Uridine Diphosphoglucuronyl Transferase Research Articles

In Silico Approach For Detection Of The Effect Of UGT1A1 Polymorphisms On Telmisartan Response

Hypertension is a cardiovascular disease that manifests itself with a continuous increase in systemic arterial blood pressure and can lead to serious complications over time. It is estimated that 37% of hypertensive patients receive treatment and one-third of them are under control. Telmisartan is an angiotensin receptor blocker used in the treatment of hypertension. Uridine 5'-diphospho-glucuronyltransferase 1 (UGT1A1) gene encodes the Uridine 5'-diphospho-glucuronyltransferase (UGT) enzyme and metabolizes the telmisartan. Single nucleotide polymorphisms cause amino acid, protein structure, and function to change. These changes affect the drug response and therapy. Polymorphisms of the UGT1A1 gene (rs4148323, rs28934877) cause telmisartan resistance. In this study, the SWISS-MODEL database and Chimera 1.15 ver. Programs were used to create homology models. The HOPE database was used to calculate the damage of mutation on protein structure and show the mutation effects on protein. The HDock server was used to demonstrate interactions between telmisartan and wild-type protein and, mutant type protein. It was detected that the mutant residue of UGT1A1 (rs4148323) is located in an important domain for protein activity. Mutation might disturb the protein function. rs28934877 was detected that this mutation is probably damaging to the protein. These mutations cause the loss of interactions and affect the drug response. By docking analysis, Telmisartan drug interactions were shown between wild and mutant types protein Possible drug conformation is designed for the effective treatment of patients carrying the common mutation.

Anti-inflammatory and Antioxidant Effect of Lycoperoside H against the 1,2-Dimethyl Hydrazine (DMH) Induced Colorectal Cancer in Rats.

In many developed countries, colorectal cancer is a leading cause of morbidity and mortality and its etiology is familiar to be a grouping of nutritional and environmental factors, less physical activity and hereditary factors. Lycoperoside H (LH) is a steroidal alkaloid saponin commonly found in the tomato and exhibited the various pharmacological effects. The aim of the current study was to scrutinized the anticancer effect of LH against 1,2‑Dimethyl Hydrazine (DMH) induced colorectal cancer (CRC) in rats. Subcutaneous injection of DMH (20 mg/kg) was used for induction the CRC and rats were received the oral administration of LH (10, 20 and 40 mg/kg) for 16 weeks. At the end of the investigation, the tumor incidence, weight, and body weight were calculated. Antioxidant enzymes (phase I and II), inflammatory cytokines, lipids and inflammatory markers were all examined. DMH induced rats exhibited the increased tumor incidence, reduced body weight and LH treatment significantly (p < 0.001) suppressed the tumor incidence, and enhanced the body weight. LH treatment significantly (p < 0.001) boosted the level of SOD, GPx, GSH, CAT and suppressed the MDA level. LH treatment suppressed the level cytochrome b5 (Cyto b5), cytochrome P450 (Cyto P450) and boosted the level of glutathione S‑transferase (GST), uridine diphosphoglucuronyltransferase (UDP‑GT) in the liver and colon tissue. LH also decreased the level of cytokines includes interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α); inflammatory mediators like Inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), prostaglandin (PGE2) and nuclear factor kappa B (NF-κB) in the hepatic and colon tissue. We can conclude that LH revealed the anticancer effect against the DMH induced CRC via suppression of inflammation and oxidative stress.

Activities of metabolizing enzymes in human placenta

In addition to the transfer across the placenta, placenta displays hormonal and xenobiotic metabolism, as well as enzymatic defense against oxidative stress. We analyzed aromatase (CYP19A1), uridine 5′-diphospho-glucuronyltransferase (UGT), glutathione-S-transferase (GST) and catalase (CAT) activities in over 70 placentas from nonsmokers stored at -80 °C from former perfusion studies. A wide interindividual variation in all activities was found. Longterm storage at -80 °C did not affect the activities. Ethoxyresorufin-O-deethylase (EROD, CYP1A1) was not detected in any of the studied placentas perfused with chemicals. Several compounds in placental perfusion changed statistically significantly the enzyme activities in placental tissue. Melamine and nicotine increased CYP19A1, melamine increased UGT and GST, PhIP with ethanol decreased CYP19A1 and increased GST, and PhIP with buprenorphine decreased CAT. Antipyrine in 100 μg/ml also changed the studied enzyme activities, but not statistically significantly. Because antipyrine is a reference compound in placental perfusions, its potential effects must be taken into account in human placental perfusion. Enzyme activities deserve further studies as biomarkers of placental toxicity.Finally, enzyme activities deserve further studies as biomarkers of placental toxicity.

Bilirubin and Stroke Risk Using a Mendelian Randomization Design.

Circulating bilirubin, a natural antioxidant, is associated with decreased risk of stroke. However, the nature of the relationship between the two remains unknown. We used a Mendelian randomization analysis to assess the causal effect of serum bilirubin on stroke risk in Koreans. The 14 single-nucleotide polymorphisms (SNPs) (<10-7) including rs6742078 of uridine diphosphoglucuronyl-transferase were selected from genome-wide association study of bilirubin level in the KCPS-II (Korean Cancer Prevention Study-II) Biobank subcohort consisting of 4793 healthy Korean and 806 stroke cases. Weighted genetic risk score was calculated using 14 SNPs selected from the top SNPs. Both rs6742078 (F statistics=138) and weighted genetic risk score with 14 SNPs (F statistics=187) were strongly associated with bilirubin levels. Simultaneously, serum bilirubin level was associated with decreased risk of stroke in an ordinary least-squares analysis. However, in 2-stage least-squares Mendelian randomization analysis, no causal relationship between serum bilirubin and stroke risk was found. There is no evidence that bilirubin level is causally associated with risk of stroke in Koreans. Therefore, bilirubin level is not a risk determinant of stroke.

Síndrome de Crigler-Najjar tipo 2 – um caso atípico

AIMS: The authors describe a case of Crigler-Najjar syndrome type 2, an inherited disorder of bilirubin metabolism resulting from a partial deficit of the enzyme uridine- diphospho-glucuronyl transferase (UDPG-T). CASE DESCRIPTION: A female infant of Asian ethnicity was admitted with five weeks of age by persistent jaundice since birth, with maternal report of progressive worsening. Upon physical examination the patient was active, reactive, and jaundiced, with mild axial hypotonia. Complementary examination showed increase in total bilirubin (32.94 mg/dL), with direct bilirubin of 0.94 mg/dL, and molecular study revealed two heterozygous mutations in the UGT1A1 gene (c.211G>A and c.1456T>G), consistent with Crigler-Najjar syndrome type 2. She was submitted to intensive phototherapy in combination with bile acid chelator, with a partial response. After reading the results of molecular studies, phenobarbital was started, leading to normal levels of bilirubin in two weeks. CONCLUSIONS: Crigler-Najjar syndrome type 2, although phenotypically similar to type 1, has different prognosis and treatment. In this case, early neonatal presentation and very high bilirubin values not fully yielded to intensive phototherapy, initially raised the suspicion of Crigler-Najjar syndrome type 1, which is the most severe form of the syndrome. With this report, the authors wish to draw attention to a rare cause of jaundice, which did not have its typical course.

Development of Radiometric Assays for Quantification of Enzyme Activities of the Key Enzymes of Thyroid Hormones Metabolism

We newly elaborated and adapted several radiometric enzyme assays for the determination of activities of the key enzymes engaged in the biosynthesis (thyroid peroxidase, TPO) and metabolic transformations (conjugating enzymes and iodothyronine deiodinases, IDs) of thyroid hormones (THs) in the thyroid gland and in peripheral tissues, especially in white adipose tissue (WAT). We also elaborated novel, reliable radiometric methods for extremely sensitive determination of enzyme activities of IDs of types 1, 2 and 3 in microsomal fractions of different rat and human tissues, as well as in homogenates of cultured mammalian cells. The use of optimized TLC separation of radioactive products from the unconsumed substrates and film-less autoradiography of radiochromatograms, taking advantage of storage phosphor screens, enabled us to determine IDs enzyme activities as low as 10(-18) katals. In studies of the interaction of fluoxetine (Fluox) with the metabolism of THs, we applied adapted radiometric enzyme assays for iodothyronine sulfotransferases (ST) and uridine 5'-diphospho-glucuronyltransferase (UDP-GT). Fluox is the most frequently used representative of a new group of non-tricyclic antidepressant drugs--selective serotonin re-uptake inhibitors. We used the elaborated assays for quantification the effects of Fluox and for the assessment of the degree of potential induction of rat liver ST and/or UDP-GT enzyme activities by Fluox alone or in combination with T(3). Furthermore, we studied possible changes in IDs activities in murine adipose tissue under the conditions that promoted either tissue hypertrophy (obesogenic treatment) or involution (caloric restriction), and in response to leptin, using our newly developed radiometric enzyme assays for IDs. Our results suggest that deiodinase D1 has a functional role in WAT, with D1 possibly being involved in the control of adipose tissue metabolism and/or accumulation of the tissue. Significant positive correlation between specific enzyme activity of D1 in WAT and plasma leptin levels was found. The newly developed and adapted radiometric enzyme assays proved to be very useful tools for studies of factors modulating THs metabolism, not only in model animals but also in clinical studies of human obesity.

Modulatory effect of troxerutin on biotransforming enzymes and preneoplasic lesions induced by 1,2-dimethylhydrazine in rat colon carcinogenesis

Colon cancer is the third most global oncologic problem faced by medical fraternity. Troxerutin, a flavonoid present in tea, coffee, cereal grains, and a variety of fruits and vegetables, exhibits various pharmacological and biological activities. This study was carried out to investigate the effect of troxerutin on xenobiotic metabolizing enzymes, colonic bacterial enzymes and the development of aberrant crypt foci (ACF) during 1,2-dimethylhydrazine (DMH) induced experimental rat colon carcinogenesis. Male albino Wistar rats were randomly divided into six groups. Group 1 served as control. Group 2 received troxerutin (50mg/kg b.w., p.o. every day) for 16weeks. Groups 3–6 received subcutaneous injections of DMH (20mg/kg b.w.) once a week, for the first four weeks. In addition, groups 4–6 received different doses of troxerutin (12.5, 25, 50mg/kg b.w., p.o. every day respectively) along with DMH injections. Our results reveal that DMH treated rats exhibited elevated activities of phase I enzymes such as cytochrome P450, cytochrome b5, cytochrome P4502E1, NADPH-cytochrome P450 reductase and NADH-cytochrome b5 reductase and reduced activities of phase II enzymes such as glutathione-S-transferase (GST), DT-diaphorase (DTD) and uridine diphospho glucuronyl transferase (UDPGT) in the liver and colonic mucosa of control and experimental rats. Furthermore, the activities of fecal and colonic mucosal bacterial enzymes, such as β-glucronidase, β-glucosidase, β-galactosidase and mucinase were found to be significantly higher in DMH alone treated rats than those of the control rats. On supplementation with troxerutin to DMH treated rats, the alterations in the activities of the biotransforming enzymes, bacterial enzymes and the pathological changes were significantly reversed, the effect being more pronounced when troxerutin was supplemented at the dose of 25mg/kg b.w. Thus troxerutin could be considered as a good chemopreventive agent against the formation of preneoplastic lesions in a rat model of colon carcinogenesis.

Polyinosinic Acid Blocks Adeno-Associated Virus Macrophage EndocytosisIn Vitroand Enhances Adeno-Associated Virus Liver-Directed Gene TherapyIn Vivo

Adeno-associated virus serotype 8 (AAV8) has been demonstrated to be effective for liver-directed gene therapy in humans. Although hepatocytes are the main target cell for AAV8, there is a loss of the viral vector because of uptake by macrophages and Kupffer cells. Reducing this loss would increase the efficacy of viral gene therapy and allow a dose reduction. The receptor mediating this uptake has not been identified; a potential candidate seems the macrophage scavenger receptor A (SR-A) that is involved in the endocytosis of, for instance, adenovirus. In this study we show that SR-A can mediate scAAV8 endocytosis and that blocking it with polyinosinic acid (poly[i]) reduces endocytosis significantly in vitro. Subsequently, we demonstrate that blocking this receptor improves scAAV-mediated liver-directed gene therapy in a model for inherited hyperbilirubinemia, the uridine diphospho-glucuronyl transferase 1A1-deficient Gunn rat. In male rats, preadministration of poly[i] increases the efficacy of a low dose (1×10¹¹ gc/kg) but not of a higher dose (3×10¹¹ gc/kg) scAAV8-LP1-UT1A1. Administration of poly[i] just before the vector significantly increases the correction of serum bilirubin in female rats. In these, the effect of poly[i] is seen by both doses but is more pronounced in the females receiving the low vector, where it also results in a significant increase of bilirubin glucuronides in bile. In conclusion, this study shows that SR-A mediates the endocytosis of AAV8 in vitro and in vivo and that blocking this receptor can improve the efficacy of AAV-mediated liver-directed gene therapy.

Polyinosinic acid blocks adeno-associated virus macrophage endocytosis in vitro and enhances adeno-associated virus liver directed gene therapy in vivo

Abstract Adeno-associated virus serotype 8 (AAV8) has been demonstrated to be effective for liver-directed gene therapy in humans. Although hepatocytes are the main target cell for AAV8, there is a loss of the viral vector because of uptake by macrophages and Kupffer cells. Reducing this loss would increase the efficacy of viral gene therapy and allow a dose reduction. The receptor mediating this uptake has not been identified; a potential candidate seems the macrophage scavenger receptor A (SR-A) that is involved in the endocytosis of, for instance, adenovirus. In this study we show that SR-A can mediate scAAV8 endocytosis and that blocking it with polyinosinic acid (poly[i]) reduces endocytosis significantly in vitro. Subsequently, we demonstrate that blocking this receptor improves scAAV-mediated liver-directed gene therapy in a model for inherited hyperbilirubinemia, the uridine diphospho-glucuronyl transferase 1A1–deficient Gunn rat. In male rats, preadministration of poly[i] increases the efficacy o...

Ameliorative effect of lycopene against 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced rat liver microsomal toxicity

The objective of the current study was to investigate the potential oxidative damage of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in hepatic microsomal fractions in vitro and to further elucidate the potential modulatory effect of lycopene. Rat liver microsomes were divided into four groups. Group I served as a control and is incubated with vehicle (toluene). Groups II and IV were incubated with 20 µM lycopene for 1 h before further incubating; groups III and IV with 15 nM of TCDD for further 1 h. Hydrogen peroxide (H2O2) production, lipid peroxidation (LPO), protein carbonyl content and activities of uridine 5'-diphospho-glucuronyltransferase (UDPGT) and P450 were significantly increased. Moreover, the activity of antioxidant enzymes superoxide dismutase, glutathione peroxidase, catalse, glutathione-S-transferase and glutathione reductase as well as the microsomal thiol content were significantly decreased. Incubation with lycopene (group IV) maintained near normal activities of the enzymes, normalized thiol and carbonyl content and significantly reduced LPO and H2O2 production. In conclusion, the findings of the study indicate that TCDD induces a significant oxidative stress in liver microsomes as manifested by increased LPO, H2O2 production, protein carbonyl content and activities of UDPGT and P450 and decreased antioxidant enzymes activities and thiol content. By the reversal of biochemical and oxidative markers toward normalcy, the protective role of lycopene is illuminated in rat liver microsomal toxicity.

Colon cancer chemopreventive efficacy of silibinin through perturbation of xenobiotic metabolizing enzymes in experimental rats

Our findings reported so far demonstrate that silibinin modulates gut microbial enzymes, colonic oxidative stress and Wnt/β-catenin signaling, to exert its antiproliferative effect against 1,2 di-methylhydrazine (DMH) induced colon carcinogenesis. Since xenobiotic metabolizing enzymes play a crucial role in carcinogen activation and metabolism, we aimed to explore the effect of silibinin on xenobiotic metabolizing enzymes during DMH induced colon carcinogenesis. Male albino rats were randomly divided into six groups. Group 1 served as control and group 2 rats received 50mg/kg body weight of silibinin p.o. every day. Groups 3–6 rats were given DMH at a dose of (20mg/kg body weight subcutaneously) once a week for 15weeks to induce colonic tumors. In addition to DMH, group 4 (initiation), group 5 (post-initiation) and group 6 (entire period) rats received silibinin (50mg/kg body weight, p.o., everyday) at different time points during the experimental period of 32weeks. Rats exposed to DMH alone showed increased activities of phase I enzymes (cytochrome b5, cytochrome b5 reductase, cytochromeP450, cytochromeP450 reductase, cytochromP4502E1) and decreased activities of phase II enzymes (Uridine diphospho glucuronyl transferase, Glutathione-S-transferase and DT-Diaphorase) in the liver and colonic mucosa as compared to control rats. Silibinin supplementation modulates the xenobiotic metabolizing enzymes favoring carcinogen detoxification. Evaluation of lipid peroxidation and antioxidants status showed that silibinin supplementation counteracts DMH induced hepatic and circulatory oxidative stress. Tumor burden in experimental animals was assessed both macroscopically and microscopically in the colon tissues. Our findings emphasize the potential chemopreventive action of silibinin against DMH induced colon carcinogenesis.

125I-labelled iodothyronines: useful tools for studies of effects of an antidepressant drug fluoxetine in the rat

Thyroid hormones (TH) are supposed to control the activity of some neurotransmitters (e.g., serotonin), which are hypothetically involved in the pathogenesis of depressive illness. A new group of non-tricyclic antidepressant drugs includes selective serotonine re-uptake inhibitors. The most frequently used representative of this group is fluoxetine (Fluox). We followed in the present paper the effects of Fluox, administered subchronicaly (for 25 days) to Wistar rats by itself, or in combination with 3,3′,5-triiodo-l-thyronine (T3). In studies of the interaction of Fluox with the metabolism of TH, we applied adapted radiometric enzyme assays for iodothyronine sulfotransferases (ST) and uridine 5′-diphospho-glucuronyltransferase (UDP-GT), as well as our newly developed radiometric assays for iodothyronine deiodinases (IDs) of types 1, 2 and 3 (D1, D2 and D3), using 125I-labelled iodothyronines of high specific radioactivity as substrates. We found about two-fold higher UDP-GT enzyme activities in samples of liver microsomes of rats treated with Fluox, in comparison with control rats. In contrast, the radiometric determination of ST activities in liver and kidney cytosolic fractions did not demonstrate any significant effects of the administration of Fluox, alone or together with T3, on the induction of these enzymes. However, profound changes in enzyme activities were determined in case of IDs, especially in the pituitary and cerebellum of treated rats. The adapted and newly elaborated radiometric enzyme assays proved to be very sensitive and rapid and, at the same time, reliable and robust.

Up- and down-modulation of liver cytochrome P450 activities and associated events in two murine malaria models

BackgroundThe mechanisms by which malaria up and down-regulates CYP activities are not understood yet. It is also unclear whether CYP activities are modulated during non-lethal malaria infections. This study was undertaken to evaluate the time course of CYP alterations in lethal (Plasmodium berghei ANKA) and non-lethal (Plasmodium chabaudi chabaudi) murine malaria. Additionally, hypotheses on the association of CYP depression with enhanced nitric oxide (NO) production, and of CYP2a5 induction with endoplasmic reticulum dysfunction, enhanced haem metabolism and oxidative stress were examined as well.MethodsFemale DBA-2 and C57BL/6 mice were infected with P.berghei ANKA or P. chabaudi and killed at different post-infection days. Infection was monitored by parasitaemia rates and clinical signs. NO levels were measured in the serum. Activities of CYP1a (ethoxyresorufin-O-deethylase), 2b (benzyloxyresorufin-O-debenzylase), 2a5 (coumarin-7-hydroxylase) and uridine-diphosphoglucuronyl-transferase (UGT) were determined in liver microsomes. Glutathione-S-transferase (GST) activity and concentrations of gluthatione (GSH) and thiobarbituric acid-reactive substances (TBARS) were determined in the liver. Levels of glucose-regulated protein 78 (GRP78) were evaluated by immunoblotting, while mRNAs of haemoxygenase-1 (HO-1) and inducible nitric oxide synthase (iNOS) were determined by quantitative RT-PCR.ResultsPlasmodium berghei depressed CYP1a and 2b and induced 2a5 in DBA-2 mice. In P.berghei-infected C57BL/6 mice CYP activities remained unaltered. In both strains, GST and UGT were not affected by P.berghei. Plasmodium c. chabaudi depressed CYP1a and 2b and induced 2a5 activities on the day of peak parasitaemia or near this day. CYP2a5 induction was associated with over-expression of HO-1 and enhanced oxidative stress, but it was not associated with GRP78 induction, a marker of endoplasmic reticulum stress. Plasmodium chabaudi increased serum NO on days near the parasitaemia peak in both strains. Although not elevating serum NO, P.berghei enhanced iNOS mRNA expression in the liver.ConclusionDown-regulation of CYP1a and 2b and induction of 2a5 occurred in lethal and non-lethal infections when parasitaemia rates were high. A contribution of NO for depression of CYP2b cannot be ruled out. Results were consistent with the view that CYP2a5 and HO-1 are concurrently up-regulated and suggested that CYP2a5 induction may occur in the absence of enhanced endoplasmic reticulum stress.

Hyperbilirubinemia in Lapatinib Treated Patients Is Associated with Gilbert's Syndrome UGT1A1 Polymorphism.

Abstract Background: Lapatinib, an oral EGFR/HER2 tyrosine kinase inhibitor, is approved in combination with capecitabine for the treatment of patients with advanced or metastatic HER2-positive breast cancer who have been treated previously with an anthracycline, taxane and trastuzumab. Treatment associated elevations in transaminases and bilirubin have been observed in lapatinib clinical trials. A common TA repeat polymorphism in the promoter region of uridine diphosphoglucuronyl transferase (UGT1A1*28, TA7 allele) reduces UGT1A1 enzyme levels and has been associated with hyperbilirubinemia for a range of drugs. We sought to determine the effect of the UGT1A1*28 variant on bilirubin elevation in lapatinib treated patients.Patients and Methods: Association between the UGT1A1 TA repeat polymorphism and total bilirubin levels (TBL) was examined in lapatinib treated patients using data pooled from 12 trials in advanced and metastatic breast cancer. A total of 899 lapatinib treated (as monotherapy or combined with chemotherapy) patients provided a germline DNA sample and were available for this analysis. UGT1A1 TA repeat genotype was determined by PCR amplification and sequencing. For case-control analysis, patients were defined as having normal TBL at baseline, with on-treatment TBL ≥1.5 and ≤1 times upper limit of normal for cases and controls, respectively, and at least 13 weeks of treatment for controls.Results: The UGT1A1 TA repeat polymorphism strongly correlated with lapatinib associated hyperbilirubinemia. A significant association (P=1.03x10-5, 60 cases and 396 controls) was observed. For hyperbilirubinemia cases, 21/60 (35%) were carriers of two TA7 alleles and 28/60 (47%) were carriers of one TA7 allele. Overall, when compared with other genotypes, the odds ratio (95% confidence interval) of the TA7/TA7 genotype for developing hyperbilirubinemia was 10.7 (5.3-21.6).Conclusion: These data suggest that UGT1A1*28 accounts for the major proportion of total bilirubin elevation observed during lapatinib treatment. This is consistent with an underlying predisposition to Gilbert's syndrome. Given the life-threatening nature of advanced and metastatic breast cancer and the benign clinical course of Gilbert's syndrome, these data support treatment continuation with lapatinib for patients with mild to moderate, isolated, indirect bilirubin elevation, when accompanied by appropriate ongoing safety evaluation (eg, bilirubin fractionation, ALT evaluation) to exclude liver injury risk in these patients. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1112.

Preparative Enzymatic Synthesis of Glucuronides of Zearalenone and Five of Its Metabolites

The resorcylic acid lactones zearalenone ( 1), alpha-zearalenol ( 2), beta-zearalenol ( 3), alpha-zearalanol (zeranol) ( 4), beta-zearalanol (taleranol) ( 5), and zearalanone ( 6) were converted to their glucuronides on a preparative scale in good yields. Reactions were conducted with bovine uridine 5'-diphosphoglucuronyl transferase (UDPGT) as catalyst and uridine 5'-diphosphoglucuronic acid (UDPGA) as cofactor. The glucuronides were isolated by column chromatography and characterized by NMR spectroscopy and mass spectrometry. Although the principal products were 4- O-glucuronides (i.e., linkage through a phenolic hydroxyl), significant quantities of the 6'- O-glucuronides (i.e., linkage through the aliphatic hydroxyl) of alcohols 2, 4, and 5 were also isolated. In the case of 3, the 2- O-glucuronide was isolated as the minor product. Overall isolated yields of glucuronides, performed on a 20-50 mg scale, were typically ca. 80% based on the resorcylic acid lactone starting material. LC-UV-MS (2) analysis of purified specimens revealed MS (2) fragmentations useful for defining the point of attachment of the glucuronide moiety to the zearalenone nucleus.

Changes of multiple biotransformation phase I and phase II enzyme activities in human fetal adrenals during fetal development

Fetal adrenal, which synthesizes steroid hormones, is critical to fetal growth and development. Our recent research showed that some xenobiotics could interfere with steroidogenesis and induce intrauterine growth retardation in rats. The study on the characteristics of biotransformation enzymes in fetal adrenals still seems to be important with respect to possible significance in xenobiotic-induced fetal development toxicity. In this study, the activities of several important xenobiotic-related phase I and phase II enzymes in human fetal adrenals were examined and compared with those in fetal livers. The activity and mRNA expression were determined by enzymatic analysis and RT-PCR. The levels of cytochrome (CYP)2A6, CYP2E1, and CYP3A7 isozymes in fetal adrenals were 82%, 92%, and 33% of those in fetal livers, respectively. There was a good positive correlation between adrenal CYP2A6 activity and gestational time. The values of alpha glutathione S-transferase (GST), pi-GST, and microGST in adrenals were 0.5, 4.4, and 8.3-fold of those in the livers, respectively, and the activity of adrenal pi-GST was negatively correlated with gestational time. The uridine diphosphoglucuronyl transferase activities, which were measured using p-hydroxy-biphenyl and 7-hydroxy-4-methylcoumarin as substrates, were 9% and 3%, respectively, of those in the fetal livers. Our investigation suggested that adrenal could be an important xenobiotic-metabolizing organ in fetal development and may play a potential role in xenobiotic-induced fetal development toxicity.

Biomarker responses in European eel ( Anguilla anguilla) exposed to persistent organic pollutants. A field study in the Vaccarès lagoon (Camargue, France)

A screening of relevant biomarkers was carried out in order to evaluate metabolic and cellular damages in European eels exposed to a non-point source contamination by persistent organic pollutants (POP) such as polycyclic aromatic hydrocarbons (PAH) and organochlorine compounds (OC) in a protected area, the Nature Reserve of Camargue (France). Investigations were focused on metabolic responses including detoxification mechanisms (biotransformation, antioxidant process), energy requirements and enzymatic membrane markers either involved in neuronal conduction (acetylcholinesterase, AChE) or in osmoregulation and energy metabolism (ATPases). The hepatic and muscular glycogen rates seemed to be suitable biomarkers as well as three hepatic activities involved in the protection against oxyradicals: catalase, glutathione peroxidase (SeGPx) and superoxide dismutases (SOD). The muscle and gill ATPases as well as the muscle and brain AChE showed more significant relevance in terms of biomarkers than the biotransformation enzymes: ethoxyresorufine- O-deethylase (EROD) and uridine diphospho-glucuronyl transferase (UDPGT). However, most of these enzymatic activities depend on numerous abiotic factors, which must be taken into account in such a biomarker assessment approach. Our study provides some conclusive elements to approve the use in situ of biomarkers developed from laboratory studies. It also raises a question regarding the location of contaminant impregnation in fish organ, in relation with age, development status or mode of contamination, and its influence on biomarker response. If the relevance of membrane indicators is confirmed, this study provides an original statement of the extent of the ecotoxicological threat for the aquatic species in a protected area, due to the occurrence of POP in the cell membranes.

The utility of in-vivo functional and genetic parameters of drug elimination for the estimation of irinotecan (Ir) clearance (CL)

2034 Background: Dosing nomograms based upon metabolic phenotype/genotypes may reduce the marked interpatient variability in the clearance (CL) of cytotoxics in cancer patients (pts). Irinotecan (Ir) is a relevant model drug, its active moiety (SN38), undergoes glucuronidation by uridine diphosphoglucuronyl transferase (UGT)-1A1, and with its metabolites is excreted via biliary membrane transporters P glycoprotein (Pgp) and MRP. The aim of this trial was to correlate functional imaging of biliary excretion, and significant polymorphisms in the genes coding for Pgp (exons 12, 21, 26) and UGT-1A1(*28) with Ir pharmacology. Methods: Eligible pts had colorectal cancer, adequate organ function, ECOG PS 0–2. Pre-therapy all pts had bloods taken for Pgp single nucleotide polymorphism (SNP) analysis and UGT 1A1*28 carriage and underwent hepatic 99mTc-DIDA and -MIBI scans, measures of biliary MRP and Pgp function, respectively. DIDA and MIBI hepatic extraction fraction (HEF), CL, elimination half-time and % retention at 1-hr were calculated. Pts received Ir 125mg/m2/90-min, Leucovorin 20mg/m2, 5FU 500mg/m2 bolus, days 1 & 8, q 3weekly. Plasma was taken for Ir and metabolite analysis on day 1 of cycle 1, using a limited sampling model. Results: 21 pts accrued, M:F = 16:5, Mean age 61 yrs. Mean Ir CL = 14.1 L.hr-1.m-2(CV: 20%), SN38 AUC = 253.9 ng.hr.ml-1 (CV: 56%) (n= 17). Ir CL failed to correlate with either hepatic imaging mode. SN38 AUC inversely correlated with DIDA hepatic clearance (R2 = 0.36). Pts hetero- and homozygous for Pgp exon 21 and 26 SNPs demonstrated a trend to increased SN38 AUC and similarly for pts hetero- (n = 7) and homozygous (n = 2) for UGT 1A1*28. The frequency of delayed diarrhea in cycle 1 was, Grade 0/1: n = 13 pts, Grade 2: 4, Grade 3: 4. A trend to increased DIDA HEF and % retention at 1-hr and MIBI HEF in pts with grade 2/3 diarrhea was noted relative to pts with grade 0/1 diarrhea. Conclusions: Functional hepatic imaging together with Pgp and UGT polymorphisms have shown potentially useful correlates with Ir and SN38 pharmacology and with further appraisal may allow for individualised dosing of this and other relevant cytotoxics. No significant financial relationships to disclose.

The utility of in-vivo functional and genetic parameters of drug elimination for the estimation of irinotecan (Ir) clearance (CL)

2034 Background: Dosing nomograms based upon metabolic phenotype/genotypes may reduce the marked interpatient variability in the clearance (CL) of cytotoxics in cancer patients (pts). Irinotecan (Ir) is a relevant model drug, its active moiety (SN38), undergoes glucuronidation by uridine diphosphoglucuronyl transferase (UGT)-1A1, and with its metabolites is excreted via biliary membrane transporters P glycoprotein (Pgp) and MRP. The aim of this trial was to correlate functional imaging of biliary excretion, and significant polymorphisms in the genes coding for Pgp (exons 12, 21, 26) and UGT-1A1(*28) with Ir pharmacology. Methods: Eligible pts had colorectal cancer, adequate organ function, ECOG PS 0–2. Pre-therapy all pts had bloods taken for Pgp single nucleotide polymorphism (SNP) analysis and UGT 1A1*28 carriage and underwent hepatic 99mTc-DIDA and -MIBI scans, measures of biliary MRP and Pgp function, respectively. DIDA and MIBI hepatic extraction fraction (HEF), CL, elimination half-time and % retention at 1-hr were calculated. Pts received Ir 125mg/m2/90-min, Leucovorin 20mg/m2, 5FU 500mg/m2 bolus, days 1 & 8, q 3weekly. Plasma was taken for Ir and metabolite analysis on day 1 of cycle 1, using a limited sampling model. Results: 21 pts accrued, M:F = 16:5, Mean age 61 yrs. Mean Ir CL = 14.1 L.hr-1.m-2(CV: 20%), SN38 AUC = 253.9 ng.hr.ml-1 (CV: 56%) (n= 17). Ir CL failed to correlate with either hepatic imaging mode. SN38 AUC inversely correlated with DIDA hepatic clearance (R2 = 0.36). Pts hetero- and homozygous for Pgp exon 21 and 26 SNPs demonstrated a trend to increased SN38 AUC and similarly for pts hetero- (n = 7) and homozygous (n = 2) for UGT 1A1*28. The frequency of delayed diarrhea in cycle 1 was, Grade 0/1: n = 13 pts, Grade 2: 4, Grade 3: 4. A trend to increased DIDA HEF and % retention at 1-hr and MIBI HEF in pts with grade 2/3 diarrhea was noted relative to pts with grade 0/1 diarrhea. Conclusions: Functional hepatic imaging together with Pgp and UGT polymorphisms have shown potentially useful correlates with Ir and SN38 pharmacology and with further appraisal may allow for individualised dosing of this and other relevant cytotoxics. No significant financial relationships to disclose.

Mise en évidence et validation de biomarqueurs écotoxicologiques dans la population d’anguilles d’un étang de la Réserve Naturelle Nationale de Camargue, le Vaccarès, exposée à des polluants organiques persistants

Though located in a rather remote area free from neighbouring industrial activities, the French National Nature Reserve of Camargue is however exposed to a non point source contamination by a number of persistant organic pollutants, particularly polycyclic aromatic hydrocarbons (PAH) and organochlorine compounds (OC) . Due to the nature of this contamination, investigations about validation of biomarkers in the eels of the Vaccarès lagoon have proven to be of the utmost importance. They have been focused on the characterization and validation of biomarkers related to metabolic responses including, not only the detoxification mechanisms (biotransformation, antioxidant process) and the energy requirements, but also some unspecific metabolic processes. These last ones are enzymatic membrane markers implicated either in the neuronal conduction (acetylcholinesterase, AChE) or in the osmoregulation and the energy metabolism (ATPases). The experimental results have been screened by the way of a multivariate analysis. It may be concluded that the rate of tissue glycogen seems to be a sensitive biomarker as well as three hepatic activities involved in the protection against oxyradicals : catalase, glutathion peroxidase (SeGPx) and superoxide dismutases (SOD). The muscle and gill ATPases as well as the muscle and brain AChE show more significant results in term of biomarkers than the biotransformation enzymes : ethoxyresorufine-O-deethylase (EROD) and uridine diphospho-glucuronyl transferase (UDPGT). However most of these enzymatic activities depend on a number of abiotic factors. This study brings some conclusive elements for the validation of the biomarkers use from data obtained in laboratory studies to their application in field ecotoxicology. It also raises the question about the tissue location of the contaminant in fish, in relationship to their age or their mode of contamination, and its influence on the biomarkers response. As a conclusion, if the validation of membrane indicators is confirmed, this study, by its innovative approach, provides the first statement on the extent of the ecotoxicological threat for the aquatic species in a protected area, stemming from the occurrence of persistent organic pollutants (POP) in the cell membranes.