CC BY
3BIOLOGICAL SCIENCES
EXPERIMENTAL METHODS IN EDUCATION: EXPERIMENTS ON PREGNANT MICE FOR THE
TREATMENT OF ALCOHOLISM
Aslanova U.
Azerbaijan State Pedagogical University https://doi.org/10.5281/zenodo.7197634
Abstract
The article discusses the inhumanity of alcohol and other experiments on pregnant mice in biological education. Use of "indicator" animals with known microbiological status. Animals are placed in the same environment as the population being tested and periodically sacrificed and examined. Naked (athymic) mice are excellent indicators because they are immunodeficient and especially susceptible to pathogens. But these mice are not suitable for detecting changes in serological titer, which may indicate the presence of viruses, since there is no antigenic response in nude mice. To determine the serological titer, it is necessary to use animals with the appropriate immune status.
Keywords: pregnant mice, experience, humanism, blood test, alcoholism, ethyl alcohol
Introduction. The World Health Organization has identified six criteria for diagnosing alcohol dependence. The main and first criterion is a person's inability to control alcohol intake. According to the second criterion, a person feels the urge to drink. This desire can manifest itself both openly and under the pretext of relieving tension and relaxation. As a result of this desire, a person forgets his family, laws and norms of social behavior. The third symptom is that the body easily "tolerates" alcohol. If a person drinks three glasses of vodka and brags that he is not drunk, it is a dangerous syndrome. The problem is that the body tries to process ethyl alcohol into the bloodstream to the best of its ability. But sooner or later the final stage is reached and the mechanism is broken. As a result, alcohol causes mental weakness. That is, the person can greet you and forget about it after a few seconds.
The fourth symptom is abstinence syndrome. This means that a person feels tired, depressed, and his hands swell the day after drinking. Such people look forward to the end of the working day to drink again, even if they do not drink early in the morning. However, those who feel unwell after drinking are also divided into two groups, and those who are poisoned by alcohol can not be considered alcoholics. They simply feel bad the next day because they have been poisoned and do not want to drink again to improve their condition. Alcoholics, on the other hand, feel the need to drink again. However, alcohol poisoning is also directly related to the culture of alcohol consumption. If a person drinks two bottles of vodka, he receives 400 milliliters of ethyl alcohol. This is a lethal dose for a person weighing 70 kilograms. The average person is resuscitated with this dose. This does not happen in alcoholics. Therefore, for drinkers, alcoholism and heroism are synonymous. The fifth symptom is various diseases. That is, a person's laboratory tests are negative or his family breaks up as a result of drinking. He can't stop drinking and can't stop drinking alcohol. The last criterion is that alcohol plays a key role in human life. In other words, according to this criterion, a person's life and personal life are
built around alcohol. Other non-alcoholic interests take second place.
Alcohol is one of the drinks that contains ethyl alcohol, and when drunk, it gives a person temporary pleasure and intoxication, as well as a harmful effect on the body, which has become a habit in many people. Those who are unable to abstain from alcohol are called alcoholics if they consume alcohol to the extent that it impairs their physical and mental health, family, social, and work life. Alcoholism is a disease that occurs as a result of regular consumption of alcohol and is addictive. This disease negatively affects a person's reputation by causing physical and mental disorders. Alcohol is mainly toxic to brain cells. A drunk person celebrates for the first moments, enjoys himself and so on. but as the effects of alcohol increase, all of this can be replaced by irritability, obscenity, irritability, and rudeness. Alcoholism causes dystrophic changes in the vascular system, the development of hypertension, serous liver, impaired renal function, as well as profound mental and somatic disorders. The disease results in mental retardation, alcoholic epilepsy, and sometimes alcohol psychosis. The patient is frightened, sleep is disturbed, hallucinations are observed. Accidents often occur among alcoholics, their ability to work is weakened. Alcoholism has a negative effect on the female psyche. Alcohol consumption during pregnancy is especially unacceptable. Thus, the alcohol consumed in this case has a very negative impact not only on the mother's body, but also on the health of the unborn child. It is more common for women with alcoholism to give birth to children with physical or mental disabilities.
Unfortunately, both in education and in science, have to experiment on animals in order to treat alcoholism and measure the scale of harm in general. Acute alcohol toxicity should be studied in several animal species, and it is imperative to use the species in which the therapeutic effect of the pharmacological substance has been shown and in which long-term toxicity was studied. Usually 2-3 species of rodents and non-rodents (mice, rats.) are used. Groups of male and female ex-
perimental animals are formed separately. For small rodents, each group should contain at least 5-6 females and the same number of males.
An experiment carried out on several lines often shows different susceptibility of the lines. The assessment of nanosafety should be based on the most sensitive line, but in some cases it is necessary to find the gene for such susceptibility. This will help clarify mechanism of nanotoxicity and obtain data for innovative drugs in the future. Unfortunately, toxicologists have never attempted such a design. If mice are used instead of rats, then more radical changes will be needed. According to foreign data, it was mice (91.4%) that were the most commonly used species for gene modification, and only then were rats (3.6%), zebrafish (2.3%), and other species, including chickens, sheep and cows (1.3%). The most frequently used for gene modification of mouse lines C57BL/6 (48.1%), 129Sv (11.1%), Balb/c (4.3%), CD1 (2.5%) and FVB (0.3%). The advantage in using mice is the wide availability of different strains and an extensive database of mouse genetics, mouse/human gene matching, and the relative ease of mouse genetic manipulation. Genetic modifications may include the "insertion" of the necessary gene that gives a response to nanosubstances.
The similarity constants show how many times faster (or slower) intoxication can develop in an animal compared to humans due to a higher (or lower) intensity of biological processes and, according to our calculations, if a person is taken as a unit: for a mouse ~9.7, rats ~5.2, guinea pig, ~4.3, rabbit, ~2.5, dogs, ~1.7, sheep, ~1.4, pigs, ~0.83 and horses~0.75.
When switching to real time, this means that, for example, intoxication or an adverse drug reaction that develops in a rat in 3 months will manifest itself in a mouse after 1.5 months, in a rabbit - after 6 months, in a dog - after 1 month, in humans - 16 months, and in the horse only after 22 months of exposure to the substance. The existence of allometric dependencies of time for the development of the effects of intoxication is necessary take into account when assessing the adequacy of the duration of pharmacological and toxico-logical experiments when extrapolating data from animals to humans.
Genetic analysis has shown that the number of newly acquired genes per neuron in the human brain is greater than in the chimpanzee brain. In addition, the adult human brain contains significantly more copies of mobile genetic L1 elements than the liver and heart, which is due to the adaptation of the nervous system to the constantly changing environment and the individual's lifelong learning. In 2006, it was found that the human genome contains 212 copies of the MGC8902 gene, which is expressed only in brain neurons and encodes the DUF1220 protein with unknown functions. At the same time, in the genome only 37 copies of this gene were found in chimpanzees, and one copy each in mouse and rat genomes. Hence, it was suggested that the MGC8902 gene may be involved in evolutionary changes in the brain. It is also assumed that the formation of a more complex neural network and, accordingly, a more complex structure of the human brain is responsible for the so-called extended transcription of
the human genome. About 20% of brain neurons normally regenerate, while at the same time, with age, up to 30 g of neurons are irretrievably lost every year.
It has been established that mice infected with toxoplasmosis "run badly" from cats and, most importantly, the parasite is transmitted to cats with great success. It is also believed that toxoplasmas "redistribute" and affect the psyche of the infected person. This is one of them examples of an amazing biological phenomenon when a parasite can be managed by a host.
In real time it has not been possible to create yet human hybrids and to obtain therapeutic antibodies are used only mouse hybrids. Imaging monoclonal antibodies represent their own powerful immunogens, which are formed in the body of patients with HAMA (from the English "human anti-mouse antibodies" -
antimyshynye antibodies of man). To reduce the immunogenicity of such antibodies are carried out with the help of different and sufficiently labor-intensive ways of their "embodiment", transferring mouse sites, determining complementarity (CDR, from English. "Complementarity determiniges" regions). Protein NR2B was found that the process of training and blood pressure are regulated by one and the same biochemical mechanism, in particular, with the participation of protein NR2B. This protein has a beneficial effect on the process of memory and training in mice. Transgenic mice with an additional copy of the NR2B protein gene instantly learned the details of the LEGO designer. This line of mice was called "Arc", in honor of the hero of the TV series "Arc Hauser - Doctor of Medicine." It is assumed that the transfer of the gene NR2B protein in human offspring will allow to create the future "wunderkinds to order". However, the addition of "smart protein" sharply increases the likelihood of stroke in the carrier of an additional copy of the gene.
In view of all this, scientists are studying the effects of alcoholism on pregnancy through various experiments. As mentioned earlier, mice, the gene closest to the human gene, are used for this purpose. Scientists keep pregnant mice in a cotton cell impregnated with ethyl alcohol and its derivatives. Some even drink ethyl alcohol to pregnant mice. Blood tests and other tests of these pregnant mice are then performed.
Let's clarify right away: the main component of any alcoholic beverage is ethyl alcohol (or ethanol). It is he who is responsible for all the changes that occur to our body after drinking alcohol. After swallowing, ethyl alcohol enters the stomach, where approximately 20% of its volume is absorbed into the blood. Most (80%) a little later enters the bloodstream already from the intestines. Once in the blood, ethanol begins to act on the body. The fact is that ethyl alcohol has a different effect on different types of nerve cells, upsetting the balance of the processes of excitation and inhibition.
All alcoholic products necessarily contain ethyl alcohol. For example, what is beer? 100 grams of beer is 6-12 grams of poison (ethyl alcohol), "dressed" in hops, rye, yeast and other ingredients.
What is wine? 100 g of wine is 20 g of poison (ethyl alcohol), "dressed" in grape, apple and other must (juices). Different varieties of grapes, apples -
these are different varieties of wine, but the main component of them is poison - ethyl alcohol, one for all. 100 g of champagne is 17 g of poison, and the rest is various extracts.
What is vodka? 100 g of vodka is 40 g of poison (ethyl alcohol) "dressed" in 60 g of water and various extracts.
What is cognac? 100 g of cognac is 40 g of poison, "dressed" in color, which is drawn from an oak tree and 60 g of water and various extracts.
What is moonshine? 100 g of moonshine is from 20 to 70 g of poison, "dressed" in water and fusel oils.
Once in the body of a mouse, ethyl alcohol is absorbed through the walls of the stomach and intestines, quickly reaches the liver and appears in the blood. The state of intoxication depends on the concentration of alcohol in the blood. The content of alcohol in the blood up to 0.5 g / l usually does not cause immediately noticeable changes. At an alcohol concentration of 0.5-1 g / l, there is no noticeable intoxication observed, but the nerve centers cease to function normally. This is a very dangerous condition, especially for car drivers. As a result of numerous medical examinations, the probability of accidents in this case increases 14 times. With the accumulation of 2 g / l in the blood, the degree of intoxication increases: the gait becomes unsteady.
In the biochemistry of ethanol, an important role is played by the fact that it forms solutions in a wide range of proportions with both water and fats. It is a byproduct of glucose metabolism; the blood of a healthy mouse can contain up to 0.01% of endogenous ethanol, which is a metabolic product. When ingested, ethanol has a narcotic and toxic effect, depending on the dose, concentration, route of entry into the body and duration of exposure, its effect varies. Any dose of alcohol harms the body, there is no safe dose. Under the narcotic effect, its ability to cause coma, stupor, insensitiv-ity to pain, depression of the central nervous system, alcoholic arousal, addiction, as well as its anesthetic effect is indicated. Under the influence of ethanol, endor-phins are released in the nucleus accumbens (Nucleus accumbens). In certain doses to body weight and concentrations leads to acute poisoning and death (lethal single dose - 4-12 grams of ethanol per kilogram of body weight).
The main metabolite of ethanol, acetaldehyde, is toxic, mutagenic, and possibly carcinogenic. There is evidence for the carcinogenicity of acetaldehyde in animal experiments; in addition, acetaldehyde damages DNA.
Long-term use of ethanol can cause diseases such as cirrhosis of the liver, gastritis, necrotizing pancreatitis, gastric ulcer, breast cancer, stomach cancer and cancer of the esophagus (that is, it is a carcinogen), hemo-lytic anemia, arterial hypertension, stroke, cause sudden death of people suffering from ischemic heart disease; can cause serious metabolic disorders. Alcohol may increase the risk of having a child with congenital anomalies of the nervous system and cause growth retardation. The use of ethanol can cause oxidative damage to brain neurons, as well as their death due to damage to the blood-brain barrier. Alcohol abuse in mice
can lead to clinical depression and alcoholism. The intake of alcoholic beverages while taking medication is highly undesirable, since alcohol perverts the effect of drugs and, as a result, becomes dangerous for the life of the mouse.
The negative effect of alcoholic beverages on the results of pharmacotherapy is diverse and depends on various factors: the individual characteristics of the patient, his sensitivity, the severity of the disease, but in all cases, in patients taking drugs and consuming alcohol, the effectiveness of pharmacotherapy is weakened, and sometimes even reduced to nothing. Ethanol can be synthesized in small amounts in the lumen of the gastrointestinal tract as a result of the fermentation of carbohydrate foods by microorganisms (conditional endogenous alcohol).
The existence of biochemical reactions with the synthesis of ethanol in the tissues of the mouse body (true endogenous alcohol) is considered possible, but has not been proven to date. The amount of endogenous alcohol rarely exceeds 0.18 ppm, which is on the border of sensitivity of the most modern devices. An ordinary breathalyzer cannot determine such quantities.
Ethanol can also be harmful to health when inhaled vapors at sufficiently high concentrations. Mutagens can be various factors that cause changes in the structure of genes, the structure and number of chromosomes. By origin, mutagens are classified into endogenous, formed during the life of the organism and exogenous - all other factors, including environmental conditions.
Chemical mutagens are the most common in the group. These include the following groups of compounds:
-some alkaloids: colchicine - one of the most common mutagens in breeding, vincamine, podophyllo-toxin;
-oxidizing and reducing agents (nitrates, nitrous acid and its salts - nitrites, reactive oxygen species);
-alkylating agents (eg, iodoacetamide, epoxyben-zanthracene);
nitro derivatives of urea: nitrosomethylurea, nitro-soethylurea, nitrosodimethylurea - often used in agriculture;
-ethyleneimine, ethyl methanesulfonate, dimethyl sulfate, 1,4-bisdiazoacetylbutane (known as DAB);
-some pesticides (pesticides of the aldrin group, hexachloran);
-some food additives (for example, aromatic hydrocarbons (benzene, etc.), cyclamates);
-oil refining products;
-organic solvents;
-drugs (eg, cytostatics, mercury preparations, im-munosuppressants).
A number of viruses can also be conditionally classified as chemical mutagens (the mutagenic factor of viruses is their nucleic acids - DNA or RNA).
The mechanism of action is based on the formation of so-called DNA adducts with nucleic bases. The more such DNA adducts are formed in a molecule, the more the native structure of DNA changes, which leads to the impossibility of the correct course of pro-
tein biosynthesis processes (transcription and replication) and thereby generates the expression of mutant proteins. Almost all chemical mutagens are sources of malignant tumors (they are carcinogenic), but not all carcinogens exhibit mutagenic properties.
The mechanism of action is based on the formation of so-called DNA adducts with nucleic bases. The more such DNA adducts are formed in a molecule, the more the native structure of DNA changes, which leads to the impossibility of the correct course of protein biosynthesis processes (transcription and replication) and thereby generates the expression of mutant proteins. Almost all chemical mutagens are sources of malignant tumors (they are carcinogenic), but not all carcinogens exhibit mutagenic properties.
Let us consider the mechanism of action of one of the mutagens, benzene epoxide.
By itself, benzene does not have mutagenic activity; is a promutagen. However, as a result of biological oxidation and biotransformation in the cells of the liver, kidneys, and especially in the myeloid tissue of the red bone marrow, it acquires mutagenic properties. Once in the hepatocyte, benzene is immediately hydroxylated by the microsomal oxidation system catalyzed by a group of enzymes of the cytochrome P450 family to epoxide. Benzene epoxide is extremely reactive due to the formation of a strained cycle between the oxygen atom and the benzene molecule. It is able to very quickly alkylate nucleic acid molecules, in particular DNA. The mechanism for the formation of a DNA ad-duct with benzene epoxide is the reaction of nucleo-philic substitution of SN2: an electrophile - in this case, it is an epoxide (due to ring breaking, it becomes electron-deficient), - which interacts with nucleophilic centers - NH2 groups (which are electron-rich) of nitrogenous bases, - forming covalent bonds with them (often very strong). This alkylation property is especially manifested in guanine, since its molecule contains the most nucleophilic centers, with the formation, for example, of N7-phenylguanine. The resulting DNA ad-duct can lead to a change in the DNA structure, thereby disrupting the proper course of transcription and replication processes, which is the source of genetic mutations. The accumulation of epoxide in liver cells leads to irreversible consequences: an increase in DNA alkyl-ation, and at the same time an increase in the expression of mutant proteins that are products of a genetic mutation; inhibition of apoptosis; transformation and even cell death. In addition to pronounced pronounced gen-otoxicity and mutagenicity, it also has strong carcinogenic activity, especially this effect is manifested in the cells of myeloid tissue (the cells of this tissue are very sensitive to this kind of xenobiotic effects).
Congenital malformations, primarily spina bifida, increased in oral doses of 150-250 mg / kg / day (approximately 1-2 times the recommended maximum human dose based on body surface comparison) in offspring of pregnant rats given ethyl alcohol during organogenesis. In the fetuses of pregnant mice treated at a dose of 50-200 mg / kg, the cleft palate was increased depending on the dose (approximately 0.2 to 0.8 times the maximum recommended human dose based on a
comparison of body surface area). Incomplete osteo-genesis and embryotoxicity have also been reported in pregnant rabbits at doses up to 200 mg / kg of ethyl alcohol per day (approximately 3 times the recommended daily human dose based on body surface area comparison). Although there is no adequate and well-controlled study in pregnant women, rifampin has been reported to cross the placental barrier and appear in cord blood.
Oral administration of ethyl alcohol to both rats and rabbits during pregnancy has been reported to have embryocidal effects, although reproductive studies in mammalian species (mice, rats, and rabbits) have not revealed congenital anomalies associated with ethyl alcohol.
Strain mice are more sensitive than, and the degree of testicular weight loss is stronger than in hybrid animals. An analysis of these indicators revealed that on days 30 and 45 after administration of ethyl alcohol, the weight of the testicles was slightly higher in the variants with the introduction of NNP. In both periods of the analysis, the frequency of was lower in the case of the introduction of the drug. When summing up the results of the analysis on days 30 and 45 after administration of ethyl alcohol.
Table 7 presents the results of the analysis of embryonic mortality in the offspring of males who received a total dose of 3 Gy, fractionated by 0.6 Gy for 5 days, which were administered after ethyl alcohol with a minimum dose of RNP 4 mg/kg for 10 days. Within 4-6 weeks after the administration of ethyl alcohol, the fertility of males in the control decreased significantly up to complete sterility. Analysis of the results as early as the 3rd week of mating revealed a positive effect of RNP on the mortality rate of embryos before the implantation period. Summing up the results of the study of the genetic action of RNP, it should be noted that in all the experiments carried out, there was a tendency towards a therapeutic effect of RNP. A statistically significant decrease in the degree of alcohol damage in terms of the percentage of effective crossings was found with the introduction of NNP (10 mg/kg) five times, after the administration of ethyl alcohol at a dose of 3 Gy of gray hybrids CBAxC57BX F1. In addition, a decrease in the frequency of AGS was found in BALB mice in a similar variant of the experiment. Three similar experiments were carried out (3 Gy + 10 mg/kg RNP x 5) on mice of three different genotypes (gray hybrids, white hybrids and BALB strain mice). The therapeutic effect was most pronounced in BALB mice. A positive effect in hybrid mice was found only in experiments on old animals. This suggests that the beneficial effect of NNP is associated with the stimulation of the cellular repair system, which may be weakened in linear and old mice. In general, the beneficial effect is most pronounced at a relatively high dose of alcohol.
The mouse genome contains the same number of genes as the human genome, with 99% of these genes appearing to be identical and 96% in the same order. This means that disease genes identified in mice can be transferred to the human gene map. You can conduct experimental crosses between mice with different
traits, and then very quickly begin to study the resulting offspring.
In view of all this, at least we should pay attention to their living conditions and good nutrition. We must follow safety rules in accordance with the norms.
The territory on which the building of biomedical research is located must be fenced and protected from external access. The order of entry into the territory is strictly defined. carry out weaning of young animals from the uterus at the end of the suckling period (mice, rats - 28-31 days; hamsters - 21-29 days; guinea pigs -27-29 days; miniature pigs - 45 days) and form groups of according to sex.
In the laboratory building, rats and mice are kept in cages in direct contact with the bedding. As bedding, it is recommended to use sawdust, shavings or small chips (length 5-20 mm, thickness 1-2 mm) from environmentally friendly hardwood. It is not allowed to use bedding made of chemically treated wood, as well as softwood. The bedding is autoclaved on trays at 118°C for 30 minutes. Bedding paper is stored and cut in a special auxiliary room. The required amount of bedding enters the housing in cut form in closed plastic bags. The closed bags are stored in the feed and litter storage room. Sanitization of the room is carried out in accordance with the "Plan of sanitary measures in the storage room for food and bedding". The control of the presence of parasites in the litter is carried out by a specialized state enterprise of the system of the sanitary and epidemiological service under a contract for disinfection work.
Methods: experimental, empirical, theoretical, comparative.
Result. In the end, not to mention that all the research proved that the mouse genome contains the same number of genes as the human genome, with 99% of these genes appearing to be identical and 96% in the same order. This means that disease genes identified in mice can be transferred to the human gene map. You can conduct experimental crosses between mice with different traits, and then very quickly begin to study the resulting offspring. It is possible to obtain mutant mice with certain gene defects, the phenotype of which can then be studied. In view of all this, at least we should pay attention to their living conditions and good nutrition. We must follow safety rules in accordance with the norms.
References:
1. Anthony B, Liang Y, et al. Facial measurements in mouse as a model for diagnosis of fetal alcohol spectrum disorders. Alcoholism: Clinical and Experimental Research. 2010;34(Suppl.):245A.
2. Balaraman S, Lunde R, et al. Circulating mi-croRNAs as biomarkers for fetal alcohol exposure. Alcoholism: Clinical and Experimental Research. 2010;34:13A.
3. Brown KL, Calizo LH, Stanton ME. Dose-dependent deficits in dual interstimulus interval classical eyeblink conditioning tasks following neonatal binge alcohol exposure in rats. Alcoholism: Clinical and Experimental Research. 2008;32(2):277-293.
4. Datta S, Turner D, Singh R, et al. Fetal alcohol syndrome (FAS) in C57BL/6 mice detected through proteomics screening of the amniotic fluid. Birth Defects Research. Part A, Clinical and Molecular Teratology. 2008;82(4):177-186.
5. Fang S, McLaughlin J, Fang J, et al. Automated diagnosis of fetal alcohol syndrome using 3D facial image analysis. Orthodontics & Craniofacial Research. 2008;11(3):162-171.
6. Goodlett CR, Horn KH, Zhou FC. Alcohol ter-atogenesis: Mechanisms of damage and strategies for intervention. Experimental Biology and Medicine (Maywood) 2005;230(6):394-406.
7. Goodlett CR, Wetherill L, Moore ES, et al. Evaluation of facial dysmorphology in an ovine model of fetal alcohol spectrum disorders. Alcoholism: Clinical and Experimental Research. 2010;34(Suppl.):100A.
8. Kraemer GW, Moore CF, Newman TK, et al. Moderate level fetal alcohol exposure and serotonin transporter gene promoter polymorphism affect neonatal temperament and limbic-hypothalamic-pituitary-ad-renal axis regulation in monkeys. Biological Psychiatry. 2008;63(3):317-324.
9. Medina AE, Krahe TE, Ramoa AS. Restoration of neuronal plasticity by a phosphodiesterase type 1 inhibitor in a model of fetal alcohol exposure. Journal of Neuroscience. 2006;26(3):1057-1060.
10. Miranda RC, Pietrzykowski AZ, Tang Y, et al. MicroRNAs: Master regulators of ethanol abuse and toxicity? Alcoholism: Clinical and Experimental Research. 2010;34(4):575-587.
