Study On The Combination Toxicity Effect Of Benzene And Formaldehyde On Mouse Embryos

Objective: To observe the combination toxicity action and to explore the combination effective type of Benzene and Formaldehyde, then to provide experimental and theoretical evidence for evaluating late injury of Benzene and Formaldehyde on organism.Methods1. The whole embryo culture system in vitro was adopted. The 8.5-day embedded KM mouse embryos were co-cultured with immediately centrifugal serum (ICS) of mouse containing Benzene and Formaldehyde for 48 hours. HE staining and light microscope was used to observe the effects of Benzene and Formaldehyde single and combination on growth and development of mouse embryo, morphodifferentiation of tissue and organ.2. Limb bud and midbrain cells were separated from mouse embryo (at 12-day stage). Micromass culture (MC) was used to investigate the effects of benzene on differentiation and proliferation of limb bud cells and midbrain.3. Fetal mouse limb bud at 12-day gestational age was cultured in a homeothermia rotating apparatus to observe the influence of benzene in different concentration on mouse limb bud development and differentiation.Results:1. Embryos after cultured in vitro for 48h in blank group and solvent group are essentially coincident with that homeochronously in vivo group (P>0.05), benzene had obvious effects on development of embryos in a dose-dependent manner: every indexes were no significant differences compared with the control (P>0.05) in 1.0μmol/L group; scores of crown-rump length, head length were decreased significantly(P<0.01) in 10μmol/L group; in 1000μmol/L group, all indexes of growth and development, and morphodifferentiantion of tissue and organs were suppressed, no normal embryos was observed, incidence of embryo malformation was increased,malformation of central nervous system that mainly included paramorphia of forebrain, hindbrain and midbrain, abnormality of trunk turning and microcephaly and also fetal death was viewed.2. Formaldehyde could affect embryonic growth and development in KM mouse on organogenesis, also have embryonic toxicity in a evident dose-dependent manner, and stimulate araphia and paramorphia of body position, chest and heart in a dose-dependent manner.3. When benzene combined with formaldehyde, development of embryo was affected, but morphodifferentiation of most tissue and organs was no influence in (10+3)μmol/L group. With the increasing concentration of benzene and formaldehyde combination, the indexes of growth and morphodifferentiation were lower than control(P<0.05), diameter of saccus vitellinus, crown-rump length, head length and value of arthromera was decreased obviously, index score of embryo morphodifferentiation was descended, abnormal embryo rate and death rate was much higher. The major appearances were cephalosome abnormal (cenencephalocele, schizencephaly, cerebtomedullary tube patency), heart dysplasia(retention on the appearance of cardiac tube stage and hydrops pericardil), limb bud was small, eyes, ear, arcus branchialis and body position was abnormal. It showed in microscope that benzene and formaldehyde could cause disordered structure and irregular arrangement of splanchnic wall VYS endodermis cells. Cell polarity was missed, degeneration and engorgement was appeared, and vacuole was encouraged in endochylema.4. Benzene and formaldehyde caused lethal effect on embryo. Death rate of embryo was 12.5% by 27μmol/L formaldehyde, the rate was 33.3% and 44.4% respectively after formaldehyde combined with 10μmol/L and 100μmol/L benzene. Abnormal embryo rate of embryo was 13.3% by 9μmol/L formaldehyde, the rate was 44.4% and 77.8% after formaldehyde combined with 10μmol/L and 100μmol/L benzene. It showed that the intensity of toxic action and teratogenic action of the combination on embryo was much stronger than the action of the mix of the two agents'solitary effect. The results illustrated that the two agents exhibited synergistic toxic effect.5. Benzene inhibited proliferation and differentiation of rat embryo limb bud and midbrain cells in vitro in a dose-dependent manner, the inhibition of benzene on differentiation was exceeded that on proliferation. The ID₅₀ and IP₅₀ of benzene on limb bud cells were 89.93μmol/L and 396.77μmol/L respectively. The ID₅₀ and IP₅₀ on midbrain cells were 116.29μmol/L and 831.46μmol/L respectively.6. The development and differentiation of mouse fore limb buds was inhibited when the concentration of benzene above 75μmol/L. With the increasing of benzene concentration, cartilagineus development and differentiation of limb was low, the Neubert score was decreased in a dose-dependent manner. Conclusions:1. Benzene and formaldehyde caused embryo toxicity and teratogenicity on cultured KM mouse embryos. Benzene could suppressed the development and differentiation of limb bud organ and inhibited proliferation and differentiation of rat embryo limb bud cells and midbrain cells.2. Benzene and formaldehyde could damage structure and function of embryo VYS, induce degeneration and death of embryo cells.3. The combination toxicity action of benzene and formaldehyde on mouse embryo was appeared to be synergistic effect.