Effect of a single treatment with the alkylating carcinogens dimethylnitrosamine, diethylnitrosamine and methyl methanesulphonate, on liver regenerating after partial hepatectomy. II. Alkylation of DNA and inhibition of DNA replication.

Experiments were carried out to determine whether replication of alkylated DNA could be involved in the initiation of hepatocellular carcinoma which results from a single administration of dimethylnitrosamine (DMN) given after partial hepatectomy. The incidence of tumours is higher when DMN is given during the wave of DNA synthesis induced by the operation than when given in the early prereplicative stage. Therefore the alkylation of DNA in the regenerating liver by DMN given at these times and the effect of DMN on DNA synthesis were investigated. The extent, duration and pattern of alkylation of DNA, including the formation of 0-6-methylguanine, were similar whether DMN was given in the early pre-replicative stage (6 h after the operation) or during the period of DNA synthesis (at 24 h). DMN given a 6 h very greatly reduced the wave of DNA replication which would otherwise have ensued. When given at 24 h, by which time DNA synthesis was already taking place, DMN reduced the rate of incorporation of (-3H)thymidine after 1-2 h delay. However, in neither case was DNA synthesis reduced to the level occurring in normal intact liver. Treatment with diethylnitrosamine (DEN) at 6 h or at 24 h had a similar effect to DMN on the wave of DNA replication induced by partial hepatectomy. Methyl methanesulphonate (MMS given in the early pre-replicative stage delayed the wave of DNA synthesis by about 8 h, but when it did take place the extent of synthesis was as great as in untreated animals. When given during the period of DNA replication, MMS rapidly reduced the rate of synthesis. As in the case of the nitrosamines, synthesis was not reduced to the level occuring in normal intact animals. The difference from the nitrosamines lies in the nature of the alkylated bases formed in DNA. The fact that a single treatment with DMN induces cancer in partially hepatectomised animals but not in intact adult animals is not considered to be due to a gross difference in the nature of the alkylation of DNA. The experiments described support the concept that replication of DNA containing bases which are likely to mispair during replication may be necessary to 'fix' the lesion and thus cause a permanent inheritable change in the genetic material.     

Effect of a single treatment with the alkylating carcinogens dimethylnitrosamine and methyl methanesulphonate on liver regenerating after partial hepatectomy. III. Effect on DNA synthesis in vivo and on DNA polymerase activity assayed in vitro.

A single treatment with dimethylnitrosamine (DMN) but not with methyl methanesulphonate (MMS) induces liver cell carcinoma if given during the period of restorative hyperplasia following partial hepatectomy, a higher incidence of tumours being induced if the carcinogen is given during the period of DNA synthesis (24 h after the operation) than if given early in the prereplicative stage (at 6 h). To study the effect of treatment with DMN and with MMS on the regenerating liver, DNA replication was measured in vivo in partially hepatectomised animals treated with the methylating agents, and DNA polymerase activity was assayed in vitro.     

Effect of a single treatment with the alkylating carcinogens dimethylnitrosamine and methyl methanesulphonate on liver regenerating after partial hepatectomy. IV. Effect on methylase-mediated methylation of DNA.

The possibility that carcinogens may affect methylase-mediated methylation of replicating DNA was investigated. A system eminently suitable for this purpose is liver regenerating after partial hepatectomy, as one injection of dimethylnitrosamine (DMN) given during the ensuing period of increased DNA synthesis induces hepatocellular carcinoma. Methylation of DNA by DNA methylase normally occurs only in proportion to DNA synthesis. Therefore simultaneous measurements were made of synthesis (incorporation of [14C]adenine into DNA adenine, or of d[5-3H]cytidine into DNA cytosine), and of methylation (incorporation of [methyl-3H]methionine into 5-methylcytosine of DNA) in liver regenerating after partial hepatectomy. After treatment with DMN, the ratio of methylation: synthesis remained within the normal range. Methyl methanesulphonate (MMS), a compound which damages DNA in regenerating liver in a similar but not identical way to DMN and which does not induce tumors in liver even when given after partial hepatectomy, caused an increase in methylation in relation to synthesis. These experiments therefore do not support the view that altered DNA methylase activity is involved in carcinogenesis.     

Effect of treatment in vivo with N,N-dimethylnitrosamine or methyl methanesulphonate on the cytoplasmic DNA polymerase of regenerating rat liver.

A 10-16 fold increase in rat liver cytoplasmic DNA polymerase (DNA polymerase-alpha) was observed by 24 hrs after two thirds partial hepatectomy. Injection of either N,N-dimethylnitrosamine (DMN) or methyl methanesulphonate (MMS) At 6 or 12 hrs after partial hepatectomy completely inhibited this increased production of polymerase, but when given at 20 hours they had less effect. Neither compound altered the molecular size distribution of the enzyme. These data indicate that the lowered levels of DNA polymerase-alpha could play a major role in the reduction in DNA synthesis which occurs after carcinogen treatment.     

Effect of triiodothyronine or etiroxate on DNA synthesis in intact and regenerating liver

An increase in liver DNA synthesis (p less than 0.01) was found in rats with an intact liver 24 h after the administration of a single dose of triiodothyronine (200 micrograms/kg i.g.) Statistically significant stimulation of DNA synthesis was also found in rats given triiodothyronine (p less than 0.01) or etiroxate (p less than 0.05) for 3 days at 24-hour intervals. When a single dose of triiodothyronine was administered immediately after partial hepatectomy (65-70% resection of the liver), increased stimulation of DNA synthesis (p less than 0.01) was found 24 h after the operation. Etiroxate partly inhibited DNA synthesis (p less than 0.05). In rats given triiodothyronine at 24-h intervals, starting at the time of partial hepatectomy, DNA synthesis 72 h after the operation was double the value in the control group. Marked stimulation of DNA synthesis by triiodothyronine (p less than 0.01) and an increase in the total DNA content of the liver (p less than 0.05) were likewise found 48 h after partial hepatectomy if the hormone was administered once, 24 h after the operation. The increase in the two indicators after the administration of etiroxate was not statistically significant.     

DNA synthesis, mitotic index, drug-metabolising systems and cytogenetic analysis in regenerating rat liver. Comparison with bone marrow test after 'in vivo' treatment with cyclophosphamide

Rat-liver cells can be used to reveal "in vivo" clastogenic activity of indirect mutagens, provided that they are stimulated to divide by partial hepatectomy. In order to characterize the rat-liver metabolic capacity in such experimental conditions, several biochemical parameters were measured during the first 54-66 h of liver regeneration in Sprague-Dawley male rats, subjected to a partial hepatectomy. The levels of cytochrome P-450, the activities of styrene monooxygenase, epoxide hydrolase and glutathione-S-epoxide transferase were chosen as markers. All the enzymatic activities and the level of cytochrome P-450 decreased during the first 12 h after the hepatectomy to about 50% of the activities of the sham-operated rats considered as controls. Subsequent recovery of the metabolic capacity was not observed. DNA synthesis and the mitotic index were measured to find the most suitable time for metaphase analysis. DNA synthesis and the number of metaphases were maximal at, respectively, 22-25 and 28-31 h after partial removal of the liver. The sensitivity to clastogenic damage induced by "in vivo" treatment with cyclophosphamide (CPA) was assayed in regenerating liver cells by chromosome-aberration analysis. Different doses, ranging from 5 to 30 mg/kg b.w., were given i.p. to the rats 17 h before or 7 h after partial hepatectomy. Liver cells were collected 31 h after surgery. Clastogenic damage was greater when the drug was administered to the animals after the hepatectomy (24 h of exposure) than before (48 h of exposure). The sensitivity to CPA-induced damage was compared with a bone marrow cell test carried out on non-hepatectomized rats treated in the same way. The results indicated that in these conditions regenerating liver cells are more sensitive than bone marrow cells to the induction of chromosome aberrations by CPA.     

Effect of the parenteral administration of energy substrates in different postoperation phases on the initiation and development of liver regeneration in rats subjected to partial hepatectomy

DNA specific activity in the liver, the total DNA content of the liver and the mitotic index of the hepatocytes were studied after the infusion of glucose or lipid emulsions in female laboratory rats with a mean pre-operation weight of 250 +/- 30 g after partial (65-70%) hepatectomy (PH). The infusions were administered in the early prereplication phase (the 1st to 6th hour after the operation), in the late prereplication phase (the 7th to 12th hour after the operation), or continuously from the 1st to the 12th, or the 1st to the 24th, hour after partial hepatectomy. The effect of these parenterally administered energy substrates on the initiation of liver regeneration was evaluated 18 and 24 hours after partial hepatectomy. The results indicate that the infusion of glucose, in any interval after the operation, inhibited the initial phases of liver DNA synthesis (18 h after PH), but not its further development (24 h after PH). Neither the mitotic index of the hepatocytes, nor the total DNA content of the liver differed from the control groups in the case of rats given a glucose infusion. In the experimental groups given lipid emulsions, inhibition of liver DNA synthesis was recorded 18 h after PH only when the infusions were given from the 1st to the 12th or the 1st to the 18th hour after PH. The total DNA content of the liver 18 h after PH was raised in all the experimental groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of realimentation after several days' isolated glucose or fructose intake on DNA synthesis in rat liver.

Marked stimulation of liver DNA synthesis was observed in rats given an isolated glucose or fructose diet for 4 days and theen refed one day on diets with different protein contents. The strongest stimulatn effect was found in rats refed, after an isolated glucose intake, with a high protein diet (81 cal%). The stimulant effect of refeeding on liver DNA synthesis was far less pronounced in rats subjected to several days' starvation before realimentation than in rats given a carbohydrate diet. The stimulant effect of realimentation after an isolated glucose intake was distinctly enhanced if triiodothyronine (50 microgram/100 g b.w., i.g.) was administered just before the change to a high protein diet. The increase in liver DNA synthesis in rats fed three days on fructose before undergoing partial hepatectomy was the same as in the controls. In rats given glucose prior to partial hepatectomy, the post-operative increase in DNA synthesis was partly inhibited.     

Effect of an inhibiting factor isolated from rat liver on DNA polymerases in regenerating rat liver.

We partially purified an inhibitory factor (LIFE), isolated from 105,000 g supernatant of a saline adult rat liver homogenate. LIF stopped in vitro cell multiplication by blocking the G1--S transition, and reduced in vivo [3H]thymidine incorporation into liver DNA in two-thirds hepatectomized rats. This reduction in DNA synthesis was observed at 24 hr after hepatectomy, even when the LIF was injected before the beginning of the S phase, 10 hr after hepatectomy, i.e. when DNA polymerase activity had not yet increased. Under these experimental conditions, LIF in vivo treatment prevented alpha DNA polymerase activity from increasing after partial hepatectomy, so that enzyme activity at 24 hr in LIF-treated rats decreased compared to the controls. No direct inhibitory effect of LIF on alpha DNA polymerase was detected. LIF did not affect beta DNA polymerase. These results suggest that LIF plays a part in controlling liver growth.     

The induction of chromosomal damage in rat hepatocytes and lymphocytes I. Time-dependent changes of the clastogenic effects of diethylnitrosamine,dimethylnitrosamine and ethyl methanesulfonate

Male Wistar rats received a single injection of diethylnitrosamine (DEN), dimethylnitrosamine (DMN) or ethyl methanesulfonate (EMS). After a number of time intervals (up to 56 days) liver cells were assayed for the presence of possible preclastogenic damage by performing partial hepatectomy and subsequent analysis of chromosomal damage (micronucleus formation) in isolated hepatocytes. Peripheral blood lymphocytes from the same animals were collected, stimulated to proliferate and assayed for the frequency of sister-chromatid exchanges (SCEs). Whereas all agents significantly increased frequencies of SCEs in lymphocytes up to at least 28 days (EMS) or 56 days (DMN, DEN) after injection, only the latter 2 compounds gave rise to significantly increased incidences of micronucleated hepatocytes. DMN-induced preclastogenic damage in hepatocytes was lost between 28 and 56 days after injection. After DEN, this type of damage was persistent over the entire experimental period (56 days).When rats treated with DEN did not undergo partial hepatectomy, the frequencies of micronuclei at different time intervals after treatment were at control level. This result, together with those from hepatectomized DEN-treated rats, suggests that it is the persistent character of the preclastogenic damage that is responsible for the occurrence of micronucleated hepatocytes at later time intervals after treatment with DEN, rather than the stability of micronuclei which might eventually have been formed soon after injection.     

Different effects of 1,10-phenanthroline on chronic liver injury induced by dimethylnitrosamine or ethionine in rats

Although 1, 10-phenanthroline (1, 10-P, 2 mg/100 g) prevents acute liver injury induced by dimethylnitrosamine (DMN), it does not protect female rats against liver damage caused by chronic treatment with DMN (2 mg/ 100 g). Liver damage was ascertained by measuring distribution and total activity of β-glucuronidase, rate of collagen synthesis, total collagen content of the liver, and amount of isocitric dehydrogenase (ICDH) in the serum. However, after simultaneous treatment with 1, 10-P and DMN for three weeks, the total amount of noncollagenous liver proteins and of microsomal protein and aniline hydroxylase activity were higher than in livers of rats receiving DMN alone. The proliferation of the smooth endoplasmic reticulum in livers of dogs treated for a 14-week period with 1, 10-P was demonstrated by ultrastructural techniques. Chronic liver injury induced by feeding female rats with a 0.3% d, 1-ethionine diet for five weeks was prevented by simultaneous administration of 1, 10-P (2 mg/100 g, i.p. 3 times weekly). It is suggested that when administered chronically, 1, 10-P acts as an inducer of the liver microsomal system and therefore increases the activity of liver mixed-function oxidases. This explains why chronic administration of 1, 10-P does not protect rats against injury caused by DMN. Ethionine hepatotoxicity, which does not seem to be related to the microsomal activity, is substantially decreased by as yet unknown mechanisms.     

Specific inhibition of the synthesis of putrescine and spermidine by 1,3-diaminopropane in rat liver in vivo.

Chronic administration of 1,3-diaminopropane, a compound inhibiting mammalian ornithine decarboxylase (EC 4.1.1.17) in vivo, effectively prevented the large increases in the concentration of putrescine that normally occur during rat liver regeneration. Furthermore, repeated injections of diaminopropane depressed by more than 85% ornithine decarboxylase activity in rat kidney. Administration of diaminopropane 60 min before partial hepatectomy only marginally inhibited ornithine decarboxylase activity at 4 h after the operation. However, when the compound was given at the time of the operation (4 h before death), or any time thereafter, it virtually abolished the enhancement in ornithine decarboxylase activity in regenerating rat liver remnant. An injection of diaminopropane given 30 to 60 min after operation, but not earlier or later, depressed S-adenosyl-L-methionine decarboxylase activity (EC 4.1.1.50) 4 h after partial hepatectomy. Diaminopropane likewise inhibited ornithine decarboxylase activity during later periods of liver regeneration. In contrast to early regeneration, a total inhibition of the enzyme activity was only achieved when the injection was given not earlier than 2 to 3 h before the death of the animals. Diaminopropane also exerted an acute inhibitory effect on adenosylmethionine decarboxylase activity in 28-h regenerating liver whereas it invariably enhanced the activity of tyrosine aminotransferase (EC 2.6.1.5), used as a standard enzyme of short half-life. Treatment of the rats with diaminopropane entirely abolished the stimulation of spermidien synthesis in vivo from [14C]methionine 4 h after partial hepatectomy or after administration of porcine growth hormone. Both partial hepatectomy and the treatment with growth hormone produced a clear stimulation of hepatic RNA synthesis, the extent of which was not altered by injections of diaminopropane in doses sufficient to prevent any enhancement of ornithine decarboxylase activity and spermidine synthesis.     

Shortening of life span caused by administration of 5-bromodeoxyuridine to neonatal rats

Newborn rats and adult animals in which liver cell replication had been induced by partial hepatectomy were injected with 5-bromodeoxyuridine (BUdR), and were kept without further treatment for the remainder of their life span. No evidence for carcinogenicity of BUdR was obtained, but a dose-dependent shortening of the life span was observed after administration to neonatal animals.     

Effect of chemical carcinogens and partial hepatectomy on "in vivo" (35S)methionine interaction with rat liver tRNA

The effect of carcinogens given by a single or multiple injections on the extent of (35S)methionine interaction with hepatic tRNA was studied in normal and partially hepatectomized rats. Either partial hepatectomy or administration of ethionine (100 or 330 mg/kg body weight) and dimethylnitrosamine (120 mg/kg body weight) by multiple i.p. injections inhibited the (35S)methionine-tRNA interaction, while administration of hepatocarcinogenic chemicals plus PH resulted rather in a stimulation. Methylnitrosourea enhanced the extent of interaction when administered in a single dose (100 mg per kg body weight) 18 h after partial hepatectomy.     

Glucosamine metabolism in regenerating rat liver.

1. Glycoprotein synthesis was investigated with [1-14C]glucosamine in vivo. [14C]Glucosamine was administered intravenously 24h after hepatectomy to rats. 2. Incorporation into the acid-soluble fraction was maximum at 15 min after injection both in sham-operated and hepatectomized rats. 3. Enhancement of incorporation into UDP-N-acetylhexosamine in regenerating liver was observed. However, its specific activity was lower, because of a greater enhancement of synthesis de novo of the amino sugar. 4. In the liver acid-insoluble fraction, maximum incorporation of [14C]glucosamine was at 30 min in sham-operated rats and 2 h in hepatectomized rats respectively. 5. In sham-operated rats, incorporation into the plasma acid-insoluble fraction followed that of the liver acid-insoluble fraction, but hepatectomy resulted in a rapid enchancement of incorporation into plasma. 6. It is concluded that synthesis of liver glycoproteins is stimulated after partial hepatectomy and that glycoproteins synthesized are released rapidly into the plasma.     

Chronic or acute administration of various dialkylnitrosamines enhances the removal of O6-methylguanine from rat liver DNA in vivo

The effect of pretreatment of rats with various symmetrical dialkylnitrosamines on the repair of O⁶-methylguanine produced in liver DNA by a low dose of [¹⁴C]dimethylnitrosamine (DMN) has been examined. DMN, diethylnitrosamine (DEN), dipropylnitrosamine (DPN) or dibutylnitrosamine (DBN) were administered to rats for 14 consecutive weekdays at a daily dose of 5% of the LD₅₀. Animals were given [¹⁴C]DMN 24 h after the last dose and were killed 6 h later. DNA was extracted from the liver and analysed for methylpurine content after mild acid hydrolysis and Sephadex G-10 chromatography. While the amounts of 3-methyladenine and 7-methylguanine were only slightly different from controls, the amounts of O⁶-methylguanine in the DNA of the dialkylnitrosamine pretreated rats were about 30% of those in control rats, indicating a considerable increase in the capacity to repair this base. Liver ribosomal RNA from control and dialkylnitrosamine pretreated rats contained closely similar amounts of O⁶-methylguanine suggesting that the induced enzyme system does not act on this base in ribosomal RNA in vivo. Pretreatment with these dialkylnitrosamines also enhanced the repair of O⁶-methylguanine in liver DNA when they were given as a single dose (50% of the LD₅₀) either 3 or 7 days before the [¹⁴C]DMN. In addition, single low doses of DMN or DEN (5% of the LD₅₀) given either 1 or 6 days before [¹⁴C]DMN increased O⁶-methylguanine repair and the magnitude of the effect after DEN was similar to that produced by the other pretreatment schedules. The possible mechanism(s) of the induction of O⁶-methylguanine repair and its relation to hepatotoxicity, DNA alkylation, carcinogenesis and the adaptive response in Escherichia coli are discussed.     

Course of liver regeneration after partial hepatectomy in rats treated with dialysates of intact organs

A number of growth phenomena observed in vitro have shown that cells, at high densities, produce and release substances which, when they have reached a given concentration, arrest further growth. In vivo, these possibilities can be studied on the model of rapid regeneration of the rat liver after 65-70% partial hepatectomy (PH). We evaluated the course of liver regeneration after PH in animals treated with dialysates (DIA) of intact rat tissues. In addition to kidney and lymph node DIA, we were particularly interested in the effect of liver and spleen DIA. The experiments were carried out on male rats weighing 210-240 g. The relevant DIA was administered 24 h prior to PH; the controls were given physiological saline. The animals were killed just before PH and 24, 48, 30 and 72 h and 14 days after. DIA obtained from intact liver tissue inhibited the regeneration process induced by PH and its effect persisted 48 h after PH. Compared with the controls and with the rats given kidney DIA, DNA synthesis in the liver 24 h after PH was reduced to 77%. After spleen DIA, several (still hypothetical) factors probably acted together synergically (factors belonging to the immune system--RES--and spleen-produced factors capable of promoting proliferation of the hepatocytes--the "portal blood factor"). We arrived at this conclusion from an evaluation of liver DNA synthesis 24 h after 24h after PH, when synthesis was altogether markedly raised, but attained far higher values after the administration of spleen DIA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nonexistence of N-acetylseryl-tRNA in rat liver.

Reports of the existence of N-acetylseryl-tRNA in rat liver were reinvestigated. Methods were developed to permit recovery of N-acetylserine in a yield of 30--40% from N-acetylseryl-tRNA added to liver homogenates and cell-free incubations. [14C]Serine and [3H]acetate were injected into rats pretreated with iron and into rats after partial hepatectomy, and aminoacyl-tRNA was isolated from their livers. The amount of radioactivity associated with N-acetylserine in the amino acids released by hydrolysis from the aminoacyl-tRNA was negligible. No formation of N-acetylseryl-tRNA could be observed in incubations of acetyl CoA and seryl-tRNA or tRNA with enzyme fractions from liver of rats pretreated with iron. It is concluded that previous reports of the existence of N-acetylseryl-tRNA in rat liver are erroneous.     

Regenerative ability of hepatocytes is inhibited in early stages of liver fibrosis.

Female Wistar rats were given three doses of carbon tetrachloride, 10.4 mmol/kg of body weight. The doses were administered within 16 days and another 16 days were allowed to pass before partial (37%) hepatectomy was done. The liver showed very mild fibrosis at that time. DNA synthesis (measured by 3H-thymidine incorporation) was decreased by 53% and mitotic activity of hepatocytes was decreased by 56% when compared to olive oil-pretreated partially hepatectomized controls. The results show that the mitotic potential of hepatocytes in early stages of liver fibrosis is impaired which may influence the course of the disease.