Hepatotoxicity and Nephrotoxicity induced by fipronil and lead acetate and their mixture as well as evaluation of therapeutic effects of camel milk against their harmful.

doi:10.21203/rs.3.rs-1975854/v1

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Hepatotoxicity and Nephrotoxicity induced by fipronil and lead acetate and their mixture as well as evaluation of therapeutic effects of camel milk against their harmful.

https://doi.org/10.21203/rs.3.rs-1975854/v1

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published 26 Jan, 2023

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Elevated environmental pollution of lead and fipronil blamed for chronic toxicity. Fipronil and lead acetate are commonly used but now no adequate data available concerning side harmful of their mixture. The present work studied the curative effects of camel milk against lead and fipronil toxicity individually and their mixture. The lead acetate or fipronil and their mixture significantly induction deteriorated kidneys and liver function parameters as well as lipid profile and oxidative stress in the both organs. Serum LDH activity increased under the same conditions. Histological examination revealed hepatic degeneration, nephropathy in intoxicated rats relative to normal health control. Both toxicants in their mixture showed more harmful than those of its individual ones. Camel milk treatments into intoxicated animals (lead, fipronil and mixture groups) attenuated the all evaluated parameters, also alleviated the harmful influences of the mixture of lead acetate and fipronil and improved the biomarkers of their oxidative stress.

Lead

Fipronil

Oxidative Stress

Toxicity

Liver

Kidneys

Fipronil is a phenyl insecticide acts by blocking the activated chloride channels in the central nervous system. The blocker of this receptor like fipronil leads to reduce neuronal inhibition, which leads to hyperexcitation of the central nervous system, convulsions and death. It has been used heavily to control insects on various crops and public health (Badgujar et.al., 2017 and Noaishi et.al., 2021). Fipronil is used close to humans. These effects included the possibilities of either oxidative stress incidence and metabolic harmful (Kartheek and David, 2016 and Kandil et.al., 2020) with the steady increase in the use of fipronil, especially in public health, there was an urgent necessity to identify the potential adverse effects long-term exposure. Lead is a multiple – source pollutant, well known for its toxicity of great risk both of the environment and human health. The main target orangs of lead are hematopoietic, liver, nervous and venal system. It is well known that most of this metal is accumulated in the blood cells and that many of the deleterious effects are related to circulating concentration. These adverse effects have been described not only in human but also in other animals. The ingestion of lead acetate into male of albino rats caused decreases in the average gain of body weight and increased orangs weight increases in liver function in enzymes (ALT and AST) Activity and changed the lipid profile but total soluble protein and albumin contents of plasma were decreased and cholinesterase activity was inhibited, in contrast alkaline and acid phosphatases as well as LDH activities were increased. The lead ingestion reduced the blood hemoglobin and RBC.s count under the same condition (Ibrahim et.al., 2011). The toxicity of lead closely related to age, sex, route of exposure, level and frequency of intake, solubility, metal oxidation state, retention percentage, and duration of exposure and efficiency of excretion. Lead has been associated with various forms of cancer (Pitot and Dragan, 1996) and (Abdel-Rahim et.al., 2011).

Camel (Camelus dromedarius) milk so called white gold of the desert is more similar to human milk than any other milk and differs from other ruminant milk because it contains low cholesterol, low sugar, high minerals, such as Na,K,Fe,Cu,Zn,Mg and others, high vitamin C, protective protein like as lactoferin, lactoperoxidase, immunoglobulins, lysozyme and insulin (Hamed et.al., 2011 and Yadav et.al.,2015). Camel is vital to daily life as source of food and means of transportation and just as importantly for the desert people in Asia and Africa. The camel milk has been used as medicines for divers' ailments since ancient times (Gader and Mlhavder ,2016). Camel can produce more milk for a longer period of time in arid zones and harsh environment than any other animals including domestic livestock species (Ahmed et.al., 2011). In this context, the present study was conducted to examine the harmful effects of Coach 20% SC (manufactured By Stuchem, Egypt ) as a commercial formulation containing 20% Fipronil active ingredient and lead acetate on body weight gain liver function, kidneys function, and serum lipid fractions profile as well as evaluated the anti-toxicant of camel milk against lead and Coach 20% SC harmful using biochemical and histological examination.

During December to February Camel milk was collected from the camel milk farm at Marsa Matroh station. Animal Production Research Institute Agricultural research center, Giza, Egypt. Milk Samples were kept in cooled boxes until transported to the laboratory. The acute oral toxicity LD₅₀ of lead Acetate and fipronil formulate (Coach 20% SC, manufactured by Stachem, Egypt ) were determined according to the method of Weil (1952). The LD₅₀ values were 5050 mg/Kg and 118 mg /Kg albino male rats body weight for lead acetate and fipronil formulation respectively. Lead Acetate was obtained from Sigma Chemical Co. Egypt, but the fipronil formulation (Coach 20% SC) was obtained from the Center of Agriculture Pesticide Laboratory ARC. The male albino rats obtained from the National Research Center (El-Dokki, Giza, Egypt) which were housed in plastic cages (two rats per cage) and acclimate for two weeks. The temperature and relative humidity were adjusted at 25–72°C and 50–60% respectively. The Experimental rats were fed a normal diet and had free access to water. These studies were approved by the institutional animal care and use committee, faculty of veterinary medicine, Cairo University (Vet Cu 011020224) and were carried out in accordance with the guidelines of care and use of Laboratory Animals stated by the National Institutes of Health, USPHS. A normal control diet according to the AIN-76 was prepared for all experimental animals.

Forty-eight male albino rats were randomly assigned to eight groups (six rats each). G1 was the normal health control. G2, G4, G6 and G8 normal health groups ingested by 2 ml of camel milk / 100 g body weight. G3 and G4 ingested by 1/30 LD₅₀ of fipronil formulation. G5 and G6 ingested by 1/30 LD₅₀ of lead acetate. G7 and G8 ingested by 1/60 LD₅₀ of lead acetate and 1/60 LD₅₀ of fipronil formulation. G1, G3, G5 and G7 were using as normal fipronil, lead acetate and fipronil + lead acetate controls respectively but the other groups were using as treated intoxicated groups. The animals were freely allowed to access the top water and were fed on basal diet composed of 15% casein, 10% corn oil, 5% cellulose, 4% salt mixture, 1% vitamins mixture and 65% starch, according to the AIN-76. All rats were weighted at the beginning and end of the experimental period (3 months) to calculate the percentage of body weight gain in each group by the following equation:

Body weight gain % = (final body weight – initial body weight) x 100

Animals were euthanized after months; serum and fresh orangs' sample were collected Each rat was weight every week and its daily food intake was determined. Feed efficiency was calculated.

Feed efficiency was calculated = Body weight gain/ Food intake

The Biochemical examinations were done by commercial enzymatic kits (obtained from Bio diagnostic, El-Dokki, Giza, Egypt) which were used for the determinations of liver function (AST and ALT activity), alkaline phosphatase (ALP) activity, bilirubin content and protein content serum according to the method of Reitman and Frankel (1957), Belfield and Goldberg (1971), Walter and Gerarde (1970) and Gonnall et.al., (1949). The kidneys' function was done by the determination of urea, uric acid and creatinine contents in serum according to Fawcett and Socctt (1960), Caraway (1955) and Schirmeister et.al., (1964) respectively, but the lactate dehydrogenase (LDH) activity was determined by King (1957). The lipid profile such as triglycerides cholesterol, high density lipoprotein (HDL-C), low density lipoprotein (LDL-C) and very low-density lipoprotein (vLDL-C) were determined according to Fossati and Prencipe (1989), Richmond (1973), Wieland (1983), Wieland and Seidel (1983) and Fiedewald et.al., (1972) respectively. The relative weight of liver and kidneys per 100g of initial body weight was determined after the animal decapitation.

The histological examination for kidneys and liver tissues for all groups was done by orangs fixed in 10% neutral buffered formalin. Specimens were then, cleaned, embeddedin paraffin, section and stained by hematoxylin and eosin stain. Tissue slides were examined by light microscopy and photographed using digital camera. Data were expressed on the Mean + SD which analyzed by one-way Analysis of Variance (ANOVA) all statistical calculations were performed using MSTAT-C (ver.210, Michigan State University, USA). The results were considered significant at P ≤ 0.05.

Elevation of unhealthy lifestyle and environmental pollution are blamed for escalatid chronic toxicity. The exposure to lead on fipronil as well as their mixture recently was suggested to have a role in the elevatirn incidence of harmful of xenobiotic toxicity. Milk and its products noe are gaining alleviation in anti-toxicant management. The body weight gain (BWG) of ratrs was significantly decreased in the all intoxicated groups with the hightest descrease in the lead acetae + fipronil groups control (G7) relative to the normal healthy contnal (G1). The BWG in the lead acetate + fipronil treated with camel milk (G8)was highr than the control (G7). The BWG recorded insignificant differences between fipronil control (G3) and lead control (G5) as well as insignifitant differences between their milk treated intoxicated groups (G4 and G6) that recorded a significant increases in the treated group compared with their intoxicated control groups. the highest BWG was recorded in the normal healthy rats treated with camel milk (G2) in recorded in table (1).

The liver function enzymes, and bilirubin contents (table 2), in serum were significantly increased in the all intoxicated groups included their milk treated groups relative to normal healthy control. The highest elevated was found in lead + fipronil intoxicated group (G7) followed by that of fipronil intoxicated control (G3) then lead acetae intoxicated control (G5). Camel ingestion treatments decresed significantly the AST, ALT and ALP activities as well as bilirubin content in serum of intoxicated animals either with lead or fipronil on their mixture. In contist, total protein was significantly decreased in the intoxicated groups which receiving lead acetate, fipronil and their mixture with the highest decrease in G7 which received the mixture of lead acetate and fipronil without treatment. The camel milk treatment ameliorated the lead and fipronil toxicity and improved the harmful in serum solulate total protein in G4, G6 and G8 which recoded insignificant difference between each other relative to their control intoxicated groups as recorded in table (2). G2 showed the best group.

The kidneys function and LDH activity are represnted in the table (3) .The higest increases in urea, uric acid and creatinines conents in serum as well as LDH activity were recorded in G7 ( lead + fipronil mixture ) intoxicated control followede by G3 (fipronil intoxicated control groups ) and G5 ( lead acetate intoxicated control group). The treatments with camel milk ingestion decreased the harmful effects of lead and fipronil as well as their mixture for kidneys function (urea, uric acid and creatinine contents ) including LDH activity. These parameters were improved under the treatments with camel milk ingestion into the different intoxicated animals. These are compared with the healthy normal contnal (G1). The best group in that of healthy normal animals Ingested with camel milk (G2).

Table (4) showed the lipid profile of the present experimental rat groups under the toxicity of lead fipronil and their mixture as well as those treated by camel milk ingestion. The total triglyceride, total cholesterol, LDL-C and vLDL-C contents were significantly increased in the intoxicated rat inducing those treated with camel milk ingestion. The highest toxicity was found in G7 (intoxicated cantonal group with lead and fipronil mixture) followed by G3 and G5 (intoxicated control groups with fipronil and lead acetate respectively). These were calculated relative to normal healthy control (G1). In G8, the total triglycerides and vLDL-C were reduced in serum compared to G7 but recorded insignificant differences. The total cholesterol contents which elevated under the effects of lead acetate and fipronil oxidation were reduced relative to their intoxicated controls by camel milk ingestion as treatments. Also, LDL-C levels of intoxicated rats' serum were significantly decreased relative their intoxicated control groups either in intoxication with lead acetate as with fipronil and this lipid profile parameter was improved by the ingestion with camel milk treatments. In case of HDL-C, its contents in serum were decreased in the all-intoxicated animals relative to that of healthy normal control (G3, G5 and G7), the highest harmful effect was observed in intoxicated group with the mixture of lead acetate and fipronil mixture control (G7), followed by groups 3 and 5 (intoxicated groups control with fipronil and lead acetate respectively. This toxicity harmful was attenuated by the treatments of camel milk ingestion on which significantly improved compared with normal healthy control group (G1). The camel milk ingestion into the normal healthy rats showed the best values, the all parameters of lipid profile.

The effects of lead acetate and fipronil as well as their mixture on liver and kidney tissues of the experimental normal rats as relative rat’s weight (g)/100g rats’ body weight are represented in table (5). The both organs weight was increased significantly under the induction of lead acetate ar fipronil as well as their mixture. The induction lead + fipronil mixture was more effective (G7) than either lead intoxicated control group (G5) or fipronil intoxicated control either group. These are relative to normal healthy control.

Histopathological finding

G7: liver of rat positive control fipronil lead acetate mixture histopathological structure of hepatic lobule (Hand E Stain 200) G8 liver of rat positive control fipronil + lead acetate mixture treated with camel milk histopathological structure of hepatic lobule (Hand E stain x 200). Figure (1) the liver histological examination of the experimental animals G1: liver of rat in control group showing normal histopathological structure of hepatic lobule (H and x 200). G2: liver of rat in healthy control group treated with camel milk showing normal histopthological structure of hepatic lobule (H and E stain x200).G3: liver of rat positive control fipronil group histopathological structure of hepatic lobule (H and E stion x 200) G4: liver of rat positive control fipronil treated with camel milk histopathological structure of hepatic lobule (Hand Ex200) G5: liver of rat positive control lead acetate group histopathological structure of hepatic lobule (Hand E stain 200) Gb : liver of rat positive control laed acetate treated with camel milk histopathological structure of hepatic lobule (H and E stain 200).

The histological analysis (Fig. 2) for liver and kidneys tissues showed that microscopic examination of liver revealed moderate hypertrophy of hepatoaytes in addition to karyomegaly, binucleation and mild solitary necrosis in the fipronil intoxicated control group (G3) compared to healthy normal control (G1) and its camel milk treated (G4). These lesions were less severe in the fipronil intoxicated rats treated with camel milk (G4). The lead acetate intoxicated groups control (G5 and G7) showed hepatic lesions which were mainly periportal hepatocytes vacuolation. The hepatic lesions were alleviated in lead intoxicated rats (G6) treated with camel milk ingestion and those of lead and fipronil mixture intoxicated animal (G8). The microscopic examination of kidneys tissue of the fipronil intoxicated rats (G3) showed mild hypertrophy and vacuolar degeneration of tubular epithelium relative to normal healthy control (G1) and it's treated by camel milk (G4). These lesions were mildly alleviated in group 4 in which fipronil intoxicated rats treated with camel milk ingestion. The lead intoxicated rats had mesangial calls, hyperplasia, beside degenerative and necrotic changes in epithelium and casts in the lumen of renal tubules. These lesions were more severe in the intoxicated rats group induced lead acetate and fipronil mixture (G7). Otherwise, those intoxicated rats treated camel milk ingestion showed lessened lesions relative to control.

In the past decade, research is directed toward the discovery of notural fundional food that would help reduce and control chronic diseases. Camel milk is, rich in many mutrients, as it as rich in mineral and anti- oxidants and they ganed high popularity in the prevention of xenobiotic toxipants (Abdel - Mobdy et.al., 2019, Abdel – Mobdy et.al., 2021 a-b and Hamed et.al., 2011). Toxicity in rats was developed using lead acetate and fipronil ingestion to evaluate the health effect of camel milk treatments. Camal milk was fed to intoxicated rats to elucidate its curative effects on the oxidative stress induced by the ingestion of lead, fipronil and their mixture in male albino rats. In the present study, fipronil seems to possess a synergistic cytotoxic influnces with lead acetate on the animal metaholism, camel milk treatments was able to decrease toxicity harmful of the present toxicants, impoved the kidneys and liver functions and lipid, alleviated histopathological alteration as well as improved the body weigh gain and organs weight, these fundings are consistent with the observations of Magjeed (2005), Khan and Alzohair (2011) and Abdel – Mobdy et.al. (2021)^a, they found that camel milk has improved the kidneys and liver functions of the intoxicated rats.

Liver is considered the key organ in metabolism, detoxification and secretory functions in the body and its disorders are numrous with no effective renders connected with other organs induding the kidneys. The increase in liver and kidneys weights (relative weight ) under the effect of lead and fipronil may be due to the tumefaction ar enlargement related to their collogen acaumalation and across such stress condition of the present toxicants exposures (E-Bahr, 2013).

The present results of hepato- and nephro-toxicity of lead and fipronil in rats as well as the camel milk protective influences were supported by the present study of the histological features evidenced by the low values in the marker parameturs of liver and kidneys.In addition, regarding the protein and lipid profile of the studied animals, lead and fipronil exposures significanty decreased the total soluble protein and HDL-C but increased LDL-C, VLDL-C,cholesterted and triglycerides in sera of intoxicated animals to normal control ones in which their protein and lipid profile were impoved as re- adjusted by camel milk treatments.These may be due to the effect of ROS (produced due to exposure to lead and fipronil) which induced oxidative stress to vital cellular molecules and structure: including protein, flududs, DNA and membranes, the harmful ROS induced physiological disorders (Latet et.al., 2010 and Abdel- Mobdy et.al., 2021^c).

In the present studies, fipronil induced oxidative stress and histopathological alterations in different organs. The lead ingestion also caused oxidative stress which was less than that of fipronil.The combination of the both (lead and fipronil ) worsened the oxidative stress, prompting elevated liver enzymes in sera and increased severity of heptocytes degradation similar to the previous studies (Ibrahim et.al.,2011 and Noaishi et.al.,2021). Camel milk treatments, decreased the oxidative stress induced by the lead and fipronil mixture. The analysis of camel milk showed that it containd vitamins (A,C,E) as anti - oxidative agents, minerals ( Na ,Fe ,K,Zn ,Mg. P and Ca ) and insulin in high amount. These compounds are potent anti -oxidants that have stronge free radical scavenging abilities ( Abdel - Mobdy et.al., 2021, and Hamed etal 2011). That camel milk may have protected animal body tissues against cytotoxic effects and oxidative stress induced by lead and fipronil. The metaholic disorder associated with the harmful of lead acetate and fipronil mixture produced harmful and toxicity higher then that of lead acetate as fipronil alone, but the intoxicated groups ingested camel milk as treatments showed a great improviment in liver and kidneys function and their histopathological and oxidative stress.

For the anaerobic metabolic pathway LDH is a crucial enzyme which catalyzes the conversion of lactate to pyruvate - reducing NAD⁺ to NADH.H⁺ ( Farhana and Lappin ,2021) LDH overexpression in some forms of toxicants hinders normal glucose metabolism and insulin secretion (Ainscow et.al., 2000 ). Camel milk was able to decrease sera LDH level significantly in intoxicated treadted groups, but it was still significantly higher than that of the normal healthy control .

The present study indicated the relative safeness of lead acetate and fipronil (which conducted oxidative stress) to be used in the public health with taking all use precautions. The incorporation of camel milk in food was able to control the lead and fipronil harmful and counteracted the oxidative stress induced by their toxicants and reduced the hepato and nephropathy associated with above toxic harmful. Lead acetate fipronil and their mixture inductions increased the relatvie weights of liver and kidneys compared with normal control. In spite of their collagen accumulation in the both organs which may be due to the stress conditions of the both xenobiotics induction .

Samples of the compounds and data used during the current study are available from the corresponding author.

Conflict of interest

The authors have no conflict of interest to declare.

Authors' contributions

Conceptualization, Yasmin E. Abdel-Mobdy; Data curation; Formal analysis, Writing – original draft, Ahmed E. Abdel-Mobdy; Investigation, Methodology, Ammar AL-Farga; Project administration; Writing – review & editing.

Acknowledgements

The authors would like to thank Entomology and Pesticide Department, Faculty of Agriculture, Cairo University, Cairo, Egypt, for the technical support.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or non-profit sectors.

Compliance with ethical guidelines

All ethical principles were considered in this article.

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Table (1): Body weight gain and feed efficiency of the experimental rats

Groups and treatments

Body Weight (g)

Food

Intake

(g)

Feed efficiency

Initial

Final

Gain

(BWG)

BWB/100g

Ratio

G1 Normal Health control

120.1

± 2.1

303.45

± 8.11

165.52

±7.96

137.93

±6.63 a

100

969

±39 a

0.171

±0.011 a

100

G2 Control and control milk

118.2

± 3.0

312.01

±13.16

169.02

±11.20

142.99

±9.48 a

104

1001

±36 a

0.169

±0.012 a

G3 Fipronil control

123.0

± 2.0

185.33

±15.00

102.22

±7.98

83.11

±6.49 c

1019

±40 a

0.100

±0.009 c

G4 Fipronil + camel milk

121.3

± 2.2

255.42

±12.91

140.00

±8.04

115.42 b

±6.63

999

±32 a

0.140

±0.010 b

G5 Lead acetate control

119.1

± 3.3

191.32

±11.11

104.00

±7.22

87.32

±6.06 c

1020

±41 a

0.102

±0.008 c

G6 lead acetate +camel milk

122.0

± 2.0

248.24

±12.63

136.42

±8.54

111.82

±7.00 b

1014

±46 a

0.135

±0.008 b

G7 Lead Acetate +fipronil control

120.1

± 2.4

114.12

± 6.66

3.02

±0.20

2.51

±0.17 d

826

±50 b

0.004

±0.001 d

G8 Lead Acetate +fipronil +camel milk

114.2

± 1.9

135.70

±10.13

11.00

±0.66

9.22

±0.55 d

812

±52 b

0.014

±0.001 d

% at normal healthy control