Canadian Journal of Chemistry Modulation of the metabolism of cis-platin in blood plasma by glutathione Journal: Manuscript ID Manuscript Type: Date Submitted by the Author: Complete List of Authors: cjc-2015-0395.R1 Article 07-Oct-2015 Sooriyaarachchi, Melani; University of Calgary, Chemistry Gibson, Matthew; University of Calgary, Chemistry Lima, Bruno; University of Calgary, Chemistry Gailer, Juergen; University of Calgary, Chemistry Dr Keyword: Canadian Journal of Chemistry Blood plasma, cis-platin, chemoprotection, glutathione, metallomics t af https://mc06.manuscriptcentral.com/cjc-pubs Page 1 of 28 Canadian Journal of Chemistry 1 2 3 4 5 6 7 8 9 10 Melani Sooriyaarachchi, Matthew A. Gibson, Bruno dos S. Lima and Jürgen Gailer* Department of Chemistry, University of Calgary, 2500 University Drive NW, Calgary, AB, T2N 1N4, Canada. Tel: 403-210-8899, Fax: 403-289-9488, e-mail: jgailer@ucalgary.ca t af Dr 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 Modulation of the metabolism of cis-platin in blood plasma by glutathione *Corresponding author: J. Gailer, Fax: 403-289-9488, e-mail: jgailer@ucalgary.ca 1 https://mc06.manuscriptcentral.com/cjc-pubs Canadian Journal of Chemistry Page 2 of 28 Abstract 46 The anticancer drug cis-platin (CP) is in worldwide clinical use to treat a variety of cancers, but is 47 inherently associated with severe toxic side-effects. Previous animal studies revealed that its 48 neurotoxicity can be significantly reduced by the co-administration of L-glutathione (GSH) without 49 affecting the anti-cancer effect. The underlying molecular mechanism, however, has remained elusive. 50 Since the bloodstream is a likely biological compartment where CP-derived hydrolysis products may 51 react with GSH, we have employed a recently developed metallomics tool to gain insight into the 52 interaction of CP and GSH in rabbit plasma in vitro. After the addition of increasing GSH:CP molar 53 ratios to plasma (25:1, 50:1 and 100:1), the determination of the Pt-distribution 5 min and 2 h later 54 revealed the formation of a Pt-GSH complex which did not bind to plasma proteins. The 55 simultaneously obtained Zn-distribution in plasma revealed a progressively more pronounced 56 perturbation of the Zn metalloproteome with increasing GSH:CP molar ratios at the 5 min time point, 57 which partially reversed at the 2 h time point. The formation of Pt-GSH species in plasma is therefore 58 likely to be directly involved in the process by which GSH protects mammalian organisms from CP- 59 induced neurotoxicity, nephrotoxicity and possibly other organ-based toxicities. t af Dr 45 60 61 62 Key words 63 Blood plasma, cis-platin, chemoprotection, glutathione, metallomics 64 65 66 67 68 69 2 https://mc06.manuscriptcentral.com/cjc-pubs Page 3 of 28 Canadian Journal of Chemistry 70 Introduction 71 The serendipitously discovered anticancer drug cis-platin (CP)1,2 heralded the dawn of metallodrugs 72 and remains one of the most effective chemotherapeutic agents more than five decades after its 73 discovery3. In fact, 50-70% of all cancer patients are treated with CP either alone or in combination 74 with other anticancer drugs.4 Despite its broad spectrum of activity against a large variety of cancers,5 75 the intravenous administration of patients with CP is associated with severe toxic side-effects, 76 including nephrotoxicity, ototoxicity and neurotoxicity6,7. Although the nephrotoxicity of CP can be 77 somewhat ameliorated by the administration of patients with hypertonic saline or mannitol, 78 clinical procedures exist to completely eliminate its ototoxicity and neurotoxicity which therefore 79 constitute the primary dose limiting factors.9 80 The aforementioned toxic side-effects of CP, however, have been shown to be significantly reduced in 81 animal studies when small-molecular-weight (SMW) compounds – also referred to as 82 ‘chemoprotective agents’ − were co-administered.9,10 Although this principle approach can adversely 83 affect the anti-tumor effect of CP,9 it has been demonstrated that this can be sometimes altogether 84 avoided if the timing between the administration of CP and the chemoprotective agent as well as their 85 route of administration is carefully optimized.11 More recent investigations aimed at better 86 understanding the biomolecular mechanisms by which chemoprotective agents ameliorate the toxicity 87 of CP revealed that sodium thiosulfate, N-acetyl-L-cysteine and D-methionine react with CP-derived 88 hydrolysis products in human blood plasma in vitro.12-14 Since some of the CP-derived hydrolysis 89 products that are formed in blood plasma are highly toxic,9 their ‘neutralization’ by chemoprotective 90 agents likely constitutes one important mechanism of action by which the latter mitigate the toxic 91 side-effects of CP.15 92 L-Glutathione (GSH) represents another SMW sulfur compound that has been demonstrated to be 93 effective in terms of reducing the toxic side-effects of CP in animals 16,17 and humans18. In contrast to 8 no t af Dr 3 https://mc06.manuscriptcentral.com/cjc-pubs Canadian Journal of Chemistry Page 4 of 28 the aforementioned chemoprotective agents, however, GSH is naturally present in human blood plasma 95 at concentrations of 1-5 µM19 and the plasma concentration of oxidized glutathione (GSSG) is ~2 µM 96 in rats.20 The administration of exogenous GSH to animals to decrease the neurotoxicity of CP without 97 diminishing the anti-tumor effect of the latter.16 Given that CP (1.0 mg/kg) was administered 98 intraperitoneally and GSH (200 mg/kg) intravenously to rats in one particular study,16 it is possible that 99 the underlying toxicologically relevant processes occurred in blood plasma.12-14 Since no direct 100 experimental evidence in support of this possibility has been obtained, we have conducted systematic 101 in vitro experiments with rabbit plasma to which pharmacologically relevant GSH:CP molar ratios – 102 defined as those close to those where the desired amelioration of the toxicity of CP was achieved in 103 vivo − were added. The analysis of the obtained mixtures with a metallomics tool − a size-exclusion 104 chromatography (SEC) system coupled on-line to an inductively coupled plasma atomic emission 105 spectrometer (ICP-AES)21 – allowed us to observe changes in the Pt-distribution in plasma between the 106 5 min and 2 h time point. Since ICP-AES allows to simultaneously monitor the emission lines of the 107 endogenous Fe, Cu and Zn-metalloproteins in addition to those of Pt, changes in the plasma 108 distribution of these metals (e.g. caused by GSH) can also be observed. The results revealed a rapid 109 formation of Pt-GSH adduct(s) in plasma, which strongly suggests their formation to be directly 110 implicated in the mechanism by which GSH can ameliorate the toxic side-effects of CP in vivo.16,17,22 t af Dr 94 111 112 Experimental 113 Chemicals and Solutions 114 Cis-platin (1 mg cis-Pt(NH3)2Cl2/mL; this solution also contained 1 mg mannitol and 9 mg NaCl; 115 sterile) was obtained from Hospira (Montreal, QC, Canada). Phosphate-buffered saline buffer (PBS) 116 tablets were purchased from Sigma-Aldrich (St. Louis, MO, USA) and the corresponding PBS-buffer 117 (10 mM phosphate, 2.7 mM KCl, 137 mM NaCl) was prepared by dissolving PBS tablets in the 4 https://mc06.manuscriptcentral.com/cjc-pubs Page 5 of 28 Canadian Journal of Chemistry 118 appropriate volume of de-ionized water (Simplicity water purification system; Millipore, Billerica, 119 MA, USA) followed by pH adjustment to 7.4. The obtained solution was filtered through 0.45 µm 120 nylon-filter membranes (Mandel Scientific, Guelph, ON, Canada) before use. A mixture of protein 121 standards which contained thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), 122 myoglobin (17 kDa), and vitamin B12 (1.35 kDa) was obtained from Bio-Rad Laboratories (Hercules, 123 CA, USA) to calibrate the employed SuperdexTM 200 Increase SEC column. GSH (≥98%) was 124 obtained from Sigma-Aldrich and an aqueous stock solution with a concentration of 142 mg/mL (0.46 125 M) was prepared by dissolving it in PBS-buffer followed by pH adjustment to 7.4 using 4 M NaOH. 126 This solution was prepared daily and was used within 6 hours during which it was stored at 5 ºC in a 127 vial with minimal head space. 128 Dr SEC-ICP-AES system 130 The SEC-ICP-AES system that was employed was comprised of a Smartline 1000 HPLC pump 131 (Knauer, Berlin, Germany) and a Rheodyne 9010 PEEK injection valve (Rheodyne, Rhonert Park, CA, 132 USA) which was equipped with a 0.5 mL PEEK injection loop (0.5 mL). A pre-packed SuperdexTM 133 200 Increase 10/300 GL high performance size-exclusion chromatography (SEC) column (30 x 1.0 cm 134 I.D., particle size 8.6 µm, fractionation range 600 - 10 kDa; GE Healthcare, Piscataway, NJ, USA) was 135 used in conjunction with a PBS-buffer mobile phase (as described in ‘chemicals and solutions’) and a 136 flow rate of 1.0 mL/min (column temperature 22º C). We note that the employed SEC column contains 137 smaller particles than the columns that we used in our previously studies,12-14,23 but that the backbone is 138 identical and resulted in a similar separation of plasma proteins (data not shown). Simultaneous 139 multielement-specific detection of Ca (317.933 nm), C (193.091 nm), Cu (324.754 nm), Fe (259.940 140 nm), Mg (280.271 nm), Pt (214.423 nm), S (180.731 nm) and Zn (213.856 nm) in the column effluent 141 was achieved with a Prodigy, high-dispersion, radial-view ICP-AES (Teledyne Leeman Labs, Hudson, t af 129 5 https://mc06.manuscriptcentral.com/cjc-pubs Canadian Journal of Chemistry Page 6 of 28 142 NH, USA) at an Ar gas-flow rate of 19 L/min, an RF power of 1.3 kW and a nebulizer gas pressure of 143 35 psi. The nebulizer gas flow rate was 1.4 L of Ar/min. A 400 s delay was implemented between 144 injection and data acquisition and data acquisition window was 2000 s. The raw data (Salsa Software) 145 were imported into Sigmaplot 13.0, smoothed using the bi-square algorithm and then the peak areas 146 were determined. 147 148 Preparation of blank plasma, CP-spiked plasma and GSH-spiked plasma 150 Rabbit plasma (Lot 0165) was obtained from Cedarlane Corporation (Burlington, ON, Canada) and 151 had been prepared by pooling plasma from 3.5 months old male New Zealand white rabbits using 152 sodium heparin (158 USP units/10 mL of whole blood) as the anticoagulant. Plasma (2.2 mL aliquots) 153 was stored at -60 ˚C and received frozen on dry ice. Individual plasma vials were thawed at room 154 temperature for 45 minutes and incubated at 37º C for 30 min before pharmacologically relevant doses 155 of CP and/or GSH were added. “Blank” plasma was prepared by spiking 1.8 mL of rabbit plasma with 156 130 µL of PBS-buffer. The solution that served as the CP control plasma was prepared by adding 75 157 µL of the CP stock solution (1 mg/mL) and 54 µL of PBS-buffer to 1.8 mL of plasma. The final 158 concentration 159 pharmacologically/therapeutically relevant24. This mixture was incubated at 37º C and samples were 160 withdrawn for analysis after 5 min and 2 h. t af Dr 149 was ~0.04 mg CP/mL of plasma (~0.13 mM), which is 161 162 Analysis of GSH and CP-spiked plasma (GSH:CP molar ratios 25:1, 50:1 and 100:1) 163 Plasma (1.8 mL) was incubated at 37° C, spiked with 54 µL of the GSH stock solution and 1 min later 164 75 µL of the CP stock solution (1 mg/mL) was added (GSH:CP molar ratio of 100:1). The obtained 165 mixture was incubated at 37° C and analyzed after 5 min and 2 h. The solution that served as the GSH 6 https://mc06.manuscriptcentral.com/cjc-pubs Page 7 of 28 Canadian Journal of Chemistry 166 control (corresponding to the GSH:CP=100:1 molar ratio) was prepared by adding 54 µL of GSH stock 167 solution and 75 µL of PBS-buffer to 1.8 mL of plasma. The obtained mixture was incubated at 37° C 168 for 6 min and 2 h + 1 min and was subsequently analyzed. 169 The solution corresponding to the GSH:CP molar ratio of 50:1 was prepared by spiking 1.8 mL of 170 plasma (that had been incubated at 37° C) with 27 µL of the GSH stock solution and 27 µL of PBS- 171 buffer. After mixing 75 µL of the CP stock solution (1 mg/mL) were added 1 min later. The solution 172 corresponding to a GSH:CP molar ratio of 25:1 was prepared in an analogous manner by spiking 1.8 173 mL of plasma with 13.5 µL of the GSH stock solution and 41 µL of PBS-buffer. After incubation at 174 37° C for 1 min, 75 µL of the CP stock solution (1 mg/mL) was added and the obtained mixture was 175 incubated at 37° C and analyzed 5 min and 2 h later. All experiments were carried out in triplicate and 176 representative chromatograms are presented. Dr 177 Analysis of CP-spiked PBS-buffer and GSH+CP-spiked PBS-buffer (GSH:CP molar ratio 100:1) 179 The CP control was prepared by spiking PBS buffer (pH 7.4, 1.8 mL) that had been incubated at 37° C 180 with 75 µL of the CP stock solution (1 mg/mL) and 54 µL of PBS-buffer. After incubation of this 181 mixture at 37° C, the latter was analyzed after 5 min and 2 h. Then, PBS buffer (pH 7.4, 1.8 mL) was 182 spiked with 54 µL of GSH stock solution, incubated at 37° C for 1 min and then spiked with 75 µL of 183 the CP stock solution (1 mg/mL). The obtained mixture was maintained at 37º C and samples were 184 withdrawn for duplicate analysis after 5 min and 2 h. All attempts to separate excess GSH from the 185 co-eluting Pt-S peak on a PRP X-100 anion-exchange HPLC column (30 x 0.46 cm I.D.) using a 186 variety of different PBS-buffer concentrations were unsuccessful. t af 178 187 188 Results and Discussion 7 https://mc06.manuscriptcentral.com/cjc-pubs Canadian Journal of Chemistry Page 8 of 28 189 After the intravenous administration of cancer patients with CP, all CP-derived metabolites will 190 interact with blood constituents, tumor cells (intended) and healthy tissue cells (unintended). Since the 191 latter interaction is associated with organ-based toxicities 192 explored to increase the tumor selectivity of metal-based drugs7. In principle, this can be achieved by 193 optimizing the tumor selectivity of novel (Pt-based) drug structures3,29,30 or by improving the selective 194 delivery of established anticancer drugs to tumor cells (e.g. using substituted ß-cyclodextrins and calix 195 [4] arenes)31-33. 196 Another feasible strategy to improve the safety of clinically used anticancer metallodrugs aims to 197 selectively reduce their toxic side-effects by the co-administration of chemoprotective agents. 198 Unfortunately, the only chemoprotective agent that has been clinically approved to date, amifostine, 199 did not result in significantly reduced ototoxicity compared to the cisplatin only group.9 Three other 200 chemoprotective agents, sodium thiosulfate, N-acetylcysteine and D-methionine, however, were 201 recently demonstrated to modulate the metabolism of CP in human plasma in vitro.12-14 The common 202 underlying biomolecular mechanism appears to involve the reaction of CP-derived hydrolysis products 203 with each chemoprotective agent to form Pt-chemoprotective agent complexes, some of which could 204 be structurally characterized.14 The fact that the parent anticancer drug CP remained in plasma for up 205 to 50 min – at least these in vitro studies − demonstrates that the co-administration of CP with 206 chemoprotective agents will not entirely inactivate the parent anticancer drug. Since this principle 207 approach has potential to improve the quality of life of patients that are being treated with CP, we 208 wanted to gain insight into the mechanism by which GSH reduced the toxic side-effects of CP in 209 previously conducted animal studies16,17 and one study involving cancer patients.18 To elucidate if and 210 in what way GSH will affect the metabolism of CP in rabbit plasma, we have conducted in vitro 211 experiments which involved the addition of increasing GSH:CP molar ratios to plasma followed by its 212 analysis with a recently developed metallomics method. The latter allows to simultaneously determine 25-28 , various approaches are currently being t af Dr 8 https://mc06.manuscriptcentral.com/cjc-pubs Page 9 of 28 Canadian Journal of Chemistry 213 essentially all CP-derived Pt-metabolites in plasma23 and all major Cu, Fe and Zn-containing 214 endogenous metalloproteins.21 215 216 Metabolism of CP in plasma 217 After the addition of a pharmacologically relevant dose of CP to plasma, the obtained mixture was 218 analyzed after 5 min and 2 h (Fig. 1A). At the 5 min time point 1.5% of total Pt eluted bound to plasma 219 proteins (Pt-PP), ~4% in form of CP-derived hydrolysis products (CPHP) and almost 95% as CP 220 (Table 1). The results that were obtained for the 2 h time point revealed a significant increase of Pt-PP 221 (26.6%), that 22.6% of total Pt eluted in form of CPHP’s and that ~51% of free CP was present in 222 plasma (Table 1). In general, these results are in good accord with previous results that were obtained 223 after the addition of the same CP-dose to human plasma.23 Dr 224 Effect of increasing GSH:CP molar ratios on the metabolism of CP in plasma 226 In order to observe the effect of GSH on the metabolism of CP, increasing amounts of GSH were 227 added to plasma first followed by a pharmacologically relevant dose of CP 1 min later to achieve 228 GSH:CP molar ratios of 25:1, 50:1 and 100:1. The obtained mixtures were analyzed after 5 min and 2 229 h (Fig. 1B-D). The average peak area counts for each detected Pt peak (as percent of the total Pt area) 230 are depicted in Table 1. For the sake of clarity, the results will be discussed first for the 5 min time 231 point, followed by those obtained for the 2 h time point. 232 At the 25:1 GSH:CP molar ratio (Fig. 1B) and compared to the control experiment (Fig. 1A), no 233 statistically significant increase was observed for Pt-PP,but 2.4% of Pt eluted in form of a Pt-peak that 234 was not detected in the control experiment and corresponds to a putative in situ formed Pt-GSH 235 complex. The retention time of this Pt-GSH complex was comparable to that of related Pt-complexes 236 that were detected in human plasma after incubation with CP and sodium thiosulfate,12 N-acetyl-L- t af 225 9 https://mc06.manuscriptcentral.com/cjc-pubs Canadian Journal of Chemistry Page 10 of 28 cysteine13 or D-methionine.14 Furthermore, 0.6% less Pt eluted in form of CPHP’s and 2.2% less Pt 238 eluted in form of CP (compared to the control group). An increase of the GSH:CP molar ratio from 239 25:1 to 100:1 marginally affected the amount of Pt that eluted as Pt-PP, but significantly increased the 240 amount of Pt that eluted in form of a Pt-GSH complex by 5.2% (Fig 1B-D, red lines). In accord with 241 these results, the amount of Pt that eluted in form of CPHP’s decreased by 0.4% and 7.2% less Pt 242 eluted in form of CP at the 100:1 GSH:CP molar ratio (compared to the control group). 243 As expected, the results that were obtained for increasing GSH:CP molar ratios on the Pt-distribution 244 in plasma at the 2 h time point were progressively more pronounced (Fig. 1B-D, blue lines) compared 245 to the control group (Fig. 1A, blue line). Increasing the GSH:CP molar ratio from 25:1 to 100:1 246 dramatically decreased the amount of Pt that eluted in form of Pt-PP by 14.4% and increased that of a 247 putative Pt-GSH complex by ~77%. The amount of Pt that eluted in form of CPHP’s decreased by 248 ~21% and that of free CP in plasma decreased by 41.2%. Overall, these results demonstrate that GSH 249 can – depending on the employed GSH:CP molar ratio − profoundly affect the metabolism of CP in 250 plasma by accelerating its hydrolysis and decreasing the amount of CPHP’s. This apparent discrepancy 251 can be rationalized in terms of the rapid formation of Pt-GSH complexes, either based on the reaction 252 of GSH with CP-derived hydrolysis products or by the direct replacement of a chloro ligand from CP 253 by GSH.34 These Pt-GSH complexes did not bind to plasma proteins and therefore resulted in a 254 decrease of the amount of total Pt that eluted in form of Pt-PP.Since the endogenous plasma GSH 255 concentration in rabbit plasma is negligible compared to the concentrations that were obtained after the 256 addition of GSH to plasma in our experiments, it is unlikely to have affected the obtained results. 257 Effect of increasing GSH:CP molar ratios on the Zn-metalloproteome in plasma 258 Since the analysis of plasma with the employed metallomics method allows to simultaneously 259 determine all major endogenous Cu, Fe and Zn metalloproteins, we noted an effect of increasing 260 GSH:CP molar ratios on the Zn-metalloproteome, but not on the Cu and Fe-metalloproteome (data not t af Dr 237 10 https://mc06.manuscriptcentral.com/cjc-pubs Page 11 of 28 Canadian Journal of Chemistry shown). The average peak area counts for PP-bound Zn and SMW Zn (as percent of the total Pt area) 262 are depicted in Table 2. The addition of a pharmacologically relevant dose of CP to plasma followed 263 by its analysis 5 min and 2 h later did not reveal any significant changes to the Zn-metalloproteome 264 (Fig. 2A, red and blue line). The fact that the obtained Zn-metalloproteome pattern for plasma spiked 265 with CP was very similar to that observed for untreated plasma (Fig. 2A, green line) indicates that CP 266 did not perturb the endogenous Zn-metalloproteome. These results are in accord with previous studies 267 that were conducted with human plasma.23 268 Strikingly different results, however, were observed for the Zn-metalloproteome in plasma with 269 increasing GSH:CP molar ratios (Fig. 2B-D, Table 2). Correspondingly, all of these detected changes 270 must be attributed to the presence of GSH. The results that were obtained for the 25:1 GSH:CP molar 271 ratio revealed a distinct perturbation of the Zn-metalloproteome at the 5 min time point (Fig. 2B, 272 orange line) and can be rationalized in terms of a GSH-mediated re-distribution of Zn from what 273 appears to be human serum albumin (HSA) to higher molecular weight (HMW) PP’s. In addition, a 274 Zn-peak eluted in the inclusion volume (~1200 s), which is indicative of a SMW Zn-species in plasma, 275 possibly a (GS)2-Zn complex 276 the 2 h time point (Fig. 2B, blue line) more closely resembled the pattern of unperturbed (native) rabbit 277 plasma (Fig. 2A, green line). This finding can be rationalized in terms of a partial reversal of the 278 binding of Zn from HMW-PP and 279 redistribution of Zn is unavailable at present, but may involve exposure to air which was not precluded 280 in our experiments. The results that were obtained for the 50:1 GSH:CP molar ratio and the 5 time 281 point (Fig. 2C, orange line) revealed an even more pronounced re-distribution of Zn from HSA to two 282 HMW-PP and the elution of a more intense Zn-peak in the inclusion volume. The Zn-metalloproteome 283 that was observed at the 2 h time point (Fig. 2C, blue line) again revealed a partial redistribution of Zn 284 from both HMW-PP to HSA, but not the SMW Zn-species. At the 100:1 GSH:CP molar ratio and the 5 35 t af Dr 261 . Interestingly, the Zn-metalloproteome pattern that was obtained for SMW moieties to HSA. A molecular explanation for this 11 https://mc06.manuscriptcentral.com/cjc-pubs Canadian Journal of Chemistry Page 12 of 28 285 min time point the comparatively most pronounced re-distribution of Zn from HSA to HMW-PP was 286 observed (we note that Zn-peak corresponding to HSA was marginally shifted to shorter retention 287 times). At the 2 h time point, a partial back distribution of Zn was observed from both HWM-PP’s and 288 the SMW Zn-species to HSA. 289 Effect of GSH on the metabolism of CP in PBS-buffer 291 To corroborate the results that were obtained with a GSH:CP molar ratio of 100:1 in rabbit plasma, the 292 same pharmacologically relevant dose of CP was added to PBS-buffer, incubated at 37°C and analyzed 293 after 5 min and 2 h (Fig. 3A, red line). Although at the 5 min time point free CP corresponded to the 294 majority of Pt that eluted from the column (97.0%), 3.0% of Pt eluted in form of a CPHP (presumably 295 the mono-aqua Pt-species)23. At the 2 h time point, the intensity of the parent CP peak had somewhat 296 decreased (84.1% of total Pt) and 15.9% of total Pt eluted in form of the same CPHP peak that was 297 detected at the 5 min time point (Fig. 3A, blue line). These dynamic changes, however, were much less 298 pronounced compared to those obtained when the same CP dose was incubated with rabbit plasma 299 (Fig. 1A, blue line). This difference can be rationalized in terms of the absence of PP in the PBS-buffer 300 experiment and the associated impact that the lack of PP will have on the dynamic equilibrium that 301 exists in plasma between CP, CPHP’s and Pt-PP.23 302 When GSH was added to PBS-buffer first followed 1 min later by CP (effective GSH:CP molar ratio 303 100:1) and the latter mixture was analyzed 5 min later (Fig. 3B, red line), a new Pt-peak (3.7% of total 304 Pt) with a retention time of ~1200 s was detected, which eluted before the CPHP (2.4% of total Pt) and 305 CP (93.9% of total Pt). At the 2 h time point, this new Pt-peak (77.9% of total Pt) was the most intense 306 Pt-peak that was detectable (Fig. 3B, blue line), while the Pt-peak corresponding to CPHP (1.1 % of 307 total Pt) was negligible compared to the second most intense Pt-peak, which corresponded to CP 308 (21.0% of total Pt) was smaller than what was observed at the 5 min time point. These results strongly t af Dr 290 12 https://mc06.manuscriptcentral.com/cjc-pubs Page 13 of 28 Canadian Journal of Chemistry suggested that a Pt-GSH complex had formed in PBS-buffer. Direct experimental evidence in support 310 of the formation of this species could be obtained by plotting Pt together with the corresponding S and 311 C-specific chromatograms for the 2 h time point (Fig. 3, pink and green lines, respectively). All 312 attempts to structurally characterize this Pt-GSH complex by electrospray-ionization mass 313 spectrometry (ESI-MS) failed, most likely due to the high salt content (>1%) of the PBS-buffer matrix. 314 The reaction between CP and GSH has been previously investigated under near physiological 315 conditions using 1H, 13C and 195Pt-NMR 34 and it was noted that although the mono-substituted species 316 cis[PtCl(GS)(15NH3)2] was formed first, several other species containing the 15NH3-Pt-S linkage were 317 present within a few minutes. The final reaction product was a HMW polymer with a 1:2 Pt:GSH 318 stoichiometry.34 In our experiments, the retention time of the Pt-GSH complex that had formed in 319 PBS-buffer was identical to that of the putative Pt-GSH complex in rabbit plasma (the presence of the 320 latter was corroborated by the co-elution of a SMW S-peak with a Pt-peak in the rabbit plasma 321 experiment; Supplementary Figure S1). Therefore, our results directly implicate the formation of Pt- 322 GSH complexes in blood plasma as the likely biomolecular basis by which GSH ameliorated the CP- 323 induced toxic side-effect in previously reported animal experiments.16,17 The fact that in previous 324 animal experiments the administration of GSH significantly reduced the neurotoxicity without 325 adversely affecting the anticancer efficacy16 may be rationalized in terms of the comparatively much 326 longer residence time of the active anticancer species CP (> 2 h) in rabbit plasma in our in vitro 327 experiments (Fig. 1). This is in contrast to our previous in vitro experiments in which free CP was 328 essentially absent in plasma when the latter was incubated with sodium thiosulfate, N-acetyl-L- 329 cysteine or D-methionine for 50 min. 330 established intracellular formation of Pt-GSH complexes,36 which effectively inactivate CP.37 Future 331 studies should be aimed at elucidating the structure of the Pt-GSH complex(es) as well as its/their 332 toxicity, biochemical fate and excretion from mammalian organisms. t af Dr 309 12-14 Our results must be clearly distinguished from the well- 13 https://mc06.manuscriptcentral.com/cjc-pubs Canadian Journal of Chemistry Page 14 of 28 333 Conclusion 335 Although CP is an effective anticancer drug, it inherently exerts severe dose-limiting toxic side-effects, 336 which can dramatically reduce the quality of life in patients, sometimes permanently. It has been 337 demonstrated in animal studies, however, that GSH can reduce the neurotoxicity and nephrotoxicity of 338 CP without notably reducing its efficacy. To possibly translate these promising results to benefits for 339 patients that are being treated with CP, it is important to elucidate the underlying mechanism of action. 340 To this end, we have conducted in vitro experiments in which increasing GSH:CP molar ratios (25:1, 341 50:1 and 100:1) were added to rabbit plasma. The determination of the Pt-distribution in plasma after 5 342 min and 2 h revealed significant changes compared to the metabolism of CP and provided evidence for 343 the formation of a Pt-GSH complex which did not bind to plasma proteins. These results were 344 corroborated by reacting GSH and CP (molar ratio 100:1) in PBS-buffer. Analysis of the obtained 345 mixture revealed the co-elution of a species which contained C, S and Pt (2 h time point) and had the 346 same retention time as a Pt-species that was detected in rabbit plasma. These findings conclusively 347 identify the formation of a Pt-GSH complex in plasma as one possible mechanism by which GSH 348 ameliorated the CP-induced toxic side-effects in animal studies without adversely affecting the 349 anticancer effect.16,17 In addition, at all investigated GSH:CP molar ratios a perturbation of the Zn 350 metalloproteome – which is attributed to GSH − was observed at the 5 min time point, while at the 2 h 351 time point the Zn distribution was more similar to that observed for untreated rabbit plasma. 352 Acknowledgements 353 MS is a Fellow of the Canadian Institutes of Health Research Training in Health Research Using 354 Synchrotron Techniques (CIHR-THRUST), MAG was supported by an NSERC-DG and BdSL by a 355 MITACS scholarship. 356 Figure Captions: t af Dr 334 14 https://mc06.manuscriptcentral.com/cjc-pubs Page 15 of 28 Canadian Journal of Chemistry 357 Figure 1: Representative Pt-specific chromatograms that were obtained after the addition of (A) cis- 358 platin (CP) to rabbit plasma and analyzed after 5 min (red line) and 2 h (blue line) of 359 incubation at 37° C. Then, L-glutathione (GSH) was added to rabbit plasma incubated for 1 360 min (37° C) and subsequently CP was added and the obtained mixtures were analyzed 5 361 min (red line) and 2 h (blue line) later by SEC-ICP-AES at GSH:CP molar ratios 25:1 (B), 362 50:1 (C) and 100:1 (D). Stationary phase: Superdex 200 Increase 10/300 GL (30 x 1.0 cm 363 I.D., 8.6 µm particle size) SEC column at 22° C; Mobile phase: PBS-buffer (0.15 M, pH 364 7.4); Flow rate: 1.0 mL/min, Injection volume: 500 µL; Detector: ICP-AES at 214.423 nm 365 (Pt). The retention times of the molecular markers are depicted on top of the figure. 366 Figure 2: Representative Zn-specific chromatograms that were obtained for blank rabbit plasma [(A) 368 green line], cis-platin (CP) spiked plasma [(A) 5 min (red line) and 2 h (blue line) of 369 incubation at 37° C after addition of CP] and plasma which was spiked with L-glutathione 370 (GSH) (incubated for 1 min at 37° C) and CP using a molar ratio of 25:1 (B), 50:1 (C) and 371 100:1 (D) [5 min (red line) and 2 h (blue line) of incubation at 37° C after addition of CP]. 372 Stationary phase: Superdex 200 Increase 10/300 GL (30 x 1.0 cm I.D., 8.6 µm particle size) 373 SEC column at 22° C; Mobile phase: PBS-buffer (0.15 M, pH 7.4); Flow rate: 1.0 mL/min, 374 Injection volume: 500 µL; Detector: ICP-AES at 213.856 nm (Zn). The retention times of 375 the molecular markers are depicted on top of the figure. HSA – human serum albumin. t af Dr 367 376 377 Figure 3: (A) Representative Pt-specific chromatograms obtained after the addition of cis-platin (CP) 378 to PBS-buffer and SEC-ICP-AES analysis after 5 min and 2 h of incubation at 37° C. (B) 379 PBS-buffer was spiked with L-glutathione (GSH), incubated at 37° C for 1 min and 380 subsequently spiked with CP (GSH:CP molar ratio 100:1). The obtained mixture was 15 https://mc06.manuscriptcentral.com/cjc-pubs Canadian Journal of Chemistry Page 16 of 28 381 analyzed after 5 min (red line) and 2 h (blue line) of incubated at 37° C by SEC-ICP-AES. 382 Simultaneously obtained S-(pink line) and C-(green line) specific chromatograms are also 383 depicted. Stationary phase: Superdex 200 Increase 10/300 GL (30 x 1.0 cm I.D., 8.6 µm 384 particle size) SEC column (22° C); Mobile phase: PBS-buffer (0.15 M, pH 7.4); Flow rate: 385 1.0 mL/min, Injection volume: 500 µL; Detector: ICP-AES at 214.423 nm (Pt), S (180.731 386 nm) and C (193.091 nm). The retention times of the molecular markers are depicted on top 387 of the figure. 388 389 Supplementary Figure S1: Representative Pt- and S-specific chromatograms that were obtained 5 min (red line) and 2 391 h (blue line) after L-glutathione (GSH) was added to rabbit plasma, the latter mixture was 392 incubated for 1 min (37° C) and cis-platin (CP) was added by SEC-ICP-AES. GSH:CP 393 molar ratio 100:1. Stationary phase: Superdex 200 Increase 10/300 GL (30 x 1.0 cm I.D., 394 8.6 µm particle size) SEC column at 22° C; Mobile phase: PBS-buffer (0.15 M, pH 7.4); 395 Flow rate: 1.0 mL/min, Injection volume: 500 µL; Detector: ICP-AES at 214.423 nm (Pt) 396 and 180.731 nm (S). The retention times of the molecular markers are depicted on top of 397 the figure. t af Dr 390 398 399 400 401 402 403 404 405 406 References (1) (2) (3) (4) Petsko, G. A. Genome Biology 2001, 3, 1. Rosenberg, B.; Van Camp, L.; Krigas, T. Nature 1965, 205, 698. Bruijnincx, P. C. A.; Sadler, P. J. Curr. Opin. Chem. 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Biochem. 1990, 38, 305. t af Dr 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 Canadian Journal of Chemistry 17 https://mc06.manuscriptcentral.com/cjc-pubs Canadian Journal of Chemistry 455 456 457 458 459 (35) (36) (37) Page 18 of 28 Fuhr, B. J.; Rabenstein, D. L. J. Am. Chem. Soc. 1973, 95, 6944. Kasherman, Y.; Sturup, S.; Gibson, D. J. Med. Chem. 2009, 52, 4319. Reedijk, J. Chem. Rev. 1999, 99, 2499. 460 461 462 463 464 465 466 467 470 t af 469 Dr 468 471 472 473 474 475 476 477 478 479 480 18 https://mc06.manuscriptcentral.com/cjc-pubs Page 19 of 28 481 Canadian Journal of Chemistry Fig.1 t af Dr 19 https://mc06.manuscriptcentral.com/cjc-pubs Canadian Journal of Chemistry Page 20 of 28 t af Dr 482 20 https://mc06.manuscriptcentral.com/cjc-pubs Page 21 of 28 483 Canadian Journal of Chemistry Fig. 2 t af Dr 21 https://mc06.manuscriptcentral.com/cjc-pubs Canadian Journal of Chemistry Page 22 of 28 t af Dr 484 22 https://mc06.manuscriptcentral.com/cjc-pubs Page 23 of 28 485 Canadian Journal of Chemistry Fig. 3 t af Dr 486 487 23 https://mc06.manuscriptcentral.com/cjc-pubs Canadian Journal of Chemistry aft Dr https://mc06.manuscriptcentral.com/cjc-pubs Page 24 of 28 Page 25 of 28 Canadian Journal of Chemistry aft Dr https://mc06.manuscriptcentral.com/cjc-pubs Canadian Journal of Chemistry t af Dr https://mc06.manuscriptcentral.com/cjc-pubs Page 26 of 28 Page 27 of 28 Canadian Journal of Chemistry Table 1: Peak areas of the Pt peaks (indicated as percentage values with respect to the total Pt area) obtained after the analysis of rabbit plasma spiked with cis-platin (CP), rabbit plasma spiked with L-glutathione (GSH) and CP using GSH:CP molar ratios 25:1, 50:1 and 100:1 by SEC-ICP-AES, after incubation of the mixture at 37 °C (n=3, Mean ± SD). Experiment Molar ratio Assigned Pt-peaks Analysis after Plasma protein bound Pt Pt-GSH complex CP-derived hydrolysis products CP 5 min 1.5 ± 0.5 - 4.1 ± 0.6 94.4 ± 1.1 10139 ± 895 2h 26.6 ± 2.6 - 22.6 ± 0.7 50.8 ± 2.4 9914 ± 536 5 min 1.9 ± 0.1 3.5 ± 0.1 92.2 ± 0.4 11094 ± 1204 2h 22.2 ± 0.9 11.3 ± 0.6 36.8 ± 1.4 10798 ± 1389 5 min 1.8 ± 0.3 3.4 ± 0.8 3.2 ± 0.5 91.6 ± 1.1 11884 ± 709 2h 16.4 ± 0.37 55.5 ± 1.8 5.3 ± 0.5 22.8 ± 1.7 11774 ± 682 5 min 2.1 ± 0.2 7.6 ± 0.5 3.1 ± 0.3 87.2 ± 0.8 11449 ± 1076 2h 12.2 ± 0.2 76.7 ± 1.9 1.5 ± 0.40 9.6 ± 1.3 11059 ± 660 GSH:CP Plasma Total Pt area N/A + CP 25:1 Plasma + GSH (1 min) + CP Dr 2.4 ± 0.3 af 29.7 ± 2.7 t 50:1 100:1 https://mc06.manuscriptcentral.com/cjc-pubs Canadian Journal of Chemistry Page 28 of 28 Table 2: Peak areas of the Zn peaks (indicated as percentage values with respect to the total Zn area) obtained after the analysis of rabbit plasma, rabbit plasma spiked with cis-platin (CP), rabbit plasma spiked with L-glutathione (GSH) and CP using GSH:CP molar ratios 100:1, 50:1 and 25:1 by SEC-ICP-AES, after incubation of the mixture at 37 °C (n=3, Mean ± SD). Assigned Zn-peaks Molar ratio Experiment GSH:CP Analysis after Plasma protein bound Zn Small molecular weight Zn-peak Total Zn area < 17 kDa Plasma 100 N/A 5 min Plasma + CP N/A 2h 24421 ± 861 100 0 22406 ± 2601 af 0 21895 ± 2997 10.1 ± 0.7 23021 ± 3050 8 ± 0.6 21073 ± 2651 Dr 100 t 5 min 89.9 ± 0.7 2h 92 ± 0.6 5 min 88.7 ± 0.8 11.3 ± 0.8 24194 ± 1868 2h 88.4 ± 0.5 11.6 ± 0.5 23165 ± 1507 5 min 86.5 ± 0.7 13.5 ± 0.7 23270 ± 2234 2h 88.9 ± 0.4 11.1 ± 0.4 22277 ± 1848 25:1 Plasma + GSH (1 min) + CP 0 50:1 100:1 https://mc06.manuscriptcentral.com/cjc-pubs