Quantitative Systems Pharmacology Approaches Applied to Microphysiological Systems (MPS): Data Interpretation and Multi-MPS Integration.

Our goal in developing Microphysiological Systems (MPS) technology is to provide an improved approach for more predictive preclinical drug discovery via a highly integrated experimental/computational paradigm. Success will require quantitative characterization of MPSs and mechanistic analysis of experimental findings sufficient to translate resulting insights from in vitro to in vivo. We describe herein a systems pharmacology approach to MPS development and utilization that incorporates more mechanistic detail than traditional pharmacokinetic/pharmacodynamic (PK/PD) models. A series of studies illustrates diverse facets of our approach. First, we demonstrate two case studies: a PK data analysis and an inflammation response--focused on a single MPS, the liver/immune MPS. Building on the single MPS modeling, a theoretical investigation of a four-MPS interactome then provides a quantitative way to consider several pharmacological concepts such as absorption, distribution, metabolism, and excretion in the design of multi-MPS interactome operation and experiments.

A novel nitroimidazole compound formed during the reaction of peroxynitrite with 2',3',5'-tri-O-acetyl-guanosine.

Peroxynitrite reacts with 2',3',5'-tri-O-acetyl-guanosine to yield a novel compound identified as 1-(2,3,5-tri-O-acetyl-beta-D-erythro-pentofuranosyl)-5-guanidino-4-nitroimidazole (6). This characterization was achieved using a combination of UV/vis spectroscopy and ESI-MS. Additionally, 1-(beta-D-erythro-pentofuranosyl)-5-guanidino-4-nitroimidazole (6a) was synthesized by an independent route, characterized by UV/vis spectroscopy, ESI-MS, and (1)H- and (13)C NMR, and shown to be identical to deacetylated 6. This product is extremely stable in aqueous solution at both pH extremes and is formed in significant yields. These characteristics suggest that this lesion may be useful as a specific biomarker of peroxynitrite-induced DNA damage. We also observed formation of 2',3',5'-tri-O-acetyl-8-nitroguanosine (2',3',5'-tri-O-acetyl-8-NO(2)()Guo), 2-amino-5-[(2,3,5-tri-O-acetyl-beta-D-erythro-pentofuranosyl)amino]-4H-imidazol-4-one (2',3',5'-tri-O-acetyl-Iz), and the peroxynitrite-induced oxidation products of 2',3',5'-tri-O-acetyl-8-oxoGuo. The formation of 6 and 2',3',5'-tri-O-acetyl-8-NO(2)()Guo was rationalized by a mechanism invoking formation of the guanine radical.

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.

Site-selective nitration of tyrosine in human serum albumin by peroxynitrite.

Peroxynitrite, which is formed in biological systems by the reaction of nitric oxide with superoxide anion, is a highly reactive molecule that can lead to cell injury or cell death. Reactions of peroxynitrite under physiological conditions include nitration of tyrosine-containing proteins or peptides, and we have been investigating the behavior of human serum albumin following exposure to peroxynitrite. Peroxynitrite, at relative concentrations ranging from 0.2 to 50 with respect to protein, was added to human serum albumin in buffer at pH 7.2. The resulting mixtures were dialyzed to remove small molecules, dried under vacuum, and then digested with trypsin. The digests were analyzed by high performance liquid chromatography with UV detection at 230 and 354 nm, the latter wavelength being selective for nitrotyrosine. At the higher relative concentrations of peroxynitrite, the 354-nm chromatograms contained a large number of peaks, including at least nine with molecular weights corresponding to nitration of nominal tryptic peptides. Following treatment with the lower relative concentrations of peroxynitrite, however, the 354-nm chromatograms were dominated by only two nitrated peptides; these were identified by comparison of LC retention times and collision-induced decomposition mass spectra as nitro-Y(411)TK(413) and nitro-Y(138)LYEIAR(144). Each of these tyrosines resides in a known reactive site within the protein, i.e., subdomains IIIA and IB, respectively.

Spiroiminodihydantoin is the major product of the 8-oxo-7,8-dihydroguanosine reaction with peroxynitrite in the presence of thiols and guanosine photooxidation by methylene blue.

[reaction: see text]. The potent oxidant, peroxynitrite, will oxidize 8-oxo-7,8-dihydroguanosine to give several products. In the presence of a thiol agent, the major final product has been determined to be a spiroiminodihydantoin compound. Additionally, we have found that the spiroiminodihydantoin, and not the previously reported 4-hydroxy-8-oxo-4,8-dihydroguanosine, is the major final product formed during the methylene blue-mediated photooxidation of guanosine.

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.

A novel nitration product formed during the reaction of peroxynitrite with 2',3',5'-tri-O-acetyl-7,8-dihydro-8-oxoguanosine: N-nitro-N'-[1-(2,3,5-tri-O-acetyl-beta-D-erythro-pentofuranosyl)- 2, 4-dioxoimidazolidin-5-ylidene]guanidine.

A novel nitration product, formed during the reaction of peroxynitrite with 2',3',5'-tri-O-acetyl-7,8-dihydro-8-oxoguanosine, has been characterized using a combination of UV/vis, CD, and NMR spectroscopy and mass spectrometry. This compound has been identified as N-nitro-N'-[1-(2,3, 5-tri-O-acetyl-beta-D-erythro-pentofuranosyl)-2, 4-dioxoimidazolidin-5-ylidene]guanidine (IV). Upon base hydrolysis, IV releases nitroguanidine (IVa) and an intermediate, 1-(2,3, 5-tri-O-acetyl-beta-D-erythro-pentofuranosyl)-5-iminoimidazolidine -2, 4-dione (IVb). This intermediate is ultimately hydrolyzed to the stable 3-(2,3,5-tri-O-acetyl-beta-D-erythro-pentofuranosyl)oxaluric acid (IVc). IV can be reduced by sodium borohydride to a pair of stable diastereomers (IV(red)()). The formation of this product is rationalized in terms of initial oxidation of 2',3', 5'-tri-O-acetyl-7,8-dihydro-8-oxoguanosine to a quinonoid diimine intermediate, 3. Nucleophilic attack at C5 of 3 by peroxynitrite leads to formation of a C5-oxyl radical species, 5, which then undergoes a series of rearrangements to yield an ylidene radical, 7. Combination of this radical species with nitrogen dioxide results in the formation of product IV.

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.

Peroxynitrite reaction products of 3',5'-di-O-acetyl-8-oxo-7, 8-dihydro-2'-deoxyguanosine.

Of the DNA bases, peroxynitrite (ONOO-) is most reactive toward 2'-deoxyguanosine (dGuo), but even more reactive with 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodGuo), requiring a 1,000-fold excess of dGuo to provide 50% protection against the reaction with 8-oxodGuo. Therefore, it seems reasonable that 8-oxodGuo is a potentially important target in DNA and that the structures of the reaction products with ONOO- should be characterized. Using 3', 5'-di-O-Ac-8-oxodGuo as a model compound, the reaction products with ONOO- have been isolated and identified under simulated physiological reaction conditions (phosphate/bicarbonate buffer at pH 7.2). The major reaction product, II, is unstable and undergoes base-mediated hydrolysis to 2,5-diaminoimidazol-4-one, IIa, and 3-(3, 5-di-O-Ac-2-deoxy-beta-D-erythro-pentofuranosyl)-5-iminoimidazolidine -2,4-dione, IIb. The latter compound further hydrolyzes to 3-(3, 5-di-O-Ac-2-deoxy-beta-D-erythro-pentofuranosyl)oxaluric acid, IIc. Other products include 3-(3, 5-di-O-Ac-2-deoxy-beta-D-erythro-pentofuranosyl)-2,4,6-trioxo-[1,3, 5]triazinane-1-carboxamidine, I, which further hydrolyzes to 1-(3, 5-di-O-Ac-2-deoxy-beta-D-erythro-pentofuranosyl)cyanuric acid, Ia. 1-(3,5-di-O-Ac-2-deoxy-beta-D-erythro-pentofuranosyl)parabanic acid, III, is a minor product that also may contribute to formation of IIc. The major products formed in these reactions are biologically uncharacterized but are similar to modified DNA bases that have been shown to be both premutagenic and blocks to DNA polymerization.

Urinary excretion of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in White, African-American, and Asian-American men in Los Angeles County.

Meats, such as beef, pork, poultry, and fish, cooked at high temperatures produce heterocyclic aromatic amines, which have been implicated indirectly as etiological agents involved in colorectal and other cancers in humans. This study examined the urinary excretion of a mutagenic/carcinogenic heterocyclic aromatic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), among 45 African-American, 42 Asian-American (Chinese or Japanese), and 42 non-Hispanic white male residents of Los Angeles who consumed an unrestricted diet. Total PhIP (free and conjugated) was isolated from overnight urine collections, purified by immunoaffinity chromatography, and then quantified by high-pressure liquid chromatography combined with electrospray ionization mass spectrometry. Geometric mean levels of PhIP in Asian-Americans and African-Americans were approximately 2.8-fold higher than in whites. The urinary excretion levels of PhIP were not associated with intake frequencies of any cooked meat based on a self-administered dietary questionnaire, in contrast to our earlier finding (Ji et al., Cancer Epidemiol. Biomark. Prev., 3: 407-411, 1994) of a positive and statistically significant association between bacon intake and the urinary level of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) among this same group of study subjects. Although there is a statistically significant association between urinary levels of PhIP and MeIQx (2-sided P = 0.001), 10 subjects (8%) displayed extreme discordance between urinary PhIP and MeIQx levels. Several factors, including variable contents of heterocyclic aromatic amines in food, enzymic and interindividual metabolic differences, and analytical methodology determine the degree of concordance between the urinary excretion levels of PhIP and MeIQx. Accordingly, urinary excretion levels of a single heterocyclic aromatic amine can only serve as an approximate measure of another in estimating exposure to these compounds in humans consuming unrestricted diets.

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.

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.

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 approximately 10-20 nmol/ml/min for 60 min, yielding a final concentration of approximately 600-1200 microM 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 approximately 10(3)-10(4). 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.

Detection and identification of carcinogen-peptide adducts by nanoelectrospray tandem mass spectrometry.

Nanoelectrospray (nanoES) tandem mass spectrometry was used to examine covalently modified peptides in crude enzymatic digests of human serum albumin (HSA) that had been exposed to either benzo[a]pyrene diol epoxide (B[a]PDE, 1), chrysene diol epoxide (CDE, 2), 5-methylchrysene diol epoxide (5MeCDE, 3), or benzo[g]chrysene diol epoxide (B[g]CDE, 4). The low flow rates of nanoES (approximately 20 nL/min) allowed several MS/MS experiments to be optimized and performed on a single sample with very little sample consumption (approximately 30 min analysis time/microL sample). Initially, nanoES was compared with conventional LC/MS/MS analysis of carcinogen-peptide adducts. For example, nanoES analysis of an unseparated digest of B[a]PDE-treated serum albumin revealed the same peptides (RRHPY and RRHPY-FYAPE) that were previously shown, by LC/MS/MS, to be adducted with B[a]PDE. In addition, nanoES could detect unstable peptide adducts that might not otherwise have been directly observable. Finally, nanoES was shown to be an effective way to screen mixtures of modified and unmodified peptides for which no chromatographic information is available.

Formation of N delta-cyanoornithine from NG-hydroxy-L-arginine and hydrogen peroxide by neuronal nitric oxide synthase: implications for mechanism.

Neuronal nitric oxide synthase (nNOS) catalyzes the oxidation of NG-hydroxy-L-arginine (NHA) by hydrogen peroxide. The amino acid products were characterized by high-performance liquid chromatography/mass spectrometry of the o-phthalaldehyde/2-mercaptoethanol derivatives and identified as citrulline and N delta-cyanoornithine (CN-orn). The assignment of CN-orn was confirmed by independent chemical synthesis and comparison of the properties of the enzyme-derived product with those of synthetic CN-orn. The inorganic products detected in the enzymatic reaction were NO2- and NO3-, presumably from oxidation of NO-. The reaction of H2O2 and NHA with nNOS was at least 10-fold slower than the reaction of NADPH, O2, and NHA (Vmax,app = 49 +/- 2 nmol min-1 mg-1 for the reactions with 10 microM added H4B). The reaction exhibited saturation kinetics with respect to hydrogen peroxide [K(m,app)(H2O2) = 10 +/- 1 mM for the reactions with 10 microM added H4B]. No H2O2-dependent reaction was observed with L-arginine as the amino acid substrate. The different products for the NADPH- and H2O2-dependent transformations of NHA are of mechanistic significance in the NOS reaction.

Identification of subdomain IB in human serum albumin as a major binding site for polycyclic aromatic hydrocarbon epoxides.

Covalent adducts between serum albumin and low molecular weight organic electrophiles are formed with a high degree of regioselectivity mostly for nucleophilic amino acid residues located in subdomains IIA and IIIA. Previous studies have indicated that diol epoxide metabolites of polycyclic aromatic hydrocarbons (PAH) may target residues in a different subdomain. The regioselectivity of PAH epoxide and diol epoxide binding was examined in this study by reaction of human serum albumin in vitro with the racemic trans,anti-isomers of 7,8-dihydrobenzo[a]pyrene-7,8-diol 9,10-epoxide (1), 2,3-dihydrofluoranthene-2,3-diol 1,10b-epoxide (2), 1,2-dihydrochrysene-1,2-diol 3,4-epoxide (5), 6-methyl-1,2-dihydrochrysene-1,2-diol 3,4-epoxide (6), 5-methyl-1,2-dihydrochrysene-1,2-diol 3,4-epoxide (7), 3,4-dihydrobenzo[c]phenanthrene-3,4-diol 1,2-epoxide (8), 11,12-dihydrobenzo[g]chrysene-11,12-diol 13,14-epoxide (9), and 11,12-dihydrodibenzo[a,l]pyrene-11,12-diol 13,14-epoxide (10) and the racemic epoxides cyclopenta[cd]pyrene 3,4-epoxide (3) and benzo[a]pyrene 4,5-epoxide (4) followed by determination of the linkage site. Adducted albumin was digested enzymatically, and digests were chromatographed by reversed-phase HPLC to purify peptide adducts, which were analyzed by electrospray ionization collision-induced dissociation (CID) tandem mass spectrometry. Product ion spectra revealed that adducts fragmented predominantly by cleavage of the peptide-PAH bond with retention of charge by the peptide as well as by the hydrocarbon. Peptide sequences were determined by MS/MS analysis of the peptide ions formed by in-source CID to cleave the adduct bond. Longer peptide sequences established site selectivity by virtue of their uniqueness, while shorter sequences revealed the reactant amino acid within the site. Epoxide 4 and diol epoxides 1, 2, 5, and 6 reacted predominantly with His146; epoxide 3 and diol epoxides 7-9 reacted predominantly with Lys137. Both residues are situated in subdomain IB. The binding site for 10 could not be determined uniquely, but one of the several possibilities was Lys159, which is also located in subdomain IB. The results, taken together with previous findings, demonstrate that the reaction of polycyclic aromatic hydrocarbon epoxides with human serum albumin is highly selective for a small number of residues in subdomain IB.

Urinary excretion of unmetabolized and phase II conjugates of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in humans: relationship to cytochrome P4501A2 and N-acetyltransferase activity.

Cooking meat, fish, or poultry at high temperature gives rise to heterocyclic aromatic amines (HAAs), which may be metabolically activated to mutagenic or carcinogenic intermediates. The enzymes cytochrome P4501A2 (CYP1A2) and N-acetyltransferase (NAT2) are principally implicated in such biotransformations. We have determined the relationship between the activity of these two enzymes and the urinary excretion of unmetabolized and Phase II conjugates of the two HAAs MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline) and PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) in individuals fed a uniform diet containing high-temperature cooked meat. The subjects in the study ate meat containing known amounts of MeIQx and PhIP, and urine collections were made 0-12 and 12-24 h after a meal. MeIQx and PhIP were measured in urine after acid treatment that quantitatively hydrolyzes the Phase II conjugates to the respective parent amine. The extracts containing the HAAs were purified by immunoaffinity chromatography and analyzed by liquid chromatography using electrospray ionization-tandem mass spectrometry. The MeIQx content in the 0-12 h urine increased after acid hydrolysis by a factor of 3-21-fold. After acid treatment, the total amount of MelQx (unmetabolized plus the N2-glucuronide and sulfamate metabolites) excreted in the 0-12 h urine was 10.5 +/- 3.5% (mean +/- SD) of the dose, whereas the total amount of PhIP [unmetabolized plus acid-labile conjugate(s)] in the 0-12 h period was 4.3 +/- 1.7% (mean +/- SD) of the dose. The total amount of PhIP in the 12-24 h urine after acid treatment was 0.9 +/- 0.4% (mean +/- SD) of the dose. Linear regression analysis of the amounts of MeIQx and PhIP excreted in the 0-12 h period expressed as a percentage of the ingested dose, for all subjects, gave a low but significant correlation (r = 0.37, P = 0.005). Linear regression analyses showed that lower total MeIQx (unmetabolized plus the N2-glucuronide and sulfamate metabolites) in urine was associated with higher CYP1A2 activity, whereas total PhIP (unmetabolized plus conjugated) in urine showed no association to CYP1A2 activity. These results indicate that in humans, MeIQx metabolism and disposition are more strongly influenced by CYP1A2 activity than are those of PhIP. Linear regression analysis found no association between NAT2 activity and the levels (unmetabolized plus acid-labile conjugates) of MeIQx or PhIP excreted in urine.

The chemistry of the S-nitrosoglutathione/glutathione system.

S-Nitrosothiols have generated considerable interest due to their ability to act as nitric oxide (NO) donors and due to their possible involvement in bioregulatory systems-e.g., NO transfer reactions. Elucidation of the reaction pathways involved in the modification of the thiol group by S-nitrosothiols is important for understanding the role of S-nitroso compounds in vivo. The modification of glutathione (GSH) in the presence of S-nitrosoglutathione (GSNO) was examined as a model reaction. Incubation of GSNO (1 mM) with GSH at various concentrations (1-10 mM) in phosphate buffer (pH 7.4) yielded oxidized glutathione, nitrite, nitrous oxide, and ammonia as end products. The product yields were dependent on the concentrations of GSH and oxygen. Transient signals corresponding to GSH conjugates, which increased by one mass unit when the reaction was carried out with 15N-labeled GSNO, were identified by electrospray ionization mass spectrometry. When morpholine was present in the reaction system, N-nitrosomorpholine was formed. Increasing concentrations of either phosphate or GSH led to lower yields of N-nitrosomorpholine. The inhibitory effect of phosphate may be due to reaction with the nitrosating agent, nitrous anhydride (N2O3), formed by oxidation of NO. This supports the release of NO during the reaction of GSNO with GSH. The products noted above account quantitatively for virtually all of the GSNO nitrogen consumed during the reaction, and it is now possible to construct a complete set of pathways for the complex transformations arising from GSNO + GSH.

Bicarbonate inhibits N-nitrosation in oxygenated nitric oxide solutions.

N-Nitrosation in oxygenated nitric oxide (NO middle dot) solutions was previously shown to be significantly inhibited by phosphate and chloride presumably by anion scavenging of the nitrosating agent nitrous anhydride, N2O3 (Lewis, R. S., Tannenbaum, S. R., and Deen, W. M. (1995) J. Am. Chem. Soc. 117, 3933-3939). Here, bicarbonate is shown to exhibit this same inhibitory effect. Rate constants for reaction of morpholine, phosphate, and bicarbonate with N2O3 relative to N2O3 hydrolysis at pH 8.9 were determined to be (3.7 +/- 0.2) x 10(4) M-1, (4.0 +/- 0.9) x 10(2) M-1, and (9.3 +/- 1.5) x 10(2) M-1, respectively. The morpholine and phosphate rate constants at pH 8.9 are similar to those reported at pH 7.4 assuring that these results are relevant to physiological conditions. The rate constant for this previously unrecognized reaction of bicarbonate with N2O3 suggests the strong scavenging ability of bicarbonate; accordingly, bicarbonate may contribute to reducing deleterious effects of N2O3. This is biologically important due to substantial bicarbonate concentrations in vivo, approximately 30 mM. Bicarbonate was previously shown to alter peroxynitrite reactivity; however, carbon dioxide is the probable reactive species. Bicarbonate is therefore potentially important in determining the fate of two reactive species generated from nitric oxide, N2O3 and ONOO-, and may thus act as a regulator of NO middle dot-induced toxicity.

Kinetics of S-nitrosation of thiols in nitric oxide solutions.

The S-nitroso adducts of nitric oxide (NO) may serve as carriers of NO and play a role in cell signaling and/or cytotoxicity. A quantitative understanding of the kinetics of S-nitrosothiol formation in solutions containing NO and O2 is important for understanding these roles of S-nitroso compounds in vivo. Rates of S-nitrosation in aqueous solutions were investigated for three thiols: glutathione, N-acetylcysteine, and N-acetylpenicillamine. Nitrous anhydride (N2O3), an intermediate in the formation of nitrite from NO and O2, is the most likely NO donor for N-nitrosation of amines as well as for S-nitrosation of thiols, at physiological pH. This motivated the use of a competitive kinetics approach, in which the rates of thiol nitrosation were compared with that of a secondary amine, morpholine. The kinetic studies were carried out with known amounts of NO and O2 in solutions containing one thiol (400 microM) and morpholine (200-5700 microM) in 0.01 M phosphate buffer at pH 7.4 and 23 degrees C. It was found that disulfide formation, transnitrosation reactions, and decomposition of the S-nitrosothiols was expressed as k7[N2O3][RSH], where RSH represents the thiol. The rate constant for S-nitrosation relative to that for N2O3 hydrolysis (k4) was found to be k7/k4 = (4.15 +/- 0.28) x 10(4), (2.11 +/- 0.11) x 10(4), and (0.48 +/- 0.04) x 10(4) M-1 for glutathione, N-acetylcysteine, and N-acetylpenicillamine, respectively. The overall (observed) rates of nitrosothiol formation reflect the fact that [N2O3] varies [NO]2[O2] and that [N2O3] also depends on [RSH] and the concentration of phosphate. Using a detailed kinetic model to account for these effects, the present results could be reconciled with apparently dissimilar findings reported previously by others.