Abstract
The efficacy of chlorine dioxide (ClO2) in detoxifying two potential bioterrorism agents, the trichothecene mycotoxins verrucarin A and roridin A, was evaluated. In the first experiment, verrucarin A (1, 5, or 10 μg) and roridin A (5 or 10 μg) were each inoculated onto square-inch sections of glass, paper, and cloth and exposed to 1000 ppm of ClO2 for either 24 or 72 h at room temperature. In the second experiment, verrucarin A and roridin A (1 or 2 ppm in water) were treated with 200, 500, or 1000 ppm ClO2 for up to 116 h at room temperature in light and dark conditions (N = 9 per treatment for test and control). A yeast assay using Kluyveromyces marxianus was used to quantify the toxicity of verrucarin A and roridin A. Additionally, high-performance liquid chromatography was performed on selected samples. Results for the first experiment showed that ClO2 treatment had no detectable effect on either toxin. For the second experiment, both toxins were completely inactivated at all tested concentrations in as little as 2 h after treatment with 1000 ppm ClO2. For verrucarin A, an effect was seen at the 500 ppm level, but this effect was not as strong as that observed at the 1000 ppm level. Roridin A toxicity was decreased after treatment with 200 and 500 ppm ClO2, but this was not significant until the 24-h exposure time was reached. These data show that ClO2 (in solution) can be effective for detoxification of roridin A or verrucarin A at selected concentrations and exposure times.
Trichothecenes are a group of toxins that are produced by several species of mold (Ueno 1984) and a few plants (Jarvis et al. 1988). Some trichothecenes are very strong inhibitors of protein synthesis and are lethal in small doses to a wide range of eukaryotic organisms. Experiments with a number of animal species have shown LD50 doses as low as 0.8 ppm (Wannemacher and Wiener 1997). Trichothecenes have been described as stable compounds that are fairly resistant to heat (Widestrand and Petterson 2001; Sharma and Kim 1991). The toxicity of some groups of trichothecenes is thought to be due to the presence of an epoxide group at positions 12 and 13. T-2 toxin, one of the trichothecene mycotoxins, is thought to be one of the active components of the biological warfare agent “yellow rain” (Mirocha et al. 1983). The topic remains controversial with evidence for and against that it has been used in areas of Southeast Asia (Tucker 2001). However, whether or not they have been used in the past, due to their stability and toxic nature, trichothecenes have potential for use as biological warfare agents (Henghold 2004). Some possible applications are exposure through aerosolization or the contamination of water supplies. Previous studies have shown that ingestion is a well-established route of exposure (Parent-Massin 2004; Revankar 2003). Note that a large amount of contamination may not be required for an effective disruption to a targeted community to take place, as was shown during the “anthrax letters” episode in 2001 (Centers for Disease Control and Prevention 2003) whereby a few people were affected by the anthrax contamination, yet the amount of distress to the community at large was highly significant.
Chlorine dioxide (ClO2) is a commonly used biocide for drinking water supplies. It is effective against a wide range of microorganisms. The gas form has also been used as a biocide. For example, the disinfection of the offices of the Hart Senate Office Building by the Environmental Protection Agency in 2002 was achieved with the help of gaseous ClO2. In our work, and based on the evidence of other studies, we have found that bleach, which uses chlorine as an active agent, is effective in inactivating trichothecene mycotoxins (Wilson et al. 2004; Wannemacher and Wiener 1997). We theorized that ClO2 could be used to neutralize trichothecene mycotoxins if they were to be used as bioterrorism agents. We set up two experiments to evaluate the efficacy of ClO2 in inactivating two potent trichothecenes (roridin A and verrucarin A) that had been added to various material surfaces and to water.
MATERIALS AND METHODS
Experiment 1
ClO2 Treatment: Materials
Stock solutions of verrucarin A and roridin A (Sigma, St. Louis, MO) were kept in high-performance liquid chromatography (HPLC)-grade methanol at 1 mg/ml. For these experiments, dilutions were made from these stock solutions. For the gas treatment, 1 square-inch sections of cloth (white 100% cotton–10 oz. 58/60″ duck/canvas; James Thompson & Co, Wilmette, IL), paper (92 brightness, 2016 weight. Hewlett Packard, Austin, TX) and 10 × 25-mm pieces of cut microscope slides (Fisher Scientific Hampton, NH) were aliquoted with 10 μl of either 1, 5, or 10 μg of verrucarin A or 5 or 10 μg of roridin A. The 1-μg level of roridin A was not used because of its low toxicity to the yeast in the yeast toxicity assay (Engler, Coker, and Evans 1999). The samples were allowed to dry to completion under a fume hood at room temperature. They were then placed in open Petri dishes inside a modified 22-L capacity pressure cooker/gas chamber.
To create the ClO2 gas, one 6-g S-Tab 10 Aseptrol tablet (Engelhard, Jackson, MS) was dissolved in 630 ml of sterile deionized water in a sealed bottle. Immediately after the tablet had dissolved (approximately 5 min), 157.5 ml was poured into a beaker with 472.5 mL sterile deionized water that was placed in the center of the gas chamber. This resulted in a final concentration of 1000 ppm ClO2 gas per allotted space. The chamber lid was immediately sealed and the entire setup was placed under a fume hood for 24 h. Additional experiments were run using samples spiked with verrucarin A whereby treatment time was increased to 72 h. After ClO2 exposure, the chamber lid was opened (under the fume hood) and the samples were individually transferred to 20 ml glass scintillation vials.
To extract the toxins from the materials, 10 ml of HPLC-grade methanol were added to each vial. The vials were allowed to sit for 30 min at room temperature, after which they were briefly vortexed followed by removal of the materials. At this point, any toxins from the materials were in solution in the vials. The extraction solutions were allowed to evaporate to completion under a fume hood (typically overnight) followed by the addition of 1 ml of methanol to resuspend any toxins in the vials. The extracts were then transferred to 1.5 ml glass vials. These were the working solutions for the yeast toxicity assay (N = 7 per test, 3 per control).
Yeast Toxicity Assay
Kluyveromyces marxianus (No. 8554; American Type Culture Collection, Manassas, VA), a yeast demonstrated to be sensitive to trichothecene mycotoxins (Engler, Coker, and Evans 1999), was routinely grown and maintained at 37°C and stored at 4°C on YPG (1% yeast extract, 1% bacteriological peptone, 2% glucose, and 2% agar). Cultures for inoculation of the bioas-say were prepared by adding a single colony from an agar plate to 5 ml of YPG-50 (1% yeast extract, 1% bacteriological peptone, and 50 mM glucose) in a culture flask. The tube was incubated in a rotary incubator for approximately 16 h at 37°C. For the bioassay procedure, YPG-50 was supplemented from a stock solution of polymixin B sulfate (PMBS; ICN Biomedicals, Seoul, Korea) to give a final bioassay concentration of 15 μg/ml. A total of 136 μl of PMBS-supplemented YPG-50 medium were added to the wells of a 96-well polystyrene microtiter plate in triplicate. A total of 8 μl of test sample or control were added to each well, followed by 16 μl of yeast inoculum to yield an initial cell density of approximately 1 × 108 cells/ml. Blank wells contained 152 μl of medium and 8 μl of water. Control wells consisted of 144 μl of medium and 16 μl of yeast inoculum. The plates were sealed and incubated on a plate shaker at 37°C for 8 h. Turbidity was measured every 2 h by measuring the absorbance at 550 nm in a microtiter plate reader. Yeast growth resulting in high turbidity and absorbance indicated that the sample was not toxic. Conversely, the lower the turbidity and absorbance, the more toxic the sample was to the yeast.
Experiment 2
ClO2 Treatment: Water
Polystyrene tubes (8-ml capacity) containing either 5 or 10 μg of verrucarin A or 10 μg roridin A received 5 ml of a solution of either 1000, 500, or 200 ppm of ClO2. This resulted in a concentration of either 1 or 2 μg/ml (1 or 2 ppm) of toxin per solution. The working concentrations of ClO2 were derived from a stock solution of 1000 ppm ClO2. This stock solution was made by using one 6-g S-Tab 10 Aseptrol tablet dissolved in 500 ml of deionized water in a flask under a fume hood. All tubes were sealed immediately following the addition of the ClO2 solution. For each treatment, two sets of tubes were prepared. One set was placed under laboratory fluorescent lights and the other in the dark for 2, 24, 36, 48, 72, or 116 h. This was followed by toxicity testing using the yeast toxicity assay as already described (N = 9 per test and control). Negative controls contained ClO2, but no toxin. Positive controls contained toxin, but no ClO2. Blank samples lacking both toxin and ClO2 were also included.
For HPLC analysis, additional samples containing 10 μg/ml verrucarin A were prepared and exposed to 1000 ppm ClO2 for 24 h. Results were compared to unexposed controls handled in a similar manner.
HPLC Analysis
HPLC analysis was performed using an Agilent Technologies 1100 Series HPLC System (Agilent, Palo Alto, CA) equipped with an ultraviolet-visible diode array detector. A 400 mm (250 mm + 150 mm) × 4.6-mm Eclipse C8, 5-μ particle size analytical column plus a 12.5-mm guard column set at 40°C was used for the analyses. The flow rate was set at 1.0 ml/min with an injection volume of 5 ml. Samples were run in a mobile phase in which the gradient changed from 35% of 5% acetonitrile in water to 85% acetonitrile in 20 min. Samples were read at 260 nm and analyzed using Chemstation software (Agilent). The method was quantitatively calibrated for verrucarin A.
Statistical Analysis
Yeast toxicity assay mean optical density (OD550) readings taken from the 8th h of incubation were used in the analysis. Mean OD550 readings for control and test samples were compared using a one way analysis of variance (ANOVA) (Sigma Stat software, version 2.0; SPSS, Inc., Chicago, IL) (N = 9 per treatment and control). Conditions of normality and equal variance were met. Test sample OD550 readings similar to negative controls, but significantly higher than positive controls, indicated that the sample was nontoxic.
RESULTS
Experiment 1
Table 1 presents data for the paper samples aliquoted with various concentration of verrucarin A and roridin A. Roridin A at the 5 and 10 μg/square-inch level on all materials was still toxic after treatment with the 1000 ppm of ClO2. However, some effect of the treatment was seen as the 8-h test OD550 values were not as low as those of the positive control. Verrucarin A was not inactivated by the treatment at any of the tested concentrations at any time point.
Experiment 2
Table 2 presents data for verrucarin A spiked samples (2 μg/ml) under light conditions. There appeared to be no effect on the efficacy of the ClO2 under light or dark conditions as results for these experiments were similar (data not shown). Verrucarin A, at the tested concentrations, was detoxified by the 1000 ppm level of ClO2 from the 2-h time point onwards. A detoxification effect was also seen with the 500-ppm treatment, but this was not as strong as that for 1000 ppm. Roridin A was completely detoxified by 1000 ppm of ClO2 from 2 h onward. At 500 ppm ClO2, the toxin was inactivated at the 24-h time period. At 200 ppm ClO2, the toxin was inactivated from 48 h onwards.
HPLC Analysis
Representative samples from the HPLC analyses are shown in Figures 1 and 2. Figure 1 shows the chromatogram of purified verrucarin A without ClO2 treatment and the effect of 24-h, 1000 ppm ClO2 treatment on verrucarin A in water. One sharp peak was identified at 11.983 min as verrucarin A. After treatment, several peaks were noted around the retention time of the purified standard, but no compound could be clearly identi-fied as verrucarin A, even with UV spectrum analysis. Figure 2 shows the signature UV spectrum of verrucarin A with a maximum absorbance at approximately 260 nm and the effect of treatment.
DISCUSSION
Our results show that various concentrations of ClO2 were successful in detoxifying verrucarin A and roridin A in drinking water at the 1 and 2 μg/ml levels. Regarding the mechanism of action, the treatment may have altered the epoxide group in the trichothecene structure, which can lead to a significant reduction in toxicity (Swanson et al. 1988). Alternatively, a major part of the trichothecene structure could have been changed as indicated by HPLC analysis. This is a likely possibility in that trichothecene mycotoxins have several chemical groups, notably oxygen and hydroxyl groups, which could act as electron donors in the oxidation reactions that take place with ClO2 treatment. A concentration of 1000 ppm chlorine dioxide gas did not have a protective effect on solid material, i.e., paper, glass, or cloth treated with aliquots of verrucarin A and roridin A. It is possible that the gaseous ClO2 treatment did not access the toxins adequately (i.e., radical formation and interaction could not take place on the dry materials). Conversely, the treatment was very effective in solution likely due to the increased free radical formation leading to the efficient inactivation of the toxins (Noss and Olivieri 1985).
With regard to the drinking water treatment, the high amounts of chlorine dioxide in the water still render it unsuitable for consumption (Couri, Abdel-Rahman, and Bull 1982; Abdel-Rahman, Couri, and Bull 1985). In particular, chlorate and chlorite ions (by-products of reactions involving chlorine dioxide) present problems. Plus, although the yeast toxicity assay does show the water to be nontoxic after treatment, other by-products may have been formed that are harmful to humans in the long term. However, strategies exist that could eliminate these products. For example, following ClO2 exposure, the chlorine dioxide by-products could be removed with substances such as ferrous ions (Katz and Narkis 2001) and other compounds removed through the use of activated charcoal. Alternatively, the water supply could be replaced with fresh water after the chlorine dioxide had been applied to the containment facilities.
There are a number of different methods for inactivating mycotoxins, particularly those associated with the food industry where the problem of mycotoxin contamination can be severe (Pitt 2000). The use of high temperatures has been employed successfully for destroying aflatoxins and ochratoxins on coffee beans (Hasan 2002; Micco et al. 1989). Other studies have shown that microwaves and ozone can be effective in reducing their toxicity (Farag, Rashed, and Abo Hggr, 1996; Chatterjee and Mukherjee 1993). Gamma-irradiation has also been employed for mycotoxin inactivation, although there have been mixed results using this technique (Wilson et al. 2004; Aziz, Attia, and Farag 1997; Halasz et al. 1989). One study showed that superactivated charcoal can neutralize T-2 toxin (Fricke and Jorge 1990). Chlorine was chosen as the active agent in this study because sodium hypochlorite has been shown to be able to inactivate trichothecene mycotoxins (Wannemacher and Wiener 1997). Additionally, chlorine is regularly used as a sterilizing agent for drinking water and has been shown to be effective in inactivating aflatoxins (Yang 1972), although it should be notedthat a carcinogenic by-product (2,3-dichloro aflatoxin B1) was formed out of this process, requiring an extra inactivation step (WHO 1980).
In conclusion, this study showed that the use of chlorine dioxide, a regularly used drinking water sterilizing agent, was successful in detoxifying two trichothecene mycotoxins, roridin A and verrucarin A, in solution. In a gaseous state, at the tested concentrations of mycotoxins on hard surfaces, ClO2 was not able to inactivate the same mycotoxins.
Footnotes
Figures and Tables
The authors would like to thank Assured Indoor Air Quality Ltd., Dallas, Texas, USA, and Texas Tech University Health Sciences Center (TTUHSC) for their financial support. Thanks are also due to Barry Speronello of Englehard for technical advice and generous supply of Aseptrol chlorine dioxide tablets. Drs. Wilson and Straus were supported by a Center of Excellence grant from TTUHSC. Dr. Straus, Mr. Brasel, and Mr. Luis Cobos were supported by a grant from the Texas Higher Education Coordinating Board.