Dinuclear Platinum Complexes as potential anticancer drugs : insights
in the intracellular distribution
Kalayda, G.V.
Citation
Kalayda, G. V. (2006, February 1). Dinuclear Platinum Complexes as potential anticancer
drugs : insights in the intracellular distribution. Retrieved from
https://hdl.handle.net/1887/4283
Version:
Corrected Publisher’s Version
License:
Licence agreement concerning inclusion of doctoral thesis in the
Institutional Repository of the University of Leiden
Downloaded from:
https://hdl.handle.net/1887/4283
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Abstract
Nine structurally distinctdinuclear platinum complexes have been evaluated in a novel model system for the investigation of renal epithelial toxicity of platinum drugs. The compounds are toxic when applied at the basolateral side of renal epithelia, whereas their toxicity on the apical side is negligible. Such a difference most likely results from the poor uptake of the complexes through the apical membrane as compared to the basolateral membrane. Toxicity of the compounds on the basolateral side varies depending on their structure. In the case of the dinuclear platinum complexes with rigid ligands, sterically hindered complexes are less toxic, due to their poor uptake and low reactivity towards glutathione.Among the dinuclear complexes with flexible ligands,cis-configured complexes are more toxic than their trans-isomers.
7.1. Introduction
Cisplatin is one of the most widely used drugs in cancer chemotherapy.1 It is used for treatment of testicular, ovarian, head and neck, bladder and small-cell lung cancer.2,3 However, the clinical application of the drug is limited due to acute and chronic kidney toxicity.4 The risk of renal failure precludes the use of higher doses in order to maximize the therapeutic effect. Furthermore, development of resistance in some tumors after repeated drug administrations presents a serious complication in chemotherapy.5 A number of new potential drug candidates were designed in order to overcome toxicity and resistance. The cisplatin analogues, carboplatin and oxaliplatin, have been approved for clinical use, and nowadays they are increasingly used in cancer treatment.6 These compounds are less toxic for kidneys, however, they do not satisfy the need for new antitumor drugs because of their limited range of activity.4,6
Dinuclear (and polynuclear) cationic platinum complexes present a class of new promising anticancer agents.7 In contrast to cisplatin and its analogues, they are water soluble, which is very convenient for clinical use. M any polynuclear platinum complexes exhibit high anticancer activity, have broad cytotoxic profiles and overcome cisplatin resistance.8-10 The dinuclear complexes with polyamines spermine and spermidine have entered Phase I clinical trials,7,11 and the trinuclear complex BBR3464 has proceeded to Phase II clinical trials.7,10 Anticancer activity and the mechanism of action of dinuclear platinum complexes have been extensively studied.12-15 However, nephrotoxicity of these complexes has not been reported. Evaluation of the toxicity of potential antitumor drugs is very important because it gives an indication of their clinical usefulness. Furthermore, development of structure-toxicity relationships might help in the design of new drugs with low toxicity.
This chapter reports nephrotoxicity of nine structurally different dinuclear platinum complexes (Figure 7.1). All of them have been previously found to exhibit high cytotoxicity in various cancer cell lines.8,9,16 These complexes represent two different types of dinuclear platinum drugs: complexes with rigid ligands and complexes with flexible bridging ligands. In this chapter, correlations between nephrotoxicity, uptake of the compounds and their reactivity are presented. In addition, structure-toxicity relationships are discussed.
7.2. Experimental section 7.2.1. Materials
K2PtCl4was obtained from Johnson&M atthey. Cis-Pt(NH3)2Cl2 (1) was synthesized
according to the literature method.17 The dinuclear platinum complexes with isomeric azines, [{cis-Pt(NH3)2Cl}2(µ-pzn)]Cl2 (2), [{cis-Pt(NH3)2Cl}2(µ-pmn)]Cl2 (3),
Pt H3N H3N Cl Cl 1 H2N(CH2)nNH2 Pt NH3 Cl NH3 Pt H3N H3N Cl H2N(CH2)nNH2 Pt NH3 H3N Cl Pt H3N Cl NH3 7(n=4) 8(n=6) 9(n=4) 10 (n=6) N N Pt O H Pt H3N H3N NH3 NH3 N N N Pt O H Pt H3N H3N NH3 NH3 5 6 Pt H3N NH3 Pt Cl NH3 NH3 Cl N N 2 3 4 H3N NH 3 Pt Cl NH3 NH3 Cl N N H3N NH 3 Pt Cl NH3 NH3 Cl N N Cl2 Cl2
(NO3)2 (NO3)2 (NO3)2
(NO3)2 (NO3)2
Figure 7.1. Schematic representation of cisplatin and the dinuclear platinum complexes used in this study.
with the azole ligands, [{Pt(NH3)2}2(µ-OH)(µ-pz)](NO3)2 (5) and [{Pt(NH3)2}2
(µ-OH)(µ-ta)](NO3)2 (6), were synthesized according to the literature procedures.9,18
[{cis-Pt(NH3)2Cl}2(µ-H2N(CH2)4NH2)](NO3)2 (7), [{cis-Pt(NH3)2Cl}2(µ-H2N(CH2)6NH2)](NO3)2
(8), [{trans-Pt(NH3)2Cl}2(µ-H2N(CH2)4NH2)](NO3)2 (9) and [{trans-Pt(NH3)2Cl}2
(µ-H2N(CH2)6NH2)](NO3)2 (10) were synthesized as previously described.19-21 Glutathione
(GSH) was obtained from Sigma.
7.2.2. Evaluation of epithelial toxicity
Nephrotoxicity of the dinuclear platinum complexes was evaluated using a recently established in vitro assay based on the measurements of transepithelial electrical resistance (TEER) of the C7 clone of Madin-Darby canine kidney (MDCK) cells.22
The C7 clone of MDCK cells resembles principal cells of the renal collecting duct and has a particularly high transepithelial electrical resistance.23 The cells were grown in minimal essential medium (MEM) with Earle’s salts, nonessential amino acids, glutamic acid, 10% fetal calf serum (Biochrom, Berlin, Germany), penicillin (100 u/ml) and streptomycin (100 µg/ml), in a 5% CO2, 95% air atmosphere.
exposed to the medium in the lower compartment, and the apical side is in contact with the upper medium.
Medium exchange and transepithelial electrical resistance measurements started three days after seeding the cells. TEER measurements have been described in detail previously.24 After C7 monolayers had developed resistance > 2 kΩ×cm2, the platinum complexes dissolved in phosphate buffered saline (PBS) containing Ca2+ and Mg2+ were added in triplicate to the upper or lower medium compartment at final concentration of 100 µM. The solvent (PBS with Ca2+ and Mg2+) was added to the cells in the control experiments. Then, transepithelial electrical resistance was measured at least once a day in each well.
7.2.3. Measurements of platinum accumulation in C7 cells
C7 cells were seeded in filter cups (2.8×105cells/cup) and allowed to grow for four days under the conditions described above. Then, the cells were exposed to the dinuclear platinum complexes for 2 h at final concentration of 100 µM (the complexes were added to the cells in triplicate). After incubation, the cells were washed twice with PBS containing Ca2+ and Mg2+; subsequently 350 µl/cup lysis buffer containing 10 mM Tris, pH 8.0, 150 mM NaCl and 0.4% Triton X-100 were added. The content of each well was collected in an Eppendorf tube, and 100 µl of 10% SDS (sodium dodecyl sulfate) was added to each sample. After that, the samples were treated for 30 min at 50 °C with 20 µg/ml of proteinase K (BV Sphaero Q, Gorinchem, the Netherlands). The samples were subsequently diluted with 20% HNO3 to a final volume of 3 ml for measuring the platinum content. Platinum
concentration was measured on Varian Vista-MPX charge-coupled simultaneous ICP-OES (inductively coupled plasma optical emission spectrometer). The uptake experiments were carried out with duplicate cultures.
7.2.4. Interaction of the dinuclear platinum complexes and cisplatin with glutathione Interaction of complexes 1 – 10 (4 mM) with glutathione (16 mM) in millipore water (pH ∼ 7) was followed at 37 °C by analytical high performance liquid chromatography. The reactions were quantified based on the relative integration values of the UV peaks of the starting complexes in the chromatograms.
HPLC analysis was carried out on a Alltima 3µ C18 reversed phase column (150 × 4.6 mm) with a flow rate of 1 ml/min, sample load of 15 µl and the following gradient conditions: 95% B for 2 min, then from 95% B to 70% B in 16 min, and subsequently from 70% B to 10% B in 3 min (eluent A: acetonitrile with 0.1% CF3COOH / eluent B: 0.1%
aqueous solution of CF3COOH). After the column separation, the flow was directed to the
7.3. Results
7.3.1. Evaluation of toxicity: transepithelial electrical resistance measurements
Epithelial toxicity of the complexes was evaluated by an in vitro assay based on the measurements of transepithelial electrical resistance of the C7 clone of MDCK cells. C7 cells are able to form tight and polarized monolayers with high transepithelial electrical resistance. This parameter is very sensitive to the changes in monolayer permeability and integrity. It has been recently shown that cisplatin induced epithelial apoptosis is accompanied by increased epithelial permeability in this model system.22 Thus, TEER measurements provide valuable information about the functional changes in epithelial monolayer permeability and viability of C7 cells.
The dinuclear platinum complexes and cisplatin (as a reference compound) were added to the apical or basolateral medium after the cells formed electrically tight monolayers (TEER > 2 kΩ×cm2). Figure 7.2 reflects the changes in TEER after addition of the dinuclear platinum complexes with rigid ligands (2 – 6) and cisplatin at the basolateral side of a monolayer. The changes in TEER after application of the dinuclear complexes with flexible ligands (7 – 10) and cisplatin to the basolateral medium are shown in Figure 7.3. As is clear from Figure 7.2 and 7.3, all platinum complexes reduce TEER already after one day of incubation, however, to different extents. Cisplatin (1) and compounds 2, 3, 7, 8 induce a complete breakdown of transepithelial resistance within 24 hours. Complexes 5, 6 and 10 lower TEER to 35 – 40% of the initial value, whereas complexes 4 and 9 only reduce resistance to 75% and 85%, respectively. Addition of the solvent to the basolateral medium does not affect electrical resistance (TEER of the controls increase by 7.5% after one day of incubation). 0 20 40 60 80 100 120 0 1 2 3 4 5 time, days T E E R (t )/ T E E R (0 ), % solvent 1 2 3 4 5 6
0 20 40 60 80 100 120 0 1 2 3 4 5 time, days T E E R (t )/ T E E R (0 ), % solvent 1 7 8 9 10
Figure 7.3. Changes in transepithelial electrical resistance during the incubation with complexes 1 and 7 – 10added to the basolateral medium. The solvent (PBS with Ca2+ and Mg2+) was added to the cells in control experiments.
Application of the complexes as well as the solvent at the apical side of C7 monolayers has no effect on TEER (Figure 7.4). Only complex 2 lowers resistance to 92.5% of the initial value. In the case of other complexes, transepithelial resistance did not significantly change after one day of incubation.
0 20 40 60 80 100 120 140 s 1 2 3 4 5 6 7 8 9 10 T E E R (2 4 h )/ T E E R (0 h ), % apical basolateral
Figure 7.4. Transepithelial electrical resistance 24 hours after application of complexes 1 – 10 to the apical or basolateral medium. The solvent (PBS with Ca2+ and Mg2+) was added to the cells in control experiments.
for cisplatin, carboplatin and oxaliplatin.22 These data indicate that the dinuclear platinum complexes as well as cisplatin and its analogues do not exhibit toxic effects at the apical side of renal epithelium. In contrast, the complexes are rather toxic when applied at the basolateral side, as is clear from the decrease of transepithelial electrical resistance. Compounds 2, 3, 7 and 8 induce a complete breakdown of TEER within 24 hours, while the other compounds partially decrease resistance.
Basolateral toxicity of the dinuclear complexes with rigid ligands is in the order: 4 < 5 ≈ 6 < 3 ≈ 2 (Figure 7.2 and 7.4). Interestingly, steric hindrance in these compounds is in the reverse order: 2 < 3 < 6 ≈ 5 < 4. The least toxic complex 4 has the most sterically constrained geometry due to the smallest distance between two platinum atoms.16 Moderately toxic complexes 5 and 6 are somewhat less sterically hindered. Relatively flexible compounds 2 and 3 show high toxicity, comparable to that of cisplatin (1). Thus, among the dinuclear platinum complexes with rigid ligands more sterically hindered compounds are less toxic.
Toxicity of the dinuclear complexes with flexible ligands at the basolateral side is in the following order: 9 < 10 < 7 ≈ 8 (Figure 7.3 and 7.4), showing that the complexes with trans-configuration are less toxic than their cis-counterparts. Both complexes of cis-geometry exhibit the same high toxicity, independent on the length of the aliphatic chain in the diamine linker. In the case of trans-complexes, complex 10 that possesses a longer 1,6-hexanediamine linker is significantly more toxic than its analogue 9 with a shorter bridging ligand (1,4-butanediamine).
7.3.2. Uptake of the platinum complexes by MDCK-C7 cells
Uptake of the dinuclear platinum complexes and cisplatin through the apical and basolateral membrane of C7 cells was studied by means of measuring platinum concentration inside the cells after 2 hours of incubation with the compounds added to the apical or basolateral medium, respectively. The results presented in Figure 7.5 show that accumulation of all the complexes through the apical membrane is lower than through the basolateral membrane.
Uptake of the dinuclear platinum complexes with rigid ligands (2 – 6) at the basolateral side as compared to each other and to cisplatin is in the order: 4 < 5 < 6 < 3 < 1 < 2. These data indicate a relationship between the structure of the complexes and their accumulation through the basolateral membrane. The steric hindrance in the complexes with rigid ligands is in the reverse order. Therefore, complexes that are more flexible are taken up better than less flexible, sterically hindered compounds.
in the following order: 10 < 9 ≈ 7 < 8. In this case, no correlation between the structure of the complexes and their uptake could be made.
0 0.5 1 1.5 2 2.5 1 2 3 4 5 6 7 8 9 10 n m o l P t/ 1 0 7 c e ll s apical basolateral
Figure 7.5. Platinum accumulation through the apical or basolateral membrane of C7 cells after 2 hours of incubation with complexes 1 – 10.
7.3.4. Reactivity of the dinuclear platinum complexes and cisplatin towards glutathione Interaction of platinum complexes with the tripeptide glutathione (GSH) plays an important role in the mechanism of their nephrotoxicity. GSH is responsible for scavenging reactive oxygen species in the cells, and thus, prevents lipid peroxidation. Binding of platinum complexes to glutathione is known to disrupt functions of some enzymes and proteins (e.g. Na+/phosphate cotransporters25 and peroxidase26). Furthermore, it results in depletion of the GSH pool, which subsequently leads to lipid peroxidation, mitochondrial damage and apoptosis of kidney cells.27,28
In order to better understand the toxic effects of the dinuclear platinum complexes, their reactivity towards glutathione was investigated. The reactions of 1 – 10 with excess of GSH were followed by analytical HPLC. The half-lives of these complexes, which are given in Table 7.1, were determined based on the relative integration values of the UV-peaks in HPLC chromatograms (spectra not shown).
Table 7.1. The times of disappearance of 50% complexes 1 – 10 (t1/2) during their
reactions with 4 equiv. of glutathione at 310 K.
Complexes 2 and 3 are the most reactive among all the compounds. Interaction of 3 with glutathione is somewhat slower as compared to 2, which is probably a result of the closer proximity of platinum atoms in 3. Other complexes with rigid ligands (4, 5 and 6) are much less reactive towards GSH, which is likely to be caused by their steric hindrance. Interestingly, more sterically constrained compound 4 reacts faster than 5 and 6. The cleavage of the stable five-membered ring containing the bridging hydroxo group in the latter complexes is likely to be more difficult than the substitution of the chloride ligand in 4. Reactivity of complex 5 is lower than that of 6. Such a difference can be explained by the presence of the intramolecular hydrogen bond between the ammine and the azole in 6 that makes the OH bridge less stable.
Cisplatin (1) and the dinuclear platinum complexes with flexible ligands (7 – 10) react with glutathione slower than 2 and 3 but faster than the sterically hindered complexes 4, 5 and 6. Among compounds 7 – 10, the cis-configured dinuclear complexes are less reactive than their trans-counterparts. It has been recently found that a dinuclear complex of cis-geometry is able to form a stable macrochelate with a bridging GS-group and a bridging diamine.29 In contrast, trans-complexes readily degrade into mononuclear species.30 Thus, the formation of stable macrochelates by cis-configured complexes may explain their lower reactivity as compared to their trans-isomers. Interestingly, the complexes with a shorter aliphatic chain of the bridging diamine (7 and 9) interact with glutathione faster than similar complexes with a longer bridging ligand (8 and 10, respectively).
7.4. Discussion and conclusions
The results presented in this chapter show that the toxic effects of the dinuclear platinum complexes on renal epithelial cells strongly depend on the site of application. The complexes exhibit negligible toxicity at the apical side, while they are rather toxic when applied at the basolateral side. The uptake of the dinuclear platinum complexes is also site-dependent. The compounds accumulate much better through the basolateral membrane than through the apical membrane. It has been earlier suggested that basolateral uptake31 and toxicity of platinum-based drugs22 are mediated by organic cation transporter, which is located in the basolateral but not in the apical membrane of MDCK-C7 cells.31,32 Thus, cell polarity plays an important role in nephrotoxicity of dinuclear platinum complexes. Higher basolateral toxicity results from the better uptake of the complexes through the basolateral membrane as compared to the apical membrane.
Among the dinuclear platinum complexes with rigid ligands, more sterically constrained compounds are less toxic. Such relationship between steric hindrance and toxicity of the complexes is determined, at least in part, by their uptake and their reactivity towards glutathione. As is clear from the above data, sterically hindered complexes accumulate in the cells less efficiently, which results in their lower toxicity. Reactivity of the complexes towards GSH is a very important factor in nephrotoxicity. Depletion of glutathione may lead to lipid peroxidation and subsequently to the mitochondrial damage and apoptosis of kidney cells.28 Therefore, complexes with lower reactivity are expected to decrease the intracellular concentration of glutathione much slower, resulting in lower toxicity. Among the complexes with rigid ligands, sterically hindered compounds have been found less reactive towards GSH. Complex 4 presents an exception, as it is the most sterically constrained but not the least reactive compound. However, it is the least toxic of all the complexes, most likely due to its poor uptake.
It is interesting to compare the toxicity of the dinuclear platinum complexes with rigid ligands and their antitumor activity. According to the previously reported data, cytotoxicity of the complexes in various cell lines is in the order: 4 < 3 < 2 < 5 ≈ 6.9,16 In the case of the azine-bridged complexes 2 – 4, the compounds showing higher anticancer activity are also more toxic. However, the moderately toxic azole-bridged complexes 5 and 6 are highly active against a number of tumors.9 Apparently, toxicity and cytotoxicity of the dinuclear complexes with rigid ligands are determined by different factors. Toxic effects of the complexes depend on their reactivity towards GSH. Anticancer activity of the azine-bridged complexes 2 – 4 is likely to be mainly determined by their reactivity towards DNA, whereas structural difference of DNA adducts probably plays a major role in cytotoxicity of the azole-bridged complexes 5 and 6.
In the case of the dinuclear platinum complexes with flexible ligands, cis-configured compounds show higher toxicity than their trans-counterparts. However, it is not clear which factors determine such a relationship between the structure of the complexes and their nephrotoxicity. No correlation between the uptake of these compounds and their structural features has been found. Reactivity studies have shown that trans-complexes are somewhat more reactive towards glutathione than cis-configured compounds. Therefore, from the reactivity point of view the complexes of trans-geometry would be expected to be more toxic, which is not the case. It must be also noted that there is no great difference in reactivity between cis- and trans-complexes, whereas the complexes significantly differ in toxicity.
than of 10. Cytotoxicity of cis-complexes 7 and 8 is approximately the same independent on the length of the aliphatic diamine.8 Nephrotoxicity of these two compounds is also not significantly different. Most likely, toxic effects and antitumor activity of the dinuclear platinum complexes with flexible ligands are determined by the same factors.
Thus, epithelial toxicity of dinuclear platinum complexes depends on the site of application. The complexes exhibit negligible toxicity at the apical side of renal epithelial cells. Toxic effects at the basolateral side vary with the structure of the compounds. In the case of the dinuclear complexes with rigid ligands, more sterically hindered complexes are less toxic due to their poorer uptake and lower reactivity towards glutathione. Among the complexes with flexible ligands, trans-configured compounds are less toxic than their cis-counterparts. However, it is not clear which factors determine structure-toxicity relationship in this case. Understanding the relationships between structure and toxicity of platinum drugs is of great importance for the design of new anticancer agents, and it deserves detailed investigation.
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