Olive leaf (Olea europaea L. folium) extract influences liver microsomal detoxifying enzymes in rats orally exposed to 2-amino-l-methyI-6-phenyI-imidazo pyridine (PhIP).

Olive tree (Olea europaea, Oleaceae) leaf extract (OLE) exerts many biological activities. One of the most common polycyclic aromatic hydrocarbons (PAHs) that pollute the environment is 2-amino-l-methyI-6-phenyI-imidazo pyridine (PhIP). It is a food-derived carcinogen that is present in fish and meat that has been cooked at high temperatures. Due to the generation of reactive electrophilic species, phase I enzymes have the potential to cause oxidative damage. In order to safely remove these reactive species from the body, phase II detoxification (conjugation) enzymes are necessary. It is not known whether OLE could influence their activities and hence reduce the carcinogenic effects of PhIP. This study evaluated whether OLE could modulate phase I detoxifying enzymes as well as phase II enzymes that metabolize PhIP in rat liver microsomes. Four groups of rats were used: group I: no treatment; group II: OLE (10 mg/kg bw orally); group III: PhIP (0.1 mg/kg bw orally); and group IV: PhIP followed by OLE. After 4 weeks, the activities of phase I enzymes such as CYP1A1 (ethoxyresorufin O-deethylase), CYP2E1 (p-nitrophenol hydroxylase), CYP1A2 (methoxyresorufin O-demethylase), UDP-glucuronyl transferase, sulphotransferase, and glutathione-S transferase were evaluated in rat liver microsomes. Analysis of OLE by gas chromatography-mass spectrometry (GC/MS) showed various active ingredients in OLE, including 3,5-Heptadienal (C10H14O), 3,4-dimethoxy benzoic acid (C8H10O3), 4-hydroxy-3-methoxy (C8H8O4), 1,3,5-Benzenetriol (C6H6O3), hexadecanoic acid (C16H32O2), and hexadecanoic acid ethyl ester (C18H36O2). Our results showed that rats given PhIP were found to have a statistically significant (p < 0.001) reduction in the activities of CYP1A1, CYP1A2, and CYP2E1 in comparison with the control group. However, treatment with OLE enhanced their activities but not to a normal level compared with untreated groups. Administration of PhIP decreased the activities of phase II enzymes (glutathione S-transferase, UDP-glucuronyltransferase, or sulphotransferase) (p < 0.01) in comparison with the control group. Histological examination of rat livers was consistent with the biochemical changes. The administration of OLE improved the phase II enzyme activities in animals injected with PhIP. We conclude that OLE influences phase I and phase II detoxification enzymes exposed to PhIP, which may represent a new approach to attenuating carcinogenesis induced by it.

Species Difference in the Metabolism of Mulberrin in Vitro and Its Inhibitory Effect on Cytochrome P450 and UDP-Glucuronosyltransferase Enzymes.

This study aimed to evaluate the interspecies difference in metabolism of mulberrin and examine the interaction between mulberrin and CYP enzymes or recombinant human uridine 5'-diphosphate (UDP)-glucuronosyltransferase (UGT) enzymes. Liver microsomes from human (HLMs), Beagle dog (DLMs), minipig (PLMs), monkey (MLMs), rabbit (RLMs), rat (RAMs), and mouse (MIMs) were used to investigate metabolic diversity among different species. Additionally, recombinant human supersomes were used to confirm that metabolic enzymes are involved in the biotransformation of mulberrin. We also evaluated the influence of mulberrin on protein expression by Western blot analysis. Mulberrin metabolism showed significant interspecies differences. We found four and two metabolites in phase I and II reaction systems, respectively. In phase I metabolism profiles of mulberrin for HLMs, PLMs and MLMs conformed to the classic Michaelis-Menten kinetics, RAMs and MIMs followed biphasic kinetics; phase II reaction of mulberrin in HLMs, DLMs, PLMs, MLMs, RLMs, RAMs and MIMs followed biphasic kinetics. UGT1A1 were the major CYP isoforms responsible for the metabolism of mulberrin. Mulberrin showed potent inhibitory effects against CYP3A4, CYP2C9, CYP2E1, UGT1A1, UGT1A3 and UGT2B7 with IC50 values of 54.21, 9.93, 39.12, 3.84, 2.01, 16.36 µM, respectively. According to Western blot analysis, mulberrin can upregulate the protein expression of CYP2C19, and downregulate the expression levels of CYP3A5 and CYP2C9 in HepG2 cells as concentration increased. The interspecies comparisons can help find other species with metabolic pathways similar to those in humans for future in vivo studies.

Calycosin alleviates hyperbilirubin nerve injury in Ugt1-/- mice by inhibiting oxidative stress, apoptosis, and mitochondrial function.

BACKGROUND AND PURPOSE: Hyperbilirubinemia is a common condition in neonates that is associated with poor neurodevelopmental outcomes. Although studies have proposed that calycosin has a neuroprotective effect, the exact molecular mechanism underlying calycosin treatment of hyperbilirubinemia remains elusive. To fill this gap, we analyzed the mechanism of calycosin treatment in hyperbilirubinemia model mice. METHOD: Thirty neonatal mice were randomly divided into wide type (WT), Ugt1-/- and calycosin treatment group. Neuronal damage was observed with Nissl staining. Immunofluorescence staining were carried out to determine DNA damage repair and neurodegeneration. Oxidative stress was investigated by immunostaining with 4-hydroxynonenal (4-HNE). Western blot (WB) and Qpcr were used to detect relative protein and mRNA expression levels. Mitochondrial CI/CII activity of mitochondria was analyzed with a spectrophotometer. RESULT: The total bilirubin concentration was significantly higher in Ugt1-/- group compared with WT, but calycosin treatment reduced concentration of bilirubin. The total bilirubin and bilirubin/albumin ratio were significantly higher at postnatal day 4 compared with day 2. Calycosin treatment reduced serum bilirubin concentration and bilirubin/albumin ratio. After calycosin treatment, Nissl body count increased, apoptosis-related protein was downregulated and 4-HNE level decreased. Compared with Ugt-/- group, calycosin treatment increased neurons (NeuN+) and calbindin positive cells and decreased fluorojade C(FJC)positive neurons in WT group. In mitochondria, calycosin alleviated mitochondrial electron transport chain dysfunction in Ugt1-/- mice. CONCLUSION: We demonstrated that the mechanism of calycosin treatment on hyperbilirubinemia-induced Ugt1-/- was associated mainly with antioxidant effects, antiapoptosis and inhibition of normal mitochondrial function.

bFGF induces changes in hyaluronan synthase and hyaluronidase isoform expression and modulates the migration capacity of fibrosarcoma cells.

BACKGROUND: Hyaluronan (HA) a glycosaminoglycan, is capable of transmitting extracellular matrix derived signals to regulate cellular functions. In this study, we investigated whether the changes in HT1080 and B6FS fibrosarcoma cell lines HA metabolism induced by basic fibroblast growth factor (bFGF) are correlated to their migration. METHODS: Real-time PCR, in vitro wound healing assay, siRNA transfection, enzyme digestions, western blotting and immunofluorescence were utilized. RESULTS: bFGF inhibited the degradation of HA by decreasing hyaluronidase-2 expression in HT1080 cells (p=0.0028), increased HA-synthase-1 and -2 expression as we previously found and enhanced high molecular weight HA deposition in the pericellular matrix. Increased endogenous HA production (p=0.0022) and treatment with exogenous high molecular weight HA (p=0.0268) correlated with a significant decrease of HT1080 cell migration capacity. Transfection with siHAS2 and siHAS1 showed that mainly HAS1 synthesized high molecular weight HA regulates HT1080 cell motility. Induced degradation of the HA content by hyaluronidase treatment and addition of low molecular weight HA, resulted in a significant stimulation of HT1080 cells' motility (p<0.01). In contrast, no effects on B6FS fibrosarcoma cell motility were observed. CONCLUSIONS: bFGF regulates, in a cell-specific manner the migration capability of fibrosarcoma cells by modulating their HA metabolism. HA metabolism is suggested to be a potential therapeutic target in fibrosarcoma.

Glucuronidation of flavonoids by recombinant UGT1A3 and UGT1A9.

Flavonoids are highlighted for their potential roles in the prevention of oxidative stress-associated diseases. Their metabolisms in vivo, such as glucuronidation, are the key points to determine their health beneficial properties. In this paper, we tested the glucuronidation of nineteen flavonoids by both recombinant human UGT1A3 and UGT1A9. Eleven compounds could be catalyzed by both enzymes. In general, both enzymes showed moderate to high catalyzing activity to most flavonoid aglycones, while the catalyzing efficiency changed with structures. Each flavonoid produced more than one monoglucuronide with no diglucuronide detected by liquid chromatography-mass spectrometry (LC-MS). Enzymatic kinetic analysis indicated that the catalyzing efficiency (Vmax/Km) of UGT1A9 was higher than that of UGT1A3, suggesting its important role in flavonoid glucuronidation. Both human UGT1A3 and UGT1A9 preferred flavonoid aglycone to flavonoid glycoside, and their metabolism to arabinoside was stronger than to other glycosides. Of the flavonoids studied, it is the first time to report isorhamnetin, morin, silybin, kaempferol, daidzein, quercetin-3',4'-OCHO-, quercetin xylopyranoside and avicularin as substrates of UGT1A3. Apigenin, morin, daidzein, quercetin-3',4'-OCHO-, quercetin xylopyranoside and avicularin were the newly reported substrates of UGT1A9.

Influence of UGT1A7 and UGT1A9 intronic I399 genetic polymorphisms on mycophenolic acid pharmacokinetics in Japanese renal transplant recipients.

UGT1A7 and UGT1A9 are uridine diphosphate-glucuronosyltransferase isoforms involved in the glucuronidation of mycophenolic acid (MPA). The aim of this study was to elucidate MPA pharmacokinetics in UGT1A7 and UGT1A9 intronic I399 genotypes in Japanese adult renal transplant recipients. Eighty recipients were given repeated doses of combination immunosuppressive therapy consisting of mycophenolate mofetil and tacrolimus every 12 hours at a designated time (9:00 am and 9:00 pm). On day 28 after renal transplantation, plasma MPA concentrations were measured by high-performance liquid chromatography. All patients had UGT1A9 98TT/-275TT/-2152CC and UGT1A10 177GG/605CC genotypes. The UGT1A7*1/*1, *1/*2, *1/*3, *2/*3, and *3/*3 genotypes were detected in 35 (43.8%), five (6.2%), 28 (35.0%), eight (10.0%), and four (5.0%) patients, respectively, and the UGT1A9 I399C/C, C/T, and T/T genotypes were detected in 12 (15.0%), 33 (41.2%), and 35 (43.8%) patients of the 80 Japanese recipients. There were no significant differences in MPA pharmacokinetics among UGT1A7 or UGT1A9 intronic I399 genotype groups. The mean dose-adjusted area under the plasma concentration-time curve from zero to 12 hours (AUC0-12) of MPA in UGT1A7*1/*1, *1/*2, *1/*3, *2/*3, and *3/*3 were 95, 98, 99, 88, and 86 ng.h/mL/mg, respectively (P = 0.9475). The mean dose-adjusted AUC0-12 of MPA in UGT1A9 I399C/C, C/T, and T/T were 87, 99, and 95 ng.h/mL/mg, respectively (P = 0.6937). The dose-adjusted trough levels of MPA in UGT1A9 I399C/C, C/T, and T/T were 5.4, 5.5, and 4.7 ng/mL/mg (P = 0.5845). Although UGT1A7*3 and UGT1A9 I399C/C are known to have low-activity variants when studied in vitro, they do not have reduced in vivo MPA glucuronidation activity. UGT1A7 and UGT1A9 I399 polymorphisms do not contribute to interindividual differences in MPA pharmacokinetics.

Cancer pharmacogenomics: international trends.

It is well known that inter-individual variability exists in the responses to many drugs. Many nongenetic factors, such as age, sex, diet, and organ function, are known to affect the therapeutic effects of drugs. However, recent advances in pharmacogenomics have revealed that genetic polymorphisms also significantly influence both the efficacy and the toxicity of drugs. Mutations in the genes encoding drug-metabolizing enzymes, transporters, and target molecules may alter their expression, activity, or affinity to drugs, thereby influencing the drugs' pharmacokinetics and pharmacodynamics. Numerous studies have reported on the correlations between therapeutic outcomes and polymorphisms in drug-metabolizing enzymes, transporters, target molecules, and DNA repair enzymes. These pharmacogenomic discoveries are expected to be useful for the individualization and optimization of cancer chemotherapy.

UGT1A1 gene variations and irinotecan treatment in patients with metastatic colorectal cancer.

SN-38 is the active metabolite of irinotecan and it is metabolised through conjugation by uridine diphosphate glucuronosyl transferase (UGT1A1). The major toxicity of irinotecan therapy is diarrhoea, which has been related to the enzymatic activity of UGT1A1. We examined the influence of the UGT1A1 gene promoter polymorphism in the toxicity profile, in the response rate and in the overall survival (OS) in 95 patients with metastatic colorectal cancer treated with an irinotecan-containing chemotherapy. Genotypes were determined by analysing the sequence of TATA box of UGT1A1 of genomic DNA from the patients. Clinical parameters and genotypes were compared by univariate and multivariate statistical methods. The more frequent adverse effects were asthenia (34 patients), diarrhoea (29 patients) and neutropenia (20 patients). Severe diarrhoea was observed in 7/10 homozygous (70%) and 15/45 heterozygous (33%) in comparison to 7/40 (17%) wild-type patients (P=0.005). These results maintained the statistical significance in logistic regression analysis (P=0.01) after adjustment for other clinical relevant variables. The presence of severe haematological toxicity increased from wild-type patients to UGT1A1(*)28 homozygotes, but without achieving statistical significance. No relationship was found between the UGT1A1(*)28 genotypes and infection, nausea or mucositis. In univariate studies, patients with the UGT1A1(*)28 polymorphism showed a trend to a poorer OS (P=0.09). In the multivariate analysis, the genotype was not related to clinical response or to OS. The role of the UGT1A1 genotype as a predictor of toxicity in cancer patients receiving irinotecan demands the performance of a randomized trial to ascertain whether genotype-adjusted dosages of the drug can help to establish safe and effective doses not only for patients with the UGT1A1(*)28 homozygous genotype but also for those with the most common UGT1A1 6/6 or 6/7 genotype.

Interaction of tributyltin with hepatic cytochrome P450 and uridine diphosphate-glucuronosyl transferase systems of fish: in vitro studies.

Hepatic microsomes of red mullet (Mullus barbatus) and flounder (Platichthys flesus) were preincubated in the presence of a concentration range of the antifouling agent tributyltin (TBT) chloride, and the interactions of TBT with cytochrome P450 and uridine diphosphate-glucuronyl transferase systems were investigated. The enzyme systems were examined in terms of cytochrome P4501A (CYP1A)-catalyzed 7-ethoxyresorufin O-deethylase (EROD) activity and benzo[a]pyrene (BaP) metabolism and in terms of glucuronidation of testosterone and 17beta-estradiol, respectively. Ethoxyresorufin O-deethylase and BaP hydroxylase (BPH) activities of both fish species were progressively inhibited by increasing concentrations of TBT, and the effects were more pronounced for EROD than for BPH (maximal inhibition at 100 microM TBT for EROD and 250-500 microM TBT for BPH). Hydroxylated metabolites of BaP (3-hydroxy-, 7,8-dihydrodiol, and 9,10-dihydrodiol), representing 95% of the total metabolites formed, were reduced up to 75% in the presence of 100 to 500 microM TBT, whereas the formation of other metabolites was less affected. This may alter BaP toxicity and carcinogenicity. Overall, the results were consistent with a specific inhibitory effect of TBT on CYP1A in the two fish species. Additionally, the conjugation of testosterone was significantly inhibited (20%) at low TBT doses (5 microM), with no effect on the glucuronidation of estradiol.

Human udp-glucuronosyltransferases: isoform selectivity and kinetics of 4-methylumbelliferone and 1-naphthol glucuronidation, effects of organic solvents, and inhibition by diclofenac and probenecid.

The glucuronidation kinetics of the prototypic substrates 4-methylumbelliferone (4MU) and 1-naphthol (1NP) by human UDP-glucuronosyltransferases (UGT) 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B7, 2B15, and 2B17 were investigated. Where activity was demonstrated, inhibitory effects of diclofenac, probenecid, and the solvents acetone, acetonitrile, dimethyl sulfoxide, ethanol, and methanol were characterized. All isoforms except UGT1A4 glucuronidated 4MU, whereas all but UGT 1A4, 2B15, and 2B17 metabolized 1NP. However, kinetic models varied with substrate (for the same isoform) and from isoform to isoform (with the same substrate). Hyperbolic (Michaelis-Menten), substrate inhibition, and sigmoidal kinetics were variably observed for both 4MU and 1NP glucuronidation by the various UGTs. K(m) or S(50) (sigmoidal kinetics) and V(max) values varied 525- (8-4204 microM) and 1386-fold, respectively, for 4MU glucuronidation, and 1360- (1.3-1768 microM) and 37-fold, respectively, for 1NP glucuronidation. The use of a two-site model proved useful for those reactions exhibiting non-Michaelis-Menten glucuronidation kinetics. The organic solvents generally had a relatively minor effect on UGT isoform activity. UGT 2B15 and 2B17 were most susceptible to the presence of solvent, although solvent-selective inhibition was occasionally observed with other isoforms. Diclofenac and probenecid inhibited all isoforms, precluding the use of these compounds for the reaction phenotyping of xenobiotic glucuronidation pathways in human tissues. Diclofenac and probenecid K(i) values, determined for selected isoforms, ranged from 11 to 52 microM and 96 to 2452 microM, respectively. Overall, the results emphasize the need for the careful design and interpretation of kinetic and inhibition studies with human UGTs.

Cancer pharmacogenetics.

The large number of active combination chemotherapy regimens for most cancers has led to the need for better information to guide the 'standard' treatment for each patient. In an attempt to individualise therapy, pharmacogenetics and pharmacogenomics (a polygenic approach to pharmacogenetic studies) encompass the search for answers to the hereditary basis for interindividual differences in drug response. This review will focus on the results of studies assessing the effects of polymorphisms in drug-metabolising enzymes and drug targets on the toxicity and response to commonly used chemotherapy drugs. In addition, the need for polygenic pharmacogenomic strategies to identify patients at risk for adverse drug reactions will be highlighted.

[Inactivation of androgens by UDP-glucuronosyltransferases]. / L'inactivation des androgènes par les UDP-glucuronosyltransférases.

Liver, kidney, and gastrointestinal tissues are well known to express UDP-glucuronosyltransférase (UGT) enzymes which conjugate exogenous compounds. This mechanism is extremely important to eliminate several compounds from the body. Recent studies have shown that, in addition to the liver and kidney, a large number of tissues, namely the prostate and skin, also express UGT which are capable of inactivating steroids. This mechanism may be a means to modulate the action of steroids in these tissues.

The human colonic microflora influences the alterations of xenobiotic-metabolizing enzymes by catechins in male F344 rats.

As other xenobiotics, polyphenols are metabolized both by the endogenous detoxication system and the gut microflora. We hypothesized that the presence of a gut microflora may account for the effect of catechins on phase I and II xenobiotic-metabolizing enzymes and that the human bacterial metabolites may be different from those of a rodent gut microflora. Therefore, the effects of 2% (+)-catechin or 2% (-)-epicatechin were studied in germ free (GF) rats and rats inoculated with the flora of a human volunteer (HFA). In addition, the catechins were administered in ethanol as a vehicle. In the liver, (+)-catechin or (-)-epicatechin decreased the total amount of CYP450 in both GF and HFA rats while the isoenzyme CYP2E1 decreased. In GF rats only, CYP2C11 increased when compared to the rats treated with the vehicle alone. (+)-catechin increased the specific activity of UGT-chloramphenicol in GF rats only and that of cytosolic glutathion-S-transferase (GST) in HFA rats only. In the intestine, (+)-catechin and (-)-epicatechin increased the specific activity of UGT-4-methylumbelliferone in both GF and HFA rats and that of UGT- chloramphenicol in HFA rats only. In conclusion, the presence of a human flora in rats is able to modify the inducing effect of catechins on the UGT and GST activities suggesting the involvement of bacterial metabolites. The alterations on CYP 450 are independent of the presence of a human gut flora.

Long-term effects of a novel phosphorothionate (RPR-II) on detoxifying enzymes in brain, lung, and kidney rats.

The effects of a phosphorothionate, 2-butenoic acid-3-(diethoxyphosphinothioyl) methyl ester (RPR-II), on the activities of glutathione S-transferase (GST) and UDP-glucuronyltransferase (UDPGT) and the level of glutathione (GSH) were evaluated in rats after administration of RPR-II at 0.014 (low), 0.028 (medium), and 0.042 (high) mgkg(-1)day(-1) for 90 days and also at 28 days (withdrawal) after stopping treatment. Brain GST activity and GSH level decreased significantly at the high dose on the 45th and 90th days of treatment. Dose- and time-dependent decreases in GST activity and GSH was level were observed in lung at medium and high doses and in kidneys at all three doses on both the 45th and 90th days. UDPGT activity increased significantly in kidneys at the medium and high doses at 45 and 90 days. Brain and lung did not display any significant variations in UDPGT activity when compared with the control. Interestingly, the withdrawal study revealed that the effect was reversible within 28 days of cessation of treatment, when enzyme activity reverted to levels close to those of controls. The study revealed that RPR-II affected the GSH- and GST-dependent detoxification system of the treated tissues of rat and its potential to modulate the enzymes is in the order kidneys>lung>>brain. The present subacute study suggests that RPR-II may bring about physiological upsets by altering GSH- and GST-dependent events in different tissues of exposed organisms.

Allelic variations in the plaice UGT1B1 gene.

We have shown that there are large internanimal variations in hepatic phenol UDP-glucuronosyltransferase (UGT) activity in the plaice which may affect their capacity to detoxify metabolites of carcinogenic PAHS such as benzo(a)pyrene with obvious implications for their health and survival. By molecular studies we have only been able to identify one copy of the UGT1B1 gene coding for the major phenol-UGT in the diploid plaice genome and found that it is highly polymorphic about this locus. Sequencing of multiple UGT1B1 cDNA clones derived from RT-PCR revealed the existence of clustered SNPs, however, from their positions we contend that this is not sufficient to explain the observed phenotypic variability. We discovered a heterozygous null genotype derived from a dinucleotide deletion (and also found preliminary evidence for a corresponding phenotype) which has a much greater implication for survival not only in polluted environments but for embryonic survival if the gene product has an essential endogenous role. We propose that the observed interanimal variations are attributable to a combination of factors including genotypic variations and differential expression.

Molecular evidence for multiple UDP-glucuronosyltransferase gene familes in fish.

The presence of multiple distinct UGT genes in fish was derived by analysis of DNA sequence data derived the zebrafish EST project, confirming indications from previous protein purification studies in another fish species, the plaice, for a diversity of isoforms in lower vertebrates. At least 10 different UGTs can be identified from nucleotide sequence data in zebrafish. Phylogenetic analysis of exon 1 sequences of the zebrafish, plaice and human UGTs indicates that six of these genes are related to the 1A, 1B and 2 families and that a further four genes were of more ancient lineage. Importantly data for the 3' sequences of the zebrafish clones, both from the database and our own sequences of the publicly available clones did not provide any evidence for elaboration of family 1A genes by alternative splicing in this lower vertebrate.

Glucuronidation of hydroxylated polychlorinated biphenyls (PCBs).

Polychlorinated biphenyls (PCBs) may be metabolized to hydroxylated compounds. While many of these metabolites are further converted to either the glucuronic acid or the sulfate conjugates by phase II enzymes, which facilitates their excretion, some hydroxylated PCBs persist in the body. This may reflect their inability to be conjugated. A possible role of uridine diphosphate glucuronosyl transferase (UGT) in the elimination of hydroxylated metabolites of PCBs was therefore investigated. Glucuronidation studies of PCB metabolites included ones which are eliminated with relative ease and also ones which are reported to be retained in blood. Liver microsomes, prepared from male Wistar rats treated by intraperitoneal injections of phenobarbital for 3 days (400 micromol/kg/day), were used as the source of UGT. Enzyme kinetics (V(max) and K(m)) were determined for each of the metabolites. The efficiency of glucuronidation (V(max)/K(m)) was found to vary from <3 to 116 microL/min/mg and was dependent on the structure of the metabolites. Substitution of chlorine atoms on the nonhydroxylated ring greatly lowered the V(max) of the enzyme, with substitution in the meta and para positions being least favorable for enzyme activity. Steric hindrance around the hydroxyl group by chlorines on adjacent carbon atoms did not play a major role. A weak relationship between the calculated dihedral angle (planarity), pK(a), log D, and enzyme activity was determined (r(2) < 0.5). However, a stronger relationship for the surface area and surface volume of the molecule was observed (r(2) >or= 0.5). This study explains in part why some PCB metabolites persist in the body.

Liver phase I and phase II enzymatic induction and genotoxic responses of beta-naphthoflavone water-exposed sea bass.

Sea bass were exposed to 0 (control), 0.1, 0.3, 0.9, and 2.7 microM beta-naphthoflavone (BNF) for 0, 2, 8, and 16 h in order to assess the chronological and concentration relationships between BNF phase I and II biotransformation responses, such as liver cytochrome P450 (P450) content, ethoxyresorufin-O-deethylase (EROD), uridine diphosphate-glucuronosyl transferase (UDP-GT), and the genotoxic effects, measured either by erythrocytic micronuclei (EMN) or erythrocytic nuclear abnormalities (ENA) tests. Liver alanine aminotransferase (ALT) activity and liver somatic index (LSI) were also measured. A significant liver EROD activity was found at 8 h exposure, respectively, to 0.1, 0.3, 0.9, and 2.7 microM BNF. Maximal liver EROD activity increase was observed at 16 h exposure to 0.9 microM BNF, whereas the highest liver P450 was reached at 8 h exposure to 2.7 microM BNF. Liver UDP-GT activity was significantly increased at 2 h exposure to 0.1 and 0.3 microM BNF and at 8 h exposure to 0.1, 0.3, and 0.9 microM BNF, decreasing at 16 h, for every exposure concentration. Significant ENA increase was observed at 2h exposure, respectively, to 0.3, 0.9, and 2.7 microM BNF. Maximal ENA increase was observed at 16 h exposure to 0.9 microM BNF. The MN was significantly increased at 8 and 16 h exposure, respectively, to 2.7 and 0.9 microM BNF. Liver ALT activity significantly increases at 8 h exposure to 0.1 and 0.3 microM BNF, whereas liver somatic index was significantly increased from 2 to 16 h exposure for every BNF concentration. A slight liver EROD activity increase with a concomitant lack of liver UDP-GT activity is able to induce significant erythrocytic genotoxic effects. Liver UDP-GT high levels are important in sea bass BNF detoxification. However, high liver UDP-GT activity is not enough to prevent the BNF metabolite genotoxic effects on sea bass erythrocytes when liver EROD activity is induced at 2 and 8 h exposure to 0.3 and 0.9 microM BNF. The genotoxic effects measured as EMN and ENA suggest that the balance between the rates of liver BNF reactive and conjugated metabolites seems to be critical.

Overexpression of hyaluronan synthase-2 reduces the tumorigenic potential of glioma cells lacking hyaluronidase activity.

OBJECTIVE: The interactions of CD44 with hyaluronan are thought to be crucial for tumor cell attachment to the extracellular matrix, migration, and invasion. For migration to occur, however, the interactions between hyaluronan and cell surface receptors need to be transient. Hyaluronidases may facilitate the degradation of hyaluronan bound to the cell surface and thus reduce the interactions of the cells with the matrix, whereas the overproduction of hyaluronan in the absence of hyaluronidase activity may prevent cells from proliferating or invading normal surrounding tissue. METHODS: We analyzed the effects in vitro and in vivo of hyaluronan synthase-2 (HAS2) overexpression on a murine glioma cell line that is deficient in hyaluronidase activity. In addition, we evaluated the expression levels of HAS and hyaluronidase genes in human glioma cell lines and in glioma specimens. RESULTS: Increased hyaluronan synthesis had no effect on the in vitro proliferation of the cells but diminished their in vivo growth rate. Several human glioma cell lines were found to overexpress hyaluronan synthases, but they did so in conjunction with hyaluronidase Hyal2 and MGEA5 expression. Similarly, all glioblastomas multiforme expressed hyaluronidases MGEA5 and Hyal2. CONCLUSION: The data suggest that an increased synthesis of hyaluronan by astrocytoma cells is only promoting tumor cell growth in vivo if the cells express hyaluronidases as well.

Hepatobiliary disposition of 3'-azido-3'-deoxythymidine (AZT) in the rat: effect of phenobarbitone induction.

Isolated liver with a recirculating perfusate was used to study 3'-azido-3'-deoxythymidine (AZT) disposition in phenobarbitone-pretreated rats at 68 microM AZT concentration in the reservoir. Clearance of AZT in the livers obtained from control animals was 0.42 +/- 0.01 (mean +/- s.d.) mL min-1/10 g liver. Over the study period of 105 min, 12.7 +/- 2.6% of the dose was excreted in bile and of this 95% was recovered as 3'-azido-3'-deoxy-5'-O-beta-D-glucopyranuronosylthymidine (GAZT). The amount of GAZT found in the perfusate after 105 min of liver perfusion was < 1% of the AZT dose introduced into the reservoir. Phenobarbitone pretreatment of rats resulted in a 5.5-fold increase of AZT clearance. In addition, the area under the perfusate concentration-time curve (AUC0-105 min) for 3'-amino-3'-deoxythymidine (AMT) and for a catabolite of unknown structure was increased 3- and 10-fold, respectively, and the amount of AZT dose excreted in the bile was nearly doubled. Thus phenobarbitone was capable of stimulating both detoxification of AZT to GAZT and bioactivation of AZT to AMT, a catabolite known to be highly toxic to human bone marrow cells. This induction was the result of enhancement of AZT catabolism rather than its transport into the cells, since on incubation of AZT (0-250 microM) with rat isolated hepatocytes, a linear relationship between concentration and amount taken up by the cells was shown. In addition, the rate of AZT uptake was not influenced by KCN, dinitrophenol, or temperature, which is consistent with a simple diffusion of AZT through the hepatocellular membrane.(ABSTRACT TRUNCATED AT 250 WORDS)