The development of methodology and valid reliable tools is aimed at revealing associations of specific outcomes of health with type, assortment of pesticides, work practice, intensity of burden, etc. Approaches are based on applying of complex of objective and subjective methods, such as in-depth interview, detailed questionnaire, direct observations, genetic testing of environmental components, biosubstrats' chemical analysis, biostatistics and mapping methodology, local pesticides' market analysis. The developed methodology and reliable tools will allow assessing the environmental chemical burden on human with purpose to reduction of rural population health risks.
In Armenia, as well as in the whole world, the amount and the variety of pesticides used in different spheres of agriculture are increasing. According to the National Statistical Service of the Republic of Armenia during 2013 the import of pesticides increased more than threefold compared with 2001. In addition, the enforcement of pesticide's use and application is very poor in Armenia and this is proven by the presence of persistent organochlorine compounds (Lindan, DDT) in women`s breast milk. These chemicals are found constantly in significant concentrations. Residues of DDT and its metabolites are evidence of the continuing contamination of the environment by these chemicals . There is no information in available references about pesticides' residues in the environment and foodstuff in Armenia. Nevertheless there are a few publications of genetic research that indicate the high load of active mutagens on environment, which might be indirect indicator of high levels of chemical contamination.
The information mentioned above stressed the importance of studies on this field. It was reported some increase of morbidity even in cases of proper use of plant protection chemicals (carbamates, chlorphenoxy acetic acid derivates, different phosphororganic compounds and their metabolites).
Recent research is focused on the mutagenic and estrogenic activity of pesticides. These mechanisms underlie carcinogenic and reproductive health impairment. The impact of pesticides is expressed by point mutations, increasing chromosomal aberrations in somatic cells and gametes. This leads to development of cancer, abortions and perinatal mortality, development of congenital abnormalities, and infertility.
The infant's health status, birth defects rates are considered the best indicators of environmental impact on population health  .
Chemical impacts on the embryo are more dangerous than on adults. They mainly accumulate in fatty tissues, during pregnancy penetrate into the flow of the blood, and pass the placenta. Fetal damage is mainly conditioned by the impact of chemicals on new developing organ systems. Actively proliferating and less differentiated tissues are more sensitive as molecular and biochemical processes get damaged and lead to problems with differentiation of tissues, cell proliferation, cells migration and so on. As has already been mentioned pesticides' hormonal impact causes congenital defects or malformations and as a consequence not only the developing fetus is affected but also the woman`s hormonal regulation during all stages . Such processes are also a problem for mature people, affecting their somatic cells (tumor cells are developed) and gametes (meiotic transfer is shattered) .
In Armenia and globally increased rates of oncological diseases and reproductive health disorders have been recorded during the last decade. For example, in 2000 the rate of cancer was 931 per 100000 population, in 2011 it was 1598. One can observe such a situation in the case of congenital malformations and pregnancy complications. In 2000 the total number of congenital malformations per 100000 population was 42.7, in 2011 it was 95. The last fact is more disturbing as we have low total fertility rate in Armenia. Instead of the recommended 2.12 we have 1.4-1.7. We have data which speaks about a high level of infertility among young couples: 17-18 %, moreover infertility among women has doubled during the last few years 2005-2009 . As research has shown the high possibility of breast cancer is positively correlated with the concentration of some persistent organochlorine pesticides (DDT, DDE) in serum and other biological fluids [7-8].
However, some research results deny the association of mean concentrations of the above mentioned pesticides with breast cancer frequency . According to the literature data, there is more evidence linking plant protection chemicals to leukemia, non-Hodgkin's lymphomas, lung cancer, bone diseases, sarcomas, etc .
Based on above mentioned we have developed methodology of study to reveal correlation between occupational exposure to pesticides and morbidity of cancer as well as influence on reproductive health.
The aim of study is development of strategies to reduce cancer morbidity and improve reproductive health based on known associations of specific outcomes with type and assortment of pesticides, work practice, etc.
The specific objectives are:
to develop and update the tools (questionnaires, database) necessary for research
to study the rates and structure of cancer morbidity among the rural population Ararat Valley rural population
to study the rates and structure of congenital abnormalities among the Ararat Valley rural population
to study the rates and structure of reproductive health disorders i. e. infertility, miscarriages
to evaluate the level of pesticide impacts by using of selected indirect indicators: the duration of work with pesticides, the application practice, etc.
to evaluate pesticide concentrations in biological tissues: fat tissues, breast milk, biopsy and autopsy substances
to evaluate the level of contamination of the environment by pesticides using genetic tests to quantitatively evaluate the negative impact of different pesticides to develop complex of preventive measures
To solve the above mentioned problems we propose to carry out a case-control study with two subgroups: one for cancer, and another for reproductive health. Rural population of two Armenian regions, i.e. Armavir and Ararat, will be involved in the research. The choice of regions was made taking into account peculiarities of climatic conditions which contribute to more growth of vegetables and fruits and use of higher amounts of pesticides.
All cases of birth defects and an equal number of controls matched by age of parents, parity, and neighborhood proximity will be included. Cases and controls will be selected from local regional maternity houses. Exclusion criteria are the following: any infectious diseases during pregnancy, known genetic disorders, family history of birth defects. A total registration of cases, which are predicted to be from 100 to150, will be completed.
In the cancer study all proven cases of cancer will be registered. After scrutiny of all patients' cards from Statistical center of Armenian national center of oncology, database with addresses of relevant patients will be created. Controls will be matched by age, sex, neighborhood proximity and duration of residency. A total registration of cases, which are predicted to be from 1300 to1500, will be completed.
The serious problem consists in the assessment of contamination level of environment by pesticides. In case of having a bunch of insecticides, herbicides, fungicides, fertilizers applied annually and many years in a row, and in addition, other persistent organic compounds and heavy metals around, the detection of certain pesticides' residues is hardly possible and worthy.
In Armenia mentioned issues are highly actual because of small sizes of plots with different crops, variety of agricultural activities in one season, etc. Thus, the important part of project is development of certain tools, indirect indicator of overall chemical burden on the environment and human. Detailed questionnaire, in-depth interview on usage of pesticides, working practice, applying of protective equipment, etc., is good source of information about possible exposure level, especially in large scale and big sample size research. Nevertheless, this type of data often is incomplete, indirect, biased, and subjective in some extensions.
To survey the target groups specific questionnaires were developed and validated. Questionnaires for cancer and reproductive health subgroups are consist of 54 and 99 questions respectively. Questions include indicators of pesticides' impact level, such as the names, amounts of pesticides used in one season, the number of application days, the duration of dealing with pesticides during a year, precautions and specific equipment used by the respondents, methods of storage of pesticides, etc. Surveys will be supplemented by nested studies.
A nested study will be conducted within each case and control group with 30-40 cases. Within these studies the load of pesticides in the environment will be assessed by testing of phytotoxicity and genotoxicity (Ames test, based on specific mutations of gens of Salmonella typhimurium, which are responsible for production of histidine, observation of meristematic cells during anaphase and metaphase, germination of seeds in the soil, chemical analysis of food and other environmental components on presence of pesticides, etc.) of environmental components sampled from pinpoint sites of respondent (soil, dust, plants) and chemical analysis of intra-operational tissues. Sampling of soil will be done by envelop method: five portions of soil taken from the cultivating area (for angles of area and the center), all this portions mixed and 1/5 part of mixture taken as a single sample. Dust will be taken from inside of house or the house backyard. Plants will be harvested from cultivating area of respondent. Biological material (fatty tissue, breast milk, umbilical cord) will be taken from appropriate healthcare facilities by acceptance of respondents. All samples will be numbered according to ID codes of respondents in database and, only after that, sent to laboratory.
Tests of phytotoxisity and genotoxicity will be conducted on Embryophytes with smaller amount of chromosomes i.e. Crepis capiliaris, Alium cepa, Taraxacum officinale, Hordeum vulgare. First of all germination of seeds in testing environment (samples from case and control groups) will be observed. Test results of both groups will be compared with germination of seeds in special control soil taken from ecologically clear areas of Armenia (mountainous uncultivated areas, far from settlements). As an indicator of genotoxicity amount of chromosomal mutations in meristematic cells of abovementioned Embryophytes will be taken. Meristematic cells from root tips will be observed during anaphase and metaphase under optical microscope after staining of preparation. Excess of mutagenic activity by two times in comparison with control samples will be interpreted as moderate mutagenic activity, 2-5 times excess as significant activity, more than 5 times – strong mutagenic activity. All tests will be duplicated [11-12].
Chosen Embryophytes have following advantage: they are wide spread in Armenia, they have small amount of chromosomes: from 6 to 24 chromosomes, they are widely used as an indicators for mutagenic activity and there are a lot of methodologies developed for fixation and staining of preparations.
Research methods will include applying of complex of objective and subjective methods, such as in-depth interview, detailed questionnaire, direct observations, genetic testing of environmental components, biosubstrats' chemical analysis, biostatistics and mapping methodology, local pesticides' market analysis.
The storage and analysis of data will be performed on personal computers using standard and special software Access, Excel, SPSS, EpiInfo, Biostat, etc. Geo-spatial mapping methodology also will be used (ARC GIS Esri software), which will allow to make morbidity data, cancer diseases and birth defects on one hand, more comparable and compatible with levels of pollution of the environment by persistent organic pollutants (POP's) on the other.
The developed methodology and reliable tools will allow to purpose to reduction of rural population health risks.
assess the environmental chemical burden on human with
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