Simple method of biologically evaluating natural and artificial chemicals by using dna injury index and apparatus therefor

Abstract

A biological evaluation method for rational and simply evaluating biological harmfulness or usefulness of a great number of natural and artificial chemicals, foods, etc. This method comprises adding a known amount of 2′-deoxyguanosine (dG) to a solution containing a test substance (drug, pesticide, functional food, etc.), optionally applying UV light and/or adding active oxygen generator, then quantifying 8-hydroxy-2′-deoxyguanosine (8OHdG) in the solution, and evaluating the toxicity or usefulness of the test substance according to the 8OhdG content (a higher 8OhdG content indicates a higher harmfulness of the test substance while a lower 8OhdG content indicates a lower harmfulness or an usefulness thereof). The invention also provide an apparatus for advantageously performing said biological evaluation method and an antioxidant preservative solution to be used in this apparatus.

1 . A biological evaluation method of natural or artificial chemical, comprising the steps of adding a known amount of 2′-deoxyguanosine to a solution containing a specific natural or artificial chemical, then quantifying 8-hydroxy-2′-deoxyguanosine in said solution, and evaluating harmfulness or usefulness of said natural or artificial chemical according to the amount of said 8-hydroxy-2′-deoxyguanosine.  
     
     
         2 . A biological evaluation method of a natural or artificial chemical, comprising the steps of adding a known amount of 2′-deoxyguanosine to a solution containing a specific natural or artificial chemical, applying UV light and/or adding active oxygen species generator to said solution, then quantifying 8-hydroxy-2′-deoxyguanosine in said solution; and evaluating harmfulness or usefulness of said natural or artificial chemical according to the amount of said 8-hydroxy-2′-deoxyguanosine by use of, as an index, presence or absence of additive, synergistic or offsetting effect to oxidation injury of gene nucleic acid attributable to the active oxygen of said natural or artificial chemical.  
     
     
         3 . A biological evaluation method of a food comprising a natural or artificial chemical, comprising the steps of adding a known amount of 2′-deoxyguanosine to a solution containing the food comprising the natural or artificial chemical, then quantifying 8-hydroxy-2′-deoxyguanosine in said solution, and evaluating harmfulness or usefulness of said food according to the amount of said 8-hydroxy-2′-deoxyguanosine.  
     
     
         4 . A biological evaluation method of a food comprising a natural or artificial chemical, comprising the steps of adding a known amount of 2′-deoxyguanosine to a solution containing the food comprising the natural or artificial chemical, then applying UV light and/or adding active oxygen species generator to said solution, quantifying 8-hydroxy-2′-deoxyguanosine in said solution, and evaluating harmfulness or usefulness of the food according to the amount of said 8-hydroxy-2′-deoxyguanosine by use of, as an index, presence or absence of additive, synergistic or offsetting effect to oxidation injury of gene nucleic acid attributable to the active oxygen of said food.  
     
     
         5 . A biological evaluation method of a food comprising a natural or artificial chemical, comprising the steps of adding a known amount of 2′-deoxyguanosine to a solution containing the food comprising the natural or artificial chemical and a substance having oxidation injury effect to gene nucleic acid, then quantifying 8-hydroxy-2′-deoxyguanosine in said solution, and evaluating effectiveness with respect to oxidation injury preventing action of gene nucleic acid in said food components according to the amount of said 8-hydroxy-2′-deoxyguanosine.  
     
     
         6 . A biological evaluation method of a test solution, comprising the steps of adding a known amount of 2′-deoxyguanosine to a test solution containing a nonspecific chemical, then quantifying 8-hydroxy-2′-deoxyguanosine in said solution, and evaluating antioxidative ability or oxidative ability of said test solution according to the amount of said 8-hydroxy-2′-deoxyguanosine.  
     
     
         7 . A biological evaluation method for a chemical or food, comprising evaluating harmfulness or usefulness of said chemical or food according to ratio between contents of 8-hydroxy-2′-deoxyguanosine and of 2′-deoxyguanosine in a living organism cell derived product collected from an animal including a human, a plant, a bacterium or a fungus administered with a specific chemical or food for a predetermined period of time.  
     
     
         8 . An apparatus for measuring a DNA oxidation injury index, comprising; 
 a) an antioxidative preservative solution to be mixed with a liquid biological sample for cool stocking, the antioxidative preservative solution having to be prepared in an amount 1 to 5 times that of the biological sample;    b) a micro dialysis apparatus for performing micro dialysis treatment by perfusing a perfusion solution comprising an aqueous glycerol solution in a dialysis membrane tube dipped in a mixed solution of the liquid biological sample and the antioxidative preservative solution to prevent a substance having a molecular weight of 20 to 50 kD or more from passing and to make target glycerol concentration of a recovered liquid 10 to 20 wt %;    c) a high performance liquid chromatograph having a column for separating a low molecular weight DNA related substance from the recovered liquid in the micro dialysis treatment;    d) a UV light absorption analyzer for measuring an amount of DNA nucleic acid by performing UV light absorption analysis of an eluate from the column; and    e) an electrochemical detector for measuring an amount of DNA oxidation injured product by electrochemically analyzing the eluate from the column; and    f) a DNA oxidation injury index of the liquid biological sample is obtained from a ratio between the amount of the measured DNA nucleic acid and the amount of the measured DNA oxidation injured product.    
     
     
         9 . An apparatus for measuring a DNA oxidation injury index, comprising: 
 a) a tube for destructing tissue cells for sealing and cool stocking a mixed solution of a biological sample containing cell components and the antioxidative preservative solution in an amount 1 to 5 times that of the biological sample;    b) an apparatus for destructing cells and removing solids for a destructing sample contained in the biological sample in the tube after the sealing/cool stocking to thereby elute free nucleic acid inside and outside the cells in the preservative solution and simultaneously to separate and sediment solids from the destructed sample;    c) a micro dialysis apparatus for performing micro dialysis treatment by perfusing a perfusion solution comprising an aqueous glycerol solution in a dialysis membrane tube dipped in a mixed solution of the liquid biological sample and the antioxidative preservative solution to prevent a substance having a molecular weight of 20 to 50 kD or more form passing and to make a target glycerol concentration of a recovered liquid 10 to 20 wt %;    d) a high performance liquid chromatograph having a column for separating a low molecular weight DNA related substance from the recovered liquid in the micro dialysis treatment;    e) a UV light absorption analyzer for measuring an amount of DNA nucleic acid by performing UV light absorption analysis of an eluate from the column; and    f) an electrochemical detector for measuring an amount of DNA oxidation injured product by electrochemically analyzing the eluate from the column; and    g) DNA oxidation injury index of the biological sample is obtained from a ratio between the amount of the measured DNA nucleic acid and the amount of the measured DNA oxidation injured product.    
     
     
         10 . An apparatus for measuring a DNA oxidation injury index according to  claim 8  or  9 , characterized in that said antioxidative preservative solution is an aqueous solution containing 0.5 to 2 mM/l of EDTA, 2 to 5 wt % of methanol, and 10 to 40 wt % of glycerol.  
     
     
         11 . An antioxidative preservative solution for a biological sample comprising an aqueous solution containing 0.5 to 2 mM/l of EDTA, 2 to 5 wt % of methanol, and 10 to 40 wt % of glycerol.