Combined use of chemically modified nucleobases and nanostructured DNA for enhanced immunostimulatory activity of CpG oligodeoxynucleotide.

Oligodeoxynucleotide (ODN) containing a cytosine-phosphate-guanine (CpG) motif, or CpG ODN, is considered suitable for treating immune diseases, including allergies. Although the phosphorothioate modification is used to enhance the stability and immunostimulatory activity of CpG ODNs, it is associated with the risk of adverse effects. Construction of nanostructured DNA assemblies, such as tripod- and hexapod-like structured DNAs, tripodna and hexapodna, respectively, were also found to increase this activity. The chemical modification of nucleobases could be another approach for enhancing CpG ODN activity. Here, we examined whether chemically modified nucleobase substitutions can enhance CpG ODN activity by measuring tumor necrosis factor α (TNF-α) release after addition to murine macrophage-like RAW264.7 cells. First, the guanine at the 18th position of phosphodiester CpG 1668 was substituted with several chemically modified guanines, and then the various guanines were substituted. Among all tested substitutions, 15,18-thdG, in which two guanines outside the CpG motif were substituted with the 2-aminothieno[3,4-d]pyrimidine guanine mimic (thdG), was the most effective. Compared to 32P-CpG 1668, 32P-15,18-thdG was taken up more efficiently by the RAW264.7 cells. Then, 15,18-thdG was incorporated into tripodna and hexapodna. 15,18-thdG/tri- or hexapodna induced higher TNF-α release from the RAW264.7 cells than PO CpG 1668/tri- or hexapodna, respectively. These results indicate that the thdG substitution is a useful effective strategy for enhancing the immunostimulatory activity of CpG DNAs in both single stranded and DNA nanostructure forms.

Highly Enhanced Antitumor Immunity by a Three-Barreled Strategy of the l-Arginine-Promoted Nanovaccine and Gene-Mediated PD-L1 Blockade.

Weak T cell responses and immune checkpoints within tumors could be two key factors for limiting antitumor efficacy in the field of cancer immunotherapy. Thus, the combined strategy of tumor vaccines and immune checkpoint blockade has been widely studied and expected to boost antitumor immune responses. Herein, we first developed a two-barreled strategy to combine the nanovaccine with a gene-mediated PD-L1 blockade. On the one hand, polyethyleneimine (PEI) worked as a vaccine carrier to codeliver the antigen ovalbumin (OVA) and the adjuvant unmethylated cytosine-phosphate-guanine (CpG) to formulate the PEI/OVA/CpG nanovaccine through electrostatic binding, which realized both dendritic cell activation and antigen cross-presentation enhancement. On the other hand, the PD-L1 silence gene was loaded by PEI to form PEI/pshPD-L1 complexes, which were further in situ shielded by aldehyde-modified polyethylene glycol (OHC-PEG-CHO) via pH-responsive Schiff base bonds. The formed pshPD-L1@NPs could decrease PD-L1 expression on the tumor cells. However, such a combined two-barreled strategy improved feebly for tumor inhibition in comparison with monotherapy, exhibiting the antagonistic effect, which might be due to the limited T cell response enhancement in the tumor microenvironment. To solve this problem, we have further developed a three-barreled strategy to combine oral administration of l-arginine, which worked as an amplifier to induce robust T cell response enhancement, without causing the upregulation of other negative immune regulators. Superior antitumor behavior and tumor rechallenge protection were realized by the three-barreled strategy in B16F10-OVA (B16-OVA)-bearing mice. The unique three-barreled strategy we developed might offer a novel clinical therapeutic treatment.

Immunological and mass spectrometry-based approaches to determine thresholds of the mutagenic DNA adduct O6-methylguanine in vivo.

N-nitroso compounds are alkylating agents, which are widespread in our diet and the environment. They induce DNA alkylation adducts such as O6-methylguanine (O6-MeG), which is repaired by O6-methylguanine-DNA methyltransferase (MGMT). Persistent O6-MeG lesions have detrimental biological consequences like mutagenicity and cytotoxicity. Due to its pivotal role in the etiology of cancer and in cytotoxic cancer therapy, it is important to detect and quantify O6-MeG in biological specimens in a sensitive and accurate manner. Here, we used immunological approaches and established an ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to monitor O6-MeG adducts. First, colorectal cancer (CRC) cells were treated with the methylating anticancer drug temozolomide (TMZ). Immunofluorescence microscopy and an immuno-slot blot assay, both based on an adduct-specific antibody, allowed for the semi-quantitative, dose-dependent assessment of O6-MeG in CRC cells. Using the highly sensitive and specific UPLC-MS/MS, TMZ-induced O6-MeG adducts were quantified in CRC cells and even in peripheral blood mononuclear cells exposed to clinically relevant TMZ doses. Furthermore, all methodologies were used to detect O6-MeG in wildtype (WT) and MGMT-deficient mice challenged with the carcinogen azoxymethane. UPLC-MS/MS measurements and dose-response modeling revealed a non-linear formation of hepatic and colonic O6-MeG adducts in WT, whereas linear O6-MeG formation without a threshold was observed in MGMT-deficient mice. Collectively, the UPLC-MS/MS analysis is highly sensitive and specific for O6-MeG, thereby allowing for the first time for the determination of a genotoxic threshold upon exposure to O6-methylating agents. We envision that this method will be instrumental to monitor the efficacy of methylating chemotherapy and to assess dietary exposures.

Inside-Out Signaling Pathways from Nuclear Reactive Oxygen Species Control Pulmonary Innate Immunity.

The airway mucosa is responsible for mounting a robust innate immune response (IIR) upon encountering pathogen-associated molecular patterns. The IIR produces protective gene networks that stimulate neighboring epithelia and components of the immune system to trigger adaptive immunity. Little is currently known about how cellular reactive oxygen species (ROS) signaling is produced and cooperates in the IIR. We discuss recent discoveries about 2 nuclear ROS signaling pathways controlling innate immunity. Nuclear ROS oxidize guanine bases to produce mutagenic 8-oxoguanine, a lesion excised by 8-oxoguanine DNA glycosylase1/AP-lyase (OGG1). OGG1 forms a complex with the excised base, inducing its nuclear export. The cytoplasmic OGG1:8-oxoG complex functions as a guanine nucleotide exchange factor, triggering small GTPase signaling and activating phosphorylation of the nuclear factor (NF)x03BA;B/RelA transcription factor to induce immediate early gene expression. In parallel, nuclear ROS are detected by ataxia telangiectasia mutated (ATM), a PI3 kinase activated by ROS, triggering its nuclear export. ATM forms a scaffold with ribosomal S6 kinases, inducing RelA phosphorylation and resulting in transcription-coupled synthesis of type I and type III interferons and CC and CXC chemokines. We propose that ATM and OGG1 are endogenous nuclear ROS sensors that transmit nuclear signals that coordinate with outside-in pattern recognition receptor signaling, regulating the IIR.

Regulation of MUTYH, a DNA Repair Enzyme, in Renal Proximal Tubular Epithelial Cells.

MUTYH is a DNA repair enzyme that initiates a base excision repair (BER) by recognizing and removing 8-Oxoguanine (8-oxoG) and its paired adenine. We demonstrated that both TGF-ß1 and H2O2 treatment led to an increased 8-oxoG in cultured human proximal tubule epithelial (HK-2) cells, while the former induced epithelial-mesenchymal transition and the latter caused cell apoptosis. Without stimulation, HK-2 cells showed MUTYH expression in mitochondria. TGF-ß1 triggered a transient upregulation of mitochondrial MUTYH and induced the expression of nuclear isoforms, while H2O2 showed no role on MUTYH expression. Ureteral obstruction (UUO) mice exhibited high 8-oxoG reactivity with tubulointerstitial lesions. After obstruction, the MUTYH expression was increased only in tubules at day 3 and decreased with obvious tubular atrophy at day 10. Particularly, MUTYH was primarily located in normal tubular cytoplasm with a dominant mitochondrial form. A few cells with nuclear MUTYH expression were observed in the fibrotic interstitium. We confirmed that increased MUTYH expression was upregulated and positively correlated with the severity of kidney fibrosis. Thus, renal fibrosis caused a cell-type-specific and time-dependent response of oxidative DNA repairs, even within the same tissues. It suggests that intervention of MUTYH might be effective for therapies.

Contrasting genome-wide distribution of 8-hydroxyguanine and acrolein-modified adenine during oxidative stress-induced renal carcinogenesis.

Oxidative stress is a persistent threat to the genome and is associated with major causes of human mortality, including cancer, atherosclerosis, and aging. Here we established a method to generate libraries of genomic DNA fragments containing oxidatively modified bases by using specific monoclonal antibodies to immunoprecipitate enzyme-digested genome DNA. We applied this technique to two different base modifications, 8-hydroxyguanine and 1,N6-propanoadenine (acrotein-Ade), in a ferric nitrilotriacetate-induced murine renal carcinogenesis model. Renal cortical genomic DNA derived from 10- to 12-week-old male C57BL/6 mice, of untreated control or 6 hours after intraperitoneal injection of 3 mg iron/kg ferric nitrilotriacetate, was enzyme digested, immunoprecipitated, cloned, and mapped to each chromosome. The results revealed that distribution of the two modified bases was not random but differed in terms of chromosomes, gene size, and expression, which could be partially explained by chromosomal territory. In the wild-type mice, low GC content areas were more likely to harbor the two modified bases. Knockout of OGG1, a repair enzyme for genomic 8-hydroxyguanine, increased the amounts of acrolein-Ade as determined by quantitative polymerase chain reaction analyses. This versatile technique would introduce a novel research area as a high-throughput screening method for critical genomic loci under oxidative stress.

Oxidative and nitrative DNA damage as biomarker for carcinogenesis with special reference to inflammation.

Reactive oxygen and nitrogen species are known to participate in a wide variety of human diseases. Oxidative DNAdamage is involved in chemical carcinogenesis and aging. Monocyclic chemicals induce mainly oxidative DNAdamage, whereas polycyclic chemicals can induce oxidative DNA damage in addition to DNA adduct formation. Recently, chronic infection and inflammation have been recognized as important factors for carcinogenesis. Nitrative DNA damage as well as oxidative DNA damage is induced in relation to inflammationrelated carcinogenesis. The authors examined the formation of 8-nitroguanine, a nitrative DNA lesion, in humans and animals under inflammatory conditions. An immunofluorescence labeling study demonstrated that 8-nitroguanine was strongly formed in gastric gland epithelial cells in gastritis patients with H. pylori infection, in hepatocytes in patients with hepatitis C, and in oral epithelium of patients with oral lichen planus. 8-Nitroguanine was also formed in colonic epithelial cells of model mice of inflammatory bowel diseases and patients with ulcerative colitis. Interestingly, 8-nitroguanine was formed at the sites of carcinogenesis regardless of etiology. Therefore, 8-nitroguanine could be used as a potential biomarker to evaluate the risk of inflammation- related carcinogenesis.

Adduct-specific monoclonal antibodies for the measurement of cisplatin-induced DNA lesions in individual cell nuclei.

The anticancer drug cisplatin executes its cytotoxic activity via formation of intra- and interstrand crosslinks in DNA. The relative contribution of structurally defined cisplatin adducts to induce apoptosis and the cellular processing of these lesions is still poorly understood mostly due to the lack of sensitive analytical tools for in vivo studies. Here we describe a new method to establish and characterize monoclonal antibodies (Mab) for structurally defined DNA adducts. The two major reaction products of cisplatin, the guanine-guanine (Pt-[GG]) and adenine-guanine (Pt-[AG]) intrastrand crosslinks are recognized by Mab R-C18 and R-B3, respectively. Both antibodies were employed in an immuno-cytological assay allowing the quantification of drug-induced lesions in individual cell nuclei at clinically relevant doses. Analyzing various tissues of cisplatin-treated C57Bl/6 mice the accumulation of Pt-(GG) was highest in kidney tubular cells compared with 30, 50 and 90% lower levels in kidney stroma, liver and peripheral blood cells, respectively. Adduct kinetics revealed that wild type mouse cells remove up to 80% of the crosslinks in contrast to their complete persistence in nucleotide excision repair-deficient (XPC-/-) mice. The aptitude of the immunoassay for human molecular dosimetry studies was demonstrated by measuring adduct levels in tumor biopsies from patients treated with cisplatin.

Analysis of urinary 8-nitroguanine, a marker of nitrative nucleic acid damage, by high-performance liquid chromatography-electrochemical detection coupled with immunoaffinity purification: association with cigarette smoking.

We have developed an analytical method to quantitate urinary 8-nitroguanine, a product of nitrative nucleic acid damage caused by reactive nitrogen species such as peroxynitrite and nitrogen dioxide. 8-Nitroguanine was purified from human urine using immunoaffinity columns with an anti-8-nitroguanine antibody, followed by quantitation by high-performance liquid chromatography-electrochemical detection. Four sequential electrodes were used to (a) oxidize interfering compounds (+250 mV), (b) reduce nitrated bases (two online electrodes at -1000 mV), and (c) quantitate reduced derivatives (+150 mV). Using this system 8-nitroxanthine can also be detected, with the detection limits being 25 and 50 fmol/injection for 8-nitroguanine and 8-nitroxanthine, respectively. The method was used to analyze both adducts in the urine of smokers (n=12) and nonsmokers (n=17). We found that smokers excrete more 8-nitroguanine [median, 6.1 fmol/mg creatinine; interquartile range (IQR), 23.8] than nonsmokers (0; IQR, 0.90) (p=0.018), and although 8-nitroxanthine was detected in human urine, its level was not related to smoking status. This is the first report to show that 8-nitroguanine is present in human urine and the methodology developed can be used to study the pathogenic roles of this adduct in the etiology of cancers associated with cigarette smoking and inflammation.

8-nitroguanine formation in the liver of hamsters infected with Opisthorchis viverrini.

Nucleic acid damage by reactive nitrogen and oxygen species may contribute to the carcinogenesis associated with chronic infection and inflammation. We examined 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation and nitric oxide (NO) production in hamsters infected with Opisthorchis viverrini (OV). Formation of 8-nitroguanine was assessed immunohistochemically with an antibody specific for 8-nitroguanine. 8-nitroguanine formation was found mainly in the cytoplasm and slightly in the nucleus of inflammatory cells and epithelial lining of bile duct at inflammatory areas in the liver. 8-nitroguanine immunoreactivity reached the highest intensity on day 30. A time profile of 8-nitroguanine formation was closely associated with that of plasma nitrate/nitrite. HPLC with an electrochemical detector revealed that the amount of 8-oxodG in the liver reached the maximal level on day 21. The mechanisms of 8-oxodG and 8-nitroguanine formation via O2*- and NO production triggered by OV infection were discussed in relation to cholangiocarcinoma development.

Molecular basis for the immunostimulatory activity of guanine nucleoside analogs: activation of Toll-like receptor 7.

Certain C8-substituted and N7, C8-disubstituted guanine ribonucleosides comprise a class of small molecules with immunostimulatory activity. In a variety of animal models, these agents stimulate both humoral and cellular immune responses. The antiviral actions of these guanosine analogs have been attributed to their ability to induce type I IFNs. However, the molecular mechanisms by which the guanosine analogs potentiate immune responses are not known. Here, we report that several guanosine analogs activate Toll-like receptor 7 (TLR7). 7-Thia-8-oxoguanosine, 7-deazaguanosine, and related guanosine analogs activated mouse immune cells in a manner analogous to known TLR ligands, inducing cytokine production in mouse splenocytes (IL-6 and IL-12, type I and II IFNs), bone marrow-derived macrophages (IL-6 and IL-12), and in human peripheral blood leukocytes (type I IFNs, tumor necrosis factor alpha and IL-12). The guanosine congeners also up-regulated costimulatory molecules and MHC I/II in dendritic cells. Genetic complementation studies in human embryonic kidney 293 cells confirmed that the guanosine analogs activate cells exclusively via TLR7. The stimulation of TLR7 by the guanosine analogs in human cells appears to require endosomal maturation because inhibition of this process with chloroquine significantly reduced the downstream activation of NF-kappaB. However, TLR8 activation by R-848 and TLR2 activation by [S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-R-Cys-S-Ser-Lys4-OH, trihydrochloride)] were not inhibited by chloroquine, whereas TLR9 activation by CpG oligodeoxynucleotides was abolished. In summary, we present evidence that guanosine analogs activate immune cells via TLR7 by a pathway that requires endosomal maturation. Thus, the B cell-stimulating and antiviral activities of the guanosine analogs may be explained by their TLR7-activating capacity.

Differential CD52 expression by distinct myeloid dendritic cell subsets: implications for alemtuzumab activity at the level of antigen presentation in allogeneic graft-host interactions in transplantation.

Alemtuzumab (anti-CD52; Campath 1-H) depletes both host and donor T cells when used in preparative regimens for allogeneic transplantation. This promotes engraftment even after nonmyeloablative conditioning and limits graft-versus-host disease (GVHD) even after unrelated or major histocompatibility complex (MHC) disparate allografts. We asked whether anti-CD52 differentially targets antigen-presenting cells (APCs), in addition to depleting T cells. Monocyte-derived dendritic cells (moDCs) expressed abundant CD52 as expected. Langerhans cells (LCs) and dermal-interstitial DCs (DDC-IDCs), however, never expressed CD52. Immunostaining of skin and gut confirmed the absence of CD52 on these resident DC populations under both steady-state and inflammatory conditions. Although anti-CD52 functions primarily by antibody-dependent cellular cytotoxicity (ADCC) in vivo, assessment of its activity in vitro included complement-dependent lysis of CD52(+) cells. Anti-CD52 did not impair DC-T-cell adhesion, diminish DC-stimulated T-cell proliferation, or alter moDC development in vitro. We propose that anti-CD52 abrogates GVHD not only by T-cell depletion, but also by removing moDCs and their precursors. This would mitigate moDC phagocytosis and presentation of host-derived antigens to donor T cells in the inflammatory peritransplantation environment, thereby limiting GVHD. The sparing of LCs and DDC-IDCs by anti-CD52, as well as the recovery of donor-derived moDCs in a less inflammatory environment later after transplantation, may allow all these DCs to exert formative roles in graft-versus-tumor (GVT) reactions and immune reconstitution. Whether these results support a separation of deleterious from beneficial graft-host interactions at the level of antigen presentation, rather than solely at the level of T cells, will require further evaluation.

Blocking neutrophil influx reduces DNA damage in hyperoxia-exposed newborn rat lung.

Hyperoxia-induced neutrophil infux in neonatal rats may contribute to impaired lung development through oxidative DNA damage. To determine whether blocking neutrophil influx prevents DNA damage, we treated newborn rats with 95% O2 beginning at birth, and at 3 and 4 d with nonimmune immunoglobulin G (IgG) (control) or anti-cytokine-induced neutrophil chemoattractant (CINC). At 8 d, lungs were inflation-fixed. Random sections were labeled using terminal transferase nick end-labeling (TUNEL), and DNA oxidation was measured using anti-8-OH-2'-deoxyguanosine (OHdG). To determine whether hyperoxia-induced TUNEL represented apoptosis, we labeled sections with anti-Bax (proapoptotic) and anti-Bcl-2 (antiapoptotic). We labled additional sections with anti-M30, directed against an epitope formed by caspase 6 digestion of cytokeratin 18 during apoptosis. Hyperoxia induced marked increases in TUNEL and OHdG signal in lung parenchymal cells, which was substantially prevented by treatment with anti-CINC. The large effects of hyperoxia on TUNEL were not accompanied by substantial effects on Bax, Bcl-2, or M30. We conclude that neutrophil influx during hyperoxia damages DNA by nicking and oxidation, and that blocking neutrophil influx can prevent this. Effects of 95% O2 on TUNEL are not primarily due to apoptosis in this model. Neutrophil-mediated oxidative DNA damage may contribute to abnormal lung development in newborns subjected to significant oxidative stress.

The influence of DNA sequence on the immunostimulatory properties of plasmid DNA vectors.

To determine the influence of DNA sequence on immunostimulatory properties of vaccine vectors, we tested the induction of in vitro and in vivo immune responses by plasmids modified to contain extended runs of dG sequences. Studies with oligonucleotides indicate that dG sequences can directly stimulate B cells as well as enhance the activity of immunostimulatory CpG motifs because of interaction with the macrophage scavenger receptor (MSR); this receptor can bind a variety of polyanions including dG sequences. To modify vectors, we introduced stretches of 20-60 dG residues into the pCMV-beta and pSG5rab.gp vectors and measured the ability of these plasmids to induce IL-12 and IFN-gamma production by murine splenocytes. The induction of in vivo antibody responses to rabies glycoprotein was also assessed with the pSG5rab.gp vectors. In in vitro cultures, cytokine production induced by plasmids with and without dG sequences was similar. Furthermore, the addition of dG sequences to pSG5rab.gp vectors failed to enhance the anti-rabies glycoprotein response to immunization. To assess further mechanisms by which plasmids stimulate macrophages, we measured the effects of MSR ligands on in vitro cytokine induction. In in vitro cultures, poly(G), dG30, and fucoidan inhibited IL-12 induction by plasmids. IL-12 induction was also inhibited by mammalian DNA but was unaffected by polyanions that are not MSR ligands. Together, these results suggest that the addition of 20 to 60-base dG sequences to plasmids does not significantly affect their properties as immunostimulators or vaccines. Furthermore, these results suggest that MSR ligands can block cytokine induction by plasmid DNA whether or not the plasmid contains extended runs of dG.

Guanine, mite, and cockroach allergens in Costa Rican homes.

Previous studies of schoolchildren in Costa Rica have shown an asthma prevalence of 23% and a high level of sensitization, particularly to mite allergens. As a continuation of these studies, some 400 dust samples were collected from various places in Costa Rica, and parts of these were analyzed for specific mite and cockroach allergens, as well as for the number of mites and amount of guanine. Guanine was quantified by a diazo, as well as an HPLC method, which were found to be highly correlated. The concentrations of guanine by the diazo method, Der p 1, Der f 1, and the number of mites were higher in bed dust than in bedroom floor dust, and it was possible to quantify mite allergens and guanine in almost all bed-dust samples. The mean levels were 2-3 times higher than the proposed risk level for elicitation of symptoms in mite-sensitive asthmatics. Bed and bedroom floor dust contained more guanine and mite allergen in humid (> 2000 mm rain) than in drier places (P < 0.05), but the number of mites in bed and bedroom floor dust was higher in less humid places (P = 0.01). The guanine content in bedroom floor dust was higher in areas with a temperate climate than in areas with a warmer climate (P < 0.001, Bartlett's chi square [BCS]), as was the number of mites (P < 0.01, Kruskal-Wallis [KW], 0.04, BCS) and the Der p 1 concentration (P = 0.01, BCS; P = 0.02, KW). The Der f 1 concentration in bedroom floor dust was higher in a warmer than in a temperate climate (P < 0.001, BCS). More guanine and mites were found in urban than in rural bed dust (P = 0.03, KW). Dust samples from the metropolitan area (temperate climate) of Costa Rica contained higher levels of guanine (P < 0.01) and Der p 1 (P = 0.07) than the coastal areas, but very little Der f 1. In these samples, guanine and Der p 1 allergen were closely related, and 2 micrograms of the allergen was equivalent to 0.49 mg of guanine. Two-thirds of bed and floor samples collected on cotton filters contained Bla g 2 allergen at mean levels of 1.6 and 2.1 units/g dust, respectively. Cockroach allergen was, however, absent in all bed samples from the metropolitan area, but did occur in very high concentrations in the coastal bed dust samples collected with tighter polyester filters. In conclusion, the concentration of guanine and Der p 1 was very high in the bed dust of Costa Rican homes. Some factors, such as humidity, small houses for large families, and type of bedding, probably favored the heavy mite infestation, which is probably related to the widespread occurrence of bronchial asthma in this country.

Formation and persistence of the miscoding DNA alkylation product O6-ethylguanine in male germ cells of the hamster.

The cellular parameters which modulate trans germ-line carcinogenesis by DNA-reactive agents have not yet been studied in detail. Therefore, we have measured in this study the formation and repair kinetics of the miscoding alkylation product O6-ethylguanine (O6-EtGua) in nuclear DNA of spermatogonial cells of the Syrian golden hamster (SGH) after exposure to either of two potent N-nitroso carcinogens, ethylnitrosourea (ENU) or diethylnitrosamine (DEN). Both compounds, the spontaneously decomposing ENU, and DEN, which has to be converted by cellular enzymes to the reactive ethyl diazonium ion, induce the same pattern of alkylation products in nuclear DNA. Adduct analyses were performed at the single-cell level by using a quantitative immunocytological assay and anti-(O6-EtGua) monoclonal antibodies. 1.5 h after intraperitoneal application of ENU (100 microg/g body weight) O6-EtGua levels in the nuclear DNA of spermatogonia were similar to those in other cell types of the same hamster. About 30% of the initially formed DNA adducts were still persistent in spermatogonial cells even 4 days after ENU exposure. The presence of O6-EtGua in DNA after exposure to DEN (100 microg/g body weight) implies the capability of hamster spermatogonial cells to convert nitrosamines into DNA-alkylating metabolites. This capability of male germ cells in combination with their limited repair capacity for a critical DNA adduct and their high rate of proliferation may be considered as a major risk factor for genetic effects including carcinogenesis in subsequent generation(s).

Phenylguanine found in urine after benzene exposure.

Comparative investigations with synthetic N7-phenylguanine were carried out to clarify whether this compound is eliminated via the urine of rats as a benzene-derived nucleic acid adduct. As sensitive methods for detecting trace amounts of the compound, gas chromatography-mass spectroscopy, high performance liquid chromatography, and two immunoassays (enzyme-linked immunosorbent assay and fluoroimmunoassay) with appropriate monoclonal antibodies were used. The results indicate the excretion of several benzene-related guanine adducts slightly different from N7-phenylguanine that may possibly be hydroxylated. These adducts differ also from (O6-, N2- and C8-phenylguanine, respectively.

Determination of 8-oxoguanine in DNA by gas chromatography--mass spectrometry and HPLC--electrochemical detection: overestimation of the background level of the oxidized base by the gas chromatography--mass spectrometry assay.

Two analytical methods, one involving the combined use of reverse-phase HPLC and electrochemical detection (HPLC-EC) and one involving a mass spectrometric detection after gas chromatography separation (GC/MS), were developed for the detection of 8-oxoguanine in DNA. In order to obtain quantitative results, 2,6-diamino-8-oxopurine, whose chemical structure and electrochemical response are very similar to 8-oxoguanine, has been employed as an internal standard in the HPLC-EC assay. In the case of the GC/MS method, an isotopically stable (M + 4) 8-oxoguanine has been employed as an internal standard. Both methods are able to detect approximately 1 modification per 10(6) DNA bases. The background level of 8-oxoguanine in DNA as determined by GC/MS is approximately 50-fold higher than that determined by the HPLC-EC assay. The discrepancy between the two methods is due to an artifactual oxidation of guanine during the derivatization reaction as demonstrated by using pure guanine. The amount of 8-oxoguanine in guanine, determined by GC/MS, increases linearly with the time of derivatization, indicating that an oxidation occurs during the silylation reaction. Derivatization under nitrogen atmosphere reduces but does not suppress the artifactual oxidation. The amount of 8-oxoguanine in DNA, quantified by GC/MS, is comparable to that obtained by HPLC-EC when 8-oxoguanine is prepurified by HPLC or by immunoaffinity chromatography, prior to the silylation reaction. The artifactual formation of 8-oxoguanine during the derivatization reaction may explain, at least in part, why the values reported for 8-oxoguanine determination by GC/MS are generally about 1 order of magnitude higher than that determined by HPLC-EC. Prepurification of 8-oxoguanine from guanine is recommended in order to obtain reliable results by GC/MS which may be compared to HPLC-EC.

Comparison of various immunochemical assays for the detection of ethylene oxide-DNA adducts with monoclonal antibodies against imidazole ring-opened N7-(2-hydroxyethyl)guanosine: application in a biological monitoring study.

Monoclonal antibodies have been developed for the analysis of the predominant lesion in DNA induced by ethylene oxide (EtOx), namely N7-(2-hydroxyethyl)guanine (N7-EtOHGua). Two monoclonal antibodies raised against imidazole ring-opened N7-(2-hydroxyethyl)guanine (RON7-EtOHGua), N7EO-E and N7EO-11, and the previously isolated antibody N7E-102 were characterized by competitive ELISA with various inhibitors. N7EO-E and N7EO-11 recognize 2-hydroxyethyl lesions better than ethyl or methyl lesions, while N7E-102 recognizes 2-hydroxyethyl and ethyl modifications equally well. All antibodies show a preference for imidazole ring-opened adducts, bind better to adducts in DNA compared to alkylated nucleosides or bases and bind 10(6)- to 3 x 10(6)-fold less well to unmodified DNA. The sensitivity of detection of RON7-EtOHGua in DNA and in the nuclei of cells in situ by antibody N7EO-E was investigated in several assays. The immunoslot blot assay was the most sensitive method (0.34 RON7-EtOHGua per 10(6) nucleotides was detectable), followed by competitive ELISA, direct ELISA and in situ detection by immunofluorescence microscopy. The immunoslot blot assay was used to analyse N7-EtOHGua levels in white blood cell DNA from individuals exposed to EtOx (2-5 p.p.m.) and of controls. This exposure did not result in a statistically significant increase in the N7-EtOHGua level.

Preparation of compound-specific and group-specific antibodies to 7-methylguanine and related 7-alkylguanines and their use in immunoaffinity purification.

The preparation and characteristics of compound-specific and group-specific antibodies against 7-alkylguanines (7-alkGua) are described. A compound-specific antibody against 7-methylguanine was prepared using a hapten bound to carrier protein through the N2 position. In a competitive enzyme-linked immunosorbent assay (ELISA) 7-methylguanine (7-MeGua) showed 50% inhibition (I50%) at 10 pmol/well at room temperature, but the inhibition was found to be 40 times better at 4 degrees C (I50% at 250 fmol/well). When the antibody was bound to protein A-Sepharose CL4B 7-MeGua was retained in immunoaffinity columns. A group-specific antibody to 7-alkGua was prepared using 7-(2-carboxyethyl)guanine (7-CEGua) bound to carrier protein via the carboxyl group. In a competitive ELISA, this antibody cross-reacted well with 7-CEGua, 7-ethylguanine (7-EtGua), 7-(2-hydroxyethyl)guanine (7-HOEtGua) and 7-(2',3'-dihydroxy)-propylguanine (7-DHPGua) and some inhibition was seen with 7-MeGua. Immunoaffinity columns prepared from this antibody retained a number of 7-alkGua of diverse structure. 7-EtGua in calf thymus DNA treated with diethylsulphate and ethylnitrosourea was isolated by immunoaffinity purification and quantified by HPLC-fluorescence. These results illustrate the potential of immunoaffinity purification for both individual DNA adducts and groups of adducts.