Urinary excretion of herbicide co-formulants after oral exposure to roundup MON 52276 in rats.

The toxicity of surfactants, which are an integral component of glyphosate-formulated products is an underexplored and highly debated subject. Since biomonitoring human exposure to glyphosate co-formulants is considered as a public health priority, we developed and validated a high-resolution mass spectrometry method to measure the urinary excretion of surfactants present in Roundup MON 52276, the European Union (EU) representative formulation of glyphosate-based herbicides. Quantification was performed measuring the 5 most abundant compounds in the mixture. We validated the method and showed that it is highly accurate, precise and reproducible with a limit of detection of 0.0004 µg/mL. We used this method to estimate the oral absorption of MON 52276 surfactants in Sprague-Dawley rats exposed to three concentrations of MON 52276 via drinking water for 90 days. MON 52276 surfactants were readily detected in urine of rats administered with this commercial Roundup formulation starting from a low concentration corresponding to the EU glyphosate acceptable daily intake. Our results provide a first step towards the implementation of surfactant co-formulant biomonitoring in human populations.

Rapid screening of low-molecular-weight phenols from persimmon (Diospyros kaki) pulp using liquid chromatography/UV-visible/electrospray mass spectrometry analysis.

BACKGROUND: Persimmon fruits have been widely used in traditional medicine owing to their phenolic composition. This research aims to perform a rapid, detailed and affordable study of the profile of low-molecular-weight phenols from persimmon pulp. RESULTS: Two different HPLC-DAD/ESI-MS(n) analyses were performed using a routine three-dimensional ion trap mass spectrometer to analyze the ethanolic extract of persimmon pulp: (1) an untargeted data-dependent analysis to identify the majority of small phenols that included full MS and MS(2) scan events; (2) a targeted data-dependent analysis to identify polymerized phenols (dimers and formic acid adducts) through a source-induced dissociation analysis that included full MS and MS(2) scan events. Thirty-two low-molecular-weight phenols were detected, comprising gallic acid and its glycoside and acyl derivatives, glycosides of p-coumaric, vanillic and cinnamic acids and different flavone di-C-hexosides, most of them reported for the first time in persimmon. CONCLUSION: The use of a straightforward and affordable methodology of analysis led to obtain an up-to-date profiling of low-molecular-weight phenols in persimmon. The results can help future actions aimed to expand the understanding of the phenolic metabolome of persimmon cultivars.

Creation of libraries of recurring mass spectra from large data sets assisted by a dual-column workflow.

An analytical methodology has been developed for extracting recurrent unidentified spectra (RUS) from large GC/MS data sets. Spectra were first extracted from original data files by the Automated Mass Spectral Deconvolution and Identification System (AMDIS; Stein, S. E. J. Am. Soc. Mass Spectrom. 1999 , 10 , 770 - 781 ) using settings designed to minimize spurious spectra, followed by searching the NIST library with all unidentified spectra. The spectra that could not be identified were then filtered to remove poorly deconvoluted data and clustered. The results were assumed to be unidentified components. This was tested by requiring each unidentified spectrum to be found in two chromatographic columns with slightly different stationary phases. This methodology has been applied to a large set of pediatric urine samples. A library of spectra and retention indices for derivatized urine components, both identified and recurrent unidentified, has been created and is available for download.

Proteome changes in tomato lines transformed with phytoene synthase-1 in the sense and antisense orientations.

The commercial cultivation of genetically engineered (GE) crops in Europe has met with considerable consumer resistance, which has led to vigorous safety assessments including the measurement of substantial equivalence between the GE and parent lines. This necessitates the identification and quantification of significant changes to the metabolome and proteome in the GE crop. In this study, the quantitative proteomic analysis of tomato fruit from lines that have been transformed with the carotenogenic gene phytoene synthase-1 (Psy-1), in the sense and antisense orientations, in comparison with a non-transformed, parental line is described. Multidimensional protein identification technology (MudPIT), with tandem mass spectrometry, has been used to identify proteins, while quantification has been carried out with isobaric tags for relative and absolute quantification (iTRAQ). Fruit from the GE plants showed significant alterations to their proteomes compared with the parental line, especially those from the Psy-1 sense transformants. These results demonstrate that MudPIT and iTRAQ are suitable techniques for the verification of substantial equivalence of the proteome in GE crops.

The surfactant co-formulant POEA in the glyphosate-based herbicide RangerPro but not glyphosate alone causes necrosis in Caco-2 and HepG2 human cell lines and ER stress in the ToxTracker assay.

The toxicity of co-formulants present in glyphosate-based herbicides (GBHs) has been widely discussed leading to the European Union banning the polyoxyethylene tallow amine (POEA). We identified the most commonly used POEA, known as POE-15 tallow amine (POE-15), in the widely used US GBH RangerPro. Cytotoxicity assays using human intestinal epithelial Caco-2 and hepatocyte HepG2 cell lines showed that RangerPro and POE-15 are far more cytotoxic than glyphosate alone. RangerPro and POE-15 but not glyphosate caused cell necrosis in both cell lines, and that glyphosate and RangerPro but not POE-15 caused oxidative stress in HepG2 cells. We further tested these pesticide ingredients in the ToxTracker assay, a system used to evaluate a compound's carcinogenic potential, to assess their capability for inducing DNA damage, oxidative stress and an unfolded protein response (endoplasmic reticulum, ER stress). RangerPro and POE-15 but not glyphosate gave rise to ER stress. We conclude that the toxicity resulting from RangerPro exposure is thus multifactorial involving ER stress caused by POE-15 along with oxidative stress caused by glyphosate. Our observations reinforce the need to test both co-formulants and active ingredients of commercial pesticides to inform the enactment of more appropriate regulation and thus better public and environmental protection.

Use of Shotgun Metagenomics and Metabolomics to Evaluate the Impact of Glyphosate or Roundup MON 52276 on the Gut Microbiota and Serum Metabolome of Sprague-Dawley Rats.

BACKGROUND: There is intense debate on whether glyphosate can inhibit the shikimate pathway of gastrointestinal microorganisms, with potential health implications. OBJECTIVES: We tested whether glyphosate or its representative EU herbicide formulation Roundup MON 52276 affects the rat gut microbiome. METHODS: We combined cecal microbiome shotgun metagenomics with serum and cecum metabolomics to assess the effects of glyphosate [0.5, 50, 175mg/kg body weight (BW) per day] or MON 52276 at the same glyphosate-equivalent doses, in a 90-d toxicity test in rats. RESULTS: Glyphosate and MON 52276 treatment resulted in ceca accumulation of shikimic acid and 3-dehydroshikimic acid, suggesting inhibition of 5-enolpyruvylshikimate-3-phosphate synthase of the shikimate pathway in the gut microbiome. Cysteinylglycine, γ-glutamylglutamine, and valylglycine levels were elevated in the cecal microbiome following glyphosate and MON 52276 treatments. Altered cecum metabolites were not differentially expressed in serum, suggesting that the glyphosate and MON 52276 impact on gut microbial metabolism had limited consequences on physiological biochemistry. Serum metabolites differentially expressed with glyphosate treatment were associated with nicotinamide, branched-chain amino acid, methionine, cysteine, and taurine metabolism, indicative of a response to oxidative stress. MON 52276 had similar, but more pronounced, effects than glyphosate on the serum metabolome. Shotgun metagenomics of the cecum showed that treatment with glyphosate and MON 52276 resulted in higher levels of Eggerthella spp., Shinella zoogleoides, Acinetobacter johnsonii, and Akkermansia muciniphila. Shinella zoogleoides was higher only with MON 52276 exposure. In vitro culture assays with Lacticaseibacillus rhamnosus strains showed that Roundup GT plus inhibited growth at concentrations at which MON 52276 and glyphosate had no effect. DISCUSSION: Our study highlights the power of multi-omics approaches to investigate the toxic effects of pesticides. Multi-omics revealed that glyphosate and MON 52276 inhibited the shikimate pathway in the rat gut microbiome. Our findings could be used to develop biomarkers for epidemiological studies aimed at evaluating the effects of glyphosate herbicides on humans. https://doi.org/10.1289/EHP6990.

A proteomic-based approach for detection of chicken in meat mixes.

A proteomic-based method has been developed for the detection of chicken meat within mixed meat preparations. The procedure is robust and simple, comprising the extraction of myofibrillar proteins, enrichment of target proteins using OFFGEL isoelectric focusing, in-solution trypsin digestion of myosin light chain 3, and analysis of the generated peptides by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Using this approach, it was possible for example to detect 0.5% contaminating chicken in pork meat with high confidence. Quantitative detection of chicken meat was done by using AQUA stable isotope peptides made from the sequence of previously selected species-specific peptide biomarkers. Linearity was observed between the amount of the peptide biomarker and the amount of chicken present in the mixture; further independent replication is required now to validate the method. Apart from its simplicity, this approach has the advantage that it can be used effectively for the detection of both raw and cooked meat. The method is robust, reliable, and sensitive, representing a serious alternative to methods currently in use for these purposes. It is amenable to highly processed foods which can be particularly problematic, as the tertiary protein structure is often affected in processed food precluding immunoassays. In addition, this proteomic analysis will permit the determination of definitive discriminatory sequence, unlike the DNA PCR based methods used presently. The present article also demonstrates the translation of the technology to routine mass spectrometry equipment, making the methodology suitable for public analysts.

Quantitation of hepcidin in serum using ultra-high-pressure liquid chromatography and a linear ion trap mass spectrometer.

Hepcidin is a peptide hormone that functions as a key regulator of mammalian iron metabolism. Biological levels are increased in end-stage renal disease and during inflammation but suppressed in hemochromatosis. Thus hepcidin levels have diagnostic importance. This study describes the development of an analytical method for the quantitative determination of the concentration of hepcidin in clinical samples. The fragmentation of hepcidin was investigated using triple quadrupole and linear ion trap mass spectrometers. A standard quantity of a stable isotopically labelled hepcidin internal standard was added to serum samples. Extraction was performed by protein precipitation and weak cation-exchange magnetic nanoparticles. Chromatography was carried out on sub 2 microm particle stationary phase, using ultra-high-pressure liquid chromatography and a linear ion trap for quantitation. The lower limit of quantitation was 0.4 nmol/L with less than 20% accuracy and precision. The mean hepcidin concentration in sera for controls was 4.6 +/- 2.7 nmol/L, in patients with sickle cell disease, 7.0 +/- 8.9 nmol/L; in patients with end-stage renal disease, 30.5 +/- 15.7 nmol/L; and patients with penetrant hereditary hemochromatosis, 1.4 +/- 0.8 nmol/L.

Itaconate controls the severity of pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease in which airway macrophages (AMs) play a key role. Itaconate has emerged as a mediator of macrophage function, but its role during fibrosis is unknown. Here, we reveal that itaconate is an endogenous antifibrotic factor in the lung. Itaconate levels are reduced in bronchoalveolar lavage, and itaconate-synthesizing cis-aconitate decarboxylase expression (ACOD1) is reduced in AMs from patients with IPF compared with controls. In the murine bleomycin model of pulmonary fibrosis, Acod1−/− mice develop persistent fibrosis, unlike wild-type (WT) littermates. Profibrotic gene expression is increased in Acod1−/− tissue-resident AMs compared with WT, and adoptive transfer of WT monocyte-recruited AMs rescued mice from disease phenotype. Culture of lung fibroblasts with itaconate decreased proliferation and wound healing capacity, and inhaled itaconate was protective in mice in vivo. Collectively, these data identify itaconate as critical for controlling the severity of lung fibrosis, and targeting this pathway may be a viable therapeutic strategy.

Blood circulation and tissue biodistribution of lipid--quantum dot (L-QD) hybrid vesicles intravenously administered in mice.

The present work describes the pharmacokinetics of recently developed liposome-quantum dot (L-QD) hybrid vesicles in nude mice following systemic administration. Hydrophobic QD were incorporated into different bilayer compositions, and the serum stability of such hybrid vesicles was evaluated using turbidity and carboxyfluorescein release measurements. L-QD hybrid blood profile and organ biodistribution were also determined by elemental (cadmium) analysis. Following intravenous administration, different tissue biodistribution profiles and tissue affinities were observed depending on the L-QD lipid bilayer characteristics. Immediate blood clearance was observed with cationic (DOTAP/DOPE/Chol) hybrid with rapid lung accumulation, while incorporation of PEG at the surface of zwitterionic vesicles dramatically prolonged their blood circulation half-life after systemic administration. Overall, the L-QD hybrid vesicle system is considered a viable platform that allows QD delivery to different tissues through facile modulation of the hybrid vesicle characteristics. In addition, L-QD offers many opportunities for the development of combinatory therapeutic and imaging (theranostic) modalities by incorporating both drug molecules and QD within the different compartments of a single vesicle.

Use of human microsomes and deuterated substrates: an alternative approach for the identification of novel metabolites of ketamine by mass spectrometry.

In vitro biosynthesis using pooled human liver microsomes was applied to help identify in vivo metabolites of ketamine by liquid chromatography (LC)-tandem mass spectrometry. Microsomal synthesis produced dehydronorketamine, seven structural isomers of hydroxynorketamine, and at least five structural isomers of hydroxyketamine. To aid identification, stable isotopes of the metabolites were also produced from tetra-deuterated isotopes of ketamine or norketamine as substrates. Five metabolites (three hydroxynorketamine and two hydroxyketamine isomers) gave chromatographically resolved components with product ion spectra indicating the presence of a phenolic group, with phenolic metabolites being further substantiated by selective liquid-liquid extraction after adjustments to the pH. Two glucuronide conjugates of hydroxynorketamine were also identified. Analysis by LC-coupled ion cyclotron resonance mass spectrometry gave unique masses in accordance with the predicted elemental composition. The metabolites, including the phenols, were subsequently confirmed to be present in urine of subjects after oral ketamine administration, as facilitated by the addition of deuterated metabolites generated from the in vitro biosynthesis. To our knowledge, phenolic metabolites of ketamine, including an intact glucuronide conjugate, are here reported for the first time. The use of biologically synthesized deuterated material as an internal chromatographic and mass spectrometric marker is a viable approach to aid in the identification of metabolites. Metabolites that have particular diagnostic value can be selected as candidates for chemical synthesis of standards.

Quantitation of hepcidin in human urine by liquid chromatography-mass spectrometry.

Hepcidin is a peptide hormone that functions as a key regulator of mammalian iron metabolism. Serum and urine levels are increased in inflammation and suppressed in hemochromatosis, and they may have diagnostic importance. This study describes the development and validation of an analytical method for the quantitative determination of the concentration of hepcidin in clinical samples. A stable, isotopically labeled internal standard, [15N,13C2]Gly12,20-hepcidin, was synthesized and a standard quantity was added to urine samples. Extraction was performed using weak cation exchange magnetic nanoparticles. An ion trap mass spectrometer was used to quantify hepcidin in the samples. The hepcidin assay was validated, and good recovery of hepcidin was obtained. The assay is accurate and precise. Urinary hepcidin levels of 3 to 9 nmol/mmol creatinine(-1) were found in healthy controls, with reduced levels in hemochromatosis (P<0.00006) and elevated levels in inflammation (P<0.00035). In sickle cell disease, a wide range was found, with the mean value not differing significantly from controls (P<0.26). In summary, a validated method has been developed for the quantitation of hepcidin using a stable, isotopically labeled internal standard and applied to determine the concentrations of hepcidin in the low nanomolar range in urine samples from patients and controls.

Quantification of hepcidin using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Hepcidin is known to be a key systemic iron-regulatory hormone which has been demonstrated to be associated with a number of iron disorders. Hepcidin concentrations are increased in inflammation and suppressed in hemochromatosis. In view of the role of hepcidin in disease, its potential as a diagnostic tool in a clinical setting is evident. This study describes the development of a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) assay for the quantitative determination of hepcidin concentrations in clinical samples. A stable isotope labeled hepcidin was prepared as an internal standard and a standard quantity was added to urine samples. Extraction was performed with weak cation-exchange magnetic nanoparticles. The basic peptides were eluted from the magnetic nanoparticles using a matrix solution directly onto a target plate and analyzed by MALDI-TOF MS to determine the concentration of hepcidin. The assay was validated in charcoal stripped urine, and good recovery (70-80%) was obtained, as were limit of quantitation data (5 nmol/L), accuracy (RE <10%), precision (CV <21%), within -day repeatability (CV <13%) and between-day repeatability (CV <21%). Urine hepcidin levels were 10 nmol/mmol creatinine in healthy controls, with reduced levels in hereditary hemochromatosis (P < 0.000005) and elevated levels in inflammation (P < 0.0007). In summary a validated method has been developed for the determination of hepcidin concentrations in clinical samples.

Liquid chromatographic-mass spectrometric analysis of glucuronide-conjugated anabolic steroid metabolites: method validation and interlaboratory comparison.

Liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method for simultaneous and direct detection of 12 glucuronide-conjugated anabolic androgenic steroid (AAS) metabolites in human urine is described. The compounds selected were the main metabolites detected in human urine after dosing of the most widely abused AAS in sports, e.g. methandienone, methenolone, methyltestosterone, nandrolone and testosterone, and certain deuterium-labeled analogs of these metabolites. Sample preparation and the LC-ESI-MS/MS method were optimized, validated, and the overall process was implemented and the results between seven laboratories were compared. All the metabolites were extracted simultaneously by solid-phase extraction (SPE) and analyzed by LC-ESI-MS/MS with positive ionization mode and multiple reaction monitoring (MRM). Recovery of the SPE for the AAS glucuronides was 89-100% and ten out of twelve compounds had detection limits in the range of 1-10 ng/ml in urine. The results for inter/intraday repeatability were satisfactory and the interlaboratory comparison with authentic urine samples demonstrated the ease of method transfer from one instrument setup to another. When equivalent triple quadrupole analyzers were employed the overall performance was independent from instrument manufacturer, electrospray ionisation (ESI) or atmospheric pressure chemical ionization (APCI) and liquid chromatohraphic (LC) column, whereas major differences were encountered when changing from one analyzer type to another, especially in the analysis of those AAS glucuronides ionized mainly as adducts.

Detection of ketamine and its metabolites in urine by ultra high pressure liquid chromatography-tandem mass spectrometry.

Current analytical methods used for screening drugs and their metabolites in biological samples from victims of drug-facilitated sexual assault (DFSA) or other vulnerable groups can lack sufficient sensitivity. The application of liquid chromatography, employing small particle sizes, with tandem mass spectrometry (MS/MS) is likely to offer the sensitivity required for detecting candidate drugs and/or their metabolites in urine, as demonstrated here for ketamine. Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) was performed following extraction of urine (4 mL) using mixed-mode (cation and C8) solid-phase cartridges. Only 20 microL of the 250 microL extract was injected, leaving sufficient volume for other assays important in DFSA cases. Three ion transitions were chosen for confirmatory purposes. As ketamine and norketamine (including their stable isotopes) are available as reference standards, the assay was additionally validated for quantification purposes to study elimination of the drug and primary metabolite following a small oral dose of ketamine (50 mg) in 6 volunteers. Dehydronorketamine, a secondary metabolite, was also analyzed qualitatively to determine whether monitoring could improve retrospective detection of administration. The detection limit for ketamine and norketamine was 0.03 ng/mL and 0.05 ng/mL, respectively, and these compounds could be confirmed in urine for up to 5 and 6 days, respectively. Dehydronorketamine was confirmed up to 10 days, providing a very broad window of detection.

Derivatization in mass spectrometry --7. On-line derivatisation/degradation.

The review describes on-line derivatization/degradation methods employed in mass spectrometry to solve some structural and analytical problems. Advantages and applications of various positions of reaction systems connected mainly to a mass spectrometer or a gas chromatograph/mass spectrometer are considered. Among these are reaction systems connected directly to the mass spectrometer (reaction mass spectrometry, pyrolysis-mass spectrometry or direct pyrolysis-mass spectrometry); flash-heaters as reactors in gas chromatography/mass spectrometry (GC/MS); in-line chemical reactors located before the chromatographic column [pre-column derivatization/degradation with the use of catalytic reactions, pyrolysis (pyrolysis-GC/MS), degradation in elemental analyzers-isotope ratio mass pectrometry (EA-IRMS)]; on-column derivatization and deuteration; reactor located between the chromatographic column and a mass spectrometer [post-column catalytic derivatization, gas chromatograph-combustion-isotope ratio mass spectrometer (GC-c-IRMS)]. Post-column derivatization in high performance liquid chromatography/mass spectro-metry is briefly mentioned. Application of such on-line methodology to structure elucidation of low molecular mass compounds and polymers, to the determination of isotope ratios of the most common elements, to the investigation of catalytic reactions is discussed..

Derivatization in mass spectrometry--8. Soft ionization mass spectrometry of small molecules.

This is the first of two reviews devoted to derivatization approaches for "soft" ionization mass spectrometry (FAB, MALDI, ESI, APCI) and deals, in particular, with small molecules. The principles of the main "soft" ionization mass spectrometric methods as well as the reasons for derivatizing small molecules are briefly described. Derivatization methods for modification of amines, carboxylic acids, amino acids, alcohols, carbonyl compounds, monosaccharides, thiols, unsaturated and aromatic compounds etc. to improve their ionizability and to enhance structure information content are discussed. The use of "fixed"-charge bearing derivatization reagents is especially emphasized. Chemical aspects of derivatization and "soft" ionization mass spectrometric properties of derivatives are considered.

Review: derivatization in mass spectrometry--5. Specific derivatization of monofunctional compounds.

The present paper is complementary to the foregoing reviews and describes some additional methods of the derivatization of particular functional groups mainly to enhance the structural information content of electron ionization and chemical ionization mass spectra. Derivatization approaches for the modification of unsaturated compounds, alcoholic, carboxylic, carbonyl, amine and other functional groups, are discussed. Derivatization for separation and quantitative determination of chiral enantiomeric compounds is also considered. Preliminary chemical and physicalchemical degradation for structure elucidation of high molecular weight compounds (biopolymers, synthetic polymers) is mentioned. Chemical aspects of derivatizations and characteristic mass spectral features of derivatives are described briefly. Some particular applications of chemical modification, in conjunction with mass spectral measurements for the analysis of various important bioorganic compounds and compounds in biological fluids, air, environmental etc., are considered.

Review: derivatization in mass spectrometry-6. Formation of mixed derivatives of polyfunctional compounds.

The review describes chemical transformations of multifunctional compounds (amino acids and peptides, amino alcohols, amino thiols, hydroxy acids, oxo acids, oxo alcohols, compounds containing simultaneously three or more different groups etc.) by using step-wise or one-step modification or protection of functional groups. Some chemical aspects of mixed derivatization performed for improving the physical-chemical properties and mass spectral characteristics are discussed. Application of mixed derivatization to qualitative and quantitative analysis of various multifunctional compounds mainly in biological fluids and other matrices by gas chromatography/mass spectrometry in electron ionization, chemical ionization, negative-ion chemical ionization and selected ion monitoring modes is considered.

NADPH oxidase 4 regulates homocysteine metabolism and protects against acetaminophen-induced liver damage in mice.

Glutathione is the major intracellular redox buffer in the liver and is critical for hepatic detoxification of xenobiotics and other environmental toxins. Hepatic glutathione is also a major systemic store for other organs and thus impacts on pathologies such as Alzheimer's disease, Sickle Cell Anaemia and chronic diseases associated with aging. Glutathione levels are determined in part by the availability of cysteine, generated from homocysteine through the transsulfuration pathway. The partitioning of homocysteine between remethylation and transsulfuration pathways is known to be subject to redox-dependent regulation, but the underlying mechanisms are not known. An association between plasma Hcy and a single nucleotide polymorphism within the NADPH oxidase 4 locus led us to investigate the involvement of this reactive oxygen species- generating enzyme in homocysteine metabolism. Here we demonstrate that NADPH oxidase 4 ablation in mice results in increased flux of homocysteine through the betaine-dependent remethylation pathway to methionine, catalysed by betaine-homocysteine-methyltransferase within the liver. As a consequence NADPH oxidase 4-null mice display significantly lowered plasma homocysteine and the flux of homocysteine through the transsulfuration pathway is reduced, resulting in lower hepatic cysteine and glutathione levels. Mice deficient in NADPH oxidase 4 had markedly increased susceptibility to acetaminophen-induced hepatic injury which could be corrected by administration of N-acetyl cysteine. We thus conclude that under physiological conditions, NADPH oxidase 4-derived reactive oxygen species is a regulator of the partitioning of the metabolic flux of homocysteine, which impacts upon hepatic cysteine and glutathione levels and thereby upon defence against environmental toxins.