Bimolane induces multiple types of chromosomal aberrations in human lymphocytes in vitro.

Bimolane has been commonly used in China for the treatment of psoriasis and various types of cancer. Patients treated with bimolane have been reported to have an increased risk of developing therapy-related leukemias. Although bimolane has been identified as a human leukemia-inducing agent, little is known about its genotoxic effects, and a systematic study of the types of chromosomal alterations induced by this compound has not been performed. In this study, a combination of immunochemical, molecular and conventional cytogenetic techniques has been used to study the chromosomal alterations induced by bimolane in cultured human lymphocytes. Immunochemical staining with the CREST antibody indicated that bimolane induces micronuclei (MN) originating primarily from chromosome breakage. Interestingly fluorescence in situ hybridization (FISH) with differentially labeled chromosomes 1 and 9 centromeric probes indicated that bimolane also caused non-disjunction and polyploidy. Consistent with this, an expedited analysis of Giemsa-stained metaphase chromosomes in bimolane-treated lymphocytes revealed a high frequency of polyploidy/hyperdiploidy as well as dicentric chromosomes, and premature centromeric division (PCD). In addition, bimolane was also found to produce binucleated cells, possibly through an interference with normal functioning of intermediate filaments. As a follow-up to these studies, three different types of commercially available bimolane formulations obtained from different Chinese manufacturers were also evaluated. The effects seen with the formulated bimolane were similar to those seen with the synthesized compound. Our studies indicate that bimolane effectively induces a variety of cellular and chromosomal changes in cultured lymphocytes and that similar alterations occurring in bone marrow stem cells could contribute to the development of the secondary cancers seen in bimolane-treated patients.

Cytotoxicity of eight cigarette smoke condensates in three test systems: comparisons between assays and condensates.

Cytotoxic properties of tobacco smoke are associated with chronic tobacco-related diseases. The cytotoxicity of tobacco smoke can be tested with short-term predictive assays. In this study, we compare eight mainstream cigarette smoke condensates (CSCs) from commercial and experimental cigarettes in three different cytotoxicity assays with unique and overlapping endpoints. The CSCs demonstrated cytotoxicity in all assays. In the multiple cytotoxicity endpoint (MCE) assay with TK-6 cells, the cigarette varieties that had the highest EC50s for reduced cell growth also showed a positive dose-response relationship for necrotic cells. In the IdMOC multiple cell-type co-culture (MCTCC) system, all CSCs reduced the viability of the cells. Low concentrations of some CSCs had a stimulatory effect in lung microvascular endothelial cells and small airway epithelial cells. In the neutral dye assay (NDA), except for a 100% flue-cured tobacco CSC, there was little consistency between CSCs producing morphological evidence of moderate or greater toxicity and the CSCs with the lowest EC50s in the MCE or MCTCC assays. Overall, cigarettes made with flue-cured tobacco were the most cytotoxic across the assays. When results were expressed on a per-mg of nicotine basis, lower tar cigarettes were the most cytotoxic in primary human respiratory cells.

Differential cell cycle-specificity for chromosomal damage induced by merbarone and etoposide in V79 cells.

Merbarone, a topoisomerase II (topo II) inhibitor which, in contrast to etoposide, does not stabilize topo II-DNA cleavable complexes, was previously shown to be a potent clastogen in vitro and in vivo. To investigate the possible mechanisms, we compared the cell cycle-specificity of the clastogenic effects of merbarone and etoposide in V79 cells. Using flow cytometry and BrdU labeling techniques, etoposide was shown to cause a rapid and persistent G2 delay while merbarone was shown to cause a prolonged S-phase followed by a G2 delay. To identify the stages which are susceptible to DNA damage, we performed the micronucleus (MN) assay with synchronized cells or utilized a combination of BrdU pulse labeling and the cytokinesis-blocked MN assay with non-synchronized cells. Treatment of M phase cells with either agent did not result in increased MN formation. Etoposide but not merbarone caused a significant increase in MN when cells were treated during G2 phase. When treated during S-phase, both chemicals induced highly significant increases in MN. However, the relative proportion of MN induced by merbarone was substantially higher than that induced by etoposide. Both chemicals also caused significant increases in MN in cells that were treated during G1 phase. To confirm the observations in the MN assay, first division metaphases were evaluated in the chromosome aberration assay. The chromosomes of cells treated with merbarone and etoposide showed increased frequencies of both chromatid- and chromosome-type of aberrations. Our findings indicate that while etoposide causes DNA damage more evenly throughout the G1, S and G2 phases of the cell cycle, an outcome which may be closely associated with topo II-mediated DNA strand cleavage, merbarone induces DNA breakage primarily during S-phase, an effect which is likely due to the stalling of replication forks by inhibition of topo II activity.

Induction of cell proliferation, micronuclei and hyperdiploidy/polyploidy in the mammary cells of DDT- and DMBA-treated pubertal rats.

The environmental estrogen, dichlorodiphenyltrichloroethane (DDT), and its metabolites have been implicated in the development of breast cancer through mechanisms that remain to be elucidated. It has been hypothesized that exposure to DDT and its metabolites, during critical periods of development, can contribute to an elevated risk for breast cancer in adults. In the present study, we have investigated the effect of o,p'-DDT on mammary gland cell proliferation and chromosomal alterations, in a rat mammary cancer model (commonly used to study human cancer), to gain insights into its potential role in the development of breast cancer. Twenty-one-day-old female Sprague-Dawley (SD) rats were administered o,p'-DDT, 7,12-dimethylbenz[a]anthracene (DMBA), genistein, DDT+DMBA, or DDT+DMBA+genistein, over a 14-day period. To determine changes in chromosome number and structure, we used the micronucleus assay as well as multicolor fluorescence in situ hybridization (FISH) region-specific DNA probes for rat chromosomes 4 and 19. Cell proliferation was evaluated using 5-bromo-2'-deoxyuridine (BrdU). Significant increases in BrdU-incorporated cells were seen in the rats treated with DDT+DMBA. Although micronucleus frequencies were somewhat elevated in several of the treatment groups, significant increases were not seen in any of them. Significant increases in numerical chromosomal aberrations were detected in all of the DDT- and DMBA-treated groups. Genistein significantly reduced BrdU incorporation and polyploidy in the DDT+DMBA-treated rats. These initial studies indicate that DDT and DMBA can induce cellular and chromosomal alterations in the rat mammary gland, which is consistent with the hypothesis that these agents can induce early events in mammary carcinogenesis.