CADMIUM STRESS AND MT GENE EXPRESSION IN THE

CADMIUM STRESS AND MT GENE EXPRESSION IN THE PLHC-1 FISH CELL LINE By: James Plymale

Introduction Cheung et al. [1] conducted studies on metal concentrations and metallothionein (MT) gene expression in the Tilapia fish cell line liver tissues. MT has been used as a biomarker of metal contaminations in polluted waters. MT is a small metalbinding protein that regulates cellular response to essential metals (Zn 2+, Cu 2+) and non-essential metal ions (Cd 2+, Hg 2+) [1]. However, only a few studies reported the study of other fish cell lines for this particular gene and its regulation by heavy metal ions in fish cell lines such as Zebrafish and Goldfish. The fish hepatoma cell line (PLHC-1) is a tool used to study cytotoxicity, but MT expression in this cell like has not been characterized. Cheung et al. [1] has reported that the metallothionein gene becomes activated once exposed to metals and induces m. RNA expression levels.

Previous Research Objectives 1) To evaluate the role of the MT gene in the PLHC-1 cell line through differential computational methods l 2) To evaluate RNA expression levels in the MT gene in PLHC-1 cell line by RTPCR l

Methods Gene Identification l Selected MT sequences were researched from NCBI Gen. Bank l Those selected sequences were grouped by similar m. RNA reference sites and aligned in Clustal. W to find a conserved sequence l An aligned sequence of the conserved region was selected and primers and a biotinylated probe was designed for the detection of the conserved region

MT RNA Expression Studies RNA Purification Step 1: Cells were grown for 3 days and treated with and without 10 u. M Cadmium for 24 hours Step 2: Cells were then washed with 5 m. L PBS and then collected by scrapping the petri dish Step 3: Purified RNA using Promega RNA Purification protocol

RNA Wet Lab Analysis Figure 1. RNA Concentration Diagram RNA concentration was measured using the Nano. Drop in Dr. Norton’s lab. The concentrations were as followed: Control (not shown) was given by Zachary Tackett, RNA 1 -black (29. 1 ng/ml; OD 260/280 1. 90), RNA 2 red ( 51. 6 ng/ml; OD 260/280 2. 02), RNA 3 green (14. 4 ng/ml; OD 260/280 1. 70).

(Reverse Transcriptase) RT-PCR Figure 2. RT-PCR Method l l l RNA samples followed the Promega protocol (Figure 2) and ran under these thermocycler conditions: l 1 cycle 45 degrees C, 45 min. l 1 cycle 94 degrees C, 2 min. l 45 cycles l 94 degrees C, 30 sec l 60 degrees C, 1 min l 68 degrees C, 2 min l 1 cycle 68 degrees C, 7 min l 1 cycle 4 degrees, hold Samples were then loaded into a 2. 5% Agarose gel in order to determine molecular weight Samples are also prepared to complete a quantitative analysis (q. PCR) (Designed by: Mia Brown)

Previous Research Results RT-PCR Analysis A B C D E Figure 3. RT-PCR Gel Electrophoresis A 157 bp RT-PCR product complementary to the MT gene in Zebrafish was amplified using designated primers and the indicated amounts of RNA. RT-PCR was performed according to the Promega System protocol. RT-PCR profile: 48°C for 45 minutes, 94°C for 2 minutes, 45 cycles (94°C for 30 seconds, 60°C for 1 minute, 68°C for 2 minutes), 68°C for 7 minutes. To analyze the reactions, 10μl of each reaction were subjected to electrophoresis on a 2. 5% agarose gel and DNA was detected by ethidium bromide staining. Lane A, DNA Ladder, Lane B, Control, Lanes C-E, RNA 1 -3 respectively.

Major Conclusions from Previous Research The MT gene in the PLHC-1 cell line has been identified computationally at around ~150 bp. l RNA Expression levels of the MT gene in the PLHC-1 cell line has not been determined specifically but has shown great potential in being the desired MT gene sequence through RT-PCR l

Future Research and Questions l l l Located gene computationally Tested gene through RT-PCR and results suggested that this could be a MT gene of interest With the PLHC-1 cell line being treated with 10µM of Cadmium at 30ºC at 24 hours which indicated that our potential gene to be over 150 base pairs At what point in time would we see the earliest expression once exposed to 10µM of Cadmium (determined over graduation of time) Results indicated that we had a maximum of 51. 6 ng per ml

Future Research and Questions (cont’d) With this data, we would like to determine the earliest form of expression (by repeating the experiment through different time points) l What is the earliest form of MT gene expression and the PLHC-1 cell line l What is the earliest form of RNA expression in the PLHC-1 cell line at 10µM of Cadmium l

Objectives l To evaluate RNA expression levels in the MT gene in PLHC-1 cell line over a supplemented time period by RT-PCR

Methods Step 1: Cells were grown for 3 days and treated with and without 10 u. M Cadmium for 6, 12, 18, and 24 hours Step 2: Cells were then washed with 5 m. L PBS and then collected by scrapping the petri dish Step 3: Purified RNA using Promega RNA Purification protocol

RNA Concentration Diagram Figure 4. RNA Concentration Diagram RNA concentration was measured using the Nano. Drop in Dr. Norton’s lab. The concentrations were as followed: 0 hours -11. 5 ng/ml (OD 260/280: 2. 29) 6 hours - 12. 5 ng/ml (OD 260/280: 1. 90) 12 hours - 16. 0 ng/ml (OD 260/280: 1. 97) 18 hours - 25. 5 ng/ml (OD 260/280 1. 94) 24 hours - 10. 2 ng/ml (OD 260/280: 1. 25)

Gel Results LANES A – Ladder B – Control C – 6 hours D – 12 hours E – 18 hours F – 24 hours G - Ladder A B C D E F G

Major Conclusions l l Previous research indicated that the expression is as originally thought (150 base pairs/size of insert) B) Control – ok C) 6 hours – ok D) 12 hours – ok E) 18 hours – gel electrophoresis suggested that there was a large amount of expression between hours 18 -24

Major Conclusions (cont’d) l Gel electrophoresis does not suggest that we indicated the MT gene specifically. But it does indicate a 150 base pair sequencer that can be sequenced for further analysis to determine whether or not we have MT gene expression

Questions/Future Research Future analysis for taking fish exposed for a specific time and amount and then looking at different pathways to the exposal of metal internal affects. l Does cadmium have a certain affect on humans such as mercury in tuna? l

References l l l Cheung, Andrews Pok Lap, Vincent Kwok Lim Lam, King Ming Chan, "Tilapia metallothionein genes: PCR-cloning and gene expression studies. " Biochimica et Biophisica Acta 1731(2005): 191 -201. Scudiero, Carginale, Capasso, Riggio, Filosa, Parisi, Rosaria, Vincenzo, Clement, Marilisa, Stefania, Elio. "Structural and functional analysis of metal regulatory elements in the promoter region of genes encoding metallothionein isoforms in the Antarctic fish Chionodraco hamatus (icefish). " Gene 274(2001): 199 -208. Li-Hua Shen, Kwok Lim Lam, Po Wai Ko and King Ming Chan, "Metal Concentrations and Analysis of Metal Binding Protein Fractions from the Liver of Tilapia Collected from Shing Mun River. " Marine Enviornmental Research 46(1998): 597 -600. Carol Hiu Mei Yan, King Ming Chan, "Cloning of zebrafish metallothionein gene and characterization of its gene promoter region in Hep. G 2 cell line. " Biochimica et Biophisica Acta 1679(2004): 47 -58. King Ming Chan, "Metallothionein: Potenial Biomarker for Monitoring Heavy Metal Pollution in Fish Around Hong Kong. " Marine Pollution Bulletin 31(1995): 411 -415.

Acknowledgements Dr. Elizabeth Murray for all her guidance l Mia Brown for taking her summer out to advise me through this experiment l Dr. Norton’s lab, Ms. Valerie, (Nano. Drop and q. PCR machine) l Yung Nahm for her PLCH-1 cell line from the previous research conducted l