Danylo Halytsky Lviv National Medical University, Lviv, Ukraine

SUMMARY. Introduction. The potential danger of combined effect of lead and fluorine to human health motivates the search and experimental substantiation of physiologically acceptable ways of bioprophylaxis directed to preventing or reducing the negative effect on the organism.

Objective: the study of the dynamics of the metabolic and functional state of the liver under conditions of combined effect of lead and fluorine without application of bioprotectors and on the background of bioprotectors application.

Material and Methods. 4 series of researches were carried out on white rats, each according to the orthogonal planning scheme 2². Lead and fluorine were administered by intragastric within 30 days: in the first series — without bioprotectors application, in the second series — on the background of pectin application, in the third series — pectin and calcium application, in the fourth series — the complex of pectin, calcium and triovit application. The level of diene conjugates, the active products of thiobarbituric acid, the activities of superoxide dis-mutase and catalase, the index of general antioxidant activity in the liver tissue, the activities of alanine aminotransferases, aspartate aminotransferases and alkaline phosphatase in blood serum, concentration of urea were determined at the 15th and 30th day of the experiments, the integral coefficient of antioxidant defense and the type of combined action were calculated.

Results. The combined effect of lead and fluorine leads to an imbalance between the indicators of lipoperox-idation and antioxidant activity of the liver tissue, the increased activities of alanine aminotransferases and aspartate aminotransferases, the phase changes in alkaline phosphatase activity, and urea concentration in serum. The application of pectin did not affect the indicators. Pectin and calcium have improved the mechanisms of antioxidant system and the activity of enzymes. The complex of pectin, calcium and triovit normalizes metabolic processes in the liver tissue and improves its functional state. The type of the combined action depends on the registered effect, the duration of the stress factor and the applied bioprotektor.

Conclusion. The optimal prophylactic effect under the combined action of lead and fluorine is achieved with the help of a complex of pectin, calcium and triovit.

Key Words: lead, fluorine, combined effect, pectin, calcium, triovit, indicators of lipid peroxidation, antioxidant defense, enzyme activity.

Combined effect (CE) of chemical substances is one of the real risk factors for environment-associated disease and syndromes of ecological maladaptation. Of all chemicals, lead is a ubiquitous heavy metal pollutant found in virtually all world regions. Principal sources of biosphere lead pollution include motor vehicles and metallurgical and chemical plants. Lead is characterized by strong cumulative properties and polytropic action, which is particularly dangerous for general health and mental development of paediatric populations [1, 9, 10, 16].

From the viewpoint of human ecology, lead is a comparatively new chemical agent, since its extensive use began in the mid-19th century. Since then, environmental levels of lead have grown substantially. The main anthropogenic sources of fluoride pollution are coal mines, coal power plants, and industrial emissions from the manufacture of aluminium, glass, ceramics, porcelain, steel and superphosphate mineral fertilizers, and household emissions. Biogeochemical regions with increased levels of fluorine are present in virtually all countries of the world. Fluorine and fluorine compounds are quite common in the biosphere, including the atmosphere, water, plants, soil and living organisms. The content of fluorine in soil, water and food products depends on local biogeochemistry and on human activity [2,9,10,12,15-17]. A. P. Avtsyn et al. [9] consider fluorine to be a conditionally essential microelement. Fluorine is one of the elements with a narrow range between safe and toxic doses. Safe levels of daily consumption are 0.1 to 2.5 mg/day for children (depending on age) and up to 4 mg/day for adults. Above safe levels, fluorine is a polyenzymatic poison, which may affect all organs and systems in the body. Lead and fluorine may enter human body concomitantly with water, food products, atmospheric air; this may happen not only in environmentally compromised areas but in relatively “clean” areas as well. There are chances of additional fluorine burden with toothpaste. With the absence of protective (prophylactic) interventions, adaptive mechanisms are always compromised with resulting environmentally-associated disease. Therefore, to ensure proper adjustment in a setting of current altered environment, there is a need for medical and biological prophylaxis (correction) in order to increase resistance of the body and to regulate adaptation and compensation mechanisms using physiologically acceptable, harmless and effective modalities aimed at specific and general modes of action of chemical stressors [4]. This directly concerns reduction (prevention) of the concomitant toxic effect of lead and fluorine. Pectin prevention is known for its preventive effects in lead exposure and calcium is beneficial with fluorides; antioxidants and adaptogens are used as general antitoxic modalities.

The objective is to study how parameters of a metabolic and functional status of the liver (as the main metabolic and detox organ) change with time in prolonged concomitant exposure to lead and fluorine with and without the use of bioprotectants.

Materials and methods of the study. The testing was conducted in albino rats weighing 170–200 g, using a 2² scheme of orthogonal planning with adherence to general ethical principles of animal experimentation. Aqueous solutions of Pb(NO₃)₂ (lead) at a dose of 36 mg/kg of body weight (1/100 of the median lethal dose) and NaF (fluorine) at the dose of 10 mg/kg of body weight were administered to fasted animals via a gastric tube for 30 days. Control group received drinking water. To prevent in vitro formation of lead fluoride, the substances were administered in a “lead–fluorine” sequence 1.5 to 2 hours apart. The following test series were assessed: Series I (functional status of the liver exposed to lead and fluorine), Series II (lead and fluorine with added dietary pectin), Series III (lead and fluorine with added dietary pectin and calcium) and Series IV (combination of pectin, calcium and Triovit). Laboratory animals were kept under standard conditions with unrestricted access to water. Investigational biological protectants included pectin (1 g/kg of body weight), calcium (officinal calcium gluconate 225 mg/kg of body weight), Triovit vitamin/mineral supplement manufactured by KRKA, Slovenia (1 capsule/kg of body weight: vit. С 100 mg, vit. Е 40 mg, β-carotene 10 mg and selenium 50 μg). On Days 15 and 30 (series І and ІІ) and on Day 30 (series ІІІ and IV) of the experiment, levels of diene conjugates (DC) [3], thiobarbituric acid (TBA) active products [11], activities of superoxide dismutase (SOD) [8] and catalase (CT) [7] and index of total antioxidant activity (І_АОА) [6] were assessed in hepatic tissue; serum tests included activities of alanine- and aspartate aminotransferase (ALT/AST) (additional testing on Day 3 of the experiment), alkaline phosphatase and urea using LACHEMA Bio-La-Test kits. Integral assessment of antioxidant status was performed using K, an integral coefficient based on the ratio of the activity indices of antioxidant system and the intensity of lipid peroxidation since it is the ratio of the aforementioned indices (not individual indices apart) to determine the intensity of metabolic processes [5]. Statistical processing was performed with the least squares method with significance assessed with Student’s t-test using Microsoft Excel software. The integral effect was assessed based on the resulting regression equations: у=b_o+b₁x₁+b₂x₂+b₁₂x₁x₂, where у stands for effect, b_o b₁, b₂ b₁₂ stand for regression coefficients and х₁ and х₂ are dose codes.

Results and discussion. In this study, the combined effect of lead and fluorine on Day 15 of the experiment caused an increase in DC- and TBA-active products in hepatic tissue with a simultaneous increase in SOD activity and a reduction in catalase activity (Fig. 1.). The processes of lipid peroxidation (LPO) are part of normal metabolic processes and LPO is one of the important adaptive responses. However, acceleration or inhibition of LPO leads to changes in

Fig. 1. The levels of lipid peroxidation products and the activity of antioxidant protection enzymes ( % deviation from control) in hepatic tissue in a combined effect of Pb(NO₃)₂ and NaF during 15–30 days with and without added pectin or pectin + calcium or pectin + calcium+ Triovit combination.

 Notes: significant (р < 0.05); 1. а = relative to controls; 2. b = relative to Day 15 of exposure Pb (NO₃)₂; 3. с = relative to combination effect without bioprotectants.

the structure, composition and functional activity of the membrane and the respective cellular activity. Counteraction against the intensive formation of free radicals is determined by the antioxidant status of cells (in part, the activity of SOD, catalase, etc.). Increased activity of SOD can be regarded as a protective response; however, the reduced activity of catalase suggests an increased formation of reactive oxygen species and hydroperoxide radicals. Inhibited synthesis of TBA-active products (compared to Day 15) and inhibited DC synthesis in a setting of reduced anti-radical and anti-(per)oxidation protection (AOP) were demonstrated to be a specific feature of prolonged concomitant exposure to lead and fluorine. This may suggest depletion of lipid peroxidation, profound dystrophic energy-deficient states in the cells, inhibition of microsomal oxygenases (lead belongs to group IV of monooxygenase system inhibitors) and accumulation of toxic underoxidized products. The patterns of changes in LPO and AOP suggest the escalation of oxidative stress with effects of lead and fluorine to the end of the experiment, impaired metabolic status and the mechanisms of adaptation of the stress-limiting system of antioxidant protection of hepatic tissue. Previously the author has found that individual effects of lead and fluorine also cause changes in the LPO/AOP system [13,14]. Taking into consideration the corresponding alterations of test parameters in a setting of individual and combined effect of lead and fluorine, it was found that in terms of DC levels in hepatic tissue on Day 15 of the experiment the CE of lead and fluorine is interdependent and unidirectional and is characterized by additivity with a trend to more than additive action, as suggested by the regression equation: у = 30.15 + 15.27х₁ + 16.53х₂ + 1.7х₁х₂, where у is the increment of DC compared to control by a corresponding percentage ( %) and х₁ and х₂ are the dose codes of lead and fluorine. The increment in TBA-active products in a setting of CE is also greater than the anticipated sum of respective individual effects of lead and fluorine. Judging by the regression equation: у = 32.40 + 18.45х₁ + 20.15х₂ + 6.20х₁х₂ the type of CE may be evaluated as “more than additive” (CE coefficient = 1.2). As for SOD activity in hepatic tissue, there is formal desensitization (reduced activity with a “lead + fluorine” CE as opposed to the intact activity when exposed to lead) and in a setting of reduced catalase activity below control levels, such pattern of combined effect has a negative impact on the maintenance of homeostasis. A formal assessment of the CE type for lead and fluorine in terms of various effects detects different CE types. However, from the pathogenetic point of view, development of processes within the LPO/AOP system should be assessed as a whole. Taking into consideration the inhibition of these processes, the CE effect may be viewed as an interdependent effect and the main type of CE is synergy with a “more than additive” effect.

Changes of ALT/AST serum activities with time were characterized by increases with undulant alterations from Day 3 to Day 30 of the experiment: ALT activity was elevated compared to controls on Day 3, Day 15 and Day 30 of the experiment: 2.1 times, 1.9 times and 4.2 times, respectively; AST was elevated 1.5, 1.4 and 2.3 times, respectively (see Table).

Table

The pattern of changes with time in parameters of the functional state of the liver in albino rats (М ± m) on Day 30 of testing in conditions of daily combined exposure to lead and fluorine both without bioprotectants and with the sequential addition of pectin, pectin + calcium and with a combination of pectin, calcium and Triovit

Note: а = significant relative to controls

The nature of CE is different at the onset of an experiment to its end; it changes from additivity and independent effect of lead to an interdependent and unidirectional mutual augmentation of effects: у = 137.0 + 96.97 х₁ + 62.33х₂ + 22.27х₁х₂ (Day 30), which speeds up the adaptation breakdown phase. The leading role in the development of combined effect belongs to lead (at virtually all follow-up time-points), which may be attributable to hepatotoxic effects of lead, its cumulative properties and accumulation in hepatic tissue. On Day 15, the activity of alkaline phosphatase (AP) increased 1.7 times compared to the control group. However, on Day 30 of the experiment, the activity of the enzyme decreased by 20.5 % of the control. Phasic nature was also inherent for patterns of changes in serum urea levels: by Day 15, the concentration decreased by 42.9 % and, contrariwise, by Day 30 it increased 70 % compared to the control group. Changes in serum activity of transaminases characterize hepatic cell damage and release of enzymes into the blood flow. The oppositely directed AP changes at various follow-up time-points suggest an aggregate of abnormalities: liver injury and impaired formation and excretion of bile, impaired calcium and phosphorus metabolism and impaired metabolism of the main components of the organic matrix of the bone system, which is typical for effects of lead and fluorine. Urea is known to be synthesized in the liver from ammonia, the latter formed via deamination of amino acids and disintegration of nucleic acids and pyrimidine nucleotides. Apparently, the reduction in serum urea levels at Day 15 of the experiment may be attributable to reduced synthesis due to liver injury; the increase in urea levels at Day 30 of the experiment may suggest renal function impairment caused by lead and fluorine.  These results suggest damage to hepatocytic cellular membranes and to hepatocytes per se, impaired metabolic and detoxification mechanisms and functional status of the liver, as well as a phasic nature of adaptive and compensatory processes with the greatest liver injury observed at the end of the experiment.

Adding pectin to the diet (test Series ІІ) produced an uneven influence on different investigational CE-effects of lead and fluorine. At Day 15 of the experiment, the level of DC has somewhat decreased, with improved catalase values in hepatic tissue; however, the nature of CE (as assessed by the levels of DC and TBA-active products) remained more than additive. The protective role of pectin was evident by the reduction in the effects of lead. At Day 30 of the experiment, the level of oxidative stress in CE of the substances remained at the same level as with no pectin prophylaxis. The use of pectin has contributed to reduced transferase activity in blood compared to Series 1 of the experiment. The mean combined effect has decreased owing to the reduced effect of lead; the nature of CE remained virtually unchanged: у = 70.75 + 39.25х₁ + 50.75х₂ + 19.25х₁х_2.  There was less pronounced AP activity at Day 15; however, pectin had no effect on the reduction of its activity at the end of the experiment. The use of pectin improved urea values, with no impact on the direction of the changes; the pattern of CE changed to a “less than additive” effect, which can be viewed as a positive trend. Therefore, the protective role of pectin (as assessed by investigational parameters) was found to be of low efficacy in a setting of CE of lead and fluorine.

The combination of pectin and calcium were conducive to the improved functional status of the liver (Series ІІІ). This combination prevents intestinal adsorption of lead, its deposition in the body and facilitate the elimination of lead from the body. Calcium ions are synergists of SOD; they act as inhibitors of the О₂ˉ-generating system by binding with the anion radical of oxygen. Calcium is known to inhibit the absorption of fluorine in the intestines and to reduce fluorine levels in the blood. Dietary supplementation of calcium partially improves metabolic processes in hepatic tissue. In this series, the levels of lipid peroxidation products have decreased. The levels of TBA-active products exceeded the respective levels in controls by not more than 18 %; the synthesis of DC remained inhibited; these substances were detected at the levels below control values. However, catalase activity was increased, but in a setting of reduced SOD. Therefore, reactive oxygen species are still being generated, although the intensity of this generation is probably already low, according to the assessment of reduced SOD activity in hepatic tissue.  The activity of transferases was reduced compared to the results obtained without added pectin and calcium. The regression equation at Day 30 of the experiment (with added pectin and calcium) suggests a unidirectional effect with a trend towards interdependent mutual attenuation: у = 26.33 + 13.03х₁ + 10.65х₂ – 2.67х₁х₂ (ALT).   The CE of lead and fluorine in a setting of calcium with pectin caused a contra-directional response on the part of alkaline phosphatase activity; namely, the AP activity was increased compared to the activity of the enzyme in a test without added calcium (when AP activity was reduced). Adding calcium had little effect on serum urea levels.

Triovit with pectin and calcium (Series ІV) regulates pro-oxidant/antioxidant balance in hepatic tissue. Owing to its four mutually balanced components, Triovit supports multiple mechanisms of antioxidant action.  Compared to test Series ІІІ, the level of TBA-active products in hepatic tissue decreased to the limits of fluctuations of control values. No DC inhibition below control level has been documented. The SOD activity in hepatic tissue still had a downward trend. Such findings may suggest an unsteady LPO-AOP balance. In turn, this is confirmed by changes in catalase activity. Catalase activity remains elevated in hepatic tissue by 23.6 % compared to the control group, which may be viewed as a protective effect. However, the use of such combination clearly manifested its bioprotectant capacities for prevention of abnormalities within the LPO-AOP system. This is confirmed by changes with time in values of K, the integral coefficient of antioxidant status, i.e. from 0.23 (without the use of bioprotectants) to 0.84 (with the addition of pectin and calcium) and to 1.06 (in the setting of a complex bioprotectant combination). Adding Triovit to the diet recovered indices of serum transaminase activity to virtually normal. In all cases, AST activity was within the level of controls; ALT was still elevated 14.8 % compared to controls. AP activity remained virtually at the level of the previous test; the increase in activity was 26.4 %, which may suggest incomplete recovery of hepatic function and calcium-phosphorus metabolism. A somewhat increased BUN was reported; however, these changes were less pronounced compared to the results obtained without added Triovit.  Improvement of impaired metabolic and functional state of the liver by using a combination of pectin, calcium and Triovit targets the toxicodynamics and toxicokinetics of lead and fluorine.

CONCLUSIONS

1. Concomitant oral exposure to lead nitrate at a dose of 36 mg/kg of body weight and sodium fluoride at a dose of 10 mg/kg of body weight for 30 days leads to metabolic and functional changes in hepatic tissue, which manifested themselves as disequilibrium between the indices of lipid peroxidation and antioxidant activity of hepatic tissue, increased activity of alanine and aspartate aminotransferases and as phasic changes in the activity of alkaline phosphatase and serum urea levels.
2. The protective role of pectin in the prolonged combined effect of lead and fluorine was shown to be insufficient. Supplementing the diet with pectin and calcium contributes to the improvement of metabolic and functional status of the liver. An optimal corrective (preventive) effect is attained with a combination of pectin, calcium and Triovit, which activates the mechanisms of the stress-limiting antioxidant system, positively impacts serum enzymatic activity, restores normal metabolic processes in hepatic tissue and adjusts its functional state.
3. Using of mathematical planning in the experiment allowed assessing the type and the changes with the time of the combined effect of lead and fluorine with and without sequential addition of bioprotectants. The type of combined effect was found to depend on the documented effect, the duration on xenobiotic exposure and the bioprotectant used; this effect may be used as an assessment criterion of how adaptive and compensatory processes develop with time in a setting of correction of maladaptive conditions caused by a combination of substances.

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Надійшла до редакції: 11.06.2018 р.