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.

Comparison of intestinal toxicity in enhancing intestinal permeability and in causing ROS production of six PPD quinones in Caenorhabditis elegans.

As the derivatives of p-phenylenediamines (PPDs), PPD quinones (PPDQs) have received increasing attention due to their possible exposure risk. We compared the intestinal toxicity of six PPDQs (6-PPDQ, 77PDQ, CPPDQ, DPPDQ, DTPDQ and IPPDQ) in Caenorhabditis elegans. In the range of 0.01-10 µg/L, only 77PDQ (10 µg/L) moderately induced the lethality. All the examined PPDQs at 0.01-10 µg/L did not affect intestinal morphology. Different from this, exposure to 6-PPDQ (1-10 µg/L), 77PDQ (0.1-10 µg/L), CPPDQ (1-10 µg/L), DPPDQ (1-10 µg/L), DTPDQ (1-10 µg/L), and IPPDQ (10 µg/L) enhanced intestinal permeability to different degrees. Meanwhile, exposure to 6-PPDQ (0.1-10 µg/L), 77PDQ (0.01-10 µg/L), CPPDQ (0.1-10 µg/L), DPPDQ (0.1-10 µg/L), DTPDQ (1-10 µg/L), and IPPDQ (1-10 µg/L) resulted in intestinal reactive oxygen species (ROS) production and activation of both SOD-3::GFP and GST-4::GFP. In 6-PPDQ, 77PDQ, CPPDQ, DPPDQ, DTPDQ, and/or IPPDQ exposed nematodes, the ROS production was strengthened by RNAi of genes (acs-22, erm-1, hmp-2, and pkc-3) governing functional state of intestinal barrier. Additionally, expressions of acs-22, erm-1, hmp-2, and pkc-3 were negatively correlated with intestinal ROS production in nematodes exposed to 6-PPDQ, 77PDQ, CPPDQ, DPPDQ, DTPDQ, and/or IPPDQ. Therefore, exposure to different PPDQs differentially induced the intestinal toxicity on nematodes. Our data highlighted potential exposure risk of PPDQs at low concentrations to organisms by inducing intestinal toxicity.

Chronic exposure to tire rubber-derived contaminant 6PPD-quinone impairs sperm quality and induces the damage of reproductive capacity in male mice.

It has been reported that N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD-Q), a derivative of the tire antioxidant, N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD), exhibits acute toxicity towards organisms. However, the possible reproductive toxicity of 6PPD-Q in mammals has rarely been reported. In this study, the effects of 6PPD-Q on the reproductive toxicity of C57Bl/6 male mice were assessed after exposure to 6PPD-Q for 40 days at 4 mg/kg body weight (bw). Exposure to 6PPD-Q not only led to a decrease in testosterone levels but also adversely affected semen quality and in vitro fertilization (IVF) outcomes, thereby indicating impaired male fertility resulting from 6PPD-Q exposure. Additionally, transcriptomic and metabolomic analyses revealed that 6PPD-Q elicited differential expression of genes and metabolites primarily enriched in spermatogenesis, apoptosis, arginine biosynthesis, and sphingolipid metabolism in the testes of mice. In conclusion, our study reveals the toxicity of 6PPD-Q on the reproductive capacity concerning baseline endocrine disorders, sperm quality, germ cell apoptosis, and the sphingolipid signaling pathway in mice. These findings contribute to an enhanced understanding of the health hazards posed by 6PPD-Q to mammals, thereby facilitating the development of more robust safety regulations governing the utilization and disposal of rubber products.

6PPDQ induces cardiomyocyte senescence via AhR/ROS-mediated autophagic flux blockage.

Recently, attention has been drawn to the adverse outcomes of N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone (6PPDQ) on human health, but its cardiac toxicity has been relatively understudied. This work aims to investigate the effects of 6PPDQ on differentiated H9c2 cardiomyocytes. Our findings demonstrated that exposure to 6PPDQ altered cellular morphology and disrupted the expression of cardiac-specific markers. Significantly, 6PPDQ exposure led to cardiomyocyte senescence, characterized by elevated ß-Galactosidase activity, upregulation of cell cycle inhibitor, induction of DNA double-strand breaks, and remodeling of Lamin B1. Furthermore, 6PPDQ hindered autophagy flux by promoting the formation of autophagosomes while inhibiting the degradation of autolysosomes. Remarkably, restoration of autophagic flux using rapamycin counteracted 6PPDQ-induced cardiomyocyte senescence. Additionally, our study revealed that 6PPDQ significantly increased the ROS production. However, ROS scavenger effectively reduced the blockage of autophagic flux and cardiomyocyte senescence caused by 6PPDQ. Furthermore, we discovered that 6PPDQ activated the Aryl hydrocarbon receptor (AhR) signaling pathway. AhR antagonist was found to reverse the blockage of autophagy and alleviate cardiac senescence, while also reducing ROS levels in 6PPDQ-treated group. In conclusion, our research unveils that exposure to 6PPDQ induces ROS overproduction through AhR activation, leading to disruption of autophagy flux and ultimately contributing to cardiomyocyte senescence.

Toxic effects and comparison of common amino antioxidants (AAOs) in the environment on zebrafish: A comprehensive analysis based on cells, embryos, and adult fish.

The ubiquity of amino antioxidants (AAOs) in the environment has attracted increasing attention, given their potential toxicity. This investigation represents a pioneering effort, systematically scrutinizing the toxicological effects of four distinct AAOs across the developmental spectrum of zebrafish, encompassing embryonic, larvae, and adult stages. The results indicate that four types of AAO exhibit varying degrees of cell proliferation toxicity. Although environmentally relevant concentrations of AAOs exhibit a comparatively circumscribed impact on zebrafish embryo development, heightened concentrations (300 µg/L) of N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) and N-isopropyl-N'-phenyl-p-phenylenediamine (IPPD) distinctly evoke developmental toxicity. Behavioral analysis results indicate that at concentrations of 20 and 300 µg/L, the majority of AAOs significantly reduced the swimming speed and activity of larvae. Moreover, each AAO triggers the generation of reactive oxygen species (ROS) in larvae, instigating diverse levels of oxidative stress. The study delineates parallel toxicological patterns in zebrafish exposed to 300 µg/L of 6PPD and IPPD, thereby establishing a comparable toxicity profile. The comprehensive toxicity effects among the four AAOs is as follows: IPPD >6PPD > N-Phenyl-1-naphthylamine (PANA) > diphenylamine (DPA). These findings not only enrich our comprehension of the potential hazards associated with AAOs but also provide data support for structure-based toxicity prediction models.

Toxicity of substituted p-phenylenediamine antioxidants and their derived novel quinones on aquatic bacterium: Acute effects and mechanistic insights.

Substituted para-phenylenediamines (PPDs) are synthetic chemicals used globally for rubber antioxidation, with their quinone derivatives (PPD-Qs) raising particular environmental concerns due to their severe toxicity to aquatic organisms. Emerging research has identified a variety of novel PPD-Qs ubiquitously detected in the environment, yet experimental proof for the toxicity of PPD-Qs has not been forthcoming due to the unavailability of bulk standards, leaving substantial gaps in the prioritization and mechanistic investigation of such novel pollutants. Here, we use synthesized chemical standards to study the acute toxicity and underlying mechanism of 18 PPD-Qs and PPDs to the aquatic bacterium V. fischeri. Bioluminescence inhibition EC50 of PPD-Qs ranged from 1.76-15.6 mg/L, with several emerging PPD-Qs demonstrating significantly higher toxicity than the well-studied 6PPD-Q. This finding suggests a broad toxicological threat PPD-Qs pose to the aquatic bacterium, other than 6PPD-Q. Biological response assays revealed that PPD-Qs can reduce the esterase activity, cause cell membrane damage and intracellular oxidative stress. Molecular docking unveiled multiple interactions of PPD-Qs with the luciferase in V. fischeri, suggesting their potential functional impacts on proteins through competitive binding. Our results provided crucial toxicity benchmarks for PPD-Qs, prioritized these novel pollutants and shed light on the potential toxicological mechanisms.

Widespread occurrence of two typical N, N'-substituted p-phenylenediamines and their quinones in humans: Association with oxidative stress and liver damage.

While N, N'-substituted p-phenylenediamines (PPDs) and their quinone derivatives (PPDQs) have been widely detected in the environment, there is currently limited data on their occurrence in humans. In this study, we conducted the first serum analysis of two PPDs and PPDQs in the healthy and secondary nonalcoholic fatty liver disease (S-NAFLD) cohorts in South China. The concentrations of four oxidative stress biomarkers (OSBs), namely, 8-iso-prostaglandin F2α (8-PGF2α), 11ß-prostaglandin F2α (11-PGF2α), 15(R)-prostaglandin F2α (15-PGF2α), and 8-hydroxy-2'-deoxyguanosine in serum samples were also measured. Results showed that N-(1,3-dimethybutyl)-N'-phenyl-p-phenylenediamine (6PPD) quinone was the predominant target analytes both in the healthy and S-NAFLD cohorts, with the median concentrations of 0.13 and 0.20 ng/mL, respectively. Significant (p < 0.05) and positive correlations were found between 6PPD concentration and 8-PGF2α, 11-PGF2α, and 15-PGF2α in both the healthy and S-NAFLD cohorts, indicating that 6PPD may be associated with lipid oxidative damage. In addition, concentrations of 6PPD in serum were associated significantly linked with total bilirubin (ß = 0.180 µmol/L, 95%CI: 0.036-0.396) and direct bilirubin (DBIL, ß = 0.321 µmol/L, 95%CI: 0.035-0.677) related to hepatotoxicity. Furthermore, 8-PGF2α, 11-PGF2α, and 15-PGF2α mediated 17.1%, 24.5%, and 16.6% of 6PPD-associated DBIL elevations, respectively. Conclusively, this study provides novel insights into human exposure to and hepatotoxicity assessment of PPDs and PPDQs.

A mini review on 6PPD quinone: A new threat to aquaculture and fisheries.

Numerous toxic substances are directly and indirectly discharged by humans into water bodies, causing distress to the organisms living on it. 6PPD, an amino antioxidant from tires reacts with ozone to form 6PPD-Q, which has garnered global attention due to its lethal nature to various organisms. This review aims to provide an understanding of the sources, transformation, and fate of 6PPD-Q in water and the current knowledge on its effects on aquatic organisms. Furthermore, we discuss research gaps pertaining to the mechanisms by which 6PPD-Q acts within fish bodies. Previous studies have demonstrated the ubiquitous presence of 6PPD-Q in the environment, including air, water, and soil. Moreover, this compound has shown high lethality to certain fish species while not affecting others. Toxicological studies have revealed its impact on the nervous system, intestinal barrier function, cardiac function, equilibrium loss, and oxidative stress in various fish species. Additionally, exposure to 6PPD-Q has led to organ injury, lipid accumulation, and cytokine production in C. elegans and mice. Despite studies elucidating the lethal dose and effects of 6PPD-Q in fish species, the underlying mechanisms behind these symptoms remain unclear. Future studies should prioritize investigating the mechanisms underlying the lethality of 6PPD-Q in fish species to gain a better understanding of its potential effects on different organisms.

Interspecies Differences in 6PPD-Quinone Toxicity Across Seven Fish Species: Metabolite Identification and Semiquantification.

N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD-Q) is a recently identified contaminant that originates from the oxidation of the tire antidegradant 6PPD. 6PPD-Q is acutely toxic to select salmonids at environmentally relevant concentrations, while other fish species display tolerance to concentrations that surpass those measured in the environment. The reasons for these marked differences in sensitivity are presently unknown. The objective of this research was to explore potential toxicokinetic drivers of species sensitivity by characterizing biliary metabolites of 6PPD-Q in sensitive and tolerant fishes. For the first time, we identified an O-glucuronide metabolite of 6PPD-Q using high-resolution mass spectrometry. The semiquantified levels of this metabolite in tolerant species or life stages, including white sturgeon (Acipenser transmontanus), chinook salmon (Oncorhynchus tshawytscha), westslope cutthroat trout (Oncorhynchus clarkii lewisi), and nonfry life stages of Atlantic salmon (Salmo salar), were greater than those in sensitive species, including coho salmon (Oncorhynchus kisutch), brook trout (Salvelinus fontinalis), and rainbow trout (Oncorhynchus mykiss), suggesting that tolerant species might detoxify 6PPD-Q more effectively. Thus, we hypothesize that differences in species sensitivity are a result of differences in basal expression of biotransformation enzyme across various fish species. Moreover, the semiquantification of 6PPD-Q metabolites in bile extracted from wild-caught fish might be a useful biomarker of exposure to 6PPD-Q, thereby being valuable to environmental monitoring and risk assessment.

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.

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.

Uptake and Biotransformation of the Tire Rubber-derived Contaminants 6-PPD and 6-PPD Quinone in the Zebrafish Embryo (Danio rerio).

N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine (6-PPD) is a widely used antioxidant in tire rubber known to enter the aquatic environment via road runoff. The associated transformation product (TP) 6-PPD quinone (6-PPDQ) causes extreme acute toxicity in some fish species (e.g., coho salmon). To interpret the species-specific toxicity, information about biotransformation products of 6-PPDQ would be relevant. This study investigated toxicokinetics of 6-PPD and 6-PPDQ in the zebrafish embryo (ZFE) model. Over 96 h of exposure, 6-PPD and 6-PPDQ accumulated in the ZFE with concentration factors ranging from 140 to 2500 for 6-PPD and 70 to 220 for 6-PPDQ. A total of 22 TPs of 6-PPD and 12 TPs of 6-PPDQ were tentatively identified using liquid chromatography coupled to high-resolution mass spectrometry. After 96 h of exposure to 6-PPD, the TPs of 6-PPD comprised 47% of the total peak area (TPA), with 4-hydroxydiphenylamine being the most prominent in the ZFE. Upon 6-PPDQ exposure, >95% of 6-PPDQ taken up in the ZFE was biotransformed, with 6-PPDQ + O + glucuronide dominating (>80% of the TPA). Among other TPs of 6-PPD, a reactive N-phenyl-p-benzoquinone imine was found. The knowledge of TPs of 6-PPD and 6-PPDQ from this study may support biotransformation studies in other organisms.

Occurrence and Fate of Substituted p-Phenylenediamine-Derived Quinones in Hong Kong Wastewater Treatment Plants.

para-Phenylenediamine quinones (PPD-Qs) are a newly discovered class of transformation products derived from para-phenylenediamine (PPD) antioxidants. These compounds are prevalent in runoff, roadside soil, and particulate matter. One compound among these, N-1,3-dimethylbutyl-n'-phenyl-p-phenylenediamine quinone (6PPD-Q), was found to induce acute mortality of coho salmon, rainbow trout, and brook trout, with the median lethal concentrations even lower than its appearance in the surface and receiving water system. However, there was limited knowledge about the occurrence and fate of these emerging environmental contaminants in wastewater treatment plants (WWTPs), which is crucial for effective pollutant removal via municipal wastewater networks. In the current study, we performed a comprehensive investigation of a suite of PPD-Qs along with their parent compounds across the influent, effluent, and biosolids during each processing unit in four typical WWTPs in Hong Kong. The total concentrations of PPDs and PPD-Qs in the influent were determined to be 2.7-90 and 14-830 ng/L. In the effluent, their concentrations decreased to 0.59-40 and 2.8-140 ng/L, respectively. The median removal efficiency for PPD-Qs varied between 53.0 and 91.0% across the WWTPs, indicating that a considerable proportion of these contaminants may not be fully eliminated through the current processing technology. Mass flow analyses revealed that relatively higher levels of PPD-Qs were retained in the sewage sludge (20.0%) rather than in the wastewater (16.9%). In comparison to PPDs, PPD-Qs with higher half-lives exhibited higher release levels via effluent wastewater, which raises particular concerns about their environmental consequences to aquatic ecosystems.

Disruption of dopamine metabolism by exposure to 6-PPD quinone in Caenorhabditis elegans.

Caenorhabditis elegans is a useful model for examining metabolic processes and related mechanisms. We here examined the effect of exposure to N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone (6-PPDQ) on dopamine metabolism and underling molecular basis in nematodes. The dopamine content was reduced by 6-PPDQ (1 and 10 µg/L). Meanwhile, dopamine related behaviors (basal slowing response and area restricted searching) were changed by 6-PPDQ (1 and 10 µg/L). Exposure to 6-PPDQ (1 and 10 µg/L) decreased expressions of genes (cat-2 and bas-1) encoding enzymes governing dopamine synthesis and cat-1 encoding dopamine transporter. Development of dopaminergic neurons was also affected by 10 µg/L 6-PPDQ as reflected by decrease in fluorescence intensity, neuronal loss, and defect in dendrite development. Exposure to 6-PPDQ (1 and 10 µg/L) altered expressions of ast-1 and rcat-1 encoding upregulators of cat-2 and bas-1. The dopamine content and expressions of cat-2 and bas-1 were inhibited by RNAi of ast-1 and increased by RNAi of rcat-1 in 6-PPDQ exposed nematodes. Using endpoints of locomotion behavior and brood size, in 6-PPDQ exposed nematodes, the susceptibility to toxicity was caused by RNAi of ast-1, cat-2, bas-1, and cat-1, and the resistance to toxicity was induced by RNAi of rcat-1. Therefore, 6-PPDQ exposure disrupted dopamine metabolism and the altered molecular basis for dopamine metabolism was associated with 6-PPDQ toxicity induction. Moreover, the defects in dopamine related behaviors and toxicity on locomotion and reproduction could be rescued by treatment with 0.1 mM dopamine.

Toxicity of 6PPD-quinone to four freshwater invertebrate species.

The antioxidant N-(1,3-Dimethylbutyl)-N'-phenyl-p- phenylenediamine (6PPD) is used to protect the rubber in tires from oxidation, which extends the life of the tire. When oxidized, 6PPD is transformed into 6PPD-quinone (6PPDQ). 6PPDQ, along with other tire ingredients, can enter aquatic ecosystems through the transport of tire wear particles in runoff during a precipitation event. The mass mortality of coho salmon following precipitation events in urban areas lead to the discovery that 6PPDQ is the likely cause due to coho salmon's relatively high sensitivity to 6PPDQ. The assessment of 6PPDQ toxicity to other aquatic species has expanded, but it has focused on fish. This study investigated the toxicity of 6PPDQ to four freshwater invertebrate species, larval burrowing mayfly (Hexagenia spp.), juvenile cladoceran (Daphnia magna), file ramshorn snail embryo (Planorbella pilsbryi), and adult washboard mussel (Megalonaias nervosa). For all four species, the highest concentration of 6PPDQ tested did not result in significant mortality. This translated into the determination of the highest concentration that did not cause significant mortality (NOEC) for Hexagenia spp., D. magna, P. pilsbryi, and M. nervosa of 232.0, 42.0, 11.7, and 17.9 µg/L, respectively. The data from this study indicate that freshwater invertebrates are not as sensitive to 6PPDQ as some salmonid species (e.g., coho salmon Oncorhynchus kisutch). This study also analyzed 6PPDQ in road runoff from around the city of Guelph in Ontario, Canada. 6PPQ was detected in all samples but the concentration was two orders of magnitude lower than the NOECs for the four tested species of freshwater invertebrate.

Tire-rubber related pollutant 6-PPD quinone: A review of its transformation, environmental distribution, bioavailability, and toxicity.

The antioxidant 6-PPD has been widely used to prevent cracking and thermal oxidative degradation and to extend the service life of tire rubber. 6-PPD quinone (6-PPDQ) is formed via the reaction of 6-PPD with O3. Due to its acute lethality in coho salmon, 6-PPDQ has become an emerging pollutant of increasing concern. In this review, we provide a critical overview of the generation, environmental distribution, bioavailability, and potential toxicity of 6-PPDQ. The transformation pathways from 6-PPD to 6-PPDQ include the N-1,3-dimethylbutyl-N-phenyl quinone diamine (QDI), intermediate phenol, and semiquinone radical pathways. 6-PPDQ has been frequently detected in water, dust, air particles, soil, and sediments, indicating its large-scale and potentially global pollution trend. 6-PPDQ is bioavailable to both aquatic animals and mammals and acute exposure to 6-PPDQ can be lethal to some organisms. Exposure to 6-PPDQ at environmentally relevant concentrations could induce several types of toxicity, including neurotoxicity, intestinal toxicity, and reproductive toxicity. This review also identifies and discusses knowledge gaps and research needs for the study of 6-PPDQ. This review facilitates a better understanding of the environmental occurrence and exposure risk of 6-PPDQ.

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.

Toxicity and mutagenicity studies of 6PPD-quinone in a marine invertebrate species and bacteria.

N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD-quinone), an oxidation product of the tire additive, 6PPD, has been associated with high mortality of salmonids (0.1 µg/L). The objective of this study was to determine the acute toxicity using neonates and mutagenicity (micronuclei in hemolymph of exposed adults) of 6PPD-quinone in the marine amphipod Parhyale hawaiensis. Also, we studied its mutagenicity in the Salmonella/microsome assay using five strains of Salmonella with and without metabolic system (rat liver S9, 5%). 6PPD-quinone did not present acute toxicity to P. hawaiensis from 31.25 to 500 µg/L. Micronuclei frequency increased after 96 h-exposure to 6PPD-quinone (250 and 500 µg/L) when compared to the negative control. 6PPD-quinone also showed a weak mutagenic effect for TA100 only in the presence of S9. We conclude that 6PPD-quinone is mutagenic to P. hawaiensis and weakly mutagenic to bacteria. Our work provides information for future risk assessment of the presence of 6PPD-quinone in the aquatic environment.

Environmental concentrations of tire rubber-derived 6PPD-quinone alter CNS function in zebrafish larvae.

N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone (6PPD-quinone) is a degradation product of 6PPD, an antioxidant widely used in rubber tires. 6PPD-quinone enters aquatic ecosystems through urban stormwater runoff and has been identified as the chemical behind the urban runoff mortality syndrome in coho salmon. However, the available data suggest that the acute effects of 6PPD-quinone are restricted to a few salmonid species and that the environmental levels of this chemical should be safe for most fish. In this study, larvae of a "tolerant" fish species, Danio rerio, were exposed to three environmental concentrations of 6PPD-quinone for only 24 h, and the effects on exploratory behavior, escape response, nonassociative learning (habituation), neurotransmitter profile, wake/sleep cycle, circadian rhythm, heart rate and oxygen consumption rate were analyzed. Exposure to the two lowest concentrations of 6PPD-quinone resulted in altered exploratory behavior and habituation, an effect consistent with some of the observed changes in the neurotransmitter profile, including increased levels of acetylcholine, norepinephrine, epinephrine and serotonin. Moreover, exposure to the highest concentration tested altered the wake/sleep cycle and the expression of per1a, per3 and cry3a, circadian clock genes involved in the negative feedback loop. Finally, a positive chronotropic effect of 6PPD-quinone was observed in the hearts of the exposed fish. The results of this study emphasize the need for further studies analyzing the effects of 6PPD-quinone in "tolerant" fish species.

Bioretention filtration prevents acute mortality and reduces chronic toxicity for early life stage coho salmon (Oncorhynchus kisutch) episodically exposed to urban stormwater runoff.

As the human population of western North America continues to expand, widespread patterns of urban growth pose increasingly existential threats to certain wild stocks of Pacific salmon and steelhead (Oncorhynchus sp.). Rainfall previously absorbed into the soils of forests and grasslands falls instead on pavement and other hardened surfaces. This creates stormwater runoff that carries toxic metals, oil, and many other contaminants into salmon-bearing habitats. These include freshwater streams where coho salmon (O. kisutch) spawn in gravel beds. Coho salmon embryos develop within a thick eggshell (chorion) for weeks to months before hatching as alevins and ultimately emerging from the gravel as fry. Untreated urban runoff is highly toxic to older coho salmon (freshwater-resident juveniles and adult spawners), but the vulnerability of the earliest life stages remains poorly understood. To address this uncertainty, we fertilized eggs and raised them under an episodic stormwater exposure regimen, using runoff collected from a high-traffic arterial roadway from 15 discrete storm events. We monitored survival and morphological development, as well as molecular markers for contaminant exposure and cardiovascular stress. We also evaluated the benefit of treating runoff with green infrastructure (bioretention filtration) on coho salmon health and survival. Untreated runoff caused subtle sublethal toxicity in pre-hatch embryos with no mortality, followed by high rates of mortality from exposure at hatch. Bioretention filtration removed most measured contaminants (bacteria, dissolved metals, and polycyclic aromatic hydrocarbons), and the treated effluent was considerably less toxic - notably preventing mortality at the alevin stage. Our findings indicate that untreated urban runoff poses an important threat to early life stage coho salmon, in terms of both acute and delayed-in-time mortality. Moreover, while inexpensive management strategies involving bioinfiltration are promising, future green infrastructure effectiveness research should emphasize sublethal metrics for contaminant exposure and adverse health outcomes in salmonids.