Migration of polycyclic aromatic hydrocarbons from a polymer surrogate through the stratum corneum layer of the skin.

In this study, we determined partition (Ksc/m) and diffusion (Dsc) coefficients of five different polycyclic aromatic hydrocarbons (PAH) migrating from squalane into and through the stratum corneum (s.c.) layer of the skin. Carcinogenic PAH have previously been detected in numerous polymer-based consumer products, especially those dyed with carbon black. Upon dermal contact with these products, PAH may penetrate into and through the viable layers of the skin by passing the s.c. and thus may become bioavailable. Squalane, a frequent ingredient in cosmetics, has also been used as a polymer surrogate matrix in previous studies. Ksc/m and Dsc are relevant parameters for risk assessment because they allow estimating the potential of a substance to become bioavailable upon dermal exposure. We developed an analytical method involving incubation of pigskin with naphthalene, anthracene, pyrene, benzo[a]pyrene and dibenzo[a,h]pyrene in Franz diffusion cell assays under quasi-infinite dose conditions. PAH were subsequently quantified within individual s.c. layers by gas chromatography coupled to tandem mass spectrometry. The resulting PAH depth profiles in the s.c. were fitted to a solution of Fick's second law of diffusion, yielding Ksc/m and Dsc. The decadic logarithm logKsc/m ranged from -0.43 to +0.69 and showed a trend to higher values for PAH with higher molecular masses. Dsc, on the other hand, was similar for the four higher molecular mass PAH but about 4.6-fold lower than for naphthalene. Moreover, our data suggests that the s.c./viable epidermis boundary layer represents the most relevant barrier for the skin penetration of higher molecular mass PAH. Finally, we empirically derived a mathematical description of the concentration depth profiles that better fits our data. We correlated the resulting parameters to substance specific constants such as the logarithmic octanol-water partition coefficient logP, Ksc/m and the removal rate at the s.c./viable epidermis boundary layer.

Commensal-Related Changes in the Epidermal Barrier Function Lead to Alterations in the Benzo[a]Pyrene Metabolite Profile and Its Distribution in 3D Skin.

Polycyclic aromatic hydrocarbons (PAH) such as benzo[a]pyrene (B[a]P) are among the most abundant environmental pollutants, resulting in continuous exposure of human skin and its microbiota. However, effects of the latter on B[a]P toxicity, absorption, metabolism, and distribution in humans remain unclear. Here, we demonstrate that the skin microbiota does metabolize B[a]P on and in human skin in situ, using a recently developed commensal skin model. In this model, microbial metabolism leads to high concentrations of known microbial B[a]P metabolites on the surface as well as in the epidermal layers. In contrast to what was observed for uncolonized skin, B[a]P and its metabolites were subject to altered rates of skin penetration and diffusion, resulting in up to 58% reduction of metabolites recovered from basal culture medium. The results indicate the reason for this altered behavior to be a microbially induced strengthening of the epidermal barrier. Concomitantly, colonized models showed decreased formation and penetration of the ultimate carcinogen B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE), leading, in consequence, to fewer BPDE-DNA adducts being formed. Befittingly, transcript and expression levels of key proteins for repairing environmentally induced DNA damage such as xeroderma pigmentosum complementation group C (XPC) were also found to be reduced in the commensal models, as was expression of B[a]P-associated cytochrome P450-dependent monooxygenases (CYPs). The results show that the microbiome can have significant effects on the toxicology of external chemical impacts. The respective effects rely on a complex interplay between microbial and host metabolism and microbe-host interactions, all of which cannot be adequately assessed using single-system studies. IMPORTANCE Exposure to xenobiotics has repeatedly been associated with adverse health effects. While the majority of reported cases relate to direct substance effects, there is increasing evidence that microbiome-dependent metabolism of xenobiotic substances likewise has direct adverse effects on the host. This can be due to microbial biotransformation of compounds, interaction between the microbiota and the host's endogenous detoxification enzymes, or altered xenobiotic bioavailability. However, there are hardly any studies addressing the complex interplay of such interactions in situ and less so in human test systems. Using a recently developed microbially competent three-dimensional (3D) skin model, we show here for the first time how commensal influence on skin physiology and gene transcription paradoxically modulates PAH toxicity.

DNA-templated native chemical ligation of functionalized peptide nucleic acids: a versatile tool for single base-specific detection of nucleic acids.

Single base-specific detection of DNA/RNA sequences is of importance in the diagnosis of disease-associated genetic disorders or early stage cancer. This chapter introduces DNA-templated native chemical PNA ligation as a potentially useful tool for the sequence specific detection of nucleic acids. The template-induced alignment of PNA-thioesters and 1,2-aminothiol-PNAs in close proximity leads to an increase in their effective molarities. This facilitates PNA ligation to proceed at concentrations where no reaction would be possible in absence of the template. Moreover, hybridization of the rather short PNA conjugates with non-complementary DNA/RNA is disfavored, which prevents PNA ligation to occur on single base-mismatched templates. Different readout strategies of the ligation reaction such as HPLC, MALDI-TOF-MS and fluorecence monitoring are discussed, and examples for the detection of a point mutation within single stranded and PCR-amplified double stranded DNA are provided.

Reducing product inhibition in nucleic acid-templated ligation reactions: DNA-templated cycligation.

Programmable interactions allow nucleic acid molecules to template chemical reactions by increasing the effective molarities of appended reactive groups. DNA/RNA-triggered reactions can proceed, in principle, with turnover in the template. The amplification provided by the formation of many product molecules per template is a valuable asset when the availability of the DNA or RNA target is limited. However, turnover is usually impeded by reaction products that block access to the template. Product inhibition is most severe in ligation reactions, where products after ligation have dramatically increased template affinities. We introduce a potentially generic approach to reduce product inhibition in nucleic acid-programmed ligation reactions. A DNA-triggered ligation-cyclization sequence ("cycligation") of bifunctional peptide nucleic acid (PNA) conjugates affords cyclic ligation products. Melting experiments revealed that product cyclization is accompanied by a pronounced decrease in template affinity compared to linear ligation products. The reaction system relies upon haloacetylated PNA-thioesters and isocysteinyl-PNA-cysteine conjugates, which were ligated on a DNA template according to a native chemical ligation mechanism. Dissociation of the resulting linear product-template duplex (induced by, for example, thermal cycling) enabled product cyclization through sulfur-halide substitution. Both ligation and cyclization are fast reactions (ligation: 86 % yield after 20 min, cyclization: quantitative after 5 min). Under thermocycling conditions, the DNA template was able to trigger the formation of new product molecules when fresh reactants were added. Furthermore, cycligation produced 2-3 times more product than a conventional ligation reaction with substoichiometric template loads (0.25-0.01 equiv). We believe that cyclization of products from DNA-templated reactions could ultimately afford systems that completely overcome product inhibition.

The role of reactivity in DNA templated native chemical PNA ligation during PCR.

DNA templated fluorogenic reactions have been used as a diagnostic tool for the sequence specific detection of nucleic acids; and it has been shown that the native chemical ligation between thioester- and 1,2-aminothiol-modified PNA probes is amongst the most selective DNA detection methods reported. We explored whether a DNA templated reaction can be interfaced with the polymerase chain reaction (PCR). This endeavor posed a significant challenge. The reactive groups involved must be sufficiently stable to tolerate the high temperature applied in the PCR process. Nevertheless, the ligation reaction must proceed very rapidly and sequence specifically within the short time available in the annealing and primer extension steps before denaturation is used after approx. 1 min to commence the next PCR cycle. This required a careful optimization of the ternary complex architecture as well as adjustments of probe length and probe reactivities. Our results point to the prime importance of the ligation architecture. We show that once suitable annealing sites have been identified less reactive probe sets may be preferable if sequence specificity is of major concern. The reactivity tuning enabled the development of an in-PCR ligation, which was used for the single nucleotide specific typing of the V600E (T1799A) point mutation in the human BRaf gene. Showcasing the efficiency and sequence specificity of native chemical PNA ligation, attomolar template proofed sufficient to trigger signal while a 1000-fold higher load of single mismatched template failed to induce appreciable signal.