Polystyrene Microplastics of Varying Sizes and Shapes Induce Distinct Redox and Mitochondrial Stress Responses in a Caco-2 Monolayer.

Currently, we lack crucial knowledge on how the physicochemical properties of particles affect cellular health, resulting in an important gap in our understanding of the human toxicity of microplastics (MPs). Our aim was to evaluate the impact of the size and the shape of MPs on uptake and the intracellular effects in a human epithelial colorectal adenocarcinoma (Caco-2) cell line. Spherical (200 nm and 2 µm) and fibre-/fragment-shaped (8.9 ± 10.1 µm by 1.14 ± 0.97 µm) polystyrene microplastics (PS-MPs) were used to study their uptake and the potential to induce redox and mitochondrial stress responses after 24 h of exposure. We demonstrated the cellular uptake of both spherical and fibre-/fragment-shaped MPs in a size-dependent manner. In response to 2 µm spheres, we observed differential expressions of redox-related genes, including HMOX1, CAT, and GPX1. All PS-MPs decreased the intracellular H2O2 levels, which can be attributed to mitochondrial stress responses, such as increased mitochondrial DNA content, footprint, and morphology. Altogether, we demonstrated uptakes and changes in redox and mitochondrial parameters for all PS-MPs, with the 200 nm spheres showing the most profound effects. This suggests that the induction of defensive responses in Caco-2 cells mainly correlates with the number of particles taken up.

Interactive toxicity of copper and cadmium in regenerating and adult planarians.

In a polluted environment, metals are present as complex mixtures. As a result, organisms are exposed to different metals at the same time, which affects both metal-specific as well as overall toxicity. Detailed information about the molecular mechanisms underlying the adverse effects of combined exposures remains limited in terms of different life stages. In this study, the freshwater planarian Schmidtea mediterranea was used to investigate developmental and physiological responses associated with a combined exposure to Cu and Cd. In addition, the cellular and molecular mechanisms underlying the provoked adverse effects were studied in different exposure scenarios. Mixed exposure resulted in a decline in survival, diverse non-lethal morphological changes, neuroregenerative impairments, altered behaviour and a limited repair capacity. Underlying to these effects, the cellular redox state was altered in all exposure conditions. In adult animals, this led to DNA damage and corresponding transcriptional changes in cell cycle and DNA repair genes. In regenerating animals, changes in hydrogen peroxide and glutathione contents led to regenerative defects. Overall, our results demonstrate that (1) developing organisms are more susceptible to metal exposures, and (2) the toxicity of an individual metal increases significantly in a mixed exposure scenario. These aspects have to be included in current risk assessment strategies.

Toxic effects of cadmium on flatworm stem cell dynamics: A transcriptomic and ultrastructural elucidation of underlying mechanisms.

Stem cells or undifferentiated cells can cope more easily with external stresses. To evaluate the impact of toxic compounds on stem cell dynamics in vivo, in relation to other biological responses, we use the carcinogenic element cadmium and the regenerating model organism Macrostomum lignano. Through both BrdU and anti-histone H3 immunostainings, cadmium-induced effects were investigated at different stages of the stem cell cycle. A 24-h exposure to 100 and 250 µM CdCl2 significantly decreased the number of stem cells (neoblasts) in mitosis, whereas the number of cells in the S phase remained unchanged. After this short-term exposure, the ultrastructure of the neoblasts was minimally affected in contrast to the epidermal tissues. These results were supported by gene expression data: transcripts of cdc2 and pig3 were significantly upregulated during all treatments. Both genes are involved in the cell cycle progression and are transcribed in the gonadal region, where stem cells are highly represented. Based on a substantial increase in gene expression of heat shock proteins (HSP) and their high activity in the gonadal region, we hypothesize that these proteins are key players in the protection of stem cells against external stresses. Apart from the strong HSP induction, other protective processes including cell division, apoptosis and anti-oxidative defence, were also activated. We, therefore, conclude that the protection of stem cells against external stressors may be based on the interplay between stem cell maintenance, i.e. repair and recovery through division, on one hand and apoptosis on the other hand. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1217-1228, 2016.

Redox-Related Mechanisms to Rebalance Cancer-Deregulated Cell Growth.

A delicate balance exists between the process of carcinogenesis and tissue regeneration. A number of malignant tumours are considered the outcome of an impaired or incomplete regeneration process, resulting in persistently dividing cells. Regeneration-competent tissues and animals are able to prevent and counteract growth abnormalities and seem to have a low vulnerability to chemical carcinogenesis. Cancer cell survival depends, among other things, on various redox-related mechanisms, which are targets of currently developed therapies. Disadvantages of these therapies are a lack of specificity and drug resistance. As the majority of these redox-related mechanisms also play an important role in successful and coordinated cell functioning and reproduction, the regeneration process offers a unique parallel context for modern cancer research. This review focuses on the interconnections between regeneration and carcinogenesis and how an understanding of regenerative forces and redox-controlled mechanisms could contribute to the identification of new therapeutic targets to block the growth and survival of cancer cells.

Physiological and molecular characterisation of cadmium stress in Schmidtea mediterranea.

The planarian Schmidtea mediterranea is a well-studied model organism for developmental research, because of its stem cell system. This characteristic also provides a unique opportunity to study stress management and the effect of stress on stem cells. In this study, we characterised the stress signature at different levels of biological organization. The carcinogenic metal cadmium was used as a model chemical stressor. We focused on stem cell activity and its interaction with other known stress parameters. Here, we have found that S. mediterranea is able to cope with high internal levels of cadmium. At endpoints such as size and mobility, cadmium-related stress effects were detected but all of these responses were transient. Correspondingly, cadmium exposure led to an elevated mitotic activity of the neoblasts, at the same time points when the other responses disappeared. At the molecular level, we observed redox-related responses that can be linked with both repair as well as proliferation mechanisms. Together, our results suggest that these animals have a high plasticity. The induction of stem cell activity may underlie this 'restoring' effect, although a carcinogenic outcome after longer exposure times cannot be excluded.

The cellular redox state as a modulator in cadmium and copper responses in Arabidopsis thaliana seedlings.

The cellular redox state is an important determinant of metal phytotoxicity. In this study we investigated the influence of cadmium (Cd) and copper (Cu) stress on the cellular redox balance in relation to oxidative signalling and damage in Arabidopsis thaliana. Both metals were easily taken up by the roots, but the translocation to the aboveground parts was restricted to Cd stress. In the roots, Cu directly induced an oxidative burst, whereas enzymatic ROS (reactive oxygen species) production via NADPH oxidases seems important in oxidative stress caused by Cd. Furthermore, in the roots, the glutathione metabolism plays a crucial role in controlling the gene regulation of the antioxidative defence mechanism under Cd stress. Metal-specific alterations were also noticed with regard to the microRNA regulation of CuZnSOD gene expression in both roots and leaves. The appearance of lipid peroxidation is dual: it can be an indication of oxidative damage as well as an indication of oxidative signalling as lipoxygenases are induced after metal exposure and are initial enzymes in oxylipin biosynthesis. In conclusion, the metal-induced cellular redox imbalance is strongly dependent on the chemical properties of the metal and the plant organ considered. The stress intensity determines its involvement in downstream responses in relation to oxidative damage or signalling.

Leaf proteome responses of Arabidopsis thaliana exposed to mild cadmium stress.

The leaf proteome of 3-week-old Arabidopsis thaliana seedlings exposed for 1 week to low, environmentally realistic Cd concentrations was investigated. The data indicated that at 1muMCd, A. thaliana plants adapted their metabolism to cope with the Cd exposure. As a result, only moderate protein changes were observed. However, at 10muMCd, severe stress was indicated by growth reduction and chlorosis of rosette leaves at the macroscopic level and by lipid peroxidation and enhanced peroxidase activity at the cellular level. Of the 730 reproducible proteins among all gels, 21 were statistically upregulated in response to Cd. These proteins can be functionally grouped into 5 classes: proteins involved in (1) oxidative stress response, (2) photosynthesis and energy production, (3) protein metabolism, (4) gene expression and finally, (5) proteins with various or unknown function. In order to provide greater insight into the mechanisms induced on Cd exposure, a working model is proposed.

Oxidative stress-related responses at transcriptional and enzymatic levels after exposure to Cd or Cu in a multipollution context.

The physiological effects of Cd and Cu have been highlighted in several studies over the last years. At the cellular level, oxidative stress has been reported as a common mechanism in both stress situations. Nevertheless, because of differences in their redox-related properties, the origin of the stress and regulation of these effects can be very different. Our results show a specific Cd-related induction of NADPH oxidases, whereas both metals induced lipid peroxidation via the activation of lipoxygenases. With respect to the antioxidative defense system, metal-specific patterns of superoxide dismutases (SODs) were detected, whereas gene expression levels of the H2O2-quenching enzymes were equally induced by both metals. Because monometallic exposure is very unusual in real-world situations, the metal-specific effects were compared with the mechanisms induced when the plants are exposed to both metals simultaneously. Combined exposure to Cd and Cu enhanced some of the effects that were induced when only one metal was applied to the medium. Other specific monometallically induced effects, such as a copper zinc superoxide dismutase (CSD2) downregulation due to Cd, were also sustained in a multipollution context, irrespective of the other monometallic effects. Furthermore, specific multipollution effects were unravelled, as iron superoxide dismutase 1 (FSD1) upregulation in the leaves was significant only when both Cu and Cd were applied. Additional relationships between these treatments and the common and specific stress induction mechanisms are discussed.

Subcellular localization of cadmium in roots and leaves of Arabidopsis thaliana.

We examined the subcellular cadmium (Cd) localization in roots and leaves of wild-type Arabidopsis thaliana (ecotype Columbia) exposed to environmentally relevant Cd concentrations. Energy-dispersive X-ray microanalysis (EDXMA) was performed on high-pressure frozen and freeze-substituted tissues. In the root cortex, Cd was associated with phosphorus (Cd/P) in the apoplast and sulfur (Cd/S) in the symplast, suggesting phosphate and phytochelatin sequestration, respectively. In the endodermis, sequestration of Cd/S was present as fine granular deposits in the vacuole and as large granular deposits in the cytoplasm. In the central cylinder, symplastic accumulation followed a distinct pattern illustrating the importance of passage cells for the uptake of Cd. In the apoplast, a shift of Cd/S granular deposits from the middle lamella towards the plasmalemma was observed. Large amounts of precipitated Cd in the phloem suggest retranslocation from the shoot. In leaves, Cd was detected in tracheids but not in the mesophyll tissue. Extensive symplastic and apoplastic sequestration in the root parenchyma combined with retranslocation via the phloem confirms the excluder strategy of Arabidopsis thaliana.