Effect of the length of alkyl chain on the cytochrome P450 dependent metabolism of N-diakylnitrosamines.

Liver microsomes from control and treated rats (P4501A, 2B, 2E1-induced) metabolize at variable metabolic rates eight N-nitroso-di-n-alkylamines, including five symmetrical (N-nitroso-dimethyl, -diethyl, -dipropyl, -dibutyl and -diamyl-amines) and four asymmetrical (N-nitrosomethylethyl, methylpropyl, methylbutyl, and methylamyl-amines), into aldehydes. Thus, the longer the alkyl chain of symmetrical N-nitrosamines, the smaller was the metabolic rate of the corresponding aldehyde formation. The chain length of the alkyl group of N-nitroso-methylalkylamines modified the oxidation of the alkyl moiety: the oxidation by CYP2E1 decreased as the n-alkyl chain length increased and conversely for the oxidation by CYP1A and CYP2B. Finally, the longer the n-alkyl chain length of asymmetrical N-nitrosamines, the greater was the oxidation of methyl groups.

Hydroxylation of carbon atoms of the alkyl chain of symmetrical N-nitrosodialkylamines by rat liver microsomes.

Liver microsomal preparations from control and treated rats (cytochromes P450 1A, 2B, 3A and 2E1-induced) metabolized at variable metabolic rates three nitrosodialkylamines (N-nitroso-dipropyl, dibutyl and diamyl-amines) into aldehydes and hydroxy-nitrosamines. The longer the alkyl chain, the smaller was the metabolic rate of the alpha-hydroxylation of alkyl chain yielding aldehyde and the greater was the metabolic rate of the corresponding (omega-1)-hydroxyl metabolite formation. Thus, the (omega-1) hydroxylation of the alkyl chain was the major metabolic pathway of N-nitrosodiamylamine (NDAA) so far as it represented 22-fold the alpha-hydroxylation. The balance between beta to omega hydroxylation and alpha-hydroxylation depends upon the alkyl chain length and also on specific P450 isoform induction.

Analysis of N-nitrosamines by high-performance liquid chromatography with post-column photohydrolysis and colorimetric detection.

N-Nitrosamines eluted from reversed-phase HPLC were quantitatively photohydrolysed in a UV photoreactor in aqueous solution to give the nitrite ion which could be determined colorimetrically with the Griess reagent. The chromatographic behavior of N-nitroso compounds (including 19 volatile dialkyl and 7 non-volatile N-nitrosamines) was studied on three octadecylsilane columns. The capacity factor varies linearly with the number of carbons atom of the n-dialkyl chains. N-nitrosamines bearing di-n-alkyl chains with the same number of carbon atoms could be separated with a highly polar mobile phase. The yield of photohydrolysis depends upon pH and time of exposure under UV light. The response was shown to be linear in the 0-200 ng range with a limit of detection of 8 pmoles injected for N-dialkyl nitrosamines. This limit was 20 pmoles for N-nitrosamines bearing two phenyl groups. Although N-nitrosamines could be detected at 230 nm without post-column reaction, such a reaction enhances the specificity of detection in biological matrices such as gastric juice or alcoholic beverages.

Cytochrome P450 hydroxylation of carbon atoms of the alkyl chain of symmetrical N-nitrosodialkylamines by human liver microsomes.

A panel of 14 human liver microsomal preparations metabolized at variable rates three symmetrical nitrosodialkylamines (N-nitroso-dipropyl, dibutyl and diamyl-amines, NDPA, NDBA, NDAA) into aldehydes and hydroxynitrosamines. Formation of linear aldehydes, convenient probes for alpha-hydroxylation of alkyl chain, and production of hydroxy metabolites of NDPA, NDBA and NDAA were simultaneously monitored by two specific HPLC detection methods. The longer the alkyl chain, the smaller the metabolic rate of the alpha-hydroxylation of the alkyl chain and the greater was the metabolic rate of the corresponding (omega-1)-hydroxy metabolite formation. Thus, the (omega-1)-hydroxylation of the alkyl chain was the major metabolic pathway of NDBA and NDAA in so far as it represented 3.3- and 86-fold of the alpha-hydroxylation. The balance between beta- to omega-hydroxylations and alpha-hydroxylation of carbon atoms of the alkyl chain depends upon its length and also upon the specific P450 isoform(s) involved. The hydroxylation site of the alkyl chain by P450 2E1 depends upon its length. For short alkyl chains, the main pathway was alpha-hydroxylation while for long alkyl chains, such as pentyl, (omega-1)-hydroxylation became the major pathway. The rate of alpha-hydroxylation was shown to be correlated with mutagenesis of 5 dialkylnitrosamines, as inferred from literature data, while the (omega-1)-hydroxylation was inversely correlated. Furthermore, other P450s than P450 2E1, such as P450 3A4 and 2C were shown to be involved in the metabolism of nitrosodialkylamines bearing long alkyl chains.

P450 2E1 expression in liver, kidney, and lung of rats treated with single or combined inducers.

Ethanol consumption combined with smoking increase the risk of cancer in many tissues. Such a mechanism implies the involvement of cytochrome P450 alcohol (CYP2E1), which is regulated by numerous xenobiotics. The combination of P450 2E1 inducers (acetone or pyridine) and 3-methylcholanthrene during rat treatment was shown to decrease the liver P450 2E1 content while it enhanced its expression in kidney. It is suggested that this differential tissue response helps explain the organotropy of nitrosamine carcinogenicity.

MICRA: a compact permanent magnet Fourier transform ion cyclotron resonance mass spectrometer.

MICRA, a compact Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer is described. The amount of miniaturisation in this device, based on a 1.24 T permanent magnet, remains compatible with genuine FT-ICR performance and analytical power in the mass range 2-1000 m/z, with a mass resolving power of 73,000 at mass 132. A first application of the transportability is the repetitive coupling of MICRA with a large-scale source of IR photons, the free electron laser CLIO.

Cytochrome P450 metabolic dealkylation of nine N-nitrosodialkylamines by human liver microsomes.

The metabolic dealkylation of nine nitrosodialkylamines, including five symmetrical (nitrosodimethylamine, nitrosodiethylamine, nitrosodipropylamine, nitrosodibutylamine and nitrosodiamylamine) and four asymmetrical nitrosodialkylamines (nitrosomethylethylamine, nitrosomethylpropylamine, nitrosomethylbutylamine and nitrosomethylamylamine), was investigated in 14 samples of human liver microsomes. All these nitrosodialkylamines were dealkytated to aldehydes that were separated by reversed phase HPLC and UV detected as dinitrophenylhydrazones. As the length of the alkyl chain increased from methyl to pentyl, dealkylation of symmetrical nitrosodialkylamines became less efficiently catalyzed by cytochrome P450. Conversely, oxidation of the methyl moiety of asymmetrical nitrosomethylalkylamines increased with the size of the alkyl moiety, while dealkylation of the longer alkyl group decreased. N-Dealkylase activities were significantly correlated with P450 activities measured in human liver microsomes. These catalytic activities involve CYP2A6 (coumarin 7-hydroxylation), CYP2C (mephenytoin 4-hydroxylation and tolbutamide hydroxylation), CYP2D6 (dextromethorphan O-demethylation), CYP2E1 (chlorzoxazone and p-nitrophenol hydroxylation) and CYP3A4 (nifedipine oxidation). By using 10 heterologously expressed P450s, it was shown that nitrosodimethylamine was mainly demethylated by CYP2E1. However, such enzyme specificity was lost with increasing size of the alkyl group. Therefore, the chain length of the alkyl group of nitrosodialkylamines determined the P450 involved in its oxidation. All these results emphasize that the catalytic site of P450 2EI has a geometric configuration such that only small molecules like nitrosodimethylamine fit favorably within the putative active site of the enzyme. Furthermore, there is good evidence that P450s other than P450 2E1, such as P450 2A6, 2C8/2C9/2C19 and 3A4, are involved in the metabolism of nitrosodialkylamines bearing bulky alkyl chains.

Gas phase infrared spectroscopy of selectively prepared ions.

The first example of direct structural characterization of polyaromatic ions by coupling a Fourier transform ion cyclotron resonance mass spectrometer with an infrared free-electron laser is presented. Measurement of the IR spectra of selectively prepared ionic reactive intermediates is allowed by the association of the high peak power and wide tunability of the laser with the flexibility of the spectrometer, where several mass selection and ion reaction steps can be combined, as demonstrated in the case of iron cation complexes of hydrocarbons. The present experimental setup opens the way to understanding chemical reaction paths.