Nicotine in breast fluid of nonlactating women.

Using a combination of gas chromatography, mass spectrometry, and selected ion recording techniques, we have identified nicotine and its major metabolite, continine, in the breast fluid of nonlactating women smokers. As little as 25 picograms could be measured by using the deuterated variants, [5',5'-2H]nicotine and [3,3-2H]cotinine, both as internal standards and as carriers in an inverse isotope dilution method.

In vitro studies of poison oak immunity. II. Effect of urushiol analogues on the human in vitro response.

Studies were performed to ascertain the effect of urushiol analogues on the in vitro lymphocyte blastogenesis elicited by urushiol in peripheral blood lymphocytes taken from individuals sensitized to poison oak or ivy. Urushiol is a mixture of alkylcatechols composed of a catechol ring coupled to mono-, di-, or tri-unsaturated C-15 or C-17 carbon side chains. Each of these two moieties, catechol ring and side chain, was tested for its role in eliciting reactivity. Analogues tested represented the catechol ring (3-methylcatechol), the mono- or di-unsaturated side chain (oleic or linoleic acid), and the saturated side chain coupled to a catechol ring (pentadecylcatechol), a blocked catechol ring (heptadecylveratrole), or a resorcinol (pentadecylresorcinol). Urushiol with a blocked catechol ring (urushiol dimethyl ether) was also included. Of these, only pentadecylcatechol evoked reactivity in sensitized lymphocytes, and this reactivity was only a fraction of that evoked by urushiol. This suggested that the system has some requirement for the side chain, and that the catechol ring is critical for reactivity. This was further investigated by testing the ability of some of these analogues to inhibit urushiol-specific blastogenesis. No inhibition was noted with compounds bearing the saturated side chain with modified ring structures (pentadecylresorcinol and heptadecylveratrole). However, both 3-methylcatechol and pentadecylcatechol (at equimolar concentrations) blocked reactivity. The results of our experiments suggested that although both the side chain and the catechol ring are required for reactivity, the latter is most critical. Unsaturation in the side chain is important for maximal reactivity because the saturated catechols were only partially as active as the urushiol oil. There may be a greater dose requirement for the catechol ring than for the side chain.

In vitro studies of poison oak immunity. I. In vitro reaction of human lymphocytes to urushiol.

Poison oak, ivy, and sumac dermatitis is a T-cell-mediated reaction against urushiol, the oil found in the leaf of the plants. This hapten is extremely lipophilic and concentrates in cell membranes. A blastogenesis assay employing peripheral blood lymphocytes obtained from humans sensitized to urushiol is described. The reactivity appears 1--3 wk after exposure and persists from 6 wk to 2 mon. The dose-response range is narrow, with inhibition occurring at higher antigen concentrations. Urushiol introduced into the in vitro culture on autologous lymphocytes, erythrocytes and heterologous erythrocytes produces equal results as measured by the optimal urushiol dose, the intensity of reaction, and the frequency of positive reactors. This suggests that the urushiol is passed from introducer to some other presenter cell. Although the blastogenically reactive cell is a T cell, there is also a requirement for an accessory cell, found in the non-T-cell population, for reactivity. Evidence is presented that this cell is a macrophage.

Neuroprotection by caffeine and A(2A) adenosine receptor inactivation in a model of Parkinson's disease.

Recent epidemiological studies have established an association between the common consumption of coffee or other caffeinated beverages and a reduced risk of developing Parkinson's disease (PD). To explore the possibility that caffeine helps prevent the dopaminergic deficits characteristic of PD, we investigated the effects of caffeine and the adenosine receptor subtypes through which it may act in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxin model of PD. Caffeine, at doses comparable to those of typical human exposure, attenuated MPTP-induced loss of striatal dopamine and dopamine transporter binding sites. The effects of caffeine were mimicked by several A(2A) antagonists (7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH 58261), 3,7-dimethyl-1-propargylxanthine, and (E)-1,3-diethyl-8 (KW-6002)-(3,4-dimethoxystyryl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione) (KW-6002) and by genetic inactivation of the A(2A) receptor, but not by A(1) receptor blockade with 8-cyclopentyl-1,3-dipropylxanthine, suggesting that caffeine attenuates MPTP toxicity by A(2A) receptor blockade. These data establish a potential neural basis for the inverse association of caffeine with the development of PD, and they enhance the potential of A(2A) antagonists as a novel treatment for this neurodegenerative disease.

Induction of tolerance to poison ivy urushiol in the guinea pig by epicutaneous application of the structural analog 5-methyl-3-n-pentadecylcatechol.

Previous studies have established that epicutaneous application of 5-methyl-3-n-pentadecylcatechol (5-Me-PDC), a synthetic analog of a poison ivy urushiol component, leads to immune tolerance to 3-n-pentadecylcatechol (PDC) in mice. The induction of tolerance by 5-Me-PDC may be mediated by a protein conjugate formed via selective reaction of thiol nucleophiles present on the carrier macromolecule with the corresponding o-quinone derived from the parent catechol. In order to examine further the tolerogenic properties of 5-Me-PDC, we have extended our studies to the guinea pig, the generally accepted experimental species for the study of contact allergy. The results have established that specific immune tolerance to poison ivy urushiol is induced following 2 epicutaneous applications of the PDC analog. Furthermore, we were able to show that the treated animals remained tolerant for at least 6 weeks, a period of time comparable to that observed following the intravenous administration of the O,O-bis-acetyl derivative of PDC. The data point to the possibility of developing a therapeutically effective topical tolerogen for poison ivy contact dermatitis.

Suppression of urushiol-induced delayed-type hypersensitivity responses in mice with serum IgG immunoglobulin from human hyposensitized donors.

Serum IgG immunoglobulin fractions from human subjects hyposensitized to poison ivy/oak by oral administration of urushiol suppressed the induction of delayed-type hypersensitivity (DTH) responses in mice to this hapten. This suppressive activity was hapten specific because it did not modify DTH responses to dinitrofluorobenzene (DNFB). Absorption of human serum with lymph node cells from urushiolsensitized but not DNFB-sensitized mice removed the suppressive activity, suggesting that anti-idiotypic antibodies reacting with T-cell receptors are involved.

Regulation of murine contact sensitivity to urushiol components by serum factors.

Mice epicutaneously painted with components of poison ivy urushiol oil exhibit contact sensitivity (as detected by ear swelling reactions) that persist for about 25 days. Sera taken from mice at times when the contact sensitization response is waning suppressed the induction of sensitization to 3-n-pentadecylcatechol (PDC), a urushiol component, in recipients. The suppressive serum factor was present in greatest amount 25 days after sensitization, but was no longer detectable 40 days post sensitization. Suppression was antigen-specific, absorbed out with PDC-immune, but not normal lymph node cells, and transferable with a single 0.6 ml dose 7 days prior to sensitization of recipients. Suppression was transferable by the purified IgG fraction of desensitized mice. Results indicate that contact sensitivity to urushiol in mice is regulated by serum factors.

Assessment of structural requirements for the monoamine oxidase-B-catalyzed oxidation of 1,4-disubstituted-1,2,3,6-tetrahydropyridine derivatives related to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

The monoamine oxidase B (MAO-B) substrate properties and distance measurements along the N1-C4 axis of 38 1,4-disubstituted-1,2,3,6-tetrahydropyridine derivatives, including seven newly synthesized MPTP analogs, were used to define the maximum size that can be accommodated by the MAO-B active site. Only those compounds measuring less than 12 A displayed significant MAO-B substrate properties. The behavior of various 4-substituted-1-cyclopropyltetrahydropyridine analogs also is discussed in terms of this N1-C4 distance parameter in an effort to understand factors which contribute to their substrate vs inactivator properties. We conclude that this distance parameter will predict the majority of substrates vs nonsubstrates with this class of compound.

Trapping of metabolically generated electrophilic species with cyanide ion: metabolism of 1-benzylpyrrolidine.

Incubations of 1-benzylpyrrolidine (4) and specifically deuterium-labeled analogues of 4 with rabbit liver microsomal preparations in the presence of cyanide ion have led to the characterization of 1-benzyl-2-cyanopyrrolidine (13), cis- and trans-1-benzyl-2,5-dicyanopyrrolidine (14a and 14b, respectively), and 1-benzyl-5-cyano-2-pyrrolidinone (15). The cyano adducts of the amine are thought to result from nucleophilic attack by cyanide ion on metabolically generated iminium species. The cyanolactam may be produced by mixed function oxidation of the dicyano compounds. Incubations of tritium-labeled 1-benzylpyrrolidine with rabbit liver microsomal preparations led to the reduced nicotinamide adenine dinucleotide phosphate dependent incorporation of the label into the macromolecular fraction isolated from the postincubates. Although the level of incorporation was low compared to the amount of cyano adducts formed, it is comparable to that reported for other metabolically activated cytotoxic agents. Attempts to identify the possible arene oxide rearrangement product 1-(4-hydroxybenzyl)pyrrolidine (24) as a metabolite of 4 were unsuccessful. The results have prompted us to postulate that metabolically generated iminium ions are capable of alkylating nucleophilic functionalities present on microsomal macromolecules.

Chemical and biological studies of 1-(2,5-dihydroxy-4-methylphenyl)-2-aminopropane, an analogue of 6-hydroxydopamine.

Autoxidation of the bis(O-demethyl)-p-hydroquinone metabolite of the psychotomimetic amine 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM) at pH 7.4 leads exclusively to a bicyclic imino quinone. This imino quinone is a good alkylating agent, forming covalent adducts via 1,4 addition to thiols. The autoxidation appears to be dependent on trace metal catalysis and is dramatically inhibited by components of the 10000g supernatant fraction of rabbit liver homogenates. Incubation of tritium-labeled hydroquinone with bovine serum albumin under oxidizing conditions leads to significant amounts of nonextractable radioactivity which presumably is dependent on imino quinone alkylation of nucleophilic functionalities present on macromolecules. Incubation of tritium-labeled DOM with rabbit microsomes in the presence of NADPH leads to irreversible binding of the label to macromolecular components of the microsomes. Since this binding is NADPH dependent, it is likely that metabolic conversion of DOM to the hydroquinone is involved. The imino quinone oxidation product is highly lypophilic and is capable of crossing the blood-brain barrier. Intravenous administration of tritium-labeled imino quinone to rats resulted in significant nonextractable radioactivity in brain tissue. These properties of the hydroquinone metabolite parallel those reported for the structurally related sympatholytic compound 6-hydroxydopamine and have led to the hypothesis that the psychotomimetic properties of DOM may be mediated through 6-hydroxydopamine-type interactions of the hydroquinone with important macromolecules in the brain.

Synthesis, redox characteristics, and in vitro norepinephrine uptake inhibiting properties of 2-(2-mercapto-4,5-dihydroxyphenyl)ethylamine (6-mercaptodopamine).

In an attempt to further characterize the structural features of 6-hydroxydopamine analogues that are associated with in vivo neuronal degeneration, the synthesis of 6-mercaptodopamine was undertaken. Although reaction conditions leading to the 1,4 addition of thiols to the model quinone 4-methyl-o-benzoquinone were achieved, attempts to obtain 6-thiolated dopamine analogues by this route failed. The synthesis of 6-mercaptodopamine was achieved by the regioselective thiocyanation of O,O-dimethyldopamine, followed by bis-O-demethylation and reductive cleavage of the S-cyano group. Unlike 6-hydroxydopamine, 6-mercaptodopamine was resistant to autoxidation at pH 7.4. Cyclic voltammometric analysis, however, indicated that electrochemically generated oxidation species of 6-mercaptodopamine are unstable and undergo spontaneous reaction, presumably intramolecular cyclization. In vivo tests revealed that 6-mercaptodopamine inhibits the uptake of tritium-labeled norepinephrine by isolated rat heart atria, although to a much lesser extent than 6-hydroxydopamine.

Regio- and stereochemical studies on the alpha-carbon oxidation of (S)-nicotine by cytochrome P-450 model systems.

Results from previous studies indicate that rabbit liver microsomal cytochrome P-450 catalyzes the C-5' two-electron oxidation of (S)-nicotine stereoselectivity with preferential loss of the pro-(E)-hydrogen atom trans to the pyridine ring. We now have examined the regio- and stereochemical features of the oxidation of (S)-nicotine by peroxides in the presence of various hemoproteins and by electrochemical and photochemical methods. None of these systems gave rise to the stereochemical outcomes observed with the cytochrome P-450 mediated reaction. The results of these studies are interpreted as additional evidence for the formation of a highly ordered complex between (S)-nicotine and cytochrome P-450 that directs the regio- and diasterioselective alpha-carbon oxidation of this substrate.

Synthesis of novel MPTP analogs as potential monoamine oxidase B (MAO-B) inhibitors.

The nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetahydropyridine (MPTP) is an excellent substrate and a weak inactivator of the flavoenzyme monoamine oxidase B (MAO-B). In an attempt to develop novel mechanism-based inactivators of MAO-B, we have synthesized analogs of MPTP bearing a variety of functional groups at either the N or the C(4) position and have examined their interactions with a purified MAO-B preparation isolated from beef liver. The substituents selected include allyl, propargyl, ethenyl, ethynyl, and cyclobutyl, that is, functionalities which were considered potential sources of enzyme generated electrophilic or radical intermediates that might alkylate and inactivate the enzyme. None of the C(4)-substituted compounds displayed significant enzyme inhibitor properties although some proved to be good substrates. In the N-substituted MPTP series only the 4-phenyl-1-propargyl analog was a good inhibitor. The time- and concentration-dependent inhibition of MAO-B displayed by this compound is consistent with a mechanism-based inactivation pathway and the catalytic mechanism currently held for monoamine oxidases. The results of these studies provide additional insights into the steric features of the active site of MAO-B and predict that the area in which the C(4) substituent of the tetrahydropyridine ring resides lacks a reactive nucleophilic group.

Studies on the monoamine oxidase (MAO)-catalyzed oxidation of phenyl-substituted 1-methyl-4-phenoxy-1,2,3,6-tetrahydropyridine derivatives: factors contributing to MAO-A and MAO-B selectivity.

The structural parameters responsible for the substrate and inhibitor selectivities of the monoamine oxidases (MAO) A and B remain poorly understood. This situation has improved somewhat with structure-activity studies that have been performed on nuclear-substituted pargyline derivatives and 4-substituted 1-methyl-1,2,3,6-tetrahydropyridine derivatives. The results of these studies suggest that the active site of MAO-A is sterically more accommodating than the active site of MAO-B. In the present work we have undertaken a more systematic structure-substrate activity analysis with the aid of a series of 4-phenoxytetrahydropyridine analogs substituted at the para, meta, and ortho positions of the phenyl ring with chloro, methoxy, methyl, nitro, and phenyl groups. All of the compounds proved to be good substrates for both MAO-A and MAO-B, and all were better MAO-A substrates than MAO-B substrates. The best defined structural parameter relating to selectivity again was the relatively better MAO-A substrate properties of tetrahydropyridine derivatives bearing bulky C-4 substituents. Attempts to identify stereoelectronic effects related to substrate properties and selectivity with this series of compounds were not successful. Although some structural correlates with substrate activity can be made, overall the present state of knowledge is inadequate to provide good descriptors of structural features that characterize MAO-A and MAO-B substrates.

In vitro O-demethylation of the psychotomimetic amine, 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane.

The possible relationship between metabolism and psychotomimetic activity among the methoxylated 1-phenyl-2-aminopropanes led to our investigation of the in vitro O-demethylation of 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (1, DOM, STP). Employing a sensitive and highly selective stable isotope dilution assay, we observed that rabbit liver homogenates biotransform the amine 1 to its 2-O-demethyl, 5-O-demethyl, and bis (O-demethyl) metabolite metabolites. Both monophenolic metabolites are enriched in their S enantiomers. The bis(O-demethyl) metabolite has structural, chemical, and electrochemical similarites to the sympatholytic agent "6-hydroxydopamine". The possible significance of metabolic O-demethylation in terms of the psychotomimetic properties of amine 1 is discussed.

Trapping of metabolically generated electrophilic species with cyanide ion: metabolism of methapyrilene.

The popular antihistamine methapyrilene [N,N-dimethyl-N'-(2-pyridyl)-N'-(2-thienylmethyl)-1,2-ethanediamine] recently has been shown to be a potent hepatocarcinogen. Metabolic studies with rabbit liver 100000g microsomal preparations have resulted in the partial characterization of the in vitro metabolic profile of methapyrilene. Evidence for the formation of the N-oxide and the three possible carbinolamines resulting from the NADPH-dependent oxidation of the (dimethylamino)ethyl side chain nitrogen and carbon atoms of methapyrilene is presented. Attempts to trap iminium ion intermediates with electrophilic alkylating potential by coincubating methapyrilene with sodium cyanide have led to the isolation of N-(cyanomethyl)normethapyrilene. The possibility of characterizing the iminium ion intermediate that would result from the oxidative deamination of the dimethylamino moiety was precluded by the chemical instability of the corresponding alpha-cyano amine, which undergoes a spontaneous retro-Michael reaction and hydrolysis to the corresponding amide. The results are discussed in terms of the metabolic activation of methapyrilene to potential alkylating species.

Studies on chiral interactions of 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane and the corresponding N-hydroxy metabolites with cytochrome P-450.

The stereoselective pharmacological behavior and metabolism of the potent psychotomimetic amine 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane have led to an investigation of the interactions of the racemic amine, its enantiomers, and the corresponding N-hydroxy metabolites with rabbit liver microsomal cytochrome P-450. An examination of the formation of cytochrome P-450 metabolic intermediate complexes with these species suggests that N-oxidation of the pharmacologically active (R)-amine in inhibited by the S enantiomer. Additionally, metabolic intermediate complex formation [favored by the (R)-amine] appears to be associated with loss of microsomal mixed function N-oxidase activity. The results have led to the prediction that N-hydroxylation of pure (R)-amine may be a qualitatively more important pathway than that observed with racemic amine even though this biotransformation may be suicidal.

Synthesis and physicochemical and neurotoxicity studies of 1-(4-substituted-2,5-dihydroxyphenyl)-2-aminoethane analogues of 6-hydroxydopamine.

In an attempt to evaluate the possible relationship between the neurotoxicity of 6-hydroxydopamine and the redox properties and electrophilic reactivity of the 6-hydroxydopamine-p-hydroquinone/p-quinone system, we have synthesized a series of 6-hydroxydopamine analogues in which the C4-hydroxy group is replaced with various electron-donating and electron-withdrawing substituents. With the aid of cyclic voltammetry, the formal oxidation potentials (E degrees ') for the p-hydroquinone/p-quinone redox couples and the rates of cyclization of the p-quinones to the corresponding p-iminoquinones were determined. As expected, electron-rich p-hydroquinones were easily oxidized to the p-quinones, which underwent cyclization slowly, whereas the oxidation of electron-poor p-hydroquinones required higher voltages and yielded p-quinones, which cyclized readily at pH 7.4. The neurotoxic potential of these compounds showed that in vivo destruction of noradrenergic terminals, as measured by inhibition of norepinephrine uptake by rat heart slices, occurred only with those analogues bearing electron-donating substituents. Potent neurotoxic properties were associated only with the 4-amino and 4-hydroxy derivatives, both of which form p-quinones, which do not cyclize readily at pH 7.4. These results support the thesis that the p-quinone derived from 6-hydroxydopamine may be an important species in the mediation of the neurodestruction caused by 6-hydrodopamine.

Metabolic oxidation of nicotine to chemically reactive intermediates.

Studies on the metabolism of nicotine by rabbit liver microsomal fractions in the presence of 0.01 M sodium cyanide have led to the characterization of two isomeric cyanonicotine compounds. The locations of the cyano groups were established by GC--EIMS analyses of the deuterium-labeled products obtained from the specifically deuterium-labeled substrates (S)-nicotine-5',5'-d2, (R,S)-nicotine-2',5',5'-d3, and (R,S)-nicotine-N-methyl-d3. One cyano adduct was shown to be 5'-cyanonicotine, a product previously isolated from similar microsomal preparations. The second cyano adduct was shown to be N-cyanomethyl)nornicotine; this structure assignment was confirmed by synthesis. Formation of N-cyanomethyl)nornicotine appears to occur, at least in part, without prior nitrogen--carbon bond cleavage, implicating the in situ generation of the N-methyleniminium species during the course of metabolic oxidative N-demethylation of nicotine.

Stereochemical studies on the cytochrome P-450 catalyzed oxidation of (S)-nicotine to the (S)-nicotine delta 1'(5')-iminium species.

Mammals metabolize the tobacco alkaloid (S)-nicotine primarily to the lactam (S)-cotinine by a pathway involving an initial cytochrome P-450 catalyzed two-electron oxidation at the prochiral 5'-carbon atom. The stereochemical course of this oxidation was examined with human microsomal preparations and the E and Z diastereomers of (S)-nicotine-5'd1. The metabolically generated delta 1'(5')-iminium ion intermediate was trapped and analyzed as the corresponding diastereomeric 5'-cyano derivatives by a capillary column GC-EIMS selected ion monitoring assay. The results of these studies established that this biotransformation proceeds with the stereoselective abstraction of the 5'-pro-E proton, that is, the C-5' proton trans to the bulky pyridyl group. The observed stereoselectivity was independent of proton vs. deuteron abstraction. Additionally, the extent of (S)-cotinine formation was minor and did not influence the stereochemical composition of the metabolically derived alpha-cyano amines. Studies with male Dutch rabbit liver microsomal preparations gave similar results. These findings suggest that the structure of the complex formed between (S)-nicotine and the active site of cytochrome P-450 is highly ordered and dictates the stereochemical course of the reaction pathway.