VIII. Mechanisms of Metal Detoxification
As already noted, individuals of the oligochaete, Limnodrilus hoffmeisteri, from metal-polluted Foundry Cove have evolved resistance to a combination of Cd, Ni, and Co (Klerks & Levinton 1989a,b). Furthermore, the data presented in Chapter VII demonstrated that this resistance was not achieved by a reduced cadmium accumulation. Since Foundry Cove worms have higher uptake rates than their non-resistant conspecifics from South Cove, resistance must be due to differences in metal detoxification.
One possible metal detoxification mechanism involves the production of metallothioneins (MTs). MTs are low molecular weight cytosolic proteins. They are induced by exposure to several metals including cadmium, mercury, zinc, copper, gold, and silver. MTs have high affinities to these metals and this binding may constitute a metal detoxification mechanism; MTs may have other roles in addition to metal detoxification, i.e.., metal detoxification may not be their primary role. The idea that binding of MTs to metals is a detoxification mechanism is supported by several lines of evidence: (1) an elevated metal resistance after an initial pre-exposure resulted in elevated MT levels, (2) protection against copper poisoning in yeast after the insertion of a monkey MT-gene, (3) reduced copper resistance in yeast after removal of the MT-gene (primary references can be found in Klerks & Bartholomew 1991).
Another possible detoxification mechanism, not exclusive from MT binding, is the sequestering of metals in vesicles, lysosomes, or other membrane-bound structures. Sequestration of a metal will constitute a detoxification mechanism if the process keeps the metal from interacting at sensitive sites in the organism. Support for this detoxification mechanism comes from a study of a copper-resistant population of the isopod crustacean, Asellus meridianus (Brown 1977), in which elevated Cu levels were found in granules and 'dense spherical inclusions'(= other round membrane-bound structures).
What was the physiological mechanism of Cd detoxification in Limnodrilus hoffmeisteri? Worms were collected and reared as described in Chapter VII. Metal accumulation was determined after exposures to either Cd-rich sediment or the radioisotope 109Cd in water. To compare cadmium detoxification, subcellular fractionation and gel permeation of 109Cd exposed worms were utilized to quantify the amounts of cadmium that are bound to MT-like proteins; electron microprobe analyses were used to determine if Cd was sequestered into granules (Klerks & Bartholomew 1991). These methods and results will be outlined and discussed below.
Methods: Subcellular fractionation
The homogenized worm samples from the 109Cd accumulation in water experiment (refer to Chapter VII for details) were individually separated by centrifugation into a 'debris' fraction (200 g pellet not used in further analyses), a particulate fraction (up to 100,000 g pellets), and a cytosol fraction (the 100,000 g supernatant). The pellets were resuspended in 0.5 ml of 50mM Tris-HCl buffer. Cadmium concentrations of the particulate fraction and the cytosol were determined by gamma counting; cytosol samples were then frozen at -80oC.
The cytosol samples from the subcellular fractionation step were separated by HPLC gel-permeation to compare the cytosolic Cd distributions. The cytosol samples were first thawed and filtered. The HPLC gel-permeation separations were done on cytosol subsamples ranging from 465 to 700 µl on a Toyo Soda column with 259 mM Tris-HCl buffer. For each sample, 65 fractions of 1ml were collected and analyzed by gamma counting. The separation characteristics of the HPLC system were then compared to proteins of known molecular weight.
Methods: Electron microprobe analyses
Fixed and embedded cross sections of Foundry Cove worms were studied with the aid of scanning electron microscopy (SEM) and electron probe microanalysis (EPMA) to examine the physical and chemical nature of Cd sequestering. Each worm cross section was coated with an electrically conductive material (in this case, aluminum). Backscattered electron imaging was used in order to identify density contrasts; such contrasts would identify the location of tissues with high concentrations of metals and other high density structures. Quantitative microanalysis methods were then employed to determine metal levels. Intensities were compared to mineral standards.
The worms from Foundry Cove accumulated more cadmium in their cytosol; the Cd distributions within the cytosol revealed that much of the Cd was associated with a protein with an apparent molecular weight of 16,000. This molecular weight as well as the high affinity for Cd are consistent with this protein being a MT; we refer to it as an MT-like protein since further characterization is required for unambiguous classification as an MT. The increased binding of Cd to an MT-like protein in Foundry Cove worms and their offspring is consistent with their elevated resistance (Klerks & Bartholomew 1991). However, these data did not show any evidence that increased binding to this protein resulted in lower Cd levels in other subcellular components. Therefore, Klerks & Bartholomew decided to look at other possible detoxification mechanisms such as sequestration of Cd into granules.
Table 1A. Cadmium Accumulation and subcellular distribution in Limnodrilus hoffmeisteri exposed to 109Cd (values are nmol Cd per g wet tissue).
|Fraction||South Cove||Foundry Cove|
|Cytosol||218 +/- 10||522 +/- 50|
|High Molecular Weight||26 +/- 5||26 +/- 3|
|Metallothionein?||55 +/- 4||229 +/- 13|
|Low Molecular Weight||96 +/- 14||208 +/- 26|
Table 1B. Cadmium Accumulation and subcellular distribution in Limnodrilus hoffmeisteri exposed to 109Cd (values are nmol Cd per g wet tissue).
|Fraction||South Cove||Foundry Cove|
|Total Homogenate||1010 +/- 157||2040 +/- 46|
|Particulate||594 +/- 98||1251 +/- 83|
|Cytosol||218 +/- 10||522 +/- 50|
Foundry Cove worms and their offspring had more Cd in the particulate subcellular pool when compared to South Cove worms; this indicates that Cd sequestration may also play a role in the resistance. The SEM and EPMA data also indicate that Cd is sequestered into granules in Foundry Cove worms. Two distinct types of granules were observed: high density granules found in most body tissues and low density granules found only in the chloragog tissue surrounding the gut. These granules were often found as aggregations; chemical analyses indicated that these large aggregations were composed of Cd and S in a 1:1 ratio.
After being exposed to cadmium, Limnodrilus hoffmeisteri from Foundry Cove had much higher levels of a Cd-binding protein than their conspecifics from the control area. Preliminary results indicate this protein to be an MT; the higher levels of the MT-like protein in worms from Foundry Cove and in their second generation offspring (reared in clean sediment) relative to the worms from South Cove indicate that the increased level of MT-like proteins could be responsible for the increased resistance of Foundry Cove worms. Microscopic analyses indicated that the higher density granules consisted primarily of Cd and S in 1:1 molar ratio which suggests cadmium sulfate. The research to date indicates that both the binding to an MT-like protein and sequestration in granules plays a role in cadmium detoxification in resistant L. hoffmeisteri.