Confirmed identities of proteins from a two-dimensional map of Syrian hamster embryo cells.

The Syrian hamster embryo (SHE) cell transformation assay is widely used to screen chemicals for carcinogenic potential. However, the biochemical mechanisms of transformation in SHE cells are incompletely understood relative to other rodent systems. Thus identification of proteins which change during transformation can provide clues to biochemical mechanisms. Previously, we published a map of SHE cell proteins based on comparisons to other maps. In this report we provide direct sequence analysis of numerous proteins which were previously identified solely by electrophoretic mobility. Protein sequencing verified original spot identifications and extended the range of identified proteins. The updated map will assist in evaluating biochemical mechanisms of morphological transformation in hamster cells.

Alpha-quartz-induced chemokine expression by rat lung epithelial cells: effects of in vivo and in vitro particle exposure.

Chemokines are chemotactic cytokines that can play a key role in leukocyte recruitment to sites of tissue injury or infection. Previous studies have demonstrated that exposure to alpha-quartz as well as other noxious particles increases chemokine gene expression in rat lung, although the cells responsible for chemokine expression and the mechanisms underlying this response have remained unclear. The present studies demonstrate that exposure of rats to alpha-quartz induced expression of mRNA for the chemokine macrophage-inflammatory protein (MIP)-2 in epithelial cells lining the terminal bronchioles and alveolar ducts as well as macrophages and alveolar type II cells in the more distal lung. Treatment of rats with an anti-MIP-2 antiserum before alpha-quartz exposure markedly attenuated neutrophilic infiltration of the lungs demonstrating an important role for MIP-2 in alpha-quartz-induced pulmonary inflammation. In vitro exposure of primary cultures of rat alveolar type II cells or the rat alveolar type II cell line RLE-6TN to tumor necrosis factor-alpha, endotoxin, or alpha-quartz increased mRNA for MIP-2 as well as the structurally and functionally similar chemokine cytokine-induced neutrophil chemoattractant but not the chemokine MIP-1 alpha. The alpha-quartz-induced increase in epithelial MIP-2 mRNA resulted, at least in part, from increased gene transcription and was associated with the release of active MIP-2 protein. Induction of RLE-6TN MIP-2 and cytokine-induced neutrophil chemoattractant mRNA expression was not unique to alpha-quartz, being also increased by crocidolite asbestus fibers but not by titanium dioxide or MMVF-10 glass fibers. These findings indicate that epithelial cells contribute to chemokine expression in rat lung after exposure to alpha-quartz and potentially other noxious particles and suggest that alpha-quartz-activated MIP-2 expression in vivo results, at least in part, from a direct action of the particles on the lung epithelium.

Optimization and validation of an ELISA to measure specific guinea pig IgG1 antibody as an alternative to the in vivo passive cutaneous anaphylaxis assay.

Assessment of the allergenic potency of enzymes involves the use of a guinea pig model in which specific IgG1 antibody titers are used as the endpoint. The in vivo passive cutaneous anaphylaxis (PCA) assay is used to measure specific IgG1 antibody. This report describes the development and validation of an enzyme-linked immunosorbent assay (ELISA) to measure guinea pig specific IgG1 antibody as an in vitro alternative to the PCA assay. Cross reactivity of various rabbit and mouse (monoclonal) anti-guinea pig IgG1 preparations were evaluated using purified IgG1 and IgG2 from serum of guinea pigs immunized with ovalbumin. The two subclasses of guinea pig IgG were purified by first using Protein A affinity chromatography, followed by anion exchange chromatography and fluid phase isoelectric focusing. Affinity-purified rabbit anti-guinea pig IgG1 was shown to have minimal cross reactivity toward IgG2, while providing a strong signal with IgG1. The ELISA was designed as an antigen capture system in which the following are added in sequence: (1) enzyme antigen (passively adsorbed to the plate), (2) diluted serum samples from guinea pigs immunized with enzyme, (3) affinity-purified rabbit anti-guinea pig IgG1, (4) alkaline phosphatase-conjugated donkey anti-rabbit IgG, and (5) p-nitrophenyl phosphate substrate. Three replicate ELISA and PCA analyses were conducted on sera samples of varying titers from guinea pigs immunized with either Alcalase (protease), BPN' (protease), and Termamyl (amylase) enzyme. The correlation coefficients (r2) between the ELISA and PCA assay for Alcalase, BPN', and Termamyl were 0.826, 0.945, and 0.755, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

A proposed screen for evaluating low-molecular-weight chemicals as potential respiratory allergens.

Allergic asthma can result when reactive low-molecular-weight chemicals (LMWCs) haptenate carrier proteins to form immunogenic conjugates, which then induce specific allergic antibodies. As part of an overall assessment process for evaluating the allergenic potential of LMWCs, an in vitro test for detecting the covalent derivatization of proteins by LMWCs was developed. In the assay, globulin-free serum albumins were incubated with increasing concentrations of a given LMWC and the mixtures separated via reversed-phase high-performance liquid chromatography (HPLC). Derivatization was monitored by shifts in the retention time of native versus modified protein. Retention time shifts were seen for most haptens when incubated with human serum albumin at a 50:1 (hapten:protein) starting molar ratio. Some haptens changed the retention time of the protein at a 5:1 initial ratio. Almost all chemicals that non-covalently bind to proteins did not change the protein retention time, even when incubated at 1500:1 molar ratios. The screen correctly identified 12/14 known human allergenic haptens and 23/24 non-allergenic LMWCs. It cannot detect sensitizers which must be metabolized into reactive haptens. This screen can be incorporated into an overall risk assessment approach for evaluating chemicals as respiratory allergens.

Induction of protein phosphorylation, protein synthesis, immediate-early-gene expression and cellular proliferation by intracellular pH modulation. Implications for the role of hydrogen ions in signal transduction.

In Syrian hamster embryo cells, intracellular acidification (but not alkalization) results in proliferation, immediate-early-gene expression and tyrosine phosphorylation. In addition, both intracellular acidification and alkalization result in serine/threonine phosphorylation and de novo protein synthesis of specific proteins. Calcium is not mobilized in response to either intracellular alkalization or acidification. Neither intracellular acidification nor alkalization altered the serum proliferative signal while intracellular alkalization (but not acidification) reduced the epidermal-growth-factor-induced proliferative signal, tyrosine phosphorylation and immediate-early-gene expression. Finally, intracellular acidification (but not alkalization) could induce immediate-early-gene expression in cells growing in the presence of serum, indicating that the pH signalling pathway is not down modulated by the serum signalling pathway. These results, while indirect, indicate that hydrogen ions may play an important role in mitogen-signal transduction in Syrian hamster embryo cells.

Macrophage inflammatory proteins 1 and 2: expression by rat alveolar macrophages, fibroblasts, and epithelial cells and in rat lung after mineral dust exposure.

Macrophage inflammatory proteins 1 alpha and 2 (MIP-1 alpha, MIP-2) are members of a growing family of cytokines thought to play a role in host defense. MIP-1 alpha and MIP-2 were previously identified in the mouse and shown to stimulate inflammatory cell recruitment. To better understand the potential role of MIP-1 alpha and MIP-2 in lung defense, we investigated the ability of rat lung cells to express mRNA for and/or secrete MIP-1 alpha and MIP-2 proteins in vitro and characterized expression of these cytokines in rat lung after in vivo exposure to silica (SiO2) or titanium dioxide (TiO2). In response to lipopolysaccharide, rat alveolar macrophages expressed increased levels of MIP-1 alpha and MIP-2 mRNA and secreted proteins (identified by N-terminal sequencing) homologous to mouse MIP-1 alpha and MIP-2. Rat alveolar macrophage MIP-1 alpha and MIP-2 mRNA expression was also increased by tumor necrosis factor-alpha (TNF) and adherence to plastic. Studies with a rat fibroblast and epithelial cell line demonstrated that MIP-2, but not MIP-1 alpha, expression can be detected in these cells after stimulation with TNF. Intratracheal instillation studies with SiO2 and TiO2 showed that inflammatory doses of these dusts increase MIP-1 alpha and MIP-2 mRNA expression in whole lung and that increased gene expression preceded the accumulation of inflammatory cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of rat alveolar macrophage fibronectin release in a cadmium chloride model of lung injury and fibrosis.

Rats were exposed to saline or cadmium chloride (CdCl2) at 25, 100, or 400 micrograms/kg body weight by intratracheal instillation. At 3, 7, 14, and 28 days after exposure five animals/treatment were euthanized, the lungs were lavaged, and bronchoalveolar lavage fluid (BALF) was analyzed for lactate dehydrogenase (LDH), total protein, N-acetylglucosamindase (NAG), and cell number, type, and viability. Lung hydroxyproline concentration was characterized as a marker of lung collagen. Alveolar macrophages (AM) obtained in BALF were cultured and the release of fibronectin and TNF was determined. Lung tissue was examined microscopically at 28 and 90 days after exposure. Exposure to CdCl2 resulted in lung injury and inflammation demonstrated by increases in BALF LDH, total protein, NAG, and inflammatory cells. AM TNF release was not significantly changed by CdCl2 treatment. All doses of CdCl2 stimulated AM fibronectin secretion, a response which persisted throughout the 28-day postexposure period examined. Pulmonary fibrosis was demonstrated biochemically and/or histologically (trichrome staining tissue) at all CdCl2 dose levels. The association of CdCl2-induced AM fibronectin release with lung fibrosis confirms and extends previous observations relating AM-derived fibronectin to the development of interstitial lung disease and provides further evidence that the persistent increase in AM fibronectin release represents an early indicator of fibrosis.

Phenotypic differences between Syrian hamster embryo cells cultured at pH 6.7 or 7.3.

We have investigated the molecular phenotypic differences between Syrian hamster embryo (SHE) cells cultured at pH 6.7 or 7.3. Multiple pH-sensitive phenotypic differences were noted including changes in cellular morphology, a unique charge differential in a major cellular protein, nine uniquely expressed proteins, two unique phosphoserine/threonine phosphoproteins, one unique phosphotyrosine phosphoprotein, and the pH dependent mRNA level of a gap junctional gene (connexin 43). These differences, combined with previously described pH-specific differences (differential transformation rates and gap junctional communication), illustrate that culturing SHE cells in media that differ by 0.6 pH units (0.3 units on either side of pH 7.0) can have a profound influence on the cellular phenotype.

Measurement of histamine in rat bronchoalveolar lavage fluid by high-performance cation-exchange chromatography coupled with post-column derivatization to o-phthaldialdehyde.

To better define involvement of mast cell derived mediators in the pulmonary response to fibrogenic dusts, a rapid and accurate method was required to analyze samples of bronchoalveolar lavage fluid for histamine. Samples of rat lung lavage were analyzed for histamine via high-performance cation-exchange chromatography coupled with post-column derivatization with o-phthaldialdehyde. The fluorescent derivative could be detected to ca. 1 ng/ml of lavage. Recoveries averaged 94.2% with an average relative standard deviation of +/- 5.3%. There were no correlations between amount or fibrogenicity of inhaled dust and subsequent release of histamine into lavage fluid.

Hyaline droplet nephropathy resulting from exposure to 3,5,5-trimethylhexanoyloxybenzene sulfonate.

Acute oral dosing of 3,5,5-trimethylhexanoyloxybenzene sulfonate (THBS) to adult male and female rats causes a male rat-specific nephrotoxicity manifested as exacerbation of hyaline droplet formation. This chemical is structurally distinct from the volatile hydrocarbons known to cause male rat-specific kidney lesions. Therefore, to classify THBS as a hyaline droplet-inducing agent, experiments were conducted to determine whether [14C]THBS equivalents bound to alpha 2 mu-globulin and caused the protein to accumulate in male rat kidney cortex. Two-dimensional gel electrophoretic separation of male rat kidney proteins indicated that alpha 2u-globulin levels in kidney increased 24 hr after a single oral dose of THBS (500 mg/kg). Furthermore, a sex-dependent retention THBS was noted as there was approximately 10 times more THBS equivalents in male rat kidney than in female rat kidney. Equilibrium dialysis experiments indicated that 40% of THBS equivalents bound reversibly to male rat kidney proteins, whereas no interaction between THBS and female rat kidney proteins was detected. Specific binding of THBS to alpha 2mu-globulin was determined by anion-exchange HPLC after which metabolites in the alpha 2u-globulin fraction were identified by gas chromatography with parallel radioactivity-mass spectrometry and mass spectrometry-matrix isolation Fourier-transform infrared analysis. Four metabolites of THBS were found in this protein fraction, and the major component (approximately 70%) was identified as the cis gamma-lactone of 3,5,5-trimethylhexanoic acid. Experiments were also conducted in mice to determine whether THBS bound to any mouse kidney proteins, particularly mouse urinary protein. The results indicated that there was no interaction between THBS and mouse urinary protein, a protein which shares significant homology with alpha 2u-globulin. These results indicate that THBS treatment exacerbates hyaline droplet formation in male rat kidneys by binding to alpha 2mu-globulin, thereby causing the protein to accumulate in the renal cortex. The interaction between THBS and alpha 2mu-globulin appears to be unique to this male rat-specific protein as THBS does not interact with a very similar protein found in mice.

Use of dye-labeled protein as spectrophotometric assay for protein precipitants such as tannin.

Bovine serum albumin has been covalently labeled with Remazol brilliant blue R to provide a substrate for a convenient spectrophotometric assay for protein precipitants. The blue protein is especially useful for measuring protein precipitation by vegetable tannins because its absorption maximum is at a wavelength where plant pigments exhibit minimum absorption. Blue BSA has been used to determine, by competition experiments, the relative affinity of various proteins for tannins. A procedure for purifying condensed tannin from commercially available quebracho extract is described.