a b s t r a c tAlthough 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 (25 M) to the highest (100 M), 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 concentrationdependent 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 (25 M) whereas for Lm(EC1) and SP a significant decrease in chl a and carotenoids was observed only at 50 and 100 M-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 50 M-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 (100 M-Cu) whereas, for Lm(EC2), this effect started to be significant after an exposure to 50 M-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.

Paraquat herbicide is toxic to animals, including humans, via putative toxicity mechanisms associated to microsomal and mitochondrial redox systems. It is also believed to act in plants by generating highly reactive oxygen free radicals from electrons of photosystem I on exposure to light. Paraquat also acts on non-chlorophyllous plant tissues, where mitochondria are candidate targets, as in animal tissues. Therefore, we compared the interaction of paraquat with the mitochondrial bioenergetics of potato tuber, using rat liver mitochondria as a reference. Paraquat depressed succinate-dependent mitochondrial Delta(psi), with simultaneous stimulation of state 4 O2 consumption. It also induced a slow time-dependent effect for respiration of succinate, exogenous NADH, and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD)/ascorbate, which was more pronounced in rat than in potato mitochondria. However, with potato tuber mitochondria, the Delta(psi) promoted by complex-I-dependent respiration is insensitive to this effect, indicating a protection against paraquat radical afforded by complex I redox activity, which was just the reverse of to the findings for rat liver mitochondria. The experimental set up with the tetraphenyl phosphonium (TPP+)-electrode also indicated production of the paraquat radical in mitochondria, also suggesting its accessibility to the outside space. The different activities of protective antioxidant agents can contribute to explain the different sensitivities of both kinds of mitochondria. Values of SOD activity and alpha-tocopherol detected in potato mitochondria were significantly higher than in rat mitochondria, which, in turn, revealed higher values of lipid peroxidation induced by paraquat.

The effects of tetrandrine (6,6 0 , 7,12-tetramethoxy-2, 2 0 -dimethyl-berbaman) on the mitochondrial function were assessed on oxidative stress, mitochondrial permeability transition (MPT), and bioenergetics of rat liver mitochondria. At concentrations lower than 100 nmol/ mg protein, tetrandrine decreased the hydrogen peroxide formation, the extent of lipid peroxidation, the susceptibility to Ca 2+ -induced opening of MPT pore, and inhibited the inner membrane anion channel activity, not significantly affecting the mitochondrial bioenergetics. High tetrandrine concentrations (100-300 nmol/mg protein) stimulated succinate-dependent state 4 respiration, while some inhibition was observed for state 3 and p-trifluoromethoxyphenylhydrazone-uncoupled respirations. The respiratory control ratio and the transmembrane potential were depressed but the adenosine diphosphate to oxygen (ADP/O) ratio was less affected. A slight increase of the inner mitochondrial membrane permeability to H + and K + by tetrandrine was also observed. It was concluded that low concentrations of tetrandrine afford protection against liver mitochondria injury promoted by oxidative-stress events, such as hydrogen peroxide production, lipid peroxidation, and induction of MPT. Conversely, high tetrandrine concentrations revealed toxicological effects expressed by interference with mitochondrial bioenergetics, as a consequence of some inner membrane permeability to H + and K + and inhibition of the electron flux in the respiratory chain. The direct immediate protective role of tetrandrine against mitochondrial oxidative stress may be relevant to clarify the mechanisms responsible for its multiple pharmacological actions.

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SummaryThe effects of the herbicides 1,1 0 -dimethyl-4,4 0 -bipyridylium dichloride (paraquat), 3,6-dichloro-2-metoxybenzoic acid (dicamba) and 2,4-dichlorophenoxyacetic acid (2,4-D) on cell growth of non-green potato tuber calli are described. We attempted to relate the effects with toxicity, in particular the enzymes committed to the cellular antioxidant system. Cell cultures were exposed to the herbicides for a period of 4 weeks. Cellular integrity on the basis of fluorescein release was strongly affected by 2,4-D, followed by dicamba, and was not affected by paraquat. However, the three herbicides decreased the energy charge, with paraquat and 2,4-D being very efficient. Paraquat induced catalase (CAT) activity at low concentrations (1 mM), whereas at higher concentrations, inhibition was observed. Dicamba and 2,4-D stimulated CAT as a function of concentration. Superoxide dismutase (SOD) activity was strongly stimulated by paraquat, whereas dicamba and 2,4-D were efficient only at higher concentrations. Glutathione reductase (GR) activity was induced by all the herbicides, suggesting that glutathione and glutathione-dependent enzymes are putatively involved in the detoxification of these herbicides. Paraquat slightly inhibited glutathione S-transferase (GST), whereas 2,4-D and dicamba promoted significant activation. These results indicate that the detoxifying mechanisms for 2,4-D and dicamba may be different from the mechanisms of paraquat detoxification. However, the main cause of cell death induced by paraquat and 2,4-D is putatively related with the cell energy charge decrease.