Effect of alkali treatment on physiological activity of cotton condensed tannin.

Cotton dusts contain condensed tannins and endotoxins, which are suspected of contributing to the development of acute and chronic biological responses in some cotton textile mill workers. Condensed tannin extracted from cotton dust was coated on to cellulose powder, and the tannin coated powder was treated with an alkali solvent system previously developed to reduce the endotoxin content and pulmonary toxicity of cotton dust. Physiological activities of the dusts and powders were compared by assaying the production of the arachidonic acid metabolites prostaglandin F2 alpha (PGF2 alpha), thromboxane A2 (TxA2) (the precursor to thromboxane B2 (TxB2], leukotriene C4 (LTC4), and prostaglandin E2 (PGE2) by guinea pig pulmonary cells obtained by lung lavage. Cotton dust stimulated the pulmonary cells to produce a total of 29 pg metabolites per 10(6) cells. Production of metabolites by cells stimulated with tannin coated cellulose powder was reduced to 8.3 pg/10(6) cells. Alkali treatment of the tannin coated cellulose powder resulted in a further decrease in its ability to stimulate the cells, producing 3.5 pg metabolites per 10(6) cells. The ability of the dusts and powders to stimulate production of metabolites of arachidonic acid by pulmonary cells from guinea pigs was highly correlated with tannin content of the materials, but not with endotoxin content as measured by the Limulus amoebocyte lysate (LAL) assay.

Inhibition of alveolar macrophage spreading and phagocytosis by cotton bract tannin. A potential mechanism in the pathogenesis of byssinosis.

One of the major host-defense functions of alveolar macrophages is the phagocytosis and clearance of inhaled particles deposited in the lower airways and alveolar spaces. Recent studies have indicated that the condensed tannins present in cotton mill dust stimulate the secretion of neutrophil chemotactic factor and arachidonic acid from resident rabbit alveolar macrophages and that these responses may contribute to the acute pulmonary inflammatory reaction associated with byssinosis. To characterize further the effect of tannin on macrophage function, the ability of tannin to modulate alveolar macrophage spreading and phagocytosis in vitro was examined. Tannin caused a dose-dependent inhibition of alveolar macrophage spreading with nearly complete inhibition occurring at concentrations of 12.5 micrograms/ml. This inhibitory effect of tannin was not reversed with removal of tannin. Furthermore addition of tannin to previously spread macrophages actively caused the macrophages to round up. Examination of the structure of alveolar macrophages exposed to tannin by scanning and transmission electron microscopy revealed blebs on the surface of the cells and the loss of most of the cellular organelle structure, as compared to control macrophages. Tannin also modulated the ability of the alveolar macrophages to phagocytize unopsonized latex microspheres. The effect of tannin was biphasic. At the lowest concentration examined (3 micrograms/ml), tannin significantly enhanced phagocytosis of the latex microspheres. However, as the concentration was increased, phagocytosis decreased almost exponentially until at 50 micrograms/ml phagocytosis was significantly inhibited compared to control macrophages. These data indicate that tannin present in inhaled cotton mill dust could significantly decrease the ability of resident alveolar macrophages to phagocytize and thereby clear inhaled dust particles. This inhibitory effect would increase the time that particles remain exposed in the lower airway and alveolar spaces and thereby increase the time that potentially toxic compounds in the dust have to exert their biologic effect. This inhibition of macrophage function may therefore contribute to the pathogenesis of byssinosis.

Integration of enzyme-linked immunosorbent assay with conventional chromatographic procedures for quantitation of aflatoxin in individual cotton bolls, seeds, and seed sections.

Integration of an enzyme-linked immunosorbent assay (ELISA) with conventional chromatographic methods proved the versatility of ELISA as a research tool and allowed for rapid assessment of aflaxtoxin in individual cottonseeds, parts of cottonseed, and composite samples of seeds taken from individual cotton bolls. Aqueous acetone was substituted for methanol in the extraction procedure prescribed by ELISA. The substitution allowed the use of a common extract for all analytical methods. An aliquot of the extract was used to screen samples by ELISA. Negative samples were identified, and toxin levels between 1 and 70 ng/g were quantitated by ELISA. Samples with toxin levels beyond the upper limit of detection by ELISA were then subjected to more time-consuming conventional cleanup prior to quantitation by liquid chromatography (LC) or thin-layer chromatography (TLC). Toxin levels detected by LC or TLC ranged from 100 to 845,000 ng/g sample. The screen by ELISA detected large numbers of toxin-negative cotton bolls or individual seeds in minimum analysis time. The combination of techniques verified the presence of seed with no toxin adjacent to toxin-containing seed in the same lock. Toxin-negative portions of individual seed with high toxin in another portion were identified for the first time. Integration of techniques provided needed information on distribution patterns of aflatoxin in cotton so that preventive measures can be developed.

Comparison of two immunochemical methods with thin-layer chromatographic methods for determination of aflatoxins.

Three different methods were compared for the determination of total flatoxins in corn and peanuts naturally contaminated with aflatoxins and in corn, peanuts, cottonseed, peanut butter, and poultry feed spiked with aflatoxins B1, B2, and G1. The 3 methods were an enzyme-linked immunosorbent assay (ELISA) screening test; a monoclonal antibody-affinity column-solid-phase separation method; and the AOAC official thin-layer chromatography (TLC) methods for all except poultry feed, for which Shannon's TLC method for mixed feed was used. The ELISA test is designed to provide only positive results for total aflatoxins at greater than or equal to 20 ng/g or negative results at less than 20 ng/g. The affinity column separation is coupled with either bromination solution fluorometry to estimate total aflatoxins or liquid chromatography (LC) to quantitate individual aflatoxins. Fluorodensitometry was used to determine aflatoxins in commodities analyzed by the TLC methods. The LC and TLC results were in good agreement for all the analyses. The results for the affinity column using bromination solution fluorometry were similar except those for cottonseed, which were about 60% higher. The ELISA screening method correctly identified naturally contaminated corn and peanut positive samples. No false positives were found for controls. The correct response for spiked corn, raw peanuts, peanut butter, and cottonseed at greater than or equal to 20 ng aflatoxins/g was about 90%. The correct response for spiked poultry feed at greater than or equal to 20 ng aflatoxins/g was about 50%.

Enzyme-linked immunosorbent assay of aflatoxins B1, B2, and G1 in corn, cottonseed, peanuts, peanut butter, and poultry feed: collaborative study.

A direct competitive enzyme-linked immunosorbent assay (ELISA) screening method for aflatoxins at 20 ng/g was studied by 12 collaborators. Test samples of peanut butter were extracted by blending with methanol-water-hexane (55 + 45 + 100) and heating the test extracts on a steam bath; test samples of the other commodities were extracted by blending with methanol-water (80 + 20). All test extracts were filtered and the filtrates were diluted with buffer to a final methanol concentration of less than 30%. Each diluted filtrate was applied to a cup containing a filter with immobilized polyclonal antibodies specific to aflatoxins B1, B2, and G1. Aflatoxin B1-peroxidase conjugate was added, the cup was washed with water, and a mixture of hydrogen peroxide and tetramethylbenzidine was added. The test sample was judged to contain greater than or equal to 20 ng aflatoxins/g when, after exactly 1 min, no color was observed on the filter; when a blue or gray color developed, the test sample was judged to contain less than 20 ng aflatoxins/g. All collaborators correctly identified naturally contaminated corn and raw peanut positive test samples. No false positives were found for controls containing less than 2 ng aflatoxins/g. The correct responses for positive test samples spiked at levels of 10, 20, and greater than or equal to 30 ng aflatoxins/g (the ratio of B1:B2:G1 was 10:1:3) were 52, 86, and 96%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Laundry parameters as factors in lowering methyl parathion residue in cotton/polyester fabrics.

Cotton/polyester fabrics were contaminated with a 1.25% solution of methyl parathion (MeP) emulsifiable concentrate formulation, then laboratory laundered. The laundering variables were detergent type, concentration, water volume, and mechanical agitation. Specimens were unfinished (UN) and soil repellent finished (SR) fabrics. Heavy duty liquid and phosphate-built powdered detergents were equally effective when used in combination with a prewash product. A negative linear relationship between detergent concentration and MeP residue remaining after laundering was established. An interaction between detergent concentration and fabric finish was observed. Soil repellent finished fabrics required detergent concentrations above the recommended amount for more efficient soil removal. A negative linear relationship between water volume and after-laundering residue was observed. Water volume played a more significant role in pesticide removal than agitation during laundering.

Relationship of byssinosis to the generation of oxygen radicals by bract tissues of cotton plants.

Byssinosis is a hazardous respiratory disorder of workers in natural fiber processing industries and, in the case of cotton, is caused by exposure to respirable dust generated from leafy trash associated with raw fibers. To understand the chemical characteristics of involucral trash components that might contribute to bysinosis, we examined the human airway constricting activity and oxygen radical generating activity of dry, frost-killed cotton bracts. In response to inhalation of aerosolized bract extracts, the expiratory flow rates of human volunteers at 40% of vital capacity during partial forced expiration decreased by 3 to 32%. These values enabled us to identify two potentially byssinogenically active bract specimens, a specimen virtually inactive, and a fourth intermediately so. Using spin trapping techniques of electron spin resonance spectrometry, we found that all specimens catalyzed the generation of hydroxyl (preponderantly) and superoxide radicals from hydrogen peroxide. However, the weakest constrictor was the most potent catalyst, and vice versa. This was consistent with transition metal content of the specimens; the most potent catalyst also contained the largest amounts of those metals, suggesting a Fenton-type reaction mechanisms. Other possibilities for the inverse relationship of airway constricting (byssinogenic) activity with oxygen radical generation are discussed. We also found that neither aflatoxin nor endotoxin, contingent contaminants of bracts, catalyzed oxygen radical production from hydrogen peroxide.

Effects of tannins from different sources on airway epithelial and platelet function.

The uniqueness of tannin isolated from aqueous extracts of cotton bracts (CBE) on the electrophysiological and ion transport properties of the canine tracheal epithelium and on 5-hydroxytryptamine (5-HT) release from platelets was investigated. The effect of CBE from Deltapine 41 cotton picked before (green, containing high-concentration, high-molecular-weight tannin) and after (brown, containing low-concentration, low-molecular-weight tannin) senescence and Darjeeling tea extracts and tannin isolated from Darjeeling tea were compared to the effects of Acala SJ-2 CBE and Acala SJ-2 tannin. Green Deltapine 41 CBE was similar to Acala SJ-2 CBE in decreasing short-circuit current (Isc) and net chloride secretion and in producing a dose-response release of 5-HT from platelets. Both Green and Brown Deltapine 41 CBE altered the paracellular pathway, but were less potent than Acala SJ-2 CBE. Brown Deltapine 41 CBE stimulated chloride transport in the airway epithelium and did not produce 5-HT release from human platelets. Darjeeling tea and tea tannin increased Isc and net chloride secretion and caused 5-HT release from platelets only at high concentrations. These studies demonstrate that all tannins are not alike; they suggest that molecular weight and concentration may be important for tannin activity and that the tannin in cotton bracts extract may be unique to cotton in its effect on the airway epithelium and on platelets.

In situ hybridization of biotinylated DNA probes to cotton meiotic chromosomes.

A modified procedure for in situ hybridization of biotinylated probes to meiotic chromosomes of cotton has been developed with high retention of squashed cells on slides, preservation of acid-fixed chromosome morphology, exceptionally low levels of background precipitate at nonspecific hybridization sites and improved photomicrographic recording. Salient features of the techniques include pretreatment of slides before squashing, cold storage of squash preparations, and use of interference filters for distinguishing precipitate from chromatin. A cloned 18S/28S ribosomal DNA fragment from soybean was biotinylated via nick-translation and hybridized to microsporocyte meiotic chromosomes of cotton (Gossypium hirsutum L. and G. hirsutum L. X G. barbadense L.). Enzymatically formed precipitate from streptavidin-bound peroxidase marked the in situ hybridization. In situ hybridization of biotinylated probes to cotton meiotic chromosomes adds the specificity and resoltion of in situ hybridization to the chromosomal and genomic perspectives provided by meiotic cytogenetic analyses. Molecular cytogenetic analyses of meiotic cells offer certain inherent analytical advantages over analyses of somatic cells, e.g., in terms of mapping, and for studying fundamental biological and genetic problems, particularly for organisms that are not amenable to somatic karyotypic analysis.

Bronchial responsiveness after inhalation of cotton bract extract.

This study examined nonspecific airway responsiveness to methacholine (MC) after inhalation of cotton bract extract (CBE). In a randomized double-blind, crossover trial, 13 healthy volunteers underwent an MC inhalation challenge test prior to inhalation of CBE and normal saline solution (NSS) aerosol sham as well as 2, 8, 24, and 168 h (7 days) later. The response parameter was the concentration of MC required to induce a 25% decrement in the maximal expiratory flow at 40% of the vital capacity below total lung capacity on the partial expiratory flow-volume curve (PC25MEF40%(P]. Five of 13 subjects demonstrated a ventilatory response to CBE with a 20% or larger decrement in the MEF40%(P); no subject demonstrated such change with NSS. For the group, the maximal decrement in MEF40%(P) was to 76.5 +/- 20.3% of baseline (mean +/- SD), occurring approximately 60 to 90 min after provocation, whereas the largest decrement after normal saline was to 88 +/- 10.6% of baseline, occurring immediately after inhalation. Changes in airway responsiveness to MC were transient. For example, the PC25MEF40%(P) for the group (mean +/- SD) was 51.3 +/- 41.1 mg/ml at baseline and 25.8 +/- 30.3 and 52.2 +/- 57.3 mg/ml at 2 and 8 h. After a pre-sham baseline of 50.4 +/- 43.2 mg/ml, PC25MEF40%(P) was 57.6 +/- 83.8 and 153.8 +/- 148 mg/ml at 2 and 8 h. Repeated measures ANOVA on these acute, same-day changes (i.e., 2 and 8 h after provocation) demonstrated a statistically significant effect of CBE on airway responsiveness (p = 0.048). These data demonstrate that inhalation of CBE, in addition to bronchospasm, causes a transient increase in airway responsiveness.

Effects of alkaline hydrogen peroxide treatment of cotton and wheat straw on cellulose crystallinity and on composition and site and extent of disappearance of wheat straw cell wall phenolics and monosaccharides by sheep.

Effects of alkaline hydrogen peroxide (AHP) treatment on cellulose crystallinity and cell wall phenolic monomer and monosaccharide composition were measured using cotton and wheat straw (WS). Two WS treatments were used in this study, Type I WS, for which pH is not regulated during AHP treatment, and Type II WS, for which pH is regulated at 11.5 +/- .2 during AHP treatment. Wheat straw had a lower degree of cellulose crystallinity than cotton, but no differences occurred between treated and untreated substrates. Alkali-labile and nitrobenzene-extractable phenolic monomer concentrations were generally lower for Type I and Type II WS compared with untreated WS. Concentrations of glucose were higher and xylose and arabinose lower in Types I and II WS than in untreated WS. Disappearance of alkali-labile phenolic monomers and cell wall monosaccharides by wethers fed diets containing Type I (Exp. 1) or Type II (Exp. 2) AHP-treated WS were determined. Apparent digestibility of glucose and xylose before the duodenum, and of glucose, xylose and arabinose in the total tract, was greatest (P less than .05) when sheep were fed AHP-treated WS diets in both experiments. In Exp. 2, disappearance of alkali-labile phenolic monomers was greatest (P less than .05) before the duodenum and in the total tract when sheep were fed AHP-treated WS diets. Treatment of WS with AHP modified cell wall composition and increased cell wall monosaccharide digestion by sheep.

Plant constituents of cotton dust and lung effects after inhalation.

Experiments were performed to assess pulmonary reactions after inhalation of cotton dusts with different levels of tannins, terpenoid aldehydes and bacterial endotoxins. Guinea-pigs were exposed to cotton dust. Free lung cells were obtained by lavage 24 h later. A dose-response relationship was found between the number of neutrophils and the amount of endotoxin in the dust. No influence of terpenoid aldehyde or tannin levels could be detected. Cotton mill workers were exposed to dust from glanded and glandless cottons in an experimental cardroom. The average decreases in forced expiratory volume in one second (FEV1) over the workday after carding the two cottons were the same, although levels of dust, tannin or terpenoid aldehydes were different. The level of airborne endotoxin was, however, equal. The results support observations from other studies on the importance of endotoxin for the development of the acute reactions observed after cotton dust exposure.

Isolation of (-)-gossypol from natural plant.

(-)-Gossypol has been isolated from cottonseed of G. barbadense L. Three Gossypium species were studied for the isolation. The study has also revealed a correlation between the optical activity of gossypol isolated and the species of cottonseed used. Gossypol isolated from the species G. barbadense is always found to be levorotatory caused by an excess of (-)-gossypol, whereas that from the species G. hirsutum is always dextrorotatory caused by an excess of (+)-gossypol. Results of most samples (cultivars) showed that optical purity of gossypol isolated from G. barbadense varied from 10-25%, while that from G. hirsutum varied from 10-20%. This is the first report of (-)-gossypol occurring naturally.

PIP: Both (+ and -)-gossypol and (+)-gossypol have been found to be naturally occurring, while no plant producing (-)-gossypol has to date been located. This study isolated (-)-gossypol from the cottonseed of Gossypium barbadense L. Cottonseed from G hirsutum L and G arboreum L was also investigated. The (-)-gossypol content was consistently higher than the (+) gossypol content for all 16 sample of G barbadense studied, while the (+)-gossypol content was higher in the 10 G hirsutum L samples and the 1 G arboreum L sample. The rotation property of gossypol was mainly correlated with the species of the cottonseed; the area where the cottonseed was cultivated had no effect. Gossypol isolated from G barbadense was always levorotatory as a result of the excess of (-)-gossypol, whereas that from the other 2 species studied was always dextrorotatory due to the excess of (+)-gossypol. The optical purity of gossypol isolated from G barbadense ranged from 10-25%, and that from G hirsutum varied from 10-20%.

3-Hydroxymyristic acid as a measure of endotoxin in cotton lint and dust.

A series of samples consisting of purified cellulose, purified cellulose spiked with endotoxin, and cotton lint and dust samples from the Human Panel Acute Exposure Studies at Clemson, South Carolina, were extracted with pyrogen-free water and with phenol-water. Phenacyl esters of the dried, hydrolyzed extract were prepared and chromatographed on a high performance liquid chromatograph. A peak assigned to the phenacyl ester of 3-hydroxymyristic acid appears in the chromatograms of extracts of celluloses that have been spiked with endotoxins and not in those of unspiked celluloses. This peak also appears in the extracts of cotton lint from samples that cause the greatest decrement in lung function in the Clemson human exposure studies. The area of this peak increases with increasing amounts of endotoxin and may serve as a measure of endotoxin concentration in cotton lint and dust, at least when fairly high levels of endotoxin (0.50 micrograms or greater) are present. The effect of extraction method on the determined amount of endotoxin is discussed.

Phosalone applied to cotton (Gossypium hirsutum L.): its deposition and persistence on foliages, soils and final residues in seeds.

Phosalone, O,O-diethyl-S-(6-chloro-1,3-benzoxazol-2(3H)-onyl)methyl phosphorodithioate, was field-applied by ground equipment to cotton (Gossypium hirsutum L.) at the rates of 1050 and 2100 g a.i./ha, respectively, to determine its dissipation on leaves and soils and the residues in seeds at harvest. The insecticide concentrations on cotton leaves and soils were measured periodically for 14 days following its application. It was found that the half lives of the insecticide on cotton leaves at the dosages of 1050 and 2100 g a.i./ha were 6.8 and 6.3 days, respectively. And the half lives on soils for the 2 dosages were 7 and 5.8 days, respectively. The residues remaining in soils at harvest time were 0.072 and 0.121 mg/kg 14 days post-application and the residues in cotton seeds were relatively low (less than 0.02-0.12 mg/kg).

Studies on Egyptian cottonseeds: fatty acid-composition and protein patterns.

Fourteen Egyptian cottonseed varieties were analysed to study some properties of their lipids and proteins. Lipid contents of the kernels ranged from 29.0 to 35.1%. The mean value of the iodine number was 110.9. The tested varieties showed little or no differences regarding to their fatty acid content. A slight higher proportion of linoleic acid was recorded for the triglycerides whereas the polar lipids components contained a lower amount of this fatty acids compared to that found in the total lipids. Protein contents in the examined varieties ranged from 32.3 to 37.9%. The amount of water-soluble protein ranged from 12.8 to 23.0% of the total protein. An almost complete recovery (94.5-100%) of the total protein was yielded when the extraction was performed with 0.02 N NaOH instead of water. The electrophoretic patterns of the water-soluble proteins gave only two bands, having a molecular weight between 14,000 and 25,000 dalton. A clear differentiation between the varieties was noticed when the alkaline soluble protein extracts were subjected to electrophoresis. Accordingly, the examined varieties were classified into five groups each of them having a similar spectrum.

Gas chromatographic determination of mecarbam and its metabolites mecarboxon, diethoate, and diethoxon in cottonseeds.

A gas chromatographic method is described for determining residues of mecarbam and 3 of its metabolites, mecarboxon, diethoate, and diethoxon, in cottonseeds. For mecarbam analysis, following Soxhlet extraction with chloroform (after blending), the oily extract is partitioned with propylene carbonate and cleaned up on a silica gel column. Metabolites are extracted by the same method, followed by cleanup of mecarboxon on a silica gel column or diethoxon on an alumina column; cleanup of diethoate can be performed on either column. All 4 compounds are determined using a flame photometric detector equipped with a phosphorus filter. Average recoveries for cottonseed samples fortified with 0.03-1.0 ppm mecarbam ranged from 80 to 88%. Average recoveries were 81-88% for mecarboxon and 90-92% for diethoate (alumina column) and diethoxon from samples fortified with 0.05-1.0 ppm. Average recovery of diethoate from samples cleaned up on the silica gel column were 84-88% in the range of 0.05-0.2 ppm. Values obtained for mecarbam residues in field-treated samples are also presented.

Capillary gas chromatography of cotton dusts associated with human pulmonary function studies.

The effects of geographical area of growth and cotton variety on pulmonary activity have been evaluated through human volunteer exposure studies conducted by NIOSH and USDA at the Cotton Quality Research Station, Clemson, S.C. These studies demonstrate that carding California cottons releases dust with less human pulmonary activity than dust released from the corresponding Mississippi cottons. Dust released from Texas cottons grown in 1982 was considerably less active than the dust from Texas cottons grown in 1983. Distinct differences in the chemical compositions of the Mississippi, Texas, and California cardroom dusts were found. Aqueous extracts of the dusts were freeze-dried and then derivatized. Capillary gas chromatography revealed that the California dust extracts and the 1982 Texas dust extract were characterized by relatively higher levels of malic acid, whereas the Mississippi dust extracts and the 1983 Texas dust extracts were characterized by relatively higher mannitol levels.