Cotton Bract Research Articles

Endothelial Cell Cytotoxicity of Cotton Bracts Tannin and Aqueous Cotton Bracts Extract: Tannin Is the Predominant Cytotoxin Present in Aqueous Cotton Bracts Extract

Using an in vitro cytotoxicity assay based on the release of 51Cr from cultured porcine thoracic aortic and pulmonary arterial endothelial cells, we have demonstrated that cotton bracts tannin is a potent endothelial cell cytotoxin. It produces dose-dependent lethal injury to both types of endothelial cells with the aortic cells being somewhat more sensitive to tannin-mediated injury than the pulmonary arterial cells. Cytotoxic injury to the cells was biphasic. During the first 3 hr of exposure to tannin, no lethal injury was detected. However, during this period, profound changes in morphology were observed suggesting sublethal injury to the cells preceded the ultimate toxic damage. Comparison of the cytotoxicity dose curves for aqueous bracts extracts with those for tannin demonstrated that tannin was the major cytotoxin present in bracts.

Pulmonary function analysis in the rabbit following bronchochallenge to causative agents and mediators of the acute byssinotic response.

New Zealand White rabbits were acutely bronchochallenged for 5 min to ascertain airway responsiveness with six potential byssinogenic agents and mediators: 0.1 g/mL cotton dust extract (CDE), 0.1 g/mL cotton bract extract (CBE), 1 mg/mL endotoxin, 1 mg/mL n-formyl methionyl peptide (n-fMet), 10 mg/mL 5-hydroxytryptamine (5-HT), and 1 mg/mL prostaglandin F2 alpha (PGF2 alpha). Methacholine (MC), 10 mg/mL, was used as a control bronchoconstrictor. Clinically objective criteria were established using increases in resistance values compared to those obtained with saline controls. Animals were classified as: mild responders (Mi) = 125-149%; moderate responders (Mo) = 150-199%; or severe responders (S) = greater than 200%. Three of five (2Mo, 1S) rabbits showed increased pulmonary resistance to CDE bronchochallenge, 3/5 (1Mi, 1Mo, 1S) to CBE, 1/5 (Mo) to purified endotoxin, 4/5 (1Mo, 3S) to n-fMet, 3/5 (1Mi, 1Mo, 1S) to 5-HT, and 2/5 (1Mo, 1S) to PGF2 alpha. All five rabbits (1Mo, 4S) responded to MC bronchochallenge. Rabbits responded minimally to saline, the common solvent of all test agents; however, when challenged with methacholine, a known bronchoconstrictor, rabbits showed significant overt symptoms of acute respiratory distress with immediate and substantial increases in resistance over saline controls. CDE, CBE, and n-fMet inhalation challenge resulted in a majority or all animals showing increased resistance. 5-HT contained in CDE and CBE, exhibited similar resistance increases; however, endotoxin, also found in cotton dust, showed little airway reactivity. The rabbit is useful for characterizing changes in pulmonary function parameters seen in the acute byssinotic reaction. This study has demonstrated that bronchochallenge in the rabbit with potential byssinogenic agents (CDE, CBE, endotoxin, and n-fMet) and mediators (5-HT and PGF2 alpha) result in measurable changes in airway function, particularly increased resistance. Since bronchoconstriction is the major clinical manifestation of the acute byssinotic reaction in man and animals, it is likely that bronchoconstriction observed in cotton mill workers may be in part or totally the result of inherent dust constrictor substances or secondarily released mediators.

Biochemical and immunologic characterization of cotton bract extract and its effect on in vitro cyclic AMP production : II. Green Bract

Cotton bract has been proposed as a probable byssinogenic agent. Mill workers are exposed to bract which has weathered in the field, but it is not known whether biologic effects of bract are due to intrinsic plant compound(s) or to contamination occurring during field weathering. Exposure of mononuclear leukocytes (MNL) to green bract extract (GBE) lowered basal cyclic AMP levels. Concomitant exposure of MNL to GBE plus agonist altered MNL cyclic AMP levels in a dose-dependent fashion. Differences between immunologic and pharmacologic test results of GBE and field-dried bract extract were mainly quantitative, suggesting the bioactive agent(s) in cotton bract is intrinsic to the plant.

Biochemical and immunologic characterization of cotton bract extract and its effect on in Vitro cyclic AMP production : I. Field-dried bract

Field-dried cotton bract, a contaminant of cotton dust, has been implicated in byssinosis pathogenesis. The results from this study demonstrated that a standard bract extract (FDBE) could be prepared. FDBE was antigenic in rabbits; however, precipitating antibodies were not detectable in the serum of cotton textile mill workers. Although exposure of mononuclear leukocytes (MNL) to FDBE did not alter intracellular cyclic AMP levels, it did induce changes in the cyclic AMP response of MNL to isoproterenol and prostaglandin E1. These changes were FDBE dose-dependent. The possible relationship of these findings to pathogenesis of byssinosis is discussed.

Effects of a cotton bract extract on guinea pig isolated airway smooth muscle.

The effects of a water extract of cotton bracts (CBE) on guinea pig isolated trachealis smooth muscle was studied. The ability of CBE to evoke tension responses, to modify tissue reactivity to excitatory and inhibitory agents, and to modify electric field stimulation-induced neurogenic responses was evaluated. CBE caused contraction in low concentrations, which were not mediated by histamine H1-, muscarinic, or 5-hydroxytryptamine (5-HT) receptors, and caused relaxation in high concentrations. In the presence of CBE, the maximum contractile response to 5-HT and the sensitivity to KCl were reduced. The maximum relaxation responses and sensitivities to adenosine and ATP were increased by CBE. In contrast, contractile responses to histamine and methacholine and relaxation responses to isoproterenol were unaffected. Neurogenic cholinergic excitatory responses and neurogenic adrenergic responses did not appear to be affected by CBE. However, the relaxant effect of nonadrenergic inhibitory nerve stimulation was increased in the presence of CBE. These findings differ from previous ones observed in our laboratory for dog isolated trachealis smooth muscle preparations, which indicates that the effects of CBE in isolated airways is species-dependent. Virtually all of the contractile activity of CBE was dialyzable (molecular weight less than 14,000) and retained in ashed samples. Inorganic constituents may contribute to the spasmogenic activity of CBE.

Interactions of a water extract of cotton bract with dog isolated airway smooth muscle.

The inhalation of cotton dust by cotton-mill workers causes byssinosis, a disease characterized by chest tightness and other symptoms indicative of bronchoconstriction. In the present study the dog isolated trachealis smooth muscle preparation was employed to examine the ability of a crude, water extract of cotton bracts (CBE) to (1) cause contractile responses mediated by receptors, (2) modify responses of the tissue to a number of excitatory and inhibitory agents, and (3) alter responsiveness of the preparations to electrical field stimulation mediated by intrinsic cholinergic, excitatory nerves and adrenergic, inhibitory nerves. CBE evoked contraction of the tissues by an action that did not involve muscarinic, histamine, or 5-hydroxytryptamine receptors. The maximum tissue responses to exogenous histamine, 5-hydroxytryptamine, and isoproterenol were enhanced significantly in the presence of CBE, while those to KCl and methacholine were unaffected. The sensitivities (EC50 values) of the tissues to these agents were not, however, altered by CBE. Contractions induced by cholinergic nerve stimulation were increased in the presence of CBE, via a mechanism that appears, at present, to result from a prejunctional facilitation of acetycholine release. Relaxations to adrenergic nerve stimulation were not affected by CBE. Results suggest that, in addition to its direct, contractile activity, CBE can alter indirectly the responsiveness of the tissue to a number of endogenous mediator substances. Intrinsic, cholinergic excitatory nerves may be a target for the acute action of CBE as well.

Effect of Area of Growth on Condensed Tannins in Cotton Bracts

Bracts of cotton plants ( Gossypium hirsutum) of three varieties grown in three states were extracted with 70% acetone. The condensed tannins in the extracts and in extracted and unextracted bracts were hydrolyzed in butanolic HCl, and the types and levels of anthocyanidins produced were compared by TLC and spectroscopy. The area of growth had more of an effect than did the variety on levels of anthocyanidins obtained.

Protection by plasma proteins against condensed tannin-mediated platelet activation

Condensed tannins isolated from cotton bracts are potent platelet agonists. They promote the secretion of 5-hydroxytryptamine from washed bovine platelets with an EC 50 of 35 μg/ml. However, when platelet-rich plasma was used in lieu of washed platelets, the potency of tannin was decreased over 50-fold. Reconstitution experiments demonstrated that some component in the plasma was responsible for the diminished potency of tannin. Fractionation of platelet-poor plasma suggested that albumin may be the inhibitory plasma component. Confirmation of this hypothesis was obtained from the finding that purified serum albumin at physiologic concentrations inhibited the tannin-mediated release of platelet 5-HT by more than 70%. These data suggest that any activation of platelets which may occur in vivo by the tannins present in inhaled cotton dust would require higher tannin concentrations than would have been predicted from our previous studies using washed platelets.

Skin testing with an aqueous extract of cotton bract

Airway challenge with cotton bract extract (CBE) causes reversible bronchospasm in most volunteers never before exposed to CBE or the textile industry. Lung function abnormalities develop slowly after inhalation with a maximum effect reached within 2 hours and lung function slowly improving thereafter. When CBE was injected intradermally in three naive subjects with negligible airway response to CBE, we observed an initial wheal-and-flare reaction within minutes followed by erythema and subsequent induration that persisted for 24 to 48 hours. Microscopic examination of skin biopsy specimens obtained during the course of these reactions revealed edema in the early phase followed by perivascular infiltration of a mixed cellular nature in the subsequent stages of the reaction. Degranulation of mast cells was noted throughout the course of the reaction. These findings indirectly suggest that a nonspecific inflammatory reaction initiated by mast cell-derived mediators and sustained by the presence of infiltrating cells may be responsible for some of the airway effects observed in byssinosis.

Byssinosis: Thromboxane Release From Human Platelets by Cotton Dust and Bract Extracts

Extracts of cotton dust and bract induced in vitro release of arachidonic acid metabolite thromboxane A2 (TxA2), a potent bronchoconstricting agent, from human platelets. TxA2 release, determined by radioimmunoassay of the stabile metabolite TxB2, was both time- and dose-dependent. Cotton green bract extract was significantly more potent in releasing TxA2 from human platelets than cotton dust extract or cotton brown bract (field-dried bract) extract, while brown bract extract was least active, which suggests that the etiological agent(s) is of plant origin and not microbial in nature. TxA2 release was shown to be enzyme-dependent by inhibition of the reaction by indomethacin (cyclooxygenase inhibitor) and 7-(1-imidazolyl) heptanoic acid (thromboxane synthetase inhibitor). This study is the first to show in vitro platelet TxA2 release by cotton extracts and may provide an explanation for increased concentrations of TxB2, a metabolite of TxA2, in the bronchoalveolar spaces of rabbits exposed to aerosolized cotton dust extract. Platelet aggregation and production of TxA2 may prove to be an important mechanism in the pathogenesis of bronchoconstriction in byssinosis.

Quantitative Determination of Indole-3 Carboxyaldehyde in Aqueous Extracts of Cotton Bracts by HPLC

A reverse phase, high performance liquid chromatbgraphy procedure for the quantification of indole-3 carboxyaldehyde in aqueous extracts of cotton bract is described. The amount of indole-3 carboxyaldehyde that could be extracted by water was 14.3 and 14.1 ppm in frost-killed (brown) and air dried green cotton bract, respectively.

Airway Reactivity and Cotton Bract-Induced Bronchial Obstruction

Most seemingly healthy persons challenged with an aerosol of cotton bract extract develop some degree of bronchospasm. The role of nonspecific reactivity of the airways in this reaction to cotton bract extract is undefined. We examined the relationship between airway responses to cotton bract extract and to methacholine, as well as between airway responses to cotton bract extract and to a bronchodilator. Twenty-two healthy subjects were screened for sensitivity to inhaled cotton bract extract. Pulmonary function was measured using partial expiratory flow-volume curves on which flow at 60 percent of the control vital capacity below total lung capacity was measured (MEF40%[P]) following ten minutes of inhalation of cotton bract extract. In the group screened, 12 were found to be responders to the extract, with drops in MEF40%(P) of 20 percent or more, and ten were found to be nonresponders. On separate days, we measured the responses of our subjects' airways to inhaled metaproterenol and methacholine. The mean threshold dose for methacholine in the responding group was 26.8 mg/ml, as compared to 55.6 mg/ml for the nonresponders (t = 2.52; p less than 0.05). Furthermore, the mean percent increase in MEF40%(P) following inhalation of metaproterenol was 41 percent in responders and 24 percent in nonresponders (t = 2.19; p less than 0.05). We conclude that some responders to cotton bract extract exhibit greater reactivity of the airways than nonresponders.

Effect of cotton bracts extract on canine tracheal epithelium and shunt pathway.

The effects of increasing concentrations (5 to 100 microliters) of an aqueous extract of cotton bracts (CBE) on the electrophysiologic properties and paracellular pathway of the canine trachea mounted in Ussing chambers were examined. The CBE produced a 50% decrease in short-circuit current and a dose-dependent decrease in transepithelial potential difference and tissue resistance when added to the mucosal bathing solution. The decrease in tissue resistance after exposure to 25 and 100 microliters was followed by an increase in mannitol flux, suggesting an effect on the paracellular pathway. No significant effect on mannitol flux was seen after 5 microliters CBE, when results were compared with control tissues from the same dog. The effects of 5 microliters CBE but not higher concentrations were reversible. Submucosal CBE had no effect on the electrophysiologic properties of the canine trachea or on mannitol flux. We conclude that in the canine tracheal epithelium, CBE inhibits active ion transport, alters the paracellular pathway, and acts only from the luminal surface.

HPLC Separation of Cotton Bract Extract

Abstract An HPLC method has been developed to separate cotton bract washwater into thirty components using two columns in series.

Characterisation of textile dust extracts: II. Bronchoconstriction in man.

Aqueous extracts of cotton bracts induce a concentration dependent decrease in flow rates on partial expiratory flow volume curves. It has been shown that the active substance(s) is stable to moderate heat and to acid or alkaline conditions. Results of dialysis and gel filtration suggest that the active component(s) has a molecular size of about 1000 daltons. The component(s) is not absorbed on to ion exchange resins but is readily removed from aqueous extract by activated charcoal. The bronchoconstrictor agent cannot be steam distilled or extracted by ether. It is readily soluble in water; a single extraction of bracts removes all biologically active material. It is proposed that healthy subjects, responsive to cotton bracts extract, can be used qualitatively to fractionate and quantitatively to assay the active substance(s) in this material.

Characterisation of textile dust extracts: I. Histamine release in vitro.

Cotton, flax, hemp, and cotton bracts extracts did not release histamine from mouse, rat, guinea pig, horse, cow, and monkey lung. Neither rat peritoneal mast cells nor mouse mastocytoma cells released histamine when incubated with textile dust extracts. Compound 48/80 caused a considerable release of histamine from all these tissues and the extracts released histamine from pig and human lung tissues. Whereas dusts that cause bronchospasm in man released histamine--for instance, cotton--those that are inactive, such as pericarps, did not. Histamine release was not quantitatively related to the concentration of extract used. Cotton bracts extract released histamine from pig lung tissue whereas in the same preparations methyl piperonylate was inactive. The active releasing agent was highly water soluble but could not be steam distilled from, nor extracted by, ether from bracts extract. These physicochemical properties are not characteristic of methyl piperonylate. There was no correlation between the induction of histidine decarboxylase and the histamine releasing capacity of the extracts. We conclude that pig lung is a useful qualitative assay tissue for the further characterisation of the histamine releasing agent(s) in textile dust extracts.

Cotton Dust Characterization: A Comparison of Sonically Separated Fine Dust (<20 μm) with Respirable (Vertical Elutriator) Dusts

Based on comparison of properties of the inorganic composition of various cotton dusts, this paper demonstrates that cotton dusts <20 μm in diameter separated from card room condenser trash material are very similar chemically to dusts collected in that same environment by a vertical elutriator cotton dust sampler. A method to estimate the fraction of cotton dust from sources such as cotton leaf and bract, humidifier water solids, soils, and background ambient air is given. Data from dusts obtained from the North Carolina State model card room and dusts reported in the literature are compared. In a study of 24 bales of cotton processed in the Model Card Room, approximately 26% of the elutriator dust was from humidifier water solids, less than 15% from ambient air and soils, and the remainder mostly from cotton bract and leaf.

Bioasssays of smooth muscle contracting agents in cotton mill dust and bract extracts: Arachidonic acid metabolites as possible mediators of the acute byssinotic reaction

Byssinosis is an occupational respiratory disease contracted by cotton mill workers who inhale cotton mill dust. The acute byssinotic reaction is characterized by a drop in the 1-sec forced expiratory volume (FEV 1.0) on Monday following a weekend's absence from work. This physiological reaction is the result of the contraction of bronchial smooth muscle, which causes narrowing of small airways. An isolated tissue bath technique was used to assay smooth muscle contractions induced by cotton dust extract (CDE) and cotton bract extract (CBE). CBE-induced contractions of rat stomach smooth muscle were blocked (82%) by 100 ng/ml methysergide (blocks 5-hydroxytryptamine (5HT) 100%); CDE was not significantly blocked by methysergide (13%). CDE-induced contractions were blocked (100%) by 25 μg/ml indomethacin and 100 μg/ml salicylic acid (blocks prostaglandin F 2α (PGF 2α) 100%). The portion of CBE contracting ability not blocked by methysergide was blocked totally by indomethacin. Blocking agents ineffective against CDE or CBE include atropine (acetylcholine blocker), pyrilamine maleate and diphenhydramine (histamine blockers), imidazole and 7-(1-imidazolyl)heptanoic acid (7IHA) (thromboxane blockers), and carboxypeptidase B 2 (kinin blocker). The data suggest that cotton bract contains 5HT, which is responsible for a majority of the CBE-induced contraction and a minority of the CDE-induced contraction. The data also suggest that cotton dust and bract contain a substance which causes the release of PGF 2α which in turn causes the contraction of smooth muscle. This substance is responsible for a majority of the CDE-induced and a minority of the CBE-induced contractions. Radioimmunoassay (RIA) confirmed PGF 2α release from rat fundal smooth muscle when exposed to CDE and CBE. Increased synthesis and release of arachidonic acid metabolites might be a major mechanism in the bronchoconstriction observed in the acute byssinotic reaction.

Associations Between Bracts and Several Agronomic Traits in Cotton1

Cotton (Gossypium hirsutum L.) bracts, as part of the trash in machine‐harvested seed cotton, have been implicated in the brown lung disease, byssinosis, in mill workers. As part of two separate investigations into the genetic nature of bract surface area (cm2 of bract area), genotypic and phenotypic correlations were calculated to determine linkages or associations with several agronomic traits. These field studies were conducted at Alexandria and Baton Rouge, La. on typic udifluvents and aquic fragiudaif soils, respectively. Significant positive associations in both studies between bract surface area and boll weight suggested little progress would be made in reducing the bract relative to the boll if only the bract surface area was considered. A better measure of heritable changes in bract surface area relative to boll weight may be the ratio of bract surface area/lint weight per boll. A low ratio of bract surface area/lint weight per boll would denote a low bract trash potential and may denote a low byssinosis potential. The ratio bract surface area/lint weight per boll appeared to be positively associated (genotypically) with 50% span length, and negatively associated with fiber micronaire and lint percent. These associations would suggest that parents with high lint percent and parents with small bracts should be selected. The potential problem with fiber length and micronaire should be considered in breeding cottons for low bract trash potential.

Quantitative Determination of Scopoletin and Lacinilene C 7-Methyl Ether in Aqueous Extracts of Frost-Killed Cotton Leaf and Bract by HPLC

A high-performance liquid chromatography procedure for the separation and quan tification of scopoletin and lacinilene C 7-methyl ether in aqueous extracts of cotton bract and leaf was developed. A procedure involving an aqueous extraction followed by an ethanol extraction is described. The amounts of scopoletin present in frost- killed bract and leaf which could be extracted with water were 22.6 ppm and 21.6 ppm, respectively. The amounts of lacinilene C 7-methyl ether present in, and water extractable from, frost-killed cotton bract and leaf were 17.8 ppm and 17.6 ppm, respectively. Samples of cotton bract and leaf picked before a frost were also examined. The amounts of lacinilene C 7-methyl ether present in cotton bract and leaf appear to be dependent on the physiological state of the plant. The amount of scopoletin present in the samples picked before a frost could not be determined due to insufficient resolution in the vicinity of the scopoletin peak in the HPLC chromatograms.