Proc. Natl. Acad. Sci. USAVol. 76, No. 12, pp. 6611-6614, December 1979Medical Sciences

Inhibition of cis-platinum nephrotoxicity by diethyldithiocarbamaterescue in a rat model

[cis-dichlorodiammineplatinum(II)IRICHARD F. BORCH AND MICHAEL E. PLEASANTSDepartment of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455

Communicated by Bryce Crawford, Jr., August 24, 1979

ABSTRACT The nephrotoxic effects of cis-dichlorodiam-mineplatinum(II) (NSC-119875) administered to male F344 ratsat the median lethal dose (LDs5; 7.5 mg/kg) were inhibited bytreatment with sodium diethyldithiocarbamate (500 or 750mg/kg) between 1 and 4 hr after cis-platinum administration.Those animals receiving cis-platinum alone had mean serumblood urea nitrogen levels of 234 mg/dl at the time of maximaltoxicity (day 5); kidney sections revealed large areas of degen-eration and necrosis. When dithiocarbamate rescue was carriedout after cis-platinum treatment, mean- blood urea nitrogenlevels were in the range 56-95 mg/dl; kidney sections weregrossly normal with a barely discernible band of degenerationat the corticomedullary junction. Gastrointestinal toxicity wasobserved in >95% of the cis-platinum-treated rats but was to-tally absent in those receiving subsequent rescue treatment. Asignificant decrease in weight loss was also observed in the di-thiocarbamate-rescued rats. Based on the chemistry of plati-num-sulfur interactions and the observed time-dependence ofthe rescue treatment, it is suggested that dithiocarbamate exertsits effects via competitive chelation and removal of platinumcoordinated to protein-bound sulfhydryl groups of the kidneytubule cells.

cis-Dichlorodiammineplatinum(II) (DDP) is an agent of es-tablished efficacy against an increasingly wide variety ofhuman tumors (1, 2). Dose-related nephrotoxicity eventuallydevelops with DDP, however, and is often dose-limiting (3, 4).Although mannitol diuresis coupled with intravenous prehy-dration ameliorates acute toxicity in both dogs (5) and humans(6), cumulative toxicity represents a real limitation on theclinical potential of the drug (4).DDP nephrotoxicity in the rat is characterized histopatho-

logically by degenerative and necrotizing lesions in the proximaltubule segments located in the outer medullary stripe. Theselesions reach a peak at 5 days after DDP administration, witha corresponding increase in serum blood urea nitrogen (BUN)and loss of body weight which peak simultaneously (7, 8).Subsequent partial recovery ensues over approximately 14-21days. In man, pathologic changes from either high-dose orlow-dose chemotherapy include acute tubular necrosis in-volving primarily the distal convoluted tubules and collectingducts, with dilatation seen primarily in the proximal tubules(9).

Although the detailed mechanism by which DDP exerts itstoxic effects on the kidney is not known, comparison of thechemistry and nephrotoxicity of Pt(II) and Hg(II) suggests botha possible mechanism and a therapeutic approach to the inhi-bition of this toxicity. Although both Pt(II) and Hg(II) have highbinding affinities to sulfhydryl ligands, the complexes of Pt(II)are kinetically much less labile than their Hg(II) counterparts.The brush border of the proximal tubule contains an abundanceof protein-bound sulfhydryl groups (10); the mechanism pos-

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tulated for mercury-induced diuresis involves reversible inhi-bition of water and electrolyte reabsorption in the proximaltubule via reversible coordination of Hg(II) to the sulfhydrylgroup of a membrane-bound transport enzyme (11).

Because of the striking histopathologic similarities betweenPt(II) and Hg(II) toxicity in the rat (3, 12) and because of theslower rates of 'igand exchange in the Pt(II) complexes, wesuggest that the mechanism for DDP nephrotoxicity in the ratinvolves a concentration-dependent, essentially irreversiblebinding of Pt(II) to the protein-bound sulfhydryl groups in theproximal tubule with resulting inhibition of one or moretransport enzymes. The fact that concentration-dependentbinding of DDP to proximal tubular membranes has been ob-served in a model system (13) is in accord with this postulate.This mechanism implies that, if a potent chelator for Pt(II) wereadministered after the platinum had accumulated and boundin the tubules but before irreversible damage had occurred,then nephrotoxicity might be inhibited. This report describesthe efficacy of sodium diethyldithiocarbamate (DDTC) as aninhibitor of nephrotoxicity in a rat model.

MATERIALS AND METHODSMale F344 (Fischer) rats (average weight, 150 g) were obtainedfrom the Charles River Breeding Laboratory. They were di-vided into groups of four rats, placed in methacrylate cages, andgiven National Institutes of Health chow for rodents and waterad lib, for 7 days. Average weight of the rats was 180 g (range,160-200 g) when injected.DDP (NSC-119875) was kindly provided by L. Pignolet

(Department of Chemistry, University of Minnesota) and bythe Drug Synthesis and Chemistry Branch (Division of CancerTreatment, National Cancer Institute). Solutions of DDP wereprepared immediately prior to injection in isotonic saline (0.9NaCl) at a concentration of 1 mg/ml; mannitol (10 mg/ml) wasadded to duplicate the formulation available for clinical use.Tetraethylthiuram disulfide (disulfiram) was obtained fromSigma. DDTC was prepared according to the procedure ofKlopping and van der Kerk (14); immediately prior to injection,it was dissolved in sterile water at a concentration of 100 mg/ml.Sodium bicarbonate was prepared in sterile water at a con-centration of 50 mg/ml. All injections were carried out underbrief ether anesthesia.

Rats in groups of 8-16 were injected intravenously with DDPat a dose of 7.5 mg/kg [the median lethal dose (LDso)] (7). Therats were then randomly divided into two groups; at times from0 to 6 hr after injection of DDP, one group was injected intra-peritoneally with DDTC (500 or 750 mg/kg), and the othergroup was injected intraperitoneally with sodium bicarbonate(370 mg/kg) as a control. One group of rats was given onlyDDTC, in a dose of 750 mg/kg intraperitoneally, to serve as a

Abbreviations: DDP, cis-dichlorodiammineplatinum(II); DDTC, so-dium diethyldithiocarbamate; BUN, blood urea nitrogen.

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control for the effects of DDTC. In one experiment, blood wasobtained from the tail vein for serial daily BUN determinations(15). Otherwise, weight and BUN were determined at 5 daysafter injection, the time of maximal observed toxicity (16).Evidence of gastrointestinal toxicity was obtained qualitativelyby noting the presence or absence of diarrhea 3-5 days afterinjection.Randomly selected animals were sacrificed at 5 days after

injection for study of histopathologic changes in the kidney andto note the presence or absence of hemorrhagic enteritis (16).Kidneys were removed, placed in Zenker's fixative, embeddedin paraffin, sectioned at 6 Atm, and stained with hematoxylinand eosin.

RESULTSDDTC, at doses of 500 and 750 mg/kg, was given at timesranging from 30 min before to 6 hr after administration of DDP(Table 1). The rats that received DDTC alone maintainednormal BUN levels throughout the 2-week period but experi-enced a mean weight loss of 6% at day 5; by 2 weeks, however,a mean weight gain of 6% was observed. The control animals,which received DDP at 7.5 mg/kg without rescue, experienceda mean weight loss of 21% at 5 days and had a mean BUN levelof 234 mg/dl. Serial BUN and body weight determinations inboth control and rescued animals confirmed that maximal BUNand weight loss occurred on day 5 (16). Survivors generallyshowed recovery of both weight and BUN from these extremelevels. Diarrhea was observed in 21 of the 22 animals examinedfor it. Necropsy revealed a distended membranous small in-testine filled with hemorrhagic fluid in every animal examined.Sections of the kidney revealed a pale zone of extensive necrosis

and degeneration of tubule cells in the outer medullary stripeextending into the cortex (Fig. 1A).Marked reduction in BUN and weight loss were noted when

DDTC was administered between 1 and 4 hr after DDP (Table1). Diarrhea was not observed in any of these rescued animals.Necropsy revealed a grossly normal small intestine; kidneysections showed a barely discernible pale band of mild tubuledegeneration in the outer zone of the outer medullary stripe(Fig. 1B). When DDTC was administered immediately afterDDP (time = 0 hr), a significant decrease in weight loss wasnoted without reduction of BUN levels. Rescue 6 hr after DDP

administration, however, failed to demonstrate significant re-duction either in BUN or weight loss, and two of the five ani-mals in this group were noted to have diarrhea. The relationshipbetween administration time and BUN is summarized graph-ically in Fig. 2. Platinum excretion kinetics were measured infour animals by measuring urinary platinum levels every 30

min for 4 hr and then every 2 hr for an additional 12 hr (17).Urinary platinum concentration declined to <10% of maximalconcentration within 1.5 hr, and >95% of the platinum excretedby the kidneys had appeared in the urine by this time.

In one experiment, disulfiram was administered to twogroups of rats at a dose of 2 g/kg via stomach tube 30 min beforeand 30 min after DDP. Fifty percent of the animals in eachgroup died before day 5, with grossly hemorrhagic fluid-filledintestine noted on necropsy. Weight loss and BUN levels in thesurvivors were highly variable (BUN, 55-180 mg/dl).

DISCUSSIONThese results demonstrate clearly the salutary effects of DDTCrescue on renal function and pathology after a toxic dose ofcis-platinum and strongly suggest a beneficial effect upongastrointestinal toxicity as well. Maximal plasma and urineplatinum levels have been observed shortly after a bolus in-jection of DDP in man and animals (17-20), with a rapid initialclearance (tl/2 = 20-60 min) followed by a second phase of slowclearance (tl/2 = 50-80 hr). Plasma and urine concentrationsgenerally decrease to <10% of maximum levels within 1-2 hr.The acute toxicity observed in a number of animal models isdose-related; this observation is consistent with our hypothesisof a concentration-dependent binding of platinum to sulfhydrylgroups in the renal tubules. A decrease in total renal sulfhydrylgroup concentration after administration of DDP to rats hasbeen reported (21).

Disulfiram, the disulfide dimer of DDTC, is reduced toDDTC in laboratory animals and in man (22-25) and thusserves as a potential source of DDTC. In the rat, DDTC israpidly metabolized, with conversion to carbon disulfide (10%),sulfate (30%), and the S-glucuronide of DDTC (60%) beingessentially complete within 1 hr; essentially no free DDTC isobserved. Twenty percent of the dose appears in the urine after1 hr, and, of this, 95% is the S-glucuronide (22). Thus, the ratappears to be a poor model in which to test the rescue effects

Table 1. Effects of DDTC rescue on BUN, weight loss, and effects in rats on day 5 after DDP(7.5 mg/kg, intravenously)

IDDTC, Rescue, BUN, Weight Incidencemg/kg hr after DDP n mg/dl* Pt loss, %* Pt of diarrhea

0 26 234 20 21 1 21/22750t 4 16± it 6 lt

500 0.5 4 152 ± 20 <0.05 4 ± 2 <0.0011 4 90± 5 <0.001 4 1 <0.0012 4 72 11 <0.001 2 2 <0.001

750 -0.5 4 418 + 43 <0.001 18 + 1 >0.10 12 214 + 21 >0.25 9 + 1 <0.001 0/60.75 5 56 ± 9 <0.001 8 ± 2 <0.001 0/51 16 63 ± 4 <0.001 13 + 1 <0.001 0/81.5 15 62 3 <0.001 5 + 1 <0.0012 14 95 9 <0.001 15 + 1 <0.001 0/143 10 77± 8 <0.001 10 1 <0.0014 7 106 8 <0.005 14 1 <0.0016 5 209 44 >0.3 18± 2 >0.1 2/5

* Mean ± SEM on day 5.t For difference from controls receiving DDP but not DDTC; Student's t test.t Treatment with DDTC in the absence of DDP.

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FIG. 1. Histologic appearance of the corticomedullary junction in the kidney (oriented with cortex on the left) 5 days after intravenousinjection of DDP (7.5 mg/kg) in F344 rats. (A) In the absence of DDTC rescue, note the widespread pale zones of degeneration and necrosisin the outer zone of the medulla and extending into the cortex. BUN on day 5, 265 mg/dl. (B) With DDTC rescue (750 mg/kg intraperitoneally)2 hr after DDP, note the grossly normal histologic appearance with a narrow pale zone at the corticomedullary junction. BUN on day 5, 96 mg/dl.(Hematoxylin and eosin; X48.)

of disulfiram administered orally; the extreme variability ofour results with disulfiram appear to confirm this.

In rabbits, 25% of disulfiram administered via stomach tubeappears in the urine as free DDTC (23). In man, disulfiramtaken orally produced substantial levels of DDTC in the plasma;although urinary excretion was normally insignificant, alka-linization of the urine by oral administration of sodium bicar-bonate produced significant urine levels of free DDTC (24).DDTC is unstable in acidic medium, decomposing to carbon

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0 1 2 3 4 5 6Rescue time after DDP, hr

FIG. 2. Effect of time interval between DDP administration (8.5mg/kg intravenously) and DDTC rescue (750 mg/kg intraperitoneally)on serum BUN levels in F344 rats at 5 days after treatment. a, Un-treated; b, treated with DDP only. Vertical bar = SEM.

disulfide and diethylamine at pH below 7; hence, these ap-parent species variations can be explained on the basis of relativestability of DDTC in the final urine, because the pH of rabbiturine is slightly alkaline (24). Presumably, all species initiallypresent free DDTC to the kidney tubule; its site and rate ofdecomposition depend upon site and degree of urine acidifi-cation. The efficacy of DDTC as a chelating agent in thetreatment of nickel carbonyl poisoning in man and animals hasbeen reported (25).On the basis of these data and our results, we suggest the

following rationale for DDP toxicity and DDTC rescue. At highplasma and urine platinum concentrations, binding occurs tosulfhydryl groups of the renal tubule; maximal platinum ac-cumulation occurs at 30-60 min. DDTC administered 30 minprior to or concurrent with DDP is rapidly metabolized beforeplatinum accumulates, and DDTC concentrations after 1 hrare inadequate to remove bound platinum. Rescue between 45min and 4 hr is effective because DDTC concentration in thetubular fluid is adequate for platinum chelation and removal.The failure of rescue at 6 hr presumably results from plati-num-induced irreversible damage having occurred prior torescue. Although inhibition of gastrointestinal toxicity generallyparallels that observed for nephrotoxicity, the beneficial resultsof concurrent administration suggest that gastrointestinal tox-icity may be more responsive to early rescue.A number of other thiol reagents have been examined for

their effects on platinum toxicity, including cysteine (26),penicillamine (13, 27), cysteamine (13), and N-acetylcysteine(13). No beneficial effects were observed when the thiol wasadministered after DDP; although some benefit was notedwhen cysteine and penicillamine were given before DDP, thepresence of the rescue agent when DDP is administered has thepotential to bind to plasma platinum and reduce its chemo-

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therapeutic effect. In fact, decreased efficacy has been observedwith both cysteine (26) and thiourea (28) when administeredprior to DDP. Thiourea has also shown promise as a rescue drugin mice (28); increased life-span was noted in a leukemia P388model for small doses administered prior to DDP or for largedoses administered 1-2 hr after DDP. Thiourea is toxic to rats(29), however, so a direct comparison of our system was notpossible. Thiourea is also known to inhibit thyroid hormoneproduction at doses below those used for rescue (30). At highconcentration, thiourea has the potential to reverse platinum-induced crosslinks and lethal lesions in an isolated DNA system(31); whether this effect can reverse DDP toxicity in tumor cellsis not known.

Because optimal timing of DDTC rescue corresponds to 4-8plasma half-lives for DDP, plasma levels of platinum at the timeof rescue are negligible, and the effect of rescue on the antitu-mor properties of DDP should be minimal. DDTC may alsohave a beneficial antitumor effect in view of its recently re-ported activity as an immunopotentiator (32). The currentavailability of disulfiram and its bioequivalence to DDTC inman suggest its possible use as a clinical rescue agent.

This research was supported by Grant CA 22925 from the NationalInstitutes of Health.

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