Abstract
Depsipeptide (FK228 or FR901228) was evaluated in the mouse bone marrow micronucleus test for its possible protective effect against chromosomal damage induced by benzo(a)pyrene and cyclophosphamide. Three doses of depsipeptide (0.5, 1, and 1.5 mg/kg body weight) were given intravenously to mice for 7 consecutive days prior to administration of genotoxins under investigation. All the three doses of depsipeptide were effective in exerting a protective effect against both benzo(a)pyrene and cyclophosamide. A significant suppression (34.9% to 67.5%) in the micronuclei formation induced by benzo(a)pyrene and (25.7% to 71.5%) cyclophosphamide was observed following intravenous administration of depsipeptide at doses of 0.5, 1, and 1.5mg/kg in Swiss albino mice.
Depsipeptide was found to exhibit significant selectivity for tumor cells (Ueda et al. 1994b). It is a unique bicyclic peptide containing a noncystine disulfide bridge, first isolated from fermentation broth of Chromobacterium violaceum Strain WB968 as an agent inducing morphological reversion of H-ras transformed NIH 3T3 cells (Shigematsu et al. 1994; Ueda et al. 1994a). Depsipeptide possesses potent antitumor activity against human tumor cell lines and significant inhibitory effects on the growth of human solid tumors implanted in mice (Ueda et al. 1994a). It has been suggested that the common use of antimutagens and anticarcinogens in every day life will be the most effective approach for preventing human cancer and slowing genetic diseases. (Chatterjee, Mukhopadhyaa, and Ghosh 1995; Morse and Stoner 1993; Rogers, Zeisel, and Groopman 1993; Ramel et al. 1986). The short-term in vivo mouse bone marrow micronucleus test, which provides information on in vivo chromosome breakage, spindle dysfunction, and mitotic nondisjunction of whole chromosomes, was used (Salamone and Heddle 1983; Schmid 1975). The development of malignancy often follows genetic damage resulting in clonal expansion of cells with chromosomal abnormalities. Micronuclei are formed as a result of chromosomal nondisjunctions after genotoxic damage (Baker et al. 1976) and are a very sensitive index of genetic damage. Hence, the present investigation was carried out to study the antimutagenic activity of depsipeptide in Swiss mice using cytogenetic end point assay.
METHODS AND MATERIALS
General
Depsipeptide was prepared by Astellas Pharma (formerly Fujisawa Pharmaceutical) and benzo(a)pyrene (B(a)P) was a gift from the National Respiratory Carcinogen Institute, MRI, Missouri, USA. May-Grunwald and Giemsa strains, fetal calf serum, and cyclophosphamide were purchased from Sigma, USA. All other chemicals used were of analytical grade.
Animals
Male Swiss mice (10 to 12 weeks old) were used throughout the study. They were maintained in a controlled environmental condition of temperature and humidity on alternatively 12-h light/dark cycles. All animals were fed standard pellet diet (Gold Mohor rat feed; Ms. Hindustan Lever, Mumbai) and water ad libitum. The experiments were conducted according to the ethical norms approved by Ministry of Social Justices and Empowerment, Government of India and Institutional Animal Ethics Committee guidelines (S.No:01/002/06 dated 28 February 2006).
Micronucleus Test
Genotoxic effects were evaluated by mouse bone marrow micronucleus test according to Schmid (1975). Animals were divided into six groups of six animals each. The group I animals were used as control and no treatment was given. The group II animals, as positive controls, one group with B(a)P and the other group with cyclophosphamide (CP) at 50 mg/kg and 40 mg/kg intraperitoneally, respectively. Group III was treated intravenously with 1.5 mg−1 kg−1day−1 for 7 consecutive days of depsipeptide for in vivo studies. Depsipeptide was dissolved in and diluted with 10% polyoxyethylated (60mol) hydrogenated castor oil in saline (HCO60 saline) (Chan, Bakhtiar, and Jiang 1997; Sasakawa et al. 2002). The animals of the groups IV, V, and VI were treated intravenously with 0.5, 1, and 1.5 mg/kg of depsipeptide, respectively, for 7 consecutive days with B(a)P and CP 1 h after the last dose of depsipeptide. Six animals from each group were killed 24 and 48 h after the single dose of mutagens. Bone marrow cells from both femurs were used for preparing slides, which were air-dried and stained with May-Grunwald and Giemsa. Two thousand and five hundred polychromatic erythrocytes (PCEs) were scored per animal for determining the frequency of micro nucleated polychromatic erythrocytes (MNPCEs). In addition, the percentage of PCEs was evaluated on the basis of the ratio of PCEs to NCEs (nonchromatic erythrocytes) in different regions of the slides prepared from control and treated animals.
Statistical Analysis
The data values are expressed as mean ± SD. The results were computed statistically on a SPSS program (SPSS 10.0; SPSS Institute, Chicago, IL). One-way analysis of variance (ANOVA) with Duncan’s multiple range test (Duncan 1955) was used to examine the difference between groups. Values of p < .05 were considered significant. All the results were expressed as mean ± SD for six animals in each group.
RESULTS
Tables 1 and 2 shows the frequencies of MNPCEs/2500 PCEs in bone marrow cells and the percentage of inhibition in mice pretreated with depsipeptide before exposure to the B(a)P and CP when compared to group II. The results revealed that mutagens when given at a single dose (group II) cause high incidence of micronuclei formation. Moreover, when pretreatment of different doses of depsipeptide was given prior to either B(a)P or CP treatment (in groups IV, V, and VI), decreased rates of micronuclei formations were observed. All the three doses of depsipeptide were effective in exerting significant effect against genotoxicity. Maximum reduction was observed in mice pretreated with depsipeptide (1.5 mg/kg at 48 h). No significant changes were observed in animals treated with depsipeptide alone.
DISCUSSION
Bone marrow cytogenetics is a useful short-term technique for elucidating as well to identify the substances for their clastogenic and anticarcinogenic activity (Renner 1990). Majority of the mutagenic and carcinogenic compounds, e.g., polycyclic aromatic hydrocarbons, act by generating electrophilic intermediates by microsomal enzymatic reaction causing mutations (Klaassen 1996; Kim, Kawack, and Lee 2000). B(a)P and CP metabolites appear to possess higher affinity for the genetic material than to other cellular components, resulting in formation of mutant cells (Jassim, Sparins, and Wattenberg 1990). Various types of blocking agents against mutagenesis and or carcinogenesis and their mechanism have been reviewed (Abraham, Singh, and Kesavan 1998). Preventing the formation of carcinogens from precursors and blocking the metabolic activation of carcinogens by increasing the activation of detoxification enzymes might inhibit initiation of cancer (Dhuley et al. 1993).
This investigation was carried out with the objective of evaluating the possible role of depsipeptide in modulating the in vivo genotoxicity of environmental mutagens and carcinogens. It has been reported that depsipeptide is effective for treating malignant T-cell lymphomas (Piekarz et al. 2001). Depsipeptide is the most promising histone deacetylase (HDAC) inhibitor, because systemic administration can achieve biologically active serum concentration according to phase I clinical trials (Sandor et al. 2003). HDAC inhibitors hyperacetylate histones, increasing the transcription activity of selected genes through relative increase of acetyltransferase activity due to inactivation of histone deacetylase enzyme (Yoshida and Horinouchi 1999; Nervi et al. 2001). Several lines of evidence suggest that histone acetylation plays a role in transcriptional regulation and repressions by allowing unfolding of the associated DNA and access by transcription factors in enhancing genomic stability (Grunstein 1997). The results show that the intravenous administration of depsipeptide can cause a significant reduction in the incidence of MNPCEs in mouse bone marrow induced by B(a)P and cyclophosphamide, which are both known genotoxins with different mechanisms of action (Willems and De Raat 1985). Because the micronuclei in young erythrocytes arise mainly from chromosomal fragments that are not incorporated into the daughter nuclei at the time of cell division in the erythropoietic blast cells (Salamone and Heddle 1983), the observed decrease in the incidence of MNPCEs can be considered an indication of an inhibitory effect of depsipeptide on the in vivo chromosomal damage induced by B(a)P and cyclophosphamide. Depsipeptide had inhibitory effects on the genotoxicity of B(a)P and cyclophosphamide even though their mechanisms of action are different. In our study, intravenous administered of depsipeptide has exerted anticlastogenic effects against the intraperitoneally injected directly acting mutagenic agents B(a)P and cyclophosphamide.
Further investigations are needed to elucidate the interactions of depsipeptide with genotoxic compounds at genetic level. The present, even if preliminary, study clearly showed that intravenous administration of depsipeptide in mice inhibits B(a)P-and CP-induced mutagenesis. The mechanism of action of depsipeptide may involve strengthening the defense against the deleterious effects of genotoxicity and carcinogens in scavenging potentially toxic mutagenic electrophiles and free radicals. Moreover, the modification of phase II enzymes and the enhancement of the detoxification pathways may be involved (Reen et al. 1996). Higher suppressive effects were observed at 48-h sampling time, indicative of greater detoxification ability in later stages. Further increase in dose did not enhance this inhibitory effect. In conclusion, the findings from the present study suggest that pretreatment with the bicyclic peptide of Chromobacterium violaceum can lead to moderate protective effects against in vivo genotoxicity.