Comparative metabolism in vitro of a novel carcinogenic polycyclic aromatic hydrocarbon, 1,2,3,4-tetrahydro-7,12-dimethylbenz[a]anthracene, and its two regioisomeric B-ring fluoro analogues.

A novel biotransformation pathway likely exists for carcinogenic 1,2,3,4-tetrahydro-7,12-dimethylbenz[a]anthracene (THDMBA), since this A-ring-reduced polycyclic aromatic hydrocarbon does not have an aromatic bay-region. The comparative metabolism of THDMBA, a non-carcinogenic 5F analogue, and a more carcinogenic 6F-THDMBA species was examined to determine potential DNA-bonding metabolites. Rat liver microsomes from phenobarbital-treated animals were incubated in the presence of THDMBA (or fluoro-THDMBA), NADPH, and O2. Metabolic products and the parent compound were extracted into organic solvent and analyzed/purified using reversed-phase high-performance liquid chromatography. Structure identification of metabolites using proton nuclear magnetic resonance, mass spectroscopy, and ultraviolet/visible spectroscopy indicated that hydroxylations at benzylic C1 and at the C7- and C12-CH3 functions are major oxidation products of THDMBA. Major metabolites for the noncarcinogenic 5F-THDMBA are the C4-hydroxy, C7-hydroxymethyl, and C12-hydroxymethyl derivatives. However, the potent carcinogen 6F-THDMBA only yielded major hydroxylation products at C1 and C12-CH3. These results together with a consideration of the electronic and steric effects of fluorine and the biological activities of these polycyclic aromatic hydrocarbons suggest that hydroxylation at the hindered benzylic C1 position or the C12-CH3 group of THDMBA is important for the biotransformation of such polycyclic aromatic hydrocarbons to DNA-bonding species.

Use of the lectin from Amaranthus caudatus as a histochemical probe of proliferating colonic epithelial cells.

A newly isolated lectin, Amaranthus caudatus agglutinin (also called amaranthin or ACA), which binds to the Thomsen-Friedenreich antigen (T-antigen) and its sialylated variants, was used as a histochemical probe for proliferating cells in sections of human colonic tissues. Binding inhibition studies revealed that ACA binds to different sites on histological sections when compared to peanut agglutinin, which also recognizes the T-antigen. ACA bound selectively to the cells at the base of the colonic crypt [46 +/- 4% (SEM) of glands] which is the zone of proliferation in this tissue and preferentially labeled cytoplasmic and apical membrane glycoconjugates. Only 7 +/- 2% of the upper portions of the colonic crypts were labeled (P less than 0.001 compared to the base), and this was largely a result of extensive labeling in 2 of 23 samples studies. A marked increase in histochemical labeling by ACA was seen in adenomatous polyps and adenocarcinomas of the colon, in which 82 +/- 7 and 97 +/- 2% of the glandular units were labeled, respectively. Transitional mucosa and connective tissue adjacent to cancers were also labeled by ACA. Neuraminidase studies indicated that removal of sialic acid residues enhanced binding by peanut agglutinin, but not ACA, to glycoconjugates in cancer specimens. Specimens of colonic tissue from patients with familial adenomatous polyposis (FAP) were examined with ACA; 83 +/- 7% of adenomatous glands and 60 +/- 7% of glands in flat, normal-appearing tissue were labeled. Colonic tissues from persons at 50% risk for hereditary nonpolyposis colorectal cancer (HNPCC), FAP, and normal colons were studied and given "weighted average" labelling scores that ranged from 0-400 to accommodate variable intensity and distribution of labeling. Normal colons had a weighted average score of 65 +/- 33; FAP tissues had a score of 224 +/- 76 (P less than 0.001 compared to normal colon) and HNPCC tissues had a score of 74 +/- 70 (P less than 0.05 compared to normal colon). A group of five HNPCC cases had scores of 203 +/- 43 (P less than 0.001 compared to normal colon). ACA labels glycoconjugates in the proliferative region of normal human colonic epithelium and neoplastic lesions of the colon. The results of FAP and HNPCC tissues suggest that it may be useful for identifying foci of abnormal proliferation in familial colorectal cancer syndromes.

Expression patterns of the T antigen and the cryptic T antigen in rat fetuses: detection with the lectin amaranthin.

The lectin amaranthin, purified from the seeds of Amaranthus caudatus, has been shown to react specifically with the Gal beta 1,3GalNAc-alpha and the NeuAc alpha 2,3Gal beta 1,3GalNAc-alpha sequence which represent the T antigen and the cryptic T antigen, respectively. We report here the development of labeling techniques that apply amaranthin to stain paraffin sections from rat fetuses. Amaranthin staining was inhibited by pre-incubation of lectin-gold complexes with 10 mM Gal beta 1,3GalNAc-alpha-O-benzyl (synthetic T antigen) or 10 mM Gal beta 1,3GalNAc-alpha-O-aminophenylethyl-human serum albumin (T antigen neoglycoprotein), asialoglycophorin, asialofetuin, and asialomucin. The beta-elimination reaction also abolished the lectin staining demonstrating specificity for O-glycosidically linked structures. A comparison with monoclonal anti-T antigen antibody immunostaining demonstrated that amaranthin detects the T antigen and its cryptic form in tissue sections. Application of the galactose oxidase-Schiff sequence abolished amaranthin (and anti-T antibody) binding to the T antigen but not to its cryptic form, and therefore permitted their differentiation in tissue sections. Histochemical evidence was obtained indicating that amaranthin is a more specific anti-T reagent than peanut lectin. Data are presented that show the differential expression of the T antigen and the cryptic T antigen in organs and cells of rat fetuses late in gestation. Therefore, amaranthin can be used for histochemical detection of the T antigen and the cryptic T antigen, and facilitates discrimination between them.

Evaluation of Cetylpyridinium Chloride-Containing Mouthwashes Using In Vitro Disk Retention and Ex Vivo Plaque Glycolysis Methods.

The Disk Retention Assay (DRA) is an in vitro method developed to measure the available level of cetylpyridinium chloride (CPC) in mouthwash formulations. This method is based on the binding of the cationic CPC molecule to the anionic surface of a cellulose filter disk. Aqueous CPC solutions demonstrate a linear response (A545) for concentrations up to 0.3%. Higher levels of CPC showed no increased response in the assay. Among common oral product ingredients, at relevant concentrations, surfactants are the primary compounds which inhibit CPC detection and hence, chemical availability. Poloxamer-407 decreased CPC availability to 60% at 0.1%, to 10% at 0.5%, and to 24-33% for 0.2-0.4%. Polysorbate-80 decreased CPC availability to 30% at 0.1% and 6% at 0.25%. A range (4-54%) of available levels of CPC were determined for several commercial products containing 0.045-0.05% nominal levels of CPC indicating significant formulation excipient influence. A plaque glycolysis (PG) assay was used to determine the biological activity of all mouthwash products analyzed by DRA. An experimental series of mouthwash formulations having nominal CPC levels of 0-0.10% demonstrated a good correlation (r2 = 0.955) between the calculated available level of CPC (DRA) and inhibition of plaque glycolysis. The calculated available level of CPC from select commercial mouthwash products, also fit the established correlation with biological activity. The combination of DRA and plaque glycolysis methods are valuable tools which can be used during development to maximize the biological activity of CPC mouthwash formulations prior to clinical evaluation.

Physicochemical properties of amaranthin, the lectin from Amaranthus caudatus seeds.

Amaranthin is the lectin present in the seeds of Amaranthus caudatus, which specifically binds the T-disaccharide (Gal beta 1,3GalNAc alpha-O-). The lectin is composed of a single type of subunit with Mr = 33,000-36,000 (Rinderle et al., 1989). Equilibrium sedimentation (Mr = 62,900) and low-angle laser light scattering (Mr = 61,400) methods have been used to unambiguously establish the native multimeric structure of amaranthin as a homodimer. These absolute molecular weight methods and the calculated Stokes radius (27.2 A) indicate that the amaranthin dimer is highly compact relative to typical globular proteins, and thus, anomalous molecular weight values are obtained when simple size exclusion chromatography is used to determine the molecular weight of amaranthin. Studies with a homobifunctional cross-linking reagent and amaranthin further support the existence of a lectin homodimer. The stoichiometry of carbohydrate binding was determined to be one T-disaccharide-binding site per amaranthin subunit (Ka = 3.6 X 10(5) M-1). Amaranthin exhibits hydrophobic-binding properties as indicated by binding of 8-anilino-1-naphthalene-sulfonate (Ka = 3.6 X 10(3) M-1) and 6-toluidinyl-2-naphthalenesulfonate (Ka = 2 X 10(4) M-1). Serological studies suggest that amaranthin does not appear to be present in the stems or leaves of the A. caudatus plant, nor were there any indications for the presence of cross-reactive material.

Isolation and characterization of amaranthin, a lectin present in the seeds of Amaranthus caudatus, that recognizes the T- (or cryptic T)-antigen.

A lectin (Amaranthin) present in the seeds of Amaranthus caudatus has been isolated by fractionation on DEAE-cellulose followed by affinity chromatography on Synsorb-T beads (Gal beta 1,3GalNAc alpha-O-R-Synsorb). The lectin appeared homogeneous by gel electrophoresis at pH 4.3 and gave a single protein band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Mr = 33,000-36,000. A native Mr = 54,000 was determined by gel filtration suggesting that amaranthin exists as a homodimer. Compositional analysis revealed high amounts of acidic and hydroxyamino acids and relatively large amounts of lysine, methionine, and tryptophan for a plant protein. Amaranthin formed a precipitate with asialo-bovine submaxillary mucin, asialo-ovine submaxillary, porcine submaxillary mucin, asialo-fetuin and asialoglycophorin. Hapten inhibition of precipitate formation between amaranthin and asialo-ovine submaxillary indicated that the T-disaccharide and its alpha-linked glycosides (Gal beta 1,3GalNAc alpha-O-R; R = OH, methyl, -(CH2)8-COOCH3, allyl, o-nitrophenyl, or benzyl) were the best inhibitors. N-Acetylgalactosamine, the only monosaccharide which inhibited precipitation, was 350-fold less effective than Gal beta 1,3GalNAc alpha-O-R. Hapten inhibition with derivatives of the T-disaccharide suggested that the C'-4 axial hydroxyl group of the galactosyl moiety, and the C-4 axial hydroxyl group, and the C-2 acetamido group of the GalNAc unit are the most important loci for lectin interaction. NeuAc alpha 2,3Gal beta 1,3GalNAc alpha-O-(CH2)8CO2CH3 was as potent an inhibitor as Gal beta 1,3GalNAc alpha-O-(CH2)8CO2-CH3, and amaranthin was precipitated by NeuAc alpha 2,3Gal beta 1,3GalNAc alpha-O-BSA (where BSA is bovine serum albumin), indicating that the amaranthin-combining site tolerates substitutions at the C'-3 hydroxyl group. Amaranthin was precipitated by a Gal beta 1,3GalNAc alpha-O-BSA glycoconjugate but not by the anomeric Gal beta 1,3GalNAc beta-O-BSA glycoconjugate illustrating that the disaccharide must be linked alpha in order to interact with the lectin. Metal ions do not appear to be required for lectin activity. A study of pH dependence showed significant precipitate formation between pH 4 to 9 with a maximum at pH 5. Hapten inhibition and glycoconjugate precipitation assays were also conducted for peanut (Arachis hypogaea) agglutinin. A comparison between the carbohydrate-binding specificities of amaranthin and peanut (Arachis hypogaea) agglutinin is discussed.

Studies on the Thomsen-Friedenreich antigen in human colon with the lectin Amaranthin. Normal and neoplastic epithelium express only cryptic T antigen.

The lectin Amaranthin has been shown to be highly specific for the galactose beta 1,3 N-acetylgalactosamine-alpha and sialic acid alpha 2,3 galactose beta 1,3 N-acetylgalactosamine-alpha sequence which represents the Thomsen-Friedenreich (T) antigen and its cryptic form, respectively. Previously, we demonstrated the usefulness of gold-labeled Amaranthin for the histochemical detection of the T antigen and its cryptic form. Application of the galactose oxidase (GO)-Schiff sequence abolished lectin binding to the T antigen but not its cryptic form, and therefore permitted their differentiation. In the present study we have analyzed by light and electron microscopy the distribution and subcellular localization of Amaranthin binding sites in normal, dysplastic and neoplastic colonic epithelium. Furthermore, a monoclonal antibody raised against synthetic galactose bera 1,3 N-acetylgalactosamine-alpha-bovine serum albumin was applied as a reagent for the T antigen. In normal colonic mucosa, two different Amaranthin staining patterns existed: (a) reactivity restricted to the lower portion of the crypts which was principally observed in the left colon, and (b) reactivity along the entire length of the crypts and in the surface epithelium with goblet cell staining in the upper portion of the crypts which was principally observed in the right colon. This Amaranthin staining was resistant to GO-Schiff treatment. No immunostaining with the monoclonal anti-T antigen was observed. Investigation of transitional mucosa, adenocarcinomas of different degrees of differentiation and mucinous carcinomas as well as adenomas with different degrees of dysplasia all revealed positive Amaranthin staining. The lectin staining was resistant to GO-Schiff treatment, and immunolabeling with the monoclonal antibody against the T antigen was absent. These results indicate that only the cryptic form of the T antigen is expressed in normal, dysplastic and neoplastic human colonic epithelium.

Structure of 6-fluoro-1,2,3,4,7,12-hexahydro-7-methyl-12-methylene- benz[a]anthracene.

The X-ray analysis confirms the structure of the 12-methylene tautomer formed by the acid catalysis of 6-fluoro-1,2,3,4-tetrahydro-7,12-dimethylbenz[a]anthracene. The central C ring is in a boat conformation, with the result that the molecule is bent about a line through atoms C(7) and C(12) with a dihedral angle of 32.5 degrees. The cyclohexene A ring is in a half-chair conformation.

Modulation of carcinogenicity in 1,2,3,4-tetrahydro-7,12- dimethylbenz[a]anthracene (THDMBA) by fluorine substitution: crystal structures of the 5- and 6-fluoro regioisomers.

1,2,3,4-Tetrahydro-7,12-dimethylbenz[a]anthracene (THDMBA) is an animal carcinogen which lacks an aromatic bay-region and shows promise as a model to investigate non-classical mechanisms of carcinogenesis. The fluorine-substituted derivatives at positions 5 and 6 on the B-ring exhibit a striking range of oncogenic potential wherein the 6F-THDMBA is twice as potent as the parent carcinogen, but the 5F-THDMBA is virtually inactive. To study structure-reactivity relationships for these fluorine regioisomers, we have determined the three-dimensional structures of the compounds by single-crystal X-ray diffraction. These crystal structures are the first such to be reported for any monofluoro anthracene (or pyrene) derivative. The partially-reduced A-ring exists in both enantiomeric half-chair conformers in the crystalline state, and the compounds have quasi-planar anthracene ring systems which exhibit a right-handed twist in the 'beta'-conformer, with the expected opposite twist in the other. A complete analysis of bond lengths, bond angles and torsion angles is presented. Preliminary electrostatic potentials have been derived from the X-ray data sets, and the results indicate significant differences in potential between 5F- and 6F-THDMBA at positions near the partially reduced bay region. Such results are likely to be of importance in the understanding of metabolic activation to reactive intermediates capable of bonding covalently to DNA.