Structural identification and comprehension of human ALDH1L1-Gossypol complex.

The folate metabolism enzyme ALDH1L1 catalyzed 10-formyltetrahydrofolate to tetrahydrofolate and CO2. Non-small cell lung cancer cells (NSCLC) strongly express ALDH1L1. Gossypol binds to an allosteric site and disrupts the folate metabolism by preventing NADP+ binding. The Cryo-EM structures of tetrameric C-terminal aldehyde dehydrogenase human ALDH1L1 complex with gossypol were examined. Gossypol-bound ALDH1L1 interfered with NADP+ by shifting the allosteric site of the structural conformation, producing a closed-form NADP+ binding site. In addition, the inhibition activity of ALDH1L1 was targeted with gossypol in NSCLC. The gossypol treatment had anti-cancer effects on NSCLC by blocking NADPH and ATP production. These findings emphasize the structure characterizing ALDH1L1 with gossypol.

The rumen-derived Lact. mucosae LLK-XR1 exhibited greater free gossypol degradation capacity during solid-state fermentation of cottonseed meal and probiotic potential.

BACKGROUND: This study aimed to isolate the rumen-derived bacteria with the ability to degrade free gossypol (FG), and to evaluate the probiotic potential in vitro for ensuring safe utilization. METHODS: The strains were anaerobically isolated from fresh rumen fluid of sheep with long-term fed cottonseed meal (CSM) with the screening agar medium containing gossypol as the sole carbon source. Afterwards, the isolated strain incubated with CSM was subjected to the determination of the FG degradation and in vitro evaluation of probiotic characteristics. RESULTS: The target strain labeled Lact. mucosae LLK-XR1 [Accession number: OQ652016.1] was obtained, and its growth on MRS Liquid medium exhibited degradation efficiency of FG up to 69.5% which was significantly greater than its growth on Man-Rogosa-Sharpe medium with glucose free for 24 h (p < 0.01). Meanwhile, LLK-XR1 showed 40.652% degradation rate of FG for unautoclaved, non-pulverized, and no additional nutrients supplementation CSM. Furthermore, LLK-XR1 presented good survivability at pH 3.0 (above 88.6%), and 0.3% bile (78.5%). LLK-XR1 showed sensitivity to broad-spectrum antibiotics except Sulfamethoxazole, Ciprofloxacin and Gentamycin and significantly inhibited E. coli CICC 10,899, Staph. aureus CICC 21,600, and Salmonella. Typhimurium CICC 21,483. LLK-XR1 demonstrated good cell surface hydrophobicity and auto-aggregation ability. CONCLUSIONS: Taken together, this study for the first time noted that rumen-originated Lact. mucosae LLK-XR1 with probiotic properties exhibited substantial FG degradation capacity when it was applied to the solid-state fermentation of CSM.

Loss of Gcn2 exacerbates gossypol induced oxidative stress, apoptosis and inflammation in zebrafish.

Gossypol, a naturally occurring compound found in cottonseed meal, shows promising therapeutic potential for human diseases. However, within the aquaculture industry, it is considered an antinutritional factor. The incorporation of cottonseed meal into fish feed introduces gossypol, which induces intracellular stresses and hinders overall health of farmed fish. The aim of this study is to determine the role of General control nonderepressible 2 (gcn2), a sensor for intracellular stresses in gossypol-induced stress responses in fish. In the present study, we established two gcn2 knockout zebrafish lines. A feeding trial was conducted to assess the growth-inhibitory effect of gossypol in both wild type and gcn2 knockout zebrafish. The results showed that in the absence of gcn2, zebrafish exhibited increased oxidative stress and apoptosis when exposed to gossypol, resulting in higher mortality rates. In feeding trial, dietary gossypol intensified liver inflammation in gcn2-/- zebrafish, diminishing their growth and feed conversion. Remarkably, administering the antioxidant N-acetylcysteine (NAC) was effective in reversing the gossypol induced oxidative stress and apoptosis, thereby increasing the gossypol tolerance of gcn2-/- zebrafish. Exposure to gossypol induces more severe mitochondrial stress in gcn2-/- zebrafish, thereby inducing metabolic disorders. These results reveal that gcn2 plays a protective role in reducing gossypol-induced oxidative stress and apoptosis, attenuating inflammation responses, and enhancing the survivability of zebrafish in gossypol-challenged conditions. Therefore, maintaining appropriate activation of Gcn2 may be beneficial for fish fed diets containing gossypol.

MiR-214_L-1R+4 regulate gossypol-induced immune response through MyD88-dependent signaling pathway in Cyprinus carpio.

MicroRNAs (miRNAs) have been demonstrated to act as crucial modulators with considerable impacts on the immune system. Cottonseed meal is often used as a protein source in aqua feed, cottonseed meal contains gossypol, which is harmful to animals. However, there is a lack of research on the role of miRNAs in fish exposed to gossypol stress. To determine the regulatory effects of miRNAs on gossypol toxicity, Cyprinus carpio were given to oral administration of 20 mg/kg gossypol for 7 days, and the gossypol concentration in the tissues was tested. Then, we detected spleen index, histology, immune enzyme activities of fish induced by gossypol. The results of miRNA sequencing revealed 8 differentially expressed miRNAs in gossypol group, and miR-214_L-1R+4 was found involved in immune response induced by gossypol. The potential targets of miR-214_L-1R+4 were predicted, and found a putative miR-214_L-1R+4 binding site in the 3'UTR of MyD88a. Furthermore, dual-luciferase reporter assays displayed miR-214_L-1R+4 decreased MyD88a expression through binding to the 3'UTR of MyD88a. Moreover, miR-214_L-1R+4 antagomir were intraperitoneally administered to C. carpio, down-regulated miR-214_L-1R+4 could increase MyD88a expression, as well as inflammatory cytokines and anti-inflammatory cytokines expression. These findings revealed that miR-214_L-1R+4 via the MyD88-dependent signaling pathway modulate the immune response to gossypol in C. carpio spleen.

Molecular and functional characterization of heat-shock protein 70 in Aphis gossypii under thermal and xenobiotic stresses.

Aphis gossypii, a globally distributed and economically significant pest of several crops, is known to infest a wide range of host plants. Heat shock proteins (Hsps), acting as molecular chaperones, are essential for the insect's environmental stress responses. The present study investigated the molecular characteristics and expression patterns of AgHsp70, a heat shock protein gene, in Aphis gossypii. Our phylogenetic analysis revealed that AgHsp70 shared high similarity with homologs from other insects, suggesting a conserved function across species. The developmental expression profiles of AgHsp70 in A. gossypii showed that the highest transcript levels were observed in the fourth instar nymphs, while the lowest levels were detected in the third instar nymphs. Heat stress and exposure to four different xenobiotics (2-tridecanone, tannic acid, gossypol, and flupyradifurone (4-[(2,2-difluoroethyl)amino]-2(5H)-furanone)) significantly up-regulated AgHsp70 expression. Knockdown of AgHsp70 using RNAi obviously increased the susceptibility of cotton aphids to 2-tridecanone, gossypol and flupyradifurone. Dual-luciferase reporter assays revealed that gossypol and flupyradifurone significantly enhanced the promoter activity of AgHsp70 at a concentration of 10 mg/L. Furthermore, we identified the transcription factor heat shock factor (HSF) as a regulator of AgHsp70, as silencing AgHSF reduced AgHsp70 expression. Our results shed light on the role of AgHsp70 in xenobiotic adaptation and thermo-tolerance.

Genetic Analysis of an F2 Population Derived from the Cotton Landrace Hopi Identified Novel Loci for Boll Glanding.

Landraces are an important reservoir of genetic variation that can expand the narrow genetic base of cultivated cotton. In this study, quantitative trait loci (QTL) analysis was conducted using an F2 population developed from crosses between the landrace Hopi and inbred TM-1. A high-density genetic map spanning 2253.11 and 1932.21 cM for the A and D sub-genomes, respectively, with an average marker interval of 1.14 cM, was generated using the CottonSNP63K array. The linkage map showed a strong co-linearity with the physical map of cotton. A total of 21 QTLs were identified, controlling plant height (1), bract type (1), boll number (1), stem color (2), boll pitting (2), fuzz fiber development (2), boll shape (3), boll point (4), and boll glanding (5). In silico analysis of the novel QTLs for boll glanding identified a total of 13 candidate genes. Analysis of tissue-specific expression of the candidate genes suggests roles for the transcription factors bHLH1, MYB2, and ZF1 in gland formation. Comparative sequencing of open reading frames identified early stop codons in all three transcription factors in Hopi. Functional validation of these genes offers avenues to reduce glanding and, consequently, lower gossypol levels in cottonseeds without compromising the defense mechanisms of the plant against biotic stresses.

Genome-wide identification and expression analysis of terpene synthases in Gossypium species in response to gossypol biosynthesis.

Cottonseed is an invaluable resource, providing protein, oil, and abundant minerals that significantly contribute to the well-being and nutritional needs of both humans and livestock. However, cottonseed also contains a toxic substance called gossypol, a secondary metabolite in Gossypium species that plays an important role in cotton plant development and self-protection. Herein, genome-wide analysis and characterization of the terpene synthase (TPS) gene family identified 304 TPS genes in Gossypium. Bioinformatics analysis revealed that the gene family was grouped into six subgroups TPS-a, TPS-b, TPS-c, TPS-e, TPS-f, and TPS-g. Whole-genome, segmental, and tandem duplication contributed to the evolution of TPS genes. According to the analysis of selection pressure, it was predicted that TPS genes experience predominantly negative selection, with positive selection occurring subsequently. RT-qPCR analysis in TM-1 and CRI-12 lines revealed GhTPS48 gene as the candidate gene for silencing experiments. To summarize, comprehensive genome-wide studies, RT-qPCR, and gene silencing experiments have collectively demonstrated the involvement of the TPS gene family in the biosynthesis of gossypol in cotton.

Transcriptome analysis reveals the effect of grafting on gossypol biosynthesis and gland formation in cotton.

BACKGROUND: Gossypol is a unique secondary metabolite and sesquiterpene in cotton, which is mainly synthesized in the root system of cotton and exhibits many biological activities. Previous research found that grafting affected the density of pigment glands and the gossypol content in cotton. RESULTS: This study performed a transcriptome analysis on cotton rootstocks and scions of four grafting methods. The gene expression of mutual grafting and self-grafting was compared to explore the potential genes involved in gossypol biosynthesis. A total of six differentially expressed enzymes were found in the main pathway of gossypol synthesis-sesquiterpene and triterpene biosynthesis (map00909): lupeol synthase (LUP1, EC:5.4.99.41), beta-amyrin synthase (LUP2, EC:5.4.99.39), squalene monooxygenase (SQLE, EC:1.14.14.17), squalene synthase (FDFT1, EC:2.5.1.21), (-)-germacrene D synthase (GERD, EC:4.2.3.75), ( +)-delta-cadinene synthase (CADS, EC:4.2.3.13). By comparing the results of the gossypol content and the density of the pigment gland, we speculated that these six enzymes might affect the biosynthesis of gossypol. It was verified by qRT-PCR analysis that grafting could influence gene expression of scion and stock. After suppressing the expression of the LUP1, FDFT1, and CAD genes by VIGS technology, the gossypol content in plants was significantly down-regulated. CONCLUSIONS: These results indicate the potential molecular mechanism of gossypol synthesis during the grafting process and provide a theoretical foundation for further research on gossypol biosynthesis.

Cobalt Ferrite-Gossypol Coordination Nanoagents with High Photothermal Conversion Efficiency Sensitizing Chemotherapy against Bcl-2 to Induce Tumor Apoptosis.

Gossypol is a chemotherapeutic drug that can inhibit the anti-apoptotic protein Bcl-2, but the existing gossypol-related nanocarriers cannot well solve the problem of chemotherapy resistance. Based on the observation that gossypol becomes black upon Fe3+ coordination, it is hypothesized that encasing gossypol in glyceryl monooleate (GMO) and making it coordinate cobalt ferrite will not only improve its photothermal conversion efficiency (PCE) but also help it enter tumor cells. As the drug loading content and drug encapsulation efficiency of gossypol are 10.67% (w/w) and 96.20%, the PCE of cobalt ferrite rises from 14.71% to 36.00%. The synergistic therapeutic effect finally induces tumor apoptosis with a tumor inhibition rate of 96.56%, which is 2.99 and 1.47 times higher than chemotherapy or photothermal therapy (PTT) alone. PTT generated by the GMO nanocarriers under the irradiation of 808 nm laser can weaken tumor hypoxia, thereby assisting gossypol to inhibit Bcl-2. In addition, the efficacy of nanocarriers is also evaluated through T2 -weighted magnetic resonance imaging. Observations of gossypol-induced apoptosis in tissue slices provide definitive proof of chemotherapy sensitization, indicating that such coordination nanocarriers can be used as an effective preclinical agent to enhance chemotherapy.

A traditional gynecological medicine inhibits ovarian cancer progression and eliminates cancer stem cells via the LRPPRC-OXPHOS axis.

BACKGROUND: Ovarian cancer (OC) is the most lethal malignant gynecological tumor type for which limited therapeutic targets and drugs are available. Enhanced mitochondrial oxidative phosphorylation (OXPHOS), which enables cell growth, migration, and cancer stem cell maintenance, is a critical driver of disease progression and a potential intervention target of OC. However, the current OXPHOS intervention strategy mainly suppresses the activity of the electron transport chain directly and cannot effectively distinguish normal tissues from cancer tissues, resulting in serious side effects and limited efficacy. METHODS: We screened natural product libraries to investigate potential anti-OC drugs that target OXPHOS. Additionally, LC-MS, qRT-PCR, western-blot, clonogenic assay, Immunohistochemistry, wound scratch assay, and xenograft model was applied to evaluate the anti-tumor mechanism of small molecules obtained by screening in OC. RESULTS: Gossypol acetic acid (GAA), a widely used gynecological medicine, was screened out from the drug library with the function of suppressing OXPHOS and OC progression by targeting the leucine-rich pentatricopeptide repeat containing (LRPPRC) protein. Mechanically, LRPPRC promotes the synthesis of OXPHOS subunits by binding to RNAs encoded by mitochondrial DNA. GAA binds to LRPPRC directly and induces LRPPRC rapid degradation in a ubiquitin-independent manner. LRPPRC was overexpressed in OC, which is highly correlated with the poor outcomes of OC and could promote the malignant phenotype of OC cells in vitro and in vivo. GAA management inhibits cell growth, clonal formation, and cancer stem cell maintenance in vitro, and suppresses subcutaneous graft tumor growth in vivo. CONCLUSIONS: Our study identified a therapeutic target and provided a corresponding inhibitor for OXPHOS-based OC therapy. GAA inhibits OC progression by suppressing OXPHOS complex synthesis via targeting LRPPRC protein, supporting its potential utility as a natural therapeutic agent for ovarian cancer.

Involvement of necroptosis in the selective toxicity of the natural compound (±) gossypol on squamous skin cancer cells in vitro.

Cutaneous basal and squamous cell carcinoma reflect the first and second most common type of non-melanoma skin cancer, respectively. Especially cutaneous squamous cell carcinoma has the tendency to metastasize, finally resulting in a rather poor prognosis. Therapeutic options comprise surgery, radiation therapy, and a systemic or targeted chemotherapy. There are some good treatment results, but overall, the response rate of newly developed drugs is still modest. Drug repurposing represents an alternative approach where already available and clinically approved substances are used, which originally intended for other clinical benefits. In this context, we tested the effect of the naturally occurring polyphenolic aldehyde (±) gossypol with concentrations between 1 and 5 µM on the invasive squamous cell carcinoma cell line SCL-1 and normal human epidermal keratinocytes. Gossypol treatment up to 96 h resulted in a selective cytotoxicity of SCL-1 cells (IC50: 1.7 µM, 96 h) compared with normal keratinocytes (IC50: ≥ 5.4 µM, 96 h) which is mediated by mitochondrial dysfunction and finally leading to necroptotic cell death. Taken together, gossypol shows a high potential as an alternative anticancer drug for the treatment of cutaneous squamous cell carcinoma.

Sequential Treatment with Temozolomide Plus Naturally Derived AT101 as an Alternative Therapeutic Strategy: Insights into Chemoresistance Mechanisms of Surviving Glioblastoma Cells.

Glioblastoma (GBM) is a poorly treatable disease due to the fast development of tumor recurrences and high resistance to chemo- and radiotherapy. To overcome the highly adaptive behavior of GBMs, especially multimodal therapeutic approaches also including natural adjuvants have been investigated. However, despite increased efficiency, some GBM cells are still able to survive these advanced treatment regimens. Given this, the present study evaluates representative chemoresistance mechanisms of surviving human GBM primary cells in a complex in vitro co-culture model upon sequential application of temozolomide (TMZ) combined with AT101, the R(-) enantiomer of the naturally occurring cottonseed-derived gossypol. Treatment with TMZ+AT101/AT101, although highly efficient, yielded a predominance of phosphatidylserine-positive GBM cells over time. Analysis of the intracellular effects revealed phosphorylation of AKT, mTOR, and GSK3ß, resulting in the induction of various pro-tumorigenic genes in surviving GBM cells. A Torin2-mediated mTOR inhibition combined with TMZ+AT101/AT101 partly counteracted the observed TMZ+AT101/AT101-associated effects. Interestingly, treatment with TMZ+AT101/AT101 concomitantly changed the amount and composition of extracellular vesicles released from surviving GBM cells. Taken together, our analyses revealed that even when chemotherapeutic agents with different effector mechanisms are combined, a variety of chemoresistance mechanisms of surviving GBM cells must be taken into account.

Gossypol and related compounds are produced and accumulate in the aboveground parts of the cotton plant, independent of roots as the source.

MAIN CONCLUSION: Use of Ultra-low gossypol cottonseed event as a scion in a graft combination confirmed that roots are not a source of terpenoids in the aboveground parts of a cotton plant. Gossypol and related terpenoids, derived from the same basic biosynthetic pathway, are present in the numerous lysigenous glands in the aboveground parts of a cotton plant. Roots, with sparse presence of such glands, do produce significant amount of gossypol and a different set of terpenoids. These compounds serve a defensive function against various pests and pathogens. This investigation was undertaken to examine whether gossypol produced in the roots can replenish the gossypol content of the cottonseed-glands that are largely devoid of this terpenoid in a genetically engineered event. Graft unions between a scion derived from the RNAi-based, Ultra-low gossypol cottonseed (ULGCS) event, TAM66274, and a rootstock derived from wild-type parental genotype, Coker 312 (Coker), were compared with various other grafts that served as controls. The results showed that the seeds developing within the scion of test grafts (ULGCS/Coker) continued to maintain the ultra-low gossypol levels found in the TAM66274 seeds. Molecular analyses confirmed that while the key gene involved in gland development showed normal activity in the developing embryos in the scion, two genes encoding the enzymes involved in gossypol biosynthesis were suppressed. Thus, the gene expression data confirmed the results obtained from biochemical measurements and collectively demonstrated that roots are not a source of gossypol for the aboveground parts of the cotton plant. These findings, combined with the results from previous investigations, support the assertion that gossypol and related terpenoids are produced in a highly localized manner in various organs of the cotton plant and are retained therein.

GhMYC2 activates cytochrome P450 gene CYP71BE79 to regulate gossypol biosynthesis in cotton.

MAIN CONCLUSION: GhMYC2 regulates the gossypol biosynthesis pathway in cotton through activation of the expression of gossypol synthesis gene CYP71BE79, CDNC, CYP706B1, DH1, and CYP82D113. Cotton is one of the main cash crops globally. Cottonseed contains fiber, fat, protein, and starch, and has important economic value. However, gossypol in cottonseed seriously affects the development and utilization of cottonseed. Nonetheless, gossypol has great application potential in agriculture, medicine, and industry. Therefore, it is very important to study gossypol biosynthesis and its upstream regulatory pathways. It has been reported that the content of gossypol in hairy roots of cotton is regulated through jasmonic acid signaling; however, the specific molecular mechanism has not been revealed yet. We found that the expression of basic helix-loop-helix family transcription factor GhMYC2 was significantly upregulated after exogenous administration of methyl jasmonate to cotton seedlings, and the content of gossypol changed significantly with the variation of GhMYC2 expression. Further studies revealed that GhMYC2 could specifically bind to the G-Box in the promoter region of CDNC, CYP706B1, DH1, CYP82D113, CYP71BE79 to activate its expression and regulate gossypol synthesis, and its activation of CYP71BE79 promoter was inhibited by GhJAZ2. Not only that GhMYC2 could also interact with GoPGF. In this work, the molecular mechanisms of gossypol biosynthesis regulated by GhMYC2 were analyzed. The results provide a theoretical basis for cultivating new varieties of low-gossypol or high-gossypol cotton and creating excellent germplasm resources.

Effective gossypol removal from cottonseed meal through optimized solid-state fermentation by Bacillus coagulans.

BACKGROUND: Cottonseed meal (CSM) is the main by-product of the cottonseed oil extraction process with high protein content, which is an important protein source for feed industry. However, CSM contains free gossypol (FG), a toxic substance that is detrimental to animal health and greatly limits its application. Microbial fermentation is currently considered to be one of the most effective methods to reduce FG and other anti-nutritional factors in CSM. Previously, yeast and bacteria species are used for degradation of FG in CSM, but showing less detoxification efficiency. Bacillus coagulans combines the properties of both lactic acid bacteria and Bacillus, producing both lactic acid and spores, and is considered a potential probiotic. In this study, we aimed to evaluate and optimize the effect of the solid-state fermentation process using a Bacillus coagulans to gossypol removal contained cottonseed meal. RESULTS: 36 B. coagulans strains were isolated and found to have the ability to remove free gossypol. Through the evaluation of strains and optimization of fermentation conditions including fermentation temperature, ratio of material to water, inoculation amount, fermentation time and pH, we have established a solid-state fermentation process using a Bacillus coagulans strain S17 on CSM substrate with 1:1 of the material-to-water ratio, 15% (v/w) seed inoculation, 2% expanded corn flour, 1% bran, and 0.3%-0.8% metal irons at 40 °C for 52 h. After fermentation, the FG content in CSM was reduced from 923.80 to 167.90 mg/kg with 81.83% detoxification efficiency. Meanwhile, the crude protein content in CSM increased from 47.98 to 52.82%, and importantly, the spore concentration of strain S17 reached 1.68 × 1010 CFU/g dry material. CONCLUSION: The study showed that B. coagulans have the potential strong ability to degrade free gossypol through cottonseed meal fermentation. This study presents a feasible process for improving the resource utilization rate and nutritional value of CSM via solid-state fermentation through B. coagulans S17.

Identification of the gossypol derivatives as androgen receptor inhibitor.

Prostate cancer (PCa) is the most frequently diagnosed male malignant tumor and remains the second leading cause of male cancer mortality in western countries. The development of novel antiandrogens to circumvent the drug resistance in anti-PCa treatment is highly demanded. Herein, we identified that gossypol (GOS) specificly inhibited the AR signaling. Further optimization of GOS derivatives led to the discovery of compound XY-32. XY-32 efficiently inhibits AR signaling with the IC50 of 1.23 µM. XY-32 downregulates both the full-length AR and the AR variable splice AR-V7 via suppressing the mRNA expression. It inhibits the proliferation of CRPC cells such as the LNCaP cells, the PC-3 cells, and enzalutamide resistance 22Rv1 cells. The work demonstrates the GOS derivatives represent a novel series of anti-androgen to conquer the acquired AR mutations or AR splice variants induced drug resistance of mCRPC.

Differential expression profiling of Oxycarenus laetus Kirby (Hemiptera: Lygaeidae) upon exposure to gossypol.

BACKGROUND: Gossypium hirsutum seeds are rich in gossypol. In addition to its diverse beneficial properties, it is a known anti-fertility inducing agent in humans. Oxycarenus laetus feeds on the cottonseeds and yet its courtship, mating and reproduction is unaffected. METHODS AND RESULTS: In this study, we performed a transcriptome profiling of O. laetus fed on Abutilon indicum (AB-no gossypol), G. hirsutum (GH-natural gossypol) and 1400 ppm commercial gossypol-soaked GH seeds (GHGO). Illumina NextSeq-500 paired-end 75 bp reads were generated and de novo assembled (48,214 genes) to identify the differentially expressed transcripts (DET) between the samples. Gene enrichment, KEGG pathway and cluster profiling of the DETs resulted in the identification of vital genes involved in the detoxification, pheromone biosynthesis, cuticle protein in the GHGO sample. Cyp4C1, Cyp6a13, Cyp6a14, Cyp4g15, Cyp4em8, Cyp303a1 were the detoxification related genes identified. Similarly, SDR dehydrogenase family 11 and fatty acid synthase in pheromone biosynthesis and cuticle proteins (RR1 and RR2) coding transcripts were found to be differentially expressed. CONCLUSION: This is the first study to report the expression of genes induced by gossypol in O. laetus. Based on the findings from the DET analysis, we conclude that the detoxification related genes of gossypol treated samples were affected.

Effects of cottonseed meal on growth performance, liver redox status, and serum biochemical parameters in goslings at 1 to 28 days of age.

BACKGROUND: Cottonseed meal (CSM), a relatively rich source of protein and amino acids, is used as an inexpensive alternative to soybean meal (SBM) in poultry diets. However, the toxicity of free gossypol in CSM has been a primary concern. The present study was conducted to investigate the effects of CSM on growth performance, serum biochemical parameters, and liver redox status in goslings at 1 to 28 days of age. Three hundred 1-day-old male goslings were randomly divided into 5 groups (10 goslings/pen, 6 replicate pens/group) and subjected to a 28-day experiment. Five isonitrogenous and isoenergetic diets were formulated such that 0% (control), 25% (CSM25), 50% (CSM50), 75% (CSM75), and 100% (CSM100) of protein from SBM was replaced by protein from CSM. The free gossypol contents in the five diets were 0, 56, 109, 166, and 222 mg/kg, respectively. RESULTS: The results showed that dietary CSM was associated with linear decreases in body weight, average daily feed intake and average daily gain and linear increases in the feed-to-gain ratio from 1 to 28 days of age (P < 0.001). As the dietary CSM concentration increased, a numerical increase was found in the mortality of goslings. According to a single-slope broken-line model, the breakpoints for the average daily gain of dietary free gossypol concentration on days 1 to 14, 15 to 28, and 1 to 28 occurred at 23.63, 14.78, and 18.53 mg/kg, respectively. As the dietary CSM concentration increased, serum albumin (P < 0.001) concentrations decreased linearly and serum uric acid (P = 0.011) increased linearly. The hydroxyl radical scavenging ability (P = 0.002) and catalase (P < 0.001) and glutathione peroxidase (P = 0.001) activities of the liver decreased linearly with increasing dietary CSM. However, dietary CSM did not affect the concentrations of reactive oxygen metabolites, malondialdehyde, or protein carbonyl in the liver. CONCLUSIONS: The increasing dietary CSM increased the concentration of free gossypol and altered the composition of some amino acids in the diet. A high concentration of CSM reduced the growth performance of goslings aged 1 to 28 days by decreasing feed intake, liver metabolism, and antioxidant capacity. From the primary concern of free gossypol in CSM, the tolerance of goslings to free gossypol from CSM is low, and the toxicity of free gossypol has a cumulative effect over time.

Induction of multiple subroutines of regulated necrosis in murine macrophages by natural BH3-mimetic gossypol.

Macrophages are critical sentinel cells armed with multiple regulated necrosis pathways, including pyroptosis, apoptosis followed by secondary necrosis, and necroptosis, and are poised to undergo distinct form(s) of necrosis for tackling dangers of pathogenic infection or toxic exposure. The natural BH3-mimetic gossypol is a toxic phytochemical that can induce apoptosis and/or pyroptotic-like cell death, but what exact forms of regulated necrosis are induced remains largely unknown. Here we demonstrated that gossypol induces pyroptotic-like cell death in both unprimed and lipopolysaccharide-primed mouse bone marrow-derived macrophages (BMDMs), as evidenced by membrane swelling and ballooning accompanied by propidium iodide incorporation and lactic acid dehydrogenase release. Notably, gossypol simultaneously induces the activation of both pyroptotic and apoptotic (followed by secondary necrosis) pathways but only weakly activates the necroptosis pathway. Unexpectedly, gossypol-induced necrosis is independent of nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, as neither inhibitor for the NLRP3 pathway nor NLRP3 deficiency protects the macrophages from the necrosis. Furthermore, necrotic inhibitors or even pan-caspase inhibitor alone does not or only partly inhibit such necrosis. Instead, a combination of inhibitors composed of pan-caspase inhibitor IDN-6556, RIPK3 inhibitor GSK'872 and NADPH oxidase inhibitor GKT137831 not only markedly inhibits the necrosis, with all apoptotic and pyroptotic pathways being blocked, but also attenuates gossypol-induced peritonitis in mice. Lastly, the activation of the NLRP3 pathway and apoptotic caspase-3 appears to be independent of each other. Collectively, gossypol simultaneously induces the activation of multiple subroutines of regulated necrosis in macrophages depending on both apoptotic and inflammatory caspases.

Knockdown of cytochrome P450 gene CYP6AB12 based on nanomaterial technology reduces the detoxification ability of Spodoptera litura to gossypol.

In insects, cytochrome P450 monooxygenases (P450s or CYPs) play an important role in the detoxification and metabolism of exogenous plant allelochemicals. In this study, a P450 gene CYP6AB12 was identified and characterized from Spodoptera litura. The cDNA contains an open reading frame (ORF) encoding 511 amino acid residues. CYP6AB12 was expressed at different ages of S. litura, with the highest levels found in the third and fourth instar larvae. Its highest expression was found in the midgut and fat body of fourth instar larvae fed with gossypol. Moreover, these expression levels were substantially increased compared with those from larvae fed with control diet. Gene silencing was then conducted by smearing dsRNA mixed with nanomaterials onto the cuticle. CYP6AB12 expression was significantly decreased in the midgut and fat body, and the net weight increase was substantially lower than that of the control group, indicating that the treatment group had more sensitivity to gossypol than the control. These results reveal that CYP6AB12 plays an important role in the detoxification and metabolism of gossypol, thus further confirming that P450s have a broad ability to detoxify and metabolize plant allelochemicals. It provides an important molecular basis for the exploration of detoxification metabolism and pest control of S. litura.