Proteomic analysis of hepatic effects of phenobarbital in mice with humanized liver.

Activation of the constitutive androstane receptor (CAR) may induce adaptive but also adverse effects in rodent liver, including the induction of drug-metabolizing enzymes, transient hepatocellular proliferation, and promotion of liver tumor growth. Human relevance of CAR-related adverse hepatic effects is controversially debated. Here, we used the chimeric FRG-KO mouse model with livers largely repopulated by human hepatocytes, in order to study human hepatocytes and their response to treatment with the model CAR activator phenobarbital (PB) in vivo. Mice received an intraperitoneal injection with 50 mg/kg body weight PB or saline, and were sacrificed after 72-144 h. Non-repopulated FRG-KO mice were used as additional control. Comprehensive proteomics datasets were generated by merging data obtained by targeted as well as non-targeted proteomics approaches. For the first time, a novel proteomics workflow was established to comparatively analyze the effects of PB on human and murine proteins within one sample. Analysis of merged proteome data sets and bioinformatics data mining revealed comparable responses in murine and human hepatocytes with respect to nuclear receptor activation and induction of xenobiotic metabolism. By contrast, activation of MYC, a key regulator of proliferation, was predicted only for mouse but not human hepatocytes. Analyses of 5-bromo-2'-deoxyuridine incorporation confirmed this finding. In summary, this study for the first time presents a comprehensive proteomic analysis of CAR-dependent effects in human and mouse hepatocytes from humanized FRG-KO mice. The data support the hypothesis that PB does induce adaptive metabolic responses, but not hepatocellular proliferation in human hepatocytes in vivo.

Pyrrolizidine alkaloid-induced transcriptomic changes in rat lungs in a 28-day subacute feeding study.

Pyrrolizidine alkaloids (PAs) are secondary plant metabolites synthesized by a wide range of plants as protection against herbivores. These toxins are found worldwide and pose a threat to human health. PAs induce acute effects like hepatic sinusoidal obstruction syndrome and pulmonary arterial hypertension. Moreover, chronic exposure to low doses can induce cancer and liver cirrhosis in laboratory animals. The mechanisms causing hepatotoxicity have been investigated previously. However, toxic effects in the lung are less well understood, and especially data on the correlation effects with individual chemical structures of different PAs are lacking. The present study focuses on the identification of gene expression changes in vivo in rat lungs after exposure to six structurally different PAs (echimidine, heliotrine, lasiocarpine, senecionine, senkirkine, and platyphylline). Rats were treated by gavage with daily doses of 3.3 mg PA/kg bodyweight for 28 days and transcriptional changes in the lung and kidney were investigated by whole-genome microarray analysis. The results were compared with recently published data on gene regulation in the liver. Using bioinformatics data mining, we identified inflammatory responses as a predominant feature in rat lungs. By comparison, in liver, early molecular consequences to PAs were characterized by alterations in cell-cycle regulation and DNA damage response. Our results provide, for the first time, information about early molecular effects in lung tissue after subacute exposure to PAs, and demonstrates tissue-specificity of PA-induced molecular effects.

Hepatotoxic pyrrolizidine alkaloids induce DNA damage response in rat liver in a 28-day feeding study.

Pyrrolizidine alkaloids (PA) are secondary plant metabolites that occur as food and feed contaminants. Acute and subacute PA poisoning can lead to severe liver damage in humans and animals, comprising liver pain, hepatomegaly and the development of ascites due to occlusion of the hepatic sinusoids (veno-occlusive disease). Chronic exposure to low levels of PA can induce liver cirrhosis and liver cancer. However, it is not well understood which transcriptional changes are induced by PA and whether all hepatotoxic PA, regardless of their structure, induce similar responses. Therefore, a 28-day subacute rat feeding study was performed with six structurally different PA heliotrine, echimidine, lasiocarpine, senecionine, senkirkine, and platyphylline, administered at not acutely toxic doses from 0.1 to 3.3 mg/kg body weight. This dose range is relevant for humans, since consumption of contaminated tea may result in doses of ~ 8 µg/kg in adults and cases of PA ingestion by contaminated food was reported for infants with doses up to 3 mg/kg body weight. ALT and AST were not increased in all treatment groups. Whole-genome microarray analyses revealed pronounced effects on gene expression in the high-dose treatment groups resulting in a set of 36 commonly regulated genes. However, platyphylline, the only 1,2-saturated and, therefore, presumably non-hepatotoxic PA, did not induce significant expression changes. Biological functions identified to be affected by high-dose treatments (3.3 mg/kg body weight) comprise cell-cycle regulation associated with DNA damage response. These functions were found to be affected by all analyzed 1,2-unsaturated PA.In conclusion, 1,2-unsaturated hepatotoxic PA induced cell cycle regulation processes associated with DNA damage response. Similar effects were observed for all hepatotoxic PA. Effects were observed in a dose range inducing no histopathological alterations and no increase in liver enzymes. Therefore, transcriptomics studies identified changes in expression of genes known to be involved in response to genotoxic compounds at PA doses relevant to humans under worst case exposure scenarios.

Cadmium in brown crab Cancer pagurus. Effects of location, season, cooking and multiple physiological factors and consequences for food safety.

Brown crab Cancer pagurus is appreciated as seafood in several European countries. However, cadmium levels in crabs can be elevated and their consumption may pose a hazard for human health. To assess if cadmium poses a threat to food safety in Norway, crabs were sampled at two different locations along the Norwegian coast: one in the South of Norway and one in the North of Norway. Cadmium levels were determined in different tissues (claw meat, hepatopancreas and inner meat). To highlight specific risk factors for cadmium, the concentration of cadmium was related to different exogenous (location, cooking and season) and physiological (size, sex, moulting stage, gonad maturation stage, condition) factors. The results confirmed previous findings of much higher cadmium levels in brown crab sampled in the North of Norway compared to the South. Cooking of crabs further led to higher concentrations in claw meat. The effect of season on cadmium levels was different in the North and South and no clear patterns could be identified, probably due to a high inter-individual variation in cadmium levels. Size showed a correlation with the total amount of cadmium for crabs in the North indicating an accumulation of cadmium over time; together with a slower growth, this may lead to the higher cadmium levels, observed in the crabs from Northern Norway. The risk connected to cadmium exposure when consuming brown crab mainly depends on the consumption pattern, the parts of the crab consumed and the origin of the crab. Regardless of origin, the consumption of claw meat does not display a consumer health risk. However, the consumption of meals consisting of inner meat only and inner meat of brown crab from Northern Norway may pose a health risk.

Phosphoprotein DIGE profiles reflect blast differentiation, cytogenetic risk stratification, FLT3/NPM1 mutations and therapy response in acute myeloid leukaemia.

Acute myeloid leukaemia (AML) is an aggressive blood cancer characterized by a distinct block in differentiation of myeloid progenitors, recurrent chromosomal translocations and gene mutations of which >50% involve signal transduction through dysregulated kinases and phosphatases. In search for novel protein biomarkers for disease stratification we investigated the phosphoproteome in leukaemic cells from 62 AML patients at time of diagnosis using immobilized metal-affinity chromatography, protein separation by two-dimensional differential gel electrophoresis (2D-DIGE) and mass spectrometry before validation by selected reaction monitoring (SRM). Unsupervised clustering found 27 phosphoproteins significantly discriminating patients according to leukaemic cell differentiation (French-American-British (FAB) classification), cytogenetic and mutational (FLT3, NPM1) status or response to chemotherapy. Monocytic differentiation (FAB M4-M5) correlated with enrichment of proteins involved in apoptosis (MOES, ANXA5 and EFHD2). TALDO, a protein associated with thrombocytopenia if down-regulated, was elevated in patients with wild type NPM1 compared to patients with NPM1 mutation. This study demonstrates the potential of quantitative proteomics in AML classification and risk stratification. BIOLOGICAL SIGNIFICANCE: Patients diagnosed with AML are currently categorized according to cellular morphology, cytogenetic alterations and mutations, although the majority of these cellular and genetic alterations have no or unsolved impact on therapy selection or prognosis. We therefore explored the phosphoproteome for abundance changes associated with traditional classifiers to unravel patterns that could stratify patients at the protein level. MOES, ANXA5 and EFHD2 were confirmed by SRM to be correlated to monocytic differentiation, whilst TALDO was elevated in NPM1 wild type patients.