Alpha-hydroxytamoxifen is a substrate of hydroxysteroid (alcohol) sulfotransferase, resulting in tamoxifen DNA adducts.

When alpha-hydroxytamoxifen (alpha-OHTAM) was incubated with rat liver hydroxysteroid (alcohol) sulfotransferase a (STa) and 3'-phosphoadenosine 5'-phosphosulfate, (E)-alpha-OHTAM was found to be a better substrate for STa than (Z)-alpha-OHTAM. To explore the formation of tamoxifen (TAM)-derived DNA adducts, DNA was incubated with STa and either (E)-alpha-OHTAM or (Z)-alpha-OHTAM in the presence of 3'-phosphoadenosine 5'-phosphosulfate. Using 32P-postlabeling analysis, the amount of TAM-DNA adducts resulting from (E)-alpha-OHTAM was 29 times higher than that observed with (E)-alpha-OHTAM alone. Using (Z)-alpha-OHTAM and STa, some TAM-DNA adducts were also detected but at levels 6.5 times lower than that observed with (E)-alpha-OHTAM and STa. When compared with standards of stereoisomers of 2'-deoxyguanosine 3'-monophosphate-N2-tamoxifen, the major tamoxifen adduct was identified chromatographically as an epimer of the trans form of alpha-(N2-deoxyguanosinyl)tamoxifen, and the minor adduct was identified as an epimer of the cis form. In the reaction mixture, a conversion from (E)-alpha-OHTAM to (Z)-alpha-OHTAM through the carbocation intermediate was also detected. These results show that sulfation of alpha-OHTAM catalyzed by STa results in the formation of TAM-DNA adducts.

Mechanism of lower genotoxicity of toremifene compared with tamoxifen.

An increased incidence of endometrial cancer has been reported in breast cancer patients taking tamoxifen (TAM) and in healthy women participating in the TAM chemoprevention trials. Because TAM-DNA adducts are mutagenic and detected in the endometrium of women treated with TAM, TAM adducts are suspected to initiate the development of endometrial cancer. Treatment with TAM has been known to promote hepatocarcinoma in rats, but toremifene (TOR), a chlorinated TAM analogue, did not. TAM adducts are primarily formed via sulfonation of the alpha-hydroxylated TAM metabolites. To explore the mechanism of the lower genotoxicity of TOR, the formation of DNA adducts induced by TOR metabolites was measured using (32)P-postlabeling/ high-performance liquid chromatography analysis and compared with that of TAM metabolites. When alpha-hydroxytoremifene was incubated with DNA, 3'-phosphoadenosine 5'-phosphosulfate, and either rat or human hydroxysteroid sulfotransferase, the formation of DNA adducts was two orders of magnitude lower than that of alpha-hydroxytamoxifen. alpha-hydroxytoremifene was a poor substrate for rat and human hydroxysteroid sulfotransferases. In addition, the reactivity of alpha-acetoxytoremifene, a model activated form of TOR, with DNA was much lower than that of alpha-acetoxytamoxifen. Thus, TOR is likely to have lower genotoxicity than TAM. TOR may be a safer alternative by avoiding the development of endometrial cancer.

Mechanistic insights into the specificity of human cytosolic sulfotransferase 2A1 (hSULT2A1) for hydroxylated polychlorinated biphenyls through the use of fluoro-tagged probes.

Determining the relationships between the structures of substrates and inhibitors and their interactions with drug-metabolizing enzymes is of prime importance in predicting the toxic potential of new and legacy xenobiotics. Traditionally, quantitative structure activity relationship (QSAR) studies are performed with many distinct compounds. Based on the chemical properties of the tested compounds, complex relationships can be established so that models can be developed to predict toxicity of novel compounds. In this study, the use of fluorinated analogues as supplemental QSAR compounds was investigated. Substituting fluorine induces changes in electronic and steric properties of the substrate without substantially changing the chemical backbone of the substrate. In vitro assays were performed using purified human cytosolic sulfotransferase hSULT2A1 as a model enzyme. A mono-hydroxylated polychlorinated biphenyl (4-OH PCB 14) and its four possible mono-fluoro analogues were used as test compounds. Remarkable similarities were found between this approach and previously published QSAR studies for hSULT2A1. Both studies implicate the importance of dipole moment and dihedral angle as being important to PCB structure in respect to being substrates for hSULT2A1. We conclude that mono-fluorinated analogues of a target substrate can be a useful tool to study the structure activity relationships for enzyme specificity.

One-electron oxidation of vindoline and 16-O-acetylvindoline catalyzed by peroxidase.

The mechanism of oxidation of the alkaloids vindoline (1) and 16-O-acetylvindoline (1a) was examined by use of the reversible redox cycle of horseradish peroxidase (HRP). Oxidation of 1 by HRP resulted in the formation of the enamine dimer 5. The highly reactive radical cation species 2 is an implied intermediate in the oxidation process. During the reaction, HRP-I was reduced to HRP-II by abstraction of an electron from vindoline. The vindoline radical thus formed eliminates a second electron and a proton to produce a highly reactive iminium derivative which undergoes intramolecular etherification and dimerization. Oxidation of 16-O-acetylvindoline (1a) by HRP-I results in the production of an iminium derivative 3a concomitant with the formation of HRP-II. The iminium 3a was isolated and characterized and was converted into monodeuterated 1a by reduction with NaBD4. The stoichiometry (HRP-II)/(substrate) was determined to be 4.77 +/- 0.17 for vindoline and 2.27 +/- 0.20 for 16-O-acetylvindoline. The enamine dimer also reduced HRP-I to form HRP-II, but the stoichiometry of this reaction was variable.

In vitro metabolic transformations of vinblastine: oxidations catalyzed by human ceruloplasmin.

The dimeric Vinca alkaloid vinblastine (VLB) undergoes metabolic transformation to three products in a reaction catalyzed by the human serum copper oxidase ceruloplasmin. The enzyme reaction requires chlorpromazine as a shuttle oxidant, and the course of the oxidation reaction appears to be subject to the nature of the shuttle oxidant used. Preparative-scale incubations have resulted in the isolation of three products, which were characterized by chemical and spectral analyses. The metabolites were identified as the ring fission product catharinine, obtained by oxidation of the Iboga ring system; an enamine/ether derivative obtained by oxidation of the Aspidosperma portion of VLB; and a metabolite embodying the same structural changes in both parts of the vinblastine dimeric structure. Catharinine is identical with the product of VLB oxidation obtained by peroxidase oxidation. The other two products are new metabolites and are derivatives of VLB. All of the metabolites are less active than VLB when tested in vitro vs the human T-cell leukemic cell line (CRFF-CEM).

In vitro metabolic transformations of vinblastine: oxidations catalyzed by peroxidase.

Vinblastine is converted to a single major metabolite during in vitro enzymatic oxidations catalyzed by horseradish peroxidase in the presence of hydrogen peroxide. Preparative-scale enzyme incubation permitted the isolation of sufficient amount of the transformation product for complete structural identification and biological evaluation. The metabolite was identified as catharinine (also known as vinamidine) by 1H and 13C NMR and by mass spectrometry. Incubations conducted in H2(18)O-enriched water gave catharinine in which a single atom of 18O was incorporated into the metabolite structure. The labeling experiment provided evidence for an unusual ring-fission pathway by which peroxidase transforms vinblastine to catharinine. Catharinine is 77 times less active than vinblastine when tested in vitro against the human T-cell leukemic cell line (CRFF-CEM).

N-Substituted sulfonamide carbonic anhydrase inhibitors with topical effects on intraocular pressure.

N-Methylacetazolamide was shown to be active topically in reducing intraocular pressure (IOP) to a small but statistically significant level in the normotensive rabbit eye. In vivo experiments with N-methylacetazolamide suggest that ocular metabolism to acetazolamide was responsible for the observed topical activity. Examination of initial rate kinetics of carbonic anhydrase catalyzed p-nitrophenyl acetate hydrolysis showed that N-methylacetazolamide was a competitive inhibitor, in contrast to noncompetitive inhibition seen with acetazolamide and other primary sulfonamide inhibitors. N-Substituted and unsubstituted 4-chlorobenzene- and 4-nitrobenzenesulfonamides were also synthesized, and their biochemical characteristics and in vivo ability to lower IOP when applied topically were determined. The primary sulfonamides were reversible noncompetitive inhibitors of carbonic anhydrase, with no effect on IOP after topical administration. 4-Nitrobenzene- and 4-chlorobenzenesulfonamides containing both N-hydroxy and N-methyl substituents were model irreversible inhibitors of carbonic anhydrase and exhibited a trend toward topical activity in reducing IOP in normotensive rabbit eyes. Therefore, this paper describes the synthesis and characterization of two types of carbonic anhydrase inhibitors; the N-methyl-substituted sulfonamides are reversible competitive inhibitors of carbonic anhydrase, while the N-hydroxy-N-methyl-substituted sulfonamides are irreversible inhibitors.

Characterization of arylamine acetyltransferase in the rabbit eye.

The activity of arylamine acetyltransferase with p-aminobenzoic acid (PABA), sulfamethazine (SMZ), and aminozolamide as substrates was studied in rabbit tissue homogenates of the corneal epithelium, stroma-endothelium, iris-ciliary process, and liver. Rabbits were classified as rapid or slow acetylators with respect to their rate of hepatic acetylation of SMZ. The ocular disposition of aminozolamide in the two phenotypes was compared using a topical ocular infusion method that permitted a constant concentration to remain in contact with the intact cornea. The effect of hepatic-acetylator phenotype on the intraocular pressure (IOP) recovery rate and drug concentrations in tissues after single-dose administration of aminozolamide also was studied. In general, the rank order of arylamine acetyltransferase activity regardless of substrate was liver greater than iris-ciliary process greater than corneal epithelium greater than stroma-endothelium. The specific activity with aminozolamide as substrate was greater than that with SMZ in each tissue homogenate and greater than with PABA as substrate in all tissues except the stroma-endothelium of slow hepatic-acetylator rabbits. Very low enzyme activity ratios for ocular acetylation between rapid and slow hepatic-acetylating rabbits indicated that acetylation in the ocular tissues did not correspond with the acetylation phenotype. At various times during and after topical infusion to the anesthetized rabbit, assay determinations of drug and metabolite in ocular tissues indicated that there were no significant differences between phenotypes in the disposition of either drug or metabolite. These results correlate with the IOP measurements after topical infusion; they also showed no difference in the effect of aminozolamide between hepatic-acetylator phenotypes. These results indicate that the ocular disposition and the decrease in IOP from topical application of aminozolamide is independent of the hepatic-acetylation phenotype in the rabbit. There are significant amounts of acetyltransferase activity in the ocular tissues of the rabbit with these three substrates, indicating that acetylation may be occurring for other arylamine drugs used in the eye.

Ocular disposition of aminozolamide in the rabbit eye.

We have previously determined that 6-amino-2-benzothiazolesulfonamide (aminozolamide) significantly lowers IOP in rabbits and, more importantly, in ocular hypertensive human subjects. Results from in vitro experiments established that both the inhibitory activity of aminozolamide against carbonic anhydrase B as well as the penetration rate across excised rabbit corneas were equivalent to ethoxzolamide. Consequently, we have investigated the ocular disposition of aminozolamide to explain its activity when instilled topically to the eye. A constant concentration of 67.4 micrograms/ml of drug was applied for 90 min to the left eye of anesthetized rabbits. Drug and metabolite were measured in both aqueous humor and iris/ciliary body over time. The metabolite was collected and purified. Identification using mass spectroscopy, high pressure liquid chromatography (HPLC) and fluorescence scans indicated that the metabolite was 6-acetamido-2-benzothiazolesulfonamide. Relatively high levels of metabolite were identified in the cornea and iris/ciliary body but were much lower in aqueous humor. Tissue concentrations over time for the metabolite in iris/ciliary body were approximately 2-fold higher than levels of metabolite measured in aqueous humor. When compared to drug levels measured in either aqueous humor or iris/ciliary body, metabolite levels in these respective tissues were much higher. It is hypothesized that topical activity is a consequence of both metabolite retention in the iris/ciliary body as well as inhibition of 99+% of carbonic anhydrase. Both of these events must occur over a sufficient time period to effect a significant lowering of IOP.

Peroxidase as a model for reduction of tertiary amine oxides catalyzed by rat hepatic supernatant and microsomal fractions.

Rat hepatic microsomal and 100,000 g supernatant fractions catalyzed an NADH- and FMN-dependent reduction of amine oxides. Horseradish peroxidase (HRP) served as a model for the amine oxide reductase located in rat hepatic 100,000 g supernatant fraction. The HRP-catalyzed reaction displayed saturation kinetics with respect to NADH and the amine oxide substrate; however, there was an optimum concentration for FMN after which inhibition was observed at increased concentrations of FMN. The reductase in the 100,000 g hepatic supernatant fraction closely paralleled HRP-catalyzed amine oxide reduction in coenzyme requirements, sensitivity to inhibitors, and substrate specificity. Moreover, the peroxidase activity of HRP and microsomal and 100,000 g supernatant fractions correlated with the NADH- and FMN-dependent amine oxide reductase activities of these enzyme preparations. The NADH- and FMN-dependent amine oxide reductase activity in 100,000 g supernatant fractions, however, did not parallel the aldehyde oxidase activity. Thus, the results indicate that there is an amine oxide reductase in rat hepatic 100,000 g supernatant fraction with catalytic properties that are modeled well by horseradish peroxidase.

Molecular specificity of aryl sulfotransferase IV (tyrosine-ester sulfotransferase) for xenobiotic substrates and inhibitors.

Studies on the interactions of benzylic alcohols, aldehydes, and carboxylic acids with homogeneous preparations of aryl sulfotransferase (AST) IV have yielded information about the nature of the active site of the enzyme. Lipophilicity and stereochemical configuration of benzylic alcohols are key factors in determining their interaction with the active site of AST IV. Furthermore, aldehydes and carboxylic acids corresponding to the subsequent oxidation states derived from benzylic alcohols are inhibitors of the enzyme. Additional investigations on the catalytic specificity of AST IV indicate that both primary and secondary N-hydroxy arylamines can serve as substrates for the enzyme. These results with benzylic alcohols, aldehydes, carboxylic acids, and N-hydroxy arylamines have yielded insight into some of the parameters important in recognition of substrates and inhibitors by the active site of the enzyme and should be useful both in understanding in vivo metabolic interactions and in designing appropriate new inhibitors to use as selective probes for the role of sulfation in metabolism of specific xenobiotics.

Studies on an affinity label for the sulfuryl acceptor binding site in an aryl sulfotransferase.

Active site-directed affinity labeling was utilized to elucidate peptide sequences at the binding site for sulfuryl acceptors in rat hepatic aryl sulfotransferase (AST) IV (also known as tyrosine-ester sulfotransferase, EC 2.8.2.9). The affinity labeling reagent, N-bromoacetyl-4-hydroxyphenylamine, was designed on the basis of substrate specificity studies with para-substituted phenols, utilization of a bromoacetamido group for reactivity with active site amino acid residues and its similarity to acetaminophen, a known substrate for aryl (phenol) sulfotransferases. AST IV utilized N-bromoacetyl-4-hydroxyphenylamine as a substrate with kinetic constants that compared favorably to those obtained with acetaminophen. Incubation of AST IV with N-bromoacetyl-4-hydroxyphenylamine at pH 7.0 in the absence of PAPS and other substrates resulted in an irreversible inactivation of the enzyme that was both time- and concentration-dependent. [14C]-N-bromoacetyl-4-hydroxyphenylamine was synthesized and used to analyze the regions of protein sequence that were involved in the binding of the affinity label. AST IV was incubated with [14C]-N-bromoacetyl-4-hydroxyphenylamine, hydrolyzed with endoproteinase Lys-C and the labeled peptides were purified by HPLC. Control incubations of AST IV with the affinity label in the presence of 4-propylphenol and PAP were utilized to ascertain the specificity of the interaction. Sequence analysis of the labeled peptides, carried out by automated Edman degradation, revealed labeling sites on cysteine (Cys-232, Cys-283 and Cys-289) and lysine (Lys-286) residues near the C-terminus of the protein. The locations of these labeling sites were further evaluated both by sequence-alignment with other sulfotransferases and by theoretical calculations on predicted secondary structure.

In vitro and ex vivo hydrolysis rates of ethacrynate esters and their relationship to intraocular pressure in the rabbit eye.

Esters of ethacrynic acid and partial structural analogs were synthesized and evaluated for topical antiglaucoma activity in rabbits. Maximum activity was shown by analogs 2 and 6 (34% and 30% reduction in intraocular pressure recovery rate, respectively). Among the esters, only the ethyl ester (2) was found to be active; the methyl and n-propyl esters (1 and 3) were inactive. Analogs 1-3 were subjected to an estimation of physicochemical properties and chemical stability. However, no correlation was found to exist between the biological activity/inactivity and the physicochemical properties of the analogs. The analogs were evaluated for ex vivo hydrolysis using rabbit aqueous humor (AH), corneal (C) homogenate and iris-ciliary body (ICB) homogenate. For all tissues, the rate of enzymatic hydrolysis increased significantly with an increasing ester chain length. The ICB-mediated hydrolysis was the fastest among the three tissues for all of the analogs. The relationship between the rate constants for the tissue-mediated hydrolyses were: analog 1, ICB>C>AH; analog 2, ICB>C=AH and analog 3, ICB>AH>C. Apparent Michaelis-Menten kinetic parameters were determined for the three analogs using corneal homogenate. Analog 2 showed the highest v0 for all substrate concentrations studied. The conventional Michaelis-Menten equation did not fit the data as well as a sigmoidal model. Both fits of the data showed the fastest enzyme-mediated hydrolysis for analog 2. The parameters of the sigmoidal fit of the data correlated with the activity/inactivity of the analogs. The data indicate that the major factors responsible for the observed activity/inactivity are the differences in the corneal enzymatic hydrolysis of the esters in conjunction with the rapid dynamics of ocular prodrug absorption.

Synthesis of Sterically Hindered Polychlorinated Biphenyl Derivatives.

A series of sterically hindered (methoxylated) polychlorinated biphenyl derivatives was synthesized using the Suzuki and the Ullmann coupling reaction. The Suzuki coupling with Pd(dba)(2)/2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl (DPDB) gave better yields (65-98%) compared to the classic Ullmann coupling reaction (20-38%). Despite the reactive catalyst system, no significant coupling with aromatic chlorine substituents was observed. Crystal structure analysis of four PCB derivatives revealed solid state dihedral angles ranging from 69.7° to 81.0°, which indicates that these highly ortho substituted PCB derivatives have some conformational flexibility.