Salud

Artículo arbitrado

Cypermethrin, deltamethrin and

glyphosate affect the activity of the

Ca -ATPase from human erythrocyte

Cipermetrina, deltametrina y glifosato afectan la actividad

enzimática de la Ca -ATPasa de eritrocito humano

JAVIER VARGAS-MEDRANO , JORGE A. SIERRA-FONSECA , MANUEL ARELLANO-CARRILLO

3,4

AND FERNANDO PLENGE-TELLECHEA

Recibido: Mayo 3, 2011

Aceptado: Julio 7, 2011

Abstract

Resumen

The extensive use of pesticides can cause many human

health problems. However, the effects of pesticides on a

biochemical level are still poorly understood. In this study we

analyzed the effect of different pesticides on the plasma

Se ha observado que la exposición de personas a plaguicidas

puede causar problemas a la salud. Sin embargo, el estudio del

efecto de plaguicidas a un nivel bioquímico ha sido pobremente

estudiado. En este estudio, analizamos el efecto de cipermetrina,

membrane Ca -ATPase (PMCA) and on the sarco/

deltametrina y glifosato sobre la actividad enzimática de las Ca -

ATPasa de membrana plasmática (PMCA) y de retículo

sarcoplásmico (SERCA). Diferentes concentraciones de estos

pesticidas fueron añadidas a los experimentos de actividad

enzimática como estrategia para determinar si estos compuestos

eran capaces de afectar la actividad enzimática de PMCA. Nuestros

resultados demuestran que la actividad enzimática de PMCA fue

parcialmente inhibida por deltametrina en un 51.85% ± 3.7 cuando

su concentración fue de 0.5 mM, mientras que cipermetrina y

glifosato estimularon la actividad enzimática de PMCA a menores

concentraciones. El máximo efecto estimulatorio de cipermetrina

fue de 155% ± 9.0 cuando el compuesto alcanzó una concentración

de 0.2 mM. Además, el herbicida glifosato fue capaz de estimular la

actividad enzimática de PMCA en un 111% ± 2.0 a concentraciones

de 0.1-0.2 mM. En conclusión, nuestros resultados demostraron

que la actividad enzimática de PMCA fue también parcialmente

inhibida con deltametrina, pero al contrario de cipermetrina y glifosato

la estimularon. Nuestros resultados sugieren que las diferencias

en las estructuras químicas de cipermetrina y deltametrina se ven

reflejadas en el efecto provocado sobre PMCA. Sin embargo, una

enzima similar, SERCA, no fue afectada ni por deltametrina ni

glifosato. Estas diferencias se pudieran ver reflejadas en disturbios

sobre la regulación celular del calcio.

endoplasmic reticulum Ca -ATPase (SERCA) activities.

Different amounts of pesticides were added to the Ca -

dependent ATPase assays in order to determine if they were

affecting the ATPase activity. The results showed that PMCA

activity was partially inhibited by deltamethrin to 51.85% ±

3.7 when its concentration in the reaction medium was 0.5

mM, while cypermethrin and glyphosate stimulated the PMCA

activity at the lowest concentrations tested. The maximum

stimulatory effect of cypermethrin was of 155% ± 9.0 at a

concentration of 0.2 mM. In addition, the herbicide glyphosate

stimulated the activity 111% ± 2.0 at a concentration of 0.2

mM. In conclusion, our results showed that PMCA activity

was partially inhibited by deltamethrin, but cypermethrin and

glyphosate stimulated their activity. Our findings suggest that

cypermethrin and deltamethrin have different structure-

activity relationships. However, SERCA was not sensitive to

deltamethrin or glyphosate. These differences may be

reflected in disturbs over cellular calcium regulation.

Keywords: PMCA (plasma membrane Ca -ATPase), SERCA

(

sarco/endoplasmic Ca -ATPase) enzymatic activity,

cypermethrin, deltamethrin, glyphosate.

Palabras clave: PMCA (ATPasa de Ca de membrana plasmática),

SERCA (ATPasa de Ca de retículo endo/sarcoplásmico), actividad

enzimática, cipermetrina, deltametrina, glifosato.

________________________________

Center of Excellence for Infectious Diseases, Biomedical Sciences Department, Texas Tech University Health Science Center and

Paul L. Foster School of Medicine. El Paso, TX, U.S.A. 79905

Department of Biological Sciences, University of Texas at El Paso, El Paso, TX, U.S.A. 79968

Departamento de Ciencias Químico Biológicas. UniversidadAutónoma de Ciudad Juárez, Juárez, Chih. México. Anillo Envolvente del

Pronaf y Estocolmo s/n. Ciudad Juárez, Chih., México. C. P. 32300. Tel/Fax: 656 688 1800 al 09

Dirección electrónica del autor de correspondencia: fplenge@uacj.mx.

•

Vol. V, No. 3 • Septiembre-Diciembre 2011 •

JAVIER VARGAS-MEDRANO, JORGE A. SIERRA-FONSECA, MANUEL ARELLANO-CARRILLO AND FERNANDO PLENGE-TELLECHEA: Cypermethrin,

deltamethrin and glyphosate affect the activity of the Ca -ATPase from human erythrocyte

Introduction

esticides are chemicals used worldwide in agriculture and in household settings, resulting

in continuous human exposure from different sources including food, water, and occupational

exposure. The effect of pesticides is targeted to specific organisms, however, pesticides

are toxic substances widely released into the environment, thus posing a threat to human health

Alavanja et al., 2004; Weiss et al., 2004). The damage caused by pesticide exposure has been a

matter of concern for many years, but the toxic effects induced by most of these chemical compounds

(

remain enigmatic (Alavanja et al., 2004).

Pyrethroids are among the most used

pesticides, and unlike first generation

pyrethroids, the second generation of

pyrethroids are more resistant to the

environmental conditions and therefore are more

persistent and toxic to living organisms. Two

members of the second generation group are

cypermethrin and deltamethrin, and they were

studied in the present work. The toxic action of

pyrethroids mainly focuses on the nervous

system by interfering with the ionic channels,

which causes membrane depolarization,

synaptic alterations and immunotoxicity (Madsen

et al., 1996; Narahashik, 1996; Soderlund et al.,

stimulated by calmodulin (CaM), a small

cytosolic Ca -binding protein (Carafoli, 1992).

Another enzyme responsible for the

maintenance of low Ca concentration in the

cytoplasm is the sarcoplasmic reticulum Ca -

ATPase (SERCA). This enzyme translocates

Ca from the cytosol to the lumen of the

sarcoplasmic or endoplasmic reticulum in an

ATP-dependent mechanism (Khan et al., 2000).

The toxic mechanisms of pesticides should

be different for each case. However, since these

molecules are generally lipophilic, the

phospholipid bilayer of biological membranes

could be a suitable toxicity target (Chefurka et

al., 1987). This could interfere with membrane

function and fluidity, thus affecting membrane-

associated enzymes such as PMCA and

SERCA. For instance, parathion, an

organophosphate pesticide, is known to alter

membrane organization, and it also has been

shown that hexachlorobenzene, a cyclic

organochlorine and hydrophobic pesticide,

affects the functionality of two membrane-bound

2002; Diel et al., 2003). Glyphosate, a frequently

used organophosphate herbicide, has been

reported to cause toxic effects such as nausea,

renal failure, hepatic alterations, and behavioral

disturbances and also affect the animal cell

cycle (Steinrucken et al,. 1980; Marc et al., 2002;

De Roos et al., 2005). Calcium (Ca ) signaling

is a key parameter for cell survival. Calcium acts

as a carrier of signals for different cellular

processes, including muscle contraction,

synaptic transmission and apoptosis. This

enzymes: 5’-nucleotidase and Na /K -ATPase

Courtney, 1976; Antunes-Madeira et al., 1994;

(

means that intracellular Ca concentration must

be strictly regulated (Carafoli, 2002). The

Randi et al., 1998). Damage to the erythrocyte

and sarcoplasmic reticulum (SR) membranes

by some pesticides has been previously

reported (Duchnowicz and Koter, 2003; Bhalla

and Agrawal, 1998; Sahib and Desaiah, 1987).

In addition, the functionality of PMCA and SERCA

can be affected by a variety of hydrophobic

molecules, including some studied pesticides

plasma membrane Ca -ATPase (PMCA) plays

a key role in the maintenance of Ca

homeostasis. PMCA is a membrane-bound

enzyme responsible for Ca transport from the

cytosol to the extracellular space using the

energy provided by theATP hydrolysis. Typically

the erythrocyte is the selected cell to obtain

PMCA preparations, but the enzyme is present

in all eukaryotic plasma membranes. The

hydrolytic activity of PMCA can be directly

(Price, 1976; Jones et al., 1985; Michelangeli et

al., 1990; Janik and Wolf, 1992; Salas and

Romero, 1996; Plenge-Tellechea et al., 1999).

The aim of the present study was to test if

• Vol. V, No. 3 • Septiembre-Diciembre 2011 •

JAVIER VARGAS-MEDRANO, JORGE A. SIERRA-FONSECA, MANUEL ARELLANO-CARRILLO AND FERNANDO PLENGE-TELLECHEA: Cypermethrin,

deltamethrin and glyphosate affect the activity of the Ca -ATPase from human erythrocyte

cypermethrin, glyphosate and deltamethrin were

propane-sulfonic acid), pH 7.4 in the last four

washes to remove EDTA (ethylenediamine-

tetraacetic acid). The erythrocyte membrane

preparations were stored in aliquots at -80°C

until they were used.

able to affect the Ca -ATPase hydrolytic activity

of PMCA; specifically the human PMCA found

in membranes from erythrocyte. Findings from

this investigation will shed light on the possible

toxicological damage induced by these widely

Preparation of SR vesicles enriched with

SERCA. Microsomal membranes were

obtained by differential centrifugation after

homogenization of low density SR of skeletal

fast-twitch muscles. They were preserved in 10

mM Mops pH 7.0 and 30% (w/v) sucrose as

previously described (Eletr and Inesi, 1972), and

stored at -80°C until they were used.

used pesticides to this human Ca -ATPase.

Material and methods

Biological materials. Plasma membrane-

containing PMCA (EC=3.6.3.8) were obtained

from packed human erythrocytes of non-

outdated human blood. Packed human blood

was a donation from the General Hospital in the

city of Juárez, México. For the isolation of SR

vesicles enriched with SERCA (EC=3.6.3.8),

female New Zealand rabbit weighing 2 kg were

obtained from the biotherium and sacrificed in a

Protein concentration. The total membrane

protein concentration was determined by the

colorimetric method described by Lowry et al.

(

1951), using bovine serum albumin as the

standard protein.

CO chamber. All the biological materials used

Free Ca concentration in the reaction

were obtained in accordance with the moral and

ethical principles of the University Autonomous

of Juárez City (UACJ).

medium. The free Ca concentration used in

the experiments was determined using a

computer program (Fabiato and Fabiato, 1979).

The computer program Calcium takes into

account the absolute stability of the constant

Chemicals.Adenosine 5’triphosphate (ATP)

and bovine serum albumin were purchased from

Sigma Inc. (Mexico). CaCl was obtained from

value for the Ca -EGTA complex, the EGTA

J. T. Baker (Mexico). Cypermethrin (alpha-

methrine) (CAS: 52315-07-8), deltamethrin

protonation equilibria (Blinks et al., 1982), the

presence of Ca ligands, and the pH in the

(CAS: 52918-63-5) and glyphosate (glyphosate-

medium.

ammonium) (CAS: 1071-83-6) were acquired

PMCA activity. The PMCA activity was

performed at 37°C in a typical reaction medium

containing 30 mM Mops buffer pH 7.0, 130 mM

KCl, 3 mM MgCl , 0.5 mM EGTA, 0.5 mM CaCl

from Supelco (USA). The Ca ionophore

A23187 (calcimycin), EGTA(ethylene glycol-bis

(

-aminoethyl ether)-N,N,N’,N’-tetra acetic acid),

Mops were purchased from Sigma-Aldrich Co.

Mexico). Other chemicals used in this

(

10 μM free Ca ) and 0.12 mg protein/ml. The

(

reaction was started with the addition of 1 mM

ofATP. The PMCAactivity was determined using

the colorimetric method described by Lanzetta

et al. (1979), following the appearance of P_i

investigation were reagent grade. Cypermethrin

and deltamethrin were dissolved in methanol.

The concentration of methanol added in the

experiments never was higher than 1% (v/v).

Glyphosate was dissolved in warm water.

(inorganic phosphate) during theATP hydrolysis

reaction. The stabilizing agent Sterox was

substituted by 0.18% (v/v) of Tween-20 as

described by Baykov et al. (1988). Different

concentrations of the pesticides cypermethrin,

deltamethrin, and glyphosate were also present

in the mixture as indicated in the corresponding

figure. The enzymatic activity was measured in

the absence of CaM.

Isolation of erythrocyte membranes

containing PMCA. Hemoglobin-free erythrocyte

membranes depleted of endogenous CaM were

obtained from non-outdated packed human

erythrocytes according to a previously described

method (Niggli et al., 1979), except for the use

of 10 mM Mops-K (Mops: 4-morpholine-

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JAVIER VARGAS-MEDRANO, JORGE A. SIERRA-FONSECA, MANUEL ARELLANO-CARRILLO AND FERNANDO PLENGE-TELLECHEA: Cypermethrin,

deltamethrin and glyphosate affect the activity of the Ca -ATPase from human erythrocyte

SERCAactivity. The hydrolytic reaction was

measured at 25°C by determining the

spectrophotometric procedure (Lanzetta et al.,

1979; Baykov et al., 1988). The pesticides tested

on PMCA activity were cypermethrin, glyphosate

and deltamethrin using the appropriate

appearance of P using a previously described

colorimetric method by Lin and Morales (1977).

The reaction mixture contained 20 mM Mops

concentrations of K , Mg , ATP, and Ca . This

study was conducted in the absence of CaM.

The second generation pyrethroids cypermethrin

and deltamethrin altered the PMCA activity as

shown in Fig. 1. Cypermethrin significantly

stimulated theATP hydrolysis at concentrations

buffer pH 7.0, 80 mM KCl, 5 mM MgCl , 1 mM

EGTA, 0.967 mM CaCl , (10 μM free Ca ), and

.75 μMA23187. The enzymatic reaction started

by the addition of 1 mM ATP. Different

concentrations of the pesticides deltamethrin

and glyphosate were also present in the mixture

as indicated in the corresponding figure.

from 0.005-0.5 mM without inhibiting the Ca -

ATPase activity (Fig. 1A).

Statistical analysis and other specifications.

Statistical analysis such as Pearson product

moment correlation, linear regression and

standard error were calculated with the

computer software Sigma Plot Graph System.

Pesticides were always included in the

incubation/reaction medium after erythrocyte

membranes or SR vesicles had been added.

The mixtures were always pre-incubated for at

least 2 minutes before the reactions were

started. The experimental data corresponds at

least to three independent measurements

performed in duplicates and using more than

one membrane preparation.

The maximal stimulation was 55% ± 9.0 over

the activity control, at a concentration of 0.2 mM.

However, deltamethrin did not stimulate PMCA

activity. Interestingly, deltamethrin significantly

inhibited the hydrolytic activity of the enzyme to

51.85% ± 3.7 at 0.5 mM (Fig. 1B). In addition, we

studied the effect of glyphosate and unexpectedly

this pesticide affected PMCA activity in a biphasic

manner. As well as cypermethrin, glyphosate

significantly stimulated the hydrolytic activity of

the enzyme over 11% ± 2.0 with respect the

control when its concentration was between

0.05-0.10 mM (Fig. 1C). However, higher

concentrations of glyphosate (0.5-1.0 mM)

significantly inhibited PMCA activity to 73% ± 3.5

when compared to the control. Our findings prove

that cypermethrin and deltamethrin, two second

generation pyrethroids, affected PMCAactivity, as

well as glyphosate.

Results

Cypermethrin, deltamethrin and glyphosate

affect PMCA activity in different manners. The

present study shows the effect of cypermethrin,

deltamethrin and glyphosate on the specificATP

The Ca -ATPase SERCA was unaffected

hydrolysis mediated by PMCA. This Ca -

dependent pump utilizes ATP as a source of

by deltamethrin and glyphosate. In order to

investigate if our previous findings were general

energy to pump Ca across the plasmatic

membrane. Our study consisted of different

independent treatments in which distinct

concentrations of pesticides where added to the

PMCA reaction medium. Randomly, different

concentrations of cypermethrin, deltamethrin

and glyphosate were tested in order to determine

effects for other Ca -ATPases, we studied the

SERCA activity as a function of the pesticide

concentration (deltamethrin and glyphosate). We

choose only one compound from the second

generation of pyrethroids, and the organophos-

phorate glyphosate to test our hypothesis. The

concentrations of deltamethrin and glyphosate

used in this study were in the same ranges as

which ones affected the Ca -ATPase activity

PMCA). Therefore, only the concentrations that

(

previously used for PMCA. The Ca -ATPase

activity was measured at 25°C, in a buffered

produced an effect are shown in this study. This

biological system is measured by following the

appearance of phosphate during the ATP

hydrolysis and it was followed using a

medium containing 10 M free Ca and 1 mM

ATP. This is a reliable and well established

procedure of measuring SERCA activity.

• Vol. V, No. 3 • Septiembre-Diciembre 2011 •

JAVIER VARGAS-MEDRANO, JORGE A. SIERRA-FONSECA, MANUEL ARELLANO-CARRILLO AND FERNANDO PLENGE-TELLECHEA: Cypermethrin,

deltamethrin and glyphosate affect the activity of the Ca -ATPase from human erythrocyte

Fig 1. The effect of cypermethrin, deltamethrin and glyphosate on the hydrolysis ofATPmediated by the Ca -ATPase from human plasma

membrane. The reactions were performed as described in Methods and they were started after the addition of 1 mMATP. The ATPase activity

was measured in the steady state of the enzyme. Different concentrations of pesticides were added into reaction media as indicated at the

x axes. Closed circles show the average of 3 ± SE independent experiments. For figure A) *, p=<0.001, **, p=0.002; for figure B) *, p=0.020,

*, p=0.003, ***, p=0.010, ****, p=<0.001, *****, p=0.002; for figure C) *, p<0.001, **, p=0.006, ***, p=0.012, ****, p=0.005 compared to the

control that correspond to the 100% of the Ca -ATPase activity. The average (n=3) of ATPase activity control was 18nmol P/min/mg of

protein and it was normalized to 100%. The chemical structures of cypermethrin and deltamethrin are shown and they differ in substitution

of two bromines in deltamethrin for two chlorines in cypermethrin (PubChem Substance ID: 24868901 and 40585 respectively).

Figure 1A

Figure 1B

Figure 1C

This data was obtained in the presence of a

membrane protein concentration of 0.01 mg SR

protein/ml. Deltamethrin (Fig. 2A) was not found

to significantly affect SERCA activity, as

demonstrated by a Pearson product-moment

correlation coefficient -0.135 whit a p=0.7283

a correlation coefficient of -0.502 with a p=0.168

(n=9), once again indicating that there was not

a statistically significant relationship between

SERCA activity and glyphosate concentration.

This conclusion was determined based on the

high p-values (p  0.05) obtained for each

corresponding analysis. Therefore, the

pesticides tested on the SERCA activity

(glyphosate and deltamethrin) had no significant

effect on theATP hydrolytic activity mediated by

this SERCA at the tested concentrations. In

conclusion, our results show that SERCA is not

sensitive to glyphosate and deltamethrin.

(

n=9), indicating the lack of a statistically

significant relationship between enzyme activity

and deltamethrin concentration. Similar results

were obtained when we assayed glyphosate

(Fig. 2B); since the compound was not found to

significantly affect SERCA activity. This was

confirmed with a correlation test, which provided

•

Vol. V, No. 3 • Septiembre-Diciembre 2011 •

JAVIER VARGAS-MEDRANO, JORGE A. SIERRA-FONSECA, MANUEL ARELLANO-CARRILLO AND FERNANDO PLENGE-TELLECHEA: Cypermethrin,

deltamethrin and glyphosate affect the activity of the Ca -ATPase from human erythrocyte

Fig 2. The effect of deltamethrin and glyphosate on the hydrolysis of ATP mediated by the Ca -ATPase of sarcoplasmic

reticulum. Ca -ATPase activity was measured as described in Methods and 1 mM ATP was used to start the reaction in the

steady state of the enzyme. The pesticides were titrated as indicated at the x axes. Each data point is the average of 3 ± SE

independent experiments. Lines correspond to a computed linear regressing showing no significant effect (p 0.05) of cypermethrin

and deltamethrin on the SERCAactivity. TheATPase activity was 2.713 ± 0.008 μmol P/min/mg of SR and it was normalized to 100%.

Figure 2A

Figure 2B

Discussion

Our research was conducted to study the

effect of cypermethrin, deltamethrin and

glyphosate on the hydrolytic activity of the

human PMCA, which is found in membranes

from erythrocytes.

as methanol are known to stimulate Ca -

ATPase activity and this was carefully evaluated

in our lab before titrating pyrethroids into our

assays (Benaim et al., 1994). The studies in the

effect of cypermethrin, deltamethrin and

glyphosate on Ca -ATPases have been

described few times in the literature, in different

animal and plant models, but not in particular

on the human PMCA isoform from membranes

of erythrocyte, therefore, the importance of our

study (Clark et al., 1987; Sahib et al., 1987;

Kodavanti et al., 1993; Souza da Silva et al.,

Pyrethroids, like cypermethrin and

deltamethrin, are widely used in combination

with organophosphates instead of chlorinated-

hydrocarbons such as DDT. In addition, it was

established that pyrethroids were designed to

target sodium channels (Cremer, 1983;

Narahashi, 1996). Later on, pyrethroids were

improved into more environmental resistant

compounds and they were called pyrethroids

of second generation and good examples of

these are cypermethrin and deltamethrin. The

second generation of pyrethroids, cypermethrin

and deltamethrin were both dissolved in

methanol; however, the amount of methanol

added together with the pyrethroids into the

reaction media never exceeded 1% as a final

concentration. The main reason for maintaining

a low concentration (lower than 1%) of methanol

in our assays was to avoid significant effects

on PMCA or SERCA activities. Alcohols such

2003). To the best of our knowledge, the effect

of these compounds have never been reported

before for human PMCA from membranes of

erythrocyte. Similar studies were done, such as

the study of esbiol and cyfluothrin effect on Ca -

ATPase using rat’s leukocyte and synaptosome

membranes (Grosmal and Diel, 2005).

According to our data, the pyrethroid

cypermethrin produced a marked increase on

the PMCAenzymatic activity, followed by a partial

inhibition on the PMCA activity (Fig. 1A). While

the hydrolytic activity was inhibited when

deltamethrin was titrated into the PMCA reaction

• Vol. V, No. 3 • Septiembre-Diciembre 2011 •

JAVIER VARGAS-MEDRANO, JORGE A. SIERRA-FONSECA, MANUEL ARELLANO-CARRILLO AND FERNANDO PLENGE-TELLECHEA: Cypermethrin,

deltamethrin and glyphosate affect the activity of the Ca -ATPase from human erythrocyte

(

Fig. 1B), on the other hand, glyphosate exhibited

ATPase from rat brain, while permethrin, esbiol,

the same biphasic effect that we observed with

cypermethrin, but the stimulation and inhibition

was lower (Fig. 3C). The adverse effect

produced by these pesticides is related to the

chemical structure of each compound

and cyfluthrin affect the activity of the Ca -

ATPase from leukocyte membranes (Kakko et

al., 2003; Grosman and Diel, 2005). This is an

important issue, because even thoughATPases

can be similar or the same in different cell types,

the lipidic composition of a membrane may

change the effect of a compound in a given

ATPase. Membrane lipid composition has been

shown to be determinant in the effect of

compounds like pyrethroids. Comparative

(

Soderlund et al., 2002). Even though the

pyrethroid molecules under study have similar

structures, they do not affect different enzymes

in the same way, hence the importance of

studying each individual compound on specific

enzymatic systems. Many structure-activity

relationship studies have been reported and it

has been shown that minor changes in the

chemical structure of a compound can

dramatically modify the effect of the molecule

on different protein systems (Plenge-Tellechea

et al., 1999). Structurally, both pyrethroids share

a similar structure with a minor difference.

Cypermethrin is a chlorinated compound, with

two chlorides in their structure, in contrast to

deltamethrin that contains two bromines instead

of chlorides (Inserts in Fig. 1A and 1B

respectively). These small modifications in the

chemical structure of both cypermethrin and

deltamethrin produced a different structure-

activity relationship. While the chlorines in

cypermethrin are making it a stimulator of PMCA

activity at the lower concentrations tested and

then an inhibitor at higher concentrations, the

bromide-bearing deltamethrin acts as a PMCA

inhibitor only.

studies using Ca -ATPase in different rat

membrane had been shown to differ in sensibility

to pyrethroids (Grosman and Diel, 2005).

Therefore, it is very interesting to see how the

effect of the same compound varies inATPases

from different derived preparations or cell

source.

Glyphosate is a pesticide that belongs to the

group of widely used organophosphates. They

produce alterations on ATPases and good

examples of them are anilofos and paraoxon

(Blasiak, 1995; Hazarika et al., 2001). In addition,

there is evidence suggesting that malathion

inhibits ATP hydrolysis in brain membranes

preparations from zebrafish (Senger et al.,

005). In agreement with our results, where

glyphosate inhibited PMCA hydrolytic activity

Fig. 1C), it has been shown that glyphosate

inhibits the ATPase of slug nervous system

Souza da Silva et al., 2003). It was interesting

(

to find out, that glyphosate affected PMCA

activity as well as the herbicide paraquat, which

is known to inhibit PMCA activity in membranes

from erythrocyte (Janik and Wolf, 1992).

However in neurons, paraquat induced the

production of ROS and this seems to inactivate

neuronal PMCA (Zaidi et al., 2009).

Different authors mentioned that

pyrethroids are highly hydrophobic pesticides

with a high-tendency to target biological

membranes from which they can disrupt

membrane organization, thus affecting the

activity of membrane proteins. In addition to our

findings, pyrethroids are known to inhibit the

ATPase activity derived from rat, squid, toad, and

cockroach brain membrane preparations

Unlike PMCA, SERCA was more resistant

to the adverse effects of the selected pesticides

in our experiments. None of the pesticides

tested (glyphosate and deltamethrin) had a

significant effect on the hydrolytic activity of the

SERCA, based on the high p-values obtained

(Michelangeli et al., 1990; Berlin et al., 1984;

Clark and Matsumura, 1987; Sahib et al., 1987;

Moya-Quiles et al., 1996). Additionally, the

pyrethroids cypermethrin and permethrin are

known to inhibit the activity of the synaptosomal

(

p  0.05) (Fig. 2A and 2B). We performed these

experiments in SERCA in order to validate our

findings in PMCA, and surprisingly they were

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Vol. V, No. 3 • Septiembre-Diciembre 2011 •

JAVIER VARGAS-MEDRANO, JORGE A. SIERRA-FONSECA, MANUEL ARELLANO-CARRILLO AND FERNANDO PLENGE-TELLECHEA: Cypermethrin,

deltamethrin and glyphosate affect the activity of the Ca -ATPase from human erythrocyte

very different. This difference may be due to the

closed-membranes (spherical shape) of SR

vesicles and to the great quantity of Ca pumps

ones found in a living model) that is required for

ROS production. In addition, there is no way for

these compounds under study to enter into a

redox cycle or be biotransformated in a redox

cycler that may lead to ROS production without

the cellular components required for ROS

production in our test tube. Moreover, several

investigations had shown that herbicides like

paraquat targets neurons, where it mediated the

generation of ROS (Corasaniti et al., 1992).

Generation of ROS mediated by these

compounds demonstrated how toxic these

compounds really are when they enter a living

cell. However, our findings contributed to show

additional toxic effects produced by these

compounds. In addition to ROS production cited

in the literature, these compounds also affect

per total protein, which represents 0.2 μM of

Ca -ATPase per 0.05 mg/ml of total membrane

protein (Deamer and Baskin, 1969; Lax et al.,

002). In contracts with SERCA, PMCA is an

open-membranes system. Plasmatic

membrane from erythrocytes also contained

less Ca -ATPase in compared to SR vesicles,

as the quantity of functional membrane protein

represents only 0.01-0.1% of PMCA from the total

erythrocyte membrane protein, the use of this

system is highly relevant, given the fact that it is

directly derived human tissue (Knauf et al.,

1974).

A second reason could be the lipid

Ca homeostasis, by affecting Ca -carriers

composition of membranes from erythrocytes

versus RS membranes from rabbit’s muscle.

The phospholipids/cholesterol ratio was

determined to be 0.96 for membrane of

erythrocyte, which means that the amount of

both lipids is equilibrated in this membrane type

such as Ca -ATPases.

Finally, SERCA and PMCA are different

proteins in terms of their pharmacological

sensitivity, as it has been demonstrated by the

use of several inhibitors. The effects caused by

pesticides on the biochemical level are still poorly

understood. However, it is clear that some

pesticides could exert their toxic effects by

interfering with membrane-bound enzymes such

(

Gottfriedg, 1967). However, in SR this ratio is

totally different and it was determined to be 0.05

Waku and Nakazawa, 1964). ATPases in

(

general, are lipid-dependent proteins and

function of the ATPase is highly dependent of

the chemical composition and physical phase

of the lipids surrounding the protein (Lee, 1998).

This may be the case of the compounds studied

here, were they may modify in some degree the

lipid environment that surround theATPase, thus

affecting ATPase function.

as PMCA and SERCA, thus disrupting Ca

homeostasis. Calcium is critical for cell function,

since cells are not able to survive in the absence

of Ca , but an excess of the ion can also be

lethal, since it can lead to apoptosis (Lam et al.,

1994).

Conclusion

It has been shown that cypermethrin,

deltamethrin and glyphosate induce the

production of reactive oxygen species (ROS) in

different model organisms; however we did not

evaluate the production of ROS in our in vitro

model and in presence of cypermethrin,

deltamethrin or glyphosate (Giray et al., 2001;

Li et al., 2007;Ahsan et al., 2008). Even though

there is a remote possibility of having ROS

production in pure and washed membranes,

which will lead to lipid peroxidation of the lipids

in our membrane model, we concluded that we

did not have an electron source (such as the

Our results do not necessarily reflect the

concentrations that may produce poisoning, but

they are helpful in understanding the effects of

cypermethrin, deltamethrin and glyphosate on the

biological systems studied in this investigation.

The present study concludes that PMCA activity

was significantly affected by the pesticides studied

and future investigations should focus on the

molecular mechanism behind this effect. SERCA

was unaffected by deltamethrin and glyphosate.

This may be due the lipidic nature of this

membrane and the closed-membrane (SR

• Vol. V, No. 3 • Septiembre-Diciembre 2011 •

JAVIER VARGAS-MEDRANO, JORGE A. SIERRA-FONSECA, MANUEL ARELLANO-CARRILLO AND FERNANDO PLENGE-TELLECHEA: Cypermethrin,

deltamethrin and glyphosate affect the activity of the Ca -ATPase from human erythrocyte

COURTNEY, K.D. 1979. Hexachlorobenzene (HCB): a review. Environ.

Res. 20(2):225-266.

CREMER, J.E. 1983. The influence in mammals of the pyrethroid

insecticides. Dev. Toxicol. Environ. Sci. 11:61-72.

DEAMER, D.W., and R.J. Baskin. 1969. Ultrastructure of sarcoplasmic

reticulum preparations. J. Cell Biol. 42(1):296-307.

DEROOS,A.J.,A. Blair, J.A. Rusiecki, J.A. Hoppin, M. Svec, M. Dosemeci,

D.P. Sandler, and M.C. Alavanja. 2005. Cancer incidence among

Glyphosate-exposed pesticide applicators in the agricultural

health study. Environ. Health Perspect. 113(1):49-54.

vesicle) system employed in this study. The effect

of pyrethroids and organophosphorates in PMCA

should affect Ca homeostasis and their

consequences still need to be elucidated. An

inhibition on SERCA and PMCA activities should

produce an increase in intracellular Ca

concentration with may kill a cell.

DIEL, F., B. Horr, H. Borck, and T. Irman-Florjanc. 2003. Pyrethroid

insecticides influence the signal transduction in T helper

lymphocytes from atopic and nonatopic subjects. Inflamm. Res.

Acknowledgements

This project was supported by the grant

CONACYT CHIH-2006 C01-57268 (Mexico).

52(4):154-163.

DUCHNOWICZ, P., and M. Koter. 2003. Damage to erythrocyte membrane

caused by chlorophenoxyacetic herbicides. Cell Mol. Biol. Lett.

(1):25-30.

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Este artículo es citado así:

Vargas-Medrano, J., J.A. Sierra-Fonseca, M. Arellano-Carrillo and F. Plenge-Tellechea. 2011: Cypermethrin,

deltamethrin and glyphosate affect the activity of the Ca2+-ATPase from human erythrocyte. TECNOCIENCIA

Chihuahua 5(3): 121-131.

• Vol. V, No. 3 • Septiembre-Diciembre 2011 •

JAVIER VARGAS-MEDRANO, JORGE A. SIERRA-FONSECA, MANUEL ARELLANO-CARRILLO AND FERNANDO PLENGE-TELLECHEA: Cypermethrin,

deltamethrin and glyphosate affect the activity of the Ca -ATPase from human erythrocyte

Resúmenes curriculares de autor y coautores

JAVIER VARGAS MEDRANO. Originario de Ciudad Juárez, ingresó al programa de Biología de la Universidad Autónoma de Ciudad Juárez

en 1999, como parte de la primera generación del naciente programa. Ahí realizó su función de técnico de investigación y su tesis

de licenciatura en el área de bioquímica, bajo la dirección del Dr. Plenge Tellechea, analizando el efecto de diferentes estructuras

químicas de diclorobenzenos (precursores de pesticidas) sobre proteínas transportadoras de calcio. Después de obtener su

licenciatura, ingresó a la Universidad de Texas en El Paso, donde comenzó su tesis doctoral estudiando como un proceso

postraduccional, la fosforilación del transportador de glicina del cerebro era capaz de regular los flujos de glicina, lo cual en la clínica

puede ser un prometedor tratamiento a esquizofrenia. Durante sus estudio doctoral fue distinguido dos veces como asistente de

investigación con fondos del National Institute of Health and National Institute of Mental Health de los Estados Unidos. Finalmente,

obtuvo el doctorado en el 2010. En el 2011, fue distinguido con el Hispanic Training Fellowship para ocupar la posición de

Postdoctoral-Research Associate en el Texas Tech Health Science Center en el Paso Texas. Actualmente, el doctor se encuentra

inmerso en varias investigaciones sobre una de las proteínas (CCR5) responsables de la entrada del virus del SIDA a las células.

JORGE ANÍBAL SIERRA FONSECA. Se graduó del programa de Licenciatura en Biología de la Universidad Autónoma de Ciudad Juárez

(UACJ) en el año 2007. Posteriormente inició sus estudios de posgrado en la Universidad de Texas en El Paso (Texas, EUA), donde

fue admitido en el programa de doctorado en Ciencias Biológicas/Patobiología. Actualmente cursa su tercer año en el programa de

doctorado, donde además de desarrollar su proyecto de investigación, también funge como instructor asistente en diversos cursos

de licenciatura. Ha asistido a diversos congresos locales, regionales, nacionales e internacionales, donde ha presentado los

resultados de diversos proyectos de investigación. Actualmente se encuentra estudiando el papel de las proteínas G heterotrimericas

en la organización del citoesqueleto durante la diferenciación neuronal y neurodegeneración, utilizando diversos modelos celulares.

Sus intereses incluyen biología celular, neurociencias, señalización celular y cáncer.

MANUEL DAVID ARELLANO CARRILLO. En el año 2009 obtuvo el grado de Licenciado en Biología en la Universidad Autónoma de Ciudad

Juárez con el tema de tesis: Interacción de plaguicidas sobre la actividad enzimática de la ATPasa de Ca2+ de eritrocito humano

(

PMCA). Además, el biólogo ha participado en diversos congresos nacionales e internacionales entre los que destacan varios

congresos nacionales de bioquímica y uno organizado por la American Chemical Society. Los trabajos de investigación donde ha

colaborado se han publicado en diversas revisas tales como en la revista Ciencia en la Frontera. Actualmente, el biólogo realiza sus

estudios en la Maestría con Orientación Genómica en la UACJ donde evalúa el efecto de deltametrina sobre la expresión de diversos

genes en Linfocitos T humanos.

LUIS FERNANDO PLENGE TELLECHEA. Desde 1990 ingresó como becario interno del laboratorio de reproducción en la Facultad de Ciencias

de la Universidad Autónoma de Baja California. En 1992 culminó sus estudios de Biología en la misma dependencia. Posteriormente

realizó sus estudios de Doctorado en Ciencias Biológicas por la Universidad de Murcia, culminando en 1998. El Dr. Plenge se ha

caracterizado por sus estudios bioquímicos en la rama de proteínas asociadas en membranas. Actualmente labora como profesor

investigador de tiempo completo en la Universidad Autónoma de Ciudad Juárez. Cuenta con múltiples publicaciones y dirección de

tesis de pregrado y de grado. Es actual director en jefe de la revista de ciencias, Ciencia en la Frontera, e imparte la cátedra de

Bioquímica en el programa de Biología y de Estructura y función proteínas en la Maestría en Ciencias con orientación en genómica

(PNP). Actualmente se encuentra de reingreso a la UACJ posterior a una estancia Posdoctoral en el Border Biomedical Research

Center de la Universidad de Texas at El Paso desde 2010 a Julio del presente, donde estudió los mecanismos bioquímicos de

neurotransportadores de dopamina y glicina así como escritura de trabajos científicos.

•

Vol. V, No. 3 • Septiembre-Diciembre 2011 •