Intention, Goal, Background
The widespread and unconcerned use of chemicals in the past has led to an accumulation of pollutants in our environment. Numerous sites are polluted with a mixture of organic chemicals and heavy metals. The future use of these sites and the safe consumption of groundwater from these areas depends on our ability to assess risk by determining the bioavailability of trace levels of pollutants in the respective soil solutions. Soil eluates containing heavy metals in mixture as well as pure heavy metals in aqueous solution were added to a spruce cell culture to set up such a test system.
The present study aims at evaluating the response of cultured spruce cells to heavy metals in aqueous solution, and at characterizing these basic cellular responses as potential biomarkers.
In order to characterize cell reactions toward heavy metals, spruce cell cultures were incubated with CdSO4 (50 to 500 µM), Na2HAsO4 (1.5 to 80 µM) or PbCl2 (10 to 150 µM). Alternatively, the cells were incubated with a standard heavy metal mixture containing 80µM Na2HAsO4, 150 µM CdSO4 and 150 µM PbCl2 in medium and with aqueous original soil eluates. Measurement of oxidative stress, antioxidants and basic detoxification enzymes involved in plant defence reactions were performed with the treated cells.
Results and Discussion
After 5 hrs of incubation, the onset of a strong oxidative burst was observed. H2O2 concentrations exceeded 40 µM in the culture media after 20 hrs. Concomitantly, glutathione levels showed drastic changes indicating the influ-ence of the metals and/or the H2O2 on antioxidative systems. Following cadmium treatment, GSH and GSSG were elevated by 50 and 200% above controls. Whereas arsenic doubled GSSG levels, treatment with lead did not cause significant changes. However, a mixture of the metals decreased both metabolites by 50%. The effect of the metals was concentration-dependent and disappeared at high concentrations. Furthermore, strong induction of glutathione S-transferase (GST) subunits was observed and, although no novel subunit was expressed, the rise of a new GST isoform occurred. The most potent inducer of plant defence reactions is cadmium, followed by arsenate and lead in descending order of effectiveness. Counter ions seem to play an important role, e.g. lead chloride influenced the investigated parameters much more than lead acetate.
The investigated metals activate gene expression through signal transduction pathways previously not associated with these metals, which points to new end points for resistance and toxicity testing. Especially a monitoring of GST subunit behaviour together with quantifying the oxidative burst seem to be promising for a biomonitoring concept. The close regulation of plant answers observed may facilitate the setup of an integrated biotest for heavy metal pollution that could be based on enzymological as well as proteome data.
Recommendations and Outlook
Heavy metals cause stress to plant cells and elicit a whole range of answers, although specific for individual metal species. The differences observed in plant answers are suitable to distinguish between metals bioavailable in soil eluates and water samples, however only at concentrations in the µM range. It will be necessary to evaluate the effects on the RNA and transcript level. We recommend that similar plant metabolic end points and enzyme reactions be screened for their suitability as biotest systems.