Content uploaded by David L Eaton
Author content
All content in this area was uploaded by David L Eaton on Sep 17, 2014
Content may be subject to copyright.
A preview of the PDF is not available
Inhibition of cell proliferation by ciprofibrate in glutathione S-transferase P1-1-positive rat hepatic hyperplastic nodules
Abstract and Figures
Previous studies have demonstrated that short-term treatment with a peroxisome proliferator (PP) decreased the size and number of genotoxic carcinogen-induced hepatic hyperplastic lesions identified by gamma-glutamyl transpeptidase (GGT) or glutathione S-transferase P1-1 (rGSTP1-1) staining. However, longer-term PP treatment of animals bearing similar hepatic hyperplastic lesions produced an increase in both the size and number of liver tumors. To characterize the hepatic hyperplastic lesions which are inhibited or promoted by PP, a unique double labeling technique was developed to determine the relative rate of cell division (e.g., DNA synthesis) in rGSTP1-1-positive nodules before and after ciprofibrate (Cip) treatment. rGSTP1-1-positive nodules were induced with the Solt-Farber resistance protocol (diethylnitrosamine-2-acetylaminofluorene partial hepatectomy). Eleven weeks after diethylnitrosamine initiation, 3 groups of rats were maintained on a control chow diet or switched to a powdered chow diet containing 0.025% Cip or 0.05% phenobarbital (PB) for the last 8 days of the experiment. A minipump implanted in the abdominal cavity released [methyl-3H]thymidine continuously for 72 h and was then removed prior to CIp or PB treatment. A second minipump was then implanted which released bromodeoxyuridine to the abdominal cavity 5 days after the start of Cip or PB administration and lasted for 72 h until the termination of the experiment. Both the [methyl-3H]thymidine and bromodeoxyuridine labeling indices (LIs) were determined in the same group of cells within individual rGSTP1-1-positive nodules in the right posterior lobes of livers. PB treatment increased both the average number of persistent GGT-positive nodules and the ratio of persistent GGT-positive to rGSTP1-1-positive nodules/cm2. In contrast, Cip treatment greatly decreased the average number and area of persistent GGT-positive nodules, as well as the ratio between persistent GGT-positive and rGSTP1-1-positive nodules/cm2. Cip treatment also resulted in a 40% decrease in the average LI in the rGSTP1-1-positive nodules. In some rGSTP1-1-positive nodules, the LI was decreased from > 40% prior to Cip treatment to < 5% afterward, suggesting that Cip treatment interrupted progression in these nodules. Such drastic changes in the LI before and after treatments were not observed in either PB- or vehicle-treated (control) animals. A number of small nodules with a high bromodeoxyuridine LI but with no or very few [methyl-3H]thymidine-labeled nuclei and negative GGT and rGSTP1-1 staining were detected only in the Cip group.(ABSTRACT TRUNCATED AT 400 WORDS)
Figures - uploaded by David L Eaton
Author content
All figure content in this area was uploaded by David L Eaton
Content may be subject to copyright.
... We consider that it is related to its antioxidative action of Boschniakia rossica [4] . Recently there are reports that GST-P is as a sensitive marker for preneoplastic hepatic foci during chemical hepatocarcinogenesis in rats [35][36][37][38][39][40] . It indicated that GST-P and GST-π are very sensitive tumor markers for basic research, prevention and cure of cancer. ...
AIM: To investigate the effect of Boschniakia rossica (BR) extract on expression of GST-P, p53 and p21ras proteins in early stage of chemical hepatocarcinogenesis in rats and its anti-inflammatory activities.
METHODS: The expression of tumor marker-placental form glutathione S-transferase (GST-P), p53 and p21ras proteins were investigated by immunohistochemical techniques and ABC method. Anti-inflammatory activities of BR were studied by xylene and croton oil-induced mouse ear edema, carrageenin, histamine and hot scald-induced rat pow edema, adjuvant-induced rat arthritis and cotton pellet-induced mouse granuloma formation methods.
RESULTS: The 500 mg/kg of BR-H2O extract fractionated from BR-Methanol extract had inhibitory effect on the formation of DEN-induced GST-P-positive foci in rat liver (GST-P staining was 78% positive in DEN+AAF group vs 20% positive in DEN+AAF+BR group, P < 0.05) and the expression of mutant p53 and p21ras protein was lower than that of hepatic preneoplastic lesions (33% and 22% positive respectively in DEN+AAF group vs negative in DEN+AAF+BR group). Both CH2Cl2 and H2O extracts from BR had anti-inflamatory effect in xylene and croton oil-induced mouse ear edema (inhibitory rates were 26%-29% and 35%-59%, respectively). BR-H2O extract exhibited inhibitory effect in carrageenin, histamine and hot scald-induced hind paw edema and adjuvant-induced arthritis in rats and cotton pellet-induced granuloma formation in mice.
CONCLUSION: BR extract exhibited inhibitory effect on formation of preneoplastic hepatic foci in early stage of rat chemical-hepato-carcinogenesis. Both CH2Cl2 and H2O extracts from BR exerted anti-inflammatory effect in rats and mice.
... In liver, most nongenotoxic carcinogens activate hepatocellular proliferation, many of them acting as direct liver mitogens inducing acute liver hyperplasia, although these agents are also mitotic inhibitors (9,42). Others induce liver cell death and provoke a regenerative response of the liver to compensate for the cell losses (20,29). ...
Peroxisome proliferators are believed to induce liver tumors in rodents due to sustained increase in cell proliferation and oxidative stress resulting from the induction of peroxisomal enzymes. The objective of this study was to conduct a sequential analysis of the early changes in cell-cycle kinetics and the dynamics of rat liver DNA synthesis after treatment with a peroxisome proliferator. Immunofluorescent detection of proliferating cell nuclear antigen (PCNA) and bromodeoxyuridine (BrdU) incorporation into DNA during S phase we used to assess rat hepatocyte proliferation in vivo during dietary administration of Wy-14,643, a known peroxisome proliferator and hepatocarcinogen in rodents. Rats were placed on diet containing 0.1% WY-14,643 and implanted subcutaneously with 5-bromo-2'deoxyuridine containing osmotic pumps 4 days prior to being sacrificed on days 4, 11, and 25 of treatment. Isolated liver nuclei labeled with fluorscein isothiocyanate (FITC)-anti-BrdU/PI and FITC-anti-PCNA/PI were analyzed for S-phase kinetics using flow cytometry. Morphometric analysis was performed to evaluate nuclear and cell size and enumeration of BrdU labeled cells, binucleated hepatocytes, and mitotic index. The BrdU labeling index increased 2-fold in livers of Wy-14,643-treated rats at day 4, but distribution of cells in G1, S phase, and G2-M did not differ significantly from controls. PCNA-positive cells decreased from 36% on day 4 to 17% on day 25, whereas the percentage of PCNA-positive cells in controls increased 2-fold from day 4 to day 11 and remained unchanged up to day 25. The differences in the number of PCNA-positive nuclei between control and Wy-14,643-treated groups were statistically significant only on day 4. Binucleated hepatocytes, determined by morphometric analysis, increased slightly on day 25 in treated rats parallel to an increase in the percentage of cells in G2-M phase. Significant shifts were noted in nuclear diameter and nuclear area after 11 and 25 days of treatment with Wy-14,643. Hepatic cell populations with nuclei > 9 microns diameter and nuclear area > 64 microns2 increased in Wy-14,643-fed rats during the treatment period compared with the control, indicating hepatic karyomegaly and hyperploidy, whereas percentage of distribution of nuclei based on diameter and area remained consistently unchanged in control animals from 4 through 25 days of sham treatment. The flow cytometric and morphometric analysis indicated an initial wave of DNA synthesis in response to Wy-14,643. The hepatomegaly was sustained over the treatment period accompanied by increase in ploidy with a significant shift toward hyperploidic hepatocytes. The increase in DNA content was almost entirely accounted for by the overall polypoidy increase rather than by an absolute increase in cells.
... In this model, activation of ERK2 in initiated cells could provide a selective growth advantage along with a protective effect against toxicity, whereas the inhibitory effects of toxicity on non-initiated cells could provide further selective pressure (48). Similarly, the PPs have inhibitory effects on cell growth (17,32) in addition to their induction of ERK activity, both of which may contribute to their tumor-promoting activity. ...
Peroxisome proliferators (PPs) are a class of nongenotoxic carcinogens in the rodent liver. The induction of immediate-early gene expression in immortalized mouse liver cells by the PPs Wy-14, 643, monoethylhexyl phthalate, ciprofibrate ethyl ester, and clofibrate suggested that they may be activating growth-regulatory signal transduction pathways. We report that incubation of quiescent ML457 cells with Wy-14,643 resulted in the appearance of two tyrosine-phosphorylated bands of approximately 44 and 42 kDa with maximal phosphorylation at 20 min. These two proteins were identified as extracellular signal-regulated kinases (ERKs) ERK1 and ERK2 (also known as mitogen-activated protein kinases, or MAPKs). Stimulation of quiescent ML457 cells with monoethylhexyl phthalate, ciprofibrate ethyl ester, and clofibrate also resulted in tyrosine phosphorylation of ERK1 and ERK2; however, the steroid PP dehydroepiandrosterone sulfate, which does not induce immediate-early gene expression, did not induce phosphorylation of ERK1 and ERK2. Kinase activity of ERK1 and ERK2 was stimulated by the PPs, consistent with their phosphorylation. The PPs also induced phosphorylation of the upstream regulator MAPK/ERK kinase (MEK). Preincubation of quiescent cells with MEK inhibitor PD98059 blocked activation of ERK1 and ERK2 by the PPs, implicating MEK activation as a requirement for PP-induced ERK activation. In addition, pretreatment with PD98059 greatly reduced the PP-induced expression of immediate-early genes c-fos, egr-1, and to a lesser extent junB. Induction of ERK phosphorylation and junB expression by Wy-14,643 was also seen in rat hepatocytes. These results attribute many of the effects of PPs on immediate-early gene expression to the activation of the MEK/ERK signal transduction pathway and add the PPs to the growing number of tumor promoters that modulate signaling proteins.
The biological activity of peroxisome proliferators (PPs) is mediated by a class of receptors, known as PPARs (PP-Activated Receptor), belonging to the nuclear receptor superfamily. Upon ligand binding, PPARs dimerize with retinoid receptors, translocate to the nucleus, recognize specific PP-responsive elements on DNA and transactivate a number of genes. Several processes are regulated by PPARs, such as mitochondrial and peroxisomal fatty acid uptake and beta-oxidation, inflammation, intracellular lipid trafficking, cell proliferation and death. In addition, PPARs have been proposed to act as tumor suppressors or as tumor promoters, depending on the circumstances. In particular, PPs have been extensively studied for their hepatocarcinogenic action in rodents, most often ascribed to their antiapoptotic action. Recent evidence, however, has been provided about the antiproliferative, proapoptotic, and differentiation-promoting activities displayed by PPAR ligands. The present review will focus on the cytotoxic effects exerted by several PPs, among which clofibrate, on different types of tumor cells, with particular reference to the mechanisms of cell death and to their relevance to cancer induction and progression.
An experiment was performed to investigate whether, during regression of the liver hyperplasia induced by a direct mitogen, apoptosis differentially affects replicated and non-replicated hepatocytes. After a single dose of the direct mitogen lead nitrate (LN), male Wistar rats were given repeated injections of tritiated thymidine, and were killed either 3 days (time of maximal hepatic DNA increase) or 15 days (complete regression of the hyperplasia) after mitogen treatment. Determination of liver DNA radioactivities and labelling indices (LIs) at the two time points revealed an approximately 40% loss in total liver DNA radioactivity, a 20% decrease in the specific activity of DNA, and a 20% reduction in the cell LI. Three days after LN administration 64% of the apoptotic bodies contained thymidine grains in their nuclear fragments. The results indicated that apoptosis affects both hepatocytes that replicated, and those that did not replicate, the former being slightly more sensitive. A second experiment was then performed to investigate whether and to what extent different types of cell death (apoptosis versus necrosis) influence the growth of hepatocytes initiated by a chemical carcinogen. Male Wistar rats were given a single dose of diethylnitrosamine, and 2 weeks thereafter either a single dose of LN, or a necrogenic dose of carbon tetrachloride (CCl4). Bromodeoxyuridine was next infused for 5 days, and some of the animals were killed at this time point, and others after an additional 3 weeks. Administration of CCl4 resulted in an increase in both the average size and the percentage area occupied by placental glutathione S-transferase-positive lesions. In contrast, administration of lead nitrate resulted in a strong reduction (50%) in the number of positive lesions with no remarkable change in the percentage area occupied by them. These differential effects occurred even though comparable LIs were observed in rats treated with the two agents. The results suggest that lead nitrate leads to a loss of initiated hepatocytes, due to the apoptosis that occurs during regression of the LN-induced hyperplasia.
A group of structurally related drugs representing diverse therapeutic classes share, among a number of pharmacological properties, enhancement of tumor growth in several rodent models of malignancy. One common action, the inhibition of histamine binding to and catalytic activity of cytochrome P450 monooxygenases, is highly correlated with potency to enhance tumor growth. Among members of this drug ensemble, the antiestrogen tamoxifen has been shown in controlled clinical studies to increase the incidence of uterine and gastrointestinal cancer and to accelerate the course of gastric cancer, and the tamoxifen analogue clomiphene has been linked to neuroblastoma and the tricyclic group of antidepressants to ovarian cancer. The determination of drug affinities for protein modulators of cell growth, proliferation, and transformation suggests a strategy for identifying at least some classes of chemicals that impart oncologic risks to humans.
Liver cell growth can be induced in two distinct patterns: compensatory regeneration and direct hyperplasia. In the former, DNA synthesis is preceded by a loss of liver cells such as seen after partial resection of the liver or cell necrosis, whereas in direct hyperplasia, DNA synthesis is stimulated without cell loss. During the past decade, considerable advances have been made in understanding molecular mechanisms of the compensatory regeneration. There is increasing evidence that hepatocyte proliferation induced by some primary mitogens is mediated by patterns of growth factor modulation and signal transduction different from those of compensatory regeneration. Indeed, whereas activation of transcription factors such as NF-kappa B and increased expression of immediate early genes such as c-fos, c-jun, egr-1, and c-myc are induced during compensatory regeneration, such changes are not observed during hyperplasia induced by certain primary mitogens. In addition, although experimental evidence suggests a critical role for growth factors such as hepatocyte growth factor and transforming growth factor-alpha for the progression into cell cycle of competent hepatocytes in compensatory regeneration, these growth factors do not appear to play a major role in direct hyperplasia. One class of primary mitogens may trigger their actions through tumor necrosis factor-alpha, and the other by activation of nuclear hormone receptors. The differences in molecular events observed between liver regeneration and direct hyperplasia may affect differently the initiation step of chemical hepatocarcinogenesis. Whereas the former supports initiation by chemicals, the latter does not. A similar lack of effect on promotion of carcinogen-altered cells has also been observed after acute treatment with some primary mitogens. Definition of the mechanisms by which primary mitogens stimulate liver cell proliferation may elucidate the nature of the signals responsible for triggering the entry into cell cycle. Furthermore, due to their low toxicity, primary liver mitogens could have significant clinical applications in gene transfer and liver transplantation.
Peroxisome proliferators (PPs) are a diverse group of nongenotoxic rodent liver carcinogens. One potential mechanism for the carcinogenicity of PPs is epigenetic modulation of growth-regulatory signal transduction pathways. We investigated the effects of PPs on growth-regulatory gene expression and cell proliferation in immortalized mouse liver cells, comparing PPs with other growth regulators and tumor promoters of known activity. The PPs Wy-14643, mono-ethylhexyl phthalate, clofibrate, and ciprofibrate ethyl-ester were found to be potent inducers of immediate-early gene expression (including c-fos, c-jun, junB, egr-1, NUP475, and to a lesser extent fosB, JE, and KC, with maximal expression seen 1 h after treatment of serum-deprived quiescent cells. The gene induction was potently inhibited by protein kinase inhibitor H7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride] but not by H8 [N-¿2-(methyl-amino)ethyl¿-5-isoquinolinesulfonamide dihydrochloride], indomethacin, or nordihydroguaiaretic acid. Compared with other growth regulators, the profile of PP-induced gene expression was most similar to that induced by arachidonic acid and eicosatetraynoic acid. The induction of immediate-early gene expression by PPs was followed by enhanced progression into S phase (DNA synthesis) when quiescent cells were treated with the PPs for only 1 h, washed, and then incubated without PPs. However, no stimulation of DNA synthesis was seen when the PPs were continually present. Furthermore, the PPs inhibited serum-induced DNA synthesis, even when they were added 6 h after serum stimulation (in late G1). Dehydroepiandrosterone-sulfate, a unique PP in being a steroid, had no detectable effect on immediate-early gene expression, did not stimulate DNA synthesis when applied for only 1 h, but did inhibit serum-induced DNA synthesis. Thapsigargin and A23187 mimicked this mitoinhibitory activity of PPs, suggesting that calcium mobilization by PPs might be involved. Our results demonstrate that PPs can modulate cell proliferation either by a stimulatory activity that functions in early G1, associated with activation of immediate-early gene expression, or by an inhibitory activity that functions in late G1; both activities could potentially play a role in tumor promotion by PPs.
Two different types of focal preneoplastic lesions, tentatively named Type I and II lesions, were recognized in the liver of rats chronically treated with clofibrate for 104 weeks. Type I lesions were characterized by mostly negative glucose-6-phosphate dehydrogenase (G6PD) activity (6 out of 10, 60%) and positive expression of succinate dehydrogenase (10 out of 10, 100%), in addition to the previously documented complete lack of expression of glutathione S-transferase, placental form (GST-P) and gamma-glutamyl transpeptidase (GGT). Furthermore, most importantly, Type I lesions exhibited a clear decrease in immunohistochemically demonstrated connexin32 (Cx32) spot counts on their hepatocyte membranes, similarly to nitrosamine-induced lesions. In contrast, Type II lesions, mostly small in size and positively expressing GST-P and/or GGT and G6PD, similarly to their previously reported nitrosamine-induced counterparts, did not exhibit a significant decrease in Cx32 count. In addition, spontaneously occurring lesions, again sharing the same enzyme phenotype, did not show a decrease in Cx32. The results indicate that: (i) a clear distinction between the two lesions, with Type I being involved in clofibrate-induced tumors and Type II being more likely to be spontaneous in nature; (ii) a decrease in Cx32 is closely linked to lesion development and possibly stage of progression, irrespective of the enzyme phenotype and the applied carcinogen; (iii) the unaltered condition of Cx32 may suggest a slow growing or non-progressive nature.
Increased expression of cyclooxygenase-2 (COX-2), the rate-limiting enzyme in prostaglandin synthesis, has been associated with growth regulation and carcinogenesis in several systems. COX-2 is known to be induced by cytokines and the skin tumor promoter 12-tetradecanoylphorbol-13-myristate (TPA). In the present study, we investigated the effects of several non-TPA-type tumor promoters on COX-2 expression in immortalized mouse liver cells. Specifically, we tested peroxisome proliferators (PPs), which are rodent liver tumor promoters that cause gross alterations in cellular lipid metabolism, the rodent liver tumor promoter phenobarbital, and the skin tumor promoters okadaic acid and thapsigargin. The PPs Wy-14643, mono-ethylhexyl phthalate, clofibrate, ciprofibrate ethyl ester, and eicosatetraynoic acid each caused large increases in COX-2 mRNA and protein, with maximal expression seen approximately 10 h after treatment of quiescent cells. COX-2 expression was also induced by thapsigargin, okadaic acid, and calcium ionophore A23187, but not by phenobarbital or the steroid PP dehydroepiandrosterone sulfate. Induction of COX-2 expression generally resulted in increased synthesis of prostaglandin E2 (PGE2). However, the PPs caused little or no increase in PGE2 levels, and they inhibited serum-induced PGE2 synthesis. Unlike non-steroidal anti-inflammatory drugs, the PPs do not directly inhibit cyclooxygenase enzyme activity in vitro. Thus, PPs regulate prostaglandin metabolism via both positive (COX-2 induction) and inhibitory mechanisms. In summary, the strong induction of COX-2 expression by PPs, thapsigargin, and okadaic acid suggests a possible role for COX-2 in the growth regulatory activity of these non-TPA-type tumor promoters.
Hyperplastic nodules appearing during the preneoplastic phase of liver carcinogenesis were divided into two types, persistent and remodeling, according to the pattern of staining for γ-glutamyltransferase. In the resistant cell model of liver carcinogenesis used in this study, hyperplastic nodules, uniformly staining for γ-glutamyltransferase, rapidly emerge by 4 weeks after a single injection of diethylnitrosamine and brief selection by dietary 2-acetylaminofluorene plus partial hepatectomy. By 6 weeks, a majority of nodules (about 75%) show an obvious irregularity and loss of uniformity in staining for γ-glutamyltransferase while the remaining nodules continue to be uniformly stained. The number of irregularly stained nodules increases over the next 18 weeks until over 95% of nodules show the nonuniform loss of enzyme activity. The progressive loss of enzyme activity is accompanied by architectural remodeling to normal-appearing liver. This is associated with the increasing disappearance of many obvious nodules from the liver as the remodeling ones blend imperceptibly with the surrounding liver. The uniformly stained nodules show the persistence of hepatocyte arrangements in plates two or more cells thick and in acini and of cytoplasmic hypertrophy characteristic of persistent hyperplastic nodules. Labeling indices are much higher in hepatocytes of the persistent uniformly stained nodules than in the remodeling ones. The possibility of exploiting this phase of the model further for in-depth analysis of the nodule-to-carcinoma sequence is discussed. © 1982, American Association for Cancer Research. All rights reserved.
Hyperplastic liver lesions which develop following administration of hepatocarcinogens have been implicated as probable precursors for the cancers which ultimately develop. Some, and possibly all, of these putative precursor lesions are resistant to the necrogenic and growth-inhibitory action of hepatocarcinogens and other hepatotoxins. An in vivo assay system based on this resistance phenomenon has been developed which encourages the rapid selective growth of carcinogen-altered hepatocytes, facilitating their early identification. The system consists of a) single carcinogenic dose of diethylnitrosamine (DEN), b) short-term dietary exposure to 2-acetylaminofluorene (2-AAF) sufficient to suppress growth of virtually all normal hepatocytes, and c) partial hepatectomy (PH) to actuate rapid growth of DEN-altered hepatocytes not suppressed by 2-AAF. Following PH, the DEN-altered hepatocytes grow out as basophilic foci which are distributed randomly throughout the 2-AAF-suppressed parenchyma. Within 1 week they can be seen as tiny, discrete, translucent nodules on the capsular and cut surface of the remaining lobes. The lesions continue to proliferate and become histologically indistinguishable from typical carcinogen-induced hyperplastic liver nodules frequently described in the literature. These in turn appear to be precursor lesions for at least some hepatocellular carcinomas. Future experimentation based on this phenomenon of "selective resistance to cytotoxicity" should prove valuable in answering specific questions about the carcinogenic process in liver and possibly in other tissues.
Dehydroepiandrosterone, a major secretory steroid hormone of the human adrenal gland, possesses mitoinhibitory and anticarcinogenic properties. It also induces peroxisome proliferation in the livers of rats and mice. Because peroxisome proliferators exhibit hepatocarcinogenic potential, it is necessary to examine the long term hepatic effects of dehydroepiandrosterone since this hormone is contemplated for use as a potential cancer chemopreventive agent in humans. Dehydroepiandrosterone was administered in the diet at a concentration of 0.45% to F-344 rats for up to 84 weeks. At the termination of the experiment, 14 of 16 rats developed hepatocellular carcinomas. Liver tumors induced by dehydroepiandrosterone lacked gamma-glutamyl transpeptidase and glutathione S-transferase (placental form); these phenotypic properties are identical to the features exhibited by liver tumors induced by other peroxisome proliferators. Dehydroepiandrosterone was also shown to markedly inhibit liver cell [3H]thymidine labeling indices, suggesting that cell proliferation is not a critical feature in liver tumor development with this agent. These results show that although dehydroepiandrosterone exerts anticarcinogenic effects in a variety of tissues, the peroxisome-proliferative property makes it a hepatocarcinogen.
This study was conducted to examine relationships between phenobarbital (PB) treatment, specific cytochrome P450 gene expression patterns and growth rates of hepatic hyperplastic nodules. Nodules were induced in 8 week old male F344 rats by a Solt-Farber resistance protocol. Six weeks after diethylnitrosamine (DEN) initiation, subgroups of rats were either kept on control chow diet or transferred to a chow diet containing 0.05% PB, then killed 2 weeks later. [3H]Thymidine was delivered continuously via osmotic minipump during the final 3 days of the experimental to label dividing cells. PB treatment resulted in a 89% increase in the number of persistent gamma-glutamyl transpeptidase (GTT) nodules per cm2 section, a 278% increase in the area of persistent GGT nodules per cm2 section, and a 116% increase in the average area per persistent nodule. PB increased the number of [3H]thymidine-labeled persistent GGT nodules but did not significantly change the labeling index (LI) distribution pattern or the average LI. A slight but uniform increase in CYP1A2 expression (relative to surrounding, non-nodular tissue) was observed in 50% (23/46) and 59% (60/102) of persistent nodules in control and PB-treated animals respectively. In contrast, for nodules undergoing remodeling, CYP1A2 expression was elevated in only 9% (2/22) and 0% (0/24) in control and PB groups respectively. In the PB group, CYP2B1/2 was underexpressed in 53% (54/102) of persistent GGT nodules and in 0% (0/24) of the remodeling nodules. Comparing LI among the persistent GGT nodules, those that displayed simultaneous increases in CYP1A2 and decreases in CYP2B1/2 had the highest LI, and were followed in level by those expressing either increases in CYP1A2 or decreases in CYP2B1/2. Nodules that expressed both CYP1A2 and 2B1/2 in a manner similar to the surrounding tissue had the lowest LI. Thus, these data suggest that expression of specific forms of cytochrome P450 may be an important factor in determining other phenotypic characteristics, e.g. rate of cell proliferation and GGT expression, within specific nodules.
Hepatocyte growth factor (HGF), mol. wt 105,000 is a potent mitogen for hepatocytes. HGF is strongly associated with compensatory regeneration in the liver after two-thirds partial hepatectomy and carbon tetrachloride administration. Plasma levels of HGF increase markedly during early stages of compensatory hyperplasia caused by these treatments. This is followed by an increase in HGF mRNA in the liver. This is in contrast to other growth factors for liver (epidermal growth factor, transforming growth factor alpha and acidic fibroblast growth factor) whose levels in plasma remain virtually undetectable during compensatory hyperplasia. We have shown that during augmentative hyperplasia caused by the tumor promoters alpha-hexachlorocyclohexane, phenobarbital and ciprofibrate, plasma levels of HGF also increase. This increase of HGF occurs during the transient wave of DNA synthesis caused by administration of these xenobiotics, providing further support for HGF as being the stimulator of DNA synthesis during both augmentative and compensatory hyperplasia.
Carcinogenesis can proceed by a variety of pathways involving the sequential mutation of normal cellular growth control genes and the clonal expansion of the resulting precancerous or cancerous cells. Chemical carcinogens may act by inducing mutations and/or altering cellular growth control. One class of chemical carcinogens are the genotoxicants. These compounds or their metabolites are DNA reactive and directly induce mutations or clastogenic changes. The observation that most mutagens are also carcinogenic is the basis for many current predictive assays and risk assessment models; however, there are different classes of nongenotoxic carcinogens that do not interact with DNA. Mitogens directly induce cell proliferation in the target tissue; cytotoxicants produce cell death followed by regenerative cell proliferation. Differential toxicity and/or growth stimulation induced by mitogens and cytotoxicants may provide a preferential growth advantage to spontaneous or chemically induced precancerous or cancerous cells. Mutagens are much more effective carcinogens at doses that also induce cell proliferation, and mutational activity may occur as an event secondary to cell proliferation. Thus, chemically induced cell proliferation is an important mechanistic consideration for both genotoxic and nongenotoxic carcinogens. The complex quantitative relationships between chemically induced cell proliferation and carcinogenic activity are under study in many laboratories. Such information should be considered in setting doses for cancer bioassays, for classifying chemical carcinogens and in providing more realistic approaches to risk assessment. Of particular concern in extrapolating cancer risk from rodent models to humans are those nongenotoxic agents that exhibit carcinogenic activity only at doses that also produce cytolethality and regenerative cell proliferation in the target organ.
Perfluorooctanoic acid (PFOA) is a peroxisome proliferator. The aim of this study was to test for its ability to act as a positive modulator of hepatocarcinogenesis, in the so-called biphasic (initiation by diethylnitrosamine 200 mg/kg ip followed by treatment with the suspected modulators) and triphasic (initiation by the same dose of diethylnitrosamine followed by a selection procedure for 2 weeks consisting of giving 2-acetylaminofluorene and in the middle of this treatment a single dose of CCl4 followed by treatment with the suspected modulators) protocols of liver carcinogenesis. In both protocols treatment with PFOA increased the incidence of malignant hepatocellular carcinoma (HCC). As compared to phenobarbital, the modulating effect of PFOA is more pronounced in a biphasic than in the triphasic protocol. In parallel with positive modulation of HCC, PFOA also selectively induced the peroxisomal acyl-CoA oxidase activity and, to a lesser extent, catalase activity.