Antioxidant response to heavy metal pollution of Regi Lagni freshwater in Conocephalum conicum L. (Dum.).

Conocephalum conicum L. is a cosmopolitan liverwort species able to respond to local environmental pollution by changing its biological features. In the present study, we assessed the different biological responses in C. conicum to heavy metal contamination of Regi Lagni channels, a highly polluted freshwater body. As for the in field experiment, we set up moss bags containing collected samples of the local wild growing C. conicum, from the upstream site (non-polluted area), and we exposed them in the three selected sites characterized by different and extreme conditions of heavy metal pollution. In addition, to better understand the contribution of heavy metals to the alterations and response of the liverwort, we performed in vitro tests, using the same concentration of heavy metals measured in the sites at the moment of the exposition. In both experimental settings, bioaccumulation, ultrastructural damage, reactive oxygen species production and localization, antioxidant enzymes activity (superoxide dismutase, catalase and glutathione S-transferases), glutathione (reduced and oxidized) levels, localization of compounds presenting thiol groups and phenolic content were investigated. The results showed that the samples from different sites and conditions (for in vitro tests) showed significant differences. In particular, the ultrastructural alterations show a trend correlated to the different exposure situations; ROS contents, glutathione, antioxidant enzyme activities, and phenolic contents were increased showing an enhancement of the antioxidant defense both by the enzymatic way and by using the synthesis of antioxidant phenolic compounds. This study confirms the ability of C. conicum to respond to heavy metal pollution and the responses studied are, at least partially, correlated to the presence of heavy metals. All the responses considered respond consistently with the pollution trend and they can be proposed as pollution biomarkers. Therefore, we suggest the use of C. conicum to identify local hot spots of pollution in further investigation.

Biological responses to heavy metal stress in the moss Leptodictyum riparium (Hedw.) Warnst.

Leptodictyum riparium, a widely distributed aquatic moss, can both tolerate and accumulate very high concentrations of toxic heavy metals, with only slight apparent damage. Here we report the effects on photosynthetic yield, glutathione (GSH), phytochelatin (PCn) synthesis, nitrogen metabolism and cellular localization of molecules rich in SH groups in L. riparium exposed in vitro to heavy metals. We simulated the concentrations of Cu, Zn, Cd, Pb detected in Regi Lagni, Italy, one of the most contaminated freshwater sites in Southern Europe, in the laboratory to test how the moss responds to heavy metal contamination. There was a steady decrease of photosynthetic efficiency correlated with the heavy metal concentrations and ultrastructural organization. All PCn levels increased significantly as the concentration of heavy metals increased, while the GSH levels did not appear to be particularly affected. A significant increase of GDH and NADH-GOGAT activities increased with increasing heavy metal concentration. Immunoblotting analysis revealed an increase of the chl-GS2 while no significant increase was detected in the cyt-GS1. These results give insight into the molecular events underlying the metal-tolerance of the aquatic moss L. riparium exposed to environmental heavy metal concentrations.

Biological Responses to Cadmium Stress in Liverwort Conocephalum conicum (Marchantiales).

Oxidative damage (production and localization of reactive oxygen species) and related response mechanisms (activity of antioxidant enzymes), and induction of Heat Shock Protein 70 expression, have been studied in the toxi-tolerant liverwort Conocephalum conicum (Marchantiales) in response to cadmium stress using two concentrations (36 and 360 µM CdCl2). Cadmium dose-dependent production of reactive oxygen species (ROS) and related activity of antioxidant enzymes was observed. The expression level of heat shock protein (Hsp)70, instead, was higher at 36 µM CdCl2 in comparison with the value obtained after exposure to 360 µM CdCl2, suggesting a possible inhibition of the expression of this stress gene at higher cadmium exposure doses. Biological responses were related to cadmium bioaccumulation. Since C. conicum was able to respond to cadmium stress by modifying biological parameters, we discuss the data considering the possibility of using these biological changes as biomarkers of cadmium pollution.

The Moss Leptodictyum riparium Counteracts Severe Cadmium Stress by Activation of Glutathione Transferase and Phytochelatin Synthase, but Slightly by Phytochelatins.

In the present work, we investigated the response to Cd in Leptodictyum riparium, a cosmopolitan moss (Bryophyta) that can accumulate higher amounts of metals than other plants, even angiosperms, with absence or slight apparent damage. High-performance liquid chromatography followed by electrospray ionization tandem mass spectrometry of extracts from L. riparium gametophytes, exposed to 0, 36 and 360 µM Cd for 7 days, revealed the presence of γ-glutamylcysteine (γ-EC), reduced glutathione (GSH), and traces of phytochelatins. The increase in Cd concentrations progressively augmented reactive oxygen species levels, with activation of both antioxidant (catalase and superoxide dismutase) and detoxifying (glutathione-S-transferase) enzymes. After Cd treatment, cytosolic and vacuolar localization of thiol peptides was performed by means of the fluorescent dye monochlorobimane and subsequent observation with confocal laser scanning microscopy. The cytosolic fluorescence observed with the highest Cd concentrations was also consistent with the formation of γ-EC-bimane in the cytosol, possibly catalyzed by the peptidase activity of the L. riparium phytochelatin synthase. On the whole, activation of phytochelatin synthase and glutathione-S-transferase, but minimally phytochelatin synthesis, play a role to counteract Cd toxicity in L. riparium, in this manner minimizing the cellular damage caused by the metal. This study strengthens previous investigations on the L. riparium ability to efficiently hinder metal pollution, hinting at a potential use for biomonitoring and phytoremediation purposes.

Functional and structural biomarkers to monitor heavy metal pollution of one of the most contaminated freshwater sites in Southern Europe.

This study evaluated the biological effects of highly polluted freshwater environment (Regi Lagni channels, S Italy) on the aquatic moss Leptodictyum riparium, exposed in bags at three sites representative of different environmental conditions and characterized by different heavy metal burdens. Bioaccumulation, ultrastructural alterations, Reactive Oxygen Species (ROS) production, antioxidant enzymes activity and DNA damage were assessed. To better evaluate the biological response of the moss species to heavy metals, the same biological parameters were assessed also in L. riparium samples cultured in vitro using metal mixtures at the same concentrations as measured at the 3 field exposure sites. Heavy metals were accumulated into the moss tissues causing severe ultra-structural damages at higher concentration case studies, and the ROS production as well as the activity of the enzyme followed a concentration-dependent increase. However, the DNA damage trend suggested a threshold effect that changed between field and in vitro experiment. The enrichment factor suggests that the concentration in the most polluted site is close to the upper limit of L. riparium to accumulate metals. Overall, combining measures of the morpho-functional traits at different level contribute to improving the knowledge about the tolerance of L. riparium to heavy metal stress, suggesting that this moss could be suitable for biomonitoring activity in field conditions.

Effect of Heat Stress on Yield, Monoterpene Content and Antibacterial Activity of Essential Oils of Mentha x piperita var. Mitcham and Mentha arvensis var. piperascens.

Heat stress affects the yield of medicinal plants and can reduce biomass and/or metabolite production. In order to evaluate the effect of heat-induced stress on the essential oil production in Mentha x piperita L. var. Mitcham (Mitcham mint) and Mentha arvensis var. piperascens Malinv. ex L. H. Bailey (Japanese mint), we studied the chemical composition of the oils of the two mint species under different heat shock stresses in growth chambers. The antibacterial activity of the essential oils was also evaluated; microscopic observation (fluorescence and electron transmission) was used to assess the effect of the tested samples on bacterial growth. The results obtained shed light on the mint essential oils composition and biological activity in relation to heat stress.

Effects of triacontanol on ascorbate-glutathione cycle in Brassica napus L. exposed to cadmium-induced oxidative stress.

The ability of exogenous triacontanol (TRIA), a plant growth regulator, to reduce Cd toxicity was studied in canola (Brassica napus L.) plants. The following biological parameters were examined in canola seedlings to investigate TRIA-induced tolerance to Cd toxicity: seedling growth, chlorophyll damage and antioxidant response. In particular, TRIA application reduced Cd-induced oxidative damage, as shown by reduction of ROS content, lipoxygenase (LOX) activity and lipid peroxidation level. TRIA pretreatment increased non-enzymatic antioxidant contents (ascorbate, AsA, glutathione and GSH), phytochelatin content (PCs) and activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX), monodehydroascorbate reductase (MDHAR), dehydro ascorbate reductase (DHAR), and glutathione reductase (GR), so reducing the oxidative stress. These results clearly indicate the protective ability of TRIA to modulate the redox status through the antioxidant pathway AGC and GSH, so reducing Cd-induced oxidative stress.

Antiproliferative, antibacterial and antifungal activity of the lichen Xanthoria parietina and its secondary metabolite parietin.

Lichens are valuable natural resources used for centuries throughout the world as medicine, food, fodder, perfume, spices and dyes, as well as for other miscellaneous purposes. This study investigates the antiproliferative, antibacterial and antifungal activity of the acetone extract of the lichen Xanthoria parietina (Linnaeus) Theodor Fries and its major secondary metabolite, parietin. The extract and parietin were tested for antimicrobial activity against nine American Type Culture Collection standard and clinically isolated bacterial strains, and three fungal strains. Both showed strong antibacterial activity against all bacterial strains and matched clinical isolates, particularly against Staphylococcus aureus from standard and clinical sources. Among the fungi tested, Rhizoctonia solani was the most sensitive. The antiproliferative effects of the extract and parietin were also investigated in human breast cancer cells. The extract inhibited proliferation and induced apoptosis, both effects being accompanied by modulation of expression of cell cycle regulating genes such as p16, p27, cyclin D1 and cyclin A. It also mediated apoptosis by activating extrinsic and intrinsic cell death pathways, modulating Tumor Necrosis Factor-related apoptosis-inducing ligand (TRAIL) and B-cell lymphoma 2 (Bcl-2), and inducing Bcl-2-associated agonist of cell death (BAD) phosphorylation. Our results indicate that Xanthoria parietina is a major potential source of antimicrobial and anticancer substances.

A Cd/Fe/Zn-responsive phytochelatin synthase is constitutively present in the ancient liverwort Lunularia cruciata (L.) dumort.

Lunularia cruciata occupies a very basal position in the phylogenetic tree of liverworts, which in turn have been recognized as a very early clade of land plants. It would therefore seem appropriate to take L. cruciata as the startingpoint for investigating character evolution in plants' metal(loid) response. One of the strongest evolutionary pressures for land colonization by plants has come from potential access to much greater amounts of nutritive ions from surface rocks, compared to water. This might have resulted in the need to precisely regulate trace element homeostasis and to minimize the risk of exposure to toxic concentrations of certain metals, prompting the evolution of a number of response mechanisms, such as synthesis of phytochelatins, metal(loid)-binding thiol-peptides. Accordingly, if the ability to synthesize phytochelatins and the occurrence of an active phytochelatin synthase are traits present in a basal liverwort species, and have been even reinforced in 'modern' tracheophytes, e.g. Arabidopsis thaliana, then such traits would presumably have played an essential role in plant fitness over time. Hence, we demonstrated here that: (i) L. cruciata compartmentalizes cadmium in the vacuoles of the phototosynthetic parenchyma by means of a phytochelatin-mediated detoxification strategy, and possesses a phytochelatin synthase that is activated by cadmium and homeostatic concentrations of iron(II) and zinc; and (ii) A. thaliana phytochelatin synthase displays a higher and broader response to several metal(loid)s [namely: cadmium, iron(II), zinc, copper, mercury, lead, arsenic(III)] than L. cruciata phytochelatin synthase.

High density polyethylene (HDPE) biodegradation by the fungus Cladosporium halotolerans.

Polyethylene (PE) is high molecular weight synthetic polymer, very hydrofobic and hardly biodegradable. To increase polyethylene bio-degradability it is very important to find microorganisms that improve the PE hydrophilic level and/or reduce the length of its polymeric chain by oxidation. In this study, we isolated Cladosporium halotolerans, a fungal species, from the gastric system of Galleria mellonella larvae. Here, we show that C. halotolerans grows in the presence of PE polymer, it is able to interact with plastic material through its hyphae and secretes enzymes involved in PE degradation.

Proteins of the fruit pulp of Acca sellowiana have antimicrobial activity directed against the bacterial membranes.

Acca sellowiana is an evergreen plant that produces edible fruit whit high nutritional properties, and also contains clinically relevant bioactive compounds, including polyphenols and essential oils. Numerous biological activities were demonstrated for A. sellowiana fruits: antifungal and antitumoral and anti-oxidant. It was also showed a strong antibacterial activity against Gram-positive and Gram-negative bacteria. Fruit are generally considered recalcitrant plant tissues for the difficulty to obtain high quality protein due to a low protein content and the presence of interfering substances. For this reason, the objective of the present work was obtain high quantity of protein extract, to determine the location in the fruit of the molecules responsible for the antibacterial activity, separate them according to molecular weight, test their thermo resistance, study the timing of action, isolate the protein fraction having activity and hypothesise a mechanism of action directed against bacterial membranes.

Fighting multidrug resistance with a fruit extract: anti-cancer and anti-biofilm activities of Acca sellowiana.

In this study, the efficacy of Acca sellowiana fruit acetonic extract on human MDR cancer cells was tested for the first time, and it was demonstrated that the fruit extract is effective on both sensitive and resistant tumor cells. The effects of A. sellowiana extract on bacterial biofilm were also examined for the first time. By crystal violet assays and confocal microscopy analyses, it was demonstrated that the plant extract is able to strongly inhibit biofilm formation of both sensitive and resistant bacterial strains. Furthermore, antimicrobial activity assays and TEM analyses clearly demonstrated the effectiveness of plant extract on planktonic bacterial cells in both sensitive and resistant strains. Altogether, these findings intriguingly expand the panel of activities of A. sellowiana fruit extract with respect to previous reports, and open interesting perspectives to its therapeutic applications.

Biological effects from environmental pollution by toxic metals in the "land of fires" (Italy) assessed using the biomonitor species Lunularia cruciata L. (Dum).

The liverwort Lunularia cruciata was collected from the town of Acerra, in the heart of the so-called 'Land of Fires' a large area in the eastern part of Campania region of Italy affected by burning of waste and fraudulent dumping and one of the vertices of the "Italian Triangle of Death" so said for the high incidence and mortality from tumors. The data obtained from these samples were compared with samples collected in two other sites representing two different environmental conditions. The soil below the samples, and gametophytes, were collected and analyzed for the concentration of Al, As, Ba, Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Se, V. DNA damage, Reactive Oxygen Species production and localization, activity of antioxidant enzymes and presence of chelating molecules were investigated. All biomarkers provided an answer closely related to the pollution conditions at the 3 sites. We discuss the data considering the possibility of using these biological changes as environmental pollution biomarkers. Finally, it is underlined the importance of phytochelatins due to of their specificity for metal pollution.

Antimicrobial and antioxidant activity of proteins from Feijoa sellowiana Berg. fruit before and after in vitro gastrointestinal digestion.

In this paper we report for the first time a method for the extraction of the protein fraction from Feijoa sellowiana Berg. fruit and its electrophoretic analysis. In addition we analyzed the protein fraction for its antioxidant activity and its effectiveness against different Gram-positive and Gram-negative bacteria both as American type culture collection (ATCC) standard and clinically isolated strains. Both antimicrobial and antioxidant activity resulted stronger respect to that previously obtained for the acetonic extract from the same fruit. Further, we study both activities also on the product of in vitro gastrointestinal digestion of F. sellowiana fruit proteins. The results showed that in vitro gastrointestinal digestion increased 10 fold the antioxidant activity, while the antimicrobial activity, tested only on ATCC strains, resulted from 2 to 4 fold increased. MTT assays showed the non-toxicity of these proteins both before and after digestion.

Antimicrobial and antioxidant activities of coumarins from the roots of Ferulago campestris (Apiaceae).

We report the isolation of several coumarins and the stereochemical assessment of some pyranocoumarins, as well as the antibacterial and antioxidant activities of the three most abundant ones (grandivittin, agasyllin and aegelinol benzoate) isolated from the roots of Ferulago campestris collected in Sicily and of the hydrolysis product (aegelinol). Aegelinol and agasyllin showed antibacterial activity against nine ATCC and the same clinically isolated Gram-positive and Gram-negative bacterial strains. At a concentration between 16 and 125 mg/mL both coumarins showed a significant antibacterial effect against both Gram-negative and Gram-positive bacteria. In particular the ATCC strains Staphylococcus aureus, Salmonella thypii, Enterobacter cloacae and Enterobacter earogenes (MIC = 16 and 32 mg/mL for aegelinol and agasyllin, respectively) were the most inhibited. Antibacterial activity was also found against Helicobacter pylori: a dose-dependent inhibition was shown between 5 and 25 mg/mL. The antioxidant activity of the coumarins was evaluated by their effects on human whole blood leukocytes (WB) and on isolated polymorphonucleate (PMN) chemiluminescence (CL), PMA-stimulated and resting.

Phenol-Rich Feijoa sellowiana (Pineapple Guava) Extracts Protect Human Red Blood Cells from Mercury-Induced Cellular Toxicity.

Plant polyphenols, with broadly known antioxidant properties, represent very effective agents against environmental oxidative stressors, including mercury. This heavy metal irreversibly binds thiol groups, sequestering endogenous antioxidants, such as glutathione. Increased incidence of food-derived mercury is cause for concern, given the many severe downstream effects, ranging from kidney to cardiovascular diseases. Therefore, the possible beneficial properties of Feijoa sellowiana against mercury toxicity were tested using intact human red blood cells (RBC) incubated in the presence of HgCl2. Here, we show that phenol-rich (10-200 µg/mL) extracts from the Feijoa sellowiana fruit potently protect against mercury-induced toxicity and oxidative stress. Peel and pulp extracts are both able to counteract the oxidative stress and thiol decrease induced in RBC by mercury treatment. Nonetheless, the peel extract had a greater protective effect compared to the pulp, although to a different extent for the different markers analyzed, which is at least partially due to the greater proportion and diversity of polyphenols in the peel. Furthermore, Fejioa sellowiana extracts also prevent mercury-induced morphological changes, which are known to enhance the pro-coagulant activity of these cells. These novel findings provide biochemical bases for the pharmacological use of Fejioa sellowiana-based functional foods in preventing and combating mercury-related illnesses.

Salicylic Acid and Melatonin Alleviate the Effects of Heat Stress on Essential Oil Composition and Antioxidant Enzyme Activity in Mentha × piperita and Mentha arvensis L.

The aim of this study was to evaluate changes in the chemical profile of essential oils and antioxidant enzymes activity (catalase CAT, superoxide dismutase SOD, Glutathione S-transferases GST, and Peroxidase POX) in Mentha × piperita L. (Mitcham variety) and Mentha arvensis L. (var. piperascens), in response to heat stress. In addition, we used salicylic acid (SA) and melatonin (M), two brassinosteroids that play an important role in regulating physiological processes, to assess their potential to mitigate heat stress. In both species, the heat stress caused a variation in the composition of the essential oils and in the antioxidant enzymatic activity. Furthermore both Salicylic acid (SA) and melatonin (M) alleviated the effect of heat stress.

The phytochelatin synthase from Nitella mucronata (Charophyta) plays a role in the homeostatic control of iron(II)/(III).

Although some charophytes (sister group to land plants) have been shown to synthesize phytochelatins (PCs) in response to cadmium (Cd), the functional characterization of their phytochelatin synthase (PCS) is still completely lacking. To investigate the metal response and the presence of PCS in charophytes, we focused on the species Nitella mucronata. A 40 kDa immunoreactive PCS band was revealed in mono-dimensional western blot by using a polyclonal antibody against Arabidopsis thaliana PCS1. In two-dimensional western blot, the putative PCS showed various spots with acidic isoelectric points, presumably originated by post-translational modifications. Given the PCS constitutive expression in N. mucronata, we tested its possible involvement in the homeostasis of metallic micronutrients, using physiological concentrations of iron (Fe) and zinc (Zn), and verified its role in the detoxification of a non-essential metal, such as Cd. Neither in vivo nor in vitro exposure to Zn resulted in PCS activation and PC significant biosynthesis, while Fe(II)/(III) and Cd were able to activate the PCS in vitro, as well as to induce PC accumulation in vivo. While Cd toxicity was evident from electron microscopy observations, the normal morphology of cells and organelles following Fe treatments was preserved. The overall results support a function of PCS and PCs in managing Fe homeostasis in the carophyte N. mucronata.

Physiological and ultrastructural effects of acute ozone fumigation in the lichen Xanthoria parietina: the role of parietin and hydration state.

The physiological and ultrastructural effects induced by acute exposure to ozone (O3) were investigated in the lichen Xanthoria parietina. Our working hypothesis was that parietin content and hydration of the thalli may play a role in the modulation of the effects of O3 exposure. Four batches of X. parietina samples, dry and wet, with (P+) and without (P-) parietin, were fumigated for 1 h with 3 ppm O3. The effects of O3 were assessed immediately after the fumigation and after one week of recovery under controlled conditions. O3 fumigation caused physiological and ultrastructural impairment both to the photobiont and the mycobiont, irrespective if samples were fumigated wet or dry, and P+ or P-. However, one week after fumigation, a recovery was observed in P+ samples for the photobiont and in dry samples for the mycobiont. We suggest that the hydration state may play a major role in determining the severity of the damage, while the presence of parietin may promote the recovery. Our results provide physiological and ultrastructural basis to explain the ecological insensitivity of lichens to high environmental levels of ozone occurring during dry Mediterranean summers.

In-field and in-vitro study of the moss Leptodictyum riparium as bioindicator of toxic metal pollution in the aquatic environment: Ultrastructural damage, oxidative stress and HSP70 induction.

This study evaluates the effects of toxic metal pollution in the highly contaminated Sarno River (South Italy), by using the aquatic moss Leptodictyum riparium in bags at 3 representative sites of the river. Biological effects were assessed by metal bioaccumulation, ultrastructural changes, oxidative stress, as Reactive Oxygen Species (ROS) production and Glutathione S-transferase (GST) activity, as well as Heat Shock Proteins 70 (HSP70s) induction. The results showed that L. riparium is a valuable bioindicator for toxic metal pollution of water ecosystem, accumulating different amounts of toxic metals from the aquatic environment. Toxic metal pollution caused severe ultrastructural damage, as well as increased ROS production and induction of GST and HSP70s, in the samples exposed at the polluted sites. To assess the role and the effect of toxic metals on L. riparium, were also cultured in vitro with Cd, Cr, Cu, Fe, Ni, Pb, Zn at the same concentrations as measured at the 3 sites. Ultrastructure, ROS, GST, and HSP70s resulted severely affected by toxic metals. Based on our findings, we confirm L. riparium as a model organism in freshwater biomonitoring surveys, and GST and HSP70s as promising biomarkers of metal toxicity.