Do heavy metals induce. HSP60 in rotifers?

Transcription

1 Do heavy metals induce HSP60 in rotifers? Edith M. Soto 1 2, Elizabeth Walsh 3, Judith Rios 45, Corresponding Author: Judith V. Rios Phone #: jvrios@utep.edu 1 Department of Biological Sciences The University of Texas at El Paso El Paso, TX Department of Biological Sciences El Paso Community College - Valle Verde Campus El Paso, TX Department of Biological Sciences The University of Texas at El Paso El Paso, TX Environmental Science and Engineering The University of Texas at El Paso El Paso, TX Department of Biological Sciences The University of Texas at El Paso El Paso, TX

2 Abstract Stress can cause many different effects on animals. It may cause evolutionary, behavioral, or physiological effects. A physiological response, in many animals, is induction of Heat Shock Protein 60. Stress induces the synthesis of this protein in rotifers as well. This can be used as a defense mechanism for cellular damage in rotifers. In this project rotifers were exposed to heat (37 C) at different time periods (10, 20, 30 and 40 minutes) and to high ( µg/µl) and low (10 µg/µl) concentrations of Arsenic, Chromium, Copper, Nickel, Lead, and Zinc, as well as to combinations of these chemicals. This project was done to find out if these elements, which are found in the Rio Grande (El Paso/Juarez area), will induce HSP60 in Plationus patulus. Introduction In this project we are trying to find out if As and heavy metals (Cr, Cu, Ni, Pb, & Zn) can induce protein production in organisms. More specifically, if these chemicals will induce a protein called Heat Shock Protein 60, in rotifers. This in turn will help us find out if we can use these organisms as bio-indicators for water criteria in aquatic systems such as the Rio Grande. Different tests such as the Bradford Method (used for protein quantification) and the Western Blot technique (used for protein identification) were performed in this project. HSP60 is a stress protein. It was previously called Heat Shock Protein 60 because it was originally discovered to be induced by heat (Ritossa, 1962). The 60 stands for its molecular weight, which is 60 kilo Daltons (R. D. Handy). It is now known as stress protein because it has been discovered to be induced by several stressors such as pollution, 2

3 chemicals etc. This protein is also known as chaperonin and Gro EL. HSP60 is located in the mitochondria and chloroplasts of eukaryotes, and in bacteria (such as E. coli). They play an important role in protein-protein interactions, including folding and assisting in establishing of proper protein conformation, and prevention of inappropriate protein aggregation (R. D. Handy). This protein is found in most organisms, including rotifers. Rotifers are micro-invertebrates that live mainly in freshwater; however, there are also some rotifers that live in salty water. They rage up to 2 mm in length and have two main characteristics (R. Wallace and T. Snell). The first main characteristic is their trophi (jaws), which they use for eating and which aids in species identification. Their second main characteristic is their ciliated corona, which they use for locomotion and for food gathering. There are over 2,000 species (R. Wallace and T. Snell) of rotifers however, in our project we will be using the Plationus patulous. The diet of a rotifer consists mainly of unicellular algae however; there are those that eat decomposing material and bacteria, and also those that eat other rotifers (R. Wallace and T. Snell). Rotifers reproduce differently according to their class. The types of rotifers we are using in this project reproduce through cyclical parthenogenesis. This means that they reproduce asexually for the most part, but under adverse environmental conditions they may produce a daughter that can reproduce sexually and then a male will more than likely be born (R. Wallace and T. Snell). We try to maintain a male-free environment in our samples, since they represent negative environmental conditions, meaning they are only present when they re over crowded or under unfavorable conditions (R. Wallace and T. Snell). The rotifers used in our project were collected from the Rio Grande. The Rio Grande, as we all probably know, has suffered by man s impact through 3

4 agricultural practice (irrigation back-flow), urban input (sewage effluent), and industrial input (smelters that emit chemicals into the river). All these things have caused a degraded environment in the Rio Grande (El Paso-Juarez area). Methods and Materials: The procedures done in this project included a survey in the Rio Grande (El Paso- Juarez area) in order to find out if there was As and heavy metals (Cr, Cu, Ni, Pb, and Zn) present in this area of the river. Rotifers were collected from the river and cultured for a year before using them for experimentation. The culturing process included: filtering the water, feeding the rotifers three types of unicellular algae (C. vulgaris, Ankis, and C. reihardtii) every other day, transferring rotifers into new petri dishes so they wouldn t be overcrowded, and keeping their environment clean. Then the Metal Exposure Experiment was performed. Exposure: First, two hundred rotifers were separated into different small dishes containing 9 ml of rotifer medium, one day before exposures, to save time. Then we exposed 4 of dishes to heat at 37 C for 10, 20 30, and 40 minutes, starting with the longest time frame. Metal dilutions of defined concentrations were used for the metal exposure and diluted from a stock solution of 100 ppm, assuming that there were 9 ml of rotifer medium in each dish. Metal solutions were then added one at a time to the dishes in ten-minute increments, starting with the lowest concentration first, leaving enough time to filter the rotifers and collect them in vials after the exposure time was completed. Timing was started as soon as the metal solution had been added. Each dish was gently swirled to make sure the metal 4

5 solution was distributed evenly. While the times of exposure were running, rotifers from the controls were collected and concentrated in vials, then homogenized and placed on ice. Metal exposure should be started with the longest exposure time, leaving the small exposure time treatments at the end in order to save time as well. Concentration: The rotifers in the vial were then concentrated, by carefully removing the excess rotifer medium, and placed in a separate dish. Any rotifers that were accidentally transferred to the petri dish were collected with the least amount of liquid and carefully placed in the vial. The liquid in the vials had to be at the same level within them. The level of medium in the vial must be labeled. Then, in a separate vial a mark is made that represents the same level of the vials medium level. With a calibrated pipette the vial was filled up to the mark with rotifer medium to determine the amount of rotifer medium of the samples. Then they were centrifuged for 2 minutes at rpm. Homogenization: 200 µl of rotifer media and 100 µl of Laemmli Buffer were added to the vials. The samples were then homogenized using the homogenizer for a total of thirty seconds. Then the samples were placed on ice. Once that was done, the samples were degraded into a hot water bath (boiling) for 4 minutes. Protein Quantification (Bradford Method): We added 5 µl of rotifer samples that had been exposed to heat and high and low concentrations of As, and heavy metals (Cr, Cu, Ni, Pb, Zn), and 1.5 ml of Bradford solution to different cuvettes and then mixed very carefully. Then we measured the protein in a 600 Optima UV-VIS spectrophotometer at a wavelength of 595nm. 5

6 Determination of HSP60: In order to determine the presence of HSP60, we used the Western Blot technique. In this technique, rotifer samples were exposed to nitrocellulose paper with a vacuum chamber. Then, the nitrocellulose paper was exposed to a blocking solution (gelatin) for 1 hour, rocking slowly. After that, the nitrocellulose paper was washed with a buffer solution (TBST 1X) for 1 hour, changing the buffer every 15 minutes. The paper was then exposed to the first antibody (Ab rabbit αgroel solution) for 1 hour (rocking slowly). After that, the paper was washed again with the same procedure. Then it was exposed to the second antibody (Ab goat-antirabbit IgG with alkaline phosphate) for 1 hour (rocking slowly). The paper was then washed again. After that, the paper was exposed to substrate for alkaline phosphatase. When a purple color started to develop, the paper was transferred to a tray with 5% acetic acid to stop the reaction (for 20 minutes). Finally, the paper was put to dry. Results In the survey done (by Judith Rios) in the Rio Grande (El Paso-Juarez area) the results were positive, meaning these chemicals (As, Cr, Cu, Ni, Pb, and Zn) were indeed found in the river water and sediments. Zn and Pb were found at higher levels than the other elements, especially Zn (seen in Figure 1). With the Bradford test we found the amount of total protein in each rotifer sample after each exposure (seen in Figure 2). The samples that were exposed to heat (37 C) showed insignificant differences compared to the controls (those not exposed). The ones exposed to 0.1 CuSO 4 (control = 0.62 µg/µl) showed an increase of total protein as the time periods increased. However, the highest peak was 2.35 µg/µl of total protein at 30 minutes of exposure. Samples exposed to CuSO 4 (control = 0.6 µg/µl) reached a peak of

7 µg/µl of total protein at 10 minutes of exposure, and then decreased somewhat with the longer treatments. Samples exposed to CuSO 4 (control = 1.75 µg/µl) went down to 0.88 µg/µl of total protein at 5 minutes and reached a peak of 255 µg/µl of total protein at 1 hour of exposure. Samples exposed to 0.02 CuSO 4 (control = 1.5 µg/µl) also seemed to increase in total protein as the time frames increased. Samples exposed to single elements (As, Cr, Cu, Ni, Pb, and Zn) at low concentrations (control = 1.28 µg/µl) seemed to show very similar amounts of total protein, but for Zn. Zn showed 0.2 µg/µl of total protein which was relatively lower than the others. Samples exposed to high concentrations of these elements (control = 1.56 µg/µl) showed a decrease in total protein in the following order: As, Pb, Cu, Ni, Cr, Zn. With the Western Blot technique, we observed the induction response of HSP60 in Plationus patulus exposed to different concentrations (63 µg/l and 100 µg/l) of CuSO 4 for varying exposure times (5, 10, 20, 30, and 60 minutes). Samples were diluted in order to discern differences in HSP60 induction. Dilutions were made based on total protein amount: 48 µg (1X), 24 µg (1/2X), 12 µg (1/4X), 6 µg (1/8X). We found that HSP60 was shown after the 1/8X dilution in both concentrations of CuSO 4 at 0 and 5 minutes exposed. However, the stains representing HSP60 (as seen on Figure 3-A&B) were much clear in the 100 µg/l CuSO 4 (A) concentration than in the 63 µg/l CuSO 4 (B) concentration. Comparison of HSP60 induction response in Plationus patulus exposed to low and high As and heavy metal concentrations are seen in Figure 3-C&D. Control samples were prepared with unexposed rotifers. Dilutions were also made based on total protein amount: 48µg (1X), 24 µg (1/2X), 12 µg (1/4X), 6 µg (1/8X). HSP60 induction was observed at 1/8X dilution of Cr, Cu, Ni and Pb, at low concentrations (as seen in Figure 3-C). HSP60 7

8 induction was also observed at 1/8X dilution at high concentrations of Cr, Cu, Ni, Pb, and also Zn (seen in Figure3-D). Discussion We found that the HSP60 induction is involved with what treatment it is being exposed to as well as the time frame and concentration. According to our Western Blot tests, Cr, Cu, Ni and Pb induced the most HSP60 in Plationus patulus at low concentrations. These metals as well as Zn also induced the most HSP60 in our rotifers at high concentrations; these results were seen much clearer. We believe that, when exposed to high concentrations of these heavy metals, Plationus patulus will induce HSP60, however, if these concentrations are too high, cellular damage may occur. Considering that high levels of Zn and Pb were found in the Rio Grande (El Paso/Juarez area), we think that they may surely have an effect on HSP60 induction in rotifers there. However, further studies should be done to make sure of this. We also think that combinations of these elements may have diverse effects on the rotifers depending on their combinations and concentrations. Further studies are also needed in order to examine this suggestion. Acknowledgements I d like to thank the Bridges to the Future program, Dr. Walsh, and Nick Lannutti for giving me the opportunity to work for them. I also want to thank Judith Rios for giving me permission to try to help her with her project and for all her help and patience. 8

9 References Ritossa, F., A new puffing pattern induced by temperature shock and DNP in Drosophila. Experiential. 18: Bradford MB (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: Cochrane BJ, Irby RB, Snell TW (1991) Effects of copper and tributylin on stress protein abundance in the rotifer Brachionus plicatilis. Comp Biochem Physiol 98C(2/3): Snell TW, Janssen CR (1995) Rotifers in ecotoxicology: a view. Hydrobiologia : Snell TW, Moffat BD, Janssen CR, Persoone G (1991) Acute toxicity test using rotifers. III. Effects of temperature, strain and exposure time on the sensitivity of Brachionus plicatilis. Ecotoxicol Toxicol Wat Qual 6:63-75 Ellis RJ (1987) Proteins as molecular chaperones. Nature (Lond) 328: Lundebye, A-K, Langston, W.J. and Deplege, M.H. (1996). Stress proteins and condition index as biomarkers of tributylin exposure and effect in mussels. Ecotoxycology 5, 1-10 Nover, L (1991). The Heat Shock Response. Boca Raton, Florida: CRC Press. Pedersen, S. N. and Lundebye, A.K. (1996). Metallothionein and stress protein levels in shore crabs (Carcinus maenus) along a trace metal gradient in the Fal Estuary System (UK). Marine Environmental Research 42, Sanders BM (1993) Stress proteins in aquatic organisms: an environmental perspective in aquatic organisms: an environmental perspective. Crit Rev Tox 23:49-75 Snell TW, Persoone G (1989) Acute toxicity bioassay using rotifers. I. A test for brackish and marine environments with Brachionus lpicatilis. Aquat Toxicol 14:65-80 Wallace RL, Snell TW (1991) Ecology and Classification of North American Fresh Water Invertebrates 8:

10 Figure Legends Figure-1: This graph represents the levels of each element (As, Cr, Cu, Ni, Pb, Zn, and Cd) found in the Rio Grande (El Paso/Juarez area). Figure-2: In this table C and H represent controls and heat exposed treatments, respectively. In single element treatments, rotifers were exposed for 20 min at low concentrations and 60 minutes at high concentrations Figure-3: This figure shows the induction response of HSP60 in rotifers exposed to different concentrations of CuSO 4 and for varying exposure times (5, 10, 20, 30, and 60 minutes). Rotifers were exposed to 63 (A), and 100 µg/l (B) of CuSO 4. Dilutions were made based on total protein amount: 48 µg (1X), 24 µg (1/2X), 12 µg (1/4X), 6 µg (1/8X). Comparison of HSP60 induction response in Plationus patulus exposed to low (C) and high (D) As and heavy metal concentrations. Samples were diluted in order to discern differences in HSP60 induction. Control samples were prepared with unexposed rotifers. Dilutions were also made based on total protein amount: 48µg (1X), 24 µg (1/2X), 12 µg (1/4X), 6 µg (1/8X). 10

11 Figure-1 Content of As and Total Metals in Rio Grande Water As Cr Cu Ni Pb Zn Cd Borderland Montoya Drain American Dam Gaging Station La Hacienda San Elizario Guayuco Sites 11

12 Figure-2 Total protein concentration in rotifer samples. Amount of Standard Amount of Standard Experiment Sample (Exposure Time) Protein(µg/µl) Deviation Sample (Exposure Time) Protein(µg/µl) Deviation Heat Exposure C (10) H (10) C (20) H (20) C (30) H (30) C (40) H (40) Copper Sulfate 0.1 CuSO 4 (0) CuSO 4 (0) Exposure 0.1 CuSO 4 (5) CuSO 4 (5) CuSO 4 (10) CuSO 4 (10) CuSO 4 (20) CuSO 4 (20) CuSO 4 (30) CuSO 4 (30) CuSO 4 (60) CuSO 4 (60) CuSO 4 (0) CuSO 4 (0) CuSO 4 (5) CuSO 4 (5) CuSO 4 (10) CuSO 4 (10) CuSO 4 (20) CuSO 4 (20) CuSO 4 (30) CuSO 4 (30) CuSO 4 (60) CuSO 4 (60) Single Element Control Control Exposure As Low As High Cr Low Cr High Cu Low Cu High Ni Low Ni High Pb Low Pb High Zn Low Zn High

13 Figure-3 Dilution Dilution Exposure Time A HSP60 Std 1X 1/2X 1/4X 1/8X Exposure Time 0 5 B HSP60 Std 1X 1/2X 1/4X 1/8X Control Control As Cr As Cr C Cu Ni D Cu Ni Pb Pb Zn Zn HSP60 Std HSP60 Std 13