Synthetic pyrethroids are a unique group of insecticides having a pyrethrin-like structure whose use has increased much in the last few years . They are preferred over organophosphates, organochlorines, and carbamates due to their high effectiveness against a wide range of insects, easy biodegradability, and low toxicity to mammals . Therefore, pyrethroids became a major pesticide in agricultural and public health applications accounting for over 30% of pesticide use . This widespread use of pyrethroids and the corresponding increase in human exposure have led to an increasing interest in studying its toxicology.
Pyrethroids act primarily on the nervous system of insect pests, and at the same time are considered as potent neurotoxicants in both vertebrates and invertebrates [4,5]. Pyrethroids are more hydrophobic than other classes of insecticides. This indicated that their site of action is the biological membranes in which pyrethroids prolong the opening duration of the voltage-dependent sodium channels . This results in altered neuronal excitability characterized by repetitive firing or depolarizing block of the neuron . Synthetic pyrethroid insecticides are toxic to the mammalian central nervous system (CNS) and peripheral nervous system in acute intoxication [7,8].
On the basis of different behavioral, neurophysiological, and biochemical profiles, two distinct classes of pyrethroids have been identified. Type I pyrethroids (allethrin and permethrin) do not contain a cyano substituent, and may cause mainly hyperexcitation and fine tremors. Type II pyrethroids (deltamethrin, cypermethrin, and fenvalerate) possess an α-cyano group and produce a more complex syndrome, including clonic seizures .
Cypermethrin is a synthetic type II pyrethroid insecticide that has been used to kill insects especially on cotton . In 1982, Egypt was the third largest consumer of cypermethrin, and was used mainly (92.5%) for the chemical control of cotton pests [11,12]. Since then, it has remained a commonly used insecticide.
Exposure to cypermethrin may occur by inhalation or by dermal and oral routes both accidentally and from the environment. The lipophilia of pyrethroids favored their absorption through the skin, gastrointestinal, and respiratory tracts to be more distributed into lipid-rich internal tissues, including body fat and elements of the CNS and peripheral nervous system . Thus, populations at high risk of exposure are producers, hygienic and pesticide workers, and farmers applying cypermethrin for plant protection.
The symptoms that are commonly observed in workers spraying pyrethroids are cutaneous paresthesia and abnormal facial sensations. In contrast, symptoms of high doses of cypermethrin toxicity in humans include twitching, drowsiness, coma, and seizures [13,14]. Pathological effects of cypermethrin were confirmed in experimental studies on different animals, and showed a picture dominated by neurotoxicity [15,16].
The metabolism of cypermethrin is quite rapid, and during its metabolism reactive oxygen species are generated. These free radicals cause oxidative stress through peroxidation of the lipid membrane. Thus, damage may occur in certain tissues and organs not only due to the direct effect of the pesticide, but also due to the generated free radicals [17–19].
Several animal studies investigated oxidative damage caused by cypermethrin [17,20,21]. Other studies evaluated the positive role of antioxidant components in the detoxification of cypermethrin [18,19,22,23].
Alpha-lipoic acid (ALA) is a powerful lipophilic antioxidant in vitro and in vivo, which plays a pivotal role as a cofactor in many mitochondrial reactions . Although there is evidence that this compound is synthesized de novo in both animal and human cells, diet seems to be the main source of this compound .
ALA differs from other antioxidants in that it neutralizes free radicals both in the fatty and in the watery regions of cells, in contrast to vitamins C and E. ALA readily scavenges hydroxyl radicals, hypochlorous acid, peroxynitrite, and singlet oxygen. Moreover, its reduced form, dihydrolipoic acid, scavenges superoxide and peroxyl radicals and can regenerate other antioxidants such as thioredoxin, vitamin C, vitamin E, and glutathione . Consequently, ALA has been proposed as a treatment for oxidative disorders of the nervous system that involve free radicals as it exerts a profound neuroprotective effect in experimental models of stroke, trauma, degenerative disorders of the CNS, and diabetes .
Previous toxicity studies of pyrethroids in general and cypermethrin in particular on the CNS and peripheral nervous system of nontarget organisms were generally concerned with their biochemical, pharmacological, and physiological. However, only few studies in the literature, were directed toward a detailed histological description of nerve lesions. To our knowledge, no available histological study addressed the effectiveness of ALA in acute cypermethrin exposure. Therefore, the aim of this study was to study the possible protective role of ALA on the sciatic nerve of albino rats intoxicated with cypermethrin by histological and morphometric methods.
Materials and methods
- (1) Cypermethrin (α-cyano-3-phenoxy-benzyl 3-(2,2-dichlorovinyl) 2,2-dimethylcyclopropane-carboxylate): a commercial grade, emulsifiable concentrate (60 g/l) was used in this study. It was kindly supplied by the Central Agricultural Pesticides Laboratory, Agriculture Research Center, Ministry of Agriculture, Dokki, Giza, Egypt.
- (2) ALA was purchased in the form of a yellow powder from Sigma chemical company (St Louis, Missouri, USA).
This study was carried out on 30 male adult albino rats, weighing 200–220 g. They were obtained from the Animal House, Faculty of Medicine, Cairo University, Egypt. Rats were housed in stainless steel cages with a 12-h light/dark cycle. They were fed standard rat chow and allowed tap water ad libitum throughout the study. Animals were treated according to the internationally accepted guidelines for care and use of laboratory animals.
Rats were divided into five equal groups (six rats each). Groups I, II, and III were controls, whereas groups IV and V were experimental groups. All groups were treated for 5 days as follows:
- (1) Group I (negative control group) received no medication.
- (2) Group II (positive control a) received 1 ml of corn oil orally by gastric gavage.
- (3) Group III (positive control b) received ALA 100 mg/kg body weight (bw) orally by gastric gavage .
- (4) Group IV (cypermethrin-treated group) received freshly prepared cypermethrin 75 mg/kg bw (approximately 1/3 LD50) dissolved in 1 ml of corn oil orally by gastric gavage .
- (5) Group V (ALA and cypermethrin-treated group) received ALA (100 mg/kg bw) orally by gastric gavage 1 h before oral cypermethrin (75 mg/kg bw) [18,28].
All animals were carefully observed for any abnormal appearance or behavior during the 5 days of treatment.
Twenty-four hours after the last dose of cypermethrin, rats were anesthetized by intraperitoneal injection of pentobarbital sodium (55 mg/kg bw). They were perfused transcardially (through a puncture at the apex of the left ventricle and a cannula inserted in the aorta) with a 150 ml fixative (containing 2.5% paraformaldehyde and 2% glutaraldehyde) in 0.1 mol/l phosphate buffer (pH 7.4) at room temperature . Perfusion continued until both the upper and lower limbs of the rat became stiff.
From each animal, both sciatic nerves were dissected out, cut into 1 mm3 pieces, and stored in the same fixative overnight at 4°C. Specimens were postfixed in 1% osmium tetroxide for 1 h, dehydrated through a graded alcohol series, and embedded in epoxy resin. Serial semithin sections (1 μm thick) were stained with 1% toluidine blue and were examined by a light microscope. Ultrathin sections (50 nm thick) were collected on copper grids and, were contrast stained with uranyl acetate and lead citrate . Then, they were examined by a JEM-1400A transmission electron microscope (JEOL, Tokyo, Japan) operated at 80 kV at the Faculty of Agriculture Research Park, Cairo University.
Quantitative morphometric analysis
The measurements were obtained using a computer-based image analysis software (Leica Qwin 500; Imaging Systems, Cambridge, UK). Semithin sections from all groups were analyzed for the following parameters :
- (1) Nerve cross-sectional area (in micrometer square): by manually outlining the nerve image at ×40 magnification.
- (2) Number of the normally appearing myelinated axons: by manually counting the normally appearing myelinated axons at ×1000 magnification in 10 randomly selected areas, each of a known area of 1311.8 μm2 marked by a standard measuring frame. Then, the number of the normally appearing myelinated axons in the entire nerve was calculated using the following equation:
- (3) Number of the abnormally appearing myelinated axons: using the previous procedure.
- (4) Percentage of the normally appearing to the abnormally appearing myelinated axons for each nerve section was calculated and the results were plotted graphically.
The cross-sectional areas, total number of axons, number (and percentage) of normally appearing myelinated axons, and number (and percentage) of abnormally appearing myelinated axons in the entire sciatic nerve among the different groups were presented as mean±standard deviation.
Statistical analysis was conducted using analysis of variance followed by post-hoc Tukey honestly significant difference test to compare variables among different groups. A P value less than 0.05 was considered to be significant.
There was no mortality in all groups during the experiment. However, salivation, slight agitation, and abnormal gait were noted in some rats treated with cypermethrin.
Light microscopic examination
All control groups (groups I, II, and III) showed the same histological features in the semithin sections of the sciatic nerve stained with toluidine blue. There was a mixture of large and small myelinated fibers appearing in transverse and longitudinal sections. They were separated by connective tissue endoneurium, and were enclosed inside fascicles bounded by connective tissue perineurium. Myelin sheath appeared as a well-preserved darkly staining rounded structure that was often rounded or elliptical in sections. The axon contained within each myelin sheath appeared pale with no structure details. Schwann cell cytoplasm stained a paler shade of blue than the myelin, and could be seen surrounding small clusters of small unmyelinated axons. The connective tissue perineurium appeared as several thin layers of collagen containing blood vessels (Fig. 1).
After cypermethrin administration (group IV), many myelinated nerve fibers appeared swollen with disrupted axoplasm and contained myelin fragments denoting Wallerian degeneration. In contrast, the unmyelinated nerve fibers appeared less affected (Fig. 2).
Administration of ALA with cypermethrin (group V) had clearly minimized the effect of cypermethrin on the sciatic nerve. Most of the myelinated nerve fibers exhibited a nearly normal appearance with only few fibers displaying disrupted axoplasm with myelin fragments denoting limited Wallerian degeneration. Swelling of Schwann cells was detected in some fibers, whereas the unmyelinated nerve fibers seemed intact. Blood vessels were also seen in the perineurium (Fig. 3).
Transmission electron microscopic examination
Ultrathin sections of groups I, II, and III showed similar structure. They revealed regular arrangement of the myelinated nerve fibers. Unmyelinated nerve fibers were also seen (Figs 4–6).
Unmyelinated fibers appeared as columns containing several axons embedded in Schwann cell processes (Fig. 5). The axons were rounded or oval separated by slender cytoplasmic processes. Similar processes separated the axons from the basal lamina surrounding the unmyelinated fibers (Fig. 6). The myelin sheath was compact and lineated. The axoplasm of both nerve fibers contained mitochondria and numerous neurofibrils (Figs 5 and 6). Schwann cells with their euchromatic nuclei were seen surrounding the myelin sheath (Figs 4 and 5). The endoneurium could be seen between the nerve fibers (Figs 5 and 6).
Cypermethrin administration (group IV) resulted in disorganization of both the myelinated and unmyelinated nerve fibers (Fig. 7). The myelinated nerve fibers were of different sizes and exhibited degeneration in their myelin sheaths. The degenerative changes were not homogeneously distributed, as some myelin sheaths were more affected than others. Some of them were not homogenous and displayed light and dark areas (Fig. 8), fragmentation, and multiple vacuolations (Figs 9–11), whereas others displayed a homogenous hyaline appearance (Fig. 10). In some sections, myelinated nerve fibers were moderately degenerated with incomplete myelin sheath that appeared thinned out at one side (Fig. 12). Sometimes the myelin sheath was irregular being larger at one pole (Fig. 10). Some myelinated nerve fibers appeared split into outer thick and inner thin layers (Figs 13 and 14) and displayed electron-lucent areas (Fig. 13) of myelin degeneration. This separation in myelin contained fragmented material (Figs 13 and 14).
Variable degrees of shrinkage of the axoplasm were observed in the sciatic nerves of cypermethrin-treated rats. The axoplasm displayed several vacuoles of different sizes (Figs 8–13) and myelin figures (Figs 9 and 12). Mitochondria are swollen with ill-distinct or ruptured cristae (Figs 8 and 11). In some axons, the axoplasm appeared fragmented, displayed membranous vacuoles, (Fig. 11) and exhibited intra-axonal wide spaces (Figs 10–12).
Some Schwann cells surrounding the myelinated nerve fibers appeared hypertrophied and swollen (Fig. 11), and exhibited multiple variable-sized cytoplasmic vacuoles (Figs 8 and 11) and membrane-bound electron-dense bodies (Figs 11 and 13).
Unmyelinated fibers were less affected, appearing as several rounded or oval axons with normal contents and surrounded by Schwann cell processes (Fig. 12). Occasionally, they were widely separated (Fig. 8).
Concomitant administration of ALA with cypermethrin (group IV) resulted in mild degenerative changes (Figs 15–18). Apart from few spots of myelin sheath vacuolations (Fig. 15), most of the myelinated and unmyelinated nerve fibers showed a picture that was similar to the control group (Figs 15–18). In isolated points, the myelin sheath was incomplete with pale areas and showed spaces containing fragments (Fig. 16). In some sections, the myelinated nerve fibers exhibited thickening and condensation of their myelin sheaths on one side and displayed multiple vacuoles containing fragments (Fig. 17).
The axoplasm was slightly shrunken and separated from the myelin sheath, thereby forming spaces that contained fragmented material (Figs 16 and 17). The axoplasm of both myelinated and unmyelinated fibers demonstrated many mitochondria and numerous neurofibrils (Fig. 18). Schwann cells encompassing the myelin sheaths were intact while the collagenous fibers of the endoneurium between the nerve fibers were regular (Fig. 18).
Quantitative morphometric examination
In semithin sections, the control sciatic nerves (group I) consisted of 1–3 fascicles, and had a total mean cross-sectional area of 74 428 μm2 (±4926) (Table 1 and Histogram 1). The mean number of myelinated nerve fibers was 7508 (±448) (Table 1), of which 7313 (±423) had a normal appearance (97.39%) whereas 196 (±39) appeared abnormal (2.61%) (Table 1 and Histogram 2).
Statistical analysis revealed no significant difference in groups II and III versus group I regarding the mean cross-sectional area of the sciatic nerve and the mean number of myelinated nerve fibers (total, normal, and abnormal).
Cypermethrin-treated semithin sections (group IV) presented a higher mean cross-sectional area of 95 686 μm2 (±5689), which was statistically significant compared with the control group (P<0.05) (Table 1 and Histogram 1). The myelinated nerve fibers had a mean number of 7339 (±504), with a statistically significant decrease (P<0.05) in the number of normally appearing myelinated nerve fibers compared with the control group to become 5601 (±419). In contrast, the mean number of abnormally appearing myelinated nerve fibers significantly increased (P<0.05) compared with the control group to become 1738 (±278) (Table 1 and Histogram 2). Abnormal appearance of myelinated nerve fibers included features of Wallerian degeneration including swelling, disrupted axoplasm, and presence of myelin fragments.
Concomitant administration of ALA with cypermethrin (group V) significantly decreased (P<0.05) the mean cross-sectional area of the sciatic nerve to 85 044 μm2 (±4938) compared with the cypermethrin-treated group (Table 1 and Histogram 1). The myelinated nerve fibers had a mean number of 7479 (±387) with a statistically significant increase (P<0.05) in the mean number of normally appearing myelinated nerve fibers to become 6952 (±455), whereas the mean number of abnormally appearing myelinated nerve fibers significantly decreased (P<0.05) compared with the cypermethrin-treated group to become 527 (±104) (Table 1 and Histogram 2).
Cypermethrin was classified as a moderately hazardous (class II) pesticide . On account of its low toxicity to mammals, cypermethrin is used to control indoor pests, but carelessness in its handling may result in serious effects .
In this study, cypermethrin neurotoxicity on sciatic nerve of the albino rat was demonstrated histologically. The Albino rat was chosen as the experimental model, because it was established that the histological structure of the rat's nerve was fundamentally indistinguishable from that of man .
The general manifestations of salivation, agitation, and abnormal gait observed in experimental rats, which received cypermethrin in this study, might be attributed to CNS toxicity. These findings coincided with the previously recorded decreased food intake, ataxia, and salivation reported in rats intoxicated by cypermethrin . In addition, the abnormal gait could be referred to sciatic nerve degeneration .
In this study, light microscopic examination of semithin sections of sciatic nerve of rats given cypermethrin for 5 consecutive days revealed nerve damage particularly affecting the myelinated nerve fibers. The lesions were manifested as axonal damage and changes in the myelin sheath. Myelinated nerve fibers were swollen with disruption of their axoplasm that enclosed myelin fragments denoting Wallerian degeneration. Unmyelinated nerve fibers appeared less affected. Morphometry and statistical analysis showed a statistically significant increase in cross-sectional diameter of the sciatic nerve and in the number of degenerated myelinated nerve fibers compared with the control. Ultrastructurally, degenerative changes were not homogeneously distributed, with some myelin sheaths affected more than others. Myelin sheaths exhibited spotty areas of fragmentation, vacuolations, and hyalinization. Some of them appeared incomplete or splitted. The splitting could be related to the accumulation of edema fluid. Axoplasm displayed shrinkage, vacuolations, fragmentations, myelin figures, and intra-axonal wide spaces that might be due to edema. Mitochondria were swollen with ill-distinct or ruptured cristae. Schwann cells around myelinated nerve fibers were swollen and filled with multiple variable-sized cytoplasmic vacuoles and membrane-bound electron-dense bodies. The latter might represent dilation of membranous organelles. Apart from few incompact and widely separated axons, unmyelinated fibers were ultrastructurally similar to the control.
Axon degeneration was described in the sciatic and tibial nerves of rats exposed to acute intoxication with cyanopyrethroid deltamethrin . The same researchers reported partially degenerated myelin sheath and intra-axonal electron-dense lamellar bodies resembling myelin figures. They also demonstrated cytoplasmic vacuolation in some Schwann cells, which were suggested to represent proliferation of rough and smooth endoplasmic reticulum and Golgi apparatus. Moreover, the sciatic nerve of cypermethrin-treated frogs showed degeneration in the myelin sheath and axon, decrease in the number of axonal microtubules and neurofilaments, and evident mitochondrial damage .
The minimal affection of unmyelinated nerve fibers compared with myelinated fibers, in this study, was explained by the fact that cypermethrin tends to be concentrated in fatty tissues. Accordingly, it affects the myelin sheath, which is lipid in nature .
Physiologically, the myelin sheath provides electrical insulation of neuronal processes and its absence leads to slowing of conduction . The normal function of myelinated nerve fibers is dependent on the integrity of not only the axon, but also of its myelin sheath . In this study, the histological findings indicated that cypermethrin affected both myelin sheaths and axons.
In contrast, no peripheral nerve affection could be demonstrated in rats fed on permethrin, which is a type I pyrethroid, lacking the α-cyano group . In contrast, type II pyrethroids such as cypermethrin, containing the α-cyano group, were shown to affect both the CNS and peripheral nervous system .
Earlier studies reported no evidence that pyrethroids acted as oxidants or free radical generators [41,42]. In contrast, the more recent studies indicated that, in addition to its direct action, cypermethrin inflicted its toxicity through oxidative stress by accumulating excess reactive oxygen species and reducing the natural antioxidative systems [17,21,43–45]. These factors might be responsible for the increased lipid peroxidation and tissue damage.
It is well known that excessive free radical production, if not effectively balanced by antioxidant systems, is responsible for aggression directed at phospholipids of cell membranes, mitochondrial, and cellular proteins . Prevention against oxidative stress can be obtained by administration of one or more chemical entities, either as individual drugs or as naturally occurring dietary constituents .
In this respect, it was noticed that concomitant administration of ALA with cypermethrin in this study resulted in less degenerative changes than those observed with cypermethrin alone. Only few nerve fibers displayed disruption of their axoplasm with myelin fragments indicating minimal Wallerian degeneration. Swelling of Schwann cells was detected in some fibers. Morphometry and statistical analysis showed a statistically significant decrease in the cross-sectional diameter of the sciatic nerve and in the number of degenerated myelinated nerve fibers compared with the cypermethrin-treated group. Ultrastructurally, only few fibers showed spots of myelin sheath affection in the form of vacuolations and irregularities. Apart from slight shrinkage in limited areas, the axoplasm of both myelinated and unmyelinated fibers, and also Schwann cells, had a comparable ultrastructure with that of the control.
ALA protected the sciatic nerve against most of the histologic changes induced by cypermethrin. Therefore, it could be proposed that oxidative stress might play a role in producing cypermethrin neurotoxicity.
This neuroprotective effect of the antioxidant ALA agreed with many previous studies that demonstrated effective protection of other antioxidants such as allopurinol, isoflavone, ascorbic acid, propolis, and α-tocopherol against toxicity of pyrethroids [18,21,22,44,45,47,48].
ALA is considered a potent free radical scavenger and a metal chelator and a very promising drug. It also plays an important role in the regeneration of the active form of other cellular antioxidants including vitamins C and E . Moreover, ALA is easily absorbed from the gastrointestinal tract and is easily transported across cell membranes. Thus, free radical protection occurs both inside and outside the cells . It is also water and fat soluble, which makes it effective against a broader range of free radicals than of vitamin C (water soluble) and vitamin E (fat soluble) alone . It can also cross the blood–brain barrier without exhibiting any serious side effects .
As a result of its potent antioxidant activity, ALA has been proposed as a treatment for oxidative disorders of the nervous system that involved free radicals, as it exerted a profound neuroprotective effect in experimental models of stroke, trauma, degenerative disorders of the CNS, and diabetes . Administration of ALA to rodents had been demonstrated to reduce the damage that occured after ischemia-reperfusion injuries in the cerebral cortex, heart, and peripheral nerves [27,52,53].
In conclusion, the results of this study revealed that the pesticide cypermethrin produced marked structural changes in the sciatic nerve of albino rats. Moreover, ALA exerted significant protection against these changes, which was confirmed morphometrically and statistically. Therefore, it is recommended that high-risk groups as farmers and other pesticide workers should take strict precautions when handling cypermethrin. Nevertheless, further long-term studies under medical observation are advised to promote the use of ALA among exposed people.
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