The effect of curcumin on the activity of glutathione-s-transferase (GST) in sheep liver

and kidney cytosolic and mitochondrial fraction isolates

Introduction
Macromolecular molecules known as enzymes are biologically important for catalysing
reactions within cells. Their ability to catalyse reactions up to an order of magnitude of 17
[Radzicka & Wolfenden 1995], make them extremely efficient and vital in bodily functions
such as metabolism [Schomburg, Chang & Schomburg 2002] and digestion. Enzymes can
be categorised into different classes depending on their functions, such as hydrolase which
is responsible for the addition of water, isomerase rearranges the atoms and transferase
transfers a functional group from donor to acceptor, with the focus of this experiment on the
latter enzyme. Extensive research and studies have suggested that deficiency of enzymes
may lead to the disruption of bodily functions and limited production of vital biological
intermediates which cascades to the build-up of toxic substances [Chaturvedi et al. 2016].

Glutathione-s-transferase (GST) is a group of metabolic enzymes with multiple forms that

uses the liver-produced antioxidant, glutathione (GSH) to act as a catalyst and together they
perform many protective roles [Townsend & Tew 2003]. GSH and GST share a dependent
relationship as depletion of one or the other significantly affects the function and abundance
of both. GST is most abundant in the liver as once blood transports to the stomach and
intestines it has to pass through the liver and since GSH is synthesised in all cells of the
body the majority of this is present in the transported blood, thus the liver receives the most
influx of GSH and GST activity. This enzyme is then further transported to the kidney to filter
waste products in the blood hence GST activity also occurs to a lesser extent in the kidney.
Like most metabolic pathways GSH is synthesised in the cytosol [Lu 2009] [Ribas, Garcia-
Ruiz & Fernndez-Checa 2014] with additional studies indicating that to some degree GST
is also present in the mitochondria [Raza et al 2002] [Meredith & Reed 1982]. The
mitochondria lack specific enzymes needed for GSH synthesis and hence depends on a
transport system of anion carriers [Raza 2011]. GSH is obtained from the cytosol and
permeated into the liver and kidney mitochondria via these carriers [Raza 2011].

GSH is composed of three amino acids connected in the order of -glutamyl, cysteinyl and
glycine [Chakravathi 2006], in which its versatility in functions arises from its redox active
amino acid, cysteine which includes the functional group sulfhydryl. Although free cysteine
has the same capability of performing these functions, it is quite toxic due to its reactive
nature. Specifically the sulphur atom [Chakravathi 2006] in cysteine acts as a scavenger for
oxidative-stress-causing molecules such as peroxidised lipids and free radicals [Ayala,
Muoz, Argelles 2014] as well as xenobiotics in order to transform them into non-toxic
compounds for elimination [Raza et al. 2002]. This mechanism of detoxification is GST
catalysed in the liver when the sulphur atom attaches itself to the substrate to increase
solubility and hence increase the ease to eliminate via kidneys. In addition to this, sulphur
also reacts with foreign disulphide bonds in proteins to ensure that they remain in optimal
chemical structure i.e. maintain correct iron charge in haemoglobin. Fluctuation of GST
levels can affect the bodys defence against free radicals from radiation, heavy metal toxicity
as well as aging by performing at a slower rate. Susceptibility to infections and lower
immune response has also been associated with depletion in GSH and hence GST levels as
the livers potential to detoxify is decreased [Lu 2009].

Another potent antioxidant is curcumin, a turmeric rhizome extracted from Curcuma longa
which has long been used in India as a spice and medicinal remedy as it has anti-
inflammatory and anticancer properties. A cancer study from Sharma et al 2001 has
indicated that tumour cells have elevated levels of GSH and GST activity which can be
induced by curcumin. This can limit the tumours resistance to chemotherapy and hence limit
cancer progression as well as improving treatment outcome. Research from Dorai et al.
 2001 and Kwon 2014 also agrees that curcumin has the potential to treat cancer, thus
curcumin has recently seen an increase in research for its promising future as a treatment
for cancer tumours. On the contrary, a study from Oetari et al 1996 found that curcumin is a
strong inhibitor of GST. Therefore, this experiment aims to determine if GST activity is
induced or decreased in the presence of curcumin by comparing GST activity present in
sheep liver and kidney in their cytosolic and mitochondrial counterparts against the same
samples but with added curcumin added directly.

Method

The method outlined in pages 48 - 57 of the 2017 Metabolic Biochemistry Subject Manual
was followed for this experiment. The following changes were made:
On page 48 step 2 outlines the use of 50 g of sheep liver and kidney. For this
experiment 5 - 10 g was used.
On page 53 step 1 outlines the dilution of the cytosol and mitochondrial fractions to
be 1 in 20. For this experiment a 1 in 10 dilution was performed.
On page 53 step 2 outlines the use of 2.15 mM stock solution of curcumin. For this
experiment a stock solution of 2.0 mM was used.

Results
Table 1. Specific activity of GST expressed as an average (U/mg of protein) in samples of
sheep liver and kidney fractions (cytosol, mitochondria) without and with curcumin.
Calculated standard deviation, number of samples and t-tests with significant p-values
asterisked are also included.

Samples of sheep Mean of GST specific Standard deviation Number T-test

liver and kidney activity (U/mg of protein) of
used samples
Without With Without With Without With
curcumin curcumin curcumin curcumin curcumin curcumin
Liver cytosol 3.02 0.627 1.96 0.752 8 0.0104*
Kidney cytosol 0.547 0.105 0.428 0.208 8 0.0251*
Liver mitochondria 0.554 0.710 0.503 1.33 8 0.764
Kidney mitochondria 0.289 0.228 0.417 0.349 8 0.756
*p0.05

Table 1 shows the average of all the collected data in this experiment along with standard
deviation. The specific activity of GST is presented in U/mg of protein and significantly low p-
values of the t-tests conducted are highlighted with asterisks. The t-test carried out for the
data of liver cytosol without curcumin versus with curcumin shows a significantly lower p-
value of 0.0104 (p< 0.05). Similarly, the t-test calculated for kidney cytosol without curcumin
and with curcumin shows a significantly lower p-value of 0.0251 (p< 0.05). Untrustworthy
data with R^2 values less than 0.95 from the Lowry assay and significant outliers have all
been removed prior to data analysis and results have been rounded to 3 significant figures.
 GST specific activity in samples without curcumin vs. samples with curcumin
3.5
3
2.5
2
1.5
1
0.5 Wi thout curcumi n
GST specific activity (U/mg protein)
Wi th curcumi n
0
l a l a C C C C
o s o d ri o s o d ri CF - F- CF - F -
t n t n L LM K KM
r Cy h o y Cy h o
ve o e o c
c
Li Mi t i d n Mi t
r K ey
ve
Li dn
Ki

Samples of sheep liver and kidney tested

Figure 1. GST specific activity (U/mg of protein) in samples of sheep liver and kidney
cytosolic and mitochondrial fractions with and without curcumin, with standard error bars.

Figure 1 illustrates the average of all the collected data in this experiment along with
standard deviation expressed as error bars. Significantly low p-values obtained from the t-
tests have also been indicated with asterisks. Histogram data values have been expressed
as averages of samples without curcumin and with curcumin in sheep liver and kidney
samples in each fraction isolate (cytosol, mitochondria) have been presented as side by side
columns to highlight difference in specific activity of GST.

Discussion
Through the course of this experiment it was hypothesised that the antioxidant curcumin
could inhibit the activity of the enzyme glutathione-s-transferase. All samples with curcumin
added directly except for liver mitochondria showed GST activity inhibition. From table 1 the
samples without curcumin all had a decrease in enzymatic activity except for the liver
mitochondrial fraction. This inhibition generated a significant p-value for the liver cytosolic
fraction, as the sample with curcumin had a significantly lower value of 0.627 than without
curcumin 3.02 (p<0.05), the same can be seen in the cytosolic kidney sample as it was
found to be 0.105 without curcumin and 0.547 with curcumin (p<0.05). Although the
mitochondrial kidney fraction did demonstrate a decrease in enzyme activity the results were
not significant. The same can be said about figure 1 where enzyme activity in samples of
liver and kidney cytosol and mitochondria with curcumin were all lower than the samples with
no curcumin, this suggests that curcumin does inhibit GST activity. As expected overall all
samples of the liver experienced more enzyme activity than kidney samples and the same
applies with their cytosolic fractions as GST synthesis is most abundant in the liver,
particularly in the cytosol as compartmentalisation is needed for complex biochemical
reactions like this [Bhagwat 1998].

From the data collected it would suggest that curcumin inhibits GST activity in the liver
cytosol, kidney cytosol and mitochondria except for liver mitochondria. This could be due to
several errors made during dilutions as they were all made up individually between groups
despite using the same stock solutions and temperature could have also affected the
enzymes [Peterson et al. 2007]. Samples of sheep liver and kidney was again obtained
individually and hence could be another source for the discrepancies. As this experiments
data was collated from many individual groups of four, human error was increased greatly as
demonstrated with the presence of negative error bar values which could possibly be from
the incorrect removal of outliers and thus multiple group results could be limited to a smaller
number for future studies. Reliability can also be increased with duplicates of the sample
being tested and using one source of sheep to obtain samples.

There have been recent studies suggesting that curcumin has the opposite effect, in that it
increases GST activity [Ye et al 2007]. This is demonstrated by a study from Piper J et al
1998 as their results indicated that curcumin increased liver GST levels in rats treated with
curcumin at doses of 1 to 500 mg kg-1 body weight for a duration of 14 days had a
maximum induction of 1.5-fold. Studies from Singh et al 1998 and Sharma et al 2001 also
suggest that curcumin has the same GST increasing capability, as mice and rats fed
curcumin for 14 days also showed levels of liver GST increase. It must be said though that
these studies were conducted in vivo with rat or mice liver as opposed to in vitro using sheep
liver, therefore that could account for the variable results obtained. On the contrary studies
from Oetari et al 1996 agree with the results of this experiment as they discovered that
curcumin is a strong inhibitor against GST and thus that would explain its anti-inflammatory,
anticancer and antioxidant properties.

The inhibition of GST activity by curcumin to limit tumour resistance to chemotherapy and
hence limit cancer progression was researched by Appiah-Opong et al. 2009 and Hayeshi et
al. 2007. It is theorised that curcumin may have a higher binding affinity and hence
outcompetes GST in binding to the cytosolic proteins and hence GST activity is inhibited
[Hayeshi et a. 2007]. Based on these studies, curcumin has the dual ability to induce and
inhibit GST activity depending on the test subjects and conditions used different outcomes
are to be expected.

Conclusion
Overall the results of this experiment indicate that curcumin can inhibit GST activity in sheep
liver and kidney with a minor discrepancy in the liver mitochondrial fraction. This can be
accounted for by the inconsistency in dilution, incubation as well as sample obtainment.
Curcumin overall has received a mixed response from many researchers as some studies
like Singh et al 1998 and Sharma et al 2001 have found that curcumin induces GST whilst
Oetari et al 1996 and Appiah-Opong et al. 2009 have suggested that curcumin has the
opposite inhibiting effect. Noting that these studies all varied in test subjects, environments
and methodology as a result it is recommended that further studies be carried out for
confirmation. As many studies have been conducted prior to this it can be safely said that
further experimentation is needed to confirm the conditions to which curcumin most
effectively inhibits or induces GST and the optimal conditions required.

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