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
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Keyword:
Canadian Journal of Chemistry
Blood plasma, cis-platin, chemoprotection, glutathione, metallomics
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Canadian Journal of Chemistry
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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
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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
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Abstract
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The anticancer drug cis-platin (CP) is in worldwide clinical use to treat a variety of cancers, but is
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inherently associated with severe toxic side-effects. Previous animal studies revealed that its
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neurotoxicity can be significantly reduced by the co-administration of L-glutathione (GSH) without
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affecting the anti-cancer effect. The underlying molecular mechanism, however, has remained elusive.
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Since the bloodstream is a likely biological compartment where CP-derived hydrolysis products may
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react with GSH, we have employed a recently developed metallomics tool to gain insight into the
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interaction of CP and GSH in rabbit plasma in vitro. After the addition of increasing GSH:CP molar
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ratios to plasma (25:1, 50:1 and 100:1), the determination of the Pt-distribution 5 min and 2 h later
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revealed the formation of a Pt-GSH complex which did not bind to plasma proteins. The
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simultaneously obtained Zn-distribution in plasma revealed a progressively more pronounced
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perturbation of the Zn metalloproteome with increasing GSH:CP molar ratios at the 5 min time point,
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which partially reversed at the 2 h time point. The formation of Pt-GSH species in plasma is therefore
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likely to be directly involved in the process by which GSH protects mammalian organisms from CP-
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induced neurotoxicity, nephrotoxicity and possibly other organ-based toxicities.
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Key words
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Blood plasma, cis-platin, chemoprotection, glutathione, metallomics
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Introduction
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The serendipitously discovered anticancer drug cis-platin (CP)1,2 heralded the dawn of metallodrugs
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and remains one of the most effective chemotherapeutic agents more than five decades after its
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discovery3. In fact, 50-70% of all cancer patients are treated with CP either alone or in combination
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with other anticancer drugs.4 Despite its broad spectrum of activity against a large variety of cancers,5
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the intravenous administration of patients with CP is associated with severe toxic side-effects,
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including nephrotoxicity, ototoxicity and neurotoxicity6,7. Although the nephrotoxicity of CP can be
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somewhat ameliorated by the administration of patients with hypertonic saline or mannitol,
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clinical procedures exist to completely eliminate its ototoxicity and neurotoxicity which therefore
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constitute the primary dose limiting factors.9
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The aforementioned toxic side-effects of CP, however, have been shown to be significantly reduced in
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animal studies when small-molecular-weight (SMW) compounds – also referred to as
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‘chemoprotective agents’ − were co-administered.9,10 Although this principle approach can adversely
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affect the anti-tumor effect of CP,9 it has been demonstrated that this can be sometimes altogether
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avoided if the timing between the administration of CP and the chemoprotective agent as well as their
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route of administration is carefully optimized.11 More recent investigations aimed at better
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understanding the biomolecular mechanisms by which chemoprotective agents ameliorate the toxicity
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of CP revealed that sodium thiosulfate, N-acetyl-L-cysteine and D-methionine react with CP-derived
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hydrolysis products in human blood plasma in vitro.12-14 Since some of the CP-derived hydrolysis
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products that are formed in blood plasma are highly toxic,9 their ‘neutralization’ by chemoprotective
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agents likely constitutes one important mechanism of action by which the latter mitigate the toxic
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side-effects of CP.15
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L-Glutathione (GSH) represents another SMW sulfur compound that has been demonstrated to be
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effective in terms of reducing the toxic side-effects of CP in animals 16,17 and humans18. In contrast to
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the aforementioned chemoprotective agents, however, GSH is naturally present in human blood plasma
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at concentrations of 1-5 µM19 and the plasma concentration of oxidized glutathione (GSSG) is ~2 µM
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in rats.20 The administration of exogenous GSH to animals to decrease the neurotoxicity of CP without
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diminishing the anti-tumor effect of the latter.16 Given that CP (1.0 mg/kg) was administered
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intraperitoneally and GSH (200 mg/kg) intravenously to rats in one particular study,16 it is possible that
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the underlying toxicologically relevant processes occurred in blood plasma.12-14 Since no direct
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experimental evidence in support of this possibility has been obtained, we have conducted systematic
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in vitro experiments with rabbit plasma to which pharmacologically relevant GSH:CP molar ratios –
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defined as those close to those where the desired amelioration of the toxicity of CP was achieved in
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vivo − were added. The analysis of the obtained mixtures with a metallomics tool − a size-exclusion
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chromatography (SEC) system coupled on-line to an inductively coupled plasma atomic emission
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spectrometer (ICP-AES)21 – allowed us to observe changes in the Pt-distribution in plasma between the
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5 min and 2 h time point. Since ICP-AES allows to simultaneously monitor the emission lines of the
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endogenous Fe, Cu and Zn-metalloproteins in addition to those of Pt, changes in the plasma
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distribution of these metals (e.g. caused by GSH) can also be observed. The results revealed a rapid
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formation of Pt-GSH adduct(s) in plasma, which strongly suggests their formation to be directly
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implicated in the mechanism by which GSH can ameliorate the toxic side-effects of CP in vivo.16,17,22
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Experimental
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Chemicals and Solutions
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Cis-platin (1 mg cis-Pt(NH3)2Cl2/mL; this solution also contained 1 mg mannitol and 9 mg NaCl;
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sterile) was obtained from Hospira (Montreal, QC, Canada). Phosphate-buffered saline buffer (PBS)
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tablets were purchased from Sigma-Aldrich (St. Louis, MO, USA) and the corresponding PBS-buffer
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(10 mM phosphate, 2.7 mM KCl, 137 mM NaCl) was prepared by dissolving PBS tablets in the
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appropriate volume of de-ionized water (Simplicity water purification system; Millipore, Billerica,
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MA, USA) followed by pH adjustment to 7.4. The obtained solution was filtered through 0.45 µm
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nylon-filter membranes (Mandel Scientific, Guelph, ON, Canada) before use. A mixture of protein
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standards which contained thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa),
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myoglobin (17 kDa), and vitamin B12 (1.35 kDa) was obtained from Bio-Rad Laboratories (Hercules,
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CA, USA) to calibrate the employed SuperdexTM 200 Increase SEC column. GSH (≥98%) was
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obtained from Sigma-Aldrich and an aqueous stock solution with a concentration of 142 mg/mL (0.46
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M) was prepared by dissolving it in PBS-buffer followed by pH adjustment to 7.4 using 4 M NaOH.
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This solution was prepared daily and was used within 6 hours during which it was stored at 5 ºC in a
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vial with minimal head space.
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SEC-ICP-AES system
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The SEC-ICP-AES system that was employed was comprised of a Smartline 1000 HPLC pump
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(Knauer, Berlin, Germany) and a Rheodyne 9010 PEEK injection valve (Rheodyne, Rhonert Park, CA,
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USA) which was equipped with a 0.5 mL PEEK injection loop (0.5 mL). A pre-packed SuperdexTM
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200 Increase 10/300 GL high performance size-exclusion chromatography (SEC) column (30 x 1.0 cm
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I.D., particle size 8.6 µm, fractionation range 600 - 10 kDa; GE Healthcare, Piscataway, NJ, USA) was
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used in conjunction with a PBS-buffer mobile phase (as described in ‘chemicals and solutions’) and a
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flow rate of 1.0 mL/min (column temperature 22º C). We note that the employed SEC column contains
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smaller particles than the columns that we used in our previously studies,12-14,23 but that the backbone is
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identical and resulted in a similar separation of plasma proteins (data not shown). Simultaneous
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multielement-specific detection of Ca (317.933 nm), C (193.091 nm), Cu (324.754 nm), Fe (259.940
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nm), Mg (280.271 nm), Pt (214.423 nm), S (180.731 nm) and Zn (213.856 nm) in the column effluent
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was achieved with a Prodigy, high-dispersion, radial-view ICP-AES (Teledyne Leeman Labs, Hudson,
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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
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35 psi. The nebulizer gas flow rate was 1.4 L of Ar/min. A 400 s delay was implemented between
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injection and data acquisition and data acquisition window was 2000 s. The raw data (Salsa Software)
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were imported into Sigmaplot 13.0, smoothed using the bi-square algorithm and then the peak areas
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were determined.
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Preparation of blank plasma, CP-spiked plasma and GSH-spiked plasma
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Rabbit plasma (Lot 0165) was obtained from Cedarlane Corporation (Burlington, ON, Canada) and
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had been prepared by pooling plasma from 3.5 months old male New Zealand white rabbits using
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sodium heparin (158 USP units/10 mL of whole blood) as the anticoagulant. Plasma (2.2 mL aliquots)
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was stored at -60 ˚C and received frozen on dry ice. Individual plasma vials were thawed at room
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temperature for 45 minutes and incubated at 37º C for 30 min before pharmacologically relevant doses
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of CP and/or GSH were added. “Blank” plasma was prepared by spiking 1.8 mL of rabbit plasma with
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130 µL of PBS-buffer. The solution that served as the CP control plasma was prepared by adding 75
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µL of the CP stock solution (1 mg/mL) and 54 µL of PBS-buffer to 1.8 mL of plasma. The final
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concentration
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pharmacologically/therapeutically relevant24. This mixture was incubated at 37º C and samples were
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withdrawn for analysis after 5 min and 2 h.
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was
~0.04
mg
CP/mL
of
plasma
(~0.13
mM),
which
is
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Analysis of GSH and CP-spiked plasma (GSH:CP molar ratios 25:1, 50:1 and 100:1)
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Plasma (1.8 mL) was incubated at 37° C, spiked with 54 µL of the GSH stock solution and 1 min later
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75 µL of the CP stock solution (1 mg/mL) was added (GSH:CP molar ratio of 100:1). The obtained
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mixture was incubated at 37° C and analyzed after 5 min and 2 h. The solution that served as the GSH
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control (corresponding to the GSH:CP=100:1 molar ratio) was prepared by adding 54 µL of GSH stock
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solution and 75 µL of PBS-buffer to 1.8 mL of plasma. The obtained mixture was incubated at 37° C
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for 6 min and 2 h + 1 min and was subsequently analyzed.
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The solution corresponding to the GSH:CP molar ratio of 50:1 was prepared by spiking 1.8 mL of
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plasma (that had been incubated at 37° C) with 27 µL of the GSH stock solution and 27 µL of PBS-
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buffer. After mixing 75 µL of the CP stock solution (1 mg/mL) were added 1 min later. The solution
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corresponding to a GSH:CP molar ratio of 25:1 was prepared in an analogous manner by spiking 1.8
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mL of plasma with 13.5 µL of the GSH stock solution and 41 µL of PBS-buffer. After incubation at
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37° C for 1 min, 75 µL of the CP stock solution (1 mg/mL) was added and the obtained mixture was
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incubated at 37° C and analyzed 5 min and 2 h later. All experiments were carried out in triplicate and
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representative chromatograms are presented.
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Analysis of CP-spiked PBS-buffer and GSH+CP-spiked PBS-buffer (GSH:CP molar ratio 100:1)
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The CP control was prepared by spiking PBS buffer (pH 7.4, 1.8 mL) that had been incubated at 37° C
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with 75 µL of the CP stock solution (1 mg/mL) and 54 µL of PBS-buffer. After incubation of this
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mixture at 37° C, the latter was analyzed after 5 min and 2 h. Then, PBS buffer (pH 7.4, 1.8 mL) was
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spiked with 54 µL of GSH stock solution, incubated at 37° C for 1 min and then spiked with 75 µL of
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the CP stock solution (1 mg/mL). The obtained mixture was maintained at 37º C and samples were
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withdrawn for duplicate analysis after 5 min and 2 h. All attempts to separate excess GSH from the
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co-eluting Pt-S peak on a PRP X-100 anion-exchange HPLC column (30 x 0.46 cm I.D.) using a
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variety of different PBS-buffer concentrations were unsuccessful.
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Results and Discussion
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After the intravenous administration of cancer patients with CP, all CP-derived metabolites will
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interact with blood constituents, tumor cells (intended) and healthy tissue cells (unintended). Since the
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latter interaction is associated with organ-based toxicities
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explored to increase the tumor selectivity of metal-based drugs7. In principle, this can be achieved by
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optimizing the tumor selectivity of novel (Pt-based) drug structures3,29,30 or by improving the selective
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delivery of established anticancer drugs to tumor cells (e.g. using substituted ß-cyclodextrins and calix
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[4] arenes)31-33.
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Another feasible strategy to improve the safety of clinically used anticancer metallodrugs aims to
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selectively reduce their toxic side-effects by the co-administration of chemoprotective agents.
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Unfortunately, the only chemoprotective agent that has been clinically approved to date, amifostine,
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did not result in significantly reduced ototoxicity compared to the cisplatin only group.9 Three other
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chemoprotective agents, sodium thiosulfate, N-acetylcysteine and D-methionine, however, were
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recently demonstrated to modulate the metabolism of CP in human plasma in vitro.12-14 The common
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underlying biomolecular mechanism appears to involve the reaction of CP-derived hydrolysis products
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with each chemoprotective agent to form Pt-chemoprotective agent complexes, some of which could
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be structurally characterized.14 The fact that the parent anticancer drug CP remained in plasma for up
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to 50 min – at least these in vitro studies − demonstrates that the co-administration of CP with
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chemoprotective agents will not entirely inactivate the parent anticancer drug. Since this principle
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approach has potential to improve the quality of life of patients that are being treated with CP, we
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wanted to gain insight into the mechanism by which GSH reduced the toxic side-effects of CP in
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previously conducted animal studies16,17 and one study involving cancer patients.18 To elucidate if and
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in what way GSH will affect the metabolism of CP in rabbit plasma, we have conducted in vitro
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experiments which involved the addition of increasing GSH:CP molar ratios to plasma followed by its
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analysis with a recently developed metallomics method. The latter allows to simultaneously determine
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, various approaches are currently being
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essentially all CP-derived Pt-metabolites in plasma23 and all major Cu, Fe and Zn-containing
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endogenous metalloproteins.21
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Metabolism of CP in plasma
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After the addition of a pharmacologically relevant dose of CP to plasma, the obtained mixture was
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analyzed after 5 min and 2 h (Fig. 1A). At the 5 min time point 1.5% of total Pt eluted bound to plasma
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proteins (Pt-PP), ~4% in form of CP-derived hydrolysis products (CPHP) and almost 95% as CP
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(Table 1). The results that were obtained for the 2 h time point revealed a significant increase of Pt-PP
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(26.6%), that 22.6% of total Pt eluted in form of CPHP’s and that ~51% of free CP was present in
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plasma (Table 1). In general, these results are in good accord with previous results that were obtained
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after the addition of the same CP-dose to human plasma.23
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Effect of increasing GSH:CP molar ratios on the metabolism of CP in plasma
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In order to observe the effect of GSH on the metabolism of CP, increasing amounts of GSH were
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added to plasma first followed by a pharmacologically relevant dose of CP 1 min later to achieve
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GSH:CP molar ratios of 25:1, 50:1 and 100:1. The obtained mixtures were analyzed after 5 min and 2
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h (Fig. 1B-D). The average peak area counts for each detected Pt peak (as percent of the total Pt area)
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are depicted in Table 1. For the sake of clarity, the results will be discussed first for the 5 min time
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point, followed by those obtained for the 2 h time point.
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At the 25:1 GSH:CP molar ratio (Fig. 1B) and compared to the control experiment (Fig. 1A), no
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statistically significant increase was observed for Pt-PP,but 2.4% of Pt eluted in form of a Pt-peak that
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was not detected in the control experiment and corresponds to a putative in situ formed Pt-GSH
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complex. The retention time of this Pt-GSH complex was comparable to that of related Pt-complexes
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that were detected in human plasma after incubation with CP and sodium thiosulfate,12 N-acetyl-L-
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cysteine13 or D-methionine.14 Furthermore, 0.6% less Pt eluted in form of CPHP’s and 2.2% less Pt
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eluted in form of CP (compared to the control group). An increase of the GSH:CP molar ratio from
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25:1 to 100:1 marginally affected the amount of Pt that eluted as Pt-PP, but significantly increased the
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amount of Pt that eluted in form of a Pt-GSH complex by 5.2% (Fig 1B-D, red lines). In accord with
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these results, the amount of Pt that eluted in form of CPHP’s decreased by 0.4% and 7.2% less Pt
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eluted in form of CP at the 100:1 GSH:CP molar ratio (compared to the control group).
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As expected, the results that were obtained for increasing GSH:CP molar ratios on the Pt-distribution
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in plasma at the 2 h time point were progressively more pronounced (Fig. 1B-D, blue lines) compared
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to the control group (Fig. 1A, blue line). Increasing the GSH:CP molar ratio from 25:1 to 100:1
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dramatically decreased the amount of Pt that eluted in form of Pt-PP by 14.4% and increased that of a
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putative Pt-GSH complex by ~77%. The amount of Pt that eluted in form of CPHP’s decreased by
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~21% and that of free CP in plasma decreased by 41.2%. Overall, these results demonstrate that GSH
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can – depending on the employed GSH:CP molar ratio − profoundly affect the metabolism of CP in
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plasma by accelerating its hydrolysis and decreasing the amount of CPHP’s. This apparent discrepancy
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can be rationalized in terms of the rapid formation of Pt-GSH complexes, either based on the reaction
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of GSH with CP-derived hydrolysis products or by the direct replacement of a chloro ligand from CP
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by GSH.34 These Pt-GSH complexes did not bind to plasma proteins and therefore resulted in a
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decrease of the amount of total Pt that eluted in form of Pt-PP.Since the endogenous plasma GSH
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concentration in rabbit plasma is negligible compared to the concentrations that were obtained after the
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addition of GSH to plasma in our experiments, it is unlikely to have affected the obtained results.
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Effect of increasing GSH:CP molar ratios on the Zn-metalloproteome in plasma
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Since the analysis of plasma with the employed metallomics method allows to simultaneously
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determine all major endogenous Cu, Fe and Zn metalloproteins, we noted an effect of increasing
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GSH:CP molar ratios on the Zn-metalloproteome, but not on the Cu and Fe-metalloproteome (data not
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shown). The average peak area counts for PP-bound Zn and SMW Zn (as percent of the total Pt area)
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are depicted in Table 2. The addition of a pharmacologically relevant dose of CP to plasma followed
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by its analysis 5 min and 2 h later did not reveal any significant changes to the Zn-metalloproteome
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(Fig. 2A, red and blue line). The fact that the obtained Zn-metalloproteome pattern for plasma spiked
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with CP was very similar to that observed for untreated plasma (Fig. 2A, green line) indicates that CP
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did not perturb the endogenous Zn-metalloproteome. These results are in accord with previous studies
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that were conducted with human plasma.23
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Strikingly different results, however, were observed for the Zn-metalloproteome in plasma with
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increasing GSH:CP molar ratios (Fig. 2B-D, Table 2). Correspondingly, all of these detected changes
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must be attributed to the presence of GSH. The results that were obtained for the 25:1 GSH:CP molar
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ratio revealed a distinct perturbation of the Zn-metalloproteome at the 5 min time point (Fig. 2B,
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orange line) and can be rationalized in terms of a GSH-mediated re-distribution of Zn from what
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appears to be human serum albumin (HSA) to higher molecular weight (HMW) PP’s. In addition, a
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Zn-peak eluted in the inclusion volume (~1200 s), which is indicative of a SMW Zn-species in plasma,
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possibly a (GS)2-Zn complex
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the 2 h time point (Fig. 2B, blue line) more closely resembled the pattern of unperturbed (native) rabbit
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plasma (Fig. 2A, green line). This finding can be rationalized in terms of a partial reversal of the
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binding of Zn from HMW-PP and
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redistribution of Zn is unavailable at present, but may involve exposure to air which was not precluded
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in our experiments. The results that were obtained for the 50:1 GSH:CP molar ratio and the 5 time
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point (Fig. 2C, orange line) revealed an even more pronounced re-distribution of Zn from HSA to two
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HMW-PP and the elution of a more intense Zn-peak in the inclusion volume. The Zn-metalloproteome
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that was observed at the 2 h time point (Fig. 2C, blue line) again revealed a partial redistribution of Zn
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from both HMW-PP to HSA, but not the SMW Zn-species. At the 100:1 GSH:CP molar ratio and the 5
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SMW moieties
to HSA. A molecular explanation for this
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min time point the comparatively most pronounced re-distribution of Zn from HSA to HMW-PP was
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observed (we note that Zn-peak corresponding to HSA was marginally shifted to shorter retention
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times). At the 2 h time point, a partial back distribution of Zn was observed from both HWM-PP’s and
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the SMW Zn-species to HSA.
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Effect of GSH on the metabolism of CP in PBS-buffer
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To corroborate the results that were obtained with a GSH:CP molar ratio of 100:1 in rabbit plasma, the
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same pharmacologically relevant dose of CP was added to PBS-buffer, incubated at 37°C and analyzed
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after 5 min and 2 h (Fig. 3A, red line). Although at the 5 min time point free CP corresponded to the
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majority of Pt that eluted from the column (97.0%), 3.0% of Pt eluted in form of a CPHP (presumably
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the mono-aqua Pt-species)23. At the 2 h time point, the intensity of the parent CP peak had somewhat
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decreased (84.1% of total Pt) and 15.9% of total Pt eluted in form of the same CPHP peak that was
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detected at the 5 min time point (Fig. 3A, blue line). These dynamic changes, however, were much less
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pronounced compared to those obtained when the same CP dose was incubated with rabbit plasma
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(Fig. 1A, blue line). This difference can be rationalized in terms of the absence of PP in the PBS-buffer
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experiment and the associated impact that the lack of PP will have on the dynamic equilibrium that
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exists in plasma between CP, CPHP’s and Pt-PP.23
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When GSH was added to PBS-buffer first followed 1 min later by CP (effective GSH:CP molar ratio
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100:1) and the latter mixture was analyzed 5 min later (Fig. 3B, red line), a new Pt-peak (3.7% of total
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Pt) with a retention time of ~1200 s was detected, which eluted before the CPHP (2.4% of total Pt) and
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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
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Pt-peak that was detectable (Fig. 3B, blue line), while the Pt-peak corresponding to CPHP (1.1 % of
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total Pt) was negligible compared to the second most intense Pt-peak, which corresponded to CP
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(21.0% of total Pt) was smaller than what was observed at the 5 min time point. These results strongly
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suggested that a Pt-GSH complex had formed in PBS-buffer. Direct experimental evidence in support
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of the formation of this species could be obtained by plotting Pt together with the corresponding S and
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C-specific chromatograms for the 2 h time point (Fig. 3, pink and green lines, respectively). All
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attempts to structurally characterize this Pt-GSH complex by electrospray-ionization mass
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spectrometry (ESI-MS) failed, most likely due to the high salt content (>1%) of the PBS-buffer matrix.
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The reaction between CP and GSH has been previously investigated under near physiological
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conditions using 1H, 13C and 195Pt-NMR 34 and it was noted that although the mono-substituted species
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cis[PtCl(GS)(15NH3)2] was formed first, several other species containing the 15NH3-Pt-S linkage were
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present within a few minutes. The final reaction product was a HMW polymer with a 1:2 Pt:GSH
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stoichiometry.34 In our experiments, the retention time of the Pt-GSH complex that had formed in
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PBS-buffer was identical to that of the putative Pt-GSH complex in rabbit plasma (the presence of the
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latter was corroborated by the co-elution of a SMW S-peak with a Pt-peak in the rabbit plasma
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experiment; Supplementary Figure S1). Therefore, our results directly implicate the formation of Pt-
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GSH complexes in blood plasma as the likely biomolecular basis by which GSH ameliorated the CP-
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induced toxic side-effect in previously reported animal experiments.16,17 The fact that in previous
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animal experiments the administration of GSH significantly reduced the neurotoxicity without
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adversely affecting the anticancer efficacy16 may be rationalized in terms of the comparatively much
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longer residence time of the active anticancer species CP (> 2 h) in rabbit plasma in our in vitro
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experiments (Fig. 1). This is in contrast to our previous in vitro experiments in which free CP was
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essentially absent in plasma when the latter was incubated with sodium thiosulfate, N-acetyl-L-
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cysteine or D-methionine for 50 min.
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established intracellular formation of Pt-GSH complexes,36 which effectively inactivate CP.37 Future
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studies should be aimed at elucidating the structure of the Pt-GSH complex(es) as well as its/their
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toxicity, biochemical fate and excretion from mammalian organisms.
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Our results must be clearly distinguished from the well-
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Conclusion
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Although CP is an effective anticancer drug, it inherently exerts severe dose-limiting toxic side-effects,
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which can dramatically reduce the quality of life in patients, sometimes permanently. It has been
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demonstrated in animal studies, however, that GSH can reduce the neurotoxicity and nephrotoxicity of
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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
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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
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ameliorated the CP-induced toxic side-effects in animal studies without adversely affecting the
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anticancer effect.16,17 In addition, at all investigated GSH:CP molar ratios a perturbation of the Zn
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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.
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Figure Captions:
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Figure 1: Representative Pt-specific chromatograms that were obtained after the addition of (A) cis-
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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),
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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.
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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
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(GSH) (incubated for 1 min at 37° C) and CP using a molar ratio of 25:1 (B), 50:1 (C) and
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100:1 (D) [5 min (red line) and 2 h (blue line) of incubation at 37° C after addition of CP].
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Stationary phase: Superdex 200 Increase 10/300 GL (30 x 1.0 cm I.D., 8.6 µm particle size)
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SEC column at 22° C; Mobile phase: PBS-buffer (0.15 M, pH 7.4); Flow rate: 1.0 mL/min,
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Injection volume: 500 µL; Detector: ICP-AES at 213.856 nm (Zn). The retention times of
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the molecular markers are depicted on top of the figure. HSA – human serum albumin.
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Figure 3: (A) Representative Pt-specific chromatograms obtained after the addition of cis-platin (CP)
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to PBS-buffer and SEC-ICP-AES analysis after 5 min and 2 h of incubation at 37° C. (B)
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PBS-buffer was spiked with L-glutathione (GSH), incubated at 37° C for 1 min and
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subsequently spiked with CP (GSH:CP molar ratio 100:1). The obtained mixture was
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analyzed after 5 min (red line) and 2 h (blue line) of incubated at 37° C by SEC-ICP-AES.
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Simultaneously obtained S-(pink line) and C-(green line) specific chromatograms are also
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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:
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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
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of the figure.
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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
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incubated for 1 min (37° C) and cis-platin (CP) was added by SEC-ICP-AES. GSH:CP
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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);
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Flow rate: 1.0 mL/min, Injection volume: 500 µL; Detector: ICP-AES at 214.423 nm (Pt)
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and 180.731 nm (S). The retention times of the molecular markers are depicted on top of
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the figure.
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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
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2.4 ± 0.3
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29.7 ± 2.7
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100:1
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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
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0
21895 ± 2997
10.1 ± 0.7
23021 ± 3050
8 ± 0.6
21073 ± 2651
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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
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