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Year : 2019  |  Volume : 30  |  Issue : 5  |  Page : 206-210

Histopathological analysis and scanning electron microscopic study of chlorpyrifos exposed kidney of neonates in pregnant rats exposed during gestation period

1 School of Health Sciences, University of Petroleum and Energy Studies, Dehradun, Uttarakhand, India
2 Department of Pharmaceutical Sciences, Bhimtal Campus, Kumaun University, Nainital, Uttarakhand, India
3 Department of Zoology, D.S.B. Campus, Kumaun University, Nainital, Uttarakhand, India

Date of Submission27-Apr-2019
Date of Decision31-May-2019
Date of Acceptance17-Jun-2019
Date of Web Publication24-Oct-2019

Correspondence Address:
Jyoti Upadhyay
School of Health Sciences, University of Petroleum and Energy Studies, Dehradun
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/UROS.UROS_27_19

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Background: Pesticide exposure has the potential for causing acute or chronic health effects at certain developmental stages of life. It can result in irreversible damage to structure and function of an organ. The exposure of females to pesticides before or after pregnancy is a matter of concern because of the possibility of having a child born with congenital disorders and developmental defects. Prenatal exposure of any toxicants can program the developing foetus to develop diseases that manifest in later life. Very few reports are available related to the pesticide exposure induced morphological changes in kidney which has prompted us to undertake this research study. Aim: The aim of this study is to evaluate the effect of pesticide, chlorpyrifos on the kidney development of rat neonates after being exposed prenatally to pesticides throughout their gestational period. Materials and Methods: Virgin female wistar rats weighing 200-250 gms were mated with adult males. It consisted of twelve, divided into two sub groups six rats per each were treated with selected pesticides, chlorpyrifos (insecticide) readily available commercially for various agricultural and non-agricultural settings. Insecticide chlorpyrifos was given at a dose of 10mg/kg per orally to the female rats throughout their gestation period. After gestation period rat neonates were sacrificed and their kidney section was processed for histopathological analysis. The SEM study of chlorpyrifos exposed kidney section of rat neonates was performed, investigating changes in morphometric and ultra-structural features of kidney in rat neonates. Results: The histopathological analysis shows morphological changes in the kidneys of prenatally pesticides exposed rat neonates markedly different from control group. Chlorpyrifos exposed neonates kidney shows damaged kidney corpuscles and glomeruli. Conclusion: This study concluded that pesticide exposure during gestation period to pregnant rats causes significant renal toxicity in their neonates.

Keywords: Chlorpyrifos, gestation period, histopathological analysis, pesticide exposure, rat neonates

How to cite this article:
Upadhyay J, Tiwari N, Rana M, Bisht SS. Histopathological analysis and scanning electron microscopic study of chlorpyrifos exposed kidney of neonates in pregnant rats exposed during gestation period. Urol Sci 2019;30:206-10

How to cite this URL:
Upadhyay J, Tiwari N, Rana M, Bisht SS. Histopathological analysis and scanning electron microscopic study of chlorpyrifos exposed kidney of neonates in pregnant rats exposed during gestation period. Urol Sci [serial online] 2019 [cited 2020 Jul 15];30:206-10. Available from: http://www.e-urol-sci.com/text.asp?2019/30/5/206/269887

  Introduction Top

Despite, ban on the use of pesticides globally, their usage continued in India, for various agricultural and nonagricultural purposes and also for vector-borne disease eradication programs. Long-term use of the pesticides and their persistence in the environment resulted in their occurrence as ubiquitous environmental contaminants. The reasons for environmental contamination by pesticides are their large production, excessive use, improper discharge, and persistence in the environment.[1] The exposure of these pesticides to living organism results in their accumulation and persistence in fatty tissues[2] and causes ill health effects in humans and other nontarget species.[3] Furthermore, pesticides are lipophilic, which causes them to go through biomagnifications in the food chain during successive trophic levels.[4],[5],[6] Chlorpyrifos (CPF), (O, O-diethyl O- [3, 5, 6-trichloro-2-pyridyl]-phosphorothioate) is the most commonly used insecticide. The active metabolite of CPF is chlorpyrifos-oxon formed by oxidative desulfuration, responsible for mammalian toxicity caused by the inhibition of cholinesterase.[7],[8],[9] Immature organisms are at much more risk to pesticide-induced toxicity than adults. Research studies showing the presence of pesticide residue in the umbilical cord and its association with reduced fetal growth during pregnancy raises a question about the sensitivity of residues parameter of pesticides.[10] The toxicity of pesticides in mammalian animals has received great attention in recent years. The major detoxication organ for many xenobiotics is kidney and is susceptible to nephrotoxic effects. Nephrotoxicity is the toxic manifestations of pesticides after its acute and chronic exposure.[11] Lesser number of the nephron is a risk factor for an adverse outcome after kidney damage. Some animal and clinical studies suggest that neonatal nephron loss is detrimental to adverse renal outcome.[12],[13] However, in some studies, it could be shown that nephron number at birth and development of cardiovascular disease and chronic kidney disease later in life correlates inversely.[14],[15],[16] Therefore, the present study investigates the effect of pesticide on the kidney development of rat neonates after being exposed prenatally to pesticides throughout their gestational period.

  Materials and Methods Top


Insecticide CPF with brand name Aladdin TC, Company Shri Ram Agro Chemicals was purchased from the local market of Bhimtal, India. Glutaraldehyde, paraformaldehyde, sodium phosphate, and osmium tetroxide were obtained from Sigma-Aldrich, India.


Ethical approval for animal experimentation

Approval of animal experimentation protocol was taken by the Institutional Animal Ethical Committee (IAEC), Department of Pharmaceutical Sciences, Kumaun University, Nainital, Bhimtal, India, and the registration number was KUDOPS/77 (approval date 11/10/2017), issued by Committee for the Purpose of Control and Supervision of Experiments on Animals. Methods performed under the relevant regulations and proper guidelines accordingly. Female virgin rats (200–250 g) within the same age range were obtained from the Animal House Facility of Department of Pharmaceutical Sciences, Kumaun University, Nainital, Bhimtal, India. All the animals were housed under laboratory conditions with temperature (22°C ± 2°C) and 55% humidity. They were kept in propylene cages (three in each cage) with light/dark conditions (12/12 h), provided standard rat chow with free access to clean drinking water ad libitum.[17] For this pesticide exposure activity, female rats were selected randomly and divided into two groups of six animals each. Female rats under proestrus cycle have been placed for overnight breeding with male rats, the day when they have copulatory plug and sperm-positive vaginal smear considered as gestational day 0. After the confirmation of gestational day, Group I served as normal control group received distilled water 1 ml, per orally. Group II received pesticide CPF at a dose of 10 mg/kg, per orally per day throughout their gestation period. The methods used in this experimental study were according to the guidelines of Organisation for Economic Co-operation and Development (OECD) for testing of chemicals and prenatal growth and development.[18]

Histopathological analysis

The rat neonates were observed, and five neonates were selected from each group and were euthanized under diethyl ether anesthesia. The kidneys of rat neonates were removed and fixed in 10% buffered formalin. Embedding of tissues was done in paraffin, and the sections were stained with dye hematoxylin and eosin). After that, they were examined under a light microscope. Histopathological changes and alterations from normal kidney section were analyzed between control and pesticide-exposed rat neonates were observed.

Scanning electron microscope analysis

The tissues of kidneys of the rat neonates exposed to CPF and fixed in glutaraldehyde (3%) and paraformaldehyde (2%) which is prepared in 0.1 M sodium phosphate buffer (pH 7.2); after that, the same is fixed in phosphate-buffered osmium tetroxide (1%). Tissues were examined using scanning electron microscope (SEM) Zeiss EVO40.

  Results Top

Body and kidney weight

Kidney and body weight of rat neonates of 1 week old were measured. A slight reduction in the body weight and kidney weight was observed in the CPF exposed group, as shown in [Table 1].
Table 1: Body and kidney weight of rats neonates 1 week old

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Histopathological study

Histopathological examination of kidney section of rat neonates revealed that no histological changes were observed in the kidney section of neonate of pregnant rat of control group [Figure 1]a and [Figure 1]b. The control group shows normal glomeruli structure, Bowman's capsule, distal convoluted tubule with low cuboidal epithelium, and distinct urinary space. Whereas in insecticide CPF exposed group, the photomicrograph section represents shrunken glomeruli, vacuolization, and marked degenerative changes within the eroded capsules [Figure 1]c and [Figure 1]d.
Figure 1: (a) Photomicrograph of kidney section of neonate of pregnant rat (control) (H and E, ×40). (b) Photomicrograph of kidney section of neonate of pregnant rat (control) showing no structural damage, i.e., normal glomeruli, Bowman's capsule, distinct urinary space, and distal convoluted tubule with low cuboidal epithelium (H and E, ×400). (c) Photomicrograph of kidney section of rat fetus treated with pesticide chlorpyrifos, (H and E, ×40). (d) Photomicrograph of section of kidney of rat fetus treated with pesticide chlorpyrifos shows marked degenerative changes and vacuolization within tubules. The glomerulus appears shrunken and shows degenerative changes, (H and E, ×400)

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Scanning electron microscope study

The SEM study provides high-resolution and three-dimensional images and topographical, morphological, and compositional information. It helps in analyzing surface fractures, microstructures, surface contaminants and also identifies crystalline structures. [Figure 2]a and [Figure 2]b represents scanning electron micrograph of kidney section of neonates of control group showing empty Bowman's capsule with tubule (T), glomerular tuft (G) and capillary network (Cap), podocytes, (P) and erythrocytes (RBC). The SEM analysis of pesticide CPF exposed group shows degenerative changes in the glomeruli of the kidney section [Figure 2]c and [Figure 2]d. Bowman's capsule within the tubule, capillary network, erythrocytes, and podocytes was also observed by the SEM study.
Figure 2: (a) Scanning electron micrograph of kidney section of neonates of control group showing empty Bowman's capsule with tubule and glomerular tuft. (b) Scanning electron micrograph of kidney section of neonates of control group showing capillary network, podocytes, and erythrocytes. (c) Scanning electron micrograph of kidney section of neonates of chlorpyrifos exposed group showing damaged empty Bowman's capsule with tubule and glomerular tuft. (d) Scanning electron micrograph of kidney section of neonates of chlorpyrifos exposed group showing capillary network, podocytes, and erythrocytes

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  Discussion Top

The primary etiology of kidney disease (acute and chronic) is hypertension and diabetes, particularly in developed countries.[19] However, most regions of the world experienced an excess of chronic kidney disease unrelated to these traditional causes particularly in Mexico and Central America (Mesoamerican nephropathy),[20] North Central Province of Sri Lanka (Sri Lanka nephropathy),[21] State of Andhra Pradesh (India) (Uddanam endemic nephropathy),[22],[23] and other countries such as Saudi Arabia.[24] Nephropathies in these regional areas occur mostly in agricultural workers, and the frequency is more among men than women.[20],[25] Many researchers believe that the etiology of chronic kidney disease is multifactorial.[26],[27],[28],[29] Several research studies of Central America suggested that both occupational and environmental factors including heavy metals, nonsteroidal anti-inflammatory drugs, pesticides, infections, alcohol, occupational heat stress-induced dehydration, intake of soft drinks rich in fructose, hyperuricosuria, and hyperuricemia.[20],[25] Previous studies reported that currently six pesticides are used which showed a strong evidence of association with acute kidney damage, and they are 2,4-D paraquat dichloride, captan, cypermethrin, glyphosate, and 1,2-Dibromo-3-chloropropane.[30] The normal kidney receives about 20%–25% of cardiac output and constitutes about 5% of total body mass. Therefore, any chemical or drug reaches into these organs from systemic circulation in very large amount causes changes in the anatomic and physiologic features of the kidney. The progressive accumulation of these pollutants in the nephron causes intraluminal precipitation of chemicals which are insoluble and produces damage to nephron as a consequence of renal failure (acute or chronic). Xenobiotics metabolism, renal transport, and accumulation are the contributing factors causing significant injuries to kidneys.[11] Degenerative changes and vacuolization of tubules observed were caused by tubular epithelium atrophy after pesticide exposure might be due to failure of the ion transport system of tubular cells. Oxidative stress caused by pesticides during the process of nephrogenesis is an important underlying factor observed that causes kidney dysfunction. Earlier studies by Ambali et al., 2011; Bhandaniya et al., 2012; and Pal et al., 2012 reported glomerulus degeneration caused by pesticide CPF.[31],[32],[33] This suggested the inefficacy of kidney cells to cope up with functional disorders caused by environmental pollutants. Any abnormality in the glomerulus leads to failure of glomerulus functioning and finally, leading to end-stage renal disease, which requires a renal transplant. The process of the formation of concentrated urine also serves to concentrate toxicants into renal tubular cells; hence, the nontoxic concentration of any chemical in the plasma reaches the toxic concentration in the kidney. Increase in the concentration of toxicants along with the nephron results in intraluminal precipitation of insoluble compounds that causes acute renal failure to tubular obstruction. The SEM analysis of kidney section of pesticide-treated rat neonates reveals the presence of podocytes, capillary network, and erythrocytes. Podocytes play an important role in the normal glomerular function, along with the endothelial cells of the glomerular capillary loop and basement membrane they form a filtration barrier. In cooperation with mesangial cells, podocytes support the structure and function of the glomerulus. The structure of podocytes can be differentiated into three segments, i.e., the major processes, foot processes, and the cell body. The major processes and foot processes play a crucial role in the proteinuria pathogenesis.[34] The biology of podocytes is an emerging science that helps in understanding the mechanistic nature of several diseases associated with proteinuria. Proteinuria, a nephritic syndrome is dysfunction of the kidney glomerulus including the podocytes epithelial cell functioning (foot process effacement). Any injury to podocytes undergoes effacement, and they lost their structure and spread out, leading to a decline in filtration barrier function. Effacement is related with the proteinuria in segmental glomerulosclerosis and diabetes.[35] This study shows the toxic effects of CPF on the kidney of neonates of pregnant rats exposed during pregnancy. As during the gestation period, the exposure of CPF affects the kidney development of the rat neonates which is responsible for acute or chronic kidney complications later in life.

  Conclusion Top

Histopathological lesions are used as biomarkers for the evaluation of health of an organism exposed to environmental pollutants and used as warning symptoms. Marked degenerative changes in the morphological structure of kidney section of pesticide-exposed rat neonates were observed through histopathological and SEM analysis. All these changes suggest tubular necrosis and glomerulonephritis, leading to renal failure, acute or chronic with increasing time. Therefore, this study brought into light, the nephrotoxicity in neonates induced by pesticide CPF exposure to pregnant females during their gestation period.

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Conflicts of interest

There are no conflicts of interest.

  References Top

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  [Figure 1], [Figure 2]

  [Table 1]


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