Blockade of CaV3 calcium channels and induction of G0/G1 cell cycle arrest in colon cancer cells by gossypol.

BACKGROUND AND PURPOSE: Gastrointestinal tumours overexpress voltage-gated calcium (CaV3) channels (CaV3.1, 3.2 and 3.3). CaV3 channels regulate cell growth and apoptosis colorectal cancer. Gossypol, a polyphenolic aldehyde found in the cotton plant, has anti-tumour properties and inhibits CaV3 currents. A systematic study was performed on gossypol blocking mechanism on CaV3 channels and its potential anticancer effects in colon cancer cells, which express CaV3 isoforms. EXPERIMENTAL APPROACH: Transcripts for CaV3 proteins were analysed in gastrointestinal cancers using public repositories and in human colorectal cancer cell lines HCT116, SW480 and SW620. The gossypol blocking mechanism on CaV3 channels was investigated by combining heterologous expression systems and patch-clamp experiments. The anti-tumoural properties of gossypol were estimated by cell proliferation, viability and cell cycle assays. Ca2+ dynamics were evaluated with cytosolic and endoplasmic reticulum (ER) Ca2+ indicators. KEY RESULTS: High levels of CaV3 transcripts correlate with poor prognosis in gastrointestinal cancers. Gossypol blockade of CaV3 isoforms is concentration- and use-dependent interacting with the closed, activated and inactivated conformations of CaV3 channels. Gossypol and CaV3 channels down-regulation inhibit colorectal cancer cell proliferation by arresting cell cycles at the G0/G1 and G2/M phases, respectively. CaV3 channels underlie the vectorial Ca2+ uptake by endoplasmic reticulum in colorectal cancer cells. CONCLUSION AND IMPLICATIONS: Gossypol differentially blocked CaV3 channel and its anticancer activity was correlated with high levels of CaV3.1 and CaV3.2 in colorectal cancer cells. The CaV3 regulates cell proliferation and Ca2+ dynamics in colorectal cancer cells. Understanding this blocking mechanism maybe improve cancer therapies.

Evaluation of ozone technology as an alternative for degradation of free gossypol in cottonseed meal: a prospective study.

Free gossypol is a toxic compound which naturally occurs in cottonseed and its derivates, affecting animal and possibly human health. Consequently, alternatives for gossypol destruction must be evaluated. This work evaluated the emerging technology of ozone processing for free gossypol destruction in cottonseed meal. Ozonation was carried out in the actual cottonseed meal and also a model system, designed to describe the involved mode of action. The model system consisted of glass pearls beads covered with free gossypol. Ozonation was performed in two ways: as a static process, i.e., without homogenising the sample after placing them in the reactor, and also homogenising it. Ozone degraded free gossypol in all the systems, but reaching different levels. Free gossypol reduction was higher in the model system than the cottonseed meal, and higher in the homogenised processing than the static one: cottonseed meal in homogenised (56%) and static (25%); model system homogenised (98%) and static (80%). The obtained differences suggest a problem of gas penetration in the solid particles, the effect of unexposed surfaces due to contact areas, and the reaction with other organic molecules further than the target. Ozonation is a promising technique for gossypol degradation in cottonseed meal, but additional strategies are needed to optimise the ozonation process and evaluate toxicological aspects.

Efeito de diferentes concentrações de ingestão de gossipol livre sobre morfometria testicular e qualidade seminal de touros da raça Nelore / Effect of free gossypol intake in different levels on the testicular morphometry and seminal quality of Nelore bulls

A cotonicultura tem forte fator de impacto na economia nacional, e o estado de Mato Grosso se destaca por ser o maior produtor de algodão herbáceo e deter o maior rebanho bovino do país, condições essas que estimulam o uso do caroço, da torta e do farelo de algodão na alimentação animal. Considerando que o gossipol está presente nos subprodutos do algodão e que seus efeitos sobre a reprodução podem reduzir a fertilidade dos animais, objetivou-se, com essa pesquisa, avaliar o efeito da ingestão de dietas com diferentes teores de gossipol livre por dia sobre a morfometria testicular e a qualidade seminal de touros da raça Nelore. Foram utilizados 28 touros, distribuídos aleatoriamente em seis tratamentos: T0, 0 grama de gossipol livre/touro/dia ; T1, 1,08 grama de gossipol livre/touro/dia; T2, 2,07 gramas de gossipol livre/touro/dia; T3, 3,24 gramas de gossipol livre/touro/dia; T4, 3,82 gramas de gossipol livre/touro/dia e T5, 5,08 gramas de gossipol livre/touro/dia. Os animais de cada tratamento foram mantidos confinados em área média de 100m2, dotada de bebedouro, cochos para mistura mineral e para volumoso/concentrado. O consumo de 3,24 gramas de gossipol livre/touro/dia alterou a qualidade espermática e a morfometria testicular de touros.(AU)

The cotton industry has a strong impact factor in the Brazilian economy and the state of Mato Grosso stands out for being the largest upland-type cotton producer and also holds the largest cattle herd in the country, conditions that stimulate the use of cottonseed pie and cottonseed meal in animal feed. Whereas gossypol is present in cotton by-products and their effects on reproduction can reduce the animal fertility, this research evaluated the effect of diets with different free gossypol contents in the testicular morphometry and semen quality of Nelore bulls. 28 bulls were randomly distributed in six treatments: T0, receiving no free gossypol; T1, receiving 1.08g / bull / day; T2, receiving 2.07g / bull / day; T3, receiving 3.24g / bull / day; T4, receiving 3.82g / bull / day and T5, receiving 5.08g / bull / day of free gossypol respectively. The animals in each treatment were kept confined in an averaged area of 100m2, having fresh water, troughs for mineral mixture and roughage / concentrate. The consumption of 3.24g of free gossypol / bull / day altered the bulls sperm quality and testicular morphometry.(AU)

Efeito de diferentes concentrações de ingestão de gossipol livre sobre morfometria testicular e qualidade seminal de touros da raça Nelore / Effect of free gossypol intake in different levels on the testicular morphometry and seminal quality of Nelore bulls

A cotonicultura tem forte fator de impacto na economia nacional, e o estado de Mato Grosso se destaca por ser o maior produtor de algodão herbáceo e deter o maior rebanho bovino do país, condições essas que estimulam o uso do caroço, da torta e do farelo de algodão na alimentação animal. Considerando que o gossipol está presente nos subprodutos do algodão e que seus efeitos sobre a reprodução podem reduzir a fertilidade dos animais, objetivou-se, com essa pesquisa, avaliar o efeito da ingestão de dietas com diferentes teores de gossipol livre por dia sobre a morfometria testicular e a qualidade seminal de touros da raça Nelore. Foram utilizados 28 touros, distribuídos aleatoriamente em seis tratamentos: T0, 0 grama de gossipol livre/touro/dia ; T1, 1,08 grama de gossipol livre/touro/dia; T2, 2,07 gramas de gossipol livre/touro/dia; T3, 3,24 gramas de gossipol livre/touro/dia; T4, 3,82 gramas de gossipol livre/touro/dia e T5, 5,08 gramas de gossipol livre/touro/dia. Os animais de cada tratamento foram mantidos confinados em área média de 100m2, dotada de bebedouro, cochos para mistura mineral e para volumoso/concentrado. O consumo de 3,24 gramas de gossipol livre/touro/dia alterou a qualidade espermática e a morfometria testicular de touros.(AU)

The cotton industry has a strong impact factor in the Brazilian economy and the state of Mato Grosso stands out for being the largest upland-type cotton producer and also holds the largest cattle herd in the country, conditions that stimulate the use of cottonseed pie and cottonseed meal in animal feed. Whereas gossypol is present in cotton by-products and their effects on reproduction can reduce the animal fertility, this research evaluated the effect of diets with different free gossypol contents in the testicular morphometry and semen quality of Nelore bulls. 28 bulls were randomly distributed in six treatments: T0, receiving no free gossypol; T1, receiving 1.08g / bull / day; T2, receiving 2.07g / bull / day; T3, receiving 3.24g / bull / day; T4, receiving 3.82g / bull / day and T5, receiving 5.08g / bull / day of free gossypol respectively. The animals in each treatment were kept confined in an averaged area of 100m2, having fresh water, troughs for mineral mixture and roughage / concentrate. The consumption of 3.24g of free gossypol / bull / day altered the bulls sperm quality and testicular morphometry.(AU)

Development of an RP-UHPLC-PDA method for quantification of free gossypol in cottonseed cake and fungal-treated cottonseed cake.

Cottonseed cake biomass, which is a residue of oil extraction, is potentially appropriate for use as animal feed, given the high mineral, fibre and protein content. The presence of free gossypol, however, a toxic pigment in the glands of the cotton plant, limits use of this biomass for monogastric livestock. A promising method to detoxify cottonseed cake relies on fermentation by fungi, which can eliminate up to 100% of gossypol. In order to quantify trace levels of free gossypol in different cotton materials, including cottonseed cake treated with macrofungi, a simple and rapid chromatographic detection method was developed and validated. Under optimized conditions, extraction was performed using 70% acetone. The extract was then analysed by Ultra High-Performance Liquid Chromatography (UHPLC), with gradient elution on a C18 reverse phase column KINETEX® (100 x 2.10 mm, 2.6 µm). Methanol-0.1% TFA aqueous solution was employed as mobile phase and PDA detection conducted at 254 nm. The optimized method was validated by analysis of specificity, linearity and range, limit of detection, limit of quantification, precision and accuracy. Detection and quantification limits were observed at 0.2 and 0.5 µg/mL, respectively. With good reproducibility, with precision (RSD)<10% and recovery greater than 94%, the developed assay was appropriate for quantification of low quantities of free gossypol. The validated method was successfully applied to determine trace levels of free gossypol cottonseed treated with a macrofungus.

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.

Gossypol promotes the degeneration of chicken ovarian follicles in vitro

Gossypol, a phenolic compound produced by the pigment glands of cotton, is known to affect male reproduction. In females, exposure to gossypol has been associated with interference with the estrous cycle and embryo development. In laying hens fed cottonseed, gossypol was found to reduce egg production and weight and to cause discoloration of the egg yolk and/or albumen. It is probable that gossypol directly affects ovarian follicles, but this has not been confirmed experimentally yet. Thus, the aim of the present study was to determine if gossypol affected cultivated ovarian follicles of the chicken. Ovarian follicles of adult female chickens were cultivated with different concentrations of gossypol (0, 5, 10 and 20 g/mL) and classified according to the stage of development as primordial, transitional, primary, secondary or antral, and as viable or atretic. The percentages of viable and atretic follicles in the ovaries cultivated for 24 hours in the presence of gossypol were significantly different from those of the controls for all types of follicles except for secondary follicles, but in all types after cultivation for 7 days. The percentage of viable follicles was higher than that of atretic follicles in ovaries cultivated without gossypol. In contrast, ovaries cultivated with gossypol showed a predominance of atretic follicles. Gossypol increased the proportion of atretic follicles at all stages of development in cultivated chicken ovaries. Thus, gossypol may affect ovarian follicular viability and maturation, which might interfere with female fertility.

Gossypol promotes the degeneration of chicken ovarian follicles in vitro

Gossypol, a phenolic compound produced by the pigment glands of cotton, is known to affect male reproduction. In females, exposure to gossypol has been associated with interference with the estrous cycle and embryo development. In laying hens fed cottonseed, gossypol was found to reduce egg production and weight and to cause discoloration of the egg yolk and/or albumen. It is probable that gossypol directly affects ovarian follicles, but this has not been confirmed experimentally yet. Thus, the aim of the present study was to determine if gossypol affected cultivated ovarian follicles of the chicken. Ovarian follicles of adult female chickens were cultivated with different concentrations of gossypol (0, 5, 10 and 20 g/mL) and classified according to the stage of development as primordial, transitional, primary, secondary or antral, and as viable or atretic. The percentages of viable and atretic follicles in the ovaries cultivated for 24 hours in the presence of gossypol were significantly different from those of the controls for all types of follicles except for secondary follicles, but in all types after cultivation for 7 days. The percentage of viable follicles was higher than that of atretic follicles in ovaries cultivated without gossypol. In contrast, ovaries cultivated with gossypol showed a predominance of atretic follicles. Gossypol increased the proportion of atretic follicles at all stages of development in cultivated chicken ovaries. Thus, gossypol may affect ovarian follicular viability and maturation, which might interfere with female fertility.(AU)

Meat characteristics of cattle fed diets containing whole cottonseed / Características da carne de bovinos alimentados com dietas contendo caroço de algodão

Sensory and instrumental analyses evaluated the meat and fat characteristics of feedlot-finished steers fed a diet containing cottonseed. Global impression, texture, meat color, and fat color were assessed. Thirty Nellore bulls with an average age of 30 ± 6 months and initial body weight of 382.7 ± 28.4kg were kept in feedlot stalls and fed the following cottonseed levels: 0; 2.22 %; 4.44 %; 6.66 %; 8.88 %; 11.11 % of the dietary dry matter. The cottonseed used in this experiment had an average free gossypol content of 4.5g/kg of cottonseed. The overall impression of the samples, assessed by the triangle test for difference, did not differ for more than 62 % of the panelists (P>0.01). The shear strength of roasted meat varied from 6.00 to 6.54kg. According to texture profile analysis (TPA), the hardness, springiness, and chewiness of roast meat ranged from 24.15 to 28.01 N, 0.52 to 0.56, 8.42 to 11.01 N, respectively; of raw meat, 9.51 to 13.86 N, 0.26 to 0.29, and 1.38 to 1.81 N, respectively. The different treatments did not affect meat texture, meat color, or fat color (P>0.05). Meat color, luminosity, and red intensity ranged from 37.71 to 42.85, 20.68 to 25.25, and 6.74 to 8.61, respectively; fat color, luminosity, and yellow intensity ranged from 62.26 to 63.78, 11.13 to 11.62, and 10.53 to 10.86, respectively. Cottonseed intake of up to 1.13kg/animal/day, equivalent to a free gossypol intake of 5.05g/animal/day, in place of soybean meal and ground corn, did not significantly change the global sensory impression, texture, and color of the meat and fat.(AU)

Avaliou-se, por meio de análise sensorial e instrumental, o efeito da adição de caroço de algodão à dieta de bovinos confinados sobre as características da carne e da gordura. Foram avaliadas a impressão global, a textura, a cor da carne e a cor da gordura. Trinta touros da raça Nelore, com médias de idade e peso vivo inicial de 30 ± 6 meses e 382,7 ± 28,4kg, foram confinados e receberam dietas com os seguintes teores de caroço de algodão: 0; 2,22%; 4,44%; 6,66%; 8,88%; 11,11% na matéria seca da dieta. O caroço de algodão utilizado neste experimento apresentou conteúdo médio de 4,5g de gossipol livre/kg de caroço de algodão. A impressão global da carne, avaliada por meio de teste triangular de diferença, mostrou que mais de 62% dos provadores não perceberam diferença significativa (P>0,01) entre as amostras. A textura da carne assada avaliada por meio do teste de força de cisalhamento variou de 6,00 a 6,54kg. Na análise do perfil de textura (TPA) da carne assada, a dureza, a elasticidade e a mastigabilidade variaram, respectivamente, de 24,15 a 28,01N, de 0,52 a 0,56N e de 8,42 a 11,01N. Na TPA da carne crua, variaram, respectivamente, de 9,51 a 13,86N, de 0,26 a 0,29N e de 1,38 a 1,81N. Na avaliação da cor da carne, a luminosidade, a intensidade de vermelho e a intensidade de amarelo da cor da carne variaram, respectivamente, de 37,71 a 42,85, de 20,68 a 25,25 e de 6,74 a 8,61. E para a cor da gordura, variaram, respectivamente, de 62,26 a 63,78, de 11,13 a 11,62 e de 10,53 a 10,86. A textura, a cor da carne e a cor da gordura não apresentaram diferenças significativas (P>0,05) entre os diferentes tratamentos. O consumo de caroço de algodão em até 1,13kg/animal/dia, que resultou no consumo de 5,05g de gossipol livre/animal/dia, em substituição ao farelo de soja e ao grão de milho triturado, não causou alterações significativas nas características da carne quanto à impressão sensorial global, à textura, à cor da carne e à cor da gordura.(AU)

Meat characteristics of cattle fed diets containing whole cottonseed / Características da carne de bovinos alimentados com dietas contendo caroço de algodão

Sensory and instrumental analyses evaluated the meat and fat characteristics of feedlot-finished steers fed a diet containing cottonseed. Global impression, texture, meat color, and fat color were assessed. Thirty Nellore bulls with an average age of 30 ± 6 months and initial body weight of 382.7 ± 28.4kg were kept in feedlot stalls and fed the following cottonseed levels: 0; 2.22 %; 4.44 %; 6.66 %; 8.88 %; 11.11 % of the dietary dry matter. The cottonseed used in this experiment had an average free gossypol content of 4.5g/kg of cottonseed. The overall impression of the samples, assessed by the triangle test for difference, did not differ for more than 62 % of the panelists (P>0.01). The shear strength of roasted meat varied from 6.00 to 6.54kg. According to texture profile analysis (TPA), the hardness, springiness, and chewiness of roast meat ranged from 24.15 to 28.01 N, 0.52 to 0.56, 8.42 to 11.01 N, respectively; of raw meat, 9.51 to 13.86 N, 0.26 to 0.29, and 1.38 to 1.81 N, respectively. The different treatments did not affect meat texture, meat color, or fat color (P>0.05). Meat color, luminosity, and red intensity ranged from 37.71 to 42.85, 20.68 to 25.25, and 6.74 to 8.61, respectively; fat color, luminosity, and yellow intensity ranged from 62.26 to 63.78, 11.13 to 11.62, and 10.53 to 10.86, respectively. Cottonseed intake of up to 1.13kg/animal/day, equivalent to a free gossypol intake of 5.05g/animal/day, in place of soybean meal and ground corn, did not significantly change the global sensory impression, texture, and color of the meat and fat.(AU)

Avaliou-se, por meio de análise sensorial e instrumental, o efeito da adição de caroço de algodão à dieta de bovinos confinados sobre as características da carne e da gordura. Foram avaliadas a impressão global, a textura, a cor da carne e a cor da gordura. Trinta touros da raça Nelore, com médias de idade e peso vivo inicial de 30 ± 6 meses e 382,7 ± 28,4kg, foram confinados e receberam dietas com os seguintes teores de caroço de algodão: 0; 2,22%; 4,44%; 6,66%; 8,88%; 11,11% na matéria seca da dieta. O caroço de algodão utilizado neste experimento apresentou conteúdo médio de 4,5g de gossipol livre/kg de caroço de algodão. A impressão global da carne, avaliada por meio de teste triangular de diferença, mostrou que mais de 62% dos provadores não perceberam diferença significativa (P>0,01) entre as amostras. A textura da carne assada avaliada por meio do teste de força de cisalhamento variou de 6,00 a 6,54kg. Na análise do perfil de textura (TPA) da carne assada, a dureza, a elasticidade e a mastigabilidade variaram, respectivamente, de 24,15 a 28,01N, de 0,52 a 0,56N e de 8,42 a 11,01N. Na TPA da carne crua, variaram, respectivamente, de 9,51 a 13,86N, de 0,26 a 0,29N e de 1,38 a 1,81N. Na avaliação da cor da carne, a luminosidade, a intensidade de vermelho e a intensidade de amarelo da cor da carne variaram, respectivamente, de 37,71 a 42,85, de 20,68 a 25,25 e de 6,74 a 8,61. E para a cor da gordura, variaram, respectivamente, de 62,26 a 63,78, de 11,13 a 11,62 e de 10,53 a 10,86. A textura, a cor da carne e a cor da gordura não apresentaram diferenças significativas (P>0,05) entre os diferentes tratamentos. O consumo de caroço de algodão em até 1,13kg/animal/dia, que resultou no consumo de 5,05g de gossipol livre/animal/dia, em substituição ao farelo de soja e ao grão de milho triturado, não causou alterações significativas nas características da carne quanto à impressão sensorial global, à textura, à cor da carne e à cor da gordura.(AU)

Effects of gossypol from cottonseed cake on the blood profile in sheep.

Cottonseed cake contains gossypol, a potentially toxic compound that, when consumed by sheep, can affect reproduction, the immune system, and the liver. Changes in hematologic and serum biochemical parameters were monitored for 63 days in 12 Santa Inês ewes, six of which received ration containing 400 g kg(-1) of cottonseed cake. Blood samples were collected at the start of the experiment and weekly thereafter for hematologic assessment and determination of serum urea, creatinine, total protein, and albumin concentrations and for measurement of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and γ-glutamyl transferase activities. No clinical signs of toxicity were observed. Evaluation of the erythron showed that sheep consuming cottonseed cake had an increased packed cell volume (p < 0.05) and increased erythrocyte counts and hemoglobin concentrations (p < 0.05) in the leukogram and serum biochemistry panel. In conclusion, consumption of 400 g kg(-1) cottonseed cake by sheep for 63 days may induce changes in the erythron but no consistent changes in serum biochemical parameters, indicating no damage to the liver or kidneys.

Impact of feeding cottonseed coproducts on reproductive system of male sheep during peripubertal period

Cottonseed coproducts contain gossypol which can have specific effects upon the male reproductive system. We evaluated the reproductive development of male lambs close to puberty fed on three cotton coproducts. Twenty-four 5-month old male lambs received four diets: 20 % of dry matter intake of whole cottonseed (WCS), high oil cottonseed meal (CSC), cottonseed meal (CSM), and a control group (CTL) without any cottonseed coproducts. Free gossypol intake was 16.32, 6.98, 5.47 and 0 mg kg1 BW1 d1 for WCS, CSC, CSM and CTL, respectively. Every 15 days, the animals were weighted and serum and semen samples were collected. After 95 days, testis samples were collected for analysis under light and transmission electron microscopes. The CTL group had higher testosterone concentrations than CSC at the end of the trial and lower total sperm defects, higher mass movement and higher scores for seminiferous epithelium than other treatments. The WCS and CSC groups showed higher levels of segmental aplasia lesion in sperm than other diets, which showed that high levels of gossypol led to higher occurrence of this lesion. Cottonseed coproducts had a negative impact on the reproductive system of pubertal lambs regardless of gossypol concentration. Therefore, the use of cottonseed coproducts to feed lambs earmarked for reproduction is not safe.

Impact of feeding cottonseed coproducts on reproductive system of male sheep during peripubertal period

Cottonseed coproducts contain gossypol which can have specific effects upon the male reproductive system. We evaluated the reproductive development of male lambs close to puberty fed on three cotton coproducts. Twenty-four 5-month old male lambs received four diets: 20 % of dry matter intake of whole cottonseed (WCS), high oil cottonseed meal (CSC), cottonseed meal (CSM), and a control group (CTL) without any cottonseed coproducts. Free gossypol intake was 16.32, 6.98, 5.47 and 0 mg kg1 BW1 d1 for WCS, CSC, CSM and CTL, respectively. Every 15 days, the animals were weighted and serum and semen samples were collected. After 95 days, testis samples were collected for analysis under light and transmission electron microscopes. The CTL group had higher testosterone concentrations than CSC at the end of the trial and lower total sperm defects, higher mass movement and higher scores for seminiferous epithelium than other treatments. The WCS and CSC groups showed higher levels of segmental aplasia lesion in sperm than other diets, which showed that high levels of gossypol led to higher occurrence of this lesion. Cottonseed coproducts had a negative impact on the reproductive system of pubertal lambs regardless of gossypol concentration. Therefore, the use of cottonseed coproducts to feed lambs earmarked for reproduction is not safe.(AU)

Influence of gossypol acetic acid on the growth of human adenoid cystic carcinoma ACC-M cells and the expression of DNA methyltransferase 1.

We investigated the effects of gossypol acetic acid (GAA) on the proliferation, apoptosis, and expression of DNA methyltransferase 1 (DNMT1) mRNA in human adenoid cystic carcinoma (ACC-M) cells in vitro. The proliferation and apoptosis of ACC-M cells after treatment with different concentrations of GAA were detected using Cell Counting Kit-8 and flow cytometry, respectively. DNMT1 mRNA expression was measured by real-time fluorescence quantitative polymerase chain reaction. The growth of ACC-M cells was inhibited after treatment with GAA for 24, 48, and 72 h. The apoptotic rates of ACC-M cells after treatment with GAA for 72 h were higher than those of control cells (without treatment) (P < 0.05). DNMT1 mRNA expression in ACC-M after treatment with GAA for 72 h was lower than that in control cells (P < 0.05). GAA had inhibitory effects on the proliferation and induced apoptosis of human ACC-M cells, while GAA also reduced the expression level of DNMT1 mRNA in ACC-M cells.

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.

Mechanisms involved in reproductive damage caused by gossypol in rats and protective effects of vitamin E.

BACKGROUND: Gossypol is a chemical present in the seeds of cotton plants (Gossypium sp.) that reduces fertility in farm animals. Vitamin E is an antioxidant and may help to protect cells and tissues against the deleterious effects of free radicals. The aim of this study was to evaluate the mechanisms of reproductive toxicity of gossypol in rats and the protective effects of vitamin E. Forty Wistar rats were used, divided into four experimental groups (n = 10): DMSO/saline + corn oil; DMSO/saline + vitamin E; gossypol + corn oil; and gossypol + vitamin E. RESULTS: Fertility was significantly reduced in male rats treated with gossypol in that a significant decrease in epididymal sperm count was observed (P < 0.05) and the number of offspring was significantly reduced in females mated with them (P < 0.05). This dysfunction was prevented by vitamin E. Gossypol caused a significant increase in the activity of the enzymes glutathione peroxidase (P < 0.01) and glutathione reductase (P < 0.01), but vitamin E did not reduce the enzyme activities (P > 0.05). The levels of reduced glutathione and pyridine nucleotides in testis homogenate were significantly reduced by gossypol (P < 0.05 and P < 0.01, respectively) and this reduction was accompanied by increased levels of oxidized glutathione (P < 0.05). Vitamin E showed a preventive effect on the changes in the levels of these substances. Gossypol significantly increased the levels of malondialdehyde (P < 0.01), a lipid peroxidation indicator, whereas treatment with vitamin E inhibited the action of the gossypol. Vitamin E prevented a decrease in mitochondrial ATP induced by gossypol (P < 0.05). CONCLUSIONS: This study suggests that the reproductive dysfunction caused by gossypol may be related to oxidative stress and mitochondrial bioenergetic damage and that treatment with vitamin E can prevent the infertility caused by the toxin.

Small-molecule BH3 mimetic and pan-Bcl-2 inhibitor AT-101 enhances the antitumor efficacy of cisplatin through inhibition of APE1 repair and redox activity in non-small-cell lung cancer.

AT-101 is a BH3 mimetic and pan-Bcl-2 inhibitor that has shown potent anticancer activity in non-small-cell lung cancer (NSCLC) in murine models, but failed to show clinical efficacy when used in combination with docetaxel in NSCLC patients. Our recent study has demonstrated that AT-101 enhanced the antitumor effect of cisplatin (CDDP) in a murine model of NSCLC via inhibition of the interleukin-6/signal transducer and activator of transcription 3 (STAT3) pathway. This study explored the underlying mechanisms for the enhanced anticancer activity of CDDP by AT-101. Our results show that, when compared with monotherapy, AT-101 significantly enhanced the inhibitory effects of CDDP on proliferation and migration of A549 cells and on tube formation and migration in human umbilical vein endothelial cells. AT-101 promoted the proapoptotic activity of CDDP in A549 cells. AT-101 also enhanced the inhibitory effect of CDDP on DNA repair and redox activities of apurinic/apyrimidinic endonuclease 1 (APE1) in A549 cells. In tumor tissues from nude mice treated with AT-101 plus CDDP or monotherapy, the combination therapy resulted in greater inhibition of angiogenesis and tumor cell proliferation than the monotherapy. These results suggest that AT-101 can enhance the antitumor activity of CDDP in NSCLC via inhibition of APE1 DNA repair and redox activities and by angiogenesis and induction of apoptosis, but other mechanisms cannot be excluded. We are now conducting a Phase II trial to examine the clinical efficacy and safety profile of combined use of AT-101 plus CDDP in advanced NSCLC patients.

Mechanisms involved in reproductive damage caused by gossypol in rats and protective effects of vitamin E

BACKGROUND: Gossypol is a chemical present in the seeds of cotton plants (Gossypium sp.) that reduces fertility in farm animals. Vitamin E is an antioxidant and may help to protect cells and tissues against the deleterious effects of free radicals. The aim of this study was to evaluate the mechanisms of reproductive toxicity of gossypol in rats and the protective effects of vitamin E. Forty Wistar rats were used, divided into four experimental groups (n = 10): DMSO/ saline + corn oil; DMSO/saline + vitamin E; gossypol + corn oil; and gossypol + vitamin E. RESULTS: Fertility was significantly reduced in male rats treated with gossypol in that a significant decrease in epididy-mal sperm count was observed (P < 0.05) and the number of offspring was significantly reduced in females mated with them (P < 0.05). This dysfunction was prevented by vitamin E. Gossypol caused a significant increase in the activity of the enzymes glutathione peroxidase (P < 0.01) and glutathione reductase (P < 0.01), but vitamin E did not reduce the enzyme activities (P > 0.05). The levels of reduced glutathione and pyridine nucleotides in testis homogen-ate were significantly reduced by gossypol (P < 0.05 and P < 0.01, respectively) and this reduction was accompanied by increased levels of oxidized glutathione (P < 0.05). Vitamin E showed a preventive effect on the changes in the levels of these substances. Gossypol significantly increased the levels of malondialdehyde (P < 0.01), a lipid peroxida-tion indicator, whereas treatment with vitamin E inhibited the action of the gossypol. Vitamin E prevented a decrease in mitochondrial ATP induced by gossypol (P < 0.05). CONCLUSIONS: This study suggests that the reproductive dysfunction caused by gossypol may be related to oxidative stress and mitochondrial bioenergetic damage and that treatment with vitamin E can prevent the infertility caused by the toxin.

Gossypol promotes degeneration of ovarian follicles in rats.

The present study aimed to determine if gossypol interferes with ovarian follicles in rats. Twenty-four female Wistar rats were assigned to two equal groups: one control group and the other dosed with gossypol (25 mg/kg/day, subcutaneously) for 15 days. Ovarian follicles were histologically classified according to the stage of development and as normal or atretic. Gossypol treatment reduced the length of estrous with an increase in the duration of the diestrus phase. This compound was responsible for reduced serum levels of T4 and progesterone. Treatment with gossypol was responsible for a significant reduction in the number of normal ovarian follicles and a significant increase in the number of atretic follicles, both in all stages of development. Thus, treatment of rats with gossypol was responsible for reduction in the number of viable follicles and changes in hormone levels that resulted in interference of the estrous cycle.