1,N2-ethenodeoxyguanosine as a potential marker for DNA adduct formation by trans-4-hydroxy-2-nonenal

The reaction of trans-4-hydroxy-2-nonenal, a major alpha, beta-unsaturated aldehyde released during lipid peroxidation, with deoxyguanosine under physiological conditions was investigated in order to assess its DNA damaging potential. This aldehyde was dissolved in tetrahydrofuran (THF) prior to addition to the reaction mixture. The results showed that structurally different adducts were formed in these reactions depending on the THF used. Using THF unprotected from light, reactions yielded adducts 1 to 6. Adduct 1 was characterized as 1,N2-ethenodeoxyguanosine (5,9-dihydro-9-oxo-3-beta-D-deoxyribofuranosylimidazo[1,2-alpha]pu rine) by its UV, proton nuclear magnetic resonance, and mass spectrum and by comparison to the corresponding guanosine and guanine adducts reported in the literature. The UV spectrum of adduct 4 was indicative of a substituted 1,N2-etheno derivative. Adducts 2,3,5, and 6 were essentially identical in UV spectra and appeared to be N2-substituted deoxyguanosine diastereomers. At room temperature adducts 2,3,5, and 6 were converted quantitatively to a single product at pH 10.5. This product was shown to be identical to 1,N2-ethenodeoxyguanosine (adduct 1). Analogous conversions to 1,N2-ethenoguanine were also observed for the corresponding guanine adducts. Using THF that had been protected from the light, however, the reactions of trans-4-hydroxy-2-nonenal with deoxyguanosine gave three major adducts, 7,8, and 9. These adducts possessed UV spectra similar to that of 1,N2-propanodeoxyguanosine and were not converted to 1,N2-ethenodeoxyguanosine upon base treatment. Evidence obtained suggests that adducts 1 to 6 were formed from the reaction of deoxyguanosine with the epoxide of trans-4-hydroxy-2-nonenal generated in the presence of hydroperoxide in the light unprotected THF, whereas adducts 7 to 9 were formed by direct Michael addition. Adducts 1 to 6 were formed presumably as a result of nucleophilic addition of the exo-amino of deoxyguanosine to the aldehydic group of the epoxide of trans-4-hydroxy-2-nonenal. Base treatment of these adducts facilitated subsequent cyclization and eliminations and finally gave 1,N2-ethenodeoxyguanosine. These results demonstrated that trans-4-hydroxy-2-nonenal readily forms adducts with deoxyguanosine either by direct Michael addition or via its epoxide formation. The facile conversion of some of these adducts to a single adduct suggests that 1,N2-ethenodeoxyguanosine may provide a simple and useful marker for assessing potential DNA damage by trans-4-hydroxy-2-nonenal and related alkenals associated with lipid peroxidation.     

Immunohistochemical detection of 1,N(6)-ethenodeoxyadenosine, a promutagenic DNA adduct, in liver of rats exposed to vinyl chloride or an iron overload

Etheno adducts in DNA bases are formed from exogenous agents such as vinyl chloride and urethane, but also via endogenous lipid peroxidation products like trans-4-hydroxy-2-nonenal. An immunohistochemical method was developed to localize the promutagenic 1,N(6)-ethenodeoxyadenosine DNA adduct in liver of rats exposed to vinyl chloride or an iron overload with or without carbon tetrachloride. Six monoclonal antibodies, previously produced through collaborative efforts, were screened for their optimal adduct recognition and low background formation. The antibody generated by clone EM-A-4 was found to be most suitable. Semi-quantitative image analysis of relative pixel intensity showed approximately 1.5 times higher adduct levels (P < 0.05) in the livers of rats treated with vinyl chloride or an iron overload when compared with untreated controls. Significantly elevated adduct levels persisted in vinyl chloride-treated rat liver 14 days after cessation of exposure, suggesting that this adduct is not rapidly eliminated from rat liver DNA. Using the new immunohistochemical method it is possible to visualize this promutagenic etheno-DNA adduct that may play a role in oxidative stress and lipid peroxidation-induced DNA damage in carcinogenesis.     

Molecular analysis of mutations induced by 2-chloroacetaldehyde, the ultimate carcinogenic form of vinyl chloride, in human cells using shuttle vectors

Vinyl chloride (VC) is a carcinogen associated with human andanimal cancers. The ultimate carcinogenic form of VC, 2-chloroacetaldehyde(CAA), has been suspected to be mutagenic and we confinned themutagenicity of CAA using a modified shuttle vector plasmid.Base sequence analyses of 109 mutant plasmids with mutationsin the supF gene, which were treated with CAA and propagatedin the cultured human cells, revealed that more than half ofthe single base substitutions were G:C to A:T transitions witheight hotspots. The majority of the mutations involving G:Cbase pairs were in 5'-AAGG-3' or 5'-CCTT-3' sequences suggestingthat these sequences are the main targets of mutagenesis causedby CAA.     

Molecular dosimetry and repair of N(2),3-ethenoguanine in rats exposed to vinyl chloride

Although the DNA adducts of vinyl chloride (VC) have been well characterized, previous studies have used single concentrations of VC that are well above contemporary human exposures. This study examined the exposure response to VC in male Sprague Dawley rats with respect to the molecular dose of the promutagenic DNA adduct N(2),3-ethenoguanine (N(2),3-epsilonG). Adult rats were exposed by inhalation to 0, 10, 100, or 1100 ppm VC for 1 or 4 weeks (6 h/day, 5 days/week). Weanling rats were similarly exposed for 5 days. The amount of N(2),3-epsilonG in hepatocyte (HEP) and nonparenchymal cell (NPC) fractions obtained from the liver was measured with a sensitive immunoaffinity/gas chromatography/high-resolution mass spectrometry assay. Endogenous N(2),3-epsilonG was present in HEPs and NPCs from all unexposed rats. The exposure response to VC in each group and cell population was supralinear, with a linear increase from 0 to 100 ppm, and a plateau between 100 and 1100 ppm. There was no statistically significant difference in N(2),3-epsilonG concentrations between HEPs and NPCs in any adult exposure group, which suggests that factors other than adduct concentrations contribute to the particular susceptibility of NPCs to VC-induced carcinogenesis. The accumulation of N(2),3-epsilonG with respect to time was nearly linear in rats exposed to 600 ppm VC for 1, 2, 4, or 8 weeks (4 h/day, 5 days/week), and no repair of N(2),3-epsilonG was detected in rats exposed to VC for 4 weeks and allowed to recover for 1 week. N(2),3-epsilonG concentrations in HEPs from weanling rats were 2-3-fold greater than those in adult rats exposed for the same time. Higher adduct concentrations in young rats may contribute to their greater susceptibility to VC-induced hepatic angiosarcoma as well as their particular susceptibility to hepatocellular carcinoma. The molecular dosimetry of N(2),3-epsilonG in liver appears to be a sensitive and informative biomarker of genotoxic effect after exposure to VC. N(2),3-epsilonG was the predominant etheno adduct measured in vivo after exposure to VC, and the saturable nature of VC metabolism was reflected in its molecular dose. The relationships between endogenous N(2),3-epsilonG and that formed by low exposures to VC were demonstrated. Conclusions drawn from these exposures may be more relevant for risk assessment purposes than those drawn from high exposures where activation, detoxication, and repair pathways may be saturated or otherwise perturbed. These data are well suited for consideration in future risk assessments of VC that incorporate nontumor mode of action data.     

Mutagenesis induced by a single 1,N6-ethenodeoxyadenosine adduct in human cells

To study the genotoxic properties of 1,N6-ethenodeoxyadenosine (epsilondA) in human cells, a novel site-specific mutagenesis approach was developed, in which a single DNA adduct was uniquely placed in either strand of a shuttle plasmid vector. The analysis of progeny plasmid derived from the modified strand shows that epsilondA, when incorporated into the position of the second A of 5'-CAA (codon 61 of the ras gene), is mutagenic in human cells, inducing A-->T, A-->G, and A-->C mutations. The efficient induction of A-->T transversions in experiments using modified double- and singlestranded DNA substrates supports the hypothesis that A:T-->T:A transversions in human and animal tumors induced by vinyl compounds reflect misinsertion of dAMP opposite this adduct. Mutagenic events were similar when the adduct was incorporated into either the leading or the lagging strand. EpsilondA was more mutagenic than 8-oxodeoxyguanosine, which induced targeted G-->T transversions in HeLa cells. In Escherichia coli, epsilondA did not significantly miscode (<0.27%) even in the presence of induced SOS functions.     

Differential induction of N(2),3-ethenoguanine in rat brain and liver after exposure to vinyl chloride

Although vinyl chloride (VC) clearly induces hepatic angiosarcoma in humans and rodents, a causal association with brain tumors has not been definitively established with the available epidemiological and experimental evidence. Because VC acts by genotoxic mechanisms, DNA adduct formation is thought to be a sensitive biomarker of early events in carcinogenesis. Adult male Sprague Dawley rats were exposed to 0 or 1100 ppm VC for 1 or 4 weeks (6 h/day, 5 days/week) by inhalation. Male weanlings were similarly exposed for 5 days. Another group of male adults was exposed to 1100 ppm [(13)C(2)]VC in a nose-only inhalation apparatus for 5 days (6 h/day). A sensitive gas chromatography high-resolution mass spectrometry assay was used to measure the major promutagenic DNA adduct, N(2),3-ethenoguanine (N(2),3-epsilonG), in rat brain and hepatocyte (HEP) DNA. The respective concentrations of N(2),3-epsilonG in control rat brain DNA at 1 and 4 weeks were 5.0 +/- 0.9 and 5.6 +/- 1.1 N(2),3-epsilonG/10(8) unmodified guanine. There was no change in N(2),3-epsilonG in adult rat brain after exposure to 1100 ppm VC for 1 or 4 weeks. In HEPs from the same animals, these adduct concentrations increased from 5.5 +/- 1.4 to 55 +/- 2.0 N(2),3-epsilonG/10(8) unmodified guanine after a 1-week exposure and from 3.0 +/- 0.3 to 110 +/- 20 N(2),3-epsilonG/10(8) unmodified guanine after a 4-week exposure. When weanlings were exposed to 1100 ppm VC for 5 days, there was a statistically significant (P = 0.04) increase in N(2),3-epsilonG in brain from 1.5 +/- 0.2 to 4.4 +/- 1.1 N(2),3-epsilonG/10(8) unmodified guanine. Weanlings exposed to 1100 ppm VC had an even greater increase in N(2),3-epsilonG in HEPs from 1.6 +/- 0.1 to 97 +/- 5.0 N(2),3-epsilonG/10(8) unmodified guanine. [(13)C(2)]N(2),3-epsilonG was not detected in brain DNA from adult rats exposed to 1100 ppm [(13)C(2)]VC for 5 days but was present in HEP DNA at 55 +/- 4.0 [(13)C(2)]N(2),3-epsilonG/10(8) unmodified guanine. The concentrations of the endogenous adduct in both organs were unchanged after this exposure. 7-(Oxoethyl)guanine (OEG), the major DNA adduct formed by VC, was reduced to 7-(2-hydroxyethyl)guanine and measured by liquid chromatography-electrospray ionization-tandom mass spectrometry in brain and HEP DNA from rats exposed to 1100 ppm VC for 1 week. Whereas 4.0 +/- 0.8 OEG/10(6) unmodified guanine were present in HEP DNA from VC-exposed rats, no adducts were detectable in brain DNA (detection limit, 0.3 OEG/10(6) unmodified guanine). These findings indicate that the genotoxic metabolite of VC is not formed in or transported to adult rat brain. Thus, it is unlikely that N(2),3-epsilonG or other VC-induced promutagenic DNA adducts play a significant role in initiating carcinogenesis in adult rat brain after exposure to VC. The data for weanling rats are less clear. Whereas a small increase in N(2),3-epsilonG in the brains of weanlings was found after exposure to 1100 ppm VC, the resulting adduct concentration was similar to that measured in unexposed adults. Future exposures of weanling rats to the stable isotopically labeled compound will be necessary to conclusively determine whether this increase was due to VC.     

1,N6-etheno-2' -deoxyadenosine and 3,N4-etheno-2'-deoxycytidine detected by monoclonal antibodies in lung and liver DNA of rats exposed to vinyl chloride

1,N⁶-Etheno-2'-deoxyadenosine (dAdo) and 3,N⁴-Etheno-2'-deoxycytidine(dCyd) are formed in vitro by reaction of DNA with the electrophilicmetabolites of vinyl chloride (VC), chloroethylene oxide andchloroacetaldehyde. To detect and quantitate these DNA adductsin vivo, we have raised a series of specific monoclonal antibodies(Mab). Among those, Mab EM-A-1 and Mab EM-C-1, respectively,were used for detection of dAdo and dCyd by competitive radioimmunoassay(RIA), following pre-separation of the etheno adducts from DNAhydrolysates by high perfonnance liquid chromatography. At 50%inhibition of tracer-antibody binding, both Mab had a detectionlimit of 187 fmol and antibody affinity constants (K) of 2 x10⁹ l/mol. The levels of dAdo and dyd were quantitated in theDNA of lung and liver tissue of young Sprague-Dawley rats exposedto 2000 p.p.m. of VC for 10 days. The dAdo/2'-deoxyadenosineand dCyd/2'-deoxycytidine molar ratios were 1.3 x 10^–7and 3.3 x 10^–7 respectively, in lung DNA, and 5.0 x 10^–8and 1.6 x 10^–7 in liver DNA. When hydrolysates of 3 mgof DNA were analyzed by RIA at 25% inhibition of tracer-antibodybinding, dAdo and dCyd were not detected in liver DNA from untreatedrats above the limiting dAdo/2'-deoxyadenosine and dCyd/2'-deoxycytidinemolar ratios of 2.2 x 10^–8 and 3.1 x 10^–8, respectively.     

Formation of the adduct 6-(deoxyguanosin-N2-yl)-3-amino-benzo[a]pyrene from the mutagenic environmental contaminant 3-nitrobenzo[a]pyrene

3-Nitrobenzo[a]pyrene (3-nitro-B[a]P) is a potent bacterialmutagen as a result of nitroreduction. Reaction of N-hydroxy-3-amino-B[a]P,prepared in situ from reduction of 3-nitro-B[a]P with calf thymusDNA, was studied. After enzymatic digestion of the DNA, theresulting modified nucleosides were analyzed by thermosprayHPLC-MS and high-resolution proton NMR spectroscopy. The majoradduct was identified as 6-(deoxyguanosin-N²-yl)-3-amino-B[a]P.The same adduct was obtained from incubation of DNA with 3-nitro-B[a]Pin the presence of the mammalian nitroreductase xanthine oxidase,and hypoxanthine. These data indicate that a mammalian nitroreductasecan metabolize 3-nitro-B[a]P to an activated derivative thatreacts with DNA to give a novel adduct distant from the siteof N-hydroxylation.     

Mutagenesis by (+)-anti-B[a]P-N2-Gua, the major adduct of activated benzo[a]pyrene, when studied in an Escherichia coli plasmid using site-directed methods.

The suspected major mutagenic adduct of benzo[a]pyrene, (+)-anti-B[a]P-N2-Gua, is built into the unique PstI recognition site of the Escherichia coli plasmid, pUC19, in order to study its mutagenic potential. The adduct can either be at G437, which is replicated during leading strand DNA synthesis, or at G438, which is replicated during lagging strand DNA synthesis. The DNA strand complementary to the strand containing the (+)-anti-B[a]P-N2-Gua adduct is saturated with UV lesions to minimize its potential to generate progeny. Although all in-frame mutations could have been detected, a G437----T transversion mutation is virtually exclusively obtained at a frequency of approximately 0.04% per adduct following transformation into Uvr+ E. coli when SOS is not induced, and approximately 0.18% when SOS is induced. The mutation frequency of the adduct in a Uvr- background is estimated to be approximately 0.2% when SOS is not induced, and approximately 0.9% when SOS is induced. The absence of G438----T mutations is rationalized. G----T mutations from (+)-anti-B[a]P-N2-Gua are compared to the mutational specificity of the ultimate mutagenic form of activated benzo[a]pyrene.     

Lack of miscoding properties of 7-(2-oxoethyl)guanine, the major vinyl chloride-DNA adduct

Chloroethylene oxide, an ultimate carcinogenic metabolite of vinyl chloride, was reacted with poly(deoxyguanylate-deoxycytidylate); the nucleic acid base adducts, 7-(2-oxoethyl)guanine and 3,N4-ethenocytosine, were analyzed by reverse-phase high-performance liquid chromatography. Chloroethylene oxide-modified poly(deoxyguanylate-deoxycytidylate) was assayed as template in a replication fidelity assay with Escherichia coli DNA polymerase I, and the newly synthesized product was subjected to nearest-neighbor analysis. Misincorporation rates of deoxyadenosine monophosphate and thymidine monophosphate were found to increase with the level of template modification. About 80% of the mispairing events were located opposite minor cytosine lesions. 7-(2-Oxoethyl)guanine, the major adduct identified (greater than 98% of the adducts), did not miscode for either thymine or adenine, failing to support an earlier hypothesis that the cyclic hemiacetal form, O6,7-(1'-hydroxyethano)guanine, could, by analogy with O6-methyl- and O6-ethylguanine, simulate adenine. Our results indicate that direct miscoding of 7-(2-oxoethyl)-guanine may contribute only slightly to the induction of mutations by chloroethylene oxide or vinyl chloride.     

Identification of the mutagenic metabolites of fluoranthene, 2-methylfluoranthene, and 3-methylfluoranthene

The metabolites of fluoranthene, 2-methylfluoranthene, and 3-methylfluorantheneobtained upon incubation with liver homogenate from Aroclorpretreated rats were assayed for mutagenicity in Salmonellatyphimurium TA100. The mutagenic metabolites of fluorantheneand 2-methylfluoranthene were identified as 2,3-dihydro-2,3-dihydroxyfluorantheneand 4,5-dihydro-4,5-dihydroxy-2-methylfluoranthene, respectively.In contrast to these results, the major proximate mutagen detectedamong the in vitro metabolites of 3-methylfluoranthene was 3-hydroxymethylfluoranthene.Comparison of the mutagenic potential of 2-hydroxymethylfluor-anthenedemonstrated that the latter was a more powerful mutagen. Quantitativeanalyses of the metabolites of fluoranthene with that of 3-hydroxymethylfluoranthenedemonstrated that the latter was a more powerful mutagen. Quantitativeanalyses of the metabolites of fluoranthene, 2-methylfluoranthene,and 3-methylfluoranthene indicated that similar amounts of dihydrodiolswere formed. 4,5-Dihydro-4,5-dihydoxy-3-methylfluoranthene,however, was not found to be a potent mutagenic metabolite.These data suggest that the activation pathway to ultimate mutagensmay differ for 2- and 3-methylfluoranthene.     

Formation of the cyclic 1,N2-glyoxal-deoxyguanosine adduct upon reaction of N-nitroso-2-hydroxymorpholine with deoxyguanosine

N-nitroso-2-hydroxymorpholine, a mutagenic metabolite of N-nitrosomorpholineand N-nitrosodiethanolamine, reacted with deoxyguanosine atpH 7, 37°C, to give 3-(2-deoxy-ß-D-erythropentofuranosyI)-6,7-dihydro-6, 7-dihydroxyimidazo-[1, 2-a]purine-9(3H)one, whichis also formed by reaction of deoxyguanosine with glyoxal. Theresults suggest that this adduct might be involved in DNA bindingby N-nitrosomorpholine and N-nitrosodiethanolamine.     

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.     

N-Nitroso-2-hydroxymorpholine, a mutagenic metabolite of N-nitrosodiethanolamine

N-Nitroso-2-hydroxymorpholine was synthesized from N-(2-hydroxyethyl)aminoacetaldehydediethylacetal by nitrosation and hydrolysis. It was detectedas a metabolite of N-nitrosodiethanolamine in rat hepatic 9000g supernatant. It was mutagenic toward Salmonella typhimuriumTA 1535, with and without activation. The results suggest thatN-nitroso-2-hydroxymorpholine, which is formed by metabolicß-oxidation of N-nitrosodiethanolamine, could be involvedin carcinogenesis by N-nitrosodiethanolamine.     

The major, N2-dG adduct of (+)-anti-B[a]PDE induces G-->A mutations in a 5'-AGA-3' sequence context

Previously, in a random mutagenesis study, the (+)-anti diol epoxide of benzo[a]pyrene [(+)-anti-B[a]PDE] was shown to induce a complex mutational spectrum in the supF gene of an Escherichia coli plasmid, which included insertions, deletions and base substitution mutations, notably a significant fraction of GC-->TA, GC-->AT and GC-->CG mutations. At some sites, a single type of mutation dominated and to understand individual mutagenic pathways these sites were chosen for study by site-specific means to determine whether the major adduct, [+ta]-B[a]P-N2-dG, was responsible. [+ta]-B[a]P-N2-dG was shown to induce approximately 95% G-->T mutations in a 5'-TGC-3' sequence context and approximately 80% G-->A mutations in a 5'-CGT-3' sequence context. (+)-anti-B[a]PDE induced principally GC-->CG mutations in the G133 sequence context (5'-AGA-3') in studies using both SOS-uninduced or SOS-induced E. coli. Herein, [+ta]-B[a]P-N2-dG is shown to induce principally G-->A mutations (>90%) either without or with SOS induction in a closely related 5'-AGA-3' sequence context (identical over 7 bp). This is the first time that there has been a discrepancy between the mutagenic specificity of (+)-anti-B[a]PDE versus [+ta]-B[a]P-N2-dG. Eight explanations for this discordance are considered. Four are ruled out; e.g. the second most prevalent adduct [+ca]-B[a]P-N2-dG also induces a preponderance of G-->A mutations (>90%), so it also is not responsible for (+)-anti-B[a]PDE-induced G133-->C mutations. The four explanations not ruled out are discussed and include that another minor adduct might be responsible and that the 5'-AGA-3' sequence context differed slightly in the studies with [+ta]-B[a]P-N2-dG versus (+)-anti-B[a]PDE. In spite of the discordance, [+ta]-B[a]P-N2-dG induces G-->A mutations in the context studied herein and this result has proven useful in generating a hypothesis for what conformations of [+ta]-B[a]P-N2-dG are responsible for G-->T versus G-->A mutations.     

A method for the amplification of chemically induced transformation in C3H/10T1/2 clone 8 cells: its use as a potential screening assay

A method has been developed by which to amplify expression of phenotypic transformation of C3H/10T1/2 clone 8 mouse embryo cells not otherwise observed in the standard transformation assay. The expression of transformed foci was amplified by subcultivating chemically treated target cells after they had reached confluence and replating them at subconfluent cell densities. Conditions leading to the expression of the highest numbers of transformed foci include a) a cell seeding density for chemical treatment of 1 X 10(4) cells/dish, b) subculture 4 weeks after treatment, and c) replating cells at a density of 2 X 10(5) cells/-dish. Agents capable of inducing transformation in the standard assay (e.g., 4,4'-bis(dimethylamino)benzophenone, benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, and others) also yielded transformation in the replating assay. The more marginal transforming activities of chemicals such as ethyl methanesulfonate, 7-(bromomethyl)-12-methylbenz[a]anthracene, and N-methyl-N'-nitro-N-nitrosoguanidine were enhanced by the amplification procedure. Compounds that failed to elicit focal transformation in the standard assay (e.g., dibenz[a,h]anthracene, Tris(2,3-dibromopropyl) phosphate, lead acetate, benzidine, propyleneimine, N-hydroxy-2-fluorenylacetamide, and numerous other compounds of various chemical classes) induced significant levels of phenotypic transformation upon amplification. Noncarcinogens (e.g., phenanthrene, anthracene, 2-aminobiphenyl, cycloheximide, and others) failed to cause significant phenotypic transformation even when cells were replated. To further enhance the applicability of this new replating system, an exogenous source of metabolic activation was added: a 9,000 X g supernatant from Aroclor 1254-induced rat hepatic S-9. This activation system was found a) to be only minimally cytotoxic by itself and b) to be able to mediate NADPH-dependent, dose-dependent toxicity, and transformation by activating the procarcinogens dimethylnitrosamine, 2-naphthylamine, 2-aminoanthracene, and aflatoxin B1. With the use of this revised assay, 14 coded and 23 model compounds were tested. Agreement with in vivo results was observed to be over 85%. The marked sensitivity and discriminatory ability of this revised assay procedure suggest its usefulness as a screen for potential carcinogens of diverse chemical structure.     

Flow cytometric chemosensitivity assay using JC-1, a sensor of mitochondrial transmembrane potential, in acute leukemia

Purpose

The purpose of the study is to establish a simple and relatively inexpensive flow cytometric chemosensitivity assay (FCCA) for leukemia to distinguish leukemic blasts from normal leukocytes in clinical samples.

Methods

We first examined whether the FCCA with the mitochondrial membrane depolarization sensor, 5, 50, 6, 60-tetrachloro-1, 10, 3, 30 tetraethyl benzimidazolo carbocyanine iodide (JC-1), could detect drug-induced apoptosis as the conventional FCCA by annexin V/7-AAD detection did and whether it was applicable in the clinical samples. Second, we compared the results of the FCCA for prednisolone (PSL) with clinical PSL response in 18 acute lymphoblastic leukemia (ALL) patients to evaluate the reliability of the JC-1 FCCA. Finally, we performed the JC-1 FCCA for bortezomib (Bor) in 25 ALL or 11 acute myeloid leukemia (AML) samples as the example of the clinical application of the FCCA.

Results

In ALL cells, the results of the JC-1 FCCA for nine anticancer drugs were well correlated with those of the conventional FCCA using anti-annexin V antibody (P < 0.001). In the clinical samples from 18 children with ALL, the results of the JC-1 FCCA for PSL were significantly correlated with the clinical PSL response (P = 0.005). In ALL samples, the sensitivity for Bor was found to be significantly correlated with the sensitivity for PSL (P = 0.005). In AML samples, the Bor sensitivity was strongly correlated with the cytarabine sensitivity (P = 0.0003).

Conclusions

This study showed the reliability of a relatively simple and the FCCA using JC-1, and the possibility for the further clinical application.     

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.