Mutagenicity in lung of big Blue((R)) mice and induction of tandem-base substitutions in Salmonella by the air pollutant peroxyacetyl nitrate (PAN): predicted formation of intrastrand cross-links.

Peroxyacetyl nitrate (PAN) is a ubiquitous air pollutant formed from NO(2) reacting with acetoxy radicals generated from ambient aldehydes in the presence of sunlight and ozone. It contributes to eye irritation associated with photochemical smog and is present in most urban air. PAN was generated in a chamber containing open petri dishes of Salmonella TA100 (gas-phase exposure). After subtraction of the background mutation spectrum, the spectrum of PAN-induced mutants selected at 3.1-fold above the background mutant yield was 59% GC-->TA, 29% GC-->AT, 2% GC-->CG, and 10% multiple mutations - primarily GG-->TT tandem-base substitutions. Using computational molecular modeling methods, a mechanism was developed for producing this unusual tandem-base substitution. The mechanism depends on the protonation of PAN near the polyanionic DNA to release NO(2)(+) resulting in intrastrand dimer formation. Insertion of AA opposite the dimerized GG would account for the tandem GG-->TT transversions. Nose-only exposure of Big Blue((R)) mice to PAN at 78ppm (near the MTD) was mutagenic at the lacI gene in the lung (mutant frequency +/-S.E. of 6.16+/-0.58/10(5) for controls versus 8.24+/-0.30/10(5) for PAN, P=0.016). No tandem-base mutations were detected among the 40 lacI mutants sequenced. Dosimetry with 3H-PAN showed that 24h after exposure, 3.9% of the radiolabel was in the nasal tissue, and only 0.3% was in the lung. However, based on the molecular modeling considerations, the labeled portion of the molecule would not have been expected to have been bound covalently to DNA. Our results indicate that PAN is weakly mutagenic in the lungs of mice and in Salmonella and that PAN produces a unique signature mutation (a tandem GG-->TT transversion) in Salmonella that is likely due to a GG intrastrand cross-link. Thus, PAN may pose a mutagenic and possible carcinogenic risk to humans, especially at the high concentrations at which it is present in some urban environments.

Quantum mechanical studies of the structure and reactivities of the diol epoxides of benzo[c]phenanthrene.

Benzo[c]phenanthrene has a crowded bay region that has been called a fjord region. As a result of the interaction between the atoms across the fjord region, it is a nonplanar molecule with a significant barrier between two helical structures. The crowding in the fjord region also affects the three-dimensional structure of the fjord region diol epoxide. Quantum mechanical studies have been performed to determine the structure and reactivities of the fjord region diol epoxides. Eight local minimum energy three-dimensional structures have been found for the trans diol of 1,2,3,4-tetrahydro-3,4-dihydroxybenzo[c]phenanthrene 1,2-epoxide. They can be characterized by three dichotomies: one between syn and anti, one between quasidiaxial and quasidiequatorial, and the third that depends on nonplanarity of the parent polycyclic aromatic hydrocarbon due to interactions in the crowded bay region, that we have named "in" and "out" based on the position of the epoxide oxygen relative to the distal ring. The structures with the epoxide oxygen on the same side of the saturated ring as the distal ring (in-) are more stable than the structures where the epoxide is on the opposite side (out-). The calculated lowest energy syn and anti structures for the diol epoxide of benzo[c]-phenanthrene are both in-quasidiequatorial, in agreement with experiment. Analysis of the results indicates that the electrostatic interaction across the fjord region could be responsible for the increased stability of the syn-in-quasidiequatorial structure compared to the syn-in-quasidiaxial structure and the stability of the in- structures in general when compared to the out- structures.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenomas induced by polycyclic aromatic hydrocarbons in strain A/J mouse lung correlate with time-integrated DNA adduct levels.

The induction of DNA adducts and adenomas in the lungs of strain A/J mice has been investigated following the single i.p. administration of each of the following polycyclic aromatic hydrocarbons (PAH): pyrene, dibenz[a,h]anthracene, benzo[a]pyrene, benzo[b]fluoranthene, 5-methylchrysene, and cyclopenta[c,d]pyrene. DNA adducts were measured by 32P-postlabeling at times between 1 and 21 days following injection, while adenomas were counted at 240 days after treatment. Pyrene did not induce either DNA adducts or lung adenomas at any of the doses examined. Each of the remaining PAH induced both adenomas and DNA adducts in a dose-dependent manner, with dibenz[a,h]anthracene > 5-methylchrysene > cyclopenta[c,d]pyrene > benzo[a]pyrene > benzo[b]fluoranthene. DNA adducts reached maximal levels between 3 and 9 days after injection, followed by a gradual decrease. The time-integrated DNA adduct level (TIDAL) was calculated by numerically integrating the areas under the adduct persistence curves extrapolated to 240 days for each PAH at each dose level. This value represents the effective total molecular dose of PAH that was delivered to the lung DNA over the entire course of tumorigenesis. A strong correlation of lung adenoma induction with the TIDAL values was observed for each PAH. The slopes of the tumors versus TIDAL value relationships were essentially identical for 5-methylchrysene, cyclopenta[cd]pyrene, benzo[a]pyrene, and benzo[b]fluoranthene. The slope of this relationship for dibenz[a,h]anthracene was markedly greater. The essentially identical induction of adenomas as a function of TIDAL values for these PAH suggests that the formation and persistence of DNA adducts determines their carcinogenic potency.

Epoxide ring opening and related reactivities of cyclopenta polycyclic aromatic hydrocarbons: quantum mechanical studies.

A series of 13 cyclopenta polycyclic aromatic hydrocarbons have been studied using quantum mechanical methods. The three-dimensional molecular structure of each carbocation that might result from the opening of a protonated epoxide ring formed between the carbon atoms completing the cyclopenta ring was computed with AM1. AM1 and ab initio calculations, using a split valence basis set, were then used to predict the direction of ring opening and obtain information about the reactivity of the carbocation. These calculations have shown that for all carbocations studied the cationic charge is well distributed throughout the molecule. The largest CH group charges are approximately 0.3 electron. If the protonated epoxide ring can open so that the nominal charge is on a CH group that is attached to the central ring of an anthracenic core, that carbocation will be greatly favored. For carbocations of this type, the unoccupied alpha' position (the CH group opposite the position of attachment to the anthracenic core) has as much or more of the cation charge as the nominally charged CH position. The group charges, and other properties related to electrostatic reactivity, clearly favor addition of nucleophiles at the unoccupied alpha' position over addition at the nominally charged position. However, when the addition of small nucleophiles at both of these positions is modeled for two such examples, the results favor addition at the nominally charged position in one case and are equivocal in the other case. The group charges and other reactivities considered characterize the electrostatic part of the interaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Genotoxicity in mouse lymphoma cells of chemicals capable of Michael addition.

Over the past several years, we have been evaluating the mutagenicity and clastogenicity of compounds capable of Michael-type reactions. These compounds, including acrylamide, several acrylate and methacrylate esters, vinyl sulfones, and phorone, have been evaluated using TK+/- -3.7.2C mouse lymphoma cells. Mutagenic chemicals induced increases in the number of small colony tk- deficient mutants. This suggested a clastogenic mechanism which was confirmed by demonstrating increases in aberrations and micronucleus frequencies in cultured cells. Vinyl sulfone was found to be the most effective chemical mutagen with induction of genotoxic effects at concentrations as low as 0.25 microgram/ml. The other compounds also produced positive results, but at higher concentrations. Since these compounds are known to deplete glutathione, phorone, a model glutathione depleter, was examined and found to produce similar effects as the other compounds in mouse lymphoma cells. These results suggest that the direct-acting Michael-type reaction has activity relevant to producing a genotoxic effect. Since acrylamide has been found to be a potent germ cell mutagen, this mechanism may be also relevant in the induction of heritable mutagenic risk.

Prediction of the reactivities of cyclopenta-polynuclear aromatic hydrocarbons by quantum mechanical methods.

1. The direction of epoxide ring opening may be predicted using the techniques of theoretical chemistry by comparing the computed total energy of the two possible carbocations formed. 2. To predict the direction of epoxide ring opening and the potential binding of aceanthrylene 1,2-epoxide to biopolymers, quantum mechanical calculations were performed on the two potential hydroxy carbocations. 3. The 2-hydroxy carbocation (II) was favoured over the 1-hydroxy carbocation by 11.8 kcal/mol. Molecule II had more positive charge at the meso carbon group than at the nominally charged 1 position. Both the lowest unoccupied molecular orbital and the molecular electrostatic potential confirm this result, and indicate the possibility of unusual adducts to biopolymers. 4. Similar calculations on the equivalent epoxides of acenaphthylene and acephenanthrylene do not show the same results. 5. Modelling the addition products of II with small nucleophiles indicates that these unusual addition products do not form, and that the interaction is controlled by electronic effects and not electrostatic effects. 6. The calculations on acephenanthrylene demonstrate the importance of including the hydroxyl group when making predictions relative to epoxide ring opening. 7. Molecular descriptors are surrogates for the interaction of that molecule with an often unknown biological target. In cases where molecular descriptors are used without information about the target, small quantitative differences may not be appropriate discriminators.

Strategies for the use of computational SAR methods in assessing genotoxicity.

The relationship between computational SAR studies and relevant data gathering and generation activities is complex. First, the chemical class to be studied is selected on the basis of information requirements for hazard identification and assessment. Membership in the class is determined by consideration of chemical structure and reactivity. Compilation of the existing bioassay data for this chemical class follows immediately from the specification of the class. Bioassay data, qualitative knowledge of general chemical reactivities in this class, and knowledge concerning potential interactions with biomolecular targets all contribute to the derivation of possible mechanisms for biological activity. Computational studies based on modeling the proposed mechanism of action and/or the existing data base can provide a quantitative basis for the differentiation between chemicals. There is the opportunity for continuing feedback between the quantitative computational studies and the development of a relevant bioassay data base for this chemical class. The qualitative and quantitative information on the potential biological responses obtained will provide a rational basis for extrapolation from the extant data base to the chemicals of interest, and to biological responses significant to the assessment for which complete data are unavailable. Knowledge concerning possible mechanisms of action and preexisting data determine the type of computational study that will be most useful.

Genetic activity profiles and pattern recognition in test battery selection.

Computer-generated genetic activity profiles and pairwise matching procedures may aid in the selection of the most appropriate short-term bioassays to be used in test batteries for the evaluation of the genotoxicity of a given chemical or group of chemicals. Selection of test batteries would be based on a quantitative comparative assessment of the past performance of similar tests applied to other chemicals of the same structural group. The information potentially available for test-battery selection through the use of this pattern-recognition technique is considerably greater than the qualitative results obtained from individual short-term tests. Application of the method should further our understanding of the relationships between chemical properties and genotoxic responses obtained in short-term bioassays and also may contribute to our knowledge of the mechanisms of complex processes such as carcinogenesis. This approach to battery selection should be augmented by careful consideration of established principles of genetic toxicity testing; that is, a chemical should be evaluated in a battery of tests representing the full range of relevant genetic endpoints.

Persistence of SCE-inducing lesions in lymphocytes of mice exposed to diaziquone.

Male C57B1/6 mice were injected i.p. with either 1.25 or 5.0 mg/kg diaziquone (AZQ) and killed at various time intervals from 1 to 99 days post treatment for examination of sister chromatid exchange (SCE) persistence in the peripheral blood lymphocytes (PBLs) and splenocytes. SCE frequencies were found to decay steeply during the first week after exposure in both PBLs and splenocytes. This pattern was followed by a slower decline to baseline over the next week. However, high-frequency cell (HFC) analysis indicates that significant numbers of HFCs persist in the PBLs through day 28 and splenocytes at day 99 post exposure. Mathematical modeling of the time-response curves indicates that the average life span of the majority of AZQ-induced SCE-producing lesions in murine PBLs and splenocytes responsive to phytohemagglutinin is between 3 and 5 days.

Analysis of the molecular electrostatic potential for the prediction of N-oxidation and biological activity of substituted pyridines.

Comparative studies on the reactivity of the heterocyclic nitrogen were carried out for pyridine and its three monosubstituted derivatives 2-aminopyridine (2-AP), 3-aminopyridine (3-AP), and 4-aminopyridine (4-AP) to reveal the structural basis for the differences in their susceptibility to N-oxidation. Molecular orbital calculations were performed to obtain the wave functions for the calculation of the molecular electrostatic potentials (MEP) generated by the molecules. The comparison of the reactivity of the cyclic nitrogen, evaluated from the depth and accessibility of the minimum in the MEP, indicates that the nitrogen in 4-AP will be most susceptible to protonation and will be the most protected from N-oxidation at physiological pH values. The MEP map for 2-AP reveals the smallest minimum in the series of compounds and a considerable reduction in the accessibility of the region near the cyclic nitrogen caused by the proximal substitution. On this basis, 3-AP becomes the most likely derivative to form the ring N-oxide. Comparison of the conclusions from the MEP analysis with available data from bioassays suggests that the mechanism responsible for the genotoxic effects of the chemicals, where only 3-AP is active, is very different from the mechanism for systemic toxicity where 3-AP is the least active, and 4-AP is most active probably due to its channel blocking properties. As the mechanisms for the biological activities of the N-oxide metabolites become clear, reliable predictions of the toxicity of the pyridines should become possible based on such reactivity characteristics.

A role for the magnetic field in the radiation-induced efflux of calcium ions from brain tissue in vitro.

Two independent laboratories have demonstrated that electromagnetic radiation at specific frequencies can cause a change in the efflux of calcium ions from brain tissue in vitro. In a local geomagnetic field (LGF) at a density of 38 microTesla (microT), 15- and 45-Hz electromagnetic signals (40 Vp-p/m in air) have been shown to induce a change in the efflux of calcium ions from the exposed tissues, whereas 1- and 30-Hz signals do not. We now show that the effective 15-Hz signal can be rendered ineffective when the LGF is reduced to 19 microT with Helmholtz coils. In addition, the ineffective 30-Hz signal becomes effective when the LGF is changed to +/- 25.3 microT or to +/- 76 microT. These results demonstrate that the net intensity of the LGF is an important variable. The results appear to describe a resonance-like relationship in which the frequency of the electromagnetic field that can induce a change in efflux is proportional to a product of LGF density and an index, 2n + 1, where n = 0,1. These phenomenological findings may provide a basis for evaluating the apparent lack of reproducibility of biological effects caused by low-intensity extremely-low-frequency (ELF) electromagnetic signals. In future investigations of this phenomenon, the LGF vector should be explicitly described. If the underlying mechanism involves a general property of tissue, then research conducted in the ambient electromagnetic environment (50/60 Hz) may be subjected to unnoticed and uncontrolled influences, depending on the density of the LGF.

Studies on the interaction of microwave radiation with cholinesterase.

The significance of the enzyme cholinesterase in studies on the biological effects of microwaves is discussed. Experiments were performed on the direct effect of microwave radiation on the enzyme activity in aqueous solution, and in rabbit blood. Microwave radiation was found to have an effect only when the temperature increase was great enough to denature the enzyme.