The administration of sorbents plays a vital role in reducing systemic toxicity after radionuclides or harmful chemicals enter the organism. This study explores the pharmacological characteristics of the newly developed ferrocyanide-bentonite sorbent, Ferbensorb. As a composite formulation, Ferbensorb integrates potassium-iron(III) hexacyanoferrate(II), bentonite, gelatin, along with a mixture of essential macro- and microelements. Experimental assessments were performed using mice and rats as animal models. The investigation evaluated its cesium sorption efficacy, strontium radionuclide retention capability, and performance under simulated associative mycotoxicosis. Autopsies were conducted to assess anatomical alterations, while variations in overall body mass and the condition of specific internal organs were documented. Blood analyses also included profiling of the leukocyte formula. The findings demonstrated that the ferrocyanide-bentonite sorbent, Ferbensorb, effectively alleviated both structural and functional disruptions induced by mycotoxins, including ochratoxin A, fumonisin B, and zearalenone. Improvements were observed in weight gain, innate immune response (indicated by a higher proportion of neutrophils), enhanced serum lysozyme and bactericidal activities, and increased survival rates in the treated animals.
Enterosorbents function as pharmacological agents that operate within the gastrointestinal tract to capture and neutralize various substances through processes such as ion exchange, adsorption, absorption, and complex formation [1]. Their administration is crucial in diminishing the organism’s exposure to toxicants, particularly when harmful chemicals or radionuclides are ingested. In scenarios where the integrity of the gastrointestinal barrier is compromised, enterosorbents serve a protective role by preventing the absorption of deleterious byproducts from the chyme, which is especially valuable in gastrointestinal pathologies [2, 3].
The therapeutic efficacy of enterosorbents arises through both immediate and systemic actions. The immediate, or direct, effects include the adsorption of toxins, xenobiotics from ingested feed, substances participating in enterohepatic and hematoenteral recirculation, intestinal breakdown products, microbial cells, microbial toxins, gases, and other harmful compounds. These effects also involve changes in chyme viscosity and stimulation of digestive organ functions. On the other hand, indirect effects result from reduced strain on organs responsible for detoxification and improved metabolic regulation [4–6].
Despite considerable progress, no universal sorbent has proven effective against the full spectrum of xenobiotics, reflecting the unexplored potential of numerous compounds with promising enterosorbent capabilities. As a result, an ongoing investigation is essential to discover and validate the most effective substances for mitigating toxic exposure from feed. Practical experience in animal nutrition has confirmed the efficacy of several organic and mineral materials in capturing diverse classes of toxins [7, 8]. Among them, clay-based minerals such as zeolites and bentonites have demonstrated consistently beneficial effects.
Bentonite is defined as a clay material predominantly composed (at least 70%) of minerals from the montmorillonite group [9]. Montmorillonite is a fine-grained, layered aluminosilicate characterized by structural cationic substitutions that produce a net negative charge, balanced by exchangeable cations situated in interlayer zones. This property underlies bentonite’s pronounced hydrophilicity [10].
Upon hydration, water molecules infiltrate the montmorillonite structure, causing interlayer swelling by interacting with surface cations. Further dilution with water transforms bentonite into a cohesive, thixotropic gel-like suspension [11]. Thanks to its exceptional cation-exchange capacity and sorption performance, bentonite is considered a beneficial feed additive that supports animal development and overall health, owing to its multi-mineral content and presence of critical trace elements [12].
Research shows bentonite positively influences digestion in both avian and mammalian species. It can effectively bind microbial toxins and alcohols, acting as a hepatoprotective agent while also aiding in the regulation of reproductive system functions [13, 14]. Supplementation with silicon-containing minerals, such as bentonite, has also been found to improve mineral metabolism. Specifically, its inclusion elevates serum levels of iron, copper, and zinc by 17.4%, 9.7%, and 42.3%, respectively [15].
This normalization of mineral homeostasis plays a significant role in promoting hematopoiesis, including both erythropoiesis and general blood formation [16]. As a result, red blood cell counts increase by 12.3%–20.2%, and hemoglobin concentration rises by 13.5%–18.9% [17].
Hexacyanoferrates, such as ferrocyanides, have been used as dyes since the 19th century. Their radioprotective potential became a subject of investigation after atmospheric nuclear tests conducted between 1954 and 1964. The mid-1960s established that administering ferrocyanides concurrently with radioactive cesium can reduce cesium absorption into body tissues and organs by 95%–98% [18]. Ferbensorb, a ferrocyanide-bentonite sorbent, is a complex formulation consisting of potassium-iron(III) hexacyanoferrate(II), bentonite, gelatin, and essential macro- and microelements.
This study explores the pharmacological characteristics of the newly developed ferrocyanide-bentonite sorbent, Ferbensorb.
An experiment was carried out to assess the sorption of strontium radionuclides using 28 outbred white rats, each weighing approximately 280 ± 12 grams. The rats were divided into 7 groups, with 4 animals per group, matched for body weight. A solution of 90Sr nitrate (197 kBq per rat) was administered to each animal through a metal probe, alongside sorbents suspended in a 1.5% starch gel. After three days, the animals were euthanized by decapitation, and bone samples were collected for radiometric analysis using the UMF-1500 system.
In another set of experiments, the sorption properties of the Ferbensorb sorbent were tested in an experimental model of associative mycotoxicosis using Wistar rats weighing 160-200 g. These rats were divided into four groups of eight, as follows: group 1 received standard food without any additives and served as the control group, group 2 rats were given a diet containing a mycotoxin mixture with 0.5% Ferbensorb (5 g per kilogram of feed), group 3 animals received the same mycotoxin mixture, but with 1% Ferbensorb (10 g per kilogram of feed), and group 4 rats were fed the mycotoxin-infused diet without any sorbent. The mycotoxin mixture was added to the feed at a concentration of 5 mg/kg.
The experiment lasted for 30 days, with animal weights recorded at the start and again after 15 days. Two rats from each group were sacrificed for a controlled necropsy at the 15-day mark. The final euthanasia and post-mortem analysis were performed after 30 days. During necropsy, pathological changes in organs and tissues were carefully noted. For the surviving animals, additional post-mortem examinations were conducted, and blood samples were collected to assess the leukocyte profile and bactericidal and lysozyme activity in serum using standardized laboratory methods.
The study aimed to assess the sorption performance of the ferrocyanide-bentonite sorbent Ferbensorb, considering varying doses. It was conducted with 20 Wistar rats weighing 180-200 g, divided into 4 groups of 5 animals each, matched for weight.
After administering a single dose of cesium-137 (370 kBq per rat), the first three groups were given Ferbensorb in the form of a 50% aqueous suspension. The sorbent doses administered to the groups were 1.0 ml, 1.5 ml, and 2.0 ml per animal, corresponding to 0.5, 0.75, and 1.0 grams per animal, respectively. The control group (group 4) did not receive any sorbent following cesium-137 injection.
Seven days after the isotope and sorbent were administered, the rats were euthanized, and their organs and tissues were analyzed for 137Cs content, as displayed in Table 1.
Table 1. The concentration of 137Cs in rat organs and tissues after administration of various amounts of Ferbensorb sorbent (in the numerator – Bq/g, in the denominator - % of control) (M ± m; n = 20)
Quantity of sorbent g/head | Name of the organ or tissue | |||||
Liver | Kidney | Lung | Spleen | Heart | Muscle | |
0.5 | 210.5 ± 24.5 22.4 | 378.6 ± 34.4 28.9 | 155.7 ± 18.7 22.3 | 205.5 ± 11.2 24.4 | 248.8 ± 30.5 25.8 | 492.0 ± 24.6 24.7 |
0.75 | 242.4 ± 30.2 26.9 | 350.7 ± 36.5 25.8 | 148.8 ± 20.2 21.4 | 199.6 ± 8.9 23.7 | 236.4 ± 21.8 24.5 | 406.6 ± 34.0 20.4 |
1.0 | 199.6 ± 23.4 22.2 | 326.4 ± 30.7 24.9 | 140.0 ± 16.7 22.1 | 190.2 ± 12.4 22.6 | 210.8 ± 32.3 21.8 | 421.3 ± 28.7 21.1 |
Control | 898.4 ± 48.8 100 | 1306.4 ± 72.2 100 | 696.4 ± 52.0 100 | 840.2 ± 54.4 100 | 962.8 ± 40.3 100 | 1990.8 ± 52.0 100 |
The findings presented in Table 1 indicate that the introduction of the sorbent into the rats resulted in a marked reduction in the distribution of 137Cs to organs and tissues, with an average decrease of 73%-80% compared to the control group. The sorption efficiency increased with increasing sorbent dose, suggesting a dose-dependent effect. However, no notable statistical differences were observed between the groups in this trend.
In a separate experiment aimed at comparing the sorption effectiveness of the ferrocyanide-bentonite sorbent Ferbensorb and the selective sorbent cesium-ferrocin, 15 rats (160-180 g) were assigned to three distinct groups. All animals were injected with 137Cs via a metal probe as a single dose in aqueous solution at 370 kBq/animal. In Group 1, rats received Ferbensorb at a dose of 1 ml per animal in a 50% aqueous suspension, immediately following the isotope injection. Group 2 rats were administered 150 mg of ferrocin per animal, while the 3rd group served as a control and received no sorbent.
After 7 days, all rats were sacrificed, and the 137Cs levels in their organs and tissues were assessed, as outlined in Table 2.
Table 2. The concentration of 137Cs in rat organs and tissues after administration of the sorbent Ferbensorb and ferrocene (in the numerator – Bq/g, in the denominator - % of control) (M ± m; n = 15)
Sorbent | Name of the organ or tissue | |||||
Liver | Kidney | Lung | Spleen | Heart | Muscle | |
Ferbensorb | 231.4 ± 32.6 20 | 348.5 ± 35.5 23.8 | 185.0 ± 21.4 23.4 | 205.7 ± 14.5 21.8 | 258.0 ± 37.4 22.6 | 124.3 ± 16.0 19.4 |
Ferrocene | 18.9 ± 2.7 1.7 | 27.6 ± 1.63 1.9 | 16.0 ± 2.2 2.2 | 19.3 ± 1.9 2.0 | 25.0 ± 1.9 2.2 | 10.2 ± 12.5 1.6 |
Ferrocene | 1110.0 ± 43 100 | 1465 ± 83.5 100 | 788.1 ± 52.9 100 | 939.8 ± 87.2 100 | 1140 ± 52.4 100 | 649.3 ± 73.9 100 |
Research findings indicate that when the ferrocyanide-bentonite sorbent Ferbensorb was administered at a dosage of 0.5 grams per animal, a significant reduction in the 137Cs concentration in the organs and tissues was observed, ranging from 81.6% to 72.7%. On the other hand, when ferrocin was provided at 0.15 g per animal, the reduction in 137Cs was even higher, ranging from 97.8% to 98.3%. These results confirm that Ferbensorb can effectively minimize the transfer of 137Cs from the digestive tract into the animal’s tissues, achieving results comparable to the selective sorbent cesium-ferrocin.
A further set of experiments focused on the absorption of strontium radionuclides, conducted on a cohort of 28 mongrel white rats weighing approximately 280 ± 12 grams each. The rats were divided into 7 groups of 4 animals each to ensure balanced body weights. The animals were injected with a solution of 90Sr nitrate (197 kBq per animal) using a metal probe, along with a suspension of sorbents in 1.5% starch gel. After three days, the rats were euthanized via decapitation, and bone samples were collected for radiometric measurements using the UMF-1500 device. The experimental setup and results are detailed in Table 3.
Table 3. The concentration of 90Sr in the rat skeleton after administration of ferrocyanide-bentonite sorbent Ferbensorb and manganese dioxide (M + m; n = 28)
Sorbent | % of the introduced isotope amount | Effectiveness (%) | |
type | Quantity (mg/animal) |
|
|
Control | - | 20.3 ± 4.2 | 100% |
Ferbensorb | 40 | 17.8 ± 4.2 | 12.9% |
Ferbensorb | 80 | 4.6 ± 0.8 | 40.9% |
Ferbensorb | 120 | 9.7 ± 0.8 | 52.6% |
manganese dioxide | 20 | 17.4 ± 2.8 | 14.6% |
manganese dioxide | 40 | 5.2 ± 1.2 | 31.8% |
manganese dioxide | 80 | 6.9 ± 0.9 | 68.7% |
The introduction of sorbents reduced the movement of 90Sr from the gastrointestinal tract to the rat skeleton. At minimal doses, both ferrocyanide-bentonite sorbent Ferbensorb and manganese dioxide showed similar levels of isotope accumulation in bone tissue. However, manganese dioxide exhibited significantly greater efficacy at 40 mg and 80 mg, reducing isotope retention by 2.5- and 1.3-fold, respectively, compared with Ferbensorb at equivalent doses.
A separate experiment evaluated Ferbensorb’s sorption properties during experimental associative mycotoxicosis in Wistar rats weighing 160-200 g. The rats were assigned to four groups of eight, with group 1 serving as the control and receiving standard food. Group 2 was fed a mixture of mycotoxins and 0.5% Ferbensorb (5 g per kg of feed), while group 3 received a mixture of mycotoxins with 1% Ferbensorb (10 g per kg of feed). Rats in the fourth group received only mycotoxins in their food. The mycotoxin mixture was added at a rate of 5 mg/kg of feed. Throughout the 30 days, animals were observed, weighed at the start and at mid-point, and euthanized for controlled analysis after 15 days. At the end of the experiment, the remaining rats were sacrificed for further examination.
The animals were carefully monitored for changes in behavior, general health, body weight, and the effects of mycotoxins, as well as the protective role of Ferbensorb. Pathological changes in the organs of deceased animals were noted, and blood samples from surviving rats were collected for analysis of immune response and enzyme activity.
The findings revealed that exposure to mycotoxins caused notable toxic effects in rats in group 4, which were fed contaminated food. Between days 6 and 9, these rats exhibited signs such as lethargy, reduced mobility, poor coordination, loss of appetite, unkempt fur, and excessive thirst. Conversely, the rats in the control group (group 1) and those fed Ferbensorb-containing food (groups 2 and 3) showed no signs of distress, maintaining normal activity levels and coat condition.
Starting on day 16, rats in group 4 developed rapid breathing, elevated heart rate, and increased body temperature (by 0.3–0.6 °C), with some individuals experiencing a 1 °C rise between days 16 and 22. After day 24, their body temperature gradually returned to normal levels. Several animals in this group exhibited poor food intake, and by days 16, 20, and 24, one rat from each of these days died. No such issues were observed in the rats in groups 1, 2, and 3, who showed no signs of toxicity.
As shown in Table 4, rats in group 4, which received mycotoxins without Ferbensorb, showed a significant delay in body weight gain, with three fatalities occurring. The rats in the control group, as well as those in groups 2 and 3, which received Ferbensorb along with the mycotoxins, gained 53.4, 58, and 64 grams, respectively. In contrast, the weight gain of rats in group 4 was 41.4 grams lower than that in group 1 and 46 grams and 52 grams lower than those in groups 2 and 3, respectively. This difference indicates the toxic impact of the mycotoxins on growth and overall body health. Rats in groups 2 and 3, however, showed no such signs and gained weight steadily, with group 2’s average daily gain exceeding that of group 1 by 4.6 grams, and group 3 exceeding group 1 by 10.6 grams.
The chronic form of associated mycotoxicosis in group 4 rats resulted in the death of three animals within 30 days, with an average survival of 20 days. However, the addition of Ferbensorb at 0.5% and 1% concentrations in the feed prevented any fatalities. Pathological examinations of the rats at the 15-day mark revealed no abnormalities in groups 1, 2, and 3. However, in group 4, rats showed significant organ changes: redness and hemorrhage in the stomach and small intestine, liver enlargement with irregular coloration, and a bile duct filled with yellow-brown bile.
Table 4. Dynamics of the course of associative mycotoxicosis in rats and the effect on their body weight gain with the introduction of the sorbent Ferbensorb, g (M + m; n = 32)
Before the introduction of toxins and sorbents | 15 days | 30 days | |||||||||
Group | Group | Group | |||||||||
1 | 2 | 3 | 4 | 1 | 2 | 3 | 4 | 1 | 2 | 3 | 4 |
157 | 175 | 150 | 125 | 193 | 225 | 197 | 186 | 210 | 221 | 211 | 151 |
161 | 175 | 151 | 131 | 194 | 207 | 195 | 257 | 215 | 212 | 226 | 190 |
142 | 144 | 155 | 185 | 189 | 245 | 217 | 201 | 237 | 248 | 242 | 172 |
153 | 190 | 165 | 181 | 187 | 186 | 183 | 170 | 201 | 190 | 197 | Fallen |
142 | 129 | 130 | 154 | 191 | 214 | 190 | 190 | 212 | 210 | 217 | Fallen |
169 | 130 | 182 | 138 | 193 | 192 | 190 | 178 | 207 | 202 | 201 | Fallen |
163 | 190 | 139 | 224 | 204 | 207 | 202 | 162 | Killed | Killed | Killed | Killed |
165 | 118 | 146 | 137 | 207 | 172 | 250 | 183 | Killed | Killed | Killed | Killed |
156.5 ± 10.2 | 156.4 ± 29.3 | 152.2 ± 15.9 | 159.4 ± 34.4 | 194.8 ± 7.07 | 206 ± 22.9 | 203 ± 21.5 | 190.9 ± 29.3 | 213.7 ± 12.4 | 213,8 ± 19,7 | 215,7 ± 16,7 | 171 ± 19,5 |
Body weight gain | +34 | +50 | +51 | +32 | +53.4 | +58 | +64 | +12 | |||
During the course of the study, rats in the 4th group experienced fatalities on the 16th, 20th, and 24th days. Upon examination of the deceased animals, notable symptoms, including bluish discoloration of the oral and nasal mucosa, redness of the conjunctiva, and fur contamination, were observed. In the post-mortem examination, the following findings were recorded:
The lungs appeared bright red, with pink frothy fluid present in the tracheal and bronchial passages. The liver was enlarged, soft, and unevenly colored, with cherry-like spots and areas of tissue death. The bile duct was filled with a yellow-brown liquid. The kidneys had slight swelling, hemorrhaging, and poorly defined boundaries between the cortical and medullary regions. The ureters were enlarged, and the bladder exhibited visible streaks of blood.
The stomach was distended, with hemorrhages along its fundal mucosa. The small intestine showed scattered hemorrhagic spots in its mucosa, while the large intestine contained air bubbles. The heart appeared enlarged, with a soft texture, and minor hemorrhages were observed on the epicardium. The heart cavities had poorly clotted blood.
Histological evaluations of liver, spleen, and heart tissues showed the most significant damage in the liver, where the normal structural arrangement was disrupted, with signs of cell death and degeneration. The lungs exhibited congestion, with capillary dilation extending into alveolar spaces. Accumulation of eosinophilic substances, along with red blood cells and epithelial cells, was observed in the alveoli.
In the spleen, vascular hyperemia and loss of differentiation between the red and white pulp were apparent, and a reduction in both the pulp size and follicle number was noted. The follicles displayed sparse lymphocytes with pale, small nuclei. Kidney tissue exhibited granular dystrophy of the renal tubule epithelium, with some areas showing sloughing.
In the control animals and those from experimental groups 2 and 3, no significant changes in gastrointestinal or parenchymal organs were observed. However, in the 4th group exposed to mycotoxins, visible changes, such as redness and hemorrhagic spots, were observed in the stomach and small intestine. The liver was enlarged, discolored, and showed bile duct congestion, while the kidneys and bladder displayed signs of hemorrhage.
The impact of the toxins on immune parameters was further assessed by analyzing blood leukocyte composition in groups 2, 3, and 4. A noticeable reduction in banded neutrophils was observed in these groups compared with the control (group 1). Group 4 also displayed a significant decrease in segmented neutrophils. Furthermore, the proportion of lymphocytes in the blood of group 4 was significantly higher than that in group 1, with a more moderate increase in the other experimental groups (Table 5).
Table 5. The effect of the sorbent Ferbensorb on the leukocyte formula of rat blood (M + m; n = 32)
Group | Neutrophils | Eosinophils | Monocytes | Lymphocytes | ||
Young | Rod-shaped | Segmented | ||||
1 Control | 0.0 | 1.5 ± 0.22* | 30.6 ± 2.48* | 4.2 ± 0.49 | 3.4 ± 0.5 | 58.3 ± 2.03 |
2 Toxins + 0.5%Ferbensorb | 0.0 | 0.2 ± 0.17х | 27.7 ± 1.61* | 3.6 ± 0.87 | 3.5 ± 0.56 | 65.0 ± 2.08 |
3 Toxins + 1% Ferbensorb | 0.0 | 0.5 ± 0.22х | 34.8 ± 1.68* | 2.7 ± 0.42 | 4.5 ± 0.4 | 57.52 ± 2.14 |
4 Toxins | 0.0 | 0.5 ± 0.22х | 13.7 ± 1.26х | 3.2 ± 0.54 | 2.9 ± 0.65 | 79.7 ± 1.08х |
The physiological norm | 0 | 2.0 (1-4) | 26.5 (20-35) | 1-5 | 1-5 | 55-75 |
Note: x 2,3,4 groups compared to the 1st group, P < 0.001; *1, 2, 3 groups compared to the 4th group, P < 0.001.
The bactericidal capacity of the blood serum from rats exposed to toxins (group 4) was significantly lower than that of the control animals (group 1), as shown in Table 6. When the sorbent was added to the feed at a concentration of 0.5% (group 2), there was no noticeable improvement in bactericidal activity. However, increasing the sorbent dose to 1% (group 3) resulted in a significant enhancement of bactericidal activity (P < 0.001).
As for lysozyme activity, rats in group 4 (treated with toxins) exhibited a decrease compared to the control group (group 1). In contrast, the lysozyme levels in rats receiving the sorbent (groups 2 and 3) remained unchanged. Notably, rats in group 3, which received feed containing 1% sorbent, showed a significant increase in lysozyme activity compared with those in group 4.
Table 6. Effect of the drug Ferbensorb on bactericidal activity and lysozyme activity of rat blood serum against the background of mycotoxicosis (M + m; n = 21)
Indicator | Group | |||
1 | 2 | 3 | 4 | |
Bactericidal activity (%) | 57.03 ± 0.38 | 47.63 ± 1.76 х | 62.58 ± 0.96х* | 49.36 ± 1.61 х |
Lysozyme activity (%) | 65.26 ± 0.74* | 61.28 ± 0.43 | 64.25 ± 0.63* | 59.27 ± 0.86 х |
Note: x 2,3,4 groups compared to group 1, P < 0.001, * 1, 3 groups compared to group 4, P < 0.001
Exposure to food contaminated with fungal toxins resulted in significant health deterioration in rats, evidenced by diminished appetite, increased water intake, gastrointestinal disturbances, a slight rise in body temperature (0.3-0.6 °C), conjunctival redness, poor fur quality, and inhibited growth.
Autopsy findings of the deceased animals revealed significant damage to various organs involved in detoxification (liver), excretion (kidneys), digestion (intestines), as well as immunological organs, the heart, and lungs.
The ferrocyanide-bentonite sorbent, Ferbensorb, played a crucial role in mitigating the adverse functional and structural changes induced by mycotoxins, including ochratoxin A, fumonisin B, and zearalenone. This intervention promoted weight gain, strengthened the immune system (as indicated by an increase in neutrophils in the leukocyte profile), enhanced bactericidal and lysozyme activities in the blood, and contributed to improved survival rates in the affected animals.
In rat models, it was demonstrated that the ferrocyanide-bentonite sorbent Ferbensorb effectively reduces the movement of 137Cs from the gastrointestinal tract into organs and tissues, with results comparable to those of the cesium-ferrocin selective sorbent.
When administered at low levels, both Ferbensorb and manganese dioxide led to similar levels of the isotope in the bones. However, manganese dioxide exhibited superior performance, being 2.5 times more efficient at a 40 mg dose and 1.3 times more effective at an 80 mg dose than Ferbensorb at equivalent amounts.
Furthermore, Ferbensorb was highly effective at mitigating the detrimental effects of mycotoxins, including ochratoxin A, fumonisin B, and zearalenone. This was reflected in enhanced body weight gain and improved immune function, as evidenced by increased neutrophil counts in the leukocyte formula and elevated bactericidal and lysozyme activity in the blood, ultimately contributing to improved survival in the test animals.
All authors contributed to the study’s design, analysis, and writing process.
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The research followed the ethical guidelines outlined in the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes.
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