Irinotecan Toxicity to Human Blood Cells in vitro: Relationship between Various Biomarkers

© 2007 The Authors

Doi: 10.1111/j.1742-7843.2007.00068.x

Journal compilation

© 2007 Nordic Pharmacological Society

. Basic & Clinical Pharmacology & Toxicology

Blackwell Publishing, Ltd.

Irinotecan Toxicity to Human Blood Cells

in vitro

: Relationship between Various Biomarkers

Nevenka Kopjar

1

, Davor

¸

elje

Ω

i

ç

1

, Ana Luci

ç

Vrdoljak

1

, Bo

Ω

ica Radi

ç

1

, Snje

Ω

ana Rami

ç

1

, Mirta Mili

ç

1

, Marija Gamulin

2

, Vesna Pavlica

3

and Aleksandra Fu

Ç

i

ç

1

1

Institute for Medical Research and Occupational Health,

2

University Hospital Zagreb, and

3

The University Hospital for Tumours, Zagreb, Croatia

(Received October 26, 2006; Accepted January 15, 2007)

Abstract:

Toxic effects of the antineoplastic drug irinotecan on human blood cells at concentrations of 9.0

µ

g/ml and4.6

µ

g/ml were evaluated

in vitro

. Using the alkaline and neutral comet assay significantly increased levels of primary DNAdamage in lymphocytes were detected. The induction of apoptosis/necrosis, as determined by a fluorescent assay, was alsonotably increased. Cytogenetic outcomes of the treatment were assessed by the analysis of structural chromosome aberrationsand fluorescence

in situ

hybridization. A significantly higher incidence of chromatid breaks and complex quadriradials wasobserved. Painted chromosomes 1, 2 and 4 were equally involved in translocations, but only the chromosome 1 was involvedin the formation of quadriradials. Sister chromatid exchange analysis was performed in parallel with the analysis oflymphocyte proliferation kinetics. The higher concentration of irinotecan caused almost seven-time increase, while thelower one caused a five-time increase of the basal sister chromatid exchange frequency, accompanied with significantlowering of the lymphocyte proliferation index. Using the cytokinesis-block micronucleus assay, a dose-dependent increasein micronucleus frequency along with the formation of nuclear buds and nucleoplasmic bridges was noticed. Inhibitoryeffects of irinotecan on enzyme acetylcholinesterase (AChE) were studied in erythrocytes. An IC

50

value of 5.0

×

10

−

7

wasestablished. Irinotecan was found to be strong inhibitor of the acetylcholine hydrolysis and to cause a continuous decreaseof catalytic activity of AChE. The results obtained on a single donor may contribute to the understanding of irinotecantoxicity, but further

in vitro

and

in vivo

studies are essential in order to clarify remaining issues, especially on possible inter-

individual variability in genotoxic responses to the drug.

Irinotecan (7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbony-loxycamptothecin; CPT-11) is clearly one of the most importantnew anticancer drugs developed in the last few decades. It isa member of the camptothecin drug family [1]. Irinotecan isa pro-drug that is biotransformed by tissue and serumcarboxylesterases to an active metabolite, SN-38 (7-ethyl-10-hydroxycamptothecin) that has a 100–1000-time higher cytotoxicand antitumour activity [2]. It acts as an inhibitor of the nuclearenzyme topoisomerase I, which is involved in cellular DNAreplication and transcription. During replication, topoisomeraseI mediates the relaxation of super-coiled DNA, and its inhibitionresults in breakage of the DNA chain and likely induces apop-tosis. Irinotecan and SN-38 both bind to the topoisomeraseI-DNA complex and prevent religation of single-strand breaks[3]. Irinotecan has undergone extensive clinical investigationworldwide and demonstrated potent activity against manytypes of human cancer, but is particularly active in the treatmentof gastrointestinal and pulmonary malignancies [1–4].

The principal dose-limiting toxicity of irinotecan is diarrhoea.It either causes acute diarrhoea related to a cholinergic surgefrom inhibition of acetyl cholinesterase, or a delayed diarrhoeasyndrome, which is possibly related to the accumulation of theactive metabolite of irinotecan in the bowel [5]. Other non-

haematological toxicities include nausea, vomiting, anorexia,fatigue, abdominal pain, alopecia, asthenia and elevated alkalinephosphatase and/or hepatic transaminases. The most commonhaematological toxicity is dose-related neutropenia. Myelo-suppression is generally not cumulative, and severe anaemiaor thrombocytopenia is less common [1,6,7].

Cytogenetic consequences of chemotherapy with irinotecanto normal cells were not extensively studied. Because con-ventional antitumour drugs are indiscriminate, the adverseconsequences of chemotherapy to non-tumour cells andtissues are almost always present. The aim of this study was toevaluate the toxicity profile of irinotecan on human non-targetcells following

in vitro

treatment with two concentrationsof the drug proportionate to its therapeutic doses. A multi-biomarker approach was used: lymphocyte viability and theinduction of apoptosis/necrosis caused by the exposure toirinotecan were studied by simultaneous use of a fluorescentassay with ethidium bromide and acridine orange; the levelsof primary DNA damage in lymphocyte genome and thedynamics of DNA repair were evaluated using the alkalineand neutral comet assay; the levels and nature of residual DNAdamage were assessed by the analysis of structural chromosomeaberrations and fluorescence

in situ

hybridisation (FISH).For the study of cytogenetic effects, the cytokinesis-blockmicronucleus (CBMN) assay and sister chromatid exchange(SCE) analysis were also employed, while the possible influencesof treatment on the progression through the mitotic cycles

Author for correspondence: Nevenka Kopjar, Mutagenesis Unit, Institutefor Medical Research and Occupational Health, Ksaverska c.2, HR-10000 Zagreb, Croatia (fax + 385-1-4673303, e-mail [email protected]).

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NEVENKA KOPJAR

ET AL

.

© 2007 The AuthorsJournal compilation

© 2007 Nordic Pharmacological Society.

Basic & Clinical Pharmacology & Toxicology

were studied by analysing lymphocyte proliferative kinetics.To further address the question of whether irinotecan actsas specific blocker of enzyme acetylcholinesterase (AChE),we performed an

in vitro

relevant experiment on humanerythrocyte AChE.

Material and Methods

Blood sampling.

Blood sample was obtained from a healthy femaledonor (age of 35 years, non-smoker) who gave informed consent forparticipation in the study. The donor had not been exposed to diagnosticor therapeutic irradiations as well as to known genotoxic chemicalsfor a year before blood sampling. Venous blood (40 ml) was collectedunder sterile conditions in heparinized vacutainer tubes (BectonDickinson, Franklin Lakes, NJ, USA) containing lithium heparinas anticoagulant.

Chemicals and reagents.

The test chemical, irinotecan was purchasedfrom Aventis Pharma Ltd. (CAMPTO

®

, Dagenham, UK). It wasused in the form of irinotecan hydrochloride trihydrate. Before use,it was dissolved in 5 ml of sterile concentrate solution for infusion toa final concentration of 20 mg/ml. The final concentrations of theantineoplastic drug in culture medium with human blood cells in our

in vitro

experiment were 9.0

µ

g/ml and 4.6

µ

g/ml. They were deducedform the therapeutic doses of irinotecan used in monotherapy(350 mg/m

2

), respectively in combination chemotherapy (180 mg/m

2

)[4] and calculated on the basis of average body weight and averagesurface area of an adult person. Other chemicals and reagents used,if not specified, were purchased form Sigma Chemical Co. (St. Louis,MO, USA).

Experimental procedure.

Two independent experiments were con-ducted on the same blood sample and the data were pooled. Theevaluation of cell viability, apoptosis measurements and comet assaywere performed on isolated lymphocytes. Six aliquots of isolatedlymphocytes were placed in sterile Falcon tubes in final concentrationof 2

×

10

6

cells/ml. Four aliquots were incubated with irinotecan for2 hr at 37

°

C, while two were negative controls. After 2 hr of exposure,culture medium containing irinotecan was carefully removed andthe cells were washed twice in fresh F-10 medium. Primary DNAdamage and cell viability was evaluated immediately after the treatment(time-point 0

′

), while the effectiveness of DNA repair was checkedat 30, 60, 90 and 120 min. after the treatment. The cell viabilityusing a fluorescence assay was studied in parallel during the wholepost-incubation period. The evaluation of A part of heparinizedblood was used for the cytogenetic analyses. For each culture 0.8 mlof heparinized blood were added to 8.0 ml of standard Ham’s F-10culture medium supplemented with 20% foetal calf serum and anti-biotics: penicillin 100 IU/ml (Pliva, Zagreb, Croatia) and streptomycin100

µ

g/ml (Krka, Novo Mesto, Slovenia). For each end-point, anuntreated control sample was also included.

Cell cultures were incubated

in vitro

at 37

°

C in humified atmospherewith 5.0% CO

2

(Heraeus Heracell 240 incubator, Langenselbold,Germany). After 2 hr of

in vitro

treatment, the incubation mediumwas removed and replaced with fresh culture medium. Then, phyto-hemagglutinin (PHA; Apogent, Hudson, NH, USA) was added (0.2 mlper each culture). Subsequent steps of cultivation, cell harvestingand slides preparation were performed as recommended for theparticular technique. One aliquot of heparinized blood was used forthe determination of the AChE activity in erythrocytes and thestudy of the inhibition of AChE by irinotecan

.

Lymphocyte isolation.

Anticoagulant-treated blood was mixed 1:1 (v/v)with balanced salts solution, layered on the Ficoll solution andcentrifuged at 375 g for 40 min. at room temperature. The layercontaining lymphocytes was carefully removed and cells were re-suspended in balanced salts solution. They were washed twice by

centrifugation at 600 r.p.m. for 10 min. The final pellet was gentlyre-suspended in culture medium F-10. Viability of cells was checkedby supravital staining with 0.1% trypan blue [8].

Assessment of cell viability, apoptosis and necrosis.

For studying celldeath and morphological changes in the nuclei, we used dye exclu-sion method [9] in which viable (intact plasma membrane) and dead(damaged plasma membrane) cells can be visualized after stainingwith the fluorescent DNA-binding dyes. Ethidium bromide and acridineorange were added to the cell suspension in final concentrationsof 100

µ

g/ml (1:1; v/v). Two parallel tests with aliquots of the samesample were performed and a total of 500 cells per sample werecounted. Quantitative assessments were made by determination ofthe percentage of apoptotic and necrotic cells.

The comet assay.

The comet assay was carried out under alkalineconditions, as described by Singh et al. [10] and neutral conditionsaccording to Wojewódzka et al. [11]. Two replicate slides per sampleper method were prepared. Agarose gels were prepared on fully frostedslides coated with 1% and 0.6% normal melting point agarose.Lymphocyte samples (5

µ

l) were mixed with 0.5% low melting pointagarose, placed on the slides and covered with a layer of 0.5% lowmelting point agarose. The slides were immersed for 1 hr in freshlyprepared ice-cold lysis solution (2.5 M NaCl, 100 mM Na

2

EDTA,10 mM Tris-HCl, 1% Na-sarcosinate, pH 10) with 1% Triton X-100 and10% dimethyl sulfoxide (Kemika, Zagreb, Croatia). Alkaline dena-turation and electrophoresis were carried out at 4

°

C under dim lightin freshly prepared electrophoretic buffer (300 mM NaOH, 1 mMNa

2

EDTA, pH 13.0). After 20 min. of denaturation, the slides wererandomly placed side by side in the horizontal gel-electrophoresistank, facing the anode. Electrophoresis at 25 V lasted another 20 min.Neutral denaturation was carried out in the dark at 8

°

C and lastedfor 1 hr in a buffer containing 300 mM sodium acetate and 100 mMTris-HCl, pH 8.5. It was followed by electrophoresis at 14 V and11–12 mA that also lasted for 1 hr. After electrophoresis, the slideswere gently washed with a neutralisation buffer (0.4 M Tris-HCl,pH 7.5) three times at 5 min. intervals. Slides were stained withethidium bromide (20

µ

g/ml) and stored at 4

°

C in humidified sealedcontainers until analysis. Each slide was examined using a 250

×

magnification fluorescence microscope (Zeiss, Oberkochen, Germany)equipped with an excitation filter of 515–560 nm and a barrier filterof 590 nm. A total of 100 comets per sample were scored (50 fromeach of two replicate slides). Comets were randomly captured at aconstant depth of the gel, avoiding the edges of the gel, occasional deadcells and superimposed comets. Using a black and white camera,the microscope image was transferred to a computer-based imageanalysis system (Comet Assay II, Perceptive Instruments Ltd.,Suffolk, UK). To avoid the variability, one well-trained scorer scoredall comets. As a measure of DNA damage, tail moment was chosen.

Analysis of structural chromosome aberrations.

The chromosomeaberration test was performed in agreement with InternationalProgramme on Chemical Safety guidelines [12]. One thousandmetaphases per sample were scored for total numbers and types ofaberrations, as well as the percentage of aberrant cells.

Fluorescence

in situ

hybridization.

Slides for metaphase FISH wereprepared according to standard International Atomic Energy Agency[13] without final incineration. FISH was performed on 3-week-oldmetaphase spreads with whole chromosome-painting probes, followingthe instructions of the supplier (Cytocell Technologies Ltd.,Cambridge, UK). Directly labelled Aquarius

®

chromosome-paintingprobes were used to paint chromosomes 1, 2 and 4. They were labelledwith a red fluorophore (Texas Red spectrum), green fluorophore (FITCspectrum) and combination of both fluorophores, respectively. Thechromosomes were counterstained with 4

′

,6-diamidino-2-phenylindole(DAPI) prepared in an antifade solution (Cytocell Technologies Ltd.).Probed slides were scored using Olympus AX70 epifluorescencemicroscope (Olympus Optical, London, UK). A triple-band pass

EVALUATION OF IRINOTECAN TOXICITY

IN VITRO

3




filter was used to permit simultaneous viewing of the probed chromo-some pairs and other chromosomes counter stained by the DAPI.Furthermore, separate specific filters for Texas Red, FITC andDAPI were used. We used PAINT nomenclature system accordingto Tucker et al. [14].

Sister chromatid exchange assay and the analysis of lymphocyteproliferation kinetics.

Cell cultures were set up according to stand-ard protocol [15]. 5-Bromodeoxyuridine (5

µ

g/ml) was added at theinitiation of the cultures. To obtain harlequin chromosomes, slideswere stained using a modified fluorescence plus Giemsa method asdescribed by Perry and Wolff [16]. A total of 100 randomly selectedsecond division metaphases (50 from each of two replicates) pereach sample were analysed blindly. The number of SCE per metaphaseand range of SCEs were determined.

In fluorescence plus Giemsa-stained preparations, cells dividing forthe first (M

1

), second (M

2

) or third time (M

3

) in culture containingbromodeoxyuridine were determined by differential staining patternof sister chromatids. Lymphocyte proliferation kinetics was studiedon 200 differentially stained metaphases per each blood sample.The proliferation rate index (PRI) was calculated according to theformula: PRI = (M

1

+ 2M

2

+ 3M

3

)/total number of cells scored, asreported by Lamberti et al. [17].

Cytokinesis-block micronucleus assay.

Lymphocyte cultures wereincubated according to standard protocol for CBMN assay [18].Cytochalasin B in final concentration 6

µ

g/ml was added in cultureat 44 hr. For MN identification the criteria of Fenech et al. [19]were used. The number of MN in 2000 binucleated (BN) cells wasscored for each treatment and the number of BN cells with MN was alsorecorded. Two thousand cells were scored to determine the percentageof cells with one to four nuclei. A nuclear division index (NDI) wascalculated according to the formula: NDI = (M

1

+ 2M

2

+ 3M

3

+4M

4

)/1000 [20].

Determination of the AChE activity.

In our experiments, the inhibitorypower of irinotecan on AChE of human erythrocytes is determinedand compared to carbamate physostigmine (eserine), which is testedas reference drug. Physostigmine salicylate salt was used at a doseof 0.1 mg/kg. Acetylthiocholine iodide (ATCh) and 5,5

′

dithiobis-2-nitrobenzoic acid (thiol reagent; DTNB) were of analytical grade.

Enzyme assay and preparation.

The activity of AChE was determinedby means of a colorimetric assay based on the enzymatic conversionof ATCh to thiocholine, which reacts with DTNB to generate thechromogen compound 5-thio-2-nitrobenzoate [21]. The assay solutionwas maintained at a temperature of 37

°

C throughout the analyticprocedure and consisted of a 0.1 mol/l phosphate buffer, pH 7.4that contained 0.01 mol/l DTNB. The source of AChE was nativehuman erythrocytes; the final dilution during enzyme assay was 400times larger. The reaction was performed in the total volume of3.0 ml. The increase in absorbance was read at 412 nm, at 15 sec.intervals; against a blank that contained the erythrocytes suspendedin buffer and DTNB.

Inhibition of AChE by irinotecan and physostigmine.

(i) The concen-tration of the compound yielding a 50% enzyme inhibition (IC

50

)was determined by incubating erythrocytes with four or moredifferent concentrations of irinotecan and physostigmine at 4

°

C andassaying for AChE activity after 15 min. Relative changes in theenzyme activity are presented as the percentage of activity of therespective of control. Only those values between 10% and 90% inhi-bition were used for calculation. (ii) For the purpose of biochemicalexperiments, we also determined catalytic activity of AChE previouslyinhibited with two different concentrations of irinotecan (9.0 and4.6

µ

g/ml) or physostigmine (0.1

µ

/ml). The reaction mixture wasincubated for 10, 30, 60, 120, 150 and 180 min. at 37

°

C after additionof each of drugs and activity of AChE was measured. Each of sampleswas assayed in triplicate, and a mean value was then calculated. The

effects of irinotecan and physostigmine were expressed as percent-ages of the AChE activity of the respective of control.

Statistical analyses.

Statistical analyses were carried out with acommercial programme Statistica 5.0 (Statsoft, Tulsa, OK, USA).

In order to normalize distribution and to equalize the variances, alogarithmic transformation of data was applied. The level of statis-tical significance was set at P < 0.05. The extent of DNA damage,as recorded by the alkaline and neutral comet assay, was analysedconsidering the mean (±S.E.M.), median and range of the comet tailmoment. Comparisons between samples were done using the one-way

and subsequently the Duncan’s test for the calculationsconcerning pair-wise comparisons. The comparisons between valuesobtained for the cell viability, apoptosis and necrosis in irinotecan-treated and control samples were made by

χ

2

-test. The same testwas applied for the statistical evaluation of data considering thestructural chromosome aberrations in lymphocytes, CBMN assayand lymphocyte proliferation kinetics. The statistical significance ofdata obtained by the SCE test was evaluated using the one-way

and the Duncan’s test for the calculations concerning pair-wisecomparisons. Each sample was characterized for the extent of DNAdamage by considering the mean (±S.D.), median and range ofSCE per cell.

Results

Cell viability, apoptosis and necrosis.

After isolation, the lymphocyte viability in the sample preparedfor the experiments was 98.8%. The percentages of viableand non-viable: apoptotic or necrotic lymphocytes after 2 hr

in vitro

exposure to irinotecan are reported in table 1.

In vitro

treatment with irinotecan caused a dose-dependent decreasein cell viability, accompanied by increases in the percentage ofapoptotic and necrotic cells. Reduced cell viability comparedto control was observed at each time-point following

in vitro

treatment with higher dose of irinotecan (P < 0.05,

χ

2

-test)and only in sample analysed at time-point 0 after treatmentwith lower dose of irinotecan. Despite of reduced cell viabilityin sample treated with a higher dose of irinotecan, differencesbetween this sample and a sample treated with a lower dose ofdrug were not statistically significant. The same was observedfor the incidence of apoptotic cells in treated samples. Duringthe 2 hr of experiment, the viability of lymphocytes in thecontrol sample gradually decreased, along with an increase inthe proportion of apoptotic cells. At time-point 120 min., thecell viability was significantly lower compared to time-points0 and 30 min. (table 1).

Alkaline comet assay.

Baseline DNA damage (mean tail moment) in freshly isolatedlymphocytes prepared for the experiments was 0.12 ± 0.02(median 0.05; min–max 0–1.87). The value of tail moment,observed at time-point 0, indicates that isolated lymphocytesduring 2 hr of incubation

in vitro

effectively repaired minorDNA lesions inflicted by isolation procedure. The values of thecomet tail moment in control cells steadily increased at latertime-points, as displayed in fig. 1, but were still significantly lowerthan in irinotecan-treated samples. Both doses of irinotecanevoked significant increase in the tail moment. Unexpectedly, theincrease was more pronounced at the lower dose applied, butthe difference was not statistically significant. The values of

4

NEVENKA KOPJAR

ET AL

.




tail moment in both irinotecan-treated samples were signific-antly increased as compared to the control sample, in mosttime-points following

in vitro

treatment (P < 0.05, analysisof variance; fig. 1). The highest level of DNA damage in bothirinotecan-treated samples was observed at time-point 60 min.after treatment. Because the comet tail moments are positivelycorrelated with the level of DNA breakage in the cell, it islikely that the amount of single strand breaks and alkali-labilesites in irinotecan-treated cells steadily increased in first60 min. after treatment. During the later 30 min. of incubation,the levels of DNA damage in both samples were significantlylowered. According to our observations, the cells exposed tothe lower dose of the drug were able to recover within the

120 min. incubation after treatment. However, in the sampletreated with a higher dose at time-point 120 min., a new peakof DNA damage was recorded, and this damage level wassignificantly higher as compared to the samples 0, 30 and90 min. (fig. 1). We assume that it was a transient increase ofDNA damage related to DNA repair and oxidative processesthat generate additional single strand breaks and alkali-labilesites detectable by the alkaline modification of the assay.

Neutral comet assay.

Baseline DNA damage (mean tail moment) in freshly isolatedlymphocytes prepared for the experiments was 0.13 ± 0.02(median 0.08; min–max 0–1.30). Contrary to the irinotecan-

Table 1.

Results of the quantitative fluorescent assay for the simultaneous identification of apoptotic and necrotic cells in samples of isolatedperipheral blood lymphocytes treated for 2 hr in vitro with irinotecan (IRI).

Time after treatment

Viable cells (%)

Non-viable cells (%)

Necrotic Σ

Apoptotic

Intact membrane Damaged membrane Σ Apo

IRI 9.0 µg/ml0′ 63.4↑ 36.6 26.4 4.8 31.2↑ 5.430′ 59.6↑ 40.4 18.2 13.6 31.8↑ 8.6↑60′ 63.0↑ 37.0 16.0 16.0 32.0 5.090′ 58.4↑ 41.6 27.6 6.0 33.6 8.0120′ 52.2↑ 47.8 23.4 12.4 35.8 12.0↑IRI 4.6 µg/ml0′ 65.8↑ 34.2 30.0 2.4 32.4↑ 1.830′ 71.0 29.0 21.0 4.0 25.0 4.060′ 67.4 32.6 17.6 9.6 27.2 5.490′ 65.0 35.0 23.8 7.0 30.8 4.2120′ 63.6 36.4 16.6 12.8 29.4 7.0Control0′ 85.8 14.2 12.2 – 12.2 2.030′ 82.4 17.6 15.2 0.8 16.0 1.660′ 77.8 22.2 17.6 3.4 21.0 1.290′ 74.6 25.4 21.0 2.4 23.4 2.0120′ 67.2* 32.8 24.2 4.8 29.0* 3.8

Evaluation was made by analysing 500 cells per sample per each experimental point. ↑significantly different as compared to control sampleanalysed at the same time; *significantly different as compared to samples 0′ and 30′ P < 0.05, χ2-test).

Fig. 1. Distribution of comet tail moments measured in peripheral blood lymphocytes throughout the post-incubation period after exposureto irinotecan (IRI) in concentrations of 9.0 and 4.6 µg/ml. In vitro treatment with irinotecan lasted for 2 hr. Alkaline comet assay was employed.Each sample was characterized for the extent of DNA damage by considering the mean ± S.E. (indicated with ) of comet tail moment.Statistical significance was evaluated on logarithmically transformed data, using the one-way and the Duncan’s test for the calculationsconcerning pair-wise comparisons; the level of statistical significance was set as P < 0.05. Significantly increased compared to sample 0′ – a;sample 30′ – b; sample 60′ – c; sample 90′ – d; sample 120′ – e; control sample – *; sample treated with lower dose of irinotecan – **.

EVALUATION OF IRINOTECAN TOXICITY IN VITRO 5

© 2007 The AuthorsJournal compilation © 2007 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology

treated samples, primary DNA damage in control lymphocytesgradually increased within first 60 min. of the incubation aftertreatment. Later on, it steadily decreased to values comparablewith those recorded in freshly isolated cells (fig. 2). Exposureto irinotecan evoked a dose-dependent increase in the tailmoment of treated lymphocytes. However, only the value,recorded in the sample exposed to a higher dose of drug, wassignificantly increased compared to control sample (P < 0.05,analysis of variance; fig. 2). The lowest level of primary DNAdamage after treatment with both doses of irinotecan wasrecorded at time-point 60 min. Later on, the levels of DNAdamage increased again (fig. 2). Statistically significantdifferences are indicated in fig. 2. It should be stressed thatthe levels of DNA damage recorded at time-point 120 min.in both irinotecan-treated samples were relatively high andstill increased compared to the control sample.

Analysis of structural chromosome aberrations.

The results obtained for the treatments with two doses ofirinotecan and corresponding control are presented in table 2.Both doses of irinotecan produced increased frequencies ofcells with chromosomal aberrations and abnormal metaphasescompared to control (P < 0.05, χ2-test). The chromosome

aberrations detected at the highest frequency were chromatidbreaks and complex quadriradial chromosome exchangefigures. Acentric chromosomes, chromosome breaks andtriradial figures followed. The least frequent aberrations weredicentric chromosomes and rings (only one ring chromosomewas found in sample treated with a higher dose of irinotecan).

Fluorescence in situ hybridization.

A dose-related increase in translocation yield was observedin lymphocytes treated with irinotecan (table 3). In the controllymphocytes, a single translocation was detected in 1183genome equivalents, whereas in the cells treated with thehigher concentration of the drug 15 in 890 genome equivalentswere observed. Complex rearrangements involving more thantwo chromosomes and more than two chromosome breakswere observed only in the lymphocytes treated with a higherdose of irinotecan. The number of acentric fragments origi-nating from one of the painted chromosomes also increasedwith the concentration (0 in the control, 1 for the lower doseand 9 for the higher dose of irinotecan). All three paintedchromosomes were equally involved in translocations.However, among them only the chromosome 1 was involvedin chromatid exchanges resulting in formation of quadriradials.In sample treated with the lower dose of irinotecan, 33.3% ofquadriradials contained the chromosome 1, and in the sampletreated with the higher dose 36.3%. Thus, there was no correla-tion between the frequency of chromosome 1 involvement inchromatid exchanges and the dose applied.

Sister chromatid exchange and lymphocyte proliferation kinetics.

The frequencies of SCEs observed in second-divisionmetaphases in irinotecan-treated and control samples are listedin table 4. In the control sample, the mean SCE frequencywas 3.11 ± 1.21 SCEs/metaphase (median 3 SCEs/metaphase;range 0–6 SCEs/metaphase). The observed value was withinthe control range normally observed in our laboratory. Thehigher concentration of irinotecan tested caused an almost

Fig. 2. Distribution of comet tail moments measured in peripheral blood lymphocytes throughout the post-incubation period after exposureto irinotecan (IRI) in concentrations of 9.0 and 4.6 µg/ml. In vitro treatment with irinotecan lasted for 2 hr. Neutral comet assay wasemployed. Each sample was characterized for the extent of DNA damage by considering the mean ± S.E. (indicated with ) of comet tailmoment. Statistical significance was evaluated on logarithmically transformed data, using the one-way and the Duncan’s test for thecalculations concerning pair-wise comparisons; the level of statistical significance was set as P < 0.05. Significantly increased compared to sample 0′– a; sample 30′ – b; sample 60′ – c; sample 90′ – d; sample 120′ – e; control sample – *; sample treated with lower dose of irinotecan – **.

Table 2.

Structural chromosome aberrations in peripheral bloodlymphocytes treated for 2 hr in vitro with irinotecan (IRI).

Treatment

Chromosomal aberrations (CA)/1000 cells Cells with CA (%) B1 B2 T Q Ac Dc R Total

IRI 9.0 µg/ml 34 14 4 24 14 2 1 93 7.6↑IRI 4.6 µg/ml 28 12 1 11 13 1 – 66 5.8↑Control 1 – – – 1 – – 2 0.2

One thousand metaphases per sample per each experimental pointwere scored. B1 – chromatid break; B2 – chromosome break; T –triradial figure; Q – quadriradial figure; Ac – acentric fragment; Dc– dicentric; R – ring; ↑significantly different as compared to controlsample (P < 0.05, χ2-test).

6 NEVENKA KOPJAR ET AL.


seven times increase of the basal SCE frequency: mean SCEfrequency in treated lymphocytes was 20.70 ± 6.36 SCEs/metaphase (median 20 SCEs/metaphase; range 9–38 SCEs/metaphase) (table 4). The observed difference was highlysignificant (P < 0.01, ). On the other hand, the lowerconcentration caused a five-time increase of the basal SCEfrequency: mean SCE frequency in treated lymphocytes was15.47 ± 7.35 SCEs/metaphase (median 13.5 SCEs/metaphase;range 6–37 SCEs/metaphase) (table 4).

The majority of cells in the control sample were in secondin vitro division (M2) at the time of analysis; moreover, theproportion of M3 cells was slightly higher than the proportionof M1 cells. The value of the PRI was 2.02 (table 4). In vitroadministration of irinotecan significantly disturbed lymphocyteproliferation kinetics. An increase in the relative proportionof M1 indicates a delay in lymphocyte cell cycle, accompaniedby decreases in the proportions of M2 and M3 cells and signifi-cant lowering of the PRI value in both irinotecan-treatedsamples (P < 0.05, χ2-test; table 4).

Cytokinesis-block micronucleus assay.

The induction of micronucleated lymphocytes by irinotecanis shown in table 5. Even at the lower dose, a significant increasein the MN frequency was observed. At this dose, irinotecaninduced a frequency of 71 MN per 1000 BN lymphocytes(control 14 MN per 1000 BN lymphocytes). In contrast,exposure to a higher dose of irinotecan produced a frequencyof 81 MN per 1000 BN cells. In the control sample, there wereonly 14 BN recorded and all of them contained only oneMN (table 5). The majority of BN in the irinotecan-treated

samples also contained only one MN. In both treated samples,a small proportion of BN with two MN was noticed, whileBN with three MN was recorded only in a sample treatedwith a higher dose of the drug (table 5). Although a dose-dependent increase in MN frequency was observed, thedifferences were not statistically significant. Exposure toirinotecan also evoked formation of nuclear buds (NB) andnucleoplasmic bridges (NPB). In the treated samples, frequencyof these types of damage was dose-dependent, but lower thanthe frequency of MN. In the control sample only NBs wererecorded. CBMN assay takes into account all cells, bothviable and non-viable, giving the better insight in actual eventswithin the culture system. We observed that apoptosis plays animportant role in the elimination of cells with DNA damage.While in the control sample only one apoptotic cell was detected,in treated samples a dose-dependent increase in frequencyof apoptotic cells was observed (table 5). Both doses of irinote-can inhibited cell proliferation, compared to control. Moreover,exposure to the higher dose of irinotecan caused lower NDIcompared to the value of the same index recorded in thesample treated with lower dose of irinotecan (table 5).

Measurement of AChE activity.

To further address the question of whether irinotecan acts asa specific AChE blocker, we performed an in vitro relevantexperiment on human erythrocyte AChE. The results obtainedin previous in vitro studies, suggest that this drug interactsdirectly with various molecular forms of AChE, leading to anon-competitive inhibition of this enzyme [22,23]. In ourexperiments, the inhibitory power (IC50) of irinotecan on

Table 3.

Results of the fluorescence in situ hybridization (FISH) performed using three-colour painting probes for chromosomes 1, 2 and 4 onperipheral blood lymphocytes treated for 2 hr in vitro with irinotecan (IRI).

Treatment Σ Cells Σ T Σ rTComplex

RR

Σ T involvingΣ deletions resulting

in acentric

Σ Q

Σ Q involving

Ch 1 Ch 2 Ch 4 Ch 1 Ch 2 Ch 4 Ch 1 Ch 2 Ch 4

IRI 9.0 µg/ml 890 15 10 4 6 6 2 – 4 5 11 4 – –IRI 4.6 µg/ml 808 5 – – 1 1 3 1 – – 6 2 – –Control 1183 1 – – – – 1 – – – – – – –

T – translocations; rT – reciprocal translocations; RR – rearrangements; Ch – chromosome; Q – quadriradials.

Table 4.

Results of the sister chromatid exchange (SCE) assay and lymphocyte proliferation kinetics on peripheral blood lymphocytes treated for 2 hrin vitro with irinotecan (IRI).

Treatment

SCE/cell Mitotic activity

Mean ± S.D. Median Range M1 (%) M2 (%) M3 (%) PRI

IRI 9.0 µg/ml 20.70 ± 6.36†,‡ 20.00 9–38 28 62 10 1.82*IRI 4.6 µg/ml 15.47 ± 7.35† 13.50 6–37 43 52 5 1.62*Control 3.11 ± 1.21 3.00 0–6 14 70 16 2.02

Data on SCE are presented as mean values obtained by analysing of 100 second metaphases; lymphocyte proliferation kinetics was evaluatedby analysing 200 cells per sample per each experimental point. M1, M2, M3 corresponded to the frequencies of cells in first, second and thirdin vitro division; PRI (proliferation rate index) was determined with the formula: 1M1 + 2M2 + 3M3/200; †significantly increased as comparedto control sample; ‡significantly increased as compared to sample treated with lower concentration of irinotecan (P < 0.01; analysis ofvariance); *significantly different as compared to control sample P < 0.05, χ2-test).



AChE of human erythrocytes is determined and comparedto carbamate physostigmine that is tested as reference drug.Namely, physostigmine has a strong inhibition potency tothe same enzyme. Under identical conditions, changes inthe enzymatic activity were detected when irinotecan orphysostigmine were incubated with human erythrocyte AChEin the assay solution for 15 min. IC50 values were 5.0 × 10−7

for irinotecan and 2.0 × 10−8 mol/l for physostigmine (figs 3and 4). The values indicate that irinotecan is an approxi-mately 10 times less potent inhibitor than physostigmine.However, irinotecan was found to be strong inhibitor of theAChE hydrolysis of acetylcholine (ACh). In addition, a con-tinuous decrease of catalytic activity of human erythrocyteAChE was obtained 10, 30, 60, 120, 150 and 180 min. afteraddition of a lower dose of irinotecan. A higher dose of iri-notecan inhibited the AChE activity with potency similar tothat estimated for physostigmine, and there was a slight reduc-tion in the inhibition of AChE by both doses of irinotecanwith increased incubation times (fig. 5).

Discussion

The present study reports for the first time the results ofsimultaneous application of alkaline and neutral cometassay on irinotecan-treated human lymphocytes. Previous

study with related drugs, camptothecin and topotecandemonstrates the power of the alkaline comet assay to detectDNA damage in treated Chinese hamster ovary cells, as wellas its repair after the drug removal [24]. Positive resultsobtained in our research indicate that in lymphocyte DNAafter treatment with irinotecan a lot of strand breaks wereinduced. Dynamics of damage infliction as observed both inalkaline and neutral modifications of the comet assay duringthe post-incubation period clearly reflects the ‘poisoning’ ofthe topoisomerase I [25]. It is known that topoisomerase Ibinds to single-strand DNA breaks. The reversible Topo I-irinotecan-DNA cleavable complex is not lethal to the cells byitself. However, by its collision with the advancing replicationforks, the formation of a double-strand DNA break occurs,leading to irreversible arrest of the replication fork and celldeath [26]. The double-strand DNA breaks caused by thetreatment are of special interest because they are more cytotoxiclesions and considered as major source of stabile chromosomeaberrations and rearrangements [27]. Furthermore, their repairis much slower and more complicated as compared withsingle-strand DNA breaks [28]. It is known that the repairprocess itself also generates additional breaks (observed inthis study as well), while a part of strand DNA breaks couldbe induced by indirect action of reactive oxygen speciesgenerated during the treatment. This assumption is also

Table 5.

Results of the cytokinesis-block micronucleus (CBMN) study of irinotecan toxicity on human peripheral lymphocytes treated for 2 hr in vitrowith irinotecan (IRI).

TreatmentΣ MNed

BN

No. of BN with

ΣMN

Total No. of Lymphocyte proliferation

1MN 2MN 3MN NB NPB APO M1 M2 M3 M4 NDI

IRI 9.0 µg/ml 74† 68 5 1 81 37 5 57 350 1507 108 64 1.958†,‡

IRI 4.6 µg/ml 68† 65 3 – 71 25 2 25 146 1661 104 89 2.068†

Control 14 14 – – 14 4 – 1 152 1567 153 128 2.129

Two thousand binucleate cells (i.e. 4000 nuclei) were scored to determinate total number of micronuclei (MN) for each experimental point.MNed – micronucleated; BN – binuclear cells; NB – nuclear buds; NPB – nucleoplasmic bridges; NDI – nuclear division index; indicates themean number of nuclei in all cells and is computed by the formula NDI = (M1+ 2M2 + 3M3 + 4M4)/N, with M1 – M4 being the number ofcells with 1–4 nuclei and N the number of screened cells. Two thousand cells per sample were screened for number of nuclei. †significantlydifferent as compared to control P < 0.05; χ2-test); ‡significantly different as compared to sample treated with lower concentration of irinotecan.

Fig. 3. Inhibition of acetylcholinesterase (AChE) by irinotecan. Fig. 4. Inhibition of acetylcholinesterase (AChE) by physostigmine.



sustained by the results of an earlier study on camptothecin-treated Jurkat cells [29]. In the present study, a positivecorrelation between the results of the comet assay and theresults obtained by quantitative determination of apoptoticand necrotic cells in the same samples was observed. Themortality of irinotecan-treated lymphocytes was primarilycaused by apoptosis. Irinotecan was established earlier as aneffective inducer of apoptosis [30]. It is assumed that theformation of DNA-protein complex stabilized by DNAtopoisomerase I inhibitor ultimately signals the onset ofapoptosis [31]. Moreover, at higher concentrations of irinote-can non-S-phase cells, besides apoptosis, could also be killeddue to transcriptionally mediated DNA damage [32].

Conventional analysis of structural chromosome aberrationsrevealed a lot of chromatid breaks and complex quadriradialchromosome exchange figures in irinotecan-treated cells.These results are also in agreement with the reports of otherresearchers who investigated the effects of camptothecin andrelated drugs [33]. There are no published data on involvementof specific chromosomes in irinotecan-induced rearrangementsyet. In the present study, three-colour painting probes forchromosomes 1, 2 and 4 were used. These chromosome pairsrepresent 22.34% of the DNA content of the human genomein a female and 22.70% in a male, as derived from Morton[34]. Thus, the labelling of chromosomes 1, 2 and 4 in cellsfrom a female donor is expected to detect as paint/non-paintinterchanges about 34.7% of the chromosomal interchangesoccurring in the complete genome [35]. According to literaturedata [14], ‘one-way’ exchanges constitute typically 20–30%of the total exchange patterns. In our study, it appeared to be33% at the highest dose of irinotecan applied. However, otherauthors using telomere probes have shown that very often,when the complete exchanges occurred, a terminal segmentwas so small that it does not register as a distinct visible signal[36,37]. This also must have been the case in our study becausethe number of ‘one-way’ translocations did no change withthe concentration. For both doses of irinotecan tested, five‘non-reciprocal’ translocations were detected. There were nosignificant differences in the frequency of involvement of

specific chromosomes in translocations. However, at the higheririnotecan concentration, chromosome 4 was under-representedin the chromosome re-arrangements. Ganguly et al. [38] observedthe same in X-irradiated human lymphocytes, whereasBraselmann et al. [39] showed over-involvement of chromosome4 in reciprocal translocations. However, all published studies oninvolvement of specific chromosomes in their re-arrangementswere done on irradiated cells. In the present study, a chemicalagent, irinotecan, was tested. Due to its chemical structure, itmay be possible that the drug exhibits certain specificity towardsspecific DNA regions on different chromosomes, which givesdifferent results on translocations distribution from thoseobtained in irradiated lymphocytes. Recently, Anderson et al.[40] reported that more breaks than expected were observedon chromosome 2 indicating a deviation from randomnessdistribution. This finding is partially in agreement with ourresults. We detected an over-involvement of chromosomes 2and 4 in acentric formation for the higher dose of irinotecan.However, when the results for involvement of the chromosomes1, 2 and 4 in acentric formation and translocation are takentogether, they appear to be in correlation with other studies[41,42]. They indicated that involvement of different chromo-somes in re-arrangements was not proportional with their DNAcontent. Satoh et al. [43] using mitomycin C treated WTK-1cell line deduced that chromosome 1 possesses significantlyhigher number of exchange sites than chromosomes 2 or 4.Thus, it is more frequently involved in quadriradials formation.In our study, only chromosome 1 was detected as the part ofquadriradials. At tested concentrations of irinotecan, the fre-quency of quadriradial formation was too low to permit detec-tion of quadriradials involving other two painted chromosomeswith a single exchange site. Testing of higher concentrationsof irinotecan for chromosomal aberration induction was notpossible due to its cytotoxicity. Furthermore, Satoh et al. [43]deduced that asymmetrical quadriradials derive unstablechromosome-type aberrations (e.g. dicentrics and acentricfragments) and symmetrical quadriradials derive stable aberra-tions (e.g. reciprocal translocations). Because we detectedinvolvement of chromosomes 2 and 4 in acentric fragmentsand reciprocal translocations, it could be presumed that some ofthose rearrangements were derived from quadriradials. Indirectly,it might suggest that also those two chromosomes are involvedin chromatid exchange and quadriradials formation.

High rates of SCE caused by both doses of irinotecan arealso indirect evidence of a large quantity of single- and double-strand DNA breaks produced by treatment. These resultsare in agreement with the reports of other authors [44–46].Besides an increased SCE frequency, irinotecan caused adelay in lymphocyte proliferation in vitro. Because topoisomeraseI is involved in the processes of chromosome segregation [47]as well as in cell transition from G0 to G1 phase and in DNAreplication [46], disturbed functions of this enzyme lead tothe retardation of the cell cycle following treatment. Previousstudies with the irinotecan metabolite SN-38 on human glio-blastoma cells indicate that cell cycle delays were induced bya decrease in the percentage of cells in the G0/G1 phase and anincrease in the percentage of cells in S and G2/M phases [48].

Fig. 5. Progressive inhibition of acetylcholinesterase (AChE) byirinotecan and physostigmine.



Formation of MN after exposure to irinotecan can mirrorclastogenic action of the drug tested. On the other hand, NPBsbetween nuclei in BN cells provide a measure of chromosomerearrangement [49]. The proportions of both MN and NPBsas detected in CBMN assay correlated positively with thetypes of chromosome aberrations recorded in the same bloodsamples. A high proportion of NBs observed after treatmentwith both doses of irinotecan indicate the possibility of geneamplification [49]. To clearly explain the origin of NBs inducedafter exposure to irinotecan, further studies are needed. Theresults obtained by CBMN assay provided comparable resultswith a fluorescence dye exclusion test and confirmed thatapoptosis plays an important role in the elimination of cellswith DNA damage.

The results of our study indicate that irinotecan as theparent drug also induces remarkable toxic effects in humanperipheral blood lymphocytes treated in vitro, both on thesubcellular and the cellular level. The same was observed byPavilliard et al. [50] in their in vitro study with two humancolorectal tumour cell lines. Therefore, irinotecan is not totallydevoid of activity, which was concluded in some earlier studies.The actual levels of DNA damage produced both in cancerand non-target cells in human evidently are much higher dueto the presence of SN-38, a metabolite with an augmentedpotency and cytotoxicity [26]. While the present study wasperformed in vitro, and the blood cells were exposed to irinote-can in a serum-free medium, there was no possibility forextensive conversion of the parent drug to SN-38, althoughsome studies indicate that human plasma esterases, especiallybutyrylcholinesterase, present in serum also possess irinotecan-activating activity [51,52].

In our study, the activity of erythrocyte AChE after in vitroexposure to irinotecan was studied simultaneously with otherend-points. The best-characterized function of AChE is thehydrolysis of ACh at cholinergic synapses [53,54]. Inhibitionof its enzyme leads to accumulation of ACh resulting inover-stimulation of the whole cholinergic system. Muscularand nerve AChE are only present in the synaptic cleft andcannot be measured directly. Because erythrocyte AChE hasa similar structure as the synaptic enzyme, it appears to be asuitable parameter to reflect the various reactions at thesynaptic site. Therefore, its measurement is of important fortherapy management, especially during the course of theintoxication with different chemicals or drugs that inhibitsthe activity of the enzyme. Clinical studies indicate thepossibility of acute cholinergic side effects after intravenousadministration of irinotecan [55–57], which, on the basis ofin vitro experimental findings, have been ascribed to a directblockade of AChE [22,23]. In this study, an attempt was madeto establish whether impairment in the activity of AChE occursin whole-blood samples previously treated with irinotecan.All our results indicate high affinity of irinotecan for AChEin vitro. The enzymatic activity decreased up to 50% whenthe enzyme was exposed to irinotecan at a concentration of5.0 × 10−7 mol/l. These findings were consistent with the resultsobtained in previous in vitro studies, suggesting that thisdrug acts as potent inhibitor of AChE [22,23]. Furthermore,

it is noteworthy that physostigmine, a well-known AChEblocker [58], inhibited the activity of AChE with a potencysimilar to that estimated for the same drug in previous studies[59,60]. Moreover, there is evidence that human erythrocyteAChE activity inhibited by irinotecan in doses comparableto those recommended in mono- or combination therapycorrelates with the activity of the same enzyme afterphysostigmine administration. Thus, irinotecan may providean interesting lead compound for the investigation of theinhibition of AChE. Judging from experimental in vitro data,we assume that measurement of AChE activity in vivo couldbe an appropriate method with possible implementation incontrol and management of acute cholinergic syndrome.However, it has to be further investigated. In addition, thecurrent evidence is insufficient to indicate whether atropinesulfate is beneficial in the management of this syndrome, thatis, whether muscarinic symptoms such as nausea, vomiting,diarrhoea and bowel movements can be stopped by adminis-tration of atropine sulfate. Future in vivo research will help todetermine whether administration of atropine or functionallyrelated compounds has a potential in medical treatment inpatients or not.

Despite of the limitations, the results of the present studymay contribute to the understanding of irinotecan toxicity andconstitute a helpful step to recognize the side effects of thedrug, even though the consequences of in vivo drug treatmentmay not be completely like those occurring in a living organism,they may indicate the existence of certain mutations in non-target cells with cancer predictive value. Using a battery of end-points in surrogate cells, we have obtained an insight into thelevels of the cyto- and genotoxicity, as well as the inhibitorypotency of irinotecan against AChE. However, further in vitroand in vivo studies are essential in order to clarify remainingissues, especially to elucidate possible inter-individual variabilityin genotoxic responses to the drug.

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Irinotecan Toxicity to Human Blood Cells in vitro: Relationship between Various Biomarkers

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