Two-color UV + VUV time-of-flight mass spectra show enhancementof fragments at m/z = 15, 42, 43,58, and 85 at the lowest UV pulse energies and depletion of the parention at m/z = 100. Ion images ofthe five major fragments are all isotropic, indicating dissociationlifetimes that are long on the timescale of molecular rotation butshorter than the laser pulse duration (<6 ns). The m/z = 15 and 85 fragments have identical momentumdistributions with moderate translational energy release, suggestingthat they are formed as a neutral product pair and likely via a Norrishtype I dissociation of the enolone to form CH3 + C(O)CH=C(OH)CH3 over a barrier on a triplet surface. The m/z = 43 fragment may be tentatively assigned tothe alternative Norrish type I pathway that produces CH3CO + CH2C(O)CH3 on S0 followingphototautomerization to the diketone, although alternative mechanismsinvolving dissociative ionization of a larger primary photoproductcannot be conclusively ruled out. The m/z = 42 and 58 fragments are not momentum-matched and consequentlyare not formed as a neutral pair via a unimolecular dissociation pathwayon S0. They also likely originate from the dissociativeionization of primary photofragments. RRKM calculations suggest thatunimolecular dissociation pathways that lead to molecular productson S0 are generally slow, implying an upper-limit lifetimeof <46 ns after excitation at 280 nm. Time-dependent measurementssuggest that the observed photofragments likely do not arise fromdissociative ionization of energized AcAc S0*.The photolysis of acetylacetone (AcAc) has been studiedusing velocity-mapion imaging with pulsed nanosecond lasers. The enolone tautomer ofAcAc (CH The initial non-radiative pathways exhibit similar timescalesto those observed in a series of simple α,β-unsaturatedcarbonyls  followingexcitation to the S2 state, where S2 →S1 IC takes place on a <200 fs timescale and subsequentS1 → S0 IC occurs on a 360–2020fs timescale, with a marked dependence on the substituents.23 ISC to triplet states appears not to be competitive,although there is evidence from the photolysis of acrolein followingexcitation to either the S2 or S1 state thata minor channel forming HCO + C2H3 occurs non-statisticallyover a barrier on a triplet surface.25 Overall, photolysisquantum yields appear to be small for α,β-unsaturatedcarbonyls.26The early time dynamics of AcAc following excitationto S10 who used acombination of photoionization mass spectrometry (PIMS), photoelectron-photoioncoincidence (PEPICO) spectroscopy, and IR laser absorption experiments,alongside supporting ab initio calculations. Six distinct one-photonchannels were identified following excitation at 248 nm (5.00 eV)and 266 nm (4.66 eV),10 most originatingon the S0 surface. The major pathway was found to be phototautomerizationto keto-AcAc on S0, with a product yield of 29%, in agreementwith earlier work in cryogenic matrices.29 The major one-photondissociation channels formed methane + acetyl ketene and water + acetylallene, with product yields of 12 and 25%, respectively. The formerchannel was attributed to dissociation over a relatively low-energytransition state (TS) on S0 identified by the ab initiocalculations. A TS for the latter channel was also identified butat a significantly higher energy. Due to the relatively large yield,it was proposed that dissociation occurred on an electronically excitedtriplet surface. Two minor channels involved the formation of ketenewith both acetone and propen-2-ol co-products resulting from the dissociationof both AcAc tautomers via accessible TSs on the S0 surface.The only radical channel observed was the Norrish type I productsacetyl + acetonyl (Φ = 0.05), which was attributed to barrierlessdissociation of keto-AcAc on S0. All other products observedwere attributed to two-photon dissociation mechanisms, including thehydroxyl radical OH that had previously been thought to be a primaryproduct resulting from one-photon dissociation.32 However, ab initio calculations at the CBS-QB3 levelfound that formation of OH should only be possible at λ <235 nm;10 the apparent one-photon signaldependence observed in earlier laser-induced fluorescence studieslikely resulted from facile saturation of the optical transition tothe S2 state.The most comprehensivecharacterization of AcAc photodissociationcomes from Antonov et al.,10 have comprehensivelycharacterized the products resulting from AcAc photochemistry, thedetailed mechanisms are still somewhat uncertain in the absence ofmeasurements performed under collision-free conditions that can alsoquantify the photofragment translational energy release. As is wellknown for simple carbonyl species, Norrish type I pathways can occurstatistically on S0 or over exit barriers on T1, leading to characteristic translational and internal energy distributions,37 while molecular dissociation via S0 transitions statesor roaming pathways can also be distinguished.40 In this paper,we present the results of a velocity-map ion imaging study of AcAcphotochemistry following UV excitation to S2 at 280 nmusing single-photon VUV ionization to detect products. The experimentsfind evidence of primary photofragments that arise from Norrish typeI dissociation on both triplet and singlet surfaces as well as productsof dissociative ionization of primary photofragments.While Antonov et al.41 and only a brief description is provided here. The apparatus consistsof source, ionization, and detector regions evacuated by turbomolecularpumps  backed by roughingpumps  to base pressures of ∼1× 10–7 Torr. During operation, the source regionpressure increased to ∼10–5 Torr. Ar carriergas at a pressure of ∼1 atm was bubbled through a liquid sampleof AcAc (Acros Organics) in a stainless-steel finger, cooled in awater ice bath, and supersonically expanded into high vacuum by asolenoid valve  operating with 500 μspulse duration. The expansion was skimmed to produce a molecular beam,which was intersected by counter-propagating and spatially overlappedUV and VUV laser beams. Based on the vapor pressure of AcAc at 273K,43 the expansion is estimated to contain 0.2%AcAc.Experiments were performed usinga DC slice velocity-map imagingapparatus that has been detailed previously,44 The UVbeam was loosely focused into the center of the ionization regionof the apparatus by an anti-reflection coated fused silica lens witha focal length of f = 500 mm. The lens was positionedto ensure that the UV beam was unfocused at the molecular beam. Theresulting UV beam diameter in the interaction region was ∼1mm. The UV pulse energy could be varied from ∼10 μJ to∼1 mJ (giving fluences ranging from ∼1.3 to ∼130mJ cm–2) using combinations of neutral-density filters.VUV pulses at 118.2 nm (10.49 eV) were generated by frequency-triplingthe 355 nm output of a Nd/YAG laser  in a Xe/Ar gas mixture. The 355 nm beam was attenuatedusing a λ/4 waveplate and Glan-Taylor polarizer to give pulseenergies ∼8 mJ and focused near the center of the 57 cm longmixing cell by a UV-grade fused silica lens (f =500 mm). A stable conversion was achieved with PXe = 25 Torr and PAr = 308 Torr,giving an Ar/Xe ratio of 12.3 that is consistent with previous studies.47 An internal MgF2 lens (f = 75 mm) actedas a window between the gas cell and the ionization region of theVMI apparatus and focused the VUV while leaving the residual 355 nmunfocused. We calculate beam diameters in the ionization region of∼3 mm for the residual 355 nm and ∼80 μm for the118 nm beam, giving fluences of 115 and 0.2 mJ cm–2, respectively. The latter value is estimated based on previouslyreported conversion efficiencies for 118 nm generation in Xe/Ar mixturesobtained using ns pulsed lasers.47 VUVgeneration required continuous convective mixing within the cell,which was maintained by cooling one limb of a circulating loop withan acetone/dry ice slush at 195 K.UV pulses at 280 nm (4.43 eV) were produced by frequency-doublingin a BBO crystal the visible output of a Nd/YAG-pumped dye laser  operating with Rhodamine590 laser dye. A double Fresnel rhomb (EKSMA Optics) was used to flipthe horizontally polarized UV beam vertically, ensuring the ejectedphotofragments would have cylindrical symmetry about an axis parallelto the plane of the detector.48 Total phosphorescence was detected using a siliconphotomultiplier (SenSL) coupled to a high-definition oscilloscope(LeCroy HDO4054), used to record time-of-flight mass spectra; the∼60 ns lifetime of the phosphor limits the obtainable resolution,although this can be overcome to some extent by step scanning thedetector gate over masses of interest. Ion images were captured bya complementary metal–oxide semiconductor (CMOS) camera (BasleracA1300-60gm). Synchronization of the pulsed valve, lasers, detectorgate, and CMOS camera exposure was achieved using a digital delaygenerator . All data acquisition and experimentalcontrol were performed using software written in LabVIEW 2013  and subsequently analyzed using procedures written inIgor Pro (WaveMetrics).Ions were accelerated towarda 40 mm diameter dual microchannelplate (MCP) and P46 phosphor screen detector assembly (Photonis).A fast high-voltage switch  was used to gate the detector to allow mass selection and DC slicing.49 Geometry optimizationsand harmonic frequency calculations were performed for various stationarypoints on the S0 surface using the B3LYP functional withDunning’s cc-pVDZ basis set. Exclusively real harmonic frequenciesconfirmed that the optimized geometries of either tautomer of AcAcand molecular or radical products were local minima on the potentialenergy surface. All transition states identified had only one imaginaryfrequency, confirming that they were first-order saddle points.All ab initio calculations were performedusing the GAMESS package(version 2020 R2).2 state of enol-AcAcalmost exclusively. The enolone is present at an almost 20 times higherconcentration than the diketone, and the transition to the S2 state has an oscillator strength that is more than 30 times greaterthan the transition to the S1 state of the minor diketoneform.8 Direct excitation to the S1(nπ*) state of enol-AcAc may also occur in this wavelengthregion21 with a cross section that is moreakin to typical α,β-unsaturated carbonyls such as MVK,although this possibility appears not to have been considered in theprevious spectroscopic analyses. Regardless, IC from S2 → S1 is expected to occur on a sub-ps timescale,20 which is effectively instantaneous on the nanosecond timescale ofthe current experiments.Excitation at 280 nm prepares the S51 The 118.2 nm (10.49 eV) VUV beamcan cause dissociative ionization of both tautomers.10 One-color VUV-only mass spectra show primarily the AcAc+ parent ion at m/z = 100with a weaker feature at m/z = 85that results from dissociative ionization of the dominant enolonetautomer. Dissociative ionization of the minor diketone tautomer producesions with m/z = 43, 58, and 72,10 but VUV-only signal at these masses is difficultto discern within the experimental signal-to-noise ratio, partiallydue to interference from ionization of background gas within the chamber.The 280 nm UV beam does not produce ions to any significant extentat pulse energies <0.5 mJ (<64 mJ cm–2). VUVgeneration requires convection in the tripling cell—withoutthe acetone/dry ice slush, the unfocused residual 355 nm causes noionization, either with or without the presence of the UV beam.The ionization energy ofenol-AcAc is 8.85 and ∼9.5 eV for the diketone form.–2, respectively). The time delay between the UV andVUV beams was 20 ns; measurements of the time profiles as the UV-VUVdelay varied are summarized in Figure S2 of the Supporting Information. The AcAc+ ion at m/z = 100 is depleted when the UV beamis introduced at earlier times, indicating loss of the neutral species.The magnitude of the depletion is ∼20–60% of the VUV-onlypeak, across the range of UV photolysis pulse energies shown. Thepresence of the UV beam also leads to additional fragment peaks at m/z ≤ 85. The major two-color fragmentsat lower UV energies give peaks at m/z = 15, 42, 43, 58, and 85 in the mass spectrum, which are indicatedwith asterisks in 0 → S2 transitionis that it is readily saturated at modest UV pulse energies, whichincreases the likelihood of multiphoton dissociation.10 Measurements of the dependences of the m/z = 15, 42, 43, and 85 signals on the UV pulseenergy over the range 40–420 μJ (fluence 5–54mJ cm–2) are summarized in Figure S4 and Table S1 of the Supporting Information. The resultsare somewhat inconclusive but suggest that there may be a contributionfrom multiphoton excitation at higher pulse energies to the m/z = 15 and 42 fragments (n > 1). In contrast, the m/z =43and 85 signals increase more slowly with UV fluence (n < 1).The time-of-flight mass spectra in m/z =39, 40, 42, and 43 are clearly resolved. While the m/z = 39 and 40 mass peaks are very weak at low UVlaser fluences, their intensity increases dramatically as the fluenceis increased, suggesting that they result from multiphoton dissociation.Similar gate scans over other regions show that the features at m/z = 15, 58, and 85 are isolated with no significant two-colorsignal in nearby mass channels.The detector phosphorescence lifetime limits themass resolution.For sequential masses, those arriving later may be lost in the phosphorescencedecay of an earlier arriving mass especially if less abundant. Usinga very short detector gate and stepping over the arrival times allowsthe individual mass peaks to be identified more clearly. The insetin m/z = 15, 42, 43, 58, and 85, for whichion images (discussed below) were recorded. While these ions may originatefrom UV photolysis of AcAc with subsequent VUV ionization of the neutralfragment, there are other potential sources. Dissociative ionizationof energized ground-state AcAc S0* following VUV absorptionmay directly produce ionic photofragments. The total excitation energyin that case is 14.92 eV (280 + 118 nm), which is comfortably greaterthan the appearance energies of all the fragment ions detected, althoughthe persistence of signals at delay times that extend beyond the expectedAcAc S0* lifetime of <50 ns  suggests that this mechanism is ofminor importance, even at a relatively short delay of 20 ns. Neutralprimary photoproducts may also undergo dissociative ionization, leadingto the formation of lower-mass daughter ions.We focus on the major featuresevident in the two-color UV + VUVmass spectra observed at the lowest UV pulse energies, namely m/z = 15 fragment can beimmediately assigned to CH3+, which can be producedby VUV photoionization of neutral methyl radicals 52 resulting from Norrishtype I dissociation of either the enolone or diketone tautomerThe 10 have found very similardissociation energies D0 for pathways(Ehν = 4.43eV). Ionization energies for both larger radical products are expectedto be <8 eV, based on ab initio calculations at the B3LYP/cc-pVDZlevel, and thus, they can be photoionized by the VUV pulse, thoughthe different m/z = 85 radical cationscannot be distinguished by VUV ionization. The magnitude of the m/z = 85 signal indicates a photoionizationcross section that is around 6–8 times larger than that ofCH3 at 118 nm (∼6 Mb).54 At higher UV pulse energies, the m/z = 15 signal increases more rapidly than the m/z = 85 signal .The n > 1 photon dependence for m/z = 15 indicates that there may be a contributionthat arises from multiphoton excitation. In contrast, the m/z = 85 channel has a photon dependence n < 1, suggesting that secondary absorption of the UVbeam and dissociation may limit the overall yield. The ion imagingexperiments described below strongly suggest that the m/z = 15 and 85 fragments are primarily formed photolyticallyas a neutral radical pair. Both m/z = 15 and m/z = 85 photofragmentsshow appearance times  thatare within the instrument resolution, limited by the 6 ns pulse durationof the laser systems.The CBS-QB3 calculations performedby Antonov et al.pathways and1b)10 have f3CO, IE = 7.0 eV)52 can also occur from either the enolone or diketonetautomerThe alternative Norrish type I dissociationpathway leading tothe formation of acetyl radicals (CH2C(O)CH3 as the co-productis energetically accessible based on the dissociation energies calculatedat the CBS-QB3 level.10 Subsequent VUVionization would give rise to peaks at m/z = 43 (CH3CO+) and m/z = 57 (CH2C(O)CH3+), although only the former is evident in the mass spectra shownin m/z = 57 signal may be attributedto dissociative ionization of acetonyl leading to m/z = 29, although that mass channel is evident mainlyat higher UV pulse energies , acetone (CH3C(O)CH3, IE = 9.70 eV), and propen-2-ol (CH2=C(OH)CH3, IE = 8.58 eV) can all be photoionized at 118 nm.52 The photoionization cross section of keteneis relatively large at 118 nm (24.8 Mb),55 and the weak signal observed at m/z = 42 at low UV pulse energies would suggest a small yield for thisproduct if it were formed via unimolecular dissociation on the S0 surface. However, the time profiles, ion imaging results,and RRKM rate calculations described below suggest that the m/z = 42 and 58 fragments are most likelynot formed by unimolecular dissociation on S0 but are morelikely to result from dissociative ionization of the C4H5O2 primary photoproduct.Two energeticallyaccessible pathways exist for reactions m/z = 15, 42, 43, 58, and 85, following subtraction of one-color VUV-onlybackground signals. All two-color UV + VUV and one-color VUV ion imageswere recorded in triplicate . Ion images recordedat low UV laser pulse energies  to minimize contributionsfrom multiphoton processes produced identical speed distributions.Fragment radial and angular distributions for each mass were extracteddirectly from the individual images and averaged. The images appearisotropic, and fitting the angular distributions to the expression P(θ) ∝ βP2(cos θ) yields values of the anisotropy parameter β thatare close to zero.56 Radial distributions were convertedinto speed distributions using a calibration factor determined fromion images of the I/I* products of CH3I photolysis (seethe Supporting Information for details),with an estimated uncertainty of ∼2%. The speed and fragmenttranslational energy distributions, P(v) and P(fET), are shownin m/z = 15 and85 ions can,in principle, be produced by VUV ionization of the neutral productsof a Norrish Type I mechanism. P(p) for these ions are identical, confirmingthat momentum-matched neutral methyl CH3 and the associatedC4H5O2 radicals are formed originallyvia either reaction P(ET), also shown in P(v) distributions for m/z = 15 and 85 usingP(ET) distributions derived in this way are identicalas expected, with average ⟨ET⟩= 0.38 eV and extending to a maximum translational energy ET,max ≈ 1 eV. As noted earlier, the CBS-QB3calculations reported by Antonov et al.10 suggest nearly identical dissociation energies for reactions m/z = 15 3 +C4H5O2 radical product pairs areformed with ET ≥ 0.61 eV, suggestingthat the true D0 value is smaller thanthat predicted by theory.We identify the maxima in the momentum distributions for each fragmentby fitting it to a Gaussian function and determining the cutoff pmax to be at least 2σ beyond the mean,which captures around 95% of the distribution. Using this value asa cutoff provides an upper limit of D0 ≤ 3.49 eV, with an estimated uncertainty of 0.13 eV. Thefraction of the available energy partitioned into translation is fT ≥ 41%. Previously, we found similar fT values for Norrish type I dissociation ofacetaldehyde and acetone on the T1 surface when the availableenergy was ∼0.5 eV above the T1 barrier.41 In contrast, barrierless dissociation on S0 is statisticaland results in slow-moving fragments with far smaller values of fT.37Approximately one-third of the CH3CO and acetonyl CH2C(O)CH3 fragments that would appear at m/z = 43 and m/z = 29  in the mass spectrum, respectively. Only theformer is clearly observed as noted above. Assuming the formationof a neutral acetyl + acetonyl product pair, the total translationalenergy distribution P(ET) has ⟨ET⟩ = 0.16 eV. Basedon the reported CBS-QB3 dissociation energy, EAVL = 1.10 eV; the fraction of available energy partitionedinto translation fT = 14% is far smallerthan the fT observed for methyl radicalsformed via either reaction 0. Dissociative ionization of the thermalized diketonetautomer has been shown to lead to the formation of m/z = 43 along with m/z = 58 and 72 fragments.10 In the absenceof collisions to stabilize any diketone produced via phototautomerization,the absence of m/z = 72 fragmentsin the mass spectra is expected.The alternative energetically accessible Norrish type I pathway,reaction m/z = 42 fragment is ketene,which can be formed along witheither acetone or its less stable isomer propen-2-ol, as shown inreactions  distributionsin v42⟩/⟨v58⟩ = 2.9 is around twice the inversemass ratio and the momentum distributions are very different .Finally, the obvious assignmentfor the eactions  and 3b)m/z = 42  5.6 see 1 is appr10 at shorter photolysis wavelengths of 248 and266 nm. Following UV excitation at 266 nm and using a similar 10.45eV VUV photon for ionization, fragments at m/z = 15, 29, 40, 42, 43, 58, 72, 82, and 84 were identified, whilewe observe m/z = 15, 40, 42, 43,58, and 85 when using low UV pulse energies to minimize multiphotondissociation. The m/z = 40 signalshows a very strong dependence on UV pulse energy and is likely aproduct of multiphoton or secondary dissociation. Ions with m/z = 82 and 84, previously assigned toacetyl allene and acetyl ketene, formed in conjunction with waterand methane, respectively, are not observed at 280 nm although theyare in principle detectable using 10.49 eV VUV radiation. The massspectra in m/z = 85in this region, which previously was observed only as the productof dissociative ionization of the enolone tautomer. The strong two-color[UV + VUV] enhancement of the signal and the momentum-matched m/z = 15 co-fragment indicates that itis formed as a neutral radical.There are some differences between the two-colordifference massspectra for 280 nm excitation shown in 10 is not entirely clear, but wenote that there are several differences in the experimental conditionsthat may be collectively responsible. The most obvious differenceis that the ion imaging experiments are performed under collision-freeconditions rather than in the collisional environment of the flowcell. Additionally, the excitation wavelengths (280 nm versus 248nm or 266 nm) and the internal state distributions of the AcAc priorto excitation  may access different regionsof the excited-state potential energy surfaces, and result in differentdynamics.The origin of the apparent differencesfrom the results of Antonovet al.2 state,the populationis returned to S0 via a series of non-radiative steps within∼330 ps.20 In this work, the VUV ionization pulse was introduced 20 ns afterUV excitation—all product ions were observed to form on a timescalefaster than the laser pulse durations . An upper-limit lifetime of energized AcAc S0* can be estimated using RRKM calculations.57 Using the Antonov et al.10 CBS-QB3calculations as a template, we have performed ab initio calculationsto optimize the geometries and calculate the harmonic vibrationalfrequencies of the seven dissociation transition states  is the sum of states evaluated at EAVL = E–ETS for the relevant transition state, and ρ(E) is the density of states evaluated at E for eitherenol-AcAc or keto-AcAc. The calculated RRKM rates and associated lifetimeswith total energy E = 35,710 cm–1 (280 nm) are compiled in 0 via TS2, which leads from enol-AcAc to methane +acetyl ketene, dominates the total loss rate and would account for97% of products formed via the molecular TSs on S0. Thesecond-fastest process, dissociation of keto-AcAc via TS7 to formketene + propen-2-ol, is more than an order of magnitude slower andwould make up the remaining 3% of S0 unimolecular dissociationproducts. The other molecular pathways contribute negligibly. Thewavelength dependence of the RRKM dissociation rates and lifetimesfor TS2 and TS7 are shown in 6 s–1, which wouldresult in a relatively long S0 lifetime of <46 ns. Thislifetime does not include contributions from barrierless radical–radicalprocesses  for which variational calculations wouldbe required. Consequently, the calculated S0 lifetime of46 ns should be regarded as an upper limit that will be reduced bycontributions from barrierless pathways, which are likely to be competitive.59Following excitation of AcAc to the S0*, but was found by Antonov et al. to have a photofragmentquantum yield of only Φ = 0.12. The VMI experiments describedhere are insensitive to methane (IE = 12.61 eV),52 and we see no evidence of acetyl ketene (IE = 9.59 eV)10 at m/z = 84.The most significant photolytic product channel identified by Antonovet al. leads to water + acetyl allene 52 with a quantum yield of Φ = 0.25. This reaction occurs onS0 via TS4 but has an exceptionally slow RRKM rate constantof only 0.10 s–1 and a lifetime of 9.9 s and consequentlyshould have a negligible yield. While the current experiments areinsensitive to water, the absence of any signal at m/z = 82 is unsurprising if the unimolecular dissociationrate were to be as slow as the RRKM calculations predict. At shorterexcitation wavelengths, the unimolecular dissociation rates increase,as shown in According to the RRKM calculations, the methane +acetyl ketenepathway, which occurs via TS2, should dominate the unimolecular dissociationof Sm/z = 42 and 58fragments appear to form energetically accessible product pairs (ketene+ acetone or ketene + propen-2-ol), they are formed with distinctlydifferent momentum distributions, indicating that they are not co-fragments.The RRKM rates for formation of ketene + acetone via TS8 and ketene+ propen-2-ol are both exceptionally slow  are inconsistent with the expectedlifetime of AcAc S0* of <46 ns, except at very shortdelays. Secondary UV photolysis of C4H5O2 radical primary photoproducts followed by VUV ionizationor direct dissociative ionization may be the source of these fragments.Although the slow see 2 implyinm/z = 15 and 85 radical pairare formed as momentum-matched neutral CH3 and either C(O)CH=C(OH)CH3 or C(O)CH2C(O)CH3 radicals that aresubsequently ionized by the VUV pulse, via a Norrish type I dissociationof either enol-AcAc or keto-AcAc shown in reactions  is consistent with dissociation over a barrier on a tripletsurface with a relatively large excess energy, as observed previouslyfor the photolysis of the monocarbonyls acetaldehyde and acetone.41 Consequently, we conclude that following initial excitation, thesystem evolves non-radiatively as deduced by the ultrafast studiesS2 → S1 → T1, whereupona subset of the population can undergo dissociation over a barrierto form CH3 + C(O)CH=C(OH)CH3, whilethe remainder undergoes ISC to repopulate S0. The dissociationrate on T1 would have to be competitive with that for T1 → S0 ISC.20 The absenceof the major S0 products predicted by the RRKM calculations(methane + acetyl ketene via TS2) suggests that dissociation on T1 may dominate under collision-free conditions. Relaxed reactionpath calculations leading to CH3 elimination by Squibbet al.19 suggest that a barrier of 4.54eV exists on the T1(ππ*) surface, which isonly slightly greater than the available energy. Further ab initiocalculations to locate and better characterize the TS for Norrishtype I dissociation on the T1 surface would be valuable.Experimentally, measurements at longer UV wavelengths would also testthe proposed mechanism. As the excess energy above the triplet barrierdecreases, partitioning to internal motion should also decrease, leadingto an increase in fT,60 as has been observed previously for acetaldehyde and acetone.41The eactions  and 1b)m/z = 15 m/z = 43fragment to CH3CO formed via the alternative Norrish typeI reaction m/z = 29 products, which appeared only as an exceptionallyweak signal in the mass spectra. Assuming acetyl + acetonyl is formed,a far smaller fraction of the available energy is partitioned intorelative translation (fT = 0.15), whichsuggests that dissociation occurs without a barrier. The most likelymechanism following excitation to S2 is non-radiative populationtransfer back to S0 on a sub-nanoscale timescale, followedby phototautomerization to keto-AcAc and subsequent barrierless dissociation,as initially proposed by Antonov et al.10 The enolone tautomer cannot dissociate to form acetyl radicals at280 nm. Based on known photoionization cross sections for CH3 and CH3CO, the two Norrish type I dissociation pathwaysleading to their formation appear to have qualitatively similar branchingfractions. Alternative mechanisms cannot be conclusively ruled out,however. As the time profile would appear to eliminate dissociativeionization of AcAc S0*, except at very short delays, andthe n < 1 photon dependence argues against secondaryUV dissociation, the most probably alternative to Norrish type I dissociationon S0 appears to be dissociative ionization of the C4H5O2 primary photoproduct.The assignment of the 62 have discussed the relative branchingbetween Norrish type I pathways and other dissociation mechanismsfor a broad range of organic carbonyls relevant to atmospheric chemistry.While Norrish type I dissociation on S0 is available toall carbonyls, the accessibility of the T1 mechanism islimited by functionality. Cleavage of either α-bond adjacentto the C=O moiety can occur, forming either larger or smalleralkyl radical products, labeled type Ia and Ib, respectively. In α,β-unsaturatedcarbonyls, the intrinsic barrier for type Ia dissociation is increasedby resonance stabilization, rendering that pathway inaccessible atactinic wavelengths, as demonstrated for both acrolein and MVK. However,the increase in barrier height is not seen for type Ib dissociation,which remains accessible under tropospheric conditions. This trendis consistent with our assignment of dissociation on T1 for the m/z = 15 and 85 radicalpair in AcAc (type Ib). In contrast, tautomerization is a competitiveground-state pathway for α,β-unsaturated carbonyls. Followingtautomerization, it is plausible that keto-AcAc would dissociate viathe type Ia mechanism to produce m/z = 43 and 57 fragments, as is tentatively assigned here. The proposedphotolysis pathways of AcAc are accordingly consistent with the knowntropospheric photochemistry of both mono- and α,β-unsaturatedcarbonyls.Rowellet al.2 at 280 nm. The major fragment mass peaks observed were m/z = 15, 42, 43, 58, and 85. All ionswere formed on a timescale that was shorter than the laser pulse duration(<6 ns) and produced isotropic images, indicating that the timescalefor dissociation was longer than the rotational period of the parentmolecule. The m/z = 15 and 85 fragmentswere momentum-matched and assigned to the Norrish type I neutral productsCH3 + C(O)CH=C(OH)CH3 resulting fromdissociation of enol-AcAc. The fraction of available energy partitionedinto relative translation fT = 0.41 suggeststhat dissociation occurs on the T1(ππ*) surface.The observed speed distributions were used to determine an upper-limitdissociation energy D0 ≤ 3.49 eV.The m/z = 43 fragment was tentativelyassigned to the alternative Norrish type I product CH3CO,presumably formed in conjunction with CH2C(O)CH3, although this fragment was not directly detected. The smaller fT value of 0.15 is consistent with dissociationof keto-AcAc following phototautomerization on S0. Alternatively,the time and power dependence of the m/z = 43 signal may indicate that it appears as a daughter ion followingdissociative ionization, most probably, of the C4H5O2 primary photofragment. The m/z = 42 and 58 fragments were not momentum-matched,indicating that they were not formed as a product pair via unimoleculardissociation on S0. Similar dissociative ionization mechanismsare most likely responsible.Velocity-map ion imaging coupled with VUVionization has been usedto study the UV photochemistry of enol-AcAc following excitation toS


Mycobacterium marinum in murine RAW264.7 macrophages. We found that, shortly after phagocytosis, DRAM1 localised in a punctate pattern to mycobacteria, which gradually progressed to full DRAM1 envelopment of the bacteria. Within the same time frame, DRAM1-positive mycobacteria colocalised with the LC3 marker for autophagosomes and LysoTracker and LAMP1 markers for (endo)lysosomes. Knockdown analysis revealed that DRAM1 is required for the recruitment of LC3 and for the acidification of mycobacteria-containing vesicles. A reduction in the presence of LAMP1 further suggested reduced fusion of lysosomes with mycobacteria-containing vesicles. Finally, we show that DRAM1 knockdown impairs the ability of macrophages to defend against mycobacterial infection. Together, these results support that DRAM1 promotes the trafficking of mycobacteria through the degradative (auto)phagolysosomal pathway. Considering its prominent effect on host resistance to intracellular infection, DRAM1 is a promising target for therapeutic modulation of the microbicidal capacity of macrophages.Damage-Regulated Autophagy Modulator 1 (DRAM1) is an infection-inducible membrane protein, whose function in the immune response is incompletely understood. Based on previous results in a zebrafish infection model, we have proposed that DRAM1 is a host resistance factor against intracellular mycobacterial infection. To gain insight into the cellular processes underlying DRAM1-mediated host defence, here we studied the interaction of DRAM1 with  Innate immune cells, including macrophages, are the gatekeepers of the immune system; they ingest microbes into internal compartments called phagosomes, they mount a range of cell-autonomous defence responses, and they alert other components of the immune system. A key cell-autonomous defence response is the phagolysosomal pathway, which delivers microbes through a phagosome maturation process to lysosomes. Upon lysosomal fusion, microbes are exposed to an acidic environment, antimicrobial peptides, and degradative enzymes . HoweverMycobacterium comprises several human pathogens that can subvert both the phagolysosomal and autolysosomal pathways, in order to facilitate their intracellular survival in macrophages [Mycobacterium tuberculosis (Mtb), which causes 10 million cases of tuberculosis (TB) and close to 1.5 million deaths every year [Mtb to a large collection of nontuberculous mycobacteria (NTM). For example, Mycobacterium avium lung infections are associated with poor clinical outcomes, and are notably on the rise [Mycobacterium marinum (Mm), known for outbreaks of a TB-like infection in fish in aquacultures, as well as for skin infections in humans [Mm is closely related to Mtb, sharing 3000 orthologous proteins with an average amino acid identity of 85% [Mm and Mtb, the bacteria require the ESX-1 system to evade phagolysosomal degradation and invade the cytosol [Mm or Mtb are targeted by xenophagy [Mtb has been shown to inhibit the autophagy-related LAP pathway [The genus rophages ,9,10,11.ery year . Howeverthe rise ,14. Anotn humans . Mm is cy of 85% . This siy of 85% . In both cytosol ). As a cenophagy ,20,21. Henophagy . Likewis pathway .Mm and Mtb infection [Mm infection, we showed that knockdown or mutation of the zebrafish ortholog of DRAM1 causes a hyper-susceptibility to infection, while its overexpression increases host resistance against Mm [Mm with LC3 and increases the lysosomal acidification of Mm-containing compartments [DRAM1, standing for damage-regulated autophagy modulator 1, is the founding member of a family of five stress-inducible autophagy regulators ,25. DRAMnfection ,24. In anfection ,27,28,29nfection . Using aainst Mm ,30. We aartments ,30. HoweMtb autophagy studies [Mm infection of this cell line leads to rapid permeabilisation of phagosomes and subsequent ubiquitination of bacteria, marking them as targets for xenophagy [Mm over the course of infection, and study this in relation to LC3, LysoTracker and LAMP1 patterns, as markers for autophagosomes and (endo)lysosomes. In addition, we generated DRAM1 knockdown macrophage cell lines to show that DRAM1 knockdown reduces the trafficking of LC3 to Mm, as well as reduces the acidification of Mm-containing vesicles. Furthermore, upon DRAM1 knockdown, the Mm infection rate increased. These results support the central role of DRAM1 in host resistance to intracellular infection, by promoting the trafficking of mycobacteria in the (auto)phagolysosomal pathway.To study DRAM1 localisation and function during mycobacterial infection in a relevant mammalian cell type, we used the mouse RAW264.7 macrophage cell line, which has been frequently employed for  studies ,32,33. Menophagy . Here, w2 atmosphere. The day before Mm infection, RAW 264.7 cells were seeded on sterile coverslips inserted in 12-well plates (2.5 × 105 cells/well for the 0–150 mpi Mm infection experiments or 4 × 105 cells/well for the Mm infection rate experiment) and incubated overnight at 37 °C with 5% CO2.The RAW 264.7 macrophage cell line was maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) with high glucose , with 10% fetal calf serum (FCS)  at 37 °C in a 5% COMm M-strain, fluorescently labelled with mWasabi [Mm was washed twice with PBS, and optical density (OD) was measured at 600 nm. An OD 600 nm value of 1 is equal to 108Mm/mL [Mm, while DRAM1 knockdown and scramble control cell lines were used for the functional analysis of DRAM1. Cells were then infected with a multiplicity of infection (MOI) of 10, immediately centrifuged at 500× g for 10 min to synchronise infection, as described in [2. After 30 min, cells were washed twice with PBS to remove extracellular Mm and then incubated with LysoTracker-enriched medium for up to 150 min at 32 °C to obtain samples for immunofluorescence at 30 min intervals. At each 30 min interval, cells were fixed with Pierce™ 4% formaldehyde (PFA)  for 20 min at room temperature (RT). To analyse infection rates, cells were infected at a MOI of 10 without centrifugation and incubated at 32 °C. After 1 h, cells were either fixed with PFA or washed with PBS twice, in order to remove extracellular bacteria, and were then incubated with fresh medium for 6 h.The day before cell infection,  mWasabi , was cul08Mm/mL . Wild-tyribed in , and incMm  in PBS for 15 min, before blocking with 1% bovine serum albumin (BSA)  in PBS for 1 h at RT. Next, cells were incubated with the appropriate primary antibody, specifically DRAM1 (1:500) , LC3 (1:500) , or LAMP1 (1:1000)  for 1 h at RT. All primary antibodies were tested for a lack of cross-reaction with in vitro-cultured Mm . For sinAcidic vesicles were visualised by incubating RAW 264.7 cells in medium that was enriched with LysoTracker™ Red DND-99 (1:2500)  for at least 1 h prior to fixation at 37 °C . For non6 RAW 264.7 cells were seeded per T25 flask in DMEM-high glucose with 10% FCS and incubated overnight at 37 °C with 5% CO2. Then, 24 h later, cells were transduced at an MOI of 4 or 8, supplemented with 8 µg/mL polybrene , and incubated at 37 °C with 5% CO2. The medium was refreshed 24 h after transduction. After 48 h of transduction, 3 µg/mL puromycin  was added. Every 2–3 days, the medium was replaced until cells were 80–90% confluent. To isolate single clones with successful DRAM1 knockdown, cells from each condition were diluted in DMEM-high glucose with 10% FCS and 3 µg/mL puromycin to achieve 1 cell/well in a 96 well plate. The following day, wells containing 1 cell were marked. Once marked wells reached 80–90% confluency, cells were transferred to 12-well plates and subsequently to T25 flasks. Antibiotic pressure was removed from the medium to confirm the stability of DRAM1 shRNA integration into cells.To knockdown DRAM1, three short hairpin RNAs (shRNAs) against DRAM1 (NM_027878) from the Mission library (Sigma-Aldrich) were used, with scrambled shRNA as a control. pLKO.1-puro shRNA constructs were transduced into RAW 264.7 cells. The day before transduction, 1 × 106 cells were dissolved in TRIzol  and then chloroform . After centrifuging at 12,000× g for 15 min at 4 °C, the aqueous phase containing the RNA was transferred to a new tube. One volume of 70% ethanol was added and then transferred to a column from the RNeasy mini kit . RNA isolation was performed according to the manufacturer’s protocol, which included on-column DNAse digestion. A total of 500 ng of each RNA sample, measured with the Nanodrop (Thermofisher), was converted to cDNA with the C1000 Touch Thermal Cycler, using the iScript cDNA Synthesis Kit according to the manufacturer’s protocol , with the following modifications: 1 µL iScript Reverse Transcriptase was modified to 0.5 µL. Samples were then subjected to quantitative PCR (qPCR) using the CFX96 Real-Time System  with the iTaq™ Universal SYBR® Green Supermix . The program was set up as follows: initial denaturation at 95 °C for 3 min, then 40 cycles of 95 °C for 15 s (denaturation), and 60 °C for 30 s . A melting curve was included to test for PCR product purity: 55–95 °C in 0.5 °C increments. Dram1 primers (Sigma-Aldrich) used are as follows: forward: 5′-CCAGCTTCTTGGTCCGACG-3′; reverse: ‘5-GGGAGAAAGGGGTTGACGTG-3’. Gapdh was used as the housekeeping gene: forward: 5′- ATGGTGAAGGTCGGTGTGAA-3′; reverse: ‘5-CTGGAACATGTAGACCATGT-3’.Cell pellets containing 1 × 10g/min. Western blotting was performed using 15% polyacrylamide gel for LC3 and 10% for polyacrylamide gels for DRAM1 and LAMP1, followed by protein transfer to commercial PVDF membranes . Membranes were blocked with 3% BSA in 1× Tris-buffered saline (TBS) solution, with 0.1% Tween-20 (TBST), before incubating with primary and secondary antibodies: polyclonal anti-rabbit DRAM1 (1:1000) , GAPDH (1:1000)(D16H11), LC3 (1:1000) , and LAMP1 (1:1000) , as well as anti-rabbit IgG, and HRP-linked antibody (1:1000) . Digital images were acquired using the Bio-Rad Universal Hood II imaging system (720BR/01565 UAS). Band intensities were quantified with ImageJ, and values were normalised to GAPDH as a loading control.Cells were harvested, lysed in RIPA buffer  containing a protein inhibitor cocktail , and centrifuged at 4 °C for 10 min at 12,000× Mm colocalisation involved the characterisation into four categories , based on visual stack-by-stack inspection of the confocal Z-stack images. ‘None’: no colocalisation between DRAM1 and Mm. ‘Membrane’: DRAM1 staining surrounds Mm in a ring. ‘Punctate’: DRAM1 colocalises with Mm, but not along Mm’s entire length. ‘Luminal’: DRAM1 completely colocalises with Mm inside the lumen of a Mm-containing vesicle. The same definitions were used to quantify LC3–Mm and LAMP1–Mm colocalisations, where we note that the membrane pattern was not observed for LC3–Mm. Mm clusters fully colocalising with LysoTracker were also quantified. For quantification we imaged different regions of interest (ROIs) of the cells that were grown on coverslips in well plates, as described by Green et al. [RAW 264.7 cells stained by immunofluorescence and/or LysoTracker dye were imaged with a TCS SP8 confocal microscope (Leica) using a 63× oil immersion objective (NA: 1.40). Image processing and the analysis of fluorescent intensity and particles were performed using ImageJ software. Quantification of DRAM1–n et al. . ROIs weMm-infected cells were serially diluted with 5 steps in 7H9 broth, and 10 µL droplets were spotted onto Middlebrook 7H10 agar plates. The plates were incubated at 32 °C until single colonies were clearly seen, and bacterial colonies were quantified.At 7 h post infection, cells were lysed in water containing 0.05% SDS for 10 min. Lysates of At 7 h post infection, SYTOX™ Green Nucleic Acid Stain  was used at a final concentration of 5 μM for 10 min, in order to quantify cell death in different cell lines. Cells were then washed 3 × 5 min before fixation.In colocalisation studies, the mean probability of colocalisation per experimental condition was estimated, using a generalised linear regression model from the glmmTMB package in R . A beta-Mm infection in RAW264.7 macrophages, we performed immunofluorescence staining. We first determined the localisation pattern of DRAM1 in non-infected conditions. We observed that DRAM1 concentrates at certain areas in the vicinity of the plasma membrane. In these highly concentrated regions, DRAM1-positive circular structures of various sizes were occasionally observed  was always LysoTracker-negative  counts from DRAM1 knockdown cells were higher than in the control group phagolysosomal pathway [Mm first resides in a DRAM1-negative non-acidic phagosome, possibly recruiting LC3 through LAP. Subsequently DRAM1 may be recruited to the phagosomal membrane. Lysosomes containing DRAM1 within the membrane may then fuse with the Mm-containing vesicle, leading to its acidification. Alternatively, Mm escapes from the phagosome into the cytoplasm and is subsequently trapped in an autophagosome. This scenario is supported by the observation that Mm in RAW264.7 macrophages is ubiquitinated, making it a substrate for ubiquitin receptor-mediated xenophagy. The continued recruitment of DRAM1 to Mm-containing vesicles, of either a phagosomal or autophagosomal nature, would result in an increase in partial DRAM1–mycobacteria colocalisation. Subsequently, we hypothesise that DRAM1 becomes luminal following the fusion of phagosomes or autophagosomes with lysosomes, resulting in full DRAM1–mycobacteria colocalisation in the acidic and degradative environment of autolysosomes and endolysosomes. Another potential explanation for the full colocalisation pattern is that it is derived from the invagination of the membrane enclosing Mm, resulting in the formation of multi-vesicular bodies (MVBs) that contain intraluminal vesicles with DRAM1 in the membrane. This would imply that lysosomes do not fuse until after complete DRAM1–mycobacteria colocalisation, as MVBs are formed at a late stage of vesicle maturation, before lysosome fusion [In the present work we focused on the role of DRAM1 in intracellular host defence. During  pathway ,30. Basee fusion .Mm-containing vesicles, suggesting that DRAM1 mediates autophagic targeting and acidic vesicle trafficking to Mm. This reflects results seen in zebrafish, where DRAM1 knockdown or mutations resulted in reduced GFP–LC3-Mm colocalisation and reduced LysoTracker–Mm colocalisation [Mm-containing compartments showing partial or no colocalisation with the remaining DRAM1 protein in the knockdown lines. In contrast, despite DRAM1 knockdown, Mm-containing vesicles that showed full colocalisation with DRAM1 remained acidic. This suggests that DRAM1 affects the trafficking of acidic vesicles to Mm-containing vesicles, primarily at the early stages of vesicle maturation. In agreement, the effect of DRAM1 knockdown on LysoTracker–Mm colocalisation was larger than the effect on LAMP1–Mm colocalisation, suggesting that DRAM1 knockdown affects not only the fusion of lysosomes with Mm-containing vesicles, but also the acidification of these vesicles, due to fusion with late endosomes at an earlier step in the maturation process. The proposed role of DRAM1 in promoting fusion between acidic vesicles and Mm-containing compartments is supported by results observed in zebrafish overexpressing DRAM1, where large vesicles containing membrane remnants were observed in transmission electron micrographs, suggesting that DRAM1 mediates multiple vesicle fusion events [Mm infection burden in RAW 264.7 macrophages.Previously, DRAM1 was shown to regulate autophagic flux through the lysosomal v-ATPase in human A549 cells, under conditions of mitochondrial stress . To detelisation ,30. Exten events . The stiMm in RAW 264.7 macrophages, support the role of DRAM1 as a host resistance factor that is conserved across zebrafish and mammals. Therefore, DRAM1 represents a putative target for host-directed anti-mycobacterial therapy that could help overcome current challenges posed by anti-microbial resistance. Therapeutic strategies targeting DRAM1 might be more broadly applicable against other intracellular pathogens, considering our recent results that DRAM1 also protects against infections with Salmonella enterica serovar Typhimurium and Aspergillus fumigatus [The results of this study, which show the requirement of DRAM1 for (auto)phagolysosomal delivery of umigatus ,48. Furt


In recent years, plastic and especially microplastic in the oceans have caused huge problems to marine flora and fauna. Recently, such particles have also been detected in blood, breast milk, and placenta, underlining their ability to enter the human body, presumably via the food chain and other yet-unknown mechanisms. In addition, plastic contains plasticizers, antioxidants, or lubricants, whose impact on human health is also under investigation. At the cellular level, the most important enzymes involved in the metabolism of xenobiotic compounds are the cytochrome P450 monooxygenases (CYPs). Despite their extensive characterization in the maintenance of cellular balance, their interactions with plastic and related products are unexplored. In this study, the possible interactions between several plastic-related compounds and one of the most important cytochromes, CYP2C19, were analyzed. By applying virtual compound screening and molecular docking to more than 1000 commercially available plastic-related compounds, we identified candidates that are likely to interact with this protein. A growth inhibition assay confirmed their cytotoxic activity on a CYP2C19-transfected hepatic cell line. Subsequently, we studied the effect of the selected compounds on the transcriptome-wide gene expression level by conducting RNA sequencing. Three candidate molecules were identified, i.e., 2,2′-methylene bis(6-tert-butyl-4-methylphenol), 1,1-bis ethane, and 2,2′-methylene bis(6-cyclohexyl-4-methylphenol)), which bound with a high affinity to CYP2C19 in silico. They exerted a profound cytotoxicity in vitro and interacted with several metabolic pathways, of which the ‘cholesterol biosynthesis process’ was the most affected. In addition, other affected pathways involved mitosis, DNA replication, and inflammation, suggesting an increase in hepatotoxicity. These results indicate that plastic-related compounds could damage the liver by affecting several molecular pathways. Despite its great importance and utility for our society, plastic poses a huge threat for the oceans ,2,3,4, mMicroplastic has recently been detected in human blood  and placCYP genes, of which only some are involved in the metabolism of drugs and xenobiotics while others are implicated in the metabolism of sterols, vitamins, and other endogenous substrates, or their functions are not yet fully elucidated [In the years to come, the amount of plastic is expected to tremendously increase . Howeverucidated . These eucidated . Among tucidated , as wellucidated .Plastic, microplastic, nanoplastic, and their related compounds are increasingly threatening human health ,34. HoweUsing the PyRx 0.8 software, we screened more than 1000 compounds downloaded from PubChem associated with plastic. As shown in 1 to 6, 1 and 2 were plasticizers, compound 3 was a UV stabilizer, and compounds 4–6 were antioxidants.To investigate the binding site of the enzyme more specifically, we performed molecular docking using AutoDock 4.2. We included only compounds (1) with their lowest binding energy (LBE) values smaller than −8.0 kcal/mol, (2) that are commonly used in the plastic industry, and (3) that were commercially available. These criteria skimmed the choice to six compounds  between 27.73 and 1087 nM. The molecular docking of these compounds was visualized in the LBE conformation. The interactions between the six candidate compounds and the enzyme are shown in 50 values of these concentrations are displayed in 1 exhibited an IC50 value of 87.99 ± 7.77 μM for the CYP2C19-overexpressing cells. Compound 3 showed a lower IC50 value (67.47 ± 2.05 μM). Compounds 4, 5, and 6 showed rather comparable IC50 values . Compound 2 did not exhibit any cytotoxic effect.To investigate the effects of the in silico identified compounds on cell viability, we used CYP2C19-overexpressing HepG2 cells . Differe4–6 on the transcriptome-wide gene expression in CYP2C19-overexpressing HepG2 cells.We performed RNA sequencing to analyze the influence of compounds 4–6 compared to the control cells treated with DMSO. Functional enrichment analysis with clusterProfiler [Differentially expressed genes were determined in the cells treated with compounds Profiler  was execProfiler , a visua4 and 6 and ‘DNA-templated DNA replication’ by compound 4. Many mitosis-related pathways were also suppressed by compounds 4 and 6 but not by compound 5 . Compound 4 suppressed ‘metaphase/anaphase transition of mitotic cell cycle’, while compound 6 down-regulated ‘nuclear chromosome segregation’, ‘mitotic nuclear division’, and ‘chromosome segregation’. The exposure of cells to compounds 4 and 5 activated cell death mechanisms such as ‘autophagy’ and ‘process utilizing autophagic mechanism’. Interestingly, angiogenesis-related pathways were activated by compound 6: ‘vasculature development’, ‘blood vessel morphogenesis’, and ‘blood vessel development’. Regarding the metabolic and biosynthetic processes in the cells, ‘response to nutrients levels’ was activated by compound 6, while many other pathways were downregulated. This was found to be true with all three compounds for ‘cholesterol biosynthetic process’, ‘sterol biosynthetic process’, and ‘secondary alcohol biosynthetic process’. Compounds 4 and 5 suppressed ‘cholesterol metabolic process’ and ‘sterol metabolic process’. Furthermore, compound 5 suppressed the ‘steroid biosynthetic process’, ‘alcohol biosynthetic pathway’, ‘alcohol metabolic process’, and ‘organic hydroxy compound metabolic process’.The pathway enrichment analyses revealed numerous pathways that may help to better explain the detrimental effects of plastic-related compounds in CYP2C19-overexpressing HepG2 cells. This was observed for the pathways related to cell division, e.g., ‘DNA replication’ was suppressed by compounds 4 and 5. No genes were in common between compounds 4 and 6 and compounds 5 and 6. Finally, two genes for compound 4, three genes for compound 5 and one gene for compound 6 were influenced by only one compound. Then, we created another heat map visualizing the log2-fold change in the genes affected by all three compounds (We highlighted certain pathways in common among the three compounds selected using signature heat maps generated by the GeneTonic software (version 2.4.0). ompounds C. Many o4 and 6 on the ‘DNA replication’ pathway are highlighted. The Venn diagram shows seventy-six genes in common between the two selected compounds  in 4, the values ranged from −8.94 kcal/mol (by hydroxylation of C.13) to −10.61 kcal/mol ). The LBE value (modifications of C.25) for compound 5 was −9.62 kcal/mol, while the LBE values for the modifications at C.2 and C.10 were −10.96 kcal/mol. For the metabolites of compound 6, the LBE was −10.48 kcal/mol (hydroxylation of C.11), while −11.00 kcal/mol was the lowest LBE (hydroxylation of C.10 and C.11).As a final step, we considered whether the metabolization of these compounds by CYP2C19 might influence their binding to this enzyme. As the metabolites of these compounds had not yet been determined, we used the SmartCYP software (version 2.3) ,38 to th4 and 6 revealed an increase in the G0/G1 population at the IC50 concentration  compared to the untreated cells (66.05 ± 1.66%). In addition, the analysis showed a decrease in the percentage of S-phase cells of the treated compounds (8.05 ± 1.92% for compound 4 and 6.76 ± 3.28 for compound 6) in contrast to the control (12.83 ± 1.54%). No significant percentage change was detected in the sub-G0 and G2/M phases.The results of the flow cytometric cell cycle analysis are shown in Despite the fact that the release of plastic-related compounds from microplastic has been previously studied ,40 and t4–6 for subsequent analyses, as they bound to this protein with good affinities and were cytotoxic at low IC50 values (<21 µM). Considering these results, we studied the possible consequences of their interactions with CYP2C19-overexpressing HepG2 cells at the gene level using RNA sequencing. Previous studies have shown that this approach is feasible, since the gene expression signatures were modulated upon exposure to environmental pollutants [Here, we focused on one of the most important CYPs, CYP2C19. As a first step, a library of plastic-related compounds was used. By molecular docking in silico and a growth inhibition assay in vitro, we selected three candidates. We selected compounds llutants ,44 as wellutants ,45. HoweTherefore, we investigated transcriptomic alterations in CYP2C19-overexpressing cells treated with the three selected plastic-related compounds. These compounds had some commonly targeted pathways: the ‘cholesterol biosynthetic process’, the ‘secondary alcohol biosynthetic process’ and the ‘sterol biosynthetic process’. However, the pathway found to be the most strongly downregulated for all the three compounds was the ‘cholesterol biosynthesis process’. By analyzing the cholesterol biosynthetic pathway in detail using heat maps of all three compounds and the Venn diagram, it was seen that all three compounds targeted both the same pathway as well as the same genes within it. These included genes that play a critical role in cellular cholesterol homeostasis  and whosMicroplastics and plastic-related compounds can cross the gastrointestinal epithelial barrier  and enteCholesterol, which is mainly produced in the liver , is a fu6 or ‘cytokine production’ and ‘regulation of cytokine production’ for compounds 4 and 5. Inflammatory processes caused by plastics, microplastics [Furthermore, inflammation-related pathways were activated such as ‘inflammatory response’ for compound plastics ,70, and plastics  have beeplastics  such as plastics .4 and 6 both affected different pathways involved in mitosis (such as ‘mitotic sister chromatid segregation’ or ‘sister chromatid segregation’) and the ‘DNA replication’ pathway. Previously, it has been shown that plastic-related compounds [4 and 6 accumulated the cells in the G0/G1 phase of the cell cycle. This behavior was also described for other compounds related to plastics (such as bisphenol A) [4 and 6 also arrested the cells in G0/G1 phase [4 and 5, the activation of pathways involved in autophagy was identified (such as ‘autophagy’ and ‘process utilizing autophagic mechanism’). This is an interesting result as compound 4 has already shown a specific influence on this exact pathway in a previous study [Our RNA-sequencing analyses additionally showed that compounds ompounds ,74,75 anompounds ,77,78 prhenol A) . Other cG1 phase . Under hG1 phase . HoweverG1 phase ,83. Regaus study .In conclusion, there is no doubt that we are frequently faced with the exposure to plastics, microplastics, and related-compounds . Not onl2: diisobutyl phthalate , compound 3: octrizole , compound 4: 2,2′-methylene bis(6-tert-butyl-4-methylphenol) , compound 6: 2,2′-methylene bis(6-cyclohexyl-4-methylphenol) . Compounds were acquired from TCI Deutschland GmbH . Compound 5, 1,1-bisethane , was purchased from abcr GmbH .Compound 1: dicyclohexyl phthalate , compound 2 in a humidified incubator. DMEM media were supplemented with 10% fetal bovine serum (FBS) and with 3 μg/mL blasticidin  to preserve the selection of transfected CYP2C19-overexpressing HepG2 cells.CYP2C19-overexpressing HepG2 cells were originated as previously described . Cells whttps://pyrx.sourceforge.io) (accessed 16 April 2020). The three-dimensional ligand structures were downloaded from PubChem  [http://www.rcsb.org/) [More than 1000 compounds associated with plastic production were screened for their interactions with CYP2C19 with the virtual screening tool PyRx   as standsb.org/)  as a PDBsb.org/) .www.ahrp.info) and the Gauss Alliance e.V. (accessed 25 January 2021).Molecular docking was run for the compounds with the lowest PyRx binding energies. The binding affinities of the top 70 compounds were read using AutoDock 4.2. The grid box was positioned around the drug binding sites of CYP2C19 with the center of the grid box at x = −99.076, y = −26.072, and z = −63.517 and with the number of grid points (npts) being 100 in x, 104 in y, and 100 in z. Molecular docking was performed with the Lamarckian genetic algorithm with 250 runs and 25 Mio evaluations. The Discovery Studio Visualizer software was used for visualizing protein–ligand interactions. Parts of this analysis were performed using the supercomputer Mogon II and the advisory services offered by Johannes Gutenberg University Mainz (hpc.uni-mainz.de), which is a member of the AHRP  was added. The fluorescence intensity was measured with an Infinite M200 Pro-plate reader . Dose–response curves were engendered for three independent experiments for each compound, and 50% inhibition concentrations (IC50) were calculated. The analysis was represented using the Prism 6 GraphPad Software (version 9.5.1) .Aliquots of 105 CYP2C19-overexpressing HepG2 cells were seeded into 6-well-plates. Cells were treated with the compounds of interest, resulting in a final concentration of IC50 calculated beforehand. Control cells were treated with 0.2% DMSO. After 24 h of incubation, the cells were harvested. RNA extraction was performed with an InviTrap® Spin Cell RNA Mini Kit  according to the manufacturer’s instructions. The cell pellet was lysed with 350 μL of Lysis Solution and treated with β-mercaptoethanol. After DNA removal, 350 μL of 70% ethanol was added, and the sample was applied onto the RNA-RTA spin filter. After several washing steps, RNA was eluted with 60 μL of Elution Buffer R, and the concentration and purity were measured with NanoDrop.A total of 24 h before treatment, 5 × 10© Ultra™ II Directional RNA Library Prep Kit , RNA sequencing of around 25 Mio PE reads  was performed with an Illumina NextSeq 2000™ system. Each treatment and control group included two replicates.RNA sequencing was performed by StarSEQ GmbH, Mainz, Germany. The quality of the extracted RNA was verified by the company with a 2100 Bioanalyzer system . After mRNA isolation and library preparation using a NEBNexthttps://www.bioinformatics.babraham.ac.uk/projects/fastqc/) (accessed 15 February 2023) was used for quality control on the sequencing data. Transcript abundance estimates were computed with Salmon (version 1.5.0) [p-value < 0.05 were chosen [The FastQC tool   with a tn 1.5.0) . Exploran 1.5.0) . Exploran 1.5.0) . Differen 1.5.0) , settingn 1.5.0) . Furthern 1.5.0)  using aln 1.5.0)  and by cn 1.5.0)  with defe chosen  for furte chosen . Gene exWe used SmartCYP ,38 to pr50 concentrations of compounds 4 and 6 for 24 h. Then, the cells were fixed using 80% cold ethanol and stored overnight at −20 °C. Successively, the cells were washed twice with PBS and resuspended in 1 mL of cold PBS containing 1mg/mL RNaseA  and 50 μg/mL PI . After 15 min of incubation, the measurements were performed using a BD LSRFortessa™ Cell Analyzer . DMSO-treated cells were used as a negative control.CYP2C19-overexpressing HepG2 cells were treated with IC4–6), a cytotoxic effect could be observed as well as a combined regulation of different gene pathways. Specifically, all three compounds caused significant under-expression in the pathway related to cholesterol biosynthesis. Furthermore, these compounds acted on several pathways related to cell replication (such as mitosis) and inflammation, causing damage to liver cells at different levels. Currently, research regarding the possible toxicity of plastic for humans, specifically concerning the compounds used to produce it, remains very limited. To fully understand their impact on human health, future in vivo research on plastic and related compounds as well as further investigations on related topics are essential. Furthermore, to mitigate the immense problem of plastic pollution, it would be useful not only to conduct scientific research but also to start effectively communicating its findings to the entire population. To this day, plastic pollution is often considered an environmental problem that exclusively concerns the oceans and marine animals; however, day by day, it is increasingly jeopardizing human health as well.We analyzed the interactions of several plastic-related compounds with cytochrome 2C19. Summarizing the results of the three selected compounds (compounds


Breast cancer is the second leading cause of cancer deaths in women worldwide; therefore, there is an increased need for the discovery, development, optimization, and quantification of diagnostic biomarkers that can improve the disease diagnosis, prognosis, and therapeutic outcome. Circulating cell-free nucleic acids biomarkers such as microRNAs (miRNAs) and breast cancer susceptibility gene 1 (BRCA1) allow the characterization of the genetic features and screening breast cancer patients. Electrochemical biosensors offer excellent platforms for the detection of breast cancer biomarkers due to their high sensitivity and selectivity, low cost, use of small analyte volumes, and easy miniaturization. In this context, this article provides an exhaustive review concerning the electrochemical methods of characterization and quantification of different miRNAs and BRCA1 breast cancer biomarkers using electrochemical DNA biosensors based on the detection of hybridization events between a DNA or peptide nucleic acid probe and the target nucleic acid sequence. The fabrication approaches, the biosensors architectures, the signal amplification strategies, the detection techniques, and the key performance parameters, such as the linearity range and the limit of detection, were discussed. Breast cancer remains one of the main causes of mortality in women; its occurrence increases with age, especially after the age of 65 ,2. The iNoncoding RNAs (ncRNAs) are a family of functional RNA molecules without the protein-coding feature that represents an important part of the human transcriptome, e.g., ribosomal RNAs, transfer RNAs, circular RNAs, long ncRNAs, and short ncRNAs. Among short ncRNAs, microRNAs (miRNAs) are a highly conserved family of transcripts with lengths of approximately 20–25 nucleotides, which primarily regulate gene expression by promoting messenger RNA degradation or repressing its translation . miRNAs Breast cancer susceptibility genes 1 and 2 (BRCA1 and BRCA2) are antioncogenes expressed in the breast and ovarian cells of women genetically predisposed to breast and ovarian cancer, where they play an important role in the repair of the chromosomal damage. Mutations in the BRCA1 lead to malfunctions and increase the risk of breast cancer. Therefore, the detection of BRCA1 biomarker allows for the characterization of the genetic features and the screening of breast or ovarian cancer patients.The usual methods for the analysis of nucleic acid breast cancer biomarkers are based on traditional molecular biology techniques, such as cloning, enzymatic ligation assays, Northern blot analysis, oligonucleotide microarray methods, quantitative real-time polymerase chain reaction (qRT-PCR), in situ hybridization, and deep sequencing ,19, but Electrochemical methods, including cyclic voltammetry (CV), differential pulse voltammetry (DPV), square wave voltammetry (SWV), amperometry (A), and electrochemical impedance spectroscopy (EIS) are highly sensitive and selective in detecting different analytes ,23,24,25According to IUPAC, an electrochemical DNA biosensor is a device consisting of an electrochemical transducer (the electrode) with a biological recognition element (the DNA film) immobilized on its surface . Its appThis review presents the recent advances on the electroanalytical methods of detection and quantification of miRNAs and BRCA1 breast cancer biomarkers using electrochemical DNA biosensors, consisting on the detection of hybridization events between a DNA or PNA (peptide nucleic acid) capture probe and the target nucleic acid sequence. The biosensors fabrication strategies, based on functional nanomaterials, oligonucleotides, and enzymes, the detection techniques, and the key performance parameters, such as the linearity range and limit of detection (LOD), are discussed.In recent years, various approaches for the development of electrochemical biosensors for miRNA analysis have been reported ,41,42, aThe biosensor electrochemical performance is closely related with its interfacial structure, which profoundly affects both the thermodynamics and kinetics of the DNA-miRNA binding and the signal transduction of the biomolecules. Therefore, to enhance the electron transfer, to improve the biosensor capture efficiency, and to amplify the electrochemical signal, various integrated nanostructured materials have been employed ,32, as s6]3−/[Fe(II)(CN)6]4− 4− , (iii) r(CN)6]4− , or (iv)(CN)6]4− ,60,61. WmiR-21 and miR-155 have been demonstrated to play a major role in breast cancer progression, which makes them especially important in breast cancer detection .−8 M to 5.0 × 10−5 M and an LOD of 5.0 × 10−9 M.An electrochemical biosensing platform for miR-21 based on the Fc-tagged DNA hydrogel self-assembled onto indium tin oxide (ITO)/polyethylene terephthalate (PET) electrode was proposed , which s−14 to 2.0 × 10−12 M and an LOD of 5.0 × 10−15 M.In a different report, a disposable biosensor for the voltammetric determination of miR-21 was reported , consist−15 to 7.0 × 10−7 M and an LOD of 1.0 × 10−15 M. In addition, based on Mb labeling, an amplification-free electrochemical biosensor was developed using DNA origami nanostructured probes self-assembled onto chitosan-modified gold electrodes  and [Ru(NH3)6]Cl3 redox probes, an electrochemical biosensor which enabled three detection modalities based on miR-21 hybridization, p19 protein binding, and protein displacement was also reported 4−/Fe(III)(CN)6]3−, redox couple, miR-21 was specifically detected at an electrochemical biosensor consisting of ssDNA capture probes immobilized onto a GCE modified by sulfonic acid deposition with subsequent chlorination 4−/Fe(III)(CN)6]3− redox couple, an electrochemical biosensor for miRNA-21 detection was developed , immobilized on the surface of a PGE [−7 to 4.3 × 10−7 M and an LOD of 8.4 × 10−8 M.A reduced miR-34a expression is strongly associated with tumor progression and indicates a worse prognosis . miR-34aof a PGE . The pro−8 to 2.0 × 10−7 M [−9 to 4.0 × 10−8 M and an LOD of 2.0 × 10−9 M [A high expression of miR-522 is correlated with poor overall survival in patients with triple-negative breast cancer . In a di× 10−7 M . Further× 10−9 M .6]4−/Fe(III)(CN)6]3− redox couple and the immobilization of SH-ssDNA capture probes onto a gold electrode modified by Au NPs and rGO, as shown in −11 to 1.0  × 10−5 M and an LOD of 1.7  × 10−12 M )-Ag NPs) and functionalized mesoporous silica  . To furt−18 to 5.0 × 10−15 M and an LOD of 1.2 × 10−18 M.In a different report, a double signal amplification strategy for the detection of miRNA of BRCA1 was developed based on a cyclic enzymatic signal amplification (CESA) with a double-strand specific nuclease (DSN) and triple-CdTe QD-labeled DNA nanocomposites (3-QD@DNA NC) as a cascade signal probe . CESA wa−15 to 1.0 × 10−9 M and an LOD of 1.0 × 10−16 M [An electrochemical biosensor based on thiol-modified tetrahedral-DNA (SH-TDNA) probes and poly-adenine (polyA)-mediated Au NPs with immobilized ssDNA showed, after hybridization with the target BRCA1, a linear range from 1.0 × 10 10−16 M .−16 to 1.0 × 10−10 mol L−1 and an LOD of 3.7 × 10−17 mol L−1; (ii) for the GCE/MWCNTs/PANHS/ssDNA configuration, a linear range from 1.0 × 10−17 to 1.0 × 10−10 mol L−1 and an LOD of 3.1 × 10−18 mol L−1; and (iii) for the GCE/rGO/MWCNTs/PANHS/ssDNA configuration, a linear range from 1.0 × 10−18 to 1.0 × 10−10 mol L−1 and an LOD of 3.5 × 10−19 mol L−1.Several reports showed the development of label-free impedimetric biosensors for BRCA1. A label-free genosensor was developed by immobilizing the ssDNA probes at the surface of the GCE modified by 1-pyrenebutyric acid-N-hydroxysuccinimide ester (PANHS), MWCNTs, and rGO . The bio−14 M to 1.0 × 10−9 M and an LOD of 1.7 × 10−15 M.Also at a GCE, complementary BRCA1 ssDNA sequences were immobilized after the modification of the electrode surface with a cross-linked polyethylene glycol (PEG) film containing amine groups, followed by the self-assembly of Au NPs . The imp−17 to 2.5 × 10− 14 M and an LOD of 5.0 × 10− 17 M [An impedimetric electrochemical biosensor based on chitosan-co-PANI (CHIT-co-PANI) copolymer coated onto indium-tin-oxide (ITO) was used for the hybridization of immobilized complementary ssDNA probes with the BRCA1 target, as shown in 10− 17 M .−19 to 1.0 × 10−7 M and an LOD of 4.6 × 10−20 M.In another report, a label-free EIS method for BRCA1 detection, based on the immobilization of SH-ssDNA onto a Au electrode and its further hybridization with a complementary BRCA1 target, was developed . The gen−16 to 1.0 × 10−11 M and an LOD of 5.0 × 10−17 M.An impedimetric biosensor platform was also developed by immobilizing the SH-ssDNA probes onto a GCE modified by PEG grafted on a Fe(III)-tannic acid (TA)/poly-dopamine (pDA) coating via a layer-by-layer technique . In the Breast cancer is associated with a high incidence rate, recurrence, and metastasis; therefore, it remains the second leading cause of cancer mortality in woman. Conventional strategies of breast cancer biomarkers’ detection are expensive, laborious, time-consuming, and require highly trained biologists.This review presents a comprehensive overview concerning the design, development, and applications of electroanalytical methods of detection of nucleic acid breast cancer biomarkers, particularly miRNAs and BRCA1. The electrochemical biosensors fabrication approaches, the signal amplification approaches, the detection techniques, and the key performance parameters, such as the linearity range and limit of detection, were presented.DNA electrochemical biosensors are considered extremely advantageous devices for evaluating the levels of nucleic acid breast cancer biomarkers due to their high sensitivity and selectivity, cost-effectiveness, short procedure time, simplicity, ability to be miniaturized, and high potential applicability in urine, blood, and tissue samples.The challenge in the development of electrochemical biosensors for nucleic acid breast cancer biomarkers is to achieve the electrochemical transduction associated with the hybridization events that do not actually involve charge transfer reactions. Nanostructured materials, such as carbon nanotubes, graphene, graphene oxide, reduced graphene oxide, graphene quantum dots, metal nanoparticles and nanostructures, magnetic beads, polymers, hydrogels, dendrimers, and nanocomposites, are playing an increasingly critical role in their fabrication and are expected to be used more in the future. Generally, they represent the most attractive approach because they not only allow the amplification of the electrochemical signal and the improvement of the biosensor sensitivity, but they can also act as signal tags or signal reporters and can form highly conductive nanostructured electrochemical platforms.Nevertheless, further studies are needed to enhance the biosensors’ stability and reproducibility, especially in biological matrixes, avoiding misleading results due to DNA and/or RNA sequences with similar structures and interferents. Moreover, challenges in the miniaturization of the biosensor devices and the integration of microfluidics technologies still need to be solved in order to develop commercially available point-of-care devices.


Cyclophosphamide (CP) is a chemotherapeutic agent that causes pulmonary damage by generating free radicals and pro-inflammatory cytokines. Pulmonary damage has a high mortality rate due to the severe inflammation and edema occurred in lung. PPARγ/Sirt 1 signaling has been shown to be cytoprotective effect against cellular inflammatory stress and oxidative injury. Protocatechuic acid (PCA) is a potent Sirt1 activator and exhibits antioxidant as well as anti-inflammatory properties. The current study aims to investigate the therapeutic impacts of PCA against CP-induced pulmonary damage in rats. Rats were assigned randomly into 4 experimental groups. The control group was injected with a single i.p injection of saline. CP group was injected with a single i.p injection of CP (200 mg/kg). PCA groups were administered orally with PCA  once daily for 10 consecutive days after CP injection. PCA treatment resulted in a significant decrease in the protein levels of MDA, a marker of lipid peroxidation, NO and MPO along with a significant increase in GSH and catalase protein levels. Moreover, PCA downregulated anti-inflammatory markers as IL-17, NF-κB, IKBKB, COX-2, TNF-α, and PKC and upregulated cytoprotective defenses as PPARγ, and SIRT1. In addition, PCA administration ameliorated FoxO-1 elevation, increased Nrf2 gene expression, and reduced air alveoli emphysema, bronchiolar epithelium hyperplasia and inflammatory cell infiltration induced by CP. PCA might represent a promising adjuvant to prevent pulmonary damage in patients receiving CP due to its antioxidant and anti-inflammatory effects with cytoprotective defenses. Pulmonary damage can result from sepsis, trauma, microbial infection, ischemia/reperfusion, or some drugs that induce acute respiratory failure with a mortality rate of approximately 40% , a synthetic alkylating agent, treats autoimmune diseases and cancer, as well as prevents organ transplantation rejection , is a polyphenolic compound, present in many vegetables and nuts  were provided by the Animal House of the National Research Centre . The rats were group-housed with free access to standard laboratory rodent chow and water under temperature−light-controlled conditions (24 ± 2 °C under a 12 h light/dark cycle). The animal experiments were performed in according to recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH No. 85:23 revised 1985) in accordance with the guidelines of the Institutional Animal Ethics Committee  of the NRC, Cairo, Egypt.Cyclophosphamide and protocatechuic acid were purchased from . Glutathione (GSH), malondialdehyde (MDA), nitric oxide (NO), and catalase colorimetric kits were determined using Biodiagnostic kit, Cairo, Egypt. Sirtuin 1 (SIRT1) was determined using ELISA kits procured from . Tumor necrosis factor alpha (TNF-α), Interleukin-17 (IL-17), nuclear factor kappa B (NF-κB), inhibitor of nuclear factor kappa beta kinase subunit beta (IKBKB), proxisome proliferator-activated receptor gamma (PPARγ), cyclooxegnase-2 (COX-2), protein kinase C (PKC), and myeloperoxidase (MPO) were determined using ELISA kits procured from Sunlong Biotech Co., Ltd, China.n = 8) as follows: Control group: Rats were injected with a single i.p injection of normal saline and received normal saline orally for 10 consecutive days. CP group: Rats were injected with a single i.p injection (200 mg/kg) (pH 7.4) as 20% (w/v) for the biochemical measurements of GSH, MDA, NO, and catalase were determined using Biodiagnostic colorimetric kits. Moreover, SIRT1, IL-17, NF-κB, IKBKB, PPARγ, COX-2, TNF-α, MPO, and PKC were determined using ELISA kits. The other lung was kept for histopathological assessment.GSH lung content was estimated according to the method that depends on the fact that protein and nonprotein SH-groups (mainly GSH) react with Ellman’s reagent  to form a stable yellow color of 5-mercapto-2-nitrobenzoic acid, which can be measured colorimetrically at 412 nm. In order to determine the GSH level in tissue, precipitation of protein SH-groups before the addition of Ellman’s reagent is necessary using precipitating solution   as MDA and catalase activity according to the methods described by Ohkawa et al and Salama and Elgohary , Ohkawa, respectLung contents of SIRT1, IL-17, NF-κB, IKBKB, PPARγ, COX-2, TNF-α, MPO, and PKC were determined using ELISA kits. Standards and samples were pipetted into wells with immobilized antibodies specific for rat SIRT1, IL-17, NF-κB, IKBKB, PPARγ, COX-2, TNF-α, MPO, and PKC and then were incubated for 30 min at 37 °C. After incubation and washing, horseradish peroxidase-conjugated streptavidin was pipetted into the wells, and incubated for 30 min at 37 °C, which was washed once again. Tetramethylbenzidine (TMB) substrate solution was added to the wells and incubated for 15 min at 37 °C; a color was developed proportionally to the amount of SIRT1, IL-17, NF-κB, IKBKB, PPARγ, COX-2, TNF-α, MPO, and PKC bound. Color development was discontinued (stop solution) and after 10-min color intensity was measured at 450 nm  with RNA sequences obtained from GenBank, using Beta Actin as a housekeeping gene  according to the manufacturers’ protocol. The extracted RNA was then quantified by spectrophotometry  at 260 nm. The obtained RNA is then used to assess the expression levels of Various groups' lung tissue was fixed in 10% formalin after being dissected. After being fixed for a day or two, the tissue was dehydrated progressively by stronger alcohol , cleared with xylene, impregnated in three changes of soft paraffin wax at 50 °C, and lastly embedded in paraffin wax to form solid blocks. A 7-μm thick transverse section was cut in a serial cross-section. Hematoxylin and eosin were used to stain paraffin slices mounted on glass slides with a layer of albumin glycerin. Light microscopy was used to qualitatively analyze the stained tissue slices  was used to carry out these statistical tests. The difference was considered significant when P value < 0.05). Meanwhile, in protocatechuic acid 50 and 100 mg/kg significantly increased PPARγ by 79 and 250% as well as reduced COX-2 levels by 24 and 39% respectively, as compared to the CP-treated group . Moreover, CP treatment significantly increased NF-κB and IKBKB lung protein levels by 105 and 70.3% respectively, in comparison with the control group. However, administration of protocatechuic acid 50 and 100 mg/kg significantly decreased NF-κB by 27 and 38%, along with IKBKB by 22 and 30% respectively, when compared with that of the CP group  . However, administration of protocatechuic acid 50 and 100 mg/kg significantly decreased IL-17 by 58 and 66%, respectively, when compared with that of the CP group . The treatment with 100 mg/kg of protocatechuic acid reduced the contents of IL-17 to their normal value, compared to the CP group . While, in protocatechuic acid 50 and 100 mg/kg caused a significant decline in the TNF-α level by 42 and 75% besides the diminution of PKC levels by 19 and 75%, respectively, as compared to CP-treated group . Treatment of protocatechuic acid 100 mg/kg reduced pulmonary levels of TNF-α to their normal value, compared to the CP group . Meanwhile, in protocatechuic acid 50 and 100 mg/kg SIRT1 protein levels were significantly increased the by 87 and 205% respectively, as compared to the CP-treated group   Fig. .Fig. 3EfP value < 0.05). The levels of MDA, NO and MPO were significantly higher in CP group by 98, 100 and 156%, respectively, than the control group (P < 0.05). Although protein levels of GSH and catalase were significantly higher in protocatechuic acid 50 and 100 mg/kg by 8, 12.3 and 371, 457% respectively, when compared with CP-treated group . Moreover, protocatechuic acid 50 and 100 mg/ kg significantly decreased the levels of MDA, NO and MPO by 20, 54 and 30%, 38, 29, 48%, respectively, when compared with CP-treated group   with a marked increase in foxo-1, when compared with the control group . Although protocatechuic acid 50 and 100 mg/kg administration resulted in a significant increase in Nrf-2 compared with cyclophosphamide group , moreover, the Foxo-1 gene was downregulated appreciably in a dose-dependent manner as compared to the CP group   are nuclear hormone and regulate the genes expression implicated in several biological processes -17 cells induced the production of pro-inflammatory IL-17  transcription factor that regulate the cellular antioxidative responses by promoting the expression of series of antioxidant genes (Tonelli et al. The current study exhibited that PCA has a vital role in cellular inflammatory, oxidative, and immune responses through modulating PPARs/IL-17/Nrf2 to mitigate CP-induced acute lung injury in the rat. In addition, PCA regulates the SIRT1/FoxO1 pathway that helps in the treatment of lung inflammatory diseases. Further investigations are required to prove PCA's ability to prevent pulmonary damage associated with CP in the clinical setting and using it in a treatment regime will be highly worthwhile.