Instabilidade cromossmica

SUMRIO
- Importncia dos estudos de instabilidade
cromossmica (IC)
- IC como fator de predisposio para o cancro
- Biomarcadores de IC
Importncia dos estudos de instabilidade cromossmica
em animais aquticos

Interesse econmico/ecolgico
(influncia de poluentes)



Interesse para a sade humana
(estudos de nutrio)

Consequncias de toxicidade ambiental
diretamente nos peixes e indiretamente
na sade humana: importante indicador
da sade ambiental
Capacidade reprodutiva em de
crescimento das populaes de peixes
comprometida por fatores de toxicidade
ambiental: importante indicador da
produtividade pisccola
Mutao somtica, provocada
por agente genotxico
Estabilidade cromossmica um estado dinmico das clulas
Mitose: responsvel pela estabilidade
gentica durante o desenvolvimento
Estabilidade cromossmica
ndice de mutao induzida depende:
- do tipo de agente indutor
- da dose
- do tempo de exposio
- da resposta celular

AGENTES GENOTXICOS MUTAES SOMTICAS
De que modo os cromossomas
se protegem destes agentes?
Mutaes espontneas
Erros na replicao; erros durante o crossing-over

Mutaes induzidas
Origem das mutaes somticas
AMBIENTAIS
Qumicos,
drogas,
radiao ionizante,
virus,
stress oxidativo (excesso de ROS)

ENDGENOS
ROS formados continuamente como co-
produtos do metabolismo aerbico normal
Recrutamento da GSH:
Regulador do potencial redox nuclear
Sinalizao de regulao da proliferao celular
Checkpoints do ciclo
celular:
Sinalizao para
repao/apoptose (memria
para reparao)
Organizao estrutural
do cromossoma
Defesa celular
mutaes
Defesa contra ROS
-Atividade enzimtica
-Antioxidantes
-Fatores de remodelao
da cromatina
ROS
Barreiras s mutaes
AGENTES GENOTXICOS
ESTABILIDADE CROMOSSMICA
defesa celular
reparao do DNA
ESTABILIDADE CROMOSSMICA
Mutaes somticas e estabilidade cromossmica
AGENTES GENOTXICOS
defesa celular
reparao do DNA
ESTABILIDADE CROMOSSMICA
Mutaes somticas e instabilidade cromossmica
Evoluo

Resposta adaptativa

Envelhecimento

Baixa taxa de crescimento

Doena
Seleo positiva de mutaes viveis
Estado ativo de reparao
Estado de reparao insuficiente
Clulas com mutaes lesivas mas no letais
Instabilidade cromossmica como fator de
Mutaes somticas e instabilidade cromossmica
Predisposio
para o cancro
Elevada taxa de apoptose
mutaes gnicas/cromossmicas sequenciais e cumulativas
afeta a expresso de genes especficos relacionados com cancro
checkpoints, genes de reparao oncogenes supressores
INSTABILIDADE CROMOSSMICA
Instabilidade cromossmica associada ao cancro pode ser definida como um
estado contnuo de formao de novas mutaes cromossmicas, a um ndice
superior ao das clulas normais (Gisselsson, 2001)
hiperproliferao
celular
proliferao
maligna
A IC como factor de predisposio para o cancro
Cancro: mutaes em genes responsveis pela regulao do ciclo de diviso
celular
Ativao sequencial de CDKs (cyclin-
dependent kinases) por ligao s
ciclinas.
FOS, JUN e MYC so genes de
resposta imediata a estmulos
extracelulares. Segue-se a
expresso sequencial das ciclinas
D1, E, A e B
O sistema de controlo da diviso
depende de:

1. Ativao cclica das protenas cinases
(Cdks)
2. Supresso da atividade Cdk por
fosforilao inibidora e protenas
inibidoras
A IC como factor de predisposio para o cancro
Genes responsveis pela regulao do ciclo de diviso celular
Mutaes e cancro
Os rearranjos cromossmicos podem dar
origem a aumento, perda ou recolocao
dos genes associados ao cancro
checkpoints, genes de reparao protoncogenes supressores
Podem sofrer mutaes:
gnicas cromossmicas
Efeitos gerais das alteraes cromossmicas
Aneuploidias:
trissomias, tetrassomias, etc.
- desequilbrio de dose (aumento de expresso de um gene ativador?)

Amplificao gnica:
HSRs (homogeneously stained regions)
DMs (double-minutes)





Aumento de material gentico ativo
Perda de material gentico ativo
Delees
deleo dos genes supressores. Exs: RB1, TP53, BRCA1, etc
Como que as translocaes ativam oncogenes?
H dois mecanismos:
1. Fuso de genes (envolve sempre um protoncogene) que d origem
formao de um novo gene (neooncogene)
2. Recolocao de um protoncogene sob controlo de um promotor ativo
(efeito de posio)

Recolocao de material gentico ativo:

Inverses e translocaes (predominantemente)
Efeitos gerais das alteraes cromossmicas
Alteraes cromossmicas no cancro
Cromossoma Philadelphia
na LMC (leucemia mieloide
crnica)
Fuso dos genes BCR e ABL
1. Exemplo de fuso de genes (envolve sempre um protoncogene) que d
origem formao de um novo gene (neooncogene)
Translocao 9;22
Alteraes cromossmicas no cancro
2. Exemplo de recolocao de um protoncogene sob controlo de um promotor
ativo (efeito de posio)
Translocao 8;14 no linfoma de Burkitt:
ativao do MYC por efeito de posio
Translocao
Oncogene
MYC
Oncogene
MYC
Cadeia pesada Ig
Cadeia
pesada Ig
Instabilidade cromossmica e cancro
Acumulao de mutaes ao longo do tempo
Mutaes gnicas espordicas Mutaes cromossmicas espordicas
Instabilidade cromossmica espordica
Aumenta a predisposio para o cancro
Evoluo do cancro colo-rectal (adaptao de Kinzler and Vogelstein, 1995)
CI
CI
CI
CI
CI
Instabilidade cromossmica (CI) e cancro
O que mais importante na caracterizao do tumor: a alterao clonal ou a IC?
Distribuio da populao em 4 categorias, quanto ao risco de
desenvolvimento de um tumor (Knudson, 1985)
1 categoria: um nvel irredutvel de cancro, a menor
incidncia possvel, inevitvel porque est
relacionada com uma instabilidade endgena

2 categoria: tumores que resultam de uma exposio
em excesso aos agentes mutagnicos.

3 categoria: inclui tumores que resultam de uma
relativa insuficincia gentica para tolerar a exposio
aos agentes mutagnicos.

4 categoria: inclui tipos de cancro onde a influncia
ambiencial insignificante. o caso de neoplasias
autossmicas dominantes, para as quais a mutao
inicial passa atravs da linha germinal.
maior influncia ambiental
(influncia da idade)
AGENTES GENOTXICOS
ESTABILIDADE CROMOSSMICA
Preveno contra a instabilidade cromossmica ambiental
Distribuio da populao em 4 categorias, quanto ao risco de
desenvolvimento de um tumor (Knudson, 1985)
1 categoria: um nvel irredutvel de cancro, a menor
incidncia possvel, inevitvel porque est
relacionada com uma instabilidade endgena

2 categoria: tumores que resultam de uma exposio
em excesso aos agentes mutagnicos.

3 categoria: inclui tumores que resultam de uma
relativa insuficincia gentica para tolerar a exposio
aos agentes mutagnicos.

4 categoria: inclui tipos de cancro onde a influncia
ambiencial insignificante. o caso de neoplasias
autossmicas dominantes, para as quais a mutao
inicial passa atravs da linha germinal.
(influncia da idade)
maior influncia gentica
reparao DNA
ESTABILIDADE CROMOSSMICA
SNDROMES DE INSTABILIDADE
CROMOSSMICA (cancer prone
syndromes)
Expls:
Ataxia Telangiectasia
- Sensibilidade radiao ionizante
(DNA repair impairment)
Sndrome de Bloom
- Sensibilidade luz UV
(DNA repair impairment)
Anemia de Fanconi
Sensibilidade a agentes alquilantes
(DNA repair impairment)
(OS impairment)
Instabilidade cromossmica de origem gentica
defesa contra ROS?
CANCROS DE ORIGEM HEREDITRIA
Citogentica tumoral
alteraes cromossmicas no clonais (NCCA)


alteraes cromossmicas clonais (CCA)
Instabilidade cromossmica
=
alteraes cromossmicas no clonais (NCCA)
Importncia das alteraes cromossmicas no cancro
Toxicidade ambiental nos organismos aquticos
Instabilidade cromossmica nos organismos aquticos

toxinas
? No existe influncia da idade?
? No existe influncia gentica?
Toxicidade ambiental nos organismos aquticos por poluentes na gua
Instabilidade cromossmica nos organismos aquticos

Toxicidade ambiental por poluentes da gua em excesso
Efeito genotxico nos organismos aquticos
Monitorizao ambiental de genotoxicidade
Taxa de sobrevivncia
Taxa de crescimento
Taxa de reproduo
Instabilidade cromossmica nos organismos aquticos

Tipos de biomarcadores
-Taxa de reproduo
-Taxa de sobrevivncia
- Biomarcadores de dose
- Biomarcadores de efeito genotxico
- efeito a nvel de DNA (comet assay, p.ex.)
- efeito a nvel cromossmico
- Apoptose (ndice de proliferao)
- Instabilidade cromossmica:
-alteraes numricas
-alteraes estruturais
-quebras e rearranjos
Deteo, a nvel cromossmico, das
mutaes somticas induzidas por
poluentes: biomarcadores citogenticos
Biomarcadores de IC
De utilizao mais generalizada:
teste dos microncleos
Alteraes a nvel de
leso no DNA:
- efeito do agente
genotxico
imediatamente a seguir
exposio
Alteraes a nvel de leso
cromossmica:
- efeito do agente genotxico
associado a exposio de
longo termo
Genotoxicity biomarkers in Mytilus galloprovincialis: wild versus
caged mussels
C Bolognesi, G Frenzilli, C Lasagna, E Perrone, P Roggieri
Mutation Research 552 (2004) 153-162
Mtodos utilizados para avaliao de genotoxicidade:
- DNA single strand breaks
-Frequncia de microncleos
Persistence of atrazine impact on aneuploidy in Pacific
oysters, Crassostrea gigas

K. Bouilly, H. McCombie, A. Leito, S. Lapgue

Widespread use of the herbicide atrazine has incited much research on its toxicity in aquatic
systems, where it is routinely detected due to runoff from cultivated fields. Moreover, the
determination of the genotoxic effect of such pollutants in the marine environment has become a
major requirement for ecosystem protection.
In the Pacific oyster, Crassostrea gigas, hypodiploid aneuploid cells have regularly been reported.
There is a negative correlation between this phenomenon and growth, as well as evidence for a
genetic basis. A positive relationship between atrazine and aneuploidy has previously been
demonstrated in C. gigas adults and juveniles. To evaluate the persistence of this impact, our study
examined the offspring of the same adult population previously treated with different atrazine doses
(10 lg l)1, representing a peak value found in a polluted environment and 100 lg l)1), and a seawater
control. We observed that these offspring exhibited significantly higher aneuploidy levels when their
parents had been exposed to atrazine (14.916.9% in comparison with the control where the levels
ranged from 11.4% to 12.8%). In addition, the present study examined the aneuploidy level of a
sample of juveniles, previously exposed for 3.5 months to the same doses of atrazine, then
transferred
to non-polluted conditions for an additional period of 2.5 months; this aneuploidy level remained
significantly different between the treatments applied.
These results demonstrate the persistence of an atrazine impact on Pacific oyster aneuploidy in time,
within and between generations, indicating that this widely used compound may represent an
important factor causing at least medium-term damage to genetic material.
Chemical pollution in water especially with heavy metals is among the most
important health significance for human beings and animals consuming such
water due to their toxicity and accumulative behavior playing a prominent role in
aquatic ecosystems (De Gregori et al., 1994).

The assessment of biological effects on aquatic vertebrate and invertebrate
species is frequently employed to monitor water pollution because it provides
meaningful information on bioavailability and effective concentration levels. Of
special concern are genotoxic agents that induce DNA alterations at sub toxic
exposure levels. Clastogenic (chromosome breaking) compounds are responsible
for altered reproductive outcomes, genetic diseases and cancer (Bunton, 1999).

Cytogenetic studies on the effect of copper sulfate and lead
acetate pollution on Oreochromis niloticus fish.

Mohamed, M.M., S.A. El-Fiky, Y.M. Soheir and A.I. Abeer, 2008.
Asian J. Cell Biol., 3: 51-60.
In this study, the detection of mutagenic-carcinogenic pollutants in water by
using cytogenetic methods in fish was examined
along with the necessity of sister chromatid exchange (SCE), anaphase
aberrations (AA) and micronucleus (MN) tests for chemical
analysis in aquatic systems. It has been reported that central mudminnow
(Umbra limi) appear to be the most suitable species
for such analysis because of its large and fewer chromosomes (2n=22) and
high cell division ratio. This species also has a wide distribution,
and can be easily captured and held for study. In such analysis, intestines,
stomach, kidney and gill tissues stand out as
giving superior numbers of usable metaphase and have been widely used.
Detection of Mutagenic-Carcinogenic Pollutants in Aquatic Systems
Using Cytogenetic Methods in Fish

M. ULUPINAR, . OKUMU
Turk J Zool, 26 (2002) 141-148
Genotoxic effects of polychlorinated biphenyls (PCB 153,
138, 101, 118) in a fish cell line (RTG-2).

Marabini L, Cal R, Fucile S.
Toxicol In Vitro. 2011 Aug;25(5):1045-52. Epub 2011 Apr 12.

Polychlorinated biphenyls (PCBs) are persistent pollutants in aquatic environments, often causing the
decline or disappearance of wild populations. The primary aim of this study was to investigate the
genotoxic effects of some PCBs (PCB153 (2,2',4,4',5,5'-hexachlorobiphenyl) and 138 (2,2',3,4,4',5'-
hexachloro-biphenyl), both non-dioxin-like compounds, and the pentachlorobiphenyls PCB118
(2,3',4,4',5-) and 101 (2,2',4',5,5'-), the former an ortho-substituted, low-affinity dioxin-like compound
and the latter a non-coplanar congener classified as non-dioxin-like) in fish cells (RTG-2). These
congeners are mostly present in surface waters and in edible aquatic organisms and the loss of DNA
integrity in vitro serves as a sensitive biomarker of cytogenetic alterations and is considered as an initial
step for the identification of genotoxic effects. The alkaline comet assay and the micronucleus test show
clear genotoxic damage after short and longer exposure (2 and 24h) to maximum soluble, non-cytotoxic
doses, evident sooner with PCBs 101 and 118. Oxidative stress situations involving ROS release,
reduction in total GSH, lipid peroxidation and alteration to superoxide dismutase, seen after exposure
with all the congeners, though with different kinetics, seem the most likely explanation for the genotoxic
damage. This appears to be confirmed by the modified comet assay (pH 10) for detection of oxidized
bases using endonuclease III. The increased generation of intracellular ROS might explain the apoptosis
seen after treatment with the single PCBs and evaluated on the basis of the rise in 3-7 caspase activity.
Therefore both the non-coplanar, non-dioxin-like PCBs (153, 138, 101) and the low-affinity dioxin-like
compound PCB118 cause evident genotoxic damage, probably as a consequence of oxidative stress
Plasmacytoid leukemia in seawater reared chinook salmon
Oncorhynchus tshawytscha
Kent, M. L.; Groff, J. M.; Traxler, G. S.; Zinkl, J. G.; Bagshaw, J. W.
Diseases of Aquatic Organisms 1990 Vol. 8 No. 3 pp. 199-209
A plasmacytoid leukaemia was observed in chinook salmon O. tshawytscha reared in seawater
netpens in British Columbia, Canada. The disease was first observed in market-size salmon (2 to 4
kg) and caused high mortality at several facilities. The disease, referred to as marine anaemia by fish
farmers, is characterized by pallor of the gills due to anaemia, enlargement of the spleen and
kidney, and ascites. Some affected fish exhibited prominent bilateral exophthalmia. Histological
examination revealed massive proliferation of plasmacytoid cells (plasmablasts) in the kidney
interstitium, spleen, intestinal lamina propria, pancreas, liver, and heart. Fish with exophthalmos
exhibited massive proliferation of the plasmablasts in the periorbital connective tissue, ocular
muscles, and choroid gland. In tissue sections, the plasmacytoid cells had large, often lobate or
clefted nuclei, and a moderate amount of amphophilic cytoplasm. In Giemsa imprints, cells had a
smooth contour, contained a large nucleus, an intense staining cytoplasm, and in some cells a
juxtanuclear hof was visible. Electron microscopy and immunohistochemistry revealed the
plasmacytoid features of the proliferating cells. The cells contained a well organized, rough
endoplasmic reticulum, the cisternae being distended with a lightly granular material.
Immunoglobulin was detected in the cells in tissue sections with a goat anti-trout immunoglobulin
peroxidase stain. Although an infectious aetiology (e.g oncogenic virus) for the disease was
suspected, no viruses have yet been detected by cell culture or electron microscopy.