Attenuating the toxicity of cisplatin by using selenosulfate with reduced risk of selenium toxicity...

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ology 226 (2008) 251–259www.elsevier.com/locate/ytaap

Toxicology and Applied Pharmac

Attenuating the toxicity of cisplatin by using selenosulfate with reduced riskof selenium toxicity as compared with selenite

Jinsong Zhang ⁎, Dungeng Peng, Hongjuan Lu, Qingliang Liu

University of Science and Technology of China, Hefei 230052, Anhui, PR China

Received 6 June 2007; revised 3 September 2007; accepted 4 September 2007Available online 19 September 2007

Abstract

It has been reported that high doses of sodium selenite can reduce side effects of cisplatin (CDDP) without compromising its antitumor activity,thus substantially enhancing the cure rate in tumor-bearing mice. However, the toxicity of selenite at high doses should be a concern. The presentstudy revealed that selenosulfate had much lower toxicity, but possessed equal efficacy in selenium (Se) utilization, as compared with selenite atsimilar doses when used for the intervention of CDDP. In addition, Se accumulation in whole blood and kidney of mice treated with selenosulfate washighly correlated with the survival rate of mice treated with CDDP (both rN0.96 and both pb0.05), suggesting that whole blood Se is a potentialclinical biomarker to predict host tolerance to CDDP. In either Se-deficient or -sufficient mice bearing solid tumors of hepatoma 22 (H22),selenosulfate did not disturb the therapeutic effect of CDDP on tumors but effectively attenuated the toxicity of CDDP. Furthermore, in a highlymalignant cancer model, with Se-sufficient mice bearing ascitic H22 cells, 8 or 10 mg/kg CDDP alone only achieved a null or 25% cure rate, whereascoadministration of selenosulfate with the above two doses of CDDP achieved cure rates of 87.5% or 75%. These results together argue forconsideration of selenosulfate as an agent to enhance the therapeutic efficacy of CDDP.© 2007 Published by Elsevier Inc.

Keywords: Cisplatin; Selenosulfate; Selenite; Toxicity

Introduction

Sodium selenite can reduce side effects of cisplatin (CDDP)without affecting its antitumor activity (Baldew et al., 1989;Satoh et al., 1989, 1992). For instance, 25.3 μmol/kg, givenintraperitoneally (i.p.) 1 h before CDDP, greatly reduced bloodurea nitrogen (BUN) and creatinine levels and morphologicalkidney damage inmice and rats (Baldew et al., 1989). Satoh et al.(1989) have developed an optimum schedule of sodium seleniteadministration for reducing lethal and renal toxicities of CDDPin mice that bear transplantable tumors. They found that the besteffect of selenium (Se) against lethal toxicity of CDDP wasobtained when sodium selenite was administered subcutaneous-ly (s.c.) to mice, simultaneously with s.c. injection of CDDP at amolar ratio of 1:3.5 (selenite:CDDP) on the first day and thesame amount of selenite was given daily for another 4 con-

⁎ Corresponding author. Fax: +86 551 3492386.E-mail address: [email protected] (J. Zhang).

0041-008X/$ - see front matter © 2007 Published by Elsevier Inc.doi:10.1016/j.taap.2007.09.010

secutive days. The coadministration of selenite and CDDP de-pressed lethal toxicity of CDDP, as well as its renal and intestinaltoxicities, but did not compromise the antitumor activity ofCDDP. Furthermore, when this optimum schedule was dupli-cated for several weeks, it yielded a 100% cure rate in micetransplanted with malignant colon adenocarcinoma 38 cells andP388 leukemia cells (Satoh et al., 1992). In contrast, CDDPalone failed to completely suppress the growth of malignantcells, and the mice died of either progressive disease or CDDP-induced toxicity (Satoh et al., 1992). It is noteworthy that highdoses of Se, in the form of sodium selenite, were given by i.p. ors.c. routes in all the studies above. Specifically, in the model ofsingle CDDP treatment, selenite at the dose of 25.3 μmol/kg wasused (Baldew et al., 1989); and in the optimum schedule, seleniteat the doses of 6.3–12.7 μmol/kg was used five times weekly for7 weeks (Satoh et al., 1992).

Since selenite has been extensively reported to be highlytoxic, long-term administration of high doses of selenite bringsout great concerns about its toxicity (Jia et al., 2005; Whanger,

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252 J. Zhang et al. / Toxicology and Applied Pharmacology 226 (2008) 251–259

2002; Wycherly et al., 2004; Zhang et al., 2005). Hopefully, adifferent Se form with significantly lower toxicity would makeSe modulation of CDDP toxicity more feasible. Selenosulfatehas been widely used as a precursor to prepare Se-containingnanoparticles, such as cadmium selenide, silver selenide andcopper selenide (Al and Islam, 2004; Raevskaya et al., 2006;Yochelis and Hodes, 2004). We previously reported thatselenosulfate could equally elevate the activity of glutathioneperoxidase (GPx) and thioredoxin reductase (TrxR) in HepG2cells, however, the bioavailability of selenosulfate in terms ofincreasing selenoenzymes and Se accumulation was slightly butsignificantly less than selenite in Se-deficient mice at a nu-tritional dose (Peng et al., 2007). Regretfully, the biologicalprofiles of selenosulfate are only limited to the above knowl-edge. In present study, the toxicological and pharmacologicalprofiles of selenosulfate have been investigated in mice ascompared with selenite; the results demonstrate that seleno-sulfate has much lower toxicity without compromising Seutilization. Then selenosulfate was used to observe its impacton CDDP in cancer therapy, in either Se-deficient or -sufficientmice, on both solid tumor and ascites models. The results showthat selenosulfate can largely attenuate the toxicity of CDDP,leading to a substantial improvement in cure rate.

Materials and methods

Chemicals and drugs. NADPH, HEPES, insulin, 5,5′-dithiobis (2-nitrobenzotic acid) (DTNB), thioredoxin (E. coli), thioredoxin reductase (E.coli), guanidine hydrochloride, reduced glutathione (GSH), bovine serumalbumin, hydrogen peroxide, and 1-chloro-2.4-dinitrobenzene (CDNB), CDDP,and sodium selenite were all purchased from Sigma (St. Louis, MO, USA).

Preparation of selenosulfate. Sodium selenosulfate was prepared by refluxing5 mM black powder of elemental Se and 20 mM sodium sulfite for 3 h at 90 °Cbased on the following reaction: Na2SO3+Se→Na2SeSO3 (Fig. 1) (García et al.,1999; Rodriguez-Lazcano et al., 2005; Yochelis and Hodes, 2004). The sulfiteions present in excess in the selenosulfate solution are of prime importance for thestability of selenosulfate (García et al., 1999; Rodriguez-Lazcano et al., 2005)because of their reduction properties [E° (SO4

2−/SO32−)=−0.90 V] (Peng et al.,

2007). Furthermore, selenosulfate was always freshly prepared before eachapplication to ensure its reduced status.

Animals and drugs administration. Male Kunming mice (body weight of 22–24 g) were used in this study unless specifically stated otherwise. Se-sufficientmice (liver Se 0.9–1.2 μg Se/g and whole blood Se 0.3–0.4 μg Se/mL) and theirdiet (0.32 μg Se/g) were purchased from Shanghai Laboratory Animal Center(SLAC). Se-deficient mice (liver Se 0.1–0.2 μg Se/g and whole blood Se–0.1 μg Se/mL) and their diet (b0.02 μg Se/g) were purchased from the animalcenter of Anhui Medical University, Hefei. The mice were housed in plasticcages in a room with controlled temperature (22±1 °C) and humidity (50±10%)

Fig. 1. Chemical structure of sodium selenosulfate and sodium selenite.

and 12 h light/dark circle. The mice were allowed to obtain diet and water adlibitum. Selenocompounds and CDDP were all administered i.p. into mice.Since there were various experimental protocols in the present study, the dosesand dose schedules of selenocompounds and CDDP have been described in thecorresponding Results section instead of here.

Murine hepatoma 22 (H22) models. H22 cells were maintained in ourlaboratory by weekly transplantation of H22 cells into the peritoneal cavity ofmice. For the experiments related to solid tumors, the ascitic fluid containingH22 cells was inoculated (s.c.) at the left axilla of mice (5×106 cells/each). Thetumor size was measured by a caliper and the volume was calculated accordingto the equation: width 2× length÷2. For the experiment related to ascites, theascitic fluid containing H22 cells was injected into the peritoneal cavity of mice(20×106 cells/each).

Blood and tissue preparation. At the end of each set of experiments, micewere sacrificed by cervical dislocation. Blood was sampled from ophthalmicveins to obtain serum after centrifugation. Liver and kidney were excised andrinsed in ice cold saline. Serum and tissue samples were stored at −30 °C beforeassay. Blood sampled from tail veins was used to observe the variation of BUNat designed times.

All protocols involved in animal treatments complied with the guidelines ofUniversity of Science and Technology of China for the care and use of laboratoryanimals.

Biochemical parameters. The serum alanine aminotransferase (ALT),aspartate aminotransferase (AST) and BUN were determined by spectropho-tometry using commercially available kits. Livers and kidneys were homoge-nized with ice cold saline and centrifuged at 15,000×g and 4 °C for 15 min. Theresulting supernatants were used for biochemical assessments. Protein level wasdetermined by the Bradford dye-binding assay with bovine serum albumin asstandard. GST activity was chemically determined using CDNB as substrate,and one unit of GST activity was expressed in terms of nmol CDNB changed/min/mg protein (Habig et al., 1974). GPx was assayed by the method of Rotrucket al. (1973), and one unit of GPx activity was calculated in terms of ìmol ofGSH oxidized/min. TrxR activity was measured using insulin as substrate,which was calculated based on the standard curve prepared with pure TrxR(Zhang et al., 2003). Se was determined by a fluorescent method (Olson et al.,1975).

Histopathological observation. Tissues were fixed in 10% neutral bufferedformalin solution, embedded in paraffin, stained with hematoxylin and eosin(H&E) and then observed under an optical microscope by an experiencedpathologist who was blind to the treatments.

Statistical analysis. Data were presented as the mean±SD. The differencesbetween the groups were examined using ANOVA. A p value of less than 0.05was considered statistically significant.

Results

Toxicity comparison

Mice were treated with selenite and selenosulfate at 25.3 and38.0 μmol/kg once daily for 14 days. The animal status, bodyweight and survival were recorded every day. Mice displayedeclampsia instantly after selenite injection at both doses; thisphenomenon remained prominent during the first few days oftreatment and gradually disappeared in the following days. Incontrast, eclampsia was not observed in mice treated withselenosulfate at both tested doses. At 38.0 μmol/kg, selenitecaused prominent 25% body weight loss (related to initial bodyweight) and 90% mortality after 9 days of treatment. Suchpotent toxicity was not found in mice treated with selenosulfate,which in fact did not cause any body weight loss and only

253J. Zhang et al. / Toxicology and Applied Pharmacology 226 (2008) 251–259

resulted in 20% mortality after 14 days of treatment (Figs. 2A,B). Significant difference in body weight between the twoselenocompounds treatment could be found when most mice inthe selenite group were alive (all pb0.01 from day 3 to day 7)(Figs. 2A, B). Difference in the final survival rate (one moreweek of observation after the last Se treatment) was noticeable(p=0.002) (Fig. 2B). At 25.3 μmol/kg, 90% of the mice were

Fig. 2. Toxicity of selenosulfate and selenite. Fifty mice were divided to five groups: tand 38.0 μmol/kg for 14 days (n=10). The first Se treatment was designed as day 0. (curve (mice in control group and 25.3 μmol/kg groups were sacrificed on day 14 for bobserved for 1 week after 14 day's Se treatment). (C) Serum ALT and AST activities(D) or selenosulfate (E), arrows indicate hydropic degeneration and ovals indicate pselenite. ⁎⁎⁎, pb0.001. ##, pb0.01. ###, pb0.001.

able to survive selenite treatment (Fig. 2B); however theirgrowth was totally suppressed from day 4 to day 13 (Fig. 2A).In contrast, all the mice survived treatment with selenosulfate(Fig. 2B); they maintained continuous growth, although at aslower rate than that of the control mice (Fig. 2A). Significantdifferences in effects on body weight between the twoselenocompounds were observed (all pb0.05 from day 4 to

he control, the groups treated (i.p.) with the selenocompounds at the dose of 25.3A) Average body weight (SD was not shown for the sake of clarity). (B) Survivaliochemical and pathological analysis, mice in 38.0 μmol/kg groups were further. (D–E) Pathological observation (H&E, ×200) of the liver treated with seleniteunctiform necrosis. (F) Liver Se. ⁎, compared with control. #, compared with


day 13) (Fig. 2A). Since most mice given 25.3 μmol/kgremained alive after Se treatment for 14 days, they weresacrificed after the last Se treatment for biochemical andpathological analysis. Selenite significantly increased serumALT and AST activities as compared with the control orselenosulfate (Fig. 2C, all pb0.001). Histological observationof liver indicated that selenite treatment caused partial puncti-form necrosis and moderate hydropic degeneration in 80% ofthe cells (Fig. 2D), whereas selenosulfate treatment only causedslight hydropic degeneration in 20% of the cells (Fig. 2E).Interestingly, although selenosulfate induced much less liverinjury than selenite, the liver Se accumulation in mice treatedwith selenosulfate was higher than that in mice treated withselenite (pb0.01, Fig. 2F), demonstrating that the lower toxicityof selenosulfate is not due to the lower accumulation of Se.Taken together, selenosulfate showed markedly lower toxicityas compared with selenite in all observed phenomena and testedbiomarkers.

Se utilization comparison

To investigate whether both Se forms had any difference intheir utilization when being administered at the levels used forimproving the therapeutic efficacy of CDDP, Se-deficient micewere treated with selenite or selenosulfate at 6.3 and 12.7 μmol/kg for 1 week. It was found that both Se forms were able tosignificantly affect the tested biomarkers (Se concentration ofserum, liver and kidney; GPx activity of serum, liver and kidney;liver GST); however, no significant difference was foundbetween the two Se forms at the same dose (Table 1). Therefore,selenosulfate appears to have lower toxicity, but without com-promising Se utilization at the levels close to the amounts usedfor modulating CDDP.

Effect of selenosulfate on CDDP toxicity in Se-deficient mice

Studies on Se toxicity and Se utilization provide a rationalbasis for the application of selenosulfate to modulate the ther-apeutic efficacy of CDDP. To investigate whether selenosulfatecould effectively attenuate CDDP toxicity, Se-deficient mice(20/each group) were treated with selenosulfate for 7 days at

Table 1Comparison of Se utilization between selenite and selenosulfate at the doses needed

Biomarkers Control Selenosulfate

6.3 μmol/kg

Serum Se 1.00±0.06 5.02±0.15Liver Se 1.00±0.27 10.9±0.18Kidney Se 1.00±0.19 2.83±0.10Serum GPx 1.00±0.22 2.97±0.37Liver GPx 1.00±0.36 19.8±0.08Kidney GPx 1.00±0.17 8.74±0.14Liver GST 1.00±0.15 1.64±0.23

Se-deficient micewere treated (i.p.) with saline (as control), selenosulfate and selenite f(n=7). Value in the control was designed as 1. The control Se level in serum, liver andThe control GPx activity in serum, liver and kidney was 525.5 U/mL serum, 88.5 U/mwas 4071.4 U/mg protein.

different doses (0, 1.3, 6.3 and 12.7 μmol/kg) to generate dif-ferent Se status. Four hours after the last selenosulfate treatment,12 mice of each group were injected with CDDP at the dose of15 mg/kg, after which survival was monitored for 10 days.Twenty-four hours after the last selenosulfate treatment, theother 8 mice in each group were sacrificed to estimate Sestatus. CDDP alone caused death in 92% of Se-deficient mice;selenosulfate dose-dependently reduced this death rate (Fig.3A). Selenosulfate increased Se level in whole blood and kidneyin a dose-dependent fashion (all pb0.001 between the doses,Figs. 3B, C). Selenosulfate also increased plasma and kidneyGPx activities as well as kidney TrxR activity (Figs. 3D, E, F).The activities of plasma GPx and kidney TrxR, but not kidneyGPx, were saturated at 6.3 μmol/kg. The correlations betweensurvival rate and the tested biomarkers were further calculated.Blood Se and kidney Se were highly correlated with the survivalrate (both rN0.96 and both pb0.05). However, the activities ofplasma GPx, kidney GPx and kidney TrxR were not signifi-cantly correlated with the survival rate (r=0.84, p=0.16 forplasma GPx; r=0.85, p=0.15 for kidney GPx; r=0.88, p=0.13for kidney TrxR). Since Se accumulation in whole blood washighly correlated with the survival rate, it could be considered asa potential clinical biomarker for predicting host tolerance ofCDDP.

Furthermore, the effect of a fixed dose of selenosulfate onthe toxicity of different doses of CDDP was investigated. Se-deficient mice were treated with selenosulfate at 12.7 μmol/kgfor 7 days. Four hours after the last selenosulfate treatment,mice were i.p. injected with a bolus dose of CDDP (12, 15and 18 mg/kg) and monitored for the next 10 days. Mean-while, mice treated with each dose of CDDP alone weredesigned as collates. The survival rate of mice treated withCDDP alone at the dose of 12, 15 and 18 mg/kg was 80%,0% and 0% respectively, whereas that of mice pretreated withselenosulfate was increased up to 100%, 100% and 66.7%respectively.

Our pilot experiments demonstrated that tumor-bearing micecould tolerate 9 mg/kg CDDP; hence, this dose was chosen toinvestigate whether selenosulfate compromises the therapeuticefficacy of CDDP. H22 cells were inoculated into left axilla ofSe-deficient mice (5×106 cells/each) on day 0, after daily

to improve the therapeutic efficacy of CDDP

Selenite Selenosulfate Selenite

6.3 μmol/kg 12.7 μmol/kg 12.7 μmol/kg

5.69±0.13 5.41±0.29 5.24±0.3911.2±0.09 15.4±0.09 15.2±0.092.85±0.10 3.50±0.12 3.90±0.143.10±0.25 4.18±0.26 4.41±0.2418.8±0.19 22.2±0.10 22.2±0.167.74±0.19 10.2±0.14 9.09±0.181.47±0.29 2.44±0.14 2.69±0.23

or 7 days at doses of 6.3 and 12.7 μmol/kg. The data were expressed asmean±SDkidney was 0.07 ìg/mL serum, 0.15 μg/g liver and 0.44 μg/g kidney, respectively.g protein and 142.8 U/mg protein, respectively. The control hepatic GSTactivity

Fig. 3. Effect of Se status on the survival of mice treated with CDDP. Eighty Se-deficient mice were divided into four groups, after being i.p. administered withselenosulfate at different doses for 7 consecutive days; 8 mice in each group were sacrificed to assess Se status and the remaining 12 mice were injected (i.p.) withCDDP at the dose of 15 mg/kg. (A) Percent survival. (B) Whole blood Se. (C) Kidney Se. (D) Plasma GPx activity. (E) Kidney GPx activity. (F) Kidney TrxR activity.⁎, compared with the control. ¶, compared with the 1.3 #, compared with the 6.3 ¶, pb0.05. ¶¶, ##, pb0.01. ⁎⁎⁎, ¶¶¶, ###, pb0.001.


treatment with saline or selenosulfate (12.7 μmol/kg) from day1 to day 7, CDDP was administered on day 7 to start thechemotherapy. CDDP with or without selenosulfate largelysuppressed the tumor growth with equal efficacy (data notshown). CDDP alone caused 34% body weight loss and 103%serum BUN increment as compared with the control, whereaspretreatment with selenosulfate decreased them to 12%( pb0.01 as compared with CDDP alone) and 25% ( pb0.01as compared with CDDP alone).

Taken together, the results demonstrated that in Se-deficientmice selenosulfate could potently attenuate CDDP toxicitywithout compromising its therapeutic efficacy. In addition, we

found that whole blood Se level was a potential clinicalbiomarker for predicting host tolerance to CDDP.

Effect of selenosulfate on CDDP toxicity in Se-sufficient mice

The above experiments indicated that whole blood Se andkidney Se but not selenoenzymes were significantly correlatedwith the survival rate of mice treated with CDDP, therebysuggesting that selenosulfate could reduce CDDP toxicity in Se-sufficient subjects with saturated selenoenzymes (Hadley andSunde, 2001) that do not further increase by supranutritional Sesupplementation (Ganther and Ip, 2001). To test this hypothesis,

Fig. 4. Effect of selenosulfate on the toxicity of CDDP in Se-sufficient micebearing H22 solid tumors. H22 cells (5×106 cells/each) were inoculated (s.c.)into left axilla of Se-sufficient mice (27–29 g) on day 2. CDDP at the dose of10 mg/kg was injected (i.p.) once weekly for 4 weeks, the first injection startedon day 0. Selenosulfate at 9.5 μmol/kg was injected (i.p.) for 5 consecutive daysweekly for 4 weeks, the first injection started on day 0 4 h prior to CDDP. Thedata were expressed as mean±SD (n=8). (A) Average tumor volume.(B) Average body weight. ##, pb0.01, when CDDP was compared withCDDP plus Se. (C) BUN level (blood was obtained via clipping approximately 5mm tail each time). ⁎, compared with control. #, compared with CDDP. #,pb0.05. ⁎⁎, ##, pb0.01. ⁎⁎⁎, pb0.001.

Table 2Effect of selenosulfate on cure rate of CDDP in mice bearing H22 ascites

Treatment Dead mice(%)

Survival time ofdead mice (day)

Cure rate(%)

Control 100 12±1 (n=8) 0CDDP (5 mg/kg) 100 20±7⁎ (n=8) 0CDDP (8 mg/kg) 100 31±12⁎ (n=8) 0CDDP (10 mg/kg) 75 21±9 (n=6) 25CDDP (8 mg/kg)/Se (7.6 μmol/kg) 12.5 29 (n=1) 87.5CDDP (10 mg/kg)/Se (9.5μmol/kg) 25 32±12⁎ (n=2) 75

⁎ indicates pb0.05 when compared with the control; n indicates the number ofdead mice. Se-sufficient mice were divided into 6 groups with 8 mice in eachgroup and were injected (i.p.) with 20 million H22 cells to their abdominalcavity. In the control group, mice were treated with saline; in the CDDP groups,CDDP (5 and 8 mg/kg) once weekly for 4 weeks or CDDP (10 mg/kg) onceweekly for 3 weeks was firstly injected (i.p.) at 48 h after the inoculation ofcancer cells; in the coadministration groups, coadministration of 8 mg/kg or10 mg/kg CDDP with selenosulfate (7.5 or 9.5 μmol/kg) at the molar ratio of1:3.5 (selenosulfate:CDDP) was applied once weekly for 4 or 3 weeks and thesame amount of selenosulfate was given daily for four subsequent days, the firstinjection of selenosulfate starting 4 h prior to CDDP injection.


the effect of selenosulfate on CDDP in Se-sufficient mice wasinvestigated.

H22 cells (5×106 cells/each) were inoculated into left axillaof Se-sufficient mice (27–29 g) 2 days prior to CDDP

chemotherapy. CDDP at the dose of 10 mg/kg was injectedonce weekly for 4 weeks. The day of the first injection wasdesignated as day 0. Selenosulfate at 9.5 μmol/kg, which waschosen based on the optimum molar ratio 1:3.5 (selenosulfate:CDDP) (Satoh et al., 1989), was injected for 5 consecutive daysweekly for 4 weeks. Selenosulfate was firstly administered 4 hprior to CDDP injection on days 0. CDDP alone substantiallysuppressed tumor development (Fig. 4A); however, it caused37.5% lethality due to its toxicity. Compared with the CDDPalone group, coadministration of selenosulfate did not disturbtumor suppression (Fig. 4A) but largely protected body weightas evidenced by data on day 7 ( pb0.01) and day 14 ( pb0.01)(Fig. 4B) and reduced CDDP nephrotoxicity indicated by BUNlevel on day 19 ( pb0.01) and day 26 ( pb0.05) (Fig. 4C).These results demonstrated that selenosulfate could effectivelydecrease CDDP toxicity even if in Se-sufficient mice, indicatingthat its benefit is not limited to Se-deficient subjects.

In addition, the protective effect of selenosulfate againstCDDP toxicity was further investigated in a highly malignantmodel in which mice bear ascitic H22 cells. Se-sufficient micewere inoculated with a great deal of H22 cells (20×106/each)for rapid ascites development. As shown in Table 2, the controlmice, which were treated without CDDP, died within 14 days(12±1). CDDP (5 and 8 mg/kg) once weekly for 4 weeks orCDDP (10 mg/kg) once weekly for 3 weeks was firstly injectedat 48 h after the inoculation of H22 cells. CDDP 5 mg/kg couldnot completely control ascites development, and all mice diedwithin 30 days (20±7). CDDP 8 or 10 mg/kg pronouncedlysuppressed ascites development; however, the toxic effect ofCDDP caused all or most mice to die, resulting in null or 25%cure rate (no observed ascites 50 days later after stopping thechemotherapy) respectively. For 8 mg/kg CDDP, coadministra-tion of selenosulfate (7.6 μmol/kg) according to the optimumschedule (Satoh et al., 1989) increased the cure rate (noobserved ascites 50 days later after stopping the chemotherapy)to 87.5%. For 10 mg/kg CDDP, coadministration of seleno-sulfate (9.5 μmol/kg) increased the cure rate to 75%. All cured


mice recovered to normal status with respect to body weight andvitality.

Discussion

Growth retardation due to Se has long been known to be amajor symptom in Se-intoxicated animals (Thorlacius-Ussinget al., 1987). In addition, animal studies have demonstrated thatliver is the major target organ of Se toxicity (Diskin et al., 1979;Zhang et al., 2005). All these symptoms were observed in thepresent study, but selenosulfate showed markedly lowertoxicity as indicated by observed phenomena and testedbiomarkers. Toxicity of Se is thought to be ascribed to itsprooxidant ability to catalyze the oxidation of thiols andsimultaneous generation of reactive oxygen species, which doharm to cellular components (Spallholz, 1994, 1997). Seleno-sulfate was different from selenite in chemical form andvalence state (Fig. 1). The difference between toxicity of thetwo selenocompounds may be due to the different metabolicprocesses to form reactive oxygen species (Spallholz, 1997;Stewart et al., 1999).

Selenite at a dose of 25.3 μmol/kg generated severe toxicityafter 2 weeks of treatment. On the other hand, to improvetherapeutic efficacy of CDDP, selenite must be repetitivelyadministered at doses ranging from 6.3 to 12.7 μmol/kg fivetimes weekly for 7 weeks, according to the optimum schedule(Satoh et al., 1992). Therefore, it is apparent that selenite used inchemotherapy has reached or is approaching its toxic level.Selenosulfate has an advantage of lower toxicity over seleniteand does not compromise Se utilization (Table 1); on thecontrary, it generates even more liver Se accumulation at25.3 μmol/kg (Fig. 2F).

Whole blood Se and kidney Se, but not the activity ofselenoenzymes, were significantly and highly correlated withthe survival rate of mice treated with CDDP. Therefore, wholeblood Se can be considered as a potential clinical biomarker forpredicting host tolerance to CDDP. Escalating whole blood Seto a level as high as 1.5 μg/mL would be particularly crucial forCDDP chemotherapy (Fig. 3A, B), which is fairly consistentwith recent reports that efficacy in increasing the therapeuticindex of irinotecan is selenomethionine and methylselenocys-teine dose-dependent (Azrak et al., 2007; Cao et al., 2004; Fakihet al., 2006). In Se-deficient mice, when the dose ofselenosulfate reached above 6.3 μmol/kg, kidney GPx activityappeared to reach the plateau, and plasma GPx and kidney TrxRactivities reached the plateau (Figs. 3D, E, F). However, eventhough selenoenzymes were saturated, Se accumulation inwhole blood and kidney still increased in proportion toselenosulfate dose (Figs. 3B, C). Accordingly, the protectiveefficacy against CDDP lethality also increased in proportion toselenosulfate dose (Fig. 3A). Therefore, it can be inferred thatSe-modulated endpoints other than selenoenzymes haveimportant impact on the protective effect. It has been reportedthat Se in some normal tissues can increase expression ofnuclear factor erythroid-2 related factor 2 (Nrf2) target genessuch as peroxiredoxin 1 (Prx1) with antioxidant and anti-apoptotic functions (Kim et al., 2007), modulate DNA repair via

thioredoxin (Fischer et al., 2006, 2007; Seo et al., 2002a) whichis one of the target genes of Nrf2 (Powis and Kirkpatrick, 2007;Tanito et al., 2007), enhance host cell reactivation of UV-damaged reporter plasmids (Seo et al., 2002b) and modulatephosphorylation of p53 (Smith et al., 2004). Indeed, in Se-sufficient mice, selenosulfate still efficiently displayed itsprotective effects against CDDP toxicity in solid tumor model(Figs. 4B, C) and prominently increased cure rate in the ascitesmodel (Table 2).

As to Se modulation on CDDP, the commonly investigatedSe form was sodium selenite (Baldew et al., 1989; Satoh et al.,1989, 1992), whereas organic Se compounds were rarely used.Satoh et al. (1989) established an optimal schedule forcoadministration of selenite and CDDP, leading to a substantialincrease in the cancer cure rate in mice (Satoh et al., 1992).Rao et al. (1998) reported that selenomethionine (SeMet)given (i.p.) 1 h before CDDP also protected against CDDP-induced nephrotoxicity without interfering with antitumoractivity. Nonetheless, since then neither appropriate dose andschedule nor escalated cure rate has been further investigatedby using SeMet. Therefore, the present study elucidated thetoxicological and pharmacological profiles of selenosulfate bycomparing with selenite. SeMet can be non-specificallyincorporated into proteins in place of methionine, therebyresulting in unwanted heavy Se accumulation in tissues atchemotherapy relevant doses (Wang et al., 2007). It has beenfound that SeMet-induced avian embryo malformation andembryotoxicity are at least partly due to excessive substitutionof methionine residues by SeMet (Spallholz and Hoffman,2002). However, Se accumulation from selenosulfate is largelysimilar to selenite as shown in Table 1. Selenosulfate andSeMet, both with lower toxicity as compared to selenite butwith different Se accumulation capacity, particularly atchemotherapy relevant doses, are warranted to be comparedin future studies to demonstrate which one would be moreappropriate for combination with CDDP and other anti-cancerdrugs.

Considering the potential application of Se in cancerpatients treated with CDDP, the Se levels administered mustfirst be taken into account. The difference between humansand mice in Se requirement may provide insight into thisissue. It is well known that 1.3 μmol/kg in diet is anutritionally adequate dose for mice, which is equivalent to0.26 μmol/kg because a mouse of 25 g body weight consumes5 g diet daily in general. Se is recommended to humans atapproximately 0.013 μmol/kg daily as a nutritional supple-mentation dose, which implies that there is a 20-folddifference between mice and humans. As to Se modulationon CDDP, Se administration by i.p. injection in the presentstudy was within the range of 6.3 to 12.7 μmol/kg. In view ofthe 20-fold difference, the recommended Se dose for cancerpatients treated with CDDP may be 0.32 to 0.64 μmol/kg,being commensurate with approximately 1.8 to 3.5 mg Se/dayfor an adult with a body weight of 70 kg. Hu et al. (1997)reported that cancer patients who received 4 mg Se per dayorally as seleno-cappacarrageean 4 days before and 4 daysafter CDDP chemotherapy showed reduced nephrotoxicity,


decreased consumption of granulocyte colony-stimulatingfactor and fewer blood transfusions as compared to patientstreated with CDDP alone. Therefore, the doses used in thepresent study are relevant to a dose that has already beendemonstrated to be beneficial in humans.

A recent study showed that Se is a highly effective modulatorof the therapeutic efficacy and selectivity of anti-cancer drugs(Cao et al., 2004); for instance, the maximum tolerated dose ofirinotecan (100 mg/kg/week×4) was totally ineffective againstA253 and HT-29 xenografts in mice, and the administration of ahigh dose of irinotecan (300 mg/kg/week×4) was required toreach 100% and 50% cure rates for A253 and HT-29 xenograftsrespectively, but only in the presence of Se. The use of such ahigh dose of irinotecan was made possible due to the protectionof normal tissues by Se; otherwise this high dose of irinotecanalone caused 100% mortality (Cao et al., 2004). Furthermore,these effects were not drug-specific as similar results wereobtained with taxanes, 5-fluorouracil, anthracyclines etc. (Caoet al., 2004).

The impressive enhancement of cure rate achieved by thecombination of irinotecan and Se raises the question whynormal tissues but not tumors are protected by Se. Firstly, p53may be an important genetic determinant that distinguishesnormal cells from cancer cells (Fischer et al., 2007). The tumorsuppressor p53 is mutated in the vast majority of cancers but isby definition wild-type in non-target tissues such as bonemarrow and gut epithelium. Se pretreatment causes a DNArepair response, which protects DNA from subsequent chal-lenge with DNA-damaging agents. The DNA repair responseand subsequent DNA damage protection are p53 dependent asneither is observed in p53-null cells (Fischer et al., 2007).Secondly and very recently, Kim et al. (2007) indicated theyhad observed that Se modulated Nrf2 activation differently intumor vs. normal tissue. After tumor-bearing mice were treatedwith SeMet at its maximum tolerated dose for 7 days, Sesuppressed Nrf2 activation and reduced the upregulation ofPrx1 in tumor tissues; conversely, Se increased expression ofPrx1 and several other Nrf2 target genes in some normal tissuesin the tumor-bearing mice. Nrf2-regulated proteins, such asglutathione S-transferase, thioredoxin and peroxiredoxin, havepotent antioxidant and antiapoptotic effects and thus protectagainst harmful insults of chemotherapeutic drugs on normaltissues. Furthermore, we infer that even though cancer cellscontain wild-type p53, the selective modulation of Se on Nrf2(Kim et al., 2007) implies that Se may only enhance DNArepair of normal tissues because an Nrf2 target gene,thioredoxin (Powis and Kirkpatrick, 2007; Tanito et al.,2007), is involved in p53 related DNA repair (Fischer et al.,2006; Seo et al., 2002a).

In summary, selenosulfate has much lower toxicity com-pared with selenite; however, its utilization as Se within thedose range for the modulation of CDDP is the same as that ofselenite. Whole blood Se level accumulated from selenosulfateis a highly useful biomarker for predicting host tolerance toCDDP. Selenosulfate effectively decreases CDDP toxicitywithout disturbing its therapeutic effect on tumors, therebylargely improving therapeutic efficacy of CDDP. These results

together argue for considering selenosulfate as an agent toenhance the therapeutic efficacy of CDDP.

Acknowledgments

The authors are grateful to Prof. Ethan Will Taylor for hishelp in the revision of the manuscript. The authors also thankDr. Huali Wang, Dr. Xufang Wang and Ms. Guilan He fortechnical assistance in this study.

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