Despite their ubiquitous distribution, the exact functions of metallothioneins (MTs) are still not known. We analyzed the expression of four novel barley (Hordeum vulgare L.) genes coding for MTs, comparing two different situations: (i) leaf senescence, and (ii) heavy metal stress. Physiological analysis of chlorophyll content and photosystem II efficiency revealed similar stress response during natural leaf senescence and short‐term heavy metal treatment with toxic concentrations of Cu and Cd. Gene expression patterns of the newly identified MTs (HvMT‐1a, HvMT‐2a, HvMT‐2b and HvMT‐3a) clearly vary under these different conditions, suggesting specific roles for the barley MTs during leaf senescence and heavy metal stress. While HvMT‐1a is induced during leaf senescence and after heavy metal treatment but not during metal deficiency, expression of HvMT‐2a is not affected under any of conditions tested. The MT HvMT‐3a is only induced in response to Cu deficiency and HvMT‐2b is downregulated in response to Cd stress.

We performed an interspecies comparison for the human hepatoma cell line HepG2 and the eukaryotic single cell organism Tetrahymena pyriformis (T. pyriformis) for 17 xenobiotics with diverse structures and four metals. The cytotoxicity was assessed by four different cell viability assays (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide reduction (MTT), neutral red uptake (NRU), resazurin dye (AlamarBlue), 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM)) for the HepG2 and by cell count and MTT for T. pyriformis. For HepG2 cells, the results revealed interassay variations depending on the compound. The highest assay conformity was found for the metal Hg(2+) and the fungicide prochloraz. The AlamarBlue assay was the most sensitive assay according to low-effect concentrations. By contrast, the NRU assay was comprised of more frequent whole concentration response relationships and was more susceptible to EC(50). For T. pyriformis the EC(50) values of the two applied assays displayed a high conformity (R(2) = 0.97). Comparing the EC(50) values obtained by the MTT assay for the two cell models, a direct correlation was absent for the xenobiotics and only present for the metals (Cd(2+), Cu(2+), and Ni(2+)). Moreover, the protozoa T. pyriformis displayed a 20 times higher sensitivity than the cell line. The highest interspecies difference of three log degrees was obtained for the polycyclic aromatic hydrocarbon fluoranthene. In addition, a correlation of the EC(50) values and octanol-water partition coefficient (log K(OW)) of the xenobiotics was performed. No correlation was found for HepG2, and a weak one for T. pyriformis. Interestingly, the interspecies difference of logarithmized EC(50) correlated positive with the log K(OW) (R(2) = 0.65). In conclusion, to obtain reliable evidence for human cytotoxicity, more than one viability/cytotoxicity assay had to be applied for cell lines. Second, the human hepatoma cell line was less affected by the organic compounds than the eukaryotic single-cell organism and was also less dependent on the log K(OW) of the xenobiotic.

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