Vinblastine-induced cytogenotoxicity in spermatogonia and its transmission in the germline cells of Swiss mice.

Vinblastine, a cytotoxic anti-neoplastic drug and a known mitotic spindle inhibitor, has reportedly induced numerical and structural alterations in chromosome complements of treated animals. In the present study, cytogenotoxic effects of three different doses of vinblastine (0.5, 1.0, and 1.5 mg/kg body weight) were assessed from mouse spermatogonia at 24 hours after treatment after a single intraperitoneal exposure. The transmission potential of such effects in the male germline of mice was also assessed from primary spermatocytic chromosome analysis and sperm morphology assay at weeks 4 and 8 after treatments, respectively. Induction of statistically significant percentages of aberrant spermatogonial metaphases (P ≤ 0.01) and chromosomal aberrations (excluding gaps) (P ≤ 0.05) in vinblastine-treated mice indicated its clastogenicity. Induction of significant percentages of aberrant primary spermatocytes with atypical bivalents (P ≤ 0.01) and different categories of abnormal sperm, although not with significant variation in frequency, indicated the transmission of vinblastine-induced cytogenotoxic effects from spermatogonia to spermatocyte to sperm. We conclude that vinblastine is cytogenotoxic to mouse spermatogonia and that such induced effects are transmissible in the male germline cells of Swiss mice. Potential transmission of such cytogenotoxic effects, from cancer survivors of reproductive age with vinblastine pretreatment through gametes, is a serious concern.

Cytosine arabinoside-induced cytogenotoxicity in bone marrow and spermatogonial cells of mice and its potential transmission through the male germline.

Cytosine arabinoside (Ara-C), a widely prescribed antineoplastic drug, especially for the treatment of acute myeloid leukaemia, is a pyrimidine analog, in which the ribose sugar of cytidine is replaced by arabinose moiety. Ara-C reportedly competes with dCTP for incorporation into DNA during synthesis and exhibits various cytogenotoxic effects. In the present study, single intraperitoneal treatment of three different doses of Ara-C, 100, 150 and 200 mg/kg b.w. of mice, selected in accordance with its human therapeutic dose, induced statistically significant (p < or = 0.01) and dose dependent increase in the percentages of aberrant metaphases and chromosomal aberrations (CAs) at 24h post-treatment, and micronuclei (MN) in polychromatic erythrocytes (PCEs) at 30 h post-treatment. However, there was no significant change in the mitotic index (MI) at 24h post-treatment, when compared to that of the control mice. In the male germline, all the three doses of Ara-C induced statistically significant (p < or = 0.05 or p < or = 0.01) and dose dependent increase in the percentages of aberrant spermatogonial metaphases and CAs at 24h post-treatment and statistically significant (p < or = 0.01) and dose dependent increase in the percentages of aberrant primary spermatocytes at week 4 post-treatment. However, the induction of abnormal sperm at week 8 post-treatment was decreased, although significantly. The results indicated that Ara-C was clastogenic to both bone marrow and spermatogonial cells of mice, and some of its cytogenotoxic effects were found transmitted through the male germline, at least up to the formation of primary spermatocytes. As the drug is not target specific, the induced cytogenotoxic effects of Ara-C on non-cancerous cells of cancer patients retain possible risk of recurrence of second malignancy among the post-chemotherapeutic cancer survivors. In addition, there is every risk of transmission of some induced genetic alterations to the next generation through the gametes of the cancer survivors pre-treated with this drug. Therefore, Ara-C essentially be made target specific.

Modulation of methotrexate-induced cytogenotoxicity in mouse spermatogonia and its transmission in the male germline by caffeine.

Apart from its own controversial cytogenotoxic effects, caffeine (CAF), one of the most commonly consumed alkaloids worldwide, is found potentiative to and so also protective from the cytogenotoxic effects of numerous chemical and physical mutagens. It also has modulated the actions of several antineoplastic agents. Additionally, it has been tested as a chemopreventive of cancer and is reportedly associated inversely with different cancer risks. Therefore, in the present study, three different sub-lethal doses of CAF, 25, 50 and 100mg/kg, were tested in mouse to assess their cytogenotoxic effects on dividing spermatogonia at 24h post-treatment, and transmission of such effects in the male germline from the primary spermatocytes and sperm at week 4 and week 8 post-treatment, respectively. CAF was found to be weakly clastogenic to mouse spermatogonia and the effects were also found transmitted in the male germline. Interestingly, such induced effects were quantitatively related to the dose of CAF tested. On the other hand, methotrexate (MTX), an antifolate antimetabolite, is prescribed frequently for the treatment of various types of cancers. However, MTX is reportedly clastogenic. Modulation of the said three different pre-treated doses of CAF on MTX 10mg/kg-induced cytogenotoxic effects, tested in the same experimental protocol, indicated that CAF pre-treatment was decreasing the MTX-induced clastogenicity in spermatogonia, and was lowering the concurrent transmission of such effects in the male germline of mice, significantly. Such decreases were related to the dose of CAF tested, i.e. higher the dose of CAF more was the decrease in the MTX-induced cytogenotoxic effects and in their transmission. The possible mechanisms that might have caused the manifestation of a weak clastogenic action of CAF on spermatogonia and in its transmission in the male germline, and the CAF modulation of MTX-induced cytogenotoxic effects in spermatogonia and in their transmission have been discussed.

Etoposide-induced cytogenotoxicity in mouse spermatogonia and its potential transmission.

As cancer chemotherapeutic agents are cytogenotoxic but not target-specific during systemic treatment, they affect all the encountered cells including the non-cancerous ones and consequently lead to the recurrence of second malignancy in post-chemotherapeutic cancer survivors. The effects would be persistent if the stem cells were affected. These drugs also may affect germline cells during therapeutic treatments. There is every chance that the effects are transmitted through the germline cells to the gametes and to the next generation if the gonadal mother cells are affected. Such transmission of effects from the post-chemotherapeutic childhood cancer survivors is of serious concern but very little attention has been given so far to such studies. Etoposide (VP-16)--a semi-synthetic epipodophyllotoxin derivative, a DNA non-intercalating agent and a topoisomerase II inhibitor--is prescribed frequently for the treatment of various types of cancers. It is a potent clastogen inducing chromosomal damage both in vitro and in vivo. Its clastogenic effect is indirect through inhibition of the catalytic activity of topoisomerase II enzymes, which maintain the topology of DNA during replication, recombination, transcription, etc. by forming a 'cleavable complex' and facilitate the cleaving and re-ligation of the cleaved DNA to relieve the torsional stress during such events. Transient stabilization of the cleavable complex by etoposide leads to illegitimate ligation of the cleaved DNA. Consequently, single- and double-strand breaks occur. In the present study, the clastogenic potential of three different doses of etoposide (10, 15 and 20 mg kg(-1)) in the male germline of mice was assessed from the dividing spermatogonia after a single exposure for one cell cycle duration at 24 h post-treatment. Transmission of such effects was assessed from the frequency of aberrant primary spermatocytes at week 4 post-treatment and of abnormal sperm at week 8 post-treatment. All three doses of etoposide were found to be clastogenic to the dividing spermatogonia of mice, and mostly chromatid breaks were induced. The effects also were transmitted through the male germline of mice, which was evident from the prevalence of statistically significant increased percentages of aberrant primary spermatocytes at week 4 posttreatment and the higher percentages of abnormal sperm at week 8 post-treatment. Thus, there is every chance that the cytogenotoxic effects of etoposide are transmitted to the next generation through the male germline of post-chemotherapeutic cancer survivors, therefore it is essential to make etoposide target-specific or modulate its effects.

Cytogenetic consequences of vinblastine treatment in mouse bone marrow.

BACKGROUND: Vinblastine (VBL), a vinca alkaloid, has very often been included in different cancer chemotherapeutic treatment regimens. Chemotherapy cures certain cancers and, at least, increases the life expectancy of cancer patients. However, in cancer survivors, a second malignancy frequently occurs after chemotherapy, which warrants detailed genotoxicity testing of the chemotherapeutic agents. The available genotoxicity test reports on VBL are self-contradictory and inconclusive. Thus, following a suitable experimental protocol, it is necessary to test the cytogenetic consequences of VBL treatment in mammals. METHODS: Swiss mice received 1 of 3 different doses of VBL (0.5, 1.0 and 1.5 mg/kg body weight) as a single intraperitoneal injection. The cytogenetic toxicity of VBL was assessed from the induced aberrant metaphases, chromosomal aberrations (CAs) excluding gaps and the mitotic index (MI) 24 h after treatment, and micronuclei (MN) 30 h after treatment. RESULTS: All 3 doses of VBL induced statistically significant (p < or = 0.01) percentages of aberrant metaphases and CAs, but there was no significant change in the MI. The induced percentage of aberrant metaphases and CAs were decreased with the increase in the dose of VBL. On the other hand, there was a dose-dependent and significant (p < or = 0.01) increase in MN induction. CONCLUSIONS: The results of this study indicate the clastogenic potential of VBL in the mouse bone marrow. In the present study, the induction of numerous relatively large-sized MN by VBL is in agreement with the reported aneugenic action of the drug. Although VBL is cytotoxic and is a spindle poison, the mechanism(s) involved in bringing about its clastogenic effects is yet to be elucidated.

Cytogenetic risk assessment of etoposide from mouse bone marrow.

Increased clinical applications of the anticancer drug etoposide (a non-intercalative epipodophyllotoxin derivative) and the frequent induction of a second malignancy, particularly leukaemia, in post-etoposide-treated cancer survivors warrant detailed genotoxicity testing of etoposide. The genotoxicity test results available on etoposide are either primarily in in vitro test systems or in lower organisms after treatment with unusually high doses, or after chronic exposures, having little extrapolative value to humans. Therefore, a cytogenetic risk assessment study on etoposide in mouse in vivo was undertaken after a low dose (in accordance with the human therapeutic dose) single exposure. The cytogenetic toxicity of etoposide was assessed from bone marrow of mouse at three separate endpoints: chromosomal aberration and mitotic index studies at 24 h post-treatment and the micronucleus test (MNT) at 30 h post-treatment. The flame drying technique using colchicine, hypotonic sodium citrate, methanol-glacial acetic acid and Giemsa was followed for the preparation of slides for the metaphase chromosomal aberration and mitotic index studies and a simple technique was followed for the MNT. Although induction of chromosomal aberrations, excluding gaps, per 100 metaphases by 10 and 15 mg kg(-1) etoposide was not significant statistically, 20 mg kg(-1) of etoposide induced a significantly higher number of chromosomal aberrations in female (P < or = 0.01) and male (P < or = 0.05) mice. There was no significant change in the induced percentages of dividing cells by any of the doses of etoposide tested. The micronucleus induction also was not significant statistically with the lowest dose but it was significant in female (P

Modulatory effects of caffeine on methotrexate-induced cytogenotoxicity in mouse bone marrow.

Caffeine (CAF), a widely used and extensively studied chemical, is known for the reports on its controversial and inconsistent genotoxic effects, potentiative and protective effects from the genotoxicity of chemical and physical mutagens, and its modulatory effects on the action of antineoplastic drugs. Methotrexate (MTX), an antifolate antimetabolite, is a widely prescribed antineoplastic drug with significant clastogenic effects. In the present study, in addition to the assessment of cytogenotoxicity of CAF 25, 50 or 100mg/kg in mouse bone marrow, their modulatory effects on the cytogenotoxicity of MTX 10mg/kg was assessed from the induced frequencies of aberrant metaphases, chromosomal aberrations (CAs) and percentages of dividing cells at 24h post-treatment and the induced frequency of micronuclei (MN) at 30h post-treatment. All the three doses of CAF induced higher percentages of aberrant metaphases, high frequency of CAs and MN and increased percentages of dividing cells, but the increase in the aberrant metaphases and CAs was statistically significant only with the highest dose of CAF. Thus, CAF was weakly clastogenic to mouse bone marrow cells. However, pre-treatment of each of the three doses of CAF reduced the frequency of MTX 10mg/kg-induced aberrant metaphases, CAs, MN and also the percentage of dividing cells, but significantly only by the two higher doses of CAF. Thus, the higher doses of CAF protected mouse bone marrow cells from the cytogenotoxicity of MTX. The possible mechanisms involved in bringing about the weak clastogenic action of CAF and its protection from the cytogenotoxic effects of MTX have been discussed, and bio-modulation of the effects of antineoplastic drugs has been highlighted.

Spermatogonial cytogenetic toxicity of vincristine and its transmission in the germline cells of Swiss mice.

We tested the anticancer drug vincristine sulfate (VCR) and cyclophosphamide for their cytogenetic toxic effects on spermatogonia in Swiss mice, and we assessed the possible transmission of such effects in the germline cells. Spermatogonial metaphase chromosome aberration study, primary spermatocytic chromosome analysis, and sperm morphology assay were examined after a single intraperitoneal exposure of VCR 0.25, 0.5, and 1.0 mg/kg and CTX 40 mg/kg body weight at 24 hours, 4 weeks, and 8 weeks posttreatment, respectively. The induction of statistically significant percentages of aberrant spermatogonial metaphases and chromosomal aberrations (excluding gaps) in the VCR-treated mice indicated its clastogenicity. The occurrence of significant percentages of aberrant primary spermatocytes with atypical bivalents and higher percentages of abnormal spermatozoa (sperm), although not statistically significant, indicated the transmission of the induced cytogenetic effects of VCR from spermatogonia to sperm. We conclude that VCR is genotoxic to the male germline cells of Swiss mice, and has the potential of transmitting the cytogenetic toxic effects to the next generation.