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Weanling male rats were fed low (LP, 7.5%), standard (SP, 15%) or high protein (HP, 45%) diet for 7 or 14 days ad libitum, and cytochrome c reductase (CYC) and UDP-glucuronosyltransferase (UGT) enzyme activities were determined in intestine, kidney and liver microsomes. HP diet increased CYC activity in intestine and kidney, while LP diet had no effect. Hepatic CYC activity declined with decreasing level of dietary protein. Liver and intestine UGT activities were higher on an LP diet, while kidney enzyme activities were higher on an HP diet. UGT activity toward alpha-naphthol, a UGT1 isoform substrate, was modulated by dietary protein in all tissues, while UGT activity toward 4-hydroxybiphenyl, a substrate for a second UGT1 isoform, was affected only in the intestine. The duration of feeding affected CYC and UGT activities in the intestine. This observation may be explained by the dynamic nature of intestinal tissue. The observation of unique tissue and enzyme responses suggests that generalizations regarding metabolic response to diets based on hepatic studies or single enzymes, may be erroneous.

The mechanism of the inhibition of glucuronidation by long-chain fatty acyl-CoAs was studied in rat liver microsomal membranes and in isolated hepatocytes. Palmitoyl- and oleoyl-CoA did not affect p-nitrophenol UDP-glucuronosyltransferase activity in native microsomes but were inhibitory in permeabilised vesicles. The extent of inhibition was dependent on the effectiveness of permeabilisation and was constant in time in fully permeabilised microsomes. Fatty acyl-CoAs mobilised calcium from calcium-loaded microsomes. Elevation of the intracellular acyl-CoA level by the addition of palmitate or oleate inhibited the glucuronidation of p-nitrophenol in isolated hepatocytes. This effect could be abolished by emptying the intracellular calcium stores. Therefore, it is concluded that fatty acyl-CoAs inhibit glucuronidation indirectly, presumably via calcium mobilisation.

Chronic administration of alachlor in the diet at a level of 126 mg/kg/day has previously been shown to cause an increase in benign thyroid follicular cell tumors in male Long-Evans rats. Studies were conducted to elucidate the mechanism of the alachlor-induced thyroid tumors in the male rat by evaluating changes in parameters that are collectively associated with a hormonally mediated mode of action for thyroid neoplasia. Male Long-Evans rats were administered 126 mg alachlor/kg body wt/day via the diet for up to 120 days. One group of animals was removed from alachlor-treated diet after 60 days and received untreated diet for an additional 60 days. Liver and thyroid weights and serum levels of triiodothyronine (T3), thyroxine (T4), and thyroid-stimulating hormone (TSH), as well as hepatic uridine diphosphate glucuronosyl transferase (UDPGT) activity, were determined at 7, 14, 28, 60, and 120 days of treatment. Liver and thyroid weights, hepatic UDPGT activity, and circulating levels of TSH were significantly increased in animals administered alachlor. These increases were seen as early as 7 days after alachlor administration. Circulating levels of T4 in alachlor-treated animals were significantly decreased compared with controls at 7 days, but had returned to control levels by 60 days. T3 levels were elevated at all time points except at 28 days. The changes in TSH and T3 levels, hepatic UDPGT activity, and liver weights were all reversible on elimination of alachlor from the diet. Thyroid weights did not completely return to control levels after removal of alachlor from the diet, although some recovery was evident. The results of this study clearly suggest that alachlor-induced thyroid neoplasia, observed in previous chronic bioassays with alachlor, was associated with increases in circulating TSH levels. Increased metabolism of T4 via hepatic enzymatic conjugation (i.e., T4-UDPGT) appeared to be responsible for the increased TSH levels. These effects were shown to be reversible on cessation of exposure to alachlor. In summary, evidence is presented for a hormonally mediated process for the development of thyroid follicular cell tumors.

Important species differences have been reported concerning the induction properties of rifampicin towards enzymes of the P-450 superfamily. Mice, rabbits and humans are far more responsive than rats and guinea pigs. In the present study a strong induction of cytochrome P-450 3A-dependent enzyme activities was observed in female rat liver microsomes after high dose treatment (> or = 250 mg/kg/day for 9 days) with rifampicin, resulting in an up to 30-fold enhanced hydroxylation rate of testosterone in the 2 beta-, 6 beta- and 15 beta-position in vitro. Other cytochrome P-450 isozyme-selective reactions were not, or only marginally, affected. A steep increase in cytochrome P-450 3A activity on a moderate elevation of the dose administered, together with the previously observed lack of efficient induction with doses below 200 mg/kg/day demonstrated that there is a threshold in enzyme induction by rifampicin. For rifabutin such a threshold was not apparent. Induction by rifabutin showed an isoenzyme-selectivity profile similar to that produced by rifampicin, but the maximally achievable induction of cytochrome P-450 3A by rifabutin was about two-fold lower compared with rifampicin. Rifampicin and rifabutin enhanced the glucuronidation of 1-naphthol, 4-hydroxybiphenyl and beta-estradiol by a factor of two to three. The potential implications of the enzyme induction by rifampicin derivatives in terms of possible drug-drug interactions are discussed.

Proinflammatory cytokines play an important role in the depression of cytochrome P450 (CYP450)-dependent drug metabolism in mammals during inflammation and infection. Although much has been learned concerning the effects and mechanisms of cytokine-mediated suppression of CYP450, there is limited knowledge about how cytokines affect UDP glucuronosyl transferases (UDPGT). The aim of the present study was to investigate the effects and dose dependency of recombinant human proinflammatory cytokines on both CYP450- and UDPGT-dependent enzyme activities in primary cultures of pig hepatocytes. A possible role of nitric oxide in cytokine-induced suppression of enzyme activities was studied by incubating hepatocytes in the presence of N G-nitro-L-arginine (L-NAME), a competitive inhibitor of nitric oxide (NO) biosynthesis. Incubation of hepatocytes with interleukin-1alpha (IL-1alpha), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) decreased both oxidation and glucuronidation activities dose dependently, in which the effects on glucuronidation activities were even more pronounced. IL-6 differed from IL-1alpha and TNF-alpha by inhibiting CYP450 and UDPFT more effectively after 24 hr of incubation, whereas the inhibition by IL-1alpha and TNF-alpha was more pronounced after 12 hr. Only at a concentration of 500 U/ml did interferon-gamma (IFN-ganna) inhibit CYP450 and UDPGT. The inhibition of CYP450 enzyme activities by cytokines was probably not due to the production of NO, because L-NAME totally blocked NO production but had no effect on the cytokine-induced suppression of CYP450 enzyme activities. However, there might be a role for NO in the decrease of glucuronidation by cytokines, as L-NAME slightly though significantly prevented the inhibition of glucuronidation.

The UDP-glucuronosyltransferase (UGT) system fulfils a pivotal role in the biotransformation of potentially toxic endogenous and exogenous compounds. Here we report that the activity of UGT in rat liver is stimulated by UDP-xylose. This stimulation was found in native microsomal vesicles as well as in the intact endoplasmic reticulum (ER) membrane, as studied in permeabilized hepatocytes, indicating the potential physiological importance of UDP-xylose in the regulation of UGT. We present evidence that UDP-xylose enhances UGT activity by stimulation of (i) the uptake of UDP-glucuronic acid across the ER membrane and (ii) the elimination of the UDP and/or UMP reaction product out of the ER lumen. UDP-xyloe produced a marked trans-stimulation of microsomal UDP-glucuronic acid uptake when it was present within the lumen of the ER. When UDP-xylose was presented at the cytosolic side of the ER, it acted as a weak inhibitor of UDP-glucuronic acid uptake. Likewise, cytosolic UDP-glucuronic acid strongly trans-stimulated efflux of intravesicular UDP-xylose, whereas cytosolic UDP-xylose was inefficient in trans-stimulating efflux of UDP-glucuronic acid. Microsomal UDP-xylose influx was markedly stimulated by UMP and UDP. Such stimulation was only apparent when microsomes had been preincubated and thereby preloaded with UMP or UDP, indicating that UMP and UDP exeted their effect on UDP-xylose uptake by trans-stimulation from the luminal side of the ER membrane.

Omeprazole induces CYP1A in the human liver and gut, which has led to concern about possible side effects. The purpose of this study was to compare the effects of omeprazole on phase 1 and phase 2 enzymes in the rat and human. Male rats were treated with intraperitoneal (40 or 80 mg/kg) or oral omeprazole (40 mg/kg) for 5 or 14 days, respectively. The activities and amounts of CYP1A, uridine diphosphate-glucuronosyltransferase, and glutathione transferase were determined in liver and gut. Enzyme activities were also determined in duodenal biopsy specimens from six healthy human volunteers before and after treatment with omeprazole (20 mg/day) for 10 days. Treatment with intraperitoneal omeprazole (40 mg/kg; 80 mg/kg) coinduced uridine diphosphate-glucuronosyltransferase (36%; 66%), glutathione transferase (22%; 50%), and CYP1A (26%; 50%) in rat liver. In rat small intestine, comparable levels of induction were observed for uridine diphosphate-glucuronosyltransferase and glutathione transferase; CYP1A was unaffected. Oral omeprazole had similar effects. Immunoblotting showed corresponding changes in the amounts of these enzymes. Omeprazole increased the activities of CYP1A (19% to 167%; p = 0.014) and uridine diphosphate-glucuronosyltransferase (11% to 68%; p = 0.04) in the duodenal biopsy specimens of all six human volunteers; glutathione transferase was unaffected. Thus, omeprazole coinduced multiple xenobiotic metabolizing enzymes in the rat and human. The pattern of induction differed in the rat and human, consistent with known differences in genetic regulatory elements in the two species.

Thirty-one differently substituted pyrimidine bases were tested for their inhibitory effect on the glucuronidation of 4-nitrophenol and phenolphthalein by rat liver microsomes. 5-Nitrouracil (compound 1) and its isomer 4,6-dihydroxy-5-nitropyrimidine (compound 2) were the most potent and selective inhibitors of 4-nitrophenol glucuronidation, without any effect on the phenolphthalein conjugating activity of UDP-glucuronyltransferase (UGT). Kinetic studies with compound 1 revealed a mixed type of inhibition toward the acceptor substrate 4-nitrophenol and an atypical competitive type of inhibition toward UDP-glucuronic acid, with apparent Ki values of 0.11 and 0.2 mM, respectively. Two benzylamino-substituted pyrimidines (compounds 10 and 12) and an orotic acid derivative (compound 25) inhibited both 4-nitrophenol and phenolphthalein UGT activities.

UDP-glucuronosyltransferase (UDP-GT) inducers have been shown to lower plasma levels of thyroxine (T4) by increasing its glucuronidation and elimination by the liver. However, there are no dose-response studies to address the relationship between induction of hepatic UDP-GT and alteration in thyroid homeostasis. Therefore, rats were fed a basal diet or a diet mixed with phenobarbital (PB: 600, 1200, 1800 or 2400 ppm), pregnenolone-16 alpha-carbonitrile (PCN: 250, 500, 1000 or 2000 ppm), 3-methylcholanthrene (3MC: 62.5, 125, 250 or 500 ppm) or a polychlorinated biphenyl mixture (Aroclor 1254, PCB: 10, 30, 100 or 300 ppm) for 15 days to determine their effects on hepatic UDP-GT induction, reduction of serum thyroid hormones and alteration of thyroid function. All the UDP-GT inducers produced a dose-dependent induction of hepatic UDP-GT activity toward T4; the increases produced by PCN (7-fold) and PCB (5-fold) were more pronounced than those produced by PB and 3MC (3-fold). Serum T4 (total and free T4) levels were reduced dramatically by the UDP-GT inducers in a dose-dependent manner (up to 50%-90%). However, they had no effect on serum free T3 and a minimal effect on decreasing serum total T3 levels (10%-20%). Reverse T3 levels were increased by all doses of PCN, by high doses of 3MC and by low doses of PCB. PCN produced a dose-dependent increase in serum thyroid-stimulating hormone (TSH) levels (up to 5-fold), and PB doubled TSH levels. Most surprisingly, even though 3MC and PCB decreased serum T4, they had minimal effects on TSH levels.(ABSTRACT TRUNCATED AT 250 WORDS)

We propose the existence in rat liver endoplasmic reticulum (ER) of two asymmetric carrier systems. One system couples UDP-N-acetylglucosamine (UDPGlcNAc) transport to UDP-glucuronic acid (UDPGlcA) transport. When UDPGlcNAc was presented at the cytosolic side of the ER, it then acted as a weak inhibitor of UDPGlcA uptake. By contrast, UDPGlcNAc produced a forceful trans-stimulation of microsomal UDPGlcA uptake when it was present within the lumen of the ER. Likewise, cytosolic UDPGlcA strongly trans-stimulated efflux of intravesicular UDPGlcNAc, whereas cytosolic UDPGlcNAc was ineffective in trans-stimulating efflux of UDPGlcA. A second asymmetric carrier system couples UDPGlcNAc transport to UMP transport. Microsomal UDPGlcNAc influx was markedly stimulated by UMP present inside the microsomes. Such stimulation was only apparent when microsomes had been preincubated and thereby preloaded with UMP, indicating that UMP exerted its effect on UDPGlcNAc uptake by trans-stimulation from the lumenal side of the ER membrane. Contrariwise, extravesicular UMP only minimally trans-stimulated efflux of intramicrosomal UDPGlcNAc. It is widely accepted that UDPGlcNAc acts as a physiological activator of hepatic glucuronidation, but the mechanism of this effect has remained elusive. Based on our findings, we propose a model in which the interaction of two asymmetric transport pathways, i.e. UDPGlcA influx coupled to UDPGlcNAc efflux and UDPGlcNAc influx coupled to UMP efflux, combined with intravesicular metabolism of UDPGlcA, forms a mechanism that leads to stimulation of glucuronidation by UDPGlcNAc.

Dietary antioxidants protect laboratory animals against the induction of tumors by a variety of chemical carcinogens. Among possible mechanisms, protection against chemical carcinogenesis could be mediated via antioxidant-dependent induction of detoxifying enzymes. Therefore, we investigated the effects of two commonly used food preservatives, butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), on the expression of UDP-glucuronosyltransferase isoforms in rat liver. Male Wistar rats were fed a control diet or diets containing BHA (0.75%) or BHT (0.5%) for 2 weeks. BHT and BHA increased UDP-glucuronosyltransferase activities in liver microsomes for p-nitrophenol (236 and 218%, respectively), 3-hydroxybenzo(a)pyrene (246 and 175%, respectively), and androsterone (269 and 152%, respectively). Immunoblots showed changes in the amounts of UDP-glucuronosyltransferase isoforms corresponding to the changes in enzyme activities. Northern blot analysis showed that the concentration of UDP-glucuronosyltransferase mRNA paralleled the concentration of enzyme proteins and their respective levels of enzyme activity. BHT, for example, caused about a 250% increase in mRNA using a probe that recognizes the common 3'-domain of bilirubin/phenol UDP-glucuronosyltransferase mRNAs. In addition to inducing hepatic enzyme activities, BHT and BHA increased the activity of UDP-glucuronosyltransferase in the small intestine and kidney.

In this study, adult female rats were treated with either estradiol, testosterone or triiodothyronine (T3). The mean +/- SD for serum levels of androstane-3 alpha,17 beta-diol glucuronide (ADIOL-G) in rats injected with estradiol (31.1 +/- 6.1 nmol/l) and T3 (28.7 +/- 6.1 nmol/l) increased three-fold in comparison to the controls (9.2 +/- 3.6 nmol/l; p < 0.01). There was no significant increase in ADIOL-G levels in rats injected with testosterone (12.0 +/- 3.4 nmol/l) even though serum testosterone levels increased more than 17-fold (1.2 +/- 0.3 nmol/l; p < 0.01). Testosterone levels remained below the detection level of the assay (0.07 nmol/l) in the control and the estradiol and T3 treatment groups. Androgen UDPGT activity in liver microsomes was not altered by treatment with testosterone, estradiol or T3. However, the UDPGT activity for p-nitrophenol increased two-fold in estradiol and T3-treated females (p < 0.01), but was not significantly altered by testosterone treatment. These results suggest that the activity of androgen UDPGT in liver is not the limiting factor in the increased serum levels of ADIOL-G observed on treatment with estradiol and T3.

Increase of N-demethylase and antioxidant activities and decrease of amount of hydroperoxides were observed in liver of rats after intragastric administration of Undevit (1/8 dragee/rats/day, 5 times a week during 2 months). Combination of Undevit with Cordiamin (5 mg/rat/day) resulted in augmentation of cytochromes P-450 and b5 content and activities of their reductases, velocity of amidopyrine and ethylmorphine N-demethylation and oxidation of HADPH. Activities of UDP-glucuronyltransferase, glutathionetransferase and hydrophobity of microsomal membranes were also increased. Antioxidant activity was increased and amount of hydroperoxides in liver microsomes and homogenates and in plasma was decreased in greater degree after administration of combination two compounds than after administration of Undevit alone.

Experimental arthritis and inflammation have been reported to reduce liver cytochrome P-450-dependent mono-oxygenase activities with subsequent impairment of drug metabolism. Interleukin-1 beta (IL-1) is among the proven mediators of both inflammation and P-450 decrease, although some paradoxical effects were sometimes reported in experimental models of arthritis. The aim of the present study was to evaluate the main liver drug-metabolizing isoenzymes during established collagen-induced arthritis in rats, and to investigate whether a systemic IL-1 treatment was able to mimic or sometimes to reverse the influence of the inflammatory process on these enzymes. Arthritis was induced on day 0 by type II collagen and a low dose (0.2 mg) of N-acetylmuramyl-L-alanyl-D-isoglutamine, and human recombinant IL-1 was administered s.c. at the daily dose of 0.02, 0.2 or 2.0 micrograms per arthritic rat, from day 21 to 25 and on day 28. Ethoxyresorufin-O-deethylation was depressed 6-fold in arthritic rat liver microsomes and the highest dosage of IL-1 potentiated this depression. Pentoxyresorufin-O-deethylation decreased by 50% in arthritic rat, a dose-dependent decrease being observed after IL-1 treatment. Progesterone 6 beta-hydroxylation and P-450 IIIA protein increased by 2-fold in both untreated arthritic rat liver microsomes and those treated by the lowest dose of IL-1. The two higher doses decreased this activity, vs. the dose, to reach the naive level. Lauric acid hydroxylation increased 2-fold in arthritic rat and was further potentiated by IL-1. UDP glucuronosyl transferase IA2 activity was increased 2-fold in arthritic rats, with subsequent decrease after 2.0 micrograms of IL-1.(ABSTRACT TRUNCATED AT 250 WORDS)

A simple and reproducible assay method for UDP-glucuronyltransferase (GT) towards 5-hydroxytryptamine (5-HT) was developed. It consists of the removal of unconjugated 5-HT by 0.6 N NH4OH-saturated n-amyl alcohol and the colorimetric estimation of 5-HT glucuronide. Using this assay method, some properties of the enzyme activity in rat liver microsomes were studied. Simple Michaelis-Menten kinetics was followed with respect to 5-HT and the apparent Km value for 5-HT was 0.1 mM. However, the deviation from this kinetics was observed with respect to UDP-glucuronic acid (UDPGA). The apparent Km values for UDPGA were 0.6 mM and 5 mM. The enzyme activity was stimulated by divalent cations. For Mg2+, the enzyme did not obey this kinetics, and the apparent Km values for Mg2+ were 1 mM and 10 mM. In the presence of Mg2+, the apparent Km value for 5-HT did not change but the Vmax value increased. On the other hand, the addition of a low concentration of Mg2+ decreased the apparent Km value for UDPGA and increased the Vmax value. The addition of a high concentration of Mg2+ did not change the apparent Km value for UDPGA but increased the Vmax value. These results indicate that the enzyme activity is stimulated by the formation of Mg(2+)-UDPGA complex which showed higher affinity for the enzyme than UDPGA at the low concentration of Mg2+ and further stimulated by the formation of Mg(2+)-enzyme complex at the higher concentration of Mg2+.

The effect of non-steroidal anti-inflammatory drugs (NSAIDs) on the activity of glycosyltransferases required for the synthesis of the polysaccharide chains of proteoglycans, was studied in human osteoarthritic cartilage in vitro. Using exogenous acceptors, salicylate and indomethacin suppressed the activity of glucuronyl- and xylosyltransferases in a concentration-dependent manner, but had little effect on N-acetylgalactosaminyl- and galactosyltransferases. When used at a concentration derived from the values found in the synovial fluid, salicylate, indomethacin and chloroquine significantly suppressed the activity of glucuronyl- and xylosyltransferases, while tiaprofenic acid, paracetamol (acetaminophen), floctafenine, ketoprofen, ibuprofen and tenoxicam had no effect on the enzymes. An alteration of some glycosyltransferases could explain the reported suppressive effect of some NSAIDs on cartilage proteoglycan synthesis.

This experiments were carried out on: 0.5-, 1-, 2-, 4-, 8-, 12-, 20- and 28-months old Wistar males rats. Before they were killed the animals received by intraperitoneal injection: phenobarbital (50 mg/kg in 0.9% saline 72- and 48-hr before the death), beta-naphthoflavone (20 mg/kg in sesame oil for 3 consecutive days prior to sacrifice), dexamethasone (10 mg/kg; see beta-naphthoflavone). All animals were decapitated after 24 hr of starvation. The livers were rinsed with cold 0.9% saline. Hepatic microsomes were prepared as described Dallner. The UDP-glucuronyltransferase activities with p-nitrophenol as aglycone were determined according to Burchell and Weatherill and protein concentration was measured as described Lowry et al. The activity of examined enzyme had maximal value in youngest rats, but in older one it was decreased. Minimum was observed in 8-month old animals and all older. Pretreatment of rats with beta-naphthoflavone significantly increased glucuronidation especially in sexually mature animals. The date obtained with phenobarbital and dexamethasone pretreatment rats suggested lower magnitude of stimulation of UDP-glucuronyltransferase than beta-naphthoflavone and lack of effect respectively.

Microsomal arylhydrocarbon (benzo[a]pyrene) hydroxylase activity in hyperplastic nodules in the livers of rats was decreased by feeding 2-acetylaminofluorene in their diet to 10% of that without 2-acetylaminofluorene feeding. By immunoblotting analysis with the antibodies raised against P-450/B[a]P, which is a form of cytochrome P-450 catalyzing benzo[a]pyrene hydroxylation in liver microsomes of untreated rats, it was shown that P-450/B[a]P content was decreased in the microsomes prepared from the nodular tissues. Microsomal UDP-glucuronyltransferase activity toward phenolic xenobiotics such as 1-naphthol and 4-nitrophenol was increased by 4.5-5.0-fold in the nodular tissues. This increase was inhibited by the addition of antibodies raised against GT-1, a form of UDP-glucuronyltransferase glucuronizing phenolic xenobiotics. Immunoblotting analysis with the antibodies against GT-1 showed that a protein band corresponding to GT-1 was increased in the microsomes from nodular tissues. The increased UDP-glucuronyltransferase also had about 4,000 daltons "high mannose" oligosaccharide(s) like GT-1. The decreases of P-450/B[a]P and increases of GT-1 were observed mainly in nodular foci of the liver tissues. These results indicate that in liver nodules, decreased cytochrome P-450-dependent benzo[a]pyrene hydroxylase activity and increased UDP-glucuronyltransferase activity toward phenolic xenobiotics result from the decrease of the P-450 corresponding to P-450/B[a]P and the increase of the GT corresponding to GT-1.