Delayed effects of tamoxifen in hepatocarcinogenesis-resistant Fischer 344 rats as compared with susceptible strains.

The anti-oestrogenic drug tamoxifen has been under investigation as a breast cancer chemopreventive agent for at least a decade. However, its use for this purpose is still debatable since it is able to induce liver tumours in rats via a mechanism involving metabolic activation to a DNA adduct-forming electrophilic intermediate. The metabolic activation and adduct-forming properties of tamoxifen are now well characterized but less is known about its ability to induce hepatic cell proliferation, which is also essential for the carcinogenic process. The effects of tamoxifen on liver weight and cell proliferation were compared in female Fischer 344 (F344), Wistar and Lewis rats given the drug in the diet for up to 26 weeks. The onset and duration of hepatic cell proliferation varied between the strains of rat. In Wistar and Lewis but not F344 rats there was a marked increase in hepatocellular proliferation during the first 4 weeks of tamoxifen administration. In the Wistar strain this was associated with an increase in DNA adduct levels; no such increase was observed in the F344 strain. The onset of the proliferative response was delayed until the 13 week time point in the F344 strain. By the 13 and 26 week time points, cell proliferation in tamoxifen-treated Wistar and Lewis rat liver had returned to normal, but the amount of apoptotic activity in these livers was elevated. This suggests that excess cells generated during the proliferative phase of tamoxifen treatment were being eliminated by apoptosis. In the F344 strain, however, increased proliferative activity was associated with relatively low apoptotic activity at the 26 week time point, suggesting that the delayed proliferative response had yet to be balanced by apoptotic deletion. This is consistent with the fact that tamoxifen-induced hepatocellular tumours develop very late, towards the end of the lifespan, in this strain. The cell proliferative activity of tamoxifen in the Wistar rat liver was compared with that of a non-mutagenic analogue, toremifene. Tamoxifen induced increased cell cycle activity in the livers of rats following gavage dosing at all sampling times (1-12 weeks), whereas toremifene had no effect on the incidence of cycling in hepatic cells, demonstrating that the hepatic cell proliferation is not a general response to anti-oestrogen treatment. These observations suggest that the rate of promotion of liver tumours by tamoxifen is a function of the rate, time of onset and duration of increased cell replication. The susceptibility of rat strains to the hepatocarcinogenic effects of tamoxifen appears to depend upon the balance between initiation via DNA adduct formation, promotion via increased cell proliferation and cell deletion via apoptosis. Our findings suggest that an early proliferative response to tamoxifen is important in this process.

Automation of mouse micronucleus genotoxicity assay by laser scanning cytometry.

BACKGROUND: The evaluation of the safety of drugs and other chemicals is an important aspect of toxicology work. The mouse micronucleus assay is a standard in vivo genotoxicity assay. Chromosomal damage is an indicator of genotoxicity, which manifests in the formation of micronuclei in polychromatic erythrocytes from bone marrow and in peripheral blood erythrocytes. The assay is laborious to perform by manual counting. The laser scanning cytometer allows automated and rapid quantitation of cellular and subcellular fluorescence in monodisperse cell samples on a microscope slide. The object of this study was to evaluate the application of this new technology in the mouse micronucleus genotoxicity assay. Materials and Methods One hundred forty-four mice of various strains were dosed with combinations of carcinogens and antioxidants. Duplicate blood films were prepared 3 days later. One set of slides was stained with acridine orange, and the proportion of micronucleated erythrocytes was counted in 5,000 cells per slide. The duplicates were stained with propidium iodide (40 microg/ml). Five thousand cells per sample were examined using a laser scanning cytometer. The proportion of micronucleated erythrocytes was measured. RESULTS: A coefficient of correlation of 0.96 was found between the data from the two assays. The automation of the assay on the LSC produced a considerable time saving and efficiency gain. CONCLUSIONS: We conclude that with further development, laser scanning cytometry is likely to become the preferred modality for the performance of standard genotoxicity assays.

Gene expression and amplification in breast carcinoma cells with intrinsic and acquired doxorubicin resistance.

The multidrug resistance (MDR) phenotype is a major cause of cancer treatment failure. Here the expressions of 4224 genes were analysed for association with intrinsic or acquired doxorubicin (DOX) resistance. A cluster of overexpressed genes related to DOX resistance was observed. Included in this cluster was ABCB1 the P-glycoprotein transporter protein gene and MMP1 (Matrix Metalloproteinase 1), indicative of the invasive nature of resistant cells, and the oxytocin receptor (OXTR), a potential new therapeutic target. Overexpression of genes associated with xenobiotic transformation, cell transformation, cell signalling and lymphocyte activation was also associated with DOX resistance as was estrogen receptor negativity. In all carcinoma cells, compared with HBL100 a putatively normal breast epithelial cell line, a cluster of overexpressed genes was identified which included several keratins, in particular keratins 8 and 18 which are regulated through the ras signalling pathway. Analysis of genomic amplifications and deletions revealed specific genetic alterations common to both intrinsic and acquired DOX resistance including ABCB1, PGY3 (ABCB4) and BAK. The findings shown here indicate new possibilities for the diagnosis of DOX resistance using gene expression, and potential novel therapeutic targets for pharmacological intervention.

Tamoxifen mutagenesis and carcinogenesis in livers of lambda/lacI transgenic rats: selective influence of phenobarbital promotion.

Administration of tamoxifen (TAM) (20 mg/kg per day p.o.) for 6 weeks to female lambda/lacI transgenic rats caused a 4-fold increase in mutation frequency (MF) at the lacI gene locus in the livers of dosed animals compared with controls. After cessation of dosing, the MF showed a further increase with time at 2, 12 and 24 weeks, respectively. Phenobarbital promotion of similarly treated animals resulted in no increase in mutation frequency compared with TAM alone. Treatment with phenobarbital or TAM+phenobarbital resulted in time-dependent increases in liver weight compared with the corresponding controls. There was an increase in cell proliferation in the phenobarbital and TAM+phenobarbital groups, and at 24 weeks in the TAM dosed animals compared with controls. There was also a progressive increase in the number of GST-P expressing foci in the livers of TAM and TAM + phenobarbital rats compared with controls. The induction of cell proliferation and GSTP foci in the rat liver by phenobarbital is consistent with its ability to promote tamoxifen-initiated liver tumours in the rat. If the lacI gene is regarded as being representative of the rat genome in general (albeit that the gene is bacterial) the above observations suggest that promotion by tamoxifen confers selective advantage on mutated genes at loci that contribute to the tumour phenotype and that promotion of rat liver tumours by tamoxifen is not dependent simply upon the enhancement of cellular proliferation.

Tamoxifen induces G:C-->T:A mutations in the cII gene in the liver of lambda/lacI transgenic rats but not at 5'-CpG-3' dinucleotide sequences as found in the lacI transgene.

Tamoxifen, a rat liver carcinogen, can induce mutations in the lacI gene in the livers of lambda/lacI transgenic rats. However, the presence of persistent tamoxifen adducts on the liver DNA raises the possibility that some contribution to the mutagenesis from ex vivo mutations during the in vitro lacI assay cannot be ruled out. To address this issue, mutagenesis at the cII gene of the transgenic shuttle vector was determined using a selection based assay which is unaffected by the presence of tamoxifen-DNA adducts. Female lambda/lacI transgenic rats were dosed orally with tamoxifen (20 mg/kg body wt) daily for 6 weeks, causing a 3.2-fold increase in the mutant frequency (MF) in the cII gene compared with that obtained with solvent treated animals. This was similar to the MF found previously at the lacI gene and confirms that tamoxifen is mutagenic in vivo. The major class of mutation induced by tamoxifen in the cII gene was G:C-->T:A transversions as was found previously in the lacI gene. However, in the one unreplicated study of mutations in the p53 gene of liver tumours induced by tamoxifen, no G:C-->T:A transversions were found; possible differences between mutagenesis in normal and tumour tissues are explored. The major proportion of the G:C-->T:A transversions occurred at 5'-CpG-3' dinucleotide (CpG) sites in the lacI gene, but not at such sites in the cII gene. The methylation of CpG sites greatly enhances the targeting of deoxyguanosine by carcinogens, thus this finding might be explained by differences in the methylation patterns at their respective CpG sites; however, nothing is known about the methylation status of either the lacI nor the cII gene in this transgenic rat. This study raises the important issue of which target genes (mammalian or transgenic) should be used as endpoints in mammalian mutagenesis assays.

Tamoxifen causes gene mutations in the livers of lambda/lacI transgenic rats.

Tamoxifen, a rat liver carcinogen, was administered to female lambda/lacI transgenic rats at a dose of 20 mg/kg body weight by gavage for 6 weeks, and the animals were sacrificed 2 weeks later. Tamoxifen induced liver DNA adducts and caused a significant increase in mutation frequency (MF) of approximately 3-fold at the lacI gene in liver DNA. Liver DNA from animals dosed with tamoxifen at 10 mg/kg also showed a similar increase in MF. The mutations were characterized by a raised proportion of: (a) G:C to T:A transversions; (b) insertions of base pairs; and (c) deletions of pairs of G:C base pairs. These observations indicate that tamoxifen induces a distinct spectrum of mutations compared with that found in controls. Toremifene, a noncarcinogenic analogue of tamoxifen with similar estrogenic/antiestrogenic properties examined at 20 mg/kg body weight using the same dosing regime as tamoxifen was not mutagenic. A single oral dose of the rat liver carcinogen aflatoxin B1 (0.5 mg/kg) also significantly raised the MF. In conclusion, although tamoxifen is not mutagenic in regulatory short-term tests, it is a gene mutagen in the rat liver.

Clastogenic and aneugenic effects of tamoxifen and some of its analogues in hepatocytes from dosed rats and in human lymphoblastoid cells transfected with human P450 cDNAs (MCL-5 cells).

Tamoxifen and its analogues 4-hydroxytamoxifen, toremifene, 4-hydroxytoremifene, clomifene and droloxifene were tested for clastogenic effects in a human lymphoblastoid cell line (MCL-5) expressing elevated native CYP1A1 and containing transfected CYP1A2, CYP2A6, CYP2E1 and CYP3A4 and epoxide hydrolase and in a cell line containing only the viral vector (Ho1). MCL-5 or Ho1 cells were incubated with 4-hydroxytamoxifen, 4-hydroxytoremifene, clomifene or droloxifene and the incidence of micronuclei estimated. With MCL-5 cells there was an increase in micronuclei with 4-hydroxytamoxifen, 4-hydroxytoremifene and clomifene but not with droloxifene. With Ho1 cells only 4-hydroxytamoxifen and 4-hydroxytoremifene caused an increase in micronuclei. MCL-5 cells were incubated with tamoxifen, 4-hydroxytamoxifen, toremifene, droloxifene, clomifene or diethylstilbestrol (0.25-10 microg/ml) for 48 h and subjected to 3 h treatment with vinblastine (0.25 microg/ml) to arrest cells in metaphase. The incidence of cells with chromosomal numerical aberrations (aneuploidy) was increased in cells treated with tamoxifen, 4-hydroxytamoxifen, toremifene, clomifene and diethylstilbestrol but not droloxifene. The frequency of cells with structural abnormalities (excluding gaps) was increased in cells treated with tamoxifen and toremifene but not 4-hydroxytamoxifen, clomifene, droloxifene or diethylstilbestrol. The clastogenic activities of tamoxifen (35 mg/kg), toremifene (36.3 mg/kg), droloxifene (35.2 mg/kg) and diethylstilbestrol (25 mg/kg) were compared in groups of four female Wistar rats. Each chemical was dissolved in glycerol formal, administered as a single dose by gavage and hepatocytes isolated by collagenase perfusion 24 h later. The cells were cultured in the presence of epidermal growth factor (40 ng/ml) for 48 h, colchicine (10 microg/ml) being added for the final 3 h of incubation. At least 100 chromosomal spreads were examined from each animal for the presence of numerical and structural abnormalities. The incidences of aneuploidy following treatment were: tamoxifen 81%, toremifene 46%, droloxifene 9.6%, diethylstilbestrol 45.7%, vehicle control 5.3%. The incidences of chromosomal structural abnormalities excluding gaps were: tamoxifen 4.3%, toremifene 0.8%, droloxifene 0.5%, diethylstilbestrol 0.8%, control 0.5%. The incidence of chromosomal structural aberrations excluding gaps in the treated animals was not statistically significantly different from controls except in the tamoxifen-treated group. Tamoxifen (35 mg/kg per os) and toremifene (36.3 mg/kg per os) were dosed to rats for 4 weeks and chromosomal spreads made from hepatocytes. The incidences of aneuploidy were: tamoxifen 94%, toremifene 57%, control 6.5%. The incidences of chromosomal aberrations excluding gaps were: tamoxifen 12%, toremifene 1%, control 0.5%. The incidence of tamoxifen-induced chromosomal structural abnormalities was significantly elevated compared with control levels. The results demonstrate that tamoxifen and toremifene are the only two drugs tested in the study that cause chromosomal structural and numerical aberrations in vitro and tamoxifen is the only drug that induces both these effects in rat liver cells stimulated to divide in culture following oral dosing. Since chromosomal mutations require cell division for their manifestation and tamoxifen is the only compound of those tested that causes hyperplasia in the rat liver, chromosomal aberrations and aneuploidy in the rat liver would only be expected to occur following treatment with tamoxifen alone, although aneuploidy could be induced by toremifene in conjunction with a promoter such as phenobarbitone.

Mutational spectra of tamoxifen-induced mutations in the livers of lacI transgenic rats.

Tamoxifen, an important drug in breast cancer treatment, causes liver cancer in rats. The standard range of in vitro tests have failed to show that it causes DNA damage, but 32P-postlabelling and DNA-binding studies have shown that tamoxifen forms DNA adducts in rat liver. In 1995 a transgenic rat (Big Blue; Stratagene, La Jolla, CA) became available which harbours the bacterial lacI gene, thereby allowing the in vivo study of tamoxifen mutagenesis. Recently, we [Styles JA et al. (1996): Toxicologist 30; 161] showed that tamoxifen caused on increase in the mutation frequency at the lacI gene in these transgenic rats. In this study, we report on our preliminary analysis of the mutational spectra of 33 control and 38 tamoxifen-induced mutant lacI genes. Plasmid DNA containing the lacI gene was isolated from the mutant phages and its DNA sequence determined. In the control animal group, 81% of the mutant lacI genes were point mutations, whilst in the tamoxifen-treated group, 62% of the mutant lacI genes were point mutations. Of the tamoxifen-induced mutants, 43% were GC-->TA transversions and 70% of point mutations. In the control group, GC-->TA transversions were 19% of all mutations and 24% of point mutations. Thus, compared with control animals, tamoxifen treatment had significantly increased the proportion of GC-->TA transversions.

Trichloroacetic acid: investigation into the mechanism of chromosomal damage in the in vitro human lymphocyte cytogenetic assay and the mouse bone marrow micronucleus test.

Trichloroacetic acid (TCA) was tested for its ability to induce chromosomal damage in cultured human peripheral blood lymphocytes and in bone marrow cells of male and female C57BL/6JfBL10/Alpk mice. Two in vitro cytogenetic assays were conducted with TCA. In the first TCA, as free acid, was added to whole blood cultures at final concentrations of 500, 2000 and 3500 micrograms/ml in the presence and absence of an auxiliary metabolic activation system (rat liver S9-mix). Statistically significant increases in the percentage of aberrant cells compared with solvent control values were observed in cultures treated with TCA at 2000 and 5000 mu/ml. Investigation into the effects of TCA on the pH of the culture medium revealed significant reductions in pH at both these TCA concentrations. Neutralized TCA was then tested at concentrations of 500, 2,000 and 5000 micrograms/ml, also in the presence and absence of S9-mix. No statistically or biologically significant increases in the percentage of aberrant cells were observed in any of these cultures. In the mouse micronucleus test, neutralized TCA was administered in two equal intraperitoneal doses 24 h apart to C57BL/6JfBL10/Alpk mice (337, 675 and 1080 mg/kg in males; 405, 810 and 1300mg/kg in females). These dose levels represent 25%, 50% and 80% of the median lethal dose (MLD) in this strain of mouse. Bone marrow samples were taken 6 and 24 h after the second dose and the chromosomal damage assessed by analysis of the bone marrow for micronuclei. No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes compared with the solvent control dosed animals were observed in either sex at the 6 h sampling time or in the females at the 24 h sampling time. A small but statistically significant increase in micronucleated polychromatic erythrocytes was observed in male mice 24 h after a dose of 675 mg/kg (50% MLD). Since no increases were noted at the 25 or 80% MLD, and the levels recorded are within the range of the concurrent solvent control values, the small increase observed in the males at the 50% MLD is considered not to be biologically significant. Flow cytometric studies on suspensions of isolated liver cell nuclei revealed that changes in FITC binding (indicating altered chromatin conformation) were induced by pH changes alone and were not caused by neutralized TCA.(ABSTRACT TRUNCATED AT 400 WORDS)

Genotoxicity of tamoxifen, tamoxifen epoxide and toremifene in human lymphoblastoid cells containing human cytochrome P450s.

The clastogenicity of tamoxifen and toremifene was tested in six human lymphoblastoid cell lines each expressing increased monooxygenase activity associated with a specific transfected human cytochrome P450 cDNA (CYP1A1, CYP1A2, CYP2D6, CYP2E1 or CYP3A4). The chemicals were also tested in a cell line (MCL-5) expressing elevated native CYP1A1 and containing transfected CYP1A2, CYP2A6, CYP2E1 and CYP3A4 and epoxide hydrolase, and in a cell line containing only the viral vector (Ho1). Dose-related increases in micronuclei were observed when cells expressing 2E1, 3A4, 2D6 or MCL-5 cells were exposed to tamoxifen. The positive responses in the cell lines were in the order MCL-5 > 2E1 > 3A4 > 2D6. Toremifene also gave positive results with 2E1, 3A4 and MCL-5 cells, although the responses were less marked and the positive effects required higher doses than with tamoxifen. A synthesized epoxide of tamoxifen was also tested in these cell lines and produced similar increases in the incidences of micronucleated cells. The increases in the responses observed with the epoxide were greater than with tamoxifen or toremifene. The P450 isoenzyme activities in these cells were in a range similar to those of human tumour-derived cell lines. Microsomes (1A1, 2A2, 2A6, 2B6, 2E1, 3A4 and 2D6) from these cells all metabolized tamoxifen. The major metabolite detected by HPLC was N-desmethyltamoxifen, and 4-hydroxytamoxifen was also detected in cells with cytochrome P450 2E1 and 2D6. These results are consistent with the following conclusions. (1) Tamoxifen requires metabolic activation to DNA-reactive species by specific CYP monooxygenases in order to exert its genotoxic effects. (2) The positive clastogenic effects elicited in lymphoblastoid cells by tamoxifen epoxide suggest that the genotoxic (and possibly the carcinogenic) effects of tamoxifen may be due to one or more epoxide metabolites that are generated intracellularly, probably in close proximity to the nucleus. (3) Tamoxifen is more genotoxic than toremifene.

Detection and quantification of ploidy, nuclearity, and DNA synthesis in rat hepatocytes after administration of a peroxisome proliferator.

The peroxisome proliferator methylclofenapate (MCP) induces species-specific liver growth and cancer in rats and mice. The acute hyperplastic effects of MCP were studied in rats given MCP (25 mg/kg by daily gavage) and injected IP (0.5 mL, 50 mM) with bromodeoxyuridine (BrdU) 6 hr before each sampling time. The animals were killed at 6-hr intervals and hepatocyte suspensions prepared from their livers by collagenase perfusion. The cells were stained with propidium iodide (PI) and BrdU antibody combined with fluorescein isothiocyanate (FITC). DNA content (PI-red fluorescence) and S phase (BrdU/FITC-green fluorescence) were analyzed simultaneously by two-parameter flow cytometry and the frequency of S phase in different ploidy classes determined. At the same time, S-phase cells from different ploidy groups were sorted onto slides by fluorescence-activated cell sorting and examined microscopically to determine the frequency of binucleated cells undergoing DNA synthesis. The results show that MCP-induced acute hyperplasia occurs mainly in a sensitive subpopulation of binucleated hepatocytes.

Measurement of ploidy and cell proliferation in the rodent liver.

In most investigations of cell proliferation in vivo, the population under study consists of mononuclear diploid cells that undergo replication via normal complete division cycles. Because the phenomena associated with the cell cycle are sequential, only one is normally measured and it is usually adequate to quantify the proliferative activity in one of two ways. The first involves labeling the cells undergoing semi-conservative DNA synthesis with a radioactive DNA precursor, preparing autoradiographs of histological sections, and counting labeled nuclei. The other commonly studied parameter of cell proliferation is mitotic activity. The livers of rats and mice, unlike those of other mammals, consist mainly of hepatocytes that contain two classes of cell with respect to nuclei and several ploidy classes. These classes of hepatocytes arise as the result of modified cell division cycles. The peculiar cytological composition of the rodent liver has, until recently, caused difficulties in the measurement and interpretation of cell ploidy and cell proliferation by the above methods. Flow cytometry and fluorescence-activated cell sorting used in conjunction with quantitative fluorescent stains for DNA and fluorescently labeled antibodies to bromodeoxyuridine have permitted the rapid and precise quantification of cell proliferative activity in the rodent liver. Studies using these techniques have revealed that proliferative activity of hepatocytes may occur in different subpopulations of cells depending on the kind of toxicological injury inflicted on the animal.

Recovery of hyperplastic responsiveness in rat liver after dosing with the peroxisome proliferator methylclofenapate.

Methylclofenapate (MCP) was administered daily by gavage (25 mg/kg) for 7 days to groups of adult male rats. Dosing was interrupted for 28, 35, 56, 70 or 84 days and then resumed (25 mg/kg by gavage at 0 and 24 h). During the second period of dosing animals were killed in groups of three at 6, 12, 18, 24, 30, 36, 42 and 48 h after the resumption of dosing. Hepatocytes in S-phase, labelled with bromodeoxy-uridine, were analysed by flow cytometry, cell sorting and microscopy. It was observed that total S-phase activity was just significantly elevated (approximately 20% of maximum) over corn oil controls after an interval of 28 days between initial and subsequent dosing periods. After an interval of 35 days total S-phase activity was approximately 65% of maximum, and full hyperplastic responsiveness, equal to that observed in naive animals given MCP, was detected after interruptions in dosing of 56, 70 and 84 days. The recovery of S-phase responsiveness during the interruptions in dosing was accompanied by an increase in the proportion of 2 X 2N hepatocytes from approximately 10% in animals dosed continuously with MCP, to approximately 11.4% after 28 days interruption, 17% after 35 days and control levels (approximately 20%) after 56, 70 and 84 days. Irrespective of the magnitude of the hyperplasia elicited by the second period of dosing with MCP, the proportion of 2 X 2N cells was reduced to the same levels as those observed in animals dosed continuously with MCP (approximately 10%). Very low S-phase activity (0.05%) was observed in animals dosed continuously with MCP, this level of activity being similar to that in animals given corn oil continuously.

Trichloroacetic acid: studies on uptake and effects on hepatic DNA and liver growth in mouse.

Trichloroacetic acid (TCA) was tested in mice for its ability to cause single-strand breaks (SSBs) in hepatic DNA in the presence and absence of liver growth induction. Male B6C3F1 mice were given 1, 2 or 3 daily doses of TCA (500 mg/kg p.o.) as a neutralized solution (sodium salt) and killed 1 h after the final dose. Some mice were given a single dose of TCA (500 mg/kg p.o.) as the acid or as a neutralized solution and killed 24 h after. Liver nuclei were prepared and the induction of DNA SSBs assayed. TCA gave no significant response. Absorption and distribution studies were conducted with radiolabelled trichloro[2-14C]acetic acid, which was administered by gavage (500 mg/kg) as aqueous free acid, neutral aqueous solution (sodium salt) or free acid in corn oil. The absorption and distribution of TCA was similar in all cases: the chemical was absorbed rapidly after dosing, maximum plasma and liver concentrations of free radiolabel being achieved in less than 1 h. Within the first 4 h following dosing there was no evidence of binding to DNA or other macromolecules in plasma and very little 'covalent' binding was detected in liver, indicating that at times when maximum DNA single-strand breakage has been reported there was no significant binding to liver cells. Studies on liver growth parameters (hyperplasia and peroxisome proliferation) with TCA revealed that the chemical induced small but significant increases in both parameters. No SSB induction was detected in association with either liver growth phenomenon elicited by TCA. We have thus found no evidence that TCA causes SSBs in the hepatic DNA of treated mice, in contrast to previous observations by other investigators.

Analysis of cytological changes in hepatocytes from rats dosed with 3'-methyl-4-dimethylaminoazobenzene: initial response appears to involve cytokinesis of binucleated cells.

The reduction in the ratio of tetraploid (4N + 2 X 2N) to diploid (2N) hepatocytes in the adult rat after treatment with the hepatocarcinogen 3'-methyl-4-dimethylaminoazobenzene (3'M) has been investigated. Analysis of isolated hepatocytes 18-28 days after treatment has confirmed that initially some of the 2 X 2N hepatocytes are converted into 2N cells by cytokinesis, and that there is no DNA synthesis during this process. Shortly afterwards nonpolyploidizing growth commences by proliferation of some 2N cells.

Mitosis and histopathology in rat liver during methylclofenapate-induced hyperplasia.

Liver hyperplasia was induced in rats by daily administration of methylclofenapate (25 mg/kg by gavage). An increase in the incidence of colchicine-arrested metaphases was observed with peaks occurring at 40 h (1.3%), 64 h (6.4%) and 84 h (6.8%) after the start of treatment. This response contrasted with the much larger (21.3%) peak in arrested metaphases at 36 h after partial hepatectomy, but was still unexpectedly large in comparison with the S-phase response to methylclofenapate reported in a previous study. Progressive hypertrophic histopathological changes were apparent during the whole course of treatment.

Studies on the hyperplastic responsiveness of binucleated rat hepatocytes.

Rats were dosed by gavage for 28 days with 25 mg/kg of 3'methyl-4-dimethylaminoazobenzene (3'M) followed by 4 days dosing with 0.5 ml/100 g corn oil. Livers from animals were taken at intervals during the 4 days of corn oil dosing and examined for S-phase activity and nuclearity. The treatment regime caused an increase in the proportion of diploid (2N) cells in the hepatocyte population, a decrease in the proportion of binucleated (2 x 2N) cells of approximately 50%, and induced an increase in cell replication. A second group of rats was dosed for 28 days with 0.5 ml/100 g corn oil followed by 4 days administration of 25 mg/kg methylclofenapate (MCP). Analysis of hepatocytes taken during the MCP treatment revealed that there was acute hyperplasia, involving mainly the 2 x 2N hepatocytes, resulting in a reduction of approximately 50% of the 2 x 2N cells and an accompanying increase in the proportion of 4N cells. When a third group was given 3'M for 28 days followed by 4 days administration of MCP, there was, at the outset of MCP dosing, a hepatocyte population typical of 3'M dosed animals, with an elevated proportion of 2N cells and a 2 x 2N fraction that was reduced to approximately 50% of control levels. During the 4 days of MCP dosing there was a wave of hyperplasia involving S-phase activity in the remaining 2 x 2N hepatocytes. The proportion of 2 x 2N cells decreased further during this period and there was a concomitant increase in 4N cells. The 2N fraction, already elevated, was not further affected. These results indicate that both genotoxic and non-genotoxic hepatocarcinogens induce acute changes in the rat hepatocyte population that involve 2 x 2N cells, and that the effects appear to involve separate sub-populations of 2 x 2N hepatocytes.

Effects produced by the non-genotoxic hepatocarcinogen methylclofenapate in dwarf mice: peroxisome induction uncoupled from DNA synthesis and nuclearity changes.

Both Snell dwarf mice (dw/dw) and their phenotypically normal heterozygotes (dw/+) were dosed with methylclofenapate (MCP) at daily intervals by gavage (25 mg/kg). Animals were killed at 12, 24, 36 and 72 h after the initial dose and the parameters of ploidy, nuclearity and DNA synthesis were measured in hepatocytes isolated by collagenase perfusion. The occurrence of peroxisome proliferation was assessed by electron microscopy after daily administration of 25 mg/kg MCP by gavage for 28 days. The hepatocytes from both phenotypes exhibited similar degrees of peroxisome proliferation but hyperplasia occurred only in the heterozygous animals. The incidence of binulceated hepatocytes in heterozygotes was approximately 50%, and at the end of acute hyperplasia this had reduced to approximately 20%; by contrast the livers of dwarf animals contained approximately 20% binucleated cells and this remained unchanged throughout the period of dosing. The hyperplasia in the wild-type mice, as measured by the occurrence of S-phase, occurred predominantly in binucleated hepatocytes. These observations are further confirmation that acute hyperplasia induced by MCP and similar liver growth inducers occurs predominantly in a sensitive sub-population of binucleated hepatocytes. The results also indicate that peroxisome proliferation and hyperplasia can occur as independent phenomena.

A species comparison of acute hyperplasia induced by the peroxisome proliferator methylclofenapate: involvement of the binucleated hepatocyte.

The acute hyperplastic response induced by methylclofenapate (MCP) was studied in several rodent species with different responsiveness to the hypertrophic and hyperplastic effects caused by this chemical. The species, in descending order of responsiveness, were: mouse, rat, hamster and guinea-pig, the latter species being non-responsive. Animals were dosed at daily intervals with MCP (25, 12 or 5 mg/kg by gavage) and killed at intervals from 12 h to 240 h. The parameters of ploidy, nuclearity and DNA synthesis were examined in isolated hepatocytes. The hyperplastic response elicited by MCP in rodent livers as detected by the occurrence of S-phase cells, was almost exclusively confined to the 2 x 2N (binucleated) hepatocyte population. At the same time the proportion of 2 x 2N cells was reduced in a time and dose-dependent manner, while the fraction of 4N cells increased. These observations indicate that the 2 x 2N cells responding to MCP undergo S-phase followed by amitotic cytokinesis to form 4N cells. The response in rats, mice and hamsters was quantitatively different but qualitatively similar, while the guinea-pig was non-responsive. In a given responsive species the areas under the curves are similar for different doses, indicating that the size of the responsive population is limited. The data also indicate that although the response in the mouse was greater than in the rat, because the total number of responsive 2 x 2N cells is larger, the percentage of responsive 2 x 2N cells is higher in the rat than in the mouse. The ploidy analysis reveals that there is no detectable change in the ratio of (4N + 2 x 2N):2N hepatocytes, but only 1-2% change would be expected, despite the number of 4N cells produced, due to the increase in total cell number.