The metabolism and toxicology of rutin on liver type cells

The metabolism and toxicology of rutin in liver-type cells Andrew Hoffman

Statement of originality:I declare that, with the exception of any statements to the contrary, the contents of this report/dissertation are my own work, that the data presented herein has been obtained by experimentation and that no part of the report has been copied from previous reports/dissertations, books, manuscripts, research papers or the internet.

Signed.............................................. Print name................................................................Date.........................................................

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Contents1. Abstract.............................................................................................................................................3

Abbreviations....................................................................................................................................3

2. Introduction.......................................................................................................................................4

3. Methods..........................................................................................................................................11

3.1 – Materials.................................................................................................................................11

3.2 – Making up the Media for the growing of the cells..................................................................11

3.3 – Splitting the cells.....................................................................................................................12

3.4 - Preparing the compounds.......................................................................................................13

3.5 – Preparing the dilutions............................................................................................................14

3.6 – Dosing the cells.......................................................................................................................17

3.7 – MTT Protocol - Running the MTT Assay..................................................................................18

4 .Results.............................................................................................................................................19

4.1 - Short Term assay (HepG2 4000 cells per well [48 hours]).......................................................19

4.2 - Long Term Assay (HepG2 2000 cells per well [1 week (168 hours)]).......................................19

4.3 - Short Term Assay Repeat (HepG2 4000 cells per well [48 hours])...........................................20

4.4 - Long Term Assay Repeat (HepG2 2000 cells per well [1 week (168 hours)])...........................20

5. Discussion........................................................................................................................................22

Results obtained..............................................................................................................................22

Problems which may have occurred and changes which could be made........................................24

Alternative approaches to the methodology...................................................................................25

MTT Assay....................................................................................................................................25

S9 Fraction...................................................................................................................................27

6. Acknowledgments...........................................................................................................................31

7. References.......................................................................................................................................32

8. Appendix..........................................................................................................................................36

Ethics Form..................................................................................................................................66

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1. Abstract

Rutin is a type of flavonoid which are a group of plant metabolites. It is found predominantly

in fruit, vegetables and in around 70 different types of plant. For medicinal use, it is found

mainly in buckwheat. A study was conducted to look at the possible anti-cancerous effects

of rutin on hepatocellular carcinoma cells (the HepG2 cell line). Out of two repeated tests,

the results were fairly inconclusive as 50% of the results found no particular correlation

between the percentage of cell viability (how many cells survived) and the concentration of

rutin. The results also showed a high EC50 in pharmacological terms. The following also

contains critical analysis of other methods which could have been used to study these

effects.

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Abbreviations17-β-E2 - Endogenousestrogen17-β-estradiolADME – Absorption, Distribution, Metabolism and ExcretionCYP – Cytochrome P450 enzymeCPZ - ChlorpromazineFBS – Foetal bovine serumMTT - 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideNADPH - Nicotinamide adenine dinucleotide phosphatePES – Phenazine ethyl sulphatePMS - Phenazine methyl sulphateRedox – Reduction and OxidationRNS – Reactive Nitrogen SpeciesRPMI 1640 – Roswell Park Memorial Institute MediumS9 Fraction – post-mitochondrial supernatant fraction


2. Introduction

Rutin is a flavonoid, which are a type of plant metabolite. When they were first discovered,

flavonoids were referred to as Vitamin P (due to their effect on vascular capillary

permeability). Rutin itself is found in many different fruits and vegetables and in over 70

different types of plant (Chua, 2013) but is most notably found for medicinal use in

Buckwheat (Fagopyrum esculentum Moench) (Kreft, et al., 1999). There is even a slight

chance that rutin can be found in Tobacco leaves (Fathiazad, et al., 2006). Rutin is the

joining of quercetin and rutinoside by a glycosidic bond, as shown in figure 1.

Figure 1 – Skeletal structure of a rutin molecule (quercetin-3-O-rutinoside).

It was believed that rutin strengthens the blood vessels and that because of this feature, it

could be used in treatment of varicose veins, internal bleeding, haemorrhoids and

haemorrhagic strokes. Since then, rutin has been acknowledged for its many medicinal

properties that it could be used for, detailed below.

Rutin has huge antioxidant capabilities as it is a wonderful free radical inhibitor (Korkmaz &

Kolankaya, 2010). Free radicals are created in vivo during respiration in mitochondria and

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are also found to be released by peroxisomes. Free radicals are known to be a catalyst for

certain redox reactions in the body and are generated either due to a persons’ diet being

unhealthy or as a result of an external stimuli, such as an infection. Excessive production of

these free radicals can lead to destruction of cells and, ultimately, the damage of tissues and

organs. High concentrations of naturally found rutin (buckwheat hull) have been found to

diminish the amount of NO2- and NO3

- (RNS) (Khan, et al., 2009). When investigated at as

little as 0.05 mg/mL, rutin showed an inhibition total of around 90%, almost as powerful as

Vitamin C which was at an inhibition rate of around 93% (Yang, et al., 2008).

Rutin also been proven to have anti-inflammation properties (Umar, et al., 2012).

Inflammation is an autonomic response to an injury that an organism might sustain. COX-2

is one of the main catalysts for the production of prostaglandins which induce the

inflammatory response. Although the inflammatory response is autonomic, it is not always

best for the body as inflammation can bring multiple problems, for example rheumatoid

arthritis and kidney failure. Rutin was proposed as a molecule to block the COX-2 pathway

(Guardia, et al., 2001) and at a concentration of 80µm there was significant inhibition on

macrophages as an in vitro model. Acting on a mouse for an in vivo model, rutin showed the

same inhibition properties when given at a 6mg dose (Shen, et al., 2002).

With its many properties, rutin is also considered to be a potent anti-adipogenic (Choi, et al.,

2006). Fatty liver is a treatable and manageable problem if and when a controlled diet and

exercise regime is implemented. If nothing is done to treat fatty liver, it can cause a whole

host of problems for the organism. In a particular study of the effect of flavonoids on fatty

liver (Choi, et al., 2006), rutin was introduced into the diet of mice that were given a high fat

diet. The mice given a high fat diet including rutin (25/ 50mg per kg of body weight on a

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daily basis) were found to have a reduced fat build up than those which had a high fat only

diet.

Previous studies on plant metabolites have shown that the compounds have similar

structures to hormones which regulate the endocrine system in a mammalian system.

Namely they have been recognised as phytoestrogens, a molecule with a similar structure to

estrogen (Guo, et al., 2012).Rutin has a similar structure to that of 17-β-E2,

endogenousestrogen17-β-estradiol (shown in figure 2), so it is possible that rutin could be

used to bind to an estrogen receptor, which would normally be occupied by 17-β-E2, and

prospectively act as estrogen (Tham, et al., 1998).

Figure 2 – Skeletal structure of 17- β-E2,

endogenousestrogen17-β-estradiol. Compared to rutin, the structures are similar due to the

3 planar benzene rings.

Flavonoids have been thought to have anti-cancerous properties and have been proven, on

an in vitro model, to inhibit a multitude of cancerous cell lines and ultimately stop and/or

minimize tumour growth and even cause it to regress (Van der Logt, et al., 2003). It has

been suggested that the size regression could be due to the inhibition of certain DNA

topoisomerases, namely topoisomerase I and topoisomerase II, which are involved in the

marking of DNA damage and chromosomal damage (Cantero, et al., 2006).

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Rutin has many other medicinal properties, although not much about the precise

mechanisms are known. Other medicinal properties include; anti-diabetes (Hao, et al.,

2012), renal protection (Kamalakkannan & Stanley Mainzen Prince, 2006), anti-asthma

(Jung, et al., 2007), gastroprotective (La Casa, et al., 2000), neuroprotection

(Tonjaroenbuangam, et al., 2011), cardioprotection (Annapurna, et al., 2009), Osteoarthritis

(only when used in combination with trypsin and bromelain) (Klein & Kullich, 2000) and

mucositis, a painful side effect of cancer treatment, characterized by the swelling and ulcer

formation in the mouth or lining of the digestive tract. It can even be used in veterinary

practice to treat animals which are suffering from idiopathic chylothorax (Kopco, 2005). It is

suggested that rutin is metabolised by microflora in the gut (Kuhnau, 1976) into smaller

metabolites including; quercetin, isoquercetin, HVA and other phenols (Arjumand, et al.,

2011).

Drug metabolism is part of drug ADME screening. ADME stands for the absorption,

distribution, metabolism and excretion. The absorption relates to how the drug is taken up,

this is why it is often referred to as the ‘administration step’ of the ADME screening. Drugs

can be administered in a number of ways, namely orally or intravenously. The amount of

absorption is dependent on factors like size, solubility, ionization and blood flow to the site

of administration. The amount of drug that was administered is not particularly the amount

that will take action. Drugs tend to be delivered in an inactivated state and the metabolism

of the drugs is what converts it or break it down into its activated materials. Distribution of

drugs is quite simply how the drugs traverse throughout the body to get to the target site.

Drugs can travel freely or can be bound to proteins in the plasma, i.e. albumin. Metabolism

of drugs, often referred to as the biotransformation, is the conversion or activation of a

drug. The main site of metabolism in a mammalian system is in the liver. The site has two

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phases of reactions; phase I and phase II. The reactions of phase I include redox and

hydrolysis which are controlled here by the CYP (Cytochrome P450) enzymes. Phase II

reactions deal with the conjugation of molecules with substrates, such as glucoronic acid,

which increase water solubility and aid renal elimination. Although renal (kidney)

elimination is the main source of drug excretion, other routes are still common e.g. faecal

matter, expiration via the lungs or sweat glands in the skin (Oh, 2002).

There are many different reactions in the liver that are involved in hepatic metabolism.

There are chemical reactions, such as protein synthesis, detoxification and the production of

digestive chemicals. The liver is also important for the metabolism of carbohydrates and is

the source of many substances that are imperative for good health. Glycogenesis,

glycogenolysis and gluconeogenesis are carbohydrate metabolism pathways which take

place in the liver as well as the conversion of carbohydrates into triglycerides. These

triglycerides are converted into free fatty acids by the liver which are released into the

bloodstream and used in other cells/tissues in the body (Sadava, et al., 2011).

Toxicology is the study of adverse effects of chemical, physical or biological agents. Viability

assays are a great way of studying this. MTT is a valuable assay for use in cell lines. It was

the first type of homogenous cell viability developed for high throughput screening

(Mosmann, 1983). MTT substrate is made up in a physiologically balanced solution, is added

to the cell culture and usually incubated for a period of 2 to 4 hours. Quantitatively, the

production of formazan is directly proportional to the number of viable cells in question.

Viable cells actively metabolise MTT to formazan, a purple coloured product. Only viable

cells can convert MTT to formazan, if the cell were to die it would lose its ability to

metabolise MTT, therefore it serves as a perfect assay to determine cell viability as dead

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cells would produce no colour change. Formazan accumulates in the cells as an insoluble

precipitate and is deposited close to the cell surface and in the culture medium. Formazan

has to be solubilised before it can be read in a photospectrometer. There are a number of

solubilisation solvents that can be used: acidified isopropanol, DMSO, dimethylformamide,

SDS and even combinations of detergent and organic solvents ( (Mosmann, 1983), (Hansen,

et al., 1989) and (Denizot & Lang, 1986)). The amount of signal generated for absorbance

readings are dependent on certain factors like the concentration of MTT, incubation period,

number of viable cells and their respective metabolic capabilities. MTT has a cytotoxic

nature, the higher its concentration the more toxic it becomes to the cells. A lower

concentration would be optimal but due to its toxicity, MTT is considered to be an ‘endpoint

assay’.

The S9 assay is an invaluable stability assay for use in mimicking a liver environment for the

hepatic cells being used for this in vitro study. The S9 contains a widespread variety of both

phase I and phase II enzymes, comprised respectively of microsomal and cytosolic enzymes

(Plant, 2004). This allows for a fairly complete metabolic profile for ADME screening. Due to

the liver being the main organ for drug metabolism, the S9 fraction is useful for monitoring

the hepatic clearance. The S9 is easy to prepare, can be stored for lengthy periods of time

and can be adapted for use in a high throughput screening like the MTT assay.

In this particular study, the main focus will be on the effect of rutin on cancerous hepatic

cells. In particular, the HepG2 cell line. The HepG2 cell line is derived from a Caucasian

teenage liver cancer patient. They were taken from this particular patient due to the well

differentiated hepatocellular carcinoma. They produce albumin, macroglobulin, antitrypsin,

transferrin and plasminogen (Sigma Aldrich, 85011430).

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The main reason for this study is to hopefully prove that rutin does in fact have anti-

cancerous properties and could be used to treat human hepatocarcinoma cells by looking at

the effects on the HepG2 cell line. This will be determined by the use of the S9 fraction and

MTT assay to monitor the metabolism and toxicology of rutin.

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3. Methods

3.1 – Materials

MTT, DMSO, Ethanol, L-Glutamine, PBS Buffer, FBS, RPMI (Fisher)

Glutamine (Bioserra)

CPZ, Flavonoid (Rutin), Trypan Blue, Non-essential amino acids, Penicillin (Sigma Aldrich)

NADPH (Apollo Scientific)

S9 Fraction (Invitrogen)

Virkon, deionised water (University of Salford)

HepG2 (Gift from cyprotex)

3.2 – Making up the Media for the growing of the cells

The media was made up using the following materials; RPMI 1640, glutamine (5ml), non-

essential amino acids (5ml), penicillin (5ml) and FBS (50ml). The media was added along

with the HepG2 cells into flasks and incubated to allow growth.

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3.3 – Splitting the cells

During the time of growth, the cells needed to be ‘split’.

The media which was already present was removed and the cells in the flask were washed

with PBS (2 times, 5ml each time). Trypsin (1ml) was added to the flask to disturb the cells

and the flask was incubated for 3 – 5 minutes. After the time had passed, the flask was

removed from incubation to see if the cells were freely moving and not stuck to the side of

the flask. If the cells are not freely moving when taken out, the flask was given a gentle tap

to ensure they dislodged from the side. Media (5ml) was reintroduced to the flask and the

mix was taken out and placed into a centrifuge tube. The tube was centrifuged at 1250 rpm

for 5 minutes. Once finished, the media was removed from the tube to leave only the pellet

which had formed (dead cells are found suspended in the supernatant and this is why the

media was discarded). Fresh media (3ml) was added to suspend the pellet and 0.5ml of the

newly suspended cells were taken and placed into a new flask. Fresh media (10ml) was

added to the flask and the flask was incubated again.

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3.4 - Preparing the compounds

The compounds were made up to be 2ml of a 10mM solution.

Mass in grams of rutin [1] needed:

M (Molarity )= MassMolecularWeight ×Volume

∴M ×MW×Vol=Mass

0.01X 160 X 0.02 = 0.122g

Amount of DMSO to be added:

Volume (¿be added∈ml )= MassMolecularWeight ×Molarity

×1000

¿0.122

160×0.01×1000

= 20 ml

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3.5 – Preparing the dilutions

Once the stock solution was prepared, the dilutions were set up as follows;

Group 1 Group 3

Tube µM (Dilution) Tube µM (Dilution)

CPZ 1 100 FLV 1 100

CPZ 2 50 FLV 2 50

CPZ 3 25 FLV 3 25

CPZ 4 12.5 FLV 4 12.5

CPZ 5 6.25 FLV 5 6.25

CPZ 6 3.125 FLV 6 3.125

Table 1 – A table to show the dilutions of CPZ and FLV

*- CPZ is Chlorpromazine and FLV is Flavonoid (in this case rutin)

The dilutions were made using the following method;

400µl of stock solution was transferred into tube 1 of both groups. 200µl of tube 1 was

transferred into tube 2 and 200µl of DMSO was added, the two were then mixed well. The

process was repeated, 200µl of tube 2 was added to tube 3 and 200µl of DMSO was also

added, the two were mixed well. This process was repeated up to the 6th tube, in which

200µl of tube 5 was added but only 100µl of DMSO was added this time. The compounds

were kept in the freezer for a period of 24 hours.

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Figure 3 - A drawing to represent the process of making up the dilutions.

a) 400µl of stock solution added to tube 1, b) 200µl taken forward from the previous tube

into the next tube, c) 200µl of DMSO added to the new tube, d) 100µl of DMSO added to

the new tube (only for the last tube).

The 12 previously made tubes were taken from the freezer and a new set of tubes was

made up by the following method;

From the first set of tubes, 20µl of group 1 CPZ 1 was transferred to the each of the new

groups, group 1 CPZ 1 and group 2 CPZ 1 + S9. 20µl of group 1 CPZ 2 was transferred to the

each of the new groups, group 1 CPZ 2 and group 2 CPZ 2 + S9. 20µl of group 1 CPZ 3 was

transferred to the each of the new groups, group 1 CPZ 3 and group 2 CPZ 3 + S9. 20µl of

group 1 CPZ 4 was transferred to the each of the new groups, group 1 CPZ 4 and group 2

CPZ 4 + S9. 20µl of group 1 CPZ 5 was transferred to the each of the new groups, group 1

CPZ 5 and group 2 CPZ 5 + S9. 20µl of group 1 CPZ 6 was transferred to the each of the new

groups, group 1 CPZ 6 and group 2 CPZ 6 + S9.

From the first set of tubes, 20µl of group 3 FLV 1 was transferred to the each of the new

groups, group 3 FLV 1 and group 4 FLV 1 + S9. 20µl of group 3 FLV 2 was transferred to the

each of the new groups, group 3 FLV 2 and group 4 FLV 2 + S9. 20µl of group 3 FLV 3 was

transferred to the each of the new groups, group 3 FLV 3 and group 4 FLV 3 + S9. 20µl of

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group 3 FLV 4 was transferred to the each of the new groups, group 3 FLV 4 and group 4 FLV

4 + S9. 20µl of group 3 FLV 5 was transferred to the each of the new groups, group 3 FLV 5

and group 4 FLV 5 + S9. 20µl of group 3 FLV 6 was transferred to the each of the new

groups, group 3 FLV 6 and group 4 FLV 6 + S9.

Once the new groups were made up, 380µl of fresh media was added to each of tubes in

Group 1 and Group 3. Groups 2 and 4 required S9 media, this was prepared by mixing

NADPH (6mg), 150µl of S9 and 6 ml of media. 380µl of this S9 media was added to each of

the tubes in groups 2 and 4.

Figure 4 – A drawing to show the how the dosing tubes are made up. This process is

repeated for each tube

*S9 media was made up using NADPH (6mg), S9 (150µl) and media (6ml).

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Group 1 Group 2 Group 3 Group 4Tube µM

(Dilution)Tube µM

(Dilution)Tube µM

(Dilution)Tube µM

(Dilution)CPZ 1 100 CPZ 1 +

S9100 FLV 1 100 FLV 1 +

S9100

CPZ 2 50 CPZ 2 + S9

50 FLV 2 50 FLV 2 + S9

50

CPZ 3 25 CPZ 3 + S9

25 FLV 3 25 FLV 3 + S9

25

CPZ 4 12.5 CPZ 4 + S9

12.5 FLV 4 12.5 FLV 4 + S9

12.5

CPZ 5 6.25 CPZ 5 + S9

6.25 FLV 5 6.25 FLV 5 + S9

6.25

CPZ 6 3.125 CPZ 6 + S9

3.125 FLV 6 3.125 FLV 6 + S9

3.125

Table 2 - A table to show the dilutions in each tube used for dosing the cells.

3.6 – Dosing the cells

Once the dilutions were complete, the cell plates could be dosed. Media, Media + S9, DMSO

and DMSO + S9 were used as controls in the experiment. The DMSO tubes were made up as

follows;

2 DMSO tubes were prepared, one with S9 and one without. The tube without S9 was made

up by mixing fresh media (570µl) with DMSO (30µl). The tube with DMSO with S9 was

prepared by mixing DMSO (30µl) with S9 media (570µl).

1 2 3 4 5 6 7 8 9 10 11 12A Media Media Media Media Media Media Media + S9 Media + S9 Media + S9 Media + S9 Media + S9 Media + S9B DMSO DMSO DMSO DMSO DMSO DMSO DMSO + S9 DMSO + S9 DMSO + S9 DMSO + S9 DMSO + S9 DMSO + S9C CPZ 1 CPZ 2 CPZ 3 CPZ 4 CPZ 5 CPZ 6 CPZ 1 CPZ 2 CPZ 3 CPZ 4 CPZ 5 CPZ 6D CPZ 1 + S9 CPZ 2 + S9 CPZ 3 + S9 CPZ 4 + S9 CPZ 5 + S9 CPZ 6 + S9 CPZ 1 + S9 CPZ 2 + S9 CPZ 3 + S9 CPZ 4 + S9 CPZ 5 + S9 CPZ 6 +S9E FLV 1 FLV 2 FLV 3 FLV 4 FLV 5 FLV 6 FLV 1 FLV 2 FLV 3 FLV 4 FLV 5 FLV 6F FLV 1 FLV 2 FLV 3 FLV 4 FLV 5 FLV 6 FLV 1 FLV 2 FLV 3 FLV 4 FLV 5 FLV 6G FLV 1 + S9 FLV 2 + S9 FLV 3 + S9 FLV 4 + S9 FLV 5 + S9 FLV 6 + S9 FLV 1 + S9 FLV 2 + S9 FLV 3 + S9 FLV 4 + S9 FLV 5 + S9 FLV 6 + S9H FLV 1 + S9 FLV 2 + S9 FLV 3 + S9 FLV 4 + S9 FLV 5 + S9 FLV 6 + S9 FLV 1 + S9 FLV 2 + S9 FLV 3 + S9 FLV 4 + S9 FLV 5 + S9 FLV 6 + S9

Table 3 - A table to show the layout of compounds in the plate wells

The wells in the cell plates were dosed with the compounds (as outlined in table 3), each

well was dosed with 25µl of said compound. Both plates were dosed at the same time, one

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plate had 4,000 cells per well and the second had 2,000 cells per well, and both plates were

incubated at 37.5oC.

3.7 – MTT Protocol - Running the MTT Assay

After dosing the cells, the plates were incubated at 37.5oC for different periods of time. The

plates which were seeded at 4,000 cells per well were only left to incubate for a period of 48

hours and this was used as a short term culture. The cell plate seeded at 2,000 cells per well

were left to incubate for a full week (168 hours) and was used as a long term culture.

The MTT assay was run by the following protocol;

MTT solution (50µl, 3mg/ml) was added to each well and was incubated for a further 3 – 4

hours at 37.5oC. After this time had passed, the media in the wells was discarded by hitting

the plate against a hard surface, to ensure that media held in the wells by water tension was

also discarded. The plates were left to dry for a further 1 – 2 hours. DMSO (100µl) was

added to each well and the plates were incubated at 37.5oC for 10 minutes. The plates were

then taken to the plate reader, which shook the plates well for approximately 15 seconds to

solubilize the formazan (a type of dye found in MTT) before reading the plates at a given

wavelength of 570nm.

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4 .Results

Below is an account of results obtained in the experiment, it should be known that all of the

graphs mentioned in the text can be found in the appendix section of the report.

4.1 - Short Term assay (HepG2 4000 cells per well [48 hours])

Graph 1 and graph 2 both show a great sigmoidal curve with regards to the cell viability.

They show that with an increased concentration of CPZ, the amount of cells which survived

decreased. This showed especially well in the S9 fraction as the viability was not over 100%,

whereas without S9 the cell viability was rather high at a low dose. Graph 3 and graph 4

show the cell viability of rutin on its own with no S9 added. Although there is no curve

shown, it is obvious that the cells have grown as the cell viability counts are at around 200%,

with some being even higher. Graph 5 and 6, like graphs 3 and 4, show the effect of rutin on

the cells but with the presence of S9. Compared to the previous graphs (3 and 4), it is again

shown that the cells grow at higher concentrations. With the presence of S9, at lower

concentrations, some of the cells have died but again, at higher concentrations it appears

that the cells have grown.

4.2 - Long Term Assay (HepG2 2000 cells per well [1 week (168 hours)])

Graph 7 and graph 8 show the cell viability for CPZ and CPZ with S9. CPZ on its own shows

another great sigmoidal curve showing that increased concentration of CPZ makes the cell

viability decrease. This shows true for CPZ with S9 also, as cell viability is below 70%, but the

results do not follow a significant pattern. The results for the long term flavonoid exposure

(graph 9 and graph 10) showed a negative correlation between cell viability and

concentration of the flavonoid. At lower concentrations the cell viability was higher than

100%, meaning cells had grown and/or replicated, but ultimately, as concentration

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increased, cell viability decreased below 100%. In the presence of rutin and the S9 fraction,

cell viability increased drastically as shown in graph 11 and graph 12. Here, it is shown that

at low concentrations (around 0.5 on the log scale) that rutin neither induces cell death or

cell growth. However, as concentrations of rutin increase, the cell viability climbs as high as

1000% in a very even pattern seemingly reaching a plateau at this point.

4.3 - Short Term Assay Repeat (HepG2 4000 cells per well [48 hours])

The cell viability for CPZ (graph 13) and CPZ with S9 (graph 14) in the repeated assays found

that viability was reduced as the concentration of CPZ increased. It should be noted that the

test of CPZ with S9 (graph 14) showed some growth at low concentrations. The results for

the cell viability in the presence of the flavonoid on its own (graph 15 and graph 16) show

that as concentrations of the flavonoid increase, the cell viability decreases. At first, cell

viability did increase slightly but increased concentrations show an overall decrease. When

the flavonoid and S9 were added together (graph 17 and graph 18), cell viability showed an

increase above 100%, but as the concentration of flavonoid and S9 increased, the cell

viability is negatively affected.

4.4 - Long Term Assay Repeat (HepG2 2000 cells per well [1 week (168

hours)])

The cell viability in the long term repeat for CPZ (graph 19) and CPZ with S9 (graph 20) again

show a negative correlation between cell viability and concentration of CPZ. In graph 20,

there is no showing of a curve however it is obvious from the plot that there is a negative

correlation. When the flavonoid was tested again, with no S9 added, results showed that

cell viability increased with concentrations of flavonoid but with no particular correlation.

Graph 21, the first flavonoid test in the repeat, shows that at a low concentration cell

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viability falls. This is likely to be an anomaly. Graphs 23 and 24 show the results for cell

viability when the flavonoid with S9 was added. The results, again, show that cell viability

increases when the flavonoid and S9 are added, however this time there is no particular

correlation between the two. All this shows is that there has been some growth, but does

not necessarily prove that the flavonoid and S9 are the cause of this.

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5. Discussion

Results obtained

The only results which follow a particular pattern of consistency are the tests which involve

CPZ. The results for CPZ across all 4 tests show a decrease in cell viability. The pattern is

shown in both the short and long term assays, the only slight difference in results are that of

lower concentrations having lower cell viability after long term exposure (whereas in the

short term the lower concentrations have slightly higher cell viability). The only obvious

outliers are shown in graph 1 and graph 20, both at the lowest concentration of CPZ. This is

likely due to some contamination as no other results go above 130%, whereas these results

show viability of around 180% (graph 20) and 300% (graph 1). The results show how CPZ is a

good positive control for this test. More specifically CPZ was used to induce cell death as it is

known to cause hepatic toxicity (MacAllister, et al., 2013). It has been previously reported

that CPZ could indirectly cause cell death by blocking Ca2+ channels, causing a build-up of

Ca2+ in the cytoplasm. An increase in the cytoplasm can increase in the nucleus and in other

compartments of the cell. If the nucleus has a build-up of Ca2+ then Ca2+ endonuclease is

activated and causes DNA fragmentation. This can lead to cell death via apoptosis or cell

death by necrosis. An increase in other compartments can activate the Ca2+ protease and

cause cell death by necrosis in this way (Ray, et al., 1993).

On a whole, in the tests where the flavonoid was added on its own, there was no strong

correlation between the results. The only possible conclusion from the results is that there

is some cell growth in both the short and long term assays. Conducting Mann-Whitney

statistical tests, it was shown that comparing the flavonoid to the control (CPZ) proved that

the flavonoid does not follow the controls pattern as p<0.05 at a 95% confidence level for

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each result (results can be found in the appendix). This shows that there is definite cell

growth over cell death. Graph’s 3 and 4 show the highest percentage of cell viability but

these results are likely to have been contaminated as the results all have similar amounts of

growth. It could be possible that rutin is not toxic enough to kill the cell, due to its high EC50

(shown throughout the tables of pharmacological dose response in the appendix), or it

could even show some possibility of cell growth inducing properties.

As it is possible for hydrolysis to occur in the liver, it could be possible that the molecule

rutin is broken down into its constituent parts, quercetin and rutinoside. Even if this is the

case, the results obtained go against other studies of flavonoids on cancerous cells.

Quercetin has previously been tested on hepatocellular carcinoma cells and had been

found to excite pathways involved with causing cell apoptosis (Granado-Serrano, et al.,

2006). It has also been shown that quercetin exerts pro-apoptotic effects on breast cancer

cells (Bulzomi, et al., 2012). Although this is a very unlikely pathway for rutin to take in the

liver, it is the only comparison to be made to the results not fitting the normal findings.

When rutin and S9 were added together, the results were conflicted. In the first run through of the

tests, all results showed a positive correlation between the dosage amount and the percentage of

cell viability. Although at low concentrations in the short term assay (graph 5 and graph 6), low

concentrations showed some cell death. The long term tests (graph 11 and graph 12) showed a

massive increase in cell viability compared to that of the short term test. From these results, it could

be concluded that rutin in fact causes cell growth rather than inhibit or reduce it. However, in the

repeated tests, it is harder to determine what happened. In the short term assay (graph 17 and 18),

results showed that as concentration of flavonoid and S9 increased, the cell viability decreased. But

on a whole the cells grew as percentage of cell viability was above 100% for all results. Finally, the

23


long term assay on the repeated test showed no specific correlation between the results but again,

the cells had ultimately grown as cell viability was about that of 100% (graph 23 and graph 24).

Again, a Mann-Whitney statistical test was run and there are conflicting results in this test also. All

statistical results showed a P value which was greater than 0.05, except for the final test (Long term

assay repeat). This shows that the results follow the controls results and cell death occurs (except in

the final test in which p<0.05 meaning the results show cell growth). It could be argued that S9

reduces the anti-cancer properties of rutin, but the evidence from the Mann-Whitney tests would

disprove this theory.

In conclusion to the results obtained, I would say that more needs to be done to study the effect of

rutin on the HepG2 cell line. The results were inconclusive on a whole as the results were too varied.

Statistical tests proved that the results were too varied to come to an absolute conclusion. This could

be due to a number of reasons outlined below. If I were to conduct the test again, I would have liked

to have a short and long term test with both 2000 cells per well and 4000 cells per well to see if the

amount of cells present had an effect on the results.

Included below are some alternate approaches to study the metabolism and the toxicology of rutin

on liver-type cells.

Problems which may have occurred and changes which could be made

During the seeding phase, it is possible that the multi-channel pipettes used for dosing were

not calibrated accurately enough and/or the tips used were not connected correctly so this

could have altered the true amount of µm added. A problem could also arise in the fact that

the dosing channels can get easily clogged, so should be changed each time. It may be

possible to use a robot to seed the cell plates for accuracy, but, although they have great

accuracy there could be mechanical problems, so there would have to be someone present

to monitor the robot and keep it maintained.

24


From the previous statement about possible contamination of the plates, it could be noted

that contamination could have occurred at points not controlled when conducting the

study. Plates are kept in communal incubators which are used by a multitude of staff and

students in the building. It may have been that the cells had been disturbed because of

another person catching the plate or moving it to retrieve another plate or flask being kept

in the incubator. Contamination could have also come from incubators not being cleaned

out properly or kept at an appropriate standard. To combat this problem, cell plates were

eventually kept sealed with tape so that it was hard to disturb the plate or cause

contamination by removing the lids by accident.

There is a chance that due to the cell line, the cells could be unhealthy and the flavonoid

could be acting differently than it would on a healthy cell. The HepG2 cells are living, which

means that the cell cycle is still progressing. This would suggest that the cells could be in

different phases (G1, G2, S or M) and this could mean that the MTT could act differently on

a different phase. It is impossible to tell which phase the cells are in unless the cells were

killed and suspended.

Alternative approaches to the methodology

MTT Assay

All the results come from how the flavonoid has acted on living cells. After incubating the

cells with the MTT, the wells of the plates were emptied. Dead cells no longer ‘stick’ to the

wells of the plate and are instantly disposed of in this step. However, just because the cells

are living, it is no evidence to support how healthy the cells may be. There are no means of

determining how healthy the cells are in the MTT assay, the cells would have to be analysed

before they are taken to be read in the photospectrometer. This could have been done by

25


looking at the cells under a microscope or even by using a FACS assay (which is detailed later

on in this section). By analysing the cells under a microscope, it could have also shown

whether there was any true contamination in the wells. Hepatic carcinoma cells (HepG2 cell

line) should form clumps. If the cells are either not in clumps or moving rapidly (cells look

like they are vibrating) then there is definite contamination.

Figure 5 – An outline of the formation of the formazan product from the mitochondrial

reduction of MTT.

The MTT assay itself could give rise to problems due to formazan crystal production causing

cell membrane punctuation (Lu, et al., 2012).

XTT is great for use in a cell proliferation assay (an assay to measure the increase in the

number of cells as a result of cell growth and cell division). XTT could be used instead as it

has a higher sensitivity and higher dynamic range compared to the MTT test. Also, the XTT

reaction gives a formazan dye which is soluble, meaning there is no need for a solubilisation

step (like the addition of DMSO in MTT) and therefore meaning a reduced handling period

and less chance of human error (e.g. air bubbles being introduced into the solution). Finally,

readings can be taken immediately after XTT has been added as there is no incubation

period, compared to the 2-3 hour incubation period in the MTT assay.

26


MTS, XTT and WTS are more recently developed tetrazolium reagents which generate an

already soluble formazan product when in the presence of viable cells. However, the

formazan species they produce are negatively charged and this can impair the permeability

(Scuderio, et al., 1988). To combat this, the cells are treated with PMS or PES which get

reduced in the cytoplasm and leave the cell. The reduced PMS or PES can convert the

negatively charged formazan product into the soluble formazan (Berridge, et al., 2005).

It could be said that the best type of assay to use is the FACS (Fluorescence activated cell

sorting) assay, commonly known as flow cytometry. Cells, living or dead, which are

suspended in a liquid medium, can be sent through the flow cytometer in single file. Each

cell can be analysed rapidly, quantitatively and under many different parameters (Sharrow,

S O, 2002). Through this, the cells can be analysed as to what stage of the cell cycle they are

in, which would have proved very beneficial in the investigation as the health of the cells

could have been determined.

S9 Fraction

S9 is a fairly weak resemblance of a true in vivo environment (Brandon, et al., 2003). The S9

is quite complex and not easily applicable but is very ethically acceptable. For an in vitro

model, better examples could be transgenic cell lines, primary hepatocytes, slices of liver

tissue or perfused liver models. After this, the tests may move onto in vivo models. Below is

the outline of what each of these models incur.

Transgenic Cell Lines:

Transgenic cell lines are an alternative to promoting the expression of phase I and phase II

enzymes. The HepG2 cell lines that were used in the experiment, have previously been

found to be able to be transfected (Caro & Cederbaum, 2001). All CYP’s that are involved in

27


the drug biotransformation pathways have been made available for stable expression within

the HepG2 cell line. ( (Gasser, et al., 1999)and (Cavin, et al., 2001)). Transfected cell lines are

easy to culture, similarly to regular, non-transfected cell lines. They are readily available

from companies who specialise in making these highly efficient transfected cell lines, for

example gentest (www.gentest.com). It is possible to use a transgenic cell line for a single

enzyme or multiple enzyme reactions and could be especially useful for this type of

experiment as they can be used to make metabolites and study drug-drug interactions on a

metabolic level. A small problem encountered with transgenic cells is the fact that only a

few iso-enzymes can be expressed at one time. Iso-enzymes are a group of enzymes which

induce the same chemical reaction but each have different amino acid sequences. This is still

not a good enough reflection of a true in vivo environment.

Primary Hepatocytes:

Hepatocytes are cells found in parental tissue in the liver and make up around 70 – 85% of

the liver. There are two sub-types of hepatocytes that could be used; primary and cultured.

Primary hepatocytes are the second best in vitro model for an in vivo liver system before

moving on to the actual in vivo system and this is why it has been used extensively for drug

biotransformation research ( (Cross & Bayliss, 2000) and (Hengstler, et al., 2000)). They are

isolated for the liver by the use of a collagenase perfusion operation, first detailed in 1967

(Howard, et al., 1967) but has since been reduced to a simple two-stage step in recent years

(Lee, et al., 2013). The perfusion is mainly undergone when a person had a partial liver

resection. If this cannot be done at the time, the liver tissue the hepatocytes are taken from,

can be taken and stored at 4oC for around 2 days (48 hours) in a UW (University of

Wisconsin) solution and will show no signs of decreased viability in this period (Guyomard,

28

http://www.gentest.com/


et al., 1990).

Cultured hepatocytes (Chenery, et al., 1987)have been proved to be great models for in

vitro – in vivo correlations like primary hepatocytes, but it has been shown that over longer

periods of time, the hepatocytes can lose the liver-specific functions they originally had,

especially in the case of CYP expression (George, et al., 1997).

Advantageously, both primary and cultured hepatocytes can be cryopreserved. Through the

process of cryopreservation, it is possible to cut out the problem of liver function loss and

make the hepatocytes commercially available (Hengstler, et al., 2000). Disadvantages can

include the fact that hepatocytes only make up a maximum of 85% of liver cells, there are

other cells that may be involved in the metabolic pathway (i.e. cells which provide

cofactors). It is possible that damage can occur during the hepatocyte extraction phase.

Another problem can come from variation between organisms. Like a fingerprint, it is

possible for hepatocytes to be completely differentiated from person to person. However

this can be overpowered by using a cocktail of hepatocytes from many donors to create an

average.

Liver Slices:

The use of liver slices was first detailed in the 1920’s but only became available for

prolonged use in more recent years when more precise tissue slicers and better

suspensions, the UW solution, became available ( (Ekins, 1999) and (Olinga, et al., 1997)).

Compared to hepatocytes, the liver slices are a better model for drug metabolism as they

contain all cell types and perfect to see what the effect would be on a three dimensional

structure. The only problems that arise from the model is that it is very hard to handle due

to its delicate state and the limited viability period.

29


Perfused liver:

Perfusion is the act of forcing blood and/or fluid through the vasculature of a certain tissue

or organ. If the liver can be isolated and perfused it could be considered as the closest in

vitro model for a representation of an in vivo model. However, there are many problems

that occur with perfused livers. The functional viability period for the liver is only around 3

hours (Wu et al, 1999) and it is not possible to induce a prolonged time period. It is also very

hard to come by a liver to use in this way, at least for a human model. It is fairly unethical so

models tend to come from animals like mice or rats which have a similar liver morphology.

The procedure is still new and there have yet been ways to suspend the viability and

cryopreservation has yet to be optimized for this (Brandon, et al., 2003).

The use of any of the above in vitro models would be great to use to determine a better

understanding of rutins action on liver-type cells. However, the cost of these models comes

much higher than that of the HepG2 cell line. The progression of in vitro models to an in vivo

model would follow a similar pattern to that which was outlined, starting with looking at the

effect of the flavonoid on liver enzymes and moving on through testing the flavonoid on

microsomes and cytosol, the S9 fraction, transgenic cell lines, primary hepatocytes, liver

slices and then finally a perfused liver. After this, testing may move onto in vivo animal

models before moving on to human trials (Brandon, et al., 2003).

Rutins fantastic qualities could also be its downfall. Rutin targets many different systems

and reactions in the body. Due to its size, there is no way of knowing if it is targeting any

other system in the cells we have used. If, for example, the target rutin is acting on has been

saturated, it could possibly start targeting other molecules. Keeping with saturation of

30


targets, it could be possible that if there are no more target left for rutin to act on and the

cancerous cells continue to grow.

31


6. Acknowledgments

A special thanks to Patricia and all the PhD students who helped with protocols and cell

splitting. Also, a special thanks to Cyprotex for the gifted HepG2 cells that we used to test

the flavonoids on.

32


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8. AppendixHepG2 4000

Graph 1 – A graph to show the percentage of cell viability in regards to the concentration of CPZ (µm) on a logarithmic scale.

Table 4 – A table to show the data found when analysing the pharmacological dose response, most importantly the EC50, of the percentage cell viability in regards to the concentration of CPZ

37


Graph 2 – A graph to show the percentage of cell viability in regards to the concentration of CPZ + S9 (µm) on a logarithmic scale.

Table 5 – A table to show the data found when analysing the pharmacological dose response, most importantly the EC50, of the percentage cell viability in regards to the concentration of CPZ + S9

38


Graph 3 – A graph to show the percentage of cell viability in regards to the concentration of flavonoid (µm) on a logarithmic scale.

Table 6 – A table to show the data found when analysing the pharmacological dose response, most importantly the EC50, of the percentage cell viability in regards to the concentration of flavonoid

39


Graph 4 – A graph to show the percentage of cell viability in a repeated test in regards to the concentration of flavonoid (µm) on a logarithmic scale.

Table 7 – A table to show the data found when analysing the pharmacological dose response, most importantly the EC50, of the percentage cell viability in regards to the concentration of flavonoid (repeat)

40


Graph 5 – A graph to show the percentage of cell viability in regards to the concentration of Flavonoid + S9 (µm) on a logarithmic scale.

Table 8 – A table to show the data found when analysing the pharmacological dose response, most importantly the EC50, of the percentage cell viability in regards to the concentration of flavonoid and S9

41


Graph 6 – A graph to show the percentage of cell viability in a repeated test in regards to the concentration of Flavonoid + S9 (µm) on a logarithmic scale.

Table 9 – A table to show the data found when analysing the pharmacological dose response, most importantly the EC50, of the percentage cell viability in regards to the concentration of flavonoid and S9

42


HepG2 2000


Table 10 – A table to show the data found when analysing the pharmacological dose response, most importantly the EC50, of the percentage cell viability in regards to the concentration of CPZ

43



*It should be noted that for graph 8, there is no value for the lowest CPZ concentration (which would be at around 0.4 on the logarithmic scale).*

Table 11 – A table to show the data found when analysing the pharmacological dose response, most importantly the EC50, of the percentage cell viability in regards to the concentration of CPZ and S9

44


Graph 9 – A graph to show the percentage of cell viability in regards to the concentration of Flavonoid (µm) on a logarithmic scale.

Table 12 – A table to show the data found when analysing the pharmacological dose response, most importantly the EC50, of the percentage cell viability in regards to the concentration of flavonoid

45


Graph 10 - A graph to show the percentage of cell viability in a repeated test in regards to the concentration of Flavonoid (µm) on a logarithmic scale.

Table 13 - A table to show the data found when analysing the pharmacological dose response, most importantly the EC50, of the percentage cell viability in regards to the concentration of flavonoid (repeat)

46


Graph 11 – A graph to show the percentage of cell viability in regards to the concentration of Flavonoid (µm) on a logarithmic scale.

Table 14 - A table to show the data found when analysing the pharmacological dose response, most importantly the EC50, of the percentage cell viability in regards to the concentration of flavonoid and S9

47


Graph 12 – A graph to show the percentage of cell viability in a repeated test in regards to the concentration of Flavonoid (µm) on a logarithmic scale.

Table 15 - A table to show the data found when analysing the pharmacological dose response, most importantly the EC50, of the percentage cell viability in regards to the concentration of flavonoid and S9 (repeat)

48


HepG2 4000 (repeat)


Table 16- A table to show the data found when analysing the pharmacological dose response, most importantly the EC50, of the percentage cell viability in regards to the concentration of CPZ

49



Table 17 - A table to show the data found when analysing the pharmacological dose response, most importantly the EC50, of the percentage cell viability in regards to the concentration of CPZ

50


Graph 15 - A graph to show the percentage of cell viability in regards to the concentration of flavonoid (µm) on a logarithmic scale.

Table 18 - A table to show the data found when analysing the pharmacological dose response, most importantly the EC50, of the percentage cell viability in regards to the concentration of the flavonoid

51


Graph 16 - A graph to show the percentage of cell viability in a repeated test in regards to the concentration of flavonoid (µm) on a logarithmic scale.

Table 19 - A table to show the data found when analysing the pharmacological dose response, most importantly the EC50, of the percentage cell viability in regards to the concentration of the flavonoid (Repeat)

52


Graph 17 - A graph to show the percentage of cell viability in regards to the concentration of flavonoid + S9 (µm) on a logarithmic scale.

Table 20 - A table to show the data found when analysing the pharmacological dose response, most importantly the EC50, of the percentage cell viability in regards to the concentration of the flavonoid with S9

53


Graph 18 - A graph to show the percentage of cell viability in a repeated test in regards to the concentration of flavonoid + S9 (µm) on a logarithmic scale.

Table 21 - A table to show the data found when analysing the pharmacological dose response, most importantly the EC50, of the percentage cell viability in regards to the concentration of the flavonoid with S9 (repeat)

54


HepG2 2000

Graph 19 – A graph to show the percentage cell viability in regards to the concentration of CPZ (µm) on a logarithmic scale.

Table 22 - A table to show the data found when analysing the pharmacological dose response, most importantly the EC50, of the percentage cell viability in regards to the concentration of the CPZ

55


Graph 20 – A graph to show the percentage cell viability in regards to the concentration of CPZ + S9 (µm) on a logarithmic scale.

Table 23 - A table to show the data found when analysing the pharmacological dose response, most importantly the EC50, of the percentage cell viability in regards to the concentration of the CPZ with S9

56


Graph 21 – A graph to show the percentage of cell viability in regards to the concentration of flavonoid (µm) on a logarithmic scale.

Table 24 - A table to show the data found when analysing the pharmacological dose response, most importantly the EC50, of the percentage cell viability in regards to the concentration of the flavonoid

57


Graph 22 – A graph to show the percentage of cell viability in a repeated test in regards to the concentration of flavonoid (µm) on a logarithmic scale.

Table 25 - A table to show the data found when analysing the pharmacological dose response, most importantly the EC50, of the percentage cell viability in regards to the concentration of the flavonoid (repeat)

58


Graph 23 – A graph to show the percentage cell viability in regards to the concentration of flavonoid + S9 (µm) on a logarithmic scale.

Table 26 - A table to show the data found when analysing the pharmacological dose response, most importantly the EC50, of the percentage cell viability in regards to the concentration of the flavonoid with S9

59


Graph 24– A graph to show the percentage cell viability in a repeat test in regards to the concentration of flavonoid + S9 (µm) on a logarithmic scale.

Table 27 - A table to show the data found when analysing the pharmacological dose response, most importantly the EC50, of the percentage cell viability in regards to the concentration of the flavonoid with S9 (repeat)

60


raw data

1 2 3 4 5 6 7 8 9 10 11 12

A0.10

60.26

40.18

70.17

40.09

90.44

40.13

10.10

51.26

90.20

90.14

8 0.259

B0.10

80.34

90.26

70.22

60.28

20.44

40.34

60.31

70.31

10.26

3 0.38 0.323

C 0.080.06

8 0.10.21

20.09

9 0.25 0.10.08

10.10

30.47

50.65

1 1.284

D0.09

80.09

1 0.110.11

40.11

80.10

90.11

30.09

50.13

90.32

70.43

4 0.428

E0.57

20.51

40.25

90.42

80.17

60.29

91.42

20.72

30.68

20.73

20.85

6 0.795

F0.55

90.49

60.14

3 0.260.39

2 1.21.42

20.78

40.77

90.89

20.81

4 0.847

G0.56

80.43

20.11

60.14

6 0.120.10

1 1.420.28

10.30

80.31

40.29

8 0.266

H0.47

60.42

40.13

60.10

20.18

20.16

41.43

10.35

30.19

7 0.250.33

3 0.308Table 28 – A table to show raw data obtained from the MTT assay that is used to calculate cell viability in the short term assay (Highlighted results are marked as possible sites of contamination)

raw data

1 2 3 4 5 6 7 8 9 10 11 12

A2.26

81.64

50.78

51.18

80.96

4 1.160.17

60.15

50.15

80.16

20.16

9 0.216

B 0.811.50

21.52

61.45

91.38

91.42

9 0.330.14

20.38

90.14

20.15

8 0.161

C0.09

5 0.08 0.080.98

1 0.871.10

40.08

40.08

60.07

80.81

90.59

6 0.951

D0.10

70.08

80.09

20.14

40.14

80.16

40.14

50.09

40.08

50.15

80.14

7 4.823

E1.76

6 0.191.17

81.70

81.40

41.58

61.41

30.89

80.73

31.99

61.46

5 2.316

F1.69

11.84

61.39

71.48

31.59

11.66

71.35

41.62

40.89

61.99

31.74

8 2.204

G0.26

21.80

32.02

41.43

31.04

60.19

91.87

21.18

10.16

90.73

80.63

2 0.166

H2.21

42.25

22.73

32.75

92.13

40.24

52.24

91.09

11.09

30.18

30.17

9 0.222Table 29 - A table to show raw data obtained from the MTT assay that is used to calculate cell viability in the long term assay (Highlighted results are marked as possible sites of contamination)

raw

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data1 2 3 4 5 6 7 8 9 10 11 12

A0.48

60.58

2 0.630.65

20.65

30.67

2 0.440.39

90.43

10.40

20.41

4 0.418

B0.57

4 0.590.62

10.69

60.69

10.64

80.38

40.41

60.37

80.36

20.42

4 0.372

C0.07

30.28

30.58

70.56

90.72

20.61

20.08

2 0.410.57

50.63

60.61

7 0.618

D0.09

60.29

80.45

20.47

10.50

10.48

20.12

80.41

90.50

50.46

50.55

1 0.431

E0.63

90.61

40.68

20.74

3 0.80.76

50.71

80.77

40.74

50.82

9 0.83 0.976

F0.59

70.64

10.70

3 0.680.77

50.76

90.66

30.83

90.45

60.80

10.88

3 0.878

G0.34

90.40

20.42

60.48

80.48

5 0.580.54

70.51

40.51

70.52

30.53

9 0.577

H0.33

60.46

90.44

20.58

2 0.50.46

10.50

40.55

70.53

20.55

70.53

1 0.574Table 30 - A table to show raw data obtained from the MTT assay that is used to calculate cell viability in the short term assay (repeat)

raw data

1 2 3 4 5 6 7 8 9 10 11 12

A 0.108 1.114 1.284 1.279 1.229 1.262 0.295 0.251 0.236 0.248 0.165 0.159

B 0.692 1.145 1.211 1.254 1.249 1.297 0.341 0.226 0.22 0.217 0.195 0.179

C 0.096 0.295 1.175 0.931 1.265 1.538 0.168 0.379 1.173 0.959 1.143 0.615

D 0.123 0.121 0.269 0.285 0.208 0.429 0.178 0.114 0.286 0.266 0.256 0.324

E 0.971 1.387 1.346 1.354 1.385 1.411 1.411 1.382 1.242 1.358 1.315 0.515

F 1.097 1.421 1.277 1.408 1.386 1.7 1.444 1.407 1.366 1.354 1.331 0.762

G 0.344 0.321 0.261 0.354 0.353 0.439 0.425 0.329 0.297 0.321 0.278 0.295

H 0.299 0.282 0.361 0.419 0.391 0.485 0.383 0.394 0.389 0.34 0.22 0.322

Table 31 - A table to show raw data obtained from the MTT assay that is used to calculate cell viability in the long term assay (repeat) (Highlighted results are marked as possible contamination)

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Statistical Test Results

Cell Plate 4000Mann-Whitney Test and CI: Cpz, Flav

N MedianCpz 6 79.7Flav 6 201.7

Point estimate for ETA1-ETA2 is -139.095.5 Percent CI for ETA1-ETA2 is (-194.8,-34.1)W = 26.0Test of ETA1 = ETA2 vs ETA1 not = ETA2 is significant at 0.0453

Mann-Whitney Test and CI: Cpz, Flav_1

N MedianCpz 6 79.7Flav_1 6 222.5


Mann-Whitney Test and CI: Cpz + S9, Flav+S9

N MedianCpz + S9 6 61.8Flav+S9 6 68.4

Point estimate for ETA1-ETA2 is -22.295.5 Percent CI for ETA1-ETA2 is (-208.6,30.5)W = 33.0Test of ETA1 = ETA2 vs ETA1 not = ETA2 is significant at 0.3785

Mann-Whitney Test and CI: Cpz + S9, Flav+S9_1

N MedianCpz + S9 6 61.8Flav+S9_1 6 76.3


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Cell plate 2000Mann-Whitney Test and CI: Cpz, Flav












64


Cell plate 4000 (repeat)Mann-Whitney Test and CI: Cpz, Flav












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Cell Plate 2000 (Repeat)Mann-Whitney Test and CI: Cpz, Flav












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Ethics Form

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The metabolism and toxicology of rutin on liver type cells

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