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V. Mode of Action of Herbicide Antidotes

V. Mode of Action of Herbicide Antidotes

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herbicide injury to corn or sorghum seedlings involve leaf or shoot deformations

such as leaf twisting or rolling, and at high rates leaves fail to emerge through the

coleoptile (Leavitt and Penner 1978b). These effects of chloroacetanilide and

thiocarbamate herbicides on grass crops are counteracted by the currently available herbicide antidotes.

Advances in our understanding of the modes of action of herbicides antidotes

are also complicated by the fact that some of the studies examining the phytotoxic effects of chloroacetanilideand thiocarbamate herbicides on plants, as well as

their counteraction by herbicide antidotes, have been conducted with broadleaved plants that are not protected against these herbicides under field conditions. Isolated spinach chloroplasts (Wilkinson and Smith, 1975), red beet disks

(Wilkinson and Smith, 1976), and tobacco suspension cultures (Rennenberg et

al., 1982) have all been used as plant systems to study the phytotoxic effects of

thiocarbamate herbicides and their counteraction by the antidotes NA and/or

R-25788. The significance of the results of these studies is difficult to assess

when attempting to explain the protective action of herbicide antidotes on grass


Extensive research on the mode of antidotal action of herbicide safeners has

been conducted primarily with the antidoteR-25788 and to a lesser extent with the

antidotes NA and CGA-43089. The findings of all these studies have resulted in a

plethora of proposed hypotheses about the mechanisms of the antidotal action of

herbicide safeners. Herbicide antidotes could protect grass crops from chloroacetanilide or thiocarbamate herbicide injury by (1) interfering with herbicide

uptake and/or translocation in the protected plant, (2) counteracting herbicide

phytotoxicity through a competitive inhibition at some common site within the

protected plant, (3) stimulatingherbicide degradation by the protected plant, and

(4) combinations of mechanisms (1)-(3). The status of our current knowledge on

the antidotal action of the herbicide safeners NA, R-25788, and CGA-43089 is

discussed briefly in the following sections. Studies on the mode of action of the

antidote MON-4606 are not yet available, whereas the antidotal action of

CGA-92194 probably resembles that of its chemical analog CGA-43089.




Early studies on the mode of antidotal action of NA revealed that its protective

effect against alachlor injury to sorghum was physiological in action and not the

result of a physical deactivation of the herbicide (Hickey and Krueger, 1974a).

Also, it was shown that the counteraction of alachlor effects on sorghum by NA

was more apparent in sorgbum shoots than in sorghum roots (Jordan and Jolliffe,

1971). Subsequent reports proposed that the protective effect of NA against

metolachlor injury to sorghum was partly caused by decreased herbicide uptake



and translocation (Ahrens and Davis, 1978). In other studies, however, NA was

not found to interfere with herbicide uptake and translocation in the protected

plants (Murphy, 1972; Holm and Szabo, 1974). Because corn seedlings treated

with NA absorbed more ['TIEPTC than nontreated ones, NA was reported to

act as a stimulator rather than as an inhibitor of herbicide uptake (Guneyli,

1971). The NA-induced stimulation of [14C]EPTCuptake by corn cell suspensions was found to be concentration dependent (Ezra et al., 1982).

In a number of other studies, interference of thiocarbamate and chloroacetanilide herbicides with plant physiological systems has been shown to be

counteracted by NA treatments. Thus, the EPTC-induced inhibition of fatty acid

synthesis in spinach chloroplasts and red beets disks was reversed by treatments

with NA (Wilkinson and Smith, 1975, 1976). Hickey and Krueger (1974b)

showed that alachlor alone increased the force needed for leaf emergence from

corn coleoptiles, but treatment with NA in a 6:l (antidotelherbicide) ratio reduced this force significantly. Hoffman (1978a) suggested that NA may act by

preventing the precocious bud dormancy of corn seedlings which is induced by

thiocarbamate and chloroacetanilide herbicides. This hypothesis was supported

by the fact that use of other well-known bud-dormancy-breaking chemicals such

as 1,Zdibromaethane resulted in the protection of corn from EPTC injury (Hoffman, 1978a). In studies monitoring the loss of 32P from onion roots it was found

that the mode of action of metolachlor involved membrane damage and that NA

was capable of protecting onion roots from permeability changes induced by this

herbicide (Mellis et al., 1982). Because metolachlor, even at high concentrations, did not induce any loss of 3*P from corn roots (Mellis et al., 1982), such a

mechanism could not explain the protective action of NA in counteracting

metolachlor or other chloroacetanilide herbicide injury to corn under field


An alternative mechanism proposed for explaining the antidotal action of NA

suggests an antidote-induced increase in the rate of metabolic detoxification of

chloroacetanilide or thiocarbamate herbicides in the protected plants. Guneyli

(1971) proposed that NA acts by activating the enzyme system(s) responsible for

EPTC breakdown in corn seedlings, whereas Holm and Szabo (1974) reported a

marked enhancement in the rate of metabolic breakdown of the herbicide

cisanilide in NA-treated corn seedlings. The significance of this mechanism,

however, has been disputed by other investigators who failed to detect significant

differences in the patterns of herbicide metabolism by NA-treated and nontreated

plants (Murphy, 1972; Hahn, 1974). Furthermore, NA was not effective in

enhancing the glutathione-S-transferaseactivity and gluthathione content in roots

of corn seedlings (Lay and Casida, 1976; Fedtke, 1981). As it will be discussed

in the next section, enhancement of the gluthathione content in corn roots has

been correlated with the protective effect of the antidote R-25788.

From the previous discussion, it is apparent that our understanding of the



antidotal action of NA is far from complete. In spite of many conflictingreports,

NA most probably exerts its antidotal action either through a stimulation of

herbicide metabolism or through a counteraction of the phytotoxic effects of

herbicides at a common site within the protected plant. The proposal that NA

may act by interfering with herbicide uptake and translocation in the protected

plants has not gained credence among researchers active in this field. However,

because of its limited botanical and chemical specificity, NA may have more

than one mode of action or it may act through a combination of all of the

proposed mechanisms. Future research is needed for a better understanding of

the antidotal action of NA.



In contrast to NA, R-25788 represents a physiologically selective antidote that

protects corn from thiocarbamate and chloroacetanilide herbicide injury (Pallos

et al., 1975; Leavitt and Penner, 1978b). Extensive research with the antidote

R-25788 during the last decade has resulted in many proposed mechanisms for

explaining its antidotal action. Early studies on the physiological behavior of

R-25788 showed that the coleoptiles of corn seedlings were the common site of

uptake and action of both the thiocarbamate and chloroacetanilideherbicides and

the antidote R-25788 (Donald and Fawcett, 1976; Gray and Joo, 1978). Because

of this, the possibility that R-25788 may protect corn from herbicide injury by

preventing the uptake and translocation of chloroacetanilide and thiocarbamate

herbicides has been examined. On the basis of present evidence, R-25788 does

not appear to prevent EPTC injury to corn seedlings by inhibiting herbicide

uptake or by altering herbicide translocation in corn (Chang et al., 1974a; Marton et al., 1978; Sagaral, 1978). On the contrary, an antidote-induced stimulation of EPTC uptake has been reported to occur in some corn cultivars that were

treated with EPTC and R-25788 (Carringer et al., 1974; Sagaral, 1978). It was

shown, however, that simultaneous applications of R-25788 and [14C]EPTCto

corn cell suspensions resulted in a rapid reduction of EPTC uptake by the corn

cells (Ezra et al., 1982). Kinetic analysis of this data indicated the existence of a

competitive inhibition of EPTC uptake by the antidote R-25788 on corn. This

competition for uptake is considered to be the first step in a series of interactions

between EPTC and R-25788 rather than a major mechanism involved in the

protective effect of R-25788 (Ezra et al., 1982).

The similarity between thiocarbamateand chloroacetanilideherbicide injury to

corn combined with the efficacy of R-25788 as an antidote for both herbicidal

classes on corn suggested that these two herbicidal classes have similar modes of

herbicidal action (Leavitt and Penner, 1978b). Furthermore, because R-25788

structurally resembles both of these herbicidal classes, the possibility that



R-25788 may counteract the effects of chloroacetanilide as well as thiocarbamate

herbicides on corn through a competitive inhibition at some active site specific to

corn has been investigated. In early studies, Wilkinson and Smith (1975) demonstrated that R-25788 could reverse an EPTC-induced inhibition of lipid synthesis

in isolated spinach chloroplasts, which suggested that R-25788 may compete

with EPTC at a site of lipid synthesis. This hypothesis was supported by studies

with corn-isolated protoplasts (Sagaral, 1978) and corn cell suspensions (Ezra

and Gressel, 1982) that demonstrated that lipid synthesis was indeed a target site

involved in the early action of EPTC. When R-25788 was added simultaneously

with EF'TC to corn protoplasts or cell suspensions, it partially prevented the

EPTC-induced inhibition of lipid synthesis (Sagaral, 1978; Ezra and Gressel,

1982), probably through a stimulation of the incorporation of [14C]acetate into

the plant tissues (Ezra and Gressel, 1982). However, the effects of EPTC as well

as of the chloroacetanilide herbicide metolachlor on lipid synthesis of corn have

been disputed by other investigators (Leavitt and Penner, 1979a). They suggested that R-25788 may act by preventing an EPTC-induced aggregation of the

epicuticular wax layer of corn rather than by overcoming a blocking of lipid

synthesis by the herbicide. The involvement of such a mechanism in the antidotal

action of R-25788 has been further supported by Gorog et al. (1982), who

demonstrated that the antidotes R-25788, R-28725, and AD-67 protect corn from

EPTC injury by preventing a herbicide-induced aggregation of epicuticular wax.

Through this mechanism, R-25788 prevents the formation of areas where the

underlying cuticle layers are exposed, resulting in a decreased transpiration

(Leavitt and Penner, 1979a; Gorog et al., 1982).

Apart from lipid synthesis or epicuticular wax distribution in corn, other active

sites that may be involved in the competitive inhibition of thiocarbamate and

chloroacetanilide herbicide effects by the antidote R-25788 include membrane

function (Bujtas, 1978), peroxidase activity (Harvey et al., 1975), and polyribosome formation (Rao and Kahn, 1975). The EPTC-induced enhancement of

membrane permeability of sugar beet root disks was counteracted by R-25788

(Bujtas, 1978), and the EPTC-induced stimulation of peroxidase activity in corn

seedlings was annulled by R-25788 (Harvey et al., 1975). Similarly, the alachlor-induced inhibition of polyribosome formation in barley roots was reversed

by R-25788, probably by a competition with the herbicide for this active site

(Rao and Kahn, 1975). In contrast to all of the aforementioned reports that

specify the plant active sites for which the herbicides and the antidote may be

competing, Stephenson et al. (1978, 1979) proposed that R-25788 or other

dichloroacetamide antidotes may apt as herbicidally inactive competitive inhibitors at an unknown site of thiocarbamate herbicide action that is specific to corn.

An additional hypothesis that offers a probable explanation for the mechanism

of antidotal action of R-25788 is the enhanced breakdown of thiocarbamate or

chloroacetanilide herbicides in antidote-treated corn plants. In early studies by



Wright et al. (1973) and Chang et al. (1974a) it was concluded that R-25788

increased the rate of butylate and EPTC metabolism in treated corn seedlings.

Because of the lack of protection by R-25788 against herbicide injury to other

grass crops, Wright et al. (1973) proposed that an alternate pathway for the

degradation of thiocarbamate herbicides could be present in corn but not in other

grasses. Lay et al. (1975) were the fmt to demonstrate that such an alternate

pathway for thiocarbamate herbicide degradation in corn indeed exists and involves two steps. In the first step the thiocarbamate herbicides are converted

through an oxidation reaction to their respective sulfoxides, which are then

conjugated to gluthathione (GSH) in the second step. The sulfoxidation of thiocarbamate herbicides has been viewed as a bioactivation reaction because the

thiocarbamate sulfoxides were found to be phytotoxic to many plants but not to

corn (Casida et al., 1974). However, it was shown that a two-step oxidation of

EF'TC (EPTC --* EPTC sulfoxide --* EPTC sulfone) rather than a single-step

oxidation (EPTC + EPTC sulfoxide) was necessary for the conjugation of EFTC

to GSH (Horvath and Pulay, 1980). In addition, Komives and Dutka (1980)

demonstrated that the phytotoxicity of EPTC to corn does not result from the

action of the EPTC sulfone but is partly the result of the action of the EPTC

sulfoxide. They suggested that both the sulfoxidation of EPTC and the subsequent conjugation to GSH are equally important for its detoxication in corn.

In a series of reports Casida and co-workers (Lay et al., 1975; Lay and Casida,

1976; 1978; Hubbel and Casida, 1977) emphasized that the conjugation of thiocarbamate sulfoxides to GSH was the most important step in thiocarbamate

herbicide detoxication by corn and proposed that R-25788 and other dichloroacetamide antidotes protect corn by increasing the rate of thiocarbamate

sulfoxide conjugation to GSH. This antidotal action of R-25788 is probably the

result of an antidote-induced elevation of the GSH content and GSH-S-transferase activity in the roots of corn seedlings (Lay et al., 1975; Lay and Casida,

1976). Subsequent studies by other investigators demonstrated that pretreatments

with R-25788 do indeed result in an elevation of GSH content of corn seedlings

(Carringer et al., 1978b; Stephenson et al., 1980), corn cell suspensions (Ezra

and Gressel, 1982), and tobacco cell suspensions (Rennenberg et al., 1982).

However, the existence of a GSH-S-transferase enzyme in corn roots that catalyzes the conjugation of thiocarbamate sulfoxides with GSH has been disputed

by some investigators (Carringer et al., 1978a; Leavitt and Penner, 1979b) who

'reported that the GSH-EPTC sulfoxide conjugation proceeds spontaneously

rather than enzymatically. It is quite possible that R-25788 protects corn from

EFTC or other thiocarbamate injury by increasing GSH production and not by

stimulating the activity of a GSH-S-transferase. This antidote-induced elevation

of GSH content in corn mots is probably the result of a direct activation of GSH

synthetase by the antidote R-25788 (Carringer et af., 1978b). However, Rennenberg et al. (1982) proposed that the stimulation of GSH synthesis by R-25788 in



heterotrophically grown tobacco cell suspensions is not caused by a direct activation of preexisting enzymes but by an enhancement of the amount of enzymes

involved in this process. According to Adams and Casida (1981), R-25788

elevates GSH content in corn by acting at an early stage in the sulfate + GSH

biosynthetic pathway.

The validity of the theory that R-25788 protects corn by increasing GSH

content has been questioned by some researchers (Ezra and Gressel, 1982;

Gressel et al., 1982). Thus Ezra and Gressel(l982) showed that although most

of the [l4C]EPTC was rapidly biotransformed by corn cell suspensions within 8

hr, measurable increases in GSH following treatment with R-25788 began after

12 hr. In addition, they showed that R-25788 added simultaneously to corn

suspension cells with EPTC reversed the inhibition of lipid synthesis induced by

EPTC. Based on these observations, Ezra and Gressel (1982) proposed that

R-25788 protection to corn may involve more than one mechanism, such as an

initial rapid effect on lipid synthesis followed by a slower effect that results in

elevations of cellular GSH content. The limited significance of the antidoteinduced elevation of GSH content for the protection of corn by R-25788 was also

emphasized in a report by Fedtke (1981), who showed that apart from R-25788 a

number of other herbicides and plant-growth regulators significantly increased

GSH content in corn and soybeans. However, none of these herbicides or growth

regulators protects corn against EPTC injury. The conclusion that the observed

elevation of GSH content of corn is not critical for the antidotal action of

R-25788 against EPTC is further supported by Casida et al. (1974), who reported

that in the absence of R-25788, corn was injured by EPTC at 3.4kg/ha but could

tolerate EPTC sulfoxide applications of as high as 27 kg/ha without significant


Leavitt and Penner (1979b) proposed that the protective effect of R-25788

against EPTC injury to corn may have a different basis than its protective effect

against chloroacetanilide herbicide injury. They suggested that an antidote-induced increase in the GSH content of corn may be important for the safening

effect of R-25788 against chloroacetanilide injury because the rate of the alachlor-GSH conjugation was less efficient than that of the GSH-EPTC sulfoxide conjugation. The significance of GSH conjugation in the metabolism of

chloroacetanilideherbicides has been reviewed by Hatzios and Penner (1982). In

addition, Leavitt and Penner (1979b) proposed that R-25788 may protect corn

from thiocarbamate herbicide injury by stimulating the rate of their sulfoxidation. The thiocarbamate sulfoxides are subsequently detoxified by conjugation to

GSH. A similar hypothesis for the mechanism of action of R-25788 has also been

proposed by Horvath and Pulay (1980).

The sulfoxidation of thiocarbamate herbicides in corn is believed to be an

enzymatic reaction mediated by mixed-function oxidases (Hubbel and Casida,

1977). Studies by Komives and Dutka (1980) showed that the insecticide syner-



gists and mixed function oxidase inhibitors, piperonyl butoxide and SKF-525A,

synergized the phytotoxic action of EPTC on corn, indicating that EPTC sulfoxidation plays a key role in the antidotal action of R-25788. Similar results have

been observed by other researchers who demonstrated that in the presence of

R-25788, EPTC interacted synergistically with the herbicide tebuthiuron

(Hatzios, 1981) or with the antioxidants, piperonyl butoxide and propyl gallate

(Hatzios, 1982b). Other studies have also demonstrated a synergistic interaction

of EPTC with the herbicide 2,4-D (Martin and Burnside, 1982) or the insecticide

fonofos (Freeman, 1978)on corn in the presence of the antidote R-25788. At the

present time, the significance of these last results cannot be easily assessed with

reference to the mode of antidotal action of R-25788. The involvement of the

oxidative metabolism of EPTC in the antidotal action of R-25788 has been

disputed by Taft (1976), who reported that mixed-functionoxidase inhibitors did

not affect either the EPTC injury to corn or the protective action of R-25788.

From the previous discussion, it is evident that our current understanding of

the antidotal action of R-25788 is not very clear. None of the presently available

theories about the mode of antidotal action of R-25788 is unequivocally accepted, and additional studies obivously are needed to elucidate the exact mode of

action of this antidote.


Because of the recent introduction of CGA-43089 as a sorghum protectant

against metolachlor injury, studies on the mode of its antidotal action are limited.

Because the main entry point of both the herbicide metolachlor and the antidote

CGA-43089 is the coleoptile of sorghum seedlings (Nyffeler et al., 1980,

Ketchersid and Merkel, 1981a), interference of CGA-43089 with metolachlor

uptake by sorghum coleoptiles could explain the protective effect of this antidote. Such a hypothesis appeared to be supported by the results of studies

conducted by Ketchersid and Merkel (1981b) and Ketchersid et al. (1982).

However, the apparent competitive effect of CGA-43089 on the absorption of

metolachlor was most evident in the roots rather than in the coleoptiles of grain

sorghum seedlings (Ketsersid el al., 1982). In addition, the decreased rate of

metolachlor uptake in the presence of CGA-43089 did not appear to be related

directly to changes in cell permeability. Ebert (1982) proposed that the safening

action of CGA-43089 against metolachlor injury to grain sorghum may result

from its ability to prevent a metolachlor-induced loss of cuticular integrity in

sorghum plants which greatly reduces the penetration of metolachlor..

Other investigatorshave disputed the theory that reduced uptake of metolachior

by sorghum seedlings in the presence of CGA-43089is the reason for the antidotal

effect of this protectant (Winkle et al., 1980; Christ, 1981). Christ (1981)



proposed that CGA-43089 is able to reduce the active amount of metolachlor at a

site of action specific to sorghum. An antidote-induced increase in the rate of the

metabolic detoxication of metolachlor by sorghum seedlings could explain such an

action of CGA-43089. More recent studies, however, demonstrated that

CGA-43089 did not influence the ability of sorghum tissues to metabolize the

herbicide metolachlor (Winkle et ul., 1980; Ketchersid and MerkIe, 1981b; Leek

and Penner, 1982). It has been observed that CGA-43089 fails to counteract

metolachlor injury to sorghum grown in nutrient solution (Leekand Penner, 1981)

or under conditions of excessive soil moisture (Ketchersid et al., 1981; Leek and

Penner, 1981). In addition, the results of another study showed that in the presence

of CGA-43089, metolachlor interacted synergistically with ozone and the antioxidants piperonyl butoxide and propyl gallate on sorghum under greenhouse conditions (Hatzios, 1983a). The results of this study combined with the observation

that CGA-43089 fails to protect sorghum against metolachlor under extremely wet

(anaerobic) conditions indicate that CGA-43089 may act by stimulating the

activity of a biological oxidation system (possibly a mixed-function oxidase) that

could be involved in the metabolic detoxication of metolachlor in sorghum.

Although such a mechanism for the protective action of CGA-43089 has been

postulated (Hatzios, 1983a), further studies are needed to establish it as a viable

theory explaining the antidotal activity of this compound. Finally, CGA-43089

was not active in increasing the GSH content of corn or soybean tissues (Fedtke,




Studies on the degradation of herbicide antidotes in plants are also limited.

Laboratory investigations with 14C-labeledNA showed that this antidote was not

metabolized or bound in corn plants, that no volatile “T metabolites were

formed, and that no residues for NA were found in mature corn plants beyond the

fifth or sixth week after emergence (Riden and Asbell, 1975). Corn seedlings

grown in soil treated with I4C-labeled R-25788 liberated 6% of the absorbed

radioactivity as 14C02in a 10-day period (Murphy et ul., 1974). Approximately

80-85% of the absorbed radioactivity could be extracted with ethanol. Four

metabolites of R-25788 were separated in corn tissues, identified as N-allyl-2,2dichloroacetamide, N,N-diallylglycolamide, N,N-diallyloxamic acid, and the

glycoside of N,N-diallylglycolamide (Murphy et al., 1974). Studies on the metabolism of the antidotes CGA-43089 and MON-4606 in sorghum plants have

not been conducted.

3 10



The concept of using herbicide antidotes offers a potential alternative for

increasing the selectivity of currently available herbicides. A desirable herbicide

antidote is a chemical agent that selectively protects crops from herbicide injury

without protecting weeds. This selectivity is the result of either a very specific

crop-herbicide-antidote interaction or a selective treatment such as the dressing

of crop seeds with the antidote. Herbicide antidotes are developed primarily

through random screening techniques that involve most combinations of important herbicides, major crops, and candidate antidotes. Chemicals that are currently used as herbicide antidotes include NA, R-25788, CGA-43089,

CGA-92194, and MON-4606.These antidotes offer adequate protection to grass

crops that are damaged but not killed by specific herbicides. Thus the presently

available herbicide antidotes can counteract, to some extent, the effects of chloracetanilide and carbamate herbicides on grass crops such as corn and grain

sorghum. Several environmental factors such as temperature, soil moisture, and

soil type may affect the field performance of herbicide antidotes and need to be

seriously considered. In addition, the timing of herbicide and antidote applications to the crop as well as the differential intraspecific tolerance of crop cultivars

to combinations of herbicides plus antidotes need to be established for optimum

effectiveness of herbicide antidotes in the field.

The mode of antidotal action of the presently available herbicide safeners is

not fully understood. It is believed that rather than merely preventing the entry of

herbicides into the plant, herbicide antidotes work inside the plant to counteract

the actions of herbicides either by competing with them for a common site of

action or by stimulating their metabolic detoxication in the protected crops. The

development of effective antidotes that could protect broad-leaved crops against

injury from herbicidal photosynthetic inhibitors represents the greatest challenge

of the pesticide industry in the near future. Advances in our understanding of

herbicide action and degradation by plants may lead to the development of more

effective herbicide antidotes in the future.


Abemthy, J. R., and Keeling, J. W. 1982. Abstr. Weed Sci. Soc. Am., No. 34.

Adams, C.A., and Casida. J. E. 1981. Abstr. Papers Am. Chem. Soc. 182nd Meet., PEST, p. 72.

Adler, E. F.,Wright, W. L., and Klingmim, G. C. 1977. In “Pesticide Chemistry in the 20th

Century” (1. R. Plimmer, ed.), pp. 39-55. h e r . Chem. Soc., Washington,D.C.

Ahle, J., and Cozart, E. 1972. Proc. North Cent. Weed Control Cot& 27,23.

Ahrens, W.H., andDavis, D. E. 1978. Proc. Souzh. Weed Sci. Soc. 31, 249.

Ali, A., and Stephenson, G. R. 1979. Abstr. Weed Sci. Soc. Am., No. 1.

Ali, M.,and Mercado, B. L. 1980. Philipp. 1. Weed Sci. 7, 26-32.

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V. Mode of Action of Herbicide Antidotes

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