Isolation and characterization of rare earth element-binding protein in roots of maize.

Rare earth element-binding protein was isolated from maize, which was grown under greenhouse conditions and characterized in terms of molecular weight, amino acid composition, and ultraviolet absorption. The molecular weight of the maize protein was determined to be 183,000, with two distinct subunits of approximately molecular weights of 22,000 and 69,000, respectively. The protein is particularly rich in asparagine/aspartic acid, glutamine/glutamic acid, glycine, alanine, and leucine and contains 8.0% of covalently bound carbohydrate. The ultraviolet absorption of the protein is low at 280 nm and no change in the adsorption was observed with a change in pH. Compared to the unique features of the metallothioneins with a molecular weight of approximately 10,000, a high cysteine content of 30%, high absorption at 254 nm and a low absorption at 280 nm, and absorption change with pH, the REE-binding protein is unlikely to be plant metallothionein in nature. It was found that an almost twofold greater concentration was found for most of the REEs in the protein isolated from the maize with REE fertilizer use than that without REE fertilizer. This study suggests that the REE-binding protein is a glycoprotein and REEs can be firmly bound with the protein of maize roots.

Speciation of rare earth elements in soil and accumulation by wheat with rare earth fertilizer application.

A greenhouse study was conducted to investigate the accumulation of rare earth elements (REEs), La, Ce, Pr and Nd, in winter wheat (Triticum aestivum L.), and the speciation of these elements in soil following the application of REE-based fertilizers. Improved crop yield was confirmed by the experiment. The accumulation behavior of La, Ce, Pr and Nd in wheat varied depending on the concentration of REE fertilizer application, i.e. increased with increasing REE concentration at low fertilization application, constant over the medium REE range, and decreased with increasing REE concentration at high fertilizer application. Significant negative correlation was obtained between REE contents in roots and soil pH (r = -0.5787 to -0.8442 for La). REEs in both the fertilized and unfertilized soils were fractionated by a three-stage sequential extraction procedure into three chemically distinct fractions: water soluble, exchangeable and carbonate bound (B1), Fe-Mn oxide bound (B2), and organic and sulfide bound (B3). REEs in fertilized soils were found mainly in the B2 and B3 fractions, with only a small amount in the B1 fraction. REEs in B1 and B2 fractions were negatively correlated with soil pH (r = -0.6892 to -0.8927 and -0.7462 to -0.9482). Significant correlation was obtained between REEs in B1 fraction and REE contents in root. The correlation coefficients ranged from 0.6159 to 0.7410 when fertilizer application was lower than 20.0 mg/kg soil. No acceptable relationship was observed between REE contents in shoot and any of the extractable fractions in soils.

Evaluation of plant availability of soil trace metals by chemical fractionation and multiple regression analysis.

Soil samples with a range of chemical and physical properties were collected from 10 different rural regions of China. Trace metals (Ni, Co, Cu, and Pb) in the soils were partitioned by a sequential extraction procedure into Mg(NO(3))(2) extractable (F1), CH(3)COONa extractable (F2), NH(2)OH.HCl extractable (F3), HNO(3)?H(2)O(2) extractable (F4), and residual (F5) fractions. Chemical fractionation showed that F1 fraction of the metals was less than 1% and residue was the dominant form for Cu and Ni in all samples, and for Co in most of the samples. Significant interrelationships of the fractions varied considerably with the different metals. Winter wheat (Triticum aestivum L.) and alfalfa (Medicago sativa L.) had been grown on the soils in a pot-culture experiment under greenhouse conditions for 40 days. Metal availability to the plants was evaluated by simple and multiple regression analysis. The Mg(NO(3))(2) extractable Co (F1) was significantly correlated with Co concentrations in different parts of wheat and in the whole of alfalfa. For the other metals, the independent variables of the multiple regression models, chosen by stepwise selection, were given as: F1 and F2 + F3 + F4 for Ni; F1, F2 + F3 and F4 for Cu; and F3 + F4 for Pb. The results of this study demonstrate that the sequential extraction procedure, in conjunction with multiple regression models using a combination of correlated fractions as an independent variable, may be useful for the prediction of plant absorption of trace metals in soils.

Factors influencing the separation of metallothioneins by capillary zone electrophoresis.

The factors influencing the separation of metallothioneins (MTs) by capillary zone electrophoresis (CZE) were studied using untreated fused-silica capillaries. A comparison was made between the various buffers of 20 mM Na2B4O7, 10 mM Na2B4O7-10 mM Na2HPO4 and 20 mM Na2B4O7-10 mM Tris for the separation of MTs in terms of migration time and UV-absorbance under the various applied voltages. The migration times for MT-I and MT-II decreased with increase in the applied voltage and column temperature. However, no significant change in UV absorbance was observed. When Na2B4O7-Tris buffer was chosen for the separation of MTs, the migration time, UV absorbance, theoretical plates and resolutions also increased with increasing buffer concentrations. The effect of buffer pH on migration time was relatively complicated. Under the optimal operating conditions the standard MTs were well separated. Determination of MTs in the real samples failed due to the adsorption of other proteins onto the capillary walls.

Determination of dietary cadmium-induced metallothioneins in rabbit kidneys and cadmium in metallothioneins by anion-exchange high-performance liquid chromatography coupled with graphite furnace atomic absorption spectrometry.

A rapid method is described for the determination of dietary cadmium-induced metallothioneins (MTs) in rabbit kidneys by anion-exchange high-performance liquid chromatography. Rabbit kidney MT-I and MT-II were eluted at ca. 15.0 and 18.8 min, respectively, from a DEAE-5PW anion-exchange column with a Tris-HCl buffer (0.01-0.25 M, pH 8.6) and detected by ultraviolet absorbance at 254 nm. A standard calibration curve was constructed using purified standard MT isoforms, which demonstrated an excellent linear correlation between UV absorbance peak heights and the amounts of MT isoforms. Feeding a dose of cadmium for some days resulted in an increase in MT concentrations in rabbit kidneys, but not in the livers. The cadmium concentrations in MT-I and MT-II elutions were determined by graphite furnace atomic absorption spectrometry. MT-I and MT-II showed some differences associated with the oral intake of cadmium. Dietary cadmium also caused zinc to accumulate in kidneys to some extent. The effects of dietary oleic acid on the synthesis of MTs were also studied. Based on the method of standard additions, the recovery of MTs exceeded 93% and replicated injection of samples yielded a relative standard deviation of 2.4% at an MT level of 280 micrograms/g.

Effect of chronic hypoxia on acetaminophen metabolism in the rat.

The effect of chronic hypoxia (10.5% O2 for 8-9 days) on acetaminophen metabolism was studied in vivo or in isolated cell or microsomal systems. Results from in vivo studies with oral administration of acetaminophen showed that in hypoxic rats, the plasma appearance of the drug was delayed and the plasma half-life was increased. Analyses of the area under the curve (AUCoral) showed that this value was higher in hypoxic rats, whereas the rate constants for elimination (kelim) and absorption (kabs) were lower in these animals. Formation of the glucuronide and sulfate conjugates was decreased significantly (P less than 0.05) in hypoxic animals. The calculated volume of distribution (Vd) after an intravenous dose was not different in either group but total clearance (CL) was 35% lower in hypoxic rats. Studies with isolated hepatocytes from both groups revealed that glucuronidation and sulfation were inhibited markedly at low O2 concentrations. The O2 concentrations required for half-maximal production (P50 values) of glucuronide (2.3 microM O2) and sulfate (1.8 microM O2) conjugates in cells from hypoxic animals were lower than for control cells (5.3 microM and 3.9 microM O2 for glucuronide and sulfate conjugates, respectively). Maximal rates of conjugation in cells from hypoxic rats were 60-70% of control rates. Similar decreases in microsomal UDP-glucuronosyltransferase and cytosolic sulfotransferase activities were found in livers of animals exposed to chronic hypoxia. These lower P50 values are consistent with a lower P50 for oxidation of mitochondrial cytochromes in hypoxic cells. In comparison, the P50 for glutathione conjugation (4.1 microM O2) was not statistically different from control (4.6 microM O2), but the maximal rate was 65% higher. The results show that chronic hypoxia causes a change of absorptive processes and decreased glucuronidation and sulfation reactions which affects the disposition of acetaminophen and potentially the disposition of a variety of other exogenous and endogenous compounds.

Glutathione-dependent protection against oxidative injury.

Functions of GSH in detoxication during radical-induced injury in specific pathological and toxicological conditions are discussed. GSH protects against oxidative damage in systems that scavenge radicals, eliminate lipid peroxidation products, preserve thiol-disulfide status of proteins, and repair oxidant damage. Several factors which affect cellular GSH homeostasis can affect these functions, including nutritional status, hypoxia and pharmacological intervention. Evidence from a variety of pathological and toxicological conditions, e.g. ischemia-reperfusion injury, chemically induced oxidative injury, radiation damage, aging, and degenerative diseases, indicate that GSH is a primary component of physiological systems to protect against oxidant and free-radical-mediated cell injury.

Drug metabolism and toxicity during hypoxia.

Oxygen concentration affects the metabolism and toxicity of various drugs. A considerable amount of information is now available on the effects of hypoxia on the major pathways of drug metabolism, including oxidation (i.e., by cytochromes P-450, NAD+-dependent dehydrogenases, and monoamine oxidase), glucuronidation, sulfation, glutathione conjugation, glycine conjugation, and acetylation. Some pathways are essentially independent of O2 concentration while others are highly dependent upon O2. Certain drugs are activated to reactive and toxic metabolites by O2-dependent pathways. This aspect of drug toxicity serves as a basis for treatment of slow-growing solid tumors which have hypoxic regions that are resistant to chemo- and radiation therapies. Recent studies have also established that hypoxic cells have increased susceptibility to oxidative injury, and this can predispose cells to other pathological processes. However, in spite of the available knowledge concerning the O2 dependence of metabolism and toxicity of drugs, relatively little is known about the effects of chronic hypoxia on the expression of drug-metabolizing enzymes or upon the absorption, elimination, or toxicity of drugs. Thus, in addition to the information presently reviewed, major gaps exist in the knowledge needed to provide optimal drug therapy in the large population of patients who experience O2 deficiency. Comments and Perspectives. Specific basic research areas which need to be studied include the effects of hypoxia on drug absorption and elimination, the changes of neahypoxia that lead to enhanced susceptibility to drug toxicity, and the effects of chronic hypoxia on the metabolic systems involved in absorption, metabolism, and elimination of drugs. At an applied level, the available data on the O2 dependences of drug metabolism pathways need to be extended to examine in detail the O2 dependence to metabolism and toxicity of relevant, currently used therapeutic agents. Such efforts can be expected to continue to improve drug therapies and reduce toxicities in hypoxic patients.