Isao Eto

Role of Vitamin B12 and Folate Deficiencies in Carcinogenesis

A significant body of experimental evidence supports the notion that a deficiency of either vitamin B12 or folic acid enhances the activity of various carcinogens. Unifying mechanisms are proposed to explain this cocarcinogenic role.

Chemopreventive efficacy of anethole trithione, N-acetyl-L-cysteine, miconazole and phenethylisothiocyanate in the DMBA-induced rat mammary cancer model

The chemopreventive efficacy of N‐acetyl‐L‐cysteine (NAC), anethole trithione, miconazole and phenethylisothiocyanate (PEITC), each of which would be expected to alter carcinogen metabolism, was examined in the dimethylbenzanthracene (DMBA) mammary carcinogenesis model. In this protocol, animals were exposed to non‐toxic doses of the chemopreventives in the diet beginning 7 days prior to DMBA administration and then continuously throughout the duration of the assay (100 days post carcinogen). Miconazole, an antifungal agent with relatively broad inhibitory activity toward a variety of cytochromes P450, increased mammary tumor latency, decreased tumor incidence at the highest dose and decreased tumor multiplicity up to 60%. Anethole trithione, a substituted dithiolthione and an analog of the relatively broad‐spectrum chemopreventive oltipraz, was administered in the diet and significantly inhibited mammary cancer multiplicity but not cancer incidence. NAC, an antimucolytic agent, failed to inhibit DMBA‐induced mammary tumorigenesis. Surprisingly, treatment with DMBA plus PEITC, a potent inhibitor of cytochrome P450 2E1, actually increased the multiplicity of tumors relative to that observed with DMBA alone. Int. J. Cancer 72:95–101, 1997.

Determination of three different pools of reduced one-carbon-substituted folates. III. Reversed-phase high-performance liquid chromatography of the azo dye derivatives of p-aminobenzoylpoly-γ-glutamates and its application to the study of unlabeled endogenous pteroylpolyglutamates of rat liver

Abstract A new reversed-phase high-performance liquid chromatographic (hplc) method is described for the separation and quantitation of picomole amounts of the azo dye derivatives of p-aminobenzoylpoly-γ-glutamates. In conjunction with our previously described procedures for the differential cleavage of one-carbon-substituted, reduced folates, this hplc method provides a rapid, sensitive, and reproducible approach to the quantitation and chain-length determination of three pools of unlabeled endogenous pteroylpolyglutamates. Analysis of rat liver (n = 9) yielded the following results ( x ∗ ± SE ): total folates 14.5 ± 1.0 nmol/g; folates of pool 1 (5,10-methylenetetrahydro- and unsubstituted tetra- and dihydrofolates) 2.65 ± 0.74 nmol/g; folates of pool 2 (5-methyltetrahydrofolates) 5.30 ± 0.36 nmol/g; and folates of pool 3 (5,10-methenyltetrahydro-, 10-formyltetrahydro-, 5-formyltetrahydro-, and 5-formiminotetrahydrofolates) 6.40 ± 1.60 nmol/g. Most of the folates of rat liver occur as penta- (7.60 ± 0.69 nmol/g) and hexaglutamates (6.00 ± 0.29 nmol/g). In pool 3 the hexaglutamates predominate. We also report experiments showing that folate patterns based on the amount of radioactive label incorporated after a pulse dose of [3H]folic acid differ at all times from the true steady-state pattern of unlabeled endogenous folates.

Determination of three different pools of reduced one-carbon-substituted folates: I. A study of the fundamental chemical reactions

Abstract The reduced one-carbon-substituted derivatives of folic acid can be grouped in three pools according to their response to acid treatment. Pool 1 is made up of N 5 , N 10 -methylene-tetrahydrofolic acid and unsubstituted dihydro- and tetrahydrofolic acid which at pH 1.0 and subsequent exposure to air cleave to p -aminobenzoylglutamic acid. Pool 2 is made up by the acid-stable N 5 -methyl-tetrahydrofolic acid, and pool 3 includes N 5 , N 10 -methenyl-tetrahydrofolic acid, N 10 -formyltetrahydrofolic acid, N 5 -formyltetrahydrofolic acid, and N 5 -formiminotetrahydrofolic acid, all of which convert to the stable N 5 , N 10 -methenyl-tetrahydro form when acid treated. Conditions are described to selectively cleave the C 9 -N 10 bond of the folates of pool 1, pools 1 + 2, and pools 1 + 2 + 3. The cleaved pools are quantitated as the Bratton-Marshall azo dyes of p -aminobenzoylglutamate. The uncleaved pools are converted to Bratton-Marshall-negative products. Pool 1 is determined by converting pool 2 to 4a-hydroxy-5-methyltetrahydrofolic acid and pool 3 to N 10 -formylfolic acid, both Bratton-Marshall negative, by 10% hydrogen peroxide oxidation at pH 6.0. Pools 1 + 2 are cleaved with 0.015% hydrogen peroxide and 0.1% potassium permanganate at pH 9.0 which convert the N 5 -methyltetrahydrofolic acid to the acid-cleavable N 5 -methyl-dihydrofolic acid. Pool 3 oxidizes to the Bratton-Marshall-negative N 10 -formylfolic acid. Pools 1 + 2 + 3 are cleaved by first reducing pool 3 to N 5 -methyltetrahydrofolic acid with sodium borohydride followed by oxidation at pH 9.0 to its acid-labile dihydro form. Determination of the poly-γ-glutamyl chain length of each pool is possible by chromatographing the azo- p -aminobenzoylpolyglutamates with authentic synthetic markers.

Effect of Canthaxanthin on Chemically Induced Mammary Carcinogenesis

Canthaxanthin, a carotenoid with no vitamin A activity, was evaluated for its efficacy in the prevention of chemically induced mammary cancers. Canthaxanthin was administered in the diet at two dose levels (3,390 or 1,130 mg/kg diet). In the dimethylbenzanthracene-induced mammary cancer model, diet supplementation with canthaxanthin for 3 weeks prior to the carcinogen resulted in a 65% reduction in the number of mammary cancers. The feeding of canthaxanthin after the administration of methylnitrosourea had no significant effect on mammary carcinogenesis. These data demonstrate that canthaxanthin, at least in these models of mammary cancer, is active in preventing cancer initiation and not promotion. Analysis of tissues by high-pressure liquid chromatography revealed that canthaxanthin levels in the liver are very high when compared to those in the mammary gland. The observation that canthaxanthin is highly effective in preventing cancer initiation without toxicity suggests that carotenoids not possessing vitamin A activity should be further evaluated as chemopreventive agents.

9cUAB30, an RXR specific retinoid, and/or tamoxifen in the prevention of methylnitrosourea-induced mammary cancers

Studies were performed in female Sprague-Dawley rats to determine the efficacy of a new RXR specific retinoid (9cUAB30) when combined with tamoxifen in the prevention of mammary cancers and to determine various pharmacokinetic parameters of the retinoid. When administered by gavage, 9cUAB30 was rapidly absorbed and had a serum t(1/2) of 13.5 h. Since the retinoid was administered in the diet for the chemoprevention study, a 28-day study in which 9cUAB30 was given at dose levels of 200, 400, and 600 mg/kg diet revealed fairly constant serum levels regardless of dose or length of treatment; possibly accounting for the observed low toxicity of this compound. When suboptimal doses of 9cUAB30 were given in the methylnitrosourea (MNU)-induced mammary cancer model, the following average number of mammary cancers were observed: 9cUAB30 (150 mg/kg diet), 4.3; tamoxifen (0.4 mg/kg diet), 4.6; 9cUAB30 (150 mg/kg diet)+tamoxifen (0.4 mg/kg diet), 2.6; and controls, 6.0. Thus, the combination of the agents resulted in an increased effect in preventing mammary cancers; suggesting that cancer cell proliferation was inhibited by the compounds blocking different pathways.

Synthesis and analysis of the pteroylpolyglutamates.

Publisher Summary This chapter discusses the synthesis and analysis of the Pteroylpolyglutamates. The biological importance of the polyglutamyl chain was recognized on the basis of its universal occurrence and by the fact that, during evolution, the ability to synthesize the peptide chain has been retained, even by organisms that have lost the pathways for folic acid synthesis. The polyglutamyl chain of the folates plays a role in the regulation of one-carbon metabolism. The polyglutamates are the best cosubstrates of the folate-requiring enzymes, and some of these enzymes show a clear preference for certain specific polyglutamyl chain lengths. The part played by the poly-γ-glutamyl chain in affecting the translocation of folates across cellular and subcellular membranes is established. This role acquires special practical significance when considering the pharmacodynamics of polyglutamyl derivatives of folic acid analogs possessing antitumor activity. Changes in the patterns of distribution of polyglutamates of different chain lengths occur under conditions, which alter the steady-state of one-carbon metabolism, a necessary effect when the polyglutamyl chain is involved in regulation. The properties of the enzymes of poly-glutamate biosynthesis and degradation are reviewed in the chapter.

Determination of three different pools of reduced one-carbon-substituted folates. II. Quantitation and chain-length determination of the pteroylpolyglutamates of rat liver

Abstract A procedure is described for the chemical quantitation of three different pools of one-carbon-substituted, reduced folates in rat liver with the simultaneous determination of their corresponding poly-γ-glutamyl chain lengths. Pool 1 is made up of 5,10-methylenetetrahydrofolates and unsubstituted tetrahydro- and dihydrofolates. Pool 2 is made up solely of 5-methyltetrahydrofolates and pool 3 includes the folates with one-carbon substituents at the formyl level of oxidation. Seventy-two hours after an intraperitoneal injection of [ 3 H]folic acid into rats, the label was incorporated predominantly into pentaglutamates in pools 1 and 2. The hexaglutamates represented about one-third or less and the pentaglutamates approximately two-thirds of these pools. In contrast, in pool 3 the proportion of hexaglutamates was approximately equal to that of pentaglutamates. The proportions of radioactivity present in pools 1, 2, and 3 were 22.8 ± 0.4, 27.3 ± 3.0, and 49.9 ± 3.1%, respectively.

Plasma and urinary levels of biopterin, neopterin, and related pterins and plasma levels of folate in infantile autism

Tetrahydrobiopterin is essential for brain cells to make monoamine neurotransmitters. It has been reported that the concentrations of tetrahydrobiopterin in plasma and urine are low in certain mental disorders and that oral supplements are beneficial. A group of Japanese investigators have been conducting clinical trials of the effect of administration of tetrahydrobiopterin to autistic children and reported that it is beneficial with no significant side effects. We, therefore, initiated a study to assess plasma and urinary levels of tetrahydrobiopterin in infantile autism to see if they are reduced. Besides tetrahydrobiopterin, we also determined plasma and urinary levels of neopterin and monapterin in these individuals in order to evaluate the status of dihydroneopterin triphosphate, a key biosynthetic precursor of tetrahydrobiopterin. Sixteen autistic children and 12 healthy controls were included in this study. Results indicated that the plasma and urinary levels of tetrahydrobiopterin are not statistically different between the two groups and, therefore, no simple explanation for the beneficial effects of administration of tetrahydrobiopterin on autistic children can be offered at the present time. In contrast, plasma and urinary levels of neopterin were depressed (.01<p<.05) and plasma monapterin was also significantly depressed (p<.01) in autistic subjects compared with controls. Levels of other pterins, including folate, were not statistically different between the two groups. The basis for this depression in neopterin and monapterin is unknown. It does not seem likely that this depression could be attributed to a difference in age or T-lymphocyte/macrophage activity. However, further studies are needed to investigate these possibilities.

A differential microdetermination for the various forms of biopterin.

Abstract Conditions for the quantitative oxidation and destruction of tetrahydrobiopterin and quinoid dihydrobiopterin and the separation of biopterin from its reduced forms by ECTEOLA-Sephadex column chromatography are described. A procedure for the quantitation of tetrahydrobiopterin plus quinoid dihydrobiopterin, 7,8-dihydrobiopterin, and biopterin using a Crithidia bioassay is presented. Using these procedures it was found that tetrahydrobiopterin plus quinoid dihydrobiopterin are the prevalent forms in liver and blood of mice and that biopterin was the predominant form in the tails of tadpoles. In human urine, approximately half of the biopterin was found as tetrahydrobiopterin plus quinoid dihydrobiopterin and the other half was 7,8-dihydrobiopterin. The presence of tetrahydrobiopterin and quinoid dihydrobiopterin was confirmed by a coenzyme assay for the hydroxylation of phenylalanine.