Demonstrating the reliability of in vivo metabolomics based chemical grouping: towards best practice.

While grouping/read-across is widely used to fill data gaps, chemical registration dossiers are often rejected due to weak category justifications based on structural similarity only. Metabolomics provides a route to robust chemical categories via evidence of shared molecular effects across source and target substances. To gain international acceptance, this approach must demonstrate high reliability, and best-practice guidance is required. The MetAbolomics ring Trial for CHemical groupING (MATCHING), comprising six industrial, government and academic ring-trial partners, evaluated inter-laboratory reproducibility and worked towards best-practice. An independent team selected eight substances (WY-14643, 4-chloro-3-nitroaniline, 17α-methyl-testosterone, trenbolone, aniline, dichlorprop-p, 2-chloroaniline, fenofibrate); ring-trial partners were blinded to their identities and modes-of-action. Plasma samples were derived from 28-day rat tests (two doses per substance), aliquoted, and distributed to partners. Each partner applied their preferred liquid chromatography-mass spectrometry (LC-MS) metabolomics workflows to acquire, process, quality assess, statistically analyze and report their grouping results to the European Chemicals Agency, to ensure the blinding conditions of the ring trial. Five of six partners, whose metabolomics datasets passed quality control, correctly identified the grouping of eight test substances into three categories, for both male and female rats. Strikingly, this was achieved even though a range of metabolomics approaches were used. Through assessing intrastudy quality-control samples, the sixth partner observed high technical variation and was unable to group the substances. By comparing workflows, we conclude that some heterogeneity in metabolomics methods is not detrimental to consistent grouping, and that assessing data quality prior to grouping is essential. We recommend development of international guidance for quality-control acceptance criteria. This study demonstrates the reliability of metabolomics for chemical grouping and works towards best-practice.

COVID-19 effects on the kidney.

Apart from pulmonary disease, acute kidney injury (AKI) is one of the most frequent and most severe organ complications in severe coronavirus disease 2019 (COVID-19). The SARS-CoV­2 virus has been detected in renal tissue. Patients with chronic kidney disease (CKD) before and on dialysis and specifically renal transplant patients represent a particularly vulnerable population. The increasing number of COVID-19 infected patients with renal involvement led to an evolving interest in the analysis of its pathophysiology, morphology and modes of virus detection in the kidney. Meanwhile, there are ample data from several autopsy and kidney biopsy studies that differ in the quantity of cases as well as in their quality. While the detection of SARS-CoV­2 RNA in the kidney leads to reproducible results, the use of electron microscopy for visualisation of the virus is difficult and currently critically discussed due to various artefacts. The exact contribution of indirect or direct effects on the kidney in COVID-19 are not yet known and are currently the focus of intensive research.

[COVID-19 effects on the kidney]. / COVID-19-Auswirkungen auf die Niere.

Apart from pulmonary disease, acute kidney injury (AKI) is one of the most frequent and most severe organ complications in severe coronavirus disease 2019 (COVID-19). The SARS-CoV­2 virus has been detected in renal tissue. Patients with chronic kidney disease (CKD) before and on dialysis and specifically renal transplant patients represent a particularly vulnerable population. The increasing number of COVID-19 infected patients with renal involvement led to an evolving interest in the analysis of its pathophysiology, morphology and modes of virus detection in the kidney. Meanwhile, there are ample data from several autopsy and kidney biopsy studies that differ in the quantity of cases as well as in their quality. While the detection of SARS-CoV­2 RNA in the kidney leads to reproducible results, the use of electron microscopy for visualisation of the virus is difficult and currently critically discussed due to various artefacts. The exact contribution of indirect or direct effects on the kidney in COVID-19 are not yet known and are currently the focus of intensive research.

Quantification of diffuse myocardial fibrosis using CMR extracellular volume fraction and serum biomarkers of collagen turnover with histologic quantification as standard of reference.

PURPOSE: To compare the assessment of diffuse interstitial myocardial fibrosis in valvular diseases using cardiac magnetic resonance (CMR) extracellular volume fraction (ECV) quantification and serum biomarkers of collagen turnover using results of myocardial biopsy as standard of reference. MATERIALS AND METHODS: This prospective monocentric study included consecutive patients before aortic valvular replacement. All patients underwent: i), 1.5T CMR with pre and post contrast T1 mapping sequence and ECV computation; ii), serum quantification of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) and iii), myocardial biopsies were collected during surgery to assess collagen volume fraction (CVF). Patients with coronary artery disease were excluded. Correlation between native T1, ECV, CVF and serum biomarkers were assessed using Pearson correlation test. Agreement between basal anteroseptal ECV with global ECV was assessed using Bland-Altman test. RESULTS: Twenty-one patients, 16 with aortic stenosis and 5 with aortic regurgitation were included. There were 12 men and 9 women with a mean age of 74.1±6.8 (SD) years (range: 32-84 years). Mean global ECV value was 26.7±2.7 (SD) % (range: 23.4-32.5%) and mean CVF value was 12.4±9.7% (range: 3.2-25.7%). ECV assessed at the basal anteroseptal segment correlated moderately with CVF (r=0.6; P=0.0026). There was a strong correlation and agreement between basal anteroseptal ECV and global ECV, (r=0.8; P<0.0001; bias 5.4±6.1%) but no correlation between global ECV and CVF (r=0.5; P=0.10). Global ECV poorly correlated with serum TIMP-1 (r=0.4; P=0.037) and MMP-2 (r=0.4; P=0.047). No correlation was found between serum biomarkers and basal anteroseptal- ECV or native T1. CONCLUSION: In patients with severe aortic valvulopathy, diffuse myocardial fibrosis assessed by anterosepto-basal ECV correlates with histological myocardial fibrosis. Anteroseptobasal ECV strongly correlates with global ECV, which poorly correlates with TIMP-1 and MMP-2, serum biomarkers involved in the progression of heart failure.

First-line treatment with R-CHOP or rituximab-bendamustine in patients with follicular lymphoma grade 3A-results of a retrospective analysis.

Based on centroblast frequency, follicular lymphoma (FL) is subdivided into grades 1-2, 3A, and 3B. Grade FL3A frequently coexists with FL1-2 (FL1-2-3A). Based on clinical trials, FL1-2 is treated with rituximab (R) or obinutuzumab plus bendamustine (B) or CHOP, while FL3B is treated with R-CHOP. In contrast, there are little data guiding therapy in FL3A. We present a retrospective, multicenter analysis of 95 FL3A or FL1-2-3A and 203 FL1-2 patients treated with R-CHOP or R-B first-line. R-CHOP facilitated a higher response rate (95% versus 76%) and longer overall survival (OS) (3-year OS 89% versus 73%, P = 0.008) in FL3A or FL1-2-3A, whereas the difference in progression-free survival (PFS) did not reach statistical significance. While transformation rates into aggressive lymphoma were similar between both groups, there were more additional malignancies after R-B compared with R-CHOP (6 versus 2 cases). In FL1-2, R-B achieved a higher 3-year PFS (79% versus 47%, P < 0.01), while there was no significant difference regarding OS or transformation. With the limitations of a retrospective analysis, these results suggest a benefit for R-CHOP over R-B in FL3A or FL1-2-3A. Confirmatory data from prospective clinical trials are needed.

The Importance of Consistent Global Forest Aboveground Biomass Product Validation.

Several upcoming satellite missions have core science requirements to produce data for accurate forest aboveground biomass mapping. Largely because of these mission datasets, the number of available biomass products is expected to greatly increase over the coming decade. Despite the recognized importance of biomass mapping for a wide range of science, policy and management applications, there remains no community accepted standard for satellite-based biomass map validation. The Committee on Earth Observing Satellites (CEOS) is developing a protocol to fill this need in advance of the next generation of biomass-relevant satellites, and this paper presents a review of biomass validation practices from a CEOS perspective. We outline the wide range of anticipated user requirements for product accuracy assessment and provide recommendations for the validation of biomass products. These recommendations include the collection of new, high-quality in situ data and the use of airborne lidar biomass maps as tools toward transparent multi-resolution validation. Adoption of community-vetted validation standards and practices will facilitate the uptake of the next generation of biomass products.

An architectural understanding of natural sway frequencies in trees.

The relationship between form and function in trees is the subject of a longstanding debate in forest ecology and provides the basis for theories concerning forest ecosystem structure and metabolism. Trees interact with the wind in a dynamic manner and exhibit natural sway frequencies and damping processes that are important in understanding wind damage. Tree-wind dynamics are related to tree architecture, but this relationship is not well understood. We present a comprehensive view of natural sway frequencies in trees by compiling a dataset of field measurement spanning conifers and broadleaves, tropical and temperate forests. The field data show that a cantilever beam approximation adequately predicts the fundamental frequency of conifers, but not that of broadleaf trees. We also use structurally detailed tree dynamics simulations to test fundamental assumptions underpinning models of natural frequencies in trees. We model the dynamic properties of greater than 1000 trees using a finite-element approach based on accurate three-dimensional model trees derived from terrestrial laser scanning data. We show that (1) residual variation, the variation not explained by the cantilever beam approximation, in fundamental frequencies of broadleaf trees is driven by their architecture; (2) slender trees behave like a simple pendulum, with a single natural frequency dominating their motion, which makes them vulnerable to wind damage and (3) the presence of leaves decreases both the fundamental frequency and the damping ratio. These findings demonstrate the value of new three-dimensional measurements for understanding wind impacts on trees and suggest new directions for improving our understanding of tree dynamics from conifer plantations to natural forests.

Added value of plasma metabolomics to describe maternal effects in rat maternal and prenatal toxicity studies.

For regulatory purposes prenatal developmental toxicity (OECD No. 414) studies are routinely performed in our laboratories. The suitability of metabolomics as technology to identify maternal toxicity in such studies was investigated. Plasma was sampled from pregnant, non-fasted rats on gestation day 20 before cesarean section. Metabolite profiling was performed by gas- and liquid-chromatography-tandem mass spectrometry techniques. The sensitivity of routinely examined maternal toxicity parameters (OECD No. 414) was compared to those of metabolome analysis. Evaluating 44 studies, the metabolome-derived NOEL was more sensitive in 45% of the cases in detecting maternal toxicity than the maternal NOAEL. Metabolome patterns indicative for liver effects and 4-hydroxyphenylpyruvate dioxygenase (HPPD) enzyme-inhibition were established in pregnant rats based on regulated metabolites using reference compounds. The HPPD inhibition and liver toxicity patterns in pregnant rats were reasonably comparable to the ones established in non-pregnant, fasted rats. Metabolomics is a useful tool for an improved and mechanism-based identification of maternal toxicity in maternal and prenatal toxicity studies. The data suggest that the current classical maternal toxicity parameters may underestimate the extent of effects of compounds on the dams.

[(Immuno­)Pathology of drug side effects in the kidney]. / (Immun­)Pathologie von Medikamentennebenwirkungen in der Niere.

Nephrotoxicity or renal side effects of drugs are frequent and may vary in their clinical presentation. Various types of acute and chronic kidney disease are known to develop as a consequence or side effect of a long list of drugs with nephrotoxicity most commonly being associated with injury in the tubulointerstitial compartment. In addition, drug-induced glomerular and vascular disease have also been reported, either as the result of direct cellular injury or immune-mediated injury to glomerular or endothelial cells. From a clinical point of view it is important to recognize such drug-induced nephropathies early in order to prevent or adequately treat them to favour kidney recovery and to avoid long-lasting negative consequences for kidney function.This article will focus on the typical morphology and pathogenesis of some frequent drug-induced renal diseases.

Impact of lincosamides antibiotics on the composition of the rat gut microbiota and the metabolite profile of plasma and feces.

The importance of the gut microorganisms and their wide range of interactions with the host are well-acknowledged. In this study, lincomycin and clindamycin were used to modulate microbial communities of Wistar rats to gain a comprehensive understanding of the implications of microbiome alterations. A metabolomics approach and taxonomic profiling were applied to characterize the effects of these antibiotics on the functionality of the microbiome and to identify microbiome-related metabolites. After treatment, the diversity of the microbial community was drastically reduced. Bacteroidetes and Verrucomicrobia were drastically reduced, Tenericutes and Deferribacteres completely disappeared, while abundance of Firmicutes and Proteobacteria were highly increased. Changes in plasma and feces metabolites were observed for metabolites belonging mainly to the class of complex lipids, fatty acids and related metabolites as well as amino acids and related compounds. Bile acid metabolism was markedly affected: taurocholic acid, glycochenodeoxycholic acid and cholic acid presented abrupt changes showing a specific metabolite pattern indicating disruption of the microbial community. In both plasma and feces taurocholic acid was highly upregulated upon treatment whereas glycochenodeoxycholic acid was downregulated. Cholic acid was upregulated in feces but downregulated in plasma. These results show that changes in the gut microbial community lead to alterations of the metabolic profile in blood and feces of the host and can be used to identify potentially microbiome-related metabolites. This implies that metabolomics could be a suitable tool to estimate the extent of changes induced in the intestinal microbiome with respect to consequences for the host.

Microbiome-related metabolite changes in gut tissue, cecum content and feces of rats treated with antibiotics.

The metabolic functionality of the gut microbiota contributes to the metabolism and well-being of its host, although detailed insight in the microbiota's metabolism is lacking. Omics technologies could facilitate unraveling metabolism by the gut microbiota. In this study, we performed metabolite profiling of different matrices of the gut, after antibiotic treatment of rats in order to evaluate metabolite changes observed at different dose levels and in different sexes, and to identify the best tissue matrix for further investigations regarding an assessment of metabolic effects of new compounds with antibiotic activity. Three different antibiotics (vancomycin, streptomycin and roxithromycin) were administered orally to rats for 28 days according to the OECD 407 guideline with a subsequent metabolic profiling in feces, cecum content and gut tissue (jejunum, ileum, cecum, colon and rectum). The data were analyzed in the MetaMap®Tox database. Treatment-related effects could be observed in the metabolite profile of feces and cecum content, but not of the different gut tissues. The metabolite profile showed compound specific effects on the microbiome. In line with the activity spectra of the antibiotics tested, vancomycin showed the largest effects, followed by roxithromycin and then by streptomycin for which changes were modest. In general, for all antibiotics the largest changes were observed for the classes of lipids (increase up to 94-fold), bile acids (increase up to 33-fold), amino acids (increase up to 200-fold) and amino acid related (increase up to 348-fold). The most relevant changes in metabolite values were similar in feces and cecum content and among sexes. The results of this targeted analysis indicate that the metabolic profiles of male and female animals in the gut microbiome are comparable. Concluding, taking other samples than feces does not add any extra information. Thus, as a non-invasive sampling method, feces provide a suitable matrix for studies on metabolism by the gut microbiota.

In-situ analysis of mast cells and dendritic cells in coronary atherosclerosis in chronic kidney disease (CKD).

AIMS: Mast cells (MC) and dendritic cells (DC) have immune modulatory function and can influence T-cell activity. Both cell types have been found in atherosclerotic plaques and are thought to play an important role for plaque stability. Compared to matched segments of the non-renal population, patients with chronic kidney disease (CKD) show a more pronounced and more aggressive course of atherosclerosis with higher plaque calcification and significantly higher complications rates. It was the aim of this study to analyze the number and localization of MCs and DCs, macrophages, T- and B-cells as well as the expression of markers of inflammation such as CRP and NFκB in calcified and non-calcified atherosclerotic plaques of patients with CKD and control patients. METHODS: Fifty coronary atherosclerotic plaques from patients with endstage CKD (CKD, n=25) and control (n=25) patients were categorized according to the Stary classification and investigated using immunohistochemistry (markers for MC, DC, T, B, macrophage and NFκB). Expression was analyzed separately for the complete plaque area as well as for the different plaque subregions and correlations were analyzed. RESULTS: We found only very few DCs and MCs per lesion area with slightly increased numbers in calcified plaques. MCs per plaque area were significantly more frequent in CKD than in control patients and this was independent of plaque calcification. MCs were most frequently found in the shoulder and basis of the plaque. DCs per plaque area were significantly less in calcified plaques of CKD compared to control patients. In control, but not in CKD patients, DCs were significantly more frequent in calcified than in non-calcified plaques. Within the plaques DCs were similarly distributed between all 4 subregions. CONCLUSIONS: Coronary atherosclerotic plaques of CKD patients showed a significantly higher number of MCs whereas DCs were less frequent compared to control patients particularly if plaques were calcified. These findings might indicate a potential proinflammatory role of MCs, but not of DCs in atherosclerotic lesions of CKD patients, adding another characteristic of advanced atherosclerosis in these patients.

[Recent development of hypertension and acute renal failure in a 59-year-old woman]. / Neu aufgetretene Hypertonie und akutes Nierenversagen bei einer 59-jährigen Patientin.

We report on a 59-year-old woman who presented with nausea, fatigue, arterial hypertension, and acute renal failure. Clinical examination, laboratory findings of blood and urine and abdominal sonography were inconclusive. Renal biopsy revealed infiltration by a diffuse large B­cell lymphoma. In fluorodeoxyglucose positron emission tomography/computed tomography tracer enrichment was demonstrated in both kidneys, skeletal system and retroperitoneal lymph nodes. Renal function improved already after the first cycle of R­CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone). After chemotherapy, a complete remission, normalization of blood pressure and renal function were achieved.

CRISPR/Cas9-The ultimate weapon to battle infectious diseases?

Infectious diseases are a leading cause of death worldwide. Novel therapeutics are urgently required to treat multidrug-resistant organisms such as Mycobacterium tuberculosis and to mitigate morbidity and mortality caused by acute infections such as malaria and dengue fever virus as well as chronic infections such as human immunodeficiency virus-1 and hepatitis B virus. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, which has revolutionized biomedical research, holds great promise for the identification and validation of novel drug targets. Since its discovery as an adaptive immune system in prokaryotes, the CRISPR/Cas9 system has been developed into a multi-faceted genetic modification tool, which can now be used to induce gene deletions or specific gene insertions, such as conditional alleles or endogenous reporters in virtually any organism. The generation of CRISPR/Cas9 libraries that can be used to perform phenotypic whole genome screens provides an important new tool that will aid in the identification of critical host factors involved in the pathogenesis of infectious diseases. In this review, we will discuss the development and recent applications of the CRISPR/Cas9 system used to identify novel regulators, which might become important in the fight against infectious diseases.

Pilot Study of Endothelin Receptor Blockade in Heart Failure with Diastolic Dysfunction and Pulmonary Hypertension (BADDHY-Trial).

BACKGROUND: In this multi-centre, randomised, placebo-controlled pilot trial, we investigated the clinical and haemodynamic effects of the endothelin-receptor blocker Bosentan in patients with heart failure, preserved ejection fraction and pulmonary hypertension (PH-HFpEF). MATERIALS AND METHODS: Eligible patients received either 12 weeks of Bosentan therapy, or a placebo drug. Patients were thereafter followed for a further period of 12 weeks without the study medication. At three points during the study (study Commencement, Week 12 and Week 24), a six-minute walk test (6MWT), echocardiographic and laboratory assessments were performed, as well as a quality of life survey. Right heart catheterisation (RHC) was undertaken at commencement only. The study was aborted early, after an interim analysis favoured the placebo. RESULTS: Six-minute walk distance (6MWD) did not change in the Bosentan group (309.7±96.3m (Commencement), 317.0±126.1m (Week 12), 307.0±84.4m (Week 24); p=0.86), but almost reached statistical significance in the placebo group from 328.8±79.6m, to 361.6±98.2m and 384.0±74.9m (Week 24); p=0.075. In the placebo group, estimated systolic pulmonary artery pressure (measured via echocardiography) significantly decreased (from 62.3±16.7mmHg [Commencement], 45.3±13.9mmHg [Week 12], to 44.6±14.5mmHg [Week 24]; p=0.014) as did right atrial pressure (13.1±5.3 [Commencement], 10.0±3.8 [Week 12], to 9.4±3.2 [Week 24]; p=0.046). CONCLUSION: Despite this study's limited sample size and premature cessation, it nevertheless suggests that endothelin receptor blockade in patients with PH-HFpEF may have no beneficial effects and could even be detrimental in comparison to a placebo.

Restoration of tumor suppression in prostate cancer by targeting the E3 ligase E6AP.

Restoration of tumor suppression is an attractive onco-therapeutic approach. It is particularly relevant when a tumor suppressor is excessively degraded by an overactive oncogenic E3 ligase. We previously discovered that the E6-associated protein (E6AP; as classified in the human papilloma virus context) is an E3 ligase that has an important role in the cellular stress response, and it directly targets the tumor-suppressor promyelocytic leukemia protein (PML) for proteasomal degradation. In this study, we have examined the role of the E6AP-PML axis in prostate cancer (PC). We show that knockdown (KD) of E6AP expression attenuates growth of PC cell lines in vitro. We validated this finding in vivo using cell line xenografts, patient-derived xenografts and mouse genetics. We found that KD of E6AP attenuates cancer cell growth by promoting cellular senescence in vivo, which correlates with restoration of tumor suppression by PML. In addition, we show that KD of E6AP sensitizes cells to radiation-induced death. Overall, our findings demonstrate a role for E6AP in the promotion of PC and support E6AP targeting as a novel approach for PC treatment, either alone or in combination with radiation.

Cell-to-cell distances between tumor-infiltrating inflammatory cells have the potential to distinguish functionally active from suppressed inflammatory cells.

Beyond their mere presence, the distribution pattern of inflammatory cells is of special interest. Our hypothesis was that random distribution may be a clear indicator of being non-functional as a consequence of lack of interaction. Here, we have assessed the implication of cell-to-cell distances among inflammatory cells in anal squamous cell carcinoma and a possible association with survival data. Thirty-eight patients suffering from anal carcinoma were studied using tissue microarrays, double staining immunohistochemistry, whole slide scanning and image analysis software. Therapy consisted of concurrent radiochemotherapy. Numbers of stromal and intraepithelial tumor-infiltrating inflammatory cells (TIC) and the distances between cells were quantified. Double-staining of FoxP3(+) cells with either CD8(+), CD1a(+) or CD20(+) cells was performed. Measured cell-to-cell distances were compared to computer simulated cell-to-cell distances leading to the assumption of non-randomly distributed and therefore functional immune cells. Intraepithelial CD1a(+) and CD20(+) cells were randomly distributed and therefore regarded as non-functional. In contrary, stromal CD20(+) cells had a non-random distribution pattern. A non-random distance between CD20(+) and FoxP3(+) cells was associated with a clearly unfavorable outcome. Measured distances between FoxP3(+) cells were distinctly shorter than expected and indicate a functional active state of the regulatory T cells (Treg). Analysis of cell-to-cell distances between TIC has the potential to distinguish between suppressed non-functional and functionally active inflammatory cells. We conclude that in this tumor model most of the CD1a(+) cells are non-functional as are the intraepithelial CD20(+) cells, while stromal CD20(+) cells and FoxP3(+) cells are functional cells.

Report on the second International Consensus on ANA Pattern (ICAP) workshop in Dresden 2015.

The second meeting for the International Consensus on Antinuclear antibody (ANA) Pattern (ICAP) was held on 22 September 2015, one day prior to the opening of the 12th Dresden Symposium on Autoantibodies in Dresden, Germany. The ultimate goal of ICAP is to promote harmonization and understanding of autoantibody nomenclature, and thereby optimizing ANA usage in patient care. The newly developed ICAP website www.ANApatterns.org was introduced to the more than 50 participants. This was followed by several presentations and discussions focusing on key issues including the two-tier classification of ANA patterns into competent-level versus expert-level, the consideration of how to report composite versus mixed ANA patterns, and the necessity for developing a consensus on how ANA results should be reported. The need to establish on-line training modules to help users gain competency in identifying ANA patterns was discussed as a future addition to the website. To advance the ICAP goal of promoting wider international participation, it was agreed that there should be a consolidated plan to translate consensus documents into other languages by recruiting help from members of the respective communities.

Metabolite profiles of rats in repeated dose toxicological studies after oral and inhalative exposure.

The MetaMap(®)-Tox database contains plasma-metabolome and toxicity data of rats obtained from oral administration of 550 reference compounds following a standardized adapted OECD 407 protocol. Here, metabolic profiles for aniline (A), chloroform (CL), ethylbenzene (EB), 2-methoxyethanol (ME), N,N-dimethylformamide (DMF) and tetrahydrofurane (THF), dosed inhalatively for six hours/day, five days a week for 4 weeks were compared to oral dosing performed daily for 4 weeks. To investigate if the oral and inhalative metabolome would be comparable statistical analyses were performed. Best correlations for metabolome changes via both routes of exposure were observed for toxicants that induced profound metabolome changes. e.g. CL and ME. Liver and testes were correctly identified as target organs. In contrast, route of exposure dependent differences in metabolic profiles were noted for low profile strength e.g. female rats dosed inhalatively with A or THF. Taken together, the current investigations demonstrate that plasma metabolome changes are generally comparable for systemic effects after oral and inhalation exposure. Differences may result from kinetics and first pass effects. For compounds inducing only weak changes, the differences between both routes of exposure are visible in the metabolome.