The effects of sublethal additions of mercuric chloride were studied in the marine diatom Skaletonema costatug (Grev.) Cleve grown in ammonium-limited chemostats and batch cultures. In the short-term Hg exposure (up to 5 hours), unexposed chemostat effluents were simultaneously perturbed with 5 μM NH₄Cl and Hg concentrations ranging from 0.04 to 5.53 nM HgCl₂. In the long-term Hg exposure (679. 5 hours), ammonium-starved effluents were only perturbed with 5 μM NH₄Cl. In the short-term Hg exposure, when the effluent from the chemostat culture was starved for 1.5 hours, Hg decreased the affinity for the substrate (increased Ks value) and the rate of ammonium assimilation or the internally controlled uptake rate, Vimax . When the effluent was starved for 30 hours, only Vimax was reduced. These effects occurred between 1.84 and 3,68, and at 0,18 nM HgCl₂ in effluents starved for 1.5 and 30 hours, respectively. The maximum rate of uptake, Vs, was not depressed. In the long-term Hg exposure, at least 0.37 nH HgCl₂ decreased the specific growth rate and the maximum cell density, while the chlorophyll a per cell increased. A period of population decline was followed by resumption of growth. Morphological alterations were observed before and after the recovery. In the long-term experiment, six days of continual exposure to 0.37 nM HgCl₂ gradually increased the Ks value without affecting Vs and Vimax. The results from exposure to 3.68 nM HgCl₂ were similar to the short-term Hg exposure, since both the substrate affinity (Ks value) and the assimilatory rate Vimax were impaired. In addition, the maximal uptake rate, Vs, was also reduced after exposure to 3.68 nM HgCl₂ for six days in the long-term experiment. After resumption of growth in the Hg-treated cultures, when a new steady-state was established, the affinity for the substrate and assimilatory rates increased in phase D (day 23) compared to phase A (day 6). The recovery of growth and nutrient uptake rates in phase D, may have been partially mediated by the acquisition of Hg tolerance and the appearance of cells of a different stage of the sexual life cycle, as suggested by differences in cell size and chemical composition. An attempt was made to determine whether a short-term physiological response (Hg induction of metallothionein synthesis) could be responsible for the recovery. The 250 nm absorbance profile, of nutrient-saturated cultures exposed for 90 to 116 hours to sublethal concentrations of mercury, showed no large absorbance peak in the medium molecular weight pool, corresponding to laetallothionein, as it occurs in animals exposed to heavy metals. The intracellular distribution and levels of Cu, Zn, and Hg in S. costatum, grown in nutrient-saturated batch cultures, were affected by 0.37 nM HgCl₂. A concentration equal to or greater than 1.84 nM HgCl₂ reduced the growth rate and cell density, possibly due to the accumulation of Hg in the high m.w. pool. Exposure to 1.84 nM HgCl₂ prior to a second addition of 5.53 nM reduced Hg levels in ths high m.w. pool. Upon Hg exposure, Zn levels decreased in the high and low m.w. fractions but gradually increased in the medium m.w. pool. Copper slightly increased in the high m.w. pool but remained constant in the medium and low m.w. pools, in relation to total intracellular levels. High levels of Cu and Zn in the low m.w. pool suggests that a substance of a lower m.w., than usually reported for metallothionein, may be involved in the storage and detoxification of heavy metals in S. costatum. / Science, Faculty of / Botany, Department of / Graduate

Splenic and lymph node lymphocytes from Balb/C mice were activated in vitro by the heavy metal cations, Zn⁺⁺ and Hg⁺⁺, as noted by the several-fold increases in ³H-thymidine incorporation at 144 hours of culture. Optimal mitogenic concentrations of Zn⁺⁺ and Hg⁺⁺ were 200 μM and 10 μM, respectively. Data from experiments in which three different methods were used to enrich for either T or B splenic lymphocytes, i.e. cell passage over nylon wool columns, use of athymic Nu/Nu mouse spleen cells, or cell lysis with monoclonal anti-Thy-1 antibody plus complement, suggested that Zn⁺⁺ and Hg⁺⁺ were mitogens for T cells. Removal of macrophages from spleen cells by treatment with carbonyl iron followed by cell passage through nylon wool eliminated the lymphocyte responses to Zn⁺⁺ and to Hg⁺⁺. Moreover, addition of these macrophage-depleted lymphocytes to monolayers of resident peritoneal macrophages restored the lymphocyte responses to these mitogens. Both Zn⁺⁺ and Hg⁺⁺ activated splenic lymphocytes to display lectin-dependent cytotoxicity and to produce gamma interferon. Furthermore, Zn⁺⁺ induced low levels of natural killer activity in spleen cells. In contrast to spleen and lymph node cells, thymocytes and bone marrow lymphocytes did not respond to either cation under standard culture conditions. However, when cultured in the presence of E. coli-derived lipopolysaccharide (LPS) and 2-mercaptoethanol for 144 hours, thymocytes were activated by Zn⁺⁺ (200 μM) but not by Hg⁺⁺. Quantities of LPS as low as 1.0 ng/ml satisfied this culture requirement. Purified interleukin 1 could not replace the helping activity mediated by LPS. Thymocyte subpopulation studies showed that Zn⁺⁺ activated enriched peanut lectin receptor-negative mature thymocytes, but LPS was required for the response. Spleen cells from mice, intraperitoneally injected with ZnCl₂ for 7 to 14 days, were not activated in vivo as assessed by ³H-thymidine incorporation in vitro, nor did they display enhanced responses to T-cell or B-cell mitogens. However, zinc administration had negative effects by decreasing spleen cell numbers by 31% and thymic weight by 59%. A theoretical model is presented in which Zn⁺⁺ and Hg⁺⁺ may mediate their stimulating effects in vitro by altering histocompatibility "self" structures on the surface of lymphocytes and macrophages via interactions with sulfhydryl groups on these structures to which T lymphocytes with receptors for "altered self" structures respond with proliferation or cytotoxicity.