New Technology and Development Trends in Toxicology

1. New Technology and Development Trends in Toxicology Content What is Toxicology ............................................................................................................................................. 1 1.Application of Gene Technology in Toxicology .............................................................................. 2 1.1. Application in Mutagenicity Detection ................................................................................. 2 1.2. Application of Transgenic Animals in Toxicology ............................................................ 3 1.2.1. General Toxicity Study Model ............................................................................................ 4 1.2.2. Mutagenesis Detection Model ........................................................................................... 4 1.2.3. Carcinogenicity Detection Model and Its Application in the Study of the Carcinogen Mechanism.......................................................................................................................... 4 1.2.4. Transgenic Animals Used for Specific Tissue Toxicity Studies ............................ 5 2.New Advances in Toxicology ................................................................................................................ 5 3. Development Trend of Toxicology in 21st Century .......................................................................... 6 3.1. The Combination of Traditional and Modern Toxicological Research Methods Will Promote Toxicology Development ..................................................................................................... 6 3.2. A Large Number of New Technologies and Methods will Further Deepen Toxicological Research ........................................................................................................................... 6 3.3. Using A Combination of Methods to Evaluate Chemical Toxicity Will Be an Important Trend ......................................................................................................................................... 7 a.Reduction in animal usage; ................................................................................................ 7 b.Shortening test period; ......................................................................................................... 7 c. Studying the actual concentration of chemicals in the environment; ................ 7 d.Using statistical or mathematical models; .................................................................... 7 e.Making effective experimental design in advance; ................................................... 7 f. Developing toxicology management. ............................................................................. 7 3.4. Toxicology Branch Has Developed Rapidly And "Polarized" Prominently. ............... 7 In a Summary ...................................................................................................................................................... 8 What is Toxicology Toxicology is a science that studies the mechanism of toxicity, severity, frequency, and toxicity of chemical substances on organisms. It is also a science for the qualitative and quantitative evaluation on toxic effects. Toxicology and pharmacology are closely related. At present, it has developed into an independent discipline with certain basic theory and experimental methods and has gradually formed some new branches. This article briefly describes the application of new technologies in molecular toxicology

2. and some development trends in toxicology. 1. Application of Gene Technology in Toxicology Molecular toxicology uses molecular biology techniques and methods to study toxicological issues. For example, cell culture assays are used to detect in vitro genotoxicity. Transgenic animal models are used in the overall animal experiments, which are important for elucidating the toxicity of exogenous chemicals and their mechanisms. Gene technology involves introducing a piece of DNA (either a complete gene or a gene fragment) into a cell or organism. The introduced DNA can change the strength of toxicity or change the way the toxicity acts. Therefore, the toxic effect of the introduced DNA can be judged by the degree of change in toxic action or manner. 1.1. Application in Mutagenicity Detection Genomic toxin are chemicals that damage DNA. They are mostly mutagens. Conventional Ame's tests are bacteria-mediated methods for detecting genotoxic agents. However, this method also has some limitations, and false positive results can sometimes occur. Both glutathione and cysteine are anticancer agents, but they show strong mutagenicity in Ame's tests. In addition, bacterial and animal cells vary a lot in their biological features, and in vitro animal cell experiments are better to reflect the role of toxins in living organism than bacteria. There are two types of cells commonly used for the detection of genotoxic agents, one being primary cells and the other being secondary cells. There are several indicators for detecting chemicals genotoxicity, such as nucleotide isotope labeling. If a chemical will damage DNA, the cells need to be repaired after treatment with the chemical. During the process, Nucleotides are used and if isotope-labeled nucleotides are added to the culture medium, the repaired DNA is labeled with isotopes. Under certain circumstances, the more damaged DNA detected, the more repair needed, which is to say the more isotopes are contained in the cell DNA. many. Therefore, the genotoxicity of the chemical is determined by detecting the isotope content in the DNA. Another way is to change the metabolism of cells based on genetic toxicity. For example, normal V79 cells have

3. hypoxanthine phosphotransferase, which is an essential enzyme for the synthesis of purine nucleotides in the normal alternative pathway. If a purine homolog (such as 8-azo guanine) is present in the medium, the enzyme can convert its homologues to the corresponding purine nucleotides and synthesize DNA, but the purine homolog does not have normal purine function, which causes cell death. Conversely, if a chemical can damage DNA and damage the hypoxanthine phosphotransferase without synthesizing normal enzymes, its damaged cells survive instead. The more cells survive, the stronger the mutagenic ability of the chemical is. Some chemicals are not toxic by themselves, but their metabolites show strong toxic effects. For these chemicals, the above methods cannot be applied. To overcome this shortcoming, liver cell extracts can be added to cells or bacterial cultures to help metabolize toxins. In some laboratories, a small number of primary cells are mixed with secondary cells, and its metabolic enzymes are provided by the primary cells. For example, nitrodimethylamine cannot show any mutation using a conventional bacterial detection system, but it shows a strong genotoxicity after addition of hepatocyte extracts to the bacterial culture solution. Of course, these experiments also have own problems, for instance, the half-life of some of metabolites are short and are inactivated by the time they enter the test cells and their DNA (the cell viability of metabolic enzymes in liver extracts or primary cells declines rapidly over time); liver extracts or primary cells contain multiple enzymes, even if they can metabolize toxins and show toxicity, there is no way to find out which enzyme is playing a leading role. It is clear that using a cell strain that synthesize a single enzyme is important for studying the toxic effects of toxins. With molecular biology techniques, the DNA that transcribes a certain metabolic enzyme is ligated to a gene carrier (mostly a plasmid or virus). This DNA, which has regulatory ability, can interact with the transcription factors of cultured in vitro cells. A gene vector encoding a DNA of a certain metabolic enzyme is introduced into cultured cells in vitro, and the cell expresses this specific enzyme. A more prominent example is multifunctional monoamine oxidase (P450). There are two types of cell lines that can express P450 in vitro, one can stably express, and the other express for a short time. Various human P450s, such as 1A1, 2A6, 2E1, 3A4, have been successfully introduced into human lymphoid cell lines, rat placental cell lines, and rat hamster liver cancer cell lines. These cell lines are capable of expressing toxic metabolic enzymes. For chemicals that need to be toxic after metabolism, their detection sensitivity is greatly increased. For example, in terms of detecting toxicity of nitrodimethylamine, cells expressing 2A6 are much sensitive than ones did not express 2A6 or 2E1 (probably 500 times). This shows that nitrodimethylamine must be metabolized before showing toxicity. It also shows that 2A6 is a metabolic enzyme that can convert nitrodimethylamine into toxic metabolites, while 2E1 is not. 1.2. Application of Transgenic Animals in Toxicology Transgenic animals are animals that contain foreign genetic material in their genome. They are widely used in scientific research. Because transgenic animals integrate genral, cellular, and molecular levels, they can better reflect the effect of life's overall research

4. and become one of the hot spots in toxicology research or labs. 1.2.1. General Toxicity Study Model C-fos-LacZ transgenic mice are used for the study of neurotoxicity. Transgene metallothionein (MT) and knockout mice are used for the study of metals and certain non-metals. Let’s say resistance to cadmium is increased in MT transgenic mice, whereas toxicity of cadmium, silver, mercury, cisplatin, and carbon tetrachloride is increased in MT-knockout mice. 1.2.2. Mutagenesis Detection Model Transgenic animals provide possibilities for solving long-standing problems in genetic toxicity studies. To give an idea, when compared to in vitro tests, the qualitative and quantitative extrapolation of living animals will consume a large number of animals and time, such as the determination of target organs and the analysis of the induced genetic changes. Since Gosen et al. established the first transgenic mutation detection model in 1989, there have been more than a dozen models. 1.2.3. Carcinogenicity Detection Model and Its Application in the Study of the Carcinogen Mechanism Transgenic animals provide a new and important way for the rapid detection of carcinogens and the mechanism of chemical carcinogenesis. Currently established detection models or research models include: a. Transgenic animal models. They overexpress oncogenes such as TG, AC mice, HK- fos transgenic mice, ras-H2 transgenic mice, oncogenes mice carrying activated H-ras and so on. The sensitivity of these transgenic animals to chemical carcinogens has been

5. increased many times. Such as tumor transgenic animals carrying Pim-l have stronger carcinogenic effect on ethyl nitro urea, compared with the non-transgenic animals. Namely its sensitivity increased 25 times; b. Gene knockout oncogenic detection models. They use homologous recombination method to integrate a piece of DNA into an anti-tumor gene so that the anti-tumor gene cannot express certain protein. An animal cultured by this method is called a gene- knockout animal. The most studied in this area is the tumor suppressor gene P53. P53 (+/-) deletion animals have the same development and growth features with P53 (+ / +) animals, but after treatment with carcinogens (such as dinitrodimethylamine), the average life span of P53 (+/-) animals is 29 weeks, while the P53 (+/+) is 42 weeks. And their occurrence and distribution of tumors are also very different. Others examples include aryl hydrocarbon receptor (AHR) mice, peroxisome proliferator-induced receptor alpha (PPARα) mice; c. Transgenic animals used for reproductive toxicity studies, for instance, ZP3 (encoded) zona pellucida glycoprotein knockout Mouse, estrogen receptor or progesterone receptor knockout mouse, DNA methyltransferase knockout mouse. 1.2.4. Transgenic Animals Used for Specific Tissue Toxicity Studies A typical example is to grow a transgenic animal with a bacteriophage containing the lactose operon. The bacteriophage with the lactose operon is blue after infection with certain bacteria. If the bacteriophage is damaged in vivo due to treatment with a chemical, normal galactosidase cannot be transcribed. After assembled in vitro, the bacterial colony of the infected bacterium is colorless. Therefore, according to the number of blue and colorless colonies, the strength of certain chemical genotoxicity can be determined. In addition, a plasmid as a vector also works to increase the sensitivity of an assay. More importantly, the gene vector can be isolated from different tissue cells in chemically treated animals, displaying cell-specific toxicological effects. 2. New Advances in Toxicology Toxicology and epidemiology, especially in combination with molecular epidemiology, are used to assess risk. Many additional amendments to the safety factor have been made in the classical method to improve the accuracy of the derivation predictions within and between species, but the consequences are that toxicologists have to correct a series of correction coefficients from various administrative regulations due to lack of scientific evidence. In recent years, new methods have been proposed to determine the safety factor as far as possible to actual data rather than artificial hypotheses. For example, assessment of carcinogens will use a quantitative model, it focuses on the derivation from high doses to low doses rather than derivation from animal to human species. The most common is the linear multi-stage LMS model. It extends the upper limit of the maximum tolerance (MTD) confidence point to the origin and uses the low-dose region to estimate the dose-effect curve of the exogenous chemical or Its toxicity

6. intensity. Instead of using zero as the origin of the dose axis, this model uses a dose value that corresponds to a small biological unit: the numerator. New advances in strategies for evaluating the safety of exogenous chemicals include the use of toxic equivalence factors (bioequivalence) or question-and-answer methods, quantitative analysis of structure-activity relationships, and pharmacodynamics and pharmacokinetic models to study complex chemicals. 3. Development Trend of Toxicology in 21st Century 3.1. The Combination of Traditional and Modern Toxicological Research Methods Will Promote Toxicology Development In the past, toxicology research was mainly combined with animal testing and human observation and it was still an important and necessary method for a considerable period of time. With the application of the theory and methods of molecular biology imploring to toxicology research, the assessment of the toxicity of exogenous chemicals was developed into a new model combining in vitro cell and molecular toxicity testing in human volunteer. Animal-based toxicology studies will be reduced. Some complex whole experiments will gradually be replaced by in vitro tests or structure-activity relationship mathematical models. The toxicity testing system currently used for harmful factors will be replaced by genetically engineered animals and cells; traditional morbidity and mortality endpoints will be replaced by biochemical indicators; toxicity studies that required several months of dosing and evaluation will be completed in hours. The toxic response of transgenic animals to exogenous chemicals will be very consistent with human body, such as taking human tissue culture cells in the division to meiosis in vitro. The interpretation and extrapolation of the current toxicity test will change. 3.2. A Large Number of New Technologies and Methods will Further Deepen Toxicological Research Some of the above-mentioned in vitro cell assays and transgenic animal or gene knockout animal models are used in toxicology studies. Other new technologies include serial analysis, DNA chip or DNA microarray can simultaneously measure the expression of thousands of genes to observe the up- and down-regulation of genes. Gene trapping or representative difference analysis, etc. will provide a possibility for the study of molecular mechanism of teratogenicity of chemicals; gene distribution maps can distinguish specific or non-specific cell damage; application of complex formation Mediated PCR studies explore the DNA damage and repair at nucleotide level; the use of biomarkers in toxicology is increasingly important, such as the use of specific DNA and protein adducts for efficient exposure of biomarkers; using NMR analyze metabolites in urine can determine the pattern of metabolic changes as a toxic response biomarker. An example, biochemical markers in peripheral blood (platelets, leukocytes, red blood cells) can be used to determine alternative markers of exogenous chemical damage to nervous system.

7. In toxicology research, the important question is how to apply the data obtained from animals to humans, from in vitro data to in vivo, from systemize complex whole systems in simple and human-controlled systems, and how to improve detection sensitivity. GM technology provides a brand-new approach to solve these problems. In the metabolic pathway, the metabolism of a certain chemical can be controlled by gene transfer. On the whole level, the expression of a certain gene can be controlled artificially to clarify the role of gene in the process of chemical toxicity. It can be predicted that transgenic animals or gene knockout animals will play a major role in elucidating the mechanism of toxic effects of chemicals. 3.3. Using A Combination of Methods to Evaluate Chemical Toxicity Will Be an Important Trend It has been doubtful whether adequate toxicological information has been obtained for most chemicals using routine toxicological methods for the study of exogenous chemicals over the past 20 years. Taking into account the impact of chemical exposure on human health, this issue becomes more prominent. Because toxicology tests consume large numbers of animals, toxicologist should consider minimizing animal use in toxicology studies. Obviously, if we want to make a reasonable change to such a large number of exogenous chemicals, we should establish new toxicological research methods that can be selected, consumed less animals, have shorter test cycles, and cost less. These methods should be measured according to the following criteria: a. Reduction in animal usage; b. Shortening test period; c. Studying the actual concentration of chemicals in the environment; d. Using statistical or mathematical models; e. Making effective experimental design in advance; f. Developing toxicology management. For example, pharmaceutical industry evaluates drugs with a combination of micronucleus tests in rodents (mainly rats) and toxicokinetic studies for two to four weeks. 3.4. Toxicology Branch Has Developed Rapidly And "Polarized" Prominently. In the past 30 years, toxicology has formed many branches due to its rapid development and crossover with related disciplines. According to task, it can be divided into clinical toxicology, environmental toxicology, industrial toxicology, management toxicology, ecotoxicology and forensic toxicology; according to research methods and end points, it can be divided into immunotoxicology, molecular toxicology, membrane toxicology, genetic toxicology and analytic toxicology; according to the research object, it can be divided into insect toxicology, veterinary toxicology, human toxicology and plant toxicology; according to different exogenous chemicals, it can be divided into metal toxicology, pesticides toxicology, food toxicology, radiation toxicology,

8. pharmacotoxicology and combustion toxicology; according to the nature of research work, it can be divided into description toxicology (referring to conventional toxicity testing and safety assessment), mechanism toxicology and management toxicology. In recent years, there have been more toxicological branches, such as comparative toxicology, geographical toxicology, and acute toxicology. It can be predicted that there will more new branches in the next century. In addition, toxicological differentiation will become more apparent. On the one hand, the toxicology software, such as management toxicology will remain one of the toxicological research hotspots, which provide a scientific basis for the management of chemical toxicants on macro level; on the other hand, the research level will become more and more sophisticated. Toxicological issues from cellular, genetic and molecular level will become basic scientific work. The above two aspects are not only differentiated but also penetrated to each other, making the combination of toxicology with software and hardware prominent. In a Summary In a word, toxicology is increasingly bonded to human daily life and production labor, such as environmental pollution, deterioration of ecological environment, adverse drug reactions, food safety, harm of veterinary drugs and pesticides, and toxic substances in the operating environment worldwide. In the 21st century, toxicology will gain greater development and make larger contributions to mankind. Although significant advances have been made in cellular and molecular studies in recent years, it is not enough to only study toxicity and mechanisms of exogenous chemicals from molecular level since organism has macroscopic side. It is necessary to link microscopic studies with macroscopic research, which is to combine the whole experiment with in vitro cell and molecular level research. Source: https://www.creative-animodel.com/blog/new-technology-and-development-trends-in- toxicology-i/ and https://www.creative-animodel.com/blog/new-technology-and- development-trends-in-toxicology-ii/