To get more insight into plant cell response to cadmium (Cd) stress, both proteomic and metabolomic "differential display" analyses were performed on Arabidopsis thaliana cells exposed to different concentrations of the toxic chemical. After a 24 h treatment, soluble proteins extracted from untreated and treated cells were separated by 2-D-PAGE and image analyses were performed to quantify and compare protein levels. Proteins up- and down-regulated in response to Cd were identified by MS and mapped into specific metabolic pathways and cellular processes, highlighting probable activation of the carbon, nitrogen, and sulfur metabolic pathways. For some of these proteins, Northern blot and RT-PCR analyses were performed to test transcript accumulation in response to Cd. In parallel, metabolite profiling analyses by LC coupled to ESI MS were initiated to better characterize the metabolic adaptation to the chemical stress. This study revealed that the main variation at the metabolite level came from the presence of six different families of phytochelatins, in A. thaliana cells treated with Cd, whose accumulation increases with Cd concentrations. Taken together these data provide an overview of the molecular and cellular changes elicited by Cd exposure.

Metabolomics is considered as an emerging new tool for functional proteomics in the identification of new protein function or in projects aiming at modeling whole cell metabolism. When combined with proteome studies, metabolite-profiling analyses revealed unanticipated insights into the yeast sulfur pathway. In response to cadmium, the observed overproduction of glutathione, essential for the detoxification of the metal, can be entirely accounted for by a marked drop in sulfur-containing protein synthesis and a redirection of sulfur metabolite fluxes to the glutathione pathway. A kinetic analysis showed sequential and dramatic changes in intermediate sulfur metabolite pools within the first hours of the treatment. Strikingly, whereas proteome and metabolic data were positively correlated under cadmium conditions, proteome and metabolic data were negatively correlated during other growth conditions, i.e. methionine supplementation or sulfate starvation. These differences can be explained by alternative mechanisms in the regulation of Met4, the activator of the sulfur pathway. Whereas Met4 activity is controlled by the cellular cysteine content in response to sulfur source and availability, the present study suggests that Met4 activation under cadmium conditions is cysteine-independent. The results clearly indicate that the metabolic state of a cell cannot be safely predicted based solely on proteomic and/or gene expression data. Combined metabolome and proteome studies are necessary to draw a comprehensive and integrated view of cell metabolism.Assimilable sulfur is essential for all living organisms. The cell requirement for sulfur can be fulfilled by the uptake of sulfur-containing amino acids or by assimilation of inorganic sulfur into organic compounds such as cysteine or homocysteine (1, 2). In yeast, homocysteine is the precursor of methionine through the methyl cycle and of cysteine through the transsulfuration pathway ( Fig. 1) (3). Cysteine is the sensor of the metabolic state in the sulfur amino acid pathway (4) and is required for the synthesis of GSH, an essential antioxidant molecule also important for detoxification.The yeast sulfur pathway has been extensively investigated at the genetic, enzymatic, and regulatory levels (3). The pools of most metabolites of the pathway have been analyzed (5, 6), and the K m values of many enzymes have been measured (3). However, some metabolic data such as the metabolite fluxes in the pathway and the concentration of the metabolites of the transsulfuration pathway (homocysteine and cystathionine) are lacking. Moreover, the levels of some sulfur metabolites are presumed to be modified in different mutants and under different physiological conditions (i.e. sulfur starvation, the presence of a sulfur metabolite, or a toxic metal in the medium), but the few quantitative data that are available are restricted to a small part of the pathway (5). Thus, it has been shown that cadmium (Cd 2ϩ ) strongly increases GSH synthesis (7), which is consistent with the primary impor...

This paper reports a liquid chromatographic/electrospray ionization mass spectrometric (LC/ESI-MS) method for profiling a wide range of structurally different sulfoconjugated compounds in urine and its application to the characterization of biomarkers for heavy metal toxicity in rat urine. Sulfoconjugates were first isolated by solid-phase extraction and the LC separation was performed on a reversed-phase column. Sulfoconjugates were detected in a triple-quadrupole mass spectrometer by simultaneously monitoring constant losses of 80 u (or 80 Th for doubly charged ions), precursors of m/z 80 (SO(3) (-*)) and precursors of m/z 97 (HSO4-). The ESI-MS detection conditions were optimized on dehydroepiandrosterone sulfate and estradiol sulfate and tested on other sulfoconjugates. The analysis of urine samples from humans and rats by using the developed method allowed the detection of about 15 peaks in each mode of detection. It was then applied to the investigation of heavy metal toxicity in rats. Comparative analysis of the chromatographic fingerprints of urine from control and uranium- and cadmium-treated rats showed several variations in the chromatographic pattern of the sulfoconjugates. Diagnostic m/z ratios were confirmed by analyzing individual urine samples and one of the observed variations seemed to be specific to uranium toxicity. The ion responsible for this variation has been identified as 4-ethylphenol sulfate by comparison of its chromatographic retention time and collision-induced dissociation mass spectra (MS(2) and MS(3) performed on a quadrupole ion trap instrument) with those of the synthesized compound.