Title of Thesis

Biological Screening of Organotin(IV) Complexes

Author(s)

MUHAMMAD SHEERAZ AHMAD

Abstract
Metal complexes generally and organotin(IV) compounds especially are used routinely in several biomedical and commercial applications like agricultural biocides, disinfectants, antitumor agents, wood preservatives, antioxidants, stabilizers for polyvinylchloride, marine antifouling coating, anti-herpes agents, flame retardants, smoke suppressants, anti-wear agents, homogenous catalysts and recycling agents. A number of organotin compounds have been shown to be toxic, and there is increasing concern regarding their widespread use in environmental and biological systems. Among organotin compounds, organotin carboxylates are highly important; these contain Sn−O
bond formed through COO¯ group and have a number of interesting structural features and biological applications.
Three groups of novel organotin(IV) carboxylates including mono-, di- and triderivatives of ligands: 3,4-methylenedioxy 6-nitrophenylpropenoic acid (L1), 3,4- methylenedioxyphenylpropenoic acid (L2) and 2,3-methylenedioxybenzoic acid (L3) were examined for their different biological activities.
In vitro antimicrobial activities of these compounds were determined against six pathogenic bacterial strains (antibacterial assay) and six pathogenic fungal strains (antifungal assay). Determined by the antimicrobial studies organotin(IV) carboxylates demonstrated a higher level of activity towards all tested bacterial and fungal strains than the respective free ligands. As a whole, antibacterial activities decreased in order of; diorganotin carboxylates > tri-organotin carboxylates > mono-organotin carboxylates > ligand acids. However, tri-organotin compounds were the best towards antifungal activities. Since the organotin(IV) carboxylates inhibited the growth of micro-organisms, it has been assumed that the production of an enzyme has been affected, hence the micro-organisms were less able to metabolize nutrients and consequently, growth ceased.
All the compounds were also investigated for their antitumor activities and cytotoxicity by crown gall tumor inhibition (potato disc) assay and brine-shrimp lethality bioassay, respectively. As determined by crown gall tumor inhibition assay and brineshrimp lethality assay; the antitumor or cytotoxic activities of these compounds fall in the order of; tri-organotin carboxylates > di-organotin carboxylates > mono-organotin carboxylates, a similar pattern is reported in literature. Such compounds may yield new leads for the development of antitumor drugs, which can display a different spectrum of antitumor activity.
A panel of different mono-, di- and tri-organotin(IV) carboxylates that have higher biological activities in previous assays were selected to investigate their effect on MCF-7 cells. Determined by the clonogenic assay, organotin(IV) compounds (0.1 µg/ml, 1.0 µg/ml or 10.0 µg/ml) were markedly cytotoxic following treatment of MCF-7 cells with ≥ 1.0 µg/ml particularly tri-organotin carboxylates.
Further investigation was aimed to determine genotoxic effect and biochemicalcell fingerprint of MCF-7 cells following treatment with selected panel of mono-, di- and tri-organotin(IV) carboxylates (0.01 µg/ml, 0.1 µg/ml or 1.0 µg/ml) that were used in clonogenic assay. Induction of DNA single-strand breaks (SSBs) were measured by using the alkaline single-cell gel electrophoresis (comet) assay and their relative micronucleus-forming activities using the cytokinesis-block micronucleus (CBMN) assay as well as alterations in biochemical-cell signatures was ascertained using attenuated total reflection fourier transform infrared (ATR-FTIR) spectroscopy.
In comet analysis, although there were marked fluctuations in the background levels of DNA SSBs measurable in control MCF-7 cell populations, a remarkable consistency in the ability of all the organotin(IV) carboxylates to be comet-forming was noted. However, with the alkaline comet assay it was more difficult to distinguish between the relative abilities of tri-organotin carboxylates, di-organotin carboxylates or mono-organotin carboxylates to induce elevations in DNA SSBs. Probably as a consequence of their lower relative cytotoxicities, higher levels of micronucleus-forming activities were observed following treatment with either mono- or di-organotins as compared to tri-organotins. Following interrogation of treated-cell populations compared to control using ATR-FTIR spectroscopy, infrared spectra (900 – 1800 cm-1) derived from organotin-treated MCF-7 cells exhibited clear alterations in their biochemical-cell fingerprint following exposures as low as 0.0001 µg/ml; this was especially evident in the DNA/RNA spectral region (≈ 1490 – 1000 cm-1). These results demonstrated a differential biocidal, cytotoxic, genotoxic and cell effects of organotin(IV) compounds.

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1.1 Bioorganic and bioorganometallic chemistry

1.2 Organotin compounds as bioorganometallics

1.3 Chemical characterization of organotin compounds

1.4 Organotin(IV) carboxylates

1.5 General preparations of organotin(IV) carboxylates

1.6 Properties and reactivity of organotin(IV) carboxylates

1.7 Applications of organotin(IV) carboxylates

1.8 Drug discover

1.9 Biological assays (bioass

1.10 FT-IR microspectroscopy

1.11 Present work

2.1 Test materials

2.2 General procedure of synthesis

3.1 Antibacterial activities of organotin(IV) carboxylates

3.2 Antifungal activities of organotin(IV) carboxylates

4.1 Crown gall tumor inhibition (potato disc) assay

4.2 Cytotoxicity determined by brine-shrimp lethality assay

4.3 Cytotoxicity of organotin(IV) carboxylates towards MCF-7 cells determined by clonogenic assay

5.1 Alkaline single cell gel electrophoresis (comet) assay

5.2 Cytokinesis-block micronucleus (CBMN) assay

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6.1 Materials

6.2 Sample preparation

6.3 Tissue preparation

6.4 ATR(FTIR) spectroscopy

6.5 Statistical analy

6.6 Results of ATR FT-IR analysis

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7.1 Antimicrobial activities of organotin(IV) carboxylates

7.2 Antitumor and cytotoxic activities of organotin(IV) carboxylates

7.3 DNA damage and genotoxic effect of organotin(IV) carboxylates on MCF-7 cells

7.4 Alterations in biochemical-cell signatures as ascertained using attenuated total reflection-fourier transform infrared (ATRFTIR)     spectroscopy

7.5 Organotin(IV) ocmpounds as cancer therapeutics

7.6 Conclusion

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