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H C Pitot

Publications and source records attributed to H C Pitot.

At least 199 records · Page 11Linked to original sources
PubMed1988

Dietary effects on initiation and promotion of hepatocarcinogenesis in rat.

Female F344/N rats were initiated with DEN (10 mg/kg) 24 h after a 70% partial hepatectomy. Groups of 10 rats were fed (a) AIN, group-1; (b) PD, group-2; or (c) NIH, group-3, for 1 week after initiation and were then fed NIH plus the promoting agent PB at a level of 0.05% in the diets for 6 months. Other groups were fed NIH for 1 week after initiation and then NIH without PB (group-4), AIN + PB (group-5), AIN without PB (group-6), PD + PB (group-7), or PD without PB (group-8) for 6 months. The numbers and volume percentages of AHF were quantified by stereologic methods from frozen serial sections, stained consecutively for GGT, ATPase, and G6Pase. For the groups fed different diets during the 1st week after initiation, the numbers and volume of AHF were significantly greater in group-2 than in groups 1 or 3. The numbers of AHF were significantly less in group-3 than in group-1. The numbers and volume of AHF were significantly greater in groups fed PB during the promotion phase, except in the case of group-7, whose focal volume did not differ from groups 6 or 8. Group-3 had significantly greater numbers of AHF than groups 5 and 7. These findings can be explained by the hypothesis that the NIH diet contained factors that acted synergistically with PB to enhance tumor promotion. The mean focal volume of both GGT positive and ATPase negative foci was significantly greater in group-5 than in all other groups; this indicates that the AIN + PB regimen selectively promoted the growth of a subpopulation of AHF. These findings show that alterations in the composition of diets fed during hepatocarcinogenesis significantly alter the effects of specific chemical agents acting during the stages of initiation and promotion in hepatocarcinogenesis.

Animals↗
PubMed1988

A method for the comparative study of replicative DNA synthesis in GGT-positive and GGT-negative hepatocytes in primary culture isolated from carcinogen-treated rats.

The presence of gamma-glutamyl transpeptidase (GGT) in focal nodules of hepatocytes is a commonly used marker for the identification of preneoplastic cell populations. Female Fischer 344 rats were initiated with a single intragastric administration of 200 mg diethylnitrosamine/kg, altered cells were selected after 0.02% 2-acetylaminofluorene was given in the diet; this was followed by a partial hepatectomy and promotion with dietary sodium phenobarbital for 4 wk. A mixed-cell population of GGT-positive and GGT-negative hepatocytes was obtained after collagenase perfusion and Percoll purification. An enriched population of GGT-positive hepatocytes was obtained by a modified "panning" technique. With quantitative scintillation spectrometry and autoradiography of [3H]thymidine incorporation, replicative DNA synthesis of GGT-positive and GGT-negative rat hepatocytes was observed in both the mixed-cell population and the enriched GGT-positive and GGT-negative cell populations. Under the culture conditions used, GGT-positive cells showed a higher level of replicative DNA synthesis than did GGT-negative cells; this indicates that such altered hepatocytes in the stage of promotion possess an inherently greater capacity for all replication, as previously suggested from studies in vivo.

Animals↗
PubMed1988

The effect of sodium butyrate on tyrosine aminotransferase induction in primary cultures of normal adult rat hepatocytes.

Treatment of primary cultures of adult rat hepatocytes with 5 mM butyrate inhibited the spontaneous decrease in basal activity and mRNA levels of tyrosine aminotransferase (TAT) that occurred during culture (Staecker et al., submitted). We report here that butyrate treatment of primary cultures of rat hepatocytes initially inhibited the induction of TAT. This inhibition was followed by a period of accelerated TAT induction. TAT induction in butyrate-treated primary cultures of adult rat hepatocytes occurred only after metabolism of butyrate by the cultured hepatocytes. The accelerated induction of TAT in hepatocyte cultures treated with sodium butyrate was reflected by increased TAT activity and mRNA levels. Cultured hepatocytes rapidly metabolized butyrate, but the addition of more butyrate into cultures after its initial metabolism resulted in a rapid reduction in TAT activity. These findings indicate that butyrate treatment can affect the expression of TAT in primary hepatocyte cultures in both a positive (increased basal TAT expression) and a negative (inhibition of the induced expression of TAT) manner.

Acetylation↗
PubMed1988

Isolation and nucleotide sequence of the cDNA for rat liver serine dehydratase mRNA and structures of the 5' and 3' flanking regions of the serine dehydratase gene.

Rat serine dehydratase cDNA clones were isolated from a lambda gt11 cDNA library on the basis of their reactivity with monospecific immunoglobulin to the purified enzyme. Using the cDNA insert from a clone that encoded the serine dehydratase subunit as a probe, additional clones were isolated from the same library by plaque hybridization. Nucleotide sequence analysis of the largest clone obtained showed that it has 1444 base pairs with an open reading frame consisting of 1089 base pairs. The deduced amino acid sequence contained sequences of several portions of the serine dehydratase protein, as determined by Edman degradation. Rat liver serine dehydratase mRNA virtually disappeared from livers of rats fed a protein-free diet for 5 days. Several genomic clones were isolated from two libraries. Determinations of the transcription start site and the structure of the 3' flanking region of the gene indicated that the coded mRNA is 1504 nucleotides long. The 5' promoter region contained a variety of sequences similar to several consensus sequences believed to be important for the regulation of specific gene expression.

Amino Acid Sequence↗
PubMed1988

Molecular cloning, sequence analysis, and expression in Escherichia coli of the cDNA for guanidinoacetate methyltransferase from rat liver.

Five cDNA clones encoding rat liver guanidinoacetate methyltransferase (S-adenosyl-L-methionine: guanidinoacetate N-methyltransferase, EC 2.1.1.2) were isolated from a lambda gt11 cDNA library by use of a polyclonal antibody to the purified enzyme. Sequence analysis of the longest cDNA indicated that it consisted of 711 base pairs (bp) of coding region, 51 bp of 5' noncoding region, and 162 bp of 3' noncoding region excluding the poly(A) tail. The amino acid sequence deduced from the cDNA contained the sequences of NH2-terminal and three tryptic peptides. The predicted amino acid composition and molecular weight were in excellent agreement with those obtained with the purified enzyme. Introduction of the cDNA into plasmid pUC118 having the lac promoter resulted in a production in Escherichia coli of a Mr 26,000 polypeptide in the presence of isopropyl beta-D-thiogalactopyranoside. This protein represented as much as 5% of the bacterial soluble protein and showed the guanidinoacetate methyltransferase activity. Sequence analysis and tryptic peptide mapping indicated that the enzyme obtained by the recombinant DNA procedures was structurally identical to the liver enzyme, except for the absence of the NH2-terminal blocking group. Also, the enzyme showed kinetic properties indistinguishable from those of the liver enzyme.

Amino Acid Sequence↗
PubMed1988

Lack of acetylaminofluorene--DNA adduct formation in enzyme-altered foci of rat liver.

Formation of the N-(deoxyguanosin-8-yl)-aminofluorene adduct was studied in enzyme-altered foci induced by four different liver carcinogenesis models. Foci were detected and scored for enzyme phenotype by a computer-aided image overlay technique. Localization of the enzymes gamma-glutamyl transpeptidase, canalicular ATPase and glucose-6-phosphatase was performed by enzyme histochemistry, allowing identification of foci of seven different phenotypes. Patterns of foci obtained by image overlay were compared to in situ 2-acetylaminofluorene--DNA adduct distribution obtained by immunofluorescence. Foci were induced by the following models: (1) chronic feeding of 0.02% 2-acetylaminofluorene (2-AAF) for 8 weeks; (2) intubation of diethylnitrosamine (DEN) (10 mg/kg) 24 h after a 70% partial hepatectomy (PH), followed 8 weeks later by a diet containing 0.05% phenobarbital for 9 months; (3) intubation of DEN (10 mg/kg) 24 h after PH, followed by a diet containing 0.01% ciprofibrate for 5 months, and after an additional 4 months a diet containing 0.05% phenobarbital for 2 months; (4) maintenance for 7.5, 16.5 or 19.5 months after transplantation of DEN/2-AAF/PH ('Solt-Farber' protocol) donor liver cells into host rats receiving a brief 2-AAF/PH selective regimen then no further treatment until sacrifice. To test the capacity of both foci and morphologically normal livers to form DNA adducts, the animals in models 2-4 received a diet containing 0.02% 2-AAF for 5 or 6 days before sacrifice. In all of the enzyme-altered foci identified in models 1-3 there were no DNA adducts visible by immunofluorescence. Scattered groups of positive cells were occasionally seen in the otherwise dark foci induced by model 4. For technical reasons some enzyme-altered foci were not identifiable on the fluorescence-stained slides. In liver serial sections from rats in models 1-4, there were 75, 304, 125 and 68 enzyme-altered foci of seven different phenotypes which were identified as AF-DNA negative. In models 1 and 4 there were some additional adduct-negative foci not associated with any of the seven identified focus phenotypes. These studies demonstrate that loss of the ability to form DNA adducts in hepatic enzyme-altered foci is a common and very early biochemical adaptation to xenobiotic exposure in different hepatocarcinogenesis models. This adaptation also is retained by the majority of foci in later stages of hepatocarcinogenesis.

2-Acetylaminofluorene↗
PubMed1988

Gene expression during multistage hepatocarcinogenesis.

Several multistage models of hepatocarcinogenesis in the rat have now been developed. In one of these models at least three distinct stages--initiation, promotion, and progression--can be delineated. The first and last are irreversible, while that of promotion is reversible. Quantitation of each stage is possible. While several variable marker enzymes have been utilized to identify and characterize preneoplastic lesions during the stage of promotion, preliminary investigations indicate that the transcriptional activation of proto-oncogenes, particularly that of c-raf-1 proto-oncogene, may be used as an indicator of those preneoplastic lesions that will potentially develop into malignant neoplasms.

Animals↗
PubMed1988

The effects of dose and duration of administration on the promotion index of phenobarbital in multistage hepatocarcinogenesis in the rat.

The relative potency of chemicals as promoting agents in multistage hepatocarcinogenesis has been previously defined as the Promotion Index through calculations of quantitative stereology. The Promotion Index is a function of the total cell population of altered hepatic foci in the liver at any given time and dose of promoting agent. When the Promotion Index was determined as a function of the dose of phenobarbital given in the diet for varying periods of time, a value of 394 was obtained for doses less than 0.01%; at doses between 0.01% and 0.1%, the Promotion Index was found to be 47. These values were obtained by the extrapolation of slopes of the Promotion Indices at various doses and durations of administration of phenobarbital. The volume percentages of the liver occupied by seven possible phenotypes using three different markers (gamma-glutamyltranspeptidase, canalicular ATPase and glucose-6-phosphatase) were relatively constant in distribution for up to one year of phenobarbital administration except at the two highest doses employed, 0.5% and 0.1%, at which a maximal effect of the promoting agent has been obtained. Possible mechanisms for the biphasic relationship of the Promotion Index of phenobarbital with the dose and time of administration are discussed.

Animals↗
PubMed1987

Effects of tumor-promoting agents 12-O-tetradecanoylphorbol-13-acetate and phenobarbital on DNA synthesis of rat hepatocytes in primary culture.

Both 12-O-tetradecanoylphorbol-13-acetate (TPA) and phenobarbital (PB) enhanced hepatocyte DNA synthesis stimulated with epidermal growth factor (EGF) by 60 to 80% in primary culture when measured by the incorporation of [3H]thymidine. This apparent increase was not due to changes in the specific activity of the deoxythymidine triphosphate (dTTP) pool. TPA enhanced DNA synthesis even at relatively high cell densities, but this was not found with the PB treatment. Although both TPA and PB enhanced DNA synthesis significantly, TPA was most effective when added during the late G1 and/or S phase of the hepatocyte cell cycle, whereas PB treatment was least effective in this period. The binding of EGF was transiently down-regulated by TPA, then restored to control values 6 h later, whereas the binding of this factor was significantly increased at both the 12th and 24th h after PB addition. These results suggest that EGF binding to hepatocytes is not correlated with the enhancement of DNA synthesis by TPA or PB and that the down-regulation of EGF binding is not causally related to the enhancement of DNA synthesis by TPA.

Animals↗
PubMed1987

Stimulation of DNA synthesis in primary cultures of adult rat hepatocytes by sodium butyrate.

Replicative DNA synthesis in primary cultures of adult rat hepatocytes is increased by the addition of the histone deacetylase inhibitors, propionate or butyrate. DNA synthesis was increased by the addition of 0.5 or 1.0 mM butyrate; in contrast 5.0 mM butyrate inhibited replicative DNA synthesis. Replicative DNA synthesis was increased only when low levels of butyrate were added as the hepatocytes entered the S phase. The observed apparent increase in replicative DNA synthesis was real, and not owing to changes in the specific activity of the dTTP precursor pool. The effects of butyrate and propionate on DNA synthesis appear to be related to their effects on histone acetylation.

Acetylation↗
PubMed1987

The control of DNA synthesis in primary cultures of hepatocytes from adult and young rats: interactions of extracellular matrix components, epidermal growth factor, and the cell cycle.

Hepatocytes from adult and 4-week-old rats cultured on one of several extracellular matrix components were stimulated to replicate by epidermal growth factor (EGF). DNA synthesis was increased at 44-48 hr in adult hepatocytes and at 24, 48, and 72 hr in hepatocytes from young rats when EGF was added 2 hr after explantation. When EGF was added at 24 hr, maximal DNA synthesis of adult hepatocytes was observed at 48 hr, whereas that of 4-week-old hepatocytes was seen at 48 and 72 hr. Ten ng EGF per ml was the optimal concentration for maximal DNA synthesis in both adult and young cells. DNA synthesis decreased with increasing cell density, but this effect was less in hepatocytes from young than in those from adults. When hepatocytes were cultured on substrata consisting of individual extracellular matrix components, neither the time that adult cells needed to respond to EGF nor the time from stimulation by EGF to the peak of maximal DNA synthesis was altered in either adult or young cells. The optimal EGF concentration for maximal DNA synthesis and the cell density control of replication were also not altered by the substrata used. Substrata made from each of the extracellular matrix components studied enhanced DNA synthesis of adult and young hepatocytes stimulated by EGF in the following decreasing order: fibronectin, type IV collagen, type I collagen, and laminin. In both adult and young hepatocytes the enhancement of DNA synthesis was greatest when cultured on fibronectin. Thus the initiation and magnitude of DNA synthesis in primary cultures of rat hepatocytes were altered both by the age of the donor and the substratum on which the cells were explanted.

Aging↗
PubMed1987

Specific gene expression during compensatory renal hypertrophy in the rat.

The compensatory growth of the kidney which is induced by unilateral nephrectomy is a highly regulated process resulting principally in hypertrophy of the remaining kidney. The events which regulate this process are unknown. We have examined the levels of transcripts for the proto-oncogenes, myc, H-ras, K-ras, and fos, and the cellular genes, H4 histone, ornithine aminotransferase, and gamma-glutamyl transpeptidase, following unilateral nephrectomy in the rat. The pattern of expression of c-myc, c-H-ras, and c-K-ras during compensatory growth of the kidney differs from the pattern of expression of these proto-oncogenes during liver regeneration, in which, unlike the kidney, hyperplasia rather than hypertrophy predominates. The lack of change in the abundance of these proto-oncogene transcripts following unilateral nephrectomy suggests a primary relationship between the expression of these proto-oncogenes and DNA synthesis and indicates there may be separate signals for cell growth, one to double cell size and one to replicate DNA. Increased mRNA transcripts for the enzymes ornithine aminotransferase and gamma-glutamyl transpeptidase were induced in the contralateral kidney after nephrectomy. The time course of expression for these two enzymes differs. The early expression of the gamma-glutamyl transpeptidase gene may indicate an involvement of this glutathione-metabolizing enzyme during renal compensatory growth, while the function of the delayed increase in ornithine aminotransferase transcripts in the remaining kidney is not apparent.

Animals↗
PubMed1987

Biological markers characterizing the development of preneoplastic and neoplastic lesions in rodent liver.

The identification of biological "markers" indicating distinctively different functions between preneoplastic and neoplastic as compared with normal cells has been a subject of intensive investigation, especially as additional technology becomes available. Although no distinct single biochemical marker is ubiquitous to the process of neoplasia or even to a single histogenetic type of neoplasm, a variety of histogenetic types of neoplasms in the human and experimental animals exhibit an extreme degree of marker or phenotypic heterogeneity. Particularly well studied are markers which occurred during the process of hepatocarcinogenesis in the rodent as well as in its final product, the hepatoma. Although phenotypic heterogeneity is characteristic of hepatocellular carcinomas in both the rat and mouse, some degree of predominant marker pattern(s) has become apparent. In multistage hepatocarcinogenesis in the rat a frequent but not completely ubiquitous marker is the enzyme gamma-glutamyltranspeptidase. In the mouse, although such markers have not been as extensively studied as in the rat, glucose 6-phosphatase is a predominant but not exclusive histochemical marker. Many preneoplastic lesions occurring during the stage of promotion exhibit reduced levels of enzymes of oxidative xenobiotic metabolism, but this pattern is not ubiquitous. Studies on the transcription of specific genes in mouse liver as well as preneoplastic and neoplastic lesions in this tissue further demonstrate the phenotypic heterogeneity characteristic of differentiated hepatocellular carcinomas. In general, current evidence supports the two theses that no single biologic marker or set of markers is uniquely characteristic of the preneoplastical and/or neoplastic phenotype and marker or phenotypic heterogeneity is by far the rule rather than the exception in hepatocarcinogenesis in the rodent and quite possibly in all histogenetic types of neoplasms in mammals.

Albumins↗
PubMed1987

Enzymes of glutathione metabolism as biochemical markers during hepatocarcinogenesis.

Enzymes of glutathione metabolism, particularly gamma-glutamyltransferase (GGT) and glutathione S-transferase (GST), play a role in multistage hepatocarcinogenesis. The enhanced expression of these enzymes in preneoplastic altered hepatic foci, nodules, and hepatocellular carcinomas has been demonstrated after treatment with a variety of initiating and promoting agents. Glutathione is necessary for the detoxification of xenobiotics and carcinogens and for cell replication. Induction of GGT in altered hepatocytes may permit these cells to utilize extracellular glutathione to preserve their internal glutathione levels. GST induction allows glutathione utilization for the protection of the altered hepatocyte in an environment of exposure to xenobiotics, such as promoting agents. Thus, the combined effects of GGT and GST, in a toxic environment, may provide for the enhanced proliferation observed in preneoplastic hepatocytes. New clinical and research opportunities may involve the use of GGT and the placental isozyme of GST (PGST) as markers of preneoplasia and neoplasia in humans. Many factors, such as hormones, diet, and exposure to initiating and promoting agents, influence GGT and GST expression. The recent cloning of cDNAs to GGT and PGST offers opportunities for the study of factors involved in the genetic expression of these two enzymes. Coupled with the use of hepatocyte culture and transplantation, the factors involved at the molecular level in the creation of hepatocellular neoplasia may be discovered.

Animals↗
PubMed1987

Effects of extracellular matrix components on the growth and differentiation of cultured rat hepatocytes.

Some effects of culturing adult rat hepatocytes on each of four different substrates--laminin (LN), collagen type I (C-I), collagen type IV (C-IV), and fibronectin (FN)--have been investigated under defined conditions. No differential effect on the attachment of the cells to the various substrates was noted; however, the spreading of hepatocytes shortly after initial plating was most strikingly enhanced by FN, whereas LN exhibited little or no such enhancement. The two collagen substrates enhanced the spreading of hepatocytes more than did LN, but less than FN. The different substrates had no differential effect on the induction of tyrosine aminotransferase by dexamethasone and glucagon for at least the first 10 d in culture. The longevity of the hepatocytes was not changed significantly by any of the substrates, at least through the 14th d of culture. During the culture periods the hepatocytes at high cell density were maintained as confluent monolayers, regardless of the substrate on which they had been cultured. After 14 d of culture, gamma-glutamyltranspeptidase activity was highest in cells cultured on C-IV, and lowest in those on FN. DNA synthesis in cultured hepatocytes at a low cell density was highest in cells cultured on FN, with decreasing levels of this parameter in cells cultured on C-IV, C-I, and LN, respectively. These results demonstrate that specific components of the extracellular matrix modulate both differentiated functions and the replication of hepatocytes cultured in serum-free medium.

Animals↗
PubMed1987

Inhibition of cyclic AMP-dependent induction of ornithine aminotransferase by simple carbohydrates in cultured hepatocytes.

Glucose administration inhibits the induction of ornithine aminotransferase (OAT) in both the whole animal and cultured hepatocytes. We have examined the ability of several hexoses and related molecules to inhibit the cAMP-dependent induction of OAT in primary cultures of adult rat hepatocytes. The hexoses (D-glucose, fructose, sorbitol, sorbose, and mannose) that were effective as inhibitors of OAT induction also resulted in accumulation of lactate in the culture medium, although lactate itself was not effective as an inhibitor. The hexoses and related 6-carbon structures (galactose, L-glucose, 2-deoxyglucose, 3-O-methylglucose, rhamnose, mannitol, and inositol) that were not effective as inhibitors of OAT induction did not result in accumulation of lactate in the culture medium. These results suggest that the carbohydrate repression of hepatic OAT requires metabolism of the carbohydrate by the liver cell. Upon addition to the culture medium of several compounds related to carbohydrate metabolism, many (ribose, xylitol, dihydroxyacetone, and glycerol) exhibited an inhibitory effect, with glycerol exhibiting the greatest effect. Fructose and glycerol inhibit OAT induction in the presence of 2-deoxyglucose, suggesting that the inhibitory effect of nonglucose carbohydrates is not occurring through conversion to glucose. The carbon sources observed to be most effective as inhibitors of OAT induction (glycerol, fructose, sorbitol, and sorbose result in more than 90% inhibition at 25 mM) all enter the glycolytic pathway at the triosephosphate level. The mechanism of the inhibitory effect of simple carbohydrates on OAT induction is not known but may involve an increase in certain glycolytic intermediates. Glucose and the related carbon sources exert their effect by inhibiting the cAMP-dependent increase in OAT synthesis. The cAMP-dependent increase in OAT mRNA was inhibited by fructose. These findings suggest that the carbohydrate inhibition of the cAMP-dependent increase in OAT synthesis occurs at a pretranslational level.

Animals↗