Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Immune system wikipedia , lookup
Polyclonal B cell response wikipedia , lookup
Lymphopoiesis wikipedia , lookup
Adaptive immune system wikipedia , lookup
Cancer immunotherapy wikipedia , lookup
Psychoneuroimmunology wikipedia , lookup
Innate immune system wikipedia , lookup
The Effects of Atrazine on Tail Regeneration in Salamanders Carlena K. Johnson Department of Biology Hartwick College Oneonta, NY This thesis is submitted in partial satisfaction of the requirements for the degree of Bachelor of Arts or Bachelor of Sciences from the Department of Biology, Hartwick College. _____________________________ Thesis Advisor _______________ Date _____________________________ Chair, Biology Department _______________ Date The Effects of Atrazine on Tail Regeneration in Salamanders Carlena Johnson Dr. Stanley K. Sessions ABSTRACT Atrazine, a widely used herbicide, is thought to be contributing to amphibian declines. In addition, it may be working as an endocrine disruptor in amphibians. To better understand if atrazine affects cell proliferation I studied the effects of atrazine on dividing cells in regenerating tails of the Red-backed Salamander (Plethodon cinereus). It was found that there was a significant difference between the percentages of dividing cells in the experimental salamanders, but not between any of the experimental groups and the control. Specifically, the tissues appear to show a dose-dependent effect in which an increase in cellular growth was seen only at the lowest concentration. This result is consistent with atrazine acting as an endocrine disruptor. Dead cells were also observed in most of the regenerating tails, but were not quantified in this study. Further research should be conducted into the effects of atrazine on both cellular growth and cell death during amphibian growth and development. INTRODUCTION Over the past decade the issue of declining amphibian populations has been in the forefront of both scientific and popular literature. Amphibians are important environmental indicators because they have thin moist skin, which is used as a respiratory surface, and because they lay their eggs in the water. In this way, they may be exposed to chemicals and other pollutants very easily. Also, the basic development and the immune system of amphibians are similar to that of humans and other vertebrates. Many hypotheses have been suggested as to why amphibian populations are declining. Some of these include: chemical pollution, UV radiation, diseases, parasite, global climate change, over-collecting and general habitat destruction. It is very likely however that it is a combination of some or all of these, or something else that has not been thought of yet. The herbicide atrazine is the most common contaminant of ground, surface, and drinking water (Hayes, 2002). About 27 million kilograms of atrazine are used each year in the US, mostly in corn growing regions (Withgott, 2002). The EPA has set a maximum limit of 3 ppb atrazine in drinking water (U.S. Environmental Protection Agency (U.S. EPA), 2006). It is therefore apparent that contamination of ground water exists, but the consequences of this contamination have been disputed (Coady et al., 2004; Filipov et al., 2005; Hayes, 2004). One study shows that atrazine affects the immune systems of juvenile mice by adversely affecting lymphocyte distribution as well as decreasing cellularity within the spleen and thymus (Filipov et al., 2005). It also shows that some effects of the herbicide remain well after exposure to it has ended. A study that looked at the effects of atrazine on embryos and larvae concludes that atrazine does not have any adverse effects on hatchability, post hatch mortality, or swimming speed and concludes that “direct toxicity of atrazine is probably not a significant factor in recent amphibian declines” (Allran and Karasov, 2001). Another study looked at the effects of atrazine on growth and gonad development (Coady et al. 2004). Coady et al. (2004) conclude that there is no difference in weight or length of the exposed vs. control individuals and also says that there is no significant difference in the gonadal development of the frogs, even though multiple testes, size irregularity, and “rudimentary hermaphroditism”, including intersex gonads and testicular oocytes, were observed. Other recent studies have shown that even low doses of atrazine, such as those found in agricultural runoff, can cause adverse effects on male African clawed frogs (Xenopus laevis), such as hermaphroditism and the demasculization of the larynges of exposed males (Hayes et al. 2003). Hayes et al. (2002), as well as others, have concluded that atrazine works as an endocrine disruptor resulting in the effects cited by these articles. Rohr et al. (2006) investigated possible persistent effects of atrazine after exposure to the herbicide was stopped on the salamander Ambystoma barbouri. They found that there was a significant difference in survival between the three experimental groups (4, 40, and 400 ppb) and the control and also cite a non-linear pattern in their results, which is characteristic of an endocrine disruptor. A study by Storrs & Keisecker (2004) also showed a non-linear rate of survivorship during a study into the effects of long-term exposure to low levels of atrazine. Brodkin et al. (2007) found that atrazine may also work as an immune disruptor in amphibians. It is obvious that there is dispute over the consequences of atrazine on amphibians, but it is clear that this issue deserves continued study to help clarify any possible effects on amphibian populations. Previous senior research theses (Wilkes, 2004 & Owens, 2005) have shown that atrazine has a negative effect on growth and development, as well as the immune system. To better understand how atrazine may cause these negative effects I choose to investigate the mechanism by which atrazine works. I chose to study the effects of atrazine on tail regeneration in salamanders to see if atrazine would affect cell proliferation in a rapidly growing tissue. MATERIALS AND METHODS A total of 18 Red-backed salamanders (Plethodon cinereus) were caught from the area surrounding Strawberry field (Hartwick College, Oneonta NY). The salamanders were brought back to the lab and acclimated for one week. Before being introduced to the treatment, about half of each salamander’s tail was removed with a clean blade. The salamanders were then randomly assigned one of four treatment groups (0 ppb control, 1ppb atrazine, 10 ppb atrazine, or 100 ppb atrazine). The atrazine treatment solutions were made from a series of dilutions from a “stock” atrazine solution. The stock solution included ethanol to dissolve the atrazine itself and aged tap water. The control solution was made with aged tap water and the same amount of ethanol as the 100 ppb treatment. Each salamander was kept in a Petri dish with filter paper and 10mls of the assigned treatment solution. Three salamanders were included in the 0 ppb control group and five salamanders were in each of the three experimental atrazine groups. The salamanders were kept at 15º C. New treatment solutions were made and replaced in each Petri dish every three days for four weeks. In addition, each salamander was fed a few fruit flies every third day. At the end of the exposure period, each salamander was injected with 0.3% colchecine twenty-five hours prior to harvesting the tail regenerate. The tails were then placed in 3:1 ethanol: acetic acid fixative. They were then embedded in wax, following standard histological procedure, and sectioned at 10µm. Slides were then stained with hematoxylin-eosin. The number of mitotic cells/total number of cells for at least 5 sections of each tail were counted and totaled to determine a proportion of mitotic cells for each salamander tail. RESULTS Over the four week period during which tail regeneration occurred, the average length of the regeneration bud for the control, 1 ppb, 10 ppb, and 100 ppb groups, respectively were: 0.4cm, 0.37cm, 0.39cm, and 0.36cm. There is no statistical significance between any of the groups regarding regeneration bud length. In addition, no adverse effects on the salamanders were noted during the one month exposure period (such as loss of appetite or death). There was no correlation between the amount of growth of the regeneration bud and the concentration of atrazine. In addition, there is no correlation between the amount of growth and the proportion of dividing cells. For each tail, 5-9 sections were used to record the proportion of mitotic cells (Fig. 1). Only one tail (in the 10 ppb treatment group) used five sections, while most tails contained six sections from which data was recorded. The number of mitotic cells per section were counted (Fig. 2) and the total for each section were added together to give a total number of mitotic cells for a given tail. In the same manner, the total number of cells per section were counted and added together for a total number of cells for a given tail. The proportion of mitotic cells was calculated from this data (Table 1). Figure 1. This shows two cells undergoing mitosis (arrows). Hematoxylin and eosin staining of 10 micrometer paraffin section. Mean Proportion of mitotic cells 6.000 5.000 4.000 3.000 2.000 1.000 0.000 Control 1 ppb 10 ppb 100 ppb Atrazine Concentration Error bars: 95% CI Figure 2. This shows the average proportion of mitotic cells for each treatment group. Note that there is a significant difference between the experimental groups 1 ppb & 10 ppb. Table 1. This shows each individual used in the four treatment groups. Each individual is separated into the total number of mitotic cells, total number of cells in the tail and the proportion of mitotic cells found for each tail. The total mitotic cells and the total cells were found by adding the numbers for each section of a given tail. Treatment Control 1 ppb 10 ppb 100 ppb Total Mitotic cells 171 224 252 257 135 310 295 392 153 200 155 186 213 163 246 349 213 Total cells 4878 7961 6319 4974 3411 6894 6670 12164 6268 5941 5909 12317 8055 6095 8152 8313 6779 Proportion of mitotic cells 3.506 2.814 3.988 5.167 3.958 4.497 4.423 3.223 2.441 3.366 2.623 1.51 2.644 2.674 3.018 4.194 3.142 One tail from the 10 ppb experimental group was not included in data analysis due to poor histology and a resultant inaccuracy in counting cells. This reduced the number of tails in the 10 ppb group to four, as compared to the 1 ppb and 100 ppb experimental groups which contain five tails each. Overall, it is apparent that the proportion of mitotic cells is largest at the 1 ppb concentration of atrazine, while it is smallest at the 10 ppb concentration of atrazine (Fig. 2, Table 2). A one-way ANOVA was preformed on the data and the results were found to be significant (F3,13 = 5.192, P = 0.014). Afterwards, post hoc tests (LSD, SNK, bonferroni, and tukey) were preformed on the data to find which treatments were significantly different. It was found that there was a statistically significant difference between the 1 ppb and 10 ppb treatment groups only. There was no difference between any of the experimental treatment groups and the control. Table 2. This table shows the average proportion of mitotic cells for each treatment group. Note that there is a significant difference between the experimental groups 1 ppb & 10 ppb. Treatment Mean Proportion of Mitotic Cells Control 3.44 ± 0.59 1 ppb 4.25 ± 0.72 10 ppb 2.49 ± 0.76 100 ppb 3.14 ± 0.63 DISCUSSION Overall, atrazine appears to have little or no effect on cell proliferation in regenerating salamander tails. There may be a small dose dependent effect (as seen by the significance between the 1 ppb and 10 ppb experimental groups), which is characteristic of an endocrine disruptor. This is consistent with other research that concludes that atrazine works as an endocrine disruptor (Hayes et al. 2002, Sessions et al. unpublished). It is also important to note the high degree of variance within the control. Due to the variance within the control, a possible pattern may have been masked. Further steps should be taken to better understand if atrazine has an effect on cell proliferation before it is ruled out as the mechanism by which the herbicide is functioning. Other protocols, such as the use of BrdU to label mitotic cells, may prove to be a better measure of mitotic activity, though preliminary tests show that background staining may be an issue that would need to be resolved to get an accurate count of cells. I also propose that similar studies should be conducted on frogs to rule out the possibility of a species-specific reaction to salamanders that may not be applicable to amphibians as a whole (though Rohr et al., 2006, showed that atrazine does have a negative effect on salamanders exposed to the chemical before metamorphosis). In addition, it was noted that many of the tail sections contained dead or dying cells. The proportion of dying cells was not quantified at this time, but the possible interrelated effects of atrazine on cell proliferation and dying cells should be looked at in future research. Studies should be done to determine the extent to which atrazine may simply be causing too much cell death and the possible effect that cell death may have on increasing the rate of mitosis, especially at low levels. It is important to consider the mechanism by which atrazine may be causing the adverse effects that many studies have found on amphibians because by better understanding how this herbicide stunts growth and development and the immune system we may be able to better understand the complex mixture of issues that may be contributing to amphibian declines. For example, a reduction in the ability of the immune system to function properly may make it easier for parasites/fungi/diseases to infect amphibians, thus causing a higher death rate in a population exposed to atrazine than one that was not exposed to the chemical. Only by better understanding how individual causes may be negatively affecting amphibian populations can we begin to understand the entire scope of these effects, not only on amphibians, but also so that we may apply this understanding to other species that are declining. ACKNOWLEDGEMENTS I would like to thank Megan Irland and Nancy Johnson for giving me advice and assisting me with my research, Dr. Mary Allen for her assistance with statistics, and Dr. Stan Sessions for all of his help with my senior research project. I would like to dedicate my research to my grandmother, Betty I. Laitsch, who’s never ending dedication to animals and the environment has inspired me to continue my career in scientific research. LITERATURE CITED Allran, John W., William H. Karasov. 2001. Effects of Atrazine on Embryos, Larvae, and Adults of Anuran Amphibians. Environmental Toxicology and Chemistry. 20(4):769775. Brodkin, Marc A., Hareth Madhoun, Muthuramanan Rameswaran, and Itzick Vatnick. 2007. Atrazine is an Immune Disruptor in Northern Leopard Frogs (Rana pipiens). Environmental Toxicology and Chemistry. 26(1): 80-84. Coady, Katherine K., Margaret B. Murphy, Daniel L. Villeneuve, Markus Hecker, Paul D. Jones, James A. Carr, Keith R. Solomon, Ernest E. Smith, Glen Van Der Kraak, Ronald J. Kendall, John P. Giesy. 2004. Effects of Atrazine on Metamorphosis, Growth, and Gonadal Development in the Green Frog (Rana clamitans). Journal of Toxicology and Environmental Health. 67:941-957. Filipov, Nikolay M., Lesya M. Pinchuk, Bobbie L. Boyd and Patrick L. Crittenden. 2005. Immunotoxic Effects of Short-term Atrazine Exposure in Young Male C57BL/6 Mice. Toxicological Sciences. 86(2):324-332. Hayes, Tyrone. 2002. Feminization of Male Frogs in the Wild. Nature. 419(6910):895-896. Hayes, Tyrone, Kelley Haston, Mabel Tsui, Anhthu Hoang, Cathryn Haeffele, and Aaron Vonk. 2003. Atrazine-Induced Hermaphroditism at 0.1 ppb in American Leopard Frogs (Rana pipiens): Laboratory and Field Evidence. Environmental Health Perspectives. 111(4): 568-575. Hayes, Tyrone B., Atif Collins, Melissa Lee, Magdelena Mendoza, Nigel Noriega, A. Ali Stuart, Aaron Vonk. 2002. Hermaphroditic, Demasculized Frogs After Exposure to the Herbicide Atrazine At Low Ecologically Relevant Doses. Proceddings of the National Academy of Sciences. 99:5476-5480. Owens, Clarence. 2005. Endocrine Disruptors in Amphibians and Mammals: Analysis of the Effects of Two Common Environmental Pollutants. Journal of Biological Research: Senior Research Theses, Department of Biology, Hartwick College. VI(May 2005): 105-112. Rohr, Jason R., Tyler Sager, Timothy M. Sesterhenn, and Brent D. Palmer. 2006. Exposure, Postexposure, and Density-Mediated Effects of Atrazine on Amphibians: Breaking Down New Effects into Their Parts. Environmental Health Perspectives. 114(1): 4650. Storrs, Sara I. and Joseph M. Kiesecker. 2004. Survivorship Patterns of Larval Amphibians Exposed to Low Concentrations of Atrazine. Environmental Health Perspectives. 112(10): 1054-1057. U.S. EPA (U.S. Environmental Protection Agency). 2006. 2006 Edition of the Drinking Water Standards and Health Advisories. EPA 822-R-06-013. Washington, DC: U.S. Environmental Protection Agency. Wilkes, Tara. 2004. The Effects of the Chemical Pollutant Atrazine on the Immune System of the Amphibian, Rana pipiens. Journal of Biological Research: Senior Research Theses, Department of Biology, Hartwick College. V(May 2004): 207-214. Withgott, Jay. 2002. Ubiquitous herbicide Emasculates Frogs. Science. 296(5567):447-448.