research article

1. Adverse effects of exposure to low
doses of chlorpyrifos in lactating rats
Sameeh A Mansour and Abdel-Tawab H Mossa
Abstract
This study was conducted to shed light on the effect of exposure of lactating rat to chlorpyrifos (CPF). CPF was
orally administered to lactating rats at 0.01 mg kg1
b.wt. (acceptable daily intake, ADI), 1.00 mg kg1
b.wt. (no
observed adverse effects level, NOAEL) and 1.35 mg kg1
b.wt. (1/100 LD50) from postnatal day 1 (PN1) until
day 20 (PN20) after delivery. Results indicated decreases in body weight and increases in relative liver and
kidney weights of exposed dams. Significant damage to liver was observed via increased plasma levels of
aminotransferases (aspartate aminotransferase (AST) and alanine aminotransferase (ALT)) lactate
dehydrogenase (LDH) and g-glutamyle transferase (g-GT) in a dose-dependent manner. At two high doses
of CPF (1.00 and 1.35 mg kg1
b.wt.), the lactating mothers showed significant decrease in the activity of cho-
linesterase (ChE). Lipid peroxidation was significantly increased, while glutathione s-transferase (GST) and
superoxide dismutase (SOD) were significantly decreased compared to control. At high dose of CPF (1.35
mg kg1
b.wt.), total protein and uric acid levels were significantly increased. CPF caused dose-related histo-
pathological changes in liver and kidney of the CPF-treated dams.
Keywords
Chlorpyrifos, lactation rats, lipid peroxidation, oxidative damage, liver and kidneys dysfunction
Introduction
Chlorpyrifos, CPF (0,0-diethyl-0-3,5,6-trichloro-2-
pyridyl phosphorothioate), is a broad-spectrum,
chlorinated organophosphate (OP) insecticide, con-
sidered as one of the largest selling OP in the world,
and has both agricultural and urban uses. Some esti-
mates for pregnant women and children indicate that
CPF exposures exceed no observable adverse effect
level (NOAEL), even in scenarios of common use
(Davis and Ahmed, 1998; Gurunathan et al., 1998).
Mothers may be exposed to lipophilic chemical (e.g.
CPF) from various sources including air, food, water
and occupational and household environments.
Lipophilic chemicals can be stored and accumulated
over time in body fat and can then be mobilized into
milk during lactation. Generally, chemicals enter
breast milk by passive transfer from plasma, and their
concentration in milk is proportional to their solubi-
lity and lipophilicity (Anderson and Wolff, 2000),
showing the relevance of the present issue with
human health concerns. Previous study reported that
OP pesticides (e.g. CPF) can be accumulated and
excreted in human milk (Marty et al., 2007; Mattsson
et al., 2000; Salas et al., 2003; Sanghi et al., 2003).
Also, it has been reported that milk CPF concentra-
tions were up to 200 times those in blood of pregnant
rats dosed with CPF (Mattsson et al., 2000). The latter
authors also made an exposure estimate of CPF to
nursing pups and reported it as roughly 0.1 mg kg1
b.wt. via nursing of dams exposed by gavage to
5 mg kg1
b.wt. The nursing dose to pups was attenu-
ated greatly from gavage dose to their dams.
In the context of chemical toxicology, it is important
to identify the chemical responsible for human
toxicity. Biological monitoring is a well-established
Environmental Toxicology Research Unit (ETRU), Pesticide
Chemistry Department, National Research Centre (NRC),
Cairo, Egypt
Corresponding author:
Abdel-Tawab H Mossa, Environmental Toxicology Research Unit
(ETRU), Pesticide Chemistry Department, National Research
Centre (NRC), Tahrir Str, Dokki, Cairo 12311, Egypt
Email: abdeltawab.mossa@yahoo.com
Toxicology and Industrial Health
27(3) 213–224
ª The Author(s) 2011
Reprints and permission:
sagepub.co.uk/journalsPermissions.nav
DOI: 10.1177/0748233710384054
tih.sagepub.com

2. technique for assessing intakes and uptakes of
toxic chemicals following either occupational or
environmental exposure. For assessing human
exposure to many of the OP insecticides and for linking
OP exposures in pregnant women to subsequent
adverse birth outcomes, the metabolite containing the
organic moiety, such as TCPy in the case of CPF, has
been used as a more specific urinary biomarker
(Berkowitz et al., 2004; Eskenazi et al., 2004; Nolan
et al., 1984).
In fact, the toxicity of many xenobiotics is associ-
ated with the production of oxygen-free radicals,
more generally known as ‘‘reactive oxygen species’’
(ROS), which are not only toxic themselves but are
also implicated in the pathophysiology of many dis-
eases (Abdollahi et al., 2004; Akhgari et al., 2003).
The harmful effects of ROS are balanced by the
antioxidant action of nonenzymatic antioxidants in
addition to antioxidant enzymes (Halliwell, 1996).
Despite the presence of the cell’s antioxidant defence
system to counteract oxidative damage from ROS,
oxidative damage accumulation during the life cycle
has been proposed to play a key role in the develop-
ment of age-dependent diseases such as cancer, arter-
iosclerosis, arthritis, neurodegenerative disorders and
other conditions (Halliwell and Gutteridge, 1999).
It has been reported that OP insecticides may induce
oxidative stress following acute exposure in humans
(Banerjee et al., 1999) and animals (Mansour and
Mossa, 2009, 2010a).
Breastfeeding is one of the most important contri-
butors to infant health and prolonged breastfeeding
protects babies from common childhood infections
through mechanisms that are interactive, adaptive and
extend into childhood (Kramer and Kakuma, 2002).
Also, breastfeeding offers a range of health benefits
for mothers, including a reduced risk of ovarian
(Rosenblatt and Thomas, 1993) and pre-menopausal
breast cancer (Collaborative Group on Hormonal
Factors in Breast Cancer, 2002). However, potential
risks associated with breast-feeding need to be
factored into the overall public health assessment
when mothers are encouraged to breast-feed their new-
born infants (Gallenberg and Vodicnik, 1989). This
could be seen as a logical demand if we considered that
lactation may pose a ‘sort of stress’ to the mothers.
Thus, such mothers will be more vulnerable, than non-
lactating mothers, to other chemical stressors. To the
best of our knowledge, toxicological information of
pesticides on lactating animals (e.g. rats) are very lim-
ited. So, the present study was undertaken on lactating
rats to evaluate the effect of exposure to CPF at low
doses. Oxidative stress, lipid peroxidation (LPO), as
well as liver and kidney dysfunction will be the criteria
of assessing the exposure effects.
Materials and methods
Chemicals
Chlorpyrifos ‘CPF’ (M.Wt. 350.6; 99% purity) was
obtained from Dow AgroSciences (Indianapolis,
Indiana, USA) and 2-thiobarbituric acid (TBA; 2,
6-dihydroxypyrimidine-2-thiol; TBA) was purchased
from Merck (Germany). All other chemicals were of
reagent grade and were obtained from the local
scientific distributors in Egypt. The kit of lactate
dehydrogenase (LDH) was obtained from Spinreact
(Santa Coloma, Spain), gamma-glutamyl transferase
(g-GT) from Greiner Diagnostic GmbH (Bahlingen,
Germany), protein from Stanbio Laboratory (Texas,
USA) and albumin from Biogamma (Roma, Italy). The
kits of superoxide dismutase (SOD), glutathione s-
transferase (GST), aminotransferases (ALT and AST),
uric acid and creatinine were obtained from Biodignos-
tic, and cholinesterase (ChE) from Diamond Diagnostic
(Egypt).
Animals and housing
The healthy male and female albino rats of the Wistar
strain Rattus norvegicus, weighing 200–220 g, were
obtained from the Animal Breeding House of the
National Research Centre (NRC), Dokki, Cairo,
Egypt. Rats were allowed to acclimate to laboratory
conditions for at least 1 week before breeding. Thirty
virgin female rats were distributed into 10 cages. In
each cage, one male was placed for overnight and the
presence of spermatozoa was checked in the vaginal
smear the following morning. This day was connoted
as gestation day 0 (GD 0). At this time, pregnant
females were individually housed in clean plastic
cages in the laboratory animal room (23
C + 2
C)
on the standard pellet diet and tap water ad-libitum,
a minimum relative humidity of 40% and a 12 h
dark/light cycle. The day of parturition, was consid-
ered day 0 of lactation, postnatal day 0 (PND 0).
Offspring of each litter were randomly reduced to 8
pups of the equal number of sex’s +1, it has been
shown that this procedure maximizes the lactation
performance (Fishbeck and Rasmussen, 1987).
The experimental work on rats was performed with
the approval of the Animal Care Experimental
214 Toxicology and Industrial Health 27(3)

3. Committee, National Research Centre, Cairo, Egypt,
and according to the guidance for care and use of
laboratory animals (NRC, 1996).
Experimental design
On the first day after parturition (PND1), 20 individu-
ally housed dams were segregated into four different
groups, five each. CPF was dissolved in corn oil and
administered by gavage at a volume of 0.5 mL/rat.
Three groups of dams were given daily, via oral route,
doses equaled to 0.01 mg kg1
b.wt. (ADI), 1.00 mg
kg1
b.wt. (NOAEL), and 1.35 mg kg1
b.wt. (1/100
LD50), of CPF according to Tomlin (2005), during the
lactation period (PND 1 to PND 20), respectively. The
fourth group of dams was used as control and received
the equivalent volume of corn oil. Dosages of CPF
administrated were adjusted daily for body weight
changes and given at approximately the same time
each morning. The animal’s cages were cleaned daily
to minimize potential contamination.
Blood collection, body weight and
organ weight ratio
Dam’s body weight was recorded daily prior to dosing.
The blood samples were drawn from all dams on
postnatal day 21 (PND 21) under ether anesthesia by
puncturing the retero-orbital venous plexus of the ani-
mals with a fine sterilized glass capillary and collected
into heparinized tubes. Within 30 min of blood collec-
tion, the plasma samples were drawn from blood after
centrifugation at 3500 rpm (600g) for 10 min at 4
C,
using Hereaeus Labofuge 400R, Kendro Laboratory
Products GmbH, Germany, to separate the plasma. The
plasma was kept in a deep freezer (20
C) until
analyzed within 10 days maximum.
After blood collection, the dams were sacrificed by
cervical dislocation. Liver and kidneys of dams were
quickly removed and weighted individually. Then, the
relative organs weights to the body weights were
calculated.
Biochemical analyses
Antioxidant enzyme assays. SOD and GST were
determined in plasma according to the manufacturer’s
instructions referred to in Woolliams et al. (1983) and
Habig et al. (1974), respectively. The activities were
expressed in terms of mmol/min/mg protein for both
enzymes.
Estimation of lipid peroxidation. Malondialdehyde
(MDA), as a marker for LPO, was determined by the
double-heating method of Draper and Hadley (1990).
The principle of the method is based on spectrophoto-
metric measurement of the color produced during the
reaction of TBA with MDA. For this purpose, 2.5 mL
of 100 gl1
trichloroacetic acid (TCA) solution was
added into 0.5 mL plasma in a centrifuge tube and
placed in a boiling water bath for 15 min. After
cooling under tap water, the mixture was centrifuged
at 600g for 10 min, and 2 mL of the supernatant was
transferred into a test tube containing 1 mL of 6.7 gl1
TBA solutions and placed again in a boiling water
bath for 15 min. The solution was then cooled under
tap water and its absorbance was measured spectro-
photometrically at 532 nm. The concentration of
MDA was calculated by the absorbance coefficient
of MDA-TBA complex 1.56 105
cm-1
M-1
and
expressed in nmol/mL.
Liver and kidneys markers. The measurement of
plasma cellular enzymes such as aminotransferases
(AST; EC 2.6.1.1 and ALT; EC 2.6.1.2), lactate
dehydrogenase (LDH; EC 1.1.1.27) and g-glutamyl-
transferase (GGT) were determined by the methods
of Reitman and Frankel (1957), Tietz (1995) and
Szasz (1969), respectively. The activity of AST, ALT,
LDH and GGT were expressed in terms of U/L. The
activity of plasma cholinesterase (BChE;
EC 3.1.1.8) was determined by the methods of Ellman
et al. (1961) and expressed as U/mL. The concentra-
tion of albumin (g/dL), total protein (g/dL), uric acid
and creatinine (mg/dL) were determined by the meth-
ods of Westgard and Poquette (1972), Gornal et al.
(1949), Barham and Trinder (1972) and Bartels
et al. (1972), respectively.
Histopathological studies
Liver and kidney samples were dissected and fixed in
10% neutral formalin, dehydrated in ascending grades
of alcohol and imbedded in paraffin wax. Paraffin
sections (5 mm thick) were stained for routine histolo-
gical study using haematoxylin and eosin (HE). For
each rat, two slides were prepared; each slide
contained two sections for each organ. Ten field areas
for each section were selected and examined for
histopathological changes (64) under light micro-
scope. The liver fields were scored as follows: nil
(normal appearance) ¼ 0%, þ ¼ mild (cellular disrup-
tion in less than 20% of field area), þþ ¼ moderate
Mansour and Mossa 215

4. (cellular disruption of 20% to less than 40% of field
area) and þþþ ¼ severe (cell disruption of 40 to less
than 70% of field area). The kidney fields were scored
based on tubular injury as described above. Such
quantitative assessment of histopathological injury has
been performed by previous investigators (Kerem
et al., 2007).
Spectrophotometric measurements
The spectrophotometric measurements were performed
by using a Shimadzu UV-VIS Recording 2401 PC
(Japan).
Statistics
The data were analyzed by using SPSS (version 14.0)
for Windows and expressed as means + SD. Paired
samples t test was used to compare between the data
of the control and those of treatments.
Results
Body and relative organs weights
There was no significant difference in body and liver
and kidney weights in lactating mothers exposed to
0.01 mg kg1
b.wt. of CPF at any time point in the
study, compared to control groups (Figure 1). The
treated groups with 1.00 mg kg1
b.wt. and 1.35 mg
kg1
b.wt. of CPF had a significant decrease in body
compared to the corresponding control group
(Figure 1A). The significant decrease in body weight
was observed from the PND 10 to the end of experi-
ment (PND 21). The reduction in body weight, if
calculated, accounted to 0.98%, 16.35% and
18.53% on PND 21 with respect to control values,
for 0.01, 1.00 and 1.35 mg kg1
b.wt. groups, respec-
tively. A dose-dependent decrease in body weight of
mothers exposed to CPF was observed. In contrast,
there was a significant increase in relative liver
weight in mothers exposed to1.00 and 1.35 mg kg1
b.wt. of CPF and significant increase in relative
kidney weight in mothers exposed to 1.35 mg kg1
b.wt. of CPF (Figure 1B).
Oxidative stress markers
Results of oxidative stress markers are shown in
Table 1. There was no significant difference in SOD
and GST activity and LPO level in mothers exposed
to 0.01 mg kg1
b.wt. of CPF. But there were signif-
icant decreases in the activity of SOD and GST and
significant increase in LPO level in mothers exposed
to either 1.00 or 1.35 mg kg1
b.wt. of CPF compared
to control groups in a dose-dependent manner. The
changes in the activity of SOD and GST (if calculated)
accounted to 5.59%, 13.14% and 19.01% and
16.0%, 46.0% and 51.33% with respect to control,
for 0.01, 1.00 and 1.35 mg kg1
b.wt. of CPF,
respectively.
Liver and kidneys markers
Treatment of lactating mothers with CPF caused
changes in the levels of AST, ALT, LDH, GGT and
ChE compared to control groups in a dose-
dependent manner (Table 2). A significant increase
(p 0.05) in the activity of AST was observed in the
treatment with 0.01 mg kg1
b.wt. At 1.0 mg kg1
b.wt. CPF, except ALT, the other major parameters
recorded significant elevation compared to the corre-
sponding control values. At high dose of CPF (1.35
mg kg1
b.wt.), the lactating mothers showed signif-
icant increase in the activity of AST, LDH, GGT
(p 0.01) and ALT (p 0.05). AST/ALT value
accounted to 1.13 for the control group and 1.51,
1.63 and 1.66 for the three tested doses of CPF,
respectively. At two high doses of CPF (1.00 and
1.35 mg kg1
b.wt.), the lactating mothers showed
significant decrease in the activity of ChE and the
change in ChE activity for the CPF groups, if
calculated, accounted to 11.11%, 15.74% and
25.46% for 0.01, 1.00 and 1.35 mg kg1
b.wt. of
CPF, respectively (Table 2). As shown in Table 3, the
high dose of CPF (1.35 mg kg1
b.wt.) caused signif-
icant increase in total protein, globulin and uric acid
(p 0.05) and decrease in albumin (p 0.01) con-
centrations in the plasma of the tested mothers. Crea-
tinine levels in exposed mothers did not show
significant alteration due to CPF-exposure at any
doses.
Histopathology changes
Histopathological findings of liver and kidney for
various treatment groups are presented in Table 4.
In light microscopic examinations, histopathologi-
cal changes were observed in liver and kidney of
all exposed groups compared to control ones. How-
ever, these changes were more frequent in CPF-
treated groups with 1.00 mg kg1
b.wt. and 1.35
mg kg1
b.wt. The liver and kidney sections of
control animals showed normal structures. With
respect to the hepatic histoarchitecture of the
216 Toxicology and Industrial Health 27(3)

5. CPF-treated animals, dilatation in central vein,
degeneration in hepatocytes, congestion of portal
vein and proliferation in bile duct, focal necrotic and
fibrosis were the main findings in CPF-treated
groups. In cases of kidney, the renal histoarchitec-
ture of the CPF-treated animals, degeneration and
necrosis in the epithelial cells lining the tubules
and proliferation of the endothelial cells lining the
tuft glomerulus were the main findings in CPF-
treated group.
Discussion
In the present study, oral administration of CPF to
lactating mothers resulted in significant decrease, in
a dose-dependent manner, in body weight and signif-
icant increase in relative liver and kidney weights.
Reduction in body weight observed in CPF-treated
dams may be a result of the combination of increased
degradation of lipids and proteins as a result of
the direct effects of CPF as an organophosphate
200
210
220
230
240
250
260
270
280
290
300
20
15
10
5
1
Days of Lactation
Body
weight
(g)
0.00 mg/kg 0.01 mg/kg 1.00 mg/kg 1.35 mg/kg
**
**
**
*
*
**
A
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
Kidney
Liver
Relative
weight
(%)
0.00 mg/kg 0.01 mg/kg 1.00 mg/kg 1.35 mg/kg
*
*
**
B
Figure 1. Body weight (A) and relative organs weights (B) of lactating mothers exposed to chlorpyrifos (CPF) during
lactation period (postnatal day 1 [PND1] to PND 20). Each value is a mean + SD; statistical difference from the control:
* significant at p 0.05 and ** highly significant at p 0.01. n ¼ 5 animals per group.
Mansour and Mossa 217

6. compound (Goel et al., 2005), as well as reduction of
food intake by lactating mothers (untabulated data),
which may cause a negative energy balance. Reduc-
tion of body weight and feed consumption in lactating
dams following oral exposure to CPF were previously
reported by Maurissen et al. (2000). In this respect, dur-
ing lactation, in many human mothers (Butte et al.,
1984) and animals (McNamara and Hillers, 1986),
decrease of the body weight and percentage body fat
is seen, presumably because the energy needs of
lactation plus normal energy needs exceed their energy
intake. The increase in relative liver weight is attributed
to the increase ofUDP-glucuronyltransferase (UDPGT)
enzyme activity, which elevates in liver, brain and kid-
ney of rats orally administered some OPs (Bulusu and
Chakravarty, 1986; Cook et al., 1997; Mahboob and
Siddiqui, 2001). The present findings related to changes
in body and internal organs weights were reported by
other investigators (Ambali et al., 2007; Mansour
et al., 2008; Mansour and Mossa, 2009). In previous
Table 1. LPO level, SOD and GST enzymes activity in plasma of lactating mothers exposed to CPF during the suckling
period (PND1 to PND 20)a
Dose (mg kg-1
b.wt.) LPO (nmoL/mL) GST (mmoL/mg protein) SOD (mmoL/mg protein)
0.00 1.43 + 0.08 1.50 + 0.12 120.51 + 7.14
0.01 1.47 + 0.10 1.26 + 0.16 114.13 + 10.11
1.00 1.66 + 0.12b
0.81 + 0.19c
105.51 + 5.22b
1.35 1.77 + 0.15b
0.73 + 0.02c
98.81 + 6.08c
Abbreviations: LPO: lipid peroxidation, SOD: superoxide dismutase, GST: glutathione s-transferase, CPF: chlorpyrifos, PND: postnatal
day.
a
Each value is a mean + SD. n ¼ 5 animals per group.
b
Statistical difference from the control: significant at p 0.05.
c
Statistical difference from the control: highly significant at p 0.01.
Table 2. Plasma AST, ALT, LDH, GGT and ChE enzymes activity in lactating mothers exposed to CPF during the suckling
period (PND1 to PND 20)a
Dose (mg kg-1
b.wt.) AST (U/L) ALT (U/L) AST/ALT value LDH (U/L) GGT (U/L) ChE (U/mL)
0.00 43.44 + 2.89 38.37 + 1.25 1.13 + 0.04 173.14 + 5.69 10.06 + 1.28 2.16 + 0.12
0.01 55.00 + 3.96b
36.36 + 1.12 1.51 + 0.06b
183.50 + 20.81 11.36 + 2.76 1.92 + 0.18
1.00 62.23 + 4.31c
38.07 + 2.24 1.63 + 0.02b
231.26 + 20.84b
23.04 + 4.21c
1.82 + 0.09b
1.35 76.51 + 4.67c
46.19 + 3.45b
1.66 + 0.02c
255.02 + 22.09c
25.09 + 3.89c
1.61 + 0.07c
Abbreviations: AST: aspartate aminotransferase, ALT: alanine aminotransferase, LDH: lactate dehydrogenase, GGT: g-glutamyl trans-
ferase, ChE: Cholinesterase, CPF: chlorpyrifos.
a
Each value is a mean + SD; n ¼ 5 animals per group.
b
Statistical difference from the control: significant at p 0.05.
c
Statistical difference from the control: highly significant at p 0.01.
Table 3. Plasma total protein, albumin, globulin, A/G ratio, uric acid and creatinine level in lactating mothers exposed to
CPF during the suckling period (PND1 to PND 20)a
Dose (mg kg-1
b.wt.)
Total protein
(g/dL)
Albumin
(g/dL)
Globulin
(g/dL)
Albumin/globulin
(A/G) ratio
Uric acid
(mg/dL)
Creatinine
(mg/dL)
0.00 7.10 + 0.68 3.40 + 0.13 3.70 + 0.32 0.92 + 0.04 3.99 + 0.41 0.61 + 0.10
0.01 7.19 + 0.71 3.29 + 0.14 3.90 + 0.45 0.84 + 0.06 3.84 + 0.56 0.65 + 0.13
1.00 8.22 + 1.19 3.15 + 0.29 5.07 + 0.61b
0.62 + 0.02b
5.12 + 0.39b
0.65 + 0.12
1.35 9.17 + 1.03b
2.85 + 0.10c
6.32 + 0.11b
0.45 + 0.01c
5.45 + 0.89b
0.81 + 0.14
Abbreviations: A/G: albumin/globulin, CPF: chlorpyrifos.
a
Each value is a mean + SD; n ¼ 5 animals per group.
b
Statistical difference from the control: significant at p 0.05.
c
Statistical difference from the control: highly significant at p 0.01.
218 Toxicology and Industrial Health 27(3)

7. studies, administration of CPF at 1.0 mg kg1
b.wt. did
not induce significant changes in body weight gain,
either in dams (Maurissen et al., 2000) or female rats
(Yano et al., 2000).
In this context, the maximum-tolerated-doses
(MTD) was initially based on a weight gain decre-
ment observed in the subchronic study; i.e., the high-
est dose that caused no more than a 10% weight gain
decrement (Interagency Staff Group on Carcinogens,
1986). It is likely to state that the decrease in body
weight gain may reach up to 20% in toxicology
studies designated for risk assessment at MTD of a
toxicant. The decrease in body weight gain in the
present study at doses 1.0 and 1.35 mg kg1
b.wt.
were far of MTD for CPF, which was administrated
in corn oil. It is documented that gavage in corn oil
enhances absorption of CPF across the gastrointest-
inal tract (GI) mucosa, thus increasing the pesticide
toxicity (Conolly et al., 1999). On the other hand,
Marty et al. (2007) reported that dietary CPF treat-
ment at 5 mg kg1
b.wt. in lactating rats had blood
CPF levels that were some 13 lower than the same
dose by oral gavage. For this reason, Conolly et al.
(1999) stressed cautions against use of corn oil gavage
administration in risk assessment of organic com-
pounds, generally. However, we cannot ignore the
effect of exposure to a toxicant in conjunction with
fats in the food.
Some studies have shown that oxidative stress
could be an important component for the mechanism
of toxicity of organophosphate insecticides (OPIs).
OPIs may induce oxidative stress leading to genera-
tion of free radicals and alteration in antioxidants or
ROS scavenging enzymes (Banerjee et al., 1999;
Mansour and Mossa, 2009, 2010a; Ranjbar et al.,
2002). Therefore, OPI-may enhance lipid peroxida-
tion (LPO) by directly interacting with the cellular
membrane and ROS. Previous studies have suggested
LPO as one of the molecular mechanisms involved in
OPIs-induced toxicity (Bagchi et al., 1995; Mansour
and Mossa, 2010a).
Plasma lipid peroxidation was estimated in the
form of thiobarbituric acid reactive substances
(TBARS) produced. Significant increase in TBARS
was observed in mothers exposed to 1.00 and 1.35
mg kg1
b.wt. of CPF when compared with control
(Table 1). Increased formation of peroxide (TBARS)
could be due to the increased peroxidation of
membrane. Literature shows the basic process by
which any and related radicals such as superoxide and
hydroxy radicals cause membrane damages in LPO
(Bachowski et al., 1997; Bagchi et al., 1995; Mansour
and Mossa, 2010a; Yamano and Morita, 1992). Our
results revealed that CPF caused a statistically signif-
icant decrease in the activity of SOD and GST and
significant increase in LPO level of mothers exposed
to CPF. The decrease in the activity of SOD in CPF-
intoxicated animals may be due to the consumption of
this enzyme in converting the O2- to H2O, which cat-
alyzed by SOD. Considering that GST are detoxifying
enzymes that catalyze the conjugation of a variety of
electrophilic substrates to the thiol group of GSH,
producing less toxic forms (Hayes and Pulford,
1995), the significant decrease of GST activity may
indicate insufficient detoxification of CPF in rats.
However, the activation of CPF to corresponding
oxons occurs through oxidative desulphuration
mediated by the cytochrom P450. CPF induces
depression in the activity of both nicotinamide ade-
nine dinucleotide phosphate (NADPH) cytochrome-
c-reductase and nicotinamide adenine dinucleotide
(NADH) cytochrome-c-reductase in intoxicated rats
(Goel et al., 2007). It was suggested that the sulphur
released during desulfuration of an OP insecticide
Table 4. Histopathological changes in the liver and kidneys of lactating-mothers exposed to CPF during the suckling
period (PND1 to PND 20), based on scoring severity of injurya
in both organs
Dose (mg
kg1
b.wt.)
Hepatic injury Renal injury
Observation Severity Observation Severity
0.00 Normal Nil Normal Nil
0.01 Congestion; degeneration; hyperplasia of bile duct þ Degeneration þ
1.00 Congestion; necrosis; inflammatory cell infiltration in
portal area; fibrosis; hyperplasia of bile duct
þþ Glomerulus hyperplasia;
necrosis renal tubules
þþ
1.35 Congestion; necrosis þþ Hyperplasia in glomerulus
tubules; degeneration
þþ
Abbreviation: CPF: chlorpyrifos.
a
Scores in terms of numerical values are mentioned in histopathological studies section, number of slides ¼ 10/group/organ.
Mansour and Mossa 219

8. may bind not only to the cytochrom P450 but also to
NADH cytochrome-c-reductase, culminating in the
overall reduced activity of this monooxygenase
(Kamataki and Neal, 1976). Alternatively, NADPH
cytochrome-c-reductase is also involved in initiating
the process of NADPH-dependent lipid peroxidation
in the microsomal membranes, and increased lipid per-
oxidation as a result of CPF intoxication may also be
the cause of depressed enzymatic activity (Sevanian
et al., 1990). These indirectly suggest an increased
production of oxygen-free radicals in rats. Highly
reactive oxygen metabolites, especially hydroxyl
radicals, act on unsaturated fatty acids of phospholipid
components of membranes to produce MDA, an LPO
product (Mansour and Mossa, 2009, 2010a).
ROS damage cells by free radical transfer from an
initial metabolic by-product to a variety of critical
biomolecules including DNA, proteins and lipids.
Free radical addition increases the likelihood of
modification through conjugation with electrophiles
or covalent bond breakage. Physical damage, mutation,
or loss of function in the short term can cause hepato-
cyte death and over the longer term can contribute to
the pathophysiology of liver disease (Murray et al.,
2006). This is in addition to the cellular stress and
energy consumption caused by redox cycling, which
on its own can overwhelm the antioxidant capacity of
hepatocytes and result in cell death. A major pathophy-
siologic concept that has emerged is that oxidative
stress contributes to many diseases, including cancer,
cardiovascular disease, liver cirrhosis, diabetes and
others related to aging (Murray et al., 2006).
Liver is the main tissue in detoxification and
metabolism of chemicals and along with kidney faces
the threat of maximum exposure to xenobiotics and
their metabolic by-products. This may impair its
regular function due to xenobiotic modification in
detoxification processes. In fact, the susceptibility of
liver and kidney tissues to this stress due to exposure
to pesticides is a function of the overall balance
between the degree of oxidative stress and the antiox-
idant capability (Khan et al., 2005). However, several
of soluble enzymes of blood plasma have been consid-
ered as indicators of hepatic dysfunction and damage.
LDH is one of the metabolic requirements of tissue and
involved in energy production. LDH activity indicates
the switching over of anaerobic glycolysis to aerobic
respiration. It can be used as an indicator for cellular
damage, clinical practice and cytotoxicity of toxic
agents (Bagchi et al., 1995). Also, g-glutamyl
transferase catalyzes the transfer of g-glutamyl group
from a g-glutamyl peptide to an amino acid or another
peptide. This enzyme is widely used as a biomarker in
preneoplastic lesions of the liver during chemical
carcinogenesis (Peraino et al., 1983). Our results
demonstrated that CPF may affect liver metabolism
and the leakage of certain intracellular enzymes,
suggesting damage in hepatocytes. Since AST/ALT
leaking to plasma is very sensitive marker of hepato-
cyte injury, increase in plasma enzymes may occur due
to minor liver injury in response to chemicals with low
histological changes (Williams and Iatropoulos,
2002). In the present study, the increment of the
activities of AST, ALT, LDH and GGT in plasma
could be attributed to the leakage of these enzymes
from the liver cytosol into the blood stream (Navarro
et al., 1993), which indicated liver damage and disrup-
tion of normal liver function (Shakoori et al., 1994; de
Boer et al., 2000; Kuester et al., 2002; El Sakka et al.,
2002). Also, AST/ALT values were higher than con-
trol of exposed mothers and ranged ‘between 1.51 to
1.66,’ which seemed to be higher than normal value
(1.00-1.50; Caglar and Kolankaya, 2007).
Cholinesterase (ChE, EC 3.1.1.8), also known as
pseudocholinesterase, has been recognized as an
enzyme that hydrolyzes choline esters. It has already
been mentioned that ChE is synthesized mainly in
hepatocytes and secreted into the blood stream
(Brown et al., 1981). ChE activity is reduced in liver
dysfunction due to reduced synthesis, in contrast to
other serum enzymes associated with the clinical
assessment of liver function whose activities increase
as a result of increased release from their cellular
sources following cell membrane damage (Moss and
Henderson, 1999). In this respect, changes in ChE
activity reflect the changes in hepatocellular functions
and have been regarded as sensitive indicators of the
diminished synthetic capacity of the hepatic parench-
yma (Adolph, 1979). In the present study, plasma ChE
activity of dams given 1.00 and 1.35 mg kg-1 b.wt. of
CPF was significantly decreased (15.74 and 25.46%)
compared to the control.
In previous studies, CPF was administered to rat
dams by gavage in corn oil (Maurissen et al., 2000)
and to female rats in feed (Yano et al., 2000). In both
studies, the administered dose of CPF was 1.0 mg
kg1
b.wt., resembling the median dose tested in the
present study. Among comparable biochemical
criteria measured in these studies, the inhibition of
ChE, which was accounted to 31.1% and 81.0%,
respectively, compared to 15.74% in the present inves-
tigation. It is known that the toxicity of a toxicant may
220 Toxicology and Industrial Health 27(3)

9. be affected by several factors, such as strain of rats,
route of administration, dose and lactation statutes.
Total protein and A/G ratio is done as a routine test
to evaluate the toxicological nature of various chemi-
cals. Increases of total protein and decrease of A/G
ratio were observed in the present study following
CPF treatment. In relation to decrease in the A/G
ratio, the albumin level was also decreased, suggest-
ing high plasma globulin levels reflecting high protein
in CPF-treated mothers. The increase of total protein
in CPF-treated groups may be due to (a) due to
production of enzymes lost as a result of tissue
necrosis (b) to meet increased demand detoxifying the
pesticide might necessitate enhanced synthesis of
enzyme proteins (Gill et al., 1990, 1991), and (c) may
be due to kidney dysfunction (Mansour and Mossa,
2005; Mansour and Mossa, 2010a).
Kidney has a relatively high level of enzyme
activities, and the role of this organ in converting
xenobiotics and endogenous substances into excretory
forms is considerable (De Kanter et al., 2002). In the
present study, exposed mothers to CPF at 1.00 mg g1
b.wt and 1.35 mg g1
b.wt. showed significant
increases in uric acid concentration in the plasma
compared to the control, while creatinine levels did
not show significant alteration. In fact, uric acid is the
end product of the catabolism of tissue nucleic acid,
i.e., purine and pyrimidine based metabolism, and the
increase in uric acid may be due to degradation of
purines and pyrimidines or to an increase of uric acid
level by either overproduction or inability of excretion
(Mansour and Mossa, 2005).
The histopathological lesions observed in the liver
and kidneys of CPF-treated animals are in corrobora-
tion with the observed biochemical changes. These
observations indicated marked changes in the overall
histoarchitecture of liver and kidney in response to
CPF, which could be due to its toxic effects primarily
by the generation of ROS, causing damage to the
various membrane components of the cell. Our results
are supported by other studies conducted on CPF and
other OP insecticides (Mansour et al., 2008; Mansour
and Mossa, 2010a, b; Wang and Zhai, 1988).
Conclusion
Exposure of lactating dams to low dose levels of the
OP insecticide, CPF, induced pronounced alterations
in LPO, oxidative damage, and liver and kidneys
dysfunctions. Compared to results of other studies
(Maurissen et al., 2000; Yano et al., 2000) conducted
at different dose levels of CPF, including 1.0 mg kg1
b.wt., the observed differences in toxicity results
would be attributed to a number of factors, such as
strain of rats, route of administration, exposure
period, dose and lactation statutes. It has been
reported that CPF exposures exceed no observable
adverse effect level (NOAEL) for pregnant women
and children, even in scenarios of common use (Davis
and Ahmed, 1998; Gurunathan et al., 1998). This lead
to paying more attention to the risks posed to lactating
women especially in developing countries, where
women are usually involved in agricultural practices.
Moreover, lactation transfers of chlorinated OPIs such
as CPF cause detrimental hazards to breast-feeding
infants (Mansour and Mossa, 2010b).
Acknowledgement
The authors are grateful to Prof. Dr Adel Mohamed Bakeer
Kholoussy, Professor of Pathology, Faculty of Veterinary
Medicine, Cairo University, for reading the histopathologi-
cal sections.
Funding
The author(s) received no financial support for the research
and/or authorship of this article.
References
Abdollahi M, Ranjbar A, Shadnia S, Nikfar S, and Rezaie
A (2004) Pesticides and oxidative stress: a review.
Medical Science Monitor 10(6): RA141–RA147.
Adolph L(1979) Diagnostische bedeutungder cholinesterase-
bestimmung im menschlichen serum. Med Wschr 121:
1527–1530.
Akhgari M, Abdollahi M, Kebryaeezadeh A, Hosseini R,
and Sabzevari O (2003) Biochemical evidence for free
radical induced lipid peroxidation as a mechanism
for subchronic toxicity of malathion in blood and
liver of rats. Human and Experimental Toxicology
22: 205–211.
Ambali S, Akanbi D, Igbokwe N, and Shittu M (2007)
Evaluation of subchronic chlorpyrifos poisoning on
hematological and serum biochemical changes in mice
and protective effects of vitamin C. Toxicology Sciences
32: 111–120.
Anderson HA, Wolff MS (2000) Environmental contami-
nations in human milk. Journal of Exposure Analysis
and Environmental Epidemiology 10: 755–760.
Bachowski S, Xu Y, Stevenson DE, Walborg EF, and
Klaunig JE (1997) Role of oxidative stress in the
mechanism of dieldrin’s heptotoxicity. Annals of
Clinical Laboratory Science 27: 196–209.
Mansour and Mossa 221

10. Bagchi D, Bagchi M, Hassoun EA, and Stohs SJ (1995) In
vitro and in vivo generation of reactive oxygen species,
DNA damage and lactate dehydrogenase leakage by
selected pesticides. Toxicology 104: 129–140.
Banerjee BD, Seth V, Bhattacharya A, Pasha ST, and
Chakraborty AK (1999) Biochemical effects of pesti-
cides on lipid peroxidation and free radicals scavengers.
Toxicology Letter 107: 33–47.
Barham D, Trinder P (1972) An improved colour reagent
for the determination of blood glucose by the oxidase
system. Analyst 97: 142–145.
Bartels H, Böhmer M, and Heierli C (1972) Serum
creatinine determination without protein precipitation.
Clinica Chimica Acta 37: 193–197.
Berkowitz GS, Wetmur JG, Birman-Deych E, Obel J,
Lapinski RH, Godbold JH, et al. (2004). In utero pesti-
cide exposure, maternal paraoxonase activity, and head
circumference. Environmental Health Perspectives
112(3): 388–391.
Brown SS, Kalow W, Pilz W, Whittaker M, and Woronick
CL (1981) The plasma cholinesterases; a new perspective.
Advances in Clinical Chemistry 22: 82–83.
Bulusu S, Chakravarty I (1986) Sub-acute administration of
organophosphorus pesticides and hepatic drug
metabolizing enzyme and malnourished rats. Bulletin of
Environmental Contamination and Toxicology 36: 73–80.
Butte NF, Garza C, Stuff JE, Smith EO, and Nichols BO
(1984) Effect of maternal diet and body composition
on lactational performance. American Journal of
Clinical Nutrition 39: 296–306.
Caglar C, Kolankaya D (2007) The effect of sub-acute and
sub-chronic exposure of rats to the glyphosate-based
herbicide Roundup. Environmental Toxicology and
Pharmacology 25: 57–62.
Collaborative Group on Hormonal Factors in Breast Cancer
(2002) Breast cancer and breastfeeding: collaborative
reanalysis of individual data from 47 epidemiological
studies in 30 countries, including 50302 women with
breast cancer and 96973 without the disease. Lancet
360: 187–195.
Conolly RB, Beck BD, and Goodman JI (1999) Stimulating
research to improve the scientific basis of risk assess-
ment. Toxicology Sciences 49: 1–4.
Cook JC, Kaplan AM, Davis LG, and O’Connor JC (1997)
Development of a Tier I screening battery for detecting
endocrine-active compounds (EACs). Regulatory
Toxicology Pharmacology 26: 60–68.
Davis DL, Ahmed AK (1998) Exposures from indoor
spraying of chlorpyrifos pose greater health risks to
children than currently estimated. Environmental Health
Perspectives 106(6): 299–301.
de Boer WB, Segal A, Frost FA, and Sterrett GF (2000)
Can CD34 discriminate between benign and malignant
hepatocytic lesions in fine-needle aspirates and thin core
biopsies? Cancer 90: 273–278.
De Kanter R, De Jager MH, Draaisma AL, Jurva, JU,
Olinga P, Meijer DK, et al. (2002) Drug-metabolizing
activity of human and rat liver, lung, kidney and intes-
tine slices. Xenobiotica 32: 349–362.
Draper HH, Hadley M (1990) Malondialdehyde determina-
tion as index of lipid peroxidation. Methods in Enzymol-
ogy 186: 421–431.
El Sakka S, Salem E, and Abdel R (2002) In vitro hepato-
toxicity of alachlor and its by-products. Journal of
Applied Toxicology 22: 31–35.
Ellman GL, Courtney KD, Andres KD, and Featherstone
RM (1961) A new and rapid calorimetric determination
of acetylcholinesterase activity. Biochemical Pharma-
cology 7: 88–95.
Eskenazi B, Harley K, Bradman A, Weltzien E, Jewell NP,
Barr DB, et al. (2004) Association of in utero organo-
phosphate pesticide exposure and fetal growth and
length of gestation in an agricultural population. Envi-
ronmental Health Perspectives 112(10): 1116–1124.
Fishbeck KL, Rasmussen KM (1987) Effect of repeated
cycles on maternal nutritional status, lactational
performance and litter growth in ad libitum fed and
chronically food. Journal of Nutrition 117: 1967–1975.
Gallenberg LA, Vodicnik MJ (1989) Transfer of persistent
chemicals in milk. Drug Metabolism Review 21:
277–317.
Gill TS, Pande J, and Tewari H (1990) Sublethal e€ects of an
organophosphorus insecticide on certain metabolite lev-
els in a freshwater fish, Puntius conchonius Hamilton.
Pesticide Biochemistry and Physiology 36: 290–299.
Gill TS, Pande J, Tewari H (1991) Individual and combined
toxicity of common pesticides to teleost Puntius concho-
nius Hamilton. Indian Journal of Experimental Biology
29: 145–148.
Goel A, Dani V, and Dhawan DK (2005) Protective effects
of zinc on lipid peroxidation, antioxidant enzymes and
hepatic histoarchitecture in chlorpyrifos-induced
toxicity. Chemico-Biological Interactions 156: 131–140.
Goel A, Dani V, and Dhawan DK (2007) Zinc mediates
normalization of hepatic drug metabolizing enzymes
in chlorpyrifos-induced toxicity. Toxicology Letter
169: 26–33.
Gornall AC, Bardawill CJ, and David MM (1949) Determi-
nation of serum protein by means of biuret reaction.
Journal of Biological Chemistry 177: 751–756.
Gurunathan S, Robson M, Freeman N, Buckley B, Roy A,
Meyer R, et al. (1998) Accumulation of chlorpyrifos on
222 Toxicology and Industrial Health 27(3)

11. residential surfaces and toys accessible to children.
Environmental Health Perspectives 106(1): 9–16.
Habig WH, Pabst MJ, and Jakoby WB (1974) Glutathione
S-transferases, the first enzymatic step in mercapturic
acid formation. Journal of Biological Chemistry 249:
7130–7139.
Halliwell B (1996) Antioxidants in human health and
disease. Annual Review Nutrition 16: 33–50.
Halliwell B, and Gutteridge JMC (1999) Free Radicals in
Biology and Medicine. 3rd ed. New York: Oxford
University Press.
Hayes D, Pulford D (1995) The glutathione S-transferase
supergene family: regulation of GST and the contribu-
tion of the isoenzymes to cancer chemoprotection and
drug resistance. Critical Reviews in Biochemistry and
Molecular Biology 30: 445–600.
Interagency Staff Group on Carcinogens (1986) Chemical
carcinogens: a reviewofthe science anditsassociatedprin-
ciples. Environmental Health Perspectives 67: 201–282.
Kamataki T, Neal RA (1976) Metabolism of diethyl
p-nitrophenyl phosphorothionate (parathion) by a recon-
stituted mixed-function oxidase enzyme system: studies
of the covalent binding of the sulfur atom. Molecular
Pharmacology 12: 933–944.
Kerem M, Bedirli N, Gurbuz N, Ekincl O, Bedirli A,
Akkaya T, et al. (2007) Effects of acute fenthion toxicity
on liver and kidney function and histology in rats.
Turkish Journal of Medical Sciences 37: 281–288.
Khan SM, Sobti RC, and Kataria L (2005) Pesticide-
induced alteration in mice hepato-oxidative status and
protective effects of black tea extract. Clinica Chimica
Acta 358: 131–138.
Kramer MS, Kakuma R (2002) Optimal duration of exclu-
sive breastfeeding. Cochrane Database of Systematic
Reviews; (7): CD003517.
Kuester RK, Waalkes MP, Goering PL, Fisher BL,
McCuskey RS, and Sipes IG (2002) Differential hepato-
toxicity induced by cadmium in Fischer 344 and
Sprague–Dawley rats. Toxicology Sciences 65: 151–159.
Mahboob M, Siddiqui MK (2001) Alterations in hepatic
detoxifying enzymes induced by neworganophosphorus
insecticides following subchronic exposure in rats.
Journal of Applied Toxicology 21: 501–505.
Mansour SA, Heikal TM, Mossa AH, and Refa A (2008)
Toxic effects of five insecticides and their mixture on
malealbinorats.JournalofEgyptianSocietyofToxicology.
39: 85–94.
Mansour SA, Mossa AH (2005) Comparative effects of
some insecticides as technical and formulated on male
albino rats. Journal of Egyptian Society of Toxicology
32: 41–54.
Mansour SA, Mossa AH (2009) Lipid peroxidation and
oxidative stress in rat erythrocytes induced by chlorpyr-
ifos and the protective effect of zinc. Pesticide Biochem-
istry and Physiology 93: 34–39.
Mansour SA, Mossa AH (2010a) Oxidative damage,
biochemical and histopathological alterations in rats
exposed to chlorpyrifos and the antioxidant role of zinc.
Pesticide Biochemistry and Physiology 96: 14–23.
Mansour SA, Mossa AH (2010b) Adverse effects of
lactational exposure to chlorpyrifos in suckling rats.
Human and Experimental Toxicology 92(2): 77–92.
Marty MS, Domoradzki JY, Hansen SC, Timchalk C,
Bartels MJ, and Mattsson JL (2007) The effect of route,
vehicle, and divided doses on the pharmacokinetics of
chlorpyrifos and its metabolite trichloropyridinol in
neonatal Sprague-dawley rats. Toxicology Science 100:
360–373.
Mattsson JL, Maurissen JP, Nolan RJ, and Brzak KA
(2000) Lack of differential sensitivity to cholinesterase
inhibition in fetuses and neonates compared to dams
treated perinatally with chlorpyrifos. Toxicology
Sciences 53: 438–446.
Maurissen JPJ, Hoberman AM, Garman RH, and Hanley
TR Jr (2000) Lack of selective developmental neuro-
toxicity in rat pups from dams treated by gavage with
chlorpyrifos. Toxicology Sciences 57: 250–263.
McNamara JP, Hillers JK (1986) Adaptations in lipid
metabolism of bovine adipose tissue in lactogenesis and
lactation. Journal of Lipid Research 27: 150–157.
Moss DW, and Henderson AR (1999) Enzymes. In:
Burtis CA, Ashwood ER (eds.) Tietz Textbook of Clinical
Chemistry. 2nd ed. Philadelphia: WB Saunders, 735–896.
Murray KF, Messner DJ, and Kowdley KV (2006) Mechan-
isms of hepatocyte detoxification. In: Johnson LR (ed.)
Physiology of the Gastrointestinal Tract. Burlington:
Elsevier Academic Press, 1483–1503.
National Research Council (NRC) (1996) Guide for the
Care and Use of Laboratory Animals. Washington,
DC: Academic Press.
Navarro CM, Montilla PM, Martin A, Jimenez J, and
Utrilla PM (1993) Free radicals scavenger and
antihepatotoxic activity of Rosmarinus. Plant Medicine
59: 312–314.
Nolan RJ, Rick DL, Freshour NL, and Saunders JH (1984)
Chlorpyrifos: pharmacokinetics in human volunteers.
Toxicology and Applied Pharmacology 73: 8–15.
Peraino C, Richards WL, and Stevens J (1983) Multi-stage
hepatocarcinogenesis. Environmental Health Perspec-
tives 56: 1–43.
Ranjbar A, Pasalar P, and Abdollahi M (2002) Inducation
of oxidative stress and acetylcholinesterase inhibition
Mansour and Mossa 223

12. in organophosphorous pesticide manufacturing workers.
Human and Experimental Toxicology 21: 179–182.
Reitman S, Frankel S (1957) A colorimetric method for the
determination of serum glutamic oxalacetic and
glutamic pyruvic transaminases. American Journal of
Clinical Pathology 28: 56–63.
Rosenblatt KA, Thomas DB (1993) WHO collaborative
study on neoplasia and steroid contraceptives. Interna-
tional Journal of Epidemiology 22: 192–197.
Salas JH, González MM, Noa M, Pérez NA, Dı́az G,
Gutiérrez R, et al. (2003) Organophosphorus pesticides
in Mexico commercial pasteurized milk. Journal of
Agriculture and Food Chemistry 51: 4468–4471.
Sanghi R, Pillai MK, Jayalekshmi TR, and Nair A (2003)
Organochlorine and organophosphorus pesticides resi-
dues in breast milk from Bhopal, Madhya Pradesh,
Indian. Human and Experimental Toxicology 22: 73–76.
Sevanian A, Nordenbrand K, Kim E, Ernster L, and
Hochstein P (19900 Microsomal lipid peroxidation:
the role of NADPH-cytochrome P450 reductase and
cytochrome P450. Free Radical Biology and Medicine
8: 145–152.
Shakoori AR, Butt U, Riffat R, Aziz F (1994) Hematologi-
cal and biochemical effects of danitol administered for
two months on the blood and liver of rabbits. Zeitschrift
fuer Angewandte Zoologie 80: 165–180.
Szasz G (1969) Kinetic photometric methods for serum
gamma glutamyltranspeptidase. Clinical Chemistry 15:
124–136.
Tietz NM (1995) Clinical Guide to Laboratory Tests. Phi-
ladelphia, PA: WB Saunders Company, 384–385.
Tomlin CDS (2005) The e-Pesticide Manual. 13th ed.
CDROM version 3.1, The British Crop Protection
Councill: Farnham, UK.
Wang X, Zhai W (1988) Cellular and biochemical
in bronchoalveolar lavage fluids of rats exposed to
fenvalerate. Zhongguo Yaolixue YuDulixue Zoghi 2:
271–276.
Westgard JO, Poquette MA (1972) Determination of
serum albumin with the ‘‘SMA 12-60’’ by a bromcresol
green dye-binding method. Clinical Chemistry 18:
647–653.
Williams GM, Iatropoulos MJ (2002) Alteration of
liver cell function and proliferation: differentiation
between adaptation and toxicity. Toxicology Pathol-
ogy 30: 41–53.
Woolliams JA, Wiener G, Anderson PH, and McMurray CH
(1983) Variation in the activities of glutathione
peroxidase and superoxide dismutase and in the concen-
tration of copper in the blood various breed crosses of
sheep. Research in Veterinary Science 34: 69–77.
Yamano T, Morita S (1992) Hepatotoxicity of trichlorfon
and dichlorvos in isolated rat hepatocytes. Toxicology
76: 69–77.
Yano BL, Young JT, and Mattsson JL (2000) Lack of
carcinogenicity of chlorpyrifos insecticide in a high-dose,
2-yeardietarytoxicitystudyinFischer344rats.Toxicology
Sciences 53: 135–144.
224 Toxicology and Industrial Health 27(3)