Demonstration of two functionally heterogenous groups within the activities of UDP-glucuronosyltransferase towards a series of 4-alkyl-substituted phenols.

1. A simple colorimetric assay for UDP-glucuronosyltransferase activities towards phenolic substrates, using Folin & Ciocalteu's phenol reagent, is described. The assay is used to measure rat liver transferase activities towards substrates from a series of 4-alkyl-substituted phenols. 2. Activities towards phenol, 4-methylphenol and 4-ethylphenol develop near-adult values before birth, are precociously stimulated by dexa methasone in utero and are stimulated 3--4-fold by 3-methylcholanthrene in adult liver. These are assigned to a "late-foetal" group of transferase activities. 3. Activities towards 4-n-propylphenol, 4-s-butylphenol and 4-t-butylphenol are negligible in late-foetal liver, developing to near-adult values in the first 4 postnatal days, and are not affected by dexamethasone or 3-methylcholanthrene. They are assigned to a "neonatal" group of transferase activities. 4. Although 4-ethylphenol and 4-n-propylphenol differ only by a single --CH2-- moiety, this is sufficient to change the acceptability of these substrates respectively from the late-foetal to the neonatal group of transferase activities. The change is distinct, with no overlapping of substrate acceptability between the two groups of transferase activities. 5. From consideration of the above and other substrates, the two groups of transferase activities do not distinguish substrates on the basis of their molecular weights or lipophilicity. The distinguishing feature appears to be the specific molecular configurations of the substrates.     

Regulation of onset of development of UDP-glucuronosyltransferase activity towards o-aminophenol by glucocorticoids in late-foetal rat liver in utero.

1. A precocious development of UDP-glucuronosyltransferase activity (EC 2.4.1.17) towards o-aminophenol is demonstrated in 15-17 day foetal rat liver in utero after dexamethasone administration to the mother. 2. This stimulation of liver transferase activity in utero is directly proportional to the dose of dexamethasone infected. 3. Precocious development of transferase activity in utero can also be effected with the natural glucocorticoid cortisol by multiple injections of large amounts of this hormone into the mother. 4. Transferase activity towards o-aminophenolin foetal lung, kidney and upper alimentary tract can also be precociously stimulated by dexamethasone in 17-day foetuses in utero. 5. Natural development of hepatic transferase activity between days 18 and 20 of gestation is retarded after foetal hypophysectomy by decapitation in utero. 6. Overall glucuronidation of o-aminophenol, as observed in foetal rat liver, is also precociously stimulated by dexamethasone. 7. From this and from evidence previously presented we suggest that glucocorticoids, which are known to increase in rat foetuses between days 17 and 20 of gestation, trigger the normal development in utero of hepatic transferase activity towards o-aminophenol which occurs at that time. We also suggest that these hormones are responsible for the rise in activity of the enzyme in foetal lung, kidney and upper alimentary tract which occurs during the same gestational period.     

Precocious development of uridine diphosphate glucuronosyltransferase activity during organ culture of foetal rat liver in the presence of glucocorticoids.

1. Precocious development of mammalian UDP-glucuronosyltransferase (EC 2.4.1.1.7) induced by endogenous compounds of known chemical composition is reported for the first time. 2. This development occurs in cultured explants of foetal rat liver when exposed to corticosteroids possessing a pregn-4'-ene structure and a hydroxy or an oxo group at C-11. 3. Explants from 14-day foetuses cultured for 3 days in a chemically defined medium containing dexamethasone exhibited transferase activities towards o-aminophenol within adult male values. Those liver transferase activities attained in utero by 17 days were still negligible. 4. Evidence from several approaches indicated that the explants required glucocorticoids for expression of the transferase, not for maintenance of viability. 5. Glucocorticoid-dependent stimulation of transferase activity required incorporation of L-[14C]leucine into protein, as judged from the pulsing of cultures with cycloheximide. 6. The relevance of these culture experiments to the situation in vivo is discussed.     

Demonstration of functional heterogeneity of hepatic uridine diphosphate glucuronosyltransferase activities after administration of 3-methylcholanthrene and phenobarbital to rats.

After the administration of 3-methylcholanthrene to adult male rats, activities of hepatic UDP-glucuronosyltransferase towards six from a group of 12 substrates were stimulated by 250-350%. Activities towards the remaining six substrates were unaffected. Conversely, after phenobarbital administration, activities formerly stimulated by 3-methylcholanthrene remained unchanged, and the other six activities were stimulated by 160-280%. The relationship of these two groups of transferase activities to other evidence suggesting the same heterogeneity of the enzyme is discussed.     

Developmental alteration of hepatic UDP-glucuronosyltransferase and sulphotransferase towards androsterone and 4-nitrophenol in Wistar rats.

Postnatal development of hepatic UDP-glucuronosyltransferase and sulphotransferase activities towards androsterone and 4-nitrophenol as well as cytochrome P-450 contents was studied in male and female Wistar rats. The rats with high and low UDP-glucuronosyltransferase activity towards androsterone were classified by the genotype of the parent animals. UDP-glucuronosyltransferase activity towards androsterone began rapidly to enhance after 30 days of age in the high-activity group, whereas the transferase activity remained low throughout in the low-activity group. Such a striking difference was not observed in UDP-glucuronosyltransferase activity towards 4-nitrophenol, sulphotransferase activity towards androsterone and 4-nitrophenol, and cytochrome P-450 contents. Sex-based difference in the sulphotransferase activity was marked after 30 days of age. Sulphotransferase activity towards androsterone was much higher in adult females than in adult males, whereas higher sulphation activity towards 4-nitrophenol was found in adult males. The results also indicate that the low level of the UDP-glucuronosyltransferase activity did not lead to compensatory stimulation of the sulphotransferase activity.     

Development of multiple activities of UDP-glucuronyltransferase in human liver.

UDP-glucuronyltransferase activities towards eight substrates were assayed in samples of foetal, term and adult human liver. Activities towards bilirubin, androsterone, testosterone, 1-naphthol, 4-nitrophenol and 2-aminophenol were present in foetal and term liver samples at less than 14% of adult values, whereas activity towards 5-hydroxytryptamine was present in foetal and term liver at 109 and 121% of adult values respectively. Thus a 'foetal' form of UDP-glucuronyltransferase may exist in human liver that is more restricted in substrate specificity than are those of the rat or rhesus monkey.     

The temporary postnatal decline in glucuronidation of certain phenols by rat liver.

A temporary but marked postnatal decline in UDP-glucuronosyltransferase activity occurs in homogenates and microsomes from rat liver. The profile of this trough and its time of occurrence (maximal over 13-16 days) are almost identical with the two substrates 2-aminophenol and 1-naphthol, whose rates of glucuronidation differ 10-fold. The trough is greatest with digitonin-activated preparations, least with fresh latent ('native') enzyme and intermediate when the native enzyme is treated with its specific activator UDP-N-acetylglucosamine (UDP-GlcNAc). Less detailed evidence supports similar conclusions with 4-nitrophenol as substrate. The trough is not due to the presence of an inhibitor of the transferase in rat liver at 15 days of age. Over the whole perinatal period, including the time of the trough, the enzyme in homogenates can be activated by UDP-GlcNAc; the microsomal enzyme is activated to a rather lesser degree perinatally, and evidence suggests this may be due to artefacts introduced during tissue fractionation. When the overall process of glucuronidation is studied in snips of intact liver offered high concentrations of the two different phenols, the trough is again evident over the same period as observed with broken cells, and of equal depth for both substrates. The infant rat is therefore probably less able to glucuronidate hepatically these phenols over the suckling or early weaning period than are the adult, late foetus or newborn, and may be especially incompetent at 13-16 days of age.     

Turkey liver xanthine dehydrogenase. Reactivation of the cyanide-inactivated enzyme by sulphide and by selenide

1. The glycogen present in the liver of rat foetuses was labelled by injecting a trace amount of [6-(3)H]glucose into the mother at 19.5 days of gestation. The radioactivity incorporated in the glycogen 4h after the administration of the label was still present 38h later. A large proportion of this radioactivity was on the outer chains of the polysaccharide. These results indicate that there is normally almost no glycogen degradation in the foetal liver. In contrast, glycogen breakdown occurs very rapidly in the livers of foetuses whose mother is anaesthetized. 2. Glycogen synthetase is present in the liver at day 16 of gestation at a concentration as high as 30% of that in the adult, but essentially as an inactive (b) enzyme. The appearance of synthetase phosphatase between days 18 and 19 corresponds to that of synthetase a and to the beginning of glycogen synthesis. From day 19 to 21.5 the amount of synthetase a present in the foetal liver is just sufficient to account for the actual rate of glycogen deposition. 3. The content of total phosphorylase in the foetal liver increases continuously from day 16 to birth. However, a precise measurement of the a and b forms of the enzyme in the liver of non-anaesthetized foetuses is not possible. Taking the rate of glycogenolysis as an appropriate index of phosphorylase activity, we conclude that this enzyme is almost entirely in the inactive form in the foetal liver under normal conditions. 4. The accumulation of glycogen in the liver during late pregnancy may therefore be explained by a relatively slow rate of synthesis and a nearly total absence of degradation.     

Liver glycogen synthase in the developing foetal rat

Kinetic constants for liver glycogen synthase (UDPglucose: glycogen 4-alpha-D-glucosyltransferase, EC 2.4.1.11) with respect to UDPglucose have been measured in foetal liver homogenates from samples taken during late gestation (days 17-22) and the first hours after birth. The V of the inactive form of glycogen synthase increased markedly in this period and there was a significant increase in V of the active enzyme to a maximum at day 20 of gestation. The Km for UDPglucose measured in the presence of glucose-6-P (total activity) did not vary greatly, mean values of 0.51 +/- 0.04 mM. Values derived for the inactive enzyme were almost identical. In contrast, Km values for active glycogen synthase in foetal livers during gestation were significantly higher than those for adult liver. Highest values were seen at day 19 of gestation (1.84 +/- 0.08 mM) followed by a steady fall to 0.55 +/- 0.05 mM in the newborn compared with a mean value of 0.48 +/- 0.04 mM for adult liver. Existence of a reduced affinity of active glycogen synthase for UDPglucose must be recognized when assaying the enzyme in foetal liver, particularly when extrapolating values to rates of glycogen synthesis in vivo. Data were obtained only after removal of an amylase-like contaminant from foetal liver samples which invalidated the radioassay of glycogen synthase. This work illustrates the care needed in the analysis of foetal tissue and the interpretation of resulting data when utilizing methods developed for adult tissue.     

Thymidine kinase in rat liver during development

1. The activity of thymidine kinase in rat liver supernatant decreased with development to a value in the adult that was 1% of that in the 17-day foetus. 2. The foetal enzyme was more stable than the adult to gel filtration on Sephadex G-25 at 0 degrees . 3. The greater stability of the foetal enzyme to incubation at 45 degrees was attributable to the presence of higher concentrations of nucleotides in foetal liver supernatant. 4. The K(m) values for foetal and adult enzymes were approx. 2.5mum- and 2.1mum-thymidine respectively. 5. The foetal enzyme was more sensitive to inhibition by thymidine triphosphate. 6. The decline in enzyme activity during the neonatal period was correlated with a shift in the enzyme properties from the foetal to the adult type, and may reflect the decrease in the proportion of haemopoietic tissue in the liver.     

Levels of metallothionein messenger RNA in foetal, neonatal and maternal rat liver

We have used translation in vitro of hepatic polyadenylated RNA to characterize the levels of metallothionein mRNA in foetal, neonatal, pregnant and nulliparous rats. The translation products of foetal hepatic metallothionein mRNA increased relative to other mRNA translation products from day 18 of gestation to birth and attained a maximum, maintained throughout suckling, which is tenfold above 17-day foetal hepatic levels and fourfold above adult levels. Maternal liver metallothionein mRNA decreased fivefold between 17 days and 20 days of gestation, rose sharply immediately before birth, and was low throughout lactation.     

UDP-glucuronyltransferase activity exhibits two developmental groups in liver from foetal rhesus monkeys.

UDP-glucuronyltransferase was assayed in liver from adult rhesus monkeys and foetuses during late gestation. Activities toward 2-aminophenol, 5-hydroxytryptamine, 1-naphthol and 4-nitrophenol in the foetal liver ranged from 46 to 114% of adult values, whereas activities toward bilirubin, oestradiol and testosterone were less than 5% of adult values. This suggests that in primates UDP-glucuronyltransferase develops differentially in two clusters analogous to that in the rat.     

Properties and prenatal ontogeny of beta-D-mannosidase in selected goat tissues.

beta-D-Mannosidase activity in selected normal adult, neonatal and foetal goat tissues and in tissues from animals affected with caprine beta-mannosidosis was examined with the use of 4-methylumbelliferyl beta-D-mannopyranoside as substrate. The enzyme in normal adult thyroid, kidney and brain exhibited a sharp unimodal pH optimum at pH 5.0, whereas the enzyme in both normal adult and mutant liver exhibited broad pH ranges of activity (pH 4.5-8.0). No residual enzyme was detectable in mutant kidney or brain; in contrast, residual activity in mutant liver was 52% of that in a neonatal control. Concanavalin A-Sepharose 4B (Con A-Sepharose) fractionation of normal adult liver beta-D-mannosidase resolved the enzyme into an unbound (non-lysosomal) from (52%) with a broad pH range of activity (pH 4.5-8.0) and a bound (lysosomal) form (48%) with a sharp pH optimum of 5.5. The enzyme in mutant liver consisted entirely of the unbound (non-lysosomal) form. Beta-D-Mannosidase activity in normal adult thyroid, kidney and brain was resolved by chromatofocusing into two major isoenzymes, with pI 5.5 and 5.9, and traces of a minor isoenzyme, with pI 5.0. In normal adult liver the enzyme was also resolved into three isoenzymes with similar pI values; however, that with pI 5.0 predominated. The predominant form of the enzyme in 60-day-foetal liver was bound by Con A, exhibited a unimodal pH optimum (5.0) and was resolved into two isoenzymes, with pI 5.4 and 5.8; only traces of an isoenzyme with pI 5.0 were detectable. Total hepatic beta-D-mannosidase activity increased progressively towards adult values during the last 90 days of gestation as a result of increasing non-lysosomal isoenzyme activity (pI 5.0). Lysosomal beta-D-mannosidase was shown to occur in all normal goat tissues studied as multiple isoenzymes, which are genetically and developmentally distinct from the non-lysosomal isoenzyme occurring predominantly, if not exclusively, in liver.     

Allosteric activation of brain hexokinase by magnesium ions and by magnesium-ion–adenosine triphosphate complex

1. The highest blood concentrations of ketone bodies were found at 5 days of age, after which time the concentration fell to reach the adult value by 30 days of age. 2. Both mitochondrial and cytoplasmic hydroxymethylglutaryl-CoA synthase activities were detected, with highest activities being found in the mitochondria at all stages of development. Activity of the mitochondrial enzyme increases rapidly immediately after birth, showing a maximum at 15 days of age, thereafter falling to adult values. The cytoplasmic enzyme, on the other hand, increased steadily in activity after birth to reach a maximum at 40 days of age, after which time activity fell to adult values. 3. Both mitochondrial and cytoplasmic aceto-acetyl-CoA thiolase activities were detected, with the mitochondrial enzyme having considerably higher activities at all stages of development. The developmental patterns for both enzymes were very similar to those for the corresponding hydroxymethylglutaryl-CoA synthases. 4. The activity of heart acetoacetyl-CoA transferase remains constant from late foetal life until the end of the suckling period, after which time there is a gradual threefold increase in activity to reach the adult values. The activity of brain 3-oxo acid CoA-transferase increases steadily after birth, reaching a maximum at 30 days of age, thereafter decreasing to adult values, which are similar to foetal activities. Although at all stages of development the specific activity of the heart enzyme is higher than that of brain, the total enzymic capacity of the brain is higher than that of the heart during the suckling period.     

Variation in the amounts of hepatic copper, zinc and metallothionein mRNA during development in the rat.

Amounts of hepatic metallothionein mRNA were assessed in RNA from foetal and neonatal rat livers by using dot-blot hybridization. Metallothionein mRNA began to increase about day 15 of gestation and reached a foetal maximum of 5-fold higher than adult values between 18 and 21 days of gestation. The amounts fell significantly for the first 3 days after parturition, and rose again to 6-fold above adult values 6 days after birth. By 15 days after birth the metallothionein mRNA had declined to adult amounts. In comparison, amounts of ornithine transcarbamoylase mRNA did not vary greatly during development. Hepatic zinc concentrations increased from day 14 of gestation to a maximum just before birth, and remained above adult values until 30 days after birth. From 14 days of gestation to 8 days after birth, hepatic copper concentrations were about 4-fold higher than in the adult, but a substantial increase (to about 9-fold higher than in the adult) occurs between 10 and 15 days after birth. CdCl2 administered to pregnant rats on day 18 of gestation was shown to block placental transfer of zinc, and we found decreased foetal hepatic zinc concentration after the CdCl2 treatment, but this failed to cause a significant decrease in metallothionein mRNA, suggesting that zinc may not be the primary inducer of hepatic metallothionein mRNA during foetal life.     

Investigation of thyroxine monodeiodinase activity in the liver, kidney and brown adipose tissue of foetal and neonatal rabbit

The maturation of the 5'- and 5-monodeiodinase system in liver, kidney and brown adipose tissue of rabbits, during the foetal period (from 21 days of gestation to birth) and the neonatal period (from birth to 3 weeks of life) was studied. A sudden increase of 5'- and 5-monodeiodinase activity in liver and kidney 3 days before birth was observed, falling to a nadir at day 3 after birth. Foetal and neonatal serum T4, T3 and rT3 concentration were very low and rose progressively with age, reaching adult values at about day 21. In the foetal brown adipose tissue high 5'-monodeiodinase and low 5-monodeiodinase activity was found. The 5'-monodeiodinase decreased during the first days of life whereas the 5-monodeiodinase activity remained at a low stable level until day 3 when the activities of both enzymes increased. The increase of conversion rate of T4 to T3 and rT3 in liver and kidney well correlate with the triiodothyronines concentration in serum from day 3 after birth.     

Development of uridine diphosphate-glucuronyltransferase activity in cultures of chick-embryo liver

1. The liver of the domestric fowl (Gallus gallus) remains capable of conjugating o-aminophenol with glucuronic acid after 8 days' culture. The pathway of o-aminophenyl glucuronide formation in cultured liver, as in fresh tissue, includes the enzyme UDP-glucuronyltransferase. 2. UDP-glucuronyltransferase activity in chick-embryo liver increases on culture from very low to adult values within 6-8 days. 3. The development of UDP-glucuronyltransferase activity in cultured chick-embryo liver requires certain serum factors in the medium. The requirements change with embryo age. Liver from embryos younger than 15 days develops enzyme activity equally well in media containing either foetal or adult serum; liver from embryos older than 16 days develops activity only with adult serum. The development of enzyme activity in liver from the older embryos appears to be stimulated by diffusible factors in adult serum and inhibited by diffusible factors in foetal serum. It is suggested that the stimulation and inhibition of enzyme formation by small, diffusible molecules may be part of the mechanism regulating UDP-glucuronyltransferase activity in vivo. 4. Liver from 19-day-old chick embryos cultured with foetal serum begins to develop UDP-glucuronyltransferase activity if transferred to an adult-serum medium. Its capacity to develop UDP-glucuronyltransferase activity in adult serum survives in a foetal-serum medium for at least 5 days, the longest period tested. 5. The activity of UDP-glucuronyltransferase reached in 19-day chick-embryo liver after 1 or 2 days with adult serum is maintained without further increase after transfer to a foetal-serum medium. After 3 days with adult serum UDP-glucuronyltransferase activity continues to increase when the tissue is transferred to a foetal-serum medium. Thus liver from 19-day-old embryos requires 3 days with adult serum before development of enzyme activity becomes independent of a continuous adult-serum environment.     

Changes in hepatic lipogenesis during development of the rat

1. Changes in the activities of ATP citrate lyase, ;malic' enzyme, glucose 6-phosphate dehydrogenase, pyruvate kinase and fructose 1,6-diphosphatase, and in the ability to incorporate [1-(14)C]acetate into lipid have been measured in the livers of developing rats between late foetal life and maturity. 2. In male rats the activities of those systems directly or indirectly concerned in lipogenesis (acetate incorporation into lipid, ATP citrate lyase and glucose 6-phosphate dehydrogenase) fall after birth and are maintained at a low value until weaning. After weaning these activities rise to a maximum between 30 and 40 days and then decline, reaching adult values at about 60 days. ;Malic' enzyme activity follows a similar course, except that none could be detected in the foetal liver. Pyruvate kinase activity is lower in foetal than in adult livers and rises to slightly higher than the adult value in the post-weaning period. Fructose 1,6-diphosphatase activity rises from a very low foetal value to reach a maximum at about 10 days but falls rapidly after weaning to reach adult values at about 30 days. 3. Weaning rats on to a high-fat diet caused the low activities of acetate incorporation, ATP citrate lyase, glucose 6-phosphate dehydrogenase and pyruvate kinase, characteristic of the suckling period, to persist. ;Malic' enzyme and fructose 1,6-diphosphatase activities were not altered appreciably. 4. No differences could be detected in hepatic enzyme activities between males and females up to 35 days, but after this time female rats gave higher values for acetate incorporation, glucose 6-phosphate dehydrogenase activity and ;malic' enzyme activity. 5. The results are discussed in relation to changes in alimentation and hormonal influences.     

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.     

Effect of maternal ethanol consumption on foetal and neonatal rat hepatic protein synthesis.

Effects of maternal ethanol consumption were investigated on the rates of protein synthehsis by livers of foetal and neonatal rats both in vivo and in vitro, and on the activities of enzymes involved in protein synthesis and degradation. The rates of general protein synthesis by ribosomes in vitro studied by measuring the incorporation of [14C]leucine into ribosomal protein showed that maternal ethanol consumption resulted in an inhibition of the rates of protein synthesis by both foetal and neonatal livers from the ethanol-fed group. The rates of incorporation of intravenously injected [14C]leucine into hepatic proteins were also significantly lower in the foetal, neonatal and adult livers from the ethanol-fed group. Incubation of adult-rat liver slices with ethanol resulted in an inhibition of the incorporation of [14C]leucine into hepatic proteins; however, this effect was not observed in the foetal liver slices. This effect of externally added ethanol was at least partially prevented by the addition of pyrazole to the adult liver slices. Pyrazole addition to foetal liver slices was without significant effect on the rates of protein synthesis. Cross-mixing experiments showed that the capacity of both hepatic ribosomes and pH5 enzyme fractions to synthesize proteins was decreased in the foetal liver from the ethanol-fed group. Maternal ethanol consumption resulted in a decrease in hepatic total RNA content, RNA/DNA ratio and ribosomal protein content in the foetal liver. Foetal hepatic DNA content was not significantly affected. Ethanol consumption resulted in a significant decrease in proteolytic activity and the activity of tryptophan oxygenase in the foetal, neonatal and adult livers. It is possible that the mechanisms of inhibition of protein synthesis observed here in the foetal liver after maternal ethanol consumption may be responsible for at least some of the changes observed in 'foetal alcohol syndrome'.