We determined the degradation rates of the herbicides 2,4-D and 2,4,5-T by two different bacterial communities. One of these originated from soil heavily contaminated with herbicides from Bien Hoa ...airbase, the other from the same soil but amended with additional carbon and Gibbs energy sources. The community from the contaminated, but untreated, soil degraded both 2,4-D and 2,4,5-T within 5 days of cultivation. The one from the amended soil, however, hardly showed any degradation of the compounds throughout 23 days of cultivation. After refreshment of the medium and prolonged culturing, however, this community degraded both herbicides within 25 days with progressively increasing rates. nMDS analysis revealed a highly significant differentiation pattern of the two communities. Cultures inoculated with amended soil showed a significant increase of
Bacillus
and
Paenibacillus
upon prolonged exposure to the herbicides. The succession in the culture from untreated soil, on the other hand, was dominated by species from the Proteobacteria. We were able to isolate two of them and they were shown to be related to
Bordetella petrii
and
Sphingomonas histidinilytica
, successively. Subsequent PCR analyses of their DNA revealed the presence of key genes involved in the degradation of the herbicides. This study provides a more fundamental understanding of the biodegradation of 2,4-D and 2,4,5-T by displaying part of the bacterial community succession during their breakdown allowing a comprehensive view on potential key degraders.
Four bacterial strains were isolated from enrichment cultures inoculated with soil from Bien Hoa military base in Vietnam contaminated with the herbicides 2,4-dichlorophenoxyacetate (2,4-D) and ...2,4,5-trichlorophenoxyacetate (2,4,5-T). They were classified as
Pseudomonas aeruginosa
BT1 2.2,
Sphingomonas histidinilytica
BT1 5.2,
Bordetella petrii
BT1 9.2, and
Achromobacter xylosoxidans
BT1 10.2. All four were able to degrade 2,4-D and 2,4,5-T, but only the last three species used them as the sole sources of carbon and energy. Mass balance analyses suggest that between 33 and 46% of the carbon in the herbicides is incorporated into dry weight (DW). We obtained insight into their degradation pathways by the genomic analysis of these strains. A
tfdCDEF
gene cluster was found in
A. xylosoxidans
BT1 10.2 with amino acid sequences of their gene products showing high identity to those in
B. petrii
DSM12804.
Bordetella petrii
BT1 9.2 has a full complement of the
tfdABCDEF
genes. Surprisingly, the gene organization along with the amino acid sequences of the gene products are virtually identical to those of
Cupriavidus pinatubonensis
JMP134, referred to as type I
tfd
genes, and different from those of
A. xylosoxidans
BT1 10.2 and
B. petrii
DSM12804. We hypothesize that some of the genetic potential to degrade the herbicides has been recruited in recent mating events between these species and other members of the proteobacteria. This is the first report showing that
B. petrii
BT1 9.2 emerges as a key player in the degradation of 2,4-D.
We present a reliable, highly sensitive, and versatile method for the simultaneous determination of endogenous polar (acidic) and apolar (retinol, retinal, and retinyl esters) retinoids in various ...biological matrices. Following a single liquid extraction of retinoids from tissues or plasma with isopropanol, polar retinoids are separated from apolar retinoids and neutral lipids via automated solid-phase extraction using an aminopropyl phase. After vacuum concentration to dryness and reconstitution of the residue in appropriate solvents, the obtained fractions are injected onto two different high-performance liquid chromatography (HPLC)-systems. Polar retinoids are analyzed on a RP18 column (2.1
mm ID) using a buffered gradient composed of methanol and water and on-column-focusing large-volume injection. Apolar retinoids are separated on a normal-bore RP18 column using a nonaqueous gradient composed of acetonitrile, chloroform, and methanol. Both HPLC systems are coupled with UV detection, and retinoids are quantitated against appropriate internal standards. The method was validated with regard to recovery, precision, robustness, selectivity, and analyte stability. Using 400
μl serum or 200
mg tissue, the limits of detection for all-
trans-retinoic acid were 0.15
ng/ml or 0.3
ng/g, respectively. The corresponding values for retinol were 1.2
ng/ml or 2.4
ng/g, respectively. This method was successfully applied to mouse, rat, and human tissue and serum samples.
We evaluated the applicability of combining in vitro bioassays with instrument analyses to identify potential endocrine disrupting pollutants in sulfuric acid-treated extracts of liver and/or blubber ...of high trophic-level animals. Dioxin-like and androgen receptor (AR) antagonistic activities were observed in Baikal seals, common cormorants, raccoon dogs, and finless porpoises by using a panel of rat and human cell-based chemical-activated luciferase gene expression (CALUX) reporter gene bioassays. On the other hand, no activity was detected in estrogen receptor α (ERα)-, glucocorticoid receptor (GR)-, progesterone receptor (PR)-, and peroxisome proliferator-activated receptor γ2 (PPARγ2)-CALUX assays with the sample amount applied. All individual samples (n = 66) showed dioxin-like activity, with values ranging from 21 to 5500 pg CALUX-2,3,7,8-tetrachlorodibenzo-p-dioxin equivalent (TEQ)/g-lipid. Because dioxins are expected to be strong contributors to CALUX-TEQs, the median theoretical contribution of dioxins calculated from the result of chemical analysis to the experimental CALUX-TEQs was estimated to explain up to 130% for all the tested samples (n = 54). Baikal seal extracts (n = 31), but not other extracts, induced AR antagonistic activities that were 8–150 μg CALUX-flutamide equivalent (FluEQ)/g-lipid. p,p′-DDE was identified as an important causative compound for the activity, and its median theoretical contribution to the experimental CALUX-FluEQs was 59% for the tested Baikal seal tissues (n = 25). Our results demonstrate that combining in vitro CALUX assays with instrument analysis is useful for identifying persistent organic pollutant-like compounds in the tissue of wild animals on the basis of in vitro endocrine disruption toxicity.
Anabolic androgenic steroids (AAS) are a class of steroid hormones related to the male hormone testosterone. They are frequently detected as drugs in sport doping control. Being similar to or derived ...from natural male hormones, AAS share the activation of the androgen receptor (AR) as common mechanism of action. The mammalian androgen responsive reporter gene assay (AR CALUX
® bioassay), measuring compounds interacting with the AR can be used for the analysis of AAS without the necessity of knowing their chemical structure beforehand, whereas current chemical–analytical approaches may have difficulty in detecting compounds with unknown structures, such as designer steroids. This study demonstrated that AAS prohibited in sports and potential designer AAS can be detected with this AR reporter gene assay, but that also additional steroid activities of AAS could be found using additional mammalian bioassays for other types of steroid hormones. Mixtures of AAS were found to behave additively in the AR reporter gene assay showing that it is possible to use this method for complex mixtures as are found in doping control samples, including mixtures that are a result of multi drug use. To test if mammalian reporter gene assays could be used for the detection of AAS in urine samples, background steroidal activities were measured. AAS-spiked urine samples, mimicking doping positive samples, showed significantly higher androgenic activities than unspiked samples. GC–MS analysis of endogenous androgens and AR reporter gene assay analysis of urine samples showed how a combined chemical–analytical and bioassay approach can be used to identify samples containing AAS. The results indicate that the AR reporter gene assay, in addition to chemical–analytical methods, can be a valuable tool for the analysis of AAS for doping control purposes.
Dithiocarbamates (DTCs) have a wide variety of applications in diverse fields ranging from agriculture to medicine. DTCs are teratogenic to vertebrates but the mechanisms by which they exert these ...effects are poorly understood. Here, we show that low nanomolar exposure to three DTCs, tetraethylthiuram (thiram), tetramethylthiuram (disulfiram), and sodium metam (metam), leads to craniofacial abnormalities in developing zebrafish embryos that are reminiscent of DTC-induced abnormalities found in higher vertebrates. In order to better understand the molecular events underlying DTC teratogenesis, we exposed embryonic zebrafish (PAC2) cells to thiram and disulfiram and measured changes in gene expression with microarrays. We found differential expression of 166 genes that were specific for exposure to DTCs and identified a network of genes related to connective tissue development and function. Additionally, we found eight downregulated genes related to transforming growth factor β-1 (TGF-β1) signaling, including an essential transcription factor for zebrafish craniofacial development, SRY-box–containing gene 9a (sox9a). Finally, we show that sox9a expression is perturbed in the ceratobranchial arches of DTC-exposed zebrafish, suggesting that this is an important event in the development of DTC-induced craniofacial abnormalities. Together, we provide evidence for a novel teratogenic endpoint and a molecular basis for a better understanding of DTC-induced teratogenesis in vertebrates.
Functional in vitro and in vivo reporter gene assays have recently been developed for the rapid determination of exposure to (xeno)estrogens. The in vitro estrogen receptor (ER)-mediated chemically ...activated luciferase gene expression (ER-CALUX) assay uses T47D human breast cancer cells stably transfected with an ER-mediated luciferase gene construct. In the in vivo assay, transgenic zebrafish are used in which the same luciferase construct has been stably introduced. In both assays, luciferase reporter gene activity can be easily quantified following short-term exposure to chemicals activating endogenous estrogen receptors. The objective of this study was to compare responses by known (xeno)estrogenic compounds in both assays. Exposure to the (xeno)estrogens estradiol (E2), estrone, ethynylestradiol (EE2), o,p‘-DDT, nonylphenol (NP), and di(2-ethylhexyl)phthalate (DEHP) revealed that EE2 was the most potent (xeno)estrogen tested and was 100 times more potent than E2 in the transgenic zebrafish assay, whereas in the in vitro ER-CALUX assay, EE2 and E2 were equipotent. Although the xenoestrogens o,p‘-DDT and NP were full estrogen agonists in the in vitro ER-CALUX assay, only o,p‘-DDT demonstrated weak dose-related estrogenic activity in vivo. To determine if differences in reporter gene activity may be explained by differential affinity of (xeno)estrogens to human and zebrafish ERs, full-length sequences of the zebrafish ER subtypes α, β, and γ were cloned, and transactivation by (xeno)estrogens was compared to human ERα and ERβ. Using transiently transfected recombinant ER and reporter gene constructs, EE2 also showed relatively potent activation of zebrafish ERα and ERβ compared to human ERα and ERβ. Zebrafish ERβ and ERγ showed higher transactivation by (xeno)estrogens relative to E2 than human ERβ.
The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that can be activated by a structurally diverse range of synthetic and natural chemicals, and it mediates the toxic and ...biological effects of environmental contaminants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The spectrum of chemicals that bind to and activate the AhR signal transduction pathway and the identity of materials containing AhR active chemicals is only now being defined. Utilizing AhR-dependent gel retardation and reporter gene bioassays, the screening of extracts of 22 dietary herbal supplements and 21 food products (vegetables and fruits) was performed to identify those containing AhR agonists. Several herbal extracts (ginseng, Fo-Ti, white oak bark, licorice, ginkgo biloba, and black cohosh) stimulated AhR DNA binding and gene expression to levels between 20 and 60% of that produced by TCDD. Although some food extracts (corn, jalapeño pepper, green bell pepper, apple, Brussels sprout, and potato) were relatively potent activators of AhR DNA binding (30−50% of TCDD), only corn and jalapeño pepper extracts induced AhR-dependent luciferase reporter gene expression. However, dilution of corn, jalapeño pepper, bell pepper, and potato extracts dramatically increased their ability to induce luciferase activity, suggesting that these extracts contained AhR antagonists whose effectiveness was overcome by dilution. Overall, these results demonstrate that dietary products can be a major source of naturally occurring AhR ligands to which animals and humans are chronically exposed. Keywords: Ah receptor; 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD; natural ligands; herbs; vegetables; fruits; natural products
The aryl hydrocarbon receptor (AhR) and glucocorticoid receptor (GR) are ligand-activated transcription factors and members of the basic helix–loop–helix Period-aryl hydrocarbon nuclear ...translocator-single minded and nuclear hormone receptor superfamilies, respectively. Besides their individual role as activators of specific gene transcription, also interplay between both transcription factors can be an important mechanism of regulation. In this study, we report that GR can strongly activate AhR-mediated transcription and consequent gene expression in rat H4IIe cells. Reporter gene assays showed an enhanced effect of dexamethasone on the dioxin response mediated by GR in rat H4IIe cells and mouse Hepa 1c1c7 cells, but not in human HepG2 cells and human T47D cells. These deviations between the rodent and human cell lines were confirmed by CYP1A1 enzyme activities. In addition, quantitative reverse transcription–PCR showed enhanced GR-mediated effects of dexamethasone on endogenous 2,3,7,8-tetrachlorodibenzo-p-dioxin target genes as well in rat H4IIe cells, but not in human HepG2 and human T47D cells. Surprisingly, AhR itself was upregulated by combined dioxin/glucocorticoid exposure in rat H4IIe cells but not in the human cells which could be explained by the presence of two putative glucocorticoid response elements in the rat AhR promoter, but not in the human AhR promoter. This GR-mediated expression of dioxin target genes through upregulation of the AhR in rat but not in human cells opens the possibility that dioxin responses in rodent-based models for toxicity differ from humans and provides new insight into the interactions of stress-related pathways, biological effects of dioxin-like compounds and may possibly have implications for risk assessment.
The total economic costs of this crisis exceeded billion Euro. ... the European Commission has established a set of instruments, including a rapid alert system, tight control measures and maximum ...limit values in food , feed and raw materials within the EU and for imported goods and products imported from non-EU countries.
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