The effect of deoxynivalenol on the secretion activity, proliferation and apoptosis of porcine ovarian granulosa cells in vitro

The aim of this in vitro study was to examine the secretion activity, markers of proliferation and apoptosis in porcine ovarian granulosa cells (GCs) after deoxynivalenol (DON) addition. Ovarian granulosa cells were incubated with DON for 24h: 10, 100 and 1000 ng/mL, while the control group received no DON. The secretion of insulin-like growth factor I (IGF-I) and progesterone was determined by radioimmunoassay (RIA) and expression of cyclin B1, PCNA and caspase-3 by immunocytochemistry. IGF-I release by GCs was inhibited by DON, while progesterone release and the expression of cyclin B1 was stimulated by DON (at 1000 ng/mL but not at 10 and 100 ng/mL). PCNA expression was stimulated by DON (at 100 and 1000 ng/mL but not at 10 ng/mL). Caspase-3 expression was not influenced by DON treatment (at all doses). In conclusion, our results indicate, (1) a direct effect of DON on secretion of growth factor IGF-I and steroid hormone progesterone, (2) expression of markers of proliferation (cyclin B1 and PCNA) but not on the (3) expression of marker of apoptosis (caspase-3) in porcine ovarian granulosa cells. This in vitro study suggests the dose-dependent association of DON on porcine ovarian functions.     

The effect of deoxynivalenol on the secretion activity,proliferation and apoptosis of porcine ovarian granulosa cells in vitro

The aim of this in vitro study was to examine the secretion activity, markers of proliferation and apoptosis in porcine ovarian granulosa cells (GCs) after deoxynivalenol (DON) addition. Ovarian granulosa cells were incubated with DON for 24h: 10, 100 and 1000 ng/mL, while the control group received no DON. The secretion of insulin-like growth factor I (IGF–I) and progesterone was determined by radioimmunoassay (RIA) and expression of cyclin B1, PCNA and caspase-3 by immunocytochemistry. IGF–I release by GCs was inhibited by DON, while progesterone release and the expression of cyclin B1 was stimulated by DON (at 1000 ng/mL but not at 10 and 100 ng/mL). PCNA expression was stimulated by DON (at 100 and 1000 ng/mL but not at 10 ng/mL). Caspase-3 expression was not influenced by DON treatment (at all doses). In conclusion, our results indicate, (1) a direct effect of DON on secretion of growth factor IGF-I and steroid hormone progesterone, (2) expression of markers of proliferation (cyclin B1 and PCNA) but not on the (3) expression of marker of apoptosis (caspase-3) in porcine ovarian granulosa cells. This in vitro study suggests the dose-dependent association of DON on porcine ovarian functions.     

Dietary bioflavonoid quercetin modulates porcine ovarian granulosa cell functions in vitro

Quercetin is a dietary bioflavonoid used widely as a food supplement and is generally recognized as safe. The aim of this in vitro study was to examine the steroid hormone (progesterone and 17- β estradiol) release, proliferation (PCNA and cyclin B1) and apoptosis (caspase 3 and p53) of porcine ovarian granulosa cells after the addition of quercetin at concentrations 0.01, 0.1, 1, 10 and 100?μmol?L^?1. Progesterone release was stimulated at the concentration 10?μmol?L^?1. Quercetin neither had any impact on 17-β estradiol secretion nor on the presence of PCNA. However, a significant enhancement of the occurrence of cyclin B1 was noted except for the lowest concentration 0.01?μmol?L^?1. Quercetin did not have any influence on the number of granulosa cells containing caspase 3, but at the concentration 10?μmol?L^?1 it inhibited p53 occurrence. Results confirm the safety of quercetin in porcine ovarian granulosa cell model and further suggest its possible concentration-dependent influence on ovarian functions through pathway that may involve progesterone, cyclin B1 and p53.     

Cobalt-induced hormonal and intracellular alterations in rat ovarian fragments in vitro

The objective of this in vitro study was to examine dose-dependent changes in the secretion activity (progesterone, 17β-estradiol and insulin-like growth factor-I) of rat ovarian fragments after experimental cobalt (Co) administration including the apoptotic potential of Co on rat ovarian fragments by evaluating the expression of apoptotic markers Bax and caspase-3. Ovarian fragments were incubated with cobalt sulphate (CoSO₄.7H₂O) at the doses 90, 170, 330 and 500 μg.mL^?1 for 24 h and compared with control group without Co addition. Release of progesterone (P₄) 17β-estradiol and insulin-like growth factor-I (IGF-I) by ovarian fragments was assessed by RIA, expression of Bax and caspase-3 by SDS-PAGE and Western blotting. Observations show that P₄ release by ovarian fragments was significantly (P < 0.05) inhibited after cobalt sulphate addition at higher doses 170–500 μg.mL^?1 used in the study in comparison to control. However, cobalt sulphate addition did not cause any significant change in the release of 17β-estradiol by ovarian fragments at all the doses used in the study (90–500 μg.mL^?1) in comparison to control. On the contrary, IGF-I release by ovarian fragments was significantly (P < 0.05) stimulated after cobalt sulphate addition at the lowest dose 90 μg.mL^?1 in comparison to control, while other doses did not cause any significant change. Also, addition of cobalt sulphate decreased the expression of both the apoptotic peptides Bax and caspase-3 at the higher doses 170, 330 and 500 μg.mL^?1, but not at the lowest dose 90 μg.mL^?1 used in the study. Obtained results suggest Co induced (1) inhibition in secretion of steroid hormone progesterone, (2) dose-dependent increase in the release of growth factor IGF-I, and (3) decrease in the expression of markers of apoptosis (Bax and caspase-3) of rat ovarian fragments.     

The influence of deoxynivalenol and zearalenone on steroid hormone production by porcine ovarian granulosa cells in vitro

Fusarium mycotoxins deoxynivalenol (DON) and zearalenone (ZEA) are frequently occurring in feed of pigs together. The aim of this study was to evaluate the possible in vitro effects of DON and ZEA, alone or their combination on steroid secretion of porcine ovarian granulosa cells (GCs). A species-specific model with porcine ovarian GCs was used to study the potential endocrine disrupting effects of DON and ZEA alone and in co-exposure. Progesterone (P4) and estradiol (E2) were determined by radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). The results of this study demonstrate that DON alone at the higher concentrations may act to stimulate P4 (at 1,000, 2,000, 3,000 and 5,000 ng mL^?1 but not 10 and 100 ng mL^?1) and E2 (at 2,000, 3,000 and 5,000 ng mL^?1 but not 10, 100 and 1000 ng mL^?1) secretion. The effects of ZEA on P4 and E2 secretion were not confirmed. DON in combination with the other fusariotoxin ZEA may impair steroidogenesis. Results aslo demonstrate different toxicological effects of fusariotoxins on follicle stimulating hormone-induced secretion of P4 and E2. All these results taken together suggest that fusariotoxin and their interactions can impact ovarian steroidogenesis, thereby demonstrating their potential reproductive effects in pigs.     

Assessment of a potential preventive ability of amygdalin in mycotoxin-induced ovarian toxicity

The possible effects of a natural substance amygdalin and its combination with the mycotoxin deoxynivalenol (DON) on the steroid hormone secretion (progesterone and 17-β-estradiol) by porcine ovarian granulosa cells (GCs) were examined in this in vitro study. Ovarian GCs were incubated without (control group) and with amygdalin (1, 10, 100, 1,000 and 10,000 μg mL¹), or its combination with DON (1 μg mL¹) for 24 h. The release of steroid hormones was determined by ELISA. The progesterone secretion by porcine ovarian GCs was not affected by amygdalin in comparison to the control. However, the highest amygdalin dose (10,000 μg mL¹) caused a significant stimulation of the 17-β-estradiol release. A combination of amygdalin with DON significantly (P < 0.05) increased the progesterone release at all concentrations. Similarly, a stimulatory effect of amygdalin co-administered with DON was detected with respect to the 17-β-estradiol secretion at the highest dose (10,000 μg mL¹) of amygdalin and 1 μg mL¹ of DON. Noticeable differences between the effects of amygdalin alone and its combination with DON on the progesterone release were detected. In contrast, no differences between the stimulatory effects of amygdalin and its combination with DON on the 17-β-estradiol synthesis by porcine GCs were observed. Findings from this in vitro study did not confirm the expected protective effect of amygdalin on mycotoxin induced reprotoxicity. Our results indicate that the stimulatory effect of amygdalin combined with DON on the progesterone release was clearly caused by the DON addition, not by the presence amygdalin per se. On the other hand, the stimulation of 17-β-estradiol production was solely caused by the presence of amygdalin addition. These findings suggest a possible involvement of both natural substances into the processes of steroidogenesis and appear to be endocrine modulators of porcine ovaries.     

Estrogenic and antiestrogenic effect of in vitro treatment of follicular cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin

OBJECTIVE: Two types of follicular cells from preovulatory ovary were cultured in vitro separately and in co-culture to test difference in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) action on particular cell types. METHODS: The accumulation of TCDD in follicular wall was analysed using coupled capillary gas chromatography mass spectrometry. Whole preovulatory follicles were isolated from ovary and incubated with prolonged exposure to 0.1 nM TCDD or single exposure to 10 nM TCDD for four days. In the second part of experiments direct effects of TCDD on steroidogenesis were investigated in porcine theca cells (Tc) and granulosa cells (Gc) cultured alone and in co-culture (GT). The media were collected after four days for steroid analysis. RESULTS: 59.3% and 81.2% of TCDD added to the culture medium was accumulated after 0.1 and 10 nM, respectively. TCDD in a dose-dependent manner increased estradiol secretion with concomitant progesterone secretion by theca interna cells. On the other hand decrease of both progesterone and estradiol secretion by granulosa cells cultured alone and in co-culture with theca cells was noted. CONCLUSION: Different cell-specific estrogenic or antiestrogenic effect of TCDD were found in ovarian follicles.     

Mycobiota and mycotoxins in bee pollen collected from different areas of Slovakia

Contamination by microscopic fungi and mycotoxins in different bee pollen samples, which were stored under three different ways of storing as freezing, drying and UV radiation, was investigated. During spring 2009, 45 samples of bee-collected pollen were gathered from beekeepers who placed their bee colonies on monocultures of sunflower, rape and poppy fields within their flying distance. Bee pollen was collected from bees' legs by special devices placed at the entrance to hives. Samples were examined for the concentration and identification of microscopic fungi able to grow on Malt and Czapek-Dox agar and mycotoxins content [deoxynivalenol (DON), T-2 toxin (T-2), zearalenone (ZON) and total aflatoxins (AFL), fumonisins (FUM), ochratoxins (OTA)] by direct competitive enzyme-linked immunosorbent assays (ELISA). The total number of microscopic fungi in this study ranged from 2.98 ± 0.02 in frozen sunflower bee pollen to 4.06 ± 0.10 log cfu.g(-1) in sunflower bee pollen after UV radiation. In this study, 449 isolates belonging to 21 fungal species representing 9 genera were found in 45 samples of bee pollen. The total isolates were detected in frozen poppy pollen 29, rape pollen 40, sunflower pollen 80, in dried poppy pollen 12, rape pollen 36, sunflower 78, in poppy pollen after UV radiation treatment 54, rape 59 and sunflower 58. The most frequent isolates of microscopic fungi found in bee pollen samples of all prevalent species were Mucor mucedo (49 isolates), Alternaria alternata (40 isolates), Mucor hiemalis (40 isolates), Aspergillus fumigatus (33 isolates) and Cladosporium cladosporioides (31 isolates). The most frequently found isolates were detected in sunflower bee pollen frozen (80 isolates) and the lowest number of isolates was observed in poppy bee pollen dried (12 isolates). The most prevalent mycotoxin of poppy bee pollen was ZON (361.55 ± 0.26 μg.kg(-1)), in rape bee pollen T-2 toxin (265.40 ± 0.18 μg.kg(-1)) and in sunflower bee pollen T-2 toxin (364.72 ± 0.13 μg.kg(-1)) in all cases in frozen samples.     

Mycobiota and mycotoxins in bee pollen collected from different areas of Slovakia

Contamination by microscopic fungi and mycotoxins in different bee pollen samples, which were stored under three different ways of storing as freezing, drying and UV radiation, was investigated. During spring 2009, 45 samples of bee-collected pollen were gathered from beekeepers who placed their bee colonies on monocultures of sunflower, rape and poppy fields within their flying distance. Bee pollen was collected from bees’ legs by special devices placed at the entrance to hives. Samples were examined for the concentration and identification of microscopic fungi able to grow on Malt and Czapek-Dox agar and mycotoxins content [deoxynivalenol (DON), T-2 toxin (T-2), zearalenone (ZON) and total aflatoxins (AFL), fumonisins (FUM), ochratoxins (OTA)] by direct competitive enzyme-linked immunosorbent assays (ELISA). The total number of microscopic fungi in this study ranged from 2.98 ± 0.02 in frozen sunflower bee pollen to 4.06 ± 0.10 log cfu.g^?1 in sunflower bee pollen after UV radiation. In this study, 449 isolates belonging to 21 fungal species representing 9 genera were found in 45 samples of bee pollen. The total isolates were detected in frozen poppy pollen 29, rape pollen 40, sunflower pollen 80, in dried poppy pollen 12, rape pollen 36, sunflower 78, in poppy pollen after UV radiation treatment 54, rape 59 and sunflower 58. The most frequent isolates of microscopic fungi found in bee pollen samples of all prevalent species were Mucor mucedo (49 isolates), Alternaria alternata (40 isolates), Mucor hiemalis (40 isolates), Aspergillus fumigatus (33 isolates) and Cladosporium cladosporioides (31 isolates). The most frequently found isolates were detected in sunflower bee pollen frozen (80 isolates) and the lowest number of isolates was observed in poppy bee pollen dried (12 isolates). The most prevalent mycotoxin of poppy bee pollen was ZON (361.55 ± 0.26 μg.kg^?1), in rape bee pollen T-2 toxin (265.40 ± 0.18 μg.kg^?1) and in sunflower bee pollen T-2 toxin (364.72 ± 0.13 μg.kg^?1) in all cases in frozen samples.     

Biologically active antimicrobial and antioxidant substances in the Helianthus annuus L. bee pollen

The objective of this study was to measure the content of flavonoids, polyphenols, and carotenoids in the Helianthus annuus L. bee pollen. It was also to evaluate the ability of the dried, frozen, and freeze-dried extracts of sunflower (H. annuus) pollen, its scavenged free radicals and reducing action. Another aim of this study was to investigate the antimicrobial in vitro action of the H. annuus pollen extracts against the Gram-positive and Gram-negative bacteria and fungi. All pollen extracts showed medium antiradical activity and reductive ability. The most effective was the freeze-dried extract in both evaluation systems. The evaluation of the protective effects of DNA using a biosensor showed an opposite trending—frozen ? dried ? freeze-dried pollen. For the evaluation of antiradical activity, the DPPH method was used, and reductive ability was assessed by means of phosphomolybdic complex formation. The comparison of the polyphenols content shows higher values in freeze-dried bee pollen than in the dried and frozen pollen. The highest content of flavonoids was found in the frozen samples and the most carotenoids were present in the dried samples. In our study, the best antibacterial effects of the dried sunflower bee pollen extracts were found against Paenibacillus larvae, Pseudomonas aeruginosa, and Enterococcus raffinosus. The best inhibitory properties of the frozen sunflower bee pollen extracts were found against Escherichia coli, Pseudomonas aeruginosa, and Paenibacillus larvae. Very good inhibitory effects of freeze-dried sunflower bee pollen were found against Paenibacillus larvae, Brochotrix thermosphacta, and Enterococcus raffinosus. The best antifungal activity of the sunflower bee pollen was found in the frozen bee pollen extracts against Aspergillus ochraceus and freeze-dried bee pollen extracts against Aspergillus niger.     

Comparison between solid-phase extraction methods for the chromatographic determination of organophosphorus pesticides in water

A solid-phase microextraction (SPME) procedure has been developed to extract eight organophosphorus pesticides (OPs) in water and the method was compared with a conventional solid phase extraction (SPE) technique. The extracted OPs were analyzed by gas chromatography using thermionic specific detection. Both extraction methods presented linear calibration at least over the concentration range investigated (100 to 1000 ng x mL(-1) for SPE and 1 to 100 ng x mL(-1) for SPME). SPME method presented higher sensitivity than SPE. The quantitation limits were between 0.1 to 1.0 ng x mL(-1) for SPME depending upon the analyte, and 100 ng x mL(-1) for SPE. The precision, as measured by the standard deviations (RSD), were in the range 3.6% to 5.8% for SPME and 2.4% to 9.2% for SPE. Along with the feature of being a solvent - free sampling technique, SPME offers additional benefits due to its high sensitivity, simplicity, and small size sample required (typically: SPE - 500 mL, SPME - 5 mL).     

Acetylcholinesterase in honey bees (Apis mellifera) exposed to neonicotinoids, atrazine and glyphosate: laboratory and field experiments

In Québec, as observed globally, abnormally high honey bee mortality rates have been reported recently. Several potential contributing factors have been identified, and exposure to pesticides is of increasing concern. In maize fields, foraging bees are exposed to residual concentrations of insecticides such as neonicotinoids used for seed coating. Highly toxic to bees, neonicotinoids are also reported to increase AChE activity in other invertebrates exposed to sub-lethal doses. The purpose of this study was therefore to test if the honey bee’s AChE activity could be altered by neonicotinoid compounds and to explore possible effects of other common products used in maize fields: atrazine and glyphosate. One week prior to pollen shedding, beehives were placed near three different field types: certified organically grown maize, conventionally grown maize or non-cultivated. At the same time, caged bees were exposed to increasing sub-lethal doses of neonicotinoid insecticides (imidacloprid and clothianidin) and herbicides (atrazine and glyphosate) under controlled conditions. While increased AChE activity was found in all fields after 2 weeks of exposure, bees close to conventional maize crops showed values higher than those in both organic maize fields and non-cultivated areas. In caged bees, AChE activity increased in response to neonicotinoids, and a slight decrease was observed by glyphosate. These results are discussed with regard to AChE activity as a potential biomarker of exposure for neonicotinoids.     

Thymol as an alternative to pesticides: persistence and effects of Apilife Var on the phototactic behavior of the honeybee Apis mellifera

Thymol is a natural substance increasingly used as an alternative to pesticides in the fight against the Varroa destructor mite. Despite the effectiveness of this phenolic monoterpene against Varroa, few articles have covered the negative or side effects of thymol on bees. In a previous study, we have found an impairment of phototaxis in honeybees following application of sublethal doses of thymol—lower or equal to 100 ng/bee—under laboratory conditions. The present work shows the same behavioral effects on bees from hives treated with Apilife Var®, a veterinary drug containing 74 % thymol, with a decrease in phototactic behavior observed 1 day after treatment. Thus, thymol causes disruption of bee phototactic behavior both under laboratory conditions as well as in beehives. The bee exposure dose in treated hives was quantified using gas chromatography coupled to mass spectrometry (GC–MS), giving a median value of 4.3 μg per body 24 h after treatment, with 11 ng in the brain. The thymol level in 20 organic waxes from hives treated with Apilife Var® was also measured and showed that it persists in waxes (around 10 mg/kg) 1 year after treatment. Thus, in the light of (1) behavioral data obtained under laboratory conditions and in beehives, (2) the persistence of thymol in waxes, and (3) the high load on bees, it would appear important to study the long-term effects of thymol in beehives.     

Preliminary study on persistence in soil and residues in maize of imidacloprid

The aim of this work was to study the distribution of imidacloprid in soil and its translocation to roots and aerial parts of maize plant. The main objective was to assess imidacloprid residues in field environment, in order to provide data on honeybees exposure level to such an active substance. Imidacloprid has been detected and quantified by Triple Quadrupole HPLC-MS-MS. Pesticide persistence in the soil and its residues in pollen and in maize plants have been evaluated during the growing of maize plants developed from seeds dressed with Gaucho 350 FS (imidacloprid: 1.0 mg/seed). The sowing has been performed by means of a pneumatic precision drill. Samples have been collected at 30, 45, 60, 80, 130 days after the sowing, as pollen samples have been collected at the tasseling. Imidacloprid presence in aerial part of maize plant declined to 2-3 μg/kg 80 days after the sowing, while concentration in kernel at harvest was <1 μg/kg. Maize pollen represents an important part of protein supply of beehives, and it is of critical importance to bee foraging. The values detected (imidacloprid residues <1 μg/kg) showed that maize pollen source should not be relevant for acute toxicity impact on honey bees.     

Development of enzyme immunoassay for detection of DDT

Dichlorodiphenyltrichloroethane (DDT) is one of the persistent organic pollutants (POPs) widely found in the environment and in the general population. In this study, a direct competitive enzyme immunoassay (EIA) has been developed for the quantitative analysis of DDT. To generate a specific polyclonal antibody for EIA, p, p'-DDT was conjugated to porcine thyroglobulin for rabbit immunization. At optimized EIA conditions, the standard curves ranged from 0.137 to 100 ng/mL with the quantification limit of 0.41 ng/mL. The coefficients of variation (CV%) were 5.42-10.53% for intra-assay and 6.04-7.26% for inter-assay. Cross-reactivities with DDT metabolites (DDTs, including o, p'-DDT, p, p'-DDD, o, p'-DDD, p, p'-DDE, o, p'-DDE, p, p'-dichlorobenzophenone (DCBP), o, p'-DCBP) were investigated. The polyclonal antibody showed relatively low and/or no cross-reactivity with these compounds, and the assay was seen to be highly selective for p, p'-DDT. Moreover, the DDTs could be ranked by their reactivity: DDT > DDD > DDE > DCBP. In addition, the characterization of the polyclonal antibody indicated that the antiserum possesses a high specificity for p, p'-isomers. The results indicated that the developed EIA using this antibody could be a convenient and supplemental analytical tool for monitoring DDT.     

Possible effects of persistent organochlorinated pollutants cocktail on thyroid hormone levels and pituitary-thyroid interrelations

In polluted district of Michalovce in East Slovakia (POLL) and two districts with background pollution (BCGR) 2046 adults (834 males and 1212 females aged 20-75 years) were examined. Serum levels of thyrotropin (TSH), free thyroxine (FT4), total triiodothyronine (TT3) and antithyroperoxidase antibodies (TPOab) were estimated by electrochemiluminiscent assay and also these of 15 polychlorinated biphenyl congeners (PCBs), p,p'-DDE, p,p'-DDT, hexachlorobenzene (HCB) and hexachlorocyclohexane were measured by high resolution gas chromatography/mass spectrometry. In addition, also dioxins, furans, coplanar- and mono-ortho-PCBs as well as selected hydroxylated and methylsulphonated PCBs and DDE metabolites were measured by appropriate methods based on gas chromatography/mass spectrometry principle. In POLL significantly higher levels of all organochlorines were found than these in BCGR. When pooled values from both areas were stratified in terms of PCBs level and treated as continuous variables, positive association of PCBs with FT4 and TT3 was found, the latter two being also mutually associated. However, within the category of PCBs level <530 ng/glipid (n=232) the association between PCBs and both the FT4 (p<0.09) and TT3 (p<0.03) was negative and any association of these was not found within the category of PCBs level of 531-1000 ng/g (n=691). In contrast, in the category of 531-2000 ng/g (n=1307) positive association appeared between PCBs and FT4 (p<0.001) as well as TT3 (p<0.05). Highly significant association of PCBs with FT4 (p<0.001) was further found in the categories with PCBs level of 1001-101414 ng/g (n=1307) and 2001-101414 (n=1123), while significant association with TT3 was observed only in the category of 531-2000 ng/g. Such findings suggest possible threshold level in positive effect of PCBs on FT4 and TT3 level which seems to be individual and located somewhere around the PCBs level of 1000 ng/g. However, highly significant negative association of both FT4 and TT3 with TSH was found in each of above indicated PCBs categories. Considerable difference in FT4 and TT3 level between large groups of subjects with the same range of PCBs level was also found suggesting different individual susceptibility to the effects of organochlorines. Among a total of 26 cases from POLL with very low TSH level (<0.5 mU l(-1)) 13 cases showed very high level of PCBs, FT4 and TT3, thus supporting a hypothesis on a novel sporadic form of high PCBs related peripheral subclinical hyperthyroidism possibly resulting from the long-term disruption of equilibrium between bound and free thyroxine in plasma by high PCBs level followed by a striking inhibition of TSH release from the pituitary.     

Ozone affects pollen viability and NAD(P)H oxidase release from Ambrosia artemisiifolia pollen

Air pollution is frequently proposed as a cause of the increased incidence of allergy in industrialised countries. We investigated the impact of ozone (O₃) on reactive oxygen species (ROS) and allergen content of ragweed pollen (Ambrosia artemisiifolia). Pollen was exposed to acute O₃ fumigation, with analysis of pollen viability, ROS and nitric oxide (NO) content, activity of nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase, and expression of major allergens. There was decreased pollen viability after O₃ fumigation, which indicates damage to the pollen membrane system, although the ROS and NO contents were not changed or were only slightly induced, respectively. Ozone exposure induced a significant enhancement of the ROS-generating enzyme NAD(P)H oxidase. The expression of the allergen Amb a 1 was not affected by O₃, determined from the mRNA levels of the major allergens. We conclude that O₃ can increase ragweed pollen allergenicity through stimulation of ROS-generating NAD(P)H oxidase.     

Leaching of trifluralin,metolachlor, and metribuzin in a clay loam soil of Louisiana

Trifluralin[2,6-dinitro-N,N-dipropyl-4-(trifluormethyl)benzenamine], metolachlor[2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl)acetamide], and metribuzin[4-amino-6-(1,1-dimethylethyl)-3-(methylthio)-1,2,4-triazin-5(4H)one] were applied in field plots located on a Commerce clay loam soil near Baton Rouge, Louisiana at the rate of 1683 g/ha, 2759 g/ha and 609 g/ha, respectively. The half-lives of trifluralin, metolachlor, and metribuzin in the top 0-15 cm soil depth were found to be 54.7 days, 35.8 days and 29.8 days, respectively. The proportion of trifluralin, metolachlor, and metribuzin in the top 0-15 cm soil depth was 94.7%, 86.6%, and 75.4%, respectively of that found in the top 0-60 cm soil depth 30 days after application. Trifluralin concentrations were within a range of 0.026 ng/mL to 0.058 ng/mL in 1 m deep well water, and between 0.007 ng/mL and 0.039 ng/mL in 2 m deep well water over a 62 day period after application. Metolachlor concentrations in the 1 m and 2 m wells ranged from 3.62 ng/mL to 82.32 ng/mL and 8.44 ng/mL to 15.53 ng/mL, respectively. Whereas metribuzin concentrations in the 1 m and 2 m wells ranged from 0.70 ng/mL to 27.75 ng/mL and 1.71 ng/mL to 3.83 ng/mL, respectively. Accordingly, trifluralin was found to be strongly adsorbed on the soil and showed negligible leaching. Although metolachlor and metribuzin were also both readily adsorbed on the soil, their leaching potential was high. As a result, in the clay loam soil studied, metribuzin concentration in groundwater with shallow aquifers is likely to exceed the 10 mg/L US Environmental Protection Agency (EPA) advisory level for drinking water early in the application season, whereas trifluralin and metolachlor concentrations are expected to remain substantially lower than their respective 2 ng/mL and 175 ng/mL EPA advisory levels.     

In vitro response of human ovarian cancer cells to dietary bioflavonoid isoquercitrin

Abstract

Isoquercitrin is a dietary bioflavonoid used as a food supplement. We studied the mechanism underlying its effect in human ovarian cancer cells using OVCAR-3 cell line. Viability, survival, apoptosis, release of human transforming growth factor-β1 (TGF-β1) and TGF-β1 receptor, and intracellular reactive oxygen species (ROS) generation by OVCAR-3 cells were examined after isoquercitrin treatment at concentrations 5, 10, 25, 50, and 100?μg mL^?1. AlamarBlue assay revealed that isoquercitrin did not cause any significant change (P?>?0.05) in cell viability as compared to control. Apoptotic assay using flow cytometry did not find any significant change (P?>?0.05) in the proportion of live, dead and apoptotic cells as compared to control. ELISA also showed that the release of human TGF-β1 and TGF-β1 receptor were not significantly (P?>?0.05) affected by isoquercitrin as compared to control. Chemiluminescence assay demonstrated that lower concentrations (5, 10, and 25?μg mL^?1) were able to exhibit beneficial effects by inhibiting the generation of intracellular ROS. In contrast, elevated concentrations of 50 and 100?μg mL^?1 led to oxidative stress (P?相似文献   

Simultaneous analysis of free and sulfo-conjugated steroid estrogens in artificial urine solution and agricultural soils by high-performance liquid chromatography

A simple and robust analytical method was developed to simultaneously detect and quantify 17β-estradiol (E2), estrone (E1), 17β-estradiol-3-sulphate (E2-3S), and estrone-3-sulphate (E1-3S) in aqueous solutions (calcium chloride and artificial urine solutions) and agricultural soils using high performance liquid chromatography and UV detection. The standards for all four compounds were linear in the range of 0.01 to 1.0 μg mL(-1) (n = 6) and 1.0 to 20 μg mL(-1) (n = 6), respectively, with correlation coefficients > 0.999. The on-column limits of detection at an injection volume of 50 μL and S/N (signal: noise) ratio of 3 were: 9.0 ng mL(-1), 10 ng mL(-1), 5.0 ng mL(-1), and 7.0 ng mL(-1) for E2-3S, E1-3S, E2 and E1, respectively. The limit of detection and quantification in artificial urine solution and CaCl(2) solution was 1.0 ng mL(-1) for all four compounds. Method detection limits for the compounds in the 3 soils ranged from 2 to 2.4 ng g(-1) (E2-3S and E1-3S), and 1.0 to 2.9 ng g(-1) (E2 and E1), respectively.