The use of modern technologies in the field of molecular biology in studying the toxicity of pesticides
The second half of the last century witnessed a great development in the field of biological sciences. This was the result of the use of basic sciences (chemistry, nature, and mathematics) in the study of life sciences, which resulted in what is known as "Molecular Biology".
Definition of Molecular Biology:
It is the study of mechanics that help to understand biological processes through understanding the chemical structures of the complex molecules that make up living cells and the changes that occur in these structures during the various biological processes at the lowest levels of the cell.
* And modern biotechnology has relied on the scientific achievements that resulted from this new concept in the study of biological sciences at the molecular level and the ability to convert those achievements into methods and materials used in industry to produce goods and services that did not exist before in various fields such as food, energy, environment ... etc.
* The technology provided the opportunity to obtain new compounds and materials that could not be produced before, whether using mutations or regular breeding methods. It also provided the opportunity to develop new methods of medicine, whether for diagnosis or treatment that did not exist before.
* Due to the natural connection of this technology with the basic sciences and the sciences of molecular biology, it has become known as Molecular biotechnology, and the important technique in molecular biology is the Recombinant DNA technique, which enables scientists to transfer part of the DNA of a particular organism to another organism in the process of Transformation and it is considered one of the important techniques in Molecular biology, which is used routinely to transfer specific genes to bacteria in the process of gene cloning or DNA cloning, which provided the opportunity for direct transfer of genes between different organisms using this technology to give the strain new productive characteristics that were not previously present in what is known as genetic engineering.
The cells of microorganisms and higher organisms of plants and animals can be used as biological plants to produce compounds such as insulin and interferons. Etc.
* According to biomolecular information, DNA is the substance through which genetic traits are transmitted and it is the one that controls the interactions that occur in the cell through the organization and construction of proteins
- the part that determines the arrangement of the different safety acids in the two protein molecules and the part of the DNA, which determines the arrangement of acids The amino is in a specific protein of the cell protein known as a gene.
* The use of microorganisms in biotechnology research:
Microbes as sources of enzymes:
Microbes are an excellent and suitable source for obtaining enzymes and they were the source of several enzymes that were used commercially for the first time in the study of molecular biology, Restriction enzymes, DNA polymerases. These enzymes were isolated from E-coli and polymerases became available for many techniques for recombinant DNA and the use of the polymerase chain reaction ( PCR) to replicate DNA and an example is Tag DNA polymer.
* The Tag is a stable enzyme when exposed to heat and is considered an essential enzyme in PCR technology. This enzyme was isolated from a microbe that lives in hot water, which is Thermopiles. There are a large number of enzymes fixed at high temperatures that are isolated from heat-loving microbes and are widely used in many From techniques such as PCR.
The different applications of biotechnology:
(1) Healthcare:
1- The use of PCR techniques in early detection
2- Genetic therapy.
3- The manufacture of medicine with modern technologies, as happened in the production of human insulin From bacteria.
4- Antibiotics.
(2)Agricultural uses:
* Food production as genetically modified foods.
* Hybridization between plant genera.
* Biocides.
Reducing the use of herbicides.
Natural protection for plants
(3)Industrial uses:
In the past, industrial products relied on non-degradable petroleum products, which led to environmental pollution and an increase in solid waste. However, biotechnology can contribute to more environmentally friendly and relevant alternatives to the field of materials and energy, as well as currently producing many biological catalysts such as enzymes with biotechnology.
There are now more than 45 enzymes as biostimulants in various industrial applications such as:
- Carbohydrates
- Peptide hydrolyzate enzymes
- Lipolytic enzymes
- Redox enzymes
- Transport enzymes
- Proteolytic enzymes
Environmental uses:
Some modern techniques are used to rid the environment of the pollutants attached to it, such as the ridding of gasoline from (MTBE) by using bacteria - as well as biotechnologies are used to get rid of oil residues in oil tanks in the Gulf countries.
Using microbes to get rid of toxic materials in the environment Bioremediation:
One of the problems that humans face at present is the problem of getting rid of toxic substances in the environment that have accumulated over the years as a result of human use of various materials.
Biological treatment technology is used to remove pollution from land, surface and subsurface water, sewage, and industrial water, and the effect of this technology is visible in the case of organic pollutants of petroleum residues, pesticides, and other organic compounds that can be destroyed by microorganisms.
* Several things must be taken into consideration when applying technology to remove pollution, including the following:
1- Specialized starch microorganisms must be available enzyme specialist for removing a contaminant.
2- These microorganisms must be effective to destroy them at a reasonable rate to reach the permissible limit of this contaminant.
3- These microorganisms must not produce toxic materials as a result of their activity to destroy the pollutant.
4- The polluted environment does not contain other compounds that have inhibitory activity for these microorganisms.
5- The target pollutants must be available for microorganisms to be destroyed.
6- That the environmental conditions are suitable for the growth and reproduction of microorganisms, such as the availability of oxygen or other electron receptors, the suitability of moisture and temperature, as well as the sources of energy and nutrients.
7 - That the cost of treatment in this way is appropriate and less expensive than any other operations that lead to the same goal.
* The most important point that controls the success or failure of Bioremediation is the extent of availability of pollutants for microorganisms (Bioavailability) because the lack of availability of this compound is a barrier to the success of the process of destruction and thus the process of removing pollutants from the environment.
The lack of availability may be either as a result of strong adsorption of the compound, such as the case of pesticide absorption on the surfaces of the clay complexes in the ground, or the presence of the pollutant in a mixture between the water or non-aqueous form, as in the case of most petroleum contamination of ground or surface water, or if the pollutant is in a place difficult for microorganisms Reach him as a result of its size.
It is considered the progress in the science of genetic engineering to produce living organisms. Genetically engineered with a high ability to break down pollutants is one of the breakthroughs in the development of Bioremediation.
Getting rid of industrial chemicals using microbes:
Microbial degradation of Xenobiotics
Xenobiotics means industrial chemical compounds that are created by humans: synthetic organic chemicals- man-made chemicals. These compounds such as organic pesticides and such as organic solvents and others. Toxic waste disposal causes a big problem, for example, the world's production of one chemical compound from the compounds that exist.
In the environment, as a result of its various uses, which is phenol pentachloride (PCPs), chemical treatments and incineration processes were used to get rid of residues of these compounds, but these processes are costly and often cause various environmental difficulties and problems.
Getting rid of industrial chemicals using microbes:
Microbial degradation of Xenobiotics
In the mid-sixties (1960), several microorganisms were discovered in the soil, soil microorganisms capable of breaking down Xenobiotics such as herbicides and other pesticides. This indicates that getting rid of the remnants of these compounds in the environment by microbes may be an important and effective method.
The predominant of these soil microorganisms that break down these compounds are of the genus Pseudomonas.
The breakdown of complex organic molecules usually requires the integration of several different enzymes, and the genes that code for these biodegradative enzymes are sometimes found on the DNA of a bacterial cell's chromosome.
* Organic aromatic compounds that do not contain halogens:
* Non-halogenated aromatic compounds:
The biological conversion or breakdown of these compounds is carried out by bacteria most of the time into catechol or protocatechuate, and then through a series of oxidative cleavage reaction, the catechol or protocols are transformed into either Succinate, Acetyl CoA, or Pyruvate, and these compounds enter the metabolism of most organisms. Then.
* Organic aromatic compounds containing halogens:
This group includes most of the organic pesticide compounds used. Such compounds are converted to Protocatechuate Catecol or Hydroquinines or the corresponding halogen derivatives of these compounds with the same enzymes that break down the non-halogenated compounds.
However, for halogen compounds, the rate of conversion of the compound by enzymes is inversely proportional to the amount of the halogens it contains, meaning that the more halogens in the compound, the faster it breaks down physiologically.
Dehalogenation process:
It is the process of removing the replaced halogen from the organic compound and replacing it with an OH group.
* Molecular biology and pesticide toxicity:
A- Carcinogenic effects:
It is a group of studies aimed at measuring the extent of the effect of pesticides or environmental pollutants in causing damage to the genetic material, whether at the cellular or at the molecular level, in which modern technologies such as PCR in the field of molecular biology are introduced to reveal the ability of these chemicals to cause mutations or tumors. Cancerous.
There are many studies on the spread of disasters that have determined the link between exposure to pesticides and the occurrence of cancer, as these pesticides produce their carcinogenic effects with mechanisms that do not affect the chromosomes, such as stimulation or the proliferation of cytoplasmic organisms or a hormonal imbalance.
On the other hand, it was found that pesticides may affect the process of cancer formation in other ways such as A change in the chromosomes or an increase in the growth of tumor cells, and chlorinated hydrocarbon compounds such as DDT are considered persistent pesticides in the environment for a long time after preventing their use with carcinogenic toxicity as they cause breast cancer as a result of their effect on estrogen.
Studies have shown that women with breast cancer contain high levels of chlorinated hydrocarbons in breast fat and serum fats, as well as many halogenated hydrocarbons, inhibit the areas of contact between cells in the cellular membranes of the normal human breast, and these compounds change the surface area. Post-transposition of the genetic code is warped and can stimulate the formation of tumors in human breast tissue.
By the interaction of organophosphorus pesticides in biological molecules by phosphorylation of the amino acid serine present in the lysing enzymes or by adding the alkyl group to the large molecules such as DNA, which are responsible for acute toxicity and stimulating the process of cancer formation
By using molecular biology, the mutations occurring in the P53 gene were detected using PCR technology and SSCP techniques. It was found that the P53 gene and the protein that encodes it is generically called "the general guardian of the genetic system" because of the critical role it plays in the continuation of the cell’s life or death.
Recent research has proven the correlation between The emergence of cancerous tumors in humans and the occurrence of the mutation in the P53 gene and on the other hand this gene in the event of damage to the DNA molecule, this gene can slow down the succession of the cell cycle or stop the cycle until the damage in the two DNA molecules can be repaired. In the case of multiple damages, the gene is repaired It also can initiate events that lead to cell suicide, which is called apoptosis
The p53 gene controls the cycle of cell division by inhibiting one of the transitions. In the life cycle of the cell, which is the stage that precedes the replication of two DNA molecules, and thus the cell stops dividing until the damage caused in the genetic material is repaired, as the gene also can stimulate genes for the synthesis of molecular repair enzymes DNA
The link between P53 and cancer:
The scientist (Hollstein et al 1991) suggested that cancer appears as a result of the accumulation of a group of mutations that lead to a defect in the cell life cycle and that there is a strong relationship between the occurring mutation and this particular gene and the emergence of cancerous tumors as this gene is in a state of rapid cell division without control (such as the accident In cancer cells) it stops this division by interfering with the processes of cell division.
And thus stops the rapid division in cancer cells, and therefore it was called the cancer suppressor gene T, and through the genebank, the nucleotide sequence of this gene was determined in humans, and its primer was made by a program specializing in that, and then duplication of this gene is done using PCR technology, and then a detection test For mutations in this gene by using single-strand conformation polymorphism in this technique (SSCP.
the double molecule of DNA is converted into a single strand in the presence of formamide, and then the separation of the polyacrylamide is done by the generation of the electrophoresis device, and by this technique, the point mutation can be determined in the gene P53 by the emergence of different pandas from the control using this technology in pesticide toxicity tests gives an early, fast and cost-effective picture of its potential to cause a carcinogenic effect on test animals, unlike the traditional cancer effect test, which takes 3 - 5 years
Evaluate mutagenicity resulting from pesticides:
It is a group of studies that aim to measure the extent of the effect of pollutants on causing genetic damage to cells, which can be detected by measuring some criteria such as:
1- Chromosomal aberration tests, which include the mutagenic ability of chemical substances through the detection of chromosomal structural changes upon exposure to the tested compound.
2- Micronucleus assay in the marrow cells of mammalian animals. It is a test that aims to detect damage resulting from exposure to the toxic compound on chromosomes or the mitotic system in cells producing red blood cells.
Immunotoxic effects
To study pesticide toxicity:
It is interested in studying the chemistry and nature of western substances that enter the body, antigens, and antibodies formed by them, as well as the interactions that take place between them. Exposure to some pesticides leads to clear changes in immunity or functions and increased sensitivity to diseases. Anemia has been observed accompanied by hypersensitivity as a result of exposure to chlorine pesticides.
Organic As some cases of allergies have been seen, especially skin sensitivity to many pesticides, studies have also shown that the decrease in immunity is due to the inhibition of ester-degrading enzymes in the osteoclasts of Western bodies, which may lead to the occurrence of lymphomas as a result of exposure to phosphorous pesticides for a long time.
It uses molecular biology to study the effects of pesticide toxicity on the immune system by Antigens: -
Antigens are chemical compounds with a large molecular weight that can stimulate the immune system in the body to form specialized antibodies and can interact with those antibodies that are formed and there are so-called complete antigens, for example, all proteins and polysaccharides if there is a chemical compound with Large molecular weight but unable to stimulate the immune system to form antibodies called incomplete antigens that can interact with antibodies formed by complete antigens. For example,
- - incomplete antigens are antibiotics
- - pesticides and steroids
- - complete antigens must be of molecular weight Greater than k.dalton8,000 and less than no response
Could the incomplete antigen turn into complete antigens?
By binding hapten to the carrier, often a protein.
The degree of specialization of the antibodies resulting from binding haptens to a protein carrier is very high - haptens have a specific composition.
When the resulting antibodies interact, they must deal with the same composition of hapten and not a different composition, as the degree of treatment differs greatly according to the different groups of substitution and the position of the group from the legacy, Para, and meta.
Two-Dimensional Immune Electrophoresis
The test is based on two steps:
The first is to electrocute the antigen in a specific direction during a generation that does not contain antibodies.
The second: is another electrical separation in a generation that contains antibodies
Immunoelectron applications: -
* application of immunoelectrophoresis:
1- Determining the degree of purity of an isolated protein.
2- Diagnostic detection of various diseases and knowledge of the efficiency of the various physiological processes in the body.
3- Detecting adulteration of proteins with other proteins.
4- Accurate quantification of chemical compounds such as hormones - pesticides and proteins - toxins - dyes.
5- It is considered one of the accurate methods of dividing and identifying different microbial strains
biomarkers a vital milestone for studying pesticide toxicity
In recent years, interest has increased in the field of biomarkers, which is any biological, biochemical, or molecular parameter that can be measured by chemical, biochemical or microbiological methods.
- The vital milestone is divided into three types:
- biomarkers of exposure: specific to exposure
- biomarkers of effect: by the effect
- biomarkers of susceptibility: by sensitivity
An overlap of biomarker types may occur. For example, some types of biomarkers for exposure such as the formation of DNA additive may also be considered a biomarker type of effect.
When the chemical substance is absorbed and distributed through the plasma, it is linked to the molecular target, either directly or after it is activated utilizing catabolism, then a series of chemical and physiological changes occur, which induce the occurrence of apparent and morphological symptoms, and therefore human exposure can be estimated at any stage of this process.
Toxic compounds and their degradation products can be measured in biological fluids. One of the best types of the biomarker for exposure is that it gives a real indication of exposure. The ideal biomarker is distinguished by its specialization and small effects can be estimated from it.
Enzymology as biomarker enzymes as biomarkers: -
Measuring the enzyme acetylcholinesterase in red blood cells is one of the oldest and best ways to detect the toxicity of phosphorous compounds. It has been found that measuring this enzyme in RBCs reflects what happens in the brain and diaphragm than if this enzyme was estimated in plasma as well as using the neurotoxic enzyme in Lymphoid tissue as a tool for monitoring late neurotoxicity, as in cases of poisoning with chloroperovos, where the rate of inhibition of this enzyme was 60%.
- Elisa determination of some enzyme
- Gst as biomarkers in detoxification of pesticides
- Superoxide dismutase in detoxification of pesticides
- Glutathione peroxidase in detoxification of pesticides
- Glutathione reductase in detoxification
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