Impact/Purpose:

The overall objective of this task is to develop rapid, cost-effective and scientifically sound techniques for measuring chemically induced DNA damage. This method is expected to provide the Agency with rapid, sensitive, and simple techniques that can be used among a panel of methods to determine the genotoxic potential of polluted samples.

Description:

Rapid and cost-effective indicator assays are being developed which may be used as a rapid screen to assess the potential for exposure to hazardous compounds that can be related to a biological target (e.g., DNA). Chemically-induced DNA damage will be measured using surrogate DNA. The approach will involve the development of rapid assays for single strand breaks. The proposed assay system will primarily measure single strand breaks in target DNA -- although this assay may also be sensitive to double strand breaks, adduct formation, and base losses. The concept for this assay is as follows: under certain conditions (i.e., high temperature or alkaline pH), double stranded DNA will unwind into single strands. Because this unwinding occurs either more rapidly or under milder conditions if the DNA backbone has been broken along one of the strands (single strand break), it can be used as an indicator of DNA damage in the form of oxidatively-induced single strand breaks.

Record Details:

Record Type:PROJECT

Start Date:01/01/1999

Completion Date:09/01/2002

Record ID: 56186

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Project Information:

Progress : ABSTRACT/ORAL: Date Cleared: 23-NOV-99

Rogers, K.R., Apostol, A.B., Madsen, S.J., and Spencer, C.W. Detection of DNA damage using a fiberoptic biosensor. ACS National Meeting, San Francisco, CA, March 26-30, 2000.

ABSTRACT/ORAL: Date Cleared: 18-FEB-00

Rogers, K.R., Apostol, A.B., Madsen, S.J., and Spencer, C.W. Detection of low dose radiation-and chemically-induced DNA damage using temperature differential fluorescence assays. 8th International Meeting on Chemical Sensors, Basel, Switzerland, July 2-5, 2000.

ABSTRACT/ORAL: Date Cleared: 18-SEP-00

Rogers, K.R., Apostol, A.B., Madsen, S.J., and Spencer, C.W. Fiber optic biosensor for DNA damage. ACS National Meeting, San Diego, CA, 2001.

ABSTRACT/ORAL: Date Cleared: 18-SEP-00

Rogers, K.R., Apostol, A. and Ramanathan, K. Screening Assay for DNA Damage. ACS National Meeting, San Diego, CA, 2001.

ABSTRACT/ORAL: Date Cleared: 28-MARCH-01

Rogers, K.R., Ramanathan, K. and Apostol, A. Detection of DNA Damage Using Melting Analysis Technique. Gordon Research Conference. Ciocco, Barga, Italy, May, 11, 2001.

BOOK CHAPTER

Rogers K. R. & Apostal, A. (1999) Optical Detection of DNA Damage. Proceedings of SPIE, vol. 3534, pp 100-104, Industrial and Environmental Monitors and Sensors. This proceedings paper describes the use of thermal denaturation of DNA followed by the use of a double strand (ds) specific fluorescent indicator to measure chemically induced damage.

JOURNAL ARTICLE: Date Cleared: 25-JULY-99

Rogers, K.R., Apostol, A., Madsen, S.J. and Spencer, C.W. (1999) Detection of Low Dose Radiation Induced DNA Damage Using Temperature Differential Fluorescence Assay. Anal. Chem. 71, 4423-4426. This paper describes the calibration of this technique using low dose gamma radiation. Radiation doses as low as 0.008 Gray yielded significant responses for amounts as small as 1 ng DNA (106 copies of the E. coli plasmid).

JOURNAL ARTICLE: Date Cleared: 17-SEPT-00

Rogers, K.R., Apostol, A., Madsen, S.J. and Spencer, C.W. (2000) Fiber Optic Biosensor for Detection of DNA Damage. Anal. Chim. Acta (in press). This paper describes the use of a hybridization-based biosensor for detection of DNA damage. The assay was sensitive and responded to both low levels of radiation- and chemically-induced oxidative damage. The assay was also rapid and could be completed in less than 30 min.

Relevance :Research being performed under this task is directed to meet the needs of the following ORD goals and objectives: (Goal 08 Sound Science) EPA will provide sound science to improve understanding of environmental risk and develop and implement innovative approaches for current and future environmental problems; (Objective 2, Research for Human Health Risk Assessment) Improve the scientific basis to identify, characterize, assess, and manage environmental exposures that pose the greatest health risks to the American public by developing models and methodologies to integrate information about exposures and effects from multiple pathways; (Subobjective S2.3 - Susceptible Subpopulations Research) By 2008, develop and verify innovative methods and models for assessing the susceptibilities of populations to environmental agents, aimed at enhancing current risk assessment and management strategies and guidance; (Subobjective S2.1Human Health Research) By 2008, Improve the scientific basis to identify, characterize, assess and manage environmental exposures that pose the greatest health risks to the American public by developing models and methodologies to integrate information about exposures and effects from multiple pathways. Significance One of the approaches for reducing uncertainties in the assessment of human exposure is to better characterize the hazardous wastes which contaminate our environment. A significant limitation to this approach, however, is that sampling and laboratory analysis of contaminated environmental and biological samples, can be slow and expensive; thus, limiting the number of samples which can be analyzed within time and budget constraints. Rapid and inexpensive indicator assays for potential exposure which can be correlated with biological dose-related effects can be of great benefit to the exposure assessment process. The use of these assays as screening tools can increase the amount of information available concerning the location, source and concentration of pollutants which may most significantly impact human health and the environment. More specifically, this proposal involves the development and characterization of rapid, sensitive and inexpensive assays for detection of molecular intercalation or damage to surrogate sequences of DNA caused by pollutants. These assays will then be correlated to (more expensive and time-consuming) classical assays for genotoxicity/mutagenicity and DNA damage.

Clients :ORD, NERL, Scientific Community

Project IDs:

ID Code :6007

Project type :OMIS