Introduction

Lead (Pb) is an environmental pollutant and well-known heavy metal which is highly poisonous and non-biodegradable trace element (Eruotor, Asiwe, & Eruotor, 2023; Elgazar, Shalaby, & Ibrahem, 2023). The main source of lead is gasoline, paints, lead-acid battery, eye cosmetics and printing of books (Ashour, Yassin, Aasi, & Ali, 2007; Yadav, Kushwaha, & Srivastav, 2023). It enters into the organisms’ body through the contaminated foods, respiratory, dermal contact, electric wastes, vehicle exhausts and mucous membranes (Ashour et al., 2007; Owumi, Arunsi, Oyewumi, & Altayyar, 2022). Lead poisoning can induce various physiological and morphological impairments including the reproductive system, central nervous system, hematopoietic system, liver and kidneys in human and animal (Azzaz, El-Kholy, & Ramadan, 2022; Inayat et al., 2022; Eivani, Zareian, Ghahari, Dadpay, & Shojaee, 2023). Lead exposure can induce abnormal increase of serum uric acid and cause the Hyperuricemia as well as dysfunction of renal calculi, metabolic syndrome and cardiovascular system (Dai et al., 2015). Pb can induce increased formation of free radicals and alter the morphology of glomerular histoarchitecture and renal tubules and provoked kidney failure (Eruotor et al., 2023). Lead toxicity induce metabolic abnormalities and alter human and animal kidney functions as well as impairments in kidney bio-markers levels in blood such as urea, creatinine and uric acid (Mohammed, Sedky, & Elsawy, 2017; Abdulshaheed, Al-Kurdy, & Abbas, 2021). Lead is bio-accumulated in mammal’s cells because it is not consumed during metabolism and affects cortical interstitial cells, renal tubules and renal parenchyma of kidney (Thuong, Lan, Van, Thanh, & Ke, 2023). Due to toxicity of lead, many countries now have strict controls on the use of lead paints and leaded gasoline (Ritchie, 2022; SunderRaj, & Ananthapadmanaban, 2024).

Syzygium cumini (family Myrtaceae) is commonly known as jamun, black plum and Indian blackberry (Srivastava et al., 2021a, b; Qamar et al., 2022). Jamun seed, fruit pulp and bark contain oleic, lauric, sterculic, myristic, vernolic acid, palmitic, stearic, linoleic, malvalic, phytosterols, corilagin, ellagitannins, ellagic acid, and gallic acid etc. It is used as antioxidant, anti-inflammatory, anti-microbial, anti-bacterial for the treatment of abnormalities such as diabetes mellitus, hepatoxicity and nephrotoxicity (Swami, Thakor, Patil, & Haldankar, 2012; Abbas et al., 2016; Kumar & Thakur, 2018).

Orange (Citrus sinensis) belongs to family Rutaceae. Orange peel contains numerous micronutrients such as rutin, cellulose, flavanone glycosides hesperidin, neohesperidin, naringin, hemi-cellulose, pectin substances, chlorophyll pigments, and other small molecules like limonene (Srivastava et al., 2021b). Orange peel extract improve the levels of kidney parameters such as, serum uric acid and serum urea,serum creatinine (Alfarajat, Mostafa, Abdel-Mogib, El-Gayar, & El-Khawaga, 2023). Orange peel extract contains specific antioxidant which protect against nephrotoxicity and improve kidney damage (Srivastava et al., 2021b).

There is no report regarding JSE and OPE regarding the ameliorative effect of the toxic effects of lead on kidney biomarkers in rats. Hence, this study evaluated the changes in kidney biomarkers such as, serum urea, serum uric acid and serum creatinine levels of rats treated with lead and evaluated the protective role of Jamun (Syzygium cumini) seed extract and orange (Citrus sinensis) peel extract on the toxic effects of lead on these parameters.

Materials and methods

One hundred twenty Wistar rat (b wt. 50-60 g; 2 months age) were purchased from Asia Scientific Emporium, Varanasi, India and were acclimatized for 2 weeks under laboratory conditions (27- 30°C with a 12 hour dark light^-1 cycle) in polypropylene cages. Standard diet and water ad libitum was provided to rats. The doses of lead, JSE and OPE given to the rats are based on the reports of the previous investigators- Lead (25,50 and 60 mg kg^-1 b wt.- Bhattacharjee, Kulkarni, Chakraborty, Habbu, & Ray, 2021; 50 mg kg^-1 b wt. - Eruotor et al., 2023; 40 mg kg^-1 b wt. - Amriza, Rita, & Elmatris, 2022; 30 mg kg^-1 b wt. - Kucukler et al., 2021); jamun peel extract (200 mg kg^-1 b wt. -Srivastava et al., 2021a; 400 mg kg^-1 b wt. - Kumar & Thakur, 2018; 400 mg kg^-1 b wt. - Sarma, 2014; 250 mg kg^-1 wt. - Sharma, Siddiqui, Kumar, Ram, & Chaudhary, 2013); orange peel extract (250 and 500 mg kg^-1 b wt.-Ekhator et al., 2022; 200 mg kg^-1 b wt. -Mohamed, Tohamy, Elgamal, & Moneim, 2014; Srivastava et al., 2021a).

Purified Lead Nitrate [Pb (NO₃)₂] was purchased from Qualigens Fine Chemicals, Mumbai, India and dissolved in distilled water to obtain the desired dose. Orange (Citrus sinensis) peel and jamun (Syzygium cumini) seed extracts were prepared according to Srivastava et al. (2021a, 2021b). This study was approved by the Research Degree Committee (RC/FSc/ZOO/2019-2020/07/22), D.D.U. Gorakhpur, Gorakhpur.

Experimental design

Acclimatized rats were grouped into A, B, C, D, E, and F groups (each with twenty rats) and treated daily (at 8 a.m.) as follow through gavages:

Group A: Control: Not treated;

Group B: (Lead): These rats were given lead (50 mg kg^-1 b wt.);

Group C: Pb + JSE: These rats were given daily lead (50 mg kg^-1 b wt.) and JSE (200 mg kg^-1 b wt.) simultaneously;

Group D: Pb + OPE: These rats received daily lead (50 mg kg^-1 b wt.) and OPE (200 mg kg^-1 b wt.) simultaneously;

Group E: OPE: Rats were given OPE (200 mg kg^-1 b wt.);

Group F: JSE: Rats were given JSE (200 mg kg^-1 b wt.).

10 rats from each group were fasted overnight and killed under light ether anaesthesia on 7 day and 14 day. Sera were separated after collection of blood by centrifugation at 3000 rpm for 5 min. Sera was stored at -20℃.Analysis of serum urea, creatinine and uric acid was performed by using kits (Beacon Diagnostics Private Ltd, India). Each sample was analyzed in duplicate.

Statistical analysis

Results of analysis are expressed as multiple group comparisons were performed by using ANOVA (One way Analysis of Variance). Student's t test and Bonferroni post hoc test was used to determined the deference between control and treated groups. The significant levels were set at p-value < 0.05.

Results

In the present study, creatinine levels increased in rat of Group B (treated with lead 50 mg kg^-1 b wt.) on 7 and 14 day as compared to control (group A) (Figure 1). Serum creatinine level significantly decreased after treatment with combination of lead and JSE (group C: Pb + JSE) and lead and OPE (group D: Pb + OPE) at 7 and 14 day as compared to Group B (only lead treated) (Figure 1). When only OPE or JSE was given to rat no significant changes were noticed in serum creatinine levels at 7 and 14 day (group E: 200 mg kg^-1 b wt. and group F: 200 mg kg^-1 b wt.). Results of ANOVA expressed that treatment is significant (7 days- F = 4.815, p < 0.002; 14 days- F = 43.419, p < 0.0001).

Serum urea levels increased significantly in rat treated with lead (group B: 50 mg kg^-1 b wt.) from 7 to 14 day as compared to control (group A; Figure 2). Serum urea levels were decreased progressively from 7 day to 14 day after treatment with lead and jamun seed extract (group C: Pb + JSE) or lead and orange peel extract (group D: Pb + OPE) as compared to Group B (lead exposed). No significant changes were recorded in serum urea levels after treatment with OPE (group E: 200 mg kg-1 b wt.) or JSE (group F: 200 mg kg-1 b wt.) at 7 and 14 day (Figure 2). Results of ANOVA expressed that treatment is significant (7 days- F = 29.475, p < 0.0001; 14 days- F = 55.954, p < 0.0001).

Increased serum uric acid levels were recorded in the rat treated with lead (group B) as compared to Group A (control) at 7 and 14 day (Figure 3). After treatment with combined dose of lead and jamun seed extract (group C: Pb + JSE) or lead and orange peel extract (group D: Pb + OPE) significant decrease in serum uric acid levels were recorded as compared to Group B (lead treated rat) at 7 and 14 day (Figure 3). The rat treated with OPE (group E: 200 mg kg^-1 b wt.) or JSE (group F: 200 mg kg^-1 b wt.), no significant changes were observed at 7 day and 14 day (Figure 3). Results of ANOVA expressed that treatment is significant (7 days- F = 11.014, p < 0.0001; 14 days- F = 32.610, p < 0.0001).

The above results clearly indicate that JSE and OPE are effective in ameliorating the effect of lead on kidney bio-markers.

Discussion

Lead exposure to rats caused alternations in the kidney bio-markers which is evident by increased urea, creatinine and uric acid levels. Previous researchers have also reported similar results in toxicants exposed rats-Acrylamide (Uthra et al., 2022), Deltamethrin (Mostafa, Atef, & el Din, 2022), Cadmium (Ilesanmi & Adeogun, 2022), Paraoxon (Sobolev, Sokolova, Jenkins, & Goncharov, 2021), Methotrexate (Alum et al., 2023), Oxazaphosphorine (Lukasz et al., 2017), Tartrazine (El-Desoky, Wabaidur, AlOthman, & Habila, 2022), Chlorpyrifos (Aung et al., 2020) and Gentamicin (Akhitha, Raghavendra, & Kumar, 2019). Few investigators have also reported alterations in kidney biomarkers after exposure to various toxicants in - (i) fishes- Bisphenol A (Srivastava & Reddy, 2020), Diazinon (Banaee et al., 2023), Imidacloprid (Qadir, Latif, Ali, & Iqbal, 2014) and Deltamethrin (Hamed, 2016); and (ii) Amphibian- Cadmium (Medina, González, Klyver, & Odstrcil, 2016) and Oxyfluorfen (El-Rahman, Ahmed, Khalil, & Abd-Elhakim, 2019). In the present study, we have observed a significant decrease in the levels of serum creatinine, serum urea, serum uric acid in the blood of rat after treatment with JSE and OPE which evident preventive effect of JSE and OPE against lead nephrotoxicity.

This study noticed an increased levels of serum creatinine in rats after lead exposure which is in agreement with observations of Eivani et al. (2023) who have also reported lead-induced increase in serum creatinine levels of rat. In group C (Pb+ JSE) and group D (Pb+ OPE), the creatinine levels decreased on 7 and 14 day in comparison to Group B (lead treated rat) this indicates that OPE and JSE are effective in protecting kidney and improved the serum creatinine levels which was increased after treatment with lead. There was no change observed in creatinine levels after treatment with orange peel extract or jamun seed extract to rat. In past, no study exists regarding the effects of JSE and OPE on creatinine levels of lead exposed rats.

In the present study, increased serum urea levels were recorded in lead treated rats at 7 and 14 day. Previous investigators have also observed increased serum urea levels in toxicants exposed rats - Tartrazine (Rahayu, Wahyuni, Fitriani, & Agung, 2022); Bisphenol A (Kobroob, Peerapanyasut, Chattipakorn, & Wongmekiat, 2018); Cypermethrin and chlorpyrifos (Ajobola et al., 2019) and Acetaminophen (Roy et al., 2015). Serum urea levels were significantly decreased after treatment with combination of lead and JSE or OPE as compared to Group B (only lead treated rat). There was no significant change observed in serum urea levels in rat which was treated with OPE and JSE. The effect of JSE and OPE on lead induced changes in serum urea of rat has not been investigated earlier.

Administration of lead provoked a significant increase in serum uric acid levels of Group B (lead treated rat) in comparison to control (group A) from 7 to 14 day. The current study derives support from studies of Ashour et al. (2007) who have also reported increased serum uric acid levels in rat after treatment with lead. In the present study, the combined dose of lead with JSE or OPE was given to the rat significantly reduced levels of serum uric acid. This clearly indicate that JSE and OPE were effective in recovering uric acid levels to nearly control values.

In the present study the recovery in lead induced kidney biomarkers e.g. creatinine, urea and uric acid by treatment with jamun seed and orange peel extracts can be attributed to the presence of phytochemicals present in the extracts. In past, few studies have reported the pharmacological properties of jamun seed, such as (i) its antidiabetic potential including the ability to prevent complications associated with diabetes e.g. nephropathy, neuropathy, gastropathy, diabetic cataract, and peptic ulceration; (ii) cardioprotective, (iii) gastroprotective, (iv) hepatoprotective, and (v) neuropsychopharmacology effects (Kumar et al., 2022). In past, dietary supplementation of orange peel powder has been reported to attenuate the toxic effect of bisphenol A-induced hepatic and splenic dysfunction, which has been attributed to its phenol and flavonoid content (AbdEl-Gwaad, El-Wahab, Mohamed, Sharaf, & Osman, 2020).

Conclusion

In conclusion, lead exposure to rat alters serum urea, serum creatinine serum uric acid levels. The jamun seed extract and orange peel extract administration showed preventive effect against renal toxicity induced by lead exposure in rat. JSE and OPE administration showed restoration of serum urea, creatinine, serum uric acid levels to near the control value. Therefore, it is advisable that the organisms exposed to heavy metals specially lead should be given dietary supplement of these botanical extracts which would ease the several toxic symptoms.

Acknowledgements

Ram Prataap Yadav is thankful to Indian Council of Medical Research, New Delhi, India for providing financial assistance [Fellowship No. 3/1/3/JRF-2021/HRD-012(1209184)] for conducting this research work. Ajai K. Srivastav and Nobuo Suzuki are thankful to Japan Society for Promotion of Science (No. L24525) for partly funding this study.

References

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