| 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
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- 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
- T. Kuritz and C.P. Wolk. 1995. “Use of cyanobacteria for biodegradation of
organic pollutants.” Appl. Environ. Microbiol. 61: 234–238.
THE OAK RIDGE COMPLEX
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capabilities of three U.S. Department of Energy facilities in Oak Ridge,
Tennessee: Oak Ridge National Laboratory, a large multipurpose research
institution; the Y-12 Plant, a defense precision manufacturing facility; and the
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remediation and waste management programs. The unique capabilities of the Oak
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P.O. Box 2009, Bldg. 9737
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(800) 356-4USA
4USA@ornl.gov
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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