A screening strategy for bioactive components of Bu-Zhong-Yi-Qi-Tang regulating spleen-qi deficiency based on "endobiotics-targets-xenobiotics" association network.

ETHNOPHARMACOLOGICAL RELEVANCE: Bu-Zhong-Yi-Qi-Tang is a famous traditional Chinese medicine formula that has been prevalent in China for over 700 years to treat spleen-qi deficiency related diseases, such as gastrointestinal and respiratory disorders. However, the bioactive components responsible for regulating spleen-qi deficiency remain unclear and have puzzled many researchers. AIM OF THE STUDY: The current study focuses on efficacy evaluation of regulating spleen-qi deficiency and screening the bioactive components of Bu-Zhong-Yi-Qi-Tang. MATERIALS AND METHODS: The effects of Bu-Zhong-Yi-Qi-Tang were evaluated through blood routine examination, immune organ index, and biochemical analysis. Metabolomics was utilized to analyze the potential endogenous biomarkers (endobiotics) in the plasma, and the prototypes (xenobiotics) of Bu-Zhong-Yi-Qi-Tang in the bio-samples were characterized using ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry. Then, these endobiotics were used as "bait" to predict targets based on network pharmacology and to screen potential bioactive components from the absorbed prototypes in the plasma by constructing an "endobiotics-targets-xenobiotics" association network. Further, the anti-inflammatory activities of representative compounds (calycosin and nobiletin) were validated through poly(I:C)-induced pulmonary inflammation mice model. RESULTS: Bu-Zhong-Yi-Qi-Tang exhibited immunomodulatory and anti-inflammatory activities in spleen-qi deficiency rat, as supported by the observation of increased levels of D-xylose and gastrin in serum, an increase in the thymus index and number of lymphocytes in blood, as well as a reduction in the level of IL-6 in bronchoalveolar lavage fluid. Furthermore, plasma metabolomic analysis revealed a total of 36 Bu-Zhong-Yi-Qi-Tang related endobiotics, which were mainly enriched in primary bile acids biosynthesis, the metabolism of linoleic acid, and the metabolism of phenylalanine pathways. Meanwhile, 95 xenobiotics were characterized in plasma, urine, small intestinal contents, and tissues of spleen-qi deficiency rat after Bu-Zhong-Yi-Qi-Tang treatment. Using an integrated association network, six potential bioactive components of Bu-Zhong-Yi-Qi-Tang were screened. Among them, calycosin was found to significantly reduce the levels of IL-6 and TNF-α in the bronchoalveolar lavage fluid, increase the number of lymphocytes, while nobiletin dramatically decreased the levels of CXCL10, TNF-α, GM-CSF, and IL-6. CONCLUSION: Our study proposed an available strategy for screening bioactive components of BYZQT regulating spleen-qi deficiency based on "endobiotics-targets-xenobiotics" association network.

Pueraria montana (Kudzu vine) Ameliorate the Inflammation and Oxidative Stress against Fe-NTA Induced Renal Cancer.

Renal tissue plays a crucial function in maintaining homeostasis, making it vulnerable to xenobiotic toxicity. Pueraria montana has more beneficial potential against the various diseases and has long history used as a traditional Chinese medicine. But its effect against the renal cancer not scrutinize. The goal of this study is to see if Pueraria montana can protect rats from developing kidney tumors caused by diethylnitrosamine (DEN) and ferric nitrite (Fe-NTA). Wistar rats was selected for the current study and DEN (use as an inducer) and Fe-NTA (promoter) for induction the renal cancer. For 22 weeks, the rats were given orally Pueraria montana (12.5, 25, and 50 mg/kg) treatment. At regular intervals, the body weight and food intake were calculated. The rats were macroscopically evaluated for identification of cancer in the renal tissue. The renal tumor makers, renal parameters, antioxidant enzymes, phase I and II enzymes, inflammatory cytokines and mediators were estimated at end of the experimental study. Pueraria montana treated rats displayed the suppression of renal tumors, incidence of the tumors along with suppression of tumor percentage. Pueraria montana treated rats significantly (p < 0.001) increased body weight and suppressed the renal weight and food intake. It also reduced the level of renal tumor marker ornithine decarboxylase (ODC) and [3H] thymidine incorporation along with suppression of renal parameter such as uric acid, blood urea nitrogen (BUN), urea and creatinine. Pueraria montana treatment significantly (p < 0.001) altered the level of phase enzymes and antioxidant. Pueraria montana treatment significantly (p < 0.001) repressed the level of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and improved the level of interleukin-10 (IL-10). Pueraria montana treatment suppressed the level of prostaglandin (PGE2), cyclooxygenase-2 (COX-2), nuclear kappa B factor (NF-κB) and transforming growth factor beta 1 (TGF-ß1). Pueraria montana suppressed the inflammatory necrosis, size the bowman capsules in the renal histopathology. Pueraria montana exhibited the chemoprotective effect via dual mechanism such as suppression of inflammatory reaction and oxidative stress.

Ficus dubia latex extract prevent DMH-induced rat early colorectal carcinogenesis through the regulation of xenobiotic metabolism, inflammation, cell proliferation and apoptosis.

Ficus dubia latex is recognized as a remedy in Asian traditional medicine with various therapeutic effects. The present study aimed to determine the preventive action of Ficus dubia latex extract (FDLE) on 1,2-dimethylhydrazine (DMH)-induced rat colorectal carcinogenesis and its mechanisms. The experiment included an initiation model in which rats were orally administered with FDLE daily for 1 week before DMH injection until the end of the experiment, while only after DMH injection until the end in the post-initiation model. The results firstly indicated that FDLE treatment could reduce the level of methylazoxymethanol (MAM) in rat colonic lumen by inhibition of the activities of both phase I xenobiotic metabolizing enzymes in the liver and ß-glucuronidase in the colon, leading to reduced DNA methylation in colonic mucosal cells, related to the number of ACF in the initiation stage. Besides, FDLE modulated the inflammation which could suppress the growth and induce apoptosis of aberrant colonic mucosal cells, leading to retardation of ACF multiplicity. Therefore, FDLE showed the ability to suppress the DMH-induced rat ACF formation and inflammation promoted growth of ACF. In conclusion, FDLE had the potential to prevent carcinogens-induced rat colorectal carcinogenesis in the initiation stage.

Gene Expression Profiling of 1α,25(OH)2 D3 Treatment in 2D/3D Human Hepatocyte Models Reveals CYP3A4 Induction but Minor Changes in Other Xenobiotic-Metabolizing Genes.

SCOPE: CYP3A4 is the most important drug-metabolizing enzyme regulated via the vitamin D receptor (VDR) in the intestine. However, less is known about VDR in the regulation of CYP3A4 and other drug-metabolizing enzymes in the liver. METHODS AND RESULTS: This study investigates whether 1α,25-dihydroxyvitamin D3 (1α,25(OH)2 D3 ) regulates major cytochrome P450 enzymes, selected phase I and II enzymes, and transporters involved in xenobiotic and steroidal endobiotic metabolism in 2D and 3D cultures of human hepatocytes. The authors found that 1α,25(OH)2 D3 increases hepatic CYP3A4 expression and midazolam 1'-hydroxylation activity in 2D hepatocytes. The results are confirmed in 3D spheroids, where 1α,25(OH)2 D3 has comparable effect on CYP3A4 mRNA expression as 1α-hydroxyvitamin D3 , an active vitamin D metabolite. Other regulated genes such as CYP1A2, AKR1C4, SLC10A1, and SLCO4A1 display only mild changes in mRNA levels after 1α,25(OH)2 D3 treatment in 2D hepatocytes. Expression of other cytochrome P450, phase I and phase II enzyme, or transporter genes are not significantly influenced by 1α,25(OH)2 D3 . Additionally, the effect of VDR activation on CYP3A4 mRNA expression is abolished by natural dietary compound sulforaphane, a common suppressor of pregnane X receptor (PXR) and constitutive androstane receptor (CAR). CONCLUSION: This study proposes that VDR or vitamin D supplementation is unlikely to significantly influence liver detoxification enzymes apart from CYP3A4.

Using QSAR Models to Predict Mitochondrial Targeting by Small-Molecule Xenobiotics Within Living Cells.

Prediction of mitochondrial targeting, or prediction of exclusion from mitochondria, of small-molecule xenobiotics (biocides, drugs, probes, toxins) can be achieved using an algorithm derived from QSAR modeling. Application of the algorithm requires knowing the chemical structures of all ionic species of the xenobiotic compound in question, and for certain numerical structure parameters (AI, CBN, log P, pK a, and Z) to be obtained for all such species. Procedures for specification of the chemical structures; estimation of the structure parameters; and application of the algorithm are described in an explicit protocol.

Comparative Studies on the Hepatoprotective Effect of White and Coloured Rice Bran Oil against Acetaminophen-Induced Oxidative Stress in Mice through Antioxidant- and Xenobiotic-Metabolizing Systems.

Rice bran oil (RBO) comprises various nutrients and phytochemicals which exhibit several health benefits. There are no studies regarding the functional effects of different colours of RBO. This study was aimed to compare the constituents and antioxidant activities of white rice bran oil (WRBO) and coloured rice bran oil (CRBO). Each RBO showed similar free fatty acid profiles. However, greater amounts of vitamin E, phytosterols, carotenoids, and chlorophylls were found in CRBO, which had lower γ-oryzanol content than WRBO. Oxidative stress was induced in male mice by an overdose of acetaminophen (APAP) at 300 mg/kg body weight. The mice were then fed with RBO at the equivalent dose to 100 mg/kg body weight of γ-oryzanol three hours later and sacrificed six hours after APAP treatment. The administration of 100 mg γ-oryzanol equivalent in CRBO ameliorated APAP-induced hepatotoxicity in mice more strongly than 100 mg γ-oryzanol equivalent in WRBO, as evidenced by the significant reduction of serum ALT, hepatocellular necrosis, and hepatic lipid peroxidation. CRBO could improve xenobiotic-metabolizing and antioxidant enzyme activities, including glutathione S-transferase, superoxide dismutase, glutathione peroxidase, and glutathione reductase, and also increase mRNA expression of various antioxidant-responsive genes. Vitamin E, phytosterols, carotenoids, and chlorophyll might be the protective compounds in CRBO that alleviate APAP-induced hepatotoxicity through the interruption of APAP metabolism and the activation of antioxidant systems at both transcriptional and enzymatic levels. These findings might provide a protective role of CRBO on oxidative stress associated with several degenerative diseases.

Metabolomic Approaches to Study Chemical Exposure-Related Metabolism Alterations in Mammalian Cell Cultures.

Biological organisms are constantly exposed to an immense repertoire of molecules that cover environmental or food-derived molecules and drugs, triggering a continuous flow of stimuli-dependent adaptations. The diversity of these chemicals as well as their concentrations contribute to the multiplicity of induced effects, including activation, stimulation, or inhibition of physiological processes and toxicity. Metabolism, as the foremost phenotype and manifestation of life, has proven to be immensely sensitive and highly adaptive to chemical stimuli. Therefore, studying the effect of endo- or xenobiotics over cellular metabolism delivers valuable knowledge to apprehend potential cellular activity of individual molecules and evaluate their acute or chronic benefits and toxicity. The development of modern metabolomics technologies such as mass spectrometry or nuclear magnetic resonance spectroscopy now offers unprecedented solutions for the rapid and efficient determination of metabolic profiles of cells and more complex biological systems. Combined with the availability of well-established cell culture techniques, these analytical methods appear perfectly suited to determine the biological activity and estimate the positive and negative effects of chemicals in a variety of cell types and models, even at hardly detectable concentrations. Metabolic phenotypes can be estimated from studying intracellular metabolites at homeostasis in vivo, while in vitro cell cultures provide additional access to metabolites exchanged with growth media. This article discusses analytical solutions available for metabolic phenotyping of cell culture metabolism as well as the general metabolomics workflow suitable for testing the biological activity of molecular compounds. We emphasize how metabolic profiling of cell supernatants and intracellular extracts can deliver valuable and complementary insights for evaluating the effects of xenobiotics on cellular metabolism. We note that the concepts and methods discussed primarily for xenobiotics exposure are widely applicable to drug testing in general, including endobiotics that cover active metabolites, nutrients, peptides and proteins, cytokines, hormones, vitamins, etc.

Nutrients, Nutraceuticals, and Xenobiotics Affecting Renal Health.

Chronic kidney disease (CKD) affects 8⁻16% of the population worldwide. In developed countries, the most important risk factors for CKD are diabetes, hypertension, and obesity, calling into question the importance of educating and acting on lifestyles and nutrition. A balanced diet and supplementation can indeed support the maintenance of a general health status, including preservation of renal function, and can help to manage and curb the main risk factors for renal damage. While the concept of protein and salt restriction in nephrology is historically acknowledged, the role of some nutrients in renal health and the importance of nutrition as a preventative measure for renal care are less known. In this narrative review, we provide an overview of the demonstrated and potential actions of some selected nutrients, nutraceuticals, and xenobiotics on renal health and function. The direct and indirect effects of fiber, protein, fatty acids, curcumin, steviol glycosides, green tea, coffee, nitrates, nitrites, and alcohol on kidney health are reviewed here. In view of functional and personalized nutrition, understanding the renal and systemic effects of dietary components is essential since many chronic conditions, including CKD, are related to systemic dysfunctions such as chronic low-grade inflammation.

Natural and environmental oestrogens induce TGFB1 synthesis in oviduct cells.

Autocrine/paracrine factors generated in response to 17ß-oestradiol (E2), within the oviduct, facilitate early embryo development for implantation. Since transforming growth factor beta 1 (TGFB1) plays a key role in embryo implantation, regulation of its synthesis by E2 may be of biological/pathophysiological relevance. Here, we investigated whether oviduct cells synthesize TGFB1 and whether E2 and environmental oestrogens (EOEs; xenoestrogens and phytoestrogens) modulate its synthesis. Under basal conditions, bovine oviduct cells (OCs; oviduct epithelial cells and oviduct fibroblasts; 1:1 ratio) synthesized TGFB1. E2 concentration-dependent induced TGFB1 levels in OCs and these effects were mimicked by some, but not all EOEs (genistein, biochanin A and 4-hydroxy-2',4',6'-trichlorobiphenyl, 4-hydroxy-2',4',6'-dichlorobiphenyl); moreover, EOEs enhanced (P < 0.05) the stimulatory effects of E2 on TGFB1 synthesis. The OCs expressed oestrogen receptors alpha and beta and aryl hydrocarbon; moreover, co-treatment with ER antagonist ICI182780 blocked the stimulatory effects of E2 and EOEs on TGFB1 synthesis. Treatment with non-permeable E2-BSA failed to induce TGFB1, thereby ruling out the involvement of membrane ERs. Cycloheximide (protein synthesis inhibitor) blocked E2-induced TGFB1 synthesis providing evidence for de novo synthesis. The stimulatory effects of E2 and EOEs, were inhibited (P < 0.05) by MAPK inhibitor (PD98059), whereas intracellular-Ca2+ chelator (BAPTA-AM) and adenylyl cyclase inhibitor (SQ22536) abrogated the effects of E2, but not EOEs, suggesting that post-ER effects of E2 and EOEs involve different pathways. Our results provide the first evidence that in OCs, E2 and EOEs stimulate TGFB1 synthesis via an ER-dependent pathway. Exposure of the oviduct to EOEs may result in continuous/sustained induction of TGFB1 levels in a non-cyclic fashion and may induce deleterious effects on reproduction.

Pharmaceutical Metabolism in Fish: Using a 3-D Hepatic In Vitro Model to Assess Clearance.

At high internal doses, pharmaceuticals have the potential for inducing biological/pharmacological effects in fish. One particular concern for the environment is their potential to bioaccumulate and reach pharmacological levels; the study of these implications for environmental risk assessment has therefore gained increasing attention. To avoid unnecessary testing on animals, in vitro methods for assessment of xenobiotic metabolism could aid in the ecotoxicological evaluation. Here we report the use of a 3-D in vitro liver organoid culture system (spheroids) derived from rainbow trout to measure the metabolism of seven pharmaceuticals using a substrate depletion assay. Of the pharmaceuticals tested, propranolol, diclofenac and phenylbutazone were metabolised by trout liver spheroids; atenolol, metoprolol, diazepam and carbamazepine were not. Substrate depletion kinetics data was used to estimate intrinsic hepatic clearance by this spheroid model, which was similar for diclofenac and approximately 5 fold higher for propranolol when compared to trout liver microsomal fraction (S9) data. These results suggest that liver spheroids could be used as a relevant and metabolically competent in vitro model with which to measure the biotransformation of pharmaceuticals in fish; and propranolol acts as a reproducible positive control.

A specialist herbivore pest adaptation to xenobiotics through up-regulation of multiple Cytochrome P450s.

The adaptation of herbivorous insects to their host plants is hypothesized to be intimately associated with their ubiquitous development of resistance to synthetic pesticides. However, not much is known about the mechanisms underlying the relationship between detoxification of plant toxins and synthetic pesticides. To address this knowledge gap, we used specialist pest Colorado potato beetle (CPB) and its host plant, potato, as a model system. Next-generation sequencing (454 pyrosequencing) was performed to reveal the CPB transcriptome. Differential expression patterns of cytochrome P450 complement (CYPome) were analyzed between the susceptible (S) and imidacloprid resistant (R) beetles. We also evaluated the global transcriptome repertoire of CPB CYPome in response to the challenge by potato leaf allelochemicals and imidacloprid. The results showed that more than half (51.2%) of the CBP cytochrome P450 monooxygenases (P450s) that are up-regulated in the R strain are also induced by both host plant toxins and pesticide in a tissue-specific manner. These data suggest that xenobiotic adaptation in this specialist herbivore is through up-regulation of multiple P450s that are potentially involved in detoxifying both pesticide and plant allelochemicals.

Xenoestrogens Ethinyl Estradiol and Zearalenone Cause Precocious Puberty in Female Rats via Central Kisspeptin Signaling.

Xenoestrogens from synthetic or natural origin represent an increasing risk of disrupted endocrine functions including the physiological activity of the hypothalamo-pituitary-gonad axis. Ethinyl estradiol (EE2) is a synthetic estrogen used in contraceptive pills, whereas zearalenone (ZEA) is a natural mycoestrogen found with increasing prevalence in various cereal crops. Both EE2 and ZEA are agonists of estrogen receptor-α and accelerate puberty. However, the neuroendocrine mechanisms that are responsible for this effect remain unknown. Immature female Wistar rats were treated with EE2 (10 µg/kg), ZEA (10 mg/kg), or vehicle for 10 days starting from postnatal day 18. As a marker of puberty, the vaginal opening was recorded and neuropeptide and related transcription factor mRNA levels were measured by quantitative real time PCR and in situ hybridization histochemistry. Both ZEA and EE2 accelerated the vaginal opening, increased the uterine weight and the number of antral follicles in the ovary, and resulted in the increased central expression of gnrh. These changes occurred in parallel with an earlier increase of kiss1 mRNA in the anteroventral and rostral periventricular hypothalamus and an increased kisspeptin (KP) fiber density and KP-GnRH appositions in the preoptic area. These changes are compatible with a mechanism in which xenoestrogens overstimulate the developmentally unprepared reproductive system, which results in an advanced vaginal opening and an enlargement of the uterus at the periphery. Within the hypothalamus, ZEA and EE2 directly activate anteroventral and periventricular KP neurons to stimulate GnRH mRNA. However, GnRH and gonadotropin release and ovulation are disrupted due to xenoestrogen-mediated inhibitory KP signaling in the arcuate nucleus.

Zinc finger nuclease-mediated gene knockout results in loss of transport activity for P-glycoprotein, BCRP, and MRP2 in Caco-2 cells.

Membrane transporters P-glycoprotein [P-gp; multidrug resistance 1 (MDR1)], multidrug resistance-associated protein (MRP) 2, and breast cancer resistance protein (BCRP) affect drug absorption and disposition and can also mediate drug-drug interactions leading to safety/toxicity concerns in the clinic. Challenges arise with interpreting cell-based transporter assays when substrates or inhibitors affect more than one actively expressed transporter and when endogenous or residual transporter activity remains following overexpression or knockdown of a given transporter. The objective of this study was to selectively knock out three drug efflux transporter genes (MDR1, MRP2, and BCRP), both individually as well as in combination, in a subclone of Caco-2 cells (C2BBe1) using zinc finger nuclease technology. The wild-type parent and knockout cell lines were tested for transporter function in Transwell bidirectional assays using probe substrates at 5 or 10 µM for 2 hours at 37°C. P-gp substrates digoxin and erythromycin, BCRP substrates estrone 3-sulfate and nitrofurantoin, and MRP2 substrate 5-(and-6)-carboxy-2',7'-dichlorofluorescein each showed a loss of asymmetric transport in the MDR1, BCRP, and MRP2 knockout cell lines, respectively. Furthermore, transporter interactions were deduced for cimetidine, ranitidine, fexofenadine, and colchicine. Compared with the knockout cell lines, standard transporter inhibitors showed substrate-specific variation in reducing the efflux ratios of the test compounds. These data confirm the generation of a panel of stable Caco-2 cell lines with single or double knockout of human efflux transporter genes and a complete loss of specific transport activity. These cell lines may prove useful in clarifying complex drug-transporter interactions without some of the limitations of current chemical or genetic knockdown approaches.

HepG2 cells as an in vitro model for evaluation of cytochrome P450 induction by xenobiotics.

Although various in vitro assays have been developed to evaluate the cytochrome P450 (CYP)-inducing potential of drug candidates, there is a continuing need for the development of a reliable model in drug discovery. The objective of the present study was to compare CYP induction by chemicals in HepG2 cells with Huh7, NKNT-3, and reverted NKNT-3 cells. HepG2 cells showed more similarity to human liver than the other cell lines in comparisons of the expression of cellular proteins. In evaluation of basal CYP activity, Huh7 cells exhibited the highest CYP1A2 and CYP3A4 activity, and HepG2 cells showed the highest CYP2B6 activity. The inducibility of CYP1A2, CYP2B6, and CYP3A4 by prototypical inducers was determined using enzyme assay, immunoblot analysis, and real-time PCR. Among the cells tested, HepG2 cells were highly responsive to CYP inducers, such as 3-methylcholanthrene for CYP1A2 and phenobarbital for CYP2B6 and CYP3A4. Moreover, HepG2 cells were responsive to various CYP1A2, CYP2B6, and CYP3A4 inducers as determined using fluorogenic and LC-MS/MS substrates. Thus, HepG2 cells may be comparable to human hepatocytes for the evaluation of CYP induction or slightly less sensitive. These results suggest HepG2 cells as a cell-based model in screening for CYP inducers in drug discovery.

Dose-dependent functionality and toxicity of green tea polyphenols in experimental rodents.

A large number of physiologically functional foods are comprised of plant polyphenols. Their antioxidative activities have been intensively studied for a long period and proposed to be one of the major mechanisms of action accounting for their health promotional and disease preventive effects. Green tea polyphenols (GTPs) are considered to possess marked anti-oxidative properties and versatile beneficial functions, including anti-inflammation and cancer prevention. On the other hand, some investigators, including us, have uncovered their toxicity at high doses presumably due to pro-oxidative properties. For instance, both experimental animal studies and epidemiological surveys have demonstrated that GTPs may cause hepatotoxicity. We also recently showed that diets containing high doses (0.5-1%) of a GTP deteriorated dextran sodium sulfate (DSS)-induced intestinal inflammation and carcinogenesis. In addition, colitis mode mice fed a 1% GTP exhibited symptoms of nephrotoxicity, as indicated by marked elevation of serum creatinine level. This diet also increased thiobarbituric acid-reactive substances, a reliable marker of oxidative damage, in both kidneys and livers even in normal mice, while the expression levels of antioxidant enzymes and heat shock proteins (HSPs) were diminished in colitis and normal mice. Intriguingly, GTPs at 0.01% and 0.1% showed hepato-protective activities, i.e., they significantly suppressed DSS-increased serum aspartate aminotransferase and alanine aminotransferase levels. Moreover, those diets remarkably restored DSS-down-regulated expressions of heme oxygenase-1 and HSP70 in livers and kidneys. Taken together, while low and medium doses of GTPs are beneficial in colitis model mice, unwanted side-effects occasionally emerge with high doses. This dose-dependent functionality and toxicity of GTPs are in accordance with the concept of hormesis, in which mild, but not severe, stress activates defense systems for adaptation and survival.

Development of a stable cell line with an intact PGC-1α/ERRα axis for screening environmental chemicals.

The estrogen-related receptor α (ERRα) and the peroxisome proliferator-activated receptor γ (PPARγ) coactivator 1α (PGC-1α) play critical roles in the control of several physiological functions, including the regulation of genes involved in energy homeostasis. However, little is known about the ability of environmental chemicals to disrupt or modulate this important bioenergetics pathway in humans. The goal of this study was to develop a cell-based assay system with an intact PGC-1α/ERRα axis that could be used as a screening assay for detecting such chemicals. To this end, we successfully generated several stable cell lines expressing PGC-1α and showed that the reporter driven by the native ERRα hormone response unit (AAB-Luc) is active in these cell lines and that the activation is PGC-1α-dependent. Furthermore, we show that this activation can be blocked by the ERRα selective inverse agonist, XCT790. In addition, we find that genistein and bisphenol A further stimulate the reporter activity, while kaempferol has minimal effect. These cell lines will be useful for identifying environmental chemicals that modulate this important pathway.

Evaluation of hydro-alcoholic extract of Eclipta alba for its multidrug resistance reversal potential: an in vitro study.

The development of multidrug resistance (MDR) causes problems in the chemotherapy of human cancer. The present study was designed to evaluate and establish the role of Eclipta alba as MDR reversal agent using multidrug resistant hepatocellular carcinoma cell line (DR-HepG2). To develop DR-HepG2, hepatocellular carcinoma cell line (HepG2) was transfected with 2-Acetylaminofluorene (AAF) and Aflatoxin B1 (AFB). Cytotoxic effects of the Eclipta alba hydroalcoholic extract (EAE) and standard anti-ancer drug Doxorubicin (DOX) were determined in DR-HepG2 and the parental cells HepG2 using MTT assay. The expression level of MDR1 gene and P-glycoprotein (P-gp) level was analyzed by RT-PCR and western blotting. From the present investigation, it was found that EAE (10 and 20 µg/ml) could significantly inhibit cell proliferation in DR-HepG2 whereas DOX (0.5 µg/ml) could not because of enhancement effect of MDR1/P-gp. This study demonstrated for the first time the antiproliferative activities of EAE in multidrug resistant DR-HepG2 cells. The findings revealed that Eclipta alba components are effective inhibitors of MDR1/P-gp.

Precision cut lung slices as an efficient tool for in vitro lung physio-pharmacotoxicology studies.

1.We review the specific approaches for lung tissue slices preparation and incubation systems and the research application fields in which lung slices proved to be a very efficient alternative to animal experimentation for biomechanical, physiological, pharmacological and toxicological approaches. 2.Focus is made on air-liquid interface dynamic organ culture systems that allow direct tissue exposure to complex aerosol and that best mimic in vivo lung tissue physiology. 3.A compilation of research applications in the fields of vascular and airway reactivity, mucociliary transport, polyamine transport, xenobiotic biotransformation, chemicals toxicology and complex aerosols supports the concept that precision cut lung slices are a very efficient tool maintaining highly differentiated functions similar to in vivo lung organ when kept under dynamic organ culture. They also have been successfully used for lung gene transfer efficiency assessment, for lung viral infection efficiency assessment, for studies of tissue preservation media and tissue post-conditioning to optimize lung tissue viability before grafting. 4.Taken all together, the reviewed studies point to a great interest for precision cut lung slices as an efficient and valuable alternative to in vivo lung organ experimentation.

Evaluation of the metabolism and hepatotoxicity of xenobiotics utilizing precision-cut slices.

1. Precision-cut liver slices are a valuable in vitro model system to study the metabolism and toxicity of xenobiotics. Liver slices retain tissue architecture so that all cell types are present and intercellular communication between the various cell types is retained. 2. Precision-cut liver slices from humans and other species have been used to study pathways of phase I (e.g. cytochrome P450-dependent biotransformations) and II (e.g. conjugation with D-glucuronic acid, sulphate and glutathione) metabolism of a wide range of xenobiotics. 3. Liver slices can also be employed to investigate the induction and inhibition of xenobiotic metabolizing enzymes and to obtain kinetic data on the rates of metabolism of xenobiotics. 4. Precision-cut liver slices from humans and other species have been used to study the toxicity of a wide variety of xenobiotics. Toxicity can be assessed by various techniques including gene expression, morphological examination and a wide range of biochemical endpoints. 5. Precision-cut liver slices can be utilized to examine species differences in hepatic xenobiotic metabolism and xenobiotic-induced toxicity, thus permitting comparisons between animal species and humans.