Enhanced protection against oxidative stress in an astaxanthin-overproduction Haematococcus mutant (Chlorophyceae)

Many unicellular green algae can become yellow or red in various natural habitats due to mass accumulation of a secondary carotenoid, such as lutein, or astaxanthin. The accumulation of secondary carotenoids is generally thought to be a survival strategy of the algae under photo-oxidative stress or other adverse environmental conditions. The physiological role of the carotenoids in stress response is less well understood at the subcellular or molecular level. In this study, a stable astaxanthin overproduction mutant (MT 2877) was isolated by chemical mutagenesis of a wild type (WT) of the green microalga Haematococcus pluvialis Flotow NIES-144. MT 2877 was identical to the WT with respect to morphology, pigment composition, and growth kinetics during the early vegetative stage of the life cycle. However, it had the ability to synthesize and accumulate about twice the astaxanthin content of the WT under high light, or under high light in the presence of excess amounts of ferrous sulphate and sodium acetate. Under stress, the mutant exhibited higher photosynthetic activities than the WT, based on considerably higher chlorophyll fluorescence induction, chlorophyll autofluorescence intensities, and oxygen evolution rates. Cell mortality caused by stress was reduced by half in the mutant culture compared with the WT. Enhanced protection of the mutant against stress is attributed to its accelerated carotenogenesis and accumulation of astaxanthin. Our results suggest that MT 2877, or other astaxanthin overproduction Haematococcus mutants, may offer dual benefits, as compared with the wild type, by increasing cellular astaxanthin content while reducing cell mortality during stress-induced carotenogenesis.

Effects of dietary ascorbic acid supplementation on the growth performance, immune and stress response in juvenile Leiocassis longirostris Gunther exposed to ammonia

This experiment was designed to investigate the effect of dietary supplemental ascorbic acid (AA) on the feed intake, growth, serum lysozyme, hepatic superoxide dismutase (SOD) and handling stress response in Chinese longsnout catfish (Leiocassis longirostris Gunther) exposed to three levels of unionized ammonia nitrogen (UIA-N). Juvenile Chinese longsnout catfish were reared in 54 fibreglass tanks with a 3 x 3 factorial design treatment consisting of three supplemental AA levels in ascorbyl 2-monophosphate (38, 364 and 630 mg AA equivalent kg(-1) diet) and three UIA-N concentrations [0.004 (the control), 0.037 and 0.292 mg L-1]. The fish were sampled on the 11th, 32nd and 60th day. On the 62nd day, the remaining fish were subjected to an acute stress by being held in a dipnet out of water for 60 s, and sampled at 30 min post handling. The results showed that the specific growth rate (SGR) in 32 days significantly decreased with increased water UIA-N (P=0.0476) but was not affected by dietary supplemental AA (P > 0.05). After 60 days, SGR, feeding rate (FR) and feed conversion efficiency (FCE) significantly increased with increased dietary supplemental AA (P < 0.001) while remaining unaffected by water UIA-N (P > 0.05). There was no significant interaction between dietary AA and UIA-N for growth responses (P > 0.05). The serum lysozyme activity on the 11th day and the hepatic SOD activity on the 32nd day were significantly affected at high (0.292 mg L-1) water UIA-N. On the 62nd day, the increase in cortisol resulting from acute stress significantly decreased by higher UIA-N (P=0.038). It is suggested that Chinese longsnout catfish displayed an adaptive response after long-term UIA-N exposure, and AA had beneficial effects on the growth and feed intake of catfish and alleviated the negative effects of chronic ammonia stress. A chronically higher ammonia level shows a tendency to inhibit the cortisol response to another acute stressor.

Inductive transcription and protective role of fish heme oxygenase-1 under hypoxic stress

Heme oxygenase-1 is the rate-limiting enzyme in the degradation of heme into biliverdin, carbon monoxide and free divalent iron. In this study, we cloned heme oxygenase isoform 1 (CaHO-1) from a hypoxia-tolerant teleost fish Carassius auratus. The full-length cDNA of CaHO-1 is 1247 bp and encodes a protein of 272 amino acids. RT-PCR and real-time PCR analysis indicated that CaHO-1 was predominantly transcribed in posterior kidney, head kidney, gill and intestine, and induction of gene transcription was observed predominantly in posterior kidney under hypoxic stress. Moreover, the hypoxia-induced transcription was confirmed in goldfish larvae and in in vitro cultured CAB cells. Fluorescence of the HO-1-GFP fusion protein revealed a cytoplasmic and plasma membrane localization, which was consistent with the putative transmembrane structure. Subsequently, we established a stably transfected CAB/pcDNA3.1-HO-1 cell line and a control CAB/pcDNA3.1 cell line, and found that the number of dead cells was obviously reduced in the pcDNA3.1-HO-1-transfected group following 4 days of hypoxic (1% O-2) treatment in comparison with numerous detached dead cells in the control pcDNA3.1-transfected cells. Furthermore, a significant cell viability difference between the two kinds of transfected cells during hypoxia-reoxygenation was revealed. Therefore, the data suggest that fish HO-1 might play a significant protective role in cells in response to hypoxic stress.

Induction of hepatic enzymes and oxidative stress in Chinese rare minnow (Gobiocypris rarus) exposed to waterborne hexabromocyclododecane (HBCDD)

The objective of this study was to evaluate the sub-lethal toxicity of hexabromocyclododecane (HBCDD) in fish. Adult Chinese rare minnows as in vivo models were exposed to waterborne HBCDD from 1 to 500 mu g/l for 14, 28 and 42 days. Hepatic CYP1A1 (ethoxyresorufin-O-deethylase, EROD) and CYP2B1 (pentaoxyresorufin-O-depentylase, PROD) activities were measured. At the same time, molecular biomarkers of oxidative stress were also assayed in the brain, including reactive oxygen species (ROS), lipid peroxidation products (thiobarbituric acid-reactive substances, TBARS), DNA damage and protein carbonyl, as well as superoxide dismutase (SOD) activity and glutathione (GSH) content. DNA damage was evaluated using the Comet assay on erythrocytes. Besides, the content of HBCDD in whole fish was determined after 42 days exposure. The results show that HBCDD could induce EROD and PROD at 500 mu g/l after 28 days exposure, and at 100 to 500 mu g/l after 42 days exposure (P < 0.05), respectively. ROS formation in fish brain was observed to be increased in both time- and dose-dependent manner due to HBCDD exposure. The significant increases in TBARS and protein carbonyl contents occurred in fish brain after 28 and 42 days exposure (P < 0.05). Significant DNA damage in erythrocytes by Comet assay was also found in the 100-500 mu g/l exposure groups (P < 0.05) after 42 days exposure. Moreover, significant depletion in brain GSH content occurred in all treated groups (P < 0.05) and apparent inhibition in SOD activity in brain was observed in the groups of 10-500 mu g/l concentrations during 42 days exposure. The results demonstrate that increasing duration of HBCDD exposure induced EROD and PROD activities, caused excess ROS formation, finally resulted in oxidative damage to lipids, proteins and DNA and decreased antioxidant capacities in fish. Chemical analysis of HBCDD in whole fish showed accumulation up to 654 mu g/g wet weight. (c) 2007 Elsevier B.V. All rights reserved.

Physiological and biochemical responses of Scytonema javanicum (cyanobacterium) to salt stress

Scytonema javanicum (Kutz.) Born et Flah (cyanobacterium) is one of the species distributed widely in the crust of desert soils regularly subjected to severe water stress. To investigate the response of the species to salt stress, many physiological and biochemical parameters, including growth rate, ratio of variable fluorescence to maximum fluorescence (Fv/Fm), reactive oxidative species (ROS), malondialdehyde (MDA), catalase (CAT), and superoxide dismutase (SOD), were determined in culture. The results showed that 50 mM NaCl inhibited growth and Fv/Fm in the medium BG-110, and that the inhibition was maximum after 1-2 days' exposure to salt stress; 50 mM NaCl also increased the contents of ROS and MDA in treated cells, which suggests that salt stress may lead to oxidative damage and lipid peroxidation in the alga. Further, changes in the antioxidative enzymes SOD and CAT in the treated alga were consistent with changes in ROS and MDA at certain extent. These observations suggest that oxidative stress resulting from salt stress in S. javanicum could result in the production of antioxidative enzymes to counteract the oxidative damage, and the enzymes may contribute to the ability of S. javanicum to survive the adverse desert environment. (c) 2007 Elsevier Ltd. All rights reserved.

Response of Microcystis to copper stress - Do phenotypes of Microcystis make a difference in stress tolerance?

To elucidate the role of phenotype in stress-tolerant bloom-forming cyanobacterium Microcystis, two phenotypes of M. aeruginosa-unicellular and colonial strains were selected to investigate how they responded to copper stress. Flow cytometry (FCM) examination indicated that the percents of viable cells in unicellular and colonial Microcystis were 1.92-2.83% and 72.3-97.51%, respectively, under 0.25 mg l(-1) copper sulfate treatment for 24 h. Upon exposure to 0.25 mg l(-1) copper sulfate, the activities of antioxidative enzyme, such as superoxide dismutase (SOD) and catalase (CAT), were significantly increased in colonial Microcystis compared to unicellular Microcystis. Meanwhile, the values of the photosynthetic parameters (F-v/F-m, ETRmax and oxygen evolution rate) decreased more rapidly in unicellular Microcystis than in colonial Microcystis. The results indicate that colonial Microcystis has a higher endurance to copper than unicellular Microcystis. This suggests that the efficient treatment concentration of copper sulfate as algaecides will be dependent on the phenotypes of Microcystis. (C) 2006 Elsevier Ltd. All rights reserved.

Induction of oxidative stress and apoptosis by PFOS and PFOA in primary cultured hepatocytes of freshwater tilapia (Oreochromis niloticus)

Perfluorinated organic compounds (PFOCs) are emerging persistent organic pollutants (POPs) widely present in the environment, wildlife and human. We studied the cellular toxicology of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) on oxidative stress and induction of apoptosis in primary cultured hepatocytes of freshwater tilapia (Oreochromis niloticus). Cultured hepatocytes were exposed to PFOS or PFOA (0, 1, 5, 15 and 30 mg L-1) for 24 h, and a dose-dependent decrease in cell viability was determined using trypan blue exclusion method. Significant induction of reactive oxygen species (ROS) accompanied by increases in activities of superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) were found, while activities of glutathione peroxidase (GPx) and glutathione-S-transferase (GST) were decreased. Glutathione (GSH) content was reduced following treatment of PFOA and PFOS. A dose-dependent increase in the lipid peroxidation (LPO) level (measured as maleic dialdehyde, MDA) was observed only in the PFOA exposure groups, whereas LPO remained unchanged in the PFOS exposure groups. Furthermore, a significant activation of caspase-3, -8, -9 activities was evident in both PFOS and PFOA exposure groups. Typical DNA fragmentation (DNA laddering) was further characterized by agarose gel electrophoresis. The overall results demonstrated that PFOS and PFOA are able to produce oxidative stress and induce apoptosis with involvement of caspases in primary cultured tilapia hepatocytes. (c) 2007 Elsevier B.V. All rights reserved.

Molecular cloning and stress-induced expression of paralichthys olivaceus heme-regulated initiation factor 2 alpha kinase

The heme-regulated initiation factor 2 alpha kinase (HRI) is acknowledged to play an important role in translational shutoff in reticulocytes in response to various cellular stresses. In this study, we report its homologous cDNA cloning and characterization from cultured flounder embryonic cells (FEC) after treatment with UV-inactivated grass carp haemorrhagic virus (GCHV). The full-length cDNA of Paralichthys olivaceus HRI homologue (PoHRI) has 2391 bp and encodes a protein of 651 amino acids. The putative PoHRI protein exhibits high identity with all members of eIF2 alpha kinase family. It contains 12 catalytic subdomains located within the C-terminus of all Ser/Thr protein kinases, a unique kinase insertion of 136 amino acids between subdomains IV and V, and a relatively conserved N-terminal domain (NTD). Upon heat shock, virus infection or Poly PC treatment, PoHRI mRNA and protein are significantly upregulated in FEC cells but show different expression patterns in response to different stresses. In healthy flounders, PoHRI displays a wide tissue distribution at both the mRNA and protein levels. These results indicate that PoHRI is a ubiquitous eIF2a kinase and might play an important role in translational control over nonheme producing FEC cells under different stresses. (c) 2006 Elsevier Ltd. All rights reserved.

Cloning and characterization of a new multi-stress inducible metallothionein gene in Tetrahymena pyriformis

A new multi-stress-inducible metallothionein (MT) gene isoform has been cloned and characterized from the ciliate Tetrahymena pyriformis. Both the 5'- and 3'-UT regions of the Tp-MT2 gene are very different from the previously reported Tp-MT1 isoform in this organism and from other described MT genes in Tetrahymena pigmentosa and Tetrahymena thermophila. The putative protein sequence of Tp-MT2 contains cysteine clusters with characteristics of the typical Tetrahymena Cd-inducible MT genes. However, the sequence has a special feature of four intragenic tandem repeats within its first half, with a conserved structural pattern x(5/8)CCCx(6)CCx(6)CxCxNCxCCK. To investigate the transcriptional activities of both Tp-MT2 and Tp-MT1 genes toward heavy metals (Cd, Hg, Cu, Zn) and H2O2, the mRNA levels of these two isoforms were evaluated by means of real-time quantitative PCR. Results showed that Tp-MT2 had a higher basal expression level than Tp-MT1 and both genes were induced by Cd, Hg, Cu, and Zn ions after short exposure (I h), although to different extents. Cd was the most effective metal inducer of both two isoforms, but the relative expression level of Tp-MT2 was much lower than that of Tp-MT1. Different expression patterns were also shown between the two genes when treated with Cd over a period of 24 h. We suggest that TpMT-1 plays the role of a multi-inducible stress gene, while TpMT-2 may have a more specific function in basal metal homeostasis although it may have undergone a functional differentiation process. The putative functional significance and evolutionary mode of the TpMT-2 isoform are discussed. (c) 2006 Elsevier GmbH. All rights reserved.

Effects of salt stress on carbohydrate metabolism in desert soil alga Microcoleus vaginatus Gom.

The effects of salt stress on carbohydrate metabolism in Microcoleus vaginatus Gom., a cyanobacterium isolated from desert algal crusts, were investigated in the present study. Extracellular total carbohydrates and exopolysaccharides (EPS) in the culture medium produced by M. vaginatus increased significantly during the growth phase and reached a maximum during the stationary phase. The production of extracellular carbohydrates also significantly increased under higher salt concentrations, which was attributed to an increase in low molecular weight carbohydrates. In the presence of NaCl, the production of cellular total carbohydrates decreased and photosynthetic activity was impaired, whereas cellular reducing sugars, water-soluble sugars and sucrose content and sucrose phosphate synthase activity increased, reaching a maximum in the presence of 200 mmol/L NaCl. These parameters were restored to original levels when the algae were transferred to a non-saline medium. Sodium and K+ concentrations of stressed cells decreased significantly and H+-ATPase activity increased after the addition of exogenous sucrose or EPS. The results suggest that EPS and sucrose are synthesized to maintain the cellular osmotic equilibrium between the intra- and extracellular environment, thus protecting algal cells from osmotic damage, which was attributed to the selective exclusion of cellular Na+ and K+ by H+-ATPase.

Exposure of spermatozoa to duroquinone may impair reproduction of the common carp (Cyprinus carpio) through oxidative stress

Toxicity of many waterborne organic contaminants to aquatic organisms is mediated through oxidative damages resulting from the production of reactive oxygen species (ROS). Using duroquinone as a model ROS inducer, we carried out in vitro and in vivo experiments to test the hypothesis that reproduction in common carp (Cyprinus carpio) can be impaired through oxidative damage of their spermatozoa. In vitro exposure of fish spermatozoa to 0, 12.5, 25, 50, 100 and 200 mu M duroquinone for 2 h showed a significant increase in the level of ROS in a dose-dependant manner. Sperm motility was significantly reduced in all exposure groups, but lipid peroxidation (LPO) and DNA strand break (measured by comet assay) were only enhanced at 50 mu M and above. A significant decrease in subsequent hatching rate was recorded in all the exposure groups, despite fertilization rate was not affected. In the in vivo experiment, spermatozoa were collected 24 and 72 h after fish received intra-peritoneal injections of 1.0 and 10 mg kg(-1) body weight duroquinone. DNA damage was clearly evident in spermatozoa of all treatment groups after 72 h exposure, and ROS was significantly enhanced in the high concentration group. LPO however, remained unchanged in both treatment groups. The overall results of both our in vitro and in vivo experiments demonstrated that duroquinone can induce ROS production in spermatozoa, which may impair sperm quality and subsequently reproductive success through oxidative stress. (c) 2006 Elsevier B.V. All rights reserved.

pkn22 (alr2502) encoding a putative Ser/Thr kinase in the cyanobacterium Anabaena sp PCC 7120 is induced by both iron starvation and oxidative stress and regulates the expression of isiA

In cyanobacteria, the isiA gene is required for cell adaptation to oxidative damage caused by the absence of iron. We show here that a putative Ser/Thr kinase gene, pkn22 (alr2052), is activated by iron deficiency and oxidative damage in Anabaena sp. PCC 7120. A pkn22 insertion mutant is unable to grow when iron is limiting. pkn22 regulates the expression of isiA (encoding CP43') but not of isiB (encoding flavodoxin) and psbC (CP43). Fluorescence measurement at 77 K reveals the absence of the typical signature of CP43' associated with photosystem I in the mutant under iron-limiting conditions. We propose that Pkn22 is required for the function of isiA/CP43' and constitutes a regulatory element necessary for stress response. (C) 2003 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.