The evaluation of methapyrilene for bacterial mutation with metabolic activation by Aroclor-induced, methapyrilene-induced and noninduced rat-liver S9.

The antihistamine methapyrilene (MP) has been shown to be a potent hepatocarcinogen in rats. However, it has demonstrated little genotoxic activity in a wide variety of short-term tests. In this study, Fischer 344 rats were fed a carcinogenic dose of 0.1% methapyrilene in the diet for 10 weeks prior to sacrifice. S9 was prepared from the livers of the control, MP-treated and Aroclor-induced Fischer 344 rats. Each type of S9 was analyzed for mixed function oxidase activity, cytochrome P-450, and protein content. MP was then evaluated for mutagenicity in 6 strains of S. typhimurium (TA1535, TA1537, TA98, TA100, TA2638 and TA104) and one strain of E. coli (WP2uvrA-) using the standard plate-incorporation assay. MP was not mutagenic in any of the 7 bacterial strains when tested at concentrations < or = 10 mg/ml in the presence of each type of S9. However, in the absence of metabolic activation, an approximate 2-fold increase in revertants was noted with strain TA1535. The data from this study show that MP was not converted to a mutagenic metabolite by any of the three S9 types examined. However, the "weak" positive response with strain 1535 in the absence of metabolic activation indicates that further research is needed to elucidate the mechanism of action of this rat carcinogen.

Metabolism of methapyrilene by Fischer-344 rat and B6C3F1 mouse hepatocytes.

1. Suspension cultures of freshly isolated F344 rat and B6C3F1 mouse hepatocytes were compared for their ability to transform various concentrations of methapyrilene (MP). 2. MP metabolites were isolated and purified by h.p.l.c., and were identified by comparing their chromatographic and mass spectral properties with those of authentic standards. 3. Both rat and mouse hepatocytes transformed MP to tentatively identified 2-thiophenecarboxylic acid (I), and definitively identified mono-N-desmethyl methapyrilene glucuronide (II), methapyrilene glucuronide (III), methapyrilene N-oxide (V), and mono-N-desmethyl methapyrilene (VII).

Effects of methapyrilene measured in mitochondria isolated from naive and methapyrilene-treated rat and mouse hepatocytes.

Methapyrilene (MP) is a rat-specific liver carcinogen that alters mitochondrial number and morphology both in vivo and in vitro. This biological phenomenon may be due to the effects of MP on mitochondrial function. To test this hypothesis, studies were conducted to examine the effects of MP on DNA and protein synthesis and respiration in isolated mitochondria. DNA and protein synthesis activities were measured using [3H]thymidine and [3H]leucine incorporation. Mouse liver mitochondria were also examined for comparison since no tumor formation or alterations in mitochondrial morphology have been associated with MP treatment in mice. A significant decrease in basal DNA and protein synthesis levels was observed in mitochondria isolated from rats and mice following in vivo MP treatment. This effect could not be reproduced when mitochondria were exposed to 0 or 100 microM MP following isolation, despite the presence of an S9 activation system. Electron microscopic examinations were performed on isolated rat mitochondria and revealed morphologic differences between mitochondria from naive and MP-treated rats. Although significant differences in State 3 and State 4 respiratory rates were noted, the respiratory control ratio, ADP/O ratio, and uncoupler-stimulated respiratory rates were unaffected. Results demonstrate that: (1) MP irreversibly depresses DNA and protein synthesis in a majority of mitochondria, despite only localized morphologic changes; (2) these changes are not reflected by a decrease in respiratory function; and (3) depression of DNA and protein synthesis does not correlate with carcinogenic susceptibility.

Species differences in the metabolism and macromolecular binding of methapyrilene: a comparison of rat, mouse and hamster.

1. The metabolism of methapyrilene (MPH) by rat, hamster and mouse liver microsomes in vitro was investigated together with the binding of 14C-MPH to calf thymus DNA after metabolic activation. 2. Both quantitative and qualitative differences in MPH metabolism were observed in these three species. Mouse liver microsomes catalyse the formation of two novel isomers of hydroxypyrdylmethapyrilene (hydroxypyridyl-MPH) as determined by mass spectral analysis. N,N'-Didesmethylmethapyrilene (didesmethyl-MPH) was formed in detectable quantities only when hamster liver microsomes were used. 3. Incubation of liver microsomes from all three species catalysed the binding of 14C-MPH to exogenous DNA, which was quantitatively similar for all three species. The effect of the cytochrome P-450 inhibitor, 2,4-dichloro-6-phenylphenoxyethylamine (DPEA), and methimazole, a flavin-dependent monooxygenase inhibitor, on binding differed significantly for the three species studied.

Lack of binding of methapyrilene and similar antihistamines to rat liver DNA examined by 32P postlabeling.

The nonmutagenic carcinogen methapyrilene, together with several noncarcinogenic analogues, was administered to rats p.o. for as long as 4 wk at concentrations of 0.1%. DNA was isolated from the liver and other organs and hydrolyzed, and the identification of covalent adducts was made using the 32P postlabeling method of Randerath. Some modified procedures were also used to deal with the possibility of very mobile adducts being formed from these hydrophilic amines. Although the rats had received as much as 2 g of amine per kg of body weight, no evidence of formation of DNA adducts in liver or other organs was seen; the level of detection was between 1 in 10(8) and 1 in 10(9) nucleotides. Adduct formation from much lower doses of the mutagenic food pyrolysis product 2-amino-3-methylimidaz(4, 5f)quinoline was detectable at a level of 1 in 10(6) nucleotides in parallel analyses. These results add to the evidence that carcinogenesis by methapyrilene is through an indirect or nonmutagenic mechanism.

A comparative in vitro metabolic study of methaphenilene and pyribenzamine.

1. In vitro metabolism of methaphenilene (MFN) and pyribenzamine (PBZ) was compared to that of methapyriline (MPH) in rat, because chronic treatment with MPH causes cancer in rats, whereas MFN and PBZ cause no cancer. 2. G.l.c. and mass spectrometry were used to identify 7 metabolites of MFN and 6 of PBZ in extracts of rat liver microsome incubations. 3. Quantification of the metabolic pathways revealed that N-oxide formation is considerably more important for both MFN and PBZ than for MPH, and only MPH forms an amide as a metabolic product. 4. Quantitative balance studies show that a lower recovery is apparent for metabolic experiments with MPH than for either MFN or PBZ under all conditions examined, indicating that significant metabolic pathways for MPH exist which are not being measured under these conditions.

Fungal transformations of antihistamines: metabolism of methapyrilene, thenyldiamine and tripelennamine to N-oxide and N-demethylated derivatives.

1. Strains of the fungus Cunninghamella elegans ATCC 9245 and 36112 were tested for their ability to transform the antihistamines methapyrilene (I), thenyldiamine (II) and tripelennamine (III). 2. Antihistamine metabolites were isolated by h.p.l.c., and identified by their 1H-n.m.r. and mass spectral properties. 3. All three drugs were transformed by both C. elegans strains to N-oxidized and N-demethylated derivatives. Metabolism during 96 h of incubation amounted to 85% for (I), 64% for (II), and 83% for (III). Metabolites soluble in organic solvents amounted to 62% to 86% of the total metabolism; approximately 88% to 95% of the organic-soluble metabolites were N-oxide derivatives of each antihistamine.

Methapyrilene is a genotoxic carcinogen: studies on methapyrilene and pyrilamine in the L5178Y/TK +/- mouse lymphoma assay.

Methapyrilene (MP), a sedating antihistamine, is a potent rat hepatocarcinogen which has been thought to be non-genotoxic on the basis of the negative results in a small number of short-term mutagenicity tests. The present studies show that MP is a moderately active mutagen in the L5178Y/TK +/-----TK-/- mouse lymphoma assay (MLA) in the presence of aroclor-induced rat-liver S9, and that it induces predominantly small-colony thymidine kinase-deficient (TK-/-) mutants of demonstrated chromosomal origin. 10 of 12 small colony TK-/- mutants analyzed by banded karyotype (230-band level of resolution) show aberrations to chromosome 11b, the known location of the single functional TK gene in these cells. The observed aberrations from nine of the mutants included insertions, deletions and translocations while the tenth mutant had highly rearranged, multiple copies of chromosome 11 segments. By varying the concentrations of the S9 protein and cofactors it was shown that our standard S9 composition was close to optimum for activating MP to a mutagen. The activity and stability of various lots of S9 prepared in-house or purchased from a contract laboratory revealed significant differences. The ability of 2 lots of in-house S9 to activate a standard concentration of MP increased rapidly over the first 4 weeks of liquid nitrogen storage then declined slowly over the next 16 weeks. Three separate lots of purchased S9 were essentially inactive for the first 2 weeks of liquid nitrogen storage then increased in activity thereafter; these were the only occasions in which MP was not mutagenic in our hands. The mutagenic activity of pyrilamine (PYR), a structurally related antihistamine which is far less carcinogenic in rats, but easily detected in short-term tests as being genotoxic, was also investigated in the MLA. PYR was slightly less mutagenic than MP over a comparable range of concentrations, and also induced predominantly small-colony mutants. These studies fail to adequately explain the great carcinogenic differences between these two compounds, but are consistent with the potent hepatocarcinogenicity of MP resulting through a mutagenic mechanism.

A comparison of in vivo and in vitro metabolites of the H1-antagonist N,N-dimethyl-N'-2-pyridyl-N'-(2-thienylmethyl)-1,2-ethanediamine (methapyrilene) in the rat.

1. The H1-antagonist N,N-dimethyl-N'-2-pyridyl-N'-(2-thienylmethyl)-1,2-ethanediamine (methapyrilene) is carcinogenic in rats. 2. The compound, which is inactive in short-term tests and does not bind to DNA, has been classified as a non-genotoxic carcinogen. 3. Studies have been made in vitro and in vivo in F344 and Sprague-Dawley rats. New metabolites included N-(N',N'-dimethylaminoethyl)-2-aminopyridine and the corresponding N'-oxide, a derivative in which methapyrilene is hydroxylated on the 5-position of the pyridine ring, 2-(N',N'-dimethylamino)-N-2'-pyridylacetamide, N-(2-pyridyl)-N-2"-thienylmethyl)aminoacetaldehyde, and 2-hydroxymethylthiophene. 4. Both strains of rat metabolize methapyrilene to reactive species which may be of importance in the carcinogenic process.

Cytochrome P-450 dependent binding of methapyrilene to DNA in vitro.

Methapyrilene ([14C]MPH) was found to bind to calf thymus DNA only after activation by both rat liver microsomes and NADPH. The cytochrome P-450 inhibitors 2,4-dichloro-6-phenylphenoxyethylamine, 2-diethylaminoethyl-2,2-diphenylvalerate and metyrapone inhibited binding, but methimazole, a flavin-dependent monooxygenase inhibitor, had no effect. However, 1,2-epoxy-3,3,3-trichloropropane, an epoxide hydrolase inhibitor, decreased binding by 30%. Pre-treatment of rats with isosafrole, pregnenolone-16 alpha-carbonitrile or phenobarbital had little or no effect on binding while 3-methylcholanthrene pretreatment decreased binding by 37%. Incubations in the presence of either N-acetylcysteine, glutathione, catalase or glutathione-peroxidase decreased binding to DNA while superoxide dismutase had no effect. These data suggest that MPH is metabolically activated to a species which binds to DNA and that this activation may be mediated by cytochrome P-450 isozymes.

Species differences in the in vitro metabolism of methapyrilene.

1. The metabolism of N,N-dimethyl-N'-(2-pyridyl)-N'-(2-thienylmethyl)-1,2- ethanediamine(methapyrilene, I) by liver microsomes from rat, guinea pig, and rabbit has been examined. 2. Methapyrilene-N-oxide, (III), normethapyrilene, (II), 2-thiophene methanol, (VI), 2-thiophene carboxylic acid, (VII), N-(2-pyridyl)-N',N'-dimethylethylenediamine, (IX), and methapyrilene amide, (XIV) were found in all species. 3. N-(2-Thienylmethyl)-2-amino pyridine, (VIII), 2-aminopyridine, (X), and (5-hydroxypridyl)-methapyrilene, (XII), were detected in rat and rabbit only. 4. N-Hydroxynormethapyrilene, (XXI), was tentatively identified by mass spectral fragmentation patterns only in rabbit liver microsomes incubations; however, it was found in 9000 g supernatant fraction incubations of rabbit, rat and guinea pig. 5. The formation of IX and XII was quantitatively more important in the rat than in either rabbit or guinea pig.

The effect of chronic methapyrilene treatment on methapyrilene metabolism in vitro.

Rats were fed 100 or 1000 p.p.m. methapyrilene (MPH) in their diet for 1, 2, 4, 8 or 16 weeks. Liver microsomes were prepared from both control and treated rats. After incubation with 1 mM MPH, eight metabolites were detected and six were quantitated. Five of the metabolites have been previously identified as 2-hydroxymethyl-thiophene (ThM), thiophene-2-carboxylic acid (ThCA), N-(2-thienylmethyl)-2-aminopyridine (TMAP), N-2-pyridyl-N',N'-dimethylethylenediamine (PMED), and 2-aminopyridine (AP). Three other metabolites have been tentatively identified based on their mass spectral fragmentation patterns as normethapyrilene (N-MPH), (5-hydroxypyridyl)methapyrilene (HP-MPH), and methapyrileneamide (MPH-A). The same metabolites were found in both control and treated animals, the most abundant being N-MPH and PMED. Pretreatment with MPH resulted in inhibition of both consumption of MPH and formation of some metabolites. However increases in the formation of all of the metabolites also occurred under different treatment conditions. In both control and treated tissue, the preliminary mass balance was less than 55%, except in incubations with tissue from rats treated with 1000 p.p.m. for 8 or 16 weeks where it was 92 and 89%, respectively. Dramatic increases in the fraction of TMAP, MPH-A, N-MPH, and HP-MPH relative to MPH consumed account for the increase in the mass balance after 8 weeks pretreatment with 1000 p.p.m. MPH, and increases in the amounts of PMED, HP-MPH and ThCA account for the higher mass balance after 16 weeks. The toxicological consequences of these complex metabolic changes may be important in the induction of cancer by MPH.

Metabolism of methapyrilene by rat-liver homogenate.

The metabolism of methapyrilene(I) in rat-liver 9000 g supernatant fraction produced four new metabolites positively identified by comparison of g.l.c. retention times and mass-spectral fragmentation patterns with those of authentic materials. These compounds are 2-thiophene-methanol(VI), 2-thiophenecarboxylic acid(VII), N-2-pyridyl-N'-dimethylethylenediamine(IX) and 2-aminopyridine(X). In addition, the previously known metabolite 2-[(2-thienylmethyl)amino]-pyridine(VIII) was also positively identified. Six other metabolites were tentatively identified by analysis of the mass-spectral fragmentation patterns of both the trimethylsilyl and the tertiary butyldimethylsilyl derivatives of each compound. These compounds are tentatively identified as: normethapyrilene(II), (hydroxypyridyl)-methapyrilene(XII), methapyrilenamide(XIV), (hydroxypyridyl)-normethapyrilene(XVI), (hydroxypyridyl)-desmethylmethapyrilenamide(XVII), and (hydroxypyridyl) methapyrilenamide(XVIII). Quantification of II, VI-X, XII and XIV account for approx. 65% of the metabolized methapyrilene.

Distribution of the liver carcinogen methapyrilene in Fischer rats and its interaction with macromolecules.

The anti-histaminic drug methapyrilene hydrochloride, which induces liver tumors in ras, was labeled with tritium by exchange and administered at a does of 21 mg containing 700 microCi to each of 15 male Fischer rats. At 1 h, 6 h, 14 h, 24 h, and 44 h after treatment three rats were killed and their livers, pancreas, kidneys, and lungs were removed. The pooled organs were homogenized and DNA, RNA, and soluble protein were isolated from each. The extent of interaction of radioactive methapyrilene with liver nucleic acids was exceedingly small and did not differ significantly from the binding to nucleic acids in kidney, lung, or pancreas, which are not target organs of this carcinogen in rats. Binding of radioactivity to soluble proteins of the liver was considerable and substantially greater than in the other organs. If the mechanism of carcinogenic action of methapyrilene involves covalent interaction with DNA this must be at a very low and highly specific level.

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.

Methapyrilene kinetics and dynamics.

A study was undertaken to characterize the H1 receptor blockade, central nervous system depressant properties, and kinetic parameters of methapyrilene in man. Eight healthy subjects received, in random order at weekly intervals, placebo and methapyrilene 20 mg intravenously and 50 mg and 25 mg orally. Methapyrilene exhibited a moderate antihistaminic effect as measured by the reduction of histamine-provoked skin wheals. Sedation and drowsiness were detected only at the first sampling time (0.75 hr) after intravenous doses. The terminal plasma half-life ranged from 1.1 to 2.1 hr, apparent volume of distribution from 2.14 to 6.61 1/kg, and plasma clearance from 0.013 to 0.048 1/min/kg. Systemic bioavailability was low and showed large interindividual differences, ranging from 4% to 46%. Recovery of unchanged drug from the 24-hr urine was under 2% of the doses.

A fatality involving methapyrilene.

Tissue concentrations of methapyrilene are given in a fatal case. There are no indications that methapyrilene had any synergistic or antagonistic effect with the patient's cardiopulmonary condition, although when taken in high overdosages the pharmacologic effects may be altered. This case was supported by other data in an attempt to elucidate as many contributing factors as possible in evaluating lethal levels.