Detection of DNA adducts using a quantitative long PCR technique and the fluorogenic 5' nuclease assay (TaqMan).

The detection of DNA adducts is an important component in assessing the mutagenic potential of exogenous and endogenous compounds. Here, we report an in vitro quantitative long PCR (XL-PCR) assay to measure DNA adducts in human genomic DNA based on their ability to block and inhibit PCR amplification. Human genomic DNA was exposed to test compounds and then a target sequence was amplified by XL-PCR. The amplified sequence was then quantified using fluorogenic 5' nuclease PCR (TaqMan) and normalized to a solvent-treated control. The extent of DNA adduction was determined based on the reduction in amplification of the target sequence in the treated sample. A 17.7kb beta-globin fragment was chosen as the target sequence for these studies, since preliminary experiments revealed a two-fold increased sensitivity of this target compared to a 10.4kb HPRT fragment for detecting hydrogen peroxide-induced DNA damage. Validation of the XL-PCR assay with various compounds demonstrated the versatility of the assay for detecting a wide range of adducts formed by direct acting or S9-activated mutagens. The same DNA samples were also analyzed using 32P-postlabeling techniques (thin-layer chromatography or high-performance liquid chromatography) to confirm the presence of DNA adducts and estimate their levels. Whereas 32P-postlabeling with nuclease P(1) enrichment was more sensitive for detecting bulky adducts induced by the compounds benzo[a]pyrene, dimethylbenzanthracene, 3-methylindole, indole 3-carbinol, or 2-acetylaminofluorene, the XL-PCR procedure was more sensitive for detecting smaller or labile DNA adducts formed by the compounds methyl methanesulfonate, diethyl nitrosamine, ethylnitrosourea, diepoxybutane, ICR-191, styrene oxide, or aflatoxin B(1). Compounds not expected to form adducts in DNA, such as clofibrate, phenobarbital, chloroform or acetone, did not produce a positive response in the XL-PCR assay. Thus, quantitative XL-PCR provides a rapid, high-throughput assay for detecting DNA damage that complements the existing 32P-postlabeling assay with nuclease P(1) enrichment.

Detection and characterization of DNA adducts of 3-methylindole.

The pneumotoxin 3-methylindole is metabolized to the reactive intermediate 3-methyleneindolenine which has been shown to form adducts with glutathione and proteins. Reported here is the synthesis, detection, and characterization of nucleoside adducts of 3-methylindole. Adducted nucleoside standards were synthesized by the reaction of indole-3-carbinol with each of the four nucleosides under slightly acidic conditions, which catalyze the dehydration of indole-3-carbinol to 3-methyleneindolenine. Following solid phase extraction, the individual adducts were infused via an electrospray source into an ion trap mass spectrometer for molecular weight determination and characterization of the fragmentation patterns. The molecular ions and fragmentation of the dGuo, dAdo, and dCyd adducts were consistent with nucleophilic addition of the exocyclic primary amine of the nucleosides to the methylene carbon of 3-methyleneindolenine. The apparent chemical preference of this addition lead primarily to dAdo and dGuo adducts, with substantially less of the dCyd adduct formed. No adduct with dThd was detected. The adducts were purified by HPLC and subsequent NMR analysis of the dGuo and dCyd adducts confirmed the proposed structures. Mass spectral fragmentation of the three adducts produced primarily two ions which were the result of the loss of either the 3-methylindole moiety or the sugar. On a triple quadrupole electrospray mass spectrometer, the neutral loss of the sugar, [M + H - 116](+), was utilized for selected reaction monitoring of the calf thymus DNA adducts, formed by incubations of 3-methylindole with various microsomes (rat liver, goat lung, and human liver). All three adducts were detected from each of the microsomal incubations, following extraction and cleavage of the DNA to the nucleoside level. The dGuo adduct was the primary adduct formed, with smaller amounts of the dAdo and dCyd adducts. Rat hepatocytes incubated with 3-methylindole produced the same three adducts, in approximately the same proportions, while no adducts were detected in untreated hepatocytes. Microsomal incubations in the presence of ([3-(2)H(3)]-methyl)indole confirmed the formation and identification of the adducts as well as the fragmentation patterns. These results demonstrate that bioactivated 3-methylindole forms specific adducts with exogenous or intact cellular DNA, and indicates that 3-methylindole may be a potential mutagenic and/or carcinogenic chemical.

Induction of chromosome aberrations in vitro by phenolphthalein: mechanistic studies.

Phenolphthalein induces tumors in rodents but because it is negative in assays for mutation in Salmonella and in mammalian cells, for DNA adducts and for DNA strand breaks, its primary mechanism does not seem to be DNA damage. Chromosome aberration (Ab) induction by phenolphthalein in vitro is associated with marked cytotoxicity. At very high doses, phenolphthalein induces weak increases in micronuclei (MN) in mouse bone marrow; a larger response is seen with chronic treatment. All this suggests genotoxicity is a secondary effect that may not occur at lower doses. In heterozygous TSG-p53((R)) mice, phenolphthalein induces lymphomas and also MN, many with kinetochores (K), implying chromosome loss. Induction of aneuploidy would be compatible with the loss of the normal p53 gene seen in the lymphomas. Here we address some of the postulated mechanisms of genotoxicity in vitro, including metabolic activation, inhibition of thymidylate synthetase, cytotoxicity, oxidative stress, DNA damage and aneuploidy. We show clearly that phenolphthalein does not require metabolic activation by S9 to induce Abs. Inhibition of thymidylate synthetase is an unlikely mechanism, since thymidine did not prevent Ab induction by phenolphthalein. Phenolphthalein dramatically inhibited DNA synthesis, in common with many non-DNA reactive chemicals that induce Abs at cytotoxic doses. Phenolphthalein strongly enhances levels of intracellular oxygen radicals (ROS). The radical scavenger DMSO suppresses phenolphthalein-induced toxicity and Abs whereas H(2)O(2) potentiates them, suggesting a role for peroxidative activation. Phenolphthalein did not produce DNA strand breaks in rat hepatocytes or DNA adducts in Chinese hamster ovary (CHO) cells. All the evidence points to an indirect mechanism for Abs that is unlikely to operate at low doses of phenolphthalein. We also found that phenolphthalein induces mitotic abnormalities and MN with kinetochores in vitro. These are also enhanced by H(2)O(2) and suppressed by DMSO. Our findings suggest that induction of Abs in vitro is a high-dose effect in oxidatively stressed cells and may thus have a threshold. There may be more than one mechanism operating in vitro and in vivo, possibly indirect genotoxicity at high doses and also chromosome loss, both of which would likely have a threshold.

Detection of DNA damage induced by human carcinogens in acellular assays: potential application for determining genotoxic mechanisms.

Positive outcomes of in vitro genotoxicity tests may not always occur as a consequence of direct reaction of a compound or a metabolite with DNA. To follow-up positive responses in in vitro tests, we developed two supplemental, cell-free assays to examine the potential of compounds and metabolites to directly damage DNA. Calf thymus DNA was used as the target for the direct detection of adducts by 32P-postlabeling/TLC and electrochemical detection, and alkaline gel electrophoresis was used to detect single-strand breakage of bacteriophage lambda DNA. To show that these assays would detect damage from relevant compounds, we examined nine human carcinogens (aflatoxin B1, busulfan, chlorambucil, cyclophosphamide, diethylstilbestrol, melphalan, 2-naphthylamine, phenacetin and potassium chromate). Each of the nine compounds produced a positive result for one or both endpoints. Using multifraction contact-transfer TLC, we detected 32P-labeled DNA adducts produced by aflatoxin B1, chlorambucil, diethylstilbestrol, melphalan, 2-naphthylamine, and potassium chromate (plus hydrogen peroxide). Aflatoxin B1, diethylstilbestrol and 2-naphthylamine required metabolic activation (induced rat liver S9) to generate DNA adducts. Although potassium chromate alone induced a slight increase in the content of 8-hydroxydeoxyguanosine (a promutagenic adduct produced by reactive oxygen species), addition of hydrogen peroxide greatly increased 8-hydroxydeoxyguanosine levels. The damage to lambda DNA by each human carcinogen (or metabolites), except diethylstilbestrol, was sufficient to generate single-strand breaks after neutral thermal hydrolysis at 70 degrees C. Chromate was a weak inducer of DNA fragmentation, but adding hydrogen peroxide to the reaction mixtures dramatically increased the DNA strand breakage. Our data suggest that these non-routine, acellular tests for determining direct DNA damage may provide valuable mechanistic insight for positive responses in cell-based genetic toxicology tests.

Mutagenic response of the endogenous hprt gene and lacI transgene in benzo[a]pyrene-treated Big Blue B6C3F1 mice.

Big Blue (BB) and generic B6C3F1 mice were given one to three i.p. injections of 50 mg/kg benzo[a]pyrene (B[a]P) in DMSO every other day to achieve cumulative doses of 50 to 150 mg/kg. Three weeks after treatment, the mutation frequency at the endogenous hprt gene and lacI transgene was measured in splenic T cells. Generic mice given 50, 100, and 150 mg/kg B[a]P displayed induced hprt frequencies (observed hprt frequency minus control frequency) of 5.5 +/- 1.0, 11 +/- 2.0, and 19 +/- 2.6 x 10(-6), respectively (average +/- SEM). In contrast, BB mice given 50 and 150 mg/kg B[a]P displayed induced hprt frequencies of 0.9 +/- 0.6 and 9.1 +/- 1.5 x 10(-6). 32P postlabelling revealed that the lower hprt response in BB mice correlated with lower amounts of BP-DNA adducts in spleen, liver, and lung 24 hours after B[a]P exposure. Western blot analysis of liver samples from B[a]P-treated mice suggests that the reduced adduct load in turn may be due to lower P450 1A1 levels in BB mice. The frequency of induced, nonsectored blue plaques (observed blue plaque frequency minus control frequency) in BB mice receiving 50 and 150 mg/kg B[a]P was 41 +/- 9 and 134 +/- 10 x 10(-6) (15- to 40-fold higher than the induced hprt frequency in the same treated animals). Sectored plaques were observed in both control and B[a]P groups but their frequency showed no relationship to dose (sectored frequency in all groups was approximately 20 x 10(-6)). To test whether persistent DNA adducts in the packaged lambda vector were contributing to the observed blue plaque frequency, purified lambda-LIZ DNA was treated in vitro with B[a]P diol epoxide (BPDE), packaged, and plated on E. coli lawn cells. Treatment with BPDE did not produce significant increases in homogeneous blue plaques, suggesting that the majority of mutants obtained from B[a]P-treated BB mice occurred in vivo. These results indicate that B[a]P exposure produces many more mutations at the lacI transgene than at the endogenous hprt locus.

Co-purification of gastric mucoproteins with DNA: an explanation for the reported 'interaction' of omeprazole with DNA in rat tissues.

Recently, Phillips et al. reported that small amounts of radioactivity derived from [14C]omeprazole were 'associated' with DNA purified from gastrointestinal tissues of treated rats (Mutagenesis 7, 277-283, 1992). We hypothesized that this radioactivity arose from omeprazole bound to contaminating protein in the DNA fraction (Mutagenesis 7, 395-396, 1992). Using rats injected with 35S-labeled amino acids, we found significant protein contamination (0.06 microgram of protein per microgram of DNA) in DNA purified from gastrointestinal tissues. Gastric mucous proteins represent likely candidates for binding of omeprazole in the rat model used by Phillips et al. To investigate this, we partially purified proteins from gastric mucus, incubated them with [14C]omeprazole, and then added these radiolabeled mucoproteins to homogenates of rat colon and duodenum before starting the DNA purification. Detectable amounts of the added mucoproteins remained in the DNA fraction, but none of the control protein, bovine serum albumin, remained with the DNA. Further characterization of the mucoproteins by hydroxyapatite chromatography indicated that a certain population of these proteins survived the DNA purification procedures. These data indicate that the association of omeprazole with DNA reported by Phillips et al. most probably is explained by binding of omeprazole to mucous glycoproteins (or other proteins present in the GI tract) that selectively survive DNA purification protocols.

Detection of DNA adducts by 32P-postlabeling and multifraction contact-transfer thin-layer chromatography.

32P-Postlabeling is a sensitive method for detecting DNA adducts. Large bulky adducts, particularly from polycyclic aromatic compounds, are readily detected using this technique. Detection of small modifications, such as methylations, has often required specific additional enrichment procedures prior to 32P-postlabeling. We report the use of a single analytical procedure that can detect DNA adducts of a wide range of sizes and hydrophobicities (exemplified by adducts produced with methyl methanesulfonate, diepoxybutane, styrene oxide, or benzo[a]-pyrene). This 32P-postlabeling/thin-layer chromatography procedure is particularly useful when examining the potential of novel compounds or their metabolites to form DNA adducts.

Phosphatase activity in commercial spleen exonuclease decreases the recovery of benzo[a]pyrene and N-hydroxy-2-naphthylamine DNA adducts by 32P-postlabeling.

Spleen exonuclease, which degrades nucleic acids into single 3'-nucleotides, is used in the detection of DNA adducts by 32P-postlabeling. Contamination of the exonuclease with phosphatase activity can reduce the recovery of benzo[a]pyrene and N-hydroxy-2-naphthylamine DNA adducts by 32P-postlabeling. Four preparations of spleen exonuclease containing varying levels of phosphatase activity (< 1-62% of the unmodified 3'-nucleotides being dephosphorylated) were used to hydrolyze the DNA. The exonuclease with the lowest phosphatase activity produced a recovery of up to 9.60 mumol of benzo[a]pyrene adducts per mole of DNA. Recovery of benzo[a]pyrene adducts was reduced to 0.56 mumol of adduct per mole of DNA using the exonuclease with the highest phosphatase activity. Phosphatase in the exonucleases also dephosphorylated N-hydroxy-2-naphthylamine DNA adducts. Surprisingly, recovery of these DNA adducts was nearly 10 times greater using nuclease P1 than when using 1-butanol extraction for adduct enrichment, since arylamine DNA adducts have previously been reported to be poorly detected by 32P-postlabeling after nuclease P1 treatment. Our data indicate that the hydrolysis of DNA by spleen exonuclease may be an important source of variability in both qualitative and quantitative analysis of adducts by 32P-postlabeling.