Avaliação dos Efeitos do Brometo de Etídio sobre os ...livros01.livrosgratis.com.br/cp032301.pdf · I. Resumo 2 I- Resumo O desenvolvimento gera milhares de novos compostos potencialmente

Avaliação dos Efeitos do Brometo de Etídio sobre os Parâmetros de Produtividade, Morfológicos e

Bioquímicos de Drosophila melanogaster (Diptera-Drosophilidae)

REJANE YURIKO OUCHI

2007

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REJANE YURIKO OUCHI

São José do Rio Preto – SP

Fevereiro de 2007

“Avaliação dos Efeitos do Brometo de Etídio

sobre os Parâmetros de Produtividade,

Morfológicos e Bioquímicos de Drosophila

melanogaster (Diptera-Drosophilidae)”

“Avaliação dos Efeitos do Brometo de Etídio sobre os Parâmetros de Produtividade,



São José do Rio Preto – SP

Fevereiro de 2007

REJANE YURIKO OUCHI

Dissertação apresentada ao curso de Pós-Graduação em Biologia Animal (Área de Concentração Biologia Estrutural) da Universidade Estadual Paulista “Júlio de Mesquita Filho”, IBILCE-UNESP, para a obtenção do Grau de Mestre.

Orientador: Prof. Dr. Gustavo Orlando Bonilla Rodriguez

Co-Orientador: Prof. Dr. Carlos Roberto Ceron

Ouchi, Rejane Yuriko.

Avaliação dos efeitos do brometo de etídio sobre os parâmetros de

produtividade, morfológicos e bioquímicos de Drosophila melanogaster

(Diptera-Drosophilidae) / Rejane Yuriko Ouchi. - São José do Rio Preto

-: [s.n.], 2007.

93 f. : il. ; 30 cm.

Orientador: Gustavo Orlando Bonilla Rodriguez

Co-orientador: Carlos Roberto Ceron

Dissertação (mestrado) – Universidade Estadual Paulista, Instituto de

Biociências, Letras e Ciências Exatas.

1. Bioquímica. 2. Biomonitoramento. 3. Drosofila – Bioindicadores.

4. Drosofila – Análise fenotípica. 5. Toxicidade – Testes. 6. Brometo de

etídio – Toxicologia. 7. Etilmetanosulfonato. 8. Carboxilesterase. I.

Bonilla-Rodriguez, Gustavo Orlando. II. Ceron, Carlos Roberto. III.

Universidade Estadual Paulista, Instituto de Biociências, Letras e Ciências

Exatas. IV. Título.

CDU – 577.1

O presente trabalho foi realizado no Laboratório de Bioquímica

de Proteínas do Departamento de Química e Ciências

Ambientais, e no Laboratório de Populações de Drosophila do

Departamento de Biologia, do Instituto de Biociências, Letras e

Ciências Exatas de São José do Rio Preto, da Universidade

Estadual Paulista, sob a orientação do Prof. Dr. Gustavo

Orlando Bonilla Rodriguez e co-orientação do Prof. Dr. Carlos

Roberto Ceron, com auxílio financeiro da CAPES.

São José do Rio Preto

15 de Fevereiro de 2007.

Banca Examinadora para Obtenção do Grau de Mestre:

- Presidente e Orientador: Prof. Dr. Gustavo Orlando Bonilla Rodriguez Universidade Estadual Paulista “Júlio de Mesquita Filho”(UNESP), Departamento de Química e Ciências Ambientais, Campus de São José do Rio Preto - SP - 2º Examinador: Prof. Dr. Paulo Roberto Petersen Hofmann Universidade Federal de Santa Catarina, Campus Universitário – Departamento de Biologia Celular, Embriologia e Genética, Florianópolis - SC - 3º Examinador: Profª. Drª. Hermione Elly Melara de Campos Bicudo Universidade Estadual Paulist “Júlio de Mesquita Filho” (UNESP), Departamento de

Biologia, Campus de São José do Rio Preto - SP

Rejane Yuriko Ouchi

“Avaliação dos Efeitos do Brometo de Etídio

sobre os Parâmetros de Produtividade,



Dedico este trabalho aos meus pais, Aguira e Claudice, e ao meu irmão Elder. Vocês, meus queridos, formam o tesouro mais precioso que tenho e, também, o alicerce e a força propulsora que me conduziram até aqui. É impossível expressar em palavras o imenso amor, carinho e orgulho que sinto de vocês. Amo-os de todo o meu coração e obrigada por tudo!!!

Rejane Yuriko Ouchi

AgradecimentosAgradecimentosAgradecimentosAgradecimentos

Ao término deste trabalho, tentarei expressar com palavras a minha eterna

gratidão, ao permanente apoio e incentivo das pessoas que convivem comigo, e que

tornam o ambiente mais agradável, cheio de calor humano e que, de alguma forma

colaboraram para concretização deste trabalho.

Primeiramente, quero agradecer a Deus pela minha vida, pelas pessoas que

fazem parte dela e pela oportunidade maravilhosa de ter realizado esse trabalho.

Agradeço ainda, pelas forças que recebi quando eu estava cansada e, paciência quando

alguns experimentos não deram certo.

Ao meu querido orientador, grande pesquisador e amigo, Prof. Dr. Gustavo

Orlando Bonilla Rodriguez, por me proporcionar crescimento e amadurecimento

científico. Admiro-o muito por seu profissionalismo e inteligência. Agradeço-te, ainda,

por estar sempre presente acompanhando-me na realização deste trabalho com muita

paciência, incentivando e oferecendo todo apoio necessário.

Ao meu querido co-orientador, Prof. Dr. Carlos Roberto Ceron, que muito

admiro desde a graduação, quando foi meu professor de bioquímica. Mesmo sem muito

tempo disponível, em razão do cargo que ocupa, tentou sempre que possível estar

presente e contribuiu satisfatoriamente com este trabalho. Agradeço-te pelo incentivo,

pela confiança, apoio em todas as etapas.

Á minha querida amiga e técnica do laboratório, Eliane Nobuco Ikeguchi Ohira

pelo incentivo do dia-a-dia, amizade e pelos valiosos conselhos. Admiro-te muito pelo

que você é como pessoa e como profissional.

Á equipe do laboratório, Patrícia Peres, Débora Noma Okamoto, Ana Lúcia

Ferrarezi, Evandro Ricardi e Luciana Puía Moro, pela convivência harmoniosa e

frutífera.

Aos integrantes mais recentes do nosso laboratório Fernanda Facchini, Bárbara

Bonine, Lílian Oliveira, Daniela Cordeiro, Maraíssa Silva Franco, Mayra Delacorte

Polotto e Giovana Gavioli Graciano, pela amizade e pelo agradável ambiente de

trabalho. À minha primeira estagiária, Luciana Delacorte Polotto, pela sua ajuda na

manutenção das linhagens, companheirismo, e amizade.

Aos meus amigos Vítor Baraldi Thomazine, Rafael Marques Paulino, Lívia

Lima e Patrícia Zazeri, todos integrantes do Laboratório de Genética - Bioquímica, pela

amizade e pelo agradável convívio diário.

Ao meu amigo Hamilton Cabral, que me co-orientou durante meu estágio básico

e iniciação científica, por ter contribuído com minha formação acadêmica.

À minha querida e grande amiga, Leliane Silva Commar, pela sua amizade

verdadeira e sincera, pelo seu companheirismo e incentivo. Agradeço por estar comigo

sempre que preciso, com uma palavra de carinho e conforto, nos momentos difíceis e

pelas suas risadas nos momentos de alegria. Agradeço-te ainda, por ter me ensinado a

fazer os meus primeiros géis de esterase e agradeço a Deus por tê-la como minha grande

amiga.

Á Profª Drª Hermione Elly Melara de Campos Bicudo e à Profª Drª Maria

Tercília Varella Azeredo de Oliveira por terem aceito meu convite para compor a minha

banca de qualificação do mestrado e pelas críticas construtivas, que muito contribuíram

para a redação final deste trabalho.

A todos meus professores da graduação e da pós-graduação pelos ensinamentos

transmitidos.

Aos técnicos e amigos Sebastião Dias Barbosa e Paulo Antônio Maziero, pela

colaboração na parte prática deste trabalho, quando realizaram com muito zelo a

preparação dos meios de cultura.

Às funcionárias Dulce Teresinha Campos Trevisan, Marta da Rocha Câmara

Carvalho, Damaris dos Santos Martins pela limpeza dos corredores e do laboratório,

proporcionando um ambiente de trabalho agradável.

À Rita Beatriz de Seixas e à Elisabete H. Habaro, pela eficiência nos momentos

em que precisei de vossa ajuda.

Ao Departamento de Biologia pela manutenção das linhagens.

Ao Prof. Antônio José Manzato pela contribuição na parte estatística.

À Profª. Mary Massumi Itoyama pela ajuda com as tabelas.

Ao Prof. Dr. Eduardo Almeida pela contribuição com protocolos e parte

experimental da carboxilesterase.

Ao MSc. Fernando Rogério Carvalho, pela paciência ao realizar as fotos digitais

de minhas Drosophila melanogaster alteradas. E ao Prof. Dr. Francisco Langeani por

ter permitido a realização das fotos em seu microscópio estereoscópico.

À Adriana Granzotto, que além de amiga e incentivadora deste trabalho, me

ajudou na identificação sistemática das espécies no início desta jornada.

Aos amigos de pós-graduação em biologia animal pela amizade.

À CAPES pela bolsa concedida.

À direção do IBILCE pelas condições materiais que proporcionaram a realização

deste trabalho.

A querida amiga Adenir da Silveira Muniz, que trabalha em minha casa à 16

anos e que diariamente torce e ora por mim.

Às minhas queridas amigas Christiane Marie Mattar Xavier Leal, Andréia

Cristina Fidélis e Fernanda Mauro Bottari pela compreensão de minhas ausências.

Ao meu namorado e amigo, Alessandro Alves Prado, pelo constante incentivo,

amor, paciência, carinho e compreensão.

Ao Misso e a Bianquinha, meus lindos e amados gatinhos, que tornam meus dias

mais felizes e dão à minha vida, um brilho especial.

Aos meus primos Ed Carlos, Elaine, Ewandro, Rafaela, Raphael, Ricardo, Lílian

Sayuri Ouchi de Melo, Ingrid Rey Coelho e Joemir Rey Coelho (in memorian); e aos

meus tios Jorge Ouchi, Meire Jacob Ouchi, Aia Ouchi, Sumie Ouchi, Alice Maria Rey

Coelho, Dirce Aparecida Rey Moura, Alicio Maganha Rey (in memorian), Manoel

Maganha Cabrera (in memorian) e Deolindo Moura, pelo carinho e incentivo.

Aos meus avós Izabel Maganha Rey Jute e Shimi Takaki Ouchi, pelo amor e

carinho. E aos meus avôs Siker Ouchi (in memorian) e Emílio Rey Jute (in memorian),

pelo eterno amor, sei que olham por mim e sentiriam grande orgulho de minha

conquista.

Aos meus pais, Aguira Ouchi e Claudice de Lourdes Maganha Rey Ouchi por

tudo, inclusive pelo incentivo no dia-a-dia, pelo amor constante e incondicional, pelas

palavras de carinho e afeto que são o alicerce de minha vida. São para mim um exemplo

de ser humano, de bondade, amor, luta, e dedicação a serem seguidos. Os ensinamentos

que recebi, e ainda recebo me tornaram o que sou hoje, por dentro (em sentimentos) e

por fora (em atitudes); ensinaram-me à importância da vida, e despertaram em mim a

vontade de crescer e progredir. Esse trabalho é uma pequena forma de retribuir tudo o

que investiram em mim, sendo que essa vitória também é mérito de vocês. Amo muito

vocês...

Ao meu amado irmão, Elder Eizo Ouchi, pelo seu incentivo, amor e carinho. É,

além de meu irmão, meu amigo e companheiro. Sempre que preciso você está ao meu

lado disposto a me ouvir, compreender e dar bons conselhos. Amo-te de todo meu

coração.

Sem minha família, meus familiares e amigos nada disso teria sentido.

“Tens contigo os companheiros certos que te

auxiliam no aperfeiçoamento a que te aspiras”

Emmanuel – Francisco Cândido Xavier.

“A cada dia que vivo mais me convenço de que o desperdício da vida está no amor que não damos, nas forças que não usamos, na prudência egoísta que nada arrisca, e que, esquivando-se do sofrimento, perdemos também a felicidade.”

Carlos Drummond de Andrade

“Quando surge um problema, você tem duas alternativas: ou fica se lamentando, ou procura uma solução. Nunca devemos esmorecer diante das dificuldades. Os fracos se intimidam. Os fortes abrem as portas e acendem as luzes.”

Dalai Lama

“O que sabemos é uma gota. O que ignoramos é um oceano.”

Isaac Newton

“...Ninguém consegue ser realmente grande, quando não aprendeu a ser pequenino .”

Emmanuel

Francisco Cândido Xavier

“Se um dia tiver que escolher entre o mundo e o amor, lembre-se: Se escolher o mundo não terá o amor, mas se escolher o amor, com ele conquistará o mundo.”

Albert Einstein

Sumário

I. Resumo 1

II. Introdução Geral 4

III. Justificativa 14

IV. Objetivos 16

V. Referências

18

Capítulo I: Evaluation of the Effects of a Single Exposure to Ethidium

Bromide in Drosophila melanogaster (Diptera-Drosophilidae).

24

Introduction 26

Materials and Methods 26

Results 28

Discussion 28

Acknowledgements 31

References 31

Capítulo II: Evaluation of Ethidium Bromide Effects in the Life Cycle and

Reproductive Behavior of Drosophila melanogaster.

33

Abstract 35

Introduction 35


Results 40

Discussion 43

Acknowledgements 45

References 45

Capítulo III: Influence of Ethidium Bromide in Daily Productivity,

Morphological and Biochemical Parameters in Ten Generations of Drosophila

melanogaster .

48

Abstract 49

Introduction 50


Results 54

Discussion 60

Acknowledgements 63

References 63

Capítulo IV: Influence of Ethidium Bromide and Ethylmethanesulfonate in

Ten Generations of Drosophila melanogaster.

67

Abstract 68

Introduction 69


Results 73

Discussion 79

Acknowledgements 82

References 82

Conclusões Gerais

87

Anexos

91

Anexo I: Tabela de Produtividade Diária das 10 Gerações da Linhagem

Isofêmea

92

Anexo II: Tabela de Produtividade Diária das 10 Gerações da Linhagem

Massal

93

I. Resumo 1

I. Resumo

I. Resumo 2

I- Resumo

O desenvolvimento gera milhares de novos compostos potencialmente perigosos

com potenciais efeitos biológicos prejudiciais, sendo as mudanças a nível celular e

bioquímico usualmente as primeiras respostas detectáveis da perturbação ambiental.

Para avaliar os efeitos em sistemas biológicos, o presente trabalho utilizou como

bioindicador a Drosophila melanogaster, verificando, ao longo de 10 gerações expostas a

três diferentes concentrações de brometo de etídio (EB): 1) efeitos morfológicos, 2)

alterações bioquímicas (no padrão de proteínas totais, esterase-6 e carboxilesterase), 3)

alterações comportamentais (tempo de cópula e pré-cópula), 4) produtividade (a

emergência diária ao longo de 15 dias) e 5) o efeito nas fases de desenvolvimento.

Na primeira fase, utilizou-se uma linhagem isofêmea de Drosophila melanogaster

exposta a três concentrações (1, 5 e 30 µM) de EB, tendo também um controle e um

controle positivo (1 µM de etilmetanosulfonato ou EMS). Os resultados demonstram que o

EB, assim como o EMS, atua no ciclo de vida desse inseto, principalmente nas primeiras

gerações. Além disso, ocorreu um deslocamento de picos de emergência para a maioria das

gerações. Os dados de produtividade diária para cada geração revelaram uma diferença

significante na quantidade de indivíduos expostos emergidos por dia com relação ao

controle (chegando até 92%, quando se considerou todos os dias de mensuração para 1µM

EB de F5). Além disso, verificou-se também que a freqüência de indivíduos apresentando

alterações morfológicas em relação ao controle foi sempre elevada. As alterações mais

freqüentes foram observadas nas asas, tergitos e pigmentação. Os experimentos com ovos e

larvas demonstram que o EB interfere na viabilidade dos mesmos. Foram também

I. Resumo 3

verificadas alterações bioquímicas, para a esterase-6, entre indivíduos tratados (e portadores

de alterações) e indivíduos controles. Para a F5, a análise da atividade catalítica da

carboxilesterase revelou que não ocorreram diferenças significantes entre machos e fêmeas.

Entretanto, essas diferenças foram verificadas entre controle e o grupo exposto a 1µM de

EB, e entre o controle e o exposto a EMS. Verificaram-se os perfis eletroforéticos de

proteínas totais de indivíduos controle e indivíduos expostos e alterados morfologicamente,

revelando que o grupo exposto apresentou um padrão protéico diferente do grupo controle.

Para a F10 realizou-se, também, a análise do tempo de cópula e pré-cópula, verificando-se

diferenças significantes no tempo de cópula entre o grupo controle e os expostos a 30µM

EB e 1µM EMS. Os grupos expostos apresentaram um efeito acumulativo ao longo das

gerações.

Em relação à linhagem massal, as análises também ocorreram ao longo de 10

gerações, com quantificação da emergência da F1, F5 e F10. A produtividade diária revelou

diferenças significantes, assim como aquelas encontradas na linhagem isofêmea. Na F5 e

F10 verificou-se que a produtividade dos tratados foi sempre menor que a do controle,

sendo para a última geração dependente da dose. Verificaram-se também elevadas

freqüências de alterações morfológicas. Este trabalho mostrou que o EB foi capaz de

induzir alterações significativas em vários aspectos, e com grande variabilidade de resposta

de cada organismo exposto, conseqüência da variabilidade genética e adaptativa existente

nas populações.

II. Introdução 4

II. Introdução

II. Introdução 5

II- Introdução:

A importância do controle da contaminação ambiental cresce a cada ano, na medida

em que as conseqüências da atividade humana assumem dimensões globais, não

circunscritas ao sítio de alteração. A poluição pode, assim, ser definida como qualquer

alteração no meio ambiente habitual, configurando um impacto biológico a curto ou longo

prazo (BONILLA-RODRIGUEZ, 1989). Neste contexto ecológico, o biomonitoramento

pode ser definido como o uso sistemático das respostas de organismos vivos para avaliar as

mudanças ocorridas no ambiente, geralmente causadas por ações antropogênicas

(MATTHEWS et al., 1982).

A utilização de espécies como uma forma de se avaliar as condições ambientais tem

sido verificada com bastante freqüência ao longo da história. Durante a Revolução

Industrial (Século XIX), canários foram colocados dentro das minas para monitorar a

qualidade do ar. Quando esses animais apresentavam algum sintoma desfavorável,

decorrente das elevadas concentrações de monóxido de carbono, as pessoas eram retiradas

do local, evitando possíveis danos à saúde (CAIRNS JR. & PRATT, 1993).

O uso das respostas dos organismos é a base dos índices biológicos. Bioindicadores

são espécies escolhidas por sua sensibilidade ou tolerância a vários parâmetros, como

poluição orgânica ou outros tipos de poluentes (WASHINGTON, 1984). Segundo Johnson

et al. (1993), um indicador biológico ideal deve possuir algumas características como ser

taxonomicamente bem definido e facilmente reconhecível por não especialistas, apresentar

distribuição geográfica ampla, ser abundante ou de fácil coleta, ter baixa variabilidade

genética e ecológica, preferencialmente possuir tamanho grande, dispor de características

ecológicas bem conhecidas e ter possibilidade de uso em estudos de laboratórios.

II. Introdução 6

A resposta biológica apresentada por esses organismos refere-se ao conjunto de

reações de um indivíduo ou uma comunidade em relação a um estímulo ou a um conjunto

de estímulos (ARMITAGE, 1995).

Na utilização do biomonitoramento para analisar os aspectos biológicos dos

ecossistemas, duas metodologias vem sendo utilizadas. Os métodos “bottom-up” utilizam

fundamentalmente dados de laboratório por meio de experimentação em sistemas simples

com subseqüente extrapolação para sistemas mais complexos. Já a metodologia “top-

down” avalia, em nível macro, os impactos ambientais por meio da medição da alteração da

organização estrutural e funcional das comunidades biológicas ou dos ecossistemas.

Os testes da metodologia bottom-up são realizados, em geral, com base nas

respostas de organismos aquáticos a agentes estressantes específicos. Nesses casos, são

utilizados como indicadores respostas bioquímicas (enzimáticas, por exemplo), fisiológicas,

metabólicas e do ciclo de vida (BUIKEMA & VOSHELL, 1993; CALOW, 1993;

ROSENBERG & RESH, 1993; BOUDOU & RIBEYRE, 1997; PIVETTA et al., 2001). O

uso das respostas fisiológicas é conhecido como teste toxicológico, e a avaliação em

laboratórios envolve a análise da exposição crônica, analisando os efeitos deletérios quanto

à genotoxicidade, carcinogenicidade e mutagenicidade (REYNOLDSON & METCALFE-

SMITH, 1992).

A abordagem top-down aplica-se ao manejo dos ecossistemas, proporcionando

controle e velocidade nas reações de testes de toxicidade (MOULTON, 1998).

A exposição contínua de qualquer organismo a agentes estressantes acarreta efeitos

adversos à saúde. Os efeitos biológicos em nível de organismo, em resposta a estes agentes,

sempre são precedidos por eventos bioquímicos e celulares, e, dessa forma, os parâmetros

celulares e bioquímicos têm um grande potencial para serem utilizados como indicadores

II. Introdução 7

de estresse a fim de avaliar as condições fisiológicas de um organismo (STEGMAN et al.,

1990). Os organismos tentam driblar estas condições estressantes ativando genes para

produzir proteínas específicas (NAZIR et al., 2003a), e a expressão de tais genes poderá

minimizar os efeitos do índice de estresse (ATKINSON & WALDEN,1985).

Em alguns casos, os mecanismos de defesa celular envolvem a indução de proteínas

adequadas denominadas de heat shock proteins (Hsp) ou proteínas de estresse térmico

(ATKINSON & WALDEN, 1985). Muitos estudos mostraram que essas proteínas

respondem a um agente estressante (poluente) mesmo em pequenas concentrações, sendo

esse um dos motivos pelo qual essas proteínas são utilizadas como bioindicadores

(BIERKENS, 2000). Atualmente, estudos demonstraram a potencialidade da Hsp70 no

monitoramento da poluição utilizando a abordagem transgênica tanto in vivo como in vitro

(MUKHOPADHYAY et al., 2003). Assim, nas últimas décadas, muitas pesquisas foram

focalizadas na Hsp como biomarcador. Elevados níveis de Hsp70 foram encontrados em

células de organismos expostos a pesticidas (CHOWDHURI et al., 1999; NAZIR et al.,

2001), solventes (NAZIR et al., 2003b), metais (GIBNEY et al., 2001), além do aumento da

temperatura (KREBS & FEDER, 1997).

Entretanto, apenas uma combinação de muitas classes de proteínas que respondam

ao estresse fornecerá bioensaios suficientemente sensitivos para a maioria das classes de

poluentes ambientais (BIERKENS, 2000), abrangendo, portanto, outras proteínas além das

Hsp. Assim, o estudo da atividade enzimática é uma alternativa como um critério de

avaliação da toxicidade de alguns resíduos químicos em sistemas biológicos.

Freqüentemente a exposição a poluentes químicos produz espécies reativas de

oxigênio (EROs), e algumas enzimas, caracterizadas como antioxidantes, protegem direta

II. Introdução 8

ou indiretamente as células contra os efeitos adversos de xenobióticos, drogas,

carcinógenos e reações com radicais tóxicos (HALLIWELL, 1995).

Mesmo no funcionamento celular normal, são produzidas as espécies reativas de

oxigênio, incluindo radicais hidroxilas (OH-), ânions superóxidos (O2-), peróxido de

hidrogênio (H2O2) e óxido nítrico (NO). Estas são espécies transitórias que devido a sua

elevada reatividade química levam à peroxidação de lipídios, oxidação de algumas

enzimas, oxidação e degradação maciça de proteínas (MATÉS & SÁNCHES-JIMÉNEZ,

1999). Esses metabólitos derivados de oxigênio podem causar patologias devido a danos ou

morte celular (TAMAGNO et al., 1998).

A prevenção da oxidação é essencial para todos os organismos aeróbios, pois o

decréscimo da proteção antioxidante levará a citotoxicidade, mutagenicidade e/ou

carcinogenicidade. Dessa maneira, moléculas pequenas que mimetizam enzimas

antioxidantes estão se tornando alternativas de tratamento de muitas doenças (MATÉS,

2000).

Além das enzimas envolvidas no metabolismo oxidativo citadas acima, os

organismos apresentam um grupo de enzimas multifuncionais, as esterases, que participam

da hidrólise de ésteres e compostos de origem xenobiótica. Nos insetos, estas enzimas estão

envolvidas em vários processos metabólicos, incluindo a degradação de inseticidas

organofosforados e carbamatos, bem como, a degradação de feromônios e da regulação dos

níveis de hormônio juvenil (KORT e GRANGER, 1981; JONES et al., 1994;

RAUSCHENBACH et al., 1994; SPACKMAN et al., 1994). As esterases encontradas nos

insetos são altamente polimórficas, o que justifica suas múltiplas funções no metabolismo

desses organismos. Nos insetos uma classe de esterases, carboxilesterases dependentes de

serina, com ampla especificidade de substratos, intervém na depuração de xenobióticos e na

II. Introdução 9

ativação de pró-drogas, de ésteres ou amidas (http://www.pdamed.com.br/diciomed/pdamed_0001_1269.php ,

acessada em 06 de janeiro de 2007).

Nas últimas décadas, a utilização de animais para pesquisas de testes toxicológicos

envolve dois conceitos fundamentais: a ciência e a ética. Assim, meios alternativos vêm

sendo utilizados na pesquisa (MUKHOPADHYAY et al., 2004).

Atualmente, espécies de Drosophila são organismos modelos para os estudos

toxicológicos por serem bem definidas geneticamente, sendo bem caracterizadas em termos

de desenvolvimento biológico, por seu genoma ser facilmente manipulado

(MUKHOPADHYAY et al., 2004), e pela sua elevada sensibilidade para detectar a

presença de substâncias tóxicas. Outros fatores que levam a sua utilização decorrem do fato

de serem insetos de fácil criação e manutenção (alimentam-se principalmente de bactérias e

leveduras que participam da fermentação de substratos ricos em carboidratos, tais como

frutos em decomposição) em condições de laboratório, apresentando, assim, vantagens

frente a outros organismos para a realização de bioensaios (ALMEIDA et al., 2001).

Além disso, a utilização de Drosophila é recomendada pelo European Center for

Validation of Alternative Methods (ECVAM), que tem por finalidade promover a aceitação

científica e regular dos métodos alternativos que reduzem, refinam e substituem o uso de

animais em laboratórios (MUKHOPADHYAY et al., 2004).

O uso de insetos, especificamente Drosophila, para monitoramento de danos

genéticos causados por agentes químicos tem tradicionalmente mais de 50 anos. Entretanto,

durante os 10 últimos anos, experimentos utilizando Drosophila estão relacionados com

atividades estruturais dos agentes genotóxicos, tendo como objetivo a identificação de

II. Introdução 10

carcinógenos e como um modelo para estudo de mecanismos de mutagenicidade de

produtos químicos (VOGEL et al., 1999).

Por exemplo, o etanol adicionado em diferentes concentrações ao meio de cultura de

Drosophila, ocasionou a malformação de patas (segmentos faltando, ausentes

completamente ou distorcidos), asas, halteres e partes bucais fundidas (RANGANATHAN

et al., 1987). A cipermetrina, um potente inseticida, promoveu o aumento significante no

dano do DNA nas células do gânglio cerebral médio e anterior (MUKHOPADHYAY et al.,

2004). Alguns metais pesados, como chumbo e cádmio, são neurotóxicos, causando um

atraso no desenvolvimento da fase larval para a pupa (AKINS et al., 1992). O solvente

Dimetilsulfóxido (DMSO) causou proporcionou tanto um efeito citotóxico como na

performance reprodutiva em Drosophila melanogaster transgênica (NAZIR et al., 2003b).

Na presença de nitrato de prata fêmeas de uma linhagem selvagem de Drosophila

melanogaster mostraram-se mais sensíveis a este agente químico do que os machos

(HEMMAT & SEMNANI, 2003). Alguns outros produtos, dentre eles acetamida,

acrilamida, benzopireno, ciclofosfamida, dietilestilbestrol, propilenoimina, tiouréia e o-

toluidina apresentaram propriedades carcinogênicas em Drosophila melanogaster

(BATISTEALENTORN et al., 1995).

Outros agentes com alta genotoxicidade foram testados em Drosophila, dentre eles:

dietilestilbestrol, difenil-hidantoína, imipramina, testosterona e tolbutamida, que têm um

elevado potencial teratogênico, cujos efeitos são evidenciados nos músculos e neurônios

(BOURNAIS-VARDIABASIS et al., 1983).

Outros estudos mostraram que em larvas de D. melanogaster, quando expostas à

azida sódica (um potente produto mutagênico), ocorre a indução de mutações somáticas e

recombinação mitótica nas células das asas (GONZÁLEZ-CÉSAR e RAMOS-MORALES,

II. Introdução 11

1997). Este mesmo efeito foi observado ao realizarem-se testes com alguns inseticidas

organofosforados, metilparation, azametifós, diclorvós e diazinon (EKEBAS et al., 2000).

Outras substâncias químicas, ou metais como cádmio (RIZKI et al., 2004), citrato de

tamoxifeno e 4-nitroquinolina-1-óxido (HERES-PULIDO et al., 2004), simazina

(TRIPATHY et al., 1995) e alguns hidrocarbonetos aromáticos policíclicos (DELGADO-

RODRIGUEZ et al., 1995) demonstraram suas propriedades genotóxicas alterando o

padrão de pintas das asas de D. melanogaster.

O processamento de substâncias com alto potencial tóxico ou mutagênico, como o

brometo de etídio (EB) e fenol, tem sido um dos grandes problemas da maioria dos

laboratórios. O EB é comumente utilizado nos laboratórios para corar ácidos nucléicos

submetidos à eletroforese em gel de agarose ou a gradiente de cloreto de césio. A ação do

EB como corante deve-se à sua capacidade de intercalar entre as bases dos ácidos nucléicos

e fluorescer sob luz ultra-violeta. Este caráter intercalante do EB também é o responsável

pelo seu alto potencial mutagênico, pois pode gerar alterações na estrutura do DNA as

quais poderão ser perpetuadas durante o processo de duplicação (Figura 1). Dessa forma, a

leitura errada das seqüências de bases do DNA pode resultar em mutações E subseqüente

alterações genéticas e bioquímicas.

Em decorrência de sua potencial toxicidade, existem várias técnicas para

descontaminação do EB, sendo que a mais simples, e de fácil aplicação, é baseada no

tratamento de soluções de EB com permanganato de potássio em condição de acidez. Este é

um método capaz de reduzir a atividade mutagênica em até 3.000 vezes. Entretanto, no

caso de soluções usadas no descoramento dos géis, onde existe uma baixa concentração de

EB, utiliza-se o hipoclorito de sódio (água sanitária comercial) na proporção de 1:2, antes

II. Introdução 12

do descarte em pia (TEIXEIRA et al., 1998), que, no entanto, se revela ineficaz, por

produzir novas substâncias mutagênicas1.

Figura 1: Representação do processo de intercalação do Brometo de Etídio (a) na dupla hélice do DNA

(b).

Apesar de ser considerado mutagênico, o EB não consta na lista da Agência

Internacional de Pesquisa do Câncer (IARC) como um dos possíveis agentes com

potencialidade carcinogênica para humanos2, de forma que decidimos testar seus efeitos em

D. melanogaster.

Contudo, alguns estudos foram encontrados justificando a razão pela qual o EB não

é muitas vezes considerado carcinogênico. Surpreendentemente, alguns trabalhos propõem

que este composto poderia ser tratado como um agente anticancerígeno (NISHIWASKI et

1 Profa. Dra. Mary Rosa Rodrigues de Marchi, Departamento de Química Analítica do Instituto de Química da UNESP de Araraquara, comunicação pessoal. 2 http://monographs.iarc.fr/ENG/Classification/index.php, acessada em 16 de janeiro de 2007

II. Introdução 13

al., 1974), embora esse potencial não seja explorado pelo fato de provocar mutações em

alguns animais (MACCANN et al., 1975).

Há, na literatura, alguns trabalhos analisando o EB em Drosophila. Entretanto, esses

não enfocam as alterações fenotípicas e bioquímicas que são o alvo de estudo deste

trabalho, de tal forma que nossa abordagem é inédita.

Os efeitos das mutações sobre as alterações fenotípicas variam enormemente.

Substituições silenciosas, bem como outros tipos podem não ter efeitos perceptíveis,

embora códons sinônimos possam ter efeitos diversos sobre a taxa de tradução de mRNA

em proteínas (FUTUYMA, 2002). Assim, testes bioquímicos refinados são uma das formas

de se avaliar os efeitos de mutágenos sobre o genoma dos indivíduos.

Ao contrário doEB, alguns mutágenos não são incorporados ao DNA, mas

sim são capazes de provocar alterações químicas diretamente nas bases nitrogenadas,

causando um mal pareamento específico. Alguns agentes alquilantes, tais como o Etil

Metanossulfonato (EMS) e o nitrosoguanidina (NG) operam por essa via. Embora tais

agentes adicionem grupos alquil (um grupo etil, no caso do EMS) em muitas posições de

todas as quatro bases, a mutagenicidade é mais bem correlacionada a uma adição ao

oxigênio, na posição seis, da guanina para criar uma O-6-alquilguanina. Isso leva a um mal

pareamento direto com timina e resultaria em transições GC→AT na próxima rodada de

replicação (GRIFFITHS et al., 1998).


III. Justificativa


III- Justificativa:

O monitoramento ambiental é absolutamente essencial para identificação de

produtos que tragam riscos à vida e para prevenir a degradação do ambiente. Quando se

leva em consideração os agentes químicos tóxicos e os seus impactos ambientais, muitos

produtos deveriam receber um tratamento específico antes de serem lançados no meio

ambiente. Entretanto, muitos dos resíduos gerados têm sua propriedade toxicológica

desconhecida, devendo ser estabelecidas abordagens de avaliação nesse sentido.

Biomonitores e biomarcadores, combinados, oferecem uma forma pela qual se pode

avaliar as condições do meio em que vivemos. A utilização de invertebrados, neste caso

Drosophila melanogaster, traz algumas vantagens nas investigações ecotoxicológicas

(impactos ambientais de poluentes) que promovem a elucidação de mecanismos vinculados

aos efeitos que ocorrerão em nível individual, assim como também suas conseqüências em

outros níveis estruturais de organização biológica (LAGADIC E CACQUET, 1998); como,

por exemplo, a bioacumulação específica de produtos químicos poluentes e seus efeitos

tóxicos em um indivíduo, numa população e na comunidade.

Sabe-se que o brometo de etídio, devido à sua capacidade intercalante no DNA,

teria o potencial de alterar o processo de leitura durante a transcrição e isto, eventualmente,

poderia causar mutações ou outras modificações em nível do DNA, perceptíveis ao nível

morfológico, de produtividade, comportamental e/ou bioquímico.

IV. Objetivos 16

IV. Objetivos

IV. Objetivos 17

IV.Objetivos:

No presente estudo objetivou-se realizar a determinação da toxicidade do brometo

de etídio (EB), utilizando-se Drosophila melanogaster como bioindicador, analisando-se o

seu efeito em duas diferentes linhagens, ao longo de 10 gerações. Foram realizadas

comparações entre grupos expostos ao EB e dois grupos controles, sendo deles um negativo

(não submetido à exposição química) e o outro positivo (submetido à exposição ao EMS –

Etilmetanosulfonato, composto mutagênico). Dessa forma analisamos:

- O efeito do EB na produtividade diária e no perfil ao longo de 15 dias de

contagem,

- Eventuais alterações morfológicas (deformações, alteração no padrão de

coloração, de posicionamento de estruturas corpóreas), dos indivíduos adultos recém

emergidos, bem como analisar a freqüência com que essas alterações ocorrem.

- A atividade de esterases, bem como o padrão de proteínas totais, utilizando

técnicas eletroforéticas.

- Paralelamente, possíveis alterações em termos comportamentais, baseando-se nas

observações do tempo médio de pré-cópula e cópula.

- A influência do EB durante o desenvolvimento embrionário, viabilidade ovo-

adulto, larva-adulto e pupa-adulto.

V. Referências 18

V. Referências

V. Referências 19

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Evaluation of the Effects of a Single Exposure to Ethidium Bromide in Drosophila melanogaster (Diptera-Drosophilidae) 24

Capítulo I

“Evaluation of the Effects of a

Single Exposure to Ethidium

Bromide in Drosophila

melanogaster (Diptera-

Drosophilidae)”

Artigo submetido à Revista: Bulletin of Environmental Contamination

and Toxicology

Evaluation of the Effects of a Single Exposure To Ethidium Bromide in Drosophila melanogaster (Diptera-Drosophilidae) 25

Evaluation of the Effects of a Single Exposure to Ethidium Bromide in

Drosophila melanogaster (Diptera-Drosophilidae).

R. Y. Ouchi1, J. A. Manzato2, C. R. Ceron3, G. O. Bonilla-Rodriguez3 1 Master degree student, 2 Department of Computing and Statistical Sciences, 3 Department of Chemistry and Environmental Sciences, IBILCE/UNESP, State University of São Paulo, Rua Cristovão Colombo 2265, São José do Rio Preto, SP, CEP 15054-000, Brazil.

Correspondence to: Gustavo O. Bonilla-Rodriguez, e-mail [email protected], Telephone (5517) 3221-2361, Fax 3221-2356


Each year new chemicals enter in the market and generate an increasing volume of residues, leading to health and environmental concerns. Although many of those compounds are known as toxic, a significant proportion do not have a proper hazard classification; they are potentially dangerous and able to generate harmful biological effects. Biochemical changes can be often translated as modifications in the morphology, behavior, or metabolic pathways, analyzed in a species known as bioindicator (Washington, 1984). In this ecological context, biomonitoring can be defined as the use of systematic responses of live organisms in order to evaluate the changes in environment, generally caused by human actions (Mathews et al., 1982). Over the past decade, issues such as animal handling and care in toxicology research and testing became one of the fundamental concerns for both science and ethics. Emphasis has been given to the use alternatives to mammals in testing, research and education. Drosophila melanogaster is the most widely used insect model because of well-elucidated genetics and developmental biology. Moreover, the use of Drosophila has been recommended by the European Centre for the Validation of Alternative Methods (ECVAM) with the purpose of reducing, refining or replacing the use of laboratory animals (Benford et al., 2000). Among thousands of residues generated by research laboratories, we have chosen to analyze the toxic effects from Ethidium Bromide (EB). That is the common name for 3,8-diamino-5-ethyl-6-phenylphenanthridinium bromide, an intercalating agent usually used in molecular genetics and in structural studies of DNA and chromatin. Heinen (1978) showed that EB inhibits cell growth in tissue culture, even at very low concentrations; but in spite of this, EB is not used as an antitumoral agent because it has mutagenic capacity in some organisms. The results in bacteria show that EB is an effective frameshift mutagen if it is metabolically activated by liver microsomes (McCann et al., 1975). Sea urchin eggs exposed to water containing 50µM or more of ethidium bromide develop chromosomal abnormalities and fail to divide normally (Vacquier and Brachet, 1969). In mice, EB apparently has little or no access to nuclear DNA, at least in

vivo, while it intercalates perfectly well with isolated nuclear DNA in vitro (Pack and Loew, 1978). In the present study we have investigated the influences of different concentrations of EB on productivity, morphological alterations and biochemical analyses based on esterase-6 activity, using Drosophila melanogaster as a bioindicator. Furthermore, we verified the action of EB in two developmental stages in this insect life cycle. MATERIALS AND METHODS

Specimens of Drosophila melanogaster were collected in May 2005, at São José do Rio Preto (State of São Paulo, Brazil). One female has originated the isofemale stock which was maintained in a temperature controlled chamber at 24°C ± 1°C.


We used three different concentrations of EB (1, 5 and 30µM) and two control groups. One µM EB corresponds to the concentration used for visualization of nucleic acids, a solution that is frequently disposed in the drain without chemical neutralization. For the positive control we used 1 µM EMS (Ethyl methanesulfonate), a mutagenic chemical, whereas the negative control was only fed with uncontaminated culture medium. The chemicals were fully mixed with warm (45ºC) 50 mL of the banana-agar culture medium, and then poured into a 250 mL glass bottle. For each treatment four replicates were prepared; three of them were used for the productivity experiments and the fourth one for the experiment of larval viability. For each bottle, twelve males of the Drosophila melanogaster stock were joined to the same number of virgin females. The treated culture medium was used as substrate for feeding and females were allowed to oviposit for 6 days. After that, the adults were removed. Ten days after the parents were added to the glass bottle, the new adult generation initiated its emergence. During fifteen days the adults were counted twice a day and morphologically analyzed with a stereoscopic microscopy (Carl Zeiss). Some adult females and males, and all the flies that displayed morphological alterations, were kept frozen at -20°C for later electrophoretical analysis. At the end of that period, pupae that did not emerged as flies and remained attached to the bottle wall were counted, and this allowed us to quantify the effect of EB on the insect viability. The proportion of flies carrying morphological alterations was calculated as the ratio of the total Drosophila emerged on each treatment. The statistical analyses were performed using the program Bioestat 4.0 (Ayres et al., 2005) in order to analyze the daily productivity. For this purpose, we applied the test of equality of two proportions (Normal approach Z) for independent samples, used for parametric data (Moore, 2005), using a p ≤0.05 significance level. The fourth replica was used to collect larvae for viability experiments. Ten glass tubes containing 7 mL of treated banana-agar culture were used for each treatment, and to each one we added ten larvae. After a few days, adults initiated their emergence and were analyzed quantitatively and morphologically in a stereoscopic microscope. After carrying out morphological and quantitative analyses, the pattern of total proteins and esterase-6 activities were verified by electrophoresis, with a specific staining for each one. For this experiment were used some males and females of the negative control and also flies displaying morphological alterations. Each fly was homogeneized in 0.2M Tris-HCl pH 8.8 buffer. For electrophoresis we used 0.1M Tris-Glicine pH 8.3, setting voltage to 180V during 4 hours. The gels were stained twice: first for esterase-6 using β-naphtyl acetate according to Galego et al. (2006) for 90 minutes. After that, it was submerged in a solution of 20% ethyl alcohol and 20% acetic acid for one hour, and subsequently stained with Coomassie Brilliant Blue R-250.


RESULTS AND DISCUSSION In order to analyze the effect of the first exposition of Drosophila melanogaster to ethidium bromide, the productivity was measured along 15 days. The total numbers of flies produced by the three replicas are shown in Table 11, where the asterisks point to significant differences between the exposed groups and the negative control. Accordingly, we can notice that most of days for the ethidium bromide and EMS treatment have demonstrated statistical differences on productivity. Remarkably, the groups exposed to 1 and 5µM EB have a different profile with delayed emergence, and suggests that it would continue beyond the 15th day. The alterations in the normal morphological patterns affected mainly tergites and wings. However, other malformations were found, such as the absence of one paw and different body pigmentation. These alterations are not shown in this work. Table 1. Proportion of daily productivity (from three replicates). The asterisks (*) indicates significant differences compared to the negative control (p<0.05).

Treatment Total Daily Productivity

(days)

Control 1µM EB 5 µM EB 30 µM EB EMS

1st 0.0023 0.0000 0.0000 0.0000 0.0017 2nd 0.0047 0.0000 * 0.0068 * 0.0061 * 0.0000 3rd 0.1707 0.0518 * 0.0408 0.0940 0.0468 4th 0.2014 * 0.1703 0.0884 * 0.2495 * 0.1875 5th 0.0632 * 0.1296 * 0.0725 * 0.0961 * 0.0642 6th 0.0913 * 0.1926 * 0.2948 * 0.1022 * 0.0642 7th 0.2529 * 0.2962 * 0.3288 0.1513 0.0920 8th 0.1428 * 0.1333 * 0.1338 0.0859 0.1302 9th 0.0304 * 0.0148 0.0227 0.1350 0.1701

11th 0.0374 * 0.0000 * 0.0068 0.0695 0.1441 13th 0.0000 * 0.0111 0.0045 0.0102 0.0764 15th 0.0023 0.0000 0.0000 0.0000 0.0226

Table 2. Viability after a first exposure to Ethidium Bromide and Ethyl methanesulfonate.

Treatment Total number of emerged

adults

Larval Viability

(%)

Pupal Viability

(%)

Frequency of Alterations

(%) Control 463 89 98.7 0.21 1µM EB 752 82 97.8 1.69 5 µM EB 917 86 97.7 1.53 30 µM EB 386 84 98.2 0.26 EMS 288 74 93.5 1.40

1 Para os números totais, veja o anexo 1


On the other hand, we cannot analyze just one parameter to infer if the chemicals are expressing their toxicity properties on this species. So, other points were verified (shown on Table 2). It is noteworthy that the group exposed to 30µM of EB has the same amount of significant days as EMS, when compared to the control. In spite of this, considering 30µM EB and control, the frequency of alterations are approximately the same; the larval viability was lower in 30µM EB compared to the negative control, and the total productivity was 17% lower. For the other treatments (1 and 5µM EB and 1µM EMS) the larval viability was always lower than for the control. However, the frequency of alterations is seven to eight times larger than for the control. Moreover, some pupae did not emerge as adult flies, and the groups exposed to 1 and 5 µM EB revealed a larger emergence than the control. These experiments revealed that the exposition of ethidium bromide was not dose-dependent, as the proportion of alterations, and larvae and pupae viability do not display a clear trend with concentration. The viability from egg to larvae was more affected by the chemical. When the curve of daily total productivity is analyzed, it is possible to verify that the profile of the curve of the group exposed to the highest EB concentration (30µM) is similar to that obtained in the presence of EMS (Figure 1). Moreover, the alterations proportion was similar in for the groups exposed to 1 and 5µM of EB and EMS. Adult specimens of male Drosophila melanogaster fed with EMS presented a high frequency of recessive lethal mutations and also polygenic mutations affecting viability (Ohnishi, 1977). This could be an explanation for the results observed in some concentrations of ethidium bromide and EMS. EMS is known to produce base-pair substitutions and chromosome changes (Mukai, 1970). Mutation and chromosome breaking effects have been reported by Alderson (1965), Epler (1966) and Jenkins (1967). The pattern of emergence in the presence of 1µM and 5µM of EB shows a significant increase compared to negative control. Mukai (1964) reported a similar effect for EMS. According to him, when all the mutations are located on the same chromosome, the viability is high, even showing overdominance of the mutants; but would be low, showing a partial dominance, when the mutants are distributed between both homologs. In order to understand biochemical changes induced by EB, the pattern of esterase-6 was analyzed by electrophoresis, of some normal and abnormal females and males (figure 2). The activity of esterase of both abnormal male and female specimens was lower than for the normal flies. According to Marcos et al. (1981), 3mM of EB can induce dominant lethals, sex-linked recessive lethals, non-disjunction, loss of X or Y chromosomes and translocations between the second and third chromosomes. Moreover, since we performed two different stains in the same gel, the presence of spots not found among normal flies suggests that some others proteins are produced or modified by the exposure to ethidium bromide. However, for the same alteration, in wings for example, different patterns are observed (specimens 6-13, figure 2). Goncharova et al. (1988) revealed that Drosophila melanogaster cells can have an individual


sensitivity to the presence of a mutagen. In order to compare data of productivity and esterase-6, we could suppose that the presence of EB has influenced in its expression, becoming inferior, so exposed males transfer to female less esterase-6, during the copula. The treated females, which have received less of this enzyme of the male, could copulate with other males, increasing the productivity, as observed in 1 and 5 µM EB. Figure 1. Total daily emergence (females and males) for a single exposure of Drosophila melanogaster larvae and pupae to EB and EMS.

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

1 2 3 4 5 6 7 8 9 11 13 15Days

Pro

po

rtio

n o

f E

me

rge

nc

e

Control

1uM EB

5uM EB

30 uM EB

EMS

Figure 2. Electrophoresis gel showing esterase activity (α and β esterase) and the pattern of total proteins for individual samples. 1,2: control males; 3: males with wings alterations; 4,5: control females; 6-13: females with wings alterations.

1 2 3 4 5 6 7 8 9 10 11 12 13

β-Est

α-Est

Total Protein

α-Est


In conclusion, ethidium bromide, even in low concentrations, can induce toxic effects in terms of productivity, morphologic and biochemical parameters, presumably due to its genotoxic properties. However, in Drosophila melanogaster these effects were not dose-dependent. The different sensibility of separate individuals to mutagens reflects the existence of cryptic genetic variability in Drosophila strains. It is relevant to take into account individual sensitivity of organisms to mutagenic factors, when conducting mutation research and studying genetic consequences of biosphere pollution. Acknowledgments. This work received financial support from FAPESP (05/02418-6) and CNPq (GOBR). R.Y. Ouchi was granted with a fellowship from CAPES.

REFERENCES Alderson TA (1965) Chemically induced delayed germinal mutations in

Drosophila. Nature 207: 164-167. Ayres M, Ayres MJr, Ayres DL, Santos AS (2005) BioEstat 4.0: Aplicações

Estatísticas nas Áreas das Ciências Biológicas e Médicas. Editora: Sociedade Civil Mamirauá/MCT/Imprensa Oficial do Estado do Pará, Brazil.

Benford DJ, Hanley BA, Bottrill K (2000) Biomarkers as predictive tools in toxicity testing. Altern Lab Anim 28: 119-131.

Epler JL (1966) Ethyl methanesulfonate induced lethals in Drosophila. Frequency-dose relations and multiple mosaicism. Genetics 54: 31-36.

Galego LG, Ceron CR, Carareto CM (2006) Characterization of esterases in a Brazilian population of Zaprionus indianus (Diptera-Drosophilidae). Genetica 106: 89-99.

Goncharova RI, Levina AB, Kuzhir TD (1988) Sensitivity of individual Drosophila to the mutagenic action of ethyl methanesulfonate. Genetika 24: 2141-2148.

Heinen E (1978) Effects of antimitotic agents either free or bound to DNA on mouse peritoneal macrophages cultivated in vitro. Virchows Arch B Cell Pathol 27: 79-87.

Jenkins JB (1967) Mutagenesis at a complex locus in Drosophila with the monofunctional alkylating agent, ethyl methanesulfonate. Genetics 57: 783-793.

McCann J, Choi E, Yamasaki E, Ames BN (1975) Detection of carcinogens as mutagens in the Salmonella/microsome test: assay of 300 chemicals. Proc Natl Acad Sci 72: 5135-9.

Marcos R, Creus A, Xamena N, López de Sepúlveda J (1981) Effect of ethidium bromide on Drosophila melanogaster and Drosophila simulans. Experientia 37: 559-560.


Mathews RA, Buikema AL, Cairns JrJ (1982) Biological monitoring part IIA: Receiving system functional methods relationships, and indices. Water Research 16: 129-139.

Moore DS (2005) A estatística básica e sua prática. LTC 3rd ed. p 419-420, Rio de Janeiro, Brazil.

Mukai T (1964) The genetic structure of nature populations of Drosophila

melanogaster. I. Spontaneous mutation rate of polygenic controlling viability. Genetics 50:1-19.

Mukai T (1970) Viability mutations induced by ethyl methanesulfonate in Drosophila melanogaster. Genetics 65: 335-348.

Ohnishi O (1977) Spontaneous and ethyl methanesulfonate induced mutations controlling viability in Drosophila melanogaster. I. Recessive lethal mutations. Genetics 87: 519-527.

Pack GR, Loew G (1978) Origins of the specificity in the intercalation of ethidium into nucleic acids. A theoretical analysis. Biochim Biophys Acta 519: 163-172.

Vacquier VD, Brachet J (1969) Chromosomal abnormalities resulting from ethidium bromide treatment. Nature 222: 193-195.

Washington HG (1984) Diversity, biotic and similarity indices. A review with special relevance to aquatic ecosystems. Water Research 18: 653-694.

Evaluation of Ethidium Bromide Effects in the Life Cycle and Reproductive Behavior of Drosophila melanogaster 33

Capítulo II

“Evaluation of Ethidium

Bromide Effects in the Life

Cycle and Reproductive

Behavior of Drosophila

melanogaster”

Artigo a ser futuramente submetido: Toxicology Letters


Evaluation of Ethidium Bromide Effects in the Life Cycle and Reproductive

Behavior of Drosophila melanogaster.

R.Y. Ouchi1, D.N. Okamoto

1, Carlos R. Ceron

2, A.J. Manzato

3, G.O. Bonilla-

Rodriguez2

1. Master degree student, 2. Department of Chemistry and Environmental Sciences, 3.

Department of Computing and Statistical Sciences, IBILCE-UNESP, State University

of São Paulo, Rua Cristóvão Colombo 2265, São José do Rio Preto SP, Brazil 15054-

000.

1 figures, 4 tables

Running headline: Ethidium bromide effects on the fruit fly

KEY WORDS: Biomonitoring, Drosophila melanogaster, Ethidium Bromide,

Development, Reproductive Behavior

ABBREVIATIONS: EB: Ethidium bromide, EMS: Ethylmethanesulfonate.

Correspondence to: Gustavo O. Bonilla-Rodriguez, Depto. de Química e Ciências

Ambientais, IBILCE-UNESP, Rua Cristovão Colombo 2265, São José do Rio Preto SP,

Brazil 15054-000. e-mail: [email protected].


ABSTRACT

Ethidium bromide (EB) is an intercalating agent of nucleic acids. For this reason it is

generally used in molecular biology and in structural studies of DNA and chromatin.

Many scientists have demonstrated that this chemical can have mutagenic properties in

some living organisms, including Drosophila melanogaster. However, most of them

used concentrations up to a thousand times higher than that used in methods of

molecular biology for nucleic acid staining after electrophoresis. In the present work we

verified the effect of Ethidium Bromide in all phases of development (egg, larva, pupa

and adult) of some generations of Drosophila melanogaster exposed to the chemical

treatment (F1, F3, F6 and F10). Moreover, we analyzed the time spent for pre-

copulation and copulation. The results show that ethidium bromide interfere in the

viability of eggs, larvae, pupae and adults of Drosophila melanogaster. On the other

hand, the behavior related to reproduction showed significant differences between the

groups exposed to 30µM EB and 1µM EMS (Ethylmethanesulfonate) and the control

group in terms of the spent time in copulation. So, the data suggest on one side that

ethidium bromide interfered in developmental genes, causing in some individuals

inviability to reach the adult phase and on the other side that it can interfere in the fruit

fly behavior, acting as a neurotoxic agent.

INTRODUCTION

Most of the chemical substances do not have a proper hazard classification. In this

context, environmental monitoring is essential for identification of toxic products.

Because of that, in the last years occurred a significant growth in the interest for

studying the effects of substances to which man is daily exposed (Itoyama et al., 1998).

The effects of those drugs have been analyzed in several organisms including bacteria,

yeast, plants and animals, besides man, whenever it is possible (Timson, 1977; Leonard

et al., 1987).

When the organisms cannot avoid the exposure to a poisonous agent, physiological

mechanisms have to face their effects. The biological effects in response to these agents

always happen after biochemical and cellular events. Accordingly, the cellular and

biochemical parameters have a great potential to be used as stress indicators to evaluate

the physiological conditions of an organism (Stegman et al., 1992). Organisms try to

overcome these stressful conditions by the activation of genes to produce specific

proteins (Nazir et al., 2003a), and the expression of such genes can minimize the stress

effects (Atkinson and Walden, 1985).

Biochemical changes can be often translated as modifications in the morphology,

behavior or metabolic pathways, analyzed in a species known as bioindicator, chosen

for its sensibility or tolerance to several parameters, as organic pollution or other kinds


of pollutants (Washington, 1984).

In the last decades, the use of animal tissues for toxicological tests involves two

fundamental concepts: science and ethics, leading to search for alternative approaches.

Nowadays, species of Drosophila are model organisms for toxicological studies, since

they are well defined in terms of their genetics, biological development, and genome

(Mukhopadhyay et al., 2004), Additionally, fruit flies have high sensibility to toxic

substances, and they are insects of easy maintenance in the laboratory, feeding mainly

of bacteria and yeast that participate in the fermentation of carbohydrate rich substrates,

such as decomposing fruits (Almeida et al., 2001). Besides, the use of Drosophila is

recommended by the European Center for Validation of Alternative Methods

(ECVAM), which promotes the scientific and regular acceptance of alternative methods

that can reduce, refine and substitute the use of animals in laboratories (Mukhopadhyay

et al., 2004).

The use of insects, specifically Drosophila, for biomonitoring of genetic damages

caused by chemical agents has traditionally more than 50 years. However, during the

last 10 years, experiments using Drosophila are related to the identification of

carcinogens and as a model for the study of mutagenicity mechanisms induced by

chemicals (Vogel et al., 1999).

Genotoxic substances such as as diethylestilbestrol, diphenylhydantoin, imipramine,

testosterone and tolbutamide have shown a high teratogenic potential, whose effects

were evident in muscles and neurons in Drosophila melanogaster (Bournais-

Vardiabasis et al., 1983). The presence of varying ethanol concentrations in the culture

medium of Drosophila caused the malformation of legs (segments lacking, absence of

the legs or deformed), wings, dumbbells and melted buccal parts (Ranganathan et al.,

1987). Cypermethrin, a potent insecticide, promoted the significant increase in DNA

damage in the cells of the medium and previous cerebral ganglia (Mukhopadhyay et al.,

2004).

The genus Drosophila is found in six of the seven zoogeographic areas of the Earth

(with exception of Antarctica). Drosophila melanogaster as the other ones from genus

Drosophila, has a complete metamorphosis, passing for all the developmental stages.

The female lays eggs, that eclode as larvae. These larvae pass for three stages and then,

get into the pupal stage. Pupae stay attached to the glass wall in an artificial system.

After a few days, from the pupae emerges the flying adult. Among thousands of

residues generated by research laboratories, we have chosen to analyze the toxic effects


of Ethidium Bromide (EB). That is the common name for 3,8-diamino-5-ethyl-6-

phenylphenanthridinium bromide, an intercalating agent used in methods of molecular

biology. There are no studies focusing on the effect of EB in the developmental stages

of Drosophila melanogaster. However, the effects of this chemical have been tested in

the other organisms.

Nishiwaki et al. (1974) pointed out that in mice EB acts as an inhibitor of RNA-

dependent DNA polymerase activity, and for this reason it can be considered as an

antitumoral agent. Furthermore, Heinen (1978) showed that EB inhibits cell growth in

tissue culture, even at very low concentrations. However, in spite of this, EB is not used

as an antitumoral agent because it has mutagenic capacity in some organisms. Results

in bacteria show that EB is an effective frameshift mutagen if it is metabolically

activated by liver microsomes (McCann et al., 1975). Sea urchin eggs exposed to water

containing 50 µM of EB developed chromosomal abnormalities and failed to divide

normally (Vacquier and Brachet, 1969). Experiments in bacteria showed that EB is an

effective frameshift mutagen if it is metabolically activated by liver microsomes

(McCann et al., 1975). Experiments reported by Nass (1972) indicated that the growth

of mouse fibroblasts and hamster kidney cells are inhibited by 0.3-13µM of ethidium,

and that mitochondrial, not nuclear DNA synthesis was inhibited by ethidium.

In Saccaromyces cerevisiae EB acts as a strong inducer of petite mutants (Slonimski et

al., 1968). Its action is based on the inhibition of mitochondrial nucleic acid and protein

synthesis and is probably due to specific intercalations between the base pairs of

mitochondrial DNA (Perlman and Mahler, 1971).

In a previous work (Ouchi et al., submitted), we have analyzed the effect of EB in

productivity, protein profile and phenotypical changes. The present work involved the

exposure of ten generations of D. melanogaster to EB and intended to analyze its effect

in the developmental phases of the insect. For specimens of F10, we also analyzed the

effect of EB in sexual behavior, measuring duration of pre-copulation and copulation.

MATERIALS AND METHODS

Stocks

Specimens of Drosophila melanogaster were collected at São José do Rio Preto (State

of São Paulo, Brazil) and identified at the Drosophila Systematic Laboratory from our

Institute. Two lines have been used in this work. One of them was originated from one


female (isofemale line). The other one was called massal line, because it has been

originated by six females, having therefore higher genetic variability. Moreover, the

isofemale line was homozygous β-esterase (Ouchi et al., submitted). Both line stocks

were maintained in a temperature-controlled chamber at 24º C ± 1ºC.

Exposure to Ethidium Bromide

We used three different concentrations of EB (1, 5 and 30µM) and two control groups.

1µM of EB corresponds to the concentration used for visualization of nucleic acids, a

solution that is frequently disposed in the drain without chemical neutralization. For the

positive control we used 1µM EMS (Ethylmethanesulfonate – Acros Organics), a

mutagenic, whereas the negative control was fed with uncontaminated culture medium.

The chemicals were fully mixed with 50 mL of warm (45ºC) banana-agar medium, and

then poured into 250 mL glass bottles. For each treatment, four replicates were

prepared; three of them were used for the productivity experiments (Ouchi et al.,

submitted) and the fourth one for an experiment of larval viability (not shown in this

article). Ethidium bromide was purchased from Promega.

Maintenance of Generations

For each bottle, twelve males of the Drosophila melanogaster stock were joined to the

same number of virgin females. The culture medium was used as substrate for feeding,

and females were allowed to oviposit for 6 days. After that, the adults were removed

from the bottle for quantification. Ten days after the parents were added to the glass

bottle, the new generation initiated its emergence (F1). In the fifth day, that corresponds

to the maximum emergence of the control group, twelve males and virgin females were

isolated from each replicate and then, transferred to a new bottle glass, maintaining the

same conditions, in order to originate F2 (the second generation). The same procedure

was repeated until the tenth generation.

Viability Egg to Adult

A couple from the fourth replicate of each treatment of F10 from the isofemale line was

separated, keeping the males separated from the females. Each Drosophila stayed

individually in glass tubes for five days, until they reached their sexual maturity. After

this period, males and females from each treatment were mixed, in the same glass tube,


where they were allowed to copulate for 24 hours. Afterwards, we removed the male

and allowed the female to lay eggs for 24 hours on a spoon containing 3 mL of agar-

sucrose medium. The eggs for the four replicates where counted with a stereoscopic

microscope (Carl Zeiss). The spoons containing eggs were transferred to a 250 mL

glass bottle, containing 50mL of banana-agar culture medium, containing or not EB or

EMS. These experiments allowed us to count how many pupae and adults were viable.

Viability Larva to Adult

The fourth replicate was also used to collect larvae for viability experiments,

accomplished for F1, F3, F6 and F10 of the isofemale line. Ten glass tubes containing

7mL of treated banana-agar culture were used for each treatment, and to each one we

added ten larvae. After a few days, adults initiated their emergence and were analyzed

quantitatively and morphologically in a stereoscopic microscope.

Viability Pupa to Adult

Three replicates were used for experiments of productivity (not shown). After fifteen

days (time reserved to collect productivity information) some pupae remained attached

to the wall of the glass without emerging as adults. These pupae were counted for F1,

F3, F6 and F10 from the isofemale line and for F1 and F10 of the massal line.

Sexual Behavior

For each treatment, 24 couples were divided in six glass tubes, containing banana-agar

culture medium. Adults from both sexes were maintained isolated for five days in order

to reach sexual maturity. For each day, a negative control was analyzed for each

treatment. At the time of measurements, every day, parameters of temperature,

brightness and period were the same. After females and males were mixed and then the

pre-copulation times were logged. When each couple started to mate, this copulation

time was logged too.

Statistical Analysis

The data of pre-copulation and copulation time were analyzed using Student´s T test

(p<0,05) by the software BioEstat 4.0 (Ayres et al., 2005, Zar, 1999).


RESULTS

In order to verify the action of ethidium bromide in the different developmental stages

of Drosophila melanogaster, we have analyzed the viability from larvae to adults of the

isofemale strain (Table 1) and also pupae that did not emerge in the isofemale and

massal lines (Table 2).

Table 1: Larval viability (%) from the isofemale strain of D. melanogaster after

exposition to EB and EMS.

Generations

Treatment

Initial number

of larvae F1 F3 F6 F10 Average

Control 100 89 93 92 84 89.5

1µM EB 100 82 94 95 73 86.0

5µM EB 100 86 97 89 79 87.8

30µM EB 100 84 92 81 71 82.0

1µM EMS 100 74 90 86 80 82.5

Table 1 shows that in the first generation, the exposed animals have their viability

smaller than the control group. Even having small differences in viability, some

morphological alterations were found in adults for the groups exposed to 1 and 5 µM of

EB: one male with morphological alteration in wings and two males with morphological

alterations in wings, respectively. This fact shows that even those insects that emerged

might have suffered some type of gene alteration. In F3, we could notice that the

viability from larva to adult was not always higher in the control group, as verified for

F1. For F6, a similar result to that described previously for F3: for the group exposed to

1 µM of EB the larval viability was higher than for the negative control. However this

fact was not observed in the same experiment for the 10th

generation, where the larval

viability was always smaller in the exposed groups. Considering the average of all

generations, the larval viability was, in all treated groups, smaller than for the control.

We also observed that, some flies started the emergence, but they stopped in the middle

of the process. So, we noticed that not only larvae were affected by the chemical

treatments, but pupae were too.

This fact was confirmed by the experiment involving the viability from pupae to adult,

showed in table 2. There we can see that the viability of pupae for the groups treated

with EB and EMS were, in all conditions, slightly lower than that from the negative

control group, in F1, F6, F10 of isofemale strain and for F1 and F10 of massal strain. If


we compare the isofemale and massal lines for F10, we notice that the massal line, for

the same concentration of EB and EMS, had a lower viability.

Table 2: Pupal viability (%) from isofemale and massal strains of D. melanogaster after

exposition to EB and EMS.

Isofemale Strain Massal Strain

Treatment F1 F3 F6 F10 F1 F10

Control 98.7 93 99.6 99.5 98.5 99.4

1µM EB 98.1 94 99.4 99.3 94.7 98.1

5µM EB 97.7 97 99.3 99.1 97.9 96.8

30µM EB 98.2 92 99.1 98.7 92.0 93.7

1µM EMS 93.5 90 98.7 97.1 97.4 93.7

In table 3, we show the effect of the chemical treatment in all the developmental stages.

This experiment shows that EB and EMS affected mainly eggs and larvae, since from

all the pupae emerged adults. Moreover, we quantified the pupae by the day that

emerged in adults, where we could notice that the adults’ emergence of the groups

exposed to 30µM EB and 1µM EMS occurred one day before to the others treatments,

and their productivity were lower to the control group, as showed by figure 1.

Table 3: Egg to adult viability for F10 from the isofemale line from D. melanogaster

exposed to EB and EMS.

F10

Treatment

Eggs laid (24h) Pupae Adults % emerged adults

Control 127 111 111 87.4

1 µM EB 161 139 139 86.3

30µM EB 109 74 74 67.8

1µM EMS 130 43 43 36.2


0

10

20

30

40

50

60

70

80

1 2 3 4 5 6 7

Days

Em

erg

en

ce

Control

1uM EB

5uM of EB

30uM EB

EMS

Figure 1: Pattern of daily emergence (egg viability).

The pre-copulation and copulation times are shown in Table 4. We can observe that the

group exposed to 30µM EB had a significant larger time for copulation when compared

to the control. However, for the group exposed to 1µM EMS the spent time was smaller

than to the control. Both differences were significant (p<0.05).

Table 4: Average time (expressed as minutes) of pre-copulation and copulation for F10.

The asterisks (*) represents the process (pre-copulation or copulation) and the treatment

that showed significant difference compared to the control.

F10 isofemale line

Treatment Pre-Copulation Copulation

Control 9’02’’ 19’36’’

1 µM of EB 7’36’’ 22’22’’

Control 13’15’’ 19’15’’

5 µM of EB 10’56’’ 20’35’’

Control 16’14’’ 18’48’’

30 µM of EB 14’08’’ 21’01’’ *

Control 17’15’’ 21’50’’

1 µM of EMS 12’15’’ 20’30’’ *


DISCUSSION

Until the decade of 1980, there are no available works related to the action of EB in

Drosophila. Marcos et al. (1981a) tested the genotoxic effects of EB in Drosophila

melanogaster, using wild-type males (Mirasol, Barcelona). The concentrations that they

used were in the range from 0.03 to 3mM, many times larger than those used in our

experiments. Toxicity tests were performed and detected that LC50 = 2.16mM, for a 48h

exposure. EB induced a significant increase in sex-linked recessive lethals (1.01% at

3mM), and induced dominant lethals to a significant extent (Marcos et al., 1981b).

Our results showed that in the experiments of viability from larvae to adults and pupae

to adults, in ten generations, some alterations had happened during the development of

the insects. These effects were observed by the higher amount of inviable larvae, which

did not complete their development or failed to emerge as adults. Moreover, some flies

emerged with wings alterations. Ranganathan et al. (1987) tested ethanol for

teratogenicity in Drosophila melanogaster, and reported malformations involving the

legs and wings. Also, by exposing larvae to ethanol, the developmental stage sensitivity

was investigated, showing also harmful effects. Genotoxic effects of griseofulvin, an

antimycotic agent widely used in dermatophytoses, were studied by Tripathy et al.

(1996) in the somatic and germ line cells, on third and second instar larvae of

Drosophila melanogaster. Second and third instar larvae, exposed to acrylamide,

considered to be a carcinogen, displayed genotoxic effects in Drosophila melanogaster,

by the wing mosaic assay and the sex-linked recessive lethals test. It was observed that

acrylamide is both mutagenic and recombinogenic in the wing disc cells and induces

sex-linked recessive lethals (Tripathy et al., 1991).

As mentioned, some pupae started their emergence, but died in the middle of the

process, leaving the body partially out of the pupal case. A similar result was observed

by Sousa-Polezzi & Bicudo (2004), analyzing the effect of Phenobarbital (PB) in the

development of Aedes aegypti (Diptera, Culicidae), suggesting that PB may affect the

nervous system.

Analyzing the viability of laid eggs for the tenth generation of the isofemale line, we

observe that it was lower in the exposed groups (table 3). Moreover, we can see that

there was a decrease of viability as EB concentration increased. Concerning the groups

exposed to EMS, viability was lower than for those treated with EB. In the present

study, EMS was used as a positive control, since it is a known mutagenic product


(Griffiths et al., 1998), being used as a parameter for the data obtained with ethidium

bromide. Marcos et al. (1981a) carried out similar experiments analyzing the influence

of EB and egg viability counting laid eggs and emerged flies. The number of viable

eggs was inferior to the number of laid eggs (when compared to the control group), in

agreement with our results. Our experiment allowed to analyze all the stages, and it is

possible to verify that the highest effect affected both eggs and larvae, because all the

pupae emerged as adults. Several cellular divisions, mitoses and meiosis characterize

the egg phase. Marcos et al. (1981a) have pointed out that EB act as a mitotic and

meiotic poison, and it even blocks the process of spermatogenesis.

Some other studies have focused on the effects of some chemical products in adults and

in the different stages of development, using Drosophila as a bioindicator. Akins et al.

(1992) reveled that some heavy metals such as lead and cadmium, caused, in

Drosophila melanogaster, a developmental delay at the phase from larva to pupa. In

larvae of D. melanogaster, exposed to sodium azide (a potent mutagenic product), it

was observed the induction of somatic mutations and mitotic recombination in the wing

cells (González-César and Branch-Morales, 1997). The same effect was observed when

the tests were performed with methyl parathion, azametyphos, dichlorvos and diazinon

(Ekebas et al., 2000). Until now, there are no studies focusing on the effect of EB in all

developmental stages of Drosophila melanogaster.

It is noteworthy that the groups exposed to 30µM EB and 1µM EMS had emergence in

the 6th

and 7th

days, whereas the others already stopped at the 5th

day.

Itoyama et al., (1995) reported similar delays, analyzing the influence of caffeine in

Drosophila prosaltans. In larvae of Telmatoscopus albipunctatus (Diptera –

Pshychodidae), Sehgal and Simões (1977) verified that caffeine caused a significant

delay of development and high mortality.

In the literature there are some works that verified the reproductive performance

through the behavior, since some drugs can act on the nervous system. Itoyama et al.

(1995) studied the effects of caffeine in mating of Drosophila prosaltans, based on the

observation of the duration of pre-copulation and copulation. Statistically, they have

found difference only in the pre-copulation time. Nazir et al. (2003b) observed that

dimethyl sulfoxide, in Drosophila melanogaster, have expressed toxic effect in

hatchability, emergence, fecundity, and in reproductive performance. In order to verify

if the behavior could be influenced by EB, we observed the duration of pre-copulation

and copulation. Our results showed that all the exposed groups had a smaller duration


for pre-copulation than the negative control. The groups treated by EB showed a larger

time for copulation, but only for 30µM EB the difference was significant. In

Drosophila, the mating movements are complex and follow a characteristic pattern of

each species. Previous works (Ouchi et al., submitted) showed that the presence of

ethidium bromide caused malformations in Drosophila’s body, which could have

influenced in sexual behavior. Besides, EB could have some influence on the neural

system.

In conclusion, our results suggest that EB influenced in phases of development, mainly

in eggs, causing in some D. melanogaster inviability to reach the adult stage and could

have some effects on the neural system.

Acknowledgements. The authors are grateful to the Department of Biology where the

insects were maintained, to FAPESP (grant 05/02418-6 for GOBR), CNPq (fellowship

for GOBR) and CAPES (fellowship for RYO).

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Influence of Ethidium Bromide in daily Productivity, Morphological and Biochemical Parameters in Ten Generations of Drosophila melanogaster 48

Capítulo III

“Influence of Ethidium

Bromide in Daily Productivity,

Morphological and

Biochemical Parameters in Ten

Generations of Drosophila

melanogaster ”

Artigo a ser futuramente submetido: Archives of Insect Biochemistry

and Physiology

Influence of Ethidium Bromide in Daily Productivity, Morphological and Biochemical Parameters in Ten Generation of Drosophila melanogaster. 49

Influence of Ethidium Bromide in Daily Productivity, Morphological and

Biochemical Parameters in Ten Generations of Drosophila melanogaster

Ouchi, R.Y. 1

; Okamoto, D.N. 1

; Almeida, E.A. 2

, Ceron, C.R. 2

;.Manzato, A.J3.; Bonilla-

Rodriguez, G.O2.

1.

Master degree student, 2.

Department of Computing and Statistical Sciences, 3.

Department of Chemistry and Environmental Sciences, IBILCE/UNESP, State

University of São Paulo, Rua Cristovão Colombo 2265, São José do Rio Preto, SP. CEP

15054-000, Brazil.

8 figures, 0 tables

Running headline: Ethidium bromide effects on the fruit fly




Abstract:

Although every day new chemicals enter the market and generate an increasing volume

of residues, a significant proportion do not have a proper hazard classification; they are

potentially dangerous and able to generate harmful biological effects. Biochemical

changes can be often translated as modifications in the morphology, behavior, or

metabolic pathways, analyzed in a species known as bioindicator. Ethidium Bromide

(EB) is a fluorescent stain used in protocols of Molecular Biology due to its ability to

intercalate between DNA nitrogenous bases. For this reason it is considered as

mutagenic, although is not classified as carcinogenic by IARC. The present work

analyzed the influence of ethidium bromide on daily emergence through 10 generations

of D. melanogaster and compared the toxic effects to a control group, not exposed to

any mutagen. EB was fully mixed with the culture medium used to feed to D.

melanogaster, in two final concentrations: 1 and 5 µM. This chemical influenced the

fruit fly development causing a delay of the life cycle; the treated groups showed

differences concerning the day the productivity reached a maximum when compared to

the control. Some morphological alterations were noticed in wings, coloration and

tergites. Furthermore, an electrophoretic gel of total protein reveled that specimens with

morphological alterations had produced some proteins not found in the control group.

One possible reason is that EB, even in low concentrations, can induce genetic damages

in this species which are translated into altered proteins, and therefore interfering in

adults' metabolism.

Key words: Biomonitoring, Drosophila melanogaster, Ethidium Bromide,

Productivity, Emergence


Introduction

Increasing environmental exposition to chemicals of unknown toxicity raises

apprehension about their effects on human health and the ecosystems as well. An

approach to gather new knowledge on this matter is known as environmental

monitoring. It is absolutely essential to identify products, which can cause risks to life

and environmental degradation. Pollution can be defined as any alteration in the

environment, causing biological impact in a short or long term (Bonilla-Rodriguez,

1989). In this ecological context, biomonitoring is the systematic use of the responses of

living beings as a way to evaluate environmental changes, generally caused by

anthropogenic actions (Mathews et al., 1982).

The use of animal species as a form to evaluate the environment started with the

Industrial Revolution (XIX Century), when canaries were left in mines to monitor the

air condition, avoiding harm to the miners' health (Cairns Jr. and Pratt, 1993).

Bioindicators are species chosen by their sensitivity and tolerance to many parameters,

such as organic pollution and to other kinds of pollutants (Washington, 1984).

According to Johnson et al. (1993), an ideal biological indicator must have some

characteristics: being a group taxonomically well defined and easy recognized by non

specialists, having a wide distribution, to be abundant or easy to collect, having low

genetic and ecological variability, well known ecological characteristics, and the

possibility to be studied in laboratories. The use of invertebrates, in this case,

Drosophila melanogaster, has some advantages in ecotoxicological investigations. For

example, promoting the elucidation of mechanisms linked to the effects, as well as their

consequences in other structural levels of biological organization (Lagadic and Cacquet,

1998). Drosophila melanogaster is the most widely used insect model because of its

well-elucidated genetics and developmental biology. Moreover, the use of Drosophila

has been recommended by the European Center for Validation of Alternative Methods

(ECVAM) with the purpose of reducing, refining or replacing the use of laboratory

animals (Benford et al., 2000).

The continuous exposition of organisms to stressful environmental, poisonous,

physiologic and metabolic agents is harmful. The organism responses occur after

cellular and biochemical events, and therefore they have a great potential to be used as a

stress indicators (Stegman et. al., 1990).

Among thousands of residues generated by research laboratories, in this work we were

interested to analyze the toxic effects from Ethidium Bromide (EB). That is the


common name for 3,8-diamino-5-ethyl-6-phenylphenantridinium bromide, an

intercalating agent usually used in molecular genetics and in structural studies of DNA

and chromatin.

EB is a common laboratory stain for double-stranded DNA and RNA (Sambrook et al.,

1989), but it is also known to possess significant anti-cancer effect (Nishiwaki et al,

1974). Furthermore, Heinen (1978) showed that EB inhibits cell growth in tissue

culture, even at very low concentrations, but in spite of this, its potential applications in

human health care have been prevented, however, due to its mutagenic activities in

model systems. Experiments reported by Nass (1972) indicated that 0.3 to 13µM of EB

inhibit the growth of both mouse fibroblasts and hamster kidney cells, and that

mitochondrial, not nuclear DNA synthesis was also inhibited by ethidium. Another

study showed that ethidium accumulates in isolated rat mitochondria and interferes with

metabolic activities related to respiration (Peña et al., 1977). Sea urchin eggs exposed to

water containing 50 µM of ethidium bromide developed chromosomal abnormalities

and failed to divide normally (Vacquier and Brachet, 1969).

Results in bacteria show that EB is an effective frame shift mutagen when metabolically

activated by liver microsomes (McCann et al., 1975). In Saccaromyces cerevisiae EB

acts as a strong inducer of petite mutants (Slonimski et al., 1968). Its action is based on

the inhibition of mitochondrial nucleic acid and protein synthesis and is probably due to

specific intercalations between the base pairs of mitochondrial DNA (Perlman and

Mahler, 1971). In Trypanosoma cruzy EB induced a synthesis of an abnormal circular

DNA molecule in the kinetoplast that represents more than 30% of kinetoplastic DNA

(Delaine & Ryo, 1969).

In mice, EB apparently has little or no access to nuclear DNA, at least in vivo, whereas

it intercalates perfectly well with isolated nuclear DNA in vitro (Pack & Loew, 1978).

Until the decade of 80, no studies were done on the mutagenic activity of EB in

Drosophila. Marcos et al. (1981) tested the mutagenic action of this compound in two

species of the genus Drosophila, Drosophila melanogaster and Drosophila simulans,

establishing the LC50 for the two species and revealed that D. melanogaster is more

sensitive to genotoxic effects of EB than D. simulans.

Ethidium bromide is also an intercalating inhibitor of topoisomerase II (topo II). Topo II

is a ubiquitous enzyme that regulates DNA topologic interconvertion during replication,

transcription and genetic recombination decreasing torsional stress in DNA by

introducing transient protein-bridged DNA breaks in both DNA strands. Through this


function, topo II plays an essential role in the maintenance of genetic material integrity

(Mo and Beck, 1999; Godard et al., 2002). Several topo II inhibitors, such as

doxorrubicin, etoposide and ellipticine have been extensively used as chemotherapic

agents in anti-cancer treatments. Rather than inhibiting the catalytic activity of the topo

II enzyme, they act by increasing levels of topo II mediated DNA cleavage. This

mechanism converts this enzyme into a cellular toxin, inducing apoptosis, cell cycle

arrest and genotoxicity (Attia et al., 2002). In fact, long-term adverse effects of this

class of chemotherapic items, such as infertility and increased incidence of secondary

malignancies, have been shown (Tiburi et al., 2002).

In the present study we have investigated the influence of different concentrations of EB

on daily productivity (in two different strains) for 10 generations of Drosophila

melanogaster, morphological alterations and biochemical analyses based on protein

electrophoretic profile (for the second generation) and carboxylesterase activity (for the

fifth generation).

Materials and Methods

Stocks

Specimens of Drosophila melanogaster were collected using traps (Medeiros and

Klaczko, 1999) at São José do Rio Preto (State of São Paulo, Brazil), and two lines have

been used in this work. One of them originated from one female (isofemale strain). The

other line was called massal, because it was originated from many females, having

higher genetic variability. Both stocks were maintained in a temperature-controlled

chamber at 24º C ± 1ºC.

Exposure to ethidium bromide

We used two different concentrations of EB (1 and 5 µM) and a negative control, fed

with uncontaminated culture medium. Ethidium bromide (Promega) was fully mixed

with warm (45ºC) 50 mL of the banana-agar medium, and then poured into 250 mL

glass bottles. For each treatment, four replicates were prepared. Three of them were

used for the productivity experiments and the fourth one for an experiment of larval

viability (not shown in this article).

Daily Productivity along 10 Generations

For each bottle, twelve males of Drosophila melanogaster from both stocks were joined

to the same number of virgin females. The treated culture medium was used as substrate

for feeding and females were allowed to oviposit for six days. After that, the adults were


removed from the bottle. Ten days after the parents were added to the glass bottle, the

new generation initiated its emergence (F1). In the fifth day, that corresponds to the

maximum emergence for that generation, twelve males and virgins females were

separated from each replicate and then, transferred to a new glass bottle, maintaining the

same conditions, in order to originate F2 (the second generation). During fifteen days

the emerged insects were counted twice a day. For the isofemale strain we analyzed all

10 generations. However, for the massal strain we analyzed the productivity of 1st, 5

th

and 10th

generations in order to compare the influence of this chemical in two different

strains. Each experimental set (1 and 5 µM EB) had thee replicates, plus a control

group, also with three replicates, fed with uncontaminated culture medium.

Morphological Alterations

During each daily counting, the flies were phenotypically analyzed with a stereoscopic

microscopy (Carl Zeiss). The insects that showed morphological alterations were

counted and then were kept frozen at -20ºC for later electrophoretical analysis. The

proportion of flies carrying morphological alterations was calculated as the ratio altered/

total number of flies emerged on each treatment.

Biochemical Analysis

A. Protein Electrophoretical Profile

After carrying out morphological and quantitative analyses, the pattern of total proteins

was studied for morphologically altered males, for the second generation, of the

isofemale line. The electrophoretical analysis of SDS-PAGE showed too many bands,

hindering the visualization of the differences between the control and the chemically

treated. So, we decided to do a non-denaturating electrophoresis, stained by silver

nitrate. For this experiment we used some males of the negative control and also flies

displaying morphological alterations. The samples were homogenized in 0.2M Tris-HCl

pH 8.8 buffer. For electrophoreses we used 0.1M Tris-Glycine pH8.3 buffer, setting the

voltage to 180V during four hours.

B. Assays of Enzimatic Activities and Total Protein Quantification

Carboxylesterase activity were measured essentially by the method of Ellman et al.

(1961), modified by Bonacci et al. (2004), using phenylthioacetate as substrate.

Carboxylesterases are able to hydrolyze phenylthioacetate yelding thioacetate, that in

combination with 5,5’-dithiobis-2-nitrobenzoic acid (DTNB) forms a yellow anion 5-

thio-2-nitrobenzoic acid which absorbs strongly at λ=412nm. Optimal assay condition

ranges were carried out using a pooled sample containing nine Drosophila


melanogaster homogenized with 200 µL of Tris-HCl buffer 100mM pH8.0 and

centrifuged for 15min at 10,000xg. 20µL from the solution were mixed with 455µL of

the same buffer. 15µL of phenylthioacetate 150mM and 10µL of DTNB 50mM were

added to start the reaction. Increasing in absorbance at 412nm was monitored during

1min. Blanks without substrates or samples were previously incubated at 25ºC for 2 min

to assess endogenous cross-reaction with DTNB. The sample protein concentration was

determined following the method of Bradford (1976). Absorbance readings were carried

out in a Varian Cary 100 spectrophotometer.

Statistical Analyses

Statistical tools were used to analyze daily emergences. For this purpose, we applied the

test of equality of two proportions (Normal approach Z) for independent samples, used

for parametric data (Moore, 2005). However in order to analyze carboxilesterase

activity and total productivity of each generation, we used Student´s t test (Zar, 1999).

We used BioEstat 4.0 (Ayres, 2005), using a p<0.05 significance level.

Results

In order to analyze if ethidium bromide has cumulating properties, the effects were

verified along ten generations of D. melanogaster. The daily productivity was measured

along fifteen days. The patterns of total numbers of flies emerging produced by the

three replicates are shown in figure 1, for the massal line, and in figure 2 for isofemale

line (A produtividade diária total pode ser verificada nos anexos 1 e 2).


0.5

0.4

0.3

0.2

0.1

*

Figure 1: Proportion of flies emerging daily for the massal line of D. melanogaster,

exposed to 1µM EB (-□-) and 5 µM EB (-▲-), and for the control (-♦-). Days with

significant differences: (*) for 1µM EB and (•) for 5µM EB.

Figure 1 shows that for 1µM EB was able to cause significant differences in

productivity on F1 and F5 of the massal strain in most of the days, with exception for

2nd

, 4th

, 9th

and 11th

for F1 and 8th

and 9th

days for F5. In F10, only two days of exposure

to EB caused significant alterations in productivity. However, the total productivity of

F10 exposed to 1µM EB is 37% smaller than that from the control. For 5µM EB, we

could notice that 70% of the days had significant differences in daily productivity in F1,

F5 and F10. For the isofemale line (figure 2), it was possible to verify that, for 1µM EB,

the differences were frequently significant compared to the control, reaching 92% in F5,

75% in F2 and F8, 67% in F1, F3, F6, F7 and F9; 60% in F4 and 34% in F10. For 5µM

EB, we could notice that for most of the days the significant differences in daily

productivity reached 75% at F2, 60% at F1, F3, F4 and F9, 50% in F7 and F8, 42% in

F6 and F10 and 34% in F5.

0

0,1

0,2

0,3

0,4

0,5

1 2 3 4 5 6 7 8 9 11 13 15

0

0,1

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F1 F5

Days

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* * * * * * * *

* * * * * * * * * *

* *

• • • • • • • •

• • • • • • •

• • • • • • •

0.5

0.4

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0.1

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0.4

0.3

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0.1

F2

F3 F8

Figure 2: Proportion of flies emerging daily for the isofemale line of D. melanogaster,

exposed to 1µM EB (-□-) and 5 µM EB (-▲-), and for the control (-♦-). The asterisks

(*) shows the significant days for 1µM EB and filled circles (•) shows significant days

for 5µM EB.

0

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Days Days

•

* * * * * * * *• • • •• •

* * * * * * * *

• • • • • • • • •

* * ** * * * *• • • • • • •

* * * * * * *• • • • • • •

* * * * * * * * * * *• • • •

* * * * * * * *

• • • • •

* * * * * * * *

• • • • • •

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• • • • • •

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Pro

port

ion

of

flie

sem

erg

ing



Besides the differences in daily emergence, the emergence profile was also affected,

increasing in F1, F2, F3, F4 and F10 of the isofemale strain (figure 2), showing a delay

of emerging flies. The control group ceased its emergence around the 9th day, whereas

the insects exposed to EB continued to emerge until the 15th day. The total productivity

of the tenth generation is shown in figure 3, for both lines. We can notice that for the

massal line, in spite of the increasing productivity, for F5 and F10, the total productivity

was smaller than for the control. Moreover, comparing F10 of both strains we can

notice that the isofemale strain had a decrease of the total productivity, although the

massal strain did not show this pattern. This could be explained by the low genetic

variability of isofemale stock, turning the flies more susceptible to the action of

mutagens. In figure 3 it is possible to verify that for 1µM EB, there was a significant

difference in total of F1 of the isofemale line and the same was verified for 5µM EB.

None significant difference was found for the massal strain.

0

200

400

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Gerations

Flies

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ing

0

200

400

600

800

1000

1200

1400

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1800

F1 F5 F10

Generations

Fli

es E

merg

ing

Figure 3: Total productivity for F10 of D. melanogaster exposed to EB. A. Isofemale line and B. massal line. Empty squares: 1 µM EB, filled triangles: 5 µM EB, filled diamonds: control. Days with significant differences: (*) for 1µM EB and (•) for 5µM EB. Furthermore, some morphological alterations (figure 4) were noticed in the pattern of

the wings (absence, malformations, non stretched), tergites (malformation) and

coloration (extra pigmentations). The frequencies of morphological alterations are

shown in figure 5, where we can observe that in the control group abnormalities are low

along the 10 generations in both lines. However, for the groups exposed to EB, the

frequency was high for the first generations, with a decrease afterwards. Besides the

high frequency of morphological alterations, these results, in most of generations were

not significant, but even so it is noteworthy that the malformation frequency was up to

eight times higher in the exposed group.

A B

* *


Figure 4: Main morphological alterations verified in Drosophila melanogaster exposed to ethidium bromide, shown as photo negatives: in wings (A-F), tergites (H, I) and pattern of coloration (G, female with a black spot in the first paw) chemically fed. The white arrows indicate the alterations.

0

0,002

0,004

0,006

0,008

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0,016

1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th

Generations

Freq

uen

cy o

f m

orp

ho

log

ical

alt

erati

on

s

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0,014

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1st 5th 10th

Generations

Freq

uen

cy o

f m

orp

ho

log

ical

alt

erati

on

s

Figure 5: Frequency of morphological alterations along all ten generations of D.

melanogaster. A- Isofemale Strain, B- Massal Strain. Empty squares: 1 µM EB, filled triangles: 5 µM EB, filled diamonds: control. The asterisks (*) shows the significant days for 1µM EB and filled circles (•) shows significant days for 5µM EB.

A B

•

•

0.016

0.014

0.012

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0.006

0.004

0.002

0

0.016

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0.008

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0.004

0.002

0


Morphological alterations are a consequence of cellular and biochemical events

affecting the insect previous to its emergence. So, searching for clues of those events,

we performed an analysis of the protein profile (figure 6). In this picture, it is possible

to notice that in samples 8, 9 and 10, there were two additional proteins that were not

present in the samples of the control group. Besides, in sample 10 the arrow shows that

one band is missing.

Figure 6: Non-denaturating polyacrylamide gel electrophoresis showing the pattern of total proteins for Drosophila from F2 generation of the isofemale line. 1-5: control males; 6-9: males with wing alterations; 10: males with tergite alterations. The arrows indicate some proteins that presented a different pattern when compared to the control group. Carboxylesterase activity was analyzed (figure 7) for males and females from F5. The

results demonstrated that there were no differences between the activities of both sexes

(data not shown), however, a significant difference was detected between the control

and the group exposed to 1µM EB.

U/m

g p

rote

in

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

control 1uM EB 5uM EB

U/m

g p

rote

in

Figure 7: Carboxylesterase activities (average ± S.D.) for control, 1µM EB and 5µM EB. The asterisk (*) shows a significant difference.

1 2 3 4 5 6 7 8 9 10

*

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0


In order to analyze the effect of EB along the generations, we calculated, for F1, F5 and

F10 from both lines, the ratio of daily productivity: number of flies emerged in the

group exposed to EB / number of flies emerged in the control group (figure 8). We can

observe that in F1 from the isofemale line, the proportion of flies emerging in the group

exposed to 1µM EB was increasing after the 5th day reaching “infinite values” for the

13th and 15th days, since in the control group emergence already stopped before.

However this proportion for the massal strain kept relatively constant around 1.0 along

the same period. For F10 the ratios were significantly lower, and for the isofemale and

massal lines only two and three days showed more productivity in the exposed group,

respectively. The isofemale line showed a more drastic reduction the productivity. The

same effect was observed for F1, F5 and F10 of 5µM EB. This effect could reflect that

along the generations, EB could be accumulating in Drosophila melanogaster.

Figure 8: Ratio of flies emerging of the group exposed to 1µM EB divided by those emerged from the control group, for F1 and F10, for the isofemale (empty circles) and massal (filled circles) lines of D. melanogaster. Discussion

In our experiments we noticed that, for both isofemale and massal strains, in most of the

days of each generation, the daily productivity were significantly different when

compared to the control reaching 92% of the days for F5 from the isofemale line

exposed to 1µM EB. Concerning the massal line, when compared to isofemale one, it

has a larger genetic variability in its population; however, in both, EB altered daily

productivity. Moreover, analyzing figure 8, the genotoxic effects affected daily

productivity, presumably by the accumulation of EB along generations, for both lines.

These were reflected by the larger ratios of 1µM EB for F1, when compared to F10. In

A B


figure 3 it was possible to analyze the interference of EB on total productivity in each

generation of the exposed groups. This effects could be explained by the action of EB as

a mitotic and meiotic poison, able to block the process of spermatogenesis (Marcos et

al., 1981), evident for F5 and F10, where the total productivity were, in all the exposed

groups, smaller than for the control. Sehgal et al. (1977) revealed that occurred a

fertility reduction in adults of T. albipunctatus, when pupae and larvae were treated with

caffeine. The interference of chemicals in the productivity was also verified by Itoyama

et al. (1995), reporting that the reduction of productivity was proportional to the

concentration of caffeine in the culture medium.

In order to analyze all the effects caused by a chemical product, several parameters need

to be verified. For this reason, besides productivity, we analyzed phenotypic alterations

caused by EB. In figure 5 it was possible to see that the frequency of alterations were in

most of the cases up to twelve times larger than those observed for the control animals

(for 1µM EB for F4). Even though in some cases, the frequency of morphological

alterations was smaller than for the control (F6 and F10 of isofemale strain). Mitchell &

Simmons (1977) studied the effect of EMS (ethyl methanesulfonate, a confirmed

mutagenic compound) on chromosome X in Drosophila melanogaster, and postulated

that one chromosome with several mutations could lead many insects to be inviable.

Accordingly, those mutations would not be observed, since the organisms do not reach

the adult stage.

The high frequency of morphological alterations for 1 and 5µM of EB decreased along

the generations. Two hypotheses could explain this effect. One of them is that the

reduction could be a result of the natural selection process, which affects the pathways

of enzymatic mechanisms responsible for DNA repair. The other one is that occurred,

along the time, a better detoxification of EB. Kuzhir et al. (1999) investigated the

modifications in repair processes caused by a chemical mutagen, EMS, in Drosophila

melanogaster, and noticed that the presence of glutapyrene induced the synthesis of

enzymes involved in repair of O6-ethylguanine. After a treatment with low doses of the

mutagen, glutapyrene increased the fertility of the parentals, but did not suppress the

mutagenic action of the chemical compound, and the frequency of altered organisms

decreased. Another effect of the chemicals product was verified in the prolongation of

life cycle of most of the generations (F1 to F4 and F10) of the isofemale strain. Fleming

et al. (1981) reported a similar effect of 0.2mM EB: it increased the developmental time

of Drosophila melanogaster by 32%. The authors suggested that oxygen consumption


was lower for flies treated with EB during their development or during their adult life.

Morel et al. (1999) verified that low concentrations of EB caused changes in

mitochondrial DNA (mtDNA), leading to reductions in the activities of the respiratory

complexes III and IV measured in treated Drosophila cells. When the figure 6 is

analyzed, we verify that some proteins are being produced or absent (samples 8, 9, 10)

or mutation that happened could cause the absence of one protein by altered organisms.

These mutations would reflect alterations at the DNA level, affecting protein synthesis,

and possibly expressing phenotypic alterations. Moreover, for the same alteration,

samples 6, 7, 8 and 9, in this case alteration in wings, they have different responses.

Goncharova et al. (1988) verified that individual sensibility was evident when they

analyzed lethal recessive mutations in germinate cells in D. melanogaster, exposed to

EMS. In terms of carboxylesterase activity, there was a significant difference between

the control and the 1µM EB groups. Carboxylesterase, a serine-dependent enzyme, is

able to hydrolyze a wide range of xenobiotic (Maxwell, 1992). The observed inhibition

of carboxylesterase was another evidence of the deleterious action of EB in Drosophila.

However, again we verify the variability of the individual sensibility of organisms to

respond to different situations, since the group exposed to 1µM EB showed differences,

but that treated with 5µM EB did not. These assays were done with a pool of flies, and

in a larger concentration of EB the selection pressure could have been enormous, so the

surviving flies used could possess some advantages concerning the others. Edwards &

Brenner (2005) studied the development of Artemia salina embryos in the presence of

EB, an inhibitor of mitochondrial transcription. The exposure results in a dose

dependent increase in the specific activity of lactate dehydrogenase, and a concomitant

decrease in the specific activity of a cyanide-resistant superoxide dismutase. The

inhibition of mitochondrial function by EB appears to exert opposite effects on the

nuclear cistrons encoding lactate dehydrogenase and superoxide dismutase

In conclusion, even at low concentrations, ethidium bromide can induce toxic effects in

terms of productivity, morphologic and biochemical parameters, presumably due to its

genotoxic properties. However, in Drosophila melanogaster these effects were not

dose-dependent. For the isofemale line, due to its smaller genetic variability, the effects

seemed to be more intense than for the massal line.The different susceptibility of

separate individuals to mutagens reflects the existence of cryptic genetic variability in

Drosophila strains.


Acknowledgements. The authors are grateful to the Department of Biology where the insects were maintained, to MSc. Fernando Rogério Carvalho and Prof. Dr. Francisco Langeani Neto for their help concerning the photographs, to FAPESP (grant 05/02418-6 for GOBR), CNPq (fellowship for GOBR) and CAPES (fellowship for RYO).

References

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Zar JH. 1999. Bioestatistical Analysis. Prentice Hall 4th ed. 663p.

Influence of Ethidium Bromide and Ethylmethanesulfonate in Ten Generations of Drosophila melanogaster 67

Capítulo IV

“Influence of Ethidium

Bromide and

Ethylmethanesulfonate in Ten

Generations of Drosophila

melanogaster”

Artigo a ser futuramente submetido: Archives of Insect Biochemistry

and Physiology

Influence of Ethidium Bromide and Ethylmethanesulfonate in ten Generations of Drosophila melanogaster 68

Influence of Ethidium Bromide and Ethylmethanesulfonate in Ten Generations of

Drosophila melanogaster

Ouchi, R.Y. 1

; Okamoto, D.N. 1

; Almeida, E.A. 2

, Ceron, C.R. 2

;.Manzato, A.J3.; Bonilla-

Rodriguez, G.O2.

1.

Master degree student, 2.

Department of Computing and Statistical Sciences, 3.

Department of Chemistry and Environmental Sciences, IBILCE/UNESP, State

University of São Paulo, Rua Cristovão Colombo 2265, São José do Rio Preto, SP. CEP

15054-000, Brazil.

7 figures, 0 tables

Running headline: Ethidium bromide effects on Drosophila




Abstract

Environmental monitoring is essential for the identification of hazards to human

health, to assess environmental cleanup efforts, and to prevent further degradation of the

ecosystems. Although many chemical residues are known as toxic, a significant

proportion do not have a proper hazard classification; they are potentially dangerous and

able to generate harmful biological effects. Biochemical changes can be often translated

as modifications in the morphology, behavior, or metabolic pathways, analyzed in a

species known as bioindicator. Ethidium Bromide (EB) is a fluorescent stain used in

protocols of Molecular Biology due to its intrinsic ability to intercalate between DNA

nitrogenous bases. For this reason it is considered as mutagenic, although is not

classified as carcinogenic by IARC. The present work analyzed the influence of EB on

daily productivity along 10 generations of D. melanogaster exposed to 30µM EB, and

the toxic effect in each generation is compared to a negative control, not exposed to any

mutagen, and a positive control: 1µM EMS (Ethylmethanesulfonate). The chemicals

were fully mixed with a banana-agar culture medium used to feed D. melanogaster, in

order to test their toxicity. EB and EMS influenced the development causing a delay of

the life cycle: for the group exposed to EB, the emergence reached a maximum after the

negative control did it. Some morphological alterations were noticed in wings,

coloration and tergites. Several effects induced by EB were very similar to those noticed

for specimens exposed to EMS. Furthermore, carboxylesterase activity was measured

and revealed that flies with morphological alterations showed a significant difference

compared to the negative control. The data are interpreted as reflecting toxic effects.

Key words: Biomonitoring, Drosophila melanogaster, Ethidium Bromide,

Productivity, Emergence, Ethylmethanesulfonate.


Introduction

Biomonitors and biomarkers allow monitoring the environment and are the best

approach to study the potential toxic effects from unknown chemicals. When behavioral

avoidance cannot prevent an animal from being exposed to novel environmental toxins,

physiological mechanisms must cope with the toxin and its effects (Etienne et al.,

2001). Biomonitors are species chosen by their sensibility and tolerance to many

parameters, such as organic pollution and to other kinds of pollutants (Washington,

1984).

An enlarged interpretation of alternatives in toxicological testing includes the use of

non-mammalian species (Lagadic & Cacquet, 1998). Over the past decade, issues such

as animal handling and care in toxicology research and testing became one of the

fundamental concerns for both science and ethics. Emphasis has been given to the use

of alternatives to mammals for testing, research and educational purposes. Drosophila

melanogaster is the most widely used insect model because of its well-elucidated

genetics and developmental biology. Moreover, the European Centre for Validation of

Alternative Methods has recommended the use of Drosophila (ECVAM) with the

purpose of reducing, refining or replacing the use of laboratory animals (Benford et al.,

2000).

Drosophila has fulfilled a dual function in the field of genetic toxicology: it has been

used for short-term tests for identifying carcinogens and also as a model for studies of

the mechanisms of mutagenesis induced by chemicals. Until the mid-1980s, the use of

Drosophila in short-term tests was restricted to assays for genetic damage in germ cells,

mostly in males (Vogel et al., 1999). The extensive knowledge of the genetics of

Drosophila melanogaster and the long experimental experience with this organism has

made it useful in mutation research and genetic toxicology (Çakir & Sarikaya, 2005).

Among thousands of chemical residues generated by research laboratories, we choose to

analyze the toxic effects of Ethidium Bromide (EB), an agent usually used in molecular

biology protocols, as a common laboratory stain for double-stranded DNA and RNA

(Sambrook et al., 1989).

Ethidium Bromide is a compound that intercalates reversibly between DNA base pairs

affecting many of its functions, including DNA and RNA synthesis and mitotic activity

(Heinen et al., 1976). Ethidium bromide was classified as an inhibitor of Topoisomerase

II (Snyder & Arnone, 2002), DNA polymerase alpha, delta and epsilon from Novikoff


hepatoma cells (Fox et al., 1996) and the unwinding DNA and ATPase activities of the

plant nuclear helicase PDH45 (Pham & Tujeta, 2002).

DNA topoisomerases (Topos) are nuclear enzymes that regulate DNA topology and are

required for DNA replication and transcription (Nelson et al., 1986; Brill et al., 1987).

These enzymes are also implicated in chromosome segregation, DNA repair, cell cycle

progression, and RNA processing (Rose & Holm, 1993; Sekiguchi & Shuman, 1997).

Eukariotic cells express two forms of topoisomerases. The type I form (Topo I) is an

ATP-independent enzyme that catalyzes DNA relaxation via transient single-stranded

DNA breaks (D’Arpa et al., 1988).

By contrast, the type II form (Topo II) is an ATP-dependent enzyme that catalyzes

knotting-unknotting and catenation-decatenation reactions by breakage, strand-passage,

and reunion of double-stranded DNA (Tsai-Pflugfelder et al., 1988).

Covalent attachment of EB to DNA in nanomolar concentrations enhances

topoisomerase II-mediated single and double-strand DNA cleavage. Therefore, the

conversion of the reversible EB-DNA complex into an irreversible adduct causes the

transformation of the drug into a catalytic topoisomerase II inhibitor (Snyder and

Arnone, 2002; Marx et al., 1997).

Stable double-strand breaks generated by EB, through inhibition of DNA topoisomerase

II, may become templates for recombination events and possibly are potentially capable

of inducing secondary malignancies mediated by mitotic crossing-over. These effects

were revealed by Becker et al., 2003, when they verified that was an increase in mitotic

recombination in diploid cells of Aspergillus nidulans in response to EB.

Several DNA topoisomerase II inhibitors are successful anticancer drugs used to treat

human malignances. However, considering EB, some studies have been done analyzing

its effects in some organisms. Nass & Ben-Shaul (1973) showed that EB has mutagenic

properties in Euglena gracilis inhibiting cell division. They also reported that the ultra-

structure is changed only in mitochondria of growing cells, not in chloroplasts, and that

the drug could develop resistence in organisms. In Saccaromyces cerevisiae, EB acts as

a strong inducer of petite mutants (Slonimski et al., 1968). Its action is based on the

inhibition of mitochondrial nucleic acid and protein synthesis, probably due to specific

intercalations between the base pairs of mitochondrial DNA (Perlman & Mahler, 1971).

The treatment of cultured mouse fibroblasts and hamster kidney cells with EB led to

breakdown of closed – circular mitochondrial DNA and greatly enlarged mitochondrial

profiles with few or no cristae (Nass, 1970).


In the present study we investigated the influence of different concentrations of EB on

daily productivity and morphological alterations along ten generations of Drosophila

melanogaster, using two different stocks. Furthermore, we performed biochemical

analyses based on carboxylesterase activities.

Materials and Methods

Stocks

Specimens of Drosophila melanogaster were collected in May 2005, at São José do Rio

Preto (State of São Paulo, Brazil) using traps (Medeiros and Klaczko, 1999). Two

stocks were used in this work; one was originated by one female (isofemale line),

whereas six females originated the other one, named massal line. Both line stocks were

maintained in a temperature-controlled chamber at 24º C ±1ºC.

Exposure to ethidium bromide and Ethyl methanesulfonate

We used 30 µM EB (Ethidium Bromide Solution - Promega), a positive control 1µM

EMS (Ethyl methanesulfonate – Acros Organics) fully mixed with warm (45ºC) 50 mL

of the banana-agar medium, and then poured into 250 mL glass bottles. For each

treatment, four replicates were prepared; three of them were used for the productivity

experiments and the fourth one for an experiment of larval viability (not shown in this

article). In addition, a negative control group was fed with uncontaminated culture

medium. 30µM of EB corresponds to thirty times the concentration used for

visualization of nucleic acids, a solution that is frequently disposed in the drain without

chemical neutralization.

Daily Productivity along 10 Generations

For each bottle, twelve males of the Drosophila melanogaster from both stocks were

joined to the same number of virgin females. The treated culture medium was used as a

substrate for feeding, and females were allowed to oviposit for six days. After that

period, the adults were removed from the bottle. Ten days after the parents were added

to the glass bottle, the new generation initiated its emergence (F1). In the fifth day, that

corresponds to the maximum emergence for that generation, twelve males and virgins

females were separated from each replicate and then, transferred to a new glass bottle,

maintaining the same conditions, in order to originate F2 (the second generation).

During fifteen days the emerged insects were counted twice a day. For the isofemale

strain we analyzed all 10 generations. However, for the massal strain we analyzed the


productivity of the 1st, 5

th and 10

th generations in order to compare the influence of this

chemical in two different strains.

Morphological Alterations

During each daily counting, the flies were morphologically analyzed with a stereoscopic

microscopy (Carl Zeiss). The insects that showed morphological alterations were

counted, photographed and then were kept frozen at -20ºC for later electrophoretical

analysis. The proportion of flies carrying morphological alterations was calculated as

the ratio altered/ total number of flies emerged on each treatment.

Biochemical Analysis

Assays of Enzimatic Activities and Total Protein Quantification

Carboxylesterase activity were measured by the method of Ellman et al. (1961),

modified by Bonacci et al. (2004), using phenylthioacetate as substrate.

Carboxylesterases are able to hydrolyze phenylthioacetate yelding thioacetate, that in

combination with 5,5’-dithiobis-2-nitrobenzoic acid (DTNB) forms a yellow anion 5-

thio-2-nitrobenzoic acid which absorbs strongly at λ=412nm. Optimal assay condition

ranges were carried out using a pooled sample containing nine Drosophila

melanogaster homogenized with 200 µL of Tris-HCl buffer 100mM pH8.0 and

centrifuged for 15min at 10,000xg. 20µL from the solution were mixed with 455µL of

the same buffer. 15µL of phenylthioacetate 150mM and 10µL of DTNB 50mM were

added to start the reaction. Increasing in absorbance at 412nm was monitored during

1min. Blanks without substrates or samples were previously incubated at 25ºC for 2 min

to assess endogenous cross-reaction with DTNB. The sample protein concentration was

determined following the method of Bradford (1976). Absorbance readings were carried

out in a Varian Cary 100 spectrophotometer.

Statistical Analyses

Statistical tools were used to analyze daily emergences. For this purpose, we applied the

parametric test of equality of two proportions (Normal approach Z) for independent

samples (Moore, 2005). However, in order to analyze carboxylesterase activity, total

productivity of each generation and frequency of morphological alterations, we used

Student´s t test (Zar, 1999). The tests were done using the software BioEstat 4.0 (Ayres

et al., 2005), using a p<0.05 significance level.


Results

The ethidium bromide effects were verified in ten generations of Drosophila

melanogaster, and daily productivity was measured along fifteen days. The total

numbers of flies emerged from the three replicates are shown in figures 1 and 2, for the

massal and the isofemale strain (A produtividade diária total pode ser verificada nos

anexos 1 e 2).

In figure 1, for F1 from the massal line exposed to 30µM, productivity from 50% of the

days showed significant differences; however this percentage goes to 60% in F5 and

67% in F10. On the other side, for EMS, 42% of the days showed differences in

productivity in F1, but in F5 raised to 50% and in F10 returned to 42%.

0

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Figure 1: Proportion of flies emerging daily for the massal stock of D. melanogaster,

exposed to 30µM EB (-*-), 1µM EMS (-◦-), and control (-♦-). The filled triangles

(▲) show significant differences for 1µM EB and the filled squares (■) for 1µM

EMS.

F10

F1 F5

Days

Days

▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲

▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲

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Figure 2: Proportion of flies emerging daily for the ten generations (F1-F10) from

isofemale line of D. melanogaster, exposed to 30µM EB (-*-), 1mM EMS (-◦-), and

control (-♦-). The filled triangles (▲) show significant days for 1µM EB and filled

square (■) for 1µM EMS.

▲

■

▲ ▲ ▲

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F2

F3 F8

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F10

Days Days

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Pro

port

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ing



In figure 2, for the isofemale strain, it was possible to verify that, for 30µM EB,

occurred significant differences in productivity: 34% of the days in F1, 60% in F2 and

F3, 75% in F4 and F6, 42% in F5, F7 and F10 and 67% in F8 and F9. For 1µM EMS,

we could notice that also for many days there were significant differences in

productivity, being the lowest value 34% of the days for F1, F6 and F9, and the highest

84% at F5.

We notice that the curve patterns for the groups exposed to 30µM EB and 1µM EMS

sometimes were very similar, as showed by F1; there was a superposition of both

curves. Moreover, for this generation, the same percentage of days with significant

differences in productivity was observed for these two treatments.

Figure 2 shows that there was, in the exposed groups, an emergence delay for some

generations. This fact was observed from F2 to F4 and for F10 too, because the control

replicates ceased their flies’ emergence around the 8th day, but for the exposed ones this

period was prorogated to the 15th of the life cycle. In other words, the maxima of flies

emerging from the groups exposed to EB and EMS are different from those showed by

the control. This fact can be seen in F2: the control group showed its maximum in the

2nd day, for the group exposed to 30µM EB this occurred in the 5th day, whereas for

EMS this event occurred in the 4th day. This effect was observed also from F3 to F6, F8

and F9.

Comparing the total productivity (figure 3) it was possible to verify that for F5 and F10

from the massal strain there was a decrease in total productivity in the exposure to EB

and EMS. The same was observed from F5 to F8 for the isofemale strain. It is

noteworthy that F10 of both strains had a very different productivity; for the isofemale

strain there was a clear decrease of total productivity, however the massal strain did not

have this behavior. The low genetic variability of the isofemale stock could account for

this, also rendering the flies more susceptible of the action of mutagens. In figure 3, for

the group exposed to EB there was a significant difference in the total productivity of

F7 and F8 of the isofemale line, while for EMS the differences were found in F8 of

isofemale line and for F10 of the massal strain.


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Figure 3: Total productivity of ten generations of D. melanogaster. A. Isofemale line and B. massal line, both exposed to 30µM EB (-*-), 1µM EMS (-◦-), and control (-♦-). The filled triangles (▲) show significant differences for 1µM EB and the filled squares (■) for 1µM EMS. Besides the effects concerning productivity, some morphological alterations (figure 4)

were noticed in the pattern of wings (absence, rib alterations, non stretched), tergites

(malformation) and coloration (extra pigmentation). The frequency of morphological

alterations is shown in figure 5, where we can notice that in the control group they were

low along the 10 generations in both lines. However, this same parameter for the insects

exposed to EB was high in the first generations along the ten generations for both

strains and treatments (EB and EMS).

A B

▲ ▲ ■

■


Figure 4: Main morphological alterations in wings (A, D), tergites (B, F) and coloration pattern (C, females with black spots in the ventral part of their abdomen) verified in Drosophila melanogaster exposed to EB (A, B, E) and EMS (C, D, F). The arrows indicate the alterations.

0

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1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th

Generations

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ns

Figure 5: Frequency of morphological alterations along ten generations of Drosophila

melanogaster. A- Isofemale strain; B- Massal Strain, both exposed to 30µM EB (-*-), 1µM EMS (-◦-), and control (-♦-). The filled triangles (▲) show significant differences for 1µM EB and the filled squares (■) for 1µM EMS.

A B C D

E F

A B 0.03

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■ ■ ■

▲

▲ ▲ ▲ ▲


Analyzing the ratio of daily productivity of the exposed groups divided by the flies

emerging in the control group (figure 6), we notice that for both stocks, most of days the

emergence of the exposed replicates were lower than for the control group, in F5 and

F10. This effect was observed for 30µM EB and for 1µM EMS and could reflect the

accumulation of toxic products along generations. Figure 10D shows that in F10, for the

group exposed to EMS, the ratio increased after the 8th day, showing a delay in the

emergence.

A

B Figure 6: Ratio of flies emerging of the group exposed divided by those emerged from the control group for the isofemale (�) and massal (�) lines of D. melanogaster. Groups exposed to 30µM EB: A. F5, B. F10. Groups exposed to 1µM EMS: C. F5, D. F10. In order to verify the influence of EB and EMS at the biochemical level, we analyzed

the activity from carboxylesterase (figure 7). This revealed that there was no difference

in the measured activities of this enzyme, considering males and females. However,

when the exposed groups were analyzed, we notice a decrease in the activity for both

EB and EMS groups, with significant differences for the last one.

A B

C D


U/m

g p

rote

in

0

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0,3

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0,5

0,6

0,7

0,8

control 30uM EB EMS

U/m

g p

rote

in

Figure 7: Carboxylesterase activities of D. melanogaster in the control and the exposed groups (to 30µM EB and 1µM EMS). The asterisk (*) shows a significant difference in activity when compared to the negative control. Discussion

In order to analyze the toxic effects induced by both chemicals, some parameters as

productivity, phenotypic alterations and carboxylesterase activities were analyzed in

Drosophila melanogaster. In this work, EMS was used as a positive control, because it

is classified as mutagenic (Griffiths et al., 1998), and the results obtained serve as

comparison parameters for the data gathered with EB. Ethylmethanesulfonate is able to

produce base-pair substitutions and chromosome changes. It also induces polygenic

mutations affecting viability (Mukai, 1970). Mutation and chromosome breaking effects

were reported by Alderson (1965), Epler (1966), Jenkins (1967), Yost et al. (1967), Lim

and Snyder (1968), Abrahamson et al. (1969), Lee et al. (1970), Brink (1970).

In our experiments we noticed that, for both isofemale and massal stocks, in most of the

days of each generation, the differences of daily productivity were significant when

compared to the control, reaching 84% of the days for 1µM EMS, for F5 of the

isofemale strain, and 75% in F4 and F6, when exposed to 30µM EB. The massal strain,

when compared to the isofemale line, has a larger genetic variability, however, in both,

EB and EMS caused differences in daily productivity. Moreover, analyzing figure 6, the

genotoxic effects were expressed in daily productivity by the accumulation of EB along

generations, for both strains. These were reflected by the larger ratio of 30µM EB and

1µM EMS to their control for F5, when compared to F10. The ratio found was in most

of the emergence period lower than 1, and that could be an effect of the cumulative

effects of both EB and EMS. Ethylmethanesulfonate mutagenesis, under standard

*

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1 0


conditions, generates mostly G/C→ A/T transitions and a small fraction of large-scale

aberrations detectable by Southern blotting (Ashburner, 1989). Ohnishi and Keightley

(1998) verified that EMS induced polygenic mutation rates affecting nine quantitative

characters in Drosophila melanogaster. Five are important fitness components:

competitive viability, egg productivity, hatchability, developmental time and longevity,

two were morphological: body length and abdominal bristle number, and two were

behavioral: phototaxis and mating speed of males.

In figure 3 it was possible to analyze the interference of EB on total productivity in

each generation of exposed groups. These effects could be explained by the action of

EB as a mitotic and meiotic poison, since it could block the process of spermatogenesis

(Marcos et al., 1981), evident for F5 and F10, where the total productivity was, in all the

exposed groups, smaller than for the control.

In order to analyze all the effects caused by the exposure to toxic products, several

parameters need to be verified. For this reason, over the productivity, we analyzed

morphological alterations caused by EB. In figure 5 it is possible to see that, analyzing

the frequency of alterations, in most of the cases those were twelve times larger than for

the control (for 1µM EMS for F2). In some cases, the frequency of malformations was

around the same of that found in the negative control (F6 and F5 of the isofemale and

massal strains, respectively). However, this number was not, in any generation, inferior

to the frequency found in the negative control. If a chemical induces toxicity and the

specimens are inviable, then the alterations would not be visible. The high frequency of

morphological alterations for 30µM of EB and 1µM EMS did not decrease along the

generations, maintaining a pattern of high frequency. These results were different than

those found by Ouchi et al. (submitted), when analyzed the effects induced by 1 and

5µM EB; the frequency of alterations decreased along the ten generations. This could

reflect the high toxicity of EB at the concentration of 30µM. Analyzing the pattern of

emerging flies, in some generations the curves were similar to EMS, as observed in F1

of the isofemale strain. However, for the same treatment, we have found different

phenotypic alterations, in wings, tergites, and coloration pattern. Goncharova et al.

(1988) verified that a significant individual variability occurred when they analyzed

lethals recessive mutations in germinate cells in Drosophila melanogaster, exposed to

EMS. Accordingly, each individual can respond in different forms, for the same

condition, and it can explain the high variability.


However, morphological alterations were not restricted to the exposed groups; they

were observed, with a low frequency, in the control group as well (F1, F2, F6 and F10).

Mutations happen randomly, in the sense that the probability of occurring is not affected

by the usefulness that the mutation can potentially have (Futuyma, 2002). For any

specific nucleotide in a DNA sequence the mutation rate is extremely low, in the order

of 1 in 100 million per generation. However, that rate, multiplied by the hundreds of

thousands of nucleotides present in a gene and for the trillion of more for an organism,

means that each individual probably holds one or more mutations (Rickfles, 2003).

Another effect of the chemicals was verified in the prolongation of the life cycle of

most of the generations (F2 to F5 and F10) of the isofemale strain. Fleming et al. (1981)

reported a similar effect of EB, although at a higher concentration: 0.2mM in the culture

medium increased the development time of Drosophila melanogaster by 32%. The

authors found that oxygen consumption was lower for flies treated with EB during their

development or during their adults’ life. Morel et al. (1999) verified that low EB

concentrations caused changes in mitochondrial DNA (mtDNA), leading to reductions

in the activities of the respiratory complexes III and IV measured in treated Drosophila

cells. A delay of development from the larval to the pupal phase was reported by Akins

et al. (1992) when they analyzed the neurotoxic effect of lead and cadmium in the life

cycle of Drosophila melanogaster. . In some generations such as F1, we have verified

that the emerging flies had a maximum in the same day for 1µM EMS and for 30µM

EB. This could be caused by the effect of both toxic products in DNA.

Looking for another evidences of the action of EMS in Drosophila, biochemical effects

were analyzed in terms of carboxylesterase activity, showing differences between the

control and the group treated with 1µM EMS. Carboxylesterase is responsible for

xenobiotic compounds elimination. In a previous work (Ouchi et al., submitted), we

found differences in carboxylesterase activity for a group exposed to 1µM EB. A

possible reason for not finding significant differences between the control and 30µM

EB groups could be the individual variability of the organisms to respond to a different

situation. Concerning the effect on other enzymes, Weiss & Zeres (1986) showed that

EB has caused alteration in the specific activity of the nuclear-gene-encoded,

mitochondrial arginine biosynthetic enzyme ornithine carbamoyltransferase (EC

2.1.3.3) in Neurospora crassa, elevating its concentrations.


In conclusion, even in low concentrations, EB and EMS can induce toxic effects in

terms of productivity, morphological and biochemical parameters, presumably due to

their genotoxic properties. The results suggest that EB, as an inhibitor of topoisomerase

II, could induce some alterations at the DNA level, which would lead to changes in the

activity of some enzymes, reflected as different phenotypic patterns. The different

susceptibility of individuals to mutagens reflects the existence of cryptic genetic

variability in Drosophila strains. It is relevant to take into account the individual

sensitivity of organisms to mutagenic factors, when conducting mutation research and

studying genetic consequences of biosphere pollution.

Acknowledgements. The authors are grateful to the Department of Biology where the insects were maintained, to MSc. Fernando Rogério Carvalho and Prof. Dr. Francisco Langeani Neto for their help concerning the photographs, to FAPESP (grant 05/02418-6 for GOBR), CNPq (fellowship for GOBR) and CAPES (fellowship for RYO). References

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melanogaster as an Alternative Animal for Studying the Neurotoxicity of Heavy Metals. Biometals 5: 111-120. Alderson TA. 1965. Chemically induced delayed germinal mutations in Drosophila. Nature 207: 164-167. Ashburner M. 1989. Drosophila: A Laboratory Manual. Cold Spring Harbor Laboratory. New York. Ayres M, Ayres JrM, Ayres DL, Santos AS. 2005. BioEstat 4.0: Aplicações Estatísticas nas Áreas das Ciências Biológicas e Médicas. Editora: Sociedade Civil Mamirauá/MCT/Imprensa Oficial do Estado do Pará, Belém, Brazil.

Becker TCA, Chiuchetta SJR, Baptista F, Castro-Prado MAA. 2003. Increase in mitotic recombination in diploid cells of Aspergillus nidulans in response to ethidium bromide. Genetics and Molecular Biology 26: 381-385.

Benford DJ, Hanley BA, Bottrill K. 2000. Biomarkers as predictive tools in toxicity testing. Altern Lab Anim 28: 119-131. Bonacci S, Browne MA, Dissanayake A, Hagger JA, Corsi I, Focardi S, Galloway TS. 2004. Esterase activities in the bivalve mollusc Adamussium colbecki as a biomarker for the pollution monitoring in the Antarctiv marine environment. Marine Pollution Bulletin 49: 445-455.


Bradford MM. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem 72: 248-254. Brill SJ, NiNardo S, Voelkel-Meiman K, Sternglanz R. 1987. Need for DNA topoismerase as a swivel for DNA replication for transcription of ribosomal RNA. Nature 326: 414-416. Brink NG. 1970. Complete and mosaic visible mutations produced by ethyl methanosulfonate in Drosophila melanogaster. Mutation Res 10: 227-236.

Çakir S, Sarikaya R. 2005. Genotoxicity testing of some organophosphate insecticides in the Drosophila wing spot test. Food and Chemical Toxicology 43: 443-450. D’Arpa P, Machlin PS, Ratrie HIII, Rothfield NF, Cleveland DW, Earnshaw WC. 1988. cDNA cloning of human DNA topoisomerase I: Catalytic activity of a 67,7kDa carboxyl-terminal fragment. Proc Natl Acad Sci 85: 2543-2547. Ellman GL, Courtney KD, Andres JrV, Featherstone RM. 1961. A new rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7: 88-95. Epler JL. 1966. Ethyl methanosulfonate induced lethals in Drosophila. Frequency-dose relations and multiple mosaicism. Genetics 54: 31-36. Etienne R, Fortunat K, Piercce V. 2001. A Mechanism of urea tolerance in urea-adapted populations of Drosophila melanogaster. A Journal of Experimental Biology 204: 2699-2707. Fleming JE, Leon HA, Miquel J. 1981. Effects of ethidium bromide on development and aging of Drosophila: Implications for the freee radical theory of aging. Experimental Gerontology 16:287-293. Fox G, Popanda O, Edler L, Thielmann HW. 1996. Preferential inhibition of DNA polymerases alpha, delta, and epsilon from Novikoff hapatoma cells by inhibitors of cell proliferation. J Cancer res Clin Oncol 122: 78-94. Futuyma D J. 2002. Biologia Evolutiva. Editora Funpec, Ribeirão Preto, Brazil. Goncharova RI, Levina AB, Kuzhir TD. 1988. Sensitivity of individual Drosophila to the mutagenic action of ethyl methanesulfonate. Genetika 24: 2141-2148. Griffiths AJF, Miller JH, Suzuki DT, Lewontin RC, Gelbart WM. 1998. Introdução à Genética. Editora Guanabara Koogan 856p. Rio de Janeiro, Brazil.

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Conclusões Gerais 87

Conclusões Gerais


Conclusões Gerais

Com a realização deste trabalho foi possível verificar os efeitos do EB em

Drosophila melanogaster, avaliados em níveis de produtividade diária ao longo de 10

gerações usando duas diferentes linhagens (massal e isofêmea), padrão de esterase-6 e

perfil de proteínas totais, assim como também a atividade de carboxilesterase. Além

disso, realizaram-se análises em nível morfológico e nas diferentes fases do

desenvolvimento de Drosophila melanogaster, das principais alterações promovidas

pela intercalação do EB no DNA. Dessa forma, através da exposição contínua ao EB,

conseguimos verificar as seguintes alterações fenotípicas:

1. Produtividade diária: Verificou-se que para as duas linhagens houve, para cada

geração, diferenças significantes em termos de produtividade. Além disso, a

análise do padrão de eclosão, ao longo dos quinze dias mensurados, nos permitiu

verificar que o EB, assim como o EMS, atua de forma a ampliar a duração do

ciclo de desenvolvimento de Drosophila melanogaster. Alguns autores

relataram um efeito semelhante de EB em desenvolvimento, sendo que com uma

concentração de 0,2mM no meio de cultura, o tempo de desenvolvimento de

Drosophila melanogaster aumentou em até 32%. Eles sugeriram que consumo

de oxigênio era mais baixo para moscas tratadas com EB durante o seu

desenvolvimento ou durante sua vida de adultos. Estudos posteriores verificaram

que em baixas concentrações de EB ocorrem mudanças no DNA mitocondrial

(mtDNA), cujas conseqüências são reduções nas atividades de complexos

respiratórios III e IV em células de moscas tratadas.

2. Análise bioquímica: Através da análise eletroforética pode-se verificar que a

presença do EB e do EMS alterou o padrão de esterase-6, em que os indivíduos

expostos e com alteração morfológica apresentaram uma concentração desta

enzima inferior àquela apresentada pelo grupo controle negativo. Além disso, a

análise do padrão de proteínas totais revelou que algumas proteínas, que não

estavam presentes no grupo controle, foram encontradas nos indivíduos expostos

e alterados morfologicamente, enquanto que outras proteínas que estavam

presentes no controle negativo não foram encontradas nos indivíduos alterados,

podendo isso representar uma alteração em nível gênico ou inibição da síntese


daquela proteína. Além disso, a atividade enzimática da carboxilesterase, uma

enzima que participa no processo de eliminação de xenobióticos, apresentou

uma diferença significante entre controle e 1µM EB e controle e EMS.

3. Análise no Desenvolvimento: A análise realizada permitiu verificar que tanto o

EB como o EMS alterou a viabilidade de ovos e larvas. A viabilidade das pupas

não foi influenciada por esses agentes.

4. Análise morfológica: Permitiu-nos observar as alterações morfológicas

decorrentes, provavelmente, de alterações no padrão de desenvolvimento. As

principais alterações foram verificadas nas asas (deformações em nervuras, asas

não esticadas, alterações no padrão de pintas das asas, infladas), no padrão de

pigmentação corpóreo (pintas ou manchas foram encontradas no escutelo, patas,

porção ventral do abdômen e entre os tergitos) e nos tergitos (malformações que

resultavam em um aspecto tortuoso ao contrário do padrão retilíneo e uniforme

normal). Com uma incidência menor, foram encontrados indivíduos

inteiramente malformados, sem asa, e sem uma das patas. A freqüência de

indivíduos alterados foi sempre mais elevada quando comparada ao controle

negativo, não exposto.

5. Análise comportamental: A análise do comportamento foi baseada na

observação do tempo de pré-cópula e cópula. Diferenças significantes foram

encontradas entre controle e 30µM EB e entre controle e 1µM EMS, para o

comportamento de cópula. Como já relatado, a presença do EB causou

malformações no corpo de Drosophila melanogaster, que podem ter

influenciado o comportamento de cópula, visto que este é compreendido por

movimentos complexos. Além disso, o EB pode ter influenciado o sistema

neural.

6. Os efeitos do EB foram perceptíveis mesmo na concentração mais baixa, igual

àquela utilizada nos laboratórios de pesquisa que usam métodos de biologia

molecular.


7. Os experimentos realizados mostraram uma elevada variação de respostas,

mostrando a variabilidade de respostas individuais. A suscetibilidade diferente

de indivíduos frente aos mutágenos reflete a existência de variabilidade genética.

É pertinente levar-se em consideração a sensibilidade individual dos organismos

para se avaliar os fatores que causam a toxicidade, bem como o seu impacto

como um poluente da biosfera.

8. Os resultados obtidos até aqui devem ser aprofundados, procurando em um

estágio mais avançado, compreender os mecanismos subjacentes às respostas

obtidas e os mecanismos de toxicidade induzidos pelo EB.

Anexos 91

Anexos

Generation Treatment 1st

2nd

3rd

4th

5th

6th

7th

8th

9th

11th

13th

15th

Total

1st

Control 6 32 44 148 191 26 6 5 2 3 0 0 463

1µM EB 3 46 53 158 202 114 44 51 26 23 25 7 752

5µM EB 0 22 96 248 199 181 63 29 14 28 26 11 917

30uM EB 0 4 25 49 226 69 10 1 2 0 0 0 386

EMS 0 1 22 52 158 47 3 1 2 2 0 0 288

2nd

Control 19 131 116 82 75 25 14 12 2 0 1 0 477

1µM EB 4 105 66 114 120 81 55 40 35 33 20 1 674

5µM EB 0 108 116 71 162 66 41 32 20 24 14 1 655

30µM EB 0 81 120 67 94 86 54 34 19 27 12 4 598

EMS 22 135 124 164 127 99 64 37 36 56 34 23 921

3rd

Control 130 173 73 76 63 98 28 11 1 3 2 0 658

1µM EB 9 101 85 77 83 32 15 35 35 136 17 1 626

5µM EB 49 163 95 54 101 98 123 67 25 8 0 0 783

30µM EB 0 79 89 22 53 104 83 74 65 46 11 1 627

EMS 22 73 73 201 146 83 123 85 50 40 14 4 914

4th

Control 174 50 35 58 82 78 39 14 5 3 0 1 539

1µM EB 14 51 95 110 108 111 80 32 13 14 28 24 680

5µM EB 69 101 86 113 134 69 46 106 99 120 43 14 1000

30µM EB 19 134 51 43 92 119 131 61 23 63 50 19 805

EMS 94 82 34 133 116 142 136 67 31 33 4 2 874

5th

Control 170 124 43 91 75 134 144 72 59 65 21 5 1003

1µM EB 161 128 85 33 35 23 28 38 15 99 57 28 730

5µM EB 131 117 64 77 121 154 150 105 58 42 22 4 1045

30µM EB 3 94 89 34 57 110 84 38 60 65 20 5 659

EMS 47 228 72 40 74 79 54 26 15 8 2 0 645

6th

Control 28 26 76 139 205 123 107 106 53 83 35 37 1018

1µM EB 29 51 14 97 138 187 165 122 48 58 59 52 1020

5µM EB 57 38 45 118 158 163 93 90 47 116 33 0 958

30µM EB 62 50 29 80 78 83 79 108 94 117 81 36 897

EMS 20 101 87 40 55 92 76 63 47 52 21 19 673

7th

Control 52 25 49 114 140 70 159 80 65 124 83 47 1008

1µM EB 84 36 33 48 85 59 130 107 91 168 132 64 1037

5µM EB 41 97 32 38 61 39 121 89 41 47 29 33 668

30µM EB 9 46 17 24 100 40 95 55 53 111 62 21 633

EMS 6 35 41 70 128 63 121 87 71 102 74 21 819

8th

Control 15 22 56 173 213 142 67 115 118 95 64 47 1127

1µM EB 50 65 31 81 127 158 77 62 30 64 79 33 857

5µM EB 28 83 51 119 130 129 87 122 78 106 115 131 1179

30µM EB 5 64 26 26 115 128 85 117 78 104 109 38 895

EMS 61 75 50 38 66 77 65 94 69 90 47 33 765

9th

Control 87 94 54 98 43 74 85 40 21 40 22 14 672

1µM EB 102 111 113 72 43 51 53 47 63 111 99 48 913

5µM EB 43 63 76 167 107 120 90 57 33 99 77 75 1007

30µM EB 27 102 39 38 68 79 82 63 36 58 41 34 667

EMS 2 38 127 69 32 58 71 33 13 14 2 0 459

10th

Control 1 2 73 86 27 39 108 61 13 16 0 1 427

1µM EB 0 0 14 46 35 52 80 36 4 0 3 0 270

5µM EB 0 3 18 39 32 130 145 59 10 3 2 0 441

30µM EB 0 3 46 122 47 50 74 42 66 34 5 0 489

EMS 1 0 27 108 37 37 53 75 98 83 44 13 576

Days

Anexo 1: Produtividade diária da linhagem isofêmea exposta ao EB e EMS, durante F1-F10

As células em negrito, correspondem aos dias significantes, em que p<0,05

Anexo 2: Produtividade diária da linhagem massal exposta ao EB e EMS, durante F1, F5 e F10

Generations Treatment 1st

2nd

3rd

4th

5th

6th

7th

8th

9th

11th

13th

15th

Total

1st

ML Control 8 92 120 77 82 52 80 73 47 136 135 58 960

1µM EB 40 82 64 62 45 73 135 103 63 129 82 35 913

5µM EB 21 171 96 61 114 107 118 85 48 131 84 28 1064

30uM EB 21 106 76 53 67 136 103 58 39 118 89 24 890

EMS 30 146 124 62 63 72 82 67 57 146 55 18 922

5th ML Control 28 63 63 64 47 131 91 115 105 118 162 57 1044

1µM EB 7 36 24 104 96 199 130 109 73 52 41 10 881

5µM EB 34 177 93 70 18 73 65 70 34 28 5 0 667

30µM EB 31 127 43 42 38 156 125 118 50 32 2 0 764

EMS 8 115 86 64 40 78 51 33 55 87 53 30 700

10th ML Control 32 135 170 430 89 139 120 76 167 100 73 58 1589

1µM EB 42 132 148 398 90 155 104 56 121 54 47 35 1382

5µM EB 16 120 80 195 66 129 137 106 158 104 63 28 1202

30µM EB 45 145 194 259 79 131 112 64 92 54 22 4 1201

EMS 103 91 66 176 66 92 79 32 103 48 27 18 901

Days

As células em negrito correspondem aos dias que foram significantes p<0,05.

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