Centrifugal elutriation of hepatocytes from 2-acetylaminofluorene-treated rats and their characterization by flow cytometry

Treatment of male Wistar rats with 2-acetylaminofluorene (2-AAF) markedly altered the ploidy distribution of liver cells. Small diploid hepatocytes first appeared after 4-5 weeks feeding of a diet containing 0.02% 2-AAF; after 9 weeks 65-70% of the hepatocytes were diploid. Approximately two-thirds of this new liver cell population persisted after termination of the treatment. The hepatocytes from 2-AAF treated animals were separated according to size and ploidy by centrifugal elutriation and stained for gamma-glutamyltranspeptidase (gamma-GTase). The percentage of gamma-GTase-positive hepatocytes did not significantly differ between the various elutriated cell fractions. Thus gamma-GTase-positive liver cells obtained by feeding of 2-AAF do not represent a distinct size class of hepatocytes. The significance of carcinogen-induced diploid hepatocytes in hepatocarcinogenesis is discussed.     

Low selection of preneoplastic hepatocytes after treatment with the 2-acetylaminofluorene diet-partial hepatectomy regimen in the liver of hepatocarcinogenesis-resistant DRH strain rats

In hepatocarcinogenesis-resistant DRH rats, preneoplastic hepatocytic lesions are smaller than those of usual rats during carcinogenesis. When preneoplastic hepatocytes from DRH and Donryu (original strain of DRH) were reciprocally transplanted into the livers of DRH and Donryu treated with 2-acetylaminofluorene (2-AAF) diet/two-thirds hepatectomy (PH), the Donryu cells formed small colonies within the DRH liver, whereas the DRH cells formed large colonies within the Donryu liver. The DRH liver showed less degree of oval cell proliferation after treatment with 2-AAF and PH, and DRH hepatocytes were more resistant to the growth-inhibitory effect of 2-AAF after PH. Furthermore, DRH hepatocytes were generally resistant to cytotoxicity of hepatotoxins. The tissue environment of the DRH liver, therefore, is less effective for selective growth of preneoplastic hepatocytes during the carcinogen treatment, which is probably a major cause of the hepatocarcinogenesis-resistance in DRH rats.     

Induction of gamma-glutamyltranspeptidase-positive altered hepatocytic lesions by combination of transplacental-initiation and postanal-selection

The purpose of this study was to induce in postnatal life the selective growth of altered hepatocytic populations which had been induced in utero by various chemicals. Pregnant rats of a Wistar strain were given a single dose of various chemical carcinogens on the 18th gestational day. From two months postpartum, both the mothers and offspring were given a diet containing 2-acetylamino-fluorene (2-AAF) at a concentration of 0.02% for two weeks. Then, at the end of the first week after the 2-AAF feeding, a two-third partial hepatectomy was performed. All the animals were killed one week after the partial hepatectomy and were examined for the incidence of gamma-glutamyltranspeptidase (GGT)-positive altered hepatocytic foci in the liver. The transplacental administration of not only a hepatic carcinogen, diethylnitrosamine (DEN) but also 7,12-dimethylbenzanthracene (DMBA), which does not usually produce hepatocellular carcinomas in adult rats, induced GGT-positive altered hepatocytes in the liver of the offspring. These hepatocytes grew into grossly visible hyperplastic nodules within a week after the two-third partial hepatectomy. The possible applicability of the transplacental initiation-postnatal selection model for the short-term assay of transplacental carcinogenicity is discussed.     

Induced drug resistance inhibits selection of initiated cells and cancer development

Compounds exerting a mitoinhibitory effect on normal hepatocytes are potent promoters in the resistant hepatocyte model of chemical carcinogenesis in combination with stimulation of regenerative growth by partial hepatectomy or treatment with carbon tetrachloride. 2- Acetylaminofluorene (2-AAF) almost completely inhibits liver cell regeneration after partial hepatectomy, allowing only resistant cells to participate in regenerative growth. After initiation by diethylnitrosamine and promotion with 2-AAF and partial hepatectomy (PH), focal growth of initiated cells generates liver lesions which occupy 40% of the hepatic volume three weeks after PH. In this work the mechanism for the anti promoting effects of phenobarbital and 3- methylcholantrene were investigated as well as their effects on the development of malignant hepatocellular carcinoma in the resistant hepatocyte model. Treatment with phenobarbital or, especially, 3- methylcholanthrene rendered normal rat hepatocytes resistant to the mitoinhibitory effect of 2-AAF. In combination with 2-AAF/PH, 3- methylcholanthrene shortened the regenerative growth period to less than one week. In the Solt-Farber protocol for experimental hepatocarcinogenesis, treatment with phenobarbital or 3- methylcholanthrene during promotion with 2-AAF/PH permitted hepatocytes surrounding the focal lesions to respond with regenerative growth. The foci and surrounding liver grew until the liver/body mass index reached the control value. With phenobarbital treatment the total focal volume was 20% of the liver volume three weeks after PH, whereas the corresponding value in the case of 3-methylcholanthrene was only 1%. Labelling index data supported the conclusion that growth of the liver lesions in the resistant hepatocyte model was dependent on differential inhibition of normal hepatocyte growth by the promoter and that the size of the foci obtained was related to the length of time after PH required to complete liver regeneration. 3-methylcholanthrene induced 2- AAF resistance prevented the development of large persistent nodules and hepatocellular carcinoma while phenobarbital delayed cancer development with several month. The data thus supports the idea that the degree of clonal expansion during promotion determines the size of the population at risk for malignant transformation, as well as the final frequency of carcinomas.      

Biotransformation in carcinogen-induced diploid and polyploid hepatocytes separated by centrifugal elutriation.

Biotransformation in carcinogen-induced diploid and polyploid hepatocytes was studied using isozyme-selective substrates for several enzyme pathways. Diploid hepatocytes were induced by partial hepatectomy, a single injection of diethylnitrosamine, and 4 weeks of 2-acetylaminofluorene (2-AAF) feeding. Then, after an additional 3-5 weeks on the control diet, diploid and polyploid hepatocytes were separated from freshly isolated hepatocytes by centrifugal elutriation. Benzo(a)pyrene hydroxylase, ethoxyresorufin O-deethylase, and methoxycoumarin O-demethylase activities were approximately 15-40% lower in the diploid hepatocyte fraction than in the polyploid cell fraction. Activities of 1-chloro-2,4-dinitrobenzene, glutathione S-transferase, 3-hydroxy-benzo(a)pyrene or 4-hydroxybiphenyl UDP-glucuronosyltransferase, and DT-diaphorase were not different in the two cell fractions. Determination of activity during the 2-AAF treatment indicated that 2-AAF increased 7-ethoxyresorufin O-deethylase and 3-hydroxybenzo(a)pyrene glucuronosyltransferase activities by 300 and 200%, respectively, in both the diploid and polyploid hepatocyte fractions. Administration of phenobarbital for 4 days at the end of the control diet period increased ethoxyresorufin and methoxycoumarin dealkylations by 2- and 4-fold, and 3-hydroxybenzo(a)pyrene glucuronidation and 1-chloro-2,4-dinitrobenzene conjugation with glutathione by 1.5- to 2-fold in both hepatocyte fractions. Slight increases in benzo(a)pyrene hydroxylation and 4-hydroxybiphenyl glucuronidation were also evident in diploid cells. Although there is a slight decrease in cytochrome P-450-dependent monooxygenase activities, these data indicate that carcinogen-induced diploid hepatocytes do not show the typical toxicant-resistant phenotype observed in preneoplastic hepatocytes of altered liver foci, which are characterized by large decreases in monooxygenase biotransformations as well as increased activities of several phase II enzymes. This finding is compatible with the hypothesis that 2-AAF-induced nonploidizing growth of diploid hepatocytes is caused by nontoxic mechanisms in the present experimental paradigm. In addition, carcinogen-induced diploid cells respond to phenobarbital in a manner similar to that of polyploid hepatocytes.     

Change in the ploidy state of rat liver cells during chemical hepatocarcinogenesis and its relationship to the increased expression of alpha-fetoprotein

The DNA content and ploidy state of cells isolated from the livers of rats exposed to the carcinogen 3'-methyl-4-dimethylaminoazobenzene for 10 and 20 weeks, as determined by flow cytometry, were correlated with the development of oval cells in the livers of treated animals and with serum levels of the oncoprotein alpha-fetoprotein (AFP). The study revealed that there was initially a steady rise in the AFP levels found in the carcinogen treated rats. Associated with this increase was a change in the ploidy pattern of the liver from an approximately equal mixture of tetraploid and diploid cells to a predominantly diploid state. Histologically, there was an increase in the number of oval cells during carcinogen treatment, and when stained immunohistochemically for AFP, these cells were positive. We conclude that the changing state of the diploid and tetraploid cell populations is due to the proliferation of oval cells and that these cells are responsible for the initial increase of serum AFP. The maintenance of the diploid population of cells at later periods of the study is a reflection of the persistence of a new cell type, possibly derived from oval cells. The effect of 3'-methyl-4-dimethylaminoazobenzene was not reversed if the animals were withdrawn from the diet at 10 weeks. In addition, in the cases of hepatocellular carcinoma that were found, a population of cells was detected by flow cytometry that contained altered DNA.     

Clonal isolation of populations of gamma-glutamyl transpeptidase-positive and -negative cells from rat liver epithelial cells chemically transformed in vitro

In a population of cultured rat liver epithelial cells transformed by 11 brief treatments with N-methyl-N'-nitro-N-nitrosoguanidine, 9% of the cells stained intensely for gamma-glutamyl transpeptidase (GGT). We have isolated from this phenotypically heterogeneous tumorigenic cell population 11 GGT-positive and 7 GGT-negative clonal subpopulations (from single cells) and have analyzed the ploidy and selected biochemical, histochemical, and growth properties of the cells in these clonal sublines. As compared to the GGT-negative strains and normal diploid rat liver epithelial cells, cells of the GGT-positive strains are larger in size, have greater DNA content, proliferate more slowly in culture, and have higher specific activities of NADH diaphorase, glucose-6-phosphate dehydrogenase, pyruvate kinase, and lactate dehydrogenase. The GGT-positive strains also show greater alteration and heterogeneity than do the GGT-negative strains in their ability to store glycogen and in their expression of lactate dehydrogenase isozymes. The results indicate that enzymatic changes commonly observed in "altered" hepatocytes in rat livers exposed to chemical carcinogens in vivo can also be produced in vitro in cultured hepatic epithelial cells by treatment with carcinogens. Moreover, treatment of a cell line with a chemical carcinogen generates a population of cells vastly heterogeneous in both their phenotypes and genotypes. Isolation of clonal subpopulations from the resulting cell line allows critical examination of the linkage and mechanistic relationship between tumorigenicity and many paratumorigenic phenotypes.     

Flow cytometric investigation of a possible precursor-product relationship between oval and parenchymal cells in the rat liver

The question of a possible precursor-product relationship betweenoval cells and hepatocytes was examined in rats treated for2 weeks with 2-acetylaminofluorene (2-AAF) with a two-thirdspartial hepatectomy (PH) performed after the first week of 2-AAFtreatment (modified Soft-Farber model). Liver cells were pulse-chaselabelled with bromodeoxynridine (BrdU) on day 6 post PH. Onday 7 post PH the nonparenchymal (NPC) fraction, which containsthe oval cells, exhibited a labelling index (LI) {small tilde}10times higher than that of the hepatocytes as analysed by flowcytometry (FCM), the majority of the proliferating cells beingoval cells. At later time points, there was no significant increasein the LI of diploid hepatocytes, and no detectable shift ofBrdU-labelled cells from the NPC fraction to the hepatocytefraction, suggesting that no extensive conversion of BrdU-labelledoval cells to hepatocytes was taking place. Throughout the experimentalperiod there was a significant increase in the diploid hepatocytecell fraction, from 12% on day 7 to 25% on day 13 post PH. Diploidhepatocytes pulse-labelled on days 7 or 9 post PH had a highU (7–8%), in contrast to the low LI (1%) of tetra- andoctoploid cells. Proliferation of diploid hepatocytes may thusexplain the large increase in the diploid hepatocyte fractionobserved from days 9 through 15 post PH. Our results, therefore,provide no reason to invoke oval cells as precursors of hepatocytesin the modified Solt-Farber carcinogenesis model.     

A precursor--product relationship exists between oval cells and hepatocytes in rat liver

Oval cell proliferation was induced in twelve male Fischer ratsby administration of 2-acetylaminofluorene (2-AAF) for 2 weeksand by performing partial hepatectomy one week after the beginningof 2-AAF administration. Albumin expression in liver was studiedby using in situ hybridization of ³H-labeled rat albumin riboprobe.Radiolabeled thy-midine was administered to a group of animalsat day 6 after partial hepatectomy. The animals were killedat 0, 3, 7, 9, 11 and 13 days after partial hepatectomy. Bothoval cells and basophilk hepatocytes showed a prominent expressionof albumin, whereas albumin expression hi acidophilic preexistinghepatocytes was decreased. Oval cell nuclei were exclusivelylabeled one day after administration of [³H]thymidine. At day9, 11 and 13 basophilic hepatocytes became labeled and the areaoccupied by these cells increased. This is the first demonstrationof the transfer of radiolabeled thymidine from oval ceDs tonewly formed hepatocytes in vivo. Thus the precursor—productrelationship between oval cells and basophilic hepatocytes hasbeen established.     

Activities of benzphetamine N-demethylase and aryl hydrocarbon hydroxylase in cells isolated from gamma-glutamyl transpeptidase-positive foci and surrounding liver

The levels of two cytochrome P-450-linked enzymes of xenobiotic metabolism, benzphetamine N-demethylase and aryl hydrocarbon hydroxylase, were determined in cells isolated from gamma-glutamyl transpeptidase (GGT)-positive foci and in cells from surrounding liver obtained from carcinogen-treated inbred F344 rats. Rat liver foci were initiated with diethylnitrosamine (CAS: 55-18-5) and promoted with sodium phenobarbital [(PB) CAS: 64038-21-7] for 4 1/2 or 12 months. The levels of both enzymes were relatively low in the GGT-positive hepatocytes, while the GGT-negative hepatocytes from the surrounding liver had elevated levels of both enzymes comparable to levels seen in rats treated with PB alone. After 12 months of promotion the PB was removed from the diet and the activities of both enzymes fell below the control levels in the GGT-positive hepatocytes and returned to the control levels in the surrounding GGT-negative hepatocytes. Therefore, the cells in the GGT-positive foci contained low levels of these two cytochrome P-450 enzymes in relation to the levels in GGT-negative cells. These levels were responsive to phenobarbital induction, although the induced levels in the GGT-positive cells were much lower than the induced levels in GGT-negative hepatocytes. The liver surrounding the foci responded to phenobarbital induction to the same degree as did the liver of noninitiated rats.     

Induction of humoral immunity toward 2-acetylaminofluorence in mice: modulation of DNA binding after 4 weeks dietary exposure to the carcinogen

In order to investigate the modulatory effects of the immuneresponse induced by recurrent carcinogen exposure, anti-2acetylaminofluorene(anti-2-AAF) IgG were elicited in Swiss mice before subsequentcarcinogen administration. The immunization schedule consideredof three weekly i.p. injections of 2-acetylaminofluorene (2-AAF)-gelatinconjugate, followed by a final immunogen injection 14 days later.At the end of treatment, the presence of specific anti-2-AAFantibodies in blood serum of all immunized animals was demonstrated.The immunization procedure did not affect liver metabolic activities,as evaluated using liver homogenates for the exogenous activationof 2-AAF to mutagen. After immunization, mice were fed 2-AAFpelleted in the diet at 50 and 150 p.p.m. for 4 weeks and killedat the end of treatment. The determination of DNA adducts byELISA in liver and spleen of treated animals revealed significantly(P< 0.01–0.001) lower 2-AAF adduct levels in both tissuesof immunized mice with respect to non-immunized animals (bothnaive and pretreated with the adjuvant alone). This result suggeststhat the specific humoral immunity elicited by repeated carcinogenexposure may be able to modulate the genotoxic effect inducedby subsequent carcinogen administration.     

Gamma-glutamyltransferase in putative premalignant liver cell populations during hepatocarcinogenesis.

The activity of gamma-glutamyltransferase, as measured quantitatively and by histochemical staining, was studied in different cell populations during the induction of liver cancer with 2-acetylaminofluorene (2-AAF) or diethylnitrosamine and compared with findings in fetal and in intact and regenerating adult liver. The enzyme activity is 20-fold higher in 12-week nodules than in control livers and 30-fold higher in 20-week nodules than in controls. A similar 30-fold increase in activity relative to control is present in hepatomas, induced by either 2-AAF or diethylnitrosamine, and in fetal hepatocytes. The enzyme shows increases in activity in foci of very early putative preneoplastic hepatocytes induced by a single dose of diethylnitrosamine and selected by low doses of 2-AAF plus partial hepatectomy. By 7 days, the foci show a 4-fold increase in enzyme activity, and by 3 weeks they are 40-fold higher than in the control liver. Histochemically, the foci are strongly positive for gamma-glutamyltransferase, especially in the bile canaliculi. By 21 days, the ductular (oval) cells induced by 2-AAF have disappeared. When stained for the enzyme activity, the foci stand out clearly against the negative background of the liver, allowing easy quantitation. It appears that gamma-glutamyltransferase is a useful marker for preneoplastic hepatocytes.     

Activation of a ribosomal S6 protein kinase in rapidly emerging diethylnitrosamine-induced gamma-glutamyltranspeptidase-positive hyperplastic liver lesions of the rat

The gamma-glutamyl transpeptidase (GGT)-positive hyperplastic liver lesions which developed in the Fisher 344 rat 7 and 60 days following a single carcinogenic dose of diethylnitrosamine (DENA, 200 mg/kg body weight), short-term dietary exposure to 0.02% 2-acetylaminofluorene (AAF) to suppress the growth of normal hepatocytes, and partial hepatectomy to actuate rapid growth of DENA altered hepatocytes not suppressed by AFF, showed an increased activity of a kinase which specifically phosphorylates the ribosomal S6 protein in vitro. Sham-operated animals showed, on the contrary, no GGT-positive cells and low S6 kinase activity, under the same conditions. After partial hepatectomy, activation of S6 kinase and elevated levels of phosphorylated S6 protein in vitro were detected in the early phases of "normal" hepatocyte proliferation, during liver regeneration, in DENA-treated, GGT-negative preparations, when the "selection" agent AAF was omitted from the diet. The observed activation of S6 kinase in GGT-positive hepatocytes and/or liver nodules could represent an early manifestation of the enhanced proliferation of altered hepatocytes during tumor induction and/or promotion under these conditions.     

Dose-dependent effects of 2-acetylaminofluorene on hepatic foci development and cell proliferation in rats

Dose-dependent development of pre-neoplastic liver cell foci induced by 2-acetylaminofluorene (2-AAF) was investigated in relation to cell-proliferative activity. Male F344 rats were initially given a single i.p. injection of diethylnitrosamine (DEN, 200 mg/kg) and starting 2 weeks later received diets containing 2-AAF at dose levels of 150, 100, 60, 45, 35 or 30 p.p.m., 500 p.p.m. phenobarbital (PB) or basal diet as a control for 6 weeks. Two-thirds partial hepatectomy (PH) was performed at week 3. The rats were sequentially killed from weeks 0 to 16 and liver sections were analysed by double staining for both BrdU incorporation and glutathione S-transferase placental form (GST-P) expression. 2-AAF increased numbers and areas of GST-P positive (GST-P+) foci in a dose-dependent manner, especially after PH. Proliferation of hepatocytes, as indicated by BrdU labelling indices (LI), was higher in GST-P+ foci than in surrounding hepatocytes in all 2-AAF-treated groups, even after cessation of carcinogen administration. Proliferative response of hepatocytes to PH was delayed in rats treated with the highest dose of 2-AAF in both foci and in surrounding areas possibly due to the 2-AAF toxicity. In the PB treated group, the results were similar to those for the lower dose 2-AAF-treated groups. It is concluded that the development of GST-P+ foci and cell proliferation in GST-P+ foci are directly related to 2-AAF treatment in a dose-dependent manner and the present assay system is reliable for detection of carcinogenicity of chemicals even at low doses.     

Decreased sensitivity to phalloidin of normal-looking rat hepatocytes after short-term 2-acetylaminofluorene feeding

Male F344/DuCrj rats were fed a diet containing 0.02% 2-acetylaminofluorene (2-AAF) for 1 or 3 weeks, and then fed a basal diet for 2 days, 2 weeks, 8 weeks, 22 weeks or 36 weeks. Hepatocytes were isolated from the liver by collagenase perfusion, and their sensitivity to phalloidin, in terms of the formation of multiple cytoplasmic blebs, was examined. The sensitivity of gamma-glutamyltransferase (GGT)-negative hepatocytes decreased on the 22nd and 36th weeks after withdrawal of 2-AAF feeding, and that of GGT-positive cells decreased on the 36th week. Induction of a small number of foci positive for the placental form of glutathione S-transferase (GSTP) was observed in the liver of all rats on the 8th, 22nd and 36th weeks after the withdrawal of the carcinogen. However, the total area of the foci was estimated to account for less than 0.2% of liver tissues even on the 36th week. Therefore, the decrease in phalloidin sensitivity of hepatocytes, particularly of GGT-negative hepatocytes, on the 22nd and 36th weeks after 2-AAF withdrawal is suggested to be a result of a decrease in the sensitivity of otherwise normal-looking hepatocytes, which may be precursors of the cells forming the preneoplastic foci.     

Decreased Sensitivity to Phalloidin of Normal-looking Rat Hepatocytes after Short-term 2-Acetylaminofluorene Feeding

Male F344/DuCrj rats were fed a diet containing 0.02% 2-acetylaminofluorene (2-AAF) for 1 or 3 weeks, and then fed a basal diet for 2 days, 2 weeks, 8 weeks, 22 weeks or 36 weeks. Hepatocytes were isolated from the liver by collagenase perfusion, and their sensitivity to phalloidin, in terms of the formation of multiple cytoplasmic blebs, was examined. The sensitivity of gamma-glutamyltransferase (GGT)-negative hepatocytes decreased on the 22nd and 36th weeks after withdrawal of 2-AAF feeding, and that of GGT-positive cells decreased on the 36th week. Induction of a small number of foci positive for the placental form of glutathione S-transferase (GSTP) was observed in the liver of all rats on the 8th, 22nd and 36th weeks after the withdrawal of the carcinogen. However, the total area of the foci was estimated to account for less than 0.2% of liver tissues even on the 36th week. Therefore, the decrease in phalloidin sensitivity of hepatocytes, particularly of GGT-negative hepatocytes, on the 22nd and 36th weeks after 2-AAF withdrawal is suggested to be a result of a decrease in the sensitivity of otherwise normal-looking hepatocytes, which may be precursors of the cells forming the preneoplastic foci.     

Fumonisin B1-induced hepatocellular and cholangiocellular tumors in male Fischer 344 rats: potentiating effects of 2-acetylaminofluorene on oval cell proliferation and neoplastic development in a discontinued feeding study

Fumonisin B1 (FB1) is a naturally occurring mycotoxin produced by Fusarium verticillioides. Dietary exposure to FB1 has been linked to human cancer in certain parts of the world, and treatment with FB1 causes oval cell proliferation and liver tumors in rats. To study the potential role of oval (liver progenitor) cells in the cellular pathogenesis of FB1-induced liver tumors, we gave male F344 rats prolonged treatment with FB1 for 25 weeks, followed by return to control diet until 50 weeks ('stop study'). The time course of FB1-induced liver lesions was followed by examination of serial liver biopsies at set time intervals and post-mortem liver tissue at the end of the study. The effects of different FB1 treatment regimens (5 versus 25 weeks), as well as the modulating effect of 2-acetylaminofluorene (2-AAF), on the kinetics of oval cell proliferation and development of liver tumors were compared. Prolonged treatment with FB1 in normal diet caused persistent oval cell proliferation and generation of both hepatic adenomas and cholangiofibromas (CFs). These liver lesions occurred in the setting of chronic toxic hepatitis and liver fibrosis/cirrhosis, similar to that seen in human hepatocarcinogenesis. Some adenomas and CFs were dysplastic, and one post-mortem liver contained a hepatocellular carcinoma. OV-6+ oval cells were noted in close relation to proliferative neoplastic liver lesions, and some of these lesions expressed OV-6, suggesting that all these cell types were derived from a common progenitor cell. 2-AAF enhanced the size of FB1-induced glutathione S-transferase pi+ hepatocellular lesions and the incidence of CFs in post-mortem liver specimens, but this was not statistically significant. In conclusion, this study supports the involvement of dietary FB1 in liver carcinogenesis in male F344 rats. Oval cells may be the source of both the hepatocellular and cholangiocellular tumors induced by prolonged treatment with FB1. 2-AAF appears to have an enhancing effect on FB1-induced liver tumors, presumably due to its potent inhibitory effects on hepatocyte regeneration.     

Inhibition of binding of 2-acetylaminofluorene to DNA by butylated hydroxytoluene and butylated hydroxyanisole in vitro

Numerous studies have shown that the food antioxidants butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA), under specific exposure conditions, can inhibit hepatocarcinogenesis induced by various carcinogens. The purpose of the present work was to study the biochemical mechanisms responsible for the anticarcinogenic activity of BHA and BHT using in vitro systems. The effects of BHA and BHT on the binding of 2-acetylaminofluorene (2-AAF) to DNA was determined in a microsomal system and in primary cultures of rat hepatocytes. It was found that both antioxidants reduce the binding of 2-AAF and that of N-OH-2-acetylaminofluorene (N-OH-2-AAF) to calf thymus DNA in the presence of liver microsomes. The inhibition was however more pronounced with the parent compound. Lower levels of DNA binding were also detected in hepatocytes incubated with 2-AAF along with BHA or BHT. These results suggest that phenolic antioxidants can exert anticarcinogenic activity through modulation of carcinogen interaction with DNA which may reflect on alteration in carcinogen metabolic activation.     

Comparison of GST-P Versus GGT as Markers of Hepatocellular Lineage During Analyses of Initiation of Carcinogenesis

INTRODUCTION AND BACKGROUND Our understanding of carcinogenesis in liver and other tissues has progressed steadily with the use of a sequential analytical approach (1-5). Persistent nodule hepatocytes which are potential precursors for cancer were shown to be resistant in vivo relative to the surrounding hepatocytes to cytotoxic and mitoinhibitory effects of hepatocarcinogens and hepatotoxins (6,7). A working hypothesis was that one population of initiated hepatocytes has this resistance property (1,3-9). To test this hypothesis of resistant hepatocytes as products of initiation with Farber and co-workers (1,9-13), we used single exposures to a variety of chemical carcinogens as initiators. This work showed that focal populations of hepatocytes were present in the liver after initiation which were resistant to cytotoxic and mitoinhibitory effects of 2-acetylaminofiuorene (2-AAF) and were manifest as nodular proliferations in response to resistance-selection with 2-AAF (9-14) or lasiocarpine (15). The resistant hepatocyte (RH) model became a useful bioassay for initiation (1,4,5). From these studies, a number of important conclusions about initiation could be reached: (a) cell proliferation of hepatocytes (within 3 days of initiating chemical exposure) is essential for initiation to occur (10-14), (b) the original clone of resistant hepatocytes after initiation is projected to be at most only a few cells in size (1,16), (c) these resistant hepatocyte nodules generated by initiation and resistance-selection are a rare event (1) or about 1 RH clone per 10⁵ or 10⁶ total hepatocytes exposed to an initiating carcinogen in vivo (1,4,5). In spite of the fact that resistant hepatocytes in persistent nodules arising after both initiation and resistant-selection could be readily studied biochemically, functionally (biologically), or histopathologically 07-31), initiated (resistant) hepatocytes induced by initiation alone without selection were not yet analyzable (1,4,5).     

In vivo differentiation of rat liver oval cells into hepatocytes

The Solt-Farber protocol, in the absence of an initiating agent, was used to examine the precursor-product relationship between oval cells and hepatocytes in rat liver. The animals were administered 2-acetylaminofluorene (AAF) by gavage for 2 wk combined with partial hepatectomy 1 wk after administering AAF Two dose levels of AAF were used: 9- and 21-mg total dose for animals in Groups I and II, respectively. [3H]Thymidine was administered i.p. to one-half of the animals at Day 6 post-partial hepatectomy. Animals were sacrificed 7, 9, 11, and 13 days after surgery. Only oval cells became labeled on Day 7 in both groups. On Day 9 both labeled oval cells and labeled basophilic hepatocytes were present in Group I, whereas in Group II only oval cells remained labeled. On Days 11 and 13 both oval cells and basophilic hepatocytes were labeled in both groups. The total amount of radioactivity in Group II livers remained the same on Day 9 when only labeled oval cells were present and on Days 11 and 13 when both labeled oval cells and labeled basophilic hepatocytes were present. The calculated half-life for basophilic hepatocytes was about 50 h. The differentiation of oval cells into basophilic hepatocytes was delayed in Group II as compared to Group I, and the higher dose of AAF also induced the formation of both intestinal metaplasia and bile duct formation. In situ hybridization with an alpha-fetoprotein probe showed a strong expression in groups of typical oval cells and in cells arranged in duct-like structures. In addition a transient expression of AFP was also observed in the areas of basophilic hepatocytes 9 to 11 days after partial hepatectomy. Administration of AAF decreased the level of albumin mRNA in preexisting hepatocytes and caused a significant decrease of serum albumin. In contrast, oval cells showed a strong albumin expression, and basophilic hepatocytes formed islands of albumin-expressing cells. Oval cells and the foci of early basophilic hepatocytes lacked glucose-6-phosphatase activity. At Day 13 significant numbers of basophilic hepatocytes were positive for glucose-6-phosphatase. Oval cells were strongly gamma-glutamyltranspeptidase positive, whereas the foci of basophilic hepatocytes were negative for gamma-glutamyltranspeptidase. Only occasionally were transiently gamma-glutamyltranspeptidase-positive hepatocytes observed in basophilic foci. In summary our data indicate that oval cells can differentiate to hepatocytes and may have an important physiological function as a source of major serum proteins when hepatocytes are unable to synthesize these proteins.