Modelling Isoniazid Hepatotoxicity on HepG2 Cells By CYP2E1 Metabolism - London Metropolitan University - Eric Garson 2011

ISONIAZID HEPATOTOXICITY ON HEPG2 CELLS│TUBERCULOSIS APRIL 2011

LONDON METROPOLITAN UNIVERSITY BY ERIC GARSON AND DR. KEN WHITE PAGE 1

MODELLING ISONIAZID

HEPATOTOXICITY ON HEPG2 CELLS

BY CYP2E1 METABOLISM

BY

ERIC GARSON1 (STUDENT)

AND

DR. KEN WHITE2 (SUPERVISOR)*

* School of Human Sciences, London Metropolitan University, 166-220 Holloway Road, London, N7 8DB. Correspondence: [email protected] (Student) [email protected] (Supervisor)

Abstract Isoniazid makes up one of the main therapies used to stop, prevent and eradicate Mycobacterium Tuberculosis (TB) from the body. However for these objectives to be met, isoniazid needs to be metabolised by the liver. This metabolic process can cause hepatotoxicity, a condition which requires immediate cessation of isoniazid medicine or fatal consequences may follow. It is thought Cytochrome P450 2E1 (CYP2E1) protein families induce toxicity in liver cells by producing Reactive Oxidative Species (ROS) when isoniazid is metabolised. We wanted to simulate this theory in vitro and to show that

CYP2E1 is responsible for the production of cell cytotoxicity through ROS production and consequently hepatotoxicity. Using HepG2 cell lines and assessing cell proliferation by MTT assays, we obtained results which showed CYP2E1 is responsible for cell death and hence hepatotoxicity. However we were unable to reproduce the initial positive results satisfactorily to conclusively justify CYP2E1’s involvement and whether ROS was the root cause. Nevertheless we have significant observations concerning the Otto Warburg effect and the Crabtree effect, adherence of galactose grown cells to flask walls and the bystander effect, the latter regarding ROS production and its effects on HepG2 cells.

Background

“Unequal development in different countries in the promotion of health and control of disease, especially

communicable disease, is a common danger.”

-World Health Organisation (WHO) Constitution 1946

A mother walks into a clinic for a check-up after giving

birth four weeks earlier. She is Hepatitis B Virus (HBV)

and Human Immunodeficiency Virus (HIV) positive, (risk

factors for hepatotoxicity) (Senousy et al 2010). Moreover

she has recently been diagnosed with a bout of primary

tuberculosis (TB) infection. Through the clinic she is able to

access the relevant treatments for her ailments, ATRIPLA

for HIV and HBV infection and Isoniazid, Rifampicin,

pyrazinamide and ethambutol cocktail therapies for her TB.

She has just received results concerning an increase in liver

function tests, particularly enzymes ALT (alanine

aminotransferase) and AST (aspartate aminotransferase), an

indication of toxicity, hepatitis or hepatotoxicity. She has

felt drowsy, weak, vomited several times and felt lethargic,

particularly in the light of having given birth recently. Her

exhaustion is compounded by her poor social conditions,

large family and improper sanitation provision within her

community.

The clinician explains the symptoms are due to her TB

regimen, in particular isoniazid. She is aware she would

need to take this medication for a further 6-9 months

depending on the effectiveness of the medicine.

She also realises that the drugs if not stopped immediately

will worsen her condition and even lead to death.

What’s more she understands that stopping her treatment

will increase the bacterium’s resistance to scarce isoniazid

medicine if retaken. What can we do to change this?

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Figure 1- Rene Laennec listening and describing a

patient’s symptoms using a stethoscope. Theobald

Chartran 1849-1907. (Roguin 2006)

“A tightly rolled sheet of paper,”

-Rene Laennec (Roguin 2006)

In the aftermath of the French revolution, the rational,

probing discipline of clinical medicine was taking a grip of

the French Universities. In 1816, one of its leading lights,

René Laennec (figure 1) placed “a tightly rolled sheet of

paper,” onto the chest of a patient treated in hospital. He

noted an unmistakable sound, “rattles.”

In comparison with other patients, Laennec noted down, “I

began to suspect the phenomenon to be due to cavities in

the lungs…I was able to confirm at autopsy that my

suspicion was correct.” Today the “tightly rolled up sheet of

paper,” is called the stethoscope. (Roguin 2006)

The 19th century put many names to the “rattles” sound

Laennec was hearing for the first time using his

stethoscope, (“stethos” meaning chest and “skopein”

meaning to explore”).

Such names included, Phthisis, (Greek for “waste away”),

Consumption, (from the Latin for to waste away”) and

Tubercles noted by Matthew Bailey and Laennec (above).

Laennec would eventually succumb to the disease at the

tragic age of 45 using his own invention to diagnose his

own impending death. (Cunningham 2007, Roguin 2006)

However it wasn’t until the germ theory of disease was set

out by visionaries, such as Joseph Lister, Louis Pasteur and

Robert Koch, that the disease took on its modern name.

Robert Koch identified the microorganism as a bacterium

(figure 2) called Mycobacterium Tuberculosis, from which

we get the disease name, tuberculosis or TB. (Saltini 2006)

TB and its aliases have been the subject of notable

literature, music, opera and art works. The latter for

instance is shown to vivid effect in the painting on page one

by Cristóbal Rojas called La Miseria (1886).

“ “If I should die”, said I to myself, “ I have left no

immortal work behind me –nothing to make my friends proud of my memory- but I have loved the principle of

beauty in all things, and if I had had time, I would have made myself remembered””

-John Keats in a letter to Fanny Brawn 1820 (J.M &M.J.

Cohen 1960)

As with many others of his generation John Keats, the

celebrated young English poet, was infected with the

bacterium and eventually died due to it. Giacomo Puccini’s

opera, La Bohème and Giuseppe Verdi’s opera, La

Traviata, also used TB as a pertinent force for their libretti

and music. (Cunningham 2007)

In fact, TB was once a creative gold mine for authors and

artists during the enlightenment and 19th century. From

Dickens, Byron and Bronte, who would kill off the heroine

right at the end with dramatic scenes exhibiting the patient

in cold sweats, exhaustion, blood in the sputum and trouble

breathing, all valid signs still used today in modern

medicine for its prospective diagnosis. (Cunningham 2007, Daniel 2006)

Figure 2- The original isolates of TB discovered by Robert

Koch, still and currently on proud display at the

Hunterian Museum, Royal College of Surgeons London.

(Royal College of Surgeons of England image library-

Surgicat)

“At all sites, where the tuberculous process was fresh and progressed rapid bacilli where found in abundance.”

-Robert Koch 1882-(Kaufmann and Schaible 2005)

Not until the 20th century did a new generation of scientists

come to battle TB. In 1927, Albert Calmette and Camille

Guérin produced the BCG vaccine (Bacille Calmette-

Guérin vaccine).

BCG is still used today, being the most widely widely

imployed of all vaccines. In fact it was the very first large-

scale task implemented by the newly formed World Health

Organisation (WHO) in 1948, vaccinating 14 million

children.

The BCG vaccine was built on the work of Edward Jenner

and Louis Pasteur’s vaccine developments in centuries gone

http://en.wikipedia.org/wiki/Crist%C3%B3bal_Rojas



by. The TB bacillus was attenuated hundreds of times until

no longer virulent.

The vaccine protects against “disseminated childhood TB.”

However, its effects on adult TB are less successful,

particularly secondary TB and latent forms of the disease.

This makes boosters necessary. (Bell 2005, Anderson 2007,

Amoils 2005, Daniel 2006)

Figure 3- Albert Calmette rounding up support for BCG

(Wellcome Trust Image Library Website 2011)

Penicillin was discovered by Ernest Duchesne and then

later Alexander Fleming. It was developed by Howard

Florey, Ernst Chain and Norman Heatley. This began a new

leap forward in fight against infectious diseases.

(Cunningham 2007, Pouillard 2002)

Following in the footsteps of penicillin, streptomycin was

discovered by Albert Schultz and Selman Waksman. This

was the first chemotherapeutic agent to demonstrate an

ability to tackle TB. (Daniel 2006)

Figure 4- The second window up left at St. Mary‘s

Hospital, Paddington, London where Penicillin’s action

was noticed by Alexander Fleming

Isoniazid

Discovered by Hans Meyer and Joseph Mally in 1912,

isoniazid was not used as an antibacterial agent against TB

until 1952. Its effectiveness was capitalised upon by

Hoffman La Roche, Bayer and Squibb pharmaceutical

companies separately. (Corey E, Czakó B and Kürti L)

Mechanism of action

The mechanism of isoniazid’s action has been unclear for

60 years. Some suggest it inhibits the formation of myolic

acids needed for the bacterium’s cell wall (figure 5).

Of particular interest is the active site of the enzyme InhA.

Moreover, for isoniazid to function actively, it is

metabolised by a family of proteins known as Cytochrome

P450 2E1. (Timmins & Deretic 2006, Timbell et al 1977)

Figure 5- Showing the present understanding of isoniazid

action

Cytochrome P450 2E1

Cytochrome P450 2E1 (CYP2E1) is a member of a diverse

“superfamily of haem-thiolate proteins” situated in

endoplasmic reticulum and mitochondria. CYP2E1 is found

in bacteria, eukaryotes and Archeae. These proteins are

responsible for a plethora of reactions including

oxidation/reduction reactions, reshaping compounds for

cleaner excretion, “dehalogenation”, “epoxication”,

metabolism of toxins, steroids, carcinogens and drug

metabolism to list a few. From an overarching viewpoint

they are “terminal oxidases”. (Cederbaum et al 2008, Danielson 2002)

Metabolism of isoniazid produces molecules which

potentially cause toxicity to the hepatocytes (liver cells)

with which these reactions occur. We call these toxins,

Reactive Oxidative Species or ROS.

ROS has been implicated in many other drugs and

conditions such as pyrazinamide and ethambutol,

Parkinson’s, Alzheimer’s and atherosclerosis conditions.

(Danielson 2002, Preziosi 2007, Senousy et al 2010)

ROS

The liver is an organ of labour performing a number of

functions and reactions, such as being the body’s heat

source, storing and releasing iron, vitamins, production of

urea, secretions of bile salts, storage of glycogen and the

production site of albumin. (Kapit et al 2001)

Besides these and many other functions, it is the centre of

drug metabolism. Consequently it suffers the most from

isoniazid therapy. However it does have various defense

mechanism, firstly Glutathione (GSH), an antioxidant

which mops up ROS and renders them harmless against the

cell. However NAPQI (N-acetyl-p-benzoquinone) can bind

to GSH reducing its volume in the hepatocyte. (Cederbaum

et al 2010)



If this and other backup mechanisms are overpowered by

the sheer scale and type of ROS, for instance OH radicals,

superoxide anion radicals, acetaminophen, N-

nitrosodimethyamine, carbon tetrachloride and benzene,

then toxicity can affect the cell. We call this hepatitis or

hepatotoxicity. (Cederbaum. et al 2010, Senousy et al 2010)

Hepatotoxicity Hepatotoxicity symptoms include tiredness, weakness,

vomiting, lethargy, ascites and abdominal pain and can

eventually lead to death. (Senousy et al 2010)

Diagnosing of hepatotoxicity revolves around studying liver

function test markers, for instance ALT or AST. Cases of

hepatotoxicity mainly occur in adults, but it would be wise

to understand what happens in children.

If hepatotoxicity is suspected or diagnosed, immediate

cessation of treatment is needed. Once liver function

markers have returned to normal, gradual reintroduction of

isoniazid therapy is possible at 50mg for the first two days,

followed by 100mg the next two, then 150mg for days 5

and 6, 200mg for days 7 and 8, 250mg for days 9 and 10

and 300mg for days 11 and 12. If the patient’s liver function

tests remain normal, then 300mg of isoniazid may continue

with the gradual reintroduction of the other main anti-TB

drugs. (Senousy et al 2010)

Figure 6- Kyphoscoliosis of the thoratic vertebrae of the

spine taken from an individual with tuberculosis. (Royal

College of Surgeons of England image library-Surgicat)

“Captain among these Men of Death”

-TB’s sobriquet (Daniel 2006)

The resurgence of antibiotic resistance, coupled with no

new antibiotics on the horizon in recent decades and the

spread of serious infectious diseases such as HIV/AIDS, has

fuelled the TB pandemic. This meant that in 2009 the

World Health Organisation (WHO) estimated 9.4 million

people had contracted the disease, with 1.3 million dying.

(WHO Global Tuberculosis Control Report 2010)

Figure 7- The link between the similarity of the TB

(above) and HIV/AIDS (below) pandemics of 2009. (WHO

Global Tuberculosis Control Report 2010)

TB is spread by droplet infection, sneezing and coughing.

Even talking or, especially, living in close quarters

exacerbates the condition.

Figure 8- TB bacilli (Toole 2004)

TB is an intracellular parasite (figure 8) affecting

primarily the lungs. It lives within macrophages which

become walled off due to inflammatory responses. This

recruits a mass of cells including neutrophils, monocytes

and giant cells. These grow in size and become walled off

from the rest of the lung tissue in a fibrous cuff. Eventually

forming a granuloma and tubercles within the lungs.

If the tubercles burst, they release TB bacilli into the blood

stream, disseminating the bacteria around the body as seen

in Addison’s hypofunction. (Marshall and Bangert 2008, Russell et al 2010)

X-rays of lungs can diagnose the presence of tuberculosis

tubercles in lungs, as seen in figure 9.



Figure 9- X-ray showing cavity (right base) of a patient

with TB. (Wellcome Trust Image Library Website 2011)

Symptoms of TB are indistinguishable from other flu-like

symptoms, including shortness of breath, blood tinged

sputum, fast pulse and respiration rate, lethargy and frailty.

Co-infection with HIV (see figure 7) is common. (Madigan

and Martinko 2006, Anderson 200, Russell et al 2010)

Diagnosis of TB is conducted by a concentrated sputum

sample, using Ziehl-Neelsen, Lowerstin-Jenson,

Middlebrook and dorset egg medias, Kinyoun techniques,

Polymerase Chain Reaction (PCR) carbol fuchsin and

auramine O laboratory methods. Some techniques may take

up to “14-21 days”. Plus sputum samples need sodium

hydroxide to reduce microbial growth. (Saltini 2006)

Due to this time delay, treating a clinically suspected patient

immediately with anti-TB drugs is often recommended until

the final results of laboratory investigations becomes

available.

“If societies can’t provide for people’s basic health, if they

can’t feed and educate people, then their populations and problems will grow and the world will be a less stable

place.”

-Bill Gates Annual letter 2011

Drugs against TB

Isoniazid is one of the major drugs which is used in

combination with ethambutol, pyrazinamide and rifampicin

to limit the potential of antibiotic resistance against TB. A

standard course of medication involves all four medicines

mentioned above for two months followed by four months

of just isoniazid and rifampicin.

Different drug regimes are tailored to different patient’s

needs depending on whether their TB is primary,

secondary/latent or reactivated, or whether they are co-

infected with another disease at the time of therapy, for

instance HIV which makes prescribing simultaneous TB

and HIV therapy far more difficult. (Senousy et al 2010)

Other medications which are effective against TB and its

various forms include streptomycin, macrolides, amikacin,

quinolones and ethionamide.

Isoniazid metabolism occurs in hepatocytes by mechanisms

that are incompletely understood. HepG2 cells are an

appropriate in vitro cell culture substitute to investigate

these mechanisms.

HepG2 cells provide particularly similar mitochondria,

organelles and expression of triglycerides and cholesterol.

And thus, HepG2 cells can provide a model for accessing

the possible deleterious and beneficial effects of isoniazid

medicine, through its metabolism in non-transfected and

transfected cells with CYP2E1. (Javitt 1990)

Warburg back on the front page

To couple this presumed effect of hepatotoxicity on HepG2

cells caused by isoniazid metabolism, we wished to further

induce cellular stress through the addition of culturing the

HepG2 cells in Dulbecco’s Modified Eagle Media (DMEM)

but, switching the standard sugar, glucose, to galactose,

therefore changing the growth ability of the cells.

This difference in growth has been suggested to be because

of the Warburg effect and the Crabtree effect. Cells grown

in galactose media are under more stress because they need

to use the oxidative phosphorylation pathway (of

respiration) as opposed to just simply using glycolysis to

provide adequate levels of ATP.

Oxidative phosphorylation provides more ATP yield, but it

also uses the mitochondria and its enzymes, therefore

producing more ROS. (Marroquin et al 2007)

It is Dr. White’s idea that forcing cells to use the oxidative

phosphorylation pathway using galactose will induce more

stress upon the cells than simply growing in glucose (which

uses glycolysis) media and hence will augment the stress

imposed upon HepG2 cells through CYP2E1 isoniazid

metabolism.

Assessing the Damage

The hepatotoxicity caused by glucose and galactose grown

HepG2 cells, both transfected with and without CYP2E1,

plus the addition of isoniazid was assessed using an MTT

assay.

Apparatus and Materials

Apparatus included HEPA biosafety cabinet/fume cabinet

appropriate for class II organisms, 37C CO2 incubator for

cell culture, -10C freezer for keeping CYP2E1 DNA

aliquots, autoclave for sterile pipette tips and PBS, phase

contrast microscope for viewing cells at x10, x40, and x100

magnification with fluorescence and camera, 1.5ml

eppendorf tubes, 15ml and 50ml centrifuge tubes,



FLUOstar Omega plate reader, 24 well plates, tin foil,

measuring scales, autoclave tape, 250 and 500ml

autoclavable screw top flasks, cold room ,waste measuring

flasks, P10, 20, 200 and 1000 eppendorf pipettes,

haemocytometer and coverslips, T25 and T75 adherent cell

culture flasks, Eppendorf 5804 centrifuge, electroporation

machine, waste autoclave bags, permanent markers, 10ml

Pasteur pipettes, 5, 10 and 20ml pipettes and pipetter and

test tube rack.

Materials included Trypsin EDTA, HepG2 cells, glucose

and galactose DMEM media (Invitrogen-GIBCO-1196-

025), RPMI media (Thermo Scientific 1640x1-CAT No.

SH3062701), DMSO (CAT-472301), MTT reagent (),

Isoniazid (Product no.133775G), autoclaved water,

autoclaved PBS using PBS tablets (calcium and magnesium

free, Trypan blue dye and RO water.

Figure 10- Clockwise from top left phase contrast

microscope and computer screen displaying live cells,

HEPA class II biosafety cabinet, electroporation machine

and an FLUOstar Omega plate reader.

Methods HepG2 cells

Glucose and galactose grown passage 1 HepG2 cells were

provided by Dr. White, and grown in glucose and galactose

DMEM media (made up by RPMI 1640, non-essential

amino acids (NEAA), 2mM glutamine, 10% fetal calf

serum (FCS), 1mM sodium pyruvate) in T25 or T75 cell

culture flasks.

T25/75 cell culture flasks are used because HepG2 cells are

adherent and stick to the surface of flasks. All cell culturing

was performed in HEPA filter cabinet for class II pathogens

and cells were incubated in a 5% CO2 incubator because to

avoid contamination stopping cell growth and killing cells.

Passaging/splits, trypsinization and harvesting

Cells were assessed under a phase contrast microscope for

health, confluence (closeness together), growth and

contamination.

To perform cell harvest, (passages/splits), DMEM media

was discarded and cells were washed with 2ml of Trypsin

EDTA for T25’s or 4ml’s in the case of the T75 flasks. The

trypsin acted as a detergent breaking the bonds which

allowed the cells to adhere to the surface of the flasks. Cells

were incubated for 5 to 10 minutes to speed up the

trypsinization process.

Glucose grown cells always trypsinized faster than

galactose grown cells. Trypsinized cells appeared round and

swam across the flask as opposed to their normal confluent

string/ rice-like shape (see figure 11).

Cells were resuspended in 2ml of fresh glucose or galactose

DMEM flasks. 1ml of fresh media above was removed and

added to 4mls of fresh cell culture media, (glucose and

galactose respectively) and into fresh T25/75 cell culture (1

in 5 dilution).

Swivelling the flask for several minutes in media meant

fully obtaining as many cells as possible from the

trypsinization process. One could see the normal transparent

DMEM pomegranate colour turn a cloudy grapefruit juice

colour.

Cells were returned to 5% CO2 incubator for 3-4 days and

the process repeated for the next passage. After passaging,

cells were periodically assessed for health, confluence,

growth and contamination under phase contrast microscope.

Figure 11 - HepG2 cells during culturing

growth/incubation phase. Cells appear confluent, healthy

and “rice” like in shape. The effect of trypsin EDTA

detaches HepG2 cells from the flask, causing cells to

become round and to “swim” across the surface of the

flask.



With CYP2E1

The addition of CYP2E1 consequently required further

additional stages to the above protocol. On day 1 normal

cell culturing continued in T25 flasks and cells were

incubated for two days as described above.

Into the growth phase

For transfection, cells were required at higher cell counts.

Therefore a stepup split into a T75 flask on day 3, induced

the cells into a growing phase prior to the transfection.

This meant normal harvesting of the cells, but resuspending

the cells after the trypsinization process in 4mls of glucose

or galactose DMEM media, then taking 2mls and adding

this to a new T75 flask containing 10mls of fresh media.

On day 4 cells were trypsinized in 4mls of trypsin EDTA

and resuspended in 10mls of glucose or galactose fresh

DMEM media. This was decanted into two respective 15ml

centrifuge tubes. The 15ml centrifuge tubes were spun at

800 RPM for 5 minutes producing a pellet of cells.

The supernatant was discarded and the pellets were flicked

to dislodge the cells in their clumps. Fresh 10mls of RPMI

media was added to each tube and again the 15ml centrifuge

tubes were spun for 5 minutes at 800 RPM. Pellets were

produced and the supernatant was discarded, cells were

flicked to dislodge clumps.

RPMI was used because standard DMEM contains FCS

which can interfere with electroporation. RPMI does not

contain FCS.

Cell viability Counts

Cells were resuspended in 10mls of fresh RPMI media;

from here a 50µl aliquot was taken and added to 50µl of

Trypan blue in a 1.5ml eppendorf tube (i.e. 1 in 2 dilution).

This was pipetted up and down to fully mix reagents. In the

region of 30-50µl of solution was added to a

haemocytometer to assess cell viability and number.

Respective counts were made and this numerical figure was

used to calculate cells at 5x104 cells per ml for a 24 well

plate assay.

Electroporation For electroporation 400µl of cells was added to a 0.2mm

transfection cuvette and 10µl of CYP2E1 DNA in H20 or

tris buffer aliquots, under ice conditions. Cuvettes were

mixed and electrodes had condensation removed in order to

aid electroporation. A charge of 180 volts and a capacitance

of 500 was passed through the cuvette in the cuvette capsule

(resistance was non applicable). This facilitated the transfer

of DNA into the HepG2 cells and hoped-for integration into

the DNA and expression.

Plating out

From here, the 410µl of electroporated cells was added to

16mls of glucose and galactose respective DMEM media in

50ml centrifuge tubes. This volume was plated out at 500µl

per well or 5x104 cells per ml in 24 well plates. Plates were

incubated overnight in a 5% C02 incubator.

New passages

Residual cells from the original 10mls were used for new

cell culture passages and resuspended at 1 in 5 dilutions

(1ml of cells +4ml of respective media) for T25 labelled

passages.

Isoniazid addition

On day 5, a 200 mM stock preparation of isoniazid was

made, using a syringe and a 0.2µl filter sterilizer.

Pre-made isoniazid concentrations were made from the 200

mM stock solution using table 1 below and all molar

concentrations were used at 5µl per well in 24 well plates.

Since only 5µl per well was pipetted, this changed the

molar concentration of the solutions below from 200, 100,

50, 20, 10 and 0mM to 2, 1, 0.5, 0.2, 0.1 and 0 mM

respectively.

Table 1-The various concentrations of isoniazid made for

addition to 24 well plates.

Figure 12- 5ul per well of isoniazid was added from left to

right at increasing molar concentrations.



MTT Assay

Plates were incubated until day 7, when 250µl of media was

discarded per well from the plates. 50µl per well of MTT

reagent was added and together plates were incubated for

3.5 hours.

After incubation the wells turned dark purple/black with

specks of precipitate. All media, cells and reagent was

removed from wells and each well was washed with 250µl

of sterile PBS.

Once the PBS was decanted from the wells, 500µl/well of

DMSO was added. This turned the wells red/purple for low

concentrations of isoniazid, whilst light purple/transparent

colourless solutions were obtained for higher concentrations

of isoniazid (figure 13 below).

Figure 13- Process of an MTT assay (left to right).

Addition of MTT, its effect after 3.5 hours, washing step

with PBS and purple colour produced due to DMSO

addition. Note in the latter three photos the 2mM isoniazid

rows are far lighter and more transparent than the rest of

the wells. This is because increased concentration

coffered by CYP2E1 metabolism caused the production of

ROS and thus toxicity to cells.

FLUOstar Omega plate reading

Hepatotoxicity due to isoniazid addition was analysed

through an FLUOstar Omega plate reader at 590 and

620nm. Results were analysed and conclusions made.

Without CYP2E1

All the sections above are followed, apart from a normal

cell count without Trypan blue and the exclusion of the

electroporation steps. Plating out, isoniazid addition and

MTT assays were the same as before.

Results

“If it disagrees with experiment, it’s wrong. In that simple

statement is the key to science”

-Richard Feynmann

The aim of this project was to access the toxicity caused by

isoniazid on non-transfected HepG2 cells compared with

HepG2 cells transfected CYP2E1, grown under different

conditions.

The first experiment pictures two sets of glucose vs

galactose non-transfected cells (figure 14).

Assessing the Effect of Isoniazid on Glucose and Galactose Grown HepG2

Cells Through MTT Assays

0.000

0.100

0.200

0.300

0.400

0.500

0.600

0.700

0.800

0.900

1.000

0 mM 0.1 mM 0.2 mM 0.5 mM 1 mM 2 mM

Isoniazid Concentration (mM)

Ab

so

rba

nc

e (

55

0-6

20

nm

)

Glucose-2 No CYP2E1

Glucose-1 No CYP2E1

Galactose-2 No CYP2E1


Figure 14-Effect of isoniazid on the growth of HepG2

cells

Cells were cultured in glucose and galactose DMEM and

treated with isoniazid, after 3 days incubation, results were

obtained by MTT assays as described in method section.

Data obtained are the mean and standard deviation of four

replicate measurements.

The lower the absorbance the greater the cytoxicity.

Growth in galactose was far lower than in glucose..

However growth in glucose was inhibited at higher

concentrations of isoniazid. Results from repeats of each

growth media mirrored each other. T-Tests and

normalisation analysis were performed, seen in Table 2

below. Only the glucose-1 1mM figure was significant, at

0.046. Normalisation values fluctuated generally.

0 mM 0.1 mM 0.2 mM 0.5 mM 1 mM 2 mM

T-TESTS 0.438 0.179 0.479 0.073 0.132

Normalisation 100% 95% 79% 98% 61% 72%

T-TESTS 0.298 0.388 0.288 0.046 0.222

Normalisation 100% 89% 97% 118% 77% 92%

T-TESTS 0.175 0.486 0.199 0.395 0.053

Normalisation 100% 122% 99% 185% 111% 147%

T-TESTS 0.188 0.264 0.376 0.141 0.135

Normalisation 100% 120% 111% 108% 80% 92%

Glucose-1 No CYP2E1

Glucose-2 No CYP2E1


Galactose-2 No CYP2E2 Table 2- Normalisation and T-Test values of figure 16

The next phase of the experiment concentrated on

transfecting the cells with CYP2E1 DNA, to see if

metabolism of isoniazid would increase cell stress.

Data from the five transfections performed are below in

figure 15a-e. Data was not pooled together because there

was variation between results. For example a) compared to

b) and c). These differences may have been due to the

efficiency of transfection, increasing age of cells (higher

passage number) and cell number.

Consequences of Isoniazid on Glucose and Galactose

grown HepG2 Cells which have been Transfected with

Cytochrome

P450 2E1

0.000

0.050

0.100

0.150

0.200

0.250

0.300

0.350

0.400

0 mM 0.1 mM 0.2 mM 0.5 mM 1 mM 2 mM

Isoniazid Concentration mM

Ab

sorb

ance

(59

0-62

0nm

)

Galactose CYP2E1 MTT Assay Glucose CYP2E1 MTT Assay Figure 15a-Effect of isoniazid on the growth of

transfected HepG2 cells. CYP2E1 cDNA in tris buffer

*

*



In figure 15a there is no trend with increasing isoniazid

concentration and CYP2E1. However there is a

considerable difference in absorbance values between

glucose and galactose grown cells. Normalisation values

were for galactose 1mM, 87% and 2mM, 93%. Whilst

glucose normalisation values were for 1mM, 102% and for

2mM, 111% .T-test values were all insignificant, bar the

0.2mM glucose bar at a p value of 0.039.

Effect of Isoniazid on CYP2E1 Transfected HepG2 Cells Analysed by

an MTT Assay

0.000

0.020

0.040

0.060

0.080

0.100

0.120

0.140

0.0 mM 0.1 mM 0.2 mM 0.5 mM 1.0 mM 2.0 mM


Ab

sorb

ance

(59

0-62

0nm

)

Glucose and CYP2E1 MTT-1 Glucose and CYP2E1 MTT-2 Figure 15b-Effect of isoniazid on the growth of

transfected HepG2 cells (Repeat 1). CYP2E1 cDNA in H20

buffer.

As opposed to the figure 15a, figure 15b CYP2E1 DNA

was in H20, this had the effect of causing a trend. As

isoniazid concentrations increased the absorbance of both

glucose replicas lowered. Only glucose results were

included because the galactose cells failed to work. The

0mM T-Test values for glucose-1 were significant at a P

value of 0.002. While the glucose-2 results were significant

for 0.5mM, 1mM and 2mM concentrations. Meanwhile the

normalisation values for glucose-1 were 100%, 86% and

36% for 0, 1 and 2mM concentrations. The normalisation

values for glucose-2 were 100%, 57% and 9% for 0,1 and

2mM concentrations.

Glucose and Galactose HepG2 Cells Transfected with CYP2E1

Analyzed by an MTT Assay

0.000

0.100

0.200

0.300

0.400

0.500

0.600

0.700

0 mM 0.1 mM 0.2 mM 0.5 mM 1 mM 2 mM


Ab

so

rba

nc

e (

63

0-5

50

nm

)

Glucose Transfected CYP2E2 Galactose Transfected CYP2E2

Figure 15c-Effect of isoniazid on the growth of

transfected HepG2 cells (Repeat 2). CYP2E1 cDNA in H20

buffer. CYP2E1 cDNA in H20 buffer.

In figure 15c, a significant trend was obtained for both

glucose and galactose grown cells. As isoniazid

concentrations increased, absorbance’s decreased

significantly. Moreover galactose values were always lower

then glucose values. All T-Test values were significant.

Normalisation values for glucose cells went from 100%,

43% and 2% for 0, 1 and 2mM concentrations respectfully.

Normalisation values for galactose cells went from 100%,

32% and 1%, for 0, 1 and 2mM concentrations respectfully.

Glucose and Galactose HepG2 Cells Transfected with CYP2E1 Analyzed by

an MTT Assay

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

0 mM 0.1 mM 0.2 mM 0.5 mM 1.0 mM 2.0 mM


Ab

so

rba

nc

e (

59

0-6

20

nm

)

Galactose Transfected CYP2E1 Glucose Transfected CYP2E1 Figure 15d-Figure 17b-Effect of isoniazid on the growth

of transfected HepG2 cells (Repeat 3). CYP2E1 cDNA in

H20 buffer. CYP2E1 cDNA in H20 buffer.

In figure 15d, there was not a significant decrease in

absorbances as isoniazid concentrations increased.

Galactose absorbances were always lower than glucose

absorbances. However results were not on the scale of

previous experiments above.

To couple this, decreasing glucose absorbances were

obtained with increasing isoniazid concentrations. The T-

Test values were significant for 0.5, 1 and 2mM at 0.012,

0.002 and 0.006 p values respectfully. Normalisation values

were 100%, 69% and 54% for 0, 1 and 2mM concentrations

respectfully.

Assessing the Cytoxicity Caused by the Metabolism of Isoniazid by

CYP2E1 on HepG2 Glucose and Galactose Grown Cells Analyzed

Through an MTT Assay

0.000

0.050

0.100

0.150

0.200

0.250

0.300

0.350

0.400

0.450

0mM 1mM 2mM


Ab

so

rba

nc

e (

59

0-6

20

nm

)

Glucose Galactose Figure 15e-Figure 17b-Effect of isoniazid on the growth

of transfected HepG2 cells (Repeat 4). CYP2E1 cDNA in

H20 buffer. CYP2E1 cDNA in H20 buffer.

There were not enough cells for a full 24 well plate, hence

figure 15e results were cut short at 0, 1 and 2mM isoniazid

concentrations. Galactose values decreased with increasing

isoniazid concentration. Glucose values fluctuated.

Galactose trend was significant with p values less than

0.005. While only glucose 1mM p value was less than

0.005. Normalisation values for glucose fluctuated from

100% to 59% and 97%. While galactose normalisation

values decreased from 100% to 81% and 52%.

P>0.005

* * *

*

* *

*

* *

* *



Figure 16-The differences obtained from galactose and

glucose cell culturing with isoniazid and CYP2E1 after 2

days.

Figure 16 shows the Crabtree Effect on galactose cells. The

media stayed the same colour due to no lactate production

and thus a neutral pH 7. This suggests the cells being forced

down the oxidative phosphorylation pathway to produce

ATP.

Meanwhile, the glucose media turned orange because of

lactate production and therefore a lowering in pH (acidic).

This suggests the cells are showing the Warburg effect, in

essence still using glycolysis for ATP production.

Figure 17- Effect of isoniazid on HepG2 cultured

galactose and glucose cells.

Figure 17 shows the effect isoniazid can have when

metabolised by transfected CYP2E1 HepG2 cells. This

suggests ROS have been produced in the 2mM wells but

not the 0mM wells. This observation was not seen in non

transfected cells.

Analysis

Galactose induces more stress upon HepG2 cells than

glucose, possibly through the Otto Warburg/Crabtree

principle. The combination of isoniazid and CYP2E1 causes

a gradual decrease in absorbances as isoniazid

concentrations increase. This may be due to the formation

of ROS from isoniazid’s metabolism by CYP2E1.

This has been supported by figures 14, 15 b) and c).

However experimental reproducibility has been

unsatisfactory. This casts doubt on the validity of the theory

that the formation of ROS by CYP2E1 metabolism of

isoniazid is the root cause of hepatotoxicity.

However there may be various explanations the lack of

reproducablity. For instance, during transfections only 1%

of cells actually takes up and expresses CYP2E1. Therefore

not enough cells are able to metabolise isoniazid and cause

the detrimental effects of hepatotoxicity. (To prove this last

statement effectively a Green Fluorescent Protein (GFP)

marker on the CYP2E1 cDNA would be needed to show

which cells had taken up CYP2E1 and which had not.)

On the other hand, the 1% efficiency of transfection may be

sufficient to cause hepatotoxicity.

And thus, this 1% of cells could be releasing large numbers

of ROS to other cells, tissues and organs, causing a chain

reaction of deleterious consequences. And as a

consequence, causing the detrimental results seen in

heptotoxicity.

This principle is known as the bystander effect. It would

have been investigated in the latter weeks of the project had

it not been for severe contamination as seen in figure 18

below. The source and cause of this are a mystery,

especially in the light of increased vigilance whilst culturing

and extra precautions taken to prevent contamination by

bacteria, fungi and yeast cells.

Figure 18- Contamination of a HepG2 cell culture.



Conclusions

“In the fields of observation, chance favours only those who

are prepared”

-Louis Pasteur (Debre 1998)

Efficiency of transfection

Efficiency of transfection may have been a factor in these

projects results. Other options of getting CYP2E1 DNA

into the cell could have included using transfection reagents

such as DOTAP, Lipofectamine, or Fugene. Even more

extravagant would be using an adenovirus particle to inject

CYP2E1 DNA into the HepG2 cells. However all of these

processes are detrimental to the cell itself either through

electricity (electroporation) or punching a hole in the cell

membrane by chemical means (DOTAP). One could even

argue it could be the transfection itself inadvertently

causing stress and therefore hepatotoxicity to the cells seen

in the results.

Other drug regimens

Isoniazid is usually prescribed with two other therapies and

is seldom a monotherapy. Thus, establishing the

hepatotoxic effects caused by these other anti-TB drugs,

plus their metabolism by different cytochrome proteins is

vital to building up a comprehensive picture of clinical and

laboratory hepatotoxicity.

To contemplate the future dimension of a complete cell

model, with the full assortment of different cytochrome

families and their varying combinations of TB drugs for the

potential of establishing their theoretical involvement in

hepatotoxicity, would be a fantastic outlook to pursue.

Galactose grown cells and trypsinization

What accounts for the time difference it took for galactose

grown cells to trypsinze faster then glucose grown cells

from the T25/75 cell culture flasks?

The only differences between the cell cultures were the

sugars.

Glucose cells use the glycolysis pathway, the Warburg

effect. Galactose cells have been forced down oxidative

phosphorylation pathway, the Crabtree effect.

Cancer cells like HepG2 cells are immortal. Cancer cells

use glycolysis, they are hypoxic. As if they were growing in

glucose.

Could there be a link between the ability of the HepG2 cells

to detach from the cell flasks and the ability of cancer cells

to migrate during metastasis. Could cellular receptors and

membranes (for example E/N-cadherins) play a role in this

possible relationship?

If so, slowing or stopping the ability of HepG2 cells to

trypsinze would have the potential of stopping or reducing

the ability of cancer cells to migrate.

The Bigger Picture- Success of an antioxidant

This project’s original goal was to establish CYP2E1 as the

principal suspect causing the source of ROS and

consequently hepatotoxicity.

If a particular ROS was isolated from within HepG2 cells

by High Pressure Liquid Chromatography (HPLC) or mass

spectrometry, then one could tailor a specific antioxidant,

such as Gentipicroside (White et al), to mop up the

production of that ROS and consign the issues of

hepatotoxicity caused by isoniazid to oblivion.

-How much would an antioxidant cost to deliver and to be

prescribed to the majority of TB sufferers who are neither

rich nor affluent?

-What effects would the antioxidant and the anti-TB drugs

together have on the body?

-Would it work in children? Hepatotoxicity is seldom seen

in children, but little data is accumulated on the overall

effects of TB drugs on child health. This must change.

With lacklustre development of antibiotics in recent

decades and antibiotic resistance growing with MRSA, C. Difficile and artemisinin resistant malaria strains, XDR-TB

and MDR-TB, new antibiotic scaffold developments and

discoveries are of greater importance then ever.

If we do not continually develop, keep abreast and educate

people about the threat of infectious diseases like TB,

humanity will face a continual and increasing threat, posing

a risk to not only the poor, but all who inhabit the globe.

Let us hope, in the light of the statement above, that the

WHO constitution quote I began this paper with, stands the

test of time.

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http://youtube.com/watch?v=b240PGCMwV0

http://people.wku.edu/charles.smith/wallace/S043.htm

http://surgicat.rcseng.ac.uk/(hkyi3345xw05pbjczp3mtcmp)/

detail.aspx#

http://surgicat.rcseng.ac.uk/(hkyi3345xw05pbjczp3mtcmp)/

detail.aspx?parentpriref=#

http://images.wellcome.ac.uk/

http://www.who.int/mediacentre/factsheets/fs104/en/

Acknowledgments

I am indebted to Dr. White, Dr. Chatterton, Dr. Searle, Mr

Armour, Dr Mclean, Gaf, Shyni, Mohammed and the

laboratory dispensary team. And finally, to persons know

and unknown, my thanks.

http://people.wku.edu/charles.smith/wallace/S043.htm

http://www.who.int/mediacentre/factsheets/fs104/en/



MODELLING ISONIAZID HEPATOTOXICITY

ON HEPG2 CELLS BY CYP2E1 METABOLISM

BY ERIC GARSON1 (STUDENT)

AND

DR. KEN WHITE2 (SUPERVISOR)*

Documents

Modelling Isoniazid Hepatotoxicity on HepG2 Cells By CYP2E1 Metabolism - London Metropolitan University - Eric Garson 2011