Recommendations for the categorization of germ cell mutagens.

Germ cell mutagens are currently classified into three categories in the German List of MAK and BAT Values. These categories have been revised and extended by analogy with the new categories for carcinogenic chemicals. Germ cell mutagens produce heritable gene mutations, and heritable structural and numerical chromosome aberrations in germ cells. The original categories 1 and 2 for germ cell mutagens remain unchanged. Two new categories 3A and 3B are proposed for chemicals suspected to be germ cell mutagens. A new category 5 is proposed for germ cell mutagens with low potency that contribute negligibly to human genetic risk provided the MAK value is observed.

Sulfotransferases: genetics and role in toxicology.

The mammalian xenobiotic-metabolizing sulfotransferases are cytosolic enzymes, which form a gene superfamily (SULT). Ten distinct human SULT forms are known. Two SULT forms represent splice variants, the other forms are encoded by separate genes. Common functional polymorphisms of the transcribed region are known for two of the forms. We have expressed 16 separate rat and human SULTs as well as some of their allelic variants, in Salmonella typhimurium TA1538 and/or V79 cells, which are target cells of commonly used mutagenicity assays. The expressed SULTs activated numerous compounds to mutagens in both assay systems. However, some promutagens were activated by only one or several of the human SULTs. Pronounced differences in promutagen activation were also detected between orthologous rat and human SULTs, and between allelic variants of human SULTs.

Human phenol sulfotransferases hP-PST and hM-PST activate propane 2-nitronate to a genotoxicant.

The industrial solvent 2-nitropropane (2-NP) is a genotoxic hepatocarcinogen in rats. The genotoxicity of the compound in rats has been attributed to sulfotransferase-mediated formation of DNA-reactive nitrenium ions from the anionic form of 2-NP, propane 2-nitronate (P2N). Whether human sulfotransferases are capable of activating P2N is unknown. In the present study we have addressed this question by investigating the genotoxicity of P2N in various V79-derived cell lines engineered for expression of individual forms of human sulfotransferases, the phenol-sulfating and the monoamine-sulfating phenol sulfotransferases (hP-PST and hM-PST) and the human hydroxysteroid sulfotransferase (hHST). Genotoxicity was assessed by measuring the induction of DNA repair synthesis and by analyzing the formation of DNA modifications. P2N induced repair synthesis in V79-hP-PST and V79-hM-PST cells, whereas induction of repair synthesis in V79-hHST cells was negligible. P2N also resulted in the formation of 8-aminodeoxyguanosine and increased the level of 8-oxodeoxyguanosine in V79-hP-PST cells, but not in the parental V79-MZ cells, which do not show any sulfotransferase activity. Acetone oxime, the tautomeric form of the first reduction product of 2-NP, 2-nitrosopropane, was inactive in all cell lines. The results show that the human phenol sulfotransferases P-PST and M-PST are capable of metabolically activating P2N (P-PST >> M-PST) and that the underlying mechanism is apparently identical to that resulting in the activation of P2N in rat liver, where 2-NP causes carcinomas. These results support the notion that 2-NP should be regarded as a potential human carcinogen.

Activation of propane 2-nitronate to a genotoxicant in V79-derived cell lines engineered for the expression of rat hepatic sulfotransferases.

2-Nitropropane (2-NP) is a genotoxic hepatocarcinogen in rats. The genotoxicity of the compound has been attributed to a sulfotransferase-mediated formation of DNA-reactive species from the anionic form of 2-NP, propane 2-nitronate (P2N). Several observations have suggested that sulfotransferases (SULTs) 1A1 and/or 1C1 may be important in the activation of P2N to a genotoxicant in rat liver, but a definite proof is lacking. In order to identify the sulfotransferase(s) of rat liver that are capable of activating P2N, we have investigated the genotoxicity of P2N in various V79-derived cell lines engineered for expression of individual forms of rat hepatic sulfotransferases. Genotoxicity was assessed by measuring the induction of DNA repair synthesis. 1-Hydroxymethylpyrene (HMP), which is metabolically activated by most sulfotransferases, served as a positive control. Neither P2N nor HMP induced DNA repair in the parental V79-MZ cells, which do not show any sulfotransferase activity. P2N was also inactive in V79-rHSTa and V79-rHST20 cells, which express specific hydroxysteroid sulfotransferases. By contrast, a clear and concentration-dependent induction of repair synthesis by P2N was observed in V79-rPST-IV and V79-rST1C1 cells, which express rat SULT1A1 and SULT1C1, respectively. HMP was genotoxic in all sulfotransferase-expressing cell lines. Acetone oxime (AO), the tautomeric form of the first reduction product of 2-NP, 2-nitrosopropane, was inactive in all cell lines. The results corroborate the essential role of sulfotransferases in the metabolic activation of P2N to genotoxic products and identify two rat sulfotransferases which are capable of catalyzing the activation step.

S-oxygenation of thiourea results in the formation of genotoxic products.

Thiourea (TU) is a thyroid carcinogen which has previously been shown to cause genotoxicity in various test systems in vitro and in vivo. The mechanism underlying these effects has not yet been elucidated. The present study addressed the question of whether the formation of oxidized products of TU might be involved in genotoxicity. Chemical oxidation of [14C]TU with hydrogen peroxide in the presence of calf thymus DNA resulted in the formation of [14C]formamidine sulfinate ([14C]FASA), [14C]cyanomide, and [14C]urea and in covalent binding of radioactivity to the DNA. Incubation of V79 Chinese hamster cells with 10-20 mM TU for 18 hr but not for 3 hr, increased the frequency of micronuclei to a slight extent. In cells depleted of glutathione, which can prevent the oxidation of TU, micronucleus induction by TU was more pronounced and detectable both after 3 and 18 hr of incubation. Exposure of the cells to 1.25 to 10 mM FASA for 3-5 hr induced micronuclei, DNA repair synthesis, and gene mutations in the cells. Flavin-containing monooxygenase (FMO], an enzyme known to catalyze the S-oxygenation of TU in liver, could not be detected in the postmitochondrial supernatant (S-9) of the V79 cells. There is evidence, however, that TU can easily autoxidize to S-oxygenated products. Both FASA and TU caused a slight induction of DNA repair synthesis in cultured rat hepatocytes, but FASA was active at lower concentrations than TU. Cyanamide did not elicit repair. The finding that FASA, a product of both the nonenzymatic and the enzymatic S-oxygenation of TU, is genotoxic in cultured mammalian cells provides for the first time a hypothesis to explain the genotoxicity of TU.

Sulfotransferase-mediated genotoxicity of propane 2-nitronate in cultured ovine seminal vesicle cells.

2-Nitropropane (2-NP) is a well-known genotoxin and carcinogen in rat liver. Several metabolic pathways, particularly cytochrome P450-, peroxidase- and sulfotransferase-dependent ones, have been suggested to lead to the formation of DNA-reactive species from 2-NP. Because rat liver cells express most types of xenobiotic-metabolizing enzymes, the role of specific pathways in the metabolic activation of 2-NP is difficult to assess in these cells. We have therefore investigated the genotoxicity of 2-NP and its anionic form, propane 2-nitronate (P2N), in cultured ovine seminal vesicle (OSV) cells. OSV cells lack cytochrome P450-dependent monooxygenase activity, but express prostaglandin-H-synthase (PHS) and, as we found out, phenol sulfotransferase. The induction of DNA repair synthesis and specific DNA modifications served as indicators for the genotoxicity of 2-NP and P2N. Both forms strongly induced repair, P2N being more active than 2-NP. The secondary nitroalkanes nitrocyclopentane and nitrocyclohexane also induced repair, whereas 1-nitropropane and the reduction product of 2-NP, acetone oxime, did not. P2N also elicited the formation of the characteristic DNA modifications 'DX1' and 8-aminodeoxyguanosine and increased the level of 8-oxodeoxyguanosine residues in the DNA. Pretreatment of OSV cells with indomethacin, an inhibitor of PHS, affected neither the induction of repair nor the formation of the DNA modifications, and P2N was not a reducing substrate for the PHS-peroxidase activity. In contrast, the sulfotransferase inhibitor pentachlorophenol strongly reduced genotoxicity. The results show that cytochrome P450-dependent monooxygenases are not required for the metabolic conversion of secondary nitroalkanes or their nitronates into DNA-damaging products, nor is PHS involved in the metabolic activation. Instead, the data corroborate an essential role of sulfotransferase(s) in the genotoxicity and carcinogenicity of secondary nitroalkanes. Moreover, it is demonstrated for the first time that these compounds can be genotoxic in cells other than hepatocytes or hepatoma cells. This implies that in species other than the rat, organs other than the liver can be targets for the genotoxicity, and possibly carcinogenicity, of secondary nitroalkanes.

DNA-damaging activity of the cyproterone acetate analogues chlormadinone acetate and megestrol acetate in rat liver.

The synthetic progestin cyproterone acetate (CPA) has been recently shown to elicit DNA repair synthesis in cultured rat hepatocytes and to form adducts with rat hepatocyte DNA in vitro and in vivo. In the present study we have examined the genotoxic potential of the structural analogues of CPA, chlormadinone acetate (CMA) and megestrol acetate (MGA) in rat liver cells. CPA strongly induced DNA repair synthesis in hepatocyte cultures from females but not from males. In contrast, CMA and MGA (2-50 microM) did not detectably increase repair synthesis in cultured hepatocytes from either gender. CMA and MGA, however, caused the formation of DNA adducts detectable by the 32P-postlabelling technique. At a concentration of 30 microM, between 30 and 50 adducts/10(9) nucleotides were found with MGA and CMA in cultured hepatocytes of female rats, and between 5 and 20 adducts/10(9) nucleotides were found in hepatocytes of males. By comparison, 30 microM CPA has been found to produce 1670 adducts/10(9) nucleotides in hepatocytes from female rats. CMA and MGA also induced low levels of DNA adducts in vivo. When female rats were treated with 100 mg/kg of CMA or MGA per os, the adduct levels were 2 and 19 adducts/10(9) nucleotides respectively. The results indicate that both CMA and MGA show some genotoxicity in rat liver cells, which is, however, much lower than that for CPA. Our findings further suggest that the high genotoxicity of CPA is associated with the presence of the 1,2 alpha-methylene group, which is absent in CMA and MGA.

DNA repair synthesis and DNA fragmentation in primary cultures of human and rat hepatocytes exposed to cyproterone acetate.

Cyproterone acetate (CPA), a synthetic steroid used in human therapy, has recently been shown to cause DNA damage in cultured rat hepatocytes and in rat liver. In the present study we have investigated whether CPA also induces genotoxic effects in human hepatocytes. Genotoxicity of CPA was determined by measuring the capability of the compound for inducing DNA repair synthesis and for causing the formation of DNA single-strand breaks. Autoradiography and alkaline elution were used to quantitate DNA repair and DNA fragmentation, respectively. Exposure of hepatocytes to CPA for 20 h induced DNA repair synthesis in two hepatocyte preparations derived from males and in four of the five preparations derived from females. In cultures from some donors, induction of repair was detected at 1 microM CPA, the lowest concentration tested. The maximum effect generally occurred at 10-20 microM. Only a very slight increase in the frequency of DNA single-strand breaks was found following exposure of the hepatocytes to 50 microM CPA for 20 h. For comparative purposes, the effects of CPA on DNA repair and DNA fragmentation were also determined in cultured rat hepatocytes. A strong induction of DNA repair synthesis, but only a slight enhancement in DNA fragmentation was observed in CPA-treated hepatocytes derived from female rats. These results indicate that the measurement of repair is a more sensitive indicator for the genotoxicity of CPA than the measurement of DNA fragmentation. No genotoxic effects of CPA were detectable in hepatocyte cultures derived from male rats. The present findings show that CPA is genotoxic in human hepatocytes and that the striking sex difference in the genotoxicity of CPA in rat cells is not observed with human cells.

DNA repair synthesis induced by azo dyes in primary rat hepatocyte cultures using the bromodeoxyuridine density-shift method.

The genotoxic activity of the benzeneamine-derived azo dyes, Disperse Red 54 (DR 54), Direct Red 81 (DR 81) and Direct Black 19:1 (DB 19:1) was studied in the in vitro DNA repair assay in primary rat hepatocyte cultures. Hepatocytes were isolated, cultured and treated with the azo dyes, bromodeoxyuridine and [3H] cytidine. DNA repair synthesis was determined as an incorporation of [3H] cytidine into unreplicated DNA strands using the bromodeoxyuridine density-shift method. Of the 3 azo dyes, only DR54 (monoazo dye) induced a weak DNA repair synthesis in rat hepatocytes in vitro. DR81 and DB 19:1 did not induce any concentration-related DNA repair synthesis expressed as cpm/micrograms DNA. The data suggest that the in vivo reduction of azo dyes is required for the genotoxicity of these azo dyes.

Recommendations for the performance of UDS tests in vitro and in vivo.

The Working Group (WG) dealt with the harmonization of routine methodologies of tests for unscheduled DNA synthesis (UDS) both in vitro and in vivo. In contrast to the existing guidelines from OECD, EPA and EC on in vitro UDS tests (there is no Japanese UDS guideline), the Working Group recommends that in general in vitro UDS tests should be performed with primary hepatocytes. For routine applications any other cell types would need special justification. Hepatocytes from male rats are preferable, unless there are contra-indications on the basis of e.g. toxicokinetic data. According to the OECD, EPA and EC guidelines, UDS may be analysed by means of autoradiography (AR) or liquid scintillation counting (LSC). The WG recommends use of AR. LSC is less suitable due to the problem of differentiation between UDS activity and replicative DNA synthesis, and the disadvantage that cells cannot be analysed individually. Since a specific cell type was recommended by the WG, methodological aspects could be described in more detail than in the present guidelines. For in vitro tests, it was agreed that the initial viability of freshly isolated hepatocytes should be at least 70%. With regard to the need for confirmatory experiments in the event of a clear-cut negative result, the majority view was that confirmation by a second (normally not identical) experiment is still needed; this is in line with the present OECD and EC guidelines. Evaluation of results from UDS tests should be based primarily on net nuclear grain (NNG) values, although it is recognised that nuclear and cytoplasmic grains result from different biological processes. Since grain counts are influenced by a number of methodological parameters, no global threshold NNG value can be recommended for discrimination of positive and negative UDS results. For in vitro assays, the criteria for positive findings go beyond those of the present guidelines and two alternative approaches are given which are based on (1) dose-dependent increases in NNG values and (2) reproducibility, dose-effect relationship and cytotoxicity. At present there is no official guideline on the performance of in vivo UDS tests. Some fundamental recommendations given for in vitro methodology also apply to the in vivo assay. For routine testing with the in vivo UDS test, again the general use of hepatocytes from male rats is recommended. However, concerning the requirement to use one or two sexes, consistency with other in vivo genotoxicity assays (e.g. the micronucleus assay) would be preferable. As for the in vitro methodology, AR is preferred rather than LSC.(ABSTRACT TRUNCATED AT 400 WORDS)

The insecticide buprofezin induces morphological transformation and kinetochore-positive micronuclei in cultured Syrian hamster embryo cells in the absence of detectable DNA damage.

The insecticide buprofezin was examined for its genotoxicity in cultured Syrian hamster embryo cells in order to better understand the mechanisms underlying the genotoxicity of the compound in mammalian cells. Exposure to buprofezin concentrations of 12.5-100 microM did not significantly affect the colony-forming ability of the cells, but did result in increased frequencies of morphologically transformed colonies. Treatment with buprofezin did not cause a detectable induction of DNA repair synthesis, an indicator of DNA damage, but significantly increased the frequency of micronuclei. Immunostaining of the cells with antikinetochore antibody (CREST antibody) showed that essentially all of the buprofezin-induced micronuclei were kinetochore-positive. The results suggest that morphological transformation of Syrian hamster embryo cells by buprofezin results from an interaction of the compound or a metabolite of it with the mitotic apparatus rather than from DNA damage.

Radical intermediates during the oxidation of nitropropanes. The formation of NO2 from 2-nitropropane, its reactivity with nucleosides, and implications for the genotoxicity of 2-nitropropane.

The chemistry of the nonenzymatic oxidation of the rat liver carcinogen, 2-nitropropane, and its anionic form, propane-2-nitronate, was investigated using pulse radiolysis and EPR/spin trapping with 3,5-dibromo-4-nitrosobenzenesulfonic acid as the trapping agent. The results suggest that, following initial oxidation to a secondary alkyl radical, propane-2-nitronate is effectively degraded in a peroxidative chain reaction with the intermediary formation of peroxyl and NO2.radicals. The latter radical was shown to react appreciably fast with ribonucleosides, deoxyribonucleosides, and guanosine nucleotides. It is proposed that nonenzymatic formation of NO2.radicals after enzymatic oxidation of propane-2-nitronate to the corresponding secondary alkyl radical accounts for the induction of DNA damage observed after exposure of rats to 2-nitropropane.

Cyproterone acetate generates DNA adducts in rat liver and in primary rat hepatocyte cultures.

Cyproterone acetate (CPA), an active component of certain contraceptive and antiandrogenic drugs, has been shown recently to induce DNA repair synthesis in rat hepatocytes in vitro. In the present study we examined whether CPA can cause the formation of DNA adducts detectable by the 32P-postlabeling technique in hepatic cells in vitro and in vivo. Incubation of primary cultures of hepatocytes from male Wistar rats with CPA resulted in the occurrence of radioactive spots in the radiochromatograms of 32P-postlabeled DNA digests indicating the formation of two DNA adducts ('A' and 'B'). At 30 microM CPA, the highest concentration tested, approximately 50 'A' adducts and five 'B' adducts were found per 10(9) nucleotides. DNA of hepatocyte cultures from female rats was found to contain adduct A and a minor adduct termed 'D', but adduct B was not observed. Between 1 and 10 microM CPA, the relative level of adduct A was approximately 20-fold higher than the level observed in male hepatocytes. In vivo DNA adducts were detected almost exclusively in hepatic DNA. Hepatic DNA from male Wistar rats treated with single doses of CPA (1-100 mg/kg) by gavage, showed the major adducts A and B and two further spots of minor intensity ('C' and 'D') in the radiochromatograms. No adducts were detectable in extrahepatic tissues. The adduct pattern of liver DNA from females exposed to single oral doses between 0.1 and 30 mg CPA/kg body wt was similar to that observed in males; however, the relative levels for adducts A and D were approximately 100-fold higher. In females, linear relationships between dose and adduct levels were observed for all four adducts. The present findings show that CPA causes damage to hepatic DNA not only in vitro, but also in vivo. Thus it appears possible that DNA adduct formation is involved in the formation of hepatic tumors during long-term treatment of rats with the synthetic steroid.

Propane 2-nitronate is more rapidly denitrified and is more genotoxic than 2-nitropropane in cultured rat hepatoma cells.

We have investigated the importance of nitronate formation from 2-nitropropane (2-NP) for the oxidative metabolism and the genotoxicity of 2-NP in 2sFou rat hepatoma cells. Treatment of the cells with 2-NP for up to 3 h resulted in the time-dependent appearance of nitrite in the culture medium and in a weak induction of DNA repair synthesis. Both nitrite formation and repair induction were markedly enhanced in cells exposed to the anionic form of 2-NP, propane 2-nitronate. These observations suggest that propane 2-nitronate, rather than 2-NP itself, is oxidized by the liver cells to yield a DNA-damaging product. The results also indicate that the nitro/nitronate equilibration in intact liver cells is slow, suggesting that nitronate formation represents the rate-limiting step in the metabolic activation of 2-NP.

Cyproterone acetate induces DNA damage in cultured rat hepatocytes and preferentially stimulates DNA synthesis in gamma-glutamyltranspeptidase-positive cells.

The synthetic anti-androgen and progestin cyproterone acetate (CPA) is known to increase the liver tumor rate in rats. The tumorigenicity of CPA has been attributed to a tumor-promoting activity of the steroid. In order to discover whether CPA acts directly on preneoplastic liver cells or via indirect effects, we investigated whether CPA stimulates replicative DNA synthesis in vitro in hepatocytes isolated from carcinogen-treated rats (two-thirds hepatectomy, 1 x 30 mg diethylnitrosamine per kg and 0.1% phenobarbital in the drinking water) and whether the degree of stimulation differs in gamma-glutamyltranspeptidase (GGT)-positive, putatively preneoplastic and GGT-negative, 'normal' hepatocytes. The possibility that CPA might also have initiating potential was investigated by studying its effects on DNA repair synthesis. Stimulation of [3H]thymidine incorporation into the DNA by CPA was only observed in the presence of epidermal growth factor (EGF) (10 ng/ml) and insulin (10 mU/ml). Maximal effects were obtained between 2 and 10 microM. DNA synthesis in the presence of EGF/insulin was reduced by the 'pure' anti-androgen flutamide, but stimulated by the 'pure' progestin promegestone. In the presence of CPA, EGF and insulin, the labelling index was twice as high in GGT-positive as in GGT-negative liver cells, regardless of whether mitogens were added at 48 or 72 h. The labelling index did not differ in the GGT-positive and negative hepatocytes when CPA was omitted. These findings are consistent with the idea that CPA has tumor-promoting activity. CPA significantly induced repair synthesis in the isolated hepatocytes from both untreated and carcinogen-treated rats. This increase was detected at a concentration as low as 2 microM and maximal effects were obtained at 20 microM. These results indicate that CPA is not only a tumor-promoting, but also a genotoxic chemical, i.e. that it might also have an initiating potential.

Nitroreduction is not involved in the genotoxicity of 2-nitropropane in cultured mammalian cells.

We have investigated the importance of nitroreduction for the genotoxicity of the carcinogen 2-nitropropane (2-NP) in primary cultures of rat hepatocytes and in V79 Chinese hamster cells. Induction of DNA repair synthesis was used as an indicator of genotoxic effects in hepatocytes. Genotoxicity in V79 cells was determined as induction of DNA repair, micronuclei and mutations to 6-thioguanine (TG) resistance. Both hepatocytes and V79 cells were found capable of reducing and oxidizing 2-NP. Reduction of 2-NP was indicated by the formation of acetone oxime, the tautomeric form of 2-nitrosopropane, the first reduction product of 2-NP. Oxidation of 2-NP was indicated by the production of acetone and nitrite. 2-NP strongly elicited repair in hepatocytes, but acetone oxime and the products of a possible further nitroreduction, isopropyl hydroxylamine (IPHA) and 2-aminopropane did not. None of the reduction products caused repair synthesis in V79 cells. However, in these cells IPHA and 2-NP increased the frequency of TG-resistant mutants. IPHA also markedly induced micronuclei. This was not seen with 2-NP. Acetone oxime was not genotoxic in V79 cells. The observations suggest that reduced metabolites are responsible neither for the induction of DNA repair synthesis by 2-NP in hepatocytes nor for the induction of gene mutations by 2-NP in V79 cells.

Involvement of different pathways in the genotoxicity of nitropropanes in cultured mammalian cells.

The metabolic pathways leading to genotoxicity of nitropropanes in mammalian cells were investigated by measuring the effects of 2-nitropropane (2-NP) and 1-nitropropane (1-NP) on various cell lines characterized for their expression of cytochrome P450-dependent mono-oxygenases. Cells used were the rat hepatoma cell lines 2sFou, H4IIEC3/G- and C2Rev7, which express various forms of cytochrome P450-dependent mono-oxygenases, and V79 Chinese hamster cells which lack these enzyme activities. Induction of DNA repair synthesis, micronuclei and, where assessable, mutations to 6-thioguanine (TG) resistance served as indicators of genotoxic effects. 2-NP elicited a positive response at all endpoints measured in the hepatoma lines after pretreatment of the cells with dexamethasone, an inducer of various liver-specific cytochrome P450 forms. Genotoxicity was much weaker or not detectable in cells not pretreated with the inducer. 1-NP was not genotoxic in the hepatoma cells irrespective of whether the cells were pretreated or not. Neither isomer elicited DNA repair synthesis in V79 cells, but both isomers caused mutations to TG resistance, and 1-NP increased the number of micronucleated and multinucleated cells. The findings show that there are different pathways in mammalian cells by which nitropropanes can be converted to genotoxic products. Presumably the induction of liver tumours by 2-NP is linked to the metabolic pathway which is characterized by the formation of genotoxic metabolites from 2-NP but not 1-NP. This pathway appears to depend on the presence of liver-specific, dexamethasone-inducible, cytochrome P450 forms. The relevance of the genotoxic effects of the nitropropanes observed in V79 cells for the situation in vivo is open to question.

2-Chlorobenzylidene malonitrile (CS) causes spindle disturbances in V79 Chinese hamster cells.

Exposure of V79 Chinese hamster cells to 2-chlorobenzylidene malonitrile (CS), a chemical used as a sensory irritant for riot control, caused a concentration-dependent increase in the incidence of spindle disturbances. A C-mitotic effect with the appearance of C-metaphases, a metaphase block and the concomitant disappearance of ana-telophase figures were observed after a 3-h treatment. The results indicate that CS might induce aneuploidy in mammalian cells by interacting with the mitotic apparatus.