Antimutagenic effect of crown ethers on heavy metal-induced sister chromatid exchanges

Mutagenesis vol.13 no.l pp.27-32, 1998 Antimutagenic effect of crown ethers on heavy metal-induced sister chromatid exchanges M.-Y.Cai and P.Arenaz1 Department of Biological Sciences, University of Texas at El Paso, El Paso, TX 79968, USA Introduction Crown ethers (macrocyclic polyethers) are characterized by having a different number of ethylene oxide (-OCH2CH2-) units, either substituted or unsubstituted, joined covalently in a macrocyclic ring (Pressman, 1976; Pedersen, 1988). Structurally crown ethers are characterized by a central hydrophilic cavity surrounded by an external hydrophobic ring. This structure enables crown ethers to form stable complexes with alkali and alkaline earth metal ions (Simon et al, 1973; Pressman, 1976; Pedersen, 1988). The metal ion is generally located at the center of the macrocyclic cavity. Because of their ability to strongly bind metal ions and incorporate into the bpid fraction of the cell membrane, crown ethers exhibit ionophoric properties (Lardy, 1968; Pedersen, 1968; Tosteson, 1968; Pedersen and Frensdorff, 1972; Tso et al, 1981). This unique ionophoric characteristic of crown ethers makes them ideal compounds for studying ion fluxes across both natural and artificial membranes (Lamb et al., 1980; Tso and Fung, 1981; Tso et al., 1981), as ion chelating resins and in ion selective electrodes (Pressman, 1976; Nishi, et al, 1997) and in organic synthesis (Botti et al, 1995). Crown ethers also have wide applications in promoting phase transfer reactions in binary solvent systems (Tso and Fung, 1981) and as pharmacological and antibacterial agents (Pressman, 1976; Brown and Foubister, 1979, 1983; Pannell et al, 1984, 1987; Szogyi et al, 1993; Borrel et al, 1995). Several ionophores, including crown ethers, have shown promise as potential antihypertensives (DeGuzman and Materials and methods Chemicals Lead nitrate [Pb(NO3)2] was purchased from Fisher Scientific Co. (Fair Lawn, NJ). Cadmium chloride (CdCy was purchased from Sigma Chemical Co. (St Louis, MO). Unsubstituted and substituted dicyclohexyl 18-crown-6 were purchased from Sigma Chemical Co. (St Louis, MO) and from Aldrich Chemical Co. (Milwaukee, WI) respectively. Unsubstituted and substituted dicyclohexyl 21-crown-7 were purchased from Parish Chemical Co. (Provo, UT). Stock solutions of heavy metals (10~3, 10"4 and 10~5 M) and crown ethers (10"' and 10~2 M) were prepared in distilled water and sterilized by filtration through a 0.2 |im filter (Acrodisc; Gelman Scientific). Cell lines CHO cell line AA8 (a gift from Dr Larry Thompson, Lawrence Livermore Laboratory) was used throughout these experiments. The AA8 cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Bioproducts, Whittaker, MA) supplemented with 10% (v/v) newborn calf serum (Bioproducts), 100 U penicillin, 100 Hg streptomycin (Bioproducts) and 2 mM Lglutamine. Cells were grown as monolayers at 37°C in an atmosphere containing 95% air and 5% COj. Cytotoxicity For survival determination CHO cells were seeded at 1-2X10* cells/100 mm dish in complete medium and incubated for 4 h prior to treatment The medium was removed, the cells washed twice with phosphate-buffered saline (PBS) to remove any residual calf serum and fresh DMEM without supplements added. The appropriate crown ether and/or metal was added directly to the DMEM and the cultures were allowed to incubate for either 3 or 12 h at 37°C. The medium containing the crown ether and/or metal was removed and 'To whom correspondence should be addressed. Tel: +1 915 747 6005; Fax: +1 915 747 6807; Email: © UK Environmental Mutagen Society/Oxford University Press 1998 27 Macrocyclic polyethers (crown ethers) are a family of compounds that possess the ability to complex with and transport metal ions across membranes. Because of their unique ionophoric characteristic, they have wide application in industry and research, chemistry and biology. In the current investigation the relationship between heavy metal mutagenesis and crown ether co-mutagenicity and/ or antimutagenicity in mammalian cells has been examined using sister chromatid exchange (SCE) as the cytogenetic end point. Chinese hamster ovary cells were treated with lead or cadmium, with and without selected crown ethers. Several genotoxic end points, including SCEs were scored and statistically compared. We report here that most of the crown ethers studied had little or no influence on leador cadmium-induced SCEs or chromosome aberrations. On the other hand, the substituted crown ether dicyclohexyl 21-crown-7 significantly decreased both spontaneous and metal-induced SCE frequencies, suggesting that this crown ether may possess antimutagenic activity. Pressman, 1974; Osborne et al, 1983; Kolbeck et al, 1984, 1992) and as antiischemics (Gurbanov et al, 1993). Both substituted and unsubstituted crown ethers have been found to be highly toxic in prokaryotic and eukaryotic organisms, including bacteria (Tso and Fung, 1980, 1981, 1982; Tso et al, 1981; Arenaz, et al, 1989), dog (Takayama et al, 1977), mice (Leong et al, 1974; Hendrixson et al, 1978; Gad et al, 1979), rat (Leong et al, 1974; Gad et al, 1979), and rabbit (Gad et al, 1979). Arenaz et al. (1989, 1992) have shown that despite the highly toxic nature of the crown ethers, these compounds were not genotoxic in prokaryotes, as measured by reversion of the His + locus in Salmonella typhimurium and in Chinese hamster ovary V79 cells, where no detectable increases in sister chromatid exchange (SCE) frequencies were observed. However, when the cells were exposed to the substituted crown ether dicyclohexyl 21-crown-7 a significant reduction in SCE frequency was observed. They suggested that because of their ionophoric properties, crown ethers may act as co-mutagens or antimutagens. In the current study we have examined the potential comutagenic and antimutagenic activity of selected crown ethers on induction of SCE in Chinese hamster ovary (CHO) cells using lead and cadmium as model metals. None of the crown ethers used appeared to have any synergistic effect on metalinduced SCEs, suggesting that they do not exhibit co-mutagenic properties. The crown ether dicyclohexyl 21-crown-7 significantly decreased Pb- and Cd-induced SCE frequency, suggesting that this crown ether may possess antimutagenic properties. M.-Y.GU and PArenaz Table L Cytotoxicity of crown ethers in combination with heavy metals on Chinese hamster ovary cells Crown ether 0 4 irr M Dicyclohexyl 21-crown-7 10" 3 M 5X10^* M KHM lO^M 5X10"7 M 10" 7 M 5X10^ M 1CH>M 5X1O"7 M 10- 7 M 5X10"^ 100" 6 30 64 96 10 23 56 100 60 72 100 8 10 11 24 26 32 61 57 58 87 92 100 8 9 13 19 26 23 45 50 54 94 89 96 62 80 100 10 8 12 30 34 27 55 62 57 85 98 91 11 13 6 21 28 25 52 46 60 85 100 100 48 65 100 22 16 11 35 30 26 60 60 53 95 100 100 4 7 10 26 31 35 50 58 63 100 92 87 61 77 100 28 15 8 35 56 22 70 75 71 100 96 88 19 13 9 36 30 15 (...truncated)