Conservation of histone carcinogen adducts during replication: implications for long-term molecular dosimetry.

The effect of cell replication on histone-carcinogen adducts was investigated by determining the specific adduct levels as a function of time following carcinogen treatment of human TK6 cells grown in culture. Core histones isolated from cells treated with aflatoxin B1 or r-7,t-8 dihydroxy-t-9,t-10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene exhibited a decrease over five generations in specific adduct level that did not exceed the decrease expected as a result of dilution with newly synthesized protein except during the early phase (< 1 generation) of the experiment when loss of chemically unstable adducts might occur. Similar kinetics without the initial, more rapid phase was observed when cells were treated with N-nitroso-N-methylurea. Multigeneration stability of aflatoxin B1 and N-nitroso-N-methylurea adducts that formed on histone H1 was also observed; in these experiments it was not possible to determine if there was an initial phase in the kinetics. These experiments indicate that cell replication does not result in the repair or removal of adducted histones, establishing the feasibility of using histone-carcinogen adducts for molecular dosimetry purposes.

Characterization of benzo[a]pyrene anti-diol epoxide adducts to human histones.

Nuclei from human lymphoblast cells grown in culture were treated with [7-14C]-(+/-)-r-7,t-8-dihydroxy-t-9,t-10-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene (anti-BPDE), and the nucleosomal core histones were isolated for adduct studies by cryogenic fluorescence line narrowing spectroscopy. The four core histones H2A, H2B, H3, and H4 were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which yielded each histone free of contamination by the others. Further purification of histones H2A, H2B, and H4 by reversed-phase HPLC also yielded a tetrahydrotetrol of benzo[a]pyrene, indicating that these three histones had some labile adducts. No tetrol was observed upon purification of histone H3. Fluorescence emission spectra of the HPLC-purified histones recorded at 4K after vibronic excitation into the S1 state were generally similar. Fluorescence line-narrowed spectra of model compounds formed by reaction of anti-BPDE with acetic acid, ethylenediamine, cysteamine, and histidine were also recorded. Only the spectra of the ethylenediamine adduct model matched consistently, at different excitation wavelengths, the spectra of the adducted histones. From this it is concluded that the stable human histone adducts of anti-BPDE are formed by reaction with lysine residues and/or the amino groups of the N-termini.

Purification and characterization of nitrous oxide reductase from Pseudomonas aeruginosa strain P2.

Nitrous oxide reductase, which catalyzes the reduction of N2O to N2, was purified in a largely oxidized form from Pseudomonas aeruginosa strain P2 by a simple anaerobic procedure to yield an enzyme with a peptide purity of 95-98%. For the native (dimeric) enzyme, Mr = 120,000 and for the denatured subunit, Mr = 73,000. The enzyme contained four Cu atoms/subunit, was purple in color, and exhibited a broad absorption band at 550 nm with an extinction coefficient of about 11,000 M-1 x cm-1 referenced to the dimer. It was nearly inactive as prepared but could be activated by incubation with 2-(N-cyclohexylamino)ethane sulfonate buffer, pH 10, to specific activities as high as 27 mumol of N2O x min-1 x mg-1.Km for N2O and benzyl viologen radical cation was about 2 and 4 microM, respectively, both before and after enzyme activation. Activation increased the t1/2 for turnover-dependent inactivation from about 30 s to 5-10 min. Reduction of the enzyme by dithionite was kinetically biphasic and resulted in the loss of the 550-nm band and ultimate appearance of a 670-nm band. Isoelectric focusing revealed five components with pI values from 5.2 to 5.7. The pI values did not change following activation. The copper CD spectrum of the enzyme as prepared was different from that for the activated enzyme, whereas those for the enzyme after exposure to air and the activated enzyme were similar. Because the activated enzyme is a mixture of activated and inactive species, the specific activity of the activated species must be substantially greater than the observed value. Molecular heterogeneity may also explain the decreased optical absorbance and CD amplitude that resulted from the activation process. The data overall reinforce the view that the absorption spectrum of nitrous oxide reductase is not a good predictor of absolute activity.

Extended x-ray absorption fine structure and electron paramagnetic resonance of nitrous oxide reductase from Pseudomonas aeruginosa strain P2.

The copper centers of nitrous oxide reductase from Pseudomonas aeruginosa strain P2 were studied by x-ray and electron paramagnetic resonance (EPR) spectroscopy. The enzyme is dimeric and contains four Cu atoms and about seven cysteine residues/subunit of Mr = 73,000. The extended x-ray absorption fine structure (EX-AFS) spectrum was analyzed for enzyme as isolated (oxidized or slightly reduced), enzyme exposed briefly to air, reduced enzyme, and enzyme at pH 7 after having been activated by standing at pH 10. The average Cu ligand environment in the first shell was best modeled for all forms of the enzyme by a combination of N/O and S atoms at a total coordination number between 3 and 4 and bond distances ranging from 1.96-2.03 A for Cu-N/O and 2.20-2.25 A for Cu-S. The data could be fit without using Cu-Cu interactions. Overall the results are similar to those reported for the enzyme for Pseudomonas stutzeri (Scott, R. A., Zumft, W.G., Coyle, C.L., and Dooley, D.M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4082-4086). The first derivative EPR spectra of the Cu(II) centers at 15 and 45 K were qualitatively similar among enzyme as isolated and enzyme exposed to N2O or air. These three nominally oxidized samples showed an axial signal with g perpendicular = 2.03 and g parallel = 2.15-2.16. Hyperfine structure was observed in both the g parallel and g perpendicular regions with splittings of 43 and 25 gauss, respectively. These hyperfine components are attributed to exchange coupled Cu(I)-Cu(II) S = 1/2 (half-met) centers. In the enzyme as isolated and after exposure to N2O, about 3/4 of the Cu was EPR silent, whereas after exposure to air the signal integrated to about half the Cu concentration. The EPR spectrum of enzyme activated at pH 10 but frozen at pH 7 was a composite of spectra from activated and inactive species. The activated species presented a complex set of narrow hyperfine components which may arise from contributions from more than one species of half-met center.

Loss of nitrous oxide reductase in Pseudomonas aeruginosa cultured under N2O as determined by rocket immunoelectrophoresis.

The mass ratio of nitrous oxide reductase to total protein in the soluble protein fraction of Pseudomonas aeruginosa P2 was highest in cells grown on nitrate, decreased in cells grown on N(2)O following the exhaustion of the initial charge of nitrate, and was nearly zero in cells exposed solely to N(2)O.

Growth of Pseudomonas aeruginosa on nitrous oxide.

Three strains of Pseudomonas aeruginosa were grown anaerobically on exogenous N2O in a defined medium under conditions that assured the maintenance of highly anaerobic conditions for periods of 1 week or more. The bacteria were observed reproducibly to increase their cell density by factors of 3 to 9, but not more, depending on the initial amount of N2O. Growth on N2O was cleanly blocked by acetylene. Cell yields, CO2 production, and N2O uptake all increased with initial PN2O at PN2O less than or equal to 0.1 atm. Growth curves were atypical in the sense that growth rates decreased with time. This is the first observation of growth of P. aeruginosa on N2O as the sole oxidant. N2O was shown to be an obligatory, freely diffusible intermediate during growth of strains PAO1 and P1 on nitrate. All three strains used this endogenous N2O efficiently for growth. For strains PAO1 and P1, it was confirmed that exogenous N2O had little effect on the cell yields of cultures growing with nitrate; thus, for these strains exogenous N2O neither directly inhibited growth nor was used significantly for growth. On the other hand, strain P2 grew abundantly on exogenous N2O when small and growth-limiting concentrations of nitrate or nitrate (2 to 10 mM) were included in the medium. The dramatic effect of these N-anions was realized in large part even when the exogenous N2O was introduced immediately after the quantitative conversion of anion-nitrogen to N2. No evidence was found for a factor in filter-sterilized spent medium that stimulated fresh inocula to grow abundantly on N2O.(ABSTRACT TRUNCATED AT 250 WORDS)