Characterization of TCHQ-induced genotoxicity and mutagenesis using the pSP189 shuttle vector in mammalian cells

Abstract

a b s t r a c tTetrachlorohydroquinone (TCHQ) is a major toxic metabolite of the widely used wood preservative, pentachlorophenol (PCP), and it has also been implicated in PCP genotoxicity. However, the underlying mechanisms of genotoxicity and mutagenesis induced by TCHQ remain unclear. In this study, we examined the genotoxicity of TCHQ by using comet assays to detect DNA breakage and formation of TCHQ-DNA adducts. Then, we further verified the levels of mutagenesis by using the pSP189 shuttle vector in A549 human lung carcinoma cells. We demonstrated that TCHQ causes significant genotoxicity by inducing DNA breakage and forming DNA adducts. Additionally, DNA sequence analysis of the TCHQ-induced mutations revealed that 85.36% were single base substitutions, 9.76% were single base insertions, and 4.88% were large fragment deletions. More than 80% of the base substitutions occurred at G:C base pairs, and the mutations were G:C to C:G, G:C to T:A or G:C to A:T transversions and transitions. The most common types of mutations in A549 cells were G:C to A:T (37.14%) and A:T to C:G transitions (14.29%) and G:C to C:G (34.29%) and G:C to T:A (11.43%) transversions. We identified hotspots at nucleotides 129, 141, and 155 in the supF gene of plasmid pSP189. These mutation hotspots accounted for 63% of all single base substitutions. We conclude that TCHQ induces sequence-specific DNA mutations at high frequencies. Therefore, the safety of using this product would be carefully examined.

Chemical and Toxicological Characterization of Halobenzoquinones, an Emerging Class of Disinfection Byproducts

“…A recent study investigated the mutagenicity of tetrachlorohydroquinone (TCHQ) using the supF shuttle vector system. The results indicated that TCHQ is a potent mutagen, with the majority (above 85%) being single-base mutations . The Ames assay (or the bacterial reverse mutation assay) is a standard method used for detecting the mutagenic potential of testing compounds .…”

“…The results indicated that TCHQ is a potent mutagen, with the majority (above 85%) being single-base mutations. 64 The Ames assay (or the bacterial reverse mutation assay) is a standard method used for detecting the mutagenic potential of testing compounds. 65 On the basis of Ames tests, DCBQ, DCMBQ, TriCBQ, and DBBQ appear to be mutagenic (unpublished work).…”

Review of current and “omics” methods for assessing the toxicity (genotoxicity, teratogenicity and nephrotoxicity) of herbal medicines and mushrooms

“…Genotoxicity can be due to many physico-chemical agents that result in a wide variety of possible damages to the genetic material, ranging from various DNA adducts to single-and double-strand breakages, DNA-DNA and DNA-protein cross-links or even chromosomal breakage (Ogura et al, 2008;Cavalcanti et al, 2010;Wang et al, 2012). The major challenge in genotoxicity testing resides in developing methods that can reliably and sensibly detect either such a vast array of damages or a general cellular response to genotoxic insult.…”

Mechanistic Study on Oxidative DNA Damage and Modifications by Haloquinoid Carcinogenic Intermediates and Disinfection Byproducts

“…In addition, the pSP189 shuttle vector of A549 human lung carcinoma cells was also used to further confirm the level of mutagenesis. 74 TCHQ was found to induce significant genotoxicity through the breakage of DNA strand and formation of DNA adduct. As shown in Table 1, DNA damage measured in the comet assay showed that more DNA breakage was induced by increasing concentration (10−50 μM, 1 h) of TCHQ.…”

“…Therefore, the comet assay, which can detect the DNA breakage and the formation of TCHQ-DNA adducts, was used to measure TCHQ genotoxicity. In addition, the pSP189 shuttle vector of A549 human lung carcinoma cells was also used to further confirm the level of mutagenesis …”