Anthrahydroquinone-2-6-disulfonate is a novel, powerful antidote for paraquat poisoning

Qian, **; Wu, Chun-Yuan; Wu, Dong-Ming; Li, Li-Hua; Li, Qi; Deng, Tang; Huang, Qi-Feng; Xu, Shuang-Qin; Wang, Hang-Fei; Wu, **n-**n; Cheng, Zi-Yi; Lv, Chuan-Zhu; Liu, **ao-Ran

doi:10.1038/s41598-021-99591-4

Anthrahydroquinone-2-6-disulfonate is a novel, powerful antidote for paraquat poisoning

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Published: 11 October 2021

Volume 11, article number 20159, (2021)
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Anthrahydroquinone-2-6-disulfonate is a novel, powerful antidote for paraquat poisoning

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** Qian¹^na1,
Chun-Yuan Wu²^na1,
Dong-Ming Wu²,
Li-Hua Li¹,
Qi Li¹,
Tang Deng¹,
Qi-Feng Huang¹,
Shuang-Qin Xu¹,
Hang-Fei Wang¹,
**n-**n Wu¹,
Zi-Yi Cheng¹,
Chuan-Zhu Lv³ &
…
**ao-Ran Liu¹

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Abstract

Paraquat (PQ) is a widely used fast-acting pyridine herbicide. Accidental ingestion or self-administration via various routes can cause severe organ damage. Currently, no effective antidote is available commercially, and the mortality rate of poisoned patients is exceptionally high. Here, the efficacy of anthrahydroquinone-2-6-disulfonate (AH₂QDS) was observed in treating PQ poisoning by constructing in vivo and ex vivo models. We then explored the detoxification mechanism of AH₂QDS. We demonstrated that, in a rat model, the PQ concentration in the PQ + AH₂QDS group significantly decreased compared to the PQ only group. Additionally, AH₂QDS protected the mitochondria of rats and A549 cells and decreased oxidative stress damage, thus improving animal survival and cell viability. Finally, the differentially expressed genes were analysed in the PQ + AH₂QDS group and the PQ group by NextGen sequencing, and we verified that Nrf2’s expression in the PQ + AH₂QDS group was significantly higher than that in the PQ group. Our work identified that AH₂QDS can detoxify PQ by reducing PQ uptake and protecting mitochondria while enhancing the body's antioxidant activity.

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Introduction

Paraquat (1,1'-dimethyl-4,4'-bipyridinium, PQ) is a fast-acting herbicide widely used for chemical weed control worldwide¹. PQ is exceptionally toxic to the human body and can cause acute poisoning by accidental or spontaneous ingestion. The vast majority of these poisonings are oral ingestions, and the adult lethal dose is 5–15 mL (20–40 mg/kg) of a 20% (w/v) aqueous solution. When PQ enters the body by various means (such as oral, local contact and injection), it is rapidly absorbed and enriched, causing an acute poisoning reaction that damages the digestive tract, kidneys, liver, lungs and other organs, resulting in multi-organ failure, with a mortality rate of 50–80%^{2,3,4,Full size image}

Discussion

In this study, we used AH₂QDS as an intervention in a rat model of PQ poisoning. Compared with those of the PQ group, the poisoning symptoms of the PQ + AH₂QDS group were significantly improved, with a lower blood drug concentration, less organ function damage, and a higher survival rate. In the PQ + AH₂QDS group, mitochondrial damage in lung tissue was alleviated, and a similar phenomenon was found in the cell test. The structure of the mitochondria was intact, the damage was significantly alleviated, and the expression of Nrf2 was significantly increased. These studies have proven for the first time that AH₂QDS is an effective treatment for PQ poisoning, and Nrf2 plays a crucial role in its detoxification process.

Previous studies have shown that activated carbon or the “white and black” scheme can effectively treat PQ poisoning^6,7,8. In contrast, our experiments only confirmed that the conventional “white and black” scheme can quickly and effectively degrade the PQ blood concentration but does not affect the survival rate of rats. Specifically, a 4X LD50 dose of PQ was used to construct the poisoning model, and the drug intervention time was as long as 2 h, during which most of the PQ may have been absorbed into the blood, while the “white and black” scheme could only absorb the residual poison in the stomach and accelerate its excretion but had no effect on the PQ already in the blood. Additionally, the results also showed that AH₂QDS is not only faster than the “white and black” scheme in removing toxins but also plays a specific role in the blood-related effects of PQ.

The toxic effect of PQ on mitochondria was proposed as early as 1968³⁶. Since then, a large number of studies on the damage of PQ to mitochondria have been published^26,27,28. Some scholars indicated that PQ could cause accumulation of the hMn-SOD precursor of human manganese-dependent peroxidase and diminish Mn-SOD activity. The conversion of GSH to GSSG leads to a decrease in GSH levels and weakens its antioxidant activity³⁷. Other studies have shown that PQ can cause the production of H₂O₂ and lessen the activity of catalase³⁸. H₂O₂ can induce changes in mitochondrial permeability and affect the mitochondrial membrane potential, resulting in the movement of cytochrome C from the mitochondria into the cytoplasm, and then induce apoptosis by activating caspase9³⁹. In our study, the microstructure of the lung tissue and A549 cells in the PQ group was observed under a projection electron microscope. It was found that the structure of the mitochondria was destroyed, vacuoles appeared in the cells, the cell walls were broken, and a large number of organelles were extruded. In contrast, the morphology of the mitochondria in the PQ + AH₂QDS group was as expected, and the lamellar structure was normal. The cell membrane potential of the PQ + AH₂QDS high-dose group was the highest, and the membrane potential was positively correlated with the concentration of AH₂QDS, while the mitochondrial membrane potential of cells treated with only PQ was the lowest. In summary, PQ can destroy the tissue structure of the mitochondria, affect the membrane potential, and eventually lead to cell rupture and death, while AH₂QDS can prevent this process and protect the function and structure of the mitochondria.

The data show that after PQ is absorbed into the blood, it causes the formation of excess reactive oxygen species (ROS), which leads to imbalance of the redox system, the consumption of NADPH, damage to mitochondria, the destruction of lipids, proteins and DNA, and a decrease in the activity of various antioxidant enzymes²⁴. After continuous oxidative stimulation, the body eventually sustains tissue damage. A large amount of ROS produced by PQ may be the leading cause of acute lung injury caused by PQ poisoning. In this study, it was found that after PQ exposure, the levels of ROS and MDA in the PQ group and conventional treatment group increased, while the level of GSH-Px decreased. In the PQ + AH₂QDS group, the ROS and MDA levels decreased, and the level of GSH-Px increased. The results show that PQ can produce a large amount of ROS to cause lipid peroxidation and oxidative stress injury. AH₂QDS can inhibit PQ's effect, improve antioxidant ability, and decrease the level of lipid peroxidation.

After further investigation of the detoxification mechanism of AH₂QDS, we found that the main differentially regulated pathway was the oxidative stress pathway, in which we found that the nuclear factor Nrf2 was significantly upregulated. Many studies have shown that Nrf2 can be used as a "guard" to protect the body against a variety of toxic effects^40,41,42. Nrf2 can be activated in a variety of processes involving oxidative stress. Nrf2 was expressed in epithelial cells, macrophages and vascular endothelial cells of normal rat lung tissue^43,44,45. MDA in the serum of rats poisoned by PQ increased significantly with the prolongation of poisoning time, while the activity of SOD decreased significantly. Nrf2 protein increased significantly in lung tissue injury induced by PQ. It has been found that the Nrf2-ARE pathway protects the lungs against dibutyl hydroxytoluene-induced acute respiratory distress syndrome (ARDS) and hyperoxia-induced lung injury by activating antioxidant enzymes^46,47. In our experiment, PQ, as a potent stressor, could activate the Nrf2 signalling pathway. Nrf2 was expressed at low levels in normal rat lung tissue and A549 cells, but the expression of Nrf2 was significantly increased after AH₂QDS treatment. These results show that Nrf2 plays a vital role in the treatment of PQ poisoning by AH₂QDS.

The direct mechanism of AH₂QDS in the treatment of PQ poisoning is that AH₂QDS enters the gastrointestinal tract and comes into contact with the PQ solution. Through a rapid redox reaction, PQ is reduced to a nontoxic green needle-like solid. Thus, detoxification is realized. Energy spectrum analysis showed that the acicular substance was stable and could not be dissolved in strong acids, strong bases, or organic solvents and was extremely stable at room temperature and pressure. At the same time, it was also found in the faeces of SD rats. We were concerned that after administration of AH₂QDS, a green needle-like solid will be formed in the blood, leading to the formation insoluble thrombi and resulting in thrombotic disease and a series of clinical symptoms. Therefore, we tested the blood and tissues of experimental animal SD rats but did not find this substance. So, after administering AH₂QDS, green needle-like solids would not be formed in the blood, tissues and organs to cause thrombotic disease.

To prove whether there is an indirect mechanism of AH₂QDS in PQ poisoning treatment, we constructed animal and cell models. ELISA, WB, and qPCR were performed to detect the levels of GSH-Px, MDA, ROS, and Nrf2, and transmission electron microscopy was performed to observe the microstructure of the mitochondria. The same trend was observed in vivo and in vitro. After the intervention with AH₂QDS, the expression of nuclear factor Nrf2 was enhanced, mitochondrial damage was relieved, and antioxidant reaction to oxidative stress was improved. Unfortunately, our experiment cannot determine whether the mechanism of AH₂QDS in the treatment of PQ poisoning is the direct mechanism or the indirect mechanism. Further research is needed.

In this paper, AH₂QDS was used as an antidote in the treatment of PQ poisoning for the first time and achieved excellent results, but this was verified only in SD rats, and it has not been tested in more advanced mammals; thus, a long and strict clinical study is needed to investigate the use of AH₂QDS in humans. Additionally, a 4X LD50 dose of PQ was given to SD rats in the poisoning model, and AH₂QDS was given for detoxification 2 h later. The 30-day survival rate of SD rats in the treatment group reached 100%, but if the time window of treatment with AH₂QDS were prolonged (2.5 h, 3 h, 4 h, or 6 h), the 30-day survival rate of SD rats in the treatment group decreases with the prolongation of intervention time. This may be because 2 h after ingestion of PQ, the rats have rapidly absorbed it into the blood and transported it to various organs through the blood flow. Even if AH₂QDS can detoxify the absorbed PQ, too high a concentration of PQ causes irreversible toxic damage to the organs in this time. In the follow-up studies, the sequencing results will be further analysed, and mechanistic research will be performed to elucidate the molecular functions of the gene, the cell location, and the biological process involved. At the same time, experiments were carried out on the Nrf2-ARE pathway through gene silencing/overexpression of related proteins to demonstrate the profound relationship between the Nrf2-ARE pathway and AH₂QDS in the treatment of PQ poisoning.

Conclusion

In summary, paraquat poisoning is still an extremely high clinical mortality disease, and conventional treatments are clinically ineffective. The new antidote we developed, AH₂QDS, can lower the concentration of PQ by binding it and protect the mitochondria and reduce the oxidative stress damage caused by PQ. The relationship between mitochondrial damage, the expression changes upstream and downstream of the Nrf2-ARE pathway, and AH₂QDS in PQ poisoning treatment must be further explored.

Methods

Animals and cell lines

All animal experiments were performed as per the protocols approved by the Animal Care and Use Committee of Hainan Medical University. All methods were performed in accordance with the guidelines and regulations of the Animal Care and Use Committee of Hainan Medical University and as per the ARRIVE guidelines 2.0. Human type II alveolar lung epithelial cells (A549) were purchased from the Shanghai Institute for Biological Sciences. The cells were maintained in a 5% CO2 incubator at 37 °C in medium (F12K) supplemented with 10% FBS and penicillin/streptomycin (100 U/ml). Sprague–Dawley (SD) rats (8 weeks, male, SPF grade) were purchased from Changsha Tianqn Biotechnology Co., Ltd., and were maintained in specific pathogen-free (SPF) facilities.

Main reagent

Paraquat solution, purchased from Nan**g Red Sun Co., Ltd., was given at a 400 mg/kg concentration. Twenty percent PQ solution was diluted into 1 mL of PQ solution with PBS. For the “white and black” scheme, 500 mg/kg activated carbon, 500 mg/kg montmorillonite powder and 5 mL mannitol were used for gastric cancer. Activated carbon was purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd. Montmorillonite powder was purchased from **ansheng Pharmaceutical Co., Ltd. Mannitol (20%) was purchased from Jiangsu Zhengda Tianqing Pharmaceutical Co., Ltd. Anthrahydroquinone-2-6-disulfonate (AH₂QDS) was synthesized by the Chinese Academy of Tropical Agricultural Sciences. The method is patented (Patent No: 2016103413306). Chemical name: anthraquinone-2-dioxo-6-disodium disulfonate, chemical formula: C14H8O8S2.2Na, molecular weight: 368.33. We prepared the AH₂QDS solution at a concentration of 40 mmol/L.

Modeling studies of PQ and AH₂QDS binding

Chemical structures of PQ and AH₂QDS were drawn with ChemDraw Pro 16.0 software. The binding conformations between PQ and AH₂QDS were simulated with AutoDock Vina²⁰.

Cell counting kit-8 (CCK8)

A549 cells were incubated with different concentrations of PQ, AH₂QDS and glutathione for 12 h. After 12, 24, 48 and 72 h, 10 μL of CCK8 solution (Do**do, Japan) was added, and the cells were incubated in the incubator for 2 h. An enzyme labelling instrument was used to measure the absorbance at 450 nm, and a formula was used to calculate the cell viability.

Mitochondrial membrane potential

The cell culture medium was removed, the cells were washed with PBS, 1 ml of medium was added, and 1 mL of JC-1 staining working solution was added and mixed well. After incubating the cells for 20 min in the incubator at 37 °C, the supernatant was removed, the cells were washed with diluted staining buffer (1x), 2 mL of medium was added, and images were captured under the fluorescence microscope.

Animal experiments

SD rats (~ 300 g) were subjected to gavage 400 mg/kg PQ, and 500 mg/kg "white and black" scheme and 400 mg/kg AH₂QDS intervention treatment were administered 2 h later. The specific methods used to establish the model is shown in Figure S3. We selected rats without collecting blood after establishing the model and observed and recorded the survival of each group over 30 days. The occurrence of death was recorded as 1, and no death was recorded as 0. Finally, a survival curve was drawn. The animal protocol passed the ethical review of the ethics committee of The First Affiliated Hospital of Hainan Medical University. (Issue number: 2020 (Research) No. (97); Review category: A Quick Review; Decision: Approval; Decision Date: July 8, 2020).

Sample collection

Blood was collected from the rats at different time points in anticoagulant tubes treated with heparin, and the plasma was separated and stored at − 80 °C for the detection of drug concentrations in the blood. The urine was left in the centrifuge tube for the detection of drug concentrations in urine. Rats were anaesthetized by intraperitoneal injection of 10% chloral hydrate (300 mg/kg), and the blood from the abdominal aorta was collected for the detection of liver, kidney and lung function. Finally, the rats were killed by exsanguination, and the lung tissue was collected, washed with PBS and stored at − 80 °C for follow-up analysis. The bodies of the animals were then incinerated.

Histopathology

SD rats were sacrificed at different times, and the lungs of the rats were harvested, fixed in 4% formalin, embedded in paraffin, sectioned, and stained with haematoxylin and eosin (H&E). The lung injury score was determined according to methods that were previously reported in the literature⁴⁸. A score of 0 means there is no alveolitis. 1 point means mild alveolitis, the lesions are limited to local and pleural lesions, accounting for less than 20% of the lung, and the alveolar structure is sound. A score of 2 indicates moderate alveolitis, and the lesion area accounts for 20–50% of the lung. Finally, a score of 3 means severe alveolitis, with diffuse alveolitis involving more than 50% of the lung.

Blood analysis

The collected venous blood samples were placed into a test tube with a coagulant and centrifuged at 3000 r/min for 5 min. Rat serum was obtained and placed into an automatic biochemical function analyser for analysis. After collecting blood from the abdominal aorta with an arterial blood gas sampler and rubbing with both hands for 1 min, 0.1 mL was injected into the blood gas analyser for analysis.

Transmission electron microscopy

Lung tissue and A549 cells were collected and placed overnight in 2.5% glutaraldehyde fixed solution that was prechilled at 4 °C, cleaned with PBS, fixed with 1 ml of 1% osmic acid for 1.5 h, dehydrated with alcohol and acetone, and impregnated with resin, and ultrathin sections were stained with uranium acetate and lead citrate. The ultrastructure was observed under a transmission electron microscope.

Ultra-high-performance liquid chromatography-tandem mass spectrometry

The concentrations of PQ and AH₂QDS were determined by ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC/Xevo TQ-S, Waters). The mobile phase was acetonitrile/100 mM ammonium formate (pH = 3.7) = 50 × 50, and the flow rate was 0.3 mL/min. An ACQUITY UPLC BEH HILIC column (100 mm × 2.1 mm, 1.7 μm) was used. PQ was quantified in the MRM mode of positive ion multireaction monitoring with an electrospray ion source. Negative ion SIR mode was used to quantify AH₂QDS. The parameters were as follows: capillary voltage: 3.2 kV, ion source temperature: 150 °C, cone hole back blowing gas flow rate: 30 L/hr, dissolvent temperature: 350 °C, and dissolvent gas flow rate: 800 L/hr.

Cytokine detection

The GSH-Px, MDA and ROS kits purchased from Nan**g Jiancheng Company were used according to the instructions to detect the levels of GSH-Px, MDA and ROS, respectively.

Western blotting

Proteins were extracted from tissues and cells with a BCA kit (Biyuntian Biotechnology Co., Ltd.), separated in SDS-PAGE gels, and transferred to cellulose membranes. After sealing, the membranes were incubated with the primary antibody overnight, then incubated with the secondary antibody for 1 h (Table S1), and finally developed by exposure.

Quantitative real-time polymerase chain reaction (RT-qPCR)

TRIzol (Biyuntian Biotechnology Co., Ltd.) was used to extract RNA, and a cDNA reverse transcription kit (Applied Biosystems, cat. no. 4368814) was used to reverse-transcribe the extracted RNA into cDNA. PCR was performed on an ABI Prism 7900HT system (Applied Biosystems, Foster City, CA, USA) using SYBR GREEN PCR Master Mix (Applied Biosystems). Primers were purchased from Sangon Biotech (Shanghai) Co., Ltd. The primer sequences are listed in Table S2.

NextGen sequencing

Total RNA was extracted from rat lung tissue in the PQ and PQ + AH₂QDS groups and enriched with eukaryotic mRNA using magnetic beads with Oligo(dT). The second cDNA strand was then purified by QiaQuick PCR kit and eluted with EB buffer, followed by end repair, the addition of poly(A) and ligation of the sequencing junction, then agarose gel electrophoresis for fragment size selection, and finally PCR amplification. After that, the library was sequenced on the Illumina NovaSeq6000 platform.

To make sure reads reliable, Illumina paired-ended sequenced Raw reads were filtered using the fastp to remove low quality reads (https://github.com/OpenGene/fastp). The filtered data is then compared to the reference sequence. Reference genome and gene model annotation files were downloaded from genome website directly. (https://www.ncbi.nlm.nih.gov/assembly/GCF_000001895.5#/def). The sequenced data were imported into Partek Flow (Partek Inc., St. Louis, MO) and principal component analysis (PCA) images were generated to visualise distribution differences.

Differential expression analysis was performed using the DESeq2⁴⁹. Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG)⁵⁰, we used the R package cluster Profiler⁵¹ to perform KEGG functional enrichment analysis of differentially expressed genes.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 8.0 or SPSS 20.0 software. Measurement data are expressed as the mean ± SEM, and significance was tested by single-factor analysis of variance (ANOVA). Kaplan–Meier survival analysis was used to analyse the survival rate of rats in different treatment groups. P < 0.05 indicates that a difference is statistically significant.

Ethical approval

The experiment was carried out according to the guiding principles for animal experiments at Hainan Medical University.

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Acknowledgements

This project supported by Hainan Province Key R&D Program (ZDYF2019125), National Natural Science Foundation of China (81960351), Hainan Provincial Natural Science Foundation of China (820QN398), the Youth Cultivation Fund Project of the First Affiliated Hospital of Hainan Medical University (HYYFYPY202012), and Hainan Province Clinical Medical Center. We would like to thank the Science experiment Center of Hainan Medical university for providing a venue for our experiment.

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These authors contributed equally: ** Qian and Chun-Yuan Wu.

Authors and Affiliations

Key Laboratory of Emergency and Trauma of Ministry of Education, The First Affiliated Hospital of Hainan Medical University, Hainan Medical University, Haikou, 571199, China
** Qian, Li-Hua Li, Qi Li, Tang Deng, Qi-Feng Huang, Shuang-Qin Xu, Hang-Fei Wang, **n-**n Wu, Zi-Yi Cheng & **ao-Ran Liu
Institute of Environment and Plant Protection, Chinese Academy of Tropical Agricultural Sciences, Haikou, 571101, China
Chun-Yuan Wu & Dong-Ming Wu
Emergency Medicine Center, Sichuan Provincial People’s Hospital, University of Electronic Science and Technology of China, Chengdu, 610072, China
Chuan-Zhu Lv

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** Qian
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Chun-Yuan Wu
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Dong-Ming Wu
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Li-Hua Li
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Qi Li
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Tang Deng
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Qi-Feng Huang
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Shuang-Qin Xu
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Hang-Fei Wang
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**n-**n Wu
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Zi-Yi Cheng
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Chuan-Zhu Lv
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**ao-Ran Liu
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Contributions

J.Q., C.Y.W. and D.M.W. carried out experimental analysis on the data and drafted the manuscript. L.H.L., Q.L., T.D., Q.F.H., S.Q.X., H.F.W. and X.X.W. performed the statistical analysis. Z.Y.C., X.R.L. and C.Z.L. conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.

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Correspondence to Zi-Yi Cheng, Chuan-Zhu Lv or **ao-Ran Liu.

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Qian, J., Wu, CY., Wu, DM. et al. Anthrahydroquinone-2-6-disulfonate is a novel, powerful antidote for paraquat poisoning. Sci Rep 11, 20159 (2021). https://doi.org/10.1038/s41598-021-99591-4

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Received: 28 April 2021
Accepted: 29 September 2021
Published: 11 October 2021
DOI: https://doi.org/10.1038/s41598-021-99591-4
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Anthrahydroquinone-2-6-disulfonate is a novel, powerful antidote for paraquat poisoning

Abstract

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Introduction

Discussion

Conclusion

Methods

Animals and cell lines

Main reagent

Modeling studies of PQ and AH2QDS binding

Cell counting kit-8 (CCK8)

Mitochondrial membrane potential

Animal experiments

Sample collection

Histopathology

Blood analysis

Transmission electron microscopy

Ultra-high-performance liquid chromatography-tandem mass spectrometry

Cytokine detection

Western blotting

Quantitative real-time polymerase chain reaction (RT-qPCR)

NextGen sequencing

Statistical analysis

Ethical approval

References

Acknowledgements

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Contributions

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Supplementary Information

Supplementary Information.

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Modeling studies of PQ and AH₂QDS binding