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  • 1. Genotoxicity of Intercalating Agents inDrosophila melanogaster

2. Drosophila melanogaster

  • The term "Drosophila", meaning "dew-loving", is a modern scientific Latinadaptation from Greek words , drsos, dew", and , phlos, "loving" with the Latin feminine suffix -a.
  • Drosophilais a genus of small flies whose members are often called small fruitflies, or more appropriately vinegar flies, wine flies, pomace flies, grape flies, and picked fruit-flies.
  • One species in particular, Drosophila melanogaster , has been heavily used in research in genetics and is a common model organism in developmental biology.

3. Morphology

  • Drosophilaare 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, about2-4 mm long
  • Life cycle
  • D . melanogasterhas a short diploid life cycle.
  • The larva forms in one day and then undergoes several stages of growth during which the imaginal disks and other precursors of adult structure proliferate.
  • These structure differentiate during pupation and the fly hatches and begins the cycle again

4. Chromosomes

  • It has only 4 pairs of chromosomes:
  • 3 autosomes (2L+2R; 3L+3R), and 1 sex chromosome (X+XorY).
  • Sex is determined by X and Y sex chromosomes.
  • XX is female and XY is male:
  • in contrast with humans, the number of Xs in relation to the number of autosomes determine sex
  • The mature larvae show giant chromosomes in the salivary glands called polytene chromosomes
  • Chromosome puffs are diffuse uncoiled regions of the polytene chromosome that are sites of RNA transcription.
  • A Balbiani ring is a large chromosome puff.
  • In addition to increasing the volume of the cells nuclei and causing cell expansion, polytene cells may also have a metabolic advantage as multiple copies of genes permits a high level of gene expression.

5. Intercalation

  • In chemistry,intercalation is the reversible inclusion of a molecule (or group) between two other molecules (or groups). Examples includeDNA intercalation.
  • There are several ways molecules also known as ligands " - can interact with DNA:Ligands may interact with DNA by covalently binding, electrostatically binding, orintercalating .
  • Intercalationoccurs 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).

6. Mechanism of Intercalation

  • Intercalation 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 byLeonard Lermanin1961 .
  • 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) andforms 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 .

7. 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 ).
  • 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.

8. 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

9. Genotoxicity of Non-covalent Interaction: DNA Inercalators

  • These DNA intercalatorsbind 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 sequencethe deletion or insertion occurs, the
  • more altered theprotein produced is.
  • Removal or addition of one or more bases shifts the reading frame A change in 1-2 bases substantially changes the output:

10. 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. somepesticides
  • Properties
  • Di-acridinesordi-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.

11. 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 byCarl GrbeandHeinrich Caro .
  • Acridine is structurally related to anthracenewith 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 derivativesbind to DNA and RNAdue to their abilities to intercalate.Acridine orange(3,6-dimethylaminoacridine)is a nucleic acid -selective metachromatic stain useful for cell cycle determination.

12. Properties

  • Acridine and its derivatives areplanar polycyclic aromatic moleculeswhich bind tightly but reversibly to DNA by intercalation, but do not usually covalently interact with it.
  • Acridines have abroad spectrum of biological activities , and a number of derivatives are widelyused as antibacterial ,antiprotozoalandanticancer drugs .
  • Simple acridines show activity asframeshift mutagens , especially in bacteriophage and bacterial assays, by virtue of their intercalative DNA-binding ability.