Tchounwou P B, Wilson B A, Ishaque A B, Schneider J
Molecular Toxicology Research Laboratory, NIH-Center for Environmental Health, School of Science and Technology, Jackson State University, 1400 Lynch Street, Box 18540, Jackson, Mississippi 39217, USA.
Environ Toxicol. 2001 Jun;16(3):209-16. doi: 10.1002/tox.1026.
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 (HepG2) by creating stable transfectants of mammalian promoter chloramphenicol acetyltransferase (CAT) gene fusions. Cytoxicity test with the parental HepG2 cells, using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]-based assay for cell viability, yielded LC50 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 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.
CAT-Tox (L) 检测法最近已被开发并验证，用于检测和量化各种外源化学物质毒性背后的特定分子机制。我们进行了该检测，以测量与2,4,6-三硝基甲苯（TNT）及其2种副产物[2,4-二硝基甲苯和2,6-二硝基甲苯（DNTs）]相关的转录反应，作用于通过创建哺乳动物启动子氯霉素乙酰转移酶（CAT）基因融合的稳定转染体从人肝癌细胞（HepG2）产生的13种不同重组细胞系。使用基于MTT [3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐]的细胞活力检测法对亲本HepG2细胞进行细胞毒性测试，在暴露48小时后，TNT在1%二甲基亚砜（DMSO）中的LC50值为105±6 mg/mL，DNTs的LC50值>300 mg/mL。TNT似乎比2,4-DNT毒性更大，而2,4-DNT与2,6-DNT相比也表现出更高的毒性。在所评估的13种重组构建体中，对于TNT、2,4-DNT和2,6-DNT，分别有8种（CYP 1A1、GST Ya、XRE、HMTIIA、c-fos、HSP70、GADD153和GADD45）、5种（c-fos、HSP70、GADD153、GADD45和GRP78）未表现出显著诱导水平（p<0.05）。对于大多数构建体，应激基因的诱导是浓度依赖性的。这些结果表明TNT和2,4-DNT有可能导致蛋白质损伤和/或蛋白质生物合成紊乱（HSP70和GRP78）、DNA序列或其螺旋结构改变（c-fos、GADD153、GADD45），以及TNT可能参与生物转化过程（CYP 1A1、GST Ya、XRE）和金属离子的毒代动力学（HMTIIA）。在测试浓度范围内（1% DMSO中0 - 300 mg TNT或DNT/mL），未发现NFKBRE、p53RE、CRE和RARE有显著诱导（p>0.05）。
