Formation of 6-dimethylcarbamyloxy-dGuo, 6-dimethylamino-dGuo and 4-dimethylamino-dThd following in vitro reaction of dimethylcarbamyl chloride with calf thymus DNA and 6-diethylcarbamyloxy-dGuo following in vitro reaction of diethylcarbamyl chloride with calf thymus DNA.

The rodent carcinogens dimethylcarbamyl chloride (DMCC) and diethylcarbamyl chloride (DECC) react with dGuo (pH 7.0-7.5, 37 degrees C, 4 h) to form the O6-acyl derivatives 6-dimethylcarbamyloxy-2'-deoxyguanosine (6-DMC-dGuo) and 6-diethylcarbamyloxy-2'-deoxyguanosine (6-DEC-dGuo), respectively. Reaction of DMCC with dThd under identical conditions yielded 4-dimethylamino-thymidine (4-DMA-dThd). Compounds 6-DMC-dGuo and 6-DEC-dGuo undergo a nucleophilic aromatic substitution reaction with dimethylamine (DMA) to form 6-dimethylamino-2'-deoxyguanosine (6-DMA-dGuo) via displacement of the C-6 dialkylcarbamyloxy moiety. The substitution reaction did not take place when diethylamine or NH3 were substituted for DMA. The structures of the new compounds 6-DMC-dGuo, 6-DEC-dGuo, 4-DMA-dThd and 6-DMA-dGuo were deduced from chemical analyses and syntheses, UV and nuclear magnetic resonance (NMR) spectra and electron impact, isobutane chemical ionization and source insertion isobutane chemical ionization mass spectra. It was postulated that 4-DMA-dThd was formed following reaction of the transient intermediate 4-DMC-dThd with DMA formed by hydrolysis of DMCC. Calf thymus DNA was reacted in vitro with DMCC (pH 7.0-7.5, 37 degrees C, 4 h) and the modified DNA hydrolyzed enzymatically to 2'-deoxynucleosides. Compounds 6-DMC-dGuo, 4-DMA-dThd and 6-DMA-dGuo were identified in the hydrolysate by high-pressure liquid chromatography (HPLC). In an identical manner 6-DEC-dGuo was identified following in vitro reaction of DECC with calf thymus DNA. Compounds 6-DEC-dGuo and 6-DMC-dGuo possess novel structures with respect to the types of adducts known to be formed between carcinogens and bases in DNA. The implications of these findings with respect to chemical mutagenesis and carcinogenesis is discussed. The structural relationship between N4-dimethyl-5-methylcytosine (4-dimethylamino-Thy) formed in DNA following in vitro reaction with DMCC and 5-methylcytosine, the only modified base found in vertebrate DNA is noted.

In vitro Dimroth rearrangement of 1-(2-carboxyethyl) adenine to N6-(2-carboxyethyl)adenine in single-stranded calf thymus DNA.

The new adduct N6-(2-carboxyethyl)adenine (N6-CEA) was prepared from 1-(2-carboxyethyl)adenine (1-CEA) by base catalyzed (Dimroth) rearrangement of 1-CEA. The structure of N6-CEA was assigned on the basis of UV spectra and electron impact and isobutane chemical ionization mass spectra. When the carcinogen beta-propiolactone was reacted in vitro with calf thymus DNA, 1-CEA but not N6-CEA was detected on paper chromatograms following acid hydrolysis of the DNA. When BPL-reacted single-stranded DNA was incubated at pH 11.7 (37 degrees C, 18 h) prior to acid hydrolysis, it was found that 1-CEA was completely converted to N6-CEA in DNA by Dimroth rearrangement, whereas no conversion occurred at pH 7.5. The extent of Dimroth rearrangement at various pHs and temperatures was determined for 1-CEA, 1-methyladenine (1-MeA), 1-(2-carboxyethyl)-deoxyadenosine-5'-monophosphoric acid (1-CEdAdo5'P) and the phosphodiester 5'-O-(2-carboxyethyl)phosphono-1-(2-carboxyethyl)deoxyadenosine (1-CE-Ado-5'-P-CE).

Tumor-promoting activity of 2,3-dihydrophorbol myristate acetate and phorbolol myristate acetate in mouse skin.

Phorbolol myristate acetate (PHMA) had been previously prepared from the potent mouse skin tumor promoter phorbol myristate acetate (PMA) by sodium borohydride reduction of the C-5 carbonyl group in PMA to a secondary alcohol. PHMA was shown to have an inflammatory effect in mouse skin equal to that of PMA. 2,3-Dihydrophorbol myristate acetate (DPMA), a new compound, was prepared from the 3-aldehyde of PMA by catalytic hydrogenation. DPMA exhibited no detectable inflammatory effect in mouse skin. Both DPMA and PHMA were tested on the dorsal skins of female ICR/Ha Swiss mice (30/group) for 433 and 380 days, respectively, in separate experiments. The tumor-promoting activity of both compounds was reduced significantly, compared with that of equimolar doses of PMA. For each treatment the number of mice with tumors per total number of tumors was: DPMA, 9/17; PMA, 29/553 at 10 microgram/mouse; PMA, 30/317; PHMA, 24/69 at 2.5 microgram/mouse. The results suggest that specific binding requirements influence the tumor-promoting and hyperplastic activity of PMA and its closely related derivatives in mouse skin.

Structure and tumor-promoting activity of analogues of anthralin (1,8-dihydroxy-9-anthrone).

Seventeen analogues of the tumor-promoting agent anthralin were tested for the same biological property by repeated skin application on mouse skin using female ICR/Ha Swiss mice, after a single application of a subcarcinogenic dose of 7,12-dimethylbenz[a]anthracene. Seven of the compounds tested are new compounds. They are 1,8-diacetoxy-9-anthrone, 1,8-dimyristoyloxy-9-anthrone, 1,8-dihydroxy-10-acetyl-9-anthrone, 1,8-dihydroxy-10-myristoyl-9-anthrone, 1,8,10-trihydroxy-9-anthrone, 1,8-dihydroxy-9,10-dihydroanthracene, and myristoyljuglone. All compounds were used in pure form for the bioassays. Of the 17 test compounds four showed notable tumor-promoting activity. They are 1,8-dihydroxy-10-acetyl-9-anthrone, 1,8-dihydroxy-10-myristoyl-9-anthrone, 1-hydroxy-9-anthrone, and juglone. In order to determine whether there is any relationship between tumor-promoting activity and metal chelation in this series, the chelating abilities of anthralin and of its inactive analogue 1,8-dihydroxyanthraquinone were examined using the bivalent metal ions Cu(II), Zn(II), Mn(II), Mg(II), and Ca(II). No relationship between chelation and tumor-promoting ability was found.

In vitro binding of beta-propiolactone to calf thymus DNA and mouse liver DNA to form 1-(2-carboxyethyl) adenine.

In vitro reaction of beta-propiolactone (BPL) with calf thymus DNA and mouse liver DNA followed by acid (HCL) hydrolyses of the BPL-reacted DNA's resulted in the isolation of a new compound, 1-(2-carboxyethyl)-adenine (1-CEA). The structure was assigned on the basis of ultraviolet spectra at acidic, alkaline and neutral pH and electron impact and chemical ionization mass spectra as well as chemical synthesis of 1-CEA from BPL and 2'-adenosine-5'-monophosphoric acid. The only other compound previously isolated from the in vitro and in vivo reactions of BPL and DNA was 7-(2-carboxyethyl)guanine (7-CEG) which we also identified as a product of our in vitro reaction. Under the conditions used the main product of alkylation was 1-CEA and the ratios of the concentrations of 1-CEA to 7-CEG was approx 3 : 1. The possible effect of the formation of 1-CEA on the structure of DNA and its role in chemical carcinogenesis is discussed.

The identification of phorbolol myristate acetate as a new metabolite of phorbol myristate acetate in mouse skin.

Aspects of the metabolism of phorbol myristate acetate (PMA) in mouse skin were investigated. Phorbolol myristate acetate (PHMA), a potential metabolite of PMA in mouse skin, was prepared from PMA by NaBH4 reduction of the C-5 carbonyl group of PMA to a secondary alcohol. The structure of PHMA was assigned on the basis of spectral and chemical evidence. PHMA had an inflammatory effect in mouse skin equal to or slightly less than PMA, on the basis of the dermal infiltration of lymphocytes and neutrophils and interfollicular epidermal hyperplasia. Application of PHMA and PMA to mouse skin resulted in equal increases in numbers of interfollicular epidermal basal layer mitotic cells after 28 hr. PHMA was found to be nearly as effective as PMA in increasing the incorporation of [3H]thymidine into interfollicular epidermal basal layer cells after 28 hr. When tritiated PMA was applied to mouse skin, PHMA was identified as a metabolite. PHMA was present in about 1.7% of the amount of PMA. When tritiated PHMA was applied to mouse skin, PMA was not detected as a metabolite in the mouse skin extracts.