EFFECT OF CYPHENOTHRIN-INDUCED SPLENIC DAMAGE AND HEMATOLOGICAL ALTERATIONS IN MALE WISTAR RATS ( RATTUS NORVEGICUS )

Objective: Cyphenothrin is a major insecticide causing toxicological implications in mammals. Several studies estimated the consequences posed by this insecticide. The present study was designed to investigate the possible pyrethroid effects of cyphenothrin-induced hematological alterations and splenic damage in male Wistar albino rats. Methods: The rats were subjected to 60 d of exposure to a sublethal concentration of cyhenothrin. Hematological analyses revealed alterations in blood indices including red blood cells, white blood cells, hematocrit, hemoglobin, and platelet count. However, increased cyphenothrin level in treated rat groups was significant in the present study. This might be attributed to cyphenothrin enhancing stress of animal physiology. Results: Histological examination of spleen resulted in rarefication of white pulp, damaged marginal zone, decreased periarteriolar lymphoid sheath (PALS) 35.33 mg/Kg BW high dose, and number of lymphoid follicles in the high concentration of cyphenothrin group. However, the treatment of cyphenothrin significantly affected the low-concentration cyphenothrin-treated group more than the high-concentration-treated group 63.6 mg/Kg BW low dose compared to the control. Conclusion : This indicates the effective property of toxicity on the immunomodulatory effects of cyphenothrin. Results of the present study suggest that the Cyphenothrin effect has a potentially key role in hematological and immunomodulatory processes that might be implemented.


INTRODUCTION
The insecticidal components of the pyrethrum essence of the chrysanthemum flower, known as pyrethrin, are naturally occurring compounds called pyrethroids, which are chemically synthesized combinations.They are one of the newest classes of pesticides now on the market and make up a significant portion of pesticides used domestically.These substances are widely used in veterinary and human medicine to manage domestic insects and ectoparasites [1].Due to implied daily exposure, this may have caused an unanticipated increase in their use [2,3].In urine samples from certain members of a group with no known exposure, studies have found chemical remnants of various pyrethroids [4,5].According to Schettgen et al. 2002, consumption of tainted food may have caused the maturity of the impurity that was seen.
Likewise, the Center for Disease Control and Prevention (CDC) Third National Report on Human Exposure to Environmental Chemicals revealed mortal exposure to these agents.These studies revealed that humans are exposed to several pyrethroids pesticides via direct use, salutary means and occupational exposure, thereby making oral route one of the major means of exposure to these chemicals [6].The vulnerable system, which also includes the blood and spleen, had been observed to be least considered in toxicological studies involving pyrethroids.This is because certain studies have previously shown that the serum of rats contained carboxylesterase, which has the capability to break down pyrethroids, hence making blood a major point of pyrethroids metabolism [7,8].Since this enzyme is set up only in the serum, there is the possibility of other organs suffering from pyrethroids toxins.Some studies have demonstrated that multitudinous of generally used pesticides can suppress the vulnerable system and affect an increased threat of contagious agents.Studies have also shown that short-term exposure to Tetrachlorodibenzo-pdioxin (TCDD) reacted in thymus and spleen atrophy as well as cellular reduction of bone gist via apoptosis [9,10].
Another synthetic pyrethroid, generally appertained to as Cyphenothrin (5% EC), was tested for effectiveness against three vector mosquitoes as a space splash or mist.The International Program on Chemical Safety has stated that cyphenothrin is dangerous (IPCS).Issues with the mosquito (Culex pipiens), housefly (Musca domestica), and German cockroach were demonstrated by nearly divergent preludes (Blatella germanica).Field trials in Malaysia with Gokilaht-S 5% EC showed encouraging goods against Aedes aegypti, Ae. albopictus, and Cx.Quinquefasciatus.
Although there is a common perception by buyers about the general safety of pyrethroids insecticides, several studies have reported their toxic goods [11][12][13].It's also believed that insecticides generally have adverse effects with prolonged exposure, mainly when they are mixed to round their modes of action, as seen in cyphenothrin insecticides.According to Gosselin, some of these agents are constantly formulated with canvases or petroleum distillates and packaged in combination with synergists analogous to piperonyl butoxide (PBO) and n-octyl bicycloheptene dicarboximide, thereby rendering them more toxic and prone to conditions.Therefore, this study aimed to probe the toxicological effects of Pyrethroids insecticides cyphenothrin on the blood and spleen of adult albino rats.

Experimental toxicant
Cyphenothrin (Type II pyrethroid) was provided with the aid of Sumitomo Chemical India Pvt.Ltd, Gujarat, India, used to be procured from the nearby agricultural market of Dharwad, Karnataka, India.The expiry date of the take a look at substance used to be established prior to the initiation of the exposure.The stock solution used to be organized by means of mixing the calculated extent of the commercialgrade solution with distilled water.

Experimental design
A test dose of 63.6 mg/kg BW (High dose) 1/5 th of LD50 and 35.33 mg/kg BW (Low dose) 1/9 th of LD50 was selected.The experimental period was 60 d and the dosing was done during daylight.Postacclimatized male albino rats were randomly selected and divided into 6 groups (n=6).The first group served as control, the second and third groups received cyphenothrin alone in high and low doses (63.6 mg and 35.33 mg/kg BW), respectively.

Hematology
Post experimental animals have been dissected on the sixtieth day and the blood has been accumulated in EDTA (helico HI-Nvac K3 EDTA, two ml) and clot activator vacutainers by using cardiac perforation [37].The accumulated blood used to be subordinated to centrifugation (3000 rpm, 5 min) to achieve a clear plasma membrane and saved in a deep freezer till further analyses.Hematological parameters such as Hemoglobin (Hb), Platelet count, Serum calcium, Cholesterol, Red blood cells (RBC), White blood cells (WBC), Hematocrit, Blood urea, Sugar, Creatinine, Neutrophils, Eosinophils, and Lymphocytes had been anatomized the usage of auto hematology analyzer (UBM, Fx-19).Mean corpuscular extent (MCV), Mean corpuscular hemoglobin awareness (MCHC) used to be calculated through the following formulae.

Histology
Rats had been autopsied on the sixtieth day of investigation and the organs have been insulated and washed with PBS (1X, pH7.0) and constant in formalin (10 percent) for 24 h.Post-constant organs have been subordinated to a dehumidification procedure with alcohol slants and bedded in paraffin wax [25].Thin sections (5 µm) of the spleen had been attained the usage of an automatic microtome (Leica RM 2255).The sections have been additionally stained with Hematoxylin and eosin and photographed with the use of an Olympus segment distinction microscope (Olympus BX51, Tokyo, Japan) with a connected photographic digital camera (ProgRes C3, Jenoptic-Germany).The slides had been anatomized in x100 exaggeration for higher histopathological findings.

Ethical statement
All experiments carried out in the investigation followed the hints of the Institutional Animal Ethics Committee (IAEC).The experimental animals used in the study had been treated with care in accordance with the pointers supplied with the aid of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), New Delhi, India, Registration number 639/GO/Re/S/02/CPCSEA.New Delhi, India.Valid for five years, from August 31, 2018, to August 30, 2023.

Statistical analyses
All the data presented in the study was performed using the Origin Pro 2019b statistical package.The data were subject to one-way analyses of variance (ANOVA) with Tukey's post-hoc test and the data are presented as mean±SD values with significance value set at P<0.05.

Haematology
The hematological information analyzed in this investigation is presented in table 1. RBC, WBC, hemoglobin, and hematocrit are hematological indices that show how the animal is doing overall.Red blood cells (RBCs) were placed in low positions in high-dose and lowdosage treated animals in the current investigation, and these positions were substantially different (P<0.05).The high-dose group showed higher numbers of white blood cells (WBC) compared to all other groups.In the insecticide-treated group, hemoglobin, hematocrit, and platelet count were all kept within normal ranges; however, in the highdose group, they declined.MCV readings did not significantly change, with the exception of the high-dose group.The high-dose group's MCHC showed a significant difference (P<0.05).Total cholesterol, blood sugar, and serum calcium conditions all differed greatly from the group in control (P<0.05).Table 1 summarizes the variations in blood urea, serum creatinine, neutrophils, lymphocytes, and eosinophils.The values (mean±SE) (n=6) bearing dissimilar letters (a to c) in column differ significantly (P<0.05).   1 shows normal histoarchitecture in the control spleen tissue of a male albino rat, with sufficient amounts of red pulp (R), white pulp (W), lymphoid follicles (LF), and marginal zone (MZ).Table 2 has detailed histology observations.The histological structures in the cyphenothrin-treated group were well terminated, just like in the control group.The red pulp in the group that received high doses of the pesticide cyphenothrin showed decreased cellularity, while the white pulp was severely destroyed, with no normal lymphoid follicle and boundary zone.White pulp and red pulp from the low-cure cyphenothrin group showed destroyed cellular integrity.This cyphenothrin-treated group, which included high and low doses, demonstrated notable modifications of histological structures in contrast.In comparison to the cyphenothrin high dosage group, the white pulp recovered effectively in the cyphenothrin insecticide low dose treatment group.

DISCUSSION
Cyphenothrin caused haematological and histological changes in the current investigation.RBCs were much lower in the high dose cyphenothrin treated group, according to haematological analyses, demonstrating that the rat's anaemia was caused by the insecticide.Still, the rat treated with the cyphenothrin showed a striking improvement in RBC count.The toxic impact of cyphenothrin, which has been shown to affect bone gist cells, may be responsible for the decreased RBC count [16].Elevated WBC levels in animals treated with high pesticide doses may signal the beginning of an infection and the development of a defense mechanism [17].Hematocrit and haemoglobin levels differed little from control.MCV levels were found to be higher in the group receiving large doses of cyphenothrin, which may indicate an attainable reason for anisocytosis.
The rats which received the dose for sixty days had the greatest effects, demonstrating that prolonged exposure to the predicted pyrethroids affects haemapoiesis.Aplastic anaemia is characterized by hypo-cellularity of the bone marrow and pancytopenia, which may result from the development of hepatitis [18].In the majority of cases of severe liver disease, decreased plasma protein synthesis may result in expansive haemorrhages.In 1996, Pereira et al.Prior research by Iteire et al. from 2005 described severe blood vessel traffic in the lungs of treated rats [19,20].Therefore, we can infer from this study that anaemia may pose a risk to rats exposed to the insecticide cyphenothrin.Additionally, the extensive proliferation of blood vessels seen, particularly in rats treated for the longest duration, is consistent with research by Luty et al. from 2000, which showed that fenvalerate and deltamethrin increased erythropoiesis and Hb conflation in male mice [21].All type II pyrethroids (allethrin, imoprothrin) contain cyano-groups, which may restrict the blood's ability to carry oxygen, resulting in a decrease in metabolism.As a result, decreased oxygen rotation in the blood will cause the production of erythropoietin, which then stimulates the production of more blood by the bone marrow until the marrow becomes overly strained and unequipped to replace lost cells, causing the bone marrow to be under-cellularized.
Increased MCV could be a sign of immature RBC due to a compensatory medium serving the cyphenothirn-affected reduction in RBC concentration [22].The high concentration of insecticide dose exhibited low position of MCHC values compared to control, which in turn indicated the reduced position of hemoglobin concentration.MCHC values represent the concentration of hemoglobin.Intoxication with insecticides also affects other hematological parameters, including blood sugar, urea, creatinine, neutrophils, lymphocytes, and eosinophils.Attenuation of lymphocytes in the group that received cyphenothrin treatment may be a sign of the drug's immunomodulatory action as a result of the low dose [23].The elevated position of serum calcium in the low dosage and high dose cyphenothrin-treated groups, respectively, indicates the calcium-promoting effort.Previous investigations with high calcium positions that accelerated bone healing capacity also produced results of a similar nature [24,25] Similar to what was seen in the bone marrow biopsies, the spleen had considerable crowding and intra-parenchyma hemorrhages.Additionally, portal hypertension, which is another source of anemia in chronic liver diseases and is accompanied with hypersplenism with typical splenomegaly, has been preliminary linked to anemia [30].As a result, the spleen's congestion could be caused by the erythrocytes' internal oxidation [31].These results are consistent with earlier research demonstrating thymus and spleen atrophy, as well as cell loss in bone marrow brought on by increased apoptosis in TCDD-treated rats [32,33].Permethrin exposure at a single dose in the tropics had previously been linked to adverse effects on immune system organs such the spleen and thymus [34].In addition, Banerjee and Hussain (1987) found that endosulfan suppressed humoral and cell-mediated immunity in rodents at remarkably low doses even in the absence of any other overt toxin-related symptoms [11].Additionally, it has been reported that profenofos and chlorpyrifos were used to treat hepatic lesions that resulted in splenic hemorrhages and congestion.Our findings thus provided more evidence that hemorrhages of rat spleens that have been pyrethroid insecticide-treated.

CONCLUSION
The current study's objective was to examine the toxin produced by pyrethroid cyphenothrin.Red blood cells (RBC), white blood cells (WBC), hemoglobin, hematocrit, and platelet count were among the haematological markers that were altered in the cyphenothrintreated groups, according to haematological research.However, cyphenothrin exposure that was successful changed the haemotoxicity in a significant way.In groups treated with cyphenothrin, histopathological examinations similar to rarefication of white pulp, diminished and damaged marginal zone, and decreased number of lymphoid follicles were clearly visible.The group that received cyphenothrin treatment in contrast, displayed the clinical signs listed below.This study demonstrates that, for the 60 d of treatment taken into account in this investigation, the cyphenothrin insecticide was harmful to rats' blood and spleens.In order to determine potential anaemic consequences from these compounds, haematological investigations demonstrate how its toxin affects blood parameters such as Packed Cell Volume (PCV) and White Blood Cell count (WBC), among others.