The effect of butylated hydroxytoluene (BHT) and its metabolites on DNA cleavage in vitro was studied with supercoiled plasmid DNA, pUC18, by agarose gel electrophoresis. Among several BHT metabolites, 2,6-di-t-butyl-p-benzoquinone (BHT-quinone) caused cleavage of supercoiled DNA (form I) at a concentration as low as 1 x 10(-6) M. The relative amount of linear form (form III) was increased with increasing concentration of BHT-quinone. 2,6-Di-t-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadienone (BHT-peroxyquinol) and 3,5-di-t-butyl-4-hydroxybenzaldehyde (BHT-CHO) also cleaved DNA, but to a lesser extent than BHT-quinone. No DNA cleavage was detected by BHT, 2,6-di-t-butyl-4-hydroxymethyl phenol (BHT-OH), 3,5-di-t-butyl-4-hydroxybenzoic acid (BHT-COOH), 2,6-di-t-butyl-4-hydroxy-4-methyl-2,5-cyclohexadienone (BHT-quinol) or 2,6-di-t-butyl-4-methylene-2,5-cyclohexadienone (BHT-quinone methide). The DNA cleavage by BHT-quinone was inhibited by oxygen radical scavengers including superoxide dismutase (SOD), catalase, polyethylene glycol, t-butyl alcohol, dimethyl sulfoxide, sodium azide, sodium benzoate, bovine serum albumin and methionine, while it was enhanced by the addition of FeCl2. The production of superoxide radical in a solution of BHT-quinone was confirmed by cytochrome c reduction assay. Superoxide was not produced by BHT or other BHT metabolites except for BHT-quinone. These results suggest that BHT-quinone, one of the principal metabolites of BHT, cleaves DNA strands via its generation of oxygen radicals.(ABSTRACT TRUNCATED AT 250 WORDS)

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Genomic DNA for Na,K-ATPase a, subunit was obtained from libraries of horse kidney genomic DNA in Charon 4A and in EMBL3 bacteriophages by screening with the full sized cDNA probe of the ai subunit of rat Na,K-ATPase as probe. The gene spans 30 kb and consists of 23 exons and 22 intervening sequences. Intron-exon boundaries were analyzed. The protein-coding nucleotide sequence encodes 1016 amino acids with an M, of 112 264. The putative amino acid sequence of horse a, is 9697% homologous to those of other mammalian species.

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Preparation of MFGMThe 8-glucuronidase inhibitory action of milk fat globule membrane (MFGM) was studied. By increasing the MFGM concentration to 0.2%, nearly 90% of the B-glucuronidase activity was inhibited. The trypsin-derived digests of MFGM inhibited 8-glucuronidase activity by = 42%. The inhibitory effect of MFGM on intestinal P-glucuronidase of mouse was studied. We administered MFGM (groups I: 5%; group II: 10%; group III: 20%) to mice and investigated the time course of changes in fecal P-glucuronidase activity. In group I and II, enzyme activity had decreased 15-20% on the 5th day of treatment, but in group III, a decrease in enzyme activity was seen with an inhibition rate of 50%, showing a marked intracolonic efficacy of MFGM. Thus MFGM may have potential in explaining, treating or preventing intestinal cancers.To prepare the MFGM substances, 50% raw cream was used. The cream was washed three times with distilled water, triple volume each time. The yield was kept at 4°C overnight for aging, then put into a small diamond-shaped stainless steel churn. The resultant buttermilk was collected, to which was added enough solid ammonium sulfate to make the resultant mixture half saturated. The mixture, after being salted out. was left ovemieht and centrifueed at 3000 mm for 30 min. The condensate in the uiper layer was-collected, dialyzed at 4°C against distilled water until the dialysate gave a negative reaction to BaCl,, and freeze-dried for storage. The recovery of MFGM substance was about 1.2% of the milk fat.