Multistage hepatocarcinogenesis in the rat: insights into risk estimation.
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Animals↗
Biomedical subjects
H C Pitot
Publications and source records attributed to H C Pitot.
Superinduction of 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible expression of aldehyde dehydrogenase by the inhibition of protein synthesis.
Inhibition of protein synthesis by cycloheximide or puromycin super-induced the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible isoform of aldehyde dehydrogenase (ALDH-3) in rat hepatoma cells. Treatment with cycloheximide did not affect the basal expression but markedly enhanced the TCDD-inducible expression of ALDH-3 mRNA. The co-treatment of cycloheximide and TCDD for 24 h caused a 10-fold greater accumulation of ALDH-3 mRNA than did TCDD alone. The transcription rate of the ALDH-3 gene in the co-treated cells was also 4- to 5-fold higher than that in the cells treated with TCDD alone. The superinduction of ALDH-3 mRNA was observed only when cycloheximide was added prior to or simultaneously with the addition of TCDD. These results suggest that the mechanism of TCDD-inducible transcription of the ALDH-3 gene involves a labile protein that modulates or represses the action of TCDD.
Actins↗
Nutritional regulation and tissue-specific expression of the serine dehydratase gene in rat.
The mechanism of dietary regulation and tissue-specific expression of the serine dehydratase gene in rat has been studied. The hepatic serine dehydratase activity and its mRNA showed a parallel increase with increasing protein content in the diet. However, when rats that had been maintained on a high protein diet were fed a protein-free diet, the mRNA level rapidly decreased to 0.5 in 3 h, whereas the enzyme activity gradually fell to a low level over a period of 5 days. With animals maintained on a high protein diet or on a protein-free diet, we examined the sites hypersensitive to DNase I in the 5'-flanking region of serine dehydratase gene in the liver chromatins. A series of DNase I-hypersensitive sites were located within 10.5 kilobase pairs upstream of the transcription start site. The DNA regions at -3050 and -3180 (region II) and -3600 to -3850 (region III) were more susceptible to the nuclease in the expressing than in the nonexpressing liver. A reverse situation obtained at -100 (region I). Kidney contained serine dehydratase mRNA at a level of 5% of liver as determined by Northern blotting. The kidney chromatin was found to be susceptible to DNase I only at region I. No conspicuous DNase I-hypersensitive sites were observed in the relevant regions of chromatins from brain and lung, in which serine dehydratase mRNA was scarcely transcribed. These results suggest that nutritional control and tissue-specific expression of the serine dehydratase gene is closely associated with the alteration of DNase I hypersensitivity at specific sites of the 5'-flanking region of the gene.
Animals↗
Effects of glucose, insulin, and cAMP on transcription of the serine dehydratase gene in rat liver.
Starvation and diabetes both caused a dramatic induction of hepatic L-serine dehydratase (SDH) (EC 4.2.1.13) in rats. Increases in the activity of the enzyme which had been demonstrated in several previous studies were found to be associated with increases in the amount of SDH protein and its mRNA in our studies reported herein. Nuclear run-on experiments with isolated liver nuclei demonstrated that the increases in SDH activity were mainly the result of increases in the rate of SDH gene transcription. Refeeding of glucose to starved rats or the administration of insulin to diabetic rats caused a marked reduction in the amount of SDH mRNA. The rates of transcription as measured in isolated nuclei were reduced to uninduced levels within 30 min of either treatment. Following the administration of Bt2-cAMP, the transcription rates of the SDH gene returned to the original induced rates within 40 min both in glucose-refed rats and in diabetic rats administered insulin. The results of these experiments indicate that the induction of SDH in rat liver in vivo is controlled predominantly at the level of gene transcription by the reciprocal action of cAMP and insulin.
Animals↗
Transcriptional regulation and localization of the tissue-specific induction of epoxide hydrolase by lead acetate in rat kidney.
Intraperitoneal administration of lead acetate to male Sprague-Dawley rats resulted in the tissue-specific transcriptional activation of the microsomal epoxide hydrolase gene in kidney. This response was followed by a 15-fold increase in the level of kidney epoxide hydrolase mRNA, while no change in mRNA level was noted in liver. Treated animals also showed no increase in mRNA levels for NADPH-cytochrome P-450 oxidoreductase, cytochrome P-450b/e, cytochrome P-450PCN (where PCN is pregnenolone 16 alpha-carbonitrile), or serum albumin in either kidney or liver. Immunoquantitation of the enzyme revealed a 16-fold increase in kidney upon treatment with lead acetate, but significant changes in epoxide hydrolase levels were not noted in liver, heart, spleen, lung, small intestine, or testis. The enzymatic activity for liver and kidney paralleled the immunochemical results; however, the activity increase in kidney was only one-third of the increase noted for total enzyme protein. Immunohistochemical analysis of epoxide hydrolase protein in sections of rat kidney demonstrated that in lead acetate-treated animals there was a marked increase in staining of the cytoplasm of the proximal tubular cells in the outer cortex as compared with kidneys from control animals. In contrast, considerable protein was also localized to collecting ducts, but no change was evident in the content of the epoxide hydrolase gene product in these structures in control and lead acetate-treated animals. Immunohistochemical differences were not noted between livers from control and lead-treated animals. Furthermore, the staining patterns for NADPH-cytochrome P-450 oxidoreductase were the same for control and treated animals in both kidney and liver. Quantitative measurements of lead uptake by various rat tissues showed liver, spleen, and small intestine reaching a maximum of approximately 12,000 ng of lead/g of dry tissue at 8, 8, and 16 h, respectively, while kidney, lung, and testis peaked (approximately 3,000 ng of lead/g of dry tissue) at 16, 16, and 12 h, respectively.
Animals↗
Amino acid-rich medium (Leibovitz L-15) enhances and prolongs proliferation of primary cultured rat hepatocytes in the absence of serum.
Multiple rounds of cell division were induced in primary cultured rat hepatocytes in serum-free, modified L-15 medium supplemented with 20 mM NaHCO3 and 10 ng/ml EGF in a 5% CO2/95% air incubator. A 150% increase in cell number and DNA content was observed between day 1 and day 5. The time course of DNA synthesis of hepatocytes cultured in L-15 medium differed from that in DMEM/F12 medium in that there were four peaks of 3H-thymidine incorporation in the L-15 medium, at 60 h, 82 h, 96 h, and 120 h, but only one peak at 48 h in modified DMEM/F12 medium. Labeling studies of the hepatocytes indicated that more than 60% of the cells were stained with antibromodeoxyuridine (BrdU) antibody in the periods of 48-72 h and 72-96 h after plating at densities between 1.5 x 10(5) and 6.0 x 10(5) cells per 35-mm dish. Even at a density of 9.0 x 10(5) cells/dish, about 40% of the cell nuclei were stained with BrdU in the periods of 48-72 h and 72-96 h. In addition, about 20% of the hepatocytes in culture initiated a second round of the cell cycle between 48 and 96 h in culture. Proliferating cells, which were mononucleate with a little cytoplasm, appeared in small clusters or colonies in the culture from day 4. These proliferating cells produced albumin. The addition of essential amino acids to the DMEM/F12 medium enhanced the DNA synthesis of hepatocytes, thus indicating that the higher level of amino acids in L-15 medium may be an important factor in its enhanced ability to support the proliferation of primary cultured rat hepatocytes.
Albumins↗
Characterization of the stage of progression in hepatocarcinogenesis in the rat.
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Aneuploidy↗
Gamma-glutamyltranspeptidase-positive (GGT+) hepatocytes from carcinogen-treated rats isolated by magnetic beads.
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Animals↗
The bicarbonate ion is essential for efficient DNA synthesis by primary cultured rat hepatocytes.
Bicarbonate in the culture medium is essential for DNA synthesis of primary cultured rat hepatocytes stimulated by epidermal growth factor (EGF). When primary cultured hepatocytes in supplemented Leibovitz L15 medium were placed in a 100% air incubator, no increase in DNA synthesis was observed even after stimulation by EGF. However, when these cells were cultured with NaHCO3 and EGF and placed in a 5% CO2:95% air incubator, a stimulus of DNA synthesis more than 10-fold greater than in cultures in air only was seen, and many mitotic figures could be identified. Furthermore, NaHCO3 added to supplemented DMEM/F12 medium enhanced the DNA synthesis of primary cultured rat hepatocytes in this medium. The ideal pH of the medium for DNA synthesis of cultured hepatocytes was in the range of 7.6 to 8.0. A dose response of NaHCO3 in several media showed that DNA synthesis of the cells increased as the concentration of NaHCO3 increased and that 25 to 30 mM NaHCO3 in the medium was optimal for the replication of DNA by primary cultured rat hepatocytes.
Animals↗
Tumor promotion as a target for estrogen/antiestrogen effects in rat hepatocarcinogenesis.
Tamoxifen is a well-tolerated palliative and adjuvant treatment for human breast cancer and requires continuous, long-term administration for optimal therapeutic effectiveness. A two-stage model of experimental hepatocarcinogenesis, based upon the natural history of cancer development, has been employed to assess the carcinogenic potential of tamoxifen. In this study, the effectiveness of tamoxifen both as an initiator and a promoter in hepatocarcinogenesis was assessed in female F-344 rats. Tamoxifen was tested as an initiator at a single intragastric dose of 40 mg/kg, followed by promotion with 0.05% phenobarbital. The number and size of the resulting altered hepatic lesions were quantified, and tamoxifen was found to lack initiating action at the dose tested. Other groups of animals were initiated with a nonnecrogenic, subcarcinogenic dose of diethylnitrosamine (10 mg/kg) and were fed tamoxifen at either 250 or 500 mg/kg in the AIN-76A purified diet for 6 months. The livers of these animals showed an increase in the size and number of altered hepatic lesions compared with those animals that were initiated but not exposed to tamoxifen; this indicates that tamoxifen acts as a tumor promoter in the rat liver. The promotion index of tamoxifen, a measure of relative potency, was less than one-tenth that of ethinyl estradiol and more than four times that of phenobarbital, an agent commonly employed as a representative promoting agent in experimental carcinogenesis. Since tamoxifen lacked initiating activity in the rat liver at the dose tested, the mechanism of tumor induction in long-term feeding studies by tamoxifen may be due to its promotion of spontaneously initiated hepatocytes. The chronic therapeutic use of tamoxifen should therefore be limited by the potential carcinogenic risk of this agent as an effective tumor promoter.
Adenosine Triphosphatases↗
An initiation-promotion assay in rat liver as a potential complement to the 2-year carcinogenesis bioassay.
Several pharmaceutical agents, manufacturing chemicals, and environmental contaminants were found to act primarily as promoting agents in an initiation-promotion paradigm. The phenotypic distribution of four enzyme markers--placental glutathione-S-transferase (PGST), gamma-glutamyl transpeptidase (GGT), canalicular ATPase (ATPase), and glucose-6-phosphatase (G6Pase)--was analyzed in altered hepatic foci (AHF) by quantitative stereology. The number and volume distribution of AHF were determined for each promoter tested. For phenobarbital and 2,3,7,8-tetrachloro-p-dioxin, PGST and GGT together scored 100% of the AHF; for 1-(phenylazo)-2-naphthol (CI solvent yellow 14) and chlorendic acid, PGST alone marked 90% of the AHF; after chronic administration of WY-14,643, ATP and G6Pase were the predominant markers. In rats fed tamoxifen, G6P scored more than half of the AHF. Differences in the number of AHF promoted by each of these agents and in their phenotypic distributions may reflect the differentially responsive nature of individual initiated hepatocytes to the action of specific promoters. Since the chronic bioassay of suspected carcinogens does not allow one to differentiate between weak complete carcinogens and those carcinogenic agents that act in a reversible manner to promote the growth of previously initiated cells, the partial hepatectomy, altered-hepatic-focus model of cancer development is proposed as a supplement to the chronic bioassay for the identification of those carcinogenic agents that are primarily, if not exclusively, promoting agents in rat liver.
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Study of the separate and combined effects of the non-planar 2,5,2',5'- and the planar 3,4,3',4'-tetrachlorobiphenyl in liver and lymphocytes in vivo.
Polychlorinated biphenyls (PCBs) are a group of industrial chemicals that are widely distributed in the environment. Because these compounds occur as mixtures, studies of their possible interactive effects are essential for an understanding of the mechanism of the toxicity of these mixtures. For the determination of a possible interaction of the effects in vivo of 2,5,2',5'-tetrachlorobiphenyl (TCB) and 3,4,3',4'-TCB, rats were exposed to a single dose of diethylnitrosamine (DEN) and subsequently to 0.1 p.p.m. 3,4,3',4'-TCB and/or 10 p.p.m. 2,5,2',5'-TCB in the feed for 1 year. The two major targets of PCB toxicity, the liver and the peripheral blood, were examined after these treatments. TCB treatment after DEN exposure caused a predominance of increased placental glutathione S-transferase (PGST) and deficiencies of ATPase as preneoplastic markers in focal hepatic lesions. When 0.05% phenobarbital (PB) was administered after DEN exposure, the distribution of markers in altered hepatic foci (AHF) was essentially equal for increased PGST and gamma-glutamyltranspeptidase (GGT) and for ATPase deficiency. Many of these AHF also exhibited increased P450 b/e expression. Our results demonstrated that the two PCB congeners interacted in vivo to produce an increase in AHF that were PGST positive and ATPase negative. PGST-positive and ATPase-negative AHF correlated best with focal areas of P450 b/e expression. The combination of the two PCBs caused a greater than additive decrease in the total number of lymphocytes and antibody-producing B-cells. Also the thymocyte-dependent T-helper cells isolated from the animals receiving the combination of TCBs demonstrated a morphologically abnormal subpopulation. The results indicate that the interaction of 2,5,2',5'-TCB and 3,4,3',4'-TCB in vivo induced much greater toxicity and mutagenicity in peripheral lymphocytes and hepatocytes than treatment with either congener alone.
Animals↗
The effect of the format of administration and the total dose of phenobarbital on altered hepatic foci following initiation in female rats with diethylnitrosamine.
The effect of changing the format of administration as well as the total dose of the promoting agent phenobarbital (PB) on the development of altered hepatic foci (AHF) was determined in an initiation-promotion protocol with female rats fed the purified AIN-76 diet. Effects on the total number of AHF and the volume percentage of liver occupied by AHF were determined for four histochemical markers, the placental form of glutathione S-transferase, gamma-glutamyl-transpeptidase canalicular ATPase, and glucose-6-phosphatase after 16 and 60 weeks of promotion with varying doses and formats of PB, as well as for a further 16-week period in which no PB was administered. At the 16-week point, animals fed 0.1% PB continuously exhibited the largest number and volume percentage of AHF, whereas rats fed 0.1% PB for 4 days followed by 10 days of no PB with continuous repetition of this pattern during the 16-week treatment period exhibited no increase in the number of AHF over control and only a slight increase in volume percentage. Rats fed a continuous repetition of 0.2% PB for 2 days followed by 12 days of no PB exhibited an intermediate increase in the number of AHF as well as the volume percentage fraction after 16 weeks of this regimen. After 60 weeks of feeding PB by these three different formats, the numbers of AHF observed in these groups were equivalent and had increased above those seen after 16 weeks of feeding. The volume percentage occupied by the AHF in these three groups was also similar, although animals receiving 0.2% PB intermittently showed a significantly lower volume percentage than animals receiving 0.1% PB continuously for 60 weeks. When animals were maintained for an additional 16 weeks without PB feeding, the numbers of AHF decreased dramatically, much more so in animals fed PB intermittently, whereas the volume percentage fraction of AHF in livers of animals receiving 0.1% PB continuously for 60 weeks almost doubled. In contrast, the volume percentage fraction of AHF in livers of animals receiving PB intermittently for 60 weeks followed by 16 weeks of no PB was slightly less. Examination of the individual size classes of AHF showed little change in their distribution at 16 and 60 weeks, but after 16 weeks of PB withdrawal (76 weeks total time), the distribution of AHF in animals that had received 0.1% PB continuously for 60 weeks exhibited a decidedly greater shift to larger AHF than animals receiving PB intermittently for the 60-week period.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Effect of dietary fat on the initiation of hepatocarcinogenesis by diethylnitrosamine or 2-acetylaminofluorene in rats.
The purpose of this study was to determine if increasing dietary fat, either as saturated fat or polyunsaturated fat, would alter initiation of hepatocarcinogenesis by diethylnitrosamine (DEN) or 2-acetylaminofluorene (AAF). Rats were fed one of three purified diets: a low-fat (LF) diet (containing 5% of calories as safflower oil), a high saturated fat (HSF) diet (containing 48% of calories as palm oil) and a high polyunsaturated fat (HPUF) diet (containing 48% of calories as safflower oil). Four weeks later, all rats were subjected to partial hepatectomy (PH). Rats were then divided into four groups and received no carcinogen, DEN (10 mg/kg, p.o., 24 h after PH) or AAF (25 or 100 mg/kg, p.o., 12 h after PH). Five days later, all rats were fed an unrefined diet, and 9 weeks later, all rats were fed phenobarbital in the diet for 26 weeks as a tumor promoter. In rats initiated with DEN, the number of gamma-glutamyl transpeptidase-positive and ATPase-negative foci was higher in the rats fed the HPUF diet, but not the HSF diet, as compared to rats fed the LF diet. The incidence of neoplastic nodules, the mean focal volume and the volume fraction, however, were not significantly altered by dietary fat in DEN-injected rats. The dietary fat content of the diet did not affect the induction of altered hepatic foci or neoplastic nodules in rats initiated with AAF or receiving no initiation. This study shows that initiation of hepatocarcinogenesis can be influenced by dietary fat, but that the effect may be carcinogen-specific.
2-Acetylaminofluorene↗
Hormonal regulation of serine dehydratase gene expression in liver and kidney of the adrenalectomized rat.
We have previously demonstrated that glucagon but not dexamethasone could induce serine dehydratase (SDH: EC.4.2.1.13) in liver, and either glucagon or dexamethasone could induce the enzyme in kidney of normal rats. The mechanism(s) of the hormonal regulation of SDH gene expression in liver and kidney was further studied using adrenalectomized rats. Simultaneous administration of glucagon and dexamethasone induced the activity, rate of SDH synthesis, and accumulation of SDH mRNA in both liver and kidney of the rat. The increased SDH activity was reflected by changes in the amount of enzyme protein and in the rate of SDH protein synthesis, both parameters closely paralleling the changes in the levels of SDH mRNA. The rates of transcription of the SDH gene as measured in run-on experiments with isolated nuclei were also increased by the administration of these hormones. These results indicate that the expression of the SDH gene was regulated primarily at the transcriptional level under these conditions. When glucagon or dexamethasone was injected separately into adrenalectomized rats, significant increases in the levels of SDH mRNA and the rate of SDH gene transcription were observed in liver. Although glucagon was more effective than dexamethasone, both hormones were required for the maximal induction of SDH gene transcription in liver. In contrast, dexamethasone alone effectively increased the rate of SDH gene transcription in kidney.(ABSTRACT TRUNCATED AT 250 WORDS)
Adrenalectomy↗
Biochemical markers associated with the stages of promotion and progression during hepatocarcinogenesis in the rat.
Specific biochemical changes occurring during hepatocarcinogenesis have been sought by many investigators. The development of multistage models for hepatocarcinogenesis in the rodent has renewed interest in such marker alterations in preneoplastic as well as neoplastic hepatocytes. Preneoplastic altered hepatic foci (AHF) exhibit specific histomorphologic changes as viewed with tinctorial stains and show a variety of biochemical changes as evidenced by enzyme and immunohistochemistry and by other histochemical markers. During the reversible stage of promotion when AHF are scored by multiple markers, the distribution of markers within these lesions differs with the use of different promoting agents. One interpretation of this finding is that each promoting agent stimulates the replication of a set of initiated cells exhibiting the phenotypic characteristics of a specific programmed phenotype. The same markers score AHF during the stage of progression, but many AHF in this stage are phenotypically heterogeneous, exhibiting in tissue sections a "focus-in-focus" pattern of marker alteration. These latter changes can be correlated with the appearance of karyotypic alterations in preneoplastic hepatocytes. On the other hand, it has been difficult to demonstrate the activation, either mutational or transcriptional, of proto-oncogenes until this stage of progression in rat hepatocarcinogenesis. Thus, a study of biochemical and molecular markers during the stages of hepatocarcinogenesis may lead to a better understanding of potential mechanisms involved in the development of neoplasia through the stages of initiation, promotion, and progression.
Animals↗
DNA damage and repair in gamma-glutamyltranspeptidase-positive and negative hepatocytes in primary culture from carcinogen-treated rats.
Chemically induced DNA fragmentation and unscheduled DNA synthesis were determined in gamma-glutamyltranspeptidase (GGT)-positive and GGT-negative hepatocytes isolated from rat livers subjected to a multistage hepatocarcinogenesis regimen (Solt-Farber), which included 0.05% phenobarbital promotion for 6 weeks (early) or 6 months (late). The results indicated that there was DNA damage in untreated GGT-positive and GGT-negative hepatocytes with either period of promotion compared with normal hepatocytes; however, no statistical difference could be seen between GGT-positive and GGT-negative hepatocytes. DNA damage induced in vitro by the activation-dependent carcinogen dimethylnitrosamine was much less in GGT-positive hepatocytes than in GGT-negative hepatocytes or normal hepatocytes. No significant difference in DNA damage was seen in both GGT-positive and GGT-negative cell populations following treatment with the activation-independent carcinogen ethylnitrosourea (ENU), although DNA damage of GGT-positive hepatocytes was less than that of normal hepatocytes. The background of unscheduled DNA synthesis in both GGT-positive and GGT-negative hepatocytes at either time of promotion was higher than that of normal hepatocytes. The capacity for DNA repair in GGT-positive hepatocytes appeared to be lower than that in GGT-negative hepatocytes. GGT-negative hepatocytes exhibited a lower capacity for DNA repair than that of normal hepatocytes in terms of the rate of unscheduled DNA synthesis elicited by dimethylnitrosamine and ethylnitrosourea in vitro.
Animals↗
Endogenous carcinogenesis: the role of tumor promotion.
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Animals↗