Dietary inhibitors of mutagenesis and carcinogenesis

Dietary inhibitors of mutagenesis and carcinogenesis are of particular interest because they may be useful for human cancer prevention. Several mutagenesis inhibitors have been demonstrated to be carcinogenesis inhibitors also, e.g., ellagic acid, palmitoleic acid, and N-acetylcysteine. This means that the search for mutagenesis inhibitors may be useful for discovering anticarcinogenic agents. Many mutagenesis inhibitors have been discovered by the use of short-term assays, particularly the Ames Salmonella test. This simple in vitro system has provided opportunities to elucidate the mechanisms of inhibition. The elucidation of the mechanism may allow us to infer the possible anticarcinogenic activity of the reagent. In this chapter, inhibitors of mutagenesis and carcinogenesis that can arise as components of diet have been reviewed. Most of the inhibitors have been demonstrated to be effective against a specific class of mutagens or carcinogens. Therefore, it may be argued that these inhibitors are antagonistic only to those particular agents. Here again, understanding of the mechanisms of these inhibitions is necessary for the assessment. Dietary inhibitors reviewed in this article include: (1) as inhibitors of mutagenesis: porphyllins, fatty acids, vitamins, polyphenols, and sulfhydryl compounds, (2) as inhibitors of carcinogenesis: vitamins A, E and C, ellagic acid, sulfhydryl compounds, fats, selenium, calcium, and fiber. Further studies in this area of science appear to help establish the recipe of a healthy diet.     

A Comparative Study of the Structures of {alpha}-Glucans from Suspension-Cultured Nonglutinous and Glutinous Rice Cells

-Glucans (average mol wt, 1.3 ? 10⁴) extracted with perchloricacid from 8-day-old suspension-cultured nonglutinous (var. Sasanishiki)and glutinous rice (var. Miyakogane) cells were compared. Theresults of hydrolysis by alpha;-, ß- and iso-amylasesand methylation analysis of the -glucans suggested that theirbasic structures are almost the same. These -glucans are highly-branchedpolysaccharides with an average chain length of about 9–10,with exterior and interior chain lengths of about 6–7and 2–3, respectively. ¹Current address: Laboratory of Food Science, Faculty of Education,Hirosaki University, Hirosaki, Aomori 036, Japan. (Received April 27, 1987; Accepted March 2, 1988)     

Analysis of 2-amino-N6-hydroxyadenine-induced mutagenesis in phage M13mp2

The mechanism of mutagenesis induced by 2-amino-N6-hydroxyadenine (AHA) and its deoxyriboside (AHAdR) was studied by determining the nucleotide sequences of phage M13mp2 mutant DNA samples. Mutations in the lac promoter-lacZ alpha region of the phage were induced by addition of this agent to culture media in which the phage was growing inside the host bacteria. The spectrum of spontaneous mutation was also investigated. The induced sequence changes were mostly base transitions (80% with AHA and 90% with AHAdR). A few single-base deletions and additions were detected, but they were ascribable to spontaneous mutations. These results are consistent with the incorporation type mechanism proposed by Janion (this issue). In the Ames Salmonella assay, both AHA and AHAdR showed strong mutagenicity in strain TA100 but no activity in TA98.     

TEI-9063, a stable and highly specific prostacyclin analogue for the prostacyclin receptor in mastocytoma P-815 cells.

The prostacyclin (PGI2) analogues, TEI-9063 and its methyl ester, TEI-1324, have been compared with another stable analogue, iloprost, with respect to binding to the PGI2 receptor, stimulation of adenylate cyclase activity and inhibition of thrombin-induced Ca2+ mobilization in mastocytoma P-815 cells. TEI-9063 displaced the [3H]iloprost binding to the membrane fraction, the IC50 value being 3 nM, but showed very low affinity for the PGE receptor. TEI-9063 dose dependently stimulated cAMP formation in the cells and GTP-dependent adenylate cyclase activity in the membrane fraction, the EC50 value being 50 and 10 nM, respectively. Furthermore, TEI-9063 prevented the thrombin-induced increase in the intracellular Ca2+ concentration, the IC50 value being 50 nM. These IC50 and EC50 values are lower than those obtained for iloprost. On the other hand, those of TEI-1324 were about two-orders higher. Although PGI2 lost its ability to stimulate cAMP formation by preincubation for 20 min at 37 degrees C, TEI-9063 completely retained its ability after 60-min preincubation. These results demonstrate that TEI-9063 is a stable and stronger agonist for the PGI2 receptor than iloprost, and that it prevents thrombin-induced Ca2+ mobilization through stimulation of the adenylate cyclase system in mastocytoma cells.     

Female-predominant expression of testosterone 16 alpha-hydroxylase ("I"-P-450(16)alpha) and its repression in strain 129/J

Using specific testosterone 16 alpha-hydroxylase activity as the basis for selection of fractions during purification, the cytochrome P-450 ("I"-P-450(16)alpha) has been isolated from livers of phenobarbital-treated 129/J female mice [K. Devore, N. Harada, and M. Negishi (1985) Biochemistry, 24, 5632-5637]. An antibody elicited in rabbits to "I"-P-450(16)alpha was used to determine the amount of hepatic microsomal 16 alpha-hydroxylase activity due to "I"-P-450(16)alpha in untreated females and males of the two mouse strains, 129/J and BALB/cJ. The activities inhibited were 0.03 and 0.3 nmol/min/mg protein in the 129/J and BALB/cJ females, respectively. No significant level of "I"-P-450(16)alpha-dependent activity was detected in the microsomes from males of either mouse strain. Immunoblotting of microsomal proteins with the antibody to "I"-P-450(16)alpha revealed approximately a 10-fold greater amount of a 54-kDa protein in the microsomes from BALB/cJ than from 129/J females (0.03 and 0.26 pmol/micrograms protein, respectively). A cDNA clone (R17) for phenobarbital-inducible rat cytochrome P-450 selected "I"-P-450(16)alpha mRNA of mice, indicating a high degree of homology between the mRNAs of mouse "I"-P-450(16)alpha and phenobarbital-inducible rat cytochrome P-450s. Northern and dot hybridization of total mouse liver poly(A)+ RNA with the R17 cDNA probe indicated that the specific content of the hybridizable mRNA was more than 10 times higher in BALB/cJ females than in males, and that the mRNA level in female 129/J mice was very similar to that of 129/J and BALB/cJ males. The repression of "I"-P-450(16)alpha in 129/J females was inherited as an autosomal recessive trait in 129/J and BALB/cJ pairs as indicated by the levels of mRNA in female F1 offspring and the "I"-P-450(16)alpha-dependent hydroxylase activity. Female and male mice of eight more inbred strains (AKR/J, DBA/2J, C57BL/6J, C3H/HeJ, NZB/J, A/J, CBA/CaJ, and P/J) were tested for levels of mRNA. The results showed that the levels of mRNA were always 5- to 10-fold greater in the females than in the corresponding males, although there was some variation in the mRNA content in the males from the different strains. 129/J females appear to be a genetic variant where the female-predominant expression of the mRNA is repressed.     

Direct-acting mutagenicity of N4-aminocytidine derivatives bearing alkyl groups at the hydrazino nitrogens.

To investigate the mechanism of N4-aminocytidine-induced mutagenesis, N'-alkyl-N4-aminocytidines and N4-alkyl-N4-aminocytidines were prepared and their mutagenicity on bacteria were assayed. N'-Methyl-N4-aminocytidine, N'-(2-hydroxyethyl)-N4-aminocytidine and N',N'-dimethyl-N4-aminocytidine showed direct-acting mutagenicity on S. typhimurium TA100 and E. coli WP2 uvrA, tester strains that are sensitive to base-pair substitutions. In contrast, N4-methyl-N4-aminocytidine, N4-(2-hydroxyethyl)-N4-aminocytidine and N4,N'-dimethyl-N4-aminocytidine were not mutagenic on these bacteria. Since N'-methyl-N4-aminocytidine does not form hydrazones, the possibility that N4-aminocytidine causes mutation due to its reactivity with carbonyl compounds has been excluded. Furthermore, the fact that only those alkyl N4-aminocytidines having a hydrogen on the nitrogen at position 4 are mutagenic is consistent with the previously proposed mechanism in which the tautomerization between the amino and the imino forms of N4-aminocytosine allowing an ambiguous base pairing is the cause of the mutagenesis.     

Determination of 9α,11β-prostaglandin F2 by stereospecific antibody in various rat tissues

In view of the recent finding that prostaglandin D₂ is stereospecifically converted to 9α,11β-prostaglandin F₂, an isomer of prostaglandin F₂α, a highly specific and sensitive radioimmunoassay for 9α,11β-prostaglandin F₂ was developed and applied to determine the content of this prostaglandin in various rat tissues. Antisera against 9α-11β-prostaglandin F₂ were raised in rabbits immunized with the bovine serum albumin conjugate, and [³H]9α,11β-prostaglandin F₂ was enzymatically prepared from [³H]prostaglandin D₂. The assay detected 9α,11β-prostaglandin F₂ over the range of 20 pg to 1 ng, and the antiserum showed less than 0.04% cross-section with prostaglandin F₂α, prostaglandin F₂β and 9β,11β-prostaglandin F₂. To avoid postmortem changes, tissues were frozen in liquid nitrogen immediately after removal. The basal level of 9α,11β-prostaglandin F₂ was hardly detectable in various tissues of the rat examined, including spleen, lung, liver and brain; although it was found to be 0.31 ± 0.06 ng/g wet weight in the small intestine. During convulsion induced by pentylenetetrazole, enormous amounts of prostaglandin D₂ (ca. 180 ng/g wet weight) and prostaglandin F₂α (ca. 70 ng/g) were produced in the brain; however, 9α,11β-prostaglandin F₂ was detected neither there nor in the blood. This result demonstrates that the conversion to 9α,11β-prostaglandin F₂ is a minor pathway, if one at all, of prostaglandin D₂ metabolism in the rat brain.     

Regulation of cyclic GMP phosphodiesterase in the submandibular gland of adult rat

Cyclic GMP phosphodiesterase was investigated in the submandibular gland of the adult rat to elucidate the regulatory mechanisms of cGMP concentration in this gland. Ca2+ sensitivity was easily demonstrated as in other tissues using EGTA in the buffer for elution from DEAE-cellulose. The presence of inhibitor proteins for basal and calmodulin-activated portions of Ca2+-dependent phosphodiesterase was suggested. The inhibitor for the basal activity of Ca2+-dependent phosphodiesterase was deemed to be a heat-labile protein, which decreased Vmax, but had no effect on the Km value for cGMP. The presence of more than one kind of inhibitor for the calmodulin-activated portion of the Ca2+-dependent phosphodiesterase was also suggested. One of these, which was not absorbed on DEAE-cellulose, was a heat-labile protein which caused an increase of Km for cGMP, but no change of Vmax.     

Location in Muscarinic Acetylcholine Receptors of Sites for [3H]Propylbenzilcholine Mustard Binding and for Phosphorylation with Protein Kinase C

Muscarinic acetylcholine receptors purified from porcine cerebra or atria were covalently labeled with [3H]propylbenzilylcholine mustard ([3H]PrBCM), and then the labeled receptors were subjected to limited hydrolysis with trypsin, V8 protease, and lysyl endopeptidase, followed by analysis involving sodium dodecyl sulfate-polyacrylamide gel electrophoresis, fluorography, autoradiography, or immunostaining. The labeled peptides were located on the basis of their reactivity with antibodies raised against three synthetic peptides with partial sequences of the m1 or m2 receptor, and of their sensitivity to endoglycosidase F, which was taken as evidence that they contain glycosylation sites near the N terminus. The [3H]PrBCM-binding site in both cerebral and atrial receptors was found to be located between the N terminus and the second intracellular loop, because the size of the smallest deglycosylated peptide that contained both the [3H]PrBCM-binding and glycosylation sites was approximately 16 kDa. Cerebral receptors were 32P-phosphorylated with protein kinase C, and the major phosphorylation sites in cerebral muscarinic receptors were found to be located in a C-terminal segment including a part of the third intracellular loop, because a 32P-labeled peptide of 12-14 kDa reacted with anti-(m1 C-terminal peptide) antiserum. The presence of an intramolecular disulfide bond, probably between Cys 98 and Cys 178 in the first and second extracellular loops, respectively, was suggested by the finding that a peptide of approximately 17 kDa containing the [3H]PrBCM-binding site, but not the glycosylation sites, was partly converted to a peptide of approximately 12 kDa on treatment with beta-mercaptoethanol.     

The chloroplastchIL gene of the green algaChlorella vulgaris C-27 contains a self-splicing group I intron

ThechiL gene product is involved in the light-independent synthesis of chlorophyll in photosynthetic bacteria, green algae and non-flowering plants. The chloroplast genome ofChlorella vulgaris strain C-27 contains the first example of a splitchiL gene, which is interrupted by a 951 bp group I intron in the coding region. In vitro synthesized pre-mRNA containing the entire intron and parts of the flanking exon sequences is able to efficiently self-splice in vitro in the presence of a divalent and a monovalent cation and GTP, to yield the ligated exons and other splicing intermediates characteristic of self-splicing group I introns. The 5 and 3 splice sites were confirmed by cDNA sequencing and the products of the splicing reaction were characterized by primer extension analysis. The absence of a significant ORF in the long P9 region (522 nt), separating the catalytic core from the 3 splice site, makes this intron different from the other known examples of group I introns. Guanosine-mediated attack at the 3 splice site and the presence of G-exchange reaction sites internal to the intron are some other properties demonstrated for the first time by an intron of a protein-coding plastid gene.