francisco cytotoxic and genotoxic …...effects of cadmium in human osteoblasts cell line mg-63....
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Universidade de Aveiro
2011
Departamento de Biologia
FRANCISCO CARVALHO VIEIRA PINHO
CYTOTOXIC AND GENOTOXIC EFFECTS OF CADMIUM IN HUMAN OSTEOBLASTS EFEITOS CITOTÓXICOS E GENOTÓXICOS DO CÁDMIO EM OSTEOBLASTOS HUMANOS
Universidade de Aveiro
2011
Departamento de Biologia
FRANCISCO CARVALHO VIEIRA PINHO
CYTOTOXIC AND GENOTOXIC EFFECTS OF CADMIUM IN HUMAN OSTEOBLASTS EFEITOS CITOTÓXICOS E GENOTÓXICOS DO CÁDMIO EM OSTEOBLASTOS HUMANOS
Dissertação apresentada à Universidade de Aveiro para cumprimento dos requisitos necessários à obtenção do grau de Mestre em Biologia Aplicada – Ramo de Biologia Molecular e Celular, realizada sob a orientação científica da Doutora Helena Cristina Correia de Oliveira, Estagiária de Pós-Doutoramento do Departamento de Biologia da Universidade de Aveiro e sob co-orientação da Prof. Doutora Maria da Conceição Lopes Vieira dos Santos, Professora Associada com agregação do Departamento de Biologia da Universidade de Aveiro.
o júri president / presidente members / vogais
Prof. Doutor João António de Almeida Serôdio professor auxiliar do Departamento de Biologia da Universidade de Aveiro Prof. Doutor Francisco Manuel Pereira Peixoto (Arguente Principal) professor auxiliar com agregação, Departamento de Química, Universidade de Trás-os-Montes e Alto Douro Doutora Helena Cristina Correia de Oliveira (Orientadora) investigadora pós-doutoramento, CESAM - Centro de Estudos do Ambiente e do Mar, Universidade de Aveiro Prof. Doutora Maria Da Conceição Lopes Vieira dos Santos (Co-Orientadora), professora associada com agregação, Departamento de Biologia da Universidade de Aveiro
agradecimentos A realização da Dissertação na Universidade de Aveiro marca uma etapa especial da minha vida. Todas as vivências nesta cidade, as pessoas que conheci e todo o convívio que daí adveio, marcaram esta fase de crescimento pessoal e académico-profissional. Por este motivo, começo por agradecer especialmente à Prof. Doutora Maria da Conceição Lopes Vieira dos Santos, por me ter recebido no seu laboratório. Agradeço-lhe ter-me proporcionado bons momentos de discussão de ideias, bons momentos de inspiração e bons momentos sobre algumas lições de vida. E ainda, como co-orientadora agradecer-lhe-ei sempre todo o apoio prestado e por me ter colocado sob a alçada da Doutora Helena Cristina Correia de Oliveira que orientou o trabalho desenvolvido. Deste modo, à Doutora Helena, agradeço veementemente, em particular por toda a sua paciência e todo o apoio ao longo do meu percurso. Assim, é com sinceridade que agradeço tudo que aprendi até agora a trabalhar nesta equipa. No laboratório tive oportunidade de fazer bons amigos e descobrir companheiros. Desta forma tenho que agradecer ao Ricardo Costa (a quem desejo os meus sinceros votos de sucesso e felicidade e a quem espero voltar a ver no laboratório a fazer trypan blue e muito mais), ao Tiago Pedrosa e ao Pedro Pinto por toda a amizade e camaradagem ao longo da saga da Dissertação nos bons e maus momentos. A todos os colegas, não posso deixar de agradecer o seu simpático acolhimento e assistência prestada em diversas ocasiões. Quero ainda agradecer a uma amiga especial e de longa data, Mariana Santos Moreda Graça, pela sua amizade e ajuda, desde sempre e, no apoio à chegada a esta cidade. Não esqueço também, a Mónica Ferreira e o Eurico Pedrosa, pela sua boa amizade e agradáveis momentos passados juntos. Em especial, a uma pessoa bela e excecional, tanto na sua forma de ser e de estar, como pela sua inteligência e companheirismo, mas também, pelo seu rigor e competência académico-profissional, agradeço assim, à Maria Cristina Monteiro. Finalmente, dedico esta Dissertação a uma pessoa muito importante. Por todo o apoio incondicional, sem a qual nada teria sido possível, expresso uma enorme gratidão. Por isto e por muito mais na minha vida, à minha Mãe o meu eterno “muito obrigado”.
“Tudo o que o homem não conhece não existe para ele. Por isso o mundo tem, para cada um, o tamanho que abrange o seu conhecimento."
Carlos Bernardo González Pecotche
keywords
Cadmium, MG-63 cell line, cell proliferation / viability, cell cycle, DNA damage, flow cytometry, comet assay
abstract
Due to industrialization, cadmium has been increasingly accumulated in soil, water and air, and consequently the food chain, thus, being responsible for many diseases. In humans, damages to several organs and carcinogenic effects take place. However, the mechanisms underlying the bone diseases remain unknown, and so, this work aims to evaluate cytotoxic and genotoxic effects of cadmium in human osteoblasts cell line MG-63. Cells were exposed to 0 µM, 20 µM and 50 µM CdCl2 for 24 and 48 hours. Cell proliferation / viability was determined by the MTT assay, cell cycle effects were evaluated by flow cytometry, and DNA damage was assessed by the comet assay. After both times of exposure, cell viability decreased in both cadmium doses, although cell cycle progression alterations were not detected. However, cadmium lead to clastogenic effects and DNA damage in cells exposed to the cadmium dose of 50 µM, for 48 h. In conclusion, at 20 µM and 50 µM and for the periods tested cadmium chloride induced cytotoxic and genotoxic effects on MG-63 cell line, as it decreased cell viability, induced DNA damage and clastogenicity, though it did not change cell cycle progression.
palavras-chave
Cádmio, linha celular MG-63, proliferação / viabilidade celular, ciclo celular, danos no ADN, citometria de fluxo, ensaio de cometas
resumo
Devido à industrialização, a contaminação ambiental por metais como o cádmio tem aumentado no solo, água e ar. Consequentemente, a cadeia alimentar é afetada e, desta forma, o cádmio surge como agente carcinogénico e como causador de algumas doenças relacionadas com lesões em vários órgãos. Contudo, os mecanismos subjacentes a doenças ósseas ainda não se encontram totalmente desvendados, e assim neste trabalho, pretende-se avaliar os efeitos citotóxicos e genotóxicos do cádmio em osteoblastos humanos, na linha celular MG-63. As células foram expostas a 0 µM, 20 µM e 50 µM de cloreto de cádmio durante 24 e 48 horas. A proliferação / viabilidade celular foi avaliada pelo ensaio MTT, os efeitos na progressão do ciclo celular por foram avaliados por citometria de fluxo e os danos no DNA pelo ensaio de cometas. Após ambos os tempos de exposição a 20 µM e 50 µM de cloreto de cádmio, as células sofreram uma diminuição da viabilidade celular e não foram observadas alterações na progressão do ciclo celular. No entanto, o cádmio conduziu a efeitos clastogénicos e danos no DNA em células expostas à concentração de 50 µM, após 48 h de exposição. Concluindo, as concentrações 20 µM e 50 µM de cloreto de cádmio para os períodos testados, induziram efeitos citotóxicos e genotóxicos nas células da linha MG-63, dado que conduziram a uma diminuição da sua viabilidade, danos no DNA e clastogenicidade, não havendo, contudo, alterações na progressão do ciclo celular.
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Index
1. Introduction .................................................................................................................................... 1
1.1. Metals ...................................................................................................................................... 1
1.1.1. Essential and non-essential metals ................................................................................... 1
1.1.2. “Heavy metals – a meaningless term?” ............................................................................ 1
1.2. Cadmium: an environmental contaminant .............................................................................. 2
1.3. Cadmium exposure and human diseases ................................................................................. 2
1.4. Cadmium, cell disturbances and cancer .................................................................................. 4
1.4.1. Oxidative stress ................................................................................................................ 4
1.4.2. Oxidative stress and cadmium induced carcinogenicity .................................................. 5
1.4.3. Molecular mechanisms ..................................................................................................... 7
1.5. Toxicity assessment................................................................................................................. 8
1.5.1. Cytotoxicity ...................................................................................................................... 8
1.5.2. Genotoxicity ..................................................................................................................... 8
1.5.3. In vitro assays and the 3Rs ............................................................................................... 9
1.5.4. MG-63 cell line ................................................................................................................ 9
1.5.5. Biomarkers ..................................................................................................................... 10
1.5.5.1. Cell proliferation / viability ..................................................................................... 10
1.5.5.2. Cell cycle analysis ................................................................................................... 10
1.5.5.3. DNA damage ............................................................................................................ 14
1.6. Aims of Dissertation.............................................................................................................. 16
2. Materials and Methods ................................................................................................................. 17
2.1. MG-63 osteoblast cell line culture ........................................................................................ 17
2.2. Cell recovery / thawing ......................................................................................................... 17
2.3. Trypsinization ....................................................................................................................... 18
2.4. Cadmium Exposure ............................................................................................................... 18
2.5. Confluence, cell morphology and contaminations ................................................................ 19
2.6. Cell proliferation and viability– MTT Assay ........................................................................ 19
2.7. Cell cycle analysis – Flow Cytometry ................................................................................... 20
2.8. DNA damage – Comet Assay ............................................................................................... 21
2.9. Statistical Analysis ................................................................................................................ 22
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3. Results .......................................................................................................................................... 23
3.1. Confluence and morphology ................................................................................................. 23
3.2. Proliferation / viability assessment – MTT Assay ................................................................ 24
3.3. Cell cycle analysis – Flow Cytometry ................................................................................... 25
3.4. DNA damage – Comet assay ................................................................................................. 28
4. Discussion .................................................................................................................................... 30
5. Conclusions and future perspectives ............................................................................................ 34
6. References .................................................................................................................................... 35
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1. Introduction
1.1. Metals
1.1.1. Essential and non-essential metals
Life itself requires metals as nutrients, which despite needed at trace levels are
essential for maintaining biological functions. These elements are present in the organisms
and are often in soluble forms, or generally associated with proteins that require their
presence to be fully functional. However, humans have also been increasingly exposed to
these essential and other non essential metals, as cadmium, since the industrial
revolution (1).
1.1.2. “Heavy metals – a meaningless term?”
In 2002, the International Union Of Pure And Applied Chemistry – IUPAC –
released a Technical Report prepared for publication by John H. Duffus (2). On the subject
matter it presented a review concluding that the use of terms such as “heavy metals”
should be kept aside because there is no terminological or scientific basis for its use. It is
also argued that a novel classification is needed. One based on their chemical properties to
allow prediction and interpretation of biochemical basis for toxicity in a rational way. As
an example, there is the known Lewis acid properties, that for their affinity for phosphate
groups and nonoxygen centers in membranes, allows the prediction that borderline and
Class B ions similar in size to calcium (II) ions are likely to cause harmful membrane
structural changes. This way, toxicity testing could be minimized for metals and their
compounds since chemical biological knowledge will allow several toxic effects to be
predicted (2).
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1.2. Cadmium: an environmental contaminant
Due to anthropogenic activities, metals have widely become environmental
pollutants and cadmium is no exception, by being utilized in the manufacture of plastic,
batteries, pigments and alloys, solders and electroplating (3,4). Soil, water and air have
been affected and, consequently, the food chain. Therefore, cadmium was designated as
“priority substance” by the European Comission (5) and by the Environmental Protection
Agency (6). This metal has been classified as a human carcinogen (group 1) by the
International Agency for Research on Cancer (7).
1.3. Cadmium exposure and human diseases
Cadmium exposure can be classified as chronic or acute. Acute exposure mainly
occurs as occupational hazards (inhaled smoke and dust, or ingestion). On the other hand,
chronic exposure occurs, in general, by air, water and by food. One of the most frequent
forms of intoxication derives from soil contamination of tobacco plant fields, mostly due to
cadmium absorption by the tobacco plant (8). Cadmium concentrations in leaves can vary
between 0 and 6.78 µg/g and each cigarette may contain 1 to 2 µg of cadmium in dry
weight. About 40% to 60% of cadmium present on inhaled smoke reaches systemic
circulation through pulmonary epithelium (8).
This way cadmium finally accumulates in the human body giving rise to health
problems, as it is not biodegradable and has a long biological half-life (9). Damage to
kidneys, liver and lungs have been associated with cadmium exposure as well as cancer
and bone disease (3,10,11). The public awareness and concern about cadmium toxicity
increased, when an outbreak in Japan reported people suffering from renal dysfunction and
osteomalacia after being exposed. People living by Jinzu river basin (see Fig.1 A, B) were
affected because of the water and rice fields industrial dumps contamination (11). This
condition designated by Itai-Itai disease “ouch-ouch” (see Fig.1 C) is a hurting result of
chronic cadmium intoxication (12).
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Fig.1 – Jinzu river basin (A, B). Patient suffering from long-term exposure to cadmium (C), adapted (13).
Besides its toxicity per se, cadmium may also have other indirect effects: for
example, it may accumulate in liver leading mostly to acute damages; also its accumulation
in kidneys and intestines results in organ malfunction eventually having negative impacts
in bone homeostasis (14,15). The accumulation of cadmium in the reproductive organs /
system and its effects in fertility are also well documented (4). Some of the main effects of
cadmium in the human body are briefly summarized in Fig.2 (16).
Fig.2 – Cadmium exposure effects in the human body, adapted (16).
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1.4. Cadmium, cell disturbances and cancer
Among the several diseases that cadmium may induce, its carcinogenic potential is
described (17). However, still remains unclear the pathways in which cadmium may
disturb cell function in order to induce carcinogenicity. Apparently this may involve direct
or indirect mechanisms (18).
In the indirect mechanisms, cadmium induction of reactive oxygen species (ROS)
in the cell, leading to oxidative stress may be one of the most important. For example, it is
described that cadmium may lead to the depletion of the antioxidant defense activity like
glutathione, and inhibit several DNA repair mechanisms (19).
1.4.1. Oxidative stress
There are three ways in which oxidative stress is imposed in cells: rise of oxidant
species generation, antioxidant defense decrease and / or stoppage / decrease of oxidative
damage repair. ROS contemplate either free radicals, reactive anions (or other molecules)
containing oxygen atoms, or other species that can produce free radicals or can be
chemically activated by them. For instance, there is the hydroxyl radical, superoxide,
hydrogen peroxide, and peroxynitrite. Aerobic respiration is the in vivo main source of
ROS. However, several other processes lead to their production, such as peroxisomal-
oxidation of fatty acids, microsomal cytochrome P450 metabolism of xenobiotic
compounds, stimulation of phagocytosis by pathogens or lipopolysaccharides, arginine
metabolism, or even tissue specific enzymes (20). The major injuries to cells come from
ROS-induced modifications of macromolecules like polyunsaturated fatty acids in
membrane lipids, proteins and DNA, which, in turn, under homeostatic conditions, are
prevented by a battery of antioxidative mechanisms. In particular, these mechanisms relay
on the activities of enzymes such as superoxide dismutase (SOD), catalase (CAT) and
glutathione reductase (GR), or peroxidase (POX) that metabolize them, therefore liberating
the cell from their effects. Other conditions such as Alzheimer´s disease, Parkinson’s
disease, cancer and aging have also been linked to oxidative stress (20–22). Fig.3
illustrates the general mechanisms involved in oxidative stress in the cell.
Pedrosa et al. (23) demonstrated that cadmium may affect some of the pathways evidenced
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in this figure, namely by stimulating SOD, CAT, and inhibiting GR in the oxidative stress
pathway.
Fig.3 – Brief description of main mechanisms involved in oxidative stress in the cell. Green and red boxes
highlights, correspond to the pathways demonstrated to be affected by cadmium in MG-63 cell line (23).
ROS – reactive oxigen species, SOD – superoxide dismutase, CAT – catalase, APX – ascorbate peroxidase,
Asa – ascorbic acid, MDHA – monodehydroascorbate, MDHAR – monodehydroascorbate reductase, DHA –
dehydroascorbate, DHAR – dehydroascorbate reductase, GSH – reduced glutathione, GSSG – oxidised
glutathione, NAD(P)H – reduced nicotinamide adenine dinucleotide (phosphate), NAD(P)+ – oxidized
nicotinamide adenine dinucleotide (phosphate), GR – glutathione reductase, GPX – glutathione peroxidase.
1.4.2. Oxidative stress and cadmium induced carcinogenicity
The general model of oxidative stress network regulation was pointed out in the
latter topic. However, cadmium implication in oxidative stress is quite a challenge yet.
Cadmium has been demonstrated by several studies to cause cancer (7). The effects of
cadmium exposure have been under study for a long time and there is an increasing data
outlining the concern in the global impact of its toxicity in organs and at the cell level, both
in vivo and in vitro, especially its carcinogenicity. Currently, a main focus relies on the
conditions of toxicity at low levels of exposure. Cadmium interacts differently with cells
according to the doses of exposure and according to the cell type / organ resistance. Most
important, cadmium exerts its effects by several ways, in particular by multiple
mechanisms of genotoxicity (all leading to impairments and ultimately cancer). As
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considered by Hartwig (18), the most promising hypothesis for cadmium induced
genotoxicity involves indirect mechanisms. There is an increased formation of ROS,
interactions with the DNA damage response system (DNA repair processes, cell cycle
control and apoptosis), and epigenetic changes like alterations in DNA methylation
patterns that cause genomic instability.
Under physiological conditions cadmium does not participate in redox reactions,
but oxidative stress related conditions are importantly linked to its carcinogenicity,
essentially at later steps, as the increased levels of ROS have been connected to tumor
formation due to mitotic stimuli and redox-sensitive transcription factors activation. This
process can be interpreted as an inhibitory effect by cadmium, of the antioxidant enzymes
(18). Although the role of ROS can be reversed by the cell its continuous elevated levels
may then lead to mutagenesis (24,25). This way, cell cycle deregulation may start to occur
depending on dose and time of exposure (18).
Other way of cadmium to exert its carcinogenic effect occurs by compromising
almost all main DNA repair pathways: Nucleotide Excision Repair – NER, Base Excision
Repair – BER, or Mismatch repair – MMR. The inhibition effects observed in the enzymes
of antioxidative DNA defenses were also observed in vivo, when rat testes were analyzed
for cadmium carcinogenicity. The levels of oxidized bases were increasing along with 8-
oxo-dGTPase activity decreased, thus showing that oxidative DNA damage lesions may be
in part a result of repair inhibition (26).
Besides the interference with DNA repair systems, cadmium also impairs the
function of p53 – tumor suppressor protein – consequently interfering with cell cycle
maintenance response. For instance, prostate epithelial cells have been shown to acquire
apoptosis resistance, which in turn, opens the possibility of cells to bypass extinction and
replicate with damaged DNA such as relevant mutations in carcinogenicity role (27).
In summary, the impact of cadmium on the stress response genes relies majorly in
those involved in the synthesis of metallothionein, heat-shock proteins, synthesis and
homeostasis of glutathione and oxidative stress response (3,19).
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1.4.3. Molecular mechanisms
The most damaging cadmium form appears to be Cd2+
. Although, investigations
conducted by Schwerdtle et al. (28) and supported by past studies (29) showed that both
particulate and soluble forms of cadmium cause relatively the same damage, including the
conformational alterations of p53 induced by cadmium.
DNA repair inhibitions, cell proliferation and cell cycle control have been observed
to be affected at low doses (18,30). This raises two points of concern: one related with the
impact of new technologies’ nanoparticles / compounds as new forms of exposure, such as
solar panels, for instance. The other one relies in the fact of why damage response
pathways are so sensitive to cadmium ions. It is known that cadmium interacts with
essential metals such as calcium and zinc. Calcium (Ca2+
) is easily replaced by Cd2+
. Zinc
(Zn2+
), on its hand, can be also replaced by Cd2+
in many enzymes and transcription
factors, being zinc-fingers an example of the high affinity of cadmium towards (thiol) SH
groups. Zinc finger proteins consist of four residues of histidine and / or cysteine to form a
domain mainly for DNA binding and also for interactions between proteins, DNA repair
and cell cycle control proteins. For instance, p53 has a zinc binding structure in its DNA
binding domain, which has a key role in its tumor suppressor job. Cadmium-induced
protein inactivation is reversed by the excess of zinc which points out to a displacement
mechanism (18,30).
Moreover, calcium can be displaced by cadmium in E-cadherin, a protein that is
responsible for cell to cell adhesion and communication. Consequently, the ability to
communicate between cells is disrupted and cell proliferation is impaired (3).
Lastly, there is some information about cadmium-transformed cells that acquired
resistance to apoptosis linked to an increase in the anti-apoptotic action of Bcl-2 altering
the pathway of JNK signal transduction pathway (27).
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1.5. Toxicity assessment
1.5.1. Cytotoxicity
The cytotoxicity term refers to the adverse effects resulting from the interaction
between an external toxic agent and cell processes essential to survival, proliferation or
normal cell maintenance (31).
Nowadays, it is hard to monitor physiological systems in vivo. This way, the
majority of trials determines effects at the cellular level (32). The development of in vitro
cytotoxicity assays has been driven by the necessity of rapidly evaluate toxicity potential
of a wide variety of substances or compounds (limiting animal experimentation – see the
principle of 3R’s into the chapter “In vitro assays and the 3Rs”).
Cytotoxicity assays evaluate drug induced alterations in metabolic pathways or
structural integrity, that may or not be related to cell death (33). The most common and fast
in vitro toxicological screening assays are the lactate dehydrogenase (LDH), neutral red (3-
amino-7-dimethylamino-2-metilphenazine hydrochloride), trypan blue and MTT (3-(4,5-
Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (34). These assays have been
optimized for the use of microplate readers allowing for a rapid throughput (35).
1.5.2. Genotoxicity
It is important to distinguish between two concepts related to in vitro assay analysis
about genotoxicity prediction. Mutagenicity concerns what is capable of leading to
permanent transmittable changes in the amount or structure of the genetic material in cells
or organisms. These changes can entail a single gene (point mutations), a block of genes or
entire chromosomes (structural or numerical chromosome aberrations). Wherein,
genotoxicity is a broader term and refers to processes that alter the structure, information
content or segregation of DNA and which are not necessarily associated with mutagenicity.
Processes that involve unscheduled DNA synthesis, sister chromatid exchange, DNA
strand breaks, DNA adduct formation, and mitotic recombination. Therefore, genotoxicity
testing is important to investigate because it can give rise to cancer development allowing
to address potential substances effects (36).
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1.5.3. In vitro assays and the 3Rs
In toxicological research there has been the effort and worry of replacing animal
laboratorial research by other strategies. Therefore, in vitro cell culture is the first and
primordial step in toxicological screen. In 1959, William Russell and Rex Burch (37)
mentioned for the first time in their book “The Principles of Humane Experimental
Technique”, the principle of 3Rs. This principle upholds methodologies towards
Reducement, Refinement and Replacement. Thus, it is necessary the development,
validation and perfectioning of alternative tests to animal experimentation. Following and
respecting this line of thought, the in vitro model was chosen to perform the study,
eventually leaving the animal experiments for later stages.
1.5.4. MG-63 cell line
The model of study relied on MG-63 cell line. MG-63 cell line was gently given by
Instituto Nacional de Engenharia Biomédica – INEB. This cell line is derived from an
osteosarcoma, a malignant bone tumor of a 14 years old caucasian male. It presents a
fibroblast morphology, being moderately differentiated and non-mineralizing in vitro
(38,39). Therefore, it is many times designated as “osteoblast-like” since it maintains
normal osteoblasts behavior such as growing in an adherent monolayer and expressing
alkaline phosphatase and osteocalcin immunocytochemical markers (40,41). MG-63 is a
continuous cell line, which benefits the workers with indefinite growth potential for a long
period of time, contrary to a primary cell line that has a finite number of replications
genetically programmed (32).
This and other osteosarcoma cell lines have been used for a variety of studies
ranging from nanoparticles, biomaterials study and toxicity assays. Cell lines like Saos-2
and U-2 OS are two of the most utilized, but MG-63 cell line is getting more and more
attention because, besides the advantages reported above, comparatively with the others
remains less investigated. Many studies have, together or separately, resorted to
proliferation assays, proteomics and genomics (42–44), immunocytochemistry (41), and so
on. However, in order to contribute for new insights about cadmium toxicity in this cell
line, until now there are no studies resorting to flow cytometry (FCM) for cell cycle
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assessment along MTT for proliferation assessment, combined with the comet assay -
DNA damage assessment.
Thus, this was a major reason for the application of FCM in the study of cadmium
effects in MG-63 cell line.
1.5.5. Biomarkers
1.5.5.1. Cell proliferation / viability
The utilization of the MTT assay described by Mosmann (45) has been widely used
as a colorimetric approach based on the activity of mitochondrial dehydrogenase enzymes
in cells. The resulting intracellular purple formazan can be solubilized and quantified by
spectrophotometric means, being widely accepted as a reliable way to examine cell
proliferation (45,46). The number of assay steps has been minimized to such an extent to
fulfill the necessity to process large numbers of samples (45–47). The MTT reagent yields
low background absorbance values in the absence of cells and for each cell type the linear
relationship between cell number and signal produced is established, thus allowing an
accurate quantification of changes in the rate of cell proliferation (45).
1.5.5.2. Cell cycle analysis
Cell cycle analysis and proliferation kinetics are two major factors in toxicological
studies and tumor progression evaluation. Due to its importance, flow cytometry brought
the possibility of performing measurements with speed and precision revolutionizing cell
biology and cell cycle studies. Cell cycle is the biological phenomenon where
undifferentiated cells originate daughter cells with same genetic characteristics. Cell cycle
involves interphase and mitosis, Fig.4. Cell division occurs in mitosis (M) separated by the
next division by interphase. Chromosome duplication (replication) occurs during a specific
period of interphase. Therefore, it was divided in three parts: the period of DNA synthesis
is denominated S phase, G1 phase occurs right before S phase and the G2 is defined as the
period between S phase and the next mitosis.
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Fig.4 – Cell cycle schematic representation, adapted (48)
The first cell cycle phase is G1 where cellular growth and protein synthesis take
place, reaching approximately 6 to 12 hours. Cells double size and mass due to the
expression of genes coding for their particular phenotype. The S phase is the next cell
cycle phase, characterized by synthesis or replication of DNA. Therefore, the nuclei
contain twice the amount of initial DNA and proteins. It has the duration of 6 to 8 hours.
The third cell cycle phase, G2 is the growth phase. It’s marked by the continuing of protein
synthesis. Lasting about 3 or 4 hours, it ends when chromatin begins to condensate at the
beginning of mitosis. In mitosis, nuclear and cytoplasmatic division occurs – citokinesis,
originating two daughter cells. There is also a stage, G0, defined as a prolonged G1
observed in cells with long periods of life rarely dividing (quiescent). However, after
adequate stimulus those cells can complete one or two complete division cycles (49–51).
To uphold the normal progression of the cell cycle and the surveillance of genomic
integrity, cells have developed a complex network of DNA repair pathways and cell cycle
checkpoints. There are cell cycle checkpoints at G1-S, at G2-M, and at the exit of mitosis.
And there is also an S-phase checkpoint that detects and responds to DNA damage and
replication stress. The checkpoints lie in biochemical signaling pathways to sense various
types of structural defects in DNA, or in chromosome function, and provoke a versatile
cellular response that activates DNA repair and delays cell-cycle progression. These
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mechanisms evolved to manage inherent errors and genotoxic stress (cellular metabolites
or xenobiotics). Hence, when DNA damage is severe, checkpoints purge such potentially
hazardous cells by cell death or induce cell-cycle arrest, to essentially offer the time
necessary to repair the damage of the DNA before cell division (52–54).
Flow cytometry
Flow cytometry was originally developed in the late 50’s of the past century for
blood cell counting. Hematology and immunology were two of the biology fields that
boosted this technology development. However, with technical development and new
fluorescent markers, the utilization of this instrumentation has been generalized among
different fields such as microbial and plant cell (55).
A flow cytometer is a system composed of 5 elements (Fig.5): light source
(mercury lamp or laser), flow chamber, optical filters for wavelength specific interval,
photodiodes or photomultipliers for sensitive detection and signal processing, and finally,
one central processing unit (56).
Fig.5 – Flow Cytometer schematics, adapted (56)
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Cell suspension is injected in the flow chamber where each cell is run through a
light beam, perpendicular to the flow. Individual cell passage is obtained by hydrodynamic
focusing of sample flow injected through a saline solution (sheath fluid) which also runs
through the flow chamber (see Fig.6). The different speeds of the fluids generate the
laminar flow. The flow speed of the sheath fluid is higher than the sample allowing the
manipulation of speed and thickness of sample solution. This way, up to 10000 cells
(events) per second can be detected. By intercepting the cell the excitation light beam
suffers a forward scatter and a side scatter. Its detection is directly made by photodiodes or
can be deviated by 90º lens, dicroic mirrors, optic filters or focused on photomultipliers.
The conjunction of the two types of light reveal information regarding cell dimension,
granularity / complexity and morphology (56).
Fig.6 – Schematic view of a flow chamber, adapted (56)
The most modern cytometers have up to 16 simultaneous detectors (fluorescence
and scattered light), allowing the analysis of a variety of cellular characteristics of elevated
number of cells individually – multiparametric analysis (56).
In brief, to perform flow cytometry it is necessary to prepare cell suspensions.
Imunofluorescent dyes or antibodies are added to the suspension. Thereafter, the mixture is
aspirated to the flow. Light is scattered or absorbed, which when in an appropriate wave
length, is reemitted as fluorescence signal if cell presents one or more fluorochrome /
antibody-fluorochrome. In turn, light scattering depends on internal cell structure, size and
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shape. Fluorescent dyes absorb light in one wave length reemitting on different
wavelength. Finally, light or fluorescent signals are detected by photodiodes and
photomultiplier tubes and aftermost amplified. Optic filters are essential to block unwanted
light, allowing that only the light with desired wave length reaches the photodetector (57).
1.5.5.3. DNA damage
The comet assay or single cell gel electrophoresis (SCGE) is widely used for visual
detection of DNA damage at the single cell level (58). It combines the simplicity of
biochemical detection techniques for DNA strand breaks, alkali-labile sites and cross-
linking sites, with the single cell level approach typical of cytogenetics.
DNA damage is assessed by visual scoring of the nucleoids with a head and a tail,
resembling a comet (by qualitative classification in five categories, based on the tail length
and shape), or using software based measurements that recognize the extension of damage.
Size, shape and DNA quantity inside the comet play a key role on the assessment of level
damage. Software analysis provide a series of different parameters, being the most relevant
the % tail DNA, tail length, giving information on the amount and size of fragmented
DNA, and tail moment, which is the result of their mathematical product (58).
DNA damage and defective DNA repair are the molecular events underlying the
cancer initiation and progress. Therefore, it’s not surprising that many studies have been
used comet assay to assess DNA damage and repair characteristics in a wide variety of
tumor cells in response to different DNA damaging agents. These studies involve
established human cell lines as well as tumor cells extracted from cancer patients (59).
Comet assay main advantages rely on the fact of collecting data cell by cell, small
number of cells are needed per sample, DNA damage detection sensibility, use of any
eukaryote single cell population both in vitro and in vivo (60). Despite great advantages,
some inconvenients are also associated with this method. Theoretically, comet assay can be
used with any type of cell line, however, data obtained are often inconsistent.
There are also reported studies about comet assay in vivo, (61,62), however, there
are not appropriate controls available to this approach.
Comet assay is not capable of detecting DNA fragments smaller than 50 kb, since
those fragments are washed away during lysis or electrophoresis. It also fails when it
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comes to detecting mitochondrial DNA damage since it is also very small. Apoptotic cells
can also be lost during lysis or electrophoresis (63).
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1.6. Aims of Dissertation
Studies are still rare in bone cells despite cadmium accumulation in bone tissues
and their full mechanism is still poorly understood. Applying flow cytometry
multiparametric analysis to the evaluation of osteoblast cell lines is not still widely
practiced for this matter. Therefore, taken together, the viability assessment combined with
the comet assay and flow cytometry employment, the work aims to:
- Assess cell viability
- Evaluate cell cycle
- Detect cytostatic effects
- Detect clastogenic effects
- Detect DNA damage
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2. Materials and Methods
2.1. MG-63 osteoblast cell line culture
Human osteoblast cell line MG-63 was kindly provided by Instituto Nacional de
Engenharia Biomédica – INEB, University of Porto, Portugal. It was in vitro cultured in a
controlled humid atmosphere of 5% CO2 at 37 ºC. Cells were manipulated under aseptic
conditions that were ensured by working on a laminar flow biological safety cabinet and
always with sterile disposables. Cells were cultured in 75 cm2 (Corning®) culture flasks
containing a final complete growth media volume of 10 mL.
The complete growth media was composed of α-MEM – Minimum Essential
Medium (Gibco), supplemented with 10% (v/v) fetal bovine serum (Gibco) and with
10000 units mL-1
penicillin-streptomycin (Gibco) and 2.5 µg mL-1
fungizone (Gibco). The
subculture routine was performed every three days: period of time cells required to reach
approximately 80% confluence – defined as the percentage of cell coverage on the surface
area of a culture vessel.
The above mentioned conditions were provided to the subcultured cells for
studying the following parameters:
Cell viability / proliferation, by the MTT reduction assay;
Cell cycle evaluation and clastogenicity, by flow cytometry;
DNA damage, by the comet assay (single cell gel electrophoresis).
Subsequent to the exposure to different concentrations of cadmium chloride (0, 20
and 50 µM) cells were harvested after 24 and 48 hours.
2.2. Cell recovery / thawing
To initiate cell culture routine, cell thawing was conducted very carefully: cells
were placed under 37 ºC in a water bath until almost 2/3 of the vial content become liquid.
Dimethyl sulfoxide (DMSO, Sigma), a preservative used in cell freezing, is cytotoxic at
room temperature, so the procedure should be done rapidly in order to minimize exposure
to DMSO at room temperature. Cells are then transferred to 5 mL of complete growth
medium very slowly, drop by drop to avoid osmotic shock. Then, cells were centrifuged at
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approximately 300 g and resuspended in fresh complete growth medium and incubated in a
culture flask. Then subculture routine would be started.
2.3. Trypsinization
A critical step in adherent cell population is trypsinization. This step involves the
addition of a proteolytic enzyme which digests cell-to-cell adhesions and also dissociates
cells from the culture vessel – culture flask or dish, Petri style.
Cells were initially observed on the inverted microscope (Leica, Germany) to verify
their status: confluence, morphology and presence of contaminants. Then, cells were
washed with sterile phosphate buffer saline (PBS) pH 7.2 (Gibco) after previously
removing culture medium. Next, trypsin was added to the culture flask in the form of
trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA 0.25% trypsin and 1mM EDTA;
from Gibco) and incubated during 5 minutes at 37 ºC. By that time, the flask was taken
under microscope observation to verify cell disaggregation. Right next, trypsin inactivation
was performed with the complete growth medium (2x the trypsin volume added to cells).
To the new culture flaks properly identified with cell passage, cell type, username and
date, complete growth culture medium following cell suspension were added to a
proportion of 1:9 – cell suspension:complete growth medium.
The cell concentration (cell density) of the newly formed cell suspension was
determined by performing a cell count on the improved Neubauer chamber under a light
microscope Nikon Eclipse 80i (Japan). To a 75 cm2 culture flask the usual procedure
consisted of 1 mL of cell suspension (1.0 x 106 cells/mL) plus 9 mL of complete growth
medium, totalizing 10 mL on this flask.
2.4. Cadmium Exposure
According to the parameter about to study cells were subcultured in multiwell
plates, incubated for 24 h in the normal culture conditions prior to cadmium exposure of 24
h and 48 h. Exposure procedure consisted of normal growth medium replacement by new
complete medium with the desired final dilutions of cadmium chloride – 0, 20, 50 µM.
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2.5. Confluence, cell morphology and contaminations
On the routinely subculturing procedure and during exposure to cadmium (e.g.
every 24-48h) cells were screened under an inverted microscope (Leica, Germany) at 40x
or 100x magnification. This way, cells were observed to quantify / qualitatively describe
parameters that are indicative of the cell functional and morphological status: confluence
(%), morphology (e.g. “fibroblast” like, dividing cells) and presence / absence of
contaminants.
2.6. Cell proliferation and viability– MTT Assay
The MTT assay was performed according to Lévesque et al. (64) with a slight
modification, and 96-well plates were used. For each time of exposure, 24 h and 48 h, 100
µL of suspended cells were added to wells (see Fig.7) 24 h prior to exposure for adhesion
and confluence achievement of 50-60 %. After the first 24 h, culture medium was replaced
by normal growth medium containing the 20 µM and 50 µM concentrations of cadmium
chloride solution, using four replicates for each condition: 0 µM, 20 µM and 50 µM. In
control wells, culture medium was replaced by an equal volume of fresh culture medium.
Fig.7 – Schematic representation of an assay in a 96 well-plate for 24 h and 48 h of cadmium exposure. Light
blue wells represent blank wells. Three clusters of cells were organized according to initial cell density for
being able to select the cluster that had the control absorbances in the range of 0.75 - 1.25.
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Cells were daily observed with an optical microscope (Nikon Eclipse 80i, Japan) to
check for contaminations, adhesion and confluence. At the end of the respective exposure
time, MTT solution (1 mg/mL in PBS pH 7.2) (Sigma Aldrich) was added to the wells’
medium and left in the incubator for four hours (37 oC, 5% CO2). Afterwards, wells’
content was replaced by DMSO for formazan crystals solubilization and left on a shaking
plate during two hours at room temperature protected from light. In the end, absorbances
were obtained by a (BioTek ®– Gen5™ software) microplate reader (570 nm wavelength)
with blank correction. The mean absorbance with blank correction was used to obtain
viability chart (in the range of 0.75 – 1.25 for control samples). The initial cell densities
selection was based on the absorbance reading in that range. Therefore, only 2.0 x 105
cels/mL and 3.0 x 105 cels/mL were used for 48 h and 24 h exposures, respectively. The
equation to obtain viability relative percentage was: relative % Viability = A570 Sample/
mean A570 Control x 100.
2.7. Cell cycle analysis – Flow Cytometry
Cell analysis was performed according to the method described by Bork et al (65)
with slight modifications. Cells were cultivated in 24 wells plates and incubated for 24 h in
the normal growth conditions. After this period, culture medium was replaced by the
respective cadmium containing growth medium solutions – 0 µM, 20 µM and 50 µM.
Three or four replicates were considered and daily observed under an inverted microscope.
After the exposure times, each well was washed with PBS pH 7.2 and then trypsinized.
After trypsin inactivation with complete growth medium cells were transferred to
microtubes to centrifuge around 300 g between three and five minutes to prevent a too
dense pellet. Cells were washed with PBS, centrifuged and ressuspended with 85% ethanol
at 4 ºC for fixation and stored at -20 ºC until further analysis.
To perform the flow cytometry analysis cells were retrieved from -20 ºC to room
temperature. Then they were centrifuged at around 300 g during 3 to 5 minutes, carefully
ressuspended in 1 mL of PBS pH 7.2, and finally, filtered through a 55 µm nylon filter to
eliminate big clusters. Then, for each sample 50 µg/mL propidium iodide (Fluka, USA)
and 50 µg/mL RNAse (Sigma Aldrich, USA) were added and samples were incubated for
20 minutes to stain and eliminate RNA from samples, respectively. Relative fluorescence
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intensity was measured in a flow cytometer (Beckman Coulter) equipped with an argon
laser (15 mW, 488 nm) having the fluorescence peak sample of G0/G1 cells adjusted to
channel 200. Acquisitions were made using SYSTEM II software (v. 3.0, Beckman
Coulter, USA). For each sample, the number of events reached approximately 5000. Cell
cycle analysis was then conducted based on the histogram outputs.
2.8. DNA damage – Comet Assay
The comet assay was performed as described by Singh et al. (66) and Tice et al.
(61) with slight modifications. Preparation of slides and solutions took place at early stage.
Slides with frosted ends were dipped into a solution of 1% normal melting point agarose
(NMPA) and then were dried overnight at room temperature. After exposure ending, cells
were harvested and transferred to microtubes. Both microtubes and NMPA slides were
previously properly identified. Then, cells were centrifuged at 300 g for 4 minutes,
carefully taking in consideration pellet thickness, therefore repeating if necessary. This
way, supernatant was removed to resuspend each sample in PBS. Positive control remained
for last, to be resuspended in 100 µM H2O2 exactly for 10 minutes. Positive controls
required strict attention to timing control. They were centrifuged for 5 minutes and
resuspended in PBS right before the end of 10 minutes of H2O2 treatment. A mixture of 50
µL of the cell suspension and 50 µL of 1% low melting point agarose (LMPA) previously
heated at 37 ºC was spread on each slide with the dried NPMA layer. A coverslip was
placed over the mixture and the agarose was allowed to solidify at 4 ºC for at least 10
minutes. The next step was the cell lysis. Lysing solution was composed of a freshly mix
of two solutions: one containing 2.5 M NaCl, 100 mM EDTA, 10mM Trizma Base, 0.2 M
NaHO, corrected pH 10; and other containing 1% Triton X-100 and 10% DMSO.
Coverslips were removed and cells were then treated with lysis solution for 2 h.
Meanwhile, electrophoresis buffer was freshly prepared with chilled distilled water. The
nuclei were immersed in an electrophoresis box with the cold electrophoretic buffer (300
mM NaOH and 1 mM EDTA, pH 13). They were left for 20 minutes to allow DNA
unwinding. Thereafter, electrophoresis ran for 30 minutes, 0.74 V/cm, 300 mA in the same
buffer. Since lysing step until the end of electrophoresis, slides were kept in the dark
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avoiding direct fluorescent light to cause DNA damage. Next, slides were neutralized three
times (5 minutes each) with 0.4 M Tris buffer pH 7.5.
Afterwards, slides were left overnight to dry and be stored for prior analysis. To
perform comet scoring, slides needed to be rehydrated for 15 minutes and stained with 10
µg/mL ethidium bromide for 3 to 5 minutes, removing the excess with distilled water.
Slides were observed under a fluorescence microscope Nikon Eclipse 80i equipped with an
excitation filter of 510-560 nm and a barrier filter of 590 nm. At the end of observation,
slides were dipped in absolute ethanol to remove staining, dehydrated and stored again. For
each treatment group, two slides from each well were screened and 50-100 randomly
visualized cells were picked. Images were captured with imaging software (NIS-Elements
F 3.00, SP7). Every nuclei photographs were run on version 1.2.2 of CASP – Comet Assay
Software Project to automatically score a comprehensive set of parameters such as % tail
DNA, tail length and tail moment, to provide a global comet description.
2.9. Statistical Analysis
For all experiments at least 3 replicates and two independent assays were
performed. Data were analyzed by two-way analysis of variance (two-way ANOVA) (p <
0.05), followed by a Holm-Sidak test (p < 0.05) to evaluate the significance of differences
in the parameters. When necessary, data were transformed to achieve normality and
equality of variances. When justified, Pearson correlation was performed and the
respective correlation coefficient was presented as r.
All statistical analyses were performed with SigmaPlot Version 11.0 for Windows.
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3. Results
3.1. Confluence and morphology
Confluence and morphology were observed during subculture routine and for each
exposure trial. Standard subculture routine and controls of the exposure trials, showed cells
with the general typical aspect documented by Fig.8A and Fig.8D. When adherent, MG-63
cells appeared oval to spindle-shaped (resembling fibroblasts), without branching cell
processes.
Fig.8 – The figure captures some of the samples obtained to visually document cadmium effects on MG-63
cell culture. A and D show pictures of control samples at 24 h and 48 h hours, respectively. B and E show
cells exposed to 20 µM cadmium at 24 h and 48 h, respectively. C and F show 50 µM cadmium exposure of
cells at 24 h and 48 h, respectively; (A to C, E, F – 100x magnification; D – 40x magnification).
Cell confluence was gradually lost along the increase of cadmium dose exposure,
with increasing appearance of detached cells typical of cell death (Fig.8 - B, C, E, F). This
was verified on both times of exposure, although the number of adherent cells was a bit
higher on the 48 h exposure trial, as shown from Fig.8D to Fig.8F as well as the
corresponding number of dead cells.
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3.2. Proliferation / viability assessment – MTT Assay
To assess the MG-63 cell response to the cadmium doses, a proliferation
assessment was performed with MTT Assay. MG-63 cell viability as response to cadmium
exposure was measured using the MTT assay as a function of initial seeding density and
values of absorbance.
As shown in Fig.9, a significant (p < 0.05) reduction in cell viability was observed
at 24 h to 50 μM cadmium and at 48 h exposure to 20 and 50 μM cadmium. For the 24 h
exposure to 50 μM dose, the viability decreased 49%. As for the 48 h exposure, to 20 μM
the viability decreased 10% and to 50 μM the viability decreased 53%. Within
concentrations no significant variations were detected between 24h and 48h of exposure.
Fig.9 – Cadmium effects in MG-63 cell viability (mean ± standard deviation) to both times of exposure for
the MTT Assay – 24h and 48h. Different letters within time represent differences between cadmium
concentrations (p < 0.05).
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3.3. Cell cycle analysis – Flow Cytometry
After the exposure, cells were collected to perform cell cycle analysis and the
global cell cycle stages are presented in Fig.10. The histograms of relative fluorescence
intensity obtained are represented in Fig.11, where cell cycle phases are depicted for
control and 50 µM cadmium of 48 h (Fig.11A and Fig.11B, respectively). Deeper analysis
revealed a trend of behavior to cadmium: for the 48 h of exposure, a statistically significant
correlation was detected between G0/G1 cell percentage and cadmium concentration
(Pearson Product Moment Correlation; r = - 0.485; p = 0.0482).
Fig.10 – Cell cycle results of MG-63 cadmium exposure after 24 h and 48 h. The given values are the mean
% of cell population (± standard deviation) along cell cycle stages of at least 3 replicates. The presence of
symbols indicates significant differences between times within a concentration at p < 0.05.
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It indicates a decrease of G0/G1 cell percentage along the increase of cadmium
concentration. However, for the same exposure time, it is possible to visualize as well an
accompanying trend of increase in G2 cell percentage along cadmium concentration
increase as visualized in Fig.10. Between times it was verified the rise of % of cell
population in G0/G1 phase in the cadmium doses 0 and 20 µM.
Fig.11 – Histograms of relative fluorescence intensity. Example of results obtained to the 48 h exposure of
MG-63. Cell cycle phases are depicted – G0/G1, S, G2. A – Control. B – 50 µM cadmium.
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Although no direct information was obtained from cell cycle analysis, other
statistical parameters were provided by the flow cytometry analysis. Half peak coefficient
of variation (HPCV) and full peak coefficient of variation (FPCV) are two of them, see
Fig.12. Samples presented a good HPCV value: under 5% indicating sample quality and
integrity. Still, FPCV showed a significant increase facing control samples: at 48 h of
exposure to 50 µM cadmium concentration.
Fig.12 – Half peak coefficient of variation (HPCV) and full peak coefficient of variation (FPCV) of MG-63
cells exposed to cadmium after 24h and 48h exposure. Different letters represent differences between
cadmium concentrations within time (p < 0.05).
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3.4. DNA damage – Comet assay
To detect DNA damage comet assay was performed. The parameters studied are in
agreement with the images taken by fluorescence microscopy. The main effects were
detected at 48 h exposure. The amount and size of fragmented DNA, given by the % tail
DNA (Fig.13A) and tail length (Fig.13B), respectively, increased significantly and it was
higher for 20 µM than 50 µM cadmium concentration (p < 0.05). As for the tail moment
(Fig.13C), it is in agreement with the latter parameters since it’s the result of their
mathematical product, and increased in cadmium exposed cells, with values statistically
different from control (p < 0.05).
Fig.13 – Comet assay assessed parameters: % Tail DNA (A), Tail Length (B) and Tail Moment (C), the latter
two represented in arbitrary units. Results are expressed as mean of medians ± standard deviation. Different
letters represent differences between cadmium concentrations within time (p < 0.05). The presence of
symbols indicates significant differences between times within a concentration (p < 0.05).
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The comet pictures obtained, as seen in the examples of Fig.14A-C, show that the
20 µM dose generated (p < 0.05) a bigger tail with a high DNA percentage on it.
Fig.14 – Comet assay representative images of MG-63 cell nuclei obtained for the 48 h exposure to cadmium
(400x). A – Typical control comet, B – example of comet obtained for the 20 µM cadmium dose, C –
example of comet obtained for the 50 µM cadmium dose.
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4. Discussion
Cadmium is a natural metallic element. It occurs naturally in the environment,
however, human technological demands have engineered to take use of cadmium
properties integrating it for instance in inks and many hardware tools such as computers
and batteries, until a few years ago. Nowadays, its use is left mostly to modern
technologies like solar panels. Its extensive use has lead to its excessive buildup in
environment, reaching the food chain and causing disease by accumulating in organisms.
Bone disease mechanisms, in particular, are not fully understood. Many studies
have shown that cadmium is cytotoxic to osteoblast cells, namely to Saos-2 (67) and
MC3T3-E3 (68), although this study aims to evaluate several toxicity parameters in a less
studied human osteoblast cell line, MG-63.
Regarding the proposed aims, in this study it has also been observed some essential
cell culture parameters. For each exposure trial confluence and morphology were observed
during subculture routine. When adherent, MG-63 cells appeared oval to spindle-shaped
(resembling fibroblasts), without branching cell processes. The expected cadmium effects
on these features were consistent with Lévesque et al. (64) which also observed these
effects on the same cell line. With increasing cadmium concentrations, detachment and
loss of any type of differentiation tends to occur. E-Cadherin is a protein responsible for
cell to cell adhesion and communication. Cadmium specifically displaces calcium in E-
cadherin disrupting this protein and thus impairing cell proliferation (69). So, confluence
was gradually lost along the increase of cadmium dose, with increasing appearance of
swelled and detached cells typical of cell death. This was verified on both times of
exposure, 24 h and 48 h. Although, probably due to have had more time to multiply
themselves, the number of adherent cells were a bit higher on the 48 h exposure trial, as
well as the corresponding number of dead cells.
This point connects with the MTT assay. Proliferation / viability measurements are
the basis of several in vitro assessments of cellular response to xenobiotic substances or
compounds. Being reduced by mitochondrial enzymes, MTT assay is essentially a measure
of metabolic activity of cells, therefore, measuring the cell proliferation rate and on the
other hand, the reduction in cell viability when metabolic events lead to apoptosis or
necrosis (70). Under these in vitro conditions the obtained results showed an affected
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viability of the human MG-63 cells in a time- and dose-dependent manner following a 24 h
and 48 h exposure. Lévesque et al. (64) interestingly exposed cells only after serum
deprivation for 24 h, suggesting that bovine serum albumin (BSA) in incubation medium
reduces cadmium availability to cells, therefore, minimizing cadmium-induced loss in cell
viability. These authors also came to show cell viability to increase with the presence of
BSA (cadmium chelating factor). This way, the authors obtained an 18 μM cadmium 50%
Lethal Concentration (LC50), while the present work shows a marked decline of cell
viability starting to occur with 50 μM for the 24 h exposure, so, these differences could
possibly result from the fact that no serum deprivation was proceeded to.
So far, the obtained results have lead to inquire about cell cycle behavior. As some
studies demonstrate cell cycle disturbances due to cadmium-induced genotoxicity
(65,71,72), for the present work, the evaluation of potential cell cycle alterations caused by
cadmium in MG-63 cell line was analyzed by flow cytometry. Cells were analyzed after 24
and 48 h of cadmium exposure. For both times, under the 20 and 50 µM cadmium no
significant effects were detected. However, it was identified a correlation between
cadmium concentration and the number of cells in G0/G1 phase. With the increase of
cadmium dose, the G0/G1 population number decreased. Also, an apparent trend stands out
relating to what can be observed in the G2 cell population. In this case, no correlation and
no significant differences were detected. Nevertheless, it would be expected to observe a
G2 cell increase which is noticeable in the histogram results – the rise of cell number on the
G2 cell cycle phase along cadmium increase of concentration. It is, for instance,
documented as a cadmium effect the occurrence of G2/M arrest in kidney proximal tubule
cells (65).
Between exposure times, it was verified the rise of cell population number of G0/G1
phase in 0 and 20 µM. This may be a result of the prolonged culture time. However, it may
be suggested that with increasing exposure times, for example until 72 h, a marked
accumulation would be clearer along the different phases. Even though no immediate
information was obtained from this cell cycle analysis due to the lack of statistical
significance, there are other important parameters provided by flow cytometry, such as
HPCV and FPCV. HPCV is measured around the mean of all cells in G0/G1 stage of the
cell cycle and can provide data in nuclear DNA content variation (73). According to
Loureiro and Santos (55), several authors mention optimal HPCV values and Galbraith
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(74) established an acceptance index under 5%. For the present study, this analysis
revealed a consistent / stable HPCV (under 5%) which is an indicating factor of quality
sample (75). FPCV showed a significant increase facing control samples of 48 h exposure
to 50 µM cadmium concentration, suggesting clastogenicity (76).
Herein, to assess DNA damage with a quick, sensitive and versatile method, the
comet assay was chosen to be used, therefore, measuring single- and double-strand breaks
(77) in MG-63 cell line. The parameters studied – % tail DNA, tail length and tail moment
- were in agreement with the images taken and visualized by fluorescence microscopy. The
main effects were detected at 48 h exposure. The amount (% tail DNA) and size (tail
length DNA) of fragmented DNA increased significantly after cadmium exposure and it
was higher for 20 µM than for 50 µM cadmium concentrations. This effect may be
explained by the following prepositions: a) cadmium is known to form DNA adducts
which lead to cross-link formations (DNA-DNA, DNA-protein) and also helical distortions
(78,79). Lesions that can disrupt replication, or cause incorporation of the wrong base (80).
This way, the highest % tail DNA and highest tail length detected appear at 20 µM instead
of occurring at 50 µM cadmium dose, possibly due to the size of the latter generated
fragments may not be fully run under agarose electrophoresis, and the 50 µM cadmium
dose generated bigger fragments that parallel FPCV results; b) at 50 µM cadmium it was
also detected significant DNA damage but not on that latter extent. Whether this damage is
due to the formation of such elevated DNA fragments and / or due to severe impairment of
DNA repair mechanisms (18,62), it would be necessary a new insight at these results with
an assessment implying lesion specific enzymes which convert damage into breaks. The
use of endonucleases as formamidopyrimidine glycosylase (FPG – which removes
oxidized and ring-opened purines, formamidopyrimidines and alkylated bases),
endonuclease III (Endo III – for oxidized pyrimidinic bases) and 3-methyladenine DNA
glycosylase II (AlkA – which recognizes alkylated bases) allows the assessment of specific
damages and, consequently, if the DNA repair mechanisms were in activity (81).
The trend to cell cycle arrest at G2 checkpoint may be a response to DNA damage
occurring in cells exposed to 20 and 50 µM cadmium after 48 h of exposure. According to
O’Connel and Cimprich (82) when this checkpoint is activated, cells have chance to repair
DNA damages preventing them from entering mitosis and culminate in catastrophic
mitosis (micronuclei formation); another pathway cells may follow after cell cycle arrest at
CYTOTOXIC AND GENOTOXIC EFFECTS OF CADMIUM IN HUMAN OSTEOBLASTS
Master in Applied Biology – Molecular and Cellular Biology
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G2 phase is apoptosis. The first pathway was observed in MG-63 cells with the cadmium
dose of 50 µM during 48 h. In these cells DNA damage has occurred, contributing to cell
cycle arrest at G2 checkpoint and, finally, leading to clastogenicity observed by the
increased FPCV detected by the flow cytometric analysis. Clastogenic effects took place in
these cells, probably because cadmium is known to cause inhibition of DNA repair (19). In
the cells exposed to 20 µM cadmium for 48 h, DNA damage was also confirmed by the
comet assay. Since DNA damages may be repairable or not, the trend to G2 arrest possibly
lead to repair pathway, otherwise entering apoptosis. Therefore, recognizing what happens
to MG-63 cells after G2 arrest would be important to clarify. For instance, resorting to
Annexin V / PI (Propidium iodide) would provide distinction between cells undergoing
apoptosis and necrosis (67).
CYTOTOXIC AND GENOTOXIC EFFECTS OF CADMIUM IN HUMAN OSTEOBLASTS
Master in Applied Biology – Molecular and Cellular Biology
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5. Conclusions and future perspectives
In conclusion it was settled that cadmium chloride exerts cyto- and genotoxic
effects in the MG-63 human cell line.
Viability is shown to decrease considerably and cell cycle revealed a tendency to
alter its pattern possibly culminating in a G2 phase arrest. The ongoing cell steps would be
clarified by Annexin V / PI to tell apart the apoptotic from the necrotic cells.
Clastogenicity was also indicated by the values obtained for the FPCV, being furthermore
supported by the DNA damage assessed and positively detected.
Therefore, in future work, it would be wise and interesting to perform a new comet
assay assessment with refinement steps such as the inclusion of lesion-specific enzymes for
excision repair pathway damage and oxidized bases detection.
Finally, micronucleus detection could be a high valuable tool to complete the
analyses and substantiate comet assay results and clastogenic effects, clarifying DNA
fragmentation or chromosomal aberrations.
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Master in Applied Biology – Molecular and Cellular Biology
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