Alan H. Rebar

Early damage indicators in the lung. III. Biochemical and cytological response of the lung to inhaled metal salts

The validity of using the enzymatic and cytologic profile of airway fluids to indicate lung damage was tested in animals exposed by inhalation to either a known toxic metallic salt (CdCl2) or a relatively innocuous salt (CrCl3). The enzymatic and cytologic response of the airways was compared to histopathological evaluation of lung damage. Syrian hamsters were exposed to an aerosol of CdCl2 (aerodynamic diameter = 1.7 μm, σg ⋍ 1.7) to achieve an initial lung burden (ILB) of 0.6 ± 0.3 and 4.4 ± 1.2 μg of CdCl2 or to an aerosol of CrCl3 (count median diameter = 1.2 μm, σg ⋍ 1.5) to achieve an ILB of 0.7 ± 0.2 or 20 ± 10 μg of CrCl3. Animals were sacrificed at 2 hr, 1, 7, and 21 days after exposure. A sample of airay fluid was obtained by bronchopulmonary lavage and examined for the enzymatic profile of the cell-free fraction and the cytological profile of the cell fraction. Lung tissue enzyme activities were also measured and histopathologic evaluations were made on lung tissue from exposed, but nonlavaged, animals. In the lavage fluid from animals exposed to CdCl2, the enzymatic and cytologic data demonstrated a dose-response pattern and the airway response preceded enzymatic changes in the lung tissue. Tissue morphological changes correlated well with the biochemical changes. The response of the lung to CrCl3 was minimal by both morphological and biochemical evaluations. Airway enzymatic and cytologic responses were shown to be potentially useful as indicators of lung damage in toxicological screening programs.

Early damage indicators in the lung: V. Biochemical and cytological response to NO2 inhalation☆

Abstract In an extension of earlier work on the usefulness of analysis of pulmonary lavage fluid as a probe to detect lung injury, we have examined lavage fluid from animals with a multifocal, terminal bronchiolitis induced by exposure to an oxidant gas. Syrian hamsters were exposed to concentrations of 0, 12, 17, and 22 ppm NO2 gas for 48 hr. Bronchopulmonary lavage fluids were profiled biochemically and cytologically to determine (1) the indicators of a multifocal, deep lung injury that could be detected in the lung washings and (2) the lowest level of this type of injury that could be detected by the lavage fluid screen. Lung homogenates were assayed for the enzymatic activities measured in lavage fluid and the lungs were evaluated histologically. Highest response for all parameters measured was at 2 days (end of the exposure) when the lavage fluid showed dose-dependent elevations in lactate dehydrogenase, alkaline phosphatase, acid phosphatase, glutathione reductase, and glutathione peroxidase activities, sialic acid, and total protein content, as well as increases in macrophage and neutrophil cell counts. By far the most sensitive indicator of this type of injury, as measured by the lavage fluid screen, was the neutrophil cell count, which showed a 10-fold increase even at the lowest level of exposure. The greatest change seen in the biochemical parameters measured in lavage fluid was the increase in sialic acid and protein content. There was good correlation between the degree of alteration of biochemical and cytologic indicators of injury seen in the lavage fluid and the morphological alterations seen in tissue.

Early damage indicators in the lungs. IV. Biochemical and cytologic response of the lung to lavage with metal salts

Abstract A rapid screening test for estimating the acute toxicity level of substances in the lung has been developed and evaluated using metal salts as the toxic agents. In the test, animals were exposed in vivo to the metal salts by bronchopulmonary lavage, and toxicity was determined by the enzymatic and cytologic response observed in the airways 1 day after exposure. The airway response was determined by analysis of bronchopulmonary lavage fluid for lactate dehydrogenase, acid and alkaline phosphatase, and β-glucuronidase activity as well as for total sialic acid and protein content and total and differential cell counts. These values showed a dose-dependent response in the lavage fluid. The enzymatic response of the lung tissue itself was not as marked as that seen in the lavage fluid. Histopathological evaluations of lung tissue were made in order to correlate morphological change with the airway response. The test allowed a rapid determination of the lung dose of metal salt which caused acute toxicity. Based on the biochemical response in the airway, the relative toxicity of the heavy-metal-containing compounds tested was: CdCl2>SeO2, NH4VO3, NiCl2>CrCl3.

Clinical Pathology Testing Recommendations for Nonclinical Toxicity and Safety Studies

Clinical pathology testing in nonclinical toxicity and safety studies is an important part of safety assessment. In recent years, clinical laboratory testing has rapidly expanded and improved. Some government regulatory agencies provide guidelines for clinical pathology testing in nonclinical toxicity and safety studies. To improve these testing guidelines and the resultant safety assessments, the American Association for Clinical Chemistrys Division of Animal Clinical Chemistry and the American Society for Veterinary Clinical Pathology formed a joint committee to provide expert recommendations for clinical pathology testing of laboratory species involved in subchronic and chronic nonclinical toxicity and safety studies. These recommendations include technical recommendations on blood collection techniques and hematology, serum chemistry, and urinalysis tests.

Microangiopathic Hemolytic Anemia Associated with Radiation- Induced Hemangiosarcomas

A retrospective study of red blood cell parameters in 53 dogs with experimental radiation-induced hemangiosarcoma showed 24 had anemia. Morphologic alterations in red blood cells in peripheral blood films from anemic dogs included signs of regeneration (anisocytosis and polychromasia), hypochromasia, red cell fragmentation and acanthocytosis. The degree and type of red cell changes varied from dog to dog and generally correlated with the principal site of tumor involvement. Blood from dogs with tumors principally involving liver had red cell regeneration, fragmentation and acanthocytosis. Blood from dogs with tumors primarily involving the heart had only red cell fragmentation. Blood films from dogs with skeletal and pulmonary hemangiosarcomas were similar to blood films from dogs with hepatic hemangiosarcoma except that red cell alterations generally were less severe. Scanning and transmission electron micrographic evaluation of neoplastic tissue showed large amounts of fibrin within neoplastic vascular sinuses and disruption and distortion of red blood cells traversing these abnormal vascular beds. The red blood cell fragmentation syndrome associated with radiation-induced hemangiosarcomas therefore was considered to be a microangiopathic hemolytic anemia of localized origin.

Comparative Study of Bronchoalveolar Lavage Fluid: Effect of Species, Age, and Method of Lavage

The analysis of bronchoalveolar lavage fluid has been used as a probe to detect lung injury in toxicological studies and to diagnose the disease state of the lung in humans. To determine how variable the content of lavage fluid from different species is, bronchoalveolar lavage fluids from normal individuals of four species (hamster, rat, guinea pig, and rabbit) were compared for enzymatic and cellular content as well as total protein and sialic acid. In addition, lavage fluid from young adult rats and hamsters was compared to that from older animals. Finally, the effect of the method of lavage on lavage fluid content was evaluated by comparing lavage fluid obtained from an excised lung with that from a lavage performed in vivo. In general, lavage fluids from the four species were similar. However, lavage fluid from guinea pigs had higher numbers of granulocytes and higher mean beta-glucuronidase activities than fluids from other species. Rats had higher mean alkaline phosphatase activities, reflecting higher serum values of this enzyme. Older hamsters had more protein in their lavage fluid than younger animals, and older rats had lower elastase inhibitory activity than young rats. Performing lavage in vivo, as compared to in vitro, did not greatly alter the lavage fluid except for a trend toward a higher level of sialic acid in fluid taken from the living animal.

Comparative Acute Toxicity of Four Nickel Compounds to F344 Rat Lung

Nickel subsulfide (Ni3S2), nickel chloride (NiCl2), nickel sulfate (NiSO4), and nickel oxide (NiO) are compounds of widely differing solubility encountered in the nickel-refining and electroplating industries. Inhalation is a common route of exposure and toxicity to the respiratory tract is possible. The purpose of this study was to evaluate the biochemical, cytological, and morphological changes in lung following administration of these compounds by intratracheal instillation. F344/Crl rats were administered a single dose of nickel compound containing 0.0, 0.01, 0.10, or 1.0 mumol Ni by intratracheal instillation. Rats were sacrificed at 1 or 7 days after compound administration, with half the animals in each exposure group taken for determination of nickel lung burden and the remaining half used for evaluation of biochemical, cytological, and histological changes. In the latter group, the right lung was lavaged and the fluid obtained was analyzed for indicators of pulmonary inflammation: lactate dehydrogenase (LDH), beta-glucuronidase (BG), total protein (TP), glutathione reductase (GR), glutathione peroxidase (GP), and sialic acid (SA). Total and differential cell counts on cells recovered in lavage fluid were also determined. The left lobe was examined for morphological changes. Clearance of nickel from the lung was most rapid for NiCl2 and NiSO4, followed by Ni3S2 and NiO. Minimal changes in all parameters were observed at 1 day after exposure. No significant changes in any parameter occurred in rats exposed to NiO, while Ni3S2, NiSO4, and NiCl2 caused increased in LDH, BG, TP, GR, SA, and total nucleated cells at 7 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Bone Marrow Alterations Associated with Canine Parvoviral Enteritis

of the glossal hemangiosarcomas also were discrete red masses, but were less than 0.5 cm in diameter. Although the larger neoplasms frequently were thinly encapsulated, the smaller lesions tended to be well-defined but unencapsulated. Microscopically the neoplasms in both the skin and tongue were similar to those commonly found in internal sites and were composed of plump, hyperchromatic spindle-shaped cells forming both irregular vascular sinusoids and solid sheets (fig. 1, 2). Mitotic figures were seen frequently. Focal areas of necrosis were found, especially in the larger neoplasms. All contained large amounts of blood either within neoplastic sinusoids or extravasated into the stroma of the neoplasm or adjacent dermis. In conclusion, hemangiosarcoma of the skin and tongue are associated with internal lesions in approximately one-third of the cases. The hemangiosarcomas reported here were first detected in the skin or tongue, or both, rather than in the internal organs. It is not known whether or not these represent primary or metastatic lesions. The frequent finding of a single skin lesion and the apparent predilection for the skin of the lower abdomen (70% of skin lesions), however, would seem to argue that this area may represent either a preferred site of primary cutaneous origin or alternatively a preferred site of concurrent origin with internal lesions rather than metastatic lesions. With cutaneous metastasis of internal primary hemangiosarcoma, one would more reasonably expect random, more widespread involvement of the skin.

Cyanide-induced neurotoxicity: calcium mediation of morphological changes in neuronal cells

Calcium channel blockade decreases the elevation of brain calcium as well as the tremors produced by cyanide in mice. To determine if cyanide-induced morphological changes could also be inhibited by calcium channel blockade, the effect of diltiazem was studied in cultured rat pheochromocytoma (PC12) cells, a neuronal model. Incubation with KCN (1 to 10 mM for 1 to 2 hr) caused depletion of secretory granules, alignment of remaining granules along the plasma membrane, and mitochondrial swelling. All these effects were inhibited by pretreatment with 0.01 mM diltiazem. Scanning electron microscopy revealed that cyanide (1 to 10 mM for 1 to 2 hr) produced loss of microvilli and bleb formation in PC12 cells. These changes were partially inhibited by preincubation with 0.01 mM diltiazem. Incubation of cells with 10 mM cyanide increased release of lactic dehydrogenase (LDH) into the culture media at 60 and 120 min. A decrease in cell viability, as determined by trypan blue dye exclusion, paralleled the release of LDH. At 120 min of cyanide incubation, 24% of the cells excluded dye. Both the release of LDH and decreased cell viability were attenuated by pretreatment with diltiazem. The results indicate that the influx of extracellular calcium is an important factor mediating cyanide-induced morphologic changes in neuronal cells.

Red Cell Fragmentation in the Dog: An Editorial Review

Red blood cell fragments in blood smears from dogs are described morphologically and pathogenetically. Categories include microangiopathic fragmentation, spherocytic fragmentation, Heinz body fragmentation, metabolic fragmentation associated with systemic disease, and artifactual fragmentation. Microangiopathic fragmentation is associated with direct physical damage to normal circulating red blood cells as they pass through abnormal capillary beds. Spherocytic fragmentation is a common feature of immune-mediated hemolytic anemia and results from the removal of portions of antibody-coated erythrocyte plasma membranes by phagocytes of the reticuloendothelial system. Heinz body fragmentation occurs when rigid particles of oxidized hemoglobin are torn from affected red cells as they circulate through the spleen. Metabolic fragmentation is an ill-defined syndrome most commonly associated with cholesterol loading of red cell membranes caused by lipid metabolism abnormalities. Resulting spiculated red cells are more susceptible to traumatic disruption. All the types of red cell fragmentation described in dogs have been observed and documented in man.