Quantitation of uracil in rodent diet.

Feeding of high levels of uracil to laboratory rodents results in the formation of calculi in the lumen of the urinary bladder. This urolithiasis stimulates cellular proliferation in the bladder and has been used in studies of two-stage carcinogenesis. Quantitation of uracil in rodent diet was achieved by extraction from the diet with ammonium hydroxide. The extract was applied to a strong anion-exchange solid-phase extraction column. Uracil is not retained on this matrix which adsorbs the majority of contaminants in the extract. The uracil was quantitated by HPLC on an ODS microbore column (100 x 2 mm internal diameter) eluted at 0.5 ml/min with 200 mM KH2PO4, pH 3.5, at 30 degrees C. Three structurally related pyrimidine bases, cytosine, uracil, and thymine, showed increasing retention on this column/solvent combination, thereby demonstrating selectivity of the analysis. Recovery of uracil was 76-90% with lower values observed when dietary levels of uracil were in excess of 4.5%.

Effect of sodium saccharin and calcium saccharin on urinary parameters in rats fed Prolab 3200 or AIN-76 diet.

The effects of the salt form of saccharin and of diet on urinary ion levels have been studied in rats. Sodium saccharin (NaS) or calcium saccharin (CaS) was fed at a level of 5% in either Agway Prolab 3200 diet or AIN-76 diet to male, 5-wk-old F344 rats for 10 wk. The AIN-76 diet contained considerably less calcium, sodium and potassium than the Prolab 3200 diet, and smaller amounts of these ions were eliminated over 24 hr in the urine of rats fed the AIN-76 diet. Although food consumption was less in the groups fed AIN-76, total urinary saccharinate ion excretion with either saccharin salt was comparable with, or even higher than, that excreted by rats fed either salt in the Prolab 3200 diet. Rats fed Prolab 3200 eliminated approximately equal amounts of saccharinate ion in the faeces and urine. Rats fed AIN-76 eliminated about 10-20 times as much saccharin in the urine as in the faeces. Total saccharin excretion (faecal and urinary) was not influenced by the salt form. Water intake and urine volume were lower in rats fed control AIN-76 diet in comparison with those fed Prolab 3200, and were increased above the control level in groups fed saccharin in the AIN-76 diet. Urine electrolyte levels and osmolality were lower in the groups fed AIN-76. In general, NaS administration in either diet resulted in increased urinary sodium compared with controls, and the pH was at, or above, the level of control rats. CaS resulted in increased urinary calcium and decreased pH. There were marked diurnal variations in the urinary excretion of the various electrolytes, pH, and urine volume over a 24-hr period in all rats. This diurnal variation was more pronounced in the rats fed the Prolab 3200 diet. These results indicate that NaS and CaS have marked effects on the excretion of urinary electrolytes, and that these effects are influenced by diet.

Implications for the formation of abasic sites following modification of polydeoxycytidylic acid by acrolein in vitro.

Polydeoxycytidylic acid (poly dC) was incubated with excess acrolein. A Nensorb 20 nucleic acid purification cartridge was used to bind the polymeric material in the poly dC/acrolein reaction mixture. The non-polymeric material eluted from this column had a UV absorbance four times higher than that of the control. The fluorescence spectrum of the eluted material did not correspond to that of unmodified cytosine. Separate aliquots of the reaction mixture were digested to deoxynucleotide 3'-monophosphates by incubation with micrococcal nuclease and spleen phosphodiesterase. The products were converted to 32P-labeled deoxynucleotide 3',5'-bisphosphates by incubation with T4 polynucleotide kinase and excess [gamma-32P]ATP. The 3'-monophosphate was selectively removed by incubation with nuclease P1. Two-dimensional thin-layer chromatography (TLC) on polyethyleneimine cellulose (PEI)-cellulose and detection of 32P-labeled deoxynucleotide 5'-monophosphates by autoradiography failed to provide evidence for the formation of an acrolein adduct of deoxycytidine 5'-monophosphate. When acrolein-modified deoxycytidine 3'-monophosphate was 32P post-labeled, a new product, which co-chromatographed with UV markers synthesized by reaction of acrolein with deoxycytidine 5'-monophosphate, was detected. These data show that acrolein-modified deoxycytidine 3'-monophosphates are substrates for 32P labeling by T4 polynucleotide kinase and are stable under the assay conditions employed. The inability to detect the acrolein-modified nucleotides after reaction with poly dC in vitro suggests that the modified bases are lost from poly dC by cleavage of the N-glycosyl bond resulting in the formation of an abasic site.

Evaluation of epidermal cell kinetics following freezing or wounding of mouse skin and their potential as initiators of carcinogenesis.

It has been shown that abrasion, and consequent regenerative hyperplasia, acts as a promoting agent in mouse skin carcinogenesis. The present experiments were designed to evaluate the possibility that ulceration and its consequent regeneration might also act as initiators. Female Sencar mice were used, and ulceration was induced either by the application of a frozen rod or by incision of the skin of the back. The time course of the ulceration, regeneration, and repair of the mouse skin following ulceration by either method was evaluated utilizing morphologic and autoradiographic techniques. The labeling index of the epidermis, using [3H]-thymidine and autoradiography, reached a maximum level 7 days after ulceration and the epidermal hyperplasia was most pronounced at days 7-14. The potential initiating activity of freeze ulceration or incision was evaluated by performing these procedures on 7-week-old female Sencar mice followed by promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA) applied twice a week to the ulcerated areas, 5.2 micrograms in each application. Extending the total experimental observation to 1 year indicated that freeze ulceration and incision did not initiate carcinogenesis in the mouse skin when promoted with TPA.