Explicar detalladamente los efectos genotóxicos que tiene el componente

seleccionado, sobre el material genético (tipo de mutaciones que puede

provocar)
Dichlorodiphenyltrichloroethane (DDT) is a wellknown
insecticide and has been used since 1940 primarily
for the prevention of malaria, yellow fever, and sleeping
sickness, and also in agriculture for the control of insects
(International Agency for Research on Cancer, 1991).

DDT has been studied extensively and shown to have a carcinogenic effect on the liver
(International Agency for Research on Cancer) as well as a neurotoxic effect
(Eriksson and Talts, 2000).
Previously, we reported that DDT and its metabolites in the brain were excreted gradually over a
few weeks and were then deposited in the adipose tissue (Tomiyama et al., 2003, 2004). Thus,
we speculate that low dose of DDT may have epigenetic effects including DNA methylation in
the young brain.
DNA methylation is the major epigenetic modification in the eukaryotic genome and occurs
predominantly at the carbon-5 position of cytosine residues when followed by a guanine in so-
called CpG dinucleotides (Bestor, 2000).

Bestor, T. H. (2000). The DNA methyltransferases of mammals. Human molecular

genetics, 9(16), 2395-2402.

DNA methylation has been studied extensively in development and has long been considered a
static process following cell differentiation (Szyf et al., 1991; Monk et al.,1987; Goto et al., 1994;
Deng and Szyf, 1999; Bestor, 2000). Consequently, the two main roles of DNA methylation are
thought to be maintenance of genome stability (Eden et al., 2003) and control of gene transcription
(Bird, 2002).
Bird, A. (2002). DNA methylation patterns and epigenetic memory. Genes & development, 16(1),
6-21.
Szyf, M., Bozovic, V., & Tanigawa, G. (1991). Growth regulation of mouse DNA methyltransferase
gene expression. Journal of Biological Chemistry, 266(16), 10027-10030.
Monk, M. A. R. I. L. Y. N., Boubelik, M. I. C. H. A. E. L., & Lehnert, S. I. G. R. I. D. (1987). Temporal
and regional changes in DNA methylation in the embryonic, extraembryonic and germ cell
lineages during mouse embryo development. Development, 99(3), 371-382.
Goto, K., Numata, M., Komura, J. I., Ono, T., Bestor, T. H., & Kondo, H. (1994). Expression of
DNA methyltransferase gene in mature and immature neurons as well as proliferating cells in
mice. Differentiation, 56(1-2), 39-44.
Deng, J., & Szyf, M. (1999). Downregulation of DNA (cytosine-5-) methyltransferase is a late
event in NGF-induced PC12 cell differentiation. Molecular brain research, 71(1), 23-31.

In higher organisms, which have functionally different cell types, DNA methylation is thought to
be important in the control of gene expression, and changes in DNA methylation can dramatically
affect chromatin structure and genome stability (Yauk et al., 2008).
Yauk, C., Polyzos, A., Rowan-Carroll, A., Somers, C. M., Godschalk, R. W., Van Schooten, F. J.,
... & Douglas, G. R. (2008). Germ-line mutations, DNA damage, and global hypermethylation in
mice exposed to particulate air pollution in an urban/industrial location. Proceedings of the
National Academy of Sciences, 105(2), 605-610.

Franco et al. (2008) indicated that reactive oxygen species multistage process of carcinogenesis
by both genetic and epigenetic mechanisms. Shahrzad et al. (2007) indicated that a low level
of the methyl group donor s-adenosyl-Lmethionine (SAM) induced by hypoxia may cause DNA
hypomethylation in solid tumors exposed to ischemia and reperfusion. These results indicate that
oxygen consumption and oxidative stress can markedly affect the methylation status of the
mammalian genome.

Shahrzad, S., Bertrand, K., Minhas, K., & Coomber, B. (2007). Induction of DNA hypomethylation
by tumor hypoxia. Epigenetics, 2(2), 119-125.
Franco, R., Schoneveld, O., Georgakilas, A. G., & Panayiotidis, M. I. (2008). Oxidative stress,
DNA methylation and carcinogenesis. Cancer letters, 266(1), 6-11.

PAPER

Shutoh, Y., Takeda, M., Ohtsuka, R., Haishima, A., Yamaguchi, S., Fujie, H., ... & Harada, T.
(2009). Low dose effects of dichlorodiphenyltrichloroethane (DDT) on gene transcription and DNA
methylation in the hypothalamus of young male rats: implication of hormesis-like effects. The
Journal of toxicological sciences, 34(5), 469-482.

o Explique el mecanismo de reparación responsable de mantener la integridad

del genoma, cuando es expuesto al genotóxico seleccionado.

ü Diseñar una metodología para la identificación del daño genotóxico en la

población en riesgo seleccionada.
Intro
Organochlorine pesticides are environmentally ubiquitous
compounds due to their long persistence in the environment.
They have the ability to bioaccumulate and
biomagnify in food chains; in addition, they also have the
capacity for a long-range atmospheric transport. Many
organochlorine compounds have been classified by the International
Agency for Research on Cancer (IARC, 1979) as
carcinogenic in animals and also suspected to be carcinogenic
in humans.
Two examples of this group of compounds
are the 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane
(DDT) and the hexachlorobenzene (HCB). Although the
Stockholm Convention on Persistent Organic Pollutants,
which came in force on 17 May 2004, outlawed the use of
DDT (United Nations Environment Programme, 2004),
excepting their use in malaria-endemic nations. In these
areas, houses are usually sprayed twice a year with DDT
to kill resting mature Anopheles (Ya`n˜ez et al., 2002); this
activity supposes the human exposure to DDT and subsequently
to DDE, its metabolite, in these areas. In addition,
DDE is the main derivative observed in people exposed to
DDT (Sunyer et al., 2005; Pe´rez-Maldonado et al., 2006).
DDT and DDE can cause greater damage to the environment
because of their continued usage in the malaria
infested parts of the world and their ability to persist for
a long time in the environment (Uppala et al., 2005).

It is considered that the potential pathways of exposure

to organochlorines are mainly by oral through consumption
of foods that contains small amounts of these compounds
that subsequently cause bioaccumulation and
biomagnification (Ritter et al., 1995). As a consequence,
significantly high levels of these compounds can be
observed in biological matrices such as adipose tissue,
serum and human milk. For example, in a study conducted
in Tunisia, significant amounts of organochlorinated compounds,
including DDE and HCB, have been found in
human breast milk, (Ennaceur, 2007), indicating a significant
exposure to such compounds and the potential risk
to mother and child, as demonstrated in other studies
(James et al., 2002).
METODOLOGÍA
The possible genotoxic potential of 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE), which is a
metabolite of dichlorobiphenyltrichloroetane (DDT), and hexachlorobenzene (HCB), which are
organochlorine pesticides have been evaluated in vitro by using human lymphocytes as test system.
Genetic damage was determined by scoring the frequency of micronuclei (MN) in primary
lymphocyte cultures obtained from different donors.
The results indicated that, under the experimental conditions used, the DDT metabolite DDE was able to
induce significant increases in the frequency of micronucleated cells, which indicate a certain
clastogenic and/or aneugenic potential. DDE was tested in the range of 10–80 mM, but the only
concentration producing a significant genotoxic effect was 80 mM. On the other hand, HCB was unable to
induce a significant increase in the MN frequency in the range of concentrations assayed,
from 0.005 to 0.1 mM. The selected concentrations of DDE and HCB were chosen according to their
toxicity in cell blood cultures; higher concentrations reduced significantly cell proliferation and produced
a low frequency of binucleated cells. In conclusion, the results
indicate that a genotoxic risk is associated with the exposure to DDE at concentrations 80 mM and
above.
According to the non-conclusive genotoxic risk associated to such compounds, we present
results on their genotoxic evaluation in human peripheral blood cultures by using the
micronucleus assay as a test system.

2.1. Chemicals
The compounds p,p0-DDE (CAS No. 72-55-9, 99% purity)
and HCB (CAS No. 118741, 99% purity) were obtained
from Polyscience Corporation Analytical Standards (Niles,
Illinois, USA). DDE was dissolved in dimethyl sulfoxide
and added to the complete culture medium in an amount
equal to the 1% of the final volume. HCB was dissolved
in acetone. Mitomicyn C (MMC), used for positive control,
was dissolved in bidistilled water to the concentration used,
just prior to treatment. Mitomycin C was the chemical
mutagen selected for positive control because of its clear
response in the micronucleus assay. The range of selected
doses was chosen according to the induced reduction on
cell proliferation; thus, the highest dose tested produced
enough high frequency of binucleated cell to obtain the
1000 binucleated cells required for the scoring of each
product concentration. HCB was much more cytotoxic
than DDE and, consequently, the final selected doses ranged
from 0.005 to 0.1 and from 10 to 80 mM for HCB and
DDE, respectively.
2.2. Lymphocyte culture and treatments
The Ethic Committee of the University approved the
study. Blood samples were collected from three healthy
volunteers from the University Department: 2 females
and 1 male aged 26, 25 and 25 years old, respectively, with
no smoking history. The male and the two females were the
donors for DDE cultures, while the male and one of the
females gave blood samples for the HCB cultures. Previous
to the analysis, all blood donors gave informed consent and
blood samples were collected and further manipulated in
accordance with ethical standards. Approximately 10 ml
of blood was drawn from each donor by venipuncture, in
heparinized syringes. Lymphocyte cultures from samples
collected were set up adding 0.5 ml of whole blood to
4.5 ml of RPMI 1640 medium supplemented with 15%
heat-inactivated fetal calf serum, 1% antibiotics (penicillin
and streptomycin) and l-glutamine. Lymphocytes were
stimulated with 1% of phytohemagglutinin and incubated
for 72 h at 37 _C. Duplicate cultures from each donor were
established. DDE and HCB were added 24 h after phytohemmaglutinin
stimulation. A final concentration of
6 lg/ml Cytochalasin B (Cyt B) was added to the cultures
44 h later, to arrest cytokinesis (Surralle´s et al., 1994). At
72 h incubation, the cultures were harvested by centrifugation
at 800 rpm for 8 min and treated with a hypotonic
solution (2–3 min in 0.075 M KCl at 4 _C). Cells were then
centrifuged and a methanol:acetic acid (3:1 v/v) solution
was added. This fixation step was repeated twice and the
resulting cells were resuspended in a small volume of fixative
and dropped onto clean slides.
2.3. MN analysis and statistical analysis
The slides were stained with 10% Giemsa in phosphate
buffer (pH 6.8) for 10 min and coded prior to scoring.
The criteria proposed by Fenech (1993) were followed to
determine the frequency of BNMN (binucleated cells with
micronuclei) and the total number of MN (micronuclei) in
lymphocytes (MN). A total of 1000 binucleated cells with
well-preserved cytoplasm (500 per replicate) were scored
per concentration, using an optical microscope. In addition, 500 lymphocytes, per each
concentration, were scored
to determine the percentage of cells with one to four nuclei
and the cytokinesis block proliferation index (CBPI) was
calculated according to Surralle´s et al., 1995. The frequency
of BNMN and the total number of MN in lymphocytes,
are parameters measuring the genotoxicity of the
tested compound. Since the BNMN is more biologically
relevant, (in terms of genotoxic potency), the statistical
analysis has been carried out using this biomarker of genotoxicity.
In addition, the CBPI is used as a measure of the
cytotoxic or cytostatic potential of the tested chemical, giving
indication on which concentrations can be selected for
the study (Surralle´s et al., 1995). Statistical differences
between control and treated cultures were determined using
the one-tailed Fisher0s exact test. A P-value of 60.05 was
considered as indicative of statistical significance.

Resultados

Table 1 show the results obtained in the genotoxic evaluation of DDE. Results indicate that
DDE induces significant increases in the frequency of binucleated cells with micronuclei
in the cultures set up with blood from the three different donors used.

In Tunisia, as in many parts of the world, DDE is present in human tissues. Thus, the levels of
DDE detected in mother’s milk reached values of 2.421 mg kg_1 fat weight, that are higher
than those referred in other populations (Jaraczewska et al., 2006; Kalanzaki et al., 2004; Konishi
et al., 2001). The world-spread detection of DDE in human tissues indicates the risk of DDT
exposure and the relevance of studies conducted to determine the possible risks, including
genotoxicity and mutagenicity, associated with such exposure.

The positive induction of micronuclei, after blood lymphocyte exposure to DDE, indicates
that this compound has some genotoxic potential. Nevertheless, it must be pointed out that
the positive induction of genotoxicity was only obtained at the highest concentration tested
(80 mM), which would mean a weak genotoxic potential.

It should be also pointed out that 80 mM DDE is a dose inducing significantly high levels of
toxicity, as observed by the CBPI value. The significant increase of genotoxic damage was
observed both for the total frequency of micronuclei scored and for the frequency of
binucleated cells with micronuclei, that are the two biomarkers of genotoxicity used.
Micronuclei are acentric fragments or complete chromosomes that fail to attach to the mitotic
spindle during cytokinesis and are excluded from the nuclei. Thus, the MN assay is a system
that allows the detection of both clastogenic and aneugenic agents (Fenech et al., 1999).
Justificación del uso de Mn assay
The detection of such effects in human has pivotal importance in human health and the use of
the in vitro MN assay is included in the UE guidelines to detect mutagenicity and genotoxicity
(Kirkland et al., 2005). The use of the MN assay in peripheral blood lymphocytes has shown to
be a good tool to determine the potential cytogenetic damage induced by different
environmental pollutants, due to its sensitivity and reliability, and its potential applicability to
different kinds of cells (Surralle`s et al., 1992; Decordier and Kirsch-Volders, 2006). In addition,
a recent study shows its usefulness as a surrogate biomarker of cancer risk (Bonassi et al., 2007).

DDT and its derivatives have usually given conflictive results in studies carried out to determine
their genotoxic potential. However, in recent studies DDE was shown to be able to induce
apoptosis in peripheral blood cells, as well as other cytogenetic changes (Lessa et al.,
1976; Pe´rez-Maldonado et al., 2004), mainly due to the production of reactive oxygen
species (Pe´rez-Maldonado et al., 2005). In addition, positive association has been found in
humans between DDT and DDE exposure and DNA damage in their blood cells, as observed in
women (Ya´nez et al., 2004) and in children (Pe´rez-Maldonado et al., 2006).

As observed, this compound gave negative

results in the cultures established from two individual
donors. These negative results extend the knowledge on
HCB as a non-genotoxic agent, as indicated by the previous
in vitro data showing that this compound is unable
to induce point mutations, as measured by the Ames test (Hawort et al., 1983) or chromosomal
aberrations, as
detected in cultured Chinese hamster cells (Ishidate,
1983).
(interpretado por mi a partir del parrafo de arriba)A positive results extend the knowledge on
DDE as a genotoxic agent, as indicated by the previous in vitro data showing that this
compound is able to induce point mutations, as measured by the Ames test

Genotoxic changes have been also reported in a biomonitoring

study on humans occupationally exposed to many
other organochlorines, including HCB (da Silva Augusto
et al., 1997). In that study, exposure to HCB was measured
in serum, and genotoxic effects were detected in peripheral
blood lymphocytes by using the micronucleus assay.

da Silva Augusto, L. G., Lieber, S. R., Ruiz, M. A., & de Souza, C. A. (1997). Micronucleus
monitoring to assess human occupational exposure to organochlorides. Environmental and
molecular mutagenesis, 29(1), 46-52.

Paper

Ennaceur, S., Ridha, D., & Marcos, R. (2008). Genotoxicity of the organochlorine pesticides 1, 1-
dichloro-2, 2-bis (p-chlorophenyl) ethylene (DDE) and hexachlorobenzene (HCB) in cultured
human lymphocytes. Chemosphere, 71(7), 1335-1339.

Por qué estos ensayos

A comprehensive evaluation of the genotoxic potential of chemicals requires the

assessment of the ability to induce gene mutations and structural chromosome
(clastogenic activity) and numerical chromosome (aneugenic activity) aberrations.
Aneuploidy is a major cause of human reproductive failure and an important contributor to
cancer and it is therefore important that any increase in its frequency due to chemical
exposures should be recognized and controlled. The in vitro binucleate cell micronucleus
assay provides a powerful tool to determine the ability of a chemical to induce chromosome
damage. The application of an anti-kinetochore antibody to micronuclei allows their
classification into kinetochore-positive and kinetochore-negative, indicating their origin by
aneugenic or clastogenic mechanisms, respectively. The availability of chromosome-
specific centromere probes allows the analysis of the segregation of chromosomes into
the daughter nuclei of binucleate cells to evaluate chromosome non-disjunction.
Quantitative relationships between the two major causes of aneuploidy, chromosome loss and
non-disjunction, can be determined. The mechanisms leading to chromosome loss and non-
disjunction can be investigated by the analysis of morphological and structural changes in the
cell division apparatus by the application of specific stains and antibodies for various cell
division components. We illustrate such analyses by the demonstration of the interaction of
the monomer bisphenol-A with the centrosome of the mitotic spindle and the folic acid
antagonist pyrimethamine with the centromeres of chromosomes. Both types of modifications
lead to the induction of aneuploidy in exposed cells. Our studies also implicate the products
of the p53 and XPD genes in the regulation of the fidelity of chromosome segregation at
mitosis.

Genetic change can be produced by gene mutations, bystructural chromosomal changes and by numerical
chromosome changes (aneuploidy). The role of genetic changes in carcinogenesis has now been established
via an extensive range of studies (for reviews see Barrett, 1995; Duesberg et al., 1999;Cowell, 2001).
In view of the role played by aneuploidy in carcinogenesis
and in human reproductive failure, this omission is perhaps
surprising. It has been estimated that at least 50% of human
conceptuses are aneuploid, although the vast majority are lost
by miscarriage (Boue´ et al., 1975). Boveri (1914) had suggested
a relationship between numerical aberrations and cancer as
early as 1914. The observation of extensive aneuploidy in
cancer cells (Mortens et al., 1997) indicates that modifications
of the fidelity of chromosome segregation leading to karyotype
instability may be critical events in the aetiology of at least a
proportion of human tumours. Chromosome instability at an
early stage in cancer progression may act as a driving force
leading to the alteration in the copy number of one or more
genes controlling cellular growth, thus affecting the expression
of oncogenes and/or tumour suppressor genes.
Recent reassessments
of the strategies for the testing of chemicals within the
European Union has resulted in the increasing recognition
of aneuploidy as an important end-point to consider when
evaluating potential genotoxicity to both somatic and germ
cells. The UK Department of Health’s Advisory Committee
on the Mutagenicity of Chemicals (COM) has recently recommended
a requirement for the measurement of aneugenic
potential in its revised guidelines for the testing of chemicals
(Committee on the Mutagenicity of Chemicals, 2000) and
it is expected that similar requirements will be introduced
throughout Europe.

Fig. 1. Diagrammatic representation of the use of the in vitro binucleate micronucleus (BNMN) assay to detect chromosome loss
and non-disjunction at
mitosis. The green bar represents a homologous pair of chromosomes; the red and green squares represent two different
chromosome-specific centromere
markers; the yellow squares represent pan-centromere markers

Paper parry 2002

o Escribir un objetivo para la identificación de los posibles daños genotóxicos.

o Describir la metodología que utilizará para la identificación del daño
genotóxico.

ü Diseñar una metodología para asociar la exposición al agente genotóxico con

mutaciones en genes.

ü Escriba un objetivo para la identificación de mutaciones en posibles genes

afectados por la exposición al agente genotóxico.

o Identifique un gen en el que se puedan presentar mutaciones por la contante

exposición a un genotóxico, justifique por qué este gen puede verse afectado
(zonas frágiles, genes de reparación o de control del ciclo celular, por ejemplo)

o Identifique el producto génico y su función en la célula.

o Explique los daños genéticos o epigenéticos que puede sufrir el gen al estar
expuesto al genotóxico seleccionado.

o Seleccione una metodología para identificar los cambios genéticos o

epigenéticos.

o Explique si al estar afectado el gen puede desencadenar una sobre

proliferación celular.

o Explique si la proliferación celular se relaciona con una sobre expresión o

baja expresión del gen.

o Diseñe una metodología para cuantificar la expresión génica del gen

seleccionado.