Permeability, Cytotoxicity, and Genotoxicity of Cr(III) Complexes and

Australia, National Occupational Health and Safety Commission (NOHSC), P.O. ... Parkville 3052, Australia, and Institute of Nuclear Physics, Cracow, P...
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Permeability, Cytotoxicity, and Genotoxicity of Cr(III) Complexes and Some Cr(V) Analogues in V79 Chinese Hamster Lung Cells Carolyn T. Dillon,† Peter A. Lay,*,† Antonio M. Bonin,‡ Marian Cholewa,§,| and George J. F. Legge§ Centre for Heavy Metals Research, School of Chemistry, The University of Sydney, NSW 2006, Australia, National Occupational Health and Safety Commission (NOHSC), P.O. Box 58, Sydney, NSW 2001, Australia, Micro Analytical Research Centre, School of Physics, The University of Melbourne, Parkville 3052, Australia, and Institute of Nuclear Physics, Cracow, Poland Received January 27, 2000

The permeabilities and genotoxicities of the Cr(III) complexes [Cr(en)3]3+, mer-[Cr(glygly)2]-, cis-[Cr(phen)2(OH2)2]3+, and trans-[Cr(salen)(OH2)2]+ and the Cr(V) analogues of cis-[Cr(phen)2(OH2)2]3+ and trans-[Cr(salen)(OH2)2]+ [en being 1,2-ethanediamine, glygly being glycylglycine, phen being 1,10-phenanthroline, and salen being N,N′-ethylenebis(salicylideneiminato)] have been studied in V79 Chinese hamster lung cells. Following exposure of ∼106 cells to 0.4 mM Cr(III) for 4 h, the Cr uptake by single cells was less than 10-14 g/cell (as determined by GFAAS analysis and as confirmed by PIXE analysis where the Cr concentration was below the limit of detection). Importantly, the Cr(V) analogue of cis-[Cr(phen)2(OH2)2] was significantly more permeable than the Cr(III) complex. The cytotoxicity of the Cr(III) complexes increased in the following order: mer-[Cr(glygly)2]- < [Cr(en)3]3+ ∼ cis-[Cr(phen)2(OH2)2]3+ < trans-[Cr(salen)(OH2)2]+. No genotoxic effects were observed following exposure to mer-[Cr(glygly)2]- or [Cr(en)3]3+ at concentrations up to 6 mM. The Cr(III) imine complexes trans-[Cr(salen)(OH2)2]+ and cis-[Cr(phen)2(OH2)2]3+, which could be oxidized to Cr(V) complexes, induced MN in vitro at rates of 13.6 and 3.3 MN/1000 BN cells/µmol of Cr, respectively. The comparative permeabilities and genotoxicities of trans-[Cr(salen)(OH2)2]+ and [CrO(salen)]+ were similar due to the instability of the Cr(V) complex at physiological pH values (7.4). There was a substantial increase in the permeability of [Cr(O)2(phen)2]+, compared to that of the Cr(III) analogue, which was accompanied by a highly genotoxic response. Consequently, any Cr(III) complex that is absorbed by cells and can be oxidized to Cr(V) must be considered as a potential carcinogen. This has potential implications for the increased use of Cr(III) complexes as dietary supplements and highlights the need to consider the genotoxicities of a variety of Cr(III) complexes when determining the carcinogenic potential of Cr(III) particularly when “high” deliberately administered doses are concerned.

Introduction The recommended dietary intake of Cr ranges from 50 to 200 µg of Cr/day (1). The endorsement of Cr stems from findings that it plays a beneficial role in glucose tolerance and diabetes (2, 3). Recently, a naturally occurring oligopeptide, low-molecular weight chromium-binding substance (LMWCr), which binds four Cr(III) ions has been identified. The protein is capable of activating insulin receptor kinase by up to 7-fold (3). Cr is also involved in lipid and carbohydrate metabolism in mammals with a Cr deficiency (1, 4, 5). Consequently, Cr, in the form of tris(picolinato)chromium(III) [Cr(pic)3] (6), is promoted as a muscle-building agent, although there is some controversy surrounding its use (7-12). While Cr(VI) has been established as a carcinogen on the basis of epidemiological evidence and animal experiments, no such evidence has been accumulated for Cr(III), which * To whom correspondence should be addressed. † The University of Sydney. ‡ National Occupational Health and Safety Commission. § The University of Melbourne. | Institute of Nuclear Physics.

has been important in its acceptance as a dietary supplement (13, 14). The main reason for this is that there is little evidence to show that Cr(III) compounds induce tumors in mice or rats (13). Furthermore, while there is overwhelming evidence to show that Cr(VI) complexes are mutagenic in bacterial and mammalian cells, most Cr(III) complexes are not mutagenic (13, 15). However, Warren et al. (15) found that Cr(III) complexes containing aromatic imine ligands, cis-[Cr(phen)2Cl2]+, cis-[Cr(bipy)2Cl2]+, and [Cr(bipy)2(ox)]I‚ 4H2O, are mutagenic in Salmonella typhimurium (TA92, TA98, and TA100). cis-[Cr(phen)2Cl2]+ also induces mutations at the HGPRT locus in V79 cells (16). The bacterial mutagenicity of the phen-containing complexes cannot be attributed directly to the ligand [i.e., phen is not mutagenic in S. typhimurium (17, 18)], although it is believed that the lipophilic nature of the phen and bipy ligands increases the permeabilities of the complexes, leading to the observed genotoxicities (16). It has been shown recently that trans-[Cr(salen)(OH2)2]+ is also mutagenic in S. typhimurium TA97a, TA98, TA100, and TA102, while the salen ligand is not mutagenic (19). In

10.1021/tx0000116 CCC: $19.00 © 2000 American Chemical Society Published on Web 07/26/2000

Permeability and Genotoxicity of Cr(III) and Cr(V)

vitro DNA studies of these complexes and their Cr(V) analogues revealed that the Cr(V) complexes interacted strongly with DNA at 37 °C and pH values of 3.3 and 7.4. Furthermore, the controversial dietary supplement (20, 21) [Cr(pic)3] is clastogenic (11), such that Cr concentrations of 0.05-1 mM induce chromosomal damage 3-18-fold above the control levels in Chinese hamster ovary cells (12). The level of DNA fragmentation is also increased by 1.2-1.6-fold when cells are incubated with [Cr(pic)3] (30-50 µg/mL) (10). Previously (22), we studied the permeabilities, cytotoxicities, and genotoxicities of Cr(V) and Cr(VI) complexes in V79 cells. PIXE (particle-induced X-ray emission) analysis using a scanning proton microprobe (1 µm beam diameter) enabled the determination of intracellular Cr levels in single cells following exposure of the V79 cells to Cr(V) and Cr(VI) complexes (0.5 µmol of Cr/ dish, 4 h). These results allowed the quantification of Cr cytotoxicity and genotoxicity at a cellular level, and revealed that the potency of Cr(V) species was similar to (if not greater than) that of Cr(VI) complexes. In this paper, the permeability, cytotoxicity, and genotoxicity of several Cr(III) complexes are reported and, where possible, those of their Cr(V) analogues. The complexes studied included [Cr(en)3]3+, a trivalent complex containing nonaromatic ligands; mer-[Cr(glygly)2]-, a biologically relevant anionic peptide-containing complex; cis-[Cr(phen)2(OH2)2]3+, a mutagenic complex and its Cr(V) analogue, [Cr(O)2(phen)2]+; and trans-[Cr(salen)(OH2)2]+, a monovalent complex containing a tetradentate imine ligand and its Cr(V) analogue, [CrO(salen)]+. The complexes were chosen because they either were among the few Cr(III) complexes that are mutagenic (15, 16, 18, 19) or represent typical classes of Cr(III) complexes that are not mutagenic. By studying this series of complexes, we anticipated that insights would be gained into the reasons why some Cr(III) complexes are genotoxic while others are not. This is important not only for understanding potential hazards of Cr(III) dietary supplements and Cr(III) complexes encountered in industry but also in the context of undergraduate and research laboratories, where such complexes are encountered frequently. The biochemistry and chemistry of the dietary supplements themselves are somewhat more complex than the systems reported here and will require further study to rationalize their bioactivity. The work is in progress and will be reported at a later date.

Experimental Procedures Caution: cis-[Cr(phen)2(OH2)2]3+, trans-[Cr(salen)(OH2)2]+, and their Cr(V) analogues are genotoxic and possible carcinogens (15, 16, 18, 19). Due care should be taken to avoid inhalation of the compounds and to avoid contact with skin. Syntheses. [Cr(en)3]Cl3 was prepared according to the method of Gillard et al. (23). Na{mer-[Cr(glygly)2]} was synthesized using the method described by Murdoch (24), and the purity was determined by electronic absorption spectroscopy in H2O: λmax (max) 550 (161 M-1 cm-1), 418 nm (37.4 M-1 cm-1) [lit., 552 (185 M-1 cm-1), 420 nm (40 M-1 cm-1) (24)]. cis-[Cr(phen)2(OH2)2](NO3)3‚5/2H2O was prepared according to the method of Inskeep et al. (25, 26). Anal. Calcd for CrC24N7H25O13.5: C, 42.42; H, 3.71; N, 14.43. Found: C, 42.60; H, 3.68; N, 14.38. trans-[Cr(salen)(OH2)2]Cl was prepared as described by Yamada and Iwasaki (27, 28). Mass spectroscopy (electron impact, Kratos MS9 upgraded to MS50 configuration) yielded a parent peak at m/z 353 and a base peak at m/z 318 corresponding to the weights of [Cr(salen)Cl]+ and [Cr(salen)]+,

Chem. Res. Toxicol., Vol. 13, No. 8, 2000 743 respectively. Electronic absorption spectroscopy in H2O resulted in a visible peak at 382 nm: max ) 4.4 × 103 M-1 cm-1 [lit., λmax ) 381 nm, max ) 5 × 103 M-1 cm-1 (29)]. The Cr(V) complexes were prepared by oxidizing the Cr(III) complexes in acetate buffer (pH 3.3) at 37 °C using a 4-fold molar excess of PbO2 (Merck). A 60 min oxidation of cis-[Cr(phen)2(OH2)2]3+ produced [Cr(O)2(phen)2]+, which was identified by an EPR signal at giso ) 1.9386 [cf. giso ) 1.937 (19, 30, 31)]. A 30 min oxidation of trans-[Cr(salen)(OH2)2]+ yielded [CrO(salen)]+, which was identified by a five-line EPR signal at giso ) 1.9755, AN ) 2.05 × 10-4 cm-1 [cf. giso ) 1.978, AN ) 2.00 × 10-4 cm-1, in acetonitrile (32)]. The features of the absorption spectrum included high absorption below 430 nm, and a peak at 590 nm, with an intensity that indicated >99% conversion to the Cr(V) analogue (32). Reactivity of Cr Complexes in Tissue Culture Medium. [Cr(en)3]Cl3 (5 µmol, 100 µL) was added to MEM tissue culture medium (2.9 mL, Eagle’s, 9.78 g L-1) to produce a final Cr(III) concentration of 1.67 mM, and the reactivity was monitored by UV/vis spectroscopy using a Hewlett-Packard 8452A diode array spectrophotometer. A Hewlett-Packard 8909A Peltier temperature control unit maintained the solution temperature at 37 °C, and the spectra were collected at 5 min intervals for 2 h. The phenol red component of the MEM interfered with the visible spectrum at approximately 550 nm (despite background subtraction of MEM), causing some difficulties with the analysis of the reactivities of mer-[Cr(glygly)2]-, cis-[Cr(phen)2(OH2)2]3+, and trans-[Cr(salen)(OH2)2]+. Consequently, the reactivities of these complexes were monitored in MEM-P, a minimal essential salt medium, which did not contain phenol red, (0.97 g/100 mL, Flow Laboratories). Solutions were prepared as 1.67 mM Cr, and the reactivities were monitored at 37 °C using the procedure mentioned above. Tissue Culture Procedures Used for V79 Cells. V79 cells were employed for the permeability, cytotoxicity, and genotoxicity studies (33). The cells, supplied initially by R. Newbold (Institute of Cancer Research, Sutton, Surrey, U.K.), were cultured from frozen stocks (maintained at -196 °C) and grown to confluency in a 5% CO2/95% air-humidified incubator (model 3029, Forma Scientific) at 37 °C. The cells were cultured in growth medium (GM) containing MEM (9.76 g L-1, Eagle’s) and sodium hydrogen carbonate (2.2 g L-1, Sigma) supplemented with 10% fetal calf serum (Multi Ser) and 50 IU mL-1 penicillin and 50 µg mL-1 streptomycin (Multi Ser) (18, 22, 34). Cytotoxicity and Genotoxicity Assays. The cytotoxicities of the Cr(III) complexes (in 5 mL of GM) following a 4 h exposure were determined using the clonal assay, and the maximum Cr(III) concentrations were established by the solubility limits of the complexes. The assays were performed in a manner identical to that described previously (18, 22), whereby the survival percentage was determined 7 days after seeding 180 treated V79 cells. The genotoxicities of the Cr complexes were determined by the in vitro micronucleus assay employing the cytokinesis block method for micronucleus detection (22, 35, 36). V79 cells (5 × 105) were seeded in GM (5 mL) contained in 60 mm tissue culture dishes. After 24 h, the cells were washed with PBS, and new GM (5 mL) was added followed by the freshly prepared Cr solutions (0.1 mL, duplicate dishes). The Cr(V) solutions were prepared by oxidation of the stock Cr(III) solution in acetate buffer (pH 3.6) at 37 °C using a 4-fold molar excess of PbO2. The solutions were filtered through a 0.2 µm filter, and the filtrates were stored on ice until dilution and cell treatment. After the 4 h treatment period, the cells were washed with PBS, and new GM (5 mL) was added. The cells were reincubated for 24 h, after which time cytochalasin B (15 µg/dish, Sigma) was added to block cytokinesis. After a further 24 h incubation, the cells were harvested and centrifuged onto slides (Shandon, cytospin 2). The cells were stained using Diff-quik stain, and the slides were scored blind at 600× magnification. The incidence of micronuclei was determined from a minimum of 1000 BN cells per duplicate dish (2000 BN cells per dose) using

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the following criteria: the micronucleus must be present in a BN cell with two discrete main nuclei, the micronucleus must be one-twentieth to one-fifth the size of the main nuclei, the micronucleus must have staining characteristics identical to those of the main nuclei, and the micronucleus must not be in contact with the main nuclei (37-39). Permeability Studies. Cells were seeded at a density of 5 ×105 cells/dish and incubated at 37 °C and 5% CO2 for 24 h. The cells were washed twice with PBS prior to treatment, and new GM (5 mL) was added, followed immediately by the freshly prepared Cr solution (2 µmol/dish, 100 µL). Control cells were prepared in an identical manner, except that 0.1 mL of sterile water (filtered through a 0.2 µm filter) was substituted for the Cr solution. Triplicate dishes were prepared for each treatment. Following the treatment period (4 h), the GM was removed and the cells were washed thoroughly using PBS solution (pH 7.4, Oxoid). Trypsin (2.5% in PBS) was added, and the cells were harvested after 5 min. The dishes were washed thoroughly with PBS to ensure that all of the cells were transferred to the polyethylene tube. The cells were centrifuged for 7 min at 1100 rpm, and the trypsin was removed. Saline solution (2 mL, 0.9% NaCl, 99.999% pure, Aldrich) was added; the suspensions were centrifuged for 7 min, and the supernatants were discarded. This procedure was repeated, and the final saline solutions were drained from the pellets. The number of cells per dish was determined by scoring cells that were grown under identical conditions but were untreated. Cr analyses of the treated cells were performed following digestion of the cells in HNO3 (TracePur, Merck, 0.9 ppb Cr batch analysis) using presoaked glassware (RBS, 3 days; 1.5 M HNO3, 3 days; distilled water, 3 days; rinsed five times in milliQ water×). The digestions were performed for approximately 6 h, and the resultant ash was dissolved in Milli-Q water in presoaked 1 mL volumetric flasks. The solutions were analyzed for Cr using a Varian GTA96 Graphite Tube Atomizer SpectrAA. The permeabilities of the Cr complexes were also investigated in individual cells by PIXE analyses by employing the scanning proton microprobe at MARC, School of Physics, The University of Melbourne, as described in detail elsewhere (22, 34). The cells were treated as described above, harvested, and freeze-dried from 200 mM ammonium acetate solution (22, 34). The PIXE data were collected as a function of the X-ray energy and the associated count frequencies at each spatial (X,Y) coordinate of the beam. The PIXE spectra of the cells were normalized to the charge, and the absolute amount of Cr was determined with the aid of a Mn standard (22). Statistical Analyses. MN data were analyzed by simple linear regressions, and a χ2 test was used to compare the frequencies of MN obtained for various treatments and the controls. A ranking of the cytotoxicities of the Cr(III) complexes was determined by Tukey-Kramer analysis of variance for the survival data obtained at 3 µmol of Cr/dish. The results of the permeability assays were assessed using ANOVA and the twotailed t test. In all cases, a P value of 0.05) as was the case for [Cr(en)3]3+ (P ) 0.13) where the linear response was poor (R2 ) 0.45) and was not dose-dependent. Furthermore, the responses of [Cr(en)3]3+ and mer-[Cr(glygly)2]- were not significantly different from each other (ANOVA). The Cr(III) imine complexes trans-[Cr(salen)(OH2)2]+ and cis-[Cr(phen)2(OH2)2]3+ were genotoxic and induced 14 MN/1000 BN/ µmol of Cr and 3 MN/1000 BN/µmol of Cr, respectively. The Cr(V) analogue of cis-[Cr(phen)2(OH2)2]3+ was the most genotoxic of all the complexes and induced 125 MN/ 1000 BN/µmol of Cr, which was significantly greater than

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Figure 5. Permeability results obtained from GFAAS analysis of cell pellets following treatment with 2 µmol of Cr(III) or Cr(V) per dish or 0.5 µmol of Cr(VI) per dish for 4 h. Data points are means ( SD (n ) 3).

the effect observed for cis-[Cr(phen)2(OH2)2]3+. While the genotoxic response of [CrO(salen)]+ appears to be more substantial than that of trans-[Cr(salen)(OH2)2]+, the slopes of the curves were not significantly different. Permeability. The GFAAS permeability results are shown in Figure 5 for cells that were treated with 2 µmol of Cr(III) or Cr(V) per dish (0.4 mM Cr) for 4 h. Also included in the graph are the results of the 4 h treatment of the cells with the positive control, [Cr2O7]2- (0.5 µmol of Cr/dish). The Cr permeability determined by GFAAS was e10-14 g/cell for all Cr(III) complexes that were tested, as was also confirmed by PIXE analyses of individual cells. Furthermore, the Cr uptake by the cells was not significantly greater than the controls for all the tested Cr(III) complexes (P > 0.5, ANOVA, TukeyKramer and Bonferroni). The Cr uptake observed following treatment of the cells with [CrO(salen)]+ was not significantly greater than the control, nor was it significantly different from that of trans-[Cr(salen)(OH2)2]+. A significant increase in the cellular Cr level was observed following exposure to [Cr(O)2(phen)2]+ (P < 0.01, TukeyKramer). The Cr uptake was also significantly greater than that observed for cis-[Cr(phen)2(OH2)2]3+ exposure. As expected, the Cr(VI) permeability following treatment with 0.5 µmol/dish was highest and was significantly greater than those of all of the tested Cr complexes. PIXE analysis of single cells treated with Cr(III) complexes confirmed that the cellular Cr concentrations were less than 10-14 g/cell. Due to the sensitivity limitations of the instrument, no distinction in the Cr content could be made for the various Cr(III) treatments above that of the control. Importantly, however, there was evidence of an increased cellular Cr level when the cells were treated with [Cr(O)2(phen)2]+ as CrKR peak magnitudes up to 8 times greater than the background were observed.

Discussion The Cr(III) complexes [Cr(en)3]3+, mer-[Cr(glygly)2]-, cis-[Cr(phen)2(OH2)2]3+, and trans-[Cr(salen)(OH2)2]+ were chosen to determine whether Cr(III) permeability, cytotoxicity, and genotoxicity were influenced by ligand specificity and/or the sign and size of the charge of the complex. [Cr(en)3]3+ and cis-[Cr(phen)2(OH2)2]3+ were added to the culture medium as triply charged cations. It is known that [Cr(en)3]3+ undergoes ligand-exchange reactions in H2O (followed by subsequent deprotonation

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of the aqua ligands at pH 7.4) to produce [Cr(en)2(OH)2]+, which is characterized by peaks at 392 and 528 nm in the UV/vis spectrum (23). This ligand-exchange reaction was quite slow in MEM, however, as determined by the 2 h study that showed that the peaks at 352 and 456 nm, indicative of [Cr(en)3]3+, did not shift (Figure 1). Inskeep et al. (25) showed that the aqua ligands of cis[Cr(phen)2(OH2)2]3+ are deprotonated at pH 7.4 (pKa1 ) 3.4 and pKa2 ) 6.0, reactions 1 and 2, respectively). This coincides with a shift of λmax from 497 nm for the diaqua species to 510 nm for the mixed aqua/hydroxo species to 519 nm for the dihydroxo species (25, 26, 41).

cis-[Cr(phen)2(OH2)2]3+ + OH- h cis-[Cr(phen)2(OH2)(OH)]2+ + H2O (1) cis-[Cr(phen)2(OH2)(OH)]2+ + OH- h cis-[Cr(phen)2(OH)2]+ + H2O (2) The observation of a peak at 519 nm (pH 7.4) shows that cis-[Cr(phen)2(OH)2]+ was produced immediately after the dissolution of the corresponding diaqua complex in MEM-P solution. The peptide complex mer-[Cr(glygly)2]represented a negatively charged structure that may mimic those found in some of the Cr(III) products of the intracellular reduction of Cr(VI). Its electronic absorption spectrum in MEM-P solution revealed a degree of instability, as shown by the 30% decay of the visible peak at λmax ) 550 nm. This was consistent with ligand-exchange reactions of glygly with the ligands present in the MEM-P solution, as the decay of the peak corresponds to the generation of Cr(III) species with lower max values (42). Finally, the addition of trans-[Cr(salen)(OH2)2]+ to the MEM results in the partial deprotonation of one of the aqua ligands (pKa1 ) 7.54) in producing a mixture of the cation and the neutral species, trans-[Cr(salen)(OH2)(OH)] (43). The small decrease in the absorption of the UV/vis peaks associated with trans-[Cr(salen)(OH2)2]+ may be due to the production of further species resulting from ligand-exchange reactions. For instance, the complex undergoes ligand-exchange reactions with azide, thiocyanate, pyridine, imidazole, and nicotinic acid to form monosubstituted products and with oxalate to form the disubstituted complex (43-45). With the exception of the glygly complex, the fact that there were only minor changes in the UV/vis spectra of all Cr(III) complexes in the medium indicated that they retained their integrities during the assay period. Therefore, some possible factors contributing to the differences in the genotoxicities between the different Cr(III) complexes may be the charge of the complexes, and the physical and/or chemical properties imparted by the ligands. The lack of a detectable Cr accumulation (GFAAS and PIXE analysis) in V79 cells exposed to 0.4 mM [Cr(en)3]3+, mer-[Cr(glygly)2]-, cis-[Cr(phen)2(OH)2]+, or trans-[Cr(salen)(OH2)2]+ for 4 h contrasts with the results obtained following exposure to Cr(VI) and Cr(V) (0.1 mM Cr for 4 h) (22). The low permeabilities of the Cr(III) complexes are consistent with those reported by a number of investigators (46-49). For example, Kortenkamp et al. (48) showed that the intracellular Cr detected in human erythrocytes following exposure to Cr(III) complexes (1 mM for 1 h) was approximately 0.5-2.0 × 10-18 mol/cell. It was not possible to detect any differences in the uptake of the complexes using GFAAS or PIXE analysis of cells

Dillon et al.

in this study due to the low permeability of V79 cells to Cr(III). Electrothermal atomic absorption studies performed by Kortenkamp et al. (48) also showed that intracellular Cr concentrations were similar when human erythrocytes were exposed to the following Cr(III) complexes: [Cr(phen)2Cl2]+, [Cr(bipy)2Cl2]+, [Cr(2,4-pentanedionato)3], [Cr(glycinato)3], [Cr(glutathionato)2]-, and [Cr(cysteinato)2]-. The study concluded that there was little discrimination among the permeability properties of the complexes based on charge and ligand lipophilicity (48). Consistent with previous studies, the cytotoxicities of the Cr(III) complexes were much lower than those of Cr(V) and Cr(VI) complexes (46, 50, 51). Following identically performed studies with V79 cells (44), negatively charged Cr(V) and Cr(VI) complexes exhibited LD50 values in the range of 0.05-0.1 µmol of Cr/dish, while the LD50 values for Cr(III) complexes were in the range of 5-30 µmol/dish. There was no evidence that cytotoxicity was enhanced by more anionic species. For instance, trans-[Cr(salen)(OH2)2]+ (existing in equilibrium with trans-[Cr(salen)(OH2)(OH)]) was the most toxic, while the negatively charged mer-[Cr(glygly)2]- was the least toxic of the complexes. There are distinct differences between the Cr(III) complexes containing aliphatic groups and those containing aromatic imine ligands. While the difference was not as significant in the cytotoxicity assays, i.e., trans-[Cr(salen)(OH2)2]+ was the most toxic, followed by cis-[Cr(phen)2(OH2)2]3+ and [Cr(en)3]3+ (both with similar toxicities), there was a marked difference in the genotoxicity assays. There was an increased incidence of MN following exposure to cis-[Cr(phen)2(OH2)2]3+ or trans-[Cr(salen)(OH2)2]+, although no well-defined dose response was observed following exposure to [Cr(en)3]3+ or mer-[Cr(glygly)2]-, even at extremely high Cr(III) concentrations (30 µmol/dish). The significantly different genotoxic responses induced by trans-[Cr(salen)(OH2)2]+ and mer[Cr(glygly)2]- suggest that a negative charge of the complex does not play a major role in producing the genotoxic effects. In previous studies, we have shown that Cr(V) mutagenicities and cytotoxicities were consistent with the chemical reactivities of the Cr(V) complexes (19, 22, 52). While there is no consistency between ligandexchange reactions of the Cr(III) complexes and their respective genotoxicity, there is one important chemical reaction that both cis-[Cr(phen)2(OH2)2]3+ and trans-[Cr(salen)(OH2)2]+ undergo. Unlike the other complexes, both of these imine complexes are readily oxidized to Cr(V) as reported previously (18, 19, 31, 32).1,2 The permeability of [Cr(O)2(phen)2]+ was significantly greater than that of cis-[Cr(phen)2(OH2)2]3+, suggesting that the Cr(V) species assisted uptake. The high genotoxicity of [Cr(O)2(phen)2]+ was consistent with the 1 Abbreviations: AAS, atomic absorption spectroscopy; ANOVA, analysis of variance; bipy, 2,2′-bipyridine; BN, binucleated; CHO, Chinese hamster ovary; en, 1,2-ethanediamine; GFAAS, graphite furnace atomic absorption spectroscopy; glygly, glycylglycine; GM, growth medium; HGPRT, hypoxanthine-guanine phosphoribosyl transferase; LMWCr, low-molecular weight chromium-binding substance; MEM, minimal essential medium; MEM-P, minimal essential medium without phenol red; MN, micronuclei; ox, oxalato(2-); phen, 1,10phenanthroline; PIXE, particle-induced X-ray emission; salen, N,N′ethylenebis(salicylideneiminato). 2 Sulfab, Y., and Nasreldin, M. Synthesis and characterization of dioxochromium(V) complexes with 1,10-phenanthroline and 2,2′-bipyridine. Electron transfer between dioxochromium(V) with an imineoxine copper(II) complex (to be submitted for publication).

Permeability and Genotoxicity of Cr(III) and Cr(V) Table 2. Summary of the Cellular Genotoxicity of the Cr Complexes

complex [Cr(glygly)2][Cr(en)3]3+ [Cr(salen)(OH2)2]+ [CrO(salen)]+ [Cr(phen)2(OH2)2]3+ [Cr(O)2(phen)2]+

genotoxicity/ cellular genotoxicity cellular Cr Cr level (MN/1000 level (MN/1000 (×10-15 g) BN/µmol of Cr) BN) (×1012) 8.2 17.3 13.3 10.3 7.2 65.7

0.01 0.1971 13.56 18.18 3.29 125

1.2 11.4 1020 1760 458 1902

increased level of Cr detected in the cells. If the genotoxicity of [Cr(O)2(phen)2]+ were solely attributed to the increased Cr uptake, however, then the genotoxicity of cellular Cr would be expected to be similar to that of cis[Cr(phen)2(OH2)2]3+, but reference to Table 2 shows that this is not the case. Furthermore, it has been shown that the Cr(V) species cleaves DNA at physiological pH values (19). Although it has been shown that trans-[Cr(salen)(OH2)2]+ is readily oxidized to [CrO(salen)]+, as evidenced by the rapid production of the dark blue coloration, this Cr(V) species is also unstable in cell medium at pH 7.4 (19, 32). Consequently, it is not surprising that the genotoxicity of [CrO(salen)]+ closely resembled that of trans-[Cr(salen)(OH2)2]+. Importantly, the permeabilities and consequently the cellular genotoxicities of the two complexes were also similar. This raises the question as to whether the uptake and genotoxicity are assisted by the ligand lipophilicity or whether they are being oxidized on the cell wall to assist in permeability and consequently genotoxicity. Previously, in vitro DNA studies have shown that [CrO(salen)]+ can interact strongly with DNA at physiological pH values in the presence of the oxidant (19). Importantly, the genotoxicity of the Cr(salen) complexes is much greater than those of [Cr(glygly)2]- and [Cr(en)3]3+ and is similar to that of [Cr(O)2(phen)2]+. The permeability of the complex is not significantly greater than those of the other Cr(III) complexes, although the genotoxicity and, consequently, cellular genotoxicity are markedly greater than those of the other Cr(III) complexes. In conclusion, if these Cr(V) species are produced intracellularly via oxidative enzymes (e.g., molybdoenzymes, oxidases), they would contribute to the observed genotoxic effects (18, 22). Furthermore, Cr(III) complexes can be oxidized to Cr(V) complexes by organic hydroperoxides, as occurs with the macrocyclic tetraamido-N ligands (53). Such organic peroxides are produced as normal metabolic byproducts of inter- and intracellular processes (54-57). The mechanism implicating the involvement of a Cr(V) species is consistent with the results reported by Sugden et al. (58), where it was shown that [Cr(bipy)2Cl2]+, which exhibits reactivities similar to that of the Cr phen analogue, was mutagenic only when bacterial cells were incubated under aerobic conditions. This difference was previously attributed to the involvement of OH• radicals, but as outlined in a recent paper, the results are more consistent with the involvement of Cr(V) (19). It was also shown by Sugden et al. (59) that both Cr(III) (cis-[Cr(phen)2Cl2]+ and cis-[Cr(bipy)2Cl2]+) and Cr(VI) complexes reverted S. typhimurium strains that were sensitive to oxidative mutations (TA102 and TA2638). This implies that both Cr(III) and Cr(VI) complexes may exert their mutagenicities by similar

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mechanisms, and it is feasible that Cr(V) intermediates are responsible. Despite the low potencies of the Cr(III) complexes, high concentrations of certain complexes, particularly those containing imine ligands, are genotoxic in mammalian cells. In conclusion, evidence shown here indicates that Cr(III) complexes that are readily oxidized to Cr(V) should be considered as potential carcinogens. This finding has potential adverse health implications with respect to the increased use of Cr(III) complexes as dietary supplements (20). It is also important in understanding the potential hazards of Cr(III) complexes that are often encountered in teaching and research laboratories.

Acknowledgment. We thank Mr. Tony Romeo for technical support. P.A.L. is grateful for support from the Australian Research Council (ARC) and the National Health and Medical Research Council. G.J.F.L. is grateful for support from the ARC. The views expressed in this article are those of the authors and do not necessarily reflect those of NOHSC.

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