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.     

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

Preneoplastic 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 and gamma-glutamyltranspeptidase-positive. Immunohistochemical detection was possible using rabbit anti-UDP-GT and peroxidase-labeled swine anti-rabbit immunoglobulins. The results of immunohistochemistry were substantiated by enzyme determination in microdissected 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.     

Preneoplastic lesions in rodent kidney induced spontaneously or by non-genotoxic agents: predictive nature and comparison to lesions induced by genotoxic carcinogens

The current literature on non-genotoxic renal carcinogens and the associated neoplastic and preneoplastic lesions has been reviewed in order to determine their occurrence and predictive nature with regard to tumor formation. In addition the mechanisms involved in the genesis of renal tumors are discussed. A more generalized classification of preneoplastic and neoplastic renal lesions was introduced, based on studies conducted with genotoxic and non-genotoxic renal carcinogens. Reports on preneoplastic lesions were found in the literature for control animals as well as animals treated with non-genotoxic carcinogens. Due to the paucity of data regarding preneoplastic lesions in control animals and animals treated with non-genotoxic carcinogens, new data were also generated by rereading kidney slides of control animals of a randomly selected NTP study and kidney slides of male rats treated with the highest dose of ochratoxin A, one of the most potent non-genotoxic renal carcinogens known. The control slides and the slides from the ochratoxin A study indicated that the cytologic and morphologic types of preneoplastic lesions characteristically observed in bioassays using genotoxic carcinogens are also present in control animals and animals treated with non-genotoxic carcinogens. The incidence of preneoplastic lesions was low in control animals and higher in animals treated with non-genotoxic carcinogens. The diverse classifications used in the literature did not allow a direct comparison of lesions and corresponding incidences with those of the newly generated data. However, three major tendencies were observed: (a) whenever a high incidence of preneoplastic lesions was reported, renal neoplasms were also found, (b) the larger the size and the further a lesion had progressed, the higher was the probability of tumor formation, and (c) not all preneoplastic lesions are irreversible, but reversibility seemed to decrease with increasing lesion size and progression. It must be emphasized that the data available for these conclusions are limited. This is not due to the lack of adequate numbers of bioassays with non-genotoxic carcinogens, but rather to the lack of consistent reporting of data. A generalized and more widely used classification which incorporates early lesions would certainly improve the current data base on renal lesions and provide future improvements in the predictive nature of these lesions.     

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).     

Modulation of calcium by the carcinogenic process in the liver induced by a choline-deficient diet

A diet devoid of choline and low in methionine (CD), without any added carcinogen, has been shown to induce 100% preneoplastic nodules and more than 50% cancer in the rat liver. Attempts to understand the mechanism by which a CD diet induces liver cell cancer revealed that like chemical carcinogens, a CD diet also appears to cause alterations in DNA, perhaps mediated by free radicals. Indeed, a CD diet induces nuclear lipid peroxidation prior to the changes in DNA. The CD diet induced DNA alterations coupled with continuing liver cell proliferation may account for the induction of initiated hepatocytes by the CD diet. To gain insight into the nature of free radicals generated by the CD diet, experiments were designed to determine whether agents that modulate free radical effects influence the CD diet induced changes in the liver. We investigated the effect of Ca2+ in the modulation of CD diet induced alterations in the liver. The results show that extra Ca2+ when added to the CD diet prevented some of the early changes due to choline deficiency, such as nuclear lipid peroxidation and DNA damage, but had little or no effect on the triglyceride accumulation in the liver. Also, the same CD diet with extra Ca2+, when used as a promoter after initiation by diethylnitrosamine, decreased the number and size of early putative preneoplastic foci and nodules.     

Radiomodification of glyoxalase I in the liver and spleen of mice: Adaptive response and split-dose effect

Glyoxalase system, particularly glyoxalase I (Gly I) plays an important role in regulation of cell division and is considered to be a metabolic indicator of cell proliferation. The glyoxalase system is likely to have a close link with cellular radiosensitivity. Therefore, we have examined the effect of adaptive and split-dose of -rays on the activity of Gly I in the liver and spleen of mice. For the adaptive response studies, mice pre-treated with a conditioning dose of 0.5 Gy were given a challenging dose of 4 Gy at varying time intervals. In the split-dose studies, a dose of 4 Gy was delivered into two equal fractions and spaced at different time intervals. The results show that pre-exposure to a conditioning dose or the fractionation of total dose decreased the specific activity of Gly I in the liver and spleen of mice. The decreased activity of Gly I was suggestive of protective action induced by the conditioning dose and fractionation of dose. The similar pattern of radiation response of Gly I probably supported the possibility of involvement of a common pathway in the radiation-induced adaptive and split-dose effect. From these observations a close link between the Gly I and the adaptive-response as well as the split-dose effect is speculated. Since, the glyoxalase system is vital for a variety of biological functions including cell division and repair, the present findings may have relevance in understanding the dose-fractionation as well as the biological defence induced by low doses of radiations.     

Control of DNA polymerase-alpha activity in regenerating rat liver by calcium and 1 alpha,25(OH)2D3

The late G1 surge of DNA polymerase-alpha activity and the initiation of DNA replication in the hepatocytes of partial hepatectomy-induced regenerating liver were severely reduced when the mitogenic partial hepatectomy was carried out in the hypocalcemic and 1,25(OH)2D3 (1 alpha,25-dihydroxycholecalciferol)-deficient environment of parathyroidectomized (PTX) or thyroparathyroidectomized (TPTX) rats. These inhibitions were prevented in TPTX rats by a postpartial hepatectomy injection of 1,25(OH)2D3, which also restored blood calcium to normocalcemic levels. Inhibition of active DNA polymerase-alpha accumulation and initiation of DNA synthesis in TPTX rats were also completely prevented by prefeeding the rats a low phosphorus diet, which stopped the lowering of the blood levels of calcium and 1,25(OH)2D3 following parathyroid removal. These studies indicate that the rise of DNA polymerase-alpha activity and the initiation of DNA replication in regenerating liver are controlled by cellular processes that rely on normal blood levels of calcium and 1,25(OH)2D3. Because DNA polymerase-alpha is the third DNA replication enzyme (the others are ribonucleotide reductase and thymidylate synthase) that has been shown to depend on parathyroid hormone and/or the circulating levels of calcium and 1,25(OH)2D3 that it controls, the authors concluded that the processes dependent on calcium and 1,25(OH)2D3 are parts of a mechanism that coordinately activates the DNA-replicating enzymes. The possibility that cyclic adenosine monophosphate (cAMP)-dependent protein kinases are involved in this replication mechanism is considered.     

Involvement of nuclear phosphatidylinositol-dependent phospholipases C in cell cycle progression during rat liver regeneration

Nuclear lipid metabolism is involved in the regulation of cell proliferation. Modulation of the expression and activity of nuclear PI-phospholipase C (PI-PLC) has been reported during liver regeneration after partial hepatectomy, although it has not been determined whether different PLC isoforms play specific roles in the regulation of cell cycle progression. Here, we report evidence that the increased activity of nuclear PLCs in regenerating rat liver occurs before the peak of DNA replication and involves the enzyme activity associated to the chromatin and not that associated to the nuclear membrane. Immunocytochemical analyses indicate that PI-PLC beta(1) isoform is exclusively localized at the chromatin level, PI-PLC beta(1) co-localizes with DNA replication sites much more than PI-PLC gamma(1), which is also present at the nuclear envelope. These findings and the increased amount of PI-PLC gamma(1) occurring after the peak of DNA replication suggest that PI-PLC beta(1) and gamma(1) play different roles in cell cycle progression during regenerating liver. The increased activity of PI-PLC beta(1) constitutively present within the hepatocyte nucleus, should trigger DNA replication, whereas PI-PLC gamma(1) should be involved in G2/M phase transition through lamin phosphorylation.     

Formation of deoxyribonucleoside 5′-monophosphates dependent on DNA synthesis in nuclei isolated from regenerating rat liver

Hydrolysis of deoxyribonucleoside 5′-triphosphate, resulting in deoxyribonucleoside 5′-monophosphate formation dependent on DNA synthesis, was observed in nuclei isolated from regenerating rat liver. The intensity of the hydrolysis in nuclei varied at different times after partial hepatectomy, showing its maximum at 48 h. The rates of DNA synthesis altered corresponding to the intensities of hydrolysis. Proportionality between decrease in DNA synthesis and decrease in dNMP production was also observed in nuclei treated with various inhibitors of DNA synthesis. The formation of dNMP was detected with the four DNA substrates, indicating no involvement of specific dNTPase. Although regenerating nuclei contained a nonspecific dNTPase activity that can cause release of dNMP, this activity was independent of DNA synthesis and not inhibited by inhibitors of DNA synthesis. These results indicated that regenerating liver nuclei had two different activities for dNMP production; one is DNA synthesis-dependent, and the other is a non-specific dNTPase activity. This paper has focused on the former activity.     

Formation of deoxyribonucleoside 5'-monophosphates dependent on DNA synthesis in nuclei isolated from regenerating rat liver

Hydrolysis of deoxyribonucleoside 5'-triphosphate, resulting in deoxyribonucleoside 5'-monophosphate formation dependent on DNA synthesis, was observed in nuclei isolated from regenerating rat liver. The intensity of the hydrolysis in nuclei varied at different times after partial hepatectomy, showing its maximum at 48 h. The rates of DNA synthesis altered corresponding to the intensities of hydrolysis. Proportionality between decrease in DNA synthesis and decrease in dNMP production was also observed in nuclei treated with various inhibitors of DNA synthesis. The formation of dNMP was detected with the four DNA substrates, indicating no involvement of specific dNTPase . Although regenerating nuclei contained a nonspecific dNTPase activity that can cause release of dNMP , this activity was independent of DNA synthesis and not inhibited by inhibitors of DNA synthesis. These results indicated that regenerating liver nuclei had two different activities for dNMP production; one is DNA synthesis-dependent, and the other is a non-specific dNTPase activity. This paper has focused on the former activity.     

Lutein presents suppressing but not blocking chemopreventive activity during diethylnitrosamine-induced hepatocarcinogenesis and this involves inhibition of DNA damage

Cancer chemopreventive agents are classified as blocking or suppressing agents if they inhibit initiation or promotion/progression phase of carcinogenesis, respectively. Two experiments were conducted in order to classify lutein as a blocking and/or suppressing agent during rat hepatocarcinogenesis. Inhibitory effects of lutein on hepatic preneoplastic lesions (PNL) and DNA strand breakage induced in Wistar rats by the resistant hepatocyte model of hepatocarcinogenesis (initiation with diethylnitrosamine and promotion with 2-acetylaminofluorene coupled with partial hepatectomy) were investigated when the carotenoid was administered specifically during initiation (experiment 1) or promotion (experiment 2) phase. Animals received by gavage during 2 (experiment 1) or six (experiment 2) consecutive weeks on alternate days 70 mg/kg body weight of lutein. Rats treated with only corn oil during these same periods and submitted to this model were used as controls. Treatment with lutein during initiation did not inhibit nor induced (P>0.05) hepatic preneoplastic lesions and DNA damage. On the other hand, treatment during promotion inhibited (P<0.05) the size of hepatic macroscopic nodules and DNA damage and increased (P<0.05) lutein hepatic levels that reached levels seen in human liver samples. Lutein presented inhibitory actions during promotion but not initiation of hepatocarcinogenesis, being classified as a suppressing agent. This reinforces lutein as a potential agent for liver cancer chemoprevention.     

Quantitative relationship between initiation of hepatocarcinogenesis and induction of altered cell islands

We have quantified the initiation of hepatocytic neoplasms and the induction of altered cell islands in regenerating livers of rats given a single treatment with one of three carcinogens before or during the peak of DNA synthesis after partial hepatectomy. For up to 20 wk after treating livers during the peak of DNA synthesis with methyl(acetoxymethyl)nitrosamine (DMN-Ac), hepatocytic neoplasms were not seen. Thereafter, in rats fed the liver tumor promoter, phenobarbital, neoplasms emerged continuously so that by 60 wk after initiation, livers held an average of 5.5 neoplasms. Islands of cellular alteration, identified by their abnormal retention of glycogen on fasting, also appeared to emerge continuously between 20 and 60 wk after initiation. By 60 wk, promoted livers contained about 10,000 islands. In DMN-Ac-initiated, phenobarbital-promoted livers, neoplasms and islands maintained a constant numerical relationship over time with about 1,450 islands emerging for every neoplasm that emerged. This ratio of islands to neoplasms differed according to the type of carcinogen used to initiate hepatocarcinogenesis and depending on whether promotion with phenobarbital was included. In livers initiated with DMN-Ac but not promoted with phenobarbital, the ratio of islands to neoplasms was about 7,750:1. In livers initiated by treatment with (+/-)-7 alpha, 8 beta-dihydroxy-9 beta, 10 beta-epoxy-7,8,9, 10-tetrahydrobenzo[alpha]pyrene at the peak of DNA synthesis and then promoted with phenobarbital, the ratio of islands to neoplasms was 7,200:1. In livers exposed to gamma rays at the peak of DNA synthesis in regenerating livers and promoted, no neoplasms were seen in our sample although islands could be enumerated.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA topoisomerases from rat liver: physiological variations

Besides the nicking-closing (topoisomerase I) activity, an ATP-dependent DNA topoisomerase is present in rat liver nuclei. The enzyme, partially purified, is able to catenate in vitro closed DNA circles in a magnesium-dependent, ATP-dependent, histone H1-dependent reaction, and to decatenate in vitro kinetoplast DNA networks to yield free minicircles in a magnesium-dependent and ATP-dependent reaction. It is largely similar to other eukaryotic type II topoisomerases in its requirements, and presumably belongs to this class of enzymes. Type I and type II activities were measured in rat liver nuclei as a function of regenerating time after partial hepatectomy: type I activity was not significantly changed during this process. In contrast, type II activity was considerably increased, suggesting a possible involvement of the enzyme in DNA replication.     

Stress-inducible overexpression of <Emphasis Type="Italic">glyoxalase I</Emphasis> is preferable to its constitutive overexpression for abiotic stress tolerance in transgenic <Emphasis Type="Italic">Brassica juncea</Emphasis>

The glyoxalase system catalyzes the conversion of cytotoxic methylglyoxal to d-lactate via the intermediate S-d-lactoylglutathione. It comprises two enzymes, Glyoxalase I (Gly I) and Glyoxalase II (Gly II), and reduced glutathione which acts as a cofactor by anchoring the substrates in the active sites of the two enzymes. The overexpression of both Gly I and Gly II, either alone or in combination, has earlier been reported to confer tolerance to multiple abiotic stresses. In the present study, we sought to evaluate the consequences of constitutive and stress-induced overexpression of Gly I on the performance and productivity of plants. Towards this end, several Gly I transgenic Brassica juncea lines (designated as R and S lines) were generated in which the glyoxalase I (gly I) gene was expressed under the control of either a stress-inducible rd29A promoter or a constitutive CaMV 35S promoter. Both the R and S lines showed enhanced tolerance to salinity, heavy metal, and drought stress when compared to untransformed control plants. However, the S lines showed yield penalty under non-stress conditions while no such negative effect was observed in the R lines. Our results indicate that the overexpression of the gly I gene under the control of stress-inducible rd29A promoter is a better option for improving salt, drought and heavy metal stress tolerance in transgenic plants.     

Enhancement of nuclear Ca2+-ATPase activity in regenerating rat liver: involvement of nuclear DNA increase

The alteration of calcium content, Ca²⁺-ATPase activity, DNA content and DNA fragmentation in the nuclei of regenerating rat liver was investigated. Liver was surgically removed about 70% of that of sham-operated rats. the reduced liver weight by partial hepatectomy was completely restored at 3 days after the surgery. Regenerating liver significantly increased Ca²⁺-ATPase activity and DNA content in the nuclei between 1 and 5 days after hepatectomy. The nuclear calcium content was clearly increased from 2 days after hepatectomy. The increase of Ca²⁺-ATPase activity in regenerating liver was clearly inhibited by the presence of trifluoperazine (10 M), staurosporine (2.5 M) and dibucaine (10 M), which are inhibitors of calmodulin and protein kinase, in the enzyme reaction mixture. However, the nuclear enzyme activity in normal rat liver was not significantly altered by these inhibitors. Meanwhile, the increase of nuclear DNA content in regenerating liver was completely blocked by the administration of trifluoperazine (2.5 mg/100 g body weight), suggesting an involvement of calmodulin. Now, the nuclear DNA fragmentation was significantly decreased in regenerating liver, suggesting that this decrease is partly contributed to the increase in nuclear DNA content. The present study clearly demonstrates that regenerating liver enhances nuclear Ca²⁺-ATPase activity and induces a corresponding elevation of nuclear calcium content. This Ca²⁺-signaling system may be involved in the regulation of nuclear DNA functions in regenerating rat liver.     

DNA-polymerase activity and DNA synthesis in the hepatocyte nuclear matrix

The comparative analysis of DNA-synthetase activity of hepatocytes, isolated nuclei and nuclear matrix from normal and regenerating rat liver was performed. The highest enrichment with newly-synthesized DNA was registered in the DNA fraction associated with the nuclear matrix both in vivo and in vitro. The functioning of DNA polymerases alpha and beta in the matrix was shown. Our results indicate that DNA polymerase beta is more firmly bound with the nuclear matrix in the cells of normal liver but this enzyme is eluted almost completely from the nuclei of regenerating liver cells. At the first moment after gamma-irradiation of rats the preferential initiation of unscheduled DNA synthesis in vivo has been observed on the nuclear skeletal structures. This may serve as an indication on the possibility that DNA repair process occurs on the nuclear matrix.     

Effects of female sex steroids on concanavalin A-mediated agglutination of hepatocytes from nonregenerating and regenerating rat liver and hepatic tumor marker enzymes

Effect of treatment of female rats with an oral contraceptive agent (OCA), Ovulen-50, for 7 weeks on agglutination of hepatocytes with concanavalin A (con A) and activities of certain tumor marker enzymes were examined to find out if OCA treatment is related to preneoplastic or neoplastic processes. Hepatocytes from regenerating and nonregenerating livers of control female rats showed negligible agglutination with Con A, whereas hepatocytes from non regenerating but not from the regenerating livers of female rats treated with a combination of 5 micrograms ethinyl estradiol and 100 micrograms ethynodiol diacetate showed agglutination. Of the tumor marker enzymes such as hepatic glucose 6-phosphatase, gamma-glutamyl transpeptidase (gamma-GT), and arginase examined in the liver, only gamma-glutamyl transpeptidase showed a significant increase in activity in the steroid-treated rats. Plasma alkaline phosphatase activity was also higher in the treated animals. However, the magnitude of the changes observed was relatively small and perhaps unrelated to the neoplastic process.     

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.     

Induction of mouse liver glyoxalase I by hypobaric hypoxia

Hypobaric hypoxia at 0.45 atm induced a reversible increase of mouse liver glyoxalase I. The levels of this enzyme increased after an exposure of 20 h and 20 + 20 h, whereas the activity decreased to the control values after 20 h at room pressure. Before the treatment, some animals received tritiated leucine (i.p.). Glyoxalase I was purified to homogeneity. The pure enzyme from the treated animals showed 20-times more radioactivity than the controls. Thus, the increase in specific activity is due to new protein synthesized in response to the treatment at 0.45 atm. The activities of glyoxalase II and glutathione S-transferase were not affected by the treatment.