Coping with gossypol
It has been suggested that bacterial infection stimulates epithelial renewal via reactive oxygen species (ROS) production . A similar stimulatory response may be triggered by low gossypol concentrations in the CBW gut, since a consequence of gossypol toxicity may involve the generation of superoxide free radicals damaging the epithelium . However, if present, such a proliferative response does not seem to be mediated by significant transcriptional changes in the midgut at the low gossypol concentration.
Gossypol, as a lipophilic compound, interacts with cellular membranes and forms bonds with the amine groups of proteins via Schiff's base formation; cross-linking of membrane proteins has been suggested to block cell-to-cell communication . Previously, it has been proposed that adaptive changes in lipid content can occur upon gossypol exposure [34, 35]. We speculate that this effect on genes involved in cell adhesion may represent a response to the cytotoxicity of gossypol at this concentration. A down-regulation of cell adhesion genes has also recently been observed as a consequence of gossypol exposure in mammalian cells . A 12-cadherin domain protein is expressed in the midgut of lepidopteran larvae where it is a binding target for Cry1A toxins from Bacillus thuringiensis (Bt) . It has recently been shown that the same cadherin is also expressed in the larval testis of the cotton specialist pink bollworm (Pectinophora
gossypiella) suggesting a role in sperm production . Mutations in this protein responsible for Bt toxin resistance reduce fertility somewhat and in turn Bt-resistant pink bollworm is more susceptible to gossypol . We observed down-regulation of the homologous 12-domain cadherin protein, not in the midgut where it abundantly expressed but in the rest of the body which includes the testis. In vertebrates, some cadherins are known to interact with compounds leading to signal transduction events such as the carcinoembryonic antigen-related cell adhesion molecules (CEACAMs); however insect homologs of the latter are unknown.
The mode of action of gossypol has been studied mostly in relation to its toxic or therapeutic effects on mammalian cells. These studies show that certain low doses of gossypol can have antitumor, antiviral and antiparasitic activities mostly due to the fact that gossypol inhibits key proteins belonging to different classes of enzymes such as oxidoreductases, hydrolases and transferases . Gossypol is not considered a bactericidal compound . Therefore, we do not consider that the hormetic effect observed at T5 is a consequence of gossypol acting as a toxin to bacteria possibly present in the artificial diet.
A stimulatory effect on energy metabolism due to gossypol has been observed in cultured mouse cells. Relatively low doses of gossypol were added to the cell medium along with glucose resulting in an increase of lactate production. Additionally, the inhibition of oxygen consumption produced by 5 mM glucose was reversed when gossypol was added to the medium . The phenomenon was explained by the ability of gossypol to uncouple oxidative phosphorylation, leading to a decrease of mitochondrial production of adenosine triphosphate (ATP). Thus, glycolysis and the production of lactate through the Embden-Meyerhof pathway were stimulated as an attempt to maintain the required ATP levels in the cell. The authors concluded that the response towards gossypol may depend on the cell's glycolytic and mitochondrial oxidative phosphorylation capacity and on its ability to maintain acid-base homeostasis . A differential susceptibility and hormetic effect in oxygen production has also been observed between somatic and germ rat cells exposed to different concentrations of gossypol . Therefore, it is possible that the gut epithelium can be maintained at low gossypol doses and even stimulated by the uncoupling of mitochondria. This may explain the fact that we do not see the biological process of glycolysis enriched by differentially regulated genes since the response to gossypol may depend on subtle adjustments in metabolism which take place as acclimatization at the protein level. However, glycolysis-related genes were affected in the RB at both gossypol concentrations examined.
It has previously been described that β-fructofuranosidases of Bombyx mori are resistant to inhibition by mulberry sugar mimic alkaloids which inhibit α-glucosidases, and therefore represent an adaptation to the mulberry host in this specialist moth . If α-glucosidases are also inhibited by gossypol, the up-regulation of β-fructofuranosidase genes by the high dose of gossypol in H. armigera might be explained by a similar compensatory mechanism. Interestingly, we found that in H. armigera, the regulation of at least one β-fructofuranosidase gene (GH32FruA-1), in response to gossypol, is tissue and dose dependent. The same seems to be true for glucose dehydrogenase genes, which are up-regulated at gossypol T7 only in the RB.
Cross-linking amino acids and proteins, phenolic compounds cause oxidative damage to the midgut cells of insects . Thus, the up-regulation of peroxisome-related genes may be part of an antioxidant response to the ROS generated by gossypol activity at such high concentration. Consistently, phenoloxidase inhibitor and oxidase peroxidase genes are respectively down-and up- regulated in both tissues, indicating that homeostasis has been compromised possibly due to major tissue wounding caused by oxidative stress.
The proteasome apparatus, composed of a proteolytic core and two regulatory particles, degrades a variety of cellular proteins involved in many essential functions, such as signal transduction pathways, stress signaling, inflammatory responses, and apoptosis. If increased proteolysis of cellular targets inactivated by binding to gossypol were required, we would expect an up-regulation of this system. The fact that the proteasome is instead down-regulated by gossypol suggests that the response of CBW to this compound may be similar to the response by an annelid towards fluoranthene, a polycyclic aromatic hydrocarbon, known to disrupt biological membranes due to its lipophilicity , characteristics shared with the gossypol molecule. This down-regulation of the ubiquitin-26S proteasome pathway is interpreted as a mechanism to reduce the proteolytic turnover of the aryl hydrocarbon receptor (Ahr) which is considered a mediator in the expression of genes involved in detoxification. In mammals, Ahr regulates cellular responses to certain polycyclic aromatic hydrocarbon toxins similar to fluoranthene and gossypol . Several pathways also mediating gene expression (i.e. mTOR, JAK-STAT, phototransduction and Notch) were affected, representing potential signaling pathways involved in the response towards gossypol which deserve further attention.
Since gossypol is a defensive chemical encountered in some but not all of the hostplants of this generalist herbivore, some transcriptional responses are expected to be directed towards its detoxification, and these can be compared with previous metabolic studies. The metabolic fate of gossypol in Heliothis virescens larvae has been examined by means of 14C-labelling the compound and adding it to an artificial diet. 25% was found to be metabolized by conjugation with six sugar molecules per mole of gossypol and excreted in the frass, whereas glutathione conjugates were not detected . This is consistent with the up-regulation of 10 UDP-glycosyltransferases but only one glutathione transferase in the midgut. If endogenous α-glucosidases were capable of hydrolyzing these glucose conjugates, their down-regulation would be favored in favor of up-regulation of β-fructofuranosidases which can still digest carbohydrates. About 33% of the labelled gossypol was found in the larval tissues, mostly in the fat body and the rest of the compound excreted as free gossypol or bound to components of the frass. About 10% of labelled gossypol was recovered as carbon dioxide but the mechanism has not been elucidated in this species. A similar study done with rats revealed that decarbonylation of gossypol is an important detoxification pathway; labeled 14CO2 appeared in expired air 1 hour after feeding of 14C-gossypol . However, in swine decarbonylation may not be the main detoxification pathway since the main products found were glucuronides, sulphates and unconjugated metabolites . These contrasts between species made the authors consider whether the degree of decarbonylation of gossypol indicates the susceptibility towards the allelochemical (i.e. rats, more tolerant to this phenolic compound, retain less of it in their tissues and detoxify it mostly by decarbonylation) .
A zinc-iron transporter was upregulated in the gut at T7 (Figure 5), which is consistent with gossypol acting as a sink for iron in the midgut lumen. Ferric ions have been shown to precipitate gossypol, while ferrous ions produce a soluble chelate which is precipitated by calcium . There is evidence that at least some of the gossypol decarbonylation observed in the rat digestive tract occurs by an auto-oxidation process catalyzed by ferrous ions through a free-radical chain mechanism similar to that proposed for benzaldehyde decarbonylation .
The oxidoreductase molecular function in the G and RB is one of the most significantly enriched at T7 and within this category P450 enzymes are prominent. In order to metabolize gossypol to gossic acid several oxidation steps are required , for which the P450 s are candidates. Consistent with previous results by others , CYP6AE14 is up-regulated in the G and the RB at T7. However, CYP6AE14 is slightly down-regulated at T5 in both tissues. Moreover, 13 additional P450 s are upregulated and two downregulated in the gut at the highest gossypol concentration. When CYP6AE14 was silenced in H. armigera by feeding dsRNA or transgenic cotton transformed with a construct expressing dsRNA, larval growth was greatly reduced in the presence of gossypol [21, 22], however there are no heterologous expression studies providing information about CYP6AE14 substrate specificity and its direct role in gossypol metabolism. Racemic gossypol promotes the formation of superoxide free-radicals when incubated with rat liver microsomes . There is evidence indicating that the damage caused by these superoxide free radicals is due to the interaction of gossypol with the iron of the P450 enzyme . It might be worth testing whether CYP6AE14 is more susceptible to this effect which may provide an alternative explanation for its up-regulation.
The strong upregulation of several carboxylesterases in the midgut poses somewhat of an enigma, since neither gossypol nor any of its known metabolites are suitable substrates for esterases. In some cases, overexpression of esterases confers resistance to organophosphorus insecticides, due to sequestration rather than metabolism (reviewed in ). If some of these esterases similarly trap gossypol, keeping it from more sensitive cellular targets, this could be an effective but expensive mechanism of tolerance. Alternatively, gossypol may interact with a regulatory molecule such as Ahr which controls a suite of different detoxicative genes, only some of which are directly involved in the detoxification of a given xenobiotic.