Jerome J. Solomon

The isolation and characterization of 3-(2-carboxyethyl)cytosine following in vitro reaction of β-propiolactone with calf thymus DNA

Abstract The new adduct 3-(2-carboxyethyl)cytosine (3-CEC) was isolated following in vitro reaction of the carcinogen β-propiolactone (BPL) with calf thymus DNA. The structure of 3-CEC was confirmed by synthesis from BPL and dCyd. Reaction of BPL with cCyd (pH 7.0–7.5, 37°C) gave 3-(2-carboxyethyl)deoxycytidine (3-CEdCyd) (9% yield) and 3, N 4 -bis(2-carboxyethyl)deoxycytidine (3, N 4 -BCEdCyd) (0.6% yield). 3-CEdCyd and 3, N 4 -BCEdCyd were hydrolyzed (1.5 N HC1, 100°C, 2 h) to 3-CEC and 3, N 4 -bis(2-carboxyethyl)cytosine (3, N 4 -BCEC), respectively. The structure of 3-CEC was assigned on the basis of UV and NMR spectra and the electron impact (EI) mass spectra of 3-CEC and a tri-trimethylsilyl (TMS) derivative of 3 CEC as well as deuterated (d 27 ) tri-TMS derivative of 3-CEC. The structure of 3, N 4 -BCEC was assigned on the basis of UV spectra and the EI mass spectra of a tri-TMS derivative. EI and isobutane chemical ionization mass spectra of 3-methylcytosine (3-MeCyt) and a di-TMS derivative of 3-MeCyt were obtained and were helpful in deducing the structures of 3-CEC and 3, N 4 -BCEC. This is the first report of the alkylation by BPL of an exocyclic atom on a base in DNA. Compound 3, N 4 -BCEC was not detected in BPL-reacted calf thymus DNA. The relative amounts of 1-(2-carboxyethyl)adenine (1-CEA), 7-(2-carboxyethyl)guanine (7-CEG), 3-(2-carboxyethyl)thymine (3-CET) and 3-CEC isolated from BPL-reacted DNA following perchloric acid hydrolysis were 0.23, 1.00, 0.39 and 0.41 respectively, when the alkylation reaction was conducted in phosphate buffer at 0–5°C and pH 7.5 and 0.10, 1.00, 0.29 and 0.28 respectively when the reaction was conducted in H 2 O at 37°C and pH 7.0–7.5.

Isolation of 3-(2-carboxyethyl)thymine following in vitro reaction of β-propiolactone with calf thymus DNA☆

Abstract 3-(2-Carboxyethyl)thymine (3-CET) was synthesized from β-propiolactone (BPL) and dThd5′P at pH 9.0–9.5 via the intermediate 3-(2-carboxyethyl)thymidine-5′-monophosphoric acid (3-CEdThd5′P). 3-CEdThd5′P was converted to 3-CET by hydrolysis in 1.5 N HCl at 100°C for 2 h. The structure of 3-CET was assigned on the basis of UV spectra, electron impact (EI) and isobutane chemical ionization mass spectra and the EI mass spectrum of a trimethylsilyl derivative of 3-CET. BPL was reacted in vitro with calf thymus DNA at pH 7.5. 100 A units of BPL-reacted DNA yielded, following perchloric acid hydrolysis and preparative paper chromatography, 3 A units of 3-CET. Reaction of BPL with the phosphodiester thymidylyl-(3′-5′)thymidine gave 3-(2-carboxyethyl)thymidylyl-(3′-5′)-3-(2-carboxyethyl)thymidine (∼3%). Phosphotriester formation was not detected.

A Simple Mathematical Model for Diffusional Sampler Operation

A simple mathematical model of the molecular basis for the function of a diffusional sampler for dilute mixtures of gaseous contaminants in supporting gases is presented. The model is based on the movement of single molecules of the contaminant between sections of a tubular diffusion path on a step-by-step basis; the length of the step and of each section of the tube are equal to the mean free path, lambda, under the specified conditions. When the model is used, the coefficient of diffusion, D, can be calculated from lambda and the average velocity, v, of the contaminant molecule. Both lambda and v were calculated independently using equations which involved the minimum number of assumptions. The value of D so estimated was of the same order as that in the literature, differing by a factor of less than 2. It should be emphasized that the model represents a statistical, thermodynamic approach to understanding diffusional samplers, and its utility is independent of the means of estimating lambda and v for specific gas pairs.

Loss of essential membrane lipids and ascorbic acid from rat brain following cryogenic injury and protection by methylprednisolone.

Previous work has shown that unsaturated fatty acid components of model membrane phospholipids in vitro, damaged via a free radical mechanism, are protected by the presence of cholesterol in these membranes. The participation of these membrane lipids in the pathogenesis of traumatic injury to brain was studied in vivo using the Klatzo method of cryogenic injury in rats. Increased edema 4 hr after cryogenic injury was noted on the lesioned side. Total cerebral cholesterol was decreased significantly in the lesioned hemispheres 10 hr following injury. In lesioned animals pretreated and post-treated with methylprednisolone, there were no significant differences in the cholesterol levels. Arachidonic acid isolated from total membrane phospholipids was significantly reduced on the injured side 24 hr after injury, but not before. Other fatty acids were not significantly affected. Methylprednisolone treatment prevented the decrease in arachidonic acid. Animals that had received a cold injury had significant decreases in ascorbic acid levels after 4 hr on the lesioned side of the brain. This decrease was significantly ameliorated by corticosteroid administration. These results support the hypothesis that the protective effect of corticosteroids in cryogenic cerebral trauma may be due to antioxidant protection of major cell membrane lipid components such as cholesterol and phospholipids.

The extent and persistence of binding to respiratory mucosal DNA by inhaled tritiated propylene oxide

In order to investigate some of the mechanisms underlying the carcinogenicity of inhaled propylene oxide (PO), the deposition and persistence of inhaled tritiated PO in the DNA of the nasal cavities, tracheae and lungs of rats were investigated. The results of dose/response exposure protocols revealed clear gradients for binding throughout the respiratory mucosa; the highest levels of binding were in the nasal mucosa and the lowest were in the lungs. Gradients became steeper as exposure concentrations were lowered. The persistence studies revealed that bound tritium declined in nasal mucosal DNA with apparent bi-exponential kinetics while clearance from the tracheal and pulmonary DNA was much slower and occurred with apparent mono-exponential kinetics. Consequently, although the initial levels of binding of inhaled PO are lower in the trachea and lungs than in the nasal cavity, the slow clearance of PO from the DNA in these organs could increase the chances of a mutational event.