Monooxygenase and UDP-glucuronyltransferase activities in established cell cultures

Cells in culture were investigated for the expression of monooxygenase and UDP-glucuronyltransferase activities as representatives of activating and inactivating pathways of drug metabolism. Most established cell lines express monooxygenase activity that is detected by the oxygenation of polycyclic hydrocarbons and appears to be a function of cytochrome P-448-dependent enzyme forms (Wiebel et al., 1977). In the hepatoma cell line, H-4-II-E, dexamethasone is capable of increasing the level of benzo(a)pyrene-monooxygenation about 10-fold and of potentiating its induction by benz(a)anthracene. The enzyme activities elicited by dexamethasone and the polycyclic hydrocarbon did not significantly differ in their response to 7,8-benzoflavone, an inhibitor of cytochrome P-448-dependent monooxygenases. Observations on the pattern of benzo(a)pyrene metabolites formed in benz(a)anthracene-treated H-4-II-E and BHK21(C13) cells indicate that established cell cultures may contain different forms of monooxygenases of the cytochrome P-448 type.The majority of cell lines tested express UDP-glucuronyltransferase activity directed toward the substrate, 3-hydroxybenzo(a)pyrene. No clear correlation appears to exist between the presence and level of monooxygenase and glucuronyltransferase activities in the various cell lines, i.e., the cultures may express any one or both enzymes. The ratio of the two enzyme levels can be modified by selective induction. Thus, at present there is a choice of established cells in culture exhibiting widely differing ratios of activating and inactivating enzymes to analyse the metabolic pathways of selected classes of foreign compounds, such as polycyclic hydrocarbons, and to determine their toxicological significance. Further efforts are likely to yield cell lines that express the enzymic functions lacking in the cultures currently used and will be suitable to study the full spectrum of foreign compounds.     

Mixed function oxidase and UDP-glucuronyltransferase activities in the human Hep G2 hepatoma cell line

In cultured human hepatoma cells phenolphthalein glucuronidation was increased 3-fold by 2 mM phenobarbitone (PB) in the culture medium but not by 25 microM benz(a)anthracene (BA), while 1-naphthol glucuronidation was not increased by either PB or BA. Ethoxyresorufin O-deethylation (EROD) was increased 15-fold by BA but not by PB, while the O-dealkylations of pentoxyresorufin (PROD) and benzyloxyresorufin (BROD) were increased by either PB or BA. The BROD activity increased by BA was sensitive to inhibition by alpha-naphthoflavone whereas that induced by PB was not. This suggests induction of different cytochrome P-450 isoenzymes. Control Hep G2 cells had similar glucuronide conjugation and cytochrome reductase activities to freshly isolated human adult hepatocytes, but had lower O-dealkylation and elevated microsomal epoxide hydrolase activities.     

Studies of UDP-glucuronyltransferase activities in human liver microsomes

Human liver samples from a "liver bank" which have been previously characterized by the ability to catalyze cytochrome P-450 dependent reactions were analyzed for various UDP-glucuronyltransferase activities. When stored at -80 degrees C, UDP-glucuronyltransferase activities and latency characteristics of the enzyme appeared to be stable for up to 2 years. The correlation of UDP-glucuronyltransferase activity (4-methylumbelliferone as substrate) with enzyme activities towards 4-nitrophenol and 1-naphthol was much higher (r greater than 0.8) than that (r less than 0.3) observed with other enzyme activities (4-hydroxybiphenyl, morphine, and chloramphenicol as substrates), suggesting the presence of multiple enzyme forms in human liver. Livers of three patients treated with phenytoin or pentobarbital showed significantly higher UDP-glucuronyltransferase activities towards 1-naphthol, 4-methylumbelliferone, and bilirubin than those from five patients who did not receive these inducing agents.     

Tooth slice organ culture and established cell line culture models for cytotoxicity assessment of dental materials.

The aim was to compare the use of different cell-material contact test methods with two different biological systems (cell line and tooth slice cultures) for cytotoxicity assessment of dental materials. Cytotoxicity of composites polymerized with two halogen-based and two light-emitting diode (LED) light-curing units (LCUs) served as the basis for comparison. Disk shaped specimens (7 x 2 mm) were fabricated using the four light sources. Composites were tested using L-929 cell line using direct/indirect/extract tests in accordance to standard protocols. Cytotoxicity was assessed using neutral red uptake. Tooth slice organ cultures were also employed to test the dental materials using direct/indirect test methods. Histomorphometric cell counting of intact odontoblasts and pulp fibroblasts and the use of tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were applied for cytotoxicity evaluation. Discrepancy in result presentation was observed in the different tests used with L-929. Sensitivity levels of the L-929 tests ranked as follows: extract test < direct contact test < indirect contact test. Tooth slice tests confirmed that L-929 direct contact test proved to be the most reliable test among the three. In conclusion, this study highlights the risk involved when relying on a single test method for cytotoxicity assessment. It would be advisable to test different culture models and then proceed using more clinically relevant biological system that stimulate the in vivo situation for confirmation.     

Induction of UDP-glucuronyltransferase and arylhydrocarbon hydroxylase activity in mouse skin and in normal and transformed skin cells in culture

Methods have been developed which allow quantitative determination of UDP-glucuronyltransferase (UDPGT) and arylhydrocarbon hydroxylase (AHH) activities in unfractionated mouse skin. These methods were used for comparative studies of basal and induced enzyme activities in whole skin and cultured skin cells. After topical application of Aroclor 1254 to the skin UDPGT activities towards 1-naphthol, 3-hydroxybenzo[a]pyrene and benzo[a]pyrene-7,8-dihydrodiol were increased 3-fold and AHH activity was increased 15-fold. Topical application of the inducer also led to a marked increase of these enzyme activities in liver. UDPGT activity towards 1-naphthol was comparable in whole skin and in cultured keratinocytes and fibroblasts. In contrast, AHH activity was higher in cultured keratinocytes than in skin. In transformed epithelial cell lines the pattern of drug metabolizing enzymes was altered: UDPGT activity was increased 4- to 6-fold whereas AHH activity was decreased. However, AHH activity was still inducible by benz[a]anthracene or 7,12-dimethylbenz[a]anthracene in cultured cells. The altered pattern of AHH and UDPGT in transformed epithelial cell lines is consistent with toxin-resistance of initiated cells, similar to the toxin-resistance phenotype characterized in liver after initiation of hepatocarcinogenesis.     

ESR and cell culture studies on free radical-scavenging and antioxidant activities of isoflavonoids

Isoflavonoids are thought to be the biologically active components in soy that play a role in the prevention of coronary heart disease and breast and prostate cancer. Mechanisms to explain how isoflavonoids mediate beneficial effects have not yet been clearly established. This study was undertaken to investigate the free radical-scavenging and antioxidant activities of various structure-related isoflavonoids including genistein, daidzein, biochanin A, and genistin in a cell-free and an endothelial cell model system. Electron spin resonance spectroscopy and spin trapping techniques were applied to evaluate the ability of isoflavonoids to scavenge hydroxyl, superoxide, nitric oxide, diphenylpicrylhydrazyl, galvinoxyl, and lipid-derived radicals. All isoflavonoids tested had no significant scavenging effects on the aforementioned radicals in concentrations up to 1.0 mM. However, at a physiologically achievable concentration of 5 nM, both genistein and daidzein slightly increased intracellular-reduced glutathione levels approximately by 10 and 30%, respectively, in human endothelial cells, whereas cellular alpha-tocopherol and uric acid remained unchanged by the isoflavonoid treatments. Present data indicate that free radical-scavenging activities of the isoflavonoids tested probably do not substantially contribute to their antioxidant properties. The ability of genistein and daidzein to increase cellular GSH (reduced glutathione) might be important for their action in biological system.     

Ascorbic acid deficiency and hepatic UDP-glucuronyltransferase

The effect of dietary ascorbic acid on hepatic microsomal UDP-glucuronyltransferase (UDPGT) activity towards p-aminophenol, bilirubin, and acetaminophen was investigated. Ascorbate deficiency produced a 33% reduction in the specific activity of UDPGT towards p-aminophenol, whereas there was no difference between microsomes from ascorbate-deficient and supplemented guinea pigs in the activity towards bilirubin and acetaminophen. This suggests that the effect of the vitamin is on a specific isozyme. This reduction was correlated with the reduced quantity of hepatic microsomal cytochrome P-450, which has been previously reported for ascorbate-deficient guinea pigs. No difference was found in the apparent affinity for the substrate, p-aminophenol, or the cofactor, UDP-glucuronic acid. Differences in microsomal UDPGT activity towards p-aminophenol occurred between the two groups with membrane-perturbing processes such as sonication and Triton X-100. Sonication and magnesium chloride were found to increase activity 329% in ascorbate-supplemented animals and 138% in the ascorbate-deficient group. The addition of ascorbate acid in vitro, or its analog d-isoascorbic acid, could protect against the detrimental effects of excess substrate by maintaining a linear enzymatic rate over a 30-min time period; there was no significant effect on the initial rate of hepatic microsomal UDPGT activity in the ascorbate-supplemented animals whereas there was a significant increase in the ascorbate-deficient group. Glutathione was as effective as ascorbic acid in protecting against the detrimental effects of excess substrate whereas cysteine and dimethyltetrapteridine were only partially effective. Ascorbyl-2-sulfate and alpha-tocopherol had no significant effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

The activities of aminopyrine N-demethylase, aniline 4-hydroxylase and UDP-glucuronyltransferase in tissues of camels, desert sheep and Nubian goats

1. The activities of the drug-metabolizing enzymes, aminopyrine N-demethylase, aniline 4-hydroxylase and UDP-glucuronyltransferase have been measured in vitro in the liver, kidney and duodenal mucosa of camels, sheep, goats and rats. 2. Enzyme activities were generally higher in the liver, followed by the duodenal mucosa, then the kidney in all species. 3. Male kids had much lower enzyme activity in the liver when compared to adult goats, and in the former animal, no measurable activity could be detected in the duodenal mucosa or kidney. 4. In general, goats seemed to have the highest and camels the lowest enzyme activity when compared to the other species. 5. Some sex differences were noted in the three enzymes studied. In sheep duodenal mucosa and rat liver and duodenal mucosa, males had higher aminopyrine N-demethylase than females. In rat liver and goat duodenal mucosa males had higher aniline 4-hydroxylase than females. Male rats had higher UDP-glucuronyltransferase in liver when compared to females.     

Inhibition and activation of UDP-glucuronyltransferase in alloxanic-diabetic rats

Short or long term alloxan diabetes produced activation of oestrone and morphine glucuronidation and inhibition of p-nitrophenol glucuronidation in rat liver microsomes. Insulin treatment restored decreased glucuronyltransferase (GT) activity for p-nitrophenol and it did not abolish diabetes activation on oestrone glucuronidation. Triton X-100 detergent activation reduced differences between normal, diabetic and insulin treated rats in the glucuronidation rates of the substrates assayed. 1,4-Benzodiazepines inhibited morphine GT activity and stimulated oestrone GT activity in normal, diabetic and insulin treated diabetic rats. Activation and inhibition of GT activities for oestrone and xenobiotics in diabetes mellitus appears to be related with membrane perturbations of liver microsomes.     

Epoxide hydrolase expression in human and rodent established cell lines

Four human, four hamster and three mouse established cell lines have been analysed for epoxide hydrolase activity, and these have been compared with activities of human and mouse tissue preparations. Activities expressed by the cell lines, using styrene oxide as substrate, varied between less than 0.005 and 0.44 nmol/min per mg total cellular protein. Human liver and human kidney expressed 12.2 nmol and 2.7 nmol/min per mg total protein respectively. Antibodies prepared against purified human- and mouse-liver epoxide hydrolase enzymes were used to characterize the enzyme in the cell lines. Antihuman antibody was able to inactivate and precipitate enzyme in human cell lines and partially inactivate and precipitate hamster enzymes. It was much less effective against mouse cell lines. Antimouse antibody was able to precipitate mouse enzymes and partially precipitate hamster enzymes but did not precipitate human enzymes. These data may reflect evolutionary divergence of the three species.     

Substrate specificity of human UDP-glucuronyltransferase in cultured lymphocytes

1. This study establishes the presence of UDP-glucuronyltransferase activity for non-steroidal as well as steroidal substrates, in cultured human B-lymphocytes. Glucuronidation of α;-naphthol and testosterone was demonstrated in homogenates of two cell lines, SN1006 and RPMI-1788, and that of phenolphthalein, 4-methylumbelliferone, p-nitrophenol and estradiol in the cell line with the higher glucuronyltransferase activity, SN1006.

2. Kinetic studies of testosterone glucuronidation in homogenates of both cell lines revealed a similarity in the behaviour of glucuronyltransferase of these cells. Thus, comparable apparent K_m values for UDPGA (0.63 mM) and for testosterone (14 μm) were observed, although apparent maximal velocities, V_max, differed several-fold (3.0 versus 05.5 pmol/10⁶ cells per min, in SN1006 and RPMI-1788 cells, respectively).

3. Kinetic studies of glucuronidation of testosterone, estradiol, phenolphthalein, α;-naphthol, 4-methylumbelliferone, and p-nitrophenol yielded comparable apparent K_m values for UDPGA (0.56-0.67mM), suggesting that the same, or similar, glucuronyltransferase(s) catalyse(s) glucuronidation of this wide range of substrates in lymphocytes. This was reinforced by the observation of competitive inhibition of testosterone glucuronidation by α;-naphthol (K_i 0.25 mM), 4-methylumbelliferone (K_i 0.8mM) and p-nitrophenol (K_i0.8mM). Thus, lymphocyte glucuronyltransferase activity with a broad substrate specificity, for steroidal and non-steroidal aglycones, is indicated.     

Xenobiotic-metabolizing enzyme activities in hybrid cell lines established by fusion of primary rat liver parenchymal cells with hepatoma cells.

1. The activities of xenobiotic-metabolizing enzymes were determined in hybrid cell lines (hepatocytoma, HPCT) which have been established by fusion of liver parenchymal cells from adult rat (PC) with cells from a Reuber hepatoma cell line (FAO). 2. Cytochrome P450 was not measurable spectrophotometrically in FAO and HPCT. P450-dependent conversion of testosterone was below the detection limit in FAO and only marginally present in HPCT. 3. Microsomal and cytosolic epoxide hydrolase, glutathione S-transferase and phenol sulphotranserase were low or even below detection limit in FAO. These enzyme activities were significantly higher in HPCT and correspond to about 1-10% the activities measured in PC. 4. 1-Naphthol UPD-glucuronosyl transferase activity was about 20% in FAO and about 100% in HPCT compared to PC. 5. Metabolic conversion of benzo[a]pyrene was low in FAO, high in PC, and intermediate in HPCT. The presented data, however, do not allow the conclusion whether this intermediate rate is catalyzed by similar P450 isoenzymes as in PC. 6. Due to the easily measurable phase II-metabolizing enzyme activities HPCT may, however, be useful for in vitro enzyme induction or repression studies.     

Functional heterogeneity of UDP-glucuronyltransferase in rat tissues

Tissue distribution of UDP-glucuronyltransferase was investigated using two substrate groups which were shown to be conjugated by two different forms of this enzyme in previous studies with rat liver. These enzyme forms were found to be differentially inducible by 3-methylcholanthrene (form 1) and phenobarbital (form 2). Group 1 substrates (conjugated by form 1) include 1-naphthol, N-hydroxy-2-naphthylamine and 3-hydroxybenzo[a]pyrene; group 2 substrates (conjugated by form 2) comprise 4-hydroxybiphenyl, morphine and chloramphenicol. Group 1 substrates are conjugated in a number of tissues, for example, liver, kidney, small intestinal mucosa, lung, skin, testes and spleen. However, conjugation of group 2 substrates is detectable only in liver and intestine to an appreciable extent. It is concluded that enzyme(s) efficient in the conjugation of group 1 substrates is ubiquitous in the investigated organs, whilst only liver and intestine possess enzyme(s) efficient in the conjugation of group 2 substrates.In contrast to 3-hydroxybenzo[a]pyrene, benzo[a]pyrene 7,8-dihydrodiol cannot be clearly associated with only one of the 2 substrate groups. Glucuronidation of benzo[a]pyrene 7.8-dihydrodiol is enhanced by both phenobarbital and 3-methylcholanthrene in liver. Conjugation of the dihydrodiol is detectable in all tissues examined. However, enzyme activity towards the dihydrodiol is much lower than that towards 3-hydroxybenzo[a]pyrene. It is disproportionately low with skin microsomes.     

建立内皮细胞系方法的研究

目的利用脐带获取内皮细胞 ,在体外建立内皮细胞系。方法用消化酶消化收集脐内皮细胞 ,扩增 ,冻存 ,复苏。结果在体外可获得大量生物学特性保持良好的内皮细胞。结论本方法简便易行 ,为临床血管内皮化研究提供保证     

Glutathione and glutathione S-transferase activities of mammalian cells in culture

Continuous cell cultures derived from various tissues of rat, mouse, hamster and man were assayed for their glutathione (GSH) content and glutathione S-transferase activities. GSH S-transferase activities were monitored toward the substrates 1-chloro-2,4-dinitro-benzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB) and 1,2-epoxy-3-(p-nitrophenoxy)propane (PO). All cell lines tested contained appreciable amounts of GSH ranging from 10 to 65 mol/mg cellular protein. Likewise, all cell lines expressed GSH S-transferase activities. However, the various cell lines differed considerably in their relative transferase activities exhibiting some degree of species-specificity.     

Microsomal UDP-glucuronyltransferase in rat liver: oxidative activation

Activation of microsomal UDP-glucuronyltransferase (UDPGT) activity by treatment of hepatic microsomes with either detergents or Fe(3+)/ascorbate pro-oxidant system has been reported; however, definite mechanisms underlying these effects have not been clarified. In this work, we characterize Fe(3+)/ascorbate-induced activation of UDPGT activity prior to solubilization with Triton X-100 and after the oxidation process provoked the solubilization of the enzyme. We observed a time-dependent increase in UDPGT activity up to 20 min. incubation of the microsomes with Fe(3+)/ascorbate (3-times); after 20 min. incubation, however, we observed a time-dependent decrease in this activity to basal levels after 4 hr incubation. Treatment of microsomes with 0.1% Triton X-100 (5 min.) lead to a similar increase in UDPGT activity; higher detergent concentrations produced a dose-dependent decrease in this activity to basal levels with 1% Triton X-100. Interestingly, UDPGT activity was susceptible to activation only when associated to microsomal membranes and the loss of activation correlated with the solubilization of this activity. UDPGT activation by either Fe(3+)/ascorbate or Triton X-100 was correlated with an increase in p-nitrophenol apparent K(m) and V(max) values. This activation was prevented or reversed by the reducing agents glutathione, cysteine or dithiothreitol when it was induced by the Fe(3+)/ascorbate. Furthermore, the latter provoked a significant decrease in microsomal thiol content, effect not observed after treatment with Triton X-100. Our results suggest that the main mechanism responsible for Fe(3+)/ascorbate-induced UDPGT activation is likely to be the promotion of protein sulfhydryl oxidation; this mechanism appears to be different from detergent-induced UDPGT activation.