Suppression of in vivo clearance of N-nitrosodimethylamine in mice by cotreatment with ethanol.

Oral cotreatment of mice with ethanol results in increased tumors in extrahepatic organs caused by some nitrosamines. This action, attributed in part to inhibition of hepatic first-pass carcinogen metabolism by ethanol, has possible relevance to the enhancing effect of alcoholic beverage consumption on human cancer risk. In this study, the effects of ethanol on clearance of N-nitrosodimethylamine (NDMA) were quantified in Swiss female and strain A male mice. In Swiss mice, a 1.6 g/kg ig ethanol dose preceding 1 or 5 mg/kg iv NDMA resulted in 20- to 30-fold increases in area-under-the-blood-concentration-vs.-time curves, mean residence times, and clearance half-times, and similar decreases in clearance. For a 0.5 mg/kg ig NDMA dose, the pharmacokinetic parameters were altered 30-fold and 450-fold by simultaneous ethanol doses of 0.08 and 0.8 g/kg, respectively. With 5 mg NDMA/kg ig, 0.4, 0.8, and 1.6 g/kg ethanol resulted in 6-, 10-, and 20-fold changes in clearance parameters. Comparison of the data with results obtained previously with patas monkeys indicated comparable effects of ethanol on tissue exposure to NDMA in the two species, confirming potential human applicability. In experiments with strain A mice, NDMA concentrations were also monitored in lung and liver. NDMA amounts in lung paralleled those in blood, and were more than sufficient to account for the previously reported increases in DNA adducts and tumors in lungs of similarly treated strain A mice.

Metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in the patas monkey: pharmacokinetics and characterization of glucuronide metabolites.

The metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was examined in the patas monkey, in order to provide further information about NNK metabolic pathways in primates. Female patas monkeys were given i.v. injections of [5-3H]NNK, and metabolites in serum and urine were analyzed by HPLC. Metabolism by alpha-hydroxylation of NNK was rapid and extensive, and the products of this pathway, 4-hydroxy-4-(3-pyridyl)butyric acid and 4-oxo-4-(3-pyridyl) butyric acid, accounted for a relatively large proportion of serum and urinary metabolites at all time points. This is significant because the formation of these products is associated with modification of DNA by NNK. The other major metabolic pathway was carbonyl reduction to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which detected both unconjugated and diastereomeric O-glucuronides. One of these glucuronides had been previously identified in rat urine, but the other diastereomer, which was the more prevalent of the two in serum and urine, had not been observed in studies of NNK metabolism in rodents. It was characterized by its spectral properties, by enzymatic hydrolysis to NNAL, and by derivatization of the released NNAL enantiomer with (R)-(+)-alpha-methylbenzylisocyanate. The two NNAL glucuronides accounted for 15-20% of the urinary metabolites in monkeys given 0.1 micrograms/kg NNK, which is similar to a smoker's dose, suggesting their use as dosimeters of NNK exposure in humans. Pharmacokinetic parameters were consistent with those observed in previous studies of nitrosamines, and varied predictably with body weight of five species. The results of this study have provided new insights relevant to assessing human metabolism of NNK.

Reduced blood clearance and increased urinary excretion of N-nitrosodimethylamine in patas monkeys exposed to ethanol or isopropyl alcohol.

Low concentrations of N-nitrosodimethylamine are metabolized in rodent and human liver by cytochrome P450IIE1, an activity competitively inhibitable by ethanol. In rodents coadministration of ethanol with N-nitrosodimethylamine results in increased tumorigenicity in extrahepatic organs, probably as a result of reduced hepatic clearance. To test this concept in a primate, the effects of ethanol cotreatment on the pharmacokinetics of N-nitrosodimethylamine were measured in male patas monkeys. Ethanol, 1.2 g/kg given p.o. before i.v. N-nitrosodimethylamine (1 mg/kg) or concurrently with an intragastric dose resulted in a 10-50-fold increase in the area under the blood concentration versus time curves and a 4-13-fold increase in mean residence times for N-nitrosodimethylamine. Isopropyl alcohol, 3.2 g/kg 24 h before N-nitrosodimethylamine, also increased these parameters 7-10-fold; this effect was associated with persistence of isopropyl alcohol and its metabolic product acetone, both IIE1 inhibitors, in the blood. While no N-nitrosodimethylamine was detected in expired air, trace amounts were found in urine. Ethanol and isopropyl alcohol pretreatment increased the maximum urinary N-nitrosodimethylamine concentration 15-50-fold and the percentage of the dose excreted in the urine by 100-800-fold. Thus ethanol and isopropyl alcohol greatly increase systemic exposure of extrahepatic organs to N-nitrosodimethylamine in a primate.

Estimation of the fraction of the dose of N-nitrosodimethylamine metabolized to methylamine in rats.

Despite many years of research on the metabolism of N-nitrosodimethylamine (NDMA) in rats, the significance of enzymatic denitrosation as a pathway remains unclear. To assess the role of this pathway of metabolism in rats, animals were administered NDMA by intravenous infusion at two infusion rates until steady state was achieved and the concentrations of NDMA (Css,NDMA) and methylamine (MA) (Css,MA), a product of the enzymatic denitrosation pathway, were determined in plasma. The clearance of NDMA (ClNDMA) from plasma was determined by dividing the infusion rate by Css,MA. The plasma clearance of MA (ClNDMA) was determined in a separate experiment. The fraction of the dose of NDMA metabolized by enzymatic denitrosation (fm) was calculated using the equation fm = (Css,MA*ClMA)/(Css,NDMA*ClNDMA). By this method it was estimated that 29% of the dose of NDMA was metabolized via the enzymatic denitrosation pathway. Thus enzymatic denitrosation is an important pathway in the metabolism of NDMA in rats.

Interspecies scaling of the pharmacokinetics of N-nitrosodimethylamine.

The pharmacokinetics of N-nitrosodimethylamine was studied in patas monkeys following i.v. doses of 0.5, 1.0, and 5.0 mg/kg and a p.o. dose of 1.0 mg/kg, and in Swiss mice at i.v. doses of 1.0 and 2.0 mg/kg. In the patas monkey the pharmacokinetics was linear over the i.v. dose range studied. The mean clearance (Cl), steady-state volume of distribution (Vss), mean residence time, and elimination half-life (t 1/2) were 103.3 +/- 26.7 (SD) ml/min, 3061 +/- 821 ml, 30.8 +/- 10.8 min, and 21.1 +/- 8.5 min, respectively. Assuming that the pharmacokinetics was linear at the p.o. dose used, the p.o. bioavailability of N-nitrosodimethylamine in the monkey was 49%. The pharmacokinetics was also linear in mice, and the average Cl, Vss, mean residence time, and t 1/2 were 3.81 ml/min, 21.0 ml, 5.5 min, and 11.9 min, respectively. These data and data for rats, hamsters, rabbits, dogs, and pigs taken from the literature were used to scale Cl and Vss to body weight using the allometric equation. The resulting equation for Cl was Cl = 49.7B0.998 and the equation for Vss was Vss = 748B1.05 where B is body weight in kg. The fit of the data to the equation was excellent in both cases. Using these equations and assuming a body weight of 70 kg for humans, the Cl and Vss for N-nitrosodimethylamine in humans are estimated to be 3450 ml/min and 64,800 ml, respectively.

Cardiovascular effects of SK&F 104078, a novel alpha-adrenoceptor antagonist, in normotensive and hypertensive rats.

SK&F 104078 is a novel alpha-adrenoceptor antagonist derived from the 3-benzazepine alpha 2-adrenoceptor antagonist SK&F 86466. SK&F 104078 will block both alpha 1- and vascular postjunctional alpha 2-adrenoceptors but does not block most prejunctional alpha 2-adrenoceptors. Intravenous (i.v.) administration of SK&F 104078 decreased blood pressure (BP) in both spontaneously hypertensive and DOCA-salt hypertensive rats. SK&F 104078 potentiated the hypotensive response to tilt in anesthetized spontaneously hypertensive rats (SHR). Although SK&F 104078 had no effect on BP in normotensive rats, the tilt-induced decrease in BP in these animals was also potentiated. In this regard, SK&F 104078 resembled the selective alpha 1-adrenoceptor antagonist prazosin, rather than the alpha 2-adrenoceptor antagonists rauwolscine or SK&F 86466. Oral administration of SK&F 104078 had no significant effect on BP in SHR unless extremely high doses were administered. This was consistent with low plasma concentrations of SK&F 104078 observed after oral administration. After i.v. administration, the clearance of SK&F 104078 from plasma was 123 ml/min/kg, the steady-state volume of distribution was 17 L/kg, and the fraction excreted unchanged in urine was less than 1%. The low oral bioavailability of SK&F 104078 did not appear to be due to high first-pass oxidative metabolism, since pretreatment of SHR with the suicide substrate inhibitor of cytochrome P-450, 1-aminobenzotriazole (ABT), did not result in increased oral efficacy. SK&F 101253, a close structural analogue of SK&F 104078, was an effective antihypertensive when administered orally. Comparison of the stability of SK&F 101253 and SK&F 104078 in acid media showed SK&F 104078, but not SK&F 101253, to be rapidly degraded.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic denitrosation of N-nitrosodimethylamine in vivo in the rat.

Enzymatic denitrosation is a potentially inactivating metabolic route that has been shown to convert carcinogenic N-nitrosodimethylamine (NDMA) to methylamine (MA) in vitro. To investigate its quantitative course in vivo, groups of 8-week-old male Fischer rats have been given small (8-15 mumol/kg) p.o. or i.v. bolus doses of 14C-labeled NDMA and the subsequent formation of radioactive MA has been monitored by high performance liquid chromatographic analysis of serially collected blood samples from each individual. Adjusting the [14C]MA fluxes observed for the previously measured rates at which MA is itself eliminated from the system after intragastric administration, denitrosation was calculated to represent a rather uniform 21.3 +/- 1.3% (SE) of total NDMA elimination in the four animals studied. By contrast, repetition of the experiment with fully deuterated NDMA (NDMA-d6) revealed a significantly wider variance in the results (39.8 +/- 8.9%). An alternative calculation using values for elimination of i.v. doses of MA and its trideuteromethyl analogue gave an even larger difference for MA formation between NDMA and NDMA-d6, the estimated extents of in vivo denitrosation in this case being 14.5 +/- 0.9% and 48.3 +/- 10.8%, respectively. The results indicate that denitrosation is a major metabolic pathway for NDMA elimination and suggest that deuteration of the carcinogen induces a shift in its metabolism toward increasing denitrosation at the expense of the competing activation pathway. Consequently, denitrosation may be the previously undefined in vivo metabolic route, the existence of which was suggested by the findings that deuteration of NDMA lowered its hepatocarcinogenicity and liver DNA alkylating ability in rats.

Pharmacokinetics of N-nitrosodimethylamine in swine.

The pharmacokinetics of N-nitrosodimethylamine (NDMA) have been studied in swine. They were studied following i.v. administration of 0.1, 0.5 and 1.0 mg/kg, and following oral doses of 1.0 and 5.0 mg/kg of NDMA. Following a bolus i.v. dose, the concentration of NDMA in blood declined biphasically with a mean distribution half-life of 7 min and a mean elimination half-life of 28 min. The areas under the blood concentration versus time curves (AUC) were roughly proportional to dose indicating that the pharmacokinetics in this dose range were first order. The mean systemic clearance from blood was 65.8 ml/min/kg, the steady-state volume of distribution was 1.4 l/kg, and the mean residence time was 20 min. Following the oral doses, the AUC and peak concentration in blood were not proportional to the dose. It is likely that the pharmacokinetics at the lower dose were first order, but at the higher dose the pharmacokinetics were no longer first order because metabolism was saturated. The bioavailability of the 1.0 mg/kg dose was 67%. Since the clearance was probably due to metabolism and the clearance from blood exceeded hepatic blood flow, the high bioavailability suggests that extrahepatic metabolism plays an important role in the systemic clearance of NDMA in swine.

Pharmacokinetics of a series of 6-chloro-2,3,4,5-tetrahydro-3-substituted-1H- 3-benzazepines in rats. Determination of quantitative structure-pharmacokinetic relationships.

To aid in the effort to discover novel agents for the treatment of cardiovascular disease, the relationships between pharmacokinetic parameters in the rat and lipophilicity and basicity were studied for a series of 6-chloro-2,3,4,5-tetrahydro-3-substituted-1H-3-benzazepines. Eight compounds, ranging in lipophilicity from log P = 1.64 to 3.50 and basicity from pKa = 6.75 to 9.36, were studied. The compounds were administered iv to rats, and the pharmacokinetic parameters were calculated from the plasma concentration-time curves. Plasma protein binding was determined in vitro using equilibrium dialysis to allow calculation of steady state volume of distribution of unbound drug, Vss,u; and tissue binding. Stepwise regression analysis with each pharmacokinetic parameter as the dependent variable and log P and pKa as the independent variables was performed. In no case was there a significant relationship between a pharmacokinetic parameter and both of the independent variables. Statistically significant linear relationships were found between pKa and Vss and t 1/2z. Lipophilicity was found to correlate with the free fraction in plasma and the free fraction in tissues. The clearance parameters did not correlate with either of the physicochemical parameters. The pharmacokinetics of the one secondary amine in the series were clearly different from those of any of the tertiary amines. The clearance of the secondary amine was lower and the volume of distribution higher than any of the tertiary amines. These results demonstrate that alteration of the lipophilicity of 3-substituted benzazepines does not alter their pharmacokinetics in a predictable fashion but that the pharmacokinetics of secondary amines may be substantially different than tertiary amines.

Pulmonary clearance of vasoactive drugs: N-oxidation of SK&F 86466 in the isolated perfused rat lung.

The contribution of the lung to the systemic clearance of the alpha receptor antagonist SK&F 86466 in rats was determined using the isolated perfused rat lung. Lungs were perfused with a blood-free medium containing SK&F 86466 at initial concentrations ranging from 0.26 to 5.1 microM at a flow rate of 104 ml/min. During the perfusion the concentration of SK&F 86466 in the perfusion medium declined monoexponentially. The half-life (8.0 +/- 1.8 min), steady state volume of distribution (205 +/- 16 ml), clearance (18.3 +/- 2.4 mL/min) and extraction ratio (0.18 +/- 0.02) were independent of the initial concentration of SK&F 86466. The free fraction of SK&F 86466 in the perfusion medium, determined by equilibrium dialysis, was 0.598. The average intrinsic clearance of SK&F 86466 in the lung, calculated using the flow rate, free fraction and clearance, was 37.4 ml/min. The concentration of the N-oxide metabolite of SK&F 86466 (SK&F 102102) in the perfusion medium increased with time, and at the end of the perfusion was approximately equal to the initial concentration of SK&F 86466. The pharmacokinetics of SK&F 86488 in lungs from rats pretreated with the suicide substrate inhibitor of cytochrome P-450 1-aminobenzotriazole were identical to the kinetics in lungs from control animals. The pretreatment regimen used reduced the activity of ethoxycoumarin-O-deethylase in lung microsomes by 70% and reduced P-450 content to below the limit of detection.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacokinetics of N-nitrosodimethylamine in beagles.

The pharmacokinetics of N-nitrosodimethylamine (NDMA) has been studied in beagles. Four male beagles were given 0.5- and 1.0-mg/kg doses of NDMA i.v. and 1.0- and 5.0-mg/kg doses p.o., and at appropriate times after dosing blood samples were drawn and the concentration of NDMA was measured. The experiments were separated by at least 1 week. Following a bolus i.v. dose, the concentration of NDMA in blood declined biphasically with a mean distribution half-life of 19 min and a mean elimination half-life of 73 min. The areas under the blood concentration versus time curves were proportional to the dose indicating that the pharmacokinetics in this dose range were first order. The mean systemic clearance was 43.3 ml/min/kg, the volume of distribution at steady state was 1.9 liters/kg and the mean residence time was 45 min. The clearance of NDMA in the dog was entirely metabolic because no NDMA could be detected in urine after i.v. dosing. The areas under the curve and maximum concentration in blood after the two p.o. doses were not proportional to dose. The evidence suggests that the pharmacokinetics of the 1.0-mg/kg dose were first order, but at the 5.0-mg/kg dose the metabolism of NDMA was saturated. The bioavailability of the lower p.o. dose (i.e., the fraction of the dose that reached the systemic circulation) averaged 93%. The high bioavailability was unexpected since, in the rat, the bioavailability of NDMA is only about 10%, and the systemic clearance in the dog exceeds hepatic blood flow. These data suggest that a substantial fraction of the systemic clearance is extrahepatic and that the pharmacokinetics of NDMA in higher species may be quite different from that observed in rodents.

Pharmacokinetics, metabolism, and disposition of 6-chloro-2,3,4,5-tetrahydro-3-methyl-1H-3-benzazepine (SK&F 86466) in rats and dogs.

The pharmacokinetics and metabolism of 6-chloro-2,3,4,5-tetrahydro-3-methyl-1H-3-benzazepine (SK&F 86466) have been studied in rats and dogs. Using radiolabeled SK&F 86466, it was shown that the compound was completely absorbed from the gastrointestinal tract following oral administration. Most of the administered radioactivity (approximately 80%) was excreted in urine with the remainder excreted in feces via the bile. Very little of the parent compound was excreted unchanged in the urine. The major urinary metabolite, accounting for about 55% of the dose in rat and 35% in dog, was the N-oxide. N-Demethylation also occurs in both species, and in the rat approximately 20% of the dose is metabolized by this route. The plasma concentration vs. time curves following iv administration were analyzed using a two-compartment open model. The distribution phase half-life was 0.24 hr in the rat and 0.37 hr in the dog. In both species the terminal half-life was approximately 2 hr. The volume of distribution at steady state in the rat was 12.1 liters/kg and in the dog was 8.2 liters/kg. About 55% of the drug in plasma was bound to protein in both species so that the volume of distribution of the free drug was 27 liters/kg in the rat and 19 liters/kg in the dog. The clearance of SK&F 86466 from blood was very high in both the dog (56 ml/min/kg) and the rat (191 ml/min/kg). Since less than 1% of the compound was excreted unchanged in urine, the clearance was almost entirely metabolic.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurement of 7-methylguanine as an estimate of the amount of dimethylnitrosamine formed following administration of aminopyrine and nitrite to rats.

We have demonstrated that there is a dose-related increase in the excretion of 7-[methyl-14C]methylguanine ( [14C]m7Gua) following p.o. administration of di[methyl-14C]methylnitrosamine to rats. Urine was collected for 24 hr after di[methyl-14C]methylnitrosamine administration, and the purines were precipitated from an aliquot of the urine with silver nitrate. Purines were released from the precipitate with HCl, and [14C]m7Gua was quantified by chromatography on an Aminex A-6 column. The excretion of [14C]m7Gua increased linearly with the dose of dimethylnitrosamine. This relationship was used to estimate the amount of di[methyl-14C]methylnitrosamine formed in the reaction of [14C]aminopyrine with sodium nitrite in rats gavaged with these compounds. The dose of dimethylnitrosamine was also estimated from the amount of alkylation of liver DNA in the same animals. These estimates usually differed by less than a factor of 2. [14C]aminopyrine and sodium nitrite were administered. The possibility of using this assay to obtain data on nitrosation in humans is discussed.

Mutation, DNA labeling, and transformation of BHK-21/CL 13 cells by MNNG, and nitrosocimetidine.

N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) has been reported to induce BHK-21/Cl 13 cell growth in agar suspension. To determine if MNNG was also mutagenic to BHK cells, an ouabain-resistance mutation assay was established using these cells. In this system MNNG was compared to nitrosocimetidine (NC). MNNG and NC did induce ouabain-resistant mutations in BHK cells. The ability of the test compounds to methylate DNA in BHK cells was also determined, and both MNNG and NC yielded detectable levels of 7-methylguanine in treated cells. MNNG and NC were tested for the ability to transform BHK cells, and did. NC was found to be as effective a mutagen and transforming agent in BHK cells as MNNG when administered at equitoxic concentrations; approx. 4-fold less effective at equimolar concentrations.

DNA-methylation by nitrosocimetidine and N-methyl-N-nitro-N-nitrosoguanidine in the intact rat.

The ability of the nitroso derivative of the drug cimetidine to interact with cellular macromolecules in the intact rat was investigated. Radio-labelled nitrosocimetidine (NC) was shown to methylate DNA in a variety of tissues in the rat after oral administration. Radioactivity was also detected in the RNA and protein extracted from these same tissues. Methylation of DNA by the parent compound, cimetidine, was not detected in any of the tissues studied. For comparison, the DNA methylation produced by the carcinogen N-methyl-N-nitro-N-nitrosoguanidine (MNNG) dosed orally was measured. DNA alkylation by MNNG was found to be approx. 2-36 times greater than that produced by NC, varying with the tissues studied. The highest yield of DNA alkylation was found in the stomach for MNNG and the small intestine for nitrosocimetidine suggesting pharmacokinetic differences.

Induction of the DNA repair enzymes uracil DNA glycosylase and 3-methyladenine DNA glycosylase in regenerating rat liver.

The capacity of eukaryotic cells to modulate the activities of DNA repair enzymes during cell proliferation was examined. Using regenerating rat liver as a model system, the specific activities of the DNA repair enzymes uracil DNA glycosylase and 3-methyladenine DNA glycosylase were determined at specific intervals after partial hepatectomy. The induction of DNA replication and the stimulation of DNA polymerase were also measured in order to relate changes in the potential for DNA repair to those observed for DNA replication. As measured in nuclear extracts, the specific activities of both the uracil DNA glycosylase and the 3-methyladenine DNA glycosylase were increased in regenerating rat liver reaching maximal levels 18--24 h after partial hepatectomy. The specific activity of each DNA repair enzyme returned to basal levels by 48 h after the hepatectomy. No increase in either enzyme activity was observed in sham operated controls. The products of the reactions were identified as 3-methyladenine or as uracil by high pressure liquid chromatography or by gel filtration on Sephadex G-10. The 2--3 fold increases in the specific activity observed for each nuclear DNA repair enzyme was comparable to the 2.7 fold increase observed for DNA polymerase activity. The stimulation of DNA repair enzymes in regenerating rat liver is a further suggestion that eukaryotic cells actively regulate excision repair pathways in the defined pattern of gene expression observed during the eukaryotic cell cycle.