The mode of toxic action of the pesticide Gliftor: The metabolism of 1,3-difluoroacetone to (-)-erythro-fluorocitrate

Article abstract of DOI:10.1002/1099-0461(2001)15:1<47::AID-JBT6>3.0.CO;2-E

The biochemical toxicology of 1,3-difluoroacetone, a known metabolite of the major ingredient of the pesticide Gliftor (1,3-difluoro-2-propanol), was investigated in vivo and in vitro. Rat kidney homogenates supplemented with coenzyme A, ATP, oxaloacetate, and Mg²⁺ converted 1,3-difluoroacetone to (-)-erythro-fluorocitrate in vitro. Administration of 1,3-difluoroacetone (100 mg kg^-1 body weight) to rats in vivo resulted in (-)-erythro-fluorocitrate synthesis in the kidney, which was preceded by an elevation in fluoride levels and followed by citrate accumulation. Animals dosed with 1,3-difluoroacetone did not display the 2-3 hour lag phase in either (-)-erythro-fluorocitrate synthesis or in citrate and fluoride accumulation characteristic of animals dosed with 1,3-difluoro-2-propanol. We demonstrate that the conversion of 1,3-difluoro-2-propanol to 1,3-difluoroacetone by an NAD⁺-dependent oxidation is the rate-limiting step in the synthesis of the toxic product, (-)-erythro-fluorocitrate from 1,3-difluoro-2-propanol. Prior administration of 4-methylpyrazole (90 mg kg^-1 body weight) was shown to prevent the conversion of 1,3-difluoro-2-propanol (100 mg kg^-1 body weight) to (-)-erythro-fluorocitrate in vivo and to eliminate the fluoride and citrate elevations seen in 1,3-difluoro-2-propanol-intoxicated animals. However, administration of 4-methylpyrazole (90 mg kg^-1 body weight) to rats 2 hours prior to 1,3-difluoroacetone (100 mg kg^-1 body weight) was ineffective in preventing (-)-erythro-fluorocitrate synthesis and did not diminish fluoride or citrate accumulation in vivo. We conclude that the prophylactic and antidotal properties of 4-methylpyrazole seen in animals treated with 1,3-difluoro-2-propanol derive from its capacity to inhibit the NAD⁺-dependent oxidation responsible for converting 1,3-difluoro-2-propanol to 1,3-difluoroacetone in the committed step of the toxic pathway.

Full text of DOI:10.1002/1099-0461(2001)15:1<47::AID-JBT6>3.0.CO;2-E

J BIOCHEM MOLECULAR TOXICOLOGY
Volume 15, Number 1, 2001
The Mode of Toxic Action of the Pesticide Gliftor:
The Metabolism of 1,3-Difluoroacetone to
(מ)-erythro-fluorocitrate
K. I. Menon, M. G. Feldwick, P. S. Noakes, and R. J. Mead
School of Biological Sciences and Biotechnology, Murdoch University, South Street, Murdoch 6150, Western Australia
Received 13 June 2000; revised 30 August 2000; accepted 06 September 2000
ABSTRACT: The biochemical toxicology of 1,3-difluo-
roacetone, a known metabolite of the major ingredient
of the pesticide Gliftor (1,3-difluoro-2-propanol), was
investigated in vivo and in vitro. Rat kidney homog-
enates supplemented with coenzyme A, ATP, oxaloac-
etate, and Mg2ם converted 1,3-difluoroacetone to (؁)-
erythro-fluorocitrate invitro. Admisntrationof
KEY WORDS: Fluoroacetate; 1080; 1,3-Difluoroacetone;
1,3-Difluoro-2-propanol; 4-Methylpyrazole; (؁)-ery-
thro-Fluorocitrate; Aconitate Hydratase
INTRODUCTION
מ1
1,3-difluoroacetone (100 mg kg body weight) to rats
The toxicity of the rodenticide fluoroacetate (Com-
pound1080) [1] is due to its conversion in vivo to fluo-
roacetyl CoA andhence, by condensation with oxalo-
acetate, to (מ)-erythro-fluorocitrate [2], which exerts its
toxic effects in two main ways. (מ)-erythro-Fluoroci-
trate is a powerful inhibitor of the TCA cycle enzyme
aconitate hydratase (citrate [isocitrate] hydrolyase, EC
4.2.1.3) [3–5]. It also irreversibly inhibits the passage of
citrate into andout of mitochondria by covalently
binding to two enzymes located in the mitochondrial
inner membrane that are involvedin citrate transport
[6,7]. As a consequence of these effects, fluoroacetate
poisoning results in citrate andfluoride accumulation,
a decrease in ATP synthesis, hyperglycemia, hypocal-
cemia, andketonemia [8,9,10]. Baiting campaigns us-
ing 1080 as a vertebrate pesticide have been conducted
in the UnitedStates to control groundsquirrels in Cali-
fornia [11], arctic foxes in Alaska [12], andblack-tailed
prairie dogs [13], while in Australia and New Zealand,
it has been usedwidely to control feral pigs, foxes,
rabbits, dingoes, and possums [14,15].
As well as being a vertebrate pesticide, fluoroace-
tate is also a natural product. In Australia it is synthe-
sizedby plants of the genus Gastrolobium, most of
which occur in the southwest of Australia [16,17]. Co-
evolution of Gastrolobium species andnative fauna in
southwestern Australia has resultedin the production
of high levels of fluoroacetate in the plants andin an
adaptive tolerance to fluoroacetate in the animals
[18,19]. Consequently, fluoroacetate can be usedas a
target-specific pesticide to control introduced 1080-
sensitive pests andpredators in southwestern Austra-
invivo resulted in( ؁)-erythro-fluorocitrate synthesis
inthe kidney, which was preceded by anelevationin
fluoride levels and followed by citrate accumulation.
Animals dosed with 1,3-difluoroacetone did not dis-
play the 2–3 hour lag phase ineither ( ؁)-erythro-fluo-
rocitrate synthesis or in citrate and fluoride accumu-
lationcharacteristic of iamnals dosed with
1,3-difluoro-2-propanol. We demonstrate that the con-
versionof 1,3-difluoro-2-propanol to 1,3-difluoroace-
ם
tone by an NAD -dependent oxidation is the rate-lim-
iting step in the synthesis of the toxic product,
(؁)-erythro-fluorocitrate from 1,3-difluoro-2-propanol.
מ1
Prior administration of 4-methylpyrazole (90 mg kg
body weight) was shown to prevent the conversion of
מ1
1,3-difluoro-2-propanol (100 mg kg body weight) to
(؁)-erythro-fluorocitrate in vivo and to eliminate the
fluoride and citrate elevations seen in 1,3-difluoro-2-
propanol-intoxicated animals. However, administra-
tionof 4-methylpyrazole (90 mg kg מ1 body weight) to
מ1
rats 2 hours prior to 1,3-difluoroacetone (100 mg kg
body weight) was ineffective in preventing (؁)-ery-
thro-fluorocitrate synthesis and did not diminish fluo-
ride or citrate accumulationinvivo. We conclude that
the prophylactic and antidotal properties of 4-methyl-
pyrazole seeninanimals treated with 1,3-difluoro-2-
ם
propanol derive from its capacity to inhibit the NAD -
dependent oxidation responsible for converting
1,3-difluoro-2-propanol to 1,3-difluoroacetone in the
committed step of the toxic pathway. ᭧ 2001 John Wiley
& Sons, Inc. J Biochem Mol Toxicol 15:47–54, 2001
Correspondence to: R. J. Mead.
Contract Grant Sponsor:
Contract Grant Number:
᭧ 2001 John Wiley & Sons, Inc.
47

48
MENON ET AL.
Volume 15, Number 1, 2001
It has been shown recently that the major ingre-
dient of Gliftor, 1,3-difluoro-2-propanol (DFP), pos-
sesses a mode of toxic action similar to that of 1080 in
that it is metabolizedafte r a 2 hour lag phase to (מ)-
erthyro-fluorocitrate, which then causes citrate and
fluoride accumulation in the kidney [29]. Gliftor is
likely, therefore, to display target specificities similar
to those of 1080, but the lag phase evident before toxic
signs are manifest may provide an opportunity to ad-
minister antidotal compounds to accidentally poi-
sonedanimals before irreversible biochemical dys-
function occurs. It has also been demonstrated [30,31]
that 4-methylpyrazole (4MP), a compoundreportedto
have minimal acute andchronic toxicity [32], is capable
of effectively combating DFP intoxication. In those
studies, pretreatment of rats with 4MP significantly de-
creased( מ)-erythro-fluorocitrate synthesis andalmost
eliminatedcitrate andfluoride accumulation in the
מ1
kidney of rats administered 100 mg DFP kg body
weight [31]. Furthermore, the DFP-treatedrats re-
mainedalert andactive anddisplayedno signs of Glif-
tor intoxication. It was suggested[31] that the prophy-
lactic and antidotal properties of 4MP derived from its
ם
capacity to inhibit an NAD -dependent oxidation re-
sponsible for the conversion of DFP to 1,3-difluoroac-
etone (DFA). It was proposed[31] that this reaction
was the initial step in the metabolism of DFP to (מ)-
erythro-fluorocitrate as shown in Scheme 1.
SCHEME 1
Though it has been established, unequivocally,
that DFP generates both DFA and( מ)-erythro-fluoro-
citrate [31], there is no direct evidence that DFA is an
intermediate in the toxic pathway or that the diminu-
tion in (מ)-erythro-fluorocitrate synthesis seen in rats
pretreatedwith 4MP is a direct result of the inhibition
lia without a significant impact on the native fauna
[20–23]. However, the extreme toxicity of fluoroacetate
andthe lack of an effective antidote to combat acci-
dental ingestion of 1080-impregnated baits by sensitive
nontarget species, including domestic and farm ani-
mals, are significant disadvantages that have restricted
its use in the UnitedStates [13] andare impediments
to its future use in Australia [21]. The development of
an alternative pesticide that possesses the target spec-
ificities of 1080 in Australia but against which an ef-
fective antidote is available would encompass signifi-
ם
of the NAD -dependent oxidation involved in the con-
version of DFP to DFA.
The purpose of this study is to evaluate the bio-
chemical toxicology of DFA andto clarify the role of
the conversion of DFP to DFA in the synthesis of (מ)-
erythro-fluorocitrate from DFP. It also aims to further
elucidate the site of action of 4MP responsible for its
prophylactic andantidotal properties.
cant advantages. Such
a pesticide would have
considerable potential as a 1080 replacement, not only
in Australia, but in any region where vertebrate pests
are a problem. One of the most promising alternatives
to 1080 is the pesticide Gliftor (70–80% 1,3-difluoro-2-
propanol and20–30% 1-chloro-3-fluoro-2-propanol),
which is used as a rodenticide in Russia and China
[24,25]. In rats it is reportedto have an LD 50 of 45.6 mg
MATERIALS AND METHODS
Origin and Housing of Experimental
Animals
מ1
kg body weight [25] but has been found to be rela-
tively nontoxic to birds that are potentially attracted to
grain baits (LD50 500–2000 mg kg body weight)
Six- to eight-week-oldmale albino Rattus norvegi-
cus (Wistar strain) were usedin all experiments. They
were housedin an animal house maintainedon a 12:12
hour photoperiodat 21–24 ЊC andwere fedRat and
Mouse cubes manufacturedby Glenn Forrest Stock-
feeders, Western Australia. Both foodandwater were
providedadlibitum.
מ1
[24,26,27]. Toxic doses of Gliftor result in signs of poi-
soning similar to those seen in fluoroacetate intoxica-
tion, but they are preceded by a longer lag phase [28].
This delayed toxic action is advantageous in that it en-
courages complete consumption of bait material [24].

Volume 15, Number 1, 2001
TOXICOLOGY OF 1,3-DIFLUOROACETONE
49
Dosing of Experimental Animals
to which aliquots of either enzyme-inactivatedkidney
supernatants derived from treated animals, or en-
zyme-inactivatedincubation mixtures, inhibitedacon-
itate hydratase activity derived from control animals.
A series of (מ)-erythro-fluorocitrate standard curves
was constructed[31], which relatedpercentage inhi-
bition of aconitate hydratase activity to known concen-
trations of authentic (מ)-erythro-fluorocitrate assayed
in the presence of a range of endogenous citrate con-
centrations. With the aidof a computer program, the
standard curves were then used to determine (מ)-er-
ythro-fluorocitrate levels in either the enzyme-inacti-
vated kidney supernatants derived from poisoned ani-
mals for which citrate levels hadbeen determined
previously, or in the enzyme-inactivatedincubation
mixtures.
Before dosing, animals were weighed and lightly
anaesthetized with a mixture of 80% carbon dioxide
and20% oxygen. Fluoroacetate (1.5 mg kg
weight), 1,3-difluoro-2-propanol (100 mg kg body
weight), 1,3-difluoroacetone (100 mg kg
weight), and4-methylpyrazole (90 mg kg
weight) were administered intraperitoneally as aque-
ous solutions.
מ1
body
מ1
מ1
body
body
מ1
Synthesis of 1,3-Difluoroacetone
1,3-Difluoroacetone was synthesizedas previously
described [33,34] by refluxing DFP with KMnO4 and
CuSO4 for 7 hours, except that dichloromethane was
usedas the solvent insteadof benzene. A yieldof 73%
DFA was obtainedafter fractional distillation with a
purity in excess of 99% as assessedby nuclear magnetic
resonance andgas chromatography-mass spectrome-
try (molecular ion [M] 94; characteristic fragments, m/
e 61 and33). 1,3-Difluoroacetone was analysedby gas
chromatography using a 12.5 m HP-1 column (30ЊC)
andan HP-MSD detector.
Preparation of Kidney Supernatants Containing
Aconitate Hydratase Activity
Untreated animals were killed by carbon dioxide
asphyxiation, and the kidneys were rapidly excised,
weighed, andhomogenizedin chilled0.1 M Tris-HCl
מ1
buffer, pH 7.4 (4.0 mL g kidney). Homogenates de-
rivedfrom three animals were pooledandcentrifuged
at 9000g for 20 minutes at 3ЊC in a Beckman J2-21 M/
E refrigeratedcentrifuge (JA20 rotor) followedby cen-
trifugation at 217,000g for 35 minutes at 3ЊC in a Beck-
man L8-70M ultracentrifuge (Ti 50 rotor). The
supernatant was storedon ice andusedwithin 12
hours as the source of active aconitate hydratase for
the (מ)-erythro-fluorocitrate assay.
Preparation of Enzyme-Inactivated Kidney
Supernatants for the Determination of
Citrate, (؁)-erythro-Fluorocitrate and
Fluoride Levels
Rats were killed by carbon dioxide asphyxiation,
and the kidneys were rapidly excised, weighed, and
homogenizedin chilled0.1M Tris-HCl buffer, pH 7.4
(3.0 mL gמ1 kidney). The homogenates were boiled for
2 minutes to eliminate enzyme activity andthen cen-
trifugedat 9000 g for 20 minutes at 3ЊC in a Beckman
J2-21 M/E refrigeratedcentrifuge (JA 20 rotor). Ali-
quots of supernatant were assayedimmediately for cit-
rate content. Residual supernatant was stored at מ5ЊC
for later determination of (מ)-erythro-fluorocitrate and
fluoride levels.
Assay of Aconitate Hydratase Activity and
Determination of (מ)-erythro-Fluorocitrate
Levels in Kidney Supernatants and Enzyme
Incubation Mixtures
Aconitate hydratase activity was determined by
measuring the rate of consumption of cis-aconitate
spectrophotometrically at 240 nm [37]. To quartz cu-
vettes with a light path of 0.5 cm, 1.055 mL, 0.1 M Tris-
HCl (pH 7.4); 0.3 mL, 0.5 M NaCl; 80 lL enzyme-in-
activatedkidney supernatant or enzyme-inactivated
incubation mixture containing (מ)-erythro-fluoroci-
trate, and20 lL aconitate hydratase-active supernatant
were added. After a 10 minute preincubation at 37ЊC,
45 lL of 10 mM cis-aconitate was added to commence
the reaction. The absorbance of the mixture was mea-
suredat 240 nm every 0.1 secondfor 3 minutes at 37 ЊC
using a Shimadzu UV-2101PC UV-vis scanning spec-
trophotometer attachedto a Shimadzu CPS-controller
andthe UV-2101PC kinetics software package (Shi-
madzu). The percentage inhibition of aconitate hydra-
tase activity relative to control values was determined
andwith reference to the endogenous citrate level of
Determination of Kidney Citrate Levels
Kidney citrate levels were determined by assaying
25, 50, or 100 lL aliquots of the supernatants, prepared
as described previously, using the citrate lyase-malate
dehydrogenase method of Mo¨llering andGruber [35]
as modified by Dagley [36], with the exception that
triethanolamine buffer was replacedby 0.1 M Tris-HCl,
pH 7.4.
Determination of (؁)-erythro-Fluorocitrate
Levels
(מ)-erythro-Fluorocitrate levels were determined
as described previously [31] by measuring the extent

50
MENON ET AL.
Volume 15, Number 1, 2001
TABLE 1. Synthesis of (מ)-erythro-Fluorocitrate In Vitro from Either Fluoroacetate or DFA by Rat Kidney Homogenates as
Determinedby Aconitate Hydratase Inhibition
Aconitate Hydratase Activity
(מ)-erythro-Fluorocitrate
Concentration in
Rate of (מ)-erythro-
מ1
(lmol cis-aconitateמ1 min
Fluorocitrate Synthesis
מ1
Incubation Mixture
mLמ1 supernatant)
% Inhibition
the Cuvette (lM)
(nmolמ1 hמ1 g kidney
)
Homogenate ם buffer
Homogenate ם FAc
Homogenate ם DFA
Homogenate ם OAA, ATP, MgCl2 & CoA
Homogenate ם OAA, ATP, MgCl2, CoA & FAc
Homogenate ם OAA, ATP, MgCl2, CoA & DFA
3.10
3.07
3.11
2.47
0.80
1.30
0
0
0
20.4
67.4
47.1
0
0
0
0
0
0
(0.13)
14.23
1.86
(11.0)
1234.4
160.7
Note: The complete incubation mixture (4.6 mL) containedhomogenate (3 mL), fluoroacetate [FAc] (5 mM), or DFA (10 mM); oxaloacetate (OAA) (5 mM), ATP
(5 mM), MgCl2 (4.2 mM), andcoenzyme A (CoA) (0.5 mM) in 0.1 M Tris-HCl buffer, pH 7.4. Incubations were carriedout for 40 minutes at 37 ЊC, andthe reaction
was stoppedby boiling a 2 mL aliquot for 2 minutes. After centrifugation the ( מ)-erythrofluorocitrate concentration in each supernatant was determined as described
in Methods.
the supernatant assayedearlier, the concentration of
(מ)-erythro-fluorocitrate was calculatedusing the ap-
propriate standard curve.
fluorocitrate, 4-methylpyrazole, NADH, andoxaloac-
etate were all obtainedfrom Sigma Chemical Co. (St
Louis, MO). Sodium fluoroacetate was obtained from
Tokyo Kasei Kogyo Co. Ltd, Tokyo. All other chemicals
were of the purest grade commercially available.
Determination of Kidney Fluoride Levels
Fluoride levels in enzyme-inactivated kidney su-
pernatants were determined with an Orion combina-
tion fluoride electrode connected to an Orion expand-
able ion-analyser, using sodium fluoride (NaF) as
standard. The electrode was calibrated using 2.0 mL
samples of 0.1, 2.5, and5 mg L מ1 fluoride to which was
added 2.0 mL of low-level Total Ionic Strength Adjust-
ment Buffer [38]. Aliquots (2.0 mL) of kidney super-
natant were similarly diluted with buffer, and the fluo-
ride concentration of each was determined.
Statistical Analysis
Kidney citrate, (מ)-erythro-fluorocitrate, andfluo-
ride concentrations were compared statistically using
a two-factor analysis of variance (ANOVA).
RESULTS
Metabolism and Mode of Toxic Action of
1,3-Difluoroacetone
Preparation of Kidney Homogenates and
Incubation Mixtures for In Vitro Synthesis
of (؁)-erythro-Fluorocitrate
To compare the biochemical characteristics of DFA
intoxication with those resulting from DFP or fluo-
roacetate administration, rats were dosed with DFA
Untreated animals were killed by carbon dioxide
asphyxiation. Kidneys were rapidly excised, weighed,
andhomogenizedin chilled0.1 M Tris-HCl buffer, pH
מ1
(100 mg kg body weight), sodium fluoroacetate (1.5
מ1
מ1
mg kg body weight), or DFP (100 mg kg body
weight), and kidney citrate levels were determined
(Figure 1). Administration of fluoroacetate, DFP, or
DFA to rats resultedin citrate accumulation in the kid-
ney, but the lag phase characteristic of DFP intoxication
was not apparent in animals dosed with DFA (Figure
1). Instead, the pattern of citrate accumulation more
closely resembledthat associatedwith fluoroacetate in-
toxication. Animals dosed with DFA also displayed
clinical signs of poisoning similar to those induced by
fluoroacetate, including cold extremities, respiratory
depression, and central nervous system irritability,
though the seizures associatedwith the central ner-
vous system dysfunction were less severe and more
infrequent. Animals treatedwith fluoroacetate (1.5 mg
kgמ1 body weight) usually died less than 5 hours after
dosing, while those receiving DFA (100 mg kgמ1 body
weight) survivedfor up to 48 hours. All animals dosed
מ1
7.4 (1.0 mL g kidney). Aliquots (3.0 mL) of the ho-
mogenate were incubatedfor 40 minutes at 37 ЊC in
flasks containing either sodium fluoroacetate or DFA
together with Mg Cl2, ATP, coenzyme A, andoxalo-
acetate in a final incubation volume of 4.6 mL. Incu-
bation mixture concentrations of the components are
shown in Table 1. A 2.0 mL aliquot of each incubation
mixture was then boiledfor 2 minutes to eliminate en-
zyme activity andcentrifugedat 9000 g for 20 minutes
at 3ЊC in a Beckman Avanti J-25 I refrigeratedcentri-
fuge (JA 25.50 rotor). Aliquots of the supernatant (80
lL) were then analyzedfor ( מ)-erythro-fluorocitrate as
described previously.
Biochemicals
ATP, cis-aconitate, citrate lyase, coenzyme A, 1,3-
difluoro-2-propanol, malate dehydrogenase, barium

Volume 15, Number 1, 2001
TOXICOLOGY OF 1,3-DIFLUOROACETONE
51
FIGURE 1. Kidney citrate levels following intraperitoneal admin-
istration of (ࡗ) sodium fluoroacetate (1.5 mg kgמ1 body wt, n ס 3).
(ꢀ) 1,3-difluoro-2-propanol (100 mg kgמ1 body wt, n ס 5); (छ) 1,3-
difluoroacetone (100 mg kgמ1 body wt, n ס 3). Results are expressed
as means ע SEM.
FIGURE 2. Kidney (מ)-erythro-fluorocitrate levels following intra-
מ1
peritoneal administration of (ࡗ) sodium fluoroacetate (1.5 mg kg
מ1
body wt). (ꢀ) 1,3-difluoro-2-propanol (100 mg kg body wt); (छ)
1,3 difluoroacetone (100 mg kgמ1 body wt). Results are expressed as
means ע SEM for three determinations in each case.
with DFP (100 mg kgמ1 body weight) survived beyond
this periodanddidnot display the seizures character-
istic of DFA or fluoroacetate intoxication.
of a fluorine atom, the effect of DFP (100 mg kgמ1 body
מ1
weight) andDFA (100 mg kg
body weight) on kid-
To assess the capacity of DFA to generate (מ)-er-
ythro-fluorocitrate in vivo, kidney (מ)-erythro-fluoro-
citrate levels were determined in rats administered
ney fluoride levels was determined in vivo. As shown
in Figure 3, treating rats with either DFA or DFP re-
sultedin an elevation in the concentration of free fluo-
ride ion in the kidney, but the extent and the rapidity
of the elevation was significantly greater with DFA (P
Ͻ 0.01).
מ1
DFA (100 mg kg body weight) and compared with
those foundin animals treatedwith sodium fluoroac-
etate (1.5 mg kg body weight) or DFP (100 mg kg
מ1
מ1
body weight). The results are shown in Figure 2. All
three compounds were metabolized to (מ)-erythro-
fluorocitrate in vivo andthe time course of ( מ)-erythro-
fluorocitrate production and citrate accumulation for
each was foundto be closely correlated(Figures 1 and
2).
The capacity of kidney tissue to convert DFA to
(מ)-erythro-fluorocitrate was also investigatedin vitro.
Both DFA andfluoroacetate were incubatedwith kid-
ney homogenates in the presence of ATP, CoASH,
Mg2ם, andoxaloacetate for 40 minutes, andthe rate of
(מ)-erythro-fluorocitrate synthesis was determined.
The results are shown in Table 1. Both DFA andfluo-
roacetate were capable of generating (מ)-erythro-fluo-
rocitrate in vitro, but the rate of its synthesis from DFA
was slower.
Effect of 4-Methylpyrazole onDFA
Metabolism and Toxicity
To determine the extent to which the antidotal and
prophylatic properties of 4MP are a function of its ca-
pacity to inhibit the oxidation of DFP to DFA, rats were
מ1
administered 4MP (90 mg kg body weight) and ei-
מ1
ther DFA (100 mg kg body weight) or DFP (100 mg
מ1
kg body weight) 2 hours later. Kidney citrate, (מ)-
erythro-fluorocitrate, and fluoride levels were then de-
terminedas shown in Figures 4, 5, and6, respectively.
4-Methylpyrazole significantly decreased (מ)-erythro-
fluorocitrate synthesis (P Ͻ 0.01) andmarkedly re-
duced citrate (P Ͻ 0.01) andfluoride (P Ͻ 0.01) accu-
mulation in rats dosedwith DFP but hadlittle effect
(P Ͼ 0.1) on these parameters in animals administered
DFA.
Because the conversion of both DFP andDFA to
(מ)-erythro-fluorocitrate necessarily involves the loss

52
MENON ET AL.
Volume 15, Number 1, 2001
FIGURE 5. Kidney (מ)-erythro-fluorocitrate levels following intra-
peritoneal administration of (ꢀ) 1,3-difluoro-2-propanol (100 mg
kgמ1 body wt) preceded by 4-methylpyrazole (90 mg kgמ1 body wt)
FIGURE 3. Kidney fluoride levels following intraperitoneal admin-
istration of (ꢀ) 1,3-difluoro-2-propanol (100 mg kgמ1 body wt, n ס
5) or (छ) 1,3 difluoroacetone (100 mg kgמ1 body wt, n ס 3). Results
are expressedas mean increases above baseline levels ע SEM.
מ1
2 hours earlier or (छ) 1,3 difluoroacetone (100 mg kg body wt)
preceded by 4-methylpyrazole (90 mg kgמ1 body wt) 2 hours earlier.
Results are expressedas means ע SEM for three determinations in
each case, except where indicated.
DISCUSSION
It has been suggested[31] that the metabolism of
DFP, the major ingredient of the pesticide Gliftor, to
highly toxic (מ)-erythro-fluorocitrate is initiatedby an
ם
NAD -dependent oxidation that generates DFA.
Though the conversions of DFP to DFA andDFP to
(מ)-erythro-fluorocitrate were demonstrated unequiv-
ocally by these authors, no direct evidence was pre-
sentedto implicate DFA in the toxic pathway. It is now
apparent that the conversion of DFP to DFA is the com-
mittedstep in the metabolism of DFP to ( מ)-erythro-
fluorocitrate. Administration of DFA to rats resulted in
(מ)-erythro-fluorocitrate synthesis in the kidney (Fig-
ure 2) preceded by a rapid elevation in kidney fluoride
levels (Figure 3) andsubsequent citrate accumulation
(Figure 1). The conversion of DFA to (מ)-erythro-fluo-
rocitrate in vivo (Figure 2) andin vitro (Table 1) pro-
vides evidence for the proposal [30,31] that DFA pro-
duction is
a prerequisite for the synthesis of
(מ)-erythro-fluorocitrate from DFP. Furthermore, the
capacity of DFA to eliminate the lag phase seen in the
patterns of citrate, (מ)-erythro-fluorocitrate, andfluo-
ride accumulation in rats dosed with DFP (Figures 1,
2, and3) suggests that the conversion of DFP to DFA
is the rate-determining step in the metabolism of DFP
to (מ)-erythro-fluorocitrate.
FIGURE 4. Kidney citrate levels following intraperitoneal admin-
istration of (ࡗ) 1,3-difluoro-2-propanol (100 mg kgמ1 body wt) pre-
ceded by 4-methylpyrazole (90 mg kgמ1 body wt) 2 hours earlier or
(छ) 1,3 difluoroacetone (100 mg kgמ1 body wt) preceded by 4-meth-
ylpyrazole (90 mg kg body wt) 2 hours earlier. Results are ex-
pressedas means ע SEM for three determinations in each case, ex-
מ1
cept where indicated.

Volume 15, Number 1, 2001
TOXICOLOGY OF 1,3-DIFLUOROACETONE
53
ם
stratedcapacity to inhibit the NAD -dependent oxi-
dation of DFP to DFA. The inability of 4MP to prevent
both (מ)-erythro-fluorocitrate synthesis from DFA (Fig-
ure 5) andthe accumulation of citrate (Figure 4) and
fluoride (Figure 6) in DFA-intoxicated animals pro-
vides direct evidence that 4MP exerts its prophylactic
andantidotal properties by inhibiting the conversion
of DFP to DFA in the rate-determining step of the toxic
pathway.
The mode of toxic action of DFP, which involves
the lethal synthesis of the aconitate hydratase inhibitor,
(מ)-erythro-fluorocitrate, is analogous to that of ethyl-
ene glycol. The metabolism of ethylene glycol is also
ם
initiatedby an NAD -dependent oxidation to gener-
ate, ultimately, oxalic acid, an inhibitor of the TCA cy-
cle enzyme, succinate dehydrogenase [39].
It is noteworthy that 4MP, marketedas fomepizole
or Antizol-Vet, has been approvedrecently by the Food
andDrug Administration in the UnitedStates as an
antidote to ethylene glycol poisoning. Its effectiveness
is due to its inhibition of the alcohol dehydrogenase
responsible for catalyzing the initial step in the toxic
pathway, thus decreasing oxalic acid production
[40,41]. Approval of 4MP for human andanimal use
provides a marketable antidote to DFP and Gliftor poi-
soning andenhances the potential for Gliftor to replace
1080 as a vertebrate pesticide.
FIGURE 6. Kidney fluoride levels following intraperitoneal admin-
istration of (ꢀ) 1,3-difluoro-2-propanol (100 mg kgמ1 body wt) pre-
ceded by 4-methylpyrazole (90 mg kgמ1 body wt) 2 hours earlier or
(छ) 1,3 difluoroacetone (100 mg kgמ1 body wt) preceded by 4-meth-
ylpyrazole (90 mg kg body wt) 2 hours earlier. Results are ex-
pressedas mean increases above baseline levels ע SEM for three
מ1
determinations in each case, except where indicated.
ACKNOWLEDGMENTS
From a chemical perspective, DFA is likely to be
more reactive than DFP. The proximity of the fluoro-
methyl groups to the carbonyl moiety is likely to ren-
der the carbonyl carbon of DFA susceptible to nucleo-
philic attack by compounds such as CoASH. Such an
attack wouldgenerate fluoroacetyl CoA, a single car-
bon entity anda fluoride ion. The rapidelevation in
kidney fluoride levels in animals administered DFA
supports this contention (Figure 3), while condensa-
tion of the fluoroacetyl CoA with oxaloacetate explains
why DFA is capable of generating (מ)-erythro-fluoro-
citrate readily (Figure 2, Table 1). The activation of DFP
to DFA provides an opportunity not only for (מ)-ery-
thro-fluorocitrate synthesis but also for other nucleo-
philic reactions, unassociatedwith the toxic pathway,
to occur. This may explain why DFA, though more
toxic than DFP, generates (מ)-erythro-fluorocitrate
more slowly than does fluoroacetate (Figure 2, Table
1).
It has been demonstrated previously [31] that prior
treatment of animals with 4MP markedly decreases
(מ)-erythro-fluorocitrate synthesis andalmost elimi-
nates citrate and fluoride accumulation in the kidney
of rats administered DFP. This finding is confirmed by
the data shown in Figures 4, 5, and 6. Furthermore,
these authors inferredthat the prophylactic andanti-
dotal properties of 4MP were a result of its demon-
We are grateful to Dr. Wayne Best, Chemistry Cen-
tre, Perth Western Australia, for his assistance with the
synthesis of 1,3 difluoroacetone.
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