Assessment of Neurotoxicity of Cultivated Plants Grown in the Neighborhood of Industrial Regions of Central Kazakhstan

 

Aiman Konkabayeva^*, Margarita Ishmuratova, Aleksandr Pudov, Rahat Bodeeva, Aidar Aitkulov, Gulmira Tykezhanova, Sayagul Tyrzhanova

E.A. Buketov Karaganda State University, Universitetskaya Str., 28, 100028, Karaganda, Republic of Kazakhstan.

 

ABSTRACT:

The article presents the results of the impact of cultivated plants grown in summer cottage areas of three industrial regions of Central Kazakhstan on the behavioral responses of rats. It was found that prolonged feeding (three months) with vegetables, grown in areas contaminated with heavy metals, caused inhibition of locomotor activity, exploratory activity, duration of freezing behavior and lack of visits to the center that is regarded as the increase of anxiety level of rats. The results showed inhibition of behavioral responses,that testifies of the neurotoxic effect of vegetables grown in the neighbourhood of industrial regions.

 

 

 

INTRODUCTION:

One of the most significant sources of environmental contamination of Central Kazakhstan with heavy metals (HM) is industrial facilities. Contamination of neighbourhoods of large industrial enterprises, significantly worsens the ecological state of the territories, causes changes in chemical composition of all natural components of urban ecosystem, and adversely affects health of the population. According to the risk coefficient, effects of heavy metals (HM) on the environmental objects are placed on one of the first places in order of priority for a number of hygiene problems in the Republic of Kazakhstan^{1, 2}.

 

The process of all migrations and dispersion causes an irreversible increase in the concentration of chemical elements in water, soil, plants and food, i.e. contamination of natural environment and biota. The prospects of studying this problem are determined by the ability of heavy metals to accumulate in the environmental objects (soil, water, plants and animals). The organisms respond to emissions by the accumulation of significant amounts of the pollutant or changes in metabolic processes. All plants most intensively accumulate metals in the period of flowering and fruiting.

 

Vegetable food is the main source of HM intake in organism of human and animals. According to various data^3,4, from 40 to 80% of HM enters the organism with it, and only 20-40% - with air and water. In the experiments with artificial models of digestion, it was found that the bioavailability of HM from soil accounted for B = Cr = Cu = Fe = Pb, and from vegetable samples - Cu = Fe = N = Pb⁵.Therefore, health of population is largely dependent on the level of accumulation of metals in plants used in food.

 

The literature data on the effects of HM on the organism are ambiguous. Thus, the study of the remote potential neurotoxic effects of HM in low doses for 1 and 3 trimester of pregnancy on the neuropsychic development of children did not detect deterioration of the cognitive development of children of preschool age⁶. Along with this, the combined effects of HM (Pb, Mn) in low doses during intake with drinking water during pregnancy affected the progeny development and manifested with neurobehavioral deficiencies⁷. There are known consequences of the ecological effects of HM on the intelligence of school children in Korea⁸.

 

The effects of HM bioaccumulation in food on the nervous system were insufficiently studied. In particular, it was shown that in HM bioaccumulation in fish and seafood, which are an important part of the diet of the inhabitants of North America, it was observed deterioration of development of the visual information processing in school children⁹.

 

In experimental studies of animals neurotoxicity of iron excess when added to the diet of rats was confirmed¹⁰. There is evidence of neurotoxicity of Cu, Zn when their intake in an organism is excessive¹¹. It is well documented in the literature that influence of excessive Cu intake on the emergence of genetic diseases involving significant neurological disorders peculiar to schizophrenia¹². Exceeding of copper level in brain tissue is often found in neurodegenerative diseases¹².

 

The experiments on fish showed that sublethal and below sublethal doses of copper chloride caused neurobehavioral changes. At the same time, transcription levels of protein of thermal shock and hypoxia inducible factor were reduced in some brain regions that may be an early indicator of ecological stress¹³.

 

Thus, there is a lot of works demonstrating the neurotoxic effects of HM on the organism. However, the issue of neurotoxicity of cultivated plants (vegetables and fruits) for the organism grown in the neighbourhood of industrial regions contaminated with HM remains unexplored.

 

In the neighbourhood of the industrial cities of Central Kazakhstan (Zhezkazgan, Balkhash, Temirtau) there is a large number of summer cottages cultivating and actively selling vegetables and fruits and berry cultures. Sources of industrial pollution are generally located at a distance of 12-18 miles. Thus, influence of industrial pollution on quality of foodstuff is not considered and possibility of its adverse effects on health remains unexplored. Therefore, the aim of our research was to explore neurotoxicity of cultivated plants, grown and harvested in the neighbourhood of three different industrial regions of Kazakhstan. The study of this issue will contribute to the development of measures aimed at reducing the risk of intoxication of population with heavy metals.

 

MATHERIALS AND METHODS:

Previously heavy metals were identified in samples of cultivated plants grown and harvested in three different adjoining to industrial enterprises territories (Balkhash, Zhezkazgan, and Temirtau) of Central Kazakhstan. The content of Cu, Fe, Zn, Mn in the ashed samples was determined by the GF AAS method SpectrAA140, Varian, Australia. Content of HM was determined in carrots and apples. Metals were distributed in the following proportions: in vegetables and fruits from summer cottage areas of Balkhash - in carrots Cu>Zn>Fe>Mn, in apples Cu>Zn>Mn>Fe; from summer cottages of Zhezkazgan - in carrots Cu>Mn>Zn>Fe, in apples Cu>Zn>Fe>Mn; from Temirtau - in carrots Fe>Mn>Zn>Cu, in apples Fe>Zn>Mn>Cu.

 

The objects of study were 48 albino rats. The weight of animals was 180-250 g. During the research the ethical principles contained in the Directive of the European Community (86/609EC) and the requirements of the World Society for the Protection of Animals (WSPA) have been complied with. The animals were kept in a free access to water and food. Experiments were carried out in the autumn-winter period in the first half of the day.

 

Rats were divided into 4 groups. Daily diet of rats in three groups included a mixture of cultivated plants (carrots, apples), grown in the respective industrial regions, 50-80 g per rat in addition to the main nutrition for 3 months. Vegetables were collected from summer cottage areas; sources of industrial pollution were generally located at a distance of 12-18 miles. The fourth group was a control group; the rats had a normal diet.

 

Testing of animals was conducted in "open field" and "elevated plus maze" with the subsequent determination of behavioral patterns. The open field test provides simultaneous measures of locomotion, and anxiety. The open field used was a square wooden arena measured (90 x 90 x 25 cm). The floor was divided by white lines into 36 smaller squares (15 x 15 cm). Test "open field", used in accordance with the protocol for screening pharmaceutical preparations and phenotyping animals, allows to reveal significant disorders in neuromuscular, sensory and vegetative systems of the organism.

 

Testing was conducted before, in a month and after 3 months of feeding with cultivated plants under standard light conditions for 5 minutes. For registration of behavior indicators technique of video fixing of the experiment was used. The following parameters were recorded: 1) horizontal activity - number of squares crossed; 2) vertical activity - number of rearing; 3) exploratory activity - number of sniffing; 4) visits to the center of the installation - number of visits; 5) freezing behavior - total time of immobility of the rat in the installation; 6) short grooming - total time; 7) long grooming; 8) defecation - number of acts; 9) urination.

The elevated plus-maze (EPM) was used for testing of anxiety and emotionality. The apparatus consists of 4 crossed arms, two open arms (50 x 10 x 30 cm) and two closed arms (50 x 10 x 30 cm). The maze was elevated 65 cm above the floor. The rat was placed in the center of the maze and the number of entries in open and closed arms, respectively, as well as the time the animal spent in the open and enclosed arms during a period of 5 min test session. All indicators were recorded in the table.

Statistical analysis of the results was performed using Student's criterion and computer program Excel. To determine the difference between the groups, Student’s “t”-test was used. P<0.05 were considered to be significant.

 

RESULTS AND DISCUSSION:

During the experiment in the "open field" test, it was found that the acts of locomotion (horizontal activity) decreased during feeding with cultivated plants for 1 month in the first group (Balkhash) by 15%, in the second group (Zhezkazgan) increased by 106%, while in the third group (Temirtau) increased by 58% compared with background and control values (table 1).

 

Table 1: Indicators of "open field" test in rats after 1 month of feeding with cultivated plants

INDICATORS	Group 1 (Balkhash)		Group 2 (Zhezkazgan)		Group 3 (Temirtau)
	Background indicators	In a month	Background indicators	In a month	Background indicators		In a month
Horizontal activity
- total number of squares	119,8±37,8	101±32,1*	86,5±26,7	179,8±59,2	113,1±44,9		178,7±30,8
Vertical activity
- number of times	13,3±7,0	11,3±6,8	8,8±6,6	14,0±2,0	11,7±7,9	16,2±6,8
Exploratory activity
- number of times	24,5±7,2	14,5±3,5*	27,2±7,9	17,3±8,0	19,8±7,5	14,7±6,4
Visits to the center
- number of times	1,6±0,7	0,0*	1,5±0,5	2,6±1,9	2,0±1,2	2,2±1,1
Freezing behavior
- seconds	76,0±47,5	33,5±14,9***	73,5±59,0	49,3±27,2	59,3±13,4	42,2±4,6
Short grooming
- seconds	15,0±10,9	10,3±2,3*	10,3±2,3	7,4±3,0	6,0±1,5	6,3±1,8
Long grooming
- seconds	27,8±12,8	27,3±10,8	27,3±10,8	25,8±9,6	16,0±6,8	36,0±11,5
Defecation
- number of acts	1,6±0,7	1,3±0,3*	2,0±1,0	1,3±0,4	1,3±0,4		1,0±0,0
Urination	1,5±0,6	2,1±0,8*	2,0±0,7	1,0±0,0*	1,4±0,5		1,0±0,0

Notes - * - p< 0.05; ** - p< 0.01; *** - p< 0.001 in comparison with controls.

 

Vertical activity rearing (standing on hind legs), also decreased by 15% in a month of feeding with vegetable mixture, in the second group increased by 55%, while in the third group - by 35%.

 

Exploratory activity of rats decreased by 40% after a month of feeding with vegetable mixture in the first group, in the second group it decreased by 34% and in the third group by 26.5%..

 

Visits to the center were not observed in the first group after feeding for 1 month, in the second group they were higher than the background and the control by 30%, in the third group changes were not significant compared to the background and the control. The most prolonged freezing response was observed in the first group ((P<0,01).

 

Grooming is a component of exploratory activity. Short grooming act in the first group of animals reduced by time during feeding with  cultivated plants by 32%, in the second group 26%, in the third group duration of grooming did not change. Duration of long grooming in the first and second group did not changed significantly, in the third group it accounted 125% of the background and the control (P<0,01).

 

Emotional manifestations are determined by urination and the amount of fecal boluses. The most significant differences compared to the background and the control in acts of defecation and urination were found in the second group (P<0,05).

 

Thus, feeding for 1 month with cultivated plants contaminated with HM showed that the greatest neurotoxicity was manifested in animals from the first group, which was accompanied by a decrease in motor activity, exploratory activity, duration of freezing, and decrease in grooming time. Neurobehavioral changes were manifested in the rats from the second and third groups but they were less expressed.

 

Prolonged feeding rats for 3 months with cultivated plants from three different industrial regions allowed to reveal significant changes in the behavioral responses of rats in the "open field" test (table 2).

 

Table 2:  Indicators of "open field" test in rats after 3 months of feeding with cultivated plants

INDICATORS	Group 1 (Balkhash)		Group 2 (Zhezkazgan)		Group 3 (Temirtau)
	Background indicators	In three months	Background indicators	In three months	Background indicators		In three months
Horizontal activity
- total number of squares	119,8±37,8	86,8±17,2*	86,5±26,7	71,8±12,4	113,1±44,9		77,1±29,7*
Vertical activity
- number of times	13,3±7,0	8,7±6,5*	8,8±6,6	7,2±4,3	11,7±7,9	8,7±5,6*
Exploratory activity
- number of times	24,5±7,2	12,8±3,9**	27,2±7,9	16,0±5,3*	19,8±7,5	13,0±5,4*
Visits to the center
- number of times	1,6±0,7	0,0**	1,5±0,5	1,0±0,0*	2,0±1,2	1,0±0,0**
Freezing behavior
- seconds	76,0±47,5	100,2±37,2*	73,5±59,0	112,2±43,5**	59,3±13,4	160,3±65,8***
Short grooming
- seconds	15,0±10,9	1,0±0***	10,3±2,3	7,0±3,6*	6,0±1,5	8,8±2,4*
Long grooming
- seconds	27,8±12,8	8,8±4,4***	27,3±10,8	19,0±10,0*	16,0±6,8	17,3±1,8
Defecation
- number of acts	1,6±0,7	1,0±0,5*	2,0±1,0	1,3±0,4*	1,3±0,4		1,0±0,0
Urination	1,5±0,6	1,2±0,5	2,0±0,7	2,0±0,0	1,4±0,5		1,8±0,4*

Notes - * - p< 0.05; ** - p< 0.01; *** - p< 0.001 in comparison with controls.

 

 

Locomotor activity of rats authentically (P <0,05) decreased in the first and third groups of animals fed with contaminated cultivated plants from industrial regions, and exploratory activity authentically (P <0,01) decreased in all studied groups.

 

Along with the decrease in the visits to the center, time of freezing response and grooming duration in all groups of animals fed with vegetables from industrial regions increased. Differences with the background and the control were significant in all groups.

 

Vegetative reactions such as urination and defecation were most expressed in the first and third groups of animals.

Thus, decrease in locomotor activity indicates the domination of inhibition processes in the CNS and reduction of behavioral activity of rats.

 

By results of more aversive test - elevated plus maze that models anxiety, deviations in behavioral reactions compared with the control and background indicators were also revealed during feeding with cultivated plants that were grown in summer cottage areas of industrial regions. More significant differences in indicators were found in prolonged 3-month vegetables feeding (table 3).

 

 

 

Table 3:  Indicators of "elevated plus maze" test in rats after 3 months of feeding with cultivated plants

INDICATORS	Balkhash		Zhezkazgan		Temirtau
	Background	In 3 months	Background	In 3 months	Background	In 3 months
Number of ОA entries	2,6±1,2	1,3±0,4*	1,4±0,5	1,3±0,4	1,6±0,6	1±0*
Number of CA entries	2,3±1,2	1,4±0,5*	1,8±0,6	1,7±0,7	1,6±0,6	1±0*
Time spent in ОA	64,7±49,5	29,0±8.2**	18,0±14,0	14,3±8,8	31,2±24,0	8,4±5,3**
Time spent in CA	252,1±47,2	282,6±21,4	285,0±13,7	290,5±9,0	268,8±24,0	295,3±5,1
Number of vertical rearing:
- in ОA	3,0±1,5	5±0,0	2,0±0,0	0,0	3,3±0,0	1,0±0,0*
Number of  leans over in OA	8,7±5,5	4±0,6*	2,5±1,5	2,0±0,0	3,3±0,4	0,0
Immobility in OA: seconds	19,7±2,3	0	7,0±5,0	13,8±7,2*	8,7±5,7	3,0±0,0**

Notes - * - p< 0.05; ** - p< 0.01; *** - p< 0.001 in comparison with controls.

 

 

 

At the same time indicators of anxiety: the number of entries into of the maze OA, duration of stay in the OA of the maze, proportion of time spent in OA and CA had significant differences with the control and the background in the first (Balkhash) and second groups. The third group showed a tendency to lowering these indicators. Locomotor and exploratory activity was also reduced in rats of groups 2 and 3, in the first group, on the contrary, a number of vertical uprights increased.  The time spent in CA greatly exceeded time spent in OA in all groups. Risk assessment (number of leans over) increased in all groups of rats, but the difference with the background and the control were significant in the first and third groups of rats. Immobility indicator in OA was absent in the first group, was higher than the background in the second group and reduced in the third group of rats. In indicators of the control group of rats having a normal diet, no significant differences with the background data were observed.

 

Decrease in exploratory and locomotor activity, duration of freezing behavior and increasing time spent in the closed arm of the maze are regarded as an increase in the anxiety level of rats. Research results showed that prolonged feeding for 3 months with cultivated plants grown in summer cottage areas of industrial cities, in which the soil and the plants were contaminated with various TM, indicators of behavioral responses were inhibited, that indicated the neurotoxicity effect of the studied foodstuff. At the same time, the longer intake of products contaminated with TM is, the more behavioral disorders are expressed.

 

CONCLUSIONS:

Trace elements are necessary for growth and brain function. Along with this, large doses of essential metals can have toxic effects. Transfer of trace elements in the brain and the intestines is strictly regulated by barrier systems of the organism. The mechanisms by which heavy metals cause neurotoxicity are not fully explored. A number of studies show that essential metals (Cu, Zn, Fe, Mn) in high doses are able to overcome barrier systems, penetrating into neurons and glial cells, and are even found in presynaptic vesicles. It is assumed that TM may be released with neurotransmitters in the synaptic cleft and interact with neurotransmitter receptors¹⁴, which may lead to changes in the behavioral responses of the organism and functions of nervous system in general.

 

Another mechanism by which excess of metals causes neurotoxicity is mediated by production of free radicals, generation of reactive oxygen species and reactive nitrogen species¹⁵. Excess of free radicals leads to the emergence of oxidative stress, DNA damage and degenerative disorders of the nervous system¹⁶.

Based on the aforesaid, it can be assumed that cultivated plants, used for food  by residents of TM contaminated industrial regions of Central Kazakhstan for a long time, may be the cause of many neurobehavioral disorders similar to those described in the literature and observed in cerebral ischemia, brain traumas and epilepsy^17,18.

 

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Accepted on 13.02.2017           © RJPT All right reserved