Biomarkers of exposure and effect of organophosphorus compounds (literature review and results of own studies)

  • Authors: O.V. Fedchenko, P.G. Zhminko
  • UDC: 615.9+615.917+616-07
  • DOI: 10.33273/2663-4570-2018-84-4-19-35
Download attachments:

State Enterprise “L. I. Medved’s Research Center of Preventive Toxicology, Food and Chemical Safety of the Ministry of Health of Ukraine”, Kyiv, Ukraine

ABSTRACT. Objective: analysis and summary of the literature data and own studies on the possibility of using biomarkers of exposure and effect of organophosphorus compounds (OPCs), determination of immunological biomarkers of the effect under exposure to OPCs that cause delayed neurotoxicity (DN).

Materials and Methods. Analytical methods were used in the work: collection of scientific information on the topic, analysis of data and scientific summary of the results. Re-assessment of the effect of OPCs causing DN was performed from the perspective of determination of the most informative immunological biomarkers of the effect of known neurotoxicants. Analysis of the study results of the effect of neurotoxic OPCs, triorthocresyl phosphate (TOCP), Afos on the immune system was performed using the most sensitive model — chicken breed Leghorn in isotoxic doses (500 mg/kg and 200 mg/kg, respectively) using common immunotoxicology methods.

Results and Conclusions. The article analyses and concludes current literary data on the justified use of biomarkers of exposure and effect of OPCs that are widespread in the environmental objects and are one of the reasons of acute and chronic poisoning in the population. We’ve reviewed aspects of the necessity for implementation of scientifically justified approach to the development and assessment of biomarkers and creation of the unified diagnostic complex that includes biomarkers of exposure, specific and non-specific action, with wider diagnostic abilities compared with determination of isolated parameters that is the basis for diagnostics, efficient treatment and prediction of consequences of poisoning with OPCs. Based on the results of own studies, we proposed the use of some parameters as the immunological biomarkers of the effect of OPCs with DN: the level of finely dispersed circulating immune complexes in the blood serum and the level of anti-brain antibodies, the number and functional activity of blood neutrophils; the number of T-and B-lymphocytes and their functional activity; the number of NK-cells, T-helper cells, and T-suppressors.

Key words: organophosphorus compounds, exposure, delayed neurotoxicity, diagnostics, biomarkers.

Organophosphorous compounds (OPCs) is a large group of chemical substances that represent esters, amides or thiol derivatives of phosphorous, alkyl phosphorous, thiophosphoric and thiophosphonic acids. OPCs are widely used for many years: in agriculture — for protection of plants and animals as insecticides, acaricides, nematocides and herbicides, in a chemical industry — as solvents and plasticisers, in a pharmaceutical industry — as components of the medicinal products, in a military sector — as chemical warfare agents [1-6].

According to WHO, 3 mln people annually suffer from acute poisoning with OPCs, and for 200,000 people poisonings are fatal [5, 7].

During the last two decades, organophosphorus pesticides are directly the main reason of acute and chronic poisoning with pesticides among the agricultural workers, among population following household use as insecticides, as well as following accidental or deliberate use that requires emergency medical care, precise diagnostics, effective treatment, and prediction of consequences of intoxication [4, 6, 8-11].

OPCs are pesticides that are most actively used all over the world, therefore their residues and metabolites are widespread in the environmental objects, worsening total burden of the negative effect of chemical factors on human health and ecological systems [2, 4, 6, 8, 12, 13].

WHO states that people are exposed to OPCs through consumption of food, beverages and during inhalation of the polluted area (Fig. 1) [13].


Fig. 1. Routes of exposure to OPCs (adapted from WHO, 2001) [13].


For example, the results of a recent study of the interaction between peculiarities of nutrition of Canadian population (5,604 subjects aged 6 to 79 years) and concentrations of OPCs metabolites (dialkyl phosphates) in urine suggest that more than 90 % of participants had at least one type of dialkyl phosphates in urine samples. Furthermore, direct interaction was found between the number of consumed vegetables and concentration of dialkyl phosphates confirming that effective regulation of the use of pesticides is a guarantee of the reduction of their negative effect and associated risk for public health [14].

Therefore, under conditions of real global threat to human livelihood, active measures are required to minimise the risk of the action of hazardous chemical factors, which including among others OPCs [4, 8, 15]. Furthermore, increased activity of international terrorism enhances the risk of use of both known organophosphorus poisoning substances and new compounds with unknown chemical structure and justifies the necessity of scientific developments of the strategies for effective protection of public health in case of possible exposure to organophosphorus agents [3, 16].

To assess the exposure and risk for exposed subjects, biomonitoring — systemic constant or regular measures of biosampling for analysis of concentrations of xenobiotics, their metabolites or determination of specific parameters of biological effects of so-called biomarkers is important [7, 17, 18].

Determination of unchanged OPCs and their metabolites in the biological matrices (blood, urine, etc.) is performed to confirm exposure in case of acute poisoning, this method, however, have limitation associated with the fact that unchanged OPCs rapidly disappear from the blood and are eliminated renally from the body [19].

Conventional biomarkers of OPCs intoxication that are widely used — determination of activity of acetylcholinesterase (AchE) and butyrylcholinesterase (BChE), are specific only in early terms of intoxication, and the number of scientific works on the determination of sensitivity, specificity and predictive value of these parameters is limited. Furthermore, under chronic intoxication with low OPCs doses, the presence of clinical symptoms may not be accompanied by the cholinesterase inhibition, therefore, the level of activity of these enzymes cannot be a universal biomarker of intoxication. Also, some age-related changes and peculiarities of the body, presence of concomitant acute and chronic diseases and effect of different exogenous factors may be accompanied by non-specific changes of the range of biochemical parameters, including the reduced activity of cholinesterase that is sometimes erroneously treated as signs of OPCs intoxication [20].

The aim of any diagnostics is the detection of impaired body condition that is suggested by changes in the complex of haematological, biochemical, immunological and other parameters-biomarkers. These data are required for further adjustment of detected impairments and prediction of consequences. In case of intoxication with xenobiotics, including OPCs, changes in various parameters (sometimes some of them may be within normal or limit values) are observed; however, analysis of these parameters-biomarkers in total may significantly improve quality of diagnostics due to the higher sensitivity and specificity in comparison with the determination of certain parameters. Since in the current science, much attention is paid to the search of new isolated markers, there is a necessity to create a complex of integral biomarkers of exposure and effect that reflect the interaction between different body systems and have higher diagnostic and predictive value [7, 20].

It is stated that the process of searching biomarkers is commonly indiscriminate since their selection and determination of validity is a complex unregulated process that differs for different biomarkers [7, 20]. For example, in 2002 National Cancer Institute (USA) proposed a new science-based approach to the development and assessment of biomarkers with gradual switch from stage I to V. Stages include non-clinical studies for identification of biomarkers, clinical study for assessment of reproducibility of the results in different laboratories, determination of sensitivity and specificity, further confirmation and clarification of these parameters using a large population of patients, then the final assessment of all advantages and disadvantages of biomarker should be performed with justification of economic expediency of its use [20].

It should be noted that inadequate attention is paid to the biomarkers of OPCs toxicity not associated with their anticholinesterase action. Under delayed neurotoxicity (DN) and chronic intoxications with OPCs, when a specific action is preceded (or manifest throughout the period of intoxication) by changes in protective body systems, namely immune system, determination of such biomarkers would have important predictive value.

Objective: Analysis and summary of the literature findings and own studies on the possibility of using biomarkers of exposure and effect of OPCs, determination of immunological biomarkers of the effect under exposure to OPCs that cause DN.

Materials and methods. The following analytical methods were used in the work: a collection of scientific information on the topic, analysis of data and scientific summary of the results. Re-assessment of the effect of OPCs causing DN was performed in previous studies from the perspective of determination of the most informative immunological biomarkers of the effect of known neurotoxicants. Results of the effect of neurotoxic OPCs on the immune system were analysed: triorthocresyl phosphate (TOCP) and O,O-diphenyl-1-acetoxy-2,2,2-trichloroethyl phosphonate (Afos) that were performed on the most sensitive model — Leghorn chickens in isotoxic doses (500 mg/kg and 200 mg/kg, respectively). In the conducted study, parameters of quantitative and morphological composition of peripheral blood, parameters of cellular and humoral immunity were measured using common methods of haematology and immunotoxicology: the number of T-lymphocytes, B-lymphocytes, T-helper cells (Th), T-suppressors (Ts) and NK-cells, functional activity of T-lymphocytes, B-lymphocytes and neutrophils; the level of circulating immune complexes (CIC) in the blood serum and their dispersive capacity were determined, auto-antibody titres to anti-nervous tissue antigens were measured by Boiden’s indirect haemagglutination test.

Results and discussion. According to the literature, organophosphorus compounds according to their chemical structure belong to thiophosphoric acid ethers (thiophos, metaphos, mercaptophos), dithio phosphorous acid ethers (carbophos, phosphamide), pyro phosphorous acid amides (octamethyl), alkyl phosphorous acid ethers (chlorophos) [3, 23, 24]. Depending on differences in a phosphorous group, three main groups of compounds are assigned: phosphates (without sulphur atom), phosphorothioates (with one sulphur atom), and phosphodithioates (with two sulphur atoms). The general schematic formula of the chemical structure of the majority of OPCs is as follows [24]:

where R 1 and R2 are alkyl, alkoxy, alkylamino, aryl or an aryloxy group; group X is the residue of non-organic or organic acid bounded directly to phosphate, or different aliphatic aromatic or heterocyclic groups that bind to phosphate via oxygen or sulphur; oxygen or sulphur may be the atom with dual bound. Compounds in which phosphorous is bound to an oxygen atom (P = O) have the highest anticholinesterase activity and toxicity than compounds with a sulphur atom (P = S). Higher toxicity of substances with P = O bound is characterised by lower lethal doses and more rapid development of their toxicity [22].

By their toxicity, OPCS are divided as follows:

1. Extremely toxic substances (LD50 — 10–50 mg/kg) — thiophos, mercaptophos, methylethylthiophos.

2. Highly-toxic substances (LD50 — 50–200 mg/kg) — methylmercaptophos, phosphamide, dichlofos, bazudin, antio, cidial, phthalophos, benzophosphate.

3. Moderately-toxic substances (LD50 — 200–1,000 mg/kg) — chlorophos, methylnitrophos, karbofos, trichlorometaphos-3, sayfos.

4. Low-toxic substances (LD50 — over 1,000 mg/kg) — vinyl phosphate, bromophos, abat, cyanox, valexon, demufos [1].

An isolated group is presented with chemical warfare agents (LD 50 is less than 10 mg/kg) — sarin, soman, VX [3, 25, 26].

Mechanism of toxic action of OPCs is realised through the similarity of their structure with neuromediator acetylcholine — natural AChE substrate. After it reaches the active region of AchE, OPC interaction with the enzyme is presented as phosphorylation of hydroxyl serine that results in AchE inactivation, accumulation of acetylcholine in cholinergic synapsis, hyperstimulation of receptors, impairment of normal neurotransmission and further blockade of neurotransmission through synaptic membrane [27] (Fig. 2).


Fig. 2 Pathophysiological mechanisms of acute toxicity of organophosphorus pesticides [27].


Acute OPC toxicity is associated with AchE inhibition. Anticholinesterase effect depends on OPC dose — the higher the dose of anticholinesterase substance is, the higher the degree of AchE inhibition in nervous cell and severity of intoxication. Clinical symptoms of acute intoxication with OPCs are manifested during the first day or within several days (in case of lipophilic OPCs) as a cholinergic crisis due to AchE inhibition and accumulation of acetylcholine in nervous endings [9, 24].

Symptoms of cholinergic crisis in OPC poisoning are the following: muscarine effects (nausea, vomiting, diarrhoea, abdominal cramps, urinary incontinence, myosis, bronchorrhoea, bronchial constriction, respiratory failure, salivation, lacrimation, hypotension, bradycardia, arrhythmia); nicotine effects (muscular weakness, tremor, weakness and paresis of diaphragm, hypertension, tachycardia, mydriasis); changes in the central nervous system (anxiety, agitation, headache, dizziness, ataxia, loss of consciousness, seizures, coma).

The first signs of cholinergic symptoms occur when blood AchE activity reduces to 50 %, and inhibition of its activity by 75 % results in the development of severe intoxication and required emergency measures [9, 24].

Some OPCs of different structure (phosphates, phosphonates, amidophosphates) are able to DN. This effect occurs after a certain latent period (14–21 days, possibly several years after previous acute poisoning), and they are characterised by the development of ataxia, muscular weakness, impaired sensitivity and numbness of the limbs, paresis and paralysis of the limbs (demyelination of the fibres of ascending spinal tracts and peripheral nerves is the morphological pattern of this condition). Upon to now, tens of thousands cases of paresis and paralysis in people as a result of OPCs (TOCP, mipafox, leptophos, chlorophos, etc.) action were reported, and TOCP is considered as a classic OPC with DN [5, 24, 28, 29].

Mechanism of DN of OPCs has not been clearly established yet. It is known that DN is not associated with AchE inhibition, and the main role in the pathogenesis of the nervous system involvement belongs to phosphorylation of protein belonging to carboxylesterase and called neurotoxic esterase (NTE) [16, 28]. Delayed neuropathies develop under conditions of NTE activity inhibition by 70–80 % and DN development is associated not only with its inhibition but with further “ageing” of NTE [24].

High OPCs doses, along with neurotoxicity, cause multiorgan dysfunction syndrome with hepatic, cardiac, renal and other organ impairment; cholinergic, cytotoxic, membranotoxic action of OPCs and oxidative stress with increased reactive oxygen species play the role in it [9].

Biomarkers: fields of application and types. Broadly defined, according to the report of the US National Academy of Sciences (1989), biomarker is any quantitative parameter that reflects the interaction between the biological system and potential hazard that may have chemical, physical or biological nature [7, 17, 19, 20]. Determination of biomarker as “a parameter that may be objectively measured and used as an indicator of physiological and pathological biological processes or pharmacological responses to the therapeutic intervention” has been provided by the working team of the National Institute of Health, USA, in 2001 [20, 30].

Fields of biomarker application:

· use in the clinical practice (for confirmation of the diagnosis of acute or chronic poisoning, assessment of the treatment efficiency and prognosis);

· assessment of the risk for health (assessment of the exposure of the people working in industries, analysis of the efficiency of protection measures on the enhancement of production processes and work environment);

· use for the monitoring (for screening at the individual and population level for the confirmation of exposure of persons in this population).

Ultimate biomarker should correspond to the following characteristics:

· sampling and analysis of the samples is simple and reliable;

· high sensitivity, specificity and predictive value;

· reflection of subclinical and reversible changes;

· there are appropriate efficient means of intervention and preventive measures;

· reliable reproduction in people of different gender and different ethnicities;

· low cost of biomarker measurement and measurement procedure is safe for patients’ health and justified by the bioethics principles [20].

The following types of biomarkers are allocated [7, 19, 31, 32, 33] (Fig. 3):

1. Biomarker of Exposure: exogenous substance, its metabolite or a product of the interaction between xenobiotic and any molecule or target cell that is located in one or another body system.

2. Biomarker of Effect: measurable biochemical, physiological, behavioural or other change in the body that may be considered as associated with proactively known or possible health problems or disease.

3. Biomarker of Susceptibility: an indicator of body inherent or acquired ability to react on the action of a certain substance-xenobiotic.

It should be noted that the distinct distribution of biomarkers by types is commonly impossible [32] (Fig. 3).


Fig. 3. Original concept of biomarkers (NRC, 1987) [32].


Biomarkers of exposure to OPCs. The following is considered as the biomarkers of exposure that confirm the existent fact of OPC action on a human:

1. Chemical substances (Table 1) and their metabolites (Table 2) in the biological matrices (blood and its components, urine, saliva, inhaled air, gastric content, tissues) [19].

2. Adducts formed in the reactions of bounding between chemical substances and cellular macromolecules (proteins and DNA).

Due to the measurement of biomarkers of exposure, it is possible to predict the risk, obtained information, however, may not be always used for prediction of toxicity (Benford et al., 2000) [19].

Measurement of unchanged OPCs in blood, urine, gastric content has a diagnostic value for confirmation of exposure in case of acute poisoning, biomonitoring of professional exposure, however, is a limitation, since OPCs rapidly disappear from the blood and are eliminated renally, and concentrations are commonly too low for detection [19].


Table 1

Measurement of organophosphorus compounds in the human biosamples [by Manel Araoud, 19].


Based on the pharmacokinetic models and biomonitoring data, it has been established that OPC concentration in human tissues higher than 100 µM (10–100 µg/mL) reflect acute (accidental or deliberate) exposure, whereas lower concentrations (0.01–1 µg/mL) is the result of the effect of environment polluted by xenobiotics. Therefore, measurement of OPCs concentration in blood and urine is used for differentiation between chronic and acute exposure to OPCs [10].

The majority of OPCs is metabolised with the formation of six final products — dialkyl phosphates (DAP) eliminated renally: dimethyl phosphates (DMP), dimethyl thiophosphates (DMTP), dimethyl dithiophosphates (DMDTP), diethyl phosphates (DEP), diethyl thiophosphates (DETP), diethyl dithiophosphates (DEDTP) [10, 19, 34]. Measurement of alkyl phosphate metabolites in urine is the most practical and common method for assessment of the internal (absorbed) dose of the majority of organophosphorus pesticides, therefore, this method is used for biomonitoring of OPC exposure in epidemiological studies [19]. At the same time, it is required to determine the optimal time for biosampling and consider the fact that in some cases DAP levels are lesser than the limit of detection [10, 19].

For exposure biomonitoring, pesticide-specific metabolites such as para-nitrophenol (PNP) and trichloro-pyridinol (TCP) are measured in urine along with DAP (Table 2.) [19].


Table 2

Main metabolites of organophosphorus compounds detected in human blood for biomonitoring [by Manel Araoud, 19].


As the biomarkers of exposure to organophosphorus poisoning substances, such as sarin, soman, VX in different biomedical samples may be metabolites provided in general in Table 3 [35].


Table 3

List of biomarkers of exposure to organophosphorus poisoning substances [35].


The attention of scientists to the determination of adducts with proteins as the biomarkers of exposure is reinforced by their ability for longer persistence in the body. In the case of OPCs and organophosphorus poisoning substances, these are adducts with butyrylcholinesterase and albumin. However it is noted, despite the abilities of current analytical methods, that determination of adducts in a delayed period after exposure is complicated due to their low concentration in the body. Furthermore, determination of adducts does not give an answer about either the severity or nature of poisoning after all [20].

It is known that complexing with proteins for the majority of xenobiotics is reversible and considered as a form of detoxification. In some cases, in particular for substances of alkalising type and easily-hydrolyzable substances, protein binding increases their toxicity [36]. Also, it has been shown that OPC binding to blood proteins contributes to the reduction of their toxicity for laboratory animals and may stipulate species-specific susceptibility to xenobiotics [37]. Under the action of OPCs, sorption on proteins is aimed at their detoxification, since blood protein binding reduces the free concentration of xenobiotic, holds it at the periphery, and reduces both anticholinesterase and toxic effect. Therefore, long-term circulation of OPC adducts in the blood may be an informative biomarker for prediction of chronic intoxication. Considering the above, this direction of toxicological studies may be considered as promising.

Esterases — biomarkers of exposure to OPCs. Since enzymes changed under the action of OPCs are more stable in the body in comparison with active substances and their metabolites that are relatively rapidly eliminated, thus measurement of enzymatic activity is an important biomarker of exposure of anticholinesterase substances [19, 20, 38-40].

The primary targets of the action of OPCs to the body are enzymes: AchE is the target of acute toxic action and NTE is the target of delayed neurotoxicity. The secondary targets of the action of OPCs: BChE and carboxylesterase (CaE), as well as other proteins with esterase activity that covalently bind OPCs (acetylcholinesterase, hydrolase of fatty acid amides, arylformamidase, serum albumin) [16, 39]. BChE and CaE are stoichiometric acceptors of OPCs, resulting in the reduced concentration of active OPCs for potential interaction with AchE. Inhibition of these so-called non-target esterases does not cause clinical effects.

The level of cholinesterase activity is not reduced every time in case of chronic intoxication with low OPC doses, therefore, it is not always a reliable biomarker of intoxication. The value of the specified biomarkers for diagnostics, assessment of treatment efficiency and prediction of consequences of OPC poisoning is questionable and it is widely discussed in the scientific literature [16, 19, 20, 38, 39].

Measurement of AchE activity. AchE in mammals is presented in CNS, peripheral nervous system (sympathetic and parasympathetic ganglia, parasympathetic nervous endings of organs, motor endings of motor neurons), blood (red blood cell membranes). Depending on the species, the plasma may contain different amounts of AchE: rat plasma may contain 30–50 % AchE, whereas this level is significantly lower in human. Obtained data suggest that the structure and pharmacodynamic similarity between AchE of the brain and red blood cells gives the possibility to use AchE inhibition in red blood cells for assessment of AchE inhibition in the brain [16, 19].

Measurement of NTE activity. Measurement of NTE activity of the nervous tissue as the biomarker of OPC DN is the reasonably established correlation between significant NTE inhibition of experimental animals during several hours after administration of neurotoxic OPCs and development of delayed neuropathy in several weeks [16, 28, 39]. The study has shown that during the first hours after administration of neurotoxicants, inhibition of NTE of lymphocytes and platelets is observed in parallel with NTE inhibition of the brain [28]. Biomonitoring of the toxic action of neurotoxic OPCs is virtually not used, although there are methods for NTE measurement in blood corpuscles.

Furthermore, the results of experimental studies showed a correlation between NTE inhibition in the brain, lymphocytes and whole blood. Currently, methods are developed with the use of highly-sensitive biosensors for measurement of NTE activity in the whole blood that are characterised by the simplicity of blood sample preparation for a biosensory test of NTE [28, 40].

Measurement of non-target esterase activity. Literary data suggest that non-target esterases are the first binding sites of OPC interaction after their penetration into the blood, furthermore, many OPCs interact in vitro with BChE and CaE more effectively than with [16, 39, 41]. Authors believe that non-target esterases are more sensitive biomarkers than AchE of the red blood cells that allows measuring the exposure of lower OPC doses.

BChE is contained in different mammalian tissues (liver, heart, vascular endothelium, nervous system, and blood plasma), participates in the wide spectrum metabolic processes of substrates of endogenous and exogenous origin and biotransformation of xenobiotics. In the presence of BChE, inactive precursors of many medicinal products pass into an active form. Measurement of BChE activity in plasma or whole blood is used as the sensitive biomarker of exposure to low OPC doses, at the same time, there is no interaction between the level of its activity and severity of intoxication [16, 19, 29, 39, 40].

Carboxylesterases (CaEs) is the large group of mammalian enzymes located in the endoplasmatic reticulum and cellular cytoplasms of many tissues, and the maximum carboxylesterase activity was detected in the liver and plasma. CaEs participate in the processes of metabolic activation of the drug products and in detoxification of some OPCs via hydrolysis of their carboxylic bounds and switch of OPC to an inactive state. Another mechanism is OPC binding in the active centre of the enzyme that leads to the reduced toxin concentration in the blood [40].

Furthermore, the ability of hydrolysis of many OPCs, including highly-toxic sarin, soman, VX, is managed by the serum enzyme paraoxonase-1 (PON1) that is the component of high-density lipoproteins. PON1 was named due to its ability to hydrolyse organophosphorus compound paraoxon. It is believed that the physiological function of PON1 is hydrolysis of homocysteine-thiolactone that prevents homocystination of proteins and development of atherosclerosis [38]. It has been established that PON1 is an important indicator of individual body susceptibility to some OPCs (as per animal studies): animals with low PON1 level (birds) were most susceptible to the action of OPCs compared to rats and rabbits. Furthermore, the level of PON1 activity is low at birth, gradually increases with the age of animal that is aligned with higher susceptibility of young animals to OPCs. Considering the above, reduced PON1 activity in the serum due to any reasons may lead to the increased susceptibility to OPC action [16, 19].

Esterase status - a complex biomarker of OPC action. The ratio of activity of 4 serine esterases (AchE, NTE, BChE, CaE), as well as serum PON1 in the scientific literature, was called “esterase status” of the body. Esterase status is an important parameter changes of which are the signal of impaired body homeostasis, and in case of OPC action, esterase status specifies subject susceptibility to their action, in addition to the confirmation of exposure [16, 38-40]. Conducted studies showed that determination of esterase status is a more informative biomarker of exposure to OPCs in comparison with standard tests on the measurement of the activity of one or another individual esterase [39, 40].

In addition to the establishment of the fact of exposure, determination of esterase status (namely simultaneous measurement of blood AchE and NTE) gives the possibility to assess the likelihood of OPC-induced delayed neurotoxicity or acute cholinergic intoxication. Since inhibition of blood esterases depends on the dose, its level allows to assess the level of action, i. e. perform dosimetry of exposure to OPCs [16, 39].

The established difference in the esterase status of different species of animals shows that different esterases are the specific biomarkers of OPC action in different species. Analysis of human and rat esterase status showed the high level of PON1 activity in all studied rats, at the same time, esterase activity in mice is higher than in rats. Rodents showed high plasma CaE activity, whereas NTE activity is significantly lower in comparison with people. Esterase activity in human blood is mainly presented with plasma PON1 (50–60 mg/mL) and plasma BChE (5 mg/mL), as well as AchE of red blood cells, and CaE activity was insignificant [38]. It has been established that the most sensitive human biomarker is BChE, whereas in mice these are CaE and BChE. [16, 39, 40].

Biomarkers of the effect of OPCs. To improve assessment of the possible risk for health, biomonitoring of exposure should be accompanied by the determination of biological effects of xenobiotics — biomonitoring of effect.

Biomarkers of the effect include certain specific markers of target tissues and should reflect early biochemical reversible changes that precede structural and functional damages. Biomarkers of the effect are the parameters that quantitatively characterise physiological, biochemical and other changes in the body, the severity of which determines actual or potential health problem or development of the disease. Biomarkers of the effect include deviations of any laboratory parameter (biochemical, immunological, cyto - and immunogenetic) that reflects functional disorders in critical organs and systems under the action of a hazardous factor or the parameter of actual morbidity by nosological forms of diseases that have an evidential relationship with the marker of exposure. The parameter of actual morbidity is used if it is not possible to quantify xenobiotic in the biological medium [31].

Biomarkers of the effect are divided into specific (specify biological effect under the action of a certain hazardous factor) and non-specific (specify general complex body response under the action of various hazardous factors).

The most informative biomarkers are based on the assessment of parameters of their specific action [19]. Biomarkers of specific OPC action is the activity of enzymes AchE and NTE.

There are several categories of the biomarkers of the effect:

- molecular products of action: DNA-adducts (products of interaction of toxicant and structural DNA fragments), glycosylated haemoglobin, products of binding electrophilic compounds with proteins and amino acids, chromosomal changes;

- endogenous biomolecules: AchE, gamma-glutamyl transferase (GGT), alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH);

- changes in the cells/tissues: the activity of macrophages, the ration of the peripheral blood corpuscles and others [31].

Some biological effects of the action of pesticides may be detected relatively early at the cellular level, at the same time they may play a simultaneous role of the indicators of exposure. Considering this, cytogenetic markers of DNA damage in the circulating lymphocytes (chromosomal aberrations, sister chromatid exchanges, micronuclei) and findings of the comet test may also be used for determination of early biological effects and as biomarkers of the exposure in the subjects exposed to pesticides. Since in the majority of cases, however, the population of people is exposed by the complex of xenobiotics, it is difficult to assign genotoxic damage to any certain chemical class or compound [10, 19, 42].

Biomarkers of the effect are used in the routine practice for clinical diagnostics, they help to establish the stage and degree of disease, prescribe appropriate therapy, and assess its efficiency.

The important role in the toxicity of many OPCs also belongs to the indirect effects, since virtually all physiological systems and organs may be involved in the pathological process under the action of OPC. Non-cholinergic mechanisms of OPC action includes the ability to change peripheral blood pattern, functional activity of the immune, monooxygenase and anti-oxidative system, and also affect liver and kidneys. Non-cholinergic mechanisms are mainly manifested after repeated exposure to small OPC doses that unable to cause pronounced cholinergic reactions and under the action of less toxic substances [2, 25].

Among non-cholinesterase parameters of OPC intoxication, the best known are:

· markers of involvement of the liver and kidneys — blood AST, ALT, GGT, LDH, creatinine, urea;

· the level of non-organic phosphorus (increased in case of chronic intoxication with OPCs);

· parameters of oxidative stress — the level of malonic dialdehyde and total oxidative status (TOS) [19, 20].

It is known that the immune system is the target for OPC toxic action [2, 3, 5, 12, 15, 23, 43, 44]. Effect of OPCs on the immune system are the following: allergenic effect (majority of OPCs are weak or moderate allergens); inhibition of antibody formation and increased susceptibility to infections, suppression of non-specific body reactivity; suppression of functional activity of immunocompetent cells (T-type immunodeficiency); autoimmune disorders (it has been shown that auto-antibodies are formed to tissues of those organs that were the most exposed to the toxic substance — liver, kidneys, brain) [2, 3, 12, 23, 44].

Mechanisms of immunosuppressive OPC action include direct pathway — inhibition of serine hydrolases or esterases in the structural elements of the immune system (complement system, lymphocyte membrane), oxidative damage of immune organs or modulation of signalling pathways that controls proliferation and differentiation of lymphocytes and their effector function. Indirect action on the immune system is associated with changed cholinergic tonus of lymphoid organs, increased production of corticosteroids, metabolic changes under chronic intoxication [2, 3, 12].

Despite the presence of a sufficient number of scientific works on exploration of OPC immunotoxic effects, there are unresolved questions and controversial data; mechanisms of OPC action at the level of organs and system have not been clearly established; biomarkers of immunotoxic effect of organophosphorus pesticides have not been established, and this justifies the necessity for further scientific researches of OPC effect on the immune system [2, 3, 12].

Immunoreactivity condition under the action of OPCs with DN and immunological biomarkers of the effect (by the results of own studies). SI “L. I. Medved’s Research Toxicology Center of the MoH of Ukraine” has studied body immune reactivity on a classic OPC DN model (Leghorn chickens) following single oral administration of isotoxic doses (by cholinesterase activity): TOCP at a dose of 500 mg/kg and afos at a dose of 200 mg/kg that have pronounced DN, cyclofos (O,S-dimethyl-O-cyclohexyl-thiophosphate) at a dose of 10 mg/kg (is not NTE inhibitor and does not cause DN) [45, 46].

It has been established that TOCP and afos action results in similar changes in the morphological composition of chicken peripheral blood, more pronounced in paralytic period (Day 21): probable reduction of the absolute (by 22 and 27 %, respectively) and relative number of white blood cells (by 22 and 37 %, respectively), reduction of the absolute number of lymphocytes (by 39 and 62 %, respectively). Throughout the experiment, the metabolic activity of neutrophils reduced, most notably under the action of afos (by 28–55 %) in comparison with TOCP. Functional activity of neutrophils under the action of TOCP and afos at Day 14 and 21 was compensated by the increase of their blood count (an increase of the relative number by 54 and 75 %, respectively) [45, 46].

Quantitative changes of immunocompetent cells developed even in the pre-paralytic period (Day 7), increasing in the paralytic period (Day 21): probable reduction of the absolute number of T-lymphocytes, B-lymphocytes, NK-cells, Ts (Fig. 4). Increase in the ratio of Th to Ts (Th/Ts = 3.4–4.4) suggests autoimmune disorders in the body [46].


Fig.  4. Changes in the absolute number of immunocompetent cells (%) in chicken at the paralysis stage under the action of afos, TOCP and cyclophosphane in isotoxic doses (* – р < 0.05) [46].


As it can be seen from Fig. 5, effect of afos in pre-paralytic period (Day 7) resulted in the reduction of functional activity of T-lymphocytes that is evidenced by the reduction of the number blast-transformed lymphocytes of peripheral blood in chickens by 59.0 %, and functional activity of T-lymphocytes in the paralytic period was at the level of control. As compared to afos, TOCP increased functional activity of T-lymphocytes at Day 21 and compared to control — by 50.4 %. Under the action of both afos and TOCP, functional activity of B-lymphocytes increased, that is evidenced by the increase of HHA titres in the blood serum of chickens at Day 21 by 61.0 % and 53.0 %, respectively.

Fig. 5 Functional activity of T- and B-lymphocytes of chickens under the acute action of afos at a dose of 200 mg/kg and TOCP at a dose of 500 mg/kg (* – Р < 0.05).


Under the action of both afos and TOCP, animals with pronounced manifestations of neuroparalytic action showed significant reduction of the total blood level of CIC, at the same time accumulation of finely dispersed pathogenic CIC took place that is evidenced by the ratio between fine dispersed and large particle CIC [47]. The highest level of pathogenic CIC was observed in the paralytic period (Day 21), and it was the most pronounced under the action of afos (Fig. 6).

Fig. 6. Autoantibody titres (AA) to antigen from the chicken brain and the ration between finely dispersed and large particle circulating immune complexes (CIC, finely dispersed/large particle ratio) under the acute action of afos at a dose of 200 mg/kg (* – Р < 0.05).


Starting from the Day 7, an increase in the titre of autoantibodies to tissue antigens of the chickens with DN clinical picture was observed. Under the action of afos (Fig. 6), the level of autoantibody titre was 3.2–4.3-fold higher in comparison with control. The level of autoantibody titre under the action of TOCP was lesser increased in comparison with afos and differed from control 1.48–3.0 times [47].

In opposition to afos and TOCP, cyclofos (isotoxic dose, 10 mg/kg) resulted in relative and absolute neutrophilia, insignificant relative lymphocytopenia, reduced relative and absolute number of B-lymphocytes, increased relative number of T-lymphocytes only at Day 7. Observed changes were unstable, and the quantitative composition of immunocompetent cells at Day 21 was at the level of control (Fig. 4), excluding an increase in the absolute number of Ts. Th/Ts ratio was 1.43 suggesting its immunotoxic action. Cyclofos resulted in a transient reduction of functional activity of neutrophils on Day 7, and this parameter did not differ from control at Day 21. Under the action of cyclofos, increase in the CIC level in the blood serum developed mainly due to the large particle forms that are easily eliminated from the body via phagocytosis. Since recovery of functional activity of neutrophils and reduction of the CIC level developed at Day 21, this fact suggests that they are eliminated from the body and have no damaging action on the tissues [46, 47].

Provided data confirm that under the action of afos and TOCP, chicken body in the pre-paralytic period forms pathogenic fine dispersed CIC, the level of which significantly increases in the paralytic period, and functional activity of neutrophils is reduced. With the development of the pathological process, changes in the immune system worsen — total number of lymphocytes, T- and B-lymphocytes, Ts, NK-cells is reduced and the level of autoantibodies to the nervous system antigen is increased that suggests the development of autoimmune disorders. Established interaction between the severity of OPC DN signs and autoimmune disorders in the chicken body suggests an important role of the immune system in the pathogenesis of this condition.

This is evidenced by the data on the prevention of complications due to afos. It was shown that haemocarboperfusion at Day 10 after afos poisoning at a dose of 200 mg/kg results in the elimination of fine dispersed (pathogenic) CIC, normalization of metabolic activity of neutrophils and functional activity of T- and B-lymphocytes, reduction of Th number [48]. The positive effect of haemocarboperfusion is aligned with clinical signs of DN: in all animals after haemocarboperfusion, clinical signs of intoxication developed later, were less pronounced, and paralysis did not develop. This fact suggests that the immune system plays a significant role in the formation of OPC DN, and parameters of the condition of the immune system may be used as biomarkers of the effect.

Considering the level of changes of immunological parameters and their interaction with the severity of clinical manifestations of OPC DN, we proposed the following parameters as biomarkers of the severity, treatment efficiency and prediction of complications: the level of finely dispersed CIC in the blood serum and the level of anti-brain antibodies, the number and functional activity of blood neutrophils; the number of T- and B-lymphocytes and their functional activity; the number of NK-cells, Th, and Ts.

Therefore, despite the presence of the sufficient scientific data on biomarkers of OPC action, there are some problems, associated with diagnostics of OPC poisoning, in particular in case of subacute and chronic intoxication. Biomarkers that are the most commonly used, namely AChE and BChE, is specific only in early terms of intoxication, the degree of inhibition of cholinesterase does not always correlate with the degree of intoxication that reduces diagnostic value of these traditional biomarkers. Determination of esterase status of the body is a more effective and informative biomarker of exposure to OPCs, although important biomarkers that are not associated with their anticholinesterase action are not taken into account [20]. Along with measurement of esterase activity, an important value in the diagnostics, assessment of the course and prediction of complications of OPC action belongs to other biomarkers of the effect that reflect the condition of different systems participating in the body homeostasis balance.

Therefore, based on the data analysis of modern literature and the results of own studies, the following conclusions can summarize this work:

1. To assess the risk of OPC for health, screening at the individual and population level and for clinical diagnostics, it is reasonable to use biomarkers of exposure and effect: active substances or their metabolites, adducts with proteins, body “esterase” status and other biomarkers not associated with their specific action.

2. As immunological biomarkers that allow to assess the severity of the course, treatment efficiency and predict complications under action of OPC with DN, the following parameters may be used: the level of finely dispersed CIC in the blood serum and the level of anti-brain antibodies, the number and functional activity of blood neutrophils; the number of T- and B-lymphocytes and their functional activity; the number of NK-cells, Th, and Ts.

3. There is a necessity to develop and justify the unified diagnostic complex that includes biomarkers of exposure, specific and non-specific action, with wider diagnostic abilities in comparison with the determination of isolated parameters that is the basis for diagnostics, assessment of treatment efficiency and prediction of consequences of poisoning with OPCs.



1. Luzhnikov E. A. Acute poisoning: Guideline for physician. 2nd edition, revised and enlarged. /E. A. Luzhnikov, L. H. Kostomarova – M.: Meditsina. – 2000. – 434 p.

2. Zhminko P. H. Immune system as a target of toxic action of chemical substances / P. H. Zhminko // Actual topics of transport medicine. – No. 1 (23). – 2011. – P.17 – 30.

3. Zabrodskii P. F. Immunotoxicology of organophosphorus compounds / P. F. Zabrodskii. – Saratov. Publishing house “Saratovskii istochnik”. – 2016. – 289 p.

4. Toxicants with anticholinesterase action: mechanism of action, clinical signs and actual topics of provision with antidotes / L. A. Ustinova, N. M. Seredynska, N. V. Kurdil [et al.] // Current problems of toxicology, food and chemical safety. – 2017. - No. 3 (79) - P. 73-82.

5. Díaz-Resendiz K. J. G. Modulation of Immune Response by Organophosphorus Pesticides: Fishes as a Potential Model in Immunotoxicology /  K. J. G. Díaz-Resendiz, G. A. Toledo-Ibarra, M. I. Girón-Pérez // Hindawi Publishing Corporation. Journal of Immunology Research. – V. 2015. – 10 p.

6. Pore N. E.  Organophosphorus poisoning / N. E. Pore, K. N. Pujari, S. P. Jadkar // International Journal of Pharma and Bio Sciences. – Vol.2. – Issue 4. – 2011. – P. 604–612.

7. Biomarkers and risk assessment: concepts and principles (Hygienic criteria of the environmental condition; 155) / Joint publication of the UN Environment Program, International Labour Organization and World Health Organization. – World Health Organization, Geneva: – 1996. – 96 p.

8. Petrov A. N. Antidotes against organophosphorus poisoning substances / A. N. Petrov, H. A. Sofronov, S. P. Nechiporenko, I. N. Somin // Russian Chemical Journal – 2004. - vol. XLVIII. – No. 2. – P. 110-116.

9. Kharchenko O. A. Acute poisoning with organophosphorus compounds: main clinical syndromes and mechanisms of their formation (literature review and data of own studies) / O. A. Kharchenko, H. M. Balan, N. N. Bubalo // Current problems of toxicology. – 2013. - No. 1-2. – P.17 – 31.

10. Biomonitoring and biomarkers of organophosphate pesticides exposure – state of the art / L. Kapka-Skrzypczak, M. Cyranka, M. Skrzypczak, M. Kruszewski // Annals of Agricultural and Environmental Medicine. – 2011. – Vol. 18. – No 2. – P. 294–303.

11. Kurdil N. V. Peculiarities of acute poisoning with pesticides in the conditions of the city: carbamates, pyrethroids, neonicotinoids / N. V. Kurdil, O. V. Ivashchenko, V. F. Struk, A. H.  Bohomol // Medicine of emergency. – 2015. – No. 7 (70).– P.43–49.

12. Galloway T. Immunotoxicity of Organophosphorus Pesticides / T. Galloway, R. Handy // Ecotoxicology. – 2003. – No. 12. – P. 345 – 363.

13. Eleršek T. Organophosphorus Pesticides – Mechanisms Of Their Toxicity / T. Eleršek, M. Filipič // Pesticides – The Impacts of Pesticides Exposure [edited by Prof. M. Stoytcheva]. – 2011. – P.243–260.

14.Associations between dietary factors and urinary concentrations of organophosphate and pyrethroid metabolites in a Canadian general population / Ye Ming, J. Beacha, J. W. Martin, A. Senthilselvan // Int. J. Hyg. Environ. Health. – 2015. [Electronic resource]. - Available at:

15. Repetto R. Pesticides and the Immune System: The Public Health Risks / R. Repetto, Sanjay S. Baliga // World Resources Institute. – 1996. – 103 p.

16. Esterase Status of Various Species in Assessment of Exposure to Organophosphorus Compounds / Natalia P. Boltneva, Еlena V. Rudakova, Larisa V. Sigolaeva, Galina F. Makhaeva // Toxicological Problems [editor: Christophor Dishovsky and Julia Radenkova-Saeva]. – Bulgarian Toxicological Society. Military Publishing House. – Sofia, Bulgaria. – 2014. – P.27–38.

17. Biomarkers and Risk Assessment: Concepts and Principles / World Health Organization, Geneva. – 1993. – 87 p.

18. Biomonitoring of Exposure in Farmworker Studies / Dana B. Barr, K. Thomas, B. Curwin [et al.] // Environmental Health Perspectives. – June 2006. – Volume 114. – № 6. – P. 936-942.

19. Araoud М. Biological Markers of Human Exposure to Pesticides / M. Araoud // Pesticides in the Modern World. Pests Control and Pesticides. Exposure and Toxicity Assessment [Edited by Dr. Margarita Stoytcheva].– 2011. – 614 p.

20. Voitenko N. H. Issues of diagnostics in intoxication with organophosphorus compounds / N. H. Voitenko, D. S. Prokofieva, N. V. Honcharov // Toxicological bulletin. – 2012. – No. 5(122).– P.2–6.

21. Laboratory test methods in clinical settings / V. V. Menshikov, L. N. Delektorskaia, R. P. Zolotnitskaia [et al.]. - Reference book [edited by V. V. Menshykov]. – M.: Meditsina. – 1987. – 368 p.

22. Biochemical, immunological and biophysical methods in toxicological experiment / Methodological guideline edited by U. A. Kuzminskaia. – K.: Rotaprint. – 1989. – P. 59-61.

23. Zabrodskii P. F. Pathogenetic mechanisms of immune status impairment with organophosphorus compounds in combination with antidotes and their correction / P. F. Zabrodskii, I. Kh. Yafarova. – Saratov. – 2009. – 185 p.

24. Kahan Yu. S. Cholinesterase blockers / Yu. S. Kahan, N. V. Kokshareva, P. H. Zhminko // General toxicology [edited by B. A. Kurliandskii, V. A. Filov]. – M.: Meditsina. – 2002. – 608 p.

25. Kutsenko S. A. Principles of toxicology: Scientific and methodological edition / S. A. Kutsenko. - St. Petersburg, Publishing house “Foliant”, LLC. – 2004. – 720 p.

26. Rosenberg Y.J. A pretreatment or post-exposure treatment for exposure to a toxic substance by pulmonary delivery (inhaler) of a bioscavenger / Y.J. Rosenberg // PCT Int. Appl. (WO 2005000195 A2). – 2005. – V.6. – No. 1. – 22 p.

27. Organophosphate Pesticides & Child Health: A Primer for Health Care Providers [Electronic resource] / 2007 Pediatric Environmental Health Specialty Unit (PEHSU), Department of  Environmental & Occupational Health Sciences. – Available at:

28. Biosensors for analysis of activity of neurotoxic esterase as a biomarker of toxic action of neuropathogenic organophosphorus compounds / L. V. Siholaieva, I. N. Kurochkin, A. V. Yeremenko [et al.] // Russian Chemical Journal – 2004. - Vol. XLVIII. – No. 4. – P. 65-72.

29. Biomarkers of organophosphorus (OP) exposure in humans / J. Marsillach, R.J. Richter, J.H. Kim [et al.] // Neurotoxicology. – 2011 Oct. – 32(5). – P. 656–660.

30. Biomarker and surrogate endpoints: preferred definition and conceptual framework / A.J. Atkinson, W.A. Colburn, V.G. De Gruttola [et al.] // Clin. Pharmacol. Ther. – 2001 (69). – P. 89-95.

31. Onyshchenko G. G. Hygienic indication of health consequences upon external environment exposure of chemical factors / H. H. Onishchenko, N. V. Zaitseva, M. A. Zemlianova [edited by H. H. Onishchenko]. – Perm, 2011. – 532 p.

32. Biological markers in environmental health research / NRC (National Research Council). –  Environ. Health Perspect. – 1987. – V.74. – P. 3-9.

33. Dietert R.R. Biomarkers for the 21st Century: Listening to the Microbiome / R.R. Dietert, E.K. Silbergeld // Toxicological Sciences. – 2015. – V.144(2). – P. 208-216.

34. Esquivel-Sentíes M. S. Organophosphorous Pesticides Metabolite Reduces Human T CD8 Homeostasis and Proliferation by Inducing Cellular Death / M. S Esquivel-Sentíes, L. Vega // J Environment Analytic Toxicol., S4:004. – 2012. [Electronic resource]. - Available at:

35. Determination of metabolites of organophosphorus poisoning substances in biomedical samples using solid-phase extraction / I. A. Berzin, V. S. Romanov, Ye. I. Savalieva [et al.] // Forensic medicine. – 2009. – Vol. 10. – P. 45-56.

36. Luik A. I. Serum albumin and biotransportation of poisons / A. I. Luik, V. D. Lukianchuk // M: Meditsina. – 1984. – 224 p.

37. Zhminko P. H. Toxicity and anticholinesterase action of some organophosphorus pesticides depending on their sorption on blood serum proteins / P. H. Zhminko, Yu. I. Loboda // Current issues of toxicology. – 2003. – No. 1. – P.18 – 21.

38. “Esterase status” of the body under the action of toxic substances and pharmaceutical products / I. D. Kurdiukov, V. I. Shmurak, A. D. Nadieiev [et al.] // Toxicological bulletin. – 2012. – No. 6 (117). – P.6–13.

39. Makhaeva G. F. Investigation of Esterase Status as a Complex Biomarker of Exposure to Organophosphorus Compounds / G.F. Makhaeva, Е.V. Rudakova, L.V. Sigolaeva // Toxicological Problems [editor: Christophor Dishovsky and Julia Radenkova-Saeva]. – Bulgarian Toxicological Society. Military Publishing House. – Sofia, Bulgaria. – 2014. – P.15–26.

40. Properties of biosensor analysers for assessment of body “esterase status” / L. H. Sokolovskaia, L. V. Siholaieva, A. V. Yeremenko [et al.] // Chemical and biological safety. – 2004. – No. 1–2 (13–14). – P.21–31.

41. Thompson C.M. Anticholinesterase insecticides / C.M. Thompson., R.J. Richardson // [edited by T.C. Marrs, B. Ballantyne]. – New York, Wiley. – 2004. – P. 89-127.

42. Biomarkers of chemical exposure: State of the art / P. Grandjean, S.S. Brown, P. Reavey, D.S.Young // Clin Chem. – 1994. – No. 40 (7). – P.1360–1362.

43. Effects of pesticide exposure on the human immune system / E. Corsini, J. Liesivuori, T. Vergieva [et al.] // Human & Experimental Toxicology. – 2008 (27). – P. 671-680.

44. Effect of organophosphorus compounds on the factors of mice congenital immunity / T. S. Zaporozhets, L. A. Ivanushko, A. K. Hazha, [et al.] // Biomeditsina. – 2013. – No. 1. - P. 36-47.

45. Zhminko P. H. Species-specific peculiarities of body immune reactivity under the action of neuroparalytic organophosphorus compounds / P. H. Zhminko, M. H. Prodanchuk, M. V. Yankevych // Current issues of toxicology. – 2002. – No. 1. – P. 46–51.

46. Zhminko P. H. Role of the immune system and non-specific body reactivity in pathogenesis of poisoning with organophosphorus pesticides and synthetic regulators of plant growth: abstract of thesis on competition for a scientific degree of the Doctor of Biological Sciences: speciality. 14.03.06 “Toxicology” / Petro Hryhorovych Zhminko, “Institute of Pharmacology and Toxicology of the NAMS of Ukraine”. – Kyiv, 2005. – 39 p.

47. Zhminko P. H. Role of the immune system in pathogenesis of delayed neurotoxicity of some organophosphorus compounds / P. H. Zhminko // Current issues of toxicology. – 1999. – No. 4. – P. 18-24.

48. Zhminko P.G. Delayed Neurotoxicity Induced by Organophosphates: Experimental Correction of Neuropathy / P.G. Zhminko. N.V.Kokshareva // Toxicological Problems  [edited by major-general prof. Stoian Tonev, prof. Kamen Kanev, prof. Christophor Dishovsky, assoc. prof. Eugenia Stankova]. – Sofia, Bulgaria, Publishing Hous Irita. – 2011. – P.175–183.


Надійшла до редакції 23.11.2018р.