https://www.sciencedirect.com/science/a ... 942032656X
Fluoride and cadmium, two typical environmental pollutants, have been extensively existed in the ecosystem and severely injured various organisms including humans. To explore the toxicological properties and the toxicological mechanism of fluoride and cadmium in silkworm, we perform a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) -based functional genomic screen, which can directly measure the genetic requirement of genes in response to the pollutants. Our screen identifies 751 NaF-resistance genes, 753 NaF-sensitive genes, 757 CdCl2-resistance genes, and 725 CdCl2-sensitive genes. The top-ranked resistant genes are experimentally verified and the results show that their loss conferred resistance to fluoride or cadmium. Functional analysis of the resistant- and sensitive-genes demonstrates enrichment of multiple signaling pathways, among which the MAPK signaling pathway and DNA damage and repair are both required for fluoride- or cadmium-induced cell death, whereas the Toll and Imd signaling pathway and Autophagy are fluoride- or cadmium-specific. Moreover, we confirm that these pathways are truly involved in the toxicological mechanism in both cultured cells and individual tissues. Our results supply potential targets for rescuing the biohazards of fluoride and cadmium in silkworm, and reveal the feasible toxicological mechanism, which highlights the role of functional genomic screens in elucidating the toxicity mechanisms of environmental pollutants.
Here are a few excerpts, with a focus on the mitogen-activated protein kinase (MAPK) signaling pathway. (Thyroid hormones play a crucial role in regulating MAPK.)
Firstly, to detect the toxicity and morphological changes of BmE cells with fluoride or cadmium exposure, we performed cytotoxicity assays for NaF and CdCl2 at different concentrations. No obvious morphological changes in BmE cells were observed after NaF and CdCl2 treatment at concentrations less than 0.1 mM and 0.1 μM, respectively, following 48 h of exposure. Following the concentrations increasing, cell morphology was altered drastically...
3.4 MAPK signaling pathways involve in fluoride- or cadmium-induced cell toxicity
Mitogen-activated protein kinases (MAPK) are protein Ser/Thr kinases responsible for converting extracellular stimuli into intracellular reactions, activating a series of cellular responses [35]. MAPK pathways exist in almost all eukaryotic cells as one of the most significant pathways in the eukaryotic signal network; they are involved in regulating cell proliferation and differentiation, metabolism, survival, and apoptosis [36-38]. Traditional MAPK mainly includes three subunits: p38 isoforms, extracellular signal-regulated kinases 1/2 (ERK1/2), and c-Jun amino (N)-terminal kinases 1/2/3 (JNK1/2/3)...
These results showed that MAPKs functioned in fluoride- or cadmium-induced cytotoxicity [7, 55, 56]. Rescue experiments using the p-JNK inhibitor, SP600125, could inhibit the fluoride- or cadmium-induced enhancement of JNK phosphorylation levels, but did not prevent cytotoxicity, in contrast to that observed in mammalian cells [7, 55]. We speculated that fluoride- or cadmium-induced cytotoxicity involved diverse biochemical pathways, which resulted in repressing the JNK pathway alone could not rescue cell viability...
In summary, the CRISPR functional genomic screening of NaF and CdCl2 in BmE cell identifies the molecular mechanism of fluoride- or cadmium-induced toxic effects, and the RSGs are found to be involved in multiple signal pathways such as MAPK signaling pathway, DNA damage, and repair.