To investigate relationships between particle (as a model of aggregates) size in a nanomaterial test suspension and its cytotoxicity, a series of eleven sizes of polystyrene (PS) particles were tested in the cytotoxicity test and the chromosome aberration test by using a Chinese hamster cell line CHL. The PS particles were spheres with defined diameters ranging from 0.1 to 9.2 µm. A series of eight sizes of particles with diameters ranging from 0.92 to 4.45 µm showed stronger cytotoxicity than the others. There was a marked difference in cytotoxicity between the 4.45- and 5.26-µm particles. The 0.92- to 4.45-µm particles did not induce structural chromosome aberrations but induced a high frequency of polyploidy in the chromosome aberration test. The 5.26-µm particles showed very weak induction of polyploidy. The incorporation of the 4.45-µm particles into CHL cells was observed by scanning electron microscopy (SEM). Some cells incorporated more than 10 particles. The semi-quantitative measurement of incorporation of particles into cells was performed by flow cytometry with a parameter of side scattered light (SSC) intensity. It showed that CHL cells preferably incorporated the 4.45-µm particles to the 5.26-µm particles. These findings suggest that CHL cells may have a kind of size-recognition ability and incorporate a particular size of particles. The particles may prevent a normal cytokinesis resulting in polyploidy induction. Nanomaterials also may show size-dependent toxicity. Data on particle (or aggregate) size distribution in the test suspension should be provided to evaluate properly the results of toxicity tests of nanomaterials.

To evaluate the role of particle size in cytotoxicity tests of nanomaterials (NMs), we exposed Chinese hamster cells to polystyrene (PS) spheres with defined diameters ranging from 0.1 to 9.2 microm. We found that the 4.45-microm PS particles were most cytotoxic while sizes 0.1 and 0.2 microm showed no cytotoxicity up to 1000 microg/ml. In the chromosome aberration test, the 4.45-microm PS particles induced polyploidy in a mass concentration-dependent manner in 24- and 48-h treatments. The 5.26-microm PS particles induced polyploidy only at 1000 microg/ml for 48 h. Next, we performed the cytotoxicity test with as-grown single walled carbon nanohorns (NHas). These were suspended in DMSO and then transferred into the culture medium followed by sonication. Six suspensions differently sonicated showed the same apparent toxicity, although the total particle size distributions differed. However, the sizes of NHas particles predicted to be most toxic from the experiments with PS particles, i.e. 1.01-4.47 microm constituted 40-60% of all particles in all six suspensions. The results suggest that the cytotoxicity of NMs in suspension depends on specific sizes of aggregates and therefore suspensions should be checked with regard to particle size distributions in assays of toxic effects. The uptake of particles into cells was confirmed by confocal microscopy.

Several studies have shown that the characteristically skewed sex ratio among the progeny of abo homozygous females, derived from heterozygous stocks and mated to attached XY males, can be modified during homozygous stock-keeping. This amelioration seems to have a complex mechanistic background, and both loss of the blood transposon from chromosome 2, where abo is located, and amplification of a specific heterochromatic element (ABO) have been suggested to work in this direction. There is also an increased frequency of non-disjunction associated with abo, beside the poor recovery of X0 males. Experiments were performed to see if there was a coordinated loss of both phenotypic expressions during homozygous stock-keeping and if non-disjunction was amenable to modification by quinacrine. We found an unexpected spontaneous amelioration of the phenotypic expressions of abo despite heterozygous stock-keeping. The spontaneous amelioration of non-disjunction and male lethality under heterozygous condition was coordinated while the process initiated by homozygosity slowly decreased non-disjunction and rapidly increased male recovery over generations, which may point to a mechanistic difference between the amelioration processes. Quinacrine was found to ameliorate the skewed sex ratio but did not affect non-disjunction. In these experiments larval and adult treatment, respectively, were employed and the respective controls revealed that also ageing before mating significantly increased male recovery and reduced non-disjunction. Ageing before mating and quinacrine seemed to act additively on male recovery, suggesting independent action, while interaction could be suspected between quinacrine and some ameliorating factor associated with brood. Some of the results also suggest that quinacrine acts indirectly and does not substitute for the abo gene product. Due to the action of quinacrine in other biological systems it is speculated that the compound compensates for a biochemical aberration in abo/abo females or their progeny, showing some relation to phospholipase activity and/or actin polymerisation state.

We conducted the recombination and sister chromatid exchange (SCE) assays with five chemicals (hydroxyurea (HU), resveratrol, 4-hydroxy-trans-stilbene, 3-hydroxy-trans-stilbene, and mitomycin C) in Chinese hamster cell line SPD8/V79 to confirm directly that SCE is a result of homologous recombination (HR). SPD8 has a partial duplication in exon 7 of the endogenous hprt gene and can revert to wild type by homologous recombination. All chemicals were positive in both assays except for 3-hydroxy-trans-stilbene, which was negative in both. HU, resveratrol, and 4-hydroxy-trans-stilbene were scavengers of the tyrosyl free radical of the R2 subunit of mammalian ribonucleotide reductase. Tyrosyl free radical scavengers disturb normal DNA replication, causing replication fork arrest. Mitomycin C is a DNA cross-linking agent that also causes replication fork arrest. The present study suggests that replication fork arrest, which is similar to the early phases of HR, leads to a high frequency of recombination, resulting in SCEs. The findings show that SCE may be mediated by HR.

Cell-penetrating peptides are regarded as promising vectors for intracellular delivery of large, hydrophilic molecules, but their mechanism of uptake is poorly understood. Since it has now been demonstrated that the use of cell fixation leads to artifacts in microscopy studies on the cellular uptake of such peptides, much of what has been considered as established facts must be reinvestigated using live (unfixed) cells. In this work, the uptake of analogs of penetratin, Tat(48–60), and heptaarginine in two different cell lines was studied by confocal laser scanning microscopy. For penetratin, an apparently endocytotic uptake was observed, in disagreement with previous studies on fixed cells found in the literature. Substitution of the two tryptophan residues, earlier reported to be essential for cellular uptake, did not alter the uptake characteristics. A heptaarginine peptide, with a tryptophan residue added in the C-terminus, was found to be internalized by cells via an energy-independent, non-endocytotic pathway. Finally, a crucial role for arginine residues in penetratin and Tat(48–60) was demonstrated.

Coordination complexes of type [Ru(L)(3)](2+), where L is a nitrogen-containing aromatic bidentate ligand, can often be photolytically reduced, making them useful in studies of DNA- or protein-mediated electron transfer and in artifical photosynthesis model systems. Upon binding to DNA some Ru(L) complexes have been found to display strongly increased fluorescence compared with when free in solution, making those compounds interesting to test as DNA probes. Thus, they are becoming widely used in the chemistry community. Here, asynchronous cultures of V79 Chinese hamster cells were exposed to the DNA bis-intercalator Delta-Delta [mu-C4(cpdppz)(2)-(phen)(4)Ru(2)](4+) at 10(-10)-10(-4) M. The extraordinarily strong binding of the compound to DNA was the reason for testing its possible interference with DNA metabolism in intact mammalian cells. Exposure for 1 h to 10(-10)-10(-4) M did not significantly decrease DNA synthesis. Cells exposed to 10(-5) M for 27 h showed no staining of the nucleus, while DNA was stained in cells electroporated in the presence of the compound. However, the Ru dimer was probably taken up by pinocytosis, because numerous minute precipitates could be observed in the cytoplasm. Treatment for 24 h at concentrations of 10(-10)-10(-5) M did not inhibit growth, as indicated by cell density and mitotic activity. Neither did it affect chromosomal arrangements during mitosis. However, at 10(-4) M the density of cultures was reduced by approximately 45% and apoptotic cells were frequent, as opposed to mitoses. We also investigated the properties of the Ru dimer as a fluorescent DNA stain. The compound appears attractive as a red DNA stain when broad excitation in the visible range is desirable and extremely low background staining is essential. The low toxicity of the compound is a favourable trait in this context.

V79 Chinese hamster fibroblasts that maintain an elongated shape in metaphase occur at a low frequency and often show the spindle asymmetrically positioned. We show here that this aberrant position is corrected in anaphase by an external force, pulling the spindle into place. The force was applied on astral microtubules because spindle motility was hampered when astral microtubules were poorly developed spontaneously, or destroyed by colcemid. Colcemid also abolished the observed downward positioning of centrosomes in anaphase. One pole of the spindle was usually dominant during correction, but occasionally both poles could become subject to pulling making the spindle move perpendicular to the long axis of the cell, which induced reshaping of the cell. The pulling force acted unevenly with short intervals of resting between the pulling. Spindle elongation also varied in rate but showed a different periodicity than translocation of the spindle, and therefore appeared independently regulated. The length of the spindle increased with the length of the cell, and the rate of spindle elongation and pole movement increased with distance moved, indicating that the forces mediated by astral microtubules increase with their length. Arp1/dynactin, not colocalising with tubulin, was more often continuous with microtubules in anaphase B than in metaphase, and was primarily located at the bottom of the cell. Further, shifts in the geometric gravity centre of the cell occurred in the same direction as migration of the spindle. To explain these results, we suggest that astral microtubles transiently anchored at the bottom of the cell are of particular importance for spindle translocation in fibroblasts.

We performed the in vitro micronucleus (MN) test on 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-1) and 2-[2-(acetylamino)-4-[N-(2-cyanoethyl)-ethylamino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-2), which are newly identified water pollutants from the Nishitakase river in Kyoto, Japan, and on their possible mother compounds (AZO DYE) and intermediates (non-ClPBTAs). We tested these compounds in the absence and presence of S9 mix in two Chinese hamster cell lines CHL and V79-MZ and scored MN, polynuclear and karyorrhectic (PN), and mitotic (M) cells. PBTA-2 in the absence of S9 mix induced the strongest responses in both cell lines. It was also a strong inducer of binucleate cells in PN cells in both cell lines, which suggested that it induced polyploidy. PBTA-1 showed clear positive results only in the absence of S9 mix and only in V79-MZ cells, inducing aneuploidy. In CHL cells AZO DYE-1 significantly induced MN cells in the presence of S9 mix, and AZO DYE-2 induced MN and PN cells, including binucleate cells and cells with a multilobed nucleus, in the absence of S9 mix. In V79-MZ cells, AZO DYE-1 and -2 induced primarily M cells in the presence of S9 mix. 9% of the M cells treated with 50 microg/ml AZO DYE-1 showed endoreduplication. AZO DYE-2 at 200 microg/ml condensed the chromatin in 100% of the cells. The non-ClPBTAs were a bit more cytotoxic than the other compounds and induced a slight increase in MN cells in both cell lines. Some of the chemicals tested induced a characteristic karyomorphology that might reflect abnormal cell division. Abnormalities of cell division could be detected in PN and M cells as well as in MN cells. Structure-activity relationships have also been discussed.

We have tested whether cytokinesis is as sensitive to hydrophobic interactions as karyokinesis, and evaluated the usefulness of the frequency of binucleated cells as end-point. Treating cultured cells for 2 or 24 h, with different lipophilic alcohols and chlorinated hydrocarbons made this possible. Colcemid and cytochalasin B were applied as positive controls for inhibition of karyokinesis and cytokinesis, respectively. Several-fold increases of binucleated cells could be seen with cytochalasin B after 2 h of treatment, while there was no increase with colcemid, which instead blocked cells in prometaphase/metaphase. The solvent acted primarily through hydrophobic interactions. For each solvent, the blocking of cells in prometaphase/metaphase and a minor increase in binucleated cells, were seen at approximately the same concentration; the binucleated cells probably emanated from cells in anaphase/telophase at the start of treatment. We conclude that the spindle function and cleavage show similar sensitivity to hydrophobic interactions. After prolonged treatment, allowing escape from the metaphase block, the solvents induced binucleated and multinucleated cells. By forming the quotient between multinucleated (MULTI) and binucleated (BIN) cells one could distinguish between effects primarily on the spindle or cytokinesis, respectively. All solvents, and a combination of colcemid and cytochalasin B, showed quotients intermediate between those observed with colcemid (high MULTI/BIN) and cytochalasin B (low MULTI/BIN), respectively. Both protocols revealed the same relationship between lowest active concentration and lipophilicity for the solvents, implying that concentration, not dose were of prime importance. The specific inhibitors acted at low concentrations in relation to lipophilicity, clearly demonstrating their chemical mechanisms. This approach can be used for rapid screening of potential aneugens, distinguishing between routes, and when lipophilicity is known, also reveal the principal mechanism of action, i.e. physico-chemical or chemical.

The polychlorinated biphenyls 2,2',5,5'- and 3,3',4, 4'-tetrachlorobiphenyl, 2,3,3',4,4'- and 3,3',4,4', 5-pentachlorobiphenyl and 2,2',4,4',5,5'-hexachlorobiphenyl were tested for spindle-disturbing activity in V79 Chinese hamster cells. Clones lacking endogenous cytochrome P450 activity or expressing rat CYP1A1 or CYP2B1 were used. Induction of abnormal chromosomal arrangements in mitosis were found to be favoured by o-chlorine substitutions, but not by co-planarity giving affinity, for example, for the Ah receptor and CYP1A isoenzymes. Only 2,2',5, 5'-tetrachloro- and 2,3,3',4,4'-pentachlorobiphenyl gave dose-response curves similar to many other compounds tested in vitro, showing an increase from the background level of 10 to 100% disturbed mitoses with nominal concentrations >10(-6) M, i.e. concentrations far above the total PCB concentrations found in human blood. Cells transfected with rat CYP2B1 were more sensitive to the most active congener, 2,3,3',4,4'-pentachlorobiphenyl, than cells lacking P450 activity or expressing CYP1A1. Induction of abnormal mitosis by PCB metabolites formed by P450 enzymes cannot be excluded, but does not seem likely because of the short treatment time and the reportedly slow metabolism of PCBs. 2,3,3',4, 4'-Pentachlorobiphenyl showed synergistic activity with the potent spindle poison triphenyltin. Inactive concentrations of both agents (10 and 50 nM, respectively) caused abnormal configurations when combined. This is an important finding since exposure to mixtures of compounds is common and it motivates further studies of subthreshold activities of highly lipophilic environmental contaminants.

The insecticide carbaryl and its metabolite 1-naphthol cause partial uncoupling of karyokinesis and cytokinesis in V79 Chinese hamster fibroblasts; karyokinesis is blocked in metaphase, the microtubules of the spindle depolymerize and the chromosomes and spindle remnants become displaced to the periphery of the cell. A high frequency of these disturbed cells elongate and a smaller fraction initiate a cleavage furrow. Here, we attempt to determine the potential targets for carbaryl and 1-naphthol in cytokinesis-specific signalling, led by the fact that the potential protein phosphatase inhibitor 1-naphthyl phosphate was previously identified in treated cells. We found that the typical cytological pattern induced by carbaryl and 1-naphthol could be obtained with tyrphostins, specific tyrosine kinase inhibitors, indicating that the carbaryl-induced effects could be due to tyrosine kinase inhibition. This was confirmed by tyrosine kinase assays showing that carbaryl, 1-naphthol and 2-naphthol were equally efficient at inhibiting tyrosine kinase activity as tyrphostin B44(-). As tyrosine kinases can act as regulatory factors in determining dephosphorylation rates, the activities of type-1 (PP1) and type-2A (PP2A) serine/threonine protein phosphatases were also determined. There was a clear up-regulation of the overall PP1/PP2A activities in cells treated with carbaryl, 1-naphthol or tyrphostin B44(-). This stimulation was shown to be indirect because these compounds had no effect on the activity of purified human PP1 in the test tube. 2-Naphthol, which has been found to be less efficient with regard to displacement of chromatin, did not cause up-regulation, but a significant decrease in PP1/PP2A activity. We suggest that a net decrease in tyrosine kinase activity in combination with a net increase in PP1/PP2A activity is a precondition for cell elongation and cytokinesis in mammalian cells and that the corresponding enzymes are targets in the network of activities serving to coordinate karyokinesis and cytokinesis.

Carbaryl causes depolymerization of spindle microtubules and apparent uncoupling of karyokinesis and cytokinesis in mitotic V79 cells. The metabolite 1-naphthol has virtually identical effects at equimolar concentrations. The closely related 2-naphthol causes similar configurations, but at a much lower frequency than 1-naphthol, showing that there are some structural requirements for these effects in mitosis. The results of the present study demonstrate that the effects of treatment are reversible; briefly (30 min) treated and thoroughly rinsed cells resume the normal appearance of cells in metaphase within 5 min, followed by anaphase approximately 15 min later. It could be demonstrated that added 1-naphthol can be converted to 1-naphthylphosphate by the cells, a recognized protein phosphatase inhibitor. With the applied method no 1-naphthylphosphate could be detected in carbaryl-treated cells, although a fraction of carbaryl was found to be converted to 1-naphthol. Carbaryl, 1-naphthol and 2-naphthol caused a decrease in protein phosphorylation of about the same magnitude. We hypothesize that 1-naphthol is a substrate for a protein kinase in mitosis and the carbaryl interferes with the same kinase. Carbaryl alone or the 1-naphthylphosphate formed may also interfere with protein phosphatase activity.

Although aneuploidy makes a significant contribution to both somatic and inherited disease the mechanisms by which environmental chemicals may induce numerical chromosome aberrations are only poorly defined. The European Union Project was aimed to further our understanding of those chemical interactions with the components of the mitotic and meiotic cell division cycle which may lead to aneuploidy and to characterise the parameters such as cellular metabolism which may influence the activity of aneugenic chemicals. C-mitosis can be induced by the highly lipophilic polychlorinated biphenyl and the completion of mitosis and cleavage can be modified by agents which deplete cellular levels of reduced glutathione. Modifications of the fidelity of chromosome segregation were produced by inhibiting the functioning of topoisomerase II during chromatid separation. In contrast, the modification of centromere integrity resulted in chromosome breakage as opposed to disturbance of segregation. Modifiers of tubulin assembly and centriolar functioning in somatic cells such as acrylamide, vinblastine and diazepam reproduced their activity in rodent bone marrow and male germ cells. The analysis of chromosome malsegregation in Aspergillus nidulans by a structurally related series of halogenated hydrocarbons was used to develop a QSAR model which had high predictive value for the results of fungal tests for previously untested related chemicals. Metabolic studies of potential aneugens in genetically engineered human lymphoblastoid cells demonstrated the detoxification of the aneugenic activity of chloral hydrate and the activation of 2,3-dichlorobutane, 1,1,2-trichloroethane and trichloroethylene by Phase I biotransforming enzymes. Cell transformation studies in Syrian hamster dermal cultures using a panel of 22 reference and or potential aneugens indicated that 15 of the 22 produced positive results following single exposures. Five of the aneugens which were negative following single exposures produced positive results where cultures were continuously exposed for up to 6 weeks to low concentrations following a single non-transforming exposure to the mutagen dimethyl sulphate. The transformation studies indicate that a significant proportion of chemical aneugens are potential complete carcinogens and/or co-carcinogens. To optimise the enumeration of chromosomes following exposure to potential chemical aneugens whole chromosome paints and centromere specific probes suitable for use in fluorescence in situ hybridisation (FISH) were developed for the rat, mouse and Chinese hamster and selected human probes evaluated for their suitability for routine use. Molecular chromosome probes were used to develop protocols for enumerating chromosomes in metaphase cells and centromeres and micronuclei in interphase cells. The analysis of segregation of specific centromeres in binucleate cells following cytochalasin B treatment was shown to be a potentially valuable system for characterising non-disjunction following chemical exposure. Whole chromosome paints and centromere specific probes were used to demonstrate the presence of dose-response thresholds following treatment with a reference panel of spindle inhibiting chemicals. These data indicate that the FISH technology is suitable for evaluating the relative hazards of low-dose exposures to aneugenic chemicals.

Denaturing gradient gel electrophoresis (DGGE) and constant denaturant gel electrophoresis (CDGE) are methods based on sequence-determined melting characteristics of DNA and thus detect different types of single base changes in the amplified fragments. We have studied detection of 19 mutations in the human N-ras oncogene and 10 mutations in exon 3 of the Chinese hamster hypoxanthine-guanine phosphoribosyltransferase (hprt) gene using GC-clamped DGGE and CDGE. After allowing formation of heteroduplexes with the corresponding wild type sequence, all the mutations separated from the wild type in at least one concentration of the denaturants used in CDGE but two of the mutations in hprt exon 3 did not show separation in any of the DGGE runs. Melting behavior of the mutant fragments was dependent, as expected, on both the type and the location of a mutation. We describe conditions allowing separation of the mutations in the fewest possible DGGE and CDGE runs.

We used high fidelity PCR and constant denaturant capillary electrophoresis (CDCE) [Khrapko et al. (1994) Nucleic Acids Res., 22, 364-369] to separate wild type and different mutant N-ras exon 1 and 2 sequences. The set of plasmids containing N-ras cDNA, wild type or mutant sequences representing all transforming amino acid-substituting single base pair changes in codons 12, 13 (exon 1) and 61 (exon 2), were amplified using Pfu polymerase in a limited cycle polymerase chain reaction. One of the primers used for the amplification of each exon included a 40 nucleotide GC rich sequence that created high and low melting domains. The amplified fragments 151 bp (exon 1) and 150 bp (exon 2) were run on the CDCE with the 'denaturant zone' temperature of the capillary corresponding to the melting temperature of 111 bp (exon 1) and 110 bp (exon 2) low melting domains. The separation was achieved between wild type and mutant sequences as homoduplexes in 15 out of 19 cases, as a single base substitution alters the electrophoretic mobility of a partially melted double stranded fragment. The denaturation and reannealing of wild type and mutant fragments together created wild type/mutant heteroduplexes. All the heteroduplexes were well resolved from wild type homoduplex. In the current form mutant sequences were detected at a frequency of 10(-3) in the presence of wild type. This study has resulted in obtaining electrophoretic spectrum of different N-ras mutants on CDCE as homoduplexes as well as heteroduplexes.

Binucleate V79 Chinese hamster cells were video time-lapse recorded during mitosis. The general pattern found was: formation of one integrated metaphase plate, most often in a tetra- or tripolar spindle, prolonged prometaphase/metaphase, and normal duration of anaphase and contraction of cleavage furrows. Cleavage, however, often showed partial regression. Reversal of cleavage usually occurred late and then was not clearly separable from fusion of daughter cells. Reversal/fusion was more common the more furrows were initiated in late anaphase. This suggests that a final part of cleavage, or establishment of daughter cell integrity, is hampered by the preceding complex chromosome/spindle arrangements in the binucleate cells. Lagging of chromosomes in anaphase and formation of micronuclei were found to be frequent events. The nuclei formed from cells with a tetra-or tripolar spindle were often uneven in size, suggesting that the cells have difficulties in distributing the chromosomes in an organized way between many poles. A comparison with previous studies of binucleate mitoses in other cell lines shows major similarities in the mitotic performance. It is suggested that induction of binucleate cells should be included as a relevant end point when screening novel compounds for toxicity and aneuploidy.

Protein kinase C (PKC) was activated in V79 Chinese hamster cells with 10 nM 12-O-tetradecanoylphorbol-13-acetate (TPA). Within 30 min soluble activity decreased concomitant with a 10-fold increase of particulate activity. The latter was still elevated after 3 h but was back to control levels after 24 h of treatment; by then soluble activity was lost. The frequency of mitotic cells with signs of abnormal spindle function increased within 15 min and reached a plateau after 45-60 min which lasted throughout the 4 h treatment. The c-mitotic effect was delayed and significantly lower when 10 nM TPA was combined with 50 microM of the protein kinase inhibitor H7. The frequency of disturbed mitotic cells decreased after 24 h of treatment but remained significantly higher than in non-treated cells. Change of medium and addition of new TPA caused a slight but significant further increase. It is suggested that PKC takes part in eliciting the c-mitotic effect of TPA. However, the sustained effect coincident with down-regulation points to significant alterations of the level or the activity of an as yet unidentified ultimate elicitor. TPA also caused a transient block in the G2 phase which was ameliorated by H7 and which could not be detected at all in TPA-pretreated cells (24 h) given new TPA. This suggests that PKC takes part in eliciting the G2/M block as well but the mechanism is different from the one(s) behind the c-mitotic effect. V79 cells were found to exit from mitosis in the presence of 0.2 microgram/ml of colcemid but TPA-pretreated cells showed a decreased exit rate. There was no sign of hampered exit among cells going into mitosis soon after the G2 block was reversed, which implies that the spontaneous reversal of the block does not involve rapid down-regulation of PKC.

A significant association between N-ras oncogene activating point mutations and testicular cancer has recently been reported. We have studied DNA samples from the blood and fresh tumor tissues of 17 Norwegian testicular cancer patients (11 seminomas/6 nonseminomas). Point mutations in K-ras-2 and N-ras exons 1 and 2 were studied by denaturing gradient gel electrophoresis (DGGE) and by oligonucleotide hybridization. No N-ras mutations were detected in these tumor samples, but two K-ras-2 exon 1 mutations were found in two of the seminoma tumors (stage I and II tumors) using the DGGE technique. The mutations were confirmed by dot blotting and oligonucleotide hybridization and identified as a G-->T and a G-->A point mutation in K-ras-2 codon 12, leading to a valine and a serine substitution, respectively. All the white blood cell DNAs were negative. As a positive control for DGGE screening, we ran two plasmid constructs carrying human N-ras exon 2 sequences with mutations. To study the role of ras gene activation in testicular cancer, a larger tumor sample population will be investigated.