U.S. patent application number 10/203553 was filed with the patent office on 2003-10-02 for simple method of biologically evaluating natural and artificial chemicals by using dna injury index and apparatus therefor.
Invention is credited to Takaki, Atsushi.
Application Number | 20030186260 10/203553 |
Document ID | / |
Family ID | 26587693 |
Filed Date | 2003-10-02 |
United States Patent
Application |
20030186260 |
Kind Code |
A1 |
Takaki, Atsushi |
October 2, 2003 |
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.
Inventors: |
Takaki, Atsushi;
(Fukuoka-shi, JP) |
Correspondence
Address: |
Oliff & Berridge
PO Box 19928
Alexandria
VA
22320
US
|
Family ID: |
26587693 |
Appl. No.: |
10/203553 |
Filed: |
August 28, 2002 |
PCT Filed: |
March 16, 2001 |
PCT NO: |
PCT/JP01/02095 |
Current U.S.
Class: |
435/6.16 |
Current CPC
Class: |
G01N 33/5014 20130101;
C12Q 1/68 20130101; G01N 33/52 20130101; C12Q 1/68 20130101; C12Q
1/68 20130101; C12Q 2523/313 20130101; C12Q 1/025 20130101; C12Q
2525/117 20130101; C12Q 2523/113 20130101; C12Q 2525/117
20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 16, 2000 |
JP |
2000-74200 |
Mar 27, 2000 |
JP |
2000-86410 |
Claims
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'-deoxyguanos- ine 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.
Description
TECHNICAL FIELD
[0001] The present invention relates to a simplified biological
evaluation method of natural and artificial chemicals by using DNA
injury index and more particularly to a method for simply
evaluating harmfulness or usefulness of natural and artificial
chemicals such as pharmaceutical preparations and agricultural
chemicals or harmfulness or usefulness of various functional foods
and the like by comparing test substance administered group and
control group with respect to reaction in which 2'-deoxyguanosine
(dG), a constituent of DNA, is converted into
8-hydroxy-2'-deoxyguanosine (8OHdG), an oxidized form, or with
respect to a ratio of 8OHdG/dG contained in living organism cell
derived products. The present invention further relates to a high
sensitive measurement apparatus for DNA oxidation injury index in
biological samples.
BACKGROUND ART
[0002] Currently, in the environment, there are various types of
artificial chemicals, for example, various carcinogenic substances,
endocrine disturbing substances and so forth originated from
pharmaceutical preparations, agricultural chemicals, detergents,
food additives, antiseptics and so forth, that have not existed in
the natural environment. Among the natural substances, there are
substances on whose toxicity must be taken care because:
[0003] 1) There is the possibility that deleterious components can
exist in natural materials;
[0004] 2) There is the possibility that in particular when it is
formulated in the form of an extract, its biological toxicity is
enhanced; and
[0005] 3) There is the possibility that various components
synergistically act to become toxic.
[0006] Genetic information is encoded by base pairs of adenine (A),
thymine (T), guanine (G) and cytosine (C). The DNA information is
injured at a certain frequency due to UV light irradiation, DNA
replication error or exposure to various carcinogenic substances or
active oxygen species, and ultimately loss, recombination, mutation
and so forth of the gene information occurs, thus causing death of
cells or the individual or on the contrary triggering naissance of
new biological species. The mechanism in which the natural and
synthetic chemicals exerts toxicity to animal including humans can
be partly explained by such DNA injury.
[0007] From this aspect, focusing attention on the oxidation of
DNA, 2'-deoxyguanosine (dG) and 2'-deoxyadenosine (dA), which are
DNAs (mononucleosides) having purine nuclei bind with hydroxy
radicals (.OH), which are active oxygen species, to be oxidized
into 8-hydroxy-2'-deoxyguanosine (8OHdG) and
2-hydroxy-2'-deoxyadenosine (2OHdA), respectively. And then the DNA
base pairs that should inherently be G:C and A:T are converted to
T:A and G:C, respectively. The frequency of occurrence of
mistranslation of gene information on the level of DNA is
considered to be important information relative to states of
various diseases and paid much attention in recent medicinal
fields.
[0008] Conventionally, biologically acceptable safe concentration
of chemicals has been quantified in consideration of
[0009] 1) Lethal dose when administered to test animals;
[0010] 2) Amount in which apparent organ disorder such as
carcinogenesis or neuropathy occurs when administered to test
animals;
[0011] 3) Amount that influences the fecundity and so forth and by
multiplying such minimum effective amount by a safety factor of 100
to 1,000 times.
[0012] However, because there are extremely many types of chemicals
which are evaluation targets; i.e., as many as at least about
100,000 types, because in the case where biological toxicity is
quantified by using mouse or rat, it takes about 2 months for
establishing a first generation and a double or more days are
necessary for evaluating influence over generations, and because a
huge cost is necessary for maintaining the evaluation system and
for some other reasons, it has conventionally been practically
impossible to evaluate the biological toxicity of all the
substances by the conventional method due to restrictions of time
and costs.
[0013] Many foods currently on the market such as health-care foods
and functional foods are in the main those extracted from natural
materials or those to which a specific substance is added in the
range of safe acceptable amount. However, no objective evaluation
system on usefulness (effectiveness) of the foods and the like has
been established.
[0014] On the other hand, as for a method for measuring
8-hydroxy-2'-deoxyguanosine (8OHdG), which is one of oxidized DNA
substances as described above, in biological samples such as
leukocytes, parenchyma organ or cell suspension, measurement has
been generally performed by extracting nuclear DNA from these cell
components and subjecting it to various enzyme treatments, passing
it through HPLC (high performance liquid chromatography), and
flowing the eluate to an electrochemical detector to measure it in
the form of mononucleoside. This method can evaluate oxidation
injury of nuclear DNA per se at measurement sensitivity in the
order of 10 pg/ml. However, the method involves steps of acid or
alkali treatment, enzyme reaction and so forth so that the
operation of it is troublesome and secondary DNA oxidation is
inevitable due to these treatments so that the method can be
practiced only in limited installment.
[0015] Likewise, in the case where 8OHdG is measured in urine that
contains it in a large amount, a method is used in which after
removal of proteins, it is passed through HPLC and an
electrochemical detector. However, since urine per se is a
biological sample that is very susceptible to oxidation, it has
been the most difficult obstacle course how to remove proteins
under conditions where oxidation is prevented.
[0016] A method for quantifying 8OHdG that is increasingly used
recently is an enzyme antibody method (EIA method) by use of
monoclonal antibody. Unlike the method by use of HPLC and an
electrochemical detector, this method is characterized in that it
can detect even when the sample is not in the form of
mononucleoside. However, it has defects that it undergoes
cross-reaction with other substance than 8OHdG and that it has low
detection sensitivity in the order of 1 ng/ml. Accordingly, there
have been many problems to be solved in order to generalize it.
[0017] On the other hand, it is relatively easy to detect
mononucleoside per se in a biological sample since the absolute
amount of it in nuclear DNA is relatively large; i.e., about 10,000
to 100,000 times that of oxidized products such as 8OHdG, and due
to the characteristics of its chemical structure, UV light
absorption measurement apparatus is effectively used in a general
detection method. However, pretreatment of analyte to be measured
is the same as in the measurement of 8OHdG by use of an
electrochemical detector and hence basically the same problems are
involved.
[0018] It is possible that the artificial chemicals and foods as
described above are daily taken by, administered to or brought into
contact with humans and therefore it is an urgent necessity to
confirm biological toxicity and usefulness, in particular toxicity.
However, the present situation is as described above and there are
problems in that a long time and high costs are necessary.
Accordingly, development of a method for evaluating biological
toxicity of artificial chemicals or harmfulness and usefulness of
foods in a simplified manner and at low costs has been
demanded.
[0019] The present invention has been made in response to such a
demand and an object of the present invention is to provide a
method for evaluating biological harmfulness or usefulness of huge
kinds of natural substances and artificial chemicals and foods by
in a rational and simplified manner.
[0020] Also, an object of the present invention is to provide a
measurement apparatus that can accurately quantify in a simplified
manner with good reproducibility the amounts of substances that
serve as indices in a biological evaluation method, more broadly,
oxidized DNA substances, in particular 8-hydroxy-2'-deoxyguanosine
(8OHdG), 2-hydroxy-2'-deoxyadenosine (2OHdA) and the like together
with the amount of nuclear DNA in the biological sample.
[0021] Furthermore, an object of the present invention is to
provide an antioxidative preservative solution being able to
prevent oxidation injury of DNA in the biological sample in the
aforementioned measurement apparatus.
DISCLOSURE OF THE INVENTION
[0022] The present inventors have made extensive studies and as a
result they have found out that the presence or absence of
oxidizing ability or antioxidant ability of a test substance and
its degree can be known by adding 2'-deoxyguanosine (dG) to a
solution containing a natural or artificial chemical or food which
is test substances and measuring after a predetermined time
concentration of 8-hydroxy-2'-deoxyguanosine (8OHdG), i.e.,
oxidized form of 2'-deoxyguanosine in the solution, and that using
it as an index biological harmfulness and nontoxicity or usefulness
of the test substance can be evaluated. As a result of further
study, the present inventors have completed the present
invention.
[0023] That is, the present invention relates to 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, quantifying
8-hydroxy-2'-deoxyguanosine in the solution, and evaluating
harmfulness or usefulness of the natural or artificial chemical
according to the amount of the 8-hydroxy-2'-deoxyguanosine.
[0024] In the biological evaluation method for the aforementioned
test solution, presence or absence of additive, synergistic or
offsetting effect to oxidation injury of gene nucleic acid
attributable to the active oxygen in the test solution by adding
dG, irradiating UV light and/or adding active oxygen species
generator to the solution, and then quantified 8OHdG in the
solution.
[0025] Therefore, the present invention relates to 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,
irradiating UV light and/or adding active oxygen species generator
to the solution, quantifying 8-hydroxy-2'-deoxyguanosine in the
solution, and evaluating harmfulness or usefulness of the natural
or artificial chemical according to the amount of the
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
the natural or artificial chemical.
[0026] Further, the present invention relates to a biological
evaluation method for 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, quantifying
8-hydroxy-2'-deoxyguanosine in the solution, and evaluating
harmfulness or usefulness of the food according to the amount of
the 8-hydroxy-2'-deoxyguanosine, and to a biological evaluation
method for 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, irradiating UV light and/or adding active
oxygen species generator to the solution, quantifying
8-hydroxy-2'-deoxyguanosine in the solution, and evaluating
harmfulness or usefulness of the natural or artificial chemical
according to the amount of the 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 the food.
[0027] The present invention also relates to a biological
evaluation method for 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, quantifying
8-hydroxy-2'-deoxyguanosine in the solution, and evaluating
usefulness with respect to oxidation injury preventing effect of
gene nucleic acid of the food components according to the amount of
the 8-hydroxy-2'-deoxyguanosine.
[0028] Furthermore, the present invention relates to a biological
evaluation method for a test solution, comprising the steps of
adding a known amount of 2'-deoxyguanosine to a test solution
containing nonspecific chemicals, quantifying
8-hydroxy-2'-deoxyguanosine in the solution, and evaluating
antioxidative ability or oxidative ability of the test solution
according to the amount of the 8-hydroxy-2'-deoxyguanos- ine.
[0029] The present invention also relates to a biological
evaluation method for a chemical or food, comprising the steps of
evaluating harmfulness or usefulness of the 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.
[0030] The present inventors have found out that by perfusion of a
biological sample mixed with an antioxidative preservative solution
through a dialysis membrane having a cutoff function whose
molecular weight is 20 to 50 kD; i.e., a so-called micro dialysis
treatment, operation of removal of protein from the biological
sample can be performed in an antioxidative environment, and that
after the perfusion, by simultaneously subjecting the eluted
component from high performance liquid chromatograph to
quantification of DNA substance by means of UV light absorption
analysis and quantification of DNA oxidation injured product by
electrochemical detection to accurately measure the both values
under the same conditions, and thereby the state of DNA oxidation
injury can be accurately detected.
[0031] Therefore, the present invention accomplishes an apparatus
for measuring a DNA oxidation injury index, characterized in
comprising
[0032] 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;
[0033] 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 %;
[0034] 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;
[0035] 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
[0036] e) an electrochemical detector for measuring an amount of
DNA oxidation injured product by electrochemically analyzing the
eluate from the column; and
[0037] 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.
[0038] The present invention further relates to an apparatus for
measuring a DNA oxidation injury index, characterized in
comprising
[0039] 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;
[0040] 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;
[0041] 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 %;
[0042] 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;
[0043] 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
[0044] f) an electrochemical detector for measuring an amount of
DNA oxidation injured product by electrochemically analyzing the
eluate from the column; and
[0045] 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.
[0046] Further, the present inventors have found out that as an
antioxidative preservative solution for a biological sample in the
aforementioned measurement apparatus, 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 is particularly useful.
[0047] Therefore, the present invention relates to the
aforementioned measurement apparatus for the DNA oxidation injury
index in which the 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, and to an antioxidative
preservative solution for a biological sample per se 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0048] FIG. 1 is a flowchart illustrating the procedure of the
biological sample measurement apparatus of the present
invention.
[0049] FIG. 2 is a graph illustrating a relationship between a
concentration ratio of NO.sub.3.sup.-/dG and that of 8OHdG/dG in
human urine measured by use of the biological sample measurement
apparatus of the present invention.
[0050] FIG. 3 is a graph illustrating a correlation of 8OHdG
concentration obtained by HPLC+electrochemical detector (ECD)
method and that obtained by EIA method.
[0051] FIG. 4 is a graph illustrating a correlation between 8OHdG
concentration and nitrate ion (NO.sub.3.sup.-) concentration in
urine.
[0052] FIG. 5 is bar graphs representing 8OHdG concentration, dG
concentration, and a ratio of 8OHdG concentration to dG
concentration for each of mainly breast-fed group, mainly powder
milk-fed group, and mixed nutrient-fed group.
BEST MODE FOR CARRYING OUT THE INVENTION
[0053] In the present invention, "evaluate (presence or absence of)
harmfulness or usefulness" means to present a criterion of judgment
as to whether or not a test substance has a biological harmfulness
(imparting oxidative stress, carcinogenicity, teratogenicity,
adverse influence on reproductive potential and so forth), or
criterion of judgment as to whether to have harmfulness or
usefulness (effectiveness), and degree of the harmfulness or
usefulness. "Evaluate antioxidant ability or oxidizing ability"
means to present a criterion of judgement as to whether or not a
test solution has an antioxidant ability, in other words, whether
or not it has a reducing power, or a criterion of judgement as to
whether or not a test solution has an oxidizing ability (oxidizing
power), and degrees of them.
[0054] 2'-Deoxyguanosine used in the present invention is a kind of
deoxypurine nucleoside, which is a constituent of DNA. Almost all
the gene information is encoded by base pairs of adenine (A) and
thymine (T), and guanine (G) and cytosine (C). This gene
information is injured at a certain frequency by spontaneous
mutations (due to UV light irradiation, DNA replication error or
the like) or mutations with artificial products (various types of
carcinogenic substances, active oxygen and so forth) and ultimately
loss, recombination, mutation and so forth of the gene information
occurs, thus causing death of cells or the individual or on the
contrary triggering naissance of new biological species.
[0055] Active oxygen is produced from oxygen incorporated in the
body and besides it is produced from natural and artificial
chemicals taken into the body that serve as sources or induction
factors of an active oxygen and is known to give oxidative injury
to proteins, lipids, gene nucleic acids and so forth, which are
living organism components. In the case of the dG, it binds with
hydroxy radicals (.OH), which is an active oxygen species, to form
oxidized 8-hydroxy-2'-deoxyguanosine (8OHdG). The 8OHdG converts
original G:C base pair into T:A base pair. That is, the conversion
from G:C to T:A means that the gene of individual cell is injured
at a certain frequency and the quantity of frequency is naturally
considered to give an important index for evaluating
self-conservation ability or species conservation ability. In fact,
there have been reported that as a typical DNA oxidative injury
index 8OHdG has a significant correlation with, for example, ratio
of carcinogenesis and occurrence of degenerative diseases of brain
("Experimental Medicine", Vol. 13, No. 15, p.31-37, 1995), a close
relationship with death of cell (apoptosis) ("Modern Medicine",
Vol. 51, No. 3, p.42-47, 1996) and the like.
[0056] The present invention further advanced use of 8OHdG as DNA
oxidative injury index and tries to evaluate harmfulness and safety
of a test substance by presence or absence of ability of the test
substance for oxidizing dG to 8OHdG and degree of them. In other
words, the present invention uses the ability of the test substance
to convert dG into 8OHdG as a criterion of judgment of presence or
absence of harmfulness, etc., of the test substance and thus
basically differs in technical idea from the conventional system
which uses the 8OHdG already present in a biological sample and the
like as a scale for the degree of risks of occurrence of cancers or
neural degenerative diseases or of senescence. The conversion from
dG to 8OHdG in the biological evaluation method of the present
invention reflects degree of injury of DNA, which is a design
drawing of biological phenomenon and may be said to be a very
rational objective index for the evaluation of influences on the
self preservation ability or species preservation ability of each
individual of organisms including humans.
[0057] The test substance on which biological evaluation is to be
made by the present invention includes natural and artificial
chemicals, for example, pharmaceutical preparations, agricultural
chemicals, detergents, food additives, antiseptics, various types
of carcinogenic substances, endocrine disturbing substances and in
addition air polluting substances, water polluting substance and so
forth, as well as foods such as health-care foods or functional
foods containing natural and artificial chemicals. The foods
include general foods and health-care foods per se and also
components thereof. The test substance of the present invention
includes aqueous solutions containing unidentified substances
(solutions whose composition is unclear, also referred to herein as
"test solution"), for example, home wastewater, industrial waste
water, sewage, city water, purified water, natural water, river
water, sea water, lake water, and so forth.
[0058] The method of present invention is usually performed by
leaving an artificial chemical or food as a test substance or a
test solution such as city water together with dG for a
predetermined time, and measuring generated 8OHdG. Besides, by
further loading an oxidation induction factor to which there is the
possibility of exposure in the nature or in vivo, through addition
of active oxygen species generator or irradiation with UV light or
the like, the interaction of such with the test substance can also
be evaluated.
[0059] Thus, the method of the present invention can evaluate
harmfulness or usefulness of artificial chemicals such as
pharmaceutical preparations and agricultural chemicals and natural
chemicals or foods in simple and rational manner at low costs.
Further, for the chemicals of which indications of approximate
allowance and effective amount have been obtained by the method of
the present invention and which are of high importance can be
further strictly evaluated by the cell cultured evaluation method,
in vivo evaluation method as described hereinbelow. The present
invention also provides such methods.
[0060] Cell Cultured Evaluation Method:
[0061] To an animal cell (for example, B9 cell line, which is a
mouse lymphocyte cell line), plant cell (for example, tobacco BY-2
cell), or fungi (for example, yeast) in culture medium which
contains a test substance is optionally added an oxidant and/or
performed irradiation with UV light, and after lapse of a
predetermined time, concentrations of dG and 8OHdG derived from the
cultured cells are measured and values before and after the
treatment are compared. The lower animal and plant cells are, the
more adaptability in various environments they have as single units
of life themselves (detoxication of artificial chemicals, active
oxygen elimination function, etc.). However, usefulness of handling
live cells is in that the degree of influence of the test substance
on the homeostasis maintaining function that the organisms have can
be evaluated thereby. In this sense, a cell evaluation method may
be said to be a biomarker (bioindex) which can be used most easily.
Cultured cells undergo alternation of generation very quickly so
that they can reflect at high sensitivity the frequency of DNA
injury that tends to occur at the time of cell fission. In
addition, for the quantification of dG and 8OHdG, sufficient
measurement can be performed when the cell number of 10.sup.7 to
10.sup.9 is available. Therefore, a system that handles a
relatively large number of analytes can be constructed by a
thermostatic chamber.
[0062] In vivo Evaluation Method:
[0063] A test substance is administered to a test animal (for
example, mouse) for a predetermined period of time and
concentrations of dG and 8OHdG in urine and genital organs (ovary
or testis) are measured. Conventionally, biological toxicity of
artificial chemicals, etc. has been evaluated from various sides
(for example, lifetime, change in body weight, biochemical data of
blood, incidence of diseases, etc.). However, it has been nearly
impossible to make a comprehensive judgment covering all individual
indices. However, DNA injury index represents the quality of design
drawing of biological activity and its production amount is
considered to be common in all the diseases. In this sense,
measurement of production amount of 8OHdG in the present invention
is simplest and rational comprehensive index that allows
presumption of degree of health in a living organism. The essence
of biological activity is concentrated on self-conservation and
species conservation, and hence evaluation of reproduction toxicity
is of great significance. According to the method of the present
invention, target is narrowed down on testis and ovary, which are
genital organs of male and female, respectively and fundamental
risk of a test substance can also be evaluated based on the degree
of injury of design drawing of life of next generation (DNA). Note
that, among a series of evaluation methods, the in vivo evaluation
method requires the most expense in time and effort and substances
whose exposition concentration is high or substances whose
exposition concentration is low but has high harmfulness must be
finally tested in animals similar to humans (mammalians). However,
the present invention is an evaluation method focused only on DNA
injury ability, so that time and costs are considerably reduced as
compared with the conventional method.
[0064] Measurement of 8OHdG concentration in the method of the
present invention can be performed, for example, by separating dG
and 8OHdG contained in a sample solution by high performance liquid
chromatography (HPLC), and by use of a UV light absorption analyzer
and an electrochemical detector connected thereto. According to
this method, concentrations of dG and 8OHdG can be simultaneously
detected. Further, the 8OHdG concentration can also be measured by
use of an antibody, preferably a monoclonal antibody that
specifically reacts with 8OHdG.
[0065] Further, in the case where a biological sample is used, it
is preferable that measurement of 8OHdG or the like be performed
after efficiently recovering low molecular weight DNA constituents
(dG, 8OHdG, etc.) by use of a micro dialysis method (dialysis
membrane perfusion method) Concretely, perfusion is conducted by
use of a dialysis membrane that can cut off a high molecular weight
components of 20 kD to 50 kD. In the case where a biological sample
is a liquid sample (plasma, urine, bone marrow, etc.), dialysis is
performed by use of a membrane as described above and a mixed
solution after the dialysis is subjected to measurement of 8OHdG,
etc. In the case of a sample containing cell components (whole
blood, cell suspension, tissue, etc.), tissue or cells are
destructed by means of a cell crashing apparatus and nucleic acid
components that exist in the cytoplasm or nucleus are eluted.
Thereafter, solid components are separated by centrifugation and
the supernatant liquor is dialyzed by use of a dialysis membrane as
described above. dG and 8OHdGin in the recovered dialyzate are
measured.
[0066] Note that, in the case where a biological sample is used as
a test substance, oxidation with active oxygen such as oxidation of
dG into 8OHdG starts immediately after the sampling, so that it may
be often the case that the state of immediately after the sampling
is not accurately reflected to the evaluation results. In such a
case, it is preferable that the progress of oxidation after the
sampling is prevented by dipping the biological sample in an
antioxidative preservative solution mainly comprised a cation
chelating agent, an antioxidant and glycerol. The cation chelating
agent has an activity of efficiently stopping various biological
reactions or enzyme reactions and, for example, sodium
ethylenediaminetetraacetate, etc. are used. The antioxidant
prevents generation and liberation of hydroxy radicals and, for
example, methanol, etc. are used. Also, glycerol has, besides an
oxidation preventing effect with a hydroxyl group, a stable
activity of preserving a biological sample under the condition of
-20.degree. C. to 4.degree. C. (conditions under which ordinary
biological sample is preserved until time of use) and, for example,
20 to 50% aqueous solution is used. Further, the antioxidative
preservative solution is added not only to the biological sample
but also to a test substance after dG is added to the test
substance and then left to stand for a predetermined time to
prevent 8OHdG oxidation until it is measured, thereby enabling
measurement of accurate (anti)oxidizing ability after lapse of a
predetermined time.
[0067] As the aforementioned antioxidative preservative solution,
the antioxidative preservative solution comprising an aqueous
solution containing 0.5 to 2 mM/l EDTA, 2 to 5 wt % methanol, and
10 to 40 wt % glycerol is preferred. The antioxidative preservative
solution specifically prevents induction of 8OHdG from dG
(deoxyguanosine) in particular. This is because, firstly EDTA, a
cation chelating agent chelates calcium to efficiently stop
biological reaction and enzyme reaction, secondly, methanol as an
antioxidant prevents production and liberation of hydroxy radicals,
and as a third factor, a glycerol solution has an oxidation
preventing effect due to an alcohol group (OH group) and at the
same time acts as an antifreeze that preserves the analyte in a
stable state at -20.degree. C. to 4.degree. C. Glycerol also
prevents proteolysis and in addition has a preventive effect for
enzyme reaction.
[0068] In order to confirm usefulness of the aforementioned
antioxidative preservative solution, the present inventors:
[0069] (1) added 20 .mu.l each of oxidant (KBrO.sub.3, 50 mg/ml
solution) to 170 .mu.g/l each of various preservative components
(candidates) and mixed them;
[0070] (2) added 10 .mu.l each of commercially available dG
standard solution (2'-deoxyguanosine, 400 .mu.g/l) to the mixed
solution to prepare an analyte;
[0071] (3) after 30 minutes, mixed 50 .mu.l each of analytes with
equal amount (50 .mu.l) of 20% aqueous glycerol solution; and
[0072] (4) examined oxidation state of each analyte (increase in
8OHdG) by quantifying 8OHdG and dG using the mixed solution as
antioxidative preservative solution.
[0073] The results are shown in the following table. Note that
KBrO.sub.3 was adopted as an oxidizing agent because this substance
is generally used as food additive such as a bleaching agent for
bread and an antiseptic.
1 Relative Substance mixed with or 80 HdG/ initial diluted to
product Oxidation conditions 80 HdG .times. 10.sup.-3 dG dG .times.
10.sup.-3 magnification Pure water dilution -- 0.2710 1.0300 0.263
1 (immediately after) Pure water dilution KBrO.sub.3 30 minutes
3.779 1.067 3.542 13.47 #1) EDTA/4Na 1 mM KBrO.sub.3 30 minutes
1.098 0.997 1.101 4.19 #2) 10% Gly. KBrO.sub.3 30 minutes 1.572
1.044 1.506 5.73 #3) 1% Meth. KBrO.sub.3 30 minutes 2.882 1.04
2.771 10.54 #4) 5% Meth. KBrO.sub.3 30 minutes 2.359 1.054 2.238
8.51 #5) 10 mg/ml Rafi. KBrO.sub.3 30 minutes 3.278 1.045 3.137
11.93 #6) 1 mg/ml Rafi. KBrO.sub.3 30 minutes 2.327 0.926 2.513
9.55 #7) 10 mg/ml Glu. KBrO.sub.3 30 minutes 2.686 1.049 2.561 9.74
#8) 1 mg/ml Glu. KBrO.sub.3 30 minutes 2.515 1.029 2.444 9.29 #9)
10% Gly + 2.5% Meth. KBrO.sub.3 30 minutes 1.335 1.045 1.278 4.86
#10) 10% Gly + 2.5% Meth. KBrO.sub.3 30 minutes 0.978 1.058 0.924
3.51 + 1 mM EDTA/4Na #11) KBrO.sub.3 10 mg/ml (KBrO.sub.3) 15
minutes 3.991 1.011 3.95 #12) KBrO.sub.3 10 mg/ml (KBrO.sub.3) 60
minutes 3.677 1.014 3.63 #13) KBrO.sub.3 1 mg/ml (KBrO.sub.3) 15
minutes 0.715 0.975 0.73 #14) KBrO.sub.3 1 mg/ml (KBrO.sub.3) 60
minutes 0.788 1.022 0.77 #15) KBrO.sub.3 1 mg/ml 15 Minutes + 90
minutes 0.727 1.018 0.71 in preservative solution #16) KBrO.sub.3
0.1 mg/ml (KBrO.sub.3) 15 minutes 0.222 1.02 0.22 #17) KBrO.sub.3
0.1 mg/ml (KBrO.sub.3) 60 minutes 0.183 1.025 0.18
[0074] On the first line of table above, there are described the
data in the case of "pure water analyte" obtained by use of pure
water instead of the mixed solution obtained in (1) above, and on
the second line, there are described the data in the case of "pure
water/oxidant analyte" obtained by use of a mixed solution
containing pure water instead of the preservative solution and
containing a prescribed amount of oxidant (KBrO.sub.3). As will be
apparent from the table above, the concentration of dG in pure
water analyte is 1.0300 VS (value expressed in terms of output of
UV absorption analyzer; in this case, standard concentration of
dissolved dG was 10 .mu.g/ml). In the stages of preparation and
measurement, the concentration of already existing 8OHdG is 0.2710
mVs (value expressed in terms of output of the electrochemical
detector; the output of 5 ng/ml standard 8OHdG corresponded to 7.0
mVs), and 8OHdG/dG ratio was 0.263.times.10.sup.-3. Then, after 30
minutes in pure water/oxidizing analyte, the 8OHdG concentration
was 3.779 mVs and the 8OHdG/dG ratio was 3.542.times.10.sup.-3,
which was 13.47 times that of the case of pure water analyte.
Hereinafter, for studying the effects of various preservative
components, 8OHdG/dG ratio is used. This is for the purpose of
reducing the influence of errors that would be expected to appear
in the same tendency in both measured values of 8OHdG and dG, for
example, errors on concentration and volume of test solutions on
data at each measurement.
[0075] Those using EDTA/4Na (concentration upon measurement: 1
mM/l), which is a cation chelating agent as a preservative solution
component showed a 8OHdG/dG of 1.101.times.10.sup.-3, and
suppressed at a level of 4.19 times that of the case of ultrapure
water analyte under the same oxidative conditions, while those
using glycerol (concentration upon measurement: 10 wt %) showed a
8OHdG/dG of 1.506.times.10.sup.-3, and suppressed at a level of
5.73 times that of the case of pure water analyte under the same
oxidative conditions. In the case of those using methanol
(concentrations upon measurement: 1 wt % and 5 wt %), which is an
antioxidant, they showed 8OHdG/dG ratios of 2.771.times.10.sup.-3
and 2.238.times.10.sup.-3, and suppressed at a level of 10.54 times
and 8.51 times, respectively, that of the case of ultrapure water
analyte under the same oxidative conditions.
[0076] The above table shows results of two cases where raffinose
and glucose were used in addition to the cases of the
aforementioned preservative solution components (EDTA/4Na,
glycerol, and methanol). Raffinose is one kind of trisaccharides
obtained from sugar beet and, because of its physical and chemical
stability, is used as a tissue stabilizing agent added to sperm
preservative solution or an organ preservative solution. On the
other hand, glucose is the most common monosaccharide and at the
same time is known to be a potent antioxidant that exists in
largest amounts in vivo (Niki et al., ed.; "Antioxidative
Chemicals" Academy Publishing Center, 1994). However, as will be
apparent form the above table, 8OHdG/dG ratios in both cases are
relatively large about 10 to 12 times as large as that of the case
of ultrapure water, so they are unsuitable as antioxidative
preservative solution components.
[0077] Additional typical substance that exhibits antioxidative
property to biological samples includes VC (ascorbic acid) and
NaN.sub.3 (sodium azide). However, in spite of extremely strong
antioxidative property (reducing property) of VC itself, it
conversely serves as a strong oxidant in the presence of a metal
cation. In fact, when it was stored for a long period of time as
added to a dG solution in such a state that it was brought in
contact with air, it was confirmed that a large amount of 8OHdG was
induced therefrom. Further, sodium azide is widely used as a
preservation solution (stabilizing agent) for components of nucleic
acid and various enzymes. This also has strong chemical reactivity
and it has been confirmed that it reacts with oxygen in the air or
in a sample to derive a large amount of nitrogen oxides.
[0078] In contrast, glycerol, methanol, and EDTA are extremely
stable chemicals; i.e., they do not induce reproduction of microbes
and showed antioxidant ability data overstriding glucose and
raffinose as explained above with respect to the above table.
[0079] Accordingly, with an expectation of synergistic effect among
the aforementioned three elements of which preferable results have
been obtained, an antioxidative preservative solution was prepared
(No. 10 analyte; a mixture of 10 wt % of glycerol, 2.5 wt % of
methanol, and 1 mM/l of EDTA/4Na, concentrations being at the time
of measurement). As a result, it was found that those using the
antioxidative preservative solution showed an 8OHdG/dG ratio of
0.924 and a magnification for initial value of 3.51 and greatly
suppressed the oxidation of dG. Therefore, it is the components of
No. 10 analyte (the aforementioned three elements) that are
suitable as components of the antioxidative preservative solution.
The above blending ratios are within the ranges of suitable
component concentrations, respectively, for being supplied to a
measurement system starting with liquid chromatograph.
[0080] Eventually, the blending ratios of components of the
antioxidative preservative solution were set such that
concentrations at the time of measurement of respective components
that vary by use of a typically 20 wt % aqueous glycerol solution
as a perfusion solution in the micro dialysis protein removal
treatment can approach the value of No. 10 analyte as described
above (provided that the concentration of glycerol at the time of
measurement may increase about 10 wt % to about 40 wt % of the
analyte, but in consideration of the relationship with the
precision of protein removal immediately before the measurement,
the upper limitation is about 20 wt %.); i.e., EDTA is set to 0.5
to 2 mM/l, methanol is set to 2 to 5 wt %, and glycerol is set to
10 to 40 wt %.
[0081] Further, in the above table, Nos. 11 to 17 analytes are
intended to show oxidation state of dG with lapse of time in the
case where pure water is used instead of the preservative solution
component in the mixed solution to which dG standard solution is
added and the concentration of oxidant (KBrO.sub.3) is changed in
three levels. Nos. 11 and 12 have the same oxidant concentration
(10 mg/ml of KBrO.sub.3) as the pure water/oxidant analyte at the
time of measurement and their 8OHdG concentrations of 3.991 after
15 minutes and of 3.677 after 60 minutes are values close to the
8OHdG concentration of 3.779 of pure water/oxidant analyte (30
minutes) These values are extremely high as compared with about 0.7
to about 0.8 and about 0.2 of Nos. 13 to 17 analytes in which the
concentrations of KBrO.sub.3 are 1 mg/ml and 0.1 mg/ml,
respectively. These indicate well that when the oxidant
(KBrO.sub.3) is added to dG without concomitant addition of
antioxidative preservative solution, oxidation proceeds depending
on the addition amount thereof.
[0082] These results suggest that 8OHdG is induced from dG
depending on the concentration of KBrO.sub.3 and since
substantially no difference in the induction amount of 8OHdG is
observed between after 15 minutes and after 60 minutes, the
oxidative reaction with KBrO.sub.3 quickly occurs and thereafter
reaches a constant level. Therefore, DNA oxidation function of
various food additives including daily taken through oral route,
for example KBrO.sub.3, can be clearly grasped by the measurement
apparatus of the present invention and can be used for restudy of
acceptable concentrations thereof.
[0083] The biological evaluation method of the present invention is
practiced, for example, as follows. First, a test substance is
diluted in multiple stages to form test solutions, into which a
known concentration of 2'-deoxyguanosine (dG) is added and left to
stand it from ice temperature to about 50.degree. C., preferably at
room temperature for a predetermined time, and then amount of
produced 8-hydroxy-2'-deoxyguanosi- ne (8OHdG) is measured (this
value is defined as A). Separately, the amount of 8OHdG after an
ultrapure water solution of dG is left to stand under the same
conditions as above for a predetermined time is measured (this
value is defined as B). If A is greater than B, the test substance
is evaluated to have oxidation induction ability and conversely if
A is smaller than B, the test substance is evaluated to have
antioxidant ability. In either case, it is judged that the greater
the difference is, the greater degree of oxidation induction
ability or antioxidant capability the test substance has.
[0084] The generation of active oxygen is a phenomenon inevitable
to perform biological activity. However, it has been clarified that
if the system for eliminating its physiological activity functions
only insufficiently, the DNA oxidation injury increases, resulting
in carcinogenesis and lowered induction of cell activity
(senescence), which in turn lead to death of cell (apoptosis). By
the present invention, evaluation as to how much the toxicity of
active oxygen is eliminated by a test substance (whether or not it
has effectiveness) on DNA nucleic acid level, single cell level,
and individual level, or as to how much the toxicity is increased
by the test substance (whether or not it is toxic) can be made in a
simple manner.
[0085] Further, the effectiveness of foods such as various
health-care foods and functional foods, can in other words, be said
to depend on how efficiently the toxicity of active oxygen that is
generated in vivo can be eliminated. Therefore, according to the
present invention, the antioxidant ability of various foods can be
rendered objective using DNA oxidation injury as an index.
[0086] UV light directly acts on nuclear DNA and is an important
factor for causing DNA oxidation injury. No living organism on the
earth can escape from the influence thereof. There is the
possibility that synthetic chemicals discharged in natural
environment or natural chemicals existing in the nature are also
activated (radicalized) by UV light irradiation, and thus,
biotoxicity that is generated by uptake of the activated synthetic
chemicals or coming into contact with the synthetic chemicals
cannot be disregarded either. According to the present invention,
additive, synergistic or canceling effect between test substances
such as the above-mentioned synthetic chemicals and UV light can
also be evaluated simply. In addition, the present invention
provides a simple evaluation method for additive, synergistic or
canceling effect between an active oxygen generator and test
substances. Further, according to the present invention, a known
amount of dG is added to a solution containing a chemical that has
been confirmed to have gene oxidation injury activity and a test
substance, and 8OHdG in the above solution is quantified to
evaluate the presence or absence of effectiveness of gene injury
preventive activity of the test substance according to the amount
thereof. Here, by selecting those having high possibilities to be
exposed in daily living as the chemical that has been confirmed to
have gene oxidation injury effect, evaluation of a test substance
can be made in a state closer to reality. In particular, by
selecting food as the test substance, the influence of mixture of
food and gene oxidation injurious chemicals can be evaluated.
[0087] In the biological evaluation method of the present
invention, living organism cell derived products collected from
animals including humans, plants, bacteria or fungi that have been
administered with chemicals or foods or that have taken up or
contacted them, for example, blood, urine, other bodily fluid,
tissue destruction fluid or cell destruction fluid and so forth as
test subjects, and content ratio of 8OHdG and dG (8OHdG/dG) in the
test subject are calculated and the harmfulness or usefulness of
the foods or chemicals is evaluated according to the magnitude of
the content ratio (a higher content of a chemical or food indicates
a higher harmfulness and a lower content of chemical or food
indicates a higher usefulness).
[0088] In the biological evaluation method of the present
invention, a substance that has excellent antioxidant ability;
i.e., that can efficiently eliminate active oxygen that has been
generated for some causes or others, for example, functional foods
containing such as various natural or artificial chemicals or
subtances whose compositions are unclear, in particular water can
treat, improve, or alleviate various diseases in animals including
humans when continuously taken up. The antioxidant ability that
such a substance should have is nearly equal to the content ratio
of 8OHdG and dG in the case where ultrapure water is used. This can
be used as one standard.
[0089] The measurement apparatus of the present invention can be
operated as described below when applied to a biological liquid
sample; i.e., an aqueous solution having dissolved therein a test
substance such as whole blood, plasma, serum, urine, cerebrospinal
fluid and tissue fluid. First, as pretreatment of a sample, a
liquid sample is mixed with the same volume of antioxidant
preservative solution and thereafter it is stored until
measurement. Immediately before measurement operation starting with
micro dialysis treatment, the stored analyte is returned to room
temperature. The test solution perfused and recovered through micro
dialysis treatment is supplied to a high performance liquid
chromatograph and an eluate containing a dG peak and an 8OHdG peak
is supplied to an 8OHdG/dG simultaneous measurement system where
measurement is performed. From measured values, DNA oxidation
injury index in a biological sample is finally calculated.
[0090] The measurement apparatus of the present invention can be
operated as described below when applied to a biological sample
containing cell components; i.e., those requiring destruction, such
as whole blood, cell suspension, organs and tissues. First, an
antioxidant preservative solution is injected in a tube for tissue
destruction in an amount of 0.5 to 0.1 ml. As the tube for tissue
destruction, a sterile tube that is physically suitable for cell
destruction and biochemically insensitive to DNAase or RNAase, for
example, "Fast DNA tube" manufactured and sold by Bio101 Corp. in
U.S.A. can be used. Then, 100 to 200 mg of a biological cell sample
is placed in the tube and immediately ice cooled. When preparations
of cell destruction and related operations are ready, tissue is
destructed by use of a special-purpose destruction apparatus, for
example, "FastPrep FP120" type apparatus manufactured and sold by
Bio101 Corp. in U.S.A. and free nucleic acid inside and outside
cells are eluted in a preservative solution. Solids are removed by
centrifugation from the preservative solution containing the free
nucleic acid and the supernatant fluid is defined as a stock
analyte. The stock analyte is returned to room temperature
immediately before measurement and subjected to micro dialysis
treatment. The test solution perfused and recovered through micro
dialysis treatment is supplied to high performance liquid
chromatograph and its elute containing a dG peak and an 8OHdG peak
is supplied to an 8OHdG/dG simultaneous measurement system where
measurement is performed. From the measured values, finally DNA
oxidation injury index of the biological sample is calculated.
[0091] The antioxidant preservative solution is basically a
solution of a cation chelating agent and an antioxidant dissolved
in a 10 to 40% aqueous glycerol solution. This is stored at a
temperature of ice cooling condition or lower and in a
light-shielded state until a biological sample is mixed therein.
Preferred blending ratios of components are as follows.
2 Glycerol: 10 to 40 wt % Methanol: 2 to 5 wt % EDTA/4Na: 0.5 to 2
mM/l
[0092] In the above blending ratios of components, methanol and
EDTA/4Na may be optionally added. Preservation of the prepared
preservative solution per se or preservation after mixing with a
biological sample can be performed in a light-shielded and sealed
state in the following manner.
[0093] Short preservation for about 2 to 3 days:
[0094] In a refrigerator (4.degree. C.)
[0095] Medium preservation for about half the year:
[0096] In a refrigerator (-10 to -20.degree. C.).
[0097] Long preservation for half the year or more:
[0098] In a refrigerator (-80.degree. C.) after purging the air in
the sample tube with nitrogen gas.
[0099] In the aforementioned micro dialysis, a dialysis membrane
tube having a cut off function of blocking a high molecular weight
component of 20 to 50 kD is dipped in an analyte and low molecular
weight DNA components are efficiently recovered from the analyte by
use of an apparatus including a perfusion system with the tube in
which a perfusion solution passes. The recovered components include
dG, C.sub.10H.sub.13N.sub.5O.sub.4, MW 267, 80 HdG,
C.sub.10H.sub.13N.sub.5O.- sub.5, MW 283, etc. The perfusion
conditions use basically the following values.
[0100] Perfusion solution: 20% Glycerol solution
[0101] Perfusion temperature: Room temperature
[0102] Perfusion rate: 0.5 to 2 .mu.l/minute
[0103] However, in order to increase the recovery rate, or improve
S/N ratio of an objective component, these conditions may be
changed as appropriate.
[0104] The low molecular weight DNA components recovered by the
micro dialysis are sent into a column of high performance liquid
chromatograph and eluted from the column by component peak such as
8OHdG, dG, etc. The eluate from the column is first sent to a
sample cell in a UV absorption analyzer to measure dG content, and
then sent to an electrochemical detector, where 8OHdG content is
measured.
[0105] The procedure of the measurement apparatus of the present
invention as stated above is arranged in accordance with the
flowchart in FIG. 1 as follows.
[0106] Step 1: Collection of a biological sample
[0107] Step 2: Preserved with cooling after mixing the biological
sample with a preservative solution
[0108] Step 3: Destruction of a sample for cell analysis mixed with
a preservative solution in the tube and separation of solids.
[0109] Step 4: Micro dialysis treatment of the preserved liquid
sample or a sample for cell analysis after destruction and
separation.
[0110] Step 5: Supply of the sample components perfused and
extracted by dialysis to high performance liquid chromatograph
(HPLC).
[0111] Step 6: Sending the eluate from the chromatograph column to
a sample cell of a UV light absorption analyzer to measure dG
content.
[0112] Step 7: Passing the eluate from the column that has passed
the above sample cell to an electrochemical detector to measure
8OHdG content.
[0113] Step 8: Processing 8OHdG/dG ratio and other data by use of
the measured values in the Steps 6 and 7.
[0114] Next, the present invention will be illustrated in more
detail by way of examples below. However, the present invention
should not be construed as being limited thereto.
EXAMPLE 1
[0115] Solutions in various concentrations (0.0005 ppm, 0.005 ppm,
0.05 ppm, 0.5 ppm and 5 ppm) of pentachlorophenol (PCP, herbicide),
bisphenol A (BPA, resin raw material), and Resveratol (RVT, one
kind of polyphenol) were prepared and 180 .mu.l portions were
injected into each well of two 99-well plastic plates. Then, 20
.mu.l of 200 .mu.g/ml dG (dissolved in ultrapure water) was added
to each well and one of the plates was left to stand as it was at
room temperature for 90 minutes and the other plate was irradiated
with UV light (254 nm, 860 .mu.W/cm.sup.2) for 90 minutes in a UV
light irradiation box. After completion of the standing or
irradiation, the solution in each well was mixed with the same
amount of 20% glycerol solution and dG and 8OHdG were separated
from each other by use of HPLC [column used: CA-50DS (manufactured
by AICOM CO.), mobile phase solution: 0.1 M phosphate buffer; 3 to
10% methanol, SOS 90 to 100 mg]. Subsequently, nucleosides in the
both were quantified by use of the UV light absorption analyzer
connected to the HPLC and the electrochemical detector. For
comparison, measurement of dG and 8OHdG was performed on ultrapure
water alone that had been treated in the same manner. The results
were shown Table 1. Although measurements were performed on both
nucleosides, the Table below shows only the concentration (ng/ml)
of 8OHdG (in examples hereinafter the same).
3TABLE 1 Concentration 90 Minutes' 90 Minutes' Test Substance (ppm)
standing UV irradiation Ultrapure 0.267 0.529 water PCP 5 0.340
5.730 0.5 0.282 0.396 0.05 0.285 0.431 0.005 0.236 0.451 0.0005
0.260 0.585 BPA 5 0.192 7.142 0.5 0.231 1.736 0.05 nd 0.614 0.005
0.315 0.794 0.0005 nd 0.689 RVT 5 nd 5.582 0.5 nd 1.378 0.05 nd
0.571 0.005 nd 0.690 0.0005 nd 1.026 (In the Table above, each
measured value is an average of two measured values, "nd" means no
measurement.)
[0116] PCP is an agricultural chemical or a softening agent for
leather and safety concentration is reported to be 5 mg/kg.
However, from the above results, it can be clearly seen that
oxidation injury activity due to UV light is extremely elevated and
therefore harmfulness is increased at 5 ppm corresponding to 5
mg/kg.
[0117] BPA is a representative endocrine disturbing substance
(environmental hormone) and at 0.5 ppm or more, it clearly elevates
oxidation injury activity due to UV light so that harmfulness is
increased. For reference, BPA concentration at which there is the
possibility that exposure will actually occur is shown as
follows.
[0118] In rivers, lakes and ponds:
[0119] About 0.001 ppm in places where it is rich
[0120] Water leaching out from general industrial wastes processing
plants:
[0121] Maximum about 20 ppm
[0122] PC made dishes (95.degree. C., 30 minutes):
[0123] 0.005 to 0.1 ppm
[0124] Elution from the coating of can for beverage:
[0125] About 0.1 ppm
[0126] Elution from the dental cement:
[0127] About 1 ppm in saliva after the treatment of dental
caries
[0128] Fish:
[0129] About 0.02 to 0.3 ppm
[0130] The standard on BPA in Japan is 2.5 ppm or less as eluted
portion and 500 ppm or less as material. Acceptable uptake amount
is 0.05 mg/kg/day, which is common in every country. The present
invention can help reviewing these standards.
[0131] RVT is a typical polyphenol and is said to be contained in
red wine in an amount of about 2 to 10 ppm and its
anti-arteriosclerosis activity, etc. have been reported in recent
years. However, the above results show that at 0.5 ppm or more,
oxidation injury activity due to UV light clearly increases to
increase harmfulness. Thus, the present invention can also simply
and rationally verify effectiveness (or harmfulness) of substances
that have conventionally been considered effective.
[0132] In the above tests, the amount of sample necessary for a
single measurement is as small as 10 to 100 .mu.l and a measurement
peak can be detected in 5 to 10 minutes from the injection of the
sample and continuous measurement is possible at a pace of 15
minutes/sample. Therefore, when the present invention is adopted
and one HPLC is operated at a pace of 120 hours per week,
measurement can be made at a pace of 480 analytes/week and about
2,000 analytes/month. This means that according to the present
invention, toxicity and so forth of test substances can be
evaluated at such a high pace that has never been reported thus
far.
[0133] In Example 1, both dG and 8 OHdG were simultaneously
measured by use of a UV light absorption analyzer and an
electrochemical detector. However, measurement of 8OHdG only may be
performed. In such a case, a large amount of sample measurement can
be done in a short time at high sensitivity by use of a measurement
kit using monoclonal antibody [for example, trade name 8-OHdG Check
(manufactured by Nippon Aging Control Laboratory).
EXAMPLE 2
[0134] Solutions of predetermined concentrations of vitamin C (VC),
vitamin E (VE), catechin (Cate) and tannic acid (Tan) were prepared
and 180 .mu.l portions were injected into each well of two 99-well
plastic plates. Then, 20 .mu.l of 200 .mu.g/ml dG (dissolved in
ultrapure water) was added to each well and one of the plates was
left to stand as it was at room temperature for 90 minutes and the
other plate was irradiated with UV light (254 nm, 860
.mu.W/cm.sup.2) for 90 minutes in a UV light irradiation box. After
completion of the standing or irradiation, the solution in each
well was mixed with the same amount of 20% glycerol solution and dG
and 8OHdG were quantified in the same manner as in Example 1. For
comparison, measurement of dG and 8OHdG was performed on distilled
water alone that had been treated in the same manner. The results
are shown in Table 2.
4TABLE 2 90 Minutes' 90 Minutes' Test Substance Concentration
standing UV irradiation distilled 0.15 1.23 water VC 0.001% 6.04
38.36 0.0001% 0.63 1.51 0.00001% 0.44 0.46 VE 0.05% 0.44 0.42
0.005% 0.45 0.18 0.0005% 0.49 0.30 Cate 100 .mu.g/ml nd nd 10
.mu.g/ml 133.71 nd 1 .mu.g/ml 9.07 nd Tan 100 .mu.g/ml 54.00 97.07
10 .mu.g/ml 5.64 19.29 1 .mu.g/ml 0.84 1.04 (In the Table above,
each measured value is an average of two measured values, "nd"
means no measurement.)
[0135] From the above results, the following conclusion can be
obtained.
[0136] Although VC is said to be a potent antioxidant, it acts as a
very strong oxidation inducing substance for water-soluble dG.
[0137] VE is a very stable antioxidant.
[0138] Cate induces 8OHdG very strongly regardless of whether or
not UV light irradiation was performed. This oxidizing ability is
considered to be associated with the bactericidal activity and
antiviral activity of Cate. There is high possibility that as in
the case of Cate, the 8OHdG inducing ability of a substance can be
used as an index (effective concentration index) for bactericidal
or bacteriostatic effect or antiviral effect.
[0139] Tan exhibits high gene injury inducing effect.
EXAMPLE 3
[0140] Solutions of predetermined concentrations of glycerol (Gly)
and methanol (Meth) were prepared and 180 .mu.l portions thereof
were injected into each well of five 99-well plastic plates. Then,
20 .mu.l of 200 .mu.g/ml dG (dissolved in ultrapure water) was
added to each well and one of the plates was left to stand as it
was at room temperature for 90 minutes and another plate was left
to stand similarly at room temperature for 24 hours. The remaining
three plates were irradiated with UV light (254 nm, 860
.mu.W/cm.sup.2) for 60 minutes, 90 minutes and 120 minutes,
respectively, in a UV light irradiation box. After completion of
the standing or irradiation, the solution in each well was mixed
with the same amount of 20% glycerol solution and dG and 8OHdG were
quantified in the same manner as in Example 1. For comparison,
measurement of dG and 8OHdG was performed on ultrapure water alone
that had been treated in the same manner. The results are shown in
Table 3.
5TABLE 3 Test Standing UV irradiation Substance Concentration 90
minutes 24 hours 60 minutes 90 minutes 120 minutes Ultrapure water
0.323 0.382 0.231 0.382 0.645 Gly 5% 0.228 0.306 0.344 0.399 0.544
20% 0.213 0.342 1.317 1.471 1.374 Meth 5% 0.146 0.336 0.181 0.209
0.332 20% 0.186 0.274 0.324 0.395 0.518
[0141] (In the Table above, each measured value is an average of
two measured values. While both Gly and Meth have been reported to
have DNA injury preventive effects, the results obtained in this
example confirmed their antioxidant ability. In particular, it was
confirmed that Gly exhibited antioxidant ability only when left to
stand at room temperature while potent antioxidant ability of Meth
was confirmed at both the time when left to stand at room
temperature and the time when irradiated with UV light.
EXAMPLE 4
[0142] Solutions of predetermined concentrations of glucose (Glu),
raffinose (Raffi) and sucrose (Suc) were prepared and 180 .mu.l
portions thereof were injected into each well of four 99-well
plastic plates. Then, 20 .mu.l of 200 .mu.g/ml dG (dissolved in
ultrapure water) was added to each well and one of the plates was
left to stand as it was at room temperature for 90 minutes. The
remaining three plates were irradiated with UV light (254 nm, 860
.mu.W/cm.sup.2) for 60 minutes, 90 minutes and 120 minutes,
respectively, in a UV light irradiation box. After completion of
the standing or irradiation, the solution in each well was mixed
with the same amount of 20% glycerol solution and dG and 80 HdG
were quantified in the same manner as in Example 1. For comparison,
measurement of dG and 8OHdG was performed on ultrapure water alone
that had been treated in the same manner. The results are shown in
Table 4.
6TABLE 4 UV irradiation Test 90 Minutes' 60 90 120 Substance
Concentration standing minutes minutes minutes Ultrapure 0.195
0.392 0.901 1.183 water Glu 1 mg/ml 0.220 0.094 0.099 0.237 100
.mu.g/ml 0.200 0.132 0.274 0.414 10 .mu.g/ml 0.240 0.291 0.458
0.277 1 .mu.g/ml 0.260 0.384 0.595 0.385 0.1 .mu.g/ml 0.295 0.399
0.702 0.410 Raffi 1 mg/ml 0.340 0.115 0.176 0.240 100 .mu.g/ml
0.455 0.158 0.370 0.503 10 .mu.g/ml 0.405 0.281 0.626 1.314 1
.mu.g/ml 0.185 0.304 0.854 1.049 0.1 .mu.g/ml 0.205 0.375 0.689
1.127 Suc 1 mg/ml 0.300 0.127 0.309 0.364 100 .mu.g/ml 0.280 nd
0.379 0.648 10 .mu.g/ml 0.235 0.333 0.690 1.279 1 .mu.g/ml 0.185
0.358 0.708 1.143 0.1 .mu.g/ml 0.235 0.299 0.756 1.073 (In the
Table above, each measured value is an average of two measured
values, "nd" means no measurement.)
[0143] From the above results, the following conclusion can be
obtained.
[0144] The potency of antioxidant ability in both cases of standing
at room temperature and UV light irradiation can be evaluated as
Glu>Raffi.apprxeq.Suc.
[0145] Assuming that normal blood sugar level is about 50 to 100
mg/dl (0.5 to 1 mg/ml), Glu existing in blood can be said to be a
very effective antioxidant substance.
[0146] Raffi is one kind of natural oligosaccharides and is added
to many foods such as soft drinks, health-care drinks and
health-care foods, and although its antioxidant ability is not so
potent as that of Glu, it is effective at a concentration of 10
.mu.g/ml or more.
[0147] Suc exhibits antioxidant ability as potent as that of
Raffi.
EXAMPLE 5
[0148] Solutions of predetermined concentrations of 1-arginine
(Arg), 1-citrulline (Cit) and spermine (Spe), each of which was
protamine related substances, were prepared and 180 .mu.l portions
thereof were injected into each well of two 99-well plastic plates.
Then, 20 .mu.l of 200 .mu.g/ml dG (dissolved in ultrapure water)
was added to each well and one of the plates was left to stand as
it was at room temperature for 90 minutes and the other plate was
irradiated with UV light (254 nm, 860 .mu.W/cm.sup.2) for 90
minutes in a UV light irradiation box. After completion of the
standing or irradiation, the solution in each well was mixed with
the same amount of 20% glycerol solution and dG and 8OHdG were
quantified in the same manner as in Example 1. For comparison,
measurement of dG and 8OHdG was performed on ultrapure water alone
that had been treated in the same manner. The results are shown in
Table 5.
7 TABLE 5 Test 90 Minutes' 90 Minutes' Substance Concentration
standing UV irradiation Ultrapure 0.219 0.422 water Arg 1 mg/ml
0.173 0.299 100 .mu.g/ml 0.150 0.305 10 .mu.g/ml 0.153 0.253 1
.mu.g/ml 0.186 0.323 Cit 1 mg/ml 0.156 0.125 100 .mu.g/ml 0.120
0.225 10 .mu.g/ml 0.179 0.305 1 .mu.g/ml 0.184 0.332 Spe 1 mg/ml
0.179 0.112 100 .mu.g/ml 0.154 0.351 10 .mu.g/ml 0.150 0.328 1
.mu.g/ml 0.140 0.297 (In the Table above, each measured value is an
average of two measured values.)
[0149] Protamine is a protein that supports a steric structure of a
gene and has an extremely high content of arginine, which is a
typical basic amino acid (20 to 70%).
[0150] Each of Arg, Cit and Spe exhibited antioxidant ability. In
the case of Cit and Spe, their antioxidant ability was particularly
high at a high concentration (1 mg/ml).
[0151] Although data were not shown, it was confirmed according to
the present invention that acidic amino acids generally had a
tendency of exhibiting oxidization induction, and basic amino acids
had a tendency of preventing oxidation.
EXAMPLE 6
[0152] Solutions of various concentrations of uric acid (dissolving
fluid: distilled water+0.025% FBS) were prepared and 180 .mu.l
portions thereof were injected into each well of two 99-well
plastic plates. Then, 20 .mu.l of 200 .mu.g/ml dG (dissolved in
ultrapure water) was added to each well and one of the plates was
left to stand as it was at room temperature for 90 minutes and the
other plate was irradiated with UV light (254 nm, 860
.mu.W/cm.sup.2) for 90 minutes in a UV light irradiation box. After
completion of the standing or irradiation, the solution in each
well was mixed with the same amount of 20% glycerol solution and dG
and 8OHdG were quantified in the same manner as in Example 1. For
comparison, measurement of dG and 8OHdG was performed on the above
dissolving fluid alone that had been treated in the same manner.
The results are shown in Table 6.
8 TABLE 6 Test 90 Minutes' 90 Minutes' Substance Concentration
standing UV irradiation dissolving 0.218 1.007 fluid uric acid 10
.mu.g/ml 0.147 15.619 1 .mu.g/ml 0.175 2.369 0.1 .mu.g/ml 0.193
0.966 0.01 .mu.g/ml 0.176 1.064 (In the Table above, each measured
value is an average of two measured values.)
[0153] Uric acid is said to be a typical in vivo antioxidant.
However, as is apparent from the above results, although it can
inhibit induction of 8OHdG at an actual in vivo plasma
concentration (40 to 80 .mu.g/ml) except for in a strong oxidative
environment, it shows a high degree of DNA oxidation injury when UV
light was coexistent and its toxicity was increased.
EXAMPLE 7
[0154] Solutions of various concentrations of potassium bromate
(KBrO.sub.3) were prepared and 180 .mu.l portions thereof were
injected into each well of two 99-well plastic plates. Then, 20
.mu.l of 200 .mu.g/ml dG (dissolved in ultrapure water) was added
to each well and one of the plates was left to stand as it was at
room temperature for 15 minutes and the other plate was left to
stand for 60 minutes. After completion of the standing, the
solution in each well was mixed with the same amount of 20%
glycerol solution and dG and 8OHdG were quantified in the same
manner as in Example 1. For comparison, measurement of dG and 8OHdG
was performed on ultrapure water alone that had been treated in the
same manner. The results are shown in Table 7.
9 TABLE 7 Test 15 Minutes' 60 Minutes' Substance Concentration
standing UV standing Ultrapure 0.155 0.289 water KBrO.sub.3 10
mg/ml 2.851 2.626 1 mg/ml 0.511 0.563 0.1 mg/ml 0.159 0.131 (In the
Table above, each measured value is an average of two measured
values.)
[0155] Potassium bromate is a food additive commonly used as a
bleaching agent for bread and antiseptic. From the above results,
it can be seen that it exhibits concentration-dependent 8OHdG
induction. Since there is no great difference between the standing
for 15 minutes and the standing for 60 minutes, the oxidation
reaction by potassium bromate is considered to occur quickly and
then reach a constant level. The method of the present invention
suggests that potassium bromate, which was considered safe
according to conventional evaluation tests, could be toxic. AS
stated above, permissible concentrations of various food additives
taken daily by oral route can be reviewed according to the present
invention.
EXAMPLE 8
[0156] 170 .mu.l portions of a 1 mM sodium
ethylenediaminetetraacetate (EDTA) solution, 10% glycerol (Gly)
solution, a 1% or 2.5% methanol (Meth) solution, a mixed solution
of 10% Gly and 2.5% Meth, a mixed solution of 10% Gly, 2.5% Meth
and 1 mM EDTA were injected into each well of a 99-well plastic
plate. Then, 10 .mu.l of 400 .mu.g/ml dG (dissolved in ultrapure
water) and 20 .mu.l of 50 mg/ml potassium bromate (KBrO.sub.3)
solution were added to each well and the plate was left to stand as
it was at room temperature for 30 minutes. Then, the solution in
each well was mixed with the same amount of 20% glycerol solution
and dG and 8OHdG were quantified in the same manner as in Example
1. For comparison, measurement of dG and 8OHdG was performed on
ultrapure water alone that had been treated in the same manner. The
results are shown in Table 8.
10 TABLE 8 30 Minutes' Test Substance standing Ultarapure water
2.699 1 mM EDTA 0.784 10% Gly 1.123 1% Meth 2.059 2.5% Meth 1.685
10% Gly + 2.5% Meth 0.954 10% Gly + 2.5% Meth + 1 mM EDTA 0.699 (In
the Table above, each measured value is an average of two measured
values.)
[0157] In the presence of potassium bromate acting as an active
oxygen generator, the mixed solution f 10% Gly, 2.5% Meth and 1 mM
EDTA exhibited the highest antioxidant ability. Based on this, it
can be seen that a solution containing these three kinds of
components is effective as an antioxidant preservative solution for
various test solutions and test samples.
EXAMPLE 9
[0158] 170 .mu.l portions of ultrapure water and predetermined
concentrations of vitamin C (VC), vitamin E (VE), and glucose (Glu)
as test substance were added to each well of two 99-well plastic
plates and, as additional solution, 20 .mu.l of ultrapure water or
predetermined concentration of bisphenol A (BPA) were injected into
each well. Then, 10 .mu.l of 400 .mu.g/ml dG (dissolved in
ultrapure water) was added to each well. One of the plates was left
to stand as it was at room temperature for 90 minutes and the other
plate was irradiated with UV light (254 nm, 860 .mu.W/cm.sup.2) for
90 minutes in a UV light irradiation box. After completion of the
standing or irradiation, the solution in each well was mixed with
the same amount of 20% glycerol solution and dG and 8OHdG were
quantified in the same manner as in Example 1. The results are
shown in Table 9.
11TABLE 9 Additional Test Substance solution 90 Minutes' 90
Minutes' (Concentration) (Concentration) standing UV irradiation
Ultrapure water Ultrapure water 0.21 0.64 BPA (50 ppm) 0.22 11.58
BPA (5 ppm) 0.31 6.19 BPA (0.5 ppm) 0.19 2.09 VC (0.001%) Ultrapure
water 16.3 5.75 VC (0.001%) BPA (50 ppm) 1.51 12.53 VC (0.0001%)
BPA (50 ppm) nd 15.95 VC (0.00001%) BPA (50 ppm) nd 14.96 VE
(0.001%) Ultrapure water 0.18 0.28 VE (0.001%) BPA (50 ppm) 0.29
3.79 VE (0.0001%) BPA (50 ppm) nd 8.27 VE (0.00001%) BPA (50 ppm)
nd 15.43 Glu (1 mg/ml) Ultrapure water 0.16 0.40 Glu (1 mg/ml) BPA
(50 ppm) 0.24 4.46 Glu (0.1 mg/ml) BPA (50 ppm) nd 7.52 Glu (0.01
mg/ml) BPA (50 ppm) nd 14.93 (In the Table above, each measured
value is an average of two measured values, "nd" means no
measurement.)
[0159] From the above results, the following conclusion can be
obtained.
[0160] By addition of BPA, 8OHdG upon irradiation with UV light
increases depending on concentration.
[0161] VE and Glu significantly inhibit the increase in 8OHdG at
the time of addition of BPA and irradiation with UV light.
[0162] 0.001% VC alone potently induces 8OHdG but the effect is
cancelled by coexisting of BPA. However, when irradiated with UV
light, VC does not cancel the induction of 8OHdG by BPA.
[0163] As stated above, gene oxidation ability of BPA undergoes
various influences by coexistent substances.
[0164] Therefore, use of the measurement method of the present
invention enables not only identifying a substance that induces
oxidation injury of a gene but also identifying antioxidant
substance that is specific to the substance concerned and can
cancel its oxidative toxicity and further quantitatively evaluating
the antioxidant ability thereof.
EXAMPLE 10
[0165] Various kinds of drinking water as test substances (test
solutions) were measured of their antioxidant ability or oxidant
ability.
[0166] dG was dissolved in various kinds of drinking water such
that their concentrations were 10 .mu.l/ml. 200 .mu.l of each of
the obtained solutions was injected into two test vessels (surface
area of the solution was 36 mm.sup.2). One of the vessels was left
to stand at room temperature for 90 minutes and the other was
irradiated with UV light (254 nm, 860 .mu.W/cm.sup.2) for 90
minutes in a UV light irradiation box. After completion of the
standing or irradiation, the solution in each vessel was mixed with
the same amount of 20% glycerol solution and dG and 8OHdG were
quantified in the same manner as in Example 1.
[0167] The kinds of drinking water tested are as follows.
[0168] Ultrapure water: Water having a specific resistance of 18
Meg.OMEGA. (manufactured by Millipore Systems), having a redox
potential of 360 mV.
[0169] City water: Ordinary city water in Kasuga City, Fukuokoka
Prefecture (collected in July, 1997), having a redox potential of
727 mV.
[0170] Processed water: Purified water obtained by passing the
above city water through a commercially available water purifier
(that purifies water by passing it through activated carbon, a
strong magnet, ceramics, etc.), having a redox potential of 518
mV.
[0171] Natural water: Groundwater collected at Nichiden City in
Ooita Prefecture (water isolated from contamination on the ground
surface such as acid rain and fertilizer, dissolved nitrogen oxides
concentration is 0.01 ppm or less), having a redox potential of 280
mV.
[0172] The results obtained are shown in Table 10.
12TABLE 10 A ratio of Test Solution UV light.sup.1) produced
8OHdG.sup.2) ultrapure water.sup.3) Ultrapure (-) 0.131 1.00 water
(+) 1.049 1.00 City water (-) 0.342 2.61 (+) 12.561 11.97 Processed
(-) 0.157 1.20 water (+) 0.584 0.56 Natural (-) 0.091 0.69 water
(+) 0.516 0.49 .sup.1)(+) indicates the case where irradiation with
UV light was performed and (-) indicates the case where no
irradiation with UV light was performed but left to stand at room
temperature for 90 minutes. .sup.2)Each measured value is an
average of measured values of two measurements. .sup.3)A ratio of
each numeral value assuming the value of produced 8OHdG with
ultrapure water as 1 is shown.
[0173] From the above results, the following conclusion can be
obtained.
[0174] The oxidizing power that city water has is judged to be very
rational in view of sterilization and an antibacterial
property.
[0175] The water processed through a water purifier (processed
water) markedly inhibited induction of 8OHdG and the induction of
8OHdG when irradiated with UV light is performed is at a low value
as compared with the case of standing at room temperature.
[0176] Natural water significantly inhibits the induction of 8OHdG
in both cases of standing at room temperature and irradiation with
UV light.
[0177] As stated above, according to the present invention, the
reducing ability (antioxidant ability) or oxidizing ability of
water can be quantified. Using this, correlation between drinking
water and various diseases (severity, progress of disease,
therapeutic effect, preventive effect, etc.) can be studied. In
addition, gene injury induction effect of an aqueous solution
containing an unknown substance can be comprehensively
evaluated.
EXAMPLE 11
[0178] Progressive urine collected from a healthy person was mixed
with an antioxidant preservative solution comprising the mixed
solution containing 10% Gly, 2.5% Meth and 1 mM EDTA described in
Example 8 in a volume ratio of 1:1 and quickly stored by
refrigeration (about -20.degree. C). Immediately before tests, the
stored mixed solution concerned was thawed and perfused (1
.mu.l/minute) through micro dialysis treatment using a dialysis
membrane having a function of cutting off a molecular weight of 50
kD. Low molecular weight DNA-related components (recovery being
about 30 to 40%) extracted by this treatment were subjected to
quantification of dG and 8OHdG in the same manner as in Example 1.
In addition to the quantification, nitrate ion (No.sub.3.sup.-)
contained in the same test sample was quantified by use of a high
sensitivity nitrogen oxides detector (ENO-10, manufactured by Eicom
Corporation).
[0179] The results obtained are shown in the graph in FIG. 2. The X
axis in the graph indicates the ratio (NO.sub.3.sup.-/dG) of the
detected NO.sup.3- concentration (.mu.mol/l) and dG concentration
(ng/ml) and the Y axis indicates logarithm of the ratio (8OHdG/dG)
of the detected 8OHdG concentration (pg/ml) and dG concentration
(ng/ml). Also, since the concentration of dG in urine correlates to
the concentration ratio of urine, errors in data attributable to
difference in the concentration ratio of urine could be excluded by
dividing the respective concentration values of NO.sub.3.sup.- and
8OHdG by the concentration value of dG. Further, by expressing the
risk of oxidation injury of gene (Y axis) in terms of oxide type
(8OHdG) /reduced type (dG), a more rational index can be obtained.
For reference, Table 11 shows approximate concentrations of 8OHdG
and dG in biological substances detected by the same measurement
apparatus as described above.
13TABLE 11 8OHdG dG Biological sample concentration concentration
Human urine 10-5000 pg/ml 50-2000 ng/ml Human plasma 1-50 pg/ml
5-25 ng/ml Human cerobrospinal fluid 2-15 pg/ml 50-200 ng/ml Mouse
brain tissue 20-50 pg/g 200-1000 ng/g Mouse testis tissue 50-100
pg/g 2000-5000 ng/g
[0180] From the results shown in FIG. 2, it is apparent that the
logarithm of 8OHdG/dG and NO.sub.3.sup.-/dG ratio are positively
correlated (p<0.05). This means that the logarithm of 8OHdG/dG
is in a positive correlation with the concentration of nitrate ion,
a typical oxide in produced in vivo, and that the logarithm of
8OHdG/dG can be an index for in vivo oxidation stress.
[0181] Although data are not shown, the studies thus far shows the
followings.
[0182] In patients with progressive cancers and congenital gene
aberration, the value of 8OHdG/dG is shifted upwards from the
approximation line plotted on healthy person (i.e., 8OHdG/dG ratio
that presumably more strongly reflects oxidation injury of gene
shows a relatively higher value than oxidation index on whole
individual expressed by NO.sub.3.sup.-/dG ratio).
[0183] In persons who smoke, there is the tendency that both
8OHdG/dG ratio and NO.sub.3.sup.-/dG ratio show high values.
[0184] Even in the same individual, when in extraordinary
conditions (for example, hangover, cold, after excessive exercise,
etc.), the values of 8OHdG/dG and NO.sub.3.sup.-/dG ratio are
shifted increasing in the upper right direction.
[0185] From the above results, the following conclusion can be
obtained.
[0186] 8OHdG/dG value can be an important index of in vivo
oxidation.
[0187] The value of 8OHdG/dG varies depending on the daily living
(eating, exercises, rest, etc.) or the like of a subject and can be
an index for stress in a broad sense as including psychosocial
factors.
[0188] By quantifying influences of natural and artificial
chemicals and foods periodically taken by living organism on these
indices, harmfulness (carcinogenicity, teratogenicity, reproduction
toxicity, etc.) or usefulness (therapeutic or preventive effects
for various diseases caused by in vivo oxidation or oxidation
injury of gene nucleic acid, refreshing effect, aging preventing
effect, etc.) of the natural and artificial chemicals and foods can
be rationally evaluated.
[0189] Measurement limit of 8OHdG is 0.5 pg/ml (sample amount 50
.mu.l), which is much superior to the measurement limit of 1 ng/ml
for the conventional EIA method. Also, measurement limit of dG is
0.2 ng/ml (sample amount 50 .mu.l).
EXAMPLE 12
[0190] The following three experiments were conducted on city
water, alkali ion water and deep sea salt mineral water and their
usefulness was evaluated.
[0191] Experiment 1 Quantification of Nitrogen Oxides
[0192] In order to measure the contents of nitric acid-form
nitrogen and of nitrous acid-form nitrogen, the nitrogen oxides in
the above three samples were quantified. High nitrogen oxides
content indicates strong contamination with exuded fertilizer
components, excrements, sewage, etc. in past and is prescribed to
be 10 ppm or less in the water quality standard. Also, it is known
that when infants (6 months old or less) take up water containing
nitrogen oxides in high concentrations, they suffer
methemoglobinemia and their respiratory action is inhibited.
[0193] Measurement was conducted by use of a high sensitivity
nitrogen oxides analyzer (manufactured by Eicom Corporation,
ENO-10) that detects NO.sub.3 and NO.sub.2 simultaneously from a
minute amount of sample, and after separating NO.sub.3 and NO.sub.2
from each other by use of a reducing column, color developed by
diazo coupling reaction was detected by absorbance at 540 nm.
[0194] The results obtained are shown in the following table.
14 NO.sub.2.sup.- NO.sub.3.sup.- Area Concentration Area
Concentration (mVs) (ppm) (mVs) (ppm) City water 1.55 0.0016
3097.88 7.4009 Alkali ion water 3.46 0.0036 2525.77 6.0341 Deep sea
salt 4.87 0.0050 3265.38 7.8011 mineral water
[0195] As a result of experiments, each sample was in the range of
the water quality standard for city water.
[0196] Experiment 2 Measurement of Redox Potential
[0197] Measurement of redox potential was conducted on the above
three samples. Also, for control, experiment was also conducted on
ultrapure water.
[0198] The results obtained are shown in the following table.
15 ORP (mV) Ultrapure water 274.6 City water 695.2 Alkali ion water
-112.4 Deep sea salt 120.5 mineral water
[0199] Antioxidant ability was high in the order of alkali ion
water>deep sea salt mineral water>city water.
[0200] Experiment 3 Experiment of Induction of Gene Oxidation
Injury
[0201] The ability of inducing oxidation injury of gene was
measured on the above three samples. In the measurement, samples
were added to glass tubes containing a solution of dG in a known
concentration and UV light was irradiated for a predetermined time
(0 (=immediately after), 0.5, 1 and2 hours) to oxidize dG.
Thereafter, the reaction was terminated with a reaction terminating
solution and dG and its oxide, 8OHdG, were separated by high
performance liquid chromatography to quantify dG and 8OHdG in the
same manner as in Example 1. Also, for control, experiment was also
conducted on ultrapure water.
[0202] The results obtained are shown in the following tables.
16 Time (hr) 8OHdG (mVs) dG (mVs) 8OHdG/dG .times. 10.sup.4
Ultrapure water 0 0.2534 1113.75 2.28 0.5 0.4563 1151.98 3.96 1
2.2136 1157.67 19.12 2 5.5788 1203.49 46.36 City water 0 1.8889
1101.03 17.16 0.5 3.8958 1127.45 34.55 1 3.8858 1145.67 33.92 2
2.0812 1201.46 17.32 Alkali ion water 0 0.1051 1107.94 0.95 0.5
0.7522 1114.67 6.75 1 0.9715 1120.04 8.67 2 1.5257 1161.09 13.14
Deep sea salt mineral water 0 0.1302 1078.80 1.21 0.5 0.5631
1117.39 5.04 1 0.5103 1125.07 4.54 2 0.9984 1157.90 8.62
[0203] From the results, the following conclusion can be obtained.
A higher 8OHdG/dG ration means a stronger effect of gene oxidation
injury.
[0204] City water exhibits the most potent effect of gene oxidation
injury in the case of 0.5 hour, and thereafter it shows a tendency
of a decrease.
[0205] As compared with ultrapure water, alkali ion water and deep
see salt mineral water both exhibit low effect of gene oxidation
injury.
[0206] Comparing the power of inhibiting the effect of oxidation
injury (antioxidant power), the order is deep see salt mineral
water>alkali ion water.
[0207] As described above, in biological evaluation method of the
present invention, the usefulness of city water, alkali ion water,
deep see salt mineral water, etc. whose composition cannot be
determined exactly can also be evaluated.
EXAMPLE 13
[0208] Three experiments were conducted on the following samples in
the same manner as in Example 12 and their toxicity was
evaluated.
[0209] Sample A: Shochu waste liquor stock solution
[0210] Sample B: electrolytically treated water obtained from
Sample A
[0211] Experiment 1 Quantification of Nitrogen Oxides
[0212] In the same manner as in Example 12, the nitrogen oxides in
Samples A and B were quantified.
[0213] The results obtained are shown below.
17 Area (mVs) Concentration (ppm) NO.sub.2-- NO.sub.3-- NO.sub.2--
NO.sub.3-- Sample A diluted 10 folds 0.80 614.32 0.0008 0.98 Sample
B diluted 10 folds 31.78 13575.81 0.0319 21.60 Sample A NO.sub.2--
0.008 ppm + NO.sub.3-- 9.8 ppm = NO.sub.x 9.808 ppm Sample B
NO.sub.2-- 0.319 ppm + NO.sub.3-- 216.0 ppm = NO.sub.x 216.319
ppm
[0214] The above results indicate the followings.
[0215] While the upper limit of the concentration of nitrogen
oxides is defined to be 10 ppm as a standard by City Water Law,
Sample A contained nitrogen oxides in an amount near the upper
limit of the standard (9.8 ppm).
[0216] Sample B contained nitrogen oxides 20 times or more the
upper limit prescribed by the City Water Law (216.3 ppm).
[0217] Experiment 2 Measurement of Redox Potential
[0218] For Sample A, Stock solution per se was used, and for Sample
B, a solution diluted to 10 ppm in terms of the concentration of
NO.sub.3.sup.- was used for measurement of redox potential
(silver/silver chloride electrode).
[0219] As a result of the measurement, the redox potential of
Sample A was 184.8 mV while that of Sample B was 715.4 mV.
[0220] Experiment 3 Experiment on Gene Oxidation Injury
[0221] 90 .mu.l of each sample was added to 10 .mu.l of dG solution
(200 .mu.g/ml) and stirred for 5 minutes. Thereafter, 100 .mu.l of
a reaction terminating solution was added and dG and 8OHdG were
simultaneously measured in the same manner as in Example 1.
[0222] The results obtained are shown below.
18 dG 200 .mu.g/mL 8OHdG/ dG (mVs) 8OHdG (mVs) dG .times. 10.sup.5
Ultrapure water 1128.40 0.0163 1.445 Sample A diluted 100 1104.00
0.0015 0.136 folds Sample A diluted 1098.00 0.0085 0.774 1000 folds
Sample B diluted 100 109.52 2.7460 2507.300 folds Sample B diluted
971.34 5.8810 605.450 1000 folds
[0223] From the above results, the followings can be deduced.
[0224] The ability of Sample A to induce gene oxidation injury is
not so high, which evidences that microbes can propagate in Sample
A.
[0225] Sample B has a toxicity strong enough to destruct dG itself
and when diluted 1,000 folds, its toxicity cannot be
neutralized.
[0226] As stated above, in the biological evaluation method of the
present invention, shochu waste liquor and shochu waste liquor
after electrolytic treatment can be evaluated for their
toxicity.
EXAMPLE 14
[0227] Treatment was conducted by adding a catalyst as indicated
below to Sample B of Example 13 to examine the content of nitrogen
oxide and a change in the ability of inducing gene oxidation
injury.
[0228] Treating conditions:
[0229] (1) Sample B+Magnetic iron ore (2 g)+TiO.sub.2 (0.5 mg)
[0230] (2) Sample B+Magnetic iron ore (2 g)+C (0.5 mg)
[0231] (3) Sample B+Magnetic iron ore (2 g)+TiO.sub.2 (0.5 mg)+C
(0.5 mg)
[0232] (4) Sample B+Magnetic iron ore (2 g)+TiO.sub.2 (0.5
mg)+magnet
[0233] (5) Sample B+Magnetic iron ore (2 g)+C (0.5 mg)+magnet
[0234] (6) Sample B+Magnetic iron ore (2 g)+TiO.sub.2 (0.5 mg)+C
(0.5 mg)+magnet
[0235] (7) Sample B+Magnetic iron ore (2 g)+TiO.sub.2 (0.5
mg)+magnet+US (42 KHz, 300 W)
[0236] (8) Sample B+Magnetic iron ore (2 g)+C (0.5 mg)+magnet+US
(42 KHz, 300 W)
[0237] (9) Sample B+Magnetic iron ore (2 g)+TiO.sub.2 (0.5 mg)+C
(0.5 mg)+magnet+US (42 KHz, 300 W)
[0238] *TiO.sub.2: Titanium dioxide, C: activated carbon, US:
Ultrasonic wave
[0239] In each of brown bottles containing respective catalysts
described above, 30 ml of Sample B was added and left to stand at
room temperature for 15 minutes. Thereafter, if ultrasonic wave is
to be applied, it was exposed to ultrasonic wave for 30
minutes.
[0240] Experiment 1 Quantification of Nitrogen Oxide
[0241] The solutions treated as described above were each diluted
20 folds and quantification of nitrogen oxide was performed in the
same manner as in Example 12.
[0242] The results obtained are shown below.
19 Magnetic Concentration iron ore TiO.sub.2 C Magnet US NO.sub.3
area (mVs) of NO.sub.3 (ppm) Sample B 4338 189 Treatment (1) + +
4217 183 Treatment (2) + + 4128 179 Treatment (3) + + + 4003 174
Treatment (4) + + + 3885 169 Treatment (5) + + + 3895 169 Treatment
(6) + + + + 3670 159 Treatment (7) + + + + 3962 172 Treatment (8) +
+ + + 3905 170 Treatment (9) + + + + + 4155 181
[0243] From the above results, the following conclusion can be
obtained.
[0244] The concentration of NO.sub.3 decreased as compared with the
electrolytically treated solution of Sample B under any one of the
treating conditions (1) to (9).
[0245] TiO.sub.2 and activated carbon gave more improved reduction
efficiency in NO.sub.3 when used in combination than when used
alone.
[0246] Addition of a magnet further improved the efficiency of
reduction in NO.sub.3.
[0247] Application of ultrasonic wave rather showed a tendency of
an increase in NO.sub.3. This is presumed to be attributable to
occurrence of various chemical reactions including oxidation
reaction by application of ultrasonic wave.
[0248] Condition (6) gave the highest efficiency of reduction in
NO.sub.3, in which NO.sub.3 is decreased by about 16% as compared
with Sample B (189 ppm.fwdarw.159 ppm).
[0249] By studying conditions such as amount of catalyst, the
efficiency of reduction in NO.sub.3 is supposed to be further
increased.
[0250] Experiment 2 Gene Oxidation Injury Test
[0251] Gene oxidation injury test was conducted on Sample B and a
solution obtained by performing treatment (9) to Sample B and a
change in the ability of inducing gene oxidation injury was
observed.
[0252] In the test, 10 .mu.l of dG (200 .mu.g/ml) was added to 90
.mu.l of the sample and the mixture was stirred for 5 minutes.
Thereafter, 100 .mu.l of a reaction terminating solution was added
thereto and dG and 8OHdG were simultaneously measured in the same
manner as in Example 1.
[0253] Each of the results obtained in the case of performing the
catalyst treatment for 2 days and the case of performing the
catalyst treatment for 1 month is shown below.
20 [Catalyst treatment for 2 days] dG 200 .mu.g/mL dG (mVs) 8OHdG
(mVs) Ultrapure water 1054 0.010 Sample B stock solution 0.29 nd
Sample B diluted 10 folds nd 32.820 Sample B diluted 100 folds
14.28 nd Sample B diluted 1000 folds 1008 2.045 Sample B diluted
10000 folds 1108 nd Catalyst (9) of sample B 2.82 nd stock solution
Catalyst (9) of sample B 946 0.135 diluted 10 folds Catalyst (9) of
sample B 1135 0.060 diluted 100 folds Catalyst (9) of sample B 1112
0.053 diluted 1000 folds
[0254]
21 [Catalyst treatment for 1 month] dG 200 .mu.g/mL dG (mVs) 8OHdG
(mVs) Ultrapure water 1101.67 1.97 Catalyst (9) of Sample B 1040.27
4.94 stock solution Catalyst (9) of Sample B 1138.57 4.09 diluted
10 folds Catalyst (9) of Sample B 1094.50 5.89 diluted 100 folds
Catalyst (9) of Sample B 1087.78 2.70 diluted 1000 folds
[0255] The above results indicate the followings.
[0256] Sample B has a toxicity strong enough to destruct dG itself
and it is necessary to dilute it 10000 folds or more in order to
neutralize its toxicity.
[0257] The solution obtained by treating Sample B according to the
treatment (9) showed a drastic reduction in its toxicity when
diluted at least 10 folds. Therefore, it is highly possible that
the treatment (9) can detoxicate the biotoxicity of the
electrolytically treated water.
[0258] By continuing the treatment according to the treatment (9)
for 1 month, the toxicity of the solution was drastically
decreased.
[0259] Therefore, it is understood that the treatment according to
the treatment (9) proceeds with time.
[0260] As described above, the biological evaluation method of the
present invention can provide an assist to determine an optimal
treating method by evaluating toxicity of solutions treated by
various methods.
EXAMPLE 15
[0261] Both quantification of nitrogen oxides and gene oxidation
injury test were performed on each of industrial wastewaters
subjected to the following treatments and the results of wastewater
treatments were examined.
[0262] Sample C: Industrial wastewater electrolytically treated on
platinum electrode
[0263] COD at completion of the treatment: 350 ppm
[0264] Concentration of hypochlorous acid at completion of the
treatment: 3,013 ppm
[0265] Sample D: Industrial wastewater electrolytically treated on
platinum/iridium electrode
[0266] COD at completion of the treatment: 2,600 ppm
[0267] Concentration of hypochlorous acid at completion of the
treatment: 1,560 ppm
[0268] Experiment 1 Quantification of Nitrogen Oxides
[0269] After diluting Samples C and D at 100 folds, respectively,
the nitrogen oxides in Samples A and B were quantified in the same
manner as in Example 12.
[0270] The results obtained are shown below.
22 Concentration Area (mVs) (ppm) NO.sub.2.sup.- NO.sub.3.sup.-
NO.sub.2.sup.- NO.sub.3.sup.- Sample C diluted 100 folds 1.99
2158.65 0.00216 2.6269 Sample D diluted 100 folds 20.65 3707.87
0.02245 4.5122
[0271] Experiment 2 Gene Oxidation Injury Test
[0272] Gene oxidation injury test was conducted on Samples C and D
and a change in the ability of inducing gene oxidation injury was
observed.
[0273] In the test, 10 .mu.l of dG (200 .mu.g/ml) was added to 90
.mu.l of each sample and the mixture was stirred for 5 minutes.
Thereafter, 100 .mu.l of a reaction terminating solution was added
thereto and dG and 8OHdG were simultaneously measured in the same
manner as in Example 1.
[0274] The results obtained are shown below.
23 dG 200 .mu.g/mL 8OHdG 8OHdG 100 ng/mL dG (mVs) (mVs) (mVs)
Ultrapure water 1114.2 0.16 9.72 Sample C stock solution 0.35 0.02
nd Sample C diluted 10 folds 0.04 0.72 4.88 Sample C diluted 100
folds 687.3 2.19 0.04 Sample C diluted 1000 folds 1149.8 2.01 0.42
Sample C diluted 10000 folds 1095.7 2.88 8.81 Sample C diluted
100000 folds 1116.3 3.67 8.83 Sample D stock solution 1085.5 4.90
9.21 Sample D diluted 10 folds 1096.1 4.36 6.70 Sample D diluted
100 folds 1078.1 4.60 8.50 Sample D diluted 1000 folds 1094.5 3.72
9.42 Sample D diluted 10000 folds 1081.4 3.94 9.74 Sample D diluted
100000 folds 1076.1 2.98 9.00
[0275] From the above results, the following conclusion can be
obtained.
[0276] The stock solution of Sample C has a very strong toxicity.
In order to neutralize the toxicity, it is necessary to dilute it
10000 folds or more.
[0277] The toxicity of Sample D mostly disappeared. The change of
treating conditions would result in a considerable difference from
Sample C.
[0278] As state above, according to the biological evaluation
method of the present invention, the usefulness of treating method
used can be evaluated by testing the solution after the
treatment.
EXAMPLE 16
[0279] To examine the correlation between milk nutrition of infants
and 8OHdG excretion amount in urine, tests were conducted in the
following procedure.
[0280] First, with respect to infants of 1 to 3 months after birth,
they were grouped into those who are grown mainly with mother's
milk (n=6), grown mainly with powder milk (n=8), and grown with
mixed nutrient (n=6). Urines progressively collected from these
infants were freeze-preserved and the concentration of 8OHdG
therein was measured by use of an 8OHdG enzyme immune antibody
(EIA) measuring kit (manufactured by Nippon Oil & Fats Co.,
Ltd., measurement sensitivity>1 ng/ml). Further, 100 .mu.l of
same frozen sample after thawing was added to a preservation tube
containing 200 .mu.l of an antioxidant preservative solution
comprising 40% of glycerol, 10% of methanol, and 2 mM EDTA to
prepare a mixed solution of test urine and the preservative
solution. Then, in the mixed solution, a micro dialysis system (50
KD cut off cellulose membrane, membrane length 10 mm, manufactured
by Eicom Corporation) was used to flow a perfusion solution (20%
glycerol solution) at a rate of 1 .mu.l/minute (37.degree. C.) and
the recovered perfusion solution was ice cooled. The recovery (30
to 40%) of the standard solutions of 8OHdG and of dG under the same
condition was confirmed for every probe of micro dialysis in
advance. 10 .mu.l of the perfusion solution was automatically
dispensed into a 8OHdG/dG simultaneous measurement system as used
in Example 1 by use of an automatic dispenser (4.degree. C.). The
mixed solution was diluted 10 folds with 100% methanol solution and
protein component was precipitated by centrifugation. The
concentration of nitrate ion in the supernatant liquid thereof was
measured by use of a high sensitivity nitrogen oxides measurement
apparatus (ENO-10, manufactured by Eicom Corporation).
[0281] The results obtained are shown in FIGS. 3 to 5.
[0282] FIG. 3 indicates the followings.
[0283] A positive correlation with a correlation coefficient of
R.sup.2=0.767 was observed between the ECD method and the EIA
method.
[0284] In the results obtained this time, the measurement values by
ECD were relatively higher than the measurement values by EIA.
Presumably, this is because there was a time lag of several months
from collection of analytes to addition of a preservative solution
thereto, which time lag have induced spontaneous oxidation of the
analytes.
[0285] Further, FIG. 4 indicates the followings.
[0286] A positive correlation with a correlation coefficient
R.sup.2=0.627 was observed between the concentrations of 8OHdG and
of NO.sup.3-.
[0287] Generally, the concentration of NO.sup.3- in urine is
considered as an index for in vivo oxidation stress. Therefore, in
consideration of the fact that the positive correlation was
observed between the concentrations of 8OHdG and of NO.sup.3-, it
is suggested that the concentration of 8OHdG is also useful as an
index for in vivo oxidation stress in general.
[0288] Further, FIG. 5 indicates the followings.
[0289] The amount of 8OHdG excreted in urine was significantly
higher in the powder milk group than in the mother's milk group.
However, the excretion amount of dG was also high, so that there
was no difference in 8OHdG/dG ratio among the groups.
[0290] Recently, reports have been made which focus only on the
amount of 8OHdG excreted in urine when evaluating the health of
subjects. However, the results obtained this time indicate that
total amount of nucleic acid excreted in urine varies greatly
according to the composition of nutrient taken up and hence the
amount of 8OHdG excreted in urine could not be a rational index for
gene oxidation injury unless the amount of excreted dG (generally
about 10 times that of 8OHdG) is simultaneously taken into
consideration.
[0291] In this connection, the measurement apparatus according to
the present invention enables one to always perform simultaneous
measurement of 8OHdG and dG in the same sample at the same time, so
that it has a great advantage over other measurement methods (such
as EIA method and LC-MAS method).
[0292] Industrial Applicability
[0293] As described in detail above, the biological evaluation
method for evaluating natural and artificial chemicals by use of a
DNA injury indey according to the present invention enables one to
evaluate biological toxicity, usefulness, or safety, etc. of test
substances such as natural or artificial chemicals or foods in
vitro in a very simple manner and at low costs, and thereafter to
perform evaluation thereof in vivo, for example cultured cells or
in animals depending on importance of the test substances.
[0294] It is virtually impossible to conduct conventional
full-scale animal experimentation or clinical experimentation for
each of artificial chemicals since over 100,000 kinds of these
exist even when only principal ones thereof are counted. Therefore,
the method of the present invention that can provide a specific DNA
injury index in a simple manner for each chemical can provide very
useful information in establishing a broad safety standard of
concentration or in considering necessity of full-scale animal
experimentation or clinical experimentation. Therefore, the
biological evaluation method of the present invention provides a
means for simply screening biological toxicity of various natural
or artificial chemicals or harmfulness/usefulness of foods, and so
forth.
[0295] The method of the present invention can objectively quantify
usefulness and harmfulness of health food or functional food on the
market or under currently development. That is, according to the
present invention, not only evaluation of usefulness, safety, etc.,
which must necessarily be conducted for foods currently under
development can be reliably and easily performed but also
reconfirmation of usefulness and safety of the foods currently on
the market can be made.
[0296] Further, also the influence of UV light irradiation to a
substance (test substance) and the influence of coexisting active
oxygen species generator or gene oxidation injury inducing
substance on that substance can be simply evaluated according to
the present invention, so that the toxicity, etc., of test
substance in nature can be more accurately evaluated.
[0297] In the method of the present invention, using an aqueous
solution containing an unknown substance as a test solution, the
gene injury inducing effect of the aqueous solution can
comprehensively evaluate; therefore, biological toxicity index for
city water, natural water, or the like, whose chemical composition
is not clear, can be obtained accurately and easily.
[0298] According to the present invention, harmfulness or
usefulness of a chemical or food can be accurately and easily
evaluated based on the ratio of contents of
8-hydroxy-2'-deoxyguanosine and of 2-deoxyguanosine in products
derived from living organism cells, such as urine, blood, etc.
collected from animals including humans administered with a
chemical or a food.
[0299] The high measurement sensitivity and reliability of
measurement results by the measurement apparatus of the present
invention are most advantageously exhibited when all of the
antioxidant preservative solution of a biological sample (in
particular nucleic acid component), deproteinization treatment
under antioxidative environment (application of a micro dialysis
method), and the 8OHdG/dG simultaneous measurement system of the
present invention are combined, and a wholly new measurement
apparatus capable of measuring DNA oxidation injury index for
almost all biological samples can be provided. That is, oxides of
nucleic acid that can be detected by an electrochemical detector,
for example, 2OHdA that causes conversion of from A:T into G:C in
the same manner as 8OHdG and its reduced form, dA, can be
simultaneously detected under substantially the same measurement
conditions as in the case of 8OHdG/dG.
[0300] The micro dialysis treatment used in the measurement
apparatus of the present invention is applicable to either of
liquid samples and cell-containing samples. Since no acid, alkali
or enzyme reaction is used for deproteinization treatment and all
the operations can be carried out at low temperatures, analytes are
susceptible to less deterioration and since extraction efficiency
of DNA related substances depends only on recovery from the
dialysis membrane, there is also an advantage such that error can
be reduced by using constant perfusion conditions. Further, when
the perfusion solution to be used is the same as the preservative
solution, a long term storage of analytes after the perfusion is
possible.
[0301] According to the 8OHdG/dG simultaneous measurement method of
the present invention, nucleosides of non-oxidized and of oxidized
types can be simultaneously quantified on the same sample, and data
processing of the both data as a ratio can exclude or reduce error
factors such as dispensing amount of analytes. In addition, cost
efficiency in service can be greatly improved because measurement
is possible even when the sample injection amount at a time is
extremely small at 10 to 100 .mu.l; useful life of the column can
be extended since the solution mainly injected into the column is a
dialysis membrane perfusion solution and thus measurement noise is
small and a higher measurement sensitivity than that attained in
the prior art can be expected, and the power saving of the system
is possible by connecting to an auto injector equipped with a
constant temperature cooler, since samples to be measured are
stable at low temperatures.
[0302] The antioxidant preservative solution of the present
invention can substantially inhibit oxidation of samples that tend
to start oxidizing immediately after their collection during
preservation, in contrast to conventional simple freezing or
cooling preservation, in which there is the fear that the
concentration of oxides in biological may become even higher. Since
it is adjusted so as to minimize variation in concentration of, in
particular, nucleoside derived from nucleic acid, and oxides
thereof, it is most suitable for the preservation of analytes whose
DNA oxidation injury indices are to be measured. Further, the
antioxidant preservative solution is of relatively simple
composition and hence it is inexpensive and the included components
are stable, so that it can be preserved for long time.
* * * * *