Liver cell proliferation induced by the mitogen ethylene dibromide, unlike compensatory cell proliferation, does not achieve initiation of rat liver carcinogenesis by diethylnitrosamine

The purpose of this investigation was to determine whether mitogen-induced cell proliferation is as effective as compensatory cell proliferation in achieving initiation of carcinogenesis in rat liver. Male Wistar rats were injected with a single non-necrogenic dose of the hepatocarcinogen diethylnitrosamine (DENA) during the peak of DNA synthesis following the administration of the hepatic mitogen ethylene dibromide (EDB) or a necrogenic dose of CCl4. After subjecting the animals to a promoting procedure, the rats were sacrificed and the initiated hepatocytes were monitored as gamma-glutamyltranspeptidase (gamma-GT) positive foci. The results indicate that while DENA administration during compensatory cell proliferation results in the formation of GT positive foci, no enzyme-altered foci were produced when the carcinogen was given during liver hyperplasia induced by EDB, despite the fact that at the time of carcinogen administration, the extent of cell proliferation, as monitored by thymidine incorporation into DNA, was the same in both the groups.     

Requirement of cell proliferation for the initiation of liver carcinogenesis as assayed by three different procedures

Experiments were designed to determine the role of cell proliferation in the initiation of liver carcinogenesis induced by chemicals. To investigate this, two methylating carcinogens, N-methyl-N-nitrosourea and 1,2-dimethylhydrazine, were used as the initiating carcinogens. The initiated hepatocytes were monitored by selectively stimulating them to grow into focal islands of presumptive preneoplastic hepatocytes. The experimental approach in brief consisted of the following. Rats received a nonnecrogenic dose of the carcinogen; at a time period when the carcinogen could no longer be detected in the system, they were subjected to either partial or sham hepatectomy. The initiated cell thus formed were selectively stimulated to grow into foci of preneoplastic hepatocytes using three different selection regimens: (a) feeding a diet containing 0.02% 2-acetylaminofluorene plus one administration of carbon tetrachloride (2 ml/kg body weight) intragastrically; (b) feeding a diet containing 0.05% phenobarbital; and (c) feeding a choline-deficient diet. The foci were quantitated by staining them for the presence of gamma-glutamyltransferase. The results obtained indicate that irrespective of the type of selection procedure used foci of preneoplastic hepatocytes were seen only in rats that received the carcinogen coupled with a cell-proliferative stimulus such as partial hepatectomy. Very few or no foci were seen in rats that received the carcinogen plus sham hepatectomy. These results suggest that cell proliferation plays an important role in the initiation of liver carcinogenesis by chemicals.     

Non-steroidol anti-inflammatory drug effect on crypt cell proliferation and apoptosis during initiation of rat colon carcinogenesis.

Sustained use of non-steroidal anti-inflammatory drugs (NSAIDs) may prevent colorectal cancer. However, the optimal drug, period of efficacy and mechanism(s) of action are unknown. Experiments were undertaken to determine which of several NSAIDs would modulate colon crypt cell proliferation or apoptosis when given during the initiation phase of 1,2-dimethylhydrazine (DMH)-induced rat colon cancer. Colon crypts located both away from and over an aggregate of lymphoid nodules (ALN) were examined. Rats were injected with aspirin, indomethacin, nabumetone, sodium salicylate, 16,16-dimethyl prostaglandin E2 or saline for 3 days and DMH or DMH vehicle on day 4 of each week for 8 weeks, then killed 3 days after the last DMH injection. At the time of killing, DMH had significantly increased crypt cell proliferation but not apoptosis. There was significantly more cell proliferation and apoptosis in crypts over the ALN than away from the ALN. Aspirin and salicylate increased proliferation and apoptosis in crypts over the ALN. Finally, the distributional peaks of cell proliferation and apoptosis were shifted significantly closer together after DMH. Thus, DMH increases proliferation and alters the distribution of proliferating and apoptotic cells in colon crypts early in carcinogenesis. Aspirin may suppress tumour incidence via salicylate by enhancing apoptosis in carcinogen-initiated cells.     

Studies on the kinetics of expression of cell cycle dependent proto-oncogenes during mitogen-induced liver cell proliferation

The present study was undertaken to determine the kinetics of DNA synthesis and expression of cell cycle dependent proto-oncogenes in response to two types of cell proliferative stimuli in male Wistar rat liver. The peak of DNA synthesis was approximately 24 h after a compensatory cell proliferative stimulus induced by 2/3 partial hepatectomy and approximately 36 h following a mitogenic stimulus obtained with a single dose of lead nitrate (10 micromol/100 g body wt, through femoral vein). Even though both proliferative stimuli induced the expression of c-fos, c-myc and c-Ha-ras, the extent of the increase in c-fos expression was 4- to 5-fold less in mitogen-induced cell proliferation. In addition, while the expression of c-myc, following partial hepatectomy returned to basal level by 4 h, the induced expression of c-myc persisted for up to 40 h during the lead nitrate-induced liver cell proliferation.     

Further evidence that mitogen-induced cell proliferation does not support the formation of enzyme-altered islands in rat liver by carcinogens

Our earlier studies have revealed that direct hyperplasia induced by liver mitogens such as lead nitrate, ethylene dibromide, nafenopin and cyproterone acetate, unlike compensatory cell proliferation induced by partial hepatectomy and CCl4, does not support the formation of enzyme-altered islands induced by chemical carcinogens in the liver. In the previous studies carcinogens were given at the peak of DNA synthesis induced by the liver mitogens. If the mitogens have altered the sensitive phase of the hepatocyte to the carcinogenic attack, administering the carcinogen at one time point following the mitogenic stimulus might have missed the sensitive phase. In order to overcome this possibility in the present study male Wistar rats weighing 200-250 g were given N-methyl-N-nitrosourea (MNU; 60 mg/kg, i.p.) at three points representing G1, S and G2/M phases of the cell cycle following different types of liver cell proliferative stimuli. In another experiment MNU (60 mg/kg, i.p.) and diethylnitrosamine (15 mg/kg, i.p.) were given prior to the administration of proliferative stimuli. The initiated hepatocytes were also assayed following promotion by two different promoting regimens, namely phenobarbital and the resistant-hepatocyte model. Further, the initiated hepatocytes were monitored not only by using the appearance of islands of enzyme-altered hepatocytes but also using the incidence of hepatocellular carcinoma. The results of this study clearly revealed that irrespective of the protocol used, only the compensatory liver cell proliferation but not the mitogen-induced direct hyperplasia supported the formation and the growth of enzyme-altered islands in the liver induced by chemical carcinogens.     

Ras-driven proliferation and apoptosis signaling during rat liver carcinogenesis is under genetic control

Fast growth and deregulation of G1 and S phases characterize preneoplastic and neoplastic liver lesions of genetically susceptible F344 rats, whereas a G1-S block in lesions of resistant BN rats explains their low progression capacity. However, signal transduction pathways responsible for the different propensity of lesions from the 2 rat strains to evolve to malignancy remain unknown. Here, we comparatively investigated the role of Ras/Erk pathway inhibitors, involved in growth restraint and cell death, in the acquisition of a phenotype resistant or susceptible to hepatocarcinogenesis. Moderate activation of Ras, Raf-1 and Mek proteins was paralleled in both rat models by strong induction of Dab2 and Rkip inhibitors. Levels of Dusp1, a specific ERK inhibitor, increased only in BN rat lesions, leading to modest ERK activation, whereas a progressive Dusp1 decline occurred in corresponding lesions from F344 rats and was accompanied by elevated ERK activation. Furthermore, a gradual increase of Rassf1A/Nore1A/Mst1-driven apoptosis was detected in both rat strains, with highest levels in BN hepatocellular carcinoma (HCC), whereas loss of Dab2IP, a protein implicated in ASK1-dependent cell death, occurred only in F344 rat HCC, resulting in significantly higher apoptosis in BN than F344 HCC. Taken together, our results indicate a control of the Ras/Erk pathway and the pro-apoptotic Rassf1A/Nore1A and Dab2IP/Ask1 pathways by HCC susceptibility genes. Dusp1 possesses a prominent role in the acquisition of the phenotype resistant to HCC by BN rats, whereas late activation of RassF1A/Nore1A and Dab2IP/Ask1 axes is implicated in the highest apoptosis characteristic of BN HCC.     

Diethylnitrosamine exposure-responses for DNA damage, centrilobular cytotoxicity, cell proliferation and carcinogenesis in rat liver exhibit some non-linearities

The exposure-responses for several effects of limited exposuresto diethylnitrosamine (DEN) in the livers of male Fischer 344rats were measured and phenobarbital promotion was used to enhanceexpression of initiation of carcino-genesis. Five doses rangingfrom a cumulative total of 0.5 to 4 mmol DEN per kg body weightwere given as weekly i.p. injections for 10 weeks. This wasfollowed by 4 weeks recovery, after which the groups were maintainedon either a basal diet or 0.05% phenobarbital (PB) to promoteliver tumor development. All doses of DEN produced ethylationin liver DNA, which increased with dose. Indicative of toxicity,the centrilobular zone of glutamine synthetase-positive hepatocyteswas reduced in relationship to exposure up to a cumulative exposureof 3 mmol/kg. The two lower exposures to DEN produced no increasein cell proliferation, whereas higher exposures resulted inmarked increases, {small tilde}4-fold between 1.0 and 2.0 mmol/kgcumulative. At the end of the recovery period (14 weeks), hepatocellularaltered foci (HAF), which expressed the placental form of glutathioneS-transferase, were induced by all exposures, with an increaseof {small tilde}4-foId between the exposures of 1.0 and 2.0mmol/kg being the greatest. In rats maintained on basal dietor PB for 24 weeks after exposure, HAF increased further andwith exposures of 2.0 mmol/kg and above, all rats developedhepatocellular carcinomas. With 1.0 mmol/ kg, no liver tumoroccurred in 12 rats without promotion, whereas in those givenPB, two adenomas and two carcinomas were present in 12 rats.At the lowest exposure of 0.5 mmol/ kg, no tumor occurred inrats on basal diet, although HAF increased {small tilde}7-fold.With PB promotion, only one adenoma developed in 12 rats andHAF increased another 2-fold. Thus, the findings document non-linearityfor some of the effects of DEN and a near no-effect level forinitiation of promotable liver neoplasms at the lowest exposurein spite of a substantial induction of HAF.     

Dietary modulation of rat liver carcinogenesis

Rat liver carcinogenesis was induced according to the resistanthepatocyte model of Solt and Farber. One week after the endof the procedure for the rapid growth of altered hepatocytes,one group of rats was submitted to a high fat (20%) regimenup to the end of the experiment. The incidence of histologicallyconfirmed malignant hepatocarcinomas was compared with thatobserved in a group that remained on a normal diet. The modulating(promoting) effect of the high fat regimen was evident sincenine out of 10 animals in this group bore macroscopically detectabletumors and eight out of 10 presented histologically confirmedhepatocellular carcinomas as early as 24 weeks after the beginningof the experiment. At that time, no malignant tumors were detectedin the group submitted to the normal fat regimen. These resultsare similar to those resulting from a porto-caval shunt or thechronic administration of liver tumor promoters. This suggeststhat at this stage of the carcinogenic process, any treatmentinducing chronically metabolic adaptation in a tissue containingpreneoplastic nodules modulates positively the progression ofthese lesions as demonstrated by the dramatic reduction of thelag period for their malignant transformation.     

Chemically induced cell proliferation in carcinogenesis.

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.     

Lysosomes of rat liver and dab carcinogenesis

The relationship between DAB carcinogenesis and changes of lysosomes in rat liver was investigated. After 40 days of treatment with DAB, nodules and an abnormal cell population, containing small, large and degenerative cells, appeared. Corresponding to these changes, the S/P ratio of lysosomal enzymes such as acid RNase and β-glucuronidase increased to two or three times the normal value. These alterations were considered along with some histochemical observations on β-glucuronidase. A possible participation of these lysosomal enzymes in carcinogenesis is discussed.     

A new protocol and its rationale for the study of initiation and promotion of carcinogenesis in rat liver

A new protocol for carcinogenesis in rat liver is describedin order that confirmatory experiments might be undertaken concurrently.The basic protocol, designated IPI (initiator + promoter + initiator),is presented in several alternative forms including the possibleuse of X-irradiation as the initiator. The rationale is discussedin terms of the two-hit somatic mutation theory of Armitageand Doll, with an initial hit produced by the first dose ofinitiator and expansion of single cells to sizable clones bypromotion thereby increasing the probability of a second hitby the second dose of initiator. The question of relevant mutationsis taken up and it is proposed that genes for chalones and forchalone receptors are logical targets for consideration in atwo-mutation sequence.     

Para-methoxyphenol strongly stimulates cell proliferation in the rat forestomach but is not a promoter of rat forestomach carcinogenesis

The modifying effects of para-methoxyphenol (PMP) second stagetreatment on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-initiatedrat forestomach carcinogenesis were investigated. Groups of15 6 week old male F344 rats were given a single intragastricadministration of 150 mg/kg body wt MNNG and starting 1 weeklater were administered powdered diet containing 2.0, 1.0, 0.5,0.25 or 0% PMP until they were killed at week 52. PMP causedepithelial damage and hyperplasia in a dose-dependent mannerin the fore-stomach epithelium, but nevertheless was not associatedwith any increase in the incidences of either papillomas orsquamous cell carcinomas. The results thus clearly indicatedthat stimulation of cell proliferation does not necessarilycorrelate with promotion in the second stage of two-stage forestomachcarcinogenesis.     

Early exposure to restraint stress enhances chemical carcinogenesis in rat liver

This study examines the effect of a stress-associated condition on chemical hepatocarcinogenesis in the rat. Rats were given diethylnitrosamine (200 mg/kg. b.w., i.p.), followed, 1 week later, by three cycles of immobilization at room temperature. Two weeks after the last cycle they were treated according to the resistant hepatocyte protocol. At 4 weeks after selection, mean size of glutathione-S-transferase 7-7 positive foci/nodules was increased in the immobilized group (0.82+/-0.22 vs. 0.25+/-0.04 mm(2) in controls). Furthermore, at the end of 1 year 10/13 animals (77%) developed hepatocellular carcinoma in the former group, while only 6/14 (43%) incidence of cancer was found in controls. These results indicate that exposure to restraint stress early during carcinogenesis enhances the development of chemically-induced hepatocellular carcinoma in the rat.     

Cell proliferation and esophageal carcinogenesis in the zinc-deficient rat

Target cell proliferation was investigated throughout the developmentof esophageal cancer induced by N-nitroso-methylbenzylamine(NMBA) in weanhing rats maintained on zinc-deficient or sufficientdiets. Deficient rats were fed ad libitum, while zinc-sufficientrats were either pair-fed to the deficient animals or fed adlibitum. After 5 weeks, half of the animals in each dietarygroup were given six intragastric doses of NMBA (2 mg/kg; twiceweekly). The remaining rats were untreated by carcinogen. Atweeks 1, 2, 3, 4, 5, 7, 9 and 11 post first dose, esophagealcell proliferation was assessed in rats from each group by invivo bromodeoxyuridine (BrDU) labeling followed by immunohistochemicaldetection of cells in S-phase. At 11 weeks, the tumor incidencewas 100, 23 and 6%, respectively, in the zinc-deficient, zinc-sufficient,ad libitum and pair-fed groups. In vivo BrDU labeling revealedthat in the NMBA-untreated groups, the labeling index (LI),the number of labeled cells, and the total number of cells percross section of entire esophagi were significantly increasedby zinc deficiency at all time points; LI was lowest in zinc-sufficient,pair-fed rats. During NMBA treatment (weeks 6, 7 and 8), increasedcell proliferation occurred in both groups of zinc-sufficientesophagi but only during week 6 in the deficient ones. In theweeks following the cessation of NMBA treatment, zinc-deficientesophagi showed significantly increased LI and greater numberof labeled cells than the carcinogen treated, zinc-sufficientpair-fed or ad libitum fed groups. On the other hand, NMBA-treatedzinc-sufficient pair-fed rats showed lower LI and smaller numberof labeled cells than their zinc-sufficient ad libitum counterparts.Most importantly, esophageal papillomas were found in two zinc-deficientanimals that had received no NMBA treatment, after 10–11weeks of experimental diet These data support a direct relationshipbetween cell proliferation and tumor incidence, and also provideevidence that zinc deficiency and its associated cell proliferationcould be carcinogenic.     

Association of chemically induced forestomach cell proliferation and carcinogenesis

A number of chemicals have been shown to cause malignant neoplasms in the forestomach of Fischer 344 rats when administered chronically by gavage. The present study was designed to identify early forestomach lesions following 2-week repeated gavage administration of some of these forestomach carcinogens. In this manner, we attempted to examine the hypothesis that early cell proliferation is associated with repetitive gavage administration of these chemicals. Groups of 8 or more male F344 rats received 1 of 6 reported forestomach carcinogens (ethyl acrylate (EtAc), diglycidyl resorcinol ether(DGRE), 1,2-dibromoethane (DBE), 1,2-dibromo-3-chloropropane (DBCP), 1-chloro-2-methylpropene (dimethylvinyl chloride, DMVC) and 3-chloro-2-methylpropene (CMP)), 1 of 2 structurally related chemicals (methyl methacrylate (MMA) and dichloroethane (DCE)) which were negative in chronic carcinogenicity studies or the vehicle (corn oil) alone 5 days/week for 2 weeks. Histopathologic examination of forestomachs of rats killed 24 h after the last dose indicated no significant difference in the incidence or severity of epithelial cell proliferation in the rat forestomach between the vehicle control group and the 2 negative control groups. In contrast, the incidence and severity of epithelial cell proliferation of the rat forestomach in every group treated with a forestomach carcinogen was significantly higher than the incidence in the vehicle or negative control groups. These results suggest that early epithelial cell proliferation of the forestomach may be associated with at least some chemicals that induce forestomach neoplasia following chronic administration by gavage.     

Balance of cell proliferation and apoptosis in breast carcinogenesis

We determined the mitotic and apoptotic index through the spectrum of preinvasive ductal breast lesions to invasive carcinoma in search of disturbances in the proliferation/cell death balance in breast carcinogenesis. Seventytwo pure preinvasive ductal breast lesions (without invasive carcinoma) and 103 invasive breast carcinomas were used. The numbers of mitotic and apoptotic cells were microscopically counted in hematoxylin and eosin stained sections (MI and Al, respectively), and the ratio of the values of MI and AI was calculated for each individual case (M/A index).A distinction was made between well differentiated and poorly differentiated breast lesions, based on histological type and nuclear grade, to arrive at two plausible progression models for breast carcinogenesis. For the well differentiated breast lesions, the MI was rather equal for hyperplasias and well differentiated DCIS, but increased 6fold from DCIS to well differentiated invasive carcinoma. The AI remained in the same range, resulting in a 4fold increase of the M/A index. For the poorly differentiated breast lesions, a significant increase in MI and AI was found from hyperplasia to poorly differentiated DCIS. From DCIS to poorly differentiated invasive carcinoma, the MI increased significantly and the AI decreased 2fold (n.s.), resulting in a 2.5fold significant increase of the M/A index.In conclusion, the net increase of the number of cells in the transition from well differentiated preinvasive to well differentiated invasive carcinoma is accompanied by an increase of cell proliferation rather than decrease in apoptosis, suggesting that in these lesions, proliferation related mechanisms are most important in carcinogenesis and progression. In contrast, in poorly differentiated breast lesions, decreased apoptosis seems to be also important in carcinogenesis and progression. At present, we are gathering patients with invasive breast cancer who had a previous biopsy with a preinvasive lesion to obtain further more direct evidence for this hypothesis.     

Cancer initiation by fumonisin B(1) in rat liver--role of cell proliferation.

Fumonisin B(1) (FB(1)), a carcinogenic mycotoxin produced by the fungus Fusarium verticillioides in corn, causes cancer initiation in rat liver in a similar manner to genotoxic carcinogens although apparently with different kinetics. The present experiment was designed to evaluate the role of regenerative cell proliferation, effected by partial hepatectomy (PH) and carbontetrachloride (CCl(4)) and direct mitogen-induced hyperplasia, induced by lead nitrate (PbNO(3)), on FB(1)-induced cancer initiation. Initiation was effected over a period of 14 days by gavage administration of FB(1) at different daily doses ranging from 0.14 to 3.5 mg FB(1)/100 g body weight while the stimuli for cell proliferation were introduced 7 days after the start of the FB(1) treatment. Based on the proliferative stimulus used, cancer promotion was effected 3 weeks after completion of the initiating treatment by 2-acetylaminofluorene (2-AAF) treatment followed by PH or carbon tetrachloride CCl(4) on day 4. Cancer initiation by FB(1) was associated with a hepatotoxic effect and an increase in lipid peroxidation. In contrast to compensatory liver cell proliferation induced by PH and CCl(4), mitogen-induced hyperplasia (PbNO(3)) failed to enhance the cancer initiating potential of FB(1) suggesting that cancer induction by a non-genotoxic carcinogen is supported by regenerative cell proliferation. Cognizance of the enhancing role of cell proliferation during cancer initiation by FB(1) is required in assessing the risks posed by this mycotoxin to humans.