L-2-Chloropropionic acid metabolism and disposition in male rats: Relevance to cerebellar injury

Article abstract of DOI:10.1007/s002040050443

L-2-Chloropropionic acid (L-CPA) produces selective necrosis to the granule cell layer of the rat cerebellum. As part of a study to understand the mechanism of selective toxicity we have investigated the metabolism and disposition of [2-¹⁴C]L-CPA in the rat, with particular emphasis on the brain. Following a single oral nontoxic dose of 250 mg/kg or a neurotoxic dose of 750 mg/ kg or 250 mg/kg per day for 3 days, L-CPA is very rapidly absorbed from the gastrointestinal tract into the blood stream. Peak plasma concentrations of 2 mM (250 mg/kg) and 6 mM (750 mg/kg) L-CPA occurred within 1 h of dosing, and the compound was readily cleared from the plasma with a half-life of 2.6 h. The only metabolite detected in the plasma was 2-S-cysteinylpropanoic acid, presumably derived from the glutathione conjugate. About 60% of the dose is excreted in the urine in the first 24 h as unchanged L-CPA, with a smaller amount excreted as the mercapturate, 2-S-N-acetylcysteinylpropanoic acid. Little radiolabel from L-CPA is excreted in the faeces; however, ~ 18% of a 250 mg/kg dose of L-CPA is eliminated as carbon dioxide. The radiolabel from [2-¹⁴C]L-CPA present in the cerebellum, forebrain and liver at all time intervals examined was L-CPA. There was some indication of retention of L-CPA in the brain relative to the plasma with a small but consistently higher concentration found in the cerebellum. Whole body autoradiography studies indicated some selective retention of radiolabel in the cerebellum after the third dose of 250 mg/kg [2-¹⁴C]L-CPA. Our findings indicate that the initial insult to the cerebellum following L-CPA administration is probably due to the parent compound however, the prolonged presence of 2-S-cysteinylpropanoic acid in the plasma and concomitant depletion of glutathione in the cerebellum may also play a role in the toxicity. The relevance of the slightly greater retention of L-CPA in the cerebellum to the selective neurotoxicity of L-CPA requires further study.

Full text of DOI:10.1007/s002040050443

Arch Toxicol (1997) 71: 668±676
Ó Springer-Verlag 1997
ORGAN TOXICITY AND MECHANISMS
Ian Wyatt á Michael Farnworth á Andrew J. Gyte
Edward A. Lock
L
-2-Chloropropionic acid metabolism and disposition in male rats:
relevance to cerebellar injury
Received: 25 March 1997 / Accepted: 21 May 1997
Abstract L-2-Chloropropionic acid (L-CPA) produces bellum to the selective neurotoxicity of L-CPA requires
selective necrosis to the granule cell layer of the rat cer- further study.
ebellum. As part of a study to understand the mechanism
of selective toxicity we have investigated the metabolism Key words
L
-2-Chloropropionic acid á Glutathione
and disposition of [2-14C]L-CPA in the rat, with partic- conjugation á Cerebellar granule cell necrosis
ular emphasis on the brain. Following a single oral non-
toxic dose of 250 mg/kg or a neurotoxic dose of 750 mg/
Introduction
kg or 250 mg/kg per day for 3 days, L-CPA is very
rapidly absorbed from the gastrointestinal tract into the
blood stream. Peak plasma concentrations of 2 mM
(250 mg/kg) and 6 mM (750 mg/kg) L-CPA occurred
within 1 h of dosing, and the compound was readily
cleared from the plasma with a half-life of 2.6 h. The only
metabolite detected in the plasma was 2-S-cyst-
einylpropanoic acid, presumably derived from the glu-
tathione conjugate. About 60% of the dose is excreted in
the urine in the ®rst 24 h as unchanged L-CPA, with a
smaller amount excreted as the mercapturate, 2-S-N-
acetylcysteinylpropanoic acid. Little radiolabel from L-
CPA is excreted in the faeces; however, ꢀ18% of a
250 mg/kg dose of L-CPA is eliminated as carbon diox-
ide. The radiolabel from [2-14C]L-CPA present in the
cerebellum, forebrain and liver at all time intervals ex-
amined was L-CPA. There was some indication of re-
tention of L-CPA in the brain relative to the plasma with
a small but consistently higher concentration found in
the cerebellum. Whole body autoradiography studies
indicated some selective retention of radiolabel in the
cerebellum after the third dose of 250 mg/kg [2-14C]L-
CPA. Our ®ndings indicate that the initial insult to the
cerebellum following L-CPA administration is probably
due to the parent compound however, the prolonged
presence of 2-S-cysteinylpropanoic acid in the plasma
and concomitant depletion of glutathione in the cere-
bellum may also play a role in the toxicity. The relevance
of the slightly greater retention of L-CPA in the cere-
The ability to identify why a speci®c population of
neurons are vulnerable to various chemicals might lead
to a greater understanding of the basis to discrete neu-
ronal cell necrosis, which occurs in neurological diseases
such as Huntington's chorea and Parkinson's disease.
The speci®c lesion obtained following an oral dose
of 750 mg/kg
L-2-chloropropionic acid (L-CPA) or
250 mg/kg L-CPA daily for 3 days is characterized by a
marked loss of cerebellar granule cells, which begins as a
multifocal lesion, and rapidly spreads to encompass the
entire granular layer of the cerebellum; there may be
resulting destruction of up to 70±80% of granule cells
(Simpson et al. 1996). Other cell types in the cerebellum
are largely una€ected by L-CPA although a small pro-
portion of Purkinje cells appear damaged (Simpson et al.
1996; Jones et al. 1997). The e€ect of a selective loss in
cerebellar granule cells is a severe impairment of normal
locomotor activity, which develops by 36 h after L-CPA
administration.
Little is known about the metabolism and disposition
of L-CPA in the rat; we have reported that L-CPA
produces a rapid and time-dependent depletion of liver
non-protein sulphydryl (NP-SH) content, mainly gluta-
thione (GSH), while in the cerebellum and forebrain
there is a slower time-dependent decrease in GSH
(Wyatt et al. 1996a). The decrease in cerebral GSH is not
accompanied by a concomitant increase in oxidized
GSH (Wyatt et al. 1996a). There is also no evidence of
increased activity of the pentose phosphate pathway in
slices of cerebellum removed from rats treated with
750 mg/kg L-CPA and examined 24 h after dosing at a
time when GSH depletion is maximal (Lock et al. 1995).
I. Wyatt á M. Farnworth á A.J. Gyte á E.A. Lock (&)
Neurotoxicology Group, Zeneca Central Toxicology Laboratory,
Alderley Park, Maccles®eld, Cheshire SK10 4TJ, UK

669
chambers were supplied by Jencons Scienti®c, Leighton Buzzard,
Beds. Halothone was obtained from Zeneca Pharmaceuticals,
Maccles®eld, Cheshire, UK. All other chemicals were of analytical
grade and were supplied by Sigma or Fisons Scienti®c Equipment,
Loughborough, Leicester.
Nor is there alteration in ascorbic acid content in the
cerebellum (Widdowson et al. 1997), indicating that
oxidative stress is not occurring prior to the onset of
granule cell neurosis.
The decrease in liver GSH following L-CPA treat-
ment appears to be due to the formation of 2-S-glut-
athionyl propanoic acid (the GSH conjugate of L-CPA),
this reaction being catalyzed by a theta class glutathi-
one-S-transferase. However, no conjugate formation
could be detected using homogenates or cytosolic frac-
tions of cerebellum (Wyatt et al. 1996a). Studies with
cerebellar slices have shown that the cysteine conjugate
Animals
Male Alderley Park rats (200±250 g body weight) were housed
in groups under controlled humidity (40±50%), temperature
(20 ‹ 2°) and a 12 h light/dark cycle (lights on at 0600 hours).
Animal care and monitoring were carried out in accordance with
strict guidelines issued by the UK Home Oce. All animal pro-
cedures and treatments were performed according to approved
animal licences and guidelines. The animals had free access to water
at all times and were allowed food (Porton combined diet) ad li-
bitum apart for 18±23 h prior to dosing.
of L-CPA (
L-2-cysteinyl propanoic acid) can inhibit cy-
stine uptake into ells, which may be a contributory
factor accounting for the depletion of cerebellar GSH,
by reducing the availability of cysteine as an essential
intermediate for GSH synthesis (Wyatt et al. 1996b).
The aim of this work was to investigate the distribution,
excretion and metabolic fate of [2-14C]L-CPA in male
rats following oral administration of a single non-toxic
dose of 250 mg/kg and toxic doses of 750 mg/kg, or
250 mg/kg per day for 3 days, with particular emphasis
on the site of toxicity, namely the cerebellum. Prelimi-
nary observation of this work has been reported in an
abstract (Wyatt et al. 1996c).
Tissue distribution of radiolabelled [2-14C]L-CPA
A stock solution of neutralized L-CPA (100 mg/ml) in deionized
water was prepared and animals were orally dosed at 10 ml/kg with
either 250 mg/kg or 750 mg/kg [2-14C]L-CPA and 250 lCi/kg.
Groups of four rats per time interval were killed with an overdose of
halothane 1, 2, 4, 8, 12, 24 and 48 h after dosing. In another study,
rats were dosed orally with 250 mg/kg [2-14C]L-CPA and 200 lCi/
kg per day for 3 days; groups of four animals were killed with an
overdose of halothane at 2, 8 and 24 h after the 2nd dose and at 1, 2,
4, 8, 12 and 48 h after the 3rd dose. Blood was taken from the heart
into lithium heparin tubes using the S-monovette system supplied by
Sarstedt (Beaumont Leys, Leicester, UK) and stored on ice prior to
centrifugation to obtain the plasma. The liver, kidneys, lung, cere-
bellum and forebrain were removed and homogenized in ice-cold
0.25 M sucrose, 5.4 mM EDTA, 20 mM TRIS, pH 7.4, to give 25%
(w/v) homogenates, which were stored on ice. Samples of plasma
(0.1 ml), in duplicate were added directly to vials containing scin-
tillation ¯uid. Duplicate samples of tissue homogenates (0.5 ml)
were digested in Soluene 350 (2 ml) and added to Hionic-Fluor
scintillant (20 ml). The amount of radioactivity present in each
sample was determined using a Packard 2500 TR scintillation
counter. The radioactivity present in the tissues and plasma was
converted to lg equivalents of L-CPA/g wet weight of tissue or ml
of plasma using the speci®c activity of the dosing solutions. The
remaining tissue homogenates and plasma were stored at )20°.
Materials and methods
Chemicals
L
-[2-14C]Chloropropionic acid (98.5% pure; 26 mCi/mmol) was
purchased from Amersham International, Little Chalfont, Bucks,
UK. L-CPA (96.1% pure) was supplied by Zeneca Specialities,
Manchester, the main impurities being D-CPA (2.1%) and 2,2¢-
dichloropropionic acid (1.0%). 2-S-Glutathionyl propanoic acid
(DL-CPA glutathione) was synthesized as described by Wyatt et al.
(1996a) and 2-S-L-cysteinyl propanoic acid (L-CPA cysteine) was
prepared by the method of Mamalis et al. (1960). 2-S-(N-Acetyl-
cysteinyl) propanoic acid (L-CPA-NAC) was prepared by an ad-
aptation of the method of Mamalis et al. (1960).
Excretion studies
Essentially, N-acetylcysteine (2.45 g) was added with clean dry
sodium to liquid ammonia (ꢀ100 ml) until the blue colour was The animals used for the 48 h tissue distribution study with 750 mg
maintained. Ammonium chloride (0.3 g) followed by L-2-chloro- /kg L-CPA were individually housed in glass metabolism cages and
propionic acid (1.63 g) was added dropwise during 10 min, the the urine and faeces collected into containers at )70° at 0±12, 12±
mixture allowed to evaporate slowly, and residual ammonia being 24, 24±36 and 36±48 h and a ®nal cage wash made at 48 h. At post-
removed under vacuum. Attempts to re-crystallize the residue from mortem the gastrointestinal tract was removed, homogenized and
aqueous solutions were unsuccessful. The residue was taken up in samples, in duplicate, taken for determination of radioactivity. In
methanol saturated with hydrogen chloride gas and stirred at am- another study, rats were given a single oral dose of 250 mg/kg
bient temperature for 2 h. The methanol was removed and the L-CPA and 250 lCi/kg and placed singly in glass metabolism cages.
residue taken up in water and extracted with ethyl acetate. The Expired air was monitored for exhaled volatiles (0±12, 12±24 h)
evaporated ethyl acetate extract was puri®ed by `¯ash' chroma- and carbon dioxide (0±12, 12±24, 24±36, 36±48 and 48±72 h) using
tography on silica eluting with 10% methanol in chloroform. The n-hexane at )70° and sodium hydroxide traps respectively. Urine
pure diester was de-esteri®ed with dilute aqueous sodium hydroxide and faeces were also collected at )70° at 12-h intervals over 72 h
to give, after extraction from the acidi®ed solution, pure 2-S-(N- and a ®nal cage wash made at 72 h. Duplicate aliquots of urine,
acetylcysteinyl)-propanoic acid. Proton NMR (CD3OD) and neg- hexane and sodium hydroxide were added to Hionic Fluor
ative ion electrospray mass spectrometry were consistent with the scintillant (10 ml) and radioactivity was determined by scintillation
expected structure.
counting. A 20% (w/v) homogenate of faeces in methanol was
Cellulose tri-acetate ®lters for micro-centrifuge tubes (mol. wt. prepared and then centrifuged at 2000 g and 4 °C for 20 min; 1 ml
cut-o€ 12 000 Da) and a Bondapak C18 HPLC column of 10 lm of supernatant, in duplicate, taken for determination of radioac-
particle size (3.9 mm ´ 150 mm) were purchased from Whatman tivity. The methanol-extracted faecal pellet was allowed to air dry,
International, Maidstone, Kent. The scintillation cocktails Ultima weighed and radioactivity present was determined in two aliquots
Gold, Hionic-Fluor and Ultima Flo 17, tissue oxidizer ingredients of known weight using a Packard sample oxidizer B 307 with col-
Carbo-Sorb and Perma¯uor and tissue solubilizer Soluene 350 were lection in carbo-sorb E and Perma¯uor E+. All fractions were
purchased from Packard, Pangbourne, Berks. Glass metabolism stored at )20°.

670
Whole body autoradiography
creas, thymus and the stomach, including both the
mucosa and contents (Fig. 1). The concentration of
radiolabel was marginally higher in the cerebellum than
in other brain regions after three doses of L-CPA, but
otherwise the radioactivity was generally distributed
throughout all tissues. The concentration of radiolabel
from [2-14C]L-CPA in the plasma peaked ꢀ1 h after
administration of 250 mg/kg L-CPA at a concentration
of 230 lg/ml (2.1 mM) and at a similar time after
750 mg/kg L-CPA at a concentration of 700 lg/ml
(6.5 mM) and was rapidly cleared from the plasma
(Figs. 2, 3).
Two rats were dosed orally with 250 mg/kg [2-14C]L-CPA and
200 lCi/kg; one was killed 24 h after the ®rst dose and the other
24 h after the third dose and the distribution of the radioactivity
determined by whole-body autoradiography. Following termina-
tion, 2% (w/v) carboxymethylcellulose (CMC) was injected into the
external nasal passages, the rats were frozen immediately in a
mixture of haxane/dry-ice and embedded in 2% (w/v) CMC using
the same coolant. Longitudinal sagittal sections of 30 lm in
thickness were taken, mounted on adhesive tape and freeze-dried
for 48 h. Autoradiographs were prepared by contact with X-ray
®lm (Hyper®lm-bmax; Amersham) and exposed for 6 weeks.
The peak concentration of radiolabel in the brain,
both forebrain and cerebellum regions, was very similar
at approx. 100 lg/g wet weight (0.93 mM) after 250 mg/
kg L-CPA and 250 lg/g wet weight (2.3 mM) after
750 mg/kg L-CPA (Figs. 2, 3). The radiolabel then de-
clined, more slowly than from the plasma, with some
retention in both brain regions at 24 and 48 h after
dosing (750 mg/kg L-CPA, 24 h: ratio of forebrain/
plasma, 1.50 ‹ 0.20, cerebellum/plasma, 2.13 ‹ 0.28;
48 h: forebrain/plasma, 3.57 ‹ 0.30, cerebellum/plas-
ma, 4.65 ‹ 0.59). The retention of radiolabel was al-
ways greater in the cerebellum than in the forebrain at
all doses examined with ratios starting at unity during
the ®rst 2 h after dosing, and increasing to ꢀ1.1 by 4 h,
1.2 by 12 h and 1.4 by 24 h after 750 mg/kg L-CPA.
The data indicate slightly more retention in the cere-
bellum than in the forebrain. In the liver, kidney and
lung the pro®le of radiolabel following either dose was
similar to that of the brain, although the peak concen-
trations were higher and some retention was observed in
the liver relative to other tissues 24 h after dosing
(Figs. 2, 3). Administration of 250 mg/kg per day for
3 days led to elimination of radiolabel from plasma,
brain and other tissues similar to that seen after a single
dose (Fig. 3). There was no obvious indication of ac-
cumulation of the chemical in the site of injury, namely
the cerebellum.
HPLC analysis of L-CPA and its metabolites
in urine, plasma and tissues
Samples of liver, forebrain and cerebellar homogenate were thawed
and centrifuged at 12 000 g for 20 min at 4 °C. Aliquots of urine,
plasma or tissue supernatant (0.4 ml) were placed in micro®lter
centrifuge tubes (mol. wt. cut-o€ 12 000 Dalton) and centrifuged at
9700 g for 90 min at 4 °C. The ®ltrate was analysed by HPLC
using the following conditions based on the method of Patel et al.
(1994). L-CPA and its glutathione-derived metabolites were sepa-
rated by injecting 50 ll of sample onto a Bondapak C18 column
(3.9 mm ´ 150 mm, 10 lm particle size) coupled to a Shimadzu
LC-10 diode-array detector operating at 205 nm. The column was
eluted at a ¯ow rate of 1 ml/min, with a gradient of solvent A
(0.1 M potassium phosphate, pH 3.0) and solvent B (40% metha-
nol in water) as follows: 0±10 min 100% A; 10±30 min linear in-
crease to 100% B; 30±35 min returned to 100% A and maintained
for up to 30 min.
The column e‚uent was monitored at 205 nm and for radio-
activity using a Radiomatic Flo-one beta (Packard) with a 3 ml
¯ow cell detector pumping Ultima-Flo 17 scintillant. Elution times
were 1.8 min for 2-S-cysteinyl propanoic acid, 5.5 min for L-CPA,
9.0 min for 2-S-glutathionyl propanoic acid, and 14 min for 2-S-
(N-acetylcysteinyl) propanoic acid. For animals dosed with
750 mg/kg [2-14C]L-CPA, plasma, urine and tissues were analysed,
whilst only plasma samples were analysed from the single and
multiple dose studies at 250 mg/kg [2-14C]L-CPA.
A portion of the urine collected 24 h after administration of
750 mg/kg [2-14C]L-CPA was incubated with b-glucuronidase
(1000 Units) for 2 h at 37 °C and submitted to HPLC analysis
as described above. Pharmacokinetic analysis of the plasma
concentration was performed using the pharmacokinetic data
analysis program PHASAR (Zeneca Pharmaceuticals, version
1.3). The area under the curve of plasma concentration versus
time (AUC) was calculated using the linear trapezoidal rule to
the last time point in the pro®le with a plasma concentration
value judged to be signi®cantly above the background value. The
elimination rate constant (kz) was calculated by log-linear re-
gression of those data points considered to represent the elimi-
nation phase. The half-lives were calculated using the equation
t1/2 = 0.693/kz.
Excretion of radioactivity, following a dose of
250 mg/kg [2-14C]L-CPA, into the urine was rapid with
almost 50% of the dose being cleared within 12 h of
dosing, followed by a further 18% in the next 12 h, with
a total of 71% being eliminated over 3 days (Table 1).
A similar pattern was seen after 750 mg/kg L-CPA ex-
cept more of the dose was cleared between 24 and 48 h
giving a total of 76% elimination in urine over 48 h
(Table 1). Excretion in the faeces was small at 3.2% and
8.8% at 250 and 750 mg/kg respectively (Table 1).
Following administration of 250 mg/kg L-CPA no ex-
haled volatile material was detected (<0.005% of the
dose); however, L-CPA was metabolized to CO2 as
shown by the collection of ꢀ11% of the dose over the
Results
Tissue distribution and excretion
of [2-14C]
L-chloropropionic acid
The distribution of radiolabel from [2-14C]L-CPA in the ®rst 24 h and a total of 16% overall (Table 1). The
rat, 24 h after a single oral dose of 250 mg/kg or after overall recovery of radiolabel following 250 mg/kg L-
250 mg/kg per day for 3 days was broadly similar as CPA was 90 ‹ 8% compared with 89 ‹ 3% following
detected by whole body autoradiography (Fig. 1). The 750 mg/kg L-CPA where expired carbon dioxide was
organs containing the highest radiolabel were the pan- not determined.

671
Fig. 1 Whole body autoradiograms of rats dosed orally with [2-14C]
Tissue and urinary concentrations of L-CPA
and metabolites
L-chloropropionic acid, A 24 h after 250 mg/kg and B 24 h after three
daily doses of 250 mg/kg. Retention of radioactivity observed in
pancreas (P), thymus (T ) and gastrointestinal tract (G.I.T.) and, after
the third dose, some retention in the cerebellum (C )
Analysis of the plasma from rats treated with either 250
or 750 mg/kg L-CPA showed the presence of the parent
compound and the cysteine conjugate derived from
GSH conjugation. The loss of L-CPA from the plasma
followed ®rst-order kinetics, the peak concentration
being 297 lg/ml after 250 mg/kg and 860 lg/ml after
750 mg/kg (Fig. 4). The area under the curve of plasma
versus time and the half-life for elimination from plasma
were 1015 lg)1 h and 2.62 h for 250 mg/kg L-CPA and
3031 lg)1 h and 2.63 h for 750 mg/kg L-CPA. The
concentration of 2-S-cysteinyl propanoic acid in plasma
following a single dose of 250 mg/kg L-CPA was ꢀ7 lg/
ml after the ®rst time interval examined, 1 h after dos-
ing, and remained at this concentration for ꢀ12 h;
however, the level was below the limit of detection at
24 h (Table 2). A similar pro®le of plasma 2-S-cysteinyl
propanoic acid was seen after the third dose of 250 mg/
kg L-CPA although the concentration was higher than
after a single dose at all time points examined, suggest-
ing some increase in the formation of the cysteine con-
jugate (Table 2). The plasma concentration of 2-S-
cysteinyl propanoic acid after a single dose of 750 mg/kg
L-CPA was higher than after a single dose of 250 mg/kg
L-CPA, but the increase was not proportional relative to
the plasma concentration of L-CPA (Table 2).
HPLC radiochemical analysis for L-CPA metabolites
in tissues following 750 mg/kg showed that L-CPA was
the only material detected in the cerebellum, forebrain

672

673
b
Fig. 2A±F Distribution of radioactivity from a single oral dose of Fig. 3A±F Distribution of radioactivity following daily oral dosing of
750 mg/kg [2-14C] -chloropropionic acid (L-CPA) in selected tissues 250 mg/kg [2-14C]
-chloropropionic acid for 3 days in selected tissues
L
L
duringthe ®rst48 h after dosing. Results are of mean ‹ SEM with four at various times after dosing. Results are of mean ‹ SEM with four
animals per time point. d±d, Total radioactivity in each tissue animals per time point. See legend to Fig. 2 for explanation of
expressed as lg equivalent of L-CPA; n±n, plasma concentration of symbols
L-CPA

674
Table 1 Excretion of radioactivity from [2-14C]
L-chloropropionic acid following oral dosing of 250 mg/kg or 750 mg/kg to rats. Results
are of mean ‹ SD with four animals per dose (ND Not determined)
Treatment
250 mg/kg
Time after
dosing (h)
Urine
(% dose)
Faeces
(% dose)
GI tract
(% dose)
CO2
(% dose)
Total recovery
(%)
0±12
12±24
24±36
36±48
48±72
47.4 ‹ 7.2
18.1 ‹ 6.3
1.9 ‹ 0.5
1.2 ‹ 0.4
2.5 ‹ 0.3a
0.3 ‹ 0.6
1.8 ‹ 1.5
0.4 ‹ 0.3
0.3 ‹ 0.1
0.2 ‹ 0.0
N.D
N.D
N.D
N.D
N.D
7.3 ‹ 2.3
4.2 ‹ 1.2
1.7 ‹ 0.5
1.2 ‹ 0.3
1.7 ‹ 0.3
90.4 ‹ 7.9
Total
71.4 ‹ 5.5
3.2 ‹ 1.5
±
15.6 ‹ 0.8
750 mg/kg
0±12
12±24
24±36
36±48
43.7 ‹ 14.7
14.2 ‹ 4.0
6.6 ‹ 4.6
5.4 ‹ 1.6
0.7 ‹ 0.3
1.5 ‹ 2.1
1.3 ‹ 0.3
N.D
N.D
N.D
4.4 ‹ 2.6
N.D
N.D
N.D
N.D
88.9 ‹ 2.5
11.2 ‹ 6.4a
Total
75.7 ‹ 4.0
8.8 ‹ 2.6
4.4 ‹ 2.6
±
a
Includes cage wash
Table 2 Plasma concentration of 2-S-cysteinyl propanoic acid
following oral dosing of [2-14C]
-chloropropionic acid (L-CPA) to
rats. Results are of mean ‹ SD with four animals per time point
L
Treatment
Time after
dosing
(h)
2-Cysteinyl
propanoic acid
(lg/ml plasma)
First dose
of 250 mg/kg L-CPA
1
2
4
8
12
24
6.8 ‹ 2.1
7.3 ‹ 2.1
7.2 ‹ 1.8
9.8 ‹ 1.8
5.8 ‹ 0.4
Not detected
Third dose
of 250 mg/kg L-CPA
1
2
4
8
12
24
10.4 ‹ 1.9
16.4 ‹ 4.6
20.9 ‹ 1.3
13.3 ‹ 6.5
12.4 ‹ 3.7
1.9 ‹ 0.7
750 mg/kg L-CPA
1
2
4
8
12
24
14.2 ‹ 4.3
17.7 ‹ 5.1
9.4 ‹ 0.7
13.0 ‹ 2.8
8.9 ‹ 1
3.0 ‹ 2.0
Fig. 4 Clearance of L-2-chloropropionic acid from plasma of rats
following a single oral dose of either 250 mg/kg or 750 mg/kg. The
solid circles are results from the 750 mg/kg dose. Results are of
mean ‹ SEM with four animals per time point
ꢀ93% of the radioactivity present in the urine was
present as these two metabolites (Table 3). Some urine
and liver. The concentration of L-CPA in the cerebellum samples had material that chromatographed with a re-
2 and 8 h after dosing was 192 ‹ 48 and 91 ‹ 40 lg/g tention time of 2 min and 9 min, similar to the 2-S-
wet weight respectively, while the concentration of ra- cysteinyl and 2-S-glutathionyl propanoic acid respec-
diolabel expressed as lg equivalents of L-CPA was tively. However, such material was not consistently de-
203 ‹ 69 and 114 ‹ 15 at 2 and 8 h respectively, tected in all animals at each time point and was <2% of
showing that 95 and 80% of the material in the brain at the material in the urine. There was also a small amount
these times is the parent compound.
of radioactivity consistently found over the ®rst 24 h
HPLC radiochemical analysis of the urine from rats with a retention time of 17±18 min, accounting for
dosed with 750 mg/kg L-CPA showed the presence of ꢀ4.5% of the radiolabel in urine which had not been
two major peaks, representing unchanged L-CPA and its identi®ed. Treatment of the urine was b-glucuronidase
mercapturic acid. During the ®rst 12 h, ꢀ85% of the did not alter the HPLC pro®le of metabolities, sug-
radioactivity present in the urine was excreted as the gesting that conjugation of L-CPA with glucuronides is
parent compound and 9% as the mercapturate; overall not a major pathway of metabolism.

675
Table 3 Main metabolites detected in urine following dosing of rat
with 750 mg/kg [2-14C]
-chloropropionic acid (L-CPA). Results
are of mean ‹ SD with four animals per time point
was retained to a signi®cant extent in the thymus and
pancreas, but was not associated with histological
damage in either of these tissues (M. Simpson, personal
communication). However, thymic lymphoid necrosis
has been reported in rats given a diet containing 1% (w/
L
Time after Total
dosing (h)
L-CPA in
urine
(% of dose)
2-S-N-acetyl-
cysteinyl
radioactivity
in urine
(% of dose)
propanoic acid in
urine (% of dose)
v) D,L
-2-chloropropionic acid (O'Donoghue 1985). There
was also some indication from the autoradiogram of the
rat given three daily doses of 250 mg/kg, of localization
of radiolabel over the granule cell layer in the cerebel-
lum. Thus there may be some, albeit small, selective
retention of L-CPA to the granule cell layer of the cer-
ebellum, the site of injury.
0±12
12±24
24±36
36±48
43.7 ‹ 14.7
14.2 ‹ 4.0
6.6 ‹ 4.6
2.4 ‹ 1.3
37.0 ‹ 14.4
7.8 ‹ 2.2
3.0 ‹ 2.4
1.3 ‹ 0.7
4.1 ‹ 0.5
4.9 ‹ 2.1
2.9 ‹ 1.8
1.1 ‹ 0.7
We have derived a hypothesis to explain the selective
toxicity of L-CPA for the granule cell layer of the cer-
ebellum. The cerebellar lesion and the cascade of sub-
sequent neurochemical events (Widdowson et al. 1996a;
Jones et al. 1997) might be initiated by L-CPA-induced
Discussion
Following oral administration L-CPA is rapidly ab-
sorbed from the gastrointestinal tract of the rat. Peak
plasma values occurred ꢀ1 h after dosing. L-CPA was
readily cleared from the plasma with a half-life of 2.6 h,
for both the non-toxic (250 mg/kg) and toxic (750 mg/
kg) dose. The bulk of the radioactivity present in the
plasma at early times after dosing was L-CPA, although
a low concentration of the cysteine conjugate of L-CPA
was present. The parent compound and its mercapturic
acid are subsequently readily excreted in the urine with
the bulk of the radiolabel being eliminated within the
®rst 24 h after dosing. Thus, L-CPA is readily absorbed
and enters the plasma.
Calculation of the initial plasma concentration from
the plasma decay curves (Fig. 4) gives initial values of
860 lg/ml and 297 lg/ml for 750 and 250 mg/kg res-
pectively, which is close to the expected value if the L-
CPA had distributed into total body water. Both the
peak plasma concentration and the area under the curve
of plasma versus time was three times greater for the
750 mg/kg dose compared to the 250 mg/kg dose. As
three doses of 250 mg/kg will produce neurotoxicity
analogous to a single dose of 750 mg/kg, the area under
the curve of plasma versus time rather than the peak
plasma concentration is likely to be important with re-
gard to the neurotoxicity. The radioactivity from L-CPA
present in the brain appeared to be mainly the parent
compound, no glutathione-derived metabolities being
detected. Presumably L-CPA can enter the brain via a
transport system such as that for propionic or lactic acid
(Oldendorf 1973). The concentration of L-CPA in both
the cerebellum and forebrain was very similar at all time
points examined, although the amount present in the
cerebellum was consistently higher than in the forebrain.
The concentration ratio of L-CPA in the cerebellum or
forebrain relative to plasma was ꢀ0.4, 1 h after dosing.
As the L-CPA was cleared from the plasma the brain/
plasma ratio increased being ꢀ1.0 at 8 h, 2.0 at 12 h and
ꢀ4.0 at 48 h after a toxic dose. Thus there appeared to
be some retention of L-CPA in the brain relative to the
plasma, the retention being slightly higher in the cere-
bellum compared to the forebrain. Whole body auto-
radiography demonstrated that radiolabel from L-CPA
activation of a particular subtype of N-methyl-
D-aspar-
tate (NMDA) receptor (NR2A and C) located primarily
in the cerebellum (Ebert et al. 1991; Akazawa et al. 1994;
Kadotani et al. 1996). Con®rmation of the involvement
of the NMDA receptor in L-CPA-induced neurotoxicity
comes from the ®ndings that non-competitive and
competitive NMDA receptor antagonists a€ord full
protection against the neurotoxicity (Widdowson et al.
1996b, c; Lock et al. 1997).
The metabolism of L-CPA appears to occur primarily
via conjugation with GSH. In the liver, extensive deple-
tion of the non-protein sulphydryl content was seen fol-
lowing doses of either 250 mg/kg or 750 mg/kg L-CPA
(Wyatt et al. 1996a). The glutathione conjugate formed
was then presumably converted to the cysteine conjugate
by peptidase and c-glutamyltransferase action with
clearance into urine following acetylation to the merc-
apturate. 2-S-N-Acetyl-cysteinyl propanoic acid was
detected in the urine, while the only metabolite detected
in plasma was 2-S-cysteinyl propanoic acid. No metab-
olites of L-CPA were detected in the brain or liver within
the limits of sensitivity of our assay. L-CPA was also
eliminated unchanged in the urine with a larger amount
being excreted at the earlier times after dosing. In addi-
tion, some 16% of the administered L-CPA was oxidized
to carbon dioxide, presumably by b-oxidation analogous
to propionic acid forming a 2-chloropropionyl-CoA es-
ter. The latter is further metabolized to methylmalonyl-
CoA and then succinyl-CoA, which can enter the Krebs
cycle and undergo metabolism to produce carbon diox-
ide. This is presumably a detoxi®cation pathway for
L-CPA, although metabolism of certain xenobiotic
compounds via CoA esters can lead to their incorpora-
tion into lipid metabolism with some pharmacological
and toxicological consequences (Dodds 1995).
The concentration of 2-S-cysteinyl propanoic acid in
the plasma remained fairly constant for ꢀ12 h after
dosing at approx. 100 lM following the third dose of
250 mg/kg L-CPA or a single dose of 750 mg/kg
L-CPA. 2-S-Cysteinyl propanoic acid has been shown to
inhibit the transport of cystine into slices of rat cere-
bellum with a Ki
of 60 lM (Wyatt et al. 1996b), cystine

676
Lock EA, Gyte A, Du€ell SI, Wyatt I (1997) Neuroprotection af-
forded by MK-801 against L-2-chloropropionic acid-induced
cerebellar granule cell necrosis in the rat. Toxicology (in press)
Mamalis P, McHale D, Green J (1960) Some derivatives of S-
carboxyalkyl-cysteines and homocysteines. J Chem Soc 2906±
2914
Murphy TH, Schnaar RL, Coyle JT (1990) Immature cortical
neurons are uniquely sensitive to glutamate toxicity by inhibi-
tion of cystine uptake. FASEB J 4: 1624±1633
O'Donoghue JL (1985) Aliphatic halogenated hydrocarbons, al-
cohols and acids and thioacids. In: O'Donoghue JL (ed) Ne-
urotoxicology of industrial and commercial chemicals, vol II
CRC Press, Boca Raton, pp 99±126
Oldendorf WH (1973) Carrier-mediated blood-brain barrier
transport of short chain monocarboxylic organic acids. Am J
Physiol 224: 1450±1452
being the source of cysteine for cerebral glutathione
synthesis. As we could ®nd no evidence for enzyme-
mediated conjugation of L-CPA with cerebellar cytosol
or whole homogenate to account for the depletion of
cerebellar GSH seen in vivo, the depletion of cerebellar
GSH could be explained by inhibition of cystine trans-
port into the brain. Immature cortical neurons in culture
have been reported to be uniquely sensitive to glutamate
toxicity when cystine uptake is inhibited (Murphy et al.
1990). Therefore, we cannot currently exclude the pos-
sibility that 2-S-cysteinyl propanoic acid may be a con-
tributory factor to toxicity of L-CPA in cerebellar
granule cells. Further studies are needed to determine
whether the slightly greater retention of L-CPA in the
cerebellum and the depletion of cerebellar glutathione
are relevant to the mechanism of selective neurotoxicity
of L-CPA to the rat.
Patel N, Birner G, Dekant W, Anders MW (1994) Glutathione-
dependent biosynthesis and bioactivation of S-(1,2,-dichloro-
vinyl) glutathione and S-(1,2-dichlorovinyl)-L-cysteine, the
glutathione and cysteine S-conjugates of dichloroacetylene, in
rat tissues and subcellular fractions. Drug Metab Dispos 22:
143±147
Simpson MG, Wyatt I, Jones HB, Gyte A, Widdowson PS, Lock
EA (1996) Neuropathological changes in rat brain following
oral administration of L-2-chloropropionic acid. Neurotoxi-
cology 17: 471±480
Widdowson PS, Gyte AJ, Simpson MG, Wyatt I, Lock EA (1996a)
Changes in cerebellar amino-acid neurotransmitter concentra-
tion and receptors following administration of the neurotoxin
L-2-chloropropionic acid. Toxicol Appl Pharmacol 136: 57±66
Widdowson PS, Wyatt I, Gyte AJ, Simpson ME, Lock EA (1996b)
L-2-chloropropionic acid-induced cerebellar neurotoxicology is
prevented by prior administration of MK-801: possible role of
NMDA receptors in neuropathology. Toxicol Appl Pharmacol
136: 138±145
Widdowson PS, Gyte AJ, Upton R, Wyatt I (1996c) Failure of
glycine site NMDA receptor antagonists to protect against L-2-
chloropropionic acid-induced neurotoxicity, highlights the
uniqueness of cerebellar NMDA receptors. Brain Res 738: 236±
242
Widdowson PS, Gyte AJ, Upton R, Foster JR, Coutts CT, Wyatt I
(1997) Calpain activation and not oxidative damage mediates
the L-2-chloropropionic acid-induced cerebellar granule cell
necrosis. Toxicol Appl Pharmacol 142: 248±255
Acknowledgements The authors thank Dick Moore for synthesis of
the mercapturic acid of L-CPA, John Nash and Edwin Howard for
the excretion-balance studies where exhaled carbon dioxide was
determined, Denise Bennett for the whole body autoradiography
and Huw Adam for valuable advice on the pharmacokinetics.
References
Akazawa C, Shigemoto R, Bessho Y, Nakanishi S, Mizuno N
(1994) Di€erential expression of ®ve N-methyl-D-aspartate re-
ceptor sub-unit mRNAs in the cerebellum of developing and
adult rats. J Comp Neurol 347: 150±160
Dodds PF (1995) Xenobiotic lipids: the inclusion of xenobiotic
compounds in pathways of lipid biosynthesis. Prog Lipid Res
34: 219±247
Ebert B, Wong EHF, Krogsgaard-Larson P (1991) Identi®cation of
a novel NMDA receptor in rat cerebellum. Eur J Pharmacol
Mol Pharmacol 208: 49±52
Jones HB, Jenkins CR, Bowdler AL, Simpson MG, Lock EA
(1997) Ultrastructural pathology and cytochemical inv-
estigations of L-2-chloropropionic acid-induced neurointoxi-
cation of the rat cerebellum. Acta Neuropathol (Berl) 93:
241±251
Wyatt I, Gyte A, Mainwaring G, Widdowson PS, Lock EA (1996a)
Glutathione depletion in the liver and brain produced by 2-
chloropropionic acid: relevance to cerebellar granule cell ne-
crosis. Arch Toxicol 70: 380±389
Wyatt I, Gyte A, Simpson MG, Widdowson PS, Lock EA (1996b)
The role of glutathione in L-2-chloropropionic acid induced
cerebellar granule cell necrosis in the rat. Arch Toxicol 70: 724±
735
Kadotani H, Hirano T, Masugi M, Nakamura K, Nakao K,
Katsuki M, Nakanishi S (1996) Motor discoordination results
from combined gene disruption of NMDA receptor NR2A and
NR2C subunits, but not from a single disruption of the NR2A
or NR2C subunit. J Neurosci 16: 7859±7867
Wyatt I, Gyte A, Farnworth M, Widdowson PS, Lock EA (1996c)
The distribution and retention of [14C]L-2-chloropropionic acid
in male rats following dosing at 250 mg/kg or 750 mg/kg. Hum
Exp Toxicol 15: 663
Lock EA, Gyte A, Widdowson PS, Simpson MG, Wyatt I (1995)
Chloropropionic acid-induced alterations in glucose metabolic
status: possible relevance to cerebellar granule cell necrosis.
Arch Toxicol 69: 640±643