Safety assessment of dicamba mono-oxygenases that confer dicamba tolerance to various crops.

Dicamba tolerant (DT) soybean, cotton and maize were developed through constitutive expression of dicamba mono-oxygenase (DMO) in chloroplasts. DMO expressed in three DT crops exhibit 91.6-97.1% amino acid sequence identity to wild type DMO. All DMO forms maintain the characteristics of Rieske oxygenases that have a history of safe use. Additionally, they are all functionally similar in vivo since the three DT crops are all tolerant to dicamba treatment. None of these DMO sequences were found to have similarity to any known allergens or toxins. Herein, to further understand the safety of these DMO variants, a weight of evidence approach was employed. Each purified DMO protein was found to be completely deactivated in vitro by heating at temperatures 55 °C and above, and all were completely digested within 30 s or 5 min by pepsin and pancreatin, respectively. Mice orally dosed with each of these DMO proteins showed no adverse effects as evidenced by analysis of body weight gain, food consumption and clinical observations. Therefore, the weight of evidence from all these protein safety studies support the conclusion that the various forms of DMO proteins introduced into DT soybean, cotton and maize are safe for food and feed consumption, and the small amino acid sequence differences outside the active site of DMO do not raise any additional safety concerns.

Cotton plants expressing a hemipteran-active Bacillus thuringiensis crystal protein impact the development and survival of Lygus hesperus (Hemiptera: Miridae) nymphs.

The plant bugs Lygus hesperus Knight (Hemiptera: Miridae) and L. lineolaris (Palisot de Beauvois) have emerged as economic pests of cotton in the United States. These hemipteran species are refractory to the insect control traits found in genetically modified commercial varieties of cotton. In this article, we report the isolation and characterization of a 35 kDa crystal protein from Bacillus thuringiensis, designated TIC807, which causes reduced mass gain and mortality of L. hesperus and L. lineolaris nymphs when presented in an artificial diet feeding assay. Cotton plants expressing the TIC807 protein were observed to impact the survival and development of L. hesperus nymphs in a concentration-dependent manner. These results, demonstrating in planta activity of a Lygus insecticidal protein, represent an important milestone in the development of cotton varieties protected from Lygus feeding damage.

Increased frequency of pink bollworm resistance to Bt toxin Cry1Ac in China.

Transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt) kill some key insect pests, but evolution of resistance by pests can reduce their efficacy. The main approach for delaying pest adaptation to Bt crops uses non-Bt host plants as "refuges" to increase survival of susceptible pests. To delay evolution of pest resistance to transgenic cotton producing Bt toxin Cry1Ac, the United States and some other countries have required refuges of non-Bt cotton, while farmers in China have relied on "natural" refuges of non-Bt host plants other than cotton. The "natural" refuge strategy focuses on cotton bollworm (Helicoverpa armigera), the primary target of Bt cotton in China that attacks many crops, but it does not apply to another major pest, pink bollworm (Pectinophora gossypiella), which feeds almost entirely on cotton in China. Here we report data showing field-evolved resistance to Cry1Ac by pink bollworm in the Yangtze River Valley of China. Laboratory bioassay data from 51 field-derived strains show that the susceptibility to Cry1Ac was significantly lower during 2008 to 2010 than 2005 to 2007. The percentage of field populations yielding one or more survivors at a diagnostic concentration of Cry1Ac increased from 0% in 2005-2007 to 56% in 2008-2010. However, the median survival at the diagnostic concentration was only 1.6% from 2008 to 2010 and failure of Bt cotton to control pink bollworm has not been reported in China. The early detection of resistance reported here may promote proactive countermeasures, such as a switch to transgenic cotton producing toxins distinct from Cry1A toxins, increased planting of non-Bt cotton, and integration of other management tactics together with Bt cotton.

Ultra-structure and histochemistry of digestive cells of Podisus nigrispinus (Hemiptera: Pentatomidae) fed with prey reared on bt-cotton.

The toxic effects of Bt proteins from the body of the prey/host to predators and parasitoids have been investigated as a route of impact on non-target insects of Bt crops. This research aimed to analyze ultrastructural changes and histochemistry in digestive cells of the middle region of the midgut of Podisus nigrispinus fed, since its second instar, with Spodoptera frugiperda reared on Bt cotton variety Acala90 B, which expresses the toxin Cry 1Ac, and its non-Bt isoline Acala 90. Fragments of the midgut of P. nigrispinus were analyzed by electron microscopy. For the histochemical analysis, Bromophenol Blue, Periodic Acid Schiff von Kossa, Alcian Blue pH 2.5 and Sudan Black were used. The Cry1Ac toxin of Bt cotton ingested by S. frugiperda promotes a disorganization in the perimicrovillar matrix of P. nigrispinus (third trophic level), thus generating ultrastructural changes in the digestive cells, as elongation of microvilli, presence of spherocrystals and granules of different electron densities, in addition to altering the distribution pattern of glycogen, lipids and calcium of these cells in the median region of the midgut. Thus, we conclude that species moderately susceptible to Cry1Ac toxin, such as S. frugiperda, can acquire this toxin and expose it to P. nigrispinus, which can interfere with your ability to predation.

Efecto ambiental de agroquímicos y maquinaria agrícola en cultivos transgénicos y convencionales de algodón / Environmental effects of agrochemicals and agricultural machinery in transgenic and conventional crops cotton

La producción de alimentos se basa en el uso de diferentes tecnologías agrícolas, que pueden derivar en conflictos entre medioambiente y agricultura. Es significativo estudiar el impacto ambiental de las nuevas tecnologías aplicadas a la agricultura, la más importante de las cuales es la transgénesis. Este trabajo se realizó en la zona algodonera del municipio del Espinal, departamento del Tolima, para la cosecha de algodón del primer semestre de 2009, usando las metodologías de Brookes y Barfoot (2006) y Kovach y colaboradores (1992). Se estudió el efecto ambiental de la aplicación de agroquímicos y el uso de maquinaria agrícola en cultivos de algodón transgénico y convencional. Se recogió información mediante encuestas en veinte fincas productoras de algodón. El análisis de las encuestas se realizó de forma descriptiva, determinando diferencias de tipo cuantitativo y cualitativo para los predios que utilizan la tecnología convencional o la tecnología transgénicas (doble gen, Bt/RR), para luego realizar una correlación con el “Enviromental Index Quotient” (EIQ). No se encontraron diferencias entre el EIQ de campo de las dos tecnologías, aunque la tecnología transgénica tiene ventajas ambientales en el control de algunas plagas de lepidópteros. En relación con el uso de maquinaria agrícola, se encontró que la tecnología convencional genera menor liberación de CO2, gas de efecto invernadero. La metodología de Brookes y Barfoot puede adaptarse para estudios comparativos de tecnologías agrícolas en países tropicales.

Food production is based on the use of various agricultural technologies, which can lead to conflicts between environment and agriculture. It is important to study the environmental impact of new technologies applied to agriculture, the most important of which is transgenesis. This work was carried out in the cotton belt of the town of Espinal, Tolima Department for the cotton crop in the first half of 2009, through methodologies Brookes & Barfoot (2006) and Kovach et al (1992). We studied the environmental impact of pesticide application and use of agricultural machinery for cultivation of transgenic and conventional cotton. Information was collected through surveys of 20 farms producing cotton. The analysis of the survey was conducted descriptively, by determining differences in quantitative and qualitative for the sites that use conventional technology, and transgenic (Bt gene and double RR / RR), and then make a correlation with the Environmental Index Quotient (EIQ). No differences were found between the fields EIQ the two technologies, although transgenic technology has environmental advantages in the control of some lepidopteran pests. In connection with the use of agricultural machinery, was found to conventional technology generates less release of CO2, greenhouse gas. The Brookes and Barfoot methodology could be adapted in comparative studies of agricultural technologies in tropical countries.

The oral toxicity of the transgenic Bt+CpTI cotton pollen to honeybees (Apis mellifera).

Transgenic insect-resistant cotton has been planted in China in a large scale and may have adverse impacts on honeybees. Pollens from the transgenic Cry1Ac+CpTI cotton Zhong-41 and the parental cotton Zhong-23 were collected from the field and their impacts on adult worker bees were assessed. Experimental results showed that Zhong-41 pollen had no acute oral toxic effect on worker bees. No significant differences were observed in the superoxide dismutase activity or in the longevity of worker bees fed with diets containing the two cotton pollens. The main reasons for the outcome may be the low expression level of the transgenic proteins Cry1Ac and CpTI in the pollen of Zhong-41 as well as the substantial equivalence in the amounts of gross protein and soluble saccharides for the two cotton pollens. The implications of these results are discussed and further work to be carried out is put forward.

TNF polymorphisms modify endotoxin exposure-associated longitudinal lung function decline.

OBJECTIVES: Endotoxin exposure induces airway inflammation, hyper-responsiveness and higher expression of tumour necrosis factor (TNF). This study was conducted to investigate whether TNF polymorphisms modify the effect of endotoxin exposure on chronic declines in lung function. METHODS: Associations between TNF and LTA polymorphisms, endotoxin exposure and lung function were analysed in 263 cotton workers and 230 silk workers as a reference group, who were prospectively followed for 20 years. Multiple linear regression models were used to assess the association, with adjustment for smoking and other covariates. RESULTS: Endotoxin exposure was associated with faster lung function decline among genotypes associated with higher TNF expression levels, with estimates of annual FEV1 change in relation to endotoxin exposure of -2.9 ml and -6.8 ml in the G/G and G/A+AA genotypes, respectively, for the TNF polymorphism; and -2.0 ml, -4.0 ml and -3.6 ml in A/A, A/G and G/G genotypes, respectively, for the LTA polymorphism. When joint effects of endotoxin exposure and smoking were considered, the effect modification of TNF and LTA polymorphisms was prominent in never smokers. CONCLUSIONS: TNF and LTA polymorphisms may modify the association between occupational endotoxin exposure and longitudinal lung function decline, which was more clearly observed in never smokers.

Calcified reticulate rind sign: a characteristic feature of gossypiboma on computed tomography.

We herein report a gossypiboma resulting from a retained surgical swab, which had been left in peritoneum for 20 years after appendectomy. CT revealed a cystic mass with a calcified reticulate rind. Subsequent surgery and pathological examination showed a gossypiboma. A simple experiment, using a barium-soaked surgical swab demonstrating similar CT appearance, supported our postulation that calcium deposition on the reticulated fibers of a surgical swab could generate such a characteristic "calcified reticulate rind" sign. We believe that identification of this CT sign facilitates the diagnosis of gossypibomas.

Endotoxin exposure and respiratory symptoms in the cotton textile industry.

One hundred fourteen male employees of a cotton spinning mill in western Germany participated in a cross-sectional study, the purpose of which was to clarify the dose effect of endotoxin exposure on respiratory symptoms. Airborne endotoxin exposures were classified as low (< or = 100 endotoxin units [EU]/m3), medium (> 100-450 EU/m3), or high (> 450 EU/m3), on the basis of endotoxin activity in the Limulus amoebocyte lysate assay. Age- and smoking-adjusted odds ratios (ORs) and confidence intervals (CIs) were estimated. The dose-response relationship between current endotoxin exposure and prevalence of wheezing (medium: OR = 2.15, 95% CI = 0.48-9.62; high: OR = 5.49, 95% CI = 1.17- 25.81) and cough (medium: OR = 2.11; 95% CI = 0.59-7.56; high: OR = 3.93; 95% CI = 1.02-15.12) was significant (test for linear trend: p values = 0.020 and 0.040, respectively). The association between exposure and wheezing was stronger among atopic workers. The higher prevalence of chest tightness and shortness of breath among workers with medium and high current endotoxin exposure did not reach statistical significance. The results suggested that there was a dose-dependent increase in bronchial symptoms, with significant effects occurring at exposures that exceeded 450 EU/m3.

Anti-inflammatory activity of Salacia oblonga Wall. and Azima tetracantha Lam.

The anti-inflammatory activity of Salacia oblonga rootbark powder and Azima tetracantha leaf powder was assayed in male albino rats using carrageenan-induced rat paw oedema (acute inflammation) and cotton pellet granuloma (chronic inflammation) methods. Both the crude drugs were maximally active at a dose of 1000 mg/kg. In the cotton pellet granuloma assay, these drugs were able to suppress the transudative, exudative and proliferative components of chronic inflammation. Furthermore, these drugs were able to lower the lipid peroxide content of exudate and liver, gamma-glutamyl transpeptidase activity in the exudate of cotton pellet granuloma. The increased acid and alkaline phosphatase activity and decreased serum albumin in cotton pellet granulomatous rats were normalised after treatment with these drugs. It is likely that these drugs may exert their activity by antiproliferative, antioxidative and lysosomal membrane stabilization.

[A method of determining trace amounts of aromatic amines in dyed cotton textiles]. / Metoda oznaczania sladowych ilosci amin aromatycznych w tkaninach bawelnianych barwionych.

A method is described for determination of trace amounts of aromatic amines present as toxic contaminants in samples of dyed cotton textiles. The study was carried out on samples of cotton textiles for children, two were dyed beige and aquamarine , one was white. A distillation-spectrophotometric method was used based on the diazo reaction of sodium nitrate + with amines and coupling reaction with alpha-naphthol. The intensity of the obtained colour was proportional to amine content in the sample. The absorbance of the colour solution was measured at 570 nm wavelength. It was shown that the tested samples contained only traces of aromatic amines calculated as aniline++--0.85-3.22 mg/kg (the acceptable amount is 40 mg/kg). The proposed method makes possible objective determination of aromatic amines in textiles, it is simple and highly effective, with detectability of the order of 10(-7) g/cm3. It may be used for amine determination in industry.

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.

Cartilage damage by a granulomatous reaction in a murine species.

The encapsulation of intact rat femoral head cartilage or discs of bovine nasal cartilage with cotton-gauze before implantation in the subcutaneous tissues of mice, results in an accelerated loss of cartilage proteoglycan. Loss of proteoglycan from bovine nasal cartilage occurred later than rat femoral head cartilage, but eventually brought about complete dissolution of the cartilage. Freeze-thaw killing of bovine nasal cartilage did not alter the amount of proteoglycan lost. Destruction of both femoral and nasal cartilage was related to the mass of cotton implanted and to the growth of connective tissue within the implanted cotton. Mice previously implanted with femoral head cartilage were able to show enhanced degradation to new implants; this was even greater if the original implants were encapsulated with cotton. Presoaking of cotton-cartilage implants with the non-specific irritant, carrageenan inhibited the breakdown of cartilage. Autoradiographs of 35sulphate pulsed femoral cartilage following implantation with cotton showed reduced incorporation of radiolabel by chondrocytes.

Chronic pulmonary effects in guinea pigs from prolonged inhalation of cotton dust.

Inhalation of cotton dust has been associated with development of byssinosis. An animal model has been described recently in which guinea pigs exposed to cotton dust for a 6-week period demonstrated acute respiratory reactions consisting of increased breathing frequency, reduced tidal volume, and airflow fluctuations most prominent on the first day of exposure following a period without exposure, often referred to as a "Monday" response (Ellakkani et al., 1984). The current study examined the effects of cotton dust inhalation for 52 weeks in order to evaluate the animal model for ability to demonstrate more chronic effects of cotton dust exposure. Twenty guinea pigs were exposed to 21 mg/m3 cotton dust for 6 hr/day, 5 days/week, for 52 weeks. Twenty control animals received sham exposure. Parameters used to indicate chronic effects included respiratory measurements, weight gain, lung volume and weight, and histopathological evaluation. Respiratory measurements were taken while animals were breathing ambient air and also while breathing a mixture of 10% CO2, 20% O2, and 70% N2. Pulmonary effects were noted to change during the 12 months of exposure. For the first 3 months experimental animals displayed an increase in breathing frequency and a decrease in breathing volume measured as whole-body plethysmographic pressure. These effects were pronounced on the "Monday" of each week. During Months 3-6, reactions occurred on each day of exposure, although Monday responses were most severe. After 6 months, respiratory reactions were pronounced daily. Other indications of a chronic effect of exposure were increased lung volume, measured by water displacement, 15.0 +/- 3.3 ml (mean +/- SD) for the exposed group, compared with 9.8 +/- 2.0 ml for the controls; increased lung weight 9.4 +/- 1.5 g vs 7.0 +/- 0.8 g; and bronchiolar epithelial hyperplasia and hyperplasia of alveolar type II cells. Additionally, a histomorphometric study of the lungs performed by others (Coulombe et al., 1986) detected changes in the peripheral conducting airways, including increased thickness of bronchiolar epithelium and increased thickness of septa at the alveolar level, denoting chronic exposure. Taken together, these results indicated chronic respiratory effects in guinea pigs as a result of 52 weeks of continued exposure to cotton dust. The parallel in development of symptoms in guinea pigs and in humans exposed to cotton dust indicates that the guinea pig is a suitable model for byssinosis.

Cotton dust-mediated lung epithelial injury.

To determine if constituents of cotton plants might play a role in byssinosis by injuring pulmonary epithelium, we added extracts of cotton dust, green bract, and field-dried bract to human A549 and rat type II pneumocytes. Injury was measured as pneumocyte lysis and detachment, and inhibition of protein synthesis. Extracts of cotton dust and field-dried bract produced significant dose- and time-dependent lysis and detachment of both target cells, while green bract extract was less damaging. Extracts treated with polyvinylpolypyrrolidone to remove tannins produced significantly less injury. In contrast, purified 5,7,3',4'-tetrahydroxy-flavan 3,4-diol (THF), a tannin in cotton dust and bract, caused substantial cell damage. Field-dried bract extract and THF also produced dose-dependent inhibition of pneumocyte protein synthesis. Endotoxin levels did not correlate with observed injury. THF added to rat tracheal explants caused epithelial disruption and desquamation, whereas endotoxin did not. Instillation of cotton dust and field-dried bract extract in rat lungs produced disruption of bronchial epithelium and smooth muscle constriction, while polyvinylpolypyrrolidone-treated cotton dust extract produced no injury. These findings suggest that extracts of cotton plants are toxic to alveolar, tracheal, and bronchial epithelium and that THF or other tannins may be the responsible agents.

Effects of endotoxin and tannin isolated from cotton bracts on the airway epithelium.

The effects of increasing concentrations of tannin isolated from cotton bracts and endotoxin prepared from Enterobacter agglomerans on the electrophysiologic and ion transport properties of the canine tracheal epithelium mounted in Ussing chambers were examined. Results were compared with those obtained using cotton bracts extract (CBE). Tannin concentrations in the isolated tannin and in the cotton bracts were analyzed spectrophotometrically. When added to the mucosal bathing solution, tannin produced a significant decrease in transepithelial potential difference and short-circuit current (lsc) with a maximal response at 25 microliter. The decrease in lsc was accounted for entirely by a decrease in net chloride secretion. The effects were reversible and specific for the mucosal bathing solution. In contrast to CBE, tannin had no effect on mannitol flux, suggesting no effect on the paracellular pathway. Endotoxin at a concentration of 10 micrograms/ml had no effect on the electrophysiologic properties of the canine trachea. We conclude that tannin in CBE is responsible for the decrease in lsc observed with aqueous extracts of CBE but is not responsible for the changes in the paracellular pathway. We also conclude that endotoxin alone has no effect on the airway epithelium.

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.

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.