Osmo Hänninen

Enhancement of drug oxidation and conjugation by carcinogens in different rat tissues

The administration of carcinogenic polycyclic hydrocarbons has been shown to enhance hepatic drug oxidation [ 1, 21. An increase takes place also in the skin [3-61, lungs and gastrointestin~ tract [7-91, kidneys [S] and placenta [ 1 O] . The oxidation step in detoxi~cation is followed in viva by conjugation. In separate studies an e~ancement of ~ucuronide synthesis has been reported in the liver [ 1 1 1 S] and skin [ 161. The time courses of the responses have not, however, received much attention. The present study was performed to clarify the responses of arylhydrocarbon hydroxylase (EC 1.14.1.1) and UDP glucuro]~yItransferase (EC 2.4.1.17) in the intestinal mucosa, liver and kidneys after an administration of 3,4-benzpyrene and 3-methylcholanthrene to rats. These two enzymes have a close functional and topochemical relationship which suggests that their synthesis might have a common control mechanism. The rise of the UDP glucuronyhransferase activity was preceded with a much more pronounced rise of the arylhydrocarbon hydroxylase activity in all tissues studied. The induction of the hepatic hydroxylase was significantly higher when the intraperitoneal administration route was used instead of the intragastric one.

Gastrointestinal Distribution of Glucuronide Synthesis and the Relevant Enzymes in the Rat

The rate of o-aminophenol glucuronide synthesis in the small intestine of the rat was found to decrease when going from the oral to the aboral end, where it was 15 per cent of the duodenal level. In the glandular stomach, caecum, and colon, the activity was 34, 15 and 30 per cent, respectively, of the duodenal value. The decrease in UDP glucuronyltransferase (p-nitrophenol) activity in the mucosal extracts was almost linear and amounted to 66 per cent from the oral to the aboral end of the small intestine. In the glandular stomach, caecum, and colon, the activities were 63, 32, and 83 per cent respectively, of the duodenal value. The β-glucuronidase activity increased linearly by 41 per cent when going from the oral to the aboral end of the small intestine. In the glandular stomach, caecum, and colon, activities of 82, 221, and 162 per cent respectively of the duodenal ones were observed.These results indicate that the distribution pattern of glucuronide synthesis, observed in tissue slices, and the UDP g...

Enhancement of Aniline p-Hydroxylation by Acetone in Rat Liver Microsomes

1. Acetone (0.8 M) enhanced the p-hydroxylation of aniline about 3-4 fold when added to the incubation mixture. Only inhibition in hydroxylation of 3,4-benzpyrene or N-demethylation of ethylmorphine was observed.2. Phospholipase C treatment of rat liver microsomes decreased aniline p-hydroxylation about 20%. The enhancing action of acetone vanished after such a treatment.3. Acetone diminished the type II binding of aniline. In the presence of acetone, low concn. of aniline produced a type I difference spectrum with rat hepatic microsomes. With higher concn. of aniline a type II spectral change was obtained. This indicates that aniline, a well-known type II substrate, can also interact with the type I binding site of cytochrome P-450. Correlation between the type I spectral change and activation of aniline p-hydroxylation is suggested. Acetone itself elicited a spectral change with rat liver microsomes resembling a type II difference spectrum.4. Acetone partly reversed the inhibition produced by aniline on...

The competitive inhibition of p-nitrophenyl-β-d-glucopyranosiduronic acid synthesis by aliphatic alcohols in vitro

Abstract The primary aliphatic alcohols, methanol, ethanol, propanol and butanol were found to compete with p -nitrophenol for the uridine diphosphate glucuronic acid transferase preparation from guinea pig liver. The K i values correlate with the water-olive oil partition coefficients of these alcohols. The inhibitory action increases exponentially with the length of the alkyl chain and linearly with the lipid solubility.

Activation of microsomal UDP glucuronyltransferase by phospholipases

Abstract The digestion of microsomal membranes (from rat liver) with purified phospholipase A or C was found to increase severalfold the measurable UDP glucuronyltransferase activity in the membranes without solubilizing the enzyme. The phospholipase A effect is at least partly mediated by the reaction products, since lysolecithin and free fatty acids also increase the enzyme activity when added as micelles into microsomal suspensions. Phospholipase A or C digestion of the membranes increased the Km for UDPglucuronic acid of the enzyme.

Enhanced glucuronide formation in different tissues following drug administration

Abstract A study was made of the o -aminophenol glucuronide synthesis in the slices of the liver, kidney and gastrointestinal tract after the administration of 3,4-benzpyrene, cinchophen, 3-methylcholanthrene, phenobarbital and salicylic acid to elucidate whether the responses of the different tissues are similar to these drugs known to enhance drug metabolism. A 1.6-fold increase of o -aminophenol glucuronide synthesis was found in liver slices 5 days after a single intraperitoneal injection of 3-methylcholanthrene whereas there was no increase in the slices from the kidney or from the various parts of the gastrointestinal tract. The i.p. administration of 3,4-benzpyrene had a similar effect. The intragastric administration of cinchophen for 3 days resulted in a two-fold increase of the synthesis in the slices from the liver, glandular stomach, caecum and colon, and a 3-fold increase was found in the kidney. In the duodenum only a slight increase of activity was observed when the analysis was carried out 24 hr after the last dose, but a 1.6-fold increase was found after 48 hr. The i.p. administration of phenobarbital for 3 days resulted in a 1.4-fold increase of the synthesis in the liver, but no change of activity was found in the kidney or in the various parts of the gastrointestinal tract. The intragastric administration of salicylic acid for 3 days resulted in a 1.9-fold increase in the kidney but only insignificant changes were found in the various parts of the gastrointestinal tract or in the liver when analysed 24 hr after the last dose. The enhancement of the glucuronide biosynthesis caused by the administration of the various drugs thus reveals marked organ specificity.

Enhancement of d-glucuronolactone and acetaldehyde dehydrogenase activities in the rat liver by inducers of drug metabolism

Abstract Three different fractions of d -glucuronolactone dehydrogenase were isolated from rat liver supernatant by means of CM and DEAE Sephadex ion exchange chromatography. These fractions also had acetaldehyde dehydrogenase activity. Intraperitoneal administration of phenobarbital, p , p ′-DDT and 3-methylcholanthrene increased the enzyme activity in two of the fractions, while the third remained unaffected by these drugs. Enhanced activity was obtained with both substrates, but it varied in degree within the group of animals. The rats could be divided into poor, medium and high response groups. A change in the ratio of the d -glucuronolactone and acetaldehyde dehydrogenase activities was also found after the drug treatment. Spironolactone, which is also an inducer of drug metabolism, did not alter the activity of any of the enzyme fractions, at the dose used.

Effect of Chyme on Mucosal Enzyme Levels in Small Intestine of the Rat

Abstract Partial jejunectomies, gastrojejunostomies (with closed pylorus), and jejunal Thiry-Vella loops were made in order to elucidate the role of chyme in the control of mucosal mass and the activities of alkaline phosphatase, ATPase, and maltase in the small intestine of the rat. After partial jejunectomy, a partially reversible mucosal hyperplasia was seen in the small intestine with the exception of distal ileum. After gastrojejunostomy a similar hyperplasia took place in the jejunum and proximal ileum. In the jejunal Thiry-Vella loops a mucosal atrophy was found in 4 wk. After partial jejunectomy the activity of alkaline phosphatase decreased slowly in 4 wk in the remaining small intestine with the duodenum as an exception. ATPase activity decreased in the duodenum. Maltase activity remained unchanged during 8 postoperative wk. In gastrojejunostomized rats the activity of alkaline phosphatase and ATPase increased slowly during 12 wk in the jejunum aborally from the gastroenterostomy. A slight depression of maltase activity was observed in the operation area and a slight increase of enzyme activity was found in the middle of the small intestine. In jejunal Thiry-Vella loops the activity of alkaline phosphatase decreased, but no change of maltase activity could be observed during 4 wk. Perfusion of a loop with maltose solution did not cause any changes in the activity of alkaline phosphatase or maltase. The results indicate that after a change in chyme passage the adaptation takes place in the small intestine primarily by the change of mucosal mass, and at least some enzyme levels in the mucosal cells are remarkably stable.

Radiochemical assay of UDP glucuronyltransferase (p-nitrophenol).

UDP glucuronyltransferas¢ (UDPglucuronate glucuronyltransfera~, EC 2.4. I. 17) catalyzing the glucuron:de formation of foreign compounds Is fimdy bound to the membranes of endoplasmtc rctlculum. The kinetics of the UDP glucuronyltransferase has therefore been studied with only crude preparations. These kinetic studies have been hampered by tl,e lack of an accurate and sensitive method for determining enzyme activity, p-Nitrophenol is very often used as an aglycone, because its spectrophotometric quantttation ts rapid and simple [ 1 ]. it has also been widely used in recent detailed kinetic studies of ttus enzyme [2-8] . This method has, howe,,ecertain disadvantages, smce the consumption of Ul¢ substrat¢ is measured and mot the formation of the product, whsch is preferable in kinetic studies. The absorption spectrum of p-mtrophenyl glucuronide and free a~lycone have different absorption maxima (glucur,~, ide at 312 and agl:,-cone at 400 nm, respectively), and this has been utilized in the direct rn.,2~.?~,tmment of IIDP #tte~srgn~,!tr,anqfera~ activity [9]. This method is, however, rather insensl. tire. In kinetic studies low substrate concentrations should also be us¢¢l due to their physiological relevance. With the old spectt~photometric methods mentioned above this has been very difficult. In the present paper we describe a radiochvmtcal method for UDP glucuronyltransferas¢ determination with IncRaae d accuracy and sensitivity, t4C-Labe!ed pnittophenol, which is now commercially available, wa~,used itSa ~UCu/onyl ac~ept0r mbstrat¢,

Action of transcription and translation inhibitors on the enhancement of drug hydroxylation and glucuronidation by 3-methylcholanthrene and phenobarbital

Abstract 1. 1. A single dose of both phenobarbital and 3-methylcholanthrene was able to induce microsomal mixed function oxidase complex and UDP glucuronyltransferase ( p -nitrophenol) in 24 hours in rat liver. The increases differed, however, in amplitude. 3-Methylcholanthrene enhanced both the aryl hydrocarbon hydroxylase and p -nitroanisole O-demethylase activities, and phenobarbital increased the 0-demethylation of p -nitroanisole without affecting aryl hydrocarbon hydroxylase activity. 2. 2. Actinomycin D, 0.4 mg./kg. of body weight, was not able to block the rise in mixed function oxidase activity, but it significantly inhibited the induction of UDP glucuronyltransferase caused by phenobarbital or 3-methylcholanthrene administration. The higher dose, 0–8 mg./kg., blocked the induction of both mixed function oxidase complex and UDP glucuronyltransferase totally. 3. 3. Cycloheximide (1 mg./kg.) was partially active in inhibiting the increase of both drug oxidation and glucuronidation; the induction caused by phenobarbital was almost totally blocked but that achieved by 3-methylcholanthrene was only slightly inhibited. 4. 4. The biosynthesis of mixed function oxidase complex and UDP glucuronyltransferase are suggested to be under separate control mechanisms.