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Effect of reductants on sodium chromate-induced cytotoxicity and morphological transformation to C3H/10T1/2 Cl 8 mouse embryo cells
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Effect of reductants on sodium chromate-induced cytotoxicity and morphological transformation to C3H/10T1/2 Cl 8 mouse embryo cells
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1
Effect of Reductants on Sodium Chromate-Induced
Cytotoxicity and Morphological Transformation to
C3H/10T1/2 Cl 8 Mouse Embryo Cells
By
Laureen Tran
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(Molecular Microbiology and Immunology)
Advisor: Dr. Joseph R. Landolph Jr., Ph.D.
December 2014
Copyright 2014 Laureen Tran
2
TABLE OF CONTENTS
TITLE PAGE……………………………………………………………………………………...1
LIST OF TABLES………………………………………………………………………………...3
LIST OF FIGURES……………………………………………………………………………….4
ABSTRACT……………………………………………………………………………………….6
CHAPTER I: INTRODUCTION………………………………………………………………….8
1.1 Epidemiological Studies of Cr(VI) Compounds……………………………………....8
1.2 Cr(VI) Carcinogenesis………………………………………………………………...8
1.3 The Role of Reductants in Cr(VI) Carcinogenesis…………………………………..11
1.4 Overall Goal of This Thesis Work…………………………………………………...12
CHAPTER II: MATERIALS AND METHODS………………………………………………...14
2.1 Chemicals…………………………………………………………………………….14
2.2 C3H/10T½ CI8 (10T½) Mouse Embryo Cell Culture Model……………………….14
2.3 Cell Culture Methods………………………………………………………………...15
2.4 Plating Efficiency Method…………………………………………………………...16
2.5 Assays for Chemically Induced Cytotoxicity………………………………………..17
2.6 Assays for Chemically Induced Morphological Transformation…………………….24
CHAPTER III: RESULTS……………………………………………………………………….28
3.1 Effect of Ascorbic Acid on the Survival of 10T1/2 Cells……………………………28
3.2 Effects of Ascorbic Acid on the Survival of 10T1/2 Cells Treated with Sodium
Chromate……………………………………………………………………………..35
3.3 Effects of Ascorbic Acid on Sodium Chromate induced Morphological
Transformation to 10T1/2 Cells……………………………………………………...44
3.4 Effect of GSH on the Survival of 10T½ Cells……………………………………….48
3.5 Effect of GSH on the Survival of 10T1/2 Cells Treated with Sodium Chromate…...61
CHAPTER IV: CONCLUSION AND DISCUSSION…………………………………………..68
4.1 Previous Work In Our Laboratory……….……….……….……….……….………..68
4.2 Effects of Ascorbic Acid on the Survival of 10T1/2 Cells Treated with Sodium
Chromate.……….……….……….……….……….……….……….……….……….68
4.3 Effects of Ascorbic Acid on Sodium Chromate-Induced Morphological
Transformation to 10T1/2 Cells……….……….……….……….……….……….….70
4.4 Effect of GSH on the Survival of 10T1/2 Cells Treated with Sodium Chromate…...72
4.5 Future Directions……….……….……….……….……….……….……….………..72
ACKNOWLEDGMENTS……….……….……….……….……….……….……….…………..74
REFERENCES…………………………………………………………………………………..75
3
LIST OF TABLES
Table 1. Survival Fractions Determined from the Effect of 0.00625mM to 0.375mM Ascorbic
Acid to 10T1/2 Cells (Cells Treated 24 Hours After Seeding)......................................................29
Table 2. Plating Efficiencies Determined from the Effect of 0.00625 mM to 0.375 mM Ascorbic
Acid to 10T1/2 Cells (Cells Treated 24 Hours After Seeding)…………………………………..29
Table 3. Plating Efficiencies and Survival Fractions Determined from the Effect of 0.00625mM
to 0.375mM Ascorbic Acid to 10T1/2 Cells (Cells Treated Upon Seeding)…..………………...33
Table 4. Survival Fractions Determined from the Effect of 0.0125 mM Ascorbic Acid on 10T1/2
Cells Treated with Sodium Chromate……….…………………………………………………...36
Table 5. Plating Efficiencies Determined from the Effect of 0.0125 mM Ascorbic Acid on
10T1/2 Cells Treated with Sodium Chromate.…………………………………………………..37
Table 6. Survival Fractions and Plating Efficiencies Determined from the Effect of 0.0125 mM
Ascorbic Acid on 10T1/2 Cells Treated with Sodium Chromate.……………………………….42
Table 7. Effect of 0.0125 mM Ascorbic Acid on the Sodium Chromate Induced Morphological
Transformation to 10T1/2 Cells.…………………………………………………………………45
Table 8. Cytotoxicity Data for the Transformation Experiment with Sodium Chromate and
Ascorbic Acid.…………………………………………………………………………………...46
Table 9. Survival Fractions Determined from the Effect of 0.001 mM to 10mM Glutathione
(GSH) to 10T1/2 Cells.…………………………………………………………………………..49
Table 10. Plating Efficiencies Determined from the Effect of 0.001 mM to 10mM Glutathione
(GSH) to 10T1/2 Cells.…………………………………………………………………………..49
Table 11. Plating Efficiency and Survival Fraction Determined from the Effect of 0.1 mM to
10mM Glutathione (GSH) to 10T1/2 Cells.……………………………………………………..54
Table 12. Survival Fractions Determined from the Effect of 0.1 mM to 0.5 mM Glutathione
(GSH) to 10T1/2 Cells.…………………………………………………………………………..56
Table 13. Plating Efficiencies Determined from the Effect of 0.1 mM to 0.5 mM Glutathione
(GSH) to 10T1/2 Cells.…………………………………………………………………………..56
Table 14. Survival Fractions Determined from the Effect of 0.1 mM GSH on 10T1/2 Cells
Treated with Sodium Chromate.…………………………………………………………………62
Table 15. Plating Efficiencies Determined from the Effect of 0.1 mM GSH on 10T1/2 Cells
Treated with Sodium Chromate.…………………………………………………………………63
4
LIST OF FIGURES
Figure 1. Experiment 1: Effect of Ascorbic Acid on the Survival of 10T1/2 Cells (Cells Treated
24 Hours After Seeding)…………………………………………………………………………30
Figure 2. Experiment 2: Effect of Ascorbic Acid on the Survival of 10T1/2 Cells (Cells Treated
24 Hours After Seeding)…………………………………………………………………………31
Figure 3. Effect of Ascorbic Acid on the Survival of 10T1/2 Cells (Cells Treated 24 Hours After
Seeding)………………………………………………………………………………………….32
Figure 4. Ascorbic Acid Cytotoxicity to 10T1/2 Cells (Cells Treated Upon Seeding)………….34
Figure 5.Experiment 1: Effect of 0.0125 mM Ascorbic Acid on the Survival of 10T1/2 Cells
Treated with Sodium Chromate (Cells Treated with Ascorbic Acid 24 hours After Seeding)….38
Figure 6. Experiment 2: Effect of 0.0125mM Ascorbic on the Survival of 10T1/2 Cells Treated
with Sodium Chromate (Cells Treated with Ascorbic Acid 24 Hours After Seeding)…………..40
Figure 7. Effect of 0.0125 mM Ascorbic Acid on the Survival of 10T1/2 Cells Treated with
Sodium Chromate (Cells Treated with Ascorbic Acid 24 Hours After Seeding)………………..41
Figure 8. Effect of 0.0125 mM Ascorbic Acid on the Survival of 10T1/2 Cells Treated with
Sodium Chromate (Cells Treated with Ascorbic Acid upon Seeding)…………………………..43
Figure 9. Effect of 0.0125 mM Ascorbic Acid on the Cytotoxicity of Sodium Chromate to
10T1/2 Cells……………………………………………………………………………………...47
Figure 10. Experiment 1: Effect of 0.001mM to 10mM GSH on the Survival of 10T1/2 Cells...50
Figure 11. Experiment 2: Effect of 0.001 mM to 10 mM GSH on the Survival of 10T1/2 Cells.51
Figure 12. Experiment 3: Cytotoxicity of 0.001 mM to 10mM GSH to 10T1/2 Cells…………..52
Figure 13. Effect of 0.001 mM to 10 mM GSH on the Survival of 10T1/2 Cells……………….53
Figure 14. Experiment 1: Effect of 0.1 mM to 1 mM GSH on the Survival of 10T1/2 Cells…...55
Figure 15. Experiment 1: Effect of 0.1 mM to 0.5 mM GSH on the Survival of 10T1/2 Cells…57
Figure 16. Experiment 2: Effect of 0.1mM to 0.5mM GSH on the Survival of 10T1/2 Cells…..58
Figure 17. Experiment 3: Effect of 0.1 mM to 0.5 mM GSH on the Survival of 10T1/2 Cells…59
Figure 18. Effect of 0.1mM to 0.5mM GSH on the Survival of 10T1/2 Cells…………………..60
5
Figure 19. Experiment 1: Effect of 0.1mM GSH on the Survival of 10T1/2 Cells Treated with
Sodium Chromate………………………………………………………………………………..64
Figure 20. Experiment 2: Effect of 0.1mM GSH on the Survival of 10T1/2 Cells Treated with
Sodium Chromate ……………………………………………………………………………….66
Figure 21. Effect of 0.1mM GSH on the Survival of 10T1/2 Cells Treated with Sodium
Chromate…………………………………………………………………………………………67
6
ABSTRACT
Hexavalent chromium compounds are known human carcinogens. Epidemiological
studies have shown that occupational exposure of humans to chromium(VI) compounds results
in an increased risk of respiratory cancers. The exact mechanisms of chromium(VI)
carcinogenesis is unknown, but chromium(VI) compounds have been shown to cause
chromosomal aberrations, mutations, and morphological and neoplastic transformation in
mammalian cells.
In this thesis, we investigated the ability of sodium chromate (Na
2
CrO
4
), a soluble
chromium(VI) compound, in its ability to induce cytotoxicity and morphological transformation
in cultured C3H/10T1/2 Cl 8 (10T1/2) mouse embryo cells. We hypothesized that intracellular
reductants, such as ascorbate, glutathione (GSH), and cysteine, can reductively activate Cr(VI)
intracellulary to proximate cytotoxins and cell transforming agents. Recent studies in our
laboratory have demonstrated the ability of ascorbate to enhance focus formation in 10T1/2 cells
treated with varying concentrations of ascorbic acid in presence of sodium chromate. In this
thesis, we performed cytotoxicity and morphological transformation assays by treating 10T1/2
cells with varying concentrations of sodium chromate in the presence of 0.0125 mM Ascorbic
acid, the concentration that yielded the greatest increase in foci from our recent studies. In
addition, we determined the highest, non-cytotoxic concentration of GSH and applied that
concentration to 10T1/2 cells in cytotoxicity assays.
Our cell survival data showed that ascorbic acid was able to modestly enhance the
cytotoxicity of sodium chromate to 10T1/2 cells when the cells were treated with ascorbic acid
24 hours prior to sodium chromate treatment. However, there was no enhancement of
cytotoxicity, but rather, a slight enhancement of survival, when 10T1/2 cells were treated with
7
ascorbic acid and sodium chromate simultaneously. Our first transformation assay with ascorbic
acid and sodium chromate showed little to no focus formation but the positive control, 3-
metylcholanthrene (a strong carcinogen), did not induce foci so this experiment is not valid.
This data is preliminary, and further assays need to be completed to obtain a conclusive result.
Our cell survival data also showed no enhancement of cell cytotoxicity when GSH was added to
our sodium-chromate treated 10T1/2 cells. However, only one method of treatment was used for
this assay and other methods need to be explored.
We need to explore different methods of treatment to observe the effects of ascorbic acid
and GSH on our sodium-chromate induced cytotoxic and transformation assays. We want to
maximize the cytotoxicity and cell transforming activity of chromium(VI) compounds so we can
eventually create transformed cell lines, characterize them, and study the aberrations in gene
expression that occur in Cr(VI)-transformed cell lines and hence, the molecular mechanisms of
chromium(VI) carcinogenesis.
8
CHAPTER I. INTRODUCTION
1.1 Epidemiological Studies of Chromium Compound-Induced Cancer in Humans
Hexavalent chromium [Cr(VI)] compounds are occupational and environmental human
carcinogens (Beaver, 2009). The hazardous exposures of humans to chromium compounds are
predominantly occupational (Norseth, 1981). Industries that expose humans to hazardous levels
of toxic, mutagenic, and carcinogenic chromium(VI) compounds include chromium compound
refining/chromate production, chrome plating, chrome pigment production, and stainless steel
production (Norseth, 1981;Shi, 1999). The highest exposures of humans to chromium(VI)
compounds occurs in the chrome plating industry and among chromate production workers and
stainless steel welders (Zhitkovich, 2005). Epidemiological studies have shown that
occupational exposure of humans to Cr(VI) compounds causes an 18-80 fold increased risk of
lung cancer (Wise, 2002). There is also an increased risk of respiratory cancers in workers in the
chromate producing industry, with many cases of lung cancer seen in workers in these industries
in a number of countries (Norseth, 1981). Many animal carcinogenicity studies and in vitro
studies support the epidemiological reports of chromium(VI) compounds as carcinogens and
show that chromium(VI) compounds a) are also mutagenic (Norseth, 1981), b) are able to induce
tumors in experimental animals (Saha, 2011) and c) are able to induce morphological and
neoplastic transformation of cultured cells (Wise, 2002).
1.2 Chromium(VI) Carcinogenicity
Chromium occurs predominantly in two valence states: hexavalent chromium
[chromium(VI)] and trivalent chromium [chromium(III)] (Bagchi, 2002). Chromate production
workers that have increased risks of lung cancer are exposed to both hexavalent and trivalent
chromium compounds (Gibb, 1989). However, only hexavalent chromium is identified as a
9
carcinogen, Trivalent chromium is not considered carcinogenic (Biedermann and Landolph,
l987; Patierno, Banh, and Landolph, l988; Landolph, 1990, l994; Shi, 1999). Animal studies
and in vitro studies show that among chromium compounds, hexavalent chromium compounds
are the most potent in causing chromosomal aberrations and cancer (Norseth, 1981). Because of
their structural and chemical similarities to sulfates and phosphates, chromium(VI) compounds
can readily cross cellular membranes on the non-specific, sulfate-phosphate, anion transport
carriers (Alexander, 1995, Bagchi, 2002, Salnikow, 2008, and Saha, 2011). Chromium(III),
however, is poorly transported across cellular membranes (Bagchi, 2002). Thus, most Cr(III)
compounds are 1,000-fold less cytotoxic/toxic, less mutagenic, and less carcinogenic than
Cr(VI) compounds (Landolph and Biedermann, l990; Shi, 1999).
Inside the cell, however, hexavalent chromium is readily reduced first to Cr(V), then to
Cr(IV), then to trivalent chromium, suggesting that the trivalent form of chromium was the
active cytotoxin, mutagen, and carcinogen at the site of action in the cell (Norseth, 1981;
Landolph and Biedermann, l990). Outside the cell, Cr(VI) can be detoxified by its reduction to
Cr(III) and excreted. It is hypothesized that the biological effects of hexavalent chromium are
due to the reduction process, leading to generation of intracellular Cr(III), which binds to DNA
and causes mutations, and to intracellular Cr(V), which reduces molecular oxygen to generate
superoxide (O
2
-
) which dismutates to
hydrogen peroxide (H
2
O
2
). Hydrogen peroxide can then
bind to DNA, leading to formation of 8-hydroxy-guanosine and other oxidized DNA bases,
which lead to generation of mutations. In addition, hydrogen peroxide can be converted to
hydroxyl radicals (OH
•
), which can also bind to DNA, leading to generation of 8-hydroxy-
deoxyguanosine and other oxidized DNA bases in the DNA, and hence mutations. However, the
overall mechanism for chromium(VI)-induced carcinogenesis is still unknown and is being
10
intensely studied (Biedermann and Landolph, l987, l990; Patierno, Banh, and Landolph, l988;
Shi, 1999).
Although the exact mechanism by which Cr(VI) compounds cause carcinogenesis is
unknown, Cr(VI) has been shown to cause chromosomal aberrations, mutations, and
morphological and neoplastic transformation in cultured mammalian cells (Biedermann and
Landolph, l987, l990; Patierno, Banh, and Landolph, l988; Shi, 1999). Cr(VI) has also been
shown to cause DNA lesions such as strand breaks, ternary Cr-DNA adducts with biological
reducers (such as ascorbate and glutathione), DNA protein cross-links, and DNA base
modification (Saba, 2011, and Shi, 1999). As mentioned, Cr(VI) can cross the cell, in the form
of a chromate anion, on the non-specific, anion transport carrier (Alexander, 1995, Bagchi, 2002,
Salnikow, 2008, Saba, 2011, and Shi, 1999). However, Cr(VI) itself does not readily react with
DNA, so the uptake of Cr(VI) into the cell and its reduction to reactive, toxic, and mutagenic
intermediates inside the cell, including Cr(V), Cr(IV), and Cr(III), and the generation of
superoxide, hydrogen peroxide, and hydroxyl radicals thereafter is key to Cr(VI) carcinogenesis
and Cr(VI)-induced DNA damage and carcinogenesis (Shi, 1999).
The reactive chromium intermediates created during the reduction of Cr(VI) by cells have
been shown to generate reactive oxygen species (ROS), leading to formation of free radicals that
may cause DNA strand breaks, base modifications, lipid peroxidation, and nuclear transcription
factor (NFkB) activation (Krumschnabel, 2004, Saha, 2011 and Shi, 1999). The formation of
ROS causes injury to cellular proteins, lipids, and DNA (Patlolla, 2009). Although all oxidation
states of chromium may generate free radicals, Cr(VI) and Cr(IV) have the highest potency in
doing so (Shi, 1999). DNA strand breaks cause DNA damage in the cell. Hydroxyl radicals
produced from the reduction of Cr(VI) can cause base modifications, by reacting with guanine
11
residues to produce 8-hydroxy-deoxyguanosine and other oxidized DNA bases, which result in
further DNA damage (Qi, 2000 and Shi, 1999). The ROS generated from the reduction of
chromium may also cause lipid peroxidation, which has implications in cancer. Lipid
peroxidation can result in lipid-hydroperoxide-derived free radicals, which can cause site-
specific cleavage of double stranded DNA (reviewed in Shi, 1999). As mentioned, Cr(III) is
incapable of entering cells through the non-specific, anion channel. However, generation of
Cr(III) inside the cell by reduction of intracellular Cr(VI) results in Cr(III) reacting with lipid
hydroperoxides to produce more lipid hydroperoxide free radicals, which can cause further DNA
damage (Shi, 1999). The ROS generated from Cr(VI) reduction can also activate NF-kB, which
is known to be a transcription activator for the c-myc proto-oncogene (Shi, 1999). The
activation of NF-kB by Cr(III) formed from reduction of Cr(VI) has implications in Cr(VI)-
induced morphological and neoplastic transformation (reviewed in Shi, 1999). The ROS
generated from reduction of intracellular Cr(VI) and subsequent reduction of molecular oxygen
leads to continuous oxidative stress. This is likely one factor contributing to Cr(VI)-induced
carcinogenesis (reviewed in Shi, 1999).
1.3 The Role of Reductants in Cr(VI) Carcinogenesis
The ROS generated from Cr(VI)-generated reactive intermediates is likely one factor
contributing to Cr(VI)-induced carcinogenesis. Many reductants have been shown to reduce
Cr(VI) at physiological pH, including glutathione (GSH), ascorbate, cysteine, lipoic acid, and
diol-containing molecules (such as NADPH, ribose, fructose, and arabinose) (reviewed in Shi,
1999). Ascorbate and glutathione are the main contributors to Cr(VI) reduction in cellular
systems, in which both may act independently or synergistically to reduce Cr(VI) (Shi, 1999).
12
However, ascorbate is kinetically favored over GSH in reducing Cr(VI) (Shi, 1999), and
ascorbate is the primary reducer that activates Cr(VI) (Stearns, 1995). Ascorbate and GSH are
both known to be anti-oxidants in the cell, but they are also key players in the reductive
activation of Cr(VI). It seems paradoxical that both ascorbate and GSH can help protect the cell
against damage, but that they also enhance the damage caused by Cr(VI). However, the
activation of chromium(VI)-induced oxidative injury depends on the balance between these
reductants acting as pro-oxidants or anti-oxidants (reviewed in Shi, 1999). If the balance
between the pro-oxidant and anti-oxidant activity two favors the pro-oxidant action, chromium
(VI)-induced oxidative injury would occur (Shi, 1999).
1.4 Overall Goal of This Thesis Work
The goal in our laboratory is to use in vitro assays to investigate the molecular
mechanisms of Cr(VI)-induced morphological and neoplastic transformation of C3H/10T1/2 Cl
8 (10T1/2) mouse embryo cell lines. To do this, we have chosen to study the effects of
intracellular reductants on the cytotoxic activity of Cr(VI) compounds and on the ability of
Cr(VI) compounds to induce morphological and neoplastic cell transformations. We next wanted
to obtain dose-response curves for Cr(VI)-induced cytotoxicity to 10T1/2 cells. We first wanted
to measure the effect of intracellular reductants on Cr(VI)-induced cytotoxicity and to determine
at what doses, these intracellular reductants would enhance Cr(VI) induced cytotoxicity. After
obtaining good cytotoxicity data, we then wanted to obtain strong, dose-dependent
morphological (focus formation) and neoplastic cell transformation in 10T1/2 mouse embryo
cells. Our goal is to study the molecular mechanisms of induction of morphological and
neoplastic transformation to 10T1/2 cells by soluble Cr(VI) compounds and to develop dose-
13
response curves for Cr(VI)-induced morphological transformation. We eventually want to ring
clone foci of Cr(VI) induced morphologically transformed cells, create transformed cell lines,
and then characterize the biological properties of these transformed cell lines. Once we were
successful in this, we would then want to use DNA microarrays to molecularly characterize the
aberrations in gene expression that occur in Cr(VI)-induced transformed 10T1/2 cell lines.
14
CHAPTER II: MATERIALS AND METHODS
2.1 Chemicals
The chemicals used in these experiments were purchased from various commercial
pharmaceutical, biotechnology, and chemical companies. The sodium chromate used in the
experiments was purchased from the J.T. Baker Chemical Co. (Phillipsburg, NJ). The ascorbic
acid, reduced glutathione (GSH), and 3-methylcholanthrene (MCA, 98% purity) were purchased
from the Sigma-Aldrich Company, St. Louis, Missouri. The sodium chromate, ascorbic acid,
and glutathione were dissolved in Dubecco’s Phosphate-Buffered Saline 1X (DPBS)
immediately before use.
2.2 The C3H/10T½ CI 8 (10T½) Mouse Embryo Cell Culture Model
In our experiments, we used the C3H/10T1/2 Cl 8 cell line (referred to as 10T1/2 cells in
our experiments) established by Reznikoff, Brankow, and Heidelberger (Reznikoff, 1973a).
C3H10T1/2 Cl8 mouse embryo cells were isolated from C3H mouse embryos (Reznikoff,
1973a). 10T1/2 cells are stable in culture, are highly sensitive to post-confluence inhibition of
cell division, and exhibit anchorage dependent growth (Reznikoff, 1973a). 10T1/2 cells also
have a very low frequency of spontaneous transformation (Reznikoff, 1073a). Carcinogenic
chemicals such as 3-methylcholanthrene (MCA) cause dose-dependent cytotoxicity and produce
a dose-dependent morphological transformation (focus formation) and neoplastic transformation
(form progressively tumors when injected into immunosuppressed or nude mice (Reznikoff,
1973b). A certain fraction of morphologically transformed cell lines derived from carcinogen-
induced, cloned foci of 10T1/2 cells also produce fibrosarcomas when injected subcutaneously
into nude mice or immunosuppressed mice (Reznikoff, 1973b). 10T1/2 cells are a good in vitro
15
cell culture model to study the molecular and cellular mechanisms of carcinogen induction of
cytotoxicity (measured by decrease in decrease in colony survival) and chemical carcinogen-
induced morphological transformation, leading to fibrosarcomas when the transformed cells are
injected into nude mice.
2.3 Cell Culture Methods
For cell culture, the C3H/10T1/2 Cl 8 (10T1/2) mouse embryo fibroblastic cells were
grown in Basal Medium Eagle (BME) supplemented with 10% heat-inactivated Fetal Bovine
Serum (FBS). BME powder was purchased from the GIBCO Company (City, State), and the
FBS was purchased from Omega Scientific Products, Irvine, California. The BME was prepared
by Ms. Nily Harel in the Bioreagents Core Facility of the USC/Norris Comprehensive Cancer
Center at the University of Southern California (USC), under the administration of Dr. Zoltan
Tokes, Professor of Biolocemistry and Molecular Biology and Director of the Bioreagents Core
Facility at USC. The BME powder was dissolved in water, the pH of the solution was adjusted
to 7.2, and then the solution was filter-sterilized. 10T1/2 cells of passages from 7-15 were
previously frozen and stored in liquid nitrogen (Reznikoff et al, 1973a). 10T1/2 cells were
thawed and used as described in Reznikoff, et al., 1973. 10T1/2 cells were thawed in a water
bath at 37 degrees Celsius. The cells were then transferred into a 15 ml centrifuge tube (Corning
Glass Works, Corning, New York) and centrifuged for 12 minutes at 3,000 rpm in an IEC-HN-S
centrifuge (Damon/IEC Division). Once centrifuged, the supernatant was aspirated and
discarded, and the pellet was resuspended in 1 ml of BME containing 10% FBS. The cell
suspension was then transferred to a 25 cm
2
vented plastic T-flask (Corning Glass Works,
Corning, New York). The centrifuge tube containing the cell suspension was then rinsed with an
16
additional 4 ml of BME containing 10% FBS, and the rinse was transferred to T-flask containing
the cells. The flask of cells was placed in a Forma Scientific humidified incubator for 24 hours
at 37 degrees Celsius with an atmosphere of 5% CO
2
. After 24 hours, the medium in the flask
was aspirated and discarded to remove any residual DMSO and dead cells. Fresh medium was
then added to the flask, and the incubation was continued, with a medium change every three
days until the cells became approximately 80 percent confluent (Reznikoff et al, l973a;
Landolph and Heidelberger, l979; Patierno et al, l988; Landolph, l994).
2.4 Method to Determine Plating Efficiency of the Cells
Plating efficiency experiments were conducted using our standard laboratory protocols,
previously published by Dr. Charles Heidelberger’s laboratory and our laboratory (Reznikoff et
al, l973a, b; Landolph and Heidelberger, l979; Miura et al, l987; Patierno et al, l988). The
plating efficiency experiments were conducted once the 10T1/2 cells from passages 7-15 reached
80 percent confluence. First, the medium from the flask of cells was aspirated, and 1ml of
Dulbecco’s Phosphate Buffered Saline 1X (DPBS) was added to the flask to remove serum
trypsin inhibitors and dead cells. The DPBS was then aspirated, and 1ml of trypsin was added to
the flask to detach the cells. Once most of the cells detached from the flask, 1 ml of BME
containing 10% FBS was added to neutralize the action of the trypsin. The cell suspension was
then transferred to a 15 ml centrifuge tube and centrifuged for 12 minutes at 3,000 rpm in the
IEC-HN-S table top centrifuge (Damon/IEC Division). After centrifugation, the supernatant was
aspirated and the pellet was resuspended in 10ml of BME containing 1% FBS. 1 ml of
resuspension was mixed with 19 ml of DPBS in a Coulter counter vial and the number of cells in
½ ml of the dilution was counted electronically in a Coulter Counter Model Zf (Coulter
17
Electronics, Hialeah, Florida). The cells were then diluted to a concentration of 200,000/5 mls,
and then three more times serially, 1/10 each time. The cells were then seeded into 60-mm
Corning Petri Dishes in 5 ml of medium (BME containing 10% FBS) at 200 cells/60 mm dish,
with five dishes for each plating efficiency of each condition assayed.
2.5 Assays for Chemically Induced Cytotoxicity
Determining the Non-Cytotoxic Concentrations of Ascorbic Acid to 10T1/2 Cells
To determine the highest, non-cytotoxic concentrations of ascorbic acid that can be used
for experiments, an assay to determine the effect of ascorbic acid on the survival of 10T1/2 cells
was first performed. 10T1/2 cells were seeded at 200 cells/60mm dish, with five dishes for each
concentration of ascorbic acid to be tested along with controls. The cells were seeded according
to the plating efficiency procedure as described previously in section 2.4.
The concentrations of ascorbic acid tested were 0.00625 mM, 0.0125 mM, 0.025 mM,
0.05 mM, 0.1 mM, 0.25 mM, and 0.375 mM. There were also two controls: a no addition
control and a DPBS control. DPBS was first filter-sterilized through 0.2 µM Nalgene filter, and
the first 50 mls was discarded in case it contained any plastic residue from the filter. Then, the
ascorbic acid was dissolved in DPBS, and the stock ascorbic acid/DPBS solution was filtered
through the same Nalgene filter before being used to treat the cells. A dilution was performed to
obtain the appropriate ascorbic acid concentrations, and each concentration was administered in
100 µL to the appropriate dishes containing 200 cells/60 mm dish and 5 ml of BME containing
10% FBS
18
There were two methods of ascorbic acid treatment. The first method was to seed the
cells, and then treat them 24 hours later with ascorbic acid concentrations of 0.00625 mM,
0.0125 mM, 0.025 mM, 0.05 mM, 0.1 mM, 0.25 mM, and 0.375 mM, along with the no addition
and DPBS controls. The dishes were incubated at 37 degrees Celsius, in constant flow
humidified carbon dioxide incubators (Forma Scientific Company) with an atmosphere of 5%
CO
2
. After 48 hours, the medium was aspirated from each dish, and 5 ml of fresh medium was
added, along with ascorbic acid to the appropriate dishes and DPBS to the DPBS control. The
dishes were then incubated again for 7 days, for 10 days of incubation time total, and then the
cells were fixed and stained (the method to fix and stain the cells will be described later).
The second method was similar to the first, but ascorbic acid was added the same day the
cells were seeded. The second method required that cells were seeded and treated with ascorbic
acid thereafter, at ascorbic acid concentrations of 0.00625 mM, 0.0125 mM, 0.025 mM, 0.05
mM, 0.1 mM, 0.25 mM, and 0.375 mM. The same controls were also used: no addition and
DPBS. The dishes were incubated at 37 degrees Celsius, in humidified incubation with an
atmosphere of 5% CO
2
. After 72 hours, the medium from each dish was removed by aspiration,
and fresh medium was added, along with ascorbic acid, to the appropriate dishes and DPBS to
the DPBS control. The dishes were then incubated again for 7 days before the cells were fixed
and stained.
19
Determining the Effect of Ascorbic Acid upon the Survival of 10T1/2 Cells Treated with Sodium
Chromate (Na
2
CrO
4
)
Once the appropriate concentration of ascorbic acid was selected for experimentation, we
tested the effect of ascorbic acid on the survival of sodium chromate-treated 10T1/2 cells.
10T1/2 cells were seeded at 200 cells/60 mm dish, with five dishes for each treatment to be
tested along with controls. Again, the cells were seeded according to the plating efficiency
procedure as described previously in section 2.4.
For this assay, cells were treated with sodium chromate alone, at concentrations of 10µM,
5µM, 4µM, 3µM, 2µM, and 1µM. There were also dishes treated with sodium chromate with
0.0125 mM ascorbic acid, using the same concentrations of sodium chromate. There were also
three controls: No addition, DPBS, and 0.0125 mM ascorbic acid only. The sodium chromate
was dissolved in DPBS that had been filtered through a 0.2 µM Nalgene filter, and the stock
sodium chromate/DPBS solution was filtered through the same Nalgene filter before being used
to treat the cells. Similarly, the ascorbic acid was also dissolved in DPBS that had been filtered
through 0.2 µM Nalgene filter, and the stock ascorbic acid/DPBS solution was filtered through
the same Nalgene filter before being used to treat the cells. Dishes that contained sodium
chromate treatments without ascorbic acid were treated with 50 µl of each treatment plus 50µl of
DPBS. Dishes treated with sodium chromate plus 0.0125 mM ascorbic acid were treated with 50
µl of each sodium chromate treatment plus 50µl of 0.0125 mM ascorbic acid. The no addition
control was left untreated, the DPBS control was treated with 100µl of DPBS, and the 0.0125
mM ascorbic acid control was treated with 50µl of 0.0125 mM ascorbic acid plus 50µl of DPBS.
20
There were two methods of performing this assay. In the first method, 10T1/2 cells were
seeded and then treated 24 hours later with the treatments described earlier. The dishes were then
incubated at 37 degrees Celsius in an atmosphere of 5% CO
2
. After 48 hours, the medium from
each dish was aspirated, and fresh medium was added. The dishes originally treated with sodium
chromate without ascorbic acid were treated with 100µl of filtered DPBS. The dishes originally
treated with sodium chromate with 0.0125 mM ascorbic acid were treated with 50 µl of 0.0125
mM ascorbic acid and 50µl of filtered DPBS. The no addition control was left untreated, the
DPBS control was treated with 100µl of DPBS, and the 0.0125 mM ascorbic acid control was
treated with 50µl of 0.0125 mM ascorbic acid and 50µl of DPBS. The cells were then incubated
for seven days before being fixed and stained.
In the second method, the 10T1/2 cells were first seeded, and treated with either 0.0125
mM ascorbic acid or DPBS thereafter. The dishes meant to be treated with sodium chromate
without ascorbic acid were treated with 50 µl DPBS upon seeding, while the dishes meant to be
treated with sodium chromate with ascorbic acid were treated with 50 µl of 0.0125 mM ascorbic
acid. The no addition control was left untreated, the DPBS control was treated with 100µl of
DPBS, and the 0.0125 mM ascorbic acid control was treated with 50µl of 0.0125 mM ascorbic
acid. This treatment allows for the dishes to be pre-treated with ascorbic acid before adding the
sodium chromate. The dishes were incubated at 37 degrees Celsius in an atmosphere of 5% CO
2
.
After 24 hours, the dishes were treated with the 50 µl of the appropriate sodium chromate
treatment. The ascorbic acid control was also treated with 50 µL of DPBS. The dishes were
then incubated for 48 hours. After 48 hours, the medium from each dish was aspirated, and the
cells were retreated with either 0.0125 mM ascorbic acid or DPBS. The dishes originally treated
with sodium chromate without ascorbic acid were re-treated with 100 µl of DPBS, and the dishes
21
originally treated with sodium chromate with 0.0125 mM ascorbic acid were re-treated with 50µl
of ascorbic acid and 50 µl of DPBS. The dishes were then incubated for seven more days before
the cells were fixed and stained and the colonies were scored.
Determining the Highest Non-Cytotoxic Concentration of Glutathione (GSH) to 10T1/2 Cells
Similarly to ascorbic acid, we decided to determine the highest non-cytotoxic
concentration of glutathione (GSH) before testing the effects of glutathione upon the cytotoxicity
of sodium chromate to 10T1/2 Cells. To determine the highest non-cytotoxic concentration of
glutathione (GSH) that could be used for experiments, assays to determine the cytotoxicity of
glutathione to 10T1/2 cells were first performed. 10T1/2 cells were seeded at 200 cells/dish, with
five dishes for each concentration of glutathione (GSH) to be tested, along with controls. Cells
were again seeded according to the plating efficiency procedure as described previously in
section 2.4
Three separate GSH cytotoxicity experiments were performed, beginning with a wide
range of GSH concentrations, and narrowing down the range after the results of each experiment
were obtained. The GSH was dissolved in DPBS that had been filtered through a 0.2 µM
Nalgene filter, and the stock GSH/DPBS solution was filtered through the same Nalgene filter
before being used to treat the cells. A dilution was performed to obtain the appropriate GSH
concentrations, and each concentration was administered in 100 µL to the appropriate dishes.
The concentrations of GSH tested in the first cytotoxicity were 0.001 mM, 0.01 mM, 0.1 mM, 1
mM, and 10mM, along with a no addition control and a DPBS control. The concentrations of
GSH tested in the second cytotoxicity experiment were 0.1 mM, 0.25 mM, 0.5 mM, 0.75 mM,
22
and 1 mM. The concentrations of GSH tested in the third cytotoxicity experiment were 0.1 mM,
0.2mM, 0.3mM, 0.4mM, and 0.5 mM.
For each GSH cytotoxicity assay, only one method was used to carry out the experiments.
10T1/2 cells were seeded and then treated 24 hours later with the treatments described earlier.
The dishes were then incubated at 37 degrees Celsius in an atmosphere of 5% CO
2
. After 48
hours, the medium from each dish was aspirated and fresh medium was added. The dishes were
then retreated with the appropriate GSH concentrations, as described above. The dishes were
incubated for seven days more, and then the cells were fixed with methanol and stained with
crystal violet.
Effects of Glutathione upon the Survival of 10T1/2 Cells Treated with Sodium Chromate
(Na
2
CrO
4
)
Once the appropriate concentration of GSH was selected for experimentation, we tested
the effect of GSH upon the survival of sodium chromate treated 10T1/2 cells. 10T1/2 cells were
seeded at 200 cells/60 mm dish, with five dishes for each treatment to be tested along with
controls. Again, the cells were seeded according to the plating efficiency procedure as described
previously in section 2.4.
For this assay, there were dishes treated with sodium chromate alone, at concentrations of
10µM, 5µM, 4µM, 3µM, 2µM, and 1µM. There were also dishes treated with sodium chromate
and 0.1 mM GSH, using the same concentrations of sodium chromate. There were also three
controls: No addition, DPBS, and 0.1 mM GSH only. Sodium chromate was dissolved in DPBS
that had been filtered through 0.2 µM Nalgene filter, and the stock sodium chromate/DPBS
23
solution was filtered through the same Nalgene filter before being used to treat the cells.
Similarly, GSH was also dissolved in DPBS that had been filtered through a 0.2 µM Nalgene
filter, and the stock GSH/DPBS solution was then filtered through the same Nalgene filter before
being used to treat the cells. Dishes that contained sodium chromate treatments without GSH
were treated with 50µl of each treatment plus 50 µl of DPBS. Dishes treated with sodium
chromate plus 0.1 mM GSH were treated with 50 µl of each sodium chromate plus 50 µl of 0.1
mM GSH. The no addition control was left untreated, the DPBS control was treated with 100µl
of DPBS, and the 0.1 mM GSH control was treated with 50 µl of 0.1 mM GSH plus 50 µl of
DPBS.
10T1/2 cells were seeded and then treated 24 hours later with the treatments described
earlier. The dishes were then incubated at 37 degrees Celsius in an atmosphere of 5% CO
2
.
After 48 hours, the medium from each dish was aspirated and fresh medium was added. The
dishes originally treated with sodium chromate without GSH were treated with 100 µl of filtered
DPBS. The dishes originally treated with sodium chromate with 0.1 mM GSH were next treated
with 50 µl of 0.1 mM GSH and 50 µl of filtered DPBS. The no addition control was left
untreated, the DPBS control was treated with 100 µl of DPBS, and the 0.1 mM GSH control was
treated with 50 µl of 0.1 mM GSH and 50 µl of DPBS. The cells were then incubated for an
additional seven days, making the duration of this experiment, at which point the cells were fixed
with methanol for one hour and stained with 1% crystal violet for 1 hour.
24
Fixing and Staining the 10T1/2 Cells to Observe Colony Formation
After a period of ten days from the day of seeding, each experiment measuring the
chemically induced cytotoxicity was ready to be fixed and stained to observe colony formation.
The medium in each dish was first aspirated. Each dish was then washed with DPBS to remove
the serum and dead and dying cells. Methanol was then added to each dish for 45 minutes to fix
the cells, then aspirated, and 1% crystal violet was added thereafter for 1 hour and 30 minutes to
stain the cells. Once the cells were stained, the dishes were washed and left to air dry. Colonies
of 20 cells or more were scored under a dissecting microscope as viable (Reznikoff et al,
1973a,b; Landolph and Heidelberger, l979; Miura et al, 1987; Patierno et al, 1988).
2.6 Assays for Chemically Induced Morphological Transformation
Determining the effect of Ascorbic Acid upon the Yield of Morphological Cell Transformation
(Foci) Induced by Sodium Chromate (Na
2
CrO
4
) in Treated 10T1/2 Cells
Next, we determined whether or not ascorbic acid could enhance morphological
transformation (focus formation) induced by sodium chromate in 10T1/2 cells. To do this we
determined the yield of type II and type III foci induced in 10T1/2 cells treated with sodium
chromate and ascorbic acid. . For this transformation assay, some dishes were treated with
sodium chromate alone, at concentrations of 0.5µM, 1µM, 2µM, and 4µM. Some dishes were
also treated with sodium chromate and also with 0.0125 mM ascorbic acid. There were also 5
controls: No addition, DPBS, 0.0125 mM ascorbic acid, MCA (3-methylcholanthrene), and
25
acetone (0.5% v/v). Sodium chromate was dissolved in DPBS that had been filtered through a
0.2 µM Nalgene filter, and the stock sodium chromate/DPBS solution was filtered through the
same 0.2 µM Nalgene filter before being used to treat the cells. Similarly, the ascorbic acid was
also dissolved in DPBS that had been filtered through a 0.2 µM Nalgene filter. The stock
ascorbic acid/DPBS solution was filtered through the same 0.2 µM Nalgene filter before being
used to treat the cells. Dishes that contained sodium chromate treatments without ascorbic acid
were treated with 50 µl of each treatment plus 50µl of DPBS. Dishes treated with sodium
chromate plus 0.0125 mM ascorbic acid were treated with 50 µl of each sodium chromate plus
50 µl of 0.0125 mM ascorbic acid. The no addition control was left untreated, and the DPBS
control was treated with 100 µl of DPBS. The 0.0125 mM ascorbic acid control was treated with
50 µl of 0.0125mM Ascorbic acid plus 50 µl of DPBS, and the 1 µg/ml MCA control was treated
with 25 µl of a solution of 0.2 µg/ml of MCA dissolved in acetone, and the acetone control was
treated with 25 µl of acetone. MCA is a strong chemical mutagen and carcinogen, and a final
concentration of 1µg/ml in the dishes was used as a positive control for the transformation assay.
To begin the transformation assay, first, 2,000 10T1/2 cells were seeded cells/5ml of
medium in each 60 mm dish, with twenty dishes for each treatment to be tested along with
controls. The cells were seeded according to the plating efficiency procedure as described
previously in section 2.4, but the calculations were done accordingly to seed 2,000 cells/5ml of
medium in each dish rather than 200 cells/5ml of medium dish. The dishes were then incubated
at 37 degrees Celsius in an atmosphere of 5% CO
2
.
Four days after seeding, some of the dishes were treated with 0.0125 mM ascorbic acid
and some with DPBS. The dishes meant to be treated with sodium chromate without ascorbic
acid were treated with 50 µl DPBS, while the dishes meant to be treated with sodium chromate
26
plus ascorbic acid were treated with 50 µl of ascorbic acid. The no addition control, MCA
control, and acetone control were left untreated. The DPBS control was treated with 100 µl of
DPBS, and the 0.0125 mM ascorbic acid control was treated with 50 µl of 0.0125 mM ascorbic
acid. This treatment allowed for the dishes to be pre-treated with ascorbic acid before adding the
sodium chromate. The dishes were incubated at 37 degrees Celsius in an atmosphere of 5% CO
2
in a humidified Forma Scientific
incubator. After 24 hours, the appropriate dishes were treated
with the 50µl of the appropriate sodium chromate treatment. The ascorbic acid control was also
treated with 50 µL of DPBS. The dishes were then incubated for 48 hours. After 48 hours, the
medium from each dish was aspirated, and the cells were re-treated with either 0.0125 mM
ascorbic acid or DPBS (negative control). The dishes originally treated with sodium chromate
without ascorbic acid were re-treated with 100 µl of DPBS. The dishes originally treated with
sodium chromate with 0.0125mM ascorbic acid were re-treated with 50µl of ascorbic acid and
50 µl of DPBS. The dishes were then incubated at the same conditions as before.
The medium change was performed twice a week until cells became confluent, then once
per week for 6 weeks for each dish. Each medium change consisted of aspirating the medium
from each dish and then adding 5 ml of fresh medium (BME containing 10% FBS) to each dish.
After each medium change, the dishes originally containing sodium chromate treatment without
ascorbic acid were treated with 100 µl of DPBS. The dishes originally containing the sodium
chromate treatment with 0.0125mM ascorbic acid were treated with 50 µl of 0.0125mM ascorbic
acid plus 50 µl DPBS. The 0.0125 mM ascorbic acid control was treated with 50 µl of
0.0125mM ascorbic acid plus 50 µl of DPBS, and the DPBS control was treated with 100 µl of
DPBS. The no addition control, 1µg/ml MCA control, and acetone control were left untreated.
At the end of the sixth week of the transformation assay, the dishes were fixed and stained.
27
The medium from each dish was aspirated, and each dish was rinsed with room temperature
DPBS. Each dish was then fixed with methanol for 45 minutes, and then stained with Giemsa
for 1 hour and 30 minutes. Each dish was scored for foci under a dissecting microscope
according to standard methods used in our laboratory and other laboratories (Reznikoff et al,
1973b).Landolph and Heidelberger, l979; reviewed in Landolph, l985; Miura et al, l987; Patierno
et al, l988; Landolph, l994).
28
CHAPTER III: RESULTS
3.1 Effect of Ascorbic Acid on the Survival of 10T1/2 Cells
Before determining the effects of ascorbic acid on the survival and morphological
transformation of 10T1/2 cells treated with sodium chromate, the highest non-cytotoxic
concentrations of ascorbic acid to 10T1/2 cells needed to be determined. By determining the
highest non-cytotoxic concentrations, we could then choose a concentration of ascorbic acid that
is not cytotoxic to be used in our sodium chromate cytotoxicity and transformation assays. As
mentioned earlier, there were two ways to perform this assay, with both methods utilizing
concentrations of ascorbic acid between 0.00625 mM to 0.375 mM.
Ascorbic Acid Cytotoxic Effects Determined with Method 1
In method 1, we seeded 10T1/2 cells and treated them with ascorbic acid 24 hours later.
Two experiments were performed using this method, in which the plating efficiencies and
survival fractions (relative plating efficiencies) are summarized in Table 1 and Table 2 below.
29
Table 1: Survival Fractions Determined from the Effect of 0.00625mM to 0.375mM
Ascorbic Acid to 10T1/2 Cells (Cells Treated 24 Hours After Seeding). The results shown are
from two experiments measuring the cytotoxicities of ascorbic acid to 10T1/2 cells using
concentrations of ascorbic acid ranging from 000625 mM to 0.375 mM. The data collected show
the individual and average (avg.) survival fractions of the treated 10T1/2 cells +/- their standard
deviations (SD).
Treatment
Experiment 1 Survival
Fraction ± SD
Experiment 2 Survival
Fraction ± SD
Average Survival
Fraction ± SD
No Addition 0.96 ± 0.03 0.90 ± 0.05 0.97 ± 0.04
0 mM Ascorbic Acid 1.00 ± 0.04 1.00 ± 0.06 1.00 ± 0.05
0.00625 mM Ascorbic Acid 0.87 ± 0.11 0.95 ± 0.10 0.91 ± 0.10
0.0125 mM Ascorbic Acid 0.85 ± 0.08 0.94 ± 0.09 0.89 ± 0.08
0.025 mM Ascorbic Acid 0.81 ± 0.09 0.93 ± 0.07 0.87 ± 0.08
0.05 mM Ascorbic Acid 0.77 ± 0.03 0.84 ± 0.05 0.81 ± 0.04
0.1 mM Ascorbic Acid 0.79 ± 0.06 0.84 ± 0.04 0.82 ± 0.05
0.25 mM Ascorbic Acid 0.73 ± 0.08 0.68 ± 0.03 0.71 ± 0.05
0.375 mM Ascorbic Acid 0.61 ± 0.11 0.59 ± 0.11 0.60 ± 0.11
Table 2: Plating Efficiencies Determined from the Effect of 0.00625 mM to 0.375 mM
Ascorbic Acid to 10T1/2 Cells (Cells Treated 24 Hours After Seeding). The results shown are
from two experiments measuring the cytotoxicities of ascorbic acid to 10T1/2 cells using
concentrations of ascorbic acid ranging from 0.000625 mM to 0.375 mM. The data collected
show the individual and average (avg) plating efficiencies (PE) of the treated 10T1/2 cells +/-
their standard deviations (SD).
Treatment Experiment 1 PE ± SD Experiment 2 PE ± SD Average PE ± SD
No Addition 38.1 ± 1.2 34.3 ± 1.8 36.2 ± 1.5
0 mM Ascorbic Acid 39.7 ± 1.6 34.7 ± 2.2 37.2 ± 1.9
0.00625 mM Ascorbic Acid 34.5 ± 4.4 33 ± 3.4 33.75 ± 3.9
0.0125 mM Ascorbic Acid 33.7 ± 3.0 32.6 ± 3.1 33.15 ± 3.0
0.025 mM Ascorbic Acid 32.1 ± 3.5 32.2 ± 2.6 32.15 ± 3.0
0.05 mM Ascorbic Acid 30.5 ± 1.4 29.3 ± 1.6 29.9 ± 1.5
0.1 mM Ascorbic Acid 31.4 ± 2.5 29.2 ± 1.5 30.3 ± 2.0
0.25 mM Ascorbic Acid 29.1 ± 3.0 23.6 ± 1.1 26.35 ± 2.1
0.375 mM Ascorbic Acid 24.4 ± 4.2 20.3 ± 4.0 22.35 ± 4.1
30
In experiment 1, the survival fractions of 10T1/2 cells treated with ascorbic acid
concentrations of 0 mM, 0.00625 mM, 0.0125 mM, 0.025 mM, 0.05 mM, 0.1 mM, 0.25 mM,
and 0.375 mM were 1.00 ± 0.04, 0.87 ± 0.11, 0.85 ± 0.08, 0.81 ± 0.09, 0.77 ± 0.03, 0.79 ± 0.06,
0.73 ± 0.08, 0.61 ± 0.11, respectively, as shown in Table 1. The survival fractions are plotted in
a graphical representation in Figure 1. As seen in Table 1 and Figure 1, the survival fractions of
the 10T1/2 cells decreased in a dose-dependent manner with increasing concentrations of
ascorbic acid. The ascorbic acid was weakly cytotoxic at lower concentrations (0.0625 mM to
0.0125 mM). The ascorbic acid was much more cytotoxic at concentrations of 0.25 mM and
0.375 mM.
Figure 1: This figure shows the graphical representation of the survival fractions +/- standard
deviations of 10T1/2 cells treated with ascorbic acid 24 hours after seeding in experiment 1. This
figure shows the survival fraction (on a logarithmic scale) of 10T1/2 cells versus the
concentration of ascorbic acid used to treat the cells.
0.5
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4
Log (Survival Fraction of 10T1/2 Cells)
Concentration of Ascorbic Acid (mM)
Experiment 1: Effect of Ascorbic Acid on the Survival of
10T1/2 Cells (Cells Treated 24 Hours After Seeding)
Ascorbic Acid Treatment (Cells Treated 24 Hours After Seeding) No Addition Control
31
In experiment 2, the survival fractions of 10T1/2 cells treated with ascorbic acid
concentrations of 0 mM, 0.00625 mM, 0.0125 mM, 0.025 mM, 0.05 mM, 0.1 mM, 0.25 mM,
and 0.375 mM were 1 ± 0.06, 0.95 ± 0.10, 0.94 ± 0.09, 0.93 ± 0.07, 0.84 ± 0.05, 0.84 ± 0.04,
0.68 ± 0.03, and 0.59 ± 0.11, respectively (Table 1). The survival fractions are plotted in
graphical representation in Figure 2 below. Again, as seen in Table 1 and Figure 2, the survival
fractions of the 10T1/2 cells decreased in a dose-dependent manner with increasing
concentrations of ascorbic acid. Ascorbic acid was weakly cytotoxic at lower concentrations
(0.0625 mM to 0.0125 mM) and more strongly cytotoxic at concentrations of 0.25 mM and
0.375 mM.
Figure 2: This figure is a graphical representation of the survival fractions +/- standard
deviations of 10T1/2 cells treated with ascorbic acid 24 hours after seeding the cells in
experiment 2. This figure shows the survival fraction (on a logarithmic scale) of 10T1/2 cells
versus the concentration of ascorbic acid used to treat the cells.
0.5
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4
Log (Survival Fraction of 10T1/2 Cells)
Concentration of Ascorbic Acid (mM)
Experiment 2: Effect of Ascorbic Acid on the Survival of
10T1/2 Cells (Cells Treated 24 Hours After Seeding)
Ascorbic Acid Treatment (Cells Treated 24 Hours After Seeding) No Addition Control
32
The survival fractions obtained from experiments 1 and 2 were averaged in Table 1 and
plotted in figure 3. Figure 3 showed that the results from experiments 1 and 2 were very
reproducible. Again, the averaged survival fractions of the 10T1/2 cells decreased in a dose-
dependent manner with increasing concentrations of ascorbic acid, as seen in Table 1 and Figure
3. Ascorbic acid was weakly cytotoxic at lower concentrations (0.00625 mM to 0.0125 mM).
Ascorbic acid was more strongly cytotoxic at concentrations of 0.25 mM and 0.375 mM. From
the average of the data produced in two separate experiments, it can be concluded that ascorbic
acid is weakly cytotoxic to 10T1/2 cells from concentrations of 0.00625 mM to 0.1 mM and is
more strongly cytotoxic at concentrations above 0.1 mM.
Figure 3: This figure is a graphical representation of the survival fractions +/- standard
deviations of 10T1/2 cells treated with ascorbic acid 24 hours after seeding from experiments 1
and 2. This figure shows the survival fraction (on a logarithmic scale) of 10T1/2 cells versus the
concentration of ascorbic acid used to treat the cells.
0.1
1
10
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4
Log (Survival Fraction of 10T1/2 Cells)
Concentration of Ascorbic Acid (mM)
Effect of Ascorbic Acid on the Survival of 10T1/2 Cells
(Cells Treated 24 Hours After Seeding)
Experiment 1 Experiment 2 Average No Addition Control
33
Ascorbic Acid Effect with Method 2
In method 2, we seeded 10T1/2 cells and treated them with ascorbic acid immediately
upon seeding the cells. One experiment was performed using this method, in which the survival
fractions (relative plating efficiencies) and plating efficiencies are summarized in Table 3 below.
Table 3: Plating Efficiencies and Survival Fractions Determined from the Effect of
0.00625mM to 0.375mM Ascorbic Acid to 10T1/2 Cells (Cells Treated Upon Seeding). The
results shown are from an experiment measuring the cytotoxicities of ascorbic acid to 10T1/2
cells using concentrations of ascorbic acid ranging from 000625 mM to 0.375 mM. The data
collected show the individual and average (avg.) plating efficiencies (PE) and survival fractions
of the treated 10T1/2 cells +/- their standard deviations (SD).
Treatment PE ± SD Survival Fraction ± SD
No Addition 20.8 ± 1.7 1.0 ± 0.1
0 mM Ascorbic Acid 20.2 ± 2.9 1.0 ± 0.1
0.00625 mM Ascorbic Acid 21.1 ± 1.1 1.0 ± 0.1
0.0125 mM Ascorbic Acid 19.7 ± 1.5 1.0 ± 0.1
0.025 mM Ascorbic Acid 18.5 ± 1.2 0.9 ± 0.1
0.05 mM Ascorbic Acid 15.7 ± 0.9 0.8 ± 0.04
0.1 mM Ascorbic Acid 16.4 ± 1.4 0.8 ± 0.1
0.25 mM Ascorbic Acid 5 ± 3.0 0.2 ± 0.1
0.375 mM Ascorbic Acid 0.8 ± 0.8 0.04 ± 0.04
The survival fractions for the 10T1/2 cells treated with ascorbic acid concentrations of 0
mM, 0.00625 mM, 0.0125 mM, 0.025 mM, 0.05 mM, 0.1 mM, 0.25 mM, and 0.375 mM were
1.0 ± 0.1, 1.0 ± 0.1, 1.0 ± 0.1, 1.0 ± 0.1, 0.9 ± 0.1, 0.8 ± 0.04, 0.8 ± 0.1, 0.2 ± 0.1 and 0.04 ±
0.04, respectively (Table 3). The data is presented in graphical representation in figure 4 below.
As noted from Table 3 and Figure 4, ascorbic acid was non-cytotoxic at concentrations from
0.00625 mM to 0.025 mM, moderately cytotoxic at concentrations from 0.025 mM to 0.1 mM,
and extremely cytotoxic at concentrations of 0.25 mM and 0.375 mM (survival fractions are 0.2
and 0.014, respectively). The survival fractions observed in method 2 are similar to method 1
when using concentrations of ascorbic acid between 0.00625 mM to 0.1 mM. However, the
34
ascorbic acid added by method 2 was much more cytotoxic to the 10T1/2 cells at the higher
concentrations, 0.25 mM and 0.375 mM, as compared to the same concentrations added to cells
by method 1.
Figure 4: This figure is a graphical representation of the survival fractions +/- standard
deviations of 10T1/2 cells treated with ascorbic acid upon after seeding. This figure shows the
survival fraction (on a logarithmic scale) of 10T1/2 cells versus the concentration of ascorbic
acid used to treat the cells.
0.001
0.01
0.1
1
10
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4
Log(Survival Fraction of 10T1/2 Cells)
Concentration of Ascorbic Acid (mM)
Ascorbic Acid Cytotoxicity to 10T1/2 Cells (Cells Treated
Upon Seeding)
Ascorbic Acid Treatment (Cells Treated Upon Seeding) No Addition Control
35
3.2 Effect of Ascorbic Acid on the Survival of 10T1/2 Cells Treated with Sodium Chromate
In section 3.1, we determined that ascorbic acid was not cytotoxic to 10T1/2 cells when
administered at concentrations between 0.00625 mM to 0.1 mM. Among these non-cytotoxic
concentrations, we decided to use an ascorbic acid concentration of 0.0125 mM as the highest
non-cytotoxic concentration of ascorbic acid. This concentration was chosen because cells
treated with this concentration in the past had shown no cytotoxicity, while this concentration of
ascorbate enhanced cytotoxicity to 10T1/2 cells when they were treated with soluble chromate
compounds, without being cytotoxic on its own (Shahin, 2014). As mentioned earlier in section
2.5, there were two methods to perform this assay.
Ascorbic Acid Effect with Method 1
In method 1, we seeded 10T1/2 cells and treated them 24 hours later with a) sodium
chromate in the absence of ascorbic acid, and b) sodium chromate in presence of 0.0125 mM
ascorbic acid. In method 1, the cells were treated with ascorbic acid and sodium chromate at the
same time. There were two experimental trials performed for this method, in which the survival
fractions and plating efficiencies are summarized in Tables 4 and 5 below, respectively.
36
Table 4: Survival Fractions Determined from the Effect of 0.0125 mM Ascorbic Acid on
10T1/2 Cells Treated with Sodium Chromate. The results shown are from two experiments
measuring the survival of 10T1/2 cells when treated with a) sodium chromate alone and b)
sodium chromate with 0.0125 mM ascorbic acid. The concentrations of sodium chromate used
ranged from 1 µM to 10 µM. The data collected showed the individual and average (avg)
survival fractions of the treated 10T1/2 cells +/- their standard deviations (SD).
Treatment
Experiment 1
Survival
Fraction ± SD
Experiment 2
Survival
Fraction ± SD
Experiment 3
Survival
Fraction ± SD
No Addition 1.1 ± 0.1 1.1 ± 0.1 1.1 ± 0.1
Sodium Chromate Treatment Without Ascorbic Acid
0 µM Na
2
CrO
4
1 ± 0.02 1 ± 0.1 1 ± 0.1
1 µM Na
2
CrO
4
0.8 ± 0.1 0.9 ± 0.2 0.9 ± 0.1
2 µM Na
2
CrO
4
0.8 ± 0.03 0.9 ± 0.2 0.8 ± 0.1
3 µM Na
2
CrO
4
0.5 ± 0.1 0.5 ± 0.1 0.5 ± 0.1
4 µM Na
2
CrO
4
0.4 ± 0.1 0.2 ± 0.06 0.3 ± 0.1
5 µM Na
2
CrO
4
0.2 ± 0.04 0.002 ± 0.004 0.1 ± 0.02
10 µM Na
2
CrO
4
<0.002 <0.002 <0.002
Sodium Chromate Treatment With Ascorbic Acid
0 µM Na
2
CrO
4
and 0.0125 mM Ascorbic Acid 0.9 ± 0.1 1.0 ± 0.1 1.0 ± 0.09
1 µM Na
2
CrO
4
and 0.012 5 mM Ascorbic Acid 0.8 ± 0.1 0.9 ± 0.1 0.8 ± 0.1
2 µM Na
2
CrO
4
and 0.0125 mM Ascorbic Acid 0.7 ± 0.04 0.9 ± 0.1 0.8 ± 0.1
3 µM Na
2
CrO
4
and 0.0125 mM Ascorbic Acid 0.6 ± 0.1 0.5 ± 0.04 0.5 ± 0.05
4 µM Na
2
CrO
4
and 0.012 5 mM Ascorbic Acid 0.7 ± 0.1 0.2 ± 0.03 0.4 ± 0.04
5 µM Na
2
CrO
4
and 0.0125 mM Ascorbic Acid 0.5 ± 0.1 0.1 ± 0.1 0.3 ± 0.1
10 µM Na
2
CrO
4
and 0.012 5 mM Ascorbic Acid 0.03 ± 0.03 <0.002 0.02 ± 0.02
37
Table 5: Plating Efficiencies Determined from the Effect of 0.0125 mM Ascorbic Acid on
10T1/2 Cells Treated with Sodium Chromate. The results shown are from 2 experiments
measuring the plating efficiencies of 10T1/2 cells when treated with sodium chromate alone and
sodium chromate with 0.0125 mM ascorbic acid. The concentrations of sodium chromate used
ranged from 0µ M to 10 µM. The data collected showed the individual and average (avg) plating
efficiencies (PE) of the treated 10T1/2 cells +/- their standard deviations (SD).
Treatment Experiment 1
PE ± SD
Experiment 2
PE ± SD
Average PE
± SD
No Addition 52 ± 3.9 57.9 ± 3.3 54.9 ± 3.6
Sodium Chromate Treatment Without Ascorbic Acid
0 µM Na
2
CrO
4
48.8 ± 1.1 53.7 ± 7.4 51.3 ± 4.2
1 µM Na
2
CrO
4
39.5 ± 2.6 49.6 ± 8.1 44.6 ± 5.4
2 µM Na
2
CrO
4
37.6 ± 1.7 47.5 ± 9.4 42.6 ± 5.6
3 µM Na
2
CrO
4
24.4 ± 3.0 24.8 ± 6.8 24.6 ± 4.9
4 µM Na
2
CrO
4
20.9 ± 2.7 8.4 ± 3.2 14.6 ± 2.9
5 µM Na
2
CrO
4
11.6 ± 1.8 0.1 ± 0.2 5.9 ± 1.0
10 µM Na
2
CrO
4
0.1 ± 0.2 0.1 ± 0.2 0.1 ± 0.2
Sodium Chromate Treatment With Ascorbic Acid
0 µM Na
2
CrO
4
and 0.0125 mM Ascorbic Acid 44.7 ± 3.2 55.1 ± 6.2 49.9 ± 4.7
1 µM Na
2
CrO
4
and 0.0125 mM Ascorbic Acid 38.2 ± 2.7 47.1 ± 7.2 42.7 ± 4.9
2 µM Na
2
CrO
4
and 0.0125 mM Ascorbic Acid 34 ± 1.9 46.5 ± 5.6 40.3 ± 3.7
3 µM Na
2
CrO
4
and 0.0125 mM Ascorbic Acid 29.9 ± 2.8 25.8 ± 2.1 27.8 ± 2.5
4 µM Na
2
CrO
4
and 0.0125 mM Ascorbic Acid 32 ± 2.6 12.3 ± 1.6 22.1 ± 2.1
5 µM Na
2
CrO
4
and 0.0125 mM Ascorbic Acid 23.9 ± 6.6 7.8 ± 3.9 15.8 ± 5.3
10 µM Na
2
CrO
4
and 0.0125 mM Ascorbic Acid 1.7 ± 1.5 0.1 ± 0.2 0.9 ± 0.9
For experiment 1, the survival fractions of the 10T1/2 cells treated with 0 µM, 1µM, 2
µM, 3 µM, 4 µM, 5 µM, 10 µM Sodium chromate in absence of ascorbic acid were 1 ± 0.02, 0.8
± 0.1, 0.8 ± 0.03, 0.5 ± 0.1, 0.4 ± 0.1, 0.2 ± 0.04, and <0.002, respectively (Table 4). The
survival fractions of the 10T1/2 cells treated with 0 µM, 1 µM, 2 µM, 3 µM, 4 µM, 5 µM, 10
µM sodium chromate in presence of 0.0125 mM ascorbic acid were 0.9 ± 0.1, 0.8 ± 0.1, 0.7 ±
0.04, 0.6 ± 0.1, 0.7 ± 0.1, 0.5 ± 0.1, and 0.03 ± 0.03, respectively (Table 4). As noted from Table
4 and Figure 5, sodium chromate definitely caused a dose dependent cytotoxicity to 10T1/2 cells.
The survival of the 10T1/2 cells decreased monotonically when cells were treated with
38
increasing concentrations of sodium chromate. However, when 10T1/2 cells were treated with
sodium chromate in the presence of 0.0125 mM ascorbic acid, there was a dose-dependent
cytotoxicity, but ascorbic acid did not enhance the cytotoxicity of sodium chromate to 10T1/2
cells. Ascorbic acid slightly enhanced the survival of 10T1/2 cells when cells were treated with
sodium chromate with method 1. Overall, ascorbic acid did not enhance the cytotoxic effect of
sodium chromate to 10T1/2 cells.
Figure 5: This figure shows the graphical representation of the survival fractions +/- standard
deviations of 10T1/2 cells. The cells were treated with either sodium chromate alone or sodium
chromate with ascorbic acid 24 hours after seeding from experiment 1. This figure shows the
survival fraction (on a logarithmic scale) of 10T1/2 cells versus the concentration of sodium
chromate used to treat the cells.
0.001
0.01
0.1
1
10
0 1 2 3 4 5 6 7 8 9 10
Log(Survival Fraction of 10T1/2 Cells)
Sodium Chromate Concentration (uM)
Experiment 1: Effect of 0.0125 mM Ascorbic Acid on the
Survival of 10T1/2 Cells Treated with Sodium
Chromate(Cells Treated with Ascorbic Acid 24 hours After
Seeding)
Sodium Chromate
Sodium Chromate with 0.0125 mM Ascorbic Acid
No Addition (Control)
39
For experiment 2, the survival fractions of 10T1/2 cells treated with 0 µM, 1 µM, 2 µM,
3µ M, 4 µM, 5 µM, 10 µM sodium chromate in absence of ascorbic acid were 1 ± 0.1, 0.9 ± 0.2,
0.9 ± 0.2, 0.5 ± 0.1, 0.2 ± 0.06, 0.002 ± 0.004, and <0.002, respectively (Table 4). The survival
fractions of the 10T1/2 cells treated with 0 µM, 1 µM, 2 µM, 3 µM, 4 µM, 5 µM, 10 µM sodium
chromate in presence of 0.0125 mM ascorbic acid were 1.0 ± 0.1, 0.9 ± 0.1, 0.9 ± 0.1, 0.5 ± 0.04,
0.2 ± 0.03, 0.1 ± 0.1, and <0.002, respectively (Table 4). From Figure 6, we observed that there
was a dose-dependent cytotoxicity of sodium chromate to 10T1/2 cells for both cells treated with
sodium chromate in absence of ascorbic acid and with sodium chromate in the presence of
0.0125 mM ascorbic acid. Overall, ascorbic acid did not increase the cytotoxicity of sodium
chromate to 10T1/2 cells at concentrations of sodium chromate up to 4 µM. However, there
appeared to be a difference in survival of the 10T1/2 cells when treated with 5µM sodium
chromate without ascorbic acid vs. cells treated with 5 µM sodium chromate with 0.0125 mM
ascorbic acid. Overall, ascorbic acid did not enhance the cytotoxicity of sodium chromate to
10T1/2 cells. In fact, at a concentration of sodium chromate of 5 µM, ascorbic acid reduced the
cytotoxicity of sodium chromate (Figure 6).
40
Figure 6: This figure shows the graphical representation of the survival fractions +/- standard
deviations of 10T1/2 cells. The cells were treated with either sodium chromate alone or sodium
chromate with ascorbic acid 24 hours after seeding from experiment 2. This figure shows the
survival fraction (on a logarithmic scale) of 10T1/2 cells versus the concentration of Sodium
chromate used to treat the cells.
The survival fractions from experiment 1 and experiment 2 were averaged, as seen in
Table 4. From Figure 7, the survival fractions of the 10T1/2 cells decreased with increasing
concentrations of sodium chromate. The survival fractions of the 10T1/2 cells also decreased
with increasing concentrations of sodium chromate in the presence of 0.0125 mM ascorbic acid.
There was also a slight enhancement of the survival of the 10T1/2 cells when they were treated
with 0.0125 mM ascorbic acid and sodium chromate concentrations greater than 4 µM. Overall,
0.001
0.01
0.1
1
10
0 1 2 3 4 5 6 7 8 9 10
Log(Survival Fraction of 10T1/2 Cells)
Sodium Chromate Concentration (uM)
Experiment 2: Effect of 0.0125mM Ascorbic on the Survival
of 10T1/2 Cells Treated with Sodium Chromate (Cells
Treated with Ascorbic Acid 24 Hours After Seeding)
Sodium Chromate
Sodium Chromate with 0.0125mM Ascorbic Acid
No Addition Control
41
ascorbic acid did not enhance the cytotoxicity of sodium chromate to 10T1/2 cells when ascorbic
acid and sodium chromate were added to cells at the same time (24 hours after seeding).
Figure 7: This figure shows the graphical representation of the survival fractions +/- standard
deviations of 10T1/2 cells. The cells were treated with either Sodium chromate alone or Sodium
chromate with Ascorbic Acid 24 hours after seeding from the averages of experiment 1 and 2.
This figure shows the survival fraction (on a logarithmic scale) of 10T1/2 cells versus the
concentration of Sodium chromate used to treat the cells.
0.001
0.01
0.1
1
10
0 1 2 3 4 5 6 7 8 9 10
Log(Survival Fraction of 10T1/2 Cells)
Sodium Chromate Concentration (uM)
Effect of 0.0125 mM Ascorbic Acid on the Survival of
10T1/2 Cells Treated with Sodium Chromate (Cells Treated
with Ascorbic Acid 24 Hours After Seeding)
Sodium Chromate
Sodium Chromate with 0.0125 mM Ascorbic Acid
No Addition (Control)
42
Ascorbic Acid Effect with Method 2
For method 2, we seeded the cells and the dishes to be treated with sodium chromate in
the presence of ascorbic acid were treated with 0.0125 mM ascorbic acid upon seeding. 10T1/2
cells were then treated 24 hours later with sodium chromate for 48 hours before a medium
change. This method allowed for the 10T1/2 cells to be exposed to and hence loaded with
ascorbic acid before sodium chromate treatment. One experiment was performed by this
method, in which the survival fractions and plating efficiencies are shown in Table 6 below.
Table 6: Survival Fractions and Plating Efficiencies Determined from the Effect of 0.0125
mM Ascorbic Acid on 10T1/2 Cells Treated with Sodium Chromate. The results shown are
from an experiment measuring the survival and plating efficiencies (PE) of 10T1/2 cells when
treated with a) sodium chromate alone and b) sodium chromate with 0.0125 mM ascorbic acid.
The concentrations of Sodium chromate used ranged from 0 µM to 10 µM.
Treatment PE ± SD Survival ± SD
No Addition 35 ± 2.5 1.2 ± 0.1
Sodium Chromate Treatment Without Ascorbic Acid
0 µM Na
2
CrO
4
28.4 ± 3.0 1 ± 0.1
1 µM Na
2
CrO
4
29.7 ± 3.6 1.0 ± 0.1
2 µM Na
2
CrO
4
27.4 ± 0.3 1.0 ± 0.01
3 µM Na
2
CrO
4
25.3 ± 3.6 0.9 ± 0.1
4 µM Na
2
CrO
4
17.1 ± 2.3 0.6 ± 0.1
5 µM Na
2
CrO
4
8.7 ± 3.5 0.3 ± 0.1
10 µM Na
2
CrO
4
0.5 ± 0.6 0.01 ± 0.02
Sodium Chromate Treatment With Ascorbic Acid
0 µM Na
2
CrO
4
and 0.0125 mM Ascorbic Acid 22 ± 0 0.8 ± 0
1 µM Na
2
CrO
4
and 0.0125 mM Ascorbic Acid 14.1 ± 1.7 0.5 ± 0.1
2 µM Na
2
CrO
4
and 0.0125 mM Ascorbic Acid 12.8 ± 1.3 0.4 ± 0.05
3 µM Na
2
CrO
4
and 0.0125 mM Ascorbic Acid 10.3 ± 3.0 0.4 ± 0.1
4 µM Na
2
CrO
4
and 0.0125 mM Ascorbic Acid 5 ± 1.5 0.2 ± 0.1
5 µM Na
2
CrO
4
and 0.0125 mM Ascorbic Acid 3.4 ± 1.5 0.1 ± 0.1
10 µM Na
2
CrO
4
and 0.0125 mM Ascorbic Acid 0.3 ± 0.3 0.01 ± 0.01
The survival fractions of 10T1/2 cells treated with 0 µM, 1 µM, 2 µM, 3 µM, 4 µM, 5
µM, 10 µM sodium chromate in absence of ascorbic acid were 1 ± 0.1, 1.0 ± 0.1, 1.0 ± 0.01, 0.9
43
± 0.1, 0.6 ± 0.1, 0.3 ± 0.1, and 0.01 ± 0.02, respectively, (Table 6). The survival fractions of
10T1/2 cells treated with 0 µM, 1 µM, 2 µM, 3 µM, 4 µM, 5 µM, and 10 µM sodium chromate
in presence of ascorbic acid were 0.8 ± 0, 0.5 ± 0.1, 0.4 ± 0.05, 0.4 ± 0.1, 0.2 ± 0.1, 0.1 ± 0.1,
and 0.01 ± 0.01, respectively. This data was plotted in figure 8 below. The survival fraction of
the 10T1/2 cells decreased with ascorbic acid treatment. The ascorbic acid treatment using this
method was able to modestly enhanced the cytotoxicity of sodium chromate to 10T1/2 cells.
Figure 8: This figure shows the graphical representation of the survival fractions +/- standard
deviations of 10T1/2 cells. The cells were treated with either Sodium chromate alone (24 hours
after seeding) or sodium chromate with ascorbic acid from experiment 1 (Ascorbic Acid treated
upon seeding and Sodium chromate treated 24 hours after seeding). This figure shows the
survival fraction (on a logarithmic scale) of 10T1/2 cells versus the concentration of Sodium
chromate used to treat the cells.
0.001
0.01
0.1
1
10
0 1 2 3 4 5 6 7 8 9 10
Log(Survival Fraction of 10T1/2 Cells)
Concentration of Sodium Chromate (uM)
Effect of 0.0125 mM Ascorbic Acid on the Survival of
10T1/2 Cells Treated with Sodium Chromate (Cells Treated
with Ascorbic Acid upon Seeding)
Sodium Chromate
Sodium Chromate With 0.0125mM Ascorbic Acid
No Addition Control
44
3.3 Effects of Ascorbic Acid on Sodium Chromate induced Morphological Transformation
to 10T1/2 Cells
Previous work in our laboratory showed that an ascorbic acid concentration of 0.0125
mM was the mos non-cytotoxic and most effective in enhancing morphological transformation of
10T1/2 cells when the cells were treated with sodium chromate (Shahin, 2014). Our previous
experiment in section 3.1 confirmed that an ascorbic acid concentration of 0.0125 mM was not
cytotoxic to cells. Our previous experiment in section 3.2 also showed that ascorbic acid
modestly enhanced the cytotoxicity of sodium chromate to 10T1/2 cells when the cells were pre-
treated with ascorbic acid 24 hours before adding sodium chromate. From the results obtained
from previous experiments, we performed an assay to determine the effect of 0.0125 mM
ascorbic acid on sodium chromate-induced morphological transformation of 10T1/2 cells. One
method was used for this assay as described in section 2.6. The results of the transformation
assay are shown in Table 7 below.
45
Table 7: Effect of 0.0125 mM Ascorbic Acid on the Sodium Chromate Induced
Morphological Transformation to 10T1/2 Cells. The results shown are from a transformation
experiment measuring the number of foci formed when 10T1/2 cells were treated with a)
Sodium chromate alone and b) Sodium chromate in presence of Ascorbic Acid. The cell survival
from the cytotoxicity control is shown for reference.
Total Number of foci/ Number of Dishes
scored (Foci/Total Dishes)*
Number of Dishes with Transformed
foci/Number of Dishes scored
Treatment Cell Survival (%) Type III Type II + Type III Type III Type II + Type III
Day 5, 24 hours
0 (medium only) 91.9 0/19 (0/20) 0/19 (0/20) 0/19 0/19
0 (0.5% acetone) 89 0/20 (0/20) 0/20 (0/20) 0/20 0/20
MCA (1.0 µg/ml) 66.9 0/20 (0/20) 1/20 (1/20) 0/20 1/20
Na 2CrO 4 (µM) only
0µM Na 2CrO 4 100 0/20 (0/20) 0/20 (0/20) 0/20 0/20
0.5µM Na 2CrO 4 93.6 0/20 (0/20) 1/20 (1/20) 0/20 1/20
1µM Na 2CrO 4 77 0/19 (0/20) 0/19 (0/20) 0/19 0/19
2µM Na 2CrO 4 78.5 0/19 (0/20) 1/19 (1/20) 0/19 1/19
4µM Na 2CrO 4 5.2 0/20 (0/20) 1/20 (1/20) 0/20 1/20
Na 2CrO 4 (µM) + 0.0125 mM Ascorbic Acid
0µM Na 2CrO 4 114.3 0/20 (0/20) 0/20 (0/20) 0/20 0/20
0.5µM Na 2CrO 4 102.9 1/20 (1/20) 2/20 (2/20) 1/20 2/20
1µM Na 2CrO 4 70.3 0/19 (0/19) 1/19 (1/20) 0/19 1/19
2µM Na 2CrO 4 34.9 0/20 (0/20) 3/20 (3/20) 0/20 3/20
4µM Na 2CrO 4 1.5 0/17 (0/20) 1/17 (1/20) 0/17 1/17
* The number of foci has been normalized to a total of 20 dishes
The acetone control (0.5% acetone, v/v) induced no foci (table 7). The MCA positive
control, a strong carcinogen, did not induce many foci (1 focus/ 20 dishes) (Table 7). Hence,
since this positive control, MCA, did not work well, this experiment was not considered valid.
Since very few foci were induced by treatment of 10T1/2 cells with sodium chromate alone and
with sodium chromate in presence of ascorbic acid (0-3 foci), the data is inconclusive as to
whether ascorbic acid is able to enhance sodium chromate induced morphological transformation
to 10T1/2 cells. This transformation experiment is preliminary, and more trials need to be
conducted to achieve conclusive results. However, when observing the cytotoxicity data for the
transformation experiment in Table 8 and Figure 9, we do see that sodium chromate was
cytotoxic to10T1/2 cells in a dose-dependent manner. It can also be observed that the ascorbic
46
acid was not cytotoxic and that the ascorbic acid modestly enhanced the cytotoxicity of sodium
chromate-treated 10T1/2 cells at concentrations of 2 µM and 4 µM of sodium chromate.
Table 8: Cytotoxicity Data for the Transformation Experiment with Sodium Chromate and
Ascorbic Acid. The results shown are from an experiment measuring the plating efficiencies
(PE) and survival fractions of 10T1/2 cells when treated with a) sodium chromate alone and b)
sodium chromate in present of 0.0125 mM ascorbic acid. The concentrations of sodium chromate
used ranged from 0 µM to 4 µM.
Treatment PE ± SD Survival Fraction ± SD
No Addition 15.8 ± 2.0 0.9 ± 0.1
Acetone 15.3 ± 1.5 0.9 ± 0.1
MCA and Acetone 11.5 ± 2.1 0.7 ± 0.1
Sodium Chromate Treatment without Ascorbic Acid
0 µM Na
2
CrO
4
17.2 ± 3.6 1 ± 0.2
0.5 µM Na
2
CrO
4
16.1 ± 1.4 0.9 ± 0.1
1 µM Na
2
CrO
4
13.3 ± 1.6 0.8 ± 0.1
2 µM Na
2
CrO
4
13.5 ± 2.0 0.8 ± 0.1
4 µM Na
2
CrO
4
0.9 ± 0.4 0.05 ± 0.02
Sodium Chromate Treatment with Ascorbic Acid
0 µM Na
2
CrO
4
and 0.0125 mM Ascorbic Acid 19.7 ± 0.2 1.1 ± 0.01
0.5 µM Na
2
CrO
4
and 0.0125 mM Ascorbic Acid 17.7 ± 2.4 1.0 ± 0.1
1 µM Na
2
CrO
4
and 0.0125 mM Ascorbic Acid 12.1 ± 1.9 0.7 ± 0.1
2 µM Na
2
CrO
4
and 0.0125 mM Ascorbic Acid 6 ± 1.8 0.3 ± 0.1
4 µM Na
2
CrO
4
and 0.0125 mM Ascorbic Acid 0.3 ± 0.3 0.01 ± 0.01
47
Figure 9: This figure shows the graphical representation of the cytotoxicity data for the
transformation experiment with sodium chromate and ascorbic acid. The data represents the
survival fractions (on a logarithmic scale) +/- standard deviations of 10T1/2 cells versus the
concentration of sodium chromate used to treat the cells. The cells were treated with either
sodium chromate alone (24 hours after seeding) or sodium chromate with 0.0125 mM ascorbic
acid (ascorbic acid treated upon seeding and sodium chromate treated 24 hours after seeding).
0.01
0.1
1
10
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
Survival Fraction of 10T1/2 Cells
Sodium Chromate Concentration (uM)
Effect of 0.0125mM Ascorbic Acid on the Cytotoxicity of
Sodium Chromate to 10T1/2 Cells
Sodium chromate
Sodium Chromate with 0.0125mM Ascorbic Acid
No Addition Control
Acetone
MCA+Acetone
48
3.4 Effect of Glutathione (GSH) on the Survival of 10T½ Cells
Before determining the effect of GSH on the survival of10T1/2 cells treated with sodium
chromate, we first determined the non-cytotoxic concentrations of GSH to 10T1/2 cells needed
to be determined. By determining the non-cytotoxic concentrations of GSH, we were able to
choose a concentration of GSH that was not cytotoxic to be used with our sodium chromate
cytotoxicity assays. There had not been previous work in our laboratory with GSH, hence we
started by testing a broad range of GSH concentrations, then narrowing the range as we obtained
results of each 10T1/2 cell survival experiment. There was only one method used in this assay,
which consisted of treating the cells with GSH 24 hours after seeding. The first range of GSH
consisted of concentrations of GSH between 0.001 mM to 10 mM. The second range of GSH
concentrations tested were between 0.1 mM to 1 mM and the third range of GSH concentrations
tested were between 0.1 mM and 0.5 mM.
Measuring Reduced Glutathione (GSH) Cytotoxicity to 10T1/2 Cells Using Concentrations of
GSH Between 0.001 mM to 10 mM
In testing the effect of 0.001 mM to 10mM GSH on the survival of 10T1/2 cells, we
conducted three experiments. The survival fractions and plating efficiencies of each experiment
as well as the average of the data are shown in Table 9 and Table 10.
49
Table 9: Survival Fractions Determined from the Effect of 0.001 mM to 10mM Glutathione
(GSH) to 10T1/2 Cells. The results shown are from three experiments measuring the cytoxicities
of reduced Glutathione (GSH) to 10T1/2 cells using concentrations of GSH ranging from 0.001
mM to 10 mM. The data collected show the individual and average (avg) survival fractions of
the treated 10T1/2 cells +/- their standard deviations (SD).
Treatment
Experiment 1
Survival
Fraction ± SD
Experiment 2
Survival
Fraction ± SD
Experiment 3
Survival
Fraction ± SD
Average
Survival
Fraction ± SD
No Addition (Control) 1.0 ± 0.1 1.0 ± 0.05 0.9 ± 0.2 1.0 ± 0.1
0 mM GSH 1 ± 0.1 1 ± 0.03 1 ± 0.6 1 ± 0.2
0.001 mM GSH 1.2 ± 0.2 1.1 ± 0.1 0.8 ± 0.1 1.0 ± 0.1
0.01 mM GSH 1.2 ± 0.04 1.05 ± 0.08 1.0 ± 0.2 1.1 ± 0.1
0.1 mM GSH 1.1 ± 0.1 1.0 ± 0.1 1.1 ± 0.1 1.1 ± 0.1
1 mM GSH 0.3 ± 0.3 0.7 ± 0.1 0.4 ± 0.1 0.5 ± 0.2
10 mM GSH 0.005 ± 0.01 0.5 ± 0.1 0.2 ± 0.1 0.2 ± 0.1
Table 10: Plating Efficiencies Determined from the Effect of 0.001 mM to 10mM
Glutathione (GSH) to 10T1/2 Cells. The results shown are from three experiments measuring
the cytoxicities of reduced Glutathione (GSH) to 10T1/2 cells using concentrations of GSH
ranging from 0.001 mM to 10 mM. The data collected show the individual and average (avg)
plating efficiencies (PE) of the treated 10T1/2 cells +/- their standard deviations (SD).
Treatment Experiment 1
PE ± SD
Experiment 2
PE ± SD
Experiment 3
PE ± SD
Average
PE ± SD
No Addition (Control) 20.8 ± 2.7 25.8 ± 1.2 17.6 ± 3.2 21.4 ± 2.4
0 mM GSH 20.3 ± 1.4 25.6 ± 0.9 19.4 ± 4.1 21.8 ± 2.1
0.001 mM GSH 23.7 ± 3.2 28.7 ± 2.6 15.8 ± 1.6 22.7 ± 2.4
0.01 mM GSH 23.4 ± 0.9 26.8 ± 2.1 20 ± 3.5 23.4 ± 2.2
0.1 mM GSH 22.8 ± 1.8 25.4 ± 3.0 20.7 ± 1.5 23.07 ± 2.1
1 mM GSH 6 ± 6.4 17.1 ± 3.3 7.9 ± 1.6 10.33 ± 3.8
10 mM GSH 0.1 ± 0.2 11.6 ± 1.9 4.6 ± 2.0 5.43 ± 1.4
In experiment 1, the survival fractions of 10T1/2 cells treated with 0 mM, 0.001 mM,
0.01 mM, 0.1 mM, 1 mM, and 10 mM GSH were 1.0 ± 0.1, 1.2 ± 0.2, 1.2 ± 0.04, 1.1 ± 0.1, 0.3 ±
0.3, and 0.005 ± 0.01, respectively, (Table 9). The survival fractions were plotted in graphical
representation in Figure 10 below. As noted from Table 9 and Figure 10, GSH was non-
cytotoxic at concentrations from 0.001 mM to 0.1 mM and was cytotoxic at concentrations
50
greater than 0.1 mM. The survival of 10T1/2 cells decreased significantly to 0.3 and 0.005 when
they were treated with GSH concentrations of 1 mM and 10 mM, respectively (Table 9 and
Figure 10).
Figure 10: This figure shows the graphical representation of the survival fractions +/- standard
deviations of 10T1/2 cells treated with GSH 24 hours after seeding from experiment 1. This
figure shows the survival fraction (on a logarithmic scale) of 10T1/2 cells versus the
concentration of GSH used to treat the cells.
In experiment 2, the survival fractions of 10T1/2 cells treated with 0 mM, 0.001 mM,
0.01 mM, 0.1 mM, 1 mM, and 10 mM GSH were 1 ± 0.03, 1.1 ± 0.1, 1.05 ± 0.08, 1.0 ± 0.1, 0.7
± 0.1, and 0.5 ± 0.1, respectively, as seen in Table 9 above. The survival fractions were plotted
in graphical representation in Figure 11 below. Consistent with experiment 1, GSH was non-
cytotoxic at concentrations from 0.001 mM to 0.1 mM and was cytotoxic at concentrations
0.001
0.01
0.1
1
10
0 1 2 3 4 5 6 7 8 9 10
Log (Survival Fraction of 10T1/2 Cells)
GSH Concentration (mM)
Experiment 1: Effect of 0.001mM to 10mM GSH on the
Survival of 10T1/2 Cells
GSH Treatment No addition (control)
51
greater than 0.1 mM. The survival of 10T1/2 cells also decreased significantly when they were
treated with GSH concentrations of 1 mM and 10 mM.
Figure 11: This figure shows the graphical representation of the survival fractions +/- standard
deviations of 10T1/2 cells treated with GSH 24 hours after seeding from experiment 2. This
figure shows the survival fraction (on a logarithmic scale) of 10T1/2 cells versus the
concentration of GSH used to treat the cells.
In experiment 3, the survival fractions of 10T1/2 cells treated with 0 mM, 0.001 mM,
0.01 mM, 0.1 mM, 1 mM, and 10 mM GSH were 1 ± 0.6, 0.8 ± 0.1, 1.0 ± 0.2, 1.1 ± 0.1, 0.4 ±
0.1, and 0.2 ± 0.1, respectively, as seen in Table 9 above. The survival fractions were plotted in
graphical representation in Figure 12 below. Consistent with experiments 1 and 2, GSH was
non-cytotoxic at concentrations from 0.001 mM to 0.1 mM and was cytotoxic at concentrations
greater than 0.1 mM. The survival of10T1/2 cells also dropped significantly when the cells were
treated with GSH concentrations of 1 mM and 10mM.
0.1
1
10
0 1 2 3 4 5 6 7 8 9 10
Log (Survival Fraction of 10T1/2 Cells)
GSH Concentration (mM)
Experiment 2: Effect of 0.001 mM to 10 mM GSH on the
Survival of 10T1/2 Cells
GSH Treatment No addition (control)
52
Figure 12: This figure shows the graphical representation of the survival fractions +/- standard
deviations of 10T1/2 cells treated with GSH 24 hours after seeding from experiment 3. This
figure shows the survival fraction (on a logarithmic scale) of 10T1/2 cells versus the
concentration of GSH used to treat the cells.
The survival fractions of the 10T1/2 cells from experiments 1, 2 and 3 were averaged
according to the data in Table 9 and Figure 13. The average survival fractions of 10T1/2 cells
treated with 0 mM, 0.001 mM, 0.01 mM, 0.1 mM, 1 mM, and 10 mM GSH were 1 ± 0.2, 1.0 ±
0.1, 1.1 ± 0.1, 1.1 ± 0.1, 0.5 ± 0.2, 0.2 ± 0.1, respectively. On average, GSH was non-cytotoxic
at concentrations from 0.001 mM to 0.1 mM and cytotoxic at concentrations greater than 0.1
mM. The survival of 10T1/2 cells also decreased significantly cells were treated with GSH
concentrations of 1 mM and 10 mM. The average shows that all three experiments were
consistent and that the data was reproducible. The data clearly shows that at some concentration
between 0.1 mM to 1 mM, GSH becomes cytotoxic.
0.1
1
10
0 2 4 6 8 10 12
Log(Survival Fraction of 10T1/2 Cells)
GSH Concentration (mM)
Experiment 3: Cytotoxicity of 0.001 mM to 10mM GSH to
10T1/2 Cells
GSH Treatment No Addition (Control)
53
Figure 13: This figure shows the graphical representation of the survival fractions +/- standard
deviations of 10T1/2 cells treated with GSH 24 hours after seeding from experiment 1, 2, and 3.
This figure shows the survival fraction (on a logarithmic scale) of 10T1/2 cells versus the
concentration of GSH used to treat the cells.
Measuring Reduced Glutathione (GSH) Cytotoxicity to 10T1/2 Cells Using Concentrations of
GSH Between 0.1 mM to 1.0 mM
From the previous experiment in which we tested the effect of 0.001 mM to 10 mM GSH
on the survival of 10T1/2 cells, we found that starting at some concentration between 0.1 mM to
1 mM, the GSH becomes toxic. Therefore, we decided to measure the effect of 0.1 mM to 1 mM
GSH on the survival of 10T1/2 cells to try to determine carefully what this concentration was.
There was only one experimental trial performed for this range of GSH concentrations, in which
the plating efficiency and survival fraction is shown in Table 11 below.
0.001
0.01
0.1
1
10
0 2 4 6 8 10 12
Log(Survival Fraction of 10T1/2 Cells)
GSH Concentration (mM)
Effect of 0.001 mM to 10 mM GSH on the Survival of
10T1/2 Cells
Experiment 1 Experiment 2 Experiment 3 Average No Addition
54
Table 11: Plating Efficiency and Survival Fraction Determined from the Effect of 0.1 mM
to 10mM Glutathione (GSH) to 10T1/2 Cells. The results shown are from an experiment
measuring the cytotoxicity of reduced Glutathione (GSH) to 10T1/2 cells using concentrations of
GSH ranging from 0.1 mM to 1 mM. The data collected show the Plating Efficiency (PE) and
the Survival Fraction of the treated 10T1/2 cells +/- their standard deviations (SD).
Treatment PE ± SD Survival Fraction ± SD
No Addition (Control) 20.1 ± 2.4 0.9 ± 0.1
0 mM GSH 21.6 ± 3.3 1 ± 0.2
0.1 mM GSH 21.3 ± 5.0 1.0 ± 0.2
0.25 mM GSH 21.3 ± 5.1 1.0 ± 0.2
0.5 mM GSH 15.5 ± 5.3 0.7 ± 0.2
0.75 mM GSH 13.6 ± 4.6 0.6 ± 0.2
1 mM GSH 11.4 ± 3.6 0.5 ± 0.2
As seen in Table 11, the survival fractions of 10T1/2 cells treated with 0 mM, 0.1m M,
0.25 mM, 0.5 mM, 0.75 mM, and 1 mM of GSH were 1 ± 0.2, 1.0 ± 0.2, 1.0 ± 0.2, 0.7 ± 0.2, 0.6
± 0.2, and 0.5 ± 0.2, respectively. Observing the trend from Table 11 and Figure 14, GSH had
no effect on the survival of 10T1/2 cells at concentrations of 0.1 mM and 0.25 mM. However, at
GSH concentrations of 0.5 mM and higher, GSH exerts some cytotoxicity, with the survival of
the 10T1/2 cells decreasing with increasing concentration of GSH. Thus, it can be concluded
that GSH becomes cytotoxic at some concentration between 0.25 mM to 0. 5 mM, as there is a
decrease in survival of 0T1/2 cells when the cells were treated with 0.5 mM of GSH. There was
only one experiment performed for these range of concentrations of GSH, and this experiment
was preliminary to test the next range of GSH concentrations.
55
Figure 14: This figure shows the graphical representation of the survival fractions +/- standard
deviations of 10T1/2 cells treated with GSH 24 hours after seeding. This figure shows the
survival fraction (on a logarithmic scale) of 10T1/2 cells versus the concentration of GSH used
to treat the cells.
Measuring Reduced Glutathione (GSH) Cytotoxicity to 10T1/2 Cells Using Concentrations of
GSH Between 0.1 mM to 0.5 mM
As mentioned from the previous experiment, testing the effect of 0.1 mM to 1 mM of
GSH on the survival of 10T1/2 cells, it was found that GSH became cytotoxic at some
concentration between 0.25 mM to 0.5 mM. To determine the cytotoxic GSH concentrations, we
narrowed the range and tested the effect of 0.1 mM to 0.5 mM GSH on the survival of 10T1/2
cells. Three experiments were performed for this assay. The survival fractions and plating
efficiencies are summarized in Tables 12 and 13 below.
0.1
1
10
0 0.2 0.4 0.6 0.8 1 1.2
Log(Survival Fraction of 10T1/2 Cells)
GSH Concentration (mM)
Experiment 1: Effect of 0.1 mM to 1 mM GSH on the
Survival of 10T1/2 Cells
GSH Treatment No Addition (Control)
56
Table 12: Survival Fractions Determined from the Effect of 0.1 mM to 0.5 mM Glutathione
(GSH) to 10T1/2 Cells. The results shown are from 3 experiments measuring the cytotoxicities
of reduced Glutathione (GSH) to 10T1/2 cells using concentrations of GSH ranging from 0.1
mM to 0.5 mM. The data collected show the individual and average (avg) survival fractions of
the treated 10T1/2 cells +/- their standard deviations (SD).
Treatment
Experiment 1
Survival
Fraction ± SD
Experiment 2
Survival
Fraction ± SD
Experiment 3
Survival
Fraction ± SD
Average
Survival
Fraction ± SD
No Addition (Control) 1.1 ± 0.2 0.9 ± 0.1 1.0 ± 0.1 1.0 ± 0.1
0 mM Glutathione 1 ± 0.1 1 ± 0.1 1 ± 0.1 1 ± 0.1
0.1 mM Glutathione 1.1 ± 0.1 0.9 ± 0.04 1.0 ± 0.1 1.0 ± 0.1
0.2 mM Glutathione 0.6 ± 0.1 0.7 ± 0.1 0.7 ± 0.05 0.7 ± 0.1
0.3 mM Glutathione 0.3 ± 0.2 03 ± 0.1 0.3 ± 0.1 0.3 ± 0.1
0.4 mM Glutathionoe 0.1 ± 0.04 0.1 ± 0.04 0.2 ± 0.04 0.1 ± 0.04
0.5 mM Glutathione 0.04 ± 0.03 0.1 ± 0.04 0.1 ± 0.1 0.08 ± 0.04
Table 13: Plating Efficiencies Determined from the Effect of 0.1 mM to 0.5 mM
Glutathione (GSH) to 10T1/2 Cells. The results shown are from 3 experiments measuring the
cytotoxicities of reduced Glutathione (GSH) to 10T1/2 cells using concentrations of GSH
ranging from 0.1 mM to 0.5 mM. The data collected show the individual and average (avg)
plating efficiencies (PE) of the treated 10T1/2 cells +/- their standard deviations (SD).
Treatment Experiment 1
PE ± SD
Experiment 2
PE ± SD
Experiment 3
PE ± SD
Average
PE ± SD
No Addition (Control) 21.4 ± 3.1 29.2 ± 3.3 35.4 ± 4.8 28.7 ± 3.7
0 mM Glutathione 20.2 ± 2.4 30.7 ± 2.5 36.3 ± 4.6 29.1 ± 3.2
0.1 mM Glutathione 21.3 ± 2.5 28.2 ± 1.4 35.8 ± 2.0 28.4 ± 1.9
0.2 mM Glutathione 12 ± 1.5 22.1 ± 3.9 24.2 ± 1.8 19.4 ± 2.4
0.3 mM Glutathione 6.5 ± 3.9 9.2 ± 2.7 12.1 ± 4.9 9.3 ± 3.8
0.4 mM Glutathionoe 1.5 ± 0.94 2.8 ± 1.1 5.6 ± 1.5 3.3 ± 1.2
0.5 mM Glutathione 0.8 ± 0.6 2.6 ± 1.1 4.6 ± 1.9 2.7 ± 1.2
In experiment 1, the survival fractions of 10T1/2 cells treated with 0 mM, 0.1 mM, 0.2
mM, 0.3 mM, 0.4 mM and 0.5 mM GSH were 1 ± 0.1, 1.1 ± 0.1, 0.6 ± 0.1, 0.3 ± 0.2, 0.1 ± 0.04,
and 0.04 ± 0.03, respectively, as seen in Table 12. Observing the trend from Figure 15 below, it
can be seen that GSH has a dose-dependent cytotoxicity starting at a concentration of 0.2 mM.
From 0.1 mM to 0.5 mM GSH, the survival of 10T1/2 cells decreased in a dose-dependent
manner as the concentrations of GSH increased. It is evident from Table 12 and Figure 15 that
57
the GSH becomes cytotoxic starting at 0.2 mM, as the cell survival decreased by 42 percent
when treated with 0.2mM as compared to when the cells were treated with 0.1mM GSH. This
experiment indicated that 0.1 mM GSH was the highest non-cytotoxic concentration to 10T1/2
cell survival.
Figure 15: This figure shows the graphical representation of the survival fractions +/- standard
deviations of 10T1/2 cells treated with GSH 24 hours after seeding for experiment 1. This figure
shows the survival fraction (on a logarithmic scale) of 10T1/2 cells versus the concentration of
GSH used to treat the cells.
In experiment 2, the survival fractions of 10T1/2 cells treated with 0 mM, 0.1 mM, 0.2
mM, 0.3 mM, 0.4 mM, and 0.5 mM GSH were 1 ± 0.1, 0.9 ± 0.04, 0.7 ± 0.1, 03 ± 0.1, 0.1 ±
0.04, and 0.1 ± 0.04, respectively, as seen in Table 12. From Table 12 and Figure 16, it was clear
that the survival of 10T1/2 cells began to decrease in a dose-dependent manner when cells were
treated with GSH concentrations greater than 0.1 mM. Consistent with experiment 1, GSH
0.01
0.1
1
10
0 0.1 0.2 0.3 0.4 0.5 0.6
Log(Survival Fraction of 10T1/2 Cells)
GSH Concentration (mM)
Experiment 1: Effect of 0.1 mM to 0.5 mM GSH on the
Survival of 10T1/2 Cells
GSH Treatment No Addition (Control)
58
started to become cytotoxic at 0.2 mM. We concluded that 0.1 mM was the highest, non-
cytotoxic GSH concentration to the survival of 10T1/2 cells.
Figure 16: This figure shows the graphical representation of the survival fractions +/- standard
deviations of 10T1/2 cells treated with GSH 24 hours after seeding for experiment 2. This figure
shows the survival fraction (on a logarithmic scale) of 10T1/2 cells versus the concentration of
GSH used to treat the cells.
For experiment 3, the survival fractions of the 10T1/2 cells treated with 0 mM, 0.1 mM,
0.2 mM, 0.3 mM, 0.4 mM, and 0.5 mM GSH were 1 ± 0.1, 1.0 ± 0.1, 0.7 ± 0.05, 0.3 ± 0.1, 0.2 ±
0.04, and 0.1 ± 0.1, respectively (Table 12). From figure 17, the survival fraction of 10T1/2 cells
again started to decrease with increasing concentrations of GSH of 0.2 mM and higher.
Consistent with experiments 1 and 2, experiment 3 showed that 0.1 mM was the highest, non-
cytotoxic concentration of GSH that could be used to treat 10T1/2 cells.
0.01
0.1
1
10
0 0.1 0.2 0.3 0.4 0.5 0.6
Log(Survival Fraction of 10T1/2 Cells)
GSH concentration (mM)
Experiment 2: Effect of 0.1mM to 0.5mM GSH on the
Survival of 10T1/2 Cells
GSH Treatment No Addition (Control)
59
Figure 17: This figure shows the graphical representation of the survival fractions +/- standard
deviations of 10T1/2 cells treated with GSH 24 hours after seeding for experiment 3. This figure
shows the survival fraction (on a logarithmic scale) of 10T1/2 cells versus the concentration of
GSH used to treat the cells.
The survival fractions of the 10T1/2 cells from experiments 1, 2, and 3 were averaged
and presented in Table 12. The average survival fractions of10T1/2 cells treated with 0 mM, 0.1
mM, 0.2 mM, 0.3 mM, 0.4 mM, and 0.5 mM GSH were 1 ± 0.1, 1.0 ± 0.1, 0.7 ± 0.1, 0.3 ± 0.1,
0.1 ± 0.04, and 0.08 ± 0.04, respectively (table 12). When looking at the average survival
fraction of 10T1/2 cells in Figure 18, the survival began to decrease with increasing
concentration of GSH starting at 0.2 mM, in which the survival fraction dropped 30%. Therefore,
0.1 mM was the highest, non-cytotoxic concentration of GSH that could be used to treat 10T1/2
cells. Figure 18 determined that all three experiments were repeatable and the data were
0.01
0.1
1
10
0 0.1 0.2 0.3 0.4 0.5 0.6
Log(Survival Fraction of 10T1/2 Cells)
GSH Concentration (mM)
Experiment 3: Effect of 0.1 mM to 0.5 mM GSH on the
Survival of 10T1/2 Cells
GSH Treatment No Addition (Control)
60
consistent. From the average data, we determined that 0.1 mM GSH would be used to treat
10T1/2 cells and observe the effect on the survival of 10T1/2 cells treated with sodium chromate,
in which the data would be shown in section 3.5.
Figure 18: This figure shows the graphical representation of the survival fractions +/- standard
deviations of 10T1/2 cells treated with GSH 24 hours after seeding for experiments 1,2, 3, and
their averages. This figure shows the survival fraction (on a logarithmic scale) of 10T1/2 cells
versus the concentration of GSH used to treat the cells.
0.01
0.1
1
10
0 0.1 0.2 0.3 0.4 0.5
Log(Survival Fraction of 10T1/2 Cells)
Concentration of GSH (mM)
Effect of 0.1mM to 0.5mM GSH on the Survival of 10T1/2
Cells
Experiment 1 Experiment 2
Experiment 3 Average
No Addition Control (Average)
61
3.5 Effects of GSH on the Survival of 10T1/2 Cells Treated with Sodium Chromate
In section 3.4, we determined that the highest, non-cytotoxic concentration of GSH that
could be used with 10T1/2 was 0.1 mM. This concentration of GSH was then used to determine
its effect on the survival of 10T1/2 cells treated with sodium chromate. Our goal was to
determine whether GSH could enhance the cytotoxicity of sodium chromate-treated 10T1/2 cells.
We wanted to determine whether adding 0.1 mM GSH and sodium chromate to 10T1/2 cells
together would further decrease the survival of 10T1/2 cells as opposed to only adding sodium
chromate to 10T1/2 cells. As mentioned earlier in section 2.5, there was only one method used
to treat 10T1/2 cells, in which we treated 10T1/2 cells with all chemicals 24 hours after seeding.
One set of 10T1/2 cells was treated with different concentrations of sodium chromate alone,
while another set was treated with different concentrations of sodium chromate in addition to 0.1
mM GSH. Two experiments were performed for this assay, in which the survival fractions and
plating efficiencies are summarized in Tables 14 and 15.
62
Table 14: Survival Fractions Determined from the Effect of 0.1 mM GSH on 10T1/2 Cells
Treated with Sodium Chromate. The results shown are from two experiments measuring the
survival of 10T1/2 cells when treated with Sodium chromate alone and sodium chromate with
0.1 mM GSH. The concentrations of Sodium chromate used range from 0 µM to 20 µM. The
data collected show the individual and average (avg) survival fractions of the treated 10T1/2
cells +/- their standard deviations (SD).
Treatment Experiment 1
Survival
Fraction ± SD
Experiment 2
Survival
Fraction ± SD
Average
Survival
Fraction ± SD
No Addition (Control) 1.1 ± 0.2 0.9 ± 0.1 1.0 ± 0.2
Sodium Chromate Treatment Without GSH
0 µM Na
2
CrO
4
1 ± 0.2 1 ± 0.1 1 ± 0.1
1 µM Na
2
CrO
4
1.1 ± 0.5 0.8 ± 0.1 0.9 ± 0.3
2 µM Na
2
CrO
4
0.5 ± 0.2 0.6 ± 0.1 0.6 ± 0.2
3 µM Na
2
CrO
4
0.4 ± 0.1 0.3 ± 0.1 0.4 ± 0.1
4 µM Na
2
CrO
4
0.2 ± 0.1 0.2 ± 0.03 0.2 ± 0.1
5 µM Na
2
CrO
4
0.2 ± 0.1 0.05 ± 0.01 0.1 ± 0.1
10 µM Na
2
CrO
4
<0.01 <0.003 <0.01
15 µM Na
2
CrO
4
<0.01 <0.003 <0.01
20 µM Na
2
CrO
4
<0.01 <0.003 <0.01
Sodium Chromate Treatment With GSH
0 µM Na
2
CrO
4
and 0.1 mM GSH 0.9 ± 0.4 1.0 ± 0.06 1.0 ± 0.2
1 µM Na
2
CrO
4
and 0.1 mM GSH 0.9 ± 0.1 0.8 ± 0.1 0.8 ± 0.1
2 µM Na
2
CrO
4
and 0.1 mM GSH 0.5 ± 0.3 0.5 ± 0.05 0.5 ± 0.2
3 µM Na
2
CrO
4
and 0.1 mM GSH 0.6 ± 0.1 0.3 ± 0.02 0.4 ± 0.1
4 µM Na
2
CrO
4
and 0.1 mM GSH 0.3 ± 0.1 0.2 ± 0.07 0.2 ± 0.1
5 µM Na
2
CrO
4
and 0.1 mM GSH 0.2 ± 0.1 0.07 ± 0.03 0.1 ± 0.1
10 µM Na
2
CrO
4
and 0.1 mM GSH <0.01 <0.003 <0.01
15 µM Na
2
CrO
4
and 0.1 mM GSH <0.01 <0.003 <0.01
20 µM Na
2
CrO
4
and 0.1 mM GSH <0.01 <0.003 <0.01
63
Table 15: Plating Efficiencies Determined from the Effect of 0.1 mM GSH on 10T1/2 Cells
Treated with Sodium Chromate. The results shown are from two experiments measuring the
plating efficiencies of 10T1/2 cells when treated with Sodium chromate alone and Sodium
chromate with 0.1 mM GSH. The concentrations of Sodium chromate used range from 0 µM to
20 µM. The data collected show the individual and average(avg) plating efficiencies of the
treated 10T1/2 cells +/- their standard deviations (SD).
Treatment Experiment 1
PE ± SD
Experiment 2
PE ± SD
Average
PE ± SD
No Addition (Control) 10.4 ± 2.2 32.2 ± 4.7 21.3 ± 3.4
Sodium Chromate Treatment Without GSH (24 hours after seeding)
0 µM Na
2
CrO
4
9.3 ± 1.6 35.5 ± 3.6 22.4 ± 2.6
1 µM Na
2
CrO
4
9.9 ± 4.7 29.1 ± 3.4 19.5 ± 4.0
2 µM Na
2
CrO
4
4.6 ± 1.5 21.8 ± 5.1 13.2 ± 3.3
3 µM Na
2
CrO
4
4.1 ± 0.9 12.3 ± 3.0 8.2 ± 1.9
4 µM Na
2
CrO
4
1.8 ± 1.2 8.5 ± 1.1 5.15 ± 1.1
5 µM Na
2
CrO
4
1.6 ± 1.2 1.6 ± 0.4 1.6 ± 0.8
10 µM Na
2
CrO
4
<0.1 <0.1 <0.1
15 µM Na
2
CrO
4
<0.1 <0.1 <0.1
20 µM Na
2
CrO
4
<0.1 <0.1 <0.1
Sodium Chromate Treatment With GSH
0 µM Na
2
CrO
4
and 0.1 mM GSH 8.7 ± 3.4 35.7 ± 2.2 22.2 ± 2.8
1 µM Na
2
CrO
4
and 0.1 mM GSH 8.1 ± 1.1 29.4 ± 4.4 18.7 ± 2.8
2 µM Na
2
CrO
4
and 0.1 mM GSH 5.1 ± 3.2 16.7 ± 1.7 10.9 ± 2.5
3 µM Na
2
CrO
4
and 0.1 mM GSH 5.2 ± 1.2 9.9 ± 0.6 7.5 ± 0.9
4 µM Na
2
CrO
4
and 0.1 mM GSH 2.5 ± 1.1 7.1 ± 2.4 4.8 ± 1.7
5 µM Na
2
CrO
4
and 0.1 mM GSH 1.6 ± 1.2 2.6 ± 0.9 2.1 ± 1.1
10 µM Na
2
CrO
4
and 0.1 mM GSH <0.1 <0.1 <0.1
15 µM Na
2
CrO
4
and 0.1 mM GSH 0.13 ± 0.5 <0.1 <0.1
20 µM Na
2
CrO
4
and 0.1 mM GSH <0.1 Na
2
CrO
4
<0.1 <0.1
For experiment 1, the survival fractions of 10T1/2 cells treated with 0 µM, 1 µM, 2 µM,
3 µM, 4 µM, 5 µM, 10 µM, 15 µM, and 20 µM sodium chromate without 0.1 mM GSH were 1 ±
0.2, 1.1 ± 0.5, 0.5 ± 0.2, 0.4 ± 0.1, 0.2 ± 0.1, 0.2 ± 0.1, <0.01, <0.01, and <0.01, respectively
(Table 14). The survival fractions of 10T1/2 cells treated with 0 µM, 1 µM, 2 µM, 3 µM, 4 µM,
5 µM, 10 µM, 15 µM, and 20 µM sodium chromate in addition to 0.1 mM GSH were 0.9 ± 0.4,
0.9 ± 0.1, 0.5 ± 0.3, 0.6 ± 0.1, 0.3 ± 0.1, 0.2 ± 0.1, <0.01, <0.01, and <0.01, respectively (Table
64
14). From Figure 19, it is apparent that 0.1 mM GSH did not enhance nor decrease the cytotoxic
effects of sodium chromate to 10T1/2 cells, as there was no decrease in survival of 10T1/2 cells
treated with both 0.1 mM GSH and sodium chromate compared to 10T1/2 cells treated with
only sodium chromate. It could be concluded from experiment 1 that when 10T1/2 cells were
treated with 0.1 mM GSH and sodium chromate at the same time, there was no effect on the
cytotoxicity of sodium chromate to 10T1/2 cells.
Figure 19: This figure shows the graphical representation of the survival fractions +/- standard
deviations of 10T1/2 cells for experiment 1. The 10T1/2 cells were treated with either sodium
chromate alone or sodium chromate plus 0.1 mM GSH 24 hours after cells were seeded. This
figure shows the survival fraction (on a logarithmic scale) of 10T1/2 cells versus the
concentration of sodium chromate used to treat the cells.
0.01
0.1
1
10
0 2 4 6 8 10 12 14 16 18 20
Log (Survival Fraction of 10T1/2 Cells)
Sodium Chromate Concentration (uM)
Experiment 1: Effect of 0.1mM GSH on the Survival of
10T1/2 Cells Treated with Sodium Chromate
Sodium Chromate Sodium Chromate with 0.1mM GSH No Addition Control
65
For experiment 2, the survival fractions of the 10T1/2 cells treated with 0 µM, 1 µM, 2
µM, 3 µM, 4 µM, 5 µM, 10 µM, 15 µM, and 20 µM sodium chromate in were 1 ± 0.1, 0.8 ±
0.1, 0.6 ± 0.1, 0.3 ± 0.1, 0.2 ± 0.03, 0.05 ± 0.01, <0.003, <0.003, and <0.003, respectively (Table
14). The survival fractions of 10T1/2 cells treated with 0 µM, 1 µM, 2 µM, 3 µM, 4 µM, 5 µM,
10 µM, 15 µM, and 20 µM sodium chromate in presence of 0.1 mM GSH were 1.0 ± 0.06, 0.8 ±
0.1, 0.5 ± 0.05, 0.3 ± 0.02, 0.2 ± 0.07, 0.07 ± 0.03, <0.003, <0.003, and <0.003, respectively
(Table 14). There was no decrease of survival when 10T1/2 cells were treated with sodium
chromate in presence of 0.1 mM GSH, compared to 10T1/2 cells treated with sodium chromate
alone (Figure 20). Consistent with experiment 1, the results from experiment 2 show that GSH
did not enhance sodium chromate-induced cytotoxicity to 10T1/2 cells when cells were treated
with 0.1 mM GSH and sodium chromate simultaneously .
66
Figure 20: This figure shows the graphical representation of the survival fractions +/- standard
deviations of 10T1/2 cells for experiment 2. The 10T1/2 cells were treated with either Sodium
chromate alone or Sodium chromate plus 0.1 mM GSH 24 hours after cells were seeded. This
figure shows the survival fraction (on a logarithmic scale) of 10T1/2 cells versus the
concentration of Sodium chromate used to treat the cells.
The survival fractions of 10T1/2 cells from experiments 1 and 2 were averaged. The
average survival fractions of 10T1/2 cells treated with 0 µM, 1 µM, 2 µM, 3 µM, 4 µM, 5 µM,
10 µM, 15 µM, and 20 µM sodium chromate were 1 ± 0.1, 0.9 ± 0.3, 0.6 ± 0.2, 0.4 ± 0.1, 0.2 ±
0.1, 0.1 ± 0.1, <0.01, <0.01, and <0.01, respectively (Table 14). The average survival fractions
of 10T1/2 cells treated with 0 µM, 1 µM, 2 µM, 3 µM, 4 µM, 5 µM, 10 µM, 15 µM, and 20 µM
sodium chromate in the presence of 0.1 mM GSH were 1.0 ± 0.2, 0.8 ± 0.1, 0.5 ± 0.2, 0.4 ± 0.1,
0.2 ± 0.1, 0.1 ± 0.1, <0.01, <0.01, and <0.01, respectively (Table 14). From Figure 21, it was
evident in the averages that the data from both experiment 1 and experiment 2 were consistent.
0.001
0.01
0.1
1
10
0 2 4 6 8 10 12 14 16 18 20
Log (Survival Fraction of 10T1/2 Cells)
Concentration of Sodium Chromate (uM)
Experiment 2: Effect of 0.1mM GSH on the Survival of
10T1/2 Cells Treated with Sodium Chromate
Sodium Chromate Sodium Chromate with 0.1mM GSH No Addition Control
67
The average survival fractions of 10T1/2 cells did not decrease when treated sodium chromate in
presence of 0.1 mM GSH, compared to the survival fractions of 10T1/2 cells treated with sodium
chromate alone. Hence, 0.1 mM GSH did not have an effect on the cytotoxicity of sodium
chromate to 10T1/2 cells when both GSH and sodium chromate were added to 10T1/2 cells
simultaneously.
Figure 21: This figure shows the graphical representation of the survival fractions +/- standard
deviations of 10T1/2 cells for the averages of experiment 1 and 2. 10T1/2 cells were treated with
either sodium chromate alone or sodium chromate plus 0.1 mM GSH 24 hours after cells were
seeded. This figure shows the survival fraction (on a logarithmic scale) of 10T1/2 cells versus the
concentration of sodium chromate used to treat the cells.
0.001
0.01
0.1
1
10
0 2 4 6 8 10 12 14 16 18 20
Log (Survival Fraction of 10T1/2 Cells)
Concentration of Sodium Chromate (uM)
Effect of 0.1 mM GSH on the Survival of 10T1/2 Cells
Treated with Sodium Chromate
Sodium Chromate Sodium Chromate with 0.1mM GSH No Addition Control
68
CHAPTER IV. DISCUSSION and CONCLUSIONS
4.1 Previous Work In Our Laboratory
Previous work done in our laboratory demonstrated a low but dose-dependent and
reproducible yield of type II + type III transformed foci induced by lead chromate in 10T1/2 cells
(Patierno et. al 1988, and Lin, 2011). The low number of type II + type III transformed foci that
we observed in 10T1/2 cells treated with lead chromate did not relect the strong carcinogenicity
of lead chromate (Patierno, 1988; Lin, 2011).
As mentioned, chromium(VI) compounds are known carcinogens, as found in
epidemiological studies, whole animal carcinogenicity studies, and in vitro cell transformation
studies. However, the low yield of type II + type III transformed foci we found that lead
chromate induced was not consistent with the large relative risk values in the epidemiological
studies of workers exposed to lead chromate and the high yield of tumors induced by lead
chromate in the animal carcinogenicity studies. To this end, we hypothesized that reduction of
Cr(VI) to Cr(V), C(IV), and Cr(III) and consequently generation of oxygen radicals (superoxide
and hydroxyl radicals) and reactive oxygen species (hydrogen peroxide) was necessary to
activate chromium(VI) compounds into cytotoxins and cell transforming agents. Thus, we
decided to add a reductive activating system to our assays to another step by supplementing our
cells with reductants, such as ascorbate (or ascorbic acid) and glutathione (GSH).
4.2 Effects of Ascorbic Acid on the Survival of 10T1/2 Cells Treated with Sodium
Chromate
Previous experimentation in our laboratory demonstrated that an ascorbic acid
concentration of 0.0125 mM was able to be the most effective concentration in enhancing
69
cytotoxicity and focus formation in sodium chromate-treated 10T1/2 cells without being
cytotoxic itself (Shahin, 2014). Thus, we used this concentration of ascorbic acid in our
cytotoxicity and transformation experiments. We confirmed from Figures 3 and 4 that 0.0125
mM ascorbic acid is non-cytotoxic to 10T1/2 cells.
We then assessed the effect of 0.0125 mM ascorbic acid on the cytotoxicity of sodium
chromate-treated 10T1/2 cells before testing the effects of this concentration of ascorbic acid on
morphological transformation of sodium-chromate treated 10T1/2 cells. Sodium chromate itself
in the absence of ascorbic acid treatment definitely caused a dose-dependent cytotoxicity to
10T1/2 cells (Figures 7 and 8), The survival of 10T1/2 cells decreased when the cells were
treated with increasing concentrations of sodium chromate.
Regarding the two methods we used to test the effect of ascorbic acid on the cytotoxicity
of sodium chromate to10T1/2 cells , one method of treatment did not show any enhancement of
the cytotoxicity of our sodium chromate-treated 10T1/2 cells, while the other method showed a
modest enhancement of toxicity. Ascorbic acid modestly enhanced the cytotoxicity of sodium
chromate to 10T1/2 cells when added 24 hours to 10T1/2 cells prior to adding sodium chromate
(Figure 8). However, when ascorbic acid and sodium chromate were added to the 10T1/2 cells
simultaneously, ascorbic acid did not enhance the cytotoxicity of sodium chromate to 10T1/2
cells (Figure 7). As mentioned previously, ascorbic acid may act as a pro-oxidant as well as an
anti-oxidant and either enhance or prevent Cr(VI)-induced damage. When 10T1/2 cells were
treated with ascorbic acid and sodium chromate simultaneously, it is likely that ascorbic acid
reduced the sodium chromate before it was able to enter the cells, resulting in Cr(III) species that
would not be able to enter the cell. Thus, ascorbic acid in this case would not be expected to
enhance the cytotoxicity of sodium chromate to10T1/2 cells. Rather, ascorbic acid would
70
enhance some of the survival, which was seen in Figure 7. However, when 10T1/2 cells were
treated with ascorbic acid 24 hours before sodium chromate treatment, the ascorbic acid would
have had time to be absorbed into the cells. Thus, when we add the sodium chromate, more
sodium chromate would be able to enter the cells and then subsequently get reduced inside the
cells by ascorbic acid, which explains the enhancement of cytotoxicity seen by this method.
Only one experiment was conducted with the method of pre-treating 10T1/2 cells with ascorbic
acid before adding sodium chromate, so further trials need to be performed to confirm these
results.
4.3 Effects of Ascorbic Acid on Sodium Chromate induced Morphological Transformation
to 10T1/2 Cells
Previous work in our laboratory showed enhancement of focus formation when 10T1/2
cells were treated with varying concentrations of ascorbic acid in presence of 1 µM of sodium
chromate (Shahin, 2014). This transformation assay was successful, as there was an increase in
the yield of transformed foci for every ascorbic acid concentration treated with sodium chromate
(Shahin, 2014). It was established that 0.0125 mM ascorbic acid was the concentration that
resulted in the highest yield of foci, without being cytotoxic itself to 10T1/2 cells. Thus, we
decided to use this concentration to observe the reductive effects of ascorbic acid to difference
concentrations of sodium chromate in our cytotoxicity assays (mentioned in section 4.2) and our
transformation assays.
As mentioned in section 4.2, ascorbic acid modestly enhanced the cytotoxicity of sodium
chromate to 10T1/2 cells when the cells were treated with ascorbic acid 24 hours prior to sodium
chromate treatment. Thus, for our transformation assay, we wanted to maximize the effect and
71
pre-treat our cells with ascorbic acid 24 hours prior to sodium chromate treatment. However, our
assay was unsuccessful. Unfortunately, our MCA positive control for focus formation showed
few or no transformed foci. MCA is a known as a strong carcinogen and has been used as a
positive control in past experiments to yield focus formation (Reznikoff et al, l973b; Landolph
and Heidelberger, l979). Hence, it is unusual that the MCA used in the transformation assay
yielded almost no foci. Second, very few to no foci formed for all treatment groups, and there
was no significant difference in focus formation between 10T1/2 cells treated with sodium
chromate in the absence or in the presence of ascorbic acid. The results of the transformation
assay are inconclusive due to the fact that our MCA control did not yield positive focus
formation. According to Dr. Landolph, who has many years of experience with this assay, there
is an occasional rare failure of the positive control, MCA, to induce morphological
transformation (foci). Hence, there is likely some factor that caused this experiment to not work.
We suspect that this may be the partial synchrony of the cells after being passaged out of the
G1/G0 state. Occasionally, there may not be many cells in the S phase, where they are most
susceptible to induction of foci. A second alternative is the failure of the cells’ cytochrome P450
to be induced and to activate MCA to MCA epoxides and diol epoxides, which cause mutations
in DNA and focus formation. However, this present transformation assay is preliminary and
more assays need to be done utilizing the same treatments with a good positive MCA control to
be able to determine the effect of ascorbic acid on the morphological transformation of sodium
chromate-induced 10T1/2 cells.
72
4.4 Effect of GSH on the Survival of 10T1/2 Cells Treated with Sodium Chromate
As documented in Table 12 and Figure 15, we observed the highest non-cytotoxic
concentration of GSH to10T1/2 cells to be 0.1 mM. GSH is known to play a role in the
cytoplasmic reduction of chromate at physiological pH, leading to chromium(V) complexes,
thiyl radicals, and reactive oxygen species that can cause cytotoxicity (O’Brien, 1992).
However, in our cell survival experiments, GSH did not enhance the cytotoxicity of sodium
chromate to10T1/2 cells (Figure 21). As mentioned, GSH is known to contribute to
chromium(VI)-induced toxicity through cytoplasmic reduction of chromate. However, the
method we used to treat our 10T1/2 cells required that we added GSH and sodium chromate
simultaneously. We need to explore different methods of treatment of 10T1/2 cells with GSH
and sodium chromate to analyze the reason as to why GSH had no effect on sodium chromate-
induced cytotoxicity to 10T1/2 cells.
4.5 Future Directions
Chromium(VI) reduction inside the cell is known to result in reactive chromium
intermediates, such as Cr(V) and Cr(IV), that generate reactive oxygen species that result in
Cr(VI)-induced damage and carcinogenesis, and also Cr(III), which binds to DNA and causes
mutations (Shi, 1999). Ascorbate and GSH are known to be the main biological reducers in the
cell to aid in the chromium(VI) reduction process (Shi, 1999). In our experiments, ascorbic acid
modestly enhanced the cytotoxicity of sodium chromate to 10T1/2 cells when cells were
pretreated with ascorbic acid 24 hours prior to sodium chromate treatment. However, our
transformation assay yielded little to no transformation with our MCA control or with our Cr(VI)
treated 10T1/2 cells. GSH also had no effect on the cytotoxicity of sodium chromate in 10T1/2
73
cells with the method of treatment we used. We definitely need to explore different methods of
treatment for our cytotoxicity assays to maximize the effect of ascorbic acid or GSH on sodium
chromate cytotoxicity to 10T1/2 cells. We also need to conduct more transformation assays to
get good MCA controls so that our results could be conclusive. Once we obtain good
transformation data, we want to create dose-response curves for the Cr(VI)-induced
morphological transformation, ring clone foci of our Cr(VI)-induced morphologically
transformed cells, create transformed cell lines, and then characterize the biological properties of
these transformed cell lines. Once we are successful in this, the next step would be to use DNA
microarrays to molecularly characterize the aberrations that occur in our Cr(VI)-induced
transformed 10T1/2 cell lines.
74
ACKNOWLEDGMENTS
I would like to thank my advisor and mentor, Dr. Joseph R. Landolph, Jr., Ph.D., for all
his support and guidance throughout my time in the Molecular Microbiology and Immunology
M.S. program at USC. He has motivated me to work through my experiments, guided me to
complete my thesis, and guided me to perform well in my courses in the M.S. program. I would
also like to thank my colleague, Mr. Qasim Akinwumi, B.S., M.S., Ph.D. student, Department of
Biochemistry, College of Medicine, in the University of Ibadan, Ibadan, Nigeria, working in Dr.
Landolph’s laboratory toward his Ph.D. degree, for training me in the laboratory and teaching me
all the techniques necessary to complete the work in my thesis. I would like to thank Mrs.
Sophia Allaf Shahin, B.S., M.S., in training me in Dr. Landolph’s laboratory. I would also like
to thank Ms. Ibukun Oluwawemitan, M.S., in working with me side by side and guiding me to
complete my experiments. I would surely not have been able to endure the long hours in the
laboratory or have completely my experiments efficiently without Ms. Oluwawemitan’s aid. I
would also like to give special thanks to Mr. William Liao, B.S. student at USC, Ms Shelly
Tseng, B.S. student at USC, Ms. Joanne Lin, B.S. student at USC, Mr. Matthew Moodie, B.S.,
and Sid Menon, B.S. in taking part to help carry out my experiments.
75
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O’Brien, P. and Wang, G. A Potentially Significant One-Electron Pathway in the Reduction of
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Patierno, S., Banh, D., and Landolph, J. Transformation of C3H/10T1/2 Mouse Embryo
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Abstract (if available)
Abstract
Hexavalent chromium compounds are known human carcinogens. Epidemiological studies have shown that occupational exposure of humans to chromium(VI) compounds results in an increased risk of respiratory cancers. The exact mechanisms of chromium(VI) carcinogenesis is unknown, but chromium(VI) compounds have been shown to cause chromosomal aberrations, mutations, and morphological and neoplastic transformation in mammalian cells. ❧ In this thesis, we investigated the ability of sodium chromate (Na₂CrO₄), a soluble chromium(VI) compound, in its ability to induce cytotoxicity and morphological transformation in cultured C3H/10T1/2 Cl 8 (10T1/2) mouse embryo cells. We hypothesized that intracellular reductants, such as ascorbate, glutathione (GSH), and cysteine, can reductively activate Cr(VI) intracellulary to proximate cytotoxins and cell transforming agents. Recent studies in our laboratory have demonstrated the ability of ascorbate to enhance focus formation in 10T1/2 cells treated with varying concentrations of ascorbic acid in presence of sodium chromate. In this thesis, we performed cytotoxicity and morphological transformation assays by treating 10T1/2 cells with varying concentrations of sodium chromate in the presence of 0.0125 mM Ascorbic acid, the concentration that yielded the greatest increase in foci from our recent studies. In addition, we determined the highest, non-cytotoxic concentration of GSH and applied that concentration to 10T1/2 cells in cytotoxicity assays. ❧ Our cell survival data showed that ascorbic acid was able to modestly enhance the cytotoxicity of sodium chromate to 10T1/2 cells when the cells were treated with ascorbic acid 24 hours prior to sodium chromate treatment. However, there was no enhancement of cytotoxicity, but rather, a slight enhancement of survival, when 10T1/2 cells were treated with ascorbic acid and sodium chromate simultaneously. Our first transformation assay with ascorbic acid and sodium chromate showed little to no focus formation but the positive control, 3-metylcholanthrene (a strong carcinogen), did not induce foci so this experiment is not valid. This data is preliminary, and further assays need to be completed to obtain a conclusive result. Our cell survival data also showed no enhancement of cell cytotoxicity when GSH was added to our sodium-chromate treated 10T1/2 cells. However, only one method of treatment was used for this assay and other methods need to be explored. ❧ We need to explore different methods of treatment to observe the effects of ascorbic acid and GSH on our sodium-chromate induced cytotoxic and transformation assays. We want to maximize the cytotoxicity and cell transforming activity of chromium(VI) compounds so we can eventually create transformed cell lines, characterize them, and study the aberrations in gene expression that occur in Cr(VI)-transformed cell lines and hence, the molecular mechanisms of chromium(VI) carcinogenesis.
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Tran, Laureen
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Effect of reductants on sodium chromate-induced cytotoxicity and morphological transformation to C3H/10T1/2 Cl 8 mouse embryo cells
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Molecular Microbiology and Immunology
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