Aquatic Toxicology 109 (2012) 59–69 Contents lists available at SciVerse ScienceDirect Aquatic Toxicology jou rnal homepage: www.elsevier.com/locate/aquatox Intracellular chromium localization and cell physiological response in the unicellular alga Micrasterias a b c a,∗ Stefanie Volland , Cornelius Lütz , Bernhard Michalke , Ursula Lütz-Meindl a Plant Physiology Division, Cell Biology Department, University of Salzburg, Hellbrunnerstr 34, 5020 Salzburg, Austria b Institute of Botany, University of Innsbruck, Sternwartestrasse 15, 6020 Innsbruck, Austria c Helmholtz Zentrum München, German Research Centre for Environmental Health, Institute of Ecological Chemistry, Ingolstädter Landstrasse 1, 85764 Neuherberg, Germany a r t i c l e i n f o a b s t r a c t Article history: Various contaminants like metals and heavy metals are constantly released into the environment by Received 22 September 2011 anthropogenic activities. The heavy metal chromium has a wide industrial use and exists in two stable Received in revised form oxidation states: trivalent and hexavalent. Chromium can cause harm to cell metabolism and develop- 21 November 2011 ment, when it is taken up by plants instead of necessary micronutrients such as for example iron. The Accepted 24 November 2011 uptake of Cr VI into plant cells has been reported to be an active process via carriers of essential anions, while the cation Cr III seems to be taken up inactively. Micrasterias denticulata, an unicellular green alga Keywords: of the family Desmidiaceae is a well-studied cell biological model organism. Cr III and VI had inhibit- Chromium ing effects on its cell development, while cell division rates were only impaired by Cr VI. Transmission Electron energy loss spectroscopy Glutathione electron microscopy (TEM) revealed ultrastructural changes such as increased vacuolization, condensed Iron cytoplasm and dark precipitations in the cell wall after 3 weeks of Cr VI treatment. Electron energy Micrasterias denticulata loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) were applied to measure intracellular Ultrastructure chromium distribution. Chromium was only detected after 3 weeks of 10 ␮M Cr VI treatment in electron dense precipitations found in bag-like structures along the inner side of the cell walls together with iron and elevated levels of oxygen, pointing toward an accumulation respectively extrusion of chromium in form of an iron–oxygen compound. Atomic emission spectroscopy (EMS) revealed that Micrasterias cells are able to accumulate considerable amounts of chromium and iron. During chromium treatment the Cr:Fe ratio shifted in favor of chromium, which implied that chromium may be taken up instead of iron. Significant and rapid increase of ROS production within the first 5 min of treatment confirms an active Cr VI uptake. SOD and CAT activity after Cr VI treatment did not show a response, while the glutathione pool determined by immuno-TEM decreased significantly in chromium treated cells, showing that glutathione is playing a major role in intracellular ROS and chromium detoxification. © 2011 Elsevier B.V. All rights reserved. 1. Introduction into the environment by processes such as electroplating, tanning, polishing, painting, pigment manufacture and wood preservation Chromium is the seventh most abundant metal in the earth’s (Peralta-Videa et al., 2009). These anthropogenic activities have led crust (Panda and Choudhury, 2005) and is naturally occurring in to a widespread contamination of the environment. soil, but can be found in all phases of the environment. In fresh Chromium is not an essential element for plant nutrition, but −1 water concentrations range from 0.1 to 117 ␮g l , while concen- may nevertheless be taken up by plants (Liu et al., 2008). Only two −1 trations in serpentine soils can reach up to 125 g kg (Shanker oxidative forms Cr III and Cr VI are stable enough to occur naturally, et al., 2005). Chromium has a wide industrial use and is released but they are drastically different in charge, physiochemical prop- erties as well as chemical and biochemical reactivity (Kotas and Stasicka, 2000). Overall, Cr VI is considered to be the more toxic than Cr III. As an anion it is negatively charged and highly soluble Abbreviations: CAT, catalase; EELS, electron energy loss spectroscopy; ESI, in water and thus has a better bioavailability and is more mobile electron spectroscopic imaging; ROS, reactive oxygen species; SOD, superoxide dis- than the cationic form Cr III. Like other heavy metals chromium mutase. ∗ is phytotoxic and can result in growth inhibition, degrade pho- Corresponding author. Tel.: +43 662 8044 5555; fax: +43 662 8044 619. tosynthetic pigments, lead to nutrient and water imbalance and E-mail addresses: [email protected] (S. Volland), induce oxidative stress (Panda and Choudhury, 2005). Terrestrial [email protected] (C. Lütz), [email protected] (B. Michalke), [email protected] (U. Lütz-Meindl). plants take up essential and non-essential elements from the soil, 0166-445X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2011.11.013 60 S. Volland et al. / Aquatic Toxicology 109 (2012) 59–69 while aquatic plants take up ions from all their surroundings. There range: 1 mM to 10 nM CrKO8S2 × 12 H2O (Cr III) and 1 mM to are many studies of the effects of chromium on higher plants (Liu 10 nM K2Cr2O7 (Cr VI). The cells were incubated for 4 h and the and Kottke, 2003; Rai and Mehrotra, 2008; Upadhyay and Panda, effects were observed after 2 and 4 h with a Univar light microscope 2010), but in respect to algae most research focuses on biosorp- (Reichert, Vienna, Austria). Pictures were acquired with a Canon tion abilities of certain species for phytoremidiation to remove Power Shot G5 camera (Tokyo, Japan). chromium from contaminated water (Sheng et al., 2004; Rai et al., Purposely a wide range of chromium concentrations was tested 2005; Gupta and Rastogi, 2008,). Only few studies investigate the to find the highest non-lethal concentrations, which had the effects of chromium on physiological processes in the algal cells strongest effect on the alga, intending to determine suitable con- (e.g. Hörcsik et al., 2007; Vignati et al., 2010) and none seem to centrations for electron microscopical investigations and long term determine where chromium is located intracellular. Nevertheless treatments of Micrasterias. it is of relevance to study not only how much metal can be accu- mulated, but also to understand how the contaminant is entering a 2.4. Cell vitality-assay plant cell, what effects it causes on cell physiology, as well as devel- opment, whether it is compartmentalized and which detoxification The percentage of living cells was determined by analyzing mechanisms exist. This is particularly important since plants are an the ability of the cells to perform plasmolysis. After chromium essential source of food to animals and humans and they are also treatment 50 cells were collected and the nutrient solution was used as resource for medical drugs and other commonly used prod- substituted with 500 mM sorbitol. Cells not undergoing plasmolysis ucts. When plants are cultivated in contaminated areas there is a within 15 min sorbitol exposure were assumed dead and counted. risk of heavy metal accumulation, allowing contaminants such as Cell vitality-assays were carried out in triplets with 1 mM Cr III and ␮ ␮ chromium to enter the food chain (Gorbi et al., 2002; Rai et al., Cr VI after 4 h treatment and with 5 M and 10 M Cr III and Cr VI 2004). Cr VI is not only considered highly toxic to plants but also after 3 weeks treatment. to mammals and humans, due to its detrimental effects on several organs and tissues, it is a potential carcinogen (Peralta-Videa et al., 2.5. Cell division rates 2009). The unicellular, fresh water, green algae Micrasterias denticulata To determine interphase cells, dividing Micrasterias cells were has been employed as a sensitive model organism and may be rep- selected from the cultures after mitosis and grown at culture con- resentative for the bottom of the food chain. Micrasterias has been ditions for 2 days before Cr treatment. The cell division rates of ␮ ␮ shown to respond in similar ways as higher plants in experiments alga cells treated with 5 M and 10 M Cr III and Cr VI were exam- and has been used for many years as a model system in cell biology ined over the course of 21 days and compared to rates of untreated (e.g. Kiermayer, 1981; Meindl, 1993; Holzinger and Lütz-Meindl, control cells. Additionally dividing cells were treated with 1 mM 2002; Eder and Lütz-Meindl, 2008; Darehshouri et al., 2008). Cr VI for 4 h, then transferred into nutrient solution for recovery, This study is intended to analyze the impact of chromium on before their cell division rates were also observed for 21 days. All the unicellular model system Micrasterias at different levels. The experiments were carried out three times starting with 10 cells at effects of Cr III and Cr VI on cell development, division rates, vital- a time. ity and photosynthesis are compared. Further ultrastructure, ROS levels, antioxidative enzyme activities and glutathione levels were 2.6. Photosynthesis and respiration investigated in Cr VI treated cells. The uptake of chromium was not only analyzed quantitatively by atomic emission spectroscopy, Micrasterias cultures were exposed to 5 ␮M and 10 ␮M Cr III but also qualitatively by TEM-coupled electron energy loss spec- and Cr VI for 3 weeks. To determine the physiological status by troscopy (EELS) and electron spectroscopic imaging (ESI) allowing means of photosynthesis and respiration, approximately 2000 cells the determination of chromium accumulation sites at a high spatial were used for each run with 3–4 light/dark cycles, which were resolution.