Open PHACTS computational protocols for in silico target validation of cellular phenotypic screens: knowing the knowns.

Phenotypic screening is in a renaissance phase and is expected by many academic and industry leaders to accelerate the discovery of new drugs for new biology. Given that phenotypic screening is per definition target agnostic, the emphasis of in silico and in vitro follow-up work is on the exploration of possible molecular mechanisms and efficacy targets underlying the biological processes interrogated by the phenotypic screening experiments. Herein, we present six exemplar computational protocols for the interpretation of cellular phenotypic screens based on the integration of compound, target, pathway, and disease data established by the IMI Open PHACTS project. The protocols annotate phenotypic hit lists and allow follow-up experiments and mechanistic conclusions. The annotations included are from ChEMBL, ChEBI, GO, WikiPathways and DisGeNET. Also provided are protocols which select from the IUPHAR/BPS Guide to PHARMACOLOGY interaction file selective compounds to probe potential targets and a correlation robot which systematically aims to identify an overlap of active compounds in both the phenotypic as well as any kinase assay. The protocols are applied to a phenotypic pre-lamin A/C splicing assay selected from the ChEMBL database to illustrate the process. The computational protocols make use of the Open PHACTS API and data and are built within the Pipeline Pilot and KNIME workflow tools.

Transcriptome analysis of peripheral blood mononuclear cells in human subjects following a 36 h fast provides evidence of effects on genes regulating inflammation, apoptosis and energy metabolism.

There is growing interest in the potential health benefits of diets that involve regular periods of fasting. While animal studies have provided compelling evidence that feeding patterns such as alternate-day fasting can increase longevity and reduce incidence of many chronic diseases, the evidence from human studies is much more limited and equivocal. Additionally, although several candidate processes have been proposed to contribute to the health benefits observed in animals, the precise molecular mechanisms responsible remain to be elucidated. The study described here examined the effects of an extended fast on gene transcript profiles in peripheral blood mononuclear cells from ten apparently healthy subjects, comparing transcript profiles after an overnight fast, sampled on four occasions at weekly intervals, with those observed on a single occasion after a further 24 h of fasting. Analysis of the overnight fasted data revealed marked inter-individual differences, some of which were associated with parameters such as gender and subject body mass. For example, a striking positive association between body mass index and the expression of genes regulated by type 1 interferon was observed. Relatively subtle changes were observed following the extended fast. Nonetheless, the pattern of changes was consistent with stimulation of fatty acid oxidation, alterations in cell cycling and apoptosis and decreased expression of key pro-inflammatory genes. Stimulation of fatty acid oxidation is an expected response, most likely in all tissues, to fasting. The other processes highlighted provide indications of potential mechanisms that could contribute to the putative beneficial effects of intermittent fasting in humans.

From nanotechnology to nanomedicine: applications to cancer research.

Scientific advances have significantly improved the practice of medicine by providing objective and quantitative means for exploring the human body and disease states. These innovative technologies have already profoundly improved disease detection, imaging, treatment and patient follow-up. Today's analytical limits are at the nanoscale level (one-billionth of a meter) enabling a detailed exploration at the level of DNA, RNA, proteins and metabolites which are in fact nano-objects. This translational review aims at integrating some recent advances from micro- and nano-technologies with high potential for improving daily oncology practice.

The role of bioinformatics in pathway curation.

Diagrams and models of biological pathways are useful tools in biology. Pathway diagrams are mainly used for illustrative purposes for instance in textbooks and in presentations. Pathway models are used in the analysis of genomic data. Bridging the gap between diagrams and models allows not only the analysis of genomics data and interactions but also the visualisation of the results in a variety of different ways. The knowledge needed for pathway creation and curation is available from three distinct sources: databases, literature and experts. We describe the role of bioinformatics in facilitating the creation and curation of pathway.

Validating nutrient-related gene expression changes from microarrays using RT(2) PCR-arrays.

Microarray technology allows us to perform high-throughput screening of changes in gene expression. The outcome of microarray experiments largely depends on the applied analysis methods and cut-off values chosen. Results are often required to be verified using a more sensitive detection technique, such as quantitative real-time PCR (qPCR or RT-PCR). Throughout the years, this technique has become a de facto golden standard. Individual qPCRs are time-consuming, but the technology to perform high-throughput qPCR reactions has become available through PCR-arrays that allow up to 384 PCR reactions simultaneously. Our current aim was to investigate the usability of a RT(2) Profilertrade mark PCR-array as validation in a nutritional intervention study, where the measured changes in gene expression were low. For some differentially expressed genes, the PCR-array confirmed the microarray prediction, though not for all. Furthermore, the PCR-array allowed picking up the expression of genes that were not measurable on the microarray platform but also vice versa. We conclude that both techniques have their own (dis)advantages and specificities, and for less pronounced changes using both technologies may be useful as complementation rather than validation.

Bioinformatic interrogation of expression array data to identify nutritionally regulated genes potentially modulated by DNA methylation.

DNA methylation occurs at CpG dinucleotide sites within the genome and is recognised as one of the mechanisms involved in regulation of gene expression. CpG sites are relatively underrepresented in the mammalian genome, but occur densely in regions called CpG islands (CGIs). CGIs located in the promoters of genes inhibit transcription when methylated by impeding transcription factor binding. Due to the malleable nature of DNA methylation, environmental factors are able to influence promoter CGI methylation patterns and thus influence gene expression. Recent studies have provided evidence that nutrition (and other environmental exposures) can cause altered CGI methylation but, with a few exceptions, the genes influenced by these exposures remain largely unknown. Here we describe a novel bioinformatics approach for the analysis of gene expression microarray data designed to identify regulatory sites within promoters of differentially expressed genes that may be influenced by changes in DNA methylation.

Genetic variation in thioredoxin interacting protein (TXNIP) is associated with hypertriglyceridaemia and blood pressure in diabetes mellitus.

AIMS: Thioredoxin interacting protein (TXNIP) is an attractive candidate gene for diabetes or diabetic dyslipidaemia, since TXNIP is the strongest glucose-responsive gene in pancreatic B-cells, TXNIP deficiency in a mouse model is associated with hyperlipidaemia and TXNIP is located in the 1q21-1q23 chromosomal Type 2 diabetes mellitus (DM) locus. We set out to investigate whether metabolic effects of TXNIP that were previously reported in a murine model are also relevant in human Type 2 DM. METHODS: The frequency distribution of a 3' UTR single nucleotide polymorphism (SNP) in TXNIP was investigated in subjects with normal glucose tolerance (NGT; n = 379), impaired glucose tolerance (IGT; n = 228) and Type 2 DM (n = 230). Metabolic data were used to determine the effect of this SNP on parameters associated with lipid and glucose metabolism. RESULTS: The frequency of the TXNIP variation did not differ between groups, but within the group of diabetic subjects, carriers of the TXNIP-T variant had 1.6-fold higher triglyceride concentrations (P = 0.015; n = 136) and a 5.5-mmHg higher diastolic blood pressure (P = 0.02; n = 212) than homozygous carriers of the common C-allele, whereas in non-diabetic subjects fasting glucose was 0.26 mmol/l lower (P = 0.002; n = 478) in carriers of the T-allele. Moreover, a significant interaction between plasma glucose concentrations and TXNIP polymorphism on plasma triglycerides was observed (P = 0.012; n = 544). CONCLUSION: This is the first report to implicate TXNIP in a human disorder of energy metabolism, Type 2 diabetes. The effect of TXNIP on triglycerides is influenced by plasma glucose concentrations, suggesting that the biological relevance of TXNIP variations may be particularly relevant in recurrent episodes of hyperglycaemia.

Gene profiling of cathepsin K deficiency in atherogenesis: profibrotic but lipogenic.

Recently, we showed that cathepsin K deficiency reduces atherosclerotic plaque progression, induces plaque fibrosis, but aggravates macrophage foam cell formation in the ApoE -/- mouse. To obtain more insight into the molecular mechanisms by which cathepsin K disruption evokes the observed phenotypic changes, we used microarray analysis for gene expression profiling of aortic arches of CatK -/-/ApoE -/- and ApoE -/- mice on a mouse oligo microarray. Out of 20 280 reporters, 444 were significantly differentially expressed (p-value of < 0.05, fold change of > or = 1.4 or < or = - 1.4, and intensity value of > 2.5 times background in at least one channel). Ingenuity Pathway Analysis and GenMAPP revealed upregulation of genes involved in lipid uptake, trafficking, and intracellular storage, including caveolin - 1, - 2, - 3 and CD36, and profibrotic genes involved in transforming growth factor beta (TGFbeta) signalling, including TGFbeta2, latent TGFbeta binding protein-1 (LTBP1), and secreted protein, acidic and rich in cysteine (SPARC), in CatK -/-/ApoE -/- mice. Differential gene expression was confirmed at the mRNA and protein levels. In vitro modified low density lipoprotein (LDL) uptake assays, using bone marrow derived macrophages preincubated with caveolae and scavenger receptor inhibitors, confirmed the importance of caveolins and CD36 in increasing modified LDL uptake in the absence of cathepsin K. In conclusion, we suggest that cathepsin K deficiency alters plaque phenotype not only by decreasing proteolytic activity, but also by stimulating TGFbeta signalling. Besides this profibrotic effect, cathepsin K deficiency has a lipogenic effect owing to increased lipid uptake mediated by CD36 and caveolins.

Hypochlorous acid is a potent inhibitor of GST P1-1.

Glutathione S-transferase is a phase II detoxification enzyme that can be inactivated by H(2)O(2). During oxidative stress various other reactive oxygen species are generated that are more reactive than the relatively stable H(2)O(2). Hypochlorous acid (HOCl) is a powerful oxidant which is highly reactive towards a range of biological substrates. We studied the influence of HOCl on the activity of GST P1-1. HOCl inhibits purified glutathione S-transferase P1-1 in a concentration dependent manner with an IC(50)-value of 0.6 microM, which is more than 1000 times as low as IC(50) reported for H(2)O(2). HOCl lowered the V(max) value, but did not affect the K(m) for CDNB. Our results show that HOCl is a potent, non-competitive inhibitor of GST P1-1. The relevance of this effect is discussed.

Inhibition of human glutathione S-transferase P1-1 by tocopherols and alpha-tocopherol derivatives.

alpha-Tocopherol inhibits glutathione S-transferase P1-1 (GST P1-1) (R.I.M. van Haaften, C.T.A. Evelo, G.R.M.M. Haenen, A. Bast, Biochem. Biophys. Res. Commun. 280 (2001)). In various cosmetic and dietary products alpha-tocopherol is added as a tocopherol ester. Therefore we have studied the effect of various tocopherol derivatives on GST P1-1 activity. It was found that GST P1-1 is inhibited, in a concentration dependent manner, by these compounds. Of the compounds tested, the tocopherols were the most potent inhibitors of GST P1-1; the concentration giving 50% inhibition (IC(50)) is <1 microM. The esterified tocopherols and alpha-tocopherol quinone also inhibit the GST P1-1 activity at a very low concentration: for most compounds the IC(50) was below 10 microM. RRR-alpha-Tocopherol acetate lowered the V(max) values, but did not affect the K(m) for either 1-chloro-2,4-dinitrobenzene or GSH. This indicates that the GST P1-1 enzyme is non-competitively inhibited by RRR-alpha-tocopherol acetate. The potential implications of GST P1-1 inhibition by tocopherol and alpha-tocopherol derivatives are discussed.

No reduction of alpha-tocopherol quinone by glutathione in rat liver microsomes.

The cell membrane is protected against lipid peroxidation by endogenous antioxidants such as vitamin E (alpha-tocopherol). The oxidised form of alpha-tocopherol (alpha-tocopherol quinone) does not have this antioxidant function. However, the literature indicates that alpha-tocopherol quinone can be reduced to alpha-tocopherol in vivo and thereby will add to the total antioxidant potential (Moore AN, Ingold KU. Free Radic Biol Med 1997;22:931-4). We found that GSH (reduced glutathione) did not mediate the reduction of alpha-tocopherol quinone, either directly in solution or in rat liver microsomes fortified with alpha-tocopherol quinone. This renders GSH a less likely candidate for alpha-tocopherol quinone reduction in vivo. In addition, alpha-tocopherol quinone did not enhance GSH-dependent protection against lipid peroxidation, either in control microsomes, or in vitamin E-extracted microsomes. Indeed, alpha-tocopherol quinone blocked GSH-dependent protection against lipid peroxidation in vitamin E-extracted microsomes. This indicates that alpha-tocopherol quinone can act as a pro-oxidant.

alpha-Tocopherol inhibits human glutathione S-transferase pi.

alpha-Tocopherol is the most important fat-soluble, chain-breaking antioxidant. It is known that interplay between different protective mechanisms occurs. GSTs can catalyze glutathione conjugation with various electrophiles, many of which are toxic. We studied the influence of alpha-tocopherol on the activity of the cytosolic pi isoform of GST. alpha-Tocopherol inhibits glutathione S-transferase pi in a concentration-dependent manner, with an IC(50)-value of 0.5 microM. At alpha-tocopherol additions above 3 microM there was no GST pi activity left. alpha-Tocopherol lowered the V(max) values, but did not affect the K(m) for either CDNB or GSH. This indicates that the GST pi enzyme is noncompetitively inhibited by alpha-tocopherol. An inhibition of GST pi by alpha-tocopherol may have far-reaching implications for the application of vitamin E.

A study on the interaction between hydroxylamine analogues and oxyhemoglobin in intact erythrocytes.

The oxidative potency of hydroxylamine (HYAM) and its O-derivatives (O-methyl- and O-ethyl hydroxylamine) is generally larger than the effects of the N-derivatives (N-methyl-, N-dimethyl-, and N,O-dimethyl hydroxylamine). The effects of the two groups of hydroxylamines also differ in a qualitative sense. To elucidate this difference in toxicity profiles we investigated the hemoglobin dependence of the toxicity, the occurrence of cell-damaging products like superoxide and H(2)O(2), and the cellular kinetics of the hydroxylamine analogues. All hydroxylamines were found to depend on the presence and accessibility of oxyhemoglobin to exert their toxicity. This did not provide an explanation for the different toxicity profiles. The interaction of some hydroxylamines with oxyhemoglobin is known to lead to the formation of radical intermediates. Differences in the stability of these radical products are known to occur, and in some cases secondary products are formed. This can contribute to the differences in toxicity. In this respect, production of superoxide radicals was demonstrated for all hydroxylamines in the reaction with oxyhemoglobin. Evidence for H(2)O(2) generation during the reaction of HYAM, O-methyl, O-ethyl-, and N-dimethyl hydroxylamine with oxyhemoglobin was also found. Next to variations in the products formed, differences in cellular kinetics are likely to be among the most important factors that explain the different toxicity patterns seen for the hydroxylamines in erythrocytes. Indeed, differences were found to exist for the kinetics of methemoglobin formation in erythrocytes. Not only was the final level of methemoglobin formed much lower for the N-derivatives, but also the reaction rate with oxyhemoglobin was slower than with HYAM and its O-derivatives. Except for N,O-dimethyl hydroxylamine (NODMH), the same pattern was seen in hemolysates. NODMH tripled its effect on hemoglobin in hemolysate compared with incubations in erythrocytes. This implies that cellular uptake is a limiting factor for NODMH. Since formation of H(2)O(2) is most likely a result of an interaction with hemoglobin, differences in kinetics of methemoglobin formation can be an explanation for the fact that NMH and NODMH did not produce H(2)O(2) to a detectable level. These results indicate that (a) the toxicity of all hydroxylamines depends on an interaction with oxyhemoglobin; (b) the interaction with hemoglobin produces radical intermediates and concomitantly superoxide radicals and H(2)O(2); and (c) differences in uptake, reaction rate with hemoglobin, and stability of the intermediates formed do exist for the different hydroxylamines and contribute to their differences in toxicity.

Erythrocyte glutathione S transferase as a marker of oxidative stress at birth.

AIMS: To determine the level of oxidative stress and cell damage as a result of exposure to O(2) at birth. METHODS: Using glutathione S transferase (GST) as an indicator of oxidative stress, GST activity in cord blood was compared with that in samples taken three hours after birth. Twenty four prematurely born infants and eight full term infants were studied. To test whether stronger effects occur under less favourable conditions, the neonates were divided in three groups: healthy premature; sick premature; and healthy full term infants. RESULTS: GST activity three hours after birth was significantly decreased compared with that at birth in all three groups tested. There were no significant differences in the magnitude of this effect among the three groups. CONCLUSIONS: These results indicate that a sudden increase in oxygenation exposes the neonate to oxidative stress. Measurement of GST activity might be useful for the evaluation of protective treatment in trials considering antioxidant strategies.

Changes in levels of catalase and glutathione in erythrocytes of patients with stable asthma, treated with beclomethasone dipropionate.

In asthmatic patients, antioxidant defence is decreased. Although inhaled corticosteroids decrease asthmatic inflammation and modulate reactive oxygen species (ROS) generation, little is known of their effect on cellular antioxidant levels. The aim of this study was to evaluate the effect of inhaled beclomethasone dipropionate (BDP; 1,000 microg x day(-1)) on erythrocyte antioxidant levels in stable asthmatic patients. Forty patients with stable, mild asthma were treated in a double-blind, placebo-controlled, parallel-group study with BDP 250 microg, two puffs b.i.d. for 6 weeks. At entry and every 2 weeks during treatment, erythrocyte antioxidant levels, haematological parameters, pulmonary function tests and asthma symptoms were determined. The results show that during treatment with BDP, erythrocyte catalase levels increased (at entry (mean +/-SEM) 41+/-4, after 6 weeks 54+/-4 micromol H2O2 x min(-1) x g haemoglobin (Hb)(-1), p = 0.05 in comparison with placebo). Erythrocyte total glutathione levels significantly decreased after 6 weeks treatment with BDP (from 7.0+/-0.4 to 6.6+/-0.3 micromol x g Hb(-1) (p = 0.04)). In the BDP-treated patients, blood eosinophil counts were higher in patients who responded with an increase in erythrocyte catalase levels during BDP treatment, as compared to those not responding ((mean +/-SEM) 340+/-39 and 153+/-52x10(6) cells x L(-1), respectively, p = 0.05). The present study shows that treatment with inhaled bedomethasone dipropionate results in changes in antioxidant levels in erythrocytes of patients with stable, mild asthma.

Only the glutathione dependent antioxidant enzymes are inhibited by haematotoxic hydroxylamines.

Hydroxylamine and some of its derivatives are known to cause oxidative effects both in vitro and in vivo. In the current study we investigated the effects of hydroxylamines on the enzymatic antioxidant defense system in human erythrocytes. The activity of catalase and superoxide dismutase was not significantly influenced by any of the hydroxylamines tested. However, the activity of glutathione peroxidase (GPX) and glutathione S-transferase (GST) was strongly inhibited by hydroxylamine and its O-derivatives (O-methyl and O-ethyl hydroxylamine). GPX was also inhibited by two N-derivatives of hydroxylamine (i.e. N-dimethyl and N,O-dimethyl hydroxylamine). This indicates that exposure to hydroxylamines not only changes the cellular oxidation-reduction status but also leads to inhibition of the glutathione dependent antioxidant enzymes. GST as well as GPX have cysteine residues at the active site of the enzymes. Such an accessible thiol group is generally susceptible to formation of protein-mixed disulphides or intramolecular disulphides. If these thiol groups are essential for activity this would be accompanied by an increase or decrease in the enzyme activity. In principle this is also true for glutathione reductase (GR), which in this study was only inhibited by N,O-dimethyl and N-methyl hydroxylamines. However, GR is capable to reduce these disulphides by taking up two electrons, either from its substrate NAPDH or from another reductant. Oxidation of these thiol groups in GR would thus not lead to impairment of GR activity. The fact that NODMH and NMH do decrease the GR activity can therefore only be explained by other modifications. The activity loss of GST and GPX on the other hand, is likely to involve oxidation of critical cysteine residues. The practical consequence of these findings is that the cellular prooxidant state that may arise in erythrocytes exposed to hydroxylamines can be further increased by activity loss of protective enzymes, which may decrease the average life span of the red blood cell.

Two mechanisms for toxic effects of hydroxylamines in human erythrocytes: involvement of free radicals and risk of potentiation.

The toxic potency of three industrially used hydroxylamines was studied in human blood cells in vitro. The parent compound hydroxylamine and the O-ethyl derivative gave very similar results. Both compounds induced a high degree of methemoglobin formation and glutathione depletion. Cytotoxicity was visible as Heinz body formation and hemolysis. High levels of lipid peroxidation occurred, in this respect O-ethyl hydroxylamine was more active than hydroxylamine. In contrast H2O2 induced lipid peroxidation was lowered after O-ethyl hydroxylamine or hydroxylamine treatment, this is explained by the ferrohemoglobin dependence of H2O2 induced radical species formation. Glutathione S-transferase (GST) and NADPH methemoglobin reductase (NADPH-HbR) activities were also impaired, probably as a result of the radical stress occurring. The riboflavin availability was decreased. Other enzyme activities glutathione reductase (GR), glucose 6-phosphate dehydrogenase (G6PDH), glucose phosphate isomerase and NADH methemoglobin reductase, were not or only slightly impaired by hydroxylamine or O-ethyl hydroxylamine treatment. A different scheme of reactivity was found for N,O-dimethyl hydroxylamine. This compound gave much less methemoglobin formation and no hemolysis or Heinz body formation at concentrations up to and including 7 mM. Lipid peroxidase induction was not detectable, but could be induced by subsequent H2O2 treatment. GST and NADPH-HbR activities and riboflavin availability were not decreased. On the other hand GR and G6PDH activities were inhibited. These results combined with literature data indicate the existence of two different routes of hematotoxicity induced by hydroxylamines. Hydroxylamine as well as O-alkylated derivatives primarily induce methemoglobin, a process involving radical formation. The radical stress occurring is probably responsible for most other effects. N-alkylated species like N,O-dimethyl hydroxylamine primarily lead to inhibition of the protective enzymes G6PDH and GR. Since these enzymes play a key role in the protection of erythrocytes against oxidative stress a risk of potentiation during mixed exposure does exist.

Oxidative effects in human erythrocytes caused by some oximes and hydroxylamine.

Both oximes and hydroxylamine (HYAM) are compounds with known oxidative capacity. We tested in vitro whether acetaldoxime (AAO), cyclohexanone oxime (CHO), methyl ethyl ketoxime (MEKO) or HYAM affect haemoglobin oxidation (into HbFe3+), formation of thiobarbituric acid reactive substances (TBARS), and glutathione (GT) depletion in human haemolysate, erythrocytes or blood. All these parameters are known to be related to oxidative stress. Glutathione S-transferase (GST) activity was measured as it may be affected by oxygen radicals. All three oximes caused a low degree of HbFe3+ accumulation in erythrocytes. This was higher in haemolysates indicating that membrane transport may be limiting or that protective mechanisms within erythrocytes are more effective. HbFe3+ accumulation was lower for the oximes than for HYAM. AAO and HYAM caused TBARS formation in blood. For HYAM this was expected as free radicals are known to be generated during HbFe3+ formation. Free radical generation by AAO and HYAM in erythrocytes was confirmed by the inhibition of GST. For the other two oximes (CHO and MEKO) some special effects were found. CHO did inhibit erythrocyte GST while it did not cause TBARS formation. MEKO was the least potent oxime as it caused no TBARS formation, little HbFe3+ accumulation and little GST inhibition in erythrocytes. However, GT depletion was more pronounced for MEKO than for the other oximes, indicating that glutathione conjugation occurs. TBARS formation, GT depletion and GST modulation caused by the oximes and HYAM were also tested in rat hepatocytes. However, no effects were found in hepatocytes. This suggests that a factor present in erythrocytes is necessary for free radical formation. Studies with proposed metabolites of the oximes (i.e. cyclohexanone, acetaldehyde or methylethyl ketone) and addition of rat liver preparations to the erythrocyte incubations with oximes, suggest that metabolism is not a limiting factor in erythrocyte toxicity.

In vitro haematotoxic effects of three methylated hydroxylamines.

Hydroxylamine (HYAM, HONH2) and some of its derivatives are known to cause erythrotoxic effects both in vitro and in vivo. Previous studies have shown that the primary in vitro effect of HYAM and O-ethyl hydroxylamine (OEH) is methaemoglobin formation, leading to liberation of free radicals which cause lipid peroxidation, enzyme inhibitions and glutathione depletion. By contrast, N-substituted N,O-dimethyl hydroxylamine (NODMH), primarily induces impairment of glucose 6-phosphate dehydrogenase (G6PDH) and glutathione reductase (GR). The oxidative potency of HYAM and the O-derivative was larger than the potency of the N,O-derivative. This seemed to indicate that attachment of an alkyl group to the nitrogen atom of hydroxylamine leads to decreased reactivity. To achieve a better understanding of the structure activity relationship for hydroxylamines three methylated derivatives were tested: N-methyl hydroxylamine (NMH). N-dimethyl hydroxylamine (NDMH) and O-methyl hydroxylamine (OMH). We were also interested in the erythrotoxic potency of OMH which recently entered industrial production. Methaemoglobin formation, high release of lipid peroxidation products, inhibition of NADPH methaemoglobin reductase and glutathione S-transferase (GST) and depletion of total glutathione (GT) were seen for OMH. The reducing enzymes G6PDH and GR were not impaired by OMH. These findings for OMH are consistent with the proposed mechanism for O-derivatives. Since both the effects caused by OMH and its potency are comparable to those of HYAM and OEH this indicates that possible occupational exposure to this compound may be approached similarly to HYAM and OEH. NMH only inhibited G6PDH and GR activity, which is fully in accord with the proposed mechanism for N-substituted derivatives of HYAM. However, NDMH a double N-substituted compound, caused a strikingly different scheme of reactivity inhibition of G6PDH but not of GR, severe methaemoglobin formation, only little lipid peroxidation and some impairment of NADPH methaemoglobin reductase. This study confirms that O-derivatives of HYAM are potent haemoglobin oxidators, leading to other oxidative effects. The main effect was confirmed for single N-derivatives as inhibition of the two protective enzymes G6PDH and GR. However, the results for NDMH indicate that this simple classification of O-derivatives and N-derivatives has to be extended for double N-substituted compounds which give a mixture of effects.

Hydroxylamine treatment increases glutathione-protein and protein-protein binding in human erythrocytes.

Hydroxylamine is a direct-acting hematotoxic agent leading to hemolytic anemia in animals and man. The effect of hydroxylamine on the morphology, sulfhydryl status and membrane skeletal proteins of human erythrocytes were studied. Loss of reduced glutathione (GSH) from the red blood cells was directly proportional to the hydroxylamine concentration used. This loss of GSH was larger than the sum of the increase in the amounts of extracellular glutathione and intracellular oxidized glutathione (GSSG). The extracellular glutathione is mainly present as GSSG, which is in agreement with the fact that only GSSG is exported from the erythrocytes by membrane bound ATPases. Lack of GSSG export was not limited by decreased ATP levels in the erythrocytes and we concluded that the GSH that disappeared did not become available as intracellular GSSG. After reduction of the erythrocyte incubates the lost GSH was almost completely recovered indicating that the lost GSH is present in the cell as protein-glutathione mixed disulfides. Glutathione thus stored within the cell can be quickly recovered by combined thioltransferase and glutathione reductase activity when conditions become more favorable again. SDS-polyacrylamide gel electrophoresis of membrane ghosts from human red cells revealed changes in skeletal proteins with a smearing of bands 1, 2 and 3 to the higher molecular weight end of the gel and the appearance of new monomeric and dimeric hemoglobin bands at about 16 and 30 kD. The observed alterations are probably a consequence of disulfide bridge formation between cellular proteins (mainly hemoglobin) and skeletal proteins as well as between hemoglobin monomers. Exposure of hydroxylamine to erythrocytes caused severe Heinz body formation but the outside morphology of the cells was only marginally altered. The described changes in sulfhydryl status of the red blood cells are likely to play a major role in the premature splenic sequestration of hydroxylamine-damaged erythrocytes.