Synergy between tobramycin and trivalent chromium ion in electrochemical control of Pseudomonas aeruginosa.

UNLABELLED: We recently demonstrated that the effectiveness of tobramycin (Tob), an aminoglycoside, against antibiotic-tolerant persister cells of Pseudomonas aeruginosa can be enhanced by electrochemical factors generated from direct currents (DC). Supplementation of Ni(II), Cr(III) and Fe(II) during carbon-mediated DC treatment revealed that these metal cations promote killing of persister cells in the presence of tobramycin, which led to our hypothesis that specific interactions between Tob and some metal ions contribute to the synergistic killing of persister cells. In this study, the interactions between selected metal cations and Tob were investigated using (1)H-(13)C HSQC NMR. Increase in the concentration of Cr(III) (in the form of [CrCl2(H2O)4](+)) in solutions containing Tob was found to shift the HSQC NMR peaks of Tob to new positions, suggesting the formation of a Cr(III)-Tob complex. Crystal field effects and electrochemical properties of the complex were further studied using UV-visible spectroscopy and cyclic voltammetry, which led to the finding that the Cr(III)-Tob complex has increased affinity with negatively charged nucleic acids. These findings are helpful for understanding the mechanism of electrochemical control of bacterial cells and for developing more effective antimicrobial therapies based on aminoglycosides and electrochemical species released from various metallic biomaterials. STATEMENT OF SIGNIFICANCE: Medical device associated infections present a major challenge to healthcare and the quality of life of affected individuals. This problem is further exacerbated by the emergence of multidrug resistant pathogens. Thus, alternative methods for microbial control are urgently needed. Recently, we reported synergy between tobramycin and low-level electrochemical currents generated using stainless steel electrodes in killing bacterial persister cells, a dormant population with high-level intrinsic tolerance to antibiotics. In this article, we describe how electrically-induced interaction between aminoglycosides and certain metal cations enhance the potency of tobramycin in bacterial killing. The findings will help design new methods for controlling infections through electrochemical disruption of cellular function and associated drug resistance.

Multifunctional DNA-gold nanoparticles for targeted doxorubicin delivery.

In this report we describe the synthesis, characterization, and cytotoxic properties of DNA-capped gold nanoparticles having attached folic acid (FA), a thermoresponsive polymer (p), and/or poly(ethylene glycol) (PEG) oligomers that could be used to deliver the anticancer drug doxorubicin (DOX) in chemotherapy. The FA-DNA oligomer used in the construction of the delivery vehicle was synthesized through the reaction of the isolated folic acid N-hydroxysuccinimide ester with the amino-DNA and the conjugated DNA product was purified using high performance liquid chromatography (HPLC). This approach ultimately allowed control of the amount of FA attached to the surface of the delivery vehicle. Cytotoxicity studies using SK-N-SH neuroblastoma cells with drug loaded delivery vehicles were carried out using a variety of exposure times (1-48 h) and recovery times (1-72 h), and in order to access the effects of varying amounts of attached FA, in culture media deficient in FA. DOX loaded delivery vehicles having 50% of the DNA strands with attached FA were more cytotoxic than when all of the strands contained FA. Since FA stimulates cell growth, the reduced cytotoxicity of vehicles fully covered with FA suggests that the stimulatory effects of FA can more than compensate for the cytotoxic effects of the drug on the cell population. While attachment of hexa-ethylene glycol PEG(18) to the surface of the delivery vehicle had no effect on cytotoxicity, 100% FA plus the thermoresponsive polymer resulted in IC50 = 0.48 ± 0.01 for an exposure time of 24 h and a recovery time of 1 h, which is an order of magnitude more cytotoxic than free DOX. Confocal microscopic studies using fluorescence detection showed that SK-N-SH neuroblastoma cells exposed to DOX-loaded vehicles have drug accumulation inside the cell and, in the case of vehicles with attached FA and thermoresponsive polymer, the drug appears more concentrated. Since the biological target of DOX is DNA, the latter observation is consistent with the high cytotoxicity of vehicles having both FA and the thermoresponsive polymer. The study highlights the potential of DNA-capped gold nanoparticles as delivery vehicles for doxorubicin in cancer chemotherapy.

Cyclodextrin capped gold nanoparticles as a delivery vehicle for a prodrug of cisplatin.

In this work, we explore the use of a quick coupling mechanism for "arming" a cyclodextrin coated gold nanoparticle (AuNP) delivery vehicle, 2, with an adamantane-oxoplatin conjugate that is a prodrug of cisplatin, 3, to produce a cytotoxic nanodrug, 4. The two-part arming system, which utilizes the well-known guest-host interaction between ß-cyclodextrin and adamantane, may be useful for rapidly constituting polyfunctional nanodrugs prior to their application in chemotherapy. The 4.7 ± 1.1 nm delivery vehicle, 2, coated with per-6-thio-ß-cyclodextrin (ßSCD), was characterized using transmission electron microscopy and absorption spectroscopy, and the density of surface-attached ßSCD molecules, ∼210 ßSCD/AuNP, was determined using thermogravimetric analysis. Because (13)C NMR spectra of ßSCD used in the study exhibited disulfide linkages and the observed surface density on the AuNP exceeded that possible for a close-packed mono layer, a fraction of the surface-attached ßSCD molecules on the particle were oligomerized through disulfide linkages. Determination of the binding constant, K, for the 3-ßCD interaction using (1)H NMR chemical shifts was complicated by the self-association of 3 to form a dimer through its conjugated adamantane residue. With a dimerization constant of K2 = 26.7 M(-1), the value of K for the 3-ßCD interaction (1:1 stoichiometry) is 400-800 M(-1), which is lower than the value, K = 1.4 × 10(3) M(-1), measured for the 2-3 interaction using ICP-MS. Optical microscopy showed that when neuroblastoma SK-N-SH cells are treated with the nanodrug, 4 (2+3), clusters of gold nanoparticles are observed in the nuclear regions of living cells within 24 h after exposure, but, at later times when most cells are dying or dead, clustering is no longer observed. Treating the cells with 4 for 72 h gave percent inhibitions that are lower than that of cisplatin, suggesting that the Pt(IV) ions in 4 may be incompletely reduced to cytotoxic Pt(II) species in the cell.

Gravitational sedimentation of gold nanoparticles.

We study the gravitational sedimentation of citrate- or ascorbate-capped spherical gold nanoparticles (AuNP) by measuring the absorption-vs.-time curve produced as the particles sediment through the optical beam of a spectrophotometer, and comparing the results with a calculated sedimentation curve. TEM showed the AuNP had gold-core diameters of 12.1±0.6, 65.0±5.2, 82.5±5.2 or 91.8±6.2 nm, and gave diameter distribution histograms. The Mason-Weaver sedimentation-diffusion equation was solved for various particle diameters and the solutions were weighted with the TEM histogram and the size-dependent extinction coefficient, for comparison with absorbance-vs.-time curve obtained from freshly prepared suspensions of the AuNP. For particles having average gold-core diameters of 12.1±0.6, 65.0±5.2 and 82.5±5.2 nm, very good agreement exists between the theoretical and observed curves, showing that the particles sediment individually and that the diameter of the gold core is the important factor controlling sedimentation. For the largest particles, observed and calculated curves generally agree, but the former shows random effects consistent with non-homogeneous domains in the sample. Unlike TEM, the simple and unambiguous sedimentation experiment detects all the particles in the sample and can in principle be used to derive the true size histogram. It avoids artifacts of TEM sampling and shear forces of ultracentrifugation. We also show how information about the size histogram can be obtained from the sedimentation curve.

Investigation of the drug binding properties and cytotoxicity of DNA-capped nanoparticles designed as delivery vehicles for the anticancer agents doxorubicin and actinomycin D.

Oligonucleotide-functionalized gold nanoparticles (AuNP) were designed and synthesized to be delivery vehicles for the clinically used anticancer drugs doxorubicin (DOX) and actinomycin D (ActD). Each vehicle contains a tailorable number of DNA duplexes, each possessing three high-affinity sequences for the intercalation of either DOX or ActD, thus allowing for control of drug loading. Drug binding was evaluated by measuring changes to DNA melting temperature, T(m), hydrodynamic diameter, D(h), and surface plasmon resonance wavelength, λ(spr), with drug loading. These studies indicate that DOX intercalates at its high-affinity sequence bound at the AuNP, and that ActD exhibits relatively weaker binding to its preferred sequence. Agarose gel electrophoresis further confirmed drug binding and revealed that particle mobilities inversely correlate with D(h). The equilibrium binding constant, K, and dissociation rate constant, ß, were determined by dialysis. Results indicate that the high negative electrostatic potential within the DNA shell of the particle significantly decreases ß and enhances K for DOX but has little effect on K and ß for ActD. The cytotoxicity of the vehicles was studied, with IC(50) = 5.6 ± 1.1 µM and 46.4 ± 9.3 nM for DOX-DNA-AuNP and IC(50) = 0.12 ± 0.07 µM and 0.76 ± 0.46 nM for ActD-DNA-AuNP, in terms of drug and particle concentrations, respectively.

Enhanced detection of gold nanoparticles in agarose gel electrophoresis.

Gel electrophoresis is a powerful tool in gold nanoparticle (AuNP) research. While the technique is sensitive to the size, charge, and shape of particles, its optimal performance requires a relatively large amount of AuNP in the loading wells for visible detection of bands. We here describe a novel and more sensitive method for detecting AuNPs in agarose gels that involves staining the gel with the common organic fluorophore fluorescein, to produce AuNP band intensities that are linear with nanoparticle concentration and almost an order of magnitude larger than those obtained without staining the gel.

Pt(IV) complexes as prodrugs for cisplatin.

The antitumor effects of platinum(IV) complexes, considered prodrugs for cisplatin, are believed to be due to biological reduction of Pt(IV) to Pt(II), with the reduction products binding to DNA and other cellular targets. In this work we used pBR322 DNA to capture the products of reduction of oxoplatin, c,t,c-[PtCl(2)(OH)(2)(NH(3))(2)], 3, and a carboxylate-modified analog, c,t,c-[PtCl(2)(OH)(O(2)CCH(2)CH(2)CO(2)H)(NH(3))(2)], 4, by ascorbic acid (AsA) or glutathione (GSH). Since carbonate plays a significant role in the speciation of platinum complexes in solution, we also investigated the effects of carbonate on the reduction/DNA-binding process. In pH 7.4 buffer in the absence of carbonate, both 3 and 4 are reduced by AsA to cisplatin (confirmed using ((195))Pt NMR), which binds to and unwinds closed circular DNA in a manner consistent with the formation of the well-known 1, 2 intrastrand DNA crosslink. However, when GSH is used as the reducing agent for 3 and 4, ((195))Pt NMR shows that cisplatin is not produced in the reaction medium. Although the Pt(II) products bind to closed circular DNA, their effect on the mobility of Form I DNA is different from that produced by cisplatin. When physiological carbonate is present in the reduction medium, ((13))C NMR shows that Pt(II) carbonato complexes form which block or impede platinum binding to DNA. The results of the study vis-à-vis the ability of the Pt(IV) complexes to act as prodrugs for cisplatin are discussed.

Adamantane-platinum conjugate hosted in ß-cyclodextrin: enhancing transport and cytotoxicity by noncovalent modification.

This work reports the synthesis of a complex of a carboplatin analog having tethered adamantane that is encapsulated in the hydrophobic cavity of ß-cyclodextrin (ßCD) and its cytotoxic activity towards human neuroblastoma cells (SK-N-SH). We found that this inclusion complex of ßCD adamantane carboplatin analog exhibited higher cytotoxicity towards SK-N-SH cells than carboplatin itself, and the inclusion complex exhibited a higher binding to plasmid pBR322 deoxyribonucleic acid (DNA) than carboplatin. Confocal fluorescence images of SK-N-SH cells treated with ßCD having an attached fluorescein isothiocyanate (FITC)-tag exhibited fluorescence in the vicinity of the nuclei of the neuroblastoma cells. Direct measurements of the platinum content in SK-N-SH cells using inductively coupled plasma mass spectrometry (ICP-MS) indicated that the uptake rate of carboplatin was about 4 times higher than ßCD adamantane carboplatin analog inclusion complex. When compared to carboplatin, we believe that the higher cytotoxicity of inclusion complex towards SK-N-SH cells is due to its higher DNA binding ability as compared to carboplatin, and more efficient delivery to the nucleus of the cell. This work suggests that the advantage of deliberate noncovalent modification with ßCD through host-guest chemistry may also be broadly applicable to other anticancer agents as well.

Stability of carboplatin and oxaliplatin in their infusion solutions is due to self-association.

Carboplatin and oxaliplatin are commonly used platinum anticancer agents that are sold as ready-to-use aqueous infusion solutions with shelf lives of 2 and 3 years, respectively. The observed rate constants for the hydrolysis of these drugs, however, are too large to account for their long shelf lives. We here use electrospray-trap mass spectrometry to show that carboplatin and oxaliplatin are self-associated at concentrations in their ready-to-use infusion solutions (~27 mM and 13 mM, respectively) and, as expected, when the drug concentration is reduced to more physiologically relevant concentrations (100 µM and 5 µM, respectively) the association equilibrium is shifted in favor of the monomeric forms of these drugs. Using (1)H NMR we measure the intensity of the NH resonance of the two symmetry-equivalent NH(3) molecules of carboplatin, relative to the intensity of the γ-methylene CH resonance, as a function of total drug concentration. Then, by fitting the data to models of different molecularity, we show that the association complex is a dimer with a monomer-dimer association constant of K (M(-1)) = 391 ± 127. The work presented here shows that carboplatin and oxaliplatin mainly exist as association complexes in concentrated aqueous solution, a property that accounts for the long term stability of their ready-to-use infusion solutions, and that these association complexes may exist, to some extent, in the blood after injection.

DNA-capped nanoparticles designed for doxorubicin drug delivery.

The anticancer drug, doxorubicin (DOX), was loaded onto DNA-capped gold nanoparticles (AuNP) designed for specific DOX intercalation. Drug binding was confirmed by monitoring DNA melting temperature, AuNP plasmon resonance maximum, and hydrodynamic radius increase, as a function of [DOX]/[DNA] ratio. The capacity for drug release to target DNA was confirmed.

Cytotoxicity of Cu(II) and Zn(II) 2,2'-bipyridyl complexes: dependence of IC50 on recovery time.

We measure the cytotoxicity of three metal complexes containing the 2,2'-bypyridine ligand, Cu(bpy)(NCS)(2), 1, [Cu(bpy)(2)(H(2)O)](PF(6))(2), 2, and Zn(bpy)(2)(NCS)(2), 3, toward neuroblastoma cells (SK-N-SH) and ovarian cancer cells (OVCAR-3) using two different cell assays. The cells were exposed to various concentrations of the compounds for 1 h and the percent inhibition of cell growth, I, measured for various times after exposure, i.e., as a function of the recovery time t. After developing the theory showing the relationship between I and t, the cytotoxicity data were analyzed to reveal that the two copper complexes, 1 and 2, cause the cells to divide at a slower rate than the controls during the recovery period, but the zinc complex, 3, had little or no effect on cell division during the recovery period. The usual metric for reporting cytotoxicity is IC(50), which is the concentration of agent required to inhibit cell growth to 50% of the control population. However, since IC(50) can depend on the recovery time, t, as is the case for 1 and 2, reporting IC(50) for a single recovery time can hide important information about the long-time effects of a cytotoxic agent on the health of the cell population. Mechanistic studies with the compounds revealed that the copper complexes, 1 and 2, cleave closed circular pBR322 DNA in the presence of ascorbate, while the zinc complex, 3, does not facilitate DNA cleavage under the same conditions. This difference in DNA cleavage activity may be related to the fact that Cu(II) is redox active and can readily change its oxidation state, while Zn(II) is redox inert and cannot participate in a redox cycle with ascorbate to break DNA.

Adsorption of the Pt(II) anticancer drug carboplatin by mesoporous silica.

MCM-41, a mesoporous silica nanomaterial with a high surface area for adsorption of small molecules, is a potential new type of delivery vehicle for therapeutic and diagnostic agents. In this report, we show that MCM-41 adsorbs the front-line anticancer drug carboplatin, [Pt(CBDCA-O,O')(NH3)2] (CBDCA=cyclobutane-1,1-dicarboxylate; 1), which is used to treat ovarian, lung, and other types of cancer. UV/Visible difference absorption spectroscopy shows that MCM-41 adsorbs 1.8+/-0.2% of its own weight of carboplatin after a 24 h exposure to 26.9 mM drug in H2O. The pseudo-first-order rate constant for adsorption of carboplatin by MCM-41, measured using [1H,15N] heteronuclear single quantum coherence (HSQC) NMR, and 15N-labeled carboplatin is k(1)=2.92+/-2.17 x 10(-6) s(-1) at ca. 25 degrees.

Formation of carbonato and hydroxo complexes in the reaction of platinum anticancer drugs with carbonate.

The second-generation Pt(II) anticancer drug carboplatin is here shown to react with carbonate, which is present in blood, interstitial fluid, cytosol, and culture medium, to produce platinum-carbonato and -hydroxo complexes. Using [(1)H-(15)N] HSQC NMR and (15)N-labeled carboplatin, we observe that cis-[Pt(CBDCA-O)(OH)(NH(3))(2)](-), cis-[Pt(OH)(2)(NH(3))(2)], cis-[Pt(CO(3))(OH)(NH(3))(2)](-), and what may be cis-[Pt(CO(3))(NH(3))(2)] are produced when 1 is allowed to react in 23.8 mM carbonate buffer. When (15)N-labeled carboplatin is allowed to react in 0.5 M carbonate buffer, these platinum species, as well as other hydroxo and carbonato species, some of which may be dinuclear complexes, are produced. Furthermore, we show that the carbonato species cis-[Pt(CO(3))(OH)(NH(3))(2)](-) is also produced when cisplatin is allowed to react in carbonate buffer. The study outlines the conditions under which carboplatin and cisplatin form carbonato and aqua/hydroxo species in carbonate media.

Influence of carbonate on the binding of carboplatin to DNA.

The reaction of aged carboplatin (reaction of carboplatin in 24 mM NaHCO(3) for 45 h, 37 degrees, pH 8.6) with pBR322 DNA at 0 < r < 2.8, where r = [drug]/[DNA-bp], in 24 mM HEPES buffer, pH 7.4, for 24 h, followed by agarose gel electrophoresis showed DNA mobility changes consistent with unwinding closed circular DNA. However, identical experiments conducted in a two-buffer system, 24 mM HEPES plus 24 mM carbonate, showed no DNA mobility changes, indicating that carbonate blocks formation of the 1,2 intrastrand cross-link on DNA. Studies with aged carboplatin and with cisplatin carried out with ca. 4.0 < r < 10.0 in the two-buffer system show that some DNA binding and unwinding occurs for both drugs. Since carbonate inhibits the binding of aged carboplatin and cisplatin to DNA, carbonate present in the body likely modulates the reactivity of these drugs with a variety of biological targets including DNA.

New extracellular resistance mechanism for cisplatin.

The HSQC NMR spectrum of 15N-cisplatin in cell growth media shows resonances corresponding to the monocarbonato complex, cis-[Pt(NH3)2(CO3)Cl](-), 4, and the dicarbonato complex, cis-[Pt(NH3)2(CO3)2](-2), 5, in addition to cisplatin itself, cis-[Pt(NH3)2Cl2], 1. The presence of Jurkat cells reduces the amount of detectable carbonato species by (2.8+/-0.7) fmol per cell and has little effect on species 1. Jurkat cells made resistant to cisplatin reduce the amount of detectable carbonato species by (7.9+/-5.6) fmol per cell and also reduce the amount of 1 by (3.4+/-0.9) fmol per cell. The amount of detectable carbonato species is also reduced by addition of the drug to medium that has previously been in contact with normal Jurkat cells (cells removed); the reduction is greater when drug is added to medium previously in contact with resistant Jurkat cells (cells removed). This shows that the platinum species are modified by a cell-produced substance that is released to the medium. Since the modified species have been shown not to enter or bind to cells, and since resistant cells modify more than non-resistant cells, the modification constitutes a new extracellular mechanism for cisplatin resistance which merits further attention.

Cytotoxicity of mesoporous silica nanomaterials.

We here measure the toxicity of MCM-41, a mesoporous silica nanomaterial, two of its functionalized analogs, AP-T, which has grafted aminopropyl groups and MP-T, which has grafted mercaptopropyl groups, and spherical silica nanoparticles (SiO(2)), toward human neuroblastoma (SK-N-SH) cells. Since the particles studied are not soluble in aqueous media, the metric used to report the cytotoxicity of these materials is a new quantity, Q(50), which is the number of particles required to inhibit normal cell growth by 50%. Determining the number of particles per gram of material applied to the cells required both the calculated and experimentally determined surface areas of these nanomaterials. This study shows that Q(50) increases in the order, MCM-41

Modification of carboplatin by Jurkat cells.

Using [(1)H,(15)N] heteronuclear single quantum coherance (HSQC) NMR and (15)N-labeled carboplatin, 1, we show that Jurkat cells affect the rate of disappearance of the HSQC NMR peak in culture medium for this Pt(2+) anticancer drug. The decay or disappearance rate constant for 1 in culture medium containing cells is k(1)=k(c)[CO(3)(2-)]+k(m)+k(u)N, where k(c) is the rate constant for reaction of 1 with carbonate in the medium, k(m) is the rate constant for reaction of 1 with all other components of the medium, and k(u) is the rate constant for reaction of 1 with cells having a number density N in the medium. Since Jurkat cells only take up a small amount of the platinum present in the medium (<1%), the observed disappearance of the HSQC NMR peak for 1 cannot be due to uptake of carboplatin by the cells.

Role of carbonate in the cytotoxicity of carboplatin.

Carboplatin, [Pt(NH3)2(CBDCA-O,O')], 1, where CBDCA is cyclobutane-1,1-dicarboxylate, is used against ovarian, lung, and other types of cancer. We recently showed (Di Pasqua et al. (2006) Chem. Res. Toxicol. 19, 139-149) that carboplatin reacts with carbonate under conditions that simulate therapy to produce carbonato carboplatin, cis-[Pt(NH3)2(O-CBDCA)(CO3)]2-, 2. We use 13C and 1H NMR and UV-visible absorption spectroscopy to show that solutions containing carboplatin that have been aged in carbonate buffer under various conditions contain 1, 2, and other compounds. We then show that aging carboplatin in carbonate produces compounds that are more toxic to human neuroblastoma (SK-N-SH), proximal renal tubule (HK-2) and Namalwa-luc Burkitt's lymphoma (BL) cells than carboplatin alone. Moreover, increasing the aging time increases the cytotoxicity of the platinum solutions as measured by the increase in cell death. Although HK-2 cells experience a large loss in survival upon exposure to carbonato forms of the drug, they have the highest values of IC50 of the three cell lines studied, so that HK-2 cells remain the most resistant to the toxic effects of the carbonato forms in the culture medium. This is consistent with the well-known low renal toxicity observed for carboplatin in therapy. The uptake rates for normal Jurkat cells (NJ) and cisplatin-resistant Jurkat cells (RJ), measured by inductively coupled plasma mass spectrometry (ICP-MS), are 16.6 +/- 4.2 and 12.3 +/- 4.8 amol of Pt h-1 cell-1, respectively, when exposed to carboplatin alone. However, when these cells are exposed to carboplatin that has been aged in carbonate media, normal Jurkat cells strongly bind/take up Pt at a rate of 14.5 +/- 4.1 amol of Pt h-1 cell-1, while resistant cells strongly bind/take up 5.1 +/- 3.3 amol of Pt h-1 cell-1. Collectively, these studies show that carboplatin carbonato species may play a major role in the cytotoxicity and uptake of carboplatin by cells.

The activation of platinum(II) antiproliferative drugs in carbonate medium evaluated by means of a DNA-biosensor.

We report on the binding of cisplatin, carboplatin and oxaliplatin to double-stranded DNA in two different (phosphate and carbonate) buffers, using an electrochemical DNA-biosensor. The propensity of the electrophilic agent produced by hydrolysis to interact with DNA was measured as a function of the decrease of guanine oxidation signal of the metal-DNA adduct immobilized on a screen-printed electrode, by using square wave voltammetry. The results obtained confirm that carbonate reacts with platinum drugs to form activated carbonato complexes, which are able to react readily with DNA.

Modification and uptake of a cisplatin carbonato complex by Jurkat cells.

The interactions of Jurkat cells with cisplatin, cis-[Pt(15NH3)2Cl2]1, are studied using 1H-15N heteronuclear single quantum coherence (HSQC) NMR and inductively coupled plasma mass spectrometry. We show that Jurkat cells in culture rapidly modify the monocarbonato complex cis-[Pt(15NH3)2(CO3)Cl]- (4), a cisplatin species that forms in culture media and probably also in blood. Analysis of the HSQC NMR peak intensity for 4 in the presence of different numbers of Jurkat cells reveals that each cell is capable of modifying 0.0028 pmol of 4 within approximately 0.6 h. The amounts of platinum taken up by the cell, weakly bound to the cell surface, remaining in the culture medium, and bound to genomic DNA were measured as functions of time of exposure to different concentrations of drug. The results show that most of the 4 that has been modified by the cells remains in the culture medium as a substance of molecular mass <3 kDa, which is HSQC NMR silent, and is not taken up by the cell. These results are consistent with a hitherto undocumented extracellular detoxification mechanism in which the cells rapidly modify 4, which is present in the culture medium, so it cannot bind to the cell. Because there is only a slow decrease in the amount of unmodified 4 remaining in the culture medium after 1 h, -1.1 +/- 0.4 microM h(-1), the cells subsequently lose their ability to modify 4. These observations have important implications for the mechanism of action of cisplatin.