Adverse Haematological and Biochemical Effects of Certain Formulated Insecticides in Male Rats
Abdel-Tawab H. Mossa
Moustafa A. Abbassy
Pesticides formulations are complex mixtures and the toxicity information on
active ingredients alone is not sufficient to evaluate the risk of adverse health
effects of commercial pesticides. So the present study was designed to investigate
the adverse effects of exposure to formulated chlorpyrifos-ethyl (9.60 mg kg-1
b.wt.), chlorpyrifos-methyl (300 mg kg-1 b.wt.) and methomyl (1.70
mg kg-1 b.wt.) on some haematological and biochemical parameters
of male rats given repetitive oral doses for 90 consecutive days. There was
significant decrease in body weight gain of chlorpyrifos-ethyl, chlorpyrifos-methyl
and methomyl and increase in relative liver and kidney weights of chlorpyrifos-ethyl
and chlorpyrifos-methyl treated rats. Chlorpyrifos-methyl caused significant
decrease in Hb conc. Haematocrit value (PCV, %) and Red Blood Cells (RCBs) counts,
while chlorpyrifos-ethyl and methomyl caused significant decrease in PCV% and
White Blood Cells (WBCs) counts. All of the tested insecticides increased significantly
serum Aspartate Aminotransferase (AST) and Alanine Aminotransferase (ALT) activities
and decreased significantly the total protein level. In addition, the tested
insecticides caused significant increase in serum uric acid and creatinine.
We can conclude that both technical and formulated methomyl and chlorpyrifos-ethyl
and its methyl analogues caused significant alteration on hematological and
biochemical parameter of mal rats. The effects of commercial form of tested
insecticides were more pronounced than its technical form.
Received: March 30, 2012;
Accepted: May 09, 2012;
Published: July 23, 2012
Organophosphorus (OPIs) and carbamate insecticides have made valuable contribution
to human health by increasing food and fiber production and by reducing occurrence
of vector-borne diseases (Blindauer et al., 1999).
The long-term application of these pesticides has resulted in residues accumulating
in soil, water and in different environmental components; thereby posing a serious
threat to public health in Egypt (Selim and El-Sebae, 1995).
According to World Health Organization report every year, 3 million serious
poisonings cases with insecticides occur worldwide and of these approximately
220,000 die (WHO, 1997).
In acute toxicity, the main mechanism of toxicity of OPIs and carbamate is
binding to the enzyme acetylcholinesterase and inhibiting its activity that
results in accumulation and prolonged effect of acetylcholine and consequently
follows with acute muscarinic and nicotinic effects (Ecobichon,
1996). Unfortunately, while the acute toxicity of most pesticides is well-documented
(Ecobichon et al., 1990), information on chronic
human illness resulting from pesticide exposure is not as sound (Wilkinson,
In fact, pesticides formulations are complex mixtures and the toxicity information
on active ingredients alone is not sufficient to evaluate the risk of adverse
health effects of commercial pesticides (Mansour and Mossa,
2005). Consequently, World Health Organization emphasized the necessity
of evaluating toxic hazard of the formulated pesticides (WHO,
1991). Therefore, this study was conducted to evaluate the effect of subchronic
exposure to formulated chlorpyrifos-methyl, chlorpyrifos-ethyl and methomyl
on blood profile and some biochemical parameters of male rats.
MATERIALS AND METHODS
Chemicals: Chlorpyrifos-ethyl (Dursban® 48% EC; O,O-diethyl-O-(3,5,6-trichloro-2-pyridinyl) phosphorothioate) and methomyl (Lannate® 90%WP; S-methyl-N-(methyl carbamoyl oxy-thio- acetimidate) were supplied from Kafr El-Zayat Pesticides and Chemicals Company, Egypt. Chlorpyrifos-methyl (Reldan® 50% EC; O,O-dimethyl-O-(3,5,6-trichloro-2-pyridinyl phosphorothioate) was supplied by National Agricultural Chemicals Company, Egypt. All other chemicals were of reagent grades and obtained from the local scientific distributors in Egypt.
Animals, groups and treatments: Male Wistar rats (weighting 100-120 g) purchased from Animal Health Research Center, Cairo, Egypt. Animals received human care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals. Animals were housed in clean plastic cages with free access to food (standard pellet diet) and tap water ad libitum, under standardized housing conditions (12 h light/dark cycle, temperature was 22±1°C and a minimum relative humidity of 40%) in the laboratory animal room. After one week of adaptation to laboratory conditions, the animals randomly divided into four groups each comprising of six animals, as follows: first group (chlorpyrifos-methyl), second group (chlorpyrifos-ethyl), third group (methomyl) and fourth group (control).
Tested insecticides were daily freshly prepared, adjusted weekly for body weight
changes and administered orally for 90 consecutive days. All treatments were
administered at a dose equal to 0.1 LD50, as reported by Tomlin
(2004), chlorpyrifos-ethyl (9.60 mg a.i. kg-1 b.wt.), chlorpyrifos-methyl
(300 mg a.i. kg-1 b.wt.) and methomyl (1.70 mg a.i. kg-1
b.wt.). The control group received an equivalent volume of distilled water (0.5
Blood collection and relative organs weights: In all groups, body weights were weekly recorded. At the end of exposure period, blood samples drawn from all rats under ether anesthesia and collected in EDTA-tubes for haematological studies and normal glass tubes to separate the sera for biochemical studies. Then, within 20 min of blood collection, the sera samples were drawn from blood after centrifugation at 3500 rpm (600 g) for 10 min at 4°C, using Universal 32 R centrifuge (Hettich-Zentrifugen GmbH, Tuttlingen, Germany). The sera was kept in a deep freezer (-20°C) until analyzed.
After blood collection, the rats were sacrificed by cervical dislocation. Liver and kidney of rats were removed and weighted. Then, the relative organs weights were calculated.
Measurement of blood constituents: Red Blood Cells (RBCs) and
White Blood Cells (WBCs) were counted according to the methods of Britton
(1963) and Seiverd (1964). Haemoglobin (Hb) measurement
was carried out according to Wintrobe method (Wintrobe, 1965).
Haematocrit value (PCV) was determined using microhaematocrit centrifuge Model
Determination of liver and kidney biomarkers: Serum ALT and AST activities
were measured as described by Reitman and Frankel (1957)
accordance with manufacturers' instructions. Total protein was determined by
the method described by Henry (1964), creatinine determination
was done according to Henry (1974) and uric acid was determined
by the methods of Barham and Trinder (1972) and Fossati
et al. (1980) using Boehringer Mannheim Gmbh Diagnostic Kits.
Spectrophotometric measurements: The Spectrophotometric measurements were performed by using a Jenway, UK, 6305 UV/Vis spectrophotometer.
Statistical analysis: The data were analyzed by using SPSS (version 11.0) for Windows and expressed as means±SE. Paired samples t-test was used to compare between the data of the control and those of treatments. The level of significance was 0.05 and 0.01.
Repeated oral administration of chlorpyrifos, chlorpyrifos-methyl and methomyl at 9.6, 300 and 1.7 mg kg day-1 did not produce any signs of toxicity and mortality during 90 days exposure. However, there was significant decrease of body weight gain of chlorpyrifos, chlorpyrifos-methyl and methomyl treated rats (Fig. 1). The body weight recorded 186.26 g in the control group and decreased to 175.80, 172.40 and 162.85 g in CPF-methyl, CPF-ethyl and methomyl treated groups, respectively. Also, the relative organ weights of liver and kidney were significantly increased in chlorpyrifos and chlorpyrifos-methyl treated rats groups compared to the control (Fig. 2). The relative liver and kidney weights accounted 2.79 and 0.63% in control and elevated to 3.42, 3.78 and 2.81% of liver and 0.77, 0.78 and 0.64% of kidney in CPF-methyl, CPF-ethyl and methomyl treated groups, respectively.
Blood profile: Results of Hb, PCV, RBCs and WBCs are shown
in Table 1. Except the treatments of chlorpyrifos-methyl,
the rest of the treatments caused insignificant decrease of haemoglobin (Hb)
and the decrease in case of chlorpyrifos-methyl accounted to 13.1 g dL-1
in comparison to control value, 22.7 g dL-1, which was statistically
highly significant (p<0.01).
||Body weights of rats exposed to tested insecticides for 90
consecutive days, Each value is a means±SE; n = 6, Statistical difference
from the control: *Significant at p<0.05 and **Highly significant at
p<0.01, CPF: Chlorpyrifos
||Relative liver and kidney weights of rats exposed to tested
insecticides for 90 consecutive days, Each value is a means±SE; n
= 6, Statistical difference from the control: *Significant at p<0.05
and **Highly significant at p<0.01. Relative weight (%) = (organ weight/body
weight)x100, CPF: Chlorpyrifos
|| Blood profile of rats exposed to test formulated insecticides
for 90 consecutive days
|Each value is a Mean±SE, n = 6; Statistical difference
from the control: *Significant at p≤0.05 and **Highly significant at
p≤0.01. Hb: Hemoglobin: RBCs: Red blood cells: WBCs: White
blood cells and PVC: Haematocrit value
||AST and ALT activities and total protein and glucose level
in the sera of rats exposed to test formulated insecticides for 90 consecutive
|Each value is a Mean±SE, n = 6; Statistical difference
from the control: *Significant at p≤0.05 and **Highly significant at
p≤0.01. Aspartate aminotransferase (AST) and alanine aminotransferase
Packed Cell Value (PCV) is a parameter for determination of the volume of
red blood cell. All formulated insecticides caused significant decrease (p<0.01)
of the PCV %, chlorpyrifos-methyl was more effective than other insecticides
and showed value accounted to 25.00% compared to control value, 48.66%. On the
other hand, RBCs counts was statistically decrease (p<0.01) after treatment
with chlorpyrifos-methyl and accounted to 2.54 compared to control value 3.97x106
mm. In contrast, chlorpyrifos-ethyl and methomyl caused decrease in WBCs
counts accounted to 12.20±0.31 and 14.10 compared to control value 15.80x103
mm which was statistically significant (p<0.05).
Liver and kidney biomarkers: The results of serum biochemical parameters
of male rats orally administered the tested formulated pesticides for 90 days
are shown in Table 2.
|| Uric acid and creatinine concentration in the sera of rats
exposed to test formulated insecticides for 90 consecutive days
|Each value is a Mean±SE, n = 6, Statistical difference
from the control: *Significant at p≤0.05 and **Highly significant at
Serum biochemical parameters, AST and ALT activities were significantly increased
than that of control rats and the formulated OPs insecticides seemed to
induce higher elevation of AST and ALT activities than carbamate insecticide,
methomyl. On the other hand, all of the tested insecticides induced significant
decreases in serum total protein as compared with the untreated group.
As shown in Table 3, chlorpyrifos-methyl (6.55 mg dL-1) and methomyl (7.41 mg dL-1) caused significant elevation of serum uric acid as compared to the control (4.06 mg dL-1). In addition, all of the tested insecticides induced significant increases in sera creatinine as compared with the untreated rats.
The present study investigated the adverse effects of exposure to formulated chlorpyrifos-ethyl (9.60 mg kg-1 b.wt.), chlorpyrifos-methyl (300 mg kg-1 b.wt.) and methomyl (1.70 mg kg-1 b.wt.) on some haematological and biochemical parameters of male rats given repetitive oral doses for 90 consecutive days.
Changes in the body weight after insecticide dosing was used as a valuable
index of insecticide-related organ damage (Lu, 1996; Mansour
and Mossa, 2010a; Mossa et al., 2011). In
the present study, results revealed that all of the tested insecticides caused
significant decrease in body weights of treated rats and increase in the relative
weight of liver and kidney. The reduction in body weight gains may be due to
the overall increased degradation of lipids and proteins as a result of the
direct effects of anti-cholinesterase compound (Goel et
al., 2005; Mansour and Mossa, 2011; Mossa
et al., 2011). Other investigations have reported the reduction in
body weight and change in relative organs weights in rats (Woolliams
et al., 1983; Mansour et al., 2001;
Mossa et al., 2011) and mice (Ambali
et al., 2007) after exposure to anti-cholinesterase insecticides.
In fact, haemoglobin concentration and haematocrit values are directly correlating
with RBCs count (El-Bakary et al., 1995).
This is due to the synergistic link among these blood parameters in all vertebrates.
This close correlation between erythrocyte count, haemoglobin concentration
and haematocrit value was also reported for other vertebrates including man
(Harris, 1972). In the present study, chlorpyrifos-methyl
caused significant decrease in haemoglobin (Hb) concentration. Packed cell value
(PCV%) is a parameter for determination of the volume of red blood cell. All
formulated insecticides caused significant decrease (p<0.01) in the PCV%,
chlorpyrifos-methyl was more effective than other insecticides. RBCs counts
was statistically decrease (p<0.01) after treatment with chlorpyrifos-methyl.
In contrast, chlorpyrifos-ethyl and methomyl caused decrease in WBCs counts
and WBCs was accounted 12.20±0.31 and 14.10 compared to control
value (15.80), which was statistically significant (p<0.05). Previous studies
found that the direct effect of pesticides is a reduction in the total number
of erythrocytes, PCV and Hb content (El-Sahhaf, 1995;
Saxena and Saxena, 1997; El-Gendy
et al., 1999; Khalaf-Allah, 1999; Yousef
et al., 1999; Mossa, 2004). It was thought
that these changes were due to an increase rate of breakdown of red cells and/or
the toxic effect of pesticides on bone marrow. The increase of WBCs may
be due to the activation of the animals defense mechanism and immune system.
Also, the reduction in Hb content may be due to increased rate of breakdown
of red cells and/or reduction in the rate of formation of RBCs (Mossa,
2004). Shakoori et al. (1990) suggested that
the decrease in RBC is either indicative of excessive damage to erythrocytes
or inhibition of erythrocyte formation in rabbits. In this work, results agree
in most cases with the results of several authors. Shakoori
et al. (1990) found decrease in erythrocytic, leukocytic counts;
PCV and Hb content in the blood of bifenthrin-treated rabbits.
Liver is the first organ to face any foreign molecule that is carried through
portal circulation and it is subjected to most damage. In addition, transaminase
(AST and ALT) are important enzymes in the biological processes. They play a
role in amino acids catabolism and biosynthesis. Consequently, they are considering
as specific indicators for liver damage (Harper, 1979)
and responsible for detoxification processes, metabolism and biosynthesis of
energetic macromolecules for different essential functions (Aly
et al., 1997). The present results showed a significant increase
in the activities of both AST and ALT in the serum of treated rats. Hayes
et al. (1989) reported that one of the most indicators for liver
damage and function is increase in the activities of serum transaminase (AST
and ALT) in the serum. This increasing may be indicative of initial cell injury
occurring in advance of gross hepatic pathology, since not only distinct cellular
damage but also any condition leading to changes in membrane permeability also
causes a generalized release of enzymes from the cell (De
Bruin, 1979). Several studies have showed that the activities of transaminases
were increased in human and animals after exposure to pesticides (Khalaf-Allah,
1999; Abbassy et al., 2000; Mossa,
2004; Mansour and Mossa, 2011; Mossa
et al., 2011).
Results revealed that the tested insecticides caused a significant decrease
in total protein and highly significant increase in creatinine in the sera of
rats compared to control. On the other hand, chlorpyrifos-methyl and methomyl
caused significant increase in serum uric acid. The protein content in different
organs was affected as a result of exposure to different insecticides (Enan
et al., 1982; Nabila et al., 1990;
El-Bakary, 1993; Khan et al.,
2003; Mossa, 2004; Mansour and
Mossa, 2010a, b; 2011; Mossa
et al., 2011). The decrease in protein content might be due to the
imbalance between the rate of protein synthesis and the rate of its degradation
in the liver. Rodwell (1979) reported that an elevated
level of urea in blood is correlated with an increase protein catabolism in
the mammalian body. It may also result due to a more efficient conversion of
ammonia to urea as a result of increased synthesis of enzyme involved in urea
production (Rodwell, 1979; El-Sebae
et al., 1981). Other investigations showed an increase of urea and
creatinine in the serum of chicks and rats-treated with acute and chronic doses
of 2,4-D and cypermethrin (Charles and Leeming, 1998;
Yousef et al., 1999). Creatinine is a metabolite
of creatine and is excreted completely in urine via glomerular filtration. An
elevation of its level in the blood is thus an indication of impaired kidney
function (Lu, 1996).
The effects of chlorpyrifos-ethyl and its methyl analogues on hematological
and biochemical parameter revealed that these compounds caused significant alteration
in blood picture , liver and kidney biomarkers in treated rats. The effect of
chlorpyrifos-ethyl was more pronounced than its methyl analogues in its commercial
form. Also, chlorpyrifos-ethyl was most toxic (acute oral LD50 for
rats 135-163 mg kg-1) and chlorpyrifos-methyl (acute oral LD50
for rats>3000). The two OP insecticides contain the same structure except
the substitution of ethyl groups in chlorpyrifos by methyl groups in chlorpyrifos-methyl,
which was less in its mammalian toxicity than chlorpyrifos. This substitution
may affect the intrinsic toxicity and/or sensitivity of the structure to the
metabolic systems in the treated rats (Mossa, 2004; Abbassy
et al., 2005).
We can conclude that both technical and formulated methomyl and chlorpyrifos-ethyl and its methyl analogues caused significant alteration on hematological and biochemical parameter of mal rats. The effects of commercial form of tested insecticides were more pronounced than its technical form.
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