Cytotoxicity and Oxidative Stress Effects of Indene on Coelomocytes of Earthworm (Eisenia foetida): Combined Analysis at Cellular and Molecular Levels.

Indene (IND) is a kind of important aromatic hydrocarbon that is extracted from coal tar and has important applications in industry and biology. In the process of production and utilization, it is easy to enter the soil and produce toxic effects on the soil or organisms. The earthworm is an important organism in the soil. The toxicity of indene on earthworm coelomocytes is rarely studied, and the oxidative stress effects of IND on earthworm coelomocytes remain unclear. In this study, coelomocytes from earthworms and antioxidant enzymes were selected as the research targets. In addition, IND caused oxidative stress, and its related toxic effects and mechanisms were systematically studied and evaluated at the cellular and molecular levels. The results showed that IND destroyed the redox balance in earthworm coelomocytes, and the large accumulation of reactive oxygen species (ROS) significantly inhibited the activities of the antioxidant system, including superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH), and caused lipid peroxidation and membrane permeability changes, resulting in a decrease in cell viability to 74.5% of the control group. At the molecular level, IND was bound to SOD by the arene-H bond, and the binding constant was 4.95 × 103. IND changed the secondary structure of the SOD and led to a loosening of the structure of the SOD peptide chain. Meanwhile, IND caused SOD fluorescence sensitization, and molecular simulation showed that IND was mainly bound to the junction of SOD subunits. We hypothesized that the changes in SOD structure led to the increase in SOD activity. This research can provide a scientific basis for IND toxicity evaluation.

Probing the biological toxicity of pyrene to the earthworm Eisenia fetida and the toxicity pathways of oxidative damage: A systematic study at the animal and molecular levels.

Pyrene (Pyr), a widely used tetracyclic aromatic hydrocarbon, enters soil in large quantities and causes environmental pollution due to its production and mining. In order to systematically study the biotoxicity of pyrene to model organisms Eisenia fetida in soil, experiments were carried out from four dimensions: animal, tissue, cell and molecule. Experimental results proved that the mortality rate increased with increasing concentration and time of exposure to pyrene, while the mean body weight and spawning rate decreased. Meanwhile, when the pyrene concentration reached 900 mg/kg, the seminal vesicle and longitudinal muscle of the earthworm showed obvious atrophy. Experimental results at the cellular level showed that pyrene induced cell membrane damage and Ca2+ influx triggered mitochondrial membrane depolarization and a surge in ROS levels. Oxidative stress causes damage to proteins and lipids and DNA inside cells. When the mortality rate was 91.67 %, the Olive Tail Movement (OTM) of the comet experiment reached 15. The results of molecular level tests showed that pyrene inhibited the activity of Cu/Zn-superoxide dismutase (Cu/Zn-SOD) mainly by changing the microenvironment and secondary structure of amino acid Tyr 108. The weakened function of direct antioxidant enzymes may be the root cause of the excessive increase of reactive oxygen species (ROS) in cells. The systematic approach used in this study enriches the network of toxic pathways in toxicological studies, and basic data on the biological toxicity of pyrene can provide support for future soil contamination detection.

Toxic mechanism on phenanthrene-induced cytotoxicity, oxidative stress and activity changes of superoxide dismutase and catalase in earthworm (Eisenia foetida): A combined molecular and cellular study.

Phenanthrene (PHE) is an important organic compound, which is widespread in the soil environment and exhibits potential threats to soil organisms. Toxic effects of PHE to earthworms have been extensively studied, but toxic mechanisms on PHE-induced cytotoxicity and oxidative stress at the molecular and cellular levels have not been reported yet. Therefore, we explored the cytotoxicity and oxidative stress caused by PHE in earthworm coelomocytes and the interaction mechanism between PHE and the major antioxidant enzymes SOD/CAT. It was shown that high-dose PHE exposure induced the intracellular reactive oxygen species (ROS) generation, mediated lipid peroxidation, reduced total antioxidant capacity (T-AOC) in coelomocytes, and triggered oxidative stress, thus resulted in a strong cytotoxicity at higher concentrations (0.6-1.0 mg/L). The intracellular SOD/CAT activity in cells after PHE exposure were congruent with that in molecular levels, which the activity of SOD enhanced and CAT inhibited. Spectroscopic studies showed the SOD/CAT protein skeleton and secondary structure, as well as the micro-environment of aromatic amino acids were changed after PHE binding. Molecular docking indicated PHE preferentially docked to the surface of SOD. However, the key residues Tyr 357, His 74, and Asn 147 for activity were in the binding pocket, indicating PHE more likely to dock to the active center of CAT. In addition, H-bonding and hydrophobic force were the primary driving force in the binding interaction between PHE and SOD/CAT. This study indicates that PHE can induce cytotoxicity and oxidative damage to coelomocytes and unearthes the potential effects of PHE on earthworms.

A review of human and animals exposure to polycyclic aromatic hydrocarbons: Health risk and adverse effects, photo-induced toxicity and regulating effect of microplastics.

Polycyclic aromatic hydrocarbons (PAHs) are one of the most widely distributed persistent organic pollutants (POPs) in the environmental media. PAHs have been widely concerned due to their significant health risk and adverse effects to human and animals. Currently, the main sources of PAHs in the environment are the incomplete combustion of fossil fuels, as well as municipal waste incineration and agricultural non-surface source emissions. In this work, the scope of our attention includes 16 typical PAHs themselves without involving their metabolites and industrial by-products. Exposure of human and animals to PAHs can lead to a variety of adverse effects, including carcinogenicity and teratogenicity, genotoxicity, reproductive- and endocrine-disrupting effects, immunotoxicity and neurotoxicity, the type and severity of which depend on a variety of factors. On the other hand, the regulatory effect of microplastics (MPs) on the bio-toxicity and bioaccumulation capacity of PAHs has now gradually attracted attention. We critically reviewed the adsorption capacity and mechanisms of MPs on PAHs as well as the effects of MPs on PAHs toxicity, thus highlighting the importance of paying attention to the joint bio-toxicity caused by PAHs-MPs interactions. In addition, due to the extensive nature of the common exposure pathway of PAHs and ultraviolet ray, an accurate understanding of biological processes exposed to both PAHs and UV light is necessary to develop effective protective strategies. Finally, based on the above critical review, we highlighted the research gaps and pointed out the priority of further studies.

Toxic mechanism of pyrene to catalase and protective effects of vitamin C: Studies at the molecular and cell levels.

Polycyclic aromatic hydrocarbons, distributing extensively in the soil, would potentially threaten the soil organisms (Eisenia fetida) by triggering oxidative stress. As a ubiquitous antioxidant enzyme, catalase can protect organisms from oxidative damage. To reveal the potential impact of polycyclic aromatic hydrocarbon pyrene (Pyr) on catalase (CAT) and the possible protective effect of Ascorbic acid (vitamin C), multi-spectral and molecular docking techniques were used to investigate the influence of structure and function of catalase by pyrene. Fluorescence and circular dichroism analysis showed that pyrene would induce the microenvironmental changes of CAT amino acid residues and increase the α-helix in the secondary structure. Molecular simulation results indicated that the main binding force of pyrene around the active center of CAT is hydrogen bonding force. Furthermore, pyrene inhibited catalase activity to 69.9% compared with the blank group, but the degree of inhibition was significantly weakened after vitamin C added into the research group. Cell level experiments showed that pyrene can increase the level of ROS in the body cavity cell of earthworms, and put the cells under the threat of potential oxidative damage. Antioxidants-vitamin C has a protective effect on catalase and maintains the stability of intracellular ROS levels to a certain extent.

Exploring the influence of silver and lead on structure and function of xylanase: spectroscopic and calorimetric methods.

Soil contamination with heavy metal could induce the alteration of soil ecological environments, and soil enzyme activities are sensitive indicators for the soil toxicology. Xylanase is one of predominant soil enzymes related to carbon nitrogen cycle. In this work, we explored the underlying mechanisms for conformational and enzymatic activity alterations of xylanase after silver and lead exposure at molecular level with systematical measurements including multiple spectroscopic methods, isothermal titration calorimetry, and enzymatic activity. Both silver and lead could loosen and unfold the skeleton of xylanase with the quenching of endogenous fluorescence. Silver interacted with xylanase forming larger-size aggregations through Van der Waals forces and hydrogen bonding, while lead interacted with xylanase forming larger-size aggregations through hydrophobic force. Silver and lead induced an obvious loss (67.1 and 56.31%) of the xylanase enzymatic activity, but silver has a greater impact on xylanase than that of lead. The xylanase enzymatic activity significantly decreased due to the conformational alterations. The negative effect of silver exposure on xylanase structure and function was more prominent than that of lead.

Toxicity assessment of Fluoranthene, Benz(a)anthracene and its mixed pollution in soil: Studies at the molecular and animal levels.

An increasing amount of Fluoranthene (Fla) and Benz(a)anthracene (BaA) is being produced and used, eventually entering the soil sediments. The accumulation of Fla and BaA will cause poisoning to typical enzymes (α-Amylase) and organisms (Eisenia fetida) in soil. However, the studies about exploring and comparing the different effects of Fla, BaA and their joint effect at different levels are rarely reported. In this paper, the different effects of Fla, BaA and their mixed pollutant on α-Amylase were evaluated and compared at the molecular level, and the effect of Fla-BaA to the antioxidant system of earthworm (Eisenia fetida) was investigated from the aspects of concentration and exposure time at the animal level. The results showed that Fla-BaA had the greatest influence on the skeleton structure and the microenvironment of amino acid residue of α-Amylase compared to Fla and BaA, and in the mixed pollutant system, the joint effect mode was additive mode. The inhibitory effect of Fla-BaA on the activity of α-Amylase was also stronger than that of the system alone. The assays at the animal level showed that low concentrations (below 5 mg/kg) of Fla-BaA increased the activity of GSH-Px and SOD while high concentrations inhibited their activity. The POD that was activated throughout the experiment period suggested its key role in the earthworm antioxidant system. Changes in T-AOC and MDA showed that long-term and high-dose of Fla-BaA exposure inhibited the antioxidant capacity of Eisenia fetida, causing lipid peroxidation and damage to cells.

Catalase and superoxide dismutase response and the underlying molecular mechanism for naphthalene.

Naphthalene, a naturally-occurring polyaromatic hydrocarbon, pose potential threats to health for its wide exposures in environment. Naphthalene could disrupt the redox equilibrium resulting in oxidative damage. Antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD) are considered to be the efficient defense barriers to protect organisms from negative impacts of toxicants. Limited information is available regarding the underlying molecular mechanism between antioxidant enzymes and naphthalene. In this paper, structural and functional alterations of CAT and SOD for low dose (1.6-25.6 mg/L) naphthalene exposure have been investigated at the molecular and cellular levels. The enzyme activity responses of CAT and SOD in hepatocytes for naphthalene were consistent with the molecular, in which the activity of CAT increased and the activity of SOD slightly inhibited. Spectroscopy methods and molecular docking were carried out to investigate the underlying binding mechanisms. Naphthalene exposure significantly changed the conformation of CAT with secondary structure alteration (α-helix increase) but only changed the skeleton structure of SOD without secondary structure alteration. Naphthalene could bind to CAT and SOD primarily via H-binding force accompanied with the particle size of CAT/SOD agglomerates decreasing. Naphthalene preferentially bound to the surface of CAT and SOD. Besides, naphthalene could also bind directly to the active center of CAT with the key residues Arg364 and Tyr 357 for activity. This paper provides a combined cellular and molecular strategy to research biomarker responses for toxicants exposure. Besides, this study offers detailed basic data for the comprehensive understanding of naphthalene toxicity.

Cytotoxicity of perfluorodecanoic acid on mouse primary nephrocytes through oxidative stress: Combined analysis at cellular and molecular levels.

Long-chain perfluoroalkyl acids (PFAAs) such as perfluorodecanoic acid (PFDA) are toxic, persistent organic pollutants. This study investigated the harmful effect of PFDA on mouse primary nephrocytes and its mechanism at cellular and molecular levels. Cellular results showed that PFDA exhibited nephrotoxicity with decreased cell viability and increased apoptosis. The increase of intracellular reactive oxygen species (ROS) content and the decrease of intracellular superoxide dismutase (SOD) activity were significant (p < 0.01) when PFDA concentration exceeded 10 µM. Additionally, the molecular results indicated that PFDA bind with Val-A98 in the surface of Cu/Zn-SOD by a 3.11 Šhydrogen bond driven by Van der Waals' force and hydrogen bonding force, which triggered the structural changes and decreased activity of Cu/Zn-SOD. Altogether, the intracellular oxidative stress is the main driver of nephrocyte apoptosis; and the interaction of PFDA and Cu/Zn-SOD exacerbated the oxidative stress in nephrocytes, which is also a nonnegligible reason of cytotoxicity induced by PDFA. This study represented a meaningful method to explore the toxic effect and mechanism of xenobiotics at cellular and molecular levels. The findings have implications for revealing the clearance of long-chain PFAAs in vivo.

Binding of copper to lysozyme: Spectroscopic, isothermal titration calorimetry and molecular docking studies.

Although copper is essential to all living organisms, its potential toxicity to human health have aroused wide concerns. Previous studies have reported copper could alter physical properties of lysozyme. The direct binding of copper with lysozyme might induce the conformational and functional changes of lysozyme and then influence the body's resistance to bacterial attack. To better understand the potential toxicity and toxic mechanisms of copper, the interaction of copper with lysozyme was investigated by biophysical methods including multi-spectroscopic measurements, isothermal titration calorimetry (ITC), molecular docking study and enzyme activity assay. Multi-spectroscopic measurements proved that copper quenched the intrinsic fluorescence of lysozyme in a static process accompanied by complex formation and conformational changes. The ITC results indicated that the binding interaction was a spontaneous process with approximately three thermodynamical binding sites at 298K and the hydrophobic force is the predominant driven force. The enzyme activity was obviously inhibited by the addition of copper with catalytic residues Glu 35 and Asp 52 locating at the binding sites. This study helps to elucidate the molecular mechanism of the interaction between copper and lysozyme and provides reference for toxicological studies of copper.

Investigations on the effects of Cu(2+) on the structure and function of human serum albumin.

Human serum albumin (HSA) is the most prominent protein in blood plasma with important physiological functions. Although copper is an essential metal for all organisms, the massive utilization of copper has led to concerns regarding its potential health impact. To better understand the potential toxicity and toxic mechanisms of Cu(2+), it is of vital importance to characterize the interaction of Cu(2+) with HSA. The effect of Cu(2+) on the structure and function of HSA in vitro were investigated by biophysical methods including fluorescence techniques, circular dichroism (CD), time-resolved measurements, isothermal titration calorimetry (ITC), molecular simulations and esterase activity assay. Multi-spectroscopic measurements proved that Cu(2+) quenched the intrinsic fluorescence of HSA in a dynamic process accompanied by the formation of complex and alteration of secondary structure. But the Cu(2+) had minimal effect on the backbone and secondary structure of HSA at relatively low concentrations. The ITC results indicated Cu(2+) interacted with HSA spontaneously through hydrophobic forces with approximately 1 thermodynamic identical binding sites at 298 K. The esterase activity of HSA was inhibited obviously at the concentration of 8 × 10(-5) M. However, molecular simulation showed that Cu(2+) mainly interacted with the amino acid residues Asp (451) by the electrostatic force. Thus, we speculated the interaction between Cu(2+) and HSA might induce microenvironment of the active site (Arg 410). This study has provided a novel idea to explore the biological toxicity of Cu(2+) at the molecular level.

Oxidative damage induced by copper in mouse primary hepatocytes by single-cell analysis.

Copper can disturb the intracellular redox balance, induce oxidative stress, and subsequently cause irreversible damage, leading to a variety of diseases. In the present study, mouse primary hepatocytes were chosen to elucidate the in vitro oxidative damage of short-term copper exposure (10-200 µM) by single-cell analysis. We evaluated the toxicity of copper by reactive oxygen species (ROS), glutathione (GSH), and oxidative DNA damage at the single-cell level. Oxidative damage induced by copper was verified by the morphological changes, persistent elevations of excessive ROS and malondialdehyde (MDA), a decrease in GSH level, and the oxidative DNA damage. Furthermore, the average ROS generation, GSH consumption, and the indicators in DNA damage did not significantly change at relatively low concentrations (10 or 50 µM), but we can find the alterations of parameters in some single cells clearly. Emphasis on the analysis of single cells is conducive to gain a better understanding on the toxicity of copper. This study will also complement studies on the environmental risk assessment of copper pollution.

Detection of glutathione within single erythrocyte of different ages and pathological state using microfluidic chips coupled with laser induced fluorescence.

As a major factor participating in the organism antioxidation and detoxification process, GSH is of vital importance to human beings. Detecting GSH content in single cells is significant to diagnosis and prevention of many diseases. In this work, the amount of GSH within single erythrocytes was detected and analyzed via statistical analysis. All erythrocytes tested were collected from people in different ages and people of different pathological states. The correlation between GSH level, age and pathological state were investigated. Results showed that the GSH level in erythrocytes decreased with the ages of patients increased. There was little difference between the GSH level in erythrocytes from people who had chronic diseases (hyperglycemia, hyperlipidemia and hypertension) and from healthy people. However, the GSH level in erythrocytes from people who had inflammation (myocarditis, nephritis and gastritis) was generally higher than that from the healthy people. This study provides basic data for researches of cell senescence and cytopathic effect and is helpful to diagnosis and prevention of diseases. In addition, it also provides a simple and effective method for rapid GSH detection within single cell.

Spectroscopy, calorimetry and molecular simulation studies on the interaction of catalase with copper ion.

In this research, the binding mechanism of Cu(2+) to bovine liver catalase (BLC) was studied by fluorescence spectroscopy, ultraviolet-visible (UV-vis) absorption spectroscopy, circular dichroism (CD) spectroscopy, isothermal titration calorimetry (ITC) and molecular docking methods. The cellar experiment was firstly carried out to investigate the inhibition effect of catalase. During the fluorescence quenching study, after correcting the inner filter effect (IFE), the fluorescence of BLC was found to be quenched by Cu(2+). The quenching mechanism was determined by fluorescence lifetime measurement, and was confirmed to be the dynamic mode. The secondary structure content of BLC was changed by the addition of Cu(2+), as revealed by UV-vis absorption and CD spectra, which further induces the decrease in BLC activity. Molecular simulation study indicates that Cu(2+) is located between two ß-sheets and two random coils of BLC near to the heme group, and interacts with His 74 and Ser 113 residues near a hydrophilic area. The decrease of α-helix and the binding of His 74 are considered to be the major reason for the inhibition of BLC activity caused by Cu(2+). The ITC results indicate that the binding stoichiometry of Cu(2+) to catalase is 11.4. Moreover, the binding of Cu(2+) to BLC destroyed H-bonds, which was confirmed by the CD result.

Detection of glutathione within single mice hepatocytes using microfluidic chips coupled with a laser-induced fluorescence system.

A rapid and accurate detection of glutathione (GSH) content in single cells is important to the early diagnosis and prevention of diseases. A microfluidic system allows the manipulation of trace amounts of reagents and single cells in a simple and cheap glass chip coupled with laser induced fluorescence (LIF) detection. 2,3-Naphthalenedicarboxaldehyde (NDA) was used as the derivatization reagent to label GSH in cells. Microchannel surface derivatization and optimization of injection and separation were investigated in detail, and then the GSH in single mice hepatocyte was separated and detected under optimum conditions with a linear range of 5×10(-4) M~5×10(-3) M and a detection limit of 4.47×10(-5) M. This study provides a simple and effective method for rapid GSH detection in single cells using few reagents.

Study on the interaction mechanism of 2-aminoanthraquinone with calf thymus DNA.

By utilizing multispectrosopic techniques, the toxic interaction of 2-aminoanthraquinone (2-AAQ) with calf thymus deoxyribonucleic acid (ctDNA) was investigated in vitro under simulated physiological conditions. The experimental results proved that 2-AAQ has a toxic interaction with ctDNA. The binding capacity of DNA with 2-AAQ is diminishing as the pH value of system increasing in the optimization of experimental condition. Moreover we selected pH 7.4, which is nearly physiological condition to enhance the practical significance. According to the Stern-Volmer equation, the quenching was the static quenching process. And the quenching constant Kq can be derived from the fluorescence quenching spectrogram. Ultraviolet absorption spectra and the change in the fluorescence intensity at different ionic strengths further indicated that there was electrostatic binding between 2-AAQ and ctDNA. The circular dichroism experiment showed that the DNA conformation varied from B to A conformation. The basic group enhanced after 2-AAQ embedding. The double helix is more compact, and the DNA conformation changes.