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Whether or not an individual's drug metabolising capacity declines with advancing age is a vexing question. There is no clear evidence that drug metabolism itself ('the biologically-assisted chemical alteration of the administered parent molecule') is less efficient in healthy old age than at younger ages, whereas a decreased capacity may be associated with ill-health and frailty. However, elderly individuals do show a reduced enzyme induction capability and are less able to tolerate overdoses. It appears that the majority of deleterious clinical outcomes related to drug therapy in an elderly (usually ill or frail) population may be ascribed to various anatomical and physiological age-related changes. These may affect both pharmacodynamics and pharmacokinetics, but not necessarily drug metabolism. Information gleaned from animal studies undertaken mainly in rodents does not seem to be of relevance to humans and studies in healthy aged human populations may not highlight possible problems. However, certain circumstances may influence metabolic competence, and phenotyping rather than genotyping is of more value in identifying those susceptible to adverse drug reactions. This short review discusses the potential contributions of four factors (inflammation, circadian rhythm, gut microbes, epigenetic aspects) which may lead to alterations in drug metabolism with increasing age.

The cytochromes P450 comprise a family of enzymes that are responsible for around three-quarters of all drug metabolism reactions that occur in human populations. Many isoforms of cytochrome P450 exist but most reactions are undertaken by CYP2C9, CYP2C19, CYP2D6 and CYP3A4. This brief review focusses on the first three isozymes which exhibit polymorphism of phenotype.If there is a wide variation in drug metabolising capacity within the population, this may precipitate clinical consequences and influence the drug treatment of patients. Such problems range from a lack of efficacy to unanticipated toxicity. In order to minimise untoward events and "personalise" a patient's treatment, efforts have been made to discover an individual's drug metabolism status. This requires knowledge of the subject's phenotype at the time of clinical treatment. Since such testing is difficult, time-consuming and costly, the simpler approach of genotyping has been advocated.However, the correlation between genotype and phenotype is not good, with values of up to 50% misprediction being reported. Genotype-assisted forecasts cannot therefore be used with confidence to replace actual phenotype measurements. Obfuscating factors discussed include gene splicing, single nucleotide polymorphisms, epigenetics and microRNA, transcription regulation and multiple gene copies.

Single-nucleotide polymorphisms (SNPs) in and around the nicotinamide N-methyltransferase (NNMT) gene are associated with a range of cancers and other diseases and conditions. The data on these associations have been assembled, and their strength discussed. There is no evidence that the presence of either the major or minor base in any SNP affects the expression of nicotinamide N-methyltransferase. Nevertheless, suggestions have been put forward that some of these SNPs do affect NNMT expression and thus homocysteine metabolism. An alternative idea involving non-coding messenger RNAs (mRNAs) is suggested as a possible mechanism whereby health is influenced. It is postulated that these long, non-coding NNMT mRNAs may exert deleterious effects by interfering with the expression of other genes. Neither hypothesis, however, has experimental proof, and further work is necessary to elucidate NNMT genetic interactions.

peer-reviewed Faecal VOCs (volatile organic compounds) were compared in horses and ponies before and after 6 weeks on Spring pasture and with and without a dietary supplement (ERME). The supplement was derived from malted barley and contained enzymes which degrade complex carbohydrates, including amylase, maltase, glucanases and fructanases. VOCs of faecal samples were analysed by selected ion flow tube mass spectrometry (SIFT-MS) and the results showed that the faecal microbiome was altered in different ways both by feeding on Spring pasture and by the dietary supplement. The composition of the equine gut microbiome (EGM) is therefore potentially a reflection of dietary constituents, both pasture and supplements; any metabolomic approach should take this into account since the EGM may have modulating effects on physiological parameters such as diabetes and obesity.

Beetroot juice (BJ), a source of nitrate, was added to the diet of thoroughbred racehorses for a four-week period. Analysis of nitrate/nitrite in plasma showed no significant differences between control and dosed groups. SIFT/MS (selected ion-flow tube/mass spectrometry) was used to analyse the faecal metabolome. The levels and types of volatile organic compounds (VOCs) were compared using headspace sampling of faeces from control and beetroot-supplemented equines, However, there were no significant differences apart from raised levels of acetonitrile in samples from the animals dosed with BJ. The conclusion was that racehorses appear not to have nitrate transporters and that addition of BJ to equine diets would not improve performance

Macro-plastic pollution is found in terrestrial and marine environments and is degraded to micro-particles (MP) and nano-particles (NP) of plastic. These can enter the human food chain either by inhalation or by ingestion, particularly of shellfish and crustaceans. Absorption across the gastrointestinal tract is relatively low, especially for MPs, which appear to have little toxicity. However, NPs are more readily absorbed and may accumulate in the brain, liver and other tissues in aquatic species and other animals. Studies using nanoparticles of other materials suggest that toxicity could potentially affect the central nervous system and the reproductive system, although this would be unlikely unless exposure levels were very high and absorption was increased by physiological factors.

Although mutations in the MECP2 gene are associated with many cases of Rett syndrome (RS), the phenotype remains unexplained. At least in the early stages, RS and autism have common features; urinary analysis of autistic children has shown an abnormal profile of excretion of sulphur-containing anions, with high levels of sulphite, sulphate and thiosulphate and low values for thiocyanate ions. These parameters were therefore studied in an RS population. Levels of urinary peptides, protein, cysteine, free sulphate, conjugated sulphate, sulphite, thiosulphate and thiocyanate were determined in female RS patients ( n = 21) and in controls ( n = 21), using standard methods. The urinary volumes were adjusted to standard creatinine levels (200 nmol/mL). Children with RS had higher urinary peptides and protein excretion. Free sulphate values and total sulphate excretion were higher in RS ( P P P

1. Sulphonation is unusual amongst the common Phase II (condensation; synthetic) reactions experienced by xenobiotics, in that the availability of the conjugating agent, sulphate, may become a rate-limiting factor. This sulphate is derived within the body via the oxygenation of sulphur moieties liberated from numerous ingested compounds including the sulphur-containing amino acids. Preformed inorganic sulphate also makes a considerable contribution to this pool. 2. There has been a divergence of opinion as to whether or not inorganic sulphate may be readily absorbed from the gastrointestinal tract and this controversy still continues in some quarters. Even more so, is the vexing question of potential absorption of inorganic sulphate via the lungs and through the skin. 3. This review examines the relevant diverse literature and concludes that sulphate ions may move across biological membranes by means of specific transporters and, although the gastrointestinal tract is by far the major portal of entry, some absorption across the lungs and the skin may take place under appropriate circumstances.

The developed world has seen an alarming increase in the incidence of gastrointestinal diseases, among the most common of which is Crohn’s disease (CD) in the young. The current “gold standard” techniques for diagnosis are often costly, time consuming, inefficient, invasive, and offer poor sensitivities and specificities. This paper compares the performances of three hyphenated instrumental techniques that have been suggested as rapid methods for the non‐invasive diagnosis of CD from urine. These techniques are gas chromatography‐mass spectrometry (GC‐MS), high performance liquid chromatography‐mass spectrometry (HPLC‐MS) and selected ion flow tube mass spectrometry (SIFT‐MS). Each of these techniques is followed by multivariate classification to provide a diagnosis based on the acquired data. The most promising results for potentially diagnosing CD was via HPLC‐MS. An overall classification accuracy of 73% (74% specificity; 73% sensitivity) was achieved for differentiating CD from healthy controls, statistically significant at 95% confidence.

Single-nucleotide polymorphisms (SNPs) in and around the nicotinamide N-methyltransferase (NNMT) gene are associated with a range of cancers and other diseases and conditions. The data on these associations have been assembled, and their strength discussed. There is no evidence that the presence of either the major or minor base in any SNP affects the expression of nicotinamide N-methyltransferase. Nevertheless, suggestions have been put forward that some of these SNPs do affect NNMT expression and thus homocysteine metabolism. An alternative idea involving non-coding messenger RNAs (mRNAs) is suggested as a possible mechanism whereby health is influenced. It is postulated that these long, non-coding NNMT mRNAs may exert deleterious effects by interfering with the expression of other genes. Neither hypothesis, however, has experimental proof, and further work is necessary to elucidate NNMT genetic interactions.

Gastrointestinal diseases such as irritable bowel syndrome, Crohn’s disease (CD) and ulcerative colitis are a growing concern in the developed world. Current techniques for diagnosis are often costly, time consuming, inefficient, of great discomfort to the patient, and offer poor sensitivities and specificities. This paper describes the development and evaluation of a new methodology for the non-invasive diagnosis of such diseases using a combination of gas chromatography mass spectrometry (GC–MS) and chemometrics. Several potential sample matrices were tested: blood, breath, faeces and urine. Faecal samples provided the only statistically significant results, providing discrimination between CD and healthy controls with an overall classification accuracy of 85 % (78 % specificity; 93 % sensitivity). Differentiating CD from other diseases proved more challenging, with overall classification accuracy dropping to 79 % (83 % specificity; 68 % sensitivity). This diagnostic performance compares well with the gold standard technique of colonoscopy, suggesting that GC–MS may have potential as a non-invasive screening tool.

Background: The aim of this study was to determine whether volatile organic compounds (VOCs) present in the headspace of feces could be used to diagnose or distinguish between chronic diseases of the gastrointestinal tract and apparently healthy volunteers. Methods: A total of 87 people were recruited, divided between 4 categories: healthy volunteers (n = 19), Crohn's disease (n = 22), ulcerative colitis (n = 20), and irritable bowel syndrome (n = 26). They each supplied fecal samples before, and except for the healthy volunteers, after treatment. Fecal samples were incubated in a sample bag with added purified air at 40[degrees]C and headspace samples were taken and concentrated on thermal sorption tubes. Gas chromatography-mass spectrometry then desorbed and analyzed these. The concentrations of a selection of high-abundance compounds were determined and assessed for differences in concentration between the groups. Results: Crohn's disease samples showed significant elevations in the concentrations of ester and alcohol derivates of short-chain fatty acids and indole compared with the other groups; indole and phenol were elevated in ulcerative colitis and irritable bowel syndrome but not at a statistically significant level. After treatment, the levels of many of the VOCs were significantly reduced and were more similar to those concentrations in healthy controls. Conclusions: The abundance of a number of VOCs in feces differs markedly between Crohn's disease and other gastrointestinal conditions. Following treatment, the VOC profile is altered to more closely resemble that of healthy volunteers.

1. Once in a while, during drug metabolism studies, an unusual or unexpected pathway is unearthed.2. Such quirky finds open a refreshing hiatus, providing a departure from the, perhaps now mundane, textbook routes.3. This brief missive draws attention to an interesting anecdote that may be unknown to some and concerns a substituted thioxanthenone drug.

The interactions between disease processes and the metabolism of therapeutic drugs have not been systematically investigated. Inflammation, with the presence of pro-inflammatory cytokines, affects Phase 1 metabolism, particularly the activity of the CYP isoforms. Inflammatory factors also alter the activity of some Phase 2 enzymes, particularly the sulphotransferases (SULT isoforms) responsible for drug sulphonation and the enzyme pathway involved in the supply of sulphate for this reaction. Being ill may, therefore, in itself make drug metabolism unpredictable.

Over the past decade, the roles of nicotinamide

1. Thianthrene is a sulfur-containing tricyclic molecule distributed widely within the macrostructure of hydrocarbon fossil fuels. Identified nearly 150 years ago, its chemistry has been widely explored leading to insights into reaction mechanisms and radical ion formation. 2. It has been claimed to have therapeutic application in the treatment of dermal infections and to interfere with enzyme and nucleic acid function, but appears to have little toxicity. 3. Following its oral administration to the rat, the majority remained within the gastrointestinal tract. After three days, about 88% was detected in the combined excreta with the remainder still within the animal. It is readily taken up into fish from the surrounding aqueous environment and has been placed within the "bioaccumulative category" to be regarded with concern. 4. Mammalian metabolism appeared to be restricted to ring carbon oxidation and subsequent glucuronic acid conjugation. Small amounts of sulfoxide and disulfoxide were also formed. No ring degradation was evident. Microorganisms similarly undertook aromatic ring hydroxylation but were able also to rupture the ring system by attacking the carbon-sulfur linkages and thereby degrading the molecule.

Summary Endocrine disruptors frequently bear little structural relationship to the hormone whose actions they disrupt. Consequently, the threat of an uninvestigated chemical cannot easily be assessed. Here three different approaches to assessment are discussed. The first presumes an endocrine-disrupting property, following which a cell model capable of responding to such a hormone is used. Although simple and cheap, it provides limited data. A second approach involves multiple assays to detect multiple hormones. Increasing the amount of data increased the difficulty in assessing the significance of results. To meet this problem, cluster analysis based on a simple mathematical matrix was adopted. The matrix was used to determine (i) a limited number of assays to identify a maximum number of endocrine disruptors and (ii) the chemicals with the most wide-ranging effects. A third approach was a whole genome expression analysis based on expression of mRNAs in human TE671 medulloblastoma cells. Expression of individual mRNAs was assessed using the Affymetrix GeneChip® Human Genome U133 Plus 2.0 chip. The significance of differential expressed genes was assessed based on gene ontology and pathways analyses using DAVID and GenMaPP programs. The results illustrated the very wide-ranging effects of these chemicals across the genome.

Summary Although some endocrine disruptors (EDs) act at steroid receptors, it is now apparent that compounds may have ED potential if they alter steroid synthesis or metabolism, particularly if they affect Phase 1 or Phase 2 pathways. In the ENDOMET project (EU-funded 5th Framework programme), 23 different assays were used on a wide range of EDs. Cluster analysis of the matrix results enabled identification of four integrated test systems that can be used to pinpoint compounds that are able to alter steroid metabolism or function. Critical pathways were shown to include oestrogen synthesis and sulphonation, synthesis of sulphate/PAPS and thyroid hormone regulation so that the activity profiles of some Phase 1 and Phase 2 reactions can be used as biomarkers for detection of compounds with ED potential.

Reports that bacteria within the Firmicutes phylum, especially the species Faecalibacterium prausnitzii, are less abundant in Crohn's disease (CD) patients and supernatants from cultures of this bacterium are anti-inflammatory prompted the investigation of the possible correlations between the abundance of F. prausnitzii and the response to treatment in patients with gut diseases and healthy controls. In a randomized, double-blind trial, faeces were collected from healthy volunteers, and from patients with active CD, ulcerative colitis (UC) and irritable bowel syndrome before and after treatment. The levels of F. prausnitzii DNA in faecal suspensions were determined by PCR. Treatment by an elemental diet was effective, resulting in decreases in both the Harvey and Bradshaw index (P

Within the field of drug metabolism, when addressing quantitative aspects, an average value is traditionally quoted, commonly the arithmetic mean with perhaps an indication of spread. Better still a range of values may be given, thereby acknowledging that various factors may precipitate differences between individuals. A single subject, however, usually only merits a single value. Nevertheless, events such as an acute illness or concurrent drug therapy serve to alert that this value may change substantially over a relatively short time-period, although any potential effects of naturally occurring phenomena, such as the female menstrual cycle, are often overlooked or disregarded. Are the biochemical and physiological changes that occur during the menstrual cycle able to influence xenobiotic metabolism? Is the idea of a stable and unwavering baseline within a single healthy individual flawed? Is it time to reassess our thinking with regards to such aspects? This brief review explores these issues and examines information available within the literature for evidence of potential influences of menstrual cycle events upon drug metabolism, defined as the actual chemical alteration of the parent molecule into another chemical species.

Alkylphenols such as nonylphenol are pollutants that are widely dispersed within our environment. They bio-accumulate within man, with levels in the μM concentration range reported in human tissues. These chemicals act as endocrine disruptors, having xenoestrogenic activity. More recently alkylphenols have also been shown to affect Ca2+ signalling pathways. Here we show that alkylphenols are potent inhibitors of sarcoplasmic–endoplasmic reticulum Ca2+-ATPase (SERCA) activity. For linear chain alkylphenols the potency of inhibition is related to chain length, with the IC50 values for inhibition ranging from 8 μM for 4-n-nonylphenol (C9) to 1.3 mM for 4-n-propylphenol (C3). Branched chain alkylphenols generally had lower potencies than their linear chain counterparts, however, good correlations for all alkylphenols were observed between their Ca2+ pump inhibition and hydrophobicity, molecular volume and flexibility, indicating that these parameters are all important factors. Alkylphenols cause abnormal elevations of intracellular [Ca2+] within TM4 Sertoli cells (cells involved in sperm maturation) depolarise their mitochondria and induce cell death in these cells, in an alkyl chain size-dependent manner.

The effects of four compounds, bis(2-ethylhexyl)phthalate (BEHP); diisodecylphthalate (DIP); 4-n-octylphenol (OP); 4- chloro-3-methylphenol (CMP), on gene expression (steady-state mRNA levels) across the whole human genome were studied in human TE671 cells. Effects were studied using the Affymetrics GeneChip® Human Genome U133 Plus 2.0, HG-U133 Plus 2.0 arrays, The array analyses the expression of 47,000 transcripts and variants, including ∼38,500 well characterised. All four compounds exerted statistically significant actions, affecting between 4 and 6.5% of all genes. Each compound had its own expression signature. In most instances where there was an effect steady-state mRNA levels were decreased, although not always. CMP reatment caused most increases in mRNA levels. .A mixture of DIP and CMP caused fewer changes in mRNA levels than either of the individual compounds. Conclusions: These plasticisers affected the steady-state mRNA levels of many human genes. Exposure to these compounds over many years has the potential to influence human health.

Some endocrine disrupting compounds such as phthalates and phenols act non-genomically by inhibiting the sulfotransferase (SULT 1E1 and SULT 1A1) isoforms which inactivate estrogens by sulfonation. A range of environmental phenolic contaminants and dietary flavonoids was tested for inhibition of the human SULT 1A1, 1E1 and 2A1 isoforms. In particular, the plasticisers 4-n-octyl- and 4-n-nonyl-phenol inhibit SULT 1E1 with IC(50) values of 0.16 microM vs. 10nM estradiol while the 2-substituted chlorophenols show similar values. Flavonoids are also SULT inhibitors; tricin is a competitive inhibitor of SULT 1E1 with a K(i) of 1.5+/-0.8 nM. In a small pilot study to determine whether ingestion of soy flavonoids would affect SULT1A1 activity in vivo as well as in vitro, sulfonation of daidzein was reduced in a group of women 'at risk' of breast cancer, as compared with controls, although the SULT 1A1*1/SULT 1A1*2 allele ratio was not different. Endocrine disrupting effects in man may be multifactorial when components from both the diet and the environment act at the same point in steroid metabolism.

The uterine environment is often viewed as a relatively safe haven, being guarded by the placenta which acts as a filter, permitting required materials to enter and unwanted products to be removed. However, this defensive barrier is sometimes breached by potential chemical hazards to which the mother may be subjected. Many of these toxins have immediate and recognisable deleterious effects on the embryo, foetus or neonate, but a few are insidious and leave a legacy of health issues that may emerge in later life. Several substances, falling into the categories of metals and metalloids, endocrine disruptors, solvents and other industrial chemicals, have been implicated in the development of long-term health problems in the offspring following maternal and subsequent in utero exposure. The mechanisms involved are complex but often involve epigenetic changes which disrupt normal cell processes leading to the development of cancers and also dysregulation of biochemical pathways.

Endocrine disruptors (EDs) have been studied quite extensively in environmental biology (Toppari et al. 1996); however, their exact effects on humans are uncertain. Rising rates of hormone-dependent cancers, such as breast, ovary, and testicular cancer, appear to be linked to rising levels of EDs contaminating the environment (Weir et al. 2000), although a causal link between observed abnormalities and chemical exposure has not been established (Baker 2001). These compounds may modulate both the endocrine, neuronal, and immune systems resulting in alteration of homeostasis, reproduction, development, and behavior (Amaral Mendes 2002). EDs are not classical poisons or carcinogens (Colborn et al. 1996); the detailed mechanisms by which EDs exert their effects are gradually being elucidated (Tapiero et al. 2002). They can directly initiate or inhibit actions mediated by members of the superfamily of zinc finger nuclear receptors, such as estrogen, androgen, and triiodothyronine receptors, although their chemical structures may not resemble those of steroids or related hormones. Alternatively, they may exert indirect genomic effects by modulating the metabolism of hormones. In this context, sulfation plays a key role because the cellular availability of steroid and thyroid hormones is modulated by their sulfation and desulfation by sulfotransferases and sulfatases, respectively. Sulfation of hydroxyl residues in these hormones renders them inactive and speeds exit from the cell, whereas desulfation regenerates their endocrine potential. A decrease in the sulfation/desulfation ratio could therefore lead to inappropriately high levels of hormones within cells (Falany et al. 2002; Kirk et al. 2001; Qian et al. 1998). Dehydroepiandrosterone (DHEA) and its sulfate-ester (DHEAS) are neurosteroids secreted mainly by the adrenal cortex and gonads, although synthesis of DHEAS can also take place in the central nervous system (CNS). In humans, plasma concentrations of DHEAS peak during the teenage years and thereafter decline by about 10% per decade (Ravaglia et al. 1996). It has been shown that any disruption of sulfation of DHEA or pregnenolone can block memory processes in rodents (Vallee et al. 2001), probably because these neurosteroids modulate acetylcholine release and γ-aminobutyric acid (GABA) and N-methyl-D-aspartate (NMDA) receptor action in the cortex and hippocampus (Dubrovsky 2005; Mayo et al. 2003). Results from studies in humans have been rather less conclusive because in vivo experiments are not possible. The situation is confounded because the brain can synthesize DHEA independently of the adrenal gland; therefore, measurement of DHEA and DHEAS in the plasma may not give a true indication of levels in the CNS (Racchi et al. 2003). Reduced levels of both plasma DHEAS and pregnenolone sulfate have been linked with decreased cognitive function (Armanini et al. 2003; Mayo et al. 2003). However, there is evidence that DHEA levels may exert subtle effects, particularly in Alzheimer’s disease (Vallee et al. 2001). DHEA sulfation is catalyzed by SULT 2A1, a member of the SULT 2 subfamily of the sulfotransferases, which mainly acts on endogenous hydroxysteroids, including pregnenolone, but can also sulfate various xenoestrogens and drugs. SULT 2A1 is unusual in that, although it has a Km for DHEA of about 2 μM (Chang et al. 2001), the physiologic concentration of DHEA ranges from 1–20 nM (Heuser et al. 1998). The essential co-factor in sulfation reactions is PAPS, which is synthesized in vivo by oxidation of sulfur-containing amino acids (Do and Tappaz 1996; Griffith 1987). Four enzymes play a key role in the synthesis of PAPS from cysteine. These are cysteine dioxygenase type I (CDO1), sulfite oxidase (SUOX), and the bifunctional enzymes 3′-phosphoadenosine 5′-phosphosulfate synthase 1 and 2 (PAPSS1 and PAPSS2). In humans, the supply of PAPS appears to be rate limiting for sulfation of both endogenous and exogenous compounds. Chemicals produced from the plastics and detergent industries, such as alkylphenols and bisphenol A, have been discovered to be estrogenic (Jobling et al. 1995). Plasticizers are ubiquitous in the environment at low levels and may be present in mixtures that result in additive, antagonistic, and/or synergistic effects (Guenther et al. 2002). In this article we show that a range of plasticizers commonly encountered in the environment act as competitive inhibitors of SULT 2A1 when sulfating physiologic concentrations of DHEA. In addition, they reduce steady-state mRNA levels of enzymes involved in PAPS synthesis.

Qusti, S.Y., Parsons, R.B., Waring, R.H. anRamsden, D.B. 2007. Cysteine-mediated excitotoxic neuronal death is an apoptosis—necrosis continuum. J. Appl. Anim. Res., 32: 7–12. To explore the mechanism whereby cysteine induces cell death this study was undertaken using a human medulloblastoma cell-line as the model system. Low levels of cysteine induced several well described phenomena associated with apoptosis, phospatidyl serine exposure, mitochondrial membrane disruption, cleavage of DNA into 50 Kb fragments and induction of caspase 3, whereas higher concentrations tended to cause necrosis. We have speculated on the role of cysteine in excitotoxicity, which occurs clinically following cerebral ischemia.

Purpose: There can be wide variation in individual responses to dietary components and also in detoxification of drugs and environmental compounds. This article seeks to explore some of the factors underlying these findings.Design: Glutathione‐S‐transferases (GST isoforms) link reactive chemical species with glutathione; they are polymorphic in human populations and are cytosolic and found in most tissues of the body. Their activity is modulated by dietary components, as is the activity of the cytosolic sulfotransferase (SULT) isoforms which catalyse sulfation of drugs and endogenous compounds such as steroids. Fruit and vegetable cytosols were incubated with cytosols from rat and human tissues and the activities of GST and SULT enzymes were measured.Materials and methods: Fresh fruits and vegetables were homogenised in distilled water to give 10% solutions. These were then added to supernatants from human and rat tissues, prepared by homogenising the tissue in ice‐cold phosphate buffer and centrifuging. ...

Endocrine disrupters (EDs) alter normal hormonal regulation and may be naturally occurring or environmental contaminants. Classically, EDs act genomically, with agonistic or antagonistic effects on steroid receptors and may alter reproductive function and/or cause feminisation by binding to oestrogen or androgen receptors; their binding to the thyroid receptor may dysregulate the neuroendocrine system. Recently, it has been shown that EDs can also act by non-genomic mechanisms, altering steroid synthesis (inhibition of cytochrome P450 isoforms) or steroid metabolism. The alkylphenol and phthalate plasticisers inhibit the inactivation of oestrogens by sulphation (via SULT 1A1 and 1E1 isoforms) and so cause a rise in levels of the free active endogenous oestrogens. A range of ED effects have been shown in mammals, fish, birds, reptiles, amphibia and aquatic invertebrates but it is not yet clear whether these processes also occur in human beings. It is evident that EDs, as well as altering reproduction, can cause changes in neurosteroid levels and so have the potential to affect immune function, behaviour and memory. This may be of long-term concern since traces of EDs such as plasticisers, brominated fire retardants, sunscreen agents and cosmetic ingredients are widely distributed in the environment and in human biofluids.

Sulphation is important in xenobiotic detoxification and in steroid and thyroid hormones synthesis, transport and metabolism. Potential endocrine disrupting actions of plasticisers were assessed by studying effects on cell viability, cell proliferation and expression of enzymes (cysteine dioxygenase, sulphite oxidase, PAPS synthase I and II) involved in the synthesis of the cofactor, PAPS, for steroid sulphotransferases. TE 671 cells were used to study the effects of exposure to alkylphenols and alkylphenolethoxylates, bisphenol A, bisphenol A methacrylate, alkyladipates, dialkyl phthalates and resorcinol. The lactate dehydrogenase assay and CellTiter 96) AQ(ueous) One Solution Cell Proliferation Assay were used to measure cytotoxicity and cell proliferation, respectively. Steady-state mRNA was assessed by semi-quantitative RT-PCR and real time RT-PCR. None of the compounds tested was cytotoxic in TE 671 cells, however, cell proliferation was significantly increased with 0.005-0.5 microM dioctyl phthalate, diisodecyl phthalate (DIP) and butylbenzyl phthalate (P

The purpose of this study was to assess the level of agreement between two techniques commonly used to measure exogenous carbohydrate oxidation (CHOEXO). To accomplish this, seven healthy male subjects (24 ± 3 yr, 74.8 ± 2.1 kg, V̇O2max 62 ± 4 ml·kg−1·min−1) exercised at 50% of their peak power for 120 min on two occasions. During these exercise bouts, subjects ingested a solution containing either 144 g glucose (8.7% wt/vol glucose) or water. The glucose solution contained trace amounts of both [U-13C]glucose and [U-14C]glucose to allow CHOEXO to be quantified simultaneously. The water trial was used to correct for background 13C enrichment. 13C appearance in the expired air was measured using isotope ratio mass spectrometry, whereas 14C appearance was quantified by trapping expired CO2 in solution (using hyamine hydroxide) and adding a scintillator before counting radioactivity. CHOEXO measured with [13C]glucose ([13C]CHOEXO) was significantly greater than CHOEXO measured with [14C]glucose ([14C]CHOEXO) from 30 to 120 min. There was a 15 ± 4% difference between [13C]CHOEXO and [14C]CHOEXO such that the absolute difference increased with the magnitude of CHOEXO. Further investigations suggest that the difference is not because of losses of CO2 from the trapping solution before counting or an underestimation of the “strength” of the trapping solution. Previous research suggests that the degree of isotopic fractionation is small (S. C. Kalhan, S. M. Savin, and P. A. Adam. J Lab Clin Med89: 285–294, 1977). Therefore, the explanation for the discrepancy in calculated CHOEXO remains to be fully understood.

The first purpose of the study was to examine whether combined ingestion of glucose and sucrose at an intake rate of 1.2 g/min would lead to higher oxidation rates compared with the ingestion of an isocaloric amount of glucose or sucrose alone. The second aim of the study was to investigate whether a mixture of glucose and sucrose when ingested at a high rate (2.4 g/min) would result in exogenous CHO oxidation rates higher than 1.2 to 1.3 g/min. Eight trained cyclists (maximal oxygen consumption: 64 +/- 2 mL . kg -1 . min -1 , mean +/- SE) performed 5 exercise trials in random order. Each trial consisted of 120 minutes of cycling at 50% maximum power output (63% +/- 2% maximal oxygen consumption), whereas subjects received a solution providing either 1.2 g/min of glucose (GLU), 1.2 g/min of sucrose (SUC), 0.6 g/min of glucose + 0.6 g/min of sucrose (M-GLU+SUC), 1.2 g/min of glucose + 1.2 g/min of sucrose (H-GLU+SUC), or water (WAT). Peak exogenous CHO oxidation rates in the H-GLU+SUC trial (1.20 +/- 0.07 g/min) were significantly higher ( P.01) compared with the GLU, M-GLU+SUC, and SUC trials (0.77 +/- 0.04, 0.90 +/- 0.07, 0.98 +/- 0.04 g/min, respectively). Furthermore, peak exogenous CHO rates in M-GLU+SUC and SUC trials were significantly higher ( P.05) compared with the GLU trial. In conclusion, combined ingestion of moderate amounts of glucose and sucrose (144 g) during cycling exercise resulted in approximately 21% higher exogenous CHO oxidation rates compared with the ingestion of an isocaloric amount of glucose. Furthermore, when a mixture of glucose and sucrose was ingested at high rates (2.4 g/min), exogenous CHO oxidation rates reached peak values of approximately 1.20 g/min.

The aim of the study was to determine whether the expression of sulphotransferase enzymes could be affected by the presence of cytokines or peptide hormones. The effects of cytokines (TNF-alpha and TGF-beta) and insulin on sulphotransferase (SULT 1A1 and 1A3) activity were studied in a human neuronal cell line (SK-N-SH) and a human gastrointestinal tract cell line (HT-29). Cells were cultured with varying concentrations of TNF-alpha, TGF-beta or insulin for 24 h; the SULT 1A1 isoform in the 2 cell lines showed different optimal substrate concentrations. There were no direct effects of cytokines on enzyme activity. Culture with TNF-alpha increased activity of both SULT 1A1 and 1A3 in the HT-29 cells; TGF-beta also increased activities of both isoforms but to a lesser extent; insulin increased activity of SULT 1A1 only. The cytokines and insulin had relatively little effect on sulphotransferase activity in the neuronal cell line. These results suggest that, unlike neuronal cells, gastrointestinal cells may respond to physiological states by altering sulphotransferase activity. As certain substrates such as diet-derived heterocyclic amines are bioactivated by sulphation to produce carcinogenic metabolites this may be a factor in the increased incidence of colorectal cancer in patients with inflammatory bowel disease or diabetes.

We investigated the ability of 37 flavonoids and flavonoid sulfoconjugates, including some abundant dietary constituents, to act as substrates and/or inhibitors of the sulfotransferase and sulfatase enzymes that interconvert active estrogens and inactive estrogen sulfates in human tissues. The enzymes studied include estrogen sulfotransferase, the thermostable phenolsulfotransferase that acts on a range of substrates including estrogens; steroid sulfatase; and two related enzymes, monoamine phenolsulfotransferase and arylsulfatase A. Several dietary flavonoids, including the soy isoflavones genistein and daidzein, were sulfated by these human sulfotransferases. Many flavonoids were potent inhibitors of thermostable phenolsulfotransferase. Genistein and equol were potent mixed inhibitors of hepatic estrogen sulfotransferase, with inhibitory constant values of 500 nM and 400 nM, respectively. Monoamine phenolsulfotransferase activity was relatively unaffected by flavonoids, but this enzyme was mainly responsible for the sulfation of flavonoids at concentrations greater than 1 micro M. Of the compounds tested, only daidzein 4,7-bisulfate, a trace metabolite in humans, significantly inhibited steroid sulfatase in the micromolar concentration range. Hence, dietary flavonoids may be able to influence the bioavailability of endogenous estrogens, and disrupt endocrine balance, by increasing the ratio of active estrogens to inactive estrogen sulfates in human tissues.

A major pathway for the production of sulphate within the mammalian body is known to be via the oxidative degradation of the sulphur moiety within the amino acid, L-cysteine. The ability of two structurally similar sulphur-containing drugs, the anti-rheumatic agent, D-penicillamine, and the mucoactive compound, S-carboxymethyl-L-cysteine, to interfere with this sulphate production was investigated. Co-administration to the male rat of D-penicillamine (p.o.) and S-carboxymethyl-L-cysteine (p.o.) with [ 3 5 S]-L-cysteine (i.p.) led to a significant decrease in the subsequent urinary elimination of inorganic sulphate whilst having no measurable effect on organic sulphate excretion. The co-administration of L-valine, an amino acid not containing sulphur, had no effect. It is not known where, within the complex sequence of events surrounding the degradation of cysteine to sulphate, that D-penicillamine or S-carboxymethyl-L-cysteine may interact.

Purpose: To explore the possible manifestations of abnormal levels of either cysteine or sulphate, whether high or low, and the ratio between them, in human subjects. Design: A case–control study of the plasma cysteine and sulphate levels and cysteine/sulphate ratio in chronically ill patients. Materials and Methods: Eighty-one chronically ill patients of a nutrition clinic were suspected of having abnormal levels of sulphate and/or cysteine. Their plasma was checked to determine their cysteine and sulphate levels and cysteine/sulphate ratio. These were compared with the results of 177 controls. They were grouped according to their results, and their symptoms were listed. Results: Only one patient had a ratio within the reference range, whereas 175 of the controls did. Patients already being treated with relevant nutritional supplements at the time of testing were less likely to have a ratio over 1000. Some close relatives shared abnormal test results, but manifested different symptoms. Conclusion: Patients with chronic conditions including myalgic encephalomyelitis, irritable bowel syndrome, migraine, arthritis, multiple chemical sensitivity and depression are likely to benefit from tests for cysteine and sulphate, and from treatment designed to improve these levels. Oral fish oil, vitamin B2, pantothenate and molybdenum, and Epsom salt baths may help patients with low sulphate. Vitamins B2 and B6, zinc and magnesium, and a low protein diet may reduce high cysteine levels. N-acetyl cysteine, zinc and vitamin C may help those with low cysteine levels. Patients with abnormal levels of sulphate might be counselled against working in polluted conditions, where efficient sulphate conjugation is required, and against using pesticides. They might be advised to be cautious in their use of drugs, and possibly vaccines too. Further work is suggested, to investigate to what extent abnormalities in cysteine and sulphate levels are genetically determined, and to test the efficacy of the treatments outlined, both at improving the cysteine and sulphate levels, and health.

Nicotinamide N-methyltransferase (NNMT) has been proposed as a link between the environmental and genetic factors of Parkinson disease (PD). Therefore, we explored the hypothesis that high levels of NNMT expression may predispose to the development of PD. Regions of high mRNA expression were shown in the spinal cord, medulla, and temporal lobe, with lowest expression in the cerebellum, subthalamic nucleus, and caudate nucleus. Using 2 NNMT antibodies, the protein was shown to be expressed in multipolar neurons in the temporal lobe, caudate nucleus, and spinal cord, granular neurons of the cerebellum, dopaminergic neurons in the substantia nigra, and in the axons of the third nerve. Expression of NNMT was compared in PD and non-PD control cerebella and caudate nucleus. PD tissue exhibited significantly increased levels of NNMT protein and activity. PD disease duration was inversely correlated with the level of expression in cerebellum. This is the first demonstration that patients with PD have higher levels of NNMT activity and protein in brain tissue than those without PD and that NNMT expression is associated with neurons that degenerate in PD.

1. Radiolabelled [(35)S]-phenothiazine has been administered orally to two healthy adult male volunteers (6 mg kg(-1) body weight). Faeces were the major route of excretion of radioactivity (68%), the remainder being eliminated via the urine (32%) with an estimated urinary half-life (biphasic) of 6-16 h. Over the 5 days of the study a complete recovery of radioactivity was achieved. 2. From urinary data, it was shown that metabolism occurred via ring carbon oxidation to form phenothiazone and thionol and via ring sulphur oxidation to form phenothiazine sulphoxide. The majority of urinary material (92%) was present in the form of conjugates of phenothiazine and phenothiazone. Only unchanged phenothiazine was detected in the faeces. Phenothiazine sulphoxide was reduced to phenothiazine during incubation with faecal homogenates.

The tungsten alloy of 91% tungsten, 6% nickel and 3% cobalt (WNC 91-6-3) induces rhabdomyosarcoma when implanted into a rat thigh muscle. To investigate whether this effect is species-specific human HSkMc primary muscle cells were exposed to WNC 91-6-3 particles and responses were compared with those from a rat skeletal muscle cell line (L6-C11). Toxicity was assessed by the adenylate kinase assay and microscopy, DNA damage by the Comet assay. Caspase 3 enzyme activity was measured and oligonucleotide microarrays were used for transcriptional profiling. WNC 91-6-3 particles caused toxicity in cells adjacent to the particles and also increased DNA strand breaks. Inhibition of caspase 3 by WNC 91-6-3 occurred in rat but not in human cells. In both rat and human cells, the transcriptional response to WNC 91-6-3 showed repression of transcripts encoding muscle-specific proteins with induction of glycolysis, hypoxia, stress responses and transcripts associated with DNA damage and cell death. In human cells, genes encoding metallothioneins were also induced, together with genes related to angiogenesis, dysregulation of apoptosis and proliferation consistent with pre-neoplastic changes. An alloy containing iron, WNF 97-2-1, which is non-carcinogenic in vivo in rats, did not show these transcriptional changes in vitro in either species while the corresponding cobalt-containing alloy, WNC 97-2-1 elicited similar responses to WNC 91-6-3. Tungsten alloys containing both nickel and cobalt therefore have the potential to be carcinogenic in man and in vitro assays coupled with transcriptomics can be used to identify alloys, which may lead to tumour formation, by dysregulation of biochemical processes.

1. It is inevitable that during some xenobiotic biotransformation studies, a certain metabolite or degradation product arises of which the identity is uncertain, the route of formation is ambiguous, or it is just a plain mystery.2. The following communication draws attention to three drugs reported in the literature, chlorphentermine, phenothiazine and aminopyrine, where after many years of investigation there still exists uncertainty over some of their metabolites. Noticeably, these three examples probably involve (potential) interaction of a nitrogen centre within the drug molecule.3. It is hoped that the resurrection and assemblage of these data will offer interesting reading and that these examples may prove sufficiently intriguing to motivate further exploration and some resolution of these lingering concerns.

Phytoestrogens are natural constituents of our diets that have been suggested to protect against hormone-dependent breast cancer. Some of the diverse effects of these compounds may be attributed to ligand-dependent differences in their interaction with oestrogen receptor sub-classes. However, phytoestrogens can also inhibit enzymes that are involved in the generation and removal of endogenous steroid hormones. Among the most potent effects of dietary phytoestrogens is their ability to inhibit the sulphotransferases that sulphate both oestrogenic steroids and a variety of environmental chemicals, including dietary pro-carcinogens. Circulating steroid sulphates are thought to be the major source of oestradiol in post-menopausal breast tumours and sulphation is a key step in the activation of some dietary procarcinogens. Hence the inhibition of sulpho-transferases by dietary phytoestrogens may have complex effects upon human susceptibility to breast cancer.

Cysteine dioxygenase (CDO) converts cysteine to cysteinesulphinic acid and is the rate-limiting step in sulphate production. Most studies have centred upon the hepatic form of the enzyme, but several studies have investigated brain CDO using activity assays and western blotting. The aim of this study was to investigate the expression of CDO in the rat brain using a combination of immunohistochemistry and in situ hybridisation. Affinity-purified anti-R and anti-H CDO antibodies were immunoprecipitated using rat brain homogenate to determine whether the antibodies could remove enzyme activity. Immunohistochemistry and in situ hybridisation were then used to determine the cellular and regional expression of both CDO protein and mRNA. Immunoprecipitation of rat brain homogenate removed up to 98% and 70% (anti-R and anti-H, respectively) of enzyme activity. Nonimmune sheep serum had no effect upon enzyme activity. CDO protein and mRNA was localised solely to the neurones of the brain, including the pyramidal cells of the hippocampus and the Purkinje cells of the cerebellum. Regional localisation varied, with high levels of expression in the hippocampus, the dentate gyrus, the outer cortices of the brain, and the substantia nigra. The relative expression of CDO activity and protein in these regions is most probably a result of the relative abundance of neurones in these regions. CDO expression in the brain may have several possibile functions, the most likely being the prevention of free radical production by the autoxidation of cysteine and dopamine. J. Neurosci. Res. 65:78–84, 2001. © 2001 Wiley-Liss, Inc.

Sulfate conjugation by sulfotransferase enzymes is an important pathway for the detoxication of xenobiotics and endogenous compounds. The large surface area of the gastrointestinal tract exposes the body to a range of potential toxins, and hence local metabolism is likely to be important. The ability of different regions of the gut to sulfate micromolar concentrations of simple phenols and catecholamines has been determined throughout the gut using 4-nitrophenol and dopamine as standard substrates. The pattern of sulfation of both compounds was similar, with activity highest in the small bowel >right colon >left colon >rectum >stomach >esophagus. High concentrations of sulfotransferases in the reservoir areas of the right and left colon indicate possible importance in detoxication by sulfation and also perhaps in activating mutagens in the same areas. Nutritional factors, such as a high-fat diet may, however, alter sulfotransferase activity.

Purpose: Previous studies in autistic children have shown that they have reduced levels of plasma sulphate as compared with age-matched control children and the aim of this study was to see if this reflected increased urinary sulphate loss. Design: Outpatient-based survey of autistic children and matched controls. Materials and methods: The children in the study were elected on the basis of ICD-10 criteria and a diagnosis of autism. Use of a behavioural questionnaire allowed children with autism to be divided into 3 subsets. Urinary excretion of sulphate, sulphite, thiosulphate and thiocyanate was measured in 232 autistic children and compared with values from 68 age-matched controls. Results: Autistic children excreted higher levels of sulphate, sulphite and thiosulphate, but reduced levels of thiocyanate. Conclusions: The significance of these altered parameters is discussed with respect to catecholamine metabolism, mucin formation, gastrointestinal hormone activation and sulphur anion metabolism.

The development of an in vitro model for cysteine dioxygenase (CDO) expression in the brain would provide a useful model for determining the mechanisms for the regulation of CDO expression that does not involve the use of animals. Here we demonstrate the screening and characterization of a cell line that expresses CDO, the primary metabolizing enzyme of cysteine and the regulatory point of sulfate production. A panel of four commercially available tumor-derived human brain cell lines, each representing one major class of brain cell, were screened using western blotting and activity assay for cysteine dioxygenase expression. One cell line, TE 671 (human medulloblastoma) was found to express both a protein of approximately 70 kDa and CDO activity. Nuclease protection assay (NPA) of mRNA isolated from TE 671 showed the expression of a CDO mRNA. Reverse transcription–polymerase chain reaction of this mRNA and sequencing of the cDNA obtained showed that this was indeed CDO. Treatment of TE 671 cells with cysteine resulted in the upregulation of CDO mRNA, whereas treatment with tumor necrosis factor α resulted in the downregulation of CDO mRNA, as evidenced using NPA. The characterization of an in vitro model for CDO expression provides a useful tool for the investigation of this important enzyme, which may have an etiological role in the pathogenesis of Parkinson's disease.

Indexed mitochondrial complex activities (MCA;) were determined in biopsies obtained from 52 donor kidneys at the end of cold ischemia (8-32 hr) to see if longer anoxia affected MCA i and accounted for the increased risk of delayed graft function (DGF) in recipients of grafts with longer cold ischemia time (CIT) or from non-heart-beating donors (NHBD). CITs were significantly different between those with and without DGF (P=0.02), being shorter in the latter, but MCA i were similar. CIT was correlated (r=0.43, P=0.003) with the time taken for creatinine concentration to fall to half the perioperative value (Cr t ½) but not with MCA i . Frequency of DGF, greater in NHBD, was significantly different from that of heart-beating donors (P= 0.04), but CIT and MCA i were similar. However, Cr t ½, was significantly different being longer in NHBD. Thus, the frequency of DGF increased and the speed of recovery diminished with longer CIT, whereas MCA, remained stable suggesting other factors determined tissue recovery.

Background: Parents of autistic children and autism support groups often report that autistic episodes are exacerbated when the children eat certain foodstuffs such as dairy products, chocolates, wheat, corn sugar, apples, and bananas. The hypothesis that autistic behavior might be related to metabolic dysfunctions has led us to investigate in a group of “low functioning” autistic children and in an age-matched control group each made up of 20 subjects, the sulphation capacity available. Methods: Utilizing the biochemical characteristics of paracetamol we evaluated by high performance liquid chromatography, the urine paracetamol–sulfate/paracetamol-glucuronide (PS/PG) ratio in all subjects following administration of this drug. Results: The PS/PG ratio in the group of autistic subjects gave a significantly lower result than the control group with p < .00002. Conclusions: The inability to effectively metabolize certain compounds particularly phenolic amines, toxic for the CNS, could exacerbate the wide spectrum of autistic behavior.

The presence and localisation of dehydroepiandrosterone sulphotransferase was assessed in adult rat brain. The activity of the enzyme was investigated and was found to be approximately four times less than that in rat liver. Rat brain dehydroepiandrosterone sulphotransferase activity was measured as 1.39 ± 0.04 nm/μg/min. The protein expression of dehydroepiandrosterone sulphotransferase was evaluated using Western blotting. The presence of a band at 35 kDa signified the presence of this enzyme in adult rat brain. Finally, the localisation of this enzyme was investigated by immunohistochemistry and by assessing activity in nine regions of brain. Immunohistochemistry showed positive staining in the hippocampus and the dendate gyrus, and in the large neurons of the midbrain, indicating the presence of dehydroepiandrosterone sulphotransferase. Western blotting showed the presence of a band in all nine regions, and enzyme activity was highest in the hippocampus, frontal cortex, thalamus and basal ganglia. These results imply that dehydroepiandrosterone and dehydroepiandrosterone sulphate (DHEAS) have a defined role in brain function and are produced in situ independently of the peripheral endocrine system.

Phenotypic differences in nicotinamide N-methyltransferase (NNMT, E. C. 2.1.1.1) activity may be due to a genetic polymorphism. We report the characterisation of the hepatic NNMT activity in cytosol from normal human livers, enzyme protein levels determined by Western blotting and ELISA and mRNA levels determined by SDS-PAGE/Northern blotting. Subjects with high NNMT activity had high levels of NNMT protein and NNMT mRNA levels in hepatic cytosol and the converse was true for individuals with low NNMT activity. No differences in sequences were seen when cDNAs of individuals with high and low NNMT activity were compared. Thus phenotypic differences in the general population are due to differences in steady-state mRNA levels and not because of a polymorphism in the coding region of the NNMT gene.