Antioxidant enzymes are induced by phenol in the marine microalga Lingulodinium polyedrum.

Knowing the impacts of different anthropogenic activities on ecosystems promotes preservation of aquatic organisms. Aiming to facilitate the identification of polluted or contaminated areas, the study of microalga Lingulodinium polyedrum in phenol-containing medium comprises the determination of toxic and metabolic phenol effects, featuring a possible use of this microorganism as bioindicator for this pollutant. Marine microalga L. polyedrum exposure to phenol increases superoxide dismutase (SOD) and catalase (CAT) activities. The 20% and 50% inhibitory concentrations (IC20 and IC50) of cells exposed to phenol were 40 µmol L(-1) and 120 µmol L(-1), respectively. Phenol biodegradation by L. polyedrum was 0.02 µmol h(-1)cell(-1), and its biotransformation was catalyzed by glutathione S-transferase (GST), phenol hydroxylase and catechol 2,3-dihydroxygenase metabolic pathways. Phenol exposure produced the metabolites 2-hydroxymuconic semialdehyde acid, 1,2-dihydroxybenzene (catechol), and 2-oxo-4-pentenoic acid; also, it induced the activity of key antioxidant biomarker enzymes SOD and CAT by three folds compared to that in the controls. Further, phenol decreased the glutathione/oxidized glutathione ratio (GSH/GSSG), highlighting the effective glutathione oxidation in L. polyedrum. Overall, our results suggest that phenol alters microalga growth conditions and microalgae are sensitive bioindicators to pollution by phenol in marine environments.

Combining P values to improve classification of differential gene expression in the HTself software.

HTself is a web-based bioinformatics tool designed to deal with the classification of differential gene expression in low replication microarray studies. It is based on a statistical test that uses self-self experiments to derive intensity-dependent cutoffs. We developed an extension of HTself, originally released in 2005, by calculating P values instead of using a fixed acceptance level α. As before, the statistic used to compute single-spot P values is obtained from the Gaussian kernel density estimator method applied to self-self data. Different spots corresponding to the same biological gene (replicas) give rise to a set of independent P values that can be combined by well-known statistical methods. The combined P value can be used to decide whether a gene can be considered differentially expressed or not. HTself2 is a new version of HTself that uses P values combination. It is implemented as a user-friendly desktop application to help laboratories without a bioinformatics infrastructure.

Antioxidant therapy attenuates oxidative stress in the blood of subjects exposed to occupational airborne contamination from coal mining extraction and incineration of hospital residues.

Coal mining and incineration of solid residues of health services (SRHS) generate several contaminants that are delivered into the environment, such as heavy metals and dioxins. These xenobiotics can lead to oxidative stress overgeneration in organisms and cause different kinds of pathologies, including cancer. In the present study the concentrations of heavy metals such as lead, copper, iron, manganese and zinc in the urine, as well as several enzymatic and non-enzymatic biomarkers of oxidative stress in the blood (contents of lipoperoxidation = TBARS, protein carbonyls = PC, protein thiols = PT, α-tocopherol = AT, reduced glutathione = GSH, and the activities of glutathione S-transferase = GST, glutathione reductase = GR, glutathione peroxidase = GPx, catalase = CAT and superoxide dismutase = SOD), in the blood of six different groups (n = 20 each) of subjects exposed to airborne contamination related to coal mining as well as incineration of solid residues of health services (SRHS) after vitamin E (800 mg/day) and vitamin C (500 mg/day) supplementation during 6 months, which were compared to the situation before the antioxidant intervention (Ávila et al., Ecotoxicology 18:1150-1157, 2009; Possamai et al., Ecotoxicology 18:1158-1164, 2009). Except for the decreased manganese contents, heavy metal concentrations were elevated in all groups exposed to both sources of airborne contamination when compared to controls. TBARS and PC concentrations, which were elevated before the antioxidant intervention decreased after the antioxidant supplementation. Similarly, the contents of PC, AT and GSH, which were decreased before the antioxidant intervention, reached values near those found in controls, GPx activity was reestablished in underground miners, and SOD, CAT and GST activities were reestablished in all groups. The results showed that the oxidative stress condition detected previously to the antioxidant supplementation in both directly and indirectly subjects exposed to the airborne contamination from coal dusts and SRHS incineration, was attenuated after the antioxidant intervention.

Occupational airborne contamination in south Brazil: 1. Oxidative stress detected in the blood of coal miners.

Reactive oxygen species and nitrogen species have been implicated in the pathogenesis of coal dust-induced toxicity. The present study investigated several oxidative stress biomarkers (Contents of lipoperoxidation = TBARS, reduced = GSH, oxidized = GSSG and total glutathione = TG, alpha-tocopherol, and the activities of glutathione S-transferase = GST, glutathione reductase = GR, glutathione peroxidase = GPx, catalase = CAT and superoxide dismutase = SOD), in the blood of three different groups (n = 20 each) exposed to airborne contamination associated with coal mining activities: underground workers directly exposed, surface workers indirectly exposed, residents indirectly exposed (subjects living near the mines), and controls (non-exposed subjects). Plasma TBARS were increased and whole blood TG and GSH levels were decreased in all groups compared to controls. Plasma alpha-tocopherol contents showed approximately half the values in underground workers compared to controls. GST activity was induced in workers and also in residents at the vicinity of the mining plant, whilst CAT activity was induced only in mine workers. SOD activity was decreased in all groups examined, while GPx activity showed decreased values only in underground miners, and GR did not show any differences among the groups. The results showed that subjects directly and indirectly exposed to coal dusts face an oxidative stress condition. They also indicate that people living in the vicinity of the mine plant are in health risk regarding coal mining-related diseases.

Occupational airborne contamination in South Brazil: 2. Oxidative stress detected in the blood of workers of incineration of hospital residues.

One of the most useful methods for elimination of solid residues of health services (SRHS) is incineration. However, it also provokes the emission of several hazardous air pollutants such as heavy metals, furans and dioxins, which produce reactive oxygen species and oxidative stress. The present study, which is parallel to an accompanied paper (Avila Jr. et al., this issue), investigated several enzymatic and non-enzymatic biomarkers of oxidative stress in the blood (contents of vitamin E, lipoperoxidation = TBARS, reduced glutathione = GSH, oxidized glutathione = GSSG, and activities of glutathione S-transferase = GST, glutathione reductase = GR, glutathione peroxidase = GPx, catalase = CAT and superoxide dismutase = SOD), in three different groups (n = 20 each) exposed to airborne contamination associated with incineration of SRHS: workers directly (ca. 100 m from the incinerator) and indirectly exposed (residents living ca. 5 km the incineration site), and controls (non-exposed subjects). TBARS and GSSG levels were increased whilst GSH, TG and alpha-tocopherol contents were decreased in workers and residents compared to controls. Increased GST and CAT activities and decreased GPx activities were detected in exposed subjects compared to controls, while GR did not show any difference among the groups. In conclusion, subjects directly or indirectly exposed to SRHS are facing an oxidative insult and health risk regarding fly ashes contamination from SRHS incineration.

Aspects of the release of superoxide by leukocytes, and a means by which this is switched off.

Although great progress has been made in understanding the respiratory burst of leukocytes that produce superoxide (O2-), it is possible that a component or components, might have been overlooked. Furthermore, O2- production and its sequels, though cardinal in bactericidal action, might ultimately be damaging to the host's own cells. It is important, therefore, that a biologic mechanism exist to turn off O2- production by stimulated leukocytes. This article offers evidence that methoxatin (PQQ), a redox-cycling orthoquinone, might be involved in O2- production by leukocytes. This is based on the fact that inhibitors of O2- production, such as diphenylene iodonium (DPI) and 4,5-dimethylphenylene diamine (DIMPDA), were shown to sequester PQQ in leukocytes, i.e., to form adducts with that substance. Addition of PQQ to cells blocked with the inhibitors partially restored O2- release. With respect to turning off cellular O2- release, a factor was observed to be released to the medium by old macrophages (14 days old, but not by those less than 7 days old). Such conditioned medium, when added to stimulated neutrophils or macrophages, blocked O2- release. This factor was sensitive to proteases, exhibited molecular sizes of 3 and 11 kDa, and its action was independent of the nature of the stimulus applied to the leukocytes. It was partially purified by column (sizing) chromatography and HPLC. It seems to be a general modulator of the release of reactive oxygen species by phagocytes and is irrespective of phagocytic cellular type, or species from which the cells were derived.

Changes in superoxide dismutase activity and photosynthetic pigment content during growth of marine phytoplankters in batch-cultures.

The ability of phytoplankton to cope with oxidative stress is one of the main factors that influence its survival in the marine environment, when senescence conditions prevail. In a first attempt to investigate the antioxidant strategies of different phytoplanktonic groups face to oxidative stress, the superoxide dismutase (SOD; EC 1.15.1.1) activity and photosynthetic pigment content along the growth curves of the dinoflagellate Lingulodinium polyedrum (Stein) Dodge, the prasinophycean Tetraselmis gracilis (Kylin) Butcher and the diatom Minutocellus polymorphus (Hargraves and Guillard) Hasle, von Stosch and Syvertsen were evaluated in batch-cultures. Total SOD activity was determined by an indirect method involving the inhibition of cytochrome c reduction. The contents of photosynthetic pigments were analysed by HPLC using a reverse phase column (RP-18), based on a ternary gradient. A peak of total SOD activity was detected at the beginning of the T. gracilis and M. polymorphus exponential growth. In L. polyedrum and M. polymorphus, SOD activity increased approximately three times by day 17 of growth, compared to the values obtained on day 3 (exponential phase) of the growth curve. All three species of microalgae had reduced SOD activity at the end of their growth. The levels of peridinin in L. polyedrum increased about 60% by day 17 of growth compared to the values obtained at exponential phase. Tetraselmis gracilis exhibited a remarkable increase (approximately 85%) in beta-carotene concentration after 10-14 days of growth whereas the beta-carotene levels in M. polymorphus decreased about 85% along its growth curve. These findings suggest that the antioxidant response during senescence in batch-cultures differ according to the species. Induction of SOD activity may occur either in the early exponential or stationary growth phases, which is important to prevent oxidative stress triggered by a number of factors that affects growth, such as nutrient and light availability.

A circadian rhythm in the activity of superoxide dismutase in the photosynthetic alga Gonyaulax polyedra.

The activity of superoxide dismutase in cell-free extracts of Gonyaulax made at different times of day and night was found to be three to four times higher during the day. This rhythm continued in cells kept in constant light, indicating that the regulation can be attributed to the cellular circadian clock.

Post-implantation mouse embryos have the capability to generate and release reactive oxygen species.

The capability of the mouse embryo to generate reactive oxygen species (ROS) was examined. Post-implantation embryos were carefully harvested on Day 8 of pregnancy and the production of ROS was quantified using luminol-sensitized chemiluminescence. The embryos were stimulated with either phorbol myristate acetate (PMA) or all-trans-retinal (retinal) and the reaction kinetics were followed over 10 min. ROS secretion was directly proportional to the number of embryos and was suppressed 56% by superoxide dismutase (SOD), 25% by mannitol and as little as 16% by catalase. Embryos deprived of trophoblast showed no light emission suggesting that the source of ROS generation is the trophoblast. Dihydronicotinamide adenine dinucleotide (NADH)-dependent oxidase activity in the plasma membrane of the trophoblast surface was demonstrated by cytochemical methods. The release of ROS into the extracellular medium during the phagocytic process has been related to the cytolytic effect exhibited by these molecules and, perhaps by this means, the trophoblast can play an active role in the phagocytosis of maternal cells during the process of embryo implantation.

Imaging oscillations in Gonyaulax: a chloroplast rhythm of nitrate reductase visualized by immunocytochemistry.

Gonyaulax polyedra is a unicellular marine photosynthetic dinoflagellate known to display numerous circadian rhythms, including bioluminescence, motility, cell division and several chloroplast-related rhythms. Due to this, Gonyaulax has become a widely used model organism for studying the cellular biological clock. In this work we describe another rhythm for Gonyaulax cells also associated with the cell's chloroplasts, a rhythm in localization of the enzyme nitrate reductase (NR). A polyclonal antibody was raised against NR purified from G. polyedra cells and used as a probe in immunogold labelling experiments on cell thin sections, comparing day- and night-phase cells. The enzyme localizes to chloroplasts in day-phase cells, while the enzyme is active, and is largely absent in night-phase cells. Counts of gold particle distribution in day- versus night-phase cells show an approximate three-fold increase in enzyme labelling in day-phase plastids. These results closely approximate the four-fold differences shown for NR activity between day and night Gonyaulax cells by biochemical studies. We conclude from the diurnal difference in labelling that NR is localized in Gonyaulax chloroplasts during the day phase and is absent (broken down) in night-phase cells. Thus NR in Gonyaulax is compartmentalized in the chloroplasts and is therefore subject to similar circadian control mechanisms exhibited for other plastid rhythms.

The effect of light on the biosynthesis of beta-carotene and superoxide dismutase activity in the photosynthetic alga Gonyaulax polyedra.

Daily oscillations of both beta-carotene and superoxide dismutase (SOD) activity are related to the intracellular control of reactive oxygen species (ROS). It is well established that ROS are present in all aerobic cells. We studied the marine dinoflagellate Gonyaulax polyedra which has been extensively used as a model to understand the biological clock at the molecular level. beta-Carotene, besides suppressing singlet molecular oxygen (1O2), may act as a photoreceptor pigment in many photosynthetic cells. The levels of beta-carotene during the day phase were shown to be twice as high as during the night phase. The dose-response curve for light-induced carotenoid synthesis was linear for up to 45 min of light exposure, after which night phase cells contained the same levels of beta-carotene as day phase cells. Cells exposed to light pulses at different times during the dark period displayed the highest beta-carotene induction in the middle of the night. SOD activity of cell-free extracts of G. polyedra was three to four times higher during the day. This rhythm continued in cells kept in constant light, indicating that the regulation can be attributed to the cellular circadian clock. No-denaturing polyacrylamide gels revealed the presence of several SOD isoenzymes in G. polyedra, including CuZnSOD and MnSOD. Furthermore, G. polyedra SOD cross-reacts with a polyclonal antibody raised against SOD. In addition to being gene regulated by ROS concentration, G. polyedra SOD expression seems also to be under the control of the biological clock.

Response of superoxide dismutase to pollutant metal stress in the marine dinoflagellate Gonyaulax polyedra.

The response of superoxide dismutase (SOD) activity in the marine dinoflagellate Gonyaulax polyedra to chronic (5.0 ppb Hg, 0.5 ppm Cd, 2.0 ppm Pb and 0.1 ppm Cu, during 30 days) and acute (10.0 ppb Hg, 1.0 ppm Cd, 5.0 ppm Pb and 0.25 ppm Cu, during 48 hours) exposure to metals was investigated. Under chronic exposure to Hg, Cd, Pb, and Cu, total SOD activity of metal-treated cells increased during the first day of exposure to plateau levels of 134, 148, 127, and 139% of control values respectively. Under acute metal exposure, SOD activity increases were of similar magnitude but much more rapid (within several hours) and of shorter duration. In addition, assays for oxidative damage to lipids revealed high levels of lipid peroxidation in cells kept in either chronic or acute exposure to metals reaching values 2-fold greater than the control group. Changes in SOD activity were dependent on the metal, its concentration, and the time of exposure. Non-denaturing polyacrylamide gels revealed induction of Fe-SOD and Mn-SOD but not Cu-Zn-SOD isoforms in cells kept under acute exposure to metals. These results suggest that oxidative stress may be an important mediator of metal toxicity in algal systems, with SOD providing antioxidant protection.

Acute and chronic effects of toxic metals on viability, encystment and bioluminescence in the dinoflagellate Gonyaulax polyedra.

Toxicity bioassays based on survival were carried out with cells of the marine dinoflagellate Gonyaulax polyedra exposed to mercury (Hg2+ ), cadmium (Cd2+), lead (Pb2+) and copper (Cu2+). The toxicity scale of these metals found was Hg2+ > Cu2+ > Cd2+ > Pb2+. Cells exposed to metals promptly underwent encystment, which is an important strategy for surviving metal exposure. Following 48 h exposure to Cu2+, complete excystment occurred within 96 h after reinoculation of cells in fresh metal-free media, and with Pb2+ partial recovery occurred in that time. Bioluminescence was affected by the metals in a dose-dependent manner primarily by increasing the frequency of flashing, but the glow emission was also altered with acute Cu2+ and Pb2+ treatments. Several physiological processes in G. polyedra are under circadian control. Chronic exposures to metals caused no substantial alterations in the circadian rhythm of bioluminescence glow, indicating that the biological clock of this dinoflagellate is not sensitive to these metals at the concentrations tested.

Molybdate:sulfate ratio affects redox metabolism and viability of the dinoflagellate Lingulodinium polyedrum.

Molybdenum is a transition metal used primarily (90% or more) as an additive to steel and corrosion-resistant alloys in metallurgical industries and its release into the environment is a growing problem. As a catalytic center of some redox enzymes, molybdenum is an essential element for inorganic nitrogen assimilation/fixation, phytohormone synthesis, and free radical metabolism in photosynthesizing species. In oceanic and estuarine waters, microalgae absorb molybdenum as the water-soluble molybdate anion (MoO4(2-)), although MoO4(2-) uptake is thought to compete with uptake of the much more abundant sulfate anion (SO4(2-), approximately 25 mM in seawater). Thus, those aspects of microalgal biology impacted by molybdenum would be better explained by considering both MoO4(2-) and SO4(2-) concentrations in the aquatic milieu. This work examines toxicological, physiological and redox imbalances in the dinoflagellate Lingulodinium polyedrum that have been induced by changes in the molybdate:sulfate ratios. We prepared cultures of Lingulodinium polyedrum grown in artificial seawater containing eight different MoO4(2-) concentrations (from 0 to 200 µM) and three different SO4(2-) concentrations (3.5 mM, 9.6 mM and 25 mM). We measured sulfur content in cells, the activities of the three major antioxidant enzymes (superoxide dismutase, catalase, and ascorbate peroxidase), indexes of oxidative modifications in proteins (carbonyl content) and lipids (thiobarbituric acid-reactive substances, TBARS), the activities of the molybdenum-dependent enzymes xanthine oxidase and nitrate reductase, expression of key protein components of dinoflagellate photosynthesis (peridinin-chlorophyll a protein and ribulose-1,5-biphosphate carboxylase/oxidase) and growth curves. We find evidence for Mo toxicity at relatively high [MoO4(2-)]:[SO4(2-)] ratios. We also find evidence for extensive redox adaptations at Mo levels well below lethal levels.

The interplay between thiol-compounds against chromium (VI) in the freshwater green alga Monoraphidium convolutum: toxicology, photosynthesis, and oxidative stress at a glance.

In this paper, the multifaceted Cr(VI) toxicity over the freshwater green alga Monoraphidium convolutum was assessed by concomitantly monitoring thiol-dependent redox balances, photosynthesis activity and growth-survival scores. Control group showed exponential growth rate at (5.78±0.29) division/day until 8th day with linear increasing chlorophyll a/protein ratios (CHLa/PROT) throughout the period. Cultures of M. convolutum were exposed for 5 days to Cr(VI) concentrations from 0 up to 100mg/L showing that CHLa/PROT ratios were sensibly affected, in agreement to the calculated LC(50,48 h) (5.38±0.72) mg/L from the concentration-response curve of cell mortality after 48 h. Regarding photosynthesis effects, Cr(VI) concentrations >1.0 mg/L showed significant increases in short-term (after 2 h) electron transfer rates (ETR) and quantum yields of photosystem II (Φ(PSII)), followed by subsequent decline of both parameters after 48 and 72 h. Biochemical analyses showed that maximal GSH concentrations in algal cultures were observed upon 1mg Cr(VI)/L and higher dichromate concentrations dramatically increased the activity of antioxidant GSH-dependent enzymes ascorbate peroxidase and glutathione reductase. However, no variation was observed in the cellular GSH levels, whereas GSSG and lipid peroxidation indexes abruptly increased upon 10 mg Cr(VI)/L exposure. Altogether, plant physiology, photosynthesis and biochemical data suggest that the GSH-dependent antioxidant system is capable to sustain M. convolutum viability through efficient photosynthesis activity and adequate antioxidant responses up to Cr(VI) concentrations of 1.0mg/L, when redox unbalances were first evidenced.

Superoxide dismutase in Cryptococcus neoformans varieties gattii, grubi, and neoformans.

Some clear dissimilarities occur among the varieties of Cryptococcus neoformans but there are few studies about the differences among individual yeast antioxidant enzymes. The total superoxide dismutase (SOD) activities and the copper, zinc-depend SOD (Cu,ZnSOD) and manganese-dependent SOD (MnSOD) isoenzymes of five reference C. neoformans strains belonged to A, B, C, AD and D serotypes (Table I) and other nine C. neoformans isolates (Table II) were determined. There were significant differences (p < 0.01 and p < 0.05) in total SOD activity among the varietie gattii (serotype C) and the other varieties. Cu,ZnSOD showed difference (p < 0.05) between A and D serotypes. These results point out a variety and serotype-independent SOD activity in C. neoformans reference strains and the other isolates that were evaluated.

A new member of the leucyl aminopeptidase family purified and identified from a marine unicellular algae.

Leucyl aminopeptidase (LAP; EC 3.4.11.1) activity was purified from crude extracts of the marine unicellular algae Gonyaulax polyedra by a combination of hydrophobic interaction with phenyl sepharose, DEAE-cellulose, and mono-Q HR5/5 ion-exchange chromatography. The undenaturated protein has a molecular mass of about 110 kD and based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme appears to be composed of two possibly identical subunits of 55 kD. The identity of the protein was confirmed by a cross-reaction of the purified protein with an antibody raised against a commercial LAP. Biochemical characterization showed that the Gonyaulax enzyme was similar to most of the previously described LAPs. Gonyaulax LAP is a metallo-enzyme since EDTA and 1,10-phenathroline significantly inhibited activity. Addition of the metal ions Zn(2+), Cu(2+) inhibited 80% of LAP activity, suggesting they are not the natural cofactors of the enzyme. Other metals, such as Ca(2+), Co(2+), Mn(2+), or Mg(2+) (concentrations up to 4 mM), caused no alteration in the total activity of Gonyaulax LAP.

Circadian oscillation of nitrate reductase activity in Gonyaulax polyedra is due to changes in cellular protein levels.

A circadian rhythm in the activity of nitrate reductase (NR; EC 1.6.6.1) isolated from the marine dinoflagellate Gonyaulax polyedra is shown to be attributable to the daily synthesis and destruction of the protein. The enzyme was purified in three steps: gel filtration on S-300 Sephacryl, an Affigel-Blue column, and a diethylaminoethyl ion-exchange column. Undenatured protein shows a molecular mass of about 310 kD; based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme appears to be composed of six possibly identical subunits. The amino acid composition of the G. polyedra NR is very similar to that reported for the NR of barley leaves, Chlorella vulgaris, and Ankistrodesmus braunii. The experiments reported indicate that the cellular expression of NR is under circadian control. In extracts of cells grown under either constant dim light or a light-dark cycle, the activity of NR exhibits a daily rhythm, peaking at midday phase, as does photosynthesis. Staining with affinity-purified polyclonal antibodies, raised in rabbits against purified NR, shows that the amount of protein changes by a factor of about 10, with the maximum occurring in midday phase.

Astaxanthin and peridinin inhibit oxidative damage in Fe(2+)-loaded liposomes: scavenging oxyradicals or changing membrane permeability?

Astaxanthin and peridinin, two typical carotenoids of marine microalgae, and lycopene were incorporated in phosphatidylcholine multilamellar liposomes and tested as inhibitors of lipid oxidation. Contrarily to peridinin results, astaxanthin strongly reduced lipid damage when the lipoperoxidation promoters-H(2)O(2), tert-butyl hydroperoxide (t-ButOOH) or ascorbate-and Fe(2+):EDTA were added simultaneously to the liposomes. In order to check if the antioxidant activity of carotenoids was also related to their effect on membrane permeability, the peroxidation processes were initiated by adding the promoters to Fe(2+)-loaded liposomes (encapsulated in the inner aqueous solution). Despite that the rigidifying effect of carotenoids in membranes was not directly measured here, peridinin probably has decreased membrane permeability to initiators (t-ButOOH > ascorbate > H(2)O(2)) since its incorporation limited oxidative damage on iron-liposomes. On the other hand, the antioxidant activity of astaxanthin in iron-containing vesicles might be derived from its known rigidifying effect and the inherent scavenging ability.

Induction of oxidative stress in the red macroalga Gracilaria tenuistipitata by pollutant metals.

Heavy metals are environmental pollutants that have the potential to induce severe stress-reactions in organisms on land as well as in the sea. We have studied effects of short term sublethal concentrations of copper (Cu2+) and cadmium (Cd2+) on the reactive oxygen metabolism of the marine red macroalga Gracilaria tenuistipitata. Additions of either 0.2 ppm Cu2+ or 1 ppm Cd2+ caused decreased growth (approximately 60%), increased oxidation of lipids and increased oxidative damage to proteins as shown by increased content of protein carbonyl groups. Together this strongly suggests an induction of oxidative stress. Cu2+ caused more oxidative damage than Cd2+. As a response to the increased oxidative stress, addition of Cu2+ induced the activities of catalase, ascorbate peroxidase, and superoxide dismutase. In contrast, Cd2+ only caused increased catalase activity. Ten-fold lower concentrations of the metals did not cause an increase in enzyme activity. Both heavy metals also increased the content of the antioxidants beta-carotene and lutein. The results show that Cd2+ and, to a larger extent, Cu2+ induce oxidative stress in short-term experiments and the seaweed responds by increasing the activity of the reactive oxygen metabolism.