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Effects of ascorbate on cytotoxicity and morphological transformation induced by insoluble chromium (VI) compounds in C3H 10T1/2 Cl 8 mouse embryo cells
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Effects of ascorbate on cytotoxicity and morphological transformation induced by insoluble chromium (VI) compounds in C3H 10T1/2 Cl 8 mouse embryo cells
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Content
EFFECTS OF ASCORBATE ON CYTOTOXICITY AND
MORPHOLOGICAL TRANSFORMATION INDUCED BY
INSOLUBLE CHROMIUM (VI) COMPOUNDS IN C3H
10T1/2 Cl 8 MOUSE EMBRYO CELLS
By
Ibukunoluwa Oluwawemitan
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfilment 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 Ibukunoluwa Oluwawemitan
2
TABLE OF CONTENTS
ABSTRACT…………………………………………………………………………………….…4
LIST OF TABLES………………………………………………………………………………...6
LIST OF FIGURES…………………………………………………………………………….…7
CHAPTER I: INTRODUCTION……………………………………………………………….…8
1.1 Epidemiological Studies on Hexavalent Chromium Compounds…………………………….8
1.2 Molecular Mechanism of Cr (VI)-induced Carcinogenicity…………………………… ........9
1.3 Role of Ascorbic Acid in Cr(VI) Reduction………………………………….………………...10
1.4 Effect of Particle Size on Cr(VI)-induced Cytotoxicity to C3H 10T1/2
Cells……………………………………………………………………………………………..….11
1.5 Goal of This Thesis……………………………………….……………………………………....12
CHAPTER II: MATERIALS AND METHODS………………………………………………...13
2.1 Chemicals ……………………..……...……………………..……………………..…….13
2.2 Cells and Cell Culture …………………………………………….…………………………..…13
2.3 Cell Culture Methods ……………………….…………………………………………………...14
2.4 Determination of Plating Efficiencies of the Cells …………………………………….…….15
2.5 Preparation of Chromium Compounds for Cytotoxicity Studies ………………………..…16
1.1 Cytotoxicity Assays …………………………………………………………..…………………..16
2.7 Determining the Effect of Ascorbic Acid on the Survival of 10T1/2 Cells Treated with
Sonicated Particulate Hexavalent Chromate………………………………...........................….17
2.8 Determining the Effect of Ascorbic acid upon the yield of Morphological Transformation
Induced by Particulate Hexavalent Chromate in 10T1/2 Cells……………………...................19
3
CHAPTER III: RESULTS ………………………………………………………………………21
3.1 Effect of Sonication of Cr(VI) Compounds on Their Cytotoxicity to 10T1/2
Cells………………………………………………………………………………………………….….21
3.2 Determination of Highest Non-Cytotoxic Concentrations of Ascorbic Acid…………..….23
3.3 Effect of Ascorbic Acid on the Survival of 10T1/2 cells Treated with Sonicated Lead
Chromate……………………………………………………………………………………….………28
3.4 Effect of Ascorbic acid on Lead Chromate-induced Morphological Transformation of
10T1/2 Cells…………………………………………………………….………………………..........40
3.5 Effect of Ascorbic Acid on the Survival of 10T1/2 cells Treated with Sonicated Barium
Chromate…………………………………………………………………………………………….....46
CHAPTER IV: DISCUSSION AND CONCLUSION……………………..................................52
4.1 Effect of Ascorbic Acid on the Survival of 10T1/2 cells Treated with Sonicated Lead
Chromate…………………………….………………………………………………………………....53
4.2 Effect of 0.0125 mM Ascorbic Acid on Sonicated Lead Chromate-Induced Morphological
Cell Transformation …………………………………………………………………….…………....54
4.3 Effect of Ascorbic Acid on the Survival of 10T1/2 cells Treated with Sonicated Barium
Chromate………………………………………………………………….…………………………....55
FUTURE DIRECTIONS………………………………………………………………………...55
ACKNOWLEDGMENTS…………………………………………………………………….…57
REFERENCES ……………………………………………………………………………….... 58
4
ABSTRACT
Hexavalent chromium [Cr(VI)] compounds are well established human carcinogens.
However, Cr(VI) compounds have different potencies as carcinogens, and the potency is
dependent on both valence and solubility of the chromium (VI) compound. It has been shown
that lead chromate particles (mean particle diameter of 80 µm) induced dose-dependent
cytotoxicity and a dose-dependent, but low, yield of morphological transformation (focus
formation) in C3H 10T1/2 Cl 8 (10T1/2) mouse embryo cells (Patierno et al, l988). It was also
observed that reduction of the particle size of lead chromate by sonication enhanced the
cytotoxicity in a dose-dependent manner, but the yield of morphological transformation (focus
formation) in C3H 10T1/2 Cl 8 (10T1/2) cells was still low (Akinwunmi, Oluwawemitan, and
Landolph, manuscript in preparation).
This thesis study sought to test the hypothesis that intracellular reductants, such as ascorbic
acid, can enhance cytotoxicity and morphological transformation in 10T1/2 cells treated with
insoluble chromate compounds, by intracellular reduction of Cr(VI) to Cr(III). In order to
enhance cytotoxicity and morphological transformation further, we investigated the effects that
ascorbic acid, an intracellular reductant, has on insoluble Cr(VI) compound-induced cytotoxicity
and morphological transformation of 10T1/2 mouse embryo cells. The concentrations of
sonicated lead chromate and barium chromate we used were 0.25 µg/ml, 0.5 µg/ml, 0.75 µg/ml,
and 1.0 µg/ml. By conducting cytotoxicity assays, we determined the highest non-cytotoxic
concentrations of ascorbic acid to 10T1/2 cells, and showed that ascorbic acid was non-cytotoxic
at concentrations of 0.00625 mM to 0.1 mM. Our results showed that pre-treatment of 10T1/2
cells with 0.0125 mM ascorbic acid enhanced the cytotoxicity and morphological transformation
of 10T1/2 cells treated with lead chromate in a dose-dependent manner. In one preliminary
5
experiment, we showed that 0.0125 mM ascorbic acid increased the yield of morphological
transformation in 10T1/2 cells treated with 0.75µg/ml or 1.0 µg/ml of lead chromate by 4-fold
and 6-fold, respectively. In summary, this thesis found that 0.0125 mM ascorbic acid enhanced
cytotoxicity and morphological transformation induced by sonicated particles of lead chromate
in 10T1/2 mouse embryo cells.
6
LIST OF TABLES
Table 1: LC50 and LC37 of Unsonicated and Sonicated Particulate Chromates.....………..…...22
Table 2: Plating efficiency and survival fraction of 10T1/2 cells treated with 0.00625 mM to
0.375 mM Ascorbic Acid upon seeding……………………………………………...….23
Table 3: Plating efficiency and survival fraction of 10T1/2 cells treated with 0.00625 mM to 0
0.375 mM Ascorbic Acid 24 hours after seeding (Experiment 1)…………………….....25
Table 4: Plating efficiency and survival fraction of 10T1/2 cells treated with 0.00625 mM to
0.375 mM Ascorbic Acid 24 hours after seeding (Experiment 2)…………………….....27
Table 5: Plating Efficiency and Survival Fraction of 10T1/2 Cells Treated with Sonicated Lead
Chromate and 0.0125 mM Ascorbic Acid (treatment at 3 different time points)……………......29
Table 6: Plating Efficiency and Survival Fraction of 10T1/2 Cells Treated with Sonicated Lead
Chromate and 0.0125 mM Ascorbic Acid (treatment at 3 different time points)……………..…35
Table 7: Effect of 0.0125mM Ascorbic Acid on Lead Chromate Induced Morphological
Transformation to 10T1/2 Cells.………………………………………………………...…….....42
Table 8: Cytotoxicity Data for Transformation Experiment with sonicated lead chromate and
ascorbic acid…………………………………………………………………………….………..44
Table 9: Plating efficiency and survival of 10T1/2 cells treated with sonicated barium chromate
and 0.0125 mM ascorbic acid (Experiment 1)………………………………………………...…46
Table 10: Plating efficiency and survival of 10T1/2 cells treated with sonicated barium chromate
and 0.0125 mM ascorbic acid (Experiment 2)…………………………………………………...49
7
LIST OF FIGURES
Figure 1: Graph showing survival fraction of 10T1/2 cells treated with 0.00625 mM to 0.375
mM Ascorbic Acid upon seeding (Experiment 1)…………….......................…………………..24
Figure 2: Graph showing survival fraction of 10T1/2 cells treated with 0.00625 mM to 0.375
mM Ascorbic Acid 24 hours after seeding (Experiment 1)……………………….……………..26
Figure 3: The survival fraction of 10T1/2 cells treated with 0.00625 mM to 0.375 mM Ascorbic
Acid 24 hours after seeding (Experiment 2)………………………………………...…………...28
Figure 4: Effect of 0.0125 mM ascorbic acid on the survival of 10T1/2 cells treated with
sonicated lead chromate (cells treated with ascorbic acid upon seeding)………………………..31
Figure 5: Effect of 0.0125 mM ascorbic acid on the survival of 10T1/2 cells treated with
sonicated lead chromate (cells treated with ascorbic acid 24 hours after seeding)…………...…32
Figure 6: Effect of 0.0125 mM ascorbic acid on the survival of 10T1/2 cells treated with
sonicated lead chromate (cells treated with ascorbic acid 24 hours after treatment with sonicated
lead chromate)…………………………………………………………………………………....33
Figure 7: Effect of 0.0125 mM ascorbic acid on the survival of 10T1/2 cells treated with
sonicated lead chromate (showing the three different treatments)………………………..……..34
Figure 8: Effect of 0.0125 mM ascorbic acid on the survival of 10T1/2 cells treated with
sonicated lead chromate (cells treated with ascorbic acid upon seeding)………………..………37
Figure 9: Effect of 0.0125 mM ascorbic acid on the survival of 10T1/2 cells treated with
sonicated lead chromate (cells treated with ascorbic acid 24 hours after seeding)……………...38
Figure 10: Effect of 0.0125 mM ascorbic acid on the survival of 10T1/2 cells treated with
sonicated lead chromate (showing the three different treatments)……………………………....40
Figure 11: Effect of 0.0125 mM ascorbic acid on sonicated lead chromate-induced
morphological transformation……………………………………………………………………43
Figure 12: Graphical representation of the cytotoxicity data for the transformation experiment
with sonicated lead chromate and ascorbic acid……………………………………………...….45
Figure 13: Effect of Ascorbic acid on survival of 10T1/2 cells treated with barium chromate
(Experiment 1)………………………………………………………………………………..….48
Figure 14: Effect of Ascorbic acid on survival of 10T1/2 cells treated with barium chromate
(Experiment 2)……………………………………………………………………..…………….51
8
CHAPTER I. INTRODUCTION
1.1 Epidemiological Studies on Hexavalent Chromium Compounds
Chromium exists largely in two valence states in nature: hexavalent chromium [Cr(VI)]
and trivalent chromium [Cr(III)] (Bagchi et al, 2007). Cr(VI) compounds are carcinogens in
animals and in humans. Animal studies and in vitro studies show that hexavalent chromium
compounds are more potent in causing chromosomal aberrations and cancer than Cr(III)
(Norseth, 1981). Chromium (III) is poorly transported across cellular membranes (Bagchi et al
2002). Thus, most Cr(III) compounds are approximately 1,000-fold less cytotoxic/toxic,
mutagenic, and less carcinogenic than Cr(VI) compounds (Landolph and Biedermann, l990; Shi,
1999).
The first association of [Cr(VI)] with respiratory cancer in humans was in 1890, when a
case report described a chrome pigment worker with an adenocarcinoma in his nasal turbinate
bone (reviewed in Wise et al, 2006). Epidemiological studies have indicated that occupational
exposure to Cr(VI) causes an 18-80-fold increased risk of lung cancer (reviewed in Biedermann
and Landolph, 1990; Wise et al, 2002). Hexavalent chromium [Cr(VI)] compounds are
commonly used in industrial chrome plating, welding, painting, metal finishing, steel
manufacturing, alloys such as stainless steel (which consists of nickel, iron, and chromium), cast
iron, and wood treatment (Bagchi et al, 2002).
Approximately 360,000 workers in the U, S. and several million workers worldwide are
currently exposed to Cr (VI) compounds in the workplace (Zhitkovich et al, 2005).
Environmental exposure of humans to Cr(VI) compounds may also occur when workers inhale
airborne particulate Cr(VI) compounds in the vicinity of smelters and Cr-waste sites (IARC
9
1990; Freeman and Lioy, 1997). Furthermore, autopsies of Cr-exposed workers show that Cr
levels accumulate and persist at bronchial bifurcation sites for as long as twenty years after
exposure to chromium (Ishikawa et al, 1994).
There are also recurrent concerns about the potential adverse effects of exposure of
residents to Cr(VI) in residential areas from contaminated water and soil. However, the extent
of exposure and the presence of toxicologically relevant biological damage in individuals
residing in the vicinity of Cr(VI)-contaminated sites has been a subject of intense debate
(Zhitkovich and Reynolds, 2007).
1.2 Molecular Mechanism of Cr (VI)-induced Carcinogenicity
Hexavalent chromium (Cr(VI)) compounds are well established human carcinogens.
However, Cr(VI) compounds appear to have different potencies as carcinogens (Wise et al,
2010). Carcinogenicity of chromium compounds depends on both valence and solubility
(Patierno et al, 1988; Landolph and Biedermann, l990). Studies have shown that the water-
insoluble (particulate) Cr(VI) salts are more potent than the water soluble Cr(VI) salts (Wise et
al, 2002).
Laboratory studies have shown that hexavalent chromium is genotoxic, mutagenic, and
clastogenic, while the trivalent chromium compounds are inactive (Wise et al, 1994) Molecular
mechanisms underlying the cytotoxic, mutagenic, and carcinogenic effects of Cr(VI)compounds
are complex, in particular due to its complicated intracellular chemistry and the involvement of
multiple intracellular targets and pathways (Zhitkovich and Reynolds, 2007).
Under physiological conditions, Cr(VI) is unreactive with DNA and requires reductive
activation to Cr(III) to induce biological damage (Zhitkovich and Reynolds, 2007). Within the
10
cell, Cr(VI) is metabolically reduced to Cr(III) by agents such as ascorbate, cysteine and
glutathione (Singh et al, 1999). The reduction process generates transient intermediate oxidation
states of chromium, such as Cr(V), Cr(IV) and reactive oxygen species (ROS), including oxygen
radicals (superoxide, hydrogen peroxide, and hydroxyl radicals) (Singh et al, 1999). This
reduction process leads to diverse cytotoxic and genotoxic effects, such as chromosomal damage,
DNA strand breaks, DNA-protein crosslinks (Wise et al., 1992, 1993), Cr-DNA adducts (Singh
et al., 1998a; Xu et al., 1996), DNA-DNA crosslinks (Singh et al., 1998b; Xu et al., 1996), and
inhibition of both DNA replication and transcription (Xu et al., 1996; Bridgewater et al., 1994;
Manning et al., 1992) decreased fidelity of DNA replication and increased mutagenesis (Snow,
1991; Singh and Snow, 1998), membrane lipid peroxidation (Susa et al, 1997), and inhibition of
mitochondrial respiration (Ryberg and Alexander, 1990). These effects may trigger diverse
cellular responses, such as cell cycle arrest, apoptosis, or carcinogenesis (reviewed by Singh et
al., 1998c).
1.3 Role of Ascorbic Acid in Cr(VI) Reduction
The reduction of Cr(VI) to Cr(III) can occur by a multiplicity of mechanisms, dependent
on the nature of the reducing agents (Shi et al., 1999). ROS, including superoxide, hydrogen
peroxide, and hydroxyl radicals, and free radicals (superoxide and hydroxyl radicals) are
generated from intermediate oxidation states of Cr [Cr(V) and Cr(IV)], and are keys to Cr(VI)-
induced carcinogenesis. At physiological pH, various cellular constituents such as glutathione
(GSH), cysteine, lipoic acid, and diol-containing molecules such as NADPH, ribose, fructose,
and arabinose, have been shown to reduce Cr(VI) in vitro (Shi et al., 1999). Reduction of Cr(VI)
to Cr(III) can be accomplished through non-enzymatic reactions with cysteine and glutathione.
However, in the target tissues of chromate toxicity (lung), ascorbate (Asc) is the primary reducer
11
of Cr(VI). Ascorbate causes the highest rate of Cr(VI) reduction among all biological reducing
agents. Asc-driven reactions generate high levels of Cr-DNA binding, which results in the
formation of highly mutagenic Asc-Cr-DNA crosslinks (Zhitkovich et al., 2005, 2007).
1.4 Effect of Particle Size on Cr(VI)-induced Cytotoxicity to C3H 10T1/2 Cells
Occupational inhalation of certain particulate Cr (VI) compounds, such as lead chromate,
has been associated with toxicity to respiratory tissue and lung cancer (Hayes, 1988; IARC,
1990; Langard, 1990). Thus, particulate Cr(VI) interacts with the epithelial cells lining the
bronchial airways to cause short-term toxicity to the lung and long-term carcinogenicity in the
lung (Singh et al, 1999). Animal and cell culture studies indicate that water solubility plays a key
role in the carcinogenicity of Cr(VI) compounds. Water-insoluble or “particulate” Cr(VI)
compounds are very potent carcinogens (Wise et al., 2006). It is unclear why insoluble
particulate Cr(VI) compounds are more carcinogenic than soluble Cr(VI) compounds. One
possibility is that insoluble Cr(VI) compounds are phagocytosed in large quantities into
mammalian cells, depositing large amounts of Cr(VI) intracellularly. In addition, human
pathology studies have shown that Cr(VI) compounds are deposited and persist at bronchial
bifurcations where Cr-associated cancers occur, which is consistent with a particulate exposure
inducing cancer of the respiratory system (Ishikawa et al, 1994; Wise et al, 2006).
Previous work in our laboratory showed that lead chromate induced cytotoxicity and
morphological and neoplastic transformation of C3H10T1/2 Cl 8 (10T1/2) mouse embryo cells
in a dose-dependent manner. However, the yield of morphological transformation (focus
formation) was low and did not correlate with epidemiological and animal studies that show
particulate lead chromate as a potent human carcinogen. Our laboratory concluded that the weak
morphological transformation caused by lead chromate in 10T1/2 cells could be a result of the
12
inefficient phagocytosis of lead chromate particles into 10T1/2 cells due to the large particle
size (mean particle size, 80 um, measured by laser diffraction).
1.5 Goal of This Thesis
The overall goal in our laboratory is to use in vitro assays to investigate molecular
mechanisms of Cr(VI) compound-induced morphological and neoplastic transformation to
10T1/2 mouse embryo cells. The specific aims of this thesis were to a) determine whether
ascorbic acid enhances the cytotoxicity and morphological transformation in 10T1/2 cells treated
with insoluble chromate compounds; and b) develop dose-response curves for insoluble
chromate-induced morphological transformation, in the presence of ascorbic acid.
13
CHAPTER II: MATERIALS AND METHODS
2.1 Chemicals
The chemicals used in these experiments were purchased from various commercial
biotechnology and chemical companies. The lead chromate (99.8% purity), ascorbic acid (97%
purity), reduced glutathione (GSH), and 3-methylcholanthrene (MCA, 98% purity) used were all
purchased from Sigma-Aldrich Company, St. Louis, Missouri. The ascorbic acid and glutathione
were dissolved in Dulbecco’s phosphate-buffered saline 1X (DPBS), purchased from Corning
Glass Company, Lake Placid, New York. Lead chromate and barium chromate were dissolved
in high pressure liquid chromatography (HPLC) grade acetone, purchased from Fisher Scientific
Company, Fair Lawn, New Jersey.
2.2 Cells and Cell Culture
The C3H/10T1/2 Cl 8 (10T1/2) cell line used in our experiments was established by
Reznikoff, Brankow, and Heidelberger (Reznikoff et al, 1973). 10T1/2 cells are an aneuploid and
immortal mouse embryonic cell line derived from the embryos of pregnant C3H mice (Reznikoff
et al, 1973; Patierno et al 1988). 10T1/2 cells form flat, even monolayers when confluent
(Reznikoff et al, 1973a). 10T1/2 cells are anchorage-dependent and highly sensitive to post-
confluence inhibition of cell division (Reznikoff et al, 1973a). 10T1/2 cells also have a very low
frequency of spontaneous transformation, and do not form tumors when injected into Balb/c
nude mice. 10T1/2 cells are stable in culture, and serve as a good in vitro model to study
chemical carcinogen-induced cytotoxicity and morphological transformation, and hence as a
good model system to study induction and characterization of fibrosarcomas (Reznikoff et al,
14
l973b; Landolph and Heidelberger, l979; reviewed in Landolph, l985; Miura et al, l987; Patierno
et al, l988).
2.3 Cell Culture Methods
10T1/2 cells were cultured in Basal Medium Eagle’s (BME), supplemented with 10%
heat-inactivated fetal bovine serum (FBS) without antibiotics. The BME powder was purchased
from GIBCO Company, Grand Island, New York, and the FBS was purchased from Omega
Scientific Products, Tarzana, California. BME was prepared in the Bio reagents Core Facility,
USC/Norris Comprehensive Cancer Center at the University of Southern California (USC), by
Ms. Nily Harel, Technician, under the supervision of Professor Zoltan Tokes (Director of the Bio
reagents Core Facility at USC). BME powder was dissolved in water, the pH of the solution was
adjusted to 7.2, and then the solution was sterilized by filtration through a 0.1 um filter.
10T½ cells of passages from 7-14 that had been previously frozen and stored in liquid
nitrogen, were thawed and used in these experiments in this thesis as described in Reznikoff et
al, 1973; Landolph and Heidelberger, l979; and Landolph, l985. 10T1/2 cells were retrieved
from the liquid nitrogen tanks and thawed quickly in a water bath set at 37
degrees Celsius. The
cells were then transferred to a 15 ml centrifuge tube and centrifuged for 12 minutes at 3,000
rpm in an IEC-HN-S centrifuge (Damon/IEC Division). After centrifugation, the supernatant was
aspirated and discarded. The cell pellet was re-suspended in 1ml of BME containing 10% (v/v)
FBS. The cell suspension was then transferred to a 25 cm
2
vented T-flask (Corning Glass Works,
Corning NY). The centrifuge tube containing the cell suspension was then rinsed with an
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
15
at 37 degrees Celsius with an atmosphere of 5% CO2. After 24 hours, the medium in the flask
was aspirated and discarded to remove residual dimethyl sulfoxide (DMSO) and dead cells.
Fresh BME containing 10% FBS was then added to the flask, and incubation was continued,
with a medium change every three days, until the cells reached approximately 80% confluence
(Reznikoff et al, 1973a; Landolph and Heidelberger, 1979; Patierno et al, 1988; Landolph, 1994).
All tissue culture flasks and dishes were purchased from Corning Glass Works, Corning, New
York, U. S. A.
2.4 Determination of Plating Efficiencies of the Cells
Once the 10T1/2 cells from passages 7-15 reached 80% confluence, plating efficiency
experiments were conducted using standard laboratory protocols published by Dr. Charles
Heidelberger’s laboratory and our laboratory (Reznikoff et al, l973a, b; Landolph and
Heidelberger, l979; Miura et al, l989; Patierno et al, l988). First, the medium from the flask of
cells was aspirated, and 5 ml 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 living cells. Once most of the cells detached
from the flask, 1ml of BME containing 10% FBS was added to neutralize the action of the
trypsin. The detached cell solution was transferred to a 15 ml centrifuge tube and centrifuged for
12 minutes at 3,000 rpm in an IEC-HN-S centrifuge (Damon/IEC Division). After
centrifugation, the supernatant was aspirated and discarded, and the pellet was re-suspended in
10 ml of BME containing 10% FBS. 1 ml of re-suspended cell suspension was mixed with 19
ml of DPBS in a Coulter counter vial, and the number of cells was then counted electronically in
a Coulter Counter Model Zf (Coulter Electronics, Hialeah, Florida). Two hundred cells were
16
seeded into five 60-mm Corning petri dishes to determine the plating efficiencies of 10T1/2 cells
in BME containing 10% FBS.
2.5 Preparation of Particulate Sonicated Hexavalent Chromium Compounds for Cytotoxicity
Studies
Particulate (insoluble) lead chromate and barium chromate were suspended in acetone to
sterilize them, added to a glass centrifuge tube, and then sonicated with a Braun Sonic Ultra
sonicator set at 150 KV for 20 minutes.
2.6 Assays for Chemically Induced Cytotoxicity Determining the Non-Toxic Concentrations of
Ascorbic acid to 10T1/2 cells
To determine the highest non-cytotoxic concentrations of ascorbic acid that could be used
for our experiments, 10T½ cells were seeded at 200 cells per 60 mm petri dish, with five dishes
being used for each concentration of ascorbic acid along with the controls. The cells were
seeded according to the plating efficiency protocol described above 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. The controls for the experiments were; a.) No addition control
(NA) and b.) DPBS control (blank). This experiment was carried out using two different
methods. The first method was to seed the cells and then treat them with the different
concentrations of ascorbic acid 24 hours after seeding. We seeded 200 cells/60 mm dish in 5 ml
of BME containing 10% FBS (five dishes per concentration) and administered 100 L of each
concentration of ascorbic acid. The dishes were incubated at 37 degrees Celsius, in a constant
flow carbon dioxide incubator with a humidified atmosphere of 5% carbon dioxide. After 48
hours (post ascorbic acid treatment), the medium was aspirated from each dish and replaced with
17
5 mls of fresh BME containing 10% FBS and retreated with the appropriate concentrations of
ascorbic acid (100 L). The dishes were incubated for another 7 days (making a total of 10
incubation days), after which the cells were fixed with 100% methanol, for 1 hour, and the
methanol was then aspirated. The cells were then stained with 10% crystal violet for 1 hour, and
then colonies containing 20 or more cells were scored under a dissecting light microscope.
For the second method, we seeded the cells and treated them with ascorbic acid at the
same time on the same day, called day 0. The dishes were incubated at 37 degrees Celsius, in a
humidified, constant flow carbon dioxide incubator with an atmosphere of 5% carbon dioxide.
After 72 hours (post seeding/ascorbic acid treatment), the medium was aspirated from each dish
and replaced with 5 ml of fresh BME containing 10% FBS and retreated with 100 L of ascorbic
acid. The dishes were incubated for another 7 days (making a total of 10 incubation days), then
the cells were fixed with 100% methanol and then stained with 10% crystal violet and colonies
containing 20 or more cells were scored under a dissecting light microscope.
2.7. Determining the Effect of Ascorbic Acid on the Survival of 10T1/2 Cells Treated with
Sonicated Particulate Hexavalent Chromate
10T1/2 cells were seeded at 200 cells per 60 mm dish, with five dishes for each
concentration, according to the plating efficiency procedure previously described in section 2.4.
The concentration and volume of ascorbic acid used in this experiment were 0.0125 mM and 100
µl, respectively. This concentration was determined from experiments carried out in section 2.6.
The effect of ascorbic acid on the survival of 10T1/2 cells treated with sonicated particulate
hexavalent chromates was determined using three different treatment methods.
18
For the first method, cells were seeded at 200 cells per 60 mm dishes, treated with 100µl
of 0.0125 mM ascorbic acid, and incubated. Twenty four hours later, we treated cells with 25 µl
of particulate hexavalent chromates (lead chromate and barium chromate) using four low
cytotoxic concentrations (0.25, 0.5, 0.75 and 1.0 µg/ml). Forty eight hours after treatment with
particulate hexavalent chromate, we aspirated the medium, replaced it with fresh BME and re-
treated the cells with 100 µl of 0.0125 mM ascorbic acid. The dishes were next incubated for
another 7 days (making a total of 10 incubation days), after which the cells were fixed with
100% methanol, then stained with 10% crystal violet, and colonies containing 20 or more cells
were then scored under a dissecting light microscope.
For the second method, we seeded 200 cells per each 60 mm dish, and incubated the cells
at 37 degrees Celsius, in a constant flow carbon dioxide incubator with a humidified atmosphere
of 5% carbon dioxide. Twenty four hours after seeding the cells, we treated cells with 100 µl
ascorbic acid and 25 µl sonicated particulate hexavalent chromate (lead chromate and barium
chromate). Forty eight hours after treatment of cells with particulate hexavalent chromate, we
aspirated the medium, replaced it with fresh BME, and retreated the cells with 100 µl of 0.0125
mM ascorbic acid. The dishes were incubated for another 7 days (making a total of 10 incubation
days) after which the cells were fixed with 100% methanol, then stained with 10% crystal violet,
and colonies containing 20 or more cells were scored under a dissecting light microscope.
For the third method, we seeded 200 cells per 60 mm dish and incubated the dishes at 37
degrees Celsius, in a constant flow carbon dioxide incubator with an atmosphere of 5% carbon
dioxide. Twenty four hours after seeding, we treated the cells with 25 µl of sonicated particulate
hexavalent chromate (lead chromate and barium chromate) and incubated the treated dishes. We
treated the cells with 100 µl ascorbic acid, twenty four hours after sonicated particulate chromate
19
treatment. Forty eight hours after treatment of cells with particulate hexavalent chromate, we
aspirated the medium, replaced it with fresh BME, and re-treated the cells with 100 µl of 0.0125
mM ascorbic acid. The dishes were incubated for another 7 days (making a total of 10 incubation
days) after which the cells were fixed with 100% methanol, then stained with 10% crystal violet,
and colonies containing 20 or more cells were scored under a dissecting light microscope.
2.8 Determining the Effect of Ascorbic acid upon the yield of Morphological Transformation
Induced by Particulate Hexavalent Chromate in 10T1/2 Cells
We designed and conducted one transformation assay to determine whether ascorbic acid
could enhance the yield of morphological transformation (focus formation) induced by sonicated
lead chromate in 10T1/2 cells. We seeded 2,000 cells per 60 mm dish according to the plating
efficiency procedure described previously in section 2.4, with twenty dishes for each treatment,
along with the appropriate controls. The dishes were then incubated at 37 degrees Celsius, in a
humidified constant flow carbon dioxide incubator with an atmosphere of 5% carbon dioxide.
Four days after seeding cells, we pre-treated some of the dishes with 100 µl 0.0125 mM
ascorbic acid and incubated the cells at 37 degrees Celsius, in a humidified, constant flow carbon
dioxide incubator with an atmosphere of 5% carbon dioxide. On day five (twenty four hours after
treating cells with ascorbic acid) we treated the cells with 25 µl sonicated lead chromate and
incubated the cells at 37 degrees Celsius, in a humidified, constant flow carbon dioxide incubator
with an atmosphere of 5% carbon dioxide. 1µg/ml of 3-Methylchloranthrene (MCA), a strong
chemical mutagen and carcinogen, served as the positive control for this morphological
transformation assay. Other controls used in this transformation assay were: a no addition
control, a DPBS control (0.1 ml/dish), a 0.0125 mM ascorbic acid only control, and an acetone
20
only (0.5%, v/v) control. Forty-eight hours later (day 7), we performed a medium change by
aspirating the medium from each dish and replacing it with fresh medium (BME with 10% FBS).
After the medium change, the dishes initially treated with ascorbic acid were retreated with 100
µl of 0.0125 mM ascorbic acid. The dishes were incubated at 37 degrees Celsius, in a
humidified, constant flow carbon dioxide incubator with an atmosphere of 5% carbon dioxide. A
medium change was performed once a week thereafter, and the appropriate dishes were retreated
with 100 µl of 0.0125 mM ascorbic acid for the remainder of the six week duration of the
experiment. After six weeks, we aspirated the medium from the dishes, rinsed the dishes with
DPBS, fixed the cells with methanol for 45 minutes, and stained the cells with Giemsa stain for1
hour and 30 minutes. The dishes were left to air-dry, after which the foci were scored under a
dissecting light microscope.
21
CHAPTER III: RESULTS
3.1 Effect of Sonication of Cr(VI) Compounds on Their Cytotoxicity to 10T1/2 Cells.
One goal in our present research was to enhance the yield of Cr(VI)-induced cytotoxicity
and morphological transformation (foci), in order to develop a credible in vitro cell system to
study molecular mechanisms of chromate-induced cytotoxicity and morphological
transformation. We designed and conducted experiments to investigate the effect of the size of
particles of lead chromate induced-cytotoxicity and morphological transformation in 10T1/2
cells. We wanted to determine the effect of particle size on the cytotoxicity of three insoluble
chromate compounds to cultured 10T1/2 cells: lead chromate, barium chromate, and strontium
chromate. In order to reduce the size of the chromate particles, we sonicated the insoluble
chromates (lead chromate, barium chromate, strontium chromate) using a Braun Sonicator set at
150 KV for 20 minutes. The concentrations of lead chromate we used ranged from 0.125 µg/ml
- 10 µg/ml. We found that treatment of 10T1/2 cells with unsonicated particulate lead chromate
resulted in a dose-dependent cytotoxicity. The LC50 values of unsonicated particulate lead
chromate, barium chromate and strontium chromate were 3.16 µg/ml, 0.93 µg/ml and 0.37
µg/ml, respectively. Treatment of 10T1/2 mouse embryo cells with sonicated particulate
chromates resulted in dose-dependent cytotoxicities, and the LC50 values of sonicated particulate
lead chromate, barium chromate and strontium chromate were reduced to 0.47 µg/ml, 0.43 µg/ml
and 0.35 µg/ml, respectively (Table 1).
22
Table 1: Effect of Sonication on Lead Chromate, Barium Chromate, and Strontium Chromate
induced cytotoxicity in 10T1/2 cells.
Particulate
Chromate
LC50 LC37 K* Cytotoxicity
Enhancement**
Lead
Chromate
Unsonicated 3.16 3.75 -0.27
Sonicated 0.47 1.65 -0.61 2,26
Barium
Chromate
Unsonicated 0.93 1.56 -0.64
Sonicated 0.43 0.69 -1.45 2.27
Strontium
Chromate
Unsonicated 0.37 0.5 -2
Sonicated 0.35 0.58 -1.72 0.86
Table 1: This table shows the LC50 and LC37 of unsonicated and sonicated particulate chromates
(lead chromate, barium chromate and strontium chromate). The LC50 value is the concentration
of the compound that reduces the survival of the cells in clonogenic assays to 50% of the control
value. The LC37 value is the concentration of the compound that reduces the survival of the cells
in clonogenic assays to 37% of the control value.
*K is the slope of the dose-response curve for cytotoxicity, and K = 1/LC37.
** The cytotoxicity enhancement is the ratio of the slope value, K, for sonicated compound/the
K value for unsonicated compound.
The survival of cells treated with cytotoxins is defined by the following equation:
S = e (-kc), where S is the survival, c is the concentration of cytotoxin, and -k is the slope of the
survival dose-response curve. When kc = 1, S = e
-1
= 0.37, or 37% survival. Hence, k = 1/LC37.
We concluded firstly, that both unsonicated and sonicated particulate chromates induced dose-
dependent cytotoxicity to 10T1/2 cells. Secondly, the cytotoxicity was enhanced by reduction in
particle size by factors of 2.26, 2.27, and 0.86 (no enhancement; slight reduction) for lead
chromate, barium chromate, and strontium chromate, respectively. Therefore, we optimized
23
conditions for inducing cytotoxicity by sonicating the particles of lead chromate, barium
chromate, and strontium chromate. We used these sonication conditions to reduce the size of
chromate particles, and used the sonciated chromate particles in cytotoxicity and cell
transformation assays.
3.2 Determination of Highest Non-Cytotoxic Concentrations of Ascorbic Acid
To determine whether ascorbic acid had any effect on sonicated particulate Cr(VI)
compound-induced cytotoxicity and morphological transformation, we first determined the
highest non-cytotoxic concentrations of ascorbic acid to use in our experiments. We designed
and conducted the experiments using two different methods, as described in section 2.6. The
concentrations of ascorbic acid used in these experiments were 0.00625 mM, 0.0125 mM, 0.025
mM, 0.05 mM, 0.1 mM, 0.25 mM, and 0.375 mM.
For the first method, we seeded 10T1/2 cells at 200 cells per 60 mm dish and treated the
cells with various concentration of ascorbic acid immediately upon seeding the cells. The plating
efficiencies and survival fractions (relative plating efficiencies) are summarized in Table 2
below.
Table 2: Plating efficiency and Survival Fractions of 10T1/2 cells Treated with 0.00625 mM to
0.375 mM Ascorbic Acid Upon Seeding.
Treatment PE ± SD Survival Fraction ± SD
No Addition
20.8 ± 1.7
1.0 ± 0.1
PBS Only
20.2 ± 2.9 1.0 ± 0.1
0.00625mM 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
24
Figure 1: Graph showing survival fraction of 10T1/2 cells treated with 0.00625 mM to 0.375
mM Ascorbic Acid upon seeding (Experiment 1).
This figure is a graphical representation of Table 2. It shows the survival fraction (on a
logarithmic scale) of 10T1/2 cells plotted against various concentrations of ascorbic acid.
0.01
0.1
1
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
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
25
The survival fractions of 10T1/2 cells treated with 0.00625 mM, 0.0125 mM, 0.025 mM,
0.05 mM, 0.1 mM, 0.25 mM, and 0.375 mM ascorbic acid were 1.0 ± 0.1, 1.0 ± 0.1, 0.9 ± 0.1,
0.8 ± 0.04, 0.8 ± 0.1, 0.2 ± 0.1, 0.04 ± 0.04, respectively (Table 2). The highest non-cytotoxic
concentrations of ascorbic acid ranged from 0.00625 mM to 0.025 mM. The slightly cytotoxic
concentrations of ascorbic acid were 0.05 mM and 0.1 mM, while 0.25 mM and 0.375 mM
ascorbic acid were highly cytotoxic.
For the second method, we seeded 10T1/2 cells at 200 cells per 60 mm dish and treated
the cells in the dishes with various concentrations of ascorbic acid twenty four hours after
seeding. Two experiments were carried out using this method. The tables and figures showing
the plating efficiencies and survival fractions (relative plating efficiency) are summarized below.
Table 3: Plating efficiency and survival fraction of 10T1/2 cells treated with 0.00625
mM to 0.375 mM Ascorbic Acid 24 Hours After Seeding (Experiment 1).
Treatment PE ± SD Survival Fraction ± SD
No Addition 38.1 ± 1.2 0.96 ± 0.03
PBS Only 39.7 ± 1.6 1.00 ± 0.04
0.00625 mM Ascorbic acid 34.5 ± 4.4 0.87 ± 0.11
0.0125 mM Ascorbic acid 33.7 ± 3.0 0.85 ± 0.08
0.025 mM Ascorbic acid 32.1 ± 3.5 0.81 ± 0.09
0.05 mM Ascorbic acid 30.5 ± 1.4 0.77 ± 0.03
0.1 mM Ascorbic acid 31.4 ± 2.5 0.79 ± 0.06
0.25 mM Ascorbic acid 29.1 ± 3.0 0.73 ± 0.08
0.375 mM Ascorbic acid 24.4 ± 4.2 0.61 ± 0.11
26
Figure 2: Graph showing survival fraction of 10T1/2 cells treated with 0.00625 mM to 0.375
mM Ascorbic Acid 24 hours after seeding (Experiment 1).
This figure is a graphical representation of the data in Table 3. It shows the survival fractions (on
a logarithmic scale) of 10T1/2 cells plotted against various concentrations of ascorbic acid.
The survival fractions for 10T1/2 cells treated with 0.00625 mM, 0.0125 mM, 0.025 mM,
0.05 mM, 0.1 mM, 0.25 mM, and 0.375 mM ascorbic acid were 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 (Table 3). We observed that
the cytotoxicity was dose-dependent with increasing ascorbic acid concentrations.
We carried out a repeat experiment as shown in Table 4 and Figure 5. The survival fractions of
10T1/2 cells treated with 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, 0.59 ± 0.11, respectively.
0.01
0.1
1
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
27
Table 4: Plating efficiency and survival fraction of 10T1/2 cells treated with 0.00625 mM to
0.375 mM Ascorbic Acid 24 hours after seeding (Experiment 2).
We found that the cytotoxicity of ascorbic acid to 10T1/2 cells to the ascorbic acid was
dose-dependent. Ascorbic acid was relatively non-cytotoxic at low concentrations of 0.00625
mM, 0.0125 mM, 0.025 mM, and moderately cytotoxic at concentrations of 0.05 mM and 0.1
mM. Ascorbic acid was highly cytotoxic to 10T1/2 cells at concentrations of 0.25 mM and
0.375 mM. We concluded that ascorbic acid was non-cytotoxic to 10T1/2 cells from
concentrations of 0.00625 mM to 0.05 mM and was cytotoxic at concentrations higher than 0.05
mM.
Treatment PE ± SD Survival Fraction ± SD
No Addition 34.3 ± 1.8 0.90 ± 0.05
PBS Only 34.7 ± 2.2 1.00 ± 0.06
0.00625mM Ascorbic acid 33 ± 3.4 0.95 ± 0.10
0.0125 mM Ascorbic acid 32.6 ± 3.1 0.94 ± 0.09
0.025 mM Ascorbic acid 32.2 ± 2.6 0.93 ± 0.07
0.05 mM Ascorbic acid 29.3 ± 1.6 0.84 ± 0.05
0.1 mM Ascorbic acid 29.2 ± 1.5 0.84 ± 0.04
0.25 mM Ascorbic acid 23.6 ± 1.1 0.68 ± 0.03
0.375 mM Ascorbic acid 20.3 ± 4.0 0.59 ± 0.11
28
Figure 3: The survival fraction of 10T1/2 cells treated with 0.00625 mM to 0.375 mM Ascorbic
Acid 24 hours after seeding (Experiment 2).
This figure is a graphical representation of Table 4. It shows the survival fraction (on a
logarithmic scale) of 10T1/2 cells plotted against various concentrations of ascorbic acid.
3.3 Effect of Ascorbic Acid on the Survival of 10T1/2 cells Treated with Sonicated Lead
Chromate
From section 3.1, we determined that the highest non-cytotoxic concentrations of
ascorbic acid were 0.00625 mM, 0.0125 mM, and 0.025 mM. We decided to use 0.0125 mM
ascorbic acid, because it is definitively non-cytotoxic to 10T1/2 cells. To determine the effect of
ascorbic acid on survival of 10T1/2 cells treated with sonicated lead chromate, we treated the
cells at different time intervals with ascorbic acid to investigate whether exposure of 10T1/2 cells
to ascorbic acid before, during or after treatment with sonicated lead chromate enhanced
0.01
0.1
1
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
29
cytotoxicity, as described in section 2.7. The plating efficiencies and survival fractions of 10T1/2
cells determined from the first experiment are shown below in Table 5.
EXPERIMENT 1
Table 5: Plating efficiency and survival fraction of 10T1/2 cells Treated with Sonicated Lead
Chromate and 0.0125 mm Ascorbic Acid (treatment at 3 different time points).
Treatment
PE ± SD
Survival fraction ±
SD
No Addition 34.5 ± 3.3
1.1 ± 0.10
PBS only 31.8 ± 3.7
1 ± 0.11
Acetone only 31.8 ± 3.5
1 ± 0.11
0.0125 mM
Ascorbic acid only 32.5 ± 2.9
1 ± 0.09
0.25µg/ml PbCrO4 only
30.1 ± 1.8
0.95 ± 0.06
0.5 µg/ml PbCrO4 only 29.5 ± 1.4 0.93 ± 0.10
0.75 µg/ml PbCrO4
only
27.8 ± 2.0 0.88 ± 0.06
1.0µg/ml PbCrO4 only 24.5 ± 3.2 0.77 ± 0.10
0.0125 mM
Ascorbic acid at
Day 0
0.25µg/ml PbCrO4 only
24.9 ± 2.1
0.78 ± 0.07
0.5 µg/ml PbCrO4 only 22.7 ± 1.2 0.71 ± 0.04
0.75 µg/ml PbCrO4
only
23.1 ± 3.9 0.73 ± 0.12
1.0µg/ml PbCrO4 only 19.8 ± 3.1 0.62 ± 0.09
0.0125 mM
Ascorbic acid at
Day 1
0.25µg/ml PbCrO4 only
28.8 ± 5.2
0.91 ± 0.16
0.5 µg/ml PbCrO4 only 26.7 ± 2.6 0.84 ± 0.08
0.75 µg/ml PbCrO4
only
24.8 ± 1.7 0.78 ± 0.05
1.0µg/ml PbCrO4 only 23.7 ± 5.6 0.75 ± 0.18
0.0125 mM
0.25µg/ml PbCrO4 only
32.7 ± 3.5
1 ± 0.11
30
For Method 1, we treated the cells with 0.0125 mM ascorbic acid upon seeding (Day 0),
and after 24 hours we treated cells separately with four concentrations of sonicated lead
chromate. The survival fractions of 10T1/2 cells treated with 0.25 µg/ml, 0.5 µg/ml, 0.75 µg/ml
and 1.0 µg/ml of sonicated lead chromate only were 0.95 ± 0.06, 0.93 ± 0.10, 0.88 ± 0.06 and
0.77 ± 0.1, respectively (Table 5). The survival fractions of 10T1/2 cells treated with 0.25 µg/ml,
0.5 µg/ml, 0.75 µg/ml and 1.0 µg/ml of sonicated lead chromate twenty four hours after prior
treatment with 0.0125 mM ascorbic acid (treatment with ascorbic acid upon seeding - day 0),
were 0.78 ± 0.07, 0.71 ± 0.04, 0.73 ± 0.12, 0.62 ± 0.09, respectively (Table 5). Sonicated lead
chromate caused a dose-dependent cytotoxicity to 10T1/2 cells. The survival of 10T1/2 cells
decreased even further when cells were treated with sonicated lead chromate in the presence of
0.0125 mM ascorbic acid (Figure 4). We concluded that pre-treatment of 10T1/2 cells with
ascorbic acid enhanced the cytotoxicity of sonicated lead chromate to10T1/2 cells.
Ascorbic acid at
Day 2
0.5 µg/ml PbCrO4 only 27.1 ± 1.5 0.85 ± 0.04
0.75 µg/ml PbCrO4
only
30.2 ± 1.6 0.95 ± 0.05
1.0µg/ml PbCrO4 only 26.5 ± 2.5 0.83 ± 0.08
31
Figure 4: Effect of 0.0125 mM ascorbic acid on the survival of 10T1/2 cells treated with
sonicated lead chromate (cells treated with ascorbic acid upon seeding)
For Method 2, we treated the cells with 0.0125 mM ascorbic acid and sonicated lead
chromate 24 hours after seeding the cells (Day 1). The survival fractions of 10T1/2 cells treated
with 0.25 µg/ml, 0.5 µg/ml, 0.75 µg/ml and 1.0 µg/ml of sonicated lead chromate only were 0.95
± 0.06, 0.93 ± 0.10, 0.88 ± 0.06 and 0.77 ± 0.1, respectively (Table 5). The survival fractions of
10T1/2 cells treated with 0.25 µg/ml, 0.5 µg/ml, 0.75 µg/ml and 1.0 µg/ml of sonicated lead
chromate and 0.0125 mM ascorbic acid (day 1), were 0.91 ± 0.16, 0.84 ± 0.08, 0.78 ± 0.05, 0.75
± 0.18, respectively (Table 5). Sonicated lead chromate caused a dose-dependent cytotoxicity to
0.1
1
0 0.25 0.5 0.75 1 1.25
Log(Relative Survival Fraction of 10T1/2 Cells)
Concentration of Lead Chromate (ug/ml)
EXPERIMENT 1: Effect of 0.0125 mM Ascorbic acid on the
survival of 10T1/2 cells treated with sonicated lead chromate (cells
treated with ascorbic acid upon seeding)
Lead Chromate only Lead Chromate + Asc at Day 0
No Addition 0.0125 mM Ascorbic acid only
32
10T1/2 cells. However, when we treated the cells with sonicated lead chromate and ascorbic acid
on day 1 (24 hours after seeding), we observed a dose-dependent cytotoxicity, but ascorbic acid
did not significantly enhance the cytotoxicity of sonicated lead chromate to 10T1/2 cells (Figure
5).
Figure 5: Effect of 0.0125 mM ascorbic acid on the survival of 10T1/2 cells treated with
sonicated lead chromate (cells treated with ascorbic acid 24 hours after seeding)
For Method 3, we treated the cells with sonicated lead chromate 24 hours after seeding
the cells (Day 1). We then treated the cells with 0.0125 mM ascorbic acid twenty-four hours after
treatment of cells with sonicated lead chromate (Day 2). The survival fractions of 10T1/2 cells
treated with 0.25 µg/ml, 0.5 µg/ml, 0.75 µg/ml and 1.0 µg/ml of sonicated lead chromate only
0.1
1
0 0.25 0.5 0.75 1 1.25
Log(Relative Survival Fraction of 10T1/2 Cells)
Concentration of Lead Chromate (ug/ml)
EXPERIMENT 1: Effect of 0.0125 mM Ascorbic acid on the survival of
10T1/2 cells treated with sonicated lead chromate (cells treated with
ascorbic acid 24 hrs after seeding)
Lead Chromate only Lead Chromate + Asc at Day 1
No Addition 0.0125 mM Ascorbic acid only
33
were 0.95 ± 0.06, 0.93 ± 0.10, 0.88 ± 0.06 and 0.77 ± 0.1, respectively (Table 5). The survival
fractions of 10T1/2 cells treated with 0.25 µg/ml, 0.5 µg/ml, 0.75 µg/ml and 1.0 µg/ml of
sonicated lead chromate and 0.0125 mM ascorbic acid (day 2), were 1 ± 0.11, 0.85 ± 0.04, 0.95
± 0.05, 0.83 ± 0.08, respectively (Table 5). Sonicated lead chromate caused a dose-dependent
cytotoxicity to 10T1/2 cells. However, when we treated the cells with sonicated lead chromate
and ascorbic acid at day 2 (24 hours after lead chromate treatment), we observed a dose-
dependent cytotoxicity, but ascorbic acid did not significantly enhance the cytotoxicity of
sonicated lead chromate to 10T1/2 cells (Figure 6).
Figure 6: Effect of 0.0125 mM ascorbic acid on the survival of 10T1/2 cells treated with
sonicated lead chromate (cells treated with ascorbic acid 24 hours after treatment with sonicated
lead chromate)
0.1
1
0 0.25 0.5 0.75 1 1.25
Log(Relative Survival Fraction of 10T1/2 Cells)
Concentration of Lead Chromate (ug/ml)
EXPERIMENT 1: Effect of 0.0125 mM Ascorbic acid on the survival
of 10T1/2 cells treated with sonicated lead chromate (cells treated with
ascorbic acid 24 hrs after PbCrO
4
treatment)
Lead Chromate only Lead Chromate + Asc at Day 2
No Addition 0.0125 mM Ascorbic acid only
34
The figure below (Figure 7) is a summary graph that shows the effect of 0.0125 mM ascorbic
acid using three different treatment methods to 10T1/2 cell survival. We observed from this
experiment that prior treatment of 10T1/2 cells with ascorbic acid upon seeding before exposure
to sonicated lead chromate enhanced the cytotoxic effect of lead chromate to 10T1/2 cells.
Figure 7: Effect of 0.0125 mM ascorbic acid on the survival of 10T1/2 cells treated with
sonicated lead chromate (showing the three different treatments)
0.1
1
0 0.25 0.5 0.75 1 1.25
Log(Relative Survival Fraction of 10T1/2 Cells)
Concentration of Lead Chromate (ug/ml
EXPERIMENT 1: Effect of 0.0125 mM Ascorbic acid on the survival of
10T1/2 cells treated with sonicated lead chromate
Lead Chromate Only Lead Chromate +Ascorbic Acid at Day 0
Lead Chromate+ Ascorbic acid at Day 1 Lead Chromate + Ascorbic acid at Day 2
No Addition Ascorbic acid only
35
EXPERIMENT 2
For Method 1, we treated the cells with 0.0125 mM ascorbic acid upon seeding (Day 0)
and after 24 hours we treated cells with four concentrations of sonicated lead chromate. The
survival fractions of 10T1/2 cells treated with 0.25 µg/ml, 0.5 µg/ml, 0.75 µg/ml and 1.0 µg/ml
of sonicated lead chromate only were 0.95 ± 0.06, 0.86 ± 0.03, 0.79 ± 0.05 and 0.66 ± 0.05,
respectively (Table 6).
Table 6: Plating efficiency and survival fraction of 10T1/2 cells treated with sonicated lead
chromate + 0.0125 mM ascorbic acid (treatment at 3 different time points).
Treatment
PE ± SD
Survival fraction ±
SD
No Addition 21.8 ± 1.1
1.1 ± 0.03
PBS only 20 ± 1.4
1 ± 0.05
Acetone only 18.5 ± 1
0.93 ± 0.05
0.0125 mM
Ascorbic acid only 19 ± 0.0
0.95 ± 0.00
0.25µg/ml PbCrO4 only
19 ± 1.8
0.95 ± 0.06
0.5 µg/ml PbCrO4 only 17.3 ± 0.9 0.86 ± 0.03
0.75 µg/ml PbCrO4
only 15.8 ± 1.4
0.79 ± 0.05
1.0µg/ml PbCrO4 only 13.1 ± 1.4 0.66 ± 0.05
0.0125 mM
Ascorbic acid at
Day 0
0.25µg/ml PbCrO4 only 12.3 ± 2.0
0.61 ± 0.07
0.5 µg/ml PbCrO4 only 12.1 ± 1.9 0.6 ± 0.06
0.75 µg/ml PbCrO4
only 10.5 ± 0.8
0.53 ± 0.07
1.0µg/ml PbCrO4 only 9.2 ± 2.4 0.46 ± 0.08
0.0125 mM
0.25µg/ml PbCrO4 only 15.7 ± 2.1
0.79 ± 0.07
36
The survival fractions of 10T1/2 cells treated with 0.25 µg/ml, 0.5 µg/ml, 0.75 µg/ml and
1.0 µg/ml of sonicated lead chromate twenty-four hours after prior treatment with 0.0125 mM
ascorbic acid (treatment with ascorbic acid upon seeding - day 0), were 0.61 ± 0.07, 0.6 ± 0.07,
0.53 ± 0.07, 0.46 ± 0.08, respectively (Table 6). Similar to Experiment 1, sonicated lead
chromate caused a dose-dependent cytotoxicity to 10T1/2 cells. The survival of 10T1/2 cells
decreased even further when the cells were treated with sonicated lead chromate in the presence
of 0.0125 mM ascorbic acid (Figure 8). We concluded that pre-treatment of 10T1/2 cells with
ascorbic acid enhanced the cytotoxicity of sonicated lead chromate to10T1/2 cells.
Ascorbic acid at
Day 1
0.5 µg/ml PbCrO4 only 13.1 ± 1.2 0.66 ± 0.04
0.75 µg/ml PbCrO4
only 12.5 ± 1.3
0.63 ± 0.04
1.0µg/ml PbCrO4 only 8.8 ± 5.6 0.44 ± 0.05
0.0125 mM
Ascorbic acid at
Day 2
0.25µg/ml PbCrO4 only 14.7 ± 2.0
0.74 ± 0.07
0.5 µg/ml PbCrO4 only 14 ± 1.1 0.7 ± 0.04
0.75 µg/ml PbCrO4
only 13.5 ± 1.1
0.68 ± 0.04
1.0µg/ml PbCrO4 only 12.6 ± 0.8 0.63 ± 0.03
37
Figure 8: Effect of 0.0125 mM ascorbic acid on the survival of 10T1/2 cells treated with
sonicated lead chromate (cells treated with ascorbic acid upon seeding)
For Method 2, we treated the cells with 0.0125 mM ascorbic acid and sonicated lead
chromate 24 hours after seeding the cells (day 1). The survival fractions of 10T1/2 cells treated
with 0.25 µg/ml, 0.5 µg/ml, 0.75 µg/ml and 1.0 µg/ml of sonicated lead chromate only were 0.95
± 0.06, 0.86 ± 0.03, 0.79 ± 0.05 and 0.66 ± 0.05, respectively (Table 6). The survival fractions
of 10T1/2 cells treated with 0.25 µg/ml, 0.5 µg/ml, 0.75 µg/ml and 1.0 µg/ml of sonicated lead
chromate and 0.0125 mM ascorbic acid (day 1), were 0.79 ± 0.07, 0.66 ± 0.04, 0.63 ± 0.04, 0.44
± 0.05, respectively (Table 5). Sonicated lead chromate caused a dose-dependent cytotoxicity to
10T1/2 cells. When we treated the cells with sonicated lead chromate and ascorbic acid at day 1
(24 hours after seeding), we observed a dose-dependent cytotoxicity, but when compared to the
0.1
1
0 0.25 0.5 0.75 1 1.25 Log(Relative Survival Fraction of 10T1/2 Cells)
Concentration of Lead Chromate (ug/ml)
Experiment 2: Effect of 0.0125 mM Ascorbic acid on the survival of 10T1/2
cells treated with sonicated lead chromate
Lead Chromate only Lead Chromate + Asc at Day 0
No Addition 0.0125 mM Ascorbic acid only
38
survival fractions from method 1, ascorbic acid did not significantly enhance the cytotoxicity of
sonicated lead chromate to 10T1/2 cells (Figure 9).
Figure 9: Effect of 0.0125 mM ascorbic acid on the survival of 10T1/2 cells treated with
sonicated lead chromate (cells treated with ascorbic acid 24 hours after seeding)
For Method 3, we treated the cells with sonicated lead chromate 24 hours after seeding
the cells (day 1). We then treated the cells with 0.0125 mM ascorbic acid twenty-four hours after
treatment with sonicated lead chromate (day 2). The survival fractions of 10T1/2 cells treated
with 0.25 µg/ml, 0.5 µg/ml, 0.75 µg/ml and 1.0 µg/ml of sonicated lead chromate only were 0.95
± 0.06, 0.86 ± 0.03, 0.79 ± 0.05 and 0.66 ± 0.05 respectively, (Table 6). The survival fractions of
10T1/2 cells treated with 0.25 µg/ml, 0.5 µg/ml, 0.75 µg/ml and 1.0 µg/ml of sonicated lead
chromate and 0.0125 mM ascorbic acid (day 2), were 0.74 ± 0.07, 0.7 ± 0.04, 0.68 ± 0.04, 0.63
0.1
1
0 0.25 0.5 0.75 1 1.25
Log(Relative Survival Fraction of 10T1/2 Cells)
Concentration of Lead Chromate (ug/ml)
Experiment 2: Effect of 0.0125 mM Ascorbic acid on the survival of 10T1/2
cells treated with sonicated lead chromate (cells treated with ascorbic acid 24
hrs after PbCrO
4
treatment)
Lead Chromate only Lead Chromate + Asc at Day 1
No Addition 0.0125 mM Ascorbic acid only
39
± 0.03, respectively (Table 6). Sonicated lead chromate caused a dose-dependent cytotoxicity to
10T1/2 cells. We observed a dose-dependent cytotoxicity of lead chromate to 10T1/2 cells when
we treated the cells with sonicated lead chromate and ascorbic acid at day 2 (24 hours after lead
chromate treatment). We observed that this method of treatment with ascorbic acid did not
significantly enhance the cytotoxicity of sonicated lead chromate to 10T1/2 cells when compared
to the previous methods above (Figure 10 and Figure 11).
40
Figure 10: Effect of 0.0125 mM ascorbic acid on the survival of 10T1/2 cells treated with
sonicated lead chromate (showing the three different treatments)
From these results, we concluded that pre-treatment of 10T1/2 cells with 0.0125 mM
ascorbic acid, twenty four hours before treatment with sonicated lead chromate, significantly
enhanced cytotoxicity when compared to the other two methods.
0.1
1
0 0.25 0.5 0.75 1 1.25
Log(Relative Survival Fraction of 10T1/2 Cells)
Concentration of Lead Chromate (ug/ml)
EXPERIMENT 2: Effect of 0.0125 mM Ascorbic acid on the
survival of 10T1/2 cells treated with sonicated lead chromate
Lead Chromate Only Lead Chromate + Ascorbic acid at Day 0
Lead Chromate + Ascorbic acid at Day 1 Lead Chromate + Ascorbic acid at Day 2
No addition Ascorbic acid
41
3.4 Effect of Ascorbic acid on Lead Chromate-induced Morphological Transformation of 10T1/2
Cells
From the experiments we carried out in sections 3.1 and 3.2, we determined that the
highest the non-cytotoxic concentration of ascorbic acid to use in our experiments was 0.0125
mM. We also established that, from the three methods of ascorbic acid treatments, pre-treatment
of 10T1/2 cells with ascorbic acid twenty four hours before exposure to lead chromate, had the
most cytotoxic effect on 10T½ cells. In line with our goals for this project, we conducted an
assay to investigate the effect of 0.0125 mM ascorbic acid on lead chromate-induced
morphological transformation to 10T1/2 cells.
We carried out this experiment using method 1, by pre-treating 10T1/2 cells with 0.0125
mM ascorbic acid, as described in section 2.8. The results of the experiment are shown in Table
7, and a graphical illustration of the results is shown in Figure 11. The survival of 10T1/2 cell
treated with 1 µg/ml of MCA, our positive control, was 93.9%, and this concentration of MCA
induced a total of 20 foci (Type II and Type III) per 20 dishes. No foci were found in dishes
treated with 0.0125 mM ascorbic acid. The number of foci found in dishes treated with 0.5
µg/ml, 0.75 µg/ml, 1.0 µg/ml, 1.25 µg/ml and 1.5 µg/ml of sonicated lead chromate only were 0,
1, 1, 3 and 1.05, respectively. Although the foci yield was low for sonicated lead chromate only
treatment, we observed a dose-dependent increase in the yield of foci when the cells were pre-
treated with 0.0125 mM ascorbic acid. The number of foci found in dishes pre-treated with
ascorbic acid twenty-four hours before treatment with 0.5 µg/ml, 0.75 µg/ml, 1.0 µg/ml, 1.25
µg/ml and 1.5 µg/ml of sonicated lead chromate were 2.1, 4, 6, 5 and 0 respectively. The
cytotoxicity data obtained from this transformation experiment (Table 8 and Figure 12) were
consistent with our previous findings that pre-treatment of 10T1/2 cells with ascorbic acid,
42
twenty-four hours before exposure to sonicated lead chromate enhanced cytotoxicity to 10T1/2
cells.
Table 7: Effect of 0.0125mM Ascorbic Acid on Lead Chromate Induced Morphological
Transformation to 10T1/2 Cells.
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) 101.4 0/20 (0/20) 0/20 (0/20) 0/20 0/20
0 (0.5% acetone) 92.6 0/20 (0/20) 2/20 (2/20) 0/20 2/20
MCA (1.0 µg/ml) 93.9 2/20 (0/20) 20/20 (20/20) 2/20 20/20
0.0125mM Ascorbic
Acid 105.9 0/20 (0/20) 0/20 (0/20) 0/20 0/20
PbCr 2O 4 (µg/ml) only
0µg/ml PbCrO 4 101 0/20 (0/20) 0/20 (0/20) 0/20 0/20
0.5µg/ml PbCrO 4 88.5 0/20 (0/20) 0/20 (0/20) 0/20 0/20
0.75µg/ml PbCrO 4 80 0/20 (0/20) 1/20 (0/20) 0/20 1/20
1.0µg/ml PbCrO 4 77.9 0/20 (0/20) 1/20 (1/20) 0/20 1/20
1.25 µg/ml PbCrO 4 83.1 0/20 (0/20) 3/20 (3/20) 0/20 3/20
1.5µg/ml PbCrO 4 77.6 0/20 (0/20) 1/19 (1.05/20) 0/20 1.05/20
PbCr 2O 4 (µg/ml) + 0.0125mM Ascorbic Acid
0µg/ml PbCrO 4 105.9 0/20 (0/20) 0/20 (0/20) 0/20 0/20
0.5µg/ml PbCrO 4 81.4 0/19 (0/20) 2/19 (2.1/20) 0/19 2.1/20
0.75µg/ml PbCrO 4 68.1 0/20 (0/20) 4/20 (4/20) 0/20 4/20
1.0µg/ml PbCrO 4 65 0/20 (0/20) 6/20 (6/20) 0/20 6/20
1.25 µg/ml PbCrO 4 64 2/20 (0/20) 3/20 (3/20) 2/20 5/20
1.5µg/ml PbCrO 4 57.5 0/20 (0/20) 0/20 (0/20) 0/20 0/20
* The number of foci has been normalized to a total of 20 dishes
43
Figure 11: Effect of 0.0125 mM ascorbic acid on sonicated lead chromate-induced
morphological transformation in 10T1/2 cells
This figure shows the enhancing effect of ascorbic acid on sonicated lead chromate induced
morphological transformation in 10T1/2 cells.
In Figure 11, it can be seen that adding 0.0125 mM ascorbic acid to 10T1/2 cells treated with 0.5
µg/ml, 0.75 µg/ml, 1.0 µg/ml, or 1.25 µg/ml, or 1.50 µg/ml of lead chromate caused the yield of
foci to rise from 0 to 2, 1 to 4, 1 to 6, 1 to 5, and 1 to 0, respectively.
0
1
2
3
4
5
6
7
0 0.5 1 1.5 2
Number of Foci/20 dishes
Concentration of Lead Chromate µg/ml
Effect of ascorbic acid on sonicated lead chromate induced
morphological transformation in 10T1/2 cells
Lead Chromate
only
Lead chromate
+ 0.0125 mM
Asc
44
Table 8: Cytotoxicity Data for Transformation Experiment with sonicated lead chromate and
ascorbic acid
Treatment
PE ± SD
Survival fraction ± SD
No Addition 29.8 ± 2.1 1.01 ± 0.07
Acetone 27.2 ± 1.2 0.92 ± 0.04
MCA 27.6 ± 2.0 0.93 ± 0.06
PBS 29.38 ± 1.4 1 ± 0.05
0.0125mM Ascorbic Acid 31.1 ± 1.1 1.05 ± 0.04
Lead Chromate Treatment
without Ascorbic Acid
0.5µg/ml PbCr2O4
26 ± 2.1 0.89 ± 0.07
0.75µg/ml PbCr2O4
23.5 ± 2.6 0.8 ± 0.08
1.0µg/ml PbCr2O4
22.9 ± 2.6 0.77 ± 0.09
1.25 µg/ml PbCr2O4
24.4 ± 3.1 0.83 ± 0.1
1.5µg/ml PbCr2O
22.8 ± 2.4 0.77 ± 0.08
Lead Chromate Treatment with
Ascorbic Acid
0.5µg/ml PbCr2O4 23.9 ± 2.0 0.81 ± 0.07
0.75µg/ml PbCr2O4
20 ± 2.0
0.68 ± 0.07
1.0µg/ml PbCr2O4
19.1 ± 2.0
0.65 ± 0.07
1.25 µg/ml PbCr2O4
18.8 ± 1.4
0.64 ± 0.05
1.5µg/ml PbCr2O4
16.9 ± 1.2
0.58 ± 0.04
45
Figure 12: Graphical representation of the cytotoxicity data for the transformation experiment
with sonicated lead chromate and ascorbic acid.
0.1
1
0 0.25 0.5 0.75 1 1.25 1.5 1.75
Log(Relative Survival Fraction of 10T1/2 Cells)
Concentration of Lead Chromate (ug/ml)
Effect of 0.0125 mM Ascorbic acid on survival of 10T1/2 cells treated
with sonicated lead chromate (Transformation Experiment)
Lead Chromate only Lead Chromate + Asc at Day 0
No Addition Ascorbate only (0.0125mM)
46
3.5 Effect of Ascorbic Acid on the Survival of 10T1/2 cells Treated with Sonicated Barium
Chromate
To determine the effect of ascorbic acid on survival of 10T1/2 cells treated with sonicated
barium chromate, we treated the cells at different time intervals with ascorbic acid to investigate
whether exposure of 10T1/2 cells to ascorbic acid before, during or after treatment with
sonicated barium chromate enhanced cytotoxicity as described in section 2.7. The plating
efficiencies and survival fractions of 10T1/2 cells determined from the first experiment are
shown below in Table 9.
Table 9: Plating efficiency and survival of 10T1/2 cells treated with sonicated barium chromate
and 0.0125 mM ascorbic acid (Experiment 1)
Treatment
PE ± SD
Survival fraction ± SD
No Addition 28 ± 2.2
0.99 ± 0.08
PBS only 28.1 ± 3.15
1 ± 0.1
Acetone only 26.7 ± 3.0
0.95 ± 0.1
0.0125 mM
Ascorbic acid only 27.9 ± 2.7
0.99 ± 0.09
0.25µg/ml BaCrO 4 only 26.6 ±12.2 0.95 ± 0.1
0.5 µg/ml BaCrO 4 only 21.6 ±1.74 0.79 ± 0.05
0.75 µg/ml BaCrO 4 only 20 ±3.1 0.71 ± 0.1
1.0µg/ml BaCrO 4 only 18.8 ±8.6 0.67 ± 0.1
0.0125 mM Ascorbic
acid at Day 0
0.25µg/ml BaCrO 4 only
22.7 ±2.8
0.85 ± 0.03
0.5 µg/ml BaCrO 4 only 20.1 ±3.9 0.86 ± 0.02
0.75 µg/ml BaCrO 4 only 17.5 ±3.9 0.64 ± 0.04
1.0µg/ml BaCrO 4 only 18.1 ±2.3 0.61 ± 0.1
0.0125 mM Ascorbic
acid at Day 1
0.25µg/ml BaCrO 4 only
22.7 ±2.8
0.81 ± 0.1
0.5 µg/ml BaCrO 4 only 20.1 ±3.9 0.71 ± 0.1
0.75 µg/ml BaCrO 4 only 17.5 ±3.9 0.62 ± 0.1
1.0µg/ml BaCrO 4 only 18.1 ±2.3 0.64 ± 0.08
47
The survival fractions of 10T1/2 cells treated with 0.25 µg/ml, 0.5 µg/ml, 0.75 µg/ml and
1.0 µg/ml of sonicated barium chromate only were, 0.95 ± 0.1, 0.79 ± 0.05, 0.71 ± 0.1, 0.67 ± 0.1
respectively. The survival fractions of 10T1/2 cells treated with 0.25 µg/ml, 0.5 µg/ml, 0.75
µg/ml and 1.0 µg/ml of sonicated barium chromate twenty four hours after prior treatment with
0.0125 mM ascorbic acid (treatment with ascorbic acid upon seeding - day 0), were 0.85 ± 0.03,
0.86 ± 0.02, 0.64 ± 0.04, 0.61 ± 0.1 respectively (Table 9). We observed that treatment with
ascorbic acid slightly enhanced barium chromate-induced cytotoxicity in a dose-dependent
manner (Figure 13).
For the cells that we treated with 0.0125 mM ascorbic acid and sonicated barium
chromate 24 hours after seeding the cells (day 1), the survival fractions of 10T1/2 cells treated
with 0.25 µg/ml, 0.5 µg/ml, 0.75 µg/ml and 1.0 µg/ml sonicated barium chromate were 0.81 ±
0.1, 0.71 ± 0.1, 0.62 ± 0.1, 0.64 ± 0.08 respectively (Table 9; Figure 13). For the cells treated
with 0.0125 mM ascorbic acid twenty-four hours after treatment with sonicated barium chromate
(day 2), the survival fractions of 10T1/2 cells treated with 0.25 µg/ml, 0.5 µg/ml, 0.75 µg/ml,
and 1.0 µg/ml of sonicated lead chromate were 0.77 +/ 0.05, 0.70 +/0.06, 0.67 +/0.10, and 0.59
+/ 0.1, respectively (Table 9, Figure 13).
0.0125 mM Ascorbic
acid at Day 2
0.25µg/ml BaCrO 4 only
21.7 ±1.4
0.77 ± 0.05
0.5 µg/ml BaCrO 4 only 19.7±1.7 0.70 ± 0.06
0.75 µg/ml BaCrO 4 only 18.7 ±3.1 0.67 ± 0.1
1.0µg/ml BaCrO 4 only 16.7 ±3.4 0.59 ± 0.1
48
Figure 13: Effect of Ascorbic acid on survival of 10T1/2 cells treated with barium chromate
(Experiment 1)
We observed that ascorbic acid slightly enhanced the cytotoxic effects of sonicated
barium chromate to 10T1/2 cells, and the enhancement was not dependent on the time at which
ascorbic acid was added to the cells. We conducted a repeat experiment using the same methods.
The plating efficiencies and survival of 10T1/2 cells treated with sonicated barium chromate are
shown in Table 10, below.
0.1
1
0 0.25 0.5 0.75 1 1.25
Relative Survival Fraction of 10T1/2 Cells
Concentration of Barium Chromate (ug/ml)
Experiment 1: Effect of Ascorbic acid on survival of 10T1/2 cells treated with
barium chromate
Barium chromate only
Barium chromate with
0.0125 mM Ascorbate at
day 0
Barium chromate with
0.0125 mM Ascorbate at
day 1
Barium chromate with
0.0125 mM Ascorbate at
day 2
0.0125 mM Ascorbate only
49
The survival fractions of 10T1/2 cells treated with 0.25 µg/ml, 0.5 µg/ml, 0.75 µg/ml and
1.0 µg/ml of sonicated barium chromate only were, 1.1 ± 0.06, 1.0 ± 0.08, 0.79 ± 0.08 and 0.63 ±
0.16, respectively. The survival fractions of 10T1/2 cells treated with 0.25 µg/ml, 0.5 µg/ml, 0.75
µg/ml and 1.0 µg/ml of sonicated barium chromate twenty four hours after prior treatment with
0.0125 mM ascorbic acid (treatment with ascorbic acid upon seeding - day 0), were 0.83 ± 0.08,
0.75 ± 0.07, 0.63 ± 0.1 and 0.70 ± 0.06 respectively (Table 10).
Table 10: Plating efficiency and survival of 10T1/2 cells treated with sonicated barium chromate
and 0.0125 mM ascorbic acid (Experiment 2)
Treatment
PE ± SD
Survival fraction
No Addition 21±1
1.2 ± 0.08
PBS only 17.25 ± 3.5
1 ± 0.06
Acetone only
18.5 ± 2.8
1.1 ± 0.05
0.0125 mM
Ascorbic acid only 12.8 ± 2.1
0.74 ± 0.04
0.25µg/ml BaCrO4 only
19.2 ± 3.3
1.1 ± 0.06
0.5 µg/ml BaCrO4 only 17.4 ± 4.4 1.0 ± 0.08
0.75 µg/ml BaCrO4 only 13.6 ± 4.4 0.79 ±0.08
1.0µg/ml BaCrO4 only 10.8 ± 8.8 0.63 ± 0.16
0.0125 mM Ascorbic
acid at Day 0
0.25µg/ml BaCrO4 only
14.3 ± 4.3
0.83 ± 0.08
0.5 µg/ml BaCrO4 only 12.9 ± 4.2 0.75 ± 0.07
0.75 µg/ml BaCrO4 only 10.9 ± 5.7 0.63 ± 0.1
1.0µg/ml BaCrO4 only 12.2 ± 3.6 0.70 ± 0.06
0.0125 mM Ascorbic
acid at Day 1
0.25µg/ml BaCrO4 only
16.3 ± 4.8
0.94 ± 0.08
0.5 µg/ml BaCrO4 only 12 ± 9.7 0.69 ± 0.1
50
For the cells that we treated with 0.0125 mM ascorbic acid and sonicated barium chromate 24
hours after seeding the cells (day 1), the survival fractions of 10T1/2 cells treated with 0.25
µg/ml, 0.5 µg/ml, 0.75 µg/ml and 1.0 µg/ml sonicated barium chromate were 0.94 ± 0.08, 0.69 ±
0.1, 0.68 ± 0.1 and 0.59 ± 0.09 respectively (Table 10; Figure 14). For the cells treated with
0.0125 mM ascorbic acid twenty four hours after sonicated barium chromate treatment (day 2),
the survival fractions of 10T1/2 cells treated with 0.25 µg/ml, 0.5 µg/ml, 0.75 µg/ml and 1.0
µg/ml of sonicated barium chromate were 0.92 ± 0.07, 0.84 ± 0.1, 0.74 ± 0.1 and 0.59 ± 0.08
respectively (Table 10; Figure 14).
0.75 µg/ml BaCrO4 only 11.8 ± 7.9 0.68 ± 0.1
1.0µg/ml BaCrO4 only 10.3 ± 5.5 0.59 ± 0.09
0.0125 mM Ascorbic
acid at Day 2
0.25µg/ml BaCrO4 only
16 ± 4.2
0.92 ± 0.07
0.5 µg/ml BaCrO4 only 14.5 ± 7.9 0.84 ± 0.1
0.75 µg/ml BaCrO4 only 12.7 ± 6.4 0.74 ± 0.1
1.0µg/ml BaCrO4 only 10.1 ± 4.3 0.59 ± 0.08
51
Figure 14: Effect of Ascorbic acid on survival of 10T1/2 cells treated with barium chromate
(Experiment 2)
0.1
1
0 0.25 0.5 0.75 1 1.25
Relative Survival Fraction of 10T1/2 Cells
Concentration of Barium Chromate (ug/ml)
Experiment 2: Effect of Ascorbic acid on survival of 10T1/2 cells treated with
barium chromate
Barium chromate only
Barium chromate with
0.0125 mM Ascorbate at
day 0
Barium chromate with
0.0125 mM Ascorbate at
day 1
Barium chromate with
0.0125 mM Ascorbate at
day 2
0.0125 mM Ascorbate
only
52
CHAPTER IV: DISCUSSION AND CONCLUSIONS
Particulate hexavalent chromium compounds are well-established human carcinogens
(reviewed in Patierno et al, l988, Wise et al, 2004). However, the mechanisms of their
carcinogenicity are poorly understood (Patierno et al, l988; Wise et al, 2004). Previous work in
our laboratory showed that lead chromate induced cytotoxicity and morphological transformation
of C3H 10T1/2 Cl 8 (10T1/2) mouse embryo cells in a dose-dependent manner. However, the
total yield of morphological transformation was weak and did not correlate with epidemiological
and animal studies which show that particulate hexavalent Cr(VI) compounds such as lead
chromate are potent human carcinogens (Patierno et al, 1988; Lin, 2011).
To this end, we hypothesized that the weak yield of morphological transformation (focus
formation) could be as a result of weak phagocytosis of lead chromate particles due to their very
large particle size (average size of 80 µm for this preparation). Particles of chromate of sizes
from 10 um or less are usually phagocytosed well by cells. We investigated the effect of particle
size on the cytotoxicity of three insoluble chromate compounds -lead chromate, barium chromate
and strontium chromate- in our laboratory prior to this thesis work and we found that reduction
in particle size down to a mean diameter of 2µm enhanced the cytotoxicity of insoluble chromate
compounds to 10T1/2 cells. However, the yield of morphological transformation was still
somewhat low.
Next, we hypothesized that, in addition to reduction of particle size by sonication, the
reduction of Cr(VI) to Cr(III) intracellularly by reductants such as ascorbic acid would enhance
the cytotoxic and cell transforming abilities of Cr(VI), in 10T1/2 cells.
53
4.1 Effect of Ascorbic Acid on the Survival of 10T1/2 cells Treated with Sonicated Lead
Chromate
First, we designed and conducted assays using three different methods to assess the effect
of ascorbic acid on survival of 10T1/2 cells treated with four different concentrations of
sonicated lead chromate. We found that treatment of 10T1/2 with sonicated lead chromate
resulted in a dose-dependent cytotoxicity, and this cytotoxicity caused by sonicated lead
chromate was further increased by ascorbic acid. We observed an enhanced, dose-dependent
cytotoxicity with sonicated lead chromate and ascorbic acid using all three methods explored for
these experiments. However, pre-treatment of 10T1/2 cells with 0.0125 mM ascorbic acid,
twenty four hours before exposure to lead chromate treatments (Method 1) increased the
cytotoxicity (i.e., decreased the survival fraction ) of 10T1/2 cells significantly (Figure 7 and
Figure 11). We believe that this enhancing effect of ascorbate on the cytotoxicity of lead
chromate could be attributed to the ability of ascorbic acid to reduce the Cr(VI) in lead chromate
to Cr(V), Cr(IV), and Cr(III). Cr(III) would then bind to guanine and other DNA bases, leading
to DNA damage and mutation. The resultant Cr(V) and Cr(IV) generated would also reduce
molecular oxygen to superoxide, which would dismute to hydrogen peroxide, bind to DNA bases
and cause formation of mutagenic products, such 8-OH-deoxyguanosine. In addition, hydrogen
peroxide would react with ferric iron in cells to produce hydroxyl radical, which would also bind
to DNA bases, leading to 8-OH-deoxyguanosine and other mutagenic DNA base products
leading to mutations and cytotoxicity. It is possible that pre-treatment of the cells with ascorbic
acid allows more uptake into the cells, which in turn will cause the reduction of Cr(VI) to Cr(III)
intracellularly, leading to DNA damage and mutations, which would lead to cytotoxicity.
54
4.2 Effect of 0.0125 mM Ascorbic Acid on Sonicated Lead Chromate-Induced Morphological
Cell Transformation
Previous work from our laboratory has shown that lead chromate induces morphological
transformation in 10T1/2 cells in a dose-dependent manner, but the yield of morphological
transformation was low (Patierno et al, l988). We then investigated the effect ascorbic acid has
on morphological transformation induced by sonicated particles of lead chromate. We found that
ascorbic acid enhanced the yield of morphological transformation (focus formation) by 4-fold to
6-fold. The number of foci per 20 dishes for 0.5 µg/ml, 0.75 µg/ml, 1.0 µg/ml, 1.25 µg/ml and
1.5 µg/ml sonicated lead chromate treatments were 0, 1, 1, 3 and 1.05, respectively.
Interestingly, the number of foci per 20 dishes for 10T1/2 cells treated with 0.5 µg/ml, 0.75
µg/ml, 1.0 µg/ml, 1.25 µg/ml and 1.5 µg/ml of sonicated lead chromate and 0.0125 mM ascorbic
acid, were 2.1, 4, 6, 5 and 0, respectively. The data presented here showed a dose-dependent
yield of transformation caused by sonicated particles of lead chromate that was enhanced when
10T1/2 cells also were pre-treated with 0.0125 mM ascorbic acid. This was a preliminary
experiment, and it would have to be repeated to determine whether is the enhancement of lead
chromate-induced morphological transformation by ascorbate a) remained dose-dependent and
b) reproducible in order to create final dose-response curves for Cr(VI)-induced morphological
transformation.
55
4.3 Effect of Ascorbic Acid on the Survival of 10T1/2 cells Treated with Sonicated Barium
Chromate
We next designed and conducted assays using these three different methods to determine
the effects of ascorbic acid on the survival of 10T1/2 cells treated with four different
concentrations of sonicated barium chromate. We found that treatment of 10T1/2 with barium
chromate resulted in a dose-dependent cytotoxicity, which was slightly enhanced by ascorbic
acid (Figures 13 and 14). There was no significant difference between the three methods of
ascorbic acid treatment. The addition of ascorbic acid to barium chromate-treated 10T1/2 cells
with all three of these methods of addition showed that ascorbic acid modestly enhanced the
cytotoxicity of barium chromate to 10T1/2 cells.
FUTURE DIRECTIONS
Future research is aimed at conducting additional transformation experiments to develop
dose-response curves for sonicated lead chromate-induced morphological transformation in
10T1/2 cells. After this, we plan to set up additional experiments using the most effective
concentration of sonicated lead chromate and ascorbate that induces morphological
transformation, ring clone the foci of transformed cells, create transformed cell lines from them,
and then biologically characterize (saturation density, focus forming efficiency, growth on soft
agar) the transformed cell line. The next step would be to use DNA microarrays to investigate
the aberrations that occur in sonicated lead chromate-induced transformed 10T1/2 cell lines.
In the future, our laboratory will study whether glutathione and cysteine separately, and
whether glutathione, cysteine and ascorbate together, could cause significant enhancement of
morphological transformation induced by lead chromate in 10T1/2 cells. An additional step
56
would be to utilize electron spin resonance (ESR) spectrometry to determine whether the
reduction of Cr(VI) intracellularly led to generation of Cr(V) radicals and to hydroxyl radicals
(by spin trapping), and whether this lead to DNA damage. Such experiments are currently being
planned in our laboratory.
57
ACKNOWLEDGEMENTS
I wish to express my deepest gratitude to my advisor and mentor, Dr. Joseph R.
Landolph, Jr., Ph.D., for his help all through my M.S program in the Department of Molecular
Microbiology and Immunology at USC. His knowledge, expertise and help were invaluable in
conducting my experiments and successful completion of my thesis. I would like to thank Mr.
Qasim Akinwumi, B.S., M.S., Ph.D. student, Department of Biochemistry, College of Medicine,
University of Ibadan, Nigeria, who worked in Dr. Landolph’s laboratory toward his Ph.D.
degree, for training me in the laboratory and teaching me all the techniques required to complete
my thesis. I would also like to thank Mrs. Sophia Allaf Shahin, B.S., M.S., for training me in Dr.
Landolph’s laboratory. I would like to specially thank Ms. Laureen Tran, M.S., for her support
and guidance on my experiments, I sincerely appreciate all the help she provided on my
coursework and for training me on how to use Microsoft Excel to compute my results. I would
also like to thanks to Mr. William Liao, B.S. student at USC, Ms. Shelly Tseng, B.S., Ms. Joanne
Lin, B.S. student at USC, Ms. Megan Trieu, B.S., and Mr. Sid Menon, B.S. for helping out on
my experiments. I would like to thank my husband, parents and siblings, for their financial
support and words of encouragement throughout the duration of my Masters’ program at USC.
Thank you God for everything, I am eternally grateful.
58
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Abstract (if available)
Abstract
Hexavalent chromium [Cr(VI)] compounds are well established human carcinogens. However, Cr(VI) compounds have different potencies as carcinogens, and the potency is dependent on both valence and solubility of the chromium (VI) compound. It has been shown that lead chromate particles (mean particle diameter of 80 µm) induced dose-dependent cytotoxicity and a dose-dependent, but low, yield of morphological transformation (focus formation) in C3H 10T1/2 Cl 8 (10T1/2) mouse embryo cells (Patierno et al, l988). It was also observed that reduction of the particle size of lead chromate by sonication enhanced the cytotoxicity in a dose-dependent manner, but the yield of morphological transformation (focus formation) in C3H 10T1/2 Cl 8 (10T1/2) cells was still low (Akinwunmi, Oluwawemitan, and Landolph, manuscript in preparation). ❧ This thesis study sought to test the hypothesis that intracellular reductants, such as ascorbic acid, can enhance cytotoxicity and morphological transformation in 10T1/2 cells treated with insoluble chromate compounds, by intracellular reduction of Cr(VI) to Cr(III). In order to enhance cytotoxicity and morphological transformation further, we investigated the effects that ascorbic acid, an intracellular reductant, has on insoluble Cr(VI) compound-induced cytotoxicity and morphological transformation of 10T1/2 mouse embryo cells. The concentrations of sonicated lead chromate and barium chromate we used were 0.25 µg/ml, 0.5 µg/ml, 0.75 µg/ml, and 1.0 µg/ml. By conducting cytotoxicity assays, we determined the highest non-cytotoxic concentrations of ascorbic acid to 10T1/2 cells, and showed that ascorbic acid was non-cytotoxic at concentrations of 0.00625 mM to 0.1 mM. Our results showed that pre-treatment of 10T1/2 cells with 0.0125 mM ascorbic acid enhanced the cytotoxicity and morphological transformation of 10T1/2 cells treated with lead chromate in a dose-dependent manner. In one preliminary experiment, we showed that 0.0125 mM ascorbic acid increased the yield of morphological transformation in 10T1/2 cells treated with 0.75µg/ml or 1.0 µg/ml of lead chromate by 4-fold and 6-fold, respectively. In summary, this thesis found that 0.0125 mM ascorbic acid enhanced cytotoxicity and morphological transformation induced by sonicated particles of lead chromate in 10T1/2 mouse embryo cells.
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Oluwawemitan, Ibukunoluwa A.
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Effects of ascorbate on cytotoxicity and morphological transformation induced by insoluble chromium (VI) compounds in C3H 10T1/2 Cl 8 mouse embryo cells
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