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Transcriptional activation of stress genes and cytotoxicity in human liver carcinoma cells (HepG 2 ) exposed to 2,4,6‐trinitrotoluene, 2,4‐dinitrotoluene, and 2,6‐dinitrotoluene
Author(s) -
Tchounwou Paul B.,
Wilson Barbara A.,
Ishaque Ali B.,
Schneider John
Publication year - 2001
Publication title -
environmental toxicology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.813
H-Index - 77
eISSN - 1522-7278
pISSN - 1520-4081
DOI - 10.1002/tox.1026
Subject(s) - cytotoxicity , toxicity , microbiology and biotechnology , trinitrotoluene , viability assay , chemistry , recombinant dna , dimethyl sulfoxide , cell culture , mtt assay , dna damage , genotoxicity , gene , biochemistry , biology , in vitro , dna , genetics , organic chemistry , explosive material
The CAT‐Tox (L) assay has recently been developed and validated for detecting and quantifying the specific molecular mechanisms that underlie toxicity of various xenobotic chemicals. We performed this assay to measure the transcriptional responses associated with 2,4,6‐trinitrotoluene (TNT) and 2 of its byproducts [2,4 and 2,6–dinitotoluenes (DNTs)] to 13 different recombinant cell lines generated from human liver carcinoma cells (HepG 2 ) by creating stable transfectants of mammalian promoter chloramphenicol acetyltransferase (CAT) gene fusions. Cytoxicity test with the parental HepG 2 cells, using the MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide]‐based assay for cell viability, yielded LC 50 values of 105±6 μg/mL for TNT in 1% dimethyl sulfoxide (DMSO), and >300 m;g/mL for DNTs, upon 48 h of exposure. TNT appeared to be more toxic than 2,4‐DNT, which also showed a higher toxicity compared to 2,6‐DNT. Of the 13 recombinant constructs evaluated, 8 (CYP 1A1, GST Ya, XRE, HMTIIA, c‐fos , HSP70, GADD153, and GADD45), 5 ( c‐fos , HSP70, GADD153, GADD45, and GRP78), and none showed inductions to significant levels ( p <0.05), for TNT, 2,4‐DNT, and 2,6‐DNT, respectively. For most constructs, the induction of stress genes was concentration‐dependent. These results show the potential for TNT and 2,4‐DNT to cause protein damage and/or perturbations of protein biosynthesis (HSP70 and GRP78), alterations in DNA sequence or its helical structure ( c‐fos , GADD153, GADD45), and the potential involvement of TNT in the biotransformation process (CYP 1A1, GST Ya, XRE), and in the toxicokinetics of metal ions (HMTIIA). Within the range of concentrations tested (0–300 mg TNT or DNT/mL in 1% DMSO), no significant inductions ( p >0.05) of NFKBRE, p53RE, CRE, and RARE were found. © 2001 John Wiley & Sons, Inc. Environ Toxicol 16: 209–216, 2001