International Journal of Medicine and

Pharmaceutical Sciences (IJMPS)

ISSN(P): 2250-0049; ISSN(E): 2321-0095
Vol. 4, Issue 6, Dec 2014, 17-28
TJPRC Pvt. Ltd.

MOSQUITO REPELLENT PYRETHROID INDUCED BIOCHEMICAL AND

BIOPHYSICAL CHANGES IN PLASMA AND ANTIOXIDANT STATUS IN HUMAN
MALE VOLUNTEERS EXPOSED TO LONG TERM ALLETHRIN AND
PRALLETHRIN INHALATION
NARENDRA MADDU1, NAGAJOTHI G2, FAREEDA BEGUM. S3 & B. SREEKANTH4
1,3

Department of Biochemistry, Sri Krishnadevaraya University, Anantapur, Andhra Pradesh, India

2

Department of Corporate Secretaryship, Queen Marys College, Chennai, Tamil Nadu, India

Department of Zoology, Sri Krishnadevaraya University, Anantapur, Andhra Pradesh, India

ABSTRACT
Allethrin and prallethrin are type-I pyrethroids commonly used for domestic and agricultural purposes to get
protection from mosquitoes and other insects. Use of this pyrethroids for longer durations (i.e. inhalation 8 hours per day
not more than 10 hours per day) and for prolonged periods in the form of coils and mats, results in biochemical and
biophysical changes in erythrocyte biomembrane has been established. The effect of allethrin and prallethrin on plasma
nitrite and nitrate concentrations on the activities of red cell enzymes acetyl cholinesterase (AChE) and antioxidant
enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) the content of reduced glutathione
(GSH) and erythrocyte membrane lipid peroxidation (LPO) were determined in human male volunteers who has been
exposed to allethrin and prallethrin for the past 7-10 years. The results were compared with controls (who did not use any
mosquito repellents). It was observed that there was an increase in plasma nitrite and nitrate, increase in activities of
defense enzymes and observed decrease levels of erythrocyte membrane lipid peroxidation (LPO), but a decreased activity
of acetyl cholinesterase (AChE) was observed in allethrin and prallethrin exposed subjects. This study revealed a modest
increase in nitric oxide production, increased antioxidant status followed by enhanced oxidative stress. A possible role of
nitric oxide in the above changes cannot be ruled out, for which further experimentation is required.

KEYWORDS: Allethrin, Prallethrin, Antioxidant Enzymes, Acetyl Cholinesterase, Lipidperoxidation, Nitrite and
Nitrate
Abbreviations: AChE: Acetyl Cholineesterase, SOD: Superoxide Dismutase, CAT: Catalase, Gpx: Glutathione
Peroxidase, GSH: Reduced Glutathione, LPO: Lipid Peroxidation

INTRODUCTION
Pyrethroids are the most commonly used insecticides in India and other countries to get protection from
mosquitoes, insects and pests for household and agricultural (Fradin, 1998; Moya-Quiles et al, 1995; Narendra et al, 2007;
2008a; 2008b; Sinha et al, 2004; Tsuji et al, 2002) purposes due to their high insecticidal activity and low toxicity in
mammals (Elliot et al, 1967; Elliot, 1977; Miyamoto, 1976). Allethrin and prallethrin are among the most widely used
pyrethroids in India and other countries including the United States (Liu et al, 2003; Ramesh and Vijayalakshmi, 2001).
Pyrethroid-induced neurotoxicity, other toxic (acute and chronic) symptoms their deleterious effects on humans and
experimental animals in recent reports aroused a concern among public regarding their chronic use (Kakko et al, 2003;
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Narendra Maddu, Nagajothi G, Fareeda Begum. S & B. Sreekanth

Motomura and Narahashi, 2001; Spencer et al, 2001; Vias et al, 2001).
Inhalation is the route of exposure in humans inhaling pyrethroids cotinuosesly for 8 hours a day leads to entry of
these compounds into circulation with maximal accumulation in all biomembranes and in specific tissues such as blood,
nerve, adipose and other tissues due to their lipophilic nature (Sinha et al, 2004; Theeraphap et al, 2003). As inhaled
pyrethroids directly enter the circulation and different tissues rapidly. Further distribution of these inhaled pyrethroids to
different tissues cause effective damage. However, if these compounds reach the nervous system of mammals, including
man in sufficient concentrations, they can cause adverse neurotoxic effects (Eriksson and Fradricksson, 1991; Malaviya et
al, 1993; Narahashi, 1992). The pyrethroids are neurotoxins, based on their structure and their toxic effects. Observed in
the rat and human beings, they are classified into two groups, namely type-I and type-II compounds. Type-I pyrethroids
may cause T syndrome with aggressive sparring, increased sensitivity to external stimuli and fine tremors progressing to
whole-body tremors and prostration (Verschoyle and Barnes, 1972; Verschoyle and Aldridge, 1980). Type II pyrethroids
produce Cs syndrome, characterized by burrowing, profuse salivation and coarse tremors progressing to choreoathetosis
and chronic seizure (Ray and Cremer, 1979; Verschoyle and Aldridge, 1980).
Pyrethroids are highly hydrophobic compounds and this suggests that their action in biological membranes might
be related to association with integral proteins and with phospholipids (Michelangeli et al, 1990). Biomembranes are
largely, if not totally, responsible for various pyrethroid induced toxicity. Since biomembranes are the known targets
because of the lipophilic nature of the pyrethroids (Kakko et al, 2003; Moya-Quiles et al, 1994; 1996a; 1996b; Narahashi et
al, 1995; 1996; Narendra et al, 2007; 2008a; 2008b). Current literature also reveals that reactive oxygen species have been
implicated in the toxicology of pyrethroids (Kale et al, 1999). Hence the present study is aimed at investigation and
evaluating the changes in antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and GPx and reduced
concentration of glutathione (GSH) in erythrocytes of exposed to inhalation of allethrin and prallethrin subjects and the
activities of red cell enzyme acetyl cholinesterase (AChE) and erythrocyte membrane lipid peroxidation (LPO) and plasma
nitrite (NO2) and nitrate (NO3) in human subjects.

MATERIALS AND METHODS

Subjects
The volunteers were using either Jet mosquito repellent coils or mats, both from Godrej Sara Lee Ltd, Mumbai,
India. The coils are composed of (w/w) 0.1% d-trans allethrin, 52.9% wood flour, 35% coconut shell powder, 12% starch,
and the mats contained (w/w) 1.6% d-trans prallethrin and 98.4% relevant ingredients as indicated by the manufacturers.
Release of the pyrethroid insecticide is either by burning the coil or placing the mat in the commercially available electric
devices. All the subjects were known to get exposed to allethrin or prallethrin for at least 8h/day but not 10h/day, and the
subjects had no known history of exposure to any other similar pyrethroids. Three groups, each group consisting of 24
male volunteers aged between 35-45 years, included in the present study were: Group I, controls who did not use mosquito
repellents; Group II, allethrin exposed subjects; Group III prallethrin exposed subjects. All the volunteers were well
explained about the experimentation and their written consent was obtained. This study was approved by the institutional
ethical committee. Blood samples from overnight fasted subjects were used for the study. All the volunteers in the present
study were free from any other chronic disease or illness, and, were teetotalers with no smoking habit and free from use of
any tranquillizers, drugs and anaesthetics.

Impact Factor (JCC): 5.1064

Index Copernicus Value (ICV): 3.0

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Mosquito Repellent Pyrethroid Induced Biochemical and Biophysical Changes in Plasma and Antioxidant
Status in Human Male Volunteers Exposed to Long Term Allethrin and Prallethrin Inhalation

Blood Collection and Sample Preparation

A 10 ml sample of blood from study subjects was obtained from the brachial vein and each 5 ml collected in vials
containing heparin. In one blood sample, erythrocytes were separated by centrifugation at 1273 g (3000 rpm) for 15 min.
The buffy coat was removed and erythrocytes were washed three times with physiological saline. Aliquots of erythrocytes
were kept at -200C except the samples for catalase assay. The other 5 ml of the blood sample was used for the study of
plasma nitrite and nitrate, erythrocyte membrane lipid peroxidation. The samples were transported on ice to the laboratory
and were processed within two hours.
Isolation of Erythrocytes
Erythrocytes were isolated by using the method of (Beutler, 1975). Anticoagulated blood were passed through the
cellulose column and the filtrate was collected to remove lymphocytes, platelets etc. The filtrate was diluted with saline
and erythrocytes were collected by centrifugation at 1000 rpm for 10 min. This washing step was repeated until the
erythrocytes for study were obtained.
Erythrocyte Membrane Preparation
Erythrocyte membranes were prepared using the method adopted by Dodge et al, (1963). Erythrocyte suspension
was washed with phosphate buffered saline (pH 7.2), and then cells were lysed with 5 mM phosphate buffer (pH 8.0) and
spun at 15000 X g for 30 min. The supernatant was removed carefully and by using the same buffer the latter step was
repeated to obtain haemoglobin-free ghosts for further analysis.
Lipid Peroxidation in Erythrocytes
The extent of lipid peroxidation was measured by the formation of malondialdehyde (MDA) by using the method
of (Buege and Aust, 1978). One ml of erythrocyte membrane was taken in a test tube to which 2 ml of reagent
(15% w/v TCA, 0.375% w/v TBA and 0.25N HCl) was added and kept in boiling water bath for 15 minutes and the
contents were allowed to cool and then centrifuged at 1000 g for 10 minutes. The supernatant was transferred into a
separate test tube and the absorbance of the sample was read at 535 nm by a UV/Visible spectrophotometer against the
reagent blank assuming the molar extinction coefficient to be 1.56x105.
Measurement of Plasma Nitrite and Nitrate
Plasma samples were treated with 30% zinc sulfate to deproteinize samples followed by centrifugation at 4000 x g
for 5 min. Nitrite was determined by the method of Sastry et al, (2002) from 1.0 ml aliquots of plasma using Griess reagent
(1% sulfanilamide, 2.5% phosphoric acid, and 0.1% 1-naphthylethylene diamine). One ml aliquots of the supernatant were
swirled for 90 min separately with activated cadmium granules for the conversion of nitrite to nitrate and then Griess
reagent was added. Nitrite concentrations were estimated using a standard curve developed with sodium nitrite.
Acetylcholinesterase Activity in Erythrocytes
Acetylcholinesterase activity in erythrocytes was measured by (George and Abernethy, 1983). The rate of
hydrolysis of acetylthiocholine iodide (substrate) in erythrocyte suspension at pH 7.6 was measured at 440 nm by the
reaction of thiocholine iodide with DTNB to give the yellow 5-thio-2-nitrobenzoate anion with UV spectrophotometer.
The enzyme activity was expressed as KU/L. Haemoglobin in blood was estimated using Drabkins reagent by the method
of (Dacie and Lewis, 1984). The protein content was determined by the method of Lowry et al, (1951).
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Narendra Maddu, Nagajothi G, Fareeda Begum. S & B. Sreekanth

Determination of Plasma Biochemical Profile

The activities of alkaline phosphatase, lactate dehydrogenase, carbonyl groups and SH groups were determined.
Erythrocyte Antioxidant Enzyme Activities
Erythrocytes were washed thrice with 0.9% Nacl and suspend in 1 volume of 0.9% Nacl. The packed cell volume
was adjusted to 5% with PBS-pH 7.5 (10 mM phosphate buffer saline). Hemoglobin content in erythrocytes was
determined. Reduced glutathione (GSH) content was estimated and expressed as mol/gHb. The superoxide dismutase
(SOD) activity was measured based on the ability of the enzyme to inhibit the autoxidantion of adrenaline and activity was
expressed as units/mg Hb/min. The catalase (CAT) activity in hemolysate was estimated and the activity of the enzyme
was calculated using the extinction coefficient of H2O2 as 0.071 cm-1 mol-1 and expressed as IU x 104/g Hb at 370C.
The glutathione peroxidase (GPx) activity was measured and the activity was expressed as mol of glutathione oxidized
min/mg Hb.
Statistical Analysis
The results of the study are expressed as mean SEM. Statistical analysis was performed using Duncans
Multiple Range (DMR) test. The significance was set at (P 0.05).

RESULTS
Data presented in table 1 suggests that a significant increase in the activity of anti oxidant enzymes such as super
oxide dismutase (+9%, +10%), catalase (+12%, +10%) GSH (+20%, +21%) and GPx (-13%, -21%) is reduction in
concentration of glutathione peroxidase (GPx) in erythrocytes of experimental subjects to pyrethroids, group II and III
when compared to controls group I. The data presented in Table 2 indicated that exposure of two different pyrethroids
allethrin and prallethrin by inhalation but significant increase in enzyme activities of plasma ALP and LDH of mosquito
repellent pyrethroids and control groups. Experimental subjects (allethrin users and prallethrin users) significantly
increased (p0.05) plasma enzyme activities when compared to control group. Plasma protein carbonyl content levels
increased significantly in exposed subjects (group II and III) when compared to controls, and total sulphydryl groups were
significantly decreased in group II and III human exposed subjects, respectively when compared to controls (group I) who
do not use any mosquito repellents.
Plasma lipid peroxidation was significantly increased in allethrin and prallethrin subjects when compared to
controls our previous report (Narendra et al, 2007, 2008a, 2008b), Data presented in Figure 1 suggests that a significant
decrease in erythrocyte membrane lipid peroxidation (-16%, -16%) in experimental subjects (group II and III) when
compared to controls, significant decrease (p0.008) our previous report (Narendra et al, 2007, 2008a, 2008b). Figure 2
shows that there was significant inhibition of acetyl cholinesterase activity (-19%, -16%) in red cell membrane in
experimental subjects (group II and III) when compared to controls. Increased concentration of nitrite (+13%, +15%) and
nitrate (+20%, +19%) in plasma suggest an increased production of nitric oxide in human volunteers exposed to mosquito
repellent pyrethroids (allethrin and prallethrin) when compared to controls Figure 3. Nitric oxide levels were increased in
plasma and red cell lysate of controls and mosquito repellent pyrethroid users. A significant increase (p0.05) in protein
carbonyl content, significant decrease (p0.05) in total sulphydryl groups were followed by a significant increase (p0.05)
in nitrites/nitrates of plasma and erythrocyte lysate of allethrin and prallethrin experimental subjects when compared to

Impact Factor (JCC): 5.1064

Index Copernicus Value (ICV): 3.0

21

Mosquito Repellent Pyrethroid Induced Biochemical and Biophysical Changes in Plasma and Antioxidant
Status in Human Male Volunteers Exposed to Long Term Allethrin and Prallethrin Inhalation

controls Figure 3.

DISCUSSIONS
Pyrethroid based allethrin and prallethrin are enter easily into humans who are exposed to them for continuously
8-10 hours a day; these compounds enter into circulation with maximal accumulation in all biomembranes and in specific
tissues such as blood, nerve, adipose and other tissues with an increased uptake due to their lipophilic nature (Sinha et al,
2004; Theeraphap et al, 2003). In the present study there was a significant decrease in erythrocyte membrane lipid
peroxidation previous report (Narendra et al, 2007; 2008) and acetyl cholinesterase activity was inhibited in red cell
membrane of allethrin and prallethrin exposed subjects when compared to controls (who did not use any mosquito
repellents).
There was an increase in activity of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT),
decreased glutathione (GSH) in erythrocytes and decreased concentration of glutathione peroxidase (GPx) in erythrocytes
of exposed subjects (group II and III) when compared to controls. Superoxide anion radicals (O20-) hydrogen peroxide
(H2O2) and hydroxyl radicals (OH) are palls of oxidative metabolism. Damage at the cell level by oxidants is attenuated by
antioxidant enzymes such as SOD, CAT and GPx. Oxidative stress, generated by xenobiotics, induces disturbances in
antioxidant enzyme systems. Recent reports show the generation of free radicals in fenvalerate and cypermethrin-induced
pyrethroid treated mice and rats an in increased activity of superoxide dismutase and catalase (Gabbinelli et al, 2002; Maiti
et al, 1995). Pyrethroids can be expected to have two modes of action: it may induce oxidative stress and, it is a
hydrophobic compound it may accumulate in cell membrane and disturb membrane structure (Gabbinelli et al, 2002).
The activities of two cytosol antioxidant enzymes, SOD, and CAT, were altered in table 1. Pyrethroids and their
esters, from cyanohydrins, which decompose to cyanides and aldehydes. Cyanide ions are mainly converted to thiocyanate
and CO2. The aldehydes and other lipophilic conjugates may produce oxidative stress in pyrethroid toxicity. Although only
few studies have investigated the effects of pyrethroids on AChE in vivo, the results of these studies are contradictory
(Bandyopadhyay, 1982; Gazula and Kosagi, 1995; He et al, 2002; Hossain et al, 2005; Reddy et al, 1991). In the present
study inhibition of AChE activity has been inhibited in pyrethroid exposed subjects (group II and III) when compared to
controls. Inhibition of AChE is believed to be the principal mode of action of pyrethroid compounds.
The inhibition in AChE activity in target tissues is often followed as a measure of pesticide intoxication
(Jayatatnam and Maroni, 1994; Kale et al, 1999). In many of the earlier studies, correlation between blood AChE inhibition
and inhibition in target tissues has been shown (Kale et al, 1999; Koshakji et al, 1973; Su et al, 1971; Yang and Dettbarn,
1996). Allethrin and prallethrin of AChE in RBC inhibited the activity, AChE is synthesized in liver and RBC (Kale et al,
1999). The present results also show significant correlation between decrease in erythrocyte membrane lipid peroxidation
and inhibition of AChE activity in RBC. Nitric oxide (NO) a labile molecule with half life of only a few seconds, is
synthesized mainly in the endothelium (Moncada et al, 1991). It is rapidly oxidized by tissue oxygen to the stable end
products, nitrate (NO3-) and nitrite (NO2-).
In the circulation, nitrite is almost converted to nitrate by hemoglobin (Stamler et al, 1992). The best index of
overall NO production, therefore, is the total concentration of both nitrate and nitrite. An increased nitric oxide levels in
plasma compartments of experimental are evident from measurements of nitrite and nitrate concentrations in plasma.
Nitric oxide is endogenously produced chiefly by endothelial cell nitric oxide synthase (NOS) and is released into blood

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Narendra Maddu, Nagajothi G, Fareeda Begum. S & B. Sreekanth

stream where it is quickly scavenged by Hb in erythrocyte or oxidized to nitrite and further oxidized to nitrate
(Ignarro et al, 1993). In addition a significant increase of nitric oxide (NO) production occurs in other tissues as different
isoforms of NOS exist.
Nitric oxide plays a principle role in basal blood flow regulation and vascular homeostasis. Besides many other
physiological processes are mediated and regulated by direct or indirect actions/of NO and/or NO derived species. NO is
capable of diffusion to great distances at physiological oxygen tension in tissue maintaining a teleological balance.
This has been observed in erythrocytic red cell mass and plasma mass (Wang et al, 2004). Though direct NO production
was not determined, plasma nitrite (NO2) and nitrate (NO3) were determined and observed in the present study asserted an
increased production and bioavailability of nitric oxide that might have rendered tolerance against haemolysis by the free
radical scavenging effect and indirectly by other possible protective mechanisms of nitric oxide (McCuskey et al, 1995;
Nanji et al, 1995; Narendra et al, 2007; 2008; Oekonomaki et al, 2004).

CONCLUSIONS
Increase in erythrocyte membrane antioxidant enzymes such as SOD, Catalase, and decreased GSH, decreased
activity of GPx exposed subjects appears to be an adaptive biochemical changes in erythrocyte membrane.
Decreased levels of erythrocyte membrane lipid peroxidation and acetyl cholinesterase activity. Increased production of
plasma nitric oxide (NO), nitrite (NO2) and nitrate (NO3) levels were observed. Further studies are needed to correlate the
toxic effects of prolonged use of allethrin and prallethrin on health status of individuals.
Table 1: Activities of Antioxidant Enzymes and Glutathione Levels
in Control and Allethrin and Prallethrin Subjects
Parameter
Superoxide Dismutase (SOD) (Units /
mg Hb/min)
Catalase (CAT) (IU/104/gm Hb)
Red Cell Reduced glutathione
(GSH)(mol/gm Hb)
Glutathione Peroxidase (GPx) (mol
of GSH xidized/min/mg Hb)

Controls

Groups
Allethrin Users

Prallethrin Users

6.242.82

9.832.76

10.122.86

12.081.74

17.061.82

17.811.86

8.820.08

10.600.08

10.650.05

17.861.62

11.581.55

12.751.48

Values are expressed as Mean SEM, in each column followed by the same letter are not significantly different
(P0.05) from each other according to Duncans Multiple Range (DMR) test, n = 24.
Table 2: Effect of Chronic Pyrethroid Inhalation on Plasma ALP and
LDH, Carbonyl Content, SH Groups in Human Male Volunteers
Parameter
Alkaline Phosphatase (ALP) (IU/L)
Lactate Dehydrogenage (LDH) (IU/L)
Carbonyl groups (nmols/mg protein)
SH groups groups (nmols/mg protein)

Controls
60.242.82
312.0410.54
0.820.08
4.340.62

Groups
Allethrin Users
86.122.06
417.0631.82
1.650.08
2.580.55

Prallethrin Users
89.152.64
470.8134.86
1.540.05
2.750.48

Values are expressed as Mean SEM, in each column followed by the same letter are not significantly different
(P0.05) from each other according to Duncans Multiple Range (DMR) test, n = 24.

Impact Factor (JCC): 5.1064

Index Copernicus Value (ICV): 3.0

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Mosquito Repellent Pyrethroid Induced Biochemical and Biophysical Changes in Plasma and Antioxidant
Status in Human Male Volunteers Exposed to Long Term Allethrin and Prallethrin Inhalation

Figure 1: Effect of Pyrethriods on Erythrocyte Membrane Lipid Peroxidation (LPO)

Values are expressed as Mean SEM, in each column followed by the same letter are not significantly different
(P0.05) from each other according to Duncans Multiple Range (DMR) test, n = 24.

Figure 2: Effect of Pyrethroids on RBC Acetyl Cholinesterase (AChE)

Values are expressed as Mean SEM, in each column followed by the same letter are not significantly different
(P0.05) from each other according to Duncans Multiple Range (DMR) test, n = 24.

Figure 3: Influence of Allethrin and Prallethrin Inhalation in Plasma Nitrite and Nitrate
Values are expressed as Mean SEM, in each column followed by the same letter are not significantly different
(P0.05) from each other according to Duncans Multiple Range (DMR) test, n = 24.

ACKNOWLEDGEMENTS
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Narendra Maddu, Nagajothi G, Fareeda Begum. S & B. Sreekanth

The authors are thankful to the Principal and authorities of Govt. Medical College & General Hospital for
providing blood samples.

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Mosquito Repellent Pyrethroid Induced Biochemical and Biophysical Changes in Plasma and Antioxidant
Status in Human Male Volunteers Exposed to Long Term Allethrin and Prallethrin Inhalation

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