Introduction

Top Abstract Introduction Experimental Procedures Results Discussion References

2-(Allylthio)pyrazine (2-AP)¹ was designed to develop a chemoprotective agent that potentially functions through selective modulation of cytochrome P-450 and other drug-metabolizing enzyme expression (Kim et al., 1997a). Previous studies have shown that 2-AP suppressed the constitutive and inducible cytochrome P-450 2E1 expression and was effective in blocking toxicant-induced liver injury (Kim et al., 1997a). A recent study demonstrated that 2-AP was active as a chemopreventive agent in reducing vinyl carbamate-induced tumorigenesis (Surh et al., 1998).

The anticarcinogenic effect of oltipraz (Olt) and dithiolethiones has been attributable to their induction of phase II detoxifying enzymes, e.g., microsomal epoxide hydrolase (mEH) and glutathione-S-transferase (GST) as well as the inhibition of certain cytochrome P-450s (e.g., P-450 1A2 and 3A4; Davidson et al., 1990; Morel et al., 1993; or Langouet et al., 1995; Primiano et al., 1997). The production of oxygen radicals by 1,2-dithiole-3-thiones has been proposed to play a role in the induction of the phase II enzymes (Hayes and Pulford, 1995). Olt and dithiolethiones mediate the conversion of molecular oxygen to reactive oxygen radicals in the presence of thiols, as monitored by the cleavage of DNA in vitro (Kim and Gates, 1997). Olt treatment elevates GSH levels in the liver of animals. Based on the observation that the biological thiols glutathione and cysteine were competent to elicit the cleavage of DNA by Olt, the role of sulfhydryl in the bioactivation of Olt and the subsequent conversion of oxygen to oxygen radicals has been raised (Kim and Gates, 1997). Nonetheless, the role of GSH in the bioactivation of Olt has not been demonstrated in vivo.

The present study was designed to establish whether 2-AP was effective in elevating mEH and GST mRNA and protein levels in the liver and to study the mechanistic basis of the detoxifying enzyme induction in vivo. We were interested in whether Olt and 2-AP were virtually bioactivated in animals and produced the reactive oxygens in the presence of GSH in vivo. The role of in vivo bioactivation of Olt and 2-AP in the induction of detoxifying enzymes was assessed under the hypothesis that production of activated oxygens may contribute to the antioxidant-responsive element (ARE)-mediated induction of anticarcinogenic phase II enzymes.

Nuclear factor-B (NF-B) activation by oxidative stress has been correlated with the cellular oxidation state, which may translate a redox sensor into a chemical signal that leads to transcriptional activation of the appropriate genes (Schreck et al., 1991; Primiano et al., 1997). The intracellular thiol level has been implicated in the regulation of transcriptional expression of a variety of genes (Rushmore et al., 1991; Pinkus et al., 1996). It has been proposed that the gene of NAD(P)H:quinone oxidoreductase, a detoxifying antioxidant enzyme, was transcriptionally activated in association with NF-B activation in certain cells by Olt or hypoxia (Yao and O'dwyer, 1995). Olt increased the expression of manganese-dependent superoxide dismutase gene and enhanced the binding of NF-B in primary-cultured hepatocytes (Antras-Ferry et al., 1997). Nonetheless, no information is available on the role of NF-B activation in the induction of detoxifying enzymes in animals by Olt or related compounds. In the current study, the NF-B factor level was monitored in the rat liver to determine the possible role of NF-B activation in the expression of mEH and rGSTA2 by these chemopreventive agents.

Whether Olt and 2-AP share the common molecular basis for thiol-dependent production of activated oxygen species was also studied in vitro using the conversion of x-174 DNA topology as an index. In addition, their radical scavenging effects were comparatively assessed in vitro.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Materials. Olt was a gift from Rhône-Poulenc Rorer (Vitry-sur-Seine, France). 2-AP was synthesized at Yuhan Pharmaceutical Co. (Kunpo, Korea). Chemical structures of 2-AP and Olt were shown in Fig. 1. [-³²P]dCTP (3000 mCi/mmol) and [-³²P]ATP (3000 mCi/mmol) were purchased from Amersham (Arlington Heights, IL). Random prime labeling and 5'-end labeling kits were purchased from Life Technologies (Gaithersburg, MD). The consensus sequence of NF-B was provided by Promega Corporation (Madison, WI). Most reagents for the molecular studies were purchased from Sigma Chemical Co. (St. Louis, MO).

View larger version (8K): [in this window] [in a new window]   Fig. 1.   Chemical structures of 2-AP and Olt.

Animal Treatment. Male Sprague-Dawley rats (200-250 g) were obtained from the Korea Food and Drug Administration (Seoul, Korea) and maintained at a temperature of 20-23°C, with a relative humidity of 50%. Animals were caged under a supply of filtered, pathogen-free air. Cheiljedang rodent chow (Seoul, Korea) and water were available ad libitum. A time course study was conducted at the dose of 100 mg/kg of 2-AP, whereas a dose-response study was carried out 24 h after treatment at the doses from 10 through 200 mg/kg of 2-AP. Animals were sacrificed at 12 h after treatment with Olt or 2-AP to assess mEH and GST mRNA levels in GSH-depleted or cysteine-treated animals. Olt was gavaged as suspended in a 0.1% carboxymethylcellulose solution, whereas 2-AP was administered as dissolved in corn oil. Buthionine sulfoximine (BSO) was i.p. injected in a 0.9% NaCl solution (De Ferreyra et al., 1979). Cysteine (1 g/kg) was gavaged as dissolved in an aqueous solution at the same time as Olt or 2-AP. For gel shift analysis, rats were treated with a single dose of Olt or 2-AP (100 or 300 mg/kg) and fasted 16 h before sacrifice. Lipopolysaccharide (LPS) was injected through the tail vein at the dose of 1 µg/kg. Rats were treated with BSO (4.5 mmol/kg) 4 h before administration of Olt or 2-AP and the nuclear extract was prepared from the rat liver at 1 h after Olt or 2-AP treatment. At least three rats were used in each treatment group. Results were confirmed using different groups of animals.

Subcellular Fractionation. Hepatic microsomal and cytosolic fractions were prepared by differential centrifugation. The microsomal and cytosolic fractions were prepared from homogenates in 0.1 M Tris acetate buffer (pH 7.4) containing 0.1 M potassium chloride and 1 mM EDTA by centrifugation at 10,000g for 30 min and subsequently at 100,000g for 90 min. Microsomes were washed in pyrophosphate buffer and stored in 50 mM Tris acetate buffer (pH 7.4) containing 1 mM EDTA and 20% glycerol. Microsomal and cytosolic preparations were stored at 70°C until use.

GST Assay. The activity of cytosolic GST was measured with 1-chloro-2,4-dinitrobenzene as a substrate, as described by Habig et al. (1974).

Immunoblot Analysis. Immunoblot analysis was performed according to previously published procedures (Kim, 1992). Microsomal and cytosolic proteins were separated by 8 and 11% SDS-polyacrylamide gel electrophoresis, respectively, and electrophoretically transferred to nitrocellulose paper (Kim, 1992).

Preparation of cDNA Probes. A cDNA probe for mEH was produced as published previously (Kim, 1992). cDNAs for major GSTs were prepared as described previously (Kim et al., 1997b).

RNA Blot Analysis. Northern blot analysis was carried out according to the procedures described previously (Kim et al., 1997b; Kim and Cho, 1996). The stripped nitrocellulose membranes were hybridized with a ³²P-end labeled poly (dT)₁₆ or a labeled glyceraldehyde-3-phosphate dehydrogenase probe to quantify the amount of mRNA loaded onto the agarose gel and transferred to the nitrocellulose paper. Each data point represents mean ± S.D. from independent measurements of three different animal experiments.

Gel Retardation Assay. A double stranded DNA probe for the consensus sequence of NF-B (5'-AGTTGAGGGGACTTTCCCAGGC-3') was used for gel shift analysis after end-labeling of the probe with [-³²P]ATP and T4 polynucleotide kinase. Nuclear extracts were obtained by a modification of the procedure published previously (Piette and Yaniv, 1988). The reaction mixtures contained 2 µl of 5X binding buffer containing 20% glycerol, 5 mM MgCl₂, 250 mM NaCl, 2.5 mM EDTA, 2.5 mM dithiothreitol, 0.25 mg/ml poly dI-dC, and 50 mM Tris-Cl (pH 7.5), 5 µg of nuclear extracts and sterile water in a total volume of 10 µl. Incubations were carried out at room temperature for 20 min by the addition of 1 µl probe (106 cpm), after 10-min preincubations. Samples were loaded onto 4% polyacrylamide gels at 100 V. The gels were removed, fixed, and dried, followed by autoradiography.

x-174 DNA Strand Breakage Assay. Conversion of the supercoiled form of x-174 DNA to the open circular form and degradation of x-174 DNA were used as indexes of DNA damage. A 20-µl volume of reaction mixture containing 50 mM sodium phosphate buffer (pH 7.0) and 0.2 µg of x-174 DNA was incubated with or without Olt or 2-AP for 1 h at 37°C, as described previously (Kim and Novak, 1990). The conversion of supercoiled form of x-174 DNA to the open circular form by Olt or 2-AP was also assessed after 16 h of incubation in the presence of -mercaptoethanol. Single strand breakage and degradation of x-174 DNA were initiated by the addition of 5 µM benzenetriol (BT) to the reaction mixture in the absence and presence of 20 µM ferrous sulfate, respectively, followed by incubation for 1 h at 37°C. The reaction was terminated by addition of 5 µl of a gel-loading buffer containing 0.25% bromophenol blue, 30% glycerol, and 0.25% SDS. Controls contained only x-174 DNA, x-174 DNA plus Olt, or x-174 DNA plus 2-AP. The supercoiled and open circular forms of x-174 DNA and its degraded products were separated on a 1% agarose gel, followed by visualization on a UV transilluminator. The data were confirmed by at least three experiments and the representative data were shown in Results.

Scanning Densitometry. Scanning densitometry was performed with a microcomputer imaging device, model M1 (Imaging Research, St. Catharines, Ontario, Canada). The area of each lane was integrated using MCID software (version 4.20, revision 1.0), followed by background subtraction.

Data Analysis. Data were analyzed using computer programs for pharmacologic calculations (Tallarida and Murray, 1987). One-way ANOVA procedures were used to assess significant differences among treatment groups. For each significant effect of treatment, the Newman-Keuls test was used for comparison of multiple group means. The criterion for statistical significance was set at  = 0.01.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Hepatic mEH and GST Gene Expression. Expression of the hepatic mEH and GST genes in response to 2-AP was assessed by Northern blot analysis. Treatment of rats with 2-AP resulted in 2- to 12-fold increases in the mRNA levels for mEH, rGSTA2, rGSTA3, and rGSTA5 in a time-dependent manner at 6, 12, and 24 h after a single dose of 100 mg/kg of 2-AP (Fig. 2). Marked increases in the mRNAs were noted at 12 or 24 h. The mRNA level for rGSTM1 was 7-fold elevated by 2-AP at 24 h, relative to control, whereas rGSTM2 mRNA was increased less (i.e., 5-fold). The mRNA levels were reduced to 25-50% of the maximal increases at 48 h, followed by returning to that in untreated animals at 72 h.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Northern blot analysis for mEH and GST mRNA levels after 2-AP treatment. The mRNA levels were determined in vehicle-treated rats (UN) or in rats at 6, 12, 24, 48, and 72 h after a single dose of 2-AP treatment (100 mg/kg po). The amount of RNA loaded in each lane was assessed by rehybridization of the stripped membrane with 32P-labeled poly(dT)16. Changes in mEH and GST mRNA levels relative to vehicle-treated animals were plotted after scanning densitometry of the blots. Each point represents the mean ± S.D. with at least three independent experiments.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Dose-dependent increases in mEH and rGSTA2 mRNA levels by 2-AP. Northern blot analysis was performed to determine the mRNA levels in rats at 24 h after treatment with 2-AP (10-200 mg/kg). Increases in mRNA levels were plotted as a function of dose of 2-AP, relative to vehicle-treated rats (UN). Each point represents the mean ± S.D. with at least three independent experiments.

mEH and GST Protein Expression. We tested the GSH conjugating activity in the liver cytosol and carried out immunoblot analyses to determine whether the increases in mRNAs were in parallel with those in proteins. A dose-response study showed that the cytosolic GSH conjugating activity toward 1-chloro-2,4-dinitrobenzene was significantly elevated after treatment of rats with 2-AP for 3 days at the dose of 10, 60, 100, and 200 mg/kg per day, resulting in 1.3-, 1.9-, 2.1-, and 3-fold increases relative to vehicle-treated animals (Table 1).

View larger version (31K): [in this window] [in a new window]   Fig. 4.   Western blot analyses for mEH and rGSTA1/2. Immunoblot analyses were carried out with hepatic microsomal and cytosolic fractions produced from vehicle (UN)- or 2-AP- treated rats (10-200 mg/kg per day for 3 days). Four micrograms of microsomal or cytosolic proteins was used for mEH and rGSTA1/2 immunoblottings, respectively.

Effects of BSO and Cysteine. A number of studies have shown that Olt induces several GST subunits with transcriptional activation (Davidson et al., 1990; Primiano et al., 1997). This is presumably mediated by ARE in the genes. In view of the potential role of reactive oxygens in the induction of mEH and rGSTA2, we were interested in establishing the molecular basis for the enzyme induction by 2-AP in comparison with that by Olt.

View larger version (55K): [in this window] [in a new window]   Fig. 5.   Effects of BSO and cysteine treatment. A, BSO effects on Olt- or 2-AP-inducible mEH and rGSTA2 mRNAs. Representative Northern blot analysis showed the hepatic mRNA levels in rats pretreated with BSO (4.5 mmol/kg) 4 h before administration of Olt or 2-AP (100 mg/kg po). The RNA was isolated at 12 h after Olt or 2-AP treatment. UN, vehicle-treated rats. B, inhibition of inducible mEH and rGSTA2 mRNA expression by cysteine. RNA was isolated at 12 h after a single dose of Olt or 2-AP (100 mg/kg po) with or without concomitant cysteine administration (1 g/kg po). Amount of RNA loaded in each lane was confirmed by rehybridization of the stripped membrane with a labeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe.

Effect on NF-B Activation. The intracellular redox potential has been implicated in the regulation of certain gene expression in association with the activation of NF-B. Olt activated the NF-B at 30 min to 1 h in primary-cultured hepatocytes, which has been claimed to be responsible for the induction of NAD(P)H:quinone oxidoreductase and for manganese-dependent superoxide dismutase (Yao and O'dwyer, 1995; Antras-Ferry et al., 1997). The effects of 2-AP and Olt on the activation of NF-B were determined in the rat liver by gel shift assays using the NF-B consensus sequence. The level of NF-B failed to be activated at 1 h after administration of 2-AP at the dose of 300 mg/kg (Fig. 6). Similar results were obtained with Olt (data not shown). Multiple analyses of three separate animal experiments showed that the agents at the doses of 100 through 300 mg/kg did not activate NF-B in the liver even at various time points (e.g., 30 min to 3 h). The agents were incapable of activating NF-B in rats pretreated with BSO (4.5 mmol/kg i.p.). Whereas treatment of rats with LPS at the dose of 1 µg/kg resulted in an increase in the nuclear p65/p50 NF-B complex, either Olt or 2-AP failed to enhance or reduce the LPS-inducible NF-B activation.

View larger version (39K): [in this window] [in a new window]   Fig. 6.   Gel shift analysis of hepatic nuclear extracts using the consensus sequence of NF-B. Nuclear extracts were isolated from rats 1 h after 2-AP treatment (300 mg/kg). Rats were intraperitonially injected with BSO (4.5 mmol/kg) 4 h before a single dose of 2-AP. Nuclear extracts were isolated 1 h after an LPS injection (1 µg/kg i.v.) to rats pretreated with 2-AP (300 mg/kg po) 2 h before. All lanes contained 5 µg of nuclear extract. Data were confirmed by at least three separate experiments.

Effects on x-174 DNA Topology. The effects of 2-AP on the topology of x-174 DNA was examined in the presence of -mercaptoethanol. Although Olt completely converted supercoiled x-174 DNA to the open circular form at the concentration of 30 µM in the presence of -mercaptoethanol², 2-AP failed to alter x-174 DNA topology at the concentration of 1 mM (Fig. 7A). Thus, in contrast to Olt, 2-AP was incapable of converting oxygen to reactive oxygens in the presence of thiol.

View larger version (34K): [in this window] [in a new window]   Fig. 7.   Effects of Olt or 2-AP on the topology of X-174 DNA. A, Reaction mixtures contained 50 mM sodium phosphate (pH 7.0), 0.2 µg of supercoiled X-174 DNA, -mercaptoethanol and Olt or 2-AP. -Mercaptoethanol was added at 5 equivalents based on the moles of either Olt or 2-AP. Incubations were carried out at 37°C for 16 h. Lane 1, no addition; lane 2, 5 mM -mercaptoethanol alone; lane 3, 1 mM 2-AP + 5 mM -mercaptoethanol; lane 4, 1 mM 2-AP alone; lane 5, 0.03 mM Olt + 0.15 mM -mercaptoethanol. B, protective effect of 2-AP against the cleavage of X-174 DNA induced by autoxidation of BT. Reaction mixtures contained 50 mM sodium phosphate buffer (pH 7.0), 0.2 µg of supercoiled X-174 DNA, and 5 µM BT in the absence or presence of 0.1 to 1 mM Olt or 2-AP. Incubations were carried out at 37°C for 1 h. Lane 1, no addition; lane 2, 5 µM BT alone; lane 3, 0.1 mM Olt + 5 µM BT; lane 4, 1 mM Olt + 5 µM BT; lane 5, 1 mM Olt alone; lane 6, 0.1 mM 2-AP + 5 µM BT; lane 7, 1 mM 2-AP + 5 µM BT; lane 8, 1 mM 2-AP alone. C, effects on the degradation of X-174 DNA induced by BT and ferrous iron. Incubation mixtures contained 50 mM sodium phosphate buffer (pH 7.0), 0.2 µg of supercoiled X-174 DNA, 5 µM BT, and 20 µM ferrous sulfate in the presence or absence of Olt or 2-AP at the concentration of 1 mM. Lane 1, no addition; lane 2, 5 µM BT alone; lane 3, 20 µM Fe2+ + 5 µM BT; lane 4, 1 mM Olt + 20 µM Fe2+ + 5 µM BT; lane 5, 1 mM 2-AP + 20 µM Fe2+ + 5 µM BT. OC, open circular; SC, supercoiled DNA.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

2-AP exerts the hepatoprotective and chemopreventive effects through selective modulation of cytochrome P-450 2E1 and other detoxifying enzyme expression (Kim et al., 1997a). The present study demonstrated that 2-AP was efficacious in inducing the hepatic mEH and GSTs with marked increases in the mRNAs, as supported by the elevation of metabolic activity, immunoblot, and Northern blot analyses. 2-AP was efficacious in inducing mEH and major GSTs including rGSTA1/2, rGSTA3/5, rGSTM1, and rGSTM2, which was comparable to the response observed after Olt treatment (Nam et al., 1997; Kim et al., 1997b). An additional dose-response study showed that 2-AP was active in inducing the detoxifying enzymes even at the dose of 10 mg/kg.

BSO inhibits the heavy subunit of -glutamylcysteine synthase, which possesses all of the catalytic activity for GSH feedback (Mulcahy et al., 1995). Stilbene oxide induced heme oxygenase-1 mRNA with concomitant decreases in the hepatic GSH level and BSO augmented the increase in heme oxygenase-1 mRNA with GSH depletion (Oguro et al., 1997). Olt has been also shown to induce heme oxygenase-1 in rat tissues (Primiano et al., 1996). The present study revealed that both Olt and 2-AP further elevated the hepatic mEH and rGSTA2 mRNA levels in the GSH-depleting animals. The hypothesis that production of activated oxygens may be responsible for the enzyme induction by the agents was supported in part by the observation. Conversely, the mRNA levels inducible by the agents were prevented by cysteine administration. Reversal of increases in mEH and GST mRNA levels by concomitant treatment with cysteine confirmed the conclusion that transcriptional induction of anticarcinogenic enzymes by Olt or 2-AP might be mediated with the production of oxygen-free radicals. Cysteine is a diffusible thiol for thiol-protein mixed disulfides regulation (Simplicio et al., 1998). The level of cysteine is the major factor that regulates the metabolism of intracellular glutathione (Stipanuk et al., 1992). Cysteine, but not GSH or N-acetylcysteine, has been shown to rapidly restore GSH and to decrease thiol-protein mixed disulfides to basal levels (Stipanuk et al., 1992; Simplicio et al., 1998).

Metabolic conversion of Olt and/or 2-AP is likely to be coupled with the steady-state consumption of reduced GSH content in the cells. However, 2-AP increases the GSH content in the liver in a dose-related manner (Kim et al., 1997a). Increases in the GSH level were also observed in animals treated with Olt (Ansher et al., 1983; Bolton et al., 1993). mEH and rGSTA2 mRNAs were elevated to greater extents in the GSH-depleting state by the agents. Hence, the increase in GSH level after treatment with Olt or 2-AP might result from the increase in the production of reactive oxygen species. A number of hepatotoxicants increase intracellular GSH levels as part of adaptive responses unless cellular viability is decreased (McMillan and Jollow, 1992; Simplicio et al., 1998). The oxidative stress from the chemopreventive agents may stimulate the synthesis of glutathione to compensate for the altered cellular oxidation state in conjunction with the transcriptional activation of phase II antioxidant enzymes. This compensatory increase in the intracellular GSH level may be associated with smaller increases in the mRNA levels at later times after multiple treatments than at early times. Hence, the elevation of the intracellular GSH content by either Olt or 2-AP is unlikely to cause the transcriptional activation of phase II enzymes. Reduction in the GSH level caused by oxidative stress would subsequently feedback-stimulate the production of GSH as well as the induction of phase II enzymes.

NF-B is activated by oxidative stress and the activation has been correlated with the cellular redox state. It has been proposed that the intracellular thiol level affects the expression of several genes after early activation of NF-B (Schreck et al., 1992; Hecker et al., 1996). Transcriptional activation of the genes of NAD(P)H:quinone oxidoreductase and manganese-dependent superoxide dismutase has been implicated with NF-B activation in cultured hepatocytes (Yao and O'dwyer, 1995; Antras-Ferry et al., 1997). In this study, however, the level of NF-B transcription factor failed to be altered by Olt or 2-AP in the rat liver, supporting the hypothesis that NF-B activation might not be involved in the transcriptional activation of mEH and GST genes by the agents in rats. This was also in accordance with our previous observation that LPS inhibited the constitutive and inducible mEH and GST expression irrespective of its activation of NF-B (Choi and Kim, 1998). The transient activation of NF-B by Olt in cultured hepatocytes might result from the altered signals in conjunction with other substances present in the culture media (e.g., serum-derived factors). Neither Olt or 2-AP activated the AP-1 nuclear factor complexes in the liver (data not shown).

The activated oxygens seem to be potentially responsible for the transcriptional induction of anticarcinogenic enzymes (Kensler et al., 1992; Hayes and Pulford, 1995). Increases in the mRNA levels of mEH and GST by Olt and presumably by 2-AP may be mediated with ARE in the genes by transcriptional activation (Hayes and Pulford, 1995). The greater increases in the hepatic mEH and rGSTA2 mRNA levels in the GSH-depleting animals provide evidence that reactive oxygen species appeared not to be produced by nonenzymatic breakdown of the agents in the presence of thiol, but presumably by bioactivation. 2-AP was effective in scavenging the reactive oxygens and in preventing iron-catalyzed DNA degradation in vitro, showing that 2-AP serves as an antioxidant in vitro. In contrast, Olt failed to scavenge oxygen species and to prevent the DNA damage. In spite of their different antioxidant effects in vitro, both agents produced reactive oxygens in vivo, which would lead to the induction of anticarcinogenic enzymes. 2-AP, in contrast with Olt, failed to convert oxygen to activated oxygen radicals in the presence of thiol in vitro. Hence, the proposed thiol-dependent production of activated oxygens by Olt (Kim and Gates, 1997) is unlikely to be responsible for the production of reactive oxygens involved in the induction of detoxifying enzymes.

In summary, 2-AP was effective in elevating the mEH and major GST gene expression in the rat liver through production of activated oxygens with no NF-B activation in vivo and that 2-AP differed from Olt in thiol-dependent conversion of DNA topology and prevented DNA damage caused by autoxidation of BT in vitro, although both agents produced reactive oxygen species in vivo.