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Nutrition Research 29 (2009) 35–40
www.nrjournal.com
Protein-energy malnutrition contributes to increased structural
chromosomal alteration frequencies in Argentinean children
a,b,c, c a
Gisel Padula ⁎, Susana A. Salceda , Analia I. Seoane
a
IGEVET, Facultad de Ciencias Veterinarias, Universidad Nacional de La Plata-CONICET, Argentina
bIDIP (MS/CIC-PBA) Hospital de Niños de La Plata, Ministerio de Salud de la Provincia de Buenos Aires, Argentina
c
División Antropología, Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, Argentina
Received 12 May 2008; revised 8 September 2008; accepted 17 September 2008
Abstract
The relationship between protein-energy malnutrition and genetic damage has been studied in
human beings and laboratory animals, but results are still conflicting. The aim of the present study
was to assess the induction of structural chromosomal aberrations in peripheral blood lymphocytes
of children with primary protein-energy malnutrition. A case-control study was performed.
Samples were obtained from 25 primary malnourished infants (mean age, 22 months; range, 1-66
months). The control group consisted of 25 eutrophic children from the same population who were
matched 1:1 by age and sex. Anthropometric and clinic evaluations were performed to assess
nutritional condition. Before blood collection, we interviewed each individual's parent to complete
a semistructural survey specifying age, dietary habits, viral or bacterial diseases; previous exposure
to diagnostic x-rays; and use of therapeutic drugs. After 48 hours, 100 cultured lymphocytes were
analyzed per patient. Statistical analysis was performed using the Epi Dat 3.0 program (P ≤ .05).
The chromosomal aberration frequency was nearly 7 times higher in malnourished infants than in
controls (14.61% vs 2.2%, respectively). This difference was statistically significant (P b .001) and
may be explained by the occurrence of achromatic lesions, breaks, and telomeric associations.
DNA damage could be attributed to several factors: severe deficiency of essential nutrients (ie
zinc, iron, and vitamin A) required in the synthesis of DNA maintenances factors; deterioration of
repair mechanisms allowing the persistence of an unusually high number of structural
chromosomal aberrations; and/or the absence of specific factors needed to protect the cell against
oxidative DNA damage.
©2009 Elsevier Inc. All rights reserved.
Keywords: Protein-energy malnutrition; Chromosomal aberrations; Peripheral blood lymphocytes; Anthropometry; Child
Abbreviations: H/A, height for age index; MI, mitotic index; MUAC/A, mid upper arm circumference for age index; PEM,
protein-energy malnutrition; SCA, structural chromosome abnormality; SS/A, subscapular skinfold for age index;
TS/A, triceps skinfold for age index; W/A, weight for age index; W/H, weight for height index.
1. Introduction
Growth is defined as a continuous process resulting from
the complex interaction between inheritance and environ-
⁎ Corresponding author. Museo de La Plata, Universidad Nacional de ment. In this context, protein-energy malnutrition (PEM)
La Plata, Paseo del Bosque S/N - La Plata - B-1900-AVW. Tel.: +54 221 results from food insufficiency as well as from poor social
4257744x125; fax: +54 221 4257744x125. and economic conditions [1,2]. Malnutrition originates from
E-mail address: giselpadula@yahoo.com.ar (G. Padula). a cellular imbalance between nutrient/energy supply and the
0271-5317/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.nutres.2008.09.013
36 G. Padula et al. / Nutrition Research 29 (2009) 35–40
body's demand to ensure growth and maintenance [3]. The collection, we interviewed each individual's parent to
term protein-energy malnutrition applies to a group of complete a semistructural survey specifying age, dietary
related disorders that develop in children and adults whose habits, viral or bacterial diseases, previous exposure to
consumption of protein and energy (measured by energy) is diagnostic x-rays, and use of therapeutic drugs. All children
insufficient to satisfy the body's nutritional needs (primary were disease-free and had not been exposed to x-rays, drug
PEM). Malnutrition affects approximately one third of therapy, or viral infection for 1 month before the study. Also,
children worldwide [4]. It continues to be an important children with anemia or signs of vitamin deficiency were not
problem in developing countries. In this sense, the total included in this study. Specific written information about the
numberofunderweightandstuntedchildrenhasnotdropped aims of the study was provided to all participants. Written
significantly since 1980 [5]. The adequate nutritional support informed consent was obtained from the participants'
of severe PEM in infants represents a great challenge [6]. parents. The Directory of the Hospital Interzonal General
Manymicronutrient deficiencies were described in PEM, de Agudos y Crónicos Dr Alejandro Korn provided the
for example, zinc, iron, and vitamin A [5,7-9]. Such essential institutional review board approval.
micronutrients are required for the synthesis of DNA
maintenance factors and repair mechanisms. 2.2. Cytogenetic analysis
The relationship between PEM and genetic damage has
been studied in human beings and laboratory animals, but Heparinized venous blood samples were used to obtain
results are conflicting [10]. It has been found that children lymphocytes from the participants and set up 2 replicate
aged 1 to 60 months with severe PEM exhibit an increased cultures, using 1 mL total blood in 9 mL RPMI 1640
frequency of chromosomal aberrations (dicentrics chromo- (Gibco-Invitrogen, Buenos Aires, Argentina) containing
somes, gaps, isogaps, and breaks) in peripheral lymphocytes 1% phytohemagglutinin (Gibco-Invitrogen), 100 IU peni-
and bone marrow cell cultures, and abnormalities persist cillin, and 100 μg/mL streptomycin (Sigma, St Louis,
even after the children have attained normal height and Mo). The cultures were incubated at 37°C and 5% CO2 for
weight [11]. In addition, low-protein diets induce chromo- 48 hours. Two hours before harvesting, colchicine was
some breaks and deletions in bone marrow cells and added at a final concentration of 0.1 μg/mL. The cells
lymphocytes of rats and female mice [12,13]. On the other were harvested by centrifugation. Metaphases were ob-
hand, it is worth mentioning that other authors did not find
any difference between normal and malnourished children
regarding chromosomal aberrations [14]. Chromosomal Table 1
aberrations have been studied over a century, and their Age of subjects participating in the study
importance in human health has been recognized [15]. Sex Age (mo)
Cytogenetic analysis in peripheral blood lymphocytes has
beenwidelyusedformonitoringhumanpopulationsexposed PEMs Cs
to mutagenic agents [16]. Female 25 26
The purpose of this study was to determine whether 36 33
children with PEM without associated infections have a 910
higher frequency of structural chromosome abnormalities 22 26
12 10
(SCAs) than normal children. Because Argentina is a 15 16
developing country, it is important to asses whether PEM, 55
per se, is able to induce enhanced chromosomal aberrations 28 31
or whether this parameter correlates with some type or 7 7.5
degree of malnutrition. 13 12
48 49
14 14
19 19
2. Methods and materials 18 18
63 62
2.1. Experimental procedure 1.5 0.5
13 12
A case-control study was performed. The first group Male 20 25
consisted of 25 primary malnourished infants (PEM sample) 12 16
attending the Consultorio del Niño Sano of the Hospital 15 16
Interzonal de Agudos y Crónicos Dr Alejandro Korn, La 55
Plata, Argentina. Children were aged 1 to 60 months. The 43 44
55 60
control group included 25 healthy eutrophic infants from the 30 33
same population who were matched 1:1 by age and sex 16 17
(Table 1). Anthropometric and clinical evaluation was Malnourished (PEMs) and control (Cs) children matched for sex and age
performed to assess nutritional condition. Before blood (in months).
G. Padula et al. / Nutrition Research 29 (2009) 35–40 37
Table 2 than −1.1 was the cut-off point to determine the prevalence
Anthropometric evaluation of malnourished children using H/A, W/A, and of stunting, underweight, and wasting, respectively. Mal-
W/Hindexes nutrition degrees were established according to Torun and
Classificationa Sex Chew [19]. For children younger than 2 years, the H/A and
Female Male W/A, stunting, and underweight indicators were used. H/A
b2 y old and W/H, stunting and wasting indicators, modified from
Underweight 1 1 1 Waterlow et al [20], were applied for the others. These
Underweight 2 2 0 classifications include the following classes: (1) normal, W/
Underweight and stunted 1 2 1 Hadequate with normal stature; (2) stunting, W/H adequate
Underweight and stunted 2 5 1 with low stature; (3) wasting, W/H low with normal stature;
Underweight and stunted 3 2 2 (4) stunting and wasting, W/H low with low stature. The
N2 y old
Wasted 1 1 0 anthropometric classification of the malnourished children is
Wasted 2 1 0 shown in Tables 2 and 3.
Wasted and stunted 1 1 0
Wasted and stunted 2 2 0
Wasted and stunted 3 0 1 2.4. Statistical analysis
Stunted 1 0 1
Stunted 2 0 1 Statistical analysis was performed using the Epi Dat 3.0
Total 17 8 program [21]. The differences were considered significant if
a For children younger than 2 years, the H/A and W/A, stunting, and the probability values were less than .05. McNemar test was
underweight indicators were used. H/A and W/H, stunting, and wasting used to compare chromosomal aberration frequencies
indicators were modified from Waterlow et al [20] and applied to the between malnourished children and paired controls. The
subjects. Malnutrition was established according to Torun and Chew [19]. same procedure was used to compare previous exposure to
genotoxic agents and chromosomal aberration frequencies.
tained by routine protocols and stained with 5% Giemsa Oddsratiowasobtainedwhenpossible.WeusedthePearson
χ2 test to compare the frequencies of SCA for global
(Spectrum Chemicals, Gardena, Calif). Structural chromo-
some aberrations were scored in 100 metaphases per
individual by using a blind analysis: one investigator
numerically identified the samples and another scored the Table 4
aberrations. Only metaphases with 46 chromosomes were Structural chromosome aberrations in malnourished and control children
considered. The identification of SCA was carried out SCAs Primary malnourished Control
following the criteria recommended by the World Health children
Organization [17]. Mitotic index (MI) was calculated. Absolute Percent Absolute Percent
frequencies frequencies frequencies frequencies
2.3. Anthropometric evaluation Monochromatid 94 4.2 ± 0.20 17 0.73 ± 0.09
Height (H), weight (W), mid upper arm circumference gaps
(MUAC), triceps (TS), and subscapular skinfold (SS) were Isochromatid 22 0.96 ± 0.10 4 0.17 ± 0.04
gaps
measured.Theheightforageindex(H/A),theweightforage Monochromatid 83 3.61 ± 0.19 7 0.3 ± 0.05
index (W/A), the weight for height index (W/H), the mid breaks
upper arm circumference for age index (MUAC/A), the Isochromatid 10 0.43 ± 0.07 1 0.04 ± 0.02
triceps skinfold for age index (TS/A), and the subscapular breaks
skinfold for age index (SS/A) were calculated. Variables Fragments 57 2.48 ± 0.16 6 0.26 ± 0.05
Dicentric 9 0.58 ± 0.06 0 0
were introduced transformed into z scores using the NCHS chromosomes
anthropometric standards as reference [18].Az score of less Rings 0 0 0 0
Telomeric 61 2.65 ± 0.16 16 0.69 ± 0.08
associations
Table 3 Total SCA 336 14.61±0.35 51 2.2 ± 0.15
Anthropometric evaluation of malnourished children using MUAC/A, Total normal 1964 85.39±0.35 2269 97.8 ± 0.15
TS/A, and SS/A indexes Values are mean frequencies of 2 cultures ± SD. Global SCAs in
2 test was used to
Nutritional status Female Male malnourished (PEM) and control children. Pearson χ
compare SCA frequencies. Total chromosomal aberration frequency was
MUAC/A TS/A SS/A MUAC/A TS/A SS/A found to be nearly 7 times greater among malnourished infants compared to
Eutrophic 0 3 4 0 3 2 controls (14.61% vs 2.2% respectively, P b.001). Chromosomes rings were
Lowreserve 2 11 7 0 2 1 not found in any groups. All the others chromosome abnormality
Very low reserve 15 3 6 8 3 5 frequencies were significantly higher in the malnourished group compared
with the controls (P b .001 for mono- and isochromatid gaps, mono-
Eutrophic indicates values between Pc10 and Pc90; low reserve, value chromatid breaks, fragments, telomeric associations; P b .01 for isochro-
between Pc3 and Pc10; very low reserve, less than Pc3. matid breaks, dicentric chromosomes).
38 G. Padula et al. / Nutrition Research 29 (2009) 35–40
Table 5
Previous exposure to potential genotoxic agents (at least 1 month before
the study)
Sample Infections Last treatment X-raysa Pesticides
Bacterial Viral Antibiotics Antiparasitics
PEMs 4 9 6 8 20 8
Cs 3 6 7 1 21 7
Absolute frequencies for children exposure in the PEM sample (PEMs) and
control sample (Cs). McNemar test was used to compare previous exposure
with genotoxic agents and chromosomal aberration frequencies. Only the
last treatment with antiparasitic drugs was significantly higher in the
Fig. 1. Pared SCAs in malnourished (PEMs) and control (Cs) children. malnourished group compared with the controls (*P b.05).
a Diagnostic x-ray (one exposure).
McNemar test was used to compare chromosomal aberration frequencies
between paired control and malnourished children. Total chromosomal
aberration frequency was significantly higher in malnourished children from P b .01 for isochromatid breaks, dicentric chromosomes).
each pair (P b.05). This is valid for all types of chromosome abnormalities
except dicentric chromosomes (P b .001 for monochromatid gaps and Most of the abnormalities were gaps and breaks.
breaks, fragments; P b .01 for telomeric associations; P b .05 for Fig. 1 shows the results of the paired case-control
isochromatid gaps and breaks). analysis. According to this analysis, global chromosomal
aberration frequency was significantly higher in malnour-
frequencies. Fisher exact test was used for MI analysis ished children (P b .05). This is valid for all types of
and Spearman range for correlation analysis between chromosomal abnormalities except for dicentric chromo-
SCA frequencies with types and degrees of malnutrition, somes (P b .001 for monochromatid gaps and breaks,
sex, and age.
Table 6
3. Results Structural chromosome abnormality absolute frequencies for sex, age, type,
and degree of malnutrition
A total of 4620 metaphases were examined; 2300 SCA frequency
belonged to the malnourished group and 2320 to the chtg chrg chtb chrb ace dic tas
controls. Table 4 shows the results of the cytogenetic
analysis. Total chromosomal aberration rate was found to be Sex
nearly 7 times higher among infants with PEM compared Female 62 14 59 6 41 4 37
Male 32 8 24 4 16 5 24
with controls (14.61% vs 2.2%, respectively). This differ- Agea
ence was statistically significant (P b .001). Chromosome 192629231852385
rings were not found in any groups. All the other 2 100 33 100 50 83 17 83
chromosome abnormality frequencies were significantly 3 100 50 100 17 83 17 100
Typeb
higher in the malnourished group as compared with the Underweight 100 100 100 25 50 0 100
controls (P b .001 for mono- and isochromatid gaps, Wasted 100 0 100 0 100 0 100
monochromatid breaks, fragments, telomeric associations; Stunted 100 50 100 0 50 0 100
Underweight-stunted 100 50 100 50 100 25 100
Wasted-stunted 90 46 100 38 92 31 77
Degreesc
1 88 75 100 13 63 25 100
2 100 42 100 42 92 17 92
3 100 40 80 40 100⁎ 20 60⁎⁎
Spearmanrangewasusedforcorrelation analysis between SCA frequencies
with types and degrees of malnutrition, sex, and age. chtg, chrg, chtb, chrb,
ace, dic, and tas indicate monochromatide gaps, isochromatide gaps,
monochromatide breaks, isochromatide breaks, fragments, dicentric chro-
mosomes and telomeric association, respectively (Mitelman F. An
international system for human citogenetic nomenclature. Recommenda-
tions of the International Standing Committee on Human Genetic
Nomenclature. Karger; 1995).
a Malnourished children were grouped into 3 age intervals: 1—0to
Fig. 2. Mitotic index of malnourished (PEMs) and control (Cs) children. 17.99 months; 2—18 to 29.99 months; 3—older than 30 months.
Fisher exact test was used for comparison. Mitotic index was higher in b Classification of Waterlow et al [20].
lymphocytes from malnourished children than in those from healthy c Torun and Chew [19].
children, but statistical analysis revealed no differences between the 2 ⁎ Significant positive correlation (R = 0.3939, P = .05).
children populations. ⁎⁎ Significant negative correlation (R = −0.3981, P = .0488).
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