THESIS
2015
xii, 63 pages : illustrations (some color) ; 30 cm
Abstract
Ambient particulate matter (PM) can cause adverse health effects via their ability to
produce reactive oxygen species (ROS). Humic-like substances (HULIS), a complex mixture
of amphiphilic organic compounds, have been demonstrated to contain the majority of redox
activity in the water-extractable organic fraction of PM. Apart from redox-active quinones,
reduced organic nitrogen compounds, such as alkaloids resulting from biomass burning
emissions, are also found among HULIS constituents.
In this study, we examined the redox activities of pyridine, imidazole and their alkyl
derivatives using a cell-free dithiothreitol (DTT) assay under simulated physiological
conditions (37 °C, pH = 7.40). These compounds were found to have little redox activity on
their own as measured by t...[
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Ambient particulate matter (PM) can cause adverse health effects via their ability to
produce reactive oxygen species (ROS). Humic-like substances (HULIS), a complex mixture
of amphiphilic organic compounds, have been demonstrated to contain the majority of redox
activity in the water-extractable organic fraction of PM. Apart from redox-active quinones,
reduced organic nitrogen compounds, such as alkaloids resulting from biomass burning
emissions, are also found among HULIS constituents.
In this study, we examined the redox activities of pyridine, imidazole and their alkyl
derivatives using a cell-free dithiothreitol (DTT) assay under simulated physiological
conditions (37 °C, pH = 7.40). These compounds were found to have little redox activity on
their own as measured by the DTT assay, but they enhanced ROS generation catalyzed by
1,4-naphthoquinone (1,4-NQ), 1,2-naphthoquinone (1,2-NQ), 9,10-phenanthrenequinone
(9,10-PQ), and HULIS isolated from multiple aerosol samples. The enhancement effect by
the individual nitrogen-containing bases was determined to be proportional to their amount in
the assay solutions. It is postulated that the underlying mechanism involves the unprotonated
N atom acting as a H-bonding acceptor to facilitate hydrogen-atom transfer in the ROS
generation cycle. The enhancement capability was found to increase with their basicity (i.e.,
pK
a of their conjugated acids, BH
+), consistent with the proposed mechanism for
enhancement. Among the imidazole homologues, a linear relationship was observed between
the enhancement factors (in log scale) of the unprotonated form of the imidazole compounds
(B) and the pKa of their conjugated acids (BH
+). This relationship predicts that the range of alkyl imidazole homologues (C
6–C
13) observed in atmospheric HULIS would be 1.5–4.4
times more effective than imidazole in facilitating HULIS-mediated ROS generation.
As for the redox activity of HULIS, it was found that the DTT consumption rate per
μg of HULIS increased with HULIS dose. A quadratic curve of zero intercept yields excellent
fitting for the relationship between DTT consumption rate and the amount of HULIS, which
is consistent with the characteristics of HULIS containing both ROS-active constituents and
components enhancing ROS activities. During these tests, the protocol of DTT assay was also
investigated. Chelex 100 resin seems to be a better choice for the treatment of phosphate
buffer, as metal chelators (e.g., DTPA and EDTA) added may cause unexpected effects on
the test. But it should be noted that Chelex 100 resin was absent in the buffer during the DTT
assay, thus cannot eliminate the DTT response from residual metals in samples. Resin
treatment is not recommended on HULIS samples as it would remove HULIS significantly.
Our work reveals that the ability of atmospheric PM organics to catalyze generation
of ROS in cells could be affected by co-existing redox inactive organic constituents, which
need to be considered when studying the dose–response relationship of atmospheric PM, and
suggests further work optimizing DTT assay protocol and deploying multiple assays be
conducted to better quantify redox capabilities and enhancement effects of the HULIS
components.
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