Faculty Bibliography

Onion and garlic essential oils were previously shown to inhibit mouse skin tumor promotion, as were the enzymes, lipoxygenase, and cyclooxygenase. In the present study, the inhibition of soybean lipoxygenase (EC 1.13.11.12) by onion and garlic components and related compounds was investigated. The IC50 values as well as the kinetic inhibition constants were determined for the most active compounds. Di-(1-propenyl) sulfide, an analog of the substrate moiety required for oxygenase action, was the only irreversible inhibitor observed with Ki = 59 microM and k3 = 0.53/min. Inhibition in the presence of substrate was uncompetitive at 88 and 132 microM linoleic acid with Ki = 129 microM. At 173 microM linoleic acid, however, inhibition was competitive with Ki = 66 microM. Dially trisulfide, allyl methyl trisulfide, and diallyl disulfide were competitive inhibitors, while 1-propenylpropyl sulfide and (E, Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide (ajoene) were mixed inhibitors. Nordihydroguaiaretic acid (NDGA), the most potent lipoxygenase inhibitor, was a competitive inhibitor with Ki = 0.29 microM. The results indicate a relative potency of inhibition for structural features in the following order: di(1-propenyl) sulfide greater than an alkenyl trisulfide greater than an alkenyl disulfide. Di(n-propyl) disulfide, a major onion oil component, inhibited neither lipoxygenase nor promotion. Di(1-propenyl) sulfide and ajoene inhibited both. This suggests that the inhibition of lipoxygenase may be involved in antipromotion

A mouse skin squamous cell carcinoma induced by topical application of the direct-acting alkylating agent beta-propiolactone contains an activated H-ras oncogene with an A----T transversion at the second nucleotide of codon 61. The mutation was detected in NIH3T3 transfectant and original tumor DNA by an XbaI restriction enzyme polymorphism and confirmed by oligonucleotide 'mismatch' hybridization. The mutation was not seen in the liver of the same animal. The activated oncogene also exhibited several restriction enzyme polymorphisms in transfectant DNA due to a reciprocal translocation 3' to the coding region of the gene, which occurred during transfection. The activating mutation was found in only 1 of 6 beta-propiolactone induced mouse skin tumors examined, the only tumor with a transforming H-ras oncogene. This is a much lower frequency of activation than that previously reported for the same tumor type induced by polycyclic aromatic hydrocarbons. The A----T transversion mutation is consistent with a potentially direct mutagenic effect of a specific beta-propiolactone-DNA adduct

Propylene oxide (PO) is a direct-acting mutagen and rodent carcinogen. We have studied how PO modifies 2'-deoxynucleosides at pH 7.0-7.5 and 37 degrees C for 10 h. PO reacts as an SN2 alkylating agent by forming the following 2-hydroxypropyl (HP) adducts: N6-HP-dAdo (7% yield), 7-HP-Gua (37%) and 3-HP-dThd (4%). Alkylation at N-3 of dCyd resulted in conversion of the adjacent exocyclic imino group at C-4 to an oxygen (hydrolytic deamination) with the formation of a dUrd adduct, 3-HP-dUrd (14%). Ultraviolet spectroscopy and mass spectrometry were used for the structural determination of these adducts. Confirmation of the unexpected 3-HP-dUrd adduct was provided by an accurate mass measurement technique where diagnostic ions in the mass spectra of 3-HP-dUrd were measured to within 0.0005 atomic mass units of the predicted mass. PO was reacted in vitro with calf thymus DNA (pH 7.0-7.5, 37 degrees C, 10 h) and yielded N6-HP-dAdo (1 nmol/mg DNA), 3-HP-Ade (14 nmol/mg DNA), 7-HP-Gua (133 nmol/mg DNA) and 3-HP-dUrd (13 nmol/mg DNA). A mechanism for the hydrolytic deamination of 3-HP-dCyd to 3-HP-dUrd involving the OH on the HP side chain is proposed. This cytosine to uracil conversion may play a role in the mutagenic and carcinogenic activity of this epoxide

A simple mathematical model of the molecular basis for the function of a diffusional sampler for dilute mixtures of gaseous contaminants in supporting gases is presented. The model is based on the movement of single molecules of the contaminant between sections of a tubular diffusion path on a step-by-step basis; the length of the step and of each section of the tube are equal to the mean free path, lambda, under the specified conditions. When the model is used, the coefficient of diffusion, D, can be calculated from lambda and the average velocity, v, of the contaminant molecule. Both lambda and v were calculated independently using equations which involved the minimum number of assumptions. The value of D so estimated was of the same order as that in the literature, differing by a factor of less than 2. It should be emphasized that the model represents a statistical, thermodynamic approach to understanding diffusional samplers, and its utility is independent of the means of estimating lambda and v for specific gas pairs

Previous studies in this laboratory have shown that the combined exposure of hydrogen chloride (HCI) and formaldehyde vapors (HCHO) elicited a significant incidence of nasal cancer in rats. In studies performed elsewhere, it has been demonstrated that exposure to formaldehyde alone induced a high nasal cancer response in rats. We wished to determine whether concurrent exposure of hydrogen chloride would enhance the tumorigenic effects of formaldehyde. Two exposure techniques were used. In one hydrogen chloride and formaldehyde were premixed at high concentrations before entry into the exposure chambers in order to maximize the formation of reactive alkylating agents. In the second the hydrogen chloride and formaldehyde were introduced separately into the exposure chamber. Appropriate control exposures consisting of formaldehyde alone or hydrogen chloride alone or air alone were also performed. The results show that nasal cancer incidences were induced in all animals receiving HCHO regardless of concurrent exposure to hydrogen chloride. The tumors were predominantly squamous cell type arising from the anterior portion of the nasal cavity. This study demonstrates that hydrogen chloride does not appreciably influence the nasal carcinogenicity of formaldehyde

Reaction of the rodent carcinogen acrylonitrile (AN) at pH 7.0 for 10 and/or 40 days with 2'-deoxyadenosine (dAdo), 2'-deoxycytidine (dCyd), 2'-deoxyguanosine (dGuo), 2'-deoxyinosine (dIno), and thymidine (dThd) resulted in the formation of cyanoethyl and carboxyethyl adducts. The adducts isolated were 1-(2-carboxyethyl)-dAdo (1-CE-dAdo), N6-CE-dAdo, 3-CE-dCyd, 7-(2-cyanoethyl)-Gua (7-CNE-Gua), 7,9-bis-CNE-Gua, imidazole ring-opened 7,9-bis-CNE-Gua, 1-CNE-dIno, and 3-CNE-dThd. Structures were assigned on the basis of UV spectra and electron impact (EI), desorption chemical ionization (DCI) and Californium-252 fission fragment ionization mass spectra. The carboxyethyl adducts resulted from initial cyanoethylation at a ring nitrogen adjacent to an exocyclic nitrogen followed by rapid hydrolysis of the nitrile moiety to a carboxylic acid. It was postulated that the facile hydrolysis is the result of an intramolecular-catalyzed reaction resulting from the formation of a transient cyclic intermediate between nitrile carbon and exocyclic nitrogen. AN was reacted with calf thymus DNA (pH 7.0, 37 degrees C, 40 days) and the relative amounts of adducts isolated was 1-CE-Ade (25.8%), N6-CE-Ade (7.6%), 3-CE-Cyt (1.3%), 7-CNE-Gua (25.8%), 7,9-bis-CNE-Gua (4.3%), imidazole ring-opened 7,9-bis-CNE-Gua (18.9%) and 3-CNE-Thy (16.3%). Thus a carcinogen once adducted to a base in DNA was shown to be subsequently modified resulting in a mixed pattern of cyanoethylated and carboxyethylated AN-DNA adducts.(ABSTRACT TRUNCATED AT 250 WORDS)

5-(Hydroxymethyl)uracil (HMUra) is a chemically stable derivative of thymine formed through the action of ionizing radiation which we previously identified in the DNA of gamma-irradiated HeLa cells [Teebor, G. W., Frenkel, K., & Goldstein, M. S. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 318-321]. In this report, we determine whether HMUra can be used as a marker of exposure of DNA to ionizing radiation. Dose-response curves for its formation in [3H]thymidine-labeled DNA were constructed by exposing the DNA to increasing amounts of gamma-radiation and measuring the HMUra content. DNA was irradiated both in solution and in intact cells. HMUra was identified as the 2'-deoxyribonucleoside 5-(hydroxymethyl)-2'-deoxyuridine (HMdU) by subjecting the irradiated DNA to enzymatic digestion and analyzing the mixture of 2'-deoxyribonucleosides by high-pressure liquid chromatography. The identity of the radiogenically formed HMdU was confirmed by acetylation and the structure of the acetyl derivative obtained by mass and nuclear magnetic resonance spectroscopies. At two different DNA concentrations in solution, the same number of thymidine moieties were converted to HMdU, indicating that within this range of concentration the formation of HMdU was mediated through the indirect action of ionizing radiation. Equal amounts of HMdU were formed in single- and double-stranded DNA at each radiation dose, indicating that DNA conformation did not affect HMdU formation. Surprisingly, the G value (number of HMdU molecules formed/100 eV) was higher in irradiated cellular DNA than in DNA irradiated in solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Reaction of the rodent carcinogen acrylamide (AM) at pH 7.0 and 37 degrees C for 10 and 40 days with 2'-deoxyadenosine (dAdo), 2'-deoxycytidine (dCyd), 2'-deoxyguanosine (dGuo), and thymidine (dThd) resulted in the formation of 2-formamidoethyl and 2-carboxyethyl adducts via Michael addition. The alkylated 2'-deoxynucleoside adducts isolated (% yield after 40 days) were 1-(2-carboxyethyl)-dAdo (1-CE-dAdo) (8%), N6-CE-dAdo (21%) (via Dimroth rearrangement of 1-CE-dAdo), 1-CE-dGuo (4%), 7-(2-formamidoethyl)-Gua (7-FAE-Gua) (6%), 7, 9-bis-FAE-Gua (1%) (formed by reaction of AM with depurinated 7-FAE-Gua during the course of the reaction), and 3-FAE-dThd (4%). The products isolated following in vitro reaction of AM with calf thymus DNA at pH 7.0 and 37 degrees C for 40 days were (nmol/mg DNA) 1-CE-dAdo (5.5), N6-CE-dAdo (1.4), 3-CE-dCyd (2.8), 1-CE-dGuo (0.3), and 7-FAe-Gua (1.6). Compound 3-FAE-dThd was not detected. Structures were assigned on the basis of chemical properties, UV spectra, and electron impact, chemical ionization, desorption chemical ionization, Californium-252 fission fragment ionization, and fast atom bombardment mass spectra. A facile hydrolysis of the amide group to a carboxylic acid was observed when AM alkylated a ring nitrogen adjacent to an exocyclic nitrogen atom. In previous studies, we had observed an analogous phenomenon when studying the in vitro reactions of acrylonitrile with DNA, i.e., a facile hydrolysis of nitrile to carboxylic acid when acrylonitrile alkylated (via Michael addition) a ring nitrogen adjacent to an exocyclic nitrogen. Since the nitrile group hydrolyzes to a carboxylic acid via an amide intermediate, we had hypothesized in the present study that the same facile hydrolysis of amide to carboxylic acid would occur under identical stereochemical conditions as had occurred with the nitrile group. Thus, in vitro alkylation of calf thymus DNA by both acrylonitrile and, in the present study, AM, resulted in mixed adduct formation.