1. Abstract
Objective. Evaluation of the changes in the
gene CYP3A4 expression in HepG2 cells
under treatment with Rifampicin or Alfa-
ketoglutarate. Methods. HepG2 cell lines were
seeded into steroids for 7 days with medium
0,1% DMSO before the treatment. Then the
cells were treated with either 10mM of
rifampicin or 4mM of alpha-ketoglutarate. The
cells were incubated at 37°C and the medium
was changed on day 3 and day 7. DNA and
RNA was extracted on day 0, day 3, and day
7. The DNA and RNA was then analyzed
through retrotranscription and qPCR with
CYP3A4 as the targeted gene. The results
were then normalized and an analysis of the
relative gene expression was made between
the treated cells and the controls. Results. The
results showed that levels of CYP3A4
increased significantly in HepG2 cells under
treatment with Rifampicin when compared to
the controls. The increase was shown clearly
the first 3 days and reached it’s peak on the
3rd day before the levels of CYP3A4 were
slightly decreased. When treated with alpha-
ketoglutarate, the gene expression of CYP3A4
in HepG2 cells were slightly decreased the first
3 days when compared to the control before
the gene expressions of CYP3A4 increase.
Conclusion. The expresssions of the gene
CYP3A4 is increased significantly when
treated with rifampicin. The expressions of the
gene CYP3A4 were increased under treatment
of alpha-ketoglutarate although it was not as
significant as the results with rifampicin.
Introduction
Epigenetics is the study of cellular and
physiological trait variations that are not
caused by changes in the DNA sequence. It is
the study of external or environmental factors
that turn genes on and off and effect how cells
read genes. It can be defined as the variation
in gene activity without changing the DNA
sequence. The term epigenetics also refers to
heritable changes in gene expression that do
not involve changes to the underlying DNA
sequence.
It is known that the gene CYP3A4 encodes a
member of the cytochrome P450 superfamily
of enzymes. The cytochrome P450 proteins
are monooxygenases that catalyze many
reactions involved in drug metabolism and
synthesis of cholesterol, steroids and other
lipids. This protein localizes to the
endoplasmic reticulum and its expression is
induced by glucocorticoids and some
pharmacological agents. This enzyme is
involved in the metabolism of approximately
half the drugs in use today. The enzyme also
metabolizes some steroids and carcinogens It1
would therefore be an interesting experiment
to see if this gene could be manipulated some
way to show more gene expression. That way
the manipulated gene could make it more
efficient to catalyze the different reactions.
5hmC is classically associated with processes
that silence or down-regulate gene expression
while increasing evidence suggest 5hmC
serves alternative roles. 5hmC is a stable
modification enriched within gene bodies,
concentrated at transcriptional regulatory
elements and positively associated with
transcriptional activity. It also recruits
transcriptional regulators, splicing factors, DNA
repair proteins and chromatin regulators that
http://www.ncbi.nlm.nih.gov/gene/1576 21/7 - 20151
1
Rifampicin and Alpha-ketoglutarate upreglates gene
expressions of the gene CYP3A4 in HepG2 cells
Farngren, AF1, Llorens, AL2, and Barragan IB
Pharmacogenetics Section, Department of Physiology and Pharmacology, Karolinska Institutet,

2. are distinct from those recruited by 5mC.
5hmC plays an essential role in embryonic
development, cellular differentiation and stem
cell reprogramming as well as it is a strong
prognostic indicator in cancer and other
diseases. 5hmC is implicates in the regulation
of neurological plasticity, immunology and
other dynamic systems by the TET enzyme
family. 2
HepG2 is a perpetual cell line derived from the
liver. They are epithelial cells with a model
chromosome number of 55, and not tumor
genetic in nude mice. The cells have been
grown successfully in large-scale cultivation
systems, and will respond to human growth
hormone. HepG2 cells are suitable in vitro
model systems for the study of polarized
human hepatocytes. They have proper culture
conditions and display robust morphological
and functional differentiation with a controllable
formation of apical and basolateral cell surface
domains that resemble the bile canalicular and
sinusoidal domains, respectively, in vivo.3
Rifampicin is used with other medications to
treat tuberculosis. Rifampicin is also used to
treat some people who have Neisseria
meningitidis (a type of bacteria that can cause
a serious infection called meningitis) infections
in their noses or throats. em from infecting
other people. Rifampicin is also sometimes
used to treat infections caused by other types
of bacteria and to prevent infection in people
who have been in close contact with a person
who has certain serious bacterial infections. 4
Previous studies (Yuhas, Yael et al. 2011)5
have shown that HepG2 cells treated with
rifampin had a significantly increase in gene
expression of the gene CYP3A4. Although, it
would be good to repeat this experiment once
again to confirm the results from the previous
experiments. This is because the previous
experiment from Yuhas, Yael et al. 2011 did
not specifically study the changes in the
evaluation of the gene expression in CYP3A4
specifically.
Alpha-ketoglutarate is a substance found
naturally in the human body. It is available in
dietary supplement form, and plays a key role
in the Krebs cycle. Alpha-ketoglutarate
supplements are purported to offer a variety of
health benefits. Alpha-ketoglutarate is
involved in the formation of glutamine, an
amino acid required for protein synthesis and
for proper functioning of the immune system.
Alpha-ketoglutarate is often used as a
medicine to treat kidney diseases. However,6
it has not been determined if alpha-
ketoglutarate can affect the gene expression
of CYP3A4 in some way. Therefore it would be
relevant if an experiment was made to see if
alpha-ketoglutarate would affect the gene
expression of CYP3A4 in some way.
Methods
A. Cell passaging
The cells were thawed and seeded in a 25
cm2 flask. Once they reached ~90%
confluency, they were washed with DPBS and
digested with 0.25 % Trypsin-EDTA.The cell
suspension was then transferred into a 50 ml
Falcon tube, then it was centrifuged at a speed
of ~1000 rpm for 5 minutes. Then the
supernatant was discarded. 9 ml of the
medium* was the added and it was pipetted up
and down several times to make a single cell
suspension. The cell suspension was then
divided into 3 new flasks. New medium was
added to make a total volume of 8 ml
approximately. Then the flask was shaken
gently back and forth, and from side to side, to
make the cells spread evenly in the flask.
http://www.cambridge-epigenetix.com/en_US/resources 21/7 - 20152
http://www.hepg2.com 21/7 - 20153
http://www.nlm.nih.gov/medlineplus/druginfo/meds/a682403.html 23/7 - 20154
Yuhas, Yael, Eva Berent, and Shai Ashkenazi. “Effect of Rifampin on Production of Inflammatory5
Mediators in HepG2 Liver Epithelial Cells.” Antimicrobial Agents and Chemotherapy 55.12 (2011):
5541–5546.
http://www.webmd.com/vitamins-supplements/ingredientmono-144-alpha-ketoglutarate.aspx?6
activeingredientid=144&activeingredientname=alpha-ketoglutarate 23/7 - 2015
2

3. *100 ml medium: 89 ml DMEM, 10 ml FBS, 1
ml PEST
B. Cell seeding
When the cells reached ~90% confluency all of
the old medium* was aspired out in the flask.
The cells were then washed with 5 ml DPBS
and the DPBS was discarded. Thereafter the
cells were digested with 1 ml 0.25 % Trypsin-
EDTA following the same procedure. The cell
suspension was then transferred into a 50 ml
Falcon tube, then it was centrifuged at a speed
of ~1000 rpm for 5 minutes. Then the
supernatant was discarded. Subsequently, 9
ml of the medium were added and it was
pipetted up and down several times to make
single cell suspension. After that, a cytometer
was used to calculate the density of the cells.
The cell suspension was diluted to 7500 cells/
ml. Then, 200 ul of the diluted cell suspension
was added into each well of a ULA 96-well
plate. The last step was to centrifuge at a
speed of 1000 rpm for 2 minutes and then
keep the plate in the incubator. 50 % of the
medium was changed 4 and 6 days after
seeding.
*100 ml medium: 89 ml DMEM, 10 ml FBS, 1
ml PEST
C. Drug incubations & treatment
solutions
For drug incubations the instructions of the
AllPrep® DNA/RNA/miRNAUniversal
Handbook were followed. DNA and RNA were
extracted from the cells on day 0, day 3, and
day 7 for PCR.
Treatment solutions
Rifampicin: Stock with the concentration 123
mM in DMSO was first diluted to the
concentrations of 2 mM and then diluted once
again to the wanted solution of 10 uM.
Alpha-ketoglutarate: Stock with the
concentration 500mM was first diluted to a
concentration of 2mM and then diluted once
again to the wanted solution of 4uM.
D. Data analysis
To analyze and calculate the results data from
the qPCR was normalized.
Results
The cells were photographed 3 days and 5
days after seeding with 10x option. The 5 days
old cells are bigger and grown more tightly
next to each other than the 3 days old cells.
This shows that the cells were forming into
proper spheroids before they were treated with
the drugs (see fig.1 and fig.2)
Fig. 1: microscopy pictures of the cells 3 days
after seeding
Fig. 2: microscopy pictures of the cells 5 days
after seeding
The results show that levels of CYP3A4
increase significantly in HepG2 cells under
treatment with Rifampicin when compared to
the controls. The increase is shown clearly the
first 3 days and reaches it’s peak on the 3rd
day before the levels of CYP3A4 is slightly
decreased. When treated with alpha-
ketoglutarate, the gene expression of CYP3A4
in HepG2 cells are slightly decreased the first
3 days when compared to the control before
the gene ’expressions of CYP3A4 increase.
(fig.3 and fig.4)
3

4. Fig 3: graph showing how the expression of
CYP3A4 changes after inducing rifampicin
Fig 4: graph showing how the expression of
CYP3A4 changes after inducing α-
ketoglutarate
When compared to each other, it is shown
clearly that the expressions of CYP3A4 are
much more in the hepatocytes treated with
rifampicin than the hepatocytes treated with
alpha-ketoglutarate no matter the time that the
treatment has gone.
Fig 5. diagram showing the expression of
CYP3A4 in HepG2 cells treated with rifampicin
and alpha-ketoglutarate 3 days after treatment
Fig.6 diagram showing the expression of
CYP3A4 in HepG2 cells treated with rifampicin
and alpha-ketoglutarate 7 days after treatment
Discussion
The results show that the gene expressions of
CYP3A4 increase significantly in HepG2 cells
when treated with Rifampicin than compared
to the controls. Meanwhile, the levels of
CYP3A4 increase less or non at all when
treated with alpha-ketoglutarate. The
expression of CYP3A4 was almost 5 times
more compared to the control 3 days after the
treatment. The alpha-ketoglutarate treatment
lead to 1,5 times more expression of the gene
CYP3A4 compare to the control 3 days after
the treatment. When comparing the rifampicin
treatment to the alpha-ketoglutarate treatment,
the rifampicin treatment showed a more
significant change in the expression of the
gene CYP3A4. The experiment with HepG2
cells treated with rifampicin have been done
before and the results of the previous studies
(Yuhas, Yael et al. 2011) can be supported by7
the results of our experiment. The alpha-
ketoglutarate treatment was, on the other
hand, the first test to be made on HepG2 cell
lines in order to study the evaluation of the
gene expression in CYP3A4. Since the results
from the alpha-ketoglutarate treatment didn’t
show a significant difference in the expression
of the CYP3A4 gene compared to the
rifampicin treatment, it would be interesting if
Yuhas, Yael, Eva Berent, and Shai Ashkenazi. “Effect of Rifampin on Production of Inflammatory7
Mediators in HepG2 Liver Epithelial Cells.” Antimicrobial Agents and Chemotherapy 55.12 (2011):
5541–5546.
4
Rifampicin
0
0,75
1,5
2,25
3
COH RIF3D RIF7D
α-Ketoglutarate
0
0,5
1
1,5
2
COH AKG3D AKG7D
3 days samples
0
1,25
2,5
3,75
5
C3D RIF3D AKG3D
7 days samples
0
1,5
3
4,5
6
C7D RIF7D AKG7D

5. more studies were done to in order to see if
alpha-ketoglutarate actually would affect the
expression of the gene CYP3A4 at all.
However, it is important to notice that the
timespan of the cell treatments only lasted for
7 days. Since the results on the rifampin
treatment showed that the expression of the
gene CYP3A4 was increased on both day 3
and day 7, it is likely that the gene expression
would still be increased a period after 7 days
of treatment since the expression of the gene
CYP3A4 never showed any tendency to
decrease during the drug incubations. A further
experiment could be made to study how the
gene expression of CYP3A4 is effected when
treated with rifampicin or alpha-ketoglutarate
during a longer period.
Importantly, this experiment was made on cell
lines and not primary cells or humans. Since
cell lines, cancerogenic cells, survive easily
and are better to experiment on. Since HepG2
are cancerogenic cells they also have different
mechanisms and metabolism than primary
cells. It would therefore be relevant to do
further experiments on primary cells to study if
the treatments would have the same results as
the cell lines. This experiment should
especially be made with alpha-ketoglutarate
since this treatment in this experiment showed
less difference in gene expression.
HepG2 cells down regulates the expression of
CYP3A4 as time passes by, which means that
the level of gene expressions in the controls
are different. This means that the difference in
the gene expressions might have been more
significant than it would have been if the
expression of the gene CYP3A4 was the same
in all controls. This also means that the
differences in the gene expression between
the controls and the treated cells would are
more significant than they should the longer
the drug incubation is. This would explain part
of the increase of the gene expression.
There were no serious complications during
this experiment that would have affected the
results in a significant way, and the methods
were followed correctly.
In summary, we have shown that the
expression of the gene CYP3A4 increase
significantly in HepG2 cells under treatment
with Rifampicin when compared to the
controls. The increase is shown clearly the first
3 days and reaches it’s peak on the 3rd day
before the levels of CYP3A4 is slightly
decreased. When treated with alpha-
ketoglutarate, the gene expression of CYP3A4
in HepG2 cells are slightly decreased the first
3 days when compared to the control before
the gene ’expressions of CYP3A4 increase.
The treatment on HepG2 cell lines with alpha-
ketoglutarate was, to our knowledge, the first
experiment to be made and needs future
experiments as support for the results.
Although the treatment with rifampicin has
been repeated and supported by previous
studies, it would be interesting if the
treatments were tested on primary cells or
humans.
References
Gene database: CYP3A4. Pubmed. [Cited
2015 Jul 21] Available from: http://
www.ncbi.nlm.nih.gov/gene/1576
5 Major Reasons why 5-
hydroxymethylcytosine is Important.
Cambridge Epigenetics. [Cited 2015 Jul 21]
Available from: http://www.cambridge-
epigenetix.com/en_US/resources
HepG2 cells, [Cited 2015 Jul 21] Available
from: http://www.hepg2.com
Drug database: Rifampicin. Pubmed. [Cited
2015 Jul 23] Available from: http://
www.nlm.nih.gov/medlineplus/druginfo/meds/
a682403.html
Yuhas, Yael, Eva Berent, and Shai Ashkenazi.
“Effect of Rifampin on Production of
Inflammatory Mediators in HepG2 Liver
Epithelial Cells.” Antimicrobial Agents and
Chemotherapy 2011; 55(12): 5541–5546.
Drug database: Rifampicin. [Cited 2015 Jul 23]
Available from: http://www.webmd.com/
vitamins-supplements/ingredientmono-144-
alpha-ketoglutarate.aspx?
activeingredientid=144&activeingredientname=
alpha-ketoglutarate
5