Comparison of the in vitro metabolism of N-nitrosohexamethyleneimine by rat liver and lung microsomal fractions.

The in vitro metabolism of N-nitrosohexamethyleneimine by lung and liver microsomes and cytosol from uninduced male Fischer rats is described. Metabolites produced by both organs appeared to be identical. The liver subcellular fractions had a lower Km (0.6 mM) than did lung fractions (3 mM) and metabolized 2.5 to 5 times as much nitrosamine per mg protein. Our results, together with those from our earlier studies, indicate that, as the size of the carbon ring increases from nitrosopyrolidine to nitrosohexamethyleneimine, lung microsomes had an increased affinity for the cyclic nitrosamines; they was only a small effect with liver enzymes. Ths suggests that microsomal enzymes that metabolize cyclic nitrosamines in rat livers and lungs are not the same. The first stable alpha-hydroxylation product, 6-hydroxyhexanal, was not detected in reactions involving microsomes alone. Apparently, this compound is rapidly converted to 1,6-hexanediol by liver or lung microsomes. The presence of cytosol was needed for the full conversion of these metabolites to xi-hydroxycaproate and maximal alpha-hydroxylation activity. xi-Aminocaproate was always found in direct proportion to the hydroxyacid, suggesting that both acids arise from the same alpha-hydroxylation event by different breakdown mechanisms. beta- and gamma-hydroxynitrosohexamethyleneimine were not metabolized significantly by rat liver enzymes and thus, in this species, may be "detoxification products" of N-nitrosohexamethyleneimine.

Relationship of rat urinary metabolites of N-nitrosomethyl-N-alkylamine to bladder carcinogenesis.

Nitrosomethylalkylamines with chain lengths from C4 (n-butyl-) to C14 (n-tetradecyl-) were each administered in three rats at doses equimolar with 12 mg of the butyl compound. All of the compounds administered to rats at this dose, twice a week for 30 weeks, induced tumors in 100% of the animals. Some of the compounds with even-numbered alkyl chains induced bladder tumors, and a connection was sought with the metabolites of these excreted in urine. The pooled 24-hr urine was extracted with ethyl acetate before and after acidification to provide a neutral fraction and a fraction containing nitrosoamino acids. The fraction containing the acids was analyzed by capillary gas chromatography and by gas chromatography-mass spectrometry after esterification with diazomethane; the neutral fraction was analyzed similarly. The principal metabolite of the nitrosoamines with odd-numbered chains was found in the acidic fraction and was identified as nitrosomethyl-2-carboxy-ethylamine. There were several acids in the mixtures derived from the nitrosamines with even-numbered chains, nitrososarcosine and nitrosomethyl-3-carboxypropylamine being the major components. There was no trend in the yields of the nitrosamino acids that could be correlated with the differences in carcinogenic potency between the nitrosamines; the maximum yield of acids was more than 30% (from the tetradecyl compound). The principal component of the neutral fraction (less than or equal to 1% of the nitrosomethylalkylamine administered) was nitrosomethyl-2-oxopropylamine. The yield of this compound increased with length of the even-numbered chain nitrosamines.

Electronic and steric factors in regioselective hydroxylation catalyzed by purified cytochrome P-450.

A reconstituted hydroxylation system consisting of electrophoretically homogeneous phenobarbital-inducible rabbit liver microsomal cytochrome P-450 (P-450 LM2), NADPH-cytochrome P-450 reductase, phospholipid, buffer, NADPH, and O2 was used to oxidize four cyclohexane derivatives: cyclohexene, methylcyclohexane, norcarane and norbornane. Cyclohexene gave only cyclohexene oxide and allylic cyclohexenol, while methylcyclohexane yielded all possible monohydric alcohols, but with 1 degrees:2 degrees:3 degrees ratios of 0.072:1:1.25. Norcarane yielded 2-norcaranol. While oxidation of norbornane produced exo-2- and endo-2-norborneols in a ratio of 3.4:1, replacement of all four exo-hydrogens by deuterium led to a reversal of the exo:endo ratio to 0.76:1. These and other observations are interpreted as evidence for a selective, hydrogen-abstracting enzyme-bound oxidant exhibiting a large intramolecular deuterium isotope effect. A transient substrate carbon radical is a probable intermediate in the hydroxylation process.

Direct quantitative analysis of indicine-N-oxide in cancer patient samples by gas chromatography using the internal standard heliotrine-N-oxide including a mass spectral comparison of their trimethylsilyl derivatives.

Indicine-N-oxide was analyzed quantitatively in biological samples using a direct partial purification method involving acetonitrite precipitation or methanol precipitation followed by ion exchange chromatography. Trimethylsilyl derivatization of the resultant provided either of two derivatives, depending on the reaction conditions used, both of which had good gas chromatographic qualities. Heliotrine-N-oxide was used as the internal standard for this work. Data are presented to show that this is a reliable and useful internal standard based on its behavior in the partial purification method and on the gas chromatographic characteristics of its two derivatives. In addition, both low and high resolution mass spectral data indicate that heliotrine-N-oxide produces two trimethylsilyl derivatives analogous to those produced by indicine-N-oxide under the same conditions. Application of this procedure to urine and blood samples from cancer patients in clinical trials indicates that over 95% of the drug is removed from the circulation and excreted in the urine over the course of 48 h.

Metabolism of nitrosomethyl-n-alkylamines in Fischer rats.

To investigate a possible connection between the pattern of the excreted metabolites of a series of nitrosomethylalkylamines and their tumour spectrum in rats, the urinary metabolites of nitrosomethylalkylamines with alkyl chain lengths from C4 (n-butyl) to C14 (n-tetradecyl) have been studied, using male Fischer rats. All of these compounds induced a high incidence of tumours in the rats, but only those with chain lengths of 8, 10, 12 or 14 carbons induced bladder tumours. The principal metabolite from the nitrosamines with an odd number of carbons in the alkyl chain was nitrosomethyl-2-carboxyethylamine. Nitrososarcosine and nitrosomethyl-3-carboxypropylamine were the principal metabolites from those compounds with even-numbered chains. No trend was discernible between the pattern or yields of these acids and the carcinogenic effectiveness. The principal component in the neutral fractions was nitrosomethyl-2-oxopropylamine (less than or equal to 1% of the dose administered). Very small amounts were obtained from the odd-numbered chain compounds, but serially increasing amounts from the even-numbered chain compounds.

alpha-Hydroxylation pathway in the in vitro metabolism of carcinogenic nitrosamines: N-nitrosodimethylamine and N-nitroso-N-methylaniline.

Evolution of 15N2-labeled molecular nitrogen was used to gauge the extent of alpha-hydroxylation during rat liver homogenate metabolism of doubly 15N-labeled N-nitrosodimethylamine (DMN) and N-nitrosomethylaniline (NMA). These measurements were correlated with the extent of total metabolism as measured by the disappearance of the nitrosamines and by the formation of formaldehyde. The results indicate that approximately 34% of DMN and 19% of NMA were metabolized by the alpha-hydroxylation pathway. Positive controls utilizing doubly 15N-labeled N-nitroso-N-methylurea yielded 96% of labeled nitrogen. These results are in variance with previously published data which claimed that either less than 5% or about 100% of DMN is metabolized by that route in vitro. Formaldehyde formation was shown to be a poor measure of the extent of metabolism. Semicarbazide gave rise to both formaldehyde and nitrogen, which makes it an undesirable component of the in vitro metabolism mixtures, particularly when those two substances are being measured.

Metabolism of carcinogenic 2-hydroxybenz.

The rates of metabolism of the carcinogenic 2-hydroxybenzo[a]pyrene (2-OH-B[a]P) and the non-carcinogenic 3- and 9-hydroxybenzo[a]pyrenes in cultured cell systems have been studied and compared. While 70-80% of the non-carcinogens are converted to water-soluble derivatives by hamster embryo fibroblasts in 24 h, carcinogenic 2-OH-B[a]P is metabolized at a slower rate (45% in 24 h), comparable to that for the parent hydrocarbon, benzo[a]pyrene (B[a]P). Analysis of extracellular organic solvent-soluble metabolites of 2-OH-B[a]P in cultured hamster embryo fibroblasts, using h.p.l.c., indicates the presence of a single major metabolite, which has been identified by mass spectroscopy as a dihydroxy derivative of B[a]P. At least one additional major organic solvent-soluble metabolite is formed in cultures of either mouse epidermal epithelial cells or human foreskin fibroblasts, indicating a different balance of metabolic pathways in these cell systems. The greater persistence of carcinogenic 2-OH-B[a]P in cells and its higher concentration in the cell cytoplasm compared with the non-carcinogenic phenols may be related to its relatively high biological activity. Differences in metabolism of 2-OH-B[a]P in several cultured cell systems indicate the importance of an appropriate choice of activating system in understanding the relationship between metabolism and carcinogenesis.