Formation of cyanamides from secondary amines in human saliva.

1-Morpholinocarbonitrile (1-cyanomorpholine) was formed from morpholine when this amine was incubated in whole human saliva. Several other secondary amines appeared to form analogous products, and this transformation may therefore represent a general metabolic pathway for amines in saliva.

Nitrosamine formation in human saliva.

Nitrosamines formed when secondary amines were added to normal human saliva. Fractionation of saliva into cells and supernatant showed that factors that accelerated and retarded the nitrosation reaction were both present. Acidification of saliva greatly increased the nitrosamine yield, but differences in nitrosamine yield among saliva fractions were still observable.

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.

Pharmacokinetic model for N-nitrosodimethylamine based on Michaelis-Menten constants determined with the isolated perfused rat liver.

A pharmacokinetic model was constructed to describe the absorption, distribution, and metabolic clearance of N-nitrosodimethylamine. The model is composed of two compartments, total body water and the liver, which are linked by blood flow. Metabolic clearance is presumed to occur only in the liver. Liver clearance kinetics was determined with isolated perfused livers. Clearance appeared to obey Michaelis-Menten kinetics with Km = 8.3 +/- 4.8 microM and Vmax = 0.15 +/- 0.02 mumol/min . liver. The observed value for Km is about 1 order of magnitude lower than any observed when clearance is determined using liver microsome preparations. The model is used to calculate whole-body clearance of N-nitrosodimethylamine and relative tissue exposure as a function of the route of administration. The calculations are compared with previously published experimental data, and it is shown that the accuracy of the model for low doses is a result of the novel value observed for Km in the perfused liver.

Molecular dosimetry of polycyclic aromatic hydrocarbon epoxides and diol epoxides via hemoglobin adducts.

Ten reactive metabolites of five polycyclic aromatic hydrocarbons and styrene were investigated to determine the generality of ester adduct formation with human hemoglobin in the form of RBC and hydrolysis to the corresponding tetrahydrotetrols or dihydrodiols. No exceptions were noted among the compounds tested, which included the anti-diol epoxides of benzo[a]pyrene (BaP), chrysene, and benz[a]anthracene; the syn-diol epoxide of BaP; a mixture of syn- and anti-diol epoxides of benzo[e]pyrene; and epoxides of styrene, benzo[e]pyrene, BaP, and cyclopenta[c,d]pyrene. A test of the propensity of the simplest benzylic epoxide, styrene oxide, to form esters that hydrolyze via a BAL1 mechanism was performed. Hydrolysis of styrene oxide-adducted hemoglobin in H2(18)O at neutral pH yielded 18O incorporation results that suggest this mechanism of hydrolysis is operant to a minor degree in styrene oxide-hemoglobin ester adducts. A method was developed for the isolation and quantification of the polycyclic aromatic alcohols, which consists of enzymatic proteolysis, immunoaffinity chromatography, and gas chromatography-mass spectrometry or fluorimetry. The method allows for routine analysis of hemoglobin from individual samples as small as 1 ml of whole blood. Analysis of blood from different human populations revealed that hemoglobin adducts of the anti-diol epoxide of BaP dominated the spectrum of adducts formed by the selected metabolites.

Urinary excretion of nitrate, N-nitrosoproline, 3-methyladenine, and 7-methylguanine in a Colombian population at high risk for stomach cancer.

Urinary excretion levels of nitrate and N-nitrosoproline were determined in 160 individuals in a Colombian population at high risk for gastric cancer. In 156 of these subjects urinary levels of 3-methyladenine and 7-methylguanine were determined. Gastric biopsy specimens were obtained from 118 individuals and were histologically characterized according to pathological criteria into the following groups: normal, superficial gastritis, chronic atrophic gastritis, chronic atrophic gastritis with intestinal metaplasia, and dysplastic. The histological changes were correlated with the four variables listed above. There were no significant differences in the excretion of nitrate, N-nitrosoproline, 3-methyladenine, or 7-methylguanine in subjects with different pathological changes. A statistically significant correlation was present between nitrate and N-nitrosoproline excretion in the total population group (r = 0.297, P = 0.0001). A highly significant correlation (r = 0.56, P = 0.0002) was noted for urinary nitrate and N-nitrosoproline excretion in individuals with intestinal metaplasia and dysplasia. An increase in the urinary excretion of 3-methyladenine and 7-methylguanine was associated with tobacco smoking in the total population group.

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.

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.

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.

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.

Urinary markers for exposures to alkylating or nitrosating agents.

Investigation of urinary markers as indices of endogenous nitrosation and of gastric cancer etiology has been a major focus of our work. As part of this effort, studies have been carried out on a Colombian population at high risk for gastric cancer. In this group, nitrosoproline excretion was highly correlated with nitrate excretion in the subpopulation with advanced gastric pathology, but not in control subpopulations with more normal stomachs. Neither urinary 7-methylguanine nor 3-methyladenine was strongly related to gastric pathology or to urinary nitrate or nitrosoproline levels. More recently, as evidence has accumulated concerning the importance of nitric oxide as a cellular messenger, we have begun research toward developing markers for the presence of nitric oxide and for endogenous nitrosation via this compound. Nitric oxide is formed from arginine by activated endothelial cells as a messenger for vasodilation. We have shown that prolonged exercise leads to increased urinary nitrate and that when 15N-arginine is ingested by humans, 15N-nitrate levels increase in 24-hr urine collections. Nitrosohydroxyethylglycine and 3-nitrotyrosine were evaluated as indices for the formation of N-nitrosomorpholine and for the nitration of protein, respectively, under experimental conditions (e.g., immunostimulation) expected to enhance nitric oxide formation. Nitrotyrosine has not proved useful as a biomarker for nitration/nitrosation reactions in immunostimulated rats. Immunostimulation of rats following administration of morpholine led to increases in urinary nitrate and nitrosohydroxyethylglycine. This procedure, however, would not be appropriate for humans due to the toxicity of morpholine and the carcinogenicity of N-nitrosomorpholine.

Characterization of various classes of protein adducts.

Analysis of the types of protein adducts formed by chemical carcinogens indicate that adducts may be categorized into various classes according to the nature of the carcinogen as well as the amino acid with which they react. Tryptophan(214) of serum albumin was previously shown to react specifically with N-sulfonyloxy-N-acetyl-4-aminobiphenyl. The same residue is now shown to also react with the sulfate esters of N-hydroxy-N-acetyl-2-aminofluorene and N-hydroxy-N,N'-diacetylbenzidine. Thus, Trp-214 appears to be a binding site for a variety of activated N-aryl hydroxamic acids. Epoxides and diol epoxides derived from polynuclear aromatic hydrocarbons alkylate carboxylic groups in hemoglobin and serum albumin. Because the esters formed are readily hydrolyzed to dihydrodiols and tetrahydrotetrols which can be determined by GC-MS, it is possible to analyze for a wide range of polyaromatic hydrocarbon (PAH) epoxide adducts. With this approach it was shown that human subjects experiencing exposure to ambient levels of environmental PAH do take up and metabolize chrysene and benzo[a]pyrene. Feral, bottom-dwelling fish inhabiting contaminated waters were also examined. Globin adducts containing certain dihydroxy groups such as those arising in anti-diol epoxide adducts were concentrated by boronate affinity chromatography and further analyzed by HPLC with diode-array UV/visible detection. Four compounds were detected that exhibited spectra characteristic of a polynuclear chromophore. Two of these appeared to be isomers. Further instrumental analysis is needed to elucidate the structure of these unknown putative adducts.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Alkylation of nucleic acids by N-nitrosodi-n-propyl-amine: evidence that carbonium ions are not significantly involved.

Administration of N-nitrosodi-n-propylamine to rats leads to the formation of 7-n-propylguanine but not 7-isopropylguanine in hepatic DNA. For RNA, a small amount of the rearranged adduct is formed. Alkylation of DNA and RNA therefore appears to occur primarily via a bimolecular reaction rather than a unimolecular pathway involving free alkyl cations.

Mechanism of alkylation by N-nitroso compounds: detection of rearranged alcohol in the microsomal metabolism of N-nitrosodi-n-propylamine and base-catalyzed decomposition of N-n-propyl-N-nitrosourea.

Metabolism of N-nitrosodi-n-propylamine by an isolated rat liver microsomal fraction yielded 17% isopropanol and 83% n-propanol (expressed as a percentage of total propanol formed). Base-catalyzed decomposition of N-n-propyl-N-nitrosourea yielded 39% isopropanol and 61% n-propanol. The values provide evidence for involvement of carbocations in both of these reactions.

Nitrosamine carcinogenicity: a quantitative relationship between molecular structure and organ selectivity for a series of acyclic N-nitroso compounds.

A mathematical model has been developed which describes the selectivity between liver and other target organs for a series of carcinogenic N-nitro-sodialkylamines. The relationship requires structural terms for the parent molecule as well as for the potential metabolites. This suggests that the nitrosamine metabolites are involved in tumor induction and that they participate directly in the mechanisms responsible for selecting the target organ.

Nitrosamine carcinogenicity: a quantitative Hansch-Taft structure-activity relationship.

Statistically significant correlations have been demonstrated between carcinogenic activity, water-hexane partition coefficients and electronic factors for an extensive series of N-nitroso compounds. Electronic factors were expressed by the Taft sigma* values of substituents on the carbon atoms alpha to the N-nitroso group. Such correlations indicate that transport of the carcinogen to its active site has an important effect on its potency. The correlations also implicate reactivity at the alpha-carbon in the determination of carcinogenic activity and point out various structural types which do not follow the general rule.

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.

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.