trimethoprim-sulfamethoxazole increase micronuclei formation in peripheral blood from weanling...

Research Article

Trimethoprim-Sulfamethoxazole IncreaseMicronucleiFormation in Peripheral Blood FromWeanling Well-

Nourished andMalnourished Rats

Roc|Ł o Ortiz,* Hilda Medina,y Edith CorteŁ s,Elsa Cervantes, and Leonor Rodr|Ł guez

Departamento de Ciencias de la Salud, Universidad AutonomaMetropolitana-Iztapalapa, Mexico DF, Mexico

The combination of trimethoprim and sulfamethox-azole (TMP-SMX) is a widely used drug. In spiteof this, there are few reports on its genotoxicity,and the results are controversial. Severe malnutri-tion is a complex condition that increases the sus-ceptibility to infections. Consequently, drugs areextensively used in malnutrition cases. Experimen-tal animal models have been widely used to studythe effects of malnutrition. Neonatal rats wereexperimentally malnourished (UN) during lacta-tion. The UN rats weighed 51.1% less than thewell-nourished (WN) controls and had lower con-centrations of serum protein and blood lipids. Themicronucleus (MN) assay is useful for detectingchromosome damage induced by nutritional defi-ciencies. In vivo rodent MN assays have beenwidely used to screen genotoxic agents. In thisstudy, we have evaluated the frequency of sponta-

neous and TMP-SMX-induced micronuclei in theperipheral blood of weanling (21 days of age)rats using a flow cytometric analysis technique.The spontaneous frequency of micronucleated re-ticulocytes (MN-RETs) was 2.7 times greater in theUN rats than in the WN rats. In rats that were nottreated with TMP-SMX, the percentage of reticulo-cytes was significantly lower (41.1%) in the UNrats than the WN controls. A therapeutic dose ofTMP-SMX (80 mg/kg (TMP), 400 mg/kg (SMX)for 48 hr) increased MN-RETs in the WN and inthe UN rats. The data demonstrate the genotoxiceffect of this drug. The results indicate that severeprotein-calorie restriction and drug treatmentenhance DNA damage in rat peripheral blood re-ticulocytes, potentially increasing the risk of nega-tive effects on health. Environ. Mol. Mutagen.52:673–680, 2011. VVC 2011 Wiley Periodicals, Inc.

Key words: reticulocytes; severe malnutrition; antibacterial drug; flow cytometry

INTRODUCTION

The combination of trimethoprim and sulfamethoxazole

(TMP-SMX) is widely used in treatment of bacterial

infections, because it has a broad spectrum of bactericidal

activity. TMP-SMX is used in the treatment of bacterial

infections including gastrointestinal tract infections, respi-

ratory tract infections, and, in particular, common urinary

tract infections. Both these drugs act synergistically and

have a pronounced antibacterial activity, as they sequen-

tially block two chemical reactions that are essential for

bacterial survival. This combination is effective against

organisms that are resistant to separate components. The

drugs are well-absorbed after oral administration, with

�95% bioavailability, reaching a peak plasma concentra-

tion in 1–4 hr [Mistri et al., 2010]. The use of TMP-SMX

has increased as a result of proven effectiveness in the

treatment of infections. Ease of administration, low cost,

and low incidence of side effects has also contributed to

its popularity.

Several studies on the effects of individual drugs, TMP

and SMX, at the genetic level have been conducted in

humans and experimental animals.

Studies on the genotoxicity of TMP were conducted in

vitro. Increases in sister chromatid exchange and micronu-

yIn memory of Hilda Medina who was an outstanding doctoral student.

This study was part of her postgraduate work. She remains in our labora-

tory and hearts.

Grant sponsor: National Council of Science and Technology (CON-

ACYT, Mexico); Grant Number 50804; Grant sponsor: Hilda Medina

and Elsa Cervantes (CONACYT, Mexico).

*Correspondence to: Rocıo Ortiz, Departamento de Ciencias de la Salud,

Universidad Autonoma Metropolitana-Iztapalapa, Mexico DF, Mexico.

E-mail: [email protected]

Received 28 November 2010; provisionally accepted 22 June 2011; and

in final form 23 June 2011

DOI 10.1002/em.20670

Published online 8 August 2011 in Wiley Online Library (wileyonlinelibrary.com).

VVC 2011Wiley Periodicals, Inc.

Environmental andMolecular Mutagenesis 52:673^680 (2011)

clei frequencies were seen after treatment with TMP in human

lymphocytes in vitro [Abou-Eisha et al., 1999]. In addition,

TMP-induced increases in micronuclei frequencies were

observed using the cytokinesis-block micronucleus (CBMN)

assay under in vitro conditions in rainbow trout gonads and

Chinese hamster ovary cell lines [Papis et al., 2011] was well

as in peripheral blood lymphocytes [Abou-Eisha, 2006].

Using the same method, small increases in sister chromatid

exchange and micronuclei were detected after treatment with

SMX, revealing genotoxicity [Abou-Eisha et al., 2004].

A limited number of manuscripts have assessed the gen-

otoxic effect of a combination of TMP-SMX. In one in

vivo study, the effect of TMP-SMX was studied in bone

marrow smears from adults (mean age 62 years) using

May–Grunwald–Giemsa staining; an increased number of

micronuclei in response to TMP-SMX treatment were

reported [Sørensen and Jensen, 1981]. Chromosomal aber-

rations in response to TMP-SMX treatment have been ana-

lyzed in human lymphocytes from adults (senior’s � 50

years) and from children, but no chromosomal damages

were detected [Stevenson et al., 1973; Gebhart, 1975].

Because of the insufficient information on the genotox-

icity of the TMP-SMX combination, we decided to pro-

vide additional genotoxicity data on the effect of TMP-

SMX in the induction of micronuclei.

Micronuclei are extranuclear chromatin resulting from

double-stranded DNA breaks or from mitotic spindle ap-

paratus dysfunction [Krishna and Hayashi, 2000]. The

MN assay is useful in detecting chromosome damage

induced by exposure to chemical agents, exposure to

physical agents, age-related alterations, and damage pro-

duced by nutritional habits [Fenech, 1993].

In vivo rodent MN assays have been widely used to

screen agents for clastogenic and aneugenic activity. Most

rodent MN assays measure micronuclei in bone marrow

or peripheral blood erythrocytes, where the visualization

of micronuclei is facilitated by the absence of a main nu-

cleus [Krishna and Hayashi, 2000].

As a measure of genotoxicity, the MN assay has advan-

tages over other methods. Unlike sister chromatid

exchange, the biological significance of micronuclei is

clear. The MN assay is devised for assessing the capacity

of test agents to induce structural and/or numerical chro-

mosomal damage. Both kinds of damage are associated

with the appearance and/or progression of tumors and

with adverse reproductive and developmental outcomes

[Krishna and Hayashi, 2000].

Moreover, micronuclei are easier to score than struc-

tural chromosome aberrations. In addition, flow cytome-

try-based methodologies have considerably improved the

speed and accuracy by which the MN assay can be per-

formed with erythrocytes [Torous et al., 2003; Cammerer

et al., 2007; Hayashi et al., 2007].

Protein-energy malnutrition is a major public health

and clinical problem in pediatric practices, and it accounts

for high child mortality and morbidity [Pelletier and Fron-

gillo, 2003; Victora et al., 2010]. Malnourished children

are immunodeficient and, therefore, are more susceptible

to infections compared to well-nourished (WN) children

[Schaible and Kaufmann, 2007; Rodrıguez et al., 2011].

Experimental animal models using rats and mice have

been widely used to study the effects of malnutrition [Alu

and Murthy, 1993]. Several studies have demonstrated the

importance of the lactation period, which is critical in the

process of growth and development [Flo et al., 1993].

Severe malnutrition is a complex condition in which

many deficiencies simultaneously occur that can alter

pharmacokinetic processes, drug responses, and increase

toxicity. It is well-recognized that diet and nutritional sta-

tus are two important variables that can alter drug metab-

olism and disposition [Krishnaswamy, 1989]. However,

malnourished (UN) children often have several concomi-

tant diseases. Therefore, drugs are more widely used in

UN children than in WN children. Consequently, it is par-

ticularly important to study the adverse effects, specifi-

cally genotoxic effects, in UN children.

The purpose of this study was to assess the effect of

TMP-SMX treatment on the frequency of micronuclei in

reticulocytes (RETs) in peripheral blood from young (21-

day-old) WN rats and rats that were experimentally UN

during the lactation period.

MATERIALS ANDMETHODS

Animals

Wistar rats were housed under a 12-hr light–dark cycle at 22–258Cand 45% relative humidity. Females that had two previous litters were

bred in acrylic boxes with a bedding of betachips (Northeastern Prod-

ucts, Warrensburg, NY). The nursing mothers were fed with a balanced

diet for rodents (Purina Mills International 5001, Richmond, VA) and fil-

tered water ad libitum.

Experimental Malnutrition and Treatment Regimen

Two methods have been used to induce malnutrition during lactation

in experimental animals. We used the method based on reducing the

quantity of milk per pup. In this method, malnutrition is induced by

increasing the number of pups per nursing mother [Ortiz et al., 1996;

Houdijk et al., 2003]. Data obtained in our laboratory showed that food

competition during lactation was a highly reproducible and efficient

method for inducing malnutrition [Ortiz et al., 1996].

The experiments were performed according to the guidelines for the

use of experimental animals of the Autonomous Metropolitan University

of Iztapalapa, which were in accordance with those approved by the

National Institutes of Health (Bethesda, MD). One-day-old Wistar rats

from different litters were randomly assigned to two groups. In the WN

group, nursing mothers were each given seven pups. In the UN group,

each nursing mother fed 15 pups. In the latter group, malnutrition was

produced in the nursing pups due to food competition [Ortiz et al.,

1996].

Rats were weaned at 21 days after birth from five different litters. At

weaning, randomly selected WN and UN male rats were treated with

single i.p. doses of TMP-SMX at levels close to the therapeutic range.

Environmental and Molecular Mutagenesis. DOI 10.1002/em

674 Ortiz et al.

Physical and Biochemical Measurements of Malnutrition

Rats were weighed throughout the nursing period (1–21 days after

birth). Animals that weighed less than 40% of WN controls at weaning

were considered severely UN. Moreover, total serum proteins were quan-

tified in 14 UN and 15 WN control rats using the method previously

described by Lowry et al. [1951]. Serum cholesterol and triglyceride lev-

els were measured in 14 UN and 15 WN rats using the Reflotron Plus

System (Roche Diagnostics, Basel, Switzerland). In addition, general

observations were made for other signs of malnutrition, such as activity

level and hair texture. These signs of malnutrition are similar to those

observed in severely UN children.

Selection of Dose

For determining the minimal dose that induced MN in RETs, rats

were injected intraperitoneally (i.p.) with the drug combination of TMP-

SMX (three rats per dose). The doses of TMP-SMX tested in the present

study were between therapeutic doses ranging from 18 mg/kg (TMP), 40

mg/kg (SMX); 80 mg/kg (TMP), 400 mg/kg (SMX); and 160 mg/kg

(TMP), 800 mg/kg (SMX). TMP-SMX was dissolved in bidistilled water

and injected i.p. in a volume of 75 ll/rat. Blood was collected 48 hr af-

ter dosing. Animals administered the vehicle alone served as the negative

control group. Three rats treated with single i.p. dose of 0.75, mg/kg of

water-soluble mitomycin C (MMC; Sigma, St. Louis, MO) administered

in saline work for positive control group (MN in RET; 3.4-fold

increase), similar to previous determined in the laboratory [Ortiz et al.,

2004].

TMP-SMX was obtained as Bactrim from Syntex (Mexico, DF), made

in Mexico for Roche products.

Treatment With Selected Dose

The dose selected for a single i.p. administration of TMP-SMX [80

mg/kg (TMP)2400 mg/kg (SMX) body weight] was half of the maximal

therapeutic oral dose for children. Five WN and five UN rats were used

for each group. Negative control rats were injected with an equal volume

(75 ll/rat) of bidistilled water (seven WN and five UN). After adminis-

tration of the drug, the animals were sacrificed at 48 hr.

Blood Sample Preparation

After administration of the drugs, �300 ll of blood was collected

from each animal by cardiopuncture with a syringe containing 100 ll ofanticoagulant solution (250 U/ml of heparin). The blood samples were

maintained at room temperature for no more than 2 hr. For fixation, the

samples were diluted 1:2 with bicarbonate-buffered saline (BBS) and

added to tubes containing ultracold (2708C) methanol. The fixed blood

samples were stored at 2708C until analysis. On the day of analysis, the

blood samples were simultaneously treated with 1 mg of RNase/ml and

anti-CD71-FITC as described by Torous et al. [2000]. For the current

experiments, a fluorescein isothiocyanate-conjugated antibody for rat

transferrin receptor (anti-CD71-FITC; Serotec, UK) was used to differen-

tially label and score cells with a high-transferrin receptor content (i.e.,

the youngest fraction of RETs). RETs also have high RNA content, and

the degradation of RETs RNA was performed to allow detection of

DNA (micronuclei)-specific fluorescence (from propidium iodide stain-

ing) [Dertinger et al., 1996]. The cells were incubated at 48C for 30 min

followed by incubation at 228C for 90 min to ensure complete degrada-

tion of cellular RNA. The cells were then kept at 48C until analysis. Im-

mediately before analysis, 1 ml of ice-cold propidium iodide staining so-

lution (1.25 lg/ml BBS) was added to each sample.

Flow Cytometry Analysis

Flow cytometry analysis was performed with a FACScalibur flow cy-

tometer (Becton Dickinson, Immunocytometry Systems, San Jose, CA)

with an excitation at 488 nm. CellQuest software (version 3.0.1, Becton

Dickinson) was used for data acquisition and analysis. Cells were ana-

lyzed at an average rate of 8,000 erythrocytes/s. Red blood cells (RBCs)

were isolated by gating on the forward light scatter and side light scatter

parameters. Electronic compensation was used to eliminate the longer-

wavelength emissions of the FITC signals. Moreover, FL2-FL1 and FL1-

FL2 compensations were set to optimally resolve the MN-RET popula-

tion [Torous et al., 1998]. To analyze the data, we first identified the

cells of interest by graphing the ‘‘forward scatter’’ against the ‘‘side

scatter.’’ Subsequently, in a plot of FL1 versus FL2, we analyzed the

DNA content to identify and exclude the nucleated cells. In the analysis

of the bivariate graph for the resolution of the various erythrocyte popu-

lations, special attention was paid to the quadrant position; in general, a

negative control was used.

The proportion of RETs in a total of 500,000 RBCs was established

by determining the proportion of RBCs with high CD71 content, and the

frequency of MN-RETs was determined by analyzing �30,000 RETs

[Torous et al., 2001]. The percentages of RETs and MN-RETs were cal-

culated according to the Micronucleus Frequency Spreadsheet from

Litron Laboratories (Rochester, NY).

Statistical Analysis

The mean weights of WN and UN rats were compared using a two-

tailed Student’s t-test. A one-way analysis of variance (ANOVA) was

performed on serum parameters, and a multiple comparison with the

controls was performed by the Dunnett test.

For the RET and MN data, statistical evaluation was done with the

NCSS software package (MultiON, Mexico, DF, 2007). Levene’s test for

homogeneity of variances was performed. If variances were not signifi-

cantly heterogeneous, an ANOVA was then performed. If the ANOVA

detected significant differences between groups, a multiple comparison

with the controls was performed by the Dunnett test. The significance

level used was P < 0.05.

RESULTS

The average body weight of the rats 1 day after birth

was similar in both groups (7.7 g 6 0.25 g). At weaning

(21 days after birth), the average body weight was 25.4 g

6 2.8 g for UN rats and 54.2 g 6 2.5 g for WN rats. UN

rats weighed 50% less than the WN rats, implying severe

protein-calorie malnutrition. Table I shows the average

body weight, serum proteins, triglycerides, and cholesterol


TABLE I. Body Weight, Serum Proteins, and Blood Lipid Con-centration of Well-Nourished and Malnourished Rats

Well-nourished Malnourished

Body weight (g) 54.2 6 2.5 25.4 6 2.8*

Body weight deficit (%) – 51.1 6 5.3

Serum proteins (mg/ml) 72.7 6 3.2 47.7 6 2.5*

Triglycerides (mg/dl) 297.3 6 27.9 182.9 6 5.5*

Cholesterol (mg/dl) 130.3 6 7.4 110.0 6 1.4*

Data are means 6 SE. Fifteen well-nourished rats and fourteen malnour-

ished rats were used. The cholesterol concentration was less than 100

mg/dl in 9 of the 14 malnourished rats.*Statistical differences from well-nourished group at P < 0.05.

TMP-SMX Increase Micronuclei Formation 675

concentrations in both groups of rats at weaning. UN rats

had significantly decreased concentrations of serum pro-

teins, triglycerides, and cholesterol. Physical observations

of the UN rats indicated that they had sparse hair growth

and sluggish movements.

TMP-SMXDose-Response

The trimethoprim and sulfamethoxazole (TMP-SMX)

treatment produced dose-related increases in the MN-RET

frequency. The MN-RET frequency was significantly

higher in WN rats treated with 160 mg/kg (TMP)–800

mg/kg (SMX) (1.87%) and 80 mg/kg (TMP)–400 mg/kg

(SMX) (1.27%) doses of TMP-SMX compared to control

rats (0.22%). No statistical difference was observed with

the 18 mg/kg (TMP)–40 mg/kg (SMX) dose (0.52%)

(data not shown).

For the analysis of RETs, MN-RETs frequencies in

WN rats compared to UN rats, the 80 mg/kg (TMP)2400

mg/kg (SMX) dose at 48 hr was selected, because it

induced MN-RETs and was half of the therapeutic dose.

Table II shows the percentages of RETs and the fre-

quencies of spontaneous and TMP-SMX-induced MN-

RETs in the peripheral blood of WN and UN rats.

Reticulocytes Frequency in Peripheral Blood FromWNandUN Rats TreatedWith TMP-SMX

The UN rats without treatment (9.3%) had a signifi-

cantly lower percentage of RETs than WN rats without

treatment (15.8%) (Table II). The RETs frequency ranged

from 11.2 to 19.1% among WN rats and 8.2–13.1%

among UN animals (Fig. 1a). No significant differences

were found for RETs percentages between the control

group and the trimethoprim and sulfamethoxazole (TMP-

SMX) group (Fig. 1b).

Micronucleated Reticulocytes Frequency in PeripheralBlood FromWNand UN Rats TreatedWith TMP-SMX

The micronucleated reticulocytes (MN-RETs) frequen-

cies in WN and UN rats with and without drug adminis-

tration are shown in Table II. The frequency of spontane-

ous MN-RET in the group of UN rats was significantly

higher (0.90%; range, 0.45–1.37%,) than the spontaneous

MN-RET frequency in WN rats (0.33%; range,

0.19–0.46%, P < 0.05) (Fig. 2a).

The MN-RET frequency was significantly lower in WN

rats without treatment (0.33%) than in WN rats that were

treated with trimethoprim and sulfamethoxazole (TMP-

SMX) (1.28%; range, 1.09–1.50%, P < 0.05) (Fig. 2a).

The MN-RET frequency was higher in UN rats treated

with TMP-SMX (1.79%; range, 1.65–2.03%) compared to

UN rats without treatment (0.90%, P < 0.05) (Fig. 2a).

The MN-RET frequency was significantly higher in UN

rats treated with TMP-SMX for 48 hr (1.79%) than in

WN rats treated with TMP-SMX (1.28%; P < 0.05), with


TABLE II. Effect of TMP-SMX on Frequencies of Reticulocytesand Micronucleated Reticulocytes in Peripheral Blood FromWell-Nourished and Malnourished Rats

Treatmeant

dose (mg/kg)

Well-nourished (WN) Malnourished (UN)

% RET % MN-RET % RET % MN-RET

0 19.07 0.33 13.05 0.91

18.68 0.26 8.37 0.45

16.42 0.36 8.56 1.37

11.19 0.36 8.30 1.04

15.31 0.46 8.17 0.73

14.50 0.35

15.28 0.19

Mean 15.78 0.33 9.29 0.90

SE 1.01 0.03 0.94 0.15

TMP-SMX 19.64 1.09 15.23 2.03

80-400 18.89 1.50 18.27 1.76

18.96 1.19 17.98 1.65

16.20 1.37 4.52 1.73

7.20 1.24 6.3 1.78

Mean 16.18 1.28 12.46 1.79

SE 2.32 0.07 2.94 0.06

Fig.1. Effect of TMP-SMX on the frequency of RETs in well-nourished

(WN) and malnourished (UN) rats. Data are based on flow cytometric

analysis of 500,000 events and are means 6 SE. (a) Spontaneous RETs

frequency. *P < 0.05 when WN rats were compared to UN rats without

treatment (7 WN and 5 UN rats without treatment). (b) RETs frequency

after TMP-SMX administration (80–400 mg/kg at 48 hr) (5 WN and 5

UN rats per treatment). RETs, reticulocytes.

676 Ortiz et al.

higher levels of MN-RETs observed in the five UN rats

(Fig. 2b). Nevertheless, by subtracting the spontaneous

MN frequency, it is evident that the observed difference

is due to the difference in baseline MN frequencies.

DISCUSSION

Malnourished children are more susceptible to bacterial

infections. The combination of TMP-SMX is widely used

in the treatment of these infections. This drug combina-

tion has several advantages; these include low cost and

effective treatment of infections, including those caused

by organisms that are resistant to the TMP and SMX indi-

vidually. Nevertheless, there is insufficient information on

the genotoxicity of TMP-SMX, and, therefore, further

studies are required to increase the knowledge about this

effect.

Numerous studies on the effects of malnutrition at the

cytogenetic level have been carried out in humans and ex-

perimental animals. Laboratory animals have been used

for both in vivo and in vitro experiments to study the

effects of malnutrition at different levels, because extra

nutritional factors can be controlled. Experimental malnu-

trition studies are complementary to those in humans, and

they provide a better understanding of the disease [Mala-

faia and Talvani, 2011]. The effect of malnutrition during

the lactation period was evaluated in this study. It is well

established that lactation is critical in the process of

growth and development. Several studies have established

that during this period, different tissues are highly vulner-

able. Using the alkaline single-cell electrophoresis assay,

increased levels of DNA damage in spleen, peripheral

blood, and bone marrow cells were observed in UN rats

at weaning (21 days of age) [Cortes et al., 2001].

In the present study, we have taken advantage of flow

cytometry-based MN analysis [Torous et al., 2000] to

determine the effect of experimentally induced malnutri-

tion on the spontaneous and TMP-SMX-induced MN fre-

quency in RETs in the peripheral blood of young rats.

The sensitivity of the in vivo micronuclei test increases

with increasing numbers of scored cells. The high-

throughput nature of flow cytometric scoring enables the

analysis of many RETs per specimen (tens of thousands

of RETs), which allows good reproducibility and rapid

results compared to manual scoring. In addition to the an-

alytical advantages of flow cytometry, the ready accessi-

bility of the small (microliter quantity) blood samples

required for the flow assay allow the integration of the

MN frequency assessment into toxicology studies. There-

fore, flow cytometry-based methodologies have consider-

ably improved the speed and accuracy by which the MN

assay can be performed with erythrocytes. Moreover, the

biological significance of micronuclei is clear, and micro-

nuclei are easy to score.

Mice are the rodent species most frequently used for

the MN assay. However, rats are the standard species

used in toxicology and toxicokinetic studies [Cammerer

et al., 2007]. Several studies indicate that the rat periph-

eral blood MN test represents a sensitive system for eval-

uating genotoxic effects of compounds [Torous et al.,

2003; MacGregor et al., 2006; Cammerer et al., 2007;

Hayashi et al., 2007].

Malnutrition has been classified as mild, moderate, or

severe, according to the degree of weight deficit or altered

weight: (1) mild or first degree, where the weight deficit

was 10–24% of that of age-matched control rats; (2) mod-

erate or second degree, where the weight deficit was 25–

40%; and (3) severe or third degree where the weight def-

icit was greater than 40% of that of age-matched control

rats (WN). In this work, only severely UN rats were stud-

ied. Therefore, in future studies, an analysis of the three

degrees of malnutrition can be conducted to identify pos-

sible malnutrition degree-related effects. It is important to

study the effect of mild and moderate malnutrition,


Fig. 2. Effect of TMP-SMX on the frequency of MN-RET in well-nour-

ished (WN) and malnourished (UN) rats. Data are based on flow cyto-

metric analysis of 500,000 events and are means 6 SE. (a) Spontaneous

MN-RET frequency. *P < 0.05 when WN rats were compared with WN

rats treated with TMP-SMX; 1P < 0.05 when spontaneous MN-RET fre-

quency in UN rats was compared to the frequency in UN rats treated

with TMP-SMX, #P < 0.05 when spontaneous MN-RET in WN was

compared to spontaneous UN rats (7 WN and 5 UN rats without treat-

ment). (b) MN-RET frequency after TMP-SMX administration (80–400

mg/kg at 48 hr). #P < 0.05 when UN-TMP-SMX rats were compared to

WN-TMP-SMX rats (5 WN and 5 UN rats per treatment). MN-RETs,

micronucleated reticulocytes.


because the prevalence of these degrees of malnutrition is

higher than severe malnutrition. In addition, poor immu-

nological response has been observed in moderate malnu-

trition [Cortes-Barberena et al., 2008].

The results of this study showed that UN rats without

treatment have a significantly lower percentage (41.1%)

of RETs compared to WN rats without treatment. This

reduction of RET frequency in the blood of UN animals

may be considered an indication of bone marrow failure,

implying the death of erythroblasts or a delay in their di-

vision. Previously, it has been reported that severe malnu-

trition affects the proportion of proliferating cells in the

bone marrow and their cell kinetics [Betancourt et al.,

1992].

Considerable variability in the percentage of RETs in

the peripheral blood of rats has been reported in previous

studies [Ortiz et al., 2004]. The relatively high level of

RETs in both groups of rats in our study may be related

to their young age (21-day-old or 3-week-old rats). This

was in agreement with previous reports indicating that the

RET frequency was highest in the 3-week-old rats and

decreased significantly from 3-week-old rats to 13-week-

old rats. These data suggest that the reduction of erythro-

poiesis with age in rats is more evident than in mice

[Hamada et al., 2003].

In the present study, we demonstrated that severe mal-

nutrition was associated with increased DNA damage in

rat peripheral blood RETs. The average spontaneous MN-

RET frequency was 2.7 times greater in UN rats than in

WN rats. The increases in MN-RETs observed in UN rats

were in agreement with previously reported results that

measured MN frequency and found that lymphocytes

from UN children have increased MN frequencies in

binucleate cells [Ortiz et al., 1997]. Another study has

shown that spleen lymphocytes from UN rats during lac-

tation have increased MN frequencies [Ortiz et al., 1995].

Furthermore, protein-calorie malnutrition experimentally

produced during lactation increases the frequencies of

spontaneous and MMC-induced MN-RETs in rat periph-

eral blood [Ortiz et al., 2004].

Several mechanisms have been proposed to account for

the induction of cellular, cytogenetic, and DNA damage

by malnutrition. The damage observed may be due to the

deficiency of several essential nutrients required for pro-

tein synthesis that are associated with DNA integrity,

impaired DNA repair mechanisms, or unavailability of

molecules necessary to protect cells against DNA oxida-

tive damage [Cortes et al., 2001].

Furthermore, the essential role of vitamins and minerals

in various DNA maintenance reactions and the relevance

of optimal micronutrient intake for the prevention of

genomic instability have been established [Fenech, 2002].

Several studies have shown that folate and vitamin B12

are required by DNA for the prevention of DNA damage.

DNA lesions may be induced by an inadequate intake of

these antimutagenic vitamins [Fenech, 2002]. In addition,

there is evidence that dietary folate deficiency enhances

the ability of arsenic to induce micronuclei in peripheral

blood of mice [McDorman et al., 2002]. In addition, pro-

tection from the induction of micronucleated erythrocytes

in peripheral blood of newborn Wistar rats born to cyclo-

phosphamide-treated mothers by folate supplementation

has been reported [Gomez-Meda et al., 2004].

Further studies will be necessary to address the rela-

tionship between levels of specific micronutrients that

may be required for genome stability, DNA repair, and

susceptibility to MN induction.

The present study showed that therapeutic doses of

TMP-SMX [80 mg/kg (TMP)2400 mg/kg (SMX) for 48

hr] induced a 3.9-fold increase in MN-RETs in WN rats.

The increases in MN-RETs observed in WN rats treated

with TMP-SMX were in agreement with previous reports

that demonstrated the significant clastogenic potential of

SMX [Abou-Eisha et al., 2004] and TMP, in cultured pe-

ripheral blood lymphocytes where increased levels of

DNA breakage and MN frequencies were detected [Abou-

Eisha, 2006].

Moreover, the data showed that UN rats treated with

TMP-SMX had a twofold increase in MN-RETs when

compared with untreated UN animals. In addition, the

MN-RET frequency in UN rats treated with TMP-SMX

was significantly higher than in WN rats also treated with

TMP-SMX, with a higher level of MN-RETs observed in

all the UN rats that were evaluated. Although the MN fre-

quency after treatment with TMP-SMX appears higher in

the UN than the WN rats, this is an artifact of the higher

baseline in the UN rats due to malnutrition. The induced

differences are not significantly different.

In this study, only one dose and time were tested.

Additional studies using consecutive treatments and dif-

ferent doses are required to supplement the knowledge

about the genotoxic effects of TMP-SMX. However, the

data obtained with the conditions used in this study dem-

onstrated the importance of evaluating the genotoxic

effect of TMP-SMX.

The data suggest that the peripheral blood MN flow

cytometry test in young rats can be used for the assess-

ment of MN induction by drugs and malnutrition. Com-

plementary studies will be necessary to analyze the effect

of other widely used drugs in severely UN children.

CONCLUSIONS

The data from this study show that severe protein-calo-

rie malnutrition that was experimentally produced during

lactation results in increased spontaneous MN frequencies

in rat peripheral blood. These data further support the pre-

mise of the greater susceptibility of cells from UN ani-

mals to chromosome damage.


678 Ortiz et al.

Furthermore, the data presented here show that the

administration of a therapeutic dose of TMP-SMX results

in increased MN frequencies in the peripheral blood of

both young, healthy rats, and UN rats. The data illustrate

the genotoxic effect of this drug. This chromosomal dam-

age increases the risk of negative effects on health and

may produce negative effects on further development of

the organism.

REFERENCES

Abou-Eisha A. 2006. Evaluation of cytogenetic and DNA damage

induced by the antibacterial drug, trimethoprim. Toxicol In Vitro

20:601–607.

Abou-Eisha A, Creus A, Marcos R. 1999. Genotoxic evaluation of the

antimicrobial drug, trimethoprim, in cultured human lymphocytes.

Mutat Res 440:157–162.

Abou-Eisha A, Marcos R, Creus A. 2004. Genotoxicity studies on the

antimicrobial drug sulfamethoxazole in cultured human lympho-

cytes. Mutat Res 564:51–56.

Alu V, Murthy PB. 1993. Chromosomal abnormalities in starved and

marginally malnourished rats and in utero upon rehabilitation.

Experientia 49:258–262.

Betancourt M, Ortiz R, Gonzalez C. 1992. Proliferation index in bone

marrow cells from severely malnourished rats during lactation.

Mutat Res 283:173–177.

Cammerer Z, Elhajouji A, Suter W. 2007. In vivo micronucleus test with

flow cytometry after acute and chronic exposures of rats to chem-

icals. Mutat Res 626:26–33.

Cortes E, Gonzalez C, Betancourt M, Ortiz R. 2001. Assessment of

DNA damage in spleen, bone marrow, and peripheral blood from

malnourished rats by single cell gel electrophoresis assay. Teratog

Carcinog Mutagen 21:231–247.

Cortes-Barberena E, Gonzalez-Marquez H, Gomez-Olivares JL, Ortiz-

Muniz R. 2008. Effects of moderate and severe malnutrition in

rats on splenic T lymphocyte subsets and activation assessed by

flow cytometry. Clin Exp Immunol 152:585–592.

Dertinger SD, Torous DK, Tometsko KR. 1996. Simple and reliable enu-

meration of micronucleated reticulocytes with a single-laser flow

cytometer. Mutat Res 371:283–292.

Fenech M. 1993. The cytokinesis-block micronucleus technique: A

detailed description of the method and its application to genotox-

icity studies in human populations. Mutat Res 285:35–44.

Fenech M. 2002. Micronutrients and genomic stability: A new paradigm

for recommended dietary allowances (RDAs). Food Chem Toxi-

col 40:1113–1117.

Flo J, Massouh EJ, Roux ME. 1993. Long term effects of malnutrition

during lactation on GALT. Reg Immunol 5:100–105.

Gebhart E. 1975. Chromosome studies in children treated with bactrim

[(author’s transl) in German]. Z Kinderheilkd 119:47–52.

Gomez-Meda BC, Zuniga-Gonzalez GM, Zamora-Perez A, Ramos-Ibarra

ML, Batista-Gonzalez CM, Torres-Mendoza BM. 2004. Folate

supplementation of cyclophosphamide-treated mothers diminishes

micronucleated erythrocytes in peripheral blood of newborn rats.

Environ Mol Mutag 44:174–178.

Hamada S, Nakajima K, Serikawa T, Hayashi M. 2003. The effect of

aging on the results of the rat micronucleus assay. Mutagen

18:273–275.

Hayashi M, MacGregor JT, Gatehouse DG, Blakey DH, Dertinger SD,

Abramsson-Zetterberg L, Krishna G, Morita T, Russo A, Asano

N, Suzuki H, Ohyama W, Gibson D;In Vivo Micronucleus Assay

Working Group, IWGT. 2007. In vivo erythrocyte micronucleus

assay III. Validation and regulatory acceptance of automated

scoring and the use of rat peripheral blood reticulocytes, with dis-

cussion of non-hematopoietic target cells and a single dose-level

limit test. Mutat Res 627:10–30.

Houdijk ME, Engelbregt MT, Popp-Snijders C, Delemarre van der Waal

HA. 2003. Long-term effects of early postnatal food restriction

on growth hormone secretion in rats. J Parent Enteral Nutr

27:260–267.

Krishna G, Hayashi M. 2000. In vivo rodent micronucleus assay: Proto-

col, conduct and data interpretation. Mutat Res 455:155–166.

Krishnaswamy K. 1989. Drug metabolism and pharmacokinetics in

malnourished children. Clinical Pharmacokinetics 17 (Suppl 1):

68–88.

Lowry OH, Rosebrough JN, Farr AL, Randall RJ. 1951. Protein meas-

urements with the Folin reagent. J Biol Chem 193:265–275.

MacGregor JT, Bishop ME, McNamee JP, Hayashi M, Asano N, Wakata

A, Nakajima M, Saito J, Aidoo A, Moore MM, Dertinger SD.

2006. Flow cytometric analysis of micronuclei in peripheral blood

reticulocytes. II. An efficient method of monitoring chromosomal

damage in the rat. Toxicol Sci 94:92–107.

McDorman EW, Collins BW, Allen JW. 2002. Dietary folate deficiency

enhances induction of micronuclei by arsenic in mice. Environ

Mol Mutag 40:71–77.

Malafaia G, Talvani A. 2011. Nutritional status driving infection by

Trypanosoma cruzi: Lessons from experimental animals. J Trop

Med 2011:981879.

Mistri HN, Jangid AG, Padage A, Shah A, Shrivastav PS. 2010. Simulta-

neous determination of sulfamethoxazole and trimethoprim in

microgram quantities from low plasma volume by liquid chroma-

tography-tandem mass spectrometry. Microchem J 94:130–138.

Ortiz R, Cortes E, Gonzalez C, Perez L, Betancourt M. 1995. Micronu-

cleus frequency in spleen lymphocytes from severely malnour-

ished rats during lactation. Environ Mol Mutag 26:55–59.

Ortiz R, Cortes E, Gonzalez C, Perez L, Betancourt M. 1996. Assess-

ment of an experimental method to induce malnutrition by food

competition during lactation. Med Sci Res 24:843–846.

Ortiz R, Cortes L, Gonzalez C, Lopez L, Perez P, Cortes E, Betancourt

M. 1997. Analysis of mitomycin C-induced micronuclei in lym-

phocytes from malnourished infected children. Environ Mol

Mutag 30:363–370.

Ortiz R, Medina H, Rodrıguez L, Gonzalez-Marquez H, Cortes E. 2004.

Spontaneous and mitomycin C-induced micronuclei in peripheral

blood reticulocytes from severely malnourished rats. Environ Mol

Mutag 43:179–185.

Papis E, Davies SJ, Jha AN. 2011. Relative sensitivity of fish and mam-

malian cells to the antibiotic, trimethoprim: Cytotoxic and geno-

toxic responses as determined by neutral red retention, Comet

and micronucleus assays. Ecotoxicology 20:208–217.

Pelletier DL, Frongillo EA. 2003. Changes in child survival are strongly

associated with changes in malnutrition in developing countries.

J Nutr 133:107–119.

Rodrıguez L, Cervantes E, Ortiz R. 2011. Malnutrition and gastrointesti-

nal and respiratory infections in children: A public health prob-

lem. Int J Environ Res Public Health 8:1174–1205.

Schaible UE, Kaufmann SHE. 2007. Malnutrition and infection: Com-

plex mechanisms and global impacts. PLoS Med 4:806–812.

Sørensen PJ, Jensen MK. 1981. Cytogenetic studies in patients treated

with trimethoprim-sulfamethoxazole. Mutat Res 89:91–94.

Stevenson AC, Clarke G, Patel CR, Hughes DT. 1973. Chromosomal

studies in vivo and in vitro of trimethoprim and sulphamethoxa-

zole (co-trimoxazole). Mutat Res 17:255–260.

Torous DK, Dertinger SD, Hall NE, Tometsko CR. 1998. An automated

method for discriminating aneugen- vs. clastogen-induced micro-

nuclei. Environ Mol Mutag 31:340–344.

Torous DK, Dertinger SD, Hall NE, Tometsko CR. 2000. Enumeration

of micronucleated reticulocytes in rat peripheral blood: A flow

cytometric study. Mutat Res 465:91–99.



Torous DK, Hall NE, Dertinger SD, Diehl MS, Illi-Love AH, Ceder-

brant K, Sandelin K, Bolcsfoldi G, Ferguson LR, Pearson A,

Majeska JB, Tarca JP, Hewish DR, Doughty L, Fenech M,

Weaver JL, Broud DD, Gatehouse DG, Hynes GM, Kwanyuen

P, McLean J, McNamee JP, Parenteau M, Van Hoof V, Vanp-

arys P, Lenarczyk M, Siennicka J, Litwinska B, Slowikowska

MG, Harbach PR, Johnson CW, Zhao S, Aaron CS, Lynch

AM, Marshall IC, Rodgers B, Tometsko CR. 2001. Flow

cytometric enumeration of micronucleated reticulocytes: High

transferability among 14 laboratories. Environ Mol Mutagen

38:59–68.

Torous DK, Hall NE, Murante FG, Gleason SE, Tometsko CR, Dertinger

SD. 2003. Comparative scoring of micronucleated reticulocytes in

rat peripheral blood by flow cytometry and microscopy. Toxicol

Sci 74:309–314.

Victora CG, de Onis M, Hallal PC, Blossner M, Shrimpton R. 2010.

Worldwide timing of growth faltering: Revisiting implications for

interventions. Pediatrics 125:e473–480.

Accepted by—A. Kligerman


680 Ortiz et al.

trimethoprim-sulfamethoxazole increase micronuclei formation in peripheral blood from weanling...

Documents