Potentiation of BCNU-induced cytotoxicity and sister chromatid exchanges by dibromodulcitol in vitro

Dibromodulcitol (DBD) is an anticancer agent that is cytotoxic against animal and human brain tumors in vivo. Clinical trials of combination therapy with radiation, DBD, and a nitrosourea have shown some efficacy, but the mechanisms that lead to enhanced cytotoxicity are poorly understood. We investigated the effects of pretreatment with DBD on cell survival and sister chromatid exchange (SCE) caused by subsequent treatment with 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) in 9L rat brain tumor cells. Pretreatment of 9L cells for 24 hr with 5 microM DBD potentiated the cell kill produced by a 1-hr treatment with BCNU; the dose enhancement ratio was 1.7 at the 10% survival level. Treatment of 9L cells for 24 hr with 1 microM DBD induced 39 +/- 12 SCEs/metaphase. There was a 50-75% increase in BCNU-induced SCEs, compared with BCNU alone, after a 24 hr pretreatment with DBD. Thus DBD potentiation of BCNU cytotoxicity appears to be related to increased DNA damage.     

Sister chromatid exchanges and chromosome aberrations induced by radiosensitizing agents in bone marrow cells of treated tumor-bearing mice

The frequency of sister chromatid exchanges (SCE) in vivo and chromosome aberrations and/or alterations were analyzed from the bone marrow cells of the treated dbrB tumor-bearing DBA/1J inbred mouse host. The results were compared with analogous data obtained from the bone marrow cells of untreated tumor-bearing mice for evaluation of the "indirect," i.e., somatic stress, effect on the normal host cells following triple-agent therapy intended for a mammary adenocarcinoma. Misonidazole (MIS), which is a known radiosensitizing drug, microwave hyperthermia (delta), and X-radiation (X) were used as therapeutic agents. Significant (P less than 0.05) numbers of SCE were induced in the bone marrow cells of the mice whose tumors received these triple-agent treatments (MIS + delta + X) simultaneously as compared with values of SCE per cell noted in bone marrow cells of untreated tumor-bearing control mice. The highest number of chromosome aberrations and alterations, including an increase in heteroploidy, was also noticed in the bone marrow cells of the mice whose tumors were treated simultaneously with MIS + delta + X. The triple-agent therapy on dbrB tumor also resulted in an unusually high polyploid metaphase plate in the bone marrow cell consisting of 320 chromosomes, indicating that this mode of therapy may act directly on the genetic material of the tumor-bearing host cells, inducing cytogenetic abnormalities as a side effect.     

Sister chromatid exchanges in Chinese hamster V79 cells treated with the trivalent chromium compounds chromic chloride and chromic oxide

The induction of sister chromatid exchanges (SCEs) in Chinesehamster V79 cells exposed to soluble CrCl₃ and insoluble Cr₂O₃,compounds of trivalent chromium (Cr³⁺), was determined. Theirability to induce SCEs was compared with those of three hexavalentchromium (Cr⁶⁺) compounds: K₂CrO₄, Na₂CrO₄ and Na₂Cr₂O₇. Boththe delay in progression through the cell cycle induced by Cr³⁺compounds and the SCE frequencies in the delayed cells werealso evaluated. The exposure for 28 h to CrCl₃ and Cr₂O₃ atconcentrations of 9.7–39 µg and of 34–136fig of Cr³⁺ per ml, respectively, induced a statistically significant(p < 0.001) dose-dependent increase in SCEs up to 1.9–fold(CrCl₃ and 4-fold (Cr₂Cl₃) over control levels. Compared withthe effective concentrations of Cr⁶⁺ compounds, which producedup to 4-fold increase of SCEs, inducing concentrations of CrCl₃and Cr₂O₃ were 300- and 1000-fold higher in terms of chromium.By prolongation of treatment time up to 48 h, a progressivedose- and time-related enhancement in SCE frequencies inducedby Cr³⁺ compounds in delayed cells was observed. Lower concentrationsof Cr₂O₃, without effect after 28 h of treatment, induced anincrease of SCEs by prolongation of exposure time.     

Sister chromatid exchanges induced in human lymphocytes by cis-diamminedichloroplatinum (II)

Sister chromatid exchanges (SCE) frequency in cultured lymphocytesfrom seven patients with lung cancer receiving cis-Diamminedichloroplatinum(II) (Pt(II)) for chemotherapy was studied. Significantly increasedSCE frequency was observed in all the patients, six of whomreceived intravenous infusion of Pt(II) and one of whom receivedthe agent as an intrapleural infusion. In addition, a dose-dependentincrease in the frequency of SCE was observed in lymphocytesfrom healthy subjects upon exposure to Pt(II) in vitro. The results of the present study revealed that Pt(1l) is a potentinducer of SCE in human lymphocytes in vivo and in vitro. Itthus appears that follow-up study of the long-term survivorswho have been treated with this agent for cancer is necessarybecause of the possible occurrence of secondary neoplasms.     

Sister chromatid exchanges and chromosomes in chronic myelogenous leukemia and cancer families

Spontaneouas sister chromatid exchanges and banded karyotypes were studied in blood lymphocytes from 96 individuals: seven patients with chronic myelogenous leukemia, 15 normal controls, and five “cancer families” comprising 12 cancer patients, 40 tumor-free blood relatives and 22 spouses. The families had: malignant melanoma; Epstein-Barr virus-associated malignancies and a birth defect syndrome; non-Hodgkin lymphoma and diverse carcinomas; Hodgkin's lymphoma and adenocarcinomas; and acute myelogenous leukemia. In addition to the Philadelphia chromosome in chronic myelogenous leukemia patients, karyotypic abnormalities, especially breaks and fragments, were found in 29% of cancer family members, but were inconsistent and usually attributable to radiotherapy. Mean sister chromatid exchange values were normal in chronic myelogenous leukemia, but low (by t-test) in tumor patients and their blood relatives in cancer-prone families. In tumor patients, mean sister chromatid exchange levels fell as age increased. After adjusting for this age effect, no significant differences remained among groups. In patients at high risk of cancer (because they have chronic myelogenous leukemia or a strong family history of cancer), spontaneous sister chromatid exchange rates were not a marker of cancer risk.     

Sister chromatid exchanges and chromosomal aberrations in lymphocytes of nurses handling cytostatic agents

A cohort study of 29 nurses who constantly handled cytostatic drugs, and 29 controls matched according to sex and age, was carried out between 1983 and 1986. Cytogenetic damage was assessed by sister chromatid exchanges (SCE) and chromosomal aberrations. No significant increase in mean number of SCE was found for nurses (7.37) as compared to matched controls (7.00), whereas a significant excess of SCE (p less than 0.001) was observed for smokers (8.23) as compared to non-smokers (6.75). The number of SCE was studied in relation to the amount and nature of cytostatics handled as well as to the duration of exposure. A significant association (p less than 0.05) was found between individual mean number of SCE and the total number of drugs handled after adjustment for confounding factors. In contrast, the number of SCE was not significantly related to the nature of drugs handled or to the duration of exposure. With regard to chromosomal damage, no significant difference was observed between nurses and controls in gap, break, dicentric and translocation frequencies.     

Sister chromatid exchanges and chromosome aberrations in lymphocytes of nurses handling antineoplastic drugs.

Chromosomal aberrations (CA) and sister-chromatid exchanges (SCE) were investigated in peripheral lymphocytes of 15 nurses and nurse's aides handling cytostatic agents in hospital oncology units. Significantly increased frequencies were noted for both CA and SCE rates when the exposed individuals were compared with 15 nurses working in other hospital units and to a control sample matched by sex and age. This points to the need for emphasizing protective measures in the handling of anti-neoplastic agents.     

Sister chromatid exchange (SCE) frequency in lymphocytes of patients with colorectal carcinoma treated with razoxane.

The increased risk of neoplasia following cytotoxic therapy for both malignant and nonmalignant disease is well known. Sister chromatid exchange (SCE) frequencies in patients receiving such chemotherapy are often elevated, and the persistence of high levels after treatment may provide an indicator for susceptibility to secondary neoplasia. The cytostatic drug razoxane has been used for the treatment of psoriasis, acute myeloid leukemia (AML), and colorectal carcinoma. Prolonged use of this drug, however, has been associated with the subsequent development of AML and, up to November 1987, 16 cases of acute leukemia following razoxane treatment have been reported. We report the SCE frequencies for 34 patients with colorectal carcinoma who were receiving or had previously been treated with razoxane. Our results show no significant increase in SCE levels in the razoxane group compared with either normal controls or untreated patients.     

Sister chromatid exchanges and chromosomal aberrations in mouse cells infected with the Abelson and Moloney leukemia viruses

‘Spontaneous’ and mitomycin C (MMC)-induced sisterchromatid exchanges (SCE) and chromatid breaks were scored inANN-1 fibroblasts, a non-producer mouse cell line transformedby the Abelson murine leukemia virus (AMuLV), a replicationdefective retrovirus whose genome contains the v-abl oncogene.Normal, non-transformed NIH3T3 fibroblasts were used as control.SCE and chromatid break frequencies in untreated or MMC-treatedANN-1 and NIH3T3 cells were compared with those observed inthe same cells after infection with the helper murine Moloneyleukemia virus (M-MuLV), which rescues the ability of A-MuLVto replicate in ANN-1 cells. The frequency of spontaneous andMMC-induced SCE were not significantly different in both ANN-1and NIH3T3 cells, independently of M-MuLV infection. After M-MuLVinfection, however, increased ‘spontaneous’ frequencyof SCE and altered susceptibilty to the induction of SCE byMMC was observed in both cell lines compared to M-MuLV-uninfectedcells. In the case of chromatid breaks, the baseline frequencywas not significantly different between the two cell lines bothin the presence or in the absence of M-MuLV infection, nor wasit significantly increased by M-MuLV, with respect to the valueobserved in uninfected cells. These results indicate that, atvariance with what occurs with SCE, viral replication is notneeded to increase the frequency of chromosomal aberrationsand that the portion of A-MuLV genome alone is sufficient to increase chromatid breaks but not SCE in ANN-1 cells. Thus,in mouse cells carrying retroviruses, SCE and chromosomal aberrationsseem to be independently generated, and influenced by differentviral genes.     

Sister chromatid exchanges induced by DNA demethylating agents persist through several cell cycles in mammalian cells

Eukaryotic DNA methylation has been extensively studied in recent years. The ability of many carcinogens to interfere with DNA methylation has not yet been directly related to their tumorigenic activity. Recent data obtained using L-ethionine and 5-azacytidine--both demethylating agents--showed a small but significant increase in the sister chromatid exchange (SCE) rate induced in mammalian cells (human lymphocytes and CHO cells). In this paper we show that the SCE increase induced by both these agents in Chinese hamster ovary (CHO) cells persists for as long as 10 cell cycles. On the other hand mitomycin-C and u.v. light-induced SCEs show a rapid decrease to the control value, as reported for all known SCE inducers. We suggest that DNA demethylation and SCEs are connected through a perturbation of the cell machinery at the level of the replication fork, producing an increase of the error-prone ligation. Since the methylation level is maintained (inherited), the SCE increase produced by these recombinational events will not be corrected through several cell cycles.     

Cytotoxicity and sister chromatid exchanges in 9L cells treated with monofunctional and bifunctional nitrogen mustards

To investigate the role of DNA interstrand crosslink formationon cytotoxicity and the induction of sister chromatid exchanges(SCEs), we treated 9L cells with the bifunctional mustard bis(2-chloroethyl)methylamine(HN2), which can alkylate DNA and form DNA interstrand crosslinks,or with two monofunctional mustards, bis(ethyl)-2-chloroethylamineand bis(methyl)-2-chloroethylamine, which can only alkylateDNA. On a molar basis, HN2 was 900—2400 times more cyto-toxicand 471—686 times more effective at inducing SCEs thanthe monofunctional mustards. HN2 induced high levels of DNAinterstrand crosslinks in 9L cells; no interstrand crosslinkswere detected in cells treated with the monofunctional mustards.Comparison of the alkylating activity of each of the mustardsby 4-(p-nitrobenzyl)pyridine reactivity showed only a 4-folddifference, which is not sufficient to account for the largedifferences in cell survival and induction of SCEs. We concludethat the effectiveness of HN2 at inducing cytotoxicity and SCEsresults from the formation of DNA interstrand crosslinks.     

Sister chromatid exchange frequency in asbestos workers

In vitro cytogenetic studies of amosite, chrysotile, and crocidolite asbestos have shown that these fibers may induce chromosome abnormalities and an elevated sister chromatid exchange (SCE) rate in mammalian cells. Twenty-five asbestos insulators (6 with radiographic asbestosis) were compared to 14 controls frequency matched for age and were found to have a marginally increased SCE rate in circulating lymphocytes with increasing years of exposure (P= 0.057). There was a significant association between SCE rate and smoking (P=0.002) after controlling for years of asbestos exposure and age. Smoking asbestos insulators had the highest SCE rate. Sister chromatid exchanges in chromosomes of group A, i.e., the group with the longest chromosomes, were significantly associated with asbestos exposure and cigarette smoking, with an interaction between the two.     

Sister chromatid exchange frequency in Hodgkin's disease patients with elevated in vivo hprt mutant frequencies

The frequency of sister chromatid exchanges (SCEs) was determinedin Hodgkin's disease (HD) patients prior to therapy, followingradiotherapy, and following combined radiotherapy and chemotherapy.The frequency of hprt^{macron} mutants in these patients hasbeen reported previously. The frequency of SCEs and hprt^{macron}mutants in the same individuals were compared. In non-HD controlsthe mean SCE frequency and the mean of high SCE frequency cells(HFCs) were significantly increased by smoking, while mutantfrequency (MF) showed no effect. Untreated HD patients had meanSCEs, mean HFCs and mean MFs that were higher than non-HD controls.In treated patients, mean SCE and HFC frequencies were lowerthan untreated patients and non-HD controls, while their MFswere significantly elevated. Overall, SCE frequency was notcorrelated with MF in control or HD patient groups, suggestingthat these biomarkers may reflect, in this case, fundamentalbiological differences between these processes.     

Increased frequency of sister chromatid exchanges in lymphocytes of breast cancer patients

Spontaneous and mitomycin-C (MMC)-induced rates of sister chromatid exchanges (SCEs) were studied in peripheral blood lymphocytes (PBLs) of 40 patients with cancer of the breast and 40 healthy female volunteers as controls. Spontaneous SCE per cell value in PBL cultures was 7.72 for the breast cancer patients, which was significantly higher (p less than 0.001) than the 6.28 SCEs per cell scored for the controls. Eight of the patients were studied a second time, 3 to 6 months following surgical removal of the tumour. A significant (p less than 0.05) reduction in SCE per cell value was observed following mastectomy. MMC-induced frequencies of SCE remained comparable in patients and controls. Cellular kinetics, calculated as average generation time (AGT) from the frequency of cells in M1, M2 and M3 cycles, were comparable in patients and controls.     

Sister chromatid exchanges induced in vivo and in vitro by chemical carcinogens in mouse lymphocytes carrying endogenized Moloney leukemia virus

The induction of sister chromatid exchanges (SCEs) was analysedin mouse spleen T lymphocytes treated in vivo or in vitro withchemical carcinogens. Lymphocytes were obtained from BALB/Momice, which carry the Moloney murine leukemia virus (M-MuLV)as endogenous type C retrovirus, and from control BALB/c mice(M-MuLV free). SCEs were determined in vitro on lymphocytesstimulated with con-canavalin A (Con A) and incubated for twogeneration cycles with bromodeoxyuridine (BUdR). The baselinefrequency of SCEs in untreated lymphocytes obtained from 2–3month old and 4 day old BALB/Mo mice was significantly higherwhen compared with that observed in control BALB/c lymphocytes.Treatments in vitro with 10^–6 M hexavalent chromium (Cr(VI)(as potassium dichromate) and 10^–7 M, or 3 x 10^–7M mitomycin C (MMC) increased the SCE frequency in BALB/c lymphocytes,even more in BALB/Mo lymphocytes, indicating that M-MuLV andchemical carcinogens interacted synergistically in inducingSCEs in the presence of BUdR. On the other hand, the frequenciesof SCEs in either BALB/c or BALB/Mo lymphocytes were not modifiedby treatment in vitro with 10^–3 M trivalent chromium (Cr(III),as chromium chloride), which is genetically inactive. Treatmentin vivo with 0.3 mg/kg MMC or 1 mg/g urethane followed by incubationin vitro with BUdR in the absence of the carcinogens producedonly additive effects on BALB/Mo lymphocytes. The same was observedwhen unstimulated BALB/Mo lymphocytes were treated in vitrowith MMC in the absence of BUdR, and then stimulated and incubatedwith BUdR but not with MMC. Treatment in vivo with a high doseof MMC (10 mg/kg), produced negative synergistic effects onthe induction of SCEs in BALB/Mo lymphocytes. The differentinteraction of M-MuLV and chemicals on the induction of SCEswas interpreted in terms of positive or negative interferenceof the oncogenic virus and chemical carcinogens with the functionsof enzymes, such as DNA topoisomerases, involved in the mechanismof SCE production, and was tentatively ascribed to the presenceof larger DNA replicons in BALB/Mo than in BALB/c lymphocytes.     

Methotrexate-related deaths in patients previously treated with cis-diamminedichloride platinum

Summary Among 106 patients treated with conventional-dose methotrexate (MTX) following prior therapy with cis-diamminedichloride platinum (CDDP), six died with clinical manifestations of MTX toxicity. Death occurred 6–13 days after the administration of 20–50 mg/m² MTX. Toxicity included severe stomatitis and myelosuppression, which appeared in all six patients, skin rash in five, and diarrhea in four. Renal failure appeared in five cases and hepatic toxicity in four. All these patients had received MTX earlier without developing any serious toxicity. At the time of the last MTX administration, all had normal blood counts and also normal kidney and liver function tests. Prior therapy with CDDP may be responsible for this relatively high incidence of MTX-related deaths.     

Cytotoxicity and induction of sister chromatid exchanges in human and rodent brain tumor cells treated with alkylating chemotherapeutic agents

Rodent (9L and 9L-2) and human (SF-126 and SF-188) brain tumor cells were treated with methylating, ethylating, and chloroethylating nitrosoureas. 9L-2 and SF-188 cells were 10-fold and 5.4-fold more resistant to the cytotoxic effects of 1-(2-chloroethyl)-1-nitrosourea (CNU) than were 9L and SF-126 cells. SF-188 cells were 2.5- to 3-fold more resistant to N-methyl-N-nitrosourea (MNU) and streptozotocin (STZ) than SF-126 cells. 9L-2 cells were slightly more resistant to the cytotoxic effects of STZ and MNU than were 9L cells, but the rodent cells were 5- to 15-fold more resistant to these agents than the human cells. There were only small differences in cytotoxicity among the four cell lines after treatment with N-ethyl-N-nitrosourea (ENU). SF-188 and 9L-2 cells were 7- to 8-fold more resistant to the induction of sister chromatid exchange (SCEs) by CNU than were SF-126 and 9L cells. SF-126 cells were 80-fold more sensitive to the induction of SCEs by both STZ and MNU than were SF-188 cells. Only small differences in SCE induction were observed in 9L and 9L-2 cells treated with MNU and STZ. After ENU treatment, SF-126, 9L, and 9L-2 cells showed similar levels of SCEs; SF-188 cells were more resistant to the induction of SCEs by ENU. Pretreatment of 9L-2 cells with MNU resulted in a dose-dependent increase in cytotoxicity and SCE induction by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Treatment with 1 mM MNU completely reversed the cellular resistance of 9L-2 cells to BCNU but did not potentiate either cytotoxicity or SCE induction in 9L cells. These results suggest that O6-alkylguanine-DNA-alkyltransferase (O6-AT) plays an important role in determining the cytotoxicity of and the induction of SCEs by CNU in both rodent and human tumor cells. O6-AT may also influence the response of human cells to both the cytotoxic effects of and the induction of SCEs by MNU and STZ. In the rodent cells, however, biochemical mechanisms other than O6-AT appear to determine the cytotoxic response to these agents. O6-AT does not appear to influence the cytotoxicity of ENU in either human or rodent cells but may have a small effect on SCE induction in human cells.     

Sister chromatid exchanges (SCE), chromosomal aberrations and micronuclei of cultured peripheral lymphocytes in patients with lung cancer, miners and non-mining workers of the tin mine in Yunnan

Determination of SCE frequency, chromosomal aberration and micronucleus rate of cultured peripheral lymphocytes in 32 patients with lung cancer, 33 miners and 40 non-mining workers in Yunnan Tin Mine was carried out. The results showed that the cancer patients had higher SCE incidence, chromosomal aberration and micronucleus rate, the non-mining workers had the least and miners on an intermediate level. There was a significant difference of SCE, chromosomal aberrations and micronucleus rate between patients and non-mining workers (P less than 0.05-0.01). We also found that miners had a significant higher SCE and chromosomal aberration rate but not micronucleus rate as compared with the non-mining workers. We proposed that some carcinogens present in the Yunnan Tin Mine be responsible for the genetic damages in the miners. Chemical drugs may be considered to contribute to the genetic damage in cancer patients who had a tendency toward an increased SCE, chromosomal aberration and micronucleus rate as compared to the miners, though without reaching a statistical significance. We suggested that combination assay of SCE with chromosomal aberration or micronucleus be an useful index for screening of the high risk population and monitoring the chemical drug prevention of lung cancers in Yunnan Tin Mine.     

Molecular dosimetry of DNA adducts and sister chromatid exchanges in human lymphocytes treated with benzo[a]pyrene

We examined the relationship between benzo[a]pyrene-DNA adducts and sister chromatid exchanges (SCEs) in human lymphocytes. Cultures of isolated phytohemagglutinin (PHA)-stimulated lymphocytes from two normal donors were treated with 0.01-5.0 microM B[a]P from 24 to 72 h of culture. Using the highly sensitive 32P-postlabeling assay, we identified seven B[a]P-DNA adducts, one of which accounted for greater than 90% of the total DNA modifications. This adduct comigrated on polyethylenimine plates with the adduct produced by (+)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10- tetrahydro-benzo[a]pyrene. B[a]P-DNA adduct levels ranged from 0.02 to 8 adducts/10(7) nucleotides. SCE frequencies measured in parallel cultures ranged from 8 to 46 SCEs/cell. At the same B[a]P concentrations, B[a]P-induced SCE frequencies and B[a]P-DNA adduct levels were higher in lymphocytes from donor 1 than in lymphocytes from donor 2. There was a linear correlation between the number of B[a]P-DNA adducts and the number of SCEs induced; slopes of the linear regressions of induced SCEs on B[a]P-DNA adducts were similar for both donors. Our data suggest that SCE induction by B[a]P in human lymphocytes results from covalent DNA modification.