Attenuation of Cytogenetic Damage by 2 ...cancerres.aacrjournals.org/content/canres/46/1/203.full.pdf · phamide in humans (8). Concentrations of up to 10 mw MESNA did not induce

[CANCER RESEARCH 46, 203-210, January 1986]

Attenuation of Cytogenetic Damage by 2-Mercaptoethanesulfonate in Cultured

Human Lymphocytes Exposed to Cyclophosphamide and Its ReactiveMetabolites1

James L. Wilmer,2 Gregory L. Erexson,3 and Andrew D. Kligerman3

Department of Genetic Toxicology, Chemical Industry Institute of Toxicology, Research Triangle Park, North Carolina 27709

ABSTRACT

Cyclophosphamide (CP) is metabolized to the reactive intermediates, phosphoramide mustard (RAM) and acrolein (AC),which have generally different molecular binding targets. Sodium-2-rnercaptoethanesulfonic acid (MESNA) has been used clinicallyto alleviate hemorrhagic cystitis caused by CP chemotherapy,has exhibited anticarcinogenic effects in rats exposed to CPduring a long-term bioassay, and acts in the urogenital tract byreacting with 4'-OH-CP and AC. The purpose of this study was

to: (a) compare the relative abilities of RAM and AC to inducecytogenetic damage and cytotoxicity in cultured human lymphocytes; (D) assess the efficacy of MESNA to attenuate the cytogenetic damage and cytotoxicity induced by CP, AC, PAM, anddiethyl-4'-hydroperoxycyclophosphamide (DEHP-CP), an acti

vated AC-generating compound; and (c) determine if concana-valin A-stimulated T-lymphocytes, which differentiate into sup

pressor cells upon lectin activation, exhibit any heightened cytogenetic sensitivity compared to a variety of cultured mammalian cells during exposure to PAM or AC as reported by otherinvestigators.

Purified mononuclear leukocytes were stimulated with concan-avalin A and exposed to CP (0.5-2.0 HIM)without an exogenousactivation system, AC (0.001-40.0 MM),PAM (0.0014-27.1 MM),or DEHP-CP (0.1-100.0 MM) in the presence or absence of

MESNA (1, 5, or 10 HIM).All four compounds induced significantconcentration-related increases in the SCE frequency, but only

PAM was clastogenic. On an induced SCE/MM basis, PAM wasabout 130 and 193 times more potent than were DEHP-CP and

AC, respectively. MESNA protected against the cytogeneticdamage and cytotoxicity induced by the four compounds, but itwas particularly effective against AC and DEHP-CP by abolishing

SCE induction completely. SCEs and chromosome aberrationsdiffered considerably in their induction kinetics in lymphocytesexposed to PAM, and these disparities suggested an uncouplingof the two phenomena. Although SCE induction was not consistently associated with cytotoxicity with the four agents, chromosome aberration induction coincided with an inhibition of cellcycle kinetics in RAM-treated cells. The exceptionally high SCE

frequency of up to 21 times baseline in cells exposed to PAM

Received 6/28/85; revised 9/17/85; accepted 10/4/85.The costs of publication of this article were defrayed in part by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This research was funded by the Chemical Industry Institute of Toxicology, a

privately funded institute, and was presented at the Environmental Mutagen Societymeetings on February 19-23,1984 (Montreal, Canada) and February 25-March 1,

1985 (Las Vegas, NV).2To whom requests for reprints should be addressed, at Bristol Laboratories,

Pathology and Toxicology Department, P. O. Box 4755, Syracuse, NY 13221.3Current address: Environmental Health Research and Testing, Inc., P. O. Box

12199, Research Triangle Park, NC 27709.

indicates that T-suppressor lymphocytes stimulated with con-canavalin A may be particularly sensitive to the DNA-damagingeffects of PAM. Finally, these data suggest that the anticarcino-

genicity of MESNA correlates with its ability to attenuate cytogenetic damage and cytotoxicity induced by reactive CP metabolites.

INTRODUCTION

There are two recent developments in the treatment of malignant diseases with oxazaphosphorine chemotherapy. The firstapproach involves treating patients with a su Ifhydry I compound,especially MESNA,4 immediately prior to or concurrently with CP

therapy to provide detoxification of activated CP metabolites inthe urogenital tract (1, 2). Because hemorrhagic cystitis duringCP therapy is a major side effect due to AC release in the urineor in urothelial cells (1-6), adjuvant chemotherapy with MESNA

makes it possible to use even higher doses of CP for directtumor cell killing (1, 3, 7). MESNA does not affect plasmaalkylating activity or, by inference, antitumor activity of isophos-

phamide in humans (8). Concentrations of up to 10 mw MESNAdid not induce SCEs or inhibit cell cycle kinetics in PHA-stimu-lated, human T-lymphocytes in vitro (9). More importantly,

MESNA has been demonstrated to abolish the induction ofurinary bladder cancer in rats on long-term exposure to CP (10).

The second approach to the treatment of malignant diseasestakes into consideration that in many instances the host's im

mune response to the tumor is suppressed by cells of T- and B-

lymphocyte or macrophage origin (11,12). Within these leukocyte populations, the pre-suppressor T-cells of the delayed-typehypersensitivity response (13), the pre-suppressor T-cells of thepokeweed mitogen-driven differentiation of B-cells (14), and B-cells (15-17) are highly sensitive to the toxicity of activated CP

and its metabolites, whereas macrophages require much higherconcentrations of activated CP in vitro before functional impairment occurs (18). Con A-stimulated T-lymphocytes have beenused as a model system to understand the cell-specific toxicityof activated CP (14,19), because these lectin-stimulated T-cellscan suppress the pokeweed mitogen-driven production of im-munoglobulin in B-lymphocytes (20). Therefore, a practical aspect of determining the differential sensitivities of lymphoid sub-

populations to activated CP by immune function or cytogeneticassays may be the possibility of treating patients with muchlower CP doses, either alone or in combination with tumorantigens, to augment a delayed-type hypersensitivity response

4The abbreviations used are: MESNA, 2-mercaptoethanesulfonic acid, sodiumsalt; SCE, sister chromatid exchange; CP, Cyclophosphamide; PAM, phosphor-amide mustard; AC, acrolein; DEHP-CP, diethyl-4'-hydroperoxycyclophosphamide;

MNLs, mononuclear leukocytes; Con A, concanavalin A; PHA, phytohemagglutinin;CHO, Chinese hamster ovary.

CANCER RESEARCH VOL. 46 JANUARY 1986

203

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CYTOGENETIC EFFECTS OF MESNA AND CYCLOPHOSPHAMIDE

against the tumor (21). In addition, clinical methods involving theadoptive transfer of immune lymphocytes require the prior administration of CP to inactivate suppressor T-cells (22). Thus, the

first method may result in severely immunosuppressed patientshaving many cells with extensive DMA damage. The secondmethod relies on selective CP toxicity to pre-suppressor andsuppressor T-cell populations while leaving a functional immune

system relatively intact and possibly having enhanced antitumoractivity through release of cytotoxic lymphocytes from suppressor factors.

CP is metabolized primarily in the liver to 4'-OH-CP, which is

in tautomerie equilibrium with aldophosphamide (23, 24). Thereis substantial evidence that, apart from hepatic activation of CP,cultured mammalian lymphocytes can metabolize CP to geno-

toxic intermediates, as evidenced by an increased SCE frequency (17, 25-27). 4'-OH-CP and aldophosphamide are

thought to be the primary transport forms of CP to tumor cellseither as unbound metabolites or bound to plasma proteins (23,24). There is some controversy as to whether PAM is also atransport form (28), because it has been detected in high concentrations (50-100 IJ.M)in human peripheral blood during CP

therapy (29). Once inside the cells, aldophosphamide decomposes to two highly toxic agents, AC and PAM, which act bykilling tumor cells directly or as a delayed effect during DNAsynthesis and cell division.

Generally, these two activated CP metabolites have differentmolecular binding targets. AC has a high binding affinity forproteins and can denature critical enzymes necessary for drugmetabolism (7, 30, 31), replicative DNA synthesis (32), RNAtranscription (33), and cell membrane integrity (34). AC alsoreacts rapidly and irreversibly with reduced glutathione (7), whichserves, among other functions, to protect critical macromole-

cules from oxidative and xenobiotic damage (35). The toxicity ofAC alone or through release during CP metabolism can beeliminated by an exogenous source of reduced sulfhydryls, including MESNA (1-7). The results of point mutation assays on

AC in prokaryotic and eukaryotic systems have generally beenequivocal, and the results of the few chromosome aberrationassays performed have been equally conflicting (36,37). AC wasreported to be a potent clastogen based on the observations oftangled chromosomes or "ball metaphases" in CHO cells (38).

AC has been shown also to react in vitro with deoxyguanosineby Michael addition to form 1,A/2-propanodeoxyguanosine ad-

ducts (39) and can induce DNA single strand breaks in L1210mouse leukemia cells (40).

The bifunctional alkylating agent PAM binds to guanosine anddeoxyguanosine In vitro, especially at the N-7 position (41)

leading to opened imidazole rings (42). Similar to nitrogen mustard (43), PAM also forms cross-links in DNA, which has been

proposed as a mechanism for its antiproliferative action (40,44).PAM has been reported to be a strong mutagen in Salmonellatyphimurium TA1535 (27) and has been shown to be a potentSCE inducer in a number of cultured rodent (27, 38, 45, 46) andhuman cells (27, 45). The only study on the comparative gene-

toxic effects of PAM and AC showed that similar frequencies ofSCEs were induced at equimolar concentrations in CHO cellsexposed in exponential growth phase to a 1-h pulse treatment

(38).The purposes of the present study were as follows: (a) to

compare the relative abilities of PAM and AC to induce cytoge-

netic damage and cytotoxicity in cultured human lymphocytes;(6) to assess the efficacy of MESNA to attenuate the cytogeneticdamage and cytotoxicity induced by CP, PAM, AC, and DEHP-CP (an activated AC-generating compound) as a means to

understand the nature of their respective molecular binding targets; and (c) to determine if Con A-stimulated T-lymphocytes,

which differentiate into suppressor cells upon lectin activation,exhibit any heightened cytogenetic sensitivity compared to avariety of cultured mammalian cells during exposure to PAM andAC as reported by other investigators.

MATERIALS AND METHODS

Chemicals. All test chemical stock solutions were prepared from 5-20 min prior to addition to the lymphocyte cultures. CP monohydrateand MESNA were obtained from Sigma Chemical Co. (St. Louis, MO)and were dissolved in RPM11640. AC (99%, Gold Label) was purchasedfrom Aldrich Chemical Co. (Milwaukee, Wl) and contained 3% water and200 ppm hydroquinone to inhibit polymerization. AC was dissolved inice-cold sterile deionized water, and all of the dilutions were kept on ice

to minimize volatilization. PAM (as the cyclohexylammonium salt) was agift from Mr. Leonard H. Kedda (Drug Synthesis and Chemistry Branch,National Cancer Institute, Bethesda, MD) and was prepared in Dulbec-co's phosphate-buffered saline (pH 7.3). DEHP-CP (Asta 7037) was a

gift from Dr. Norbert Brock (Asta-Werke AG, Degussa Pharma Gruppe,Bielefeld, Federal Republic of Germany) and was dissolved in ice-colddeionized water. DEHP-CP undergoes spontaneous hydrolysis or enzymatic reduction to diethyl-4'-OH-CP (47), which then can release AC and

A/, W-bis(ethyl)phosphorodiamidic acid without the alkylating activity nor

mally associated with the 2 chloroethyl side chains (48). Stock solutionsof CP, MESNA, PAM, and DEHP-CP were sterilized using 0.22 MMillex-

GS filter units (Millipore Corp., Bedford, MA). All test chemicals wereadded to the cultures in 2CM aliquots, and the final culture volumeswere the same.

Blood Processing, Lymphocyte Culture Technique, and ChemicalExposure. Whole blood (~50 ml) was drawn by venipuncture into a

heparinized syringe. The same healthy adult male (34 years old) wasused as a source of the blood over the course of 9 months. MNLs wereseparated on Ficoll-Paque (Pharmacia Fine Chemicals, Piscataway, NJ)

density gradients and cultured as described previously (49). Erythrocyteswere removed because AC has been shown to have a high affinity forthe cytoskeletal protein spectrin (34), which could interfere with thedetermination of the antagonistic effects of MESNA. The cultures wereestablished by inoculating 106 MNLs into 1.9 ml of complete mediumcomposed of RPMI 1640 plus 25 mw A/-2-hydroxyethylpiperazine-A/'-2-

ethanesulfonic acid as buffer, 10% heat-inactivated fetal bovine serum,

100 units of penicillin and 100 ng of streptomycin sulfate/ml, and anadditional 292 Â¿igof L-glutamine/ml. Two to 4 MNL cultures were usedfor each treatment group. T-lymphocytes were stimulated mitogenicallywith Con A (4 /ig/ml). The cultures were incubated at 37Â°in a humidified

atmosphere containing 5% CO2 for 22 or 24 h. In the CP experiment,MESNA (1, 5, or 10 mw), CP (0.5,1, or 2 rtiM), and bromodeoxyuridine(5 Â¿/M)were added concurrently at 24 h. In the experiments using AC,PAM, and DEHP-CP, MESNA (1 mw) was added at 22 h after culture

initiation to allow equilibration. Bromodeoxyuridine (5 ^M) was added 2h later, and then the test compounds were added separately to thecultures over the following concentration ranges: AC (0.001-40 UM);PAM (excluding cyclohexylamine, 0.0014-27.1 pu); and DEHP-CP (0.1-

100 fiM). The culture tubes were loosely capped during the subsequentincubation. The cultures were harvested at 72 h following a 4-h exposure

to demecolcine (1.35 /IM). The procedure for cell harvest was as described previously (49).

Slide Preparation. The microscope slides were prepared as describedpreviously, coded, and stained using a modified fluorescence-plus-


204




Giemsa technique (50, 51). Nomarski phase contrast optics were usedduring cytogenetic analysis. Twenty-five second division metaphases,

100 consecutive metaphases, and 1000 nuclei were scored from eachculture for SCE frequency, cell cycle kinetics, and mitotic index, respectively. Only metaphases with 46 centromeres were included in the finaltabulations, and SCEs occurring at the centromere were counted unlessthere was obvious twisting of the chromatids. The replicative index (Rl)was calculated from the cell cycle kinetic data by the formula: Rl = ([1

x percentage of first division cells] + (2 x percentage of second divisioncells] + [3 x percentage of third division cells] + [4 x percentage offourth division cells] (52). Chromosome aberrations were analyzed from100 first division metaphases per culture on the same slides using thecriteria of Evans and O'Riordan (53).

Statistical Analyses. All cytogenetic data, with the exception of thechromosome aberration data, were tested for normality and then subjected to a one-way analysis of variance (54). A square root transfor

mation of individual means was used to equalize the variances in all ofthe experiments (55). A one-tailed Dunnett's multiple comparison test

was used to compare the various treatment groups to the concurrentcontrol if warranted (54, 56). Another method of statistical analysis wasalso used and involved the cell-to-cell variation in the SCE frequencyrather than culture-to-culture variation in the mean SCE frequency. By

using the pooled SCE counts from each treatment group, a potentiallymore stable estimate of variance could be obtained by larger degrees offreedom. The pooled SCE counts were subjected to a one-way analysisof variance, and then Dunnett's multiple range test was used to compare

the various treatment groups to the concurrent control. However, thesetwo statistical methods gave essentially the same results for the in vitroexperiments described here. A one-tailed Student's f test was used to

compare the mean SCE frequencies of cultures treated with the testchemicals in the presence or absence of MESNA to determine at whichpoint a significant attenuation of cytogenetic damage appeared. A \2

test was used to analyze the incidence of chromosome aberrations inthe first division metaphases. The level of significance was chosen as0.05 for all of the determinations.

RESULTS

CP induced significant concentration-related increases in the

SCE frequency of up to 3 times baseline at 2 mu in the absenceof any exogenous metabolizing system (Table 1). However, CPdid not depress mitotic activity or inhibit cell cycle progressionsignificantly (Table 1). When MESNA was added to the lymphocyte cultures containing CP, the SCE frequency was reducedsignificantly by 27-45% (Table 1) for all 3 MESNA concentra

tions. Although MESNA alone did not induce SCE or perturb cellcycle kinetics, there was some evidence of cell cycle inhibition incultures exposed to a combination of 2 mw CP and 1 mw MESNA(Table 1).

Because MNLs were apparently capable of metabolizing CPat a slow rate, as shown by an increase in the SCE frequency,it was important to know the relative capabilities of the activatedCP metabolites to induce cytogenetic damage and cytotoxicity.AC induced significant concentration-related increases in theSCE frequency of up to 1.6-fold at 20 U.M,but the inductionoccurred over a narrow concentration range (5-20 ?M) (Table 2).

AC was not clastogenic at the highest analyzable concentration(20 UM) (data not shown). Forty U.MAC was lethal to MNLs, asevidenced by many pyknotic nuclei and no mitoses. AlthoughAC did not significantly depress the mitotic index, it slowed thecell cycle kinetics significantly at 10 and 20 UM (Table 2). 1 muMESNA protected completely against AC-induced SCEs and cell

lethality (Table 2) up to 40 U.MAC; however, significant cell cycle

Table 1

Effect of MESNA on the SCE frequency, mitotic activity, and cell cycle kinetics incultured human lymphocytes exposed to CP

CPconcentration

(mu)Control0.51.02.0Control0.51.02.0Control0.51.02.0Control0.51.02.0MESNA

concentration(Ã•TIM)00001111555510101010SCE

frequency/metaphase8.5

Â±0.5*(0.36)Â°13.8Â±0.8e(0.53)17.0Â±0.2Â°(0.46)25.6Â±1.4C(0.56)7.7

Â±0.3"(0.37)8.9Â±0.4Â°(0.42)1

4.7 Â±0.5C(0.51)20.2Â±0.2e(0.54)7.9

Â±0.2"(0.33)10.9Â±0.5Â°(0.44)12.2Â±0.4C(0.45)18.3Â±0.2Â°(0.53)8.5

Â±0.6"(0.38)10.0Â±1.1C(0.51)13.0Â±0.3C(0.57)1

8.0 Â±1.4C (0.60)Mitotic

index(%)6.6

Â±0.35.5Â±1.13.9

Â±0.64.6Â±0.75.4

Â±0.34.9Â±0.75.7Â±0.55.0Â±0.56.0

Â±0.56.4Â±0.66.8Â±0.35.8

Â±1.17.1

Â±0.95.2Â±0.34.5Â±1.24.6Â±0.9Replicative

index2.58

+0.102.53Â±0.022.67Â±0.052.48

Â±0.102.71

Â±0.022.73Â±0.212.64Â±0.052.34Â±0.19C2.52

Â±0.292.66Â±0.112.61

Â±0.142.52Â±0.132.44

Â±0.312.36Â±0.272.33Â±0.401.97Â±0.12

8 Mean Â±SD of triplicate cultures.

Numbers in parentheses, estimated standard error of the mean for pooledSCE counts.

c Significantly different from the concurrent control using Dunnett's multiple

comparison test (P < 0.05)." Significant reductions in the SCE frequency at all CP concentrations compared

to cultures without MESNA.

inhibition occurred at 10 and 40 Â¿<MAC.Since the extracellular exposure of lymphocytes to AC is

unlikely to occur in vivo due to rapid scavenging by nucleophilicplasma components, it was necessary to use an activated cyclo-phosphamide analogue, DEHP-CP, to mimic the intracellular

release of AC during CP degradation to reactive intermediates.DEHP-CP was comparable to AC in its SCE-inducing capabilityover a similar narrow range (10-20 nu) where a 1.8-fold increasein the SCE frequency was seen at 20 U.M(Table 3). DEHP-CP

concentrations of 15 and 20 UM decreased the mitotic activitysignificantly, and the cell cycle progression was inhibited significantly at concentrations >0.5 J/M (Table 3). No mitoses wereobserved at DEHP-CP concentrations >40 U.M.One mM MESNA

protected completely against SCE induction at 10 and 15 U.MDEHP-CP and provided partial protection up to 100 U.MDEHP-

CP (Table 3). For example, the SCE frequency of the cellsexposed to MESNA and 100 u.u DEHP-CP was about equal tocells exposed to 20 UM DEHP-CP alone. MESNA protectedcompletely against cytotoxicity up to 40 UMDEHP-CP (Table 3).

Although a significant depression in mitotic activity and inhibitionof cell cycle kinetics was observed in cultures containing >80UM DEHP-CP and MESNA, the extent of cytotoxicity was comparable to that seen at >15 JIMDEHP-CP alone.

PAM induced significant concentration-related increases in theSCE frequency over a 500-fold range (Table 4). A significant

increase in the SCE frequency was detected at 0.0068 U.MPAM(1.5 ng/ml), and a 21-fold increase in the SCE frequency (185.4

Â±4.3 SCEs/cell) was observed at 3.39 U.MPAM. PAM alsoinduced a 33-fold increase above baseline in the number of

damaged first division metaphases at 3.39 UM(Table 5). Definiteincreases in the number of damaged cells occurred at 0.339 UMPAM, a concentration which is 50 times higher than that requiredto see a significant elevation in SCE induction. The disparity inthe kinetics of SCE and chromosome aberration induction can


205




Table 2Effect of MESNAon SCEinduction, mitotic activity, and cell cycle kinetics in cultured human lymphocytes exposed to acrolein

Acroleinconcentration

(MM)Control0.0010.010.10.51.05.010.015.020.040.0SCE

frequency-1

rriMMESNA8.4

Â±0.7a(0.30)"8.0

Â±0.1(0.46)8.1Â±0.3(0.49)9.0Â±1.4(0.48)9.5Â±0.2(0.58)9.5Â±0.3(0.53)10.6Â±0.1C(0.49)9.8

+ 0.5(0.59)10.8Â±0.6Â°(0.65)13.3Â±0.7Â°(0.70)No

metaphases+1

muMESNA7.3

Â±0.5(0.26)7.7Â±0.7(0.49)8.1Â±0.5(0.52)8.4Â±1.6(0.48)8.2Â±0.4(0.44)8.0+ 0.3(0.47)7.2Â±0.4"(0.36)8.1

+ 0.5(0.53)7.6Â±0.3d(0.46)8.5Â±2.0"(0.46)8.4

Â±0.0 (0.41)Mitotic

index(%)-MESNA2.5

+0.33.8Â±1.03.4

Â±0.22.9Â±0.33.9Â±0.34.0Â±0.83.1Â±0.13.2

Â±0.72.6Â±0.21.5

Â±0.3Nomitoses+MESNA

(1HIM)3.5

Â±0.53.6Â±0.93.8Â±0.83.0Â±0.45.4Â±0.53.1Â±0.73.4Â±0.63.7Â±0.93.5Â±0.94.0Â±0.13.5Â±1.5Replicative

index-MESNA2.18

Â±0.122.13+0.112.32Â±0.142.15+0.062.24+0.012.35Â±0.042.02+0.031.91

+0.01C2.04

Â±0.081.74Â±0.14CNo

mitoses+MESNA

(1mM)2.30

Â±0.122.34Â±0.012.41

Â±0.112.16+0.012.42Â±0.042.16+0.122.37Â±0.092.02

Â±0.11Â°2.14

Â±0.022.04+0.061.97+ 0.25Â°

" Mean Â±SD between duplicate cultures except the control (n = 4)." Numbers in parentheses,estimated standard error of the mean for pooled SCE counts.Â°Significantlydifferent from control using Dunnett's multiple comparison test (P < 0.05)." Significantlydifferent from the respective cultures without MESNA using a one-tailed Student's i-test (P < 0.05).

Table 3Effect of MESNAon SCEinduction, mitotic activity, and cell cycle kinetics in cultured human lymphocytes exposed to DEHP-CP

DEHP-CPconcentration

(MM)Control0.10.51.05.010.015.020.040.080.0100.0SCE

frequency-1

mMMESNA9.0+ 0.8s(0.41)"9.7

Â±0.7(0.56)9.8Â±0.9(0.54)10.7Â±0.3(0.66)10.5Â±0.6(0.47)12.6+ 0.3Â°(0.66)12.2+ 1.4Â°(0.60)16.4Â±1.1Â°(0.86)No

mitosesNomitosesNo

mitoses+1

mMMESNA8.4

Â±0.58.7+0.08.7

Â±1.78.7+0.38.8

Â±1.28.6Â±1.410.3

+0.111.1Â±0.8Â°'10.4

Â±0.214.4Â±2.1Â°15.3Â±1.1C(0.38)(0.49)(0.48)(0.52)(0.57)(0.46)(0.50)"(0.65)(0.56)(0.60)(0.71)Mitotic

index(%)-MESNA6.1

Â±1.36.6Â±0.05.0

Â±1.86.5Â±0.45.7

Â±1.24.0Â±0.13.5

Â±0.1Â°1.9Â±1.0Â°No

mitosesNomitosesNo

mitoses+MESNA

(1mM)7.3

Â±0.49.7Â±3.68.1

Â±1.16.9Â±0.26.3

Â±1.25.1Â±0.37.1Â±0.07.2

Â±1.16.0Â±0.62.6

Â±2.1Â°2.0Â±0.1Â°Replicative

index-MESNA2.64

Â±0.802.56Â±0.042.41Â±0.03C2.55

Â±0.062.47Â±0.13Â°2.14Â±0.04Â°2.05Â±0.06Â°1.91

Â±0.11Â°No

mitosesNomitosesNo

mitoses+MESNA

(1mM)2.54

Â±0.112.63Â±0.182.76Â±0.072.61

Â±0.172.55Â±0.142.65Â±0.472.32+0.192.34Â±0.032.20Â±0.181.86

Â±0.19Â°1.86Â±0.16Â°

* Mean Â±SD between duplicate cultures except the control (n = 3)." Numbers in parentheses,estimated standard error of the mean for pooled SCE counts.Â°Significantlydifferent from control using Dunnett's multiple comparison test (P < 0.05).tf Significantlydifferent from the respective cultures without MESNA using a one-tailed Student's f-test (P< 0.05).

Table 4Effect of MESNAon SCEinduction, mitotic activity, and cell cycle kinetics in cultured humanlymphocytes exposed to PAM

PAMconcentration

(MM)Control

0.00140.00680.0680.3390.683.39>6.8SCE

frequency-1

mMMESNA9.3+ 0.9a(0.33)"

9.4 + 0.1 (0.55)11.7 Â±0.1Â°(0.63)15.7 Â±0.8Â°(0.53)42.4 Â±2.9Â°(1.06)61.6 Â±4.2Â°(1.19)

185.4 Â±4.3Â°(4.25)

No mitoses+1

mMMESNA7.7

Â±0.9 (0.34)9.3 Â±0.6 (0.61)9.7 Â±0.2Â°'" (0.64)

12.0 Â±0.4Â°'"(0.53)24.7 Â±0.1Â°'"(0.77)49.2 Â±3.5Â°'"(1.1 5)

117.4 Â±7.8Â°'"(2.13)

No mitosesMitotic

index(%)-MESNA5.3

+ 0.76.0 + 2.75.9 Â±0.86.3 Â±0.53.7 Â±0.13.8 + 0.81.1 Â±0.4Â°

No mitoses+

MESNA (1mM)5.7

Â±0.55.4 Â±0.55.2 Â±0.85.2 Â±1.75.0 Â±0.14.5 + 0.12.9 Â±0.6Â°

No mitosesReplicative

index-MESNA2.38

+ 0.062.27 Â±0.012.42 Â±0.082.28 Â±0.011.98 Â±0.03Â°1.84 Â±0.01Â°1.29 Â±0.03Â°

No mitoses+MESNA

(1mM)2.37

Â±0.082.18 Â±0.01Â°2.22 + 0.02Â°

2.31 Â±0.032.27 + 0.01Â°1.69 Â±0.00Â°1.56 + 0.09Â°

No mitosesÂ°Mean Â±SD of duplicate cultures except the control (n = 4)." Numbers in parentheses,estimated standard error of the mean for pooled SCE counts.Â°Significantlydifferent from control using Dunnett's multiple comparison test (P < 0.05).* Significantlydifferent from respective cultures without MESNA using a one-tailed Student's i-test (P < 0.05).

also be seen at concentrations of 0.339 and 0.68 U.MPAM. Whilethe number of cells with chromosome damage, as well as theextent of damage within each cell, remained the same at thesetwo concentrations (Table 5), an increase of about 19 SCEs/cellwas observed (Table 4). This plateau phase in chromosomeaberration induction was also apparent in the cultures treatedwith MESNA (Table 5), whereas there was a difference of about25 SCEs/cell between these two PAM concentrations. PAMdepressed the mitotic activity significantly only at 3.39 MM andinhibited cell cycle kinetics at >0.339 UM(Table 4). The inhibitionof cell cycle kinetics coincided with a distinct increase in chro

mosome aberrations. One mu MESNA decreased the RAM-induced SCE frequency significantly by 21 -49% at PAM concentrations >0.0068 UM(Table 4). Similarly, MESNA decreased the

percentage of cells with chromosome aberrations by a factor of3.8 at PAM concentrations 20.339 /Â¿M(Table 5). If an estimatednumber of breaks required for the formation of a particularaberration is calculated as shown in the legend of Table 5, thenthe addition of 1 mM MESNA to the cultures containing 3.39 ^MPAM can be shown to have reduced the number of breaks percell by a factor of 6.5. MESNA did not protect consistentlyagainst the cytotoxicity of PAM (Table 4).


206




Table 5Effect of MESNAon structural chromosomeaberration induction in first division metaphasesof cultured

human lymphocytes exposed to PAM

PAMconcentrationControl0.00140.00680.068

0.3390.683.39a6.8Control0.00140.00680.0680.3390.68

3.39>6.8%of

Â±MESNA damaged(1 HIM) metaphases2.05.53.55.5

11.0*10.56

65.5"-Nomitoses+

3.5+4.0+2.5+1.5+5.0+

30.5b-c+

No mitosesFrequency

of aberrations per 100cells"CB'1.54.03.05.0

10.010.0

104.03.03.01.51.03.53.5

24.0CB"0.02.00.50.52.5

2.019.50.51.01.00.51.01.5

6.5ID0.5

0.52.00.51.5DM

CIG'0.5

0.52.02.54.5

4.51.0

1.5 36.53.52.01.56.00.51.53.01.0

4.0 4.0G"0.50.51.01.5

0.00.53.00.00.01.50.51.50.52.5

" A total of 200 metaphases were analyzed per treatment group. The chromosome aberration categoriesand an estimated numberof breaks in parenthesesrequired for their formation were as follows: CB', chromatidbreak (1); CB", isolocus break (2); ID, interstitial deletion (2); DM, double minute (2);CI, chromatid interchange(2); G', chromatid gap; G", isolocus gap. One dicentric without paired fragments was detected in the cultures

containing 3.39 MMPAM plus MESNA but was not included in the table, as it indicatesa derived chromosomeaberration. Gaps were excluded in the tabulation of percentageof damaged metaphases.

b Significantlydifferent from the control using x2 test (P < 0.05).0 Significantlydifferent from the respective cultures without MESNA using the x2 test (P < 0.05).

DISCUSSION

These data show that human lymphocytes can activate CP togenotoxic intermediates, as shown by a 3-fold increase in the

SCE frequency, and that both CP metabolites, AC and PAM,can induce cytogenetic damage and cytotoxicity. These datademonstrate also that MESNA attenuates the cytogenetic damage induced by CP, AC, DEHP-CP, and PAM and provide an

insight into the molecular mechanism of action of these agents.The kinetics of SCE and chromosome aberration induction in thePAM-exposed cells provides evidence for the uncoupling of these

two cytogenetic endpoints and for the closer relationship ofchromosome aberration induction to cytotoxicity. The unusuallyhigh SCE and chromosome aberration frequencies induced byPAM indicate not only that PAM is one of the most potentchromosomal mutagens known but also that Con A-stimulatedT-lymphocytes are highly sensitive to PAM-induced DMA dam

age.The ability of MESNA to provide protection against the geno-

toxicity and cytotoxicity of AC and PAM indicates the nature andaffinity of their respective molecular binding targets. One mwMESNA protected completely against AC-induced SCEs and cell

lethality at a molar ratio of 25:1 (Table 2). When a different meansof delivering AC to the lymphocytes' nuclei was used, 1 mw

MESNA protected completely against DEHP-CP-induced SCEs

at a molar ratio of 67:1 and provided partial protection at a ratioof 10:1 (Table 3). The cytotoxicity of DEHP-CP was inhibited

completely by MESNA at a ratio of 25:1, and lower molar ratioswere partially effective (Table 3). In contrast, although the decreases in SCE induction by addition of MESNA to the lymphocyte cultures exposed to PAM were dramatic, MESNA did notcompletely attenuate the PAM-induced SCEs, even at a molar

ratio of 147,600:1. This large dichotomy in the protective effectsof MESNA indicates that the affinity of DEHP-CP and AC for

reduced sulfhydryls is about 2200 and 5900 times, respectively,

that of PAM. These results support the observations of a highreactivity of AC with proteins and a significantly lower affinity ofPAM for proteins in two separate studies. Wildenauer and Oehl-mann (34) showed that there was preferential binding of [14C]AC

to microsomes and erythrocytes during CP metabolism, but littlebinding of 3H-chloroethyl side chains was detected. Gurtoo ef al.(57) demonstrated that 2100 times more [14C]AC than [3H]PAM

was bound to liver microsomes, whereas 22 times more[3H]PAM than [14C]AC was bound to native calf thymus DNA

during metabolism of radiolabeled CP in vitro. Thus, besides thenature of their respective induced DNA lesions (39-42, 44), the

ability of PAM and AC to induce cytogenetic damage might bedue primarily to their divergent affinities for low molecular weightthiols and proteins compared to nucleic acids.

An important aspect of the antagonistic effects of MESNA onAC-induced toxicity was that MESNA inhibited cell lethality observed at 40 fiM AC and 40-100 MM DEHP-CP. These results

obtained from using lymphocytes as a model cell type supportand amplify the hypothesis of Cox (58) regarding the relationshipof CP-induced urinary bladder cystitis and bladder cancer. The

cause of cystitis during CP chemotherapy has been shownconclusively to be mediated by AC rather than PAM (2, 4, 5).Because the urine is normally low in reduced thiols, urothelialcells represent the major target for CP metabolites. The decomposition of activated CP releases AC in the urine or in urothelialcells, which damages cell membranes (34) and cytoplasmiccomponents (7, 30, 31 ) and induces SCEs (38; present study).The rapid death of urothelial cells induced by AC leads tocompensatory regeneration of the underlying diploid cell populations (59, 60). Because PAM is also released simultaneouslywith AC, it induces DNA lesions which are expressed as SCEsand chromosome aberrations during S-phase. Thus, fixation of

cytogenetic damage during induced cell turnover may be animportant first step in cell transformation to a malignant pheno-type in urothelium and possibly in lymphoid cells as well.


207




The administration of MESNA concurrently with oxazaphos-

phorine therapy has been shown to abolish hemorrhagic cystitisin humans (1, 2) and rodents (2, 3), as well as to attenuate theinduction of urinary bladder cancer in rats during long-term

exposure to CP (10). Besides binding to AC and RAM, MESNAalso reacts with 4'-OH-CP to form the condensation product, 4-

(2-sulfonatoethylthio)-CP, which is thought to be a temporary

inactivation product in the urine (4). MESNA was shown to bethe least toxic thiol compound in rats and to provide the greatestamount of regional protection to the urogenital tract among manyexperimental compounds tested (3). The present study hasconfirmed the observations of Becher ef al. (9) that concentrations of up to 10 mw MESNA are not genotoxic or cytotoxic.The present study suggests that the anticarcinogenicity ofMESNA correlates with its ability to attenuate the cytogeneticdamage and cytotoxicity induced by reactive CP metabolites.

The results of the present study with SCE induction by CP inpurified human lymphocyte cultures support the findings in anumber of other studies that mammalian lymphocytes can metabolize CP to genotoxic intermediates (17, 25-27). However,

the rate of CP metabolism in blood cultures is slow, as evidencedby only a 3-fold increase in the SCE frequency at 2 mw CP (Table1). Because PAM was such a potent SCE inducer at low concentrations relative to AC and DEHP-CP, most of the genotox-

icity of CP in lymphocyte cultures is probably originating fromPAM. This assumption is partly supported by the fact thatMESNA did not protect completely against CP-induced SCEs

but reduced the SCE frequency by about the same percentageas in PAM-treated cells; that is, a 27-45% reduction in the SCEfrequency by MESNA was observed in the CP-exposed cells(Table 1) and a 21-49% reduction was seen in the PAM-exposed

cells (Table 4).The findings of the present study that AC induces SCEs over

a narrow concentration range (5-20 UM)(Table 2) agree well with

the results of Au ef a/. (38). They showed that AC induced abouta 2.6-fold increase in the SCE frequency in CHO cells exposedfor 1 h, whereas a 1.6-fold increase was seen in the present

study. However, they reported that AC induced chromosometangling at concentrations >40 UM, which was taken as anindication of "potential" clastogenicity. There was no evidence of

clastogenicity or tangled chromosomes at 20 fiu AC in thepresent study. The depression in mitotic activity by 20 ^M ACwas much less in human lymphocytes (Table 2) compared to thecomplete inhibition by 10 UM AC in CHO cells exposed for 5 h(38). These results suggest that human lymphocytes may beless susceptible than CHO cells to the cytotoxic effects of AC.

The present study demonstrated that PAM is one of the mostpotent chromosome mutagens known and is comparable toanother bifunctional alkylating agent, mitomycin C, in its SCE-

inducing capability (61). On an induced SCE//tw basis, PAM isabout 130 and 193 times more potent than are DEHP-CP and

AC, respectively. The form of structural chromosome aberrationsof the chromatid-type support the conclusions of Winckler ef a/.(27) that PAM is an S-phase dependent agent (Table 5). The

present results contrast sharply with the findings of Au ef al.(38), who reported that the SCE-inducing ability of PAM is similar

to that of AC over equimolar concentrations. These large discrepancies might be explained by a species or cell-type differ

ence, but a more likely possibility is that a 1 h pulse treatmentwith PAM is not a long enough exposure for a compound that is

ionized at physiological pH (23) and would be taken up slowlyby cells.

Although PAM proved to be a strong chromosome mutagen,the magnitude of the cytogenetic response suggested that ConA-stimulated lymphocytes are more sensitive than any mamma

lian cell tested to date. In the present study a significant increasein the SCE frequency was detected at 6.8 nw, whereas the otherstudies reported significant increases at concentrations fromabout 14.8-33 times higher (27, 45). Treatment of Con A-

stimulated lymphocytes with 3.39 UM PAM for 48 h resulted in a21-fold increase in the SCE frequency. Dearfield (45) observeda 9.5-fold increase in SCE frequency in human IMR-90 lung

fibroblasts exposed to 4.52 UMPAM for two cell cycles. Similarly,Winckler ef a/. (27) saw a 11.4-fold increase in the SCE frequencyin PHA-stimulated, whole blood T-lymphocytes exposed to 10

tÃMPAM for 54 h using a virtually identical treatment procedureto the present study. Con A is a non-specific, polyclonal activatorof T-lymphocytes which differentiate into cells exhibiting suppressor phenotypes (14,20). These suppressor T-cells and their

progenitors are highly sensitive to the toxicity of activated CP(14,19). Generally, PHA-stimulated lymphocytes are affected to

a lesser extent by the toxicity of activated CP, as measured bythe incorporation of [3H]thymidine into DNA (62). Deknudt (63)

has shown nearly a 2-fold higher SCE response in Con-A-stimulated whole blood lymphocytes compared to PHA-stimulated lymphocytes during a 1-h exposure to S-9 activated CP at48 h post-culture initiation. The inhibition of differentiation by

activated CP can occur without the loss of mitogen responsiveness or cell viability and in the absence of detectable DNA crosslinks (14) at comparable concentrations to PAM in the presentstudy where SCEs, but not chromosome aberrations, wereinduced. The significantly higher SCE frequency observed in ConA-stimulated lymphocytes compared to other mammalian cells

may reflect a cytogenetic basis for their greater susceptibility toinhibition of differentiation by activated CP.

The kinetics of SCE and chromosome aberration inductionwere different in the experiment with PAM (Tables 4 and 5). Oneobvious difference was that a significant increase in SCEs wasinduced at a concentration of PAM about 50 times less than theconcentration required to induce a significant increase in chromosome aberrations. This disparity between the two endpointshas been observed many times with alkylating agents (64). Thesecond difference was that chromosome aberrations reached aplateau phase from 0.339-0.68 tiM PAM in the presence or

absence of MESNA and then rose dramatically at 3.39 UM,whereas there was no evidence of a plateau phase in SCEinduction. These two differences in the kinetics of inductionindicate that SCEs and chromosome aberrations arise fromeither different mechanisms of formation or different DNA lesions,as suggested by others (65, 66). The relatively greater decline inchromosome aberrations compared to SCEs in cultures treatedwith MESNA suggests that MESNA reacts with one of thechloroethyl side chains of PAM and partially abolishes the cross-

linking capability, while the monofunctional alkylating activityremains. These data support the hypothesis that cross-links are

more likely to lead to chromosome aberration induction throughthe processes of misrepair and deletion (67), while monoadductsare efficient stimulators of SCE induction (66).

Although previous investigations have shown a correlation ofSCE induction and cytotoxicity (68, 69), there did not appear to


208




be a consistent correlation of SCE to cytotoxicity by the agentsused in the present study. SCE induction could occur in theabsence of depressed mitotic activity and inhibition of cell cyclekinetics. SCE induction reached 2-3 times baseline, especially

with CP (Table 1) and RAM (Table 4), without significant inhibitionof cell cycle progression. However, the induction of chromosomeaberrations coincided with a distinct inhibition of cell cycle kinetics, which was evident at 0.339 MM RAM (Tables 4 and 5). Thecoincident occurrence of cell cycle inhibition and chromosomeaberrations reinforces the concept that chromosome aberrationsgenerally lead to cell death (66). The results of the present studysupport the findings of Kaina (70) and Nishi ef al. (71) that SCEinduction by mono- and bifunctional alkylating agents is not

related to cytotoxicity. Additionally, because AC has been shownto induce DNA single strand breaks in cultured mouse leukemiacells (40), single strand breaks might be relatively inefficientstimuli for SCE induction (72). Because nitrogen mustard, ananalogue of RAM, exhibits many of the characteristics of RAMin its interactions with DNA (43) and is a poor inducer of specific-

locus mutations compared to SCEs in mammalian cells (71), it isdoubtful that SCE induction by RAM can be strictly equated withspecific-locus mutagenesis. The results of the present study and

other studies on immunotoxic effects at low RAM or activatedCP concentrations suggest that SCE induction is more closelyrelated to inhibition of differentiation in lymphoid cells and is notan innocuous event, but further experimentation will be neededto test this hypothesis.

In conclusion this study has demonstrated that SCE inductionis probably not closely related to cytotoxicity or chromosomeaberration formation. This study has also shown a possiblecytogenetic basis for the heightened sensitivity of Con A-stimu-lated, suppressor T-lymphocytes to activated CP and RAM and

may offer a means of understanding differential toxicity in lymphoid subpopulations using cytogenetic techniques. The attenuation of cytogenetic damage by MESNA provides insights into themolecular mechanisms of induced toxicity by CP and its reactivemetabolites and lends support for the concept of regional chem-

oprotection during CP therapy to abrogate the induction ofcystitis and urinary bladder cancer.

ACKNOWLEDGMENTS

The authors thank Robert 0. Beauchampfor helpful discussions on the chemistry of thiol compounds, Beth Williamsonfor library research, Dr. Norbert Brock(Asta-Werke, Bielefeld, Federal Republic of Germany) for providing the generousgift of diethylhydroperoxycyclophosphamide,Leonard H. Kedda (Drug Synthesisand Chemistry Branch, the NationalCancer Institute, Bethesda, MD) for providingthe gift of phosphoramide mustard, and Diane Doach for typing the manuscript.The present study is dedicated to the memory of the senior author's (J. L. W.)

mother, who received cyclophosphamide chemotherapy without the benefit ofMESNA adjuvant therapy.

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1986;46:203-210. Cancer Res James L. Wilmer, Gregory L. Erexson and Andrew D. Kligerman Exposed to Cyclophosphamide and Its Reactive Metabolites2-Mercaptoethanesulfonate in Cultured Human Lymphocytes Attenuation of Cytogenetic Damage by

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Attenuation of Cytogenetic Damage by 2 ...cancerres.aacrjournals.org/content/canres/46/1/203.full.pdf · phamide in humans (8). Concentrations of up to 10 mw MESNA did not induce