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Wide application of perfluoroalkyl acids (PFAAs) has raised great concerns on their side-effects on human health. PFAAs have been shown to accumulate mainly in the liver and cause hepatotoxicity. However, PFAAs can also deposit in lung tissues through air-borne particles and cause serious pulmonary toxicity. But the underlying mechanisms are still largely unknown. Autophagy is a type of programmed cell death parallel to necrosis and apoptosis, and may be involved in the lung toxicity of PFAAs. In this study, lung cancer cells, A549, were employed as the model to investigate the effects of three PFAAs with different carbon chain lengths on cell autophagy. Through Western blot analysis on LC3-I/II ratio of cells exposed to non-cytotoxic concentration (200 µM) and cytotoxic concentration (350 µM), we found concentration-dependent increase of autophagosomes in cells, which was further confirmed by TEM examination on ultra-thin section of cells and fluorescence imaging on autophagosomes in live cells. The abundance of p62 increased with the PFAAs concentration indicating the blockage of autophagy flux. Furthermore, we identified the mitochondrial autophagy (mitophagy) and endoplasmic reticulum autophagy (ER-phagy) morphologically as the major types of autophagy, suggesting the disruption on mitochondria and ERs. These organelle damages were confirmed by the overgeneration of ROS, hyperpolarization of mitochondrial membrane potential, as well as the up-regulation of ER-stress-related proteins, ATF4 and p-IRE1. Further analysis on the signaling pathways showed that PFAAs activated the MAPK pathways and inhibited the PI3K/Akt pathway, with potencies following the order of PFDA > PFNA > PFOA. Anti-oxidant (NAC) treatment did not rescue cells from death, indicating that oxidative stress is not the reason of cytotoxicity. Inhibition of autophagy by Atg5 siRNA and chloroquine even increased the toxicity of PFAAs, suggesting that PFAAs-autophagy was induced as the secondary effects of organelle damages and played a protective role during cell death.

We reported an unprecedented chemiluminescence (CL) behavior of polyamine-functionalized carbon dots induced by Fe3+–H2O2 Fenton-like system. The first-step CL intensity increased with the increasing of the concentration of H2O2 and Fe3+, when the Fe3+ concentration came to 10−3 M, the unprecedented two-step CL behavior appeared. The CL intensity of BPEI-CDs induced by Fenton-like system was about 10 times stronger than that of naked CDs. The possible two-step CL mechanism was speculated based on the photoluminescence spectra, CL emission spectra, and the effects of radical scavengers on the CL intensity. Radiative recombination of the injected holes by strong oxidant perferrate formed through Fe3+–H2O2 reaction and the ·OH generated from successive Fenton reaction with the thermally excited electrons was proposed, which further facilitate full understanding about the optical properties of carbon dots.

Cellular uptake and exocytosis of SWCNTs are fundamental processes determining their intracellular concentration and effects. Despite the great potential of acid-oxidized SWCNTs in biomedical field, understanding of the influencing factors on these processes needs to be deepened. Here, we quantitatively investigated uptake and exocytosis of SWCNTs in three lengths-630 (±171) nm (L-SWCNTs), 390 (±50) nm (M-SWCNTs), and 195 (±63) nm (S-MWCNTs) in macrophages. The results showed that the cellular accumulation of SWCNTs was a length-independent process and non-monotonic in time, with the most SWCNTs (3950 fg/cell) accumulated at 8 h and then intracellular SWCNTs dropped obviously with time. The uptake rate of SWCNTs decreased with increasing concentration, suggesting that intracellular SWCNTs accumulation is a saturable process. After refreshing culture medium, we found increasing SWCNTs in supernatant and decreasing intracellular SWCNTs over time, confirming the exocytosis occurred. Selective inhibition of endocytosis pathways showed that the internalization of SWCNTs involves several pathways, in the order of macropinocytosis> caveolae-mediated endocytosis> clathrin-dependent endocytosis. Intriguingly, clathrin-mediated endocytosis is relatively important for internalizing shorter SWCNTs. The dynamic processes of SWCNTs uptake and exocytosis and the mechanisms revealed by this study may render a better understanding on SWCNT toxicity and facilitate the design of CNT products with mitigated toxicity and desired functions.

Perfluoroalkyl compounds (PFCs) have been shown to disrupt thyroid functions through thyroid hormone receptor (TR)-mediated pathways, but direct binding of PFCs with TR has not been demonstrated. We investigated the binding interactions of 16 structurally diverse PFCs with human TR, their activities on TR in cells, and the activity of perfluorooctane sulfonate (PFOS) in vivo. In fluorescence competitive binding assays, most of the 16 PFCs were found to bind to TR with relative binding potency in the range of 0.0003-0.05 compared with triiodothyronine (T3). A structure-binding relationship for PFCs was observed, where fluorinated alkyl chain length longer than ten, and an acid end group were optimal for TR binding. In thyroid hormone (TH)-responsive cell proliferation assays, PFOS, perfluorohexadecanoic acid, and perfluorooctadecanoic acid exhibited agonistic activity by promoting cell growth. Furthermore, similar to T3, PFOS exposure promoted expression of three TH upregulated genes and inhibited three TH downregulated genes in amphibians. Molecular docking analysis revealed that most of the tested PFCs efficiently fit into the T3-binding pocket in TR and formed a hydrogen bond with arginine 228 in a manner similar to T3. The combined in vitro, in vivo, and computational data strongly suggest that some PFCs disrupt the normal activity of TR pathways by directly binding to TR.

Nanomaterials have been used increasingly in a wide variety of applications, and some of them have shown toxic effects on experimental animals and cells. In this study, a previously established photoelectrochemical DNA sensor was employed to rapidly detect DNA damage induced by polystyrene nanosphere (PSNS) suspensions. In the sensor, a double-stranded DNA film was assembled on a semiconductor electrode, and a DNA intercalator, Ru(bpy)2(dppz)2+ (bpy = 2,2′-bipyridine, dppz = dipyrido[3,2-a:2′,3′-c]phenazine) was used as the photoelectrochemical signal indicator. After the DNA-modified electrode was exposed to 2.0 mg/mL PSNS suspension, photocurrent of DNA-bound Ru(bpy)2(dppz)2+ decreased by about 20%. The decrease is attributed to the chemical damage of DNA and consequently less binding of Ru(bpy)2(dppz)2+ molecules to the electrode. Gel electrophoresis of DNA samples incubated with PSNS suspension confirmed DNA damage after the chemical exposure. However, in both photoelectrochemical and gel electrophoresis experiments, extensively washed PSNS did not induce any DNA damage, and the supernatant of PSNS suspension exhibited comparable DNA damage as the unwashed PSNS suspension. Furthermore, UV-visible absorption spectrum of the supernatant displayed a pattern very similar to that of styrene oxide (SO), a compound which has been shown to induce DNA damage by forming covalent DNA adducts. It is therefore suggested that styrene oxide and other residual chemicals in the PSNS may be responsible for the observed DNA damage. The results highlight the importance of full characterization of nanomaterials before their toxicity study, and demonstrate the utility of photoelectrochemical DNA sensors in the rapid assessment of DNA damage induced by chemicals and nanomaterials.

Strategies for electrochemical sensing of DNA can be classified into label-free and label-based approaches, categories of which include enzyme-, nanomaterial- and redox labels that are attached to DNA either by covalent or non-covalent means. Metallointercalators represent one group of small molecule redox labels that non-covalently enter the groove of a DNA. The metallointercalator plays a dual-role in acting as a structure indicator (for hybridization) and a signal generator. Labeling is not needed, and electrochemical measurements can be carried out in a label-free solution of an electrolyte. However, such metallointercalators lack the option of catalytic signal generation as in the case of enzyme- and nanomaterial-based labels. Therefore, signal amplification becomes crucial. We first survey here recent progress in this area. A signal-amplifying system is presented that relies on the electroatalytic oxidation of a metallointercalator ruthenium(II)bipyridine/phenoxazine complex in the presence of electron donor species such as oxalate, DNA bases, or tripropylamine. Recent work on such DNA sensors is discussed. Results suggest that such metallointercalator-based DNA sensors represent a viable platform for developing high-throughput and automated PCR/lab-on-a-chip devices as well as visualized multifunctional DNA sensors.

DNA films immobilized on an indium tin oxide (ITO) electrode surface were detected and determined employing a high-affinity intercalator, Ru(bpy)2(dppz)2+ (bpy = 2,2′-bipyridine, dppz = dipyrido[3,2-a:2′,3′-c]phenazine), as a redox indicator, and oxalate as a sacrificial electron donor in solution to chemically amplify the voltammetric signal of the indicator. Nucleic acids were immobilized on ITO by layer-by-layer electrostatic adsorption, using avidin as the first layer and nucleic acid as the second layer. In quartz crystal microbalance (QCM) measurements on an avidin-coated gold surface, the amount of adsorption from a 200 µg mL−1 nucleic acid solution was found to be 3.2 ng mm−2 for both double-stranded (ds-) and single-stranded (ss-) calf-thymus DNA as well as polycytidylic acid (Poly-C). After binding with Ru(bpy)2(dppz)2+ (Ru-dppz), voltammetry of the ds-DNA film on ITO was carried out in an indicator-free phosphate buffer. An anodic peak at about 1.15 V was observed, and it was assigned to Ru-dppz oxidation. When measured in an oxalate buffer, however, a catalytic current was observed due to the oxidation of oxalate by electrochemically generated Ru(bpy)2(dppz)3+, resulting in a 120-fold increase in the signal. Since oxalate itself produces a very low oxidation current on ITO, catalytic voltammetry produces about a 14-fold improvement in the signal-to-blank ratio over the non-amplified determination. As a result, ds-DNA adsorbed from 20 ng mL−1 solution could be detected, which was estimated by QCM to be 160 pg mm−2 on the surface. The catalytic current of ds-DNA was substantially higher than that of ss-DNA and poly-C, indicative of selective binding of the redox indicator to ds-DNA. The results serve as a basis for the catalytic voltammetric detection of DNA hybridization in future work.

Perfluoroalkyl acids (PFAAs) are carcinogens,and elucidating their DNA binding properties is crucial forunderstanding PFAA genotoxicity. We have investigated thebinding mode and affinity of five PFAAs to seven DNAmolecules using fluorescence displacement and moleculardockinganalysis. DNA conformational changes uponPFAAbinding were also examined by circular dichroism (CD).The data revealed that DNA intercalation was the dominantinteraction mode of the PFAAs; however, these moleculesalso bound to grooves. The dissociation constants for thePFAAs ranged between 0.11 and 1,217.14 μM, and between3.46 and 2,141.21 μM for DNA intercalation and groovebinding, respectively. PFAAs that contain longer carbonchains had stronger DNA intercalation affinities. Bindingto DNA was stronger for perfluoroalkyl sulfonates than forperfluorcarboxyl acids that contain the same number ofcarbons. This observation is postulated to arise from thepresenceofmorefluorineandoxygenatomsinperfluoroalkylsulfonates acting as hydrogen bond donors that facilitatestronger DNA intercalation. The binding of the PFAAs toDNA showed some CT-DNA sequence selectivity. Moleculardocking analysis confirmed the DNA binding mode andaffinities of the PFAAs. CD analysis revealed that thePFAAs weakened DNA base stacking and loosened DNAhelicity.Thepresentstudyhasimprovedourunderstandingofthe formation of PFAA–DNA adducts.Keywords Perfluoroalkylacids .DNAbinding .Intercalation .Minorgroovebinding .DNAconformationalchange .DNAsequenceselectivityIntroductionPerfluoroalkyl acids (PFAAs) have been produced and usedfor over 50 years. Because of their specific physical andchemical properties, chemical and thermal inertness, lowsurface energy, and special surface-active properties,PFAAs have applications in numerous industrial and con-sumer products, ranging from carpet protection to fire-fighting foams (Lehmler 2005; Martin et al. 2004). PFAAspersist ubiquitously in the environment, resisting hydrolysis,photolysis, and biodegradation because of the high energyof the carbon–fluorine bond (Hekster et al. 2003; Martin etal. 2004; Quinete et al. 2010). Although the toxicologicaleffects of these chemicals are still under investigation, manystudieshavereportedontheirdevelopmentalandreproductivetoxicity, neurotoxicity, immunotoxicity, and carcinogenicityinanimalsbothinvivoandinvitro(Heksteretal.2003;Lauetal. 2004; Slotkin et al. 2008). PFAAs can cause peroxisomalproliferation, mitochondrial dysfunction, disturbance of fattyacid metabolism, hepatocellular hypertrophy, changes in