The effect of some pesticides on lipid peroxidation and on the content of cytochrome p-450 of mitochondrial membranes and liver microsomes of pregnant rats and their embryos

. The article presents data obtained in experimental studies on the effect of pesticides - butylcaptax and droppa on lipid peroxidation of mitochondrial membranes and microsomes of liver cells of pregnant rats and their embryos. It has been shown that inoculation with pesticides intensifies NADP.H-and ascorbate-dependent LPO both in mitochondria and in liver microsomes of pregnant rats and their embryos. The level of MDA is higher in case of poisoning with butylcaptax on the 3rd day of pregnancy. A decrease in the content of the microsomal enzyme P-450 in the microsomal fraction of the liver of pregnant rats was also found with the introduction of butylcaptax and dropp. This decrease is more pronounced on butylcaptax poisoning on the 3rd and 19th days of pregnancy.


Introduction
One of the central non-specific mechanisms of stress damage is the stimulation of free radical processes (oxidative stress).
The imbalance caused by the stimulation of free radical oxidation (CO) and a decrease in the activity of antioxidants (AO) often leads to the accumulation of CO products, which have an extremely high reactivity, causing modification of the structure of lipids, proteins and nucleoproteins, as well as other important groups of biological molecules, which leads to subsequently to the induction and progression of various pathological processes in the human body [14].
The pathogenesis of oxidative stress is based on specific changes in cellular structures, activities of a number of cytosolic and mitochondrial enzymes, depletion and damage of antioxidant defense systems, changes in the spectrum of metabolites, and some other changes that are mainly destructive in nature.The mechanisms causing the destructive effect of ROS include lipid peroxidation of all cell membranes, oxidation of cell proteins at tyrosine, cysteine and serine residues, oxidative damage to cellular and mitochondrial deoxyribonucleic acid (DNA), a decrease in the redox potential of the cell due to the oxidation of glutathione and NAD (P)H [15].
Detoxification processes in the body are carried out by all organs, but mainly by the liver.Under the action of various chemical pollutants, systemic enzymatic disorganization and labialization of membranes, the most important intracellular organelles (mitochondria, endoplasmic reticulum, lysosomes) occur [1].
It has been established that pesticides penetrate the cell, accumulate in it, and change the functioning of enzyme systems [3].The results of the research showed that the activity of alkaline phosphatase increased by 2-3 times compared to the norm on the 30th and 50th days after the poisoning of karate rats.The results of determining catalase activity show that catalase activity sharply decreases on the 5th day of karate poisoning, the activity is 55.5% of the activity from the control, and by the 5th day 75% of the activity is detected, which is 24% below the norm.It was also shown that experimental rats inoculated with the pesticide on the 5th day after poisoning showed a sharp increase in the activity of SOD and glutathione reductase.The observed increase in enzyme activity can be considered as a protective, adaptive reaction of the body against lipid peroxidation, which disrupts the structure and function of cell membranes [4,5].
It is necessary to note the effect of pesticides on the structure and functions of mitochondrial and microsomal membranes.Studies at the cellular level make it possible to characterize the effect of pesticides on a specific process, the role of a foreign substance in certain structures.This is especially important for determining the interaction of a foreign substance with cell components at the early stages.
A large amount of experimental data has been obtained, confirming the assumption of B.N. Tarusov about the leading role of LPO processes in the development of various pathological conditions of cells [6,12].
Many pesticides used in agriculture and the national economy adversely affect the liver, brain, heart and blood system of mammals.The mechanisms of the molecular action of pesticides on cells and their organelles (mitochondria, microsomes) are different and, as a rule, lead to a sharp increase in the amount of free radicals from the mitochondrial respiratory chain [14,15].The intensity of ROS formation under the action of pesticides causes an increase in lipid peroxidation of the inner and outer mitochondrial membranes [15].
At the early stages of ontogenesis in mitochondria and microsomes of the liver of rats, the rate of non-enzymatic and enzymatic lipid peroxidation increases markedly.The rate of ascorbate-dependent LPO reaches maximum values both in microsomes and in mitochondria of the liver of 2.5-month-old rats (an increase of almost 5 times compared to the liver of newborns).The maximum rate of non-enzymatic LPO was noted in the liver of 3-day and 2month-old rats.According to different authors, the rate of non-enzymatic lipid peroxidation in the membrane structures of the embryonic liver is higher or lower than in adult animals [7,8].
The intensity of lipid peroxidation in rat fetal liver homogenates is higher than in adult animals, and after birth up to the age of 6-10 days it gradually decreases [9,13].
As shown by a number of researchers [16], the intensification of free radical processes, PUFA peroxidation is observed during the development of a general nonspecific adaptation syndrome (stress), that is, in almost most acute diseases and conditions, exacerbation of chronic diseases, intoxications, burns, injuries, operations .The biological expediency of this intensification is based on the enhancement of eicosanoid synthesis, membrane renewal, and detoxification processes under emerging extreme conditions.The accumulation of reactive oxygen species, peroxides in significant amounts (as observed under the action of radiation, ultraviolet radiation, hyperbaric oxygenation, intoxication, including alcohol) can be accompanied by a number of negative changes.

Purpose and task
The results of the above studies make it possible to judge the degree of disturbances in metabolic processes caused by pesticides.These data show the need to study the functional state of cells and cell organelles under the influence of pesticides.The aim of this work was to investigate the effects of some pesticides on the functional state of mitochondrial membranes and liver microsomes in pregnant rats and their embryos.Based on this, we set the task to investigate the effects of pesticides -butyl captax and dropp on lipid peroxidation of mitochondrial membranes and microsomes of liver cells of pregnant rats and their embryos at different stages of pregnancy.

Materials and methods
White female Wistar rats weighing 180-200 g served as the objects of research.Animals were priming with butylcaptax and dropp at a dose of 1/10 LD50 on the 3rd, 13th and 19th days of pregnancy intragastrically with a special probe for 5 days.Animals were slaughtered on the 20th day of pregnancy, when the embryo reached a significant size, at the end of organogenesis.In the experiments, mitochondria and microsomes of the liver of embryos and the maternal organism were used.
Mitochondria were isolated by the differential centrifugation method proposed by Parsons, M.Y.Simson [13].
The microsomal fraction from the liver was isolated by differential centrifugation [13].
The activity of ascorbate-dependent and NADPH-dependent lipid peroxidation in mitochondria and microsomes was determined by the content of malondialdehyde (MDA).The MDA content was determined using 2-thiobarbituric acid [11].
The determination of cytochrome P-450 was carried out according to the method of T. Omuro, Sato [13,17], based on the change in the absorption value of the complex reduced by carbon monoxide.Measurements of the content of cytochrome P-450 in a suspension of microsomes 1-2 mg / ml of protein in an incubation medium containing 0.1 M K-phosphate buffer, pH 7.4 were carried out on SPECORD UV VIS [Germany].

Research results and discussion
Unsaturated fatty acids of biological membrane phospholipids are optimal substrates of peroxidation in terms of their structure.In recent years, lipid peroxidation of biological membranes has been given a special role in the pathogenesis of various diseases.With advanced LPO process in phospholipids of biomembranes, the amount of unsaturated fatty acids decreases with a simultaneous increase in the amount of saturated ones.
We have studied the effect of pesticides -butylcaptax and dropp on the content of malondialdehyde (MDA), one of the end products of lipid peroxidation, in the liver of pregnant rats and their embryos (Fig. 1 and 2).It was found that with the introduction of butylcaptax on the 3rd day of pregnancy, NADP.H and ascorbate-dependent LPO in pregnant rats increased by 61 and 75%, respectively.The content of LPO products in the microsomal fraction increases by 60 and 72%.Inoculation of animals with this pesticide on the 13th day of pregnancy also enhances enzymatic and non-enzymatic lipid peroxidation.In mitochondria and microsomes, NADP-H-dependent LPO increases by 50 and 75%, respectively, and ascorbate-dependent -by 74 and 66%, but these figures are lower than after priming on the 3rd day.A similar picture is observed in case of poisoning on the 19th day of pregnancy.Intensification of NADP.H and ascorbate-dependent LPO in mitochondria is 62 and 58%, respectively, in microsomes -67 and 62%.Consequently, in microsomes, the process of formation of LPO products is much more intense than in mitochondria.
The same picture was revealed in embryos on the 3rd, 13th and 19th day of their development.In particular, when priming on the 3rd day, the intensification of NADP.H and ascorbate-dependent LPO in mitochondria was 86 and 57%, in microsomes -74 and 47%.When seeded on the 13th day of development, the MDA level increased by 57 and 44%, respectively, in mitochondria and by 55 and 36% in microsomes.On the 19th day, enzymatic and non-enzymatic lipid peroxidation increased both in mitochondria (by 69 and 41%) and in microsomes (54 and 39%).The high content of enzymatic LPO in liver microsomes can be explained by the fact that in case of NADP.H-dependent LPO, the substrate in microsomes is polyunsaturated fatty acids -phospholipid acyls localized near the components of the electron transport chain, and in ascorbate-dependent -other fatty acids [8,12].

Mitochondria Microsomes Ascorbate-dependent LPO pathway
The effect of dropp was studied at the same time of pregnancy and embryonic development.On the 3rd day of pregnancy in rats, an increase in enzymatic and non-enzymatic lipid peroxidation was observed, respectively, by 36 and 43% in mitochondria and by 60 and 50% in microsomes.Poisoning of rats on the 13th day of pregnancy led to an increase in the content of MDA in mitochondria and microsomes by 29 and 45, 50 and 37%, respectively.when pregnant rats were inoculated on the 19th day of pregnancy, the level of MDA in mitochondria increased by 50 and 79%, and in microsomes by 47 and 45%.
Similar changes in the level of oxidation products were noted in the mitochondria and microsomes of the liver of embryos poisoned with dropp on the 3rd, 13th, and 19th days of development.
On the 3rd day of development, the level of enzymatic and non-enzymatic lipid peroxidation in the mitochondria of the liver of embryos increased by 66 and 42%, in microsomes -by 60 and 50%.On the 13th day, it increased by 41 and 50% in mitochondria and by 44 and 37% in microsomes.On the 19th day, enzymatic LPO in mitochondria and microsomes increased by 57 and 47%, respectively, ascorbate-dependent -by 52 and 33%.At the same time, the content of enzymatic LPO in mitochondria, ascorbate-dependent LPO in mitochondria and microsomes was significantly lower than in butyl captax lesions.NADP.H-dependent LPO in microsomes was at the same level as in the liver of pregnant rats after poisoning with butylcaptax.
Thus, inoculation with pesticides marked intensification of NADP.H-and ascorbatedependent LPO both in mitochondria and in liver microsomes of pregnant rats and their embryos.The level of MDA is higher in case of poisoning with butylcaptax on the 3rd day of pregnancy.
It has been shown that the oxidation products of butylcaptax and dropp stimulate peroxidation reactions in the membranes of mitochondria and microsomes of the liver of the maternal organism and the embryo, which leads to their damage.
It is necessary to note the influence of pesticides on the function of microsomal membranes.Studies at the cellular level make it possible to characterize the effect of pesticides on a particular process, the role of a foreign substance in certain structures.This is especially important for determining the interaction of a foreign substance with cell components at the early stages.
Most of the metabolic transformations that foreign compounds undergo in the body are provided by enzymes localized in the membranes of the endoplasmic reticulum of liver cells.Microsomal enzymes catalyze hydroxylation reactions of foreign compounds, as well as important endogenous substrates (saturated and unsaturated fatty acids, phospholipids, etc. [2,6].
The endoplasmic reticulum of the liver contains a physiologically important system of biological oxidation of xenobiotics and endogenous substrates in the sense of "protecting the internal environment of the body" [18,19,20].Most of the metabolic transformations that foreign compounds undergo in the body are provided by enzymes localized in the membranes of the endoplasmic reticulum of liver cells.
The activation of molecular oxygen in the membranes of the endoplasmic reticulum is carried out by an enzyme complex in which the hemoprotein cytochrome P-450 acts as an enzyme that binds the substrate and activates molecular oxygen.The enzyme system uses NADP.H as an electron donor required for oxygen activation [25].
At high concentrations of xenobiotics, that is, when cytochrome P-450 is fully saturated with substrates, mitochondrial oxidation is inhibited and prerequisites arise for the regulatory switching of the flow of reduced equivalents from mitochondria to the endoplasmic reticulum.At the same time, cytochrome P-450 is activated and detoxification of foreign substances occurs faster [26,27].
Microsome monooxygenase, which is characterized by wide specificity due to the existence of multiple forms of cytochrome P-450, has the ability to oxidize hundreds of substances of various chemical nature, preventing the accumulation of toxic hydrophobic compounds in the body [28,29].
The total content of cytochrome P-450 hemoprotein in liver microsomes of pregnant rats is significantly higher than that of non-pregnant rats [30,31].
In the embryonic liver of rats, the endoplasmic reticulum is predominantly represented by a rough subfraction.At first, it looks like bubbles, which then turn into tanks.Smooth membranes appear in the postnatal period.There is very little endoplasmic reticulum in the third phase of pregnancy, its monooxygenase activity is very low.GER appears in significant numbers at the age of one week [32,33].On the 20th day of pregnancy, cytochrome P-450 was not detected spectrophotometrically in the embryonic liver of intact rats.In the following days until the moment of delivery, the concentration of hemoprotein gradually increases.The peak of the differential spectrum of cytochrome P-450 in these periods is stably fixed at a wavelength of 450-450.5 nm [33,35].In the rat embryo, cytochrome P-450 apoprotein is present just before delivery and remains approximately at the same level for several days after delivery.Since cytochrome P-450 was not found in the liver, it was assumed that heme does not attach to the protein before birth [13].A characteristic feature of the organization of microsome membranes is their high content of polyunsaturated fatty acids (PUFAs), which can undergo intense oxidation by the free radical peroxide mechanism [35,36] introduction of lysophosphatidylcholine into rat liver microsomes causes the conversion of cytochrome P-450 to P-420, which is reflected in their differential spectra.Phospholipids are necessary components of the monooxygenase system of microsomes that promote the binding of metabolizing substrates by cytochrome P-450 [37,38].
The enzyme system of microsomal oxygenases plays an important role in the metabolism of chemical carcinogens.The terminal site of this system is the cytochrome P-450 enzyme, which plays the main role in the biotransformation of foreign substances [38].The changes we found in the lipid matrix of microsomal membranes under the action of pesticides suggest changes in the properties of the cytochrome P-450 enzyme complex.
In the next experiment, the effect of dropp on the content of cytochrome P-450 in the microsomal fraction of the liver of pregnant rats was studied.When the organism of pregnant rats was poisoned with butylcaptax on the 3rd, 13th and 19th days of pregnancy, the content of cytochrome P-450 in the microsomal fractions of the liver decreased.On the 3rd day of pregnancy, the content of cytochrome P-450 decreased by 42%, on the 13th day -by 25%, on the 19th day -by 36%.With the introduction of droppa into the body of pregnant rats on the 3rd, 13th and 19th days of pregnancy, the content of cytochrome P-450 in the microsomal fractions of the liver decreased.Under the action of dropp, the content of cytochrome P-450 decreased by 24% on the 3rd day of pregnancy, by 13% on the 13th day, and by 23% on the 19th day (Fig. 3).

Conclusion
Thus, from the above data, the activation of LPO processes causes the degradation of cytochrome P-450 and the weakening of microsomal oxidation processes.Metabolic activation of xenobiotics, leading to the formation of free radicals, is carried out in the electron transport chain of the endoplasmic reticulum.The key role is played by cytochrome P-450.With the introduction of butylcaptax and dropp, the content of cytochrome P-450 in the microsomal fraction of the liver of pregnant rats decreases.This decrease is more pronounced on butylcaptax poisoning on the 3rd and 19th days of pregnancy.In this regard, our data are consistent with the results of earlier studies, according to which the processes of microsomal oxidation and lipid peroxidation are alternative: activation of one of them leads to suppression of the other.Inactivation of cytochrome P-450 confirms the disturbance of lipid peroxidation processes in microsome membranes and, in our opinion, plays a leading role in the mechanism of pathology development.

Fig. 1 .
Fig. 1.The content of lipid peroxidation products in mitochondria and microsomes of the liver pregnant rats under the action of butylcaptax and dropp (mgmol/mg protein)

Fig. 2 .
Fig. 2. The content of lipid peroxidation products in mitochondria and microsomes of the liver of embryos under the action of butylcaptax and dropp (mgmol/mg protein) /doi.org/10.1051/e3sconf/20234520101717 452

Fig. 3 .
Fig. 3.The effect of butylcaptax and dropp on the content of cytochrome P-450 in microsomal fractions of the liver of pregnant rats, nmol/mg of protein /doi.org/10.1051/e3sconf/20234520101717 452