Polycyclic aromatic hydrocarbon components contribute to the mitochondria-antiapoptotic effect of fine particulate matter on human bronchial epithelial cells via the aryl hydrocarbon receptor.

epithelial cells to increasing amount of PM 2.5-AW from 1 to 50 μg/cm2.

Gives the exposure series this assay records: PM2.5-AW from 1 to 50 ug/cm2 on 16HBE cells.

2 μM hydroethidine (HE, Molecular Probes) for superoxide anion generation

Identifies the ROS probe and concentration recorded in ros_probe_type.

16HBE cells were exposed to PM 2.5-AW (1 to 50 μg/cm2)o rH 2O2 (500 μM) for 48 h or 72 h

Gives the 48 h and 72 h kinetic conditions recorded above, alongside the hydrogen peroxide positive control.

even when 16HBE cells were exposed for longer times to PM 2.5-AW (1-50 μg/cm2), no significative increase of apoptotic parameters was observed

The assay's result: no significant rise in any apoptotic parameter, including superoxide anion generation, at any dose out to 72 h.

In this study, we showed that low levels of Parisian PM2.5 are not cytotoxic for three different cell lines and primary cultures of human bronchial epithelial cells.

The paper's own summary of this negative cytotoxicity result across all four bronchial epithelial models.

by four parameters and expressed as percentage of 16HBE cells showing ΔΨm drop (DiOC6(3) low) and ROS production (HE->Eth high) or PS exposure (Annexin V+) and plasma membrane permeabilization (PI high).

Confirms ROS production was one of the four parameters quantified in this pretreatment experiment, which is what makes it an oxidative stress assay.

After a 4 h pretreatment with PM 2.5-AW (10 μg/cm2), 16HBE cells were exposed to different inducers of cell death for another 20 h such as mitochondrial respiratory chain inhibitors (rotenone (Rot, 5 μM), antimycin A (AMA, 25 μg/ml) and oligomycin (Omy, 5 μM)), calcium ionophores (ionomycin (Iono, 0.5 μM) and A23187, 3 μM), protein kinases inhibitor (staurosporine STS, 1 μM) and oxidative stress activator

Gives the exposure design recorded in the conditions above: 4 h PM2.5-AW at 10 ug/cm2, then 20 h with A23187 at 3 uM.

Conversely, a 4 hour-pretreatment with PM2.5 prevent mitochondria-driven apoptosis triggered by broad spectrum inducers (A23187, staurosporine and oligomycin) by reducing the mitochondrial transmembrane potential loss, the subsequent ROS production, phosphatidylserine externalization, plasma membrane permeabilization and typical morphological outcomes

The assay's result on the ROS parameter: PM2.5 pretreatment lowered the ROS production that follows the mitochondrial transmembrane potential loss.

mitochondrial and plasma membranes alterations of apoptosis at concentrations as high as 5 μM of A23187

The protection held across the A23187 dose range, up to 5 uM - the inducer dose-response arm of this assay.

Particle pretreatment was performed with different doses (1-50 μg/cm2) 4 h prior to induction of apoptosis by 3 μM of A23187.

Gives the particle dose-response pretreatment arm recorded above, alongside the standard 10 ug/cm2 condition.

Subconfluent 16HBE cells were exposed to PM 2.5-AW for 4 h or 24 h and supernatants were recovered

Gives the two exposure durations and the sample type this assay records.

of Amphiregulin (AR) and GM-CSF released were evaluated with an enzyme-linked immunosorbent assay kit (ELISA, R&D Systems Europe; Abingdon, UK)

Names the two analytes and the ELISA method recorded in this assay's protocols.

Results showed that AR and GM-CSF secretion occur only after a 24 h exposure, which is in agreement with previous studies published on PM 2.5-VW and PM 2.5-AS

The assay's result: amphiregulin and GM-CSF rose at 24 h but not at 4 h, which is why the 4 h and 24 h output records are kept apart.

Then, 10 nM of AhR siRNA (Hs_AHR_6 AlexaFluor-647, Ref SI03043971, Qiagen) or control non-silencing siRNA (AllStars Neg AlexaFluor-647, Ref 1027287, Qiagen)

Gives the siRNA concentration and the two constructs recorded as this assay's two exposure conditions.

validation of AhR silencing by western blot (Figure 7B upper panel)

The assay's result: the Western blot confirmed that the siRNA lowered AhR protein.

the primary antibodies used were: mouse-monoclonal anti-AhR (WH0000196M2, Sigma) and anti-Actin (A5441, Sigma).

Names the AhR antibody and the actin normalization reference recorded in this assay's Western blot protocol.

even when 16HBE cells were exposed for longer times to PM 2.5-AW (1-50 μg/cm2), no significative increase of apoptotic parameters was observed

Superoxide anion generation (hydroethidine) was one of the apoptotic parameters quantified, so this result shows that PM2.5-AW at 1-50 ug/cm2 did not increase oxidative stress in 16HBE cells even at 48-72 h.

Conversely, a 4 hour-pretreatment with PM2.5 prevent mitochondria-driven apoptosis triggered by broad spectrum inducers (A23187, staurosporine and oligomycin) by reducing the mitochondrial transmembrane potential loss, the subsequent ROS production, phosphatidylserine externalization, plasma membrane permeabilization and typical morphological outcomes

PM2.5 pretreatment reduced rather than increased ROS production downstream of the apoptosis inducers, which is the opposite direction to this key event.

Finally, the activation or silencing of the aryl hydrocarbon receptor (AhR) showed that it is involved into the molecular mechanism of the antiapoptotic effect of PM2.5 at the mitochondrial checkpoint of apoptosis.

Both pharmacological activation and genetic silencing of AhR changed the PM2.5 response, placing AhR activation in the mechanism.

Note that the particle-coupled PAH are bioavailable in our system since CYP1A1 mRNA and its enzymatic activity were increased

Induction of CYP1A1, a canonical AhR target gene, is direct evidence that the particle-bound PAH reached and activated the AhR pathway in these cells.

beta-NF (20 μg/ml) used prior to A23178 significantly reduced the amount of apoptotic cells (31% reduction of DiOC6(3) low), and further improved the protection conferred by PM 2.5 exposure (54% vs. 37% reduction of DioC6(3) low).

The AhR agonist beta-naphthoflavone reproduced and amplified the PM2.5 effect, showing that activating this pathway is sufficient to protect the mitochondrial checkpoint.

exposure (25% vs. 37% reduction of DiOC6(3) low), although it did not noticeably modify the apoptotic effect of A23187.

The AhR antagonist alpha-naphthoflavone cut the PM2.5 protection from 37% to 25% without affecting A23187 on its own, so the loss is specific to the AhR-dependent arm.

Then, intracellular PAH can target and activate the aryl hydrocarbon receptor (AhR).

The authors' mechanistic model names PAH desorbed from endocytosed particles as the AhR ligands.

Results showed that AR and GM-CSF secretion occur only after a 24 h exposure, which is in agreement with previous studies published on PM 2.5-VW and PM 2.5-AS

PM2.5 exposure does elicit secretion of the EGFR ligand amphiregulin from 16HBE cells, confirming EGFR pathway engagement - but as a late (24 h) event.

The use of recombinant EGF and specific inhibitor led us to rule out the involvement of the classical EGFR signaling pathway as well as the proinflammatory cytokines secretion.

Neither adding recombinant EGF nor inhibiting the receptor altered the response, so EGFR activation is not the step through which PM2.5 suppresses apoptosis here.

Conversely, a 4 hour-pretreatment with PM2.5 prevent mitochondria-driven apoptosis triggered by broad spectrum inducers (A23187, staurosporine and oligomycin) by reducing the mitochondrial transmembrane potential loss, the subsequent ROS production, phosphatidylserine externalization, plasma membrane permeabilization and typical morphological outcomes

A 4 h PM2.5 pretreatment suppressed every measured hallmark of mitochondria-driven apoptosis, which is this key event.

(33% reduction of DiOC6(3) low), the calcium ionophore A23187 (35% reduction of DiOC6(3) low) and staurosporine (44% reduction of DiOC6(3) low), but not by ionomycin, rotenone,

Quantifies the inhibition in 16HBE cells - 33% for oligomycin, 35% for A23187, 44% for staurosporine - and shows it is selective, with no protection against ionomycin, rotenone, antimycin A or H2O2.

experiments performed in NCI-H292 and NHBE cells showed that PM 2.5-AW also reduced apoptosis induced by A23187 or STS but not by H2O2

The inhibition is not a quirk of one immortalized line: it reproduces in the NCI-H292 carcinoma line and in primary NHBE cells.

the antiapoptotic effect of PM 2.5 -AW was partially efficient at 10 μg/cm 2 and totally effective for concentrations beyond 25 μg/cm 2

Gives the particle dose-dependence of the inhibition: partial at the standard 10 ug/cm2 dose, complete above 25 ug/cm2.

but PM 2.5 -AW (50 μg/cm 2) totally counteracted the cellular volume decrease

The morphological arm of the same inhibition: A23187 alone shrank the cells to 66.2% of control volume, and PM2.5-AW at 50 ug/cm2 fully blocked that apoptotic cell-volume loss.

Instead, the water-soluble fraction, washed particles and organic compounds such as polycyclic aromatic hydrocarbons (PAH) could mimic this antiapoptotic activity.

Identifies which particle fractions carry the inhibitory activity: the water-soluble fraction, the washed particles themselves, and the adsorbed organic PAH.

Except for B(b)F, all the other PAH reduced the amount of apoptosis induced by A23187, similar to that of PM 2.5 pretreatment and B(a)P seems to be the most efficient

Four of the five heavy PAH tested reproduced the inhibition on their own, with benzo[a]pyrene the most potent.

The PM2.5-antiapoptotic effect in addition to the well-documented inflammatory response might explain the maintenance of a prolonged inflammation state induced after pollution exposure and might delay repair processes of injured tissues.

The authors place their finding alongside the established inflammatory response to PM2.5, proposing that apoptosis resistance is what sustains airway inflammation after exposure.

Thus, mechanisms involved in airway remodelling might be the excessive cell proliferation as well as the resistance to the apoptotic cell death.

The paper names resistance to apoptotic cell death as one of the mechanisms behind airway remodeling, which is why this tissue-level key event is relevant here.

Finally, the activation or silencing of the aryl hydrocarbon receptor (AhR) showed that it is involved into the molecular mechanism of the antiapoptotic effect of PM2.5 at the mitochondrial checkpoint of apoptosis.

States the causal link directly: AhR status determines whether PM2.5 suppresses apoptosis at the mitochondrial checkpoint.

AhR silencing significantly reduced the protection triggered by PM 2.5 (18% vs. 27% reduction of DiOC(6)3 low) alike the antagonist (alpha-NF) did.

Genetic loss of AhR weakened the downstream inhibition of apoptosis, the key necessity test for this relationship.

these results suggest that AhR partially contributes to the antiapoptotic effect of PM 2.5 exposure.

The authors' own summary of the relationship's strength: real but partial, which is why evidence_support is moderate.

some of them might be mitochondrial MMP regulators (dotted arrow), thus might protect the cell from apoptosis induced by A23187, staurosporin (STS) or oligomycin (Omy).

Proposes the intermediate step - AhR target genes acting as mitochondrial membrane permeabilization regulators - linking AhR activation to the apoptosis block.

we used a recombinant EGF ligand (rEGF) or the inhibitor of EGF receptor (AG1478, Figure 5B) to show that none of the two compounds modifies the reduction of A23187-induced apoptosis.

Driving the receptor with ligand and blocking it with AG1478 both left the apoptosis inhibition unchanged, so EGFR activation is not the upstream step.

Results showed that AR and GM-CSF secretion occur only after a 24 h exposure, which is in agreement with previous studies published on PM 2.5-VW and PM 2.5-AS

Timing argues against the link: the EGFR-ligand response is a late event while the apoptosis inhibition is established within the 4 h pretreatment.

The use of recombinant EGF and specific inhibitor led us to rule out the involvement of the classical EGFR signaling pathway as well as the proinflammatory cytokines secretion.

The authors state the rule-out explicitly for both the EGFR pathway and the proinflammatory cytokine arm.

The PM2.5-antiapoptotic effect in addition to the well-documented inflammatory response might explain the maintenance of a prolonged inflammation state induced after pollution exposure and might delay repair processes of injured tissues.

States the hypothesised causal chain from the antiapoptotic effect to prolonged airway inflammation and delayed tissue repair.

Thus, mechanisms involved in airway remodelling might be the excessive cell proliferation as well as the resistance to the apoptotic cell death.

Names resistance to apoptotic cell death as a mechanism of airway remodeling, which is the relationship asserted here.