Use of biomarker data and metabolite relative potencies to support derivation of noncancer reference values based on the reproductive and developmental toxicity effects of 1,3-butadiene.

Subchronic and chronic reference values (RfVs) were derived for 1,3-butadiene (BD) based upon its ability to cause reproductive and developmental effects observed in laboratory mice and rats. Metabolism has been well-established as an important determinant of the toxicity of BD. A major challenge to human health risk assessment is presented by large quantitative species differences in the metabolism of BD, differences that should be accounted for when the rodent toxicity responses are extrapolated to humans. The methods of Fred et al. (2008)/Motwani and Törnqvist (2014) were extended and applied here to the noncancer risk assessment of using data-derived extrapolation factors to account for species differences in metabolism, as well as differences in cytotoxic potency of three BD metabolites. This approach made use of biomarker data (hemoglobin adducts) to quantify species differences in the internal doses of BD metabolites experienced in mice, rats and humans. Using these methods, the dose-response relationships in mice and rats exhibit improved concordance, and result in subchronic and chronic inhalation reference values of 29 and 10 ppm, respectively, for BD. Confidence in these reference values is considered high, based on high confidence in the key studies, medium-to-high confidence in the toxicity database, high confidence in the estimates of internal dose, and high confidence in the dose-response modeling.

[Secondary form of Takotsubo syndrome, developed on pulmonary embolism].

The clinical case of the Takotsubo syndrome (TT) developed in process of treatment pulmonary embolism (PE) in old woman is described. A feature of this observation is the occurrence of TT not in the acute phase of PE, but in a month after the debut of the disease and in a week after the start of successful treatment.

Can 5-methylcytosine analogues with extended alkyl side chains guide DNA methylation?

5-Methylcytosine (MeC) is an endogenous modification of DNA that plays a crucial role in DNA-protein interactions, chromatin structure, epigenetic regulation, and DNA repair. MeC is produced via enzymatic methylation of the C-5 position of cytosine by DNA-methyltransferases (DNMT) which use S-adenosylmethionine (SAM) as a cofactor. Hemimethylated CG dinucleotides generated as a result of DNA replication are specifically recognized and methylated by maintenance DNA methyltransferase 1 (DNMT1). The accuracy of DNMT1-mediated methylation is essential for preserving tissue-specific DNA methylation and thus gene expression patterns. In the present study, we synthesized DNA duplexes containing MeC analogues with modified C-5 side chains and examined their ability to guide cytosine methylation by the human DNMT1 protein. We found that the ability of 5-alkylcytosines to direct cytosine methylation decreased with increased alkyl chain length and rigidity (methyl > ethyl > propyl ∼ vinyl). Molecular modeling studies indicated that this loss of activity may be caused by the distorted geometry of the DNA-protein complex in the presence of unnatural alkylcytosines.

Locating nucleobase lesions within DNA sequences by MALDI-TOF mass spectral analysis of exonuclease ladders.

The location of carcinogen-modified nucleobases (DNA adducts) within DNA sequences is a critical factor affecting their promutagenic properties and persistence in DNA. We now report the use of controlled exonuclease digestion followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to directly map modified nucleobases within DNA. The DNA sequence is determined by mass spectral analysis of the DNA ladders produced by sequential removal of nucleotides with either 5'-->3' or 3'-->5' exonuclease. Individual mononucleotides are identified from the mass differences between adjacent peaks corresponding to singly charged ions of the products of enzymatic cleavage. Chemically modified nucleotides are detected and identified by their molecular weight. The resolution and mass accuracy of this approach are sufficient to identify nucleobase modifications differing in mass by as little as 2 Da. No a priori information on the DNA sequence or adduct type is required. We demonstrate the general applicability of this method by sequencing synthetic oligonucleotides containing a range of nucleobase modifications: O(6)-methylguanine, peroxynitrite-induced oxidative lesions (oxaluric acid, oxazolone, cyanuric acid), and the N(2)-guanine adduct of (+,-)-7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydribenzo[a]pyrene. Sequence information is also obtained for DNA oligodeoxynucleotides containing O(6)-pyridyloxobutylguanine, despite the ability of this lesion to block 3'-phosphodiesterase.

Fragmentation of 3,7-dialkyl-1,5-diphenyl-3,7-diazabicyclo[3.3. 1]nonan-9-ones under electron ionization.

A series of 3,7-dialkyl-1,5-diphenyl-3,7-diazabicyclo[3.3. 1]nonan-9-ones was prepared, and the details of their fragmentation under electron ionization (EI) were elucidated. The molecular ions of each compound under consideration were quite abundant in their EI spectra. Full-scan spectra exhibited a number of fragment ions which were clearly assigned using MS/MS and accurate mass measurements. The basic fragmentation of 3,7-dialkyl-1,5-diphenylbispidinones was due to the cleavage of C(1)-C(2) bond followed by a hydrogen migration similar to an odd-electron McLafferty rearrangement. Alternatively, the C(1)-C(2) bond cleavage was followed by the elimination of an imine molecule, Alk-N=CH(2). Further fragmentation resulted in ions at m/z 234 and 103, present in the spectra of all the compounds under study. The fragmentation pathways proposed in this paper are based on the substituent shifts, accurate mass measurements and collision-induced dissociation spectra of selected ions. The results of the present work can be useful in selecting the fragment ions suitable for identification and quantitation of bispidinones in biological matrices.

Peroxynitrite-induced secondary oxidative lesions at guanine nucleobases: chemical stability and recognition by the Fpg DNA repair enzyme.

Synthetic oligodeoxynucleotides containing secondary oxidative lesions at guanine nucleobases have been prepared by the site-specific oxidation by ONOO(-) of oligomers containing 8-oxoguanine (8-oxo-G). The oligomers have been tested for their stability to the standard hot piperidine treatment that is commonly used to uncover oxidized DNA lesions. While DNA containing oxaluric acid and oxazolone was cleaved at the site of modification under hot piperidine conditions, the corresponding cyanuric acid and 8-oxo-G lesions were resistant to piperidine. The recognition of the oxidative lesions by formamidopyrimidine glycosylase (Fpg enzyme) was examined in double-stranded versions of the synthetic oligodeoxynucleotides. Fpg efficiently excised 8-oxo-G and oxaluric acid and to some extent oxazolone, but not cyanuric acid. These data suggest that some DNA lesions formed via ONOO(-) exposures (cyanuric acid) are not repaired by Fpg and are not uncovered by assays based on piperidine cleavage at the site of lesion. Our results indicate that cryptic secondary and tertiary oxidation products arising from 8-oxo-G may contribute to the overall mutational spectra arising from oxidative stress.

DNA adducts: effects of low exposure to ethylene oxide, vinyl chloride and butadiene.

Dose-response relationships of genotoxic agents differ greatly depending on the agent and the endpoint being evaluated. Simple conclusions that genotoxic effects are linear cannot be applied universally. The shape of the molecular dose of DNA adducts varies from linear, to supralinear, to sublinear depending on metabolic activation and detoxication, and repair of individual types of DNA adducts. For mutagenesis and other genotoxicity endpoints, the dose-response reflects the molecular dose of each type of DNA adduct, cell proliferation, as well as endogenous factors that lead to mutagenesis such as the formation and repair of endogenous DNA adducts. These same factors are important when interpreting the shape of dose-response data for carcinogenesis of genotoxic agents, however, tumor background variability adds additional complexity. Endogenously formed DNA adducts may be identical to those formed by chemicals, as in the case of vinyl chloride and ethylene oxide, or they may be those associated with oxidative stress. Data presented in this paper demonstrate that the exogenous number of adducts induced by 5 days of exposure to 10 ppm vinyl chloride is only 2. 2-fold greater than that present as a steady-state amount in unexposed control rats. Similar data are shown for ethylene oxide. Extremely sensitive methods have been developed for measuring the molecular dose of genotoxins. These methods can detect DNA adducts as low as 1 per 10(9) to 10(10). However, in view of the high number of endogenous DNA adducts that are present in all cells, it is unlikely that causal relationships can be attributed to very low numbers of such DNA adducts. Effects of both exogenous and endogenous DNA adducts need to be factored into the interpretation of chemical exposures.

Peroxynitrite-induced DNA damage in the supF gene: correlation with the mutational spectrum.

Tissue inflammation and chronic infection lead to the overproduction of nitric oxide and superoxide. These two species rapidly combine to yield peroxynitrite (ONOO(-)), a powerful oxidizing and nitrating agent that is thought be involved in both cell death and an increased cancer risk observed for inflamed tissues. ONOO(-) has been shown to induce single-strand breaks and base damage in DNA and is mutagenic in the supF gene, inducing primarily G to T transversions clustered at the 5' end of the gene. The mutagenicity of ONOO(-) is believed to result from chemical modifications at guanine nucleobases leading to miscoding DNA lesions. In the present work, we applied a combination of molecular and analytical techniques in an attempt to identify biologically important DNA modifications induced by ONOO(-). pUC19 plasmid treated with ONOO(-) contained single-strand breaks resulting from direct sugar damage at the DNA backbone, as well as abasic sites and nucleobase modifications repaired by Fpg glycosylase. The presence of carbon dioxide in the reaction mixture shifted the ONOO(-) reactivity towards reactions at nucleobases, while suppressing the oxidation of deoxyribose. To further study the chemistry of the ONOO(-) interactions with DNA, synthetic oligonucleotides representing the mutation-prone region of the supF gene were treated with ONOO(-), and the products were analyzed by liquid chromatography-negative ion electrospray ionization mass spectrometry (LC-ESI(-) MS) and tandem mass spectrometry. 8-Nitroguanine (8-nitro-G) was formed in ONOO(-)-treated oligonucleotides in a dose-dependent manner with a maximum at a ratio of [ONOO(-)]: [DNA]=10 and a decline at higher ONOO(-) concentrations, suggesting further reactions of 8-nitro-G with ONOO(-). 8-Nitro-G was spontaneously released from oligonucleotides (t(1/2)=1 h at 37 degrees C) and, when present in DNA, was not recognized by Fpg glycosylase. To obtain more detailed information on ONOO(-)-induced DNA damage, a restriction fragment from the pSP189 plasmid containing the supF gene (135 base pairs) was [32P]-end-labeled and treated with ONOO(-). PAGE analysis of the products revealed sequence-specific lesions at guanine nucleobases, including the sites of mutational "hotspots." These lesions were repaired by Fpg glycosylase and cleaved by hot piperidine treatment, but they were resistant to depurination at 90 degrees C. Since 8-nitro-G is subject to spontaneous depurination, and 8-oxo-guanine is not efficiently cleaved by piperidine, these results suggest that alternative DNA lesion(s) contribute to ONOO(-) mutagenicity. Further investigation of the identities of DNA modifications responsible for the adverse biological effects of ONOO(-) is underway in our laboratory.

Molecular dosimetry of N-7 guanine adduct formation in mice and rats exposed to 1,3-butadiene.

1,3-Butadiene (BD) is a high-volume chemical used in the production of rubber and plastic. BD is a potent carcinogen in mice and a much weaker carcinogen in rats, and has been classified as a probable human carcinogen. Upon metabolic activation in vivo, it forms DNA-reactive metabolites, 1,2-epoxy-3-butene (EB), 1,2:3, 4-diepoxybutane (DEB), and 3,4-epoxy-1,2-butanediol (EBD). The molecular dosimetry of N-7 guanine adduct formation by these metabolites of BD in liver, lung, and kidney of B6C3F1 mice and F344 rats exposed to 0, 20, 62.5, or 625 ppm BD was studied. The adducts, racemic and meso forms of N-7-(2,3,4-trihydroxybut-1-yl)guanine (THB-Gua), N-7-(2-hydroxy-3-buten-1-yl)guanine (EB-Gua I), and N-7-(1-hydroxy-3-buten-2-yl)guanine (EB-Gua II), were isolated from DNA by neutral thermal hydrolysis, desalted on solid-phase extraction cartridges, and quantitated by LC/ESI(+)/MS/MS. The number of adducts per 10(6) normal guanine bases for a given adduct was higher in mice than rats exposed to 625 ppm BD, but generally similar at lower levels of exposure. The THB-Gua adducts were the most abundant (6-27 times higher than EB-Gua) and exhibited a nonlinear exposure-response relationship. In rats, the exposure-response curves for the formation of THB-Gua adducts reached a plateau after 62.5 ppm, suggesting saturation of metabolic activation. The number of THB-Gua adducts continued to increase in mice between 62.5 and 625 ppm BD. In contrast, the less common EB-Gua adducts had a linear exposure-response relationship in both species. Combining the information from this study with previous data on BD metabolism, we were able to estimate the number of THB-Gua that resulted from DEB and EBD, and conclude that most of the THB-Gua is formed from EBD. We hypothesize that most of the EBD arises from the immediate conversion of DEB to EBD within the endoplasmic reticulum. This study highlights the need for measurements of the levels of EBD in tissues of rats and mice and for the development of a unique biomarker for DEB that is available for binding to DNA.

Peroxynitrite-induced reactions of synthetic oligonucleotides containing 8-oxoguanine.

8-Oxoguanine (8-oxo-G) is one of the most common DNA lesions present in normal tissues due to exposure to reactive oxygen species. Studies at this and other laboratories suggest that 8-oxo-G is highly susceptible to secondary oxidation, making it a likely target for endogenous oxidizing agents, such as peroxynitrite (ONOO-). Synthetic oligonucleotides containing 8-oxoguanine were treated with ONOO-, and the reaction products were analyzed by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI--MS). CCACAACXCAAA, CCAAAGGXAGCAG, CCAAAXGGAGCAG, and TCCCGAGCGGCCAAAGGXAGCAG (X is 8-oxo-G) were found to readily react with peroxynitrite via the same transformations as those observed for free 8-oxo-2'-deoxyguanosine. The composition of the reaction mixtures was a function of ONOO- concentration and of the storage time after exposure. The oligonucleotide products isolated at low [ONOO-]/[DNA] ratios (<5) were tentatively assigned as containing 3a-hydroxy-5-imino-3,3a,4,5-tetrahydro-1H-imidazo[4, 5d]imidazol-2-one, 5-iminoimidazolidine-2,4-dione, and its hydrolytic product, oxaluric acid. At a [ONOO-]/[DNA] ratio of >10, 2,4,6-trioxo[1,3,5]triazinane-1-carboxamidine- and cyanuric acid-containing oligomers were the major products. The exact location of a modified base within a DNA sequence was determined using exonuclease digestion of oligonucleotide products followed by LC/ESI--MS analysis of the fragments. For all 8-oxo-G-containing oligomers, independent of the sequence, the reactions with ONOO- took place at the 8-oxo-G residues. These results suggest that 8-oxo-G, if present in DNA, is rapidly oxidized by peroxynitrite and that oxaluric acid is a likely secondary oxidation product of 8-oxo-G under physiological conditions.

Identification and quantitation of DNA adducts from calf thymus DNA exposed to 3,4-epoxy-1-butene.

3,4-Epoxy-1-butene (EB) is the major mutagenic metabolite of butadiene (BD), an important industrial chemical classified as a probable human carcinogen. Although the mechanism of carcinogenicity of EB is not known, its reactions with nucleophilic sites of DNA giving pro-mutagenic lesions are likely to constitute the early crucial step in multistage carcinogenesis. This study was conducted to characterize the adducts formed from reactions of EB with the most nucleophilic DNA nucleobases, adenine (Ade) and guanine (Gua), as free nucleobases, 2'-deoxyribonucleosides and constituents of calf thymus DNA (CT DNA) in order to provide insight into the nature of DNA modification by EB. The adducts were isolated using HPLC separation coupled with diode array detection (DAD) and structurally characterized from their electronic, mass- and nuclear magnetic resonance spectra. Four EB-adenine products were identified as N-1-(2-hydroxy-3-buten-1-yl) adenine (EB-Ade I), N-1-(1-hydroxy-3-buten-2-yl) adenine (EB-Ade II), N-3-(2-hydroxy-3-buten-1-yl) adenine (EB-Ade III) and N-3-(1-hydroxy-3-buten-2-yl) adenine (EB-Ade IV). Two previously reported guanine adducts: N-7-(2-hydroxy-3-buten-1-yl) guanine (EB-Gua I) and N-7-(1-hydroxy-3-buten-2-yl) guanine (EB-Gua II) were also collected. The purified adducts were used as reference compounds to detect and quantitate the corresponding adduct species formed in calf thymus DNA incubated with EB. All six adducts were detected in treated DNA. The N-7 position of guanine was the most reactive in DNA followed by N-3 of adenine and N-1 of adenine. The formation of N-1 and N-3-adenine adducts (EB-Ade I, 1.2 +/- 0.36; EB-Ade II, 0.8 +/- 0.27; EB-Ade III, 2.7 +/- 0.38; EB-Ade IV, 5.9 +/- 0.68 nmol/micromol Ade) in CT DNA was approximately one-tenth that of EB-guanine adducts (50.7 +/- 2.37 and 47.9 +/- 3.6 nmol/micromol Gua, respectively). The N-1-EB-Ade adducts detected in this study are likely to be the precursors of previously reported N6-EB-adenine adducts (Koivisto et al., 1995) through Dimroth rearrangement. Since BD and EB induce significant numbers of point mutations at A:T base pairs, the EB-adenine adducts may represent important lesions involved in BD-induced mutagenesis and carcinogenesis.

Adenine adducts with diepoxybutane: isolation and analysis in exposed calf thymus DNA.

1,3-Butadiene (BD) is a high-volume industrial chemical and a common environmental pollutant. Although BD is classified as a "probable human carcinogen", only limited evidence is available for its tumorigenic effects in occupationally exposed populations. Animal studies show a surprisingly high sensitivity of mice to the carcinogenic effects of BD compared to rats (approximately 10(3)-fold), making interspecies extrapolations difficult. Identification and quantitation of specific BD-induced DNA adducts are important for improving our understanding of the mechanisms of BD biological effects and for explaining the observed species differences. Covalent binding of BD to DNA is probably due to its two epoxy metabolites: 3,4-epoxy-1-butene (EB) and 1,2:3,4-diepoxybutane (DEB). Both EB and DEB are direct mutagens producing frameshift and point mutations at both A:T and G:C base pairs. DEB is 100 times more mutagenic than EB and is found in quantity only in tissues of the most sensitive species (mouse). This has led to the suggestion that the higher sensitivity of mice to BD could be due to greater exposure to DEB. The present work was initiated in order to isolate and structurally characterize DEB-induced adenine adducts. The adducts were formed by reacting DEB with free adenine (Ade), 2'-deoxyadenosine (2'-dAdo), and calf thymus DNA followed by HPLC separation and analysis of the products by UV spectrophotometry, electrospray ionization mass spectrometry, and nuclear magnetic resonance. The adenine reaction resulted in three products which were identified as N-3-, N-7-, and N-9-(2'-hydroxy-3',4'-epoxybut-1'-yl)adenine. These adducts underwent acid-catalyzed hydrolysis to their corresponding (2',3',4'-trihydroxybut-1'-yl)adenines upon heating or storage. The 2'-dAdo reaction with DEB followed by acid hydrolysis yielded a single adduct, N6-(2',3',4'-trihydroxybut-1'-yl)adenine (N6-DEB-Ade). N-3-DEB-Ade and N6-DEB-Ade were also found in hydrolysates of calf thymus DNA exposed to DEB. The amounts of N-3-DEB-Ade (13/10(3) normal Ade) and N6-DEB-Ade (5/10(3) normal Ade) were slightly lower than those of the corresponding EB-induced adducts in similar experiments, suggesting comparable reactivity of the two epoxy metabolites of BD toward adenine in DNA. The findings of this study provide a basis for future analyses of BD-induced adenyl DNA adducts in vitro and in vivo.