Titration of tannin via alkaline phosphatase activity.

An enzyme assay for tannin is described. It is based on the following steps: (i) bovine serum albumin (BSA) is absorbed onto polystyrene microplates; (ii) tannin is bound to the BSA-coated plates; (iii) alkaline phosphatase is then interacted with the free tannin-binding sites. The method takes advantage of the multiple hydroxyl groups of tannin which can associate more than one ligand, e.g., proteins. A pH-dependent dynamic equilibrium sets up between bound and unbound tannin and is controlled only by its initial concentration.

Chiral and structural discrimination in binding of polypeptides with condensed nucleic acid structures.

In biological systems nucleic acids are invariably found in highly compact forms. These rather intricate forms raise questions of basic importance which are related to the various factors involved in the condensation processes, the chemical, physical, and structural features revealed by the packed species, and the effects of the extremely tight packaging upon interactions of the DNA molecules with proteins and drugs. A means for addressing these questions on a molecular level is provided by various procedures known to induce in vitro condensation of DNA molecules into highly compact species which, in turn, may serve as a model for the in vivo physical organization of nucleic acids. A study of the optical properties of the tightly packed DNA molecules indicates that the interactions of these species with polypeptides are characterized by distinct, hitherto unobserved, chiral and structural discrimination. Specifically, the polypeptides found to be selected against are composed of those amino acids that are not normally used in protein biosynthesis, such as D-lysine or ornithine. These findings provide new clues to long debated topics such as the specific universal chirality of amino acids in proteins or the correlation between conformational flexibility of polypeptides and their ability to form stable compact complexes with nucleic acids.

Metabolism of benzo[c]phenanthrene by rat liver microsomes and by a purified monooxygenase system reconstituted with different isozymes of cytochrome P-450.

Metabolism of the environmental pollutant and weak carcinogen benzo[c]-phenanthrene (B[c]Ph) by rat liver microsomes and by a purified and reconstituted cytochrome P-450 system is examined. B[c]Ph proved to be one of the best polycyclic aromatic hydrocarbon substrates for rat liver microsomes. It is metabolized by microsomes from control rats and by rats treated with phenobarbital or 3-methylcholanthrene at 3.9, 4.2 and 7.8 nmol/nmol cytochrome P-450/min, respectively. Principal metabolites are dihydrodiols along with small amounts (less than 10%) of phenols. The K-region 5,6-dihydrodiol is the major metabolite and accounts for 77-89% of the total metabolites. The 3,4-dihydrodiol with a bay-region 1,2-double bond is formed in much smaller amounts and accounts for only 6-17% of the total metabolites, the highest percentage being formed by microsomes from control rats. Highly purified monooxygenase systems reconstituted with cytochrome P-450a, P-450b and P-450c and epoxide hydrolase form predominantly the 5,6-dihydrodiol (95-97% of total metabolites) and only a small percentage of the 3,4-dihydrodiol (3-5% of total metabolites). The 3,4-dihydrodiol is formed with higher enantiomeric purity by microsomes from 3-methylcholanthrene-treated rats (88%) than by microsomes from control rats (78%) or phenobarbital-treated rats (60%). In each case the (3R,4R)-enantiomer predominates. B[c]Ph 5,6-dihydrodiol formed by all three microsomal preparations is nearly racemic.

Monoclonal IgA J539 binds galactopyranosyl antigens on its surface.

Murine monoclonal IgA J539 binds to methyl beta-D-galactopyranoside. With nearly the same affinity, it binds to methyl 6-O-pivaloyl-beta-D-galactopyranoside (4) and to methyl 6-O-beta-D-gentiobiosyl-beta-D-galactopyranoside (7). These observations confirm that the combining area of J539 is of the surface-, and not of the cavity-type.

Nitroimipramines - synthesis and pharmacological effects of potent long-acting inhibitors of [3H] serotonin uptake and [3H] imipramine binding.

A series of nitro derivatives of imipramine have been prepared by nitration of imipramine. Several of the products, especially 2-nitroimipramine (2) and 2,8-dinitro-imipramine (4) were found to be very potent inhibitors of [3H] serotonin uptake and high affinity [3H]imipramine binding in human platelets. In contrast to the parent antidepressant, imipramine, the inhibition of platelet [3H] serotonin uptake and [3H] imipramine binding by the nitro derivatives of imipramine was long-acting and essentially irreversible at low temperatures. These compounds should prove to be valuable tools for subsequent studies on the purification and characterization of the transport protein(s) involved in serotonin uptake and may have novel behavioral and clinical effects.

Preparation of two methyl deoxyfluoro-beta-D-galactopyranosides, and their interaction with galactan-specific immunoglobulin A J539 (Fab').

Methyl 2-deoxy-2-fluoro-beta-D-glactopyranoside (2) and methyl 4-deoxy-4-fluoro-beta-D-glactopyranoside (7) have been prepared, and the possibility of their binding to (1 leads to 6)-beta-D-galactopyranan-specific immunoglobulin A J539 (Fab') has been investigated. Compound 2 does not show binding, whereas 7 does. It appears that the 2-hydroxyl group of methyl beta-D-galactopyranoside may take part in hydrogen bonding to the protein.

Highly tumorigenic bay-region diol epoxides from the weak carcinogen benzo[c]phenanthrene.

In the four years since its inception, the bay-region theory has proved highly successful in predicting which diol epoxide of a polycyclic aromatic hydrocarbon would have the highest tumorigenic activity. The present studies on benzo[c]phenanthrene have shown this hydrocarbon to be unique. It is the first hydrocarbon for which the bay-region diol epoxide that has its benzylic hydroxyl group and epoxide oxygen cis (isomer-1 series) has significant tumorigenic activity. Additionally, its bay-region diol epoxides are the most tumorigenic diol epoxides yet tested on mouse skin despite their expected and observed very low chemical reactivity. Perhaps some unique feature of the shape of benzo[c]phenanthrene can account for the remarkable biological activity of its bay-region diol epoxides. The high degree of crowding in the bay-region of benzo[c]phenanthrene may be such a contributing factor. It is know, for example, that methyl-substitution in the bay-region but not on the critical benzo-ring enhances the tumorigenic activity of 7,12-dimethylbenzo[a]anthracene relative to 7-methylbenzo[a]anthracene (Newman, 1976), of 5-methylchrysene relative to chrysene (Hecht et al., 1974), and of 11-methylbenzo[a]pyrene relative to benzo[a]pyrene (Iyer et al., 1980). Steric crowding in the bay-region of benzo[c]phenanthrene (Hirshfeld, 1963) and 7,12-dimethylbenzo[a]anthracene has been shown by x-ray crystallography to cause out-of-plane deformation of their aromatic ring systems.

Mutagenicity and tumor-initiating activity of cyclopenta(c,d)pyrene and structurally related compounds.

The biological activities of benzo(a)pyrene, cyclopenta(c,d)pyrene, and 12 other structurally related compounds were assessed by mutagenicity studies with bacterial and mammalian cells and/or skin tumorigenicity studies with mice. The ability of the parent hydrocarbons to be metabolically activated to mutagenic products was examined in strains TA98 and TA100 of Salmonella typhimurium, using 3 experimental protocols. In each case, cyclopenta(c,d)pyrene was metabolically activated to products mutagenic to the bacteria to a greater extent than was benzo(a)pyrene. However, 7,8-dihydrobenzo(a)pyrene and 0,10-dihydrobenzo(e)pyrene were the best substrates for metabolic activation to bacterial mutagens. Highly purified epoxide hydrase added to a purified and reconstituted monooxygenase system readily abolished the mutagenic activity observed in strain TA100 of S. typhimurium when cyclopenta(c,d)pyrene was the substrate, but not when benzo(a)pyrene was the substrate. Inherent mutagenicity of several epoxides of the hydrocarbons generally paralleled the ability of their potential metabolic precursors to be activated to mutagens. 1-Pyrenyloxirane and 10,11-dihydrocycloheptapyrene 8,9-oxide were highly mutagenic in strains TA98 and TA100 of S. typhimurium, and in the former strain these activities were comparable to that observed with 9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, 4-Pyrenyloxirane was significantly less mutagenic than was 1-pyrenyloxirane in both strains of bacteria and in mammalian cells. Benzo(a)pyrene was over 20 times more tumorigenic than was cyclopenta-(c,d)pyrene, and it was the most potent of the 11 compounds tested for tumor-initiating activity in 2-stage initiation-promotion experiments on the skin of mice. Cyclopenta(c,d)pyrene had tumor-initiating activity comparable to that of benzo-(a)anthracene, but it was significantly less active than chrysene. Thus, contrary to inferences made from its high mutagenic activity, cyclopenta(c,d)pyrene is a weak tumor initiator on mouse skin.

Mutagenicity of phenanthrene and phenanthrene K-region derivatives.

Phenanthrene and 9 K-region derivatives, most of them potential metabolites of phenanthrene, were tested for mutagenicity by the reversion of histidine-dependent Salmonella typhimurium TA1535, TA1537, TA1538, TA98 and TA100 and the rec assay with Bacillus subtilis H17 and M45. The strongest mutagenic effects in the reversion assay were observed with phenanthrene 9,10-oxide, 9-hydroxyphenanthrene and N-benzyl-phenanthrene-9,10-imine. Interestingly, the mutagenic potency of the arene imine was similar to that of the corresponding arene oxide. This is the first report on the mutagenicity of arene imine. The mutagenic effects of all these phenanthrene derivatives were much weaker than that of the positive control benzo[a]pyrene 4,5-oxide. Even weaker mutagenicty was found with cis-9,10-dihydroxy-9,10-dihydrophenanthrene and with trans-9,10-dihydroxy-9-10-dihydrophenanthrene. The other derivatives were inactive in this test. However, 9-10-dihydroxyphenanthrene and 9,10-phenanthrenequinone were more toxic to the rec- B. subtilis M45 strain than to the rec+ H17 strain. This was also true for phenanthrene 9,10-oxide and 9-hydroxyphenanthrene, but not with the other test compounds that reverted (9,10-dihydroxy-9,10-dihydrophenanthrenes; N-benzyl-phenanthrene 9,10-imine; benzo[a]pyrene 4,5-oxide) or did not revert (phenanthrene, 9,10-bis-(p-chlorophenyl)-phenanthrene 9,10-oxide, 9-10-diacetoxyphenanthrene) the Salmonella tester strains. Although the K region is a main site of metabolism and although all potential K-region metabolites were mutagenic, phenanthrene did not show a mutagenic effect in the presence of mouse-liver microsomes and an NADPH-generating system under standard conditions. However, uhen epoxide hydratase was inhibited, phenanthrene was activated to a mutagen that reverted his- S. typhimurium. This shows that demonstration of the mutagenic activity of metabolites together with the knowledge that a major metabolic route proceeds via these metabolites dose not automatically imply a mutagenic hazard of the mother compound, because the metabolites in question may not accumulate in sufficient quantities and therefore the presence and relative activities of enzymes that control the mutagenically active metabolites are crucial. N-Benzyl-phenanthrene 9.10-imine was mutagenic for the episome-containing S. typhimurium TA98 and TA100 but not for the precursor strains TA1538 and TA1535. This arene imine would therefore be useful as a positive control during routine testing to monitor in the former strains the presence of the episome which is rather easily lost.