans as well as left ventriculography and coronary angiography was demonstrated. At autopsy, heart weight/body weight ratio was significantly elevated in infected animals. The heart of all but two animals showed active myocarditis associated with fibrosis and focal calcification in the later stages. In general, the cardiovascular manifestations were parallel with those seen in acute and subacute myocarditis in humans. It is concluded that encephalomyocarditis infection in the pig is a large animal model of viral myocarditis suitable for assessing alterations in the structure and function of the cardiovascular system and the effects of interventions.

Methyl prednisolone acetate modulation of infection and subsequent pulmonary pathology in hamsters exposed to parainfluenza-1 virus (Sendai).

Parainfluenza-1 virus (PI-1) has a wide host range; the disease process observed varies with the age, previous exposure, and species. This study was performed to determine possible effects of the corticosteroid methyl prednisolone acetate (MPA) on PI-1 infection in hamsters. Hamsters serologically negative for PI-1 were exposed to virus alone or were exposed to virus the day after pretreatment with a single subcutaneous injection of MPA. Serum antibodies to PI-1 were present in virus-only exposed hamsters by Day 8 and increased up to Day 20. PI-1 was recovered from lungs of virus-only exposed hamsters on Day 2 to 8. Virus antigen was detected by immunocytochemistry on Days 2 to 10 in lungs of virus-only exposed hamsters. Virus-associated lesions in these hamsters began as acute bronchiolar epithelium degeneration and necrosis on Day 4 and were foci of fibrosis by Days 12 to 20. Hamsters exposed to virus after MPA treatment developed no antibodies to virus, had no virus detectable by plaque assays or immunocytochemistry, and had no pulmonary lesions. Hamsters treated with MPA had decreased total lymphocyte counts up to Day 20 after treatment. Treatment of hamsters with MPA one day prior to PI-1 virus exposure is associated with no detectable evidence of viral infection. Humoral and cellular immunity mediated by MPA-sensitive lymphocytes may mediate some of the manifestations of PI-1 pulmonary disease.

Idiopathic cardiomyopathy in C3Hf/Bd mice.

Male and female 16 to 18 month old C3Hf/Bd mice in a dermal carcinogenicity study were moribund or died at earlier time points than the expected 24 to 30 months. Clinical signs observed in both treated and control animals included dyspnea, lethargy, and death. Lesions seen in treated as well as control mice were cardiomegaly with myocardial degeneration and necrosis, hydrothorax and pulmonary edema, and ascites and chronic passive congestion of the liver. Mice were negative for serologic, bacteriologic and microscopic evidence of viruses, bacteria and protozoa which can induce heart lesions. Possible causes of the cardiomyopathy include metabolic, degenerative, genetic or undetermined infectious disease.

Effect of ibuprofen on the pulmonary and systemic response to repeated doses of endotoxin.

We infused 10 doses of Escherichia coli endotoxin, 1 microgram/kg, during a 5-day period, into eight unanesthetized sheep with lung and systemic lymph fistulas. The animals were then monitored for an additional 5 days. We noted an attenuation of the lung microvascular permeability changes with the later endotoxin doses. However, a 50% increase in cardiac index and oxygen consumption and a leukocytosis were seen beginning with the ninth endotoxin injection; these persisted throughout the 15-day postendotoxin period, as did an increase in pulmonary artery pressure. The hyperdynamic state was present when plasma prostanoids were only modestly increased, and there was no evidence of increased lung or systemic vascular permeability. Postmortem lung findings, 5 days after endotoxin administration, showed a marked interstitial inflammatory response, with infiltration of macrophages, neutrophils, and some lymphocytes and an increase in interstitial fibrous tissue. Six sheep were then given ibuprofen, 12.5 mg/kg, intravenously before the ninth and tenth doses and on the subsequent day. Ibuprofen significantly attenuated the hyperdynamic state and the pulmonary hypertension. In addition, the lung inflammation and fibrous tissue deposition was markedly attenuated. We conclude that a systemic hyperdynamic state develops that corresponds in time with lung inflammation but not with increased permeability. The lung and systemic changes may be blocked by ibuprofen. The ibuprofen effect may be due to a response other than prostanoid production.

Serum cortisol radioimmunoassay values in the normal ferret and response to ACTH stimulation and dexamethasone suppression tests.

Cortisol values were obtained from 39 ferrets, Mustela putorius furo, by using a commercial radioimmunoassay. Sera from 25 males (18 intact, 7 neutered) and 14 females (7 intact, 7 spayed) were assayed. Resting serum cortisol values ranged from 0.13 to 2.70 micrograms/dl for males (mean = 0.93 +/- 0.63 micrograms/dl), and 0.55 to 1.84 micrograms/dl for females (mean = 0.86 +/- 0.29 microgram/dl). The resting cortisol values of both males and females were comparable to those of the cat (1.0 to 3.0 micrograms/dl). A 7 year old male ferret with suspected hyperadrenocorticism and an adrenal mass had a cortisol level of 8.1 micrograms/dl. Adrenal cortical carcinoma was the histologic diagnosis. One adult female ferret had a cortisol level of 3.30 micrograms/dl. This animal also had proliferative colitis upon necropsy. An ACTH stimulation test (1 U/kg IM) and a low-dose dexamethasone suppression test (0.1 mg/kg) were performed on 10 ferrets. Post-ACTH serum cortisol levels increased by an average of 89%. Post-dexamethasone serum cortisol values decreased by an average of 18% 6 hours post-injection.

Serum thyroxine and triiodothyronine radioimmunoassay values in the normal ferret.

The European ferret, Mustela putorius furo, has become increasingly popular as an animal model in biomedical research. However, certain important normal clinical data have not been established for the ferret. In this study, serum thyroxine (T4) and 3,3',5-triiodothyronine (T3) values were obtained from ferrets by the use of commercial radioimmunoassays. Sera from 44 animals, 31 males (27 intact and 4 castrated) and 13 females (10 intact and 3 spayed) were assayed. Serum T4 values ranged from 1.01-8.29 micrograms/dl for males (mean = 3.24 +/- 1.65 micrograms/dl), and 0.71-3.43 micrograms/dl for females (mean = 1.87 +/- 0.79 micrograms/dl). Serum T4 values of adult female ferrets, juvenile ferrets (less than 1 year old) of either sex, and castrated males were similar to the normal T4 values of the cat, 1.20-3.80 micrograms/dl. Intact adult male ferrets had higher serum T4 values which were more comparable to those of the normal dog 1.52-3.60 micrograms/dl. Serum T3 values ranged from 0.45-0.78 ng/ml for males (mean = 0.58 +/- 0.09 ng/ml), and 0.29-0.73 ng/ml for females (mean = 0.53 +/- 0.13 ng/ml). These values are comparable to those of dogs and cats which are 0.50-1.50 ng/ml.

Tissue inflammation without bacteria produces increased oxygen consumption and distant organ lipid peroxidation.

An inflammatory focus was produced by implantation of gauze below the hide in the flanks of six sheep with flank lymph fistulas. Physiologic and metabolic parameters were monitored in the unanesthetized animals for 7 days, after which the gauze was removed and monitoring continued for another 5 days. Animals were then killed. Lung and liver tissue was inspected and analyzed for lipid peroxide content. Data were compared with those of six controls in which gauze was not implanted. We noted a transient significant increase (on day 1 only) in wound lymph, thromboxane B2, and 6-keto-PGF1 alpha from baseline values of 190 +/- 70 and 20 +/- 10 pg/ml to 1000 +/- 240 and 420 +/- 70 pg/ml, respectively. Plasma values were also significantly increased on day 1. Body temperature increased by 1 degree C and cardiac index increased by 30% during this period, whereas oxygen consumption, VO2, was not significantly increased. The VO2 and cardiac index increased by 50% over baseline, beginning on day 5, whereas systemic vascular resistance decreased. Body temperature was not increased. These changes corresponded with an increase in wound lymph monocyte count from 0% to 15% of total. The VO2 and cardiac index remained increased after gauze removal. No bacteria were found in the wound. Postmortem analysis revealed a marked monocyte-macrophage infiltration in both lung and liver. Lung water, represented as water content over dry weight, was normal. Lung and liver lipid peroxidation, measured by the by-product malondialdehyde content, increased 300% and 90% over control values, respectively. We conclude that a local, nonbacteria-induced wound inflammation increases VO2, with the increase not corresponding to increase in body temperature. Distant organ changes, namely, changes in lung and liver, were also evident 5 days after gauze removal.