Structure-activity relationships in the mutagenicity and cytotoxicity of putative metabolites and related analogs of benzene derived from the valence tautomers benzene oxide and oxepin.

A series of putative metabolites and related analogs of benzene, derived from the valence tautomers benzene oxide and oxepin, was tested for mutagenicity (reversions to histidine prototrophy and forward mutations to resistance to 8-azaguanine) and for cytotoxicity by the Ames Salmonella mutagenicity test. Benzene was not mutagenic in either assay. The benzene oxide-oxepin system and benzene dihydrodiol induced point mutations but not frameshifts. 4,5-sym-Oxepin oxide, which is a putative metabolite of the oxepin valence tautomer; 3,6-diazo-cyclohexane-1,6-3,4-dioxide, a synthetic precursor of sym-oxepin oxide; and transoid-4,11-dioxatricyclo(5.1 0)undeca-1,6-diene, a stable bridge-head diene analog of sym-oxepin oxide, were toxic but not mutagenic in both assays. 4H-Pyran-4-carboxaldehyde, a stable acid catalyzed rearrangement product of sym-oxepin oxide, was not mutagenic and much less cytotoxic than sym-oxepin oxide. Stable analogs of the valence tautomer benzene oxide, namely syn-indan-3a,7a-oxide and syn-2-hydroxyindan-3a,7a-oxide, were mutagenic and induced point mutations. All compounds were cytotoxic to Salmonella. Firstly, the apparent decay times of these chemicals, especially that of sym-oxepin oxide, were surprisingly longer than expected, as judged by quantitative plate diffusion assays. Secondly, it is concluded that if benzene oxide is further metabolized in its oxepin tautomeric form, toxic but not mutagenic products are formed. Thirdly, the relatively weak mutagenicity of benzene oxide may be mainly due to its instability and corresponding low probability to reach intracellular polynucleotide targets, whereas stable analogs of benzene oxide are relatively more potent mutagens.

Microsomal activation of fluoranthene to mutagenic metabolites.

The in vitro metabolism of fluoranthene (FA) was assessed by incubating 3-[3H]FA, the synthesis of which is described, with rat hepatic microsomal enzymes. Several metabolites including the FA 2,3-diol, FA 2-3,-quinone, 3-OH-FA, 1-OH-FA, and 8-OH-FA were isolated by high-pressure liquid chromatography and identified by comparison of chromatographic properties and uv-visible spectra with those of synthetic standards. The major metabolite produced over the FA concentration range studied (23-233 microM) was FA 2,3-diol, accounting for 29-43% of the total extractable metabolites. This diol was characterized further by high-resolution mass spectroscopy and H-NMR and determined to be identical in structure to the trans-2,3-dihydroxy-2,3-dihydrofluoranthene. The FA 2,3-diol, syn and anti 2,3-diol-1,10b-epoxides, FA 2,3-quinone, and FA 7,8-diol were all shown to be mutagenic toward Salmonella typhimurium TM677. The FA 1,10b-diol and syn and anti FA 1,10b-diol-2,3-epoxides were not mutagenic. The epoxide hydrolase inhibitor, 3,3,3-trichloropropylene oxide, markedly reduced the mutagenic potency of FA while concurrently inhibiting FA 2,3-diol production but not overall FA metabolism. These results suggests that a major metabolic activation pathway of FA resulting in the production of mutagenic species involves the formation of the FA 2,3-diol and the subsequent oxidation of this diol to a FA 2,3-diol-1,10b-epoxide. Another minor activation pathway with mutagenic endpoints may involve the formation of the 7,8-diol.

In vitro DNA-binding of microsomally-activated fluoranthene: evidence that the major product is a fluoranthene N2-deoxyguanosine adduct.

Incubation of 3-[3H]fluoranthene with a rat liver microsomal activation system in the presence of calf thymus DNA resulted in radioactivity bound to the DNA. The fluoranthene-modified DNA was enzymatically digested and a DNA adduct elution profile developed using h.p.l.c. The fraction containing the major fluoranthene--DNA adduct was further purified by h.p.l.c. and separated into two subfractions. Treatment of these with perchloric acid liberated guanine in both cases. Evidence that both were N-2 guanine derivatives was based on the pK values of these adducts determined before and after treatment with nitrous acid. The major adduct (70% of total modified deoxyribonucleosides) was further characterized by high resolution, fast atom bombardment mass spectroscopy which yielded a molecular ion consistent with a fluoranthene triol bound to the N-2 position of deoxyguanosine. Synthetic syn and anti 3-[3H]2,3-dihydroxy-1,10b-epoxy-1,2,3-trihydrofluoranthene were reacted directly with DNA and the 8-[3H]-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrofluoranthene was incubated with DNA and microsomes. The DNA from each of these reactions was enzymatically hydrolyzed and h.p.l.c. adduct profiles were developed. The major adduct formed from reaction of the anti 2,3-dihydroxy-1,10b-epoxy-1,2,3-trihydrofluoranthene with DNA and the major N-2 fluoranthene derived adduct had identical elution times on two different h.p.l.c. systems, similar pK values (before and after nitrous acid treatment) and the same u.v. spectra. In addition, derivatization of both adducts with ethyl methanesulfonate yielded identical products, as determined by h.p.l.c. analysis.

Enzyme engineering.

Enzyme research and development efforts have been shaped by the tools and concepts available for enzyme production and utilization. A new phase of enzymology characterized by the production of modified protein catalysts has begun, made possible by recombinant DNA technology.

Stereospecificity of the ferric enterobactin receptor of Escherichia coli K-12.

Synthetic enterobactin and enantioenterobactin (D-seryl enterobactin) have been examined for the ability to transport iron in Escherichia coli. Failure of the unnatural, D-serine-derived material to support growth of E. coli mutants indicates outer membrane receptor specificity for the naturally occurring complex having an L-seryl backbone and the delta-cis configuration of the Fe(III).catecholate center. Enantioenterobactin was markedly less effective in protecting cells against colicin B compared to synthetic or natural enterobactin.