Simulating real-life exposures to uncover possible risks to human health: A proposed consensus for a novel methodological approach

Abstract

In real life, consumers are exposed to complex mixtures of chemicals via food, water and commercial products consumption. Since risk assessment usually focuses on individual compounds, the current regulatory approach doesn’t assess the overall risk of chemicals present in a mixture. This study will evaluate the cumulative toxicity of mixtures of different classes of pesticides and mixtures of different classes of pesticides together with food additives (FAs) and common consumer product chemicals using realistic doses after long-term exposure. Groups of Sprague Dawley (CD-SD) rats (20 males and 20 females) will be treated with mixtures of pesticides or mixtures of pesticides together with FAs and common consumer product chemicals in 0.0, 0.25 × acceptable daily intake (ADI)/tolerable daily intake (TDI), ADI/TDI and 5 × ADI/TDI doses for 104 weeks. All animals will be examined every day for signs of morbidity and mortality. Clinical chemistry hematological parameters, serum hormone levels, biomarkers of oxidative stress, cardiotoxicity, genotoxicity, urinalysis and echocardiographic tests will be assessed periodically at 6 month intervals. At 3-month intervals, ophthalmological examination, test for sensory reactivity to different types of stimuli, together with assessment of learning abilities and memory performance of the adult and ageing animals will be conducted. After 24 months, animals will be necropsied, and internal organs will be histopathologically examined. If the hypothesis of an increased risk or a new hazard not currently identified from cumulative exposure to multiple chemicals was observed, this will provide further information to public authorities and research communities supporting the need of replacing current single-compound risk assessment by a more robust cumulative risk assessment paradigm.

Keywords

Pesticides food additives long-term toxicity acceptable daily intake tolerable daily intake combined exposure

Introduction

Approximately 5.2 billion of plant protection products (PPPs) are used worldwide every year.¹ The term pesticide not only refers to PPPs but also includes substances (or mixture of substances) used as insecticides, fungicides, herbicides, acaricides, rodenticides, nematicides, growth regulators, repellents and biocides to prevent, destroy or control harmful organisms (pests) or vector-borne diseases.² Thus, many active substances used as PPPs are also used as biocides, which are defined by Regulation European Union (EU) no. 528/2012 as any substance or mixture used with the intention of destroying, deterring, inactivating, preventing the action of, or exerting or controlling the effect of any harmful organism using physical or mechanical action.³ PPPs are regulated in the EU under Regulation (EC) no. 1107/2009 and are defined as products that contain active substances used to protect plants or plant products, before or after harvest, against pests or diseases, to preserve plant products, to influence the life process of plants or to destroy or prevent undesired growth of plants or parts of plants.⁴ These active substances can be marketed in a commercial formulation together with other chemicals, such as safeners or synergists.

Although pesticide operators, farm workers and bystanders undergo the highest exposure, pesticide residues in food and water represent a potential risk for the general population.^5

–8 According to the 2013 EU report on pesticide residues in food, pesticide residues were found in all food samples analysed: unprocessed and processed food products, baby food, organic food and food products of animal origin (Table 1).⁹ Multiple residues (more than one pesticide) were found in 27.3% of the samples analysed with strawberries, peaches, apples and lettuce among the products with multiple residues.

Table 1.

Pesticide residues in food samples (EFSA, 2015).⁹

Food samples analysed	Samples with pesticide residues below MRLs (%)	Samples with pesticide residues that exceeded MRL (%)
All food products	42.8	2.6 unprocessed products more frequent in: guava, lychee, passion fruit, tea leaves, okra, basil, parsley, spinach, turnips, papaya, cassava and pomegranates; processed products more frequent in: wild fungi, tea leaves, peas with pods, peppers, herbal infusions, beans with pods and rice
Organic products	15.5	0.8
Food products of animal origin	11.7	0.3 more frequently in: honey, chicken eggs, poultry meat, fat of swine, bovine, poultry, bovine meat and liver and kidney of swine and bovine
Baby food products	7.3	0.7

MRL: maximum residue level.

In the last half-century, with the expansion and development of the global food industry, the number of substances used as food improvement agents, such as additives, enzymes, flavouring agent and so on, has increased dramatically. Additives are defined by EU Regulation (EC) no. 1333/2008 as any substance not normally consumed as a food itself and not normally used as a characteristic ingredient of food, whether or not it has nutritive value that is intentionally added to food for a technological purpose in the manufacture, preparation, treatment, processing, packaging, transport or storage and became directly or indirectly a component of such foods.¹⁰

In addition, industrial chemicals represent another source of risk not only for workers and professional users but also for the general population that is exposed to these agents through common commercial products. In the EU, two ambitious regulations^11,12 have entered into force in order to improve the protection of human health and the environment from the risks posed by chemicals while enhancing the competitiveness of the EU chemicals industry. At the same time, the EU legislation on chemicals ensures that the hazards presented by chemicals need to be clearly communicated to EU workers and consumers through appropriate classification and labelling of chemicals.^11,12

A number of human biomonitoring studies^{13

–20} reported that populations from different world areas are exposed to chemical mixtures associated with a wide spectrum of adverse health effects. The presence of phthalates, parabens metabolites and bisphenol A in urine or hair of adults indicates exposure via personal care products and contaminated food and water.^21

–27 The exposure of the general population to artificial sweeteners and potential health risks was also suggested by other biomonitoring studies.^28,29 Consumers of all ages are exposed to such compounds every day not only from direct contact with product items but also through the food chain, water consumption and other indirect environmental sources.

The EU legislation has set safe doses for all of the substances that will be addressed in this article.^30

–33 Regulatory terms for PPPs food additives (FAs) and lifestyle chemicals (common chemicals in commercial products) are reviewed in Table 2.

Table 2.

Regulatory terms for plant protection products, FAs and lifestyle chemical.

Abbreviation	Definition	Explanation
ADI	Acceptable daily intake	ADI is defined as the amount of a specific substance in food expressed as milligrams per kilograms body weight/day on which exposure over a lifetime would not cause any appreciable health risk to any consumer, taking into account sensitive groups within the population such as children and the unborn. ³¹ The ADI is derived from the NOAELs determined in animal toxicity tests, divided by appropriate arbitrary or data-derived safety or uncertainty factors.³² This term is generally used for FAs and PPPs.
DNEL	Derived no-effect level	DNEL represents the level of exposure to a substance above which human should not be exposed.³⁰
TDI	Tolerable daily intake	TDI is an estimate of the amount of a substance expressed on a body weight basis that can be ingested daily over a lifetime without appreciable health risk. This term is generally used for food contaminants.³³
MRL	Maximum residue level	MRL represent the upper legal level of a concentration for a pesticide residue in or on food or feed based on good agricultural practice.³¹
NOAEL or LOAEL	No- (or lowest) observed-adverse-effect-level	NOAEL represents the higher tested dose of a substance in a subchronic or chronic study reported to have no adverse health effects. LOAEL is the lowest concentration or amount of a substance found by experiment or observation that causes an adverse alteration of morphology, function, capacity, growth, development, or lifespan of a target organism distinguished from normal organisms of the same species under defined conditions of exposure.

FA: food additive; PPP: plant protection product.

A number of publications have suggested possible associations between exposure to chemical mixtures and adverse health outcomes.^34

–38 For example, the US National Health and Nutrition Examination Surveys has reported urinary levels of different xenobiotics, such as arsenic phthalates, phenols, parabens, pesticides, heavy metals, nitrate, perchlorate, polyaromatic hydrocarbons and polyfluorinated compounds being associated with adult hearing disturbance,³⁹ depression,⁴⁰ oral health,⁴¹ emotional support needs⁴² and memory.⁴³ One of the limitations regarding the existing safe doses, like acceptable daily intakes (ADIs) and tolerable daily intakes (TDIs), is that they are based on classic risk assessment approaches that focus on ‘one-exposure-one-health-effect (the health effect with the lowest level of risk)’ assumption and on studies of individual chemicals. Since consumers are exposed to complex chemical mixtures, this assumption is not expressing realistic exposure scenarios and could lead to underestimation of safety issues. Studies reporting interactions between pesticides from different classes, or within the same class, have shown that toxicokinetic interactions may occur and can lead to potentiation and synergism that may determine an unpredicted toxicological response.³⁵ However, these interactions are unlikely at low doses (around ADI or TDI).³⁵ The presence of multiple endocrine active chemicals could have additive effects, and thus, may produce non-linear dose–response curves, even when low doses alone do not produce untoward effects.⁴⁴

The number of existing studies on chemical mixtures is limited, and the majority have been conducted with high doses as proposed by international protocols.⁴⁵ Consequently, these studies do not simulate accurately the real exposure scenarios of doses around the ADIs.

A limited number of in vivo studies conducted on chemical mixtures have shown additive effects; however, these studies have been limited to a relatively few endpoints. In one case, the authors studied a mixture of 13 chemicals for nipple retention and prostate weight in male rats.^34,46,47 However, most studies typically use relatively high doses, as already mentioned, and are directed toward examining a limited number of endpoints, which represent another limitation of the classical approach. No study has assessed so far the toxic effect of low-dose chemical mixtures monitoring different endpoints at the same time.

Aim

Consumers of all ages are continuously exposed via food, water and other commercial products to a wide range of pesticide residues, FAs and chemicals used in lifestyle/consumer products. Even if these substances are found at very low concentrations, long-term exposure of combinations of ingredients may have the potential to induce adverse health effects.

In an attempt to contribute to a better understanding of the potential cumulative risk of such mixtures, the following study protocol has been designed to address the hazard assessment of some selected mixtures:

mixtures of pesticides along with food and lifestyle additives,

under a chronic exposure framework,

at more realistic low doses and

by monitoring an expanded number of endpoints addressing different toxicities in a single study.

The EU has recognized the need of conducting more research on risks resulting from cumulative exposures to combinations of toxicants, and European, Food and Safety Agency has started to pay more attention to cumulative risk assessment from exposure to mixtures of pesticides eliciting common outcomes on the same target organ/system.⁴⁸

The major objective of this study will be to assess the hazards from long-term exposure to low doses of combined pesticides from different classes, alone and in combination with low doses of selected FAs and common commercial products chemicals. If new hazards were identified, these chemical combinations may represent a risk for human health after long-term low-doses exposures. Therefore, new information will be delivered, supporting the change from single-compound risk assessment to a new era of cumulative risk assessment. Three dose levels will be used following a long-term exposure protocol to assess whether the same or different outcomes are induced. The mid dose will be the ADI or TDI dose for each chemical in the mixture in an effort to simulate real-life exposures, since it is expected that most commercial products and activities comprising chemicals conform to regulatory limits. The low dose and high dose will be 0.25 × ADI/TDI and 5 × ADI/TDI, respectively.

Additional endpoints (cardiotoxicity, oxidative stress, genotoxicity and endocrine disruption) will be added to the Organization for Economic Co-Operation and Development (OECD) TG 452 protocol design⁴⁵ in order to collect more information on health hazards from a single chronic study.

The following major strengths of this study are:

to provide a mechanistic interpretation of any observed effect and to link outcomes with exposure to the tested chemical mixture and

to translate the results of the present experimental protocol into a cost-effective targeted testing strategy that can support policy interventions towards public health protection.

Methods/design

Design/setting

This study will evaluate if mixtures of pesticides from different classes and mixtures of pesticides from different classes together with FAs and common chemicals from commercial products in doses of 0.25 × ADI/TDI, ADI/TDI and 5 × ADI/TDI induce similar or different health outcomes after long-term exposure. Animal experiments will be performed in laboratories from Romania, Greece, Iran, Turkey, Italy, Spain, Korea and Russia (Moscow and Vladivostok); part of the genetic and oxidative stress testing will be done in laboratories from Greece, Turkey and Italy.

Description of the study

The workflow of the protocol is depicted in Figure 1.

Figure 1.

Workflow of the protocol.

Selection of chemicals to be tested will be based on the following series of criteria such as:

use patterns and expected levels found in everyday life;

identified health hazards and existing health concerns;

potential of toxicokinetic interactions that could lead to a synergistic or potentiating effect, or inhibition of a protective mechanism, thus resulting in increased toxicity or an unpredicted toxicological response;

potential endocrine disruptor properties; and

current knowledge on the mode of action.

Selected mixtures will be tested for chemical stability in drinking water and then tested in a long-term in vivo experiment.

Ethical considerations

All ethical issues and implications related to this study will be in accordance with EU Directive 2010/63/EU⁴⁹ for animal experiments and Laboratories Code of Ethics on the proper conduct of research.

Administration of complex mixtures to rats

Female and male Sprague Dawley (CD-SD) rats (Crl: CD(SD) rats (Charles River)) will be used as experimental animals. Sex-based differences will also be assessed. The study will be conducted according to the OECD Guidance Document 452⁴⁵ for chronic toxicity testing of chemicals with the innovation of using a mixture of different substances instead of only a single chemical. Groups of 20 male and 20 female rats will be treated daily for 104 weeks with three different dose levels of mixtures consisting of (a) six pesticides (PPPs), (b) six pesticides and three FAs and three chemicals commonly found in commercial products life style additives (LSAs) as follows:

LD Mixture of PPPs (0.25xADI/TDI),

MD Mixture of PPPs (ADI/TDI),

HD Mixture of PPPs (5xADI/TDI),

LD Mixture of PPPs + FAs + LSAs (0.25xADI/TDI),

MD Mixture of PPPs + FAs + LSAs (ADI/TDI) and

HD Mixture of PPPs + FAs + LSAs (5xADI/TDI).

Animals in the control group will receive only drinking water/animal feed.

The substances will be given in the drinking water/animal feed, depending on the physicochemical properties of the mixtures, according to the OECD guidelines. The concentrations of the chemical mixtures in drinking water/animal feed will be adjusted accordingly to water/feed consumption that will be monitored and recorded every day at the same hour. Animals’ body weight will be registered every week. Both exposed and control group animals will receive commercial animal feed and water ad libitum.

Observation during the study

Animals will be examined at least once daily for signs of morbidity and mortality. Adverse clinical signs, such as assessment of the skin, fur, eyes, mucous membranes, secretions and excretions, as well as checking the autonomic activity (lacrimation, piloerection, pupil size and unusual respiration), stereotypic behaviour (excessive grooming and repetitive circling), bizarre behaviour (self-mutilation and walking backward), changes in gait, posture, and the response to handling and the presence of clonic or tonic convulsions will be observed as part of the daily monitoring. Feed and water consumption will be monitored and recorded daily.

Ophthalmological examination and evaluation of neurotoxic effects

Ophthalmological examination will be performed on all animals before starting the study and at time intervals of 3, 6, 9, 12, 15, 18, 21 and 24 months. The neurotoxic potential will be assessed by testing sensory reactivity against auditory, visual and proprioceptive stimuli. This assessment will be made before the initiation of the study and then repeated at 3, 6, 9, 12, 15, 18, 21 and 24 months intervals during the study. The influence of the mixtures on the learning abilities and memory performance of adult and ageing rats will be checked from 15 months onwards at intervals of 3 months.

Blood chemistry parameters, hematological parameters, hormones, cardiotoxicity, genotoxicity and oxidative stress markers

Blood samples will be collected from the tail vein from all animals at 6 months (between weeks 24 and 26), at 12 months (between weeks 50 and 52), at 18 months (between weeks 102 and 104) and at 24 months (before the end of the study). Clinical chemistry parameters, hematological parameters, hormone levels and biomarkers of cardiotoxicity, genotoxicity and oxidative stress will be determined in these samples. Blood samples will be collected every 2 days according to the guidelines for blood collection in animals.⁵⁰ Each animal will be restrained (manually or using an animal restrainer), and then the tail vein will be punctured using a 23G needle to collect blood.⁵¹ Blood samples that will not be used immediately will be kept frozen at −80°C after centrifugation to separate plasma/serum and erythrocyte package for latter determinations.

A BC-5000 Vet auto hematology analyser (Mindray, North America) will be used to determine the following hematological parameters: total red blood cell erythrocyte count (RBC), hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin (MCH), MCH concentration, platelets, total white blood cell count (WBC) and differential WBC counts (including neutrophils, lymphocytes, monocytes, eosinophils and basophils).

A SPOTCHEM EZ SP-4430 automated analyser (Arkray, Japan) will be used to determine the serum levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total bilirubin, blood urea nitrogen, creatinine, total protein, albumin, globulin, total cholesterol, triglycerides, glucose, calcium, inorganic phosphorus, sodium, potassium, chloride and total bile acids. Cholinesterase activity will be assessed as well using Ellman’s assay.⁵²

Fresh urine samples will be collected from all exposed and control rats at every 6 months for the total length of treatment. Urine will be collected by holding the rat over a petri plate and encouraging it to urinate by the application of gentle transabdominal pressure over the bladder.⁵³ The following parameters will be measured: pH, protein, glucose, bilirubin, ketone, specific gravity, occult blood, urobilinogen, nitrite, leukocytes, colour and turbidity (insight urine test strips).

Hormone determinations

Thyroid function will be assessed in all groups by measuring the serum levels of triiodothyronine (T3), thyroxine (T4) and thyroid-stimulating hormone (TSH). Testosterone will be analysed in male rats, and estradiol, luteinizing hormone and follicle-stimulating hormone in female rats. These determinations will be done using quantitative enzyme-linked immunosorbet assay method.⁵⁴

Genotoxicity

For genotoxicity evaluation, the following test will be used: in vivo micronuclei test, comet assay and random amplified polymorphic deoxyribonucleic acid (DNA) (RAPD) assay. The in vivo micronuclei test will be conducted according to OECD guidelines.⁵⁵ In brief, whole blood will be harvested with an anticoagulant (e.g. heparin) and then separated on gradient centrifugation to yield lymphocytes. The supernatant fraction will be removed by aspiration, and portions of the pellet will be spread on slides and air-dried. The slides will be fixed in methanol, stained in Giemsa and May–Grünwald solutions and protected by mounting with coverslips. At least two replicated are mounted for each sample. Using a standard Nikon microscope, at least 2000 cells/animal are counted equally divided between replicates.

Comet assay will test the influence of the compounds on DNA damage and repair. Whole blood drawn from the animals or separated lymphocytes will be mixed with low-melting point agarose and layered onto a slide that will be subjected to electrophoresis after cells lysing and stained with a fluorescent DNA-binding dye. Cells with increased DNA damage will have a typical migration pattern and will display images in the form of a comet.⁵⁶

For RAPD assay, the protocol in brief will be the following: after DNA extraction from biological samples, fragments generated by RAPD are visualized after agarose gel electrophoresis and ethidium bromide staining. The DNA profiles are compared to controls and band shifts, and the missing bands or the appearance of new bands will be assessed in the treated samples. ⁵⁷

Cardiotoxicity

The cardiotoxic potential of the mixtures will be assessed at weeks 26, 52, 78 and 104 by means of an echocardiographic evaluation of all animals from every study group. The echocardiographic evaluation of the rats will take place under pentobarbital anaesthesia (25 mg/kg intraperitoneal) as this showed to lead to similar data obtained in conscious rats.⁵⁸ From the sampled blood, biochemical indices, such as lactate dehydrogenase-1 isoenzyme, creatine phosphokinase isoenzyme MB and troponin C, will be determined.

Assessment of oxidative stress

Oxidative stress markers will be determined in blood at weeks 26, 52, 78, 104 and before the end of the study as well as from tissue samples after euthanasia of the animals. Total antioxidant capacity (TAC), protein carbonyl (CARB) levels and thiobarbituric reactive species (TBARS) will be determined in plasma and tissue samples. In turn, glutathione (GSH) levels and catalase activity (CAT) will be determined in erythrocytes and tissue samples. After blood collection, samples will be centrifuged immediately at 1370 g for 10 min at 4°C, and then, the plasma will be collected and used for measuring TAC, TBARS and CARB levels. The packed erythrocytes will be lysed with distilled water (1:1 v/v), inverted vigorously, centrifuged at 4020 g for 15 min at 4°C, and the erythrocyte lysate will be collected for the measurement of GSH and CAT levels.

After all tissues collection, the samples are snapped frozen in liquid nitrogen. The procedure for tissue biochemical analysis is briefly described as follows: mortar and pestle will be used for crushing and grinding the samples with the assistance of liquid nitrogen. One part of tissue powder is then homogenized with two parts (w/v) of 0.01-M phosphate-buffered saline pH 7.4 (138-mM sodium chloride, 2.7-mM potassium chloride and 1-mM Ethylenediaminetetraacetic acid [EDTA]) and a cocktail of protease inhibitor tablet (complete mini inhibitor protease cocktail tablet; Roche, Munich, Germany) is added. The homogenate is vigorously vortexed, and a brief sonication treatment on ice is applied. The homogenate is then centrifuged at 12,000 × g for 30 min at 4^°C, and the supernatant fraction is collected. Tissues are stored at −80^°C until analysed.

Histological examination

Animals will be killed by exsanguinations from the abdominal aorta under isoflurane anaesthesia after 12 h of fasting. Animals found dead will be placed in a freezer at −80°C until necropsied, if appropriate. A detailed macroscopic post-mortem examination will be performed on all rats, including the external surfaces of the carcass and the internal organs. All grossly visible abnormalities will be recorded. Organs will be weighed (paired organs: kidney and adrenal, both organs will be weighed separately), and the organ to body weight (final body weight) ratios will be calculated based on the fasting weight. Other organs also to be weighed will be the brain, thymus, heart, liver, kidney, spleen, adrenal, testis, thyroid (weighed postfixation, with parathyroid glands), ovary and uterus. A full histological examination will be performed on all study animals. The following tissues will be preserved in 10% neutral buffered formalin for histopathological examination: all gross lesions, adrenal gland, aorta, brain (including sections of cerebrum, cerebellum and medulla/pons), caecum, cervix, coagulating gland, colon, duodenum, epididymis, eye (including retina), harderian gland, heart, ileum, jejunum, kidney, lacrimal gland (exorbital), liver, lung, lymph nodes (both superficial and deep), female mammary gland, oesophagus, ovary, pancreas, parathyroid gland, peripheral nerve (distal sciatic nerve), pituitary, prostate, salivary gland, seminal vesicle, skeletal muscle, skin, spinal cord (at three levels: cervical, mid-thoracic and lumbar), spleen, stomach, testes, thymus, thyroid gland, trachea, urinary bladder, uterus (including cervix), vagina and a section of bone marrow and/or a fresh bone marrow aspirate.

The routine paraffin embedding technique will be the method used for processing tissues and tissue fragments. Tissues will be cut to 3–5-μm-thick histopathological sections, which are then attached on slides. Staining and mounting procedures will follow via haematoxylin and eosin staining.

Strengths and limitations of the study protocol

The experimental protocol described herein has important strengths. The study design, to the best of the authors’ knowledge, is the first one that takes into consideration the realistic scenario of long-term low-doses exposure to mixtures containing different chemicals of everyday life. Simultaneous monitoring of multiple endpoints by, for example, the determination of hormones, oxidative stress markers, liver enzymes, cardiac enzymes, hematological parameters and echocardiographic and histopathological evaluation of various organs, together with neurotoxicity and genotoxicity assessment, adds further value to the study power. In addition, the combination of the selected chemicals will allow the identification of potential interactions that could lead to new hazard identification and a possible different risk assessment approach. One of the main limitations, that at the same time could also be a challenge for the researchers, is attributing the identified hazards to the correct chemical, since in the framework of toxicodynamic interactions, it is anticipated difficult to ascertain which of the substances administered reaches its target and causes the primary toxic effect and at the same time to identify whether another co-substance also reaches the same target, causing a second toxic effect, or exert a toxicokinetic interaction, thus potentiating or antagonizing the effect of the first chemical.⁵⁹

The ADI is derived from no-observed-adverse-effect-levels determined in animal toxicity tests and divided by appropriate safety or uncertainty factors.³² Therefore, in the lowest dose used (0.25 × ADI), it is possible to have no effect in rats even if the mixtures at specific dose could produce unexpected effects in humans.

Conclusion

This protocol design represents the first study that takes into consideration the realistic scenario of long-term exposure to low doses of mixtures containing different chemicals, such as pesticides alone or in combination with FAs and common chemicals from commercial products. In this way, the expected everyday exposure of the general population to multiple chemicals commonly used at doses normally below the regulatory limits is thus adequately represented. Adding additional endpoints (cardiotoxicity, neurotoxicity, oxidative stress, genotoxicity and endocrine disruption) to the OECD TG 452 protocol design will allow the researchers to collect more information from a single chronic study and, therefore, adopt a more holistic approach. Depending on the outcome of these studies and if the hypothesis of an increased risk from cumulative exposure of chemicals in ADI levels were shown to be true or even a new unknown hazard of the tested mixture was identified, useful information to regulatory authorities and research communities will provide support to the decision of shifting from the current single-compound risk assessment to a new era of cumulative risk assessment. As a result, the incidences of different chronic diseases as well as the cost for their treatment might be reduced.

Footnotes

Acknowledgements

The authors express their gratitude for partial support to the following research programs and organisations: Special Account for Research of the University of Crete ELKE KA 3392; ERA.NET RUS PLUS NABUCO; Grant of the President of Russian Federation for young doctors of science MD-7737.2016.5; Mashhad University of Medical Science, Iran; Research Funds and Agreement Contracts of Erisman Institute, Moscow; University of Granada, Research Group BIO-253; Scientific and Technological Research Council of Turkey, 214S112; Romanian Ministry of Education and Research – Research Grant PN-II-PT-PCCA-2013-4-1386 (Nanopatch); Romanian Ministry of Education and Research – Research Grant PN-II-176/01.07.2014; Internal Research Grant of the University of Medicine and Pharmacy Craiova, Romania, no. 15/8.01.2015.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/orpublication of this article: The authors received support from the programs and grants listed in the acknowledgements for the research, authorship, and/or publication of this article.

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