G. J. Dutton

Evidence that d-glucaro-1,4-lactone shortens the pharmacological action of drugs being disposed via the bile as glucuronides

Abstract Depression of locomotor activity caused by phenobarbital (80 mg/kg, i.p.) or progesterone (160 mg/kg, i.p.) was shortened in male rats pretreated with d -glucaro-1,4-lactone (1.2 g/kg, p.o.), which did not exert any effect when given alone. The duration of the zoxazolamine paralysis (75 mg/kg, i.p.) was not affected, however, by the presence of d -glucaro-1,4-lactone. After oral administration of d -glucaro-1,4-lactone, β-glucuronidase was greatly inhibited within 6 hr, when measured in luminal contents throughout the intestinal tract, at pH values of 6.5, 7.8 and 7.0, which were those found to exist in the jejunum, the ileum and the colon respectively. d -Glucaro-1,4-lactone itself, added in vitro , inhibited β-glucuronidase from intestinal epithelial cells and luminal contents at the forementioned physiological pH values. The results suggest that inhibition by d -glucaro-1,4-lactone of the intestinal bacterial β-glucuronidase could enhance the elimination of substances excreted in the bile as d -glucuronic acid conjugates. This is probably mediated by suppressing the enterohepatic circulation of the pharmacologically active aglycones.

Comparison between o-aminophenol glucuronidation in liver slices and homogenates from control and phenobarbital-treated Wistar and Gunn rats

Abstract Glucuronidation rates were studied in sliced and homogenised liver from control and phenobarbital-treated Gunn and Wistar rats of both sexes, aglycone concentration not being significantly rate-limiting. Kinetic parameters of o -aminophenol UDP-glucuronyltransferase (EC 2.4.1.17) were determined in fresh (“native”) homogenates and those activated by UDP- N -acetylglucosamine, digitonin and diethylnitrosamine added singly or together. Apparent K UDP-glucuronate -values were increased by both UDP- N -acetylglucosamine and digitonin, but not by the specific activator diethylnitrosamine; values were similar for both strains and not significantly affected by phenobarbital pretreatment; sex differences were encountered. Apparent V max -values were increased by 30- to 65-fold for Wistar and 400- to 1600-fold for Gunn rats in maximally-activated homogenates. Phenobarbital pretreatment approximately doubled V max of maximally-activated enzyme in all animals, but had little effect on V max of “native” or partially-activated preparations. Slices of Gunn rat liver glucuronidated o -aminophenol at rates 30–44 per cent those of Wistar rats; rates in both strains exhibited a consistent sex difference and increased after phenobarbital treatment. Comparison of results from slices and homogenates containing physiological concentrations of UDP-glucuronate suggested that in both sexes and strains UDP-glucuronyltransferase activity in vivo is higher than that observed in “native” unactivated homogenates, presumably because of endogenous activators; however, as glucuronidation in similar, but maximally-activated, homogenates was well above that in slices, the enzyme may still remain partially “latent” in vivo .

Relation between the induction of hydroxylation and of glucuronidation in chick liver

Hydroxylation of drugs or drug-metabolites frequently precedes their glucuronidation [l] . Both processes occur in liver endoplasmic reticulum and both are increased in vivo following administration of barbiturates or polycyclic hydrocarbons [2-41. Their metabolic relationship is therefore of interest [5-71; their induction has been thought of as coupled, hydroxylation providing inducers of subsequent glucuronidation [5,6] . Enzymes for both hydroxylation and glucuronidation can be induced in cellor organ-cultured liver. Induction of hydroxylating enzymes requires phenobarbital [8] in the culture medium. UDP-glucuronyltransferase (EC 2.4.1.17) is induced spontaneously ln cultures of chick embryo liver [9,10] ; phenobarbital enhances this induction in cultures [ 1 l] and increases the enzyme’s activity in ovo from zero to very high levels [ 121 . Hitherto the relationship of hydroxylating and glucuronidating enzymes has not been examined in cultured systems. These systems allow manipulation of the environment, and we report conditions which appear clearly to separate induction of glucuronidation from that of hydroxylation.

Precocious development in utero of certain UDP-glucuronyltransferase activities in rat fetuses exposed to glucocorticoids.

Abstract The first precocious development of UDP glucuronyltransferase in the mammalian fetus in utero by a known compound of endogenous origin is described. Intraperitoneal injection of cortisol (8 mg) into maternal rats on days 14 and 15 of gestation stimulated fetal-liver transferase activity from near zero to 1 2 maternal levels by day 17; 0.3 mg dexamethasone, possessing a longer biological half-life, raised activity to full maternal level by day 16. In controls, injected with solvent only, fetal-liver transferase remained low on day 16. With both glucocorticoids, transferase stimulation was dose-dependent. Transferase activities were assayed in a range of digitonin concentrations from zero to above optimal for enzyme activation. Activities stimulated were towards o -aminophenol, p -nitrophenol, 1-naphthol and serotonin. Activities towards bilirubin, morphine and testosterone were not stimulated. The former group of activities are stimulated by glucocorticoids in culture and normally reach approximate adult levels just before birth; the latter group are not so stimulated on culture and normally reach adult levels after birth. Implications of these findings are discussed.

Biochemistry for medical students: a flexible student-oriented approach.

This is a description of some of our experiences in the Department of Biochemistry at the University of Dundee during a radical revision of our course for medical students. It is a personalized account, given with the hope that it may be useful to others in their own situation. Hindsight can colour one’s orginal motives and intentions, and an account such as this can border on complacency if the experiment has been a success. We think we have been honest in our presentation and it is our sincere wish that the feasibility of this kind of approach will encourage others not to copy us directly but to construct their own course, designed for their own situation and subject. When we constructed our own learning method, we borrowed the ideas and experiences of others and adapted them to our requirements (Chignell & Macqueen, 1974). Our motivation for change was not originally based on a sound knowledge of educational psychology. We were ignorant of learning theories, educational objectives and the like. Basically we had an unsatisfactory course because it had not changed substantially for some years whereas the students and their needs had. Any changes had been in the more scientific presentation of our subject and had

Induction of UDPglucose dehydrogenase during development, organ culture, and exposure to phenobarbital. Its relation to levels of UDPglucuronic acid and overall glucuronidation in chicken and mouse.

Liver UDPglucose in early chick-enbryo has, by the 19th day of incubation, reached levels existing in young hatched (White Leghorn) chicks. In developing ASH/TO mouse liver, the dehydrogenase is low, but increases sharply at late foetal and weaning stages; adult activity is greater in females than males. The UDPglucuronic acid content of embryo liver from at least 12 days resembles that of adult chicken; in mouse liver it rises over birth and infancy. These differences in relative rates of development of enzyme and nucleotide in the 2 species can explain why overall glucuronidation by liver appears in chick rapidly after hatching, but in mouse only gradually during infancy. UDPglucose dehydrogenase increases in embryo liver, probably by induction, 2-3-fold during culture with phenobarbital and some 5-fold when exposed to the drug in ovo. Phenobarbital treatment also increases the enzyme in late foetal and adult mice, abolishing the sex difference. Differences between induction of UDPglucose dehydrogenase and UDPglucuronyl transferase during development, culture and phenobarbital treatment indicate that control mechanism for these two enzymes are not directly linked.

Release by phenobarbital of the repression of UDP-glucuronyltransferase activity in ovo.

Abstract UDP-Glucuronyltransferase activity in chick-embryo liver and kidney, known to be induced by hatching or by culture, appears to be repressed in ovo . Injection of phenobarbital into the egg overcomes this repression. Chick-embryo liver and kidney cells can thus exhibit high transferase levels whilst still in ovo . Response to phenobarbital by liver tissue occurs in situ or when grafted on to the chorioallantoic membrane, and can be elicited by 96 hr of age. The ability of phenobarbital to over-ride the natural regulation of liver UDP-glucuronyltransferase development in ovo is contrasted with its inability in utero . Overall glucuronidation, as well as the transferase, is elicited by phenobarbital in ovo ; differences between regulation of glucuronidating and hydroxylating enzymes are noted.

Differential effects of hormones on precocious yolk sac retraction in chick embryos following administration by a new technique

Abstract A simple new technique allowing continuous application of hormones to the chorioallantoic membrane of the embryonated chicken egg is described. By use of this technique, by grafting the pars distalis, and by monitoring the hormonal response, with inhibitors and with induction of glucocorticoid-dependent liver enzymes, yolk sac retraction has been shown to be linked with thyroidal and not with adrenocortical or several other hormones. Evidence is also presented that the pars distalis, from chickens up to at least 18 months old, is capable of autonomous performance of certain adrenocorticotrophic or thyrotrophic functions when grafted on the chorioallantoic membrane of embryonated eggs.