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The role of nickel-induced ROS in nickel (II)-induced cytotoxicity

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The role of nickel-induced ROS in nickel (II)-induced cytotoxicity

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Content Copyright 2023 i Sheldy SoJeong Shin
The Role of Nickel-Induced ROS in Nickel (II)-Induced Cytotoxicity
By
Sheldy SoJeong Shin
A Thesis Presented to the
FACULTY OF THE USC KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR, MICROBIOLOGY, AND IMMUNOLOGY)
December 2023

Copyright 2023 ii Sheldy SoJeong Shin
Acknowledgements
I would like to express my deepest gratitude to my supervisor, Dr. Joseph R.
Landolph, for his unwavering support, insightful guidance, and invaluable mentorship
throughout this research journey. His expertise and dedication have been instrumental in
shaping this thesis.
I am also grateful to the members of my thesis committee, Dr. Ha Youn Lee and Dr.
Leslie Khawli, for their valuable feedback and constructive criticism that significantly
improved the quality of this work.
Sepcial thanks are due to Dr. Axel Schönthal, for his generous collaboration in
sharing laboratory space with our research team. This collaboration has been instrumental in
facilitating our research efforts and advancing our scientific endeavors.
I want to acknowledge my friends and family for their unwavering support and
encouragement during this challenging endeavor. Their belief in me has been a constant
source of motivation
Lastly, I want to thank my partner, Zijie Jin, for his patience, love, and understanding
during the long hours and late nights dedicated to this project. Your unwavering support
sustained me through this journey.
I am deeply appreciative of the contributions and support of everyone mentioned
above, without whom this thesis would not have been possible.

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TABLE OF CONTENTS
Acknowledgements……………………………………………………………………………ii
List of Tables………………………………………………………………………………….iv
List of Figures………………………………………………………………………………....v
Abstract..……………………………………………………………………………………..vii
Introduction…..………………………………………………………………………………..1
Chapter 1: Materials and Methods..…………………………………………………………...8
Chapter 2: Results..…………………………………………………………………………..10
Chapter 3: Discussion...………………………………………………………………………40
References......………………………………………………………………………………..41

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List of Tables
Table1a. Plating Efficiency of BEAS 2B using the non-optimized method…………………10
Table1b. Plating Efficiency of BEAS 2B using the optimized method……………………...10
Table2a. Cytotoxicity of Black NiO to BEAS 2B cells……………………………………...11
Table2b. Cytotoxicity of Nickel Subsulfide to BEAS 2B cells……………………………...12
Table3a. Seeding Dilution of BEAS 2B cells using H2DCFDA……………………………..14
Table3b. Sensitivity test of BEAS 2B cells using H2DCFDA……………………………….14
Table4a. Total ROS measurement on Black NiO to BEAS 2B cells using H2DCFDA……...15
Table4b. Normalization of total ROS measurement on Black NiO to BEAS 2B cells using
H2DCFDA……………………………………………………………………………………15
Table4c. Total ROS measurement on Green NiO treated BEAS 2B cells using H2DCFDA..16
Table4d. Normalization of total ROS measurement on Green NiO treated BEAS 2B cells
using H2DCFDA……………………………………………………………………………..17
Table4e. Total ROS measurement on Ni2S3 treated BEAS 2B cells using H2DCFDA……...18
Table4f. Normalization of total ROS measurement on Ni2S3 treated BEAS 2B cells using
H2DCFDA……………………………………………………………………………………19
Table5a. Plating Efficiency of C3H 10T1/2 Cl8 mouse embryo cells using the non-optimized
method………………………………………………………………………………………..20
Table5b. Plating Efficiency of C3H 10T1/2 Cl8 mouse embryo cells using the optimized
method………………………………………………………………………………………..20
Table6a. Cytotoxicity of Black NiO to C3H 10T1/2 Cl8 mouse embryo cells………………21
Table6b. Cytotoxicity of Nickel Subsulfide to C3H 10T1/2 Cl8 mouse embryo cells………22
Table7a. Total ROS measurement on Black NiO to C3H 10T1/2 Cl8 mouse embryo cells
using H2DCFDA……………………………………………………………………………..24
Table7b. Normalization of total ROS measurement on Black NiO to C3H 10T1/2 Cl8 mouse
embryo cells using H2DCFDA……………………………………………………………….24
Table7c. Total ROS measurement on Green NiO treated C3H 10T1/2 Cl8 mouse embryo
cells using H2DCFDA………………………………………………………………………..25
Table7d. Normalization of total ROS measurement on Green NiO treated C3H 10T1/2 Cl8
mouse embryo cells using H2DCFDA……………………………………………………….25

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List of Figures
Figure 1. The plating efficiency of BEAS 2B with 200 cells vs. 2000 cells seeded per 60mm
dish…………………………………………………………………………………………...26
Figure 2. The plating efficiency of BEAS 2B with 200 cells seeded by optimized method vs.
non-optimized method………………………………………………………………………..27
Figure 3. The plating efficiency of 10T1/2 cells with 200 cells vs. 2000 cells seeded per
60mm dish……………………………………………………………………………………27
Figure 4. The plating efficiency of 10T1/2 cells with 200 cells seeded by optimized method
vs. non-optimized method……………………………………………………………………28
Figure 5. Cytotoxicity of Black NiO treated 10T1/2…………………………………………28
Figure 6. Cytotoxicity of Nickel Subsulfide treated 10T1/2…………………………………29
Figure 7. Cytotoxicity of Black NiO treated BEAS2B cells…………………………………29
Figure 8. Cytotoxicity of Nickel Subsulfide treated BEAS2B cells…………………………30
Figure 9. Seeding Dilution of 10T1/2 cells using H2DCFDA………………………………..30
Figure 10. Seeding Dilution of BEA 2B cells using H2DCFDA…………………………….30
Figure 11. Sensitivity test using different concentrations of H2O2 in BEAS 2B cells with
H2DCFDA……………………………………………………………………………………31
Figure 12. Total ROS measurement as a function of the duration using different Black NiO
concentrations………………………………………………………………………………...32
Figure 13. Total ROS measurement as a function of the concentration Black NiO…………32
Figure 14. Total ROS measurement as a function of the duration using different Green NiO
concentrations………………………………………………………………………………...33
Figure 15. Total ROS measurement as a function of the concentration Green NiO…………34
Figure 16. Total ROS measurement as a function of the duration using different Nickel
Subsulfide concentrations……………………………………………………………………35
Figure 17. Total ROS measurement as a function of the concentration Nickel Subsulfide….35
Figure 18. Total ROS measurement as a function of the duration using different Black NiO
concentrations on 10T1/2…………………………………………………………………….36
Figure 19. Total ROS measurement as a function of the concentration Black NiO on
10T1/2………………………………………………………………………………………37
Figure 20. Total ROS measurement as a function of the duration using different Green NiO
concentrations on 10T1/2…………………………………………………………………….38
Figure 21. Total ROS measurement as a function of the concentration Green NiO on
10T1/2………………………………………………………………………………………..39

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Abstract
Nickel is the 24th most abundant transition metal in the Earth's crust. It is also an
essential material in modern industry, used in stainless steel for electroplating, welding, alloy
production, and more. Nickels are announced as occupational and environmental
carcinogens. Nickel compounds are presented in two states- one in water-soluble states, such
as Nickel dichloride (NiCl2), and the other in water-insoluble states, such as green Nickel
oxide (NiO). Insoluble nickel (II) compounds are known to be more carcinogenic than
soluble nickel (II) compounds. According to the International Agency for Research on
Cancer, insoluble Nickel (II) compounds are known as group I carcinogens. In culture and
morphological neoplastic cell transformation, nickel compounds cause chromatin damage in
mouse and human cells. A small amount of data shows that nickel compounds generate
reactive oxygen species. Reactive oxygen species include superoxide, hydrogen peroxide,
and hydroxyl radicals. Here, we showed cytotoxicity of insoluble nickel (II) compounds
(Black NiO and Nickel Subsulfide) and measured total reactive oxygen species (ROS) using
H2DCFDA on insoluble nickel (II) compound treated C3H Cl8 10T1/2 embryonic mouse
cells and BEAS 2B cells. Moreover, after discussing the results, we share further research to
accomplish the role of Nickel-induced ROS in Nickel-induced cytotoxicity and
morphological transformation.

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Introduction
Nickel and respiratory cancer
Baron Axel Fredrik of Sweden first identified nickel, a silver-white metal, in 1751
(Ostendrop, 2001). Nickel is the fifth of the most abundant transition metals in the Earth's
crust (Huang, 2002). Nickel is liberated into the environment through volcanic eruptions,
rock weathering, and refinery of nickel dust (Huang, 2002). Since the nineteenth century,
nickel has been broadly used due to its corrosion resistance and durability (Savin, 2003).
Nickel is essential for modern industrial activities, such as electroplating, welding, and nickel
alloy production (Savin, 2003). The frequency of use of nickel alloys has increased since,
when nickel is combined with other metal compounds, the metals gain the advantage of the
properties of nickel (Savin, 2003). Even fossil fuels contain a Nickel-Chromium-Cobalt alloy
(Altin, 2021). Unfortunately, the combustion of fossil fuels releases nickel into the
environment (Costa, 1996; Govindarajan, 2002). The release of nickel compounds leads to
environmental pollution (Merian, 1984). The routes by which humans are exposed to nickel
include inhalation (smoking cigarettes), cutaneous absorption (jewelry), and oral intake
(foods like spinach) (Oller, 1997; Nielson, 1987).
Nickel was first recognized as a possible carcinogen because many nickel refinery
workers who also smoked cigarettes developed nasal and lung cancer in 1932 at Clydach,
Wales (Norseth, 1977; Bridge, 1933). Interestingly, most nickel refinery workers also smoke
cigarettes (Landolph, 1985). Since 1901, workers from nickel refineries have reported various
respiratory cancers, such as nasal and lung cancers in the workers (Landolph, 1985). After the
10-fold reduction of the contaminant arsenic, in sulfuric acids and thereby nickel arsenide
(Ni5As) in nickel refineries in 1923, respiratory cancer decreased substantially in the workers
(Landolph, 1985). Then, in 1990, the International Agency for Research on Cancer (IARC) in
Lyon, France designated nickel compounds as human carcinogen (IARC, 1990).
The mechanism of nickel carcinogenesis is still not fully understood. However,
water-insoluble nickel compounds, and possibly water-soluble nickel compounds, are
carcinogens that lead to lung and nasal cancer when inhaled (Doll, 1990; Grimsrud, 2003).
Insoluble nickel compounds enter cells via phagocytosis, and soluble nickel enters the cell via
the Divalent Metal Ion Transporter (DMT1) (Costa, 1981; Fletcher, 1994). When animals
inhaled insoluble Ni3S2 and black or green NiO, they developed lung cancer, while no cancer
development was observed when they inhaled soluble NiSO4 (NTP, 1994).
In vitro, cell transformation experiments in our laboratory found that orcelite (Ni5As2)
and green nickel induced in dose-dependent morphological transformation of C3H 10T1/2
Cl8 mouse embryo cells (Clemens and Landolph, 2003, reviewed Landolph, 1985). Clemens
and Landolph studied Clydach nickel refinery samples obtained from the nickel refinery there
in 1920 and 1929. They found that the 1920 sample induced substantial morphological cell
transformation and chromosomal aberrations in 10T1/2 cells. The 1920 samples contain
37.4% nickel, and the 1929 samples contain 26.6% nickel. Both samples had green NiO as a
significant component (Clemens and Landolph, 2003, reviewed in Landolph, 1985).
Nevertheless, the 1920 sample had 25% orcelite (Ni5As2), while the 1929 sample had only
2.5% orcelite (Ni5As2), a 10-fold decrease, due to use of purified sulfuric acid contains only
10% of the orcelite in the 1929 sample (reviewed in Clemen and Landolph, 2003). This
correlated with the story ability of teh 1920 sample to induce morphological transformation
in 10T1/2 cells, while the 1929 sample did not induce morphological transformation in

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human cells. (Clemens and Landolph, 2003). Clemens and Landolph (2003) postulated that
the combination of green nickel oxide adn orcelite induced morphological transformation in
10T1/2 cells of hence also nasal and respiratory tumors in human.
Some investigators postulate that nickel compounds, especially Ni2+ ions, decrease
histone H4 acetylation by binding to his N-terminal histone-18, producing reactive oxygen
species (ROS) by affecting the Histone acetyltransferases (HAT) enzyme (Kang, 2003).
Histones have four subtypes (Klochemdler-Veivin, 2001). H2A, H2B, H3, H4. H4 plays a
vital role in histone acetylation (Klochemdler-Veivin, 2001). Using Western blotting and 3
Hacetate, Kang and his colleagues found Ni2+ had an inhibitory effect on H4 acetylation. Using
antioxidants, reactive oxygen species (ROS) generation decreases and further decreases
histone acetylation inhibition activity (Kang, 2003). However, nickel compound induced
hypoacetylation shows less evidence of how nickel compounds could be carcinogenic, since
histones rarely cause mutations in DNA (Kang).
Another possible mechanism for nickel-induced ROS besides histones is ROS
activating hypoxic signaling (Salnikow, 2000). With exposure to nickel (II) compounds, cells
had increased expression of HIF-1 alpha protein and HIF-1 dependent transcription factors,
while p53 had no significant change. HIF-1 alpha protein is an oxygen-sensitive
transcriptional activator that increases as oxygen concentration decreases in the cell (Ke,
2006), indicating increased ROS in vitro.
On the other hand, Hernandez‐Boussard and his colleagues claimed that the nickel (II)
compounds mutate the p53 gene (Hernandez‐Boussard, 1999). The p53 gene mutation could
be due to DNA damage directly caused by nickel (II) compounds. However, many studies
show that the levels of p53 are not significantly changed by nickel (II) exposure (Weghorst,
1994; Lin, 1994; Rani, 1993). Therefore, the hypothesis could be neglected.
Nickel Compound induced Cytotoxicity and Genotoxicity
Water-insoluble nickel compounds, such as nickel subsulfide (Ni3S2), black and
green nickel oxide, are phagocytosed into the cell’s nucleus, whereas soluble nickel
compounds, such as nickel sulfate, are not phagocytosed into mammalia cells (Evans, 1982).
The higher cytotoxicity of insoluble nickel compounds is likely due to their higher turnover
of reactive oxygen species (ROS) (Ahamed, 2015). ROS could cause apoptosis,
inflammation, and DNA damage (Guo, 2015).
When cells are exposed to nickel (I), and nickel (II) ions, chromatin remodeling,
DNA methylation, and histone acetylation are observed (Sutherland, 2003). A change in
chromatin structure can alter gene expression without directly altering the genome
(Sutherland, 2003). Sutherland et al. (2003) found that even in yeast cells, where no DNA
methylation occurs, Nickel +2 ion changes lysine acetylation to regulate gene expression
further. More finding of nickel-induced DNA methylation and histone acetylation was
explained through gpt gene silencing in the G12 transgenic cell line (Yan et al., 2003).
When insoluble nickel, such as green NiO, enters the body, it is phagocytosed into the
cells and is released intracellularly as Ni2+ ions (Landolph, 2002). Released Ni2+ ions bind to
histone, in the nucleus and may contact DNA bases (Landolph, 2002). According to
Landolph et al., when Ni2+ is bound to DNA-bound proteins, H2O2 and OH* radicals could
react with the DNA bases, oxidity them, and leading to DNA damage. Incorrect repair of

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these Nickel (II)- induced DNA lesions could lead to cytotoxicity, morphological, and
neoplastic transformation of 10T1/2 mouse embryo cells. The induction of cytotoxicity and
morphological transformation in C3H/10T/1/2 Cl8 mouse embryo cells was dose-dependent
upon the amount of insoluble Nickel (II) compound added to the cells (Miura et al., 1989).
Interestingly, soluble nickel compounds, such as nickel sulfate and nickel chloride, only
caused cytotoxicity in 10T1/2 cells but no morphological transformation, while insoluble
nickel compounds, such as nickel subsulfide and nickel monosulfide, induced both
cytotoxicity and morphological transformation (Miura et al., 1989).
Nickel (II) compounds cause effects similar to hypoxic signaling (Maxwell, 2004).
Hypoxia is a condition commonly associated with the tumor microenvironment because it
requires a large amount of oxygen for the rapid growth of cells (Maxwell, 2004). Nickel
compounds stabilize HIF alpha proteins, increasing HIF-1-dependent transcription as if
hypoxic signaling is activated (Maxwell, 2004). ROS plays an essential role in HIF-1
stabilization (Chandel, 1998). HIF-1 requires ROS to bind to DNA to stabilize it (Chandel,
1998).
Morphological cell transformation occurs when C3H10T1/2 Cl8 (10T1/2) mouse
embryo cells are exposed to crystalline nickel monosulfide (NiS) (Salnikow, 1999; Miura,
1989). (Miura et al., 1989) found that insoluble nickel compounds caused dose-dependent
cytotoxicity and those - dependent morphological transformation in 10T1/2 mouse embryo
cells (Miura et al., 1989). Transformed cell lines derived from foci induced by nickel
compounds show an amplification of the proto-oncogene ECT-2 and overexpression of Ect-2
mRNA and protein (Clemens et al., 2005). A possible reason for Ect-2 amplification is nickel
ions binding to DNA polymerase (Clemens et al., 2005). The increased Ect-2 protein could
lead in part to morphological transformation, anchorage-independent transformation, and
neoplastic transformation via microtubule disassembly (Tatsumoto, 1999).
Interestingly, do soluble nickel compounds are likely to affect tumor suppressors. At
the same time, soluble nickel compounds not have genotoxicity (Goodman, 2009), as nickel
sulfate hypermethylated the DNA and inactivated the tumor suppressor gene (Benson, 2002).
Soluble nickel compounds may have affected histone acetylation or methylation to inactive
the tumor suppressor genes (Ke, 2006). According to Rivedal (1981), nickel compounds may
also act as tumor promoters. When nickel sulfate was added after benzo(a)pyrene treatment,
the transformation frequency increased from 0.4% to 4.9%. When the tumor promoter,
TPA, was replaced with nickel sulfate, the transformation frequency was increased to
10.6%. An increase in transformation frequency suggests that soluble nickel compounds are
not cancer initiators but promoters.
Nickel compounds also inhibit DNA repair systems (Snyder, 1989). Alkyl DNA
dioxygenases, an iron- and ascorbate-dependent DNA repair enzyme, hydroxylate alkyl
groups on 1-methyladenine and 3-methylcytosine (Duncan, 2002). Nickel exposure inhibited
the activity of Alkyl DNA dioxygenases, increasing DNA mutation. The finding of nickel
inhibiting DNA repair system that Nickel compounds are not only carcinogens but also
cocarcinogens due to their synergic result with other carcinogens (Rivedal, 1980; Salnikow,
2007).
DNA mutation and morphological changes

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Nickel compounds generate specific morphological changes in chromosomes of
mammalian cells. Both insoluble Ni3S2 and soluble NiCl2 damage the heterochromatin of
Chinese hamster X chromosome and Chinese hamster ovary (CHO) cells. Nickel compounds
induced sister chromatid exchanges (SCE) and deletion mutations in CHO cells (Salnikow,
2000; Rosetto, 1994). SCE induced by nickel compounds were also observed in human
lymphocytes (Sahu, 1989). Rosetto showed gene deletion increased in the following order:
NiSO4 > Ni(OH)2> Ni3S2.
Multiple studies with diverse cell types showed that nickel compounds induce
morphological transformation (Costa,1979; Saxholm, 1981). When Syrian hamster fetal cells
were exposed to nickel subsulfide (Ni3S2), the cells under went dose-dependent
morphological transformation, and 0.1 or 1μg/mL Ni3S2 showed no decreased cell plating
efficiency at low concentration. Also, in 10T1/2 cells, a moderate concentration (0.1μg/mL)
of nickel subsulfide caused morphological transformation to type I, II, and III foci, while high
concentrations (10 and 100 μg/mL) of nickel subsulfide lead to cell lysis after a lag period.
Insoluble nickel compounds also induce anchorage independence in human diploid
fibroblasts (Biedermann and Landolph, 1987). According to Bidermann and Landolph(1987),
induction of anchorage independence in human cells had the optimal expression time of 12
days with dose-dependent cytotoxicity - with Ni3S2 showing a linear does-dependent
relationship to induction of anchorage independent relationship. Even though Ni3S2 increased
anchorage independence 200-fold, it did not increase ouabain-resistant or 6-thioguanineresistant colonies. V79 hamster cells also failed to show 6-thioguanine-resistant colonies
when treated with NiS or NiO black (Kargacin, 1993). However, Nickel-induced DNA
mutation increased 20-fold when G12 transgenic cell lines were treated with insoluble nickels
(Lee, 1995).
Nickel compounds are not mutagenic in prokaryotic cells, but DNA mutation was also
not observed in cells when treated with nickel compounds (Costa, 1991 Miura et al., 1989).
However, oxidative DNA damage at the G site induced by nickel compounds leads to G/C to
T/A transversion in the K-ras gene (Higinbotham, 1992). G-to-T transversion mutations are
also commonly found with DNA mutation caused by ROS (Du, 1994). The finding increased
the possibility that nickel can directly oxidize DNA bases to damage using reactive oxygen
species, not only breaking the DNA strand and causing chromosomal aberrations (Sen,
1985).
DNA strand breaks, DNA-protein cross-links, nucleotide excision, single gene
mutations, sister chromatid exchanges, and more are observed in cells treated with nickel
compounds (Das, 2008). Some mutations were non-repairable with certain nickel compounds
(nickel subsulfide and nickel oxide), leading to cell proliferation (Dunnick, 1995). Many
studies revealed that nickel compounds indirectly mutate the genes by generating free
radicals and oxidative stress (Dunnick, 1995).
ROS-induced DNA mutation
Reactive oxygen species can cause DNA mutation by inducing DNA base damage
(Evans, 2004). Hydroxyl radical, an electrophilic compound, prefers to add itself to the
highest electron density. When hydroxyl radical adds itself to the highest electron density, it
changes the chemical structure of the pyrimidines, cytosine, and thymine, and the purines,
guanine, and adenine (Evans, 2004). To overcome various DNA base damage, various

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enzymes are induced to repair base mutations (Evans, 2004). Oxygen radicals react with
Guanine and produce 8-hydroxyguanine (Evans, 2004). 8-OH-Gua is found in blood and
urine as a marker of oxidative stress (Cooke, 2002). 8-OH-Gua tends to substitute G toT in
mammalian cells (Moriya, 1993). 8-OH-Gua glycosylase (OGG1) is an enzyme to repair 8-
OH-Gua. Hung et al. suggested that mutation in OGG1 and lung cancer risk are positively
related. With the OGG1 knockout mice model, the frequency of lung adenoma increased
(Hirano, 2008). Cadmium, a heavy carcinogenic metal, down-regulated OGG1 expression
(Hirano, 2008). Manganese (Mn2+) decreased the enzymatic activity of OGG1. Arsenic
inhibited OGG1 activity (Hirano, 2008).
Microsatellite mutations were also observed in human cancer cells treated with the
hydrogen peroxide (Zienolddiny, 2000). Hydrogen peroxide induced a 27-fold higher
mutation frequency than spontaneous mutation frequency (Zienolddiny, 2000). When cells
were treated with hydrogen peroxide as oxidative stress, the stability of the microsatellite
sequence was affected (Zienolddiny, 2000). DNA base alterations, abasic sites, and strand
breaks within the microsatellite sequences (Zienolddiny, 2000). Possible mechanisms could
be the mispairing of altered bases, strand displacement, and/ or misalignment (Zienolddiny,
2000).
ROS have been shown to suppress DNA repair enzymes (Hu, 1995). Hu et al. found
that H2O2 has an inhibitory effect on DNA repair enzymes due to its influence on cellular
redox states. H2O2 may oxidize cellular glutathione and protein thiols, which are important
for DNA repair. Cellular glutathione repairs radical damaged DNA by hydrogen transfer
from thiol groups (Alvarez et al., 2012).
Nickel-induced Reactive Oxygen Species (ROS)
Oxidative stress is one of the well-known factors contributing to carcinogenesis (Das,
2008). Nickel (II) produces free radicals when humans are exposed to it. Nickel compounds
generate free radicals by reducing molecular oxygen to superoxide anion (Das, 2008).
Insoluble nickel could develop soft tissue sarcomas, when it is injected ….. soluble
nickel could induce epithelial malignancies. However, the mechanisms of these are not fully
understood. To understand how nickel compounds lead to carcinogenesis, Govindarajan et al.
used wild-type C57BL/6 and heterozygous p53 mice with exposure to nickel sulfide. This
experiment found that nickel sulfide led to the activation of the MAP kinase signaling
pathway and hypermethylation of the p16Ink4a promoter (Govindarajan, 2002). There was also
a structural mutation in p53, the tumor suppressor gene in p53 heterozygous mice, via
deletion of exon 5. Wild-type mice also showed point mutation of A to C in intron 7.
Mutation in the intron could lead to a splice site or interruption of gene expression. This
paper showed how nickel-induced sarcomas were on the legs of the mice. Since this article
revealed the activation of the MAP kinase signaling pathway and the silence of the p16Ink4a
promoter was revealed through this article, the mechanism of other nickel-induced cancer
could likely be found.
Activation of the MAP kinase signaling pathway could lead to pancreatic cancer and
more through mutation of Ras. According to Dhillon et al., when the ERK pathway, a wellknown mammalian MAP kinase pathway, is activated, tyrosine kinase initiates guanosine
triphosphate (GTP) loading of Ras GTPase is necessary to activate Ras. Mutated Ras will
remain activated since it prevents GTP hydrolysis and keeps Ras GTPase in an active state.

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The active Ras GTPase also activates ERK signaling, which continues the loop. Ras does not
function solely but has Raf as an effector. They explain this more in-depth in the literature.
Hypermethylation of p16Ink4a leads to the silencing of tumor suppressor genes.
According to LaPak et al., the silence of p16Ink4a was found in urothelial carcinoma,
glioblastoma multiforme, and more. The p16Ink4a has the function of inhibiting RB
phosphorylation, a tumor suppressor pathway. The ratio of p16Ink4a to RB phosphorylation
results in different cancers. If there is more p16Ink4a, they detect lung cancer. They found that
kidney cancer was detected when more RB phosphorylation was presented. Increased level of
RB phosphorylation shows that p16Ink4a is not affecting carcinogenesis alone, but the RB
pathway influences tumor formation.
Fluorescence of H2DCFDA to detect ROS
H2DCFDA (2',7'-Dichlorodihydrofluorescein diacetate; Invitrogen, MA, USA) is a
widely used fluorescent probe for detecting total reactive oxygen species (ROS) levels in
cells (Aitken, et al., 2013). H2DCFDA is enzymatically cleaved by intracellular esterases to
form non-fluorescent DCFH-DA (2',7'-dichlorodihydrofluorescein diacetic acid) (Eruslanov,
2010). In the presence of ROS, DCFH-DA is oxidized by ROS to yield the highly fluorescent
compound DCF (2',7'-Dichlorofluoresein) (Rota, et al. 1999). This oxidation occurs through
the removal of acetate groups by ROS (Eruslanov. Et al., 2010). The fluorescence emitted by
DCF when excited with light of the appropriate wavelength is directly proportional to
intracellular ROS levels, allowing the detection of total ROS (Mahfouz, et al., 2010). By
quantifying the intensity of emitted fluorescence, researchers can estimate the relative
abundance of ROS in a cell population (Sieprath, et al., 2015). It should be noted that
H2DCFDA is not specific to individual ROS species but can detect a variety of ROS
including hydrogen peroxide, superoxide anions, and hydroxyl radicals (Lü, et al., 2010).
Cell lines used in these studies
BEAS-2B cells are a human bronchial epithelial cell line derived from normal
bronchial tissue (Kim, 2011). These cells are commonly used in biomedical research to study
the effects of environmental toxins and carcinogens on the respiratory system, as they provide
a physiologically relevant model of the human airway. What is unique about BEAS-2B cells
is that they have retained many characteristics of normal bronchial epithelial cells, including
forming a polarized monolayer and differentiating into ciliated and secretory cells (Oh,
2011). This makes them a valuable tool for studying respiratory diseases such as asthma,
chronic obstructive pulmonary disease (COPD), and lung cancer.
Furthermore, BEAS-2B cells are relatively easy to culture and maintain in the
laboratory, which makes them a popular choice for in vitro studies (Hiemstra, 2918). They
have also been extensively characterized, and their gene expression profile has been welldefined, which allows researchers to study the effects of different treatments or
environmental exposures on specific genes or pathways. Overall, BEAS-2B cells are a
valuable tool for studying the effects of environmental toxins and carcinogens on the
respiratory system (Oh, 2011). They have contributed significantly to understanding
respiratory disease pathogenesis and developing new therapeutics.
C3H 10T1/2 Cl8 mouse embryo cells (10T1/2) are a mouse embryonic fibroblast cell
line commonly used in biomedical research (Landolph, 1985). One unique feature of 10T1/2

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cells is their ability to differentiate into multiple cell types, including adipocytes,
chondrocytes, and osteoblasts. This makes them a useful model system for studying
developmental processes and tissue regeneration. 10T1/2 cells also have been wellcharacterized in their gene expression profile and signaling pathways, making them valuable
for studying various biological processes such as cell cycle regulation, DNA damage
response, and cellular senescence. Overall, the versatility of 10T1/2 cells, their ability to
differentiate into multiple cell types, and their amenability to genetic manipulation make
them a valuable tool for studying a wide range of biological processes, including tissue
regeneration, developmental biology, and cancer.

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Materials and Methods
Chemicals
Basal Medium Eagle (BME) was purchased from the Sigma-Aldrich Company, St.
Louis, MO, USA. Fetal Bovine Serum (FBS) was purchased from the Thermofisher
Company, Waltham, MA, USA. L-glutamine (100X) was purchased from Thermofisher
Company, Waltham, MA, USA. Penicillin was purchased from the Gibco Media Network,
New York, NY, USA. Dulbecco’s Modified Eagle Medium (DMEM) supplemented with Lglutamine, 1g/L glucose, and sodium pyruvate was purchased from the Corning Company,
Corning, New York, USA. Dulbecco’s Phosphate Buffered Saline (DPBS) was purchased
from Sigma-Aldrich Company, St. Louis, MO, USA. Methanol was purchased from the
Fisher Scientific International L.L.C, Waltham, MA, USA. Trypsin Solution derived from
Porcine Pancreas was purchased from Sigma-Aldrich Company, St. Louis, MO, USA.
Acetone was purchased from the company. Phosphate-buffered saline (PBS) was purchased
from the Fisher Scientific International L.L.C, Waltham, MA, USA. 70% ethanol was
purchased from Thermofisher Company, Waltham, MA, USA. Dimethyl Sulfoxide (DMSO)
was purchased from the Sigma-Aldrich Company, St. Louis, MO, USA.
Cells and Cell Culture
C3H/10T1/2 Cl 8 (10T1/2) cells were grown in Basal Medium Eagles (BME, SigmaAldrich Company, St. Louis) supplemented with 10% heat-inactivated fetal calf serum (FCS,
Thermofisher Company, Waltham) and 1% glutamine (the Thermofisher Company,
Waltham) on Cell culture dish (Corning Company, Upstate New York). When cells are
subconfluent (about 80% confluent) on the flask, cells are trypsinized to be passaged. After
trypsinizing cells, cells were seeded at 5x104 per 25cm2 T flask (Fisher Scientific Company,
Waltham). The medium was changed every five to seven days, and cells were passaged every
ten days.
Human bronchial epithelial cells, BEAS-2B, were originally purchased from the
America Tissue Culture Collection (ATCC) and generously provided to our laboratory by Dr.
Schönthal Axel, Associate Professor of Molecular, Microbiology, and Immunology (MMI),
Keck School of Medicine, USC, Los Angeles, CA, USA. BEAS-2B cells were cultured in
Dulbecco’s Modified Eagle’s Medium (DMEM, Corning Company, Corning, NY, USA) with
10% fetal bovine serum (FBS, Thermofischer Company, Waltham, MO, USA). When cells
are subconfluent on the 25cm3 flask, cells are trypsinized and passaged. After trypsinizing
cells, cells were seeded at 5x104 per 25cm T flask. The medium was changed every three
days, and cells were passaged every six days.
Plating Efficiency
Cells were trypsinized, counted with a hemocytometer, and seeded at 200 cells into
each 60mm culture dish (Corning Company, Corning, NY, USA). For 10T1/2 cells, the
medium was changed on day 5. For BEAS 2B cells, the medium was changed on Days 3 and
6. Between Days 8 and 10, when colonies were large, the medium was aspirated off, and the
cells were fixed with methanol, stained with 1% Crystal violet, rinsed with tap water, and air
dried. The number of colonies containing greater than 20 cells was counted under a dissecting
microscope (Miura et al., 1989, Landolph, 1979). The number of colonies was used to
calculate plating efficiency (# of colonies) / (# of cells seeded) x 100 (%). Optimized and
non-optimized plating efficiency have been conducted. For the optimized method, the

Copyright 2023 9 Sheldy SoJeong Shin
medium was changed a day before plating, while for the non-optimized method, cells were
plated without changing the medium a day before.
Cytotoxicity Assay
For 10T1/2 cells, 10T1/2 cells were treated with nickel compounds one day after
seeding. Nickel solutions were made with acetone. For insoluble nickel compounds, such as
green NiO, black NiO, nickel subsulfide (Ni3S2), 2μg/mL, 1.5μg/mL, 1μg/mL, and 0.5μg/mL
concentrations are used. For soluble nickel compounds, such as NiSO4, NiCl2, and Ni(CO)3,
100μg/mL, 75μg/mL, 50μg/mL, and 25μg/mL concentration are used. 25μL of suspension of
insoluble nickel compouds on 0.5% of acetone was added for insoluble nickel solutions, and
0.5mL was added for soluble nickel solutions. For negative control, cells were treated with
0.5% acetone (25μL added to 5L of medium) for insoluble nickel compounds and PBS
(0.5mL) for soluble nickel compounds. After 48 hours of exposure, the treatments were
removed, and the medium was changed.
For BEAS 2B cells, the mediums were changed on day 5. Plating efficiency was
measured on Day 8 after staining with 1% crystal violet. The number of colonies determined
the survival rate after the treatment over the number of colonies treated with positive
controls.
Methods to detect ROS
Cells were grown and treated with nickel compounds just as was done on the
Cytotoxicity Assay. On Day 8, cells were collected for fluorescence measurement. To
measure overall reactive oxygen species (ROS), DCFDA/ H2DCFDA (H2DCFDA), provided
by Dr. Schönthal. On Day 8, 25,000 cells were plated on each wall of 96-well, Cell CultureTreated Flat-Bottom microplate. The cells were put in the incubator for 24 hours to allow
adherence. On Day 9, medium was removed and 100μL 1x Dilution Buffer (what is included)
was added to each well to wash. 1x Buffer was removed and 100μL of diluted DCFDA
solution was added to each well to stain the cells. The plates were incubated in the incubator
for 45 minutes at 37°C in the dark. DCFDA solution was removed and then 100μL of 1x
Buffer was added to each well. Nickel compounds and 50μM tert-butyl hydroperoxide
(TBHP, positive control) solution were added to the assigned well and incubated for an
additional 4 hours. H2DCFDA was measured at 485 nm (Ab)/ 535 nm (Em).

Copyright 2023 10 Sheldy SoJeong Shin
Results
BEAS 2B cells
Table1a. Plating Efficiency of BEAS 2B using the non-optimized method.
Number of cells seeded Number of colonies
(mean±SEM)
Plating efficiency(%)
(mean±SEM)
Expt. 1
2000 181±18 > 9±1% (TNTC)
200 44±3 22±2%
Expt. 2
2000 TNTC TNTC
200 92±4 46±2%
Expt. 3
2000 182±15 > 9±1% (TNTC)
200 88±8 44±4%
Average of three experiments
2000 199±14 10±0.6%
200 75±7 37±3%
Each experiment had four plates for each conditions (200 cells or 2000 cells) and used the
same passaged cells on the same date. The medium was not changed on the day before the
experiment. TNTC= Too Numerous To Count; the colonies were emerging into one big
colony; therefore, one colony was used to calculate overall plating efficiency.
Table1b. Plating Efficiency of BEAS 2B using the optimized method.
Number of cells seeded Number of colonies
(mean±SEM)
Plating efficiency(%)
(mean±SEM)
Expt. 1
2000 261±6 (TNTC) > 13.0±0.3% (TNTC)
200 106±5 53±2%

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Expt. 2
2000 182±15 (TNTC) > 9.1±0.8% (TNTC)
200 100±7 50±4%
Expt. 3
2000 175±8 (TNTC) > 8.7±0.4% (TNTC)
200 64±7 32±4%
Average of three experiments
2000 206±13 (TNTC) > 10±0.6% (TNTC)
200 90±3 45±3%
Each experiment had four plates and used the same passaged cells on the same date. The
medium was changed on the day before the experiment to boost cell growth.
Table 2a. Cytotoxicity of Black NiO to BEAS 2B cells
Concentration Number of colonies
(mean±SEM)
Acetone treated
cells
(0μg/mL)
(mean±SEM)
Survival Fraction
(mean±SEM)
Exp.1
No treatment 99±7 (PE)
100±8 (PE)
0.99±0.07
0.5μg/mL 83±7 (PE) 0.83±0.07
1μg/mL 86±6 (PE) 0.87±0.06
1.5μg/mL 80±6 (PE) 0.8±0.06
2μg/mL 60±6 (PE) 0.6±0.06
Exp.2
No treatment 154±7 (PE) 0.96±0.05

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0.5μg/mL 150±3 (PE)
160±5 (PE)
0.93±0.02
1μg/mL 147±1 (PE) 0.69±0.01
1.5μg/mL 110±2 (PE) 0.68±0.01
2μg/mL 88±3 (PE) 0.55±0.02
Exp.3
No treatment 124±2 (PE)
128±3 (PE)
0.97±0.01
0.5μg/mL 114±3 (PE) 0.9±0.03
1μg/mL 112±2 (PE) 0.88±0.02
1.5μg/mL 109±1 (PE) 0.85±0.01
2μg/mL 84±5 (PE) 0.66±0.04
Average of three experiments
No treatment 110±4 (PE)
129±8 (PE)
0.88±0.05
0.5μg/mL 118±8 (PE) 0.89±0.02
1μg/mL 112±6 (PE) 0.88±0.02
1.5μg/mL 101±4 (PE) 0.78±0.03
2μg/mL 79±4 (PE) 0.6±0.02
Each experiment was set by four plates of each concentration used the same passaged cells on
the same date. The medium was changed on the day before the experiment. The concentration
of the treatment was calculated in m/v, where the volume was 5 mL of the medium. The
survival rate of each treatment was calculated according to the positive control, acetone
treatment. Percent Survival= (number of colonies of the treatment)/ (number of colonies of
positive control) x100(%). PE= plating efficiency.
Table 2b. Cytotoxicity of Nickel Subsulfide to BEAS 2B cells
Concentration
Number of colonies
(mean±SEM)
Acetone treated cells
(0μg/mL)
(mean±SEM)
Percent Survival
(mean±SEM)

Copyright 2023 13 Sheldy SoJeong Shin
Exp.1
No treatment 89±3 (PE)
80±2 (PE)
1.11±0.03
0.5μg/mL 47±5 (PE) 0.59±0.07
1μg/mL 42±5 (PE) 0.53±0.06
1.5μg/mL 35±5 (PE) 0.44±0.06
2μg/mL 13±1 (PE) 0.17±0.01
Exp.2
No treatment 106±5 (PE)
116±6 (PE)
0.92±0.04
0.5μg/mL 59±2 (PE) 0.51±0.01
1μg/mL 49±3 (PE) 0.42±0.02
1.5μg/mL 20±2 (PE) 0.17±0.02
2μg/mL 10±2 (PE) 0.09±0.02
Average of two experiments
No treatment 98±4 (PE)
98±7 (PE)
1.01±0.04
0.5μg/mL 53±3 (PE) 0.55±0.04
1μg/mL 46±3 (PE) 0.47±0.04
1.5μg/mL 27±4 (PE) 0.3±0.06
2μg/mL 12±1 (PE) 0.13±0.02
Each concentration had four plates and used the same passaged cells on the same date. The
medium was changed on the day before the experiment. The concentration of the treatment
was calculated in m/v, where the volume was 5 mL of the medium. The survival rate of each
treatment was calculated according to the positive control, acetone treatment. Percent
Survival= (number of colonies of the treatment)/ (number of colonies of positive
control)x100(%). PE= plating efficiency.

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Table 3a. Seeding Dilution of BEAS 2B cells using H2DCFDA
Number of cells Fluorescence Arbitrary Units (mean±SEM)
0 0±0
100 4063±81
200 4186±32
1000 4023±27
2000 3961±50
10000 4055±64
30000 4141±42
60000 4152±36
Different number of cells were seeded on the 96-well plate on four wells. Fluorescence
arbitrary unit was found by subtracting the average data of empty well (background).
Table 3b. Sensitivity test of BEAS 2B cells using H2 DCFDA
H2O2 Concentration (μM) Fluorescence Arbitrary Units (mean±SEM)
0 (spontaneous ROS) 3577±585
5 2391±1014
10 1055±434
20 318±30
25 2764±335
50 15761±6753
100 885±515
200 561±94
250 7560±1344
Sensitivity test was done by adding different concentration of H2O2 (0, 5, 10, 20, 25, 50, 100,
200, and 250μM). 60,000 cells were seeded to each well on 96-well plate. After 5 days of
seeding, the wells were washed with 1x Buffer solution (Abcam, Cambridge, UK) and then
H2DCFDA was added. The cells were incubated for 45 minutes. After the incubation,
H2DCFDA was removed and 1x Buffer was added. Then different concentration of H2O2 was
added to each four wells and incubated for four hours. After four hours, H2DCFDA was
measured at 485 nm (Ab)/ 535 nm (Em).

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Table 4a. Total ROS measurement on Black NiO treated BEAS 2B cells using H2DCFDA
Fluorescence Arbitrary Unit (mean±SEM)
Cell
+DCFDA
50μM
H2O2
0 μg/mL
(acetone
treated)
0.5μg/mL 1μg/mL 1.5μg/mL 2μg/mL
Exp.1
Day 1 2188±320 3051±216 2607±63 2037±145 2065±81 2327±149 2214±254
Day 2 2712±130 4416±146 2276±186 2279±154 2258±170 2432±209 2968±177
Day 3 1873±125 3685±137 2001±120 1833±52 1762±235 2066±254 1699±79
Day 4 973±16 2881±199 954±13 1056±17 978±14 1535±19 968±13
Day 5 1269±17 3612±53 1206±12 1195±11 1249±17 1149±25 1572±111
Exp.2
Day 1 948±8 1646±235 993±11 973±9 984±6 993±14 975±10
Day 2 1442±146 3619±283 1681±163 1832±54 2039±89 2232±99 2382±236
Day 3 990±17 3159±213 1424±136 1256±37 1386±47 1465±171 1408±56
Day 4 932±15 4334±262 1324±71 1820±142 1542±95 2463±172 1357±27
Day 5 874±18 3399±119 1185±40 1072±58 1193±86 1249±133 1467±196
Total reactive oxygen species (ROS) were measured using CM-H2DCFDA. Cells with
fluorescence were used as a negative control, and 50μM H2O2 was used as a positive control.
0μg/mL of treatment indicates 0.05% acetone-treated cells. 0.5μg/mL, 1μg/mL, 1.5μg/mL,
and 2μg/mL of Black NiO were added to each 4 wells. The number of days indicates
fluorescence measured after treatments were removed.
Table 4b. Normalization of total ROS measurement of Black NiO treated BEAS 2B cells using
H2 DCFDA
DCFDA fluorescence (% of control) [mean±SEM]
Cell
+DCFDA
50μM
H2O2
0 μg/mL
(acetone
treated)
0.5μg/mL 1μg/mL 1.5μg/mL 2μg/mL

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Exp.1
Day 1 100±15% 139±10% 119±3% 93±7% 94±4% 106±7% 101±12%
Day 2 100±5% 163±6% 84±7% 84±6% 83±6% 90±8% 109±7%
Day 3 100±7% 197±8% 107±7% 98±3% 94±13% 110±14% 91±4%
Day 4 100±2% 296±21% 98±2% 109±2% 101±2% 158±2% 100±2%
Day 5 100±2% 285±4% 95±1% 94±1% 98±2% 91±2% 124±9%
Exp.2
Day 1 100±1% 174±25% 105±1% 103±1% 104±1% 105±2% 103±1%
Day 2 100±10% 251±20% 165±17% 127±4% 141±6% 155±7% 165±17%
Day 3 100±2% 319±22% 144±14% 127±4% 140±5% 148±18% 142±6%
Day 4 100±2% 465±28% 142±8% 195±15% 165±10% 264±19% 146±3%
Day 5 100±2% 389±14% 136±5% 123±7% 137±10% 143±15% 168±23%
Average of two experiments
Day 1 100±6% 157±14% 112±3% 98±4% 111±11% 106±3% 102±5%
Day 2 100±5% 192±17% 98±8% 102±9% 112±12% 127±13% 137±13%
Day 3 100±3% 244±24% 126±11% 111±6% 117±11% 129±12% 117±10%
Day 4 100±1% 355±34% 115±8% 140±14% 128±13% 211±22% 115±8%
Day 5 100±1% 324±22% 113±8% 104±5% 118±8% 113±12% 146±14%
Total reactive oxygen species (ROS) were measured using C M-H2DCFDA. Cells with
fluorescence were used as a negative control, and 50μM H2O2 was used as a positive control.
0μg/mL of treatment indicates 0.05% acetone-treated cells. 0.5μg/mL, 1μg/mL, 1.5μg/mL,
and 2μg/mL of Black NiO were added to each 4 wells. The number of days indicates
fluorescence measured after treatments were removed. The data was normalized by dividing
with cells with fluorescence (% of control).
Table 4c. Total ROS measurement on Green NiO treated BEAS 2B cells using H2DCFDA

Copyright 2023 17 Sheldy SoJeong Shin
Fluorescence Arbitrary Units (mean±SEM)
Cell
+DCFDA
50μM
H2O2
0 μg/mL
(acetone
treated)
0.5μg/mL 1μg/mL 1.5μg/mL 2μg/mL
Exp.1
Day 1 2188±320 3051±216 2607±63 1305±34 2457±258 2417±185 1947±63
Day 2 2712±130 4416±146 2276±186 1664±104 2039±151 2203±142 2354±46
Day 3 1873±125 2685±137 2001±120 1352±32 1441±141 1849±179 1720±84
Day 4 973±16 2881±199 954±13 1261±30 1551±20 1366±119 964±11
Day 5 1269±17 3612±53 1206±12 1609±8 1110±16 1149±29 1155±20
Exp.2
Day 1 948±8 1646±235 993±11 979±7 960±14 964±9 967±20
Day 2 1442±146 3619±283 1681±163 1386±75 1591±119 1721±221 2011±172
Day 3 990±17 3159±213 1424±136 1197±31 1181±63 1149±45 1523±98
Day 4 932±15 4334±262 1324±71 1453±88 2332±264 1783±226 1276±56
Day 5 874±18 3399±119 1185±40 1151±68 1116±62 1165±109 1163±120
Total reactive oxygen species (ROS) were measured using CM-H2DCFDA. Cells with
fluorescence were used as a negative control, and 50μM H2O2 was used as a positive control.
0μg/mL of treatment indicates 0.05% acetone-treated cells. 0.5μg/mL, 1μg/mL, 1.5μg/mL,
and 2μg/mL of Green NiO were added to each 4 wells. The number of days indicates
fluorescence measured after treatments were removed.
Table 4d. Normalization of total ROS measurement on Green NiO treated BEAS 2B cells
using H2DCFDA
DCFDA fluorescence (% of control) [mean±SEM]
Cell
+DCFDA
50μM
H2O2
0 μg/mL
(acetone
treated)
0.5μg/mL 1μg/mL 1.5μg/mL 2μg/mL
Exp.1

Copyright 2023 18 Sheldy SoJeong Shin
Day 1 100±15% 139±10% 119±3% 60±2% 112±12% 110±9% 89±7%
Day 2 100±5% 163±6% 84±7% 61±4% 75±6% 81±5% 87±2%
Day 3 100±7% 197±8% 107±7% 72±2% 77±8% 99±10% 92±5%
Day 4 100±2% 296±22% 98±2% 130±3% 159±2% 140±12% 99±1%
Day 5 100±2% 285±4% 95±1% 127±1% 88±2% 91±2% 91±2%
Exp.2
Day 1 100±1% 174±25% 105±1% 103±1% 101±2% 102±1% 102±2%
Day 2 100±10% 251±20% 117±12% 96±5% 110±8% 119±16% 139±12%
Day 3 100±2% 319±22% 144±14% 121±3% 119±7% 116±5% 154±10%
Day 4 100±2% 465±28% 142±8% 156±10% 250±29% 191±24% 137±6%
Day 5 100±2% 389±14% 136±5% 132±8% 128±7% 133±13% 133±14%
Total
Day 1 100±6% 157±14% 112±3% 81±8% 107±6% 105±4% 96±4%
Day 2 100±5% 192±17% 98±8% 77±8% 93±8% 100±10% 113±11%
Day 3 100±3% 244±24% 126±11% 93±10% 98±9% 107±66% 123±13%
Day 4 100±1% 355±34% 115±8% 144±7% 206±21% 166±16% 118±8%
Day 5 100±1% 324±21% 113±8% 130±4% 108±8% 112±10% 112±10%
Total reactive oxygen species (ROS) were measured using CM-H2DCFDA. Cells with
fluorescence were used as a negative control, and 50μM H2O2 was used as a positive control.
0μg/mL of treatment indicates 0.05% acetone-treated cells. 0.5μg/mL, 1μg/mL, 1.5μg/mL,
and 2μg/mL of Green NiO were added to each 4 wells. The number of days indicates
fluorescence measured after treatments were removed. The data was normalized by dividing
with cells with fluorescence (% of control).
Table 4e. Total ROS measurement on Ni2S3 treated BEAS 2B cells using H2DCFDA
Fluorescence Arbitrary Unit (mean±SEM)

Copyright 2023 19 Sheldy SoJeong Shin
Cell
+DCFD
A
50μM
H2O2
0 μg/mL
(acetone
treated)
0.5μg/mL 1μg/mL 1.5μg/mL 2μg/mL
Exp.1
Day 1 948±8 1646±235 993±11 722±40 783±72 832±4 820±9
Day 2 1442±14
6
3619±283 1681±163 984±47 1110±86 1157±64 1445±81
Day 3 990±17 3159±213 1424±136 2640±127 1604±44 2510±76 2339±204
Day 4 932±15 4334±262 1324±71 2565±192 1529±88 2760±287 2589±373
Day 5 874±18 3399±119 1185±40 1219±84 1084±25 1337±56 1157±95
Total reactive oxygen species (ROS) were measured using CM-H2DCFDA. Cells with
fluorescence were used as a negative control, and 50μM H2O2 was used as a positive control.
0μg/mL of treatment indicates 0.05% acetone-treated cells. 0.5μg/mL, 1μg/mL, 1.5μg/mL,
and 2μg/mL of Ni2S3 were added to each 4 wells. The number of days indicates fluorescence
measured after treatments were removed.
Table 4f. Normalization of total ROS measurement on Ni2S3 treated BEAS 2B cells using
H2DCFDA
DCFDA fluorescence (% of control) [mean±SEM]
Cell
+DCFDA
50μM
H2O2
0 μg/mL
(acetone
treated)
0.5μg/mL 1μg/mL 1.5μg/mL 2μg/mL
Exp.1
Day 1 100±1% 174±25% 105±1% 76±4% 83±8% 88±1% 86±1%
Day 2 100±10% 251±20% 117±12% 68±3% 77±6% 80±5% 100±6%
Day 3 100±2% 319±22% 144±14% 267±13% 162±5% 254±8% 236±21%
Day 4 100±2% 465±28% 142±8% 275±21% 164±10% 296±31% 278±40%
Day 5 100±2% 389±14% 136±5% 132±11% 153±7% 124±3% 140±10%
Total reactive oxygen species (ROS) were measured using CM-H2DCFDA. Cells with
fluorescence were used as a negative control, and 50μM H2O2 was used as a positive control.
0μg/mL of treatment indicates 0.05% acetone-treated cells. 0.5μg/mL, 1μg/mL, 1.5μg/mL,
and 2μg/mL of Ni2S3 were added to each 4 wells. The number of days indicates fluorescence

Copyright 2023 20 Sheldy SoJeong Shin
measured after treatments were removed. The data was normalized by dividing with cells
with fluorescence (% of control).
10T1/2 cells
Table 5a. Plating Efficiency of C3H 10T1/2 Cl8 mouse embryo cells using the non-optimized
method.
Number of cells seeded Number of colonies
(mean±SEM)
Plating efficiency(%)
(mean±SEM)
Expt. 1
2000 188±6 (PE) 9.4±0.3%
200 127±14 (PE) 63±7%
Expt. 2
2000 152±7 (PE) 7.6±0.4%
200 67±3 (PE) 34±2%
Average of two experiments
2000 170±8 (PE) 8.5±0.4%
200 97±13 (PE) 36±2%
Each experiments had four plates of each condition (200 cells or 2000 cells seeded) and used
the same passaged cells on the same date. The medium was not changed on the day before the
experiment.
Table 5b. Plating Efficiency of C3H 10T1/2 Cl8 mouse embryo cells using the optimized
method.
Number of cells seeded Number of colonies
(mean±SEM)
Plating efficiency(%)
(mean±SEM)
Expt. 1
2000 42±4 (PE) 2±0.2%
200 62±3 (PE) 31±2%
Expt. 2
2000 118±13 (PE) 6±0.5%

Copyright 2023 21 Sheldy SoJeong Shin
200 136±7 (PE) 68±3%
Expt.3
2000 80±22 (PE) 4±1%
200 99±21 (PE) 50±10%
Expt. 3
2000 118±13 (PE) 6±0.5%
200 136±7 (PE) 68±3%
Average of three experiments
2000 90±11 (PE) 5±1%
200 68±2 (PE) 34±1%
Each experiment had four plates and used the same passaged cells on the same date. The
medium was changed on the day before the experiment to boost cell growth.
Table 6a. Cytotoxicity of Black NiO to C3H 10T1/2 Cl8 mouse embryo cells
Concentration Number of colonies
(mean±SEM)
Acetone treated
cells
(0μg/mL)
(mean±SEM)
Survival Fraction
(mean±SEM)
Exp.1
no treatment 62±3 (PE)
70±6 (PE)
0.89±0.05
0.5μg/mL 22±3 (PE) 0.31±0.04
1μg/mL 5±2 (PE) 0.07±0.02
1.5μg/mL 4±1 (PE) 0.05±0.01
2μg/mL 0±0 (PE) 0.0±0.0
Exp.2
no treatment 28±3 (PE) 0.97±0.11

Copyright 2023 22 Sheldy SoJeong Shin
0.5μg/mL 22±3 (PE)
29±4 (PE)
0.75±0.10
1μg/mL 16±2 (PE) 0.53±0.07
1.5μg/mL 18±5 (PE) 0.63±0.16
2μg/mL 1±2 (PE) 0.04±0.03
Exp.3
no treatment 33±4 (PE)
30±6 (PE)
1.11±0.12
0.5μg/mL 20±5 (PE) 0.69±0.12
1μg/mL 12±1 (PE) 0.41±0.04
1.5μg/mL 6±2 (PE) 0.19±0.05
2μg/mL 4±2 (PE) 0.13±0.06
Average of three experiments
no treatment 41±5 (PE)
129±8 (PE)
0.99±0.05
0.5μg/mL 21±2 (PE) 0.58±0.08
1μg/mL 11±1 (PE) 0.33±0.06
1.5μg/mL 9±2 (PE) 0.29±0.09
2μg/mL 2±1 (PE) 0.06±0.03
Each concentration had four plates and used the same passaged cells on the same date. The
medium was changed on the day before the experiment. The concentration of the treatment
was calculated in m/v, where the volume was 5 mL of the medium. The survival rate of each
treatment was calculated according to the positive control, acetone treatment. Percent
Survival= (number of colonies of the treatment)/ (number of colonies of positive
control)x100(%).
Table 6b. Cytotoxicity of Nickel Subsulfide to C3H 10T1/2 Cl8 mouse embryo cells
Concentration Number of colonies
(mean±SEM)
Acetone treated cells
(0μg/mL)
(mean±SEM)
Survival Fraction
(mean±SEM)

Copyright 2023 23 Sheldy SoJeong Shin
Exp.1
no treatment 28±2 (PE)
29±2 (PE)
0.97±0.11
0.5μg/mL 19±3 (PE) 0.66±0.09
1μg/mL 13±1 (PE) 0.47±0.04
1.5μg/mL 15±1 (PE) 0.52±0.02
2μg/mL 9±2 (PE) 0.25±0.1
Exp.2
no treatment 27±5 (PE)
30±6 (PE)
0.89±0.17
0.5μg/mL 25±5 (PE) 0.89±0.16
1μg/mL 21±4 (PE) 0.72±0.13
1.5μg/mL 15±3 (PE) 0.52±0.1
2μg/mL 8±2 (PE) 0.26±0.05
Average of two experiments
no treatment 27±2 (PE)
129±8 (PE)
0.94±0.08
0.5μg/mL 22±2 (PE) 0.77±0.08
1μg/mL 16±2 (PE) 0.55±0.08
1.5μg/mL 15±1 (PE) 0.52±0.04
2μg/mL 8±1 (PE) 0.25±0.04
Each concentration had four plates and used the same passaged cells on the same date. The
medium was changed on the day before the experiment. The concentration of the treatment
was calculated in m/v, where the volume was 5 mL of the medium. The survival rate of each
treatment was calculated according to the positive control, acetone treatment. Percent
Survival= (number of colonies of the treatment)/ (number of colonies of positive
control)x100(%).

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Table 7a. Total ROS measurement on Black NiO treated C3H 10T1/2 Cl8 mouse embryo cells
using H2DCFDA
Fluorescence Arbitrary Unit (mean±SEM)
Cell
+DCFDA
50 μM
H2O2
0 μg/mL
(acetone
treated)
0.5
μg/mL
1 μg/mL 1.5
μg/mL
2 μg/mL
Exp.1
Day 1 2051±147 3912±452 2291±193 1815±40 1906±82 1955±49 2244±193
Day 2 1702±195 3548±206 1481±219 1366±148 1765±36 1390±65 3714±550
Day 3 1819±195 4791±444 2270±260 1421±144 2469±182 1785±144 2454±117
Day 4 1724±214 3303±93 1487±87 1200±54 1324±155 2350±225 4725±590
Total reactive oxygen species (ROS) were measured using CM-H2DCFDA. Cells with
fluorescence were used as a negative control, and 50μM H2O2 was used as a positive control.
0μg/mL of treatment indicates 0.05% acetone-treated cells. 0.5μg/mL, 1μg/mL, 1.5μg/mL,
and 2μg/mL of Black NiO were added to each 4 wells. The number of days indicates
fluorescence measured after treatments were removed.
Table 7b. Normalization of total ROS measurement on Black NiO treated C3H 10T1/2 Cl8
mouse embryo cells using H2DCFDA
DCFDA fluorescence (% of control) [mean±SEM]
Cell
+DCFDA
50 μM
H2O2
0 μg/mL
(acetone
treated)
0.5
μg/mL
1 μg/mL 1.5
μg/mL
2 μg/mL
Exp.1
Day 1 100±0.5% 191±22% 112±10% 88±2% 93±4% 95±3% 109±11%
Day 2 100±12% 208±12% 87±13% 80±9% 104±2% 82±4% 218±30%
Day 3 100±11% 263±25% 125±15% 78±8% 136±10% 98±8% 135±7%
Day 4 100±13% 192±6% 86±5% 70±3% 77±9% 136±13% 274±34%
Total reactive oxygen species (ROS) were measured using CM-H2DCFDA. Cells with
fluorescence were used as a negative control, and 50μM H2O2 was used as a positive control.
0μg/mL of treatment indicates 0.05% acetone-treated cells. 0.5μg/mL, 1μg/mL, 1.5μg/mL,
and 2μg/mL of Black NiO were added to each 4 wells. The number of days indicates

Copyright 2023 25 Sheldy SoJeong Shin
fluorescence measured after treatments were removed. The data was normalized by dividing
with cells with fluorescence (% of control).
Table 7c. Total ROS measurement on Green NiO treated C3H 10T1/2 Cl8 mouse embryo
cells using H2DCFDA
Fluorescence Arbitrary Unit (mean±SEM)
Cell
+DCFDA
50 μM
H2O2
0 μg/mL
(acetone
treated)
0.5
μg/mL
1 μg/mL 1.5
μg/mL
2 μg/mL
Exp.1
Day 1 2051±147 3912±452 2291±193 1940±36 2011±189 1742±120 2010±239
Day 2 1702±195 3548±206 1481±219 2679±344 2286±278 1716±200 1791±257
Day 3 1819±195 4791±444 2270±260 1110±77 1378±111 1320±173 4050±402
Day 4 1724±214 3303±93 1487±87 1916±182 1470±103 1513±215 1419±30
Total reactive oxygen species (ROS) were measured using CM-H2DCFDA. Cells with
fluorescence were used as a negative control, and 50μM H2O2 was used as a positive control.
0μg/mL of treatment indicates 0.05% acetone-treated cells. 0.5μg/mL, 1μg/mL, 1.5μg/mL,
and 2μg/mL of Green NiO were added to each 4 wells. The number of days indicates
fluorescence measured after treatments were removed.
Table 7d. Normalization of total ROS measurement on Green NiO treated C3H 10T1/2 Cl8
mouse embryo cells using H2DCFDA
DCFDA fluorescence (% of control) [mean±SEM]
Cell
+DCFDA
50 μM
H2O2
0 μg/mL
(acetone
treated)
0.5
μg/mL
1 μg/mL 1.5
μg/mL
2 μg/mL
Exp.1
Day 1 100±1% 191±22% 112±10% 95±2% 98±9% 85±6% 98±12%
Day 2 100±12% 208±12% 87±13% 157±20% 134±17% 101±12% 105±15%
Day 3 100±11% 263±25% 125±15% 61±4% 76±6% 73±10% 223±22%
Day 4 100±13% 192±6% 86±5% 97±9% 74±5% 76±11% 71±2%
Total reactive oxygen species (ROS) were measured using CM-H2DCFDA. Cells with
fluorescence were used as a negative control, and 50μM H2O2 was used as a positive control.

Copyright 2023 26 Sheldy SoJeong Shin
0μg/mL of treatment indicates 0.05% acetone-treated cells. 0.5μg/mL, 1μg/mL, 1.5μg/mL,
and 2μg/mL of Green NiO were added to each 4 wells. The number of days indicates
fluorescence measured after treatments were removed. The data was normalized by dividing
with cells with fluorescence (% of control).
Optimized method of cell seeding for cytotoxicity
In BEAS 2B cells, the plating efficiency of 200 cells seeded into the 60 mm culture
dish increased significantly compared to seeding 2000 cells into the 60 mm culture dish. (45%
and 10% respectively) (Figure 1). 2000 cells had a lower plating efficiency because there were
too many cells and they started to fuse, making different colonies into one big colony. When
the medium was changed the day before seeding (optimized method), the plating efficiency
increased, but not significantly more than in the non-optimized method (45% and 37%
respectively) (Figure 2).
In C3H 10T1/2 Cl8 (10T1/2) mouse embryo cells, plating efficiency of 200 cells
seeded into the 60mm culture dish increased significantly compared to 2000 cells seeded (34%
and 5% respectively) (Figure 3). However, 10T1/2 cells showed decreased in optimized method
compared to non-optimized method, but not significantly (34% and 36% respectively) (Figure
4).
Figure 1. The plating efficiency of BEAS 2B with 200 cells vs. 2000 cells seeded per 60mm
dish.
200 BEAS 2B cells were plated on the 60 mm cell culture dish with the optimized method
(left), and 2000 BEAS 2B cells were plated on the plates with the optimized method (right).
For the optimization method, the medium was changed one day before seeding. The bar
graph shows the mean plating efficiency ± the standard error of the mean (SEM).

Copyright 2023 27 Sheldy SoJeong Shin
Figure 2. The plating efficiency of BEAS 2B with 200 cells seeded by the optimized method
vs. non-optimized method.
200 BEAS 2B cells were plated on the 60 mm cell culture dish with optimized method (left)
and with the non-optimized method (right). For optimized method, the cell culture medium
was changed one day before seeding, while the non-optimized method, no medium was
changed one day before seeding. The bar graph shows the mean plating efficiency ± the
standard error of the mean (SEM).
Figure 3. The plating efficiency of 10T1/2 cells with 200 cells vs. 2000 cells seeded per
60mm dih.
200 10T1/2 cells were plated on the 60 mm cell culture dish with the optimized method (left),
and 2000 10T1/2 cells were plated on the plates with the optimized method (right). For the
optimization method, the medium was changed one day before seeding. The bar graph shows
the mean plating efficiency ± the standard error of the mean (SEM).

Copyright 2023 28 Sheldy SoJeong Shin
Figure 4. The plating efficiency of 10T1/2 with 200 cells seeded by the optimized method vs.
non-optimized method.
200 10T1/2 cells were plated on the 60 mm cell culture dish with optimized method (left) and
with the non-optimized method (right). For optimized method, the cell culture medium was
changed one day before seeding, while the non-optimized method, no medium was changed
one day before seeding. The bar graph shows the mean plating efficiency ± the standard error
of the mean (SEM).
Cytotoxicity of Insoluble Nickel (II) compounds
One day after seeding, 10T1/2 cells and BEAS 2B cells were exposed to insoluble
nickel (II) compounds for 48 hours in increasing concentrations from 0 μg/mL to 2 μg/mL
Black NiO and Nickel Subsulfide (Ni3S2). Following the exposure of 10T1/2 cells to the
insoluble nickel (II) compounds, there was a dose dependent increase in the cytotoxicity of
Black NiO to 10T1/2 cells (Figure 5 and Figure 6). The plot of Ln(S) vs. concentration was
linear, defined by the equation, S=e-kc to Ln(S)=-kc.
Figure 5. Cytotoxicity of Black NiO treated 10T1/2 cells.
Average of three experiments was used (Table 6a). Cytotoxicity of Black NiO was measured
and documented by ln of survival (S). The survival fraction was obtained by dividing the
number of colonies treated with each concentration of Black NiO by the mean number of
colonies with acetone. Then applied ln on the survival fraction. Each dot plot shows the mean
ln(S) of cells treated with each concentration of Black NiO ± the standard error of the mean
(SEM).

Copyright 2023 29 Sheldy SoJeong Shin
Figure 6. Cytotoxicity of Nickel Subsulfide treated 10T1/2 cells.
Average of two experiments was used (Table 6b). Cytotoxicity of Nickel Subsulfide was
measured and documented by ln of survival (S). The survival fraction was obtained by
dividing the number of colonies treated with each concentration of Nickel Subsulfide by the
mean number of colonies with acetone. Then applied ln on the survival fraction. Each dot
plot shows the mean ln(S) of cells treated with each concentration of Nickel Subsulfide ± the
standard error of the mean (SEM).
Following a 48 hour exposure of BEAS 2B to increasing concentrations of insoluble
Nickel (II) compounds (Black NiO and Nickel Subsulfide (Ni3S2)) from 0 μg/mL to 2 μg/mL,
there was a dose-dependent decrease in cell survival (Figure 7 and Figure 8). The plot of
Ln(S) vs. concentration was linear, defined by the equation, S=e-kc to Ln(S)=-kc.
Figure 7. Cytotoxicity of Black NiO treated BEAS 2B cells.
Average of three experiments was used (Table 2a). Cytotoxicity of Black NiO was measured
and documented by ln of survival (S). The survival fraction was obtained by dividing the
number of colonies treated with each concentration of Black NiO by the mean number of
colonies with acetone. Then applied ln on the survival fraction. Each dot plot shows the mean
ln(S) of cells treated with each concentration of Black NiO ± the standard error of the mean
(SEM).

Copyright 2023 30 Sheldy SoJeong Shin
Figure 8. Cytotoxicity of Nickel Subsulfide treated BEAS 2B cells.
Average of two experiments was used (Table 2b). Cytotoxicity of Nickel Subsulfide was
measured and documented by ln of survival (S). The survival fraction was obtained by
dividing the number of colonies treated with each concentration of Nickel Subsulfide by the
mean number of colonies with acetone. Then applied ln on the survival fraction. Each dot
plot shows the mean ln(S) of cells treated with each concentration of Nickel Subsulfide ± the
standard error of the mean (SEM).
Determining seeding number and positive control
The fluorescence of H2DCFDA was used to measure total ROS in the cell. Increased
number of cells seeded to 96-well plate showed increased fluorescence in 10T1/2 cells and
BEAS 2B cells (Figure 9 and Figure 10, respectively).
Figure 9. Seeding Dilution of 10T1/2 cells using H2DCFDA.
Fluorescence of H2DCFDA was measured using different numbers of cells per well: 0, 100,
200, 1000, 2000, 10000, 30000, and 60000 cells. Each dot plot shows the mean fluorescence
in arbitrary units with the standard error of the mean (SEM).
Figure 10. Seeding Dilution of BEAS 2B cells using H2DCFDA.

Copyright 2023 31 Sheldy SoJeong Shin
This experiment measured the fluorescence of H2DCFDA using different numbers of cells
per well: 0, 100, 200, 1000, 2000, 10000, 30000, and 60000 cells. Each dot plot shows the
mean fluorescence in arbitrary units with the standard error of the mean (SEM).
When 10T1/2 cells and BEAS 2B cells were treated with increasing concentrations
of tert-Butyl hydroperoxide from 5 μM to 250 μM. The fluorescence of H2DCFDA was used
to detect total ROS measurement. Compared to the control, which had no H2O2 added, 50μM
H2O2 had significant increase in total ROS measurement (Figure 11).
Figure 11. Sensitivity test using different concentrations of H2O2 in BEAS 2B cells with
H2DCFDA.
Different concentrations of H2O2 (0, 5, 10, 20, 25, 50, 100, 200, and 250μM) on the
fluorescence of H2DCFDA in BEAS 2B cells were measured. BEAS 2B cells without nickel
salt treatment but with DCFDA fluorescence were used as a negative control. The data is
expressed as the means± the standard error of the mean (SEM) of four independent
experiments. *p<0.05, **p<0.01, ***p<0.001 vs the control (0μM H2O2).
Total ROS measurement on insoluble Nickel treated cells
10T1/2 cells and BEAS 2B cells were each treated with insoluble Nickel (II)
compounds (Black NiO, Green NiO, and Nickel Subsulfide). The total ROS generated in
cells by each insoluble nickel (II) compound was measured after the nickel compounds were
removed from the medium. Day 1 was one day after the nickel compounds were removed.
After averaging two experiments, a high increase of H2DCFDA fluorescence was detected
four days (Day 4) after 0.5 μg/mL (Figure 12A) and 1.5 μg/mL (Figure 12C) of Black NiO
treatments were removed from BEAS 2B cells seeded on the wells. While 1 μg/mL of Black
NiO treated BEAS 2B cells showed relatively similar ROS measurements from Day 1 to Day
5 (Figure 12B), 2 μg/mL of Black NiO treated BEAS 2B cells showed increased ROS
measurement on Day 2 (Figure 12D). After the high increase of ROS, the following ROS
measurement showed a decrease (Figure 12).
The total ROS measurement in Black NiO treated BEAS 2B cells in dose-dependent
manner was graphed (Figure 13) using the data on Day 4 from two experiments. 1.5μg/mL of

Copyright 2023 32 Sheldy SoJeong Shin
Black NiO treated BEAS 2B cells had significantly higher fluorescence than the spontaneous
ROS from BEAS 2B cells (control).
Figure 12. Total ROS measurement as a function of the duration using different Black NiO
concentrations.
Two experiments were combined. Total ROS was measured using H2DCFDA after the
treatment was removed. Day 1 indicates one day after the nickel treatment was removed. Day
2 is two days after and so on. The control is the fluorescence as a function of arbitrary units
from cells with H2DCFDA only. The data is expressed as the means± the standard error of
the mean (SEM). (A) Total ROS measurement using H2DCFDA on BEAS 2B cells treated
with 0.5μg/mL of Black NiO. (B) Total ROS measurement using H2DCFDA on BEAS 2B
cells treated with 1μg/mL of Black NiO. (C) Total ROS measurement using H2DCFDA on
BEAS 2B cells treated with 1.5μg/mL of Black NiO. (D) Total ROS measurement using
H2DCFDA on BEAS 2B cells treated with 2μg/mL of Black NiO.
Figure 13. Total ROS measurement as a function of the concentration of Black NiO.
Two experiments were combined. Different concentrations of Black NiO (0.5, 1, 1.5, and 2
μg/mL) on the H2DCFDA fluorescence in BEAS 2B cells were measured. 0.05% Acetone
treated cells were used as 0 μg/mL Black NiO concentration. BEAS 2B cells without
treatment but with DCFDA fluorescence were used as a negative control. 50 μM of tert-butyl

Copyright 2023 33 Sheldy SoJeong Shin
hydroperoxide (TBHP) was used a positive control. The data is expressed as the means± the
standard error of the mean (SEM). *p<0.05, **p<0.01, ***p<0.001 vs the control (cells with
DCFDA only).
After averaging two experiments, a high increase of H2DCFDA fluorescence was
detected four days (Day 4) after 0.5μg/mL (Figure 14A), 1μg/mL (Figure 14B), and
1.5μg/mL (Figure 14C) of Green NiO treatments were removed from BEAS 2B cells seeded
on the well, while 2μg/mL of Green NiO treated BEAS 2B showed relatively similar ROS
concentrations from Day 1 to Day 5 (Figure 14D). After a high increase of ROS, the
following ROS measurements showed a decrease (Figure 14).
The total ROS measurement in Green NiO treated BEAS 2B cells in dose-dependent
manner was graphed (Figure 15) using the data on Day 4 from two experiments. All
concentrations of Green NiO treated BEAS 2B cells had significantly higher fluorescence
than the spontaneous ROS from BEAS 2B cells (control). In Green NiO treated BEAS 2B
cells, the total ROS measurement showed increases from 0.5μg/mL to 1μg/mL, then drops
from 1.5μg/mL to 2μg/mL.
Figure 14. Total ROS measurement as a function of the duration using different Green NiO
concentrations.
Two experiments were combined. Total ROS was measured using H2DCFDA after the
treatment was removed. Day 1 indicates one day after the nickel treatment was removed. Day
2 is two days after and so on. The control is the fluorescence as a function of arbitrary units
from cells with H2DCFDA only. The data is expressed as the means± the standard error of
the mean (SEM). (A) Total ROS measurement using H2DCFDA on BEAS 2B cells treated
with 0.5μg/mL of Green NiO. (B) Total ROS measurement using H2DCFDA on BEAS 2B
cells treated with 1μg/mL of Green NiO. (C) Total ROS measurement using H2DCFDA on
BEAS 2B cells treated with 1.5μg/mL of Green NiO. (D) Total ROS measurement using
H2DCFDA on BEAS 2B cells treated with 2μg/mL of Green NiO.

Copyright 2023 34 Sheldy SoJeong Shin
Figure 15. Total ROS measurement as a function of the concentration of Green NiO.
Two experiments were combined. Different concentrations of Green NiO (0.5, 1, 1.5, and 2
μg/mL) on the H2DCFDA fluorescence in BEAS 2B cells were measured. 0.05% Acetone
treated cells were used as 0 μg/mL Green NiO concentration. BEAS 2B cells without
treatment but with DCFDA fluorescence were used as a negative control. 50 μM of tert-butyl
hydroperoxide (TBHP) was used a positive control. The data is expressed as the means± the
standard error of the mean (SEM). *p<0.05, **p<0.01, ***p<0.001 vs the control (cells with
DCFDA only).
After averaging two experiments, a high increase of H2DCFDA was detected four
days (Day 4) after all concentration of Nickel Subsulfide (Ni3S2) treatments were removed
from BEAS 2B cells seeded on the well (Figure 16). After a high increase of ROS, the
following ROS measurements showed a decrease (Figure 16).
The total ROS generation in Nickel Subsulfide treated BEAS 2B cells as a function
of the concentrations at Ni3S2 treatments were next graphed (Figure 17) using the data on
Day 4 from two experiments. All concentrations of Ni3S2 treated BEAS 2B cells had
significantly higher ROS concentration than the spontaneous ROS from BEAS 2B cells
(control). There was a slight decrease in 1μg/mL of Ni3S2 treated BEAS 2B cells, likely due
to the cytotoxicity of Ni3S2.

Copyright 2023 35 Sheldy SoJeong Shin
Figure 16 Total ROS measurement as a function of the duration using different Nickel
Subsulfide concentrations.
Two experiments were combined. Total ROS was measured using H2DCFDA after the
treatment was removed. Day 1 indicates one day after the nickel treatment was removed. Day
2 is two days after and so on. The control is the fluorescence as a function of arbitrary units
from cells with H2DCFDA only. The data is expressed as the means± the standard error of
the mean (SEM). (A) Total ROS measurement using H2DCFDA on BEAS 2B cells treated
with 0.5μg/mL of Nickel Subsulfide. (B) Total ROS measurement using H2DCFDA on
BEAS 2B cells treated with 1μg/mL of Nickel Subsulfide. (C) Total ROS measurement using
H2DCFDA on BEAS 2B cells treated with 1.5μg/mL of Nickel Subsulfide. (D) Total ROS
measurement using H2DCFDA on BEAS 2B cells treated with 2μg/mL of Nickel Subsulfide.
Figure 17. Total ROS measurement as a function of the concentration of Nickel Subsulfide.

Copyright 2023 36 Sheldy SoJeong Shin
Two experiments were combined. Different concentrations of Nickel Subsulfide (0.5, 1, 1.5,
and 2 μg/mL) on the H2DCFDA fluorescence in BEAS 2B cells were measured. 0.05%
Acetone treated cells were used as 0 μg/mL Nickel Subsulfide concentration. BEAS 2B cells
without treatment but with DCFDA fluorescence were used as a negative control. 50 μM of
tert-butyl hydroperoxide (TBHP) was used a positive control. The data is expressed as the
means± the standard error of the mean (SEM). *p<0.05, **p<0.01, ***p<0.001 vs the control
(cells with DCFDA only).
On 10T1/2 cells, a high increase of H2DCFDA fluorescence was detected four days
(Day 4) after 1.5 μg/mL (Figure 18C) and 2 μg/mL (Figure 18D) of Black NiO treatments
were removed from 10T1/2 cells seeded on the wells. While 0.5 μg/mL of Black NiO treated
10T1/2 cells showed relatively similar ROS measurements from Day 1 to Day 4 (Figure
18A), 1 μg/mL of Black NiO treated 10T1/2 cells showed increased ROS measurement on
Day 3 (Figure 18B). After the high increase of ROS, the following ROS measurement
showed a decrease (Figure 18). For 10T1/2 cells, the fluorescence was measured only up to
Day 4.
The total ROS measurement in Black NiO treated 10T1/2 cells in dosedependent manner was graphed (Figure 19) using the data on Day 4. All concentrations of
Black NiO treated 10T1/2 cells had significantly higher fluorescence than the spontaneous
ROS from 10T1/2 cells (control).
Figure 18. Total ROS measurement as a function of the duration using different Black NiO
concentrations on 10T1/2.
Two experiments were combined. Total ROS was measured using H2DCFDA after the
treatment was removed. Day 1 indicates one day after the nickel treatment was removed. Day
2 is two days after and so on. The control is the fluorescence as a function of arbitrary units
from cells with H2DCFDA only. The data is expressed as the means± the standard error of

Copyright 2023 37 Sheldy SoJeong Shin
the mean (SEM). (A) Total ROS measurement using H2DCFDA on 10T1/2 cells treated with
0.5μg/mL of Black NiO. (B) Total ROS measurement using H2DCFDA on 10T1/2 cells
treated with 1μg/mL of Black NiO. (C) Total ROS measurement using H2DCFDA on 10T1/2
cells treated with 1.5μg/mL of Black NiO. (D) Total ROS measurement using H2DCFDA on
10T1/2 cells treated with 2μg/mL of Black NiO.
Figure 19. Total ROS measurement as a function of the concentration of Black NiO on
10T1/2.
Two experiments were combined. Different concentrations of Black NiO (0.5, 1, 1.5, and 2
μg/mL) on the H2DCFDA fluorescence in 10T1/2 cells were measured. 0.05% Acetone
treated cells were used as 0 μg/mL Black NiO concentration. 10T1/2 cells without treatment
but with DCFDA fluorescence were used as a negative control. 50 μM of tert-butyl
hydroperoxide (TBHP) was used a positive control. The data is expressed as the means± the
standard error of the mean (SEM). *p<0.05, **p<0.01, ***p<0.001 vs the control (cells with
DCFDA only).
A high increase of H2DCFDA fluorescence was detected two days (Day 2) after
0.5μg/mL (Figure 20A), 1μg/mL (Figure 20B), and 1.5μg/mL (Figure 20C) of Green NiO
treatments were removed from BEAS 2B cells seeded on the well, while 2μg/mL of Green
NiO treated 10T1/2 showed high increase of fluorescence on Day 4 (Figure 20D). After a
high increase of ROS, the following ROS measurements showed a decrease (Figure 20).
The total ROS measurement in Green NiO treated 10T12 cells in dose-dependent
manner was graphed (Figure 20) using the data on Day 2. 0.5 μg/mL and 1 μg/mL of Green
NiO treated 10T1/2 cells had significantly higher fluorescence than the spontaneous ROS
from 10T1/2 cells (control). In Green NiO treated 10T1/2 cells, the total ROS measurement
showed decreases as the concentration of Green NiO increases from 0.5 μg/mL to 2 μg/mL.

Copyright 2023 38 Sheldy SoJeong Shin
Figure 20. Total ROS measurement depending on the time duration using different Green
NiO concentration on 10T1/2.
Total ROS was measured using H2DCFDA after the treatment was removed. Day 1 indicates
one day after the nickel treatment was removed. Day 2 is two days after and so on. Control
is the fluorescence arbitrary unit from cells with H2DCFDA only. The data is expressed as the
means±SEM of four independent experiments. (A) Total ROS measurement using H2DCFDA
on 10T1/2 Cl8 treated with 0.5μg/mL of Green NiO. (B) Total ROS measurement using
H2DCFDA on 10T1/2 Cl8 treated with 1μg/mL of Green NiO. (C) Total ROS measurement
using H2DCFDA on 10T1/2 Cl8 treated with 1.5μg/mL of Green NiO. (D) Total ROS
measurement using H2DCFDA on 10T1/2 Cl8 treated with 2μg/mL of Green NiO.

Copyright 2023 39 Sheldy SoJeong Shin
Figure 21. Total ROS measurement as a function of the concentration of Green NiO on
10T1/2.
Two experiments were combined. Different concentrations of Green NiO (0.5, 1, 1.5, and 2
μg/mL) on the H2DCFDA fluorescence in 10T1/2 cells were measured. 0.05% Acetone
treated cells were used as 0 μg/mL Green NiO concentration. 10T1/2 cells without treatment
but with DCFDA fluorescence were used as a negative control. 50 μM of tert-butyl
hydroperoxide (TBHP) was used a positive control. The data is expressed as the means± the
standard error of the mean (SEM). *p<0.05, **p<0.01, ***p<0.001 vs the control (cells with
DCFDA only).

Copyright 2023 40 Sheldy SoJeong Shin
Discussion
From the experiment, our lab had found dose dependent cytotoxicity of insoluble
Nickel (II) compounds (Black NiO and Nickel Subsulfide) on C3H Cl8 10T1/2 mouse
embryo cells and BEAS 2B cells. As the concentration of insoluble nickel (II) compounds
increased, lower survival fraction was shown. By plotting the survival fraction (S) into Ln(S),
the slope (-k) shows the rate of cytotoxicity in dose-dependent manner. For Black NiO
treated 10T1/2 cells, the slope (-k) is -0.49 with linear regression (R2
) of 0.897 (Figure 5). For
Black NiO treated BEAS 2B cells, the slope (-k) is -0.181 with linear regression (R2
) of 0.917
(Figure 7). For Nickel Subsulfide (Ni3S2) treated 10T1/2 cells, the slope (-k) is -0.39 with
linear regression (R2
) of 0.875 (Figure 6). For Black NiO treated BEAS 2B cells, the slope (-
k) is -0.47 with linear regression (R2
) of 0.922 (Figure 8). Interestingly, total ROS
measurement using the fluorescence of H2DCFDA showed higher percent of control in 1.5
μg/mL of Nickel subsulfide treated BEAS 2B cells (296±31% on Day 4, Table 4e) than 1.5
μg/mL of Black NiO treated BEAS 2B cells (211±22% on Day 4, Table 4b). Unfortunately,
we couldn’t measure the fluorescence of Nickel subsulfide treated 10T1/2 cells, so no further
interpretation is possible. However, with future studies on Green NiO cytotoxicity and Nickel
subsulfide total ROS measurement will give more information.
After the highest increase of ROS, the following ROS measurement showed a
decrease (Figure 12, 14, 16, 18, and 20). One possible reason for the decreased ROS is
cytotoxicity of nickel compounds to 10T1/2 and BEAS 2B cells. With the time nickel
compounds induce more ROS, cells are killed due to oxidative stress. We could see an
increase of ROS measurement until it reaches the highest measurement. After the oxidative
stress, less cell number leads to decrease of ROS measurement after the highest ROS
measurement point. After the drop survived cells mitoses and increase cell number again
increasing ROS.

Copyright 2023 41 Sheldy SoJeong Shin
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Abstract (if available)

Abstract Nickel is the 24th most abundant transition metal in the Earth's crust. It is also an essential material in modern industry, used in stainless steel for electroplating, welding, alloy production, and more. Nickels are announced as occupational and environmental carcinogens. Nickel compounds are presented in two states- one in water-soluble states, such as Nickel dichloride (NiCl2), and the other in water-insoluble states, such as green Nickel oxide (NiO). Insoluble nickel (II) compounds are known to be more carcinogenic than soluble nickel (II) compounds. According to the International Agency for Research on Cancer, insoluble Nickel (II) compounds are known as group I carcinogens. In culture and morphological neoplastic cell transformation, nickel compounds cause chromatin damage in mouse and human cells. A small amount of data shows that nickel compounds generate reactive oxygen species. Reactive oxygen species include superoxide, hydrogen peroxide, and hydroxyl radicals. Here, we showed cytotoxicity of insoluble nickel (II) compounds (Black NiO and Nickel Subsulfide) and measured total reactive oxygen species (ROS) using H2DCFDA on insoluble nickel (II) compound treated C3H Cl8 10T1/2 embryonic mouse cells and BEAS 2B cells. Moreover, after discussing the results, we share further research to accomplish the role of Nickel-induced ROS in Nickel-induced cytotoxicity and morphological transformation.

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Creator Shin, Sheldy SoJeong (author)

Core Title The role of nickel-induced ROS in nickel (II)-induced cytotoxicity

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Degree Conferral Date 2023-12

Publication Date 11/20/2023

Defense Date 07/20/2023

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

Tag BEAS 2B cells,C3H Cl8 10T1/2 embryonic mouse cells,carcinogens,chromatin damage,cytotoxicity,group I carcinogens,H2DCFDA,hydrogen peroxide,hydroxyl radicals,insoluble nickel (II) compounds,morphological transformation,nickel,nickel compounds,nickel dichloride (NiCl2),nickel oxide (NiO),nickel subsulfide,nickel-induced cytotoxicity,OAI-PMH Harvest,reactive oxygen species (ROS),role of nickel-induced ROS,superoxide,transition metal,water-insoluble nickel compounds,water-soluble nickel compounds

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