Journal of Neuroscience Research 47:90–97 (1997)
Cytotoxic Effects of Repin, a Principal
Sesquiterpene Lactone of Russian Knapweed
M. Robles, N. Wang, R. Kim, and B.H. Choi*
Division of Neuropathology, Department of Pathology, University of California, Irvine
Repin is the principal sesquiterpene lactone isolated
from Russian knapweed (Centaurea repens), a peren-
nial weed found in many parts of the United States.
Ingestion of Centaurea repens by horses has been
reported to cause a movement disorder simulating
Parkinson’s disease (PD) and nigrostriatal degenera-
tion, called equine nigrostriatal encephalomalacia
(ENE). To understand the mechanisms whereby inges-
tion of Centaurea repens induces ENE and a PD-like
disorder, repin cytotoxicity was examined to explore
its pathogenetic relationship to ENE and to PD. Repin
was highly cytotoxic to both PC12 cells and mouse
astrocytes in a dose- and time-dependent manner. The
cytotoxic effects were accompanied by depletion of
glutathione (GSH), a rise in the level of reactive
oxygen species (ROS) and damage to cellular mem-
branes. Although repin is a highly reactive electro-
phile that can readily conjugate GSH, GSH depletion
may not be the sole mechanism underlying repin
cytotoxicity as shown by our study using buthionine
sulfoximine, in which severe GSH depletion did not
result in a parallel increase in cell death. However,
pre-treatment with GSH-glycoside or with lipoic acid
provided significant protection from repin-induced
cell death. These data suggest that oxidative stress
plays a major role in repin cytotoxicity. Since oxida-
tive stress is considered to play a major role in
neuronal degeneration accompanied by depletion of
mitochondrial GSH and an increase in lipid peroxides
in the substantia nigra of PD, further elucidation of
mechanisms of repin neurotoxicity may generate clues
regarding not only the mechanisms of neuronal degen-
eration but also the possible role of environmental
factors in the pathogenesis of PD. J. Neurosci. Res.
47:90–97, 1997. r 1997 Wiley-Liss, Inc.
Key words: repin; GSH; oxidative stress; Parkinson’s
disease
INTRODUCTION
Sesquiterpene lactones (SQLs) are a large group of
biologically active plant constituents that possess diverse
pharmacological and toxicological properties (Robles et
r 1997 Wiley-Liss, Inc.
al., 1995; Rodriguez et al., 1976). Early interest in SQLs
stems from the use of many of the Asteracea in folk
medicine as herbal remedies (Heptinstall et al., 1992).
More recently, investigators have identified a number of
plants consumed by chimpanzees that appeared to be
used for their medicinal properties (Nishida, 1990; Rod-
riguez and Wrangham, 1993; Wrangham et al., 1991).
Most of these plants contain significant quantities of
SQLs. The cytotoxic properties of SQLs have also
attracted considerable attention, not only because of their
potential toxicity to grazing animals, but also because of
their potential use as chemotherapeutic agents (Dollahite
et al., 1964; Kupchan et al., 1969; Rodriguez et al., 1976;
Ruffner and Weidner, 1977).
Repin is the most abundant SQL in Russian knap-
weed (Centaurea repens), a perennial weed that is rapidly
taking over pastures in many parts of the United States
due to its capacity to withstand harsh environmental
conditions. Chronic ingestion of large amounts of Centau-
rea repens by horses has been reported to cause equine
nigrostriatal encephalomalacia (ENE), characterized by
degeneration and necrosis of the substantia nigra and
corpus striatum and by signs and symptoms similar to
those of Parkinson’s disease (PD) (Cordy, 1954, 1978;
Fowler, 1965; Young et al., 1970). An almost identical
disorder develops in horses after ingestion of a related
weed called yellow star thistle (Mettler and Stern, 1963).
The biologically active ingredient common to both of
these plants is SQL. The functional group in repin is
represented by an a-methylenebutyrolactone moiety (Fig.
1) that can readily undergo conjugation with various
biological nucleophiles such as DNA, proteins and gluta-
thione (GSH).
Sponsored by NIEHS, grant number ES 02928.
Presented in parts at American Association of Neuropathologists, San
Antonio, Texas, June 1995, and Society of Toxicology, Anaheim,
California, March 1996.
*Correspondence to: B.H. Choi, Department of Pathology, Room D
440, Medical Sciences I, College of Medicine, University of California,
Irvine, CA 92697–4800.
Received 16 July 1996; Revised 15 August 1996; Accepted 15
September 1996.

Fig. 1. Chemical structure of repin. Note the presence of
exocyclic double bonds (empty arrow) and epoxides (arrows).
Although it is likely that PD is multifactorial in
origin, encompassing the combined effects of genetic and
environmental factors and age, the search for environmen-
tal neurotoxins that may cause or contribute to the
development of PD continues, fueled by the 1-methyl-4-
phenyl-1,2,3,6-tetrahydropyridine (MPTP) experience
(Langston et al., 1983, 1987).
The precise mechanism by which ingestion of
Centaurea repens induces a movement disorder in horses
simulating PD remains unclear, but the observation that
exposure to a natural product in our environment can lead
to a movement disorder associated with degeneration of
nigrostriatal pathways is intriguing. The purpose of this
study was to elucidate the mechanisms of repin cytotoxic-
ity and to explore its pathogenetic relationship to ENE
and to PD.
MATERIALS AND METHODS
Isolation of Repin
Repin was isolated by the method of Robles (1995).
Briefly, the aerial parts of Centaurea repens (250 g) were
extracted twice with 2.0 L of chloroform, filtered and
concentrated in a rotatory evaporator. The crude extract
was then dissolved in methanol, cooled to 4°C for 20 min
and filtered to remove waxes and other non-polar sub-
stances. This extract was placed onto a Sephadex LH-20
gel and eluted with methanol. Fractions were collected
and combined according to their respective thin-layer
chromatography (TLC) Rf values in a 7:3 chloroform-
methanol solvent system. Further purification was
achieved using silica gel column chromatography with an
eluting gradient of chloroform to 1% methanol. Prepara-
tory TLC was used for final purification with a 6:4
chloroform-acetone solvent. Dimethylsulfoxide was used
to dissolve repin for experimental use.
Cell Cultures
PC12 cells, obtained from the American Type
Culture Collection (Rockville, MD), were maintained in
Repin Toxicity In Vitro 91
75 cm2 tissue culture flasks in RPMI 1640 medium
supplemented with 10% fetal calf serum, 5% horse
serum, 50 units/ml penicillin and 100 mg of streptomycin
under 5% CO2 at 37°C. The cell number averaged
approximately 105 per ml. PC12 cells were grown for 7
days prior to experimental use.
Mouse astrocytes were cultured from the forebrains
of C57BL/6J mice at postnatal day 1 by a method
modified from McCarthy and de Vellis (1980). Briefly,
cerebral cortical tissues were finely minced after remov-
ing the meninges, suspended in 0.1% trypsin-EDTA
buffer with trituration and placed into an incubator for 10
min at 37°C. Following incubation, the cell suspensions
were further triturated, centrifuged and plated onto poly-
L-lysine-coated T-flasks in complete culture media, which
consisted of Dulbecco’s minimum essential medium
supplemented by 10% fetal calf serum, 1% 200 mM
glutamine, 75 µg penicillin-streptomycin/ml and 0.5%
glucose. Seven days after establishment of the primary
culture, the T flasks were shaken on a rotary shaker to
detach oligodendrocytes (OC). Three days after the
removal of OC, astrocytes were dissociated from the
original flasks and plated onto T-flasks. After 3 days in
culture, the cells were treated with 40 µM cytosine
arabinoside for 24 hr. Cultures were then fed with
complete culture media and grown for an additional 5
days before use.
Cytotoxicity Assay
Both PC12 cells and mouse astrocytes dissociated
using trypsin-EDTA in calcium- and magnesium-free
phosphate-buffered saline were suspended into T-flasks
(105 cells/ml) in complete culture media and slowly
shaken on a rotary shaker at approximately 60 rpm. The
cell survival rate was determined by the dye exclusion
test, using 0.4% trypan blue in 0.9% NaCl solution and
counting in a hemocytometer. Cell survival rates at
various repin concentrations ranging from 1.0 to 20 µM
for 3 hr were measured in triplicate samples obtained
from at least three flasks for each repin concentration, and
the approximate EC50 (effective concentration to induce
50% cell death) determined.
To examine the effects of repin on GSH, both PC12
cells and mouse astrocytes were exposed to 10 and 20 µM
repin and the level of GSH determined at 1, 2 and 3 hr. To
study further the relationship between GSH and cytotox-
icity, PC12 cells were preincubated with 1.0 mM buthio-
nine sulfoximine (BSO) (a specific inhibitor of glutamyl-
cysteinyl synthetase), 1.0 mM lipoic acid (a powerful
antioxidant) or 1.0 mM GSH-glycoside (GSH-glyc) [a
compound newly synthesized in our laboratory that
permits the cellular uptake of GSH (Choi et al., 1996)]
prior to repin (20 µM) exposure. The rate of cell death and
92 Robles et al.

TABLE I. Experimental Groups oxidized to fluorescent 82,87-dichlorofluorescein (DCF)

No. of Pre-incubation in the presence of hydrogen peroxide (Bondy et al.,
Group flasks (8 hr) Exposure (3 hr) 1992). Superoxide may also dismutate to hydrogen
I 3 Control medium — peroxide, thus contributing to the total DCF fluorescence.
II 3 Control medium Repin Initial fluorescence was recorded after loading cells with
III 3 BSO — DCFH-DA. Fluorescence was then recorded after the
IV 3 BSO Control medium addition of repin in concentrations ranging from 10 to 80
V 3 BSO Repin µM. PC12 cells were also exposed to 10 and 20 µM repin,
VI 3 Lipoic acid Repin
VII 3 GSH-glyc Repin and ROS production was determined every 5 min for 1 hr.
In addition, the cells were incubated in 1.0 mM lipoic
1.0 mM of each of BSO, lipoic acid, or GSH-glyc was used for
acid or 1.0 mM BSO for 18 hr prior to repin exposure, and
preincubation for 8 hr. Repin exposure (20 µM) was for 3 hr. BSO,
buthionine sulfoximine; GSH-glyc, GSH-glycoside. the formation of ROS was determined 15 min after repin
exposure. The formation of DCF was monitored at an
excitation wavelength of 488 nm and an emission wave-
length of 525 nm, using a spectrophotofluorometer. The
the GSH levels were determined in different experimental cuvette holder was thermostatically maintained at 37°C.
groups, as shown in the Table I. DCF formation was quantified from a standard curve
using Locke’s buffer (0.05–0.1 µM).
Morphological Assessment
Both control and repin-exposed PC12 cells were Statistical Analyses
fixed on coverslips in 10% formalin and stained with Differences between groups were assessed by the
hematoxylin and eosin. In addition, PC12 cells on cover two-tailed, unpaired Student’s t test throughout this study.
slips were fixed in Karnovsky’s fluid and processed for The data are expressed as value 6 standard error of the
examination by both scanning (Philips EM 500) and mean. The acceptance level of significance was P , 0.05.
transmission (Philips EM 400) electron microscopy.

GSH Assay RESULTS

Total GSH was determined using the Tietze method
(1969). Briefly, samples of cell homogenates were mixed
with GSH reductase (100 IU/mg) in the presence of 200
µM reduced triphosphopyridine nucleotide and Ellman’s
reagent. Measurements of absorbance changes at 412 and
340 nm were made for 3 min using a Gilford spectropho-
tometer. All reactions were carried out at 25°C. Total
GSH concentrations were expressed as mM/g of protein.
Protein content was measured by the method of Lowry et
al. (1951).
Repin-GSH Complex Formation
Repin was reacted nonenzymatically with GSH in
various molar ratios (1:0.5, 1:1, 1:2, 1:4) in test tubes.
Following the reaction (in min), assays for free GSH were
carried out over time using Ellman’s reagent. Because of
the rapidity of the reaction the experiments were carried
out both at room temperature and at 2°C.
Determination of ROS Generation
PC12 cells were incubated with 5 µM 82,87-
dichlorofluorescein diacetate (DCFH-DA) for 15 min. A
stable, non-fluorescent DCFH-DA molecule is hydro-
lyzed by intracellular esterases to DCFH, which is then
Repin was found to be highly cytotoxic to both
PC12 cells and mouse astrocytes in a dose-dependent
manner (Fig. 2). EC50 in PC12 cells and astrocytes were
determined to be approximately 14 µM and 6.2 µM,
respectively. Significant depletion of GSH was already
apparent 1 hr after exposure to 10 or 20 µM repin and
declined progressively during the subsequent 2 hr in both
cell types (Fig. 3). Non-enzymatic complex formation
between repin and GSH in vitro was extremely rapid in a
1:1 molar ratio at both room temperature and at 2°C.
As shown in Figure 4, 3-hr exposure to repin led to
an increase in cell death rate to 27% (control:14%) and to
a moderately severe (28% of control) depletion of GSH.
BSO also induced severe (22% of control) depletion of
GSH, but the cell death rate did not increase in a parallel
fashion, remaining at approximately 18% (control:14%).
When BSO was washed and replaced with complete
control medium the cell death rate improved significantly
to 15%, but GSH remained low at 19% of the control
level. Incubation with both BSO and repin resulted in a
62% cell death rate with marked depletion of GSH to
22% of control levels. Incubation with lipoic acid or
GSH-glyc prior to repin exposure improved not only the
survival rate but also the GSH content (Fig. 4).
The rate of ROS production in PC12 cells was
significantly enhanced in a dose- and time-dependent

Fig. 2. A: Repin cytotoxicity in PC12 cells. The cells were
exposed to repin for 3 hr at 37°C. Each point represents the
mean for three independent experiments (P 5 0.05). Note the
dose-dependent decrease in the mean cell survival rate, with the
EC50 being approximately 14 µM. B: Repin cytotoxicity in
mouse astrocytes. The cells were exposed to repin for 3 hr at
37°C. Each point represents the mean for three independent
experiments (P 5 0.05). Note higher vulnerability of astrocytes
to repin toxicity, the approximate EC50 being 6.2 µM.
manner following repin exposure (Fig. 5) at all concentra-
tions. The highest level of DCF fluorescence was ob-
served at 15 min. With extended exposure to 10 to 20 µM
repin, the rate of ROS generation began to decline
gradually at about 40 min, and enhancement of ROS
generation was no longer apparent by 60 min (data not
shown). When cells were preincubated with 1.0 mM
Repin Toxicity In Vitro 93
Fig. 3. A: GSH depletion in repin-exposed PC12 cells. The
cells were incubated with 10 or 20 µM repin at 37°C and the
GSH levels determined at 1, 2 and 3 hr. Each point represents
the mean for three independent experiments (*P,0.05). Note
the dose- and time-dependent reduction of GSH. B: GSH
depletion in repin-exposed mouse astrocytes. The cells were
incubated with 10 or 20 µM repin at 37°C and the GSH levels
determined at 1, 2 and 3 hr. Each point represents the mean for
three independent experiments (*P , 0.05).
lipoic acid overnight prior to repin exposure, a 62%
reduction in ROS generation was observed. Conversely,
overnight incubation of BSO enhanced the generation of
ROS by 68% (Fig. 6).
Figure 7A depicts the typical light microscopic
appearance of PC12 cells with their long extended
processes, moderate amounts of cytoplasm and ruffling of
the surface membrane. The nucleus was often eccentric
and showed an intact nuclear membrane with finely
granular chromatin and a prominent nucleolus. Repin
94 Robles et al.
Fig. 4. GSH levels and cell death rate in repin-exposed PC12
cells. The cells were preincubated for 8 hr with either control
culture medium (Con), BSO, lipoic acid (Lip) or GSH-glyc
(GSH-glyc) followed by 3 hr of exposure to repin (Rep) or
control culture medium (Con). Note the significant increase in
cell death rate (*P , 0.05) associated with a marked reduction
in GSH levels (**P , 0.05) following repin exposure. BSO
preincubation enhanced cell death rate as well as GSH deple-
Fig. 5. ROS formation in repin-exposed PC12 cells. Note the
dose- and time-dependent rise in the rate of ROS production.
The highest level of DCF fluorescence was noted at 15 min.
exposure caused immediate retraction of the processes,
rounding and vacuolization of the cytoplasm, loss of
surface ruffling and nuclear pyknosis (Fig. 7B).
Scanning EM of control PC12 cells showed the cell
surface to be studded with numerous microvilli (Fig. 8A),
whereas repin-exposed PC12 cells demonstrated smooth-
ing of the cell surface with almost complete loss of
microvilli (Fig. 8B). An interesting phenomenon was the
tion. When cells were exposed to BSO for 8 hr and then
replaced with control culture medium, cell survival improved to
normal, but GSH reduction was still highly significant. Lipoic
acid preincubation improved cell survival and GSH concentra-
tions. GSH-glyc preincubation improved the cell survival and
greatly increased the GSH content of cells, even in the presence
of repin.
Fig. 6. Effects of BSO and lipoic acid (Lip) on repin-induced
ROS generation. PC12 cells were incubated in BSO or lipoic
acid for 18 hr and then exposed to repin. DCF fluorescence was
determined 15 min after repin exposure. Repin exposure
enhanced DCF fluorescence in PC 12 cells significantly (*) as
compared to that of control. Although BSO or lipoic acid alone
did not cause an increase in ROS generation, a marked
enhancement of ROS generation was observed when BSO
preincubation was followed by repin exposure. Preincubation in
lipoic acid, on the other hand, significantly reduced ROS
generation in the presence of repin. *P , 0.05.
presence of what appeared to be a punched hole in the cell
membrane of each repin-exposed cell (Fig. 8B). Transmis-
sion EM of control PC12 cells showed intact cytoplasmic
organelles and surface membranes, well preserved nuclear
 Repin Toxicity In Vitro 95

Fig. 7. Photomicrographs of control (A) and repin-exposed (B)

PC12 cells. Note the long extended processes (arrows) of
normal PC12 cells containing an eccentric nucleus with a
nucleolus (thick arrow) and ruffling of the surface membrane
(small arrows). Repin-exposed cells show complete withdrawal
of processes with smoothing of the cell surface (arrows) and
loss of ruffling. Note also pyknosis of nuclei (thick arrow) and
vacuolization of the cytoplasm. H&E 3 560.

membranes and prominent nucleoli (Fig. 9A), whereas

repin-exposed cells showed rupture of both cytoplasmic
and nuclear membranes, dilatation of the endoplasmic
reticulum, swelling of mitochondria and loss of mitochon-
drial cristae (Fig. 9B).

DISCUSSION
Repin, a novel compound isolated and purified from
a plant commonly found in our environment, is demon-
strated to be highly cytotoxic to both PC12 cells and
mouse astrocytes. Marked depletion of cellular GSH is a
prominent feature associated with repin cytotoxicity.
Since repin contains an a-methylenebutyrolactone moi-
ety, a Michael-type addition with GSH could readily have
taken place, as shown by others (Rodriguez et al., 1976)
and by us, with very rapid non-enzymatic 1:1 complex
formation between repin and GSH in vitro. Because the
GSH redox cycle represents one of the principal endog-
enous protective systems against toxic chemicals and the
oxidative products of normal cellular metabolism, GSH
depletion probably plays a significant role in repin
cytotoxicity. On the other hand, as evidenced by experi-
ments using BSO, simple depletion of GSH may not have
been the sole mechanism. Nevertheless, once depleted of
protective GSH, susceptibility to secondary insults by the
other toxic actions of repin would have been greatly
enhanced. For example, the epoxide moieties in the repin
molecule may open under certain conditions to induce an
additional series of oxidative events.
As shown by both light and EM, toxic actions of
repin appear to be exerted predominantly on cytoplasmic,
Fig. 8. Scanning EM of control (A) and repin-exposed (B) PC
12 cells. Note control cell surface studded with numerous
microvilli (arrowhead). Repin-exposed cells show almost com-
plete loss of surface microvilli and smoothing of the cell surface
(arrow). Note also the presence of what appears to be a punched
hole in the cell membrane (arrowheads).
nuclear and mitochondrial membranes. The extended
cytoplasmic processes of PC12 cells immediately became
withdrawn, with loss of surface ruffling, vacuolization of
the cytoplasm and pyknosis of nuclei. Rupture of both
cytoplasmic and nuclear membranes was prominent. An
apparent improvement in the cell survival rate following
preincubation with lipoic acid further suggests that lipid
peroxidation may be of importance in the development of
injury. Lipid peroxidation may have been caused by ROS,
since repin exposure induced a significant rise in the
generation of ROS in a dose- and time-dependent manner.
Furthermore, overnight pre-incubation of 1.0 mM lipoic
96 Robles et al.
Fig. 9. Transmission EM of control (A) and repin-exposed (B)
PC12 cells. Control cells show well-defined nuclei with clear
nuclear membranes (thick arrows) and prominent nucleoli. The
organelles are distributed evenly within the cytoplasm and the
cell membranes (arrow) are intact. Small arrows point to
microvilli on the cell surface. Repin-exposed cells show rupture
of cell (arrows) and nuclear membranes (short arrows), with
partial extrusion of nuclear material. The endoplasmic reticu-
lum shows marked dilatation (empty arrows) and swelling of
mitochondria, with loss of cristae (thick arrow).
acid or GSH-glyc alleviated ROS production, whereas
incubation with BSO prior to repin exposure greatly
enhanced the rate of ROS generation. These data suggest
that oxidative stress plays a major role in repin-induced
cytotoxicity.
Our previous findings, in C57BL/6J mice, of signifi-
cant GSH depletion in the striatum as compared to other
regions of the brain, with an increase in thiobarbiturate
reactive substances (TBARS) and in striatal dopamine
levels (suggestive of modification of striatal dopamine
metabolism) within hours of repin exposure (Robles and
Choi, 1995), indicate that repin can induce selective
biochemical alterations in striatal dopaminergic path-
ways. It appears, therefore, that repin toxicity may have
played a significant role in the pathogenesis of the
PD-like movement disorder that followed chronic inges-
tion of Russian knapweed in horses.
The concept that oxidative stress plays a major role
in the degeneration of nigral neurons in PD is supported
by reports showing depletion of mitochondrial GSH
in the substantia nigra and an increase in nigral iron
and lipid peroxides, findings that correlate with the
severity of illness (Jenner, 1991; Riederer et al., 1989).
The sudden onset of a parkinsonian syndrome following
intoxication with MPTP is believed to be mediated
by oxidized 1-methyl-4-phenylpyridinium ion (MPP1)
via unstable 1-methyl-4-phenyl-2,3-dihydropyridine
(MPDP) by monoamine oxidase B in astrocytes (Castag-
noli et al., 1985; Langston et al., 1984). MPP1 is then
taken up specifically by dopaminergic neurons and accu-
mulates within mitochondria, disturbing the electron
transport chain (ETC) and leading to depletion of energy
stores and to formation of free radicals (Langston et al.,
1983, 1987; Singer et al., 1987; Sinha et al., 1986; Vyas et
al., 1986).
Based on our observations, oxidative stress in repin
neurotoxicity appears to result not only from GSH
depletion but also from excessive generation of ROS.
Possible mechanisms of ROS formation in repin toxicity
include the direct action of repin on mitochondrial
respiratory system, oxidative events created by instability
of epoxides in the parent repin molecule and possible
effects of methacrylate side chain of the repin molecule.
Mitochondrial ETC has been shown to be a common
target for epoxides and their phenol derivatives (Naka-
gawa et al., 1993), and toxic effects of methyl methacry-
late, an analog of methacrylic acid, are thought to be
mediated through disturbances in mitochondrial ETC
(Bereznowski, 1994). Since the acute cytotoxic effects of
repin appear to be exerted through oxidative stress in
vitro, and since repin appears to induce oxidative stress
on the striatal dopaminergic pathways in vivo (Robles
and Choi, 1995), we propose that repin toxicity could be
used as a model for studying the pathogenesis of PD.
Repin is a purified compound, extracted from a common
plant in our environment, that is capable of inducing a
PD-like disease in horses; therefore, studies of the
mechanisms of repin neurotoxicity may generate further
clues regarding not only the mechanisms of neuronal
degeneration but also the possible role of environmental
factors in the pathogenesis of PD.

ACKNOWLEDGMENTS
The authors thank Simon Yee, Bora Han and Joyce
Shin for their expert technical assistance. This study was
supported in part by NIEHS grant ES 02928 (BHC).
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