[32P]ATP mediates formation of 8-hydroxy-2'-deoxyguanosine from 2'- deoxyguanosine, a possible problem in the 32P-postlabeling assay

8-Hydroxy-2'-deoxyguanosine (8-OH-dG) is a biomarker for oxidative stress on DNA, a common lesion in mammalian cells. A correlation between increased levels of 8-OH-dG and diseases like diabetes, infections and cystic fibrosis has been found in humans. 8-OH-dG levels have been shown to be decreased by antioxidants, an indication of the importance of dietary habits. 8-OH-dG is used as a biomarker for oxidative stress in vivo as well as in vitro and is suggested to be a mutagenic DNA lesion. Different methods are used for the analyses of 8- OH-dG, i.e. GC-MS, HPLC-EC and 32P-postlabeling. The most commonly used method is HPLC-EC. In the analysis of 8-OH-dG, the work-up procedure for DNA, as well as the preparation for analysis, are of critical importance as there is a risk for auto-oxidation of deoxyguanosine (dG), which would result in false high background levels and low sensitivity in analysis. 32P-Postlabeling has recently been applied to the analysis of 8-OH-dG and has shown to be a very sensitive method for the detection of DNA adducts. It is shown here that after extrapolation to normal 32P-postlabeling conditions, [32P]ATP generated 8-OH-dG to levels of 25 8-OH-dG/10(5) dG. [32P]ATP mediated the formation of 8-OH- dG from dG in a dose-dependent manner at all dose levels (0.13-12 microCi). The reaction occurred immediately and increased with time in a linear dose-response fashion. At high doses (6.0 and 12 microCi) the dose-response declined after 24 h, which indicates a possible decomposition or rearrangement of 8-OH-dG. A repeated experiment with 5 microCi [32P]ATP during 2 h resulted in a linear formation of 8-OH-dG and a level of 19 8-OH-dG/10(5) dG. The results indicated that awareness of the auto-oxidation generated by 32P[ATP] in the postlabeling assay is of utmost importance and that dG must be separated before 32P-postlabeling of 8-OH-dG.      

Formation of DNA adduct 8-hydroxy-2'-deoxyguanosine induced by man-made mineral fibres

Two man-made mineral fibres, rockwool and glasswool, were found to mediate hydroxylation of deoxyguanosine and calf thymus DNA to form the DNA adduct 8-hydroxy-2'-deoxyguanosine. The modification of the nucleoside is probably mediated by hydroxyl radicals and may play a role in fibre-induced carcinogenesis.     

Formation of cyclic adducts of deoxyguanosine with the aldehydes trans-4-hydroxy-2-hexenal and trans-4-hydroxy-2-nonenal in vitro

trans-4-Hydroxy-2-hexenal (t-4HH), a reactive metabolite isolated from the pyrrolizidine alkaloid senecionine, and trans-4-hydroxy-2-nonenal (t-4HN), a product of lipid peroxidation, reacted nonenzymatically with deoxyguanosine at pH 7.4 at 37 degrees C in vitro with each compound yielding two pairs of diastereomeric adducts. Adducts were isolated using reverse phase high-performance liquid chromatography and were characterized by their mass spectra and proton magnetic resonance spectra. Adducts 1 and 2 from t-4HH were assigned the structures 3-(2-deoxy-beta-D-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8R-hydroxy- 6S[1- (R and S)hydroxypropyl]pyramido[1,2-a]purine-10-(3H)one and Adducts 3 and 4 were assigned the structures 3-(2-deoxy-beta-D-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8S-hydroxy- 6R-[1- (R and S)hydroxypropyl]pyramido[1,2-a]purine-10-(3H)one. Similar 6-hydroxyhexyl adducts were isolated in the reaction of deoxyguanosine with t-4HN. The reactions appear to involve Michael additions of the N2 amino group of deoxyguanosine followed by cyclization at the 1-N site. This reaction mechanism is similar to that reported for deoxyguanosine adduct formation with the nonhydroxylated alpha, beta-unsaturated aldehydes crotonaldehyde and acrolein. Total adduct formations following 16-h incubations were 0.91% for t-4HH and 0.85% for t-4HN. These results demonstrate that t-4HH and t-4HN possess the ability to alkylate deoxyguanosine in vitro and suggest possible mechanisms for 4-hydroxyalkenal and pyrrolizidine alkaloid genotoxicity.     

Capability of human blood cells to form the DNA adduct, C8-(N2-aminofluorenyl)-deoxyguanosine-3'-5'-diphosphate from 2-aminofluorene

Human blood cells, separated by Ficoll-Hypaque centrifugation, were tested for their ability to catalyze the formation of DNA adducts of 2-aminofluorene (AF), using the 32P-postlabeling procedure for adduct analysis. Incubation of neutrophils with AF, hydrogen peroxide and exogenous DNA yielded a single DNA adduct identified as C8-(N2-aminofluorenyl)-deoxyguanosine-3'-5'-diphosphate (AFdG) by cochromatography with a standard sample. AFdG levels in intact cells, lysed cells and in the granule fraction prepared from cell lysates were 102, 894 and 240 AFdG adducts/10(9) nucleotides/30 min respectively. AFdG levels corresponded to the activity of neutrophil peroxidase in these preparations. The monocyte/lymphocyte fraction yielded a low amount of 30 and 40 AFdG/10(9) nucleotides/30 min in the presence of hydrogen peroxide and of NADPH respectively. Erythrocytes did not generate a detectable level of AFdG, neither as intact cells nor as cell lysates. Whole blood samples likewise did not generate AFdG. Our findings reveal that, among blood cells, only neutrophils are capable of forming a biologically significant DNA adduct of aminofluorene in reasonable amounts and suggest that myeloperoxidase was the catalyzing enzyme.     

Enhanced levels in neonatal rat liver of 7,8-dihydro-8-oxo-2'- deoxyguanosine (8-hydroxydeoxyguanosine), a major mutagenic oxidative DNA lesion

The purpose of this study was to determine whether the level of 7,8- dihydro-8-oxo-2'-deoxyguanosine (8-hydroxy-2'-deoxyguanosine) (8-oxo- dG), a major mutagenic DNA oxidation product, is enhanced in newborn rat liver DNA as a consequence of oxidative stress incurred during the early postnatal period. 32P-postlabeling showed this adduct to increase approximately 2-fold from the 20th day of gestation (2 days before birth) to a peak level at 50-53 h after birth. Postnatal levels exceeded fetal levels at all time points investigated, i.e. 0.5-1, 8, 24, 50-53, 100, 216 and 432 h after birth. Increased formation of this mutagenic DNA lesion during the critical postnatal phase when there is rapid cell proliferation in all tissues is proposed to contribute to carcinogenesis in susceptible tissues later in life.      

X-radiation induces 8-hydroxy-2'-deoxyguanosine formation in vivo in rat mammary gland DNA

Ionizing radiation is a carcinogen that induces oxidative DNA damage. 8- Hydroxy-2'-deoxyguanosine (8-OHdG) is a relatively abundant, mutagenic lesion that is widely regarded as a reliable index of oxidative DNA damage. The purpose of this study was to examine the effects of X- radiation on levels of 8-OHdG in the context of an experimental model for breast cancer in which chronic radiation exposure has been shown to be carcinogenic in Sprague-Dawley rats. A secondary objective of this study was to determine if the use of phenol during DNA isolation affected the concentration of 8-OHdG subsequently measured. Our results indicate that a profoundly carcinogenic dose of radiation induced a small but significant increase in 8-OHdG concentration in mammary gland DNA, and that the use of a phenol-based versus a salt-based method of DNA isolation had no significant impact on the levels of 8-OHdG detected in either control or irradiated tissue.      

Immunoaffinity isolation of urinary 8-hydroxy-2'-deoxygunosine and 8-hydroxyguanine and quantitation of 8-hydroxy-2'deoxyguanosine in DNA by polyclonal antibodies

An immunoaffinity column is described that facilitates the analysisof oxidative DNA adducts excised from DNA are excreted in urineand can assayed as a measure of DNA damage in individuals. Polyclonalantibodies that recognize 8-hydoroxy-2'-deoxyguanosine (oh⁸dG),a biiomarker of oxidative damage to DNA, have been producedand their binding properties characterized. The antibodies,raised in rabbits following immunization with protein carrier-haptenconjugates prepared by covalently linking periodate-treated8-hydroxyguanosine (oh⁸dG to bovine serum albumin (BSA) or casein,bind ohn⁸dG with high affinity and selectivity, as measuredby a competitive radio-immunoassy (RIA). Antibodies oboitainedfrom the rabbits immunized with the casein conjugate exhibiteda binding affinity for oh⁸dG of 6.9 10⁸ x M^–1. Studieson the relative binding affinities of these polyclonal antibodiesfor oh⁸dG, unmodified nucleosides, or derivatives of guanineindicate that the antibodies are suitable for the preparationof immuno-affinity dolumns that permit us to rapidly isolateoh⁸dG and 8-hydroxyguanine (oh⁸dG) from urine. The high selectivityof the antibodies for oh⁸dG and oh⁸dG reduces the amount ofurinary contaminants previsouly observed in samples preparedby solid phase extraction, thus greatly facilitating the isolationof these damage products from urine. The relative binding affinityof these antibodies for oh⁸dG. The antiboty can be used to quantitateoh⁸dG in enzymatic hydrolyzated of DNA with values comparableto those obtained by HPLC with electrochemicaldetection (HPLC-EC).     

Activation of 4-hydroxytamoxifen and the tamoxifen derivative metabolite E by uterine peroxidase to form DNA adducts: Comparison with DNA adducts formed in the uterus of Sprague-Dawley rats treated with tamoxifen

Daily intraperitoneal treatment of female Sprague-Dawley ratswith either 5, 10 or 20 mg/kg tamoxifen (TAM) for 1 weeks increasedthe level of peroxidase activity in the uterus 2- to 10-foldcompared to the control level. Using uterine extracts preparedfrom control and TAM treated animals, we investigated the activationof 4-hydroxytamoxifen (4-HO-TAM) and (E, Z)-1, 2-dipheny-1-(4-hydroxyphenyl)-but-1-ene(cis/trans-metabolite E) to form DNA adducts. Activation of4-HO-TAM by uterine extracts prepared from either control orTAM-treated rats produced one major (a) and Two minor DNA (band c) adducts. A similar activation of cis/trans-metaboliteE produced two adducts (d and e). There was good correlationbetween levels of uterine peroxidase activity and levels ofDNA adducts formed by 4-HO-TAM and cis/trans-metabolite E. Activationof 4-HO-TAM and cis/trans-metabolite E with horseradish peroxidase(HRP) produced the same adducts as observed by activation withuterine extract Treatment of Sprague-Dawley rats with 5 and10 mg/kg for 7 days produced eleven DNA adducts in the liverwith no adducts detected in the uterus. However, treatment ofrats with 20 mg/kg of TAM for 7 days produced the same adductpattern in the liver and also one major adduct (1) in the uteruswith a relative adduct level of 6.4 ±4.1x10^–9.Tamoxifen-DNA adduct 1 detected both in the liver and in theuterus of treated rats was similar to adducts produced by activationof 4-HO-TAM with either uterine extract or HRP. The resultsof these studies suggest a general model whereby the tamoxifenmetabolite 4-HO-TAM is further activated in the uterus by peroxidaseenzymes to form DNA adducts.     

5-Aminolevulinic acid mediates the in vivo and in vitro formation of 8-hydroxy-2'-deoxyguanosine in DNA

5-Aminolevulinic acid (ALA), a heme precursor accumulated inchemical and inborn porphyrias, may behave as an endogenouspro-oxidant In chronically treated rats (40 mg ALA/kg body wtevery 2 days for 15 days) the steady-state level of 8-hydroxy-2'-deoxyguanosine(8-OHdG) in liver DNA (94.5 ± 233 residues/106 dG) was4.5 times higher than in non-treated rats (21 ± 7.5 residues/10⁶ dG). In vitro exposure of calf thymus DNA to ALA (0.05-5mM) in the presence of 10 uM Fe²+ caused the formation of 8-OHdG.The amount of 8-OHdG rose from 135 ± 15 residues/10⁶dG in the control system to 1140 ± 150 residues/106 dGafter incubation with 5 mM ALA and 10 µM Fe²1. Diethylenetriaminepentaaceticacid (5 mM) or mannitol (100 mM) inhibited the formation of8-OHdG by 63 and 69% respectively, evidencing the involvementof both H2O2 and HO in this process. Hydrogen peroxide (100µM) or Fe²+ alone did not cause DNA oxidation. The presentdata support the hypothesis that ALA-generated reactive oxygenspecies can oxidize DNA and may be involved in the developmentof primary liver cell carcinoma in individuals with symptomaticacute intermittent porphyria.     

Formation of C8-modified deoxyguanosine and C8-modified deoxyadenosine as major DNA adducts from 2-nitropyrene metabolism mediated by rat and mouse liver microsomes and cytosols

2-Nitropyrene, the geometric isomer of the most studied nitropolycyclic aromatic hydrocarbon (nitro-PAH), 1-nitropyrene, is an environmental contaminant detected in ambient air and a potent direct-acting mutagen. Its metabolic activation leading to the formation of DNA adducts was studied. The activated metabolite, N-hydroxy-2-aminopyrene, was prepared and reacted with calf thymus DNA. Upon enzymatic hydrolysis of the DNA, the resulting nucleosides were separated by HPLC, and the adducts were characterized by mass and proton NMR spectral analysis. Both N-(deoxyguanosin-8-yl)-2-aminopyrene and N-(deoxyadenosin-8-yl)-2-aminopyrene, in a 5:2 ratio, were identified. These adducts were then utilized as standards to identify the DNA adducts formed from reaction of [3H]2-nitropyrene with DNA mediated by liver microsomes and cytosols of mouse and rat. In all cases, both adducts were formed. The quantities of the two adducts formed in each system were: mouse liver microsomes (11.3 pmol [3H]2-nitropyrene/mg DNA), rat liver microsomes (23), mouse liver cytosol (11.4) and rat liver cytosol (5.1). Thus, these adducts were formed in highest yield from rat liver microsomes and the lowest from rat liver cytosol. The deoxyguanosine/deoxyadenosine adduct ratio was higher from rat and mouse liver microsomes (7.8:9.2) than from rat and mouse liver cytosols (2.5:3.1). Our results represent the first direct demonstration of a C8-deoxyadenosine adduct being formed as a major product from the reaction of a nitro-PAH metabolite with DNA.     

Role of sulfation in the formation of DNA adducts from N-hydroxy-2-acetylaminofluorene in rat liver in vivo. Inhibition of N-acetylated aminofluorene adduct formation by penta-chlorophenol

N-Hydroxy-2-acetylaminofluorene (N-OH-AAF) is metabolicallyconverted into reactive N, O-esters which are capable of formingcovalent adducts with DNA in rat liver in vivo. The effect ofinhibiting one of the proposed pathways, N-O-sulfation, on DNAadduct formation was studied by using a specific sulfotransferaseinhibitor, pentachlorophenol. Rats were pretreated with pentachlorophenoland, after 45 min, N-OH-AAF was administered. Four hours afterdosing the animals were sacrificed and hepatic DNA was isolated.In DNA from control livers two acetylaminofluorene-and one amino-fluorene-substituteddeoxyguanosine adducts were found. The acetylaminofluorene derivatives,N-(deoxy-guanosin-8-yl)-2-acetylaminofluorene and 3-(deoxy-guanosin-N²-yl)-2-acetylaminofluorene,accounted for 40% of the total binding in the hydrolyzed DNA.The aminofluorene adduct, N-(deoxyguanosin-8-yl)-2-aminofluorene,accounted for the remainder. In rats that were pretreated withpentachlorophenol, total DNA binding was decreased by 26%. Thesame three adducts were found, but the acetylaminofluorene adductswere now only 13% of the total, while the aminofluorene adductaccounted for 87%. The absolute amount of aminofluorene adductwas not altered as compared to control rats. These data demonstratethe involvement of N-O-sulfation in carcinogen - DNA bindingand indicate that at least 70% of the acetylaminofluorene boundto deoxyguanosine in rat liver DNA, in vivo, is formed throughN-O-sulfation of N-OH-AAF.     

DNA adduct formation in primary rabbit tracheal epithelial cells following treatment with 1,8-dinitropyrene and its partially reduced derivative, 1-nitro-8-nitrosopyrene

Formation of DNA adducts, following treatment of primary rabbit tracheal epithelial cells (RTEC) with 1,8-dinitropyrene (1,8-DNP) and its partially reduced derivative, 1-nitro-8-nitrosopyrene (1,8-NONO2), was examined using the 32P-post-labelling technique. Treatment of aerobic cells with 1,8-DNP or 1,8-NONO2 produced qualitatively similar results. Cochromatography showed that the major adduct observed corresponded to the major adduct seen following treatment of poly(dG.dC) with N-hydroxyl-1-amino-8-nitropyrene, generated from 1,8-NONO2. A minor adduct migrated to the same area on the TLC plate as the major compound observed following a similar treatment with poly(dA.dT). Relative adduct labelling (RAL) values were consistently an order of magnitude higher with 1,8-NONO2 than with 1,8-DNP, suggesting that reduction of a nitro group of 1,8-DNP to a nitroso group may be a rate-limiting step in the cells. In studies on the formation and persistence of the 1,8-NONO2 adduct in RTEC maximum binding was observed at 1 h. Fifteen hours later the RAL value was less than 15% of this maximum level.     

Microsomal and peroxidase activation of 4-hydroxy-tamoxifen to form DNA adducts: comparison with DNA adducts formed in Sprague-Dawley rats treated with tamoxifen

Using rat liver microsomal preparations and peroxidase enzymes,we have investigated the formation of DNA adducts by the antiestrogencompound tamoxifen (TAM) and its metabolite 4-hydroxy-tamoxifen(4-OH-TAM). When reduced nicotinamide-adenine dinucleotide phosphate(NADPH) was used as a cofactor in microsomal activation of either4-OH-TAM or TAM, one DNA adduct and relative DNA adduct levelsof 4.6 and 3.1x10^–8, respectively were detected by 32P-postlabeling.The DNA adduct produced by microsomal activation of 4-OH-TAMand TAM was the same. With cumene hydroperoxide (CuOOH) as thecofactor for the microsomal activation of either 4-OH-TAM orTAM, three to six DNA adducts were produced; the relative adductlevels were 8.0 and 20.6x10^–8, respectively. Comparisonof the DNA adduct patterns produced by 4-OH-TAM and TAM showedthat they were distinct However one of the DNA adducts (a) producedby microsomal activation of 4-OH-TAM using CuOOH was the sameas adduct a produced by microsomal activation of 4-OH-TAM withNADPH. Activation of 4-OH-TAM with horseradish peroxidase resultedin the formation of a single DNA adduct and a relative adductlevel of 20.7x10^–8. Rechromatography analysis of thisDNA adduct showed that it was identical to that produced bymicrosomal activation of 4-OH-TAM with NADPH and one of theadducts produced using CuOOH as the cofactor. Ten DNA adductsand a relative adduct level of 15.3x10^–8 were detectedin the liver of female Sprague-Dawley rats treated daily with20 mg/kg of TAM for 7 days. The DNA adduct pattern in the liverof the treated animals was similar to that produced by microsomalactivation of TAM using CuOOH as the co-factor. The principalDNA adduct (no. 6) formed in the livers of rats treated withTAM was the same as the principal DNA adduct formed followingmicrosomal activation of TAM using CuOOH as a cofactor. TheDNA adduct formed following microsomal activation of eitherTAM or 4-OH-TAM using NADPH was also present as one of the adducts(1^*) formed in vivo following TAM treatment These studies demonstratethat 4-OH-TAM can be activated to form DNA adducts and thatit contributes to the formation of DNA adducts in the liverof rats treated with TAM.     

Cyclooxygenase-2, malondialdehyde and pyrimidopurinone adducts of deoxyguanosine in human colon cells

Cyclooxygenases (COX) catalyse the oxygenation of arachidonic acid to prostaglandin (PG) endoperoxides. Activity of one of the COX isoforms, COX-2, results in production of prostaglandin E(2) (PGE(2)) via the endoperoxide PGH(2). COX-2 has been implicated in the pathogenesis of colorectal cancer. Malondialdehyde (MDA) is a mutagen produced by spontaneous and enzymatic breakdown of PGH(2). MDA reacts with DNA to form adducts, predominantly the pyrimidopurinone adduct of deoxyguanosine (M(1)G). Here the hypothesis was tested that COX-2 activity in human colon cells results in formation of MDA and generation of M(1)G adducts. M(1)G was detected in basal cultures of human non-malignant colon epithelial (HCEC) and malignant SW48, SW480, HT29 and HCA-7 colon cells, at levels from 77 to 148 adducts/10(8) nucleotides. Only HCA-7 and HT29 cells expressed COX-2 protein. Levels of M(1)G correlated significantly (r = 0.98, P < 0.001) with those of intracellular MDA determined colorimetrically in the four malignant cell types, but neither parameter correlated with expression of COX-2 or PG biosynthesis. Induction of COX-2 expression by phorbol 12-myristate 13-acetate in HCEC cells increased PGE(2) production 20-fold and MDA concentration 3-fold. Selective inhibition of COX-2 activity in HCA-7 cells by NS-398 significantly inhibited PGE(2) production, but altered neither MDA nor M(1)G levels. Malondialdehyde treatment of HCEC cells resulted in a doubling of M(1)G levels. These results show for the first time in human colon cells that COX-2 activity is associated with formation of the endogenous mutagen, MDA. Moreover, they demonstrate the correlation between MDA concentration and M(1)G adduct levels in malignant cells.     

Application of an immunoassay for cyclic acrolein deoxyguanosine adducts to assess their formation in DNA of Salmonella typhimurium under conditions of mutation induction by acrolein

Acrolein has been shown to form cyclic deoxyguanosine adductswhen it reacts with DNA in vitro. In this study, we have useda recently developed immunoassay for these adducts to studytheir formation in DNA from Salmonella typhimurium exposed toacrolein. Acrolein-deoxyguanosine adducts were formed in a dose-dependentfashion in Salmonella tester strains TA100 and TA104, reachinglevels as high as 5 µmol adduct per mol deoxyguanosine.Using the liquid pre-incubation assay acrolein-induced mutationswere also found in strains TA100 and TA104. The correlationbetween acrolein-deoxyguanosine adduct concentration and acrolein-inducedmutations in TA100, which contains GC base pairs at the siteof reversion, suggests that the acrolein-deoxyguanosine adductis a promutagenic lesion. That mutations are also seen in TA104which contains AT base pairs at the site of reversion suggestthat adducts of bases other than deoxyguanosine may also beimportant in the mutagenic activity of acrolein.     

Quantitative immunoanalysis of promutagenic 8-hydroxy-2'-deoxyguanosine in oxidized DNA

The modified DNA base 8-hydroxyguanine has been implicated inspontaneous mutagenesis, carcinogenesis and cellular aging.Polyclonal antibodies specific for the 8-hydroxy-2'-deoxyguanosinemoiety in oxidized DNA were used for sensitive detection andquantitation of this biomarker of oxidative damage to cellularDNA. The analysis was performed with immunoslot blot assay (ISB)of oxidized DNA modified in vitro with methylene blue plus lightand upon H₂O₂ treatment of cultured human cells. The level of8-OHdG in DNA exposed to 90 and 120 min light in the presenceof 100 µM methylene blue showed 15.96 ± 2.4 and22.65 ± 3.65 pmol/µg DNA compared to 0.107 ±0.024 pmol/µg in commercial calf thymus DNA control. Inherentdamage, due to cellular endogenous oxidation of DNA, increasedsignificantly upon inhibition of catalase activity in humancells with 10 mM azide. The damage increased further on exposureof azide-treated cells to H₂O₂. The amounts of 8-OHdG followingtreatment of cells with 10 and 100 µM H₂O₂ were determinedto be 205 ± 42 and 333 ± 17.5 pmol/µg DNArespectively. Very low but quantifiable antibody binding wasseen with the ‘control unoxidized’ human nuclearDNA. This DNA, obtained under controlled conditions to restrictthe induction of 8-OHdG during isolation, provides a backgroundlevel of 0.022 ± 0.005 pmol 8-OHdG/µg DNA. Thequantitative assessment of 8-OHdG by ISB assay, with fmol sensitivityand direct analysis using unhydrolyzed DNA, should prove a highlyvaluable alternative to currently used approaches to detecting8-OHdG in enzymatic DNA hydrolysates.     

Glutathione S-transferase mu in human lymphocyte and liver: role in modulating formation of carcinogen-derived DNA adducts.

Glutathione transferase (GT) activity towards trans-stilbene oxide (tSBO), benzo[a]pyrene-4,5-oxide (B[a]PO) and 1-chloro-2,4-dinitrobenzene (CDNB) was measured in human liver and lymphocytes. GT-tSBO activity is catalyzed by GT mu which has polymorphic expression in human lymphocytes. Our results show that activity of GT-tSBO in lymphocytes correlates with its activity in liver (r = 0.7, P less than 0.001). GT activity towards BPO (GT-BPO) also correlated with GT-tSBO in lymphocytes and liver. However, interindividual variation of GT-BPO is less than that of GT-tSBO, suggesting that BPO may not be as specific a substrate for GT mu and therefore other GT isozymes may contribute to BPO conjugation. Conjugation of CDNB by GT was not different using cytosols from either high or low GT mu individuals. The functional significance of the GT-mu polymorphism was evaluated by measuring its effect on benzo[a]pyrene (B[a]P)- and aflatoxin B1 (AFB1)-DNA adduct formation in vitro. Human liver cytosols prepared from persons having low or high GT-tSBO activity were incubated with human liver microsomes, calf thymus DNA and B[a]P or AFB1. HPLC analysis revealed that the major B[a]P adduct was dG(N2)-7 beta, 8 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-dG). BPDE-dG adducts were decreased equally by cytosols from either low or high conjugators. In contrast, AFB1-DNA binding was inhibited to a greater extent in high conjugators than low conjugators. HPLC analysis demonstrates that adducts formed were AFB1-FAPyr and AFB1-N7-Gua. The correlation between AFB1-DNA adduct concentrations and GT mu activity was highly significant with a correlation coefficient of r = 0.88 at P less than 0.001. These results suggest that GT mu plays an important role in detoxifying DNA reactive metabolites of AFB1 and this enzyme may be a susceptibility marker for AFB1 related liver cancer. Moreover, our data demonstrate that lymphocytes are a reliable surrogate tissue for detecting liver GT mu polymorphisms.     

Radiation-induced formation of 8-hydroxy-2'-deoxyguanosine and its prevention by scavengers

Reactive oxygen species (ROS) induce 8–hydroxy–2'-deoxyguanosine(8-OHdG) formation, which has been proposed as a key biomarkerrelevant to carcinogenesis. 8-OHdG has been induced in a numberof different ways, most often without knowledge of the specifictype and amount of ROS generated. We have measured 8-OHdG formationin calf thymus DNA exposed to ionizing radiation under conditionsgenerating either hydroxyl radicals (OH·), superoxideanions (O2^–) or both. Additionally, we investigated therelationship between the scavenger effect of the drug 5-aminosalicylicacid (5-ASA) and increasing OH· exposure toward 8-OHdGformation. The effect of this drug was compared to those ofthe physiological scavengers ascorbate and reduced glutathione(GSH). We found that OH· generated 8-OHdG in a dose-dependentmanner, whereas O₂^– did not cause 8-OHdG formation. 5-ASA,ascorbate and GSH all acted as hydroxyl radical scavengers,although with different concentration-effect curves, emphasizingthe importance of using relevant pharmaco-/physiological concentrationsin studies focusing on therapeutic applications of scavengers.The scavenger effect of 5-ASA at concentrations     

Peroxidative in vitro metabolism of diethylstilbestrol induces formation of 8-hydroxy-2'-deoxyguanosine

The generation of superoxide and hydroxyl radicals is knownto be implicated in the hydroxylation of 2'-deoxyguanosine (dG)at the C-8 position and of guanine base residues in DNA. Itwas also shown previously that in the presence of horseradishperoxidase, hydrogen peroxide and Fe³⁺-EDTA complex, diethylstilbestrol(DES) induces single strand breaks in DNA, caused by the productionof superoxide anion (O^–) and hydroxyl (OH) radicals. Bymeans of high-pressure liquid chromatography and electrochemicaldetection a strong indication is adduced that dG is oxidizedto 8-hydroxy- 2'-deoxyguanosine during peroxidative in vitrometabolism of DES, which might be at the basis of DES inducedcell transformation.