Isolation of Chinese hamster cells hyposensitive to ethyl methanesulfonate and their characterization in induction and antagonization of sister-chromatid exchanges

Dominant lethal tests were performed on female mice injected intraperitoneally with cyclophosphamide (200 mg/kg) or with mitomycin C (0.2 or 5 mg/kg) at the preovulatory stage of oogenesis. Complementary experiments were undertaken to clarify the results obtained. Embryo culture showed that sterility found after treatment with cyclophosphamide or with the high dose of mitomycin C was the reflection of true dominant lethal effects. Mortality after cyclophosphamide treatment occurred predominantly at the 2- and 3-cell stages, while it was reported in all preimplantation stages after treatment with the high dose of mitomycin C. Embryos treated with the low dose of mitomycin C developed normally to the blastocyst stage, confirming the absence of preimplantation effects found with this dose in the dominant lethal test. Cytogenetic analysis of female pronuclei at the first cleavage division were performed after mating treated females with males homozygous for one Robertsonian translocation. This method allowed one to distinguish easily the female pronuclei from the male ones, which exhibited one translocated 'marker' chromosome. After treatment with cyclophosphamide, most female pronuclei showed multiple chromatid exchanges or shattering of the entire genome. After treatment with the high dose of mitomycin C, various types of premature chromosome condensation were found, and they were often accompanied by important interchromosome associations. After treatment with the low dose of mitomycin C, no structural chromosome aberrations were found, and the number of numerical anomalies was not significantly different from that found in control embryos. These last results suggest that the increase in rate of postimplantation loss obtained in the dominant lethal test with the low dose of mitomycin C was not due to clastogenic effects of this compound in the female germ cells, but rather to indirect effects on the maternal organism.     

Evaluation of spermatogenic response of mice to the induction of mutations by combined treatment with X rays and antineoplastic drugs

Combined treatment with low doses of X-rays plus cyclophosphamide (0.25 Gy+25 mg/kg body weight) or X-rays plus mitomycin C (0.25 Gy+1.75 mg/kg body weight) did not induce significant dominant lethal effects in any stage of spermatogenesis when a parameter representing pre- and postimplantation loss, such as the decrease of live implants per female, was applied. After combined exposure to high doses of X-rays plus cyclophosphamide (1.00 Gy+100 mg/kg body weight) an increase of dominant lethal mutations (DLMs) was observed in differentiating spermatogonia, spermatids, and spermatozoa with the same parameter. Combined treatment with high doses of X-rays plus mitomycin C (1.00 Gy+5.25 mg/kg body weight) produced DLMs in differentiating spermatogonia and late spermatocytes. A calculation of enhanced risk was applied to the data of DLMs from the combined treatment regimen and was based on the proportion of dead implants (postimplantation loss only). Enhanced risk could be shown not only after high but also after low combined exposure to X-rays plus cyclophosphamide and X-rays plus mitomycin C. With low doses this enhanced risk was observed in spermatids for X-rays plus cyclophosphamide and in differentiating spermatogonia to early spermatocytes for X-rays plus mitomycin C.     

Use of the cytokinesis-block micronucleus method in mouse splenocytes

Mouse splenocytes have been used in the cytokinesis-block method for the evaluation of micronuclei induced by mutagenic agents in vitro as well as in vivo. Stimulation with concanavalin A for 48 h followed by 16-24-h treatment with 5 micrograms/ml cytochalasin B was found to be an optimum condition to obtain micronuclei in the binucleated splenocytes after the cells were cultured in vitro. Under the above conditions splenocytes from mice pretreated with a single i.p. injection of cyclophosphamide gave a significant increase in micronucleus production. This increase was dependent on the dose of cyclophosphamide (r = 0.99). A dose of 50 mg/kg resulted in 22% of the binucleated cells producing micronuclei, more than 20 times the level in the untreated control. The increase was also dependent on the time of cyclophosphamide injection before removal of the spleen. A duration of 4-8 h after cyclophosphamide injection gave rather sharp optimum values for the production of micronuclei. When splenocytes from non-treated mice were treated with mitomycin C together with cytochalasin B in the above in vitro condition, there was a significant increase in micronucleus production in the binucleated cells. It was also dependent on the dose of mitomycin C (r = 0.975) and a dose of 0.5 micrograms/ml resulted in a more than 20-fold increase over the untreated control. Thus, the use of mouse splenocytes in the cytokinesis-block micronucleus assay was shown to be sensitive enough for testing mutagenic agents in vivo as well as in vitro.     

Selective enhancement of bleomycin cytotoxicity by local anesthetics

The cytotoxic effect of the antitumor antibiotic bleomycin toward cultured mouse FM3A cells was greatly enhanced by exposure of the cells to local anesthetics either before or together with treatment with bleomycin. Such local anesthetics include dibucaine, tetracaine, butacaine, lidocaine and procaine. Dibucaine-induced cell sensitization to bleomycin cytotoxicity produced a decrease in cell survival that became dependent on dose and time of bleomycin treatment. This effect of local anesthetics seems to be selective to bleomycin, since dibucaine and lidocaine do not enhance the cytotoxic effect of other antitumor agents including adriamycin, mitomycin C and $\text{cis}$-diamminedichloroplatinum(II).     

Potentiating effects of caffeine on teratogenicity of alkylating agents in mice

Teratogenic to subteratogenic doses of x-ray, mitomycin C, MNNG, thio-TEPA, cyclophosphamide, and chlorambucil were administered to pregnant ICR mice together with caffeine at doses of 12.5, 25, or 50 mg/kg on day 11 of gestation. Fetuses were examined for gross malformations on day 18 of gestation. The teratogenicity of mitomycin C was significantly potentiated by caffeine at a dose as low as 12.5 mg/kg. The teratogenicity of chlorambucil was also significantly potentiated by caffeine at 50 mg/kg, but similar potentiation was not observed for x-ray, MNNG, thio-TEPA, and cyclophosphamide.     

Interactions of drugs and radiation in haemopoietic tissue assessed by lethality of mice after whole-body irradiation

Drug-radiation interactions in haemopoietic tissue were assessed as the lethality of mice within 7-28 days after whole-body irradiation. The investigated drugs were adriamycin (ADM), bleomycin (BLM), cyclophosphamide (CTX), 5-fluorouracil (5-FU), methotrexate (MTX), mitomycin C (MM-C) and cis-diamminedichloroplatinum II (cis-DDP). The drugs were administered as single doses 15 min before graded doses of whole-body irradiation or at different intervals from 7 days before to 7 days after fixed radiation doses. ADM, CTX, 5-FU, MM-C and cis-DDP enhanced the radiation response when administered 15 min before irradiation. The dose effect factor (DEF) was 9.11 for 5-FU and in the range 1.25-1.59 for the other drugs. MTX administration 15 min before irradiation had no effect (DEF 1.00). However, MTX increased lethality if given 1-3 days after irradiation (DEF 1.21-1.76) and protected against lethality if given 1-3 days before irradiation (DEF 0.83). A similar time dependence was observed for ADM, CTX, 5-FU, MM-C and cis-DDP. Protection against lethality was not observed but in all these cases the lethality was significantly lower at administration 1-3 days before than 1-3 days after irradiation. A proper investigation of the effect of BLM was not possible as the combination of this drug and whole-body irradiation caused a high rate of gastrointestinal deaths.     

Formation of micronuclei in erythrocytes of the fathead minnow (Pimephales promelas) after acute treatment with mitomycin C or cyclophosphamide

Despite the widespread use of the fathead minnow in ecotoxicology, there have been relatively few studies on genotoxicity biomarkers in this small, warm-water fish species. Consequently, we investigated the effect of two known genotoxins, mitomycin C and cyclophosphamide, on micronucleus induction in spleen and peripheral blood erythrocytes of this species. Initially, 96-h experiments after intra-peritoneal (i.p.) injections of mitomycin C and cyclophosphamide were undertaken to determine the maximum tolerated dose (MTD). From these studies, MTDs of 10 and 400 mg/kg, respectively, were obtained: doses that were higher than those reported for other fish species. Next, an assessment of micronucleus induction at 1, 2, 4, 8 and 14 days after injection was undertaken for each compound at the MTD. Mitomycin C at 10 mg/kg significantly induced micronuclei in erythrocytes from the spleen, but not from the peripheral blood, at 8 and 14 days. In addition, the overall levels of micronuclei observed were lower than most previously published data from other fish species. In contrast to mitomycin C, treatment with 400 mg/kg cyclophosphamide failed to significantly induce micronuclei in erythrocytes from any of the tissues employed, in contrast to previous reports of significant induction in other species. The reasons for the apparent relative insensitivity of the fathead minnow to these clastogens, with respect to both MTDs and micronucleus induction, are not clear. The fathead minnow, however, has previously been described as relatively insensitive compared to other fish species with respect to selected carcinogens and cytochrome P450 inducers; the latter suggesting that the lack of a significant induction following cyclophosphamide exposure may be due to low metabolic activation in vivo. Consequently, further clarifying work is required to delineate the response shown, considering the extensive use of this species in ecotoxicology research and regulatory testing.     

Resistance of the suppressor function of T-cells to agents possessing an antiproliferative action]

Administration of cyclophosphamide in a dose of 50 to 400 mg/kg to mice immunized with sheep red blood cells failed to decrease significantly the capacity of the splenic cells of these mice to suppress the primary immune response in transplantation to intact syngeneic recipients. Irradiation of the donors of immune splenic cells (ISC) in a dose of 900 r or treatment of ISC in vitro with mitomycin C failed to influence their suppressor activity. Supernatant obtained after the ultracentrifugation of ISC treated with ultrasound inhibited the primary immune response of intact mice. A conclusion was drawn that the suppressor effect of ISC was caused by the factor produced by T-cells. Active proliferation of these cells was not necessary for the realization of its action.     

The effect of chemotherapy on the in vivo frequency of glycophorin A 'null' variant erythrocytes

A human in vivo somatic cell assay based on the enumeration of variant erythrocytes lacking expression of an allelic form of the cell-surface sialoglycoprotein, glycophorin A, was applied to the study of blood samples from patients obtained prior to, during, and following chemotherapy for malignant disease in order to determine the effect of mutagenic chemical agents on the frequency of variant cells. In 22 patients assayed prior to therapy, the mean variant cell frequency was 11.9 per million, which was not significantly different from that observed in healthy controls. In an initial cross-sectional survey, blood samples were obtained at various times during and after therapy from 30 patients diagnosed with a variety of malignancies who were treated with one or more known mutagenic agents including adriamycin, bleomycin, cis-platinum, cyclophosphamide, dacarbazine, etoposide, lomustine, mechlorethamine, melphalan, mitomycin C, and procarbazine. Significant elevations in the mean frequency of variant cells over pre-therapy and normal levels were observed in samples obtained during and after therapy. In a time-series study, 14 breast cancer patients treated with CAF (cyclophosphamide, adriamycin, 5-fluorouracil), CMF (cyclophosphamide, methotrexate, 5-fluorouracil), or VMF (vinblastine, methotrexate, 5-fluorouracil) adjuvant chemotherapy were sampled repeatedly during and after therapy. For the CAF and CMF patients an increase in the frequency of variant cells was observed with a lag in the appearance of induced variants after initiation of therapy; variant frequencies gradually increased during therapy reaching a maximum at or shortly after the end of therapy, then declined to near pre-therapy levels within 6 months. The maximum level of induced variants ranged from 2- to 7-fold over pre-therapy or normal levels depending on the combination of agents used. The breast cancer patients treated with both adriamycin and cyclophosphamide showed consistent elevations in the frequency of variant cells; patients treated only with cyclophosphamide showed lower and more variable elevations. The data demonstrate that mutagenic chemotherapy agents induce elevated levels of glycophorin A variant erythrocytes consistent with the hypothesis that variant cells result from somatic mutation. The elevations in variant cells were transient, suggesting that these agents primarily affect the rapidly cycling committed erythroid cell population.     

The effect of chemotherapy on the in vivo frequency of glycophorin A ‘null’ variant erythrocytes

A human in vivo somatic cell assay based on the enumeration of variant erythrocytes lacking expression of an allelic form of the cell-surface sialoglycoprotein, glycophorin A, was applied to the study of blood samples from patients obtained prior to, during, and following chemotherapy for malignant disease in order to determine the effect of mutagenic chemical agents on the frequency of variant cells. In 22 patients assayed prior to therapy, the mean variant cell frequency was 11.9 per million, which was not significantly different from that observed in healthy controls. In an initial cross-sectional survey, blood samples were obtained at various times during and after therapy from 30 patients diagnosed with a variety of malignancies who were treated with one or more known mutagenic agents including adriamycin, bleomycin, cis-platinum, cyclophosphamide, dacarbazine, etoposide, lomustine, mechlorethamine, melphalan, mitomycin C, and procarbazine. Significant elevations in the mean frequency of variant cells over pre-therapy and normal levels were observed in samples obtained during and after therapy. In a time-series study, 14 breast cancer patients treated with CAF (cyclophosphamide, adriamycin, 5-fluorouracil), CMF (cyclophosphamide, methotrexate, 5-fluorouracil), or VMF (vinblastine, methotrexate, 5-fluorouracil) adjuvant chemotherapy were sampled repeatedly during and after therapy. For the CAF and CMF patients an increase in the frequency of variant cells was observed with a lag in the appearance of induced variants after initiation of therapy; variant frequencies gradually increased during therapy reaching a maximum at or shortly after the end of therapy, then declined to near pre-therapy levels within 6 months. The maximum level of induced variants ranged from 2- to 7-fold over pre-therapy or normal levels depending on the combination of agents used. The breast cancer patients treated with both adriamycin     

Synthesis of recA protein and induction of bacteriophage lambda in single-strand deoxyribonucleic acid-binding protein mutants of Escherichia coli.

We investigated the capacity of Escherichia coli mutants defective in the single-strand deoxyribonucleic acid (DNA)-binding protein to amplify the synthesis of the recA protein, induce prophage lambda, and degrade their DNA after treatment with ultraviolet radiation, mitomycin C, or bleomycin. The thermosensitive ssbA1 strain induced recA protein and lambda phage normally at 30 degrees C, but no induction was observed at 42 degrees C when ultraviolet radiation or mitomycin C was used. The lexC113 mutant did not amplify recA protein synthesis or induce phage lambda at either 30 or 42 degrees C with those agents. Bleomycin was able to elicit induction of recA and phage lambda in both mutants at any temperature. After induction with ultraviolet radiation at the elevated temperature, no DNA degradation was observed for 40 min, but at later times there was increased degradation in the lexC113 strain, compared with the wild type, and even greater degradation in the ssbA1 mutant. We discuss the role of single-strand DNA-binding protein in induction and the possibility that the lexC product may exert its influence on recA and lambda induction at the level of the single-strand DNA gap.     

A comparison between mouse and fish micronucleus test using cyclophosphamide,mitomycin C and various pesticides

A comparative analysis between mouse and fish erythrocyte micronuclei (MN) assays was carried out using cyclophosphamide, mitomycin C and various pesticides such as alliete, brestanid, decis 25 CE (deltamethrin), kelthane 480 CE (dicofol), roundup (glyphosate), imazapyr and thiram. The aim of this study was to evaluate the fish species Tilapia rendalli as a suitable organism for the detection of genotoxicants in water. The clastogens cyclophosphamide and mitomycin C induced MN in both test-systems. Insecticides: decis 25 CE increased T. rendalli MN frequencies at doses of 1.0 and 5.0mg/kg, but not at the highest dose, and in mice there was no MN induction. Kelthane 480 CE also induced a significant MN frequency in T. rendalli, but not in mice. Fungicides: alliete and brestanid induced MN only in T. rendalli, while thiram was negative in both assays. Herbicides: imazapyr induced MN in T. rendalli at the maximum tolerated dose only, while roundup induced MN at three dosed levels. In mice both herbicides presented negative results. This study revealed that fish MN assay can be used as a genotoxicological test-system since some methodological particularities were observed.     

Comparison of DNA Strand Break Induction in CHO Cells Measured by Alkaline Elution and by Fluorometric Analysis of DNA Unwinding (FADU)

DNA damage in X-irradiated CHO cells was measured by alkaline filter elution and compared to fluorometric analysis of DNA unwinding (FADU). The FADU method proved to be as sensitive as the alkaline filter elution technique in detecting X-ray induced DNA breaks. Strand break induction was also measured after treatment with four radical generating chemicals (hydrogen peroxide, bleomycin, mitomycin C and methyl viologen) using the FADU technique.     

Comparison of DNA Strand Break Induction in CHO Cells Measured by Alkaline Elution and by Fluorometric Analysis of DNA Unwinding (FADU)

DNA damage in X-irradiated CHO cells was measured by alkaline filter elution and compared to fluorometric analysis of DNA unwinding (FADU). The FADU method proved to be as sensitive as the alkaline filter elution technique in detecting X-ray induced DNA breaks. Strand break induction was also measured after treatment with four radical generating chemicals (hydrogen peroxide, bleomycin, mitomycin C and methyl viologen) using the FADU technique.     

Genetic analysis of mitomycin C-induced interchange in Drosophila melanogaster females

A genetic analysis of an array of mitomycin-induced rearrangements in immature Drosophila oocytes is reported. Induced aberrations were recovered representing detachments of the compound-X chromosome, Y chromosome fragments, X chromosome loss and mosaicism. The spectrum of rearrangements induced by mitomycin C was very similar to that induced by X-ray treatment of immature oocytes. This work suggests that mitomycin C has two modes of action. The drug is radiomimetic for it induces the types of aberrations recovered after X-irradiation. Mitomycin C also seems to have a delayed effect which is reflected in the relatively high recovery of mosaics.     

Expression of the SOS system in Escherichia coli growing under nitrate respiration conditions

Induction of several SOS functions by mitomycin C, bleomycin or thermal treatment of a recA441 mutant growing under nitrate respiration conditions was studied in Escherichia coli. Mitomycin C caused inhibition of cell division, induction of prophages and expression of umuC gene but like in aerobically growing cells, it did not trigger the cessation of cell repiration. On the contrary, both recA⁺ and recA441 cultures either treated with bleomycin or incubated at 42°C failed to induce any of the different SOS functions cited above.Furthermore, after bleomycin addition or thermal treatment both recA⁺ and recA441 cultures did not present any variation in the cellular ATP level, contrary to what happens under aerobic growth. The blocking of the expression of some SOS functions under nitrate respiration conditions is not an irreversible process because cells incubated under these anaerobic conditions were able to induce the SOS system when changed to an aerobic medium 30 min after the SOS-inducing treatment had been applied.     

Influence of metabolic factors on the mutagenic effectiveness of cyclophosphamide in Drosophila melanogaster

This paper describes the influence of changes in metabolic activity on the in-vivo mutagenic effectiveness of cyclophosphamide in Drosophila melanogaster. A dose-dependent increase in mutagenicity was observed until a plateau value is reached which was increased only slightly after enzyme induction with Aroclor 1254, whereas induction with phenobarbital resulted in a decrease, especially when cyclophosphamide was applied by injection. Treatment of the adult males with inhibitors of the monoamine oxidase (MAO, EC 1.4.3.4), such as iproniazid (Ipr), benzimidazole or tryptamine, led to a marked increase of the mutagenic effectiveness of cyclophosphamide especially in spermatocytes. This indicates the importance of metabolic de-activation processes for the limited mutagenicity of cyclophosphamide in Drosophila. The principal active metabolite of cyclophosphamide, phosphoramide mustard, is extensively de-activated by enzymes that can be inhibited by 1-phenylimidazole (PhI), presumably cytochrome P-450 (EC 1.14.14.1), but not by those blocked by MAO inhibitors. Inhibition of the FAD-containing dimethylaniline monooxygenase (FDMAM, EC 1.14.13.8) by N,N-dimethylbenzylamine (N,N-DMB) resulted in some increase in cyclophosphamide mutagenicity only in spermatids. The marginal mutagenicity of cyclophosphamide in Drosophila larvae could not be increased either by cytochrome P-450 induction with phenobarbital or by MAO inhibition with Ipr. In contrast to the failure of cyclophosphamide to induce rod-chromosome loss, a considerable activity was found when a ring-shaped chromosome was used. Similar to the sex-linked recessive lethal (SLRL) test, ring-X loss frequency could be enhanced by simultaneous treatment with MAO inhibitors. The observed ring-X loss frequency declined when males treated with cyclophosphamide were mated to DNA-repair deficient mei-9L1 females. Cyclophosphamide produces chromosome breaks, detected as 2-3 translocations, in Drosophila spermatocytes, the stage in spermatogenesis that is also the most sensitive to the induction of SLRL mutations.     

The Intercellular Distribution of Mutations Induced in Oocytes of DROSOPHILA MELANOGASTER by Chemical and Physical Mutagens

When females of Drosophila melanogaster are treated with chemical or physical mutagens, not only in one but also in both of the two homologous X chromosomes of a given oocyte, a recessive sex-linked lethal mutation may be induced. A method is described that discriminates between such "single" and "double mutations". A theory is developed to show how a comparison between the expected and the observed frequency of double mutations yields an indication of the intercellular distribution (random or nonrandom) of recessive lethal mutations induced by mutagenic agents in oocytes and, consequently, of the distribution (homogeneous or nonhomogeneous) of those agents.--Three agents were tested: FUdR (12.5, 50.5 and 81.0 micrograms/ml), mitomycin C (130.0 micrograms/ml) and X rays (2000 R, 150 kV). After FUdR feeding, no increase in the mutation frequency usually observed in D. melanogaster without mutagenic treatment was obtained (u = 0.13%, namely three single mutations among 2332 chromosomes tested). After mitomycin C feeding, 104 single and three double mutations were obtained. All of the 50 mutations observed after X irradiation were single mutations. The results obtained in the mitomycin C and radiation experiments favor the assumption of a random intercellular distribution of recessive lethal mutations induced by these two agents in oocytes of D. melanogaster. Reasons are discussed why for other types of mutagenic agents nonrandom distributions may be observed with our technique.     

Investigations into sister chromatid exchange in patients under cytostatic therapy

Summary In vivo sister chromatid exchange (SCE) determinations were carried out in patients with Psoriasis vulgaris under methotrexate therapy and patients with histologically verified bronchial carcinoma under cyclophosphamide, methotrexate or a combined chemotherapy. A pronounced increase in SCE rate was only found after cyclophosphamide treatment.Abbreviation Cyclo cyclophosphamide - MTX methotrexate - FU fluorouracil - Pred prednisolone - VB vinblastine - VC vincristine - BM bleomycin     

Effect of 5-bromodeoxyuridine substitution on sister chromatid exchange induction by chemicals

The fluorescence-plus-Giemsa (FPG) technique for analysis of sister chromatid exchange (SCE) is widely used as an assay for mutagenic carcinogens. There is very little information, however, on whether incorporation of the bromodeoxyuridine (BrdU) necessary for visualization of SCEs affects the sensitivity of the SCE test system to different chemical agents. We have investigated the effect of BrdU incorporation on SCE induction by labeling cells with BrdU for either the first cell cycle or the first and second cell cycles. The cells were then treated with bleomycin, which produces DNA strand breakage; proflavine, which intercalates into DNA; mitomycin C, which produces monoadducts and DNA crosslinks; or aphidicolin, which inhibits DNA polymerase . Chemicals were added before BrdU exposure or during the first, second, or both cell cycles. Only mitomycin C, which induces long-lived lesions, elevated the SCE frequency when cells were treated before BrdU labeling. When bleomycin, proflavine, or mitomycin C was present concurrently with BrdU, the frequency of SCEs was increased independently of the BrdU labeling protocol. Aphidicolin, on the other hand, induced more SCEs when present for the second cell cycle, when DNA replicates on a template DNA strand containing BrdU. We also examined the induction of SCEs in the first cell cycle (twins) and in the second cell cycle (singles) after continuous treatment of cells with BrdU and the test chemicals. Only aphidicolin increased SCE frequency in the second cell cycle. These results indicate that aphidicolin, but not bleomycin, proflavine, or mitomycin C, affects BrdU-substituted DNA and unsubstituted DNA differently. This type of interaction should be taken into consideration when the SCE test is used as an assay system.