Metabolomics: a tool for early detection of toxicological effects and an opportunity for biology based grouping of chemicals-from QSAR to QBAR.

BASF has developed a Metabolomics database (MetaMap(®) Tox) containing approximately 500 data rich chemicals, agrochemicals and drugs. This metabolome-database has been built based upon 28-day studies in rats (adapted to OECD 407 guideline) with blood sampling and metabolic profiling after 7, 14 and 28 days of test substance treatment. Numerous metabolome patterns have been established for different toxicological targets (liver, kidney, thyroid, testes, blood, nervous system and endocrine system) which are specific for different toxicological modes of action. With these patterns early detection of toxicological effects and the underlying mechanism can now be obtained from routine studies. Early recognition of toxicological mode of action will help to develop new compounds with a more favourable toxicological profile and will also help to reduce the number of animal studies necessary to do so. Thus this technology contributes to animal welfare by means of reduction through refinement (2R), but also has potential as a replacement method by analyzing samples from in vitro studies. With respect to the REACH legislation for which a large number of animal studies will need to be performed, one of the most promising methods to reduce the number of animal experiments is grouping of chemicals and read-across to those which are data rich. So far mostly chemical similarity or QSAR models are driving the selection process of chemical grouping. However, "omics" technologies such as metabolomics may help to optimize the chemical grouping process by providing biologically based criteria for toxicological equivalence. "From QSAR to QBAR" (quantitative biological activity relationship).

The use of metabolomics for the discovery of new biomarkers of effect.

Will metabolomics have a greater chance of success in toxicology and biomarker assessment than genomics and proteomics? Metabolomics has the advantage that (1) it analyses the last step in a series of changes following a toxic insult, (2) many of the metabolites have a known function and (3) changes are detectable in blood. If the analysis of a great number of individual organs can be replaced by one matrix then this will provide significant advantages (less invasive method, no need to kill animals, time course analysis possible). We have chosen to perform the analysis of blood metabolites in such a way as to minimize the risk of artifacts and to have a high number of known metabolites. We have also reduced the amount of variation in the biological system as well as during analysis. In a series of proof of concept studies it could be demonstrated that (1) the metabolome of control animals was stable of a period of nearly 1 year, with a remarkable differentiation between males and females, (2) a dose response relationship in metabolome changes was induced by phenobarbital and that (3) different modes of action could be distinguished by blood metabolome analysis. To investigate the potential of metabolomics to find biomarkers or specific patterns of change we have analyzed the blood metabolome of rats treated with HPPD inhibitors, a novel class of herbicides. The results demonstrated that a single metabolite, tyrosine, can be used as a biomarker. In addition to tyrosine we also found a specific pattern of change that involved nine metabolites. Though the extent of change was less than for tyrosine the consistent change of these metabolites is diagnostic for this (toxicological) mode of action.

Extracellular matrix structure after heart transplantation.

Following heart transplantation remodeling of the donor heart causes changes in the extracellular myocardial matrix. We investigated 20 right ventricular endomyocardial biopsies taken 17+/-4 days (group I, n=9) and 63+/-13 days (group II, n=11) after heart transplantation from 16 patients transplanted for end-stage cardiomyopathy (15 dilated/1 ischemic). Immunohistochemical staining for collagen I, collagen III, collagen IV, and fibronectin was used. Evaluation was performed at a magnification of 400x using a computer-assisted image analyzing system measuring the relative area stained by the immunoperoxidase method, the number of cells in the given area, and the total area. Collagen I per cell was 13.9+/-5.9 microm2 in group I and increased significantly 66+/-13 days after heart transplantation in the perimysium around the myocardial cells as well as in the endocardium to 49.9+/-15.1 microm2 (P<0.05). No quantitative change in collagen III was noted (75.7+/-12.4 versus 75.5+/-16.0 microm2 n.s.). Collagen IV was found in the perimysial, in the capillary bed and in the vascular network. Significant quantitative change in the amount of collagen IV was not found (64.1+/-12.6 versus 61.0+/-8.9 microm2). Fibronectin was found in the entire perimysial extracellular matrix and in the endocardium in relationship with collagen I and III. An increased amount of fibronectin from 87.09+/-9.9 microm2 (group I) to 140.8+/-17.9 microm2 (group II, P<0.05) was found. The cell area and cell diameters were not significantly different (group I; cell area 772+/-227 microm2, diameter 31.3 microm; group II; cell area 776+/-224 microm2, diameter 31.4 microm). It is concluded that remodeling of the donor heart after transplantation is characterized by a specific increase in collagen I and fibronectin, whereas a change in other collagen subtypes was not observed.

Informatics united: exemplary studies combining medical informatics, neuroinformatics and bioinformatics.

OBJECTIVES: Medical informatics, neuroinformatics and bioinformatics provide a wide spectrum of research. Here, we show the great potential of synergies between these research areas on the basis of four exemplary studies where techniques are transferred from one of the disciplines to the other. METHODS: Reviewing and analyzing exemplary and specific projects at the intersection of medical informatics, neuroinformatics, and bioinformatics from our experience in an interdisciplinary research group. RESULTS: Synergy emerges when techniques and solutions from medical informatics, bioinformatics, or neuroinformatics are successfully applied in one of the other disciplines. Synergy was found in 1. the modeling of neurophysiological systems for medical therapy development, 2. the use of image processing techniques from medical computer vision for the analysis of the dynamics of cell nuclei, and 3. the application of neuroinformatics tools for data mining in bioinformatics and as classifiers in clinical oncology. CONCLUSIONS: Each of the three different disciplines have delivered technologies that are readily applicable in the other disciplines. The mutual transfer of knowledge and techniques proved to increase efficiency and accuracy in a manifold of applications. In particular, we expect that clinical decision support systems based on techniques derived from neuro- and bioinformatics have the potential to improve medical diagnostics and will finally lead to a personalized delivery of healthcare.

[When lipids increase in dialysis: the role of heparin! Standard heparin increases, low-molecular-weight heparin lowers triglycerides]. / Wenn bei der Dialyse die Lipide steigen: Auf's Heparin kommt's an! Standardheparin erhöht, niedermolekulares Heparin senkt Triglyzeride.

Dyslipoproteinemia in patients on hemodialysis is characterized by a decrease in high density lipoprotein (HDL), cholesterol, hypertriglyceridemia, increased triglyceride-rich lipoproteins, such as very low density lipoprotein (VLDL) and intermediate density lipoprotein (IDL), a higher proportion of the small dense low density lipoprotein (small dense LDL) subfraction, and higher lipoprotein(a) concentration. The reason for the changes in triglyceride metabolism is an increase in the production of apolipoprotein B, and a decrease in the metabolism of VLDL as a consequence of decreased endothelial cell delipidation. The endothelial lipoprotein lipase, which plays a major role in this process, is released by heparin, which is essential for the function of the enzyme. Repeated administration of heparin for anticoagulation during hemodialysis apparently leads to an LPL depletion in the endothelium. This results in further exhaustion of lipolysis. Clinical studies in hemodialysis patients with high triglyceride and cholesterol levels indicate that a change from standard heparin to low-molecular-weight heparin improves the lipid profile by lowering triglycerides and cholesterol.

Reproducibility and robustness of metabolome analysis in rat plasma of 28-day repeated dose toxicity studies.

BASF has developed a rat plasma metabolomics database (MetaMap®Tox) containing the metabolome of more than 500 chemicals, agrochemicals and drugs, for which the toxicity is well known, derived from 28-day repeated dose toxicity studies in rats. The quality/reproducibility of data was assessed by comparing the metabolome of 16 reference compounds tested at least twice under identical experimental conditions at three time points (day 7, day 14 and day 28). Statistical correlation analysis showed that the repeated treatment induced very similar changes to the metabolome. For all repetitions the modes of action of the compounds were always correctly identified. Moreover, when compared against the metabolome of all compounds available in the MetaMap®Tox database, the repetitions showed in most cases the highest degree of overall similarity with the metabolome of the original study. In addition, we also evaluated the robustness of our metabolomics technique, displayed by constancy of variability in control groups over time. Based on these results, it can be concluded, that metabolomics can reproducibly be applied during toxicological in vivo testing in rats under the conditions applied here.

Increased toxicity when fibrates and statins are administered in combination--a metabolomics approach with rats.

Combination therapies with fibrates and statins are used to treat cardiovascular diseases, because of their synergistic effect on lowering plasma lipids. However, fatal side-effects like rhabdomyolysis followed by acute renal necrosis sometimes occur. To elucidate biochemical changes resulting from the interaction of fibrates and statins, doses of 100 mg/kg fenofibrate, 50mg/kg clofibrate, 70 mg/kg atorvastatin and 200 mg/kg pravastatin as well as combinations thereof were administered to Crl:Wi(Han) rats for 4 weeks. Plasma metabolome profile was measured on study days 7, 14 and 28. Upon study termination, clinical pathology parameters were measured. In a separate experiment plasmakinetic data were measured in male rats after 1 week of drug administration in monotherapy as well as in combinations. Lowering of blood lipid levels as well as toxicological effects, like liver cell degradation (statins) and anemia (fibrates) and distinct blood metabolite level alterations were observed in monotherapy. When fibrates and statins were co-administered metabolite profile interactions were generally underadditive or at the utmost additive according to the linear mixed effect model. However, more metabolite levels were significantly altered during combination therapy. New effects on the antioxidant status and the cardiovascular system were found which may be related to a development of rhabdomyolysis. Accumulation of drugs during the combination therapy was not observed.

Application of in vivo metabolomics to preclinical/toxicological studies: case study on phenytoin-induced systemic toxicity.

BASF and Metanomics have built-up the database MetaMap(®)-Tox containing rat plasma metabolome data for more than 500 reference compounds. Phenytoin was administered to five Wistar rats of both sexes at dietary dose levels of 600 and 2400 ppm over 28 days and metabolome analysis was performed on days 7, 14 and 28. Clinical pathology did not indicate clear evidence for liver toxicity, whereas liver histopathology revealed slight centrilobular hepatocellular hypertrophy. The metabolome analysis of phenytoin shows metabolome changes at both dose levels and the comparison with MetaMap-Tox indicated strong evidence for liver enzyme induction, as well as liver toxicity. Moreover, evidence for kidney and indirect thyroid effects were observed. This assessment was based on the metabolite changes induced, similarities to specific toxicity patterns and the whole metabolome correlation within MetaMap-Tox. As compared with the classical read-out, a more comprehensive picture of phenytoin's effects is obtained from the metabolome analysis, demonstrating the added value of metabolome data in preclinical/ toxicological studies.

Nutritional impact on the plasma metabolome of rats.

Metabolite profiling (metabolomics) elucidates changes in biochemical pathways under various conditions, e.g., different nutrition scenarios or compound administration. BASF and metanomics have obtained plasma metabolic profiles of approximately 500 compounds (agrochemicals, chemicals and pharmaceuticals) from 28-day rat studies. With these profiles the establishment of a database (MetaMap(®)Tox) containing specific metabolic patterns associated with many toxicological modes of action was achieved. To evaluate confounding factors influencing metabolome patterns, the effect of fasting vs. non-fasting prior to blood sampling, the influence of high caloric diet and caloric restriction as well as the administration of corn oil and olive oil was studied for its influence on the metabolome. All mentioned treatments had distinct effects: triacylglycerol, phospholipids and their degradation product levels (fatty acids, glycerol, lysophosphatidylcholine) were often altered depending on the nutritional status. Also some amino acid and related compounds were changed. Some metabolites derived from food (e.g. alpha-tocopherol, ascorbic acid, beta-sitosterol, campesterol) were biomarkers related to food consumption, whereas others indicated a changed energy metabolism (e.g. hydroxybutyrate, pyruvate). Strikingly, there was a profound difference in the metabolite responses to diet restriction in male and female rats. Consequently, when evaluating the metabolic profile of a compound, the effect of nutritional status should be taken into account.

The individual and combined metabolite profiles (metabolomics) of dibutylphthalate and di(2-ethylhexyl)phthalate following a 28-day dietary exposure in rats.

Metabolite profiles (metabolomics) of plasma samples of Wistar rats dosed with di(2-ethylhexyl)phthalate (DEHP - 3000ppm) and dibutylphthalate (DBP - 150, 1000 and 7000ppm) were individually determined in 28 days dietary studies. In addition, profiles of combined exposure to 3000ppm DEHP and either 150, 1000 or 7000ppm DBP were determined. High dose levels induced more profound metabolite changes in males than in females for both compounds. At 150ppm DBP (NOEL for toxicity) there were very few (

Influence of strain and sex on the metabolic profile of rats in repeated dose toxicological studies.

The impact of the strain on the metabolite profile of plasma samples in rats dosed with 2500 ppm 2-methyl-4-chlorophenoxyacetic acid (MCPA acid) or 45 mg/kg bw/day 4-chloro-3-nitroaniline (4C3N) for 4 weeks was evaluated. Four different strains were used: two Wistar strains (Crl:WI(Han), Han:RCC:WIST(SPF)), one Sprague-Dawley (Crl:CD) and one Fisher strain (F-344/Crl). The metabolite profiles in the plasma were measured by LC-MS and GC-MS. The profound changes of the metabolite values induced by the MCPA acid treatment outweighed slight deviations caused by physiological variations between the different rat strains. The metabolome changes of the MCPA acid in all strains could be related to toxicological "mode of action" patterns (peroxisome proliferator, renal organic anionic transporter inhibition) with Crl:WI(Han) rats as reference strain. 4C3N administration led to extravascular hemolytic anemia with a small number of metabolome changes, which were strain dependent. The metabolome pattern associated with "hemolytic anemia" established with the reference strain (Crl:Wi(Han)) was not sufficiently similar in other strains. Thus, comparable metabolome profiles were obtained in different rat strains for a compound inducing profound metabolite changes. For a compound with a weak profile the results were more variable and appeared to be strain dependent.

The influence of a new cardiotonic drug, acrihellin, on sinus node and the current underlying pacemaker activity of cardiac Purkinje fibres.

The spontaneous beating frequency of guinea pig sino-atrial preparations was observed under the influence of 3 beta, 5, 14-trihydroxy-19-oxo-5 beta-bufa-20,22-dienolide-3-(3-methylcrotonate (D 12316, acrihellin), a new cardiotonic drug. There was a slight acceleration of spontaneous frequency after administering rather high doses (10(-6) mol/l) of the drug. Voltage clamp experiments were performed in sheep cardiac Purkinje fibres. AcriheLlin strongly affected the current underlying pacemaker activity, ik2. The kinetic parameter s infinity of this current system was shifted in the hyperpolarizing direction by 4-13 mV. The amplitude of the pacemaker current in Purkinje fibres was reduced by up to 25% in voltage clamp experiments, but the reversal potential and the inward going rectification were left unchanged. After exposure to adrenaline (epinephrine) the well-known depolarizing shift of s infinity was partially restored by acrihellin. This can be interpreted as an antiadrenergic effect of the drug similar to that of beta-adrenoceptor antagonists. However, beta-blockers do not affect the pacemaker kinetics of fibres untreated with adrenaline as does acrihellin. This suggests that the interplay of acrihellin and adrenaline is not that of an agonist and antagonist at a membrane receptor but rather the overlapping of two separate membrane effects.

Effects of acrihellin on cardiac contractility in comparison to various inotropic interventions.

The inotropic effects of the new cardiotonic drug 3 beta,5,14-trihydroxy-19-oxo-5 beta-bufa-20,22-dienolide-3-(3-methylcrotonate) (D 12316, acrihellin) were investigated using twitch contractions time to peak and twitch lengths did not change. Peak tension increased by 140% on an average in 12 experiments at 10(-6) mol/l (acrihellin). In the dose-response curve half maximal positive inotropism occurred at approx. 10(-7) mol/l. Experiments on length tension relationships of resting and twitch tension confirmed that resting tension does not change at any stretch length after administering acrihellin. The position Lmax of maximal twitch contraction force also was not changed but in normalized length-tension cones the positive slope region showed an increase relative to maximal tension which is a feature of positive inotropic interventions. Measurements of contractility in various external calcium solutions between 0.45 mmol/l and 7.2 mmol/l gave evidence that the response of contraction force of fibres exposed to acrihellin to variation of external calcium differs from untreated fibres which respond more strongly. This behaviour of the cardiosteroid acrihellin was very similar to that of adrenaline (epinephrine) whereas digoxin and ouabain did not affect the response of twitch force to changes in external calcium.

Voltage clamp study of the action of the new cardiotonic compound acrihellin on fast and slow inward currents in cardiac Purkinje fibres.

The action of 3 beta,5,14-trihydroxy-19-oxo-5 beta-bufa-20,22-dienolide-3-(3-methylcrotonate) (D 12316, acrihellin) on the fast and slow inward currents of sheep cardiac Purkinje fibres was analyzed by means of a conventional double microelectrode voltage clamp technique. Acrihellin diminished action potential duration and the plateau phase. Effective refractory periods were shortened in relation to the decrease in action potential duration. The upstroke velocity was slightly decreased; a systematic study of dV/dtmax, however, did not reveal any specific local anaesthetic side-effect of the drug. The decrease in the plateau phase of the action potential was due to a reduction in time-dependent slow inward current. The kinetics and the reversal potential of the slow inward current was not affected by acrihellin, but only the current voltage relationship, which was decreased.

Excitation-contraction coupling in voltage-clamped cardiac myocytes.

Myocytes isolated from guinea pig ventricles were voltage-clamped using patch pipettes in the whole-cell configuration. For proper voltage control fast Na+ current was blocked by TTX or inactivated by an appropriate prepulse. Zero-load cell shortening was monitored by a photoelectric device. The mechanical response to a short depolarizing clamp was mainly a phasic (transient) contraction. Long-lasting depolarizations caused a tonic (sustained) shortening of a cell. Different clamp patterns were used to study the mode of activation of phasic contraction. 1) With a constant Ca2+ preload established by a train of conditioning pulses, the shortening-voltage relation measured with test pulses of varying height was a bell-shaped curve reflecting the slow inward current (ICa)-voltage relation. The test pulse had a striking influence on the first contraction of the following conditioning series, resulting in an S-shaped relation between post-test contraction and test potential. 2) With series of identical clamps of varying height, steady-state contraction was maximal around 40 mV and not in proportion to ICa. In these measurements Ca2+ preload was likely to increase with increasing potential. It is concluded that ICa initiates phasic contraction by inducing a release of Ca2+ from internal stores while replenishment of the stores is largely determined by an electrogenic transsarcolemmal Na+-Ca2+ exchange. The data suggest that Na+-Ca2+ exchange is not only involved in long-term changes of cardiac contractility but also in beat-to-beat regulation.

Photoelectric recording of mechanical responses of cardiac myocytes.

A method to monitor contraction of isolated myocytes by transmicroscopic photometry is illustrated. Two photodiodes are mounted inside an inverse microscope used for visual control of a cell. Illumination of one diode varies in proportion to changes in cell length. The contraction signal is amplified in a comparator circuit. Spatial resolution of the device is in the order of 1 micron which corresponds to about 5% of cell shortening in the fully activated state of contraction. The method was tested on isolated myocytes from guinea-pig ventricle. Optical records of contraction in response to action potentials or during voltage clamp compare well with the contractile behavior of multicellular preparations.

Haemodialysis monocytopenia: differential sequestration kinetics of CD14+CD16+ and CD14++ blood monocyte subsets.

In peripheral blood the majority of circulating monocytes present a CD14highCD16- (CD14++) phenotype, while a subpopulation shows a CD14lowCD16+ (CD14+CD16+) surface expression. During haemodialysis (HD) using cellulosic membranes transient leukopenia occurs. In contrast, synthetic biocompatible membranes do not induce this effect. We compared the sequestration kinetics for the CD14+CD16+ and CD14++ monocyte subsets during haemodialysis using biocompatible dialysers. Significant monocytopenia, as measured by the leucocyte count, occurred only during the first 30 min. However, remarkable differences were observed between the different monocyte subsets. CD14++ monocyte numbers dropped to 77 +/- 13% of the predialysis level after 15 min, increasing to > or = 93% after 60 min. In contrast, the CD14+CD16+ subset decreased to 33 +/- 15% at 30 min and remained suppressed for the course of dialysis (67 +/- 11% at 240 min). Approximately 6 h after the end of HD the CD14+CD16+ cells returned to basal levels. Interestingly, the CD14+CD16+ monocytes did not show rebound monocytosis while a slight monocytosis of CD14++ monocytes was occasionally observed during HD. A decline in CD11c surface density paralleled the sequestration of CD14+CD16+ monocytes. Basal surface densities of important adhesion receptors differed significantly between the CD14+CD16+ and CD14++ subsets. In conclusion, during HD the CD14+CD16+ subset revealed different sequestration kinetics, with a more pronounced and longer disappearance from the blood circulation, compared with CD14++ monocytes. This sequestration kinetics may be due to a distinct surface expression of major adhesion receptors which facilitate leucocyte-leucocyte, as well as leucocyte-endothelial, interactions.

Biochemical and immunological properties of urinary angiotensinase A and dipeptidylaminopeptidase IV. Their use as markers in patients with renal cell injury.

Dipeptidyl peptidase IV (EC 3.4.14.5) and angiotensinase A (EC 4.4.11.7) were purified to homogeneity from pooled urine concentrate of patients with renal damage, using ultrafiltration, ammonium sulphate precipitation, lectin affinity chromatography, FPLC-ion-exchange(Mono-Q-)chromatography, and FPLC-gel filtration (Superdex). Based on the specific enzyme activity of the starting material, dipeptidyl peptidase IV was enriched 1629 fold, angiotensinase A 1183 fold. The relative molecular masses, Michaelis constants and isoelectric points were determined. Negative staining of the purified enzymes revealed globular proteins (5-7 nm). Antisera raised against dipeptidyl peptidase IV and angiotensinase A reacted specifically with tubular and, in the case of anti-angiotensinase A sera, with tubular and glomerular structures. In addition, urinary membrane vesicles of proximal tubule origin were eluted with the void volume (Superdex-gel filtration), indicating heavy epithelial cell disintegration. Both soluble tissue enzymes (dipeptidyl peptidase IV, angiotensinase A) and vacuolar blebs shed from epithelia contribute to proteinuria, as was shown in patients with glomerulonephritis, interstitial nephritis, diabetic nephropathy and, for angiotensinase A, in patients with essential arterial hypertension.

Self-organizing maps for visual feature representation based on natural binocular stimuli.

We model the stimulus-induced development of the topography of the primary visual cortex. The analysis uses a self-organizing Kohonen model based on high-dimensional coding. It allows us to obtain an arbitrary number of feature maps by defining different operators. Using natural binocular stimuli, we concentrate on discussing the orientation, ocular dominance, and disparity maps. We obtain orientation and ocular dominance maps that agree with essential aspects of biological findings. In contrast to orientation and ocular dominance, not much is known about the cortical representation of disparity. As a result of numerical simulations, we predict substructures of orientation and ocular dominance maps that correspond to disparity maps. In regions of constant orientation, we find a wide range of horizontal disparities to be represented. This points to geometrical relations between orientation, ocular dominance, and disparity maps that might be tested in experiments.

Tl+-ions: effects on the automaticity of sinoatrial tissue and dV/dtmax and iK2 of cardiac Purkinje fibres.

The disrhythmic effects of thallium were investigated in various cardiac tissues to determine the primary site of intoxication with respect to ensuing arrhythmias. In isolated cardiac tissue Lameijer and van Zwieten [1] had contended that arrhythmias arise from the sinus node after thallium poisoning. To test this hypothesis we administered concentrations of Tl+ between 10(-7) and 10(-4) M to guinea pig sinoatrial preparations, to guinea pig papillary muscles and to sheep cardiac Purkinje fibres. In sinoartial preparations thallium provoked increases and decreases of spontaneous beat frequency which were not linked to corresponding changes in contractile force. In conductive tissue, Purkinje fibres, the inactivation kinetics of the fast sodium current and the pacemaker current iK2 were investigated by voltage clamp experiments. Here, thallium was seen to be essentially without toxic effects which could account for arrhythmias. In ventricular muscle actions potentials and contractile force were recorded simultaneously. Here again, ventricular arrhythmias are not to be expected from thallium intoxication in rather high concentrations. The findings support the view that arrhythmogenic effects of thallium are restricted to the sinus node.