Chang Kee Lim

Species differences in the covalent binding of [14C]tamoxifen to liver microsomes and the forms of cytochrome P450 involved

Species differences in the NADPH-dependent covalent binding of [14C]tamoxifen to liver microsomes have been studied using preparations from humans, female F344 rats and DBA/2 mice. Protein binding has been used as an index of metabolic activation and as a surrogate for DNA binding in order to establish which forms of cytochrome P450 are responsible for genotoxicity. A panel of 12 human liver microsomes has been characterized and immunoquantified for nine cytochrome P450 isoenzymes. Binding of tamoxifen (45 microM) (25 +/- 2.5 pmol/15 min/mg protein, mean +/- SE) correlated (P < 0.05) with CYP3A4 and CYP2B6 content. Covalent binding of [14C]tamoxifen to microsomal preparations from human breast tumour tissue could also be detected but at levels 7-fold lower than in liver. The covalent binding of tamoxifen to mice, rat or human liver microsomal preparations increased with increasing substrate concentration. Covalent binding of [14C]tamoxifen (45 microM) in rats was 3.8-fold and mice 17-fold higher than in human liver microsomal preparations. In mice, the apparent Km (9.6 +/- 1.9 microM) was very much lower than for rats (119 +/- 41 microM). Pretreatment of female rats with phenobarbitone or dexamethasone resulted in a 4- to 5-fold increase in [14C]tamoxifen binding, relative to controls, consistent with the involvement of CYP2B1 and CYP3A1 in the metabolic activation. It cannot be distinguished at present if the same reactive metabolites are involved in protein and DNA binding. The greater potential of mouse liver microsomes to activate tamoxifen, relative to rats, does not reflect DNA damage or hepatocarcinogenicity seen following dosing with tamoxifen in vivo. It is concluded that covalent binding of tamoxifen to protein in vitro cannot be directly related to the carcinogenic potential of this compound. However, in the three species investigated, results suggest that the rat is a better model than the mouse for human liver microsomal activation of tamoxifen both with respect to kinetic parameters and the pattern of metabolic products.

N-(4-nitro-2,1,3-benzoxadiazoyl-7-yl)-N-methyl-2-aminoacetohydrazide (NBD-CO-Hz) as a precolumn fluorescent derivatization reagent for carboxylic acids in high-performance liquid chromatography

A new fluorescent reagent for carboxylic acids, N-(4-nitro-2,1,3-benzoxadiazoyl-7-yl)-N-methyl-2-aminoacetohydr azide (NBD-CO-Hz) was synthesized and its applicability as a precolumn derivatization reagent in high-performance liquid chromatography was examined. NBD-CO-Hz reacted with 2-arylpropionic acids (2-APAs), a group of non-steroidal antiinflammatory drugs (NSAIDs) in the presence of a condensing agent, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and pyridine at room temperature for 2 h to give fluorescent adducts. The reaction solution was subjected to a reversed phase or a chiral stationary phase HPLC and the derivatives were detected fluorometrically at a wavelength of 530 nm with an excitation of 475 nm. The detection limits were in the fmol range on column.

Identification and mechanism of formation of potentially genotoxic metabolites of tamoxifen: study by LC-MS/MS

On-line high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI MS) and tandem mass spectrometry (MS/MS) have been applied to the study of tamoxifen metabolism in liver microsomes and to the identification of potentially genotoxic metabolites. The results showed that the hydroxylated derivatives, including 4-hydroxytamoxifen and alpha-hydroxytamoxifen are detoxication metabolites, while arene oxides, their free radical precursors or metabolic intermediates, are the most probable species involved in DNA-adduct formation.

Tamoxifen metabolism in rat liver microsomes: identification of a dimeric metabolite derived from free radical intermediates by liquid chromatography/mass spectrometry

Tamoxifen has been shown to be a potent liver carcinogen in rats, and generates covalent DNA adducts. On-line high performance liquid chromatography/electrospray ionisation mass spectrometry (HPLC/ESI-MS) has been used to further study the metabolites of tamoxifen formed by rat liver microsomes in the presence of NADPH with a view to identifying potential reactive metabolites which may be responsible for the formation of DNA adducts, and liver carcinogenesis. A metabolite has been detected with a protonated molecule at m/z 773. The mass of this compound is consistent with a dimer of hydroxylated tamoxifen (m/z 388). Analysis of 4-hydroxytamoxifen incubated with a rat liver microsomal preparation showed the formation of a similar metabolite with an apparent MH+ ion at m/z 773, believed to be a dimer of 4-hydroxytamoxifen formed by a free radical reaction. The retention time for this metabolite from 4-hydroxytamoxifen is identical to that of the tamoxifen metabolite, suggesting that these two compounds are the same. The levels of the dimer were higher when 4-hydroxytamoxifen was used as substrate and, in addition, two isomers were detected. It is proposed that tamoxifen was first converted to arene oxides which react with DNA or to 4-hydroxytamoxifen, either directly or via 3,4-epoxytamoxifen, which then undergoes activation via a free radical reaction to give reactive intermediates which can then react with DNA and protein, or with themselves, to give the dimers (m/z 773).

Analysis of urinary and faecal porphyrin excretion patterns in human porphyrias by fast atom bombardment mass spectrometry

We report a new method for obtaining urinary and faecal porphyrin excretion patterns in human porphyrias based on fast atom bombardment mass spectrometry (FAB-MS). Porphyrins were esterified and extracted from urine or faeces as their methyl esters for analysis by FAB-MS. The protonated pseudo-molecular ion [M + H]+ observed for each porphyrin is characteristic of that porphyrin, thus allowing a mixture of porphyrins to be analysed without the need for chromatographic separation. By using tandem MS, identification and characterisation of unknown porphyrins can be achieved. The urinary and faecal porphyrin excretion patterns from various porphyric patients obtained by FAB-MS are in good agreement with those analysed by TLC or HPLC methods.