CA2125778A1 - Dna biomarkers of cancer and genotoxic injury - Google Patents

Abstract

A method for diagnostic and prognostic monitoring of genotoxic changes in tissue arising from oxidative modifications of the genus consisting of the DNA nucleotide bases containing purine or pyrimidine or structurally or metabolically similar surrogates by analyzing the altered bases or surrogates and comparing those results to baseline levels. [See In re Harmisch, 206 USPQ 300 (1980)].
This method is directed towards diagnosing or predicting the occurrence of neoplasia (cancerous growth), assaying the impact of genotoxic effects brought about by exposure to environmental chemicals, and tracking the effects of such modifications over time in response to oxidative repressing agents.

Description

W093/12258 2 1 2 ~ 7 7 8 P~T/US92/10669 DNA BIOMARKERS OF CANCER AND GENOTOXIC INJURY

FIELD OF THE INVENTION

This invention relates to the field of detection and assessment of oxidatively mcdified DNA nucleotide bases or their functional equivalents in eukaryotic systems and more particularly to the comparison of levels of such modified molecules to normal levels in eukaryotic systems as a means to determine genot~xic effects of environmental influences.

- SUBSTITUTE SHEET

2 1 2 ~ 7 7 8 PCT/US92/10669 DESCRIPTION OF THE RELATED ART

There is abundant evidence to indicate that reactive oxygen species, formed in the body as a consequence of aerobic metabolism, can produce damage to somatic cells (reviewed in Ann. Rev.
Pharmacol. Toxicol. 25, 509-528 (1985), Ciba Found. SvmP. 67, 301-328 (1978), Science 221, 1256-1264 (1983~). The reduction of ` molecular oxygen in all aerobic eukaryotic cells results in the formation of intermediates that are highly toxic. These include .
the superoxide ion (2~ H2O2 and OH- While O- and H O
individually may not be particularly damaging, their combined action leads to the formation of the highly reactive OH:
2 + H2O2 ---> OH + OH-+ 2 This reaction can be relatively slow; however, when catalyzed by metal ions [e.g., Fs+2], it is substantially accelerated and becomes e8pecially relevant in the initiation of biological damage (Science 24Q, 640-642 (1988)). H2O2 itself is converted to OH through the iron (Fe~2)-catalyzed Fenton reaction (Science 240, 640-642 (1988)).,The proliferation of OH may then reæult in an attack on most molecules in living cell~s with deleterious consequences (,S~cience 227, 375-381 (1985)). The primary defense again6t such radical-induced damage i8 provided by enzymes that catalytically scavenge the intermediates of oxygen reduction and by antioxidants, such as glutathione. For example, 2- iS
2S eliminated by superoxide dismutase (SOD) which catalyses a dismutation reaction leading to the formation of 2 and H2O. In W093/12258 212 ~ 7 7 ~ PCTIUS92/10669 addition, the latter structures are destroyed by catalases and glutathione peroxidase (Ann. Rev. Pharmacol. Toxicol. 25, 509-528 (1985)).
This type of damage may be an important factor in the etiology of cancer and other genetically-related diseases (Science 221, 1256-1264 (1983), Nature 327, 77-79 (1987)).
However, a major factor that hamper~ a clear understanding of the 8ignificance of reactive oxygen species in biological injuries is a lack of adequate information on the critical cellular targets involved (e.g. DNA). Obviously, DNA is critical because of the pivotal role that it plays in information transfer between generations of somatic cells.
In response to this desire to provide a method for determining the role of oxidative modification in genetically related disea6es, research has been conducted relating to DNA
lesion~. A number of DNA lesions have been identified, mostly wlth in vitro systems, and putatively associated with the interaction of the hydroxy radical (-OH) with nucleotide deriva-tives. The~e include thymine glycol, 5,6-dihydroxythymine ~Journ. Biol. Chem. 264(22), 13025-13028 (1989)) and 8-hydroxy-deoxyguanos~ne (8-OH-dG) (Nature 327, 77-79 (1987), Biochem. J.
238, 247-254 (1986), Carcinoaenesi~ 8, 1959-1961 (1987)).
Moreover, a clear advance in understanding the association between OH-induced modifications in DNA and carcinogenesis, for example, was indicated when it was shown that 8-OH-dG was mi6read in a DN~ synthesis system in vitro with E. coli (Nature 327, ~ 12g 7 ~8 PCT/US92/10669 77-79 (1987)). In fact, the presence of the 8-OH-dG in DNA was viewed as ... an important cause of mutation and carcinogenesi~"
(Nature 327, 77-79 (1987)). These examples represent a growing body of evidence broadly implicating the OH in DNA injury (Science 227, 375-381 (1985)).
The inventor has been exploring the relationship between the generation of the O~ and the occurrence of disease conditions.
In a 1983 report (Environ. Sci._Technol., 17: 679-685 (1983)), co-authored by the inventor, it was shown that free radicals are produced in the neoplastic livers of ~nglish sole from polluted environments. Further support for this finding was provided in subsequent publications (Aquat. Toxicol., 6: 87-105 (1985), Marine Environ. Res. 17: 205-210 (1985)). ~oreover, it was ulti-mately shown that free radical damage to the DNA had occurred in the tumor-bearing liver~ of the exposed fish ~Aquatic Toxicol., 11, 43-67 (1988)). The DNA alterations appeared to involve the formation of aromatic adducts and the free radical portion of the aromatic molecule was likely to have been initiated by a reaction involving the OH. Thu~, in the early 1980s, some indication exi8ted about the involvement of the OH in DNA modification and attendant biological damage.
More recently, the inventor narrowed his research to determine the presence of a DNA modified nucleotide base -- 2,6-diamino-4-hydroxy-5-formamidopyrimidine or FapyGua. Using liver tissue of five English sole bottomfish that were exposed in the wild to carcinogenic chemicals, the inventor excised the hepatic WO93/12258 2 1 2 ~ 7 7 8 PCT/US92/10669 tumors together with the surgical margin tissue. In this study, the tumor tissues, but not the surgical margin, showed substan-tially elevated levels of FapyGua, thus indicating that reaction of the ~OH had resulted in this nucleotide base modification. It was not obvious, however, from this limited study whether other modifications existed in the DNA or, indeed, whether the FapyGua or any other DNA lesion that may exi~t would ~erve a diagno~tic or prognostic role in relation to the causation of genetically~
related patholo~y or disease.
Thus, a need existed to determine whether the observed damage to the DNA nucleotide bases would serve as a sentinel or biomarker for diagnosing or predicting the existence or likely onset of pathology or genetically related disease in living systems.
Heretofore, no method existed that could demonstrate that oxidative damage to the nucleotide bases of DNA could be 6tructurally and quantitatively elucidated in tissues an body fluids in relationship to the diagnosis and prediction of genetically altered pathologic conditions or diseases, such as cancer. As a consequence of the methods described in this pstent, an invention is disclosed that for the first time identifies and quantifies biochemical changes in livin~ systems, or alterations in these systems though exposure to environmental chemicals, that result in the modification of DNA nucleotide-like molecules. In addition, this invention provides methods to W~ 7 ~ PCT/US~2/10669 demonstrate ~hat DNA modifications in chemically-exposed living systems are related to pathological alterations in cells.

WO93/12258 2 1 ~ 5 7 7 8 PCT/USg2/~0669 SUMMARY OF THE INVENTION

The present invention provides methods for determining oxidative modifications to DNA nucleotide bases or their functional equivalents in relation to their si~nificance as biomar~ers or sentinels for the diagnostic and prognostic monitoring of genotoxic changes in tissues, body fluids, and cell cultures of eukaryotic systems that are associated with pathologic or disease conditions. The modifications of DNA bases may occur through disruptions in normal biochemical processes in vivo (e.~., alterations in enzyme activities), or through the influence of chemical exposures, such as those that occur through the acquisition of toxic substances from the environment.

Through the use of Gas Chromatography/Mass Spectrometry with Selected Ion Monitoring (GC/MS-SIN), methods have been discovered whereby quantities of altered DNA nucleotide bases from subject tis~ues can be assessed and compared to base-line or normal lovel~ thereby providing important information about the condition of the test tissues or subject as detailed in this ~pocification. The ba6is of the disclosed methods stems from the ~nventors discovery that the mechanism for the hydroxyl radical ~-OH) attack on nucleotide bases in vivo forms specific radical base8 which have a unique and quantifiable structure. This di8covery, in conjunction with the discovery that in vivo alterations of these base~ are inextricably linked to neoplastic ,r~ "~, "~ " ;~ "~ "~ ,~

WO93/12258 PCT/US92/10~69 21ZS~8 and pre-neoplastic tissues provides the foundation for this patent.

Therefore, one object of this invention is to provide a method for determining the presence and quantity of altered DNA
nucleotide bases. By providing for this determination, a comparison can be made between these levels and those obtained from a baseline. By applying these findings with the disclosed knowledge that elevated levels of altered nucleotide ba6es are an indicator of genotoxic changes in tissue and body fluids, an evaluation can be made relating to the presence or likelihood of pathologic or disease conditions of that tissue or body fluid.
As a corollary, it i8 also an object of this invention is to provide a method whereby a 6ubject is administered structurally equivalent "surrogate" compounds (biomarkers) for diagnosing or lS predicting the occurrence of genotoxic effects brought about by exposure of animal or plant systems to environmental chemicals.
A further object of this invention is to provide a method whereby the presence and quantity of such altered nucleotide bases or biomarkers can be used to assay the degree of exposure of a 8ystem to external chemicals and the rate and degree of decline in 8uch alterations upon introduotion of therapeutically-applied antioxidants or other radical trapping agents.
Because the invention reIates to the presence and quantification of altered DNA nucleotides or biomarkers, the method of determining such alteration is not limited to those employed by the inventor. Other methods such as the use of ~ WO93/12258 212 $ 7 7 8 PCTJUS92/10669 monoclonal or polyclonal antibodies, or adducts utilizing uniquely identifiable labels are equally plausible, depending upon the needs of the analyst.

:

wo 93/i~2s8 rCr,~uss2/lo~i,6~i - BRIEF DESCRIPTION OF THE DRAWINGS

Fi~ An explanation is given for the formation of the cleav-age product of adenine (FapyAde) resulting from the attack of the OH at C-8 of the purine ring.

Fia. 2: Elevated concentrations in 8-hydroxyguanine in DNA are shown in neoplastic hepatic tissue of feral fish, compared to normal tissue.
1~
Fia. 3A: Elevated concentrations of 8-hydroxyadenine in DNA are ~hown in neoplastic hepatic tissue of feral fish, compared to normal tissue.

Fia. 3B: Elevated concentrations of 4,6-diamino-5-formamido-pyrimidine (FapyAde) in DNA are shown in neoplastic tissue of feral fish, compared to normal tissue.

F~g. 4: Compari60ns are made between English sole from "clea~'~
referenco areas and fish with normal livers from a mildly pollut-ed area having microscopically normal livers. These are also compared to the DNA lesions found in tumor tissue from fish living in a heavily polluted area.

Fig. 5: Comparisons of invasive ductal carcinoma tissue to normal DNA from calf thymus indicate that substantial changes in , ' 10 .. WO93/1225X 212 5 7 7 8 PCT/US92/10669 the nucleotide bases have taken place in the cancer tissue as a result of the attack of the OH on the base structures.

,,~

.' ~ .

WO93/12258 PCT/US92/106~.9 ~ ~2~
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention provides for a method to quantitative-ly and structurally identifying oxidatively modi~ied nucleotide bases or their functional equivalents, and the effect that such modified nucleotide bases have on the ability of the DNA to euccessfully act to self-replicate or the implications arising from alterations to functional equivalents. To better understand the preferred embodiments, it is helpful to understand the events leading to this invention. Therefore, the following is a brief discus~ion of the research conducted in conjunction with this ~nvention.
Studies published in 1983 on the presence of free radicals in the livers of English sole with liver tumors ~rom polluted environments of Puget Sound, Washington, demonstrated that the formation of free radicals was associated with the pregence of the liver tumors. The radicals were aromatic in nature; and appeared to be formed through the action of the OH. It wa6 lster established that DNA in the liver tumor tissues had been modified by the addition of a radical functional group. This group also likely arose from a reaction involving the OH.
However, these findings provided no definitive information on the biological 6ignificance of the radical interactions with the cellular biochemi~try, but did established that oxyradicals are associated with environmental chemical exposures in the English sole.
Pre~i~usly, W from neopla ic hep~tl~ - s~ue~ of feral W093/122~8 2 1 2 ~ 7 7 8 PCT/~S~2/10669 fish environmentally exposed to carcinogens was investigated by the inventor and it was f ound that an abnormally high concentra-tion of an oxidatively modified DNA nucleotide base was present.
The results of this research confirmed for the first time that at least one modified nucleotide ba~e -- FapyGua or 2,6-diamino-4-hydroxy-5-formamidopyrimidine -- was present in the DNA of the carcinogenic hepatic ti~sue of feral fish. The inventor hypothe-8ized that an oxygen radical, the OH, was responsible for the cleavage of the purine ring of guanine. It was further hypothe-~ized that the OH attacked the electron deficient C-8 of the purine ring, leadin~ to the cleavage product of FapyGua (Figure l). Based on the presence of thi6 DNA le~ion în carcinogenic tissue, the inventor concluded that the free radical-induced DNA
modifications were intimately associated with the neoplastic lS tis6ue. However, there was no evidence to ~upport the conclusion that the single DNA modification observed with just five fi h wa~
a precondition to tumorigenesis and not merely a product of tumorigene8is or that such an alteration would occur in other t~8~ue~ or other ~pecies. Neither was it apparent whether the ~ttack of the OH had occurred broadly, involving a number of modifications of the nucleotide bases, or indeed whether any evidence could be obtained experimentally on the occurrence of ~imilar types of modifications in the other three ba~es compris-ing DNA. Thus, any pos6ible diagnostic or prognostic value that the DNA modification may have represented was both elusive and unpredictable.

. 13 WO93/12258 PCT/US92~10~9 ~2~78 It was during this research effort that the inventor used for the first time the Gas Chromatograph/Mass Spectrograph with Selected Ion Monitoring (5CJMS-SIM) method of analysis for in vivo studies of carcinogenic tissue. Prior to this effort, the GC/MS-SIM method of analy6is had been limited to studies of modifications of normal and irradiated DNA nucleotides in vitro in an in vitro system involving DNA base damage to neutrophil~.
Ba~ed on the above described hypothesis, a second research effort was then undertaken to investigate whether other oxida-tively modified DNA nucleotides were present in hepaticneoplastic tissues of feral fish taken from a chemically polluted 8ite and absent from feral fish obtained from non-polluted sites.
By hypothesizing the method of attack of the OH on the DNA
nucleotides, the selected ions 6ignifying other potential base modifications were chosen for analysis by the GC/MS-SIM method.
The findings of the research supported the hypothesi6.
Elevated concentrations of three new modified DNA nucleo-tide8 were discovered to exist in the neoplastic tissue sample~:
FapyAde or 4,6-diamino-4-hydroxy-5-formamidopyrimidine, 8-OH-Gua or 8-hydroxyguanine, and 8-OH-Ade or 8-hydroxyadenine. Moreover, the research indicated that the tis~ue ~amples of feral fish obtained from non-polluted sites did not contain an increased level of modified nucleotides. Based on these findings, a probable mechanism for the formation of the modified nucleotides was proposed (see legend to Figure l).
Subsequent to these initial feral fish analyses, the inven-tor conducte~ further research to investigate whether apparentlynormal hepatic liver tissue of f eral f ish obtained from the same contaminated ~ite as the fish exhibiting neoplastic growth would exhibit the same elevated levels of these four modified nucleo-tide bases. The findings indicated for the first time thatincreased levels of these modified bases, with the exception of FapyAde, occurred in histologically normal liver tissues that were obtained from a tumor-bearing population. The increased levels of modified nucleotide bases were intermediate between the neoplastic hepatic tissue samples and hepatic tissue samples obtained from feral fish from non-contaminated sites.
The impact of these findings was profound: The discovery of this intermediate-concentration of altered nucleotides in histo-logically normal hepatic tissue indicated that such elevated lS concentrations, occurring prior to tumor formation, were causally l~nked to tumorigenesis and that the presence of the modified nucleotide6 were not ju~t a product of neoplastic~growth. Thus, by snalyzing histologically normal ti6sue for the presence of elevatod levels of modified nucleotide base6, one could determine prognostically whether that tis6ue wa~ likely to exhibit subse-quent histological/pathological change, such as tumorigenesis. In fact, it wa6 sugge6ted that the concentrations in the histologi-cally normal fish ~may be close to thre~hold concentrations for the development of liver cancer in the population." It was further concluded that ~...the DNA lésions appear to represent readily evinced alterations at the molecular level that are W093/12258 PcT/uss2/loF~s ' 212~8 highly relevant biomarkers for cytogenetic change (Aquatic Toxicol., 20, 123-130 (1991)).~ -To extend these findings and conclusicns to mammalian models, the inventor analyzed tissue from female breast cancer S patients using the same methods as described immediately above.
The analysis demonstrated that the same oxidative modifications present in hepatic tissue samples from feral fish, with the exception of FapyAde, were also present in substantially elevated concentrations in the breast tissue. Thus, it was concluded that whSle the cause of breast cancer remains uncertain, the presence of these modified nucleo~ides was intrinsically linked to the DNA
self-replication process which is the basis of tumorigenesis.
The results from this study, using the GC/MS-SIM method of analyzing oxidatively modified nucleotide bases represented the first time that the presence of these bases had been quantita-tively linked to the formation and presence of cancer in mammali-~n tissues.
- Sub~equent to the publication of this work (unpublished re~ults, 1991), the inventor demonstrated using the GC-MS/SIM
methodology, that 6ubstantially higher concentrations of the oxidatively derived pyrimidine base, 5-hydroxymethyluracil, existed in the DNA from the breast carcinoma tissue compared to ~hat of either the surgical margin or normal DNA from caIf thymus. It was thus demonstrated that an additional biomarker existed for at least cancer of the breast.
To further establish the link between environmental toxins WO93~12258 ~1 2 ~ 7 7 8 PCT/US92/10669 and genotoxic injury to DNA (e.g., neoplasia), the inventor conducted additional research utilizing Medaka (o. Latipes) fish chronically exposed to ground water contaminated with complex mixtures of toxic substances. By employing the same method of analysis as previously used w-th feral fish and breast cancer tissues, it wa~ found that Medaka exposed to contaminated ground water and certain of its components (e.g., trichloroethylene, a genotoxic agent) exhibited the same elevated occurrences of modified DNA nucleotides as in other studies of carcinogenic and pre-carcinogenic tissues. The research also indicated that once the environmental toxic influences were removed, the levels of modified nucleotids bases significantly decreased to virtually ~backgroundH levels (Table 1), suggesting that early proces~es of tumorigenesis are likely to be reversible. Overall, the research again established and confirmed the significant role played by the oxidative modifications in genotoxic injury to DNA in rela-tion to environmental chemical exposures.
From the foregoing, it is apparent that a method of analyzing the oxidative modification of DNA nucleotides and comparing those findings with a ba6e-line or normal level of alterations provides the analyst with important information relating to the condition of the DNA, and hence the tissue from which it was obtained.
For example, it is po6sible to correlate both the type and concentration of the DNA modifications with the development of pathologic changes in organi6ms. This is true whether the 2125~78 ~
organisms are exposed to external chemical stresses or are undergoing biochemical changes internally (e.g., through the actions of hormones) that lead to pathologic conditions or disease, as may be the case with carcinogenesis which, in ~ome cases, has no apparent external causation.
It is also possible to follow changes in the DNA modifica-tions in relation to a recovery process or the cessation of exposure--conditions which are likely to result in a decrease in the biomarker concentrations in tissue~ and body fluids, as the ~nventor hss demonstrated with Medaka as describe in an example contained in this patent.
Biopsy specimens and blood samples (e.g., to include isolated fractions, such as leukocytes) are additional examples of materials that may be subjected to the DNA base determinations and used in conjunction with hi~tolo~ic, pathologic and other ~nformation relating to disease or health status.
Moreover, ~6urrogate~ compounds (biomarkers) that metabolically mimic the essential 6tructure of the nucleotide b~8, or otherwise serve as sentinels for the threat posed to~
tho nucleotido bases from the attack of reactive oxygen co~pound8~ may be admini~tered under therapeutic or other conditions. Under these circumstances, the modifications in the surrogate compound~ may be followed as undertaken with the normal DNA.
Additionally, the use of the DNA biomarkers can be extended to "tracking" the effects of administered or dietary substances WO93/12258 2 I 2 S 7 7 8 PCT/~S92/10669 `

that have the ability to inhibit or essentially nullify the injurious effects of the oxidative injury to DNA by interacting with reactive oxygen species. such therapeutically-applied trapping agents include antioxidants (e.g., vitamin E, indoles, and glutathione).
Further, no restriction i8 made in the embodiment of this invention to confine the DNA biomarker6 to use with intact cells in living organi~ms. Clearly, an important aspect of the embodiments of this invention i~ the use of cells isolated from ~ntact living organisms, maintained in cell culture (Cell Culture, Methods in EnzYmoloaY, Vol. LVIII, Academic Pres6 ~l979)), and exposed to virtually any substance that i8 perceived aQ having or promoting an effect resulting in the formation of the DNA modifications. As implied, the types, classes and lS mixtures of substances are vast and diver6e, including soil, ~ediment, water, water surface microlayer, food chain organisms, air individual chemical6, chemical mixture~, extracts derived from biological material, drugs, pharmaceutical, carcinogenic matorials, co~metic6, food product6.
The ~ethods described in thi~ invention are clearly an important tool in establishing risk with regard to a variety of ` gonotoxic change~. Given that cancer, for example, is initiated at the level of DNA where oxidative base modifications are known to occur, DNA modifications a6certained by the present methodolo-2S gy would provide an early warning or prognosis of the likelihoodof future pathologic changes. In this same context, the opportu-: ', 19 2 nlty exists for assessing risk from both exposure to toxic substances and internal ~iochemical changes, such as regulated by genetic makeup (e.g., hormone effects). In that DNA is a compo-nent of all living systems, no restrictions are placed on whether the materials analyzed are from the animal or plant kingdoms.
The following is an example of the preferred method for employing the GC/MS-SIM methodology when identifying and quantifying altered DNA nucleotide bases or biomarker~. This method was employed by the inventor in much of his research.
Approximately 70 mg of tissue to be tested is removed from the subject. In addition, surgical margin tissue or other suitable "control" tissue, is also analyzed to determine whether differences exist between ~normal" and pathologic or diseased tissue. Immediately after removal, the tissues are placed in liquid nitrogen and maintained at or below -70C prior to extrac-tion of the DNA.
Extracting the DNA from the tissue is accomplished by :
` d~gesting the ti~sue with proteinase K and sodium dodecylsulfate, followed by hydrolysis with a-amylase for DNA and RNase A for ~ A
~Cance~ ReQ., 47:6543-6548, (1987)). [The hydrolysis with the a-~myla8e i8 u~ually only necessary when substantial glycogen deposits exist, such as in fish livers]~ The solution i6 then deproteinized with phenol/chloroform/isoamyl alcohol and chloro-form/isoamyl alcohol. The DNA is recovered by precipitation with 200 proof ethoxyethanol and washed with 70% ethoxyethanol. The purity of the DNA is established by spectrometric measurement WO 93/12258 PCr/US92/10669 212~778 using the UV absorbance ratio of 260 nm/280 nm and 1 absorbance unit = 50 ~g/ml (Cancer Res., 47:6543-6548, (1987)).
The DNA solution was then placed in an evacuated sealed tube at a temperature of 140 ~c and allowed to react with concentrated S formic acid (88%) for 30 minutes. This procedure did not alter the ~tructure of the nucleotide bases being studied and achieved the goal of preparing trimethylsyl (TMS) derivatives for the GC-MS/SIM. The solution was then dried in a desiccator under vacuum and allowed to react with acetonitrile -bis(trimethylsilyl)tri-fluoracetamide (BSTFA) (2:1 v.v) and acetonitrile (1:1) in polytetrafluorethylene-capped hypovials for 45 minutes at 80 C
~n an atmosphere of pure nitrogen.
Reference standards for the GC/MS-SIN analysis were pre-pared. These 6tandards were synthesized or purchased from commercial 60urce6. For example, the inventor purcha&ed FapyAde, 8-hydroxymethyluracil, wherea6 8-hydroxyadenine, and FapyGua were synthe~ized in his laboratory.

Once the sample wa~ ready, the injector port and interface of tho GC-MS equipment were maintained at 250C. The column of the GC/MS-SIM unit was a fused 6ilica capillary column (15.0 m., 0.2 mm inner diameter) coated with cro~s-linked 5% phenylmethyl-~ilicone gum phage (film thickness, 0.33 um). The column temper-ature was increased from 120C to 176C at a rate of 3C/min, and from 176C to 250C at a rate of 6C/min., after initially being held for 1.5 min. at 120C. A carrier gas of helium wa~ used wos3~l22s8 PCTtUS92/10669 ~t~
with a linear velocity of 23.5 cm/s through the column. Approxi-mately 0.7 ~g of TMS hydrolysate was in jected onto the column .
Quantification of DNA base derivatives was undertaken on the basi~ of the principal ions for the oxidized nucleotide ba~es, such as m/z 442, 440, 354 and 352 for FapyGua, 8-hydroxyguanine, FapyAde, and 8-hydroxyadenine, respectively. All ~pectra were compared to those from commercially obtained standards and authentic samples of TMS derivatives synthesized in the inven-tor's laboratory. The area counts for the principal ion~ were intograted and the data obtained included SIM plots and derived ma~ spectra.
The GC/MS-SIM equipment i~ sensitive enough to analyze the presence and quantity of trace concentrations of modified nucleo-tide bases in normal tissue~ and body fluids. By analyzing this baseline level against the level observed in the biological 8~mple in question, one can accurately determine the percentage ~ncrease or decrease in the modified biomarker analyzed. The re~ult~ from the analysis can then be u~ed in conjunction with pathological and histological data that reflect the health sta~us o~ the tiBsUes ~ cells or body fluids examined. For example, in tho case of expo~ed fish, ample documentation exists in the lit~rature for the types of morphological changes that occur in relation to a variety of environmental contaminants (J. Natl.
Cancer. Inst. 78, 333-363 (1987)).
The ~uccess obtained from the use of this method i~ apparent from the results of the inventor's extended research efforts.

WO93/12258 21 2 S 7 7 ~ PCT/US92/10669 This method is by no means exclusive, however. Becau~e the invention discovered the presence and related association of altered DNA nucleotide ba~es with carcinogenic and pre-carcinogenic tissue, the methods available for assaying this condition extend beyond the preferred method. It is well known in the biochemical community that additional methods exist for detecting altered nucleotide bases. Use of alternate means to define the extent and nature of oxidative DNA base modifications i~ therefore unrelated to the essential i~sue of whether this .-d~mage exists or not and, what is the nature of the resultantpotent~al or real impact on the living organism. Therefore, the8e examples are not intended to limit or provide an exhaustive list of alternate methods but are provided as further examples of 8uch alternative methods.
V-e of Monoclonal or Polyclonal Antibodies to Monltor Oxldatl~ Modlflcatlo~.
One ~uch alternative method for as6aying altered DNA nucleo-tide ba8e~ utilizes ~onoclonal or polyclonal antibodies with high ~pec~f~city for the modified nucleotide base~. Such antibodiés c~n be prepared using de~cribed procedures and applied in a quantitative a~6ay using the ELISA (Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (l988~) or radioimmunoas~ay ~Monoclonal Antibody TechnolooY, Elsevier Publishers (1984)) procedures. Examples of production of monoclonal and polyclonal antibodies follow this discussion.
This altsrnative method could be applied to native DNA

W093/12258 PcT/vss2/lo669 21~S~8 ....
-extracted as described above or to hydroly~ed DNA. This method would provide an advantage due to the comparative ease in sample preparation and analysis. In 8uch an approach, DNA to be te~ted could be coupled to a solid ~upport or coated onto plastic plates. The samples would be blocked with a 5~ ~SA solution in PBS for l hour prior to incubation with appropriate mono- or polyclonal antibodies for l to 2 hours. These primary antibodies could be used individually for a specific modified ba~e or mixed together to broadly define the extent of base modification.
After treatment with the primary antibody, detection could be 8fforded through treatment with a secondary antibody conjugated to a chromophore generating enzyme as in an ELISA assay (Antibod-ies: A LaboratorY Manual, Cold Spring Harbor Laboratory (1988)) or w~th a radioisotope such a6 ~25I-protein A binding (Antibodies:
lS A Laboratory Manual, Cold Spring Harbor Laboratory (1988)). This m~thod would sfford rapid quantitative information relating to the ~ltered purine and pyrimidine bases.
Monoclonal ~nt1bodios can be prepared in mice immunized as described for polyclonal antibody production prior to fusion. Cell fusion i8 conducted according to the method previously described (Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988)). X63-Ag8 cells grown in RPMI 1640 medium containing 10%
FCS are used as the fu6ion partner. X63-Ag8 cells, 5x107, are mixed in a ratio of l:4 with mou6e spleen cells prior to fu~ion with PEG at room temperature. After removal of the PEG and ` 24 WO93/12258 2 1 2 S 7 7 ~ PCT/US92/10~69 washing the fused cells with fresh medium, the fusion from each spleen is mixed with thymocytes derived from a single thymus in HAT containing RPMI 1640, 10~ FCS and plated into 4-96 well culture plates. In order to maintain humidity, the outer wells of each 96 well plate contain serum free medium.
Screening of hybrid cells i~ conducted by binding assays with modified nucleotide bases. Positive clones are moved to 24 well plates and further tested by reactivity to unmodified bases.
The clones showing proper specificity were then further cloned with a thymocyte feeder layer in a ratio of 50 cells per 96 well plate to achieve a uniform antibody producing cell population.
Example of Polyclonal AntibodY Production Polyclonal antibodies ~pecific for oxidatively modified nucleotide bases can be prepared in New Zealand White rabbits by immunization with modified bases conjugated to a protein such as keyhole limpet hemocyanin using the procedures previously described (Antibodies:
A ~aboratory Manual, Cold Spring Harbor Laboratory (1988)). The modified base is either chemically prepared or extracted from preparative quantities of appropriate DNA. The antigen i8 thén ~ixed with 1 ml of Freund' 6 incomplete adjuvant and emulsified.
Thi8 mixture i8 injected subcutaneously into multiple ~ites. The above procedure i8 repeated after two weeks and the animals bled sfter an additional two weeks. The pooled serum can be purified by removing antibodies specific for the conjugation protein by 2S chromatography on an affinity column containing the immobilized protein. The resulting 6erum is then assayed for reactivity with ?~.2S~
n~ otide base~ and modified bases using binding assays as described (Antibodies: A Laboratory Manual, Cold spring Harbor Laboratory (1988)).
USE OF ADDUCTS COMPRISING UNIQUELY IDENTIFIABLE LABELS
Another means could employ reacting the mixture of DNA bases ¦ obtained from in vivo DNA after isolation and hydrolysis with w -ab60rbing, radioactively labelled, or fluorescent molecules taking advantage of the reactive groups on the DNA bases. Such ia vitro generated adducts could then be conveniently separated by thin layer chromatography (~LC), or other convenient chromato-graphic methods, and quanti.tated ba~ed on the properties of the chromophore used or specific radioactivity of the labelled compound used. TLC conditions capable of ~eparating modified ba8e8 from unmodified bases would be employed. Such an analysis would provide a fingerprint for each specimen in terms of modi-~iod bases. In thi~ 6ame way, alternate means a~ defined above ¦ for analyzing DNA base modification~ could be applied to surro-I gate compo~nds which would be administered to a living organism, ~nd their fate followed with time to determine the rate and amount of oxidative modification generated. This approach would util~2e a non-physiological analog compound capable of undergoing the same type~ of in vivo generated damage as occur~ in the DNA
~nd wo~ld have the advantage of simplifying the preparative and potentially analytical procedures used.
Overall, the embodiments of this invention are wide in ~cope with application~ ranging from the diagno~tic and progno~tic WO93/12258 ~ 212 ~ 778 PCT/US92/10669 evaluation of human disease and pathologic conditions to the a6sessment of toxicity and health.risks at hazardous wastes sites where feral eukaryotic organisms inhabiting these sites, as well a8 te8t animals intentionally maintained there, can be studied or otherwise evaluated through the use of the DNA biomarkers.
Moreover, the opportunity exists to isolate both normal and abnormal cells and grow them in cultures, subjecting them to a variety of chemical and other stimuli and then evaluate changes ~n the proportions and concentrations of the DNA biomarkers. In essence then, the embodiments of this patent are not restricted to the direct measurement of the biomarkers in excised tissues ~nd body fluids, but incorporate usage in isolated cell systems maintained under suitable culture conditions.

W093/l22s8 PCT/US92/10669 2 ~ ~ ~LES
The following examples were obtained during research con-ducted by the inventor and associates. These examples are presented to illu~trate the application of the invention, and are not intended to limit the scope of the disclosure or the protec-tion granted by the Letters Patent. For convenience, the following abbreviation~ are used: 8-OH-Gua for 8-hydroxyguanine, 8-OH-Ade for 8-hydroxyadenine, FapyGua for 2,6-diamino-4-hydroxy-5-formamidopyrimidine, FapyAde for 4,6-diamino-5-formamidopyrimi-iO dine, and 5-OHUra for 5-hydroxymethyluracil.

ALTERED DNA NUCLEOTIDE BASES IN NEOPLASTIC AND PRE-NEOPLASTIC TISSUES.

Radical-induced alterations in the hepatic DNA of English 801e exposed to carcinogens.
English sole were collected from Eaqle Harbor and Elger Bay ~n Wa8hington State. The same species was obtained from Newport, Oregon. Five individual fish were obtained from each site. Each of the fi8h from Ea~le Harbor contained hepatic neoplasms (liver coll adenoma and hepatocellular carcinoma) which were revealed hi8tologically. Eagle Harbor, in Puget Sound, is heavily con-taminated with creosote hydrocarbons that have been linked in a number of studies (reviewed in Environ. Health_Perspectives 71, 5-16 (1987)) to hepatic tumors in the English sole. Elger Bay is relatively free of contamination, a~ is Newport. The livers of WO93/12258 21 2 S 7 7 8 PCT/US92/1~69 these "reference~l fis~ from the uncontaminated areas were found to be hi6tologically normal.
Areas of the liver characterized by grossly visible raised tumor nodules were excised from the tumor areas of expo~ed fi~h, as were sections of livers from the normal fish. After exci~ion, the tissues were preserved in liquid nitrogen prior to extraction of DNA.
The extraction procedure involved hydrolysis of the DNA with formic acid, followed by trimethylsilylation under a closed system of pure nitrogen. The DNA was quantified by measuring the W absorbance to determine its purity. The TMS derivatives were th~n ~nalyzed by GC/MS-SIM~ The inlet pres~ure of helium was at 7 kpa and the column temperature was increased from 120 to 176C
~t 3C/min. and from 176 to 250C at 6/min., after initially being held for 1.5 min at 120C. Mass ~pectra were obtained with 70 eV ion~zing energy.
The concentrations of oxidatively modified nucleotides in norm~l DNA are severely restricted metabolically, such as through tho glycosylase~ and other enzymes that participate in the ~20 ~x~8ion repair process. In this regard, the concentrations of DNA losions from Elger Bay fish did not differ significantly from th~ value8 of the Newport fish. The values obtained varied from 0.02 ~ 0.01 nmol/mg DNA for 8-hydroxyadenine (Elger Bay) to 0.13 ~ 0.06 nmol/mg DNA for 8-hydroxyguanine (Newport) (Figure~ 3A, 3B). These values are ~omewhat lower than tho~e reported for the same three nucleotide modification~ in normal calf thymus DNA

r~ rl 8 ~`
- (Journ. Biol. Chem. 264(22), 13025-13028 (1989), Anal. siochem.
156, 182-188 (1986)). However, the previously obtained value for FapyGua from normal English sole liver (Carcinoqenesis 11, 1045-1047 (1990)) was < 0.01 nmol/mg, which is the same as that obtained with calf thymus DNA (Journ. siol. Chem. 264(22), 13025-13028 (1989), Anal. Biochem. 156, 182-188 (1986)).
The average values for each of the modified nucleotide~ from the tumor-bearing Eagle Harbor fish were substantially higher than those from either Elger say or Newport (Figures 2, 3A, 3B).
They ranged from 0.17 + 0.12 nmol/mg DNA for FapyAde to 1.38 +
0.35 nmol/mg DNA for 8-OH-Gua. The previous value of 2.08 ~ 1.75 nmol/mg DNA for FapyGua from the tumor tissue was 208 times that for the normal tissue (Carcinogenesis 11, 1045-1047 (1990)). In the present work, the tumor tissue values were about 7, 12, and 20 times higher for FapyAde, 8-OH-Gua and 8-OH-Ade, respectively, comparod to the average values for the normal tissues. The findings thus indicated that a variety of nucleotides were modi-f~ed by the attack of the hydroxyl radical on the hepatic DNA.
For the first time these results suggested that oxidative dam~ge to DNA (e.g. through OH) has a putative association with tumori-gonesi8 in vivo.
In a sub~equent study, five English sole were obtained from Port Madison, Washington State, a site where these and other bottom fish have been extensively studied in relation to ~5 contaminant effects, including tumor formation (Environ. Health Perspectives 71, 5-16 (1987)) and other genotoxic changes W093/12258 212 ~ 7 7 8 PCT/US92/10669 (Aquatic Toxicol., 6, 165-177 (1985)). The livers of the Port Madison fish were excised and immediately frozen in liquid nitrogen. DNA was isolated from each of the livers separately.
The DNA was hydrolyzed with formic acid in 6ealed, evacuated ampules and trimethylsilylated under an atmosphere of pure nitrogen, as undertaken previously.
TMS derivatives of the nucleotide bases were analyzed by GC-MS/SIN, essentially as previously described herein. Briefly, as before, the nucleotide bases were allowed to react with acetoni-trile-bi8(trimethylsilyl)trifluoracetamide (BSTFA) (2:1 v.v) for 45 min. at 80C. Quantification of DNA base der'ivatives was undert8ken on the basis of the mass to charge ratio (m/z): 354, 352, 442, and 440 for FapyAde, 8-OH-Ade, FapyGua and ~-OH-Gua, respectively. Analyses were performed with a Hewlett Packard 5890A gas chromatograph e~uipped with an auto-sampler interfaced to a Hewlett Packard mass-selective detector model 5970B. A
fu8ed 6ilica capillary column coated with 5% phenylmethylsilicone gum phase (lSm; 0.2mm i.d., and 0.3ym film thickness) was used for the separation of the DNA ba~e derivatives. The column ~' tomperature was maintained at 120C for 1.5 min., increased to 176C at 3/min., and then to 250C at 6/min. The injection port and ion source were kept at 250C throughout the analysis.
Helium was the carrier gas and mass spectra were obtained with 70 eV ionizing energy.
The DNA lesions in hepatic tissues from the Port Madison fish were statistically compared to tho~e from the tumor-bearing 21~7~8 ,,~....
~ fish from Eagle Harbor and normal reference fish from the essen-tially uncontaminated sites of Newport OR and Elger Bay WA (Table II).
English sole from Port Madison have an incidence of about 3 hepatic neoplasms and 3% preneoplastic ~oci (Aquatic Toxicol., 11, 43-67 (1988), Aauatic Toxicol., 11, 143-162 (1988)). Both of these changes have been associated with exposure to a variety of environmental chemicals, including genotoxic agents (Aquatic ~oxicol., 6, 165-177 (1985)). The low incidence of liver lesions in the fi8h from Port Madison allowed samples to be obtained from a sub-population of English sole in one catching effort that 8howed no evidence of preneoplastic or neoplastic changes in the liver, as demonstrated on the basis of well-established histolog-ical criteria (J. Natl. Cancer. Inst. 78, 333-363 ~1987))--that i8, the livers were considered normal. However, examination of the hepatic DNA by GC-MS/SIM provided a different perspective: 8-OH-Gua, 8-OH-Ade and FapyGua were present in concentrations that were decidedly higher that those previou81y obtained from h~8tologically normal 801e from uncontaminated reference area~s~
~.o., Elger Bay WA and Newport OR). FapyAde was higher only with ro~p~ct to the Newport control. Moreover, with the exception of FapyAde, the concentrations of these DNA lesions were also significantly lower than the previously determined concentrations in hepatic tumors from Eagle Harbor, where the tumor incidence was about 25%. Most significantly the 8-OH-Gua, 8-OH-Ade and FapyGua concentrations were intermediat~ with respect to those of W093/12258 2 1 2 ~ 7 7 ~ PCT/US92/1066~

normal DNA from the reference fish from Newport and Elger Bay and the hepatic tumors of fish from Eagle Harbor. This is illu~trated in Figure 4 where the present data from Port Madison are compared with previously obtained results from tumor-bearing fish from Eagle Harbor and the reference sites. Specifically, the relationships between the concentrations of the nucleotide base modifications in the hepatic DNA and the site of capture are 8hown. Statistical evaluation using single factor analysis of variance (ANOVA) revealed that, with the exception of FapyAde, .
8ignificant differences existed between the concentrations of DNA
le8ions with respect to Eagle Harbor, Port Madison and ~he reference sit~s (Table 2).
The finding that the histologically normal fish from Port Madi60n had significantly higher concentrations of the DNA
le8ions compared to normal fish from the uncontaminated sites and significantly lower concentrations than fish from Eagle Har~or support8 the concept that the eOH-induced DNA lesions progres-8~vely accumulate in the liver. Importantly, the fact that the DNA lesions occurred at lntormedlate concentrations in appare'ntly normal fish from the tumor-bearing population at Port Madison lend~ 8upport to the inventor' 8 initial concept that the changes n DNA are causally related to tumorigenesis.
Overall, the presen~ work is consistent with the inventor's hypothesis that the OH-induced modification of the hepatic DNA
2s in English sole is a progressive event initiated by exposure to environmental chemicals, the ultimate result being tumorigenesis.

2 1~ ~q ~ PCT/US92/10669 Considering the fact that Port Madison has a low incidence of tumor-bearing fish, it seems likely that the 8-OH-Gua, 8-OH-Ade, FapyGua and FapyAde may be close to threshold concentrations for the development of liver cancer in the population. In this respect, it i8 suggested that the DNA lesions have an important u8e in the future for predicting the occurrence of cancer in organisms exposed to carcinogens. Moreover, as implied previou81y, the DNA le8ion8 represent readily evinced alterations at the molecular level that are highly relevant biomarkers for cytogenetic change in a variety of animal systems.

WO93/12258 2 I 2 ~ 7 7 8 PCT/US92/106~9 PRESENCE OF ALTERED DNA NUCLEOTIDES IN CARCINOMIC MAMMALIAN
TISSUES.

Radical-induced Alterations in DNA in Invasive Ductal Carcinogenic of the Female Breast.
Microscopic examination of excised carcinoma ti~sue from the five female patient~ was ~hown to contain invasive ductal carci-nomas; however, examination of the excised surgical margin tissue -revealed no evidence for neoplasia, altho~gh there wa~ some evidence for other microscopic changes (e.g. fibrocystic).
Ro~idual carcinoma-containing and excised surgical margin tissue w~ placed in liquid nitrogen immediately after removal and maintained at -70C prior to extraction of the DNA, which was undertaken as previously described. The DNA was then hydrolyzed and TMS derivatives were prepared under an atmosphere of pure nitrogen. The ~MS derivatives were analyzed by GC-MS/SIM as de~cr~bed previously, using a Hewlett-Packard Model 5890 m~croproce~sor-controlled gas chromatograph interfaced to a RP
Mod~l 5970~ mass ~electiv~ detector. The injector port and int~rfsce were both maintained at 250C. The column was a fused sil~ca capillary column (15.0 m., 0.2 mm inner diameter) coated ~ith cross-linked 5~ phenylmethylsilicone gum phase (film thickness, 0.33 ym). The column temperature was increased from 120 to 176C at 3C/min. and from 176 to 250C at 6/min., after initially being held for 1.5 min. at 120C. Helium wa~ used a~

21~S~77 8 - the carrier gas with a linear velocity of 23.5 cm./s through the column. The amount of TMS hydrolysate injected onto the column wa~ about 0.7 yg. Quantitation of the modified nucleotide derivatives was undertaken on the basis of the principal ions, such as m/z 442 for the TMS deriva~ive of FapyGua.

Analysis of the tis~ue6 revealed dramatic differences in the concentrations of 8-OH-Gua, FapyGua and 8-OH-Ade with respect to the control and the carcinoma tis~ue~. The values for 8-OH-Gua, FapyGua and 8-OH-Ade in the control were 0.13 + 0.03, 0.08 +
0.08, and 0.22 ~ 0.05 nmol/mg, respectively (Figure 5). The respective values for the carcinoma tissues were 8- to 17-fold higher--1.26 + 0.78, 1.33 + 0.97, and 1.67 + 1.86 nmol/mg DNA.
In both the control and the carcinoma tissues, FapyAde was present only at low levels near the limits of detection of the GC-MS/SIM technique (0.04 nmol/mg DNA). Accordingly, Fapy~de is not a prominent indicator of altered DNA in breast cancer in contrast to the other ba~e modifications~ There was not a ~gnificant differences between the calf thymus and surgical '~
m~rgin DNA with respect to any of the base modifications;
however, a 8ignificant difference did exist between the DNA from the surgical margin and the carcinoma ti~ue with respect to 8-OH-Gua (p S 0.01), FapyGua (p < 0.03) and 8-OH-Ade (p ~ 0.05).
On a matched pair ba~is (surgical margin v~. carcinoma), the concentrations of each of the above base modifications were substantially higher in the carcinoma/ with the exception of FBT-5 which had relatively low concentrations of the base lesions(see legend to Figure 5).
In studies with the English sole carcinogenesis model, the inventor and colleagues found that the relatively low concentra-tions of base modifications in normal tissues were within arelatively narrow ranye, close to the threshold of detection of the GC/MS-SIM method. In this regard, the present values with calf thymu6 DNA were not appreciably different from tho~e ob-tained by the inventor and colleagues and other workers (Anal.
Biochem. 156, 182-188 (1986)). Moreover, in an initial attempt to under~tand base level concentrations of the modified nucleo-tide derivatives in human tissues, leukocytes from the blood of two apparently normal individuals were studied. The values obtained were consi~tently low: 0.20 and 0.23; 0.12 and 0.14;
0.01 and 0.07; and 0.04 and 0.04 nmol/mg DNA for 8-OH-Gua, 8-OH-Ade, FapyGua, and FapyAde, respectively. In each sample, the concentration of Fapy-A was ~ 0.04 nmol/mg DNA. The surgical margin ti6sue may not be microscopically normal. Nevertheless, ~ indicatod, in terms of the DNA ba6es examined, the surgic~
margin DNA wa~ not significantly different from the calf thymus DNA. Accordingly, the calf thymu6 data which have previou~ly ~rved as a standard for Hnormal DNA" (Anal. Biochem. 156, 182-188 (1986)) are compared to the carcinoma data in Figure 5.
Overall, the present findings provide persuasive evidence for substantial OH-induced alterations having taken place in the purine nucleotides. Moreover, it seems unlikely that the radical - attack on the DNA was essentially confined to 8-OH-Gua, Fapy-Gua and 8-OH-Ade. By utilizing the methodology and teachings of this patent, the inventor was able to identify and quantify a modified pyrimidine base. The results of this application was a determination that an elevated concentration of 5-OHUra was present in the carcinomic female breast tissues.
This study was the first to examine DNA base modifications in any mammalian tissue on a structural and quantitative basis and link them to pa~hologic or disease conditions. Thus, the findings provided a unique opportunity to evaluate their signifi-cance in relation to the pathobiology of breast cancer. In this re8pect, the presence of the relatively high concentration of 8-OH-Gua in the DNA of the carcinoma tissues seems especially relevant in view of the evidence demonstrating that 8-OH-dG has an overwhelming effect in causing misreplication in template-directed DNA synthesis (Nature 327, 77-79 (1987)). Considering the 8pecial need for maintaining the ~tructural integrity of DNA
through enzymatic and other processes, the substantial OH-~nduced modification8 in thi8 molecule are likely to be causa~
rolated to the neoplastic transformations in the breast.
However, the origin of the OH that potentially initiates the - ba8e modifications is unclear, although one possibility i8 that thi~ radical arises from H202 generated through the cytochrome P-450 - mediated redox cycling of estrogen during the formation of DNA-binding metabolites (J. Exp. Med, 153:766-782 ~1981)).

.

WO 93/12258 2 1 ~ S 7 7 8 PCI/I~S92/10669 EXPOSURE AND RECOVERY OF SUBJECT EXPOSED TO ENvIRoNMENTAL
TOXINS BY ASSAYING DEGREE OF NUCLEOTIDE ALTERATIONS.

Radical-induced alterations in DNA of Medaka.
S A study wa~ conducted in which Medaka were chronically exposed to trichloroethylene (TCE), diethylnitrosamine (DEN) and groundwater ( Final Report: u.S. Army siomedical Research and Development Laboratory, Fort Detrick, MD; grant no. DAMD17-88-Z-8043). In this experiment khe two control values differed ~ignificantly (Tsble 1). However, despite this difference, 10~
groundwater and 100% groundwater appeared to have a particularly dramatic effect in rai~ing the concentrations of the DNA base le B ~ ons. For example, the 10% groundwater exposure resulted in 1.03 nmol/mg DNA total lesions compared to the average value for the controls of 0.424 nmol/mg. The 100% groundwater exposure produced an even greater value (1.81 nmol/mg). The values for groundwater + 10 mg/L DEN 8Ugqe8t that DEN tends to suppress the oxpression of the base modifications, although further work is roquired to verify this ob~ervation. Of particular interest ~n the pre~ent experiment i8 the fact that 5 ppm of TCE (with and w~thout DEN) resulted in a substantial increa~e in the total base le~ion concentrations.
A companion experiment was conducted in which the exposed fish were allowed to recover in clean water (Table 1). The findings were most revealing in that evidence was provided for the first time to indicate that the base lesion concentrations 3~

2 ~ PCT/US92/10669 produced through the chemical exposures are substantially reduced when the exposures are terminated. A salient example of this phenomena can been seen from examination of the data from the ¦ Medaka that were originally exposed to 100% groundwater (1.81 nmol/mg DNA total lesions). After "recovery" the value was 0.S60 nmol/mg. Similarly, the total lesion value of 1.01 nmol/mg for the Medaka exposed to 5 ppm TCE was reduced to 0.289 nmol/mg in the "recovered" fish.
Overall, it is clear that exposure of the Medaka to environ-mental chemicals results in significant increases in the DNA base l~sion8. The groundwater exposures are notable in that substan-tlal increases in the DNA base lesions occur at low concentra-tions and that these increases appear to be reversible when the animals are placed in "recovery" conditions. Thus, the potential lS clearly exists to use the base lesions as biomarkers of the dogree of exposure, as well as with regard to tracking the rocovery process after clean-up operations at contaminated cnvironmental sites. The influence of DEN on the exposures is pro8ently unclear; however, the DEN appears to generally lower~
tbo tendency of the groundwater to increase the base concentra-tions, 8ugge8ting that competitive factors are influential.

Claims

What is claimed:
1) A method for detecting an increased likelihood of genotoxic injury occurring in vivo in a test subject comprising the steps of:
a) obtaining a first specimen from a test subject;
b) obtaining a second specimen from a second subject known not to have or be at risk of genotoxic injury, for the purpose of establishing a reference;
c) analyzing in vitro said first and said second specimen to determine the quantity of DNA nucleotides oxidatively modified in vivo; and d) comparing the quantity of said oxidatively modified nucleotides in said first specimen to the quantity of said oxidatively modified nucleotides in said second specimen wherein an increased likelihood of genotoxic injury occurring in vivo is associated with an increased quantity, relative to said second specimen, of said oxidatively modified nucleotides in said first specimen.

2) The method of claim 1 wherein said oxidatively modified nucleotides are selected from the group consisting of oxidatively modified purine or pyrimidine.

3) The method of claim 1 wherein said oxidatively modified nucleotides are selected from the group consisting of 8-hydroxyguanosine, 2,6-diamino-4-hydroxy-5-formanidopyrimidine, 8-hydroxyadenine, and 4,6-diamino-5-formanidopyrimidine.

4) The method of claim 1 wherein said genotoxic injury is neoplasia and said test subject is human and said oxidatively modified nucleotides are selected from the group consisting of 8-hydroxyguanosine, 2,6-diamino-4-hydroxy-5-formanidopyrimidine, 8-hydroxyadenine, and 5-hydroxymethlyuracil.

5) The method of claim 1 wherein said specimens comprise tissue or fluids obtained from said test subjects.

6) The method of claim 1 wherein the step of analyzing said first and said second specimen is by Gas Chromatography/Mass Spectrometry with Selected Ion Monitoring which comprises the steps of:
a) isolating DNA from said specimens;
b) hydrolyzing said DNA;
c) preparing trimethylsilyl derivatives of said hydrolyzed DNA; and d) determining presence of modified nucleotide bases and quantity thereof by introducing said derivatives into a Gas Chromatography/Mass Spectrometry Selected Ion Monitoring apparatus having Selected Ion Monitoring capabilities.

7) The method of claim 1 wherein the step of analyzing said first and said second specimen is by monoclonal antibody assay which comprises the steps of:
a) isolating DNA from said specimens;
b) coating a solid surface with said DNA;
c) blocking said solid surface with a suitable protein;
d) incubating said DNA with monoclonal antibodies specifically reactive with oxidatively modified nucleotides; and e) quantitating the amount of specific antibodies bound to said DNA.

8) The method of claim 7 wherein said quantitating comprises use of ELISA procedures.

9) The method of claim 7 wherein said quantitating comprises use of radioimmunoassay procedures.

10) The method of claim 1 wherein the step of analyzing said first and said second specimen is by polyclonal antibody assay which comprises the steps of:
a) isolating DNA from said specimens;
b) coating a solid surface with said DNA;
c) blocking said solid surface with a suitable protein;

d) incubating said DNA with polyclonal antibodies specifically reactive with oxidatively modified nucleotides; and e) quantitating the amount of specific antibodies bound to said DNA.

11) The method of claim 10 wherein said quantitating comprises use of ELISA procedures.

12) The method of claim 10 wherein said quantitating comprises use of radioimmunoassay procedures.

13) The method of claim 1 wherein the step of analyzing said first and said second specimen is by chromatographic analysis which comprises the steps of:
a) extracting DNA from the specimens and hydrolyzing said DNA;
b) introducing a tag to said hydrolyzed DNA; and c) separating and quantitating the amount of oxidatively modified nucleotides.

15) The method of claim 13 wherein chomophoric tags are used.

16) The method of claim 13 wherein radioactive tags are used.

17) A method for assaying the toxicity of an environment or compound to determine health risks to a test subject comprising the steps of:
a) exposing said test subject to an environment or compound;
b) obtaining a first specimen from said test subject wherein said specimen does not exhibit manifest tumorigenesis;
c) obtaining a second specimen from a similar subject known not to have or be at risk of cancer, for the purpose of establishing a reference;
d) analyzing in vitro said first and said second specimen to determine the quantity of DNA nucleotides oxidatively modified in vivo; and e) comparing the quantity of said oxidatively modified nucleotides in said first specimen to the quantity of said oxidatively modified nucleotides in said second specimen wherein an increased health risk to said test subject exposed to the environment or compound is associated with an increased quantity, relative to said second specimen, of said oxidatively modified nucleotides in said first specimen.

18) The method of claim 17 wherein said oxidatively modified nucleotides are selected from the group consisting of oxidatively modified purine or pyrimidine.

19) The method of claim 17 wherein said oxidatively modified nucleotides are selected from the group consisting of 8-hydroxyguanosine, 2,6-diamino-4-hydroxy-5-formanidopyrimidine, 8-hydroxyadenine, 4,6-diamino-5-formanidopyrimidine, and 5-hydroxymethlyuracil.

20) The method of claim 17 wherein said specimens comprise tissue or fluids obtained from said test subjects.

21) The method of claim 17 wherein the step of analyzing said first and said second specimen is by Gas Chromatography/Mass Spectrometry with Selected Ion Monitoring which comprises the steps of:
a) isolating DNA from said specimens;
b) hydrolyzing said DNA;
c) preparing trimethylsilyl derivatives of said hydrolyzed DNA; and d) determining presence of modified nucleotide bases and quantity thereof by introducing said derivatives into a Gas Chromatography/Mass Spectrometry apparatus having Selected Ion Monitoring capabilities.

22) The method of claim 17 wherein the step of analyzing said first and said second specimen is by monoclonal antibody assay which comprises the steps of:
a) isolating DNA from said specimens;

b) coating a solid surface with said DNA;
c) blocking said solid surface with a suitable protein;
d) incubating said DNA with monoclonal antibodies specifically reactive with oxidatively modified nucleotides; and e) quantitating the amount of specific antibodies bound to said DNA.

23) The method of claim 22 wherein said quantitating comprises use of ELISA procedures.

24) The method of claim 22 wherein said quantitating comprises use of radioimmunoassay procedures.

25) The method of claim 17 wherein the step of analyzing said first and said second specimen is by polyclonal antibody assay which comprises the steps of:
a) isolating DNA from said specimens;
b) coating a solid surface with said DNA;
c) blocking said solid surface with a suitable protein d) incubating said DNA with polyclonal antibodies specifically reactive with oxidatively modified nucleotides; and e) quantitating the amount of specific antibodies bound to said DNA.

26) The method of claim 25 wherein said quantitating comprises use of ELISA procedures.

27) The method of claim 25 wherein said quantitating comprises use of radioimmunoassay procedures.

28) The method of claim 17 wherein the step of analyzing said first and said second specimen is by chromatographic analysis which comprises the steps of:
a) extracting DNA from the specimens and hydrolyzing said DNA;
b) introducing a tag to said hydrolyzed DNA; and c) separating and quantitating the amount of oxidatively modified nucleotides.

30) The method of claim 28 wherein chomophoric tags are used.

31) The method of claim 28 wherein radioactive tags are used.

33) A method for determining a long term risk factor for exposure to an environment or compound comprising the steps of:
a) exposing a test subject to said environment or compound;
b) obtaining a first specimen from said test subject;

c) analyzing in vitro said first specimen to determine the quantity of DNA nucleotides oxidatively modified in vivo;

d) removing said test subject from exposure to said environment or compound;
e) obtaining a second specimen from said test subject;
f) analyzing in vitro said second specimen to determine the quantity of DNA nucleotides oxidatively modified in vivo;
g) comparing the quantity of said oxidatively modified nucleotides in said fist specimen to the quantity of said oxidatively modified nucleotides in said second specimen wherein a decreased health risk exists for a subject exposed to said environment or compound but subsequently removed from exposure thereto when a decreased quantity, relative to said first specimen, of said oxidatively modified nucleotides in said second specimen is detected.

34) The method of claim 33 wherein steps e) through g) are periodically repeated to obtain statistical data to obtain a rate of recovery of said test subject once removed from exposure to said environment or compound.