PMC

a Crocidolite-induced cytotoxicity of lung epithelial cells LA-4, dose response. Crocidolite was added at a final concentration of 5, 10, or 20 μg/cm². After 24 h of exposure, viability was evaluated as described in the Methods section. b LDH release by LA-4 cells after crocidolite exposure. Crocidolite was added at a final concentration of 20 μg/cm². After 24 h of exposure, cytotoxicity was evaluated by detecting LDH release. c 6-Keto PGF_1α production in LA-4 cells after crocidolite exposure. Crocidolite was added at a final concentration of 10 μg/cm². After 24 h of exposure, 6-keto PGF_1α release was measured as described in the Methods section. d 6-Keto PGF_1α production in LA-4 cells after crocidolite exposure in the presence of COX inhibitors. COX-1/COX-2 inhibitor indomethacin and specific COX-2 inhibitor indomethacin heptyl ester were added at a dose 10 times their respective IC₅₀ simultaneously with crocidolite (10 μg/cm²). After 24 h of exposure, 6-keto PGF_1α release was measured as described in the Methods section

The flow cytometric analysis of crocidolite fibers and MeT5A cells. (A) The cytogram of MeT5A cells without incubation with crocidolite. (B) The cytogram of crocidolite fibers alone revealed that most of these fibers displayed FSC values of less than 200. The amplification gain of the FSC photodiode detector was 8.50. (C) The cytogram without a threshold of forward scatter (FSC) included crocidolite fibers that had detached from the cell surface. To generate this cytogram, MeT5A cells at 70% confluence were incubated with 5 µg/cm² crocidolite for 2 h.

Differential MAP kinase activation is necessary for optimal TNF-α gene expression. (A) Blood monocytes were cultured in the presence or absence of SB203580 (SB) for 30 minutes before exposing to crocidolite asbestos for 3 hours. Total RNA was reverse transcribed to cDNA. Real-time PCR amplification was performed as described in Materials and Methods. Data are expressed as fold induction of TNF-α mRNA expression from control. For statistical analysis, * denotes a comparison of crocidolite asbestos to control (P < 0.0037), and ** denotes a comparison of crocidolite to crocidolite + SB203580 (P < 0.0046). (B) THP-1 cells were transiently transfected with the -600 TNF-α-CAT reporter plasmid and either an empty vector (shaded bars) or pcDNA-HA-ERK2 (K/A) (solid bars) expression vector. After 24 hours, cells were exposed to crocidolite asbestos for 24 hours. Whole cell lysates, which were normalized to protein, were incubated with 0.1 μCi of [¹⁴C] chloramphenicol and 1.0 mM acetyl coenzyme A for 2 hours at 37°C. Acetylated derivatives (CM3-AC and CM1-AC) were separated from nonacetylated chloramphenicol (CM) by ascending thin layer chromatography in chloroform/methanol (95:5) solvent. *P < 0.0175, **P < 0.0050. (C) THP-1 cells were transiently transfected with the −600 TNF-α-CAT reporter plasmid and either an empty vector (shaded bars) or the constitutive active pCMV-MEK1 (solid bars) expression vector. After 24 hours, cells were exposed to crocidolite asbestos for 24 hours, and the CAT assay was performed as described in B. *P < 0.012, **P < 0.038. In B and C, densitometry of three separate experiments was performed and is expressed graphically in arbitrary units. For statistical analysis, * denotes a comparison of control to crocidolite (vector); and ** denotes a comparison of crocidolite (vector) to (B) crocidolite (pcDNA-HA-ERK2 [K/A]) and (C) crocidolite (pCMV-MEK1). (D) THP-1 cells were transiently transfected with pTRE-luc and pTET-ATF and either an empty vector (solid bars) or the constitutive active MEK1 (shaded bars). After 24 hours, cells were exposed to crocidolite asbestos for 6 hours. Luciferase activity, which was normalized to Renilla luciferase, is expressed in real light units. For statistical comparisons, * denotes a comparison of vector to pCMV-MEK1 (P < 0.0031).

The cell-block sections of MeT5A cells with uptake of crocidolite fibers. Images were taken at ×1000 magnification (oil-immersion). (A, B, C and D) MeT5A cells at 70% confluence were incubated with 5 µg/cm² crocidolite for 2 h. (E and F) MeT5A cells without crocidolite. Compared with routine HE staining (A), crocidolite fibers can be identified more easily by Kernechtrot staining (B) in bright field observation. By observing the same area of the sample with Kernechtrot staining, crocidolite fibers contrasted better in dark field observation (C), but for identification and counting of intracellular fibers, dark field observation appeared not to have enough advantage even by using unstained sections (D), partly because MeT5Acells themselves without crocidolite fibers showed focal brightness, especially nearby plasma membrane both in the unstained section (E) and in the section with Kernechtrot staining (F).