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 Oce. 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°.