University of Southern California Dissertations and Theses

Ascorbate and dehydroascorbate enhance the cytotoxicity and morphological transformation of C3H/10T½ C1 8 mouse embryo cells induced by soluble chromium (VI) compounds

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Ascorbate and dehydroascorbate enhance the cytotoxicity and morphological transformation of C3H/10T½ C1 8 mouse embryo cells induced by soluble chromium (VI) compounds

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ASCORBATE AND DEHYDROASCORBATE ENHANCE THE CYTOTOXICITY AND

MORPHOLOGICAL TRANSFORMATION OF C3H/10T½ C1 8 MOUSE EMBRYO CELLS

INDUCED BY SOLUBLE CHROMIUM (VI) COMPOUNDS

By

SOPHIA ALLAF SHAHIN

___________________________________________________________

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
(Pathology)

May 2014

Copyright 2014 Sophia Allaf Shahin

2

TABLE OF CONTENTS
TITLE PAGE……………………………………………………………………………………...1
TABLE OF CONTENTS…...……………………………………………………………………..2
LIST OF FIGURES…...…………………………………………………………………………..4
LIST OF TABLES………………………………………………………………………………...6
ABSTRACT………………………………………………………………………………….……8
INTRODUCTION…………………………………………………………………………….....10
1.1 Epidemiological Studies of Cr(VI) Compounds……………………………………………….10
1.2 Carcinogenesis of Cr(VI) compounds……………………………………………………..…...12
1.3 The Dual Roles of Ascorbate in Cr(VI) Carcinogenesis……………………………………..16
1.4 Cytotoxicity of Ascorbate and Dehydroascorbate and Morphological Transformation...18
MATERIALS AND METHODS………………………………………………………………...21
2.1 Chemicals………………………………………………………………………………...………..21
2.2 C3H/10T½ Cl 8 (10T½) Mouse Embryo Cell Culture Model…………….…………………22
2.3 Cell Culture Methods…………………………………………………………………………..…23
2.4 Determination of Plating Efficiencies of the Cells…………………………………………...24
2.5 Assays for Chemically Induced Cytotoxicity……………………………………………….....25
RESULTS………………………………………………………………………………………..29
3.1 Cytotoxicity of Sodium Chromate Survival to 10T ½ Cells……………………..……….….29
3.2 Effects of Ascorbate and Dehydroascorbate on the Survival of 10T ½ Cells treated with
Sodium Chromate….……………………………………………………………………………….…36
3.3 Cytotoxicity of Calcium Chromate to 10T ½ Cells……………………………….…….….…49
3

3.4 Effects of Ascorbate and Dehydroascorbate on the Survival of 10T ½ Cells treated with
Calcium Chromate……………………………………..……………………………………………..55
3.5 Potassium Dichromate Survival of 10T1/2 Cells…………….…...……………………….…63
3.6 Effects of Ascorbate on the Transformation of 10T ½ Cells with Sodium Chromate…….68
CONCLUSION AND DISCUSSION…………………………………………………………...72
4.1 Soluble Cr(VI) Compounds Carcinogenesis…………………………………….………..72
4.2 Effect of Ascorbate on Cytotoxicity……………………………………………….…….….74
ACKNOWLEDGMENTS……………………………………………………………………….76
REFERENCES…………………………………………………………………………..………77

4

LIST OF FIGURES
Figure 1. Theoretical Model for Dose-Response Cr(VI)-Induced Transformation….………..…19
Figure 2. Cytotoxicity of sodium chromate to 10T ½ Cells: Experiment #1……….……….…...31
Figure 3. Cytotoxicity of sodium chromate of 10T½ Cells: Experiment #2……….……….…...32
Figure 4. Cytotoxicity of sodium chromate to 10T ½ Cells: Experiment #3………………….....33
Figure 5. Average of all three sodium chromate cytotoxicity experiments to 10T ½ Cells…......35
Figure 6. Effect of Ascorbate on the Survival of 10T1/2 Cells Treated with Sodium Chromate:
Experiment #1……………………………………………………………………….....37
Figure 7. Effect of Ascorbate on the Survival of 10T1/2 Cells Treated with Sodium Chromate:
Experiment #2…………………………………………………………….……….…...38
Figure 8. Effect of Ascorbate on the Survival of 10T1/2 Cells Treated with Sodium Chromate:
Experiment #3……….……………………………………………………..…...……...39
Figure 9. Effect of Ascorbate on 10T1/2 Cells Treated with Sodium Chromate: Average of
Experiments #1, #2, and #3……………………………….…………………………....41
Figure 10. Effect of varying concentrations of Dehydroascorbate on the Survival of 10T1/2 Cells
treated with Sodium Chromate: Experiment #1…………..……………………………44
Figure 11. Effect of varying concentrations of Dehydroascorbate on the Survival of 10T1/2 Cells
treated with Sodium Chromate: Experiment #2…………..……………………………45
5

Figure 12. Effect of varying concentrations of Dehydroascorbate on the Survival of 10T1/2 Cells
treated with Sodium Chromate: Experiment #3……………………..…………………46
Figure 13. Effect of varying concentrations of Dehydroascorbate on the Survival of 10T1/2 Cells
treated with Sodium Chromate: Experiment #1, 2 and 3………………………………48
Figure 14. Cytotoxicity of Calcium Chromate to 10T ½ Cells: Experiment #1……….………...50
Figure 15. Cytotoxicity of Calcium Chromate of 10T½ Cells: Experiment #2……….………....51
Figure 16. Cytotoxicity of Calcium Chromate to 10T ½ Cells: Experiment #3……………......52
Figure 17. Average of 3 Cytotoxicity Experiments for Calcium Chromate 10T ½ Cells……….54
Figure 18. Effect of Ascorbate on Calcium Chromate Cytotoxicity: Experiment #1……………56
Figure 19. Effect of Ascorbate on Calcium Chromate Cytotoxicity: Experiment #2……………57
Figure 20. Effect of Dehydroascorbate on Calcium Chromate Cytotoxicity: Experiment #1…..60
Figure 21. Effect of Dehydroascorbate on Calcium Chromate Cytotoxicity: Experiment #2…..61
Figure 22. Cytotoxicity of Potassium Dichromate: Experiment #1……………………………..64
Figure 23. Cytotoxicity of Potassium Dichromate: Experiment #2……………………………..65
Figure 24. Cytotoxicity of Potassium Dichromate: Experiment #3……………………………..66
Figure 25. Average of all 3 Cytotoxicity Experiments for Potassium Dichromate……..……... 67
Figure 26. Cytotoxicity Experiments for Transformed 10T ½ Cells treated with Ascorbate and
Sodium Chromate…………………………………………………………………….71
6

LIST OF TABLES
Table 1. Average of the 3 Sodium Chromate Plating Efficiency Cytotoxicities of 10T1/2
Cell……………………………………………………………………………………..30
Table 2. Average of the 3 Sodium Chromate Cytotoxicities of 10T1/2 Cells…………………...30
Table 3. Average of the 3 Sodium Chromate Cytotoxicities with Varying Concentrations of
Ascorbate……………………………………………...……………………………….36
Table 4. Average Effect of Ascorbate on 10T1/2 Cells Treated with Sodium Chromate……….37
Table 5. Average of the 3 Sodium Chromate Cytotoxicities with Varying Concentrations of
Dehydroascorbate………………………………...…………………………………….42
Table 6. Average Effect of Dehydroascorbate on 10T1/2 Cells Treated with Sodium
Chromate………………………………………………………………………….……43
Table 7. Average of the 3 Calcium Chromate Plating Efficiency Cytotoxicities of 10T1/2
Cells…………………………………………………………………….…………..….49
Table 8. Average of the 3 Calcium Chromate Survival Fractions of 10T1/2 Cells ……………..50
Table 9. Calcium Chromate Plating Efficiencies with Varying Concentrations of Ascorbate….55
Table 10. Calcium Chromate Survival Fraction with Varying Concentrations of Ascorbate…..56
Table 11. Calcium Chromate Cytotoxicities Plating Efficiencies with Varying Concentrations of
Dehydroascorbate…………………………………………………………….……….59
7

Table 12. Calcium Chromate Survival Fractions with Varying Concentrations of
Dehydroascorbate……………………………..……………………………………..60
Table 13. Average of the 3 Potassium Dichromate Plating Efficiency Cytotoxicities of 10T1/2
Cells………………………………………………………………………………….63
Table 14. Average of the 3 Potassium Dichromate Cytotoxicities of 10T1/2 Cells….……….64
Table 15. Transformation Data for Transformation Experiment #1 with Sodium Chromate….68
Table 17. Transformation Data for Transformation Experiment #2 with Sodium Chromate….69
Table 18. Cytotoxicity Data for Transformation #1 Experiment with Sodium Chromate…...70
Table 18. Cytotoxicity Data for Transformation #2 Experiment with Sodium Chromate…...70

8

ABSTRACT
Hexavalent chromium [Cr(VI)]-containing compounds are human carcinogens. They
cause cancers in the respiratory system when inhaled, and stomach, kidney/other internal cancers
when ingested. Soluble and insoluble hexavalent chromium [Cr(VI)] compounds induce base
substitution, deletion, addition, and frameshift mutations. Cr(VI) compounds also induce DNA-
DNA cross links and DNA-protein cross-links in mammalian cells.
In this thesis, we examined the ability of the soluble chromium compounds, sodium
chromate (Na
2
CrO
4
), calcium chromate (CaCrO
4
) and potassium dichromate (K
2
Cr
2
O
7
), to
induce cytotoxicity and morphological transformation in cultured C3H/10T½ Cl 8 (10T
1/2
)
mouse embryo cells. We tested the hypothesis that the intracellular reductants, ascorbate and
dehydroascorbate, can reduce Cr(VI), to Cr(V), Cr(IV), and Cr(III) intracellularly, making
Cr(VI) a strong cytotoxin in mammalian cells. Ascorbate is present in serum at concentrations in
the mM range under physiological conditions in humans, but is only present in the μM range in
mouse embryo cells grown in BME cell culture medium plus 10% fetal calf serum.
We hypothesized that the relatively weak responses for induction of morphological
transformation of mammalian cells by Cr(VI) compounds in culture (dose-dependent but weak
induction of foci by lead chromate, and no induction of foci by calcium chromate, potassium
dichromate , and sodium chromate) is due to the small amounts of ascorbate in cultures of
mammalian cells that are insufficient to reduce Cr(VI) to Cr(V), Cr(IV), and Cr(III). Therefore,
we investigated the cytotoxic effects of ascorbate on 10T½ mouse embryo cells, and the effects
of the highest non-cytotoxic concentrations of ascorbate on Cr(VI)-induced cytotoxicity in 10T½
cells, using reduction in plating efficiency as our cytotoxicity assay. Cell survival data showed
that ascorbate exerted significant cytotoxic effects at concentrations of 0.00625 mM and higher
9

on 10T½ mouse embryo cells. Furthermore, when 10T½ cells were treated with both Cr(VI) and
ascorbate, ascorbate played dual roles. It served as a pro-oxidant (enhancer of the cytotoxicity of
chromate) at concentrations up to 0.1 mM, and as an anti-oxidant (reducer of the cytotoxicity of
chromate) at concentrations of 0.25 mM and higher. This information is important for our
ongoing and future experiments, where we designed a protocol to incorporate ascorbate into our
assays assessing the cytotoxicity and cell transforming activity of Cr(VI) compounds. At
concentrations of 0.1 mM and lower, ascorbate enhanced the cell transforming ability of Cr(VI)
compounds to induce morphological transformation.

10

CHAPTER ONE
INTRODUCTION
1.1 Epidemiological Studies of Cr(VI) Compounds
Chronic occupational exposure of workers to Cr(VI) compounds by the inhalation route
has led to many epidemiological reports showing a strong correlation to the development of
nasal and respiratory cancers. Nasal and respiratory cancers occurred in workers engaged in the
manufacture of chrome-containing pigments and in workers involved in chromium electroplating
(reviewed in Leonard A. and Lauwreys R., 1980; reviewed in Biedermann and Landolph, l988,
l990; reviewed in Landolph, l994; Gibbs, et al, 2000; IARC). Many animal carcinogenicity
experiments, and in vitro cell transformation and mutagenicity experiments, have further
supported these epidemiology reports by showing that Cr(VI) compounds induce DNA damage,
cytotoxicity, mutation, and morphological and neoplastic transformation of mammalian cells
(Biedermann et Landolph, l988, l990; reviewed in Landolph, l994). Hence, the carcinogenesis
and cell transformation/genotoxicity studies showing that Cr(VI) compounds are mutagenic,
clastogenic, genotoxic, and carcinogenic are consistent with the epidemiological studies.
Most regulatory agencies say Cr(VI) carcinogenesis in humans follows a linear, no-
threshold dose-response curve (De Flora et al., 2000). Furthermore, even at the permissible
exposure levels of 52 μg/m
3
in 1971, statistical studies have found that humans have a 25% risk
of dying from lung cancer when exposed occupationally to Cr(VI) (Gibbs, H. et al., 2000).
Therefore, this standard was lowered to 5 μg/m
3
by U.S. Occupational Safety Health
Administration in 2006 (OSHA, 2006). Unfortunately, even with this new standard, scientists
still expect that there will be 10-45 deaths for every 1,000 exposed workers exposed to Cr(VI)
11

compounds at this concentration of 5 ug/m
3
(Gibbs, H. et al., 2000). The epidemiological
studies, together with the in vivo and in vitro studies, and the ranking of Cr(VI) compounds in
Category 1 (known human carcinogens) by the International Agency for Research on Cancer
(IARC) in Lyons, France, have led many scientists to study the molecular mechanisms by which
Cr(VI) compounds are carcinogenic (IARC, 1982; Patierno, S., et al 1988: Biedermann, K., and
Landolph, J. R., l987, l990; Zhitkovich, A., Voitkun, V., and Costa, M., 1996).

12

1.2 Carcinogenicity of Cr(VI) compounds
Under the conditions of physiological pH (7.14) and temperature (37 degrees
Centigrade), Cr(VI) is unreactive with DNA. Cr(VI) requires a biological reducer(s) for the
activation of its cytotoxic, mutagenic, and carcinogenic activity (Yuann, J., et al 1999). There are
two types of Cr(VI) based on its solubility in water: 1) the soluble Cr(VI) compounds, such as
potassium dichromate (K
2
Cr
2
O
7
), and 2) the insoluble Cr(VI) compounds, including lead
chromate (PbCrO
4
). Studies conducted on K
2
Cr
2
O
7
indicate its high cytotoxicity. K
2
Cr
2
O
7
may
also be involved in the apoptosis of Cr(VI)-induced cytotoxicity, and the induction of DNA
inter-strand cross-links and single-strand breaks in DNA (Flores A. and Perez J., 1999). Our
laboratory has shown that Cr(VI) compounds can induce strong cytotoxicity, and that lead
chromate can induce low but dose-dependent levels of morphological and neoplastic
transformation in the C3H/10T
1/2
Cl 8 (10T½) mouse embryo cell line (Patierno, S., et al,
1988). Other studies from our laboratory have demonstrated that Cr(VI) compounds, both
soluble and insoluble, are highly cytotoxic to cultured human diploid foreskin fibroblasts, and
can induce mutation to 6-thioguanine resistance and anchorage-independent transformation in
cultured diploid human fibroblasts (Biedermann and Landolph, l987, l990). Cr(VI) compounds
induce cytotoxicity, diploid mutation, and anchorage independence at 1,000-fold lower levels
than Cr(III) compounds, indicating that they are 1,000-fold stronger cytotoxins and genotoxins
than Cr(III) compounds (Biedermann and Landolph, l987; Biedermann and Landolph, l990).
These studies demonstrate that both insoluble and soluble Cr(VI) compounds are highly
cytotoxic and carcinogenic in diploid human fibroblasts (Biedermann and Landolph, l988, l990;
Nickens, K. et al, 2010). Due to their differing water solubility and the different mechanisms by
which the soluble and insoluble Cr(VI) compounds enter cells, they have differing cytotoxic and
13

carcinogenic efficiencies. Furthermore, due to their structural and chemical mimicry of sulfates
and phosphates, soluble chromates gain entry inside cells via the relatively non-specific sulfate,
phosphate anion transporter on the plasma membrane of mammalian cells (Alexander J., et al,
1995, Salnikow et al., 2008). However, before soluble Cr(VI) compounds could arrive at the
anion transporters, a fraction of the soluble chromates will be reduced extracellularly from
Cr(VI) to Cr(III) by biological reducers, such as ascorbate, cysteine, and glutathione. This
prevents a fraction of soluble Cr(VI) compounds from entering cells, since Cr(III) does not enter
cells efficiently, and detoxifies Cr(VI) compounds (Biedermann and Landolph, l990). The
insoluble chromates, such as lead chromate, escape extracellular reduction (Nickens, K., et al,
2010) and are internalized into the cells in large quantities via phagocytosis (Patierno, S., et al,
1988). Once both soluble and insoluble Cr(VI) compounds enter the cells, they are still
unreactive with DNA and unable to cause any damage until they are intracellularly reduced into
other Cr intermediates, such as Cr(V), Cr(IV) and Cr(III). During which reduction process,
Cr(V) and Cr(IV) also generate reactive oxygen species (ROS), including superoxide, hydrogen
peroxide, and hydroxyl radicals, which can also cause DNA damage and mutation.
Throughout the reduction process, there is a mixture of Cr species, such as Cr(V), Cr(IV)
and Cr(III) (Stearns, D., et al, 1993). Any of these Cr intermediates could potentially cause
damage to the DNA, Cr(V) and Cr(IV) because they cause generation of ROS. Cr(III) is very
important inside cells. Cr(III) ions can bind to DNA to form coordination complexes with DNA
bases which result in mutation (Shu, X., 1999). In addition, during the reduction of Cr(VI) to
Cr(V) and Cr(IV), these reduced species [Cr(V) and Cr(IV)] can reduce molecular oxygen to
superoxide, which can then dismute to hydrogen peroxide. Hydrogen peroxide can react further
with Cr(VI) or Fe(II) to generate hydroxyl radicals. These hydroxyl radicals can cause oxidative
14

damage to DNA, such as formation of 8-hydroxy-deoxyguanosine, thymine glycol, and damage
to other DNA bases, resulting in mutations. The most abundant Cr-induced damage is Cr-DNA
adducts (Peter-Roth, E, et. al., 2005). The major form of Cr-DNA adducts are the ternary
adducts, which usually form ternary complexes with biological reducers, such glutathione,
ascorbate, and cysteine (Quievryn, G., et al 2002 and Zhitkovich A., et al 1996). There are also
other types of genotoxic damage documented in cells treated with Cr(VI) compounds, such as
DNA-protein cross-links (Macfie, A., et al, 2010), DNA inter-strand cross-links, DNA breaks,
and base substitution and deletion mutations (Casadevall, M., et al, 1999).
Cr(VI) compounds may also cause complete loss of function of the MMR mismatch
repair system (Reynolds, M., et al, 2006). One interesting common feature of Cr(VI)-induced
carcinogenesis is the presence of microsatellite instability, which usually suggests that mismatch
repair (MMR) is impaired, and the cells are no longer able to correct errors arising from the
replication process (Hirose, T., et al, 2002). In Cr(VI)-induced carcinogenesis, other studies have
found that the MMR system has been either mutated or deleted in cancer cells, promoting the
accumulation of mutations (Takahashi, Y., et al 2005). Peterson-Roth et al. have shown
development of resistance to apoptosis is induced by Cr(VI) in MMR-deficient cells. In 2007,
Reynolds et al. showed that in cells in which MutS or MutL has been silenced by shRNA, there
is resistance to Cr(VI)-induced cytotoxicity. Without the proper functioning of MMR, mutations
accumulate and will eventually lead to carcinogenesis. Collectively, this has led to a selection
model that explains the development of Cr(VI)-induced carcinogenesis. This model suggests that
once cells are chronically exposed to a toxic dose of Cr(VI), only the resistant clones that lack
the MMR will outgrow, and the continuous exposure of Cr(VI) might no longer be needed, since
the absence of MMR will generate more mutations by itself (Salnikow, K., and Zhitkovich, A.,
15

2008). As a result, the resistant clone will eventually develop many mutations, and some of the
mutated, resistant cells will grow into tumors.

16

1.3 The Dual Roles of Ascorbate in Cr(VI) Carcinogenesis
Even though soluble and insoluble Cr(VI) compounds are well-documented carcinogens,
it is important to understand that reductive activation is absolutely necessary to initiate their
carcinogenic activities. There are many biological reducers in mammals, including glutathione,
cysteine, and ascorbate, that maintain reducing capability in the microenvironment in mammals
and in mammalian cells. Among these, ascorbates are the dominant biological reducers that
perform most of the reducing activities in vivo in mammals (Salnikow, K., and Zhitkovich, A.,
2008). Ascorbate, also known as vitamin C, is a water-soluble vitamin that is best known for its
anti-oxidative activities (Corti, A., et al, 2010). Ascorbate scavenges the reactive and toxic
hydroxyl radicals, and thereby prevents cancer development. Paradoxically, ascorbate is also the
principle reducer that activates Cr(VI) compounds by reducing them and thereby triggers their
carcinogenic activities (Ding, M., and Shi, X., 2002).
It has been shown that ascorbate acts as a two-electron donor to Cr(VI), yielding Cr(III)
(Quievryn, G., et al, 2002 and Ahmet, N., et al, 2003). Furthermore, there is some evidence
which shows that the generation of first superoxide radicals, then hydrogen peroxide, then
reactive hydroxyl radicals occurs upon reduction of Cr(VI) (Martin, B., et al, 2006, Zhitkovich,
A., et al, 2008). Therefore, the duality of ascorbates allows them to act as anti-oxidants at low
concentrations as well as pro-oxidants at higher concentrations. Depending on where the
reduction occurs, and the concentration of ascorbate, one role predominates over the other. If the
reduction of Cr(VI) occurs extracellularly, ascorbate acts as an anti-oxidant and detoxifies
Cr(VI) by generating Cr(III) extracellularly. This is because Cr(III) cannot enter mammalian
cells to any significant extent, and because the cytotoxicity of Cr(III) is only 1/1000 the
cytotoxicity of Cr(VI) (Biedermann and Landolph, l990). Inside the cell, ascorbate acts as a pro-
17

oxidant by generating Cr(III) and other reactive oxygen species (ROS, including superoxide
anion, hydrogen peroxide, and hydroxyl radicals) (Poljsak, B., et al, 2005 and Martin, B., et al
2006). Interestingly, the ratio of ascorbate: Cr(VI) could also affect the overall action of
ascorbate. A low ratio of ascorbate: Cr(VI) [low ascorbate, high Cr(VI)] gives an overall pro-
oxidative activity. Conversely the high ratio of ascorbate: Cr(VI) confers anti-oxidation
(Poljsak, B., et al, 2005). In cell culture, although ascorbate is an essential component in
activating Cr(VI) compounds, it has been shown to be present at undetectable or low
concentrations (Quievryn, G., et al, 2002). Therefore, to reproduce in vitro the conditions in
mammalian cells in vivo, it is important to introduce a supplement of ascorbate into the cell
culture medium.

18

1.4 Cytotoxicity of Ascorbate and Dehydroascorbate and Morphological Transformation
The overall goal of this project in our laboratory is to investigate the in vitro cytotoxic
activity of Cr(VI) compounds, and then the effect of intracellular reductants on the ability of the
Cr(VI) compounds to induce morphological and neoplastic cell transformation. To do this, we
began with the soluble chromium compounds, sodium chromate (Na
2
CrO
4
), calcium chromate
(CaCrO
4
), and potassium dichromate (K
2
Cr
2
O
7
), in the absence of ascorbate/dehydroascorbate.
In order to accomplish this, the first part of this thesis work is dedicated to explore the cytotoxic
effects of Cr(VI) compounds on C3H/10T½ Cl 8 (10T½) mouse embryo cells. The second part
of this thesis is to define the highest non-cytotoxic concentration of ascorbate/dehydroascorbate
that we can use in cell cultures. Next, the third part of this thesis work is to determine the pro-
oxidant (enhancing) and anti-oxidant (inhibitory) effect of ascorbate/dehydroascorbate on
Cr(VI)-induced cytotoxicity in 10T½ cells and to determine the maximum concentration of
ascorbate/dehydroascorbate that would confer the pro-oxidative activity without being cytotoxic
itself. Furthermore, in the third part of this thesis, we explored the most effective way to deliver
ascorbate/dehydroascorbate intracellularly, so we could investigate the pro-oxidant and anti-
oxidant aspects of ascorbate/dehydroascorbate on Cr(VI) cell induced transformation.
In future work, we eventually want to design a protocol maximizing the pro-oxidant
activity of ascorbate/dehydroascorbate that would provide the maximal transforming activity of
Na
2
CrO
4
, K
2
Cr
2
O
7
and CaCrO
4
, so that we could demonstrate dose-response curves for Cr(VI)
compound-induced morphological and neoplastic transformation of C3H/10T½ cells as shown in
the theoretical model demonstrated in Figure 1 (Lin, 7). We would then work to gain insight into
the molecular mechanisms of induction of morphological and neoplastic transformation of 10T
1/2

cells by soluble Cr(VI) compounds, and then develop dose-response curves for Cr(VI)-induced
19

morphological transformation. We would then ring clone foci of Cr(VI)-induced
morphologically transformed 10T
1/2
cells, expand the foci into transformed cell lines, and
characterize the biological properties of the Cr(VI)-induced transformed cell lines. Following
this, we would then work to molecularly characterize the aberrations that occur in Cr(VI)-
induced transformed cell lines by employing DNA microarrays.

In vitro cell culture experiments show that Cr(VI) compounds induce morphological and
neoplastic transformation in cultured mouse and hamster fibroblasts. Cr(VI) carcinogenesis in
animals and in humans is believed to follow a linear, no-threshold dose-response curve. In our
prior experiments, we found that lead chromate (PbCrO
4
) induced a low but dose-dependent
yield of morphological transformation (Patierno et al, l988). We suspected that we were missing
a component of the reductive activation of Cr(VI) →Cr(V) →Cr(IV) →Cr(III). We repeated our
earlier experiments, and found again a very low induction of morphologically transformed foci
induced by PbCrO
4
in 10T
1/2
cells when compared with the high yields of foci induced by
1g/mL of the strong mutagenic and clastogenic carcinogen, 3-methylcholanthrene (MCA) (Jim
20

K. Lin, M. S. Thesis, Dept. of Molecular Microbiology and Immunology, USC, 2011). This was
surprising, considering that Cr(VI) compounds are strong human carcinogens to the respiratory
system and also to internal organs when administered by the ingestion/drinking water routes.
However, we realized from reading the scientific literature, that ascorbate, a dominant biological
reducing agent, is required for reductive activation of the carcinogenic activity of Cr(VI).
Therefore, we began experiments to determine a) the cytotoxicity of ascorbate and
dehydroascorbate to 10T½ cells, and b) the effects of ascorbate and dehydroascorbate on Cr(VI)-
induced cytotoxicity and morphological transformation.

21

CHAPTER TWO
MATERIALS AND METHODS
2.1 Chemicals
All chemicals used in this thesis were purchased from commercial chemical,
pharmaceutical, or biotechnology companies. The soluble chromates used in all the
experimentations were purchased from J.T. Baker Chemical Co. (Phillipsburg, NJ). The
chemical carcinogen, 3-methylcholanthrene (MCA), of 98% purity, was purchased from Aldrich
Chemical Company, St. Louis, Missouri. Ascorbate, of 97% purity, was purchased from Sigma
Chemical Company, St. Louis, Missouri.
Basal Medium Eagles (BME) in powdered form was purchased in powder from GIBCO
Company and was prepared in the Bioreagent Core Facility of the USC/Norris Comprehensive
Cancer Center at the University of Southern California (USC), by Ms. Nily Harel under the
authority of Professor Zoltan Tokes, Director of the Bioreagents Core Facility at USC. They
dissolved BME power in water, adjusted its pH to 7.2 and filter-sterilized it for us so we could
use it to support the growth of C3H/10T½ Cl 8 (10T½) mouse embryo cells.

22

2.2 C3H/10T½ Cl 8 10T½ Mouse Embryo Cell Culture Model
The C3H/10T½ Cl 8 (10T½) mouse embryo cell line is employed as the cell culture
model for all the experimentation in this thesis. 10T½ cells are a cloned, aneuploid (4n = 81)
mouse embryonic cell line that was derived from the embryos of pregnant C3H mice (Reznikoff
et al, l973a). Under the microscope, they are morphologically elongated and stellate (cigar-
shaped) at low cell densities. At confluence, the cells are compressed into diamond or cuboidal
shapes. Studies have shown that 10T½ cells are highly sensitive to post confluence inhibition of
cell division, and that they are also anchorage dependent (Reznikoff et al, l973a). Most
importantly, 10T½ cells have a very low frequency of spontaneous focus formation and a very
low frequency of spontaneous anchorage independence. 10T½ cells possess many
characteristics that make this cell line a good in vitro cell culture model for conducting
cytotoxicity and transformation assays induced by various chemicals (Reznikoff et al, l973b;
Landolph and Heidelberger, l979; Miura et al, l988; reviewed in Landolph, l985; l994).

23

2.3 Cell Culture Methods
10T½ cells of passages from 7-15 were previously frozen and stored in liquid nitrogen
(Reznikoff et al, l973a). Cells were thawed and used as described in Resnikoff et al., 1973a, b.
The cells were first thawed in a water bath that was set at 37 degrees C and were then transferred
to a centrifuge tube for centrifugation. After centrifugation for 12 minutes at 3,000 rpm on an
IEC-HN-S centrifuge (Damon/IEC Division), the supernatant was aspirated and discarded. The
cell pellet was resuspended in 1ml of Basal Medium Eagle’s (BME) containing 10% fetal calf
serum (FCS). The resulting cell suspension was then transferred into a 25 cm
2
vented T-flask
(Corning Glass Works, Corning, New York), followed by rinsing the plastic vial containing the
original cells with an additional 4 mLs of BME, which was then also transferred into the flask.
The cells were then maintained in a Forma Scientific CO
2
humidified incubator with an
atmosphere of 5% CO
2
at 37 degrees C for 24 hours. The medium was then changed to fresh
medium the next day to remove any residual DMSO and dead and dying cells. C3H/10T½ Cl 8
(10T½) mouse embryo fibroblastic cells were grown in Basal Eagle Medium (BME),
supplemented with 10% heat-inactivated fetal bovine serum (Omega Scientific Products, Irvine,
California). Incubation was then continued, with a medium change every three days, until the
cells reached approximately 80% confluence (Reznikoff et al, l973a; Landolph and Heidelberger,
l979; Patierno et al, l988; Landolph, l994).

24

2.4 Determination of the Plating Efficiencies of the Cells
Once 10T½ cells from passages 7-12 had grown to 80% confluence, the plating
efficiencies of the cells were measured using standard protocols in use in our laboratory, as
follows: The medium was aspirated off the cells, and 1ml of Dulbecco’s Phosphate Buffered
Saline 1X (DPBS) was added to the cells to remove serum containing trypsin inhibitors so the
cells could be easily trypsinized. The DPBS was aspirated and discarded, and then 1 ml of
trypsin was added back to each flask containing cells. Once most of cells were observed
detaching from the vented flask, after approximately 5 minutes of trypsinization, we added 1ml
of medium containing 10% FCS to neutralize the further action of the trypsin on the cells. The
cell suspension was then transferred to a centrifuge tube, which was centrifuged for 12 minutes
at 3,000 rpm. Next, the supernatant was aspirated, and the cell pellet was re-suspended in 10 ml
of BME containing 10% FCS. 1 mL of re-suspended cell suspension was then aliquoted and
diluted with 19 mls of isoton buffer, and the diluted cell suspension was counted using a Coulter
Counter Model Zf (Coulter Electronics, Hialeah, Florida). The cells were then diluted to a
concentration of 200,000/5 mls, and then three times more serially, 1/10 each time, and then
seeded onto 60-mm Falcon Petri dishes in 5 ml of medium at 200 cells/dish, five dishes for each
plating efficiency determination performed. These methods have previously been published by
our laboratory and others (Reznikoff et al, l973a, b; Landolph and Heidelberger, l979; Miura et
al, l987; Patierno et al, l988).

25

2.5 Assays to Determine Chemically Induced Cytotoxicity

Measurement of the Cytotoxicity of Ascorbate and Dehydroascorbate

10T
1/2
cells were seeded at 200 cell/dish, five dishes for each concentration of ascorbate
or dehydroascorbate studied, according to the plating efficiency protocol described above.
Twenty four hours later, cells were treated with chemicals at different concentrations, for various
exposure times, to best explore the most effective method to deliver ascorbate intracellularly.
The concentrations of ascorbate tested were 0.01 mM, 0.03 mM, 0.05 mM, 0.07 mM., 0.1
mM, and 0.2 mM. Ascorbate was first dissolved in PBS. We next rinsed the 0.2 µM Nalgene
filters with 50 of DPBS, and discarded this to remove any residues of toxic compounds on the
plastic filters. Next, we then filter-sterilized and collected a second 50 ml of rinsate to use as a
negative control and as a diluent for the initial stock solution. Next, we filtered the initial
ascorbate stock solutions in PBS through 0.2 µM Nalgene filters to sterilize them, diluted them
to the appropriate stock concentrations, and then administered 25 ul of the appropriate stock
solution to the designated dishes.
The first method was to seed 200 cells per 60 mm dish, 5 dishes per each concentration of
compound tested. Then, we treated the cells 24 hours later with ascorbate at concentrations of
0.01 mM, 0.03 mM, 0.05 mM, 0.07 mM, 0.1 mM, and 0.2 mM, and exposed the cells to
ascorbate for 24 hours. We then removed the ascorbate by aspiration with a Pasteur pipette, then
added back fresh BME medium containing 10% fetal calf serum (FCS) without ascorbate. Eight
days later, we fixed the cells with methanol for thirty minutes, stained them with crystal violet
for one hour, rinsed the dishes with tap water, and allowed the dishes to air dry. We then
counted colonies containing twenty or more cells under a dissecting microscope by standard
26

methods currently in use in our laboratory (Reznikoff et al, l973b; Miura et al, l987; Patierno et
al, l988).

Effects of Ascorbate and Dehydroascorbate Upon the Cytotoxicity of the Soluble Chromium
Compounds: Sodium Chromate (Na
2
CrO
4
), Calcium Chromate (CaCrO
4
) and
Potassium Dichromate (K
2
Cr
2
O
7
)

We next seeded 60 mm dishes with 200 10T
1/2
cells per dish, five dishes for each
concentration of ascorbate or dehydroascorbate, the appropriate soluble chromium compound for
that particular experiment, or various combinations of the two to be tested. Twenty-four hours
later, without medium change, we added the appropriate concentration of soluble chromate
compound into each dish at a constant concentration of 12 μg/ml (LC
50
), and exposed the cells to
these treatment conditions for 48 hours, then changed the medium.
The second method was similar to the first method, except after the exposure to ascorbate
or dehydroascorbate, there was a medium change before treating the cells with Cr(VI). In the
third method, the order was done in reverse. The cells were treated with Cr(VI) for 48hrs first.
Without a medium change, we then added ascorbate or dehydroascorbate at concentrations that
were mentioned previously for 24 hrs. Then, a medium change was performed. The cultures
were incubated for a total period of 10 days. Then, we aspirated the medium off, rinsed the
cultures with isotonic saline (0.9% NaCl), fixed the cells with methanol, stained the cells with
1% crystal violet, and left them to air dry on the dishes. We then scored the colonies containing
20 or more cells under a dissecting microscope (Reznikoff et al, l973a,b; Miura et al, l987;
Patierno et al, l988).

27

Cytotoxicity of 3-Methylcholanthrene
The chemical mutagen and carcinogen, 3-methelchloanthrene (MCA), was used at a
concentration of 1 µg/ml for the cytotoxicity assays. Due to its nature as a strong mutagenic
carcinogen, it is used as a positive control to induce cytotoxicity and morphological
transformation (focus formation) in cell transformation assays. MCA was dissolved in acetone
and administered as 25 µl volume of a stock solution of 0.2 mg/ml to the medium on top of the
cells, such that 25 µl was added to 5.0 mls of culture medium, so we achieved a final acetone
concentration of 0.5% (v/v), which is the highest concentration of acetone that is not cytotoxic to
10T½ cells Reznikoff et al, l973b; Landolph and Heidelberger, l979; Miura et al, l988; Patierno
et al, l988)
Assay for Chemically Induced Morphological Transformation
In this paper, we have compared the incidence of focus formation induced by ascorbate
and soluble chromium compounds. 10T
1/2
cells were grown to 80% confluence, trypsinized and
seeded into 60-mm dishes in 5 ml medium at 2,000 cell/dish, 20 dishes per concentration of each
treatment condition used. Four days after seeding, we treated the cells with ascorbate at
concentrations of 0.0125 mM, 0.025 mM, 0.05 mM and 0.1 mM, respectively. Five days after
seeding, we treated the cells with the soluble chromate compound of either sodium chromate
(Na
2
CrO
4
), calcium chromate (CaCrO
4
) or potassium dichromate (K
2
Cr
2
O
7
) at a concentration of
1µM. The medium was then changed 48 hours after being treated with the soluble chromate
compound and retreated with the ascorbate. The medium was changed once a week for 6 weeks.
At the end of the sixth week of the transformation assay, the dishes were rinsed with 0.9% NaCl
saline, fixed with methanol, stained with crystal violate and scored for foci under a dissecting
microscope, according to standards methods in others (Reznikoff et al, l973b) and in our
28

laboratory (Landolph and Heidelberger, l979; Miura et al, l987; Patierno et al, l988; Landolph,
l994).

29

CHAPTER 3
RESULTS
3.1 Survival of 10T ½ Cells Treated With Sodium Chromate
Many epidemiological, animal, and cell culture studies have repeatedly concluded that
Cr(VI)-containing compounds are strong carcinogens that cause cancers in the respiratory system
(reviewed in Leonard A. and Lauwreys R., 1980; Patierno et al, l988; Biedermann and Landolph,
l987, l990; Landolph, l994; Salnikow and Zhitkovich, 2008; Shi, 2010). Therefore, in vitro cell
transformation studies with Cr(VI)-containing compounds are necessary to determine the
molecular mechanisms by which Cr(VI) compounds induced morphological and neoplastic cell
transformation. Before attempting to understand the molecular actions of Cr(VI) in transforming
10T
1/2
cells, we needed to generate transformed cell lines induced by Cr(VI). The following
experiments conducted with our three soluble chromium compounds, sodium chromate
(Na
2
CrO
4
), calcium chromate (CaCrO
4
) or potassium dichromate (K
2
Cr
2
O
7
) were completed to
induce cytotoxicity and morphological transformation of C3H/10T½ Cl 8 (10T
1/2
) mouse
embryo cells. From our previous work on PbCrO
4
, we showed we could induce a small number
of foci in a dose-dependent manner when we treated 10T
1/2
cells with lead chromate (Patierno et
al, l988). Here, we attempt to show similar results with sodium chromate.
Sodium chromate was the first soluble compound in our series of three compounds that
we determined the cytotoxicity of in 10T
1/2
cells. In experiment #1, the cytotoxicity of sodium
chromate of 10T ½ cells was found to be cytotoxic at a concentration of 1.0 μM and greater
(Table 1). At sodium chromate concentrations of 1.0 μM, 2.0 μM, 5.0 μM, 10.0 μM, and 20 μM,
the survival fractions of the cells were reduced to 0.81, 0.72, 0.01, 0, and 0, respectively (Table
30

2). Hence, the cytotoxicity of sodium chromate was dose-dependent in 10T
1/2
cells in
experiment #1. This data is also plotted graphically on a semi-log plot in Figure #2. As shown,
there is a dramatic drop in survival from 2 μM to 5 μM, where the compound becomes highly
toxic (Figure 2).

Table 1: Individual and average plating efficiencies +/- standard deviations from
three experiments determining the 10T
1/2
cells as a function of the concentrations
of sodium chromate used treat the cells.

Table 2: Average of the Sodium Chromate Cytotoxicity Survival Fractions of 10T
1/2
Cells

Concentration
(µM)
Survival Fraction 1
± SD
Survival Fraction 2
± SD
Survival Fraction 3
± SD
Avg Survival Fraction
± SD
0 1 ± 0.13 1 ± 0.15 1 ± 0.17 1 ± 0.15
1 0.81 ± 0.06 1 ± 0.14 0.88 ± 0.09 0.90 ± 0.10
2 0.72 ± 0.05 0.82 ± 0.13 0.78 ± 0.05 0.78 ± 0.08
5 0.01 ± 0.01 0.01 ± 0.02 0.01 ± 0.01 0.01 ± 0.01
10 0.00 ± 0.01 0.01 ± 0.01 0.00 ± 0.01 0.00 ± 0.01
20 0.00 ± 0.01 0.00 ± 0.01 0.00 ± 0.01 0.00 ± 0.01

Table 2: Individual and average survival fractions +/- standard deviations from
three experiments determining the 10T ½ cells as a function of the concentrations
of sodium chromate used to treat the cells.

Table 1: Average of the Sodium Chromate Plating Efficiency Cytotoxicities of 10T
1/2
Cells

Concentration Plating Efficiency 1
± SD
Plating Efficiency 2
± SD
Plating Efficiency 3
± SD
No Addition 40.6 ± 5.3 31.5 ± 4.8 37.7 ± 6.3
PBS 37.7 ± 2.2 32.1 ± 3.0 35.4 ± 3.8
1µM 32.8 ± 2.3 31.5 ± 4.4 33.1 ± 3.5
2 µM 29.4 ± 2.0 25.9 ± 4.2 29.4 ± 2.0
5 µM 0.3 ± 0.3 0.3 ± 0.7 0.4 ± 0.2
10 µM 0.2 ± 0.4 0.2 ± 0.4 0.1 ± 0.2
20 µM 0.1 ± 0.2 0 ± 0 0 ± 0
31

Figure 2: Survival of 10T
1/2
cells treated with various concentrations of sodium
chromate in experiment 1. The figure shows the survival fraction versus the
concentration of sodium chromate administered to 10T
1/2
cells.

0.0001
0.001
0.01
0.1
1
10
0 5 10 15 20 25
Log
10
(Survival Fraction)
Concentration of Sodium Chromate (uM)
Experiment 1: Cytotoxicity of Sodium Chromate
to 10T1/2 Cells
32

In experiment #2, sodium chromate was shown to be cytotoxic at a concentration of 1.0
µM and greater (Table 1). When the cells were treated with concentrations of sodium chromate
at 1.0 μM, 2.0 μM, 5.0 μM, 10 μM, and 20 μM, the Survival Fractions decreased from 1.0 to 1.0,
0.82, 0, 0, and 0, respectively (Table 2 and Figure 3). As shown in Figure 3, there is a dramatic
drop in survival fraction where 10T
1/2
cells were treated with sodium chromate with
concentrations from 2.0 μM to 5.0 μM

Figure 3: Survival of 10T
1/2
cells treated with various concentrations of sodium
chromate in experiment 2. The figure shows the survival fraction of 10T
1/2
cells
as a function of the concentrations of sodium chromate used to treat the cells.

0.001
0.01
0.1
1
10
0 5 10 15 20 25
Log
10
(Survival Fraction)
Concentration of Sodium Chromate
Experiment 2: Cytotoxicity of Sodium
Chromate to 10T1/2 Cells
33

In experiment #3 to measure the cytotoxicity of sodium chromate, the cytotoxicity of
sodium chromate to 10T½ cells was again manifest at concentrations of 1.0 μM and greater
(Table 1). The survival fractions for cells treated with 1.0 μM, 2.0 μM, 5.0 μM, 10 μM, and 20
μM were 0.88, 0.78, 1.1, 0.3, and 0, respectively (Table 2). Therefore, the survival of 10T
1/2

cells decreased in a dose-dependent manner as the concentration of sodium chromate was
increased (Table 2 and Figure 4). As shown, there is a dramatic drop in cell survival from 2.0 μM
to 5.0 μM, although the survival curve appears to follow a semi-logarithmic function, i.e., S=e-
kc
.

Figure 4: Survival of 10T ½ Cells as a function of the concentration of sodium
chromate used to treat the cells for experiment 3. This figure shows the survival
fraction of the 10 ½ cells as a function of the concentration of sodium chromate
used to the treat the cells. Results are the mean +/- standard deviation of values
from five dishes of cells treated at each concentration.

0.001
0.01
0.1
1
10
0 5 10 15 20 25
Log
10
(Survival Fraction)
Concentration of Sodium Chromate (uM)
Experiment 3: Cytotoxicity of Sodium
Chromate to 10T1/2 Cells
34

After all three experiments on the cytotoxicity of sodium chromate to 10T
1/2
cells were
completed, all the survival fractions at each concentration of sodium chromate were averaged
(Table 2). These survival fraction average values were 0.90, 0.78, 0.01, 0, and 0 in cells treated
with 1.0 μM, 2.0 μM, 5.0 μM, 10 μM, and 20 μM, respectively. This data averaged over three
experiments was plotted graphically, and is shown in Figure 5. Again, the data shows 1) that
these three experiments are very reproducible and the data can easily be averaged, and 2) that the
cytotoxicity (reduction in cell survival fractions) is dose-dependent.

35

Figure 5: Average survival +/- standard deviation of 10T
1/2
cells as a function of the
concentration of sodium chromate used to treat the cells. This data is collected from the three
experiments shown in Table 2.

0.0001
0.001
0.01
0.1
1
10
0 5 10 15 20 25
Log
10
(Survival Fraction)
Concentration of Sodium Chromate (uM)
Average of the Cytotoxicities of Sodium Chromate
to 10T1/2 Cells
Experiment 1
Experiment 2
Experiment 3
Average
36

3.2 Effects of Ascorbate and Dehydroascorbate on the Survival of 10T ½ Cells Treated with
Sodium Chromate
In experiment #1 for ascorbate, the effect of ascorbate on the cytotoxicity of sodium
chromate at 1µM to 10T
1/2
cells was measured. The effect of ascorbate was manifested at
concentrations of 0.00625 mM and greater. The survival fractions for 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 0.048, 0.048,
0.067, 0.188, 0.683, 0.269, 0.067, respectively (Table 4 and Figure 6). It appears that at ascorbate
concentrations of 0.00625 mM to 0.1 mM, ascorbate acts as an anti-oxidant, while at ascorbate
concentrations of 0.1 mM and above, ascorbate acts as an pro-oxidant (Figure 6).

Table 3: Average of the Sodium Chromate Cytotoxicities with Varying Concentrations of Ascorbate

Concentration (mM) Plating Efficiency 1 ± SD Plating Efficiency 2 ± SD Plating Efficiency ± SD
PBS 18.4 ± 1.9 18.3 ± 4.8 9.2 ± 2.8
No Addition 20.8 ± 1.6 25.3 ± 2.0 10 ± 3.2
0.00625 mM 1.0 ± 1.5 0 ± 0 0 ± 0
0.0125 mM 1.0 ± 1.1 0.1 ± 0.2 0 ± 0
0.025 mM 1.4 ± 1.3 0 ± 0 0.3 ± 0.7
0.05 mM 3.9 ± 2.2 0.4 ± 0.2 3.3 ± 1.6
0.1 mM 14.2 ± 2.5 8.9 ± 1.6 4.1 ± 2.0
0.25 mM 5.6 ± 1.3 1.1 ± 0.7 0 ± 0
0.375 mM 1.4 ± 0.8 1.3 ± 0.8 0 ± 0

Table 3: Averages of the plating efficiencies +/- standard deviations from three
experiments determining the effect of ascorbate on 10T
1/2
cells treated with 1µM
sodium chromate. The plating efficiencies of 10T
1/2
cells treated with 1µM
sodium chromate were measured at varying concentrations of ascorbate.

37

Table 4: Average Effect of Varying Concentrations of Ascorbate on 10T
1/2
Cells Treated with Sodium Chromate

Concentration
(mM)
Survival Fraction 1
± SD
Survival Fraction 2
± SD
Survival Fraction 3
± SD
Average Survival Fraction
± SD
0 1 ± 0.075 1 ± 0.079 1 ± 0.320 1 ± 0.158
0.00625 0.048 ± 0.070 0.004 ± 0.009 0.010 ± 0.023 0.021 ± 0.034
0.0125 0.048 ± 0.051 0.004 ± 0.009 0.010 ± 0.022 0.021 ± 0.027
0.025 0.067 ± 0.065 0.004 ± 0.009 0.030 ± 0.067 0.034 ± 0.047
0.05 0.188 ± 0.104 0.016 ± 0.009 0.330 ± 0.160 0.178 ± 0.091
0.1 0.683 ± 0.121 0.352 ± 0.065 0.413 ± 0.197 0.482 ± 0.128
0.25 0.269 ± 0.062 0.043 ± 0.026 0.010 ± 0.022 0.108 ± 0.037
0.375 0.067 ± 0.039 0.051 ± 0.030 0.010 ± 0.022 0.043 ± 0.031

Table 4: Averages of the survival fractions +/- standard deviations from three
experiments determining the effect of ascorbate on 10T
1/2
cells treated with 1µM
sodium chromate. The survival fractions of 10T
1/2
cells treated with 1µM sodium
chromate were measured at varying concentrations of ascorbate.

Figure 6: Effect of varying concentrations of ascorbate on the survival of 10T
1/2

Cells treated with 1µM of sodium chromate for experiment 1. The survival
fraction was plotted as a function of the concentration of ascorbate used to treat
the cells. The average survival fraction of 10T
1/2
cells for sodium chromate at
1µM was also plotted for comparison.

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
10
(Survival Fraction)
Concentration of Ascorbate (mM)
Exp 1: Effect of Ascorbate on the Survival of 10T1/2 Cells
Treated with Sodium Chromate
Experiment 1
Sodium Chromate at
1uM (Control)
38

In experiment #2, the effect of ascorbate on the cytotoxicity of sodium chromate at 1µM
to 10T
1/2
cells was measured. The effect of ascorbate was manifested at concentrations of
0.00625 mM and greater (Table 3). The survival fractions for 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 of ascorbate were 0.004,
0.004, 0.004, 0.016, 0.352, 0.043, 0.051, respectively (Table 4 and Figure 7). It appears that at
ascorbate concentrations of 0.00625 mM to 0.1 mM, ascorbate acts as an anti-oxidant (Figure 6),
while at concentrations of 0.1 mM and above, ascorbate acts as a pro-oxidant (Figure 6).

Figure 7: Effect of varying concentrations of ascorbate on the survival of 10T
1/2

cells treated with 1µM of Sodium chromate for experiment 2. The survival
fraction was plotted as a function of the concentration of ascorbate used to treat
the cells. The average survival fraction of 10T
1/2
cells for sodium chromate at
1µM was also plotted for comparison.

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
10
(Survival Fraction)
Concentration of Ascorbate
Experiment 2: Effect of Ascorbate on 10T1/2 Cells
Treated with Sodium Chromate
Experiment 2
Sodium Chromate at
1uM (Control)
39

In experiment #3, the effect of ascorbate on the cytotoxicity of sodium chromate at 1 μM
to 10T
1/2
cells was again measured. The effect of ascorbate was manifested at concentrations of
0.00625 mM and greater (Table 3). The survival fractions for 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 0.010, 0.010, 0.030,
0.330, 0.413 0.010, 0.010, respectively (Table 4 and Figure 8). It appears that at ascorbate
concentrations of 0.00625 mM to 0.1 mM, ascorbate acts as an anti-oxidant (Figure 6), while at
concentrations of 0.1 mM and above, ascorbate acts as a pro-oxidant (Figure 6).

Figure 8: Effect of varying concentrations of ascorbate on the survival of 10T
1/2

cells treated with 1µM of sodium chromate for experiment 3. The survival
fraction was plotted as a function of the concentration of ascorbate used to treat
the cells. The average survival fraction of 10T
1/2
cells for sodium chromate at
1µM was also plotted for comparison.

0.01
0.1
1
10
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4
Log
10
(Survival Fraction)
Concentration of Ascorbate (mM)
Experiment 3: Effect of Ascorbate on 10T1/2
Cells Treated with Sodium Chromate
Experiment 3
Sodium Chromate at
1uM (Control)
40

After all three experiments testing the effect of ascorbate on the cytotoxicity of sodium
chromate to 10T
1/2
cell were completed, all the survival fractions at each concentration of
ascorbate were averaged. The survival fraction average values at 0.00625 mM, 0.0125 mM,
0.025 mM, 0.05 mM, 0.1 mM, 0.25 mM, and 0.375 mM of cells treated with ascorbate, were
0.021, 0.021, 0.034, 0.178, 0.482, 0.108, 0.043, respectively (Table 4). This data averaged over
three experiments was plotted graphically, as shown in figure 9. Again, from figure 9, the data
shows that 1) at concentrations of 0.00625 mM to 0.1 mM, ascorbate acts as an anti-oxidant and
2) at concentrations greater than 0.1 mM ascorbate acts as a pro-oxidant.

41

Figure 9: Average survival +/- standard deviation of 10T
1/2
cells treated with
1µM sodium chromate as a function of the concentration of ascorbate used to treat
cells. This data is collected from three experiments as shown in table 4. The effect
of ascorbate on 10T
1/2
cells without sodium chromate treatment was also plotted
for comparison. The average survival fraction of 10T
1/2
cells for sodium chromate
at 1µM was also plotted for comparison.

0.001
0.01
0.1
1
10
0 0.1 0.2 0.3 0.4 0.5 0.6
Log
10
(Survival Fraction)
Concentration of Ascorbate (mM)
Effect of Ascorbate on 10T1/2 Cells Treated with
Sodium Chromate
Experiment 1
Experiment 2
Experiment 3
Average
Ascorbate Control
Sodium Chromate at
1uM (Control)
42

In experiment #1 for dehydroascorbate, the effect of varying concentrations of
dehydroascorbate on the cytotoxicity of sodium chromate at 1µM to 10T
1/2
cells was measured.
The effect of dehydroascorbate was manifested at concentrations of 0.00625 mM and greater.
The survival fractions for cells treated with 0.00625 mM, 0.0125 mM, 0.025 mM, 0.05 mM, 0.10
mM, 0.25 mM, 0.375 mM, and 0.5 mM were 0.058, 0.034, 0.010, 0.091, 0.314, 0.365, 0.341,
0.404, respectively (Table 6). At concentrations of dehydroascorbate below 0.025 mM,
dehydroascorbate appears to act as a pro-oxidant (Figure 10). At concentrations of
dehydroascorbate above 0.025 mM, dehydroascorbate appears to act as an anti-oxidant (Figure
10).

Table 5: Average of the Sodium Chromate Cytotoxicities with Varying Concentrations of Dehydroascorbate

Concentration Plating Efficiency 1 ± SD Plating Efficiency 2 ± SD Plating Efficiency 3 ± SD
PBS 18.4 ± 1.9 18.3 ± 4.8 9.2 ± 2.8
No Addition 20.8 ± 1.6 25.3 ± 2.0 10 ± 3.2
0.00625 mM 1.2 ± 0.6 0 ± 0 0 ± 0
0.0125 mM 0.7 ± 0.3 0 ± 0 0 ± 0
0.025 mM 0.2 ± 0.3 0 ± 0 0 ± 0
0.05 mM 1.9 ± 1.1 0 ± 0 0 ± 0
0.1 mM 7.1 ± 2.0 0 ± 0 1.4 ± 1.2
0.25 mM 7.6 ± 1.7 0.2 ± 0.4 5.8 ± 0.8
0.375 mM 7.1 ± 2.9 1.2 ± 1.1 7.6 ± 1.5
0.5 mM 8.4 ± 2.5 0.8 ± 0.6 3.3 ± 1.0

Table 5: Averages of the plating efficiencies +/- standard deviations from three
experiments determining the effect of dehydroascorbate on 10T
1/2
cells treated
with 1µM sodium chromate. The plating efficiencies of 10T
1/2
cells treated with
1µM sodium chromate were measured at varying concentrations of
dehydroascorbate.

43

Table 6 : Average Effect of Dehydroascorbate on 10T
1/2
Cells Treated with Sodium Chromate

Concentration Survival Fraction 1 ± SD Survival Fraction 2 ± SD Survival Fraction 3 ± SD Average Survival Fraction ± SD
No Addition 1 ± 0.075 1 ± 0.079 1 ± 0.079 1 ± 0.158
0.00625 0.058 ± 0.027 0.004 ± 0.009 0.010 ± 0.009 0.024 ± 0.020
0.0125 0.034 ± 0.013 0.004 ± 0.009 0.010 ± 0.009 0.016 ± 0.015
0.025 0.010 ± 0.013 0.004 ± 0.009 0.010 ± 0.009 0.008 ± 0.015
0.05 0.091 ± 0.055 0.004 ± 0.009 0.010 ± 0.009 0.035 ± 0.029
0.1 0.341 ± 0.098 0.004 ± 0.009 0.140 ± 0.009 0.162 ± 0.075
0.25 0.365 ± 0.084 0.008 ± 0.018 0.580 ± 0.018 0.318 ± 0.059
0.375 0.341 ± 0.137 0.048 ± 0.043 0.760 ± 0.043 0.383 ± 0.109
0.5 0.404 ± 0.108 0.032 ± 0.023 0.330 ± 0.023 0.255 ± 0.078

Table 6: Averages of the survival fractions +/- standard deviations from three
experiments determining the effect of dehydroascorbate on 10T
1/2
cells treated
with 1µM sodium chromate. The survival fractions of 10T
1/2
cells treated with
1µM sodium chromate were measured at varying concentrations of
dehydroascorbate in mM.

44

Figure 10: Effect of varying concentrations of dehydroascorbate on the survival
of 10T
1/2
cells treated with 1µM of sodium chromate for experiment 1. The
survival fraction was plotted as a function of the concentration of
dehydroascorbate used to treat the cells. The average survival fraction of 10T
1/2

cells for Sodium chromate at 1µM was also plotted for comparison.

0.001
0.01
0.1
1
10
0 0.1 0.2 0.3 0.4 0.5 0.6
Log
10
(Survival Fraction)
Concentration of DehydroAscorbate (mM)
Experiment 1: Effect of DehydroAscorbate on
10T1/2 Cells Treated with Sodium Chromate
Exp 1
Sodium Chromate
at 1uM (Control)
45

In experiment #2, the effect of dehydroascorbate on the cytotoxicity of sodium chromate
at 1µM to 10T
1/2
cells was measured. The effect of dehydroascorbate was again manifested at
concentrations of 0.00625 mM and greater. The survival fractions for cells treated with 0.00625
mM, 0.0125 mM, 0.025 mM, 0.05 mM, 0.10 mM, 0.25 mM, 0.375 mM, and 0.5 mM were 0.004,
0.004, 0.004, 0.004, 0.004, 0.008, 0.047, 0.032, respectively (Table 6). At concentrations of
dehydroascorbate below 0.10 mM, dehydroascorbate seems to act as a pro-oxidant (Figure 11).
At concentrations of dehydroascorbate above 0.10 mM to 0.375 mM, dehydroascorbate appears
to act as an anti-oxidant (Figure 11).

Figure 11: Effect of dehydroascorbate on the survival of 10T
1/2
cells treated with
1µM of sodium chromate for experiment 2. The survival fraction was plotted as a
function of the concentration of dehydroascorbate used to treat the cells. The
average survival fraction of 10T
1/2
cells for sodium chromate at 1µM was also
plotted for comparison.

0.001
0.01
0.1
1
10
0 0.1 0.2 0.3 0.4 0.5 0.6
Log
10
(Survival Fraction)
Concentration of DehydroAscorbate (mM)
Experiment 2: Effect of DehydoAscorbate on
10T1/2 Cells Treated with Sodium Chromate
Exp 2
Sodium Chromate at
1uM (Control)
46

In experiment #3, the effect of varying concentrations of dehydroascorbate on the
cytotoxicity of sodium chromate at 1µM to 10T
1/2
cells was measured. The effect of
dehydroascorbate was manifested at concentrations of 0.00625 mM and greater. The survival
fractions for cells treated with 0.00625 mM, 0.0125 mM, 0.025 mM, 0.05 mM, 0.10 mM, 0.25
mM, 0.375 mM, and 0.5 mM were 0.010, 0.010, 0.010, 0.010, 0.140, 0.580, 0.760, 0.330,
respectively (Table 6). At concentrations of dehydroascorbate below 0.05 mM, dehydroascorbate
seems to act as a pro-oxidant (Figure 12). At concentrations of dehydroascorbate at 0.05 mM to
0.375 mM, dehydroascorbate appears to act as an anti-oxidant (Figure 12).

Figure 12: Effect of dehydroascorbate on the survival of 10T
1/2
cells treated with
1µM of sodium chromate for experiment 3. The survival fraction was plotted as a
function of the concentration of dehydroascorbate used to treat the cells. The
average survival fraction of 10T
1/2
cells for sodium chromate at 1µM was also
plotted for comparison.

0.01
0.1
1
10
0 0.1 0.2 0.3 0.4 0.5 0.6
Log
10
(Survival Fraction)
Concentration of DehydroAscorbate (mM)
Exp 3: Effect of DehydroAscorbate on 10T1/2
Cells Treated with Sodium Chromate
Exp 3
Sodium Chromate at
1uM (Control)
47

After all three experiments testing the effect of dehydroascorbate on the cytotoxicity of
sodium chromate to 10T
1/2
cell were completed, all the survival fractions at each concentration
of sodium chromate were averaged. The survival fraction average values at 0.00625 mM, 0.0125
mM, 0.025 mM, 0.05 mM, 0.1 mM, 0.25 mM, 0.375 mM, and 0.50 mM of dehydroascorbate
were 0.024, 0.016, 0.008, 0.035, 0.162, 0.318, 0.383, 0.255, respectively (Table 6). This data
averaged over three experiments was plotted graphically, as shown in figure 13. From figure 13
and table 6, the average of all three experiments showed a pro-oxidant effect of
dehydroascorbate at concentrations of 0.025 mM and below, and its anti-oxidant effect at
concentrations of 0.05 mM to 0.375 mM.

48

Figure 13: Average survival +/- standard deviation of 10T
1/2
cells treated with
1µM sodium chromate as a function of the concentration of dehydroascorbate
used to treat cells. This data is collected from three experiments as shown in table
6. The effect of dehydroascorbate on 10T
1/2
cells without sodium chromate
treatment was also plotted for comparison. The average survival fraction of 10T
1/2

cells for sodium chromate at 1µM was also plotted for comparison.

0.001
0.01
0.1
1
10
0 0.1 0.2 0.3 0.4 0.5 0.6
Log
10
(Survival Fraction)
Concentration of DehydroAscorbate (mM)
Effect of DehydroAscorbate on 10T1/2 Cells Treated with Sodium
Chromate
Experiment 1
Experiment 2
Experiment 3
Average
DehydroAscorbate
Control
Sodium Chromate
at 1uM (Control)
49

3.3 Calcium Chromate Survival of 10T ½ Cells
Calcium chromate was the second soluble Cr(VI) compound that we studied to determine
its cytotoxicity in 10T
1/2
cells. In experiment #1, the cytotoxicity of calcium chromate on 10T½
cells became manifest at a concentration of 1.0 µM and greater (Table 7). At concentrations of
1.0, 2.0, 5.0, 10.0, and 20.0 μM, the survival of 10T
1/2
cells decreased to 0.85, 0.75, 0.25, 0.02
and 0.00 (Table 8 and Figure 14). The cell survival decreased in a dose-dependent manner.

Table 7: Average of the Calcium Chromate Plating Efficiency Cytotoxicities of 10T
1/2
Cells

Concentration PE 1 ± SD PE 2 ± SD PE 3 ± SD Avg PE ± SD Survival 1 Survival 2 Survival 3 Avg Survival
No Addition 45.0 ± 1.4 40.7 ± 2.6 42 ± 2.6 42.6 ± 2.2 100.0% 100.0% 100.0% 100.0%
PBS 44.2 ± 1.5 41.4 ± 2.3 40.8 ± 3.3 42.1 ± 2.4 98.2% 101.7% 97.1% 99.0%
1µM CaCrO4 38.1 ± 1.9 28.9 ± 5.7 31.6 ± 1.8 32.9 ± 3.1 84.7% 71.0% 75.2% 77.0%
2 µM CaCrO4 33.7 ± 2.8 26 ± 3.6 23.7 ± 5.3 27.8 ± 3.9 74.9% 63.9% 56.4% 65.1%
5 µM CaCrO4 11.1 ± 3.5 8.25 ± 2.5 7.9 ± 2.0 9.1 ± 2.7 24.7% 20.3% 18.8% 21.2%
10 µM CaCrO4 0.9 ± 0.9 0.7 ± 0.5 0.2 ± 0.3 0.6 ± 0.5 2.0% 1.7% 0.5% 1.4%
20 µM CaCrO4 0 ± 0 0.1 ± 0.2 0.1 ± 0.2 0.1 ± 0.1 0.0% 0.2% 0.2% 0.2%

Table 7: Individual and average plating efficiencies +/- standard deviations from three
experiments determining the 10T
1/2
cells as a function of the concentrations of calcium chromate
used treat the cells.

50

Table 8: Average of the Calcium Chromate Cytotoxicity Survival Fractions of 10T
1/2
Cells

Concentration Survival
Fraction 1
Survival
Fraction 2
Survival
Fraction 3
Avg Survival
Fraction
Error 1 Error 2 Error 3 Average Error
No Addition 1 1 1 1 0.03 0.06 0.06 0.05
1µM CaCrO4 0.85 0.71 0.75 0.77 0.04 0.14 0.04 0.08
2 µM CaCrO4 0.75 0.64 0.56 0.65 0.06 0.09 0.13 0.09
5 µM CaCrO4 0.25 0.20 0.19 0.21 0.08 0.06 0.05 0.06
10 µM CaCrO4 0.02 0.02 0.00 0.014 0.02 0.01 0.01 0.01
20 µM CaCrO4 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.01

Table 8: Individual and average survival fractions +/- standard deviations from three
experiments determining the 10T ½ cells as a function of the concentrations of calcium
chromate used to treat the cells.

Figure 14: Survival fraction +/- standard deviation of 10T
1/2
cells treated with
various concentrations of calcium chromate for experiment 1. The figure shows
the survival fraction versus the concentration of calcium chromate administered to
10T
1/2
cells.

0.0001
0.001
0.01
0.1
1
10
0 5 10 15 20 25
Log
10
(Survival Fraction)
Concentration of Calcium Chromate (uM)
Effect of Calcium Chromate on the Survival
of the 10T 1/2 Cells
51

In experiment #2, the cytotoxicity of sodium chromate to 10T½ cells also began at a
concentration of 1.0 µM, and continued to increase in a dose-dependent manner with the
concentrations of calcium chromate applied to the cells (Table 8 and Figure 15). At calcium
chromate concentrations of 1.0, 2.0, 5.0, 10.0, and 20.0 µM, the survival of 10T
1/2
cells
decreased to 0.71, 0.64, 0.20, 0.02, and 0.00, respectively (Table 8). The curve for survival
fraction as a function of dose of calcium chromate appeared to follow the semi-logarithmic
relationship, S = E (-kc).

Figure 15: Survival fractions (relative plating efficiencies) +/- standard deviation
of 10T ½ cells treated with varying concentrations of calcium chromate. The
figure shows the survival fraction versus the concentration of calcium chromate
administered to 10T
1/2
cells.

0.001
0.01
0.1
1
10
0 5 10 15 20 25
Log
10
(Survival Fraction)
Concentration of Calcium Chromate (uM)
Effect of Calcium Chromate on the Survival of the
10T 1/2 Cells
52

In experiment #3, the cytotoxicity of calcium chromate to 10T½ cells again was
dependent upon the dose of calcium chromate added to the cells, and again appeared to follow
the relationship S = e (-kc). In cells treated with 1.0, 2.0, 5.0, 10.0, and 20.0 µM calcium
chromate, the survival of 10T
1/2
cells again decreased in a dose-dependent manner to 0.71, 064,
0.20, 0.017, and 0.0025, respectively (Table 8). When this data was plotted, it again appeared to
follow a function of the form S = e(-kc) (Figure 16).

Figure 16: Survival fractions +/- standard deviations of 10T ½ cells treated with
varying concentrations of calcium chromate. The figure shows the survival
fraction versus the concentration of calcium chromate administered to 10T
1/2

cells.

0.001
0.01
0.1
1
10
0 5 10 15 20 25
Log
10
(Survival Fraction)
Concentration of Calcium Chromate (uM)
Effect of Calcium Chromate on the Survival
of the 10T 1/2 Cells
53

After all three cytotoxicity experiments with calcium chromate were completed, an average
survival fraction at each concentration of calcium chromate was calculated from the three experiments
(Table 8). The average survival fraction was plotted against the concentrations of calcium chromate used
to treat the 10T
1/2
cells (Figure 17). This cumulative data for the three experiments indicated that in cells
treated with 1.0, 2.0, 5.0, 10.0, and 20 µM, the survival fractions decreased 1.0 in control 10T
1/2
cells, to
0.75, 056, 0.19, 0.005, and 0.002, respectively (Table 8). The results have been plotted and graphed in
Figure 17 below, which again shows that the survival curve for 10T
1/2
cells treated with calcium
chromate follows the relationship S = e(-kc), and that the data are very reproducible among the three
experiments.

54

Figure 17: Survival fractions of 10T½ cells shown in from three experiments
shown in Figures 6-8 along with the determined standard deviations. These data
were averaged and plotted in Figure 9.

0.0001
0.001
0.01
0.1
1
10
0 5 10 15 20 25
Log
10
(Survival Fraction)
Concentration of Calcium Chromate (uM)
Average of Calcium Chromate 10T
1/2
Cell Survival
Experiments
Experiment 1
Experiment 2
Experiment 3
Average
55

3.4 Effects of Ascorbate and Dehydroascorbate on Survival of 10T ½ Cells treated with Calcium
Chromate
In experiment #, the effect of varying concentrations of ascorbate on the cytotoxicity of
calcium chromate at 1µM to 10T
1/2
cells was measured. The effect of ascorbate was manifested
at concentrations of 0.00625 mM and greater. The survival fractions for 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 0.60,
0.42, 0.81, 0.78, 0.61, 0.13, 0.03, respectively (Table 10 and Figure 18).

Table 9: Calcium Chromate Cytotoxicity Plating Efficiency with Varying Concentrations of Ascorbate

Concentration Plating Efficiency 1 ± SD Plating Efficiency 2 ± SD

No Addition 21.7 ± 3.9 17.2 ± 1.0

0.00625 mM 12.9 ± 2.8 12.3 ± 2.8

0.0125 mM 9.1 ± 4.1 14.6 ± 2.6

0.025 mM 17.6 ± 2.2 14.2 ± 2.6

0.05 mM 16.9 ± 2.9 18.8 ± 1.6

0.1 mM 13.1 ± 1.7 11.1 ± 2.7

0.25 mM 2.8 ± 2.9 7.1 ± 4.7

0.375 mM 0.6 ± 0.7 4.9 ± 3.7

Table 9: Averages of the plating efficiencies +/- standard deviations from two
experiments determining the effect of ascorbate on 10T
1/2
cells treated with 1µM
calcium chromate. The plating efficiencies of 10T
1/2
cells treated with 1µM
calcium chromate were measured at varying concentrations of ascorbate.

56

Table 10 : Effect of Ascorbate on Survival of 10T
1/2
Cells Treated with Calcium Chromate

Concentration

Survival Fraction 1 ± SD

Survival Fraction 2 ± SD

No Addition 1 ± 1 0.17 ± 0.06
0.00625 mM
0.58 ± 0.71 0.12 ± 0.16
0.0125 mM
0.42 ± 0.84 0.19 ± 0.15
0.025 mM
0.82 ± 0.82 0.10 ± 0.15
0.05 mM
0.78 ± 1.09 0.14 ± 0.09
0.1 mM
0.61 ± 0.66 0.08 ± 0.16
0.25 mM
0.13 ± 0.41 0.13 ± 0.27
0.375 mM
0.03 ± 0.28 0.03 ± 0.21

Table 10: Averages of the survival fractions +/- standard deviations from two experiments
determining the effect of ascorbate on 10T
1/2
cells treated with 1µM calcium chromate.
The survival fractions of 10T
1/2
cells treated with 1µM calcium chromate were measured
at varying concentrations of ascorbate.

Figure 18: Effect of ascorbate on the survival of 10T
1/2
cells treated with 1µM of
calcium chromate for experiment 1. The survival fraction was plotted as a
function of the concentration of ascorbate used to treat the cells. The survival
fraction of 10T
1/2
cells treated with only calcium chromate at 1µM is shown for
comparison.
0.01
0.1
1
10
0 0.1 0.2 0.3 0.4
Log
10
(Survival Fraction)
Concentration of Ascorbate (mM)
Effect of Ascorbate on 10T1/2 Cells treated
with Calcium chromate
Exp 1
Calcium Chromate
without Ascorbate
(control)
57

In experiment #2, the effect of varying concentrations of ascorbate on the cytotoxicity of
calcium chromate at 1µM to 10T
1/2
cells was measured. The effect of ascorbate was manifested
at concentrations of 0.00625 mM and greater. The survival fractions for 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 0.72,
0.85, 0.83, 1.09, 0.65, 0.41, and 0.28, respectively (Table 10 and Figure 19).

Figure 19: Effect of ascorbate on the survival of 10T
1/2
cells treated with 1µM of
calcium chromate for experiment 2. The survival fraction was plotted as a
function of the concentration of ascorbate used to treat the cells. The survival
fraction of 10T
1/2
cells treated with only calcium chromate at 1µM is shown for
comparison.

0.01
0.1
1
10
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4
Log
10
(Survival Fraction)
Concentration of Ascorbate (mM)
Effect of Ascorbate on 10T1/2 Cells treated
with Calcium chromate
Ascorbate
Calcium chromate
without Ascorbate
(Control)
58

After both experiments on the effect of ascorbate to the survival of calcium chromate to
10T
1/2
cells were completed, the survival fractions at each concentration of calcium chromate
were averaged. The survival fraction average values at 0.00625 mM, 0.0125 mM, 0.025 mM,
0.05 mM, 0.1 mM, 0.25 mM, 0.375 mM, and 0.50 mM of ascorbate were 0.35, 0.31, 0.46, 0.85,
0.35, 0.13 and 0.03 respectively (Table 10). This data averaged and showed a pro-oxidant effect
of ascorbate at concentrations of 0.025 mM and below, and its anti-oxidant effect at
concentrations of 0.05 mM to 0.375 mM.

59

In experiment #1 for dehydroascorbate, the effect of varying concentrations of
dehydroascorbate on the cytotoxicity of calcium chromate at 1µM to 10T
1/2
cells was measured.
The effect of dehydroscorbate was manifested at concentrations of 0.00625 mM and greater. The
survival fractions for cells treated with 0.00625 mM, 0.0125 mM, 0.025 mM, 0.05 mM, 0.1 mM,
0.25 mM, 0.375 mM, and 0.5 mM were 0.19, 0.14, 0.24, 0.20, 0.36, 0.53, 0.55, 0.55, respectively
(Table 12 and Figure 20).

Table 11: Calcium Chromate Cytotoxicity Plating Efficiency with Varying Concentrations of Dehydroscorbate

Concentration Survival Fraction 1 ± SD Survival Fraction 2 ± SD
No Addition 21.7 ± 3.9 17.2 ± 1.0
0.00625 mM 4.1 ± 1.2 15.8 ± 2.6
0.0125 mM 3.1 ± 2.4 15.2 ± 2.7
0.025 mM 5.1 ± 2.9 14.6 ± 1.8
0.05 mM 4.4 ± 2.0 14.3 ± 1.4
0.1 mM 7.8 ± 3.4 15.1 ± 2.1
0.25 mM 11.4 ± 2.5 18.6 ± 2.2
0.375 mM 11.8 ± 1.4 12.6 ± 1.9
0.5 mM 12.0 ± 2.1 12.4 ± 1.4
Table 11: Averages of the plating efficiencies +/- standard deviations from two
experiments determining the effect of dehydroascorbate on 10T
1/2
cells treated with 1µM
calcium chromate. The plating efficiencies of 10T
1/2
cells treated with 1µM calcium
chromate were measured at varying concentrations of dehydroascorbate.

60

Table 12: Calcium Chromate Survival Fraction with Varying Concentrations of Dehydroscorbate
Concentration Survival Fraction 1 ± SD Survival Fraction 2 ± SD
No Addition 1 ± 1 0.2 ± 0.1
0.00625 mM 0.2 ± 0.9 0.1 ± 0.2
0.0125 mM 0.1 ± 0.8 0.1 ± 0.2
0.025 mM 0.2 ± 0.8 0.1 ± 0.1
0.05 mM 0.2 ± 0.8 0.1 ± 0.8
0.1 mM 0.4 ± 0.8 0.2 ± 0.1
0.25 mM 0.5 ± 1.1 0.1 ± 0.1
0.375 mM 0.5 ± 0.7 0.1 ± 0.1
0.5 mM 0.6 ± 0.7 0.1 ± 0.1
Table 12: Averages of the survival fractions +/- standard deviations from three
experiments determining the effect of dehydroascorbate on 10T
1/2
cells treated with 1µM
calcium chromate. The survival fractions of 10T
1/2
cells treated with 1µM calcium
chromate were measured at varying concentrations of dehydroascorbate.

Figure 20: Effect of dehydroascorbate on the survival of 10T
1/2
cells treated with
1µM of calcium chromate for experiment 1. The survival fraction was plotted as a
function of the concentration of dehydroascorbate used to treat the cells. The
survival fraction of 10T
1/2
cells treated with only calcium chromate at 1µM is
shown for comparison in red.
0.01
0.1
1
10
0 0.1 0.2 0.3 0.4 0.5 0.6
Log
10
(Survival Fraction)
Concentration of Dehydroascorbate (mM)
Exp 1: Effect of Dehydroascorbate on 10T
1/2
Cells
Treated with Calcium Chromate
Exp 1
61

In experiment, the effect of varying concentrations of dehydroascorbate on the
cytotoxicity of Calcium chromate at 1µM to 10T
1/2
Cells was measured. The effect of
dehydroscorbate was manifested at concentrations of 0.00625 mM and greater. The survival
fractions for cells treated with 0.00625 mM, 0.0125 mM, 0.025 mM, 0.05 mM, 0.1 mM, 0.25
mM, 0.375 mM, and 0.5 mM were 0.92, 0.88, 0.85, 0.83, 0.88, 1.08, 0.73, 0.72, respectively
(Table 12 and Figure 21).

Figure 21: Effect of dehydroascorbate on the survival of 10T
1/2
cells treated with
1µM of calcium chromate for experiment 2. The survival fraction was plotted as a
function of the concentration of dehydroscorbate used to treat the cells. The
survival fraction of 10T
1/2
cells treated with only calcium chromate at 1µM is
shown for comparison in red.

0.1
1
10
0 0.1 0.2 0.3 0.4 0.5 0.6
Log
10
(Survival Fraction)
Concentration of Dehydroascorbate (mM)
Exp 2: Effect of Dehydroascorbate on 10T
1/2
Cells
Treated with Calcium Chromate
Dehydroascorbate
62

After both experiments on the effect of dehydroascorbate to the survival of calcium
chromate to 10T
1/2
cells were completed, the survival fractions at each concentration of calcium
chromate were averaged. The survival fraction average values at 0.00625 mM, 0.0125 mM,
0.025 mM, 0.05 mM, 0.1 mM, 0.25 mM, 0.375 mM, and 0.50 mM of ascorbate were 0.15, 0.1,
0.15, 0.15, 0.3, 0.3 and 0.35 respectively (Table 12). This data averaged and showed a pro-
oxidant effect of ascorbate at concentrations of 0.025 mM and below, and its anti-oxidant effect
at concentrations of 0.05 mM to 0.375 mM.

63

3.5 Potassium Dichromate Survival of 10T ½ Cells
Potassium dichromate was the third soluble Cr(VI) compound in our series of three
compounds that we studied to determine its cytotoxicity in 10T
1/2
cells. In experiment #1, the
cytotoxicity of potassium dichromate on 10T½ cells became manifest at a concentration of 1.0
µM and greater (Table 13). At concentrations of 1.0, 2.0, 5.0, 10.0, and 20.0 μM, the survival of
10T
1/2
cells decreased to 0.28, 0.15, 0.04, 0.01, and 0.00, respectively (Table 14 and figure 20).
The cell survival decreased in a dose-dependent manner as shown in figure 20.

Table 13: Average Potassium Dichromate Plating Efficiency of the Cytotoxicity to 10T
1/2
Cells
Concentration PE 1 ±
SD
PE 2 ±
SD
PE 3 ±
SD
Average
PE ± SD
Survival
1
Survival
2
Survival
3
Average
Survival

No Addition

40.6 ± 1.1

42.5 ± 2.2

45.6 ± 1.7

42.9 ± 1.7

100.0%

100.0%

100.0%

100.0%
PBS 38.8 ± 2.5 39.7 ± 1.8 47 ± 1.8 41.8 ± 2.0 95.6% 93.4% 103.1% 97.3%
1µm 11.2 ± 2.0 11.7 ± 1.3 15.3 ± 1.6 12.7 ± 1.6 27.6% 27.5% 33.6% 29.6%
2µm 5.9 ± 2.6 7.1 ± 2.1 9.9 ± 2.2 7.6 ± 2.3 14.5% 16.7% 21.7% 17.6%
5µm 1.6 ± 2.2 1.0 ± 1.2 1.5 ± 1.7 1.4 ± 1.7 3.9% 2.4% 3.3% 3.2%
10µm 0.5 ± 0.6 0.2 ± 0.3 0 ± 0 0.2 ± 0.3 1.2% 0.5% 0.0% 0.6%
20µm 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0.0% 0.0% 0.0% 0

Table 13: Individual and average plating efficiencies +/- standard deviations from
three experiments determining the 10T
1/2
cells as a function of the concentrations
of potassium dichromate used treat the cells.

64

Table 14: Average Survival Fraction of Potassium Dichromate Cytotoxicities to 10T
1/2
Cells

Concentration Survival
Fraction 1
Survival
Fraction 2
Survival
Fraction 3
Avg
Survival
Fraction
Error 1 Error 2 Error 3 Avg
Error

No Addition

1

1

1

1

0.03

0.05

0.04

0.04
1 0.28 0.28 0.34 0.30 0.05 0.03 0.04 0.04
2 0.15 0.17 0.22 0.18 0.061 0.05 0.05 0.05
5 0.04 0.02 0.03 0.03 0.05 0.03 0.04 0.04
10 0.01 0.00 0.00 0.01 0.02 0.01 0.00 0.01
20 0.00 0.00 0.00 0.00 0.01 0.01 0.00 0.01

Table 14: Individual and average survival fractions +/- standard deviations from
three experiments determining the 10T ½ cells as a function of the concentrations
of potassium dichromate used to treat the cells.

Figure 22: Survival of 10T
1/2
cells treated with various concentrations of
potassium dichromate in experiment 1. The figure shows the survival fraction
versus the concentration of potassium dichromate administered to 10T
1/2
cells.

0.001
0.01
0.1
1
10
0 5 10 15 20 25
Survival Fraction
Concentration of Potassium Dichromate (uM)
Cytotoxicity 1: Potassium Dichromate Survival
65

In experiment #2, the cytotoxicity of potassium dichromate to 10T½ cells also began at a
concentration of 1.0uM, and continued to increase in a dose-dependent manner with the
concentrations of potassium dichromate applied to the cells (Table 13 and Figure 21). At
concentrations of 1.0, 2.0, 5.0, 10.0, and 20.0 μM, the survival of 10T
1/2
cells decreased to 0.28,
0.17, 0.02, 0.00, and 0.00, respectively (Table14). The curve for survival fraction as a function of
dose of calcium chromate appeared to follow the semi-logarithmic relationship, S = E (-kc)
(Figure 21)

Figure 23: Survival of 10T
1/2
cells treated with various concentrations of
potassium dichromate in experiment 2. The figure shows the survival fraction
versus the concentration of potassium dichromate administered to 10T
1/2
cells.

0.001
0.01
0.1
1
10
0 5 10 15 20 25
Survival Fraction
Concentration of Potassium Dichromate (uM)
Cytotoxicity 2: Potassium Dichromate Survival
66

In experiment #3, the cytotoxicity of potassium dichromate to 10T½ cells also began at a
concentration of 1.0uM, and continued to increase in a dose-dependent manner with the
concentrations of potassium dichromate applied to the cells (Table 13 and Figure 22). At
concentrations of 1.0, 2.0, 5.0, 10.0, and 20.0 μM, the survival of 10T
1/2
cells decreased to 0.34,
0.22, 0.03, 0.00, 0.00, respectively (Table 14). Again, the curve for survival fraction as a
function of dose of calcium chromate appeared to follow the semi-logarithmic relationship, and
the survival fraction of 10T
1/2
cells seems to be dose-dependent (Figure 22).

Figure 24: Survival of 10T
1/2
cells treated with various concentrations of
potassium dichromate in experiment 3. The figure shows the survival fraction
versus the concentration of potassium dichromate administered to 10T
1/2
cells.

0.001
0.01
0.1
1
10
0 5 10 15 20 25
Survival Fraction
Concentration of Potassium Dichromate (uM)
Cytotoxicity 3: Potassium Dichromate Survival
67

After all three experiments on the cytotoxicity of potassium dichromate to 10T
1/2
cells
were completed, all the survival fractions at each concentration of potassium dichromate were
averaged. These survival fraction average values were 0.30, 0.18, 0.03, 0.01, 0.00 in cells
treated with 1.0 μM, 2.0 μM, 5.0 μM, 10 μM, and 20 μM, respectively (Table 14). This data
averaged over three experiments was plotted graphically, and is shown in Figure 23. Again, the
data shows 1) that these three experiments are very reproducible and the data can easily be
averaged, and 2) that the cytotoxicity (reduction in cell survival fractions) is dose-dependent.

Figure 25: Average survival +/- standard deviation of 10T
1/2
cells as a function of
the concentration of potassium dichromate used to treat the cells. This data is
collected from the three potassium dichromate cytotoxicity experiments as shown
in Table 14.

0.001
0.01
0.1
1
10
0 5 10 15 20 25
Survival Fraction
Concentration of Potassium Dichromate (uM)
Average of 3 Potassium Dichromate Cytotoxicity
Survival Experiments
Experiment 1
Experiment 2
Experiment 3
Average
68

3.6 Effects of Ascorbate on the Transformation of 10T ½ Cells with Sodium Chromate
Transformation experiments were performed to determine the effects of ascorbate to
10T½ cells with sodium chromate. In experiment #1, the transformation data showed numerous
transformed foci. For 10T ½ cells treated with ascorbate only, there were Type II foci in both
0.025mM and 0.05mM concentrations of ascorbate (Table 15). However, when cells where
treated with both sodium chromate and ascorbate, there was a 600 times increase in the amount
of transformed foci per dish (Table 15) and found in each concentration of treatment.
_______________________________________________________________________________________

Total Number of foci/Number of Dishes scored Number of dishes with transformed foci/
(Foci/Total Dishes)* Number of dished scored
Treatment Cell Survival
(%) Type III Type II + Type III Type III Type II + Type III
Day 5, 24 hours
0 (medium only )

124.42

0/20 (0.0)

0/20 (0.0)

0/20

0/20
0 (0.5% acetone) 100.00 0/20 (0.0) 0/20 (0.0) 0/20 0/20
M CA (1.0 µg/mL)

103.86 25/20 (1.25) 86/20 (4.3) 16/20 20/20

NaCrO4 Only 75.58 1/15 (1.33) 1/15 (1.33) 1/15 1/15
Ascorbate Only (mM)

0.125mM 104.11 0/15 (0.0)
0/20 (0.0)
0/15 (0.0)
0/20 (0.0)
0/15
0/20
0/15
0/20 0.025mM 128.28 0/15 (0.0) 1/15 (1.33) 0/15 1/15
0.05mM 93.06 0/17 (0.0) 1/17 (1.18) 0/17 1/17
0.1mM 87.40 0/18 (0.0) 0/18 (0.0) 0/18 0/18
0.25mM 0.00 0/5 (0.0) 0/5 (0.0) 0/5 0/5
NaCrO4 (µg/mL)
+0.125mM 77.12 0/20 (0.0) 10/20 (0.5) 0/20 8/20
+0.025mM 74.29 0/15 (0.0) 5/15 (6.66) 0/15 5/15
+0.05mM 44.73 0/20 (0.0) 5/20 (0.25) 0/20 5/20
+0.1mM 68.64 0/20 (0.0) 12/20 (0.6) 0/20 9/20
+0.25mM 0.00 0/5 (0.0) 2/5 (8.0) 0/5 2/5

* The number of foci has been normalized to a total of 20 dishes.

Table 15. Experiment #1 showing the effects of ascorbate on the transformation of
10T½ cells with sodium chromate.

69

Transformation experiments were performed again to determine reproducibility of the
effects of ascorbate to 10T½ cells with sodium chromate. In experiment #2, the transformation
data showed numerous transformed foci. For 10T ½ cells treated with ascorbate only, there was
both Type II and Type III foci in both 0.025mM and 0.05mM concentrations of ascorbate (Table
16). However, when cells where treated with both sodium chromate and ascorbate, there was a
1000 times increase in the amount of transformed foci per dish (Table 16) and found in each
concentration of treatment.
_______________________________________________________________________________________

Total Number of foci/Number of Dishes scored Number of dishes with transformed foci/
(Foci/Total Dishes)* Number of dished scored
Treatment Cell Survival
(%) Type III Type II + Type III Type III Type II + Type III
Day 5, 24 hours
0 (medium only )

119.92

0/20 (0.0)

0/20 (0.0)

0/20

0/20
0 (0.5% acetone) 100.00 0/20 (0.0) 0/20 (0.0) 0/20 0/20
M CA (1.0 µg/mL)

104.07 22/20 (1.) 92/20 (4.6) 17/20 20/20

NaCrO4 Only 78.98 1/15 (1.33) 1/15 (1.33) 1/15 1/15
Ascorbate Only (mM)

0.125mM 114.91 0/15 (0.0)
0/20 (0.0)
0/15 (0.0)
0/20 (0.0)
0/15
0/20
0/15
0/20 0.025mM 129.31 0/15 (0.0) 1/15 (1.33) 0/15 1/15
0.05mM 89.99 0/17 (0.0) 1/17 (1.18) 1/17 2/17
0.1mM 86.91 0/18 (0.0) 0/18 (0.0) 0/18 1/18
0.25mM 0.00 0/5 (0.0) 0/5 (0.0) 0/5 0/5
NaCrO4 (µg/mL)
+0.125mM 81.10 0/20 (0.0) 10/20 (0.5) 0/20 9/20
+0.025mM 77.19 0/15 (0.0) 5/15 (6.66) 1/15 4/15
+0.05mM 45.04 0/20 (0.0) 5/20 (0.25) 9/20 7/20
+0.1mM 69.00 0/20 (0.0) 12/20 (0.6) 0/20 9/20
+0.25mM 0.00 0/5 (0.0) 2/5 (8.0) 0/5 1/5

* The number of foci has been normalized to a total of 20 dishes.

Table 16. Transformation Data for Transformation Experiment #2 with Sodium
Chromate

70

The cytotoxicity data for both transformation experiments were performed to determine
the platting efficiencies for the effect of ascorbate to 10T½ cells with sodium chromate.

Treatment PE 1 ± SD
No Addition 48.4±2.88
Acetone Only 38.9±1.78
NaCrO4 Only 29.4±7.56
MCA Only 40.4±2.77
Ascorbate Only
0.0125mM 40.5±4.73
0.025mM 49.9±8.95
0.05mM 36.2±2.02
0.1mM 34±1.41

NaCrO4 and Ascorbate
+Ascorbate 0.0125mM 30±5.22
+Ascorbate 0.025mM 28.9±2.99
+Ascorbate 0.05mM 17.4±2.95
+Ascorbate 0.1mM 26.7±3.42

Table 17. Cytotoxicity Data for Transformation #1 Experiment with Sodium Chromate

Treatment PE 1 ± SD
No Addition 49.1±1.99
Acetone Only 37.9±2.89
NaCrO4 Only 30.1±3.20
MCA Only 42.3±1.97
Ascorbate Only
0.0125mM 42.1±3.11
0.025mM 42.9±6.90
0.05mM 33.9±1.97
0.1mM 32.9±1.01

NaCrO4 and Ascorbate

+Ascorbate 0.0125mM 32.9±4.92
+Ascorbate 0.025mM 27.9±1.89
+Ascorbate 0.05mM 20.1±2.14
+Ascorbate 0.1mM 24.9±4.44

Table 18. Cytotoxicity Data for Transformation #2 Experiment with Sodium Chromate
71

The cytotoxicity data for both transformation experiments performed were then graphed
to determine the reproducibility of the effect of ascorbate to 10T½ cells with sodium chromate.

Figure 26. Cytotoxicity Data for Transformation Experiment with Sodium Chromate and
Ascorbate

72

CHAPTER 4
DISCUSSION AND CONCLUSIONS
4.1 Soluble Cr(VI) Compounds Carcinogenesis
Numerous epidemiological and in vitro studies have noted that the insoluble Cr(VI)
compounds are the most cytotoxic and carcinogenic among all types of chromium compounds
(Patierno et al, l988; Biedermann and Landolph, l988, l990; Nickens, K., et al, 2010). It is
believed that this is due to the phagocytosis of particles of the insoluble Cr(VI) compounds into
cells, leading to deposition of a large bolus of Cr(VI) into the cells (Patierno et al, l988). For
soluble chromium compounds, the influx of these compounds into cells on the relatively non-
specific sulfate-phosphate anion transport carrier is believed to be an early step in cytotoxicity
and carcinogenesis caused by soluble Cr(VI) compounds.
The detailed molecular mechanisms of Cr(VI)-induced cell transformation and
carcinogenesis still remain unknown. A number of published reports have suggested the
mechanism by which the chromate particle enters the cell may be able to explain this
observation. It has been documented that insoluble chromate particles adhere to the cell surface,
and dissolve slowly and chronically within its microenvironment. Consequently, this allows the
released chromate oxyanions to escape from the extracellular reduction and be absorbed into the
cells via phagocytosis (Nickens K., et al, 2010). Our laboratory has previously taken electron
micrographs that revealed the internalization of many PbCrO
4
particles in the cytoplasm of
10T
1/2
cells (Patierno, S., et al, l988), further documenting phagocytosis as a likely initial step in
the overall mechanism of carcinogenesis caused by particulate chromate compounds.
73

Once the Cr(VI) compounds are inside the cells, the Cr(VI) is believed to undergo a
series of reductive activations and to arrest at the most thermodynamically stable form, Cr(III)
(Ding, M., and Shi, X. 2002). Strong evidence has shown that Cr(III) induces many types of
genotoxic damage, including Cr-DNA adducts, DNA-protein cross-links, DNA inter-strand
cross-links, DNA single-strand breaks, and base substitution mutations (Casadevall, M., et al,
1999, Macfie, A., et al, 2010, Capellmann, M., et al, 1995). Indirect genotoxic damage caused by
Cr(VI) is also documented, most notable being the generation of reactive hydroxyl radicals
during the reduction process of Cr(VI) (Ding M., and Shi, X., 2002). All these types of DNA
damage have the potential to induce morphological and neoplastic cell transformation if they are
not properly repaired intrinsically. It has been estimated that 95-99% of DNA damage is
repaired correctly, and that the residual 1% - 5% of DNA damage is either not repaired or is
repaired incorrectly, leading to mutations in DNA (reviewed in Salnikow and Zhitkovich, 2008).
These published reports conquer with the date we acquired with soluble chromate compounds as
well.
From Table 15, at concentrations of 0.025 mM and 0.05 mM, Type II foci were observed.
However, when the 10T
1/2
cells were treated with both sodium chromate and ascorbate, there was
a 600 times increase in transformed foci formation in every treatment of concentration. For the
concentrations 0.125 mM, 0.025 mM, 0.05 mM, 0.1 mM and 0.25 mM, there was an observation
of 8, 5, 5, 9, 2 transformed foci per dish, respectively. Furthermore, from Table 16, at
concentrations of 0.025 mM and 0.05 mM, Type II and Type III foci were observed. However,
when the 10T
1/2
cells were treated with both sodium chromate and ascorbate, there was a 1000
times increase in transformed foci formation in every treatment of concentration. For the
concentrations 0.125 mM, 0.025 mM, 0.05 mM, 0.1 mM and 0.25 mM, there was an observation
74

of 9, 4, 7, 9, 1 transformed foci per dish, respectively. This data is conclusive upon the
hypothesis that ascorbate and dehydroascorbate enhance the cytotoxicity and morphological
transformation of C3H/10T½ Cl 8 mouse embryo cells induced by soluble chromium (VI)
compounds. In order to validate the transformation induced by sodium chromate, we want to
show a smooth curve in which the transformation frequency increases in a dose-dependent
manner and in which a large number of foci were induced. Afterward molecular characterization
studies will proceed.
This data conquers with epidemiological evidence that chromium is a strong carcinogen.
There are many unknown factors that justify this phenomenon. We hypothesize that it is
necessary to oxidatively activate Cr(VI) in our cell culture system before we will observe
significant numbers of foci. We are in the process of discovering a key component that could be
crucial for the reductive activation of Cr(VI) compounds, such as glutathione.
4.2 Effect of Ascorbate on Cytotoxicity
The carcinogenicity of Cr(VI) compounds needs to be activated through series of
intracellular reduction process (Salnikow, K., and Zhitkovich, A. 2008). There are many
reducing agents present in the mammals and in mammalian cells that could perform the reductive
action, including glutathione, cysteine, lipoic acid, NAD(P)H, ribose, fructose, and ascorbate.
Among these natural reducing agents, ascorbate is the dominant biological reducer and is the
principle reducer that activates Cr(VI) (Stearns, D., et al, 1995). This essential component,
however, is significantly reduced in quantity in cell culture. Reports have consistently shown that
physiological ascorbate levels are in the millimolar range, e.g. approximately 1.3 mM ascorbate
has been quantified in human lung tissue (Slade, R., et al, 1985). On the contrary, the ascorbate
75

concentrations in cell culture are either undetectable or in the micromolar range (Reynolds, M.,
and Zhitkovich, A. 2007).
Under in vitro conditions, the reductive activation of Cr(VI) compounds is a slow thiol-
dependent process (Quievryn, G., et al, 2002). Therefore, a simple supplement of ascorbate may
be the missing piece that would confer a successful transformation assay. Because of the dual
nature of ascorbate, it has both pro-oxidative and anti-oxidative properties. Polisak et al. has
shown the production of reactive and toxic hydroxyl radicals and other harmful toxic Cr species,
such as Cr(III), upon the reduction of Cr(VI). Reynolds et al. showed that the restoration of
ascorbate levels in human lung cells has increased clonogenic lethality and apoptosis. However,
generally in physiology, ascorbate serves as an antioxidant by reducing the concentration of
hydroxyl radicals and also serves as a detoxifying agent by reducing the extracellular Cr(VI) and
preventing the Cr(VI) from entering the cells (Salnikow, K., et al, 2008).
Prior to a transformation assay, we needed to determine the maximum concentration of
ascorbate that would be pro-oxidative and also minimize the cytotoxic effect of ascorbate on
10T
1/2
cells. We also wanted to design a method that delivers the ascorbate intracellularly most
efficiently. First we examined the cytotoxic effect induced by ascorbate alone. Generally, all
three figures show a dose-dependent cytotoxic effect as the concentration of ascorbate increases.
There is not too much fluctuation in cell survival, because the cells could be relatively insensitive
to the uptake of ascorbate one day after seeding. Overall, ascorbate and dehydroascorbate
enhance the cytotoxicity and morphological transformation of C3H/10T½ C1 8 mouse embryo
cells induced by soluble chromium (vi) compounds .
.
76

ACKNOWLEDGMENTS
I would like to extend my heartfelt gratitude to my Advisor, Mentor, and Group Leader,
Dr. Joseph R. Landolph Jr., Ph. D. His support and encouragement is never-ending and is the
reason for the success of this thesis. I would also like to thank my colleagues, Mr. William Liao,
Ms. Shelly Tseng and Mr. Matthew Moodie, undergraduate students at USC majoring in
Biological Sciences, Ms. Laureen Tran, M. S. Student in the Dept. of Molecular Microbiology
and Immunology at USC, and Mr. Qasim Akinwumi, B. S., M.S., Ph. D. Student in
Biochemistry, University of Ibadan, Ibadan, Nigeria for their skillful research practices and
encouraged me to bring my research project forward. Thank you for all your research and
assistance to me in completion of this thesis.

77

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Phosphate Group. Biochemistry. 35, 7275-7282, 1996.

Abstract (if available)

Abstract Hexavalent chromium [Cr(VI)]‐containing compounds are human carcinogens. They cause cancers in the respiratory system when inhaled, and stomach, kidney/other internal cancers when ingested. Soluble and insoluble hexavalent chromium [Cr(VI)] compounds induce base substitution, deletion, addition, and frameshift mutations. Cr(VI) compounds also induce DNA‐DNA cross links and DNA‐protein cross-links in mammalian cells. ❧ In this thesis, we examined the ability of the soluble chromium compounds, sodium chromate (Na₂CrO₄), calcium chromate (CaCrO₄) and potassium dichromate (K₂Cr₂O₇), to induce cytotoxicity and morphological transformation in cultured C3H/10T½ Cl 8 (10T1/2) mouse embryo cells. We tested the hypothesis that the intracellular reductants, ascorbate and dehydroascorbate, can reduce Cr(VI), to Cr(V), Cr(IV), and Cr(III) intracellularly, making Cr(VI) a strong cytotoxin in mammalian cells. Ascorbate is present in serum at concentrations in the mM range under physiological conditions in humans, but is only present in the μM range in mouse embryo cells grown in BME cell culture medium plus 10% fetal calf serum. ❧ We hypothesized that the relatively weak responses for induction of morphological transformation of mammalian cells by Cr(VI) compounds in culture (dose‐dependent but weak induction of foci by lead chromate, and no induction of foci by calcium chromate, potassium dichromate , and sodium chromate) is due to the small amounts of ascorbate in cultures of mammalian cells that are insufficient to reduce Cr(VI) to Cr(V), Cr(IV), and Cr(III). Therefore, we investigated the cytotoxic effects of ascorbate on 10T½ mouse embryo cells, and the effects of the highest non‐cytotoxic concentrations of ascorbate on Cr(VI)‐induced cytotoxicity in 10T½ cells, using reduction in plating efficiency as our cytotoxicity assay. Cell survival data showed that ascorbate exerted significant cytotoxic effects at concentrations of 0.00625 mM and higher on 10T½ mouse embryo cells. Furthermore, when 10T½ cells were treated with both Cr(VI) and ascorbate, ascorbate played dual roles. It served as a pro‐oxidant (enhancer of the cytotoxicity of chromate) at concentrations up to 0.1 mM, and as an anti‐oxidant (reducer of the cytotoxicity of chromate) at concentrations of 0.25 mM and higher. This information is important for our ongoing and future experiments, where we designed a protocol to incorporate ascorbate into our assays assessing the cytotoxicity and cell transforming activity of Cr(VI) compounds. At concentrations of 0.1 mM and lower, ascorbate enhanced the cell transforming ability of Cr(VI) compounds to induce morphological transformation.

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University of Southern California Dissertations and Theses

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Asset Metadata

Creator Shahin, Sophia Allaf (author)

Core Title Ascorbate and dehydroascorbate enhance the cytotoxicity and morphological transformation of C3H/10T½ C1 8 mouse embryo cells induced by soluble chromium (VI) compounds

School Keck School of Medicine

Degree Master of Science

Degree Program Experimental and Molecular Pathology

Publication Date 04/28/2014

Defense Date 04/12/2014

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemical,chromium,OAI-PMH Harvest,physical carcinogenesis

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Landolph, Joseph R., Jr. (committee chair)

Creator Email sophiaallaf@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-385505

Unique identifier UC11296582

Identifier etd-ShahinSoph-2417.pdf (filename),usctheses-c3-385505 (legacy record id)

Legacy Identifier etd-ShahinSoph-2417.pdf

Dmrecord 385505

Document Type Thesis

Format application/pdf (imt)

Rights Shahin, Sophia Allaf

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA