Joaquim A.F. Vicente

Ecophysiological tolerance of duckweeds exposed to copper

Although essential for plants, copper can be toxic when present in supra-optimal concentrations. Metal polluted sites, due to their extreme conditions, can harbour tolerant species and/or ecotypes. In this work we aimed to compare the physiological responses to copper exposure and the uptake capacities of two species of duckweed, Lemna minor (Lm(EC1)) and Spirodela polyrrhiza (SP), from an abandoned uranium mine with an ecotype of L. minor (Lm(EC2)) from a non-contaminated pond. From the lowest Cu concentration exposure (25microM) to the highest (100microM), Lm(EC2) accumulated higher amounts of copper than Lm(EC1) and SP. Dose-response curves showed that Cu content accumulated by Lm(EC2) increases linearly with Cu treatment concentrations (r(2)=0.998) whereas quadratic models were more suitable for Lm(EC1) and SP (r(2)=0.999 and r(2)=0.998 for Lm(EC1) and SP, respectively). A significant concentration-dependent decline of chlorophyll a (chl a) and carotenoid occurred as a consequence of Cu exposure. These declines were significant for Lm(EC2) exposed to the lowest Cu concentration (25microM) whereas for Lm(EC1) and SP a significant decrease in chl a and carotenoids was observed only at 50 and 100microM-Cu. Electric conductivity (EC) and malondialdehyde (MDA) content increased after Cu exposure, indicating oxidative stress. Significant increase of EC was observed in Lm(EC2) for all Cu concentrations whereas the increase for Lm(EC1) and SP became significant only after an exposure to 50microM-Cu. On the contrary, for Lm(EC1), SP, and Lm(EC2), MDA content significantly increased even at the lowest concentration. Protein content and catalase (CAT) activity showed a decrease with an increase in Cu concentration. For the species Lm(EC1) and SP, a significant effect of copper on CAT activity was observed only at the highest concentration (100microM-Cu) whereas, for Lm(EC2), this effect started to be significant after an exposure to 50microM-Cu. Superoxide dismutase (SOD) activity increased with increasing concentrations of Cu, with a very similar trend between the three populations of duckweed. However, due to the facts that enzyme activity is expressed as units of activity per gram of protein and that protein content decreased with Cu exposure, the increase in SOD activity might partly result from a relative increase of this enzyme inside the pool of proteins. Consequently, the results obtained in our experimental conditions strongly suggest that duckweed species from the uranium-polluted area have developed mechanisms to cope with metal toxicity and that this tolerance is based on the existence of protective mechanism to limit the metal uptake rather than on an enhancement of the antioxidative metabolism.

Inoculation of Ni-Resistant Plant Growth Promoting Bacterium Psychrobacter sp. Strain SRS8 for the Improvement of Nickel Phytoextraction by Energy Crops

This study was conducted to elucidate effects of inoculating plant growth-promoting bacterium Psychrobacter sp. SRS8 on the growth and phytoextraction potential of energy crops Ricinus communis and Helianthus annuus in artificially Ni contaminated soils. The toxicity symptom in plants under Ni stress expressed as chlorophyll, protein content, growth inhibition, and Fe, P concentrations were studied, and the possible relationship among them were also discussed. The PGPB SRS8 was found capable of stimulating plant growth and Ni accumulation in both plant species. Further, the stimulation effect on plant biomass, chlorophyll, and protein content was concomitant with increased Fe and P assimilation from soil to plants. Further, the induction of catalase and peroxidase activities was also involved in the ability of SRS8 to increase the tolerance in both plant species under Ni stress. The findings suggest that strain SRS8 play an important role in promoting the growth and phytoextraction efficiency of R. communis and H. annuus, which may be used for remediation of metal contaminated sites.

Cisplatin impairs rat liver mitochondrial functions by inducing changes on membrane ion permeability: prevention by thiol group protecting agents.

Cisplatin (CisPt) is the most important platinum anticancer drug widely used in the treatment of head, neck, ovarian and testicular cancers. However, the mechanisms by which CisPt induces cytotoxicity, namely hepatotoxicity, are not completely understood. The goal of this study was to investigate the influence of CisPt on rat liver mitochondrial functions (Ca(2+)-induced mitochondrial permeability transition (MPT), mitochondrial bioenergetics, and mitochondrial oxidative stress) to better understand the mechanism underlying its hepatotoxicity. The effect of thiol group protecting agents and some antioxidants against CisPt-induced mitochondrial damage was also investigated. Treatment of rat liver mitochondria with CisPt (20nmol/mg protein) induced Ca(2+)-dependent mitochondrial swelling, depolarization of membrane potential (DeltaPsi), Ca(2+) release, and NAD(P)H fluorescence intensity decay. These effects were prevented by cyclosporine A (CyA), a potent and specific inhibitor of the MPT. In the concentration range of up to 40nmol/mg protein, CisPt slightly inhibited state 3 and stimulated state 2 and state 4 respiration rates using succinate as respiratory substrate. The respiratory indexes, respiratory control ratio (RCR) and ADP/O ratios, the DeltaPsi, and the ADP phosphorylation rate were also depressed. CisPt induced mitochondrial inner membrane permeabilization to protons (proton leak) but did not induce significant changes on mitochondrial H(2)O(2) generation. All the effects induced by CisPt on rat liver mitochondria were prevented by thiol group protecting agents namely, glutathione (GSH), dithiothreitol (DTT), N-acetyl-L-cysteine (NAC) and cysteine (CYS), whereas superoxide-dismutase (SOD), catalase (CAT) and ascorbate (ASC) were without effect. In conclusion, the anticancer drug CisPt: (1) increases the sensitivity of mitochondria to Ca(2+)-induced MPT; (2) interferes with mitochondrial bioenergetics by increasing mitochondrial inner membrane permeabilization to H(+); (3) does not significantly affect H(2)O(2) generation by mitochondria; (4) its mitochondrial damaging effects are protected by thiol group protecting agents. Based on these conclusions, it is possible to hypothesise that small changes on the redox-status of thiol groups, affecting membrane permeability to cations (Ca(2+) and H(+)) underlie CisPt-induced liver mitochondrial damage, putatively responsible for its hepatotoxicity. Therefore, we propose that CisPt-induced mitochondrial damage and consequent hepatotoxicity could be prevented by using thiol group protecting agents as therapeutic adjuvants.

Effects of 1,4-dihydropyridine derivatives (cerebrocrast, gammapyrone, glutapyrone, and diethone) on mitochondrial bioenergetics and oxidative stress: a comparative study.

The potential protective action of 1,4-dihydropyridine derivatives (cerebrocrast, gammapyrone, glutapyrone, and diethone) against oxidative stress was assessed on mitochondrial bioenergetics, inner membrane anion channel (IMAC), Ca2+-induced opening of the permeability transition pore (PTP), and oxidative damage induced by the oxidant pair adenosine diphosphate (ADP)/Fe2+ (lipid peroxidation) of mitochondria isolated from rat liver. By using succinate as the respiratory substrate, respiratory control ratio (RCR), ADP to oxygen ratio (ADP/O), state 3, state 4, and uncoupled respiration rates were not significantly affected by gammapyrone, glutapyrone, and diethone concentrations up to 100 microM. Cerebrocrast at concentrations higher than 25 microM depressed RCR, ADP/O, state 3, and uncoupled respiration rates, but increased three times state 4 respiration rate. The transmembrane potential (deltapsi) and the phosphate carrier rate were also decreased. At concentrations lower than 25 microM, cerebrocrast inhibited the mitochondrial IMAC and partially prevented Ca2+-induced opening of the mitochondrial PTP, whereas gammapyrone, glutapyrone, and diethone were without effect. Cerebrocrast, gammapyrone, and glutapyrone concentrations up to 100 microM did not affect ADP/Fe2+-induced lipid peroxidation of rat liver mitochondria, while very low diethone concentrations (up to 5 microM) inhibited it in a dose-dependent manner, as measured by oxygen consumption and thiobarbituric acid reactive substances formation. Diethone also prevented deltapsi dissipation due to lipid peroxidation initiated by ADP/Fe2+. It can be concluded that: none of the compounds interfere with mitochondrial bioenergetics at concentrations lower than 25 microM; cerebrocrast was the only compound that affected mitochondrial bioenergetics, but only for concentrations higher than 25 microM; at concentrations that did not affect mitochondrial bioenergetics (< or = 25 microM), only cerebrocrast inhibited the IMAC and partially prevented Ca2+-induced opening of the PTP; diethone was the only compound that expressed antioxidant activity at very low concentrations (< or = 5 microM). Cerebrocrast acting as an inhibitor of the IMAC and diethone acting as an antioxidant could provide effective protective roles in preventing mitochondria from oxidative damage, favoring their therapeutic interest in the treatment of several pathological situations known to be associated with cellular oxidative stress.

Toxicity assessment of the herbicide metolachlor comparative effects on bacterial and mitochondrial model systems.

Metolachlor is one of the most intensively used chloroacetamide herbicides. However, its effects on the environment and on non-target animals and humans as well as its interference at a cell/molecular level have not yet been fully elucidated. The aim of this study was: firstly, to evaluate the potential toxicity of metolachlor at a cell/subcellular level by using two in vitro biological model systems (a strain of Bacillus stearothermophilus and rat liver mitochondria); secondly, to evaluate the relative sensibility of these models to xenobiotics to reinforce their suitability for pollutant toxicity assessment. Our results show that metolachlor inhibits growth and impairs the respiratory activity of B.stearothermophilus at concentrations two to three orders of magnitude higher than those at which bacterial cells are affected by other pesticides. Also at concentrations significantly higher than those of other pesticides, metolachlor depressed the respiratory control ratio, membrane potential and respiration of rat liver mitochondria when malate/glutamate or succinate were used as respiratory substrates. Moreover, metolachlor impaired the respiratory activity of rat liver mitochondria in the same concentration range at which it inhibited bacterial respiratory system (0.4-5.0 micromol/mg of protein). In conclusion, the high concentration range at which metolachlor induces toxicity in vitro suggests that this compound is safer than other pesticides previously studied in our laboratory, using the same model systems. The good parallelism between metolachlor effects on both models and the toxicity data described in the literature, together with results obtained in our laboratory with other compounds, indicate the suitability of these systems to assess toxicity in vitro.

4,6=Dinitro=o=cresol Uncouples Oxidative Phosphorylation and Induces Membrane Permeability Transition in Rat Liver Mitochondria

The effect of the herbicide 4,6-dinitro-o-cresol (DNOC), a structural analogue of the classical protonophore 2,4-dinitrophenol, on the bioenergetics and inner membrane permeability of isolated rat liver mitochondria was studied. We observed that DNOC (10-50 microM) acts as a classical uncoupler of oxidative phosphorylation in rat liver mitochondria, promoting both an increase in succinate-supported mitochondrial respiration in the presence or absence of ADP and a decrease in transmembrane potential. The protonophoric activity of DNOC was evidenced by the induction of mitochondrial swelling in hyposmotic K(+)-acetate medium, in the presence of valinomycin. At higher concentrations (> 50 microM), DNOC also induces an inhibition of succinate-supported respiration, and a decrease in the activity of the succinate dehydrogenase can be observed. The addition of uncoupling concentrations of DNOC to Ca(2+)-loaded mitochondria treated with Ruthenium Red results in non-specific membrane permeabilization, as evidenced by mitochondrial swelling in isosmotic sucrose medium. Cyclosporin A, which inhibits mitochondrial permeability transition, prevented DNOC-induced mitochondrial swelling in the presence of Ca2+, which was accompanied by a decrease in mitochondrial membrane protein thiol content, owing to protein thiol oxidation. Catalase partially inhibits mitochondrial swelling and protein thiol oxidation, indicating the participation of mitochondrial-generated reactive oxygen species in this process. It is concluded that DNOC is a potent potent protonophore acting as a classical uncoupler of oxidative phosphorylation in rat liver mitochondria by dissipating the proton electrochemical gradient. Treatment of Ca(2+)-loaded mitochondria with uncoupling concentrations of DNOC results in mitochondrial permeability transition, associated with membrane protein thiol oxidation by reactive oxygen species.

The herbicide dicamba (2-methoxy-3,6-dichlorobenzoic acid) interacts with mitochondrial bioenergetic functions

The effects of dicamba, a widely used broad-leaf herbicide, on rat liver mitochondrial bioenergetic activities were examined. The results obtained for state 4 respiration indicate not only an uncoupling effect, the result of an increase on the permeability of inner mitochondria membrane to protons, but also a strong inhibitory effect on the redox complexes. State 3 and respiration uncoupled by FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone) were inhibited to approximately the same extent, i.e. by about 70%. Depression of respiratory activity is essentially mediated through partial inhibition of mitochondrial complexes II and III. ATPase activity was much less depressed by dicamba than ATP synthase activity. Therefore, a considerable part of the inhibition observed on ATP synthase is the result of an inhibition on the redox complexes. The loss of phosphorylation capacity, induced by dicamba, was in the last analysis not only the result of a direct effect of dicamba on the enzymatic complex (F0–F1 ATPase) but also the result of a deleterious effect on the integrity of the mitochondrial membrane, which can promote an inhibition of the respiratory complexes and an increase of the proton permeability of the inner membrane.

Comparative effects of the herbicide dinitro-o-cresol on mitochondrial bioenergetics

The herbicide dinitro-o-cresol (DNOC) was evaluated for its effects on bioenergetic activities of potato tuber mitochondria to elucidate its mechanism of action and to compare its toxicological properties with those of the chemically related uncoupler dinitrophenol (DNP). DNOC acts as a typical uncoupler, similarly to the classical uncouplers DNP and carbonylcyanide p-trifluoromethoxyphenyl-hydrazone (FCCP). Low concentrations of DNOC ( 100 μM) act also more efficiently than DNP simultaneously as a protonophore and inhibitor of respiration, especially when respiration is supported by substrates that are transported to the matrix. The efficiency of DNOC is decreased with increase of mitochondrial protein, BSA and exogenous orthophosphate. Although similar effects were observed for animal and plant mitochondria, rat-liver mitochondrial respiration was more sensitive to DNOC than plant mitochondria. Furthermore, in the presence of DNOC, liver mitochondria exhibited a higher state 3 respiratory coupling level than potato tuber mitochondria, as a result of a considerable stimulation (60%) of state 3 respiration. In conclusion, DNOC is a more potent mitochondrial uncoupler and respiratory chain inhibitor than DNP, although their chemical structures are very similar. Apparently, the additional methyl group of DNOC increases its efficiency as an uncoupler and as an inhibitor, as compared to DNP. Plant mitochondria were shown to be as useful as animal mitochondria in evaluating the toxicity of these xenobiotics.

Herbicides: the Face and the Reverse of the Coin. An in Vitro Approach to the Toxicity of Herbicides in Non-Target Organisms

I. Historical aspects, benefits and disadvantages of herbicide use During thousands of years up to about a hundred years ago, man expended most of required energy in arable farming with mechanical operations aiming to remove weeds and providing suitable conditions for the efficient growth of crop plants, considering that weeds compete with beneficial and desired vegetation, which means that weeds are plants growing where man does not which them to grow. With the dawn of industrialization, labor to the factories decreased manpower on the farms, which forced to think of more efficient mechanical means of weed control. The need of weed management is as old as agriculture itself. Six stages in the evolution of weed control practices can be considered: 1) 10,000 B.C.— removing weeds by hand; 2) 6,000 B.C.— the use of primitive hand tools to till the land and destroy weeds; 3) 1,000 B.C.— animal-powered implements like harrows; 4) 1920 A.D.— mechanically-powered implements like cultivators, blades, harrows, finger-weeders, rotary-hoes, rod-weeders, etc.; 5) 1930 A.D.— biological control and; 6) 1947 A.D.— chemical control, with the commercial development of organic herbicides such as 2,4-D and MCPA (Hay, 1974). Especially in the last century, for various reasons among which the population explosion, in his effort to produce adequate supplies of food, man needed to combat efficiently the attacks of various pests on agricultural and horticultural crops. Pesticides, falling into three major classes: insecticides, fungicides, and herbicides (or weed killers), are required. Herbicides, specifically, are used for control of weeds. At the end of the twentieth century, with an estimated world population of 6 billion people, some 700 million were undernourished and 1.3 billion exist on an inadequate diet. In 2009 FAO says that 1.02 billion people are undernourished, corresponding to 15 percent of the estimated world population of 6.8 billion. Undoubtedly, the first problem of Humankind is the lack of food, which affects especially underdeveloped countries. So, the urgent need for much greater application of herbicides and other agrochemicals is essential to increase food supply. Crops can duplicate or increase even more at the expenses of the agrochemicals use.

Comparative effects of 3,4-methylenedioxymethamphetamine and 4-methylthioamphetamine on rat liver mitochondrial function

Ecstasy, which is used as a recreational drug, is a common street name for 3, 4-methylenedioxymethamphetamine (MDMA). Another drug of abuse chemically related, though less common than MDMA, is the amphetamine derivative 4-methylthioamphetamine (MTA). MDMA and MTA induce different systemic and organ-specific effects, including neurotoxicity, hyperthermia, nephrotoxicity, cardiotoxicity and hepatotoxicity. Therefore, it is clear that MDMA and MTA are responsible for inducing organ toxicity. The mechanisms underlying MDMA and MTA-induced hepatotoxicity are multifactorial, and therefore not completely understood. Recent findings indicate interference with cellular bioenergetics as an important toxicological feature of ecstasy. However, less is known about the involvement of mitochondria in MTA-induced hepatotoxicity. Thus, we compared the direct influence of MDMA and MTA on rat liver mitochondrial function [mitochondrial permeability transition (MPT), mitochondrial oxidative stress, and mitochondrial bioenergetics]. It was shown that MTA (from 0.025 up to 0.1mM) was more potent than MDMA (from 0.2 up to 0.5mM) in decreasing the sensitivity of rat liver mitochondria to MPT. However, higher concentrations of MTA (from 0.5 up to 2mM) were highly toxic to mitochondria. MTA simultaneously increased H(2)O(2) production in a monoamine oxidase (MAO)-dependent way, and uncoupled and inhibited mitochondrial respiration. In contrast, MDMA had only limited or no effects on these mitochondrial parameters. According to these results, it is possible to postulate that, depending on the concentration, MTA can potentially be more efficient in its effects on liver mitochondria than MDMA and, also, that its harmful effects may contribute to its hepatotoxicity.