Transcriptomics analysis of hepatotoxicity induced by the pesticides imazalil, thiacloprid and clothianidin alone or in binary mixtures in a 28-day study in female Wistar rats.

Co-occurrence of pesticide residues in food commodities raises a potential safety issue as their mixture effects on human health are largely unknown. In a previous study, we reported the toxicological effects (pathology and histopathology) of imazalil (IMZ), thiacloprid (THI), and clothianidin (CTD) alone and in binary mixtures in a 28-day oral gavage study in female Wistar rats. Five dose levels (up to 350 mg/kg body weight/day) ranging from a typical toxicological reference value to a clear effect dose were applied. In the present study, we undertook a transcriptomics analysis of rat livers by means of total RNA sequencing (RNA-Seq). Bioinformatic data analysis involving Ingenuity Pathway Analysis (IPA) was used to gain mechanistic information on hepatotoxicity-related pathways affected after treatment with the pesticides, alone and in mixtures. Our data show that 2986 genes were differentially regulated by CTD while IMZ and THI had effects on 194 and 225 genes, respectively. All three individual compounds shared a common subset of genes whose network is associated with xenobiotic metabolism and nuclear receptor activation. Similar networks were retrieved for the mixtures. Alterations in the expression of individual genes were in line with the assumption of dose addition. Our results bring new insight into the hepatotoxicity mechanisms of IMZ, THI, and CTD and their mixtures.

Pyrrolizidine Alkaloids Disturb Bile Acid Homeostasis in the Human Hepatoma Cell Line HepaRG.

1,2-unsaturated pyrrolizidine alkaloids (PAs) belong to a group of secondary plant metabolites. Exposure to PA-contaminated feed and food may cause severe hepatotoxicity. A pathway possibly involved in PA toxicity is the disturbance of bile acid homeostasis. Therefore, in this study, the influence of four structurally different PAs on bile acid homeostasis was investigated after single (24 h) and repeated (14 days) exposure using the human hepatoma cell line HepaRG. PAs induce a downregulation of gene expression of various hepatobiliary transporters, enzymes involved in bile acid synthesis, and conjugation, as well as several transcription regulators in HepaRG cells. This repression may lead to a progressive impairment of bile acid homeostasis, having the potential to accumulate toxic bile acids. However, a significant intracellular and extracellular decrease in bile acids was determined, pointing to an overall inhibition of bile acid synthesis and transport. In summary, our data clearly show that PAs structure-dependently impair bile acid homeostasis and secretion by inhibiting the expression of relevant genes involved in bile acid homeostasis. Furthermore, important biliary efflux mechanisms seem to be disturbed due to PA exposure. These mole-cular mechanisms may play an important role in the development of severe liver damage in PA-intoxicated humans.

Pyrrolizidine Alkaloids Induce Cell Death in Human HepaRG Cells in a Structure-Dependent Manner.

Pyrrolizidine alkaloids (PAs) are a group of secondary metabolites produced in various plant species as a defense mechanism against herbivores. PAs consist of a necine base, which is esterified with one or two necine acids. Humans are exposed to PAs by consumption of contaminated food. PA intoxication in humans causes acute and chronic hepatotoxicity. It is considered that enzymatic PA toxification in hepatocytes is structure-dependent. In this study, we aimed to elucidate the induction of PA-induced cell death associated with apoptosis activation. Therefore, 22 structurally different PAs were analyzed concerning the disturbance of cell viability in the metabolically competent human hepatoma cell line HepaRG. The chosen PAs represent the main necine base structures and the different esterification types. Open-chained and cyclic heliotridine- and retronecine-type diesters induced strong cytotoxic effects, while treatment of HepaRG with monoesters did not affect cell viability. For more detailed investigation of apoptosis induction, comprising caspase activation and gene expression analysis, 14 PA representatives were selected. The proapoptotic effects were in line with the potency observed in cell viability studies. In vitro data point towards a strong structure-activity relationship whose effectiveness needs to be investigated in vivo and can then be the basis for a structure-associated risk assessment.

Role of Vitamin D in Preventing and Treating Selected Extraskeletal Diseases-An Umbrella Review.

Evidence is accumulating that vitamin D may have beneficial effects on respiratory tract, autoimmune, neuro-degenerative, and mental diseases. The present umbrella review of systematic reviews (SRs) of cohort studies and randomised controlled trials (RCTs), plus single Mendelian randomisation studies aims to update current knowledge on the potential role of vitamin D in preventing and treating these extraskeletal diseases. Altogether, 73 SRs were identified. Observational data on primary prevention suggest an inverse association between vitamin D status and the risk of acute respiratory tract infections (ARI), dementia and cognitive decline, and depression, whereas studies regarding asthma, multiple sclerosis (MS), and type 1 diabetes mellitus (T1DM) are scarce. SRs of RCTs support observational data only for the risk of ARI. No respective RCTs are available for the prevention of chronic obstructive pulmonary disease (COPD), MS, and T1DM. SRs of RCTs indicate beneficial therapeutic effects in vitamin D-deficient patients with asthma and COPD, while effects on major depression and T1DM need to be further elucidated. Mendelian randomisation studies do not consistently support the results of SRs. Since several limitations of the included SRs and existing RCTs do not permit definitive conclusions regarding vitamin D and the selected diseases, further high-quality RCTs are warranted.

Hepatotoxicity of the pesticides imazalil, thiacloprid and clothianidin - Individual and mixture effects in a 28-day study in female Wistar rats.

Humans are exposed to pesticide residues through various food products. As these residues can occur in mixtures, there is a need to investigate possible mixture effects on human health. Recent exposure studies revealed the preponderance of imazalil, thiacloprid, and clothianidin in food diets. In this study, we assessed their toxicity alone and in binary mixtures in a 28-day gavage study in female Wistar rats. Five dose levels (up to 350 mg/kg bw/day) ranging from a typical toxicological reference value to a clear effect dose were applied. Data show that the liver was a target organ of all pesticides and their mixtures. Increases in liver weight were observed and histopathological examination revealed centrilobular hepatocellular hypertrophy and cytoplasm degeneration for all treatment conditions. No accumulation of hepatic triglycerides was reported. Tissue residue analysis showed altered pesticide residues in the liver and the kidney when being in mixture as compared to the levels of pesticide residues for the single compound treatment, indicating possible toxicokinetic interactions. Overall, all mixtures appeared to follow the additivity concept, even though quantitative analysis was limited for some endpoints due to the semi-quantitative nature of the data, raising no specific concern for the risk assessment of the examined pesticides.

Structure-dependent induction of apoptosis by hepatotoxic pyrrolizidine alkaloids in the human hepatoma cell line HepaRG: Single versus repeated exposure.

Pyrrolizidine alkaloids (PA) are secondary plant compounds. PA intoxication in humans causes severe acute and chronic hepatotoxicity. However, the molecular mechanisms of PA hepatotoxicity in humans are not well understood yet. Therefore, we investigated cell death parameters in human HepaRG cells following either single (24 h) or repeated dose treatment (14 d) with structurally different PA of the retronecine (echimidine, senecionine), heliotridine (heliotrine), and otonecine type (senkirkine). After 24 h of exposure only retronecine-type PA were cytotoxic in HepaRG cells and induced apoptosis indicated by a loss of membrane asymmetry, disruption of the mitochondrial membrane potential, and increased pro-caspase and PARP cleavage. In contrast, after 14 d all four PA exerted the aforementioned effects. Furthermore, the apoptotic events caspase 3, 8 and 9 activation as well as nuclear condensation and DNA fragmentation were only detected for the retronecine-type PA after single exposure (6 h). Overall, our studies revealed a time- and structure-dependent apoptosis after PA exposure, suggesting that retronecine-type PA seem to be more potent apoptosis inducers than heliotridine- or otonecine-type PA. Furthermore, our results suggest that PA-induced apoptosis in HepaRG cells occur most probably by involving both, the extrinsic death receptor pathway as well as the intrinsic mitochondrial pathway.

Dose-dependent induction of signaling pathways by the flavonoid quercetin in human primary hepatocytes: A transcriptomic study.

SCOPE: Quercetin is widespread in plant kingdom and consumed regularly with human diet (16 mg/day). Due to reported positive effects on health, quercetin supplements with recommended doses up to 2 g/day are offered. However, molecular effects of such high doses on human liver have not been assessed yet. Therefore, molecular effects on human hepatocytes were analyzed to help assessing the risk of quercetin supplementation. METHODS AND RESULTS: Molecular effects of three different quercetin concentrations on gene expression in human hepatocytes were investigated by microarray analysis. Possible new signaling pathways were investigated using reporter gene assays. Quercetin concentrations representing the normal intake showed weak effects on mRNA expression in liver cells. In contrast, supplemental doses affect immune response and p53 signaling and might be associated with cancer. Additionally, quercetin showed inhibition of transcriptional activation and mRNA-expression of HNF4α and its target genes. Inhibitory effects were also found for FXR, LXRα, and PXR. CONCLUSION: Normal intake of quercetin seems to play a minor regulatory role, while supplement doses may have great effects on gene expression in hepatocytes. However, since it is not clarified whether such high doses of quercetin exert positive or negative effects, a careful handling of quercetin supplements is advised.