Formation of quaternary amines by N-methylation of azaheterocycles with homogeneous amine N-methyltransferases.

Catalytic activities of two amine N-methyltransferases were documented for the following azaheterocycles: isomeric phenyl- and bispyridyls; 2-, 3- and 4-mono-substituted pyridines; and a miscellaneous group of azaheterocycles that included mono- and diazabenzenes and mono- and diazanaphthalenes. The broad substrate specificities of the two amine N-methyltransferases for primary and secondary amines are here extended to a large number of aromatic azaheterocycles in which N-methylation results in the formation of quaternary ammonium metabolites. Pyridine was the best substrate for both enzymes. Substitution in the ring at the 2-position sterically hindered methylation of the pyridyl nitrogen; 2-phenylpyridine and 2,2'-bispyridyl were not substrates.

In vivo depletion of S-adenosyl-L-homocysteine and S-adenosyl-L-methionine in guinea pig lung after chronic S-(-)-nicotine administration.

Guinea pig lung tissue is dramatically depleted of S-adenosyl-L-homocysteine (SAH) after chronic exposure (600 micrograms/h s.c. for 21 days) of animals to either R-(+)- or S-(-)-nicotine enantiomers; S-(-)-nicotine decreased lung SAH levels by 60-fold, while R-(+)-nicotine caused an 11-fold reduction of SAH, relative to control values. Lung S-adenosyl-L-methionine (SAM) levels were also reduced (15-fold and 9-fold reductions with R-(+)- and S-(-)-isomers, respectively) in nicotine-treated animals, compared to controls. These depletions are more pronounced with the S-(-)-enantiomer. Liver tissue levels of SAH and SAM are less affected, in fact, a 4-fold increase in liver SAH levels was observed after exposure of animals to R-(+)-nicotine. These results indicate that chronic exposure to nicotine may perturb important endogenous methyltransferase reactions, which could be a contributory factor in the toxicological effects produced by cigarette smoking.

Effect of propylene glycol 1,2-dinitrate on cerebral blood flow in rats: a potential biomarker for vascular headache?

Two measurable indices of toxicity that can be correlated with exposure to propylene glycol dinitrate (PGDN) were evaluated along with its metabolism. Propylene glycol dinitrate was administered by rapid i.v. injection to male Fischer-344 rats. These rats demonstrated a dose-response of blood pressure (BP) to doses of PGDN ranging from 0.1 to 30 mg/kg; the maximum fall in systolic BP occurred within 1 min of dosing. The i.v. administration of PGDN to separate groups of animals resulted in an increase in cerebral blood flow that was correlated with the dose, but a clear dose-response was not obtained.

Lack of detectable metabolism for solubilized 2,3,4-trimethylpentane by rat kidney proximal tubules.

Primary proximal tubule suspension cultures exposed to solubilized 2,3,4-trimethylpentane (2,3,4-TMP) resulted in a linear dose response, as determined by cellular lactate dehydrogenase leakage. The EC50 for 2,3,4-TMP was 16.3 mM. Metabolite analysis by gas chromatography/mass spectrometry of supernate and cell extracts from cultures exposed to 2,3,4-TMP (12.0 mM) failed to detect the presence of metabolites. Electron-microscopic examination of proximal tubules exposed to 2,3,4-TMP indicated ultrastructural changes that included increased mitochondrial swelling, increased vesiculation, decreased microvilli and pyknotic nuclei. This study indicates that kidney proximal tubules do not appear to metabolize 2,3,4-TMP.

Effect of continuous administration of nicotine on urinary histamine and N tau-methylhistamine levels in the guinea pig.

The effect of continuous subcutaneous administration of S-(-)- and R-(+)-nicotines on urinary excretion levels of histamine and N tau-methylhistamine in guinea pigs, over a 23-day period, has been studied. Urinary levels of these endogenous compounds were measured utilizing paired-ion reversed-phase high performance liquid chromatography with flow-through electrochemical detection. Urinary histamine levels of animals that had been administered either of these nicotine isomers were not significantly different from control values. Initial levels of urinary N tau-methylhistamine (days 2-3) in R-(+)- and S-(-)-nicotine-treated animals were, 2-fold and 8-fold higher, respectively than control levels but in both cases these levels returned to control values over the remainder of the time course examined (days 6-23). These results suggest that exposure to S-(-)-nicotine results in initial histamine release and/or inhibition of histamine uptake. However, longer term exposure to S-(-)-nicotine may not result in significantly altered levels of circulating histamine.

N-methylation of phenylpyridines and bispyridyls as a potential toxication route: tissue distribution of azaheterocycle N-methyltransferase activity in the rabbit.

The in vitro N-methylating capability of rabbit tissue cytosolic preparations, utilizing S-adenosyl-L-[methyl-3H]methionine as methyl donor, is described. Dialysed preparations from the lung, liver and kidney are capable of N-methylating 3- and 4-phenylpyridines and 3,3'- and 4,4'-bispyridyls; the corresponding 2-substituted derivatives are not substrates. With all the four substrates, azaheterocycle N-methyltransferase activity decreased in the order lung greater than kidney greater than liver; the activity in the lung was manifold greater than in the kidney and the liver. Azaheterocycle N-methyltransferase activity could not be detected in the brain cytosol under the enzyme assay conditions used. The production of N-methylpyridinium ion metabolites in the lung, from 4-phenylpyridine, 4,4'-bispyridyl and other pyridino-compounds present in tobacco smoke, may contribute to the observed pulmonary toxicity in tobacco users.

Effect of exposure route on measurement of blood pressure by tail cuff in F-344 rats exposed to OTTO Fuel II.

Male Fischer-344 rats demonstrated a dose-response of blood pressure (BP) to increasing doses of propylene glycol dinitrate (PGDN), the major constituent of OTTO Fuel II (OFII) following administration by subcutaneous injection. Dermal application of the same doses to separate groups of rats resulted in variable responses of BP that were unrelated to dose. A nose-only exposure system was developed but no effect on BP was observed in rats exposed to a nearly saturated atmosphere of PGDN (approx. 750 mg/m3 at 25 degrees C). This study has indicated both the difficulties associated with the use of tail cuff measurement of BP and the need for either a more sensitive or more specific biomarker of effect for exposure to nitrate esters.

Evaluation of the promotion potential of chlorotrifluoroethylene trimer acid in male Sprague-Dawley rats.

The promoting effect of chlorotrifluoroethylene trimer acid (TRA), a metabolite of the 6-carbon oligomer of Halocarbon 3.1 oil, was investigated using a bioassay designed to detect enzyme-altered foci. These oligomers, as well as their carboxylic acid metabolites, have been shown to cause hepatomegaly and an increased rate of hepatic peroxisomal fatty acid beta-oxidation following administration by oral and inhalation routes. Groups of 2/3 partially hepatectomized male Sprague-Dawley rats were initiated with a single dose of diethylnitrosamine (10 mg/kg). Two weeks later phenobarbital (0.5% in the drinking water) was provided to animals in the positive control group. At the same time, three other groups received an initial dose of TRA by intraperitoneal injection (98, 9.8 and 0.98 mg/kg). Biweekly intraperitoneal injections of TRA (12.3, 1.2, and 0.12 mg/kg) were continued for 9 months. Quantitative sterological analysis revealed that TRA exposure resulted in a significant dose-dependent increase in the number of gamma-glutamyltranspeptidase-positive foci.

In vitro inhibition of histamine metabolism in guinea pig lung by S-(-)-nicotine.

S-(-)-Nicotine competitively inhibits the metabolism of histamine to its N tau-methylated derivative in guinea pig lung homogenates. S-(-)-Nicotine exhibited a dissociation constant of the enzyme:inhibitor complex, Ki, of 9.4 X 10(-5) M compared with Km's for histamine and co-factor, S-adenosylmethionine, of 4.74 X 10(-5) M and 1.76 X 10(-5) M, respectively. This demonstrates the first reported involvement of nicotine in histaminergic mechanisms.

N-methylation of nicotine enantiomers by human liver cytosol.

Incubation of human liver cytosol with either R-(+)-[3H-N'CH3]nicotine or S-(-)-[3H-N'CH3]nicotine results in the formation of the corresponding N-methyl quaternary ammonium metabolite. A substrate stereoselectivity was observed in that the turnover number for the methylation of the S-(-)-isomer was 0.25 pmol mg-1 protein h-1, whereas that for the R-(+)-isomer was 2.11. The latter substrate exhibited an apparent Km value of 20.1 microM. Nicotine N-methylation appears to be species-dependent, since rat liver homogenates contained no 'nicotine N-methyltransferase' activity, whereas with guinea-pig liver homogenates, a substrate specificity for only R-(+)-nicotine was observed.

Inhibition of histamine N-methyltransferase activity in guinea-pig pulmonary alveolar macrophages by nicotine.

Both S-(-)- and R-(+)-nicotine enantiomers are inhibitors of histamine N tau-methylation activity in guinea-pig pulmonary alveolar macrophage cultures, exhibiting IC50 values of 7 and 8 microM, respectively. S-(-)-Nicotine is not biotransformed under the conditions of the experiment, however, R-(+)-nicotine undergoes significant N-methylation to produce N-methylnicotinium ion. S-(-)-Nicotine appears to inhibit the N-methylation of its optical antipode by the alveolar nicotine N-methyltransferase. The results indicate that a contributing factor in the toxicology of cigarette smoke inhalation may be due to the inhibition of pulmonary metabolism of histamine by nicotine.

Enantioselective metabolism during continuous administration of S-(-)- and R-(+)-nicotine isomers to guinea-pigs.

The S-(-)- and R-(+)-nicotine isomers were administered subcutaneously via Alzet osmotic pumps to male Hartley guinea-pigs (n = 5 with each isomer) over a 23-day period. Estimated dosage rate throughout the experiment was 0.6 mg-1. Urine samples were collected over this time and the levels of urinary oxidative and N-methylated nicotine metabolites were measured by cation-exchange HPLC analysis. S-(-)-Nicotine formed only oxidative metabolites, whereas the R-(+)-isomer formed both oxidative and N-methylated metabolites. 3'-Hydroxycotinine and nicotine-1'-oxide were major metabolites of both enantiomers; cotinine and nornicotine were only minor metabolites. The major N-methylated metabolite of R-(+)-nicotine was N-methylnicotinium ion; N-methylcotininium ion and N-methylnornicotinium ion were also identified as metabolites of this nicotine isomer. Total N-methylated quaternary ammonium metabolites accounted for 15 to 20% of the administered dose of R-(+)-nicotine. An interesting enantioselective reduction in the percent of oxidative urinary metabolites formed S-(-)-nicotine was observed over 23 days. This may indicate the enantioselective induction of an uncharacterized metabolic pathway for this nicotine isomer.

The ability of amine N-methyltransferases from rabbit liver to N-methylate azaheterocycles.

The substrate specificity of two homogeneous amine N-methyltransferases from rabbit liver has been demonstrated to extend to the azaheterocycles pyridine, R-(+)-nicotine and S-(-)-nicotine. Both enzymes methylate R-(+)-nicotine at the pyridyl nitrogen to afford the N-methylnicotinium salt, whereas S-(-)-nicotine does not act as a substrate for either enzyme. Surprisingly, R-(+)-nicotine is methylated at either the pyridyl nitrogen, or the pyrrolidine nitrogen, to afford the two isomeric monomethylate nicotinium ions when an enzymic preparation containing both methyl transferase activities was used. Under similar conditions S-(-)-nicotine was methylated only at the pyridyl nitrogen. The production of charged metabolites in-vivo, from the large number of pyridino-compounds that are used as drugs, or are present in the environment, may be of toxicological significance, in view of the reported toxicities of several such quaternary ammonium compounds.

Safety of selamectin in dogs.

Selamectin is a broad-spectrum avermectin endectocide for treatment and control of canine parasites. The objective of these studies was to evaluate the clinical safety of selamectin for topical use in dogs 6 weeks of age and older, including breeding animals, avermectin-sensitive Collies, and heartworm-positive animals. The margin of safety was evaluated in Beagles, which were 6 weeks old at study initiation. Reproductive, heartworm-positive, and oral safety studies were conducted in mature Beagles. Safety in Collies was evaluated in avermectin-sensitive, adult rough-coated Collies. Studies were designed to measure the safety of selamectin at the recommended dosage range of 6-12mgkg(-1) of body weight. Endpoints included clinical examinations, clinical pathology, gross and microscopic pathology, and reproductive indices. Selected variables in the margin of safety and reproductive safety studies were subjected to statistical analyses. Pups received large doses of selamectin at the beginning of the margin of safety study when they were 6 weeks of age and at their lowest body weight, yet displayed no clinical or pathologic evidence of toxicosis. Similarly, selamectin had no adverse effects on reproduction in adult male and female dogs. There were no adverse effects in avermectin-sensitive Collies or in heartworm-positive dogs. Oral administration of the topical formulation caused no adverse effects. Selamectin is safe for topical use on dogs at the recommended minimum dosage of 6mgkg(-1) (6-12mgkg(-1)) monthly starting at 6 weeks of age, and including dogs of reproducing age, avermectin-sensitive Collies, and heartworm-positive dogs.

Safety of selamectin in cats.

The safety of the avermectin, selamectin, was evaluated for topical use on the skin of cats of age six weeks and above, including reproducing cats and cats infected with adult heartworms. All studies used healthy cats. Acute safety was evaluated in domestic cross-bred cats. Margin of safety was evaluated in domestic-shorthaired cats, starting at six weeks of age. Reproductive, heartworm-infected, and oral safety studies were conducted in adult, domestic-shorthaired cats. Studies were designed to measure the safety of selamectin at the recommended dosage range of 6-12mgkg(-1) of body weight. Assessments included clinical, biochemical, pathologic, and reproductive indices. Selected variables in the margin of safety study and the reproductive studies were subjected to statistical analyses by using a mixed linear model. Cats received large doses of selamectin at the beginning of the margin of safety study when they were six weeks of age and at their lowest body weight, yet displayed no clinical or pathologic evidence of toxicosis. Similarly, selamectin had no adverse effect on reproduction in adult male and female cats. There were no adverse effects in heartworm-infected cats. Oral administration of the topical formulation, which might occur accidentally, caused mild, intermittent, self-limiting salivation and vomiting. Selamectin is a broad-spectrum avermectin endectocide that is safe for use in cats starting at six weeks of age, including heartworm-infected cats and cats of reproducing age, when administered topically to the skin monthly at the recommended dosage to deliver at least 6mgkg(-1).

Gas chromatographic determination of propylene glycol dinitrate in rodent skin.

A gas chromatographic (GC) method was developed for the detection of propylene glycol dinitrate (PGDN) in rodent skin following extraction with ethyl acetate. Known quantities of PGDN contained in the torpedo fuel Otto Fuel II were added to homogenates of rat skin, which were subsequently extracted with two 10-mL portions of ethyl acetate. An aliquot of each extract was analyzed by GC with a flame ionization detector. With this method, concentrations ranging from 0.0042 to 11.2 mg/mL were determined by comparison with a standard curve. The extraction efficiencies ranged from 85.7% for the lowest concentration to 101% for the highest concentration.

N-methylation as a toxication route for xenobiotics. II. In vivo formation of N,N'-dimethyl-4,4'-bipyridyl ion (paraquat) from 4,4'-bipyridyl in the guinea pig.

The biotransformation of 4-phenylpyridine and 4,4'-bipyridyl to N-methylated quaternary ammonium metabolites in guinea pig and rabbit has been examined. Neither animal species excreted the neurotoxin N-methyl-4-phenylpyridinium ion as a urinary metabolite after ip administration of 4-phenylpyridine. However, treatment of rabbits with 4,4'-bipyridyl resulted in the formation of N-methyl-4,4'-bipyridinium ion in the urine (1.2% of the administered dose), and ip administration of 4,4'-bipyridyl to guinea pigs afforded both N-methyl-4,4'-bipyridinium ion and N,N'-dimethyl-4,4'-bipyridinium ion (paraquat) as urinary metabolites (0.8% and 2.9%, respectively, of the administered dose). The detection of the lung toxin paraquat as a urinary metabolite of 4,4'-bipyridyl is a significant finding, in that it represents the first documented report of the formation of a toxic metabolite via the N-methylation pathway.

Evidence of stereospecificity in the in vivo methylation of [14C](+/-)-nicotine in the guinea pig.

In vivo metabolism of [14C]nicotine to N-methylnicotinium ion in the guinea pig appears to be a stereospecific biotransformation, involving only the R-(+)-isomer. The detection and quantitation of urinary radiolabeled nicotine metabolites after ip injection of either [N-14CH3](+/-)-nicotine or [2'-14C](+/-)-nicotine, was carried out on an HPLC cation-exchange analytical system. Utilizing the recently discovered phenomenon of differential enantiomeric association, the stereo-chemistry of the methylation pathway was determined. Significant differences in the tissue distribution of 14C label from the [N-14CH3]-vs. [2'-14C]nicotine-treated animals were observed 24 hr after nicotine administration. An unidentified metabolite with high affinity on cation-exchange chromatography, was detected as a metabolite of [2'-14C](+/-)-nicotine, but was not observed as a metabolite of [N-14CH3](+/-)-nicotine.