Influence of polymorphisms of the human glutathione transferases and cytochrome P450 2E1 enzyme on the metabolism and toxicity of ethylene oxide and acrylonitrile

A cohort of 59 persons with industrial handling of low levels of acrylonitrile is being studied as part of a medical surveillance programme. Previously, an extended haemoglobin adduct monitoring (N-(cyanoethyl)valine and N-(hydroxyethyl)-valine) was performed regarding the glutathione transferases hGSTM1 and hGSTT1 polymorphisms but no influence of hGSTM1 or hGSTT1 polymorphisms on specific adduct levels was found. A compilation of case reports of human accidental poisonings had pointed to significant individual differences in human acrylonitrile metabolism and toxicity. Therefore, a re-evaluation of the industrial cohort included known polymorphisms of the glutathione transferases hGSTM3 and hGSTP1 as well as of the cytochrome P450 CYP2E1. A detailed statistical analysis revealed that exposed carriers of the allelic variants of hGSTP1, hGSTP1*B/hGSTP1*C, characterized by a single nucleotide polymorphism at nucleotide 313 which results in a change from Ile to Val at codon 104, had higher levels of the acrylonitrile-specific haemoglobin adduct N-(cyanoethyl)valine compared to the carriers of the codon 113 alleles hGSTP1*A and hGSTP1*D. The single nucleotide polymorphism at codon 113 of hGSTP1 (hGSTP1*A/hGSTP1*B versus hGSTP1*C/hGSTP1*D) did not show an effect, and also no influence was seen on specific haemoglobin adduct levels of the polymorphisms of hGSTM3 or CYP2E1. The data, therefore, point to a possible influence of a human enzyme polymorphism of the GSTP1 gene at codon 104 on the detoxication of acrylonitrile which calls for experimental toxicological investigation. The study also confirmed the impact of GSTT1 polymorphism on background N-(hydroxyethyl)-valine adduct levels in haemoglobin which are caused by endogenous ethylene oxide.

Interaction of metal salts with cytoskeletal motor protein systems

Interactions of chemicals with the microtubular network of cells may lead to genotoxicity. Micronuclei (MN) might be caused by interaction of metals with tubulin and/or kinesin. The genotoxic effects of inorganic lead and mercury salts were studied using the MN assay and the CREST analysis in V79 Chinese hamster fibroblasts. Effects on the functional activity of motor protein systems were examined by measurement of tubulin assembly and kinesin-driven motility. Lead and mercury salts induced MN dose-dependently. The no-effect-concentration for MN induction was 1.1 μM PbCl2, 0.05 μM Pb(OAc)2 and 0.01 μM HgCl2. The in vitro results obtained for PbCl2 correspond to reported MN induction in workers occupationally exposed to lead, starting at 1.2 μM Hg(II) (Vaglenov et al., 2001, Environ. Health Perspect. 109, 295-298). The CREST Analysis indicate aneugenic effects of Pb(II) and aneugenic and additionally clastogenic effects of Hg(II). Lead (chloride, acetate, and nitrate) and mercury (chloride and nitrate) interfered dose-dependently with tubulin assembly in vitro. The no-effect-concentration for lead salts in this assay was 10 μM. Inhibition of tubulin assembly by mercury started at 2 μM. The gliding velocity of microtubules along immobilised kinesin molecules was affected by 25 μM Pb(NO3)2 and 0.1 μM HgCl2 in a dose-dependent manner. Our data support the hypothesis that lead and mercury genotoxicity may result, at least in part, via disturbance of chromosome segregation via interaction with cytoskeletal proteins.

Determination of urinary thymidine glycol using affinity chromatography, HPLC and post-column reaction detection : a biomarker of oxidative DNA damage upon kidney transplantation

Reactive oxygen species are generated during ischaemia-reperfusion of tissue. Oxidation of thymidine by hydroxyl radicals (HO) leads to the formation of 5,6-dihydroxy-5,6-dihydrothymidine (thymidine glycol). Thymidine glycol is excreted in urine and can be used as biomarker of oxidative DNA damage. Time dependent changes in urinary excretion rates of thymidine glycol were determined in six patients after kidney transplantation and in six healthy controls. A new analytical method was developed involving affinity chromatography and subsequent reverse-phase high-performance liquid chromatography (RP-HPLC) with a post-column chemical reaction detector and endpoint fluorescence detection. The detection limit of this fluorimetric assay was 1.6 ng thymidine glycol per ml urine, which corresponds to about half of the physiological excretion level in healthy control persons. After kidney transplantation the urinary excretion rate of thymidine glycol increased gradually reaching a maximum around 48 h. The excretion rate remained elevated until the end of the observation period of 10 days. Severe proteinuria with an excretion rate of up to 7.2 g of total protein per mmol creatinine was also observed immediately after transplantation and declined within the first 24 h of allograft function (0.35 + 0.26 g/mmol creatinine). The protein excretion pattern, based on separation of urinary proteins on sodium dodecyl sulphate-polyacrylamide gel electrophorosis (SDS-PAGE), as well as excretion of individual biomarker proteins, indicated nonselective glomerular and tubular damage. The increased excretion of thymidine glycol after kidney transplantation may be explained by ischaemia-reperfusion induced oxidative DNA damage of the transplanted kidney.

Determination of glutathione transferase (GSTT1-1) activities in different tissues based on formation of radioactive metabolites using 35S-glutathione

A new system has been developed to determine enzyme activities of glutathione transferase θ (GSTT1-1) based on radiometric product detection resulting from the enzymic reaction of methyl chloride with 35S-labelled glutathione. In principle, the method is universally applicable for determination of glutathione transferase activities towards a multiplicity of substrates. The method distinguishes between erythrocyte GSTT1-1 activities of human 'non-conjugators', 'low conjugators' and 'high conjugators'. Application to cytosol preparations of livers and kidneys of male and female Fischer 344 and B6C3F1 mice reveals differential GSTT1-1 activities in hepatic and renal tissues. These ought to be considered in species-specific modellings of organ toxicities of chlorinated hydrocarbons.

Biological monitoring in workers in a nitrobenzene reduction plant : Haemoglobin versus serum albumin adducts

The high priority of monitoring workers exposed to nitrobenzene is a consequence of clear findings of experimental carcinogenicity of nitrobenzene and the associated evaluations by the International Agency for Research on Cancer. Eighty male employees of a nitrobenzene reduction plant, with potential skin contact with nitrobenzene and aniline, participated in a current medical surveillance programme. Blood samples were routinely taken and analysed for aniline, 4-aminodiphenyl (4-ADP) and benzidine adducts of haemoglobin (Hb) and human serum albumin (HSA). Also, levels of methaemoglobin (Met-Hb) and of carbon monoxide haemoglobin (CO-Hb) were monitored. Effects of smoking were straightforward. Using the rank sum test of Wilcoxon, we found that very clear-cut and statistically significant smoking effects (about 3-fold increases) were apparent on CO-Hb (P = 0.00085) and on the Hb adduct of 4-ADP (P = 0.0006). The mean aniline-Hb adduct level in smokers was 1.5 times higher than in non-smokers; the significance (P = 0.05375) was close to the 5% level. The strongest correlation was evident between the Hb and HSA adducts of aniline (rs = 0.846). Less pronounced correlations (but with P values < 0.02) appeared between aniline-Hb and 4-ADP-Hb adducts (rs = 0.388), between 4-ADP and 4-ADP-HSA adducts (rs = 0.373), and between 4-ADP-Hb and aniline-HSA adducts (rs = 0.275). In view of the proposal for additional use of the aniline-HSA adduct for biological monitoring, particularly in cases of acute overexposures or poisonings, the strong correlation of the Hb and HSA conjugates is noteworthy; the ratio aniline-HSA:aniline-Hb was 1:42 for the entire cohort.

Re-evaluation of the effect of smoking on the methylation of N-terminal valine in haemoglobin

In view of the established extrapulmonary cancer sites targeted by smoking a multiplicity of compounds, and mechanisms might be involved. It has been debated that smoking caused increased incidence of N-methylvaline at the N-terminus of haemoglobin. Because this could indicate a relevance of methylating nitrosamines in tobacco smoke, data are presented from an industrial cohort of 35 smokers and 21 non-smokers repeatedly monitored between 1994 and 1999. In general, N-methylvaline adduct levels in haemoglobin of smokers were approximately 50% higher than those of non-smokers. The smoking-induced methylation of haemoglobin is likely to be caused by dimethylnitrosamine (N-nitroso-dimethylamine), a major nitrosamine in side-stream tobacco smoke. The biomonitoring data emphasise the potential value of N-methylvaline as a smoking-related biomarker and call for intensified research on tobacco smoke compounds that lead to macromolecular methylation process.

Possible impact of human CYP2E1 polymorphisms on the metabolism of acrylonitrile

Case reports of human accidental poisonings point to significant individual differences in human acrylonitrile metabolism and toxicity. A cohort of 59 persons with industrial handling of low levels of acrylonitrile has repetitively been studied from 1994 through 1999 as part of a medical surveillance programme. The analyses included adduct determinations of N-terminal N-(cyanoethyl)valine in haemoglobin and genotypings of the following cytochrome P-450 2E1 (CYP2E1) polymorphisms: G-1259C and C-1019T (two subjects heterozygous), A-316G (three subjects heterozygous), T-297A (15 subjects heterozygous), G-35T (eight subjects heterozygous), G4804A (two subjects heterozygous), T7668A (six subjects heterozygous). N-(Cyanoethyl)valine adduct levels were, if any, only slightly influenced by smoking and mainly determined by the external acrylonitrile exposures. The individual means and medians of N-(cyanoethyl)valine levels over the entire observation period were compared with the CYP2E1 variants (Wilcoxon rank sum test). No influences of the investigated CYP2E1 polymorphisms on the N-(cyanoethyl)valine levels appeared at the 5% level. However, there was a trend, at a level of P≃0.1, pointing to higher acrylonitrile-specific adduct levels in persons with the A-316G mutation. Higher adduct levels would be compatible with a slower CYP2E1-mediated metabolism of acrylonitrile and with lower extents of toxification to cyanide.

Human glutathione S-transferase T1-1 enhances mutagenicity of 1,2-dibromoethane, dibromomethane and 1,2,3,4-diepoxybutane in Salmonella typhimurium

The rat theta class glutathione S-transferase (GST) 5-5 has been shown to affect the mutagenicity of halogenated alkanes and epoxides. In Salmonella typhimurium TA1535 expressing the rat GST5-5 the number of revertants was increased compared to the control strain by CH2Br2, ethylene dibromide (EDB) and 1,2,3,4-diepoxybutane (BDE); in contrast, mutagenicity of 1,2-epoxy-3-(4'-nitrophenoxy)propane (EPNP) was reduced. S.typhimurium TA1535 cells were transformed with an expression plasmid carrying the cDNA of the human theta ortholog GST1-1 either in sense or antisense orientation, the latter being the control. These transformed bacteria were utilized for mutagenicity assays. Mutagenicity of EDB, BDE, CH2Br2, epibromohydrin and 1,3-dichloroacetone was higher in the S.typhimurium TA1535 expressing GSTT1-1 than in the control strain. The expression of active enzyme did not affect the mutagenicity of 1,2-epoxy-3-butene or propylene oxide, GSTT1-1 expression reduced the mutagenicity of EPNP. Glutathione S-transferase 5-5 and GSTT1-1 modulate genotoxicity of several industrially important chemicals in the same way. Polymorphism of the GSTT1 locus in humans may therefore cause differences in cancer susceptibility between the two phenotypes.

Differential substrate behaviours of ethylene oxide and propylene oxide towards human glutathione transferase theta hGSTT1-1

The transformation of ethylene oxide (EO), propylene oxide (PO) and 1- butylene oxide (1-BuO) by human glutathione transferase theta (hGSTT1-1) was studied comparatively using 'conjugator' (GSTT1 + individuals) erythrocyte lysates. The relative sequence of velocity of enzymic transformation was PO > EO >> 1-BuO. The faster transformation of PO compared to EO was corroborated in studies with human and rat GSTT1-1 (hGSTT1-1 and rGSTT1-1, respectively) expressed by Salmonella typhimurium TA1535. This sequence of reactivities of homologous epoxides towards GSTT1-1 contrasts to the sequence observed in homologous alkyl halides (methyl bromide, MBr; ethyl bromide, EtBr; n-propyl bromide, PrBr) where the relative sequence MeBr >> EtBr > PrBr is observed. The higher reactivity towards GSTT1-1 of propylene oxide compared to ethylene oxide is consistent with a higher chemical reactivity. This is corroborated by experimental data of acid-catalysed hydrolysis of a number of aliphatic epoxides, including ethylene oxide and propylene oxide and consistent with semi-empirical molecular orbital modelings.

Species differences in the glutathione transferase GSTT1-1 activity towards the model substrates methyl chloride and dichloromethane in liver and kidney

Glutathione transferase (GST) GSTT1-1 is involved in the biotransformation of several chemicals widely used in industry, such as butadiene and dichloro methane DCM. The polymorphic hGSTT1-1 may well play a role in the development of kidney tumours after high and long-term occupational exposure against trichloroethylene. Although several studies have investigated the association of this polymorphism with malignant diseases little is known about its enzyme activity in potential extrahepatic target tissues. The known theta-specific substrates methyl chloride (MC) dichloromethane and 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) were used to assay GSTT1-1 activity in liver and kidney of rats, mice, hamsters and humans differentiating the three phenotypes (non-conjugators, low conjugators, high conjugators) seen in humans. In addition GSTT1-1 activity towards MC and DCM was determined in human erythrocytes. No GSTT1-1 activity was found in any tissue of non-conjugators (NC). In all organs high conjugators (HC) showed twofold higher activity towards MC and DCM than low conjugators (LC). The activity in human samples towards EPNP was too close to the detection limit to differentiate between the three conjugator phenotypes. GSTT1-1 activity towards MC was two to seven-times higher in liver cytosol than in kidney cytosol. The relation for MC between species was identical in both organs: mouse > HC > rat > LC > hamster > NC. In rats, mice and hamsters GSTT1-1 activity in liver cytosol towards DCM was also two to seven-times higher than in the kidney cytosol. In humans this activity was twice as high in kidney cytosol than in liver cytosol. The relation between species was mouse > rat > HC > LC > hamster > NC for liver, but mouse > HC > LC/rat > hamster/NC for kidney cytosol. The importance to heed the specific environment at potential target sites in risk assessment is emphasized by these results.

The hereditary transmission of the glutathione transferase hGSTT1-1 conjugator phenotype in a large family

The polymorphism of human glutathione transferase hGSTT1-1 is expressed in three phenotypes. Experimentally, individuals can be classified as non-conjugators, low conjugators and 'high' conjugators depending on the enzyme activity in blood towards methylene chloride using a gas chromatographic assay. Non-conjugators do not have a functional hGSTT1 gene; however, little is known about the molecular basis of the three conjugator phenotypes. The higher hGSTT1-1 activity in high conjugators may be the result of enzyme induction or be genetically determined. Twenty-nine members of a large family, including three generations were phenotyped and genotyped with respect to hGSTT1-1. The hGSTT1-1 enzyme activity of high conjugators was twice as high as that of low conjugators. The distribution of hGSTT1-1 phenotypes strongly indicates a Mendelian intermediary inheritance, in which a gene-dosage effect results in a doubled enzyme expression in the presence of two functional alleles. The Mendelian intermediary inheritance is further supported by the findings of a semiquantitative polymerase chain reaction method designed to distinguish the three genotypes of hGSTT1 for rapid screening of large study groups.

Menstrual effluent induces epithelial-mesenchymal transitions in mesothelial cells

Background Menstrual effluent affects mesothelial cell (MC) morphology. We evaluated whether these changes were consistent with epithelial-mesenchymal transitions (EMT). Methods Monolayer cultures of MC were incubated overnight in conditioned media, prepared from cells isolated form menstrual effluent, with or without kinase and ATP inhibitors. Changes in cell morphology were monitored using time-lapse video microscopy and immunohistochemistry. Effects on the expression of EMT-associated molecules were evaluated using real-time RT-PCR and/or Western blot analysis. Results Incubation in conditioned media disrupted cell-cell contacts, and increased MC motility. The changes were reversible. During the changes the distribution of cytokeratins, fibrillar actin and α-tubulin changed. Sodium azide, an inhibitor of ATP production, and Genistein, a general tyrosine kinase inhibitor, antagonized these effects. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, and SU6656, an Src tyrosine kinase inhibitor, only partially antagonized the effect. The expression of Snail and vimentin was markedly up-regulated, whereas the expression of E-cadherin was decreased and cytokeratins were altered. Conclusions In MC, menstrual effluent initiates a reversible, energy-dependent transition process from an epithelial to a mesenchymal phenotype. Involvement of the (Src) tyrosine kinase signalling pathway and the changes in the expression of cytokeratins, Snail, vimentin and E-cadherin demonstrate that the morphological changes are EMT.

Saliva proteome research: current status and future outlook

Human saliva harbours proteins of clinical relevance and about 30% of blood proteins are also present in saliva. This highlights that saliva can be used for clinical applications just as urine or blood. However, the translation of salivary biomarker discoveries into clinical settings is hampered by the dynamics and complexity of the salivary proteome. This review focuses on the current status of technological developments and achievements relating to approaches for unravelling the human salivary proteome. We discuss the dynamics of the salivary proteome, as well as the importance of sample preparation and processing techniques and their influence on downstream protein applications; post-translational modifications of salivary proteome and protein: protein interactions. In addition, we describe possible enrichment strategies for discerning post-translational modifications of salivary proteins, the potential utility of selected-reaction-monitoring techniques for biomarker discovery and validation, limitations to proteomics and the biomarker challenge and future perspectives. In summary, we provide recommendations for practical saliva sampling, processing and storage conditions to increase the quality of future studies in an emerging field of saliva clinical proteomics. We propose that the advent of technologies allowing sensitive and high throughput proteome-wide analyses, coupled to well-controlled study design, will allow saliva to enter clinical practice as an alternative to blood-based methods due to its simplistic nature of sampling, non-invasiveness, easy of collection and multiple collections by untrained professionals and cost-effective advantages.

DNA methylation at the novel CpG sites in the promoter of MED15/PCQAP gene as a biomarker for head and neck cancers

Head and neck cancers (HNCs) represent a significant and ever-growing burden to the modern society, mainly due to the lack of early diagnostic methods. A significant number of HNCs is often associated with drinking, smoking, chewing beetle nut, and human papilloma virus (HPV) infections. We have analyzed DNA methylation patterns in tumor and normal tissue samples collected from head and neck squamous cell carcinoma (HNSCC) patients who were smokers. We have identified novel methylation sites in the promoter of the mediator complex subunit 15 (MED15/PCQAP) gene (encoing a co-factor important for regulation of transcription initiation for promoters of many genes), hypermethylated specifically in tumor cells. Two clusters of CpG dinucleotides methylated in tumors, but not in normal tissue from the same patients, were identified. These CpG methylation events in saliva samples were further validated in a separate cohort of HNSCC patients (who developed cancer due to smoking or HPV infections) and healthy controls using methylation-specific PCR (MSP). We used saliva as a biological medium because of its non-invasive nature, close proximity to the tumors, easiness and it is an economically viable option for large-scale screening studies. The methylation levels for the two identified CpG clusters were significantly different between the saliva samples collected from healthy controls and HNSCC individuals (Welch's t-test returning P, 0.05 and Mann-Whitney test P, 0.01 for both). The developed MSP assays also provided a good discriminative ability with AUC values of 0.70 (P, 0.01) and 0.63 (P, 0.05). The identified novel CpG methylation sites may serve as potential non-invasive biomarkers for detecting HNSCC. © the authors.

Design and test of a custom instrumented leg press for injury and recovery intervention

There is an increasing desire and emphasis to integrate assessment tools into the everyday training environment of athletes. These tools are intended to fine-tune athlete development, enhance performance and aid in the development of individualised programmes for athletes. The areas of workload monitoring, skill development and injury assessment are expected to benefit from such tools. This paper describes the development of an instrumented leg press and its application to testing leg dominance with a cohort of athletes. The developed instrumented leg press is a 45° reclining sled-type leg press with dual force plates, a displacement sensor and a CCD camera. A custom software client was developed using C#. The software client enabled near-real-time display of forces beneath each limb together with displacement of the quad track roller system and video feedback of the exercise. In recording mode, the collection of athlete particulars is prompted at the start of the exercise, and pre-set thresholds are used subsequently to separate the data into epochs from each exercise repetition. The leg press was evaluated in a controlled study of a cohort of physically active adults who performed a series of leg press exercises. The leg press exercises were undertaken at a set cadence with nominal applied loads of 50%, 100% and 150% of body weight without feedback. A significant asymmetry in loading of the limbs was observed in healthy adults during both the eccentric and concentric phases of the leg press exercise (P < .05). Mean forces were significantly higher beneath the non-dominant limb (4–10%) and during the concentric phase of the muscle action (5%). Given that symmetrical loading is often emphasized during strength training and remains a common goal in sports rehabilitation, these findings highlight the clinical potential for this instrumented leg press system to monitor symmetry in lower-limb loading during progressive strength training and sports rehabilitation protocols.