ATP synthesis plays a pivotal role in maintaining physiological state of organisms, which is controlled by mitochondrial complex V. In Caenorhabditis elegans, we found that 6-PPD quinone (6-PPDQ) inhibited complex V activity. Expressions of atp-2, F58F12.1, asb-1, and Y82E9BR.3 encoding V-F1 and V-F0 subunits were also increased by 6-PPDQ, and RNAi of these genes increased ATP content after 6-PPDQ exposure. Meanwhile, 6-PPDQ-induced mitochondrial UPR was enhanced by atp-2, F58F12.1, asb-1, and Y82E9BR.3 RNAi, and expression of these 4 genes and complex V activity were influenced by RNAi of genes encoding components of complex I-IV. atp-2, F58F12.1, asb-1, and Y82E9BR.3 functioned in intestine, neurons, and germline to control the 6-PPDQ caused decrease in ATP content and induction of mitochondrial UPR. Moreover, RNAi of atp-2, F58F12.1, asb-1, and Y82E9BR.3 increased expressions of genes governing citric acid cycle and elevated NADH and FADH2 contents in 6-PPDQ exposed nematodes. 6-PPDQ has potential to bind to ATP-2, F58F12.1, ASB-1, and Y82E9BR.3. Therefore, potential direct effect of 6-PPDQ exposure in disrupting complex V was suggested to decrease the ATP content in organisms.

6-PPDQ readily accumulates in the liver, disrupting hepatic glucose and lipid homeostasis and precipitating liver injury. Whether it can also trigger the chronic metabolic disease metabolic dysfunction-associated fatty liver disease (MAFLD) remains unknown. Here we integrate network toxicology with molecular docking to explore the possibility that 6-PPDQ induces MAFLD and the underlying toxicity targets and molecular mechanisms. By mining public toxicological and disease databases we identified 45 target genes associated with 6-PPDQ and MAFLD. STRING and Cytoscape analyses pinpointed four hub genes—TNF, IL1B, IL6 and TP53. In addition, GEO datasets GSE63067 and GSE89632 were used to define the key phenotypes involved. Construction of a 6-PPDQ-genes-phenotypes-MAFLD network revealed the potential core targets, biological processes and pathways. Single gene GSEA indicated that these hubs modulate downstream hypoxia, inflammation, apoptosis and fatty acid metabolism, thereby influencing MAFLD progression, while molecular docking confirmed stable binding between the hubs and 6-PPDQ. Finally, we assembled an adverse outcome pathway (AOP) framework from 6-PPDQ to MAFLD. Our findings not only deepen understanding of 6-PPDQ toxicity but also provide a methodological template for assessing adverse health outcomes of emerging environmental pollutants.

In Caenorhabditis elegans, suppression in innate immunity could be induced by 6-PPD quinone (6-PPDQ); however, underlying mechanisms remain unknown. RNA interference (RNAi) of antimicrobial genes (lys-7 and spp-1) enhanced 6-PPDQ-induced lifespan reduction and increased 6-PPDQ accumulation. Accompanied with these, expressions of daf-16 encoding FOXO transcriptional factor and pmk-1 encoding p38 MAPK were decreased by 6-PPDQ exposure. In 6-PPDQ exposed nematodes, daf-16 and pmk-1 RNAi caused more severe inhibition in expression of antimicrobial genes. Additionally, 6-PPDQ caused lifespan reduction and 6-PPDQ accumulation were accelerated by daf-16 and pmk-1 RNAi. 6-PPDQ induced decrease in expression of antimicrobial genes and lifespan reduction and 6-PPDQ accumulation could be suppressed by pharmacological treatment with cuminaldehyde. Moreover, these beneficial effects of cuminaldehyde treatment were inhibited by daf-16 and pmk-1 RNAi, which further suggests crucial functions of DAF-16 and PMK-1. Our results highlight association of immunosuppression with 6-PPDQ-induced lifespan reduction, which was controlled by molecular signals of DAF-16 and PMK-1.

As a tire derived emergent contaminant, 6-PPD quinone (6-PPDQ) has received increasing attention. In the current study, we aimed to examine association of disruption in flavin adenine dinucleotide (FAD) metabolism with 6-PPDQ toxicity. In Caenorhabditis elegans, FAD content and expressions of flad-1 governing FAD synthesis and riboflavin transporter genes were decreased by 0.1-10 mu g/L 6-PPDQ. After 6-PPDQ exposure, flad-1 expression was further decreased by RNAi of riboflavin transporter genes. RNAi of flad-1 and riboflavin transporter genes resulted in susceptibility to 6-PPDQ caused immunosuppression. TORC1/DAF-15 was identified as target of FLAD-1, and FLAD-1 regulated 6-PPDQ caused immunosuppression by inhibiting DAF-15. Moreover, DAF-15 controlled 6-PPDQ caused immunosuppression by suppressing transcriptional factors (DAF-16, SKN-1, ATF-7, and ELT-2) and their downstream proteases (ASP-13 and ASP-14). Furthermore, 6-PPDQ caused immunosuppression could be suppressed by FAD treatment. Therefore, our results suggest that 6-PPDQ at environmentally relevant concentrations potentially disrupts FAD synthesis and riboflavin transporters, which is linked to immunosuppression induction.

6-PPD quinone (6-PPDQ) is the derivative from N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6-PPD) after reaction with environmental ozone. Pentose phosphate pathway (PPP) serves as a principal source of nicotinamide adenine dinucleotide phosphate (NADPH). However, association between disruption in PPP and 6-PPDQ neurotoxicity remains largely unclear. In Caenorhabditis elegans, we first examined the role of PPP derived NADPH in controlling 6-PPDQ neurotoxicity. Exposure to 0.1-10 µg/L 6-PPDQ decreased NADPH level and inhibited expression of gspd-1 and T25B9.9 encoding PPP related enzymes. Meanwhile, NADPH-dependent antioxidant defense was inhibited by 6-PPDQ, which was reflected by reduced glutathione (GSH) content and elevated H₂O₂ level. Additionally, expressions of glutathione peroxidase genes (gpx-1, gpx-2, gpx-3, gpx-4, gpx-5, and gpx-6) were decreased by 6-PPDQ. RNAi of gspd-1, T25B9.9, and glutathione peroxidase genes exacerbated 6-PPDQ induced inhibition in locomotion and degeneration of D-type neurons. Moreover, RNAi of these genes strengthened activation of neurodegeneration related genes (deg-3, crt-1, itr-1, and tra-3) and suppressed expression of genes related to stress response (jnk-1, glb-10, and dbl-1) in 6-PPDQ exposed nematodes. Therefore, our results suggested the possibility of disruption in PPP by 6-PPDQ in intensifying damage on NADPH-dependent antioxidant defense and thereby causing the neurotoxicity in nematodes.

The 6-PPDQ is an emerging contaminant ubiquitously detected in the environment and showing multiple aspects of toxicity on organisms. SIRT1 is a conserved NAD+-dependent deacetylase required for controlling stress response. In Caenorhabditis elegans, 0.1-10 mu g/L 6-PPDQ reduced nuclear NAD+ level, and decreased expressions of sir-2.1 encoding SIRT1 and nuclear SIR-2.1::mCherry, which could be reversed by treatment with NAD+ precursors, such as nicotinic acid (NA) and nicotinamide (NAM). Intestinal RNAi of sir-2.1 exacerbated 6-PPDQ caused immunosuppression, whereas intestinal sir-2.1 overexpression prevented 6-PPDQ induced immunosuppression. The sir-2.1 RNAi upregulated insulin ligand genes and daf-2, and inhibited daf-16 and its target genes. Genetic analyses confirmed that both DAF-16 and insulin ligands acted downstream of intestinal SIR-2.1 to regulated 6-PPDQ caused immunosuppression. Therefore, 6-PPDQ exposure disrupted intracellular NAD+ homeostasis and inhibited nuclear SIR-2.1, which in turn enhanced 6-PPDQ caused immunosuppression by modulating the insulin signals.

The 77PD quinone (77PDQ), a member of PPDQs family, can be frequently detected in environment and bioavailable to organisms. In nematodes, toxicity of 77PDQ on longevity and healthspan and underlying mechanism were determined. Exposure to 0.1-10 μg/L 77PDQ reduced lifespan and inhibited healthspan indicated by change of locomotion behavior during the aging. 77PDQ was accumulated in mitochondrion, and caused mitochondrial dysfunction. Activities of mitochondrial complex I/II and expression of component genes for complex I/II were inhibited by 77PDQ, and RNAi of component genes of gas-1 and mev-1 strengthened 77PDQ toxicity on longevity and healthspan. Additionally, expressions of hsp-6/60, mitochondrial UPR (mt UPR) marker genes, were inhibited by 10 μg/L 77PDQ, suggesting induction of suppression in mt UPR. 77PDQ toxicity on longevity and healthspan was also exacerbated by hsp-6/60 RNAi. Pharmacological treatment with cuminaldehyde inhibited 77PDQ toxicity on longevity and healthspan and in causing suppression in mt UPR. This beneficial effect of cuminaldehyde could be disrupted by gas-1, mev-1, hsp-6, and hsp-60 RNAi, which further confirmed role of these mitochondrial signals in controlling 77PDQ toxicity. Therefore, risk of 77PDQ exposure in inhibiting longevity and healthspan was suggested, which was associated with dysregulation of mitochondrial signals.

6-PPD quinine (6-PPDQ) affects intestinal barrier function; however, its underlying mechanisms remain largely unknown. In the current study, we examined the role of reduction in phosphatidic acid synthesis in mediating the toxicity of 6-PPDQ in affecting intestinal barrier function. In Caenorhabditis elegans, 6-PPDQ exposure reduced the phosphatidic acid content, which was accompanied by the decreased expression of acl-5 and acl-6 encoding glycerol-3-phosphate acyltransferase. The RNAi of acl-5 and acl-6 lowered the phosphatidic acid content, enhanced intestinal permeability, and resulted in the increased accumulation of 6-PPDQ. Meanwhile, acl-5 and acl-6 RNAi caused susceptibility to 6-PPDQ toxicity by upregulating the expressions of insulin ligands and receptor genes and downregulating the expressions of daf-16 and its target genes. Moreover, the RNAi of acl-5 and acl-6 elevated the expression of let-363, and the RNAi of let-363 could reduce the expressions of insulin ligand genes and confer resistance to 6-PPDQ toxicity. The double RNAi of acl-5 and acl-6 caused more severe enhanced intestinal permeability and 6-PPDQ toxicity. Therefore, 6-PPDQ exposure potentially disrupts phosphatidic acid synthesis to affect intestinal barrier function by downregulating acl-5 and acl-6 expressions.

Glutamate synthesized from the proline can serve as a precursor for key intermediate metabolites of citric acid cycle. Recently, we observed reduced glutamate content and expression of alh-6 controlling glutamate synthesis by 6-PPD quinone (6-PPDQ) in Caenorhabditis elegans. However, possible effect of 6-PPDQ on proline synthesis and the association with 6-PPDQ toxicity induction remain unclear. After 0.1-10 μg/L 6-PPDQ exposure, proline content was further reduced, and expression of pycr-1 governing proline biosynthesis was decreased. In 6-PPDQ exposed nematodes, RNA interference (RNAi) of pycr-1 decreased α-ketoglutarate content, enhanced mitochondrial dysfunction, reduced nicotinamide adenine dinucleotide (NADH) and reduced flavine adenine dinucleotide (FADH₂) contents, inhibited mitochondrial complex I/II activities, and decreased expressions of gas-1 and mev-1. Moreover, compared to single RNAi, double RNAi of pycr-1 and alh-6 exacerbated the 6-PPDQ toxicity in reducing α-ketoglutarate, NADH, and FADH₂ contents, and suppressing mitochondrial complex I/II activities and gas-1 and mev-1 expressions. Additionally, double RNAi of pycr-1 and alh-6 intensified toxicity of 6-PPDQ on longevity and caused upregulation of insulin ligand and receptor genes and downregulation of daf-16 and its targeted genes in 6-PPDQ exposed nematodes. Furthermore, after 6-PPDQ exposure, daf-16 RNAi suppressed pycr-1 and alh-6 expressions, suggesting formation of a regulatory feedback loop between pycr-1/alh-6 and daf-16. Our findings highlight involvement of disrupted proline and glutamate metabolisms in 6-PPDQ-induced mitochondrial dysfunction and reduced longevity.