Dinesh Rao

Characterization of biliary metabolites of 4-n-nonylphenol in rainbow trout (Oncorhynchus mykiss)

1. [R-2,6-3H]-4-n-nonylphenol was synthesized and a single dose (5 mg, 1850 KBq) orally administered to rainbow trout. After 48 h, the radioactivity present in the bile amounted 5.5%. More than ten biliary metabolites were separated by hplc and collected for subsequent mass spectrometry analysis. The metabolic profile was totally modified by beta-glucuronidase hydrolysis, showing that most of the metabolites were glucuronic acid conjugates. 2. Conjugated metabolites were identified by lc-ms analysis and their aglycones were analysed by gc-ms analysis as TMS and acetyl derivatives. 3. The major metabolite accounted for 52+/-11% of the biliary radioactivity and was identified as nonylphenol-glucuronide. 4. Nonylphenol was hydroxylated at both omega and omega-1 positions of the alkyl chain, giving 9-hydroxynonylphenol and 8-hydroxynonylphenol. 5. 9-Hydroxynonylphenol was oxidized to the corresponding acid, and subsequently beta-oxidized, yielding 7-(4-hydroxyphenyl)heptanoic acid, 5-(4-hydroxyphenyl)pentanoic acid, 3-(4-hydroxyphenyl)propionic acid and 3-(4-hydroxyphenyl)-2-propenoic acid.

Urinary metabolites of 4-n-nonylphenol in rainbow trout (Oncorhynchus mykiss)

Nonylphenol is present in surface water and aquatic sediments and because of its lipophilic characteristics shows a considerable potential to bioaccumulate in aquatic organisms. Nonylphenol inhibits testicular growth and induces vitellogenin synthesis in male rainbow trout. In order to better understand the effects of nonylphenol on fish and its impact in the aquatic environment, it is essential to elucidate the metabolic fate of this compound. A single oral dose (5 mg, 1850 KBq) of [3H]4-n-nonylphenol resulted in 1.1% and 3.0% of the ingested radioactivity eliminated in urine after 24 and 48 h, respectively. Four metabolites were separated by radio-HPLC and tentatively identified by mass spectrometry. Urinary metabolites likely resulted from the initial omega-oxidation of 4-n-nonylphenol to the putative 9-(4-hydroxyphenyl)-nonanoic acid which subsequent beta-oxidation led to 4-hydroxybenzoic acid as major metabolite. Intermediary metabolites, namely 3-(4-hydroxyphenyl) propionic acid and 3-(4-hydroxyphenyl)-2-propenoic acid confirmed the occurrence of this beta-oxidative pathway. Urinary metabolites identified in this study were quite different from biotransformation products previously described in bile of trout treated with 4-n-nonylphenol.

Metabolic fate of 2,4-dichloroaniline, prochloraz and nonylphenol diethoxylate in rainbow trout: a comparative in vivo/in vitro approach.

The metabolism and distribution of 2,4-dichloroaniline (2,4-DCA), prochloraz and 4-n-nonylphenol diethoxylate (NP2EO) were investigated in vivo and in vitro in rainbow trout (Oncorhynchus mykiss). Each compound was administered p.o. (10 mg/kg wet weight) and urine was collected during 48 h (2,4-DCA, prochloraz) or 72 h (NP2EO). Fish were sacrificed, the gall bladder was excised and radioactivity was measured in tissues, viscera and carcasses. Metabolic profiles were performed by radio-HPLC and when possible metabolites were identified by LC/MS. For comparison, the biotransformation of these xenobiotics was also investigated in freshly isolated hepatocytes. The metabolic pathways of 2,4-DCA have been identified leading to the glucuronide conjugate (in vivo) and to the glucuronide conjugate and the hydroxylamine metabolite (in vitro). This difference highlights the usefulness of the hepatocyte system in metabolic studies, since the formation of the hydroxylamine reactive metabolite cannot be demonstrated in vivo. For prochloraz, we observed that residue levels are significantly higher in males than in females for gill, fat, brain and carcasses, however, the reasons for this difference remain unclear. Although, the presence of glucuronide conjugates was detected in vivo and in vitro, the chemical structure of isolated metabolites has to be determined. However, the comparison of the in vivo versus in vitro metabolic profiles indicates that several peaks, probably corresponding to intermediate metabolites, were present only in hepatocyte incubations. Biotransformation of NP2EO occurred in vivo and in vitro in rainbow trout, but did not result in the formation of 4-n-NP. The major metabolite present in bile corresponded to the NP2EO-glucuronide but this metabolite was not found in vitro. It is concluded that hepatocytes may produce a different metabolic pattern than in the whole fish, but may also give evidence of a metabolic pathway difficult to apprehend in vivo.

Disposition and metabolism of [3H]-4-n-nonylphenol in rainbow trout

Abstract In order to better understand the fate of nonylphenols consumed by aquatic vertebrates, the fate of low (6 μg kg −1 ) and high (25 mg kg −1 ) doses of [ 3 H]4-n-nonylphenol was followed 48 h after a single oral dose. In the low dose experiments 7.6 and 7.2% of the dose was excreted into urine or bile whereas 46.5% was still present in other tissues of the fish. In the high dose experiment these compartments comprised 3.0, 5.5 and 11.0% of the dose respectively. Radioactivity was widely distributed in trout tissues and organs, the highest concentration being in the digestive tract, liver and kidney. No identification of the nature of residues was performed on these tissues. A radio-HPLC system was developed for the analysis of biliary metabolites. Based on this method, more than 10 metabolites were separated and tentatively identified by mass spectrometry. A major part of metabolites were glucuronic acid conjugates of nonylphenol and related hydroxylated compounds. Hydroxylation and oxidation occur on the alkyl chain whereas indirect evidence exists of an hydroxylation on the phenol ring of the molecule. No trace of unchanged nonylphenol was found in bile and urine.

Simple gas chromatographic method for the determination of medicagenic acid in alfalfa (medicago sativa)

A gas chromatographic method for the determination of medicagenic acid in alfalfa leaves, stems, entire plant (tops) and roots and also in leaf protein concentrates is described. The method is based on extraction of lucerne saponins followed by hydrolysis of the triterpene glycosides. After derivatization of the liberated sapogenins to silylated products, the trimethylsilylated medicagenic acid was determined by gas chromatography. The sensitivity of the method permits the detection of 50 ng of medicagenic acid.

Tandem mass spectrometric investigation on 17β-estradiol palmitate in negative ion chemical ionization

Abstract In negative ion mass spectrometry (NI-MS) of bifunctional compounds (e.g. 17β-estradiol palmitate, containing two acidic sites), the orientation of the deprotonation process is greatly influenced by the (MH) − preparation mode [i.e. in solution for fast atom bombardment (FAB) and in the gas phase for chemical ionization (CI)]. Under negative ion FAB conditions, deprotonation is regioselective and takes place at position C 3 of a phenolic A ring whereas, in negative ion CI, competitive deprotonation occurs from both acidic sites of the molecule. Indeed, collisional activated dissociation (CAD) spectra of (MH) − present different fingerprints according to the preparation mode used. Using deuterium labelling in NI-FAB, no deuterium incorporation is observed on the deprotonated molecule [i.e. exclusive (M d D) − formation]. The situation is not the same as in NICI-ND 3 experiments since both (M d D) − and (M d H) − are produced in similar abundance. The observed differences in behavior of these (M d D) − and (M d H) − species towards high-energy collision (eV range, in triple quadrupole instrument) demonstrate that both ions were deprotonated from the C 3 A ring and from the enolizable position of the C 17 side chain, respectively. These CAD spectra provide evidence that these deprotonated forms are not convertible. Furthermore, the diagnostic daughter ions produced under CAD conditions indicate that, prior to collision, two different ion-dipole complexes are formed from the precursor (M d D) − and (M d H) − ions.

Liquid chromatographic separation and gas chromatographic—mass spectrometric determination of 17α-methyltestosterone residues extracted from rainbow trout tissues

Abstract The residues of [3 H]-17-methyltestosterone (17MT) in rainbow trout after a single intragastric dose (200 μg, 0.37 MBq) were investigated. The metabolites were extracted from liver and carcass with chloroform—methanol (2 + 1, v/v). After enzymatic hydrolysis of the glucuronides, unconjugated steroids and aglycones were chromatographed by reversed-phase liquid chromatography. Each labelled fraction was collected and analysed by gas chromatography—mass spectrometry. After 24 h, the liver contained 4% of the administered dose. In the hepatic unconjugated and aglycone fractions the major metabolite was an isomer of 17α-methylandrostane-3ξ,16ξ,17β-triol. 7α-Hydroxy-17α-methylandrostane-3α,17β-diol was also observed in the unconjugated fraction. In addition to these metabolites, unchanged 17MT, 17α-methyl-5β-androstane-3α,17β-diol and 17β-hydroxyandrosta4,6-dien-3-one were identified in the unconjugated fraction extracted from carcass samples. After 72 h, 7α-hydroxy-17α-methylandrostane-3α,17β-diol persisted to a significant extent in carcass samples whereas no trace of 17MT was detected. This metabolite could therefore be used as an indicator of the administration of 17MT in fish.