Experimental study on the genotoxic effect of lithium chloride on mouse somatic cells
Lithium salts are widely used in cutting-edge industries such as batteries, chemicals, ceramic manufacturing, metallurgy, and national defense, and are in direct contact with The number of lithium workers continues to increase, and the resulting occupational hazard problems are also increasing day by day; lithium salts are widely used in clinical settings because of their anti-manic therapeutic effects [1]. The world uses up to 25,000 tons of lithium every year. According to WHO statistics, at least 500 million people in the world suffer from various mental disorders, accounting for 10% of the world's population. Since the safety margin of therapeutic and toxic doses of lithium salts is small, various toxic effects often occur, and the treatment course for mental disorders is long, it is necessary to study the hazards of long-term chronic exposure and provide scientific basis for its safe use. At present, except for the effect of lithium on the bone marrow micronucleus rate of mice in Ilonzan, there are no other reports on the genotoxicity of lithium salts in mice. Therefore, this study intends to investigate the reproductive development and reproductive development of lithium chloride in our laboratory. Based on the immunotoxicity and teratogenicity tests, the genetic toxicity of lithium chloride to somatic cells in different tissues of Kunming mice was observed.
1 Materials and Methods
1.1 Materials
1.1.1 Experimental Animals Xinjiang Medical Experimental Animal Center provided healthy Kunming mice 18-25 g, 25-25 g 30g.
1.1.2 Main reagents in the experiment Lithium chloride: LiCl. H2O, molecular weight 60.41, analytical grade, provided by Shanghai Reagent Factory No. 2. Cyclophosphamide (CP): provided by Shanghai No.12 Pharmaceutical Factory. Colchicine: C22H25O6N, molecular weight 399.43, produced by Kunming Pharmaceutical Factory.
1.2 Methods and observation indicators
1.2.1 For the chromosomal aberration test of mouse bone marrow cells, 40 healthy sexually mature Kunming mice, 18 to 25 g, were selected randomly. Divided into 5 groups, 3 exposure groups and negative and positive control groups. The dose selection is based on the LD50 obtained by this laboratory. The high-dose group is 1/8 LD50, the medium-dose group is 1/24 high-dose group, and the low-dose group is 1/80 high-dose group. That is, the doses of each group are: 225, 75, 22.5 mg/kg lithium chloride solution, and the negative control group was given distilled water. The poison was administered by gavage continuously for 5 days, once a day. The amount of gavage was adjusted according to body weight every day. The positive control group was given intraperitoneal injection of CP40 mg/kg 1 day before the animals were killed. The animals in each group were injected 2 to 3 hours before being killed. Colchicine 4 mg/kg was intraperitoneally injected, and the animals were sacrificed 6 hours after the last exposure. Bilateral femoral bone marrow was harvested and prepared according to conventional procedures to observe the chromosomal aberration rate of bone marrow cells.
1.2.2 Micronucleus test of mouse bone marrow cells. Select 50 healthy sexually mature Kunming mice of 18-25 g, half male and half male. The groups and doses of each group are the same as above. The mice were sacrificed 24 hours after the last exposure, and the bone marrow from both femurs was collected and smeared according to conventional methods to observe the micronucleus rate of polychromatic erythrocytes in the bone marrow.
1.2.3 For the micronucleus test of mouse fetal liver cells, healthy sexually mature Kunming mice of 25 to 30 g were selected, and the male and female were caged together at a ratio of 2:1. The day when vaginal plug was detected was recorded as the day of conception. On day 0, 50 pregnant rats were randomly divided into 5 groups, and the grouping and exposure dose were the same as above. On the 14th day of pregnancy, the drug was administered once by gavage. The mice were killed 12 to 18 hours after the exposure. At least two fetal livers were taken from each litter and prepared according to the literature method [2]. Polychromatic erythrocytes in the fetal liver were observed. Micronucleus rate.
1.2.4 Statistical methods: Use the PEMS software package to conduct statistical analysis of data using analysis of variance and pairwise comparisons of q test and rank sum test.
Discussion
3.1 The effect of lithium chloride on the chromosomal aberration rate of mouse bone marrow cells
About the effect of lithium on the chromosomal aberration rate of bone marrow cells, there have been studies abroad Scholars have reported that Bille et al. in 1975 did not find any chromosomal aberrations in the chromosomes of bone marrow cells after studying patients treated with lithium salts [3]. Sobti studied the chromosomal aberrations caused by three kinds of lithium salts: lithium chloride, lithium carbonate, and lithium acetate in mouse bone marrow cells. The results showed that these three kinds of lithium salts have a damaging effect on mouse chromosomes. The results of this test are consistent with this. Consistently, gene mutations induced by many compounds are highly related to chromosomal aberrations. Studies have shown that lithium can selectively bind to DNA and can also compete with Mg2+ to weaken DNA synthesis and repair, thus inducing chromosomal aberrations [4]. The test results showed that cracks, fractures and rings were the most common distortion types.
Among them, the broken ring is a chromosomal aberration, which is an "unstable" aberration and often leads to cell death. If the crack can be determined to be a chromatid aberration, the damage caused by it will easily lead to genetic damage if it is repaired incorrectly and can be used as a Sensitive genetic toxicological parameters. The results of this experiment show that lithium chloride at a dose of 225.0 mg/kg has a genotoxic effect on mouse bone marrow cells.
3.2 Effect of lithium chloride on the micronucleus rate of mouse bone marrow cells
Detecting the micronucleus rate of mammalian bone marrow cells is one of the commonly used mutagenic short-term detection methods to determine induced chromosomal damage. , Micronuclei are centromere-lost chromosome fragments or chromatid fragments that remain in the cytoplasm during the late stages of mitosis due to damage. The results of this experiment show that lithium chloride at a dose of 225.0 mg/kg can cause chromosome breakage and damage in mouse bone marrow cells.
3.3 Effect of lithium chloride on micronucleus rate in mouse fetal liver
Experiments have shown that many chemicals cause chromosome damage to fetal mouse liver cells much more than to maternal bone marrow cells Damage to chromosomes. Therefore, only in vivo experiments in adult animals may not reliably predict the harm to the fetus caused by exposure to genetic toxicants during the embryonic period, and the stem cells that supply new cells throughout the animal's life are formed during embryonic development. Therefore, chromosomal damage during the embryonic period can increase the susceptibility of offspring to tumors and other diseases after birth, and the effects of mutagens during the embryonic period can also increase the rate of neonatal malformations. Therefore, when evaluating the potential genetic harm of chemical substances, sufficient attention must be paid to the impact on the fetus. Because the metabolism of the placenta in mammals is active during pregnancy, the mother's sensitivity to mutagens is increased. Immature embryonic cells divide vigorously and are also quite sensitive to mutagens. Some pro-mutagens and pro-carcinogens are metabolized and activated in the liver, leading to an increase in the concentration of mutagens in the liver. Therefore, the chromosomes of fetal cells are broken under the action of certain poisons, causing an increase in micronuclei. Therefore, the embryonic cell micronucleus test can be used to detect promutagens and procarcinogens that are insensitive to the bone marrow micronucleus test and to screen for chemical mutagens that can pass through the placental barrier and be passed to offspring and that are prone to accumulate in the fetus. While measuring the micronucleus rate of polychromatic erythrocytes in the fetal liver of fetal rats, we also observed the micronucleus rate of maternal bone marrow cells. The experimental results showed that lithium chloride has obvious genotoxic effects on fetal rats. At the same time, the micronucleus rate of bone marrow cells and the micronucleus rate of fetal liver cells were compared at the same dose, indicating that maternal bone marrow cells were not as sensitive to chromosome clastogens as fetal mouse liver polychromatic erythrocytes.
Based on the above test results, it can be concluded that oral administration of lithium chloride has a genotoxic effect on somatic cells of Kunming mice, and its genotoxic effect on somatic cells of fetal mice is more obvious. It is necessary to further Study the genotoxic effects of lithium chloride on mouse germ cells.