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Phytotoxicity and metabolism of ethofumesate in transgenic rice plants expressing the human CYP2B6 gene
Pesticide Biochemistry and Physiology 74 (2002) 139–147 www.elsevier.com/locate/ypest
Phytotoxicity and metabolism of ethofumesate in transgenic rice plants expressing the human CYP2B6 gene Hiroyuki Kawahigashi,a,* Sakiko Hirose,a Etsuko Hayashi,b Hideo Ohkawa,b and Yasunobu Ohkawaa a
b
Plant Biotechnology Department, National Institute of Agrobiological Sciences, 2-1-2, Kannondai, Tsukuba, Ibaraki 305-8602, Japan Research Center for Environmental Genomics, Kobe University, Rokkodaicho 1-1, Nada, Kobe 657-8501, Japan Received 18 July 2002; accepted 24 November 2002
Abstract Transgenic rice plants expressing human CYP1A1, CYP2B6, or CYP2C19 show strong cross-tolerance to various herbicides. However, these plants showed susceptibility to the herbicides ethofumesate and benfuresate. Both herbicides inhibited germination of transgenic rice plants at a concentration of 2.0 lM in the culture medium, whereas control Nipponbare plants showed normal growth in their presence. The CYP2B6 rice plants metabolized ethofumesate to produce the de-ethylated metabolite DHDBM (2,3-dihydro-2-hydroxy-3,3-dimethyl-5-benzofuranyl methanesulfonate), which was accumulated to levels up to 60 times higher than in control plants. Germination of both control and CYP2B6 rice plants was inhibited completely with 0.75 lM DHDBM in the culture medium. The phytotoxicity of DHDBM to rice plants was at least four times greater than that of ethofumesate. Because both ethofumesate and benfuresate are metabolized to give DHDBM or analogous metabolites, we consider that DHDBM was the major phytotoxic metabolite in these rice plants. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Cytochrome P450; Benzofuranyl alkanesulfonate; Benfuresate; Herbicide tolerance; Germination; Transgenic rice; Phytoremediation
1. Introduction Cytochrome P450 (P450) mono-oxygenases play important roles in oxidative reactions in secondary metabolism and xenobiotic metabolism in higher plants. Genome sequencing has revealed that P450 species constitute the largest family of *
Corresponding author. Fax: +81-298-38-8397. E-mail address: shiwak@affrc.go.jp (H. Kawahigashi).
enzymes in higher plants [1]. Most of the enzymes are involved in the biosynthesis of secondary metabolites, including fatty acids, steroids, phenylpropanoids, terpenoids, and alkaloids. A number of P450 species are involved in the metabolism of xenobiotic compounds in the microsomes of mammalian livers. These P450 species show a broad and overlapping substrate specificity toward lipophilic xenobiotics, including herbicides. In humans, 11 P450 species seem to cover
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more than 90% of P450-dependent xenobiotic metabolism in the liver [2]. Shiota et al. [3] attempted to generate transgenic plants expressing mammalian P450 species. Transgenic tobacco plants harboring the rat CYP1A1 gene showed high tolerance toward the herbicide chlortoluron [4]. The human CYP1A1 gene has been introduced into tobacco, potato, and rice [5,6]. These transgenic plants showed a remarkable cross-tolerance toward herbicides, including chlortoluron, atrazine, and norflurazon. The CYP2B6 gene has been also introduced into rice plants. Transgenic CYP2B6 rice plants showed strong herbicide tolerance, particularly toward the acetanilide herbicides, including acetochlor, alachlor, and metolachlor [7]. It was shown that CYP2B6 rice plants produced the P450 enzyme in their leaves and metabolized these herbicides more effectively than control plants [8]. The herbicides ethofumesate and benfuresate are benzofuranyl alkanesulfonate herbicides. Ethofumesate is used in pre- and post-emergence control of grasses and broad-leaved weeds in sugar beet and other crops, and in turf, ryegrass, and other pasture grasses [9–11]. Benfuresate is
used for the post-emergence control of grasses and broad-leaved weeds in paddy rice, fruit, beans, maize, sugarcane, and perennial crops, and for pre-sowing weed control in cotton and tobacco [12–14]. Particularly in Japan, benfuresate is used to control Eleocharis kuroguwai in paddy fields, a plant that is difficult to control with other herbicides. Both herbicides inhibit the growth of meristems, retard cell division, and limit cuticle formation [15]. In a study of the fate of ethofumesate in sugar beet and weeds, it was found that rapid metabolism was the basis for the development of tolerance to ethofumesate [16]. The metabolic pathway of benfuresate in 2 varieties of rice has been reported [17]. Benfuresate is hydroxylated at the C2 site, the ring is opened, and the resulting aldehyde is oxidized to the acid as shown in Fig. 1. In contrast, oxidative dealkylation would be the initial reaction for ethofumesate. The metabolites of ethofumesate and benfuresate, DHDBM and DHDBE (2,3-dihydro-2-hydroxy-3,3-dimethyl-5-benzofuranyl ethanesulfonate), respectively, are both 2-hydroxy derivatives (Fig. 1). Then these metabolites are hydroxylated to HMSPP (2-(hydroxy-4-methyl-
Fig. 1. Schematic diagram of the metabolic pathways of ethofumesate and benfuresate in transgenic rice plants expressing P450 species. Black arrows indicate the points at which each herbicide is attacked by P450s.
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sulfonyloxyphenyl)-2-methylpropanoic acid) and HESPP (2-(hydroxy-4-ethylsulfonyloxyphenyl)-2methylpropanoic acid), respectively. We attempted to examine the phytotoxicity and metabolism of ethofumesate and benfuresate in transgenic rice plants expressing the human CYP2B6 gene, because P450 species have been found to metabolize both herbicides [18] and human P450s are well characterized about their enzymatic activities to avoid potential risks of transgenic plants. We also examined the phytotoxicity to rice plants of CYP2B6 metabolites of ethofumesate.
2. Materials and methods
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mosaic virus (CaMV) 35S promoter containing a 7-tandem enhancer region and a 50 -untranslated region from the coat protein mRNA of alfalfa mosaic virus (AMV). The plasmids were each used for transformation of rice plants in the same manner. 2.3. Germination tests Germination tests were carried out in a 2.5-cmdiameter 15-cm-high test tube. R2 seeds of transgenic and non-transgenic control (Nipponbare) rice plants were embedded into 10 mL MS solid medium [19] containing up to 2.0 lM ethofumesate, benfuresate or DHDBM and cultured for 10 days under 16 h light conditions ð40 lmol= m2 sÞ at 27 °C.
2.1. Chemicals Ethofumesate and benfuresate were purchased from Hayashi Pure Chemical Industries (Osaka, Japan). 14 C-ring-labeled ethofumesate, ðÞ-2-ethoxy2,3-dihydro-3,3-dimethyl-5-benzofuranyl methanesulfonate (sp. act. 2.33 GBq/mmol, radiochemical purity > 99.5%), was purchased from Amersham Pharmacia Biotech (Buckinghamshire, England). The de-ethylated metabolite of ethofumesate, 2, 3-dihydro-2-hydroxy-3,3-dimethyl-5-benzofuranyl methanesulfonate (DHDBM), was synthesized at Nacalai Tesque (Kyoto, Japan). 2.2. Preparation of rice plants expressing CYP2B6 Human CYP1A1, CYP2B6, and CYP2C19 cDNA clones were provided by Dr. Inui of Faculty of Agriculture in Kobe University. The expression plasmid pIJ2B6 was constructed by the insertion of human CYP2B6 cDNA into the pIG121 vector. The constructed expression plasmid was introduced into Agrobacterium strain EHA101, which was subsequently used for transformation of Oryza sativa cv. ÔNipponbare.Õ Plants (R0 ) regenerated on medium containing 50 mg/L hygromycin were analyzed by PCR using human-CYP2B6-specific primers. Progeny of the CYP2B6 rice were selected by a germination test with 2.5 lM metolachlor. On the basis of the tolerance of the progeny toward metolachlor, homozygotes (lines A11, A1181) were selected and used for further study. Human CYP1A1 and CYP2C19 cDNA clones were each introduced into the expression vector pIES6, which carries the chimeric cauliflower
2.4. Thin-layer chromatography (TLC) analysis Rice seeds were planted in MS solid medium [19] and incubated at 27 °C for 6 days under 16 h light conditions ð40 lmol=m2 sÞ. Six-day-old plants were each transferred into 3 mL Hyponex 5-10-5 (Hyponex, Osaka, Japan) solution containing 40,000 dpm ½14 Cethofumesate at a concentration of 10 lM in a 2.5-cm-diameter 15-cm-high test tube. The plants were incubated under 24 h light conditions ð40 lmol=m2 sÞ, and both the plants and the culture medium were sampled at 0, 12, 24, and 48 h of incubation. Three samples were analyzed at each point. Radioactive chemicals were extracted from plants with a mixture of methanol and water (9:1, v/v). The extract and the culture medium were dried and dissolved in 90% methanol. Radioactive extracts of 2000 dpm were applied to each lane on a silica gel 60F254 TLC plate (Merck, Darmstadt, Germany). The TLC plates were developed with dichloromethane. Radioactivity was measured in an FLA-2000 Bio-Imaging Analyzer (Fuji Photo Film, Tokyo, Japan). 2.5. HPLC and LC/MS analysis Ethofumesate metabolites produced in CYP2B6 rice plants and recombinant yeast microsomes were dissolved in 20 lL of 90% methanol and subjected to high-performance liquid chromatography (HPLC) (Model L-7000, Hitachi, Tokyo, Japan) or liquid chromatography/mass spectrometry (APCI-LC/MS; Model L-8000T, Hitachi, Tokyo, Japan). The solvent system for
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HPLC was water containing 0.1% acetic acid and acetonitrile (65:45, v/v). Metabolites were separated at a flow rate of 1.0 mL/min on a 4:6- 150-mm Comosil 5C18-AR column (Nacalai Tesque, Kyoto, Japan) and detected at a wavelength of 230 nm. The conditions for MS were as follows: needle voltage, 3500 V; focus voltage, 30 V; nebulizer temperature, 140 °C; desolvator temperature, 360 °C; aperture temperature, 150 °C; polarity, negative; drift voltage, 30–70 V.
3. Results The CYP2B6 rice plants have been reported to show a remarkable tolerance to the herbicides alachlor, acetochlor, and metolachlor [7]. However, they did not grow in culture medium containing 2.0 lM ethofumesate, in which the untransformed rice Nipponbare grew. Similarly, the CYP2B6 rice plants were sensitive to 2.0 lM benfuresate in the culture medium (Fig. 2). Transgenic rice plants expressing human CYP1A1 or CYP2C19 also showed higher sensitivity to both ethofumesate and benfuresate than the control plant Nipponbare (Fig. 2). Because similar results were obtained in CYP1A1, CYP2B6, and
CYP2C19 rice plants, further analyses were performed on CYP2B6 rice plants only. After ½14 Cethofumesate was applied to both the control and CYP2B6 rice plants, ½14 C-metabolites from the plants were analyzed by TLC. Both the control Nipponbare and the CYP2B6 rice plants metabolized ethofumesate, and similar metabolites were produced in both plants (Fig. 3). The CYP2B6 rice plants metabolized ethofumesate more actively than Nipponbare. Two metabolites were detected on TLC analysis, and one of these metabolites was identified as DHDBM by co-chromatography in HPLC and LC–MS with authentic standards. From the results of HPLC and LC–MS analysis we were reasonably certain that the other metabolite was HMSPP. About 10% of the ½14 C-radioactivity added was detected in the extracts from both Nipponbare and the CYP2B6 rice plants. Ethofumesate accounted for 70–90% of the radioactivity in the whole-plant extracts of Nipponbare (Fig. 4A). The metabolite DHDBM was hardly detectable. In contrast, water-soluble conjugated compounds (indicated as ori) accounted for 75–95% of the radioactivity in the CYP2B6 rice plants. The metabolite DHDBM accounted for 12.5% of the radioactivity in the CYP2B6 rice plants after 12 h of incubation and 8.7% after 24 h. In CYP2B6 rice
Fig. 2. Phytotoxicity of ethofumesate and benfuresate in transgenic rice plants expressing P450 species. Lane CN, control Nipponbare without herbicides; lane N, Nipponbare; lane A, CYP1A1 rice plants; lane B, CYP2B6 rice plants; lane C, CYP2C19 rice plants.
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Fig. 3. TLC analysis of ethofumesate metabolites produced in CYP2B6 rice plants. (A) TLC analysis of extracts from whole plants and (B) extracts from culture medium treated with [14 C]ethofumesate. Equal counts (2000 dpm) of radioactive extracts were applied to each lane on a TLC plate and analyzed. EF, HMSPP, DHDBM, and ori indicate the migrating points of ethofumesate, HMSPP, and DHDBM and the origin of the TLC plates, respectively.
plants, DHDBM accumulated to a level 60 times higher than that in Nipponbare after 12 h and 10 times after 24 h. In the culture medium of Nipponbare, most of the radioactivity consisted of ethofumesate (Fig. 4B), the level of which was about 60% of the initial level of radioactivity even after 48 h of incubation. DHDBM accounted for only 5.7% of the initial radioactivity after 48 h. In contrast, in the culture medium of the CYP2B6 rice plants, ethofumesate was metabolized rapidly and accounted for only 8.9% of the initial radioactivity after 12 h incubation (Fig. 4C). The metabolite DHDBM increased rapidly to a level of 47% of the initial radioactivity after 12 h and decreased to 17% after 48h. HMSPP increased to levels of about 16.7% and 19.4% of the initial radioactivity after 24 and
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48 h, respectively. Therefore, we considered that in the CYP2B6 rice plants, ethofumesate was metabolized to produce DHDBM, which was then hydroxylated to HMSPP. HMSPP was finally conjugated to give water-soluble metabolites. Because we considered that DHDBM was the initial metabolite in the CYP2B6 rice plants, we performed germination tests with Nipponbare and the CYP2B6 rice plants using chemically synthesized DHDBM. Germination of both Nipponbare and the CYP2B6 rice plants was almost inhibited with 0.75 lM DHDBM in the culture medium. The inhibitory effect of DHDBM was nearly the same on both Nipponbare and the CYP2B6 rice plants (Fig. 5). Retarded germination of Nipponbare was observed with 0.5 lM DHDBM in the culture medium. Similar retarded germination of Nipponbare was observed with 2.0 lM ethofumesate in the culture medium (Fig. 2). Therefore, the phytotoxicity of DHDBM to rice plants was at least four times greater than that of ethofumesate (Figs. 2 and 5). In contrast, the CYP2B6 rice plants were susceptible to a 0.75-lM concentration of both ethofumesate and DHDBM in the culture medium. Germination was inhibited with 0.5 lM ethofumesate in the culture medium (Fig. 5B), but better growth was observed with 0.5 lM DHDBM.
4. Discussion In humans, 11 P450 species involved in xenobiotic metabolism in the microsomes of the liver have been reported to cover more than 90% of P450-dependent xenobiotic metabolism [2]. These P450 species exhibit a broad and overlapping substrate specificity toward xenobiotics. They enable us to metabolize a large number of chemicals, although some chemicals are activated by the metabolism of P450 species in the liver [20,21]. We found that the CYP2B6 rice plants showed a remarkable tolerance towards several herbicides so far [7]. However, transgenic rice plants expressing human CYP1A1, CYP2B6, or CYP2C19 showed greater susceptibility to ethofumesate and benfuresate than control Nipponbare plants. It has been reported that recombinant yeast microsomes expressing human CYP2B6 or CYP2C19 metabolize ethofumesate, whereas recombinant yeast microsomes expressing human CYP1A1, CYP2B6, CYP2C9, CYP2C18, or CYP2C19 metabolize benfuresate [18]. Therefore, CYP2B6 ex-
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Fig. 4. Time course of metabolism of ethofumesate in control (Nipponbare) and CYP2B6 rice plants (A1181). (A) Relative amounts of ethofumesate and its metabolites in extracts from whole plants. (B) Degradation of ethofumesate in culture medium of Nipponbare and (C) in culture medium of the CYP2B6 rice plants. Radioactive spots in TLC analysis were measured with a bio-imaging analyzer. The quantity of original radioactivity applied to the tube was set at 100%. These values are the averages of three experiments performed independently.
pressed in the transgenic rice plants could metabolize both ethofumesate and benfuresate. Both herbicides are in the same group. Therefore, we expected that their related metabolites would cause phytotoxicity. We examined the metabolism of ethofumesate in rice plants. DHDBM was the major metabolites and accumulated to a level 60 times greater in extracts from CYP2B6 rice plants than in extracts from Nipponbare controls after 12 h of incubation. Similarly, the DHDBM level was 9.7 times higher in the culture medium of the transgenic plants after 12 h of incubation. These results indicate that CYP2B6 worked effectively to metabolize ethofumesate, giving rise to much greater levels of the de-ethylated metabolite DHDBM than in the Nipponbare control. In the germination test, DHDBM showed stronger phytotoxicity than ethofumesate to Nipponbare (Fig. 5). Neither Nipponbare nor the
CYP2B6 rice plants showed normal growth in 0.5 lM DHDBM, whereas Nipponbare grew up in 2.0 lM ethofumesate. The TLC analysis showed the accumulation of DHDBM in CYP2B6 rice plants but not in Nipponbare. CYP2B6 rice plants were susceptible to both ethofumesate and DHDBM (Fig. 5). Therefore, we conclude that the susceptibility of the CYP2B6 rice plants to ethofumesate was caused by the presence of DHDBM produced from ethofumesate by the action of CYP2B6. The phytotoxicity of DHDBM to Nipponbare was more than four times greater than that of ethofumesate when they were added to the culture medium. The results that CYP2B6 rice plants were more sensitive to the related herbicides ethofumesate and benfuresate can be explained by the kinetics. If the conversion of HMSPP to the conjugates is slower than the formation of DHDBM enhanced by the introduced CYP2B6 and the further metabolite HMSPP, then accumulation of these
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Fig. 5. Phytotoxicity of ethofumesate and DHDBM in Nipponbare and CYP2B6 rice plants. (A) Germination tests of Nipponbare in MS culture medium containing 0.25–1.0 lM DHDBM. (B) Germination tests of CYP2B6 rice plants A11 (right) and A1181 (left) in MS culture medium containing 0.5 or 0.75 lM ethofumesate or DHDBM.
compounds would lead to the enhanced phytotoxicity in CYP2B6 rice plants. In the control plants, Nipponbare, the formation of DHDBM and HMSPP is similar in rate to the formation of the conjugates. Therefore DHDBM and HMSPP do not significantly accumulate during the course of conversion to the conjugates. It was clear that the accumulation of DHDBM leads the rice plants to the loss of tolerance in our experiments. In a study of the fate of ethofume-
sate in sugar beet and weeds, however, the rapid metabolism was the basis for the tolerance to ethofumesate [16]. It is difficult to compare the differences in tolerance between the sugar beets and rice plants because the tolerance highly depends on growth stage and plant condition. Considering the amount of herbicides applied to the practical field, rice plants seems to be more sensitive than sugar beet (benfuresate to rice, 5 g/a; ethofumesate to sugar beet, 220 g/a).
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In tolerant plants such as sugar beet, ethofumesate was rapidly metabolized to water-soluble and non-extractable compounds, which are presumed to be non-toxic [16]. Thus, toxic compounds ethofumesate and DHDBM do not seem to accumulate enough in the tolerant plants to show toxicity. On the other hand, ethofumesate accumulated in sensitive plants such as redroot pigweed [16] and non-transgenic rice plants (Fig. 4). In the CYP2B6 rice plants, the first step of metabolism of ethofumesate was accelerated by introduced CYP2B6, toxic DHDBM accumulated more in the plants to show severe toxicity (Fig. 4). A combination of the bacterial P450 gene and the sulfonylurea compound R7402 has been used as a negative selection marker in transgenic barley [22]. Similarly, the combination of CYP2B6 and ethofumesate or benfuresate may be useful as a negative selection system in transgenic plant technologies such as homologous recombination systems. Moreover, human P450 species and herbicide systems would be useful for large-scale screening of transgenic plants by germination testing. Although plants have been found that they can naturally take up and metabolize chemicals and pollutants, transgenic plants have enhanced the capabilities including 2,4,6-trinitrotoluene [23,24], trichloroethylene [25], and heavy metals [26,27]. Recombinant yeast microsomes expressing human P450 species can metabolize lipophilic chemical compounds such as herbicides and potential endocrine disrupters [18]. Therefore transgenic plants expressing P450 species would be useful not only for combating herbicide tolerance but also for phytoremediation of environmental contaminants in soil as well as in ground and surface waters.
Acknowledgments This study was supported by a program for Promotion of Basic Research Activities for Innovative Biosciences of Bio-oriented Technology Research Advancement Institution (BRAIN). Agrobacterium EHA101 was kindly provided by the Plant Biotechnology Institute in Ibaraki, Japan.
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Phytotoxicity and metabolism of ethofumesate in transgenic rice plants expressing the human CYP2B6 gene Hiroyuki Kawahigashi,a,* Sakiko Hirose,a Etsuko Hayashi,b Hideo Ohkawa,b and Yasunobu Ohkawaa a
b
Plant Biotechnology Department, National Institute of Agrobiological Sciences, 2-1-2, Kannondai, Tsukuba, Ibaraki 305-8602, Japan Research Center for Environmental Genomics, Kobe University, Rokkodaicho 1-1, Nada, Kobe 657-8501, Japan Received 18 July 2002; accepted 24 November 2002
Abstract Transgenic rice plants expressing human CYP1A1, CYP2B6, or CYP2C19 show strong cross-tolerance to various herbicides. However, these plants showed susceptibility to the herbicides ethofumesate and benfuresate. Both herbicides inhibited germination of transgenic rice plants at a concentration of 2.0 lM in the culture medium, whereas control Nipponbare plants showed normal growth in their presence. The CYP2B6 rice plants metabolized ethofumesate to produce the de-ethylated metabolite DHDBM (2,3-dihydro-2-hydroxy-3,3-dimethyl-5-benzofuranyl methanesulfonate), which was accumulated to levels up to 60 times higher than in control plants. Germination of both control and CYP2B6 rice plants was inhibited completely with 0.75 lM DHDBM in the culture medium. The phytotoxicity of DHDBM to rice plants was at least four times greater than that of ethofumesate. Because both ethofumesate and benfuresate are metabolized to give DHDBM or analogous metabolites, we consider that DHDBM was the major phytotoxic metabolite in these rice plants. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Cytochrome P450; Benzofuranyl alkanesulfonate; Benfuresate; Herbicide tolerance; Germination; Transgenic rice; Phytoremediation
1. Introduction Cytochrome P450 (P450) mono-oxygenases play important roles in oxidative reactions in secondary metabolism and xenobiotic metabolism in higher plants. Genome sequencing has revealed that P450 species constitute the largest family of *
Corresponding author. Fax: +81-298-38-8397. E-mail address: shiwak@affrc.go.jp (H. Kawahigashi).
enzymes in higher plants [1]. Most of the enzymes are involved in the biosynthesis of secondary metabolites, including fatty acids, steroids, phenylpropanoids, terpenoids, and alkaloids. A number of P450 species are involved in the metabolism of xenobiotic compounds in the microsomes of mammalian livers. These P450 species show a broad and overlapping substrate specificity toward lipophilic xenobiotics, including herbicides. In humans, 11 P450 species seem to cover
0048-3575/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0048-3575(02)00153-0
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more than 90% of P450-dependent xenobiotic metabolism in the liver [2]. Shiota et al. [3] attempted to generate transgenic plants expressing mammalian P450 species. Transgenic tobacco plants harboring the rat CYP1A1 gene showed high tolerance toward the herbicide chlortoluron [4]. The human CYP1A1 gene has been introduced into tobacco, potato, and rice [5,6]. These transgenic plants showed a remarkable cross-tolerance toward herbicides, including chlortoluron, atrazine, and norflurazon. The CYP2B6 gene has been also introduced into rice plants. Transgenic CYP2B6 rice plants showed strong herbicide tolerance, particularly toward the acetanilide herbicides, including acetochlor, alachlor, and metolachlor [7]. It was shown that CYP2B6 rice plants produced the P450 enzyme in their leaves and metabolized these herbicides more effectively than control plants [8]. The herbicides ethofumesate and benfuresate are benzofuranyl alkanesulfonate herbicides. Ethofumesate is used in pre- and post-emergence control of grasses and broad-leaved weeds in sugar beet and other crops, and in turf, ryegrass, and other pasture grasses [9–11]. Benfuresate is
used for the post-emergence control of grasses and broad-leaved weeds in paddy rice, fruit, beans, maize, sugarcane, and perennial crops, and for pre-sowing weed control in cotton and tobacco [12–14]. Particularly in Japan, benfuresate is used to control Eleocharis kuroguwai in paddy fields, a plant that is difficult to control with other herbicides. Both herbicides inhibit the growth of meristems, retard cell division, and limit cuticle formation [15]. In a study of the fate of ethofumesate in sugar beet and weeds, it was found that rapid metabolism was the basis for the development of tolerance to ethofumesate [16]. The metabolic pathway of benfuresate in 2 varieties of rice has been reported [17]. Benfuresate is hydroxylated at the C2 site, the ring is opened, and the resulting aldehyde is oxidized to the acid as shown in Fig. 1. In contrast, oxidative dealkylation would be the initial reaction for ethofumesate. The metabolites of ethofumesate and benfuresate, DHDBM and DHDBE (2,3-dihydro-2-hydroxy-3,3-dimethyl-5-benzofuranyl ethanesulfonate), respectively, are both 2-hydroxy derivatives (Fig. 1). Then these metabolites are hydroxylated to HMSPP (2-(hydroxy-4-methyl-
Fig. 1. Schematic diagram of the metabolic pathways of ethofumesate and benfuresate in transgenic rice plants expressing P450 species. Black arrows indicate the points at which each herbicide is attacked by P450s.
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sulfonyloxyphenyl)-2-methylpropanoic acid) and HESPP (2-(hydroxy-4-ethylsulfonyloxyphenyl)-2methylpropanoic acid), respectively. We attempted to examine the phytotoxicity and metabolism of ethofumesate and benfuresate in transgenic rice plants expressing the human CYP2B6 gene, because P450 species have been found to metabolize both herbicides [18] and human P450s are well characterized about their enzymatic activities to avoid potential risks of transgenic plants. We also examined the phytotoxicity to rice plants of CYP2B6 metabolites of ethofumesate.
2. Materials and methods
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mosaic virus (CaMV) 35S promoter containing a 7-tandem enhancer region and a 50 -untranslated region from the coat protein mRNA of alfalfa mosaic virus (AMV). The plasmids were each used for transformation of rice plants in the same manner. 2.3. Germination tests Germination tests were carried out in a 2.5-cmdiameter 15-cm-high test tube. R2 seeds of transgenic and non-transgenic control (Nipponbare) rice plants were embedded into 10 mL MS solid medium [19] containing up to 2.0 lM ethofumesate, benfuresate or DHDBM and cultured for 10 days under 16 h light conditions ð40 lmol= m2 sÞ at 27 °C.
2.1. Chemicals Ethofumesate and benfuresate were purchased from Hayashi Pure Chemical Industries (Osaka, Japan). 14 C-ring-labeled ethofumesate, ðÞ-2-ethoxy2,3-dihydro-3,3-dimethyl-5-benzofuranyl methanesulfonate (sp. act. 2.33 GBq/mmol, radiochemical purity > 99.5%), was purchased from Amersham Pharmacia Biotech (Buckinghamshire, England). The de-ethylated metabolite of ethofumesate, 2, 3-dihydro-2-hydroxy-3,3-dimethyl-5-benzofuranyl methanesulfonate (DHDBM), was synthesized at Nacalai Tesque (Kyoto, Japan). 2.2. Preparation of rice plants expressing CYP2B6 Human CYP1A1, CYP2B6, and CYP2C19 cDNA clones were provided by Dr. Inui of Faculty of Agriculture in Kobe University. The expression plasmid pIJ2B6 was constructed by the insertion of human CYP2B6 cDNA into the pIG121 vector. The constructed expression plasmid was introduced into Agrobacterium strain EHA101, which was subsequently used for transformation of Oryza sativa cv. ÔNipponbare.Õ Plants (R0 ) regenerated on medium containing 50 mg/L hygromycin were analyzed by PCR using human-CYP2B6-specific primers. Progeny of the CYP2B6 rice were selected by a germination test with 2.5 lM metolachlor. On the basis of the tolerance of the progeny toward metolachlor, homozygotes (lines A11, A1181) were selected and used for further study. Human CYP1A1 and CYP2C19 cDNA clones were each introduced into the expression vector pIES6, which carries the chimeric cauliflower
2.4. Thin-layer chromatography (TLC) analysis Rice seeds were planted in MS solid medium [19] and incubated at 27 °C for 6 days under 16 h light conditions ð40 lmol=m2 sÞ. Six-day-old plants were each transferred into 3 mL Hyponex 5-10-5 (Hyponex, Osaka, Japan) solution containing 40,000 dpm ½14 Cethofumesate at a concentration of 10 lM in a 2.5-cm-diameter 15-cm-high test tube. The plants were incubated under 24 h light conditions ð40 lmol=m2 sÞ, and both the plants and the culture medium were sampled at 0, 12, 24, and 48 h of incubation. Three samples were analyzed at each point. Radioactive chemicals were extracted from plants with a mixture of methanol and water (9:1, v/v). The extract and the culture medium were dried and dissolved in 90% methanol. Radioactive extracts of 2000 dpm were applied to each lane on a silica gel 60F254 TLC plate (Merck, Darmstadt, Germany). The TLC plates were developed with dichloromethane. Radioactivity was measured in an FLA-2000 Bio-Imaging Analyzer (Fuji Photo Film, Tokyo, Japan). 2.5. HPLC and LC/MS analysis Ethofumesate metabolites produced in CYP2B6 rice plants and recombinant yeast microsomes were dissolved in 20 lL of 90% methanol and subjected to high-performance liquid chromatography (HPLC) (Model L-7000, Hitachi, Tokyo, Japan) or liquid chromatography/mass spectrometry (APCI-LC/MS; Model L-8000T, Hitachi, Tokyo, Japan). The solvent system for
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HPLC was water containing 0.1% acetic acid and acetonitrile (65:45, v/v). Metabolites were separated at a flow rate of 1.0 mL/min on a 4:6- 150-mm Comosil 5C18-AR column (Nacalai Tesque, Kyoto, Japan) and detected at a wavelength of 230 nm. The conditions for MS were as follows: needle voltage, 3500 V; focus voltage, 30 V; nebulizer temperature, 140 °C; desolvator temperature, 360 °C; aperture temperature, 150 °C; polarity, negative; drift voltage, 30–70 V.
3. Results The CYP2B6 rice plants have been reported to show a remarkable tolerance to the herbicides alachlor, acetochlor, and metolachlor [7]. However, they did not grow in culture medium containing 2.0 lM ethofumesate, in which the untransformed rice Nipponbare grew. Similarly, the CYP2B6 rice plants were sensitive to 2.0 lM benfuresate in the culture medium (Fig. 2). Transgenic rice plants expressing human CYP1A1 or CYP2C19 also showed higher sensitivity to both ethofumesate and benfuresate than the control plant Nipponbare (Fig. 2). Because similar results were obtained in CYP1A1, CYP2B6, and
CYP2C19 rice plants, further analyses were performed on CYP2B6 rice plants only. After ½14 Cethofumesate was applied to both the control and CYP2B6 rice plants, ½14 C-metabolites from the plants were analyzed by TLC. Both the control Nipponbare and the CYP2B6 rice plants metabolized ethofumesate, and similar metabolites were produced in both plants (Fig. 3). The CYP2B6 rice plants metabolized ethofumesate more actively than Nipponbare. Two metabolites were detected on TLC analysis, and one of these metabolites was identified as DHDBM by co-chromatography in HPLC and LC–MS with authentic standards. From the results of HPLC and LC–MS analysis we were reasonably certain that the other metabolite was HMSPP. About 10% of the ½14 C-radioactivity added was detected in the extracts from both Nipponbare and the CYP2B6 rice plants. Ethofumesate accounted for 70–90% of the radioactivity in the whole-plant extracts of Nipponbare (Fig. 4A). The metabolite DHDBM was hardly detectable. In contrast, water-soluble conjugated compounds (indicated as ori) accounted for 75–95% of the radioactivity in the CYP2B6 rice plants. The metabolite DHDBM accounted for 12.5% of the radioactivity in the CYP2B6 rice plants after 12 h of incubation and 8.7% after 24 h. In CYP2B6 rice
Fig. 2. Phytotoxicity of ethofumesate and benfuresate in transgenic rice plants expressing P450 species. Lane CN, control Nipponbare without herbicides; lane N, Nipponbare; lane A, CYP1A1 rice plants; lane B, CYP2B6 rice plants; lane C, CYP2C19 rice plants.
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Fig. 3. TLC analysis of ethofumesate metabolites produced in CYP2B6 rice plants. (A) TLC analysis of extracts from whole plants and (B) extracts from culture medium treated with [14 C]ethofumesate. Equal counts (2000 dpm) of radioactive extracts were applied to each lane on a TLC plate and analyzed. EF, HMSPP, DHDBM, and ori indicate the migrating points of ethofumesate, HMSPP, and DHDBM and the origin of the TLC plates, respectively.
plants, DHDBM accumulated to a level 60 times higher than that in Nipponbare after 12 h and 10 times after 24 h. In the culture medium of Nipponbare, most of the radioactivity consisted of ethofumesate (Fig. 4B), the level of which was about 60% of the initial level of radioactivity even after 48 h of incubation. DHDBM accounted for only 5.7% of the initial radioactivity after 48 h. In contrast, in the culture medium of the CYP2B6 rice plants, ethofumesate was metabolized rapidly and accounted for only 8.9% of the initial radioactivity after 12 h incubation (Fig. 4C). The metabolite DHDBM increased rapidly to a level of 47% of the initial radioactivity after 12 h and decreased to 17% after 48h. HMSPP increased to levels of about 16.7% and 19.4% of the initial radioactivity after 24 and
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48 h, respectively. Therefore, we considered that in the CYP2B6 rice plants, ethofumesate was metabolized to produce DHDBM, which was then hydroxylated to HMSPP. HMSPP was finally conjugated to give water-soluble metabolites. Because we considered that DHDBM was the initial metabolite in the CYP2B6 rice plants, we performed germination tests with Nipponbare and the CYP2B6 rice plants using chemically synthesized DHDBM. Germination of both Nipponbare and the CYP2B6 rice plants was almost inhibited with 0.75 lM DHDBM in the culture medium. The inhibitory effect of DHDBM was nearly the same on both Nipponbare and the CYP2B6 rice plants (Fig. 5). Retarded germination of Nipponbare was observed with 0.5 lM DHDBM in the culture medium. Similar retarded germination of Nipponbare was observed with 2.0 lM ethofumesate in the culture medium (Fig. 2). Therefore, the phytotoxicity of DHDBM to rice plants was at least four times greater than that of ethofumesate (Figs. 2 and 5). In contrast, the CYP2B6 rice plants were susceptible to a 0.75-lM concentration of both ethofumesate and DHDBM in the culture medium. Germination was inhibited with 0.5 lM ethofumesate in the culture medium (Fig. 5B), but better growth was observed with 0.5 lM DHDBM.
4. Discussion In humans, 11 P450 species involved in xenobiotic metabolism in the microsomes of the liver have been reported to cover more than 90% of P450-dependent xenobiotic metabolism [2]. These P450 species exhibit a broad and overlapping substrate specificity toward xenobiotics. They enable us to metabolize a large number of chemicals, although some chemicals are activated by the metabolism of P450 species in the liver [20,21]. We found that the CYP2B6 rice plants showed a remarkable tolerance towards several herbicides so far [7]. However, transgenic rice plants expressing human CYP1A1, CYP2B6, or CYP2C19 showed greater susceptibility to ethofumesate and benfuresate than control Nipponbare plants. It has been reported that recombinant yeast microsomes expressing human CYP2B6 or CYP2C19 metabolize ethofumesate, whereas recombinant yeast microsomes expressing human CYP1A1, CYP2B6, CYP2C9, CYP2C18, or CYP2C19 metabolize benfuresate [18]. Therefore, CYP2B6 ex-
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Fig. 4. Time course of metabolism of ethofumesate in control (Nipponbare) and CYP2B6 rice plants (A1181). (A) Relative amounts of ethofumesate and its metabolites in extracts from whole plants. (B) Degradation of ethofumesate in culture medium of Nipponbare and (C) in culture medium of the CYP2B6 rice plants. Radioactive spots in TLC analysis were measured with a bio-imaging analyzer. The quantity of original radioactivity applied to the tube was set at 100%. These values are the averages of three experiments performed independently.
pressed in the transgenic rice plants could metabolize both ethofumesate and benfuresate. Both herbicides are in the same group. Therefore, we expected that their related metabolites would cause phytotoxicity. We examined the metabolism of ethofumesate in rice plants. DHDBM was the major metabolites and accumulated to a level 60 times greater in extracts from CYP2B6 rice plants than in extracts from Nipponbare controls after 12 h of incubation. Similarly, the DHDBM level was 9.7 times higher in the culture medium of the transgenic plants after 12 h of incubation. These results indicate that CYP2B6 worked effectively to metabolize ethofumesate, giving rise to much greater levels of the de-ethylated metabolite DHDBM than in the Nipponbare control. In the germination test, DHDBM showed stronger phytotoxicity than ethofumesate to Nipponbare (Fig. 5). Neither Nipponbare nor the
CYP2B6 rice plants showed normal growth in 0.5 lM DHDBM, whereas Nipponbare grew up in 2.0 lM ethofumesate. The TLC analysis showed the accumulation of DHDBM in CYP2B6 rice plants but not in Nipponbare. CYP2B6 rice plants were susceptible to both ethofumesate and DHDBM (Fig. 5). Therefore, we conclude that the susceptibility of the CYP2B6 rice plants to ethofumesate was caused by the presence of DHDBM produced from ethofumesate by the action of CYP2B6. The phytotoxicity of DHDBM to Nipponbare was more than four times greater than that of ethofumesate when they were added to the culture medium. The results that CYP2B6 rice plants were more sensitive to the related herbicides ethofumesate and benfuresate can be explained by the kinetics. If the conversion of HMSPP to the conjugates is slower than the formation of DHDBM enhanced by the introduced CYP2B6 and the further metabolite HMSPP, then accumulation of these
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Fig. 5. Phytotoxicity of ethofumesate and DHDBM in Nipponbare and CYP2B6 rice plants. (A) Germination tests of Nipponbare in MS culture medium containing 0.25–1.0 lM DHDBM. (B) Germination tests of CYP2B6 rice plants A11 (right) and A1181 (left) in MS culture medium containing 0.5 or 0.75 lM ethofumesate or DHDBM.
compounds would lead to the enhanced phytotoxicity in CYP2B6 rice plants. In the control plants, Nipponbare, the formation of DHDBM and HMSPP is similar in rate to the formation of the conjugates. Therefore DHDBM and HMSPP do not significantly accumulate during the course of conversion to the conjugates. It was clear that the accumulation of DHDBM leads the rice plants to the loss of tolerance in our experiments. In a study of the fate of ethofume-
sate in sugar beet and weeds, however, the rapid metabolism was the basis for the tolerance to ethofumesate [16]. It is difficult to compare the differences in tolerance between the sugar beets and rice plants because the tolerance highly depends on growth stage and plant condition. Considering the amount of herbicides applied to the practical field, rice plants seems to be more sensitive than sugar beet (benfuresate to rice, 5 g/a; ethofumesate to sugar beet, 220 g/a).
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In tolerant plants such as sugar beet, ethofumesate was rapidly metabolized to water-soluble and non-extractable compounds, which are presumed to be non-toxic [16]. Thus, toxic compounds ethofumesate and DHDBM do not seem to accumulate enough in the tolerant plants to show toxicity. On the other hand, ethofumesate accumulated in sensitive plants such as redroot pigweed [16] and non-transgenic rice plants (Fig. 4). In the CYP2B6 rice plants, the first step of metabolism of ethofumesate was accelerated by introduced CYP2B6, toxic DHDBM accumulated more in the plants to show severe toxicity (Fig. 4). A combination of the bacterial P450 gene and the sulfonylurea compound R7402 has been used as a negative selection marker in transgenic barley [22]. Similarly, the combination of CYP2B6 and ethofumesate or benfuresate may be useful as a negative selection system in transgenic plant technologies such as homologous recombination systems. Moreover, human P450 species and herbicide systems would be useful for large-scale screening of transgenic plants by germination testing. Although plants have been found that they can naturally take up and metabolize chemicals and pollutants, transgenic plants have enhanced the capabilities including 2,4,6-trinitrotoluene [23,24], trichloroethylene [25], and heavy metals [26,27]. Recombinant yeast microsomes expressing human P450 species can metabolize lipophilic chemical compounds such as herbicides and potential endocrine disrupters [18]. Therefore transgenic plants expressing P450 species would be useful not only for combating herbicide tolerance but also for phytoremediation of environmental contaminants in soil as well as in ground and surface waters.
Acknowledgments This study was supported by a program for Promotion of Basic Research Activities for Innovative Biosciences of Bio-oriented Technology Research Advancement Institution (BRAIN). Agrobacterium EHA101 was kindly provided by the Plant Biotechnology Institute in Ibaraki, Japan.
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