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Biomedical subjects

S R Tannenbaum

Publications and source records attributed to S R Tannenbaum.

At least 37 records · Page 2Linked to original sources
PubMed1999

Biomonitoring of heterocyclic aromatic amine metabolites in human urine.

Human exposure to heterocyclic aromatic amines such as MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline) may be monitored by measuring the levels of the heterocyclic aromatic amine in urine. In order to investigate the contribution of N-oxidation to the metabolism of MeIQx in vivo, we developed a biomonitoring procedure for the analysis and quantification of the N2-glucuronide conjugate of 2-hydroxyamino-3,8-dimethylimidazo[4,5-f]quinoxaline in human urine. Subjects (n = 66) in the dietary study ingested a uniform diet of cooked meat containing known amounts of MeIQx, and urine was collected after consumption of the test meal. A method based on solid-phase extraction and immunoaffinity separation was used to isolate N2-(beta-1-glucosiduronyl)-2-hydroxyamino-3,8-dimethylimidazo++ +[4,5-f]quinoxaline and its stable isotope-labeled internal standard from urine. The isolated conjugate was converted to the deaminated product 2-hydroxy-3,8-dimethylimidazo[4,5-f]quinoxaline by treatment with acetic acid under moderate heating. 2-Hydroxy-3,8-dimethylimidazo[4,5-f]quinoxaline and the [2H3]methyl analog were derivatized to form the corresponding 3,5-bis(trifluoromethyl)benzyl ether derivatives and quantified by capillary gas chromatography-negative ion chemical ionization mass spectrometry employing selected ion monitoring procedures. The amounts of N2-(beta-1-glucosiduronyl)-2-hydroxyamino-3,8-dimethylimidazo++ +[4,5-f]quinoxaline recovered in urine collected 0-12 h after the test meal accounted for 2.2-17.1% of the ingested dose, with a median value of 9.5%. The variability in the proportion of the dose excreted among the subjects may be reflective of several factors, including interindividual variation in the enzymic activity of CYP1A2 and/or conjugation reactions of the N-hydroxylamine metabolite with N-glucuronosyltransferase(s).

Humans↗
PubMed1999

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.

Animals↗
PubMed1999

DNA adduct formation by secondary metabolites of cyclopenta[cd]pyrene in vitro.

In this study, calf thymus DNA was reacted in vitro with cyclopenta[cd]pyrene 3,4-epoxide (CPPE) or its metabolites, 3,4-dihydroCPP-3,4-diol (CPP-3,4-diol) and 4-hydroxy-3,4-dihydroCPP (4-OH-DCPP), activated with sulfotransferase. The adducts formed were analyzed by HPLC with fluorescence detection following enzymatic digestion of DNA to deoxynucleosides. We have shown previously that the major CPPE-reacted DNA adducts are cis-3-(deoxyguanosin-N2-yl)-4-hydroxy-3,4-dihydroCPP. Sulfotransferase activation of trans-CPP-3,4-diol yielded two adducts that were identical to the products resulting from the reaction of CPPE with DNA, while cis-CPP-3,4-diol gave very low covalent binding. Two adducts formed by sulfotransferase activation of 4-OH-DCPP were identical to the products of the reaction of synthetic 4-NaO3S-O-DCPP or sulfotransferase-activated 4-OH-DCPP with deoxyguanosine. These results indicate that guanine is the predominant site of CPP adduct formation in DNA, and that the 4-hydroxy-3-dGuo adducts can arise by reaction of DNA with either CPPE or sulfotransferase-activated trans-CPP-3,4-diol.

Animals↗
PubMed1999

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.

Chromatography, Liquid↗
PubMed1999

DNA damage in deoxynucleosides and oligonucleotides treated with peroxynitrite.

Peroxynitrite (ONOO-) is a powerful oxidizing agent that forms in a reaction of nitric oxide (NO*) and superoxide (O2-*). We have investigated ONOO--induced DNA damage using deoxynucleosides and oligonucleotides as model substrates, with particular attention paid to the oxidation of 8-oxodG by ONOO-. With regard to deoxynucleosides, ONOO- was found to have significant reactivity only with dG; dA, dC, and dT showed minimal reactivity. However, two of the major products of ONOO--induced oxidation of dG (8-oxodG and 8-nitroG) were both found to be significantly more reactive with ONOO- than with dG. In the context of an oligonucleotide, we observed a concentration-dependent oxidation of 8-oxodG to at least two types of products, one appearing at ONOO- concentrations of </=100 microM and the other at concentrations of >/=500 microM. We also examined the susceptibility of these oxidation products to repair by FaPy glycosylase, endonuclease III, uracil glycosylase, and MutY. FaPy glycosylase, which recognizes 8-oxoG as its primary substrate, was the only enzyme that exhibited an efficient reaction with 8-oxodG oxidation products at low ONOO- concentrations (</=100 microM); the product(s) formed at ONOO- concentrations of >/=500 microM either was not recognized or was poorly repaired by the enzymes. While processing of the lesions was inefficient with endonuclease III and not apparent with uracil glycosylase, the excision of A opposite an 8-oxoG lesion by the enzyme MutY was not affected by the reaction of 8-oxoG with ONOO-. In addition to demonstrating the complexity of ONOO- DNA damage chemistry, these results suggest that 8-oxodG may be a primary target of ONOO- in DNA.

DNA Damage↗
PubMed1999

Molecular and genetic damage from environmental tobacco smoke in young children.

To assess the risks of early life exposure to environmental tobacco smoke (ETS), we tested whether four biomarkers in peripheral blood were associated with home ETS exposure in Hispanic and African-American children. The biomarkers included cotinine (a metabolite of nicotine) and three indicators of molecular and genetic damage from mutagens/carcinogens, protein adducts formed by the carcinogens 4-aminobiphenyl (4-ABP) and polycyclic aromatic hydrocarbons (PAHs), and sister chromatid exchanges (SCEs). We also explored possible ethnic differences in biomarkers. The study cohort comprised 109 Hispanic and African-American preschool children (1-6 years of age). Plasma cotinine was analyzed by gas chromatography, 4-ABP-hemoglobin adducts by gas chromatography-mass spectroscopy, PAH-albumin adducts by ELISA, and SCEs by cytogenetic techniques. Data on the amount of smoking by mothers (average 10.5 cigarettes per day) and other household members and regular visitors (average 6.5 cigarettes per day) were obtained by interview-administered questionnaires. Cotinine, 4-ABP-hemoglobin adducts, and PAH-albumin were significantly higher (P < 0.05) in the ETS-exposed children compared with the unexposed. SCEs were marginally higher (P = 0.076). African-American children had higher levels of cotinine (P = 0.059) and PAH-albumin (P = 0.02) than Hispanic children, after controlling for exposure to ETS. These results indicate molecular and genetic damage in minority children with

Aminobiphenyl Compounds↗
PubMed1999

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.

Adult↗
PubMed1998

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.

Cytosine↗
PubMed1998

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.

Benzo(a)pyrene↗
PubMed1998

Etheno adducts in spleen DNA of SJL mice stimulated to overproduce nitric oxide.

In order to investigate specific DNA damage caused by nitric oxide (NO) induced lipid peroxidation, levels of promutagenic etheno adducts 1,N6-ethenodeoxyadenosine (epsilondA) and 3,N4-ethenodeoxycytidine (epsilondC) were measured in spleen DNA of SJL mice induced to produce high levels of NO by injection of RcsX (pre-B-cell lymphoma) cells. epsilondA and epsilondC levels were quantified by an ultrasensitive immunoaffinity-32P-post-labeling method. Spleen DNA of control mice (n = 5) had background levels of 9.2+/-5.4 epsilondA adducts per 10(9) dA and 13.1+/-5.7 epsilondC adducts per 10(9) dC. In RcsX cell-injected mice (n = 7), levels of these adducts were elevated approximately 6-fold, i.e. 53.9+/-39.4 epsilondA per 10(9) dA and 83.5+/-57.8 epsilondC per 10(9) dC (P < 0.05). Mice injected with RcsX cells and also treated with NG-methyl-L-arginine (NMA), an inhibitor of inducible nitric oxide synthase (n = 6), had significantly reduced levels (P < 0.05) of both epsilondA and epsilondC (13.5+/-5.7 epsilondA per 10(9) dA and 28.2+/-15.7 epsilondC per 10(9) dC). These findings constitute the first available evidence of formation of etheno adducts associated with NO overproduction in vivo. The adducts were presumably formed from lipid peroxidation products such as trans-4-hydroxy-2-nonenal (HNE), generated via oxidation of lipids by peroxynitrite. The results suggest that etheno-DNA adducts, among other types of damage, may contribute to the etiology of cancers associated with chronic infection/inflammation in which NO is overproduced.

Aldehydes↗
PubMed1998

Absence of the glutathione S-transferase M1 gene increases cytochrome P4501A2 activity among frequent consumers of cruciferous vegetables in a Caucasian population.

The cancer protective effect of cruciferous vegetables has been attributed to induction of phase II enzymes. But cruciferous vegetables also induce cytochrome P4501A2 (CYP1A2), which catalyzes the metabolic activation of various procarcinogens, including aromatic amines in tobacco. Thus, frequent intake of cruciferous vegetables could also result in cancer-enhancing effects. GSTM1 is involved in the detoxification of various carcinogens, but it also enhances the excretion of isothiocyanates and possibly other enzyme inducers in cruciferous vegetables. We, therefore, hypothesized that GSTM1 null genotype might be associated with increased CYP1A2 activity among frequent consumers of cruciferous vegetables because the excretion of CYP1A2 inducers contained in these vegetables may be partially inhibited in the absence of the GSTM1 enzyme. Three hundred twenty-eight non-Hispanic white residents of Los Angeles County (265 males and 63 females) were genotyped for the presence or absence of GSTM1 alleles and phenotyped for CYP1A2 activity. Information on usual dietary habits was obtained from these subjects through in-person interviews. Among frequent (at least once a week) consumers of broccoli, GSTM1 null individuals exhibited a 21% higher geometric mean level of CYP1A2 activity relative to GSTM1 non-null individuals (5.24 versus 4.32, two-sided P = 0.01). No such difference was observed in subjects who consumed broccoli less frequently (two-sided P = 0.39). This interactive effect of GSTM1 genotype and vegetable intake on CYP1A2 activity also was observed when overall intake of the five cruciferous vegetables under study (broccoli, cabbage, cauliflower, Brussels sprouts, and mustard greens) was examined. Among weekly consumers of cruciferous vegetables, GSTM1 null individuals showed a 16% higher geometric mean level of CYP1A2 activity relative to GSTM1 non-null individuals (5.03 versus 4.33, two-sided P = 0.02), whereas no difference was evident among those who consumed cruciferous vegetables less frequently (two-sided P = 0.35). Our results suggest that cruciferous vegetables contain CYP1A2 inducers, which are deactivated in the presence of GSTM1.

Brassicaceae↗
PubMed1997

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.

Animals↗
PubMed1997

Nitrotyrosine formation, apoptosis, and oxidative damage: relationships to nitric oxide production in SJL mice bearing the RcsX tumor.

In SJL mice, growth of RcsX lymphoma cells results in activation of macrophages in the spleen and lymph nodes to produce high levels of NO radical (NO.). We used this experimental model system to study the toxicology of NO. in vivo. To characterize spatial relationships between sites of NO. production and tissue damage, immunohistochemical techniques were developed for simultaneous detection of inducible NO. synthase (iNOS), 3-nitrotyrosine, and apoptosis in spleen and lymph nodes of tumor-bearing animals. Elevated expression of iNOS, presumed to reflect increased NO. production, was associated with a significant increase in frequency of apoptotic nuclei. Both apoptotic nuclei and 3-nitrotyrosine staining were found in cells juxtaposed to iNOS-expressing (ie., NO.-producing) macrophages and also within the macrophages themselves. To assess the extent of DNA damage associated with the response, 8-oxoguanine levels were quantified in DNA extracted from spleens of tumor-bearing mice. No increase in levels of this marker of oxidative DNA damage was found in tissues in which apoptosis and 3-nitrotyrosine levels were highly elevated within specific subsets of cells. Collectively, our results indicate that under the pathophysiological conditions existing in the RcsX tumor-bearing SJL mouse, cellular damage caused by NO. and/or other reactive species produced by activated macrophages is highly localized within cells in close proximity to the activated macrophages.

Animals↗
PubMed1997

A mechanistic analysis of nitric oxide-induced cellular toxicity.

Nitric oxide (NO.)-induced toxicity was investigated in two different cell lines, Chinese hamster ovary (CHO-AA8) and human lymphoblastoid (TK6), over a range of NO. doses (0-9 mM) delivered for an exposure of 2 h. To determine both short-term and delayed effects leading to death, a range of assays was employed to decipher the major mechanisms of cytotoxicity. Examples of damage parameters measured in this study include inhibition of DNA synthesis, damage to mitochondria, loss of cell membrane integrity, apoptosis, changes in cell cycle distribution, and the occurrence of DNA strand breaks. Our results indicate that NO.-induced toxicity is an extremely complex process involving multiple pathways generally leading to apoptotic cell death. Results consistently demonstrate that TK6 cells are much more susceptible to NO.-induced toxicity than CHO-AA8 cells. This difference in sensitivity could be seen for all types of cellular damage examined. The earliest observable effect of NO. exposure is inhibition of DNA synthesis which is not the result of inhibition of ribonucleotide reductase but may be the result of DNA damage leading ultimately to cell cycle arrest.

Animals↗
PubMed1997

Quantitation of 8-oxoguanine and strand breaks produced by four oxidizing agents.

Reactive oxygen species, produced endogenously or by exposure to environmental chemicals and ionizing radiation, induce a wide range of DNA lesions. The variety of chemistries associated with different oxidants suggests that each will produce a unique spectrum of DNA damage products. To extend our efforts to relate genotoxin chemistry to DNA damage, we measured both strand breaks and 8-oxoguanine (8-oxoG) in DNA after exposure to gamma-radiation, Fe(II)-EDTA/H2O2, Cu(II)/H2O2, and peroxynitrite at concentrations approaching physiological relevance. We found that the ratio of 8-oxoG to strand breaks varied more than 10-fold depending on the oxidizing agent: approximately 0.4 for Cu(II)/H2O2 and peroxynitrite and approximately 0.03 for Fe-(II)-EDTA/ H2O2 and gamma-radiation. In the case of Cu(II)/H2O2, the relative proportion of 8-oxoG and strand breaks was found to vary more than 2-fold (0.14-0.37) for different Cu(II) concentrations, consistent with other studies. We were able to detect 8-oxoG formation by peroxynitrite by using low peroxynitrite concentrations in conjunction with a sensitive immunoaffinity/HPLC-ECD methodology. The level of 8-oxoG relative to strand breaks produced by peroxynitrite was higher than that produced by Fe(II)-EDTA/H2O2 and gamma-radiation, which is consistent with the altered reactivity or accessibility of a non-hydroxyl radical species produced by peroxynitrite.

Copper↗
PubMed1997

Characterization of DNA adducts formed by cyclopenta[cd]pyrene epoxide.

Cyclopenta[cd]pyrene (CPP) is a ubiquitous environmental pollutant whose 3,4-epoxide (CPPE) is generally regarded as its ultimate carcinogenic metabolite. The present study was undertaken to determine the structures of major DNA adducts formed by CPPE in vitro. Incorporation of specific radiolabeled bases into calf thymus DNA prior to reaction with CPPE demonstrated that the major adducts were formed by guanine, while minor adducts were formed by adenine and cytosine. Unmodified DNA was reacted with [3H]CPPE and the deoxynucleoside adducts obtained were compared chromatographically with the products obtained by reaction of CPPE with 2'-deoxyguanosine (dGuo). Two dGuo adducts from the latter reaction were identified by 1H-NMR, fast atom bombardment mass spectrometry, and circular dichroism as diastereoisomers of cis-3-(deoxyguanosin-N2-yl)-4-hydroxy-3,4-dihydroCPP. Other products that may have included the isomeric trans-N2-dGuo adduct were formed in the reaction. The major adduct fraction in the DNA digest, accounting for over 70% of the total, was chromatographically indistinguishable from the two cis dGuo-N2 adducts. A second DNA adduct fraction was observed, which appeared also to be formed by reaction with guanine as indicated by experiments in which DNA containing [3H]guanine was reacted with unlabeled CPPE. The results confirm that guanine is the major target in DNA for reaction with CPPE and are the first proof of structure for a CPPE-deoxynucleoside adduct.

Animals↗
PubMed1997

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.

Binding Sites↗