Hepatotoxicity of nonsteroidal anti-inflammatory drugs (NSAIDs) in mice
Mindi Clegg
Advisor: Dr. Brown
Analyzing ALT levels
Hepatotoxicity is one of the common side effects of nonsteroidal antiinflammatory drugs. Individual NSAIDs differ in the incidence, severity, and type
of liver damage they produce (Wade et al., 1997, 546). Symptoms of
hepatotoxicity can range from mild elevation in serum aminotransferases to
severe hepatocellular injury (Wade et al., 1997, 546). Drug induced liver
damage is the most common cause for drugs to be withdrawn from the market
(Obach et al., 2008, 1814).
Studies have shown that acetaminophen forms a reactive metabolite (NAPQI)
that is an oxidizing agent that depletes the cell of glutathione (Gibson et al.,
1996, 581). The biochemical functions of glutathione are recognized in
maintaining cellular protein sulfhydryl groups and in detoxifying substances
including metals, hydrogen peroxide, and oxygen radicals (Kuralay et al., 1998,
224). A depletion of glutathione has been used to evaluate toxicity, because it is
involved in the response to toxins.
The second assay used in this experiment was the alanine aminotransferases
(ALT) assay. ALTs are normally found in large concentrations in the liver. The ALT
blood test is typically used to detect liver damage, because it is released into the
bloodstream due to liver injury. ALT values are often compared to the results of
other tests to determine which form of liver disease is present (Abboud and
Kaplowitz, 2007, 278-280).
This research attempted to determine if other NSAIDs, such as naproxen and
ibuprofen, have the same hepatotoxic effects on mice livers as acetaminophen.
Mice were used as the study organism, because previous experiments had used
them and their metabolism is similar to that of humans. The hypothesis tested
in this experiment is that the hepatotoxicity of naproxen and ibuprofen will be
less than that of acetaminophen, since naproxen and ibuprofen are metabolized
by different enzymes in the liver. Glutathione levels and ALT levels were
analyzed to determine the amount of liver damage.
Dosing of mice
Materials and methods
Experiments were performed with 12 male mice, which were divided into
four groups with three mice in each group. Mice in the same testing group were
housed together in Dr. Lustofin’s office and kept on a natural light cycle during
the month of February. Mice were moved to individual cages with no food or
bedding and fasted 2 hours before dosing.
Group 1 (control) received 200 mg of peanut butter. Group 2 received 200
mg of peanut butter with 4 mg of acetaminophen. Group 3 received 200 mg of
peanut butter with 3.2 mg of ibuprofen. Group 4 received 200 mg of peanut
butter with 1.6 mg of naproxen. These doses were calculated using the weight
of the mouse and the half-life of the drug. Mice were dosed daily for ten days.
Methods
After sacrificing the mice, whole blood samples were collected. The samples
were centrifuged and the serum was collected. The assay protocol listed in the
Bioo Scientific Instruction Manual for the ALT Assay Kit was followed.
ALT Levels in Mice Serum
200
180
160
140
120
100
80
60
40
20
0
ALT (units/L)
Introduction
167.490
Control
189.598
Acetaminophen
108.716
52.441
Ibuprofen
Naproxen
Figure 1. Average ALT (units/L) levels in mice serum for each testing group.
Results
Statistically the only difference found in the ALT test was between the control
and the naproxen group. The acetaminophen and ibuprofen were not
statistically different from the control.
Analyzing glutathione levels
Methods
After sacrificing the mice, livers were dissected and stored in a -80o freezer.
Glutathione (GSH) level analysis was conducted to determine the concentration
of GSH in the mice livers (Brown, 2009, 1-10). When GSH is converted to GSSG
using DTNB, TNB is formed. A set amount of GSSG-reductase was added to the
samples to measure the formation of TNB. The rate of TNB production was used
as the measure of GSH present. The absorbance of TNB was measured at 412
nm.
Figure 2. Shows the chemical process of converting GSH to GSSG producing
TNB, which was used as a measure of GSH.
Concentration of GSH in Mice Livers
5
Acknowledgements
4
[GSH] in µM
I would like to thank Dr. Brown for being my capstone advisor and my
capstone classmates for their support and feedback. I would like to thank Dr.
Lustofin for letting me use her office to house my mice. I would also like to
thank the Marietta College Biology Department for their funding and the
Investigative Studies for their supplies grant to purchase the ALT Assay Kit.
Finally, I would like to thank my friends and family for their encouragement
during this experiment.
3
2
1
0
3.503
2.517
3.997
3.830
Control Acetaminophen Ibuprofen Naproxen
Figure 3. Average concentration of GSH in mice livers for each testing group.
Results
The acetaminophen did cause a statistically significant decrease in the GSH
levels in group 2. The GSH levels in the other test group were not statistically
different from the control.
Conclusions
This research attempted to determine if other NSAIDs, such as naproxen and
ibuprofen, have the same hepatotoxic effects on mice livers as acetaminophen.
It was hypothesized that the hepatotoxicity of naproxen and ibuprofen would be
less than that of acetaminophen. Although GSH levels in the acetaminophen
testing group were depleted, the ALT tests show that no significant liver damage
occurred, so the hypothesis was not supported.
In the ALT experiment, liver damage was not observed for the dosed groups
of mice. The naproxen was significantly lower than the control. However, the
group that was dosed with acetaminophen, did show elevated ALT levels in the
blood serum, but levels were not statistically significant.
In the glutathione experiment, liver damage was observed in the group dosed
with acetaminophen. The GSH levels in the livers of the mice dosed with
acetaminophen were depleted. The groups dosed with ibuprofen and naproxen
did not show depleted levels of GSH present.
Research was not found that directly compared the three drugs tested in this
experiment. However numerous amounts of research showed that a similar
dose of acetaminophen caused liver damage in mice (Agarwal et al., 2010, 8).
Liver damage due to acetaminophen has become more common, because it is a
frequently used anti-inflammatory drug (Gibson et al., 1996, 580). It may be
possible to reduce the risk of drug induced liver damage by using other NSAIDs
to treat patients. The use of these other NSAIDs would still reduce the pain and
inflammation in the patient, but would not cause the same degree of damage to
the liver (Wade et al., 1997, 547).
It would be beneficial to repeat this experiment with a larger sample size in
order to determine if the ibuprofen and naproxen damage the liver less than
acetaminophen. Different doses may need to be tested in order to see
distinguishable results.
Literature Cited
Abboud G, Kaplowitz N. (2007) Drug-Induced Liver Injury. Drug Safety.
30(3):277-294.
Agarwal R, MacMillan-Crow LA, Rafferty TM, Saba H, Roberts DW, Fifer K, James
LP, Hinson JA (2010) Acetaminophen-Induced Hepatotoxicity in Mice Occurs
with Inhibition of Activity and Nitration of Mitochondrial Manganese
Superoxide Dismutase. American Society for Pharmacology and Experimental
Therapeutics. 1-34.
Brown D. (2009) Measuring Glutathione and Glutathione Disulfide Levels.
Toxicology Lab Manual. Marietta College: 38-42.
Gibson JD, Pumford NR, Samokyszyn VM, Hinson JA. (1996) Mechanism
of Acetaminophen-Induced Hepatotoxicity: Covalent Binding versus
Oxidative Stress. Chemical Research in Toxicology. 9:580-585.
Kuralay F, Akarca US, Ozutemiz AO, Kutay F, Batur Y. (1998) Possible
Role of Glutathione in Prevention of Acetaminophen-Induced Hepatotoxicity
Enhanced by Fish Oil in Male Wistar Rats. Journal of Toxicology and
Environmental Health. 53:223-229.
Obach RS, Kalgutkar AS, Soglia JR, and Zhao SX. (2008) Can In Vitro MetabolismDependent Covalent Binding Data in Liver Microsomes Distinguish
Hepatotoxic from Nonhepatotoxic Drugs? An Analysis of 18 Drugs with
Consideration of Intrinsic Clearance and Daily Dose. Chemical Research in
Toxicology. 21:1814-1822.
Wade LT, Kenna G, Caldwell J. (1997) Immunochemical Identification of Mouse
Hepatic Protein Adducts Derived from the Nonsteroidal Anti-Inflammatory
Drugs Diclofenac, Sulindac, and Ibuprofen. Chemical Research in Toxicology.
10:546-555.