Fragmentation Pattern-Based Screening Strategy Combining Diagnostic Ion and Neutral Loss Uncovered Novel para-Phenylenediamine Quinone Contaminants in the Environment.

Identifying transformed emerging contaminants in complex environmental compartments is a challenging but meaningful task. Substituted para-phenylenediamine quinones (PPD-quinones) are emerging contaminants originating from rubber antioxidants and have been proven to be toxic to the aquatic species, especially salmonids. The emergence of multiple PPD-quinones in various environmental matrices and evidence of their specific hazards underscore the need to understand their environmental occurrences. Here, we introduce a fragmentation pattern-based nontargeted screening strategy combining full MS/All ion fragmentation/neutral loss-ddMS2 scans to identify potential unknown PPD-quinones in different environmental matrices. Using diagnostic fragments of m/z 170.0600, 139.0502, and characteristic neutral losses of 199.0633, 138.0429 Da, six known and three novel PPD-quinones were recognized in air particulates, surface soil, and tire tissue. Their specific structures were confirmed, and their environmental concentration and composition profiles were clarified with self-synthesized standards. N-(1-methylheptyl)-N'-phenyl-1,4-benzenediamine quinone (8PPD-Q) and N,N'-di(1,3-dimethylbutyl)-p-phenylenediamine quinone (66PD-Q) were identified and quantified for the first time, with their median concentrations found to be 0.02-0.21 µg·g-1 in tire tissue, 0.40-2.76 pg·m-3 in air particles, and 0.23-1.02 ng·g-1 in surface soil. This work provides new evidence for the presence of unknown PPD-quinones in the environment, showcasing a potential strategy for screening emerging transformed contaminants in the environment.

Presence of N, N'-Substituted p-Phenylenediamine Quinones in Tap Water: Implication for Human Exposure.

Monitoring studies have demonstrated the wide presence of N, N'-substituted p-phenylenediamine-derived quinones (PPDQs) in environmental matrices. The general population may be potentially exposed to PPDQs through the consumption of tap water. While, the existence of PPDQs in tap water has not been well examined. To fill this gap, in this study we collected tap water samples from Hangzhou, China, and examined seven homologues of PPDQs in collected samples. All target PPDQs were identified in the collected tap water samples, with distinct detection frequencies (38-89%). PPDQs detected in tap water was dominated by N-(1, 3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPDQ; mean 0.56 ng/L, < LOD-4.0 ng/L). The profiles of PPDQs concentrations in tap water from the four districts of Hangzhou city were slightly different. The daily intake (DI) was found highest for 6PPDQ (mean 14-22 pg/kg bw/day, median 10-15 pg/kg bw/day) through tap water intake. The relatively higher DIs of various PPDQs were displayed for infants (mean 10-22 pg/kg bw/day, median 6.5-15 pg/kg bw/day), relative to the children (8.0-18 pg/kg bw/day, 5.4-12 pg/kg bw/day) and adults (6.7-14 pg/kg bw/day, 4.5-10 pg/kg bw/day). These data are crucial for assessing the overall human exposure to PPDQs. This study first, to our knowledge, reveals the concentrations and profiles of PPDQs in tap water.

Environmental fate of tire-rubber related pollutants 6PPD and 6PPD-Q: A review.

To enhance tire durability, the antioxidant N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) is used in rubber, but it converts into the toxic 6PPD quinone (6PPD-Q) when exposed to oxidants like ozone (O3), causing ecological concerns. This review synthesizes the existing data to assess the transformation, bioavailability, and potential hazards of two tire-derived pollutants 6PPD and 6PPD-Q. The comparative analysis of different thermal methods utilized in repurposing waste materials like tires and plastics into valuable products are analyzed. These methods shed light on the aspects of pyrolysis and catalytic conversion processes, providing valuable perspectives into optimizing the waste valorization and mitigating environmental impacts. Furthermore, we have examined the bioavailability and potential hazards of chemicals used in tire manufacturing, based on the literature included in this review. The bioavailability of these chemicals, particularly the transformation of 6PPD to 6PPD-Q, poses significant ecological risks. 6PPD-Q is highly bioavailable in aquatic environments, indicating its potential for widespread ecological harm. The persistence and mobility of 6PPD-Q in the environment, along with its toxicological effects, highlight the critical need for ongoing monitoring and the development of effective mitigation strategies to reduce its impact on both human health and ecosystem. Future research should focus on understanding the chronic effects of low-level exposure to these compounds on both terrestrial and aquatic ecosystems, as well as the potential for bioaccumulation in the food chain. Additionally, this review outlines the knowledge gaps, recommending further research into the toxicity of tire-derived pollutants in organisms and the health implications for humans and ecosystems.

Neurotoxicity from long-term exposure to 6-PPDQ: Recent advances.

The recent acceleration of industrialization and urbanization has brought significant attention to N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone (6-PPDQ), an emerging environmental pollutant from tire wear, due to its long-term effects on the environment and organisms. Recent studies suggest that 6-PPDQ can disrupt neurotransmitter synthesis and release, impact receptor function, and alter signaling pathways, potentially causing oxidative stress, inflammation, and apoptosis. This review investigates the potential neurotoxic effects of prolonged 6-PPDQ exposure, the mechanisms underlying its cytotoxicity, and the associated health risks. We emphasize the need for future research, including precise exposure assessments, identification of individual differences, and development of risk assessments and intervention strategies. This article provides a comprehensive overview of 6-PPDQ's behavior, impact, and neurotoxicity in the environment, highlighting key areas and challenges for future research.

Acute toxicity testing of 6PPD-quinone on the estuarine-dependent sport fish, Sciaenops ocellatus.

Recently, large-scale fish kills in the Pacific Northwest were linked to tire wear particles (TWPs) left on roadways, with the lethality attributed to 6PPD-quinone. which has a median lethal concentration of <1 µg/L for selected salmonids. However, there remains a paucity of 6PPD-quinone toxicity values developed for estuarine fish species, which is particularly significant because estuaries receiving inflows from highly urbanized watersheds are especially vulnerable to TWP contamination. Therefore, the present study aimed to determine the toxicity of 6PPD-quinone to an economically and ecologically important estuarine-dependent fish-red drum (Sciaenops ocellatus). Here, we examined the relative sensitivities of three early life stages within red drum: embryonic, larval, and post-settlement for 24-72 hours, depending on the life stage. Exposure concentrations ranged from 10 µg/L to 500 µg/L. We also assessed the sub-lethal impacts of 6PPD-quinone exposure on development during embryonic and larval stages, including body and organ sizes. Our results indicate that red drum are not acutely sensitive to 6PPD-quinone at each early life stage tested. We also found that yolk-sac larvae did not exhibit sub-lethal morphological impacts in a dose-dependent manner, regardless of exposure during embryonic and larval stages. These data are the first to assess the impacts of 6PPD-quinone on estuarine-dependent non-model fishes.

Exposure to 6-PPD Quinone at Environmentally Relevant Concentrations Inhibits Both Lifespan and Healthspan in C. elegans.

N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine (6-PPD), one of the most common additives used in rubber, enters the environment due to significant emissions of tire wear particles. 6-PPD quinone (6-PPDQ) is an important derivative of 6-PPD after ozonization. With concentrations ranging from nanograms per liter to µg/L, 6-PPDQ has so far been identified in a series of water samples. Acute lethality of 6-PPDQ in coho salmon (LC50 < 1 µg/L) was lower than environmental concentrations of 6-PPDQ, highlighting the environment exposure risks of 6-PPDQ. It is becoming increasingly necessary to investigate the potential toxicity of 6-PPDQ at environmental concentrations. Here, we examined the effect of 6-PPDQ exposure on lifespan and healthspan and the underlying mechanism in Caenorhabditis elegans. Exposure to 6-PPDQ (1 and 10 µg/L) shortened the lifespan. Meanwhile, during the aging process, 6-PPDQ (0.1-10 µg/L) could decrease both pumping rate and locomotion behavior, suggesting the 6-PPDQ toxicity on healthspan. For the underlying molecular mechanism, the dysregulation in the insulin signaling pathway was linked to toxicity of 6-PPDQ on lifespan and healthspan. In the insulin signaling pathway, DAF-2 restricted the function of DAF-16 to activate downstream targets (SOD-3 and HSP-6), which in turn controlled the toxicity of 6-PPDQ on lifespan and healthspan. Additionally, in response to 6-PPDQ toxicity, insulin peptides (INS-6, INS-7, and DAF-28) could activate the corresponding receptor DAF-2. Therefore, exposure to 6-PPDQ at environmentally relevant concentrations potentially causes damage to both lifespan and healthspan by activating insulin signaling in organisms.

Exposure to 6-PPD Quinone at Environmentally Relevant Concentrations Causes Abnormal Locomotion Behaviors and Neurodegeneration in Caenorhabditis elegans.

6-PPD quinone (6-PPDQ) can be transformed from 6-PPD through ozonation. Nevertheless, the potential neurotoxicity of 6-PPDQ after long-term exposure and the underlying mechanism are largely unclear. In Caenorhabditis elegans, we here observed that 0.1-10 µg/L of 6-PPDQ caused several forms of abnormal locomotion behaviors. Meanwhile, the neurodegeneration of D-type motor neurons was observed in 10 µg/L of 6-PPDQ-exposed nematodes. The observed neurodegeneration was associated with the activation of the Ca2+ channel DEG-3-mediated signaling cascade. In this signaling cascade, expressions of deg-3, unc-68, itr-1, crt-1, clp-1, and tra-3 were increased by 10 µg/L of 6-PPDQ. Moreover, among genes encoding neuronal signals required for the control of stress response, expressions of jnk-1 and dbl-1 were decreased by 0.1-10 µg/L of 6-PPDQ, and expressions of daf-7 and glb-10 were decreased by 10 µg/L of 6-PPDQ. RNAi of jnk-1, dbl-1, daf-7, and glb-10 resulted in the susceptibility to 6-PPDQ toxicity in decreasing locomotory ability and in inducing neurodegeneration, suggesting that JNK-1, DBL-1, DAF-7, and GLB-10 were also required for the induction of 6-PPDQ neurotoxicity. Molecular docking analysis further demonstrated the binding potential of 6-PPDQ to DEG-3, JNK-1, DBL-1, DAF-7, and GLB-10. Together, our data suggested the exposure risk of 6-PPDQ at environmentally relevant concentrations in causing neurotoxicity in organisms.

Reactive Oxygen Species and Chromophoric Dissolved Organic Matter Drive the Aquatic Photochemical Pathways and Photoproducts of 6PPD-quinone under Simulated High-Latitude Conditions.

The photochemical degradation pathways of 6PPD-quinone (6PPDQ, 6PPD-Q), a toxic transformation product of the tire antiozonant 6PPD, were determined under simulated sunlight conditions typical of high-latitude surface waters. Direct photochemical degradation resulted in 6PPDQ half-lives ranging from 17.5 h at 20 °C to no observable degradation over 48 h at 4 °C. Sensitization of excited triplet-state pathways using Cs+ and Ar purging demonstrated that 6PPDQ does not decompose significantly from a triplet state relative to a singlet state. However, assessment of processes involving reactive oxygen species (ROS) quenchers and sensitizers indicated that singlet oxygen and hydroxyl radical do significantly contribute to the degradation of 6PPDQ. Investigation of these processes in natural lake waters indicated no difference in attenuation rates for direct photochemical processes at 20 °C. This suggests that direct photochemical degradation will dominate in warm waters, while indirect photochemical pathways will dominate in cold waters, involving ROS mediated by chromophoric dissolved organic matter (CDOM). Overall, the aquatic photodegradation rate of 6PPDQ will be strongly influenced by the compounding effects of environmental factors such as light screening and temperature on both direct and indirect photochemical processes. Transformation products were identified via UHPLC-Orbitrap mass spectrometry, revealing four major processes: (1) oxidation and cleavage of the quinone ring in the presence of ROS, (2) dealkylation, (3) rearrangement, and (4) deamination. These data indicate that 6PPDQ can photodegrade in cool, sunlit waters under the appropriate conditions: t1/2 = 17.4 h tono observable decrease (direct); t1/2 = 5.2-11.2 h (indirect, CDOM).

Enhanced Formation of 6PPD-Q during the Aging of Tire Wear Particles in Anaerobic Flooded Soils: The Role of Iron Reduction and Environmentally Persistent Free Radicals.

Rapid urbanization drives increased emission of tire wear particles (TWPs) and the contamination of a transformation product derived from tire antioxidant, termed as N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD-Q), with adverse implications for terrestrial ecosystems and human health. However, whether and how 6PPD-Q could be formed during the aging of TWPs in soils remains poorly understood. Here, we examine the accumulation and formation mechanisms of 6PPD-Q during the aging of TWPs in soils. Our results showed that biodegradation predominated the fate of 6PPD-Q in soils, whereas anaerobic flooded conditions were conducive to the 6PPD-Q formation and thus resulted in a ∼3.8-fold higher accumulation of 6PPD-Q in flooded soils than wet soils after aging of 60 days. The 6PPD-Q formation in flooded soils was enhanced by Fe reduction-coupled 6PPD oxidation in the first 30 days, while the transformation of TWP-harbored environmentally persistent free radicals (EPFRs) to superoxide radicals (O2•-) under anaerobic flooded conditions further dominated the formation of 6PPD-Q in the next 30 days. This study provides significant insight into understanding the aging behavior of TWPs and highlights an urgent need to assess the ecological risk of 6PPD-Q in soils.

Urine Excretion, Organ Distribution, and Placental Transfer of 6PPD and 6PPD-Quinone in Mice and Potential Developmental Toxicity through Nuclear Receptor Pathways.

The rubber antioxidant 6PPD has gained significant attention due to its highly toxic transformation product, 6PPD-quinone (6PPDQ). Despite their detection in urines of pregnant women, the placental transfer and developmental toxicity of 6PPD and 6PPDQ are unknown. Here, we treated C57Bl/6 mice with 4 mg/kg 6PPD or 6PPDQ to investigate their urine excretion and placental transfer. Female and male mice exhibited sex difference in excretion profiles of 6PPD and 6PPDQ. Urine concentrations of 6PPDQ were one order of magnitude lower than those of 6PPD, suggesting lower excretion and higher bioaccumulation of 6PPDQ. In pregnant mice treated with 6PPD or 6PPDQ from embryonic day 11.5 to 15.5, 6PPDQ showed ∼1.5-8 times higher concentrations than 6PPD in placenta, embryo body, and embryo brain, suggesting higher placental transfer of 6PPDQ. Using in vitro dual-luciferase reporter assays, we revealed that 6PPDQ activated the human retinoic acid receptor α (RARα) and retinoid X receptor α (RXRα) at concentrations as low as 0.3 µM, which was ∼10-fold higher than the concentrations detected in human urines. 6PPD activated the RXRα at concentrations as low as 1.2 µM. These results demonstrate the exposure risks of 6PPD and 6PPDQ during pregnancy and emphasize the need for further toxicological and epidemiological investigations.

Toxic Tire Wear Compounds (6PPD-Q and 4-ADPA) Detected in Airborne Particulate Matter Along a Highway in Mississippi, USA.

Tire wear particles (TWPs) are a major category of microplastic pollution produced by friction between tires and road surfaces. This non-exhaust particulate matter (PM) containing leachable toxic compounds is transported through the air and with stormwater runoff, leading to environmental pollution and human health concerns. In the present study, we collected airborne PM at varying distances (5, 15 and 30 m) along US Highway 278 in Oxford, Mississippi, USA, for ten consecutive days using Sigma-2 passive samplers. Particles (~ 1-80 µm) were passively collected directly into small (60 mL) wide-mouth separatory funnels placed inside the samplers. Particles were subsequently subjected to solvent extraction, and extracts were analyzed for TWP compounds by high resolution orbitrap mass spectrometry. This pilot study was focused solely on qualitative analyses to determine whether TWP compounds were present in this fraction of airborne PM. The abundance of airborne TWPs increased with proximity to the road with deposition rates (TWPs cm-2 day-1) of 23, 47, and 63 at 30 m, 15 m, and 5 m from the highway, respectively. Two common TWP compounds (6PPD-Q and 4-ADPA) were detected in all samples, except the field blank, at levels above their limits of detection, estimated at 2.90 and 1.14 ng L-1, respectively. Overall, this work suggests airborne TWPs may be a potential inhalation hazard, particularly for individuals and wildlife who spend extended periods outdoors along busy roadways. Research on the bioavailability of TWP compounds from inhaled TWPs is needed to address exposure risk.

Bioaccessibility of Organic Compounds Associated with Tire Particles Using a Fish In Vitro Digestive Model: Solubilization Kinetics and Effects of Food Coingestion.

Tire and road wear particles (TRWP) account for an important part of the polymer particles released into the environment. There are scientific knowledge gaps as to the potential bioaccessibility of chemicals associated with TRWP to aquatic organisms. This study investigated the solubilization and bioaccessibility of seven of the most widely used tire-associated organic chemicals and four of their degradation products from cryogenically milled tire tread (CMTT) into fish digestive fluids using an in vitro digestion model based on Oncorhynchus mykiss. Our results showed that 0.06-44.1% of the selected compounds were rapidly solubilized into simulated gastric and intestinal fluids within a typical gut transit time for fish (3 h in gastric and 24 h in intestinal fluids). The environmentally realistic scenario of coingestion of CMTT and fish prey was explored using ground Gammarus pulex. Coingestion caused compound-specific changes in solubilization, either increasing or decreasing the compounds' bioaccessibility in simulated gut fluids compared to CMTT alone. Our results emphasize that tire-associated compounds become accessible in a digestive milieu and should be studied further with respect to their bioaccumulation and toxicological effects upon passage of intestinal epithelial cells.

Organic Markers of Tire and Road Wear Particles in Sediments and Soils: Transformation Products of Major Antiozonants as Promising Candidates.

Tire and road wear particles (TRWPs) are one of the main sources of particulate traffic emissions, but measured data on TRWP contents in the environment are scarce. This study aims at identifying organic compounds suitable as quantitative markers for TRWPs by a tiered multistep selection process involving nontarget screening and subsequent identification by liquid-chromatography high-resolution mass spectrometry. Starting from several thousands of signals recorded in the extract of tire particles, the rigorous selection process considered source specificity, tendency of leaching, analytical sensitivity and precision, and stability during aging. It led to three transformation products of N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6-PPD) as the most suitable marker candidates: N-formyl-6-PPD, hydroxylated N-1,3-dimethylbutyl-N-phenyl quinone diimine, and 6-PPD-quinone. A linear response in standard addition experiments with tire particles and the correlation with TRWP contents in a diverse set of environmental samples imply that these compounds are promising candidates as markers for the quantification of TRWPs. Organic markers for TRWP contents in the environment would allow TRWP quantification with the traditional tandem MS (LC-MS/MS) equipment of an organic trace analytical laboratory and, thus, allow easy generation of data on TRWP occurrence in sediments and soils and other environmental matrices.

Exposure to 6-PPD quinone enhances glycogen accumulation in Caenorhabditiselegans.

Glycogen metabolism is an important biological process for organisms. In Caenorhabditis elegans, effect of 6-PPD quinone (6-PPDQ) on glycogen accumulation and underlying mechanism were examined. Exposure to 6-PPDQ (1 and 10 µg/L) increased glycogen accumulation. Meanwhile, exposure to 6-PPDQ (1 and 10 µg/L) increased expression of gsy-1 encoding glycogen synthase and decreased expression of pygl-1 encoding glycogen phosphorylase. In 6-PPDQ exposed animals, glycogen content and glycogen accumulation were inhibited by RNAi of gsy-1 and enhanced by RNAi of pygl-1. RNAi of gsy-1 increased pygl-1 expression, and RNAi of pygl-1 increased gsy-1 expression after 6-PPDQ exposure. In 6-PPDQ exposed nematodes, daf-16 and aak-2 expressions were decreased and glycogen accumulation was suppressed by RNAi of daf-16 and aak-2, suggesting alteration in daf-16 and aak-2 expressions did not mediate glycogen accumulation. Moreover, resistance to 6-PPDQ toxicity on locomotion and brood size was observed in gsy-1(RNAi) animals, and susceptibility to 6-PPDQ toxicity was found in pygl-1(RNAi) animals. Therefore, glycogen accumulation could be enhanced by exposure to 6-PPDQ in nematodes. In addition, alteration in expressions of gsy-1 and pygl-1 governing this enhancement in glycogen accumulation mediated induction of 6-PPDQ toxicity.

Exposure to 6-PPD quinone causes damage on mitochondrial complex I/II associated with lifespan reduction in Caenorhabditis elegans.

N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone (6-PPDQ) is an emerging pollutant transformed from 6-PPD. However, the effect of 6-PPDQ exposure on mitochondrion and underlying mechanism remains largely unclear. Using Caenorhabditis elegans as animal model, exposed to 6-PPDQ at 0.1-10 µg/L was performed form L1 larvae to adult day-1. Exposure to 6-PPDQ (1 and 10 µg/L) could increase oxygen consumption rate and decease adenosine 5'-triphosphate (ATP) content, suggesting induction of mitochondrial dysfunction. Activities of NADH dehydrogenase (complex I) and succinate dehydrogenase (complex II) were inhibited, accompanied by a decrease in expressions of gas-1, nuo-1, and mev-1. RNAi of gas-1 and mev-1 enhanced mitochondrial dysfunction and reduced lifespan of 6-PPDQ exposed nematodes. GAS-1 and MEV-1 functioned in parallel to regulate 6-PPDQ toxicity to reduce the lifespan. Insulin peptides and the insulin signaling pathway acted downstream of GAS-1 and MEV-1 to control the 6-PPDQ toxicity on longevity. Moreover, RNAi of sod-2 and sod-3, targeted genes of daf-16, caused susceptibility to 6-PPDQ toxicity in reducing lifespan and in causing reactive oxygen species (ROS) production. Therefore, 6-PPDQ at environmentally relevant concentrations (ERCs) potentially caused mitochondrial dysfunction by affecting mitochondrial complexes I and II, which was associated with lifespan reduction by affecting insulin signaling in organisms.

Exposure to 6-PPD quinone disrupts glucose metabolism associated with lifespan reduction by affecting insulin and AMPK signals in Caenorhabditis elegans.

Glucose metabolism plays an important role for formation of normal physiological state of organisms. However, association between altered glucose metabolism and toxicity of 6-PPD quinone (6-PPDQ) remains largely unknown. In 1-100 µg/L 6-PPDQ exposed Caenorhabditis elegans, we observed increased glucose content. After 6-PPDQ exposure (1-100 µg/L), expressions of F47B8.10 and fbp-1 governing gluconeogenesis were increased, and expressions of hxk-1, hxk-3, pfk-1.1, pyk-1, and pyk-2 governing glycolysis were decreased. Under 6-PPDQ exposure condition, glucose content could be changed by RNAi of F47B8.10, hxk-1, and hxk-3, key genes for gluconeogenesis and glycolysis. In 6-PPDQ exposed nematodes, RNAi of daf-16 and aak-2 elevated glucose content, increased expressions of F47B8.10 and/or fbp-1, and decreased expressions of hxk-1, hxk-3, and/or pfk-1.1. Additionally, lifespan and locomotion during aging were increased by RNAi of F47B8.10 and decreased by RNAi of hxk-1 and hxk-3 in 6-PPDQ exposed nematodes. Moreover, after 6-PPDQ exposure, RNAi of F47B8.10 decreased expressions of insulin peptide genes (ins-7 and daf-28) and insulin receptor gene daf-2 and increased expressions of daf-16 and aak-2. In 6-PPDQ exposed nematodes, RNAi of hxk-1 and hxk-3 further increased expressions of ins-7, daf-28, and daf-2 and decreased expressions of daf-16 and aak-2. Our results demonstrated important association between altered glucose metabolism and toxicity of 6-PPDQ in inducing lifespan reduction in organisms.

Exposure to 6-PPD quinone causes ferroptosis activation associated with induction of reproductive toxicity in Caenorhabditis elegans.

Exposure to N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone (6-PPDQ) caused toxicity on Caenorhabditis elegans, including reproductive toxicity. However, the underlying mechanisms for this induced reproductive toxicity by 6-PPDQ remain largely unclear. We examined possible association of ferroptosis activation with reproductive toxicity of 6-PPDQ. In 1-100 µg/L 6-PPDQ exposed nematodes, Fe2+ content was increased, which was accompanied with enhanced lipid peroxidation, increased malonydialdehyde (MDA) content, and decreased L-glutathione (GSH) content. Exposure to 1-100 µg/L 6-PPDQ decreased expressions of ftn-1 encoding ferritin, ads-1 encoding AGPS, and gpx-6 encoding GPX4 and increased expression of bli-3 encoding dual oxidase. After 6-PPDQ exposure, RNAi of ftn-1 decreased ads-1 and gpx-6 expressions and increased bli-3 expression. RNAi of ftn-1, ads-1, and gpx-6 strengthened alterations in ferroptosis related indicators, and RNAi of bli-3 suppressed changes of ferroptosis related indicators in 6-PPDQ exposed nematodes. Meanwhile, RNAi of ftn-1, ads-1, and gpx-6 induced susceptibility, and RNAi of bli-3 caused resistance to 6-PPDQ reproductive toxicity. Moreover, expressions of DNA damage checkpoint genes (clk-2, mrt-2, and hus-1) could be increased by RNAi of ftn-1, ads-1, and gpx-6 in 6-PPDQ exposed nematodes. Therefore, our results demonstrated activation of ferroptosis in nematodes exposed to 6-PPDQ at environmentally relevant concentrations, and this ferroptosis activation was related to reproductive toxicity of 6-PPDQ.

Evaluation of 6-PPD quinone toxicity on lung of male BALB/c mice by quantitative proteomics.

N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone (6-PPDQ), a transformation product of tyre-derived 6-PPD, has been frequently detected in different environments. After 6-PPDQ exposure, we here aimed to examine dynamic lung bioaccumulation, lung injury, and the underlying molecular basis in male BALB/c mice. After single injection at concentration of 4 mg/kg, 6-PPDQ remained in lung up to day 28, and higher level of 6-PPDQ bioaccumulation in lung was observed after repeated injection. Severe inflammation was observed in lung after both single and repeated 6-PPDQ injection as indicated by changes of inflammatory cytokines (TNF-α, IL-6 and IL-10). Sirius red staining and hydroxyproline content analysis indicated that repeated rather than single 6-PPDQ injection induced fibrosis in lung. Repeated 6-PPDQ injection also severely impaired lung function in mice by influencing chord compliance (Cchord) and enhanced pause (Penh). Proteomes analysis was further carried out to identify molecular targets of 6-PPDQ after repeated injection, which was confirmed by transcriptional expression analysis and immunohistochemistry staining. Alterations in Ripk1, Fadd, Il-6st, and Il-16 expressions were identified to be associated with inflammation induction of lung after repeated 6-PPDQ injection. Alteration in Smad2 expression was identified to be associated with fibrosis formation in lung of 6-PPDQ exposed mice. Therefore, long-term and repeated 6-PPDQ exposure potentially resulted in inflammation and fibrosis in lung by affecting certain molecular signals in mammals. Our results suggested several aspects of lung injury caused by 6-PPDQ and provide the underlying molecular basis. These observations implied the possible risks of long-term 6-PPDQ exposure to human health.

Polyethylene nanoparticles at environmentally relevant concentrations enhances neurotoxicity and accumulation of 6-PPD quinone in Caenorhabditis elegans.

The exposure risk of 6-PPD quinone (6-PPDQ) has aroused increasing concern. In the natural environment, 6-PPDQ could interact with other pollutants, posing more severe environmental problems and toxicity to organisms. We here examined the effect of polyethylene nanoplastic (PE-NP) on 6-PPDQ neurotoxicity and the underling mechanisms in Caenorhabditis elegans. In nematodes, PE-NP (1 and 10 µg/L) decreased locomotion behavior, but did not affect development of D-type neurons. Exposure to PE-NP (1 and 10 µg/L) strengthened neurotoxicity of 6-PPDQ (10 µg/L) on the aspect of locomotion and neurodegeneration induction of D-type motor neurons. Exposure to PE-NPs (10 µg/L) caused increase in expressions of mec-4, asp-3, and asp-4 governing neurodegeneration in 10 µg/L 6-PPDQ exposed nematodes. Moreover, exposure to PE-NP (10 µg/L) increased expression of some neuronal genes (daf-7, dbl-1, jnk-1, and mpk-1) in 6-PPDQ exposed nematodes, and RNAi of these genes resulted in susceptibility to neurotoxicity of PE-NP and 6-PPDQ. 6-PPDQ could be adsorbed by PE-NPs, and resuspension of PE-NP and 6-PPDQ after adsorption equilibrium exhibited similar neurotoxicity to co-exposure of PE-NP and 6-PPDQ. In addition, exposure to PE-NP (1 and 10 µg/L) increased 6-PPDQ accumulation in body of nematodes and increased defecation cycle length in 6-PPDQ exposed nematodes. Therefore, 6-PPDQ could be adsorbed on nanoplastics (such as PE-NPs) and enhance both neurotoxicity and accumulation of 6-PPDQ in organisms.

Potential human health risk of the emerging environmental contaminant 6-PPD quinone.

The tire antioxidant 6-PPD has been widely used to enhance tire performance and extend tire lifespan. 6-PPD quinone (6-PPDQ), a quinone derivative derived from 6-PPD in the presence of ozone, has been recognized an emerging environmental contaminant. In addition to causing acute lethality to coho salmon, 6-PPDQ exhibits toxic effects on other aquatic species and mammals. Based on the existing evidence, we provide a critical overview on the human internal exposure, potential adverse effects on health, and prediction of human health risk of 6-PPDQ. 6-PPDQ could be detected in human samples, including human urine, blood, and cerebrospinal fluid. Human exposure to 6-PPDQ in the environment is inevitable and may lead to adverse health effects, including hepatotoxicity, enterotoxicity, pulmonary toxicity, neurotoxicity, reproductive toxicity, and cardiotoxicity. Additionally, potential human health risk to 6-PPDQ through exposure routes and human samples were predicted. This review is helpful to identify the existing knowledge gaps and future research directions regarding the human health effects of 6-PPDQ.