The aryl hydrocarbon receptor and retinoid receptors cross-talk at the CYP1A1 promoter in vitro

The epithelium of the small intestine plays an important role in detoxification processes due to the presence of various xenobiotic-metabolizing enzymes from phase I and II, as well as transport proteins of the ATP-binding cassette superfamily. Exposure to xenobiotics induces the expression of these proteins in the small intestine, with multiple signaling pathways stimulated by exogenous compounds converging at individual gene promoters by mechanisms which have not been fully understood yet. In this context the promoter region of the CYP1A1 gene, encoding the phase I monooxygenase cytochrome P450 1A1, was analyzed by chromatin immunoprecipitation with regard to binding of xeno-sensing receptors following stimulation of Caco-2 cells with agonists of the aryl hydrocarbon receptor (AHR) and retinoid receptors. Histone acetylation in the regulatory region of CYP1A1 was enhanced by treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or all-trans retinoic acid (at-RA). Binding of retinoid-X-receptor (RXR) α to the promoter region was detected in response to at-RA, while AHR bound to the gene promoter following its activation by TCDD. Of note, enhanced RXRα binding was also detected after AHR stimulation, and increased AHR binding was observed after retinoid receptor activation by at-RA. Exposure of Caco-2 cells to mixtures of AHR and retinoid receptor agonists yielded synergistic induction of CYP1A1 mRNA. In conclusion, the present data improve our knowledge on retinoic acid-dependent effects on CYP1A1 expression and demonstrate unexpected mixture effects by cross-talk of the different receptors

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.publishedVersio

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.publishedVersio

The pyrrolizidine alkaloid senecionine induces CYP-dependent destruction of sinusoidal endothelial cells and cholestasis in mice

Pyrrolizidine alkaloids (PAs) are widely occurring phytotoxins which can induce severe liver damage in humans and other mammalian species by mechanisms that are not fully understood. Therefore, we investigated the development of PA hepatotoxicity in vivo, using an acutely toxic dose of the PA senecionine in mice, in combination with intravital two-photon microscopy, histology, clinical chemistry, and in vitro experiments with primary mouse hepatocytes and liver sinusoidal endothelial cells (LSECs). We observed pericentral LSEC necrosis together with elevated sinusoidal marker proteins in the serum of senecionine-treated mice and increased sinusoidal platelet aggregation in the damaged tissue regions. In vitro experiments showed no cytotoxicity to freshly isolated LSECs up to 500 µM senecionine. However, metabolic activation of senecionine by preincubation with primary mouse hepatocytes increased the cytotoxicity to cultivated LSECs with an EC50 of approximately 22 µM. The cytochrome P450 (CYP)-dependency of senecionine bioactivation was confirmed in CYP reductase-deficient mice where no PA-induced hepatotoxicity was observed. Therefore, toxic metabolites of senecionine are generated by hepatic CYPs, and may be partially released from hepatocytes leading to destruction of LSECs in the pericentral region of the liver lobules. Analysis of hepatic bile salt transport by intravital two-photon imaging revealed a delayed uptake of a fluorescent bile salt analogue from the hepatic sinusoids into hepatocytes and delayed elimination. This was accompanied by transcriptional deregulation of hepatic bile salt transporters like Abcb11 or Abcc1. In conclusion, senecionine destroys LSECs although the toxic metabolite is formed in a CYP-dependent manner in the adjacent pericentral hepatocytes.</p

RNA-protein correlation of liver toxicity markers in HepaRG cells

The liver is a main target organ for the toxicity of many different compounds. While in general, in vivo testing is still routinely used for assessing the hepatotoxic potential of test chemicals, the use of in vitro models offers advantages with regard to throughput, consumption of resources, and animal welfare aspects. Using the human hepatoma cell line HepaRG, we performed a comparative evaluation of a panel of hepatotoxicity marker mRNAs and proteins after exposure of the cells to 30 different pesticidal active compounds comprising herbizides, fungicides, insecticides, and others. The panel of hepatotoxicity markers included nuclear receptor target genes, key players of fatty acid and bile acid metabolism-related pathways, as well as recently identified biomarkers of drug-induced liver injury. Moreover, marker genes and proteins were identified, for example, S100P, ANXA10, CYP1A1, and CYP7A1. These markers respond with high sensitivity to stimulation with chemically diverse test compounds already at non-cytotoxic concentrations. The potency of the test compounds, determined as an overall parameter of their ability to deregulate marker expression in vitro, was very similar between the mRNA and protein levels. Thus, this study does not only characterize the response of human liver cells to 30 different pesticides but also demonstrates that hepatotoxicity testing in human HepaRG cells yields well comparable results at the mRNA and protein levels. Furthermore, robust hepatotoxicity marker genes and proteins were identified in HepaRG cells

An expeditive and green chemo-enzymatic route to diester sinapoyl- l -malate analogues: sustainable bioinspired and biosourced UV filters and molecular heaters †

Sinapoyl malate, naturally present in plants, has proved to be an exceptional UV filter and molecular heater for plants. Although there are nowadays industrially relevant sustainable synthetic routes to sinapoyl malate, its incorporation into certain cosmetic formulations, as well as its adsorption on plant leaves, is limited by its hydrophilicity. To overcome these obstacles, it is important to find a way to effectively control the hydrophilic–lipophilic balance of sinapoyl malate to make it readily compatible with the cosmetic formulations and stick on the waxy cuticle of leaves. To this end, herein, we describe a highly regioselective chemo-enzymatic synthesis of sinapoyl malate analogues possessing fatty aliphatic chains of variable length, enabling the lipophilicity of the compounds to be modulated. The potential toxicity (i.e., mutagenicity, carcinogenicity, endocrine disruption, acute and repeated-dose toxicity), bioaccumulation, persistence and biodegradability potential of these new analogues were evaluated in silico, along with the study of their transient absorption spectroscopy, their photostability as well as their photodegradation products