Genetic diversity of mitomycin C-hypersensitive Chinese hamster cell mutants: a new complementation group with chromosomal instability.

A Chinese hamster cell mutant (V-C8) isolated previously, which is approximately 100 fold more sensitive to mitomycin C (MMC) than its parental wild-type V79 cells (judged by D10 values), was further characterized. V-C8 cells exhibit an increased sensitivity towards other cross-linking agents, such as cis-DDP (approximately 40-fold), DEB (approximately 30-fold), and also to adriamycin (approximately 5-fold), and the monofunctional alkylating agents: MMS (approximately 5-fold) and EMS (approximately 6-fold). V-C8 cells show a higher level induction of chromosomal aberrations by cross-linking agents (MMC, cis-DDP, and DEB) and an increased level of spontaneous chromosomal aberrations in comparison to the wild-type V79 cells. To determine whether the V-C8 mutant represents a new complementation group among Chinese hamster cell mutants that also display the extreme sensitivity to MMC, V-C8 cells were fused with irs1, irs1SF, UV20, UV41, and V-H4 cells. In all cases, the derived hybrids regained the MMC sensitivity similar to wild-type cells, indicating that the V-C8 mutant belongs to a new sixth complementation group.

A Chinese hamster ovary cell mutant (EM-C11) with sensitivity to simple alkylating agents and a very high level of sister chromatid exchanges.

We have isolated a Chinese hamster ovary cell mutant hypersensitive to monofunctional alkylating agents. The mutant, designed as EM-C11, showed hypersensitivity to ethyl methanesulfonate (EMS), methyl methanesulfonate and ethylnitrosourea (8-, 7- and 2-fold, respectively, based on D10 values). About 2-fold increased sensitivity towards 4-nitroquinoline-1-oxide and only slightly increased sensitivity to X-rays (1.4-fold) and mitomycin C treatment (1.6-fold) were found in this mutant. EM-C11 was not hypersensitive to UV irradiation nor to adriamycin. The EM-C11 cells showed approximately 10-fold higher level of spontaneous sister chromatid exchange. The level of spontaneous chromosomal aberrations was 2- to 3-fold higher, but the frequency of EMS-induced chromosomal aberrations was approximately 10-fold higher in the mutant cells, in agreement with the increased sensitivity to killing. As measured by alkaline elution, EM-C11 cells showed a defect in the rejoining of single-strand DNA breaks after exposure to X-rays and even more so after the EMS treatment. Genetic analysis revealed that the EM-C11 mutant belongs to the same complementation group as the EM9 mutant described earlier. The XRCC1 gene which complements the defect in EM9 also complements the defect in EM-C11, confirming that these two independently isolated mutants are defective in the same gene.

The Chinese hamster V79 cell mutant V-H4 is phenotypically like Fanconi anemia cells.

It has been shown by genetic complementation analysis that a mitomycin C-sensitive mutant (V-H4) of Chinese hamster V79 cells is the first rodent equivalent of Fanconi anemia (FA) group A. The V-H4 mutant shows many typical characteristics of cells derived from FA patients. V-H4 cells exhibit increased sensitivity towards cross-linking agents as MMC (approximately 30-fold), cis-DDP (approximately 10-fold), DEB (approximately 10-fold), and PUVA (approximately 1.6-fold), but an only slightly increased sensitivity to monofunctional alkylating agents (EMS and MMS) and actinomycin D. V-H4 cells are also moderately sensitive to adriamycin (1.6-fold), and not sensitive to H2O2. The levels of chromosomal aberrations induced by MMC and cis-DDP treatment are higher (4- to 6-fold) in V-H4 cells than in the wild-type V79 cells. Genetic complementation analysis with other Chinese hamster mutants hypersensitive to MMC (irs1, irs1SF, UV20 and UV41) indicates clearly that V-H4 belongs to a different, new complementation group. This unique mutant is very stable and can serve as a vehicle to isolate the complementing FA-A gene from normal human DNA.

Persistence of preclastogenic damage in hepatocytes of rats exposed to ethylnitrosourea, diethylnitrosamine, dimethylnitrosamine and methyl methanesulphonate. Correlation with DNA O-alkylation.

Male Wistar rats received a single injection of ethylnitrosourea (ENU; 140 mg/kg), dimethylnitrosamine (DMN; 10 mg/kg) or methyl methanesulphonate (MMS; 80 mg/kg). After 1, 6, 28 and 56 days liver cells were assayed for the presence of preclastogenic lesions. This was done by analysis of micronuclei in hepatocytes isolated at 2, 3 and 4 days after partial hepatectomy. Treatment with MMS did not give rise to a statistically significant increased micronucleus frequency after the two time intervals tested (1 and 6 days). In contrast, frequencies of micronuclei were significantly enhanced at all time intervals after treatment with either ENU or DMN. These results support our previous conclusion that only those alkylating agents which give rise to substantial DNA O-alkylation are able to induce long-lived preclastogenic damage in rat liver cells. Both after ENU and DMN treatment, frequencies of micronuclei were significantly higher at day 6 than at days 1, 28 and 56. It is postulated that part of the primary preclastogenic lesions (supposed to be DNA adducts) are converted into secondary preclastogenic lesions during the first period after exposure to the chemicals. Apparently, both primary and secondary preclastogenic lesions are gradually lost after day 6. This loss is rather low (less than 25%) after ENU, but more impressive after DMN (approximately 60%). The persistent nature of preclastogenic damage induced by diethylnitrosamine was demonstrated in an experiment which showed that a single dose of this agent (50 mg/kg) had the same clastogenic effect as a fractionated dose (five weekly injections of 10 mg/kg). This result also implies that a threshold dose for induction of micronuclei, if present, must be very low, appreciably below 10 mg/kg.

Changes in erythropoiesis due to radiation or chemotherapy as studied by flow cytometric determination of peripheral blood reticulocytes.

Flow cytometric determination of time dependent changes of numbers of reticulocytes in peripheral blood were investigated as a parameter for changes in erythropoiesis induced by radiation- or chemotherapy. Rats irradiated or treated with drugs (such as e.g. cyclophosphamide 100 mg/kg, vincristin 0.2 mg/kg, or mitomycin C 1.0 mg/kg) showed clear changes in erythropoietic activity. Reticulocyte numbers decreased rapidly until day 3-4 after treatment; this period was followed by a gradual increase and normal control values were seen at day 8-11. Radiation effects of doses as low 0.5 Gy could be detected in such a way. Similar studies were performed with patients with ovarian tumors treated with cis-platinum, a drug that may cause non-immune haemolysis. During prolonged treatment some patients showed increasing numbers of reticulocytes, measured at the first day of each hospitalization period, whereas leucocyte and platelet counts stayed more or less constant. Increasing numbers of reticulocytes generally indicates stimulation of erythropoietic activity of the bone marrow (due to increased blood loss); in this study increasing numbers often preceeded a decrease in hemoglobin values later on. Flow cytometric analysis of reticulocytes is therefore a potentially useful tool to detect changes in erythropoiesis, and considered more sensitive for the early recognition of patients that develop anemia, than hemoglobin measurements only.

A micronucleus method for detection of meiotic micronuclei in male germ cells of mammals.

A new rapid micronucleus method is presented for the detection of chromosomal damage induced in spermatocyte stages of mammals. Analysis of micronuclei is done in early spermatids that have been isolated from testis tubules in a special testis isolation medium supplemented with enzymes (collagenase, trypsin and DNAase).

The induction of chromosomal damage in rat hepatocytes and lymphocytes. I. Time-dependent changes of the clastogenic effects of diethylnitrosamine, dimethylnitrosamine and ethyl methanesulfonate.

Male Wistar rats received a single injection of diethylnitrosamine (DEN), dimethylnitrosamine (DMN) or ethyl methanesulfonate (EMS). After a number of time intervals (up to 56 days) liver cells were assayed for the presence of possible preclastogenic damage by performing partial hepatectomy and subsequent analysis of chromosomal damage (micronucleus formation) in isolated hepatocytes. Peripheral blood lymphocytes from the same animals were collected, stimulated to proliferate and assayed for the frequency of sister-chromatid exchanges (SCEs). Whereas all agents significantly increased frequencies of SCEs in lymphocytes up to at least 28 days (EMS) or 56 days (DMN, DEN) after injection, only the latter 2 compounds gave rise to significantly increased incidences of micronucleated hepatocytes. DMN-induced preclastogenic damage in hepatocytes was lost between 28 and 56 days after injection. After DEN, this type of damage was persistent over the entire experimental period (56 days). When rats treated with DEN did not undergo partial hepatectomy, the frequencies of micronuclei at different time intervals after treatment were at control level. This result, together with those from hepatectomized DEN-treated rats, suggests that it is the persistent character of the preclastogenic damage that is responsible for the occurrence of micronucleated hepatocytes at later time intervals after treatment with DEN, rather than the stability of micronuclei which might eventually have been formed soon after injection.

Differential persistence of chromosomal damage induced in resting rat-liver cells by X-rays and 4.2-MeV neutrons.

Rats were exposed to 400 rad X-rays or 100 rad 4.2-MeV neutrons and subjected to partial hepatectomy at 1 day, 1 week, 1 month or 3 months after irradiation. 2 or 3 days later the remaining liver was perfused and isolated hepatocytes were screened for micronuclei in the cytoplasm. Frequencies of micronucleated hepatocytes following X-irradiation decreased with increasing time intervals after irradiation, the frequency at 3 months being not significantly different from the control value. Following neutron irradiation, frequencies of micronuclei remained constant at all time intervals tested. In the case of X-irradiation, the progressive decrease in frequencies of micronuclei may be explained by 2 mechanisms operating simultaneously: (i) slow long-term repair of chromosomal damage and (ii) mitotic selection against cells carrying substantial amounts of chromosomal damage. Both mechanisms do not seem to operate in the case of neutron irradiation. For both types of radiation, the dose-effect relationship was linear in the ascending part of the dose-effect curves. The dose-effect curve plateaud at 400 rad X-rays and 50 rad neutrons. The RBE value for 4.2 MeV neutrons in the ascending part of the dose-effect curves was about 4.

A micronucleus technique for detecting clastogenic effects of mutagens/carcinogens (DEN, DMN) in hepatocytes of rat liver in vivo.

A micronucleus assay in vivo has been developed that utilizes freshly isolated hepatocytes from livers of hepatectomized rats. In a small validation study, increased frequencies of micronuclei were detected in rats exposed to DEN and DMN before or after hepatectomy. The method is suitable for the detection of clastogenic effects of compounds or their metabolites that are too short-lived to reach the classical target cells used in cytogenetic studies. Because the non-hepatectomized liver shows very low levels of mitosis, the liver-micronucleus assay can also be used for the study of storage effects and the clastogenic effects of chronic exposures to mutagens/carcinogens.