Absolute bioavailability in man of N-acetylprocainamide determined by a novel stable isotope method.

Absorption of a single oral dose of N-acetylprocainamide (NAPA) was studied in 3 normal subjects. Approximately 85% of the oral dose was absorbed and peak plasma NAPA concentrations were reached in 45 to 90 min. In 2 subjects, NAPA was absorbed at a fast initial rate, then more slowly, prolonging the apparent elimination phase half-life. Absolute bioavailability was determined by a new stable isotope method that entailed intravenous injection of NAPA 13C at the same time that an unlabeled NAPA capsule was given orally. Plasma levels and urine excretion of both compounds were determined by mass fragmentography. Bioavailability was assessed by deconvoluting the plasma level vs time curves resulting from intravenous and oral drug administration, and also by comparing the relative percentage of NAPA and NAPA-13C excreted unchanged in the 24 hr after simultaneous administration.

Antiarrhythmic efficacy of N-acetylprocainamide in patients with premature ventricular contractions.

Oral administration of a 1.5-gm dose of N-acetylprocainamide (NAPA) to 9 patients with premature ventricular contractions (PVCs) confirmed previous indirect evidence that this metabolite of procainamide has antiarrhythmic efficacy and potency comparable to those of procainamide. Although the mechanism by which NAPA acts as an antiarrhythmic drug is not known, it was found that the 6 patients with coupled PVCs responded to NAPA therapy and that the 3 patients without coupled PVCs failed to respond. Coupling interval prolongation also occurred during NAPA therapy in 4 of the 6 responding patients. These observations suggest that NAPA may terminate coupled PVCs by slowing and then interrupting conduction of re-entrant impulses, as has been proposed for procainamide. NAPA plasma concentrations of 7.4-17.2 mug/ml were well tolerated by the patients and produced an average fall of 3 mm Hg in mean arterial pressure and a 7.6% mean increase in corrected QT interval.

Procainamide and N-acetylprocainamide kinetics investigated simultaneously with stable isotope methodology.

The pharmacokinetics of procainamide (PA) and N-acetylprocainamide (NAPA) were compared in 3 normal subjects after simultaneous intraveous injection of PA and NAPA-13C. The distribution kinetics of both compounds were modeled with a 3-compartment mamillary system, and it was found that their steady-state distribution volumes were not significantly different, averaging 1.41 L/kg for PA and 1.46 L/kg for NAPA. However, the intercompartmental clearances of NAPA were slower than those of PA. In these normal subjects, the average elimination t1/2 and total elimination clearance for PA were 2.5 hr and 589.8 ml/min, and for NAPA were 6.2 hr and 233.7 ml/min. Mean renal clearances of PA (346.7 ml/min) and of NAPA (199.5 ml/min) exceeded the usual rate of glomerular filtration, which suggests that both compounds are eliminated in part by renal tubular secretion. All subjects were phenotypic rapid acetylators of isoniazid and converted approximately one fourth of the administered PA dose to NAPA-12C. The fate of 15.4% of the administered PA and 14.5% of the administered NAPA-13C was not determined.

Binding of nitropyrenes and benzo[a]pyrene to mouse lung deoxyribonucleic acid after pretreatment with inducing agents.

In assessing the biological effects of exposure to a complex chemical mixture, it is important to determine how the behavior of one compound may be influenced by the presence of other compounds in the mixture. In this study the effect of pre-exposure to an organic extract of diesel exhaust or to selected compounds in diesel exhaust on the binding of diesel exhaust compounds to DNA was determined. The amount of radiolabel covalently bound to mouse lung DNA following intratracheal administration of radiolabeled benzo[a]pyrene (BaP), 1-nitropyrene, 1,3,6-trinitropyrene, or a mixture of dinitropyrene was determined following pretreatment with benzo[a]pyrene, 1-nitropyrene, and diesel exhaust extract. Male CD-1 mice, 15-18 weeks of age, received 10 mg/kg of putative inducing agents by intratracheal instillation and, after 24 hr, 0.03 to 1.2 mg/kg radiolabeled putative DNA binding agents. Lung DNA was extracted, and covalent binding was quantitated by liquid scintillation spectroscopy. 1-Nitropyrene was a potent lung DNA binding agent in the absence of inducing agents [Covalent Binding Index (CBI) = 970] and was extremely potent after benzo[a]pyrene pretreatment (CBI = 21,540, comparable to the CBI for aflatoxin B1). Similar results were obtained for DNA binding of dinitropyrene and trinitropyrene with and without BaP pretreatment. DNA binding of BaP was lower (CBI = 40) and less inducible (BaP-pretreatment CBI = 230). Pretreatment with diesel extract caused an elevation in the binding of benzo[a]pyrene but little or no elevation in the binding of the nitropyrenes. Pretreatment with 1-nitropyrene did not increase significantly DNA binding of any of the agents tested. These results indicate that nitropyrenes bind readily to lung DNA and this binding may be increased in the presence of respirable mixtures, especially those containing inducing agents such as BaP.

Mutagenic screening of marker grenade dyes by the Salmonella reversion assay, L5178Y/TK+/- mouse lymphoma assay, and in vivo sister chromatid exchange analysis in mice.

Two dyes (C.I. Solvent Yellow No. 33 and a mixture of C.I. Solvent Yellow No. 33 and C.I. Solvent Green No. 3) were tested for mutagenicity in the Salmonella reversion assay and the L5178Y/TK+/- mouse lymphoma assay, and also for sister chromatid exchange (SCE) induction in vivo in C57B1/6J mice. In addition, a greater than 99.9% pure sample of the yellow dye [2-(2'-quinolyl)-1,3-indandione] was tested with and without exogenous activation in the Salmonella reversion assay and the L5178Y/TK+/- mouse lymphoma assay. Neither C.I. Solvent Yellow No. 33 nor the C.I. Solvent Yellow No. 33 and Solvent Green No. 3 mixture was positive for inducing SCEs in vivo. All three dyes were tested in the standard plate incorporation test in seven Salmonella strains TA98, TA100, TA102, TA104, TA1535, TA1537, and TA1538. The dyes were negative with and without exogenous activation in TA98, TA1535, and TA1538. One test with TA1537 was positive with the greater than 99.9% purified yellow dye. All three dyes gave weakly positive results (less than a twofold increase) with S-9 in TA100 and were clearly positive in TA102 and TA104 both with and without S-9. They also induced mutation at the thymidine kinase locus in mouse lymphoma cells, produced both large- and small-colony trifluorothymidine-resistant mutants, and were clastogenic. The purified yellow dye was further tested for SCE induction in mouse lymphoma cells and was determined to give a slightly positive response in the presence of S-9.

Disposition of [14C]methyl bromide in Fischer-344 rats after oral or intraperitoneal administration.

Methyl bromide is used as a disinfectant to fumigate soil. The intent of our study was to determine the disposition of methyl bromide following a single acute administration. Male Fischer-344 rats were given 250 mumol of [14C] methyl bromide/kg body wt by either oral or i.p. administration. Urine, feces and expired air were collected and at the end of 72 h the rats were sacrificed and tissues analyzed to determine 14C excretion and tissue distribution. After i.p. administration of methyl bromide, the dominant route of excretion was exhalation of 14CO2, with 46% of the dose exhaled as 14CO2. In contrast, urinary excretion of 14C was the major route of elimination (43% of the dose) when methyl bromide was given orally. Very little of the 14C appeared in the feces (less than 3% of the dose) regardless of route of administration. In rats with bile duct cannulations, 46% of an oral dose appeared in the bile over a 24-h period. Collection of bile significantly decreased the exhalation of 14CO2 and 14C excreted in urine compared to controls. At 72 h after oral or i.p. administration, 14-17% of the 14C remained in the rats, with liver and kidney being the major organs of retention. Results indicate that route of administration can affect the pathways for excretion. In addition, excretion of 14C in bile, coupled with the low levels of radioactivity found in the feces, indicates that reabsorption of biliary metabolites from the gut plays a significant role in the disposition of [14C] methyl bromide.

Disposition of [14C]2,3-dichloropropene in Fischer-344 rats after oral or intraperitoneal administration.

2,3-Dichloropropene (2,3-DCP), a component of commercial fumigants and nematocides, was mixed with [14C]2,3-DCP and given to rats by peroral (p.o.) or intraperitoneal (i.p.) administration. Urine, feces, and expired air were collected over 72 h. Excretion of radioactivity in urine predominated over other routes, with 66% to 75% of the dose excreted in 72 h. Feces contained from 13% to 21% of dose. 8% of the dose was exhaled as 14CO2. At the end of 72 h, only 2% to 3% of the dose remained in the carcass with the highest concentrations of 14C in liver, kidney, testes, and lung. Approx. 91% of the p.o. dose was absorbed from the gastrointestinal (GI) tract.

Analysis of procainamide hydrochloride and acecainide hydrochloride in rat feed.

An extraction and GLC assay procedure was developed for quantitation of procainamide hydrochloride and acecainide hydrochloride in rat feed. 4-Amino-N-[2-(dipropylamino)ethyl]benzamide hydrochloride was synthesized and utilized as an internal standard. The assay has good precision and accuracy and was used to establish the stability of acecainide hydrochloride and procainamide hydrochloride in rat feed.

Mutagenicity of mono-, di- and tri-nitropyrenes in Chinese hamster ovary cells.

1-Nitropyrene (1-NP), 1,3-dinitropyrene (1,3-DNP), 1-6-dinitropyrene (1,6-DNP), 1,8-dinitropyrene (1,8-DNP) and 1,3,6-trinitropyrene (1,3,6-TNP) were tested for mutagenicity in cultured Chinese hamster ovary (CHO) cells. Mutation at the hypoxanthine-guanine phosphoribosyl transferase gene locus was quantified. While 1-NP and 1,3-DNP had only marginal direct-acting mutagenicity, 1,6-DNP, 1,8-DNP and 1,3,6-TNP showed definite mutagenicity, with specific mutagenic activities of 8.1, 21 and 54 mutants/10(6) survivors/micrograms . ml-1 respectively. The mutagenicity of 1-NP increased with increasing concentrations of Aroclor-1254 induced liver homogenate (S9) in the treatment medium. However, S9 at all concentrations tested decreased the mutagenicity of 1,6-DNP and 1,8-DNP. S9 at low concentrations enhanced the mutagenicity of 1,3-DNP and 1,3,6-TNP and that at high concentrations decreased their mutagenicity. The positive mutagenic response of the nitropyrenes suggests that they are potentially carcinogenic, and that further research into their possible human health risk should be performed.

Contribution of primary aromatic amines to the mutagenicity of gasifier tars and coal oils.

Two reactions that chemically alter primary aromatic amines (PAA) were used to assess the contribution of these compounds to the indirect bacterial mutagenicity of tar from an experimental low Btu gasifier. The first reaction, nitrosation, effectively eliminated the mutagenicity of several PAA standards and a coal oil when run in a low pH media (1.2). When applied to gasifier tar, extensive direct (not requiring metabolic activity) mutagenicity was generated. This direct mutagenicity limited the interpretation of results. When the pH of the reaction media was raised to 2.5, the mutagenicity of PAA standards and the coal oil were still greater than 90% eliminated, however, no direct mutagenicity was observed for the gasifier tar. Furthermore, only 61% of the indirect (requiring metabolic activation) mutagenicity was eliminated. Acetylation reduced the indirect activity of most primary amine standards by greater than 79%. Acetylation of the tar likewise eliminated part, but not all, of the activity, whereas most of the activity of the coal oil was eliminated. These results indicated that a much lower percentage of the mutagenic activity of low Btu coal tar samples was due to primary aromatic amines than was the case for coal oil.

Distribution and elimination of inhaled phenanthridone in Fischer-344 rats.

Aza-arenes are widely distributed in the environment. Certain members of this chemical class are biologically active and therefore could pose health hazards to humans if inhaled or ingested. Since inhalation is the most likely route for significant exposure for aza-arenes that are air pollutants, knowledge of the absorption, distribution, and excretion after inhalation is necessary to understand mechanisms of toxicity and predict health hazards associated with exposure to aza-arenes. In this study, rats were exposed nose-only to an aerosol of [14C]phenanthridone, a mutagenic aza-arene found in coal tar. Tissues, urine, feces, and expired air were collected at specified times and assayed for radioactivity. Radioactivity was rapidly absorbed from the respiratory tract and distributed to all tissues examined. It was rapidly eliminated from tissues (greater than 80% in 12 h), being concentrated only in the large intestine at later timepoints. Excretion of radioactivity in both feces and urine was complete within 4 d. Virtually none of the dose was eliminated as [14C]CO2. [14C] Phenanthridone was also administered orally to assess its absorption from the gastrointestinal tract. Absorption was nearly complete (83 +/- 6%), indicating that phenanthridone ingested as a result of mucociliary clearance and swallowing, grooming, or coprophagy could also contribute to tissue exposure. These results are similar to those obtained from inhalation studies of other polycyclic aromatic hydrocarbons, indicating that as a class, inhaled polycyclic aromatic hydrocarbons may be rapidly absorbed from the respiratory tract, widely distributed throughout the body, and readily eliminated in both urine and feces.

Renal toxicity due to reactive metabolites formed in situ in the kidney: investigations with 4-ipomeanol in the mouse.

The in vitro metabolism and covalent binding of the furan derivative, 4-ipomeanol, was mediated by oxygen-requiring, NADPH-dependent, CO-inhibitable microsomal enzymes present in the livers, lungs and kidneys of adult male mice. These activities were inhibitable by piperonyl butoxide and they were markedly enhanced in hepatic microsomes from C57/6J mice, but not DBA/2J mice, pretreated with 3-methylcholanthrene. The i.p. administration of 4-ipomeanol to adult male mice resulted in the covalent binding of large amounts of its metabolite(s) in the lungs and kidneys. The material bound in the kidneys was located predominantly in the proximal renal cortical tubules. The covalent binding and toxicity of 4-ipomeanol to the renal tubules could be prevented by pretreatment of the animals with piperonyl butoxide. The hepatic covalent binding and toxicity of 4-ipomeanol were enhanced and the pulmonary and renal covalent binding and toxicity were decreased in C57BL/6J mice pretreated with 3-methylcholanthrene; however, this pretreatment did not significantly alter the tissue covalent binding or toxicity of 4-ipomeanol in noninducible DBA/2J mice. These results support the view that renal damage by 4-ipomeanol in the mouse is caused by reactive 4-ipomeanol metabolite(s) formed in situ in the kidney.

Determination of plasma procainamide and N-acetylprocainamide concentration by high-pressure liquid chromatography.

We describe a routine method for determining concentrations of the antiarrhythmic drug procainamide and its active metabolite, N-acetylprocainamide, in plasma. A simple extraction of 1.0 ml of plasma is followed by separation and chromatographic analysis by use of a column containing microparticulate silica. p-nitro-N-(2-diethylaminoethyl)benzamide hydrochloride was synthesized and used as the internal standard. Total chromatographic time is only 7 min. The day-to-day CV during three months of daily use was less than 4% of the mean for each compound, and we saw no deterioration in column performance during this time. Phenobarbital, phenytoin, lidocaine, primidone, methsuximide, quinidine, and their metabolites do not interfere.

A sensitive and specific assay for pentamethylmelamine in plasma: applicability to clinical studies.

A sensitive and specific procedure for measuring plasma levels of pentamethylmelamine (PMM), a chemotherapeutic agent currently undergoing phase I trials, has been developed. PMM was isolated (recovery greater than 95%) from 3-ml plasma samples by cation exchange chromatography followed by solvent extraction, and was quantitated by selected ion monitoring with a gas chromatograph/mass spectrometer. Deuterium-labeled PMM was synthesized and used as an internal standard; standard curves were prepared by plotting m/e 196/199 ratios versus concentrations, and were linear in the range of 0.05-50.0 microM. The coefficient of variation for repeated measurements was less than 1% at all concentrations studied. To test the applicability of this method to clinical studies, plasma clearances of PMM were measured in two studies in each of three cancer patients receiving 80-mg/m2 doses of PMM as 1-hour iv infusions. Peak plasma concentrations, observed immediately after the end of the infusions, averaged 4.1 microM (SE = 0.6 microM). Plasma elimination was found to be biphasic; a short initial half-life (approximately 27 minutes) was followed by a longer elimination half-life of 133 minutes (SE = 35 minutes). Measurements in two patients indicated that less than 0.1% of the administered PMM was excreted unchanged in the urine in 24 hours, suggesting that hepatic metabolism of PMM may be a major contributor to plasma clearance of the drug in man.

Mutagenicity of used crankcase oils from diesel and spark ignition automobiles.

The Salmonella mutagenicity assay was used to compare the mutagenic activity of used crankcase oil (UCO) from diesel and spark-ignition (gasoline) engine passenger cars. UCO samples were obtained during periodic oil changes from 9 spark-ignition and 10 diesel-powered vehicles. Five samples of unused motor oil were also tested. Direct tests of UCO did not detect mutagenic activity in Salmonella typhimurium strain TA-98. Therefore, an extraction procedure was used to concentrate the mutagens and remove interfering chemicals. Extracts were tested both with and without Aroclor-1254-induced rat liver homogenate fraction (S-9). Dose-dependent mutagenicity with and without S-9 was observed in both diesel and spark-ignition engine UCO extracts. Mutagenic activity was also found in unused oil extracts, but it was lower than that in UCO extracts and generally required addition of S-9. The mutagenic potency of diesel UCO extracts was similar to that of gasoline UCO extracts, both with and without addition of S-9. This indicated that potential health risks associated with disposal, handling, and recycling of diesel UCO may not be significantly different from those of UCO from gasoline engines.

Metabolism of 1-[14C]nitropyrene in isolated perfused rat livers.

1-Nitropyrene (1-NP), a constituent of diesel exhaust, is carcinogenic to rats and is a bacterial and mammalian mutagen. Biliary and fecal excretion of 1-NP metabolites are the major routes of excretion in rats, suggesting that hepatic metabolism plays a dominant role in determining the biological fate of 1-NP. The purpose of this investigation was to quantitate 1-[14C]NP metabolites formed in isolated perfused rat livers and excreted in bile from rats. Perfused rat livers displayed a capacity for oxidation, reduction, acetylation, and conjugation of 1-NP (or its metabolites). Reduction of 1-NP followed by N-acetylation was the major metabolic pathway observed in the perfused livers. Acetylaminopyrene (AAP) was the major metabolite detected, with total quantities (150 nmol) accounting for about 60% of the total 1-[14C]NP dose (258 nmol) added to the perfusate. Considerably smaller quantities of aminopyrene and hydroxynitropyrenes were also detected. Livers perfused with 1-[14C]NP excreted about 36 nmol equivalents of 1-[14C]NP (12% of the total 1-NP dose) in bile after 60 min. Some of the biliary metabolites were tentatively identified as metabolites of the mercapturic acid pathway. The spectrum of biliary metabolites was qualitatively identical to that seen in bile from intact rats. Quantities of 14C covalently bound to hepatic macromolecules from perfused livers were 0.4 nmol 1-NP eq/g liver. The data from this study indicate that the liver may be an important site for metabolism of 1-NP.

Excretion and metabolism of 1-nitropyrene in rats after oral or intraperitoneal administration.

Many nitro-substituted polycyclic aromatic hydrocarbons (NPAHs) have been identified as environmental pollutants and have been found to be mutagens and carcinogens in bacteria and mammalian systems. They require metabolism to express their biological activity. The metabolism and excretion of 1-nitropyrene (NP), a prevalent NPAH, by Fischer-344 rats after intraperitoneal (ip) or oral administration was studied. Radiolabeled NP was administered to rats (10 mg NP/kg body wt), and urine and feces were collected for 7 days. After ip administration of [14C]NP, 60% of the radioactivity was found in the urine and 20% in the feces. Likewise, 55 and 35% of the orally administered 14C was found in urine and feces, respectively. Both urine and feces were analyzed by high-pressure liquid chromatography for metabolites. The majority of the radioactivity in both urine and feces was associated with very polar metabolites, none accounting for more than 10% of the dose. Small amounts (less than 1% of the dose) of aminopyrene (AP), acetylaminopyrene, and NP were detected. A urinary metabolite (3-8% of the dose) was found that converted to acetylaminopyrene phenol (two isomers) when urine was heated overnight at 37 degrees C at pH 4.5. More of this metabolite (2.2 times) as well as AP (1.8 times), was excreted after oral than after ip administration of NP. The NP metabolites found in this study demonstrate that reduction of the nitro group is a significant route of NP metabolism in rats. Since nitroreduction appears to be necessary in the activation of NPAHs to bacterial mutagens, this indicates that similar metabolic pathways are present in rats (catalyzed by mammalian and/or gut bacterial enzymes) and that activation of NPAHs to carcinogens or toxins by nitroreduction is possible.

Ipomeanol 4-glucuronide, a major urinary metabolite of 4-ipomeanol in the rat.

Urinary excretion and metabolism of 4-ipomeanol was studied in rats injected ip with the radiolabeled compound. There was a rapid elimination of radioactivity in the urine, amounting to 47% of the administrated dose within 4hr. One major metabolite was isolated and purified by HPLC. Analysis by analytical HPLC, beta-glucuronidase hydrolysis, and mass spectrometry identified this material as ipo-meanol 4-glucuronide. The large amount of this metabolite excreted suggests that 4-glucuronidation is an important detoxication reaction in vivo for 4-ipomeanol in rats.

In vitro cytotoxicity to alveolar macrophages of tar from a low-Btu coal gasifier.

The cytotoxicity of fractions, subfractions, and some individual components present in tar from a low-Btu coal gasifier was determined in vitro with cultured dog alveolar macrophages. A subfraction of the tar which contained phenols and neutral nitrogen heterocycles was found to be a major contributor to the cytotoxic activity of the tar. Four compounds present in this subfraction were tested individually. Ranked from the most to the least toxic they were: 9-phenanthrol, 2-fluorenol, phenanthridone, and carbazole.