APPENDIX II-AQ: Malkiewicz, et al, Cypermethrin
alters Glial Fibrillary
Acidic Protein levels in the rat brain,Environmental Toxicology and
21, Issue 1, January 2006, Pages 51-55
Katarzyna Malkiewicza, b, ,
Marcin Koterasa, Ronnie Folkessonb, Jacek Brzezinskia, Bengt Winbladb, Miroslaw
Szutowskia and Eirikur
aDepartment of Toxicology, Medical University of Warsaw, Pharmaceutical Faculty, Warsaw, Poland
bKarolinska Institutet, Neurotec, Section of Experimental Geriatrics, KFC, Novum, 141 86 Stockholm, Sweden
Received 24 January 2005; accepted 18 June 2005. Available online 2 August 2005.
Pyrethroids, widely used insecticides, are biologically active in neurons. Whether they act on the non-neuronal brain cells remains an open question. Thus, the aim of this study was to examine whether Cypermethrin intoxication affects astroglial cells in the rat brain. The levels of Glial Fibrillary Acidic Protein (GFAP) in different brain regions were measured by ELISA following oral treatment with 5 or 10% of LD50 of Cypermethrin per day for 6 days. A significant decrease of GFAP was observed in different brain regions of treated animals. The cerebral cortex showed the most pronounced effect with GFAP levels reduced to 81% of the controls 2 days after treatment and 77% 21 days after treatment. Although we did not find profound changes in the morphology of astrocytes in Cypermethrin treated animals, the decrease in GFAP suggests that astrocytes were affected by low doses of pyrethroids. The possible consequences were discussed.
Cypermethrin is one of the widely used type II pyrethroids that are regarded as safe insecticides because they are rapidly metabolized in mammals and the ecosystem. However, some of them, particularly neurotoxic isomers of potent type II compounds, have acute oral toxicities comparable to organophosphorus insecticides (Soderlund et al., 2000). Little data is available on repeated or chronic exposure to low doses of pyrethroids. This data is needed as the majority of the population is exposed to low doses of pesticides through residues in the food and beverages (Schettgen et al., 2002).
It has been shown that the neurotoxic effect of pyrethroids is based on their effect on the neuronal cell membrane. The voltage-dependent sodium channels were found to be the principal target for these compounds (Narahashi et al., 1998). Pyrethroids bind to a unique site on voltage-dependent sodium channels and prolong sodium currents, leading to repetitive bursts of action potentials or use-dependent nerve-block (Smith and Soderlund, 1998). The ATP-hydrolyzing enzymes are also sensitive to the action of pyrethroid insecticides (Dorman and Beasley, 1991). The neuronal membrane ATPase is a target for pyrethroids in the invertebrate cockroach (Clark and Matsumura, 1987), in rat synaptosomes (Rao et al., 1984) and in the developing rat brain (Malaviya et al., 1993 and Husain et al., 1994).
The effects pyrethroids have on non-neuronal cells in the brain remains an open question, but there is evidence suggesting that astroglia might be involved in their neurotoxic mechanism. Pyrethroids cause oxidative stress and were shown to disturb blood–brain barrier (Giray et al., 2001, Gupta et al., 1999a, Gupta et al., 1999b and Gupta et al., 2000).
There is increasing evidence that astrocytes may be involved in the etiology of the neurotoxicity of many xenobiotics (Aschner and Kimelberg, 1991). Disturbances in astrocyte ATPase activity and changes in membrane fluidity have been found to mediate the neurotoxicity of many lipid-soluble compounds, such as organic solvents (Vaalavirta and Tahti, 1995). Furthermore astroglial cell death caused by an excessive influx of sodium ions after treatment with veratridine (Na+ channel opener) and ouabain (inhibitor of Na+, K+ -ATPase) was reported (Takahashi et al., 2000).
The aim of this study was to examine if Cypermethrin at low, repeated doses affects astroglial cells in the rat brain. The Glial Fibrillary Acidic Protein (GFAP) has been widely used as an astroglial marker (O’Callaghan, 1991a and O’Callaghan, 1991b) and therefore we used a quantitative ELISA to determine if Cypermethrin intoxication alters the concentration of GFAP in rat brain. The results show that oral exposure to low, repeated doses of Cypermethrin results in decreased expression of GFAP in the rat brain.
Cypermethrin (Promochem), GFAP standard (American Research Project), monoclonal anti-GFAP antibody (Boerhinger Mannheim), polyclonal anti-GFAP antibody (DAKO), BCA protein assay kit (Pierce), alkaline phosphatase conjugated anti-mouse IgG (Jackson ImmunoResearch), serum-free protein block (Dako), biotinylated anti rabbit IgG antibody and avidin–biotin–peroxidase reagent (ABC kit, Vector) from Vectastain, DAB from Sigma.
Four weeks old male Wistar rats were housed under a 12-h light/12-h dark cycle and given 1 week for acclimatization prior to the experiments. Standard rodent chow and tap water were available ad libitum. All procedures were conducted in accordance with the Guide for Care and Use of Laboratory Animals and previously approved by the Animal Care and Use Committee. Six animals in each group were treated by gavage with Cypermethrin dissolved in a corn oil. The animals treated with Cypermethrin received a daily dose 5% of LD50 (12.5 mg/kg) or 10% of LD50 (25 mg/kg) for 6 days and were sacrificed 2 or 21 days after treatment. Control animals received only corn oil and were terminated at the same time as the treated animals. The animals were weighed twice, before treatment and before termination. The brains were rapidly excised, weighed and the following brain regions were dissected from 5 animals from each group: cerebral cortex, hippocampus, hypothalamus, cerebellum and medulla oblongata. Additionally, the brain from one animal from each group was used for immunohistology.
Dissected brain tissue was weighed, immediately frozen on a block of dry ice, and stored at −70 °C. Tissue samples were homogenized by sonication in 10 vol. (w/v) of hot (90–95 °C) 1% SDS/PBS and stored at −20 °C until analyzed. Before use the samples were heated (100 °C) and vortexed, diluted (1/20) in PBS containing 0.5% Triton X-100 (PBS-T) and centrifuged (10 min at 10,000 × g). The supernatants were collected and total soluble protein determined using the BCA protein assay. The supernatants were further diluted (1/25) in PBS-T before GFAP determination. GFAP was determined in duplicates using a sandwich ELISA method according to O’Callaghan (O’Callaghan, 1991a and O’Callaghan, 1991b) with minor modifications (Malkiewicz et al., 2002). Values from duplicates were averaged for further analysis. Samples from both control and treated animals were measured simultaneously on the same plate. GFAP standards ranging from 0.25 to 20 ng GFAP/100 μl were run on every plate. The concentration of GFAP was calculated in nanograms of GFAP per microgram of total protein.
Brain samples were fixed in 10% neutral-buffered formalin, dehydrated and embedded in the paraffin wax. Six micrometer-thick sections were cut, placed on glass slides and incubated at 37 °C overnight. Dewaxed sections were incubated with protein block for 30 min, and then incubated overnight at 4 °C with the polyclonal anti-GFAP antibody (1:1000). Sections were then incubated with biotinylated anti-rabbit IgG antibody (1:100) and finally with the avidin–biotin–peroxidase reagent for 30 min at room temperature. The peroxidase reaction was visualized with DAB. Samples were counterstained with haematoxylin. To visualize neurons, sections were stained with Nissl staining.
The concentrations of GFAP in the brains of the control rats were determined by quantitative ELISA at the postnatal age 43 and 62 days. These correspond to the experimental time-points of 2 and 21 days post-treatment. A small but statistically significant increase in GFAP was observed during this period in the hypothalamus (increase by 23%, p < 0.005) and cerebellum (increase by 13%, p < 0.05). A similar but statistically not significant tendency was observed in all other examined brain regions (Table 1).
For rats the LD50 of Cypermethrin dissolved in corn oil is 250 mg/kg (Desi et al., 1986). The animals treated orally with Cypermethrin received a daily dose 5% of LD50 (12.5 mg/kg) or 10% of LD50 (25 mg/kg) for 6 days and were sacrificed 2 or 21 days after treatment. No signs of general toxicity, as determined by survival index, body and brain weight were observed after treatment with Cypermethrin in our study. None of the choreothetosis with salivation syndromes that are characteristic for acute intoxication with pyrethroids type II were seen.
The concentration of GFAP in the treated animals after 21 days was significantly decreased in all regions of the brain compared to the controls (Table 1). Two days after treatment with Cypermethrin at both the lower and higher doses, the GFAP levels in the cortex were 81% of the controls. At 21 days post-treatment the levels were 77% of the control levels. A similar pattern was observed in hippocampus, although the changes were less prominent. In the hypothalamus significant decreases to 87 and 83% of the control were observed 21 days post-treatment with the lower and higher dose, respectively. In the medulla a significant decrease of GFAP was found, 76% with low dose and 78% with the high dose after 21 days. The changes in the cerebellum were small but reached significance at 21 days.
The distribution of GFAP immunoreactivity in the rat brain was examined. Morphologically, the astrocytes in the brains of Cypermethrin treated animals did not show any profound changes. The decrease in GFAP determined by ELISA was probably too small to be detected by immunohistochemistry. Nissl staining did not reveal any profound changes in neurons after treatment with Cypermethrin.
It is well established that the acute neurotoxic effect of pyrethroids is due to their effect on the neuronal cell membrane. The voltage-dependent sodium channels and ATP-hydrolyzing enzymes are the principal targets for these compounds. Based on the fact that astrocytes are sensitive to compounds that display similar mechanism of neurotoxicity, we hypothesized, that astroglial cells would also be affected by pyrethroid insecticides. In order to test this hypothesis we used quantitative ELISA to determine the concentration of the astrocytic marker GFAP in the brains of rats exposed to the Cypermethrin.
GFAP is an intermediate filament protein widely used as an astroglial marker. During rat brain development the concentration of GFAP rises sharply as neural precursor cells differentiate into astrocytes with a rapid increase in the second to third postnatal week in the most brain regions. At 5 weeks of age the rapid glial development is completed. We therefore used 5 weeks old animals to study the effect of Cypermethrin on GFAP expression.
Our results show that the levels of GFAP in the brain of adolescent rats between 43 and 62 days of age are increasing slightly. Small changes were found in all examined brain regions but statistically significant differences were only observed in the hypothalamus and cerebellum. This suggests that the astrocytes were still undergoing a late maturation process. Others have reported that a maturation pattern from the 11th to the 30th postnatal day consisting of higher cellular expression of GFAP and an increase in overall cell size and expanding arborisation is followed by stabilization of these parameters until the 90th day of life, and a subsequent decrease. Specifically, the hippocampal astrocytes undergo rapid maturation in the first month of postnatal life, followed by a slow consolidation of this process until the third month of life. At 5 months of age, there are still dynamic changes in the mature astrocytes, which become slender and thinner (Catalani et al., 2002 and Bushong et al., 2004).
In our experimental model a statistically significant decrease of GFAP, measured by ELISA, was observed in Cypermethrin treated animals when compared to the controls. In the cerebral cortex the effect was observed already 2 days after treatment and was not dose dependent at the doses used here. The same tendency was observed in other brain regions but was not statistically significant. After 21 days the effect intensified in the cerebral cortex and became significant in other brain regions. This indicates that the astrocytes in the cerebral cortex were the most sensitive to Cypermethrin at both of the time points examined whereas delayed and weaker effects were observed in the other brain regions. It is not clear if these differences are due to the different distribution and kinetics of the Cypermethrin in the brain since no such a data is available for repeated treatment with low doses. The results from a study on rabbits, using a single acute dose, suggest two patterns of Cypermethrin turnover depending on the tissue lipid concentration. While the highest concentration of Cypermethrin is reached in white matter followed by a rapid decline, in gray matter the uptake and release is slower. Therefore, even if the maximum concentration is lower in gray matter, the effect may be prolonged (Khanna et al., 2002). Interestingly, pyrethroids induced elevation in levels of peroxidation products is greater in the cerebral cortex than, e.g. in the brainstem (Rao and Rao, 1995).
Morphologically, astrocytes in the brains of Cypermethrin treated animals did not show profound changes when compared to the controls. This suggests that determination of GFAP by ELISA is a sensitive method for detection of the changes in astrocytes that might not be visible by histological observation.
Typically, when neurons are injured, GFAP rises almost immediately as a forerunner to gliosis. Decrease of GFAP, although less common, has been observed in several studies. Treatment of rats with chlorpyrifos during gliogenesis resulted in a significant decrease of GFAP across all brain regions (Garcia et al., 2002). Also nicotine treatment during adolescence leads to decrease in GFAP in the hippocampus. Hormones regulate GFAP synthesis and glucocorticoids may positively or negatively regulate GFAP, depending on the target brain structure (O’Callaghan et al., 1989, Laping et al., 1994, Maurel et al., 2000 and Nichols et al., 1990). It has been shown that toluene-induced decrease of GFAP in experimental studies was accompanied by increased corticosterone in serum (Little et al., 1998). There are a number of studies where GFAP decreases as well as increases were observed depending on the dose used and the brain regions examined (Barone, 1993, Evans and Little, 1993 and Henriksson and Tjalve, 2000).
It is difficult to predict the significance of the Cypermethrin induced decreases in GFAP for the brain metabolism and neurotoxicity of this xenobiotic but neuron-astrocyte interactions are important factors optimizing neuron function and limiting neuron death due to a number of stressors including excitotoxins and oxidants (Ye and Sontheimer, 1998 and Dringen et al., 2000). Therefore, functional impairment of astrocytes has the potential to induce and/or exacerbate neuronal dysfunction.
It has been previously shown that chlorpyrifos, an organophosphate pesticide targets developing glia in vivo and inhibits their replication and differentiation in vitro and elicits oxidative stress (Garcia et al., 2002). Our results suggest that the pyrethroid insecticide Cypermethrin also affects astrocytes in a similar manner during their late maturation. It has been also shown that low doses of pyrethroids, like the organophosphate pesticides, cause oxidative stress (Giray et al., 2001) and formation of reactive oxygen species (ROS) (Gupta et al., 1999b) along with lipid peroxidation in various tissues and decreased the GSH content in the brain of neonatal rats. There is considerable evidence suggesting that astrocytes are required to maintain optimal thiol status of neurons, which protects these cells from oxidative damage. Generally, astrocytes have more efficient GSH synthesis systems and higher levels of GSH than neurons. They buttress GSH content of neurons by the export of GSH into the intracellular milieu. Although exported GSH likely plays a direct antioxidant role in the extracellular compartment, it also increases neuronal GSH synthesis by increasing cellular cysteine levels and possibly by supporting the neuronal uptake of the CysGly dipeptide (reviewed Watts et al., 2005). Astrocytes are also involved in the formation and maintenance of blood-brain barrier and impairment of astrocytes could lead to dysfunction of the BBB. Exposure to Cypermethrin alone or in combination with quinalphos during the early postnatal period indeed caused significant impairment in the development and maturation of the blood-brain barrier (Gupta et al., 1999a, Gupta et al., 1999b and Gupta et al., 2000).
In summary, the results from our study show that oral
treatment of the rats with Cypermethrin at low doses
affects astrocytes. This effect is manifested by a
decrease of GFAP that is most pronounced in the cortex. Further studies are
required in order to elucidate the mechanisms of Cypermethrin
induced decrease in GFAP and its consequences for the nervous system.
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