Abstract
As a widely grown economic crop, cotton is the major oil and protein resource for human and livestock. But the highly toxic of gossypol in cottonseed severely restricts its effective utilization, consequently creating huge resource waste. Previous studies have shown the male germline stem cells were the most vulnerable cells in gossypol damages, but the mechanism was still unclear. We found gossypol induced cell viability decline resulted from apoptosis. And the increase of Caspase-9 activity in gossypol treatment hinted the mitochondrial apoptosis. So the mitochondrial dysfunction was confirmed by the decreased mitochondrial membrane potential and ATP concentration. We found the higher intracellular H2O2 level did not accompany with the O2·- associated increase in gossypol-treated, which indicated that gossypol obstructed the intracellular reactive oxygen species (ROS) elimination. Manipulated gossypol-induced H2O2 level by H2O2 and α-lipoic acid, we demonstrated that the mitochondrial dysfunction resulted from the excessive intracellular H2O2. Treated with Apogossypolone (ApoG2), an aldehyde group removed derivative of gossypol, the GSH/GSSG ratio and H2O2 did not decrease. ApoG2 also did not cause the mitochondrial apoptosis. So the aldehyde group is key factor in gossypol cytotoxicity. We respectively detected the NAD+/NADH ratio, SIRT1 activity, the relative protein level and apoptosis. Comparing with the specific inhibitors groups, the data illustrated that gossypol induced apoptosis through SIRT1-P53-PUMA pathway. This study helped to overcome barriers of gossypol cytotoxicity, which is crucial in feed and food use of cottonseed. This also provides a reference for the gossypol derivatives using in male contraception and anticancer.
Highlights
As one of the world’s major economic crops, global cotton acreage is basically stable at 34 million hectares, corresponding a huge cotton by-product output in recent years [1]
We found the cell viabilities and numbers decrease was proportional to the increased of gossypol concentration, and the results showed that it started occurrence the statistical difference in 5 μM (Figure 1A and 1B)
We focused on analyzing the mechanism of gossypol reproductive toxicity
Summary
As one of the world’s major economic crops, global cotton acreage is basically stable at 34 million hectares, corresponding a huge cotton by-product output in recent years [1]. China’s cotton byproduct output was 3127 tons including 31% cottonseed in 2014. The highly toxic of (±)-gossypol (gossypol) in cottonseed severely restricts its effective use as food and fodder, creating a huge waste of resources [3,4,5]. As a natural product isolated from the cotton (Gossypium) seeds [7], gossypol is a yellow phenolic aldehyde that promotes several toxic effects in vertebrates but provides the cotton plant with resistance to pests [8]. The cottonseed may contain concentrations greater than 14,000 mg/kg total gossypol and 7,000 mg/kg free gossypol [9, 10]
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