A 36-nucleotide oligomer containing a single O2-ethyldeoxythymidine (O2-Et-dT) adduct at a specific site was synthesized. The oligomer, which corresponds to a specific DNA sequence in gene G of bacteriophage phi X174, was used as a template by T7 DNA polymerase to investigate the in vitro mutagenic specificity of O2-Et-dT. At 10 microM dNTP and 5 mM Mg++, the progress of T7 DNA polymerase was interrupted by O2-Et-dT: 80% 3' to O2-Et-dT and 14% after incorporating a nucleotide opposite O2-Et-dT (incorporation-dependent blocked product). DNA synthesis past the lesion was low (6%). Incorporation of a nucleotide opposite O2-Et-dT and subsequent postlesion synthesis were enhanced by increasing the dNTP concentration, with postlesion synthesis reaching 30% at 200 microM. Postlesion synthesis was further increased to 45% by addition of 10 mM dAMP to the polymerization reactions. DNA sequencing revealed that both dA and dT were incorporated opposite O2-Et-dT with dA incorporation impeding the progress of DNA synthesis. dT incorporation was efficiently extended implicating O2-Et-dT in transversion mutagenesis in vivo. These studies provide a basis for understanding the molecular mechanisms by which ethylating agents contribute to cytotoxicity, A.T transversion mutagenesis and activation of the oncogene neu by an A.T----T.A transversion event in rat neuroblastomas.

N3-Ethylthymidine (N3-Et-dT) was site specifically incorporated into a 17-nucleotide oligomer to investigate the significance of DNA ethylation at the central hydrogen-bonding site (N3) of thymine. The 5'-(dimethoxytrityl)-protected N3-Et-dT was converted to the corresponding 3'-phosphoramidite and used to incorporate N3-Et-dT at a single site in the oligonucleotide during synthesis by the phosphite triester method. The purified N3-Et-dT-containing oligomer was ligated to a second 17-mer to yield a 34-nucleotide template with N3-Et-dT present at position 26 from the 3'-end. The template DNA, which corresponds to a specific sequence at gene G of bacteriophage phi X174, was used to study the specificity of nucleotide incorporation opposite N3-Et-dT. At 10 microM dNTP and 5 mM Mg2+, N3-Et-dT blocked DNA synthesis by Escherichia coli polymerase I (Klenow fragment): 96% immediately 3' to N3-Et-dT and 4% after incorporation of a nucleotide opposite N3-Et-dT (incorporation-dependent blocked product). DNA replication past the lesion (postlesion synthesis) was negligible. Incorporation opposite N3-Et-dT increased with increased dNTP concentrations, reaching 35% at 200 microM. Postlesion synthesis remained negligible. DNA sequencing of the incorporation-dependent blocked product revealed that dA is incorporated opposite N3-Et-dT consistent with the "A" rule in mutagenesis. Formation of the N3-Et-dT.dA base pair at the 3'-end of the growing chain terminated DNA synthesis. These results implicate N3-Et-dT as a potentially cytotoxic lesion produced by ethylating agents.

Schiff bases were prepared from all-trans-retinal (I) and pyrrolidine perchlorate (11) and from I and n-butyl m i n e , protonated with anhydrous hydrogen chloride gas, 111. Initial quantum yields of rrunr cis photoisomerization (+'PI) were determined and primary photoproducts and product ratios were measured in aerated methanol. +'PI of all-fruns-111 is independent of excitation energy. All analyses were made using high-pressure liquid chromatographic (LC) methods. It was necessary to hydrolize the Schiff bases to corresponding retinals prior to LC analysis.

A comatose 46-year-old woman, admitted to the emergency room, had isopropanol and acetone concentrations of 2000 and 120 mg/L, respectively, in her serum. She had no known history of acute isopropanol intoxication and was otherwise physically healthy. Pharmacokinetic analysis showed that the elimination of both isopropanol and its major metabolite acetone obeyed apparent first-order kinetics with half-lives of 6.4 and 22.4 h, respectively. These data contrast with the commonly held view that isopropanol is slowly metabolized. Concentrations of these analytes in cerebrospinal fluid 6 h after admission were similar to those in serum. This is the first report of the pharmacokinetics of both agents in a nonalcoholic person, and it gives the first data on concentrations of these substances in cerebrospinal fluid.

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