Health risk assessment of atmospheric polycyclic aromatic hydrocarbons over the Central Himalayas

Carcinogenic risk assessments of polycyclic aromatic hydrocarbons (PAHs) in four sites from the Central Himalayas (Bode, Lumbini, Pokhara, and Dhunche) were performed. Lifetime Average Daily Dose (LADD), Lifetime lung cancer risk (LLCR) and Incremental lifetime cancer risk (ILCR) were calculated in order to evaluate the cancer risk. PAHs levels were converted to BaP equivalent concentrations (B[a]Peq), and models of health risk assessment were applied. B[a]Peq concentrations exceeded the standard limited value (1 ng/m³) in all the four sites. The human health risk assessment (HHRA) demonstrated high carcinogenic risk on residents of Bode and Lumbini. Further, LLCR in all sites were over the acceptable range (1.15E-03, 7.90E-04, 1.40E-04 and 9.96E-05, respectively); however, ILCR ranking exhibited acceptable range in Lumbini, Pokhara, and Dhunche (7.10E-06, 1.26E-06, and 8.95E-07). The risk variation among the sites is due to the difference in pollution status. The study shows health risk due to atmospheric PAHs via inhalation prevails all the seasons throughout, differing only seasonally; nevertheless, the concentration and carcinogenic risk decreased remarkably from south-north transect of the central Himalaya. Keeping some uncertainties aside, this study provides noble insights and helps to formulate new advance assessment on the carcinogenic risk of atmospheric PAHs over the Central Himalayas.     

Benzo[a]pyrene-7,8-quinone-3'-mononucleotide adduct standards for 32P postlabeling analyses: detection of benzo[a]pyrene-7,8-quinone-calf thymus DNA adducts

Benzo[a]pyrene-7,8-quinone (BPQ) is one of the reactive metabolites of the widely distributed archetypal polycyclic aromatic hydrocarbon, benzo[a]pyrene (B[a]P). The formation of BPQ from B[a]P through trans-7,8-dihydroxy-7,8-dihydroB[a]P by the mediation of aldo-keto reductases and its role in the genotoxicity and carcinogenesis of B[a]P currently are under extensive investigation. Toxicity pathways related to BPQ are believed to include both stable and unstable (depurinating) DNA adduct formation as well as reactive oxygen species. We previously reported the complete characterization of four novel stable BPQ-deoxyguanosine (dG) and two BPQ-deoxyadenosine (dA) adducts (Balu et al., Chem. Res. Toxicol. 17 (2004) 827-838). However, the identification of BPQ-DNA adducts by 32P postlabeling methods from in vitro and in vivo exposures required 3'-monophosphate derivatives of BPQ-dG, BPQ-dA, and BPQ-deoxycytidine (dC) as standards. Therefore, in the current study, BPQ adducts of dGMP(3'), dAMP(3'), and dCMP(3') were prepared. The syntheses of the BPQ-3'-mononucleotide standards were carried out in a manner similar to that reported previously for the nucleoside analogs. Reaction products were characterized by UV, LC/MS analyses, and one- and two-dimensional NMR techniques. The spectral studies indicated that all adducts existed as diastereomeric mixtures. Furthermore, the structural identities of the novel BPQ-dGMP, BPQ-dAMP, and BPQ-dCMP adducts were confirmed by acid phosphatase dephosphorylation of the BPQ-nucleotide adducts to the corresponding known BPQ-nucleoside adduct standards. The BPQ-dGMP, BPQ-dAMP, and BPQ-dCMP adduct standards were used in 32P postlabeling studies to identify BPQ adducts formed in vitro with calf thymus DNA and DNA homopolymers. 32P postlabeling analysis revealed the formation of 8 major and at least 10 minor calf thymus DNA adducts. Of these BPQ-DNA adducts, the following were identified: 1 BPQ-dGMP adduct, 2 BPQ-dAMP adducts, and 3 BPQ-dCMP adducts. This study represents the first reported example of the characterization of stable BPQ-DNA adducts in isolated mammalian DNA and is expected to contribute significantly to the future BPQ-DNA adduct studies in vivo and thereby to the contribution of BPQ in B[a]P carcinogenesis.     

Combination of elevated CO<Subscript>2</Subscript> concentration and elevated temperature and elevated temperature only promote photosynthesis of <Emphasis Type="Italic">Quercus mongolica</Emphasis> seedlings

One-year-old oak (Quercus mongolica Fisch.) seedlings were grown in growth chambers for 30 days to investigate the effects of the combination of elevated CO₂ concentration ([CO₂], 700 μmol/mol) and temperature (ambient T + 4°C) and only elevated temperature (ambient T +4°C) on leaf gas exchange, chlorophyll a fluorescence, and chlorophyll content. In the growth chambers, natural conditions of the Maoershan mountain regions of Heilongjiang Province (45–46°N, 127–128°E) of China for the average growth season were simulated. The results showed that the maximum net photosynthetic rate (P _Nmax) was ≈ 1.64 times greater at elevated temperature than at ambient temperature. The irradiance saturation point (I _s), apparent quantum yield (AQY), maximum photosystem II efficiency (F _v/F _m), and chlorophyll content significantly increased, while irradiance compensation point (I _c) was not affected by elevated temperature. The combination of elevated [CO₂] and temperature also significantly increased P _Nmax by approximately 34% but much lower than that under elevated temperature only. In the case of factor combination, dark respiration (R _d), I _c, F _v/F _m, and total chlorophyll content increased significantly, while I _s and AQY were not affected. Moreover, under elevated [CO₂] and temperature, R _d and I _c, F _v/F _m were significantly higher than under elevated temperature only. The results indicated that the combination of elevated [CO₂] and temperature expected in connection with the further global climate change may affect carbon storage of the coenotype of Q. mongolica in this region of China. This text was submitted by the authors in English.     

Role of cell signaling in B[a]P-induced apoptosis: characterization of unspecific effects of cell signaling inhibitors and apoptotic effects of B[a]P metabolites

Here we show that several cell signaling inhibitors have effect on cyp1a1 expression and the metabolism of benzo[a]pyrene (B[a]P) in Hepa1c1c7 cells. The CYP1A1 inhibitor alpha-naphthoflavone (alpha-NF), the p53 inhibitor pifithrin-alpha (PFT-alpha), the ERK inhibitors PD98059 and U0126, and the p38 MAPK inhibitors SB202190 and PD169316 induced the expression and level of cyp1a1 protein. On the other hand, during the first h the inhibitors appeared to reduce the metabolism of B[a]P as measured by the generation of tetrols and by covalent binding of B[a]P to macromolecules. In contrast, the phosphatidylinositol-3 (PI-3) kinase inhibitor wortmannin, had neither an effect on the cyp1a1 expression nor the B[a]P-metabolism. In order to avoid these unspecific effects, we characterized the mechanisms involved in the apoptotic effects of B[a]P-metabolites. B[a]P and the B[a]P-metabolites B[a]P-7,8-DHD and BPDE-I induced apoptosis, whereas B[a]P-4,5-DHD had no effect. B[a]P, B[a]P-7,8-DHD and BPDE-I induced an accumulation and phosphorylation of p53, while the Bcl-2 proteins Bcl-xl, Bad and Bid were down-regulated. Interestingly, the levels of anti-apoptotic phospho-Bad were up-regulated in response to B[a]P as well as to B[a]P-7,8-DHD and BPDE-I. Both p38 MAPK and JNK were activated, but the p38 MAPK inhibitors were not able to inhibit BPDE-I-induced apoptosis. PFT-alpha reduced the BPDE-I-induced apoptosis, while both the PI-3 kinase inhibitor and the ERK inhibitors increased the apoptosis in combination with BPDE-I. BPDE-I also triggered apoptosis in primary cultures of rat lung cells. In conclusion, often used cell signaling inhibitors both enhanced the expression and the level of cyp1a1 and more directly acted as inhibitors of cyp1a1 metabolism of B[a]P. However, studies with the B[a]P-metabolite BPDE-I supported the previous suggestion that p53 has a role in the pro-apoptotic signaling pathway induced by B[a]P. Furthermore, these studies also show that the reactive metabolites of B[a]P induce the anti-apoptotic signals, Akt and ERK. Neither the induction nor the activity of p38 MAPK and JNK seems to be of major importance for the B[a]P-induced apoptosis.     

Heat shock protein 70 response to physical and chemical stress in Chamelea gallina

Intertidal area is characterized by several fluctuations in natural agents and anthropogenic factors (oxygen levels, temperature, salinity, B[a]P presence) that cause a noticeable increase in the expression rate of heat shock protein 70 (HSP70). HSPs acting as molecular chaperones and their induction represent a specific cellular defence mechanism in response to several stress.Chamelea gallina specimens from the North Adriatic coast were exposed to different experimental conditions: varying oxygen levels (48 h of anoxia followed by 24 h of normoxic recovery), temperatures (20, 25, 30 °C for 7 days), salinity (28, 34, 40‰ for 7 days) and B[a]P concentrations (0.5 mg/L for 24 h, 7 and 12 days). Following the extraction of the digestive gland and gills, HSP70 levels were identified in the cytosolic fraction by immunoblotting using primary monoclonal antibodies. An increase in the rate of HSP70 expression under anoxic conditions in the digestive gland was observed at high temperatures, at low salinity and in the presence of B[a]P. The protein was overexpressed in the absence of oxygen and after 12 days of B[a]P exposure, while it was underexpressed in hyposaline conditions in the gills.HSP70 induction can be considered an adaptation mechanism associated with changes in environmental parameters, but also with xenobiotic presence. The overexpression of HSP70 is therefore induced by protein damage due to stressogenic factors. HSP recruitment renders them available for the processes of folding and refolding of denatured proteins or for their transport to a degradation system. The evident sensitivity of HSP70 to natural and anthropogenic stressogenic agents was examined in the present research.The results of this research revealed an interesting response of heat shock protein 70 in C. gallina, underlining the sensitivity of this important commercial species to natural and anthropogenic stress agents.     

Hoogsteen-paired homopurine [RP-PS]-DNA and homopyrimidine RNA strands form a thermally stable parallel duplex

Homopurine deoxyribonucleoside phosphorothioates possessing all internucleotide linkages of R(P) configuration form a duplex with an RNA or 2'-OMe-RNA strand with Hoogsteen complementarity. The duplexes formed with RNA templates are thermally stable at pH 5.3, while those formed with a 2'-OMe-RNA are stable at neutrality. Melting temperature and fluorescence quenching experiments indicate that the strands are parallel. Remarkably, these duplexes are thermally more stable than parallel Hoogsteen duplexes and antiparallel Watson-Crick duplexes formed by unmodified homopurine DNA molecules of the same sequence with corresponding RNA templates.     

On stabilization of a neutral aromatic ligand by π-cation interactions in monoclonal antibodies

It has been shown that anti-PAH mAb can bind a particular cross-reactant by adopting two distinct “red” and “blue” conformations of its binding sites [N.M. Grubor et al. PNAS 102, 2005, 7453-7458]. In the case of red conformation of pyrene (Py)/anti-PAH mAb (with a broad fluorescence (0,0)-band with fwhm ~ 140 cm⁻¹), the central role in complex formation was played by π-π interactions. The nature of the blue-shifted conformation with very narrow fluorescence (0,0)-band (fwhm ~ 75 cm⁻¹) was left unclear due to the lack of suitable data for comparison. In this work, we suggest spectroscopic and modeling results obtained for the blue conformation of Py in several mAb (including 4D5 mAb) are consistent with π-cation interactions, underscoring the importance of π-cation interaction in ligand binding and stabilization in agreement with earlier modeling studies [J-L. Pellequer, et al. J. Mol. Biol. 302, 2000, 691-699]. We propose considerable narrowing of the fluorescence origin band of ligand in the protein environment could be regarded as a simple indicator of π-cation interactions. Since 4D5 mAb forms only the blue-shifted conformation, while anti-PAH and 8E11 mAbs form both blue- and red-shifted conformations, we suggest mAb interactions, with Py molecules lacking H-bonding functionality, may induce distinct conformations of mAb binding sites that allow binding by π-π and/or π-cation interactions.     

Chemical rescue of a site-modified ligand to a [4Fe-4S] cluster in PsaC, a bacterial-like dicluster ferredoxin bound to Photosystem I

Chemical rescue of site-modified amino acids using externally supplied organic molecules represents a powerful method to investigate structure-function relationships in proteins. Here we provide definitive evidence that aryl and alkyl thiolates, reagents typically used for in vitro iron-sulfur cluster reconstitutions, serve as rescue ligands to a site-specifically modified [4Fe-4S]^1+,2+ cluster in PsaC, a bacterial dicluster ferredoxin-like subunit of Photosystem I. PsaC binds two low-potential [4Fe-4S]^1+,2+ clusters termed F_A and F_B. In the C13G/C33S variant of PsaC, glycine has replaced cysteine at position 13 creating a protein that is missing one of the ligating amino acids to iron-sulfur cluster F_B. Using a variety of analytical techniques, including non-heme iron and acid-labile sulfur assays, and EPR, resonance Raman, and Mössbauer spectroscopies, we showed that the C13G/C33S variant of PsaC binds two [4Fe-4S]^1+,2+ clusters, despite the absence of one of the biological ligands. ¹⁹F NMR spectroscopy indicated that the external thiolate replaces cysteine 13 as a substitute ligand to the F_B cluster. The finding that site-modified [4Fe-4S]^1+,2+ clusters can be chemically rescued with external thiolates opens new opportunities for modulating their properties in proteins. In particular, it provides a mechanism to attach an additional electron transfer cofactor to the protein via a bound, external ligand.     

In vitro evolution of a hyperstable Gbeta1 variant

An in-vitro selection strategy was used to obtain strongly stabilized variants of the beta1 domain of protein G (Gbeta1). In a two-step approach, first candidate positions with a high potential for stabilization were identified in Gbeta1 libraries that were created by error-prone PCR, and then, after randomization of these positions by saturation mutagenesis, strongly stabilized variants were selected. For both steps the in-vitro selection method Proside was employed. Proside links the stability of a protein with the infectivity of a filamentous phage. Ultimately, residues from the two best selected variants were combined in a single Gbeta1 molecule. This variant with the four mutations E15V, T16L, T18I, and N37L showed an increase of 35.1 degrees C in the transition midpoint and of 28.5 kJ mol(-1) (at 70 degrees C) in the Gibbs free energy of stabilization. It was considerably more stable than the best variant from a previous Proside selection, in which positions were randomized that had originally been identified by computational design. Only a single substitution (T18I) was found in both selections. The best variants from the present selection showed a higher cooperativity of thermal unfolding, as indicated by an increase in the enthalpy of unfolding by about 60 kJ mol(-1). This increase is apparently correlated with the presence of Leu residues that were selected at the positions 16 and 37.     

4S polycyclic aromatic hydrocarbon receptor (glycine N‐methyltransferase) and the aryl hydrocarbon receptor nuclear translocator (hypoxia inducible factor‐1β) interaction in Chinese hamster ovary and rat hepatoma cells: 4S PAH‐R/ARNT hetero‐oligomers?

Rat CYP1A1 promoter‐luciferase, transiently transfected wild‐type and 4S PAH receptor (glycine N‐methyl transferase, GNMT)‐transformed Chinese hamster ovary (CHO) cells were exposed to benzo[a]pyrene and assayed for luciferase activity as an indicator of CYP1A1 promoter activity. CHO cells transformed with the rat 4S PAH receptor/GNMT expression vector had twice the induction level of luciferase activity with respect to wild‐type CHO cells in concert with previously published reports that the 4S PAH receptor/GNMT mediates benzo[a]pyrene induction of CYP1A1 gene expression. Lysates of GNMT‐transformed CHO cells and wild‐type H4IIE rat hepatoma cells exposed to benzo[a]pyrene were immuno‐precipitated with anti‐GNMT antibodies, separated by SDS–polyacrylamide gel electrophoresis and transferred to PVDF membrane for Western blot analysis with anti‐aryl hydrocarbon receptor nuclear translocator (ARNT, HIF‐1β) antibodies. Results of this analysis indicated that the 4S PAH receptor/GNMT forms a hetero‐oligomer (dimer?) with ARNT/HIF‐1β which dissociates in the presence of B[a]P. These observations further indicate the role of GNMT (which has been shown to be multifunctional) and B[a]P in the induction of CYP1A1 and also a potential role of GNMT in the modulation of hypoxia inducible factor‐1 function with respect to the HIF‐1β subunit (ARNT). J. Cell. Biochem. 112: 2015–2018, 2011. © 2011 Wiley‐Liss, Inc.     

Searching for multiple folding pathways of a nearly symmetrical protein: temperature dependent phi-value analysis of the B domain of protein A

The B domain of protein A (BdpA) is a popular paradigm for simulating protein folding pathways. The discrepancies between so many simulations and subsequent experimental testing may be attributable to the protein being highly symmetrical: changing experimental conditions could perturb the subtle interplay between the effects of symmetry in the native structure and the effects of asymmetry from specific interactions in a given sequence. If the protein folds via multiple pathways, perturbations, such as temperature, denaturant concentration, and mutation, should change the flux of micro pathways, leading to changes in the bulk properties of the transition state. We tested this hypothesis by conducting a Phi-analysis of BdpA as a function of temperature from 25.0 degrees C to 60.0 degrees C. The Phi-values had no significant dependence on temperature and the values at 55.0 degrees C (denaturing conditions) are very similar to those at 25.0 degrees C (folding conditions), indicating the structure of the transition state does not significantly change although the experimental conditions are considerably altered. The results suggest that BdpA folds via a single dominant folding pathway.     

Assignment of a kinetic component to electron transfer between iron-sulfur clusters FX and FA/B of Photosystem I

We studied the kinetics of reoxidation of the phylloquinones in Chlamydomonas reinhardtii Photosystem I using site-directed mutations in the PhQ_A-binding site and of the residues serving as the axial ligand to ec3_A and ec3_B chlorophylls. In wild type PS I, these kinetics are biphasic, and mutations in the binding region of PhQ_A induced a specific slowing down of the slow component. This slowing allowed detection of a previously unobserved 180-ns phase having spectral characteristics that differ from electron transfer between phylloquinones and F_X. The new kinetic phase thus reflects a different reaction that we ascribe to oxidation of F_X⁻ by the F_A/B FeS clusters. These absorption changes partly account for the differences between the spectra associated with the two kinetic components assigned to phylloquinone reoxidation. In the mutant in which the axial ligand to ec3_A (PsaA-Met688) was targeted, about 25% of charge separations ended in P₇₀₀⁺A₀⁻ charge recombination; no such recombination was detected in the B-side symmetric mutant. Despite significant changes in the amplitude of the components ascribed to phylloquinone reoxidation in the two mutants, the overall nanosecond absorption changes were similar to the wild type. This suggests that these absorption changes are similar for the two different phylloquinones and that part of the differences between the decay-associated spectra of the two components reflect a contribution from different electron acceptors, i.e. from an inter-FeS cluster electron transfer.     

Isolated epsilon subunit of Bacillus subtilis F1-ATPase binds ATP

Previously, we demonstrated ATP binding to the isolated epsilon subunit of F1-ATPase from thermophilic Bacillus PS3 [Kato-Yamada Y., Yoshida M. (2003) J. Biol. Chem. 278, 36013]. However, whether it is a general feature of the epsilon subunit from other sources is yet unclear. Here, using a sensitive method to detect weak interactions between fluorescently labeled epsilon subunit and nucleotide, it was shown that the epsilon subunit of F1-ATPase from Bacillus subtilis also bound ATP. The dissociation constant for ATP binding at room temperature was calculated to be 2 mM, which may be suitable for sensing cellular ATP concentration in vivo.     

Key role of coulombic interactions for the folding transition state of the cold shock protein

The cold shock protein CspB shows a five-stranded beta-sheet structure, and it folds rapidly via a native-like transition state. A previous Phi value analysis showed that most of the residues with Phi values close to one reside in strand beta1, and two of them, Lys5 and Lys7 are partially exposed charged residues. To elucidate how coulombic interactions of these two residues contribute to the energetic organisation of the folding transition state we performed comparative folding experiments in the presence of an ionic denaturant (guanidinium chloride) and a non-ionic denaturant (urea) and a double-mutant analysis. Lys5 contributes 6.6 kJ mol(-1) to the stability of the transition state, and half of it originates from screenable coulombic interactions. Lys7 contributes 5.3 kJ mol(-1), and 3.4 kJ mol(-1) of it are screened by salt. In the folded protein Lys7 interacts with Asp25, and the screenable coulombic interaction between these two residues is fully formed in the transition state. This suggests that long-range coulombic interactions such as those originating from Lys5 and Lys7 of CspB can be important for organizing and stabilizing native-like structure early in protein folding.     

Steady-state and transient polarized absorption spectroscopy of photosytem I complexes from the cyanobacteria Arthrospira platensis and Thermosynechococcus elongatus

Core antenna and reaction centre of photosytem I (PS I) complexes from the cyanobacteria Arthrospira platensis and Thermosynechococcus elongatus have been characterized by steady-state polarized absorption spectroscopy, including linear dichroism (LD) and circular dichroism (CD). CD spectra and the second derivatives of measured 77 K CD spectra reveal the spectral components found in the polarized absorption spectra indicating the excitonic origin of the spectral forms of chlorophyll in the PS I complexes. The CD bands at 669-670(+), 673(+), 680(−), 683-685(−), 696-697(−), and 711(−) nm are a common feature of used PSI complexes. The 77 K CD spectra of the trimeric PS I complexes exhibit also low amplitude components around 736 nm for A. platensis and 720 nm for T. elongatus attributed to red-most chlorophylls. The LD measurements indicate that the transition dipole moments of the red-most states are oriented parallel to the membrane plane. The formation of P700⁺A₁⁻ or ³P700 was monitored by time-resolved difference absorbance and LD spectroscopy to elucidate the spectral properties of the PS I reaction centre. The difference spectra give strong evidence for the delocalization of the excited singlet states in the reaction centre. Therefore, P700 cannot be considered as a dimer but should be regarded as a multimer of the six nearly equally coupled reaction centre chlorophylls in accordance with structure-based calculations. On the basis of the results presented in this work and earlier work in the literature it is concluded that the triplet state is localized most likely on P_A, whereas the cation is localized most likely on P_B.     

Increased folding stability of TEM-1 beta-lactamase by in vitro selection

In vitro selections of stabilized proteins lead to more robust enzymes and, at the same time, yield novel insights into the principles of protein stability. We employed Proside, a method of in vitro selection, to find stabilized variants of TEM-1 β-lactamase from Escherichia coli. Proside links the increased protease resistance of stabilized proteins to the infectivity of a filamentous phage. Several libraries of TEM-1 β-lactamase variants were generated by error-prone PCR, and variants with increased protease resistance were obtained by raising temperature or guanidinium chloride concentration during proteolytic selections. Despite the small size of phage libraries, several strongly stabilizing mutations could be obtained, and a manual combination of the best shifted the profiles for thermal unfolding and temperature-dependent inactivation of β-lactamase by almost 20 °C to a higher temperature. The wild-type protein unfolds in two stages: from the native state via an intermediate of the molten-globule type to the unfolded form. In the course of the selections, the native protein was stabilized by 27 kJ mol^− 1 relative to the intermediate and the cooperativity of unfolding was strongly increased. Three of our stabilizing replacements (M182T, A224V, and R275L) had been identified independently in naturally occurring β-lactamase variants with extended substrate spectrum. In these variants, they acted as global suppressors of destabilizations caused by the mutations in the active site. The comparison between the crystal structure of our best variant and the crystal structure of the wild-type protein indicates that most of the selected mutations optimize helices and their packing. The stabilization by the E147G substitution is remarkable. It removes steric strain that originates from an overly tight packing of two helices in the wild-type protein. Such unfavorable van der Waals repulsions are not easily identified in crystal structures or by computational approaches, but they strongly reduce the conformational stability of a protein.     

Consequences of Domain Insertion on the Stability and Folding Mechanism of a Protein

SlyD, the sensitive-to-lysis protein from Escherichia coli, consists of two domains. They are not arranged successively along the protein chain, but one domain, the “insert-in-flap” (IF) domain, is inserted internally as a guest into a surface loop of the host domain, which is a prolyl isomerase of the FK506 binding protein (FKBP) type. We used SlyD as a model to elucidate how such a domain insertion affects the stability and folding mechanism of the host and the guest domain. For these studies, the two-domain protein was compared with a single-domain variant SlyDΔIF, SlyD* without the chaperone domain (residues 1-69 and 130-165) in which the IF domain was removed and replaced by a short loop, as present in human FKBP12. Equilibrium unfolding and folding kinetics followed an apparent two-state mechanism in the absence and in the presence of the IF domain. The inserted domain decreased, however, the stability of the host domain in the transition region and decelerated its refolding reaction by about 10-fold. This originates from the interruption of the chain connectivity by the IF domain and its inherent instability. To monitor folding processes in this domain selectively, a Trp residue was introduced as fluorescent probe. Kinetic double-mixing experiments revealed that, in intact SlyD, the IF domain folds and unfolds about 1000-fold more rapidly than the FKBP domain, and that it is strongly stabilized when linked with the folded FKBP domain. The unfolding limbs of the kinetic chevrons of SlyD show a strong downward curvature. This deviation from linearity is not caused by a transition-state movement, as often assumed, but by the accumulation of a silent unfolding intermediate at high denaturant concentrations. In this kinetic intermediate, the FKBP domain is still folded, whereas the IF domain is already unfolded.     

Different mechanisms involved in apoptosis following exposure to benzo[a]pyrene in F258 and Hepa1c1c7 cells

The present study compares and elucidates possible mechanisms why B[a]P induces different cell signals and triggers apparently different apoptotic pathways in two rather similar cell lines (hepatic epithelial cells of rodents). The rate and maximal capacity of metabolic activation, as measured by the formation of B[a]P-tetrols and B[a]P-DNA adducts, was much higher in mouse hepatoma Hepa1c1c7 cells than in rat liver epithelial F258 cells due to a higher induced level of cyp1a1. B[a]P increased intracellular pH in both cell lines, but this change modulated the apoptotic process only in F258 cells. In Hepa1c1c7 cells reactive oxygen species (ROS) production appeared to be a consequence of toxicity, unlike F258 cells in which it was an initial event. The increased mitochondrial membrane potential found in F258 cells was not observed in Hepa1c1c7 cells. Surprisingly, F258 cells cultured at low cell density were somewhat more sensitive to low (50nM) B[a]P concentrations than Hepa1c1c7 cells. This could be explained partly by metabolic differences at low B[a]P concentrations. In contrast to the Hepa1c1c7 model, no activation of cell survival signals including p-Akt, p-ERK1/2 and no clear inactivation of pro-apoptotic Bad was observed in the F258 model following exposure to B[a]P. Another important difference between the two cell lines was related to the role of Bax and cytochrome c. In Hepa1c1c7 cells, B[a]P exposure resulted in a "classical" translocation of Bax to the mitochondria and release of cytochrome c, whereas in F258 cells no intracellular translocation of these two proteins was seen. These results suggest that the rate of metabolism of B[a]P and type of reactive metabolites formed influence the resulting balance of pro-apoptotic and anti-apoptotic cell signaling, and hence the mechanisms involved in cell death and the chances of more permanent genetic damage.