Irving T. Salmeen

Contribution of 1-nitropyrene to direct-acting Ames assay mutagenicities of diesel particulate extracts

Projections of future increases in the number of diesel powered U.S. passenger cars have raised concerns about potential adverse human health effects associated with diesel engine particulate exhaust (Pepelko et al., 1980). One concern has been carcinogenic polycyclic aromatic hydrocarbons (PAH), known for many years to be associated with diesel exhaust particles. A more recent concern, raised by experiments with the Ames Salmonella mutagenesis assay, has been the discovery that PAH may not be the only potential carcinogens in the diesel exhaust particles (Huisingh et al., 1978; Wang et al., 1978; Pepelko et al., 1980). These experiments have shown that organic solvent extracts of diesel exhaust particles are directly mutagenic (i.e., mutagenic without activation by mammalian tissue homogenate), whereas the predominant PAH identified in the extracts are mutagenic in the Ames assay only with activation (Searle, 1976; McCann and Ames, 1976; Gibson et al., 1978). We (Schuetzle et al., 1981) and others (Erickson et al., 1979) have identified many oxygenated derivatives of PAH in these extracts. Many of these derivatives could be direct-acting mutagens, but little is known yet about their mutagenicities or their concentrations in the extracts. One of the PAH derivatives identified was l-nitropyrene (I-NP) (Schuetzle et al., 1982), recently shown to be an extremely potent direct-acting mutagen in the Ames assay (Wang et al., 1980; LOfroth et al., 1980, Rosenkranz et al., 1980). Herein we report a rigorous determination of the percentage contribution of 1-NP to the direct-acting Ames assay mutagenicities of extracts of exhaust particles collected from 3 different diesel powered passenger vehicles. The percentage contribution, P, of 1-NP is given by:

Ames assay chromatograms and the identification of mutagens in diesel particle extracts.

Dichloromethane extracts of passenger car diesel exhaust particles were separated into 65 fractions by normal-phase high-performance liquid chromatography (HPLC), and Ames assays (TA98 without activation) were carried out on each fraction. The only prominently mutagenic fractions were ones with elution times approximately coincident with those for 1,3-, 1,6-, and 1,8-dinitropyrenes, 1-nitropyrene, and 3- and 8-nitrofluoranthene. These compounds were quantified in one extract sample by gas chromatography-mass spectrometry and gas chromatography coupled with a nitrogen-phosphorus detector. Among five extract samples studied, 30-40% of the total mutagenicity recovered from the columns could be attributed to these six nitroarenes, 15-20% was recovered in the as yet uncharacterized polar fraction, and the remainder was distributed roughly equally over 63 fractions. About 70% of the mutagenicity applied to the HPLC columns was recovered from the columns. The Ames assay mutagenicities of the unfractionated extracts were shown to be additive in the mutagenicities of the components. 23 references.

Resonance Raman spectroscopy of cytochrome c oxidase and electron transport particles with excitation near the Soret band

Abstract We report the resonance Raman spectra of cytochrome c oxidase, both solubilized and in electron transport particles using laser excitation near the Soret band. As in the spectra of other hemoproteins, such as cytochrome c , the shape and intensity of a number of bands change when the oxidation state is varied. However, one of the hemes of solubilized cytochrome c oxidase shows redox behavior which is anomalous. Spectra of electron transport particles are dominated by cytochrome c oxidase. There are, however, definite differences between spectra of solubilized cytochrome c oxidase and electron transport particles in the oxidized states.

Fluorescence spectroscopic study of reactions between gaseous ozone and surface-adsorbed polycyclic aromatic hydrocarbons.

Kinetics of reactions between gaseous ozone (O/sub 3/) and two polycyclic aromatic hydrocarbons (PAH), perylene and benzo(a)pyrene (BaP), adsorbed on fused silica plates were studied by observation of PAH fluorescence spectra as functions of PAH concentration, reaction time, and O/sub 3/ concentration. Significant improvement of the fluorescence signal to scattered light ratio was achieved by using small-angle edge-on illumination configuration. In addition, the photoinduced reactions of PAH by excitation light was minimized. The PAH in aggregated states, as inferred from the excimer fluorescence, exhibited a much slower reaction with O/sub 3/ than those in dispersed forms. With highly dispersed samples, the decays were exponential and the rates proportional to O/sub 3/ concentrations. From these data, the second-order rate constants were determined with the corresponding half-lives of 132 and 2.6 min in 0.2 ppm of O/sub 3/ for perylene and BaP, respectively.

Some effects of bacteria populations on quantitation of ames salmonella-histidine reversion mutagenesis assays

We show from simple theory that the number of mutants observed in the Ames assay should be approximately proportional to alpha Cn0P where alpha is the mutation rate per concentration of mutagen, C; n0 is the initial inoculum; and P is the average number of bacteria per colony in the background lawn. We tested this theory by carrying out Ames assays for the directly acting mutagen 2-nitrofulorene on TA98 as a function of initial inoculum over the range 10(4)-10(8) bacteria/plate. P was assumed to be proportional to the average volume of the background colonies and was estimated from 100X photomicrographs of the background lawn. The initial inoculum was determined by counting background colonies in the photomicrography, by dilution-plating, and by electronic particle counting. We found that the number of mutants depended weakly on n0, but the dependence could be quantitatively accounted for by the simple theory. These data and the theory explain the least some of the reported variations in quantitation of the Ames assay; show that the slopes of dose response curves depend on n0; explain the range observed for spontaneous revertants; suggest some limitations to quantifying the Ames assay and, within these limits, suggest a method for normalizing independently obtained Ames assay data.

Contact-Shifted NMR of Spinach Ferredoxin: Additional Resonances and Partial Assignments

We have extended the study of the contact-shifted resonances in the nmr spectra of oxidized and reduced spinach ferredoxin to lower magnetic field than reported by others. We have found two resonances not observed previously: one at 37 ppm, corresponding to as many as eight protons, in the oxidized protein; and the other at 43 ppm, corresponding to two protons, in the reduced protein. Assignments are proposed for these resonance and for those previously reported. We conclude that amino acid residues other than cysteine may participate in coordinating the iron atoms. By comparing the line widths in our 60 MHz spectra with those in the published 220 MHz spectra, by evaluating hyperfine coupling constants, and by an independent measurement, of the electron paramagnetic resonance line width at 230” K, we estimate the electronic spin-relaxation time of the reduced protein to be ca. 1 X 10-n sec. With this value for T we determine the dipolar contribution to the line width and hence estimate the distances between the contact-shifted protons and the paramagnetic center.

The temperature dependence of Raman intensities of DNA: Evidence for premelting changes and correlations with ultraviolet spectra

Abstract A detailed study was made of the temperature dependence of the intensities of certain bands in the Raman spectra of double-stranded calf thymus DNA. The temperature dependent Raman bands were assigned to structural components of the macromolecule by comparison with the behavior of temperature dependent intensities of simple model polymers and nucleotides. In addition to changes occurring near the melting temperature, complex changes were observed in Raman spectra at temperatures between 40 and 55°C, well below the melting temperature of this DNA. To determine the correlation between these Raman spectral changes and ultraviolet absorption changes in the pre-melting temperature range, we studied the temperature dependence of ultraviolet difference spectra for calf thymus DNA. Using a simple model we can account for the variety of Raman temperature dependences and qualitatively correlate these with the temperature dependences observed in the ultraviolet spectra. These new findings were obtained at a temperature range sufficiently low to be of significance for the study of DNA in vitro.

Urban and regional ozone air quality: Issues relevant to the automobile industry

Abstract Of the six criteria pollutants for which National Ambient Air Quality Standards (NAAQS) have been established in the U.S., ozone has been the most difficult to control. This report assesses the technical issues and control measures relevant to the ozone air quality standard, including health and welfare effects, the effect of past control efforts, the current understanding of ozone‐precursor relationships, Federal and California attainment strategies, and implications of the 1990 Clean Air Act Amendments and California clean air programs. Despite progress in reducing volatile organic compounds (and oxides of nitrogen) emissions over the last 2 decades, improvement in ozone air quality has been slow. It is clear now that in the worst areas even the most costly and stringent of all available control measures will not lower emissions levels sufficiently to meet the ozone air quality standard. A central issue is how rapidly to proceed to reduce ozone levels, i.e., how to balance the urgency of attain...

1-Nitropyrene reduction by Salmonella typhimurium, V79 Chinese hamster and primary rat liver cells

1-Nitropyrene is an unusually potent direct-acting (in the sense that tissue homogenate is not required for activation) mutagen in the Ames assay (Mermelstein et al., 1981). Recently, there has been considerable interest in 1-nitropyrene as an environmental mutagen because the compound has been found at low concentrations (1-100 ppm) in particles collected from ambient urban air (Gibson, 1982); from fireplace emissions (Gibson, 1982); and from diesel engine exhaust (Pederson and Siak, 1981; Salmeen et al., 1982). In some cases 1-nitropyrene accounted for a few to 10°70 of the direct-acting mutagenicities observed for organic solvent extracts of the particles (Gibson, 1982; Salmeen et al., 1982). Despite its potency in the Ames assay, 1-nitropyrene is not a direct-acting mutagen in mammalian cell culture assays so far tested (I(arpinski et al., 1982; Li and Dutcher, 1983; our data, unpublished). (It may be a weak mutagen for some cells with

Influence of sampling filter type on the mutagenicity of diesel exhaust particulate extracts

9 activation (Li and Dutcher, 1983).) One conjecture to explain the difference between the mutagenicity of 1-nitropyrene in bacteria and in mammalian cells is that the bacteria have a unique nitroreductase activity (Karpinski et al., 1982; Mermelstein et ah, 1981). This conjecture was based on reasonably strong evidence that 1-nitropyrene reduction is necessary for the bacterial mutagenicity of the compound. Herein we describe experiments in which we tested this conjecture with measurements of nitropyrene reduction by intact Salmonella bacteria which are sensitive to mutation by 1-nitropyrene; by Salmonella bacteria which are resistant to mutation by 1-nitropyrene; and by two types of mammalian cells V79 Chinese hamster cells (not sensitive to mutation by 1-nitropyrene), and primary rat liver cells (non-dividing cells). We found that all of the bacterial strains and both types of mammalian cells reduced 1-nitropyrene to 1-aminopyrene. We conclude that reduction of nitropyrene is not unique to bacteria which are sensitive to mutation by 1-nitropyrene and that, although 1-nitropyrene reduction may be necessary, it is not sufficient, for the mutagenicity of 1-nitropyrene.