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.

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.

Activity of 2,4,6-trinitrotoluene in an in vitro mammalian gene mutation assay.

In the present study both 2,4,6-trinitrotoluene (TNT) and pure 2,4-dinitrotoluene (2,4-DNT) gave positive responses in the P388 mouse lymphoma gene mutation assay in the absence of auxiliary metabolic activation. Both chemicals gave negative results when an activation system was included. Technical grade DNT (consisting of an 80:20 mixture of 2,4- and 2,6-DNT) and pure 2,6-DNT gave negative responses in the assay both in the presence and absence of auxiliary metabolism. These observations in mammalian cells support the bacterial mutagenesis data indicating the TNT is a potential rodent liver carcinogen and suggest that the activity of TNT should be investigated in vivo to assess whether its potential hepatocarcinogenicity is realised in rats.

Effects of hydroxyurea consistent with the inhibition of repair synthesis in ultraviolet irradiated HeLa cells.

Many studies on DNA repair using established in vitro cell cultures employ conditions of low serum, nutrient deprivation, and blockade with hydroxyurea (HU) to reduce the background levels of DNA replication. There are some reports in the literature which indicate that HU inhibits DNA repair. In the present study the effects of HU on strand breaks and repair synthesis in UV irradiated Hela cells were investigated using a combined strand break and UDS assay. In conditions of low serum and arginine deprivation, HU produced effects consistent with the inhibition of the repair synthesis step in excision repair. Although the conditions used in this study are severe the results suggest that HU may have qualitatively similar effects in other studies which employ its use to detect repair synthesis.

Norharman and ellipticine: a comparison of their albilities to interact with DNA in vitro.

The anti-tumor agent ellipticine has been compared in vitro with the bacterial co-mutagen norharman, a compound which it resembles superfically in chemical structure. Ellipticine was shown to stabilize the structure of double stranded calf-thymus DNA, to induce mutations in strain TA153 of Salmonella tryhimurium and to cause BHK cells to transform. Further, the major absorbance in its visible spectrum underwent a red shift of approximately 40 nm in the presence of native DNA. It is concluded that ellipticine intercalates with dna, and from this, that its action as an anti-tumor agent may, as has been previously suggested, be dependent upon this property. In contrast, norharman, a chemical suspected initially of being an intercalating agent, failed to stabilize the structure of DNA, was non-mutagenic to the same strain of S. typhimurium and was inactive as cell-transforming agent. In addition, its visible spectrum was not affected by the presence of DNA. The last observation is contrary to the conclusion of other workers, and an explanation of this difference is given. It is concluded that norharman is not capable of intercalating with DNA, and consequently, its mode of action as a co-mutagen is probably dependent upon its ability to inhibit certain mixed-function oxidase enzymes present in the liver activation system employed with in vitro mutagenicity assays.

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.

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.

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.

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.

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.

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.

The mutagenic and cell transforming properties of shikimic acid and some of its bacterial and mammalian metabolites.

Known and proposed metabolites of shikimic acid were synthesised, characterised and tested for genotoxic activity using the Salmonella/mammalian microsome mutagenicity test, the bacterial fluctuation mutagenicity test and the BHK 21 cell transformation test. Under the conditions used, none of these compounds showed any activity. However, shikimic acid itself was active in the BHK 21 cell transformation assay. It therefore seems unlikely that shikimic acid is a carcinogenic initiating agent, but it may act as a carcinogen-promoting agent in the bracken fern (Pteridium aquilinum).

Cytogenetic effects of benzene: dosimetric studies on rats exposed to benzene vapour.

Rats were exposed to benzene vapour at nominal concentrations in air of 1, 10, 100 and 1000 ppm acutely for 6 h. Bone marrow cells from each animal were examined for chromosomal abnormalities 24 h after the end of the exposure period. This analysis was carried out on 250 metaphases per animal where possible and showed a significant increase in the percentage of cells with chromosomal abnormalities, excluding gaps, in the groups of animals exposed to 100 and 1000 ppm benzene. In the 10-ppm and 1-ppm exposure groups there were elevated levels of cells with abnormalities which showed evidence of being dose-related, although they were not statistically significant.

The mouse spot test. Evaluation of its performance in identifying chemical mutagens and carcinogens.

The published results on 60 chemicals and X-rays investigated in the mouse spot test were compared with data on the same chemicals tested in the bacterial mutation assay (Ames test) and lifetime rodent bioassays. The performance of the spot test as an in vivo complementary assay to the in vitro bacterial mutagenesis test reveals that of 60 agents, 38 were positive in both systems, 6 were positive only in the spot test, 10 were positive only in the bacterial test and 6 were negative in both assays. The spot test was also considered as a predictor of carcinogenesis; 45 chemicals were carcinogenic of which 35 were detected as positive by the spot test and 3 out of 6 non-carcinogens were correctly identified as negative. If the results are regarded in sequence, i.e. that a positive result in a bacterial mutagenicity test reveals potential that may or may not be realized in vivo, then 48 chemicals were mutagenic in the bacterial mutation assay of which 38 were active in the spot test and 31 were confirmed as carcinogens in bioassays. 12 chemicals were non-mutagenic to bacteria of which 6 gave positive responses in the spot test and 5 were confirmed as carcinogens. These results provide strong evidence that the mouse coat spot test is an effective complementary test to the bacterial mutagenesis assay for the detection of genotoxic chemicals and as a confirmatory test for the identification of carcinogens. The main deficiency at present is the paucity of data from the testing of non-carcinogens. With further development and improvement of the test it is probable that the predictive performance of the assay in identifying carcinogens should improve, since many of the false negative responses may be due to inadequate testing.

A comparison of the incidence of micronuclei in blood and bone marrow in 3 strains of mouse dosed with cyclophosphamide or hexamethylphosphoramide (HMPA).

The response of 3 strains of mouse (C57Bl/6J, C3H/C57 hybrid and BALBC/CBA) to cyclophosphamide (75 mg/kg) and hexamethylphosphoramide (HMPA) (1.28 ml/kg) were compared in the micronucleus test. Each compound was administered by intraperitoneal injection on two consecutive days and samples of bone marrow and blood taken for examination at 48 and 72 h after the first injection. Both test chemicals produced a statistically significant increase (P 0.001) in the incidence of micronuclei in bone marrow cells in all strains at both sampling times but the response with HMPA in C57Bl/6J mice appears to occur earlier than in the other two strains. Significant increases in micronuclei were seen in circulating erythrocytes only at 48 h in C57Bl/6J mice with both test chemicals and in C3H/C57 mice only with cyclophosphamide.

Evaluation of some formaldehyde-release compounds and other biocides in the mouse micronucleus test.

A number of biocidal chemicals were tested for clastogenic activity in the micronucleus test using C57Bl/6J mice. The materials tested were: 5-chloro-2-methyl-4-isothiazolin-3-one (I), N-methyl-isothiazolone hydrochloride (II), Glokill 77 and Parmetol A23. Two of the biocides (Glokill and Parmetol) depend on the release of formaldehyde for their activity while the other two compounds are the active chemicals in the biocide Kathon. Hexamethylphosphoramide (HMPA) was tested as the positive control for the series and N,N-dinitrosopentamethylenetetramine (DNPT) as the negative control. HMPA produced significant dose-related increases in the incidence of micronuclei whereas DNPT, I, II, Glokill and Parmetol A23 were without effect.

Activity of vinyl chloride monomer in the mouse micronucleus assay.

C57Bl/6J mice of both sexes were exposed to 50 000 ppm vinyl chloride monomer (VCM) for 6 h. Animals were killed 24 and 48 h after cessation of exposure and examined for the presence of micronuclei in bone marrow cells. At 24 h the control incidences of micronuclei per 1000 polychromatic erythrocytes (PCEs) were 2.6 (male) and 1.2 (female), while in animals exposed to VCM the incidences were 24.6 (male) and 25.0 (female). At 48 h the control incidences were 2.2 (male) and 1.6 (female) and in the VCM exposed animals 7.2 (male) and 4.4 (female).

Synthesis of N7-hydroxyethylguanine and O6-hydroxyethylguanine. Markers for the reaction of ethylene oxide (EO) with DNA.

O6-Hydroxyethylguanine has been synthesized by reaction of mono-sodium glycolate with 6-chloroguanine. The crystalline product has been characterized using a variety of analytical techniques and compared with a sample of the corresponding N7-hydroxyethyl derivative. These 2 chemicals may prove useful as standards when studying the reaction of ethylene oxide (EO) with DNA.

Comparisons between carcinogenic potency and mutagenic potency to Salmonella in a series of derivatives of 4-dimethylaminoazobenzene (DAB).

8 derivatives of the rodent liver carcinogen 4-dimethylaminoazobenzene (DAB), all of known carcinogenicity in rodents, have been evaluated in the 3 major variants of the Salmonella mutation assay; the standard plate test of Ames et al., the pre-incubation assay of Yahagi et al. and the fluctuation assay of Gatehouse. Although 4 of these chemicals were reported to be non-carcinogenic, and 4 to be of greater carcinogenic potency than DAB, each was mutagenic in a least 2 of the assays. Further, no quantitative correlation between carcinogenic and mutagenic potency was evident in any of the assay employed. The parent carcinogen DAB, 5-dimethylaminophenylazoindazole (a non-carcinogenic bacterial mutagen) and 6-dimethylaminophenylazobenzthiazole (a carcinogenic bacterial mutagen) were administered to rats via intraperitoneal injection, followed, 26 h later, by a sub-acute dose of [14C] dimethylnitrosamine. The histopathological condition of the livers of the treated animals was assessed together with a determination of the extent and nature of methylation by DMN of the DNA in the livers according to the method of O'Connor. Disturbances in both the pathological and DNA-related parameters were observed for the 2 carcinogens while control levels were seen for the non-carcinogen. Within this context the value of short-term assays conducted in vivo is discussed, especially their potential to identify potent mammalian carcinogens from among a collection of structurally related bacterial mutagens.