analysis of mitomycin c-induced micronuclei in lymphocytes from malnourished infected children

Environmental and Molecular Mutagenesis 30:363–370 (1997)

Analysis of Mitomycin C-Induced Micronuclei inLymphocytes From Malnourished Infected Children

RocıBo Ortiz,1* Leticia Cortes,2 Cristina Gonzalez,1 Laura Lopez,3

Patricia Perez,4 Edith Cortes,1 and Miguel Betancourt11 Departamento de Ciencias de la Salud, Universidad Autonoma

Metropolitana-Iztapalapa, Mexico D.F. Mexico2 CINVESTAV-IPN, Mexico D.F. Mexico

3 Hospital Infantil Iztapalapa D.D.F., Mexico D.F. Mexico4 Instituto Nacional de Pediatrıa, Mexico D.F. Mexico

The purpose of this study was to determine if periph- ished infected children and 1.0‰ in well-nourishederal blood lymphocytes from malnourished children infected children. In MMC-exposed cultures thewith gastrointestinal or respiratory bacterial infec- mean induced micronucleus frequency was 32.6 {tion show increased frequencies of Mitomycin C 6.1 vs. 12.9 { 2.3; 68.6 { 12.1 vs. 21.0 { 5.1,(MMC)-induced micronuclei as compared to well- and 88.1 { 16.2 vs. 41.7 { 5.0 for malnourishednourished, infected children. The results indicate and well-nourished children at 20, 40, and 60 ng/that cells from malnourished, infected children had ml MMC, respectively. The number of binucleatedgreater chromosome damage. This may indicate cells with more than one micronucleus was alsothat such children would be more susceptible to en- higher in malnourished, infected children at allvironmental damage and malignant transformation. doses tested, including cells with two micronuclei

Micronucleus frequencies were analyzed in binu- in MMC-free cultures from malnourished, infectedcleate cells produced by the cytokinesis block children. This increase was not found in peripheralmethod; the overall micronucleus frequency was sig- blood lymphocytes from well-nourished infectednificantly higher in binucleate cells from malnour- children. Environ. Mol. Mutagen. 30:363–370,ished, infected children. The mean micronucleus fre- 1997 q 1997 Wiley-Liss, Inc.quency in MMC-free cultures was 4.3‰ in malnour-

Key words: malnourished children; micronuclei; Mitomycin C; cytokinesis block micronucleusmethod; human lymphocytes; bacterial infections

INTRODUCTION The MN assay is a useful technique to assess chromo-some damage; the induction of MN in peripheral bloodlymphocytes has been proposed as an alternative to meta-In developing countries, malnutrition is a public healthphase analysis for biomonitoring human populations. MNproblem with a high and increasing prevalence due toare formed by malsegregated acentric chromosome frag-poverty, unemployment, and ignorance [DeMello, 1994].ments or by whole chromosomes that were not integratedIt occurs as a consequence of deficient food intake and/into the main nucleus during cell division and appear inor low-protein diets. Children are the most affected group,the cytoplasm as small nuclei [Fenech and Morley,and its effects can be particularly devastating in the first1985a]. The cytokinesis block (CB) method improves theyears of life, when the body is growing rapidly and thereadability and sensitivity of the assay because MN areneed for nutrients is greatest. These children also havescored only in those cells that have completed one nuclearan increased frequency of gastrointestinal and respiratorydivision following chemical treatment. The MN assay isinfections. Several studies on the effects of malnutritionuseful in detecting chromosome damage induced by sev-at the cellular and genetic levels have been carried out ineral factors, such as exposure to chemical or physicalhumans and experimental animals, revealing that malnu-

trition increases susceptibility to chromosome damage,as analyzed by different methods, such as chromosomalaberrations [Alu and Murthy, 1993], sister chromatid ex- Contract grant sponsor: National Council of Science and Technology

(CONACyT, Mexico); Contract grant number: 3005P-M9608.changes (SCE) [OrtıB z et al., 1994], and micronuclei (MN)[OrtıB z et al., 1995]. In the latter study, the total number * Correspondence to: Dr. RocıBo Ortiz, Departamento de Ciencias de la

Salud, Universidad Autonoma Metropolitana-Iztapalapa, Apdo. Postalof MN was increased in lymphocytes obtained from the55-535, C.P. 09340. Mexico D.F. Mexico.spleens of malnourished as compared to well-nourished

rats. Received 16 December 1996; revised and accepted 2 June 1997.

q 1997 Wiley-Liss, Inc.

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364 Ortiz et al.

TABLE I. Characteristics of Well-Nourished Children

Age Weight/heightChild (months) Sex deficit (%) Infection type

1) DMC 2 M 16.1 Gastrointestinal2) MAO 9 F 14.1 Respiratory3) APE 9 F 0.0 Respiratory4) SAA 11 F 15.1 Gastrointestinal5) YSN 21 F 15.0 Respiratory6) APA 24 F 0.7 Gastrointestinal7) CEF 36 M 0.0 Gastrointestinal8) ELG 54 M 5.0 Respiratory

mented with 0.2 ml of phytohemagglutinin (5 mg/ml) (Microlab). Theagents, age-related alterations, and nutritional habits [Fen-cells were incubated at 377C and MMC (Sigma, St. Louis, MO.) wasech, 1993].added after 24 hr of culture at the following doses: 20, 40, and 60 ng/

Mitomycin C (MMC) is a clastogenic agent that has ml. At 44 hr of incubation, 3 mg/ml cytochalasin-B (Sigma) were addedbeen used to study the susceptibility of cells to chromo- to cultures, which were incubated for an additional 28 hr for a total

culture time of 72 hr. Afterwards, cells were fixed with methanol-coldsomal damage and cytotoxic effects [FrıB as et al., 1996].acetic acid (3:1) and slides were prepared and stained with May Grun-It acts as a bifunctional or trifunctional alkylating agentwald-Giemsa dyes (Sigma-Merck, Mexico).and forms intercatenary and intracatenary cross-links

which lead to chromosome aberrations [Lown, 1979].Micronucleus AssayMMC is capable of inducing chromosome aberrations in

vitro [Gupta and Sharma, 1981], increased SCE [Lit- The slides were coded and the same person scored the slides for thetlefield et al., 1981], DNA damage [Pfuhler and Wolf, entire study in order to minimize variation due to operator differences.

MN were analyzed in CB-induced binucleated cells in order to score1996], and MN in a dose-dependent fashion [Channaray-only cells which had divided. Binucleated cells were identified accordingappa et al., 1990]. The purpose of the present study wasto: a) equal size and separation of both nuclei, and b) integrity of the

to determine if lymphocytes from malnourished, infected cytoplasm. The most representative criteria to identify MN were: 1) thechildren show increased frequencies of MMC-induced morphology of MN should be similar to that of the normal nucleus, 2)micronuclei compared to lymphocytes from well-nour- the diameter should be 1

3 to 116 that of the main nucleus, 3) the MN

should not be refringent, and 4) MN should be located near the nucleus,ished, infected children.but not touch it [Fenech, 1993].

Slides were analyzed with a light microscope at 1,000 1 magnifica-MATERIALS AND METHODStion. MN were scored in 1,000 binucleated cells with well-preserved

Subjects cytoplasm and the number of cells showing MN and the number of MNper cell were quantified (Fig. 1). The number of induced micronucleiTwo groups of pediatric patients were studied. Both groups includedwas measured by subtracting the micronucleus frequency in controlseverely infected children who had been hospitalized. Group 1 includedcultures from that in treated cultures. Cell cycle kinetics were assessedeight well-nourished children between the ages of 2 and 54 months, ofusing the nucleation index (NI) analyzing 2,000 consecutive cells perwhom four had bacterial respiratory infections and four had bacterialchild. The NI was estimated as described by Erexson et al. [1991]:gastroenteritis. Each child had normal weight and height for its age

(deficit less than 20%). Group 2 included seven malnourished childrenNI Å [1(% mononucleate cells) / 2(% binucleate cells)between the ages of 2 and 47 months, of whom four had gastrointestinal

bacterial infections, one had an upper airway bacterial infection, one / 3(% trinucleate cells) / 4(% tetranucleate cells)]/100had an infection whose type could not be determined, and one was notinfected. Two had second-degree or moderate malnutrition, and five had

Statistical Analysisthird-degree or severe malnutrition.Age, sex, weight/height deficit, degree of malnutrition, and the type Total MN counts and NI at different MMC doses were compared

of infection of the children from both groups are shown in Tables I and between well-nourished and malnourished children using the non-para-II. Blood samples were drawn on admission to the emergency service metric Mann-Whitney U test for two independent samples. The sameof the Iztapalapa Pediatric Hospital in Mexico City only from children test was used to compare MN frequencies at different doses in childrenwho had received no previous drug treatment. Weight/height deficits with second- and third-degree malnutrition, and to evaluate the effectwere estimated according to the data reported in Mexican children of gastrointestinal and respiratory infection. The dose-response effect[Ramos-Galvan, 1976] and malnourished children were classified ac- of MMC on MN frequencies was tested by estimating the correlationcording to the criteria reported by Gomez et al. [1956]. The study was coefficient. The number of MN per cell was compared in both groupsapproved by the Medical Ethics Committee of the General Direction of using the chi-square test.Medical Services (Federal District Department, Mexico).

RESULTSLymphocyte CulturesTable III shows the number of MN in cells exposed toFrom each patient, 0.3 ml of heparinized peripheral blood was cul-

tured in modified McCoy’s 5a medium (Microlab, Mexico) supple- different doses of MMC in both groups. The number

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MMC-Induced MN in Malnourished Children 365

TABLE II. Characteristics of Children with 2nd and 3rd Degree Malnutrition

Age Weight/height Degree ofChild (months) Sex deficit (%) malnutrition Infection type

1) RGN 2 F 26.8 2nd Gastrointestinal2) LHL 2 M 25.0 3rd Not determined3) RSV 3 M 44.9 3rd Gastrointestinal4) MSS 11 F 42.5 3rd Gastrointestinal5) GRL 14 M 41.8 3rd Gastrointestinal6) JAG 44 M ND 3rd No infection7) ARL 47 F 26.7 2nd Respiratory

2nd/Second degree or moderate malnutrition.3rd/Third degree or severe malnutrition.

of MN increased with the addition of MMC in a dose- The decrease of NI was dose-dependent and more evidentin well-nourished children.dependent fashion, and a clear difference between two

groups of children was evident at all doses tested. In The NI in MMC-free cultures was higher in malnour-ished than in well-nourished children (1.52 { 0.12 andview of the significant difference (P õ 0.001) observed

in baseline micronuclei between malnourished and well- 1.30 { 0.09, respectively, mean { 1 SE; P õ 0.02).When cultures were exposed to MMC, the estimated NInourished children, the results for induced micronuclei

(Table III) are shown after subtraction of the values for decreased (1.31 { 0.08 and 1.15 { 0.02, P õ 0.03; 1.17 {0.05; and 1.09 { 0.02, P õ 0.03) for well-nourishedbaseline micronuclei. MMC produced a smaller increase

in micronuclei in cells from well-nourished children than and malnourished children at 20 and 40 ng/ml MMC,respectively. At the highest MMC dose tested (60 ng/ml),from malnourished children (Fig. 2). Mean MN frequen-

cies showed a positive correlation with the MMC dose the NI was also significantly different (1.13 { 0.02 and1.10 { 0.03, P õ 0.05). Additionally, in two well-nour-in both groups (malnourished r Å 0.994, well-nourished

r Å 0.981), which were not significantly different. How- ished children it was not possible to determine the NI,perhaps due to a pronounced cytotoxic effect of MMCever, in MMC-free cultures, the mean MN frequency was

significantly greater in malnourished children. on these cultures.Table V shows the frequency of cells found with 1, 2,In MMC-free cultures, three of the eight well-nourished

children did not show MN, two showed 1, and three and 3 MN, and the total number of MN in control andMMC-exposed cultures. The number of cells with 1 MNshowed 2 MN (1.0 { 0.3, mean { 1 SE). By contrast,

all malnourished children showed a baseline MN range was clearly greater in malnourished children at all dosestested. Cells with 2 MN were present in MMC-free cul-from 1 to 10 (4.3 { 1.2). When cultures were exposed

to 20 ng/ml MMC, cells from well-nourished children tures from malnourished children, but not found in controlcultures from well-nourished children. Also, a higher in-showed 9 to 21 MN (12.9 { 2.3), except one child, who

did not show induced MN. In comparison, malnourished crease of these cells was observed in MMC-exposed cul-tures in cells from malnourished children. Similarly, al-children showed 8 to 55 MN (32.6 { 6.1). The same

effect was observed when cells were exposed to 40 ng/ though cells with 3 MN were scarce, they were morefrequent in cultures from malnourished children exposedml MMC: well-nourished children showed 6 to 49 MN

(21.0 { 5.1) and malnourished showed 22 to 114 MN to 60 ng/ml MMC, as compared to well-nourished chil-dren. The number of cells with more than 1 MN was(68.6 { 12.1). At the highest MMC dose tested (60 ng/

ml), the MN scored in cells from well-nourished children significantly higher in lymphocytes from malnourishedchildren (P õ 0.001).ranged from 29 to 60 (41.7 { 5.0), while malnourished

children had 30 to 153 MN (88.1 { 16.2). The differencesbetween well-nourished and malnourished at all doses DISCUSSIONtested were statistically significant (P õ 0.007; P õ0.001; and P õ 0.037). MN frequencies were not signifi- The results of the present study show that cells from

malnourished, infected children have an increased suscep-cantly different between children with gastrointestinal andrespiratory infection or between children with second- tibility to present MN as compared to well-nourished,

infected children. In the present study, the increased MNand third-degree malnutrition (P ú 0.05).The NI estimations showed significant differences be- frequency could be related to the severe infection. At least

a part of the chromosomal damage observed could be duetween malnourished and well-nourished children in bothunexposed and exposed cells to MMC (Table IV). In each to alterations in cellular metabolism resulting in increased

levels of endogenous toxic agents, or to genotoxic sub-case, the NI was decreased in cultures exposed to MMC.

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366 Ortiz et al.

Fig. 1. Photomicrographs of cytokinesis-blocked lymphocytes. a) Binucleated cell without MN. b) Binu-cleated cell with one MN. The arrow indicates the MN. c) Binucleated cell with two MN (arrows).Magnification 1 1000.

stances released by the infection agent itself. Several re- [Ahuja, 1991]. Additionally, the damage may be associ-ated with free radicals released by the lymphoid cellsports have demonstrated that bacterial infection can pro-

duce genetic damage [D’Souza and Das, 1994]. It is during their attempt to control the infection.The presence in the malnourished group of only oneknown that certain bacterial enzymes can act as DNAses

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MMC-Induced MN in Malnourished Children 367

TABLE III. Number of Micronuclei (MN) in MMC-Exposed Peripheral Blood Lymphocytes From Well-Nourishedand Malnourished Infected Children

MMC ng/ml 0 20 40 60

Child WN MN WN MN WN MN WN MN

Number of micronuclei per child

1 0 2 14 55 32 114 60 962 2 5 0 43 8 56 51 1063 2 1 21 25 19 97 ND 1534 0 6 15 44 49 82 35 1085 1 2 20 21 14 41 31 366 2 4 12 32 6 68 ND 887 0 10 12 8 27 22 44 308 1 9 13 29

Mean 1.0 4.3 12.9 32.6 21.0 68.6 41.7 88.1{ 1 SE 0.3 1.2 2.3 6.1 5.1 12.1 5.0 16.2

WN Å well nourished, MN Å malnourished, ND Å not determined, SE Å standard error. Data obtained from the analysis of 1,000 cells per child.Induced micronuclei are shown after subtraction of the values for baseline micronuclei.

Fig. 2. Frequency of micronuclei induced by different doses of Mitomycin C in lymphocytes from well-nourished and malnourished children.

child with respiratory infection type, of one child whose with the severity of malnutrition. For example, the twochildren with second-degree malnutrition showed similarinfection type could not be determined, and one child

without infection, preclude an accurate assessment of how values of MN as that observed in third-degree malnutri-tion children. Clearly, further studies involving a largethe infection type is related to the increase in MN fre-

quency. However, the lack of difference between the dif- number of patients to address the relation between levelsof MN induction and specific kinds of infection, and typeferent infection types, and the fact that the child without

infection showed an induced frequency similar to infected and severity of malnutrition, will be necessary. Cells frommalnourished children clearly showed a greater chromo-children, indicate that the differences observed may be

due to malnutrition. some damage; the MN frequencies of MMC-exposedlymphocyte cultures were higher in malnourished chil-The MN induction frequency did not appear to correlate

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368 Ortiz et al.

TABLE IV. Nucleation Index (NI) in MMC-Exposed Peripheral Blood Lymphocytes From Well-Nourishedand Malnourished, Infected Children

MMC ng/ml 0 20 40 60

Child WN MN WN MN WN MN WN MN

1 1.60 1.15 1.29 1.12 1.19 1.11 1.18 1.112 1.04 1.61 1.11 1.39 1.08 1.35 1.08 1.133 1.21 1.50 1.12 1.43 1.04 1.39 ND 1.264 1.18 1.84 1.19 1.46 1.18 1.15 1.15 1.145 1.29 1.77 1.20 1.11 1.08 1.10 1.12 1.126 1.76 1.69 1.05 1.62 1.03 1.14 ND 1.147 1.22 1.07 1.13 1.05 1.09 0.98 1.05 1.058 1.11 1.13 1.04 1.00

Mean 1.30 1.52 1.15 1.31 1.09 1.17 1.10 1.13{ 1 SE 0.09 0.12 0.02 0.08 0.02 0.05 0.03 0.02

WN Å well-nourished, MN Å malnourished, ND Å not determined, SE Å standard error. Data obtained from the analysis of 2,000 cells per child.

TABLE V. MN Frequencies in MMC-Exposed Peripheral Blood Lymphocytes of Well-Nourishedand Malnourished Children*

Well-nourished children Malnourished children

MMC N Distribution of MN Total N Distribution of MN Total

Dose** 1 2 3 MN 1 2 3 MN

0 8 8 0 0 8 7 22 4 0 3020 8 104 3 0 110 7 226 16 0 25840 8 146 15 0 176 7 454 28 0 51060 6 200 22 4 256 7 547 38 8 647

*Data obtained from the analysis of 1,000 cells per child.**ng/ml.N Å Number of children studied.

dren at all doses tested. However, this increase was more edly, cells from well-nourished children showed a sig-nificantly lower NI than cells from malnourished children.evident when cultures were treated with 20 and 40 ng/ml

MMC as compared to the highest dose tested (60 ng/ml). This may be due to the greater sensitivity of lymphocytesfrom these children to the cytotoxic effect of MMC.This may have been due to a cytotoxic effect of MMC.

Fenech and Morley [1985b] found a lesser increase in the Clearly, further studies will be necessary to address thesensitivity to MMC, and the relation between cellularfrequency of MN in lymphocytes from old individuals

after exposure to MMC, presumably in relation to cell kinetics and MN induction in lymphocytes from well-nourished and malnourished children.cycle kinetics factors.

The NI estimate in MMC-free cultures was similar to The mean baseline MN frequency in lymphocytes fromwell-nourished children was 1.0‰, which is lower thanthose reported by Erexson et al. [1991] in human periph-

eral blood lymphocytes. The higher NI obtained in mal- that previously reported in peripheral blood lymphocytesof young adults (7.3‰) [Fenech and Morley, 1985b]. Innourished children is in agreement with the previous ob-

servation that a fraction of lymphocytes from malnour- general, MN studies in humans have been performed inlymphocytes and other cell types from adults. Therefore,ished children had a heightened response to PHA,

manifested by a higher replication index. This may be a more studies on baseline frequencies and different re-sponses to genotoxic agents are needed in children.consequence of the responsive lymphocytes from mal-

nourished children having a shorter lag period between The number of cells with 1, 2, and 3 MN was greaterin malnourished children at all doses tested. Although thePHA exposure and transition from the G0 to the G1 phase

in the cell cycle, which may occur when the ‘‘starving majority of binucleated cells from both groups showedonly 1 MN per cell, malnourished children had binucle-cells’’ from malnourished children are placed in a richated cells with 2 MN even in MMC-free cultures, reveal-culture medium [OrtıB z et al., 1994]. The decreased dose-ing considerable baseline chromosome damage. In addi-dependent NI estimate in MMC-treated lymphocyte cul-

tures indicates the cytotoxic effect of MMC. Unexpect- tion, there was a significant increase with increasing doses

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MMC-Induced MN in Malnourished Children 369

in the number of binucleated cells containing multiple chromosome damage in malnutrition. In addition, the datafrom this study also suggests that malnourished childrenMN. This effect has also been observed in dose-dependentmay be more susceptible to environmental damage. Fol-X-ray-induced damage [Erexson et al., 1991].low-up of these children would be of interest to study theIt has been established that the cytokinesis block micro-adverse effects of malnutrition on lymphocyte functionnucleus assay is a sensitive method to detect the mecha-and chromosome damage. It would be interesting to studynism of action (aneugenic/clastogenic) of genotoxicif micronutrient supplements such as folate and vitaminagents. There are many strategies to discriminate betweenB12 might reduce chromosome damage in these childrenMN harboring whole chromosomes and those containing[Fenech and Rinaldi, 1995]. In malnourished children, theacentric fragments: area, DNA content, C-banding, kinet-greater chromosome damage observed could be relatedochore detection with antibodies, and fluorescence in situto protein deficiency, and to the deficiency of severalhybridization [Surralles et al., 1995]. These studies haveessential micronutrients, such as folates and vitamins B12revealed that the majority of MN induced by MMC inand E [Fenech and Rinaldi, 1994]. These deficiencies mayperipheral blood lymphocyte cultures are related to chro-have an additive effect and may be involved in incorrectmosome breaks. Based on these observations, we suggestDNA replication, deficient synthesis of DNA integritythat the majority of the MN observed in this study wereproteins, and/or defective DNA repair mechanisms [Be-acentric fragments. Nevertheless, it would also be im-tancourt et al., 1995].portant to analyze this characteristic of MN in MMC-

In conclusion, the results of this study complementfree and treated lymphocyte cultures from malnourishedthose of previous studies of cellular and cytogenetic dam-children. The quantification of the DNA content of MNage in malnourished children. In addition, this studyand the differences in DNA content between the two ma-shows that cells from malnourished children had greatercronuclei in the binucleated cells without MN, as mea-susceptibility to chromosome damage, which is evidencedsured by image analysis [Van-Hummelen et al., 1995] orby the increased frequency of baseline and MMC-inducedanalysis of DNA content by flow cytometry [Schreiber etmicronuclei in peripheral blood lymphocyte cultures. Thisal., 1992] may be useful for this purpose.greater susceptibility could be related to predisposition toIt has been suggested that lymphocyte subsets differ indamage induced by several environmental agents, possi-sensitivity with respect to induction of MN and cytotoxicbly causing malignant transformation, functional defi-

effects. Holmen et al. [1994], obtained an increased fre-ciency, and/or increased cell death, which could interfere

quency of MN with increasing doses of MMC, and alsowith the development of these children, who should be

found that T4 (helper/inducer cells) and B-cells hadsubmitted for follow-up and medical control.

higher MN frequencies than T8 (suppressor/cytotoxic)cells. But Wuttke et al. [1993], found a higher level of

ACKNOWLEDGMENTSMN in T-cells than B-cells. In this study with PHA stimu-lation, the MN frequencies obtained are related mainly

We are grateful for the participation of the personnelto pan-T (T-lymphocytes). For this reason, it would befrom the Iztapalapa Pediatric Hospital (Mexico City).interesting to study MN frequencies in different subpopu-This work formed part of a collaborative project betweenlations of lymphocytes.the Universidad Autonoma Metropolitana and the GeneralThe increase in MN frequencies observed in this studyDirection of Medical Services (DDF). We are grateful tois supported by other studies, which have found increasedErnesto RodrıBguez Aguilera for his statistical work.frequencies of sister chromatid exchanges in lymphocytes

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