The hypothalamus-pituitary-thyroid axis in teleosts and amphibians: endocrine disruption and its consequences to natural populations.

Teleosts and pond-breeding amphibians may be exposed to a wide variety of anthropogenic, waterborne contaminants that affect the hypothalamus-pituitary-thyroid (HPT) axis. Because thyroid hormone is required for their normal development and reproduction, the potential impact of HPT-disrupting contaminants on natural teleost and amphibian populations raises special concern. There is laboratory evidence indicating that persistent organic pollutants, heavy metals, pharmaceutical and personal care products, agricultural chemicals, and aerospace products may alter HPT activity, development, and reproduction in teleosts and amphibians. However, at present there is no evidence to clearly link contaminant-induced HPT alterations to impairments in teleost or amphibian population health in the field. Also, with the exception of perchlorate for which laboratory studies have shown a direct link between HPT disruption and adverse impacts on development and reproductive physiology, little is known about if or how other HPT-disrupting contaminants affect organismal performance. Future field studies should focus on establishing temporal associations between the presence of HPT-disrupting chemicals, the occurrence of HPT alterations, and adverse effects on development and reproduction in natural populations; as well as determining how complex mixtures of HPT contaminants affect organismal and population health.

The evaluation of surface and wastewater genotoxicity using the Allium cepa test.

Screening for mutagens in complex environmental mixtures, such as surface water or industrial wastewater, is gradually being accepted as a routine method in environmental monitoring programs. In the present work, the simplified Alliumcepa root assay was utilized to evaluate the possible cyto- and genotoxic effects of surface and wastewaters collected near the Sava River (Croatia) over a three-month monitoring period. Physicochemical characterization of the water samples included measurements of conductivity, chemical and biological oxygen demand, levels of suspended matter and salts, nitrate, nitrite, ammonium, total nitrogen and total phosphorus. Morphological modifications of the A. cepa roots, inhibition of root growth, cell division and induction of mitotic and chromosomal aberrations were observed. The most highly polluted water samples (industrial effluents) caused an inhibition of root growth of over 50%, a decrease in the mitotic index of over 40%, and a considerable increase in chromosomal aberrations compared to the control. The measured biological effects of some water samples appeared related to the physicochemical characteristics. Therefore, mutagenicity/genotoxicity assays should be included, along with conventional chemical analysis, in water quality monitoring programs. Their use would allow the quantification of mutagenic hazards in surface and wastewaters.

Gene expression profiling in the neuroendocrine brain of male goldfish (Carassius auratus) exposed to 17alpha-ethinylestradiol.

17-alpha ethinylestradiol (EE2), a pharmaceutical estrogen, is detectable in water systems worldwide. Although studies report on the effects of xenoestrogens in tissues such as liver and gonad, few studies to date have investigated the effects of EE2 in the vertebrate brain at a large scale. The purpose of this study was to develop a goldfish brain-enriched cDNA array and use this in conjunction with a mixed tissue carp microarray to study the genomic response to EE2 in the brain. Gonad-intact male goldfish were exposed to nominal concentrations of 0.1 nM (29.6 ng/l) and 1.0 nM (296 ng/l) EE2 for 15 days. Male goldfish treated with the higher dose of EE2 had significantly smaller gonads compared with controls. Males also had a significantly reduced level of circulating testosterone (T) and 17beta-estradiol (E2) in both treatment groups. Candidate genes identified by microarray analysis fall into functional categories that include neuropeptides, cell metabolism, and transcription/translation factors. Differentially expressed genes verified by real-time RT-PCR included brain aromatase, secretogranin-III, and interferon-related developmental regulator 1. Our results suggest that the expression of genes in the sexually mature adult brain appears to be resistant to low EE2 exposure but is affected significantly at higher doses of EE2. This study demonstrates that microarray technology is a useful tool to study the effects of endocrine disrupting chemicals on neuroendocrine function and suggest that exposure to EE2 may have significant effects on localized E2 synthesis in the brain by affecting transcription of brain aromatase.

Risk assessment of the National Institute of Standards and Technology petroleum crude oil standard water accommodated fraction: further application of a copepod-based, full life-cycle bioassay.

The U.S. National Institute of Standards and Technology (NIST) petroleum crude oil was used to generate NIST water-accommodated hydrocarbon fractions (WAFs) for standardized assessment of crude oil effects on the copepod Amphiascus tenuiremis. Effects were assessed using a 96-well microplate, full life-cycle test. Briefly, nauplii (age, 24 h) were reared individually to adults (n > or =120 nauplii/treatment) in microplate wells containing 200 microl of treatment solution (seawater control [0%] or 10, 30, 50, or 100% NIST-WAF). Nauplii were monitored through development to adulthood, and mature virgin male:female pairs mated in wells containing original treatments (<30 d). A second bioassay using 0, 10, 30, and 50% WAFs (n > or =60 nauplii/treatment) was conducted to assess the effects of ultraviolet (UV) light on naupliar endpoints (<16 d). In the first experiment, nauplius-to-copepodite survival in exposures to 100% WAF was 27% +/- 6% lower than in controls (92% +/- 1%), but copepodite-to-adult survival was greater than 90% across all treatments. Analysis of development curves showed that nauplii in the 10% WAF developed into copepodites 25% faster, whereas nauplii in the 50 and 100% WAFs developed 17% slower, than controls. Copepodite development into male and female copepods was significantly delayed (2 and 4 d, respectively) in the 100% WAF compared to controls. Although none of the WAF exposures had significant effects on fertilization success or total viable production (p > 0.05), embryo hatching in the 100% WAF was significantly less (70.0% +/- 21.2%) than that in controls (87.0% +/- 19.4%). Results from the UV bioassay showed that relatively short exposures (<14 d) to 30 and 50% WAFs in the presence of UV light caused negative effects on copepod survival and development. Naupliar-stage survival and developmental endpoints were the most sensitive indicators of exposure to the NIST crude oil WAF

Quantitative structure-activity relationships for estrogen receptor binding affinity of phenolic chemicals.

The estrogen receptor (ER) binding affinities of 25 compounds including 15 industrial phenolic chemicals, two phytoestrogens, three natural steroids and one man-made steroid were detected by a binding competition assay. The 17 industrial phenolic chemicals were selected as objective compounds because they are possibly released from epoxy and polyester-styrene resins used in lacquer coatings of concrete tank and lining of steel pipe in water supply system. A quantitative structure-activity relationship (QSAR) for structurally diverse phenols, nine alkylphenols with only one alkyl group, four hydroxyl biphenyls, bisphenol A and four natural and man-made estrogens was established by applying a quantum chemical modeling method. Logarithm of octanol-water coefficient (logPow), molecular volume (V(m)), and energies of the highest occupied molecular orbital ( epsilon (HOMO)) and lowest unoccupied molecular orbital ( epsilon (LUMO)) were selected as hydrophobic, steric (V(m)), and electronic chemical descriptors, respectively. Chemicals capable of ER binding had large V(m) and high epsilon (HOMO), while the effects of logPow and epsilon (LUMO) on the binding affinity could not be identified. The QSAR made successful predictions for the three phytoestrogens. Also, the successful prediction of ER-binding affinity for biochanin A, another phytoestrogen, two indicators of pH (phenolphthalin and phenolphthalein) and one alkylphenolic chemical with three alkyl groups (4-methyl-2,6-di-butyl-phenol), by amending the V(m) in the above-mentioned QSAR according to the electron-density distribution (or HOMO density) is an additional step in the elucidation of chemical steric and electronic parameters for predicting the binding affinities of phenolic compounds.

Characterization of manganese-dependent peroxidase isoenzymes from the ligninolytic fungus Phanerochaete flavido-alba.

Phanerochaete flavido-alba is able to decolorize and detoxify olive oil wastewater (OMW) in a process in which simple and polymeric phenols are removed. An unusual acidic MnP is accumulated during the degradation course. This microorganism produces two families of MnPs. MnP1 has an apparent molecular weight of 45 kDa and is secreted as a mixture of isoenzymes with pI ranging from 5.6 to 4.75. MnP2, which is produced as an unique isoenzyme, has an apparent molecular weight of 55.6 Mr and an unusual acidic pI lower than 2.8. The higher specific peroxidase activity for purified MnP2 was for Mn2+ oxidation. Hydroquinone and methylhydroquinone oxidation by MnP2 was Mn2+ dependent, in reaction mixtures without exogenous H2O2. Conversely, ABTS oxidation was Mn2+ independent. Two different DNA fragments (mnpA and mnpB), amplified by PCR, using MnP2 N-terminal sequence and oligonucleotides deduced from two conserved sequences of other MnPs, code for MnPs that belong to the P. chrysosporium mnp2 subfamily on the basis of intron position. The structure of mnpA and mnpB seems to be related to known manganese peroxidase genes, but mnpA encodes an Alanine instead of a Serine (Ser168) regarded as invariant within typical MnPs.

Effect of olive oil mill wastewater on extracellular ligninolytic enzymes produced by Phanerochaete flavido-alba.

Our previous results have demonstrated that Phanerochaete flavido-alba decoloration, dephenolization and detoxification of olive oil mill wastewater (OMW) were associated with changes in the ligninolytic major exoenzymes accumulated in the cultures. This paper describes the effect of the two main OMW components (monomeric aromatic compounds and a major brownish polymeric pigment), on extracellular P. flavido-alba ligninolytic enzymes. Laccase was the sole ligninolytic enzyme detected in cultures containing monomeric aromatic compounds. Laccase and an acidic manganese-dependent peroxidase (MnPA, pI<2.8) were accumulated in cultures with OMW or polymeric pigment. Also, modified manganese-dependent peroxidases were observed mainly in OMW-supplemented cultures. Laccase was more stable to the effect of OMW toxic components and was accumulated in monomeric aromatic-supplemented cultures, suggesting a more important role than manganese-dependent peroxidases in OMW detoxification. Alternatively, MnPA accumulated in cultures containing the polymeric pigment seems to be more essential than laccase for degradation of this recalcitrant macromolecule by P. flavido-alba.

The role of humic substances in drinking water in Kashin-Beck disease in China.

We conducted in vitro and in vivo assays in a selenium-deficient system to determine if organic matter (mainly fulvic acid; FA) is involved in a free radical mechanism of action for Kashin-Beck disease. Cartilage cell culture experiments indicated that the oxy or hydroxy functional groups in FA may interfere with the cell membrane and result in enhancement of lipid peroxidation. Experiments with rats demonstrated that toxicity from FA was reduced when the hydroxy group was blocked. Induction of lipid peroxidation by FA in liver and blood of rats was similar to that exhibited by acetyl phenyl hydrazine. FA accumulated in bone and cartilage, where selenium rarely concentrates. In addition, selenium supplementation in rats' drinking water inhibited the generation of oxy-free radicals in bone. We hypothesized that FA in drinking water is an etiological factor of Kashin-Beck disease and that the mechanism of action involves the oxy and hydroxy groups in FA for the generation of free radicals. Selenium was confirmed to be a preventive factor for Kashin-Beck disease.

Effect of oil and dispersant on growth and chlorophyll a content of the marine microalga Tetraselmis suecica.

Low hydrocarbon concentrations stimulated the growth of Tetraselmis suecica, whereas higher concentrations (200 ppm) inhibited growth. The content of chlorophyll a in this microalga was affected in a similar way. Crude oil had the most marked effects. Dispersant SEAKLIN-101-NT and mixtures of oil and SEAKLIN-101-NT did not show selective toxicity for the microalga, although inhibitory effects could be observed at high concentrations.

The effect of an in situ-produced oil shale processing water on metabolism.

The effects of in situ-produced oil shale retort water on the metabolism of various substrates was studied both in vivo and in vitro. The induction observed in rats was classified as Type I due to an increase in metabolism of hexobarbital and ethylmorphine without subsequent increases in zoxazolamine metabolism. The maximal absorption of the cytochrome-P450-CO complex was observed to be 450 millimicron, also consistent with Type I inducers. Cytochrome P-450 levels were also significantly increased over controls.