Biomedical subjects

D S Sarma

Publications and source records attributed to D S Sarma.

At least 55 records · Page 3Linked to original sources

PubMed1992

Abnormal hepatic nucleotide pools in sparse fur (spf) mutant mice deficient in ornithine transcarbamylase.

Sparse fur hemizygous male mice are over 90% deficient in ornithine transcarbamylase and exhibit increased synthesis of orotic acid. Because our earlier studies have demonstrated that orotic acid is a liver tumor promoter in the rat, it was of interest to determine whether this genetic disorder also increases the risk of tumor promotion. The results revealed that the livers of mutant mice showed a fourfold increase in uridine nucleotides and a 50% decrease in adenosine nucleotides compared to corresponding controls, a pattern of nucleotide pool imbalance similar to that seen in the livers of rats exposed to orotic acid under promoting conditions. Creation of such an imbalance appears to be important for orotic acid to exert its promotional effects. Sparse fur mutant mouse may, therefore, be an ideal animal model to study the tumor-promoting effects of orotate.

Animals↗

PubMed1992

Inhibition of DNA synthesis by phenobarbital in primary cultures of hepatocytes from normal rat liver and from hepatic nodules.

One of the many hypotheses put forward to explain the mechanism by which phenobarbital (PB) promotes hepatocarcinogenesis is by differential mitoinhibition of surrounding hepatocytes while allowing the initiated hepatocytes to respond to growth stimuli and form foci and nodules. Given the similarity in structures between PB and orotic acid (OA), another rat liver tumor promoter, the present investigation was designed to determine (i) whether PB, like OA, exerts its mitoinhibitory effect at a site beyond the growth factor receptor and receptor mediated early events; and (ii) whether PB exerts a differential mitoinhibitory effect by selectively inhibiting the non-initiated hepatocytes but not the initiated hepatocytes in vitro. Our studies demonstrate that, like OA, PB also inhibits DNA synthesis in hepatocytes from normal rat liver in a dose dependent manner with 80-90% at a dose of 6 mM. One target site may lie beyond the growth factor receptor mediated early events because PB inhibited DNA synthesis in hepatocytes primed with the growth factor 24 h earlier. Interestingly, PB inhibited DNA synthesis not only in hepatocytes from non-nodular surrounding liver but also in hepatocytes from persistent hepatic nodules initiated with 1,2-dimethylhydrazine and promoted with OA. Therefore, our results suggest that although PB is a mitoinhibitor of DNA synthesis in hepatocytes, it does not appear to create as strong a differential mitoinhibition between non-nodular surrounding and initiated hepatocytes as is evident in the resistant hepatocyte and OA models. These results raise the question whether differential mitoinhibition is the major contributing factor in the PB mediated rat liver tumor promotion.

Animals↗

PubMed1992

Rat hepatocyte nodules are resistant to the necrogenic effect of D-galactosamine.

D-Galactosamine is a known hepatotoxin which induces liver cell necrosis via depletion of UTP and other uridine nucleotides. Our previous work indicated that nodular hepatocytes have higher levels of total uridine nucleotides compared to normal liver, and in the present study we investigate the effect of galactosamine treatment on hepatocyte nodules and surrounding liver. Hepatic nodules were generated in male Wistar rats according to the Solt and Farber protocol. Six months after initiation animals received a single injection of D-galactosamine (500 mg/kg i.p.) and were then killed 1, 2, 4 or 7 days later. Histological analysis of liver revealed the presence of extensive liver cell necrosis in normal tissue 1 and 2 days after galactosamine treatment. However, very little or no necrosis was detectable inside hepatic nodules at any time point, indicating that these focal areas are resistant to the cytotoxic effect of galactosamine. This type of resistance could be the expression of a new component in the resistant phenotype of hepatic nodules.

Animals↗

PubMed1992

Mitogen-induced liver hyperplasia does not substitute for compensatory regeneration during promotion of chemical hepatocarcinogenesis.

Experiments were designed to determine the efficacy of different types of liver cell proliferative stimuli given during exposure to several liver tumor-promoting regimens, on the formation of foci of enzyme-altered hepatocytes. Male Wistar rats were initiated with diethylnitrosamine (150 mg/kg body wt). After a 2 week recovery period animals were subjected to promoting regimens, the resistant hepatocyte model, the phenobarbital model and the orotic acid model. While the rats were on these regimens they were given liver cell proliferative stimulus, either a compensatory type (two-thirds partial hepatectomy or a necrogenic dose of carbon tetrachloride) or a direct hyperplastic stimulus such as that induced by the primary mitogen, lead nitrate. Initiated cells so promoted by these regimens were monitored as foci of enzyme-altered hepatocytes positive for gamma-glutamyltransferase and placental glutathione S-transferase or deficient for adenosine triphosphatase. While carbon tetrachloride and partial hepatectomy-induced compensatory regeneration stimulated the promoting ability of the regimens used, direct hyperplasia could not stimulate the formation of foci and/or nodules from initiated hepatocytes. Evaluation of thymidine incorporation indicated that there was no significant difference in the extent of DNA synthesis in both the proliferative stimuli irrespective of the promoting procedure used.

2-Acetylaminofluorene↗

PubMed1992

Hypomethylation of beta-hydroxy-beta-methyl-glutaryl coenzyme A reductase gene and its expression during hepatocarcinogenesis in the rat.

Our earlier studies had demonstrated that inhibition of DNA methylation following carcinogen treatment potentiated initiation of the carcinogenic process in the rat liver system. The hepatic nodules developed by initiation-promotion protocols showed a characteristic hypomethylation in the cell-cycle-related genes c-fos, c-myc and c-Ha-ras. In the present study we have found that the gene for beta-hydroxy-beta-methyl glutaryl coenzyme A reductase, a major rate-limiting enzyme in the biogenesis of mevalonate, is also hypomethylated at both CCGG and GCGC sites and expressed in hepatic nodules. This gene, however, did not exhibit hypomethylation in CCGG sequences in non-nodular surrounding liver, livers from rats subjected to two-thirds partial hepatectomy, or exposed to initiator alone (1,2-dimethylhydrazine given 18 h after partial hepatectomy) or to diets containing 1% orotic acid alone (promoting regimen). The activity of the enzyme and mevalonate formation are positively correlated with DNA synthesis and cell proliferation--two key components of the carcinogenic process. Taken together, the results suggest that hypomethylation of specific genes occurs in the carcinogenic process and this altered pattern of DNA methylation may play a role in the growth of the nodules.

1,2-Dimethylhydrazine↗

PubMed1991

Chronic mitoinhibition during promotion of hepatocarcinogenesis.

We have reported previously that orotic acid (OA), a precursor for pyrimidine nucleotide biosynthesis, is able to promote carcinogenic process in both liver and duodenum of rats. The present study investigates the possible role of mitoinhibitory effects of OA as being responsible for its promotional effects. Male Fischer 344 rats were given a semisynthetic basal diet (BD) or a diet containing 1% OA for four weeks coupled with 2/3 partial hepatectomy (PH), and all animals were then continued on BD for an additional four weeks. This protocol is known to exert a promoting effect on the initiated rat liver. Livers were perfused, and the labeling index (LI) of isolated cultured hepatocytes was monitored. Hepatocytes isolated from livers of rats fed a BD or 1% OA exhibited in vitro an LI of 39 +/- 2 and 24 +/- 1%, respectively. The lowered in vitro LI was seen even upon exposure to epidermal growth factor (EGF) (67 +/- 2% in OA-treated livers compared to 91 +/- 2% in hepatocytes from control rat liver). A similar four-week exposure to OA coupled with PH decreased hepatic DNA synthesis induced by a choline-deficient diet in vivo by about 50%. These results indicate that OA is able to decrease the response of normal hepatocytes to growth factors and suggest a possible mechanism of chronic differential mitoinhibition as a basis for promotion induced by OA.

Animals↗

PubMed1991

Diploid growth pattern of hepatocellular tumours induced by various carcinogenic treatments.

Hepatocellular carcinomas from rats of different strains, subjected to a variety of carcinogenic treatment regimens in different laboratories (initiation by diethylnitrosamine or dimethylhydrazine, promotion by phenobarbital, 2-acetylaminofluorene, nafenopin, orotic acid or deoxycholic acid, growth stimulation by partial hepatectomy or necrogenic CCl4 treatment), were all found to be predominantly diploid by flow cytometric analysis, in contrast to normal liver tissue in which polyploid nuclei were predominant. A switch from polyploidization to diploid growth would thus seem to be a common property of malignant liver tumours. Benign neoplastic liver nodules were likewise predominantly diploid, with the exception of nodules induced by long-term deoxycholic acid treatment in Fischer rats. In addition to containing a majority of polyploid cells, the latter nodules failed to progress to the carcinoma stage.

Animals↗

PubMed1990

Studies on the effect of dietary orotic acid on mouse liver carcinogenesis induced by diethylnitrosamine.

The present study was designed to determine whether orotic acid, a liver tumor promoter in the rat, also promotes liver carcinogenesis in the mouse. Eight-week-old male BALB/c mice were initiated with diethylnitrosamine (90 mg/kg i.p.). One week later they were divided into 2 groups and given either a basal diet or the basal diet containing 1% orotic acid (OA). They were killed at 6 or 10 months after the administration of the carcinogen. At 6 months, no nodular lesions were seen in mice whether or not they were exposed to OA. However by 10 months 100% of mice in both groups developed hepatic nodules. OA neither shortened the latent period for the appearance of the nodular lesions not did it increase the size of the nodules. Although BALB/c mice exhibited an increase in uridine nucleotides and a decrease in adenosine nucleotides in the liver upon exposure to OA, the magnitude of the change was less compared with that seen in the rat liver. The resistance of BALB/c mouse to the tumor-promoting effects of OA may reflect in part the resistance of the mouse to OA-induced nucleotide pool imbalance.

Animals↗

PubMed1990

Liver hyperplasia is not necessarily associated with increased expression of c-fos and c-myc mRNA.

Experiments were designed to investigate the expression of three cell-cycle-dependent proto-oncogenes in response to two different types of proliferative stimuli: compensatory cell proliferation after partial hepatectomy (PH) or CCl4 and liver hyperplasia induced by the mitogens ethylene dibromide (EDB) and cyproterone acetate (CPA). Steady-state levels of messenger RNAs for c-fos and c-myc were found to be elevated after PH or CCl4 with a maximum increase between 0.5 and 2 h for c-fos and at 2-3 h for c-myc and a rapid decline after 3 h. However, when liver cell proliferation was induced by mitogens, no increase in the expression of c-fos mRNA was observed with both EDB or CPA during the first 24 h. In addition, elevated expression of c-myc was found only in liver hyperplasia induced by EDB, but not with CPA. While the expression of c-myc mRNA and c-fos mRNA was different in the two types of proliferative stimuli, that of c-Ha-ras and c-Ki-ras was similar in all the experimental groups. Cell proliferation monitored by means of incorporation of labelled thymidine into DNA or mitotic index at 24 h following PH, EDB and CPA occurred at a similar extent in all the experimental groups. Our data indicate that the transient and sequential expression of cell-cycle-related genes may vary in response to proliferative stimuli of different nature and suggest that increased expression of cell-cycle-related genes may not be a necessary prerequisite for the entry of the cells into the cell cycle.

Animals↗

PubMed1990

Studies on the mitoinhibitory effect of orotic acid on hepatocytes in primary culture.

Orotic acid (OA), a promoter of liver carcinogenesis, inhibited proliferation of primary hepatocytes in culture as monitored by labelling index, mitotic index and total DNA content. The mitoinhibitory effect of OA was seen even in the presence of a strong mitogen such as epidermal growth factor (EGF). The growth inhibitory effect of OA was not due to cell killing. Upon exposure to OA the hepatocytes exhibited an increase in the ratio of uridine nucleotides to adenosine nucleotides, and as this ratio increased the response of hepatocytes to proliferate in the presence or absence of EGF decreased. Washing the hepatocytes free of added OA resulted in a gradual decrease in the ratio of uridine nucleotides to adenosine nucleotides, paralleled by an increase in hepatocytic proliferation. Adenine, an agent that inhibits the metabolism of OA to uridine nucleotides, not only inhibited the increase in the ratio of uridine nucleotides to adenosine nucleotides but also counteracted the OA-induced mitoinhibitory effect. These results, together with our earlier observations, suggest that an imbalance in nucleotide pools composed of an increase in uridine nucleotides and a decrease in adenosine nucleotides appears to be important for OA-induced mitoinhibition.

Animals↗

PubMed1990

Lack of DNA alterations induced by orotic acid in rat liver as evaluated with two DNA unwinding methods.

One percent orotic acid supplemented diet is a promoting treatment in the rat model of liver carcinogenesis. After treatment with this type of diet, DNA alterations were observed using alkaline sucrose gradients and alkaline elution methods. In this work we have utilized two unwinding methods for the detection of DNA fragmentation. One method is a viscosimetric method in which the rate of increase in DNA viscosity with time is related to the rate of alkaline DNA unwinding. The second method measures fluorimetrically the amount of renatured and denatured DNA after different times allowed for alkaline DNA unwinding. These two methods are very sensitive in detecting DNA breaks induced by typical alkylating agents, X-rays and H2O2. The two unwinding methods were clearly negative for the orotic acid supplemented diet. We suggest that the DNA alterations detected with alkaline sucrose gradients and alkaline elution methods, after promoting treatment with orotic acid, are probably different from the DNA breaks induced by typical alkylating agents, X-rays and H2O2.

Animals↗

PubMed1989

Studies on the kinetics of expression of cell cycle dependent proto-oncogenes during mitogen-induced liver cell proliferation.

The present study was undertaken to determine the kinetics of DNA synthesis and expression of cell cycle dependent proto-oncogenes in response to two types of cell proliferative stimuli in male Wistar rat liver. The peak of DNA synthesis was approximately 24 h after a compensatory cell proliferative stimulus induced by 2/3 partial hepatectomy and approximately 36 h following a mitogenic stimulus obtained with a single dose of lead nitrate (10 micromol/100 g body wt, through femoral vein). Even though both proliferative stimuli induced the expression of c-fos, c-myc and c-Ha-ras, the extent of the increase in c-fos expression was 4- to 5-fold less in mitogen-induced cell proliferation. In addition, while the expression of c-myc, following partial hepatectomy returned to basal level by 4 h, the induced expression of c-myc persisted for up to 40 h during the lead nitrate-induced liver cell proliferation.

Animals↗

PubMed1989

Further evidence that mitogen-induced cell proliferation does not support the formation of enzyme-altered islands in rat liver by carcinogens.

Our earlier studies have revealed that direct hyperplasia induced by liver mitogens such as lead nitrate, ethylene dibromide, nafenopin and cyproterone acetate, unlike compensatory cell proliferation induced by partial hepatectomy and CCl4, does not support the formation of enzyme-altered islands induced by chemical carcinogens in the liver. In the previous studies carcinogens were given at the peak of DNA synthesis induced by the liver mitogens. If the mitogens have altered the sensitive phase of the hepatocyte to the carcinogenic attack, administering the carcinogen at one time point following the mitogenic stimulus might have missed the sensitive phase. In order to overcome this possibility in the present study male Wistar rats weighing 200-250 g were given N-methyl-N-nitrosourea (MNU; 60 mg/kg, i.p.) at three points representing G1, S and G2/M phases of the cell cycle following different types of liver cell proliferative stimuli. In another experiment MNU (60 mg/kg, i.p.) and diethylnitrosamine (15 mg/kg, i.p.) were given prior to the administration of proliferative stimuli. The initiated hepatocytes were also assayed following promotion by two different promoting regimens, namely phenobarbital and the resistant-hepatocyte model. Further, the initiated hepatocytes were monitored not only by using the appearance of islands of enzyme-altered hepatocytes but also using the incidence of hepatocellular carcinoma. The results of this study clearly revealed that irrespective of the protocol used, only the compensatory liver cell proliferation but not the mitogen-induced direct hyperplasia supported the formation and the growth of enzyme-altered islands in the liver induced by chemical carcinogens.

Adenosine Triphosphatases↗

PubMed1989

Studies on hypomethylation of liver DNA during early stages of chemical carcinogenesis in rat liver.

Our finding that the inhibitors of DNA methylation, 5-azacytidine, 5-azadeoxycytidine or adenosine dialdehyde, given after a carcinogen all potentiated initiation suggested that hypomethylation of DNA during repair synthesis of DNA might play a role in the initiation of the carcinogenic process. To examine this aspect further, we have asked the question, do the nodules which develop from initiated cells after promotion with 1% orotic acid exhibit an altered methylation pattern in their DNA? The methylation status of the DNA from nodules has been examined using the restriction endonucleases HpaII/MspI and HhaI which distinguish between methylated and unmethylated cytosines in their nucleotide recognition DNA 5'-CCGG and 5'-GCGC respectively. The proto-oncogenes, c-myc, c-fos and c-Ha-ras, in the DNA were primarily studied in this investigation because of their possible involvement in cell proliferation and/or in cell transformation and tumorigenesis. The results indicate that in the nodule DNA, c-myc and c-fos are hypomethylated in the sequence of CCGG while the c-Ha-ras shows hypomethylation in the alternating GCGC sequence. This methylation pattern seen in the nodule DNA is not found in the DNA of the non-nodular surrounding liver or liver tissue after exposure to promoter or carcinogen alone. It is also not found in the DNA of regenerating liver. It is particularly significant that the methylation patterns in the c-myc and c-Ha-ras regions are similar to those found in several cancer tissues. The results suggest that this methylation pattern is acquired early in the carcinogenic process and raises the question whether it has any bearing on the process.

1,2-Dimethylhydrazine↗

PubMed1988

Rapid and transient induction of c-fos, c-myc and c-Ha-ras in rat liver following glycine administration.

Administration of glycine (2.5 mmoles/100 g., i.p.) results in an increased expression of several cell cycle dependent genes such as c-fos, c-myc and c-Ha-ras in the rat liver. The increased expression could be noticed as early as 20-40 minutes and declined by 2 hours following glycine administration. The rapid rise and decline in the mRNA levels of c-fos, c-myc and c-Ha-ras in response to glycine is of significance because in response to a wide variety of growth stimuli, these proto-oncogenes exhibit a temporal sequence in their expression; for example, the expression of c-fos precedes that of c-myc, which in turn precedes the increased expression of c-Ha-ras. The experimental model using a simple amino acid such as glycine will be useful in exploring some of the mechanisms of regulation of expression of these proto-oncogenes.

Animals↗

PubMed1988

Inhibition of DNA synthesis in primary cultures of hepatocytes by orotic acid.

Orotic acid has been shown to promote carcinogenesis in the liver and the intestine of the rat. In an attempt to determine whether orotic acid promotes liver carcinogenesis by creating differential mitoinhibition, experiments were designed to study the effect of orotic acid on the labeling index of isolated hepatocytes in response to epidermal growth factor. The results indicated that orotic acid added in vitro inhibited epidermal-growth-factor-induced labeling index of isolated hepatocytes. In addition, isolated hepatocytes from rats exposed to orotic acid under promoting conditions also exhibited a decreased response to epidermal growth factor. These data suggest that orotic acid may exert its promoting effect by differentially inhibiting the response of normal hepatocytes to one or more endogenous growth stimuli while permitting the initiated hepatocytes to respond to such stimuli and grow to form hepatic nodules.

Animals↗

PubMed1988

Modulation of the activity of hepatic gamma-glutamyl transpeptidase, adenosine triphosphatase, placental glutathione S-transferase and adenylate cyclase by acute administration of lead nitrate.

The effect of a single administration of lead nitrate on the activity of gamma-glutamyltranspeptidase (gamma-GT), adenosine triphosphatase (ATPase), the placental form of glutathione S-transferase (GST-P) and adenylate cyclase (AC), four enzymes widely used as phenotypic markers for preneoplasia, was investigated in the liver of male Wistar rats. The results of the histochemical enzymatic staining indicated that an acute treatment with lead nitrate induces the activity of gamma-GT, mainly in the hepatocytes located around zone I of the liver acinus, with a maximum seen between 72-96 hours. On the other hand, the activity of ATPase was found to be severely inhibited at 2-3 days after treatment, as shown by a strong decrease in the staining of the bile canaliculi of zones II and III. Immunohistochemical analysis revealed that lead nitrate administration also resulted in the appearance in most of the hepatocytes of GST-P, an enzyme whose activity is almost undetectable in normal rat liver, but is elevated in preneoplastic liver lesions. Finally, lead nitrate treatment resulted in an inhibition of AC activity which was maximal after 24 hours.

Adenosine Triphosphatases↗

PubMed1987

Inability of mitogen-induced liver hyperplasia to support the induction of enzyme-altered islands induced by liver carcinogens.

Experiments were designed to determine whether liver cell proliferation induced by direct mitogens is as effective as compensatory cell proliferation consequent to previous cell loss, in supporting the growth of enzyme-altered islands in the liver induced by chemical carcinogens. Male Wistar rats were given injections of a single nonnecrogenic dose of N-methyl-N-nitrosourea or benzo(a)pyrene during the S phase following the administration of four different liver mitogens, namely, lead nitrate, ethylene dibromide, nafenopin, and cyproterone acetate, or during compensatory cell proliferation following partial hepatectomy or a necrogenic dose of CCl4. The carcinogen-altered hepatocytes were monitored as gamma-glutamyltransferase- or placental glutathione S-transferase-positive foci using a 2-wk promoting regimen consisting of 0.03% 2-acetylaminofluorene coupled with a necrogenic dose of CCl4. The results indicate that, unlike compensatory cell proliferation induced by partial hepatectomy or CCl4, the mitogen-induced cell proliferation did not result in a significant number of enzyme-altered islands, despite the fact that the extent of cell proliferation at the time of carcinogen administration, as monitored by the examination of labeled cells, is similar with both types of proliferative stimuli.

Animals↗