Change in intracellular pH of rat liver during azo-dye carcinogenesis.

The change in intracellular pH of rat liver during 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) feeding was examined, contrasting with that during 2-methyl-4- dimethylaminoazobenzene (2-Me-DAB) feeding. Intracellular pH of liver was measured by the DMO method.The intracellular pH decreased markedly until the 5th week after the beginning of 3'-Me-DAB feeding, and then somewhat recovered. After 11 weeks, however, it decreased rapidly again with a lower point in the 15th week. When rats were returned to a basal diet after the dye had been fed for various periods, the pH value returned to the normal range. No significant change in rat liver pH was found during 2-Me-DAB feeding. Although it is not obvious what causes the decrease in intracellular pH of rat liver fed on the 3'-Me-DAB diet, or what role it plays in hepatocarcinogenesis, this alteration in cellular environment seems to be associated with biochemical changes accompanied by carcinogenesis.     

Enhancement of aflatoxin B1-induced hepatocarcinogenesis in rats by partial hepatectomy

The effect of enhanced cell replication induced by partial hepatectomy (PH) in aflatoxin B1 (AFB1)-induced hepatocarcinogenesis has been studied in rats of the inbred As2 strain. Animals were given 0.25 mg/kg body weight of AFB1 as a single intraperitoneal dose 24 h after PH. Non-hepatectomized animals given the same dose of AFB1 served as controls. Neoplastic nodules and hepatocellular carcinoma (HCC) were detected respectively in 100% and 90% of hepatectomized animals sacrificed between 55 and 65 weeks after AFB1 administration. None of the ten non-hepatectomized rats sacrificed at this time interval showed HCC or neoplastic nodules. On histochemical staining the tumour population was found to be heterogeneous. Thus PH resulted in enhancement of AFB1-induced hepatocarcinogenesis in rats of the AS2 strain.     

Control of catabolism of mitochondria in oncogenesis. I. Increase of hepatic mitochondrial population during feeding inactive or marginally active amino azo dyes: absence of de novo biosynthesis

The mechanism of the well established phenomenon that the number of liver mitochondria increases during administration of 2-methyl-4-dimethylaminoazobenzene (2-Me-DAB) has been investigated. Fed to rats, both 2-Me-DAB (0.06%) and 4-diethylaminoazobenzene (4-DEAB) (0.0635 %) increase the amount of liver mitochondria by 47% and 31%, respectively. It was established that this is not due to de novo mitochondriogenesis. The increase in the amount of mitochondria correlates with an ~ 10% decrease of total liver protein per g of tissue. Mitochondrial ATP synthesis, which is a prerequisite of any anabolic situation, is drastically impaired following feeding of 2-Me-DAB beyond 1 week as indicated by a very substantial decrease of State 3 respiration, the respiratory control index, and the $\text{ADP}\text{O}$ ratio. Determination of the polysome profile and polysome/monosome ratio at intervals during 2-Me-DAB administration showed no change, despite the fact that mitochondrial components are coded for in both nuclear and mitochondrial DNA. During 4-DEAB administration there was, however, a small but definite rise of the polysome/monosome ratio. Administration of 2-Me-DAB up to 42 days brought about drastic inhibition of the incorporation of [³H]thymidine into both DNA's (approx. 59% with mitochondrial DNA and approx. 77% with nuclear DNA), indicating that these templates could not possibly be involved in the substantial increase of the mitochondrial population. The data suggest that the increase results from a steady accumulation due to increase of the half-life of mitochondria, owing possibly to an inhibition of lysosomal catabolic enzymes.     

Alterations in turnover of labelled precursors incorporated into rat liver nuclear macromolecules during azo dye feeding

Continual feeding of either 4-dimethylaminoazobenzene (DAB) or 2-methyl-4-dimethylaminoazobenzene (2-MeDAB) to rats resulted in an increase in the uptake but a decrease in the turnover of [³H]lysine in all the nuclear proteins of rat liver. The pattern of lysine turnover in acidic nuclear proteins from DAB-fed animals was more similar to that of normal acidic nuclear proteins than that from the 2-MeDAB-fed animals. The histone fractions showed an increase in uptake after dye feeding which was greater in the lysine-rich fractions than in the arginine-rich fractions. During DAB feeding both the uptake and rate of turnover of [³H]thymidine were greatly increased, but with the noncarcinogenic 2-MeDAB the uptake of the precursor was lower and the rate of turnover slower than in normal animals. These differences in metabolism in response to azo dye feeding are discussed in relation to azo dye carcinogenesis.     

Effect of saline solutions administered parenterally in different postoperation phases on the regeneration of rat liver after partial hepatectomy

Two series of experiments were carried out on female laboratory rats with a mean pre-operation weight of 250 +/- 30 g, fed up to the time of the experiment on a standard laboratory diet with water ad libitum. In the first series the rats were subjected to 65-70% partial hepatectomy (PH) or to laparotomy (LAP) and their serum Na+, K+, Cl- and total calcium concentrations were determined 6, 12, 18 and 24 h after the operation. At given postoperation intervals the serum Na+, Cl- and total calcium concentrations in hepatectomized animals were lower than in the intact controls, while the K+ concentration was higher. In the second series of experiments, the rats were given infusions of physiological saline or Ringer solution at different intervals (1-6, 7-12, 1-12 and 1-24 h) after PH. Specific DNA activity in the liver, the hepatocyte mitotic index, the total DNA content of the liver and other indicators show that physiological saline infusions had an inhibitory, or at most a neutral effect on the initiation of liver regeneration, while the effect of the infusion of Ringer solution on the initiation of liver regeneration, in most of the given intervals, was indifferent. The regeneration response depends on the post-PH phase in which the solution is infused.     

The effect of prolonged dietary administration of the non-carcinogen, 4-acetylaminofluorene on the liver of the rat. An electron microscope study

A diet containing 0.05 % of the non-carcinogen 4-acetylaminofluorene (4-AAF) was fed to male Leeds strain rats for periods of up to 10 months. Some animals were killed after 8–12 weeks, 6 months and 8–10 months of 4-AAF feeding, while further groups were returned to a normal diet after 10 months of treatment and then killed 2, 5, 9 and 12 months later. The hepatic tissues were removed and prepared for electron microscopy. The main fine structural changes induced by 4-AAF were a prominent hypertrophy of the agranular endoplasmic reticulum, glycogen depletion and lipid accumulation. It was noted that these changes persisted following withdrawal of dietary 4-AAF, for the duration of the experiment. The effects of 4-AAF are compared with those of its carcinogenic isomer, 2-acetylaminofluorene (2-AAF) and the possibility is discussed that the clear differences revealed in this study may be directly related to the relative carcinogenicities of these two compounds.     

Effect of dietary vitamin E and Santoquin on regenerating rat liver

The influence of dietary vitamin E and Santoquin on lipid peroxidation and liver regeneration in partially-hepatectomized rats was studied. Rats were fed either a basal 10% tocopherol-stripped corn oil diet, the basal diet plus 40 mg dl-alpha-tocopheryl acetate/kg, or the basal diet plus 2 g Santoquin (6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline)/kg. After 6 weeks, rats fed the antioxidant-deficient diet produced more of the lipid peroxidation product, pentane, than did the rats fed antioxidants. Partial hepatectomy was performed after six and one-half weeks or ten weeks of feeding the diets. At 3 and 6 days after surgery, pentane production was significantly elevated over pre-surgery levels in rats fed the antioxidant-deficient or vitamin E-supplemented diets, but not in rats fed the Santoquin-supplemented diet. Six days after surgery, there were fewer thiobarbituric acid reactants in regenerating liver of Santoquin-fed rats than of vitamin-E fed rats or antioxidant-deficient rats. There was no increase in the 6-day level of thiobarbituric acid reactants over the 3-day level in livers of rats fed Santoquin, while there was an increase in livers of the antioxidant-deficient and vitamin E-supplemented rats. Liver sulfhydryl levels were higher at 3 and 6 days post surgery in the Santoquin-fed rats than in the antioxidant-deficient or vitamin E-supplemented rats. Plasma gamma-glutamyl-transpeptidase activity was not different among the groups of rats. Between the third and sixth day following surgery, liver regeneration was significantly stimulated in Santoquin-fed, but not vitamin E-fed rats. After 11 days, a stimulatory, but not statistically significant, effect of vitamin E was found. Although DNA content of liver was higher at 6 days than at 3 days post surgery, it was not different among the dietary groups, indicating that cell proliferation rather than hypertrophy had occurred. Partial hepatectomy could have altered the ability of the liver to metabolize pentane, thus explaining part of the increased production of pentane. However, the results obtained support the interpretation that elevated levels of dietary antioxidants can be beneficial in terms of reduced lipid peroxidation and increased rates of liver regeneration following liver surgery.     

Changes in the proliferative activity of the corneal epithelium and the regenerating liver in partial splenectomy in mice

The mitotic activity in epithelial cells of the mouse cornea was studied 4 h, 1, 2, 5, 8 and 14 days after a sham operation or partial (2/3) splenectomy. The decrease in the number of dividing cells in the corneal epithelium was observed within two days after a sham operation and within five days after partial splenectomy. On the contrary, partial hepatectomy increased the number of mitoses in the corneal epithelium. Liver regeneration against the background of a sham operation or partial splenectomy was accompanied by a lesser number of mitoses (by a factor of 2.5-4) in hepatocytes than in the animals subjected to partial hepatectomy only.     

Effect of realimentation after several days' pure carbohydrate intake on DNA synthesis in regenerating rat liver

The authors studied the effect of realimentation after several days' isolated glucose or fructose intake on DNA synthesis in liver regenerating after partial hepatectomy (PH) (65-70%) or after carbon tetrachloride (CCl4) poisoning 1.5 ml/kg. Two days before PH or the administration of CCl4 and two days after, the experimental rats were given glucose (50% solution) of fructose (50% solution) as the only source of energy. Rats with PH were then fed for one day on a standard laboratory diet (25 cal% protein) or a high protein diet (81 cal% protein). Rats with CCl4 liver damage were fed for one day on the standard laboratory diet only. In the rats given glucose, liver DNA synthesis and the total amount of these nucleic acids in the liver 48 hours after CCl4 administration was lower than in the controls or the rats given fructose. In all the experimental groups (PH and CCl4), stimulation of liver DNA synthesis was observed after one day's realimentation. The total DNA content of the liver of rats with PH rose markedly during realimentation. The experiments indicate that the regenerative activity of damaged liver can be influenced by the nutritional regimen.     

Effect of the parenteral administration of amino acid solutions in different phases after partial hepatectomy on the initiation and development of liver regeneration in rats

Amino acid solutions enriched with branched-chain amino acids or pure branched-chain amino acid solutions were administered parenterally to female laboratory rats (pre-operative weight 230 +/- 30 g) which had been subjected to 65-70% partial hepatectomy (PH), and specific liver DNA activity, the hepatocyte mitotic index and other indicators of the initiation of liver regeneration were studied. Both solutions were infused in an hourly dose of 3.3 ml/kg body weight, during the following postoperative intervals: 1-6, 7-12, 1-12, 1-18 and 1-24 hours. The control rats continued to be fed on the standard laboratory diet after the operation. The results show that the infusion of an amino acid solution enriched with branched-chain amino acids had an inhibitory effect on the onset of DNA synthesis in the liver 18 hours after partial hepatectomy whatever the administration interval. The situation in the case of pure branched-chain amino acid solutions was the same. Twenty-four hours after PH, neither type of solution, irrespective of the infusion interval, was followed by an increase in DNA synthesis compared with the controls fed on the standard laboratory diet. Neither the hepatocyte mitotic index, nor the total liver DNA concentration, showed any changes indicative of stimulation of the initiation of liver regeneration. An infusion stress effect, evaluated from the decrease in the weight of the thymus, was found chiefly in the case of infusions lasting 12 h or longer.     

Vascular endothelial growth factor and nitric oxide in rat liver regeneration

In this work we investigated the role of nitric oxide (NO) in the angiogenesis mediated by vascular endothelial growth factor (VEGF) during rat liver regeneration after two-thirds partial hepatectomy. Sham operated (Sh) and partially hepatectomized (PH) male Wistar rats were randomized in three experimental groups: control (treated with vehicle); pre-treated with sodium nitroprusside (SNP: 0.25 mg/kg body weight, i.v. at a rate of 1 ml/h) and pre-treated with the preferential iNOS inhibitor, aminoguanidine (AG, 100 mg/kg body weight, i.p.). Animals were killed at 5, 24 and 72 h after surgery. At 5 h post-surgery, NO production was estimated by EPR (Sh-Control: 37.65+/-10.70; PH-Control: 88.13+/-1.60(); Sh-SNP: 90.35+/-3.11(); PH-SNP: 119.5+/-12.10()(#); Sh-AG: 33.27+/-5.23, PH-AG: 36.80+/-3.40(#)) (p<0.05 vs Sh-Control; (#)p<0.05 vs PH-Control). At 24 h after PH, VEGF levels showed no difference between PH-Control and PH-SNP animals. However, after 72 h, VEGF protein levels in PH-SNP animals were found to be increased (above 300%) with respect to PH-Control. On the other hand, aminoguanidine (AG) pre-treatment blocked the rise of inhibition of NO generation and decreased VEGF expression. Our results demonstrated that NO plays a role in modulating VEGF protein expression after hepatectomy in rats.     

Effect of propylthiouracil on liver regeneration in rats after partial hepatectomy.

The effect of propylthiouracil (PTU) on the growth activity of intact liver and liver regenerating after partial (65-70%) hepatectomy (PH) was studied in rats. PTU (Propycil, Kali-Chemie, FRG) was dissolved in drinking water (1 g PTU per litre) and this was given to the rats, as their sole source of fluids, three days before PH and then up to the end of the experiment. In rats given PTU, marked inhibition of liver DNA synthesis and the mitotic activity of hepatocytes was found after PH. This effect was potentiated to some extent by partial inanition of the experimental animals given PTU, as demonstrated in a paired feeding test in control rats. PTU inhibition of DNA synthesis in intact and regenerating liver also took effect in thyroidectomized rats, even with substitution (thyroid hormone) therapy. The experiments demonstrated that the effect of propylthiouracil on DNA synthesis in the liver is mediated primarily by way of its direct effect on the liver.     

Albumin synthesis and catabolism following partial hepatectomy in the rat. The effects of amino acids and adrenocortical steroids on albumin synthesis after partial hepatectomy.

Plasma albumin levels were measured in partially hepatectomized, sham operated and control rats. The levels fell in both the partially hepatectomized and sham operated groups; while the latter group returned to normal within a few days, the low plasma albumin in the partially hepatectomized animals was sustained. Albumin synthesis rates in the isolated perfused rat liver were measured in the three groups of animals at varying intervals after partial hepatectomy. There was a significant depression of albumin synthesis rate in terms of both liver and whole animal weights when compared to the sham operated and control animals. This depression was almost completely reversed by the addition of arginine, asparagine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan and valine added together to 10 times their normal plasma concentrations. The addition of hydrocortisone had no effect on the albumin synthesis rate after partial hepatectomy. Studies in vivo in the three groups of animals (partially hepatectomized, sham operated and control animals) revealed a fall in the albumin catabolic rate after partial hepatectomy coinciding with the fall in the albumin synthesis rate. An hypothesis whereby the amino acids may have their stimulatory effect is proposed.     

Relationship between parathyroid hormone and adenosine 3′,5′-monophosphate metabolism in the kidney of vitamin D-deficient rats

The effect of vitamin D administration on cyclic AMP metabolism in the kidney was examined in rats fed a vitamin D-deficient, low Ca diet. The renal cyclic AMP level in vitamin D-deficient rats was higher than that in normal rats fed a laboratory chow, and in significantly decreased after thyroparathyroidectomy. Parathyroid hormone administered in vitro and in vivo did not cause as great a cyclic AMP response in vitamin D-deficient rats as that seen in the normal rats. The response to calcitonin, however, was not blunted in vitamin D-deficient animals. The blunted cyclic AMP accumulation in the kidney seemed to be related to formation, rather than degradation, of the nucleotide. The rats fed the low Ca diet were still hypocalcemic even after supplementation of the diet with a daily dose of either 0.625 μg of vitamin D-3 for 3 weeks or 2.5 μg of vitamin D-3 for the last 3 days. Vitamin D supplementation did not influence either the basal level or parathyroid hormone-stimulated increase of cyclic AMP in the kidney. On the contrary, when animals maintained on the vitamin D-deficient, low Ca diet were switched to the vitamin D-deficient, high Ca diet containing lactose for several days, they recovered normocalcemia and a normal response. These results suggest that the blunted cyclic AMP response to parathyroid hormone in vitamin D deficiency is due to hypocalcemia or associated secondary hyperparathyroidism and not due to deficiency of vitamin D action.     

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.     

The effect of diet and 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane (DDT) on microsomal hydroxylating enzymes and on sensitivity of rats to carbon tetrachloride poisoning

1. Protein-depleted rats are resistant to the lethal effects of carbon tetrachloride. The LD₅₀ is 6·4ml./kg. in stock rats and 14·7ml./kg. in rats fed on protein-free diets. 2. Protein-depleted rats are resistant to carbon tetrachloride in its effect on the liver as judged by histology, accumulation of liver water, and plasma enzyme and bilirubin measurement. 3. The protection is present after feeding rats on a no-protein diet for 4 days. It is present after feeding rats on a 3%-casein diet, and partly found after feeding rats on a 6%-casein diet. 4. The activities of the microsomal enzymes that demethylate Pyramidon and hydroxylate benzopyrene in the liver fall by over 80% in rats fed on the no-protein diet for 4 days or more, or in rats fed on a 3%-casein diet. A 50% fall is found in rats fed on a 6%-casein diet. 5. A single dose of DDT or three doses of phenobarbitone cause increased microsomal enzyme activity in protein-depleted rats. 6. The animals are then sensitive to the lethal and liver-damaging effects of carbon tetrachloride. 7. DDT dosage also leads to increased sensitivity to carbon tetrachloride in rats fed on stock diets. 8. These findings support the hypothesis that carbon tetrachloride is metabolized by microsomal enzymes to form the true toxic compound.     

Protective effects of Ginkgo biloba against rat liver carcinogenesis

Ginkgo biloba (EGb) has been proposed as a promising candidate for cancer chemoprevention and has shown protective effects on the liver against chemically induced oxidative injury and fibrosis. The potential beneficial effects of EGb were investigated in two rat liver carcinogenesis bioassays induced by diethylnitrosamine (DEN). In a short-term study for anti-initiating screening, male Wistar rats were fed a basal diet or supplemented diet with 500 or 1000 ppm EGb and initiated 14 days later with a single dose of DEN (100 mg/kg i.p.). The respective groups were killed 24h or 2 weeks after DEN-initiation. Liver samples were collected for the analysis of proliferating cell nuclear antigen (PCNA), transforming growth factor alpha (TGF-alpha), p53, apoptosis and induction of single hepatocytes and minifoci positive for the enzyme glutathione S-transferase P-form (GST-P). In a medium-term study for anti-promoting screening, the animals received a single dose of DEN (200 mg/kg i.p.) and, 2 weeks later, were fed a basal diet or supplemented diet with 500 or 1000 ppm EGb for 6 weeks. All animals underwent 70% partial hepatectomy (PH) at week 3 and killed at week 8. Liver samples were collected to analyze development of preneoplastic foci of altered hepatocytes (FAH) expressing GST-P. In the short-term study, pretreatment of rats with 1000 ppm EGb significantly reduced the rates of cell proliferation, apoptosis and p53, TGF-alpha immunoreactivity and the number of GST-P-positive hepatocytes. In the medium-term study, EGb treatment during the post-initiation stage failed to reduce the development of DEN-induced GST-P-positive foci. Thus, EGb presented inhibitory actions during initiation but not promotion of rat liver carcinogenesis induced by DEN.     

Regulation of hepatic lipogenesis in starved and diabetic animals by thyroid hormone

The effects of intragastric feeding with glucose and of the administration of L-triiodothyronine (T₃) on in vivo rates of hepatic lipogenesis were investigated in control (fed ad libitum on norrnal diet), diabetic (fed ad libitum on normal diet), fat-fed (fed ad libitum on high-fat diet), and starved (food removed for 48 h) rats. Two days of T₃ treatment increased hepatic lipogenesis in control and fat-fed animals but not in the diabetic or starved animals, although increases in lipogenesis in diabetic animals were observed after 4 days of T₃ treatment. Intragastric glucose feeding increased hepatic lipogenesis in the livers of control animals and T₃-treated control animals. Such increases are mediated by an increase in the circulating insulin concentration, as increases are not observed in diabetic rats or T₃-treated diabetic rats. Glucose feeding failed to increase hepatic lipogenesis in fat-fed rats or starved rats. Insulin injection together with glucose feeding increased lipogenesis in the fat-fed group but not the starved group; i.e., impaired insulin secretion following an oral glucose load may in part explain the lack of response in the fat-fed but not the starved animals. Marked increases in hepatic ]ipogenesis after glucose feeding were, however, observed if either the starved or the fat-fed animals were treated with T₃, The physiological implications of these observations are discussed.