Increase In Β-glucuronidase Activity Research Articles

Phylogenetic distribution of genes encoding β‐glucuronidase activity in human colonic bacteria and the impact of diet on faecal glycosidase activities

Bacterial β-glucuronidase in the human colon plays an important role in cleaving liver conjugates of dietary compounds and xenobiotics, while other glycosidase activities are involved in the conversion of dietary plant glycosides. Here we detected an increase in β-glucuronidase activity in faecal samples from obese volunteers following a high-protein moderate carbohydrate weight-loss diet, compared with a weight maintenance diet, but little or no changes were observed when the type of fermentable carbohydrate was varied. Other faecal glycosidase activities showed little or no change over a fivefold range of dietary NSP intake, although α-glucosidase increased on a resistant starch-enriched diet. Two distinct groups of gene, gus and BG, have been reported to encode β-glucuronidase activity among human colonic bacteria. Degenerate primers were designed against these genes. Overall, Firmicutes were found to account for 96% of amplified gus sequences, with three operational taxonomic units particularly abundant, whereas 59% of amplified BG sequences belonged to Bacteroidetes and 41% to Firmicutes. A similar distribution of operational taxonomic units was found in a published metagenome dataset involving a larger number of volunteers. Seven cultured isolates of human colonic bacteria that carried only the BG gene gave relatively low β-glucuronidase activity that was not induced by 4-nitrophenyl-β-D-glucuronide. By comparison, in three of five isolates that possessed only the gus gene, β-glucuronidase activity was induced.

Evaluation of the effects of dietary organic germanium, ge-132, and raffinose supplementation on caecal flora in rats.

Poly-trans-[(2-carboxyethyl) germasesquioxane] (Ge-132) is the most common organic germanium compound. The ingestion of Ge-132 promotes bile secretion. We assessed the rat caecal characteristics after the administration of Ge-132 and raffinose, a prebiotic oligosaccharide, because both Ge-132 and some prebiotics can change the fecal color to yellow. We also compared the changes in the caecal flora caused by the two compounds. In addition, we evaluated the simultaneous administration of Ge-132 and raffinose and their effects on β-glucuronidase activity, which is known to be a factor related to colon cancer. Male Wistar rats (three weeks old) were given one of the following diets: 1) a control diet (control group), 2) a diet containing 0.05% Ge-132 (Ge-132 group), 3) a diet containing 5% raffinose (RAF group) or 4) a diet containing 0.05% Ge-132 + 5% raffinose (GeRAF group). The Bifidobacterium, Lactobacillus and total bacteria counts were significantly increased by the dietary raffinose, and Ge-132 did not suppress this increase. The raffinose intake increased caecal acetic acid production significantly. The activity of β-glucuronidase in the caecal contents was increased by dietary Ge-132, whereas dietary raffinose decreased the β-glucuronidase activity significantly. These results indicate that the simultaneous intake of dietary raffinose and Ge-132 does not inhibit the effects of either compound on intestinal fermentation and bile secretion. Additionally, the simultaneous intake of both raffinose and Ge-132 could abrogate the increase in β-glucuronidase activity induced by Ge-132 alone.

Experimentally induced mastitis and metritis modulate soy bean derived isoflavone biotransformation in diary cows

The present study compared the changes in isoflavone (daidzein and genistein) and their metabolite (equol and para-ethyl-phenol) concentrations in the blood plasma of cows with induced mastitis and metritis after feeding with soy bean. Sixteen cows were divided into four groups: control for mastitis group, cows with induced mastitis group, control for metritis group, and cows with induced metritis group. All cows were fed a single dose of 2.5 kg of soy bean and then blood samples were taken from the jugular vein for 8 h at predetermined intervals. The concentrations of soy bean-derived isoflavones and their active metabolites were measured in the blood plasma on HPLC system. β-Glucuronidase activity in the blood plasma of cows was measured by fluorometric method. In the blood plasma of cows with induced mastitis and metritis, we found considerably higher concentrations and time-dependent increase in isoflavone metabolites (equol and para-ethyl-phenol) with reference to cyclic cows (P < 0.05). Moreover, we noticed significant decrease of genistein in the blood plasma of the cows with induced metritis compared with control cows (P < 0.05). In addition, in the blood plasma of the cows with induced metritis, we found an increase in β-glucuronidase activity compared with control cows (P < 0.05). In conclusion, health status of the females influenced the concentrations of isoflavone metabolites in the blood plasma of the cows. Experimentally induced mastitis and metritis increased isoflavone absorption, biotransformation and metabolism. Therefore, we suggest that cows with induced mastitis and metritis are more exposed to active isoflavone metabolite actions than healthy cows.

Tomato mitogen activated protein kinases regulate the expression of extracellular invertase Lin6 in response to stress related stimuli.

Activation of mitogen-activated protein kinases (MAPKs) is a common reaction of plant cells in defence-related signal transduction pathways. Since the downstream events after the activation of MAPKs are largely unknown in plants, the role of MAPKs in the coordinate regulation of defence reactions and primary carbon metabolism by stress related stimuli has been analysed in tomato (Lycopersicon peruvianum Mill.). Thus, the relationship between MAPK, LpMPK2 and LpMPK3 and extracellular invertase Lin6, as the key enzyme of an apoplasmic phloem unloading pathway, has been analysed. It was observed that the mRNAs of LpMPK3 and Lin6 are sequentially induced by the same set of stress related stimuli, wounding, a fungal elicitor derived from Fusarium oxysporum lycopersici, the endogenous plant derived elicitor PGA and salt stress, while LpMPK2 transcripts are constitutively expressed. In a gain of function approach, a His-tagged version of LpMPK2 and a HA-tagged version of LpMPK3 were transiently and functionally expressed in leaves of transgenic tobacco (Nicotiana tabacum L.) plants expressing the β-glucuronidase reporter gene under control of the Lin6 promoter via agro-infection. The induction of the Lin6 promoter, as revealed by an increase in β-glucuronidase activity after 24 h, was dependent both on the expression and activation of both LpMPK2 and LpMPK3. These data suggest that the induction of extracellular invertase Lin6 by stress-related stimuli requires LpMPK2 and LpMPK3, and thus demonstrate that MAPK signalling might be involved in the regulation of primary carbon metabolism in general and sink metabolism in particular.

Effect of coconut cake on the bacterial enzyme activity in 1,2-dimethyl hydrazine induced colon cancer

Background: Colon cancer is one of the most common forms of malignant tumors in humans, and its incidence is increasing. Since the intestinal microflora is directly in contact with the colonic cells, the enzymes of the bacterial microflora may also play a role in colon carcinogenesis. We studied the activity of bacterial enzymes in experimental colon cancer. Methods: Twenty milligrams per kilogram body weight of 1,2-dimethyl hydrazine (DMH) was administered subcutaneously once a week for first 15 weeks and then discontinued. Coconut cake (25%) was mixed in the diet and given to 30 rats to study the diet effect throughout the experimental period. After 30 weeks, the macroscopic findings in the colon as well as the incidence of tumors in 30 rats was recorded in each group and the activity of β-glucuronidase and mucinase was estimated in the tissues, colon and fecal contents of 10 rats per group . Results: Average number of tumors in the colon as well as the incidence of cancer was significantly increased in DMH-treated rats which was markedly reduced on supplementing coconut cake. DMH injections significantly elevated both the activities of β-glucuronidase (distal colon, distal intestine, liver and colon contents) and mucinase (colon and fecal contents) as compared to the control rats. The increase in β-glucuronidase activity may augment the hydrolysis of glucuronide conjugates, liberating the toxins, while the increase in mucinase activity may enhance the hydrolysis of the protective mucins in the colon. Coconut cake supplementation to DMH-treated rats significantly decreased the incidence and number of tumors as well as the activity of β-glucuronidase and mucinase. Conclusions: Coconut cake has a protective effect against DMH induced colon cancer by virtue of its ability to lower the activities of the microbial enzymes β-glucuronidase and mucinase.

Influence of spices on the bacterial (enzyme) activity in experimental colon cancer

In the presence of a known colon carcinogen, 1,2-dimethyl hydrazine (DMH), the activity of β-glucuronidase was found to be significantly increased in the distal colon, distal intestine, liver and colon contents and the activity of mucinase was increased in both the colon and fecal contents when compared to control rats. Chilli (Capsicum annum L., Solanaceae) administration also showed an increase when compared to control rats, whereas supplementation with cumin (Cuminum cyminum L., Apiaceae) and black pepper (Piper nigrum L., Piperaceae) in the presence of DMH, showed more or less similar values as that of the control rats. The increase in β-glucuronidase activity may increase the hydrolysis of glucuronide conjugates, liberating the toxins, while the increase in mucinase activity may enhance the hydrolysis of the protective mucins in the colon. Thus cumin and black pepper may protect the colon by decreasing the activity of β-glucuronidase and mucinase. Histopathological studies also showed lesser infiltration into the submucosa, fewer papillae and lesser changes in the cytoplasm of the cells in the colon in cumin and black pepper groups when compared to the DMH and chilli treated animals.

Modulation of degranulation and superoxide generation in human neutrophils by unsaturated fatty acids of odd carbon numbers

Unsaturated fatty acids of odd carbons, 13:1(12), 17:1(10trans), 19:1(7) and 19:1(10) inhibited release of myeloperoxidase (MPO) from fMet-Leu-Phe-cytochalasin B-treated neutrophils. The inhibitory effect was smaller than that of aseanostatins which have been isolated as microbial-derived free fatty acids with a methyl blanch (i-14:0 and ai-15:0) (Journal of Antibiotics (1991) 44, 524–532). These unsaturated fatty acids also inhibited lactoferrin release by the same treatment. On the other hand, 13:1(12), 15;1(10) and 19:1(10) inhibited fMet-Leu-Phe-stimulated superoxide generation of neutrophils, and the fatty acids 15:1(10), 17:(10) and 19:2(10,13) induced superoxide generation in both unstimulated cells and the cell-free system. However, none of unsaturated fatty acids of odd carbons tested inhibited β-glucuronidase release, whereas 15:1(10), 17:1(10), 19:1(7), 19:1(10) and 19:2(10,13) rather enhanced an increase in β-glucuronidase activity liberated from cells at high concentrations over 35 μM, indicating cellular damages by these fatty acids. These observations suggest that unsaturated free fatty acids having odd carbons such as 13, 15, 17 and 19 may act as modulators of neutrophil functions including degranulation and superoxide generation.

Correction of β-glucuronidase deficiency in mice by intrasplenic transplantation of isolated hepatocytes

Correction of enzyme activity was successfully accomplished in β-glucuronidase deficient C3H/HeJ mice after intrasplenic transplantation of isolated hepatocytes obtained from normal Balb/C donors mice. Enzyme activity increased from a mean of 1.1 ± 0.2 nmol/h/ml on day 0 to 15.8 ± 3.3 nmol/h/ml on day 55 post-transplantation. Examination of enzyme activity in liver tissue by repeated liver biopsies revealed an increase in β-glucuronidase activity from a mean of 1.85 ± 0.6 nmol/h/mg protein on day 0 up to 42.6 ± 7.3 nmol/h/mg protein on day 35 post-transplantation. β-Galactosidase and β- N-acetyl-glucosaminidase activities were not significantly changed throughout the study. Intrasplenic transplantation of hepatocytes isolated from β-glucuronidase-deficient mice did not induce a significant change in three examined hydrolases. These results suggest that transplantation of isolated hepatocytes may be used as an effective tool for therapy of lysosomal enzyme deficiency in mice.

Acquisition of a lysosomal enzyme by myoblasts in tissue culture

Skeletal muscle myoblasts from different sources acquired high levels of the lysosomal enzyme beta-glucuronidase, when they were cultured together with mitogen-activated lymphocytes. Immunofluorescent staining, thermal stability, and electrophoretic mobility showed that the increase in enzyme activity in the myoblasts was due to the presence of the lymphocyte form of the enzyme. Although myoblasts were able to take up exogenous beta-glucuronidase from the culture medium by mannose 6-phosphate receptor-mediated endocytosis, enzyme acquisition during co-culture with lymphocytes was independent of this pathway. Enzyme transfer from the lymphocytes was found to require direct cell-cell contact with the muscle cells, and was accompanied by an increase in beta-glucuronidase activity in the lymphocytes themselves. Since this additional activity was also due to the presence of the lymphocyte form of the enzyme, these results indicate that interaction with the muscle cells induced the de novo synthesis of beta-glucuronidase in the lymphocytes.

Neurotoxic effects of the combined exposure to carbon disulphide and ethanol in rats

Neurotoxic effects of the combined exposure of rats to carbon disulphide (CS 2) and ethanol (EtOH) were studied. Biochemical and ultrastructural evaluation of the central nervous system (CNS) and peripheral nervous system (PNS) was performed. Male Wistar rats were exposed to CS 2 vapour (0.8 mg/l air) and to 10% alcohol in the drinking water for 8 months. EtOH elevated the increase in β-glucuronidase activity caused by CS 2 in the hippocampus and in the cerebral cortex. No effect on the high-affinity synaptosomal uptake of l-glutamate and GABA was observed and no marked ultrastructural changes in the tested brain regions were found. In the peripheral nerves CS 2 alone evoked axonal degeneration whereas CS 2 combined with EtOH caused disturbances in myelin. Ultrastructural changes preceded biochemical alterations in the PNS and the biochemical indicators of peripheral neuropathy such as β-glucuronidase activity and cholesterol ester content were not significantly affected. It is suggested that CS 2 and EtOH combined affect both PNS and CNS to a higher extent than each of these substances alone.

The antiandrogenic action of gonadotropin-releasing hormone and its agonists on the mouse kidney.

It was recently demonstrated that GnRH and its agonists can inhibit the growth-promoting effects of testosterone on the prostate and seminal vesicle of the rat. In the present study, the kinetics and mechanism of this antiandrogenic response were investigated on the mouse kidney, an organ that has been used extensively to investigate androgen action. GnRH and its agonist, [(imBzl)-D-His6,Pro9-NEt]GnRH (D-His), inhibited the increase in kidney β-glucuronidase activity induced by testosterone propionate (TP) in castrated male mice. D-His also inhibited the action of TP on this enzyme in castrated hypophysectomized male mice. When D-His (10 μgday) was administered with various doses of TP (20–160 μg/day), the dose-dependent increase in β-glucuronidase activity was blunted. Similarly, D-His (0.1–100 μg/day) produced a dose-dependent decrease in the renal β-glucuronidase activity of castrated male mice treated with TP (80 μg/day). Even though GnRH and its agonist inhibited the action of TP in male NCS and Balb/C mice, these peptides slightly potentiated the action of TP on renal β-glucuronidase activity in female mice of these strains. D-His did not compete with [3H]methyltrienolone for binding sites on the androgen receptor as measured in an in vitro assay. In addition, the administration of D-His with TP in vivo did,not affect the total cytosol receptor activity as measured with an exchange assay. It is concluded that the antiandrogenic actions of GnRH and its agonists are not mediated via the androgen receptor in a manner comparable to that of steroid antagonists since 1) no competition between peptide and steroid could be demonstrated for the androgen receptor, and 2) the inhibitory actions of GnRH peptides were not uniform between the kidneys from male and female mice, which are equally sensitive to testosterone. The finding that GnRH has antisteroidal activity on the kidney and other tissues raises the possibility that this, or a similar peptide, could act as a local regulator of hormone action on extragonadal organs.

Acid hydrolases and nitrogen mustard cytotoxicity

Acid Hydrolases and Nitrogen Mustard Cytotoxicity. Cohen, A. M. (1978). Toxicol. Appl. Pharmacol. 43 , 85–97. The role of acid hydrolases in the cytocidal action of nitrogen mustard (HN 2 ) in rat intestine crypt epithelial cells was studied. An eight-fold increase in crypt cel β-glucuronidase activity was observed at 24 hr after HN 2 injection (2 mg/kg, ip). The activity of acid phosphatase and cathepsin D was not significantly altered by HN 2 administration. Inhibition of DNA synthesis and widespread pycnosis and karyorrhexis of crypt cells preceded significant increases in β-glucuronidase activity. However, loss of intestinal DNA prelabeled with tritiated 5-iodo-2′-deoxyuridine occurred subsequent to significant increases in β-glucuronidase activity. A single injection of cycloheximide (1 mg/kg, ip) given at either 30 min before or 30 min after HN 2 delayed the development of crypt cell histopathology. A second injection of cycloheximde at 8 hr after HN 2 prevented further development of crypt histopathology and completely blocked the increase in β-glucuronidase activity and the loss of prelabeled DNA observed at 24–30 hr after HN 2 alone. These studies suggest that acid hydrolases do not play a major role in the initiation of the processes responsible for the death of proliferating crypt cells exposed to HN 2 . However, the results are consistent with a possible role for certain of these enzymes in the degradation and removal of dead cells from the crypt epithelium.

Effect of androgens and progestins on mouse kidney cells: An in vitro model distinct from that of intact kidney

Androgen metabolism in cultured mouse kidney cells was studied. Kidneys from Balb/C mice were dispersed with trypsine and the resulting cell suspension grown to confluent monolayers by 12–14 days in the presence of fetal calf serum. Cultured kidney cells had sufficient 5α-reductase to metabolize testosterone to dihydrotestosterone and 5α-androstanediols. This is in marked contrast to the mouse kidney in vivo which contains little or no 5α-reductase activity. The presence of an androgen receptor was suggested by the saturable uptake of testosterone in cultured kidney cells. When testosterone was added to the growth medium, total DNA, protein and β-glucuronidase activity increased 2–3-fold in a dose-dependent fashion with a half-maximal response at 2 × 10 −9 M testosterone. A specific effect on β-glucuronidase activity was shown by the increase of the β-glucuronidase/DNA ratio. Dihydrotestosterone and, to a lesser extent, the androstanediols had a similar effect. This is in marked contrast to the in vivo effects of androgens which produce a 20–50-fold increase in β-glucuronidase activity and no increase in DNA synthesis. In addition to androgens, 3 progestational steroids which are known to bind to the androgen receptor (progesterone, medroxyprogesterone acetate and cyproterone acetate) also stimulated renal cell cultures. It is concluded that primary cultures of mouse kidney cells metabolize testosterone and respond to both androgens and progestins. However, the metabolism and actions of androgens in primary kidney cells were markedly different from in vivo effects. The presence of the following features distinguishes the cultured renal cells from those in the intact kidney: presence of 5α-reductase; androgen-induced DNA synthesis; and blunted β-glucuronidase response.

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.

Preputial gland growth and protein synthesis in the androgen-insensitive male pseudohermaphroditic rat.

The androgen-dependent growth of the preputial gland was studied in normal (Nl) male and female rats as well as in the androgen-insensitive Stanley-Gumbreck male pseudohermaphroditic (Ps) rat. Preputial gland weight was 3 times higher in the Nl males than Nl females or Ps rats. Castration of Nl males reduced preputial gland weight to that of females. Administration of testosterone enanthate, 6 mg daily for 10 days, to intact Nl female and castrated Nl males produced 4-fold increases in preputial gland weight, total soluble protein and total RNA; a doubling of total DNA; and a 9-fold increase in β-glucuronidase activity. By contrast, this dose of testosterone enanthate had little effect on the preputial gland of Ps rats, but when treatment was increased 100-fold (60 mg daily) several indexes of preputial gland growth were stimulated, indicating that the genetically determined androgen insensitivity is relative rather than absolute. Since the androgen-dependent control of DNA, RNA and protein synthesis were...

EFFECTS OF ANDROGENS AND ESTROGENS ONβ-GLUCURONIDASE IN INBRED MICE1,2

In recent years, Morrow et al. (1949, 1950) reported that certain pure inbred strains of mice exhibited strikingly low concentrations of β-glucuronidase in the liver, spleen and kidney. Cohen and Bittner (1951) found that mammary tissue β-glucuronidase activity bore a relationship to the strain of mice employed, extremely low concentrations being found in the C3H strain. Among the interesting questions which these findings provoked is the one of whether or not tissue β-glucuronidase in these low-glucuronidase strains is responsive to extrinsic factors. It is known that certain glucuronidogenic drugs will produce an increase in β-glucuronidase activity in the non-sex organs (Fishman, 1940)) and that estrogen will restore uterine β-glucuronidase activity of the castrated mouse to normal levels Fishman and Fishman, 1944; Fishman, 1947) with no alteration in the non-sex organ glucuronidase. (Fishman, 1947; Harris and Cohen, 1951). An exception to this lack of effect in the non-sex organs was reported by Kerr,...