Biomedical subjects
H C Pitot
Publications and source records attributed to H C Pitot.
A method to quantitate the relative initiating and promoting potencies of hepatocarcinogenic agents in their dose-response relationships to altered hepatic foci.
The relative response to various initiating doses of diethylnitrosamine (DEN) and dimethylbenz[a]anthracene of the induction of numbers and size (vol. % of liver) of altered hepatic foci (AHF) in livers of adult female rats of the Sprague-Dawley and Fischer 344 (F-344) strains was studied by methods of quantitative stereology in the presence and absence of the promoting agent, phenobarbital (PB, 0.05% in the diet). In all cases, a relatively linear response with dose, even at the lowest doses employed, was obtained except for the numbers of AHF at the highest dose of DEN (30 mg/kg), which was not significantly different from that at a dose of 10 mg/kg in F-344 female rats. Similar dose-response data were obtained at various doses of two promoting agents effective in hepatocarcinogenesis, PB and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), in livers of F-344 female rats following initiation with DEN (10 mg/kg) 24 h post-70% hepatectomy. The response to these agents exhibited threshold levels below which no increase in number or vol. % of liver of AHF was noted in comparison with that in livers of animals not treated with the promoting agents. At several subthreshold doses of both PB and TCDD an inhibition of AHF formation and growth (measured as vol. % of liver) was observed. Based on quantitative stereologic calculations, parameters for the estimation for the relative potency of chemicals as initiating or promoting agents have been established. These are defined as: initiation index = no. of foci induced X liver-1 X [mmol/kg body wt]-1 and promotion index = Vf/Vc X mmol-1 X weeks-1, where Vf is the total volume fraction (%) occupied by AHF in the livers of rats treated with the test agent and Vc is the total volume of AHF in control animals which have only been initiated. These parameters were calculated for a number of agents based on data published in the literature and from those reported herein. Neither parameter varied significantly with the dose of the initiating agent based on the data in this paper. The range of promotion indices extended over more than eight orders of magnitude, whereas that of initiation indices was much less variable. Such parameters may be useful as quantitative estimates of the potency of hepatocarcinogenic agents, such values having potential application to risk estimations.
Quantitative stereological evaluation of four histochemical markers of altered foci in multistage hepatocarcinogenesis in the rat.
Female F344/N rats dosed with diethylnitrosamine (DEN) 24 h after partial hepatectomy were treated with the promoting agents, phenobarbital (PB) or 3,4,7,8-tetrachlorodibenzo-p-dioxin (TCDD), or the peroxisome proliferating agent, WY 14,643, for 6 months. Another group was subjected to the Solt-Farber protocol. Altered hepatic foci (AHF) were analyzed by quantitative stereology from frozen serial sections stained for gamma-glutamyl transferase (GGT), canalicular adenosine triphosphatase (ATPase), glucose-6-phosphatase (G6Pase) and the placental isozyme of glutathione S-transferase (PGST). PGST scored more foci in all groups than GGT and ATPase. PGST marked greater focal volume than GGT or ATPase, and PGST marked focal volume equal to or greater than G6Pase in rats treated with PB, TCDD or the Solt-Farber protocol. However, after treatment with WY 14,643, GGT and PGST marked much less focal volume than ATPase or G6Pase, and PGST scored fewer foci than G6Pase. Numerical estimations of foci scored by those markers on the basis of area of the entire tissue section (per cm2) were relatively different from those values determined by quantitative stereology. While these results confirm earlier studies, they demonstrate the importance of quantitative stereologic analysis of AHF during multistage hepatocarcinogenesis.
An approach to the determination of the relative potencies of chemical agents during the stages of initiation and promotion in multistage hepatocarcinogenesis in the rat.
The potency of carcinogenic agents in eliciting neoplastic lesions has long been a concern of investigators in the field of oncology. This paper describes a method, based on quantitative stereologic calculations, to estimate the relative potency of chemicals as initiating and/or promoting agents. The parameters defined in this paper are: (a) Initiation index = no. foci induced X liver-1 X [mmole/kg body weight]-1; and (b) Promotion index = Vf/Vc X mmol-1 X wk-1. These parameters have been calculated for a number of chemical agents, based both on data from this laboratory and others published in the literature. Neither parameter varied significantly with the dose of two different initiating agents used in this study. The range of promotion indices extended over more than eight orders of magnitude, whereas that of the initiation indices was much less variable. Such parameters may be useful as quantitative estimates of the potency of hepatocarcinogenic agents not only in rodents, but potentially in quantitative risk estimations in the human.
Biochemical and immunologic studies on the native and deglycosylated forms of gamma-glutamyltranspeptidase of rat kidney.
We have deglycosylated the enzyme gamma-glutamyl transpeptidase by treatment of the protein with anhydrous hydrofluoric acid at 0 degree C. After deglycosylation, the heavy and light subunits showed a molecular weight of 43 and 23 Kd respectively. Whereas the antiserum against the native enzyme recognized both proteins, the antiserum against the deglycosylated enzyme failed to recognize the native enzyme, indicating that some of the determinants of the native enzyme are masked by the carbohydrate moiety.
Characterization and sequence of a cDNA clone of gamma-glutamyltranspeptidase.
We have isolated several cDNA's complementary to gamma-glutamyltranspeptidase (GGT) mRNA by screening a rat kidney library constructed in lambda gtll with antibodies specifically reactive to the enzyme protein. The clone selected an mRNA that was translated into a 62 Kd peptide, corresponding to the GGT precursor. The longest clone isolated was 1842 bp long with an open reading frame coding for 565 amino acids. The length of the mRNA coding for GGT was estimated to be 2.2 kb long. The amino acid sequence derived from the nucleotide sequence matched the short sequences determined by us as well as by other authors.
Use of a low-speed, iso-density percoll centrifugation method to increase the viability of isolated rat hepatocyte preparations.
A simple yet effective method (iso-density percoll centrifugation) has been developed for consistently preparing isolated rat liver parenchymal cells with over 98% initial viability. The method has been applied to cells isolated by a variety of collagenase digestion techniques. This procedure involves the low-speed centrifugation (50 X g) of the initial cell suspension through a percoll medium having a density of 1.06 g/ml and results in the separation of single and viable parenchymal cells from cell aggregates, debris, and nonparenchymal cells. The enriched parenchymal cells have been shown to be superior to untreated cells by a number of criteria including: preparation homogeneity, cell morphology, maintenance of cytochrome P-450, hormonal responsiveness (measured by the induction of tyrosine aminotransferase after treatment with glucagon or dexamethasone, or both), plasma membrane integrity (determined by both trypan blue exclusion and leakage of glutamic-oxaloacetic transaminase), and the DNA repair capability after treatment with benzo[a]pyrene or 2-acetylaminofluorene.
Extracellular matrix components influence DNA synthesis of rat hepatocytes in primary culture.
The effects of several extracellular matrix components (EMCs)--fibronectin (Fn), laminin (Ln), type I (C-I) and type IV (C-IV) collagen--on DNA synthesis in rat hepatocytes in primary culture were examined by both quantitative scintillation spectrometry and autoradiography of [3H]thymidine incorporation. Hepatocytes cultured on Fn showed the most active DNA synthesis initiated by epidermal growth factor (EGF) with decreasing levels of [3H]thymidine uptake exhibited in the cells cultured on C-IV, C-I, and Ln, respectively. The decreasing level of DNA synthesis in hepatocytes cultured on Fn, C-IV, C-I, and Ln respectively was not influenced by cell density. The number of EGF receptors of hepatocytes was also not influenced by EMCs. These data suggest that EMCs modify hepatocyte DNA synthesis by means of post-EGF-receptor mechanisms which are regulated by both growth factors and cell density.
Tumor-promoting barbiturates act on DNA repair of cultured hepatocytes.
We have observed that two promoters of liver carcinogenesis, i.e. phenobarbital and barbital, markedly increase DNA-repair synthesis of cultured hepatocytes following treatment with the ultimate carcinogens methyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, N-acetoxy-2-acetylaminofluorene, and UV light of 254 nm. Phenobarbital also increased the incorporation rates of deoxynucleoside triphosphates into nuclear DNA of permeabilized hepatocytes following carcinogen treatment. The action of these barbiturates apparently correlates with their potential to promote hepatocarcinogenesis in vivo, since the non-promoting agent barbituric acid did not modify carcinogen-induced repair synthesis. Moreover, the mechanisms of action of tumor-promoting barbiturates is different from the known enhancing action on repair synthesis of inhibitors of nuclear poly(ADP-ribose) biosynthesis.
The phenotypic stability of altered hepatic foci: effect of the short-term withdrawal of phenobarbital and of the long-term feeding of purified diets after the withdrawal of phenobarbital.
The effects of the short-term withdrawal of phenobarbital (PB) and of the feeding of purified diets during the long-term withdrawal of dietary PB on the stability of altered hepatic foci (AHF) were studied. In both experiments female CD rats initially received an intragastric dose of diethylnitrosamine (10 mg/kg) 20 h after being subjected to partial hepatectomy. In the short-term study rats were fed 0.05% PB in a cereal-based diet for 6 months; at this time half of the rats were killed, whereas the other half were withdrawn from PB for 10 days before sacrifice. Withdrawing PB for 10 days resulted in a decrease in the number and volume of AHF, particularly those which stained positively for gamma-glutamyltranspeptidase (GGT). In the long-term experiment rats were fed 0.05% phenobarbital in a cereal-based diet for 3 months; they were then withdrawn from the cereal-based diet containing PB and fed either a low-fat or a high-fat purified diet without PB for 8 months. At this time the number and volume of AHF were much less than that seen at the time of PB withdrawal. In addition, the distribution of phenotypes was altered: the percentage of foci containing GGT as a marker decreased dramatically. These results indicate that certain phenotypic characteristics of AHF, specifically their observable number and total volume, rapidly decrease after the withdrawal of PB from rats fed a cereal-based diet and that the feeding of purified diets after such PB withdrawal does not result in the reappearance of AHF. These studies suggest that the phenotype, volume and observable number of AHF may change in the absence of PB as compared with its presence in the diet and that some cereal-based diets may contain unspecified agents which 'stabilize' or enhance the phenotypic appearance of AHF.
The phenotypic stability of altered hepatic foci: effects of withdrawal and subsequent readministration of phenobarbital.
Previous studies from this and a number of laboratories have demonstrated that the majority of both focal and nodular lesions which occur during the early phase of multistage hepatocarcinogenesis in the rodent disappear or 'remodel' upon removal of the promoting agent. Utilizing techniques of quantitative stereology we have found that many if not the majority of altered hepatic foci induced by initiation with diethylnitrosamine/partial hepatectomy and promoted for 4 months with phenobarbital disappear with little or no morphologic evidence of remodeling within 10 days after withdrawal from phenobarbital and do not reappear for a 6-month period thereafter. However, administration of phenobarbital in the diet following an interval period of 10, 20, 30 or 90 days in the absence of the promoting agent resulted in the reappearance of these focal lesions at the same or an increased numerical level. The volume occupied by these focal lesions, which reflects the total numbers of altered cells present in the liver, increased somewhat even during the interval absence of phenobarbital but increased dramatically upon readministration of the promoting agent. Rats fed only a semi-purified diet for 180 days following a 4-month period of phenobarbital promotion of initiated animals exhibited a greater number but only half the total volume of altered hepatic foci, as did rats fed a cereal-based diet only for 180 days. The cereal-based diet (NIH-07) may contain some unknown initiating and/or promoting factors responsible for this phenomenon. These studies present further evidence both for the reversible nature of the stage of promotion of hepatocarcinogenesis and the irreversible characteristic of the stage of initiation, as well as the importance of the composition of the diet utilized in models of multistage hepatocarcinogenesis.
Application of quantitative stereology to the evaluation of phenotypically heterogeneous enzyme-altered foci in the rat liver.
Quantitative stereologic relationships are applied in this report to the evaluation of F344 rat liver foci where the tissue sections exhibit congruent enzyme-altered areas of the several different phenotypes as well as enzyme-altered areas within a larger area of another enzyme alteration, that is, a "focus within a focus.' Quantitation of both the numbers and volume occupied by each of the phenotypes of the enzyme-altered foci was accomplished by the unique logic described in this report. The application of this logic to four representative experimental protocols with the use of three phenotypic markers demonstrated all possible congruent phenotypes as well as a small number of "foci within foci.' The variance of the quantitation of the experimental data was shown to depend on the number of focal transections identified in the sections, the number of sections examined, and the distribution of phenotypic alterations among foci.
Models of hepatocarcinogenesis in the rat--contrasts and comparisons.
A variety of approaches for the induction of altered hepatic foci, hyperplastic nodules, and hepatocellular carcinomas in rat liver have been developed. These protocols, aided by the appearance of preneoplastic lesions during the carcinogenic process, have proven to be very useful for examining many of the characteristics of events involved in rat hepatocarcinogenesis. A number of models have demonstrated distinct steps or stages in the progression of the carcinogenic process. These protocols are currently employed in the classification and distinction of agents effecting hepatocarcinogenesis at one or more of its stages. This review presents an overview of the present-day multistage hepatocarcinogenesis model systems in the rat, with contrasts and comparisons of these systems. The potential uses of the model systems in the identification of complete carcinogens, initiating agents, and promoting agents devoid of initiating action are discussed.
Influence of dietary fat on the promotion of diethylnitrosamine-induced hepatocarcinogenesis in female rats.
The effect of varying the amount and type of dietary fat on the promotion of gamma-glutamyltranspeptidase (GGT)-positive foci and hepatocarcinomas in female rats was studied. In the first study, two-thirds of the rats were first intubated with diethylnitrosamine (DEN, 10 mg/kg) 20 hr after partial hepatectomy; 1 week later, rats were fed one of three purified diets (a low-fat diet similar to the AIN-76 diet, a high saturated fat diet, or a high polyunsaturated fat diet) with or without 0.05% phenobarbital in the diet for 10 months. Increasing the fat level of the diet did not increase the number of GGT-positive foci arising spontaneously or induced by DEN alone. When phenobarbital was present in the diet, feeding the high polyunsaturated fat diet slightly increased the number of GGT-positive foci and the incidence of tumors. The low-fat diet, however, increased the incidence of fatty liver. We therefore reexamined the effect of diet on promotion by phenobarbital, using an additional low-fat diet with cornstarch rather than sucrose as the carbohydrate source. In this experiment, both high-fat diets slightly enhanced the induction of GGT-positive foci; the carbohydrate source had no effect. The incidence of tumors was not affected by diet in this experiment, but the incidence of fatty liver was again enhanced by feeding a diet high in sucrose. We conclude that increasing the fat level of the diet does not promote the development of DEN-initiated GGT-positive foci or carcinomas in female rats. Increasing the dietary fat level, however, may enhance promotion of liver foci by phenobarbital. Finally, increasing the sucrose content of the diet increases the incidence of fatty liver.
Expression of albumin messenger RNA detected by in situ hybridization in preneoplastic and neoplastic lesions in rat liver.
The process of chemical hepatocarcinogenesis is characterized by the appearance of preneoplastic lesions showing changes in the expression of various marker enzymes. We have analyzed the phenotype of small preneoplastic foci and expansively growing nodules in liver sections obtained from rats treated with various carcinogens. Changes within the lesions in canalicular adenosine triphosphatase, gamma-glutamyl transpeptidase, NADPH-(cytochrome P-450) reductase, cytochrome P-450 PB2, epoxide hydrolase, and glycogen content were detected by means of enzyme histochemical and immunohistochemical staining procedures. In parallel sections the expression of albumin messenger RNA was investigated by in situ hybridization using a 35S-labeled albumin specific complementary DNA probe. In general, small preneoplastic lesions showed unchanged levels of albumin messenger RNA. In contrast, the expression of albumin messenger RNA was found to be reduced to varying degrees in large hepatic nodules. An expression of alpha-fetoprotein messenger RNA could not be detected in any of the nodules. No direct correlation between the enzyme phenotype of the lesions and the degree in reduction of albumin messenger RNA could be established except that the reduction was most pronounced in nodules which had lost their ability to store glycogen. Since the synthesis and excretion of albumin is a typical function of the differentiated hepatocyte in the adult animal, the observed decrease in albumin messenger RNA expression in large hepatic nodules is in accordance with the hypothesis of a gradual dedifferentiation or retrodifferentiation of the cell population during carcinogenesis. Hyperplastic nodules produced by continuous treatment of rats with 4-dimethylaminoazobenzene showed increased rather than decreased albumin levels. The analysis of albumin messenger RNA expression might therefore be used as a tool to discriminate between nodules of differing biological nature and fate.
Expression of H-ras and c-myc protooncogenes in isolated gamma-glutamyl transpeptidase-positive rat hepatocytes and in hepatocellular carcinomas induced by diethylnitrosamine.
The appearance of gamma-glutamyl transpeptidase (GGT) in focal areas of hepatocytes is a widely used histochemical marker for the identification of preneoplastic cell populations. The characterization of these GGT-positive preneoplastic cells in relation to possible alterations in protooncogene expression may help define cellular changes occurring during the early stages of hepatocarcinogenesis. Female Sprague-Dawley rats were subjected to a two-thirds partial hepatectomy, followed 18 h later by a single intragastric administration of 30 mg of diethylnitrosamine per kg and subsequent feeding of a diet containing 0.05% phenobarbital for 6 or 11 mo. Primary cell suspensions were obtained after the perfusion of liver with collagenase. Cell debris and nonviable cells were removed with multiple washes and a Percoll gradient step. GGT-positive hepatocytes were enriched from the cell suspension by adherence to an affinity-purified GGT antibody affixed to Petri dishes. These dishes allowed the selective adherence and collection of up to 2.28 X 10(6) GGT-positive cells per liver. The starting cell population and the isolated GGT-positive and -negative cells were then used for subsequent analysis. RNA was prepared from the cell isolates and from hepatocellular carcinomas induced with the same diethylnitrosamine and phenobarbital regimen as that used to induce GGT-positive foci; 10 micrograms of total cellular RNA were used for Northern blot hybridizations. The blots were probed with 32P-labeled c-myc, H-ras, and albumin DNAs. The results indicate that GGT-positive hepatocytes do not differ from the other hepatocyte populations in either the size or amount of mRNA transcripts for the c-myc and H-ras protooncogenes. Increased expression of c-myc and H-ras was observed in some malignant lesions and may represent a secondary alteration occurring during the multistage process of hepatocarcinogenesis.
Induction of altered hepatic foci in rats by the administration of hypolipidemic peroxisome proliferators alone or following a single dose of diethylnitrosamine.
The effect of feeding hypolipidemic peroxisome proliferators on the induction of altered hepatic foci (AHF) in Fischer rats was studied in order to determine whether such agents can induce or promote the development of AHF. In the first study, rats were fed ciprofibrate (10 mg/kg/day) for 1 yr. AHF, neoplastic nodules, and hepatocellular carcinomas were induced. The presence of putative gamma-glutamyltranspeptidase (GGT) activity was numerically the most common marker, although it was absent in larger foci and nodules. A deficiency in canalicular ATPase and glucose-6-phosphatase provided the best markers for the larger foci and nodules. In the second study, rats were subjected to partial hepatectomy, and half of the animals were then intubated with diethylnitrosamine (10 mg/kg). One wk later, rats were fed Wy-14,643 at concentrations of 0, 0.05, and 0.1% in the diet for 6 mo. At 6 mo, the number and volume of foci were increased by the feeding of Wy-14,643 after partial hepatectomy alone and were greatly increased when Wy-14,643 was fed after partial hepatectomy/diethylnitrosamine administration. Canalicular adenosine triphosphatase and glucose-6-phosphatase deficiencies were the most common markers of AHF, and AHF of these phenotypes occupied practically all of the focal volume. The larger AHF did not express GGT, and those foci exhibiting GGT were much less common and occupied very little volume. The absence of the GGT protein itself, as opposed to an inhibition of GGT activity, was verified by immunohistochemical staining using an antibody to GGT. These studies show that hypolipidemic peroxisome proliferators can stimulate an increase in AHF following a single dose of diethylnitrosamine and a mitotic stimulus, and they thus can act as promoters in two-stage liver carcinogenesis. GGT is a poor marker for identifying AHF induced by peroxisome proliferators during the early, premalignant phase of hepatocarcinogenesis.
The molecular determinants of carcinogenesis.
This monograph has emphasized the molecular aspects of both the differences between cancer and normal cells and the development of a cancer cell from a normal cell. Multistage carcinogenesis is now well known in a variety of histogenetic circumstances, and definable stages of initiation, promotion, and progression have been identified. Activation of proto-oncogenes in neoplasms may occur at any time during the development of malignancy, but most examples to date have shown this occurrence during the final stage of progression. A new form of carcinogenesis, that induced by specific genetic constructions in vivo or in vitro, has been accomplished. The specificity of genetic carcinogenesis can reside in the specific construct itself, especially in tissue-specific enhancer regions. On the other hand, the specific molecular genetic characteristics of at least one human genetic condition resulting in a high incidence of a specific histogenetic type of neoplasia, hereditary neuroblastoma, have been partially characterized. All of the various types ov carcinogenesis--genetic, viral, chemical, and radiation--exhibit, or at one time or another or in one situation or another, multiple stage ion their development.