Koli Taghizadeh

A Quantitative Systems Approach Reveals Dynamic Control of tRNA Modifications during Cellular Stress

Decades of study have revealed more than 100 ribonucleoside structures incorporated as post-transcriptional modifications mainly in tRNA and rRNA, yet the larger functional dynamics of this conserved system are unclear. To this end, we developed a highly precise mass spectrometric method to quantify tRNA modifications in Saccharomyces cerevisiae. Our approach revealed several novel biosynthetic pathways for RNA modifications and led to the discovery of signature changes in the spectrum of tRNA modifications in the damage response to mechanistically different toxicants. This is illustrated with the RNA modifications Cm, m5C, and m2 2G, which increase following hydrogen peroxide exposure but decrease or are unaffected by exposure to methylmethane sulfonate, arsenite, and hypochlorite. Cytotoxic hypersensitivity to hydrogen peroxide is conferred by loss of enzymes catalyzing the formation of Cm, m5C, and m2 2G, which demonstrates that tRNA modifications are critical features of the cellular stress response. The results of our study support a general model of dynamic control of tRNA modifications in cellular response pathways and add to the growing repertoire of mechanisms controlling translational responses in cells.

AlkB reverses etheno DNA lesions caused by lipid oxidation in vitro and in vivo.

Oxidative stress converts lipids into DNA-damaging agents. The genomic lesions formed include 1,N6-ethenoadenine (εA) and 3,N4-ethenocytosine (εC), in which two carbons of the lipid alkyl chain form an exocyclic adduct with a DNA base. Here we show that the newly characterized enzyme AlkB repairs εA and εC. The potent toxicity and mutagenicity of εA in Escherichia coli lacking AlkB was reversed in AlkB+ cells; AlkB also mitigated the effects of εC. In vitro, AlkB cleaved the lipid-derived alkyl chain from DNA, causing εA and εC to revert to adenine and cytosine, respectively. Biochemically, εA is epoxidized at the etheno bond. The epoxide is putatively hydrolyzed to a glycol, and the glycol moiety is released as glyoxal. These reactions show a previously unrecognized chemical versatility of AlkB. In mammals, the corresponding AlkB homologs may defend against aging, cancer and oxidative stress.

Infection-induced colitis in mice causes dynamic and tissue-specific changes in stress response and DNA damage leading to colon cancer

Helicobacter hepaticus-infected Rag2-/- mice emulate many aspects of human inflammatory bowel disease, including the development of colitis and colon cancer. To elucidate mechanisms of inflammation-induced carcinogenesis, we undertook a comprehensive analysis of histopathology, molecular damage, and gene expression changes during disease progression in these mice. Infected mice developed severe colitis and hepatitis by 10 wk post-infection, progressing into colon carcinoma by 20 wk post-infection, with pronounced pathology in the cecum and proximal colon marked by infiltration of neutrophils and macrophages. Transcriptional profiling revealed decreased expression of DNA repair and oxidative stress response genes in colon, but not in liver. Mass spectrometric analysis revealed higher levels of DNA and RNA damage products in liver compared to colon and infection-induced increases in 5-chlorocytosine in DNA and RNA and hypoxanthine in DNA. Paradoxically, infection was associated with decreased levels of DNA etheno adducts. Levels of nucleic acid damage from the same chemical class were strongly correlated in both liver and colon. The results support a model of inflammation-mediated carcinogenesis involving infiltration of phagocytes and generation of reactive species that cause local molecular damage leading to cell dysfunction, mutation, and cell death. There are strong correlations among histopathology, phagocyte infiltration, and damage chemistry that suggest a major role for neutrophils in inflammation-associated cancer progression. Further, paradoxical changes in nucleic acid damage were observed in tissue- and chemistry-specific patterns. The results also reveal features of cell stress response that point to microbial pathophysiology and mechanisms of cell senescence as important mechanistic links to cancer.

N-formylation of lysine in histone proteins as a secondary modification arising from oxidative DNA damage

The posttranslational modification of histone and other chromatin proteins has a well recognized but poorly defined role in the physiology of gene expression. With implications for interfering with these epigenetic mechanisms, we now report the existence of a relatively abundant secondary modification of chromatin proteins, the N6-formylation of lysine that appears to be uniquely associated with histone and other nuclear proteins. Using both radiolabeling and sensitive bioanalytical methods, we demonstrate that the formyl moiety of 3′-formylphosphate residues arising from 5′-oxidation of deoxyribose in DNA, caused by the enediyne neocarzinostatin, for example, acylate the N6-amino groups of lysine side chains. A liquid chromatography (LC)–tandem mass spectrometry (MS) method was developed to quantify the resulting N6-formyl-lysine residues, which were observed to be present in unperturbed cells and all sources of histone proteins to the extent of 0.04–0.1% of all lysines in acid-soluble chromatin proteins including histones. Cells treated with neocarzinostatin showed a clear dose–response relationship for the formation of N6-formyl-lysine, with this nucleosome linker-selective DNA-cleaving agent causing selective N6-formylation of the linker histone H1. The N6-formyl-lysine residue appears to represent an endogenous histone secondary modification, one that bears chemical similarity to lysine N6-acetylation recognized as an important determinant of gene expression in mammalian cells. The N6-formyl modification of lysine may interfere with the signaling functions of lysine acetylation and methylation and thus contribute to the pathophysiology of oxidative and nitrosative stress.

Quantification of DNA damage products resulting from deamination, oxidation and reaction with products of lipid peroxidation by liquid chromatography isotope dilution tandem mass spectrometry

The analysis of damage products as biomarkers of inflammation has been hampered by a poor understanding of the chemical biology of inflammation, the lack of sensitive analytical methods and a focus on single chemicals as surrogates for inflammation. To overcome these problems, we developed a general and sensitive liquid chromatographic tandem mass spectrometry (LC/MS-MS) method to quantify, in a single DNA sample, the nucleoside forms of seven DNA lesions reflecting the range of chemistries associated with inflammation: 2′-deoxyuridine, 2′-deoxyxanthosine and 2′-deoxyinosine from nitrosative deamination; 8-oxo-2′-deoxyguanosine from oxidation; and 1,N2-etheno-2′-deoxyguanosine, 1,N6-etheno-2′-deoxyadenosine and 3,N4-etheno-2′-deoxycytidine arising from reaction of DNA with lipid peroxidation products. Using DNA purified from cells or tissues under conditions that minimize artifacts, individual nucleosides are purified by HPLC and quantified by isotope-dilution, electrospray ionization LC/MS-MS. The method can be applied to other DNA damage products and requires 4–6 d to complete depending upon the number of samples.

Hepatocellular Carcinoma Associated with Liver-Gender Disruption in Male Mice

Hepatocellular carcinoma (HCC) is a male-predominant cancer associated with chronic hepatitis. Like human viral hepatitis, murine Helicobacter hepaticus infection produces inflammation and HCC with a masculine bias. We used this model to identify potential mechanisms of male HCC predisposition. Male weanling A/JCr mice (n = 67) were gavaged with H. hepaticus or vehicle. At 1 year, mice were distributed into four groups: surgical castration, chemical castration, castration followed by dihydrotestosterone supplementation, or sexually intact controls. Responses to infection were compared with IFN-gamma challenge alone. At 21 months, there was no significant difference in hepatitis between groups. Neither castration nor androgen receptor agonism altered tumor incidence. Infected mice with severe, but not mild, disease exhibited a mosaic of alterations to sexually dimorphic genes and microsomal long-chain fatty acids. By microarray, tumorigenic hepatitis was strongly associated with liver-gender disruption, defined as the loss of a gender-identifying hepatic molecular signature. IFN-gamma alone produced similar changes, demonstrating a role for proinflammatory cytokines in this process. In conclusion, hepatocarcinogenesis in male mice with chronic hepatitis is maturationally imprinted and androgen-independent. Proinflammatory cytokines may promote HCC in a male-predominant fashion due to high sensitivity of the masculinized liver to loss of sex-specific transcriptional balance. Liver-gender disruption has pleiotropic implications for hepatic enzyme activity, lipid processing, nuclear receptor activation, apoptosis, and proliferation. We propose a multistep model linking chronic hepatitis to liver cancer through cytokine-mediated derangement of gender-specific cellular metabolism. This model introduces a novel mechanism of inflammation-associated carcinogenesis consistent with male-predominant HCC risk.

Characterization of flame-generated C10 to C160 polycyclic aromatic hydrocarbons by atmospheric-pressure chemical ionization mass spectrometry with liquid introduction via heated nebulizer interface

Complex mixtures of polycyclic aromatic hydrocarbons (PAHs) generated from fuel-rich combustion of ethylene-naphthalene mixtures in a jet-stirred-plug-flow reactor were chemically characterized by combined mass spectrometric techniques to yield product composition data that cover the molecular mass region from simple PAHs (naphthalene, 128 u) to large molecules comparable in molecular size (1792 u) to nanoparticles of soot. Two techniques based on atmospheric-pressure chemical ionization mass spectrometry (APCI-MS) were investigated: (1) APCI-MS combined with high-performance liquid chromatography through a heated nebulizer interface was found suitable for PAHs up to C36 (448 u). (2) For the characterization of larger PAHs beyond C36, direct liquid introduction (DLI) of sample into an atmospheric-pressure chemical ionization mass spectrometer through a heated nebulizer gave protonated molecular ions for PAHs over the m/z 400–2000 range. Although unequivocal elemental composition information is unattainable from the unit-resolution DLI/APCI-MS data, by starting with structural data from identified C16 to C32 PAHs, and applying PAH molecular growth principles, it was possible to generate PAH molecular maps from the DLI/APCI-MS data from which values for the elemental composition could be derived for all major peaks.

DNA phosphorothioation is widespread and quantized in bacterial genomes

Phosphorothioate (PT) modification of DNA, with sulfur replacing a nonbridging phosphate oxygen, was recently discovered as a product of the dnd genes found in bacteria and archaea. Given our limited understanding of the biological function of PT modifications, including sequence context, genomic frequencies, and relationships to the diversity of dnd gene clusters, we undertook a quantitative study of PT modifications in prokaryotic genomes using a liquid chromatography-coupled tandem quadrupole mass spectrometry approach. The results revealed a diversity of unique PT sequence contexts and three discrete genomic frequencies in a wide range of bacteria. Metagenomic analyses of PT modifications revealed unique ecological distributions, and a phylogenetic comparison of dnd genes and PT sequence contexts strongly supports the horizontal transfer of dnd genes. These results are consistent with the involvement of PT modifications in a type of restriction-modification system with wide distribution in prokaryotes.

Effects of PAH isomerizations on mutagenicity of combustion products

Abstract Most of the mutagenicity of mixtures of polycyclic aromatic hydrocarbons (PAH) mixtures found in combustion exhaust gases is contributed by a relatively small number of the many PAH present. Since PAH mutagenicity is structure and hence isomer sensitive, changes in the distribution of isomers can change the mutagenicity of the mixture. Whether isomerization reactions in combustion play a significant role in determining the distributions of PAH isomers and the mutagenicity of product mixtures is assessed here for the following pairs of isomers: 1. (1) fluoranthene-pyrene 2. (2) fluoranthene-acephenanthrylene 3. (3) cyclopenta[ cd ]pyrene-benzo[ ghi ]fluoranthene 4. (4) benzo[ k ]fluoranthene-benzo[ a ]pyrene Concentration ratios of the isomer pairs were measured in ethylene combustion with naphthalene injection using a plug flow reactor at equivalence ratios of 1.2 and 2.2 and temperatures of 1520, 1620, and 1705 K, and compared with equilibrium ratios based on properties computed from molecular mechanics and semiempirical quantum mechanical programs [MM3; MNDO, AM1, and PM3 in both restricted- and unrestricted-Hartree-Fock forms]. Bacterial mutagenicity was measured by a forward mutation assay using Salmonella in the presence of rat liver supernatant, and found to vary significantly among the above compounds. The measured concentration ratios for isomer pairs (2) and (3) are near the equilibrium values and becoming more so as temperature increases, but the measured ratios for isomer pairs (1) and (4) are far from the equilibrium values at all the temperatures. From kinetics estimations, the characteristic isomerization time for isomer pairs (2) and (3) at 1705 K and perhaps at 1620 K is less than the experimental residence times, while the only isomerization mechanisms envisioned for isomer pairs (1) and (4) would not be kinetically viable at these temperatures. These results indicate that isomerizations affecting mutagenicity are significant under these conditions for isomer pairs (2) and (3), which involve only compounds containing both five- and six-membered rings, but not for isomer pairs (1) and (4), each of which includes a compound containing only six- membered rings.

Defects in purine nucleotide metabolism lead to substantial incorporation of xanthine and hypoxanthine into DNA and RNA

Deamination of nucleobases in DNA and RNA results in the formation of xanthine (X), hypoxanthine (I), oxanine, and uracil, all of which are miscoding and mutagenic in DNA and can interfere with RNA editing and function. Among many forms of nucleic acid damage, deamination arises from several unrelated mechanisms, including hydrolysis, nitrosative chemistry, and deaminase enzymes. Here we present a fourth mechanism contributing to the burden of nucleobase deamination: incorporation of hypoxanthine and xanthine into DNA and RNA caused by defects in purine nucleotide metabolism. Using Escherichia coli and Saccharomyces cerevisiae with defined mutations in purine metabolism in conjunction with analytical methods for quantifying deaminated nucleobases in DNA and RNA, we observed large increases (up to 600-fold) in hypoxanthine in both DNA and RNA in cells unable to convert IMP to XMP or AMP (IMP dehydrogenase, guaB; adenylosuccinate synthetase, purA, and ADE12), and unable to remove dITP/ITP and dXTP/XTP from the nucleotide pool (dITP/XTP pyrophosphohydrolase, rdgB and HAM1). Conversely, modest changes in xanthine levels were observed in RNA (but not DNA) from E. coli lacking purA and rdgB and the enzyme converting XMP to GMP (GMP synthetase, guaA). These observations suggest that disturbances in purine metabolism caused by known genetic polymorphisms could increase the burden of mutagenic deaminated nucleobases in DNA and interfere with gene expression and RNA function, a situation possibly exacerbated by the nitrosative stress of concurrent inflammation. The results also suggest a mechanistic basis for the pathophysiology of human inborn errors of purine nucleotide metabolism.