John S. Wishnok

ANALYSIS OF NITRATE, NITRITE AND 15N NITRATE IN BIOLOGICAL FLUIDS

Abstract A new automated system for the analysis of nitrate via reduction with a high-pressure cadmium column is described. Samples of urine, saliva, deproteinized plasma, gastric juice, and milk can be analyzed for nitrate, nitrite, or both with a lower limit of detection of 1.0 nmol NO 3 − or NO 2 − /ml. The system allows quantitative reduction of nitrate and automatically eliminates interference from other compounds normally present in urine and other biological fluids. Analysis rate is 30 samples per hour, with preparation for most samples limited to simple dilution with distilled water. The application of gas chromatography/mass spectrometry for the analysis of 15 NO 3 − in urine after derivatization to 15 NO 2 -benzene is also described.

The chemistry of DNA damage from nitric oxide and peroxynitrite

Nitric oxide is a key participant in many physiological pathways; however, its reactivity gives it the potential to cause considerable damage to cells and tissues in its vicinity. Nitric oxide can react with DNA via multiple pathways. Once produced, subsequent conversion of nitric oxide to nitrous anhydride and/or peroxynitrite can lead to the nitrosative deamination of DNA bases such as guanine and cytosine. Complex oxidation chemistry can also occur causing DNA base and sugar oxidative modifications. This review describes the different mechanisms by which nitric oxide can damage DNA. First, the physiological significance of nitric oxide is discussed. Details of nitric oxide and peroxynitrite chemistry are then given. The final two sections outline the mechanisms underlying DNA damage induced by nitric oxide and peroxynitrite.

Nitrate, nitrite and N-nitroso compounds

A risk assessment has been made on nitrate, nitrite and N-nitroso compounds encountered in the human diet. Vegetables constitute a major source of nitrate providing over 85% of the average daily human dietary intake. Nitrite and N-nitroso compounds present in the diet contribute relatively small amounts to the body burden and the major source of these biologically reactive compounds is derived from the bacterial and mammalian metabolism of ingested nitrate. Additionally, endogenous synthesis provides an important source contributing to the body burden of nitrate. Data from animal toxicological studies, human effects and epidemiological surveys have been reviewed and evaluated. It is concluded that there is no firm scientific evidence at present to recommend drastic reductions beyond the average levels of nitrate encountered in vegetables grown in keeping with good agricultural practice. Recommendations have also been made for further animal and human studies to be carried out to elucidate the potential risks to man from ingested nitrate.

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.

Arsenic exposure perturbs the gut microbiome and its metabolic profile in mice: an integrated metagenomics and metabolomics analysis.

Background: The human intestine is host to an enormously complex, diverse, and vast microbial community—the gut microbiota. The gut microbiome plays a profound role in metabolic processing, energy production, immune and cognitive development, epithelial homeostasis, and so forth. However, the composition and diversity of the gut microbiome can be readily affected by external factors, which raises the possibility that exposure to toxic environmental chemicals leads to gut microbiome alteration, or dysbiosis. Arsenic exposure affects large human populations worldwide and has been linked to a number of diseases, including cancer, diabetes, and cardiovascular disorders. Objectives: We investigated the impact of arsenic exposure on the gut microbiome composition and its metabolic profiles. Methods: We used an integrated approach combining 16S rRNA gene sequencing and mass spectrometry–based metabolomics profiling to examine the functional impact of arsenic exposure on the gut microbiome. Results: 16S rRNA gene sequencing revealed that arsenic significantly perturbed the gut microbiome composition in C57BL/6 mice after exposure to 10 ppm arsenic for 4 weeks in drinking water. Moreover, metabolomics profiling revealed a concurrent effect, with a number of gut microflora–related metabolites being perturbed in multiple biological matrices. Conclusions: Arsenic exposure not only alters the gut microbiome community at the abundance level but also substantially disturbs its metabolic profiles at the function level. These findings may provide novel insights regarding perturbations of the gut microbiome and its functions as a potential new mechanism by which arsenic exposure leads to or exacerbates human diseases. Citation: Lu K, Abo RP, Schlieper KA, Graffam ME, Levine S, Wishnok JS, Swenberg JA, Tannenbaum SR, Fox JG. 2014. Arsenic exposure perturbs the gut microbiome and its metabolic profile in mice: an integrated metagenomics and metabolomics analysis. Environ Health Perspect 122:284–291; http://dx.doi.org/10.1289/ehp.1307429

Inhibition of nitrosamine formation by ascorbic acid.

Nitrosation occurs under a wide variety of conditions by reaction of most types of amines with any of a large number of nitrosating species. Nitrite can be formed in vivo via bacterial reduction of nitrate and by activated macrophages and endothelial cells. The mechanism of nitrite formation by mammalian cells is via enzymatic oxidation of arginine to NO followed by oxidation to N2O3 and N2O4. Nitrosatable amines are found in many foods and some, eg, dimethylamine, are synthesized in the body. Precursors of N-nitroso compounds are thus almost constantly present together under favorable reaction conditions in vivo and there is, consequently, considerable interest concerning possible human health risks arising from endogenous formation of this class of compounds. Among many nitrosation inhibitors, most attention has focused on ascorbic acid, which reacts with many nitrosating agents and which is virtually nontoxic. This presentation discusses the chemistry of ascorbic acid inhibition of nitrosation reactions.

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.

Nitric Oxide-induced Deamination of Cytosine and Guanine in Deoxynucleosides and Oligonucleotides

The autoxidation of nitric oxide (NO⋅) forms the nitrosating agent N2O3, which can directly damage DNA by deamination of DNA bases following nitrosation of their primary amine functionalities. Within the G:C base pair, deamination results in the formation of xanthine and uracil, respectively. To determine the effect of DNA structure on the deamination of guanine and cytosine, the NO⋅-induced deamination rate constants for deoxynucleosides, single- and double-stranded oligonucleotides, and a G-quartet oligonucleotide were measured. Deamination rate constants were determined relative to morpholine using a Silastic membrane to deliver NO⋅ at a rate of ∼10–20 nmol/ml/min for 60 min, yielding a final concentration of ∼600–1200 μm NO2 −. GC/MS analysis revealed formation of nanomolar levels of deamination products from millimolar concentrations of deoxynucleosides and oligomers. Deamination rate constants for cytosine and guanine in all types of DNA were lower than the morpholine nitrosation rate constant by a factor of ∼103–104. Xanthine was formed at twice the rate of uracil, and this may have important consequences for mechanisms of NO⋅-induced mutations. Single-stranded oligomers were 5 times more reactive than deoxynucleosides toward N2O3. Double-stranded oligomers were 10-fold less reactive than single-stranded oligomers, suggesting that Watson-Crick base pairing protects DNA from deamination. G-quartet structures were also protective, presumably because of hydrogen bonding. These results demonstrate that DNA structure is an important factor in determining the reactivity of DNA bases with NO⋅-derived species.

L-Arginine is a precursor for nitrate biosynthesis in humans

Nitrogen from L-arginine was incorporated into urinary nitrate in human subjects. Two subjects given an oral dose of [15N2]L-arginine excreted 24 and 17 umol [15N]nitrate/24 hr, respectively, in their urine in the 24 hr period following the dose. This work demonstrates that L-arginine, a nitrogen source for biosynthesized nitrate in cultured cells and research animals, is a precursor for endogenously synthesized nitrate in humans.

Analysis of nitrated proteins by nitrotyrosine-specific affinity probes and mass spectrometry

Tyrosine nitration is a well-established protein modification that occurs in disease states associated with oxidative stress and increased nitric oxide synthase activity. Nitration of specific tyrosine residues has been reported to affect protein structure and function, suggesting that 3-nitrotyrosine formation may not only be a disease marker but may also be involved in the pathogenesis of some diseases and in normal regulatory processes. It has been, however, difficult to identify sites of nitration. We describe a method that combines specific isolation of nitrated proteins with mass spectrometric determination of the amino acid sequence and the site of nitration of individual proteins. A complex protein mixture, e.g., serum or cell lysate, was enriched for nitrotyrosine-containing proteins by immunoprecipitation with antinitrotyrosine antibodies. The nitrotyrosines were then reduced to aminotyrosines with a strong reducing agent in parallel in-gel and in-solution procedures. Using nitrated human serum albumin as a model, we reduced the disulfide bonds with dithiothreitol and alkylated the free sulfhydryl groups with iodoacetamide. The nitrotyrosines were next reduced to aminotyrosines with sodium dithionite, and-at pH 5.0-cleavable biotin tags were selectively attached to the aminotyrosines and the albumin was then digested with trypsin. The biotinylated tryptic peptides were purified on a streptavidin affinity column and identified by mass spectrometry. We have also purified nitrated human serum albumin from an enriched sample of SJL mouse plasma and confirmed its identity by peptide mass fingerprinting and MASCOT.