Transcriptional activation of stress genes and cytotoxicity in human livercarcinoma cells (HepG(2)) exposed to 2,4,6-trinitrotoluene, 2,4-dinitrotoluene, and 2,6-dinitrotoluene

The CAT-Tox (L) assay has recently been developed and validated for detecti ng and quantifying the specific molecular mechanisms that underlie toxicity of various xenobotic chemicals. We performed this assay to measure the tra nscriptional responses associated with 2,4,6-trinitrotoluene (TNT) and 2 of its byproducts [2,4 and 2,6-dinitotoluenes (DNTs)] to 13 different recombi nant cell lines generated from human liver carcinoma cells (HepG(2)) by cre ating stable transfectants of mammalian promoter chloramphenicol acetyltran sferase (CAT) gene fusions. Cytoxicity test with the parental HepG(2) cells , using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromi de]-based assay for cell viability, yielded LC,, values of 105 +/- 6 mg/mL for TNT in 1% dimethyl sulfoxide (DMSO), and > 300 mg/mL for DNTs, upon 48 h of exposure. TNT appeared to be more toxic than 2,4-DNT, which also showe d a higher toxicity compared to 2,6-DNT. Of the 13 recombinant constructs e valuated, 8 (CYP 1A1, GST Ya, XRE, HMTIIA, c-fos, HSP70, GADD153, and GADD4 5), 5 (c-fos, HSP70, GADD153, GADD45, and GRP78), and none showed induction s to significant levels (p < 0.05), for TNT, 2,4-DNT, and 2,6-DNT, respecti vely. For most constructs, the induction of stress genes was concentration- dependent. These results show the potential for TNT and 2,4-DNT to cause pr otein damage and/or perturbations of protein biosynthesis (HSP70 and GRP78) , alterations in DNA sequence or its helical structure (c-fos, GADD153, GAD D45), and the potential involvement of INT 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% DMS O), no significant inductions (p > 0.05) of NFK BRE, p53RE, CRE, and RARE w ere found. (C) 2001 by John Wiley & Sons, Inc.