Carbonic anhydrase is a ubiquitous metalloenzyme, which catalyzes the reversible hydration of CO₂ to HCO₃⁻ and H⁺. Metals play a key role in the bioactivity of this metalloenzyme, although their relationships with CA have not been completely clarified to date. The aim of this review is to explore the complexity and multi-aspect nature of these relationships, since metals can be cofactors of CA, but also inhibitors of CA activity and modulators of CA expression. Moreover, this work analyzes new insights and perspectives that allow translating new advances in basic science on the interaction between CA and metals to applications in several fields of research, ranging from biotechnology to environmental sciences.

Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a hydrophilic analog of vitamin E, is known for its strong antioxidant activity, being a high radical scavenger of peroxyl and alkoxyl radicals. Under particular conditions, Trolox may also exhibit prooxidant properties. The present work aimed at studying the dual antioxidant/prooxidant behavior of Trolox over a wide range of concentrations (from 2.5 to 160 µM) in HeLa cells. In particular, the study addressed the dose-dependent effects of Trolox on the oxidative cell status and vitality of HeLa cells, focusing on the potential role of the vitamin E analog in the induction of one of the first steps of the apoptotic process, Apoptotic Volume Decrease (AVD). In HeLa cells, Trolox showed significant antioxidant activity, expressed as the ability to reduce the endogenous ROS production detected by the ROS-sensitive probe 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA), at low concentrations (range: 2.5–15 µM), but exerted a dose-dependent prooxidant effect at higher concentrations after 24 h exposure. The prooxidant effect was paralleled by the reduction in cell viability due to the induction of the apoptotic process. The dual behavior, antioxidant at lower concentrations and prooxidant at higher concentrations, was evident also earlier after 2 h incubation, and it was paralleled by the isotonic shrinkage of the cells, ascribed to AVD. The use of SITS, known Cl⁻ channel blocker, was able to completely inhibit the Trolox-induced isotonic cell shrinkage, demonstrating the involvement of the vitamin E analog in the alteration of cell volume homeostasis and, in turn, in the AVD induction. In conclusion, the study shed light on the concentration dependence of the Trolox antioxidant/prooxidant activity in HeLa cells and revealed its role in the induction of one of the first events of apoptosis, AVD, at high concentrations.

Long and short-term exposure to atmospheric particulate matter (PM) has detrimental effects on human health. The effective mechanisms leading to PM toxicity are still not fully understood, even if it is known that physical-chemical properties, strongly influenced by sources and atmospheric processes, are known to play an important role. In this work, PM₁₀ samples were collected, at an urban background site in southern Italy, to determine cytotoxicity (using MTT test on A549 cells), genotoxicity (using the comet assay), and intracellular oxidative stress on A549 cells exposed for 24 h to aqueous extracts of PM₁₀ samples. Organic carbon (OC) and elemental carbon (EC) content of PM₁₀ and acellular determination of oxidative potential with DTT assay were performed to compare results of acellular and cellular biological assays. Cellular (OSGC_V and MTT_V) and acellular (OP^DTT_V) outcomes, normalized in volume, are well correlated (statistically significant results) with carbon content suggesting that combustion sources play an important role in determining cellular oxidative stress and cytotoxicity of PM₁₀. Even if the number of data is limited, genotoxicity results are well correlated (Pearson r > 0.95) with OSGC_V and MTT_V, and a weaker, but statistically significant correlation was observed with OP^DTT_V. OSGC_V is well correlated with the cell mortality observed with the MTT_V test and a lower, but still statistically significant correlation is observed between MTT_V and OP^DDT_V. A statistically significant correlation was found between OP^DTT_V and OSGC_V results. When the outcomes of the cellular and acellular assay are compared normalized in mass (i.e., intrinsic values), the correlations become significantly weaker suggesting that the different sources acting on the site produces particulate matter with different toxicological potential influencing differently the biological tests studied.

The measurement of cellular and sub-cellular responses to chemical contaminants (referred to as biomarkers) in living organisms represents a recent tool in environmental monitoring. The review focuses on carbonic anhydrase, a ubiquitous metalloenzyme which plays key roles in a wide variety of physiological processes involving CO₂ and HCO₃⁻. In the last decade a number of studies have demonstrated the sensitivity of this enzyme to pollutants such as heavy metals and organic chemicals in both humans and wildlife. The review analyses these studies and discusses the potentiality of this enzyme as novel biomarker in environmental monitoring and assessment.

Marine biodiversity is threatened by several anthropogenic pressures. Pollution deriving from the discharge of chemical contaminants in the sea represents one of the main threats to the marine environment, influencing the health of organisms, their ability to recover their homeostatic status, and in turn endangering biodiversity. Molecular and cellular responses to chemical pollutants, known as biomarkers, are effect-based methodologies useful for detecting exposure and for assessing the effects of pollutants on biota in environmental monitoring. The present review analyzes and discusses the recent literature on the use of biomarkers in the framework of biodiversity conservation. The study shows that pollution biomarkers can be useful tools for monitoring and assessment of pollution threat to marine biodiversity, both in the environmental quality monitoring of protected areas and the assessment of the health status of species at risk. Moreover, key areas of the research that need further development are suggested, such as the development of omics-based biomarkers specifically addressed to conservation purposes and their validation in the field, the extension of the biomarker study to a wider number of endangered species, and the development of organic guidelines for the application of the biomarker approach in support to conservation policies and management.

Exposure to atmospheric particulate matter (PM) has detrimental effects on health, but specific mechanisms of toxicity are still not fully understood. In recent years, there has been a growing evidence that oxidative stress is an important mechanism of toxicity; however, when acellular oxidative potential (OP) data are correlated with the outcomes of in vitro (or in vivo) toxicological tests there are contrasting results. In this work, an analysis of PM₁₀ health effect indicators was done, using the acellular Dithiotreitol (DTT) assay to retrieve OP^DTT, the Microtox^® test on Vibrio fischeri bacterium to assess the ecotoxicological potential, and the in vitro MTT assay on the human cell line A549 to estimate the cytotoxicological potential. The objective was to evaluate the correlation among acellular OP^DTT and the results from toxicological and ecotoxicological bioassays and how these health-related indicators are correlated with atmospheric PM₁₀ concentrations collected at an urban background site in Southern Italy. Results indicated that both bioassays showed time-dependent and dose-dependent outcomes. Some samples presented significant ecotoxic and cytotoxic response and the correlation with PM₁₀ concentration was limited suggesting that these health endpoints depend on PM₁₀ chemical composition and not only on exposure concentrations. OP^DTT showed a statistically significant correlation with PM₁₀ concentrations. MTT and Microtox outcomes were not correlated suggesting that the two toxicological indicators are sensitive to different physical-chemical properties of PM₁₀. Intrinsic oxidative potential OP^DTT_M (DTT activity normalised with PM₁₀ mass) was correlated with mortality observed with MTT test (normalized with PM₁₀ mass); however, it was not correlated with Microtox outcomes.