Dirigent gene editing of gossypol enantiomers for toxicity-depleted cotton seeds.

Axial chirality of biaryls can generate varied bioactivities. Gossypol is a binaphthyl compound made by cotton plants. Of its two axially chiral isomers, (-)-gossypol is the bioactive form in mammals and has antispermatogenic activity, and its accumulation in cotton seeds poses health concerns. Here we identified two extracellular dirigent proteins (DIRs) from Gossypium hirsutum, GhDIR5 and GhDIR6, which impart the hemigossypol oxidative coupling into (-)- and (+)-gossypol, respectively. To reduce cotton seed toxicity, we disrupted GhDIR5 by genome editing, which eliminated (-)-gossypol but had no effects on other phytoalexins, including (+)-gossypol, that provide pest resistance. Reciprocal mutagenesis identified three residues responsible for enantioselectivity. The (-)-gossypol-forming DIRs emerged later than their enantiocomplementary counterparts, from tandem gene duplications that occurred shortly after the cotton genus diverged. Our study offers insight into how plants control enantiomeric ratios and how to selectively modify the chemical spectra of cotton plants and thereby improve crop quality.

[Short-term exposure to gossypol causes reversible reproductive toxicity and nephrotoxicity in mice].

OBJECTIVE: To study the toxic effects of short-term exposure to gossypol on the testis and kidney in mice and whether these effects are reversible. METHODS: Twenty 7 to 8-week-old male mice were randomized into blank control group, solvent control group, gossypol treatment group and drug withdrawal group. In the former 3 groups, the mice were subjected to daily intragastric administration of 0.3 mL of purified water, 1% sodium carboxymethylcellulose solution, and 30 mg/mL gossypol solution for 14 days, respectively; In the drug withdrawal group, the mice were treated with gossypol solution in the same manner for 14 days followed by treatment with purified water for another 14 days. After the last administration, the mice were euthanized and tissue samples were collected. The testicular tissue was weighed and observed microscopically with HE and PAS staining; the kidney tissue was stained with HE and examined for mitochondrial ATPase activity. RESULTS: Compared with those in the control group, the mice with gossypol exposure showed reduced testicular seminiferous epithelial cells with rounded seminiferous tubules, enlarged space between the seminiferous tubules, interstitium atrophy of the testis, and incomplete differentiation of the spermatogonia. The gossypol-treated mice also presented with complete, non-elongated spermatids, a large number of cells in the state of round spermatids, and negativity for acrosome PAS reaction; diffuse renal mesangial cell hyperplasia, increased mesangial matrix, and adhesion of the mesangium to the wall of the renal capsule were observed, with significantly shrinkage or even absence of the lumens of the renal capsules and reduced kidney mitochondrial ATPase activity. Compared with the gossypol-treated mice, the mice in the drug withdrawal group showed obvious recovery of morphologies of the testis and the kidney, acrosome PAS reaction and mitochondrial ATPase activity. CONCLUSIONS: Shortterm treatment with gossypol can cause reproductive toxicity and nephrotoxicity in mice, but these toxic effects can be reversed after drug withdrawal.

Short-term exposure to gossypol causes reversible reproductive toxicity and nephrotoxicity in mice / 南方医科大学学报

OBJECTIVE@#To study the toxic effects of short-term exposure to gossypol on the testis and kidney in mice and whether these effects are reversible.@*METHODS@#Twenty 7 to 8-week-old male mice were randomized into blank control group, solvent control group, gossypol treatment group and drug withdrawal group. In the former 3 groups, the mice were subjected to daily intragastric administration of 0.3 mL of purified water, 1% sodium carboxymethylcellulose solution, and 30 mg/mL gossypol solution for 14 days, respectively; In the drug withdrawal group, the mice were treated with gossypol solution in the same manner for 14 days followed by treatment with purified water for another 14 days. After the last administration, the mice were euthanized and tissue samples were collected. The testicular tissue was weighed and observed microscopically with HE and PAS staining; the kidney tissue was stained with HE and examined for mitochondrial ATPase activity.@*RESULTS@#Compared with those in the control group, the mice with gossypol exposure showed reduced testicular seminiferous epithelial cells with rounded seminiferous tubules, enlarged space between the seminiferous tubules, interstitium atrophy of the testis, and incomplete differentiation of the spermatogonia. The gossypol-treated mice also presented with complete, non-elongated spermatids, a large number of cells in the state of round spermatids, and negativity for acrosome PAS reaction; diffuse renal mesangial cell hyperplasia, increased mesangial matrix, and adhesion of the mesangium to the wall of the renal capsule were observed, with significantly shrinkage or even absence of the lumens of the renal capsules and reduced kidney mitochondrial ATPase activity. Compared with the gossypol-treated mice, the mice in the drug withdrawal group showed obvious recovery of morphologies of the testis and the kidney, acrosome PAS reaction and mitochondrial ATPase activity.@*CONCLUSIONS@#Shortterm treatment with gossypol can cause reproductive toxicity and nephrotoxicity in mice, but these toxic effects can be reversed after drug withdrawal.

Intestinal Microbiota Mediates Gossypol-Induced Intestinal Inflammation, Oxidative Stress, and Apoptosis in Fish.

Gossypol, the main antinutritional factor in cottonseed protein concentrate (CPC), could affect the growth conditions of fish, but the underlying mechanism remains unclear. In this study, an 8-week feeding trial was carried out to investigate the effects of gossypol on Nile tilapia (Oreochromis niloticus). Three experimental diets were designed, including control diet (CON), control diet supplemented with 150 mg/kg gossypol (ML), and 300 mg/kg gossypol (MH). 16S rRNA gene sequencing showed that gossypol significantly reduced the richness and diversity of the gut microbiota. Untargeted metabolite analysis revealed that most metabolites were down-regulated by gossypol, and riboflavin was the key metabolite with significant difference between CON-treated and gossypol-treated groups. Gossypol caused intestinal inflammation, oxidative stress, and apoptosis. Through fecal bacteria transplantation experiments, we demonstrated that intestinal microbiota mediated gossypol-induced negative effects, suggesting that intestinal microbiota and its metabolite may account for the harmful effects of gossypol.

Transcriptomic analysis of salivary gland and proteomic analysis of oral secretion in Helicoverpa armigera under cotton plant leaves, gossypol, and tannin stresses.

Gossypol and tannin are involved in important chemical defense processes in cotton plants. In this study, we used transcriptomics and proteomics to explore the changes in salivary gland functional genes and oral secretion (OS) proteins after feeding with artificial diet (containing gossypols and tannins) and cotton plant leaves. We found that dietary cotton plant leaves, gossypols and tannins exerted adverse impacts on the genes that regulated the functions of peptidase, GTPase, glycosyl hydrolases in the salivary glands of the Helicoverpa armigera (H. armigera). However, GST, UGT, hydrolases, and lipase genes were up-regulated to participate in the detoxification and digestive of H. armigera. The oral secretory proteins of H. armigera were significantly inhibited under the stress of gossypol and tannin, such as enzyme activity, but some proteins (such as PZC71358.1) were up-regulated and involved in immune and digestive functions. The combined analysis of transcriptomics and metabolomics showed a weak correlation, and the genes and proteins involved were mainly in digestive enzyme activities. Our work clarifies the deleterious physiological impacts of gossypols and tannins on H. armigera and reveals the mechanism by which H. armigera effectively mitigate the phytotoxic effects through detoxification and immune systems.

The BH3 mimetic (±) gossypol induces ROS-independent apoptosis and mitochondrial dysfunction in human A375 melanoma cells in vitro.

A major challenge in current cancer therapy is still the treatment of metastatic melanomas of the skin. BH3 mimetics represent a novel group of substances inducing apoptosis. In this study, we investigated the cytotoxic effect of (±) gossypol (GP), a natural compound from cotton seed, on A375 melanoma cells and the underlying biochemical mechanisms. To prevent undesired side effects due to toxicity on normal (healthy) cells, concentrations only toxic for tumor cells have been elaborated. Viability assays were performed to determine the cytotoxicity of GP in A375 melanoma and normal (healthy) cells. For the majority of experiments, a concentration of 2.5 µM GP was used resulting in a ROS-independent but caspase-dependent cell death of A375 melanoma cells. At this level, GP was non-toxic for normal human epidermal melanocytes. GP has a very short half-life, however, it was demonstrated that only the "parent" compound and not decomposition products are responsible for the cytotoxic effect in A375 melanoma cells. GP significantly decreased mitochondrial membrane potential accompanied by a Drp1-dependent loss of mitochondrial integrity (fragmentation) in tumor cells. Taken together, GP induced a ROS-independent intrinsic apoptosis leading to the conclusion that within a specific concentration range, GP may work as effective anticancer drug without harmful side effects.

Taurine attenuates gossypol-induced apoptosis of C2C12 mouse myoblasts via the GPR87-AMPK/AKT signaling.

Gossypol, a toxic polyphenol extracted from cotton seeds, is hazardous to human and animal health. Taurine is considered as an essential or semi-essential amino acid and has diverse cytoprotective effects. This study was aimed to investigate the protective effect and molecular mechanism of taurine against apoptosis of C2C12 mouse myoblasts induced by gossypol. C2C12 mouse myoblasts were exposed to gossypol (0, 1 nM, 10 nM, 100 nM, 1 µM, and 10 µM). Cell numbers were rapidly decreased with increasing concentrations of gossypol. Gossypol significantly induced apoptosis, decreased Bcl2 expression, and increased the protein levels of Bax and the cleaved caspase 3. Taurine (0.24 mM) treatment largely rescued the cell number decreased by gossypol, attenuated gossypol-induced cell apoptosis. GPR87 knockdown abolished the inhibition by taurine of cell apoptosis. Furthermore, GPR87 overexpression attenuated cell apoptosis induced by gossypol. Both taurine treatment and GPR87 overexpression stimulated AKT phosphorylation but inhibited AMPK phosphorylation, whereas gossypol had the opposite effects. Taurine treatment promoted GPR87 expression and subcellular localization and partially rescued the inhibition of gossypol on this expression. In summary, these data reveal that taurine attenuates gossypol-induced apoptosis of C2C12 mouse myoblasts via the GPR87-AMPK/AKT signaling.

Gestational and lactational exposure to gossypol alters the testis transcriptome.

BACKGROUND: Reproductive capacity can be altered by challenges experienced during critical periods of development, including fetal development and early neonatal life. Gossypol is a polyphenolic compound, commonly found in cotton seeds, that impairs male reproduction. Here, we investigated whether the exposure to gossypol in utero and during lactation alters male reproductive function in sheep. From conception until 60 days postpartum, ewes were randomly assigned to a control diet or a gossypol-rich diet based on cottonseed. Lamb testicles were removed at 60 days of age and subjected to RNA-sequencing. RESULTS: Lambs derived from the maternal cottonseed diet showed significantly lower growth and lower testis weight as a proportion of the total body weight, and reduced testosterone levels. In addition, the testis transcriptome was significantly altered by the maternal cottonseed diet. Most of the altered genes are directly implicated in testis development and sperm biology, cell communication, iron ion metabolism, calcium homeostasis and signaling, among other functions. Interestingly, network analysis revealed that exposure to gossypol significantly disturbed coexpression patterns among spermatogenesis-related genes, suggesting a disruption in coregulation mechanisms. CONCLUSIONS: Our findings provide evidence that maternal exposure to gossypol alters male reproductive function in the offspring, with potential lasting or lifelong negative consequences.

Establishment of a glioblastoma in vitro (in)complete resection dual co-culture model suitable for drug testing.

BACKGROUND: The treatment of glioblastomas (GBM) is still a clinical challenge. Current GBM therapeutic plans focus on the development of new strategies for local drug administration in the tumor cavity to realize an efficient long-term treatment with small side-effects. Here, different amounts of residual GBM cells and healthy brain cells define the microenvironment of the tumor cavity after individual surgical GBM resection (complete or incomplete). METHODS: We evaluated available in vivo data and determined the required amounts and numerical ratios of GBM and healthy brain cells for our in vitro (in)complete resection dual co-culture model. We applied a generic two-drug treatment [Temozolomide (TMZ) in combination with AT101, followed by single AT101 treatment] strategy and analyzed the results in comparison with appropriate mono-culture systems to prove the applicability of our model. RESULTS: We established a suitable GBM dual co-culture model, mimicking the complete and incomplete resection in vitro, giving stable and reliable results on drug testing. Both dual co-culture conditions protectively influenced on cell death and growth rates of primary GBMs when treated with TMZ+AT101/AT101, although the treatment strategy per se was still efficient. Cell death of astrocytes correlated with amounts of increasing GBM cell numbers in the incomplete resection model upon drug treatment, and probably GBM-released chemokine and cytokines were involved in this interplay. CONCLUSIONS: Our results suggest that this dual co-culture model provides a biologically relevant platform for the discovery and compound screening of local GBM treatment strategies.

Toxicity and sublethal effects of two plant allelochemicals on the demographical traits of cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae).

Plant allelochemicals are a group of secondary metabolites produced by plants to defend against herbivore. The mortality of two plant allelochemicals (tannic acid and gossypol) on the cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae), were investigated using feeding assays and the sublethal effects were evaluated using the age-stage, two-sex life table approach. Tannic acid and gossypol have deleterious effects on A. gossypii, and as the concentrations increased, the mortality of cotton aphid increased. The life history traits of A. gossypii including the developmental duration of each nymph stage, the longevity, oviposition days, total preadult survival rate and adult pre-oviposition period were not significantly affected by sublethal concentration of tannic acid (20 mg/L) and gossypol (50 mg/L), while the population parameters (r, λ and R0) were significantly affected by these two plant allelochemicals. Furthermore, tannic acid can increase the pre-adult duration time and TPOP but reduce the fecundity of A. gossypii significantly compared to the control and gossypol treatment groups. These results are helpful for comprehensively understanding the effects of plant allelochemicals on A. gossypii.

Adaptive regulation of detoxification enzymes in Helicoverpa armigera to different host plants.

Cotton plants produce gossypol as a major secondary metabolite to resist insect herbivores and pathogens. Helicoverpa armigera may employ multigene families of detoxification enzymes to deal with this metabolite. So far, the strength of the transcriptional response to gossypol detoxification in the cotton bollworms remains poorly understood. Here, we investigated the genomewide transcriptional changes that occur in cotton bollworm larvae after one generation feeding on various host plants (cotton, corn, soybean and chili) or an artificial diet. Six genes potentially involved in detoxification of xenobiotics were highly upregulated in bollworms fed on cotton, and the expression of five of these differed significantly in insects that fed on gossypol diet compared with the artificial diet. When these six genes were downregulated using RNA interference, downregulation only of CYP4L11, CYP6AB9 and CCE001b led to reduced growth of bollworm larvae feeding on gossypol diets. These data suggest that the three genes are involved in response of H. armigera to gossypol of cotton. Our results proved that H. armigera may have a broad mechanism for gossypol detoxification.

Gossypol Induces Disruption of Spermatogenesis and Steroidogenesis in Male Mice.

Gossypol, commonly found in cotton seeds, is hazardous to male reproductive physiology. Although several studies have indicated the toxicity of gossypol in human and animal reproduction, the mechanism of gossypol action in testes has not yet been elucidated. In the present study, we investigated the effects of gossypol in normal mouse testis cells, TM3 and TM4 cells, and in gossypol-treated C57BL/6 mice. We confirmed the antiproliferative effects of gossypol using cell viability assays, with PCNA as a proliferation marker, and cell cycle analysis. We also verified mitochondrial dysfunction and Ca2+ dysregulation in the cytosol of TM3 and TM4 cells, using JC-1 and Fluo-4 dyes. To confirm the cellular signaling mechanisms in testis cell lines, we performed Western blot analysis to assess the changes in MAPK and PI3K/Akt signal transduction, using their pharmacological inhibitors. Moreover, we screened the mRNA expression of genes involved in spermatogenesis and steroidogenesis in TM3 and TM4 cells. We also confirmed the mRNA expression and localization of genes regulating testis function in gossypol-treated and untreated mice testes. Collectively, we suggest that gossypol induces negative effects on testis function by reducing cell viability, mitochondrial membrane potential, and testis development-related genes in vitro and in vivo as well as by modulating the MAPK and PI3K signaling pathways.

Differences in Active Defense Responses of Two Gossypium barbadense L. Cultivars Resistant to Fusarium oxysporum f. sp. vasinfectum Race 4.

A highly virulent race 4 genotype of Fusarium oxysporum f. sp. vasinfectum (Fov) was identified for the first time in the western hemisphere in 2002 in cotton fields in the San Joaquin Valley of California. The Gossypium barbadense L. cotton cultivars 'Seabrook Sea Island 12B2' ('SBSI') and 'Pima S-6' are resistant to Fov race 4. Active defense responses were quantitated by monitoring the accumulation of antimicrobial terpenoids (i.e., phytoalexins) in inoculated stem stele tissue in these cultivars. The increase in the concentration of the most toxic phytoalexins was statistically faster after 24 h in 'SBSI' compared to 'Pima S-6'. The sesquiterpenoid hemigossylic acid lactone, which was observed for the first time in nature, also accumulated in diseased plants. Neither hemigossylic acid lactone nor the disesquiterpenoids gossypol, gossypol-6-methyl ether, and gossypol-6,6'-dimethyl ether showed toxicity to Fov. Segregation of F2 progeny from 'SBSI' × 'Pima S-6' crosses gave a few highly susceptible plants and a few highly resistant plants, indicating separate genes for resistance in the two cultivars.

In vitro study of gossypol's ovarian toxicity to rodents and goats.

Gossypol interferes with reproduction, causing damage to sperm, disrupting the estrous cycle and resulting in embryonic lethality. In females, gossypol administration promotes degeneration of ovarian follicles, but it is unknown whether this effect is direct or indirect. Thus, the aim of this study is to determine whether gossypol interferes with folliculogenesis in vitro in rats, mice and goats. Ovaries from rats and mice and fragments of goat ovaries were grown in cell culture for 24 h or 7 days. Four groups were tested: 0 (control), 5, 10 and 20 µg gossypol/ml. After incubation, the ovaries were fixed and processed for histological analysis. Follicles were classified according to their stage of development as either viable or atretic. It was found that the ovaries of rats, mice and goats cultured with gossypol showed an increase in the proportion of atretic follicles and a consequent reduction in the proportion of viable follicles at all stages of follicular development. Compared to the control group, the viability of all ovarian follicles in the rat, mouse and goat groups was reduced after cultivation for 24 h by 56.9%, 56.5% and 68.0%, respectively, with the highest concentration of gossypol (20 µg/mL), and after seven days, the respective reductions were 65.4%, 65.3% and 88.2%. Thus, it is possible that gossypol may directly affect follicular maturation, and consequently female fertility.

Free gossypol supplementation frequency and reproductive toxicity in young bulls.

The aim of the present study was to analyze seminal quality of young bulls subjected to different frequencies of gossypol supplementation. Forty-eight Nellore bulls, with 19 months of age and weighing 357.8 ±â€¯7.2 kg, were used in this study. Animals were fed with 10.5 kg of standard supplement containing free-gossypol from whole cottonseed (WCS) at the following frequency: 3x/week (G3x), 5x/week (G5x) or 7x/week (G7x - Control). Additionally, a negative control was provided, and the treated animals received only mineral supplement (MM) ad libtum. The experiment lasted for 84 days and semen was collected at the beginning and at the end for analysis and cryopreservation. Fresh semen was used for initial analysis and plasma membrane integrity and sperm morphology were also determined. General motility using computer assisted sperm analysis (CASA), plasma and acrosomal membranes integrity, mitochondrial activity, and induced oxidative stress were assessed in post-thawed semen. The study design was completely randomized. Parametric data were analyzed by ANOVA and non-parametric data by the Wilcoxon test, using the statistical program SAS. Level of significance was set at 5%. Supplementation with WCS, regardless the frequency, increased total (P = .009) and head (P = .005) defects in comparison to animals receiving only forage and mineral supplement. Infrequent supplementation, particularly 5 times in the week (G5X), increased head (P = .026) and midpiece (P = .014) abnormalities. Sperm motility in fresh semen was lower in animals that received daily supplementation than those supplemented on alternate days (P = .021). Additionally, animals supplemented daily showed lower percentage of spermatozoa with intact acrosome compared to those supplemented on alternate days (P = .005). Thus, regardless the frequency of supplementation, free-gossypol supplementation affects sperm quality. Although the amount of free gossypol supplied weekly was the same among treatments, daily supplementation compromised sperm kinetics, differently from infrequent supplementation that led to sperm defects developed during spermatogenesis.

The genotoxic effects of anti-cancer drug gossypol on human lymphocytes and its mitigation by melatonin.

PURPOSE OF STUDY: To determine melatonin as a potential natural antioxidant to mitigate the genotoxic effects of promising anti-cancer drug gossypol in human lymphocytes. INTRODUCTION: Gossypol, is a polyphenolic compound naturally occurring in cotton seed, was originally identified as a male contraceptive but it has several proposed clinical applications. Gossypol has anti-proliferative effects on cancer cell lines. However, its genotoxic effects on normal cells are not much studied. Hence, there is a paucity of data available. Hence, the study was conducted to investigate gossypol-induced genotoxic effects on lymphocytes. METHODS: Peripheral blood lymphocyte cultures (PBLC) were done and exposed by two different doses of an anti-cancer drug, gossypol (0.274 mM, 1.645 mM) to check genotoxic effects. Melatonin (0.2 mM) is used as an antioxidant. Genotoxic indices such as sister chromatid exchanges (SCEs), cell cycle proliferative index (CCPI), average generation time (AGT), population doubling time (PDT) were assayed in the cultures. RESULT: Gossypol-treated groups indicated significant increases in frequency of SCEs calculated for SCE/plate and SCE/chromosome. Furthermore, CCPI showed a remarkable reduction and increased AGT and PDT levels were found in exposed cultures. When the higher dose of gossypol cultures was treated along with melatonin, these indices were found to be declined and comparable to control. CONCLUSION: Gossypol, an anti-cancer drug, induces genotoxicity on lymphocyte cells and co-supplementation of melatonin antioxidant ameliorates these toxic effects of gossypol.

Potential detoxification of gossypol by UDP-glycosyltransferases in the two Heliothine moth species Helicoverpa armigera and Heliothis virescens.

The cotton bollworm Helicoverpa armigera and the tobacco budworm Heliothis virescens are closely related generalist insect herbivores and serious pest species on a number of economically important crop plants including cotton. Even though cotton is well defended by its major defensive compound gossypol, a toxic sesquiterpene dimer, larvae of both species are capable of developing on cotton plants. In spite of severe damage larvae cause on cotton plants, little is known about gossypol detoxification mechanisms in cotton-feeding insects. Here, we detected three monoglycosylated and up to five diglycosylated gossypol isomers in the feces of H. armigera and H. virescens larvae fed on gossypol-supplemented diet. Candidate UDP-glycosyltransferase (UGT) genes of H. armigera were selected by microarray studies and in silico analyses and were functionally expressed in insect cells. In enzymatic assays, we show that UGT41B3 and UGT40D1 are capable of glycosylating gossypol mainly to the diglycosylated gossypol isomer 5 that is characteristic for H. armigera and is absent in H. virescens feces. In conclusion, our results demonstrate that gossypol is partially metabolized by UGTs via glycosylation, which might be a crucial step in gossypol detoxification in generalist herbivores utilizing cotton as host plant.

Gossypol induces pyroptosis in mouse macrophages via a non-canonical inflammasome pathway.

Gossypol, a polyphenolic compound isolated from cottonseeds, has been reported to possess many pharmacological activities, but whether it can influence inflammasome activation remains unclear. In this study, we found that in mouse macrophages, gossypol induced cell death characterized by rapid membrane rupture and robust release of HMGB1 and pro-caspase-11 comparable to ATP treatment, suggesting an induction of pyroptotic cell death. Unlike ATP, gossypol induced much low levels of mature interleukin-1ß (IL-1ß) secretion from mouse peritoneal macrophages primed with LPS, although it caused pro-IL-1ß release similar to that of ATP. Consistent with this, activated caspase-1 responsible for pro-IL-1ß maturation was undetectable in gossypol-treated peritoneal macrophages. Besides, RAW 264.7 cells lacking ASC expression and caspase-1 activation also underwent pyroptotic cell death upon gossypol treatment. In further support of pyroptosis induction, both pan-caspase inhibitor and caspase-1 subfamily inhibitor, but not caspase-3 inhibitor, could sharply suppress gossypol-induced cell death. Other canonical pyroptotic inhibitors, including potassium chloride and N-acetyl-l-cysteine, could suppress ATP-induced pyroptosis but failed to inhibit or even enhanced gossypol-induced cell death, whereas nonspecific pore-formation inhibitor glycine could attenuate this process, suggesting involvement of a non-canonical pathway. Of note, gossypol treatment eliminated thioglycollate-induced macrophages in the peritoneal cavity with recruitment of other leukocytes. Moreover, gossypol administration markedly decreased the survival of mice in a bacterial sepsis model. Collectively, these results suggested that gossypol induced pyroptosis in mouse macrophages via a non-canonical inflammasome pathway, which raises a concern for its in vivo cytotoxicity to macrophages.

Toxicity of Gossypol from Cottonseed Cake to Sheep Ovarian Follicles.

Gossypol, a polyphenol compound produced by cotton plant, has proven reproductive toxicity, but the effects of gossypol on sheep ovaries are unknown. This study was aimed to determine the in vitro and in vivo effects of gossypol on the ovarian follicles of sheep. This trial was divided into two experiments. In the first one, we used twelve non-pregnant, nulliparous, Santa Inês crossbred ewes, which were randomly distributed into two equal groups and fed diets with and without cottonseed cake. Feed was offered at 1.5% of the animal's body weight for 63 days. The concentrations of total and free gossypol in the cottonseed cake were 3.28 mg/g and 0.11 mg/g, respectively. Throughout the trial period, no animal showed clinical signs of toxicity and no effects on body weight were observed. However, there was a significantly lower number of viable ovarian follicles (20.6%) and higher number of atretic follicles (79.4%) in the gossypol-fed sheep compared to the control (85.1 and 34.9%, respectively). These findings were observed at all stages of follicular development. In the second experiment, eight ovaries from slaughterhouse were cultured with different concentrations of gossypol acetic acid (0, 5, 10 and 20 µg/mL) for 24 hours or seven days. The in vitro action of gossypol resulted in a significant decrease in viable ovarian follicles, especially the primary and transition follicles, and a significant increase in the number of atretic follicles after 24 hours of culture. These follicles were greatly affected when cultured with gossypol for seven days. It is concluded that gossypol present in cotton seeds directly acts on ovarian follicles in sheep to increase atresia.

The response of meat ducks from 15 to 35 d of age to gossypol from cottonseed meal.

The objective of this study was to investigate the responses of meat ducks of 15 to 35 d of age to free gossypol (FG) from cottonseed meal (CSM) and to establish the maximum limits of dietary FG concentration based on growth performance, blood parameters, and tissue residues of gossypol. Nine hundred 15-d-old ducks were randomly allocated to 5 treatments with 10 cages/treatment and 18 ducks/cage on the basis of BW. Five isonitrogenous and isocaloric experimental diets were formulated on a digestible amino acid basis to produce diets in which 0% (without FG), 25% (36 mg FG/kg), 50% (75 mg FG/kg), 75% (111 mg FG/kg), and 100% (153 mg FG/kg) of protein from soybean meal were replaced by that from CSM. Increasing dietary FG content, BW, and ADG decreased (linearly, P<0.05, except for ADG of days 29 to 35), and F/G linearly increased (P<0.05). At 35 d, blood hemoglobin, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration linearly decreased (P<0.05), while serum total protein, albumin, and globulin content linearly decreased (P<0.05), and the residue of gossypol in liver, kidney, heart, breast, and leg muscle linearly increased (P<0.001) with increases in dietary FG concentration. Ducks fed 36 mg FG/kg (5.83% CSM of diet) diet had a normal histological structure of liver, and muscle (breast and leg) had no residue of gossypol. The maximum limit of dietary FG concentration was estimated to range from a low of 36 mg/kg to maximize serum globulin concentration to a high of 124 mg/kg to minimize feed intake for 22 to 28 d on the basis of a quadratic broken-line model.