Insight into the relationship between metabolic enzymes and oxadiazon degradation in Oryza sativa for reducing environmental risks.

Oxadiazon (ODZ) is extensively utilized in agricultural fields for weed control owing to its strong effectiveness. However, excessive loading of ODZ in water bodies and agricultural soils can lead to various environmental concerns. Therefore, it is crucial to understand the ODZ metabolic process and associated mechanisms in crops to assess the likelihood of ODZ contamination in the environment. This study aimed to assess the effects of ODZ on the growth and toxicological responses of rice (Oryza sativa). The growth of rice tissues was notably compromised with the increase in ODZ concentrations. RNA sequencing in combination with liquid chromatography-quadrupole-time-of-flight-high-resolution mass spectrometry/mass spectrometry (LC-Q-TOF-HRMS/MS) analysis allowed for the identification of numerous transcriptional components associated with ODZ metabolism. Four libraries comprising rice roots and shoots exposed to ODZ were RNA-sequenced in triplicate. The application of environmentally realistic ODZ concentrations upregulated the expression of 844 genes in shoots and 1476 genes in roots. Gene enrichment analysis revealed the presence of multiple enzymes involved in ODZ metabolism and detoxification. These enzymes play a critical role in mitigating environmental stress and facilitating xenobiotic metabolism. Notably, among differentially expressed genes, several key enzymes were identified, including cytochrome P450s, protein kinases, aminotransferases, and ATP-binding cassette transporters involved in the metabolic process. Using LC-Q-TOF-HRMS/MS, 3 metabolites and 13 conjugates were identified in multiple metabolic pathways involving oxidation, hydrolysis, glycosylation, acetylation, and methylation. This study successfully established a potential link between the specific metabolic products of ODZ and increased activities of their corresponding enzymes. Moreover, this study considerably elucidates the detailed pathways and mechanisms involved in ODZ metabolism. The study findings provide valuable insights into the development of genotypes for reducing ODZ residues in paddy fields and minimizing their accumulation in rice crops.

Uptake, translocation, accumulation, and metabolism of fluroxypyr-meptyl and oxidative stress induction in rice seedling.

Fluroxypyr-meptyl (FLUME) is heterocyclic herbicide with internal absorption and transmission characteristics. Owing to its low cost and rapid efficacy, it has been widely used to control broad-leaved weeds in wheat, corn, and rice fields. However, the uptake, translocation, accumulation, and metabolism of FLUME in rice seedlings and the extent of oxidative stress induced by it remain largely unknown, which consequently restricts the comprehensive risk assessment of FLUME residues in the environment during rice production. Hence, we systematically investigated the growth and physiological responses of rice to FLUME and analyzed its uptake, translocation, accumulation, and metabolism in rice seedlings. The results indicated that under 0-0.12 mg/L FLUME treatment, only a small proportion of FLUME was translocated upward and accumulated in rice shoots following absorption via roots, with all the translocation factor values being < 1. Moreover, the distribution and enrichment ability of FLUME in rice seedlings were greater in roots than in shoots. Furthermore, we revealed that FLUME accumulation in rice seedlings evidently inhibited their growth and activated the defense system against oxidative stress, with an increase in the activity of antioxidant and detoxifying enzymes. In addition, multiple metabolic reactions of FLUME were observed in rice seedlings, including dehalogenation, hydroxylation, glycosylation, acetylation, and malonylation. Our study provides systematic insights into the uptake, translocation, accumulation, and metabolism of FLUME in rice seedlings as well as the oxidative stress induced by FLUME accumulation, which can help improve FLUME applications and environmental risk assessments in crops.

Multiple Metabolism Pathways of Bentazone Potentially Regulated by Metabolic Enzymes in Rice.

Bentazone (BNTZ) is a selective and efficient herbicide used in crop production worldwide. However, the persistence of BNTZ residues in the environment has led to their increasing accumulation in farmland and crops, posing a high risk to human health. To evaluate its impact on crop growth and environmental safety, a comprehensive study was conducted on BNTZ toxicity, metabolic mechanism, and resultant pathways in rice. The rice growth was compromised to the treatment with BNTZ at 0.2-0.8 mg/L (529.95-1060.05 g a.i./ha), while the activities of enzymes including SOD, POD, CAT, GST, GT, and CYP450 were elevated under BNTZ stress. A genome-wide RNA-sequencing (RNA-Seq) was performed to dissect the variation of transcriptomes and metabolic mechanisms in rice exposed to BNTZ. The degradative pathways of BNTZ in rice are involved in glycosylation, hydrolysis, acetylation, and conjugation processes catalyzed by the enzymes. Our data provided evidence that helps understand the BNTZ metabolic and detoxic mechanisms.

Acetyltransferase OsACE2 acts as a regulator to reduce the environmental risk of oxyfluorfen to rice production.

The constant use of the pesticide oxyfluorfen (OFF) in farmland contaminates the soil, posing threats to crop growth and human health. To avoid the contamination of food crops with OFF, it is critically important to understand its absorption and degradation mechanisms. In this study, we characterized a new functional locus encoding an acetyltransferase (OsACE2) that can facilitate OFF degradation in rice. OsACE2 was drastically induced by OFF at 0.04-0.2 mg L-1 for 6 days and the rice growth was significantly inhibited. To demonstrate the regulatory role of OsACE2 in resistance to OFF toxicity, we generated OsACE2 overexpression (OE) and knockout mutant using genetic transformation and gene-editing technologies (CRISPR/Cas9). The OE plants grown in the hydroponic medium showed improved growth (plant elongation and biomass), increased chlorophyll content, and reduced OFF-induced oxidative stress. The OsACE2-improved growth phenotypes of rice were attributed to the significantly lower OFF accumulation in OE plants. Conversely, knocking out OsACE2 resulted in compromised growth phenotypes compared to the wild-type (WT). Using LC-LTQ-HRMS/MS, five mono-metabolites and eleven conjugates of OFF were characterized through various canonical pathways, such as hydrolysis, oxidation, reduction, glycosylation, acetylation, malonylation, and interaction with amino acids. These metabolites increased in the OE plants, and five acetylated conjugates were reported for the first time. Collectively, OsACE2 plays a primary role in catabolizing OFF residues in rice through multiple degradation pathways and reducing OFF in its growth environment.

OsBR6ox, a member in the brassinosteroid synthetic pathway facilitates degradation of pesticides in rice through a specific DNA demethylation mechanism.

This manuscript described a comprehensive study on a pesticide degradation factor OsBR6ox that promoted the degradation of pesticides atrazine (ATZ) and acetochlor (ACT) in rice tissues and grains through an epigenetic mechanism. OsBR6ox was transcriptionally induced under ATZ and ACT stress. Genetic disruption of OsBR6ox increased rice sensitivity and led to more accumulation of ATZ and ACT, whereas transgenic rice overexpressing OsBR6ox lines (OEs) showed opposite effects with improved growth and lower ATZ and ACT accumulation in various tissues, including grains. OsBR6ox-mediated detoxification of ATZ and ACT was associated with the increased abundance of brassinolide (one of the brassinosteroids, BRs), a plant growth regulator for stress responses. Some Phase I-II reaction protein genes for pesticide detoxification such as genes encoding laccase, O-methyltransferase and glycosyltransferases were transcriptionally upregulated in OE lines under ATZ and ACT stress. HPLC-Q-TOF-MS/MS analysis revealed an enhanced ATZ/ATC metabolism in OE plants, which removed 1.21-1.49 fold ATZ and 1.31-1.44 fold ACT from the growth medium but accumulated only 83.1-87.1 % (shoot) and 71.7-84.1 % (root) of ATZ and 69.4-83.4 % of ACT of the wild-type. Importantly, an ATZ-responsive demethylated region in the upstream of OsBR6ox was detected. Such an epigenetic modification marker was responsible for the increased OsBR6ox expression and consequent detoxification of ATZ/ACT in rice and environment. Overall, this work uncovered a new model showing that plants utilize two mechanisms to co-regulate the detoxification and metabolism of pesticides in rice and provided a new approach for building up cleaner crops and eliminating residual pesticides in environments.

Expression of CYP76C6 Facilitates Isoproturon Metabolism and Detoxification in Rice.

Agricultural chemical residues in farmland and crops is one of the serious public issues that constantly threatens crop production, food security, and human health. Understanding their decay mechanism in crops for accelerating their degradative metabolism is important. In this study, a rice uncharacterized cytochrome P450 gene encoding CYP76C6 was functionally identified in rice exposed to isoproturon (IPU). To verify the role of CYP76C6 in rice resistance to IPU toxicity, CYP76C6 overexpression (OEs) and knockout mutant rice by CRISPR/Cas9 were generated through genetic transformation and gene-editing technologies. Assessment of growth and physiological responses revealed that the growth of OE lines was improved, the IPU-induced cellular damage was attenuated, and IPU accumulation was significantly repressed, whereas the Cas9 lines displayed a contrasting phenotype compared to the wild-type. Both relative contents of IPU metabolites and conjugates in OE lines were reduced and those in Cas9 line were increased, suggesting that CYP76C6 plays a critical role in IPU degradation. Our study unveils a new regulator, together with its mechanism for IPU decay in rice crops, which will be used in reality to reduce environmental risks in food safety and human health.

Metabolomics and integrated network pharmacology analysis reveal attenuates cardiac hypertrophic mechanisms of HuoXin pill.

ETHNOPHARMACOLOGICAL RELEVANCE: Cardiac hypertrophy (CH) is maladaptive and contributes to the pathogenesis of heart failure. Huoxin pill (HXP), a Chinese herbal prescription, is widely applied in the treatment of cardiovascular disease (CAD). Its mechanism, however, is unclear. AIM OF THE STUDY: This study investigated the mechanism of action for Huoxin pill in the treatment of CH, an important stage of CAD. MATERIALS AND METHODS: A total of 60 rats were injected with isoprenaline (ISO) to establish a model of CH. Echocardiography and histopathologic evaluation were performed to evaluate the disease severity, whereas ELISAs were conducted to determine the expression of oxidative stress. Network pharmacology and metabolomic analyses were conducted to identify the key compounds, core targets and pathways that mediate the effects of HXP against CH. Western blotting and immunohistochemistry were used to test apoptosis protein levels. RESULTS: HXP administration in ISO-treated rats decreased hypertrophy indices, alleviated cardiac pathological damage, and downregulated oxidative stress levels when compared to those of rats subjected to ISO treatment only. Moreover, network pharmacology results suggested that the PI3K-Akt pathway is a main mechanism by which HXP inhibits cardiac hypertrophy, and experimental verification showed that HXP inhibited cardiomyocyte apoptosis via activation of the PI3K-Akt pathway. The results of metabolomic analysis identified 21 differential metabolites between the HXPH group and ISO group, which were considered to be metabolic biomarkers of HXP in the treatment of CH. Among them, 6 differential metabolites were significantly upregulated, and 15 were significantly downregulated. CONCLUSIONS: The present study presents an integrated strategy for investigating the mechanisms of HXP in the treatment of CH and sheds new light on the application of HXP as a traditional Chinese medicine.

Identification, characterization and expression of rice (Oryza sativa) acetyltransferase genes exposed to realistic environmental contamination of mesotrione and fomesafen.

The plant acetyltransferases (ACEs) belong to a super family of proteins that contribute to secondary metabolisms and involve various abiotic and biotic stress responses. However, how rice ACEs respond to toxic agrochemicals is largely unknown. This study demonstrates that 86 and 83 genes coding ACEs in the transcriptome profiling were expressed under mesotrione (MTR) and fomesafen (FSA) exposure, respectively. Of these, 18 and 8 ACE differentially expressed genes (DEGs) were identified in MTR- and FSA-exposed rice transcriptome datasets. Some of the ACE genes were validated by quantitative RT-PCR analysis. Analysis of biochemical properties of ACEs revealed that many genes have various cis-elements and structural domain which may cope with a variety of biotic and abiotic stress responses and detoxification of xenobiotics. Moreover, the ACE activities in rice were induced under MTR and FSA exposure and reached out to the highest value at the 0.1 mg L-1. The ACE activities in the MTR and FSA treated roots were 2.6 and 3.5 fold over the control and those in shoots with MTR and FSA were 4.0 and 26.1 fold over the control, respectively. These results indicate that the ACE-coding genes can respond to the MTR and FSA stress by increasing their transcriptional level, along with the enhanced specific ACE protein activities in rice tissues.

Insight into metabolism pathways of pesticide fomesafen in rice: Reducing cropping and environmental risks.

Fomesafen (FSA) is widely used in soybean fields for weed control. However, the persisting characteristics of FSA in the agricultural soil or water may become a hidden danger causing environmental pollution and phytotoxicity to succession crops. In this study, the growth and physiological responses of rice to FSA were investigated. It was found that the growth of rice seedlings was obviously inhibited by FSA exposure especially at over 0.1 mg L-1. To gain an insight into the molecular mechanisms for the potential ecotoxicology, four libraries of rice roots and shoots exposed to FSA were created and subjected to the global RNA-sequencing (RNA-Seq) combined with HRLC-Q-TOF-MS/MS analytical technologies to comprehensively characterize the biochemical processes and catalytic reactions involved in FSA metabolism in rice. Compared with those without FSA, 499 and 450 up-regulated genes in roots and shoots with FSA were detected. Many of them were closely correlated with the tolerance to environmental stress, detoxification of xenobiotics and molecular metabolism process including cytochrome P450, glutathione S-transferases and acetyltransferase. A total of eight metabolites and fourteen conjugates in the reactive pathways of hydrolysis, substitution, reduction, methylation, glycosylation, acetylation, and malonylation were characterized by HRLC-Q-TOF-MS/MS. The relationship between the metabolized derivatives of FSA and enhanced expression the corresponding enzymatic regulators was established. This study will help understand the mechanisms and pathways of FSA metabolism and inspire the further research on FSA degradation in the paddy crops and environmental or health risks.

Comprehensive analyses of degradative enzymes associated with mesotrione-degraded process in rice for declining environmental risks.

Mesotrione (MTR) is a highly effective pesticide widely used for weeding in farmland. Overload of MTR in agricultural soils may result in environmental problems. To evaluate the potential contamination of MTR in environments, a better understanding of the MTR degradation process and mechanisms in crops is required. This study investigated the impact of MTR on growth and toxicological responses in rice (Oryza sativa). The growth of rice tissues was significantly compromised with increasing MTR concentrations. RNA-sequencing combined with HRLC-Q-TOF-MS/MS analysis identified many transcriptional components responsible for MTR degradation. Four libraries composed of root and shoot tissues exposed to MTR were RNA-sequenced in biological triplicate. Compared to -MTR, treatment with environmentally realistic MTR concentration upregulated 1995 genes in roots and 326 genes in shoots. Gene enrichment revealed many MTR-degradative enzymes functioning in resistance to environmental stress and molecular metabolism of xenobiotics. Specifically, many differentially expressed genes are critical enzymes like cytochrome P450, glycosyltransferases, methyltransferase, glutathione S-transferases and acetyltransferase involved in the process. To evidence MTR degradative metabolisms, HRLC-Q-TOF-MS/MS was used to characterize eight metabolites and five conjugates in the pathways involving hydrolysis, reduction, glycosylation, methylation or acetylation. The precise association between the specific MTR-degraded products and enhanced activities of its corresponding enzymes was established. This study advanced our understanding of the detailed MTR degradative mechanisms and pathways, which may help engineer genotypes to facilitate MTR degradation in the paddy crop.

Biodegrading Two Pesticide Residues in Paddy Plants and the Environment by a Genetically Engineered Approach.

Accumulating pesticide (and herbicide) residues in soils have become a serious environmental problem. This study focused on identifying the removal of two widely used pesticides, isoproturon (IPU) and acetochlor (ACT), by a genetically developed paddy (or rice) plant overexpressing an uncharacterized glycosyltransferase (IRGT1). IRGT1 conferred plant resistance to isoproturon-acetochlor, which was manifested by attenuated cellular injury and alleviated toxicity of rice under isoproturon-acetochlor stress. A short-term study showed that IRGT1-transformed lines removed 33.3-48.3% of isoproturon and 39.8-53.5% of acetochlor from the growth medium, with only 59.5-72.1 and 58.9-70.4% of the isoproturon and acetochlor remaining in the plants compared with the levels in untransformed rice. This phenotype was confirmed by IRGT1-expression in yeast ( Pichia pastoris) which grew better and contained less isoproturon-acetochlor than the control cells. A long-term study showed that isoproturon-acetochlor concentrations at all developmental stages were significantly lower in the transformed rice, which contain only 59.3-69.2% (isoproturon) and 51.7-57.4% (acetochlor) of the levels in wild type. In contrast, UPLC-Q-TOF-MS/MS analysis revealed that more isoproturon-acetochlor metabolites were detected in the transformed rice. Sixteen metabolites of isoproturon and 19 metabolites of acetochlor were characterized in rice for Phase I reactions, and 9 isoproturon and 13 acetochlor conjugates were characterized for Phase II reactions in rice; of these, 7 isoproturon and 6 acetochlor metabolites and conjugates were reported in plants for the first time.

Development and evaluation of topotecan loaded solid lipid nanoparticles: A study in cervical cancer cell lines.

The study aims at statistical development of solid lipid nanoparticles (SLNs) loaded with topotecan hydrochloride for avoiding the drawbacks of conventional drug therapies used in cervical cancer. Twenty SLN batches were prepared using organic solvent evaporation method to provide response surface curves. Thereafter, optimized SLNs were obtained using numeric method based on desirability functions providing maximum drug loading and appropriate particle size. Physical characterization of optimized TPH loaded SLNs was performed in terms of particle size, zeta potential, transmission and scanning electron microscopic evaluation. Cytotoxicity studies were performed against cervical cancer cell lines, including cervical squamous cell carcinoma cell line (HeLa) and human squamous cell carcinoma cell line (SiHa). Also, Swiss mouse embryo fibroblast cells (3T3-L1) and African green monkey kidney epithelial (Vero) cells were used to evaluate biocompatibility in normal cells. As pronounced from the results, optimized SLNs may provide an attractive alternative to conventional cervical cancer drug products.

[Expression of hypoxia-inducible factor 1 alpha and vascular endothelial growth factor in prostate cancer and its significance].

OBJECTIVE: To study the expression of hypoxia-inducible factor 1 alpha (HIF-1 alpha) and vascular endothelial growth factor (VEGF) in prostate cancer (PCa) and its biological significance. METHODS: Specimens from 75 patients with PCa (32 cases), benign prostatic hyperplasia (BPH, 16 cases), high-grade prostate intraepithelial neoplasia (PIN, 15 cases) and normal prostate (NP, 12 cases) were comprised in the study. The PCa was divided into high Gleason score group (Gleason score > or = 7) and low Gleason score group (Gleason score < 7). Immunohistochemistry was performed with antibodies against HIF-1 alpha, VEGF and CD34. Microvessel density (MVD) was analyzed by staining with antibodies to CD34. RESULTS: The positive rates of HIF-1alpha were significantly higher in PCa (62.5%) and PIN (60.0%) than those in BPH (6.3%) and NP(0) (P < 0.05), respectively. The positive rates of VEGF were significantly higher in PCa (78.1%) and PIN (73.3%) than those in BPH (18.7%) and NP (8.3%) (P < 0.05), respectively. A higher concentration of MVD was observed in PCa (66.9 +/- 18.0) compared with those in BPH (28.3 +/- 6.9) and NP (15.3 +/- 2.9) (P < 0.05). MVD and the positive rates of HIF-1alpha and VEGF were significantly higher in high Gleason score group than those in low Gleason score group. CONCLUSION: The results suggest that the overexpression of HIF-1alpha and VEGF are closely related to PCa, and the son score group. up-regulation of HIF-1alpha and VEGF are early events in PCa.