Dieter Ullrich

Immunohistochemical and biochemical detection of uridine-diphosphate-glucuronyltransferase (UDP-GT) activity in putative preneoplastic liver foci

SummaryPreneoplastic liver foci were produced in female Wistar rats by the administration of 2-acetylaminofluorene (0.03% w/w) in the diet for 174 days. Increased UDP-glucuronyltransferase (UDP-GT) could be visualized immunohistochemically in the same focal areas which were ATPase-negative andγ-glutamyltranspeptidase-positive. Immunohistochemical detection was possible using rabbit anti-UDP-GT and peroxi-dase-labeled swine anti-rabbit immunoglobulins. The results of immuno-histochemistry were substantiated by enzyme determination in micro-dissected material. UDP-GT activity was 5-fold higher in focal areas in comparison with the surrounding liver tissue.Increased UDP-GT activity in conjunction with the altered pattern of other drug-metabolizing enzymes is consistent with increased resistance of preneoplastic cells to the cytotoxicity of carcinogens. Immunohistochemical detection of UDP-GT may provide a new marker for preneoplastic lesions which, in conjunction with other markers, may prove useful in analyzing the various stages of liver carcinogenesis and the remodeling of preneoplastic lesions after cessation of carcinogenic stimuli.

Glucuronide formation of various drugs in liver microsomes and in isolated hepatocytes from phenobarbital- and 3- methylcholanthrene-treated rats

Various substrates of rat liver microsomal UDP-glucuronosyltransferase were classified in vitro as preferred substrates of either 3-methylcholanthrene- or phenobarbital-inducible enzyme forms. Microsomal UDP-glucuronosyltransferase activities towards a third group of substrates (including oestrone, phenolphthalein, paracetamol and oxazepam) are not markedly altered by treatment with either 3-methylcholanthrene or phenobarbital. Some substrates of the 3-methylcholanthrene- and phenobarbital-inducible enzyme activities were selected to evaluate the importance of multiple enzyme forms for glucuronide formation in the intact cell. The metabolism of these compounds was compared in isolated hepatocytes from untreated controls and from rats treated with 3-methylcholanthrene (MC-hepatocytes) or phenobarbital (PB-hepatocytes). Glucuronidation of 1-naphthol and 3-hydroxybenzo[a]pyrene was chiefly enhanced in MC-hepatocytes (greater than 2-fold), whereas glucuronidation of chloramphenicol and bilirubin was chiefly enhanced in PB-hepatocytes. These observations are in agreement with differential induction of UDP-glucuronosyltransferase activities in vitro suggesting that, besides other factors such as cofactor supply, physiological activators, etc., the levels of the multiple enzyme forms are critically determining glucuronide formation in the intact cell.

Intralobular distribution of UDP-glucuronosyltrans-ferase in livers from untreated, 3-methylcholanthrene- and phenobarbital-treated rats

A 3-methylcholanthrene-inducible enzyme form of UDP-glucuronosyltransferase has been localized within the liver lobule both immunohistochemically and enzymatically in microdissected centrilobular and periportal liver tissue. Livers of untreated, 3-methylcholanthrene- and phenobarbital-treated rats have been compared. The enzyme was detected in hepatocytes throughout the liver. However both immunohistochemical determination of the enzyme level and biochemical determination of its activity towards 1-naphthol revealed a heterogeneous distribution of the enzyme. In untreated controls and 3-methylcholanthrene-treated rats both enzyme activity and histochemical staining was highest in centrilobular hepatocytes. However, after phenobarbital-treatment enzyme staining and activity was highest in periportal hepatocytes, suggesting that the differentially inducible enzyme activities may be localized in different zones of the liver lobule. The results demonstrate that the 3-methylcholanthrene-inducible UDP-glucuronosyltransferase is preferentially expressed in centrilobular hepatocytes.

Gilbert's syndrome: diagnosis by typical serum bilirubin pattern

Analysis of serum unconjugated and conjugated bilirubin fractions by routine diazo procedures does not allow a definite diagnosis of Gilberts syndrome. By the alkaline methanolysis procedure of Blanckaert followed by thin-layer chromatography we were able to discriminate Gilberts syndrome even in the presence of normal serum bilirubin concentrations from healthy subjects, patients with chronic persistant hepatitis and patients with chronic hemolysis. The relative proportion of unconjugated bilirubin in serum was 95 +/- 2% in patients with Gilberts syndrome (n = 28), 84 +/- 5% in healthy subjects (n = 29), 75 +/- 6% in patients with chronic persistant hepatitis (n = 7) and 85 +/- 3% in patients with chronic hemolysis (n = 9). The difference between Gilberts syndrome and the control groups with normal or elevated serum bilirubin was highly significant (p less than 0.001). In Gilberts syndrome, unconjugated bilirubin ranged between 90 and 99%, in healthy subjects between 72 and 90%, in patients with chronic persistant hepatitis between 68 and 85% and in patients with chronic hemolysis between 81 and 89% of total. An overlap was only seen in one patient with Gilberts syndrome and in 2 healthy subjects at the 90% level. We conclude that in most patients with Gilberts syndrome provocation tests are no longer necessary.

Activating and inactivating reactions controlling 2-naphthylamine mutagenicity

Factors controlling 2-naphthylamine mutagenicity were studied using the Ames test. 1) Both rat liver microsomes and cytosolic proteins were required for generation of mutagenic metabolites. 2) 1-Hydroxy-2-naphthylamine, the major metabolite of 2-naphthylamine, was not mutagenic but cytotoxic to bacteria. 3) Ascorbic acid, reduced glutathione and conjugation reactions, such as glucuronidation, were strongly inhibiting 2-naphthylamine mutagenicity. 4) When isolated hepatocytes were used as the activating system mutagenic metabolites could not be detected. However cytotoxicity was detectable at doses of 2-naphthylamine greater than 0.2 μmol/106 cells.The results suggest that the formation of genotoxic metabolites of 2-naphthylamine is largely prevented in the intact, non-dividing rat hepatocyte.

Immunohistochemical demonstration of increased UDP-glucuronyltransferase in putative preneoplastic liver foci

0.01% (V/V) H202 in 0.12 M cacodylate buffer (pH 5.45) for 30 min. With the method described above we were able to stain very few and under optimal circumstances even one single cell. From our experience best results in filling one single cell with its dendritic branching and the efferent axon were obtained, if a very clear single-cell recording had occurred indicating that the electrode tip was as close as possible to one single cell (Fig. 1 b). In some preparations we could even find afferent cells to the recorded and injected neuron which were filled retrogradely.

Renal clearance of bilirubin conjugates in newborns of different gestational age

The renal excretion of bilirubin conjugates was analysed in 22 newborns. Bilirubin monoconjugate was the only metabolite detectable in urine samples and its renal excretion correlated with the creatinine excretion rate (r=0.91). The renal clearance of bilirubin mono-conjugates in newborns ranged between 380 and 2160 ml/1.73 m2 per 24 h (median: 790). According to the present findings the renal function should be monitored in newborns and infants with conjugated hyperbilirubinaemia.