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AV7909 is a next-generation anthrax vaccine under development for post-exposure prophylaxis following suspected or confirmed Bacillus anthracis exposure, when administered in conjunction with the recommended antibacterial regimen. AV7909 consists of the FDA-approved BioThrax ® vaccine (anthrax vaccine adsorbed) and an immunostimulatory Toll-like receptor 9 agonist oligodeoxynucleotide adjuvant, CPG 7909 . The purpose of this study was to evaluate the potential systemic and local toxicity of AV7909 when administered via repeat intramuscular injection to the right thigh muscle (biceps femoris) to male and female Sprague Dawley rats. The vaccine was administered on Days 1, 15, and 29 and the animals were assessed for treatment-related effects followed by a 2-week recovery period to evaluate the persistence or reversibility of any toxic effects. The AV7909 vaccine produced no apparent systemic toxicity based on evaluation of clinical observations, body weights, body temperature, clinical pathology, and anatomic pathology. Necrosis and inflammation were observed at the injection sites as well as in regional lymph nodes and adjacent tissues and were consistent with immune stimulation. Antibodies against B. anthracis protective antigen (PA) were detected in rats treated with the AV7909 vaccine, confirming relevance of this animal model for the assessment of systemic toxicity of AV7909. In contrast, sera of rats that received saline or soluble CPG 7909 alone were negative for anti-PA antibodies. Overall, 3 intramuscular immunizations of Sprague Dawley rats with AV7909 were well tolerated, did not induce mortality or any systemic adverse effects, and did not result in any delayed toxicity.

A critical global health need exists for a Zika vaccine capable of mitigating the effects of future Zika epidemics. In this study we evaluated the antibody responses and efficacy of an aluminum hydroxide adjuvanted purified inactivated Zika vaccine (PIZV) against challenge with Zika virus (ZIKV) strain PRVABC59. Indian rhesus macaques received two doses of PIZV at varying concentrations ranging from 0.016 µg - 10 µg and were subsequently challenged with ZIKV six weeks or one year following the second immunization. PIZV induced a dose-dependent immune response that was boosted by a second immunization. Complete protection against ZIKV infection was achieved with the higher PIZV doses of 0.4 µg, 2 µg, and 10 µg at 6 weeks and  with 10 ug PIZV at  1 year following vaccination. Partial protection was achieved with the lower PIZV doses of 0.016 µg and 0.08 µg. Based on these data, a neutralizing antibody response above 3.02 log 10 EC50 was determined as a correlate of protection in macaques. PIZV elicited a dose-dependent neutralizing antibody response which is protective for at least 1 year following vaccination.

Bacille Calmette-Guérin (BCG), the only licensed vaccine for the prevention of tuberculosis (TB), provides only limited protection against certain forms of Mycobacterium tuberculosis (Mtb) infection. While infection with Mtb can be treated with antibiotics, the therapy is expensive, toxic, and requires several months for treatment. In addition, the emergence of drug resistant strains limits the impact of antibiotics and underlines the importance of developing a more effective vaccine to control this disease. Given that pulmonary TB is the most common form of the disease, a vaccine capable of inducing lung-resident immunity may be advantageous for combating this infection. New advances in pulmonary delivery make this route of vaccination feasible and affordable. Here, we evaluate the safety and immunogenicity of an aerosolized Ad35-based vaccine, AERAS-402, delivered to the lungs in nonhuman primates as part of a GLP acute and chronic toxicology and safety study. In this study, animals received three high doses (1 x 10(11) vp) of AERAS-402 by inhalation via a nebulizer at 1-week intervals. Aerosol delivery of AERAS-402 resulted in an increase in relative lung weights as well as microscopic findings in the lungs, mediastinal lymph nodes, bronchus-associated lymphatic tissue, and the naso-oropharynx that were consistent with the induction of an immune response during the acute phase. These findings resolved by the chronic phase and were considered to be non-adverse. Furthermore, we observed transient vaccine-specific immune responses in the peripheral blood as well as sustained high-level polyfunctional CD4(+) and CD8(+) T cell responses in the bronchoalveolar lavage fluid of vaccinated nonhuman primates. The data suggest that pulmonary delivery of Ad35-based vaccines can be safe and can induce potent lung-resident immunity.

Coordinated interactions between helper and cytotoxic T-lymphocytes (HTL and CTL) are needed for optimal effector cell functions and the establishment of immunological memory. We, therefore, designed a mixed format vaccine based on the use of highly conserved HIV-derived T-lymphocyte epitopes wherein the HTL epitopes were delivered as a recombinant protein and the CTL epitopes which were encoded in a DNA vaccine plasmid. Immunogenicity testing in HLA transgenic mice and GLP preclinical safety testing in rabbits and guinea pigs were used to document the utility of this approach and to support Phase 1 trial clinical testing. Both vaccine components were immunogenic and safely co-administered.

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).

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.

Environmental and occupational exposures are typically to mixtures of chemicals, although most toxicity information is for individual compounds. Interactions between chemicals may involve pharmacokinetic and/or pharmacodynamic effects resulting in modulation of toxicity. Therefore, physiologically based pharmacokinetic modeling has been used to analyze data describing the metabolism of vinyl chloride (VC) and trichloroethylene (TCE) mixtures in rats. A single saturable pathway was modeled, representing cytochrome P450 2E1. This was partially validated using preexposure to trans-1,2-dichloroethylene (tDCE) which virtually eliminated in vivo metabolism of both VC and TCE at low concentrations. Microsomes from tDCE-exposed animals showed inhibition of metabolism of P450 2E1 substrates (chlorzoxazone, p-nitrophenol, and TCE) and no effect on 7-ethoxycoumarin deethylation. Studies with liver microsomes from VC-exposed animals found that neither suicide inhibition nor induction occurred during 6-hr exposures to high concentrations. Therefore, these effects were not modeled. Modeling of mixtures of VC and TCE was successful only using competitive inhibition, as might be predicted for cytochrome P450 2E1 substrates, and not uncompetitive or noncompetitive inhibition. These results were further confirmed by determining the depletion of glutathione due to VC metabolism. The validation of a detailed model for the inhibition kinetics of metabolism of these two compounds permits better understanding of the implications of coexposures for toxicity. It is notable that competitive inhibition only becomes significant at relatively high concentrations (tens of ppm), while at typical low environmental concentrations (ppb), absorption is perfusion limited and enzyme is in excess so that the chemicals will be metabolized independently.

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.

The subchronic (26 day) oral toxicities of two AF hydraulic fluids (MIL-H-5606 [H5], MIL-H-83282 [H8]), a commercial phosphate ester (PE), and two candidate hydraulic fluids (low temperature version of MIL-H-83282 [LT] and chlorotrifluorethylene oligomers [polyCTFE]) were compared in male F-344 rats. Oral dosing was used in order to quickly compare these fluids to PolyCTFE, the only fluid at the time to have been tested in a 90-day inhalation study. Rats were initially dosed with 1.0 g/kg/day of each fluid. H8 increased alkaline phosphatase (ALKP) while LT produced an anemia and leukocytosis. Exposure to H5 fluid resulted in lymphocytopenia and persistent diuresis. Due to their greater toxicity, resulting in lethality in the first dosing study, only 0.5 g/kg/day of PE and PolyCTFE were administered in the second study. Exposure to PE (0.5 g/kg) resulted in an anemia and decreases in BW (day 10 until day 25), spleen/BW ratio, blood urea nitrogen (BUN), and creatinine (CREAT). PolyCREAT (0.5 g/kg) decreased BW (day 11 to the end of the study) and testicular weight. PolyCTFE (0.5 g/kg) increased relative spleen weights, various clinical chemistry parameters, and triggered a reversible diuresis. PolyCTFE (0.5 g/kg), PE (0.5 g/kg), and H5 produced an increase in absolute and relative liver weights compared to control livers. Peroxisomal beta oxidation, an indicator of peroxisomal proliferation, was significantly increased above control levels in the livers of all rats except the PE (0.5 g/kg) group, where the increase was not significant. Hydrocarbon nephropathy, indicated by increased levels of hyaline droplets in kidney tubules, was severe in H5, mild in H8, LT, and PolyCTFE (0.5 g/kg), and minimal in PE (0.5 g/kg). The MIL-H-83282 fluids (H8 and LT) were the least toxic hydraulic fluids. PolyCTFE and PE were the most toxic, with H5 intermediate.

Currently, N-methyl-N'-nitroguanidine (MNG) is being considered by the U.S. Air Force Armament Laboratory for use in explosive formulations. A mammalian toxicity profile has been performed which includes the analysis of chemical impurities and an assessment of the potential for the metabolism of MNG to 1-methyl-3-nitro-1-nitrosoguanidine (MNNG). Potential in situ gastric conversion of MNG to MNNG is a toxicological concern because MNNG is both mutagenic and carcinogenic. The compound was also evaluated in several bioassays to assess its potential genotoxic activity. The acute oral toxicity was determined in male and female Fischer 344 rats administered a single dose of MNG in corn oil. The maximum suspension of MNG that could be delivered, 1 mg MNG/kg body weight, produced no signs of toxic stress during the 14-day observation period. The primary eye and skin irritation potential of MNG was determined in female New Zealand white rabbits using the Draize technique. MNG produced no irritation to intact skin but did produce mild conjunctival irritation. The response of a single guinea pig to the dermal sensitization evaluation indicated that MNG is a weak sensitizer. The results of three genetic tests indicated that MNG does not interact with genetic material. Gastric contents and feces from treated animals showed no evidence of conversion of MNG to MNNG.

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.

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.

Perfluoro-n-decanoic acid (PFDA) is a perfluorinated fatty acid that produces hepatomegaly and increased peroxisomal beta-oxidation when administered to rodents. Chlorotrifluoroethylene (CTFE) trimer acid and CTFE tetramer acid are metabolites of the six- and eight-carbon oligomers of CTFE, respectively. They are structurally related to PFDA, and CTFE tetramer acid has caused toxic effects in rodents that are similar to those observed following PFDA administration. Because of the correlation between peroxisome proliferation and hepatocarcinogenesis, CTFE trimer acid, CTFE tetramer acid, and PFDA were evaluated in in vitro and in vivo/in vitro bioassays to assess their potential genotoxic activity. The assays conducted were the Ames Salmonella/microsomal mutagenicity assay, the hypoxanthineguanine phosphoribosyltransferase (HGPRT) locus Chinese hamster ovary gene mutation assay, the sister chromatid exchange (SCE) assay, chromosomal aberration assay, and an in vivo/in vitro unscheduled DNA synthesis (UDS) and S-phase DNA synthesis assay. All test articles were negative in the Ames assay, the HGPRT assay, and the SCE assay. In the chromosomal aberration assay CTFE trimer acid and CTFE tetramer acid were negative in cultures with and without S9 metabolic activation. PFDA was also negative in the absence of metabolic activation, but chromosomal aberrations were observed when PFDA was incubated in the presence of S9 fraction. All test articles were negative for inducing UDS but all induced S-phase replicative DNA synthesis 16 hr after administration of the test article to the test animals; only CTFE tetramer acid and PFDA induced S-phase synthesis 48 hr after dosing: the usual timepoint examined for this response.

Polychlorotrifluoroethylene (3.1 oil) is a nonflammable hydraulic fluid composed of chlorotrifluoroethylene (CTFE) oligomers of different carbon chain lengths (C5 to C9), primarily six (trimer) and eight (tetramer) carbons. Four test groups of Fischer 344 rats (16 rats/group) were orally gavaged daily over a 2-week period at doses of 1.25 g/kg with 3.1 oil containing a 55:45 ratio of trimer and tetramer (3.1 oil-C6:C8), 3.1 oil composed of 95% trimer (3.1 oil-C6), pure tetramer, and pure trimer. Four rats per treatment group were terminated after 1, 3, 7, and 14 doses. Rats dosed with either 3.1 oil-C6:C8 or pure tetramer demonstrated significant weight losses, increased liver weights, increased rates of liver fatty acid beta-oxidation, pronounced hepatomegaly and altered hepatocellular architecture, and elevated serum liver-associated enzymes. Rats dosed with either 3.1 oil-C6 or only pure trimer demonstrated significant increase in liver weight and moderate liver histopathologic changes. Compositional analyses of the ratio percentage of trimer to tetramer present in 3.1 oil-C6:C8 (55:45) were found to be altered when measured in the liver (32:68). Differential CTFE oligomer toxicity was indicated by effects on liver, body weight, and peroxisomal beta-oxidation and may allow for less toxic formulations of 3.1 oil to be generated by reducing or eliminating the tetramer component.

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.

The N-methylation of pyridine in vitro, using dialysed and undialysed hepatic, pulmonary, renal and brain preparations from rabbits, is described. Analysis of the quaternary metabolite, N-methylpyridinium ion, was carried out by selective ion-pair extraction and cation-exchange high-performance liquid chromatography (h.p.l.c.) using a u.v. detector, and also by direct cation-exchange h.p.l.c. of incubates containing S-adenosyl-L-[methyl-3H]methionine using a flow-through radioactivity detector. N-Methylation of pyridine could be readily demonstrated with dialysed homogenates, 9000 g and 100 000 g supernatant fractions from lung, kidney and liver, but not with any of the brain preparations. 'Pyridine N-methyltransferase' activity was confined to the tissue cytosol, and this enzyme utilized S-adenosyl-L-methionine as the methyl donor. Since the activity of the 'pyridine N-methyltransferase' in rabbit tissues is increased many fold by dialysis, this enzyme, in common with most other N-methylating enzymes, is subject to inhibition by a low-molecular-weight endogenous substance.

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.

The kinetics of nicotine methylation by guinea pig lung homogenates has been investigated. An interesting stereospecificity has been observed for nicotine enantiomers. R-(+)-Nicotine is a substrate Km = 1.42 X 10(-5)M for an SAM-dependent guinea pig lung aromatic azaheterocycle N-methyltransferase, whereas S-(-)-nicotine acts as a competitive inhibitor (Ki = 6.25 X 10(-5)M) of the N-methylation of its antipode.

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.

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.

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.

Primary rat hepatocyte suspension cultures (approximately 2 X 10(6) cells) exposed to solubilized 2,3,4-trimethylpentane at concentrations ranging from 7.9 to 31.5 mM under two different culture conditions resulted in a linear dose response, as determined by lactate dehydrogenase leakage and viability data. A significant increase in the 2,3,4-trimethylpentane effective concentration 50 for primary hepatocytes occurred when exposures were implemented in medium containing 0.05% albumin. The effective concentration 50 for hepatocytes exposed to 2,3,4-trimethylpentane in medium lacking and containing albumin were 17.1 and 20.7 mM, respectively. Metabolite analysis by gas chromatography-mass spectrometry of supernatant (lacking or containing albumin) and cell extracts from hepatocyte cultures exposed to 2,3,4-trimethylpentane for 4 h indicated the presence of three metabolites: 2,3,4-trimethyl-1-pentanol, 2,3,4-trimethyl-2-pentanol, 2,3,4-trimethyl-2-pentanol, and 2,3,4-trimethyl-1-pentanoic acid. Electron microscopic examination of 2,3,4-trimethylpentane-exposed primary hepatocytes indicated ultrastructural changes which included abnormal condensed chromatin association with the nuclear membrane, swollen mitochondria, increased amounts of cytoplasmic lipid, significant loss of microvilli from the cell surface, increased vacuolation, and increased numbers of peroxisomes. Although these changes were observed under both culture conditions, they were more severe in cultures lacking albumin. This study indicates that primary hepatocyte suspension cultures provide a useful system for rapidly identifying liver metabolites of selected test compounds of interest.

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.

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.

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.

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.

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.

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.

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.