By-pass of the major aminofluorene-DNA adduct during in vivo replication of single- and double-stranded oX174 DNA treated with N-hydroxy-2-aminofluorene

To examine the effects of aminofluorene-DNA adduct formationon the biological activity of DNA, single-stranded (ss) øX174DNA and øX174 replicative form (RF) DNA were modifiedto different extents with ³H-labeled N-hydroxy-2-aminofluoreneand subsequently transfected to Escherichia coli spheroplastswith different repair capabilities. When the fraction of activess øX174 DNA molecules was measured as a function ofthe mean number of adducts per molecule, exponential survivalcurves were obtained from which it could be deduced that inwild-type, uvrA^– and recA^– cells at least 86%, andin uvrC^– cells at least 82% of the introduced adductsdo not cause inactivation. In the case of RF DNA the survivalcurves are non-exponential, but they nevertheless show thatan exceptionally high number of adducts per RF molecule mustbe introduced to destroy its biological activity. On average52 adducts per RF molecule were needed to reduce the survivalto 37%, irrespective of whether wild-type, uvrA^– or recA^–cells were used. On the other hand, the survival of the uvrC^–cells was considerably lower, but even in these cells a majorityof the adducts is not lethal. By h.p.l.c. analysis of the modifiedDNA after hydrolysis with trifluoro-acetic acid, 81 and 84%of the adducts in ss- and RF DNA, respectively, could be identifiedas N-(guanin-8-yl)-2-aminofluorene. The results strongly indicatethat this type of major modification product is very frequentlyby-passed during replication of both single- and double-strandedDNA. The results together with the data obtained by sucrosegradient analysis both before and after an alkali treatmentand those obtained by h.p.l.c. analysis suggest that inactivationof ssDNA is mainly due to minor modifications such as secondarylesions consisting of chain breaks and alkali-labile sites togetherwith unidentified interaction products.     

Detection of surface free radical activity of respirable industrial fibres using supercoiled {varphi}X174 RF1 plasmid DNA

The ability of a number of respirable industrial fibres, amositeand crocidolite asbestos, refractory ceramic fibres (RCFs) andman-made vitreous fibres (MMVFs), to cause free radical injuryto plasmid     

DNA adduct formation after oral administration of 2-nitrofluorene and N-acetyl-2-aminofluorene, analyzed by 32P-TLC and 32P-HPLC

DNA adducts have been detected in laboratory animals after exposureto carcinogens as well as in human populations with known orsuspected risk of developing cancer. Examples are smokers, cokeand aluminium workers, urban citizens and roofers. The formationof DNA adducts is an early event in carcinogenesis which canbe used for measuring target dose and as a biomarker for genotoxkrisk. A method of analyzing ³²P-postlabeled DNA adducts on reverseHPLC with on-line detection of ³²P has been developed. The methodpermits direct injection of the ³²P-postlabeling mixture intothe analytical system without prior purification with backgroundradioactivity on a low level. The method can be used in parallelwith TLC analyses of ³²P-postlabeled DNA adducts to improvethe analytical capacity. The time for analysis of a typicalsingle sample by HPLC and TLC is 30–60 min and 6–24h respectively. A high (2 M) salt concentration in the HPLCeluent reduces the ³²P background considerably. Also the peaktailing was substantially diminished, giving an ability to separateDNA adducts equal to or better than the TLC method. The methodhas been applied to 2-nitrofluorene (NF), a carcinogenic airpollutant, and N-acetyl-2-aminofluorene (AAF), a model carcinogenwhich is also a metabolite of NF. A number of DNA adducts areformed in the livers of rats. After oral administration of AAFand NF, DNA adducts in the liver have been characterized asdG-C₈-AF and dG-C₈-AAF. The major DNA adduct found in both NF-and AAF-administered animals was dG-C₈-AF. The described HPLCmethod can, with minor adjustments, generally be used to analyze³²P-postlabeled DNA adducts.     

Characterization of the major DNA adduct formed by the food mutagen 2-amino-3-methyl-9H-pyrido[2, 3-b] indole (MeA{alpha}C) in primary rat hepatocytes

Cooking of proteinous food results in the formation of heterocyclicamines. Among these, 2-amino-3-methyl-9H-pyrido[2, 3-b]indole(MeA     

The essential role of microsomal deacetylase activity in the metabolic activation, DNA-(deoxyguanosin-8-yl)-2-aminofluorene adduct formation and initiation of liver tumors by N-hydroxy-2-acetylaminofluorene in the livers of infant male B6C3F1 mice

Deacetylation of N-hydroxy-2-acetylaminofluorene. (N-hydroxy-AAF) to N-hydroxy-2-aminofluorene (N-hydroxy-AF) has been proposedas one of the critical metabolic steps in the formation of hepaticDNA adducts and the initiation of liver tumors in 12-day-oldmale B6C3F₁ mice. In this study, the importance of the microsomaldeacetylase activity for N-hydroxy-AAF in the initiation ofhepatocarcinogenesis in these mice was demonstrated by usinga carboxylesterase and amidase inhibitor, bis(p-nitrophenyl)phosphate(BNPP), that is much less toxic in vivo than is paraoxon. Pre-incubationof liver microsomes from 12-day-old male B6C3F₁ mice with 10^–3M BNPP reduced the deacetylase activity by 80% while paraoxoninhibited the deacetylase activity completely at a concentrationof 10_–4 M. Pretreatment of 12-day-old male B6C3F₁ micewith 4 x 75 µg doses of BNPP/g body weight before theadministration of n-hydroxy-AAF reduced the hepatic N-(dGuo-8-yl)-AFadduct levels to 1.09 and 0.68 pmol/mg DNA compared with 2.87and 1.64 pmol/mg DNA for mice treated once with 0.06 or 0.03µmol of N-hydroxy-AAF/g body weight respectively. However,BNPP pretreatments did not affect the levels of the acetylatedDNA adducts, N-(dGuo-8-yl)-AAF and 3-(dGuo-N²-yl)-AAF, formedby these doses of N-hydroxy-AAF. The initiation of liver tumorsby n-hydroxy-AAF was also inhibited by BNPP pretreatment. Thus,for mice that received single doses of 0.12, 0.06 and 0.03 µmolof N-hydroxy-AAF/g body weight, the multiplicities of livertumors at 10 months were reduced by BNPP pretreatments to 5.6,1.0 and 0.3 compared with multiplicities of 11.8, 4.8 and 1.7without pretreatment respectively. On the other hand, BNPP pretreatmentshad no significant inhibitory effects on the levels of the hepaticDNA-N-dGuo-8-yl)-AF adduct or on the liver tumor multiplicitiesinduced by comparable doses of N-hydroxy-AF. It is concludedthat deacetylation of n-hydroxy-AAF to N-hydroxy-AF is essentialfor the metabolic activation, DNA-N-(dGuo-8-yl)-AF adduct formationand liver tumor initiation in infant male B6C3F₁ mice by N-hydroxy-AAF.     

Transforming activity of human c-Ha-ras-1 proto-oncogene generated by the binding of 2-amino-6-methyl-dipyrido[1,2-a:3',2'-d]imidazole and 4-nitroquinoline N-oxide: direct evidence of cellular transformation by chemically modified DNA

An activity that transforms NIH 3T3 cells was generated by the in vitro modification of plasmids containing the human c-Ha-ras-1 proto-oncogene with the synthesized ultimate carcinogen, 2-acetoxyamino-6-methyldipyrido[1,2-a:3',2'-d]-imidazole (N-OAc-Glu-P-1). DNAs isolated from the transformed cells were analyzed by restriction fragment length polymorphism (RFLP) assay using the restriction enzyme Msp I. Of fourteen transformants studied, six contained a mutation in the region of the CCGG sequence of the eleventh and the twelfth codons, in which GG corresponds to the first two nucleotides of the twelfth codon. Transforming activity was also generated by the chemical modification of the plasmids with 4-acetoxyaminoquinoline N-oxide (N-OAc-4AQO). The results clearly indicate that formation of DNA adducts with N-OAc-Glu-P-1 or N-OAc-4AQO causes the induction of transformation of mammalian cells.     

DNA binding by [2, 5-14C]N7-nitrosopyrrolidine in excision-repair proficient and deficient strains of Salmonella. Evidence for a major premutagenic adduct

Little is known about the nature and possible genotoxic effectsof the DNA adducts formed by N-nitrosopyrrolidine (NPYR) inwhole animals. DNA binding in DNA isolated from [2, 5-¹⁴C]NPYR-treatedSalmonella was studied and attempts were made to monitor DNAadducts and correlated DNA binding with mutagenesis. NPYR wasmetabolized by hamster liver S-9 fraction in the presence ofS.typhimurium TA1535 (uvrB^–) or TA1975(uvrB⁺ DNA isolatedfrom TA1535 contained about three times as much radioactivityas that isolated from TA1975, and NPYR-induced mutagenesis wasseveral-fold higher in TA1535. The fraction of radioactivityincorporated into TA1535 was {small tilde}10^-5. Thermal hydrolysisof the ¹⁴C-containing DNA at neutral pH, followed by precipitation,released {small tilde}2/3 of the radioactivity into the supernatant.HPLC analysis of the supernatant revealed one major peak. Thispeak was absent in DNA from TA1975. Acid hydrolysis of the DNAprecipitate after neutral hydrolysis released most of the residualradioactivity. Several small peaks were observed after HPLCanalysis of the TA1535 acid hydrolysate or the TA1975 acid hydrolysate.These results demonstrate that NPYR is capable of binding toSalmonella DNA yielding one major product after hydrolysis andthis DNA binding product appears to be repaired by the excisionrepair system. The fact that the major peak of radioactivityreleased from Salmonella is only found in the strain which isefficiently reverted by NPYR suggests that mutagenesis is dependenton the DNA modification leading to this peak.     

Absence of {gamma}-glutamyl transpeptidase activity in neoplastic lesions induced in the liver of male F-344 rats by di-(2-ethylhexyl)phthalate, a peroxisome proliferator

Male F-344 rats were fed a diet containing 2% di-(2-ethylhexyl)phthalate(DEHP) for 95 weeks. Liver nodules and/or hepatocellular carcinomas(HCC) developed in 6/10 rats fed DEHP and none were found incontrols (P<0.005 by x² test). All the nodules and HCC werenegative for -glutamyl transpeptidase. In the non-tumorous portionsof liver, the hepatocytes contained an increased number of peroxisomesand extensive accumulation of lipofuscin. By immunocyto-chemicalanalysis, the liver peroxisomes in rats treated chronicallywith DEHP had visually detectable decrease in the H₂O₂-degradingcatalase and increase in H₂O₂-producing fatty acyl-CoA oxidase.These results show that higher dietary level of DEHP, whichcauses substantially greater degree of peroxisome proliferationthan the 1.2% dietary level used in the National ToxicologyProgram bioassay (1982, Publication no. NTP-80-37, Tech. ReportSeries No. 217), can induce liver tumors in male rats.     

Identification of N2-(deoxyguanosin-8-yl)-2-amino-3,8-dimethylimidazo[4,5-f)quinoxaline 3',5'-diphosphate, a major DNA adduct, detected by nuclease P1 modification of the 32P-postlabeling method, in the liver of rats fed MeIQx

The carcinogenic heterocyclic amine 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline(MeIQx) is widely distributed in cooked foods. The nucleaseP1 method increased the sensitivity of the standard ³²P-postlabelinganalysis about 1000-fold for detection of MeIQx-DNA adducts.The recovery of MeIQx-DNA adducts by the nuclease P1 methodwas determined to be about 50% using liver DNA of a rat treatedwith [¹⁴C]MeIQx intragastrically. By the nuclease P1 methodfive adducts were detected in the liver DNA of rats fed MeIQxand two of them, including the most abundant one, were identifiedas MeIQx-deoxyguanosine adducts by comparison with the adductsformed in in vitro reactions of N-acetoxy-2-amino-3,8-dimethylimidazo[4,5-f)quinoxalinewith the four 2'-deoxyribonucleotides. The most abundant adductin vivo was identified as N²-(deoxyguanosin-8-yl)-MeIQx 3',5'-diphosphate(3',5'-pdGp-C8-MeIQx). MeIQx-DNA adduct levels in human tissuescould be determined by the nuclease P1 modification of the ³²P-postlabelingmethod in combination with HPLC, and thus provide informationon the roles of MeIQx in human carcinogenesis.     

A nuclear protein complex containing high mobility group proteins B1 and B2, heat shock cognate protein 70, ERp60, and glyceraldehyde-3-phosphate dehydrogenase is involved in the cytotoxic response to DNA modified by incorporation of anticancer nucleoside analogues

Thiopurine treatment of human leukemia cells deficient in components of the mismatch repair system (Nalm6) initiated apoptosis after incorporation into DNA, as revealed by caspase activation and terminal deoxynucleotidyl transferase-mediated nick end labeling assay. To elucidate the cellular sensor(s) responsible for recognition of DNA damage in cells with an inactive mismatch repair system, we isolated a multiprotein nuclear complex that preferentially binds DNA with thioguanine incorporated. The components of this nuclear multiprotein complex, as identified by protein mass spectroscopy, included high mobility group proteins 1 and 2 (HMGB1, HMGB2), heat shock protein HSC70, protein disulfide isomerase ERp60, and glyceraldehyde 3-phosphate dehydrogenase. The same complex was also shown to bind synthetic oligodeoxyribonucleotide duplexes containing the nonnatural nucleosides 1-beta-D-arabinofuranosylcytosine or 5-fluoro-2'-deoxyuridine. Fibroblast cell line derived from Hmgb1(-/-) murine embryos had decreased sensitivity to thiopurines, with an IC(50) 10-fold greater than Hmgb1-proficient cells (P < 0.0001) and exhibited comparable sensitivity to vincristine, a cytotoxic drug that is not incorporated into DNA. These findings indicate that the HMGB1-HMGB2-HSC70-ERp60-glyceraldehyde 3-phosphate dehydrogenase complex detects changes in DNA structure caused by incorporation of nonnatural nucleosides and is a determinant of cell sensitivity to such DNA modifying chemotherapy.