Carcinogenicity assessment of the pan-caspase inhibitor, emricasan, in Tg.rasH2 mice.

Emricasan, formerly IDN-6556, is a small molecule currently being evaluated in clinical trials to reduce hepatic injury and liver fibrosis. Since emricasan is an irreversible pan-caspase inhibitor that potently inhibits caspase-mediated apoptosis and inflammation, its carcinogenic potential was evaluated in a humanized mouse model. Tg.rasH2 mice received LabDiet formulated with 0, 10, 25, and 75mg/kg/day of emricasan, for 26weeks. At terminal sacrifice, blood was collected for clinical pathology analysis and tissues were collected, processed, and evaluated microscopically. There were no treatment related deaths or overt signs of toxicity for the duration of the study. There was no evidence of a carcinogenic effect in the peripheral blood leukocyte counts. Liver microgranulomas, which are background lesions, were slightly increased, especially in males. Increases in the incidence of the activated germinal centers were seen in the spleens and mesenteric lymph nodes of males and females, and in the mandibular lymph nodes of male mice. Atrophy of ovaries and testicular degeneration were also seen in emricasan treated animals. Although several non-neoplastic lesions were observed, there was no evidence of emricasan-related tumor formation in any tissue. In addition, the non-neoplastic lesions were not considered pre-neoplastic. Thus, emricasan is not considered carcinogenic.

In vitro and ex vivo toxicological testing of sildenafil-loaded solid lipid nanoparticles.

The aim of this study was to investigate the potential cytotoxicity of solid lipid nanoparticles (SLN) loaded with sildenafil. The SLNs were tested as a new drug delivery system (DDS) for the inhalable treatment of pulmonary hypertension in human lungs. Solubility of sildenafil in SLN lipid matrix (30:70 phospholipid:triglyceride) was determined to 1% sildenafil base and 0.1% sildenafil citrate, respectively. Sildenafil-loaded SLN with particle size of approximately 180 nm and monomodal particle size distribution were successfully manufactured using a novel microchannel homogenization method and were stable up to three months. Sildenafil-loaded SLN were then used in in vitro and ex vivo models representing lung and heart tissue. For in vitro models, human alveolar epithelial cell line (A459) and mouse heart endothelium cell line (MHEC5-T) were used. For ex vivo models, rat precision cut lung slices (PCLS) and rat heart slices (PCHS) were used. All the models were treated with plain SLN and sildenafil-loaded SLN in a concentration range of 0-5000 µg/ml of lipid matrix. The toxicity was evaluated in vitro and ex vivo by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Median lethal dose 50% (LD50) values for A549 cells and PCLS were found to be in the range of 1200-1900 µg/ml while for MHEC5-T cells and precision cut heart slices values were found between 1500 and 2800 µg/ml. PCHS showed slightly higher LD50 values in comparison to PCLS. Considering the toxicological aspects, sildenafil-loaded SLN could have potential in the treatment of pulmonary hypertension via inhalation route.

"Sontaneous" neoplastic transformation in vitro: a form of foreign body (smooth surface) tumorigenesis.

Explants of subcutaneous connective tissue from adult BALB/c mice into plastic petri dishes were serially subcultured and tested for tumorigenicity in two ways: by the subcutaneous implantation of cells attached to plastic plates (1 by 5 by 10 millimeters), and by the subcutaneous injection of cells suspended in saline. Cells grown in vitro for 18 or more days before being implanted attached to a plastic plate (2.4 x 10(4) to 3.4 x 10(5) cells per plate) formed tumors after 24 to 79 weeks. The latent period before tumor appearance correlated inversely with the time spent by the cells in tissue culture. Cells inoculated in saline suspension (10 to 100 times the above number per plate) did not form tumors until after 84 days in vitro; plates alone did not induce tumor formation within more than 1 1/2 years of implantation. The tumors arising from the plate-attached cells were transplantable without plates and histologically appeared to be undifferentiated sarcomas. It is well established that smooth-surfaced foreign bodies, regardless of their chemical composition, will produce sarcomas when transplanted subcutaneously in rodents. We interpret our data, particularly the decrease in tumor latent period with time spent in tissue culture, as indicating that a smooth surface was acting as a carcinogen first in vitro (the surface of the tissue culture dish) and then in vivo (the surface of the plastic plate).

Specific depression of the antitumor cellular immune response with autologous tumor homogenate.

A momogenate of an SV40-transformed firbosarcoma of BALB/c mice (E4 tumor) injected i.p. into E4, tumor-immune syngeneic mice specifically depressed their cell-mediated immune responses to autologous tumor cells, as measured by a radioisotopic foot pad assay. The fraction of the tumor homogenate that brought about this depression was present in the high-speed supernatant and pellet of a 3 M KCl extract of the tumor. The specificity of the depression was shown in three ways: (a) the serum of E4 tumor-immune mice, but not of normal mice, given injections of E4 tumor homogenate 24 hr previously, suppressed antitumor immunity in vitro, as measured by the release of 51Cr from labeled E4 tumor cells incubated with spleen cells from tumor-immune animals; (b) the i.p. inoculation of E4 tumor homogenate did not alter the cellular immune response of tuberculin-sensitized mice to tuberculin; and (c) the i.p. injection of a homogenate of antigenically unrelated tumor did not depress the cellular immune response of E4 tumor-immune mice to E4 tumor cells.

Vasoformative sarcomas arising from BALB/3T3 cells attached to solid substrates.

The BALB/3T3 mouse embryo cell line, noted for its marked postconfluence inhibition of proliferation, anchorage dependence, and high serum requirement, and frequently studied as a prototype nontumorigenic "fibroblast" line that is compared with tumorigenic sublines transformed with various agents, produced tumors within 2 to 3 months when an average of 3 X 10(4) cells were implanted s.c. attached to 1- X 5- X 10-mm polycarbonate platelets. Plastic platelets alone produced no tumors after 1 year of observation. The tumors, as well as others arising from implants of BALB/3T3 cells attached to 3-mm glass beads, were given the histological diagnosis of "vasoformative saroma" because the tumor cells frequently formed vascular channels. The vasoformative pattern and the results of specific staining for reticulin and collagen support the likelihood that BALB/3T3 cells originated from endothelial cells rather than from fibroblasts. That the tumors were derived from BALB/3T3 cells and not host cells was proved when tumors arising in BALB/c X C57BL/6 F1 hybrids were shown to be transplantable to BALB/c but not to C57BL/6 mice. The cultured tumor cells showed loss of both postconfluence inhibition of proliferation and anchorage dependence. Evidence of the induction of endogenous oncornaviruses was obtained in only one of four tumors tested. These tumors also exhibited tumor-unique transplantation rejection antigens. We conclude that BALB/3T3 cells are preneoplastic and give rise to different spontaneously transformed clones bearing unique tumor rejection antigens when implanted in vivo attached to a solid substrate.

Discussion paper: specific paralysis of the antitumor cellular immune response produced by growing tumors studied with a radioisotope footpad assay.

The kinetics of the antitumor cellular immune response of mice with progressively growing syngeneic tumors were determined in vivo using a quantitative radioisotopic footpad assay. A close correlation was found between the size of the tumor and the degree of the cellular immune response. An initial phase of cellular immune stimulation was followed by specific suppression and subsequent immunologic paralysis as the tumor grew larger. This immune paralysis was attributed to increased tumor load since a homogenate of an SV40 transformed fibrosarcoma injected intraperitoneally into tumor-immune mice specifically depressed their cellular immune response. The fraction of the tumor homogenate that brought about this depression was present in the high speed supernatant and pellet of a 3M KCl extract of the tumor. The specificity of the depression was determined in vivo by the radioisotopic footpad assay and in vitro by a 51Cr cytolysis assay. Unwashed spleen cells harvested from mice bearing large tumors were unreactive in a local adoptive footpad assay. However, reactivity could be restored by repeatedly washing the spleen cells.

Calcification of the urinary bladder in the wild cotton rat (Sigmodon hispidus).

Calcification of the urinary bladder epithelium was observed in 19 of 30 and 18 of 30 wild cotton rats from control and petrochemical-contaminated sites, respectively. The rats in the two sites did not differ significantly in respect of serum calcium and phosphorus concentrations. The calcification was considered to be dystrophic in nature. An unidentified factor common to both control and petrochemical-contaminated sites was considered to be responsible for this syndrome.

Spontaneous altered hepatocellular foci (AHF) in captive aged male cotton rats (Sigmodon hispidus).

Spontaneous altered hepatocellular foci (AHF) of basophilic type were observed in adult male cotton rats. The histological features of these foci were similar to those observed in Fischer 344 rats. However, an immunohistochemical technique with antibody to glutathione S-transferase placental form (GST-P) failed to stain these foci. Also normal bile ducts were not immunoreactive for GST-P. The presence of a gall bladder and of non-GST-P immunoreactive liver foci and bile ducts suggests that these cotton rats are phylogenetically closer to mice than rats.

Physicochemical characterization of sildenafil-loaded solid lipid nanoparticle dispersions (SLN) for pulmonary application.

For the development of any colloidal system, thorough characterization is extremely essential. This article discusses the physicochemical characterization of sildenafil-loaded solid lipid nanoparticle dispersions (SLN) including stability analysis over 6 months time period for possible pulmonary administration for the treatment of pulmonary arterial hypertension (PAH). SLN consisting of phospholipid and triglycerides were manufactured using a novel microchannel homogenization method. These sildenafil-loaded SLN were then subjected to physicochemical characterization namely, particle size and distribution over shelf life, differential scanning calorimetry (DSC), wide angle X-ray diffraction (WAXD) and analysis of nebulization performance of these SLN by the means of next generation impactor (NGI). Additionally, the morphology of nebulized particles was assessed by transmission electron microscopy using negative staining technique. The solubility of sildenafil citrate and base in the lipid matrix was determined and was 0.1% w/w and 1% w/w, respectively. From the particle size measurements, it was observed that SLN without sildenafil demonstrated consistent particle sizes over 6 months. For the sildenafil-loaded SLN, increased particle sizes were found after manufacturing and further increased within weeks. From WAXD studies, after 6 months high intensity reflections corresponding to the stable ß modification were observed. From DSC results, the peak minimum temperatures increased upon storage, hinting at a transformation to the stable ß modification of triglycerides in the case of sildenafil-loaded SLN. Hence, it can be concluded that even small drug concentration influences particle size and stability.

Neoplasms produced from C3H/10T 1/2 cells attached to plastic plates; saturation density, anchorage dependence and serum requirement of in vitro lines correlated with growth aggressiveness in vivo.

The C3H/10T 1/2 embryo cell line, which is nontumorigenic when inoculated subcutaneously in saline suspension, produces tumors when implanted subcutaneously attached to 1 X 5 X 10 mm plastic plates. Under these in vivo conditions there is direct selection for "spontaneous" transformants that have undergone the specific cellular alterations required for neoplastic behavior. This is in contrast to the conventional situation where transformants are obtained in vitro and are only secondarily tested in vivo for neoplastic behavior. Early passages of cell lines from four different C3H/10T 1/2 tumors explanted back in culture were quantitatively examined for tumorigenicity and for alteration in the properties of density inhibition, anchorage dependence, serum requirement, and plasminogen activator production. A fairly consistent quantitative relationship was found between the degree of growth aggressiveness in vivo and the degree of expression of these phenotypic markers of the transformed state in vitro during early passages of the cell lines after tumor explantation.

Immunohistochemical and clinical evaluation of p53 in canine cutaneous mast cell tumors.

One hundred twenty-six cutaneous mast cell tumors obtained by excisional biopsy from 106 dogs were evaluated using immunohistochemical staining for the presence of p53 protein. A standard avidin-biotin immunohistochemical protocol was used incorporating a polyclonal antibody of rabbit origin (CM-1) as the primary antibody. Histopathologic grading of tumors was performed on hemotoxylin and eosin-stained samples. There was a significant difference in the percentage of cells staining positive for p53 for the histopathologic grades (P = 0.0005). Grade III tumors had a significantly greater p53 content than did grade I or II tumors (P < 0.05). Clinical data obtained retrospectively was available for 54 dogs. Tumor recurred in 19 of 54 (35.2%) dogs. Twenty-nine dogs died by the end of the study; 9 of 29 (31.0%) died of mast cell tumor disease. Histopathologic grade showed a significant negative association with survival time. Both clinical stage and histopathologic grade showed a significant negative association with time to recurrence. The percentage of cells staining positive for p53 did not significantly improve the forward analysis. Immunohistochemical detection of p53 did not appear useful in characterizing the clinical association between cutaneous mast cell tumor cellular features and survival time or time to tumor recurrence in dogs.

Fluorosis in a wild cotton rat (Sigmodon hispidus) population inhabiting a petrochemical waste site.

We have developed an in situ mammalian model for evaluating environmental contamination using wild cotton rats. In a series of experiments, 200 male cotton rats were captured during 4 collection periods (spring 1991 = 35; fall 1991 = 60; spring 1992 = 53; fall 1992 = 52). A total of 103 of these cotton rats were captured from control sites, and the remaining 97 were captured from an abandoned oil refinery. All sites were located in the vicinity of Cyril, Oklahoma. There were alterations in the incisors of cotton rats captured from the refinery site. Normal color of cotton rat incisors is deep yellow-orange, which is imparted by a pigment normally produced by ameloblasts. Grossly, the upper incisors of 37 of 97 rats and lower incisors of 54 of 97 rats were affected. The affected incisors were white, chalky, and thin with striations and erosions of the enamel. Microscopic examination revealed that there were dysplastic and necrotic changes in the ameloblasts. The bone fluoride levels were significantly higher in rats captured from the refinery as compared to the rats captured from the control sites.