Biomedical subjects

Richard N Loeppky

Publications and source records attributed to Richard N Loeppky.

8 recordsLinked to original sources

PubMed2006

Genotoxicity of glycidamide in comparison to (+/-)-anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide and alpha-acetoxy-N-nitroso-diethanolamine in human blood and in mammalian V79-cells.

Genotoxic activity of glycidamide (GA) was investigated in comparison to that of the known carcinogens (+/-)-anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide ((+/-)-BPDE) and alpha-acetoxy-N-nitroso-diethanolamine (alpha-A-NDELA), using the hypoxanthine-phosphoribosyl-transferase (hPRT) gene mutation assay with V79 mammalian cells and modified alkaline single cell gel electrophoresis (alkaline comet assay with and without treatment of cells with formamido-pyrimidine-DNA-glycosylase (FPG)) in lymphocytes from human whole blood. As shown earlier, GA induced significant DNA damage in lymphocytes from treated whole blood at > or = 300 microM (4 h) (Baum et al., Mutat. Res. 2005, 580, 61-69). In the present study, using the alkaline comet assay with FPG treatment, increased formation of DNA strand breaks was observed in lymphocytes treated with GA (10 microM; 4 h). alpha-A-NDELA and (+/-)-BPDE were genotoxic at 10-30 microM (1 h). Genotoxic activity of these compounds was not enhanced after FPG treatment. FPG treatment thus offers an enhanced sensitivity of DNA damage detection for genotoxic compounds with preference for N(7)- resp. N(3)-purine alkylation. In the hPRT assay with V79 cells, mutagenic activity of (+/-)-BPDE became significant at > or = 3 microM (24 h). For alpha-A-NDELA significant activity was observed at greater, not dbl 10 microM (24 h). As previously observed, GA was considerably less effective, inducing significant mutagenicity roughly at about 80-300-fold higher concentrations (800 microM; 24 h) (Baum et al., Mutat. Res. 2005, 580, 61-69).

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed2005

Nitrosation chemistry of pyrroline, 2-imidazoline, and 2-oxazoline: theoretical Curtin-Hammett analysis of retro-ene and solvent-assisted C-X cleavage reactions of alpha-hydroxy-N-nitrosamines.

The results are presented of a theoretical study of the nitrosation chemistry of pyrroline 1 (X = CH2), imidazoline 2 (X = NH), and 2-oxazoline 3 (X = O). Imines 1-3 are converted to the alpha-hydroxy-N-nitrosamines 7-9 via the N-nitrosoiminium ions 4-6. The NN-cis isomers of 7-9 may undergo retro-ene reactions to the delta-oxoalkyl diazotic acids 10-12. With the opportunity for microsolvation, C-X cleavage becomes possible for 8 and 9 and leads to the formation of N-(2-aminoethyl)- and N-(2-hydroxyethyl)-N-nitrosoformamides 15 and 16, respectively. The NN-isomerization barriers are comparable to the barriers for the ring-opening reactions, and the consideration of two Curtin-Hammett scenarios is required: CH-I for the NN-trans-rotamers of 7-9 to undergo C-X cleavage or NN-isomerization and CH-II for the NN-cis-rotamers to undergo C-X cleavage, C-N cleavage, or NN-isomerization. We determined all stereoisomers of the substrates, the products, and of all transition states structures for the retro-ene reactions of 7-9, the C-X cleavages of microsolvated 8 and 9, and the NN-isomerizations of 8 and 9. The potential energy surfaces were explored at the B3LYP/6-31G level, and the results are discussed with emphasis on the comparison of the kinetics and thermodynamics of C-N versus C-X cleavage. The study shows all decompositions to be very fast with activation barriers below 21 kcal.mol(-1), and the comparative analysis predicts that the chemical toxicologies of 1 and 3 should be similar and remarkably different from that of 2.

Hydrolysis↗

PubMed2005

The mechanistic origin of regiochemical changes in the nitrosative N-dealkylation of N,N-dialkyl aromatic amines.

The regioselectivity of the nitrous acid mediated dealkylation of 4-substituted-N-ethyl-N-methylanilines is a function of the acidity of the reaction mixture. At high acidity deethylation predominates, whereas demethylation is the predominant reaction in nitrosamine formation at pH 2 and above. In some cases the regioselectivity of nitrosative dealkylation changes as the run proceeds. Through the use of the corresponding 4-nitroaniline as the primary substrate, CIDNP, kinetics, kinetic deuterium isotope effects and other transformations involving nitrosations with NO2 or NOBF4 in aprotic solvents, a new mechanism of tertiary amine nitrosation has been deduced and proposed to explain regioselective deethylation. The mechanism involves the oxidation of the substrate to the amine radical cation by NO+. This is followed by the abstraction of a hydrogen atom from the carbon adjacent to the amine nitrogen by NO2 to produce an iminium ion which reacts further to produce the corresponding aldehyde and the nitrosamine. Depending upon the acidity, this process competes with three other mechanistic pathways, two of which give the nitrosamine through the iminium ion, and one leads to the formation of C-nitro compounds. The competing pathways to nitrosamine formation involve NOH elimination from a nitrosammonium ion and deprotonation of the radical cation to give an alpha-amino radical which rapidly oxidized to the iminium ion. Predominant, but not highly regioselective demethylation occurs by these pathways. Nitro compound formation principally arises from the reaction of NO2 with the radical cation followed by deprotonation, but also occurs by para C-nitrosation followed by oxidation.

Amines↗

PubMed2005

Mass spectrometric methodology for the determination of glyoxaldeoxyguanosine and O6-hydroxyethyldeoxyguanosine DNA adducts produced by nitrosamine bident carcinogens.

N-Nitrosodiethanolamine (NDELA) is a bident carcinogen that undergoes both P-450 mediated alpha-hydroxylation and beta-oxidation, leading ultimately to the formation of two prominent DNA adducts, glyoxaldeoxyguanosine (gdG) and O6-2-hydroxyethyldeoxyguanosine (OHEdG), in rat liver. HPLC coupled with electrospray ionization (ESI) and tandem mass spectrometry was used for both detection and quantification of gdG and OHEdG. The method, which is fast, sensitive, and unambiguous, is a significant improvement over the previous 32P-postlabeling methodology. A rapid procedure for the enzymatic hydrolysis of the DNA under acidic conditions preserved the integrity of the pH sensitive gdG adducts. Glyoxal and 3-nitroso-2-oxazolidinone generated gdG and OHEdG adducts, respectively, in calf thymus DNA (ct-DNA) in a concentration (range of 10(4)) dependent manner permitting optimization. Isotopomeric internal standards were prepared from the modified guanine derivatives by enzymatic trans-glycosylation. Quantitative HPLC-ESI-MS/MS analysis employing selective reaction monitoring (SRM) for the loss of the deoxyribose fragment was utilized. Both adducts could be detected in the liver DNA of rats that were administered NDELA in a dose range of 0.4-0.8 mmol/kg. At the highest dose, gdG adducts (4.4-11 adducts/10(6) nuc.) were more abundant than OHEdG adducts (0.35-0.87 adducts/10(6) nuc.). Conversely, OHEdG adducts were produced in higher yields in ct-DNA than were gdG adducts at the same reagent concentrations.

Animals↗

PubMed2005

The carcinogenic significance of reactive intermediates derived from 3-acetoxy- and 5-acetoxy-2-hydroxy-N-nitrosomorpholine.

N-Nitroso-2-hydroxymorpholine (NHMOR), a relatively reactive metabolite of two potent carcinogens, N-nitrosodiethanolamine (NDELA) and N-nitrosomorpholine (NMOR), has been reported to not be carcinogenic. Two isomeric acetate esters of the alpha-hydroxynitrosamines expected to be produced from the cytochrome P450-mediated metabolism of NHMOR have been synthesized, and their hydrolytic decomposition products, hydrolysis rates, and deoxyguanosine (dG) reaction adducts have been determined. N-Nitroso-3-acetoxy-2-hydroxymorpholine was prepared in high yield from the reaction of N-nitroso-2,3-dehydromorpholine with dry peracetic acid in glacial acetic acid or by the reaction of its dimethyldioxirane-produced epoxide with glacial acetic acid. The hydrolysis of this alpha-acetoxynitrosamine gave acetaldehyde (10%), ethylene glycol (55%), glyoxal (95%), and acetic acid. The pH rate profile for the hydrolysis of this nitrosamine was abnormal in that it exhibited pronounced base-catalyzed hydrolysis beginning at pH 5. The mechanism of hydrolytic decomposition is proposed to involve neighboring group participation with the formation of a reactive epoxide intermediate. N-Nitroso-3-acetoxy-2-hydroxymorpholine reacted with dG to give these guanine adducts after acidic deglycosylation: 1,N2-glyoxal (65%), 7-(2-hydroxyethyl)guanine (9%), and O6-hydroxyethylguanine (3%). N-Nitroso-5-acetoxy-2-hydroxymorpholine was synthesized from 2-hydroxyethylvinylnitrosamine by its oxidative conversion to the corresponding aldehyde followed by reaction with dry peracetic acid in glacial acetic. The hydrolytic decomposition products of this nitrosamine were 2-acetoxyacetaldehyde (65%), a rearrangement product, glycol aldehyde (15%), a trace of glyoxal, and acetic acid. The pH rate profile for the hydrolysis of this acetate is similar to other alpha-acetoxynitrosamines in that it exhibits a pH-independent region which gives way to base-catalyzed ester hydrolysis beginning at pH 7. The lower pH ( approximately 7 < 9) onset of base catalysis is proposed to involve base-catalyzed opening of the hemiacetal and intramolecular acyl transfer to give an unstable alpha-hydroxynitrosamine. N-Nitroso-5-acetoxy-2-hydroxymorpholine was less reactive toward dG and gave the 1,N2-etheno-dG adduct (44%). The products from both of the isomeric alpha-acetoxy nitrosamines were judged to arise from diazonium ions produced from unstable alpha-hydroxynitrosamine intermediates. The high yield of the rearrangement product 2-acetoxyacetaldehyde could explain the low carcinogenic potential of NHMOR if it is mainly alpha-hydroxylated at the 5 carbon. Hydroxylation of NHMOR at carbon 3 is expected to yield a carcinogenic outcome.

Acetates↗

PubMed2004

Amidine nitrosation.

The acidic nitrosation chemistry of nine acyclic secondary and tertiary amidines (Ph-N=C(R(1))NR(2)R(3); R(1) = H, CH(3), Ph; R(2), R(3) = H, Ph or (CH(3))(2) or C(CH(2))(4)) and several N-acylamidines was investigated. The principal nitrosation products were amides derived from the amino moiety and compounds derived from the benzenediazonium ion, which was independently trapped for quantitation in several cases. Tertiary amidines also produce nitrosamines in minor, but significant, yields. The benzamidines did not react, and the N-acylamidines hydrolyzed much more rapidly than they nitrosated. The data support the hypothesis that the reaction occurs by nitrosation on the imino nitrogen, followed by the addition of H(2)O to give a tetrahedral intermediate (alpha-hydroxynitrosamine) for which the main decomposition pathway generates an amide and a diazonium ion. In the case of the pyrrolidine-derived amidines, about 25% of the decomposition results in cleavage of the amine moiety, which nitrosates to give N-nitrosopyrrolidine. Pseudo-first-order rate constants for amidine nitrosation in aqueous acetic acid with excess nitrite at 25 degrees C ranged from (3 to 106) x 10(-5) s(-1), while the amidine basicity ranged over 5 pK(a) units. Rate constants corrected for amidine basicity showed the pyrrolidine derived amidines to be most reactive. The lack of benzamidine nitrosative reactivity is attributed to a very slow rate of H(2)O additon to the N-nitrosoamidinium ion and reversible nitrosation.

Amides↗

PubMed2002

Microsome-mediated oxidation of N-nitrosodiethanolamine (NDELA), a bident carcinogen.

N-Nitrosodiethanolamine (NDELA), an environmentally prevalent, potent carcinogen, undergoes competitive rat liver microsome-mediated oxidation at both the alpha (adjacent to N)- and beta-positions of the 2-hydroxyethyl chains. The former process, alpha-hydroxylation, is detected by the formation of glycolaldehyde (determined as its 2,4-dinitrophenylhydrazone DNP) that is assumed to arise from the decomposition of the corresponding alpha-hydroxynitrosamine, which is also the progenitor of the 2-hydroxyethyldiazonium ion. This finding refutes prior published work that states that the alpha-hydroxylation of NDELA does not occur. Competitive microsomal oxidation at the beta-position gives the hemiacetal N-nitroso-2-hydroxymorpholine (NHMOR) at a rate 1.5 times alpha-hydroxylation. Glycolaldehyde is oxidized in this system to glyoxal at a rate 39 times the conversion of NDELA to glycolaldehyde. The alpha-hydroxylation of NHMOR at either C-3 or C-5 to give glyoxal or glycolaldehyde, respectively, occurs at respective rates 3-6 times that of the alpha-hydroxylation of NDELA. Ethylene glycol, a hydrolysis product of the 2-hydroxyethyldiazonium ion is shown to undergo microsome mediate oxidation to glyoxal. Ethyl-2-hydroxyethylnitrosamine (NEELA) undergoes a similar set of microsome-mediated oxidations at alpha-position of the ethyl (fastest) and 2-hydroxyethyl groups, as well as beta-oxidation of the 2-hydroxyethyl group, a process which is slightly more rapid than alpha-hydroxylation of the same chain. Comparisons of oxidations rates of these substrates, as manipulated by preinducers, isoniazid, streptozocin, and phenobarbital, and enzyme inhibitors diethyldithiocarbamate and 4-methylpyrazole, with that of dimethylnitrosamine, a substrate for cytochrome P450 2E1, strongly suggest that this isozyme is also responsible for the oxidations reported here. alpha-Deuteration of NDELA practically eliminates its alpha-hydroxylation by microsomes from isoniazid induced rats, but doubles beta-oxidation, while beta-deuteration of this substrate significantly reduces beta-oxidation and enhances alpha-hydroxylation. Since both glyoxal-guanine and 2-hydroxyethyl-DNA base adducts are known to arise from the in vivo administration of NDELA and because this work demonstrates that these two fragments can come from the microsomal oxidation of a single nitrosamine molecule containing the 2-hydroxyethyl group, NDELA and related nitrosamines are bident (two-toothed) carcinogens, a process which is likely to enhance their carcinogenic potency.

Animals↗

PubMed2002

DNA adducts from N-nitrosodiethanolamine and related beta-oxidized nitrosamines in vivo: (32)P-postlabeling methods for glyoxal- and O(6)-hydroxyethyldeoxyguanosine adducts.

The mechanism by which environmentally prevalent N-nitrosodiethanolamine (NDELA) and related 2-hydroxyethyl- or other beta-oxidized nitrosamines initiate the carcinogenic process has remained obscure. (32)P-Postlabeling assays for the pH sensitive glyoxal-deoxyguanosine (gdG) and the O(6)-2-hydroxyethyldeoxyguanosine (OHEdG) DNA adducts have been developed as probes in this mechanistic investigation and used in both in vitro and in vivo experiments. The ready cleavage of the glyoxal fragment from gdG at pH 7 and greater has required methods of optimization in order to achieve a detection limit of 0.05 micromol/mol of DNA. Nuclease P1 treatment enhances the detection of gdG adducts but does not increase the detection limit for OHEdG. For OHEdG, best results were achieved using fraction collection from HPLC (0.3 micromol/mol of DNA). Using radiochemical methods, both adducts could be detected either by HPLC or 2D TLC. NDELA, N-nitrosomorpholine (NMOR), N-nitrosomethyethanolamine (NMELA), and N-nitrosoethylethanolamine (NEELA) all produce both gdG and OHEdG adducts in rat liver DNA in vivo and are called bident carcinogens because fragments from both chains of the nitrosamine are incorporated into DNA. N-Nitroso-2-hydroxymorpholine (NHMOR), a metabolite of NDELA and NMOR, generates gdG in DNA in vitro and in vivo. gdG DNA adducts were found in the range 1.1-6.5 micromol/mol of DNA. OHEdG DNA adducts were produced from equimolar amounts of nitrosamines in rat liver in vivo over the range 4-25 micromol/mol of DNA and in the order NMELA > NEELA > NDELA > NMOR. Deuterated isotopomers of NDELA showed a marked isotope effect on DNA OHEdG adduct formation. alpha-Deuteration markedly decreased OHEdG adduct formation while beta-deuteration had the opposite effect. These data support the hypothesis that NDELA and related nitrosamines are activated by both enzyme mediated alpha-hydroxylation and beta-oxidation. The formation of OHEdG adducts from NDELA requires alpha-hydroxylation of the 2-hydroxyethyl chain, and formation of gdG necessitates a beta-oxidation as well. The bident nature of these carcinogens may explain why they are relatively potent carcinogens despite the fact that major proportions of doses are excreted unchanged.

Animals↗