Estelle Rathahao

Biotransformation of bisphenol F by human and rat liver subcellular fractions.

Bisphenol F [4,4-dihydroxydiphenyl-methane] (BPF) has a broad range of applications in industry (liners lacquers, adhesives, plastics, coating of drinks and food cans). Free monomers of this bisphenol can be released into the environment and enter the food chain, very likely resulting in the exposure of humans to low doses of BPF. This synthetic compound has been reported to be estrogenic. A study of BPF distribution and metabolism in rats has demonstrated the formation of many metabolites, with multiple biotransformation pathways. In the present work we investigated the in vitro biotransformation of radio-labelled BPF using rat and human liver subcellular fractions. BPF metabolites were separated, isolated by high-performance liquid chromatography (HPLC), and analysed by mass spectrometry (MS), MS(n), and nuclear magnetic resonance (NMR). Many of these metabolites were characterized for the first time in mammals and in humans. BPF is metabolised into the corresponding glucuronide and sulfate (liver S9 fractions). In addition to these phase II biotransformation products, various hydroxylated metabolites are formed, as well as structurally related apolar metabolites. These phase I metabolic pathways are dominant for incubations carried out with liver microsomes and also present for incubations carried out with liver S9 fractions. The formation of the main metabolites, namely meta-hydroxylated BPF and ortho-hydroxylated BPF (catechol BPF) is P450 dependent, as is the formation of the less polar metabolites characterized as BPF dimers. Both the formation of a catechol and of dimeric metabolites correspond to biotransformation pathways shared by BPF, other bisphenols and estradiol.

4-Hydroxynonenal in foodstuffs: heme concentration, fatty acid composition and freeze-drying are determining factors

Abstract 4-Hydroxynonenal (HNE) is a product of lipid peroxidation. It has been often used as a biomarker of endogenous lipid peroxidation and its concentration is increased in several diseases. But HNE is not only formed during lipid peroxidation occurring in the body. Some authors have shown that it is also present in oxidized oils and in meats. The aim of this study is to compare the effect of food composition (heme iron, fatty acid composition) or freeze-drying on HNE formation in foodstuffs. The methodology is based on extraction/purification procedure followed by HPLC separation with UV detection. As HNE is chemically very reactive and binds easily to proteins, we used radiolabeled HNE to calculate extraction efficiency, so total HNE can be estimated as only free HNE can be measured. The concomitant presence of both heme iron and omega 6 fatty acids, such as linoleic acid, is important for HNE formation in foodstuffs. Freeze-drying increases this formation.

Structural characterization by both positive and negative electrospray ion trap mass spectrometry of oligogalacturonates purified by high-performance anion-exchange chromatography.

The off-line coupling of high-performance anion-exchange chromatography to electrospray ion trap mass spectrometry (ESI-IT-MS) is described. Two sets of isocratic conditions were optimised for the semi-preparative purification of oligogalacturonates of degree of polymerisation from 4 to 6 by monitoring eluates with either pulsed amperometric detection or evaporative light scattering detection in the presence of an online Dionex Carbohydrate Membrane Desalter (CMD). In these conditions, purified oligogalacturonate solutions were suitable, without further desalting steps, for infusion ESI-IT-MS experiments. This paper provides some interesting features of positive and negative ESI-IT-multiple MS (MSn) of these acidic oligosaccharides. The spectra acquired in both ion modes show characteristic fragments resulting from glycosidic bond and cross-ring cleavages. Under negative ionization conditions, the fragmentation of the singly-charged [M-H]- ions, as well as the Ci-, and Zi-, fragment ions through sequential MSn experiments, was always dominated by product ions from C- and Z-type glycosidic cleavages. All spectra also displayed 0.2 A-type cross-ring cleavage ions which carry linkage information. Collision-induced dissociation (CID) spectra of sodium-cationized species obtained under positive ionization conditions were more complex. Successive MSn experiments also led to the 0.2 A-type cross-ring cleavage ions observed together with B- and Y-type ions. The presence of the 0.2 A ion series was related to Mr 60 (C2H4O2) losses. Combined with the absence of the Mr 30 (CH2O) and the Mr 90 (C3H6O3) ions, these ions were indicative of 1-4 type glycosidic linkage.

In vitro aromatic bioactivation of the weak estrogen E2α and genesis of DNA adducts

Abstract Specific A-ring hydroxylated metabolites of 17β-estrogens are known to be endogenous pro-carcinogens, more particularly the 4-hydroxylated forms of estrogens produced by cytochrome P4501B1. In this study, we investigated whether estradiol-17α, the main hepatic residue of estradiol-17β in cattle treated for anabolic purposes with estradiol containing implants, could be significantly metabolized by human cells, and whether its aromatic metabolites could induce the formation of DNA adducts as estradiol-17β and estrone do. First, using a human kidney adenocarcinoma cell line, which expresses specifically the cytochrome P4501B1, we showed that estradiol-17α is bioactivated into a mixture of 2- and 4-catechol estrogens leading to the corresponding methoxyestrogens unambiguously identified by LC–APCI–MS/MS. We then demonstrated that the 2- and 4-hydroxylated derivatives of estradiol-17α incubated under oxidative conditions with calf thymus DNA gave stable DNA adducts and abasic sites, respectively. From these results, we can consider that human cells expressing CYP1B1-dependent hydroxylation activities metabolize estradiol-17α at the same magnitude as estradiol-17β and estrone, and that in oxidative conditions, the resulting aromatic metabolites can lead to the formation of both stable and unstable DNA adducts.

Stereochemical effects enhanced by using selective “self-ionization” under electron ionization conditions in a quadrupole ion trap mass spectrometer

Abstract From a previous study on the reactivity of the cis and trans (4-hydroxy-4-methyl-cyclohexyl)-benzoates (Mw = 234) under ammonia chemical ionization conditions in high pressure source, it was demonstrated that competitive decompositions, have specific orientations. In particular, highly stereospecific benzoic acid loss from the trans isomer takes place, suggesting that protonation by NH 4 + regioselectively occurred at the benzoate site rather than at the hydroxyl group. From the cis epimer elimination of water was only observed. This behavior could be explained by (1) possible proton transfer to both the functional groups and (2) decomposition occurring via water loss due to a higher rate constant compared with that of the benzoic acid loss (PA C 6 H 5 COOH > PA H 2 O ), which is not observed. These epimers are now studied under low pressure chemical ionization (CI) and electron impact (EI) conditions by using an ion trap mass spectrometer. Epimer differentiation from the stereospecific benzoic acid loss can be achieved independently of the gas-phase reagent used for chemical ionization, even if quasimolecular ions are entirely decomposed (i.e. in low pressure isobutane CI and methane CI). More spectacular differentiation is displayed in the ammonia CI mass spectra of epimers. Among the various quasimolecular species, the product [MNH 4 –H 2 O] + ions, termed substituted ions, usually containing a covalent C–NH 3 + bond are herein in fact a noncovalent form similar to the ammonia solvating protonated(4-hydroxy-4-methyl-cyclohexyl)-benzoate structure, as shown by deuterium labeling. Other product ions such as C 7 H 11 NH 3 + and {NH 3 , C 6 H 5 COOH 2 + } are detected in contrast to those observed under high pressure source conditions. Collision induced dissociation spectra of the adduct MNH 4 + ions in addition to those of protonated molecules are investigated to obtain information about the location of ammonium (or proton) attachment. It appears for the trans isomer, a regioselective approach to the benzoate group, yielding [MH–C 6 H 5 COOH] + , takes place rather than attack at the OH site resulting into [MH–H 2 O] + . This difference is at the origin of the observed stereospecific decomposition, which occurs via anchimeric assistance. Alternatively, these epimers give similar EI mass spectra, masking all the stereochemical effects, which reappear when using a residence time of 80 ms prior to apply the analytical scan. Under such conditions, ion–molecule reactions are enhanced, yielding formation of the epimeric MH + ions. These “ self-ionization ” processes are induced through exothermical proton transfers from many EI fragment ions. Competitive decompositions of the product even-electron MH + species provide a direct cis / trans differentiation. The diagnostic cleavages involved are comparable to those observed under low pressure CI conditions except that the loss of water occurs from both the precursors. The latter loss indicates that protonation is in competition and takes place at either of the basic sites, in contrast with that observed in chemical ionization. Furthermore, the exothermicity of proton transfer is preserved as internal energy of MH + which allows consecutive decompositions. At higher m/z ratio ranges, several adduct species are stereoselectively observed in the self-ionization mass spectrum of the trans epimer. These specific processes provide enhancement of the stereochemical cis / trans differentiation.

Investigation of the regio- and stereo-selectivity of deoxyguanosine linkage to deuterated 2-hydroxyestradiol by using liquid chromatography/ESI-ion trap mass spectrometry

From previous studies on the reactivity of estradiol 2,3-quinone towards deoxyribonucleosides, it was demonstrated that several isomeric adducts were formed. Although adduction on steroid ring A or B has been evidenced using sequential MSn experiments, in some cases attachment positions are difficult to identify unambiguously. In this work, 2-hydroxyestradiol labeled with deuterium at various positions [6β (1); 6α–7α (2); 6α-6β-7α (3)] have been used. Isomeric adduct differentiation could be achieved using LC-ESI-MSn. The m/z shift of the quasi-molecular ions as well as the fragmentation pathways suggested that adduction could occur on both C6 and C9 sites of the steroid B ring: Nucleophilic attack of the base on the C6 position of the steroid led to major adducts and addition of the base on the activated C9 site gave minor adducts that were found to be unstable. LC-MSn experiments carried out under deuterated medium provided information about some fragmentation processes by studying the m/z shift of fragment ions: (1) the loss of deoxyribose from the quasi-molecular ions took place according to a process involving a deuterium transfer from the deoxyribose alcohol function; (2) the cleavage of the steroid-base linkage involved a deuterium transfer from the hydroxy group of the catechol and likely occurred via the formation of an ion-dipole complex. The model studies conducted in this work provide new information on the fragmentation mechanisms of covalent adducts formed from estrogen quinones and deoxyguanosine, the most reactive DNA base. Besides, the first unequivocal characterization of adducts involving the steroid C9 position is shown by using deuterium labeled estrogen quinones.

Oligonucleotide covalent modifications by estrogen quinones evidenced by use of liquid chromatography coupled to negative electrospray ionization tandem mass spectrometry.

Liquid chromatography coupled to tandem mass spectrometry has been used for the detection and the structural characterization of T-rich model oligonucleotides covalently modified by estradiol-2,3-quinone. After separation by gradient elution, adducts were analyzed by negative electrospray mass spectrometry, enabling to evidence and localize the modifications in the oligonucleotide sequence. Modifications by one molecule of estrogen were evidenced on purines (A, G) whereas no reaction was observed on pyrimidic bases (T). Isomeric adducts were differentiated using tandem mass spectrometry, and energy resolved mass spectrometry allowed to underline differences in the behavior of the adducts towards collisional excitation into an ion trap device.

Adduction of catechol estrogens to nucleosides.

We report the formation, detection, quantitation and structural characterization of products resulting from the adduction of deoxynucleosides (deoxyadenosine, deoxyguanosine, deoxycytidine and 5-methyldeoxycytidine) to the catechol estrogens (CE) of estrone, estradiol-17beta and estradiol-17 alpha. The crude products are obtained in a one-pot synthesis through oxidation of catechols to quinones and subsequent Michael-type reaction with the deoxynucleosides in acidic medium. In all experiments, adducts are detected by electrospray ionization mass spectrometry analysis after HPLC separation (LC/ESI/MS(n)). The two pyrimidines deoxycytidine and 5-methyldeoxycytidine yield only CE adducts to deoxynucleosides, which correspond to stable adducts on DNA. For purines, the results depend on the CE (2,3- or 3,4-catechols) used, the function and configuration on carbon 17 (ketone for estrone, alcohol for alpha and beta isomers of estradiol), and on the purine itself (deoxyadenosine or deoxyguanosine). Both stable adducts and deglycosylated adducts are formed, and therefore formation of stable adducts on DNA as well as the loss of purines from the DNA strands could be possible. MS(2) and MS(3) experiments prove to be relevant for further structural determinations, enabling in some cases the elucidation of the regiochemistry of adduction on the A and B rings of the steroid moiety.

High potency of bioactivation of 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) in mouse colon epithelial cells with ApcMin mutation

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a prominent heterocyclic aromatic amine (HAA) found in meat and fish cooked at moderate to high temperature. It is considered as a potent dietary factor promoting colon carcinogenesis. However, the role of intestinal cells in PhIP bioactivation has not been fully explained, particularly when cells are pre-malignant. Loss of function of the adenomatous polyposis coli (APC) gene product is an early and frequent event in human colorectal carcinogenesis. Normal (Apc(+/+)) and pre-malignant (Apc(Min/+), where Min=multiple intestinal neoplasia) colonic epithelial cells of mice can be used to study promotion of carcinogenesis, but these cells have not been characterized for bio-activation of HAA. We investigated the metabolism of (14)C-PhIP in these two murine cell lines. Cells induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) metabolized PhIP into 4-OH-PhIP as the main metabolite in PhiP detoxification. Besides, 5-OH-PhIP was identified, revealing the formation of intermediary reactive metabolites, since it results from a degradation of conjugates of N-acetoxy-PhIP. Apc(Min/+) cells produce significantly higher amounts of these metabolites. Demethylated metabolites are also observed, indicating that the colon contains a significant CYP1 family dependent metabolic activity. A minor hydroxy-glucuronide-PhIP metabolite is observed in Apc(Min/+) cells, the glucuronidation being known as an important step in the detoxification pathway. Quantitative real-time reverse transcription polymerase chain reaction experiments demonstrate that induction by TCDD has prevailing effects in gene expression of CYP1A1, CYP1A2 and CYP1B1 in Apc(Min/+) cells. In these cells, N-acetyltransferase-2 is also expressed at higher levels. So, the more important potency to metabolically bio-activate PhIP, as measured in Apc(Min/+) cells, can be linked to a higher probability to generate new in situ mutations.

Metabolic Fate of [14C] Chlorophenols in Radish (Raphanus sativus), Lettuce (Lactuca sativa), and Spinach (Spinacia oleracea)

Chlorophenols are potentially harmful pollutants that are found in numerous natural and agricultural systems. Plants are a sink for xenobiotics, which occur either intentionally or not, as they are unable to eliminate them although they generally metabolize them into less toxic compounds. The metabolic fate of [ (14)C] 4-chlorophenol (4-CP), [ (14)C] 2,4-dichlorophenol (2,4-DCP), and [ (14)C] 2,4,5-trichlorophenol (2,4,5-TCP) was investigated in lettuce, spinach, and radish to locate putative toxic metabolites that could become bioavailable to food chains. Radish plants were grown on sand for four weeks before roots were dipped in a solution of radiolabeled chlorophenol. The leaves of six-week old lettuce and spinach were treated. Three weeks after treatments, metabolites from edible plant parts were extracted and analyzed by high performance liquid chromatography (HPLC) and characterized by mass spectrometry (MS), and nuclear magnetic resonance spectroscopy (NMR). Characterization of compounds highlighted the presence of complex glycosides. Upon hydrolysis in the digestive tract of animals or humans, these conjugates could return to the toxic parent compound, and this should be kept in mind for registration studies.