Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/483—Physical analysis of biological material
  • G01N33/487—Physical analysis of biological material of liquid biological material
  • G01N33/49—Blood
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/50—Determining the risk of developing a disease
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/70—Mechanisms involved in disease identification
  • G01N2800/7004—Stress
  • G01N2800/7009—Oxidative stress
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/70—Mechanisms involved in disease identification
  • G01N2800/7023—(Hyper)proliferation
  • G01N2800/7028—Cancer

Definitions

• the present invention relates to a method of determining free radical induced DNA damage. More particularly to a method of determining the predisposition of a subject to cancer or other disease states characterised by free radical induced DNA damage such as COPD or asthma.
• the invention also relates to a method of determining the current status of the subject in relation to certain cancers.
• the invention also relates to a method of determining the sensitivity of a subject to electromagnetic (EM) radiation induced cellular damage.
• the invention relates to a method of determining the sensitivity of a subject to ultraviolet radiation (UV) induced cellular damage.
• EM electromagnetic
• UV ultraviolet radiation
• the invention also relates to an assay product or interrelated kit for use in performing the methods of the invention.
• Oxidative DNA damage to cells is an unavoidable consequence of cellular metabolism; however, interactions with exogenous sources such as carcinogenic compounds, redox-cycling drugs, ionising and UV radiation also contribute to oxidative damage.
• UV can be used as a physical generic mutagen to induce DNA damage in vitro. Compared to a chemical genotoxin, the advantage of using UVA light is the exact setting of exposure time. Chemical genotoxins require removal by centrifuging and washing procedures.
• UVA is part of the sunlight with its electromagnetic spectrum at sea level (290-5000 nm) not only including the visible (56%) and infrared (39%) part but also ultraviolet (UV) light (5%) Usually UVA (320-400 nm) and to a lesser extent due to atmospheric absorption UVB (290-320 nm) reaches the earth's surface, while the germicide UVC part is completely filtered off.
• the UVA/B light has been commonly characterised as an environmental human carcinogen being also responsible for erythema (sun-burn), tanning, photo-aging and immune-suppression.
• artificial UV sources mainly emitting UVA, can also be found in tanning studios and for the treatment of psoriasis.
• UVA UVA cyclobutane pyrimidine dimers
• UVB UVA induced oxidative DNA damage like 8-oxol0 guanine depends on non-DNA chromophores present in the cells.
• UV absorption generates oxygen derived free radicals inducing DNA damage via the production of a range of photoproducts. This process can change the base pairing abilities of normal DNA resulting in mutations. It is these mutations that may lead to cancers because they disrupt tumour suppressor genes such as P53, and INK4A. Free radicals play a role in numerous types of cancers. There is evidence that free radical related mutations of the ATM (Ataxia-telangiectasia mutated) and ATR (Rad3- related) genes play a role in lymphomagenesis.
• Electrophoresis at high pH results in structures resembling comets, observed by fluorescence microscopy in which the intensity of the comet tail relative to the head reflects the number of DNA strand breaks and alkali labile sites. Loops containing a break lose their supercoiling and freely move towards the anode.
• the Comet assay provides a means of determining genomic damage. Calculation of fluorescence to determine the extent of DNA damage can be performed by manual scoring using imaging software.
• a method of screening subjects for a predisposition to diseases characterised by DNA damage comprising the steps:
• the method of screening includes a final step of;
• the present invention shows a clearer separation between groups than prior art methods. It also allows greater reproducibility of results.
• the step of exposing each sample to a different level of electromagnetic radiation is carried out by altering the distance between the source of radiation and the sample to produce the correct intensity for each sample.
• the plurality of samples may be taken as a single sample from a subject provided that it can be separated into a plurality of samples for testing.
• samples are exposed to electromagnetic radiation for between 10 minutes and 30 minutes
• the samples are exposed to electromagnetic radiation 15 minutes.
• the step exposing the samples to varying levels of electromagnetic radiation is carried out varying the levels of electromagnetic radiation emitted by adjusting the height.
• no additional layer of barrier material is placed between the samples and the source of electromagnetic radiation.
• the whole blood comprises peripheral whole blood.
• the method includes the step of:
• ⁇ of diluted whole blood is mixed with an equal amount of cell culture medium (RPMI) and 10% of DMSO (storage and freezing medium), and this is mixed with ⁇ of 0.5% low melting point agarose ( ⁇ 40°C) and applied to dry agarose-coated slides.
• RPMI cell culture medium
• DMSO storage and freezing medium
• ⁇ of 0.5% low melting point agarose ⁇ 40°C
• 35 ⁇ , 40 ⁇ or 45 ⁇ 1 of blood would be preferred volumes.
• Cell numbers in these cases would be 7-10, 10-15 or 20-25 cells respectively according to blood volume. Density of cells would be 0.0015, 0.0020 or 0.0030 respectively according to blood volume.
• the mounting medium is 0.5% low-melting point agarose ( ⁇ 40°C).
• the EM radiation is UV radiation.
• the sample is provided on a substrate without any additional barrier layer being provided on top, it is possible to lyse the whole blood cells after they have been exposed to EM radiation, thus removing the need for laborious pre-treatment steps.
• the method may also include the step of:
• the method could be used to screen subjects for a predisposition to cancers including but not limited to skin cancer, lung cancer, breast cancer, bowel cancer, prostate cancer, colo-rectal cancer.
• Figure 1 is a graphical representation of data obtained from examples using the method of the invention, showing olive tail moments of cells at varying height of UV light (i.e. varying distances between sample and UV source, resulting in varying intensities) and showing the difference in profiles between healthy controls, precancerous states and cancers.
• Figure 2 is a graphical representation of data obtained utilising a standard normal (conventional) Comet Assay protocol as published in the literature, using variable heights of UV light. Notably, the profile here differs from profiles obtained using the new methodology. Differential responses of cells to UV, a physical generic mutagen, have been found by the inventors to provide an accurate indication of a subject's predisposition to cancer.
• the present invention provides a simple and accurate method for testing a sample for sensitivity of cells in the sample to UV and using the results to provide an accurate indication of a subject's predisposition to cancer.
• the first step is to obtain a sample or samples from a patient.
• peripheral whole blood samples can be easily collected from a subject by venepuncture.
• the samples (which can be derived from a single blood sample taken from a subject which has been divided appropriately) can the be used in the following assaying method (generally using an assay product or interrelated kit).
• Preferred Protocol (based on UVA, whole blood and modified standard Comet assay methodology, i.e. the New assay) Glass slides from BDH (SuperfrostTM) to be used are coated with 1% agarose
• the first slide in the treated group is the positive control and the intensity is ⁇ 1.20 mW/crr and the rest of the slides are at different intensities.
• the second is ⁇ 0.80 mW/crr
• the third is ⁇ 0.50 mW/crr
• the fourth is ⁇ 0.20 mW/cm ⁇
• Group 1 There are two series of slides: Group 1:
• Slide number two is covered with ⁇ of LMP 0.5% agarose and the UVA intensity is ⁇ 1.20 mW/cm* (distance from UVA lamp is 15cm) (positive control).
• Slide number three is covered with ⁇ of LMP 0.5% agarose and the UVA intensity is ⁇ 0.80 mW/cm 2 .(distance from UVA lamp is 20cm).
• Slide number four is covered with ⁇ of LMP 0.5% agarose and the UVA intensity is ⁇ 0.50 mW/cm 2 . (distance from UVA lamp is 25cm).
• the slides are immersed after treatment laterally in a container with cold lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 10% DMSO, 1% Triton X-100, pH 10). The slides are incubated at 4°C for 1 hour or overnight. After lysis, the slides are placed horizontally on the tray of an electrophoresis tank, filled with cold electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH ⁇ 13) The slides are incubated for 30 minutes at 4°C in the dark to allow the unwinding of DNA and expression of the single strand breaks (SSB) or double strand breaks (DSB) alkali labile damage. Electrophoresis is conducted at the same temperature for 30 minutes at 25 volts and an adjusted current of 300 mA (by raising or lowering the buffer level).
• cold lysing solution 2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 10% DMSO,
• the slides are removed from the tank and soaked three times each with neutralising buffer (400 mM Tris, pH 7.5) for a period of five minutes (Tice, Agurell et al. 2000; Anderson, Schmid et al. 2003).
• neutralising buffer 400 mM Tris, pH 7.5
• Cells are stained with 20 ⁇ g/ml ethidium bromide or an appropriate alternative e.g. SYBR Green and examined using a fluorescence microscope equipped with a monochrome CCD-camera.
• Figure 1 shows that cells of an individual having cancerous or precancerous conditions follow a similar profile, which is very distinct from that observed for cells of healthy individuals.
• the healthy controls show a very different profile to either cancerous or pre-cancerous cells. This allows clear identification of the subjects whose samples indicate a predisposition to cancer.
• the profile shown in Figure 2 using standard normal (conventional) comet assay could not be used in this manner as it is not sufficiently distinguishable from healthy controls to provide a clear and accurate result.
• the second layer of agar 100 ⁇ of 0.5% LMP cannot cover the entire slide at the time of applying.
• the present invention aims to mitigate these problems.

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• Pathology (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Investigating Or Analysing Biological Materials (AREA)

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• 2012
  • 2012-09-11 GB GBGB1216174.1A patent/GB201216174D0/en not_active Ceased
• 2013
  • 2013-09-10 WO PCT/GB2013/052365 patent/WO2014041340A1/en not_active Ceased
  • 2013-09-10 IN IN2685DEN2015 patent/IN2015DN02685A/en unknown
  • 2013-09-10 CN CN201380047021.2A patent/CN104755927A/zh active Pending
  • 2013-09-10 EP EP13760109.2A patent/EP2895853A1/en not_active Withdrawn
  • 2013-09-10 US US14/426,444 patent/US20150219619A1/en not_active Abandoned
  • 2013-09-10 JP JP2015530497A patent/JP2015529077A/ja active Pending

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