Mutagenicity studies of methyl-tert-butylether using the Ames tester strain TA102.

Methyl-tert-butylether (MTBE) is an oxygenate widely used in the United States as a motor vehicle fuel additive to reduce emissions and as an octane booster [National Research Council, Toxicological and Performance Aspects of Oxygenated Motor Vehicle Fules, National Academy Press, Washington, DC, 1996]. But it is the potential for MTBE to enter drinking water supplies that has become an area of public concern. MTBE has been shown to induce liver and kidney tumors in rodents but the biochemical process leading to carcinogenesis is unknown. MTBE was previously shown to be non-mutagenic in the standard Ames plate incorporation test with tester strains that detect frame shift (TA98) and point mutations (TA100) and in a suspension assay using TA104, a strain that detects oxidative damage, suggesting a non-genotoxic mechanism accounts for its carcinogenic potential. These strains are deficient in excision repair due to deletion of the uvrB gene. We hypothesized that the carcinogenic activity of MTBE may be dependent upon a functional excision repair system that attempts to remove alkyl adducts and/or oxidative base damage caused by direct interaction of MTBE with DNA or by its metabolites, formaldehyde and tert-butyl alcohol (TBA), established carcinogens that are mutagenic in some Ames strains. To test our hypothesis, the genotoxicity of MTBE-induced DNA alterations was assayed using the standard Ames test with TA102, a strain similar to TA104 in the damage it detects but uvrB + and, therefore, excision repair proficient. The assay was performed (1) with and without Aroclor-induced rat S-9, (2) with and without the addition of formaldehyde dehydrogenase (FDH), and (3) with human S-9 homogenate. MTBE was weakly mutagenic when tested directly and moderately mutagenic with S-9 activation producing between 80 and 200 TA102 revertants/mg of compound. Mutagenicity was inhibited 25%-30% by FDH. TA102 revertants were also induced by TBA and by MTBE when human S-9 was substituted for rat S-9. We conclude that MTBE and its metabolites induce a mutagenic pathway involving oxidation of DNA bases and an intact repair system. These data are significant in view of the controversy surrounding public safety and the environmental release of MTBE and similar fuel additives.

Spermine inhibition of the 2,5-diaziridinyl-1,4-benzoquinone (DZQ) crosslinking reaction with DNA duplexes containing poly(purine). poly(pyrimidine) tracts.

Upon reduction, 2,5-diaziridinyl-1,4-benzoquinone (DZQ) can form an interstrand guanine to guanine crosslink with DNA duplexes containing a d(GC).d(GC) dinucleotide step. The reaction is enhanced by a thymine positioned 5[prime] to each guanine [i.e. in a d(TGCA). d(TGCA) duplex fragment]. Here we show that spermine can inhibit DZQ crosslink formation in duplexes of sequence d[C(N6)TGCA(M6)C]. d[G(M[prime]6)TG-CA(N[prime]6)G]. For N6= M6= GGGGGG, N6= M6= a 'random' sequence and N6= GGGGGG and M6= a 'random' sequence, spermine concentrations of 20, 1 and 3 microM, respectively, were required for 50% inhibition of the DZQ crosslink. This suggests that spermine is more strongly bound to the polyguanosine tract than the random sequence, making it less available for crosslink inhibition. When the polyguanosine tract is interrupted by N 7-deazaguanine (D) located three bases, d(CGGGDGGTGCAGGDGGGC), and four bases, d(CG-GDGGGTGCAGGGDGGC), from the d(TGCA).d(TGCA) site, 30 and 3 microM spermine, respectively, were required for 50% crosslink inhibition. We suggest that this difference is due to the relative proximity of the three-guanosine tract to the d(TGCA).d(TGCA) site. We were able to confirm these conclusions with further experiments using duplexes containing three-guanosine and two-guanosine tracts and from computer simulations of the spermine-DNA complexes.

Convenient separation of high-purity C60 from crude fullerene extract by selective complexation with AlCl3.

A purification procedure for C60 from the fullerene extract has been developed using the differential complexation of C60 and C70 with AlCl3 (or its conjugate acid) in CS2 solution. In addition to pure C60 (>99.8%), the procedure also provides C70-enriched fullerene mixture.