Faculty of Science
THE ANTIMUTAGENIC EFFECTS OF ARCEUTHOBIUM AMERICANUM
2019 | DRAYDEN ARISTOTTLE DEVON KOPP

B.Sc. Honours thesis – Biology

THE ANTIMUTAGENIC EFFECTS OF ARCEUTHOBIUM AMERICANUM
by
DRAYDEN ARISTOTTLE DEVON KOPP
A THESIS SUBMITTED IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
BACHELOR OF SCIENCE (HONS.)
in the
DEPARTMENT OF BIOLOGICAL SCIENCES
(Cellular Molecular and Microbial Biology)

This thesis has been accepted as conforming to the required standards by:
Joanna Urban (M.Sc.), Thesis Supervisor, Dept. Biological Sciences
Dr. Kingsley Donkor (Ph.D), Co-supervisor, Dept. Physical Sciences
Dr. Jonathan Van Hamme (Ph.D.), Examining Committee member, Dept. Biological Sciences

Dated this 20th day of July, 2018, in Kamloops, British Columbia

© Drayden Aristottle Devon Kopp, 2018

ABSTRACT
Extracts from European mistletoe (Viscum album) have been used in alternative cancer treatments
for nearly a century, and the extracts have modest antimutagenic properties. The dwarf mistletoe
(Arceuthobium americanum) and V. album are from the same family and share similar
characteristics. Both are plant-plant parasites, taking water and minerals from their hosts. However,
it remains unknown as to whether A. americanum extracts are also antimutagenic. The objective
of this research is to determine if extracts of A. americanum have antimutagenic properties when
tested against some standard mutagens. Methanol extraction of A. americanum’s fruits and stem
were conducted, and the extracts were tested against histidine dependent Salmonella typhimurium
strains via the Ames test, a standard method of detecting mutagenicity/antimutagenicity. The S.
typhimurium strains contain different mutations in assorted genes in the histidine operon and are
more easily back mutated than others in the presence of mutagens. The increased S. typhimurium
growth can then be an indicator of increased genetic mutations that resulted in histidine reversions.
The same test was conducted with modifications to test for antimutagenic activity. This research
revealed modest A. americanum antimutagenic properties similar to V. album; however, the results
were not statistically significant and difficult to get replicates. This result is a first for the
Arceuthobium genus. Future research into this field could find more concrete evidence into the
potential for the plant.

Thesis Supervisor: Associate Professor Joanna Urban, PHD.

ii

ACKNOWLEDGEMENTS
I would like to express my gratitude to Joanna Urban, Dr. Kingsley Donkor and Dr. Cynthia Ross
Friedman for the support and experience gained throughout my Honours research. I would also
like to express gratitude to Dr. Jonathan Van-Hamme for accepting the role of an external
committee member and Dr. Louis Gosselin for advice regarding organization of research and
development of the thesis. Thank you to Thompson Rivers University for the funding provided via
the Undergraduate Research Experience Award Program (UREAP).

iii

TABLE OF CONTENTS
Abstract ........................................................................................................................................... ii
Acknowledgements ........................................................................................................................ iii
List of figures ................................................................................................................................. vi
List of Tables ................................................................................................................................ vii
Introduction ..................................................................................................................................... 1
Cancer and mutagenesis .............................................................................................................. 1
Antimutagens & Mutagens ......................................................................................................... 3
The Principle of the Ames Test .................................................................................................. 4
European Mistletoe & cancer treatment ..................................................................................... 5
Dwarf Mistletoe .......................................................................................................................... 5
Materials and Methods .................................................................................................................... 6
Site Description Sample Collection ............................................................................................ 6
Methanol Extraction and Filtration ............................................................................................. 6
Rotary Evaporation and Nitrogen Evaporation........................................................................... 8
Rotary Evaporation ................................................................................................................. 9
Nitrogen Evaporation ............................................................................................................ 10
Ames Test for Antimutagenicity............................................................................................... 11
Growth Procedure ................................................................................................................. 11
Flash Freezing ....................................................................................................................... 11

iv

Traditional Ames Test........................................................................................................... 12
Modified Xenometrix™ Ames Assay: ................................................................................. 13
Results ........................................................................................................................................... 16
Methanol Extraction.................................................................................................................. 16
Rotary & Nitrogen Evaporation ................................................................................................ 16
Ames Test for Antimutagenicity............................................................................................... 18
Growth and Competency ...................................................................................................... 18
Flash Freezing ....................................................................................................................... 18
Traditional Ames Test........................................................................................................... 18
Xenometrix™ Ames Test ..................................................................................................... 19
Discussion ..................................................................................................................................... 20
Extraction & Evaporation ......................................................................................................... 20
Traditional Ames Tests ............................................................................................................. 20
Xenometrix™ Ames Tests ........................................................................................................ 20
Appendix ....................................................................................................................................... 22
Appendix A. Plant collections figures. ..................................................................................... 22
Appendix B. Flash Freezing Conditions. .................................................................................. 22
Appendix C. Results of Traditional Ames Test. ....................................................................... 22
Appendix E. Results of Xenometrix™ Ames Test. .................................................................. 23
References: .................................................................................................................................... 25
v

LIST OF FIGURES
Figure 1. Two kinds of mutations shown in blue, the codons are indicated within the red lines,
Single base pair substitution shows the replacement of a A=T base pair with a C≡G base pair
(Habibi Najafi and Pezeshki 2013. The Frameshift mutation shows an insertion of the highlighted
C≡G base pair which shifts the codons out of their original state (Habibi Najafi and Pezeshki 2013).
......................................................................................................................................................... 2
Figure 2. Extraction methods used for A.americanum. A. shows the initial steps of collecting and
crushing the whole plant in a mortar and pestle and then placing the plant in a 500 mL flask with
450 mL of methanol. B shows sonication via probe sonicator which is done for roughly 2-3 hours
at a time; afterwards, the methanol extract is poured into a container which is stored at roughly
room temp in a dark room. C is the solid remnants of the extract after the methanol is poured off,
this solid extract have 450 mL of methanol added and step B is repeated. Step C is repeated at
most 2 times. ................................................................................................................................... 8
Figure 3. Flowchart of evaporation techniques used to remove all methanol. A shows transfer of
the extracts to a round bottom flask which is then used to evaporate the fluids off; afterwards, the
remaining fluids are collected and in B are transferred to several small test tubes. Nitrogen
evaporation was used to evaporate the remaining extracts to get a solid brown mass. ................ 10
Figure 4. Flowchart for the Traditional Ames Test. A shows the top agar in a hot water bath
(~50℃). B shows incubation of the appropriate bacteria into the top agar after it has cooled slightly.
C and D show the pouring and spreading of the top agar on the low glucose concentration plate. E
shows expected growth results for positive control (+) and negative control (-), with more growth
on the positive control than the negative. ..................................................................................... 12
Figure 5. Flowchart of the modified Xenometrix™ Ames Test. A shows the addition of the
antimutagen with the mutagen and the addition of the positive and negative control. B shows the
dilution of the mutagen and antimutagen from the stock, downwards. C shows the incubation of
the bacteria into each well, exposure media is added during this step as well. D shows the transfer
from the 24 well plate to the 96 well plate. E shows the 48 hour incubation and the expected colour
changes. Yellow indicates reversion and purple indicates no reversion. ...................................... 14
Figure 6. Dwarf Mistletoe extraction in 500 mL of methanol. The left shows a new extraction with
the first addition of 450 mL methanol, the right shows an exhausted extract with its third addition
of 450 mL of methanol. Both have been sonicated for two hours. ............................................... 17
Figure 7. The mean results for the Xenometrix™ Ames Test with the average number of revertants
shown, lower indicates less mutagenicity. .................................................................................... 19
Figure 8. Dwarf mistletoe plants, female is on the right and male is on the left. ......................... 22

vi

LIST OF TABLES
Table 1. Flash freezing results of cells for three different trials. The O.D of the first cell sample
grown from the frozen culture is shown. ...................................................................................... 22
Table 2. Colony counts from the traditional Ames Testing. The * marks contamination. ........... 22
Table 3. Results of three Xenometrix Ames Test trials labeled A, B and C. ............................... 23
Table 4. Anova single factor test between trials of three of the Xenometrix Ames Test trials. ... 24

vii

INTRODUCTION
Cancer and mutagenesis
Cancer is a disease which occurs in eukaryotic cells when cell replication occurs at an uncontrolled
rate (Stadler 2014). Many eukaryotes normally derogate cancer cells but in cases where the body
cannot correct these cancer cells, treatments such as chemotherapy or surgery must be used (Kastan
and Bartek 2004). Cancer can be the result of carcinogens that may occur in the diet, through
smoke or other forms of exposure, which can cause mutations (Lou et al. 1998). The uncontrolled
replication occurs when cellular phases (G1, G2, S and M) no longer are correctly regulated by the
regulatory checkpoints (Kastan and Bartek 2004). The mutations in the genome often effect the
proteins that regulate the cell cycle (Stadler 2014). Therefore, it appears that the underlying cause
of cancers is due to extensive mutations in the genome (Casás-Selves and DeGregori 2011).
Although these genetic mutations are essential evolutionarily, at excessive rates they show to cause
damage to exposed cells (Casás-Selves and DeGregori 2011). There are several types of mutations
which effect the genome which are caused by either an insertion, deletion, replacement or
duplication of a base pair (Habibi Najafi and Pezeshki 2013; Paulson 2018). The results of these
can be a single base pair substitution, a frameshift mutation or a repeat expansion (Habibi Najafi
and Pezeshki 2013). A frameshift mutation shifts the genomic DNA generally as a result of an
insertion or deletion of a base pair – this may ultimately result in the genetic codons being
completely different, as shown in Figure 1 (Habibi Najafi and Pezeshki 2013). A single-base pair
substitution is caused by a replacement of a single base pair which may result in the amino acid
for the subsequent codon being changed, this is also shown in Figure 1 (Habibi Najafi and Pezeshki
2013). Repeat expansion on the other hand is when a small section of DNA is repeated several
times (Paulson 2018); however, this is not relevant to this study.

1

Single Base Pair Substitution

Frameshift Mutation

Figure 1. Two kinds of mutations shown in blue, the codons are indicated within the red lines,
Single base pair substitution shows the replacement of a A=T base pair with a C≡G base pair
(Habibi Najafi and Pezeshki 2013. The Frameshift mutation shows an insertion of the highlighted
C≡G base pair which shifts the codons out of their original state (Habibi Najafi and Pezeshki 2013).

2

Antimutagens & Mutagens
Different chemical carcinogens can cause different mutations – often through a unique mechanism
(Słoczyńska et al. 2014). They may act directly or indirectly on the genome through oxidation,
generation of free radicals or other mechanisms (Słoczyńska et al. 2014). Some substances can
inhibit the activity of mutagens – these are termed antimutagens (Kusamran et al. 1998). While
antimutagens prevent the occurrence of mutagens, anticarcinogens more broadly impede cancer
directly (Kusamran et al. 1998). Therefore, some anticarcinogens work by killing the host cell
containing the cancer via apoptosis (Stewart et al. 2003); but, this does not work on the actual
mutation itself (Kusamran et al. 1998). Antimutagens can be thought as a category of
anticarcinogen (Lou et al. 1998). There are a variety of antimutagens, bio-antimutagenics describe
antimutagens that effect the cellular process, while desumutagen inactivates the mutagen itself
(Lou et al. 1998). There are several compounds which contain antimutagenic properties
(Słoczyńska et al. 2014). For example, substances such as magnesium salts assist in prevention of
oxidative damage. (Bronzetti et al. 2000). A commonly used antimutagen which assists in
preventing exposure to the genetic mutations is sunscreen which prevents the penetration of UV
light which causes mutations within the epithelial cells (Lucas 2011). Other antimutagens may
help prevent the formation of free radicals or also act as an anti-oxidant (Słoczyńska et al. 2014).
The use of practical antimutagenics can help with prevention and treatment of cancer (Zhang et al.
2014). This is very important due to the lethality of cancer (Stadler 2014). Therefore, the topic of
antimutagenics may be a relatively unexplored resource for cancer treatment and prevention
(Słoczyńska et al. 2014).

3

The Principle of the Ames Test
The Ames test is a tool which can assess the rate and type of mutations (Ames 1973; Kilbey et al.
2012). When done correctly the Ames test can be done in quick and effective manner (Kilbey et
al. 2012). The type of mutations that are assessed are single base pair substitutions and frameshift
mutations achieved with two particular strains of Salmonella typhimurium (Ames 1973; Kilbey et
al. 2012). Strain TA98 is used for frameshift mutation assessment and TA100 is used for single
base pare substitution assessment (Kilbey et al. 2012). This is achieved by a modification to the
genome of the S. typhimurium’s gene which produces histidine – an essential protein for growth
(Kilbey et al. 2012). Normal S. typhimurium cells produce histidine when needed; however, the
TA98 and TA100 have the histidine genes knocked out (Kilbey et al. 2012). Thus, the cells no
longer produce their own histidine and it needs to be added for the cells to grow (Kilbey et al.
2012). Therefore, the cells are incubated with limited histidine and a potential mutagen. If this
mutagen causes a reversion to the histidine producing genes then the cells should be able to grow
when the provided histidine runs out by producing their own histidine (Kilbey et al. 2012). This
process is however entirely by chance; therefore, some cells will grow in a negative control due to
spontaneous mutations resulting in reversion (Hebert et al. 2015). Therefore, the Ames Test has
shown success in indicating substances which are mutagenic to cells and has been used often as a
tool to assess the toxicity of substances (Ames 1973; Kilbey et al. 2012). Tests have also been
done to test the opposite effect of a substance – which is if it prevents the mutagenic effects of a
substance when included with the mutagen (Hong and Lyu 2012). This allows for testing of
antimutagens (Hong and Lyu 2012).

4

European Mistletoe & cancer treatment
In Germany and other European countries, chemotherapy is occasionally combined with mistletoe
injections (Ernst 2006). Some studies show that the use of mistletoe as a combined treatment in
cancer does show clinical benefits (Kienle et al. 2006). In contrast, there are several clinical trials
showing no benefit of using mistletoe treatment (Kleijnen and Knipschild 1994). Regardless, past
research has shown antimutagenics appearing in the Viscum album primarily by studying the
lectins extracted from the plants utilizing the Ames Test (Hong and Lyu 2012). The reason for the
exact mechanism for this reported antimutagenic effects is unknown (Hong and Lyu 2012). Other
research has shown anti oxidant activity within the mistletoe (Vicas et al. 2012). Thus, there is
potential that this anti oxidant activity is blocking the oxidizing effects of the mutagen. It may be
these antioxidant properties which allows for the antimutagenic behaviour of the extracts (Hong
and Lyu 2012; Vicas et al. 2012).
Dwarf Mistletoe
A. americanum also known as Lodgepole Pine Dwarf Mistletoe, which is parasitic to the Pinus
contorta (Fort Collins et al. 2002). This mistletoe shares the same family as the Viscum album –
the European mistletoe. Due to this close phylogeny – the two share similar characteristics as
regarding their parasitic nature; however, there are some distinct differences. The A. americanum
can retrieve a significant amount of carbohydrates from its parasitic host (Hawksworth and Wiens
1998); whereas, the Viscum album primarily obtains water and minerals from its host (Kahle-Zuber
2008). Therefore the additional carbohydrates within the A.americanum may provide new
substances to be investigated. Research in 2012 showed that Viscum album appears to produce
antimutagenic activity from its lectins (Hong and Lyu 2012). With the different nutrient
requirements (Hawksworth and Wiens 1998) – there is potential for other antimutagenic products

5

and potentially more potent products in the A. americanum. The research into the existence of
lectins within A. americanum has yet to be done – therefore analyzing the plant’s potentially
antimutagenic activity would provide a novel starting place.
MATERIALS AND METHODS
Site Description Sample Collection
Samples were collected two times from the same location to have enough material for the project.
The site of collection was near Stake lake at (50.573659, -120.434551). The host trees appeared to
be primarily of the Pinus genus and had no notable swelling that is associated with the parasitism.
Host trees were not chosen in a systematic approach as it was desired to collect a high raw biomass.
Male and female plants were identified and collected into separate bags for each sex. Sex was
identified based on notable differences between the male and female morphologies shown in
Figure 8 in Appendix A. Bags were driven back to TRU campus and stored in the Science building
in room S365 for roughly 2 days in a -20 ℃ freezer for the initial collection. This was done for
preservation. No plant samples were stored in -80℃.
Methanol Extraction and Filtration
The samples that had been collected underwent methanol extraction. This technique involved
weighing out the mass of the plant and crushing it in a mortar and pestle. It should be noted that
the plant is not frozen to -80℃; thus, the plant didn’t shatter. The entire plant was used in the
extraction. The plant was then transferred into a 500 mL Erlenmeyer flask which was filled with
450 mL of methanol. Afterwards, the flask was sonicated utilizing a probe sonicator which was
set to roughly 20V. Sonication was done from roughly 2-3 hours and afterwards the methanol was
poured off into a storage container and 450 mL of fresh methanol was added. Sonication was
repeated 2-3 times, each time removing and re-adding the same volume of fresh methanol. The
6

color of the extract changed throughout the process, from a dark green to a near clear. This
indicated that the pigments were extracted and likely other organic solvents as well; therefore, the
extracts were combined and filtered. There were 3 conditions: male extracts, female extracts and
male and female extracts planned to be tested separately. The extracts were stored at room
temperature until evaporation of the methanol was conducted. An overview of the extraction
procedure is shown in Figure 2. The retrieved extracts still contained a considerable amount of
solid plant debris. This was removed using simple gravity filtration. The filter used was cheese
cloth to allow for a quicker extraction with high volumes; however, the same can be achieved
utilizing paper filters. The cheese cloth was packed into a funnel and a beaker was placed beneath
for extraction. Afterwards, the filtration was repeated several times to achieve a homogeneous
mixture. Originally, the extract had much more opacity from the solid debris which were removed,
and the mixture was green and clear. This was stored at room temperature in a closed container
until evaporation was conducted.

7

A.

B.

C.
Filtration and Storage

Figure 2. Extraction methods used for A.americanum. A. shows the initial steps of collecting and
crushing the whole plant in a mortar and pestle and then placing the plant in a 500 mL flask with
450 mL of methanol. B shows sonication via probe sonicator which is done for roughly 2-3 hours
at a time; afterwards, the methanol extract is poured into a container which is stored at roughly
room temp in a dark room. C is the solid remnants of the extract after the methanol is poured off,
this solid extract have 450 mL of methanol added and step B is repeated. Step C is repeated at
most 2 times.
Rotary Evaporation and Nitrogen Evaporation
After methanol extraction was conducted, rotary evaporation and nitrogen evaporation was
conducted to remove all the methanol to have pure extracts. Originally rotary evaporation was
going to be utilized solely; however, it was difficult to evaporate all the methanol and as a result
nitrogen evaporation was utilized as well. An overview for both methanol and nitrogen evaporation
is shown in Figure 3.

8

Rotary Evaporation
The filtered methanol extracts were collected from storage. They were placed within a 500 mL
round bottom flask. The flask was cleaned thoroughly with ethanol, and methanol to remove any
residue. The flask was filled until roughly half full, thus not all extracts could be evaporated off at
a single time. Rotary evaporation was conducted at approximately 40℃ which is below the boiling
point of methanol. This is done because the vacuum created by the rotary evaporator depresses the
boiling point of methanol. While evaporating, a noticeable amount of bumping occurred. Bumping
refers to sudden volatile boiling of a substance resulting a large air bubble to be released. This is
an issue as bumping often shoots substance into the bump suppressor of the rotary evaporator
which may be contaminated. Thus, any substance in the bump suppressor was discarded. Strangely,
bumping occurred the most at low methanol volumes at approximately under 100 mL - while a
smooth boil occurred more often at higher volumes of around 250 mL. Once the majority of the
methanol was boiled off, the remaining fluid did not appear to boil off. Thus, this was stored in a
small container and transferred for low flow nitrogen evaporation.

9

Nitrogen Evaporation
Nitrogen evaporation was conducted on the remaining extracts which remained after rotary
evaporation. The extracts were distributed among approximately 10 test tubes. These tubes
contained a low amount of the extract in each tube of approximately 1-2 mL. The nitrogen
evaporation apparatus was cleaned with ethanol and the tips were placed near the middle of the
test tubes. Flow was attempted to be kept high enough that the evaporation would be efficient but
not too high as to cause splashing of the fluids. Evaporation was conducted for roughly an hour
and the vials were assessed to confirm that they lacked methanol. This was tested by increasing
the flow of nitrogen slightly and checking for any splashing. When there appeared to be no liquid
left the test tubes were covered with Parafilm and stored in a fume hood at room temperature for
future use.
A.

B.

Figure 3. Flowchart of evaporation techniques used to remove all methanol. A shows transfer of
the extracts to a round bottom flask which is then used to evaporate the fluids off; afterwards, the
remaining fluids are collected and in B are transferred to several small test tubes. Nitrogen
evaporation was used to evaporate the remaining extracts to get a solid brown mass.

10

Ames Test for Antimutagenicity
There were two variations of the Ames test which were run over the course of the study. The first
test described as the “Traditional Ames Test” utilized a protocol similar to the one described by
Ames in 1973 (Ames 1973). The Xenometrix Ames Assay was utilized later in the research due to
difficulty with results from the traditional Ames Test. In both cases, the strains of bacteria used
were TA98 and TA100.
Growth Procedure
Both TA100 and TA98 were grown under the same conditions and stored in the same freezer. A
stock TA98 and TA100 was obtained from a -80℃ freezer. The stock retrieved from the suppliers
was stored in DMSO, the self-created stock was stored in glycerol. These stocks were retrieved
from the freezer and left to thaw at room temperature. Afterwards the stock was incubated. The
media was nutrient broth with 5 g/L NaCl. This media was 10 mL either in a test tube or
Erlenmeyer flask. Growth was always done at 37℃ and the rotations per minute (RPM) were
adjusted between 150 RPM and 250 RPM to achieve the best growth. After overnight growth, the
optical density (OD) was measured, and tests were conducted depending on if the OD was
sufficient. Typically, an OD of greater than 2.0 is needed but 1.5 was still utilized typically at
higher bacterial volumes. Negative controls of pure growth media were implemented after
contamination was found in early tests.
Flash Freezing
Cells retrieved from the manufacturer stock were flash frozen in glycerol. This was done in three
separate trials. The third trial achieved the best results. Trial 1, 2 and 3 are shown in Table 1.
However only trial 3 will be discussed. This trial used 9 mL of the overnight culture and
centrifuged it at 2000 rpm for 10 minutes. This resulted in a cell pellet. Most of the supernatant
11

was removed, and the pellet was resuspended in roughly 1 mL of media. 200 μL of the resuspended
cells were transferred into microcentrifuge tubes and 200 μL of glycerol was added. The two
components were mixed, and the cells were flash frozen with liquid nitrogen. Afterwards, these
cells were stored in a -80℃ freezer for future use.
Traditional Ames Test
The traditional Ames test was conducted as per chapter 6 in the Handbook of Mutagenicity Test
Procedures (Kilbey et al. 2012). This utilized a top agar and a low glucose concentration plates.
The top agar contained 0.6% Difco agar and 0.5% NaCl. The top agar used was stored from
previous a research project. Top agar was melted in a microwave and kept a liquid in a water bath
of approximately 50℃. Biotin and histidine are added to the top agar along with the positive and
negative controls. The top agar is left to cool slightly prior to the addition of the bacteria.
Afterwards the top agar is quickly mixed and poured over top of the low concentration glucose
plate. The plate is swirled to allow for even distribution of top agar and left to cool for
approximately 10 minutes. Afterwards, the plates are incubated at 37℃ for 48 hours. The number
of colonies were counted after the 48 hours have passed.
A.

B.

C.

D.

E.

Figure 4. Flowchart for the Traditional Ames Test. A shows the top agar in a hot water bath
(~50℃). B shows incubation of the appropriate bacteria into the top agar after it has cooled slightly.
C and D show the pouring and spreading of the top agar on the low glucose concentration plate. E
shows expected growth results for positive control (+) and negative control (-), with more growth
on the positive control than the negative.

12

Modified Xenometrix™ Ames Assay:
The protocol for the Xenometrix™ Ames Assay was modified to utilize 96 well plates entirely
versus the 384 well plates. This used a 24 well plate which was used to dilute the antimutagen with
the mutagen. This is shown below in Figure 5. After the dilution is concluded, a positive control
of 4-nitroquinoline N-oxide (2-NQO) and 2 nitrofluorene (2-NF) is used. The positive control
stock contained a 100 ug/mL of 2-NF and 5 ug/mL of 4-NQO. The positive control was solely the
mutagens with no antimutagen. The test and controls use an exposure media included with the kit
and the negative control is exposure medium solely. The ingredients of the exposure media are not
specified in the kit. 240 μL of the mistletoe sample was mixed with the 40 μL of the mutagen. This
underwent serial dilution shown in Figure 5 B by taking 140 μL and adding it to another well. This
receives 140 μL of exposure media and is further diluted utilizing the same steps. For TA98 – 6.3
mL of exposure media were added to 0.7 mL of bacteria culture. For TA100 – 6.65 mL of exposure
media were added to 0.35 mL bacteria culture. 240 μL of these stocks were added to each of the
corresponding wells. Afterwards, 1 mL of indicator media was added to each of the wells.
Afterwards, 50 μL of each well was transferred to a 98 well plate as shown in Figure 5 D. The
plates were inoculated at 37℃ for 48 hours and wells were counted. Yellow indicated growth and
purple indicated no growth. If the well was in between colours it was marked as growth. Statistical
analysis via SINGLE factor Anova was conducted to measure the significance between the data
sets.

13

A.

B.

C.

Addition of chemicals

Dilution of
chemicals

Inoculation
of bacteria

D.

Distribution into 96 well plate
E.

Results of the Assay

Figure 5. Flowchart of the modified Xenometrix™ Ames Test. A shows the addition of the
antimutagen with the mutagen and the addition of the positive and negative control. B shows the
dilution of the mutagen and antimutagen from the stock, downwards. C shows the incubation of
the bacteria into each well, exposure media is added during this step as well. D shows the transfer
14

from the 24 well plate to the 96 well plate. E shows the 48 hour incubation and the expected colour
changes. Yellow indicates reversion and purple indicates no reversion.

15

RESULTS
Methanol Extraction
Soaking the crushed plant in methanol with sonication showed the release of pigments as the
methanol changed to a dark green fluid. As more extraction runs were completed, the methanol
became less green and the solid plant also lost much of its pigments. This is demonstrated in Figure
6, which shows the difference between three extraction runs versus one extraction run. The
filtration of the extraction material still left a green tinted fluid, this indicates that the pigments
were solubilized within the methanol. Therefore, an assumption was made that the organic
compounds were also solubilized.
Rotary & Nitrogen Evaporation
Rotary evaporation resulted in incomplete evaporation of the substance. There was residue left on
the inside of the round bottom flask which may have slightly decreased the yield. The remaining
methanol was transported to test tubes tubes and was slightly cloudy and dark green. When this
methanol had undergone nitrogen evaporation, it had turned to a brown waxy solid. When water
was added for Ames testing – it appeared to be a brown liquid.

16

Figure 6. Dwarf Mistletoe extraction in 500 mL of methanol. The left shows a new extraction with
the first addition of 450 mL methanol, the right shows an exhausted extract with its third addition
of 450 mL of methanol. Both have been sonicated for two hours.

17

Ames Test for Antimutagenicity
Growth and Competency
When growing cells overnight, generally an OD of 2.0 or greater is desired for the bacteria;
however, this was difficult to achieve with the bacteria used. Generally, the count would get close
to 2.0 but never surpassed it. The exact reason for this is uncertain but may have affected the results
of the Ames test. Typical values were seen between 1.5 – 2.0. Testing for standard Ames Test did
not use a negative control in the growth conditions. There was one notable example were S.auereus
contamination appeared to be present in gram staining. Therefore, negative controls were used for
the later Ames tests to ensure easy recognition of contamination. In most cases, the negative
control had a O.D. of less than 0.1 indicating no bacterial growth; however, there was a notable
exception and the growth media was believed to be contaminated; therefore, these cells were
discarded.
Flash Freezing
Flash freezing of the cells were attempted three separate times. Each occurrence of flash freezing
utilized glycerol to prevent the formation of ice crystals. The methodology was similar for each
process; however, slight modifications were made to get the best cell growth results. The three
trial conditions are listed in Table 1. Each trial utilized a 50% mixture of glycerol. Trial 1 and 2
did not pellet the cells before freezing, as a result it appeared that the growth may have been less
optimal compared to trial 3 which had the bacteria pelleted before glycerol was added.
Traditional Ames Test
The traditional Ames Test was attempted only with controls to attempt to get correct positive and
negative results. This appeared to not be the case most of the time. Two of the trials are shown in
Table 2 which demonstrate the incorrect growth values. The O.D. for these cells used had an O.D
18

of greater than 1.5. Trials that had a low O.D of below 1.5 were not tested and were discarded.
Some contamination was also seen during these trials on a TA98 plate. Issues with the traditional
Ames Test lead to the utilization of the Xenometrix™ Ames Testing plate.
Xenometrix™ Ames Test
The Xenometrix™ Ames test showed the most substantial results of any antimutagenic activity.
Due to the 96 well trial assay, each group has a maximum of 4 revertants for each trial. Therefore,
the average values are taken among the three trials used. The results are shown in Figure 7 and
show that when the mistletoe and antimutagen are diluted to 1:8, this results in least amount of
revertants with an average of 1.6 out of 4 wells containing revertant among the trials. The 1:16
dilution showed an average of 2.6 revertants. Single factor ANOVA was run for the 1:8 dilution
to determine if the results within the three trials were significantly close to one another. This

Average Number of Revertants (n=3)

appeared to not be so with a P-value of 0.32.

4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
Negative
1:32
1:16
1:8
1:4
1:2
Control Dilution Dilution Dilution Dilution Dilution

Extract Positive
Control
and
Mutagen

Experiemntal conditons

Figure 7. The mean results for the Xenometrix™ Ames Test with the average number of revertants
shown and standard error plotting on the error bars, lower indicates less mutagenicity/

19

DISCUSSION
Extraction & Evaporation
The extraction process appeared to be successful based on the transfer of pigment into the methanol.
The pigment extracted was likely chlorophyll A and chlorophyll B. This indicates that the cells
appeared to be successful extraction of organic solvents. Based off this information it was deduced
that lectins may have also been extracted along with other organic compounds. However, no
analytical testing was done to confirm this. Storage of the extraction was at room temperature –
this may have lead to break down of the organic compounds over time. The extracts were stored
for approximately 6 months prior to being utilized in the Ames testing – this likely also influence
the results. In future work it would likely be good to store the extracts in a freezer for stability, or
to test the extracts much sooner after the extraction process has been completed.
Traditional Ames Tests
The results of the traditional Ames tests appeared to have a variety of issues with the controls
incorrectly working. There may be several reasons for this. Some of the materials were re-used
from previous testing. This includes the top Agar; therefore, there may have been issues not noticed
with this trial. There may have also been issues with the cell viability – as the cells were grown
from a frozen stock and then attempted to be reflash frozen using a small volume of nitrogen which
may not have been sufficient. The results indicate that the cell viability may be a big issue as
negative control values sometimes had more growth than the positive control plates. This may be
the result of these cells being selected against slowly during the freezing and melting processes.
Xenometrix™ Ames Tests
The Xenometrix™ Ames Assay showed to give a difference in growth between the mistletoe trials
particularly at 1:8 and 1:16 dilutions. The reason for this is uncertain and it should be dually noted
20

that the antimutagen and mutagen were both diluted with one another. However, the dilution of
the mutagen is unlikely a cause of any alteration of results since lower concentrations did not see
the same low revertant rates that would be expected if this were the case. It must also be noted that
among the three trials for these dilutions – the results were significantly different from one another
based on the ANOVA tests; thus, it cannot be said with certainty that this is not an anomaly. These
results differ much from past research by (Hong and Lyu 2012), research on the Viscum album and
indicate that there is an overall less antimutagenic effect at higher dilutions. Therefore, it is
uncertain as to why the increased dilution caused greater antimutagenicity.
The A. americanum demonstrated modest antimutagenic properties; however, more vigorous
testing would have to be done to assess any significant findings. A major issue was getting controls
to work correctly with the antimutagenic assays. Future testing with a calibrated Ames Test
procedure would allow for a greater understanding of any underlying antimutagenicity.

21

APPENDIX
Appendix A. Plant collections figures.

Figure 8. Dwarf mistletoe plants, female is on the right and male is on the left.
Appendix B. Flash Freezing Conditions.
Table 1. Flash freezing results of cells for three different trials with variations in reagents and cell
culture conditions. The O.D. was taken after a sample was grown for 14 hours.
Trial
Number:

Reagents:

1

•
•
•

2

•
•
•
•

3

500 μL dH2O
500 μL glycerol
200 μL of cells
in media
500 μL media
500 μL glycerol
200 μL media
200 μL glycerol

Cell culture conditions:
200 μL of cells incubated into
mixture

cells in growth media and glycerol
added
Cells pelleted and frozen with
glycerol

O.D. After
growing for 14
hours
1.4

1.2
1.6

Appendix C. Results of Traditional Ames Test.
Table 2. Colony counts from the traditional Ames Test plates. The * marks contamination.
22

Trial

Strain

1

TA100
TA98
TA100
TA98*

2

Positive
Control
(colony
count)
28
8
173
77

Negative
Control
(colony count)
150
33
141
66

Appendix E. Results of Xenometrix™ Ames Test.
Table 3. Results of three Xenometrix™ Ames Test trials labeled A, B and C. The values show the
average number of revertants with a maximum of 4 and minimum of 0 for trials A, B and C.
Category

A

B

C

Sum:

Avg:

Revertants:

Negative Control

4

0

3

7

2.3

1.7

1:32 Dilution

0

0

0

0

0

4

1:16 Dilution

4

0

0

4

1.3

2.7

1:8 Dilution

4

4

0

8

2.7

1.3

1:4 Dilution

0

0

0

0

0

4

1:2 Dilution

0

0

0

0

0

4

Extract and

0

0

0

0

0

4

0

0

0

0

0

4

Mutagen
Positive Control

23

Table 4. Anova single factor test between trials of three of the Xenometrix™ Ames Test trials.

SUMMARY
Groups
A
B
C

Count
8
8
8

Sum
12
4
3

Average
1.5
0.5
0.38

Variance
4.3
2
1.1

24

LITERATURE CITED
Ames BN. 1973. Carcinogens are mutagens: their detection and classification. Environ Health
Perspect. 6:115.
Bronzetti G, Aretini P, Ambrosini C, Cini M, Fiorio R, Caltavuturo L, Della Croce C, Panunzio
M. 2000. Antimutagenesis studies of magnesium and calcium salts.
Casás-Selves M, DeGregori J. 2011. How Cancer Shapes Evolution and How Evolution Shapes
Cancer. Evol Educ Outreach. 4:624–634. doi:10.1007/s12052-011-0373-y.
Ernst E. 2006. Mistletoe as
doi:10.1136/bmj.39055.493958.80.

a

treatment

for

cancer.

BMJ.

333:1282–1283.

Habibi Najafi MB, Pezeshki P. 2013. Bacterial Mutation; Types, Mechanisms and Mutant
Detection Methods: a Review. In: 1st Global Multidisciplinary eConference.
Hawksworth FG, Wiens D. 1998. Dwarf mistletoes: biology, pathology, and systematics. Diane
Publishing.
Hebert A, Bishop M, Bhattacharyya D, Gleason K, Torosian S. 2015. Assessment by Ames test
and comet assay of toxicity potential of polymer used to develop field-capable rapid-detection
device to analyze environmental samples. Appl Nanosci. 5:763–769. doi:10.1007/s13204-0140373-7.
Hong C-E, Lyu S-Y. 2012. The Antimutagenic Effect of Mistletoe Lectin (Viscum album L. var.
coloratum agglutinin). Phytother Res. 26:787–790. doi:10.1002/ptr.3639.
Kahle-Zuber D. 2008. Biology and evolution of the European mistletoe (Viscum album).
Kastan MB, Bartek J. 2004. Cell-cycle checkpoints and cancer. Nature. 432:316.
Kienle GS, Kiene H, Albonico HU. 2006. Anthroposophische Medizin: Health Technology
Assessment Bericht – Kurzfassung. Complement Med Res. 13(suppl 2):7–18.
Kilbey BJ, Legator M, Nicholson W, Ramel C. 2012. Handbook of Mutagenicity Test Procedures.
Elsevier Science.
Kleijnen J, Knipschild P. 1994. Mistletoe treatment for cancer review of controlled trials in humans.
Phytomedicine. 1:255–260. doi:10.1016/S0944-7113(11)80073-5.
Kusamran WR, Tepsuwan A, Kupradinun P. 1998. Antimutagenic and anticarcinogenic potentials
of some Thai vegetables. Mutat Res Mol Mech Mutagen. 402:247–258. doi:10.1016/S00275107(97)00304-7.

25

Lou J, Lenke LG, Ludwig FJ, O’brien MF. 1998. Apoptosis as a mechanism of neuronal cell death
following acute experimental spinal cord injury. Spinal Cord. 36:683–690.
Lucas RM. 2011. An epidemiological perspective of ultraviolet exposure—public health concerns.
Eye Contact Lens. 37:168–175.
Paulson H. 2018. Repeat expansion diseases. In: Handbook of Clinical Neurology. Vol. 147.
Elsevier.
p.
105–123.
[accessed
2018
Jul
11].
https://linkinghub.elsevier.com/retrieve/pii/B9780444632333000099.
Słoczyńska K, Powroźnik B, Pękala E, Waszkielewicz AM. 2014. Antimutagenic compounds and
their possible mechanisms of action. J Appl Genet. 55:273–285. doi:10.1007/s13353-014-0198-9.
Stadler WM, editor. 2014. Cancer biology review: a case-based approach. New York: Demos
Medical Publishing, LLC.
Stewart BW, Kleihues P, International Agency for Research on Cancer, editors. 2003. World
cancer report. Lyon: IARC Press.
Vicas SI, Rugina D, Socaciu C. 2012. Antioxidant activity of European mistletoe (Viscum album).
In: Phytochemicals as Nutraceuticals-Global Approaches to Their Role in Nutrition and Health.
InTech.
Zhang H-M, Zhao L, Li H, Xu H, Chen W-W, Tao L. 2014. Research progress on the
anticarcinogenic actions and mechanisms of ellagic acid. Cancer Biol Med. 11:92–100.
doi:10.7497/j.issn.2095-3941.2014.02.004.

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