Project Ppt

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Genotoxicity of Intercalating Agents in Drosophila melanogaster

Transcript of Project Ppt

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Genotoxicity of Intercalating Agents in Drosophila melanogaster

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Drosophila melanogaster

The term "Drosophila", meaning "dew-loving", is a modern scientific Latin adaptation from Greek words δρόσος, drósos, “dew", and φίλος, phílos, "loving" with the Latin feminine suffix -a.

DrosophilaDrosophila is a genus of small flies whose is a genus of small flies whose members are often called small fruitflies, members are often called small fruitflies, or more appropriately vinegar flies, wine or more appropriately vinegar flies, wine flies, pomace flies, grape flies, and picked flies, pomace flies, grape flies, and picked fruit-flies. fruit-flies.

One species in particular,One species in particular, DrosophilaDrosophila melanogastermelanogaster, has been heavily used in , has been heavily used in research in genetics and is a common research in genetics and is a common model organism in developmental model organism in developmental biology. biology.

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Morphology Drosophila are small flies, typically pale yellow to

reddish brown to black, with red eyes. Many species, including the noted Hawaiian picture-wings, have distinct black patterns on the wings. Most are small, about 2-4 mm long

Life cycle

DD..melanogastermelanogaster has a short diploid life cycle. has a short diploid life cycle.

The larva forms in one day and then undergoes The larva forms in one day and then undergoes several stages of growth during which the several stages of growth during which the imaginal disks and other precursors of adult imaginal disks and other precursors of adult structure proliferate.structure proliferate.

These structure differentiate during pupation and These structure differentiate during pupation and the fly hatches and begins the cycle againthe fly hatches and begins the cycle again

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Chromosomes • It has only 4 pairs of chromosomes:It has only 4 pairs of chromosomes: 3 autosomes (2L+2R; 3L+3R), and 1 sex 3 autosomes (2L+2R; 3L+3R), and 1 sex

chromosome (X+XorY).chromosome (X+XorY).• Sex is determined by X and Y sex Sex is determined by X and Y sex

chromosomes.chromosomes.• XX is female and XY is male:XX is female and XY is male: in contrast with humans, the number of X’s in contrast with humans, the number of X’s

in relation to the number of autosomes in relation to the number of autosomes determine sexdetermine sex

The mature larvae show giant chromosomes in the The mature larvae show giant chromosomes in the salivary glands called polytene chromosomessalivary glands called polytene chromosomes

• Chromosome puffs are diffuse uncoiled regions Chromosome puffs are diffuse uncoiled regions of the polytene chromosome that are sites of of the polytene chromosome that are sites of RNA transcription. RNA transcription.

• A Balbiani ring is a large chromosome puff.A Balbiani ring is a large chromosome puff.• In addition to increasing the volume of the cells In addition to increasing the volume of the cells

nuclei and causing cell expansion, polytene cells nuclei and causing cell expansion, polytene cells may also have a metabolic advantage as may also have a metabolic advantage as multiple copies of genes permits a high level of multiple copies of genes permits a high level of gene expression.gene expression.

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IntercalationIn chemistry, intercalation is the reversible inclusion of a molecule (or group) between

two other molecules (or groups). Examples include DNA intercalation.

There are several ways molecules also known as “ligands" - can interact with DNA: Ligands may interact with DNA by covalently binding, electrostatically binding, or intercalating.

Intercalation occurs when ligands of an appropriate size and chemical nature fit themselves in between base pairs of DNA. These ligands are mostly polycyclic, aromatic, and planar, and therefore often make good nucleic acid stains. Intensively studied DNA intercalators include ethidium bromide, provlavine, daunomycin, doxorubicin, and thalidomide.

Intercalation induces structural distortions. Left: unchanged DNA strand. Right: DNA strand intercalated at three locations (red areas).

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Mechanism of IntercalationIntercalation as a mechanism of interaction between

cationic, planar, polycyclic aromatic systems of the correct size (on the order of a base pair) was first proposed by Leonard Lerman in 1961.

One proposed mechanism of intercalation is as follows: in aqueous isotonic solution, the cationic intercalator is attracted electrostatically to the polyanionic DNA. The ligand displaces a sodium and/or magnesium cation that always surround DNA (to balance its charge) and forms a weak electrostatic bond with the outer surface of DNA. From this position, the ligand may then slide into the hydrophobic environment found between the base pairs and away from the hydrophilic outer environment surrounding the DNA. The base pairs transiently form such openings due to energy absorbed during collisions with solvent molecules.

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Contd…

In order for an intercalator to fit between base pairs, the DNA must dynamically open a space between its base pairs by unwinding.

The degree of unwinding varies depending on the intercalator, for example, ethidium cation (the ionic form of ethidium bromide found in aqueous solution) unwinds DNA by about 26° while proflavine unwinds it by about 17°.

This unwinding causes the base pairs to separate,

creating an opening of about 0.34 nm (3.4 Å). This unwinding induces local structural changes

to the DNA strand, such as lengthening of the DNA strand, or twisting of the base pairs.

These structural modifications can lead to functional changes, often to the inhibition of transcription and replication and DNA repair processes, which makes intercalators potent mutagens.

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Intercalation: Two Types• Non-covalent Bonding• Covalent Bonding

Non-covalent Bonding: A noncovalent bond is a type of chemical bond, typically between macromolecules, that does not involve the sharing of pairs of electrons, but rather involves more dispersed variations of electromagnetic interactions

Example: The most extensively studied DNA intercalating agents are acridine and its derivatives, that bind reversibly but non-covalently to DNA. These are frameshift mutagens, especially in bacteria and bacteriophage, but do not otherwise show a wide range of mutagenic properties.

Covalent Bonding: A covalent bond is a form of chemical bonding, that is characterized by the sharing of pairs of electrons between atoms, or between atoms and other covalent bonds. In short, attraction-to-repulsion stability that forms between atoms when they share electrons is known as covalent bonding.

Example: The DNA intercalating agent Ethidium bromide, bind covalently to DNA

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Genotoxicity of Non-covalent Interaction: DNA InercalatorsThese DNA intercalators bind reversibly but non-covalently to DNA. These are frameshift

mutagens, especially in bacteria and bacteriophage, but do not otherwise show a wide range of mutagenic properties.

Frameshift Mutation: A frameshift mutation (also called a

framing error) caused by insertion or deletion of a number of

nucleotide from a DNA sequence. Due to the triplet nature of

gene expression by codons, the insertion or deletion can

disrupt the reading frame, or grouping of the codons, resulting

in a completely different translation from the original. The

earlier in the sequence the deletion or insertion occurs, the

more altered the protein produced is.

• Removal or addition of one or more bases shifts the reading frame A change in 1-2 bases substantially changes the output:

THE FAT CAT ATE HIS HAT      THE FAA TCA TAT EHI SHA T        THE FAT AAA CAT ATE  HIS HAT

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Causes of Insertions & Deletions: • Misalignment of the template & strands during DNA replication • Chemicals: Intercalating Agents- can insert into a DNA strand • Usually similar in size & shape to nucleotides • E.g. some pesticides

Properties Di-acridines or di-quinolines may be either mono- or bis-intercalators, depending upon the

length of the alkyl chain separating the chromophores. Those which monointercalate appear as either weak frameshift mutagens in bacteria, or as non-mutagens. However, some of the bisintercalators act as "petite" mutagens in Saccharomyces cerevisiae, suggesting that they may be more likely to target mitochondrial as compared with nuclear DNA.

A number of flavonoids appear to intercalate into DNA. However, their mutagenic properties may be dominated by the fact that many of them are also able to inhibit topoisomerase II enzymes, and this property implies that they will be potent recombinogens and clastogens. DNA intercalation may serve to position other, chemically reactive molecules, in specific ways on the DNA, leading to a distinctive (and wider) range of mutagenic properties, and possible carcinogenic potential.

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Acridine As DNA intercalating Agent

• Acridine, C13H9N, is an organic compound and a nitrogen heterocycle. Acridine is also used to describe compounds containing the C13N tricycle.

• Acridine was first isolated in 1871 by Carl Gräbe and Heinrich Caro.

• Acridine is structurally related to anthracene with one of the central CH groups is replaced by nitrogen. Acridine, a colorless solid, was first isolated from coal tar. It is a raw material used for the production of dyes and some valuable drugs. Many acridines, such as provlavine, also have antiseptic properties. Acridine and related derivatives bind to DNA and RNA due to their abilities to intercalate. Acridine orange (3,6-dimethylaminoacridine) is a nucleic acid -selective metachromatic stain useful for cell cycle determination.

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Properties Acridine and its derivatives are planar polycyclic aromatic molecules which bind

tightly but reversibly to DNA by intercalation, but do not usually covalently interact with it.

Acridines have a broad spectrum of biological activities, and a number of derivatives are widely used as antibacterial, antiprotozoal and anticancer drugs.

Simple acridines show activity as frameshift mutagens, especially in bacteriophage and bacterial assays, by virtue of their intercalative DNA-binding ability.

Acridines bearing additional fused aromatic rings (benzacridines) show little activity as frameshift mutagens, but interact covalently with DNA following metabolic activation (forming predominantly base-pair substitution mutations).

Compounds where the acridine acts as a carrier to target alkylating agents to DNA (e.g. the ICR compounds) cause predominantly frameshift as well as base-pair substitution mutations in both bacterial and mammalian cells.

Nitroacridines may act as simple acridines or (following nitro group reduction) as alkylating agents, depending upon the position of the nitro group.

Acridine-based topoisomerase II inhibitors, although frameshift mutagens in bacteria and bacteriophage systems, are primarily chromosomal mutagens in mammalian cells.

These mutagenic activities are important, since the compounds have considerable potential as clinical antitumour drugs.

Although evidence suggests that simple acridines are not animal or human carcinogens, a number of the derived compounds are highly active in this capacity.

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Anthracene Anthracene is a solid polycyclic aromatic

hydrocarbon consisting of three fused benzene rings derived from coal tar. Anthracene is used in the artificial production of the red dye alizarin. It is also used in wood preservatives, insecticides, and coating materials. Anthracene is colorless but exhibits a blue (400-500 nm peak) fluorescence under ultraviolet light.

ToxicologyIn spite of other polycyclic aromatic

hydrocarbons (PAH), anthracene is not carcinogenic but has been recently included in the Substances of Very High Concern list (SVHC) by the European Chemicals Agency (ECHA) because being considered Persistent, Bio-accumulative and Toxic for freshwater and marine ecosystems within the REACH framework. Anthracene, as many others PAH is generated during combustion processes: exposure to human happens mainly through tobacco smoke and ingestion of food contaminated with combustion products.

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Anthracene As DNA intercalating AgentThere are two carcinogens, derivatives of anthracene 7-chloromethyl-12methylbenz[a]anthracene and 7-chloromethylbenz[a]anthracene

Both compounds are carcinogenic and are believed to act by alkylating DNA. However, the first has a nonplanar ring system, whereas the second has a planar ring system. The non-planarity of 7-chloromethyl- 12-methylbenz[a]anthracene results from steric hindrance between a hydrogen atom of the 12-methyl group and a hydrogen atom on the [a] ring.

It is concluded that the carcinogenic activity of thesecompounds does not correlate with planarity of the ringsystem. This implies that, if DNA is the critical target ofattack by these carcinogens, complete intercalation of thearomatic ring system of the carcinogen between the basesof DNA is not a likely mechanism of carcinogenic action inthis system of compounds.

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Clastogenic Effects of DNA Intercalating AgentsBioflavonoids are naturally occurring polyphenols with intriguing and varied

therapeutic and chemo-protective activities generally ascribed to their antioxidant properties. However, many flavonoids have also been shown to be genotoxic in a variety of prokaryotic, eukaryotic, and in vivo systems.

Five of the flavonoids examined, luteolin, quercetin, genistein, apigenin, and acacetin, were strongly clastogenic.

This clastogenicity requires DNA intercalation and was substantially reduced by catalytic inhibitors of DNA topoisomerase II. The transition metals Cu(II) and Mn(II) formed chelates with and/or modified the structure and biological activity of some flavonoids but no consistent relationship could be demonstrated between metal reactivity and clastogenicity.

There was no clear association between generation of ROS and clastogenicity.

Genotoxicity of most flavonoids arises via DNA intercalation and topo II poisoning, likely mediated through metabolism to flavonoid quinones. Interestingly, other flavonoids such as myricetin, daidzein, baicalein, fisetin,

and galangin were catalytic topo II inhibitors, rather than poisons.

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Genotoxicity of Covalent Interaction: DNA Inercalators

A covalent bond is a form of chemical bonding, that is characterized by the sharing of pairs of electrons between atoms, or between atoms and other covalent bonds. In short, attraction-to-repulsion stability that forms between atoms when they share electrons is known as covalent bonding.

Ethidium Bromide: Ethidium bromide (sometimes abbreviated

as "EtBr", the abbreviation also confusingly used for bromoethane)

is an intercalating agent commonly used as a fluorescent tag (necleic

acid) in molecular biology laboratories for techniques such as

agarose gel electrophoresis. When exposed to ultra violet, it will

fluoresce with an orange color, intensifying almost 20-fold after

binding to DNA.

Ethidium bromide may be a strong mutagen. It is

also widely assumed to be a carcinogen or tetragen

although this has never been carefully tested.

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Health Hazards:

Ethidium bromide is a mutagen, suspected carcinogen and at high concentrations is irritating to the eyes, skin, mucous membranes and upper respiratory tract.

The health effect of ethidium bromide exposure have not been thoroughly investigated. It is suspected to be carcinogenic and teratogenic because of its mutagenicity, although there is no direct evidence of either effect. The toxic effects of ethidium bromide may be experienced if swallowed, inhaled or absorbed through the skin. However, ethidium bromide is not easily absorbed through the skin because of positive charge and bulky structure.

Ethidium bromide is thought to act as a mutagen because it intercalates into double stranded DNA, thereby deforming the molecule. This is believed to block or trip biological processes occurring on DNA, like DNA replication and transcription.

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Mechanism: Ethidium binds by inserting itself bewteen the stacked bases in double-stranded

DNA. Note that the ring structure of ethidium is hydrophobic and resembles the rings of the bases in DNA. Ethidium is capable of forming close van der Walls contacts with the base pairs and that's why it binds to the hydrophobic interior of the DNA molecule.

In doing so, they distort the double helix and

interfere with DNA replication, transcription,

DNA repair, and recombination. This is why

intercalating agents are often potent mutagens.

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Cyclopent[a]anthraquinones as DNA intercalating agents with covalent bond formation potential

A series of mitomycin C (MMC) analogues, namely cyclopentanthraquinone derivatives.

These new compounds are planar structures, like MMC, and bear an aziridine ring and a methyl carbamate side chain. After bioreduction, they are anticipated to be capable of intercalating into double-stranded DNA and bind covalently.

• Mitomycin C is a well characterized antitumor antibiotic which forms a covalent interaction with DNA after reductive activation. The activated antibiotic forms a cross-linking structure between guanine bases on adjacent strands of DNA thereby inhibiting single strand formation (this is essential for mRNA transcription and DNA replication).

• Anthramycin is an antitumor antibiotic which bind covalently to N-2 of guanine located in the minor groove of DNA. Anthramycin has a preference of purine-G-purine sequences (purines are adenine and guanine) with bonding to

the middle G.

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Quinacrine

The influence of cuprum ions on the interaction between

the quinacrine (QA) and DNA takes place at molecular

and cellular levels. An alteration of quinacrine

luminescence intensity in complex with DNA caused by

cuprum ions is explained in terms of redistribution of

QA molecules from quenching GC- to fluorescent AT-

DNA binding sites due to the competition of Cu2+ with

the dye. Mechanisms of component interactions in the

triplex "DNA-QA-Cu2+" in model and cellular systems

are shown to be in qualitative agreement. QA

photodynamic activity change caused by Cu2+ action is

explained on the basis of the ideas being developed.

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Effects of Intercalating Agents In Drosophila melanogaster

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Mitotic Recombination And Sex Chromosome Loss In Somatic Cells

The compounds tested were the intercalating agent adriamycin (AD), the alkylating compound chlorambucil (CAB) and the spindle poisons demecolcine (DEM), paclitaxel (taxol, TX) and vinblastine (VBL).

Genotoxic profiles obtained for these drugs indicate direct and indirect effects.

• While AD seems to be clastogenic due to its induction of X chromosome loss in XrX females.

• DEM, CAB and TX produced both structural chromosome aberrations through clastogenic activities and mitotic recombination through DNA interactions.

• The cytotoxic VBL induced rX loss only in XrY and intra-chromosomal recombination (XY) males, probably due to sister strand recombination, forward mutations or small deletions at the white locus.

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Genotoxic Activity of Four inhibitors of DNA

Topoisomerases in Larval Cells of Drosophila melanogaster Four inhibitors of DNA topoisomerases namely nalidixic acid, camptothecin, m-

amsacrine and etoposide, have been evaluated for genotoxic effects in the wing spot test of Drosophila melanogaster. This assay assesses somatic recombination and mutational events. We studied nalidixic acid as an inhibitor of bacterial DNA gyrase, camptothecin as a topoisomerase I inhibitor, as well as m-amsacrine and etoposide as topoisomerse II inhibitors.

The genotoxic effects were determined from the appearance of wing spots in flies

Nalidixic acid and m-amsacrine were compounds that did not increase the incidence of mutant clones, camptothecin and etoposide proved to be significantly genotoxic in this test, being camptothecin more effective than etoposide.

A significant proportion of the total spot induction was due to mitotic recombination

On the other hand, the cotreatments of each topoisomerase inhibitor with the alkylating agent ethyl methanesulfonate (EMS) indicate that, while nalidixic acid, m-amsacrine and etoposide show a tendency to an antagonistic interaction, camptothecin shows an additive effect

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A Mutation With Major Effects on Drosophila

melanogaster Sex Pheromones

Sex pheromones are intraspecific chemical signals that are crucial for mate attraction and discrimination. In Drosophila melanogaster, the predominant hydrocarbons on the cuticle of mature female and male flies are radically different and tend to stimulate or inhibit male courtship, respectively. This sexual difference depends largely upon the number of double bonds (one in males and two in females) added by desaturase enzymes.

A mutation was caused by a PGal4 transposon inserted in the desat1 gene that codes for the desaturase crucial for setting these double bonds. Homozygous mutant flies produced 70–90% fewer sex pheromones than control flies, and the pheromonal

difference between the sexes was almost abolished.