ISSN 1563-034Х; еISSN 2617-7358 Экология сериясы. №2 (67). 2021 https://bulletin-ecology.kaznu.kz

IRSTI 34.23.51;87.25.00 https://doi.org/10.26577/EJE.2021.v67.i2.07

A.M. Kalimagambetov1 , A.A. Sarsenovа1* , S.S. Zhumagulovа2 , N.A. Musrepova2 1Al-Farabi Kazakh National University, , Almaty 2Aktobe Regional Prenatal Center, Clinical Diagnostic Laboratory, Kazakstan, *e-mail: [email protected] CHROMOSOMAL ANOMALIES OF THE FETUS OF PREGNANT WOMEN FROM SETTLEMENTS PRONE TO TECHNOGENIC POLLUTION

The article presents the analysis results of the spectrum and frequency of distribution of the fetus chromosomal set of 590 pregnant women living in the city of Aktobe and various districts of the in Kazakhstan. Biological material was obtained by biopsy of the chorion and placenta, amnio- centesis and cordocentesis. For the preparation of chromosome formations, the conventional “direct” method and the classical method of cell cultivation were used. Metaphase cells were stained with GTG and FISH methods. In total, 64 (10.9%) cases of fetal karyotype abnormalities were detected. Of these, genomic abnormalities accounted for 59 (10.0%), and structural – 5 (8.5%). Among the genomic ab- normalities in 39 (66.1%) cases, the fetus had a Down syndrome karyotype. The frequency of chromo- somal abnormalities in the fetus of pregnant women in Aktobe, where industrial facilities of ferrous and nonferrous metallurgy are located, was 7.9%. The frequency of fetal chromosomal abnormalities in 3 districts of Aktobe region (fetal karyotypes of 39 pregnant women were studied) without large industrial facilities was 12.8%, and with the presence of large industrial complexes in 5 districts (fetal karyotypes of 131 pregnant women were studied) – 19,8%, which is 1.5 times more. Thus, it can be assumed that technogenic pollution by large industrial complexes (mining, cement, chemical, oil and gas processing industries) of places of residence contributes to the appearance of genomic and structural disorders of the genetic apparatus of human cells. Key words: prenatal diagnosis, fetal karyotype, chromosomal abnormalities, technogenic factor.

А.М. Қалимағамбетов1, А.А. Сәрсенова1*, С.С. Жумагулова2, Н.А. Мусрепова2 1Әл-Фараби атындағы Қазақ ұлттық университеті, Қазақстан, Алматы қ. 2Ақтөбе облыстық пренаталды орталығы, клинико-диагностикалық зертхана, Қазақстан, Ақтөбе қ. *e-mail: [email protected] Техногендік ластануға ұшыраған елді мекендерде тұратын жүкті әйелдер ұрығының хромосомалық бұзылыстары

Қазақстанда Ақтөбе қаласы мен Ақтөбе облысындағы экологиялық жағдай ең қолайсыз жағдайлардың бірі болып саналады. Бұл тау-кен, химия, мұнай-газ өңдеу, қара және түсті металлургия, құрылыс материалдар өндірісі кәсіпорындарының болуына байланысты. Ақтөбе қаласында және Ақтөбе облысының 8 ауданында тұратын жүкті әйелдер ұрығының хромосомалық жиынтығының спектрі мен таралу жиілігіне 2018 жылдан 2020 жылға дейін талдау жасалды. Ақтөбе облыстық пренатальды орталығының клиникалық диагностикалық зертханасында 590 жүкті әйелдің ұрықтың хромосома жиынтығына цитогенетикалық зерттеулер жүргізілді. Биологиялық материал хорион мен плацентаның биопсиясы, амниоцентез және кордоцентез бойынша алынды. Хромосома препараттарын дайындау үшін кәдімгі «тікелей» әдіс және клеткаларды өсірудің классикалық әдісі қолданылды. Метафазалық клеткалары GTG және FISH әдістерімен боялған. ISCN 2013 жүйесі бойынша хромосомалардың сандық және құрылымдық ауытқулары тіркелді. Ұрық кариотипінің ауытқулары 64 (10,9%) жағдайларда анықталды. Олардың ішінде геномдық ауытқулар 59 (10,0%) жағдайда, ал құрылымдық – 5 (8,5%) жағдайда байқалды. Геномдық ауытқулардың арасында Даун синдромының кариотипі 39 (66,1%) ұрықтарда кездесті. Жүкті әйелдердің тұру аймақтарына байланысты ұрықтың хромосомалық патологиясының жиілігінде өзгергіштік байқалды. Қара және түсті металлургияның өндірістік нысандары орналасқан Ақтөбе қаласындағы жүкті әйелдердің ұрығындағы хромосомалық ауытқулардың жиілігі 7,9% құрады. Ірі өндірістік кешендері орналаспаған Ақтөбе облысының 3 ауданындағы (39 жүкті әйелдің ұрық кариотиптері зерттелді) ұрықтың хромосомалық жиынтығындағы ауытқулардың жиілігі 12,8%

68 © 2021 Al-Farabi Kazakh National University A.M. Kalimagambetov et al.

құрады, ал ірі өндірістік кешендері орналасқан 5 ауданында (131 жүкті әйелдің ұрық кариотиптері зерттелген) – 19,8%, яғни 1,5 есе артуын көрсетті. Осылайша, ірі өнеркәсіптік кешендердің (тау- кен, цемент, химия, мұнай және газ өңдеу өнеркәсібі) техногендік ластануы адам клеткаларының генетикалық аппараттарының геномдық және құрылымдық бұзылыстарының пайда болуына мүмкіндік тудырады деп болжауға болады. Түйін сөздер: пренаталды диагностика, ұрықтың кариотипы, хромосомалық бұзылыстар, техногендік фактор.

А.М. Калимагамбетов1, А.А. Сарсенова1*, С.С. Жумагулова2, Н.А. Мусрепова2 1Казахский национальный университет имени аль-Фараби, Казахстан, г. Алматы 2Актюбинский областной пренатальный центр, клинико-диагностическая лаборатория, Казакстан, г. Актобе *e-mail: [email protected] Хромосомные аномалии плода беременных женщин из населенных пунктов, подверженных техногенному загрязнению

Экологическая обстановка в г. Актобе и Актюбинская области Казахстана считается одной из самых неблагополучных регионов. Это обусловлено наличием предприятий горнодобывающей, химической, нефтегазоперерабатывающей промышленности, черной и цветной металлургии, производства строительных материалов. В работе проведен анализ спектра и частоты распределения нарушении хромосомного набора плода беременных женщин, проживающих в г. Актобе и в 8 районах Актюбинской области. За период с 2018 по 2020 г.г. в клинико-диагностической лаборатории Актюбинского областного пренатального центра проведены цитогенетические исследования хромосомного набора плодов 590 беременных женщин. Биологический материал получен путем биопсии хориона и плаценты, амниоцентеза и кордоцентеза. Для приготовления препаратов хромосом использовались общепринятый «прямой» метод и классический метод культивирования клеток. Метафазные клетки окрашивались GTG- и FISH-методами. Численные и структурные нарушения хромосом учитывались по системе ISCN 2013. Всего обнаружено 64 (10,9%) случая нарушений кариотипа плода. Из них геномные нарушения составили – 59 (10,0%), а структурные – 5 (8,5%). Среди геномных отклонений в 39 (66,1%) случаях у плода выявлен кариотип синдрома Дауна. Частота хромосомных нарушений плода беременных женщин г. Актобе, где располагаются промышленные объекты черной и цветной металлургии, составила 7,9%. Частота хромосомных нарушений плода в 3-х районах Актюбинской области (исследованы кариотипы плода 39 беременных женщин) без крупных промышленных объектов составила 12,8%, а с наличием крупных промышленных комплексов в 5-ти районах (исследованы кариотипы плода 131 беременной женщины) – 19,8%, что больше в 1,5 раза. Таким образом, можно предположить, что техногенное загрязнение крупными промышленными комплексами (горнодобывающей, цементной, химической, нефтегазоперера­ батывающей промышленности) мест проживания способствует появлению геномных и структурных нарушений генетического аппарата клеток человека. Ключевые слова: пренатальная диагностика, кариотип плода, хромосомные нарушения, техногенный фактор.

Introduction intolerance. It is known that the consequences of chemical carcinogenesis and mutagenesis lead to the Currently, the study of congenital diseases emergence and development of genetic diseases in related to the number and structure of chromosomes the body and increase the presence of birth defects in is a considerable issue. At the same time, along regions with poor ecology [2]. with the type of hereditary diseases, it is necessary Studies have demonstrated that the population to determine the cause of its occurrence. In most of Vladikavkaz residents has a genotoxin effect of regions, the cause of congenital malformations environmental mutagens. A link between an increase may be the consequences of man-made pollution of in the level of chromosomal disorders in individuals various concentrated production facilities [1]. associated with harmful production and those who The connection between atmospheric air pollution have nothing to do with it was found. Indeed, the and genetic diseases was revealed. The mutagenicity incidence of congenital malformations, stillbirths, of chemicals released into the air from production sites and intrauterine mortality among residents of this leads to chromosomal diseases, which in turn leads city is enlarging [3]. to neoplasms, spontaneous miscarriage, perinatal According to data researched in this field, fetal death, developmental abnormalities and child environmental pollution by chemical and radioactive

69 Chromosomal anomalies of the fetus of pregnant women from settlements prone to technogenic pollution waste developes the chromosomal instability of the the combustion of polyvinyl chloride, polyethylene human genome, increasing the risk of chromosomal and other polymers have significant mutagenic aberrations in the blood lymphocytes of people of activity. This is typical for cities with a developed different age groups among the population [4]. metallurgical and chemical industry [13]. In Spain, among the workers working at the According to the Ministry of the World Health Prestige oil production complex, there was a break Organization, the development of the fetus during in the chromosomal strands and it was found that perinatal time is 25% dependent on the environment. the impact of oil does not extend to the human body The greatest danger is the first 14-60 days at the even after 6 years [5]. The chromosomes exposed embryo stage. After all, it is at this moment that the to the oil are not only interrupted in certain places, nervous system and internal organs that are sensitive but are also considered precursors of such tumor to toxins develop [14]. diseases as leukemia, lymphoma [6]. In Korea, due According to cytogenetic studies, the frequency to the largest influx of oil on the beach of the city of cells with chromosomal abnormalities in the of Taean, an environmental disaster occurred, as a peripheral blood lymphocytes of pregnant women result of which 27% of the population (adults and is 2.5 times higher when exposed to chemical children) were affected, i.e. for 3 years, problems factors with mutagenic activity (lead, tin, and with the respiratory, mental, and sexual organs were cadmium in the foreground). Most of the congenital observed [7]. B. In the literature review of Laffon malformations found in newborns are characteristic (2016), the relationship between the toxicological of de novo mutations, which indicates the instability properties of oil components and the impact of of the genome and indicates the influence of negative risks to human health (mental, physical/ chemical mutagens [15]. physiological) (genotoxin, immune and endocrine Numerous studies have indicated that the toxicity) is clarified [8]. atmospheric air of large industrial cities annually If we consider the studies conducted in receives about 500 thousand tons of harmful substances Kazakhstan, then in the city of Shymkent, South (about 450 names) with dust and gas emissions, while Kazakhstan region, in newborns born on the territory the volume of emissions of pollutants from stationary contaminated with lead and other heavy metal salts, sources of pollution and vehicle exhaust gases is the structure of congenital malformations revealed comparable to each other [16]. pathology of the nervous system by 28% [9]. Pregnant women living in regions contaminated According to statistics, chromosomal diseases in with various xenobiotics at a high level of the the Aktobe region are a phenomenon that is currently ecosystem are exposed to them during pregnancy. widespread among newborns. Studies have shown This process contributes to the disruption of the that the Aktobe region ranks 5th in the country in physiological / normal course of pregnancy, and, in terms of the number of congenital malformations particular, the negative effect of xenobiotics is very and the incidence of chromosomal diseases among dangerous during implantation, placentation and children aged 0 to 14 years [10]. There are many organogenesis in the sensitive period of pregnancy factors that influence their occurrence, and one of [17]. their bases is technogenic factors. After all, more A molecular relationship (DNA adducts) than 50 production facilities are concentrated between somatic mutation in a newborn and in the Aktobe region. In particular, most of the transplacental exposure to common air pollutants enterprises are directly connected with the oil and has been established [18]. gas industry. According to the literature, it is known According to the information bulletin of that reproductive dysfunction occurs in men who the Republic of Kazakhstan on the state of the directly interact with natural gas condensate [11]. environment, environmental pollution of the Aktobe Oil and gas, the process of development and region occupies a leading position. The indicators operation of fields, transportation of hydrocarbon identified in 2018-2020 indicate a high level of raw materials, as a rule, is accompanied by intensive atmospheric air pollution. In these years, the pollution of the environment with petroleum average air pollution index (ALI) =6* (high level), products, petroleum gases and their combustion the standard index (SI)=25 (very high level) and the products, hydrogen sulfide and sulfur oxide, maximum repeatability (SPO), =21% (high level). mineralized professional wastewater and drilling Surface water pollution and soil contamination with mud, drilling waste. Such large-scale environmental heavy metals in the territory of the Aktobe region pollution directly affects the key health indicators in some cases exceeds the maximum permissible of the population [12]. The products formed during concentrations (MPC) [19,20].

70 A.M. Kalimagambetov et al.

The aim of the work is to analyze the frequency in a thermostat for 72 hours in a special nutrient of violations of the set of fetal chromosomes in medium. Metaphase cells in preparations stained pregnant women living in settlements exposed to by GTG and FISH methods [21-24]. The set man-made pollution. of chromosomes was performed according to the “international system of human cytogenetic Materials and methods of research. nomenclature” (ISCN 2013) [25]. In a cytogenetic study, 20-25 metaphase cells of each fetus were For the study, the fetal karyotypes of 590 pregnant examined under a light microscope (Axio Imager women who were examined in the regional perinatal A2, Hyperlink, Germany, 2015). center of Aktobe in 2018-2020 were examined. The age of pregnant women ranged from 17 to 47 years, Results and discussion the average age was 33.0±0.3 years. The following methods of prenatal diagnostics were used for the Invasive prenatal diagnostic methods to production of chromosomal preparations of fetal determine the fetal karyotype were used. Among biomaterial: chorionic fiber biopsy, placentobiopsy, them there were 37 chorionic fiber biopsies, which amniocentesis, cordocentesis. accounted for 6.3%, 70 (11.9%) placentobiopsies, The chromosomal preparations were 188 (31.9%) amniocentesis, 295 (50.0%) developed using the “direct” method from the cordocentesis. A total of 590 fetal biomaterials were chorion and placenta, after the fetal umbilical obtained. The number and frequency of disorders in cord blood lifocytes and amniocytes were grown the chromosome set are shown in Table 1.

Table 1 – Number and frequency of chromosomal anomalies in the fetal karyotype

N Chromosomal abnormalities Quantity Frequency (%) 1 Trisomy on chromosome 13 2 3,2 2 Trisomy on chromosome 18 10 15,6 3 Trisomy on chromosome 21 39 60,9 4 Monosomy on the X chromosome 5 7,8 5 Polisomy on the X chromosome 2 3,1 6 Disomy on the Y chromosome 1 1,6 7 Structural abnormalities 5 7,8 Total 64 100

In the cytogenetic study according to the first found. The number of fetuses with the karyotype table, 64 cases showed chromosomal abnormalities of Shereshevsky-Turner syndrome was observed of the fetal karyotype, which was 10.8%. Quantitative in 5 (0.8%) cases, and the karyotype of Klinefelter abnormalities of 13, 18, and 21 autosomal syndrome-in 2 (0.3%) cases. There were cases when chromosomes and sex X, Y chromosomes were some defects (for example, Edwards and u dysomy) observed. Their overall frequency of occurrence was occurred in parallel in 1 (0.2) cases. Karyotypes of 92.2%. Among the chromosomal disorders, the most Patau, Edwards, and Down syndromes, respectively, common is the karyotype of Down syndrome in 39 2 (0,3%), 3 (0,5%) 39 (6,6%) identified in cases. In (60.9%) cases. Structural disorders of chromosomes the types of chromosomal aberrations, inversion were detected in 5 (7.8%) cases. The spectrum was observed on chromosomes 7 and 9, deletion and and frequency of all chromosomal disorders are duplication-on the long arm of chromosomes 18. The presented in Table 2. results reveal that changes related to the structure of According to the second table, quantitative and chromosomes are about 12 times less common than structural chromosome disorders in both sexes were changes related to the number of chromosomes.

71 Chromosomal anomalies of the fetus of pregnant women from settlements prone to technogenic pollution

Table 2 – Spectrum and frequency of fetal chromosomal disorders

N Chromosomal changes Fetal karyotype Quantity Frequency (%) 46,ХУ 255 43,2 1 Normal 46,ХХ 271 45,9 Numerical chromosome changes – 59 2 Trisomy on chromosome 13 47,ХҮ,+13 2 0,3 47,ХҮ,+18 3 0,5 3 Trisomy on chromosome 18 47,ХХ,+18 7 1,2 47,ХҮ,+ 19 3,2 4 Trisomy on chromosome 21 47,ХХ,+21 20 3,4 5 Monosomy on the X chromosome 45,ХО 5 0,8 6 Polisomy on the X chromosome 47,ХХҮ 2 0,3 Disomy on the Y chromosome and extra 18 7 48,ХҮҮ,+18 1 0,2 chromosome Structural changes in chromosomes – 5 46,ХҮ,inv(9) 1 0,2 8 Inversion 46,ХХ,inv(7)(q11.1q11.23) 1 0,2 46,XX,inv(9)(p22q13) 1 0,2 9 Duplication 46,ХҮ,dup18(q11.2q25) 1 0,2 10 Deletion 46,ХХ,del18(qh) 1 0,2 All chromosomal changes 64 10,8 Total 590 100

Aneuploidy occurs as a result of a violation of - -without large industrial the segregation of chromosomes during mitosis complexes, production of alcohol and poultry meat; and meiosis. It can be noted that the reason lies - Baiganin district-construction cement plant, in the high degree of sensitivity of microtubules asphalt concrete plant; to mutagens in relation to cell division. Dynamic - Kargaly district-KazTransOil-oil pipeline instability of microtubules [26] can lead to an company; incorrect distribution of chromosomes in the cells - -KazakhOil-oil production of the offspring. company; In the course of the work, the correspondence - Oil district-without large industrial complexes, of the frequency of violations of the set of fetal borders with Munailinsky districts of Atyrau region; chromosomes of pregnant women living in - district-Kozhasay gas processing plants settlements subject to man-made pollution to (oil drilling complex), Zhanazhol oil production the presence of large industrial complexes was complex, Gas plant branch-Kazakhoil, coal water considered (Table 3). raw material extraction; The location of production facilities in the - district: Molodezhnaya mine- city of Aktobe and the districts of the Aktobe chromite ore mining mine, 10 years of independence region: mine-chromite ore mining mine, Don Mining and – In Aktobe-a plant of chrome mixtures (a plant Processing Plant-chromite ore mining plant, – for processing chromium), a plant of ferroalloys Izbes chromium mining plant, chromium mining (production of chromium, titanium alloys, etc. complexes; metals), a plant for processing copper metals, a - The district is an ecologically plant for the production of polymers (polymer unfavorable area that does not have large industrial pipes), etc.; complexes, due to its proximity to the island .

72 A.M. Kalimagambetov et al.

Table 3 – The number of fetuses that underwent cytogenetic research on industrial complexes in the city of Aktobe and districts of the Aktobe region

Number of fetuses with The number of fetuses with N Living Districts Description of large industrial enterprises normal karyotype abnormalities in the karyotype 1 Aktobe city Ferrous and non-ferrous metallurgy 387 33 There are no large manufacturing 2 Alga district 18 2 enterprises 3 Baiganin Cement production 7 6 4 Kargaly Oil and gas processing plant 9 3 5 Mugalzhar Oil and gas processing plant 34 5 There are no large manufacturing 6 Oil 6 1 enterprises 7 Теmir Oil and gas processing plant 9 3 Khromtau Mining industry, Ferrous and non-ferrous 8 46 9 metallurgy There are no large manufacturing 9 Shalkar 10 2 enterprises Total 526 64

According to the third table, large industrial fetus with deviations in the karyotype was observed complexes are located in Aktobe and 5 regional in Aktobe and districts in 33 and 26 cases, and in districts. Three districts (Alga, Oyil and Shalkar) areas without large industrial complexes-in 5 cases. do not have large production areas, but their The frequency of fetal karyotype in regions with environmental situation remains low. In regions and without large industrial complexes is shown in with large industrial complexes, the number of Table 4.

Table 4 – Frequency of fetal karyotype in regions with and without large industrial complexes

Fetuses with normal Fetuses with karyotype karyotype disorders Number of Number of N Description of regions Frequency Frequency regions fetuses Quantity Quantity (%) (%) 1 Aktobe city 1 420 387 92,1 33 7,9 Regions with large industrial 2 5 131 105 80,2 26 19,8 complexes There are no large 3 3 39 34 87,2 5 12,8 manufacturing enterprises

Analytical work on the fourth table was carried considered as social and economic conditions, i.e. it out in three groups. The frequency of fetuses with is known that the condition of the urban population is deviations in the karyotype was 7.9%, 19.8% and much better. The most common disorders in the fetal 12.8% in Aktobe and in nearby regions with and chromosome set are the places of oil production and without large industrial complexes, respectively. It processing, gas-and cement-producing complexes. was found that the frequency of karyotype disorders in pregnant women living in settlements of regional Conclusion districts is 1.6 and 2.5 times higher, respectively, compared with chromosomal abnormalities among Due to the presence in Kazakhstan of enterprises pregnant women. One of the reasons for this can be of mining, chemical, oil and gas processing,

73 Chromosomal anomalies of the fetus of pregnant women from settlements prone to technogenic pollution ferrous and non-ferrous metallurgy, production of was 12.8%, while in pregnant women living in 5 building materials in Aktobe and nearby region, districts, where large production complexes are the environmental situation is considered one of located, karyotype deviations were 19.8%, i.e., the most unfavorable. During the study, karyotypes the number of cases in which there are no major of fetuses of pregnant women were extracted for production complexes. They showed an increase analysis. The total deviations of the karyotypes were of 1.5 times. It was noted that the frequency 10.8%. Of these, genomic anomalies were observed of chromosomal abnormalities in the fetus of in 92.2%, and structural anomalies-in 7.8%. Among pregnant women in Aktobe is 1.6 and 2.5 times the genomic abnormalities, the karyotype of Down lower than in pregnant women living in settlements syndrome was 60.9%. Depending on the areas of of other regions, compared with violations of residence of pregnant women, there was a variability the fetal karyotype. It can be assumed that such in the frequency of fetal chromosomal pathologies. large differences relate to social and economic The frequency of chromosomal abnormalities in conditions. the fetus of pregnant women in Aktobe, where the To sum up, it can be concluded that technogenic production facilities of ferrous and non-ferrous pollution of large industrial complexes (enterprises metallurgy are located, was 7.9%. The frequency of ferrous and non-ferrous metallurgy, mining, of abnormalities in the chromosome set of pregnant chemical, oil and gas processing, cement industry, women living in 3 districts of the Aktobe region, etc.) causes genomic and structural disorders of the where large production complexes are not located, genetic apparatus of human cells.

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