In vitro & molecular toxicology最新文献

Contact sensitivity is a T-cell-mediated immune disease that can occur when low-molecular-weight chemicals penetrate the skin. In vivo topical application of chemical sensitizers results in morphological modification of Langerhans cells (LC). Moreover, within 18 h, LC increase their major histocompatibility complex (MHC) class II antigens expression and migrate to lymph nodes where they present the sensitizer to T lymphocytes. We wanted to determine if such an effect could also be observed in vitro. However, because of the high genetic diversity encountered in humans, assays were performed with dendritic cells (DC) obtained from a Balb/c mouse strain. The capacity of a strong sensitizer, DNBS (2,4-dinitrobenzene sulfonic acid), to modulate the phenotype of bone marrow-derived DC in vitro, was investigated. A specific and marked increase of MHC class II molecules expression was observed within 18 h. To eliminate the use of animals in sensitization studies, the XS52 DC line was tested at an immature stage. A 30-min contact with the strong sensitizers DNBS and oxazolone, or the moderate mercaptobenzothiazole, resulted in upregulation of MHC class II molecules expression, analyzed after 18-h incubation. This effect was not observed with irritants (dimethyl sulfoxide and sodium lauryl sulfate) nor with a neutral molecule (sodium chloride). These data suggested the possibility of developing an in vitro model for the identification of the sensitizing potential of chemicals, using a constant and non animal-consuming material.

The consumption of plants containing atractyloside, a diterpenoid glycoside, causes selective proximal tubule injury leading to renal failure and death in humans. The underlying mechanisms responsible for its toxicity are still not well understood. The present study was therefore carried out to determine the mechanism and the exact sequence of events that lead to molecular toxic injury. A comparative study using renal cortical slices, suspension of freshly isolated renal proximal tubular fragments and glomeruli of male Wistar rat was made. These in vitro systems were exposed to 100-1000 mM atractyloside for 2-3 h at 37 degrees C. Atractyloside caused a significant alteration in various toxicity parameters in a concentration- and time-dependent manner in renal cortical slices and proximal tubular fragments, but not in glomeruli. The earliest change following exposure to atractyloside (1000 microM) was a significant reduction of intracellular adenosine 5'-triphosphate (ATP) content occurring within 1 h in the tubules and 2 h in slices. The significant depletion of reduced glutathione (GSH) inhibitor of p-aminohippuric (acid) (PAH) uptake and gluconeogenesis occurred simultaneously following loss of cellular energy. These events were only limited to the renal cortical slices and proximal tubular fragments. Increased severity of cellular injury resulted in cytotoxicity with the significant increase in the leakage of alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) in proximal tubular fragments (occurring at 2 h) and renal cortical slices (occurring at 3 h). There were, however, no alterations in oxidized glutathione (GSSG) levels or in the ratio of GSH/GSSG. Only limited lipid peroxidation in proximal tubular fragments and glomeruli was observed at atractyloside concentrations of 500 microM and above. In all cases of toxicity, the glomeruli were unaffected. Pretreatment of slices or fragments with probenecid (1.0 mM) failed to completely abolish atractyloside toxicity. These data demonstrate dose- and time-dependent toxicity of atractyloside and clearly confirmed the proximal tubular fragments as the target tissue. Atractyloside exhibits a toxicity profile that indicates early alteration in mitochondrial function and consequently loss of cellular energy, followed by reduced metabolic function and transport processes and ultimately cell death. This appears to be the most likely mechanism by which atractyloside exerted its acute cytotoxicity. Renal cortical slices, which maintain proximal tubule and glomeruli in their anatomic relationship, responded similarly to atractyloside toxicity as the proximal tubular fragments, and might be suggested as the most suitable in vitro model system for studying the mechanisms of atractyloside toxicity as they are more likely to mirror changes seen in the whole organ.

In vitro effect of mercury (Hg2+), cadmium (Cd2+), and arsenic (As3+) on adenylate cyclase (AC) and phosphodiesterase (PDE) activity in relation to platelet aggregation (PA) was studied in rats. Cd(2+) significantly elevated cAMP (p < 0.005) in a dose-dependent (5, 10 and 20 pmoles) manner while Hg(2+) and As(3+) significantly reduced the cAMP level (p < 0.01 and p < 0.005, respectively). Our studies further reveal that Hg21 and As(3+) inhibit AC and stimulate PDE activity with a concomitant increase in the rate of PA. On the other hand, Cd(2+) stimulates AC and inhibits PDE activity with a decrease in the rate of PA. The present investigation suggests that cellular cAMP is a regulatory molecule in the event of PA and the disruption of its homeostasis is directly correlated to xenobiotic effects on PA. It is concluded that other than divalent heavy metal cations, As(3+) appears to be one of the most toxic xenobiotics to platelet function.

Chronic inhalation of hard metal (WC-Co) particles causes alveolitis and the eventual development of pulmonary fibrosis. The initial inflammatory response includes a change in the alveolar epithelial cell-capillary barrier, which has been shown to be regulated by the state of assembly and organization of the actin cytoskeletal network. The objective of this study was to evaluate the effect WC-Co particles have on F-actin organization of lung epithelial cells in an in vitro culture system. Rat lung epithelial (L2) cells were exposed to 5, 25, and 100 microg/mL of WC-Co particles, as well as the individual components (Co and WC) of the hard metal mixture particles for 24 h. The effect on F-actin organization was visualized by laser scanning confocal microscopy (LSCM) following Bodipy-Phallacidin staining. Minimal changes in the F-actin microfilaments of L2 cells were observed by LSCM after exposure to WC and WC-Co at 5 and 25 microg/mL, while at 100 microg/mL, there was a noticeable disruption in the uniform distribution of L2 cell F-actin microfilaments. After exposure to Co, a dose-dependent change in the F-actin organization of the L2 cells was observed. Little change in F-actin assembly was observed after treatment with 5 microg/mL of Co (the concentration equivalent to the 5% amount of Co commonly present in 100 microg/mL of the WC-Co sample mixture). However, at 100 microg/mL of Co, the microfilaments aggregated into homogeneous masses within the cells, and a significant loss in the organization of L2 F-actin was observed. These dramatic alterations in F-actin organization seen after exposure to the higher doses of Co were attributed to an increase in L2 cell injury as measured by lactate dehydrogenase and trypan blue exclusion. We conclude the pulmonary response evoked in the lung by inhalation of high levels of WC-Co particles is unlikely due to alterations in the F-actin microfilaments of lung-epithelial cells.

The ability of a variety of compounds to disrupt normal endocrine homeostasis, and potentially, the physiological and reproductive capacity of an organism, has gained worldwide attention in recent years. In an attempt to identify such compounds, and to examine the mechanism(s) by which they may exert their actions, we have constructed reporter plasmid vectors that contain the firefly luciferase gene under hormone-inducible control of estrogen-, androgen-, or retinoic acid-responsive DNA enhancer elements. Transient transfection of these vectors into appropriate receptor-containing cell lines revealed their ability to respond to their respective hormones with the induction of luciferase. Here, we describe development and optimization of a recombinant human ovarian carcinoma (BG-1) line, which has been stably transfected with the estrogen responsive luciferase reporter plasmid. The resulting recombinant cell line (BG1Luc4E(2)) responds to 17beta-estradiol at concentrations as low as 1 pM. The utility of BG1Luc4E(2) cells as a bioassay screening system for environmental estrogens was demonstrated by their response to known xenoestrogens, and also by the putative identification of two polychlorinated biphenyls (2,3',4, 4,'-tetrachlorobiphenyl and 2,2',3,5',6-pentachlorobiphenyl) as novel estrogenic chemicals. These cell bioassay systems have applications for rapid screening, identification, and characterization of endocrine disrupting chemicals.

Cultured murine macrophages (RAW 264.7) were used to evaluate the temporal relationships between cytotoxicity, phagocytosis, tumor necrosis factor-alpha (TNF-alpha), and nitric oxide (NO) production, and alterations in expression of stress proteins after exposure to cadmium oxide (CdO) or cadmium chloride (CdCl(2)), particulate and soluble forms of cadmium, respectively. Macrophages were exposed in vitro to CdO (25 or 50 microg) or CdCl(2) (30 or 40 microM) for 2 to 72 h. Cytotoxicity was not evident until 18 h when exposed to 30 microM CdCl(2) or 25 microg CdO, but occurred as early as 12 h after exposure to 40 microM CdCl(2) or 50 microg CdO. Relative to untreated controls, phagocytic activity decreased progressively from 2 to 24 h after exposure to both forms of cadmium. TNF-alpha levels increased to 2- to 3-fold after 4 h and remained elevated until 24 h after exposure to 25 and 50 microg CdO and 30 microM CdCl(2), but decreased by 18-24 h at 40 microM CdCl(2). CdCl(2) or CdO alone did not induce NO; however, both cadmium species reduced lipopolysaccharide (LPS)-stimulated NO production in a dose-dependent manner. Enhanced de novo synthesis of 70- and 90-kD heat shock, or stress, proteins was observed 2 to 8 h after exposure to both CdCl(2) and CdO; however, synthesis of these proteins returned to control levels by 24 h. Stress protein synthesis was enhanced by CdCl(2) or CdO prior to cytotoxicity, but coincided with a decrease in phagocytic capacity and an increase in TNF-a levels. The data suggest that cultured macrophages respond similarly in vitro to a particulate form and a soluble form of cadmium in a cell type that plays a pivotal role in inflammatory and immune responses.

Sulfur mustard provokes an acute inflammatory response in skin. To determine if keratinocytes regulate this response and whether three potential vesicant antagonists can counteract adverse changes, specimens of EpiDerm (MatTek Corp., Ashland, MA), a human skin model of differentiating keratinocytes, were exposed 2 h to humidified air with or without 2-chloroethyl ethyl sulfide (CEES, 1.72-1.73 mg/L/min) with or without 10 mM niacinamide, a poly (ADP-ribose) polymerase (PARP) inhibitor, 25 microM CGS9343B (calmodulin antagonist), or 8.4 mM leupeptin (cysteine protease inhibitor). After a 22-h incubation, levels of interleukin-1 alpha (IL-1alpha), its receptor antagonist (IL-1Ra), soluble type II receptor (sIL-1RII) and prostaglandin-E(2) (PGE(2)) were determined. Methylthiazole tetrazolium (MTT) viability tests and histological observations were also conducted. PGE(2) levels were abundant but unaffected by CEES regardless of antagonist presence. Total amounts (media plus lysate) of IL-1alpha, IL-1Ra, and sIL-1RII were reduced with CEES irrespective of antagonist. CEES promoted the release of IL-1Ra. Exposure of EpiDerm to CEES in the presence of the vesicant antagonists did not improve viability or counteract histological damage. We conclude CEES depresses total IL-1alpha and related cytokines, does not affect PGE(2) release, and adverse changes associated with CEES-exposed EpiDerm are not ameliorated by these particular antagonists. Dramatically increased (5- to 10-fold) release of IL-1Ra may provide a useful marker for cytotoxicity. The high level of IL-1Ra and increased release with injury suggest a primary function in down-regulating IL-1 inflammatory responses in skin.