Molecular Mechanisms And Protection Strategies For Toxicity Of Graphene Oxide In Caenorhabditis Elegans

Background:Graphene oxide (GO), a single layer of carbon atoms in a honeycomb lattice, offers superior qualities including optical transmittance and electronic properties, which make it a promising material for a wide range of applications, particularly in biomedicine, electronic devices and composite material. Nevertheless, the potential health and environmental risks will be formed along with the huge economic benefits and technical breakthrough of GO. Nematode Caenorhabditis elegans has a relatively short and prolific life cycle, small and transparent body, ease of culture and maintenance and well-characterized genome. C. elegans has been successfully used in the study of environmental toxicology or nanotoxicology. In the present study, we employed the in vivo assay system of C. elegans to perform the systematic study on the adverse effects of GO and the underlying molecular mechanisms. Moreover, with the aid of nitrogen-doped graphene quantum dots (N-GQDs), Glycyrrhizae radix (GR) and its active components, we investigated the prevention strategies based on both the chemical modification and the pharmacological administration.Methods:We employed the endpoints of lifespan, locomotion behavior, brood size, intestinal ROS induction, germ cell apoptosis cell number in the in vivo assay system of C. elegans to perform the systematic study on the underlying molecular mechanism of GO toxicity. To examine the translocation and biodistribution of GO, Rho B was baded onto the GO. We employed the SOLiD and Illumina HiSeqTM 2000 sequencing techniques, respectively, to examine the dysregulated miRNAs and mRNAs profiling in GO exposed nematodes. The techniques of bioinformatics analysis, molecular biology, and genetics were used to determine the biological functions of dysregulated miRNAs and mRNAs in the control of GO toxicity. N-GQDs, GR and its active components were used to investigate the possible prevention effects of chemical modification or pharmacological administration in being against the adverse effects of GO.Results:1. miRNAs control of in vivo toxicity from GOWith the aid of SOLiD sequencing, we identified 23 up-regulated and 8 down-regulated miRNAs in GO-exposed nematodes. Gene ontology and KEGG pathway database analysis implied that these identified miRNAs might be involved in control of many biological processes. Functions of the identified miRNAs in regulating the GO toxicity on lifespan and locomotion behavior during aging were confirmed in the available miRNAs mutants. mir-244 and mir-235 mutants exhibited hypersensitive property to GO toxicity than wide type, whereas mir-247/797, mir-73/74 and mir-231 mutants exhibited GO toxicity resistance. GO might reduce lifespan through influencing the functions of insulin/IGF signaling, TOR signaling, and germline signaling pathways controlled by miRNAs.2. miRNAs-mRNAs network involved in the control of toxicity of GOEmploying the HiSeq 2000 sequencing technique, we identified 970 up-regulated and 995 down-regulated mRNAs induced by GO exposure. Considering the feet that miRNAs usually act to post-transcriptionally inhibit target genes expression, we here raised a miRNAs-mRNAs network based on the identified dysregulated miRNAs and mRNAs profiling induced by GO. This miRNAs-mRNAs network is helpful for explaining the molecular basis for the important roles of oxidative stress, intestinal development and function, and defecation behavior in regulating GO toxicity. In this miRNAs-mRNAs network, some dysregulated miRNAs might function with genes encoding JNK signaling pathway as their targeted genes to regulate GO toxicity. In the JNK signaling pathway, JKK-1 and MEK-1 functioned upstream of JNK-1 to regulate GO toxicity.3. GO induced reproductive toxicity and the underlying molecular mechanismGO exposure induced a significant increase in the germ cell apoptosis. GO induced cell death required the involvement of core apoptotic machinery genes of ced-3 and ced-4. The apoptosis might be triggered by DNA damage, thereby inducing germ cell cycle arrests and leading to a decrease in the number of mitotic cells in GO exposed nematodes. miRNAs were involved in the regulation of reproductive toxicity induced by GO through influencing functions of genes encoding apoptosis signaling pathway and cell cycle checkpoint proteins. Mutation of mir-360 was hypersensitive in response to GO induced reproductive toxicity. Thus, miRNAs may play an important role in GO induced reproductive toxicity through influencing functions of the core cell death signaling pathway and the cell cycle checkpoint proteins.4. Involvement of p38 signaling in the control of GO toxicity and the underlying molecular mechanismGO exposure altered expression patterns of genes encoding the p38 signaling pathway, and caused to the transbcation of PMK-1 protein from cytoplasm to nucleus, sek-1, mek-1, and pmk-1 mutants showed the sensitive phenotype to GO toxicity compared with wide type, and more GO accumulation were found in sek-1, mek-1, and pmk-1 mutants compared with wild-type. Intestine-specific RNAi of pmk-1 gene resulted in the similar phenotypes to those in nematodes with whole body RNAi ofpmk-1 gene. Intestine-specific expression of pmk-1 gene rescued the sensitive phenotype of pmk-1 mutants. Under the GO exposure condition, double mutants of pmk-1;mek-1 and pmk-1;sek-1 showed the similar phenotypes to those in pmk-1 single mutant, suggesting that MEK-1 and SEK-1 may act upstream of PMK-1 in regulating the GO toxicity. Similarly, pmk-lskn-1 double mutant showed the similar phenotypes to those in skn-1 single mutant, pmk-1gst-4 double mutant showed the similar phenotypes to those in gst-4 single mutants, and skn-1 gst-4 double mutant exhibited the similar phenotypes to those in gst-4 single mutants, suggesting that PMK-1 may function upstream of SKN-1 and GST and SKN-1 may further function upstream of GST-4 to regulate the GO toxicity.5. Transgenerational safety of nitrogen-doped graphene quantum dots and the underlying cellular mechanismProlonged exposure to N-GQDs did not induce lethality, lifespan reduction, or change the functions of primary and secondary targeted organs in the wild-type nematode and in nematodes with mutations of the sod-2 or sod-3 genes that encode Mn-SODs. Moreover, no adverse effects were detected in progeny of N-GQD-exposed wild-type and mutant nematodes. N-GQDs were only distributed in the intestine of both wild-type and mutant nematodes. No N-GQDs accumulation was observed in embryos and progeny of exposed nematodes. After N-GQDs exposure, the normal biological state of the intestinal barrier and defecation behavior were found in wide-type and mutant nematodes. We hypothesis that the physiological states of intestinal barrier and defecation behavior, together with the decrease in C=O=C group compared with GO, may contribute greatly to the lack of transbcation of N-GQDs into the secondary target organs and the progeny of exposed nematodes.6. Glyrrhizic acid, active component from Glycyrrhizae radix, prevents toxicity of GO by influencing functions of miRNAsPretreatment with GR (62.5-125 mg/mL) prevented the adverse effects of GO on function of both primary and secondary targeted organs. Among the active components (glyrrhizic acid, liquiritin, or isoliquiritin) in GR, glyrrhizic acid mainly accounted for the beneficial effects of GR in being against the adverse effects of GO on nematodes. Pretreatment with glyrrhizic acid also suppressed the translocation of GO through the intestinal barrier into the secondary targeted organs of nematodes. Pretreatment with glyrrhizic acid further inhibited the induction of intestinal ROS production and recovered the expression patterns of dysregulated genes required for the control of oxidative stress in GO exposed nematodes. Moreover, pretreatment with glyrrhizic acid recovered the expression patterns of dysregulated miRNAs induced by GO, and genes required for the control of oxidative stress act as the targeted genes for some of these miRNAs. Mutation of mir-360 enhanced the beneficial effects of glyrrhizic acid in being against the adverse effects of GO. We hypothesis that glyrrhizic acid may prevent GO toxicity and transbcation by influencing functions of miRNAs which upstream regulate functions of their targeted genes required for the control of oxidative stress.Conclusions:In the present study, we obtained the dysregulated miRNAs and mRNAs profiling induced by GO exposure. Moreover, we raised a miRNAs-mRNAs network involved in the molecular control of GO toxicity. For the detailed molecular mechanisms, our data suggest that apoptosis signaling pathway and cell cycle checkpoint proteins were involved in the control of GO induced reproductive toxicity, and p38 signaling pathway (SEK-1/MEK-1-PMK-1-SKN-1-GST-4) played a crucial role in regulating GO toxicity and translocation. We provide the evidence to show the potentials of chemical modification and pharmacological administration in preventing GO toxicity.