Integrated data from intravital imaging and HPLC-MS/MS analysis reveal large interspecies differences in AFB1 metabolism in mice and rats.

Large interspecies differences between rats and mice concerning the hepatotoxicity and carcinogenicity of aflatoxin B1 (AFB1) are known, with mice being more resistant. However, a comprehensive interspecies comparison including subcellular liver tissue compartments has not yet been performed. In this study, we performed spatio-temporal intravital analysis of AFB1 kinetics in the livers of anesthetized mice and rats. This was supported by time-dependent analysis of the parent compound as well as metabolites and adducts in blood, urine, and bile of both species by HPLC-MS/MS. The integrated data from intravital imaging and HPLC-MS/MS analysis revealed major interspecies differences between rats and mice: (1) AFB1-associated fluorescence persisted much longer in the nuclei of rat than mouse hepatocytes; (2) in the sinusoidal blood, AFB1-associated fluorescence was rapidly cleared in mice, while a time-dependent increase was observed in rats in the first three hours after injection followed by a plateau that lasted until the end of the observation period of six hours; (3) this coincided with a far stronger increase of AFB1-lysine adducts in the blood of rats compared to mice; (4) the AFB1-guanine adduct was detected at much higher concentrations in bile and urine of rats than mice. In both species, the AFB1-glutathione conjugate was efficiently excreted via bile, where it reached concentrations at least three orders of magnitude higher compared to blood. In conclusion, major differences between mice and rats were observed, concerning the nuclear persistence, formation of AFB1-lysine adducts, and the AFB1-guanine adducts.

Comparative metabolism of aflatoxin B1 in mouse, rat and human primary hepatocytes using HPLC-MS/MS.

Aflatoxin B1 (AFB1) is a highly hepatotoxic and carcinogenic mycotoxin produced by Aspergillus species. The compound is mainly metabolized in the liver and its metabolism varies between species. The present study quantified relevant AFB1- metabolites formed by mouse, rat, and human primary hepatocytes after treatment with 1 µM and 10 µM AFB1. The use of liquid chromatographic separation coupled with tandem mass spectrometric detection enabled the selective and sensitive determination of phase I and phase II metabolites of AFB1 over incubation times of up to 24 h. The binding of AFB1 to macromolecules was also considered. The fastest metabolism of AFB1 was observed in mouse hepatocytes which formed aflatoxin P1 as a major metabolite and also its glucuronidated form, while AFP1 occurred only in traces in the other species. Aflatoxin M1 was formed in all species and was, together with aflatoxin Q1 and aflatoxicol, the main metabolite in human cells. Effective epoxidation led to high amounts of DNA adducts already 30 min post-treatment, especially in rat hepatocytes. Lower levels of DNA adducts and fast DNA repair were found in mouse hepatocytes. Also, protein adducts arising from reactive intermediates were formed rapidly in all three species. Detoxification via glutathione conjugation and subsequent formation of the N-acetylcysteine derivative appeared to be similar in mice and in rats and strongly differed from human hepatocytes which did not form these metabolites at all. The use of qualitative reference material of a multitude of metabolites and the comparison of hepatocyte metabolism in three species using advanced methods enabled considerations on toxification and detoxification mechanisms of AFB1. In addition to glutathione conjugation, phase I metabolism is strongly involved in the detoxification of AFB1.

Polyphenols Cause Structure Dependent Effects on the Metabolic Profile of Human Hepatocarcinogenic Cells.

SCOPE: Although many beneficial health effects are attributed to polyphenols their influence on the human metabolome has not been elucidated yet. The ubiquitous occurrence of polyphenols in the human diet demands comprehensive knowledge about physiological and toxicological effects of these compounds on human cells. METHODS AND RESULTS: The human hepatocarcinogenic cell line HepG2 is used to elucidate the effects of 13 polyphenols and three respective phenolic degradation products on the human metabolome using HPLC-MS/MS. To investigate structure-activity-relationships, structurally related examples of polyphenols from different compound classes are selected. The analysis of catechins points toward a relation between the degree of hydroxylation and the extent of metabolic effects particularly on the urea cycle and the pentose phosphate pathway (PPP). A correlation between the modulation of the PPP and the stability of the compounds is demonstrated, which may be caused by reactive oxygen species (ROS). The incubation of flavones and alkenylbenzenes demonstrates reduced activity of methoxylated compounds and no impact of the B-ring position. CONCLUSION: In general, polyphenols induce a multitude of metabolic effects, for example, on energy metabolism, PPP, and urea cycle. These metabolic alterations may be related to the widely reported bioactivity of these compounds such as the anticarcinogenic effects.

Dose-dependent expression of GFI1 alters metabolism in the haematopoietic progenitors and MLL::AF9-induced leukaemic cells.

Growth factor independence 1 (GFI1) is a transcriptional repressor protein that plays an essential role in the differentiation of myeloid and lymphoid progenitors. We and other groups have shown that GFI1 has a dose-dependent role in the initiation, progression, and prognosis of acute myeloid leukaemia (AML) patients by inducing epigenetic changes. We now demonstrate a novel role for dose-dependent GFI1 expression in regulating metabolism in haematopoietic progenitor and leukaemic cells. Using in-vitro and ex-vivo murine models of MLL::AF9-induced human AML and extra-cellular flux assays, we now demonstrate that a lower GFI1 expression enhances oxidative phosphorylation rate via upregulation of the FOXO1- MYC axis. Our findings underscore the significance of therapeutic exploitation in GFI1-low-expressing leukaemia cells by targeting oxidative phosphorylation and glutamine metabolism.

Glycolytic interference blocks influenza A virus propagation by impairing viral polymerase-driven synthesis of genomic vRNA.

Influenza A virus (IAV), like any other virus, provokes considerable modifications of its host cell's metabolism. This includes a substantial increase in the uptake as well as the metabolization of glucose. Although it is known for quite some time that suppression of glucose metabolism restricts virus replication, the exact molecular impact on the viral life cycle remained enigmatic so far. Using 2-deoxy-d-glucose (2-DG) we examined how well inhibition of glycolysis is tolerated by host cells and which step of the IAV life cycle is affected. We observed that effects induced by 2-DG are reversible and that cells can cope with relatively high concentrations of the inhibitor by compensating the loss of glycolytic activity by upregulating other metabolic pathways. Moreover, mass spectrometry data provided information on various metabolic modifications induced by either the virus or agents interfering with glycolysis. In the presence of 2-DG viral titers were significantly reduced in a dose-dependent manner. The supplementation of direct or indirect glycolysis metabolites led to a partial or almost complete reversion of the inhibitory effect of 2-DG on viral growth and demonstrated that indeed the inhibition of glycolysis and not of N-linked glycosylation was responsible for the observed phenotype. Importantly, we could show via conventional and strand-specific qPCR that the treatment with 2-DG led to a prolonged phase of viral mRNA synthesis while the accumulation of genomic vRNA was strongly reduced. At the same time, minigenome assays showed no signs of a general reduction of replicative capacity of the viral polymerase. Therefore, our data suggest that the significant reduction in IAV replication by glycolytic interference occurs mainly due to an impairment of the dynamic regulation of the viral polymerase which conveys the transition of the enzyme's function from transcription to replication.

Metabolic profiling as a powerful tool for the analysis of cellular alterations caused by 20 mycotoxins in HepG2 cells.

Mycotoxins are secondary fungal metabolites which exhibit toxic effects in low concentrations. Several mycotoxins are described as carcinogenic or immunosuppressive, but their underlying modes of action especially on molecular level have not yet been entirely elucidated. Metabolic profiling as part of the omics methods is a powerful tool to study the toxicity and the mode of action of xenobiotics. The use of hydrophilic interaction chromatography in combination with targeted mass spectrometric detection enables the selective and sensitive analysis of more than 100 polar and ionic metabolites and allows the evaluation of metabolic alterations caused by xenobiotics such as mycotoxins. For metabolic profiling, the hepato-cellular carcinoma cell line HepG2 was treated with sub-cytotoxic concentrations of 20 mycotoxins. Moniliformin and citrinin significantly affected target elements of the citric acid cycle, but also influenced glycolytic pathways and energy metabolism. Penitrem A, zearalenone, and T2 toxin mainly interfered with the urea cycle and the amino acid homeostasis. The formation of reactive oxygen species seemed to be influenced by T2 toxin and gliotoxin. Glycolysis was altered by ochratoxin A and DNA synthesis was affected by several mycotoxins. The observed effects were not limited to these metabolic reactions as the metabolic pathways are closely interrelated. In general, metabolic profiling proved to be a highly sensitive tool for hazard identification in comparison to single-target cytotoxicity assays as metabolic alterations were already observed at sub-toxic concentrations. Metabolic profiling could therefore be a powerful tool for the overall evaluation of the toxic properties of xenobiotics.

Pharmacy interns on the ward--a pilot study.

OBJECTIVE: To assess the feasibility and impact of the activities of pharmacy interns on German hospital wards as well as the acceptance of their activities by medical and pharmaceutical staff. METHOD: The project was carried out for 36 months in six hospitals on different wards. Seventeen interns spent three months first in the pharmacy followed by another three months in the wards. Information about their tasks on different wards was obtained through reporting by interns on a standardized data collection form. Questionnaires regarding acceptance and impact of pharmacy interns were answered by medical and pharmaceutical staff. RESULTS: After having adjusted to their tasks, the interns investigated and developed solutions for structural and process-related slacks in the handling of medicines in the wards. They focused on drug information, on the detection and prevention of medication and documentation errors and storage of medicines in the wards. One hundred and forty six questionnaires regarding acceptance, impact and possible tasks of the interns were answered. Ninety percent of the surveyed medical staff considered the work of the interns as useful and 89% were in favour of permanent interns in the wards. The acceptance by pharmaceutical staff was slightly lower. CONCLUSION: This pilot study represents a landmark for the implementation of clinical pharmacy in daily practice especially on medical wards in Germany. Working in wards offers interns a possibility to extend their knowledge and skills. The project demonstrates that pharmacy interns can play an important role in drug safety in hospital wards. The acceptance by physicians and nurses is high. The majority of them requested the continuation of the project.

Plasma levels of advanced glycation end products during haemodialysis, haemodiafiltration and haemofiltration: potential importance of dialysate quality.

BACKGROUND: Advanced glycation end products (AGEs) accumulate in patients with end-stage renal disease (ESRD). The aim of this study was to investigate the potential influence of different modalities of renal replacement therapies on plasma AGE levels. METHODS: The removal of AGEs by high-flux haemodialysis (HD) using standard and ultrapure dialysis fluid (SDF and UDF), by haemodiafiltration (HDF) and by haemofiltration (HF) was studied by fluorescence spectroscopy and by a carboxymethyllysine (CML)-specific ELISA. In addition, molecular weight distribution of fluorescent AGE products in serum of several patients was analysed by gel filtration. RESULTS: The highest AGE-typical fluorescence was found in the serum of patients on HD using SDF (114,667+/-18,967 arbitrary units (AU)), followed by patients on HDF (86,912+/-24,411 AU, P<0.005), by patients on HD using UDF (74,953+/-21,152 AU, P<0.0001) and by patients on HF (74 039+/-17 027 AU, P<0.0001). Similar results were found for serum CML levels with the highest values in HD patients on SDF (1609+/-504 ng/ml), followed by patients on HF (1354+/-614 ng/ml, P<0.001), then by HD patients on UDF (1310+/-403 ng/ml, P<0.001) and by patients on HDF (1132+/-338 ng/ml, P<0.001). The removal rate of AGEs, as evaluated by the determination of the pre-/post-dialysis AGE differences, was comparable across all groups. CONCLUSION: These findings suggest that factors other than removal are responsible for the lower pre-dialysis AGE levels found in patients on convective dialysis as well as on HD with UDF. A role of water quality is assumed. This is corroborated by the finding that the high molecular weight AGE-fraction is preferentially lowered in comparison with patients on HD with SDF, as analysed by gel filtration chromatography. These findings could be best explained by a less severe oxidative stress (i.e. resulting in decreased AGE generation) with HF and HDF, as well as with ultrapure HD.