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H C Pitot

Publications and source records attributed to H C Pitot.

At least 181 records · Page 10Linked to original sources
PubMed1989

Human gamma-glutamyl transpeptidase cDNA: comparison of hepatoma and kidney mRNA in the human and rat.

gamma-Glutamyl transpeptidase (GGT) is a glutathione-metabolizing enzyme that has been extensively studied in relation to hepatocarcinogenesis. Using a cDNA for rat kidney GGT as a probe, we have isolated a full-length cDNA for human GGT from a hepatoma cell-line library. Nucleotide sequence analysis of the clone revealed a 2326-bp insert that includes a 5'-untranslated region of 487 nucleotides (nt), an open reading frame (ORF) of 1707 nt, and a 3'-untranslated region of 132 nt. The ORF encodes a protein with an amino acid sequence that is highly similar to that of the rat GGT precursor peptide, with an overall identity of 79%. The cDNA clone was used to probe Northern blots of hepatoma and kidney RNA from both human and rat. In both species, the GGT mRNA is longer in hepatoma than in kidney. In addition, the human mRNAs were longer than their counterparts in the rat. None of three human hepatocellular carcinomas examined showed a marked elevation in GGT mRNA levels relative to surrounding liver tissue.

Amino Acid Sequence↗
PubMed1989

The ornithine aminotransferase gene in gyrate atrophy of the retina: analysis of expression and gross structure of this gene in cultured fibroblasts.

Gyrate atrophy (GA), a degenerative disease of the human chorioretina, is associated with a deficiency of ornithine aminotransferase (OAT) activity, hyperornithinemia, and ornithinuria. We have characterized a cDNA clone for OAT (HLOAT) that was isolated from a cDNA library constructed from mRNA prepared from Hep G2 cells, a human hepatoma cell line. We have used HLOAT and a nearly full length OAT cDNA clone isolated from a rat liver library (RLOAT) to examine in cultured fibroblasts from individuals with GA and control individuals, the expression of OAT mRNA and the gross structure of the OAT gene. Northern blot analyses of total cellular RNA indicated that 3 of 3 control cell lines and 5 of 6 GA cell lines are capable of expressing an OAT related mRNA of approximately 2100 bases, the size of OAT mRNA. To date, this is the only case of GA in which a complete lack of OAT mRNA has been observed. Southern blot analyses of DNA isolated from these cell lines indicated that the gross structure of the OAT gene is usually not detectably altered in individuals with GA. However, a unique pattern of restriction fragments was observed upon digestion with Eco RI or Hind III of DNA from the GA cell line that does not express OAT mRNA. These unique Eco RI and Hind III fragments arise from the OAT structural gene and will serve as useful molecular markers that allow this particular defective OAT allele to be identified. When the cellular DNAs were digested with Hinf I and examined with a probe that corresponds to at least a portion of the active site of the enzyme, i.e., the pyridoxal phosphate binding site, identical patterns of fragments were detected in all samples. Therefore, it appears unlikely that the loss of OAT activity associated with these GA cases, 4 of which are pyridoxal phosphate responders, is the result of insertions or deletions in this region of the OAT gene. This study indicates that the lack of OAT enzyme activity associated with GA is the result of a variety of different molecular defects within the OAT gene.

Alleles↗
PubMed1989

Regulation of the expression of some genes for enzymes of glutathione metabolism in hepatotoxicity and hepatocarcinogenesis.

The reversible stage of tumor promotion, which follows the stage of initiation and precedes that of progression in multistage carcinogenesis, is a unique example of reversible toxicity in biological systems. In order to study the molecular mechanisms involved in the action of promoting agents during this stage, the regulation of the expression of genes for two enzymes of glutathione metabolism, gamma-glutamyl transpeptidase (GGT) and the placental isozyme of glutathione S-transferase (GST-P), was studied under several different conditions of promotion during multistage hepatocarcinogenesis in the rat. Promotion by phenobarbital caused an increased expression of both of these genes in altered hepatic focal lesions, although this was somewhat more variable in the case of the GGT gene. C.I. Solvent Yellow 14, an industrial dye, served as an effective promoting agent. Feeding this dye resulted in a dramatic increase in the expression of GST-P, but not that of GGT in altered hepatic foci. Factors in crude, cereal-based diets inhibited the stage of promotion by diethylnitrosamine, but enhanced promotion by phenobarbital in a synergistic manner. In contrast, at least one purified diet had the converse effect during this stage. The mRNA levels of GST-P were uniformly elevated dramatically in reversible nodules and neoplasms of rat liver that had been induced by diethylnitrosamine and phenobarbital promotion. In contrast, the level of GGT mRNA was somewhat variable, with an occasional neoplasm exhibiting almost a background level of expression of this gene. Therefore, the altered regulation of multiple genes in hepatocytes during the stage of promotion can vary with the promoting agent itself; this process may be related to the heterogeneous gene expression seen in hepatic neoplasms. A possible role for specific DNA sequences in the 5' flanking regions of such genes is considered. In addition, a cDNA clone to the mRNA of human liver GGT was isolated and sequenced. The homology of the coding sequence of the human liver GGT mRNA to that of rat kidney GGT mRNA was striking.

Animals↗
PubMed1989

Proto-oncogene activation during chemically induced hepatocarcinogenesis in rodents.

The liver is a frequent site for the development of chemically induced cancer in rodents. This is primarily owing to the capability of the liver to activate a large variety of exogenous chemicals metabolically to reactive electrophilic species that can covalently interact with cellular DNA and other macromolecules (Miller and Miller, 1966; Miller, 1978). It is the potential alteration of the hepatocellular genome by mutational events that forms the theoretical basis for the heritable nature of cancer as well as, at least in part, the altered phenotype of neoplastic cells; however, our understanding of the exact nature of these heritable genetic alterations remains fragmentary. Within the last decade the delineation of the molecular basis of viral oncogenesis, especially by retroviruses, has revealed potential targets in the cell genome for the reactive forms of chemical agents in relation to their carcinogenic action (Bishop, 1987). Primary among such potential targets are proto-oncogenes, homologous to the transforming genes of oncogenic retroviruses from which they have evolved (Temin, 1974). The objective of this brief review is to consider the evidence that induced alterations in the structure and/or regulation of expression of proto-oncogenes may play one or more roles in rodent hepatocarcinogenesis, especially in relation to the stages of initiation, promotion, and progression (Pitot et al., 1988).

Animals↗
PubMed1989

Effect of the antihistamine, methapyrilene, as an initiator of hepatocarcinogenesis in female rats.

The antihistamine methapyrilene was examined for its ability to initiate hepatocarcinogenesis in rats. Rats were first subjected to partial hepatectomy and then were intubated with one of four doses (30, 100, 200 or 300 mg/kg) of methapyrilene hydrochloride (or an equivalent amount of water for controls, or 10 mg diethylnitrosamine/kg for positive controls). Rats were then fed 0.05% phenobarbital in the diet for 3, 6 or 9 months. The number and volume of altered hepatic foci were quantified with the histochemical markers gamma-glutamyl transpeptidase, glucose-6-phosphatase, and ATPase. The number of foci induced was increased 2- to 4-fold by the highest dose of methapyrilene at all 3 time points, but the only statistically significant increase was produced by the 200 mg/kg dose after 3 months of promotion. This study shows that methapyrilene may act as a weak initiator.

Adenosine Triphosphatases↗
PubMed1989

A semipurified diet that suppresses phenobarbital promotion of hepatocarcinogenesis in the rat.

Six groups of F344/N female rats were fed either a modified AIN-76 diet (20% casein, 5% corn oil, 65% cornstarch, 5% cellulose) (AIN) or a diet formulated by Dr. M. Pariza (PD) (30% casein, 10% partially hydrogenated corn oil, 40% sucrose, 15% cornstarch) beginning four days before 70% partial hepatectomy. One day after the surgery, one group fed each diet was intubated with 10 mg/kg diethylnitrosamine (DEN). One week later, these groups plus one control group fed each diet were given 0.05% phenobarbital in the diet for 6 or 14 months. After the rats were killed, blocks of liver tissue were frozen on dry ice and stored at -70 degrees C. Three frozen serial sections were stained for gamma-glutamyltransferase, ATPase, and glucose-6-phosphatase. Numbers and volume of altered hepatic foci (AHF) were analyzed by stereological techniques. After 14 months of feeding these regimens, rats initiated with DEN and fed the AIN + PB had significantly greater numbers and a higher percent volume of the liver of most phenotypes of AHF than all other groups, including those fed PD + PB following initiation with DEN. The numbers of AHF exhibiting more complex phenotypes (i.e., scored by more than one marker) remained unaltered between 6 and 14 months. These findings indicate that the effectiveness of PB as a promoting agent in multistage hepatocarcinogenesis is significantly altered when fed with two different diets of known composition. Therefore, dietary composition can be a significant factor in studies of the stage of promotion in hepatocarcinogenesis.

Animals↗
PubMed1989

Ploidy and karyotype of hepatocytes isolated from enzyme-altered foci in two different protocols of multistage hepatocarcinogenesis in the rat.

The ploidy and karyotypes of hepatocytes isolated from the livers of rats subjected to the protocols of Peraino et al. and of Solt and Farber were determined by the examination of such cells in primary culture. A study of 100 or more metaphases from each of five rats on each protocol revealed that 75-80% of gamma-glutamyltranspeptidase-positive (GGT+) hepatocytes isolated from livers of rats in either protocol were diploid, whereas only 23-33% of GGT- cells were diploid. Fifty percent or more of the karyotypes of hepatocytes from livers of rats receiving the Solt-Farber protocol exhibited one or more chromosomal breaks, whereas hepatocytes from livers of rats subjected to the Peraino protocol showed no increase in chromosomal breakage over that in normal controls. These studies demonstrate that the majority of GGT+ cells from altered hepatic foci are diploid and that the greater toxicity of the Solt-Farber protocol over that of Peraino is correlated with marked chromosomal breakage of GGT+ hepatocytes.

Animals↗
PubMed1989

Critical parameters in the quantitation of the stages of initiation, promotion, and progression in one model of hepatocarcinogenesis in the rat.

Critical parameters in the quantitation of altered hepatic foci (AHF) developing during multistage hepatocarcinogenesis in the rat include: 1) the enumeration of AHF induced by test agents as well as those AHF occurring spontaneously in livers of untreated animals; 2) the volume percentage or fraction of the liver occupied by all AHF as a reflection of the total number of altered cells within the liver and the degree of tumor promotion which has occurred; and 3) the phenotype of individual AHF as determined by multiple markers with serial sections. These parameters, especially the number of AHF, should be corrected by the presence of spontaneous AHF which increase with the age of the animal, more so in males than females. While accurate estimation of the background level of spontaneous AHF can be important in demonstrating that a carcinogenic agent does not possess the ability to increase the numbers of AHF above the background level, a better method to distinguish the effectiveness and relative potencies of agents as initiators or promoters is reviewed. The relative effectiveness of four different markers--gamma-glutamyltranspeptidase (GGT), a placental form of glutathione S-transferase (GST), canalicular ATPase, and glucose 6-phosphatase (G6Pase)--was described for the chemicals C.I. Solvent Yellow 14 and chlorendic acid as promoting agents in males and females. C.I. Solvent Yellow 14 is a more effective promoting agent in females than males, and AHF exhibit extremely low numbers scored by GGT. On the other hand, the numbers of AHF present in livers of male rats promoted by this agent are more than twice those seen in livers of female animals, possibly owing to the effectiveness of this agent as an initiator in the male but not the female. Very few AHF, especially in the male, are scored by GGT during chlorendic acid promotion. The distribution of phenotypes with these markers also differs in the spontaneous AHF appearing in the livers of animals fed 0.05% phenobarbital on either a crude NIH-07 or AIN-76 purified diet. Such studies emphasize the extreme dependence of the promoting stage of hepatocarcinogenesis on environmental factors of sex, diet, and the molecular nature of the promoting agent itself. The hallmark of the final stage of progression in the development of hepatocellular carcinomas is aneuploidy, which may be reflected by phenotypic heterogeneity within individual AHF, termed foci-in-foci. The implications of such quantitative analyses during hepatocarcinogenesis induced by specific agents in relation to the specific action of the agent at one or more of the stages of hepatocarcinogenesis are discussed.

Animals↗
PubMed1989

Use of rat liver altered focus models for testing chemicals that have completed two-year carcinogenicity studies.

Partial hepatectomy (PH) and neonatal rat short-term liver focus models were used to examine the effects of selected chemicals that had been previously tested in the National Toxicology Program (NTP) 2-yr carcinogenicity studies. C.I. Solvent Yellow 14, monuron, chlorendic acid, and 4-hydroxyacetanilide were tested for initiating and promoting activity in the PH model. Chlorendic acid, 4,4'-oxydianiline, 1-amino-2,4-dibromoanthraquinone (ADBAQ), and 4-hydroxyacetanilide were similarly tested in a neonatal rat liver focus model. With the exception of 4-hydroxyacetanilide which was not carcinogenic in the NTP studies, all chemicals tested showed clear evidence of hepatocarcinogenicity. While none of the chemicals showed initiating activity in either the PH or neonatal models, promoting activity, as indicated by increased number, size, or volume fraction of histochemically detected hepatic foci of cellular alteration, was evident for all chemicals with previously demonstrated hepatocarcinogenicity. Liver tumor incidence was documented at 14 months in the PH model and at 300 days in the neonatal model. On the basis of the results obtained from these few chemicals, it is suggested that the use of short-term rat liver focus models may represent a reliable means for identifying chemicals with hepatocarcinogenic potential.

Animals↗
PubMed1988

Lack of expression of glutathione-S-transferase P, gamma-glutamyl transpeptidase, and alpha-fetoprotein messenger RNAs in liver tumors induced by peroxisome proliferators.

Many structurally unrelated nonmutagenic peroxisome proliferators induce altered areas, neoplastic nodules, and hepatocellular carcinomas in rats. Unlike the lesions induced by genotoxic hepatocarcinogens, these lesions do not stain positively for the phenotypic markers gamma-glutamyl transpeptidase (GGT) and glutathione-S-transferase P (GST-P). To ascertain whether the absence of immunocytochemically detectable GST-P and GGT proteins in peroxisome proliferator-induced neoplastic lesions is due to the absence of specific mRNAs, we analyzed the total RNA isolated from hepatocellular carcinomas induced by three different peroxisome proliferators (ciprofibrate, Wy-14643, and BR-931) and the genotoxic carcinogens, 2-acetylaminofluorene and aflatoxin B1 (AFB), for the presence of GST-P, GGT, and alpha-fetoprotein (AFP) mRNAs. Northern and dot blot analysis of total RNA isolated from liver tumors induced by three different peroxisome proliferators revealed no detectable GST-P, GGT, and AFP mRNAs. GST-P mRNA was also not detected in a transplantable hepatocellular carcinoma established from a liver tumor induced by ciprofibrate. In contrast, GST-P mRNA levels were high in primary liver tumors induced by both 2-acetylaminofluorene and AFB and the two transplantable hepatocellular carcinomas established from such tumors. By immunoblot method, GST-P protein was found to be abundant in both primary and transplantable liver tumors induced by genotoxic carcinogens but not in those derived from peroxisome proliferator treatment. The GGT and AFP mRNAs were also not found in all 18 liver tumors induced by peroxisome proliferators that were analyzed and also in the ciprofibrate-derived transplantable liver tumor. The expression of GGT and AFP genes in liver tumors induced by 2-acetylaminofluorene and AFB was variable. These studies with peroxisome proliferators show that the GST-P and GGT gene derepression is not essential for the hepatocarcinogenesis or successful tumor transplantation. Further characterization of the molecular basis for the differential expression, particularly of the GST-P gene in liver tumors, may help identification of the critical event(s) in hepatocarcinogenesis by genotoxic carcinogens and nongenotoxic peroxisome proliferators.

Animals↗
PubMed1988

Differential gene expression in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Noncoordinate regulation of a TCDD-induced aldehyde dehydrogenase and cytochrome P-450c in the rat.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) regulates the transcription of a specific subset of genes through a receptor-mediated mechanism. We have isolated a cDNA to a TCDD-inducible rat liver aldehyde dehydrogenase and have shown that its induction by TCDD differs from that of another TCDD-induced gene, cytochrome P-450c, with regard to dose-response relationship, induction kinetics, and tissue specificity. At least a 10-fold higher dose of TCDD was required for half-maximal induction of TCDD-inducible rat liver aldehyde dehydrogenase in rat liver than the dose that half-maximally induced cytochrome P-450c. Further, the kinetics of induction of TCDD-inducible rat liver aldehyde dehydrogenase by TCDD in rat liver was delayed compared with that of cytochrome P-450c. Striking discrepancies were found in the capacity of various organs to induce both TCDD-inducible rat liver aldehyde dehydrogenase and cytochrome P-450c in a coordinated manner in response to TCDD. Organs that were able to evoke one of these responses to TCDD were not necessarily able to evoke coordinately the other response. The capacity of an organ to exhibit either of these two responses to TCDD did not correlate stringently with reported Ah receptor abundance. Our data suggest that TCDD can modulate the expression of specific genes in different ways and that regulatory pathways in addition to the classically defined Ah receptor may be involved.

Aldehyde Dehydrogenase↗
PubMed1988

The expression and metabolic activity of cytochrome P-450 isozymes in control and phenobarbital-induced primary cultures of rat hepatocytes.

The expression and activity of the phenobarbital (PB)-inducible P-450 isozymes, P-450b and P-450e, and the major 3-methylcholanthrene (MC)-inducible form, P-450c, were studied in primary cultures of adult rat hepatocytes in T1, Leibovitz L-15 (L-15), and a modification of Waymouth 752/1 (Way) media. P-450 isozymes in initially isolated hepatocytes and control and PB-treated cultures were quantitated by Western blot analysis, and activity was determined with 7,12-dimethylbenz[a]anthracene (DMBA) as substrate. Data from the Western blot analysis correlated well with the metabolic activity toward DMBA. P-450b was consistently induced by PB in hepatocytes in T1 and to a lesser extent in Way. P-450e protein was constitutive in initially isolated cells, expressed in control cultures at a reduced level, and increased or maintained by PB in all three media. DMBA metabolite formation associated with P-450b and P-450e activity was induced by PB in hepatocytes in T1 and Way and was inhibited by antibodies to P-450b. P-450c was only infrequently expressed in freshly prepared hepatocytes, but was detected in all control and PB-treated cultures although at a much higher level in T1. Thus, the amounts of P-450 isozymes, their inducibility by PB, and their activity toward DMBA were found to be dependent on the medium. We have demonstrated enzyme induction and increased activity of the major PB-inducible isozymes in hepatocytes in T1; these are also associated with a change in the control of P-450c expression leading to enhanced constitutive expression and inducibility by phenobarbital.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1988

Expression of the c-raf protooncogene, gamma-glutamyltranspeptidase, and gap junction protein in rat liver neoplasms.

Female Harlan Sprague-Dawley and F-344 rats were subjected to a 70% hepatectomy and 18 h later given one dose of 30 mg diethylnitrosamine/kg body weight. Beginning 1 week later, the animals were fed a diet containing 0.05% phenobarbital. Groups of rats were sacrificed 6 and 15 months later, and the livers were either frozen for cryostat sectioning or used to isolate RNA. Primary liver tumors present in these animals were used for RNA isolation, and a portion was taken for histopathological analysis. Eleven of 13 primary lesions, consisting of either neoplastic nodules or hepatocellular carcinomas, showed elevated levels of mRNA for the c-raf protooncogene. Increased c-raf mRNA in these tumors appeared to be unrelated to their cellular proliferative status inasmuch as the levels of c-raf mRNA did not correlate with levels of H4 histone mRNA. Decreased expression of the major rat liver gap junction protein mRNA was observed in all of the primary tumors. Immunocytochemical analysis using an anti-gap junction antibody revealed a decrease in gap junction immunoreactivity in some but not all preneoplastic focal lesions. All preneoplastic foci having positive gamma-glutamyltranspeptidase enzyme staining also exhibited a marked increase in gamma-glutamyltranspeptidase mRNA as determined by in situ hybridization. The possible relation of alterations of the mRNA levels of c-raf and the gap junction protein to the further development of preneoplastic foci is discussed.

Animals↗
PubMed1988

Sodium butyrate preserves aspects of the differentiated phenotype of normal adult rat hepatocytes in culture.

We have determined that sodium butyrate and, to a lesser extent, dimethylsulfoxide (DMSO) and 3-aminobenzamide (3-AB) preserve aspects of the differentiated phenotype of primary cultures of adult rat hepatocytes. The histone deacetylase inhibitor, butyrate, inhibits the increase in gamma-glutamyltranspeptidase (GGT) activity and the decrease in basal tyrosine aminotransferase (TAT) activity normally observed when hepatocytes are cultured under appropriate conditions. The effects of butyrate on GGT and TAT activities are accompanied by parallel changes in GGT and TAT mRNA levels. The poly(ADP)ribose-synthetase inhibitor, 3-aminobenzamide, has effects similar to butyrate on GGT activity and mRNA levels, while both 3-AB and DMSO increase basal TAT activity in cultured hepatocytes. Under appropriate conditions all three agents--butyrate, 3-AB, and DMSO--extend the length of time cultured hepatocytes can be maintained as confluent monolayers. However, under all the conditions studied, butyrate extended the length of time hepatocytes could be maintained as monolayers more than any other treatment used. Butyrate-treated hepatocytes maintained ultrastructural features that were more similar to those of hepatocytes in vivo than hepatocytes treated with any other of the agents tested. Histone acetylation levels of primary cultures of adult rat hepatocytes declined concomitant with the loss of the differentiated phenotype of the cells. These results suggest that histone acetylation may play a role in the changes in gene expression observed when hepatocytes are placed in culture.

Animals↗