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PubMed1998

Inhibition of intestinal microflora beta-glucuronidase modifies the distribution of the active metabolite of the antitumor agent, irinotecan hydrochloride (CPT-11) in rats.

PURPOSE: SN-38, a metabolite of irinotecan hydrochloride (CPT-11), is considered to play a key role in the development of diarrhea as well as in the antitumor activity of CPT-11. We have previously found that the inhibition of beta-glucuronidase, which hydrolyzes detoxified SN-38 (SN-38 glucuronide) to reform SN-38, in the lumen by eliminating the intestinal microflora with antibiotics, markedly ameliorates the intestinal toxicity of CPT-11 in rats. In this study we compared the disposition of CPT-11 and its metabolites in rats treated with and without antibiotics. METHODS: Rats were given drinking water containing 1 mg/ml penicillin and 2 mg/ml streptomycin from 5 days before the administration of CPT-11 (60 mg/kg i.v.) and throughout the experiment. CPT-11, SN-38 glucuronide and SN-38 concentrations in the blood, intestinal tissues and intestinal luminal contents were determined by HPLC. RESULTS: Antibiotics had little or no effect on the pharmacokinetics of CPT-11, SN-38 glucuronide or SN-38 in the blood, or in the tissues or contents of the small intestine, which has less beta-glucuronidase activity in its luminal contents. In contrast, antibiotics markedly reduced the AUC1-24 h of SN-38 (by about 85%) in the large intestine tissue without changing that of CPT-11, and this was accompanied by a complete inhibition of the deconjugation of SN-38 glucuronide in the luminal contents. CONCLUSIONS: These results suggest that SN-38, which results from the hydrolysis of SN-38 glucuronide by beta-glucuronidase in the intestinal microflora, contributes considerably to the distribution of SN-38 in the large intestine tissue, and that inhibition of the beta-glucuronidase activity by antibiotics results in decreased accumulation of SN-38 in the large intestine.

Administration, Oral↗
PubMed1989

Use of antisense RNA to help identify a genomic clone for the 5' region of mouse beta-glucuronidase.

It was possible to gauge the inhibition of mouse beta-glucuronidase expression by injecting RNA, made from both strands of subclones of a cosmid containing the complete gene, into mouse blastomeres at the four-cell stage. Although our initial screen did not identify the 5' region, we were able to isolate a subclone containing homology to 20 bp coding for N-terminal amino acids of rat and human beta-glucuronidase structural genes. Antisense RNA prepared from one strand of the 350 bp Pst I subclone inhibited beta-glucuronidase expression by 89% while RNA prepared from the other strand had little effect. The subclone appears to correspond to the 350 bp fragment identified by others as one including the ATG start site of mouse beta-glucuronidase.

Animals↗
PubMedDate not supplied

Hydrolysis of glycyrrhizin to 18 beta-glycyrrhetyl monoglucuronide by lysosomal beta-D-glucuronidase of animal livers.

Glycyrrhizin (GL), a main constituent of liquorice, was hydrolysed to 18 beta-glycyrrhetic acid mono-beta-D-glucuronide (GAMG, glycyrrhetyl monoglucuronide) by rat liver homogenate, and the hydrolytic activity was localized in the lysosomes among the same subcellular fractions as acid beta-D-glucuronidase activity (p-nitrophenyl beta-D-glucuronide (pNPG)-hydrolysing activity). Rat liver lysosomes hydrolysed GAMG to 18 beta-glycyrrhetic acid (GA) at only 30% rate compared with the rate of GL to GAMG. GA was also produced slowly from GL after time lag by the lysosomes. Thus, GL seems to be first hydrolysed to GAMG, which was successively hydrolysed slowly to GA. GL-hydrolysing activity was released together with acid beta-D-glucuronidase activity from the lysosomes by sonication. Both activities from the sonicated lysosomes were eluted coincidentally on Sephacryl S-300 and butyl-Toyopearl 650M column chromatography, indicating that both activities are exhibited by the same enzyme. Moreover, GL-hydrolysing activity was inhibited strongly with D-saccharic acid 1,4-lactone, a specific inhibitor beta-D-glucuronidases of various origins. pH optimum of GL-hydrolysing activity was found to be 5.6, different from that (less than 4.0) of pNPG-hydrolysing activity. Km for GL was found to be 2 x 10(-5) M. Although hepatic lysosomes from mouse and cattle hydrolysed GAMG to GA similarly to those from rat, the hydrolysis of GAMG was not detected in lysosomes of human and porcine livers. Accordingly, lysosomal beta-D-glucuronidases from human and porcine livers converted GL to GAMG only.

Animals↗
PubMed1981

beta-Glucuronidase-resistant bilirubin glucuronide isomers in cholestatic liver disease--determination of bilirubin metabolites in serum by means of high-pressure liquid chromatography.

"Direct reacting bilirubin" in serum of patients with cholestatic liver disease and in serum of bile duct-ligated rats consists of a complex mixture of bilirubin metabolites. These metabolites were studied by means of high-pressure liquid chromatography. Bilirubin glucuronides in normal bile are beta-glycosidic 1-O-acyl conjugates which are completely hydrolyzed on incubation with beta-glucuronidase. Cholestatic serum contains glucuronide and non-glucuronide bilirubin metabolites. The glucuronides were only partially hydrolyzable with beta-glucuronidase. Compernolle et al. [11] showed that the 1-O-acyl bond of bilirubin glucuronides is labile and prone to migrate from the C1 position at the glucuronosyl residue to positions C2, C3 and C4. The isomerisation products are non-beta-glycosidic, beta-glucuronidase-resistant conjugates. The main beta-glucuronidase-resistant conjugates in cholestatic serum were characterized as: non-beta-glycosidic bilirubin monoglucuronide, non-beta-glycosidic diglucuronide and a diglucuronide isomer with beta-glycosidic and non-beta-glycosidic glucuronosyl groups. Moreover, a substantial amount of bilirubin monoglucoside monoglucuronide was detected in cholestatic human serum.

Animals↗
PubMed1985

Cerebrospinal fluid beta-glucuronidase activities in patients with central nervous system metastases.

beta-Glucuronidase activities were determined in cerebrospinal fluid from 249 patients suffering from various neurological diseases. Reference values were established as 9-27 mU/l. Marked elevations of cerebrospinal beta-glucuronidase activities were observed in patients with bacterial and carcinomatous meningitis. Slight elevations of cerebrospinal beta-glucuronidase activities were observed in epidural and parenchymal metastases from solid tumours. Comparison was made with the determination of total protein, glucose and lactate dehydrogenase in cerebrospinal fluid. Cerebrospinal beta-glucuronidase activity appeared the most useful test in monitoring patients at risk in developing meningeal metastases from solid primary tumours.

Adolescent↗
PubMed1990

Beta-glucuronidase mediated pathway essential for retinal pigment epithelial degradation of glycosaminoglycans. Disease expression and in vitro disease correction using retroviral mediated cDNA transfer.

A beta-glucuronidase mediated pathway for the degradation of glycosaminoglycans is present in the retinal pigment epithelium. The pathway has been defined using ocular tissues and cultured cells from mutant animals having a recessively inherited deficiency of the lysosomal enzyme. In situ, storage products accumulate in secondary lysosomes of the retinal pigment epithelium, the cytoplasm fills with inclusions and the cells hypertrophy; severity of the disease increases with aging. Deficient activity of beta-glucuronidase is present in primary and second passage cultures. Radiolabel studies with 35SO4 show a significant retention of cell layer label by mutant retinal pigment epithelial cells during a 72-hr pulse or 24-hr chase period. The labels is in newly synthesized chondroitin sulfate and heparan sulfate, which are natural substrates for the deficient enzyme. There is no difference from normal in the total radioactivity and electrophoretic profile of the glycosaminoglycans that are synthesized and released into the media. A retroviral vector was used to transfer normal rat beta-glucuronidase cDNA into the mutant cells. The vector treatment results in restoration of enzyme activity and correction of the degradative defect; 35SO4 labeling shows that chondroitin sulfate and heparan sulfate levels return to normal. The vector treatment studies indicate that a single gene defect determines the abnormal beta-glucuronidase mediated pathway in the mutant retinal pigment epithelium.

Animals↗
PubMed1985

Human beta-glucuronidase. Studies on the effects of pH and bile acids in regard to its role in the pathogenesis of cholelithiasis.

Human bile contains a considerable amount of endogenous beta-glucuronidase. The effects of pH and bile acids on its activity have been studied in regard to its role in the pathogenesis of cholelithiasis. beta-Glucuronidase, purified from human liver to homogeneity, was structurally stable between pH 4 and 10, but was active only over a much narrower range of pH, with a pH optimum of 5.2. The inactivation below pH 4 was due to its irreversible denaturation, whereas the inactivation at higher pH was due to a true reversible pH effect on the enzyme velocity. Kinetic studies revealed that hydrogen ion acted as a substrate-directed activator of the free enzyme, but not the enzyme-substrate complex, with a molecular dissociation constant of 4 X 10(-6). The enzyme activity was not affected by unconjugated bile acids, primarily due to their extremely low water solubility. Conjugated bile acids, on the other hand, exerted heterogeneous and pH-dependent effects on the enzyme. At pH 5.2, taurocholic acid and glycocholic acid were substrate-directed activators of the enzyme; taurochenodeoxycholic acid and taurodeoxycholic acid, competitive inhibitors; and glycochenodeoxycholic acid and glycodeoxycholic acid, mixed inhibitors. At pH 7.0 all taurine and glycine conjugates behaved as substrate-directed activators. Though beta-glucuronidase activity at pH 7 was only 23% of its maximal activity at pH 5.2, conjugated bile acids tended to restore its activity to a certain extent at pH 7. Thus, endogenous beta-glucuronidase could play a significant role in pigment cholelithiasis.

Bile Acids and Salts↗
PubMed1988

Pancreatobiliary and biliary control of fecal beta-glucuronidase activity in the rat.

Bile duct ligation experiments suggest that bile is an important regulator of intestinal beta-glucuronidase, an enzyme thought to be involved in colon carcinogenesis. Exclusion of pancreatobiliary secretions from the rat intestine significantly decreases glucuronidase activity (Roberton et al., Cancer Res., 42 (1982) 5165-5166). However, the separate roles of pancreatic and biliary secretions have not been examined. We ligated the bile ducts of rats at the hepatic duct, allowing pancreatic juice but not bile to enter the intestine, or at the Sphincter of Oddi, excluding pancreatic juice as well as bile. Fecal beta-glucuronidase activity was lowered in both cases, indicating that bile itself, and not pancreatic juice, is a major factor modulating beta-glucuronidase activity.

Animals↗
PubMed2000

Beta-glucuronidase latency in isolated murine hepatocytes.

The physiological function of microsomal beta-glucuronidase is unclear. Substrates may be either glucuronides produced in the lumen of endoplasmic reticulum (ER) or those taken up by hepatocytes. In the latter case, efficient inward transport of glucuronides at the plasma membrane and the ER membrane would be required. Therefore, the potential role of beta-glucuronidase in ER was investigated. Isolated mouse hepatocytes and mouse and rat liver microsomal vesicles were used in the experiments. Selective permeabilization of the plasma membrane of isolated hepatocytes with saponin or digitonin resulted in an almost 4-fold elevation in the rate of beta-nitrophenol glucuronide hydrolysis, while the permeabilization of plasma membrane plus ER membrane by Triton X-100 caused a further 2-fold elevation. In microsomal vesicles, the p-nitrophenol glucuronide or phenolphthalein glucuronide beta-glucuronidase activity showed about 50% latency as revealed by alamethicin or Triton X-100 treatment. A light-scattering study indicated that the microsomes are relatively impermeable to both glucuronides and to glucuronate. On the basis of our results, the role of liver microsomal beta-glucuronidase in the deconjugation of glucuronides taken up by the liver seems unlikely. Hydrolysis of the glucuronides produced in the ER lumen may play a role in substrate supply for ascorbate synthesis or in "proofreading" of glucuronidation.

Animals↗
PubMed2002

Synthesis and biological activity of the prodrug of class I major histocompatibility peptide GILGFVFTL activated by beta-glucuronidase.

The first synthesis of a prodrug of HLA-A2.1 associated antigenic influenza peptide 2a was accomplished. Two methods for synthesis of prodrugs of antigenic peptides activated by beta-glucuronidase and comprising a self-immolative 3-nitrobenzyloxycarbonyl moiety were investigated. Reaction of beta-glucuronic acid glycoside of 4-hydroxy-3-nitrobenzyl alcohol (3) with N,N'-disuccinimidyl carbonate (DSC) followed by conjugation with AlaOMe, Gly, Thr, Phe-Leu, and Leu-Arg gave carbamates 4a-4f. Deacetylation of 4b and 4e with MeONa/MeOH gave beta-glucuronides 5b and 5e. Compound 5e was converted to beta-glucuronic acid conjugate 6e by the action of pig liver esterase (PLE). Compound 6e is a substrate for beta-glucuronidase. Method of a direct introduction of the prodrug residue into antigenic nonapeptide GILGFVFTL (2b) failed. Alternately, glycine conjugate 5b was activated to pentafluorophenyl ester 10. Model coupling of 10 with Phe-Leu gave tripeptide conjugate ester 11a which was hydrolyzed by PLE to uronic acid 12. Condensation of 10 with octapeptide ILGFVFTL (9) gave prodrug precursor 11b. Octapeptide 9 was prepared by de novo synthesis using a racemization-free fragment coupling method. Ester hydrolysis with Ba(OH)(2)/MeOH gave the target prodrug 2a which is a substrate for beta-glucuronidase. Prodrug 2a does not bind to HLA-A2.1 of T2 human cells defective in major histocompatibility complex I (MHC I)-associated peptide processing. Addition of beta-glucuronidase restored the binding to the level observed with parent nonapeptide 2b although higher concentrations of prodrug 2a and enzyme were necessary.

Glucuronidase↗
PubMed2005

Beta-glucuronidase-cleavable prodrugs of O6-benzylguanine and O6-benzyl-2'-deoxyguanosine.

Glucuronic acid linked prodrugs of O(6)-benzylguanine and O(6)-benzyl-2'-deoxyguanosine were synthesized. The prodrugs were found to be quite stable at physiological pH and were more than 200-fold less active as inactivators of O(6)-alkylguanine-DNA alkyltransferase (alkyltransferase) than either O(6)-benzylguanine or O(6)-benzyl-2'-deoxyguanosine. Beta-glucuronidase from both Escherichia coli and bovine liver cleaved the prodrugs efficiently to release O(6)-benzylguanine and O(6)-benzyl-2'-deoxyguanosine, respectively. In combination with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), the prodrugs were not effective adjuvants for HT29 cell killing. However, as expected, incubation of these prodrugs with beta-glucuronidase in the culture medium led to much more efficient cell killing by BCNU as a result of the liberation of the more potent inactivators, O(6)-benzylguanine and O(6)-benzyl-2'-deoxyguanosine. These prodrugs may be useful for prodrug monotherapy of necrotic tumors that liberate beta-glucuronidase or for antibody-directed enzyme prodrug therapy with antibodies that can deliver beta-glucuronidase to target tumor cells.

Alkyl and Aryl Transferases↗
PubMed2022

Characterizing the metabolic effects of the selective inhibition of gut microbial β-glucuronidases in mice.

The hydrolysis of xenobiotic glucuronides by gut bacterial glucuronidases reactivates previously detoxified compounds resulting in severe gut toxicity for the host. Selective bacterial β-glucuronidase inhibitors can mitigate this toxicity but their impact on wider host metabolic processes has not been studied. To investigate this the inhibitor 4-(8-(piperazin-1-yl)-1,2,3,4-tetrahydro-[1,2,3]triazino[4',5':4,5]thieno[2,3-c]isoquinolin-5-yl)morpholine (UNC10201652, Inh 9) was administered to mice to selectively inhibit a narrow range of bacterial β-glucuronidases in the gut. The metabolomic profiles of the intestinal contents, biofluids, and several tissues involved in the enterohepatic circulation were measured and compared to control animals. No biochemical perturbations were observed in the plasma, liver or gall bladder. In contrast, the metabolite profiles of urine, colon contents, feces and gut wall were altered compared to the controls. Changes were largely restricted to compounds derived from gut microbial metabolism. This work establishes that inhibitors targeted towards bacterial β-glucuronidases modulate the functionality of the intestinal microbiota without adversely impacting the host metabolic system.

Mice↗
PubMed2002

A fully human anti-Ep-CAM scFv-beta-glucuronidase fusion protein for selective chemotherapy with a glucuronide prodrug.

Monoclonal antibodies against tumour-associated antigens could be useful to deliver enzymes selectively to the site of a tumour for activation of a non-toxic prodrug. A completely human fusion protein may be advantageous for repeated administration, as host immune responses may be avoided. We have constructed a fusion protein consisting of a human single chain Fv antibody, C28, against the epithelial cell adhesion molecule and the human enzyme beta-glucuronidase. The sequences encoding C28 and human enzyme beta-glucuronidase were joined by a sequence encoding a flexible linker, and were preceded by the IgGkappa signal sequence for secretion of the fusion protein. A CHO cell line was engineered to secrete C28-beta-glucuronidase fusion protein. Antibody specificity and enzyme activity were retained in the secreted fusion protein that had an apparent molecular mass of 100 kDa under denaturing conditions. The fusion protein was able to convert a non-toxic prodrug of doxorubicin, N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-beta-glucuronyl carbamate to doxorubicin, resulting in cytotoxicity. A bystander effect was demonstrated, as doxorubicin was detected in all cells after N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-beta-glucuronyl carbamate administration when only 10% of the cells expressed the fusion protein. This is the first fully human and functional fusion protein consisting of an scFv against epithelial cell adhesion molecule and human enzyme beta-glucuronidase for future use in tumour-specific activation of a non-toxic glucuronide prodrug.

Animals↗
PubMed1968

Behavior of the NO-beta-D-glucosiduronic acid of N-acetyl-N-phenylhydroxylamine as a substrate for beta-glucuronidase.

1. Biosynthetic sodium (N-acetyl-N-phenylhydroxylamine NO-beta-d-glucosid)-uronate is hydrolysed completely by purified mouse urinary beta-glucuronidase into the products N-acetyl-N-phenylhydroxylamine and glucuronic acid. The hydrolysis is inhibited by saccharo-(1-->4)-lactone. These results not only confirm the identity and purity of the substrate but also establish it as a substrate for beta-glucuronidase. 2. Mammalian and bacterial beta-glucuronidase preparations hydrolysed the substrate at a rate one-fifth of that for (phenolphthalein beta-d-glucosid)uronic acid under the optimum conditions of hydrolysis for each source. 3. The pH optimum is 4.1 and the Michaelis constant, K(m), is 3.3x10(-4)m with purified mouse urinary beta-glucuronidase as the enzyme source acting on the NO-beta-d-glucosiduronic acid. The aglycone after extraction into chloroform was quantitatively determined spectrophotometrically at its absorption maximum (256mmu). 4. The hydrolysis was studied as a function of time and temperature. 5. From a consideration of the chemical and enzymic properties of this NO-beta-d-glucosiduronic acid it is possible to suggest its catabolism in vivo.

Animals↗
PubMed1972

Intracellular transport of mouse kidney -glucuronidase induced by gonadotrophin.

1. The response of renal beta-glucuronidase with time to the injection of gonadotrophin was investigated in each submicrosomal fraction of rough and smooth microsomal fractions of mouse kidney homogenate. 2. The increase in beta-glucuronidase activity appeared initially in membranes of the rough microsomal fraction, 24h after injection. 3. Afterwards the newly synthesized enzyme appeared in the contents of the rough microsomal fraction and was subsequently found in the smooth microsomal fraction, reaching a maximum concentration in this fraction at 72h. 4. At this juncture, a decrease in the enzyme activity was observed in rough microsomal contents whereas the lysosomal fraction had reached its maximum value. 5. The time-course of the appearance of beta-glucuronidase in the submicrosomal fractions after the gonadotrophin stimulation suggests that the newly synthesized enzyme at the site of membrane-bound ribosomes is transferred across the membrane into cisternae of the rough endoplasmic reticulum, and then is transported into lysosomes via the smooth endoplasmic reticulum. 6. The properties of microsomal and lysosomal beta-glucuronidases were compared.

Animals↗
PubMed1997

Glycosaminoglycan storage in cultured neonatal murine mucopolysaccharidosis type VII neuroglial cells and correction by beta-glucuronidase gene transfer.

The inherited deficiency of beta-glucuronidase activity causes the lysosomal storage disorder mucopolysaccharidosis (MPS) type VII (Sly disease). The sequential catabolism of glycosaminoglycans in lysosomes is blocked, and undegraded substrates accumulate in cells of many tissues, including neurons and glia in the brain. To evaluate the deficient metabolic pathway, primary cultures of mixed brain cells were established from newborn MPS VII mice. beta-Glucuronidase levels and glycosaminoglycan accumulation were studied in normal, carrier, and MPS VII cells. Retroviral vector-mediated transfer of a normal beta-glucuronidase cDNA corrected the enzymatic deficiency in MPS VII cells and restored glycosaminoglycan catabolism to normal. High levels of beta-glucuronidase expression were sustained in vector-corrected nondividing glial cell cultures for >2 months. These studies provide an in vitro model for evaluating somatic gene transfer in neural cells affected in mucopolysaccharidoses.

Animals↗
PubMed2000

Beta-glucuronidase inhibitory activity and hepatoprotective effect of 18 beta-glycyrrhetinic acid from the rhizomes of Glycyrrhiza uralensis.

An inhibitor of beta-glucuronidase from the rhizomes of Glycyrrhiza uralensis was isolated and its hepatoprotective activity on CCI4-induced hepatotoxicity of rats was investigated. From the water-soluble extract of G. uralensis, glycyrrhizin was isolated as a potent inhibitor of beta-glucuronidase. When glycyrrhizin was orally administered, it had a hepatoprotective activity. However, when glycyrrhizin was intraperitoneally administered, it did not have a hepatoprotective activity. 18 beta-Glycyrrhetinic acid, which is a major metabolite of glycyrrhizin by human intestinal bacteria, was also a potent inhibitor of beta-glucuronidase. When 18 beta-glycyrrhetinic acid was intraperitoneally administered, it also had some hepatoprotective activity. These results suggest that glycyrrhizin may be a natural prodrug for the observed hepatoprotective effect in rats and that serum beta-glucuronidase levels have implications for the liver injury, as reductions of its activity by administration of inhibitors such as G. uralensis or its derived products and silymarin correlate with reductions in biochemical indices of liver injury.

Animals↗
PubMed2000

[The effect of midazolam and flunitrazepam on the liberation of lysozyme and beta-glucuronidase from neutrophil granulocytes in vitro].

OBJECTIVE: Polymorphonuclear neutrophile leucocytes (PMNL) play an important role in the defence against bacterial infections. It is known that some anaesthetics are able to disturb PMNL functions. We examined the influence of midazolam and flunitrazepam on the activity of the bactericidal enzymes lysozyme and beta-glucuronidase released from PMNL in vitro. METHODS: As described before [4], PMNL were isolated from venous blood samples obtained from 10 healthy male volunteers. PMNL stimulation and measurement of lysozyme and beta-glucuronidase activities were conducted according to the description by Metcalf et al. [5]. The BIOMED-system [8] was used for statistical evaluation. RESULTS: Neither midazolam nor flunitrazepam caused any statistically important alteration of lysozyme activity. However, clinically relevant concentrations of both benzodiazepines significantly enhanced beta-glucuronidase activity. The additives of flunitrazepam did not play any role. CONCLUSION: Surprisingly enough, midazolam and flunitrazepam increased the activity of beta-glucuronidase released from PMNL in vitro. At present, this result can neither be explained nor can its importance be estimated. On the other hand, the benzodiazepines did not relevantly influence lysozyme activity.

Anesthetics, Intravenous↗