2014 CMA Instabilidade cromossomica.pdf

8/12/2019 2014 CMA Instabilidade cromossomica.pdf

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Instabilidade cromossmica

SUMRIO

- Importncia dos estudos de instabilidadecromossmica (IC)- IC como fator de predisposio para o cancro- Biomarcadores de IC


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Mutao somtica, provocadapor agente genotxico

Estabilidade cromossmica

um estado dinmico das clulas

Mitose: responsvel pela estabilidadegentica durante o desenvolvimento

Estabilidade cromossmica


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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 espontneasErros na replicao; erros durante o crossing-over

Mutaes induzidas

Origem das mutaes somticas

AMBIENTAISQumicos,drogas,radiao ionizante,virus,

stress oxidativo (excesso de ROS)

ENDGENOSROS formados continuamente como co-produtos do metabolismo aerbico normal


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Recrutamento da GSH:Regulador do potencial redox nuclear

Sinalizao de regulao da proliferao celular

Checkpointsdo ciclo

celular:Sinalizao pararepao/apoptose (memriapara reparao)

Organizao estruturaldo cromossoma

Defesa celular

mutaes

Defesa contra ROS-Atividade enzimtica

-Antioxidantes-Fatores de remodelaoda cromatina

ROS

Barreiras s mutaes


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AGENTES GENOTXICOS

ESTABILIDADE CROMOSSMICA

defesa celular

reparao do DNA


Mutaes somticas e estabilidade cromossmica


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AGENTES GENOTXICOS defesa celular

reparao do DNA


Mutaes somticas e instabilidade cromossmica


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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


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mutaes gnicas/cromossmicas sequenciais e cumulativas

afeta a expresso de genes especficos relacionados com cancro

checkpoints, genes de reparaooncogenes supressores

INSTABILIDADE CROMOSSMICA

Instabilidade cromossmica associada ao cancropode ser definida como um

estado contnuo de formao de novas mutaes cromossmicas, a um ndice

superior ao das clulas normais (Gisselsson, 2001)

hiperproliferaocelular

proliferaomaligna

A IC como factor de predisposio para o cancro


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Cancro: mutaes em genes responsveis pela regulao do ciclo de divisocelular

Ativao sequencial de CDKs (cyclin-dependent kinases) por ligao s

ciclinas.

FOS, JUN e MYC so genes deresposta imediata a estmulos

extracelulares. Segue-se aexpresso sequencial das ciclinasD1, E, A e B

O sistema de controlo da divisodepende de:

1. Ativao cclica das protenas cinases(Cdks)

2. Supressoda atividade Cdk porfosforilao inibidora e protenas

inibidoras

A IC como factor de predisposio para o cancro


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Genes responsveis pela regulao do ciclo de diviso celular

Mutaes e cancro

Os rearranjos cromossmicos podem darorigem a aumento, perda ou recolocao

dos genes associados ao cancro

checkpoints, genes de reparaoprotoncogenes supressores

Podem sofrer mutaes:

gnicas cromossmicas


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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


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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


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Alteraes cromossmicas no cancro

Cromossoma Philadelphia

na LMC (leucemia mieloide

crnica)

Fuso dos genesBCReABL

1. Exemplo de fuso de genes (envolve sempre um protoncogene) que d

origem formao de um novo gene (neooncogene)

Translocao 9;22


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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 MYCpor efeito de posio

Translocao

OncogeneMYC Oncogene

MYC

Cadeia pesada IgCadeiapesada Ig


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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


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Evoluo do cancro colo-rectal (adaptao de Kinzler and Vogelstein, 1995)

CICI

CICI

CI

Instabilidade cromossmica (CI) e cancro

O que mais importante na caracterizao do tumor: a alterao clonal ou a IC?


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Distribuio da populao em 4 categorias, quanto ao risco dedesenvolvimento de um tumor(Knudson, 1985)

1 categoria: um nvel irredutvel de cancro, a menorincidncia possvel, inevitvel porque estrelacionada com uma instabilidade endgena

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

3 categoria: inclui tumores que resultam de umarelativa insuficincia gentica para tolerar a exposioaos agentes mutagnicos.

4 categoria: inclui tipos de cancro onde a influnciaambiencial insignificante. o caso de neoplasiasautossmicas dominantes, para as quais a mutaoinicial passa atravs da linha germinal.

maior influncia ambiental

(influncia da idade)


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AGENTES GENOTXICOS


Preveno contra a instabilidade cromossmica ambiental


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Distribuio da populao em 4 categorias, quanto ao risco dedesenvolvimento de um tumor(Knudson, 1985)

1 categoria: um nvel irredutvel de cancro, a menorincidncia possvel, inevitvel porque estrelacionada com uma instabilidade endgena

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

3 categoria: inclui tumores que resultam de umarelativa insuficincia gentica para tolerar a exposioaos agentes mutagnicos.

4 categoria: inclui tipos de cancro onde a influnciaambiencial insignificante. o caso de neoplasiasautossmicas dominantes, para as quais a mutaoinicial passa atravs da linha germinal.

(influncia da idade)

maior influncia gentica


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reparao DNA


SNDROMES DE INSTABILIDADECROMOSSMICA (cancer prone

syndromes)

Expls:Ataxia Telangiectasia- Sensibilidade radiao ionizante

(DNA repair impairment)

Sndrome de Bloom

- Sensibilidade luz UV(DNA repair impairment)

Anemia de FanconiSensibilidade a agentes alquilantes

(DNA repair impairment)

(OS impairment)

Instabilidade cromossmica de origem gentica

defesa contra ROS?

CANCROS DE ORIGEM HEREDITRIA


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Citogentica tumoral

alteraes cromossmicas no clonais (NCCA)

alteraes cromossmicas clonais (CCA)

Instabilidade cromossmica=alteraes cromossmicas no clonais (NCCA)

Importncia das alteraes cromossmicas no cancro


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Toxicidade ambiental nos organismos aquticos

Instabilidade cromossmica nos organismos aquticos

toxinas

? No existe influncia da idade?? No existe influncia gentica?


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Toxicidade ambiental nos organismos aquticos por poluentes na gua



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Toxicidade ambiental por poluentes da gua em excesso

Efeito genotxico nos organismos aquticos

Monitorizao ambiental de genotoxicidade

Taxa de sobrevivnciaTaxa de crescimentoTaxa de reproduo



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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 rearranjosDeteo, a nvel cromossmico, dasmutaes somticas induzidas porpoluentes: biomarcadores citogenticos

Biomarcadores de IC

De utilizao mais generalizada:

teste dos microncleos


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Alteraes a nvel deleso no DNA:- efeito do agentegenotxicoimediatamente a seguir exposio

Alteraes a nvel de lesocromossmica:- efeito do agente genotxicoassociado a exposio delongo termo

Genotoxicity biomarkers in Mytilus galloprovincialis: wild versuscaged mussels

C Bolognesi, G Frenzilli, C Lasagna, E Perrone, P RoggieriMutation Research552 (2004) 153-162

Mtodos utilizados para avaliao de genotoxicidade:

- DNA single strand breaks-Frequncia de microncleos


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Persistence of atrazine impact on aneuploidy in Pacificoysters, Crassostrea gigas

K. Bouilly, H. McCombie, A. Leito, S. LapgueWidespread use of the herbicide atrazine has incited much research on its toxicity in aquaticsystems, where it is routinely detected due to runoff from cultivated fields. Moreover, thedetermination of the genotoxic effect of such pollutants in the marine environment has become amajor 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 agenetic basis. A positive relationship between atrazine and aneuploidy has previously beendemonstrated in C. gigas adults and juveniles. To evaluate the persistence of this impact, our studyexamined 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 seawatercontrol. We observed that these offspring exhibited significantly higher aneuploidy levels when theirparents 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 asample of juveniles, previously exposed for 3.5 months to the same doses of atrazine, thentransferredto non-polluted conditions for an additional period of 2.5 months; this aneuploidy level remainedsignificantly 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 animportant factor causing at least medium-term damage to genetic material.


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Chemical pollution in water especially with heavy metals is among the mostimportant health significance for human beings and animalsconsuming suchwater due to their toxicity and accumulative behavior playing a prominent role inaquatic 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 providesmeaningful information on bioavailability and effective concentration levels. Ofspecial concern are genotoxic agents that induce DNA alterations at sub toxicexposure levels. Clastogenic (chromosome breaking) compounds are responsiblefor altered reproductive outcomes, genetic diseases and cancer (Bunton, 1999).

Cytogenetic studies on the effect of copper sulfate and lead

acetate pollution on Oreochromis niloticusfish.Mohamed, M.M., S.A. El-Fiky, Y.M. Soheir and A.I. Abeer, 2008.Asian J. Cell Biol., 3: 51-60.


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In this study, the detection of mutagenic-carcinogenic pollutants in water by

using cytogenetic methods in fish was examinedalong with the necessity of sister chromatid exchange (SCE), anaphaseaberrations (AA) and micronucleus (MN) tests for chemicalanalysis in aquatic systems.It has been reported that central mudminnow(Umbra limi) appear to be the most suitable speciesfor 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 asgiving superior numbers of usable metaphase and have been widely used.

Detection of Mutagenic-Carcinogenic Pollutants in Aquatic SystemsUsing Cytogenetic Methods in Fish

M. ULUPINAR, . OKUMUTurk J Zool, 26 (2002) 141-148


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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 thedecline or disappearance of wild populations. The primary aim of this study was to investigate thegenotoxic 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 compoundand the latter a non-coplanar congener classified as non-dioxin-like) in fish cells (RTG-2). Thesecongeners are mostly present in surface waters and in edible aquatic organisms and the loss of DNAintegrity in vitro serves as a sensitive biomarker of cytogenetic alterations and is considered as an initialstep for the identification of genotoxic effects. The alkaline comet assay and the micronucleus test showclear genotoxic damage after short and longer exposure (2 and 24h) to maximum soluble, non-cytotoxicdoses, 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 genotoxicdamage. This appears to be confirmed by the modified comet assay (pH 10) for detection of oxidizedbases using endonuclease III. The increased generation of intracellular ROS might explain the apoptosisseen 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-likecompound PCB118 cause evident genotoxic damage, probably as a consequence of oxidative stress
http://www.ncbi.nlm.nih.gov/pubmed?term=%22Marabini%20L%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Cal%C3%B2%20R%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Fucile%20S%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Fucile%20S%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Fucile%20S%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Fucile%20S%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Cal%C3%B2%20R%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Cal%C3%B2%20R%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Cal%C3%B2%20R%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Marabini%20L%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Marabini%20L%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Marabini%20L%22%5BAuthor%5D


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Plasmacytoid leukemia in seawater reared chinook salmonOncorhynchus tshawytscha

Kent, M. L.; Groff, J. M.; Traxler, G. S.; Zinkl, J. G.; Bagshaw, J. W.

Diseases of Aquatic Organisms1990 Vol. 8 No. 3 pp. 199-209

A plasmacytoid leukaemia was observed in chinook salmon O. tshawytscha reared in seawaternetpens in British Columbia, Canada. The disease was first observed in market-size salmon (2 to 4kg) 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 andkidney, and ascites. Some affected fish exhibited prominent bilateral exophthalmia. Histologicalexamination revealed massive proliferation of plasmacytoid cells (plasmablasts) in the kidneyinterstitium, spleen, intestinal lamina propria, pancreas, liver, and heart. Fish with exophthalmosexhibited massive proliferation of the plasmablasts in the periorbital connective tissue, ocularmuscles, 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 asmooth contour, contained a large nucleus, an intense staining cytoplasm, and in some cells ajuxtanuclear hof was visible. Electron microscopy and immunohistochemistry revealed theplasmacytoid features of the proliferating cells. The cells contained a well organized, roughendoplasmic reticulum, the cisternae being distended with a lightly granular material.Immunoglobulin was detected in the cells in tissue sections with a goat anti-trout immunoglobulinperoxidase 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
http://www.cabdirect.org/search.html?q=au:%22Kent,+M.+L.%22http://www.cabdirect.org/search.html?q=au:%22Groff,+J.+M.%22http://www.cabdirect.org/search.html?q=au:%22Traxler,+G.+S.%22http://www.cabdirect.org/search.html?q=au:%22Zinkl,+J.+G.%22http://www.cabdirect.org/search.html?q=au:%22Bagshaw,+J.+W.%22http://www.cabdirect.org/search.html?q=do:%22Diseases+of+Aquatic+Organisms%22http://www.cabdirect.org/search.html?q=do:%22Diseases+of+Aquatic+Organisms%22http://www.cabdirect.org/search.html?q=au:%22Bagshaw,+J.+W.%22http://www.cabdirect.org/search.html?q=au:%22Zinkl,+J.+G.%22http://www.cabdirect.org/search.html?q=au:%22Traxler,+G.+S.%22http://www.cabdirect.org/search.html?q=au:%22Groff,+J.+M.%22http://www.cabdirect.org/search.html?q=au:%22Kent,+M.+L.%22

2014 CMA Instabilidade cromossomica.pdf

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