Reproductive effects of long-term exposure to Bisphenol A in the fathead minnow (Pimephales promelas)

Bisphenol A (BPA), a high-volume chemical used to make polycarbonate plastic, epoxy resins, and other chemicals has been reported to be weakly estrogenic. To investigate the effects of long-term exposure to Bisphenol A, a multigeneration study was conducted in which fathead minnows (Pimephales promelas) were exposed to water concentrations of BPA in the range from 1 to 1280 micrograms/L. In this paper, we report the growth and reproductive effects of BPA on sexually mature adults in the F0 generation (after 43, 71, and 164 d of exposure) and the effects on hatchability in the F1 generation. Mean measured concentrations of BPA in the water for all doses, over a 164-d exposure period, were between 70% and 96% of nominal. An inhibitory effect of BPA on somatic growth (length and weight) occurred in adult male fish exposed to 640 and 1280 micrograms/L (after 71 and 164 d). BPA induced vitellogenin synthesis (VTG; a biomarker for estrogen exposure) in males at concentrations of 640 and 1280 micrograms/L after 43 d and 160 micrograms/L after 71 d. In females, plasma VTG concentrations were elevated above controls only after 164-d exposure to 640 micrograms/L. Inhibition of gonadal growth (as measured by the gonadosomatic index) occurred in both males and females at concentrations of 640 and 1280 micrograms/L after 164 d. In males, a concentration of 16 micrograms/L altered the proportion of sex cell types in the testis, suggesting inhibition of spermatogenesis. Concentrations of BPA that induced VTG synthesis and affected gonadal development were lower than those that resulted in discernible effects on reproductive output. Egg production was inhibited at a BPA concentration of 1280 micrograms/L, and hatchability in the F1 generation was reduced at a BPA concentration of 640 micrograms/L (there were not enough eggs spawned in the 1280 micrograms/L group for hatchability studies to be conducted). The results demonstrate that BPA acts as a weak estrogen to fish when administered via the water, with effects on breeding at and above 640 micrograms/L.     

Tissue lead concentration during chronic exposure of Pimephales promelas (fathead minnow) to lead nitrate in aquarium water

The fathead minnow is a useful species for evaluating the toxicity of wastewater effluents. While this fish is widely used for "survival" studies of metal toxicity, little or no work has been done on the tissue distribution of metals in fathead minnows. To determine the distribution of tissue lead, aquarium studies were conducted for several weeks with fish maintained in soft synthetic freshwater. Lead- (II) nitrate was added to three aquaria attaining concentrations of 20-30 ppb (aquarium B), 100-140 ppb (aquarium C), and roughly 200 ppb (aquarium D). Results were compared to controls (aquarium A). During the initial week, the majority of aquarium D fish died, whereas few deaths occurred in the other groups. Lead accumulation was dose- and tissue-dependent, with highest uptake by the gills. Gill concentrations of aquarium D fish averaged about 4-fold higherthan in skeleton or skin and muscle. In vitro, lead (2.5-25 ppm) caused dose-dependent reductions in the ratio of reduced glutathione/oxidized glutathione (GSH/GSSG) in gills incubated in physiological buffer. These findings demonstrate that fathead minnow gills bind and accumulate waterborne lead rapidly and preferentially and raise the possibility that gill lipid peroxidation contributes to lead toxicity at low water hardness.     

Assessing effects of metal mining effluent on fathead minnow (Pimephales promelas) reproduction in a trophic-transfer exposure system

Assessment of effects of metal mine effluent (MME) on aquatic organisms in lab-based settings predominantly evaluates contaminant transfer through the water only with little emphasis on food-borne exposure. The effects of MME on fathead minnow (Pimephales promelas) (FHM) have been reported downstream of metal mine discharges in the Junction Creek system, Sudbury, ON, but to date, no study has investigated the significance of trophic transfer in this system. Our objective was to develop a self-sustaining trophic-transfer bioassay, using Chironomus tentans and FHM, that allowed assessment of the effects of not only water-borne (FHM-only) but also food- and water-borne (trophic-transfer) exposure to MME on FHM reproduction. Reproductive performance of FHM was assessed for 21 days under controlled laboratory conditions to obtain baseline data of various endpoints, including egg production and hatching success. Exposure to 45% (v/v) Copper Cliff mine effluent (CCME) and control treatments for both systems was then conducted for a further 21 days. It was evident that reproductive output in both the water-only and the trophic-transfer system was reduced compared to controls. It was only in the trophic-transfer system that a significant reduction in larval hatching and an increase in deformities occurred after exposure to CCME. This would suggest that contaminated food was a route of exposure causing effects on larval survival.     

Evaluation of the model anti-androgen flutamide for assessing the mechanistic basis of responses to an androgen in the fathead minnow (Pimephales promelas)

In this study, we characterized the effects of flutamide, a model mammalian androgen receptor (AR) antagonist, on endocrine function in the fathead minnow (Pimephales promelas), a small fish species that is widely used for testing endocrine-disrupting chemicals (EDCs). Binding assays with whole cells transiently transfected with cloned fathead minnow AR indicated that flutamide binds competitively to the receptor. However, as is true in mammalian systems, a 2-hydroxylated metabolite of flutamide binds to the AR with a much higher affinity than the parent chemical. Mixture experiments with flutamide and the androgen 17beta-trenbolone demonstrated that the anti-androgen effectively blocked trenbolone-induced masculinization (nuptial tubercle production) of female fathead minnows, indicating antagonism of an AR receptor-mediated response in vivo. Conversely, reductions in vitellogenin in trenbolone-exposed females were not blocked by flutamide, suggesting that the vitellogenin response is not directly mediated through the AR. The results of these studies provide data demonstrating the validity of using the fathead minnow as a model species for detecting EDCs that exert toxicity through interactions with the AR.     

Oxygen flux as an indicator of physiological stress in fathead minnow (Pimephales promelas) embryos: a real-time biomonitoring system of water quality

The detection of harmful chemicals and biological agents in real time is a critical need for protecting freshwater ecosystems. We studied the real-time effects of five environmental contaminants with differing modes of action (atrazine, cadmium chloride, pentachlorophenol, malathion, and potassium cyanide) on respiratory oxygen consumption in 2-day postfertilization fathead minnow (Pimephales promelas) eggs. Our objective was to assess the sensitivity of fathead minnow eggs using the self-referencing micro-optrode technique to detect instantaneous changes in oxygen consumption after brief exposures to low concentrations of contaminants. Oxygen consumption data indicated that the technique is indeed sensitive enough to reliably detect physiological alterations induced by four of the five contaminants. After 2 h of exposure, we identified significant increases in oxygen consumption upon exposure to pentachlorophenol (100 and 1000 microg/L), cadmium chloride (0.0002 and 0.002 microg/L), and atrazine (150 microg/L). In contrast, we observed a significant decrease in oxygen flux after exposuresto potassium cyanide (44 and 66 microg/L) and atrazine (1500 microg/L). No effects were detected after exposures to malathion (200 and 340 microg/L). Our work is the first step in development of a new technique for physiologically coupled biomonitoring as a sensitive and reliable tool for the detection of environmental toxicants.     

A method for the determination of genetic sex in the fathead minnow, Pimephales promelas, to support testing of endocrine-active chemicals

Certain endocrine-active toxicants have been reported to completely sex reverse both male and female individuals in amphibian, avian, fish, invertebrate, and reptile species, resulting in a phenotype indistinguishable from unaffected individuals. Detection of low-level sex reversal often requires large numbers of organisms to achieve the necessary statistical power, especially in those species with predominantly genetic sex determination and cryptic/homomorphic sex chromosomes. Here we describe a method for determining the genetic sex in the commonly used ecotoxicological model, the fathead minnow (Pimephales promelas). Analysis of amplified fragment length polymorphisms (AFLP) in a spawn of minnows resulted in detection of 10 sex-linked AFLPs, which were isolated and sequenced. No recombination events were observed with any sex-linked AFLP in the animals examined (n=112). A polymerase chain reaction (PCR) method was then developed that determined the presence of one of these sex-linked polymorphisms for utilization in routine toxicological testing. Analyses of additional spawns from our in-house culture indicate that fathead minnows utilize a XY sex determination strategy and confirm that these markers can be used to genotype sex; however, this method is currently limited to use in laboratory studies in which breeders possess a defined genetic makeup. The genotyping method described herein can be incorporated into endocrine toxicity assays that examine the effects of chemicals on gonad differentiation.     

Oral exposure of PBDE-47 in fish: toxicokinetics and reproductive effects in Japanese Medaka (Oryzias latipes) and fathead minnows (Pimephales promelas)

The toxicokinetics of 2,2,4,4-tetrabromodiphenyl ether (PBDE-47) was studied in the Japanese Medaka (Oryzias latipes) after a single oral exposure followed by termination at specific time points. The effects of repeated oral exposure to PBDE-47 on reproductive performance was assessed using a pair breeding experimental design with fathead minnows (Pimephales promelas) given daily PBDE-47 exposures for 25 days, during which fecundity was measured as an indicator of reproductive performance. Medaka and fathead minnows were orally exposed to PBDE-47 by bioencapsulation in brine shrimp, Artemia sp. In the medaka studies, measurable levels of PBDE-47 were detected in the carcass within 0.25 h with peak levels occurring at 8 h. The body levels of PBDE-47 slowly declined and were still 25% of peak levels at 624 h after dosing. Assimilation of the bioencapsulated dose was at least 80% and may well approach 100%. The PBDE-47 concentration-time profile was fitted to a one-compartment clearance-volume toxicokinetic model and the model-predicted value for elimination half-life was determined to be 281 h and the first-order absorption rate constant was Ka = 0.26 hr(-1). In the fathead minnow study, egg laying in the PBDE-treated breeding pairs stopped after 10 days. The condition factor of PBDE-treated males was significantly reduced (P <0.011) compared with control males, whereas no significant difference was observed in females. Histological examination revealed a greater than 50% reduction in mature sperm in PBDE-47 exposed minnows compared to controls. Collectively, these results suggest PBDE-47 is selectively toxic to sexually mature male fathead minnows.     

Cloning and in vitro expression and characterization of the androgen receptor and isolation of estrogen receptor alpha from the fathead Minnow (Pimephales promelas)

In vitro screening assays designed to identify hormone mimics or antagonists typically use mammalian (rat, human) estrogen (ER) and androgen receptors (AR). Although we know that the amino acid sequences of steroid receptors in nonmammalian vertebrates are not identical to the mammalian receptors, a great deal of uncertainty exists as to whether these differences affect interactions of potential endocrine-disrupting chemicals (EDC) with the receptors. This leads to substantial uncertainty with respect to the utility of mammalian-based screening assays to predict possible effects of EDCs in nonmammalian wildlife. This paper describes preparation of a cDNA library from a small fish model commonly used in ecological risk assessments, the fathead minnow (Pimphales promelas). The cDNA library was subsequently used to isolate and sequence both AR and ERalpha. In addition, the fathead minnow (fh)AR was expressed and characterized with respect to function using saturation and competitive binding assays in COS monkey kidney cells. Saturation experiments along with subsequent Scatchard analysis determined that the Kd of the fhAR for the potent synthetic androgen R1881 was 1.8 nM, which is comparable to that for the human AR in the same assay system. In COS whole cell competitive binding assays, potent androgens such as dihydrotestosterone and 11-ketotestosterone were also shown to be high affinity ligands for the fhAR. We also report affinity of the receptor for a number of environmental contaminants including the AR agonists androstenedione and 17a- and 17beta-trenbolone;AR antagonists such as p,p'-DDE, linuron, and vinclozolin; and the ER agonist 17beta-estradiol. Future plans include comparison of binding affinities of the fhAR to those of the human AR, also expressed in COS cells, using a range of EDCs.     

Untargeted metabolite profiling of serum in rats exposed to pyrraline

Food Science and Biotechnology - Pyrraline, one of advanced glycation end-products, is formed in advanced Maillard reactions. It was reported that the presence of pyrraline was tested to be...     

Volatile metabolite profiling to discriminate diseases of McIntosh apple inoculated with fungal pathogens

Gas chromatography–mass spectrometry (GC/MS) technology was used to profile volatile metabolites from the headspace gas of McIntosh apple (Malus domestica Borkh.), which were non‐inoculated or inoculated with four different fungi, Botrytis cinerea Pers, Penicillium expansum Link, Mucor piriformis Fischer and Monilinia sp. The study yielded a total of 498 different volatile metabolites. Among them only 35 occurred relatively consistently in six replicates over three incubation periods. Of the consistent metabolites, 20 were specific to one or more inoculation agents/diseases, including seven that were unique to apples inoculated with different pathogens. Fluoroethene and 3,4‐dimethyl‐1‐hexene were specific for Penicillium, while butanoic acid butyl ester, 4‐methyl‐1‐hexene and 2‐methyltetrazole were specific for Mucor. Similarly, acetic acid methyl ester and fluoroethane were specific to Botrytis and Monilinia, respectively. The method developed in this study can be used by storage managers to detect apple diseases at an early stage of disease progress and use this to manage apple diseases in storage, after further validation under commercial conditions. Copyright © 2004 Society of Chemical Industry     

Bioactive compounds and antioxidant activities of the Korean lotus leaf (Nelumbo nucifera) condiment: volatile and nonvolatile metabolite profiling during fermentation

Recent studies have shown that lotus (Nelumbo nucifera) leaf has various pharmacological effects. However, there have been no scientific investigations into these leaves as a fermented crude liquid. This study compared the in vitro antioxidant capacity of lotus leaf‐fermented broth (LLFB) with that of 57° Brix sugar broth (SB) as a control over a period of 6 months based on 1,1‐diphenyl‐2‐picrylhydrazyl radical scavenging activity, ferric ion reducing power, superoxide dismutase‐like activity, tyrosinase inhibition and nitrite scavenging activity. The dominant species during fermentation process were Leuconostoc lactis and Saccharomyces cerevisiae for LLFB. A total of thirty‐six metabolites such as alkaloids, fatty acids, organic acids, phenolics, sugar and sugar derivatives, ethyl esters and monoterpenoids differed between SB and LLFB using gas chromatography–mass spectrometry. Meanwhile, nine volatile compounds in LLFB contributed pleasant, slightly sweetish and fruity odour of condiment and sensory evaluation score of 4.06 ± 1.52 in the proportion of 1:9 LLFB/water.     

抗（耐）甜菜丛根病新品种中甜—双丰17号（张甜201）的选育

抗（耐）甜菜丛根病新品种中甜一双丰１７号（张甜２０１）是以抗（耐）丛根病品系抗１和高糖，抗褐斑病品种双丰８号杂交而成的普通二倍体甜菜新品种。该品种块根严重，含糖中等，工艺品质好，抗（耐）丛根病及褐斑病性强，在１９９４～１９９５两年国家“八五”科技攻关抗（耐）甜菜丛根病品种联合区试中达标，平均块根产量为３２３８．７ｋｇ／亩，比对照品种增产３６０．５％，含糖率（１４．７８％）提高３．３９个百分点，产糖     

Global gene expression profiling in larval zebrafish exposed to microcystin-LR and microcystis reveals endocrine disrupting effects of Cyanobacteria

Microcystis blooms occur worldwide and threaten aquatic ecosystems and human health. Sublethal effects on early developmental stages of fish are largely unknown, and research has mainly focused on microcystin toxins (such as MC-LR) rather than Microcystis cells. We exposed (96 h) zebrafish larvae to purified MC-LR (0-1000 μg/L) or lyophilized Microcystis aeruginosa containing 4.5 μg/L MC-LR and evaluated changes in global gene expression (Affymetrix GeneChip zebrafish genome arrays). Significant changes in gene expression (≥ 1.7-fold change, p < 0.0001) were determined with Rosetta Resolver 7.0, and ontology analysis was conducted with the DAVID bioinformatics tool. The number of differentially expressed genes relative to control increased with MC-LR concentration and included genes related to known mechanisms of action for MC-LR in mammals and older life stages of fish, as well as genes unique to larval zebrafish. Up-regulation of vitellogenin genes (vtg) (19.2-fold to >100-fold on arrays; 619.3-fold confirmed by quantitative PCR) was observed in Microcystis-exposed larvae but not in larvae exposed to MC-LR. Up-regulation of vtg indicates exposure to estrogenic substance(s) and suggests that Microcystis may be a natural source of environmental estrogens. Concerns about effects of Microcystis blooms may extend beyond those associated with the microcystin toxin.     

Comparative metabolite profiling of raw milk from subclinical and clinical mastitis cows using 1H-NMR combined with chemometric analysis

The objectives of this study were to characterise and compare non-volatile metabolite profiles of raw milk from healthy, subclinical and clinical mastitis cows using a non-targeted proton nuclear magnetic resonance (¹H-NMR) metabolomic approach. A total of 46 metabolites were identified. Multivariate analysis allowed discrimination of milk from healthy and mastitis cows according to their ¹H-NMR metabolite profiles. Significant increases in acetate, formate, lactate, benzoate, hippurate, β-hydroxybutyrate, valerate, alanine, histidine, isoleucine, leucine, N-acetylamino acid, phenylalanine, threonine and valine content were associated with extremely high somatic cell count and clinical mastitis status of the cows. Furthermore, significant rises in hippurate, valerate, N-acetylglucosamine, histidine, isoleucine and leucine content could be used as potential biomarker for indicating subclinical mastitis infection of the udders. Beside the existing biomarkers for diagnosing mastitis in literatures, we found additional candidate metabolites, such as alanine, valerate and N-acetylglucosamine, in correlation with mastitis status of Thai crossbred Holstein dairy cows.     

Investigating Dried Distiller's Grains with Solubles vulnerability to Tribolium castaneum (Herbst) infestation by using choice and no-choice experiments

Demand for Dried Distiller's Grains with Solubles (DDGS) in international markets and the United States has increased during the past few years. Knowledge of DDGS supplemented animal feed vulnerability to insect infestation is critical for safe feed storage. To assess this vulnerability, it is necessary to know how DDGS is susceptible to insect infestation, while stored as raw ingredient. This research focused on the susceptibility of different types of DDGS (raw and ground) to red flour beetle, T. castaneum, infestation under 30% and 50% relative humidity (r.h.) regimes. Larval period at 30% r.h. increased 2–3 fold on raw DDGS diets with larger particle sizes (PSs) compared with their normal laboratory diet, a mixture of flour and yeast (9:1) (F/Y). However, grinding DDGS samples and increasing the r.h. to 50% decreased the amount of time required for insect development thus increasing DDGS vulnerability to T. castaneum infestation compared with raw DDGS at r.h. of 30%. As was expected, T. castaneum egg and pupal development were not affected by diet or humidity. The results suggested that DDGS as a raw ingredient at 30% r.h. was not a suitable food source for T. castaneum and given a choice, the majority of T. castaneum adults prefer laboratory diet over DDGS. Additionally, fecundity was significantly lower on DDGS compared with the control diets (F/Y and ground corn (GCORN)). These results indicated that these types of DDGS were not suitable developmental diets compared with the F/Y diet if stored at 30% r.h. with larger PSs.