Comparative whole-genome transcriptome analysis in renal cell populations reveals high tissue specificity of MAPK/ERK targets in embryonic kidney.

BACKGROUND: MAPK/ERK signaling is a well-known mediator of extracellular stimuli controlling intracellular responses to growth factors and mechanical cues. The critical requirement of MAPK/ERK signaling for embryonic stem cell maintenance is demonstrated, but specific functions in progenitor regulation during embryonic development, and in particular kidney development remain largely unexplored. We previously demonstrated MAPK/ERK signaling as a key regulator of kidney growth through branching morphogenesis and normal nephrogenesis where it also regulates progenitor expansion. Here, we performed RNA sequencing-based whole-genome expression analysis to identify transcriptional MAPK/ERK targets in two distinct renal populations: the ureteric bud epithelium and the nephron progenitors. RESULTS: Our analysis revealed a large number (5053) of differentially expressed genes (DEGs) in nephron progenitors and significantly less (1004) in ureteric bud epithelium, reflecting likely heterogenicity of cell types. The data analysis identified high tissue-specificity, as only a fraction (362) of MAPK/ERK targets are shared between the two tissues. Tissue-specific MAPK/ERK targets participate in the regulation of mitochondrial energy metabolism in nephron progenitors, which fail to maintain normal mitochondria numbers in the MAPK/ERK-deficient tissue. In the ureteric bud epithelium, a dramatic decline in progenitor-specific gene expression was detected with a simultaneous increase in differentiation-associated genes, which was not observed in nephron progenitors. Our experiments in the genetic model of MAPK/ERK deficiency provide evidence that MAPK/ERK signaling in the ureteric bud maintains epithelial cells in an undifferentiated state. Interestingly, the transcriptional targets shared between the two tissues studied are over-represented by histone genes, suggesting that MAPK/ERK signaling regulates cell cycle progression and stem cell maintenance through chromosome condensation and nucleosome assembly. CONCLUSIONS: Using tissue-specific MAPK/ERK inactivation and RNA sequencing in combination with experimentation in embryonic kidneys, we demonstrate here that MAPK/ERK signaling maintains ureteric bud tip cells, suggesting a regulatory role in collecting duct progenitors. We additionally deliver new mechanistic information on how MAPK/ERK signaling regulates progenitor maintenance through its effects on chromatin accessibility and energy metabolism.

The role of UDP-glycosyltransferases in xenobioticresistance.

Uridine diphosphate sugar-utilizing glycosyltransferases (UGTs) are an enzyme superfamily that catalyzes glycosyl residues transfer from activated nucleotide sugars to acceptor molecules. In addition to various endogenous compounds, numerous xenobiotics are substrates of UGTs. As the glycosides formed are generally less active/toxic and more hydrophilic than aglycones, UGTs effectively protect organisms from potentially harmful xenobiotics. Therefore, increased UGT expression and/or activity improve the protection of the organism and may contribute to the development of individuals that become more resistant to certain xenobiotics. While the function of UGTs in the resistance of human cancer cells to chemotherapy is now well known, other organisms and other xenobiotics have attracted much less attention. This review was designed to fill this knowledge gap by presenting complex information about the role of UGTs in xenobiotic-resistance in various organisms. This summarization and evaluation of the available information reveals that UGTs play an important role in defense against xenobiotics not only in humans, but in countless other organisms such as parasites, insects, and plants. Moreover, many recent studies clearly show the participation of UGTs in the resistance of nematodes to anthelmintics, insects to insecticides, weeds to herbicides as well as humans to various drugs (not only those used in cancer therapy but also in the treatment of epilepsy, psychiatric disorders, hypertension, hypercholesterolemia, and HIV infection). Nevertheless, although the contribution of UGTs to xenobiotic resistance in diverse organisms has become obvious, many pieces of information remain missing, for example with regard to the mechanisms of UGT regulation.

In vitro metabolism of helenalin and its inhibitory effect on human cytochrome P450 activity.

Sesquiterpene lactone helenalin is used as an antiphlogistic in European and Chinese folk medicine. The pharmacological activities of helenalin have been extensively investigated, yet insufficient information exists about its metabolic properties. The objectives of the present study were (1) to investigate the in vitro NADPH-dependent metabolism of helenalin (5 and 100 µM) using human and rat liver microsomes and liver cytosol, (2) to elucidate the role of human cytochrome P450 (CYP) enzymes in its oxidative metabolism, and (3) to study the inhibition of human CYPs by helenalin. Five oxidative metabolites were detected in NADPH-dependent human and rat liver microsomal incubations, while two reduced metabolites were detected only in NADPH-dependent human microsomal and cytosolic incubations. In human liver microsomes, the main oxidative metabolite was 14-hydroxyhelenalin, and in rat liver microsomes 9-hydroxyhelenalin. The overall oxidation of helenalin was several times more efficient in rat than in human liver microsomes. In humans, CYP3A4 and CYP3A5 followed by CYP2B6 were the main enzymes responsible for the hepatic metabolism of helenalin. The extrahepatic CYP2A13 oxidized helenalin most efficiently among CYP enzymes, possessing the Km value of 0.6 µM. Helenalin inhibited CYP3A4 (IC50 = 18.7 µM) and CYP3A5 (IC50 = 62.6 µM), and acted as a mechanism-based inhibitor of CYP2A13 (IC50 = 1.1 µM, KI = 6.7 µM, and kinact = 0.58 ln(%)/min). It may be concluded that the metabolism of helenalin differs between rats and humans, in the latter its oxidation is catalyzed by hepatic CYP2B6, CYP3A4, CYP3A5, and CYP3A7, and extrahepatic CYP2A13.

MicroRNAs mediated regulation of glutathione peroxidase 7 expression and its changes during adipogenesis.

Glutathione peroxidase 7 (GPx7) acts as an intracellular stress sensor/transmitter and plays an important role in adipocyte differentiation and the prevention of obesity related pathologies. For this reason, finding the regulatory mechanisms that control GPx7 expression is of great importance. As microRNAs (miRNAs) could participate in the regulation of GPx7 expression, we studied the inhibition of GPx7 expression by four selected miRNAs with relation to obesity and adipogenesis. The effect of the transfection of selected miRNAs mimics on GPx7 expression was tested in three cell models (HEK293, SW480, AT-MSC). The interaction of selected miRNAs with the 3'UTR of GPx7 was followed up on using a luciferase gene reporter assay. In addition, the levels of GPx7 and selected miRNAs in adipose tissue mesenchymal stem cells (AT-MSC) and mature adipocytes from four human donors were compared, with the changes in these levels during adipogenesis analyzed. Our results show for the first time that miR-137 and miR-29b bind to the 3'UTR region of GPx7 and inhibit the expression of this enzyme at the mRNA and protein level in all the human cells tested. However, no negative correlation between miR-137 nor miR-29b level and GPx7 was observed during adipogenesis. Despite the confirmed inhibition of GPx7 expression by miR-137 and miR-29b, the action of these two molecules in adipogenesis and mature adipocytes must be accompanied by other regulators.

The Modulation of Phase II Drug-Metabolizing Enzymes in Proliferating and Differentiated CaCo-2 Cells by Hop-Derived Prenylflavonoids.

Prenylflavonoids in the human organism exhibit various health-beneficial activities, although they may interfere with drugs via the modulation of the expression and/or activity of drug-metabolizing enzymes. As intestinal cells are exposed to the highest concentrations of prenylflavonoids, we decided to study the cytotoxicity and modulatory effects of the four main hop-derived prenylflavonoids on the activities and mRNA expression of the main drug-conjugating enzymes in human CaCo-2 cells. Proliferating CaCo-2 cells were used for these purposes as a model of colorectal cancer cells, and differentiated CaCo-2 cells were used as an enterocyte-like model. All the tested prenylflavonoids inhibited the CaCo-2 cells proliferation, with xanthohumol proving the most effective (IC50 8.5 µM). The prenylflavonoids modulated the activities and expressions of the studied enzymes to a greater extent in the differentiated, as opposed to the proliferating, CaCo-2 cells. In the differentiated cells, all the prenylflavonoids caused a marked increase in glutathione S-transferase and catechol-O-methyltransferase activities, while the activity of sulfotransferase was significantly inhibited. Moreover, the prenylflavonoids upregulated the mRNA expression of uridine diphosphate (UDP)-glucuronosyl transferase 1A6 and downregulated that of glutathione S-transferase 1A1/2.

The Hepatotoxicity of Alantolactone and Germacrone: Their Influence on Cholesterol and Lipid Metabolism in Differentiated HepaRG Cells.

The sesquiterpenes alantolactone (ATL) and germacrone (GER) are potential anticancer agents of natural origin. Their toxicity and biological activity have been evaluated using the differentiated HepaRG (dHepaRG) cells, a hepatocyte-like model. The half-maximal inhibitory concentrations of cell viability after 24-h treatment of dHepaRG cells are approximately 60 µM for ATL and 250 µM for GER. However, both sesquiterpenes induce reactive oxygen species (ROS) formation in non-toxic concentrations and significantly dysregulate the mRNA expression of several functional markers of mature hepatocytes. They similarly decrease the protein level of signal transducer and activator of transcription 3 (STAT3), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and their transcription target, intercellular adhesion molecule 1 (ICAM-1). Based on the results of a BATMAN-TCM analysis, the effects of sesquiterpenes on cholesterol and lipid metabolism were studied. Sesquiterpene-mediated dysregulation of both cholesterol and lipid metabolism was observed, during which these compounds influenced the protein expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and sterol regulatory element-binding protein 2 (SREBP-2), as well as the mRNA expression of HMGCR, CYP19A1, PLIN2, FASN, SCD, ACACB, and GPAM genes. In conclusion, the two sesquiterpenes caused ROS induction at non-toxic concentrations and alterations in cholesterol and lipid metabolism at slightly toxic and toxic concentrations, suggesting a risk of liver damage if administered to humans.

The Selection and Validation of Reference Genes for mRNA and microRNA Expression Studies in Human Liver Slices Using RT-qPCR.

The selection of a suitable combination of reference genes (RGs) for data normalization is a crucial step for obtaining reliable and reproducible results from transcriptional response analysis using a reverse transcription-quantitative polymerase chain reaction. This is especially so if a three-dimensional multicellular model prepared from liver tissues originating from biologically diverse human individuals is used. The mRNA and miRNA RGs stability were studied in thirty-five human liver tissue samples and twelve precision-cut human liver slices (PCLS) treated for 24 h with dimethyl sulfoxide (controls) and PCLS treated with ß-naphthoflavone (10 µM) or rifampicin (10 µM) as cytochrome P450 (CYP) inducers. Validation of RGs was performed by an expression analysis of CYP3A4 and CYP1A2 on rifampicin and ß-naphthoflavone induction, respectively. Regarding mRNA, the best combination of RGs for the controls was YWHAZ and B2M, while YWHAZ and ACTB were selected for the liver samples and treated PCLS. Stability of all candidate miRNA RGs was comparable or better than that of generally used short non-coding RNA U6. The best combination for the control PCLS was miR-16-5p and miR-152-3p, in contrast to the miR-16-5b and miR-23b-3p selected for the treated PCLS. Our results showed that the candidate RGs were rather stable, especially for miRNA in human PCLS.

Sesquiterpenes Are Agonists of the Pregnane X Receptor but Do Not Induce the Expression of Phase I Drug-Metabolizing Enzymes in the Human Liver.

Sesquiterpenes, the main components of plant essential oils, are bioactive compounds with numerous health-beneficial activities. Sesquiterpenes can interact with concomitantly administered drugs due to the modulation of drug-metabolizing enzymes (DMEs). The aim of this study was to evaluate the modulatory effects of six sesquiterpenes (farnesol, cis-nerolidol, trans-nerolidol, α-humulene, ß-caryophyllene, and caryophyllene oxide) on the expression of four phase I DMEs (cytochrome P450 3A4 and 2C, carbonyl reductase 1, and aldo-keto reductase 1C) at both the mRNA and protein levels. For this purpose, human precision-cut liver slices (PCLS) prepared from 10 patients and transfected HepG2 cells were used. Western blotting, quantitative real-time PCR and reporter gene assays were employed in the analyses. In the reporter gene assays, all sesquiterpenes significantly induced cytochrome P450 3A4 expression via pregnane X receptor interaction. However in PCLS, their effects on the expression of all the tested DMEs at the mRNA and protein levels were mild or none. High inter-individual variabilities in the basal levels as well as in modulatory efficacy of the tested sesquiterpenes were observed, indicating a high probability of marked differences in the effects of these compounds among the general population. Nevertheless, it seems unlikely that the studied sesquiterpenes would remarkably influence the bioavailability and efficacy of concomitantly administered drugs.

Antiproliferative Effects of Hop-derived Prenylflavonoids and Their Influence on the Efficacy of Oxaliplatine, 5-fluorouracil and Irinotecan in Human ColorectalC Cells.

Beer, the most popular beverage containing hops, is also frequently consumed by cancer patients. Moreover, non-alcoholic beer, owing to its nutritional value and high content of biological active compounds, is sometimes recommended to patients by oncologists. However, the potential benefits and negatives have to date not been sufficiently evaluated. The present study was designed to examine the effects of four main hop-derived prenylflavonoids on the viability, reactive oxygen species (ROS) formation, activity of caspases, and efficiency of the chemotherapeutics 5-fluorouracil (5-FU), oxaliplatin (OxPt) and irinotecan (IRI) in colorectal cancer cell lines SW480, SW620 and CaCo-2. All the prenylflavonoids exerted substantial antiproliferative effects in all cell lines, with xanthohumol being the most effective (IC50 ranging from 3.6 to 7.3 µM). Isoxanthohumol increased ROS formation and the activity of caspases-3/7, but 6-prenylnaringenin and 8-prenylnaringenin exerted antioxidant properties. As 6-prenylnaringenin acted synergistically with IRI, its potential in combination therapy deserves further study. However, other prenylflavonoids acted antagonistically with all chemotherapeutics at least in one cell line. Therefore, consumption of beer during chemotherapy with 5-FU, OxPt and IRI should be avoided, as the prenylflavonoids in beer could decrease the efficacy of the treatment.

Inter-Individual Variability in Acute Toxicity of R-Pulegone and R-Menthofuran in Human Liver Slices and Their Influence on miRNA Expression Changes in Comparison to Acetaminophen.

Monoterpenes R-pulegone (PUL) and R-menthofuran (MF), abundant in the Lamiaceae family, are frequently used in herb and food products. Although their hepatotoxicity was shown in rodent species, information about their effects in human liver has been limited. The aim of our study was to test the effects of PUL, MF and acetaminophen (APAP, as a reference compound) on cell viability and microRNA (miRNA) expression in human precision-cut liver slices. Slices from five patients were used to follow up on the inter-individual variability. PUL was toxic in all liver samples (the half-maximal effective concentration was 4.0 µg/mg of tissue), while MF and surprisingly APAP only in two and three liver samples, respectively. PUL also changed miRNA expression more significantly than MF and APAP. The most pronounced effect was a marked decrease of miR-155-5p expression caused by PUL even in non-toxic concentrations in all five liver samples. Our results showed that PUL is much more toxic than MF and APAP in human liver and that miR-155-5p could be a good marker of PUL early hepatotoxicity. Marked inter-individual variabilities in all our results demonstrate the high probability of significant differences in the hepatotoxicity of tested compounds among people.

Hepatotoxicity of monoterpenes and sesquiterpenes.

Public interest in natural therapies has increased significantly over past decades. Herbs and herbal products are extensively consumed worldwide and they are generally considered as safe. However, this may not always be true as many cases of herb-induced liver injury are reported every year. The liver is a frequent target tissue of toxicity from all classes of toxicants as liver structure and function predispose it to high sensitivity to xenobiotics. The present review is focused on the hepatotoxic properties of monoterpenes and sesquiterpenes, plant secondary metabolites that represent the major components of essential oils wildly used in folk medicines, pharmaceutical industry and cosmetics. Most of these terpenes easily enter the human body by oral absorption, penetration through the skin, or inhalation leading to measurable blood concentrations. Several studies showed that some monoterpenes (e.g., pulegone, menthofuran, camphor, and limonene) and sesquiterpenes (e.g., zederone, germacrone) exhibited liver toxicity, which is mainly based on reactive metabolites formation, increased concentration of reactive oxygen species and impaired antioxidant defense. There is a high probability that many other terpenes, without sufficiently known metabolism and effects in human liver, could also exert hepatotoxicity. Especially terpenes, that are important components of essential oils with proved hepatotoxicity, should deserve more attention. Intensive research in terpenes metabolism and toxicity represent the only way to reduce the risk of liver injury induced by essential oils and other terpenes-containing products.

Sulforaphane Alters ß-Naphthoflavone-Induced Changes in Activity and Expression of Drug-Metabolizing Enzymes in Rat Hepatocytes.

Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, exerts many beneficial effects on human health such as antioxidant, anti-inflammatory, and anticancer effects. The effect of SFN alone on drug-metabolizing enzymes (DMEs) has been investigated in numerous in vitro and in vivo models, but little is known about the effect of SFN in combination with cytochrome P450 (CYP) inducer. The aim of our study was to evaluate the effect of SFN on the activity and gene expression of selected DMEs in primary cultures of rat hepatocytes treated or non-treated with ß-naphthoflavone (BNF), the model CYP1A inducer. In our study, SFN alone did not significantly alter the activity and expression of the studied DMEs, except for the glutathione S-transferase (GSTA1) mRNA level, which was significantly enhanced. Co-treatment of hepatocytes with SFN and BNF led to a substantial increase in sulfotransferase, aldoketoreductase 1C, carbonylreductase 1 and NAD(P)H:quinone oxidoreductase 1 activity and a marked decrease in cytochrome P450 (CYP) Cyp1a1, Cyp2b and Cyp3a4 expression in comparison to the treatment with BNF alone. Sulforaphane is able to modulate the activity and/or expression of DMEs, thus shifting the balance of carcinogen metabolism toward deactivation, which could represent an important mechanism of its chemopreventive activity.

Essential Oil from Myrica rubra Leaves Potentiated Antiproliferative and Prooxidative Effect of Doxorubicin and its Accumulation in Intestinal Cancer Cells.

Essential oil from the leaves of Myrica rubra, a subtropical Asian fruit tree traditionally used in folk medicines, has a significant antiproliferative effect in several intestinal cancer cell lines. Doxorubicin belongs to the most important cytostatics used in cancer therapy. The present study was designed to evaluate the effects of defined essential oil from M. rubra leaves on efficacy, prooxidative effect, and accumulation of doxorubicin in cancer cell lines and in non-cancerous cells. For this purpose, intestinal adenocarcinoma CaCo2 cells were used. Human fibroblasts (periodontal ligament) and a primary culture of rat hepatocytes served as models of non-cancerous cells. The results showed that the sole essential oil from M. rubra has a strong prooxidative effect in cancer cells while it acts as a mild antioxidant in hepatocytes. Combined with doxorubicin, the essential oil enhanced the antiproliferative and prooxidative effects of doxorubicin in cancer cells. At higher concentrations, synergism of doxorubicin and essential oil from M. rubra was proved. In non-cancerous cells, the essential oil did not affect the toxicity of doxorubicin and the doxorubicin-mediated reactive oxygen species formation. The essential oil increased the intracellular concentration of doxorubicin and enhanced selectively the doxorubicin accumulation in nuclei of cancer cells. Taken together, essential oil from M. rubra leaves could be able to improve the doxorubicin efficacy in cancer cells due to an increased reactive oxygen species production, and the doxorubicin accumulation in nuclei of cancer cells.

Influence of diet supplementation with green tea extract on drug-metabolizing enzymes in a mouse model of monosodium glutamate-induced obesity.

PURPOSE: Consumption of dietary supplements with green tea extract (GTE) is popular for weight management, but it may be accompanied by various side effects, including interactions with drugs. The aim of the present in vivo study was to evaluate the effect of defined GTE (Polyphenon 60) in three dosage schemes on insulin, leptin and drug-metabolizing enzymes in obese mice. METHODS: Experimental obesity was induced by repeated s.c. application of monosodium glutamate to newborn mice. Green tea extract was administered in three dosage schemes in chow diet. The plasmatic levels of insulin and leptin were assayed using enzyme-linked immunosorbent assay. Enzyme activities and mRNA expressions of drug-metabolizing enzymes (totally 13) were analyzed in liver and small intestine using spectrophotometric and HPLC assays and RT-PCR, respectively. RESULTS: GTE-treatment decreased insulin and leptin levels. Eleven enzymes were significantly affected by GTE-treatment. Long-term administration of 0.01% GTE caused increase in the activity and mRNA level of cytochrome P450 3A4 (CYP3A4) ortholog in the liver as well as in the small intestine. Interestingly, short-term overdose by GTE (0.1%) had more pronounced effects on enzyme activities and mRNA expressions than long-term overdose. CONCLUSIONS: GTE-mediated induction of CYP3A4 ortholog, the main drug-metabolizing enzyme, could result in decreased efficacy of simultaneously or subsequently administered drug in obese individuals.

The modulation of carbonyl reductase 1 by polyphenols.

Carbonyl reductase 1 (CBR1), an enzyme belonging to the short-chain dehydrogenases/reductases family, has been detected in all human tissues. CBR1 catalyzes the reduction of many xenobiotics, including important drugs (e.g. anthracyclines, nabumetone, bupropion, dolasetron) and harmful carbonyls and quinones. Moreover, it participates in the metabolism of a number of endogenous compounds and it may play a role in certain pathologies. Plant polyphenols are not only present in many human food sources, but are also a component of many popular dietary supplements and herbal medicines. Many studies reviewed herein have demonstrated the potency of certain flavonoids, stilbenes and curcuminoids in the inhibition of the activity of CBR1. Interactions of these polyphenols with transcriptional factors, which regulate CBR1 expression, have also been reported in several studies. As CBR1 plays an important role in drug metabolism as well as in the protection of the organism against potentially harmful carbonyls, the modulation of its expression/activity may have significant pharmacological and/or toxicological consequences. Some polyphenols (e.g. luteolin, apigenin and curcumin) have been shown to be very potent CBR1 inhibitors. The inhibition of CBR1 seems useful regarding the increased efficacy of anthracycline therapy, but it may cause the worse detoxification of reactive carbonyls. Nevertheless, all known information about the interactions of polyphenols with CBR1 have only been based on the results of in vitro studies. With respect to the high importance of CBR1 and the frequent consumption of polyphenols, in vivo studies would be very helpful for the evaluation of risks/benefits of polyphenol interactions with CBR1.

Cranberry extract-enriched diets increase NAD(P)H:quinone oxidoreductase and catalase activities in obese but not in nonobese mice.

Consumption of antioxidant-enriched diets is 1 method of addressing obesity, which is associated with chronic oxidative stress and changes in the activity/expression of various enzymes. In this study, we hypothesized that the modulation of antioxidant enzymes and redox status through a cranberry extract (CBE)-enriched diet would differ between obese and nonobese mice. The CBE used in this study was obtained from the American cranberry (Vaccinium macrocarpon, Ericaceae), a popular constituent of dietary supplements that is a particularly rich source of (poly)phenols and has strong antioxidant properties. The present study was designed to test and compare the in vivo effects of 28-day consumption of a CBE-enriched diet (2%) on the antioxidant status of nonobese mice and mice with monosodium glutamate-induced obesity. Plasma, erythrocytes, liver, and small intestine were studied concurrently to obtain more complex information. The specific activities, protein, and messenger RNA expression levels of antioxidant enzymes as well as the levels of malondialdehyde and thiol (SH) groups were analyzed. Cranberry extract treatment increased the SH group content in plasma and the glutathione S-transferase activity in the erythrocytes of the obese and nonobese mice. In addition, in the obese animals, the CBE treatment reduced the malondialdehyde content in erythrocytes and increased NAD(P)H: quinone oxidoreductase (liver) and catalase (erythrocytes and small intestine) activities. The elevation of hepatic NAD(P)H: quinone oxidoreductase activity was accompanied by an increase in the corresponding messenger RNA levels. The effects of CBE on the activity of antioxidant enzymes and redox status were more pronounced in the obese mice compared with the nonobese mice.

The Influence of Sesquiterpenes from Myrica rubra on the Antiproliferative and Pro-Oxidative Effects of Doxorubicin and Its Accumulation in Cancer Cells.

The sesquiterpenes ß-caryophyllene, ß-caryophyllene oxide (CAO), α-humulene (HUM), trans-nerolidol (NER), and valencene (VAL) are substantial components of the essential oil from Myrica rubra leaves which has exhibited significant antiproliferative effects in several intestinal cancer cell lines, with CaCo-2 cells being the most sensitive. The present study was designed to evaluate the effects of these sesquiterpenes on the efficacy and toxicity of the anticancer drug doxorubicin (DOX) in CaCo-2 cancer cells and in primary culture of rat hepatocytes. Our results showed that HUM, NER, VAL and CAO inhibited proliferation of CaCo-2 cancer cells but they did not affect the viability of hepatocytes. CAO, NER and VAL synergistically potentiated the efficacy of DOX in cancer cells killing. All sesquiterpenes exhibited the ability to selectively increase DOX accumulation in cancer cells and did not affect DOX concentration in hepatocytes. Additionally, CAO and VAL were able to increase the pro-oxidative effect of DOX in CaCo-2 cells. Moreover, CAO mildly ameliorated DOX toxicity in hepatocytes. Based on all results, CAO seems to be the most promising compound for further testing.

Altered cytochrome P450 activities and expression levels in the liver and intestines of the monosodium glutamate-induced mouse model of human obesity.

UNLABELLED: Cytochromes P450 (CYPs) are enzymes present from bacteria to man involved in metabolism of endogenous and exogenous compounds incl. drugs. Our objective was to assess whether obesity leads to changes in activities and expression of CYPs in the mouse liver, small intestine and colon. MAIN METHODS: An obese mouse model with repeated injection of monosodium glutamate (MSG) to newborns was used. Controls were treated with saline. All mice were sacrificed at 8 months. In the liver and intestines, levels of CYP mRNA and proteins were analyzed using RT-PCR and Western blotting. Activities of CYP enzymes were measured with specific substrates of human orthologous forms. KEY FINDINGS: At the end of the experiment, body weight, plasma insulin and leptin levels as well as the specific content of hepatic CYP enzymes were increased in obese mice. Among CYP enzymes, hepatic CYP2A5 activity, protein and mRNA expression increased most significantly in obese animals. Higher activities and protein levels of hepatic CYP2E1 and 3A in the obese mice were also found. No or a weak effect on CYPs 2C and 2D was observed. In the small intestine and colon, no changes of CYP enzymes were detected except for increased expression of CYP2E1 and decreased expression of CYP3A mRNAs in the colon of the obese mice. SIGNIFICANCE: Results of our study suggest that the specific content and activities of some liver CYP enzymes (especially CYP2A5) can be increased in obese mice. Higher activity of CYP2A5 (CYP2A6 human ortholog) could lead to altered metabolism of drug substrates of this enzyme (valproic acid, nicotine, methoxyflurane).

Effect of oral administration of green tea extract in various dosage schemes on oxidative stress status of mice in vivo.

Green tea is a favorite beverage and its extracts are popular components of dietary supplements. The aim of the present in vivo study was to obtain detailed information about the effect of a standard green tea extract (Polyphenon, P), at different doses, on antioxidant enzymes and oxidative stress markers in murine blood, liver, small and large intestine. In all doses, P improved the oxidative stress status via an increased content of plasmatic SH-groups (by 21-67 %). Regarding antioxidant enzymes in tissues, the low dose of P had the best positive effect as it elevated the activity of NADPH quinone reductase in liver and small intestine, thioredoxin reductase in small intestine and hepatic superoxide dismutase. Based on these facts, consumption of green tea seems to be safe and beneficial, while consumption of dietary supplements containing high doses of catechins may disturb oxidative balance by lowering the activity of thioredoxin reductase, glutathione S-transferase, glutathione reductase and superoxide dismutase.

Drug-metabolizing and antioxidant enzymes in monosodium L-glutamate obese mice.

The prevalence of obesity is rapidly increasing across the world. Physiologic alterations associated with obesity are known to alter enzyme expression and/or activities. As drug-metabolizing and antioxidant enzymes serve as defense system against potentially toxic compounds, their modulation might have serious consequences. In this work, we studied selected antioxidant and drug-metabolizing enzymes (DME) in monosodium glutamate-mouse model of obesity. Specific activities, protein, and mRNA expressions of these enzymes in liver as well as in small intestine were compared in obese male mice and in their lean counterparts. Furthermore, expression of the NF-E2-related factor 2 (Nrf2) and its relation to obesity were tested. Obtained results showed that obesity affects expression and/or activities of some DME and antioxidant enzymes. In obese mice, upregulation of UDP-glucuronosyltransferases 1A (UGT1A), NAD(P)H:quinone oxidoreductase 1 (NQO1), nuclear transcription factor Nrf2, and downregulation of some isoforms of glutathione S-transferases (GST) were observed. Most of these changes were tissue and/or isoform specific. NQO1 seems to be regulated transcriptionally via Nrf2, but other enzymes might be regulated post-transcriptionally and/or post-translationally. Enhanced expression of Nrf2 in livers of obese mice is expected to play a role in protective adaptation. In contrast, elevated activities of NQO1 and UGT1A may cause alterations in drug pharmacokinetics in obese individuals. Moreover, decreased capacity of GST in obese animals indicates potentially reduced antioxidant defense and weaker chemoprotection.