ABSTRACT
Doxorubicin (DOX) is a common chemotherapy drug used for its high anti-tumor efficiency, but its use is limited due to the toxicity it causes to healthy cells through mechanisms like reactive oxygen species (ROS) formation. The oxidative stress caused by increased ROS levels damages kidney tissues and causes nephrotoxicity. Under normal biological system, antioxidant enzymes within the body counteract ROS, in order, to prevent damage to biological systems. Although extensive research exists surrounding DOX-induced oxidative stress, finding interventions to mediate the damage caused by DOX could lead to better patient outcomes. Creatine has been more extensively researched due to its potential antioxidant properties. METHODS: 28 rats were randomly placed in four groups: Control + DOX, n=7, Cr + DOX, n=8, C + Sal, n=7, and Cr + Sal, n=6. Two groups received regular chow while the other ate chow supplemented with 3% creatine. After two weeks, all groups were injected with equivalent volumes of either DOX or saline. Kidney tissue samples were collected from the four different groups, and the proteins collected, glutathione peroxidase (GPx), CAT, and b-actin, were analyzed using a Western blotting technique. GPx and CAT were set to values relative to b-actin values. RESULTS: For GPx and CAT, Cr + DOX were significantly lower than Control + Sal, and for GPx, Control + DOX was significantly lower than Control + Sal. The change in body mass of the rats in the control + DOX and creatine + DOX groups were significantly different than the control + saline group, and the differences in relative kidney mass had no significant differences. CONCLUSION: Creatine supplements could potentially be used as an intervention in nephrotoxicity for patients experiencing chemotherapy. Creatine was suggested to act as an antioxidant while functioning alongside the other antioxidant enzymes. Future studies involve gaining a better understanding of creatine’s antioxidant properties and its effects on the other antioxidant enzymes.
INTRODUCTION
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The oxidative stress caused within the kidneys of those treated with DXR can damage the cells membranes and increase glomerular capillary permeability, which further damages the kidney [3]. Increasing antioxidant enzyme activity within these tissues can counteract these effects. Recently, creatine has become an important research topic due to its antioxidant and anti-inflammatory abilities, and its potential protective effects, which increase cell viability [5]. Other studies have demonstrated creatine’s potential in protecting nuclear and mitochondrial DNA and RNA and reducing chronic and acute inflammation [6]. Sestili and colleagues (2011) also discussed how creatine supplements can upregulate the antioxidant enzyme pathways for glutathione and thioredoxin. Since creatine was found to exhibit only mild antioxidant activity, its ability to upregulate other antioxidants is beneficial in conditions involving ROS. The protective effects of creatine pose as a potential therapeutic alternative for cancer patients undergoing chemotherapy.
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Globally, cancer is a prominent and growing issue that affects 1 in 6 people and is the second leading cause of death [1]. Although there have been significant advancements in the treatment of cancer, chemotherapy has severe adverse effects like nephrotoxicity that limit their use in patients.
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Doxorubicin (DOX), a highly effective chemotherapy drug, has been shown to cause acute kidney injury in patients [2]. Much of this damage is due to the oxidative stress associated with DOX. In oxidative stress, free radicals are highly reactive molecules that cause a chain reaction which disrupts biological systems and damages tissues. Many of these free radicals are reactive oxygen species (ROS) or oxygen radicals. Although the mechanism behind DOX-induced nephrotoxicity is not completely understood, models have tested that this drug promotes an increase in free radical formation, tissue damage, and inflammation, but ROS produced in normal conditions are inactivated through antioxidant enzymes, such as glutathione peroxidase [3][4]. Mediating the effects of DOX can improve the lifestyle of patients and allow them a higher chance of combating the cancer.
Figure 1. Pathway of DOX-induced nephrotoxicity.
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This research project aimed to use creatine to attenuate DOX-induced nephrotoxicity in a rat model. Animals were randomly distributed in four groups (Saline + control, Saline + Creatine, DOX + Control, and DOX + Creatine). The body mass of the rats before and after treatments of DOX or saline were collected to consider the health of the animal. Animals were anesthetized and sacrificed and kidney tissue samples were taken; the mass of the kidney was measured and reported relative to the body mass. By taking tissue samples and analyzing the proteins through gel electrophoresis, an antioxidant enzyme profile was determined. Using this information, the activity level of an antioxidant, glutathione peroxidase, was observed which indicate creatine’s ability to reduce ROS levels within the kidney and thus, decrease tissue damage. A statistical analysis was performed, and a p-value < 0.05 was considered significant.
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The projected the hypothesized that creatine would be able to increase the activity of glutathione peroxidase which would reduce the oxidative stress within the kidneys.
METHODOLOGY
Animal Treatment
The Institutional Animal Care and Use Committee at the University of Northern Colorado approved all procedures which were followed in accordance with the Animal Welfare Act. 28 Sprague-Dawley male rats were obtained and kept in a controlled environment. The animals were either fed regular food as a control diet or regular food supplemented with 3% creatine. The body mass of the rats was collected before creatine chow was introduced and three more times for the two and half weeks of treatment. After two weeks, they were injected with equivalent volumes (15 mg/kg) of either DOX (Control + DOX, n=7 and Cr + DOX, n=8) or saline (C + Sal, n=7 and Cr + Sal, n=6) as the control. Five days following the injection, the rats were euthanized and the kidney was excised.
Biochemical Analysis
Samples of the kidney were homogenized with RIPA lysis buffer (Santa Cruz Biotechnology). From the homogenized solution, the protein concentrations were prepared with laemmli buffer (Sigma) for Western blotting. A milk solution that consisted of 1.7 g of non-fat dry milk and 40 mL of TBSt was used to block the PVDF membranes, and they were incubated with b-actin antibodies (Santa Cruz Biotechnology). After the incubation period was over, the antibodies were removed and a secondary mouse antibody was used for another incubation period. After each incubation period, the antibodies were removed and the membranes were washed with TBSt three times for five minutes.
An image of the membrane was collected using an imaging device (Li-cor). A chemiluminescent solution was prepared with a 1:1 concentration using peroxide buffer and luminal substrate in a 2 mL centrifuge container; it was poured into the center of the imaging device. The membrane was placed face down and using Image Studio Software, the image was developed and the one with the best clarity was chosen. Using the software ImageJ, the protein concentration of the bands was quantified by finding the optical density. Once the images were collected, the membranes were stripped using a buffer (Thermo Fischer Scientific) and the procedure was repeated for Glutathione Peroxidase (GPx) using a secondary rabbit antibody. Using b-actin as a loading control, GPx was adjusted to values that were relative to b-actin.
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Statistical Analysis
The data was displayed as mean ± SEM, and parameters were analyzed using a one-way analysis of variance (ANOVA), in order, to determine the differences between groups. Tukey post hoc tests were performed to ascertain where differences existed when F-values were found to be significant; α= .05 was set as significant.
Results
All data was presented as mean ± SEM. The expression of the antioxidant enzyme, GPx was significantly lower in rats treated with control and DOX and creatine and DOX when compared to the group treated with control and saline (Figure 1.). While the C+S group showed an optical density of 1.297 ± 0.55, C + D and Cr + D has a significantly lower optical density at 0.6324 ± 0.54 and 0.2229 ± 0.13 respectively (Table 1.).
The average difference in the body mass of the rats before and after an injection of their respective treatments was measured. The change in body mass of the rats in the control + DOX and creatine + DOX groups were significantly different than the control + saline group (Figure 2.). Control + DOX and Cr + DOX had an average change of mass -68.85 ± 16.5 g and -70.375 ± 19.74 g respectively which was significantly different when compared to the Cr + Sal group which changed 2.166 ± 7.332 g. (Table 2.). No significant differences existed between the groups for the relative kidney mass of the rats (Figure 4.).
Figure 4. Difference in Relative Kidney Mass
There was no significant difference found (p > .05)
DISCUSSION
DOX is a toxic chemotherapy drug that causes ROS production which damages both healthy and cancerous cells. Although the mechanism of DOX-induced nephrotoxicity is not fully understood, providing an intervention method capable of diminishing the kidney damage of those affected by DOX would be beneficial.
Under normal biological conditions, ROS production is counteracted through a variety of antioxidants. GPx and CAT are both major enzymatic antioxidants responsible for reducing hydrogen peroxide, a physiologically significant ROS, into water [7]. Previous studies have studied the antioxidant enzyme activity of models experiencing oxidative stress, and they have reported decreased levels of both GPx and CAT which aggravates the damage and stress within the body [4]. In the present study, GPx was significantly lower in rats treated with regular chow and DOX when compared to those in the control group (control + saline). This follows other studies findings that DOX reduces the activity level of antioxidant enzymes. Although GPx levels of creatine and DOX were significantly lower than the control group, creatine has the potential to act as an antioxidant and have a positive effect on the kidneys in conditions with high levels of ROS.
As an antioxidant, creatine has demonstrated the potential to act as antioxidant in conditions that require its use. Similar to this project, another study presented that creatine had mild free radical scavenging abilities, but it had no significant effect on the activity levels of GPx or CAT [8]. More specifically, creatine had the ability to counteract charged reactive species like superoxide anion radicals which are the most prevalent ROS produced in the metabolic process, and it would work alongside the other enzymes rather than influence them [8 ], [9], [10]. In the current study, the creatine supplements provided to the group being treated with DOX (Cr + DOX) could potentially be acting in a similar manner found in other studies, and the creatine could be used as an intervention method against oxidative stress caused by DOX. The significantly lower levels of GPx could also suggest that creatine is playing an antioxidant role and reducing ROS levels and therefore antioxidant enzymes levels.
The project tested the hypothesis that GPx would be upregulated by creatine supplements to alleviate oxidative stress. Creatine’s antioxidant properties might explain the lower GPx levels for groups given the supplement. If creatine has the properties reported in other studies, the reduction of GPx could be due to this effect. As a therapeutic technique, creatine has a lot of potential in alleviating the damage caused by DOX and the oxidative stress that results from using this drug. Investigating this supplement as an intervention method for patients being treated with chemotherapy should be studied further, in order, gain a better understanding of creatine’s antioxidant properties, and its effects on other antioxidant enzymes.
LITERATURE REVIEW
Oxidative Stress within the Kidneys
In oxidative stress, free radicals are highly reactive molecules that react with others to form more free radicals; this chain reaction disrupts biological systems and damages tissues. Many of these free radicals are reactive oxygen species (ROS) or oxygen radicals. ROS damages tissues through different mechanisms like DNA damage and protein modification, but ROS produced in normal conditions are inactivated through defense mechanisms like antioxidants. Ozbek (2012) summarized how oxidative stress can cause diseases like diabetes and infection. Some experimental studies showed that in diabetic rat kidneys there was a decrease in the antioxidant enzymes glutathione peroxidase (GPx) and catalase (CAT) which induced oxidative stress. This imbalance in the antioxidant-oxidant system was due to high levels of glucose in the blood. In kidney infections when toxic substances are circulated through the kidneys, they produce ROS; when the antioxidants are defective, the system cannot provide a proper defensive mechanism. Due to this, the kidneys experience cellular damage which can lead to renal diseases [4]. The kidney is a vulnerable organ to ROS; issues that exacerbate the problem pose a high risk to patients affected by it.
Doxorubicin’s Toxicity
Doxorubicin (DOX) is a highly efficient chemotherapy drug, but its use is limited due to the to its renal, pulmonary, and cardiac toxicity. DOX causes oxidative stress: an imbalance of free oxygen radicals and antioxidants. In the kidneys, the stress can damage the membranes and increase glomerular capillary permeability which further damages the kidney. DOX is suggested to play a role in NO production which is a free radical gas that can act as a cytotoxic agent. In rat models injected with DOX, the results demonstrated that there was increased NO production in the renal tissue which contributed to the inflammatory response. The NO released by DOX is responsible for the toxicity caused by this chemotherapy drug. In these tissues, the activity of the antioxidant enzymes glutathione peroxidase (GPx) and catalase (CAT) were reduced [3]. Both of these enzymes help degrade hydrogen peroxide, an ROS, into water: an easier and safer molecule for the body. Reduced levels of these enzymes cause further damage and increase the oxidative stress affecting these kidneys as more free radicals are produced. Ayla and colleagues (2011) tested the use of nicotinamide (NAD), a vitamin used to reduce skin inflammation in the medical field, to reduce the toxicity of DOX through its anti-inflammatory and antioxidant properties. NAD was shown to reduce the damage caused by oxidative stress. Looking at this study, NAD could act as an effective intervention in DOX-induced kidney toxicity.
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Creatine Supplements’ Abilities as a Therapeutic Drug
Creatine is naturally synthesized within the body and a majority is stored within the skeletal muscles. A dietary supplement of creatine has been shown to increase phosphocreatine levels in the muscles, and it is responsible for the phosphorylation of ADP to ATP during short term high intensity workouts. The objective of this research project was to find the effects creatine supplementation can have on disease and inflammation. Riesberg (2016) observed creatine’s ability as a neuroprotector and an anti-inflammatory agent. Using mouse models affected by neurological disorders like Huntington’s Disease and traumatic brain injuries, they observed the effect creatine has on the permeability of the mitochondrial transition pore. If these pores are defective, it can cause more reactive oxidative species (ROS) to form and damages the mitochondria; this reduces ATP production levels which leads to developmental problems and other mental issues. Results demonstrated that creatine was able to inhibit mitochondria permeability, stabilize ATP levels, and reduce tissue damage; creatine’s antioxidant properties also protect against mitochondrial and nuclear DNA damage caused by ROS and increase cell viability. As an anti-inflammatory agent, creatine was found to have reduced formaldehyde-induced arthritis and carrageenan-induced inflammation of the foot paw indicating that it was productive in reducing chronic and acute inflammation. In the airways of mouse models affected by asthma and allergies, creatine supplements exacerbated the problem [6]. Creatine is a cost- effective alternative to many other neuroprotective and immunomodulatory drugs, and it should be further investigated to gain a better understanding of the in vivo actions of this supplement.
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Creatine’s Antioxidant Properties
Creatine monohydrate is widely used in the sports industry and has gained more attention due to its therapeutic abilities in degenerative diseases and conditions. Different studies have shown creatine’s ability to reduce the RNA damage caused by DOX; this is due to its antioxidant properties which scavenges for free radicals and counteracts them. It has also demonstrated protective effects within mtDNA which stabilized ATP production; buffering of cellular ATP levels reduces the accumulation of Ca2+ which decreases the formation of ROS and further tissue damage [5]. Creatine can also increase cellular resistance to oxidative stress. Sestili and colleagues (2011) discussed how creatine supplements can upregulate the antioxidant enzyme pathways for glutathione and thioredoxin. Since creatine was found to exhibit only mild antioxidant activity, its ability to upregulate other antioxidants is beneficial in conditions involving ROS. Increasing antioxidant levels and acting like an antioxidant make creatine a viable therapeutic agent in preventing oxidative stress within the kidneys.
REFERENCES
[1] Cancer. World Health Organization. http://www.who.int/news-room/fact-sheets/detail/cancer. Accessed June 29, 2019.
[2] Refaie, M. M., Amin, E. F., El-Tahawy, N. F., & Abdelrahman, A. M. (2016). Possible Protective Effect of Diacerein on Doxorubicin-Induced Nephrotoxicity in Rats. Journal of toxicology, 2016, 9507563. doi:10.1155/2016/9507563
[3] Ayla, A., Seckin, I., Tanriverdi, G., Cengiz, M., Eser, M., Soner B. C., Oktem, G. (2011). Doxorubicin Induced Nephrotoxicity: Protective Effect of Nicotinamide. International Journal of Cell Biology. doi:10.1155/2011/390238
[4] Ozbek, E. (2012). Induction of Oxidative Stress in Kidney. International Journal of Nephrology. doi:10.1155/2012/465897
[5] Sestili, P., Martinelli, C., Colombo, E., Barbieri, E., Potenza, L., Sartini, S., Fimognari, C. (2011). Creatine as an Antioxidant. Amino Acids. 40(5). 1385-1396. DOI 10.1007/s00726-011-0875-5
[6] Riesberg, L., Weed, S., McDonald, T., Eckerson, J., Drescher, K. (2016). Beyond muscles: The untapped potential of creatine. International Immunopharmacol0gy. 37. 31-42. https://doi.org/10.1016/j.intimp.2015.12.034
[7] Birben, E., Sahiner, U. M., Sackesen, C., Erzurum, S., & Kalayci, O. (2012). Oxidative stress and antioxidant defense. The World Allergy Organization journal, 5(1), 9–19. doi:10.1097/WOX.0b013e3182439613
[8] Sestili, P., Martinelli, C., Bravi, G., Piccoli, G., Curci, R., Battestelli, M., Falcieri, E., Agostini, D., Gioacchini, A. G., Stocchi, V. (2006). Creatine supplementation affords cytoprotection in oxidatively injured cultured mammalian cells via direct antioxidant activity. Free Radical Biology and Medicine. 40(5). 839-847. https://doi.org /0.1016 /j.freeradbiomed.2005.10.035
[9] Lawler, J. M., Barnes, W. S., Wu, G., Song, W., Demaree, S. (2002). Direct antioxidant properties of creatine. Biochemical and Biophysical Research Communications, 290, 47–52. doi:10.1006/bbrc.2001.6164.
[10] Phaniendra, A., Jestadi, D. B., & Periyasamy, L. (2015). Free radicals: properties, sources, targets, and their implication in various diseases. Indian journal of clinical biochemistry : IJCB, 30(1), 11–26. doi:10.1007/s12291-014-0446-0
ACKNOWLEDGMENTS
First, I would like to thank Ms. Lori K. Ball and the FSI staff for providing me with this opportunity to participate in this program. I would like to thank Matt DeSelm for being my advisor and helping me with my research paper, and I would like to thank Raquel Busekrus for providing me with the lab and equipment to be able to conduct this study and for guiding me through this research. Lastly, I would like to thank Xcel Energy and the Maya-Maes Johnson family for sponsoring my research and my stay at this program.
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