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Microbiology

 

Influence of early life-stage exposure to oxidative stressors on antioxidant enzymes, oxidative damage and whole animal growth in turbot (Scophthalmus maximus L.)

D R Livingstone, S C M O'Hara, A Frettsome, D M Lowe & J Rundle
CCMS, Plymouth Marine Laboratory, Citadel Hill, Plymouth, Devon, PL1 2PB

Molecular oxygen is vital for animal life, but potentially toxic reactive oxygen species (ROS), such as the superoxide anion radical, hydrogen peroxide (H2O2) and hydroxyl radical, are continually produced in biological systems as unwanted by-products of normal oxidative metabolism. ROS are detoxified by antioxidant defences, but increased production by various biological and environmental factors can lead to oxidative damage to key molecules (lipid, protein, DNA etc.), oxidative stress and disease. Using S. maximus as a model the primary aims of the project are to investigate whether oxidative stress in early life-stages of fish is a) a common mechanism of toxicity for a number of natural (H2O2, iron, UV) and pollutant (nickel, 1.4-naphthoquinone, polychlorobiphenyls (PCBs) lindane) environmental stressors; and b) has consequences for subsequent growth and development of the organism. Aquaculture tank systems were set up for the rearing of S. maximus larvae from early free feeding to post-metamorphosis stages (23- to 143-day and later post-hatch stages), including the development of culture facilities for the required food organisms (alga, Pavlova Luther; Baker's yeast; Saccharomyces cerevisiae; rotifer, Brachionus plicatilis; crustacean, Artemia salina). Toxicity range-finding experiments were carried out on 30-day old larvae exposed for 24 hours to water-borne PCB-mixture (Arochlor 1254), the organochlorine pesticide lindane (gamma-1,2,3,4,5,6-hexachlorocyclohexane) and the polycyclic aromatic hydrocarbon quinone 1,4-naphthoquinone (concentration range: 1 to 1000 ppb). Mortalities were seen at the higher concentrations of lindane and 1,4-naphthoquinone. 100% mortality was observed, within 30 minutes for 1000 ppb lindane and within 8 hours for 100 ppb lindane, 1000 and 100 ppb 1,4-naphthoquinone. In other treatments the larvae survived the exposure period. Moribund or live larvae were frozen in liquid nitrogen and stored at -80oC prior to analysis of whole-body 1000g supernatants for lipid peroxidation (reaction with thiobarbituric acid (TBA), HPLC resolution of malonaldehyde-TBA adduct and spectrofluorometric quantitation) and potential for NAD(P)H-dependent ROS production (oxidation of 2-keto-4-methiolbutyric acid to ethylene and gas chromatographic resolution and quantitation). Increased lipid peroxidation with increasing contaminant concentration was indicated for all three contaminants (maximal at 1000 ppb Arochlor 1254, 100-1000 ppb lindane and 100 ppb 1,4-naphthoquinone). In contrast, NADH- and/or NADPH-dependent ROS production were indicated to increase with Arochlor 1254 concentration, but decrease with lindane and 1,4-naphthoquinone concentration.

Subsequent experiments have been carried out exposing 30-day larvae to combinations of water borne H2O2 and Ni2+, and investigating the long-term biochemical and growth consequences of 24-hour exposure to Arochlor 1254, lindane and 1,4-naphthoquinone. Additionally, attention is being focused on the determination of oxidised protein (Western blot analysis) and oxidised DNA damage (8-hydroxy-deoxyguanosine formation).

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Charoy CP, Janssen CR, Persoone G, Clement P. 

The swimming behaviour of Brachionus calyciflorus (rotifer) under toxic stress. I. The use of automated trajectometry for determining sublethal effects of chemicals. 

Aquat Toxicol 1995;32(4):271-82.
BIOSIS COPYRIGHT: BIOL ABS. Changes in the locomotory behaviour of the freshwater rotifer Brachionus calyciflorus were used as sublethal indicators of toxic stress. To that end, the swimming behaviour of this rotifer was analysed using an automated tracking system. The swimming speed (temporal factor), the swimming sinuosity (spatial factor), and the periods of swimming were measured and the influence of four chemicals, each representing a distinct chemical class (copper, pentachlorophenol, lindane and 3,4-dichloroaniline), on the rotifer's swimming characteristics were examined. The three test parameters exhibit different sensitivities depending on the chemical tested. The 2-h EC50s obtained with the behavioural test were of the same order of magnitude as the 24-h LC50s resulting from conventional acute toxicity tests with the same test species. This potential use of behavioural test criteria for sublethal toxicity testing with rotifers is briefly discussed.


J Bacteriol 1999 Nov;181(21):6712-9
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Cloning and sequencing of a novel meta-cleavage dioxygenase gene whose product is involved in degradation of gamma-hexachlorocyclohexane in Sphingomonas paucimobilis.

Miyauchi K, Adachi Y, Nagata Y, Takagi M

Department of Biotechnology, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.

Sphingomonas (formerly Pseudomonas) paucimobilis UT26 utilizes gamma-hexachlorocyclohexane (gamma-HCH), a halogenated organic insecticide, as a sole source of carbon and energy. In a previous study, we showed that gamma-HCH is degraded to chlorohydroquinone (CHQ) and then to hydroquinone (HQ), although the rate of reaction from CHQ to HQ was slow (K. Miyauchi, S. K. Suh, Y. Nagata, and M. Takagi, J. Bacteriol. 180:1354-1359, 1998). In this study, we cloned and characterized a gene, designated linE, which is located upstream of linD and is directly involved in the degradation of CHQ. The LinE protein consists of 321 amino acids, and all of the amino acids which are reported to be essential for the activity of meta-cleavage dioxygenases are conserved in LinE. Escherichia coli overproducing LinE could convert both CHQ and HQ, producing gamma-hydroxymuconic semialdehyde and maleylacetate, respectively, with consumption of O(2) but could not convert catechol, which is one of the major substrates for meta-cleavage dioxygenases. LinE seems to be resistant to the acylchloride, which is the ring cleavage product of CHQ and which seems to react with water to be converted to maleylacetate. These results indicated that LinE is a novel type of meta-cleavage dioxygenase, designated (chloro)hydroquinone 1, 2-dioxygenase, which cleaves aromatic rings with two hydroxyl groups at para positions preferably. This study represents a direct demonstration of a new type of ring cleavage pathway for aromatic compounds, the hydroquinone pathway.

PMID: 10542173, UI: 20011338

 
J Bacteriol 1999 Sep;181(17):5409-13
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Two different types of dehalogenases, LinA and LinB, involved in gamma-hexachlorocyclohexane degradation in Sphingomonas paucimobilis UT26 are localized in the periplasmic space without molecular processing.

Nagata Y, Futamura A, Miyauchi K, Takagi M

Department of Biotechnology, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan. aynaga@hongo.ecc.u-tokyo.ac.jp

gamma-Hexachlorocyclohexane (gamma-HCH) is one of several highly chlorinated insecticides that cause serious environmental problems. The cellular proteins of a gamma-HCH-degrading bacterium, Sphingomonas paucimobilis UT26, were fractionated into periplasmic, cytosolic, and membrane fractions after osmotic shock. Most of two different types of dehalogenase, LinA (gamma-hexachlorocyclohexane dehydrochlorinase) and LinB (1,3,4,6-tetrachloro-1,4-cyclohexadiene halidohydrolase), that are involved in the early steps of gamma-HCH degradation in UT26 was detected in the periplasmic fraction and had not undertaken molecular processing. Furthermore, immunoelectron microscopy clearly showed that LinA and LinB are periplasmic proteins. LinA and LinB both lack a typical signal sequence for export, so they may be secreted into the periplasmic space via a hitherto unknown mechanism.

PMID: 10464214, UI: 99395050

FEBS Lett 1999 Mar 5;446(1):177-81
Identification of the catalytic triad in the haloalkane dehalogenase from Sphingomonas paucimobilis UT26.

Hynkova K, Nagata Y, Takagi M, Damborsky J

Department of Environmental Chemistry and Ecotoxicology, Masaryk University, Brno, Czech Republic.

The haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB) is the enzyme involved in the gamma-hexachlorocyclohexane degradation. This enzyme hydrolyses a broad range of halogenated aliphatic compounds via an alkyl-enzyme intermediate. LinB is believed to belong to the family of alpha/beta-hydrolases which employ a catalytic triad, i.e. nucleophile-histidine-acid, during the catalytic reaction. The position of the catalytic triad within the sequence of LinB was probed by a site-directed mutagenesis. The catalytic triad residues of the haloalkane dehalogenase LinB are proposed to be D108, H272 and E132. The topological location of the catalytic acid (E132) is after the beta-strand six which corresponds to the location of catalytic acid in the pancreatic lipase, but not in the haloalkane dehalogenase of Xanthobacter autotrophicus GJ10 which contains the catalytic acid after the beta-strand seven.

PMID: 10100638, UI: 99198737

Characterization of Cyanobacteria (Blue-green Algae) for Removal of Organic Pollutants from Surface Waters

SUMMARY

Cyanobacteria, also known as blue-green algae, are photosynthetic microorganisms that derive their energy from sunlight. Some species are capable of fixing atmospheric nitrogen. Although cyanobacteria are present in various environments, including those with extreme conditions or pollutants, their ability to degrade organic xenobiotics is yet to be studied in detail.

Earlier research by a current researcher at Oak Ridge National Laboratory showed that two filamentous, nitrogen-fixing forms of cyanobacteria, Anabaena sp. and Nostoc ellipsosporum, can degrade the pesticide lindane (gamma -hexachlorocyclohexane) and can be engineered to degrade a common paper mill runoff chemical, p-chlorobenzoic acid. Furthermore, the ability to degrade lindane has been evaluated for 15 species of cyanobacteria that belong to 7 different genera, including filamentous and unicellular types. The rate of lindane degradation varied among the species. Eight strains were able to reduce lindane concentration by 98% or more in 48 hours. Preliminary evidence was obtained for the ability of Anabaena to degrade trichlorethylene. These results point to a potential use of cyanobacteria for degrading organic pollutants in surface waters.

PUBLICATION

  1. T. Kuritz and C.P. Wolk. 1995. “Use of cyanobacteria for biodegradation of organic pollutants.” Appl. Environ. Microbiol. 61: 234–238.

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340: 9/3/96 


Biorestoration of PCB and Lindane Contaminated Soil.

T. PHILLIPS1*, L.A. BEAUDETTE2, K. SHAW1, A. SEECH1, H. LEE2 AND J.T. TREVORS.2  1Grace Bioremediation Technologies, Mississauga, Ontario and 2University of Guelph, Guelph, Ontario. Email: tphillip@uogeulph.ca

Polychlorinated biphenyl- (PCB) and hexachlorocyclohexane (alpha, beta, delta-HCH; gamma-HCH or Lindane) were tested for their biodegradability in contaminated soils using Grace Bioremediation Technologies patented soil amendment technology. Soil microcosms were prepare in mason jars with the soil being cycled through oxic/anoxic cycles.

--HCH-contaminated soil amended with Daramend® technology and Fe resulted in a total HCH loss of 92%. Forty percent greater then the control.
--Lindane (gamma-HCH), a known persistent pesticide, was reduced by 95% in the total HCH loss.
--No significant loss of PCBs was observed using Grace Bioremediation Technologies patented amendment technology as compared to the controls.

NIOSH Medical tests

HCH References

 

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