Identification of a family of species-selective complex I inhibitors as potential anthelmintics.

Soil-transmitted helminths (STHs) are major pathogens infecting over a billion people. There are few classes of anthelmintics and there is an urgent need for new drugs. Many STHs use an unusual form of anaerobic metabolism to survive the hypoxic conditions of the host gut. This requires rhodoquinone (RQ), a quinone electron carrier. RQ is not made or used by vertebrate hosts making it an excellent therapeutic target. Here we screen 480 structural families of natural products to find compounds that kill Caenorhabditis elegans specifically when they require RQ-dependent metabolism. We identify several classes of compounds including a family of species-selective inhibitors of mitochondrial respiratory complex I. These identified complex I inhibitors have a benzimidazole core and we determine key structural requirements for activity by screening 1,280 related compounds. Finally, we show several of these compounds kill adult STHs. We suggest these species-selective complex I inhibitors are potential anthelmintics.

Heat stress-induced NO enhanced perylenequinone biosynthesis of Shiraia sp. via calcium signaling pathway.

Perylenequinones (PQs) are natural photosensitizing compounds used as photodynamic therapy, and heat stress (HS) is the main limiting factor of mycelial growth and secondary metabolism of fungi. This study aimed to unravel the impact of HS-induced Ca2+ and the calcium signaling pathway on PQ biosynthesis of Shiraia sp. Slf14(w). Meanwhile, the intricate interplay between HS-induced NO and Ca2+ and the calcium signaling pathway was investigated. The outcomes disclosed that Ca2+ and the calcium signaling pathway activated by HS could effectively enhance the production of PQs in Shiraia sp. Slf14(w). Further investigations elucidated the specific mechanism through which NO signaling molecules induced by HS act upon the Ca2+/CaM (calmodulin) signaling pathway, thus propelling PQ biosynthesis in Shiraia sp. Slf14(w). This was substantiated by decoding the downstream positioning of the CaM/CaN (calcineurin) pathway in relation to NO through comprehensive analyses encompassing transcript levels, enzyme assays, and the introduction of chemical agents. Concurrently, the engagement of Ca2+ and the calcium signaling pathway in heat shock signaling was also evidenced. The implications of our study underscore the pivotal role of HS-induced Ca2+ and the calcium signaling pathway, which not only participate in heat shock signal transduction but also play an instrumental role in promoting PQ biosynthesis. Consequently, our study not only enriches our comprehension of the mechanisms driving HS signaling transduction in fungi but also offers novel insights into the PQ synthesis paradigm within Shiraia sp. Slf14(w). KEY POINTS: • The calcium signaling pathway was proposed to participate in PQ biosynthesis under HS. • HS-induced NO was revealed to act upon the calcium signaling pathway for the first time.

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.

In Vitro and in Vivo Study of a Photostable Quinone Compound with Enhanced Therapeutic Efficacy against Chagas Disease.

Chagas disease, a neglected tropical disease caused by the protozoan Trypanosoma cruzi poses a significant health challenge in rural areas of Latin America. The current pharmacological options exhibit notable side effects, demand prolonged administration, and display limited efficacy. Consequently, there is an urgent need to develop drugs that are safe and clinically effective. Previously, we identified a quinone compound (designated as compound 2) with potent antiprotozoal activity, based on the chemical structure of komaroviquinone, a natural product renowned for its antitrypanosomal effects. However, compound 2 was demonstrated considerably unstable to light. In this study, we elucidated the structure of the light-induced degradation products of compound 2 and probed the correlation between the quinone ring's substituents and its susceptibility to light. Our findings led to the discovery of quinones with significantly enhanced light stability, some of which exhibiting antitrypanosomal activity. The most promising compound was evaluated for drug efficacy in a mouse model of Chagas disease, revealing where a notable reduction in blood parasitemia.

Dihydrotanshinone I exhibits antitumor effects via ß-catenin downregulation in papillary thyroid cancer cell lines.

Thyroid cancer is the most common endocrine carcinoma and, among its different subtypes, the papillary subtype (PTC) is the most frequent. Generally, PTCs are well differentiated, but a minor percentage of PTCs are characterized by a worse prognosis and more aggressive behavior. Phytochemicals, naturally found in plant products, represent a heterogeneous group of bioactive compounds that can interfere with cell proliferation and the regulation of the cell cycle, taking part in multiple signaling pathways that are often disrupted in tumor initiation, proliferation, and progression. In this work, we focused on 15,16-dihydrotanshinone I (DHT), a tanshinone isolated from Salvia miltiorrhiza Bunge (Danshen). We first evaluated DHT biological effect on PTC cells regarding cell viability, colony formation ability, and migration capacity. All of these parameters were downregulated by DHT treatment. We then investigated gene expression changes after DHT treatment by performing RNA-seq. The analysis revealed that DHT significantly reduced the Wnt signaling pathway, which plays a role in various diseases, including cancer. Finally, we demonstrate that DHT treatment decreases protein levels of ß-catenin, a final effector of canonical Wnt signaling pathway. Overall, our data suggest a possible use of this nutraceutical as an adjuvant in the treatment of aggressive papillary thyroid carcinoma.

Aromatic amine antioxidants (AAs) and p-phenylenediamines-quinones (PPD-Qs) in e-waste recycling industry park: Occupational exposure and liver X receptors (LXRs) disruption potential.

Recently, evidence of aromatic amine antioxidants (AAs) existence in the dust of the electronic waste (e-waste) dismantling area has been exposed. However, there are limited studies investigating occupational exposure and toxicity associated with AAs and their transformation products (p-phenylenediamines-quinones, i.e., PPD-Qs). In this study, 115 dust and 42 hand wipe samples collected from an e-waste recycling industrial park in central China were analyzed for 19 AAs and 6 PPD-Qs. Notably, the median concentration of ∑6PPD-Qs (1,110 ng/g and 1,970 ng/m2) was significantly higher (p < 0.05, Mann-Whitney U test) than that of ∑6PPDs (147 ng/g and 34.0 ng/m2) in dust and hand wipes. Among the detected analytes, 4-phenylaminodiphenylamine quinone (DPPD-Q) (median: 781 ng/g) and 1,4-Bis(2-naphthylamino) benzene quinone (DNPD-Q) (median: 156 ng/g), were particularly prominent, which were first detected in the e-waste dismantling area. Occupational exposure assessments and nuclear receptor interference ability, conducted through estimated daily intake (EDI) and molecular docking analysis, respectively, indicated significant occupational exposure to PPD-Qs and suggested prioritized Liver X receptors (LXRs) disruption potential of PPDs and PPD-Qs. The study provides the first evidence of considerable levels of AAs and PPD-Qs in the e-waste-related hand wipe samples and underscores the importance of assessing occupational exposure and associated toxicity effects.

A facile cellulose finishing strategy through in-situ growth of sliver-doped manganese dioxide assisted by amine-quinone for improving indoor living quality.

Nowadays, various harmful indoor pollutants especially including bacteria and residual formaldehyde (HCHO) seriously threaten human health and reduce the quality of public life. Herein, a universal substrate-independence finishing approach for efficiently solving these hybrid indoor threats is demonstrated, in which amine-quinone network (AQN) was employed as reduction agent to guide in-situ growth of Ag@MnO2 particles, and also acted as an adhesion interlayer to firmly anchor nanoparticles onto diverse textiles, especially for cotton fabrics. In contrast with traditional hydrothermal or calcine methods, the highly reactive AQN ensures the efficient generation of functional nanoparticles under mild conditions without any additional catalysts. During the AQN-guided reduction, the doping of Ag atoms onto cellulose fiber surface optimized the crystallinity and oxygen vacancy of MnO2, providing cotton efficient antibacterial efficiency over 90 % after 30 min of contact, companying with encouraging UV-shielding and indoor HCHO purification properties. Besides, even after 30 cycles of standard washing, the Ag@MnO2-decorated textiles can effectively degrade HCHO while well-maintaining their inherent properties. In summary, the presented AQN-mediated strategy of efficiently guiding the deposition of functional particles on fibers has broad application prospects in the green and sustainable functionalization of textiles.

Hydroxysafflor yellow A induced ferroptosis of Osteosarcoma cancer cells by HIF-1α/HK2 and SLC7A11 pathway.

Osteosarcoma is a very serious primary bone cancer with a high death rate and a dismal prognosis. Since there is no permanent therapy for this condition, it is necessary to develop a cure. Therefore, this investigation was carried out to assess the impacts and biological functions of hydroxysafflor yellow A (HYSA) in osteosarcoma cell lines (MG63). In this investigational study, MG63 cells were utilized. Microarray experiments, quantitative polymerase chain reaction (qPCR), immunofluorescent staining, extracellular acidification rate (ECAR), oxygen consumption rate (OCR), glucose consumption, lactate production, and ATP levels, proliferation assay, 5-Ethynyl-2'-deoxyuridine (EDU) staining, and Western blot were performed. In MG63 cells, HYSA lowered cell proliferation and metastasis rates, suppressed EDU cell number, and enhanced caspase-3/9 activity levels. HYSA reduced the Warburg effect and induced ferroptosis (FPT) in MG63 cells. Inhibiting ferroptosis diminished HYSA's anti-cancer activities in MG63 cells. The stimulation of the HIF-1α/SLC7A11 pathway decreased HYSA's anti-cancer activities in MG63 cells. HIF-1α is one target spot for HYSA in a model of osteosarcoma cancer (OC). HYSA altered HIF-1α's thermophoretic activity; following binding with HYSA, HIF-1α's melting point increased from ~55°C to ~60°C. HYSA significantly enhanced the thermal stability of exogenous WT HIF-1α while not affecting Mut HIF-1α, suggesting that ARG-311, GLY-312, GLN-347, and GLN-387 may be involved in the interaction between HIF-1α and HYSA. Conclusively, our study revealed that HYSA induced FPT and reduced the Warburg effect of OC through mitochondrial damage by HIF-1α/HK2/SLC7A11 pathway. HYSA is a possible therapeutic option for OC or other cancers.

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.

Quinones-enhanced humification in food waste composting: A novel strategy for hazard mitigation and nitrogen retention.

The harmless and high-value conversion of organic waste are the core problems to be solved by composting technology. This study introduced an innovative method of promoting targeted humification and nitrogen retention in composting by adding p-benzoquinone (PBQ), the composting without any additives was set as control group (CK). The results indicated that the addition of exogenous quinones led to a 30.1% increase in humic acid (HA) content during the heating and thermophilic phases of composting. Spectroscopic analyses confirmed that exogenous quinones form the core skeleton structure of amino-quinones in HA through composting biochemical reactions. This accelerated the transformation of quinones into recalcitrant HA in the early stages of composting, and reduced CO2 and NH3 by 8% and 78%, respectively. Redundancy analysis (RDA) revealed that the decrease in carbon and nitrogen losses primarily correlated with quinones enhancing HA formation and greater nitrogen incorporation into HA (P < 0.05). Furthermore, the compost treated with quinones demonstrated a decrease in phytotoxicity and earthworm mortality, alongside a significant increase in the relative abundance of actinobacteria, which are associated with the humification process. This research establishes and proposes that co-composting with quinones-containing waste is an effective approach for the sustainable recycling of hazardous solid waste.

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.

A nation-wide study for the occurrence of PPD antioxidants and 6PPD-quinone in road dusts of China.

N,N'-substituted p-phenylenediamines (PPDs) are widely used antioxidants in rubber tires, which could be released and accumulated in road dusts with rubber tires wear. As ozonation product of N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD), 6PPD-quinone (6PPD-Q) exhibited higher toxicity to coho salmon. However, studies on their environmental behaviors are still limited. Road dust is the major medium PPDs exist, which significantly affects the levels of PPDs in other mediums, especially surface water and particulate matter. In this study, road dust samples were collected in 55 major cities of China to explore the distribution characteristics of PPDs and 6PPD-Q. The concentrations of total PPDs (ΣPPDs) and 6PPD-Q in urban trunk road dust samples were in the ranges of 7.90-727 and 3.00-349 ng/g, with median concentrations of 68 and 49 ng/g, respectively. 6PPD and 6PPD-Q are the dominant components in most road dusts. The functional region-dependent pollution characteristics of PPDs and 6PPD-Q give the first finding that urban tunnel road was the highly polluted region, followed by urban trunk roads. Suburban road dusts had a lower pollution level. Moreover, the estimated daily intake (EDI) of PPDs and 6PPD-Q for children was much higher than adults.

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.

Engineering substrate specificity of quinone-dependent dehydrogenases for efficient oxidation of deoxynivalenol to 3-keto-deoxynivalenol.

The oxidative reaction of Fusarium mycotoxin deoxynivalenol (DON) using the dehydrogenase is a desirable strategy and environmentally friendly to mitigate its toxicity. However, a critical issue for these dehydrogenases shows widespread substrate promiscuity. In this study, we conducted pocket reshaping of Devosia strain A6-243 pyrroloquinoline quinone (PQQ)-dependent dehydrogenase (DADH) on the basis of protein structure and kinetic analysis of substrate libraries to improve preference for particular substrate DON (10a). The variant presented an increased preference for substrate 10a and enhanced catalytic efficiency. A 4.7-fold increase in preference for substrate 10a was observed. Kinetic profiling and molecular dynamics (MD) simulations provided insights into the enhanced substrate specificity and activity. Moreover, the variant exhibited stronger conversion of substrate 10a to 3-keto-DON compared to the wild DADH. Overall, this study provides a feasible protocol for the redesign of PQQ-dependent dehydrogenases with favourable substrate specificity and catalytic activity, which is desperately needed for DON antidote development.

Mixture toxicity of 6PPD-quinone and polystyrene nanoplastics in zebrafish.

Plastic pollution, including micro- and nanoplastics, is a growing concern. Tyre-wear particles (TWPs) are the second largest source of microplastics in the ocean following abrasion of synthetic fibres. In addition to the particles themselves, TWPs contain many harmful chemicals, including 6PPD. This chemical reacts with atmospheric ozone and forms the toxic compound 6PPD-quinone (6PPDq), which poses a danger to aquatic life. There is a knowledge gap in understanding risks associated with the combined toxicity of nanoplastics (NPs) and 6PPDq. The present study aimed to investigate the toxicity of NPs and 6PPDq on adult zebrafish using phenotypic (behaviour, histology) and transcriptomic endpoints. Zebrafish were exposed to four treatments: control (contaminant-free), 50 µg/L 6PPDq, 3 mg/L polystyrene (PS)-NPs, and a combination of 50 µg/L 6PPDq and 3 mg/L PS-NPs. We did not observe locomotory dysregulation in zebrafish exposed to NPs. However, we found significant hyperlocomotion in zebrafish exposed to 6PPDq and this effect was even more substantial after co-exposure with PS-NPs. This study explores the molecular mechanisms behind these effects, identifying genes associated with neurotransmitters and fatty acid metabolism that were dysregulated by the co-exposure. Transcriptomic analysis further showed that both 6PPDq and PS-NPs impacted cellular processes associated with sterol biosynthesis, cholesterol metabolism, and muscle tissue development. The effects on these mechanisms were stronger in co-exposed zebrafish, indicating a heightened risk to cellular integrity and mitochondrial dysfunction. These results highlight the significance of mixture toxicity when studying the effects of NPs and associated chemicals like 6PPDq.

Electrochemical Analysis for Total Alkalinity of Water by the Measurement of Cathodic Prepeak of Quinone Caused by Surplus Acid.

In this study, an electrochemical analysis, coupled with the concept of back neutralization titration and the voltammetric determination of surplus acid, is proposed for determining the total alkalinity of water samples. When linear sweep voltammetry of 3,5-di-tert-butyl-1,2-benzoquinone (DBBQ) with H2SO4 in a water and ethanol (44 : 56, v/v) mixture was carried out using a bare glassy carbon working electrode, a cathodic prepeak of DBBQ caused by H2SO4 was observed on the voltammogram at a more positive potential than when compared with the original cathodic peak of DBBQ. When similar voltammetry was carried out in the presence of Na2CO3 and H2SO4, the cathodic prepeak height of DBBQ was decreased with an increase in the Na2CO3 concentration. The decrease of the cathodic prepeak height of DBBQ was found to be linearly related to the Na2CO3 concentration ranging from 0.025 to 2.5 mM (r2 = 0.998). The total equivalent concentrations of inorganic bases in samples of mineral water and tap water were determined, and then the results were converted to the total alkalinities of the water samples (mg/L CaCO3). The total alkalinities of the water samples determined by the present electrochemical analysis were essentially the same compared with those by the neutralization titration method. From these results, we were able to demonstrate that the present electrochemical analysis with accuracy and precision could be applied to determine the total alkalinity, which is one of the indicators to examine water quality. The present electrochemical analysis would contribute to achieving the sustainable development goals (SDGs) of #6 and #14.

Visualized sensing of erythritol using a simple enzyme-free catechol-based hydrogel film.

Based on the versatile properties of bio-derived materials, non-enzymatic assays in combination with electronic devices have attracted increasing interest. Here, we report a novel enzyme-free visualization approach for the detection of erythritol, which is a zero-calorie natural sweetener and serves as an ideal sucrose substitute for diabetics or overweight people who need sugar control. The recognition element of the electrochemical biosensor was constructed by catechol modification on a chitosan-based hydrogel film. The signal transduction was achieved by the competitive binding assay of sweeteners. The results show that 2-fluorophenylboronic acid (FPBA) can form a cyclic boronate ester with the ortho-hydroxyls of both reduced catechol and oxidized quinone, impeding the electron transfer and leading to redox signal attenuation. The addition of sweeteners caused a competitive reaction resulting in bonding between the 1,2-diols and FPBA moieties, and in the recovery of the redox signals. Importantly, the pattern of redox signal changes of catechol can be detected optically, as the oxidized quinone state is darker in color than the reduced catechol state. Using a simple cell phone imaging application, we demonstrate that erythritol can be distinguished from other sweeteners in real samples using the oxidized catechol-Chit0/agarose hydrogel film. Thus, we envision that this method could allow diabetics and people who need to control their sugar intake to detect whether the product contains only erythritol in the field or at home. In addition, this work further illustrates the potential of bio-derived materials for performing redox-based functions and enzyme-free visualization assays.

Mitochondrial quinone redox states as a marker of mitochondrial metabolism.

Mitochondrial and thus cellular energetics are highly regulated both thermodynamically and kinetically. Cellular energetics is of prime importance in the regulation of cellular functions since it provides ATP for their accomplishment. However, cellular energetics is not only about ATP production but also about the ability to re-oxidize reduced coenzymes at a proper rate, such that the cellular redox potential remains at a level compatible with enzymatic reactions. However, this parameter is not only difficult to assess due to its dual compartmentation (mitochondrial and cytosolic) but also because it is well known that most NADH in the cells is bound to the enzymes. In this paper, we investigated the potential relevance of mitochondrial quinones redox state as a marker of mitochondrial metabolism and more particularly mitochondrial redox state. We were able to show that Q2 is an appropriate redox mediator to assess the mitochondrial quinone redox states. On isolated mitochondria, the mitochondrial quinone redox states depend on the mitochondrial substrate and the mitochondrial energetic state (phosphorylating or not phosphorylating). Last but not least, we show that the quinones redox state response allows to better understand the Krebs cycle functioning and respiratory substrates oxidation. Taken together, our results suggest that the quinones redox state is an excellent marker of mitochondrial metabolism.

Ongoing evolution of the Mycobacterium tuberculosis lactate dehydrogenase reveals the pleiotropic effects of bacterial adaption to host pressure.

The bacterial determinants that facilitate Mycobacterium tuberculosis (Mtb) adaptation to the human host environment are poorly characterized. We have sought to decipher the pressures facing the bacterium in vivo by assessing Mtb genes that are under positive selection in clinical isolates. One of the strongest targets of selection in the Mtb genome is lldD2, which encodes a quinone-dependent L-lactate dehydrogenase (LldD2) that catalyzes the oxidation of lactate to pyruvate. Lactate accumulation is a salient feature of the intracellular environment during infection and lldD2 is essential for Mtb growth in macrophages. We determined the extent of lldD2 variation across a set of global clinical isolates and defined how prevalent mutations modulate Mtb fitness. We show the stepwise nature of lldD2 evolution that occurs as a result of ongoing lldD2 selection in the background of ancestral lineage-defining mutations and demonstrate that the genetic evolution of lldD2 additively augments Mtb growth in lactate. Using quinone-dependent antibiotic susceptibility as a functional reporter, we also find that the evolved lldD2 mutations functionally increase the quinone-dependent activity of LldD2. Using 13C-lactate metabolic flux tracing, we find that lldD2 is necessary for robust incorporation of lactate into central carbon metabolism. In the absence of lldD2, label preferentially accumulates in dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P) and is associated with a discernible growth defect, providing experimental evidence for accrued lactate toxicity via the deleterious buildup of sugar phosphates. The evolved lldD2 variants increase lactate incorporation to pyruvate while altering triose phosphate flux, suggesting both an anaplerotic and detoxification benefit to lldD2 evolution. We further show that the mycobacterial cell is transcriptionally sensitive to the changes associated with altered lldD2 activity which affect the expression of genes involved in cell wall lipid metabolism and the ESX- 1 virulence system. Together, these data illustrate a multifunctional role of LldD2 that provides context for the selective advantage of lldD2 mutations in adapting to host stress.

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.