Arbuscular mycorrhizal (AM) fungi establish a mutualistic symbiosis with most land plants. AM fungi regulate plant copper (Cu) acquisition both in Cu deficient and polluted soils. Here, we report characterization of RiCRD1, a Rhizophagus irregularis gene putatively encoding a Cu transporting ATPase. Based on its sequence analysis, RiCRD1 was identified as a plasma membrane Cu (+) efflux protein of the P(1B1)-ATPase subfamily. As revealed by heterologous complementation assays in yeast, RiCRD1 encodes a functional protein capable of conferring increased tolerance against Cu. In the extraradical mycelium, RiCRD1 expression was highly up-regulated in response to high concentrations of Cu in the medium. Comparison of the expression patterns of different players of metal tolerance in R. irregularis under high Cu levels suggests that this fungus could mainly use a metal efflux based-strategy to cope with Cu toxicity. RiCRD1 was also expressed in the intraradical fungal structures and, more specifically, in the arbuscules, which suggests a role for RiCRD1 in Cu release from the fungus to the symbiotic interface. Overall, our results show that RiCRD1 encodes a protein which could have a pivotal dual role in Cu homeostasis in R. irregularis, playing a role in Cu detoxification in the extraradical mycelium and in Cu transfer to the apoplast of the symbiotic interface in the arbuscules.

International audience | Arbuscular mycorrhizal (AM) fungi establish a mutualistic symbiosis with most land plants. AM fungi regulate plant copper (Cu) acquisition both in Cu deficient and polluted soils. Here, we report characterization of RiCRD1, a Rhizophagus irregularis gene putatively encoding a Cu transporting ATPase. Based on its sequence analysis, RiCRD1 was identified as a plasma membrane Cu (+) efflux protein of the P(1B1)-ATPase subfamily. As revealed by heterologous complementation assays in yeast, RiCRD1 encodes a functional protein capable of conferring increased tolerance against Cu. In the extraradical mycelium, RiCRD1 expression was highly up-regulated in response to high concentrations of Cu in the medium. Comparison of the expression patterns of different players of metal tolerance in R. irregularis under high Cu levels suggests that this fungus could mainly use a metal efflux based-strategy to cope with Cu toxicity. RiCRD1 was also expressed in the intraradical fungal structures and, more specifically, in the arbuscules, which suggests a role for RiCRD1 in Cu release from the fungus to the symbiotic interface. Overall, our results show that RiCRD1 encodes a protein which could have a pivotal dual role in Cu homeostasis in R. irregularis, playing a role in Cu detoxification in the extraradical mycelium and in Cu transfer to the apoplast of the symbiotic interface in the arbuscules.

Cadmium (Cd) is being frequently detected in marine organisms. However, dose-dependent effects of Cd challenged unraveling the toxicological mechanisms of Cd to marine organisms and developing biomarkers. Here, the dose-dependent effects of Cd on clams Ruditapes philippinarum following exposure to 5 doses of Cd (3, 9, 27, 81, 243 μg/L) were investigated using benchmark dose (BMD) method. By model fitting, calculation of BMD values was performed on transcriptomic profiles, metals concentrations, and antioxidant indices. Cd exposure induced not only significant Cd accumulation in clams, but also marked alterations of essential metals such as Ca, Cu, Zn, Mn, and Fe. Gene regulation posed little influence on essential metal homeostasis, indicated by poor enrichment of differentially expressed genes (DEGs) associated with metal binding and metal transport in lower concentrations of Cd-treated groups. BMD analysis on biological processes and pathways showed that peptide cross-linking was the most sensitive biological process to Cd exposure, followed by focal adhesion, ubiquitin mediated proteolysis, and apoptosis. Occurrence of apoptosis was also confirmed by TUENL-positive staining in gills and hepatopancreas of clams treated with Cd. Furthermore, many DEGs, such as transglutaminases (TGs), metallothionein (MT), STEAP2-like and laccase, which presented linear or monotonic curves and relatively low BMD values, were potentially preferable biomarkers in clams to Cd. Overall, BMD analysis on transcriptomic profiles, metals concentrations and biochemical endpoints unraveled the sensitiveness of key events in response to Cd treatments, which provided new insights in exploring the toxicological mechanisms of Cd in clams as well as biomarker selection.

Ionic liquids (ILs), also known as green solvents, are widely acknowledged in several fields, such as chemical separation, synthesis, and electrochemistry, owing to their excellent physiochemical properties. However, their poor biodegradability may lead to environmental and health risks, posing a severe threat to humans, thus requiring further research. In this study, the biotoxicities of the imidazolium-based ILs were evaluated in Tetrahymena pyriformis. Moreover, IL detoxification was investigated by addition of glutathione (GSH), cysteine, and nicotinamide adenine dinucleotide (NADH). Reactive oxygen species (ROS) initiated by different IL types caused damage to Tetrahymena, while glutathione, cysteine, and NADH eliminated ROS, achieving the detoxification purposes. Detoxification results showed that NADH exhibited the best detoxification ability, followed by glutathione and cysteine. Finally, RT-PCR results suggested that metallothionein might have participated in IL detoxification.

Cigarette smoke (CS) affects immune functions, leading to severe outcomes in smokers. Robust evidence addresses the immunotoxic effects of combustible tobacco products. As heat-not-burn tobacco products (HNBT) vaporize lower levels of combustible products, we here compared the effects of cigarette smoke (CS) and HNBT vapor on Jurkat T cells. Cells were exposed to air, conventional cigarettes or heatsticks of HNBT for 30 min and were stimulated or not with phorbol myristate acetate (PMA). Cell viability, proliferation, reactive oxygen species (ROS) production, 8-OHdG, MAP-kinases and nuclear factor κB (NFκB) activation and metallothionein expression (MTs) were assessed by flow cytometry; nitric oxide (NO) and cytokine levels were measured by Griess reaction and ELISA, respectively. Levels of metals in the exposure chambers were quantified by inductively coupled plasma mass spectrometry. MT expressions were quantified by immunohistochemistry in the lungs and liver of C57Bl/6 mice exposed to CS, HNBT or air (1 h, twice a day for five days: via inhalation). While both CS and HBNT exposures increased cell death, CS led to a higher number of necrotic cells, increased the production of ROS, NO, inflammatory cytokines and MTs when compared to HNBT-exposed cells, and led to a higher expression of MTs in mice. CS released higher amounts of metals. CS and HNBT exposures decreased PMA-induced interleukin-2 (IL-2) secretion and impaired Jurkat proliferation, effects also seen in cells exposed to nicotine. Although HNBT vapor does not activate T cells as CS does, exposure to both HNBT and CS suppressed proliferation and IL-2 release, a pivotal cytokine involved with T cell proliferation and tolerance, and this effect may be related to nicotine content in both products.

It is estimated that approximately 0.4% of the total leachate produced in a landfill is destined for treatment plants, while the rest can reach the soil and groundwater. In this context, this study aimed to perform leachate toxicity evaluations through immune system cytotoxic assessments, genotoxic (comet assay) appraisals and antioxidant system (superoxide dismutase - SOD; catalase - CAT, glutathione-S-transferase - GST; reduced glutathione - GSH and metallothionein - MT) evaluations in Eisenia andrei earthworms exposed to a Brazilian leachate for 77 days. The leachate sample contained high organic matter (COD - 10,630 mg L⁻¹) and ammoniacal nitrogen (2398 mg L⁻¹), as well as several metals, including Ca, Cr, Fe, Mg, Ni and Zn. Leachate exposure resulted in SOD activity alterations and increased CAT activity and MT levels. Decreased GST activity and GSH levels were also observed. Antioxidant system alterations due to leachate exposure led to increased malondialdehyde levels as a result of lipid peroxidation after the 77 day-exposure. An inflammatory process was also observed in exposed earthworms, evidenced by increased amoebocyte density, and DNA damage was also noted. This study demonstrates for the first time that sublethal effect assessments in leachate-exposed earthworms comprise an important tool for solid waste management.

Heavy metals have been found in increasing concentrations in the aquatic environment. Fishes exposed to such metals have altered gene expression, serum profiles, tissue histology and bioindices that serve as overall health biomarkers. The heavy metals (Ni, Cd, and Cr) accumulated in water and fish tissues, were beyond the permissible limits defined by the Central Pollution Control Board/World Health Organization. Metallothionein (MT) and glutathione peroxidase (GPX) genes expression patterns highlighted the metal-specific exposure of fish. An increased fold change of genes against beta-actin serves as a potential feature for toxicity. Metal toxicity is also reflected by an increased level of digestive enzymes (amylase and lipase) in the serum and alterations in values of reproductive hormones (11-Ketotestosterone and progesterone). Total serum bilirubin attribute to the liver and biliary tract disease in fishes. Histopathological studies show cellular degeneration, breakage, vacuolization signifying the chronic stress.

Bulk Cu compounds such as Cu(OH)₂ are extensively used as pesticides in agriculture. Recent investigations suggest that Cu-based nanomaterials can replace bulk materials reducing the environmental impacts of Cu. In this study, stress responses of alfalfa (Medicago sativa L.) seedlings to Cu(OH)₂ nanoparticle or compounds were evaluated. Seeds were immersed in suspension/solutions of a Cu(OH)₂ nanoform, bulk Cu(OH)₂, CuSO₄, and Cu(NO₃)₂ at 25 and 75 mg/L. Six days later, the germination, seedling growth, and the physiological and biochemical responses of sprouts were evaluated. All Cu treatments significantly reduced root elongation (average = 63%). The ionic compounds at 25 and 75 mg/L caused a reduction in all elements analyzed (Ca, K, Mg, P, Zn, and Mn), excepting for S, Fe and Mo. The bulk-Cu(OH)₂ treatment reduced K (48%) and P (52%) at 75 mg/L, but increased Zn at 25 (18%) and 75 (21%) mg/L. The nano-Cu(OH)₂ reduced K (46%) and P (48%) at 75 mg/L, and also P (37%) at 25 mg/L, compared with control. Confocal microscopy images showed that all Cu compounds, at 75 mg/L, significantly reduced nitric oxide, concurring with the reduction in root growth. Nano Cu(OH)₂ at 25 mg/L upregulated the expression of the Cu/Zn superoxide dismutase gene (1.92-fold), while ionic treatments at 75 mg/L upregulated (∼10-fold) metallothionein (MT) transcripts. Results demonstrated that nano and bulk Cu(OH)₂ compounds caused less physiological impairments in comparison to the ionic ones in alfalfa seedlings.