Summary:
The effects of atmospheric pollutants on human health constitute an open
research field for many decades. Today, the great majority of the relevant
toxicological and epidemiological studies is mainly focused on particles with
an aerodynamic diameter 10μm (particulate matter, PM), which are considered to
be the most health threatening. Due to their size, these particles are able to
penetrate and accumulate in the respiratory system, while the high
«surface/mass» ratio allows the adsorption of various toxic compounds on their
surface. Transition metals, polycyclic aromatic hydrocarbons (PAHs), inorganic
ions and various biogenic compounds are thought to be responsible for the toxic
effects of air pollution. The effects of air pollution on human health include
a large number of pathophysiological conditions ranging, from simple skin
irritations and mild allergic reactions, to chronic respiratory problems,
cardiovascular dysfunction and increased cancer risk. Additionally, apart from
the aforementioned physicochemical properties of PM, additional factors, such
as specific conditions of exposure (duration and frequency of exposure
episodes, concentration of pollutants) and individualized sensitivity (age,
pathological background), play a key role in the determination of the final
outcome.
The aim of this Ph.D. thesis was to assess the effects of micro- and
nano-particulate pollutants, of variable composition and complexity, on lung
epithelial cells (namely of the human cell line A549), in order to identify
early changes in cell physiology and signaling, which could contribute to cell
transformation overtime. The responses of A549 were compared to those of human
skin epithelial cells (A431), as well as of primary cultures of normal lung and
dermal fibroblasts. Among the signaling molecules and transcription factors
studied, we particularly focused on Akt kinase and transcription factor HIF-1α,
which promote cell survival and are typical biomarkers of early stages of tumor
development. A second transcription factor, NrF2, was used as a reliable
oxidative stress marker. The three categories of atmospheric pollutants studied
were: 1) a complex mixture of PM2,5-10 collected from an industrial workplace
near Athens, b) a Standard Reference Material (SRM2975) of diesel nanoparticles
(Diesel Exhaust Particles, DEP) and c) the common PAH, benzo[α]pyrene (B[α]P).
B[α]P was selected as a representative component of the organic fraction of the
previous mixtures, in order to investigate, in more detail, its molecular
mechanism of action.
Urban PM were found to significantly decrease cell survival, in a way inversely
related to particle aerodynamic diameter. The cytotoxic effect of the inorganic
fraction was more pronounced compared to the organic fraction, a result that
could be partly attributed to the high concentrations of SO42- and NH4+ ions
detected in the PM mixture. In all cases, reduced cell survival was accompanied
by a parallel decrease in Akt phosphorylation and HIF-1α protein levels.
The main component of the organic fraction of urban PM is DEP nanoparticles.
Low DEP concentrations produced mild oxidative stress conditions, as revealed
by the induction of NrF2, and resulted in an immediate accumulation of HIF-1α.
Repetitive 2-hour exposure of A549 cells to DEP, induced a cumulative increase
of HIF-1α levels, which was maintained for 22 hours, even after DEP removal.
Although, NrF2 was not affected under the same culture conditions, the
concomitant upregulation of its target protein, HO-1, indicates the induction
of NrF2, at earlier time points. Coculture with normal lung fibroblasts, did
not alter the profile of A549 responses. However, using the same experimental
setup, the responses of lung fibroblasts, which were not in direct contact with
the nanoparticles, were of particular interest. Specifically, the paracrine
communication between the two cell types resulted in a substantial induction of
all the survival markers investigated in fibroblasts. Based on these results,
in conjuction with supportive literature data, we propose that pro-inflammatory
cytokines, such as IL-1β, may play a key role to the observed responses.
B[α]P cytotoxicity was mild and detected only at high concentrations. Its
genotoxic effects were confirmed by the formation of stable adducts and by DNA
fragmentation. Interestingly, combined exposure of A549 cells to B[α]P and
manufactured, biocompatible PLGA nanoparticles enhanced the toxic potential of
the pollutant, possibly by increasing its bioavailability. However, low doses
of B[α]P promoted cell proliferation, which was accompanied by upregulation of
NrF2, HIF-1α and Akt. The induction of total protein levels of both
transcription factors was followed by their nuclear accumulation and
transcriptional activation. The response of HIF-1α to B[α]P was confirmed in
the epidermoid carcinoma cell line, A431, as well as in both lung and skin
primary fibroblast cultures. Also, similar results were obtained during
exposure of A549 to B[α]P in the presence of stress conditions, such as
hypoxia, that characterize various lung diseases. Therefore, it seems that
HIF-1α is an early marker of B[α]P effects and, interestingly, its induction
was – at least partially – mediated by the PI3K/Akt pathway. In order to
elucidate the mechanism of HIF-1α induction by B[α]P, we investigated Ca2+
homeostasis. Indeed, we showed that B[α]P disturbs intracellular Ca2+
homeostasis, increasing basal cytoplasmic ion levels, decreasing ion ER content
and, thus, enhancing the potential of extracellular ion uptake. It should be
noted that Ca2+ influx, through the plasma membrane channels, directly triggers
Akt phosphorylation, contributing to the early accumulation of HIF-1α.
In conclusion, the most important results of this doctorate thesis are
summarized as follows:
The cancer biomarker HIF-1α is directly upregulated in the presence of low,
non-cytotoxic concentrations of organic pollutants. This response appears to be
consistent among different cell types and tissues, is mediated – at least in
part – by the PI3K/Akt survival pathway and results in the transcriptional
activation of HIF-1α.
Short-term but repetitive exposure to atmospheric pollutants can possibly cause
long-term alterations in molecular pathways of the cell. In the case that the
affected pathways are implicated in the promotion of cell survival, an
environment which favors gradual cell transformation, could be generated.
The paracrine interaction between epithelial cells and fibroblasts contributes
to the modulation of the final cell response to pollutants. Therefore, under in
vivo conditions, paracrine communication can possibly affect cell types that
are not even in direct contact with the particles.
Calcium homeostasis is disturbed by organic pollutants at low, non-cytotoxic
doses. In particular, B[α]P increases the potential of Ca2+ uptake through the
plasma membrane. This enhanced Ca2+ influx contributes to HIF-1α upregulation
by inducing upstream Akt phosphorylation.
