spontaneous mutation

induced mutation

Chemical substances and other environmental factors cause biological remains The ability to transmit changes in matter, and the change process can be transmitted during cell division

The occurrence and process of mutation

direct mutagen

indirect mutagen

Mutation caused by change or loss of a certain base pairing performance (a certain base is replaced by another base)

One or more pairs (except pairs of 3 or multiples of 3) of bases are reduced or added, and the base sequence is completely changed from the damaged point, forming an incorrect code and being translated into incorrect amino acids.

Stable chromosomal aberrations: clefts, deletions, inversions, duplications, etc.

Unstable chromosome aberrations: dicentric chromosomes, acentric fragments, ring chromosomes, etc.

base mismatch

Planar macromolecules embedded in DNA strands

base analog substitution

The chemical structure of the base is changed or destroyed

Pyrimidine dimer formation

DNA adduct formation

DNA-protein cross-link formation

Binds to tubulin dimers

Binds to tubulin sulfhydryl group

Destroy assembled microtubules

Centriole movement is blocked

Other functions

Enzymes involved in the DNA replication process

Enzymes that act in the DNA repair process.

somatic cells

germ cells

DNA repair

Includes multiple genetic endpoints

Test indicator organisms include prokaryotes and eukaryotes

Including in vivo and in vitro tests

somatic cells and germ cells

Use auxotrophic mutant strains to observe whether the test substance can Correct or compensate for the mutational changes carried by the mutant and determine its mutagenicity

Histidine auxotroph Salmonella typhimurium

Tryptophan auxotrophic E. coli

Forward mutation testing refers to mutations that inactivate the wild-type gene. This mutation Changes in enzymes and functional proteins caused by mutations

Commonly used cell lines

Commonly used mutation markers

Micronucleus formation: Acentric fragments of chromosomes or chromatids or entire chromosomes lost due to damaged spindle fibers are left in the cytoplasm during the late stages of cell division and form one or several regular subnuclei after telophase, including in the cytoplasm of daughter cells

Mouse bone marrow polychromatic erythrocyte micronucleus test

Other micronucleus test methods

The chromosome shape of cells staying in the metaphase phase of division was observed with an optical microscope. State structure and number changes

The mutual exchange of DNA replication products at homologous loci on chromosomes, May be related to DNA breakage and rejoining

Dominant lethality refers to the genetics of developing sperm or eggs Damage, which does not affect fertilization, but causes the fertilized egg to or death of the developing embryo

Dominant lethality is mainly the result of chromosomal damage (structure, quantity)

DLT uses early embryonic death as the observation endpoint to detect the effect of test substances on chromosome damage to animal germ cells.

Only expose male animals to the poison, mate with untreated female animals, and observe embryonic death.

Commonly used animals: adult sexually mature mice and rats

Utilizing the cross-heritage characteristics of recessive genes in sex-linked inheritance, Drosophila melanogaster is selected as the experimental animal, and the test substance is given to the male fly. If the male fly has a mutation in the X chromosome, it is passed to the F1 generation female fly, and then through the F1 generation female fly. The fly is passed to the F2 generation male fly, so that the recessive gene located on the X chromosome is expressed in the hemizygous male fly. The lethal mutation does not appear, but the obviously marked wild-type male fly appears.

Principle: Normal cells only perform DNA replication and synthesis in the S phase during mitosis. When DNA is damaged, DNA repair and synthesis can occur in other phases besides the normal replication and synthesis phase.

Synchronous culture blocks cells in G phase and blocks normal DNA half-retained replication. The cells are treated with the test substance and cultured in a culture medium containing H-thymidine. If the test substance causes DNA damage and activates the DNA damage repair mechanism, 3H-thymidine in the culture medium will be incorporated into the DNA chain, and the DN radioactivity is measured, which indirectly reflects the degree of DNA damage.

Methods to detect DNA damage in nucleated cells at the single cell level

Principle: When DNA is damaged, broken DNA fragments migrate faster than large fragments of DNA, and the comet shape appears after electrophoresis, which can be used to determine DNA damage. The more serious the damage to the DNA, the more fragments are generated, and the smaller the fragments, the greater the amount of migrated DNA during electrophoresis, and the longer the migration distance. Under a fluorescence microscope, it is observed that the tail length increases and the fluorescence density intensity of the tail increases. By measuring the light of the migrated part Density and migration length quantification of DNA damage in individual cells

advantage:

Sperm maturation and morphological development are regulated by genes. Gene mutations lead to an increase in sperm deformity rates. By examining morphological changes such as sperm heads and tails, the mutagenic effects of chemicals can be detected.

New developments in observation methods

Negative control: Blank control or solvent control. Except for no treatment factors, other conditions should be exactly the same as the experimental group.

Positive control: using a substance known to produce a positive reaction as a control

S9: Liver homogenate prepared after treatment with enzyme inducer, 9000g The supernatant obtained by centrifugation is added with appropriate buffer and auxiliary Factors, mainly containing mixed function oxidase (MFO)

shortcomings

Some tissues differ in their reactivity to activating or degrading chemicals

The normal flora of the digestive tract also participates in metabolic activation in animals

Substances that induce enzyme systems and substances that alter physiological states can alter toxicant metabolism

The balance between activation and detoxification in vitro is different from that in animals

In vivo testing:

In vitro testing:

QC

Mutagenicity tests detect potential carcinogens

Uncertainties in mutagenicity testing