In vitro evaluation of the cytotoxic and mutagenic potential of the 5-aminolevulinic acid hexylester-mediated photodynamic therapy.

Photodynamic therapy (PDT) of tumors with 5-aminolevulinic acid hexylester (h-ALA) causes photo-oxidative reactions in treated tissues. In order to study cytotoxic and/or mutagenic effects, cells of the tumor cell line RPMI 2650 as well as fibroblasts of the cell line WS 1 were given photodynamic treatment in vitro. The cells were photosensitized with a 1mM h-ALA-medium solution for 5h and illuminated with different light doses (0.5, 1.0, 1.5 and 2.0 J/cm2) using red light (633+/-20 nm). PDT-induced cytotoxic effects were determined by measurement of the mitotic index (MI) and the nuclear division index (NDI). Chromosome aberrations (CA) and micronuclei (MN) were recorded to study mutagenicity. After treatment of the photosensitized RPMI 2650 cells with a light dose of 2.0 J/cm2, the MI was significantly decreased to 16.9 per thousand in comparison with that of the h-ALA control (33.8 per thousand ). In photosensitized WS 1 cells, light doses up to 2.0 J/cm2 showed no significant effect. The NDI of photosensitized RPMI 2650 cells was significantly decreased by light doses from 1.0 to 2.0 J/cm2, whereas no significant effect was seen in WS 1 cultures. Thus, h-ALA-PDT only induced desirable cytotoxic effects in tumor cells, but not in the fibroblasts. After application of light doses from 0.5 to 2.0 J/cm2, photosensitized RPMI 2650 cultures showed CA in 7.0-7.5% of the metaphases, which was not a significant increase (h-ALA control: 5.5%). In WS 1 cultures metaphases containing CA varied non-significantly from 5.0 to 7.5%. The MN rates were approximately the same in illuminated RPMI 2650 cultures and in the corresponding h-ALA control (4.4-4.9 per thousand ). The MN rates of the illuminated WS 1 cultures also varied non-significantly from 4.5 to 5.0 per thousand in comparison with the h-ALA control (5.5 per thousand ). In the mutagenicity tests the h-ALA-PDT had no significant effect, neither on the tumor cells nor on the fibroblasts. In addition to the cytogenetic analysis, spectral karyotyping (SKY) was used to characterize the cell lines and gain more detailed information on possibly PDT-induced CA. The SKY evaluation also showed no significant increase of the CA rate, but confirmed the result of the CA test. Thus, within the scope of the experiments performed, a mutagenic potential of the h-ALA-PDT can be excluded.

Mutagenic and cytotoxic effectiveness of diisopropyl xanthogen polysulphide in human lymphocyte cultures.

The mutagenic and cytotoxic effectiveness of the new rubber vulcanisation accelerator diisopropyl xanthogen polysulphide (Robac AS 100) was tested in human lymphocyte cultures of four healthy probands. The concentrations of Robac AS 100 were 0.57, 5.7 and 57.0 microg/ml. Higher concentrations showed too high cytotoxicity to be evaluable. Without external activation, incubation time with Robac AS 100 was 21 h. In the presence of rat liver microsomes from aroclor-induced rats (2mg microsomal protein/ml), incubation of the test compound was 2h. Mutagenicity testing was performed by analysis of micronuclei (MN), structural chromosome aberrations (CAs) and sister chromatid exchanges (SCEs). The MN-rate was determined using the cytochalasin B (cyt B) block method. For evaluation of cytotoxicity, mitotic index (MI) and nuclear division index (NDI) were determined. The validity of the test methods was ascertained by positive controls: mitomycin C (MMC) and bleomycin (BLM) were used in experiments without exogenous activation and cyclophosphamide (CP) in experiments with exogenous activation. The presence of rat liver microsomes increased the mutagenic effect of Robac AS 100 in the SCE- and MN-test. But only the highest Robac AS 100-concentration (57.0 microg/ml) showed significantly increased mutagenic activity in all tests. However, cytotoxicity at this concentration was already substantial. Therefore, we consider the evidence for mutagenicity of Robac AS 100 as limited.

Elimination of doxepin isomers from the horse following intravenous application.

The tricyclic antidepressant doxepin, representing a 5:1 mixture of trans- and cis-isomers, owns tranquilizing properties. This compound has been associated with illicit medication of racing horses, and therefore should be considered in doping control. Because analysis of doxepin in equine body fluids has not been documented in the literature, a highly sensitive analytical method was developed to individually monitor the doxepin isomers in blood and urine of horses by the use of gas chromatography/mass spectrometry. Following a dose of 1 mg doxepin-HCl/kg intravenously (i.v.), both the isomers were quantified for up to 24 h in serum of horses (n=4). The beta-half-lives of the trans- and cis-isomers were 3.5 and 3.1 h, respectively. The ratio of the trans/cis-isomers was found to be constant (4.7:1) during drug elimination and thus corresponded to the original composition of the antidepressant. Up to 12 h following administration low trans-isomer concentrations in an average range of 2-6 ng/mL were detected in urine of each of the horses, while the cis-isomer was only present in two of four horses for up to 8 and 12 h, respectively. In serum, mean trans-isomer concentrations exceeded urine levels maximally 120-fold after 3 h and at least sixfold after 12 h. As serum exhibits considerably higher concentrations of the doxepin isomers as compared with urine, blood of horses is the recommended body fluid when screening for the antidepressant.

Mutagenic and cytotoxic effectiveness of zinc dimethyl and zinc diisononyldithiocarbamate in human lymphocyte cultures.

The mutagenic and cytotoxic effectiveness of the vulcanisation accelerators zinc dimethyldithiocarbamate (ZDMC; ziram) and zinc diisononyldithiocarbamate (ZDINDC; arbestab Z) was tested in lymphocyte cultures of five healthy probands. ZDMC and ZDINDC (c=0.1, 1.0 and 10.0microg/ml) were studied in lymphocyte cultures without external metabolic activation. Additionally, incubation of the compounds (c=10.0microg/ml) was performed in the presence of liver microsomes from aroclor-induced rats (1 and 2h, 1 and 2mg microsomal protein). Genotoxicity testing was performed by analysis of chromosomal aberrations (CA), sister chromatid exchanges (SCEs) and micronuclei (MN). For evaluation of antiproliferative effects, mitotic index (MI) and cell cycle kinetics (CCK) were determined. In contrast to earlier investigations we found no significantly increased mutagenic or cytotoxic activity of ZDMC; ZDINDC also was inactive under these conditions.

Protective effects of vitamins C and E on the number of micronuclei in lymphocytes in smokers and their role in ascorbate free radical formation in plasma.

Cigarette smoke is widely believed to increase free radical concentrations causing subsequent oxidative processes that lead to DNA damage and hence, to several diseases including lung cancer and atherosclerosis. Vitamin C is a reducing agent that can terminate free-radical-driven oxidation by being converted to a resonance-stabilized free radical. To investigate whether short-term supplementation with the antioxidants vitamin C and E decreases free-radical-driven oxidation and thus decreases DNA damage in smokers, we determined the frequency of micronuclei in lymphocytes in 24 subjects and monitored the electron paramagnetic resonance signal of ascorbate free radical formation in plasma. Further parameters comprised sister-chromatid exchanges and thiobarbituric acid-reactive substances. Twelve smokers and twelve non-smokers took 1000 mg ascorbic acid daily for 7 days and then 1000 mg ascorbic acid and 335.5 mg RRR-alpha-tocopherol daily for the next 7 days. Baseline concentrations of both vitamins C and E were lower and baseline numbers of micronuclei were higher (p < 0.0001) in smokers than in non-smokers. After 7 days of vitamins C and E, DNA damage as monitored by the number of micronulei was decreased in both, smokers and non-smokers, but it was more decreased in smokers as indicated by fewer micronuclei in peripheral lymphocytes (p < 0.05). Concomitantly, the plasma concentrations of vitamin C (p < 0.001) as well as the ascorbate free radical (p < 0.05) were increased. The corresponding values in non-smokers, however, did not change. Our findings show that increased ascorbate free radical formation in plasma after short-term supplementation with vitamins C and E can decrease the number of micronuclei in blood lymphocytes and thus DNA damage in smokers.

Inhibition of cyclin-dependent kinase 1 (CDK1) by indirubin derivatives in human tumour cells.

The bisindole indirubin has been described, more than 30 years ago, as being clinically active in the treatment of human chronic myelocytic leukaemia. However, the underlying mechanism of action has remained unclear. We have reported previously that indirubin and its analogues are potent and selective inhibitors of cyclin-dependent kinases (CDK). In this study, we investigated the influence of indirubin and derivatives on CDK1/cyclin B kinase in human tumour cells at concentrations known to induce growth inhibition. Cells of the mammary carcinoma cell line MCF-7, synchronized by serum deprivation, after serum repletion stay arrested in the G(1)/G(0)phase of the cell cycle in the presence of 2 microM indirubin-3'-monoxime. At higher drug concentrations (> or = 5 microM) an increase of the cell population in the G(2)/M phase is additionally observed. Cells synchronized in G(2)/M phase by nocodazole remain arrested in the G(2)/M phase after release, in the presence of indirubin-3'-monoxime (> or =5 microM). After 24 h treatment with 10 microM indirubin-3'-monoxime a sub-G(2)peak appears, indicative for the onset of apoptotic cell death. Treatment of MCF-7 cells with growth inhibitory concentrations of indirubin-3'-monoxime induces dose-dependent inhibition of the CDK1 activity in the cell. After 24 h treatment, a strong decrease of the CDK1 protein level along with a reduction of cyclin B in complex with CDK1 is observed. Taken together, the results of this study strongly suggest that inhibition of CDK activity in human tumour cells is a major mechanism by which indirubin derivatives exert their potent antitumour efficacy.

Chromosome painting reveals specific patterns of chromosome occurrence in mitomycin C- and diethylstilboestrol-induced micronuclei.

Cultures of human blood lymphocytes from three subjects were incubated with the clastogen mitomycin C (MMC, 500 ng/ml) and the aneugen diethylstilboestrol (DES, 80 microM) 23 h before harvesting, to induce formation of micronuclei (MN) and numerical and structural alterations in metaphase chromosomes. We used fluorescence in situ hybridization (FISH) with painting probes for all human chromosomes to determine which chromosomes had contributed material to the induced MN. MMC treatment induced an approximately 18-fold increase in MN and led to a significant increase in hypodiploidy and structural chromosome aberrations in metaphase preparations. Undercondensation of pericentromeric heterochromatin of chromosomes 9 and 1 occurred in 20-75% of metaphases and FISH disclosed an abundance of material from these chromosomes in induced MN (62-69% from chromosome 9 and 7-12% from chromosome 1). DES treatment of lymphocytes induced a seven-fold increase in MN frequency and four-fold increase in the frequency of numerical aberrations; structural aberrations were not significantly increased. FISH analysis showed that material from all chromosomes was present in DES-induced MN, with material from chromosome 1 present in 16% of MN and material from each other chromosomes being present in 2-10% of MN. Material from chromosomes 14, 19 and 21 was significantly more frequent material from chromosome Y significantly less frequent in DES-treated cells than in controls. The findings of the MMC studies indicate that the heterochromatin block of chromosome 9 is a specific target for MMC-induced undercondensation, which induces a preferential occurrence of chromosome 9 material in MN. DES, in contrast, does not trigger heterochromatin decondensation and fails to induce such a significant appearance of material of particular chromosomes in MN.

Double minutes and c-MYC amplification in acute myelogenous leukemia: Are they prognostic factors?

A case of acute myelogenous leukemia (AML) with double minutes (dmin) and X chromosome loss is presented. Using comparative genomic hybridization (CGH), a region of high-level DNA amplification was detected at 8q24, the locus of the c-MYC proto-oncogene. Fluorescence in situ hybridization (FISH) with a DNA probe specific for the human c-MYC gene confirmed the extrachromosomal amplification of this proto-oncogene in the dmin of the leukemic cells. During the course of the disease, three relapses occurred; two complete remissions could be achieved by treatment with various chemotherapy regimens. The patient's survival time of 25 months was considerably longer than in most reported cases of AML with extrachromosomal c-MYC amplification. Therefore, the present case challenges the view that the occurrence of dmin in AML is generally an indication of poor prognosis.

Methadone screening of racehorses.

The misuse of opiates in racehorses relates to their effect of increasing locomotor activity. Because methadone, a narcotic analgesic, has been suspected of use as a doping compound in the past, it was added to the list of banned drugs and should be considered in doping control. Because the literature fails to provide information on detection of methadone in blood or urine of horses, an enzyme-linked immunosorbent assay was developed to monitor this narcotic in equine body fluids. Combined with high-performance liquid chromatography, the immunoassay also served to confirm positives indicated by screening. Following intravenous administration of methadone (0.1 mg/kg), the drug was found for up to 24 h in blood but was never identified in urine (10-pg/mL detection limit). Thus, urine is dismissed as a source of methadone control, and the use of blood to screen racehorses for this narcotic analgesic is suggested.

Comparison of spontaneous and idoxuridine-induced micronuclei by chromosome painting.

Fluorescence in situ hybridisation (FISH) technique with chromosome specific library (CSL) DNA probes for all human chromosomes were used to study about 9000 micronuclei (MN) in normal and idoxuridine (IUdR)-treated lymphocyte cultures of female and male donors. In addition, MN rates and structural chromosome aberrations were scored in Giemsa-stained chromosome spreads of these cultures. IUdR treatment (40 microg/ml) induced on the average a 12-fold increase of the MN rate. Metaphase analysis revealed no distinct increase of chromosome breaks but a preferential decondensation at chromosome 9q12 (28-79%) and to a lower extend at 1q12 (8-21%). Application of FISH technique with CSL probes to one male and one female untreated proband showed that all human chromosomes except chromosome 12 (and to a striking high frequency chromosomes 9, X and Y) occurred in spontaneous MN. In cultures containing IUdR, the chromosomal spectrum found in MN was reduced to 10 chromosomes in the male and 13 in the female proband. Eight chromosomes (2, 6, 12, 13, 14, 15, 17 and 18) did not occur in MN of both probands. On the contrary chromosomes 1 and especially 9 were found much more frequently in the MN of IUdR-treated cultures than in MN of control cultures. DAPI-staining revealed heterochromatin signals in most of the IUdR-induced MN. In an additional study, spontaneous and IUdR-induced MN were investigated in lymphocytes of another female donor using CSL probes only for chromosomes 1, 6, 9, 15, 16 and X. The results confirmed the previous finding that chromosomes 1 and 9 occur very often in MN after IUdR-treatment. The results indicate that decondensation of heterochromatic regions on chromosomes 1 and 9 caused by IUdR treatment strongly correlates with MN formation by these chromosomes.

Frequencies of occurrence of all human chromosomes in micronuclei from normal and 5-azacytidine-treated lymphocytes as revealed by chromosome painting.

Chromosome painting with library DNA probes specific for all human chromosomes was used to study the chromosomal content of micronuclei (MN) in normal and 5-azacytidine (5-aza-C)-treated lymphocyte cultures. More than 60,000 normal lymphocytes were screened for associated MN after in situ hybridization. At least 50 MN were scored for each probe. With the exception of chromosomes 12 and 19, which did not occur in MN, all other chromosomes were detected in MN at frequencies varying from 1 to 11.5%. Treatment of lymphocyte cultures with 5-aza-C induced preferential exclusion of chromosomes 1 (34%), 9 (32%) and 16 (20%) material in MN, whereas chromosome 8, 10, 12-15 and 21 material was not detected in MN. The results obtained from normal lymphocytes allow for the first time an estimation of the frequency of occurrence of all chromosomes in spontaneously occurring MN in human cells. Data derived from 5-aza-C-treated lymphocytes are furthermore consistent with the view that undermethylation of heterochromatin may be associated with loss of specific chromosomes at metaphase.

Induction of apoptosis by an inhibitor of cAMP-specific PDE in malignant murine carcinoma cells overexpressing PDE activity in comparison to their nonmalignant counterparts.

In order to study potential changes in phosphodiesterase (PDE) activity associated with malignant transformation, normal primary keratinocytes and cells corresponding to different stages of epidermal tumor development in mouse skin were analyzed with respect to their 3',5'-cyclic adenosine monophosphate (cAMP) hydrolyzing activity. Expression of cAMP-specific PDE-4, intracellular cAMP content, and the sensitivity to the growth inhibitory effect of the PDE-4-specific inhibitor 7-benzylamino-6-chloro-2 piperazino-4-pyrrolidino-pteridine (DC-TA-46) were studied in the two papilloma cell lines, MSCP6 and 308, and in the highly malignant carcinoma cell line CarB. No significant difference in soluble PDE activity and in intracellular cAMP was found in the two papilloma cell lines when compared to primary keratinocytes. In contrast, the spindle-cell carcinoma cell line CarB exhibited significantly higher PDE activity, concomitant with the lowest cAMP level. In all cell lines and also in the primary keratinocytes, rolipram-sensitive PDE-4 activity accounted for the major cAMP-hydrolyzing activity. In primary keratinocytes and in MSCP6 cells, the PDE-4 inhibitor DC-TA-46 induced at best marginal growth inhibition, whereas cell growth of 308 cells was markedly affected at concentrations > 2 microM. The carcinoma cell line CarB showed the highest sensitivity to DC-TA-46 (IC50 = 0.8 +/- 0.3 microM). Treatment of CarB cells with DC-TA-46 strongly inhibits intracellular PDE activity, resulting in a marked and long-lasting rise of cAMP. After 24 h of treatment, arrest in the G0/G1 phase of the cell cycle is induced. Treatment with concentrations > 2 microM of this highly effective PDE inhibitor results in induction of apoptotic cell death, as detected by fluorescence microscopy, flow cytometry, and ELISA-based determination of fragmented DNA in intact cells.

Genotoxic effects of 2-trans-hexenal in human buccal mucosa cells in vivo.

In 7 non-smoking healthy volunteers, the number of micronuclei (MN) was determined in exfoliated buccal mucosa cells before and after rinsing the mouth with an aqueous 10 ppm solution of 2-trans-hexenal during 3 consecutive days. All individuals showed at least a doubling of the MN frequency during one of the next 4 days. An increase of the mean group MN frequency was observed on the fourth day, becoming significant between the sixth and the seventh day. During the next 2 days, the MN frequency dropped down to nearly the control level. In a second study, 7 other volunteers were examined before and after eating 3-6 bananas per day over a period of 3 days. The bananas contained about 35 ppm of hexenal. Six of the 7 individuals showed at least a doubling of the MN frequency during one of the next 6 days. An increase in the mean MN counts was also observed, but the difference to the control value become non-significant during the test period. The results show for the first time that the flavoring constituent 2-trans-hexenal, which is present in many human foods exerts genotoxic effects on human buccal mucosa cells in vivo.

Excretion of the anabolic steroid boldenone by racing pigeons.

OBJECTIVES: To detect the anabolic steroid bolden-one and to monitor its elimination in the droppings (hereafter referred to as feces) of pigeons treated with the drug. ANIMALS: 8 female pigeons ("Texas" race, 500 +/- 20 g). PROCEDURE: 4 pigeons were given boldenone-17-undecylenate (10 mg/kg of body weight, IM). Feces were collected over defined periods, freeze-dried, extracted with buffer (pH 7.2), and centrifuged. Total immunoreactivity in the supernatant was determined directly by use of an ELISA, and individual boldenone concentration was measured by use of a high-performance liquid chromatography (HPLC)/ELISA after solvent extraction of the aqueous phase. An additional 4 pigeons received a 1-mg/kg dose of the drug. RESULTS: Screening of feces from boldenone-treated pigeons revealed detection of the drug up to 49 days after its administration. The free parent compound was detected during the same period at a constant value of 12 ng/g of lyophilized feces. Positive results predicted by screening were reliably confirmed by the HPLC/ ELISA. Treatment of pigeons with a lower dosage (1 mg/kg) yielded positive results for 31 days. CONCLUSIONS: Illegal medication of pigeons with the anabolic steroid boldenone can be uncovered by screening of feces, using a specific ELISA. Confirmation analysis by use of 2 HPLC systems combined with ELISA reliably yields evidence of drug misuse. Owing to the multiple immunoreactive material, the apparent boldenone concentrations registered by screening markedly exceeded the immunoreactivity attributed to boldenone recovered by HPLC/ELISA. Because the test yields positive results in pigeons for at least 31 days after a single treatment, even at a low dosage of the 17-undecylenate preparation (1 mg/kg), the proposed boldenone test procedure is recommended for doping control in racing pigeons.

Exhaustive physical exercise increases frequency of micronuclei.

Micronucleus (MN) tests were performed on PHA-stimulated blood lymphocytes of six healthy volunteers before and after two exhaustive sprints, causing lactate concentrations in the peripheral blood between 9.6 and 12.4 mmol/l. The number of micronuclei was significantly increased after 24-48 h in all six subjects. The mean of the total group increased from 37 MN per 3000 binucleated cells before exercise to 56 and 55 MN 24 and 48 h after exercise, respectively. These differences were also significant. These results indicate that exhaustive physical exercise causes severe mutations at the chromosome level in blood lymphocytes.

[Detection of doping compounds in the racing pigeon]. / Nachweis von Dopingsubstanzen in der Brieftaube.

To overcome the doping problems in racing pigeons it requires reliable methods to detect illegally medicated drugs. Difficulties applying specifically to pigeons caused the present investigation, since urine and blood commonly analyzed cannot be gathered from pigeons. Drug detection is complicated by those compounds exhibiting an extremely high potency such as the anabolic boldenone, the glucocorticoid prednisolone, and the bronchodilator clenbuterol. The material for analysis selected was faeces of pigeons treated with the doping substances under investigation. The freeze-dried material was subjected to liquid/liquid extraction (Extrelut), and the extracts were submitted to high performance liquid chromatography. Identification of drugs present in the fractions was carried out by specific antibodies (ELISA), and their relative chromatographic retentions were calculated by means of internal standards. The results obtained provide an overall information about elimination kinetics of the three drugs examined. Thus, boldenone, prednisolone, and clenbuterol were detected in the faeces up to 7 weeks, 2 and 1 day, respectively (limits of detection amounted to 0.1-4 ng per g of the freeze-dried faeces.) The detection periods correspond to the periods of pharmacological action of the individual compounds. The results reported here exemplify the proof of doping by examination of faeces from pigeons treated with very potent drugs. High performance liquid chromatography combined with ELISA turned out to be a suitable technique to detect illegal medication in racing pigeons.

Genotoxic effects of the alpha, beta-unsaturated aldehydes 2-trans-butenal, 2-trans-hexenal and 2-trans, 6-cis-nonadienal.

The genotoxic effects of the 2-alkenals crotonaldehyde, 2-trans-hexenal and 2-trans-6-cis-nonadienal were studied by cytogenetic methods, analyzing frequencies of sister-chromatid-exchanges, numerical and structural chromosome aberrations and micronucleus induction in human blood lymphocytes and cells of the permanent Namalva line. Crotonaldehyde and hexenal were tested in concentrations of 5 microM to 250 microM and nonadienal from 5 microM to 70 microM. Significant dose-related increases of sister-chromatid-exchanges and micronuclei were found for all three compounds. Structural chromosomal aberrations were significantly increased only by crotonaldehyde, but not by hexenal and nonadienal. In contrast numerical chromosome aberrations were not induced by crotonaldehyde whereas hexenal and nonadienal were potent inducers of aneuploidy. The micronuclei were classified by using a centromere-specific DNA probe in a fluorescence in situ hybridization assay. Hexenal and nonadienal increased the percentage of centromere-positive micronuclei, nonadienal being considerably more potent than hexenal. From these results it was concluded that crotonaldehyde acts more as a clastogen whereas hexenal and nonadienal preferentially show aneugenic effects.

Telomere length variation in normal and malignant human tissues.

Tissue and tumor specific length variation of telomere (TTAGGG)n repeats was studied in DNAs from various normal and malignant tissues. DNA was isolated from bone marrow and blood cells, malignant tissues, and established tumor cell lines. Nonisotopic Southern hybridization revealed a reduction of telomere repeat arrays in 14 of the 35 tumors analyzed. However, other cases (60%) showed no reduction, or even an increase, in telomeric length. Our finding of elongated telomere stretches in several tumors of different origin compared with normal tissue is in contrast to previous reports describing a general shortening of terminal repeat length in colorectal cancer and neuroblastoma. We tentatively conclude that there is no general tendency to telomere reduction in malignant tissues.

Characterization of marker chromosomes in Namalva cells by chromosomal in situ suppression (CISS) hybridization and R-banding.

Chromosomal in situ suppression (CISS) hybridization was used to investigate the distribution of material of chromosomes 1 and 5 present in marker chromosomes of Namalva cells. The Namalva cell line, established from a Burkitt's lymphoma, exhibits a highly variable female karyotype with a large number of marker chromosomes. Libraries from sorted human chromosomes 1 and 5 were used to delineate material of these chromosomes present in the Namalva karyotype. We used the DAB/peroxidase reaction and reflection contrast microscopy for detection of biotinylated hybrid molecules. Identification of chromosomes was achieved by fluorescent R-banding after CISS hybridization, which allowed the assignment of hybridized regions to the particular marker chromosomes. After CISS hybridization with a chromosome I library, the normal chromosome I was labelled as well as a large marker MI and the long arm of marker M3. Using a chromosome 5 library it could be shown that the distal part of the long arm of one chromosome 5 was translocated to marker M2. The normal chromosome 5 was completely labelled. The present investigation demonstrates the advantage of combining CISS hybridization and banding for the identification of complex, rearranged tumor karyotypes.