1. We used chicken embryos as models for this experiment. Chicken embryos were injected on day 13 with the following treatments for a 2 day treatment period: Arsenic (n = 7) Estrogen (n = 7) Tamoxifen (n = 7) Arsenic & estrogen (n = 7) The chick embryos were extracted on day 15 and weights were taken in order to check viability after injection of treatments. Liver were removed and frozen with liquid nitrogen and placed at -80 o C for storage. Livers were taken from storage and RNA extraction began. Livers were suspended in trizol (Invitrogen) and were homogenized using a sonicator to break the tissue apart. The samples were incubated at room temperature for 5 minutes followed by the addition of 200 µl of chloroform and centrifugation at 12000g. The supernatant was removed and transferred to a new 1.5 ml RNAse-free tube. 500 µl of isopropyl alcohol was added to the tubes to form a pellet of RNA and the tubes were allowed to set for 10 minutes at room temperature. Another centrifugation was done followed by a wash of the pellet using 1 ml of ethanol and a centrifugation at 7500g. The pellet was then allowed to dry and 50ml of nuclease free water was added and quantification using absorbance in a UV spectrometer was done by adding the RNA to RNAse-free water and running it through the instrument. After quantification, the RT-PCR was done. During the RT, a master mix of 4 µl of DNTPs, 2 µl of random decamers, and 5 µl of RNAse-free water was made for each sample and was added to 0.5 ml tubes. By using the absorbance of each of the samples from the UV spectrometer, a calculation of how many µl of each sample would be added to the master mix was determined. The total volume of the amount added would be raised to 5 µl by adding RNAse-free water to the sample amount. A total volume of 16 µl of master mix and sample was heated at 70 o C for 3 minutes and placed on ice for 1 minute. The RT mixture that was added after this step contained 2 µl of 10x RT buffer, 1 µl of RT, and 1 µl of RNAse inhibitor bringing the total volume of the sample to 20 µl. The sample was incubated at 42 o C for 60 minutes and then heated to 94 o C for 10 minutes and stored at 4 o C. PCR followed the RT by making a supermix of 22.5 µl of PCR mastermix and 0.5 µl of both forward (5’-GCACAAGTGAAGCTGGAGTGG-3’ and 5’- TTCACCACCACAGCCGAGAG-3’ for vitellogenin and β-actin, respectively) and reverse (5’-AATTCTCGAGCACGGCAGAGG-3’ and 5’-CACCAGAAGGCACTGTGTTG-3’ for vitellogenin and β-actin, respectively) primers. A total of 23.5 µl of this supermix was added to 1.5 µl of sample. The PCR was run with various annealing temperatures of 52- 63 o C for 33 cycles. Gels were run using a 2% agarose gel, in 1x SB buffer (Media LLC), and stained with GelStar gel stain (Cambrex). Samples were run at 300 volts for approximately 15 minutes. After the run, pictures of the gels were taken and analyzed for possible bands. Cytoplasm Introduction Arsenic, a known carcinogen, has been shown to cause liver, skin, and bladder cancer in mammals (Abernathy et al. 1999). However, its carcinogenic mechanism is unclear because it does not affect DNA directly. Arsenic has been shown to inhibit the response of the glucocorticoid receptor (GR) by inhibiting gene expression (Kaltreider et al. 2001). Glucocorticoids are ligands that bind to the GR, which is a transcription factor that regulates gene expression. Glucocorticoids are steroid hormones that interact with the glucocorticoid receptors to regulate glucose homeostasis. Because glucocorticoids reside in the same hormone family as estrogen and have similar sequence homologies, we were interested if arsenic had the same effects on estrogen. In order to observe arsenic’s effect on estrogen, we examined vitellogenin, known to be an estrogen sensitive gene, would be the best way to observe the effects. The vitellogenin protein is used in chicken embryos to break down the yolk in the eggs for nutrients. The gene has an estrogen response element (ERE) and has been shown, when in the presence of estrogen, to produce a large amount of m- RNA for the protein. Our study examined the effect of arsenic (III) on the estrogen responsiveness of this gene. Materials and Methods By Jason Schafer Biology Department, York College of Pennsylvania Effect of Arsenic (III) on the Estrogen Responsiveness of Vitellogenin in Chick Embryos Figure 1: Graph of weight compared to treatment for chick embryos after 15 days of incubation. Error bars shown one standard deviation from the mean. The asterisk indicates a significant difference for the chick weights of arsenic treatment compared to estrogen and arsenic and estrogen (unpaired t-test, p = 0.0101 and 0.0207, respectively). 5 4 3 2 1 100 BP Figure 2: β-actin amplified from chick samples. Samples were amplified through PCR with beta actin primers from literature. A 2% agarose gel stained with GelStar gel stain was run for 15 minutes for treatment groups of arsenic, estrogen, arsenic and estrogen, water, and ethanol (1, 2, 3,4,5 respectively). 5 4 3 2 1 100 BP Figure 3: Vitellogenin amplified from chick samples at 54 o C. Samples were amplified through PCR using chicken vitellogenin primers from literature. A 2% agarose gel stained with GelStar gel stain was run for 15 minutes for treatment groups of arsenic, estrogen, arsenic and estrogen, water, and ethanol (1, 2, 3,4,5 respectively) at the annealing temperature of 54 o C. 5 4 3 2 1 100 BP β-actin Vitellogenin Figure 4: Vitellogenin amplified from chick samples at 57 o C. Samples were amplified through PCR using chicken vitellogenin primers from literature. A 2% agarose gel stained with GelStar gel stain was run for 15 minutes for the treatment groups of water, arsenic, estrogen, arsenic and estrogen, and tamoxifen and estrogen (1, 2, 3,4,5 respectively) at the annealing temperature of 57 o C. Vitellogenin Figure 5: Vitellogenin amplified from chick samples at 60 o C. Samples were amplified through PCR using chicken vitellogenin primers from literature. A 2% agarose gel stained with ETBR was run for 15 minutes for treatment groups of water, arsenic, estrogen, arsenic and estrogen, and tamoxifen and estrogen (1, 2, 3,4,5 respectively) at the annealing temperature of 60 o C. Results Arsenic’s treatment group had a significantly lowered chick weight when compared to estrogen and arsenic + estrogen (p= 0.0101 and p=0.0207 respectively). Other treatment groups produced no significant difference in chick weight (Figure 1). A band at 267 base pairs was shown for the beta actin. m-RNA was present in the samples (Figure 2). Vitellogenin band appears on the gel at 286 base pairs with an annealing temperature of 54 o C. There was a brighter band for the estrogen group compared to the arsenic + estrogen group. No other bands appeared for other treatment groups (Figure 3). Vitellogenin band appears on the gel at 286 base pairs with an annealing temperature of 57 o C. Vitellogenin induction did not occur (Figure 4). Vitellogenin band appears on the gel at 286 base pairs with an annealing temperature of 60 o C. Vitellogenin induction did not occur (Figure 5). Vitellogenin Est Est Receptor ERE Vitellogenin Est Receptor Est AS Conclusions Non-toxic arsenic significantly reduced chick weight compared to the estrogen and arsenic + estrogen groups. Though sick, the chicks for the arsenic group did not show a reduction in viability. Other treatment groups had no significant effect on chick weight. With the beta actin gel working, m-RNA was present in the sample and we were able to amplify the m-RNA with RT-PCR. However, due to lack of correct bands for the c-DNA product through the PCR, it is likely that the vitellogenin primers were not working as reported in the literature. T m was reported in literature as 64 o C, but, when synthesized, the actual T m was much lower at 54 o C. Different annealing temperatures were used to find out which would be best for the vitellogenin primers. It appears that the best annealing temperature was 60 o C, which can now be used to address whether or not arsenic (III) alters estrogen-induced vitellogenin gene expression. It is possible that we may have missed the window of induction for the vitellogenin gene. Time of treatment and isolation should be reduced. Literature Cited Abernathy CO, Liu YP, Longfellow D, Aposhian HV, Beck B, Fowler B, Goyer R, Menzer R, Rossman T, Thompson C, et al. 1999. Arsenic: health effects, mechanisms of actions, and research issues. Environ Health Perspect. 107:593–597. Kaltreider, R. C., Davis, A. M., Lariviere, J. P., and Hamiliton, J. W. 2001. Arsenic alters the function of the glucocorticoid receptor as a transcription factor. Environ. Health Perspect. 109, 245-251. Lorenzen, A. Casley, W. L. and Moon, T. W. 2001. A reverse transcription-polymerase chain reaction bioassay for avian vitellogenin mRNA. Toxicology and Applied Pharmacology. 176, 169-180. Acknowledgements Dr. Ronald C. Kaltreider Dr. Jeffrey P. Thompson Nucleus Cytoplasm Tamoxifen & estrogen (n = 3) No injection (n = 3) Ethanol (control) (n = 3) Water (control) (n = 3) Scheme 1 : The induction of vitellogenin through the estrogen response pathway with arsenic (III) as an inhibitor. Estrogen (Est), Arsenic (AS), and estrogen response element (ERE) are shown. Objective: To determine if arsenic (III) alters estrogen-induced vitellogenin gene expression.