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

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

Effects of the multi-stress-resistant strain Zygosaccharomyces parabailii MC-5K3 bioaugmentation on microbial communities and metabolomics in tobacco waste extract.

Industrial tobacco waste was mainly treated via a reconstituted tobacco process using the paper-making method, which involves aqueous concentrated tobacco waste extract (cTWE) fermentation (aging). The fermentation was done to improve the quality of reconstituted tobacco. However, cTWE is a multi-stress environment that is characterized by low pH (about 4), as well as high sugar (above 150 g/L) and nicotine (above 15 g/L) content. In this study, a specific selection strategy was used to successfully isolate multi-stress-resistant bacterial or fungal strains, that exhibited positive effects on cTWE fermentation, thereby improving the quality of final products. A potential strain Zygosaccharomyces parabailii MC-5K3 was used for the bioaugmentation of cTWE fermentation and it significantly influenced the microbial diversity of the fermented cTWE. Zygosaccharomyces was observed to be the only dominant fungal genus instead of some pathogenic bacterial genera, with an abundance of over 95% after four days, and still more than 80% after a week. Meanwhile, metabolomics profiling showed significant concentration decrease with regard to some flavor-improving relative metabolites, such as 3-hydroxybenzoic acid (log2FC = - 5.25) and sorbitol (log2FC = - 5.54). This finding is extrapolated to be the key influence factor on the quality of the fermented cTWE. The correlation analysis also showed that the alterations in microbial diversity in the fermented cTWE led to some important differential metabolite changes, which finally improved various properties of tobacco products.

Four-week repeated exposure to tire-derived 6-PPD quinone causes multiple organ injury in male BALB/c mice.

N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone (6-PPDQ) is the ozonation product of tire antioxidant 6-PPD. 6-PPDQ can be detected in different environments, such as roadway runoff and dust. Although 6-PPDQ toxicity has been frequently assessed in aquatic organisms, the possible toxic effects of 6-PPDQ on mammals remain largely unclear. We here aimed to perform systematic assessment to evaluate 6-PPDQ toxicity on multiple organs in mice. Male BALB/c mice were intraperitoneally injected with 6-PPDQ for two exposure modes, single intraperitoneal injection and repeated intraperitoneal injection every four days for 28 days. Serum, liver, kidney, lung, spleen, testis, brain, and heart were collected for injury evaluation by organ index, histopathology analysis and biochemical parameters. In 0.4 and 4 mg/kg 6-PPDQ single injected mice, no significant changes in organ indexes and biochemical parameters were detected, and only moderate pathological changes were observed in organs of liver, kidney, lung, and brain. Very different from this, in 0.4 and 4 mg/kg 6-PPDQ repeated injected mice, we observed the obvious increase in organ indexes of liver, kidney, lung, testis, and brain, and the decrease in spleen index. Meanwhile, the significant pathological changes were formed in liver, kidney, lung, spleen, testis, and brain in 0.4 and 4 mg/kg 6-PPDQ repeated injected mice. Biochemical parameters of liver (alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP)) and kidney (urea and creatinine) were all significantly upregulated by repeated injection with 0.4 and 4 mg/kg 6-PPDQ. After repeated exposure, most of 6-PPDQ was accumulated in liver and lung of mice. Therefore, our results suggested the risk of repeated exposure to 6-PPDQ in inducing toxicity on multiple organs in mice.

Sulfonate-Modified Polystyrene Nanoparticle at Precited Environmental Concentrations Induces Transgenerational Toxicity Associated with Increase in Germline Notch Signal of Caenorhabditis elegans.

Recently, the transgenerational toxicity of nanoplastics has received increasing attention. Caenorhabditis elegans is a useful model to assess the transgenerational toxicity of different pollutants. In nematodes, the possibility of early-life exposure to sulfonate-modified polystyrene nanoparticle (PS-S NP) causing transgenerational toxicity and its underlying mechanisms were investigated. After exposure at the L1-larval stage, transgenerational inhibition in both locomotion behavior (body bend and head thrash) and reproductive capacity (number of offspring and fertilized egg number in uterus) was induced by 1-100 µg/L PS-S NP. Meanwhile, after exposure to 1-100 µg/L PS-S NP, the expression of germline lag-2 encoding Notch ligand was increased not only at the parental generation (P0-G) but also in the offspring, and the transgenerational toxicity was inhibited by the germline RNA interference (RNAi) of lag-2. During the transgenerational toxicity formation, the parental LAG-2 activated the corresponding Notch receptor GLP-1 in the offspring, and transgenerational toxicity was also suppressed by glp-1 RNAi. GLP-1 functioned in the germline and the neurons to mediate the PS-S NP toxicity. In PS-S NP-exposed nematodes, germline GLP-1 activated the insulin peptides of INS-39, INS-3, and DAF-28, and neuronal GLP-1 inhibited the DAF-7, DBL-1, and GLB-10. Therefore, the exposure risk in inducing transgenerational toxicity through PS-S NP was suggested, and this transgenerational toxicity was mediated by the activation of germline Notch signal in organisms.

Taurine alleviates ochratoxin A-induced pyroptosis in PK-15 cells by inhibiting oxidative stress.

Ochratoxin A (OTA) is one of the most harmful mycotoxins, which can cause multiple toxicological effects, especially nephrotoxicity in animals and humans. Taurine is an essential amino acid with various biological functions such as anti-inflammatory and anti-oxidation. However, the protective effect of taurine on OTA-induced nephrotoxicity and pyroptosis had not been reported. Our results showed that OTA exposure induced cytotoxicity and oxidative stress in PK-15 cells, including reactive oxygen species (ROS) accumulation, increased mRNA levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2), and decreased mRNA levels of catalase (CAT), glutathione peroxidase 1 (GPx1), and glutathione peroxidase 4 (GPx4). In addition, OTA treatment induced pyroptosis by increasing the expressions of pyroptosis-related proteins NLRP3, GSDMD, Caspase-1 P20, ASC, Pro-caspase-1, and IL-1ß. Meanwhile, taurine could alleviate OTA-induced pyroptosis and cytotoxicity, as well as reduce ROS level, COX-2, and iNOS mRNA levels, and increase the mRNA levels of the antioxidant enzyme in PK-15 cells. Taken together, taurine alleviated OTA-induced pyroptosis in PK-15 cells by inhibiting ROS generation and altering the activity of antioxidant enzymes, thereby attenuating its nephrotoxicity.

Low doses of fumonisin B1 exacerbate ochratoxin A-induced renal injury in mice and the protective roles of heat shock protein 70.

Fumonisin B1 (FB1) and ochratoxin A (OTA) possess nephrotoxicity to animals and widely co-exist in food and feedstuffs. FB1 rarely, while OTA often, causes toxicosis in animals. Heat shock protein 70 (Hsp70) resists lung injury induced by pneumolysin, but whether Hsp70 could remission mycotoxins-induced renal injury is still unknown. The present study aims to explore the impacts of nontoxic doses of FB1 on OTA-induced nephrotoxicity and the protective roles of Hsp70. In the mycotoxins-challenge experiment, ICR mice were co-exposed to nontoxic doses of FB1 (0, 0.2, 0.5, 1.0 mg/kg bw, IP) and toxic dose of OTA (0.4 mg/kg bw, IP) for 16 d. The results showed that the levels of BUN, Cr, MDA in serum, the Cyto C in renal tubes or glomerulus, pro-apoptosis genes and p-JNK protein expression in kidney were significantly increased. Histopathological results revealed the glomerular swelling. The above all indexes were dose-dependent. In the protection experiment, the mice were pretreated with the eukaryotic plasmid of pEGFP-C3-Hsp70, these increasing parameters in the mycotoxins-challenge experiment were reversed. In vitro, after pK-15 cells were treated with 8 µM FB1 and 5 µM OTA for 48 h, the mitochondrial membrane potential was significantly reduced, mitochondrial ROS was remarkably increased, more Cyto C was leaked from mitochondria into cytoplasm, and pro-apoptosis genes were significantly up-regulated. After the Hsp70 level was up-regulated by pEGFP-C3-Hsp70 or ML346 in pK-15 cells, these above indexes were reversed. However, activation of JNK by anisomycin significantly suppressed the protective effects of Hsp70. Our results demonstrate that the nontoxic doses of FB1 exacerbate the toxic dose of OTA-induced renal injury, while Hsp70 alleviates renal injury by inhibiting the JNK/MAPK signaling pathway. Hsp70 up-regulation may be an efficient strategy for protecting against tissue damage and bio-function impairment induced by co-exposure to FB1 and OTA.

Characterization of the complete chloroplast genome of Veronica arvensis and its phylogenomic inference in plantaginaceae.

Veronica arvensis, which is an annual flowering plant in the plantain family Plantaginaceae, has commonly used as a Chinese herbal medicine to treat malaria in China. Here, the complete plastome of V. arvensis was successfully assembled based on genome skimming sequencing. The plastome of V. arvensis was 149,386 bp in length, comprising a pair of inverted repeats (IR; 24,946 bp) separated by a large single-copy (LSC) region (82,004 bp) and a small single-copy (SSC) region (17,490 bp). The plastid genome encoded 113 unique genes, consisting of 79 protein-coding genes, 30 tRNA genes, and four rRNA genes, with 19 duplicated genes in the IR regions. Six plastid hotspot regions (trnH-psbA, trnK-rps16, atpI-rps2, ndhF-rpl32, ccsA-ndhD and rps15-ycf1) were identified within Veronica. Phylogenetic analysis showed that the representative species from Veronica was monophyletic. V. persica and V. polita formed a maximum clade, followed by sister to V. arvensis.

Crucial Function of Caveolin-1 in Deoxynivalenol-Induced Enterotoxicity by Activating ROS-Dependent NLRP3 Inflammasome-Mediated Pyroptosis.

Deoxynivalenol (DON) is one of the most pervasive contaminating mycotoxins in grain, and exposure to DON is known to cause acute and chronic intestinal damage. As the gut is the most important target organ of DON, it is essential to identify the pivotal molecules involved in DON-induced enterotoxicity as well as the potential regulatory mechanisms. In the present study, we found that DON treatment dramatically decreased the jejunal villus height and increased the crypt depth in mice. DON exposure induced oxidative stress and NLRP3 inflammasome activation while increasing the levels of pyroptosis-related factors GSDMD, ASC, Caspase-1 P20, and IL-1ß and inflammatory cytokines IL-18, TNF-α, and IL-6. In vitro, 0.5-2 µM DON caused cytotoxicity and oxidative stress, as well as NLRP3-mediated pyroptosis in IPEC-J2 cells. Furthermore, DON treatment substantially improved the expression of Caveolin-1 (Cav-1) in vitro and in vivo. Interestingly, Cav-1 knockdown effectively attenuated DON-induced oxidative stress and NLRP3-mediated pyroptosis in IPEC-J2 cells. Meanwhile, treatment with the antioxidant NAC significantly alleviated DON-induced cytotoxicity and pyroptosis in IPEC-J2 cells. Likewise, after inhibiting NLRP3 inflammasome activation with the inhibitor MCC950, DON-induced cytotoxicity, pyroptosis, and inflammatory response were attenuated. However, NLRP3 inhibition did not affect Cav-1 expression. In conclusion, our study demonstrated that pyroptosis may be an underlying mechanism in DON-induced intestinal injury, and Cav-1 plays a pivotal role in DON-induced pyroptosis via regulating oxidative stress, which suggests a novel strategy to overcome DON-induced enterotoxicity.

Deoxynivalenol aggravates the immunosuppression in piglets and PAMs under the condition of PEDV infection through inhibiting TLR4/NLRP3 signaling pathway.

Mycotoxins are toxic metabolites produced by fungi, which are ubiquitous in cereals and feed worldwide and threaten human and animal health. Deoxynivalenol (DON) is one of the most prevalent mycotoxins and causes a series of toxicities, especially enterotoxicity and immunotoxicity. Porcine epidemic diarrhea virus (PEDV) is a destructive enteropathogenic animal coronavirus, is often accompanied with DON contamination in the swine herd. Previous studies have shown that PEDV infection leads severe immunosuppression in pigs. However, whether DON exposure aggravates the PEDV-induced immunosuppression remains unclear. In this study, weaned piglet and porcine alveolar macrophage cell (PAM) models were established to explore the effects of DON on the PEDV-induced immunosuppression and to clarify its underlying mechanism. The in vivo results showed that 2.25 mg/kg feed DON significantly exacerbated the immunosuppressive effects on the PEDV-infected piglets, as demonstrated by the decreases in growth performance, the numbers of goblet cells and CD3+T cells, as well as the protein expressions of ZO-1, Claudin1 and Muc2, in addition to the increases in anti-inflammatory factors levels and the intestinal injury. Similarly, the in vitro results demonstrated that 3-4 µM DON markedly aggravated apoptosis, enhanced the expressions of anti-inflammatory factors, but reduced the migration and phagocytosis abilities of the PEDV-infected PAMs. Furthermore, DON significantly suppressed the expressions of TLR4/NLRP3 in vivo and in vitro. To contrast, lipopolysaccharide (LPS), the corresponding activator, obviously alleviated the DON-exacerbated immunosuppression. Our findings suggest that DON could aggravate host immunosuppression under the condition of PEDV infection through inhibiting TLR4/NLRP3 signaling pathway, and provide novel theoretical insights into the further studies on the immunotoxicity of DON contamination and PEDV-induced immunosuppression.

Selenium and Taurine Combination Is Better Than Alone in Protecting Lipopolysaccharide-Induced Mammary Inflammatory Lesions via Activating PI3K/Akt/mTOR Signaling Pathway by Scavenging Intracellular ROS.

Mastitis is mainly induced by gram-negative bacterial infections, causing devastating economic losses to the global cattle industry. Both selenium (Se) and taurine (Tau) exhibit multiple biological effects, including reducing inflammation. However, no studies have reported the protective effect of the combined use of Se and Tau against mastitis, and the underlying mechanisms remain unclear. In this study, lipopolysaccharide (LPS), the vital virulence factor of gram-negative bacteria, was used to construct the in vivo and vitro mastitis models. The results of in vivo model showed that Se and Tau combination was more effective than either substance alone in reducing tissue hyperemia, edema, and neutrophil infiltration in the mammary acinar cavity, improving the blood-milk barrier in LPS-induced mice mastitis, and decreasing the expression of proinflammatory factors and the activity of MPO. Moreover, Se and Tau combination significantly increased the levels of LPS-induced reduction in PI3K/Akt/mTOR, but the expressions of TLRs and NLRP3 were not significantly changed in the mammary tissue. In the in vitro experiments, the effects of Se and Tau combination or alone on inflammatory factors, inflammatory mediators, MPO activity, and blood-milk barrier were consistent with those in vivo. The Se and Tau combination has also been found to increase the survival rate of BMECs compared with each substance alone via promoting cellular proliferation and inhibiting apoptosis. Also, it has been confirmed that this combination could restore the LPS-induced inhibition in the PI3K/Akt/mTOR signaling pathway. Inhibition of mTOR by Rapamycin counteracted the combined protection of SeMet and Tau against LPS-induced inflammatory damage, the inhibition of PI3K by LY294002 blocked the activation of mTOR, and the accumulation of ROS by the ROS agonist blocked the activation of PI3K. In conclusion, these findings suggested that Se and Tau combination was better than either substance alone in protecting LPS-induced mammary inflammatory lesions by upregulating the PI3K/Akt/mTOR signaling pathway.

Mannan Oligosaccharide Could Attenuate Ochratoxin A-Induced Immunosuppression with Long-Time Exposure Instead of Immunostimulation with Short-Time Exposure.

Our previous study showed that ochratoxin A (OTA), one of the most common mycotoxins in feed, could induce immunosuppression with long-time exposure but immunostimulation with short-time exposure. However, limited studies for the control of OTA-induced two-way immune toxicity were carried out. This study explored the effects of mannan oligosaccharide (MOS), a glucomannoprotein complex with immunoregulatory capability derived from the yeast cell wall, on OTA-induced immune toxicity and its underlying mechanisms. Surprisingly, the results showed that MOS significantly attenuated immunosuppression induced by long-time OTA treatment but did not provide protection against immunostimulation induced by short-time OTA treatment on porcine alveolar macrophages (PAMs), as demonstrated by the expressions of inflammatory cytokines and the capability of migration and phagocytosis. Further, MOS increased the OTA-inhibited autophagy level and the JNK phosphorylation level on PAMs with long-time OTA treatment. In addition, the inhibition of autophagy by 3-MA or the inhibition of JNK phosphorylation by SP600125 could partly block the protective effects of MOS on OTA-induced immunosuppression. Importantly, the inhibition of JNK phosphorylation down-regulated the MOS-promoted autophagy level. In conclusion, MOS could attenuate OTA-induced immunosuppression with short-time exposure on PAMs through activating JNK-mediated autophagy but had no significant effects on OTA-induced immunostimulation with short-time exposure. Our study provides new insights into the application of MOS as an immunoregulator against mycotoxin-induced immune toxicity.

Two-way immune effects of deoxynivalenol in weaned piglets and porcine alveolar macrophages: Due mainly to its exposure dosage.

Mycotoxins are toxic metabolites produced by fungal species that occur frequently in cereals and animal forages throughout the world, posing a serious threat to humans and animals. Although some studies showed the immunotoxicity of mycotoxins, little research focused on the two-way effects of mycotoxins on immune response in vitro and vivo. Here, we explored the effects of deoxynivalenol (DON), one of the most widely distributed mycotoxins, on immune function of piglets and porcine alveolar macrophages (PAMs), and found it exhibited bidirectional immune effects due to different exposure doses. Our results revealed that low doses of DON increased the expressions of TNF-α and IL-6 in piglets and PAMs, promoted the chemotaxis and phagocytosis of PAMs and transformed macrophages to M1 phenotype (P < 0.05). Conversely, high doses of DON increased the expressions of TGF-ß and IL-10 in piglets and PAMs, inhibited the chemotaxis and phagocytosis of PAMs and induced macrophages M2-type polarization (P < 0.05). Mechanistically, DON exposure significantly activated the TLR4/NFκB pathway at low doses and induced mitophagy-mediated mitochondrial dysfunction at high doses in vitro and vivo. TLR4 interference and mitophagy activator, CCCP, were used to further confirm their roles. Therefore, we concluded that DON exposure at low doses caused immunostimulation via activating TLR4/NFκB, whereas it was immunoinhibitory at high doses through blocking mitophagy. Our study suggested that both high and low doses mycotoxins contamination might be harmful, and further back up the necessity to take a vigilant attitude to minimize humans and animals intake of mycotoxins in the environment.

Quality evaluation of flue-cured tobacco by near infrared spectroscopy and spectral similarity method.

Herein we propose near infrared (NIR) spectroscopy as a rapid method of evaluating the quality of agricultural products. Unlike existing quantitative or qualitative models, quality similarity is characterised using spectral similarity. Key factors of the spectral similarity method were investigated, including variable selection, pre-processing and similarity measures. Sophisticated techniques were developed to ensure the reliability of similarity algorithm. The proposed method was tested by quality similarity of flue-cured tobacco samples. The results demonstrated that the quality-related factors between the target and the similar samples (determined by spectral similarity), showed high similarities. This new method has the potential to characterise product quality effectively and could be a useful new alternative to the widely used PLS models.