Non-Invasive Biomarkers of DNA Damage Related to Red Meat Intake and Risk of Colorectal Cancer

  • Topic: Colorectal Cancer
  • Institution: The Open University
  • Country: United Kingdom
  • Status: Completed

Scientific abstract

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Background

Colorectal cancer is one of the most common cancers found in developing countries.

Red meat has been found to be associated with high risks of colorectal cancer. This is mainly because high red meat consumption is known to enhance the endogenous formation of N-nitroso compounds, which are potent carcinogens. Nitrosation of glycine is followed by the endogenous formation of the DNA adduct O6-carboxymethylguanine (O6-CMG) which may be a potential urinary biomarker of colorectal cancer risk.

Aim

The aim of our research is to develop methods for the measurement of DNA damage in urine samples in order to identify those people at highest risk of developing cancer. Specifically, liquid chromatography tandem mass spectrometry (LC-MS/MS) analytical methods will be used for the measurement of O6-CMG in urine samples and the levels of urinary DNA adducts will be correlated to different diets.

Methods

A highly selective and sensitive LC-MS/MS method was developed to analyze O6-CMG in synthetic urine with a limit of detection of 0.38 ng/ml. This analytical method was extended to the simultaneous analysis of the corresponding nucleoside O6-carboxymethyldeoxiguaning (O6-CMdG) and other adducts of interest in urine. The LC-MS/MS method was then used to investigate the level of adducts in urine samples from a small cohort of healthy human volunteers in order to gain insight into the mode of DNA repair in-vivo. 24 hours urine samples from volunteers on different diets (with vegetarian diet as the control) were analysed following these approaches: LC-MS/MS with direct injection, LC-MS/MS with injection after purification by solid phase extraction (SPE) and LC-MS/MS with injection after acidic depurination and purification by SPE.

Results

The LC-MS/MS direct injection analysis of the clinical samples offered low sensitivity. However the LC-MS/MS analysis of urine samples after SPE purification and the analysis after acidic depurination followed by SPE purification did not show adduct quantities in those clinical samples above the limit of detection.

Conclusions

The validated LC-MS/MS method was shown not to have sufficient sensitivity to quantify the low levels of DNA adducts excreted in urine related to high red meat diets. Further studies will be required to analyse a large number of clinical samples of healthy volunteers and cancer patients.

Plain language abstract

Hypothesis

We are investigating the hypothesis that vegetarian diets help reduce cancer risk by reduction of DNA damage levels. The analysis of urine samples from healthy volunteers on different diets could provide evidence of a relationship between the levels of DNA damage derived from read meat in the gastrointestinal tract and the products of DNA repair excreted in urine.

Background

A diet high in red meat has been found to be linked with colorectal cancer in healthy volunteer studies. This is due to DNA damage in the cells that line the colon. DNA is intrinsically chemically unstable at physiological temperature and pH. This instability is crucial for biological evolution however there is a risk that part of the DNA damage escape repair and give rise to mutation.

Glycine is one of the structurally simplest aminoacids and a common constituent of dietary proteins. It appears that endogenous nitrosation products of glycine constitute a major source of alkilating agents in the human gastrointestinal tract that lead to the formation of DNA adducts. DNA adducts are pieces of DNA covalently bonded to a cancer-causing chemical. If the DNA damage is repaired, the measurement of these DNA adducts in biological fluids could be a very useful indicator not only of exposure but also of risk of disease.

Our research team at the OU have already shown that this damage related to consumption of red meat can be detected in colorectal cells and in blood. We are now extending these results to a non-invasive approach for the detection of urinary DNA adducts; this kind of methodology will be more suitable for routine screening studies in the general population.

Methods

Liquid chromatography methods are used to separate the biochemical mixtures into pulses of pure DNA adducts. Identification and quantification of each DNA adduct will be carried out by mass spectrometry methods. Each DNA adduct will create a unique pattern depending on if and how it breaks into ions of different mass.

In order to detect very low levels of DNA adducts, samples are concentrated and interferences from the urine matrix are removed by purifying the samples before analysis using solid phase extraction.

Key findings

We have established a new liquid chromatography-mass spectrometry analytical method for the measurement of DNA adducts related to high red meat diets. The method was validated and used to analyse a small number of urine samples from healthy volunteers on different diets.

However, the levels of these DNA adducts have proven to be quite low in urine, below the limit of detection, despite there is strong evidence that the adducts are indeed formed. Further studies will be required to analyse a large number of clinical samples of healthy volunteers and cancer patients.

Grant publications