774 Journal of Health Science, 52(6) 774–780 (2006)

The Pyrethroid Cypermethrin- INTRODUCTION

Induced Biochemical and The undesired effects of pesticides have been

Histological Alterations in Rat recognized as a serious public health concern dur-
ing the past decades. Synthetic pyrethroids such as
Liver cypermethrin, permethrin, and deltamethrin are in-
creasingly used for indoor pest control because of
Altug Yavasoglu,a Ferah Sayım,b their high insecticidal activity and considerably
Yigit Uyanıkgil,a Mehmet Turgut,*, c lower mammalian toxicity compared with other pes-
and Nefise Ülkü Karabay-Yavasoglub ticides.1) In 1977, cypermethrin was allowed for turn-
over as a very active synthetic pyrethrin insecticide,
a
Department of Histology and Embryology, Ege University, effective in the control of many pest species in agri-
Faculty of Medicine, TR-35100 Izmir, Turkey, bDepartment of culture, animal breeding and the household.2,3) After
Biology, Ege University, Faculty of Science, TR-35100 Izmir,
household treatments, it persists in air and on walls
Turkey, and cDepartment of Neurosurgery, Adnan Menderes
and furniture for about three months.4) In spite of
University, Faculty of Medicine, TR-09100 Aydın, Turkey
(Received November 14, 2005; Accepted May 25, 2006)
the low toxicity of pyretroids, persistence of these
compounds in mammalian tissues may be danger-
Cypermethrin, a synthetic pyrethroid, has broad- ous.5) Permanence of cypermethrin and its fatty acid
spectrum use in agriculture, domestic and veterinary conjugates in adipose tissue, brain and liver was re-
applications due to its high bioefficacy, enhanced sta- ported in rats.6,7) Several studies have demonstrated
bility and considerably low mammalian toxicity. The that cypermethrin has hepatotoxic potential in ro-
objective of this study was to investigate the dents and it also acts as a neurotoxin in mammals
cypermetrin-induced alterations in the liver tissue of and insects and suppresses immune system.1,4,8–12)
Wistar male rats, based on the histopatological, The data in the literature about cypermethrin
enzymological analyses and apoptotic changes. The were obtained in different experimental conditions
animals of the experimental groups were orally fed with such as different doses, animals and different treat-
laboratory chow combined 60, 150, and 300 mg/kg Kral ment schedule. This study is a repeated dose 28-day
250 EC during 28 consecutive days. At the end of the oral toxicity study in rodent (OECD 407)13) and it
treatment, no significant change was found in relative has been designed to assess histopathological, bio-
liver weights, liver total proteins and cholinesterase
chemical, and organ weight endpoints and changes
enzyme activities of cypermethrin treated rats, when
in hepatocyte apoptosis. The aim of the current study
compared with control animals. Histopathological
was to analyze the subacute hepatotoxic effect of
changes such as vacuolar degeneration, enlargement
of the sinusoids, degeneration in hepatic cords and
the orally administered cypermetrin in Wistar albino
hepatocytes, vacuole formations in hepatocytes, pleo- male rats, based on histopathological and biochemi-
morphism in nucleus, and congestion were observed cal findings. The TUNEL assay was used to detect
in liver tissues of only 150 and 300 mg/kg cypermethrin apoptotic cells in liver.
treated rats. Mononuclear cell infiltration and an in-
crease in the Kupffer cells in liver parenchymatous tis-
sue were also determined. In all cypermethrin treated MATERIALS AND METHODS
groups, the apoptotic index in livers of rats was sig-
nificantly increased compared to control group (p < Chemicals —–— Commercial formulation of
0.001). These results suggest that cypermethrin might cypermethrin (250 g of cypermethrin/l), Kral 250
cause hazardous effects in different levels to non-tar- EC (Safa Agriculture, Türkiye) was used. It was in
get organisms. the form of emulsion and adequate dilutions were
done in water in order to reach test concentrations
Key words —–— cypermethrin, histopathology, apoptosis,
(60, 150, and 300 mg/kg). The test concentration of
cholinesterase activity, liver, rat
cypermethrin was calculated from the percentage of
the active ingredient of commercial formulation of
cypermethrin. Solutions were freshly made imme-
*To whom correspondence should be addressed: Cumhuriyet
Mahallesi, Cumhuriyet Caddesi, No:6 Daire:7, TR-09020 Aydın,
diately before usage. All the other reagents used were
Turkey. Tel.: +90 256 2134874; Fax: +90 256 2120146; E-mail: of analytical reagent grade and obtained from Sigma
drmturgut@yahoo.com (St Louis, U.S.A.).
 No. 6 775

Animals and Experimental Design —–— The pro- transcardially with 100 ml heparinised saline fol-
tocol was approved by the Animal Ethical Commit- lowed by 300 ml of 4% para-formaldehyde in
tee of Ege University, Faculty of Medicine. The study 0.1 mol/l phosphate buffer (pH: 7.4). Livers were
was conducted on 80 adult male (8-week old) Wistar removed, post-fixed for 24 hr in the same fixative,
albino rats weighting 120–160 g obtained from and processed for paraffin embedding. After routine
Breeding Center of Experimental Animals in Ege processing, paraffin sections of each tissue were cut
University, Faculty of Medicine. After 10 days of into 5–6 µm thickness and stained with haematoxylin
acclimation, the rats were assigned randomly to ei- and eosin (H&E).
ther the exposure groups (60, 150, and 300 mg/kg) Analysis of Apoptosis in Tissue Sections —–—
or the control group, each containing 20 male rats Apoptosis in the liver was defined and quantitated
and housed individually in labelled cages (19 × 19 × as previously described by Promega DeadEnd™
12 cm) with solid plastic sides and stainless-steel Colorimetric Apoptosis Detection System (TUNEL)
grid tops and floors. They were maintained in con- (Promega Corp., Madison, U.S.A., Cat No: G7130).16)
trolled laboratory conditions of 12 hr dark/ light For TUNEL, cells were fixed in 4% paraformalde-
cycle, 21 ± 1°C temperature and 45–75% humidity. hyde solution for 25 min at room temperature, rinsed
Animals of the control group were orally fed daily in phosphate buffered saline (PBS) and
with a normal diet in standard laboratory chow (10 g/ permeabilized by immersing the slides in 20 µg/ml
rat/day), while the animals of the treated groups were Proteinase K solution. Cells were incubated with ter-
fed with laboratory chow (10 g/rat/day) combined minal deoxynucleotidyl transferase (rTdT) reaction
cypermethrin during 28 consecutive days as de- mixture containing biotinylated nucleotides and
scribed in OECD guideline 407.13) Tap water was rTdT at 37°C for 60 min, rinsed with sodium chlo-
also available ad libitum. All animals were weighed ride-sodium citrate buffer (SSC) and PBS.
weekly throughout the study. Streptavidin horseradish peroxidase (HRP) was
Biochemical Assays —–— After 28 days of the ex- added to each slide and incubates for 30 min at room
periment, ten animals of each group were killed by temperature. Slides were then stained with diamino
cervical dislocation and the livers them were dis- benzidyne system (DAB). In this study, TUNEL (+)
sected out, weighed and stored at –70°C until analy- immunoreactivity was assessed by light microscopy
sis. The liver tissues (1 g) homogenized with Ultra (Olympus BX-51 light microscope, Olympus C-
Turrax homogeniser in 5 ml of 50 mM phosphate 5050 digital camera) at a magnification of ×100.
buffer (pH: 7.4). The particle free supernatant was TUNEL (+) cells were counted with the use of Im-
obtained by centrifugation at 5000 g for 20 min at age-Pro Express (Media-Cybernetics, 2002, U.S.A.)
4°C and used as enzyme source. Liver cholinesterase image software in rat liver. The number of apoptotic
(ChE) activities were determined by the hepatocytes (at any morphological phase) was di-
spectrophometric method of Ellman et al.14) The as- vided by the total number of hepatocytes and multi-
say mixture contained 0.259 mM 5,5-dithiobis-2- plied by 100 to achieve and apoptotic index. For each
nitrobenzoic acid (DTNB) in 67 mM phosphate group, a minimum of 2000 hepatocytes was counted.
buffer, pH: 7.4, 0.298 mM buthirylthiocholine Statistics —–— The results of biochemical analysis,
iyodide and 20 µl of 250-fold dilution of the en- liver and relative liver weights were presented as
zyme source in a total volume of 3.02 ml. Reaction the mean ± standard error of mean (S.E.M.). Com-
was followed at 410 nm for 10 min intervals at 37°C parisons were made between control and treatment
against blank containing buthirylthiocholine iyodide groups using one-way analysis of variance
and phosphate buffer. The extinction coefficient of (ANOVA) followed by Dunnett’s test. Values of p <
the product of the chemical reaction, 5-thio-2- 0.05 were regarded as statistically significant.
nitrobenzoate is 13.61 mM–1 cm–1. The protein con-
tent was estimated, described previously by Lowry
et al.15) On the other hand, the liver weights were RESULTS
recorded and relative liver weights of each animal
were calculated. Body and Liver Weights
Histopathological Examination —–— For light mi- Mean changes in body weight, liver and relative
croscopic examination, other 10 animals of each liver weights are summarized in Table 1. At the end
group were anaesthetized (0.10 mg/kg Ketalar® + of the experiment, no statistically significant differ-
0.02 mg/kg Rompun ® , i.p.) and perfused ences were occurred in any treatment group com-
 776 Vol. 52 (2006)

pared with control group for absolute change in soids, degeneration in hepatic cords and hepatocytes,
weight gain (p > 0.05). When compared with the vacuole formations in hepatocytes were determined
control animals, while a significant decrease was in the liver of the rats in 150 and 300 mg/kg
determined in the liver weight of the rats treated with cypermethrin treatment (Fig. 1B). Mononuclear cell
150 and 300 mg/kg cypermethrin (p < 0.05), no sig- infiltration and an increase in the number of Kupffer
nificant change was found in any treatment group cells (hyperplasia) were designated in the liver par-
for relative liver weights. enchymatous tissue of rats (Fig. 1C). In addition,
congestion was noted in the liver of all treated rats
Biochemical Changes (Fig. 1D). Vacuolar degeneration as a result of an
Table 2 shows the effect of cypermethrin on liver increase in the number of lipid vacuoles in cytoplasm
total protein values and ChE activities of experimen- and therefore a cytoplasmic damage were observed
tal rats. There was no significant change in liver to- in the liver tissues of 150 and 300 mg/kg
tal protein values among control and treatment cypermethrin treatment.
groups. Liver ChE activities were increased in all
treatment groups compared with control values but Apoptotic Index in Rat Liver
these inductions were not statistically significant When compared with control animals, there were
(p values for 60, 150, and 300 mg/kg doses are 0.448, significant increases on apoptotic index in liver tis-
0.307, and 0.059, respectively). sues of cypermethrin treated rats (p < 0.001). Table 3
and Fig. 2A–2D is represented apoptotic nuclei of
Histopathological Changes rat hepatocytes. In comparison with the control
The liver of control animal is showed in Fig. 1A. group, 91, 253, and 595% dose-dependent increases
The administration of cypermethrin for 28 days re- of apoptotic nuclei in liver tissues were observed in
sulted in dose-dependent degenerative changes of rats treated with 60, 150, and 300 mg/kg
variable degrees in many areas of the liver. Histo- cypermethrin, respectively.
pathological effects of 150 and 300 mg/kg
cypermethrin on the liver of treated animals are pre-
sented in Fig. 1B–1D. An enlargement of the sinu-

Table 1. Mean Changes in Body Weight, Liver and Relative Liver Weights of Rats in Control and Cypermethrin Treated Groups

Parameters Control 60 mg/kg 150 mg/kg 300 mg/kg

(n = 10) (n = 10) (n = 10) (n = 10)
Initial Weight (g) 157.1  24.2 120.0  28.7 121.0  15.9 126.3  14.3
Final Weight (g) 180.0  25.1 173.1  21.4 154.8  13.7 155.9  11.3
Weight Change (g) 22.9 53.1 33.8 29.6

Liver Weight (g) 6.56  0.65 6.59  0.66 4.75  0.19a) 5.30  0.92a)

Relative Liver Weight 0.030  0.002 0.038  0.003 0.031  0.003 0.034  0.002
Values are given as mean  S.E.M. a) Statistically significant difference from control by Dunnett test (p < 0.05). n is total number of animal
in each group.

Table 2. Liver Total Protein Values and ChE Enzyme Activities of Rats in Con-
trol and Treatment Groups

Groups n Total Protein ChE

(mg/ml) (mol/min/mg protein)
Control 10 16.87  2.93 0.0045  0.0009
60 mg/kg 10 22.35  4.70 0.0066  0.0013
150 mg/kg 10 26.00  1.28 0.0073  0.0017
300 mg/kg 10 23.48  5.29 0.0099  0.0030
Values are given as mean  S.E.M. n is total number of animal in each group.
 No. 6 777

Fig. 1. The Administration of Cypermethrin for 28 days (150 and 300 mg/kg) Resulted in Damage of Architecture of Liver Cells along
with Disarrangement of Hepatic Cords
A: The liver of control animal. B–D: An enlargement of sinusoids (S), vacuole formation in hepatic paranchyma (arrow), degenerative changes in
hepatocyte (H) could only be observed in 150 and 300 mg/kg dosed animals. 1D also exhibit erythrocytes in liver paranchyma (C). All such changes some
time also appears when animals are killed using faulty way of killing. H&E Stain; Original magnification, × 40 (A, B, and D), × 10 (C).

Table 3. Apoptotic Index of Rats in Control and Treatment Long-term feeding studies with laboratory ani-
Groups mals have shown adverse effects of cypermethrin: it
Groups n Apoptosis Increase (%) caused reduced growth rate and increased liver
Control 10 4.85  0.26 — weight in rats.19) However, our study is a repeated
60 mg/kg 10 9.28  0.35a) 91.3 dose 28-day oral toxicity study in rat and it is a short-
150 mg/kg 10 17.14  0.50a) 253.4 term feeding study. Therefore, in this study, while
300 mg/kg 10 33.71  1.22a) 595.0 there was no significant change in relative liver
Values are given as mean  S.E.M. a) Statistically significant weight of rats of all cypermethrin treated groups,
<
difference from control by LSD by Dunnett test (p 0.001). is n there was a significant decrease in the liver weight
total number of animal in each group.
of the rats treated with only 150 and 300 mg/kg per
day cypermethrin. Body weight gain of rats in ex-
perimental groups did not change throughout the ex-
DISCUSSION perimental period when compared to control. It was
reported that some synthetic pyrethroids such as
The prolonged and indiscriminate use of permethrin, deltamethrin had no effect on body
cypermethrin is reported to cause both acute and weight gain of rats.20,21) These results support with
chronic toxicity in non-target species including hu- ours.
mans.17,18) Most toxic chemicals are metabolized in There is limited research about the effects of
liver and these processes may cause liver injuries. cypermethrin on ChE enzyme activity in liver. In
In our study, the evaluation of liver tissue using bio- our study, cypermethrin had no significant effect in
chemical and histopathological assays indicated that total protein and ChE enzyme values of liver. Simi-
subacute doses of cypermethrin induce only dose- larly, in another study, no statistically significant
dependent histopathological changes in the tissue; changes were detected in total protein levels during
however, in biochemical examination, no significant 7 days of i.p. doses of 300 mg/kg cypermethrin treat-
change in total protein and ChE enzyme levels was ment.22) However, in chronic studies it was observed
determined. that cypermethrin caused an increase in protein con-
 778 Vol. 52 (2006)

Fig. 2. A: The Liver Tissue of Rats in Control Group, B–D: The Liver Tissue of Rats Treated with 60, 150, and 300 mg/kg Cypermethrin,
Respectively
The arrows points apoptotic nuclei of rat hepatocytes. Slides were stained with DAB. Original magnification, × 10.

tent.23) parison with the control group, dose-dependent in-

Our histological results showed that histopatho- creases of apoptotic nuclei in liver tissues were ob-
logical changes such as mononuclear cell infiltra- served in rats treated with 60, 150, and 300 mg/kg
tions, an enlargement of the sinusoids, degeneration cypermethrin, respectively. Programmed cell death
in hepatic cords and hepatocytes in liver of rats ex- or apoptosis is important during embryonic devel-
posed to 150 and 300 mg/kg cypermethrin. Similar opment, maintaining tissue homeostasis, and for re-
histolopathological changes such as mononuclear moving damaged or infected cells. However,
cell infiltration and parenchymatous degenerations apoptosis has been shown to be triggered by several
of hepatocyte in liver were observed in Wistar rats factors, including exposure to pesticide.33–36) Our
exposed 250 mg/kg orally alpha-cypermethrin.3) results showed that cypermethrin produced cell in-
However, it was reported that slight histopathologi- jury and apoptosis in rat hepatocytes. Also
cal changes in liver was observed in dermally cypermethrin-induced apoptosis in the telencepha-
cypermethrin applied rats.24–26) Daily oral cyhalothrin lon of tadpoles has been reported by Izaguirre et al.
administration in mice at levels up to 2000 mg/kg (2000).37) Permethrin, another synthetic pyrethroid,
diet for 28 days showed dose-related histopathologi- caused concentration-dependent increase in both
cal changes in liver of rats exposed to dosages of apoptotic and necrotic cell death in thymocytes.38)
100 mg/kg and above.27) Histopathological liver Though these findings indicate a specific role for
damages caused by cypermethrin were determined apoptosis in cypermethrin induced toxicity, it is still
in other studies as well.22,28,29) premature to draw definite conclusions on the role
In vivo and in vitro studies of cypermethrin have of apoptosis in environmental pollutant-induced tox-
shown that it causes necrosis, inflammation and cy- icity.
toplasmic hypertrophy in hepatocytes.30,31) Previous The overall results of this study showed that oral
studies demonstrated cypermethrin-induced hepato- exposure to cypermethrin introduces significant his-
toxicity in rats.22,24,32) We have also observed that sub- topathological alteration in liver of rats. Based on
acute hepatotoxicity of cypermethrin on the rats. our results and literature data, we suggest that
There were significant increases on apoptotic index cypermethrin usage might cause hazardous effects
in liver tissues of cypermethrin treated rats. In com- in various levels to non-target organisms, including
 No. 6 779

human being. 14) Ellman, G. L., Courney, K. D., Andres, V. and

Featherstone, R. M. (1961) A new and rapid colori-
metric determination of acetylcholinesterase activ-
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