WO1993012258A1

WO1993012258A1 - Dna biomarkers of cancer and genotoxic injury

Info

Publication number: WO1993012258A1
Application number: PCT/US1992/010669
Authority: WO
Inventors: Donald C. Malins
Original assignee: Pacific Northwest Research Foundation; CytoChem Inc
Current assignee: Pacific Northwest Research Foundation; CytoChem Inc
Priority date: 1991-12-13
Filing date: 1992-12-10
Publication date: 1993-06-24
Anticipated expiration: 1994-06-13
Also published as: CA2125778A1; AU3275493A

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

DNA BIOMARKERS OF CANCER AND GENOTOXIC INJURY

FIELD OF THE INVENTION

This invention relates to the field of detection and assessment of oxidatively modified 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 genotoxic effects of environmental influences.

SUBSTITUTESHEET 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. Pharπtaco1. Toxico1. 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

-Λ the superoxide ion (0₂ ^"-), H₂0₂ and »OH. While 0₂ ^"- and H₂0₂ individually may not be particularly damaging, their combined action leads to the formation of the highly reactive •OR:

0_a ^" + H₂0₂ > *»0H + OH^" + 0₂

This reaction can be relatively slow; however, when catalyzed by metal ions [e.g., Fe⁺²], it is substantially accelerated and becomes especially relevant in the initiation of biological damage (Science 240, 640-642 (1988)). H₂0₂ itself is converted to «0H through, the iron (Fe⁺²)-catalyzed Fenton reaction (Science 240, 640-642 (1988)). The proliferation of »OH may then result in an attack on most molecules in living cells with deleterious consequences (Science 227, 375-381 (1985)). The primary defense against such radical-induced damage is provided by enzymes that catalytically scavenge the intermediates of oxygen reduction and by antioxidants, such as glutathione. For example, 0₂ ^~- is eliminated by superoxide dismutase (SOD) which catalyses a dismutation reaction leading to the formation of 0₂ and H₂0. In 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 hampers a clear understanding of the significance of reactive oxygen species in biological injuries is a lack of adequate information on the critical cellular targets

-Λ 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 diseases, research has been conducted relating to DNA lesions. A number of DNA lesions have been identified, mostly with in vitro systems, and putatively associated with the interaction of the hydroxy radical (»OH) with nucleotide deriva¬ tives. These include thymine glycol, 5,6-dihydroxythymine (Journ. Biol. Chem. 264(22), 13025-13028 (1989)) and 8-hydroxy- deoxyguanosine (8-OH-dG) (Nature 327, 77-79 (1987), Biochem. J. 238, 247-254 (1986), Carcinoσenesis 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 misread in a DNA synthesis system in. vitro with E. coli (Nature 327, 77-79 (1987)). In fact, the presence of the 8-OH-dG in DNA was viewed as "...an important cause of mutation and carcinogenesis"

(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 »OH 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 ~-t produced in the neoplastic livers of English 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)). Moreover, it was ulti¬ mately shown that free radical damage to the DNA had occurred in the tumor-bearing livers 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 #0H. Thus, in the early 1980s, some indication existed 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 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 exist would serve a diagnostic or prognostic role in relation to the causation of genetically- related pathology or disease.

-Λ 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 structurally 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 patent, an invention is disclosed that for the first time identifies and quantifies biochemical changes in living 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 demonstrate that DNA modifications in chemically-exposed living systems are related to pathological alterations in cells.

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 significance as biomarkers 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.g., 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-SIM), methods have been discovered whereby quantities of altered DNA nucleotide bases from subject tissues can be assessed and compared to base-line or normal levels thereby providing important information about the condition of the test tissues or subject as detailed in this specification. The basis of the disclosed methods stems from the inventors discovery that the mechanism for the hydroxyl radical (•OH) attack on nucleotide bases in vivo forms specific radical bases which have a unique and quantifiable structure. This discovery, in conjunction with the discovery that in vivo alterations of these bases are inextricably linked to neoplastic and pre-neoplastic tissues provides the foundation for this paten .

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 bases are an indicator of genotoxic changes in tissue and body fluids, an -.t 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 is also an object of this invention is to provide a method whereby a subject is administered structurally equivalent "surrogate" compounds (biomarkers) for diagnosing or 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 system to external chemicals and the rate and degree of decline in such alterations upon introduction of therapeutically-applied antioxidants or other radical trapping agents.

Because the invention relates 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 monoclonal or polyclonal antibodies, or adducts utilizing uniquely identifiable labels are equally plausible, depending upon the needs of the analyst.

BRIEF DESCRIPTION OF THE DRAWINGS

Fiα. 1: 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.

Fig. 2: Elevated concentrations in 8-hydroxyguanine in DNA are shown in neoplastic hepatic tissue of feral fish, compared to normal tissue.

Fig. 3A: Elevated concentrations of 8-hydroxyadenine in DNA are shown in neoplastic hepatic tissue of feral fish, compared to normal tissue.

Fig. 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.

Fig. 4: Comparisons are made between English sole from "clean" reference 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 the nucleotide bases have taken place in the cancer tissue as a result of the attack of the »OH on the base structures.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides for a method to quantitative¬ ly and structurally identifying oxidatively modified nucleotide bases or their functional equivalents, and the effect that such modified nucleotide bases have on the ability of the DNA to successfully 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 -.t discussion of the research conducted in conjunction with this invention.

Studies published in 1983 on the presence of free radicals in the livers of English sole with liver tumors from polluted environments of Puget Sound, Washington, demonstrated that the formation of free radicals was associated with the presence of the liver tumors. The radicals were aromatic in nature; and appeared to be formed through the action of the »OH. It was later 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 *0H.

However, these findings provided no definitive information on the biological significance of the radical interactions with the cellular biochemistry, but did established that oxyradicals are associated with environmental chemical exposures in the English sole.

Previously, DNA from neoplastic hepatic tissues of feral fish environmentally exposed to carcinogens was investigated by the inventor and it was found that an abnormally high concentra- s tion of an oxidatively modified DNA nucleotide base was present. The results of this research confirmed for the first time that at 5 least one modified nucleotide base — FapyGua or 2,6-diamino-4- hydroxy-5-formamidopyrimidine — was present in the DNA of the carcinogenic hepatic tissue of feral fish. The inventor hypothe¬ sized that an oxygen radical, the •OH, was responsible for the cleavage of the purine ring of guanine. It was further hypothe-

-Λ

10 sized that the »0H attacked the electron deficient C-8 of the purine ring, leading to the cleavage product of FapyGua (Figure 1). Based on the presence of this DNA lesion in carcinogenic tissue, the inventor concluded that the free radical-induced DNA modifications were intimately associated with the neoplastic

15 tissue. However, there was no evidence to support the conclusion that the single DNA modification observed with just five fish was a precondition to tumorigenesis and not merely a product of tumorigenesis or that such an alteration would occur in other tissues or other species. Neither was it apparent whether the

20 attack of the *0H 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 similar types of modifications in the other three bases compris¬ ing DNA. Thus, any possible diagnostic or prognostic value that 25 the DNA modification may have represented was both elusive and unpredictable. It was during this research effort that the inventor used for the first time the Gas Chromatograph/Mass Spectrograph with Selected Ion Monitoring (GC/MS-SIM) method of analysis for in vivo studies of carcinogenic tissue. Prior to this effort, the GC/MS-SIM method of analysis 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 neutrophils. Based on the above described hypothesis, a second research effort was then undertaken to investigate whether other oxida- tively modified DNA nucleotides were present in hepatic neoplastic tissues of feral fish taken from a chemically polluted site and absent from feral fish obtained from non-polluted sites. By hypothesizing the method of attack of the «0H on the DNA nucleotides, the selected ions signifying other potential base modifications were chosen for analysis by the GC/MS-SIM method. The findings of the research supported the hypothesis.

Elevated concentrations of three new modified DNA nucleo¬ tides were discovered to exist in the neoplastic tissue samples: 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 tissue samples 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 1) .

Subsequent to these initial feral fish analyses, the inven- tor conducted further research to investigate whether apparently normal hepatic liver tissue of feral fish obtained from the same contaminated site 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 that increased 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

-Λ 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 concentrations, occurring prior to tumor formation, were causally linked to tumorigenesis and that the presence of the modified nucleotides were not just a product of neoplastic growth. Thus, by analyzing histologically normal tissue for the presence of elevated levels of modified nucleotide bases, one could determine prognostically whether that tissue was likely to exhibit subse¬ quent histological/pathological change, such as tumorigenesis. In fact, it was suggested that the concentrations in the histologi¬ cally normal fish "may be close to threshold concentrations for the development of liver cancer in the population." It was further concluded that "...the DNA lesions appear to represent readily evinced alterations at the molecular level that are highly relevant biomarkers for cytogenetic change (Aquatic

Toxicol., 20, 123-130 (1991))."

To extend these findings and conclusions to mammalian models, the inventor analyzed tissue from female breast cancer 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 -.t while the cause of breast cancer remains uncertain, the presence of these modified nucleotides 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¬ an tissues.

Subsequent to the publication of this work (unpublished results, 1991), the inventor demonstrated using the GC-MS/SIM methodology, that substantially higher concentrations of the oxidatively derived pyrimidine base, 5-hydroxymethyluracil, existed in the DNA from the breast carcinoma tissue compared to that of either the surgical margin or normal DNA from calf 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 and genotoxic injury to DNA (e.g., neoplasia), the inventor conducted additional research utilizing Medaka (0. Latipes) fish ^~ chronically exposed to ground water contaminated with complex mixtures of toxic substances. By employing the same method of

•*

5 analysis as previously used with feral fish and breast cancer tissues, it was 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

10 pre-carcinogenic tissues. The research also indicated that once the environmental toxic influences were removed, the levels of modified nucleotide bases significantly decreased to virtual! - "background" levels (Table 1), suggesting that early processes of tumorigenesis are likely to be reversible. Overall, the research

15 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

20 comparing those findings with a base-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 possible to correlate both the type and 25 concentration of the DNA modifications with the development of pathologic changes in organisms. This is true whether the 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 some 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 tissues and body fluids, as the inventor has 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 hiεtologic, pathologic and other information relating to disease or health status.

Moreover, "surrogate" compounds (biomarkers) that metabolically mimic the essential structure of the nucleotide bases, or otherwise serve as sentinels for the threat posed to the nucleotide bases from the attack of reactive oxygen compounds, may be administered under therapeutic or other conditions. Under these circumstances, the modifications in the surrogate compounds may be followed as undertaken with the normal DNA. Additionally, the use of the DNA biomarkers can be extended o "tracking" the effects of administered or dietary substances 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,

* 5 and glutathione) .

Further, no restriction is made in the embodiment of this invention to confine the DNA biomarkers to use with intact cells in living organisms. Clearly, an important aspect of the embodiments of this invention is the use of cells isolated from ^*-.t 10 intact living organisms, maintained in cell culture (Cell

Culture, Methods in Enzymology, Vol. LVIII, Academic Press

(1979)), and exposed to virtually any substance that is perceived as having or promoting an effect resulting in the formation of the DNA modifications. As implied, the types, classes and

15 mixtures of substances are vast and diverse, including soil, sediment, water, water surface microlayer, food chain organisms, air individual chemicals, chemical mixtures, extracts derived from biological material, drugs, pharmaceutical, carcinogenic materials, cosmetics, food products.

20 The methods described in this invention are clearly an important tool in establishing risk with regard to a variety of genotoxic changes. Given that cancer, for example, is initiated at the level of DNA where oxidative base modifications are krown to occur, DNA modifications ascertained by the present methodolo-

"25 gy would provide an early warning or prognosis of the likelihood of future pathologic changes. In this same context, the opportu- nity exists for assessing risk from both exposure to toxic substances and internal biochemical 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 biomarkers. This method was employed by the inventor in much of his research.

-Λ 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 -70°C prior to extrac¬ tion of the DNA.

Extracting the DNA from the tissue is accomplished by digesting the tissue with proteinase K and sodium dodecylsulfate, followed by hydrolysis with α-amylase for DNA and RNase A for RNA (Cancer Res. , 47:6543-6548, (1987)). [The hydrolysis with the α-amylase is usually only necessary when substantial glycogen deposits exist, such as in fish livers]. The solution is then deproteinized with phenol/chloroform/isoamyl alcohol and chloro- fαrm/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 using the UV absorbance ratio of 260 nm/280 nm and 1 absorbance unit = 50 μg/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 * 5 formic acid (88%) for 30 minutes. This procedure did not alter the structure 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-

-Λ

10 fluoracetamide (BSTFA) (2:1 v.v) and acetonitrile (1:1) in polytetrafluorethylene-capped hypovials for 45 minutes at 80 °C in an atmosphere of pure nitrogen.

Reference standards for the GC/MS-SIM analysis were pre¬ pared. These standards were synthesized or purchased from

15 commercial sources. For example, the inventor purchased FapyAde, 8-hydroxymethyluracil, whereas 8-hydroxyadenine, and FapyGua were synthesized in his laboratory.

Once the sample was ready, the injector port and interface 20 of the GC-MS equipment were maintained at 250°C. The column of the GC/MS-SIM unit was a fused silica capillary column (15.0 m., 0.2 mm inner diameter) coated with cross-linked 5% phenylmethyl- silicone gum phase (film thickness, 0.33 um) . The column temper¬ ature was increased from 120°C to 176°C at a rate of 3°C/min, and 25 from 176°C to 250°C at a rate of 6°C/min., after initially being held for 1.5 min. at 120°C. A carrier gas of helium was used with a linear velocity of 23.5 cm/s through the column. Approxi¬ mately 0.7 μg of TMS hydrolysate was injected onto the column.

Quantification of DNA base derivatives was undertaken on the basis of the principal ions for the oxidized nucleotide bases, such as m/z 442, 440, 354 and 352 for FapyGua, 8-hydroxyguanine, FapyAde, and 8-hydroxyadenine, respectively. All spectra 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 ions were integrated and the data obtained included SIM plots and derived mass spectra.

The GC/MS-SIM equipment is sensitive enough to analyze the presence and quantity of trace concentrations of modified nucleo¬ tide bases in normal tissues and body fluids. By analyzing this baseline level against the level observed in the biological sample in question, one can accurately determine the percentage increase or decrease in the modified biomarker analyzed. The results from the analysis can then be used in conjunction with pathological and histological data that reflect the health status of the tissues, cells or body fluids examined. For example, in the case of exposed fish, ample documentation exists in the literature 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 success obtained from the use of this method is apparent from the results of the inventor's extended research efforts. This method is by no means exclusive, however. Because the invention discovered the presence and related ssociation of altered DNA nucleotide bases 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 is therefore unrelated to the essential issue of whether this damage exists or not and, what is the nature of the resultant potential or real impact on the living organism. Therefore, these examples are not intended to limit or provide an exhaustive list of alternate methods but are provided as further examples of such alternative methods. Use of Monoclonal or Polyclonal Antibodies to Monitor Oxidative Modifications.

One such alternative method for assaying altered DNA nucleo¬ tide bases utilizes monoclonal or polyclonal antibodies with high specificity for the modified nucleotide bases. Such antibodies can be prepared using described procedures and applied in a quantitative assay using the ELISA (Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988)) or radioimmunoassay (Monoclonal Antibody Technology, Elsevier Publishers (1984)) procedures. Examples of production of monoclonal and polyclonal antibodies follow this discussion.

This alternative method could be applied to native DNA extracted as described above or to hydrolyzed DNA. This method would provide an advantage due to the comparative ease in sample preparation and analysis. In such an approach, DNA to be tested could be coupled to a solid support or coated onto plastic plates. The samples would be blocked with a 5% BSA solution in PBS for 1 hour prior to incubation with appropriate mono- or polyclonal antibodies for 1 to 2 hours. These primary antibodies could be used individually for a specific modified base or mixed together to broadly define the extent of base modification.

-Λ After treatment with the primary antibody, detection could be afforded 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 with a radioisotope such as ¹²⁵I-protein A binding (Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988)). This method would afford rapid quantitative information relating to the altered purine and pyrimidine bases.

Example of Monoclonal Antibody Production: Monoclonal antibodies can be prepared in mice immunized as described for polyclonal antibody production prior to fusion. Cell fusion is 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 fusion partner. X63-Ag8 cells, 5xl0⁷, are mixed in a ratio of 1:4 with mouse spleen cells prior to fusion with PEG at room temperature. After removal of the PEG and 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 is 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 specific 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 Laboratory Manual, Cold Spring Harbor Laboratory (1988)). The modified base is either chemically prepared or extracted from preparative quantities of appropriate DNA. The antigen is then mixed with 1 ml of Freund's incomplete adjuvant and emulsified.

This mixture is injected subcutaneously into multiple sites. The above procedure is repeated after two weeks and the animals bled after an additional two weeks. The pooled serum can be purified by removing antibodies specific for the conjugation protein by chromatography on an affinity column containing the immobilized protein. The resulting serum is then assayed for reactivity with nucleotide bases 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 UV- absorbing, radioactively labelled, or fluorescent molecules taking advantage of the reactive groups on the DNA bases. Such in vitro generated adducts could then be conveniently separated by thin layer chromatography (TLC), or other convenient chromato- graphic methods, and quantitated based on the properties of the chromophore used or specific radioactivity of the labelled compound used. TLC conditions capable of separating modified bases from unmodified bases would be employed. Such an analysis would provide a fingerprint for each specimen in terms of modi¬ fied bases. In this same way, alternate means as defined above for analyzing DNA base modifications could be applied to surro¬ gate compounds which would be administered to a living organism, and their fate followed with time to determine the rate and amount of oxidative modification generated. This approach would utilize a non-physiological analog compound capable of undergoing the same types of i vivo generated damage as occurs in the DNA and would have the advantage of simplifying the preparative and potentially analytical procedures used. Overall, the embodiments of this invention are wide in scope with applications ranging from the diagnostic and prognostic evaluation of human disease and pathologic conditions to the assessment of toxicity and health risks at hazardous wastes sites where feral eukaryotic organisms inhabiting these sites, as well as test 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 in the proportions and concentrations of the DNA biomarkers. In

Λ essence then, the embodiments of this patent are not restricted to the direct measurement of the biomarkers in excised tissues and body fluids, but incorporate usage in isolated cell systems maintained under suitable culture conditions.

EXAMPLES

The following examples were obtained during research con¬ ducted by the inventor and associates. These examples are presented to illustrate 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 abbreviations 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- ~_t 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 sole exposed to carcinogens.

English sole were collected from Eagle Harbor and Elger Bay in Washington State. The same species was obtained from Newport, Oregon. Five individual fish were obtained from each site. Each of the fish from Eagle Harbor contained hepatic neoplasms (liver cell adenoma and hepatocellular carcinoma) which were revealed histologically. 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, as is Newport. The livers of these "reference" fish from the uncontaminated areas were found to be histologically normal.

Areas of the liver characterized by grossly visible raised tumor nodules were excised from the tumor areas of exposed fish, -. 5 as were sections of livers from the normal fish. After excision, 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 10 system of pure nitrogen. The DNA was quantified by measuring the UV absorbance to determine its purity. The TMS derivatives were then analyzed by GC/MS-SIM. The inlet pressure of helium was at 7 kpa and the column temperature was increased from 120 to 176°C at 3°C/min. and from 176 to 250°C at 6°/min., after initially 15 being held for 1.5 min at 120°C. Mass spectra were obtained with 70 eV ionizing energy.

The concentrations of oxidatively modified nucleotides in normal DNA are severely restricted metabolically, such as through the glycosylases and other enzymes that participate in the 20 excision repair process. In this regard, the concentrations of

DNA lesions from Elger Bay fish did not differ significantly from the values 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) (Figures 3A, 25 3B). These values are somewhat lower than those reported for the same three nucleotide modifications in normal calf thymus DNA (Journ. Biol. Chem. 264(22), 13025-13028 (1989), Anal. Biochem. 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. Biol. Chem. 264(22), 13025- 13028 (1989), Anal. Biochem. 156, 182-188 (1986)).

The average values for each of the modified nucleotides from the tumor-bearing Eagle Harbor fish were substantially higher than those from either Elger Bay or Newport (Figures 2, 3A, 3B) .

-A 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, compared to the average values for the normal tissues. The findings thus indicated that a variety of nucleotides were modi¬ fied by the attack of the hydroxyl radical on the hepatic DNA. For the first time these results suggested that oxidative damage to DNA (e.g. through »OH) has a putative association with tumori¬ genesis in vivo.

In a subsequent 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 contaminant effects, including tumor formation (Environ. Health Perspectives 71, 5-16 (1987)) and other genotoxic changes (Aouatic 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 sealed, evacuated ampules and trimethylsilylated under an atmosphere of pure nitrogen, as undertaken previously.

TMS derivatives of the nucleotide bases were analyzed by GC- MS/SIM, essentially as previously described herein. Briefly, as before, the nucleotide bases were allowed to react with acetoni-

-Λ trile-bis(trimethylsilyl)trifluoracetamide (BSTFA) (2:1 v.v) for 45 min. at 80°C. Quantification of DNA base derivatives was undertaken on the basis of the mass to charge ratio (m/z): 354, 352, 442, and 440 for FapyAde, 8-OH-Ade, FapyGua and 8-OH-Gua, respectively. Analyses were performed with a Hewlett Packard 5890A gas chromatograph equipped with an auto-sampler interfaced to a Hewlett Packard mass-selective detector model 5970B. A fused silica capillary column coated with 5% phenylmethylsilicone gum phase (15m; 0.2mm i.d., and 0.3μm film thickness) was used for the separation of the DNA base derivatives. The column temperature was maintained at 120°C for 1.5 min., increased to 176°C at 3°/min., and then to 250°C at 6°/min. The injection port and ion source were kept at 250°C 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 those from the tumor-bearing 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- foci" (Aquatic Toxicol., 11, 43-67 (1988), Aguatic Toxicol. , 11, 143-162 (1988)). Both of these changes have been associated with exposure to a variety of environmental chemicals, including genotoxic agents (Aquatic

Toxicol. , 6, 165-177 (1985)). The low incidence of liver lesions **.** in the fish from Port Madison allowed samples to be obtained from a sub-population of English sole in one catching effort that showed 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 is, 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 previously obtained from histologically normal sole from uncontaminated reference areas (i.e., Elger Bay WA and Newport OR). FapyAde was higher only with respect 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 inteirmediate with respect to those of normal DNA from the reference fish from Newport and Elger Bay and the hepatic tumors of fish from Eagle Harbor. This is illustrated 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 shown. Statistical evaluation using single factor analysis of variance (ANOVA) revealed that, with the exception of FapyAde, significant differences existed between the concentrations of DNA lesions with respect to Eagle Harbor, Port Madison and the reference sites (Table 2).

The finding that the histologically normal fish from Port Madison had significantly higher concentrations of the DNA lesions compared to normal fish from the uncontaminated sites and significantly lower concentrations than fish from Eagle Harbor supports the concept that the •OH-induced DNA lesions progres¬ sively accumulate in the liver. Importantly, the fact that the DNA lesions occurred at intermediate concentrations in apparently normal fish from the tumor-bearing population at Port Madison lends support to the inventor's initial concept that the changes in DNA are causally related to tumorigenesis.

Overall, the present work is consistent with the inventor's hypothesis that the *OH-induced modification of the hepatic DNA in English sole is a progressive event initiated by exposure to environmental chemicals, the ultimate result being tumorigenesis. 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 is suggested that the DNA lesions have an important use in the future for predicting the occurrence of cancer in organisms exposed to carcinogens. Moreover, as implied previously, the DNA lesions represent readily evinced alterations at the molecular level that are highly relevant biomarkers for cytogenetic change in a variety of animal systems.

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 tissue from the five female patients was shown to contain invasive ductal carci¬ nomas; however, examination of the excised surgical margin tissue revealed no evidence for neoplasia, although there was some evidence for other microscopic changes (e.g. fibrocystic) . Residual carcinoma-containing and excised surgical margin tissue was placed in liquid nitrogen immediately after removal and maintained at -70°C 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 TMS derivatives were analyzed by GC-MS/SIM as described previously, using a Hewlett-Packard Model 5890 microprocessor-controlled gas chromatograph interfaced to a HP Model 5970B mass selective detector. The injector port and interface were both maintained at 250°C. The column was a fused silica capillary column (15.0 m., 0.2 mm inner diameter) coated with cross-linked 5% phenylmethylsilicone gum phase (film thickness, 0.33 μm) . The column temperature was increased from 120 to 176°C at 3°C/min. and from 176 to 250°C at 6°/min., after initially being held for 1.5 min. at 120°C. Helium was used as the carrier gas with a linear velocity of 23.5 cm./s through the column. The amount of TMS hydrolysate injected onto the column was about 0.7 μg. Quantitation of the modified nucleotide derivatives was undertaken on the basis of the principal ions, such as m/z 442 for the TMS derivative of FapyGua.

Analysis of the tissues revealed dramatic differences in the concentrations of 8-OH-Gua, FapyGua and 8-OH-Ade with respect to the control and the carcinoma tissues. The values for 8-OH-Gua,

-A 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, FapyAde is not a prominent indicator of altered DNA in breast cancer in contrast to the other base modifications. There was not a significant differences between the calf thymus and surgical margin DNA with respect to any of the base modifications; however, a significant difference did exist between the DNA from the surgical margin and the carcinoma tissue with respect to 8- OH-Gua (p < 0.01), FapyGua (p < 0.03) and 8-OH-Ade (p < 0.05). On a matched pair basis (surgical margin vs. 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 a relatively narrow range, close to the threshold of detection of the GC/MS-SIM method. In this regard, the present values with calf thymus DNA were not appreciably different from those ob¬ tained by the inventor and colleagues and other workers (Anal.

-A Biochem. 156, 182-188 (1986)). Moreover, in an initial attempt to understand 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 consistently 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 tissue may not be microscopically normal. Nevertheless, as indicated, in terms of the DNA bases examined, the surgical margin DNA was not significantly different from the calf thymus DNA. Accordingly, the calf thymus data which have previously served as a standard for "normal 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 pathologic or disease conditions. Thus, the

^~-Λ findings provided a unique opportunity to evaluate their signifi¬ cance in relation to the pathobiology of breast cancer. In this respect, 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 special need for maintaining the structural integrity of DNA through enzymatic and other processes, the substantial •0H- induced modifications in this molecule are likely to be causally related to the neoplastic transformations in the breast.

However, the origin of the •OH that potentially initiates the base modifications is unclear, although one possibility is that this 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)). EXPOSURE AND RECOVERY OF SUBJECT EXPOSED TO ENVIRONMENTAL TOXINS BY ASSAYING DEGREE OF NUCLEOTIDE ALTERATIONS.

Radical-induced alterations in DNA of Medaka. A study was conducted in which Medaka were chronically exposed to trichloroethylene (TCE), diethylnitrosamine (DEN) and groundwater (Final Report: U.S. Army Biomedical Research and Development Laboratory, Fort Detrick, MD; grant no. DAMD17-88-Z- 8043). In this experiment the two control values differed

-A significantly (Table 1). However, despite this difference, 10% groundwater and 100% groundwater appeared to have a particularly dramatic effect in raising the concentrations of the DNA base le¬ sions. 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 suggest that DEN tends to suppress the expression of the base modifications, although further work is required to verify this observation. Of particular interest in the present experiment is the fact that 5 ppm of TCE (with and without DEN) resulted in a substantial increase in the total base lesion 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 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 0.560 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- -.t mental chemicals results in significant increases in the DNA base lesions. The groundwater exposures are notable in that substan¬ tial 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 clearly exists to use the base lesions as biomarkers of the degree of exposure, as well as with regard to tracking the recovery process after clean-up operations at contaminated environmental sites. The influence of DEN on the exposures is presently unclear; however, the DEN appears to generally lower the tendency of the groundwater to increase the base concentra¬ tions, suggesting that competitive factors are influential.

Claims

What is claimed:

1) A method for detecting genotoxic injury occurring in vivo in a test subject comprising the steps of: a) obtaining a first in vivo specimen from a test subject; b) obtaining a second specimen from a second subject known not to have or be at risk of cancer, for the purpose of establishing a reference; c) analyzing in vitro sa_-- first and said second specimen to determine the quantity of DNA nucleotides

-A oxidatively modified in vivo; and d) comparing the quantity of each species of said oxidatively modified nucleotides in said first specimen to the quantity of each species of said oxidatively modified nucleotides in said second specimen wherein an increased likelihood of in vivo genotoxic injury 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 11 wherein said species of oxidatively modified nucleotides are selected from the group consisting of oxidatively modified purine or pyrimidine.

3) The method of claim 11 wherein said species of oxidatively modified nucleotides are selected from the group consisting of 8- OH-Gua, Fapy-Gua, 8-OH-Ade, and Fapy-Ade. 4) The method of claim 11 for detecting pre-neoplastic or neoplastic conditions in humans wherein said specimen comprises body tissue or body fluid and said species of oxidatively modified nucleotides is selected from the group consisting of 8- OH-Gua, Fapy-Gua, 8-OH-Ade, and 5-OHUra.

5) The method of claim 11 wherein said specimens comprise tissue or fluids obtained from said test subjects.

6) The method of claim 11 wherein the step of analyzing said first and said second specimen is by GC/MS-SIM which comprises the steps of: a) isolating DNA from said specimens; b) hydrolyzing said DΝA; c) preparing trimethylsilyl derivatives of said hydrolyzed DΝA; and d) determining presence of modified nucleotide bases and quantity thereof by introducing said derivatives into a GC/MS apparatus having SIM capabilities.

7) The method of claim 11 wherein the step of analyzing said first and said second specimen is by monoclonal antibody assay which comprises the steps of: a) isolating DΝA from said specimens; b) coating a solid surface with said DΝA; 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 17 wherein said quantitating comprises use of ELISA procedures.

9) The method of claim 17 wherein said quantitating comprises use of radioimmunoassay procedures.

10) The method of claim 11 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 20 wherein said quantitating comprises se of ELISA procedures. 12) The method of claim 20 wherein said quantitating comprises use of radioimmunoassay procedures.

13) The method of claim 11 wherein the step of analyzing said first and said second specimen is by spectrum analysis which comprises the steps of: a) extracting DNA from the specimens and hydrolyzing said DNA; b) introducing an appropriate tag to said hydrolyzed DNA;

-A and c) separating and quantitating the amount of oxidatively modified nucleotides.

14) The method of claim 23 wherein the step of separating and quantitating oxidatively modified nucleotides is by chromatography.

15) The method of claim 23 wherein chomophoric tags are used.

16) The method of claim 23 wherein radioactive tags are used.

17) A method for assaying the toxicity of an environment or compound to determine health risks to eukaryotes comprising the steps of: a) exposing an eukaryotic system to a subject environment; b) obtaining a first specimen from said eukaryotic system; c) obtaining a second specimen from a similar eukaryotic system 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 each species of said oxidatively modified nucleotides in said first specimen to the quantity of each species of said oxidatively modified nucleotides in said second specimen wherein an increased health risk to eukaryots exposed to the subject 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 27 wherein said species of oxidatively modified nucleotides are selected from the group consisting of oxidatively modified purine or pyrimidine.

19) The method of claim 27 wherein said species of oxidatively modified nucleotides are selected from the group consisting of 8- OH-Gua, Fapy-Gua, 8-OH-Ade, Fapy-Ade, and 5-OHUra.

20) The method of claim 27 wherein said specimens comprise tissue or fluids obtained from said test subjects. 21) The method of claim 27 wherein the step of analyzing said first and said second specimen is by GC/MS-SIM 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

-A GC/MS apparatus having SIM capabilities.

22) The method of claim 27 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 32 wherein said quantitating comprises se of ELISA procedures. 24) The method of claim 32 wherein said quantitating comprises use of radioimmunoassay procedures.

25) The method of claim 27 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;

-A 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 35 wherein said quantitating comprises use of ELISA procedures.

27) The method of claim 35 wherein said quantitating comprises use of radioimmunoassay procedures.

28) The method of claim 27 wherein the step of analyzing said first and said second specimen is by spectrum analysis which comprises the steps of: a) extracting DNA from the specimens and hydrolyzing said DNA; b) introducing an appropriate tag to said hydrolyzed DNA; and c) separating and quantitating the amount of oxidatively modified nucleotides.

29) The method of claim 38 wherein the step of separating and quantitating oxidatively modified nucleotides is by chromatography.

-.t 30) The method of claim 38 wherein chomophoric tags are used.

31) The method of claim 38 wherein radioactive tags are used.

32) A method for detecting pre-neoplastic conditions in a test subject comprising the steps of: a) administering a nucleotide analog to a first test subject and a second test subject, said second test subject known not to have or be at risk of neoplasia; b) exposing said first test subject to a desired environment for a desired period; c) obtaining a first and a second in vivo specimen from said first test subject and said second test subject; d) analyzing in vitro said first and said second specimen to determine the quantity of analog oxidatively modified in vivo; and e) comparing the quantity of each species of said oxidatively modified analog in said first specimen to the quantity of each species of said oxidatively modified analog in said second specimen wherein an increased likelihood of neoplasia is associated with an increased quantity, relative to said second specimen, of said oxidatively modified analog in said first specimen.