Gut microbiome affects the metabolism of metronidazole in mice through regulation of hepatic cytochromes P450 expression.

Microbiome is now considered as a significant metabolic organ with an immense potential to influence overall human health. A number of diseases that are associated with pharmacotherapy interventions was linked with altered gut microbiota. Moreover, it has been reported earlier that gut microbiome modulates the fate of more than 30 commonly used drugs and, vice versa, drugs have been shown to affect the composition of the gut microbiome. The molecular mechanisms of this mutual relationship, however, remain mostly elusive. Recent studies indicate an indirect effect of the gut microbiome through its metabolites on the expression of biotransformation enzymes in the liver. The aim of this study was to analyse the effect of gut microbiome on the fate of metronidazole in the mice through modulation of system of drug metabolizing enzymes, namely by alteration of the expression and activity of selected cytochromes P450 (CYPs). To assess the influence of gut microbiome, germ-free mice (GF) in comparison to control specific-pathogen-free (SPF) mice were used. First, it has been found that the absence of microbiota significantly affected plasma concentration of metronidazole, resulting in higher levels (by 30%) of the parent drug in murine plasma of GF mice. Further, the significant interaction between presence/absence of the gut microbiome and effect of metronidazole application, which together influence mRNA expression of CAR, PPARα, Cyp2b10 and Cyp2c38 was determined. Administration of metronidazole itself influenced significantly mRNA expression of Cyp1a2, Cyp2b10, Cyp2c38 and Cyp2d22. Finally, GF mice have shown lower level of enzyme activity of CYP2A and CYP3A than their SPF counterparts. The results hence have shown that, beside direct bacterial metabolism, different expression and enzyme activity of hepatic CYPs in the presence/absence of gut microbiota may be responsible for the altered metronidazole metabolism.

Metabolism of 2,3-Dehydrosilybin A and 2,3-Dehydrosilybin B: A Study with Human Hepatocytes and Recombinant UDP-Glucuronosyltransferases and Sulfotransferases.

2,3-Dehydrosilybin A and 2,3-dehydrosilybin B are a pair of enantiomers formed by the oxidation of the natural flavonolignans silybin A and silybin B, respectively. However, the antioxidant activity of 2,3-dehydrosilybin molecules is much stronger than that of their precursors. Here, we investigated the biotransformation of pure 2,3-dehydrosilybin A and 2,3-dehydrosilybin B in isolated human hepatocytes, and we also aimed to identify human UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) with activity toward their respective enantiomers. After incubation with hepatocytes, both 2,3-dehydrosilybin A and 2,3-dehydrosilybin B were converted to hydroxyl derivatives, methylated hydroxyl derivatives, methyl derivatives, sulfates, and glucuronides. The products of direct conjugations predominated over those of oxidative metabolism, and glucuronides were the most abundant metabolites. Furthermore, we found that recombinant human UGTs 1A1, 1A3, 1A7, 1A8, 1A9, and 1A10 were capable of catalyzing the glucuronidation of both 2,3-dehydrosilybin A and 2,3-dehydrosilybin B. UGTs 1A1 and 1A7 showed the highest activity toward 2,3-dehydrosilybin A, and UGT1A9 showed the highest activity toward 2,3-dehydrosilybin B. The sulfation of 2,3-dehydrosilybin A and B was catalyzed by SULTs 1A1*1, 1A1*2, 1A2, 1A3, 1B1, 1C2, 1C4, and 1E1, of which SULT1A3 exhibited the highest activity toward both enantiomers. We conclude that 2,3-dehydrosilybin A and B are preferentially metabolized by conjugation reactions, and that several human UGT and SULT enzymes may play a role in these conjugations.

Methods for simultaneous and quantitative isolation of mitochondrial DNA, nuclear DNA and RNA from mammalian cells.

The aim of this study was to assess two protocols for their capacities to simultaneously isolate RNA, mtDNA and ncDNA from mammalian cells. We compared the Invitrogen TRIzol-based method and Qiagen DNeasy columns, using the HepG2 cell line and human primary glioblastoma stem cells. Both methods allowed the isolation of all three types of nucleic acids and provided similar yields in mtDNA. However, the yield in ncDNA was more than tenfold higher on columns, as observed for both cell types. Conversely, the TRIzol method proved more reproducible and was the method of choice for isolating RNA from glioblastoma cells, as demonstrated for the housekeeping genes RPLP0 and RPS9.

Identification of Human Sulfotransferases Active towards Silymarin Flavonolignans and Taxifolin.

Natural phenolic compounds are known to be metabolized by phase II metabolic reactions. In this study, we examined the in vitro sulfation of the main constituents of silymarin, an herbal remedy produced from the fruits of the milk thistle. The study focused on major flavonolignan constituents, including silybin A, silybin B, isosilybin A, isosilybin B, silychristin, and silydianin, as well as the flavonoid taxifolin. Using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS), individual flavonolignans and taxifolin were found to be sulfated by human liver and human intestinal cytosols. Moreover, experiments with recombinant enzymes revealed that human sulfotransferases (SULTs) 1A1*1, 1A1*2, 1A2, 1A3, 1B1, 1C4, and 1E1 catalyzed the sulfation of all of the tested compounds, with the exception of silydianin, which was not sulfated by SULT1B1 and SULT1C4. The sulfation products detected were monosulfates, of which some of the major ones were identified as silybin A 20-O-sulfate, silybin B 20-O-sulfate, and isosilybin A 20-O-sulfate. Further, we also observed the sulfation of the tested compounds when they were tested in the silymarin mixture. Sulfates of flavonolignans and of taxifolin were produced by incubating silymarin with all of the above SULT enzymes, with human liver and intestinal cytosols, and also with human hepatocytes, even though the spectrum and amount of the sulfates varied among the metabolic models. Considering our results and the expression patterns of human sulfotransferases in metabolic tissues, we conclude that flavonolignans and taxifolin can potentially undergo both intestinal and hepatic sulfation, and that SULTs 1A1, 1A3, 1B1, and 1E1 could be involved in the biotransformation of the constituents of silymarin.

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.

Identification of UDP-glucuronosyltransferases involved in the metabolism of silymarin flavonolignans.

Silybum marianum (milk thistle) is a medicinal plant used for producing the hepatoprotective remedy silymarin. Its main bioactive constituents, including silybin and related flavonolignans, can be metabolized directly by phase II conjugation reactions. This study was designed to identify UDP-glucuronosyltransferases (UGTs) involved in the glucuronidation of six silymarin flavonolignans, namely silybin A, silybin B, isosilybin A, isosilybin B, silychristin, and silydianin. UHPLC-MS analyses showed that all of the tested compounds, both individually and in silymarin, were glucuronidated by human liver microsomes, and that glucuronidation was the main metabolic transformation in human hepatocytes. Further, each compound was glucuronidated by multiple recombinant human UGT enzymes. UGTs 1A1, 1A3, 1A8 and 1A9 were able to conjugate all of the tested flavonolignans, and some of them were also metabolized by UGTs 1A6, 1A7, 1A10, 2B7 and 2B15. In contrast, no glucuronides were produced by UGTs 1A4, 2B4, 2B10 and 2B17. With silymarin, we found that UGT1A1 and, to a lesser extent UGT1A9, were primarily responsible for the glucuronidation of the flavonolignan constituents. It is concluded that the metabolism of silymarin flavonolignans may involve multiple UGT enzymes, of which UGT1A1 appears to play the major role in the glucuronidation. These results may be relevant for future research on the metabolism of flavonolignans in humans.

Effect of bilberry extract (Vaccinium myrtillus L.) on drug-metabolizing enzymes in rats.

Vaccinium myrtillus L. (bilberry) fruit is a blue-colored berry with a high content of anthocyanins. These bioactive secondary metabolites are considered to play a major role in the health-promoting properties of bilberries. Our in vivo study was designed to assess the possible influence of bilberry extract on drug-metabolizing enzymes (DMEs). Rats were exposed to bilberry extract in drinking water at two concentrations (0.15 and 1.5 g/L). Selected DMEs were determined (mRNA expression and enzymatic activity) after 29 and 58 days in rat liver. In addition, a panel of antioxidant, physiological, biochemical and hematological parameters was studied; these parameters did not demonstrate any impact of bilberry extract on the health status of rats. A significant increase in activity was observed in cytochrome P450 (CYP) 2C11 (131% of control) and CYP2E1 (122% of control) after a 29-day administration, while the consumption of a higher concentration for a longer time led to a mild activity decrease. Slight changes were observed in some other DMEs, but they remained insignificant from a physiological perspective. According to our results, we conclude that the consumption of bilberries as a food supplement should not pose a risk of interacting with co-administered drugs based on their metabolism.

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.

The metabolism of flubendazole in human liver and cancer cell lines.

Flubendazole (FLU), a benzimidazole anthelmintic drug widely used in veterinary medicine, has been approved for the treatment of gut-residing nematodes in humans. In addition, FLU is now considered a promising anti-cancer agent. Despite this, information about biotransformation of this compound in human is lacking. Moreover, there is no information regarding whether cancer cells are able to metabolize FLU in order to deactivate it. For these reasons, the present study was designed to identify all metabolites of Phase I and Phase II of FLU in human liver and in various cancer cells using ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. Precision-cut human liver slices and 9 cell lines of different origin (breast, colon, oral cavity) were used as in vitro model systems. Our study showed that FLU with a reduced carbonyl group (FLUR) is the only FLU metabolite formed in the human liver. All human cancer cell lines were able to form FLUR. In addition, methylated FLUR was detected in breast cells MCF7 and intestinal SW480 cells. The accumulation of FLU and its reduction to FLUR markedly differed among cells. The extent of FLU reduction was in a good correlation with the detected expression level of carbonyl reductase 1. In most cases, FLU entered in a higher amount and was reduced to a lesser extent in proliferating (metastatic) cells than in differentiated (non-cancerous, non-metastatic) ones. These results support the promising potential of FLU in anti-cancer therapy.

Induction of xenobiotic-metabolizing enzymes in hepatocytes by beta-naphthoflavone: Time-dependent changes in activities, protein and mRNA levels.

In the present study, time-dependency of the induction effect of a selective inducer on the activity, protein and mRNA levels of cytochromes P450 1A1/2 (CYP1A1/2), NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione S-transferases (GSTA), in primary culture of rat hepatocytes was tested and evaluated. To show the differences in responses of tested enzymes, the common aryl hydrocarbon receptor (AhR) ligand agonist, beta-naphthoflavone (BNF), was used. Induction of CYP1A1/2 by BNF was detected at all time intervals and at all levels (i.e., mRNA, protein, enzyme activity). Different responses of NQO1 and GSTA upon BNF treatment were observed. Our results demonstrate that the responses of different xenobiotic-metabolizing enzymes to the inducer vary in time and depend on the measured parameter. For these reasons, an induction study featuring only one-time interval treatment and/ or one parameter testing could produce misleading information.

The impact of sesquiterpenes ß-caryophyllene oxide and trans-nerolidol on xenobiotic-metabolizing enzymes in mice in vivo.

1. Sesquiterpenes, constituents of plant essential oil, are popular bioactive compounds due to the positive effect on human health, but their potential toxicity and possible herb-drug interactions are often omitted. In our in vivo study, we followed up the effect of p.o. administration of two sesquiterpenes ß-caryophyllene oxide (CAO) and trans-nerolidol (NER) on various xenobiotic-metabolizing enzymes in mice liver and small intestine. 2. To spot the early effect of studied compounds, enzymatic activity and mRNA levels were assessed 6 and 24 h after single dose. 3. CAO and NER markedly increased cytochromes P450 (CYP2B, 3A, 2C) activity and mRNA levels in both tissues. Liver also showed elevated activity of aldo-ketoreductase 1C and carbonyl reductase after treatment. Contrary, sesquiterpenes decreased NAD(P)H:quinone oxidoreductase 1 activity in small intestine. Among conjugation enzymes, only liver sulfotransferase activity was increased by sesquiterpenes. 4. Our results document that single dose of sesquiterpenes modulate activities and expression of several xenobiotic-metabolizing enzymes.

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.

The inhibitory effects of ß-caryophyllene, ß-caryophyllene oxide and α-humulene on the activities of the main drug-metabolizing enzymes in rat and human liver in vitro.

Sesquiterpenes, the main components of plant essential oils, are often taken in the form of folk medicines and dietary supplements. Several sesquiterpenes possess interesting biological activities but they could interact with concurrently administered drugs via inhibition of drug-metabolizing enzymes. Therefore, the present study was designed to test the potential inhibitory effect of tree structurally relative sesquiterpenes ß-caryophyllene (CAR), ß-caryophyllene oxide (CAO) and α-humulene (HUM) on the activities of the main drug-metabolizing enzymes. For this purpose, rat and human hepatic subcellular fractions were incubated with CAR, CAO or HUM together with specific substrates for oxidation, reduction and conjugation enzymes and their coenzymes. HPLC, spectrophotometric and spectrofluorimetric analyses of product formations were used. All tested sesquiterpenes significantly inhibited cytochromes P4503A (CYP3A) activities in rats as well as in human hepatic microsomes, with CAO being the strongest inhibitor. A non-competitive type of inhibition was found. On the other hand, none of the tested sesquiterpenes significantly affected the activities of carbonyl-reducing enzymes (CBR1, AKRs, NQO1) or conjugation enzymes (UGTs, GSTs, SULTs, COMT). As CYP3A enzymes metabolize many drugs, their inhibition by CAO, CAR and HUM might affect the pharmacokinetics of concurrently administered drugs. Similar results obtained in rat and human hepatic microsomes indicate that rats could be used for further testing of possible drug-sesquiterpenes interactions in vivo.

Nerolidol and Farnesol Inhibit Some Cytochrome P450 Activities but Did Not Affect Other Xenobiotic-Metabolizing Enzymes in Rat and Human Hepatic Subcellular Fractions.

Sesquiterpenes, 15-carbon compounds formed from three isoprenoid units, are the main components of plant essential oils. Sesquiterpenes occur in human food, but they are principally taken as components of many folk medicines and dietary supplements. The aim of our study was to test and compare the potential inhibitory effect of acyclic sesquiterpenes, trans-nerolidol, cis-nerolidol and farnesol, on the activities of the main xenobiotic-metabolizing enzymes in rat and human liver in vitro. Rat and human subcellular fractions, relatively specific substrates, corresponding coenzymes and HPLC, spectrophotometric or spectrofluorometric analysis of product formation were used. The results showed significant inhibition of cytochromes P450 (namely CYP1A, CYP2B and CYP3A subfamilies) activities by all tested sesquiterpenes in rat as well as in human hepatic microsomes. On the other hand, all tested sesquiterpenes did not significantly affect the activities of carbonyl-reducing enzymes and conjugation enzymes. The results indicate that acyclic sesquiterpenes might affect CYP1A, CYP2B and CYP3A mediated metabolism of concurrently administered drugs and other xenobiotics. The possible drug-sesquiterpene interactions should be verified in in vivo experiments.

Catechins Variously Affect Activities of Conjugation Enzymes in Proliferating and Differentiated Caco-2 Cells.

The knowledge of processes in intestinal cells is essential, as most xenobiotics come into contact with the small intestine first. Caco-2 cells are human colorectal adenocarcinoma that once differentiated, exhibit enterocyte-like characteristics. Our study compares activities and expressions of important conjugation enzymes and their modulation by green tea extract (GTE) and epigallocatechin gallate (EGCG) using both proliferating (P) and differentiated (D) caco-2 cells. The mRNA levels of the main conjugation enzymes were significantly elevated after the differentiation of Caco-2 cells. However, no increase in conjugation enzymes' activities in differentiated cells was detected in comparison to proliferating ones. GTE/EGCG treatment did not affect the mRNA levels of any of the conjugation enzymes tested in either type of cells. Concerning conjugation enzymes activities, GTE/EGCG treatment elevated glutathione S-transferase (GST) activity by approx. 30% and inhibited catechol-O-methyltransferase (COMT) activity by approx. 20% in differentiated cells. On the other hand, GTE as well as EGCG treatment did not significantly affect the activities of conjugation enzymes in proliferating cells. Administration of GTE/EGCG mediated only mild changes of GST and COMT activities in enterocyte-like cells, indicating a low risk of GTE/EGCG interactions with concomitantly administered drugs. However, a considerable chemo-protective effect of GTE via the pronounced induction of detoxifying enzymes cannot be expected as well.

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.

Effect of standardized cranberry extract on the activity and expression of selected biotransformation enzymes in rat liver and intestine.

The use of dietary supplements containing cranberry extract is a common way to prevent urinary tract infections. As consumption of these supplements containing a mixture of concentrated anthocyanins and proanthocyanidins has increased, interest in their possible interactions with drug-metabolizing enzymes has grown. In this in vivo study, rats were treated with a standardized cranberry extract (CystiCran®) obtained from Vaccinium macrocarpon in two dosage schemes (14 days, 0.5 mg of proanthocyanidins/kg/day; 1 day, 1.5 mg of proanthocyanidins/kg/day). The aim of this study was to evaluate the effect of anthocyanins and proanthocyanidins contained in this extract on the activity and expression of intestinal and hepatic biotransformation enzymes: cytochrome P450 (CYP1A1, CYP1A2, CYP2B and CYP3A), carbonyl reductase 1 (CBR1), glutathione-S-transferase (GST) and UDP-glucuronosyl transferase (UGT). Administration of cranberry extract led to moderate increases in the activities of hepatic CYP3A (by 34%), CYP1A1 (by 38%), UGT (by 40%), CBR1 (by 17%) and GST (by 13%), while activities of these enzymes in the small intestine were unchanged. No changes in the relative amounts of these proteins were found. Taken together, the interactions of cranberry extract with simultaneously administered drugs seem not to be serious.