Rapid decrease in N-hydroxy-2-acetylaminofluorene sulfotransferase activity of liver cytosols from rats fed carcinogen

Sulfation of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) by N-OH-AAF sulfotransferase yields a candidate for ultimate carcinogen in hepatocarcinogenesis in rats. We have monitored this pathway during the initial phase(s) of hepatocarcinogenesis produced by feeding male Holtzman rats a diet containing 0.05% 2-acetylaminofluorene (AAF). Our studies revealed an immediate and precipitous decrease in N-OH-AAF sulfotransferase activity beginning after 1 day on the AAF diet and decreasing 4- to 5-fold after 5 days on the AAF diet. This decrease in activity remained at low values during continuous administration of AAF throughout 4 weeks but was shown to be both reversible and AAF dose dependent. Parallel monitoring of rat serum glutamic oxaloacetic acid transaminase activity during the administration of AAF indicated that no appreciable hepatocellular toxicity occurred during the period of sulfotransferase activity lowering. Other known carcinogenes, i.e. 3′-methyl- and 4′-fluoro-4-dimethylaminoazobenzene, aflatoxin B₁, thioacetamide, ethionine, and diethylnitrosamine, and the hepatotoxin α-naphthylisothiocyanate, also caused decreases in N-OH-AAF sulfotransferase activity after 7 and 28 days of administration. In contrast, very weak or non-carcinogens, i.e. p-aminoazobenzene, fluorene, and barbital, failed to reduce N-OH-AAF sulfotransferase activity during 28 days of feeding. Data from these studies on the short-term chronic administration of xenobiotics suggest (a) reduced likelihood for the direct involvement of the sulfotransferase pathway in providing sufficient cytotoxic AAF metabolites to cause compensatory hyperplasia and its putative promotion-effect for AAF-mediated carcinogenesis, and (b) the possible use of the rapid loss in sulfotransferase activity as an early indicator of hepatocarcinogenesis.     

Sequence analysis, in vitro translation, and expression of the cDNA for rat liver minoxidil sulfotransferase.

A cDNA encoding minoxidil sulfotransferase (Mx-ST), a rat liver cytosolic sulfotransferase that catalyzes the 3'-phosphoadenosine 5'-phosphosulfate-dependent sulfate conjugation of minoxidil and p-nitrophenol, has been isolated from a lambda gt11 cDNA library constructed from poly(A)+ RNA isolated from female Sprague-Dawley rat liver. The largest cDNA, designated Mx-STb, consists of 1245 base pairs and contains an open reading frame of 291 amino acids. The predicted size of the protein translated by Mx-STb is 33,909 Da; however, the molecular mass of the pure protein [Biochem. J. 270:721-728 (1990)] is estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 35,000 Da. The size of the protein obtained by in vitro translation of Mx-STb is identical to that of the pure protein. Results of initial studies of the expression of Mx-STb in COS-1 cells indicate that the expressed protein displays characteristic Mx-ST and p-nitrophenol sulfotransferase activity, is recognized by rabbit polyclonal antibodies raised against pure rat liver Mx-ST, and migrates at approximately 35,000 Da during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This paper presents the cloning and expression of a rat phenol sulfotransferase for which the physical, immunological, and kinetic properties are known. Isolation of the cDNA for Mx-ST will aid in the investigation of the heterogeneity, the tissue localization, and the characterization of the kinetic properties of this important drug-metabolizing enzyme, with respect to other similar phenol sulfotransferases present in rat liver cytosol.     

Effect of microsomal enzyme inducers on the soluble enzymes of hepatic phase II biotransformation

Numerous xenobiotics induce microsomal enzymes such as cytochrome P-450-dependent monooxygenases, epoxide hydrolase, and UDP-glucuronyltransferase by causing an increase in enzyme synthesis. Since induction of soluble enzymes involved in phase II biotransformation has not been thoroughly studied, effects of the following microsomal enzyme inducers on three important soluble enzymes were examined: phenobarbital (PB), 3-methylcholanthrene (3-MC), butylated hydroxyanisole (BHA), isosafrole (ISF), pregnenolone-16α-carbonitrile (PCN), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and trans-stilbene oxide (TSO). Representative microsomal enzymes of phase I pathways were examined simultaneously to indicate effective induction. The inducers selected produced the expected increases in hepatic cytochrome P-450 (75–170%), ethylmorphine (200–260%), and benzphetamine (100–260%) N-demethylation, benzo[a]pyrene hydroxylation (300%), ethoxyresorufin O-deethylation (2700%), and styrene oxide hydration (100–270%). The soluble conjugative enzymes studied were glutathione S-transferase, N-acetyltransferase, and sulfotransferase. Glutathione conjugation of 1,2-dichloro-4-nitrobenzene, 1-chloro-2,4-dinitrobenzene, and sulfobromophthalein was increased by TSO (100–160%), BHA (60–80%), ISF (60–80%), and PB (60–80%). β-Naphthylamine N-acetyltransferase activity was increased by PCN and 3-MC (60 and 40%, respectively). Only PCN was able to enhance sulfotransferase. Sulfation of 2-naphthol, taurolithocholate, and dehydroepiandrosterone was increased by 28, 111, and 140%, respectively. In conclusion, while microsomal enzymes could be readily induced, activity of soluble phase II enzymes was increased to a much lesser extent. Of the inducers studied, PCN was the most effective at increasing activity of soluble enzymes.     

Studies of glucuronidation and sulfation in tumor-bearing rats

Rats bearing subcutaneous Walker 256 carcinoma or intramuscular Reuber H-35 hepatoma were tested for their ability to metabolize ¹⁴C-p-nitrophenol into its sulfate and glucuronide. Amounts of p-nitrophenyl glucuronide (PNPGA) and p-nitrophenyl sulfate (PNPS) excreted by the Walker hosts did not differ significantly from those excreted by the controls. Hepatoma hosts, compared with controls, excreted comparable amounts of PNPS but significantly higher amounts of PNPGA. To clarify the basis for the changes in the conjugate levels in urine of the hepatoma hosts, sulfotransferase and glucuronyl transferase activities of the hepatoma and of host and control livers were assayed. The hepatoma possessed significantly higher glucuronyl transferase activity than either type of liver, but had a very low sulfotransferase activity. Microsomal glucuronyl transferase of the hepatoma obeyed Michaelis-Menten kinetics; K_m value for p-nitrophenol is 0.22 mM. Liver transferase(s) gave different and irregular kinetics, suggesting qualitative differences between the transferases of the hepatoma and liver.     

The hepatic metabolism of ethidium bromide to reactive mutagenic species: biochemical and structural requirements

Ethidium bromide, which was known to undergo N-acetylation in rats, is shown to be metabolized by rat liver cytochrome P448 and a soluble protein to one or several reactive species which are detected in the Salmonella assay. This biotransformation depends on the presence of a free aromatic amino group at position-3, on the basis of a structure-mutagenic activity study of eleven phenanthridine derivatives. Using the Salmonella assay as a biological test for the production of reactive intermediates, we have attempted to purify this enzyme and demonstrated its lack of N-acetyl- or sulfotransferase activity. Ethidium bromide thus behaves as a mutagenic aromatic amine, the bioactivation of which differs from previously described mechanisms.     

Rat phenol sulfotransferase: Assay procedure,developmental changes,and glucocorticoid regulation

Phenol sulfotransferase (PST) catalyzes the sulfate conjugation of phenolic monoamines and phenolic drugs. It has been difficult to measure PST activity in tissue homogenates accurately because of the presence of potent endogenous PST inhibitors. Optimal conditions were determined for the assay of rat PST in very dilute tissue homogenates. These conditions negated the effects of endogenous enzyme inhibitors. Apparent K_m values for 3-methoxy-4-hydroxyphenylglycol, the sulfate acceptor substrate used, were 0.15, 0.14, and 0.02 mM for liver, kidney, and brain homogenates respectively. Apparent K_m values in the same tissues for 3'-phosphoadenosine-5'-pnosphosulfate, the sulfate donor, were 0.11, 0.07, and 0.07 μM respectively. Rat PST activity expressed per mg protein increased 6.3-fold in the liver, 6.6-fold in the brain, and did not change in the kidney between birth and 10 weeks of age. There was a 5-fold increase in kidney PST activity in both adrenalectomized and sham-operated Sprague-Dawley rats after treatment with dexamethasone (7 μmoles/kg daily for 3 days). Brain enzyme activity was unchanged and liver PST activity increased only 41% during 72 hr of daily treatment with dexamethasone. Basal enzyme activities in all three tissues were no different in adrenalectomized and sham-operated animals. The increase in rat kidney PST activity in response to dexamethasone was dose dependent, and treatment of animals with cycloheximide, a protein synthesis inhibitor, blocked the elevation of kidney PST activity after dexamethasone. Treatment of eight inbred and two outbred rat strains with dexamethasone resulted in striking increases in renal PST, smaller increases in liver PST, and no changes in brain enzyme activity in all ten strains.     

Separation of water-soluble metabolites of benzo[a]pyrene formed by cultured human colon.

A method has been developed to separate conjugated metabolites of benzo[a]pyrene into three major fractions: sulfate esters, glucuronides and glutathione conjugates. In cultured human colon, formation of sulfate esters and glutathione conjugates is the major conjugation pathway, while formation of glucuronides accounts for only 6 per cent of the water-soluble metabolites. Hydrolysis of the sulfate esters with arylsulfatase and the glucuronides with β-glucuronidase released metabolites of benzo[a]pyrene that were extractable with organic solvent. Separation of these metabolites by high-pressure liquid chromatography indicated that trans-4,5-dihydro-4,5-dihydroxybenzo[a]pyrene,7,8,9, 10-tetrahydro-7,8,9, 10-tetrahydroxybenzo[a]pyrene and trans-9, 10-dihydro-9, 10-dihydroxybenzo[a]pyrene were the major substrates for UDP-glucuronic acid transferase, while trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene and 9-hydroxybenzo[a]pyrene were the major substrates for sulfotransferase in cultured human colon.     

N-oxidation of N,N-dimethylaniline in the rabbit and rat lung

We have reported previously that chlorpromazine (CPZ) and imipramine (IMP) are metabolized via N-oxidation by the rat lung, while they are not appreciably metabolized by the rabbit lung. Indeed, marked species differences exist in the pulmonary N-oxidation of these pneumophilic drugs. In the present studies, the isolated, ventilated and perfused lung (IPL) preparations as well as in vitro preparations of the rabbit and rat lungs were used to examine the pulmonary disposition of [¹⁴C]-N,N-dimethylaniline (DMA) which has been used frequently as a substrate for N-oxidation. Although the IPLs of both species were active in DMA N-oxidation, the rabbit lung was more active in DMA N-oxidation than the rat lung on the basis of per g lung. The gradual decline in radiolabel concentration in the perfusate was more marked in the rat than in the rabbit IPL. This decline was not due to the drug accumulation in the lung, but to its volatility. There was no dose dependency in the tissue/medium DMA concentration ratios (approximately 1.60), indicating uptake by simple diffusion and low affinity for the lung tissue. In vitro lung preparations showed higher DMA N-oxidase activity in the rabbit than in the rat, regardless of whether whole homogenate, post-mitochondrial supernatant fraction or microsomal fraction was used, or how the activities were expressed (per mg protein or per g tissue). These results suggest that, although DMA is not highly concentrated in the lung, it is N-oxidized by the lung and that DMA N-oxidase is different from CPZ or IMP N-oxidase reported previously.     

The formation and metabolism of N-hydroxymethyl compounds—I: The oxidative N-demethylation of N-dimethyl derivatives of arylamines,aryltriazenes, arylformamidines and arylureas including the herbicide monuron

The metabolism of the N-methyl moieties of aryldimethylamines and N-methyl compounds of the general formula Aryl-X-N(Me)₂, where X is either —N=N— (3-aryl-1,1-dimethyltriazenes), —NHCO— (N′-aryl-N,N-dimethylureas) or —N=CH— (N′-aryl-N,N-dimethylformamidines) was studied using mouse liver microsomes. Products of microsomal metabolism were reincubated with mouse liver homogenate devoid of microsomes and assayed colourimetrically for formaldehyde. This allows metabolically generated formaldehyde to be distinguished from formaldehyde precursors. Whereas the N-methyl moieties of the aryldimethyltriazenes, formamidines and amines were metabolised to formaldehyde, the aryldimethylureas formed stable formaldehyde precursors upon metabolism. The products of metabolism of one such aryldimethylurea, the herbicide monuron (N′-(4-chlorophenyl)-N,N-dimethylurea) were investigated using a high pressure liquid chromatographic method. Two metabolites were found on incubation of monuron with microsomes, one of which was identified as the N-desmethyl compound by mass spectrometry. The other product showed chromatographic properties similar to 4-chlorophenylurea but resembled the monomethylaryl urea on mass spectral analysis. It is concluded that this metabolite is likely to be N′-(4-chlorophenyl)-N-hydroxymethyl-N-methylurea. A urinary product of conjugative metabolism obtained after the administration of monuron to mice also gave the mass spectrum of the monomethyl compound after deconjugation which suggests that a conjugated N-hydroxymethyl compound may have been formed in vivo.     

Radioisotopic assay for rat liver sulfotransferase activity.

A hepatic sulfotransferase enzyme catalyzing the transfer of sulfate-(³⁵S) from 3′-phosphoadenosine-5′-phosphosulfate-(³⁵S) to p-nitrophenol was partially purified by differential centrifugation and (NH₄)₂SO₄ fractionation. Employing this enzyme, a radioisotopic procedure for assaying rat liver sulfotransferase activity is described. The enzyme activity was linear for the first 10 min of incubation and with a protein concentration up to 0·2 mg/ml of incubation mixture. Enzyme activity was present within a pH range of 5·4 to 8·4; however, maximum sulfate transfer activity occured around pH 6·4. Seventy-one per cent of the initial enzyme activity was present after almost 2 months of storage at ?75°, while the activity of enzyme stored at 4 and ?20° had decreased to 32 and 30 per cent of the original activity respectively. In addition, the Michaelis constant (K_m) and maximal velocity were determined for p-nitrophenol, salicylamide and dehydroepiandrosterone.     

Simultaneous effects of tocopheryl polyethylene glycol succinate (TPGS) on local hair growth promotion and systemic absorption of topically applied minoxidil in a mouse model

In this study, topical minoxidil solutions supplemented with TPGS in cosolvent systems of various compositions consisting of water, alcohol, and polyethylene glycol 400 were designed to evaluate the efficacy of promoting hair growth after topical application and the safety in terms of the amount of minoxidil absorbed through the skin into the circulation using C57BL/6J mice as a model. The commercial product of 2% Regaine) was used as the positive control. The role, which sulfotransferase activity plays in hair growth with treatment using minoxidil, was determined as well. The results revealed that the addition of 0.5% TPGS was able to enhance the proliferation of hair, but an increase in the amount of TPGS to 2% led to deterioration in the enhancement of hair growth. At the higher added amount (2.0%) of TPGS, the promotion of hair growth was slightly reduced for both cosolvent formulations F1 (100% water) and F3 (100% PEG 400), whereas it was reduced to a greater extent for the cosolvent formulations F8-F10. In comparison, the influences of cosolvent compositions with TPGS amounts of 0.0 and 2.0% on the promotion of hair growth were similar. On the contrary, variability in the promotion of hair growth by different solvent formulations was minimal when the added amount of TPGS was 0.5%. In general, a relationship between hair growth and sulfotransferase activities after topical application of 2% Regaine and minoxidil formulations containing various amounts of TPGS was not demonstrated. Plasma concentrations of minoxidil with 2% Regaine were found to be greater than those of 2% minoxidil in those cosolvent formulations containing various amounts of TPGS, while showing insignificant differences among those 10 cosolvent formulations with a fixed amount of TPGS. A tendency for the plasma concentration of minoxidil to increase after the topical administration of minoxidil formulations containing the higher amount of TPGS (2%) was noted.     

Identification of metabolites from salts of pyridine-2-thiol-1-oxide following intravenous and dermal adminstration to swine

Mature female Yorkshire swine were given ¹⁴C-labeled zinc pyrithione (ZPT), sodium pyrithione (NPT), and the magnesium sulfate adduct of 2,2′-dithio-bis(pyridine-1-oxide) (MDS) iv and by dermal application. The urine from the iv study contained one major and two minor metabolites. Analyses of these by gas chromatography-mass spectrometry indicated that the major metabolite was the S-glucuronide of 2-mercaptopyridine-N-oxide and one of the minor metabolites was the S-glucuronide of 2-mercaptopyridine. The metabolic pattern after the dermal application was considerably different. Chromatographic evidence indicated that the major metabolites were 2,2′-pyridyldisulfide and 2-(pyridyl-N-oxide) sulfonic acid.     

Pharmacology of [3H]apomorphine binding to bovine brain tissue

Activity against high-affinity (nM), and over 80 per cent dopamine (DA)-agonist-displaceable binding of [³H]apomorphine (APO) to a “subsynaptosomal” membrane fraction of bovine caudate nucleus was evaluated using amino-6,7-dihydroxytetralin (ADTN) as a blank. Aporphines with 10,11-catechol and small N-alkyl groups were potent inhibitors ($\text{IC}{}_{50}\text{< 25}\text{nM}$) with parallel inhibition curves (n_h = 0.8 to 1.0). For phenethylamines, catechol and amine moieties were important structural requirements; α or β substitution markedly reduced potency. Thus, epinephrine and isoproterenol were weak while DA and its N-methylated congeners were potent; (? )norepinephrine (NE) and fluoro-DAs were intermediate in potency and ( + )NE, other catechols and hydroxylated phenethylamines were virtually inactive; m-tyramine, while weak, was more active than p-tyramine. Other DA agonists [ADTN > lergotrile >N-n-propyl-3-(3-hydroxyphenyl)-piperidine (3-PPP) > bromocriptine] were active. Neuroleptics competed relatively weakly, with imperfect correspondence to in vivo activities. Stereoselectivity was found with several aporphines, phenethylamines, and antipsychotic drugs. Many other neuropharmacologically active agents, including inhibitors of amine uptake and adrenergic receptors, were inactive. These characteristics strongly suggest that APO interacts with a DA agonist binding site in mammalian brain tissue.     

Metabolic activation of 2-aminofluorene by isolated rat liver cells through different pathways leading to hepatocellular DNA-repair and bacterial mutagenesis

The aromatic amine 2-aminofluorene (2-AF) is metabolized by isolated rat liver cells to reactive species, thereby causing mutagenic effects in Salmonella typhimurium TA 1538 and evoking DNA-excision repair within the liver cells. The pathway leading to the production of metabolites mutagenic in Salmonella is likely to proceed via direct N-hydroxylation of 2-AF to N-hydroxy-2-aminofluorene (N-OH-2-AF). On the other hand, the formation of intermediates giving rise to hepatocellular DNA-repair is shown to depend upon N-acetylation of 2-AF to 2-acetylaminofluorene(2-AAF), whereas a subsequent conjugation reaction, most likely to be sulfate ester formation, is also essentially involved.     

Functional nephrotoxicity of gentamicin in the rat

Gentamicin sulfate (GM), 100 mg/kg, was administered once daily for up to 4 days to adult male rats and was found to concentrate in cortical regions of the kidney. Uptake of the organic base N-methylnicotinamide (NMN) by rental cortical slices was reduced by GM treatment, whereas uptake of the organic acid p-aminohippurate (PAH), the sugar analog α-methyl-d-glucopyranoside (AMG), and the amino acid analog α-aminoisobutyrate (AIB) were unaffected. Gentamicin reduced sulfate renal slice formation of glucose and ammonia and the rate of slice respiration. In addition, GM increased urinary excretion of glucose and β-d-galactosidase (β-d-Gal), a proximal tubular cytoplasmic enzyme. These results have led to the hypothesis that GM is a mitochondrial toxin which exerts deleterious effects specifically on the kidney because of selective uptake in this organ.     

Mutagenesis of certain benzo(a)pyrene phenols in vitro following further metabolism by mouse liver.

Low concentrations of 1-hydroxy- and 3-hydroxybenzo[a]pyrene in the presence of NADPH and liver S-9 fraction from 3-methylcholanthrene-treated C57BL/6N mice are as much as 3-fold more mutagenic than benzo[a]pyrene in the bacteria Salmonella typhimurium LT₂ tester strain TA98. The level of mutagenicity rises with increasing phenol or S-9 protein concentration. In this system, 9-hydroxy-benzo[a]pyrene is slightly mutagenic, while 2-hydroxy-, 7-hydroxy- and 12-hydroxybenzo[a]pyrene are not mutagenic at low concentrations. The S-9 fraction from 3-methylcholanthrene-treated DBA/2N mice or phenobarbital-treated C 5 7BL/6N mice does not support significant levels of mutagenesis. The high level of mutagenicity by 1-hydroxy- or 3-hydroxybenzo[a]pyrene is inhibited by α-naphthoflavone but is not inhibited by metyrapone, 1, 2-epoxy-3, 3, 3-trichloropropane or glutathione. The substrate for UDP-glucuron-osyltransferase, UDP-glucuronic acid, prevents more than half of the mutagenicity caused by the further metabolism of 1-hydroxy- and 3-hydroxybenzot alpyrene. The combination of UDP-glucuronic acid and UDP-N-acetylglucosamine provides an even higher level of protection. The addition of the substrate for sulfotransferase(s), 3'-phosphoadenosine 5'-phosphate sulfate, also prevents about half of the mutagenesis caused by 1-hydroxy- or 3-hydroxybenzo[a]pyrene.     

Metabolism of chlorpromazine by pulmonary microsomal enzymes in the rat and rabbit

Pulmonary metabolism of chlorpromazine (CPZ) was compared using isolated microsomes of rat and rabbit lungs. CPZ-metabolizing activity of the rat lung was found to be 10-fold higher than that of the rabbit lung. The principal metabolic pathways were N-oxidation in the rat lung and N-demethylation in the rabbit lung. Kinetic analyses revealed that, although the values for apparent K_m were roughly similar for both pathways, V_max for N-oxidation by the rat lung was approximately ten times greater than that for N-demethylation by the rabbit lung. N-Oxidation by the rat lung had a broad range of pH optimum of 7–8, whereas N-demethylation by the rabbit lung had a pH optimum 8–9. SKF525-A, piperonyl butoxide, n-octylamine and CO did not inhibit N-oxidation by the rat lung, but inhibited N-demethylation by the rabbit lung. SKF525-A and n-octylamine stimulated the CPZ-N-oxidation by the rat lung. Hg²⁺ and Mg²⁺ inhibited N-oxidation by the rat lung. These results indicate that pulmonary metabolism of CPZ in the rat is catalyzed by a microsomal flavoprotein monooxygenase, while pulmonary metabolism in the rabbit is catalyzed by a cytochrome P-450 monooxygenase system, and that a marked species variation exists with respect to pulmonary metabolism of CPZ.     

Inhibition of brain epinephrine synthesis by 3,4-dichlorophenylethanolamine,a competitive substrate for norepinephrine N-methyltransferase

3,4-Dichlorophenylethanolamine (DCPE) is a substrate for norepinephrine N-methyltransferase (NMT) from rat brain stem or from rabbit adrenal glands and competitively inhibits the methylation of (?)norepinephrine by NMT in vitro. The K_i value for inhibition of rat brain NMT by DCPE was 6 × 10^?5 M. When DCPE was injected i.p. into rats at a dose of 50 mg/kg, epinephrine concentration in hypothalamus was reduced, although NMT activity measured in hypothalamic homogenates in vitro was not inhibited. The findings contrast in several ways to those with 8,9-dichloro-2,3,4,5-tetrahydro-1H-2-benzazepine (LY134046), a competitive inhibitor of rat brain NMT with an in vitroK_i of 2.4 × 10^?8 M. Although the potency of DCPE was 1/2500th that of LY134046 in vityro, it lowered hypothalamic epinephrine concentrations at similar doses in vivo. The NMT activity measured in vivo in homogenates of hypothalamic tissue from LY134046-treated rats was reduced by a greater percentage than was epinephrine concentration. The reduction of epinephrine concentration in vivo by DCPE may relate to its ability to be a substrate for NMT, which would result in conversion of S-adenosylmethionine (SAMe) to S-adenosylhomocysteine (SAH) in NMT-containing neurons. The resulting increase in the SAH/SAMe ratio may inhibit norepinephrine N-methylation, since SAH is a competitive inhibitor of NMT when SAMe is the variable substrate. This mechanism is similar to that suggested for the decrease in epinephrine concentration in rat brain following l-dopa injection. l-Dopa injection increases SAH and decreases SAMe concentration measured in hypothalamus, due presumably to extensive O-methylation of dopa and its decarboxylated metabolites. In contrast, SAH and SAMe concentrations in hypothalamus were not measurably altered after DCPE injection, a not-unexpected finding since SAH/SAMe changes would occur only in NMT-containing neurons, which make up a negligibly small percentage by weight of hypothalamic tissue. DCPE and other NMT substrates may be able to inhibit epinephrine synthesis in vivo as effectively as non-substrate, competitive inhibitors having much higher affinity for NMT because their N-methylation elevates SAH/SAMe ratios specifically within NMT-containing neurons.     

Mixed-function amine oxidase of the rat hepatocyte nuclear envelope: Demonstration and effects of phenobarbital and 3-methylcholanthrene

Mixed-function amine oxidase (EC 1.14.13.8) has been demonstrated in highly purified rat hepatocyte nuclear envelope. The enzyme was present in the nuclear envelope at a level 20 percent of that observed in microsomes. Induction studies indicated that nuclear envelope amine oxidase as well as its microsomal counterpart were refractory to the effects of phenobarbital and 3-methylcholanthrene. Phenobarbital administration increased the specific activity of the microsomal N,N-dimethylanilineN-demethylase and benzo[a]pyrene hydroxylase by 600 and 190 percent, respectively, but decreased the specific activity of the nuclear enzymes by 30–50 percent. In contrast, 3-methylcholanthrene increased the specific activity of benzo[a]pyrene hydroxylase in nuclear envelope and microsomes by 42- and 11-fold, respectively. The hydrocarbon also increased the microsomal and nuclear N,N-dimethylanilineN-demethylase by 40 and 60 percent, respectively, but the specific activity of microsomal and nuclear aniline 4-hydroxylase was decreased by 50 percent. Demonstration of amine oxidase in rat hepatocyte nuclear envelope implicates this enzyme in the toxicity and carcinogenicity of certain drugs and chemicals.     

Studies on the cyanamide-ethanol interaction: Dimethylcyanamide as an inhibitor of aldehyde dehydrogenase in vivo

Administration of dimethylcyanamide (DMC) to rats caused a marked elevation in ethanol-derived blood acetaldehyde (AcH) and depressed the specific activity of the low K_m mitochondrial aldehyde dehydrogenase (AlDH) by 90% at 12–24 hr, coincident with depletion of hepatic glutathione levels. Comparison of the relative efficacy of DMC and cyanamide in elevating blood AcH measured at 2hr and 1 hr post-drug treatment, respectively, indicated that DMC was at least one-fifth as active as cyanamide. However, since the comparison was not made at optimal times for DMC (12–24 hr), it is likely that its activity in vivo approaches that of cyanamide itself. DMC was essentially inactive in vitro as an inhibitor of the low K_m AlDH isozyme in intact rat liver mitochondria. Although methylcyanamide, the product of N-demethylation of DMC, was too unstable to be prepared for this evaluation, the higher monoalkyl cyanamide, n-propylcyanamide, was synthesized chemically and was shown to be a good inhibitor of the mitochondrial enzyme in vitro. These results suggest that DMC must be N-demethylated before being converted to a reactive species that inhibits AlDH activity.