V. Strauss

Short term inhalation toxicity of a liquid aerosol of CdS/Cd(OH)2 core shell quantum dots in male Wistar rats

Colloidal quantum dots (QD) show great promise as fluorescent markers. The QD used in this study were obtained in aqueous medium rather than the widely used colloidal QD. Both methodologies used for the production of QD are associated with the presence of heavy metals such as cadmium (Cd). Here we investigate the short-term inhalation toxicity of water-soluble core-shell CdS/Cd(OH)₂ QD. Male Wistar rats were head-nose exposed for 6 h/day on 5 days at the technically maximum concentration (0.52 mg Cd/m³). Histological examination was performed directly after the last exposure. Additional rats were used for Cd organ burden determinations. Clinical parameters in blood, bronchoalveolar lavage fluid and lung tissue were determined 3 days after the last exposure. To analyze the reversibility or progression of effects, the examinations were performed again after a recovery period of 3 weeks. The results of the study indicate that CdS/Cd(OH)₂ QD caused local neutrophil inflammation in the lungs that partially regressed after the 3-week recovery period. There was no evidence that QD were translocated to the central nervous system nor that a systemic acute phase response occurred.

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 (<false positives rate), inconsistent changes, demonstrating a metabolomic NOEL. A part of the total metabolite profile was consistent with a pattern of changes indicative of peroxisome proliferation, confirmed by increased cyanide-insensitive Palmitoyl-CoA oxidation. Simultaneous administration of 3000ppm DEHP and 150ppm DBP did not result in relevant changes when compared to the metabolite profile of 3000ppm DEHP alone. Co-administration of 1000ppm DBP induced marginal additional changes relative to the profile of 3000ppm DEHP alone. Simultaneous exposure to high dose levels of DEHP and DBP resulted in a profile that was significantly different compared to the individual compounds. A quantitative statistical analysis of the data revealed that the effect of combined treatment on the metabolites was less than additive.

Detection of hepatotoxicity potential with metabolite profiling (metabolomics) of rat plasma

While conventional parameters used to detect hepatotoxicity in drug safety assessment studies are generally informative, the need remains for parameters that can detect the potential for hepatotoxicity at lower doses and/or at earlier time points. Previous work has shown that metabolite profiling (metabonomics/metabolomics) can detect signals of potential hepatotoxicity in rats treated with doxorubicin at doses that do not elicit hepatotoxicity as monitored with conventional parameters. The current study extended this observation to the question of whether such signals could be detected in rats treated with compounds that can elicit hepatotoxicity in humans (i.e., drug-induced liver injury, DILI) but have not been reported to do so in rats. Nine compounds were selected on the basis of their known DILI potential, with six other compounds chosen as negative for DILI potential. A database of rat plasma metabolite profiles, MetaMap(®)Tox (developed by metanomics GmbH and BASF SE) was used for both metabolite profiles and mode of action (MoA) metabolite signatures for a number of known toxicities. Eight of the nine compounds with DILI potential elicited metabolite profiles that matched with MoA patterns of various rat liver toxicities, including cholestasis, oxidative stress, acetaminophen-type toxicity and peroxisome proliferation. By contrast, only one of the six non-DILI compounds showed a weak match with rat liver toxicity. These results suggest that metabolite profiling may indeed have promise to detect signals of hepatotoxicity in rats treated with compounds having DILI potential.

The Use of Metabolomics in Cancer Research

The use of metabolite profiling techniques (metabonomics or metabolomics) in toxicology is a relatively new branch of this science. Due to their unique biochemical properties, cancer cells should, in principle, be an ideal field of application for metabolite profiling. However, due to technical and study design limitations there are only a few reliably metabolite profiles for human tumors. This chapter provides examples for the recognition of metabolic changes in animals induced by exposure to (carcinogenic) chemicals. In two major projects (COMET and MetaMapTox), data bases have been developed which are sufficiently large to evaluate the full potential of metabolite profiling in toxicology and cancer research. In both projects blood and urine were used as matrices which can be easily obtained with minimally-invasive methods. Based on a high degree of standardization and a large-scale controlled data collection, consistent patterns of metabolite changes have been identified which are associated with different toxicological modes of action, some of which are known to enhance tumor development in rodents.

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.

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 phenytoins effects is obtained from the metabolome analysis, demonstrating the added value of metabolome data in preclinical/ toxicological studies.

Mechanistic analysis of metabolomics patterns in rat plasma during administration of direct thyroid hormone synthesis inhibitors or compounds increasing thyroid hormone clearance

For identification of toxicological modes of action (MoAs) a database (MetaMap(®)Tox) was established containing plasma metabolome consisting of approximately 300 endogenous metabolites. Each five male and female Wistar rats per groups were treated with >500 reference compounds over a period of 28 days. More than 120 specific toxicity patterns of common metabolite changes associated with unique MoAs were established. To establish patterns predictive effects on the thyroid, animals have been treated with reference compounds directly acting on the thyroid hormone formation (such as methimazole, ethylenethiourea) as well as liver enzyme inducers leading to an increased excretion of thyroid hormones and therewith to a secondary response of the thyroid (such as aroclor 1254 and boscalid). Here we present the plasma metabolite changes which form the patterns for direct and indirect effects on the thyroid. It is possible to identify metabolites which are commonly regulated irrespective of an indirect or direct effect on the thyroid as well as groups of metabolites separating both MoAs. By putting the metabolite regulations in the context of affected pathways helps to identify thyroid hormone inhibiting MoAs even when the hormone levels are not consistently changed. E.g., direct thyroid hormone synthesis inhibitors affect some enzymes in the urea cycle, increase the ω-oxidation of fatty acids and decrease glutamate and oxoproline levels, whereas indirect thyroid hormone inhibiting compounds interact with the lipid mediated and liver metabolism.

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.

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.

Prediction of Clinically Relevant Safety Signals of Nephrotoxicity through Plasma Metabolite Profiling

Addressing safety concerns such as drug-induced kidney injury (DIKI) early in the drug pharmaceutical development process ensures both patient safety and efficient clinical development. We describe a unique adjunct to standard safety assessment wherein the metabolite profile of treated animals is compared with the MetaMap Tox metabolomics database in order to predict the potential for a wide variety of adverse events, including DIKI. To examine this approach, a study of five compounds (phenytoin, cyclosporin A, doxorubicin, captopril, and lisinopril) was initiated by the Technology Evaluation Consortium under the auspices of the Drug Safety Executive Council (DSEC). The metabolite profiles for rats treated with these compounds matched established reference patterns in the MetaMap Tox metabolomics database indicative of each compounds well-described clinical toxicities. For example, the DIKI associated with cyclosporine A and doxorubicin was correctly predicted by metabolite profiling, while no evidence for DIKI was found for phenytoin, consistent with its clinical picture. In some cases the clinical toxicity (hepatotoxicity), not generally seen in animal studies, was detected with MetaMap Tox. Thus metabolite profiling coupled with the MetaMap Tox metabolomics database offers a unique and powerful approach for augmenting safety assessment and avoiding clinical adverse events such as DIKI.