Dr Raj Singh

Contact Details

Tel: 0116 223 1827
Email: rs25@le.ac.uk 
Office: Room 001, Biocentre, University of Leicester, University Road, Leicester LE1 7RH

Education

  • B.Sc. (Hon), Biochemistry, University of Liverpool
  • M.Sc., Toxicology, University of Birmingham
  • Ph.D., MRC Toxicology Unit, University of Leicester

Research Interests

 

Keywords – DNA adducts, Ribonucleosides, Carcinogenesis, Chemoprevention, Adductomics, Metabonomics, Epigenetics

1. Background

DNA adducts are formed by the covalent interaction of genotoxins (carcinogens) with the purine and pyrimidine bases that are present in DNA. A mutation may result following DNA replication if the DNA adduct is not repaired and subsequent initiation of the carcinogenic process. The objective is to investigate the formation of DNA adducts in humans following exposure to carcinogenic chemical compounds from occupational, lifestyle, environmental and dietary sources. Human urine contains levels of ribonucleosides that reflect RNA turnover and metabolism in the body. Evidence from numerous reports in the literature suggests that urinary ribonucleosides can serve as biomarkers of cancer with elevated levels being detected in cancer patients having different types of tumours, however analytical methods for their detection need further improvement. The epigenetic alteration in DNA, 5-methyl-deoxycytidine plays an important role in many biological processes such as cellular differentiation, gene expression and maintaining genome integrity. Furthermore aberrant methylation has been found to be associated with carcinogenesis. The recent discovery of the so-called “sixth DNA base”, 5-hydroxymethyl-deoxycytidine has lead to substantial interest and speculation in the role of this new modified DNA base, although its precise function has not yet been elucidated. The assessment of DNA adducts and epigenetic modifications or ribonucleosides in humans may serve as useful diagnostic biomarkers for cancer allowing early detection, diagnosis and monitoring of therapeutic intervention, thus allowing the correct clinical decisions to be made for patient treatment.

rajfig1
Figure 1 Exposure to DNA damaging agents from occupational, lifestyle, environmental and dietary sources

2. Questions under investigation

By developing sensitive analytical techniques for detecting DNA adducts arising from exposure to both endogenously formed eg. by oxidative stress and exogenous genotoxic agents as well as epigenetic modifications we would like to investigate the biological significance of DNA damage and gain further insight into the mechanism of the carcinogenic process. One of the major requirements for improvement of cancer therapy is the detection of early small volume tumours, at the stage before clinical detection is possible, so that appropriate treatment may be initiated at an earlier time. Therefore DNA adducts and epigenetic modifications or ribonucleosides as biomarkers of early cancer detection would have a huge impact in controlling the manifestation of the disease and also allow the investigation of the efficacy or potential toxicity of putative chemopreventive agents.

3. Research strategy

New methods employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) have been developed for the detection of DNA adducts derived from a variety of genotoxic agents, such as polycyclic aromatic hydrocarbons, heterocyclic amines, alkylating agents as well as oxidative stress. Significant achievements over the last few years has been the development of new LC-MS/MS methods for the determination of DNA adducts such as benzo[a]pyrene-deoxyguanosine, 3-Nitrobenzanthrone-deoxyguanosine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-deoxyguanosine, 2-Amino-3,8-dimethylimidazo-[4,5-f]quinoxaline-deoxyguanosine, 8-oxo-deoxyguanosine, 8-oxo-deoxyadenosine, N2-ethyl-deoxyguanosine and N-7 ethylguanine, which have been applied to the detection and quantitation of these adducts in various studies. We have developed similar methods using LC-MS/MS constant neutral loss scanning to screen for ribonucleosides in human urine. The future strategy will involve the development of DNA adductomics approaches using LC-MS/MS for the simultaneous screening of multiple forms of DNA damage. DNA adductomics will allow the analysis of DNA damage in derived from a wide spectrum of genotoxic agents including those derived from endogenous and exogenous sources. The intention is to apply these newly developed adductomic and metabonomic methods for the analysis of non-invasively obtained human biological matrices such as blood, urine, sputum and saliva obtained from clinical trials. It is anticipated that methods which will allow the simultaneous determination of different epigenetic modifications in genomic DNA will also be integrated into the DNA adductomic approach.

rajfig2
Figure 2 Determination of B[a]PDE-N2dG adducts by LC-ESI-MS/MS SRM and 32P postlabelling in mice liver DNA at different time points following ip. dosing with A) tricaprylin, control B) 50 mg/kg B[a]P (28 days) and C) 200 mg/kg B[a]P (1 day). The internal standard (lower traces) is [15N5] B[a]PDE-N2dG.

rajfig3
Figure 3 DNA adductomic maps and typical online column-switching LC-MS/MS total ion chromatograms for the analysis of deoxynucleoside adducts in a mixture consisting of a combination of calf thymus DNA modified with different PAH-dihydrodiol epoxides using (A) SRM transitions for the most abundant product ions (B) SRM transitions resulting from the neutral loss of 116 u and (C) CNL scanning for the neutral loss of 116 u.

rajfig4
Figure 4 Schematic representation of the immunoslot-blot procedure used to detect malondialdehyde-deoxyguanosine (M1dG) adducts in human DNA samples.

To view the scientific publications of Dr Singh, please use the link: Publications.

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NEWS

SEND Study Success

Congratulations to Chris Avery, Honorary Associate Professor and Consultant Maxillofacial Surgeon, who with collaborating colleagues published the findings of the significant SEND study, a multi-centre randomised trial using two different surgical protocols for  mouth cancer treatment.  A nationwide first for this type of trial, using real-world data.  The paper, published in the British Journal of Cancer, is available by clicking this link: SEND Paper

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Congratulations from the LCRC to Bethan Rogoyski and Aleksandra Bzura.

Bethan won best oral presentation at the AMS Midlands Academy of Medical Sciences Festival held on 27th March 2019 for her presentation on "Redeployment: The potential of repurposed drugs in preventing mesothelioma".

Aleks won the University PGR poster fair competition on 18th March 2019 for her poster entitled "New hope for treating mesothelioma cancer using PARP inhibitors".

Also, a huge well done to Grandezza Aburido who was in the semi-finals of the PGR poster fair.

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New: Wellcome Trust Institutional Strategic Support Fund

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Centre Health and Wellbeing Group
Join up for a walk on the park Friday lunchtime - details here.

More information from Jenny McNair jm65@le.ac.uk

Contact Details

Leicester Cancer Research Centre
Clinical Sciences Building
University of Leicester
Leicester Royal Infirmary
Leicester LE2 7LX

T: +44 (0) 116 252 3170
E: cancerstudies@le.ac.uk

(Please note this email address should not be used for clinical referrals or patient correspondence)

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