Effects of leonardite and succimer treatment on enzyme activity in rats exposed to lead acetate

The study aimed to research in vivo changes in the enzyme activity of the blood and liver of laboratory rats under continuous exposition to a moderate dose of lead acetate while receiving succimer together with humic feed additive madden out of leonardite. Two-month-old white rats were used to assess the detoxifying properties of the humic compounds leonardite and lignite. To conduct the study, a total of 32 male laboratory rats were divided into four groups, each consisting of eight animals, using the analog pair approach. Rats received a pre-meal injection of lead acetate at a dose of 7 mg/100 g of animal weight (1/110 LD 50 ) using a veterinary feeding needle. Humic feed additives were supplied to animals by creating solutions from leonardite at a dosage of 18 mg/kg depending on the active ingredient. Succimer was prescribed in an amount of 30 mg/kg, previously dissolved in apple juice using a feeding tube 3 hours after eating one time in a day. Upon analyzing the collected data, it can be deduced that the treatment involving succimer and the humic-based feed additive leonardite alongside lead acetate poisoning yielded improved protein metabolism and activity of enzymes in poisoned rats. Notably, in all statistically significant instances (P < 0.05) of improvement in indicators among animals in that research group, the numerical values closely approximated those of intact animals. The key distinction between treating animals with succimer alone and the combination of succimer and leonardite primarily manifested in alterations in the activity of liver enzymes involved in the antioxidant system such as: catalase, superoxide dismutase


Introduction
Lead compounds rank highly among the most prevalent environmental pollutants, particularly among heavy metals.Water-soluble variants like lead acetate pose significant toxicity risks.Pollution often stems from emissions by industries and the combustion of fuels, affecting water bodies, pastures, and areas where farm animals and their feed are cultivated.These factors contribute to animal poisoning (Kalia & Flora, 2005).
Lead acts as a thiol poison, engaging with the SH groups of different enzymes within the body.This toxic compound exhibits astringent, irritating, and cauterizing effects on cellular protoplasm (Nemsadze et al., 2009).Heavy metal poisoning induces oxidative stress, leading to an elevation in free radicals such as oxygen species.This increase in free radicals damages target tissues, affecting their function, and results in decreased antioxidant levels.Heavy metals tend to accumulate in certain soft tissues like the liver, causing alterations in enzyme and hormone levels (Ilesanmi et al., 2022).
Well-known efficient oral lead chelator is succimer.However, some studies suggest it is not recommended to use with ongoing lead exposure (Aaseth et al., 1995).In contrast, a study on suckling rats showed that use in such condition is possible and does not affect the growth and development of animals in lead-affected enviroments (Varnai et al. 2001).
All drugs that remove lead from the body act slowly and have serious side effects.Since chelating drugs also remove beneficial minerals from the body -zinc, copper, and ironthe victim is often prescribed supplements.Since chelating drugs also remove beneficial minerals from the body -zinc, copper, and iron -the victim is often prescribed supplements to reduce side effects (Vorvolakos et al., 2016).Humates are potential active ingredients found in feed additives that may mitigate the toxic effects on animal organisms.Typically, humates comprise three components: humic ac-ids, fulvic acids, and mineral humic residue (Jarukas et al., 2021).
Last studies on piglets demonstrate the significant antioxidant effectiveness of humic feed additives derived from leonardite and lignite in managing diarrhea (Trckova et al., 2018).Researchers suggest that leonardite, owing to its oxidative properties, exhibits greater efficacy.This outcome is likely attributed to the abundant presence of fulvic acids in its composition.Fulvic acids, with their lower molecular weight and well-developed donor-acceptor properties, are more actively involved in oxidative reactions compared to humic acids (Szabó et al., 2017).Additionally, a theoretical study indicates the capacity of fulvic acids to form chelate complexes with heavy metal ions, rendering them neutrally charged and preventing their absorption in the gastrointestinal tract (Glynn, 1995).The beneficial impact of humic acids on lead redistribution within affected rats' organs, its elimination from the body, and the synthesis of the antioxidant Glutathione is well-documented (Žatko et al., 2014;Vašková et al., 2019).
Recent studies indicate that the biochemical and hematological parameters of rat blood, following exposure to the toxic agent lead acetate along with a feed additive containing leonardite, showed values more closely resembling those of intact rats compared to rats treated solely with lead acetate.These observed improvements are likely attributed to the substantial presence of fulvic acids.Leveraging their donor-acceptor abilities, fulvic acids can potentially bind a portion of lead ions at specific metabolic stages.Additionally, the adaptogenic effects of other humic substances present in the composition may contribute to these beneficial outcomes (Varava, 2023).
Thus, this study aimed to show changes in the protein metabolism, enzyme activity of the blood and liver of laboratory rats under the constant influence of a moderate dose of lead acetate with the succimer treatment and administration of humic feed additive based on leonardite.

Materials and methods
The evaluation of the detoxification properties of leonardite combined with succimer treatment was carried out using two-months old white rats.The study was conducted within the vivarium facilities of Dnipro State Agrarian and Economic.Male laboratory rats were allocated into four groups of eight animals each by using the analog pair method.All groups were housed in individual cages under identical conditions, with ad libitum access to water and a balanced diet.The duration of the study was 21 days.
The administration of factors that influence groups of animals occurred per os every day of the study.Dietary treatments of rats were as follows: Group І -intact animals with standard vivarium diet; Group ІІ -standard vivarium diet together with the toxic agent -lead acetate (Pb(C2H3O2)2); Group ІІІ -standard vivarium diet together with toxic agent and succimer (C 4 H 6 O 4 S 2 ); Group IV -standard vivarium diet together with toxic agent, succimer and leonardite based humic feed additive.Lead acetate was administered via a veterinary feeding needle (UNO Life Science Solutions, Netherlands) for rats prior to feeding at a dose of 7 mg/100 g of animal weight (1/110 LD 50 ) (Tkachenko & Melnikova, 2008).Feed additive of humic nature was fed by forming solutions from leonardite at dosage based on the active substance of 18 mg/kg.The resulting solution was mixed with feed.Succimer was prescribed in an amount of 30 mg/kg, previously dissolved in apple juice using a feeding tube 3 hours after eating one time in a day (Smith et al., 1998).Table 1 depicts the experiment's layout.Each day, all animals underwent a daily assessment for clinical signs of toxicity and were checked twice for mortality.Blood samples for research purposes were obtained once, nearing the conclusion of the in-life phase of the study, from the right ventricle of the heart while the subjects were under thiopental narcosis (60 μg/kg).
In order to assess the activity of antioxidant enzymes and peroxidation level, liver samples were homogenized (1:10, wt/vol) in 100 mM phosphate buffer, pH 7.4, using an electrically driven pestle.Subsequently, the homogenate underwent centrifugation at 4 °C for 20 minutes at 7,000 × g, and the resulting supernatant was collected for the determination.ELISA test was carried out separately for CAT (catalase), SOD (superoxide dismutase), GPx (glutathione peroxidase), MDA (malondialdehyde) with specific for each substance assay kit (Cayman Chemical Company, USA).
All handling and scientific inquiries concerning animals adhered to the guidelines outlined in the "European Convention for the Protection of Vertebrate Animals used for Experimental or other Scientific Purposes" (Strasbourg, France, March 18, 1986, ETS No. 123), and the Law of Ukraine "On Protection of Animals from Cruel Treatment" (Kyiv, February 21, 2006, No. 3447-IV).
The experimental data underwent statistical analysis using Student's t-test through MS Office Excel 2019.Significance was determined at P < 0.05.The results are expressed in the tables as mean values accompanied by standard error (m ± SE).

Results and discussion
No deaths related to treatment or noticeable clinical symptoms, such as hair loss, scabbing, soft or mucoid excrements, decreased defecation, smaller-than-usual feces, or vocalization upon handling, were observed in any of the treated groups throughout the study.Furthermore, at the conclusion of the study, animals in all treatment groups did not exhibit significant disorders.
Table 2 displays the results of the serum protein metabolites research.There was an increase in the concentration of serum protein in the blood of rats for group ІІІ that received succimer treatment by 3.9 % (P < 0.05), especially for group IV, which also received a feed additive from leonardite by 4.8 % (P < 0.01) compared to the control group ІІ.The main changes in this parameter in animals of these groups could be explained by an increase in albumin concentration in relation to animals of the control group by 15.7% (P < 0.001) and 17.7 % (P < 0.001) for group ІІІ and IV, respectively..07$ Note: Probability of the difference experiment groups to the intact group: * − P < 0.05; ** − P < 0.01; *** − P < 0.001 Probability of the difference experiment groups to the control group: $ − P < 0.05; $$ − P < 0.01; $$$ − P < 0.001 Protein metabolites of rats of the studied groups, such as urea and creatinine, also changed compared to the control group.There was a decrease in these indicators in rats in relation to animals of the control group to group ІІІ by 9.2 % (P < 0.001), and 3.6 % (P < 0.05) for urea and creatinine, respectively.More expressive decrease in the concentration of these metabolites was observed in animals of group IV by 12.4 % (P < 0.001), and 5.8 % (P < 0.001).
Indicators of oxidative stress in rats of the studied groups were also changed when exposed to succimer and leonardite.Thus, the concentration of SH-groups was closer to that of the animals of the intact group by 8.4 % (P < 0.05) for group ІІІ and by 10.9 % (P < 0.001) for group IV compared to the control group.Against this background, glutathione, a protein that determines oxidative properties in rats of groups ІІІ and IV, was higher by 16.1 % (P < 0.01) and 17.2 % (P < 0.001) compared to animals in the control group.These changes suggest a slight effect of leonardite on general antioxidant processes in the body of rats.
The concentration of Ceruloplasmin (copper-containing metalloprotein) was reduced in animals of both study groups compared to the control group, in which animals received only lead acetate, in group ІІІ by 12.0 % (P < 0.01), and in group IV by 14.2 % (P < 0.001).There was also a decrease in the concentration of osteocalcin for rats of the above listed groups relative to the control group by 7.8 % (P < 0.05) and 8.9 % (P < 0.05), which indicates a decrease in the overall oxidation of the body.The C-reactive protein was slightly detected in the blood of animals in the control group and was absent in the experimental groups, which may indicate the absence of inflammatory reactions.
Effect of Lead acetate on blood enzymes has been shown in Table 3.The concentration of the ALT enzyme in groups III and IV animals was lower compared to those treated solely with lead acetate by 26.8 % (P < 0.01) and 31.9 % (P < 0.001), respectively.Additionally, a decrease in AST activity was observed in these groups by 15.0 % (P < 0.01) and 25.0 % (P < 0.001), respectively.Conversely, the ALP enzyme concentration increased by 12.3 % (P < 0.05) and 19.4 % (P < 0.01), respectively, in these compared groups of animals.The measurement approached the value observed in the intact animals.

Table 3
Effect of Lead acetate on blood enzymes in laboratory rats under the influence of leonardite and succimer on the 21 st day of the study (m ± SE, n = 8)

Group ІІІ (Succimer)
A significant decrease in GGT activity by 50.2 % (P < 0.001) was observed in group III and by 58.7 % (P < 0.001) in group IV of animals in relation to animals poisoned with lead acetate.Another positive factor is that there was an increase in amylase levels for rats of these groups by 19.7 % (P < 0.05) and 26.0 % (P < 0.05).The noted changes indicate a rapid recovery of the rats' body as a whole, while a significant effect of leonardite was noted.
In addition, there was an increase in CPK closer to the level of intact animals for rats of group III by 23.4 % (P < 0.05) and rats of group IV by 31.6 % (P < 0.01) compared to the control group.ACE plays an important role in the control of blood pressure.It was shown that this hormone decreased in animals by 21.9 % (P < 0.001) and 29.8 % (P < 0.001) for groups III and IV relative to the control group.
A significant decrease in the level of activities of SOD, GPx, and CAT; as well as an increase in MDA content were observed in lead-treated rats.Administration of leonardite and succimer brought along with lead acetate increased the levels of SOD (P < 0.01), GPx (P < 0.001), and CAT (P < 0.05) and decreased the liver MDA (P < 0.001) content when compared with lead-alone treated rats in Table 4.After analyzing the obtained data, it could be concluded that the treatment of rats from lead acetate poisoning in combination with succimer and the feed additive of humic nature leonardite has increased the effectiveness.So, in all statistically significant cases of improvement in indicators in animals of group 4, the numerical values were close or approximately equal to intact animals.At the same time, the main difference between the treatment of animals with succimer and the combination of succimer and leonardite manifested itself in changes in the activity of enzymes primarily included in the antioxidant system.

Conclusions
In summary, this study illustrates that lead acetate induces considerable oxidative damage and suppresses the activities of antioxidant enzymes.Treatment with succimer combined with leonardite shows promise in mitigating these risks, suggesting its potential usefulness and rationality in lead toxicity treatment.In particular, the activity of liver enzymes in rats poisoned with lead acetate and cured with this treatment regimen was close to that of intact animals.

Table 1
The experiment's layout

Table 2
Effect of Lead acetate on blood protein metabolites in laboratory rats under the influence of leonardite and succimer on the 21 st day of the study (m ± SE, n = 8)

Table 4
Effect of Lead acetate on blood protein metabolites in laboratory rats under the influence of leonardite and succimer on the 21 st day of the study (m ± SE, n = 8)