The effects of copper and ascorbic acid on The effects of copper and ascorbic acid on D. melanogasterD. melanogasterJennifer RockholdJennifer Rockhold

Advisor: Dr. BrownAdvisor: Dr. Brown

INTRODUCTION Oxidation is a naturally-occurring process in the body that can be caused by normal cellular functions or environmental sources. Copper is one of the environmental sources that can cause oxidation. Copper is an essential trace element found in humans, though the threshold for copper poisoning is as little as 10mg/day. Copper is taken into the body through the lungs, gastrointestinal tract, or the skin. It is then sequestered by the body in the bones, eyes, liver, kidneys, or brain, and long-term overexposure to copper can cause damage to the organs it is being stored in. Though it is necessary for human health and function, copper can lead to oxidative damage. This occurs when Copper undergoes reaction that produces a hydroxyl and hydroxyl radical.

The hydroxyl radical is a reactive oxygen species, meaning that it has an unpaired valence electron in its outer shell. This causes the hydroxyl to be very destructive to many things it comes into contact with. Antioxidants function to donate an electron to the hydroxyl radical to make it less reactive. The importance of antioxidants is undisputed, but the question of how effective they are under certain situations remains. There a number of antioxidants that may be ingested or produced by the body, such as vitamin E or glutathione, respectively. Ascorbic acid, or vitamin C, is an antioxidant that must be ingested. The test organism in this experiment is Drosophila melanogaster, or the common fruit fly. D. melanogaster has an average lifespan of approximately 30 days and takes about 12-14 days to develop from conception to adult fly. D. melanogaster is an excellent test mechanism to use for research because of its quick development, cost effectiveness, and ease of reproduction. The fruit fly lifecycle is illustrated in figure 1. The research hypothesis is that ascorbic acid, when given to flies who are being concurrently dosed with copper, will lessen the detectable oxidative damage caused by copper. Also, flies who are given ascorbic acid only will show less oxidative damage than a control group.

METHODOLOGY Flies of uniform age were put into either a group dosed with copper, ascorbic acid, a mixed solution, or a control solution. The solutions were mixed using 50ppm measurements (1.00g/100ml for the ascorbic acid and 3.91g/100ml for the copper, diluted by 20x). At the end of either the 21-day or 14-day trials the flies were sacrificed using the freezer method. The type of container the flies were held in as they were tested can be seen in figure 2. A carbonyl assay (as described in Fagan et al.) was performed to determined the amount of oxidative damage present in the flies after a 21-day period of dosing. All four groups of 150 flies were uniformly dosed as previously indicated. After the 21-day test period, the flies in each group were homogenized, centrifuged, and placed into 1.5ml tubes. The procedures called for 1.0ml of supernatant for each tube; however, due to an error in methodology, the procedures were altered to accommodate for 0.1ml of supernatant. The flies were centrifuged and washed as described in Fagan et al., and read in a spectrophotometer at 380nm. The results are shown in figure 3. Atomic absorption spectroscopy (AAS) was used to determine the concentration of copper in groups of 150 flies dosed with copper, mixed, and control solutions for a 14-day period. The flies were solubilized using Nitric acid, HCl, and H2O2, then read by an AAS machine. The results are shown in figure 4. A survivorship curve (figure 5) was created using data collected from groups of 125 flies from all four aforementioned dosing groups. The number of dead flies was counted up every other day and then plotted in a chart to visually determine if one group had a notably greater or lesser survival than the other groups.

RESULTS Using the carbonyl assay it was found that the ascorbic acid group exhibited the lowest amount of oxidative damage. The copper-dosed group showed a slightly greater amount of oxidative damage, and the mixed and control groups showed the most oxidative damage. The results (figure 3) are shown as a bar graph with error bars. There was a statistically-significant difference found between the four test groups. Using the atomic absorption spectroscopy it was found that there was the most copper present in the copper dosed groups, then approximately half as much in the mixed group, and the littlest copper found in the control group. The results are shown in table 1. The survivorship curve (figure 4) shows that the copper-dosed group experienced the most shorted lifespan, while the mixed group showed slightly better survivorship rates. The control group and ascorbic acid group both showed roughly the same affect on survivorship. The survivorship curve illustrates that all of the flies, even the control group, showed an shortened lifespan from the natural fly lifespan average of 30 days.

CONCLUSIONS After experimentation, it was determined that the hypothesis was partially supported. The carbonyl assay showed that the ascorbic acid group did exhibit the lowest amount of protein oxidation, which supports the hypothesis; however, the results of the carbonyl assay must be taken with a grain of salt, for the diluting from 1.0ml to 0.1ml may have skewed the results to some degree. The fact that the mixed and control groups had more detectable protein oxidation in them than the copper group implies that there is a possibility of some skewing of results. The exact reason for this difference is not know, though, it could potentially be because the copper-dosed flies died earlier than the other groups and thus had less time to ingest enough copper or undergo enough natural bodily processes to come up with a comparable amount of oxidative damage. The results of the AAS show that the copper-dosed fly group did indeed ingest and retain the copper they were being exposed to. The mixed group, given the same amount off copper-in-solution as the copper-dosed group, showed approximately half as much copper in the sample as the copper-dosed group. Interestingly, the control group also showed detectable copper, though reason for this is not known. The survivorship curve partially supports the hypothesis, as the copper-dosed group did lead to a markedly shorter lifespan in the flies, as opposed to the control and ascorbic acid groups which lived, comparably, much longer. However, the hypothesis is not completely supported by the survivorship curve because the control and ascorbic acid flies lived approximately the same amount of time, whereas the hypothesis supposed that the ascorbic acid-dosed flies would have lived longer than the control. In this experiment, it was found that ascorbic acid does have some positive effect on copper-dosed flies, though it does not have a positive lifespan effect on flies not being dosed with copper.

FUTURE RESEARCH I would like to look at other antioxidants. I only used vitamin C (ascorbic acid) in my research, but it would be interesting to study the efficacy of other antioxidants such as beta-carotine or vitamin E. It is possible that a different antioxidant may be more effective against copper-induced oxidation. I would also be interested in looking at other ways to dose the flies, such as using food scraps or peels. It would be a more natural way for the flies to ingest the antioxidant, however, it would be more difficult to quantify the amount of antioxidant present in the food sample.

LITERATURE CITEDAgarwal S, Sohal RS. 1994. DNA oxidative damage and life expectancy in houseflies. Proceedings of the National Academy of Sciences- Biochemistry 91: 12332-12335.Baumeister P, Huebner T, Reiter M, Schwenk-Zieger S, Harreus U. 2009. Reduction of oxidative DNA fragmentation by ascorbic acid, zinc and N-Acetylcysteine in nasal mucosa tissue cultures. Anticancer Research 29(11): 4571-4574.Fagan JM, Sleczka BG, Istvan S. 1999. Quantitation of oxidative damage to tissue proteins. International Journal of Biochemistry and Cell Biology 31(7): 751-757Hanafy S, Soltan M. 2007. Comparative changes in absorption, distribution and toxicity of copper and cadmium chloride in toads during the hibernation and the role of vitamin C against their toxicity. Toxicological and Environmental Chemistry 89(1-4): 89-110.Hoffman R, Garewal H. 1995. Antioxidants and the prevention of coronary heart disease. Archives of Internal Medicine 155(3): 241-246.Kovacic P, Somanathan R. 2010. Dermal toxicity and environmental contamination: electron transfer, reactive oxygen species, oxidative stress, cell signaling, and protection by antioxidants. Reviews of Environmental Contamination and Toxicology 2010: 119-138. Kumar VH, Patel A, Swartz D, Wang H, Wynn KA, Nielsen Lori C, Ryan RM. 2010. Exposure to supplemental oxygen and its effects on oxidative stress and antioxidant enzyme activity in term newborn lambs. Pediatric Research 67(1): 66-71.Lima C, Pereira A, Silva J, Oliveira L, Resck M, Grechi C, Bernardes M, Olímpio F, Santos A, Incerpi E, Garcia J. 2009. Ascorbic acid for the healing of skin wounds in rats. Brazilian Journal of Biology 69(4): 1195-1201.Niemiec T, Sawosz E, Grodzik M, Chwalibog A. 2009. High L-ascorbic acid content of diet modulates the non-specific immunity in rats. Animal Science Papers and Reports 27(2): 149-157Paital B, Chainy G. 2010. Antioxidant defenses and oxidative stress parameters in tissues of mud crab (Scylla serrata) with reference to changing salinity. Comparative Biochemistry and Physiology 151(1): 142-151.Verhamme C, de Haan RJ, Vermeulen M, Baas F, de Visser M, Van Schaik I. 2009. Oral high dose ascorbic acid treatment for one year in young CMT1A patients: a randomized, double-blind, placebo-controlled phase II trial. BMC Medicine 7(1): Article 70.

ACKNOWLEDGEMENTSI would like to thank Dr. Brown, my capstone advisor. I would also like to thank my classmates in the 2011 Capstone class and the Marietta College Biology Department for providing me with the necessary materials and instruments.

O2- + Cu(II) O2+ Cu(I)

Cu(I) + H2O2 Cu(II) + OH + OH-

http://www.seop.yale.edu/supplementary/supp_drosophila.html

Figure 2. The flies were dosed using plastic vials filled with the respective dosing solution, with a

cotton ball used to control the flow of solution.

Figure 1. The lifecycle of the Drosophila fly is shown. Testing was done upon the

flies as adults.

Figure 4. The survivorship

curve shows the number of dead

flies in each group, taken in

2-day increments for 21 days.

CopperCopper Plus Ascorbic Acid Control

44.44 μg/g 27.77 μg/g 15.87 μg/g

Figure 3. The results of the carbonyl

assay are shown. Each of the four groups is plotted

against the average absorbance at

380nm (with error bars).

Table 1. The results of the AAS are shown. The measurements are in μg of copper

per gram fly weight.