J. Pausch

The regulation of pyrimidine nucleotide level and its role in experimental Hepatitis

Abstract The GalN-induced liver injury is presented as an experimental tool for studies on the regulation of uridylate biosynthesis and on the metabolic consequences of changes in uracil nucleotide contents of the liver. The primary biochemical lesion consists of a deficiency of uridine phosphates and UDP-hexoses for a defined minimum period of time. Evidence is presented for a stimulation of uridylate de novo synthesis by release of feedback inhibition during the deficit period. Orotate administration results in a cycloheximide-sensitive increase of the activities of OMP pyrophosphorylase and OMP decarboxylase. The stimulated rate of uridylate biosynthesis is positively correlated to the activities of both enzymes under a variety of conditions. The intensity and the duration of the deficiency of uridine phosphates depend on the relative capacities of uridylate producing and consuming processes. The quantitation of the deficit period and its relation to changes of UMP biosynthesis and of UTP- and UDP-hexose-dependent processes enable an understanding of the effect of measures which protect against GalN hepatitis, of the existence of GalN-refractory conditions, and of the different susceptibilities of various species and organs to this amino sugar. If a critical value of the uridylate deficit period is surpassed, irreversible liver cell damage is consistently observed several hours later. Under these conditions, RNA synthesis is strongly depressed. The reduction of guanosine incorporation into RNA can be relieved within a short time by uridine administration. The concept of the deficit period appears to be a valuable approach to biochemical pathology.

Activity and distribution of the enzymes of uridylate synthesis from orotate in animal tissues

Abstract 1. 1.|Activity and distribution of orotidine 5′-monophosphate pyrophosphorylase (EC 2.4.2.10) and orotidine 5′-monophosphate decarboxylase (EC 4.1.1.23), were studied. The activities of both enzymes catalyzing uridylate biosynthesis from orotate were measured by the formation of 14 CO 2 from [ carboxy - 14 C]orotate and orotidine 5′-monophosphate (OMP), respectively. Improved conditions for their determination are given, including the use of excess EDTA, for the assay of OMP pyrophosphorylase with endogeneous OMP decarboxylase as indicator enzyme. 2. 2.|The subcellular distribution of both enzymes in rat liver indicates an exclusive localization in the cytosol. 3. 3.|The specific activity of OMP pyrophosphorylase in different rat tissues was found to decrease in the following order: regenerating liver > spleen > liver > gut > kidney > brain > skeletal muscle. The order of OMP decarboxylase activity was: spleen > regenerating liver > liver > kidney > gut > brain > skeletal muscle. 4. 4.|The hepatic activities of the two enzymes were decreasing during the post-natal development. 5. 5.|In the livers of different animal species the specific activities of both enzymes were highest in mice, lowest in guinea pigs, and intermediate in chicken and rats. 6. 6.|Under a variety of conditions, including fast growing tissues, OMP pyrophosphorylase and OMP decarboxylase showed closely parallel, possibly coordinate changes in activity.

First clinical experiences with a selective chemotherapy of hepatocellular carcinoma with 5-fluorouridine in combination with other antipyrimidines

Sixteen unselected patients with non-resectable hepatocellular carcinoma were treated in a phase I study with 261 cycles of D-galactosamine and 6-azauridine prior to 5-fluorouridine. Thirty % of the patients survived for more than one year without signs of tumor progression and with an unchanged performance status. The compatibility of this chemotherapeutical method was quite satisfactory. The only extrahepatic side effect was a leucopenia and/or thrombocytopenia which was reversible upon reduction of the 5-fluorouridine dose. The heterogeneity of the 16 patients treated to date does not allow a definite statistical evaluation of the reported clinical observations and results. A final decision about the clinical applicability of this concept of a selective chemotherapy of hepatocellular carcinoma requires further experience.

Ansätze zur selektiven Chemotherapie des hepatozellulären Karzinoms

Summary1. An improvement of the chemotherapy of hepatocellular carcinoma with adriamycin or 5-fluorouracil and a reduction of side effects has been achieved by intra-arterial administration of the drugs. This treatment provides a somewhat extended survival but no cure.2. The treatment of hepatocellular carcinoma in patients by reduction of an inactive precursor of a cytocidal alkylating agent by azoreductase of the tumor showed no therapeutic effect.3. A selective hepatocellular uptake of drugs coupled to asialoglycoproteins has been described. An application of this concept for the chemotherapy of hepatocellular carcinoma seems doubtful since a loss of binding proteins for desialylated glycoproteins during experimental hepatocarcinogenesis has been demonstrated.4. The increased uptake of 5-fluorouridine in hepatomas after induction of a tissue-specific depletion of uridine 5′-triphosphate and cytidine 5′-triphosphate provides an effective experimental chemotherapy with limited side effects. A clinical use of this new concept for the chemotherapy of hepatocellular carcinoma may serve as a useful approach.Zusammenfassung1. Die Chemotherapie des hepatozellulären Karzinoms mit Adriamycin oder 5-Fluoruracil, deren Wirksamkeit und Verträglichkeit durch intraarterielle Applikation verbessert werden kann, ermöglicht nur eine kurzfristige Lebensverlängerung und keine Heilung.2. Der klinische Versuch einer gewebespezifischen Chemotherapie des hepatozellulären Karzinoms durch Reduktion der unwirksamen Vorstufe einer zytocidalen alkylierenden Substanz mit Hiife der Azoreduktase des Tumors hatte keinen lebensverlängernden Effekt.3. Die Kopplung von Medikamenten an Asialoglykoproteine ermöglicht eine selektive Aufnahme von Antimetaboliten in die Leber. Eine Anwendung dieses Konzepts zur selektiven Chemotherapie des hepatozellulären Karzinoms scheint wegen des Verlusts von Plasmamembranrezeptoren bei der experimentellen Hepatokarzinogenese fragwürdig.4. Die selektive Einschleusung von 5-Fluoruridin in Hepatome nach Erzeugung eines gewebespezifischen Uridintriphosphat- und Cytidintriphosphatmangels ermöglicht im Tierexperiment einen guten Chemotherapieeffekt bei geringen Nebenwirkungen, sodaß gute Erfolgschancen für eine klinische Anwendung dieses neuen Therapiekonzepts für das hepatozelluläre Karzinom gegeben sind.

Zur selektiven Chemotherapie des hepatozellulären Karzinoms

Die bisherigen Moglichkeiten einer Chemotherapie hepatozellularer Karzinome sind unbefriedigend. Nur durch eine operative Entfernung des Tumors kann ein geringer Prozentsatz der Patienten uberleben. Eine Resektion des Tumors ist nur in etwa 30% der Falle moglich und mit einer betrachtlichen Mortalitat verbunden [14]. Eine Chemotherapie mit 5-Fluoruracil [3] oder Adriamycin [15] kann lebensverlangernd wirken [3, 15]; beiden Cytostatika fehlt jedoch eine Selektivitat fur das hepatozellulare Karzinom. Die extrahepatischen Nebenwirkungen konnten durch Cytostatika-Infusion in die Arteria hepatica verringert werden [1, 4].

Zur Bedeutung der Aktivität von Enzymen der Pyrimidinbiosynthese für die Entwicklung der Galaktosaminschädigung der Leber

Der Aminozucker D-Galaktosamin (GalN) lost in der Leber verschiedener Tierspecies eine Sequenz von Reaktionen aus, die schlieslich zu einem Hepatitisahnlichen Leberzellschaden fuhrt [1, 2, 3] Am Anfang dieser Reaktionsfolge steht eine starke und langdauernde Verminderung von Uridinphosphaten [3, 4, 5, 6], die fur wichtige Biosynthesen in der Leber (z. B. RNS-Synthese, Glykogensynthese, Glykosylierungen, Glucuronidierungen) essentiell sind. Dieses Uridinphosphatdefizit entsteht durch Bildung und Anhaufung von UDP-Derivaten des GaIN bei unzureichender Nachsynthese der Uridinphosphate [3, 4, 5, 6]. Die Nachsynthese erfolgt uber die de-novo-Pyrimidinbiosynthese aus Orotat und z.dT. durch Phosphorylierung von Uridin. Der Schweregrad der Galaktosaminschadigung ist abhangig von Ausmas und Dauer des Uridinphosphatdefizits [6]. Der modifizierende Einflus weiterer Faktoren auf das Schadigungsbild widerspricht dieser Beziehung nicht.

The glutamine analog acivicin as antipyrimidine. Studies on the interrelationship between pyrimidine and urea synthesis in liver.

The inhibition of cytosolic carbamoyl-phosphate synthetase II by acivicin was used to study the role of the cytosolic carbamoyl phosphate pool as the exclusive substrate source for de novo pyrimidine synthesis in rat hepatocytes. De novo pyrimidine synthesis was stimulated: 1. by uridine triphosphate deficiency (incubation with D-galactosamine) leading to a stimulation of cytosolic carbamoyl phosphate synthesis, and 2. by accumulation and efflux of mitochondrial carbamoyl phosphate (incubation with ammonium ions and L-norvaline). The stimulated orotate formation from cytosolic carbamoyl phosphate in UTP depleted cells was completely blocked by acivicin. It was not influenced by an inhibition of mitochondrial carbamoyl phosphate synthesis mediated by 4-pentenoate, since mitochondrial carbamoyl phosphate did not participate in cytosolic pyrimidine synthesis even in the presence of ammonium ion concentrations maintaining physiological rates of urea synthesis. An excess of ammonium ions led to an artificial accumulation and efflux of mitochondrial carbamoyl phosphate, which could be avoided by 4-pentenoate. The non-regulated stimulation of pyrimidine synthesis from surplus mitochondrial carbamoyl phosphate was not inhibited by acivicin. Utilization of mitochondrial carbamoyl phosphate for de novo pyrimidine synthesis presumably does not occur under physiological conditions because mitochondrial CP efflux depends on the accumulation of this metabolite in the mitochondria under experimental or pathological circumstances. Acivicin inhibition of CPS II thus cannot be bypassed by mitochondrial CP. It is suitable as inhibitor of the physiological de novo pyrimidine synthesis.