Antipyrine clearance,aminopyrine N-demethylase,and bilirubin udp-glucuronyl transferase activity in patients with amoebic liver abscess

Three indices of drug metabolism, antipyrine clearance in vivo, and aminopyrine N-demethylase and bilirubin UDP-glucuronyl transferase activity in liver biopsies, were studied in fifteen patients with amoebic liver abscess (with and without jaundice). The mean (± S.E.) antipyrine half-life in patients with jaundice was 21.64 (±1.52)h and in patients without jaundice was 19.36 ( ± 0.93)h. It was significantly prolonged in both groups of patients as compared to controls (11.63 ± 0.86 h). It showed a good correlation with serum albumin (p<0.01), prothrombin time index (p<0.01), and aminopyrine N-demethylase (p<0.05). Aminopyrine N-demethylase was found to be decreased in patients without jaundice but no significant change could be observed in patients with jaundice. Bilrubin UDP-glucuronyl transferase showed no significant change in either of the groups.     

Selective inhibitory effect of organophosphates on UDP-glucuronyl transferase activities in rat liver microsomes

The effects of acute and subacute administration of diisopropylfluorophosphate (DFP) and acute administration of Soman, Sarin and Tabun on UDP-glucuronyltransferase (GT) activity towards 4-nitrophenol, 4-methylumbelliferone, phenolphthalein and testosterone in rat liver microsomes were investigated. Twenty-four hours after a single injection of DFP, the activity of GT towards 4-nitrophenol and 4-methylumbelliferone was inhibited, and the inhibitory effect continued for 3 days. The activity had recovered by 7 days after injection. The activity of GT towards phenolphthalein and testosterone was not affected at any time after injection. Soman, Sarin and Tabun showed the same effect as DFP after a single injection. After daily DFP injections, the activity of GT towards 4-nitrophenol and 4-methylumbelliferone was decreased to the same level as found following acute treatment with DFP. The in vitro addition of DFP to liver microsomes did not affect GT activity towards 4-nitrophenol. It is suggested that these changes are not due to a direct effect of DFP. Furthermore, the effects of two enzyme inducers on GT activity in the presence and absence of DFP were investigated. In the 3-methylcholanthrene (MC) pretreatment group, DFP inhibited only the GT activity towards 4-nitrophenol and 4-methylumbelliferone. On the other hand, in the phenobarbital (PB) pretreatment group, DFP did not inhibit the GT activity towards 4-nitrophenol and 4-methylumbelliferone. It was also demonstrated that MC pretreatment increased the mortality in the DFP-treated rats but that PB pretreatment suppressed it. These results suggest that DFP and other organophosphorus agents may be useful agents for studies on the heterogeneity of GT.     

Effect of asbestos fibers on aryl hydrocarbon hydroxylase and aminopyrine N-demethylase activities of rat liver microsomes

Chrysotile asbestos fibers impair the activities of rat liver microsomal aryl hydrocarbon hydroxylase (AHH), aminopyrine (AP) N-demethylase and dimethylnitrosamine (DMN) demethylase in vitro. This inhibition is concentration-dependent. Preincubation of 3-methylcholanthrene (3-MC)-pretreated rat liver microsomes with chrysotile depresses the overall metabolism of [G-3H]benzo[a]pyrene (BaP). Various forms of asbestos employed inhibit AHH activity to the same extent. However, other types of asbestos are not as effective as chrysotile in diminishing AP demethylase activity. Chrysotile and crocidolite fibers are not found to significantly change the apparent Km of AHH activity, from 3-MC-pretreated rat liver microsomes, for BaP. Increasing the microsomal protein concentration partially abolishes the inhibition of AHH activity caused by chrysotile fibers. Inhibition of AP demethylase and AHH activities is attenuated by bovine serum albumin (BSA) or ferritin. Depression of AHH activity by crocidolite is significantly reversed by ferritin. Since polymers such as ferritin override enzyme inhibition by chrysotile as well as crocidolite, surface chemical groups of the fibers may be involved in enzyme modification.     

Induction of liver microsomal epoxide hydrolase, UDP-glucuronyl transferase and cytosolic glutathione transferase in different rodent species by 2-acetylaminofluorene or 3-methylcholanthrene

Control activities vary 12-fold for microsomal epoxide hydrolase, two-fold for UDP-glucuronyl transferase and five-fold for cytosolic glutathione (GSH) transferase among the different rodents (rat, hamster, guinea-pig, mouse) examined. For all three enzymes the activities in rat liver are towards the lower values. In these rodents, except for a 100% increase in microsomal epoxide hydrolase in guinea-pig liver, 2-acetylaminofluorene induces the three phase 2 enzymes only in rat. Treatment with 3-methylcholanthrene also produces the largest effects on these three enzyme activities in rat liver; exceptions are its failure to induce microsomal epoxide hydrolase in female rat and the large induction of cytosolic GSH transferase in hamster liver. Quantitatively, hepatic microsomal epoxide hydrolase, UDP-glucuronyl transferase and cytosolic GSH transferase activities, and their inducibility by 2-acetylaminofluorene or 3-methylcholanthrene, in male Sprague-Dawley rats are not representative for other rodent species or even, in all cases, for female rat.     

Direct measurement of aminopyrine N-demethylase and antipyrine hydroxylase activities in a monolayer rat primary isolated hepatocyte system

This paper describes a simple method for monitoring changes in aminopyrine N-demethylase and antipyrine hydroxylase activities in isolated primary hepatocyte monolayer culture. Aminopyrine N-demethylase activity was determined by monitoring the rate of formation of 14CO2 derived from the N-demethylation of [dimethylamino-14C]aminopyrine (AP). The rate of AP N-demethylation increased linearly with time for 60 min and proportionately with cell concentrations between 4.1 x 10(5) to 1.67 x 10(6) cells/incubation. As expected, non-linear AP N-demethylase kinetics were observed with hepatocytes as well as with microsomal preparations derived from control rats. Hepatocytes prepared from phenobarbital (PB)-pretreated animals exhibited increased AP N-demethylase activity and typical Michaelis-Menten kinetics. In contrast, microsomal preparations from PB-treated animals exhibited non-linear N-demethylase kinetics that differed from the kinetics of preparations derived from control animals. Antipyrine hydroxylase activity was determined by monitoring the rate of formation of non-extractable conjugated 4-hydroxyantipyrine from [N-14C-methyl]antipyrine. Antipyrine hydroxylase activity was found to increase linearly for 120 min and proportionately with cell concentrations. Antipyrine hydroxylation by hepatocytes prepared from control and PB-pretreated animals followed typical Michaelis-Menten kinetics. AP N-demethylase activity immediately after plating was 10 per cent lower than at 4 hr, whereas antipyrine hydroxylase activities were similar. Culturing hepatocytes for 24 hr resulted in a decline to 40 and 60 per cent of control for AP N-demethylase activity and antipyrine hydroxylase activity respectively.     

Purification and aminopyrine monooxygenase activity of liver microsomal cytochrome P-450 from alloxan-induced diabetic rats.

We purified two diabetes-inducible and insulin-sensitive forms of cytochrome P-450, named P-450AL-1 and AL-2, from the liver microsomes of alloxan-diabetic male rats, using sodium cholate solubilization, octylamino-Sepharose 4B chromatography, and HPLC with diethylaminoethyl-5PW and hydroxyapatite columns. The purified forms gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular weight of 50,000 for P-450AL-1 or 48,500 for P-450AL-2. The CO-reduced spectral maximum of these forms was at 452 nm for P-450AL-1 and 451 nm for P-450AL-2. The two purified forms had the low-spin state of heme in the oxidized form. Both P-450AL-1 and AL-2 were active in the metabolism of aniline, benzphetamine, and 7-ethoxycoumarin. However, the catalytic activity of P-450AL-2 for these substrates was obviously higher than that of AL-1. The NH2-terminal sequences of P-450AL-1 and AL-2 differed from each other, and did not agree with those of the other P-450 forms purified from diabetic rats previously. Furthermore, we examined the metabolism of aminopyrine in a reconstituted system with the purified cytochromes P-450. The diabetes-inducible forms of P-450 had high aminopyrine 3-hydroxylation and low N-demethylation activities. These findings provide clear evidence supporting our previous results, which have shown an increase in 3-hydroxymethyl-2-methyl-4-dimethylamino-1-phenyl-3-pyrazolin-5-on e and a decrease in 4-monomethylaminoantipyrine in intact diabetic rats.     

In vitro metabolism of azasqualene derivatives and their effects on aminopyrine N-demethylase activity in rat liver microsomes

The metabolism of squalene dimethylamine (I), a potent inhibitor of 2,3-oxidosqualene (SO) cyclase, and of sixteen other squalene derivatives was investigated in rat liver microsomes. N-oxidation was the only metabolic pathway observed, squalene dimethylamine N-oxide being the only metabolite isolated from incubation of I. The azasqualane and quaternary ammonium derivatives did not form N-oxides during their metabolism. The inhibition of aminopyrine N-demethylase activity was also studied and the IC50, for compound I, which shows weak competitive inhibition, was determined. At 1 mM concentration the other squalene derivatives showed a range of inhibition activity possibly due to their different lipophilicity.     

Human liver morphine UDP-glucuronyl transferase enantioselectivity and inhibition by opioid congeners and oxazepam.

1. Morphine uridine diphosphate glucuronyl transferase (UDP-GT) was studied in human liver microsomes. The (-)- and (+)-morphine enantiomers were used as substrates and inhibitors, such as oxazepam and various opioid congeners were employed to characterize the different glucuronidation pathways. The kinetics of the oxazepam inhibition were studied in the rat liver. 2. The overall glucuronidation of (+)-morphine was higher than that of (-)-morphine. The morphine congeners tested, potently inhibited the formation of (-)-morphine-3-glucuronide ((-)-M3G), except for normorphine and codeine. The formation of (+)-morphine-6-glucuronide [+)-M6G) was potently inhibited by only dextromethorphan and (+)-naloxone. All drugs except normorphine inhibited the formation of (+)-M3G by 18-50%. 3. The metabolism of (-)-morphine to (-)-M3G was more sensitive to oxazepam inhibition than the formation of (+)-M3G from (+)-morphine in the rat liver. 4. The glucuronidation of natural morphine is subject to in vitro interaction with oxazepam and several opiate drugs. Our study supports the theory of more than one type of UDP-GT being involved in morphine glucuronidation.     

Sex and age-related differences in aminopyrine N-demethylase activity in DA- and Wistar-strain rat liver microsomes. Effect of ovariectomy

1. Liver microsomal mixed-function oxidase components were studied in Wistar and Dark Agouti (DA) rats (4-45 weeks) with regard to sex- and age-related differences. Total cytochrome P-450 ranged from 0.29 to 1 nmol/mg in Wistar rats and from 0.21 to 1.27 nmol/mg in DA rats, males had higher levels than females (P<0.0025). Cytochrome b₅ ranged between 0.42-1.37 nmol/mg and 0.42-1.56 nmol/mg in Wistar and DA strains, respectively, and NADPH-reductase activity ranged between 14-43 and 11-46 nmol/min per mg (Wistar and DA respectively).

2. Significant age-related differences were found in DA rats with four- to six-fold increase in N-demethylase activity from young to adult rats. Sex-related differences were found in both Wistar- and DA-strain rats, with males having higher (about twice) metabolic activity than females. In contrast, no significant sex- or age-related differences in cytochrome ₅ content, or NADPH-reductase activity, were found.

3. Ovariectomy of 10-13-week-old females did not affect N-demethylase activity, cytochrome P-450, cytochrome b₅ or NADPH-reductase activity in Wistar or DA rats.

4. Cytochrome P-450 content did not correlate (r = 0.35) with aminopyrine N-demethylase activity.

5. Results indicate that sex- and age-related differences are due to changes in the isozymic composition of cytochrome P-450, and that these changes are not subject to oestrogen regulation.     

Sex and age-related differences in aminopyrine N-demethylase activity in DA- and Wistar-strain rat liver microsomes. Effect of ovariectomy

1. Liver microsomal mixed-function oxidase components were studied in Wistar and Dark Agouti (DA) rats (4-45 weeks) with regard to sex- and age-related differences. Total cytochrome P-450 ranged from 0.29 to 1 nmol/mg in Wistar rats and from 0.21 to 1.27 nmol/mg in DA rats, males had higher levels than females (P less than 0.0025). Cytochrome b5 ranged between 0.42-1.37 nmol/mg and 0.42-1.56 nmol/mg in Wistar and DA strains, respectively, and NADPH-reductase activity ranged between 14-43 and 11-46 nmol/min per mg (Wistar and DA respectively). 2. Significant age-related differences were found in DA rats with four- to six-fold increase in N-demethylase activity from young to adult rats. Sex-related differences were found in both Wistar- and DA-strain rats, with males having higher (about twice) metabolic activity than females. In contrast, no significant sex- or age-related differences in cytochrome b5 content, or NADPH-reductase activity, were found. 3. Ovariectomy of 10-13-week-old females did not affect N-demethylase activity, cytochrome P-450, cytochrome b5 or NADPH-reductase activity in Wistar or DA rats. 4. Cytochrome P-450 content did not correlate (r = 0.35) with aminopyrine N-demethylase activity. 5. Results indicate that sex- and age-related differences are due to changes in the isozymic composition of cytochrome P-450, and that these changes are not subject to oestrogen regulation.     

Caffeine demethylase activity in human and Dark Agouti rat liver microsomes. Comparison with aminopyrine N-demethylase activity.

Human and Dark Agouti rat liver microsomes were analyzed for caffeine metabolism focusing on demethylase activity by using a simplified isocratic HPLC method. Results showed that, despite interspecies quantitative and qualitative differences on caffeine metabolism, mainly demethylated caffeine metabolites were detected in both species. Humans showed interindividual quantitative differences in caffeine demethylase activity in contrast to rats and, in both cases, the proportion of metabolites remained constant. In addition, aminopyrine N-demethylase activity was assayed, showing a direct correlation with caffeine demethylase activity in humans (r = 0.71) that is reported here for the first time, but not in rats (r = 0.21). These results indicate that, in human liver microsomes, caffeine and aminopyrine could be demethylated by the same or by closely related enzymes; whereas in Dark Agouti rats, there is no apparent relation between caffeine and aminopyrine demethylation pathways.     

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.     

Precocious induction of hepatic aniline hydroxylase and aminopyrine N-demethylase with hydrocortisone in neonatal rat

The pre- and postnatal development of hepatic aniline hydroxylase and aminopyrine N-demethylase activities was studied in rats in order to identify the natural trophic factors, if any, responsible for early neonatal formation of the enzymes. The postnatal development of these two enzymes up to 3 weeks of age was comparable with that of cytoplasmic tyrosine aminotransferase. They could be precociously induced in 7-day-old rats by hydrocortisone. Cycloheximide inhibited their induction.     

Nuclear and nucleolar glutathione reductase, peroxidase, and transferase activities in livers of male and female Fischer-344 rats.

The present studies were to test the hypotheses that glutathione reductase (GR), glutathione peroxidase (GPX), and glutathione S-transferase (GST) activities are expressed in nuclei and nucleoli of rat liver cells, and that differences in activities of these enzymes would correlate with the greater resistance of female than of male Fischer-344 rats to hepatic necrosis in vivo, mediated by reactive oxygen species generated by redox-cycling metabolism of diquat. Adult male and female Fischer-344 rats were treated with comparably hepatotoxic doses of diquat (0.1 or 0.2 mmol/kg, respectively), or equal volumes of saline, ip. Six hours later, the livers were harvested, and purified nuclei and nucleoli were isolated by differential centrifugation. Nuclear GR activities in male and female rats were 12 and 15 mU/mg protein, and nucleolar activities were 30 and 51 mU/mg protein, respectively, p < 0.05. Some differences between male and female rats in nuclear and nucleolar activities of GPXs and GSTs were observed, as were some differences in the respective diquat-treated animals, but implications of these differences for susceptibility to diquat-induced oxidant stress effects are not apparent. Nuclear GR, GPX, and GST probably contribute to antioxidant defense mechanisms, but the functions served by localization of GR and GPX in nucleoli are less evident.