Effects of Arsenite on Incorporation of Mevalonate-2-<SUP>14</SUP>C into Squalene-<SUP>14</SUP>C in Rabbit Dental Pulp
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There are many reports showing that arsenite may play an important role as one of the dental pulp devitalizators. It is well known that arsenite is an inhibitor for the SH-enzymes, and several enzymes of the dental pulp are inhibited by arsenite [1, 2, 3]. Goodman et al. [4] reported that squalene synthesis from farnesyl pyrophosphate in the rat liver was inhibited by sodium arsenite. It is well known that lipids in the cell membrane may play an important role in the active transport. However, the effects of arsenite on lipid synthesis in the dental pulp have not been reported in detail. Therefore, in the present work the effects of arsenite on squalene synthesis from mevalonate in the dental pulp were investigated. <BR>DL-Mevalonic acid-2-<SUP>14</SUP>C lactone (specific activity : 5.58 mCi/mM) was obtained from Daiichi Pure Chemical Co., Ltd., and before use, lactone was converted to the acid by alkali. Male rabbits weighing about 2.5 Kg were used. The animals were sacrificed by decapitation, and their dental pulps were quickly excised. The dental pulps (2 g) were homogenized with 4.0 ml of Krebs-Ringer phosphate buffer (pH 7.4) containing 16 mg of nicotinamide, and the homogenate was centrifuged for 10 minutes at 800 × g. The supernatant fraction was used for the enzyme assay of the squalene biosynthesis. The components of the incubation mixture were prepared according to the method of Kelly et al. [5] except 1μ Ci of mevalonic acid-2-<SUP>14</SUP>C was used as substrate. Incubation was carried out for 2 hours at 37°C, and the reaction was stopped by the addition of 3.0 ml of water-ethanol (1:1) containing 15% KOH and 20μg of carrier squalene, after which the mixture was heated for 1 hour at 90°C. The mixture was added 6ml of distilled water and extracted three times each time with 20ml of petroleum ether. The combined petroleum ether was washed three times each time with 10ml of distilled water and evaporated to dryness. The dried nonsaponifiable extract obtained was dissolved in a small volume of benzene, and applied on a silica gel plate for thin-layer chromatography (TLC). n-Hexene-ethyl acetate (9:1) was used as an ascending solvent. After development, the plate was sprayed with 0.1% 2.7'-dichlorofluorescein ethanol solution to detect the carrier. The spot thesis in the dental pulp by arsenite is similar to that occurring in the rat liver. As the inhibition of squalene biosynthesis by arsenite in the rat liver could be almost completely abolished by the addition of an excess of glutathion [4], arsenite may play as SH-inhibitor for the enzymes of the squalene synthesizing system in the dental pulp.
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日本大学歯学部 | 論文
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