Studies On Physiological And Toxic Effects Induced By Endogenous And Exogenous Formaldehyde And Mechanisms Of Formaldehyde Toxicity Using CRISPR

Formaldehyde(FA)plays an important role in economic development,and millions of people around the world are exposed to formaldehyde environmentally and occupationally.Some of these occupational environments often induce high levels of FA,such as the wood processing industry,the antiseptic industry and pathology laboratories.The lower levels of formaldehyde exposure caused by household materials such as car engines,tobacco smoke,wooden furniture,and carpets have affected the health of more people.In addition,similar to nitric oxide(NO),carbon monoxide(CO),and hydrogen sulfide(H2S),FA can also be produced endogenously through many biochemical pathways and play a variety of physiological roles in biological systems.As a ubiquitous environmental pollutant,the health effects caused by FA have received widespread attention.A large number of studies have shown that FA can cause a variety of toxic effects such as acute toxicity and irritation,oxidative stress and inflammation,genotoxicity,neurotoxicity,cardiovascular effects and carcinogenicity.Among them,some studies have shown that endogenous FA has some cardiovascular effects,such as being associated with atherosclerosis and causing the damage to vascular endothelial cells as well as vasodilation.In the early days,a study proposed the hypothesis that endogenous FA may act as a new type of gas signaling molecule in the body.However,the relevant researches are very limited and there is a big gap in the cardiovascular effect of endogenous FA and the hypothesis of it being as a gas signaling molecule is still needed to be explored.On the other hand,FA has been classified as a Class ?human carcinogen by the International Agency for Research on Cancer(IARC),which can cause nasopharyngeal cancer.In addition,National Toxicology Program(NTP)in the United States has classified FA as a myeloid leukemogen,which is associated with myeloid leukemia.However,the conclusion about FA-induced leukemia is still controversial,because many classic leukemogens can directly destroy the hematopoietic stem/progenitor cells(HSC/HPC)in the bone marrow.while as the smallest and highly reactive aldehyde,FA cannot reach the bone marrow via inhalation.A recent breakthrough study found that the lungs of mice contain functional HSC/HPC,which can produce blood cells and transport them between the lungs and bone marrow bidirectionally.Therefore,this latest discovery forms the basis of the hypothesis of this study that inhaling formaldehyde does not directly induce bone marrow toxicity but causes HSC/HPC toxicity in the lungs and/or nose of mice.On the other hand,some previous studies used yeast models for functional toxicogenomic screening and identified many genes and cellular pathways related to the modulation of FA toxicity by many eukaryotic cells.Nevertheless,the specific genes,pathways,and mechanisms of formaldehyde toxicity regulation in human cells are still unclear.Therefore,on the basis of exploring the toxic effects of endogenous and exogenous formaldehyde,this study further used loss-of-function genome-wide CRISPR screening to identify the regulatory mechanism of K562 cells(a human leukemia cell line)for formaldehyde toxicity.The following are the three main aspects of the current study:(1)A preliminary study mimicking the internal environment of human body was conducted to investigate the relaxation effect of endogenous FA on rat aortas and its possible mechanisms.We found that FA induced vasorelaxant effects on rat aortic rings in a concentration-dependent manner.The NO/cGMP pathway was up-regulated when the rat aortas were treated with FA.The expression of large-conductance Ca2+-activated K+(BKCa)channel subunits ? and ? of the rat aortas was increased by FA.Similarly,the levels of ATP-sensitive K+(KATP)channel subunits Kir6.1 and Kir6.2 were also up-regulated when the rat aortas were incubated with FA.In contrast,levels of the L-type Ca2+ channel(LTCC)subunits,Cav1.2 and Cav1.3,decreased dramatically with increasing concentrations of FA.It was demonstrated that the regulation of FA on vascular contractility may be via the up-regulation of the NO/cGMP pathway and the modulation of ion channels,including the upregulated expression of the KATP and BKCa channels and the inhibited expression of LTCCs.Further study is needed to explore the in-depth mechanisms of FA-induced vasorelaxation.(2)To test the hypothesis mentioned above,we successfully cultured both burst-forming unit-erythroid(BFU-E)and colony-forming unit-granulocyte,macrophage(CFU-GM)colonies in mouse lung and nose and further reported that in vivo exposure to FA at 3 mg/m3 or ex vivo exposure to 400?M FA decreased the formation of both two colony types in the mouse lung and nose.The result was also directly compared with the hematotoxicity induced in the bone marrow and spleen.These findings,to the best of our knowledge,are the first to show FA exposure can damage mouse pulmonary/olfactory HSC/HPC distant to the bone marrow.What's more,we proposed the lungs and/or nose is the target site of FA to trigger leukemia.It suggests that FA-induced HSC/HPC toxicity in the mouse lung and nose could be a potential biological plausibility of leukemogenesis associated with FA inhalation though further confirmation is needed.(3)In the current study,a genome-wide,loss-of-function CRISPR screening was applied to identify modulators of FA toxicity in the human hematopoietic K562 cell line.The cellular genetic determinants of susceptibility and resistance to FA(40,100 and 150 ?M)was assessed at at two time points,day 8 and day 20.Multiple candidate genes were identified to increase sensitivity(e.g.ADH5,ESD and FANC family)or resistance(e.g.FASN and KMD6A)to FA when disrupted.Pathway analysis revealed a major role for FA metabolism and the DNA homologous repair(HR)pathway in FA tolerance,consistent with findings from previous studies.More network analyses revealed potentially important roles for one-carbon metabolism,fatty acid synthesis and mTOR signaling in modulating FA toxicity.Validation of these novel findings will further enhance our understanding of FA toxicity in human cells.The current findings may support the utility of CRISPR-based functional genomics screening on studying environmental chemicals toxicity.In summary,this study confirmed through experiments that endogenous formaldehyde may act as another gas signaling molecule in the body.In addition,the current study was the first to culture colonies from.hematopoietic stem and progenitor cells in the lung and nose of mice.It's proposed that the lungs/nose may be the target site of formaldehyde to induce leukemia,which provides a strong proof for the biological possibility of formaldehyde-induced leukemia.Further,in this study,CRISPR screening was used to investigate the susceptible genes and biological pathways related to formaldehyde-induced cytotoxicity and new molecular mechanisms and pathways were discovered at the genome-wide level,which also provides a new study point for a deeper understanding of formaldehyde toxicity in the future.