Reactivity of Haloketenes and Halothioketenes with Nucleobases:  Reactions in Vitro with DNA

Abstract

Ketenes are important and highly reactive intermediates. Thioketenes are formed by cysteine conjugate beta-lyase-dependent biotransformation of 1-halovinylcysteine S-conjugates which are metabolites of several halogenated olefins. Nucleic acid constituents react with haloketenes and halothioketenes in vitro. Thioketenes induce DNA strand breaks in incubations of 1,2-dichlorovinyl 2-nitrophenyl disulfide, a thioketene precursor, with pBr 322 plasmid DNA. After treatment of single-stranded or native calf thymus DNA with chlorothioketene generated by the hydrolysis of 1,2-dichlorovinyl 2-nitrophenyl disulfide, the formation of 3,N4-thioacetylcytosine could be demonstrated. N6-(Dichloroacetyl)adenine and N4-(dichloroacetyl)cytosine, however, adducts formed by dichloroketene in vitro, are labile to hydrolysis. Therefore, the binding of this compound to DNA constituents in intact DNA is difficult to demonstrate. Substitution of one chlorine atom by fluorine allowed us to use 19F NMR as a tool to demonstrate the formation of adducts by dihaloketenes in intact DNA. N6-(Chlorofluoroacetyl)adenine and N4-(chlorofluoroacetyl)cytosine were synthesized (yields 77%, 15%, respectively) as references and characterized by LC/MS, 1H, 13C, and 19F NMR, FT-IR, and elemental analysis. To demonstrate the ability of dihaloketenes to bind to DNA, poly-dA (1 mg) and calf thymus DNA (10 mg) were suspended in DMF and treated with different concentrations of chlorofluoroketene (50-200 micromol). Analysis of the polymeric DNA by 19F NMR showed one doublet at -137.2 ppm downfield from the reference (CFCl3). A doublet at -146.9 ppm, characteristic for chlorofluoroacetic acid, an expected product of DNA adduct hydrolysis, was not detected. These results demonstrate the formation of a stable adenine adduct by dihaloketenes in intact calf thymus DNA.

Occupational Trichloroethylene Exposure and Renal Carcinoma Risk: Evidence of Genetic Susceptibility by Reductive Metabolism Gene Variants

“…This mechanism is biologically plausible in humans as: 1) GSTT1 is the most active, highly expressed glutathione S-transferase in the kidney (40,41), 2) GST theta enzyme expression is directly related to GSTT1 genotype (42), 3) the GST theta enzyme metabolizes small halogenated compounds like TCE (40), 4) the renal CCBL1 enzyme is expressed primarily in the kidney and 5) GST conjugation is required prior to formation of mutagenic isomers in the kidney. The major isomer, S-(1,2,-dichlorovinyl)-L-cysteine is significantly more toxic than S-(2,2,-dichlorovinyl)-L-cysteine (12,13). The importance of these genetic polymorphisms in the general public with regard to activation of small halogenated compounds such as TCE could have public health importance since both alleles associated with increased risk in the presence of TCE exposure are not uncommon.…”

“…Toxicological studies in animal models suggest that TCE-associated kidney damage occurs only after bioactivation through the reductive metabolic pathway, that requires prior hepatic and renal glutathione S-transferase (GSH) conjugation and subsequent cleavage by renal cysteine conjugate β-lyase (CCBL1), to form cysteine S-conjugates; S-(1,2,dichlorovinyl-L-cytseine) and S-(1,2,2-trichlorovinyl L-cysteine) (9–12). These metabolites are highly reactive and have been shown experimentally to form DNA adducts, strand breaks, bacterial mutagenicity, and renal cell genotoxicity and cytotoxicity (11–13). Therefore, the second aim of this study was to evaluate the significance of the reductive pathway in human carcinogenicity, and whether common variation in genes involved in reductive metabolism would modify TCE-associated RCC risk.…”

Trichloroethylene: Mechanistic, epidemiologic and other supporting evidence of carcinogenic hazard

“…Second, DCVC can generate the reactive thiolate S -(1,2-dichlorovinyl)-thiol (DCVT) via action of cysteine conjugate β-lyase. DCVT spontaneously rearranges to form either chlorothioketene or chlorothionoacetyl chloride (Volkel & Dekant, 1998), both of which are chemically unstable and reactive and form covalent adducts with nucleic acids (Muller, et al, 1998), proteins (Hayden, et al, 1991), and phospholipids (Hayden, et al, 1992). Third, DCVC can yield a reactive S -(1,2-dichlorovinyl)-L-cysteine sulfoxide by flavin-containing monooxygenase.…”

Trichloroethylene biotransformation and its role in mutagenicity, carcinogenicity and target organ toxicity

“…DCVT spontaneously rearranges to form either chlorothioketene (CTK) or chlorothionoacetyl chloride (CTAC) [104, 105]. Both of these species are chemically unstable and reactive and are believed to be responsible for formation of covalent adducts derived from DCVC with nucleic acids [106], proteins [107], and phospholipids [108]. CCBL activity has been detected not only in the kidneys, but in liver and other tissues as well.…”