Hematological and serum biochemical changes induced by individual and combined exposure to lithium carbonate and lead acetate in rats

Introduction

Rapid industrialization and urbanization associated with mining, agriculture, waste management, and automobile industries have significantly increased heavy metal contamination in the environment. Heavy metals are persistent contaminants that bioaccumulate through the food chain and tend to concentrate in higher organisms, including humans and animals (Jarup, 2003) [16] . Their accumulation in soil and water occurs mainly through leaching processes, resulting in long-term exposure risks (Luoma and Rainbow, 2008) [22] . Due to their extended biological half-lives, heavy metals pose serious global public health concerns (Jaishankar et al. 2014) [15] . Among heavy metals, lithium (Li) and lead (Pb) are increasingly prevalent due to industrial expansion and rising demand for batteries, electric vehicles, and pharmaceuticals (Livingstone, 2003) [20] . Lithium is naturally present in soil and water, with reported concentrations of up to 200 µg/g in soil and 1-10 µg/ml in surface water, reaching as high as 5 mg/L in certain regions (Choubey et al. 2017) [8] . The earth's crust contains approximately 50-65 mg/kg of lithium, while soil concentrations range from 10-100 mg/kg in the United States and 10-50 mg/kg in Russia (Weiner, 1993; Léonard et al. 1995; Lagerkvist and Lindell, 2002) [41,19,18] . Elevated lithium levels have also been reported in drinking water, reaching 78 mg/L in parts of Texas (Trieff et al. 1973) [37] . Lithium carbonate is widely used in the treatment of bipolar disorder; however, its narrow therapeutic index and slow excretion may result in accumulation and toxicity upon prolonged exposure (Grof, 2010; Warick, 1979; Yip & Yeung, 2007) [13,40,44] . Excess lithium exposure induces oxidative stress, inflammation, and apoptosis, affecting multiple organ systems including the hematopoietic, renal, hepatic, and cardiovascular systems (Sahin et al. 2006; Chadha et al. 2008; Ostadhadi et al. 2018) [31,7,27] . Lithiuminduced toxicity has been associated with altered antioxidant enzyme activities, increased lipid peroxidation, and disruption of mitochondrial function, leading to biochemical and hematological abnormalities (Kiełczykowska et al. 2015) [17] . Lead is another highly toxic, non-biodegradable heavy metal with widespread environmental presence. It causes extensive physiological and biochemical dysfunctions across multiple organ systems, with the hematological system being particularly vulnerable due to lead's affinity for erythrocytes and hemoglobin (Goyer, 1991; Andjelkovic et al. 2019) [12,3] . Despite declining blood lead levels globally, occupational workers, infants, and children remain at high risk (Godwin, 2001) [11] . Lead exposure contributes significantly to morbidity and mortality, especially in developing countries (Smith and Flegal, 1995) [35] . Major sources of lead contamination include paints, batteries, cosmetics, medicines, fuel, and industrial effluents. Lead concentrations exceeding permissible limits have been reported in Indian water bodies, including Gujarat lakes, where values far exceeded WHO and Indian standards (Moore, 1988; Smith and Smith, 2004; Vediya and Patel, 2011; Patel and Vediya, 2012) [25,34,29,38] . After ingestion, lead is absorbed via the gastrointestinal tract and distributed through the bloodstream to vital organs, causing hematological disturbances, oxidative stress, and biochemical alterations (Barbosa et al. 2005; Flora et al. 2012) [4,10] [23,32] . Recent studies indicate that combined exposure to heavy metals may result in synergistic toxicity. Luo et al. (2023) [21] demonstrated enhanced toxicity of lithium and lead combinations in mouse brain stem cells, while Cunha et al. (2023) [9] reported amplified stress responses following combined exposure in Mytilus galloprovincialis. These findings suggest that simultaneous exposure to lithium and lead may exert greater toxicological effects than individual exposures. However, limited information is available regarding the combined haematological and serum-biochemical effects of lithium and lead exposure in mammalian systems. Therefore, the present study was designed to evaluate the toxicological interaction of lithium carbonate and lead acetate, administered individually and in combination, on haematological and serum-biochemical parameters in rats following daily oral exposure for 28 days.

Materials and Methods

Test compounds

Lithium carbonate (≥99.5% purity, CAS No. 554-13-2) and lead acetate (≥97.0% purity, CAS No. 6080-56-4) were procured from Himedia, India. Stock solutions were prepared by dissolving 400 mg of each compound separately in 50 mL of Milli-Q water. The powders were accurately weighed using a calibrated analytical balance (Model: MS 204S/A01, Mettler Toledo, US). The prepared stock solutions were further diluted as required to obtain the desired concentrations for administration to rats.

Experimental animals

The study was conducted using 24 male Wistar rats aged 7-8 weeks, obtained from the Zydus Research Centre, Ahmedabad, Gujarat. Experimental procedures were approved by the Institutional Animal Ethics Committee (IAEC) under Protocol No. KU-JVC-IAEC-SA-123-23 and were carried out in compliance with the Committee for Control and Supervision of Experiments on Animals (CCSEA, 2003) guidelines. Animals were housed in polypropylene cages equipped with stainless steel grills and provided with corn cob bedding, which was changed twice weekly. Standard laboratory conditions were maintained, including a 12 h light/dark cycle, ambient temperature ranging from 23.19 ± 0.13 °C to 25.76 ± 0.13 °C, and relative humidity between 48 ± 0.53% and 65 ± 0.74%. Rats were allowed ad libitum access to standard pellet feed (VRK Nutritional Solutions, Maharashtra) and filtered reverse osmosis drinking water supplied in 300 mL polypropylene bottles. Prior to treatment, animals were acclimatized for 7 days, during which they were individually identified by tail marking using a black marker. The experiment followed a Completely Randomized Design (CRD), with animals randomly allocated into four treatment groups (n = 12 per group) based on body weight, ensuring a variation within ± 5%.

Exposure

Rats in the control group (C) received reverse osmosis (R.O.) water in volumes ranging from 0.75 to 1.12 mL, adjusted according to individual body weights. Lithium carbonate and lead acetate were administered separately at 20 mg/kg body weight (BW) daily for 28 consecutive days to the Li and Pb groups, respectively. The Li + Pb group received both lithium carbonate and lead acetate at the same dose rate of 20 mg/kg BW each, administered daily for 28 days. The selected dose levels were chosen based on earlier studies reporting clear toxicological effects without inducing overt mortality, thereby making them appropriate for sub-chronic exposure assessments (Sahin et [31,36,5,30] .

Haematology and Serum biochemical analysis

On the 29 th day, blood samples (3-4 mL) were collected from the retro-orbital plexus under light anesthesia. Samples intended for hematological analysis were collected in sterile K₃EDTA tubes, while those for serum biochemistry were collected in anticoagulant-free vials, allowed to clot at room temperature, and centrifuged at 10,000 g for 10 min (Eppendorf 5430 R, Germany). The separated serum was stored at -80 °C and analyzed within 2 days. Hematological parameters, including hemoglobin (Hb), total erythrocyte count (TEC), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), total leukocyte count (TLC), and differential leukocyte count (DLC), were measured using an automated hematology analyzer (Abacus Junior Vet 5, Diatron, Hungary). Biochemical parameters, namely alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), albumin (ALB), globulin, albumin/globulin ratio, alkaline phosphatase (ALP), lactate dehydrogenase (LDH), serum creatinine (CK), urea, and triglycerides, were determined using standard diagnostic kits on an automated biochemistry analyzer (LANDWIND C100, Diatek, China).

Statistical analysis

Statistical analyses of all data were carried out using GraphPad Prism 9.0. The Kolmogorov-Smirnov test was used to evaluate the normality of data, along with Bartlett's test to confirm the equal variance. Data with normal distribution and homogeneous variance were analyzed by parametric one-way analysis of variance (ANOVA) followed by Tukey's HSD test. The data that didn't have either a normal distribution or equal variances were analyzed by the Kruskal-Wallis test followed by Dunn's test. The value of p < 0.05 was considered statistically significant.

Results

Haematology and serum biochemical analysis

A significant reduction in platelets and an increase in WBCs were observed in the Li + Pb-treated groups compared to the control group. Platelet levels were significantly lower in the Li + Pb-treated group than in the Li-treated group, possibly due to the specific effects of Pb exposure. In contrast, a significantly increased WBC was observed in the Li + Pbtreated group compared to the Pb-treated group, which might be associated with the Li exposure. Other haematological parameters in all toxicity groups were not significantly different. The effects of daily oral administration of Li, Pb, and Li + Pb for 28 days on hematological parameters in rats are shown in Tables 1 and illustrated in Figures 1 &2. A significant increase in ALT and urea was observed in the Li and Li + Pb-treated groups compared to the control group. The AST level in the Pb-treated group was significantly lower than that of the Li-treated group. A significant increase in ALP and LDH was observed in all toxicity groups compared to the control group. A significant increase in triglyceride was observed in the Li-treated group compared to the control group; whereas AST, TP, albumin, globulin, A/G Ratio, and creatinine were not significantly altered by all treatments compared to the control group. The LDH and the triglyceride levels in the Pb and Li + Pb-treated groups were significantly lower than those of the Li-treated group, which shows the interaction of both metals. The effects of daily oral administration of Li, Pb, and Li + Pb for 28 days on serumbiochemical parameters in rats are shown in Tables 2 and illustrated in Figures 3 &4.

Discussion

The present study investigated the toxicological effects of lithium carbonate (Li), lead acetate (Pb), and their combined exposure in adult Wistar rats following 28 days of oral administration. Control rats were unexposed, while treatment groups received Li, Pb, or Li + Pb (20 mg/kg BW each). A comprehensive evaluation was performed to assess hematological and biochemical parameters in male rats. Combined Li and Pb exposure significantly reduced platelet counts and elevated WBC levels compared to controls, indicating distinct toxicant interactions. The more pronounced thrombocytopenia in the Li + Pb group than in Li alone appears largely Pb-driven, consistent with reports of dosedependent lead-induced hematological toxicity (Andjelkovic et [3,1,28,6] . The elevated WBC counts in Li + Pb rats, greater than in Pb alone, suggest lithium's modulatory influence. Previous studies show variable hematological effects of lithium ranging from leukocytosis with reduced monocytes (Malhotra & Dhawan, 2008) [24] , to no changes (Kiełczykowska et al. 2014) [17] , or reductions in MCV, MCH, and platelets with concurrent WBC increases (Ben Saad et al.

2017;

Alamri & Alfaifi, 2024) [5,2] . Such discrepancies likely reflect dose, duration, and experimental differences. Lithium's effects on blood cells are linked to altered RBC membrane dynamics and stimulation of leukopoiesis, leading to leukocytosis (Malhotra & Dhawan, 2008) [24] , while Pb induces leukocytosis via toxic injury to lymphoid organs and inflammatory stimulation of germinal centers (Yagminas et al. 1990) [42] . Lithium has also been reported to enhance phagocytic activity, increasing WBC counts with reduced monocytes (Yildirim et al. 2018) [43] . Collectively, these findings suggest that Pb predominantly drives thrombocytopenia, while Li modulates leukopoietic and immune responses. Their combined exposure produces distinct hematological alterations decreased platelet counts and increased WBC levels reflecting both toxic and adaptive immunomodulatory effects. Lithium and lead exposures produced distinct and interactive effects on liver and kidney function markers. Lithium significantly increased ALT and urea, indicating hepatocellular stress and impaired renal clearance, consistent with lithium-induced hepatotoxicity and nephrotoxicity (Vijaimohan et al. 2010; Mwaheb et al. 2016) [39,26] . Elevated urea likely reflects lithium accumulation in proximal tubules causing nephron damage and reduced filtration (Zhang et al. 2022) [45] , while increased ALT, LDH, and triglycerides indicate oxidative stress-mediated hepatocellular injury and disrupted lipid metabolism. Lead exposure similarly elevated ALP and LDH, supporting its role in hepatocellular necrosis and oxidative stress (Ibrahim et al. 2012; Bidanchi et al. 2022) [14,6] . Raised ALP reflects impaired detoxification and membrane integrity, LDH indicates widespread cellular injury (Seddik et al. 2010) [33] , and elevated urea confirms renal impairment. Combined Li + Pb exposure showed unique interactions: ALT and urea elevations mirrored lithium's effect, but LDH and triglycerides were lower than in Li alone, suggesting Pb modulates lithium's impact on lipid metabolism. Lead-induced oxidative stress and enzymatic inhibition may suppress triglyceride synthesis or enhance breakdown, counteracting lithium's hypertriglyceridemic effect. Overall, lithium primarily impairs hepatic and renal function through oxidative stress and membrane disruption, whereas lead exerts broader enzymatic and renal effects; their co-exposure modifies these outcomes, particularly in lipid metabolism, indicating toxicodynamic interactions.

Conclusion

Hematological analysis showed thrombocytopenia and leukocytosis in the Li + Pb group, with Pb primarily affecting platelets and Li influencing white blood cells. Liver and kidney function markers (e.g., ALT, ALP, LDH, BUN) were significantly altered, especially in the combined exposure group, indicating hepato-renal toxicity. Overall, the study confirmed that while both Li and Pb are toxic individually, co-exposure leads to more severe hematological and biochemical underlining the need to consider combined exposures in toxicological risk assessments.

Consent for publication: All authors approved the manuscript for publication.

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