Environ. Sci. Technol. 2000, 34, 2249-2253
Arsenic and Antimony Biomethylation by Scopulariopsis brevicaulis: Interaction of Arsenic and Antimony Compounds PAUL ANDREWES,† W I L L I A M R . C U L L E N , * ,† A N D ELENA POLISHCHUK‡ Environmental Chemistry Group and Biological Services Facility, Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1
The biomethylation of arsenic by the filamentous fungus Scopulariopsis brevicaulis is well documented, and the biomethylation of antimony by this fungus was recently established. However, in all the previous studies each metalloid was studied in isolation. Arsenic and antimony are often associated in the environment, and so an understanding of interactions between these elements is necessary. To this end, S. brevicaulis was grown in media containing mixtures of arsenic and antimony compounds in various proportions, and the principle nonvolatile biomethylation products (trimethylantimony and trimethylarsenic species) in the medium were quantified by using HGGC-AAS. It was found that the yield of trimethylantimony compounds, obtained from the biomethylation of potassium antimony tartrate, was increased in the presence of sodium arsenite. The production of trimethylarsenic species from sodium arsenite was significantly inhibited in the presence of antimony (either as potassium antimony tartrate or antimony trioxide) at antimony concentrations too low to inhibit growth. This is although arsenic(III), in the absence of antimony, is much more readily biomethylated. That is 1.25.3% of added arsenic is biomethylated by S. brevicaulis whereas only 0.0006-0.008% of added antimony(III) is biomethylated over 1 month. Potassium hexahydroxyantimonate had no effect on arsenic biomethylation. The addition of potassium tartrate to cultures did not inhibit arsenic biomethylation. The biomethylation of sodium arsenate was not inhibited as much by antimony compounds. The inhibitory effect of antimony was found to be a function of the ratio of antimony to arsenic rather than the absolute amount of antimony.
Introduction Arsenic biomethylation in the environment and in humans is well established (1), and this is significant because the chemical properties and toxicities of methylarsenic species are different from those of inorganic arsenic species. Methylantimony species have been detected in the environment (2-7), and the biomethylation of antimony by some microorganisms has been demonstrated in laboratory cultures * Corresponding author phone: (604)822-4435; fax: (604)822-2847; e-mail:
[email protected]. † Environmental Chemistry Group. ‡ Biological Services Facility. 10.1021/es991269p CCC: $19.00 Published on Web 05/02/2000
2000 American Chemical Society
(8-15). However, antimony has not been studied to the same extent as arsenic, and little is known about the biological properties of organoantimony species. In almost all studies of arsenic and antimony biomethylation, each metalloid was studied in isolation even though the two elements are usually associated in the environment. In addition, arsenic and antimony are both in Group 15 of the periodic table and so have similar chemistries; therefore, it is possible that biological processes involving one of these elements are likely to be affected by the presence of the other. The simplest interactions that might be expected are direct competition, where, for example, the metalloids might compete for a binding site, a process that would be controlled by the concentration of each metalloid and the strength of binding. A few studies of antimony and arsenic interactions in mammalian systems have been reported. Bailly et al. (16, 17) reported that arsenic biomethylation, in rat liver cytosol, is completely inhibited at antimony concentrations of 10-5 M (as SbCl3), but few details were given. The result of sister chromatid exchange tests (for genotoxicity) revealed that the combined effects of arsenic and antimony compounds on human lymphocytes are less than predicted from addition of the individual genotoxicities of arsenic and antimony compounds (18). By using the micronucleus tests with V79 cells, it was found that chromosome mutagenicity induced by arsenic(III) was significantly suppressed by antimony(III) (19). In both of these genotoxicity studies the subadditive interaction of arsenic and antimony was not attributable to inhibition of cell uptake. Another study reported that human tumor cells that were resistant to arsenite were also resistant to potassium antimony tartrate and vice versa. This cross resistance could be partially attributed to reduced uptake of arsenic and antimony (20). Thus, it seems that predicting the effects of arsenic contamination may be significantly complicated by the presence of antimony. In light of this situation, Gebel, in a letter to Science, called for the measurement of antimony co-contamination in arsenic contaminated waters and also for “the inclusion of antimony as a putative confounding variable in the toxicity of arsenic in future investigations” (21). Research on arsenic biomethylation in mammals is currently very topical in order to establish if methylation increases or decreases the carcinogenicity of inorganic arsenic species, but usually co-exposure to other contaminants is not considered (22). A significant increase or decrease in arsenic biomethylation could be the result of co-exposure to antimony. Arsenic biomethylation by the filamentous fungus Scopulariopsis brevicaulis has been studied in detail, and this fungus has served as a useful model in the study of other systems (1, 23). Recently, S. brevicaulis was shown to biomethylate antimony (10-15). The present paper reports some studies on antimony and arsenic biomethylation by S. brevicaulis to see if the yields of biomethylation products from one would be increased or decreased in the presence of the other. In this study we concentrated on the production of nonvolatile biomethylation products that are found in the medium, i.e., the trimethylarsenic and trimethylantimony species that produce trimethylarsine and trimethylstibine, respectively, on hydride derivatization. These are the principle products (>90%) of biomethylation under the conditions of this study. For antimony, the yields of volatile trimethylstibine [10 ng of Sb (14)] are always much lower than the yields of the trimethylantimony species in the media [300-3000 ng of Sb VOL. 34, NO. 11, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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(12)]. For arsenic, the yields of trimethylarsine are much lower (1 mg of As/L the yield of trimethylarsine is directly proportional to the concentration of trimethylarsenic species in the medium (24). The average concentration of arsenic in the earths crust (1.8 mg of As/kg) is an order of magnitude greater than that of antimony (0.2 mg of Sb/kg). However, in local environments the ratio of arsenic to antimony can vary tremendously. For example, the Rapahannock (Fredricksburg, VA) and Roanoke (Weldon, NC) Rivers, in the United States have arsenic to antimony ratios of 0.1:1, whereas the Sri Nakarin River (Si Sawat, Thailand) has a ratio of approximately 100:1 (25). These variations reflect differing geochemistry in each river and anthropogenic influences. The ratios of arsenic to antimony employed in this study ranged from 100:1 to 1:1 (for studying antimony biomethylation) and from 2:1 to 0.1:1 (for studying arsenic biomethylation). Thus, the present investigation is environmentally relevant.
Materials and Methods Submerged cultures of S. brevicaulis (ATCC 7903) mycelial balls were prepared by adding 40 mL of a seed culture to 400 mL of a minimal salts/glucose medium (26) in 1-L Erlenmeyer flasks. Appropriate amounts of solutions of sodium arsenite, sodium arsenate, potassium tartrate, potassium antimony tartrate, and potassium hexahydroxyantimonate were added to cultures via 0.2-µm syringe filters to give the mixtures of arsenic and antimony compounds, as summarized in Tables 1 and 2. Solid powdered antimony trioxide was added directly to the cultures (0.2 g) to give a saturated solution (∼4 mg of Sb/L). The Erlenmeyer flasks were shaken horizontally (∼135 rpm, 1.75 in. displacement) and maintained at 26 °C for 1 month [previous studies had shown that the production of trimethylantimony or trimethylarsenic species, in significant amounts, ceases after 1 month (12, 23)]. After being incubated, the cultures were autoclaved (19 psi, 121 °C, 20 min) before analysis (previous studies had shown that the carbonantimony and carbon-arsenic bonds of trimethylantimony and trimethylarsenic species, respectively, are stable at the high temperatures encountered in the autoclave). Inorganic arsenic and antimony compounds (>99.5%) were removed from the medium by using solid-phase extraction (>90% of the trimethylantimony and trimethylarsenic species were recovered): In a 60-mL syringe was placed basic alumina (20 g; 80-200 mesh, Brockman activity I), held in place by a small glass wool plug. Ammonium carbonate (40 mL, pH 12, 50 mM) was passed through the alumina to prime the column. Medium (40 mL) was decanted off the autoclaved cultures and passed through the basic alumina column; the eluate was collected. The ammonium carbonate and the medium flowed through the column under the force of gravity. No attempt was made to rinse the column after the medium had passed through. The eluate was analyzed for trimethylarsenic species or trimethylantimony species by using HG-GC-AAS procedures that have been described in previous publications (12, 23). Quantification was performed by using trimethylarsine oxide or trimethylantimony dichloride to perform standard additions.
Results Influence of Antimony Compounds on the Biomethylation of Arsenic Compounds. Cultures of S. brevicaulis were grown in media containing arsenic (either as sodium arsenite or sodium arsenate) and various antimony compounds. The concentration of trimethylarsenic species in the medium, 2250
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FIGURE 1. Chromatograms obtained when media samples (1 mL) from the cultures of experiment 4 (Table 1) were analyzed by using solid-phase extraction and HG-GC-AAS. Cultures were sampled and analyzed after 1 month of incubation. after 1 month of incubation, was determined as trimethylarsine by using HG-GC-AAS. A representative series of HGGC-AAS chromatograms is shown in Figure 1; these are from experiment 4 (Table 1). The only significant peak in these chromatograms is that of trimethylarsine because almost all (>99.5%) of the inorganic arsenic was removed by using solidphase extraction. The concentration of trimethylarsenic in each sample was determined by standard additions, and these results are reported in Table 1. In the biomethylation controls (no antimony compounds added), between 1.2 and 5.3% of the arsenic substrate is biomethylated to trimethylarsenic species. The final column in Table 1 gives the relative concentration of trimethylarsenic species, and this is obtained by taking the average of the biomethylation controls (i.e., cultures with no antimony compounds added) in each experiment as being 100%. Some of these results are also plotted in Figure 2. Error bars are not shown on the figure because the error in analysis is insignificant as compared to the variation between culture replicates. Because inorganic arsenic and inorganic antimony species were removed before analysis, by using solid-phase extraction, and quantification was done by standard additions, the observed reduction in the levels of trimethylarsenic species is not a result of matrix (inorganic antimony) effects during the analysis. In each experiment all cultures were prepared from the same batch of medium, antimony/arsenic substrates, and seed culture. Also, all cultures were incubated and analyzed under identical conditions. However, there were still large variations in the amounts of biomethylation products for replicate cultures. This variation is most likely the result of the inhomogeneity of the seed culture. But the observed effects presented here are greater than the variation from culture inhomogeneity. In the presence of 1000 mg of Sb/L, as potassium antimony tartrate, little inhibition of S. brevicaulis growth was visually observed. This is consistent with other work on two strains of S. brevicaulis that determined the EC50 (median effective
TABLE 1. Influence of Antimony Compounds on Arsenic Biomethylationa substrate concentrations expt 1
2
3 4
5
biomethylation product concentration
culture
antimony species (mg of Sb/L)
arsenic species (mg of As/L)
trimethylarsenic species (µg of As/L)
relatived trimethylarsenic concn (%)
A, control B C D E F A, control B, control C D A, control B C A, control B, control C D E F A, control B, control C, control D, control E F G H I J
PATb (0) PAT (1) PAT (2) PAT (4) PAT (6) PAT (8) 0 (0) PAT (10) PAT (10) 0 PHHAc (10) PHHA (100) 0 0 Sb2O3 (4) Sb2O3 (4) PAT (50) PAT (50) 0 0 0 0 PAT (50) PAT (50) PAT (50) PAT (5) PAT (5) potassium tartrate (50)
NaAsO2 (1) NaAsO2 (1) NaAsO2 (1) NaAsO2 (1) NaAsO2 (1) NaAsO2 (1) Na2HAsO4 (1) Na2HAsO4 (1) Na2HAsO4 (1) Na2HAsO4 (1) NaAsO2 (1) NaAsO2 (1) NaAsO2 (1) NaAsO2 (10) NaAsO2 (10) NaAsO2 (10) NaAsO2 (10) NaAsO2 (10) NaAsO2 (10) NaAsO2 (10) NaAsO2 (10) Na2HAsO4 (10) Na2HAsO4 (10) Na2HAsO4 (10) Na2HAsO4 (10) NaAsO2 (10) NaAsO2 (10) NaAsO2 (10) NaAsO2 (10)
34 21 7 2 3 2 27 16 9 8 38 45 30 153 124 64 62 17 19 266 205 388 536 187 229 27 88 161 286
100 62 21 6 9 6 126 74 42 37 100 118 79 110 90 46 45 12 14 113 87 84 116 40 50 11 37 68 121
a Concentration of antimony and arsenic substrates in cultures and concentration of trimethylarsenic species after 1 month of incubation of cultures. b PAT, potassium antimony tartrate. c PHHA, potassium hexahydroxyantimonate. d Average concentration of trimethylarsenic species in controls for each experiment taken as 100%.
FIGURE 2. Influence of antimony on arsenic biomethylation. Relative concentration of trimethylarsenic species (%) in cultures of S. brevicaulis after 1 month of incubation. All cultures contained 10 mg of As/L either as sodium arsenite (panels a and b) or as sodium arsenate (panel c). Cultures contained between 0 (controls) and 50 mg of Sb/L either as potassium antimony tartrate or as antimony trioxide (panel a, cross-hatched box). PAT, potassium antimony tartrate. concentration for inhibition of hyphal extension) values to be >300 mg of Sb/L (27). In the results presented below, reduction of arsenic biomethylation, in the presence of antimony, occurs at antimony concentrations significantly lower than the EC50, so the observed inhibition must be a result of more than just reduced growth. Influence of Antimony(III) on the Biomethylation of Arsenic(III) (Experiments 1, 4, and 5). In preliminary experi-
ments (results not shown) involving sodium arsenite (all cultures: 1 mg of As/L) and potassium antimony tartrate (0, 1, 10, 100, and 1000 mg of Sb/L), the cultures that contained 10 mg of Sb/L or greater produced no detectable (
