Research Article

Hepato-Nephrotoxic Effects of Induced Fluorosis in Rabbits and Broilers

Barirah Rehman Talpur1, Zaheer Ahmed Nizamani1*, Imdad Hussain Leghari2, Mansoor Tariq1, Aisha Rehman3, Shahnawaz Kumbhar1

1Department of Veterinary Pathology, Sindh Agriculture University Tandojam-70060, Pakistan; 2Department of Poultry Husbandry, Sindh Agriculture University Tandojam-70060, Pakistan; 3Department of Pharmacology & Toxicology, College of Veterinary Medicine, China Agriculture University, Beijing, China.

Received | August 13, 2021; Accepted | November 01, 2021; Published | June 01, 2022

*Correspondence | Zaheer Ahmed Nizamani, Department of Veterinary Pathology, Sindh Agriculture University Tandojam-70060, Pakistan; Email: [email protected]

Citation | Talpur BR, Nizamani ZA, Leghari IH, Tariq M, Rehman A, Kumbhar S (2022). Hepato-nephrotoxic effects of induced fluorosis in rabbits and broilers. J. Anim. Health Prod. 10(2): 214-220.

DOI | http://dx.doi.org/10.17582/journal.jahp/2022/10.2.214.220

ISSN | 2308-2801

 

 

Copyright: 2022 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Fluoride is a micronutrient naturally found in ground water, soil and plants in variable concentrations but is also sometimes added in small amounts in drinking water to maintain desired level (Takizawa, 2008). In the last few decades, due to water shortages the use of poor-quality groundwater having high fluoride contamination has become a serious threat to public health, wildlife, livestock and poultry. The World Health Organization (WHO) recommended Fluoride level in drinking water is 1.5 mg/l. Li et al. (2021), in their studies showed that more than recommended level causes health problems in animals. Fluoride contaminated groundwater has been reported in various areas of Pakistan and India with concentration ranging from 0.93 to 49.3 mg/L (Brahman et al., 2013; Farooqi et al., 2007; Kisku & Sahu, 2020; Rasool et al., 2018; Shah & Danishwar, 2003) .

Fluoride is good for dental and bone but excess amount of (>1.5 mg/L) in long term causes dental and skeletal fluorosis; renal and neuronal disorders along with myopathy (Ayoob & Gupta, 2006). Humans, animals and birds are severely affected in fluoride contaminated areas. The fluoride concentration of up to 1.5 ppm in water causes chronic fluorosis in animals (Ranjan & Ranjan, 2015). Several studies have been conducted on fluorosis in birds, cattle, rabbits and sheep while outbreaks of fluorosis in pigs have also been reported in china (Kazi et al., 2018; Park et al., 2021; Tao et al., 2006).Excess fluoride intake in drinking water results decrease in milk production of cows and an increase in calving interval (Shupe et al., 1972).

In Pakistan, the groundwater quality is poor in many areas due to high fluoride levels. Most affected areas are Indus deltaic plain and Thar Desert in Sindh province (Rafique, et al., 2008; Rafique et al., 2015). As a result, people and animals are suffering from fluorosis in these areas (Farooqi et al., 2007). Keeping in view the above facts; the study was designed to evaluate the hepato-nephrotoxicity produced by induced fluorosis in mammalian (rabbit) and avian (chicken) models.

MATERIALS AND METHODS

Animal management and experimental protocol

The animals and birds were kept at the Animal House of Department of Veterinary Parasitology, Sindh Agriculture University Tandojam. A total of 24 rabbits (~ 1.5 kg BW) and twenty-four broilers (04 weeks age) were purchased from local market. An approval for the study was granted by the ethics committee, Faculty of Animal Husbandry and Veterinary Sciences, Sindh Agriculture University Tandojam. Rabbits and broilers were divided into four subgroups, each having six animals, viz; A1, A2, A3, A4 and B1, B2, B3, B4. The animals in group A1 and B1 were kept as control and given normal water. The Groups A2-B2, A3-B3 and A4-B4 were given 50, 100 and 200 mg Sodium Fluoride/litter in drinking water respectively for 18 days.

Serum Biochemistry

The 3ml blood was collected on days 0, 5, 10, 15 and 18 of experiment. For evaluation of various biochemical parameters relating to liver and kidney functions, serum levels of Alanine aminotransferase (GPT), Alkaline phosphatase (ALP), Gamma glutamyl transferase (γGT), Creatinine, Uric acid along with calcium were analyzed spectrophotometrically by using commercially available kits (HUMAN Diagnostic Co., Wiesbaden, Germany) through kinetic method as recommended by IFCC (International Federation of Clinical Chemistry) (Ceriotti et al., 2008).

Recording morbidity and mortality

All animals and birds were observed daily for development of clinical findings, morbidity and mortality.

Gross and Histopathological Examination

For studying lesions on various visceral organs necropsies were performed on dead while surviving broilers and rabbits were euthanized and dissected. For histopathological examination, the kidney and liver samples were cut into small pieces through sharp scalpel blade and were preserved in 10% formalin. Tissue samples were dehydrated in ethanol, cleared in Xylene, infiltrated / embedded in hard paraffin wax, sectioned and stained with Hematoxylin and Eosin stains. The histological lesions were recorded at 10 and 40X.

Statistical Analysis

Data was analyzed using two-way analysis of variance (ANOVA) to observe the difference between control group and experimental groups at different time intervals. Statistical differences among different treatment groups were determined by Duncan’s Multiple Range test (DMR) at 5% level of significance (Akdogan et al., 2004).

RESULTS

Serum biochemical profile

Serum biochemical profile of rabbits and broilers treated with various concentrations of Sodium fluoride in drinking water and showing serum Alanine aminotransferase (GPT), Alkaline phosphatase (ALP), Gamma glutamyl transferase (γGT), Serum creatinine, uric acid and calcium levels are shown in Table 1-3. Serum levels of Alanine aminotransferase (GPT), Alkaline phosphatase (ALP), Gamma glutamyl transferase (γGT),in all treatment groups of both rabbits and broilers significantly (p<0.05) increased in time and dose dependent manner as compared to control (Table 1). Serum creatinine and uric acid levels were significantly higher (p<0.05), in all treatment groups of both rabbits and broilers, as compared to control group (Table 2). The increases were dose dependent and time dependent. Moreover, time and dose dependent significant (p<0.05) decrease was seen in serum calcium levels in the groups treated with Sodium Fluoride as compared to control group (Table 3).

Clinical findings

There were no clinical signs observed in Rabbits during experiment, while in broiler chickens, paralysis of legs in few birds, decreased feed intake, increased water intake and intermittent diarrhea were observed in treatment groups (B2, B3, and B4) with signs being more pronounced with higher dose. There were only three deaths, one in each treatment group of broilers.

 

Table 1: Serum ALP, GPT and γGT levels of rabbits and broiler chicken given different concentrations of Sodium Fluoride in drinking water.

Test	Group	Day 0	Day 5	Day 10	Day 15	Day 18
ALP (IU/L)	A1 (0)	70.67±0.0aA	70.91±0.01dA	70.9±0.02dA	70.9 ±0.04dA	70.9 ±0.03dA
	A2(50)	70.8±0.03aD	72.5±0.14cCD	73.3±0.05cC	78.6 ±0.14cB	83.3 ±0.15cA
	A3 (100)	70.9±0.0aE	77.51±0.23bD	80.5±0.07bC	83.3±0.29bB	86.3±0.23bA
	A4 (200)	70.9±0.0aE	79.45±0.06aD	86.6±0.13aC	90.5 ±0.12aB	94.24±0.84aA
	B1 (0)	76.2±0.08aA	76.10±0.02dA	76.2±0.03dA	76.1±0.07dA	76.2±0.06dA
	B2(50)	75.4±0.54abE	77.4±0.04cD	79.6±0.14cC	82.38±0.04cB	85.05±0.15cA
	B3 (100)	74.5±1.03bE	78.2±0.08bD	80.3±0.12bC	84.5±0.15bB	88.17±0.17bA
	B4 (200)	75.4±0.18abE	83.3±0.1aD	85.1±0.03aC	88.62±0.19aB	92.42 ±0.15aA
GPT (IU/L)	A1 (0)	25.8±0.01aA	25.8±0.01dA	25.8±0.01dA	25.8±0.01dA	25.8±0.01dA
	A2(50)	25.7±0.03aE	28.3±0.02cD	30.8±0.02cC	33.8±0.06cB	35.2±0.07cA
	A3 (100)	25.6±0.09aE	31.7±0.01bD	34.3±0.13bC	36.62±0.05bB	38.6±0.06bA
	A4 (200)	25.6±0.14aE	35.6±0.05aD	37.4±0.13aC	42.33±0.08aB	45.58±0.12aA
	B1 (0)	37.5±0.13aA	37.6±0.11dA	37.6±0.14dA	37.7±0.06dA	37.7±0.01dA
	B2(50)	37.4±0.16aE	39.1±0.02cD	41.7±0.77cC	43.2±0.11cB	46.7±0.08cA
	B3 (100)	37.8±0.08aE	42.5±0.05bD	44.4±0.16bC	46.6±0.12bB	48.5±0.15bA
	B4 (200)	37.6±0.07aE	45.7±0.07aD	47.3±0.08aC	49.3±0.2aB	49.3±0.24aA
γGT (IU/L)	A1 (0)	6.2±0.02aA	6.1±0.01dA	6.2±0.02dA	6.2±0.14dA	6.3±0.1dA
	A2(50)	6.2±0.05aE	10.3±0.16cD	13.8±0.29cC	16.5±0.08cB	18.4±0.1cA
	A3 (100)	6.1±0.05aE	15.3±0.05bD	18.3±0.05bC	19.6±0.14bB	21.7±0.13bA
	A4 (200)	6.1±0.03aE	20.1±0.04aD	20.7±0.01aC	21.2±1.33aB	24.3±0.95aA
	B1 (0)	17.5±0.12aA	17.5±0.12dA	17.5±0.13dA	17.5±0.13dA	17.5±0.13dA
	B2(50)	17.3±0.09aE	18.5±0.16cD	21.6±0.17cC	24.5±0.06cB	26.5±0.05cA
	B3 (100)	17.4±0.2aE	20.4±0.02bD	24.3±0.16bC	28.6±0.12bB	31.1±0.33bA
	B4 (200)	17.6±0.07aE	45.7±0.07aD	47.3±0.08aC	49.3±0.2aB	49.3±0.24aA

Means±SD with different lowercase superscripts, for each species, show significant differences among the treatments for each time point (comparison per column), whereas different capital letters show significant differences among the time points for each treatment (comparison per line) (p<0.05).

Table 2: Serum creatinine and uric acid levels of rabbits and broiler chicken given different concentrations of Sodium Fluoride in drinking water.

Test	Group	Day 0	Day 5	Day 10	Day 15	Day 18
Creatinine (mg/dl)	A1 (0)	1.9±0.05aA	1.9±0.11dA	1.9±0.52dA	1.9±0.17dA	1.9±0.31dA
	A2(50)	1.9±0.0aD	3.3±0.1cC	4.4±0.17cCB	5.4±0.24cB	7.6±0.13cA
	A3 (100)	1.9±0.0aD	4.8±0.02bC	5.4±0.1bCB	6.6±0.16bB	8.9±0.02bA
	A4 (200)	1.9±0.0aE	8.6±0.1aD	10.2±0.08aC	13.9±0.09aB	17.4±0.42aA
	B1 (0)	1.9±0.02aA	1.9±0.04dA	1.9±0.34dA	1. 9±0.13dA	1.9±0.27dA
	B2(50)	1.8±0.04aE	2.8±0.03cD	3.3±0.11cC	3.6±0.12cB	5.2±0.15cA
	B3 (100)	1.7±0.04aE	4.5±0.09bD	5.9±0.28bC	8.4±0.14bB	9.5±0.09bA
	B4 (200)	1.8±0.08aE	5.4±0.08aD	7.2±1.12aC	9.2±0.19aB	14.7±1.54aA
Uric Acid (mg/dl)	A1 (0)	1.6±0.02aA	1.6±0.04dA	1.6±0.01dA	1.6±0.01dA	1.6±0.16dA
	A2(50)	1.6±0.04aE	3.5±0.25cD	4.6±0.04cC	6.3±0.15cB	7.6±0.1cA
	A3 (100)	1.6±0.01aE	4.1±0.04bD	7.1±0.22bC	8.4±0.15bB	10.3±0.25bA
	A4 (200)	1.7±0.06aE	9.4±0.09aD	14.5±0.18aC	18.4±0.19aB	19.7±0.04aA
	B1 (0)	12.1±0.05aA	12.2±0.13dA	12.1±0.1dA	12.1±0.03dA	11.9±0.18dA
	B2(50)	12.1±0.08aE	14.1±0.22cD	23.3±1.15cC	28.1±0.64cB	29.9±0.49cA
	B3 (100)	12.1±0.04aE	16.7±0.10bD	26.7±0.1bC	36.4±2.16bB	35.1±0.57bA
	B4 (200)	12.1±0.05aE	18.9±0.33aD	39.1±2.53aC	58.5±2.4aB	68.1±4.57aA

Means±SD with different lowercase superscripts, for each species, show significant differences among the treatments for each time point (comparison per column), whereas different capital letters show significant differences among the time points for each treatment (comparison per line) (p<0.05).

 

Table 3: Serum calcium levels of rabbits and broiler chicken given different concentrations of Sodium Fluoride in drinking water.

Test	Group	Day 0	Day 5	Day 10	Day 15	Day 18
Calcium (mg/dl)	A1 (0)	21.4±0.03aA	21.4±0.33aA	21.4±0.46aA	21.4±0.07aA	21.4±0.61aA
	A2(50)	21.5±0.01aA	21.2±0.05aA	20.4±0.1abB	19.2±0.04bC	18.8±0.01bD
	A3 (100)	21.5±0.01aA	20±0.11abB	19.4±0.09bC	18.6±0.06cbD	16.4±0.17cE
	A4 (200)	21.5±0.01aA	19.8±0.02bB	16.3±0.12cC	15.61±0.02cD	11.4±0.14dE
	B1 (0)	13.4±0.07aA	13.5±0.01aA	13.3±0.18aA	13.3±0.07aA	13.5±0.01aA
	B2(50)	13.6±0.06aA	12.4±0.01bB	10.5±0.06bC	8.5±0.03bD	6.9±0.22bE
	B3 (100)	13.6±0.07aA	8.6±0.03cB	8.3±0.04cC	6.7±0.06cD	5.5±0.09cE
	B4 (200)	13.6±0.06aA	6.5±0.05dB	5.5±0.12dC	4.3±0.06dD	3.5±0.18dE

Means±SD with different lowercase superscripts, for each species, show significant differences among the treatments for each time point (comparison per column), whereas different capital letters show significant differences among the time points for each treatment (comparison per line) (p<0.05).

Gross and Histopathological Examination

In rabbits, postmortem lesions observed in kidney (Figure 1) included enlarged and inflamed kidneys, while in liver (Figure 2) there were inflammation and discoloration. The most common postmortem lesions observed in broilers were the toxic lesions on mucus membranes of proventriculus, swollen and congested spleen and enlarged gallbladder. The kidneys and liver were discolored and enlarged.

 

The kidney tissues, in both species had mild inflammatory cellular infiltration, hyperemia in intertubular space, shrinkage of glomeruli with widened bowman’s spaces (Figure 1). Sections of liver tissue of rabbit showed dilation of central vein and sinusoids and fatty degeneration of hepatocytes (Figure 2). On other hand, group of broilers showed hepatocytes surrounding the central vein were observed to possess darkly staining condensed nuclei and necrosis of the hepatocytes around the peri-portal and central vein area (Figure 2).

 

DISCUSSION

The fluorosis affects multiple organ systems of body including teeth, bones, gastrointestinal tract, nervous and muscular tissues. Among various modes of injury, reducing blood calcium levels, involvement of free radicals, increased generation of oxygen radicals and enhanced lipid peroxidation have been shown to play an important role in fluorosis (Faccini & Teotia, 1974; Guo et al., 2003; Rzeuski, 1998). Most nutrients and toxins upon absorption, first pass through and are metabolized by liver and are excreted by kidneys. Therefore, these two organs are commonly affected in most toxicosis. While subtle damage is only reflected in changes of serum biochemical parameters, more severe damage is seen in on gross and microscopic levels. The present study was, therefore, designed to evaluate the effect of oral administration of high dose Sodium Fluoride in drinking water on liver and kidney structure and function.

In current study, both species belonging to two different taxonomic classes, showed structural and functional disturbance of liver and kidneys. The clinical signs of fluorosis and mortalities occurred only chicken, indicating greater susceptibility to fluoride toxicity. One probable reason could be the much higher growth and metabolic rate along with greater feed and water requirement in chickens.

Liver plays a major role in detoxification of various compounds. Serum ALP, γGT, GPT levels are important markers of liver function. In present study, increased serum levels of ALP, γGT and GPT show that liver is significantly affected in fluorosis. Similar results showed in rats and rabbits (Akinrinde et al., 2021; Al-safei & Al-Mashhadane, 2021). It is in line with various other studies (Guo et al., 2003). Similarly, Faccini and Teotia (1974) have also reported the high plasma alkaline phosphatase in the patients affected with fluorosis. Livers in both rabbits and broiler chicken tissues of rabbit showed dilation of central vein and sinusoids and fatty degeneration of hepatocytes while in broiler showed hepatocytes surrounding the central vein having degenerative changes and necrosis in some areas (Figure 2). Similar lesions observed in liver of rats, mouse and ducks (Akinrinde et al., 2021; Chen et al., 2019; Ouyang et al., 2021). Shashi et al. (2001) have also reported degenerative changes in liver of rabbits but changes were much more severe along with necrosis as duration of fluoride administration was 15 weeks. Similar findings have also been reported in fluoride exposed mice and rats (Akdogan et al., 2004; Akinrinde et al., 2021; Basha & Rao, 2014; Chinoy et al., 1993).

The kidney is the major route for removal of fluoride from the body, and consequently, this organ is sensitive to damage because of excessive fluoride exposure. In our study, serum levels of uric acid and creatinine in both rabbits and broilers increased with dose as well time duration. This study is also in agreement with Al-safei & Al-Mashhadane, (2021) and Dembińska-Kieć et al. (2017) who reported that in fluorosis produced significant increase in the concentration of creatinine in the blood due to impaired renal function. Similarly, Appleton (1995) also reported that high doses of sodium fluoride injection into rats resulted in increases in the concentration of urea, and creatinine in the plasma. In rat, kidney is exposed to concentrations of fluoride about five times higher than in other organs, as the tissue/plasma ratio for the kidney is approximately 5 to 1. In current study, mild inflammatory cellular infiltration, hyperemia in intertubular space, shrinkage of glomeruli with widened bowman’s spaces were observed. Other researchers have found more or less severe gross and histopathological changes in kidneys as fluoride doses, routes of administration, duration of treatment and species vary. Basha and Rao (2014) found necrosis in glomerulus, degenerative changes in Bowman’s capsule and alterations in glomerulus’s tubular region in fluoride-treated albino mice. Similarly, Shashi et al. (2002) also reported same investigation necrotic and degenerative changes in kidney on rabbit however duration of treatment was fifteen weeks. Xu et al. (2006) reported similar the hypertrophy and hyperplasia in the renal tubules and damage kidney structure in fluoride-treated rats.

In the present study, rabbits and broilers were tested for serum calcium levels. The serum concentration of calcium in both rabbits and broilers decreased with fluoride dose as well time duration of treatment. Similar decrease has also been reported by various other researchers as well (Chandra, 1997; Madan et al., 2009; Tao et al., 2006; Wang et al., 1992). Similar observations were also reported by Faccini and Teotia (1974) who reported that increased urinary fluoride, a low urinary calcium excretion along with increased Ca and F in bone in the patients affected with fluorosis. Similarly, Appelton (1995) reported calcium decreased in blood due to renal insufficiency and cause in hypocalcemia in fluoride- treated rats. The decrease in serum calcium is related to a decrease of intestinal absorption of calcium by fluorine (Tao et al., 2006).

CONCLUSION

It is concluded that, there are dose and time dependent hepato-nephrotoxic effects of fluorosis in the rabbits and broilers as is reflected by gross and histopathological changes along with significant increase in serum ALP, γGT, GPT, creatinine, uric acid levels and decrease in calcium concentration.

acknowledgements

The authors thank the department of Veterinary Parasitology, Faculty of Animal Husbandry and Veterinary Sciences, Sindh Agricutlure University Tandojam for providing the facility of Animal House for experiments.

Conflict of Interest

The authors declare that there is no conflict of interests regarding the publication of this article.

novelty statement

Livestock and poultry farming industries in many parts of Pakistan have difficulties due to high fluoride content of groundwater. In the manuscript we have used a mammalian (rabbit) and avian (chicken) model to investigate the hepato-nephropathic effects of induced fluorosis. We report that dose-dependent subacute fluoride toxicity affects both function as well structure of liver and kidneys of rabbits and chicken.

Authors Contributions

Barirah Rehman Talpur conducted all experiments as part of MPhil research. Dr. Zaheer Ahmed Nizamani designed experiments and analyzed the data. Dr. Mansoor Tariq, Dr. Imdad Hussain Leghari and Aisha Rehman helped in data collection. Dr. Shahnawaz Kumbhar assisted in histopathological evaluation of slides.

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