The enzymatic mechanism of glucuronidation catalyzed by two purified rat liver steroid UDP-glucuronosyltransferases

A kinetic analysis of two homogeneous rat liver steroid (3 alpha-hydroxysteroid and 17 beta-hydroxysteroid) UDP-glucuronosyltransferases was conducted using bisubstrate kinetic analysis, product inhibition studies, and dead-end competitive inhibition studies. Double reciprocal plots of initial velocity versus substrate concentration, using bisubstrate kinetic analysis, gave a sequential mechanism. Product inhibition studies were compatible with either a rapid equilibrium, random-order kinetic mechanism or an ordered Theorell-Chance mechanism. Results of dead-end competitive inhibition studies excluded an ordered Theorell-Chance mechanism. The cumulative results are consistent with a rapid equilibrium random-order sequential kinetic mechanism for the glucuronidation of testosterone by purified 17 beta-hydroxysteroid UDP-glucuronosyltransferase and of androsterone by purified 3 alpha-hydroxysteroid UDP-glucuronosyltransferase.     

The kinetic basis for age-associated changes in quercetin and genistein glucuronidation by rat liver microsomes

The dietary bioavailability of the isoflavone genistein is decreased in older rats compared to young adults. Since flavonoids are metabolized extensively by the UDP-glucuronosyltransferases (UGTs), we hypothesized that UGT flavonoid conjugating activity changes with age. The effect of age on flavonoid glucuronidation was determined using hepatic microsomes from male F344 rats. Kinetic models of UGT activity toward the flavonol quercetin and the isoflavone genistein were established using pooled hepatic microsomal fractions of rats at different ages, and glucuronidation rates were determined using individual samples. Intrinsic clearance (V_max/K_m) values in 4-, 18- and 28-month-old rats were 0.100, 0.078 and 0.087 ml/min/mg for quercetin-7-O-glucuronide; 0.138, 0.133 and 0.088 for quercetin-3′-O-glucuronide; and 0.075, 0.077 and 0.057 for quercetin-4′-O-glucuronide, respectively. While there were no differences in formation rates of total quercetin glucuronides in individual samples, the production of the primary metabolite, quercetin-7-O-glucuronide, at 30 μM quercetin concentration was increased from 3.4 and 3.1 nmol/min/mg at 4 and 18 months to 3.8 nmol/min/mg at 28 months, while quercetin-3′-O-glucuronide formation at 28 months declined by a similar degree (P≤.05). At 30 and 300 μM quercetin concentration, the rate of quercetin-4′-O-glucuronide formation peaked at 18 months at 0.9 nmol/min/mg. Intrinsic clearance values of genistein 7-O-glucuronide increased with age, in contrast to quercetin glucuronidation. Thus, the capacity for flavonoid glucuronidation by rat liver microsomes is dependent on age, UGT isoenzymes and flavonoid structure.     

Comparison of cytochrome P-450 levels in adult rat liver, postnatal rat liver, and primary cultures of postnatal rat hepatocytes

A system of primary cultures of postnatal rat hepatocytes has been developed to serve as an experimental model for drug metabolism and toxicity investigations. The purpose of this study was to examine the reported loss of cytochrome P-450 of hepatocytes when placed in culture and to compare activity in culture to intact liver and freshly isolated hepatocytes. A medium enriched with several hormones and a system of floating filters as a substratum for cell attachment were investigated as methods to reduce the expected loss of cytochrome P-450. When compared to initial values of cytochrome P-450 in whole liver and isolated hepatocytes, these methods failed to prevent the reduction of cytochrome P-450 in culture. However, our results compare favorably with other values reported in the literature.     

The oxidation of different substrates by liver mitochondria from rat pups in early postnatal development

We have studied the respiratory ratio of liver mitochondria during oxidation of substrates of the carbohydrate and lipid metabolism in newborn rats and during early postnatal development. High rate of respiration coupled with the synthesis of ATP from ADP and phosphate has been found during oxidation of carbohydrate substrates (pyruvate + malate); however, caprilate, a substrate of lipid metabolism, does not support such respiration. However, in the young rats aging from 2 to 30 days utilization of carbohydrate and lipid substrates via the phosphorylating pathway proceeds with similar efficacy.     

The metabolism of benzo[a]pyrene phenols by rat liver microsomal fractions

The oxidative metabolism of benzo[a]pryrene (B[a]P) phenols catalyzed by liver microsomes in vitro leads to multiple products. High-pressure liquid chromatography analysis of the organic-soluble products formed indicates that regardless of the animal pretreatment regime, 3-hydroxy-B[a]P is metabolized to the 3,6-quinone and to a hydroxylated derivative tentatively identified as 3,9-dihyroxy-B[a]P. However, the distribution of products obtained with 9-hydroxy-B[a]P varied with animal pretreatment. A maximum of three distinct metabolites was obtained when the 9-phenol was metabolized in vitro with microsomes from phenobarbital-pretreated rats and the tentative 3,9-dihydroxy derivative was a common metabolite for all pretreatment regimes. Physical characterization, including mass spectrometry, indicates that all three products have an extra oxygen atom incorporated into their molecular structure from molecular oxygen. Studies utilizing specific inhibitors of the cytochrome P-450-dependent monooxygenase clearly suggest that the formation of dihydroxy or phenol-oxide derivatives is catalyzed by the hemoprotein, cytochrome P-450. These metabolites of the benzo[a]pyrene phenols are most likely related to the putative phenol-oxides of benzo[a]pyrene which have been demonstrated to alkylate DNA and protein. Repetitive scan difference spectrophotometric analysis of incubation mixtures containing rat liver microsomes, 3- or 9-hydroxy-B[a]P, NADPH, and oxygen shows the conversion of the phenols into products which absorb in the region from 400 to 500 nm. During and after the steady state of the reaction, it can be seen that certain of the hydroxy compounds produced are in equilibrium with their respective quinone form and may be involved in an oxygen-coupled redox cycle.     

Dicoumarol-sensitive glucuronidation of benzo(a)pyrene metabolites in rat liver microsomes

The effect of dicoumarol on glucuronidation of 3-OH-benzo(a)pyrene (BP) appears to be due to inhibition of UDPglucuronosyltransferase (UDPGT) and not to an inhibited DT-diaphorase (NAD(P)H:quinone oxidoreductase); to date the only enzyme known to be inhibited by dicoumarol. This dicoumarol-sensitive form of UDPGT does not seem to be identical to the major form catalyzing the glucuronidation of p-nitrophenol or methylumbelliferone, nor to the isozyme involved in the formation of phenolphthalein glucuronides. These conclusions are based on the following observations: In solubilized microsomes, devoid of DT-diaphorase, a 3-OH-BP glucuronidation activity is found which is very similar to that observed in microsomes before passing through an azodicoumarol Sepharose 6B column that binds more than 98% of DT-diaphorase; in the eluate from this column the inhibition by dicoumarol of 3-OH-BP glucuronidation is the same as in microsomes containing DT-diaphorase; other coumarin derivatives, which are either modified or substituted in the methylene bridge between the two coumarin entities in dicoumarol, are potent inhibitors of DT-diaphorase but not of UDPGT; a concentration of 10(-6) M dicoumarol is sufficient to inhibit 3-OH-BP glucuronidation 50%. In contrast, to inhibit glucuronidation of p-nitrophenol or methylumbelliferone the concentration of dicoumarol must be raised to the substrate level: i.e., 10(-4) M. Phenolphthalein glucuronidation is almost unaffected even by this high concentration of dicoumarol. The present investigation also reveals that DT-diaphorase and NADPH-cytochrome P-450 reductase can both catalyze the reduction of BP-3,6-quinone for the formation of BP-3,6-quinol glucuronides. In the eluate from the azodicoumarol Sepharose 6B column, no NADH-supported glucuronidation of BP-3,6-quinone can be detected unless DT-diaphorase is added. However, NADPH-supported formation of BP-3,6-quinol glucuronides can still be observed. The rate of the latter reaction is sufficient enough to allow studies on the effect of dicoumarol on BP-3,6-quinone glucuronidation. These results show that glucuronidation of BP-3,6-quinols is also catalyzed by a dicoumarol-sensitive UDPGT. However, not only is the formation of BP-3,6-quinol monoglucuronides inhibited by dicoumarol, but the conversion of monoglucuronides to diglucuronides is inhibited as well. The former reaction is inhibited 50% by 3.5 X 10(-6) M dicoumarol (close to the I50 for 3-OH-BP glucuronidation), whereas 10 times less dicoumarol (2 X 10(-7) M) is sufficient for 50% inhibition of the latter reaction.(ABSTRACT TRUNCATED AT 400 WORDS)     

Enhancement of microsomal drug oxidation and glucuronidation in rat liver by an environmental chemical, polychlorinated biphenyl

A polychlorinated biphenyl (PCB) compound, Clophen A 50, enhanced both hepatic aryl hydrocarbon hydroxylase and p-nitroanisole O-demethylase activities (7.5-fold and 16-fold, respectively), after treating the rats for 6 days with consecutive daily injections of Clophen A 50 (15 mg/kg i.p.). The treatment increased 3-fold the content of the carbon monoxide binding hemoprotein in liver microsomes, causing a concomitant shift in its reduced carbon monoxide absorbance peak to 448 nm. NADPH cytochrome c reductase, another component reaction of the microsomal mixed-function oxidase, was enhanced 1.5-fold in 6 days. A slight enhancement in the overall hydroxylation reactions was already observable 24 h after a single injection of Clophen A 50.The UDPglucuronosyltransferase activity of native liver microsomes was enhanced 3-fold in 6 days by the Clophen A 50 treatment of rats. The enhancement was, however, more pronounced, if the microsomes were treated in vitro with membrane-perturbing agents to activate the latent UDPglucuronosyltransferase before measuring its activity. After treatment for 6 days, the enhancement was about 6-fold in digitonin-treated, 5-fold in phospholipase C-treated and about 10-fold in trypsin-digested microsomes. No enhancement could be detected 24 h after a single Clophen A 50 injection.Aryl hydrocarbon hydroxylase activity was also enhanced in lung (5-fold), and kidney (8-fold) microsomes, whereas the microsomes from the duodenal mucosa exhibited no enhancement by a Clophen A 50 treatment of rats for 3 days.The data obtained support the assumption that PCBs form a new type of inducer group in enhancing the microsomal drug biotransformation. Both the monooxygenase complex and UDPglucuronosyltransferase differ in their properties from those after enhancement with the known types of inducers, exemplified by phenobarbital and 3-methylcholanthrene, respectively.     

An organ culture of postnatal rat liver slices

Summary A technique for the organ culture of postnatal and adult rat liver has been developed. Liver slices, 0.3 mm thick, were maintained in Conway units at the interphase between medium and a 95% O₂:5% CO₂ atmosphere. Postnatal liver in culture for up to 72 h had healthy hepatocytes throughout the explants; if adult liver was used the upper 0.2 mm was healthy after 24 h. These slices incorporated tritiated orotate and leucine into trichloroacetic acid-precipitable material. Incorporation of orotate was shown to be spread over the entire slice of neonatal liver. Culturing did not alter the potassium ion content of postnatal liver. Tyrosine aminotransferase activity in liver slices from postnatal, adult, and adrenalectomized adult rats was stimulated by glucocorticoids and dibutyryl cyclic AMP. Cycloheximide and actinomycin D prevented this response. Further, cortisol exerted a permissive effect on the stimulation of tyrosine aminotransferase activity by dibutyryl cyclic AMP in slices from adrenalectomized rats. Induction of urea cycle enzymes by cortisol was demonstrated in cultures of liver from adrenalectomized adult animals. Deceased October 1, 1983. This research was supported in part by a grant from the South African Council for Scientific and Industrial Research.     

Co-regulation of the mixed-function oxidation of p-nitroanisole and glucuronidation of p-nitrophenol in the perfused rat liver by carbohydrate reserves

Maximal rates of mixed-function oxidation of p-nitroanisole and the glucuronidation of p-nitrophenol in perfused livers from phenobarbital-treated rats varied directly with the nutritional state of the rat (i.e., fasted < fed < fasted-refed). Rates correlated with intracellular concentrations of NADPH, UDP-glucuronic acid, and glycogen but not with amounts of cytochrome P-450 or glucuronyltransferase activity. These data support the hypothesis that mixed-function oxidation and glucuronidation are coregulated in intact cells by carbohydrate-dependent cofactor synthesis.     

Peroxisome proliferator-activated receptor-alpha expression in rat liver during postnatal development

The expression of the peroxisome proliferator-activated receptor-alpha (PPARalpha) as well as of some related genes was studied in rat liver at different stages of development (from 19-day-old fetuses to 1 month-old rats). The level of PPARalpha mRNA appeared higher in neonates than in fetuses or 1 month-old rats. Whereas the pattern for phosphoenolpyruvate carboxykinase (PEPCK) mRNA level was similar to that of PPARalpha, the mRNA level of both acyl-CoA oxidase (ACO) and apolipoprotein CIII (apo CIII) showed diverse profiles. Western blotting analysis also revealed an increased level of PPARalpha protein in liver of suckling rats. Similarities of mRNA PEPCK and PPARalpha expression indicate a common control mechanism, where both nutritional and hormonal factors may be involved.     

In vitro glucuronidation of xanthohumol, a flavonoid in hop and beer, by rat and human liver microsomes

Xanthohumol (XN) is the major prenylated flavonoid of hop plants and has been detected in beer. Previous studies suggest a variety of potential cancer chemopreventive effects for XN, but there is no information on its metabolism. The aim of this study was to investigate in vitro glucuronidation of XN by rat and human liver microsomes. Using high-performance liquid chromatography, two major glucuronides of XN were found with either rat or human liver microsomes. Release of the aglycone by enzymatic hydrolysis with beta-glucuronidase followed by liquid chromatography/mass spectrometry and nuclear magnetic resonance analysis revealed that these were C-4' and C-4 monoglucuronides of XN.     

The esterification of dolichol by rat liver microsomes.

The incubation of 1-[3H)dolichols with cell-free preparations from various rat tissues resulted in the formation of a labeled material which possessed the characteristics of synthetic dolichol palmitate. Rat liver microsomes were found to be a good source of the acyltransferase activity, and the properties of the reaction were investigated using microsomal preparations. The reaction did not require ATP, CoA, or Mg2+ and was stimulated by the addition of phosphatidylcholine. The esterification of dolichol appears to be similar to the esterification of retinol. The fact that the esterification of dolichol is not depressed even in the presence of a several-fold excess of retinol is evidence that the two reactions are catalyzed by different enzymes.     

The uptake of choline by rat liver mitochondria.

1. Rat liver mitochondria can accumulate choline against a concentration gradient. Maximally about 30 nmol choline per mg mitochondrial protein are found in the matrix space. 2. The process of choline uptake is biphasic. After a rapid uptake of 1.5-15 nmol per mg protein, a slower uptake occurs if an energy supply is present. In the absence of energy, only the rapid uptake is found. 3. The inhibition of uncoupler-stimulated choline oxidation by cations is the result of an inhibition of choline uptake.