Environ. Sci. Technol. 2007, 41, 1777-1782
Predicting Copper Toxicity with Its Intracellular or Subcellular Concentration and the Thiol Synthesis in a Marine Diatom AI-JUN MIAO AND WEN-XIONG WANG* Department of Biology, The Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon, Hong Kong
The accumulation and subcellular distribution of copper (Cu) in a marine diatom Thalassiosira weissflogii were examined under different nutrient conditions [nitrogen- (-N) and phosphorus-starved (-P) conditions as well as nutrientenriched conditions with nitrate (+NO3-) and ammonium (+NH4+) as the nitrogen source]. Cu accumulation was induced in the NO3--exposed (+NO3- and -P) cells, suggesting that Cu may be directly or indirectly required for intracellular NO3- reduction. The relationships between the response of the cell-specific growth rate or photosynthetic system II maximum quantum yield (ΦM) to Cu exposure in different nutrient-conditioned cells and the free copper ion concentration, the intracellular Cu concentration (intra-Cu), and the distribution of Cu in different subcellular compartments were also examined. We found that the intraCu was the best Cu toxicity predictor, as it accounted for most of the Cu-induced ΦM response between different nutrient-conditioned cells. The synthesis of intracellular cysteine and five low molecular weight (LMW) thiols was not notably induced at high Cu levels possibly because of the existence of some other Cu detoxification mechanisms. This was further demonstrated by the much lower cysteine and LMW thiol contents in the -N cells with a similar Cu tolerance to the nutrient-enriched cells. Overall, our results suggest that Cu toxicity could be better predicted with the intra-Cu and its subcellular distribution as compared with the currently used free ion activity model and biotic ligand model. However, the LMW thiols had unexpectedly little contribution to Cu detoxification in T. weissflogii.
Introduction Effects of copper (Cu) on marine phytoplankton (e.g., uptake, toxicity, and physiological function) have received extensive attention over the past few decades. As an essential metal, Cu is an important cofactor in plastocyanin, cytochrome C, and ascorbate oxidase (1). However, Cu is toxic at high concentrations because of nonspecific competition for important coordination sites with other essential metals. Several models have been proposed to predict the metal accumulation and toxicity to phytoplankton as a function of free metal ion concentrations (free ion activity model, FIAM) (2, 3) or metals adsorbed at the sensitive sites of algal surface * Corresponding author phone: (852) 23587346; fax: (852) 23581559; e-mail:
[email protected]. 10.1021/es0613963 CCC: $37.00 Published on Web 01/20/2007
2007 American Chemical Society
(biotic ligand model, BLM) (4, 5). However, these models are based on several assumptions and many exceptions have been documented (6, 7). Exploration of a more accurate model is thus warranted. De Schamphelaere et al. (8) attempted to relate intracellular or cell-surface-adsorbed metal concentration to the cell growth inhibition. Recently, we demonstrated that cadmium (Cd) subcellular distribution in the soluble fraction can better explain the toxicity difference of photosynthetic system II (PS II) maximum quantum yield (ΦM) and the cell-specific growth rate (µ) for phytoplankton under different nutrient conditions (9). Whether it is possible to extend this modeling idea to other metals remains unknown. Phytochelatins (PCs) are low molecular weight (LMW) thiols with a general formula (γ-Glu-Cys)n-Gly, where n is the number of γ-Glu-Cys groups and the carboxyl in the functional chain of glutamate is bound with the amino group of the cysteine (γ linkage) (10). PC2-4 (n ) 2-4) are the predominant PCs in phytoplankton. PC synthesis can be strongly induced by several metals and Cd is the most effective activator (11), suggesting that PCs may play an important role in metal detoxification in algae. Trace metals are bound to PCs through coordination with the sulfhydryl group in cysteine with a sulfur-to-metal ratio ranging from 2 to 4. However, both the induction of PCs and their metal-binding stoichiometry are specific to metal and phytoplankton species. Accordingly, their contribution to metal detoxification may be different for different metals or algal species (11-14). Glutathione (GSH) is another important LMW thiol involved in the alleviation of intracellular oxidative stress as well as the metal sequestration and is the substrate for PC synthesis (13, 15). Examination of intracellular PC and GSH contents as well as their precursors (i.e., cysteine and γ-GluCys [γGC]) would be helpful in determining the potential Cu tolerances of different nutrient-conditioned algae. Nitrogen (N) and phosphorus (P) limitations are both frequently observed in marine waters (16), which may influence metal assimilation by phytoplankton. N limitation can strongly suppress metal accumulation in both laboratory cultures and natural phytoplankton communities while P has little effect (9, 17, 18). Meanwhile, the algal biochemical composition varies under different nutrient conditions (19). Therefore, different nutrient-conditioned phytoplankton may have different metal sensitivities. The algal tolerance to metal may be affected not only by the ambient nutrient concentration but also by different N sources (nitrate [NO3-] vs ammonium [NH4+]). As NH4+ is preferred over NO3- by phytoplankton and its assimilation could save energy used for NO3- reduction (20), more energy may be available for metal detoxification, which leads to the hypothesis that phytoplankton exposed to NH4+ are more metal-tolerant. In this study, we examined the intracellular Cu accumulation and its subcellular distributions in a marine diatom Thalassiosira weissflogii under different macronutrient conditions. The main objectives of our study were to examine (1) the effects of macronutrients on Cu inhibition of ΦM and µ, (2) whether the Cu inhibition toxicity can be better predicted by the intracellular or subcellular Cu concentration, and (3) whether or not PCs and GSH play an important role in Cu detoxification in T. weissflogii.
Materials and Methods Phytoplankton and Culture Conditions. An axenic culture of the diatom Thalassiosira weissflogii (CCMP 1048) was obtained from the Provasoli-Guillard Center for the Culture of Marine Phytoplankton, Bigelow Laboratory, West Boothbay VOL. 41, NO. 5, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
1777
Harbor, ME. The cells were maintained under sterile conditions in f/2 medium (21) at 19 °C with a light illumination of 170 µmol photons m-2 s-1 in a 14:10 light-dark (LD) cycle. The pH value of the medium was 8.2 ( 0.1. Seawater for the culture was collected 10 km off East Hong Kong to minimize any anthropogenic influences and was filtered through a 0.22µm Poretic membrane before use. Cu-Exposure Media. The preparation of Cu-exposure media was similar to that of the Cd-exposure media in our previous study (9). Briefly, artificial seawater was used for all the Cu-exposure experiments. N, P, silicate, and vitamins were spiked at the f/2 levels (e.g., 882.4 and 36.2 µmol L-1 for N and P, respectively), while the trace metals were at the f/10 levels except for Cu. N and P were not added in the nitrogen- (-N) and phosphorus-starved (-P) experiments, respectively. NO3- was used as the N source in the +NO3(nutrient enriched with NO3- as the N source) and -P experiments while it was replaced by the same amount of NH4+ in the +NH4+ (nutrient enriched with NH4+ as the N source) experiments. There were six treatments (i.e., treatments A-F) with two replicates each in every Cu toxicity test and the dissolved Cu concentration (nominal) was 0.008, 5, 10, 50, 100, and 110 µmol L-1, respectively. Treatment A served as the control for the experiments. One hundred micromole L-1 nitrilotriacetate (NTA) was added to keep the free Cu ion concentration ([Cu2+]) constant. The pH value of the media was adjusted to 8.2 ( 0.1 with 1 mol L-1 Suprapure NaOH (Merck, Darmstadt, Germany). [Cu2+] was calculated by the MINEQL+ software package (Version 4.5 from Environmental Research Software, Hallowell, ME) with the updated thermodynamic constants and the calibration set on ionic strength. It was 1.00 × 10-13, 7.94 × 10-11, 2.00 × 10-10, 2.00 × 10-9, 7.94 × 10-8, and 2.51 × 10-6 mol L-1 for treatments A-F, respectively. The high [Cu2+], although less environmentally realistic, had to be used to observe a complete dose response from 0 to 100% inhibition. Pulse Amplitude Modulated Fluorometry (PAM) Measurements. Cu-exposure experiments were performed on the four different nutrient-conditioned (i.e., +NO3-, -N, -P, and +NH4+) cells. After reaching the midexponential phase in their development, the cells were gently filtered (
