Iron and the liver. Acute and long-term effects of iron-loading on hepatic haem metabolism.

We have determined the dose-response curves (100-900 mg of Fe/kg body wt.) and the time course over 84 days for the effects of a single injection of iron-dextran on rat hepatic 5-aminolaevulinate synthetase, cytochrome P-450, iron content, and GSH (reduced glutathione). Porphyrins in liver and urine have also been measured. (1) At 2 days after treatment, a dose of 500 mg of Fe/kg produced a 20-fold increase in iron concentration, which was maintained for 14 days. Total hepatic iron remained constant over 63 days, falling slightly by 84 days. (2) The activity of 5-aminolaevulinate synthetase was maximally increased (6-fold) 12-24 h after iron treatment. By 48 h the activity fell to less than twice the control value and thereafter remained slightly above the control value (1.1-1.5-fold) until 84 days after iron treatment. Liver GSH concentrations were unaffected by iron. Porphyrins in liver and urine were either unchanged or decreased. (3) Hepatic cytochrome P-450 decreased after iron treatment to a minimum (63% of control) at 48 h after iron administration and gradually returned to the control value by 28 days. (4) Iron-dextran potentiated 2 allyl-2-isopropyl-acetamide-induced synthesis of hepatic 5-aminolaevulinate. Potentiation occurred if the drug was given at the same time or 36 h after iron administration, but did not occur if the drug was given 14 or 64 days after iron administration. (5) The results are discussed in relation to proposed mechanisms for the effects of iron on hepatic haem metabolism.     

Induction of (2'-5')oligoadenylate synthetase in serum-starved HeLa S3 cells by growth factors and its role in growth regulation

When serum-starved HeLa S3 cells were stimulated to proliferate by addition of fetal calf serum (FCS), (2'-5')oligoadenylate synthetase (2-5A synthetase) activity was induced. Although no interferon (IFN) activity was detectable in the HeLa S3 cell-conditioned culture medium after growth stimulation, addition of anti-IFN-beta monoclonal antibody inhibited both the expression of the 2-5A synthetase gene and the production of the enzyme, suggesting that endogenous IFN-beta was involved in 2-5A synthetase induction. Purified preparations of three growth factors, epidermal growth factor, platelet-derived growth factor, and insulin, also induced 2-5A synthetase through IFN-beta. When serum-starved HeLa S3 cells were treated with FCS, DNA synthesis was initiated synchronously, with peaks after 12 and 32 h, although the level of 2-5A synthetase reached a maximum after the first peak of DNA synthesis. Inhibition of 2-5A synthetase induction by anti-IFN-beta antibody enhanced the second, but not the first cycle of DNA synthesis. These results suggested that in HeLa S3 cells, after stimulation with growth factors the IFN/2-5A synthetase system played a role in cell growth negative regulatory mechanisms.     

Protoporphyrin IX and oxidative stress

The short- and long-term pro-oxidant effect of protoporphyrin IX (PROTO) administration to mice was studied in liver. A peak of liver porphyrin accumulation was found 2 h after the injection of PROTO (3.5 mg/kg, i.p.); then the amount of porphyrins diminished due to biliar excretion. After several doses of PROTO (1 dose every 24 h up to 5 doses) a sustained enhancement of liver porphyrins was observed. The activity of δ-amino-levulinic acid synthetase was induced 70-90% over the control values 4 h after the first injection of PROTO and stayed at these high levels throughout the period of the assay. Administration of PROTO induced rapid liver damage, involving lipid peroxidation. Hepatic GSH content was increased 2 h after the first injection of PROTO, but then decreased below the control values which were maintained after several doses of porphyrin. After a single dose of PROTO, Cu-Zn superoxide dismutase (SOD) was rapidly induced, suggesting that superoxide radicals had been generated. Increased levels of hydrogen peroxide coming from the reaction catalyzed by SOD and lipid peroxides as a consequence of membrane peroxidation, induced the activity of catalase and glutathione peroxidase (GPx), while decreased GSH levels induced glutathione reductase (GRed) activity. However after 5 doses of PROTO, the activity of SOD was reduced reaching control values. GPx and catalase activities slowly went down, while GRed continued increasing as long as the levels of GSH were kept very low. TBARS values, although lower than those observed after a single dose of PROTO, remained above control values; Glutathione S-transferase activity was instead greatly diminished, indicating sustained liver damage.

Our findings would indicate that accumulation of PROTO in liver induces oxidative stress, leading to rapid increase in the activity of the antioxidant enzymes to avoid or revert liver damage. However, constant accumulation of porphyrins provokes a liver damage so severe that the antioxidant system is compromised.     

Protoporphyrin IX and oxidative stress

The short- and long-term pro-oxidant effect of protoporphyrin IX (PROTO) administration to mice was studied in liver. A peak of liver porphyrin accumulation was found 2 h after the injection of PROTO (3.5 mg/kg, i.p.); then the amount of porphyrins diminished due to biliar excretion. After several doses of PROTO (1 dose every 24 h up to 5 doses) a sustained enhancement of liver porphyrins was observed. The activity of δ-amino-levulinic acid synthetase was induced 70–90% over the control values 4 h after the first injection of PROTO and stayed at these high levels throughout the period of the assay. Administration of PROTO induced rapid liver damage, involving lipid peroxidation. Hepatic GSH content was increased 2 h after the first injection of PROTO, but then decreased below the control values which were maintained after several doses of porphyrin. After a single dose of PROTO, Cu-Zn superoxide dismutase (SOD) was rapidly induced, suggesting that superoxide radicals had been generated. Increased levels of hydrogen peroxide coming from the reaction catalyzed by SOD and lipid peroxides as a consequence of membrane peroxidation, induced the activity of catalase and glutathione peroxidase (GPx), while decreased GSH levels induced glutathione reductase (GRed) activity. However after 5 doses of PROTO, the activity of SOD was reduced reaching control values. GPx and catalase activities slowly went down, while GRed continued increasing as long as the levels of GSH were kept very low. TBARS values, although lower than those observed after a single dose of PROTO, remained above control values; Glutathione S-transferase activity was instead greatly diminished, indicating sustained liver damage.

Our findings would indicate that accumulation of PROTO in liver induces oxidative stress, leading to rapid increase in the activity of the antioxidant enzymes to avoid or revert liver damage. However, constant accumulation of porphyrins provokes a liver damage so severe that the antioxidant system is compromised.     

Influence of glucagon, 6-N,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate and triamcinolone on the arginine synthetase system in perinatal rat liver

1. The administration of triamcinolone (19-190mug/animal) to postnatal rats increased the arginine synthetase system activity 1.2-2.5-fold above control values 24h after exposure to the hormone. Cortisol (hydrocortisone), however, increased the arginine synthetase system activity only when larger (190mug/animal) or repeated daily doses were given. Glucagon (100mug/animal) stimulated arginine synthetase system activity only after the second postnatal day. None of these agents increased the activity in 19.5-21.5-day foetuses after intrauterine administration. 2. The viability of foetal rat liver explants maintained in organ culture for up to 54h was validated both by ultramicroscopic examination and by incorporation of radioactive leucine and orotic acid. 3. In organ cultures of foetal rat liver explants (18.5 days to term), triamcinolone (20mug/ml of medium) evoked a 2.8-4.3-fold increase after 24h of incubation. This increase was completely inhibited by actinomycin D (25mug/ml) or cycloheximide (10mug/ml). Cortisol (5-50mug/ml) or glucagon (0.067-67mug/ml) also increased the arginine synthetase system activity above the respective control values, but there was no increase in activity with insulin (0.05-0.25i.u./ml). 4. Maximum concentrations of glucagon (67mug/ml), dibutyryl cyclic AMP (6-N,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate) (0.1mm) and triamcinolone (20mug/ml) incubated for 24h with foetal rat liver explants each produced between a two-and three-fold increase in the activity of the arginine synthetase system. Combinations of maximum amounts of glucagon and the cyclic nucleotide did not produce a greater effect than either agent alone. However, the combination of dibutyryl cyclic AMP with triamcinolone appeared to produce somewhat less than additive effects. 5. The effects of the cyclic nucleotide and triamcinolone were evident after 12h of incubation and increased steadily throughout the 24h of observation. This time-course of increased enzyme activity is very much slower than that reported for the induction of other enzymes in explant cultures of foetal rat liver.     

Reversibility of the antiproliferative effect of interferon

The reversibility of the antiproliferative effect of interferon (IFN) and its correlations to the induction of (2',5') oligoadenylate synthetase (2-5A synthetase) activity was studied on NIH/3T3 cells transformed by Moloney murine sarcoma virus. The cells were treated with various doses of mouse beta-IFN. At 72 h after treatment, the cultures were subdivided. While half received fresh doses of IFN, the second half received no IFN. Reversibility of the IFN effect was then followed. Three different parameters as indicators for cell proliferation were used: cell growth, protein synthesis and cloning efficiency. In parallel, the IFN-induced activity of 2-5A synthetase was determined. The data obtained led to the following conclusions. (1) The antiproliferative effect of IFN increases with increased IFN concentration (90-1,800 IU/ml) and with time of treatment, up to 72 h after treatment. (2) The induced activity of 2-5A synthetase increases with a much faster rate, reaching maximum activity at 24 h after treatment with 450 IU/ml. This means that the induction of the enzyme precedes the antiproliferative effects of IFN. (3) There is almost no recovery of the IFN antiproliferative effect following treatment for 72 h with high doses of IFN (1,200-1,800 IU/ml). However, at lower doses, recovery is evident. (4) Removal of IFN after treatment for 3 days with 450 IU/ml resulted in a gradual decrease of 2-5A synthetase activity, reaching the basal level at 72 h after removal. However, there is no reduction of enzyme activity following treatment for 72 h with 1,800 IU/ml of IFN.     

Regulation of protein synthesis in lymphoblastoid cells during inhibition of cell proliferation by human interferons

Treatment of human lymphoblastoid (Daudi) cells with interferons inhibits cell proliferation in culture within 24 h. The failure of cell growth has been shown to be associated with impaired processing and decreased stability of newly replicated DNA. Because there is a close relationship between DNA replication and protein synthesis we have measured protein synthesis in intact Daudi cells. Protein synthesis declined steadily between 24 and 96 h after interferon treatment to a value which is only 20-30% of the rate in control cells. The enzyme 2',5'-oligo(A) synthetase is induced but our data do not support a role for the 2',5'-oligo(A)-activated ribonuclease in the control of translation in this system.     

Action of insulin on liver carbamoyl-phosphate synthetase II (glutamine-hydrolyzing) activity

Carbamoyl-phosphate synthetase II (glutamine-hydrolyzing) (EC 6.3.5.5) (synthetase II), is the first and rate-limiting enzyme in the de novo UMP biosynthetic pathway. The present investigation showed that insulin has a regulatory action on hepatic synthetase II activity. When diabetes was induced with injection of different doses of alloxan the plasma insulin concentrations decreased in a dose-dependent fashion to 72, 38, 31 and 28% and concurrently the liver synthetase II activity decreased to 75, 43, 29 and 22% of the normal values. In diabetic rats dose response studies showed that with insulin injections of 4, 6, 8 or 10 U/day for 48 h the hepatic synthetase II activity increased to 81, 95, 99 and 103% of the control liver values. In the diabetic rats the insulin-induced rise in liver synthetase II activity was prevented by treatment of the rats with actinomycin.     

Vasoactive intestinal peptide induces 2',5'-oligoadenylate synthetase and antiviral state in cells of HT-29 colonic cancer]

Vasoactive Intestinal Peptide (VIP) is able at the concentration 10 to 100 nM to induce in HT-29 cells 2'5' oligoadenylate (2'5' A) synthetase activity. The kinetics of this induction show that the maximal effect is attained after 24 hrs. VIP induces 2'5' A synthetase parallel to inhibition of vesicular stomatitis virus growth. The levels of these two induced activities after VIP treatment are comparable to those induced by the poly (I).poly (C), an inducer of IFN beta/alpha in mammalian cells. Moreover the anti-IFN beta/alpha antibodies abolish the VIP-induced 2'5' A synthetase whereas anti-IFN gamma antibodies are ineffective. The fact that VIP establishes an antiviral state in HT-29 cells potentiates new pharmaceutical applications for this neuropeptide.     

Glutathione correlates with lipid peroxidation in liver mitochondria of triiodothyronine-injected hypophysectomized rats

The temporal relationship of changes in state 3 respiration, lipid peroxidation, and glutathione (GSH) content was investigated in liver mitochondria of hypophysectomized rats after an injection of 3,3',5-triiodo-L-thyronine (T3). Lipid peroxidation induced by ADP/Fe3+/NADPH was determined by the amount of malondialdehyde formed. Hypophysectomy decreased respiration and lipid peroxidation (from 19.88 +/- 3.04 to 14.19 +/- 1.14 nmol malondialdehyde.mg protein-1.10 min-1) but increased GSH content (from 7.06 +/- 2.08 to 12.46 +/- 3.58 nmol/mg protein). Daily injections of a low dose (5 micrograms/100 g) of T3 for 7 days restored the parameters. Time course (up to 96 h) of these changes was followed after one injection of a moderate (100 micrograms/100 g) and high (1000 micrograms/100 g) dose of the hormone. Respiration showed a significant increase at 24 h and declined slightly at 96 h. There was a slow loss of respiratory control ratio after 24 h. Lipid peroxidation remained unchanged at 24 h and showed a gradual increase, becoming significantly higher at 72-96 h depending on the hormone dosage. Changes in GSH content followed a time course similar to that of lipid peroxidation except that it showed a decrease instead of an increase. There was a high degree of inverse linear correlation between lipid peroxidation and GSH (correlation coefficient = 0.95). Because GSH is required for detoxification of hydroperoxides generated by the respiratory chain, it is suggested that lipid peroxidation may play a major role in the modulation of intramitochondrial GSH.     

Effects of varying doses of methionine sulfoximine on liver glutamine synthetase activity and time courses of blood and urinary nitrogenous compounds in the chicken (Gallus domesticus)

1. The activity of liver glutamine synthetase was inhibited to 7-12% of the control activity by an intracardiac injection with methionine sulfoximine (MSM) at dosages of 20, 50, 75 and 100 mg/kg body wt. 2. Plasma glutamine concentrations in all the MSM treatments decreased sharply, then reached steady-state levels within 0.5-2.5 hr, which were almost proportional to a dose of MSM. 3. Blood ammonia concentration sharply increased to a steady-state level attained at 4.5 hr, which was proportional to a dose of MSM. The excretion rate of urinary ammonia augmented linearly up to the dose dependent maximum rates within 2-5 hr. 4. Plasma uric acid concentration dropped linearly by about 6.4 mg/100 ml at doses of 50, 75 and 100 mg MSM and by 3.7 mg/100 ml at a dose of 20 mg MSM within 2.5 hr, then recovered a little. 5. The decreases in excretion rates of urinary uric acid for the first 4 hr were almost the same at doses of 50 mg and larger, being twice as large as that of the control chicken. 6. Any doses of MSM affected neither the time course of excretion rate of total urinary nitrogen nor its total amounts for 7 hr after MSM treatment.     

Hormonal regulation of fatty acid synthetase in chick embryo liver.

Fatty acid synthetase activity in chick embryonic liver is negligible compared to that in newly hatched, fed chicks. The enzyme activity is prematurely induced 5–50-fold in 20-day-old embryos and in newly hatched chicks by the administration of insulin, hydrocortisone, growth hormone, glucagon or dibutyryl cyclic AMP. The induction of the enzyme activity is blocked by the administration of cycloheximide, indicating that new protein synthesis is required. Immunochemical titrations of different enzyme preparations from 5-day-old chicks, adult chicken and various inducer-treated embryos gave an identical equivalence point, indicating that the changes in synthetase activity after hormonal induction in embryos are related entirely to changes in content of enzyme. The increase in liver synthetase content after administration of insulin, glucagon or dibutyryl cyclic AMP is directly related to an increase in the rate of synthetase synthesis. The induction of the synthetase activity by suboptimal doses of glucagon or cyclic AMP is potentiated by the phosphodiesterase inhibitory theophylline. There is a very rapid decay of synthetase activity, with a half-life of about 4 h after elevation to higher levels following administration of insulin, glucagon or dibutyryl cyclic AMP. Glucagon and dibutyryl cyclic AMP induction of the synthetase activity is observed early in the embryonic development, whereas insulin induction is noted 2 days before hatching. Insulin, glucagon and cyclic AMP are potentially capable of altering the levels of glycolytic intermediates which may be involved in the induction of synthetase.     

Relative effects of S-adenosylmethionine depletion on nucleic acid methylation and polyamine biosynthesis.

Treatment of cultured L1210 cells with 1 mM-L-2-amino-4-methoxy-cis-but-3-enoic acid (L-cisAMB), a methionine-analogue inhibitor of S-adenosylmethionine (AdoMet) synthetase (EC 2.5.1.6), produced a rapid and near-total depletion of AdoMet by 4 h. After this, the pools recovered to 60% of control by 48 h, apparently because of an increase in AdoMet synthetase activity. Both AdoMet depletion and the accompanying increase in synthetase activity were substantially enhanced by lowering methionine concentrations in the media from 100 microM to 30 microM, the minimal concentration that supports cell growth at control values. During a 4 h incubation in media containing 30 microM-methionine, 1-5 mM-L-cisAMB depleted cellular AdoMet to undetectable values, and inhibited nucleic acid methylation by 44-72% and RNA methylation by 60-87%. Under these same treatment conditions, putrescine pools increased by about 3-fold, whereas spermidine pools decreased by only 20% and spermine pools remained the same. Pool changes were accompanied by a 2-4-fold increase in ornithine decarboxylase activities and AdoMet activities. Thus the rapid depletion of AdoMet pools by L-cisAMB results immediately in a decrease in methyl-transfer reactions involving nucleic acids, whereas, by contrast, biosynthesis of higher polyamines appears to be minimally affected, owing to compensatory increases in key enzyme activities.     

Effects of liver regeneration on tRNA contents and aminoacyl-tRNA synthetase activities and sedimentation patterns.

The tRNA content and aminoacyl-tRNA synthetases of regenerating liver in the phase of rapid growth were compared with those of livers from both intact and sham-operated rats. At 48 h after hepatectomy, the amount of active tRNA (called 'total acceptor capacity') is significantly higher in regenerating liver than in control livers, owing to a general, possibly not uniform, increase in the various tRNA families, which suggests that it may contribute to the increased protein synthesis and to decreased protein degradation as well. The activities of most, but not of all, aminoacyl-tRNA synthetases in cell sap of regenerating liver tend to be greater than normal. Increased activity of histidyl-tRNA synthetase fits in with the possibility that the mechanisms that control the rate of protein degradation through aminoacylation of tRNAHis in cultured cells [Scornik (1983) J. Biol. Chem. 258, 882-886] also operate in the liver and play a role in regeneration. Sedimentation analysis of cell sap in sucrose density gradients shows a shift of prolyl-tRNA synthetase activity toward the high-Mr form in regenerating liver. This change might be related to the positive protein balance and to growth in vivo, since it is also observed in the anaplastic Yoshida ascites hepatoma AH 130.     

Pharmacokinetic study of a human recombinant interferon (Re-IFN-alpha A) in cynomolgus monkeys by 2'-5' oligoadenylate synthetase assay

We evaluated 2'-5'Oligoadenylate (2-5 A) synthetase assay for pharmacokinetic study of human interferon (IFN) in cynomolgus monkeys. The enzyme was induced in primary cultures of cynomolgus monkey kidney (PMK) cells as well as in FL cells in response to human recombinant IFN-alpha A treatment. The enzyme activity increased with IFN dose and, in parallel with the enzyme elevation, developed the antiviral state of the cells. The enzyme activity induced in the peripheral blood lymphocytes peaked at 6 to 12 hr after iv or im administration. The peak level of the enzyme activity depended on the IFN concentration of the blood and the activity rapidly decreased as serum IFN was cleared from the blood. These results indicate that human recombinant IFN-alpha A induces 2-5 A synthetase in monkey cells both in vitro and in vivo, and that the enzyme assay can be used to quantitatively monitor the host response after IFN administration.     

Induction and function of 2',5'-oligoadenylate synthetase in differentiation of mouse myeloid leukemia cells

The activity of 2′,5′-oligoadenylate synthetase (2-5A synthetase), known to be induced by interferon, was detected in mouse myeloid leukemic M1 cells only when they differentiated to phagocytic cells after incubation with conditioned medium (CM) from rat embryo cells. However, no interferon activity occurred in culture fluids of CM-treated M1 cells, although some activity was detected in the cell extracts. When anti-interferon serum was added to M1 cell cultures, the induction of 2-5A synthetase by CM was suppressed. These results suggest that CM stimulated the M1 cells to produce a minute amount of interferon, which was reponsible for induction of the 2-5A synthetase activity. On the other hand, development of the phagocytic activity of M1 cells could not be influenced by addition of antiserum. Interferon added exogenously per se neither induced phagocytic activity of M1 cells, nor did it enhance the CM-induced differentiation of the cells. Moreover, dexamethasone, which induced differentiation of M1 cells, was not capable of inducing 2-5A synthetase. These results indicate that interferon and/or 2-5A synthetase plays no essential part in the differentiation of M1 cells.     

Immunochemical evidence for glutamine-mediated degradation of glutamine synthetase in cultured Chinese hamster cells.

The specific activity of glutamine synthetase in cultured Chinese hamster cells is inversely related to the concentration of glutamine in the surrounding solution. Enzyme specific activity increases 8- to 10-fold when glutamine is removed from serum-free F12 growth media. The induction of glutamine synthetase activity occurs only after glutamine removal and not after the removal of other amino acids (methionine, leucine, or isoleucine). The analysis of the glutamine-mediated decrease in glutamine synthetase activity has been simplified by the finding that depression proceeds in nutrient-free buffered saline solution (141 mM NaCl, 5.4 mM KCl and 30 mM Tricine (pH 7.4). Under these conditions, 0.1 mM cyanide blocks glutamine-mediated depression. The cyanide inhibition is reversed by the addition of 1.0 mM glucose which suggests that ATP is required for depression. Glutamine-mediated depression is temperature-dependent, occurring between 25 and 45 degrees with an optimum rate at 37 degrees. Studies of the time course of induction and depression as a function of glutamine concentration suggest that glutamine regulates the rate at which the enzyme is either modified or degraded. We have employed an antibody prepared against homogeneous Chinese hamster liver glutamine synthetase to measure the amount of glutamine synthetase protein in extracts of cells containing induced or depressed levels of enzyme activity. A highly sensitive immunoprecipitation procedure enables quantitation of nanogram amounts of glutamine synthetase protein. Glutamine synthetase in cell extracts containing induced levels of enzyme activity possesses the same molecular specific activity (ratio of activity to antigenicity) as homogeneous Chinese hamster liver glutamine synthetase. The molecular specific activity of glutamine synthetase is almost the same in extracts of cells with depressed levels of enzyme obtained by growth for short (2 hours) and long (24 hours) times in the presence of glutamine. These data suggest that glutamine-mediated depression of glutamine synthetase results from degradation of enzyme molecules.     

The 2'-5' adenylate (2-5A) system in erythropoiesis

The trimer and tetramer core forms of 2'-5' Adenylate (2-5A) were tested in vitro for their effect on mouse liver CFU-E. Both forms of 2-5A core partially inhibited the CFU-E response to erythropoietin when given as a single dose at time 0 hours. Approximately 25% fewer colonies were seen after 2-days growth following exposure of the CFU-E to 0.1 mM 2-5A core. A 0.01 mM concentration of exogenous 2-5A core was not inhibitory. Endogenous 2-5A synthetase activity was assayed in spleen cell lysates from mice made anemic by several injections of phenylhydrazine. This method of treatment stimulated erythropoiesis, and this was directly correlated with an increase in the rate of enzyme activity measured by lysate conversion of 14C-ATP to 2-5A (our in press data suggests that the same may occur with hypoxic stimulation). This suggested to us that endogenous 2-5A synthetase and its 2-5A, a known inhibitor of DNA synthesis and protein synthesis, may help to regulate some of the late events in erythropoiesis.     

Expression of N-acetylglucosaminyltransferase III in hepatic nodules during rat liver carcinogenesis promoted by orotic acid

The activity of N-acetylglucosaminyltransferase III, which adds a "bisecting" GlcNAc in beta 1,4 linkage to the beta-linked Man of the core of Asn-linked oligosaccharides (Narasimhan, S. (1982) J. Biol. Chem. 257, 10235-10242), was determined in hepatic nodules of rats initiated by administration of a single dose of carcinogen 1,2-dimethylhydrazine.2HCl (100 mg/kg, intraperitoneal) 18 h after partial hepatectomy and promoted by feeding a diet supplemented with 1% orotic acid for 32-40 weeks. N-Acetylglucosaminyltransferase III was assayed using glycopeptide GlcNAc beta 1,2Man alpha 1,6(GlcNAc beta 1,2Man alpha 1,3)Man beta 1, 4GlcNAc beta 1,4GlcNAc-Asn as substrate and, as enzyme sources, microsomal membranes of the hepatic nodules, surrounding liver, regenerating liver, and age- and sex-matched control liver. The nodules had significant N-acetylglucosaminyltransferase III activity (0.78-2.18 nmol GlcNAc transferred/h/mg of protein), while the surrounding liver, the regenerating liver (24 h after partial hepatectomy), and the control liver had negligible activity (0.02-0.03 nmol/h/mg of protein). Product isolated from a large scale N-acetylglucosaminyltransferase III incubation with hepatic nodules as enzyme source showed the presence of the bisecting GlcNAc residue by 500 MHz proton NMR spectroscopy. Concomitant with the appearance of N-acetylglucosaminyltransferase III activity in the preneoplastic nodules, the activities of N-acetylglucosaminyltransferase I and II were decreased in these membranes when compared to those from surrounding liver, regenerating liver, and control liver. These results suggest that N-acetylglucosaminyltransferase III is induced at the preneoplastic stage in liver carcinogenesis promoted by orotic acid and are consistent with the reported presence of bisecting GlcNAc residues in the Asn-linked oligosaccharides of rat and human hepatoma gamma-glutamyl transpeptidase and their absence in enzyme from normal liver of rats and humans (Kobata, A., and Yamashita, K. (1984) Pure Appl. Chem. 56, 821-832).