L.F. De Filippis

The Effect of Sub-Lethal Concentrations of Mercury and Zinc on Chlorella: II. Photosynthesis and Pigment Composition

Summary The effects of either 1 mM ZnCl 2 , 1 μm HgCl 2 or 0.1 μM phenyl mercuric acetate solutions on photosynthesis, pigment composition and respiration are described for cultures of Chlorella . Although photosynthesis and respiration are initially inhibited in young cultures, there is a recovery towards control values as the cultures proceed in their growth. In young cultures the inhibition in photosynthesis is associated with a loss in chlorophyll. In cultures grown for several days in metal solutions, photosynthesis per unit mass of chlorophyll may exceed control values - in phenyl mercuric acetate solutions in particular. Mercury solutions markedly enhance protochlorophyll levels in young cultures, while decreasing chlorophyll a/b and pheophytin a/b ratios. These observations are interpreted in terms of the action of metals on the oxidative and reductive steps in the biosynthetic pathway of pigments.

The Effect of Sub-Lethal Concentrations of Mercury and Zinc on Chlorella I. Growth Characteristics and Uptake of Metals

Summary The effects of either 1 mM ZnCl 2 , 1 μM HgCl 2 or 0.1 μM phenylmercuric acetate solutions on a variety of growth parameters are described for cultures of the Emerson strain of Chlorella . A significant finding is the comparative gain in the net carbon balance of heavy metal treated cells due to the inhibition of export of carbon compounds such as glycollate from the cells. There is also a stimulation of RNA, DNA and protein levels in HgCl 2 treated cells. After an initial rapid uptake of mercury and zinc by cells, it appears that the daughter cells are able to regulate and prevent the further entry of these heavy metals into the biomass.

The Effect of Sub-Lethal Concentrations of Mercury and Zinc on Chlorella: III. Development and Possible Mechanisms of Resistance to Metals

Summary Chlorella cultures maintained continuously in replenished solutions of heavy metals (1 mM ZnCl 2 , 1 μM HgCl 2 or 0.1 μm phenyl mercuric acetate) initially exhibit symptoms of metal toxicity, which are characterised by a sharp reduction in the pigment content and in the rates of cell division and metabolic activity. There is a concurrent gain in the net carbon balance of the culture due to the inhibition of export of photosynthates from the cells by heavy metals ( De Filippis and Pallaghy, 1976a , De Filippis and Pallaghy, 1976b ). After a period of 40 days (approximating 40 series of cell division) the cultures develop resistance and almost fully regain the rates of cell division displayed by control cultures. In attempting to elucidate the mechanism of resistance, we studied the fate of the heavy metals in cultures of sensitive and resistant cells. Evidence from 65 Zn uptake studies indicate that resistance is accompanied by an inhibition of the temperature sensitive component of zinc uptake and by a reduction in the number of exchange sites available for zinc in the free space (cell walls) per unit dry weight of the cells. Tolerance is therefore characterised by the development of a typical exclusion mechanism. Mercury resistance is primarily associated with an increased capacity for resistant cells to volatilize mercury out of solution by enzymic means. The ethylene generated by metal stressed cultures (up to 1.3 pmol/mg dry wt/hr) is sufficient to account for approximately 25%, and a mercury-reducing enzyme system for the remainder of the mercury volatilized.

Localization of zinc and mercury in plant cells

Abstract The retention of zinc and mercury was examined in radioactively labelled plant tissues following either freeze-substitution or glutaraldehyde fixation. There were large losses of both 65Zn and 203Hg from glutaraldehyde fixed tissues, which could be largely prevented in the case of 65Zn only by saturating the fixation medium with sulphide ion. On the other hand, freeze-substitution resulted in the total retention of zinc and led to only a 4% loss of label from mercury treated tissues. Energy dispersive X-ray analysis of both freeze-substituted and glutaraldehyde fixed tissues enabled the subcellular localization of mercury and zinc in tissues treated with either 0.1mM HgCl2 or 50mM ZnCl2. In view of the findings described, it will be necessary to exercise caution if reliance is placed only on conventional procedures of electron microscopy when localizing mercury and zinc in environmentally contaminated biological materials. Physiological implications of this work are discussed.

The Effect of Sub-Lethal Concentrations of Mercury and Zinc on Chlorella IV. Characteristics of a General Reducing Enzyme System for Metallic Ions

Summary Chlorella cultures which have an induced resistance to mercuric chloride (HgCl 2 ) and phenyl mercuric acetate (PMA) exhibit greatly elevated levels of an enzyme system which apparently reduces both these mercury compounds to metallic mercury (Hg°). Therefore, such mercury resistant cells volatilize more mercury out of solution into the atmosphere than do untreated (sensitive) cells. Sensitive cells and zinc chloride (ZnCl 2 ) resistant cells show only a trace amount of the reducing enzyme. However, HgCl 2 resistant cells are also resistant to PMA and vice versa, while ZnCl 2 resistant cells are not resistant to either of these mercury compounds. The enzyme system has a requirement for sulphydryl group (-SH), but can operate at a reduced rate upon the addition of -SCH 3 groups under both aerobic or anaerobic conditions. The system can utilize NADPH or NADH as an energy source, although it appears to utilize NADH at only about half efficiency. The enzyme system is not only able to reduce mercury ions, but also a number of other metallic ions (depending on their electrode potentials), which suggests that the system is a general reducing enzyme for metallic ions (GR Enz.). The volatilization of mercury was demonstrated not to be due to a vitamin B 12 mediated mercury methylation process.

The Effect of Heavy Metals on the Absorption Spectra of Chlorella Cells and Chlorophyll Solutions

Summary The effect of 12 heavy metal compounds on the absorption spectrum of whole cells of Chorella , and of chlorophyll extracted in four different solvent systems is investigated. The study included metals already known to effect chlorophyll such as zinc chloride (ZnCl 2 ), mercuric chloride (HgCl 2 ), cupric chloride (CuCl 2 ), lead nitrate (Pb[NO 3 ] 2 ), methylmercuric chloride (MMC) and phenylmercuric acetate (PMA). All of the metal compounds tested decreased the level of chlorophyll in cells or in solvent extracts of chlorophyll, although the amount of degradation was highly solvent dependent with the greatest decrease being in methanol extracts. In whole cells of Chorella , MMC and PMA decreased the levels of chlorophyll more than in extracted chlorophyll, and this may be related to their greater ability to cross cell membranes. Cupric chloride was the only heavy metal ion tested that caused a shift in the chlorophyll spectrum in both whole cells and in solvent extracts. The most likely explanation for this is that copper is able to displace the magnesium from the chlorophyll molecule. The amount of chlorophyll degradation by metals was strongly correlated to their standard redox potentials, and the differences observed in the four solvent systems was correlated to their dielectric constants.

The Effect of Sub-Lethal Concentrations of Mercury and Zinc on Chlorella: V. The Counteraction of Metal Toxicity by Selenium and Sulphhydryl Compounds

Summary Sodium selenite (Na2SeO3) and some sulphydryl (-SH) compounds, including the amino acid cysteine protect Chlorella against the sub-lethal effects of mercuric chloride (HgCl2) and phenylmercuric acetate (PMA). However just the addition of selenium and the sulphur compounds to the culture medium containing zinc chloride (ZnCl2), HgCl2 or PMA provided little or no protection. But when Chlorella was incubated for six days in selenium and the sulphydryl compounds prior to metal treatments, then cells were markedly protected against mercury toxicity (but not zinc toxicity) as measured by increased cell division rates. Cells incubated in methionine (-SCH3) were only slightly protected, and cells incubated in the disulphide compound, cystine (-SS) were not protected at all. The increase in rates of cell division (protection) was strongly correlated to the amount of sulphydryl groups in the cells. Physiological implications of the work are discussed.

Localization of Organomercurials in Plant Cells

Summary The retention of zinc and mercury was examined in radioactively labelled plant tissues following either freeze-substitution or glutaraldehyde fixation. There were large losses of both 65Zn and 203Hg from glutaraldehyde fixed tissues, which could be largely prevented in the case of 65Zn only by saturating the fixation medium with sulphide ions. On the other hand, freeze-substitution resulted in the total retention of zinc and over 96% retention of label from mercury treated tissues. Energy dispersive X-ray analysis (EDAX) of freeze-substituted tissues enabled the subcellular localization of mercury in tissues treated with either 10 μM phenyl mercuric acetate (PMA) or 10 μM methyl mercuric chloride (MMC). In view of the findings described, it will be necessary to exercise caution if reliance is placed only on conventional procedures of electron microscopy when localizing zinc and mercury ions, as well as organomercurials, in environmentally contaminated biological materials. Physiological implications of this work are discussed.

A simple model for the non-enzymatic reduction and alkylation of mercuric salts in biological systems.

Data are given that confirm that mercury may be vaporized from 1.5 ..mu..M solutions of HgCl/sub 2/ either by air, or by a purified airstream containing 0.1 ..mu..l per liter (0.1 ppm) of ethylene or acetylene. The reaction is light sensitive, pH dependent, inhibited by 20 ..mu..M AgNO/sub 3/, but is largely independent of oxygen concentration. It is hypothesized that unsaturated hydrocarbons may play an important role in the reduction or alkylation of mercury compounds in biological systems and in the environment in general.

Non-enzymatic reduction of organomercurial salts in biological systems.

Mercury redox transformation reactions in nature may be biological (enzymic) or nonbiological (nonenzymic). Biological mercury transformation has usually been explained in terms of the action of a number of specialized enzymes. While nonbiological transformation has been explained in terms of the action of certain biological products such as methylcobalamin (methyl vitamin B/sub 12/), methionine, ascorbate, humic acid and some proteins. It has been shown that mercuric salts (Hg/sup + +/) can be reduced and alkylated by the action of ethylene and acetylene (two known reducing agents in air), and it has been established that mercuric salts are reduced by propene and other higher molecular weight unsaturated hydrocarbons (olefins).