Isolation and characterization of a cadmium binding protein from the planarian, Dugesia dorotocephala, and evaluation of its protective effects against DNA damage caused by cadmium and radiation

Abstract

Cadmium contamination of the environment occurs as a result of mining and smelting operations, industrial use of the metal and inappropriate disposal of cadmium-containing products. The health effects related to cadmium exposure are numerous and varied, with manifestations of neuropathy, hepatotoxicity, bone mineralization disorders, neurotoxicity, cardiovascular disease, immunological disorders, genetic mutations, teratogenesis and carcinogenesis. The planarian, Dugesia dorotocephala, has been shown to have a high incidence of malignant post-pharyngeal tumors following exposure to cadmium and the tumor-promoting phorbol ester, TPA. This makes the planarian an attractive model to study the mechanisms of carcinogenesis in a simple multicellular organism. Mammalian species exposed to cadmium respond with the induction of a low molecular weight, heat stable protein, metallothionein, which has a high cysteine content and is capable of binding large amounts of cadmium. Metallothionein has never been isolated nor characterized in planaria. This provided the basis for the initial hypothesis that the planarian, D. dorotocephala, produces metallothionein or a metallothionein-like protein that is capable of binding cadmium. In addition, the protein should be inducible with exposure to zinc and cadmium, as has been shown for mammalian metallothionein. It was further hypothesized that planarian metallothionein would be capable of binding cadmium and would thus reduce DNA damage caused by cadmium exposure. Following exposure to sub-lethal doses of either cadmium or zinc, planaria were homogenized. The resulting homogenate was then subjected to centrifugation to isolate cytosol, which was then heat treated and re-centrifuged to isolate heat stable cytosolic proteins. The heat stable cytosolic proteins were then separated using gel chromatography. Separated proteins were then subjected to spectrophotometric and atomic absorption analysis (AA) to identify a cadmium-binding protein that had high 254 nm and low 280 nm absorbance. The molecular weight of this cadmium-binding protein was determined to be approximately 7400 Da by gel filtration chromatography. The isolated protein was found to have a high thiol content. The protein was further purified by diethylaminoethylcellulose (DEAE) ion exchange high-performance liquid chromatography (HPLC). The amino acid content of the purified protein was determined and was found to contain 28% cysteine. These results are consistent with the cysteine content of mammalian metallothionein. Thus, planaria were shown to produce a low molecular weight protein like mammalian metallothionein that was heat stable, bound cadmium and had a high thiol content primarily in the form of cysteine. Because mammalian metallothionein is inducible by exposure to a variety of metals, including zinc and cadmium, planaria were exposed to non-lethal doses of different metals with the planarian metallothionein levels determined by AA analysis of the cadmium-saturated protein. Planarian metallothionein was shown to be inducible following exposure to metals and hydroperoxides like benzoyl peroxide. To test the hypothesis that planarian metallothionein could reduce DNA damage, planaria were pre-treated with zinc for 5 days to increase planarian metallothionein levels. Pre-treated and non pre-treated planaria were then exposed to different concentrations of Cd ranging from 100 to 2,500 μg/L for varying periods of time up to 48 hours. DNA fragmentation was assessed using a modified alkaline agarose gel electrophoresis assay. Damage to DNA was quantified using densitometric analysis of the mean migration distance. DNA from planaria pre-treated with zinc before cadmium exposure had less DNA fragmentation than planaria not pre-treated with zinc before cadmium exposure. Decreases in electrophoretic migration of DNA of 2.4- to 2.6-fold were associated with increases of planarian metallothionein of 2.5- to 4.5-fold due to zinc treatment. Similar results were noted in planaria pre-treated with a cocktail of buthionine sulfoximine (BSO) and diethylmaleate (DEM) to deplete glutathione. Planaria treated with BSO, DEM and zinc had less DNA fragmentation than planaria treated with BSO and DEM but not zinc. Using the same techniques for measuring DNA fragmentation in planaria, the hypothesis that ionizing radiation both induced planarian metallothionein and protected against DNA fragmentation was tested. Three days after planaria were irradiated with 1.0 to 6.0 Gy from a cesium -137 source, there was a significant increase in metallothionein levels that ranged between 12.8- and 14.4-fold over protein levels measured in controls. There was also a significant decrease (ranging from 1.4- to 3.2-fold) in the amount of DNA fragmentation in planaria pre-exposed to the radiation source before being exposed to varying doses of ionizing radiation compared with DNA damage measured in planaria not pre-exposed to ionizing radiation. In summary, the planarian, D. dorotocephala, was shown to have a metallothionein that is a low molecular weight protein, that is heat stable and binds cadmium. Furthermore, the protein was shown to be inducible with exposure to metals, hydroperoxides and radiation, as is mammalian metallothionein. Furthermore, it was shown that induction of the planarian metallothionein significantly reduced cadmium- and radiation-initiated DNA fragmentation. Although the planarian, D. dorotocephala, is an invertebrate, isolation and characterization of metallothionein from this organism show that its cells react to cadmium exposure like mammalian cells. Moreover this discovery helps to further validate the organism as an alternative model to study Cd-induced cancer.