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Ui:tv'EF:SITY OF HAWAI'I LIBRARY
KAVA-ASSOCIATED HEPATOTOXICITY
A THESIS SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAW AI'I AT MANOA IN PARTIAL
FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
MASTERS OF SCIENCE
IN
MOLECULAR BIOSCIENCES AND BIOENGINEERING
AUGUST 2006
By Steven Tai Shun Lim
Thesis Committee:
Pratibha V. Nerurkar, Chairperson H.C. Bittenbender
Gemot Presting Dulal Borthakur
We certify that we have read this thesis and that, in our opinion, it
is satisfactory in the scope and quality as a thesis for the degree of
Masters of Science in Molecular Biosciences and Bioengineering.
THESIS COMMITIEE
ii
ACKNOWLEDGEMENTS
I would like to thank my advisor, Dr. Pratibha V. Nerurkar, for
guiding and pushing me to become the best student that I could be. I have
gained more knowledge than I could have ever imagined. I would also like
to thank my committee members Dr. Skip Bittenbender, Dr. Gemot Presting
and Dr. Dulal Borthakur for their encouragement and help with my project.
I would like to thank my lab mates for their encouragement and making lab
enjoyable. Finally, I would like to thank my family for supporting me
throughout my degree. All of you made this experience one that I will never
forget.
This study was partially supported by USDA CSREES (2003-34135-
14033), and partially by U.S. Public Health Service grants (G12RR003061
and P20RROll091) from the Research Centers in Minority Institutions
Program, National Center for Research Resources, National Institutes of
Health awarded to Dr. Pratibha V. Nerurkar.
iii
ABSTRACT
Kava is a shrubby herb that has been used for centuries by the Pacific
Island societies as a ceremonial, medicinal, social and recreational beverage.
Kava, in the form of aqueous drinks prepared from rhizome and root, was
considered "safe" with only mild but reversible adverse effects. However,
between 1990 and 2001, there were 82 case reports of severe liver toxicity
and even a few deaths among patients consuming kava extract preparations.
Due to the continued usage of kava in the U.S. as well as its importance in
the Pacific Islands, it is essential to delineate any negative effects that may
occur with kava intake. Kava stem peelings, which contain high amounts of
the cytotoxic alkaloid pipermethystine (PM), were reportedly incorporated
into commercial extract preparations and were hypothesized to have
contributed to the toxic effects of kava observed in humans. PM was shown
induced oxidative stress and cell death in the human hepatoma cell line,
HepG2, while the active components of the kava plant called kavalactones
(KL) failed to induce any changes. However, lO-week treatment of PM
(2mg/kg) and kava stem peeling extract (8.7Smg/kg) in CS7BL6 mice failed
to induce liver toxicity. Short term administration of PM (10mg/kg) and
acetonic kava extracts (lOOmg/kg), individually and in combination, also
failed to induce liver toxicity in F-344. However, an initial antioxidant
iv
stress response was noted in addition to increased CYP4S0 enzyme
expression. From the data obtained, kava toxicity observed in humans are
likely due to a number of factors including drug interactions rather than
direct liver toxicity from PM and kavalactones alone.
v
TABLE OF CONTENTS
Acknowledgements II •••••••••••••••••••••• 1 ••••••••••• II ••••• 1 •••••••••• 1 •••••••••••••••••• 1 •••••••••••••• iii
Abstract .......................................................................................................... iv
List of Tables ................................................................................................. xi
T ! f F· .. L.l.st 0 19ures II •••• II •••••••••• I •••••••••• II •••••••••••• I •••• I ••••••••••••••• II ••••••••••• II •••••• II •••••••• Xll
Ust of Abbreviations ................................................................................... xiv
1. Chapter 1: Introduction ................................................................... 1
1.1. General introduction .......................................................................... 1
1.2. Is kava safe? ....................................................................................... 2
1.3. History of kava use in the west .......................................................... 4
1.4. Case reports and kava ban ................................................................. 5
1.5. Kavalactones ...................................................................................... 6
1.6. Metabolism and Pharmacokinetics .................................................... 7
1.7. Kava-drug interactions ...................................................................... 8
1.8. Cytochrome P450 enzymes ............................................................... 9
1. 9. Differences in extraction method ........ ..... ... ......... ...... .... ... .... .......... 10
1.10. Kava toxicity and dosage ............................................................... 11
1.11. The liver and hepatotoxicity .......................................................... 12
1.12. Oxidative stress and mitochondria ................................................ 14
1.13. Importance of kava research ......................................................... 14
vi
1.14. Hypothesis and objectives ............................................................. 15
2. Chapter 2: Effects of kava acetonic extract, kava alkaloid
pipermethystine and kavalactones dihyromethysticin and
desmethoxyyangonin on the human hepatocellular
carcinoma cell Hne, HepG2 .............................................................. 17
2.1. Introduction ................................................................................... 17
2.2. Results ........................................................................................... 18
2.2.1. Effects of PM, DMY, DHM and KRE on cytotoxicity ........... 18
2.2.2. Effects of PM, DMY, DHM and KRE on markers of
mitochondrial dysfunction ....................................................... 19
2.2.3. Effects of Cyclosporin A on PM induced cytotoxicity ............ 20
2.2.4. Effects of PM on protein expression of cytochrome c,
AIF and HNE ........................................................................... 20
2.3. Discussion ..................................................................................... 30
3. Chapter 3: Long term effects of kava alkaloid
pipermethystine and kava stem peeling extract on CS7BL6
mice ..................•..............................•...............................................•..... 33
3.1. Introduction ................................................................................... 33
3.2. Results ........................................................................................... 33
vii
3.2.1. Effects of PM and KSPE on body weight, feed intake and
liver weight ............................................................................... 33
3.2.2. Effects of PM and KSPE on serum AST, ALT and LDH ....... 34
3.2.3. Effects of PM and KSPE on hepatic triglyceride levels .......... 34
3.2.4. Effects of PM and KSPE on CYP2El expression ................... 34
3.3. Discussion ..................................................................................... 41
4. Chapter 4: Short term effects of pipermethystine and kava
rhizome extract on F ·3~ rats ............................................................ 43
4.1. Introduction ................................................................................... 43
4.2. Results ............................................................................................ 43
4.2.1. Effects of PM and KRE on markers of liver injury ................. 43
4.2.2. Antioxidant status and oxidative stress .................................... 44
4.2.3. Effects of PM and KRE on mRNA expression of Bc1-2,
Bax and lNF-alpha .................................................................. 44
4.2.4. Hepatic protein expression of UCP-2 ...................................... 45
4.2.5. Effects of PM and KRE on drug metabolizing enzymes,
CYP450 .................................................................................... 45
4.3. Discussion ..................................................................................... 53
5. Chapter 5: General Discussion ......................................................... 57
6. Chapter 6: Materials and Methods .................................................. 59
viii
6.1. Isolation of PM, DHM and DMY ................................................. 59
6.2. Preparation of Kava Stem Peeling Extract (KSPE) and Kava
Rhizome Extract (KRE) ................................................................ 59
6.3. HepG2 cell cultures ....................................................................... 60
6.4. C57BL6 mouse treatment procedure ............................................ 60
6.5. F-344 rat treatment procedure ....................................................... 61
6.6. HepG2 cell treatments ................................................................... 62
6.7. Cytotoxicity assay (LDH release) ................................................. 63
6.8. ROS production ................................................................................... 63
6.9. Mitochondrial membrane potential (A'l') ...................................... 64
6.10. Cellular ATI levels ......................................................................... 64
6.11. Serum liver injury markers ............................................................ 65
6.12. Total Hepatic ATP levels ............................................................... 65
6.13. Hepatic Glutathione (GSH) ........................................................... 66
6.14. Hepatic Superoxide Dismutase Activity (SOD) and Lipid
Peroxidation (LPO) ........................................................................ 66
6.15. Hepatic Aconitase activity ............................................................ 67
6.16. Cellular Uncoupling Protein-2 mRNA Expression ....................... 67
6.17. Semiquantitation of Bax, Bc1-2 and TNF-alpha Gene
Expression ..................................................... I ••••• I ••••••••••••••••••••••••• 68
ix
6.18. Hepatic Lipids extraction ............................................................. ~ 68
6.19. Triglyceride (TG) assay ................................................................ 69
6.20. Mitochondrial and cytosolic extract preparation for HepG2 ........ 69
6.21. Liver Microsome preparation ........................................................ 70
6.22. Mitochondria and Cytosol Preparation ......................................... 70
6.23. Protein determination .................................................................... 71
6.24. Western blotting for mitochondrial and cytosolic Apoptosis-
inducing Factor (AlP) and cytochrome c ...................................... 71
6.25. Western blotting for Hydroxy-nonenol (HNE) adduct ................. 72
6.26. Western blotting of microsomal Cyp2El.. ................................... 72
6.27. Western Blot Analysis of CYP450 Proteins, UCP-2 and
HSP-70 .......................................................................................... 73
6.28. Statistical Analysis ........................................................................ 74
References .................................................................................................... 79
x
UST OF TABLES
Table
1. Body weight, feed intake and liver weight changes of PM and KRE
treated F-344 rats .......................................................................................... 46
2. Effects of PM and KRE on hepatic injury markers in rat serum ............. 46
xi
liST OF FIGURES
Figure
2. Chapter 2 Figures: Effects of kava alkaloid, lactones and rhizome extract
on human hepatoma cell line, HepG2 .............................................. 21-29
2.1. Cytotoxicity assay for pipermethystine ......................................... 21
2.2. Cytotoxicity assay for desmethoxyyangonin ................................ 22
2.3. Cytotoxicity assay for dihydromethysticin ................................... 23
2.4. Cytotoxicity assay for kava root extract.. ...................................... 24
2.5. Cytotoxicity assay for combination treatment of PM, DMY and
DHM ................................................................................................ 25
2.6. Reactive oxygen species production for pipermethystine and
kavalactones DMY and DHM ......................................................... 26
2.7. Mitochondrial membrane potential for pipermethystine and
kavalactones DMY and DHM ......................................................... 27
2.8. Total cellular ATP levels for KRE and PM .................................. 28
2.9. Attenuation of PM-induced cytotoxicity by Cyclosporin A ......... 29
3. Chapter 3 Figures: Long term effects of pipermethystine and kava stem
peeling extract on C57BL6 mice ...................................................... 35-40
3.1. Weekly body weight change ......................................................... 35
xii
3.2. Weekly feed intake change ............................................................ 36
3.3. Final liver weight ........................................................................... 37
3.4. Serum liver injury markers ............................................................ 38
3.s. Serum lactate dehydrogenase levels .............................................. 39
3.6. Hepatic triglyceride levels ............................................................. 40
4. Chapter 4 Figures: Short term effects of pipermethystine and kava
rhizome extract on F-344 rats ........................................................... 47-52
4.1. Total hepatic ATP and ADP .......................................................... 47
4.2. Hepatic antioxidant status ............................................................. 48
4.3. Total liver aconitase activity ......................................................... 49
4.4. Hepatic mRNA expression of TNF-alpha ..................................... 50
4.5. Mitochondrial protein expression of UCP-2 ................................. 51
4.6. Microsomal cytochrome P450 protein expression ........................ 52
xiii
liST OF ABBREVIATIONS
ADP adenosine diphosphate
AlF apoptosis-inducing factor
ALP alkaline phosphatase
ALT alanine aminotransferase
AST aspartate aminotransferase
ATP adenosine triphosphate
BSA bovine serum albumin
BMI body-mass index
BW body weight
cDNA complementary DNA
CNS central nervous system
CsA cyclosporine A
cyt c. cytochrome c
CYP cytochrome P450
DCF 2', 7'-dichlorofluorescin diacetate
DHM dihydroxymethysticin
DLS Diagnostic Laboratory Services
DMSO dimethylsulfoxide
DMY desmethoxyyangonin
xiv
DNA
EC50
ECL
EDTA
ETOAc
FBS
FDA
g
GABA
GC-FID
GC-MS
GGT
GSH
h
HAE
HDL
HEPES
HNE
HPLC
HRMS
deoxyribonucleic acid
median effective concentration
enhanced chemiluminescence
ethylenediaminetetraacetic acid
ethyl acetate
fetal bovine serum
Food and Drug Administration
gram
gamma-amino-butyric-acid
gas chromatography-flame ionization detector
gas chromatography-mass spectroscopy
gamma-glutamyl transferase
reduced glutathione
hour
hydroxyalkenals
high-density lipoprotein
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
hydroxynonenol
high-performance liquid chromatography
high resolution mass spectroscopy
xv
HRP
IACUC
IgG
JC-l
kb
KCI
kDa
kg
KL
KRE
KSPE
L
LDH
MEME
MDA
mg
MPT
mRNA
mt
horseradish peroxidase
Institutional Animal Care & Use Committee
immunoglobulin G
5,5',6,6'- tetrachloro
tetraethylbenzinidazolylcarbocyanide iodide
kilobase
potassium chloride
kilo Dalton
kilogram
kavalactone
kava root extract
kava stem peeling extract
liter
lactate dehydrogenase
minimum essential medium eagle
malondialdehyde
milligram
mitochondrial permeability transition
messenger RNA
mitochondria
xvi
NaCI
NTP
PBS
PM
RNA
ROS
SD
SDS-PAGE
SOD
TCA
TG
Tris-HCI
UCP
v/v
xg
sodium chloride
National Toxicology Program
phosphate buffered saline
pipermethystine
ribonucleic acid
reactive oxygen species
standard deviation
sodium dodecyl sulfate polyacrilarnide gel
superoxide dismutase
trichloroacetic acid
triglyceride
Trizma-hydrochloride
uncoupling protein
volume to volume
gravitational force
xvii
CHAPI'ERI
INTRODUCTION
General Background
Kava (Piper methysticum Forst. f.) or 'awa (in Hawaiian) is a shrubby herb
that is a member of the pepper family, Piperaceae. Kava has been used for
centuries by the Pacific Island societies as a ceremonial, medicinal, social and
recreational beverage (Lebot et al., 1992). Kava infusions were an important part
of ceremonies welcoming guests and in rituals where it aids in connecting with
gods and ancestors (Lebot et al., 1997). Traditionally, both fresh and dried
rhizome and roots were ground into an aqueous suspension with coconut milk or
water and the strained product was consumed. Kava's effects are considered mild
and not drastically mind-altering like commonly consumed drugs such as alcohol.
In addition, when sensible amounts are consumed there are no after effects such as
"hangovers". Kava drinks are said to give the user a sense of well-being and are
associated with anxiolytic, muscle relaxant, sedative and diuretic properties,
among others (Lebot et aI., 1997). Kava's taste has been characterized as earthy,
unpleasant, bitter, astringent, spicy or pungent. Kava is one of the most important
traditional beverages to the Pacific Island people, being one of the first indigenous
plants to be domesticated in those areas (Lebot et al., 1997). To this day, it still has
1
importance in religious, social and economic life for many of the Pacific Island
communities.
Is kava "safe"?
Kava has long been characterized as very safe (Singh, 1992; Singh and
Singh, 2002), supported by the time tested consumption in the Pacific Islands over
the last 2000 years (Steiner, 2000). In the Pacific Islands, males drink kava, often
in quantities much higher than in Hawaii, mainland U.S. and Europe. However,
the rate of liver toxicity in the islands is similar between sexes (Singh and Singh,
2002). Therefore, toxic responses to traditional kava drinks are not very
pronounced. However, milder forms of liver toxicity have not been systematically
studied so less severe cases could have been present without being documented
(Moulds and Malani, 2003). Up until 1998, side effects associated with kava
consumption were generally thought to be negligible. A survey performed in the
Arnhem Land aboriginal communities in Australia reported that ''very heavy" kava
use was associated with low BMI, dry and scaly skin and decreased lymphocyte
counts, as well as reduced total blood protein, albumin and bilirubin (Mathews et
aI., 1988). In addition, kava users showed abnormally high levels of gamma
glutamyl transferase (GG1) and high density lipoprotein (HDL)-cholesterol
(Mathews et aI., 1988). Subsequent studies in the same region reported similar
2
fmdings, with recent kava use associated with abnormalities in liver function test
enzymes, GGT and alkaline phosphatase (ALP), present in 61% and 50% of kava
drinkers, respectively (Clough et aI., 2003b). Interestingly, these surveys report
normal levels of alanine aminotransferase (ALT), which is inconsistent with acute
inflammation and the changes seen in kava-associated hepatotoxicity (Currie and
Clough, 2003). However, one study conducted in New Caledonia concluded that
traditional kava preparations are the most likely cause of hepatitis in two women
with markedly increased transaminases and hyperbilirubinaemia (Russmann et al.,
2003). It must be noted that these cases are very rare and have confounding factors
that may have contnbuted to the toxicity. Moreover, clinical observation of
aboriginal communities in the Northern Territory spanning a 20 year period has not
seen severe hepatic injury with kava consumption, even at levels 50 times higher
than the recommended pharmaceutical dosage (Clough et al., 2003a). Other side
effects associated with kava consumption include yellowing of the skin, allergic
reactions, red eyes, gastrointestinal complaints and lethargy hindering the drinker's
ability to sufficiently eat (Singh, 1992; Lebot et al., 1997). However, reactions to
traditional kava extracts were for the most part reversible upon discontinuation of
the drug (Singh, 1992).
3
IDstory of kava use in the west
British explorer Captain James Cook was the first European to observe kava
some 200 years ago on his first voyages to the Polynesian islands and since then its
use has spread throughout the western world (Lebot et al., 1997). Kava
preparations and extracts have been used in Germany since the late 1800's (Barsky
et al., 2002). Over the years, kava has been greatly studied and many beneficial
properties have been exploited. In the 1990's, commercial kava products gained
immense popularity in Europe and North America as a treatment option for anxiety
and nervous disorders due to studies showing it to be non-addictive and clinically
effective (O'Sullivan and Lum, 2001; Singh and Singh, 2002; Pittler and Ernst,
2003; O'Sullivan and Lum, 2004). The most common prescription medication used
to treat anxious disorders is benzodiazapines, which are associated with adverse
effects such as dependence, sedation and memory impairment (Pittler and Ernst,
2000). As a result, kava became a low risk and less-expensive alternative to the
current treatments for anxiety disorders (Schmidt, 2002). It was readily available
as an over-the-counter herbal supplement in Europe and the u.S. and was
commonly used as an anxiolytic and sleep aid. By 2000, sales of kava products
totaled about $15 million in the US, ranking it ninth in sales among all herbal
products (Gruenwald, 2002).
4
Case reports and kava ban
Between 1990 and 2001, there were 82 case reports of alleged kava
associated liver toxicity and even a few deaths among patients consuming kava
extract preparations (Schmidt, 2002; Oouatre, 2004). By the end of 2001, kava
containing products were banned in many Western countries such as Germany,
France, Switzerland, Australia and Canada (Russmann et al., 2001; Schmidt et al.,
2002; Clouatre, 2004). In addition, warnings were put out by the U.S. Food and
Drug Administration (FDA) advising of the possible adverse effects associated
with consumption of kava preparations (Food and Drug Administration, 2002;
Center for Disease Control and Prevention, 2003). Cases involving kava extract
ingestion reported patients with greatly heightened AST and ALT, liver necrosis,
cholestatic hepatitis, lobular hepatitis, acute liver failure and liver cirrhosis, with
some cases resulting in fulminant hepatic failure, liver transplantation and death
(Denham et al., 2002; Schmidt, 2002; Gow et aI., 2003; Humberston et aI., 2003;
Stickel et al., 2003). Interestingly, this severe liver injury associated with kava
extracts has not been seen in the Pacific Islands with traditional kava use, even at
much higher doses than consumed in the West (Denham et aI., 2002; Moulds and
Malani, 2003). Moreover, there were many confounding factors that could have
contributed to the toxicity, making it difficult to form a direct link between kava
and severe liver toxicity.
5
None of the five cases in which kava was linked to a fatal outcome, exhibit
an absolute causal relationship, as these individuals either had pre-existing liver
conditions or consumed other medications that likely contributed to the observed
toxicity (Schmidt, 2002). Thirteen reported cases required liver transplantation as
a consequence of kava extract consumption, but the effects may have been
attributable to previous conditions or other medications (Schmidt, 2002). In
addition, many of the less severe case reports are inconclusive due to lack of
evidence. Overall, causality of liver toxicity from kava extracts and the need for a
ban are still highly controversial (Moulds and Malani, 2003). The interpretation of
the toxicity is difficult as it is likely caused by a combination of factors rather than
a single factor.
Kavalactones (KL)
Steinmetz (1960) was the first to point out that the psychoactive properties
of kava extracts are highly dependent on the dissociation of the resinous
components of the root into the extraction solution. It was later found that these
resinous components of the kava plant are in fact the active ingredients, called
kavalactones or styryl a-pyrones. Kavalactones are lipophilic compounds that
make up 3-20% of the dry root weight depending on the age of the plant (Lebot
and Uevesque, 1989). Fifteen kavalactones have been isolated and together make
6
up approximately 96% of the lipid soluble fraction of extracts (Lebot and
Laevesque, 1989). These compounds are thought to be responsible for kava's
physiological effects, with the following six being the most important: kavain (K),
dihydrokavain (DHK), methysticin (M), dihydromethysticin (DHM), yangonin (Y)
and desmethoxyyangonin (DMY) (Lebot et al., 1997). A half a coconut shell of
kava infusion, containing about 1-1.5 g of resin, is said to be strong enough to
induce "deep" and "dreamless" sleep (Lebot and Cabalion, 1988). The strength of
the kava drink varies· considerably, depending on the plant age, cultivar and
extraction method.
Metabolism and Pharmacokinetics
In vitro studies have reported that the parent compounds of kava are
responsible for its cellular effects rather than activated metabolites (Klohs, 1967;
Zou et al., 2004a). Interestingly, individual kavalactones do not produce the same
physiological effects as whole kava extracts. Studies have shown that mixtures of
kavalactones work synergistically to produce a greater psychoactive outcome
(Steinmetz, 1960).
A recent study by Mathews et al. (2005) in rats provides evidence that drug
interaction may cause a decreased plasma clearance of kava. They reported that
7
kavain, co-administered with kava extract, resulted in a marked increase in plasma
concentration and half-life as compared to kavain alone (Mathews et al., 2005).
Kava-drug interactions
In a study performed in the Arnhem Land Aboriginal communities in
Australia, there was a high prevalence of co-ingestion of drugs with kava, where
82% of current kava users smoked tobacco, 45% drank alcohol, 37% smoked
marijuana and 37% previously sniffed petrol (Clough et al., 2003b). This multi
substance use is also highly prevalent in the Western world. Overall, there is a
lack of knowledge about the possible drug interactions that can occur with herbal
remedies as many of them are taken as a concoction of herbs, often in combination
with other drugs. Patients over 50 years of age are typically diagnosed with one
chronic disease every decade which requires long term drug treatment (Bressler,
2005). This means the elderly are usually taking several drugs concomitantly to
control their health conditions. In the U.S., the use of complementary and
alternative medications including many herbs has steadily increased over the past
few decades, reaching 36% in 2002 (Bressler, 2005). Kava is widely used in the
Western world as a sleep aid and for treatment of anxiety and nervous disorders
(Wheatley, 2001). Kava is thought to act on gamma-amino-butyric acid (GABA)
receptors making its anxiolytic and sedative effects an indirect one (Wheatley,
8
2001). Consequently, it has been suspected that benzodiazepines, which bind
GABA receptor, may have an additive or synergistic effect with kava. Interactions
have aIso been reported with aIcohol, barbiturates, narcotics, anesthetics and
anaIgesics (Bressler, 2005). A recent review concluded that kava has a propensity
to cause pharmacokinetic and pharmacodynamic interactions with other drugs that
may lead to adverse effects (Anke and Ramzan, 2004)
Cytochrome P450 (CYP450) enzymes
Cytochrome P450 enzymes, responsible for the metabolism of over 90% of
therapeutic drugs (Mathews et aI., 2002), have been shown to be strongly inhibited
by kavalactones in human liver microsomes and recombinant CYP450 proteins
(Mathews et aI., 2002; Unger et aI., 2002; Zou et aI., 2002; Zou et aI., 2004b).
Inhibition of the most important CYP450 isoforms, specifically CYP 2C9, 2C19,
3A4, 2D6, 4A9/11 and lA2A, was observed at kavaIactone concentrations between
10-1001lM (Mathews et aI., 2002; Unger et aI., 2002; Zou et aI., 2002). These
studies suggest that interactions between kava and other drugs may occur through
CYP450 enzymes.
A case study also reported that a poor-metabolizer phenotype in the
CYP2D6 enzyme may be a risk factor in kava-associated liver injury (Russmann et
aI.,2001). Interestingly, this deficiency occurs in 10% of Europeans while Pacific
9
Islanders lack this genetic defect. This suggests a possible genetic factor that can
lead to a susceptibility to a toxic response (Wanwimolruk et aI., 1998).
Differences in extmction methods
In recent years, kava extracts and preparations have been widely available in
U.S. and European markets. Unlike traditional beverages prepared by aqueous
extraction methods, commercial preparations were usually solvent-based extracts
standardized for kavalactone content (Strahl et aI., 1998; Escher et aI., 2001; Kraft
et aI., 2001; Campo et aI., 2002). Ethanol (>60%) or acetone (>60%) at different
concentrations have been commonly used for preparations depending on the
desired extract potency (Whitton et aI., 2003). The extracts obtained contain much
higher concentrations of the lipophilic components of the kava plant, with
kavalactone content between 30-70%. It has been hypothesized that extraction
techniques using less polar solvents may result in a much higher proportion of
kavalactones and other lipophilic components extracted from the plant as
compared to protective compounds such as glutathione (Whitton et aI., 2003).
Traditional extracts have a kavalactone:glutathione ratio of 1:1 whereas
commercial standardized extracts contain no glutathione and have 30-fold higher
kavalactone concentrations (Whitton et aI., 2003). The enhanced concentration of
kavalactones with simultaneous decrease in phase II detoxification enzyme
cofactor glutathione may produce an extract capable of causing functional changes
10
in the liver (Whitton et al., 2003). A recent in vitro study reported that kava
fractions extracted using more polar solvents had greater cytotoxic effects (Jhoo et
aI., 2006). Thus, the differences in commercial extraction methods compared to
traditional extraction methods may be factor in the toxicity observed with kava
extracts.
Kava toxicity and dosage
In vitro studies on the toxic effects of kavalactones and kava extracts using
human and rat hepatocytes have reported ECso values that were much higher than
levels that would be expected in human plasma after kava ingestion (Teschke et al.,
2003). Between 60-240 mg of kavalactones (KL) per day is the dosage
recommended for kava use (Humberston et al., 2003; Clouatre, 2004). Assuming
the average human weighs 70 kg, the dosage range would be the equivalent of 1-4
mg of KL/kg body weight/day (Russmann et al., 2005). Thus far, animal studies
testing the effects of kavalactones and kava extract on liver function and health at
much higher doses than would have been ingested by humans have failed to cause
toxicity (Singh and Devkota, 2003) (National Toxicology Program, 2005).
Although rodents have a faster metabolism than humans, studies in mice and rats
have exceeded the theoretical dosage of 10-40 mg KL/kg body weight/day,
factoring in an allometric scaling of lO-fold. Traditional aqueous extracts
11
administered at daily doses of up to 500 mg KLlkg body weight/day to Sprague
Dawley rats for 4 weeks had no effect on markers of liver injury (Singh and
Devkota, 2003). Serum enzymes aspartate aminotransferase (AST), ALT, ALP
and lactate dehyrogenase (LDH) were not elevated nor were hepatic
malondialdehyde (MDA) levels (Singh and Devkota, 2003). The National
Toxicology Program (2005) has conducted 2 and 13-week studies in mice and rats
testing the effects of kava extracts at doses of up to 2.0 g/kg/day. Some mortality
was observed at doses of 1.0 and 2.0 g/kglday, mostly during the first week.
CentriIobuiar and hepatocyte hypertrophy were also observed at doses of 1.0 and
2.0 g/kglday, however, no hepatic necrosis was seen (National Toxicology
Program, 2005). The effects ranged from minimal to mild and lacked the severe
liver toxicity that was associated with kava consumption in humans (National
Toxicology Program, 2005). Based on these studies, it is unlikely that
kavalactones alone are the cause of the severe liver damage seen in humans.
The liver and hepatotoxicity
The liver, which is the largest internal organ in the body, plays a major role
in digestion, metabolism, glycogen storage and xenobiotic detoxification (Webb,
1999; Baynes and Dominiczak, 2005). It is involved in many of the biochemical
pathways required for growth, fighting diseases, providing energy and even
12
reproduction (Baynes and Dominiczak, 2005). Liver cells, or hepatocytes, perform
thousands of biochemical reactions every second to perform all of its required
functions (Webb, 1999). Since the liver is involved with so many biochemical
processes it is not surprising that there are numerous different diseases that will
affect it.
Drug detoxification involves phase I or first-pass metabolism and phase II or
conjugation. Phase I involves the conversion of the inactive drug to an active
metabolite via the cytochrome P450 enzymes (Webb, 1999). The active metabolite
circulates through the blood stream and elicits an effect on the body (Webb, 1999).
Phase II metabolism converts the active metabolite to a more water soluble
product, rendering it is easier to be taken up by the kidneys and excreted through
the urine (Webb, 1999). The dysfunction or overload of this drug metabolizing
system in the liver can lead to chemical stress within hepatocytes leading to
progression towards the diseased state (Webb, 1999). Oxidative stress and
subsequent mitochondrial dysfunction in hepatocytes are thought to be critical in
the early progression of chemical-induced liver injury (Lemasters and Nieminen,
2001; Caro and Cederbaum, 2004; Van Houten et al., 2006).
13
Oxidative stress and mitochondria
Tissue homeostasis and organ health rely on the tightly regulated elimination
of damaged cells through apoptosis or programmed cell death (Kroemer and Reed,
2000). When there is too much cell death over time, organ damage and failure can
occur. The mechanisms underlying the progression of cell death leading to a
diseased state greatly relies on the mitochondria and the selective mitochondrial
permeability transition (MPT) (Kroemer and Reed, 2000). Mitochondria are
thought to be the earliest targets of cell stress and are responsible for signaling the
cell to undergo apoptosis or necrosis (Kroemer et aI., 1998; Bras et al., 2005).
Xenobiotic exposure can lead to an increased production of reactive oxygen
species (ROS) within the cell resulting in oxidative damage to cellular proteins,
DNA and lipids (Van Houten et al., 2006).
Importance of kava research
In the United States, kava tinctures, extract capsules, teas and dried powders
continue to be sold in health food stores, ethnic markets and kava "cafe" regardless
of a consumer advisory issued by the Food and Drug Administration (FDA)
concerning the potential hepatotoxicity of kava products. Therefore, it is critical to
delineate any possible adverse side effects that could cause toxicity for the safety
of the kava-consuming population. Kava cultivation and export has also been a
14
significant part of the economy of many Pacific Island nations. Therefore, it is
essential to determine kava toxicology to revitalize the kava market and aid the
agriculture economy in the Pacific region. Kava is also a natural alternative to
highly addictive, prescription anti-anxiety medications. It is possible that
continuation of this kava ban could cause greater harm than benefit, as it creates a
therapeutic gap between alternative and prescription medications.
Hypothesis and objectives
Although the kava drink is traditionally prepared from the roots and
rhizome, industrial preparations have included stem peelings into the raw material,
due to easy availability and high demand by pharmaceutical companies (Dragull et
aI., 2003). A higher presence of kava alkaloids such as pipermethystine (PM) has
been demonstrated in the above-ground organs such as stem peelings and leaves as
compared to roots and rhizome (Dragull et al., 2003) and is more toxic to human
hepatoma cells, HepG2, than KL, the physiologically active components of kava
(Nerurkar et al., 2004). Studies in our laboratory have demonstrated that in vitro
toxicity of PM was associated with a significant increase in the production of
reactive oxygen species (ROS), loss of mitochondrial membrane potential,
reduction in cellular ATP levels and ultimately cell death (Nerurkar et al., 2004).
Studies thus far have failed to conclusively link kavalactones and kava extracts to
15
the liver toxicity seen in humans. Based on these observations, we hypothesize
that a synergistic interaction between PM and kava contributed to the liver toxicity
seen in humans: Therefore, the objective of this research was to determine the
effects of the alkaloid pipermethystine, solvent-based kava rhizome extract, stem
peeling extract and kavalactones, in combination or individually, using in vitro and
in vivo models. Specifically, human hepatoma cell line HepG2, C57BL6 mice and
F-344 rats were used to delineate any possible adverse effects of kava on the liver.
16
CHAPTER 2
EFFECTS OF KAVA ACETONIC EXTRACT, KAVA ALKALOID
PIPERMETHYSTINE AND KA V ALACTONES DIHYROMETHYSTICIN
AND DESMETHOXYYANGONIN ON THE HUMAN
HEPATOCELLULAR CARCINOMA CELL LINE, HEPG2
Introduction
Previous studies in our laboratory have shown that PM is associated with a
significant increase in the production of reactive oxygen species (ROS), loss of
mitochondrial membrane potential, reduction in cellular ATP levels and significant
cell death after 24 h of treatment (Nerurkar et al., 2004). However, nothing is
known about the initial effects or synergistic interactions between PM and
kavalactones. Therefore, the aim of this study was to determine the early effects of
PM, kavalactones and kava rhizome extract, individually and in combination, on
markers of oxidative stress, mitochondrial dysfunction and cell death.
Results
The recommended dosage of kava extracts for humans have been reported
between 60 and 240 mg kavalactones/day. Assuming complete oral absorption of
all kavalactones and a theoretical blood volume of 6 L, the plasma concentration of
17
kavalactones would be in the range of 40-160 ,uM. Previous experiments done in
our laboratory established that concentrations below 1 .aM failed to cause
significant changes in HepG2 cell function or health. Earlier studies on PM have
obtained data after 24 and 48 h of treatment. Therefore, concentrations between 1-
200,uM were chosen for treatment of HepG2 cells with of DHM, DMY, PM and
KRE at time points between 1 and 24h.
Effects of PM, DMY, DHM and KRE on cytotoxicity. Lower
concentrations of PM (1-25 ,uM) had no effect on cell death of HepG2 cells, as
measured by the presence of LDH in the culture medium, a marker of cell
membrane integrity. However, treatment of cells with 50,uM concentrations of PM
exhibited a 200% and 1000% increase in LDH at 6 and 24 h, respectively (Figure
2.1, p<0.05). In contrast to PM, treatment with kavalactones DMY and DHM at
concentrations of 50,uM failed to cause any changes in LDH leakage at both 6 and
24 hr time points. DMY and DHM produced only a slight increase in LDH at the
highest concentration tested (100 ,uM) of 120% and 140%, respectively (Figure
2.2, 2.3, p<0.05). Similar results were obtained from the whole kava rhizome
extract, where only the higher concentrations of KRE (100, 200,uM) caused an
increase in LDH leakage (130% and 140%, respectively) after 24 h of treatment
(Figure 2.4, p<0.05). A subsequent study investigated the synergistic interaction
between sub-toxic levels of kavalactones DMY and DHM with PM by
18
measurement of LOH released into the culture media. Individual treatment of
OMY (100uM), OHM (100,uM) and PM (25,uM) caused a 150-200% increase in
LOH release as compared to the controls after 24h of treatment (Figure 2.5). The
combination of OMY and OHM displayed LDH levels similar to individual
treatment (200%), however, the addition of 25 ,uM PM further increased in LDH
release by 200% (400% increase total).
Effects of PM, DMY, DHM and KRE on markers of mitochondrial
dysfunction. The molecular mechanisms of cell death induced by PM were further
examined by cell based assays measuring reactive oxygen species production,
mitochondrial membrane potential and total cellular ATP. The effects of OMY,
OHM and KRE were also tested for these common markers of mitochondrial
dysfunction. The highest concentrations of kavalactones (KL) tested had no effect
on ROS production and mitochondrial membrane potential after 1, 2, 3, 4 and 6 h
of treatment (Figure 2.6, 2.7). However, 50,uM PM caused a significant increase in
cellular ROS production by 150% at 1hr and 300% at 6h, as compared to the
control (Figure 2.6, p<0.05). Mitochondrial membrane potential simultaneously
decreased over time with 50,uM PM treatment, showing a 25% and 65% decrease
after 2 and 4 h, respectively (Figure 2.7, p<0.05). In addition, total cellular ATP
showed a significant decrease (25%) after 6 h of treatment with 50,uM PM (Figure
2.8, p<0.05). Interestingly, 100,uM concentrations of KRE caused a 35% and 40%
19
decrease in total cellular ATP after 3 and 6 h of treatment, respectively (Figure 2.9,
p<0.05).
Effects of Cyclosporin A on PM induced cytotoxicity. Reactive oxygen
species are one of many agents that cause the mitochondrial permeability transition
(MPT). The opening of permeability transition pores leads to mitochondrial
depolarization and ATP depletion, which can eventually lead to cell death. Since
PM significantly altered these markers associated with the MPT, we examined
whether the PT pore inhibitor Cyc\osporin A (CsA) could ameliorate cell death
caused by PM. Figure 2.9 shows that CsA (l,uM) co-treatment reduced PM (50
,uM) induced LDH leakage by about 50% (p<0.05) after 24 h of treatment.
Effects of PM on protein expression of cytochrome c, AlF and HNE
The translocation of cytochrome c from the mitochondria into the cytosol
and increases in cytosolic AlF are common marker of apoptosis; therefore,
cytochrome c and AlF protein expression was determined for both mjtochondrial
and cytosolic fractions. Treatment of HepG2 with 50,uM PM for 24h, failed to
induce the any changes in cytochrome c and AlF expression (data not shown).
HNE protein levels, which are indicative of oxidative stress, were also unaffected
by PM treatment after 24 h (data not shown).
20
(A)
g 250
8 200
'0 ~ 150 Q)
:a' 100
m 50 J: 9
6hr
*
O~~~~~~~~~~-r-L~~~~----~~~ Untreated DMSO 1 10 25 50
Pipermethystlne (tIM)
(8) 1400 24hr * = .g 1200
c 8 1000 '0 ~ 800 Q)
I 600
400 .!!l J: 9 200
0 Untreated DMSO 1 10 25 50
Pipermethystine (tIM)
Figure 2.1. Cytotoxicity assay for pipermethystine (PM). HepG2 cells were treated with PM at concentrations up to 50pM. The levels of lactate dehydrogenase were assayed in the culture media after 6h (A) and 24h (B) incubations with PM. Each value represents the mean + SD from one experiment done in triplicate, n=3. Data is represented as a percentage of the untreated control; *p < 0.05.
21
(A) 120
""" o g100 8 '0 80 ~ CD 60 i 1 40
9 20
o 4--J= lntreated
(8)
& 80
i 60 - 40 J:
::I 20
o +--''-'" Untreated DMSO
Shr
Desmethoxyyangonln (l./M)
24hr
*
1 10 25 50 100 Desmethoxyyangonln {J.tM)
Figure 2.2. Cytotoxicity assay for desmethoxyyangonin (DMY). HepG2 cells were treated with DMY at concentrations up to 100J.lM. The levels of lactate dehydrogenase were assayed in the culture media after 6h (A) and 24h (8) incubations with DMY. Each value represents the mean + SD from one experiment done in triplicate, n=3. Data is represented as a percentage of the untreated control; *p < 0.05.
22
(A)
140 '0 ~ 120
~ 100
~ 80 III
fi 60 ~ ~ 40 J:
:3 20
c::o
o
(B)
140
~ 120
~ 100
~ 80
f 60
40 J:
:3 20
o
6hr
Untreated DMSO 1 10 25 50 100
Dlhydroxymethysticln (.11M)
24hr *
Dlhydroxymethystlcln (.11M)
Figure 2.3. Cytotoxicity assay for dihydromethysticin (OHM). HepG2 cells were treated with OHM at concentrations up to 100JIM. The levels of lactate dehydrogenase were assayed in the culture media after 6h (A) and 24h (B) incubations with OHM. Each value represents the mean ± SO from one experiment done in triplicate, n=3. Data is represented as a percentage of the untreated control; *p < 0.05.
23
(A)
o Untreated DMSO 10
(B)
160
0' 140 .::. 8 120
'5 100
~ 80
f 60
:r: 40
:3 20
o
6hr
25 50 100 200
Kava rhizome extract (PM)
24hr *
Kava rhizome extract (PM)
Figure 2.4. Cytotoxicity assay for kava rhizome extract (KRE). HepG2 cells were treated with KRE at concentrations up to 200,uM. The levels of lactate dehydrogenase were assayed in the culture media after 6h (A) and 24h (8) incubations with KRE. Each value represents the mean + SD from one experiment done in triplicate, n=3. Data is represented as a percentage of the untreated control; *p < 0.05.
24
600
==- 500
~ 8 400 '0 ~ 300 Q)
~ 1 200
::c 9 100
*
* *
Untreated DMSO DMY100 DHM100 PM25 DMY100 DMY100 +DHM100 +DHM100
+PM25
Figure 2.5. Cytotoxicity assay for combination treatment of desmethoxyyangonin (OMy), dihydromethysticin (OHM) and pipermethystine (PM). HepG2 cells were treated with different combinations of PM, OMY and OHM at concentrations up to 100pM. The levels of lactate dehydrogenase were assayed in the culture media after 24h incubations with OMY, OHM and PM. Each value represents the mean + SO from one experiment done in triplicate, n=3. Oata is represented as a percentage of the untreated control; *p < 0.05.
25
400
.:-~ 350
8 '5 300
fP. ~ 250 c o
g 200
~ Q. 150 en o a: 100
50
*
•
--control --KL200 --&- PM50
* *
* *
; I
O+-------,-------,-------,-------,-------~ 1 hr 2hr 3hr 4hr Shr
Figure 2.6. Reactive oxygen species (ROS) production for pipermethystine (PM) and kava lactones DMY and OHM (KL) in HepG2. ROS production was measured in HepG2 treated with pipermethystine (50 JIM) and kava lactones (200JIM) for 1, 2, 3, 4, 6 hrs. Each value represents the mean + SO from 3 separate experiments, n=9. Data is represented as a percentage of the untreated control; *p < 0.05.
26
-+- control --KL50 --.- PM50
140
~ 120 Ql '0 c.
100 Ql
faa * ........
..01: 80 ~ 8
Eo * iiI"g .~ .e- 60 '0 c: 0 .c 40 0 0 :t: ~
20
0 1hr 2hr 3hr 4hr 6hr
Figure 2.7. Mitochondrial membrane potential for pipermethystine (PM) and kava lactones DMY and DHM (KL) in HepG2 . Mitochondrial membrane potential was measured in HepG2 treated with pipermethystine (50 tIM) and kava lactones (50 tIM) for 1, 2, 3, 4, 6 hrs. Each value represents the mean ± SD from 3 separate experiments, n=9. Data is represented as a percentage of the untreated control; *p < 0.05.
27
• control • KRE100 £!I PM50
140
120
~
(5 100 ~ -c 0 '-' - 80 0
~ (/) 60 Q) > ~ CL 40 ~
20
0
3hr Shr
Figure 2.8. Total cellular ATP for kava rhizome extract (KRE) and pipermethystine (PM) treated HepG2 cells . Total ATP was measured in HepG2 treated with KRE (100J.1M) and PM (50J.1M) for 3 and 6 hrs. Each value represents the mean ± SO from 2 separate experiments, n=6. Data is represented as a percentage of the untreated control; *p < 0.05.
28
1600 II Cyclosporin A II OMSO
1400 ~
(5 ~
1200 .... c 0 <.> ..... 1000 0
~ Q) 800 Cl «l .:.: 600 «l .!!1 I 400 0 --I
200
0 Pipermethystine (11M) 0 25 50
Cyclosporine (1J1M) + + +
Figure 2.9. Attenuation of pipermethystine (PM)-induced cytotoxicity by Gyclosporin A (GsA). HepG2 cells were treated with 0, 25 and 50J.1M pipermethystine in the presence or absence of 1J.1M Gsa for 24 hours and the levels of lactate dehydrogenase were assayed in the culture media. Each value represents the mean + SO from one experiment done in triplicate, n=3. Data is represented as a percentage of the untreated control.
29
Discussion
Since kava-associated liver toxicity surfaced in the late 1990's, many studies
have been conducted to determine the mechanisms of this recent phenomenon. As
discussed earlier, differences in extraction methods and Western usage of kava
preparations compared to traditional customs as well as genetic polymorphisms,
drug interactions and health compromised states in kava-consuming individuals,
may all have contributed to this rare liver toxicity.
Traditional kava beverages were prepared exclusively using kava roots and
rhizome while above ground portions (stems, branches, leaves and flowers) were
mainly used in folk medicine and topical application (Dragull et al., 2003). In
recent years, due to the heightened demand for kava raw material, it is known that
stem peelings (bark) were being sold to pharmaceutical companies for
incorporation into kava preparations (Dentali, 1997; Dragull et al., 2003). It has
been reported that kava leaf extracts have a higher binding inhibition of eNS
receptors in vitro compared to root extracts (Dinh et al., 2001). It was also found
that above ground portions of the kava plant contain higher concentrations of
alkaloids such as PM in relation to the roots (Smith, 1979; Dragull et al., 2003).
The effects of piperdine alkaloids on humans are unknown, however, pyridine
30
alkaloids with similar structures have been reported to cause cytotoxic effects (Duh
et al., 1990).
A recent study compared the effects of the kava alkaloid PM to kavalactones
DHM and DMY (Nerurkar et aI., 2004). Treatment with 50,uM PM in hepatoma
cell line HepG2 resulted in significant mitochondrial dysfunction, caspase-3
release and cell death within 24 h of treatment (Nerurkar et aI., 2004). The lack of
toxicity observed for kavalactones, even at higher concentrations and longer
incubations, suggests that PM is more likely to have contributed to the severe liver
toxicity seen in humans.
. Oxidant-induced damage to proteins, DNA or lipid membranes is a major
cause of dysfunction in mitochondrial bioenergetics (Van Houten et al., 2006). We
observed that the toxicity seen with PM resulted in markedly increased ROS
production after 1 h of treatment. In addition, PM caused a decrease in
mitochondrial membrane potential after 2 h and total cellular ATP reductions after
6 h of treatment. The changes observed are common events of MPT which is a key
step in the progression towards cell death (Kroemer and Reed, 2000). Moreover,
MPT pore inhibitor, Cyclosporin A, attenuated PM induced cell death by 50%.
This study corroborates previous studies showing that PM is considerably
more toxic than kavaIactones in HepG2 cells. It must be noted that kavalactones
31
showed a slight toxic effect at the higher concentrations. An additive toxicity was
also observed with PM and kavalactones combination treatment. Therefore,
further studies should address the possibility of a synergistic or additive toxic
effect between kava alkaloids and lactones as a possible cause for liver toxicity
seen in humans.
32
CHAPTER 3
LONG TERM EFFECTS OF KA VA ALKALOID PIPERMETHYSTINE
AND KAVA STEM PEELING EXTRACT ON C57BL6 MICE
Introduction
As discussed earlier, stem peelings of the kava plant, known to contain high
concentrations of the cytotoxic alkaloid Pipermethystine (PM), were incorporated
into preparations during the height of the demand for kava from pharmaceutical
companies. Therefore, the aim of this study was to test the effects of PM and kava
stem peeling extracts in C57BL6 mice to delineate any adverse effects on the liver
function.
Results
Effects of PM and KSPE on body weight, feed intake and liver weight.
The relative changes in body weight, feed intake and liver weight were monitored
during the course of the treatment period as a sign of liver toxicity. Body weight
gain and feed intake was 10-15% lower in the vehicle control, PM, KSPE and
PM+KPSE groups as compared to the untreated control; however, these changes
were likely due to the handling and administration of treatments (Figure 3.1, 3.2).
33
Interestingly, there was an increase in liver weight in the PM+KSPE treatment
group as compared to the controls (Figure 3.3).
Effects of PM and KSPE on serum AST, ALT and WH. At the time of
sacrifice, activities of AST, ALT and LDR were determined in C57BL6 mouse
serum as a marker of liver injury. Treatments of PM, KSPE and PM+KSPE failed
to significantly change AST, ALT and LDR levels in all treatment groups as
compared to the controls (Figure 3.4, 3.5).
Effects of PM and KSPE on hepatic triglyceride levels. As shown in
Figure 3.6, the total hepatic triglyceride levels in all treatment groups were
unchanged as compared to the controls.
Effects of PM and KSPE on CYP2EI expression. Microsomal protein
expression of the enzyme CYP2El showed no significant change in all treatments
as compared to the controls (data not shown), although there was a slight induction
in the PM+KSPE treatment group.
34
--untreated --- vehicle --.- PM --- KSPE --- PM + KSPE
32
30 § 1: OJ .~ 28
i CD 26
24
o 12 16 20 26 33 40 47 55 62 68 75
Day
Figure 3.1. Weekly body weight change of pipermethystine (PM) and Kava stem peeling extract (KSPE) treated C57BL6 mice. PM (2mg/kg) and KSPE (8.75 mg/kg), individually and in combination, was orally administered daily for 10 weeks. Body weight was measured every 6-7 days except for the first two weeks.
35
~.~ .. ::-.~. untreated --vehicle
5.5
5 :§
4
PM KSPE-iII~ PM + KSPE
........ "'y
o 12 16 20 26 33 40 47 55 62 68 75
Day
Figure 3.2. Weekly feed intake of pipermethystine (PM) and kava stem peeling extract (KSPE) treated C57BL6 mice. PM (2mg/kg) and KSPE (8.75 mg/kg), individually and in combination, was orally administered daily for 10 weeks. Feed intake was measure every 6-7 days except for the first two weeks.
36
~
Cl ~
1: Cl 'iii == ... CD
:3
50
45
40
35
30 untreated vehicle PM KSPE PM+ KSPE
Treatment
Figure 3.3. Final liver weight of Pipermethystine (PM) and kava stem peeling extract (KSPE) treated C57BL6 mice. PM (2mg/kg) and KSPE (8.75 mg/kg), individually and in combination, was orally administered daily for 10 weeks. Liver weight was measured at sacrifice. Values are expressed as mean ± standard deviation (n=5).
37
250 (A)
200
~ 150 ::J ~
t; <C
100
50
o untreated
150 (8)
120
vehicle PM Treatment
KSPE PM + KSPE
~ 2. 90
!:i <C 60
30
o untreated vehicle PM KSPE PM + KSPE
Treatment
Figure 3.4. Serum aspartate aminotransferase (AS1) and alanine aminotransferase (AL 1) levels of PM and KSPE treated C578L6 mice. PM (2mg/kg) and KSPE (8.75 mg/kg), individually and in combination, was orally administered daily for 10 weeks. Serum activity of AST (A) and AL T (8) were assay after sacrifice. Values are expressed as mean ± standard deviation (n=5).
38
150
""'" 120
~ 8 'l5 90 <;e. ~
~ ~ 60 :c 9 E 2 30 CD en
untreated vehicle PM KSPE PM + KSPE
Treatment
Figure 3.5. Serum lactate dehydrogenase (LDH) levels of pipermethystine (PM) and kava stem peeling extract (KSPE) treated C57BL6 mice. PM (2mglkg) and KSPE (S.75 mglkg), individually and in combination, was orally administered daily for 10 weeks. Serum activity of LDH was assayed after sacrifice. Values are expressed as mean + standard deviation (n=5).
39
2 ~ .E-m "C '1:
~ ~ '1: -.Y 1i1 c. CD
::c
60
45
30
15
o untreated vehicle PM KSPE PM+ KSPE
Treatment
Figure 3.S. Hepatic triglyceride levels of piperrnethystine (PM) and kava stem peeling extract (KSPE) treated C57BLS mice. PM (2mg/kg) and KSPE (8.75 mg/kg), individually and in combination, was orally administered daily for 10 weeks. Total lipids were extracted from livers and assayed for triglyceride levels. Values are expressed as mean + standard deviation (n=5).
40
Discussion
The present experimental results obtained from PM and KSPE treatment of
C57BL6 mice failed to reproduce the effects observed previously using an in vitro
model. Although PM caused cytotoxic effects in human hepatocellular carcinoma
cell line HepG2, lO-week treatment in mice failed to induce changes in all markers
tested for liver injury and function. In addition, KSPE failed to induce any liver
dysfunction, even in combination with PM.
It is important to note that nothing is known about the phamacokinetics and
pharmacodynamics of PM or the kava stem peeling extract. There is no
information on the expected plasma levels of PM in someone who ingested
extracts contaminated with stem peelings. Therefore, the doses chosen were based
on theoretical plasma levels that would be expected. It may be possible that
differences between human and mouse metabolic rates may have contributed to a
faster detoxification and elimination of PM from the body. Hence, the doses tested
were too low to induce a toxic response.
Finally, the treatments were administered in coconut milk as a carrier for the
lipophilic kava stem peeling extracts and PM. However, studies have shown that
saturated fat consumption exhibits hepatoprotective effect against alcoholic liver
41
disease (Ronis et aI., 2004). Therefore, the lack of toxicity may be in part due to
amelioration of PM and KSPE induced toxicity by the high saturated fat content of
the coconut milk vehicle used.
42
CHAYI'ER4
SHORT TERM EFFECTS OF KAVA ALKALOID PIPERMETHYSTINE
AND KAVA RHIZOME EXTRACT IN F·344 RATS
Introduction
We have demonstrated that in vitro toxicity of pipermethystine (PM) was
associated with a significant increase in the production of ROS, loss of
mitochondrial membrane potential and reduced cellular ATP levels leading to cell
death. In addition, there was an additive toxic effect between PM and KL.
Therefore, the aim of this study was to determine whether short term
administration of PM and kava rhizome extract (KRE) could synergistically affect
hepatic mitochondrial function and drug metabolism, in vivo.
Results
Effects of PM and KRE on markers of liver injury. Overall, PM and KRE
had no effect on body weight, daily food intake and liver weight as compared to
the vehicle control rats (Table 1). Although, rats in all treatment groups lost 20·
25g of body weight, this was probably due to gavage feeding of treatments (Table
1). PM, alone or in combination with KRE, failed to elicit any changes in liver
enzymes such as AST and ALT, as compared to vehicle control group (Table 2).
43
Total hepatic ATP levels showed a slight increasing trend in all treatment groups;
however, the change was not significant (Figure 4.1).
Antioxidant status and oxidative stress. The antioxidant reduced
glutathione (GSH) showed an insignificant increase in all treatment groups, and to
a greater extent in the combination treatment (Figure 4.2A). Cytosolic (Cu/Zn
SOD) and mitochondrial (Mn-SOD) superoxide dismutase (SOD) activity
significantly increased with both PM and PM+KRE treatments as compared to
controls (p<O.05, Figure 4.2B and 4.2C, respectively), which may be a protective
response to increased ROS production. However, hepatic levels of lipid
peroxidation markers MDA and HNE were unchanged in all treatments indicating
a lack of oxidative stress (Figure 4.2D). In addition, aconitase activity, which is
considered a sensitive marker of oxidative stress, was unaffected by PM and KRE
(Figure 4.3).
Effects of PM and KRE on mRNA expression of Bcl-2, Bax and TNF
alpha.
Treatment with PM and KRE failed to cause changes in the hepatic mRNA
expression of anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax as
compared to the control (data not shown). An increase in TNF-alpha expression
was seen with all treatments but only the PM group reached significance (p<O.05).
44
Hepatic protein expression of UCP-2. Based on previous studies where
ATP levels were modulated by PM and KRE, UCP-2 mitochondrial protein
expression was determined. Figure 4.5 indicates that KRE treatment reduces
hepatic protein levels of UCP-2 by about 20% but due to variability between
groups, the changes were not significant.
Effects of PM and KRE on drug metabolizing enzymes, CYP450. Figure
4.6 represents the changes in hepatic microsomal CYP450 protein levels in rats
treated with PM and KL. PM and KL both increased hepatic CYPIA2 protein
levels, however, increases were significant only in the KL receiving animals
(p<0.05, Figure 4.6A). Hepatic CYP2El and CYP2D6 protein expression were
non-significantly increased in all treatment groups, while CYP3A4 protein levels
remained unchanged (Figure 4.6A, B, C).
45
TABLE 1
Body weight, Feed Intake and Liver weight changes for Pipermethystine (PM) and Kava
Group
Control Pipermethystine Kava Rhizome Extract PM + KRE
rhizome extract (KRE) treated F-344 rats
Body Weight Initial (g)
346.6±17.0 348.6±16.9 361.4±9.0 347.8+11.6
Final (g) 322.8±19.8 321.2±24.6 323.2±31.7 324.4+20.5
Feed Intake (g(day)
11.44±3.51 1O.84±2.18 1O.16±3.19 11.58+1.47
Liver weight (mg(gBW) 36.01±5.87 35.14±2.61 36.84±3.02 37.86+4.65
Values are expressed as means ±S.D. for five rats in each treatment group.
TABLE 2
Effect of Pipermethystine (PM) and Kava root extract (KRE) on
Group
Control Pipermethystine Kava Rhizome Extract KRE+PM
hepatic injury markers in rat serum
AST(U/L)
100.6±105
96.4±15.9
102.8±27.2 108.2+36.9
ALT(U/L)
78.2±21.5 72.8±4.2
62.6±5.6
58.8+8.8 Values are expressed as means ±S.D. for five rats in each treatment group.
46
::::::-e -c: 0 (,)
'0 #. -
.ATP IlEADP 160
140
120
100
80
60
40
20
0 Control PM KRE PM+KRE
Figure 4.1. Total hepatic ATP and ADP levels in F-344 rats treated with doses of PM (10mg/kg) and/or KRE (100mg/kg) for 11 days. The bar graph represents ATP and ADP values expressed as % of control. Values are expressed as the mean + S.D for 5 rats in each treatment group.
47
120 (A) (B) • 0.25
:;:- f:g- 0.2 .<: .2' 90
ffi~ ",l!' · . ., Cl'5 · . C ~ 0.15 · . 0'5 lii ~ 60 · .
cn~ ~,g C", 0.1
0 :liE E 30 ~ 2 0.05 a.
0 · . 0 Control PM KRE PM+KRE Control PM KRE PM+KRE
(C) (0) B • ~ * 4
:fi~ :;:-.<: tIlt 6 < l!' 3 C'a; C~ O~ :li'5 ".ll 4 lii ~ 2
~'" ~,g o.€. 2 0
~::J E 1 .,~ a. ~ 0 0
Control PM KRE PM+KRE Control PM KRE PM+KRE
Figure 4.2. Hepatic reduced glutathione (A), Mn-superoxide dismutase activity (B), Cu/Zn-superoxide dismutase activity (C) and lipid peroxidation markers MOA+HNE (0) levels in F-344 rats were treated with doses of PM (10mg/kg) and/or KRE (1 OOmg/kg for 11 days. Bar graphs are expressed as the mean +5.0. for 5 rats in each treatment group, *p<O.05.
48
:::-e 'E
160
8 120 '0 ~
Control PM KRE PM+KRE
Figure 4.3. Total liver aconitase activity in F-344 rats treated with doses of PM (10mg/kg) and/or KRE (100mg/kg) for 11 days. The bar graph represents aconitase activity expressed as % of control. Values are expressed as the mean ± S.D for 5 rats in each treatment group.
49
TNF-a -+
l3-actin -+
250
.::-E! 200 -o c
'ti!8 "''0 c .- ~ 150 tie., 'l' UI
a:l. :t:::: .. c 9 ::::J u.~ 100 Zas 1-"-:t::::
~ 50
Control PM
.... " ... '..' .•. -....•... ; ....• ' .•........•. ~. .; -, " : . -" _. .
,- ,.. '-
*
o ..L-_
Control PM
KRE PM+KRE
KRE PM+KRE
Figure 4.4. Hepatic mRNA expression of TNF-a in F-344 rats treated with doses of PM (10 mg/kg) and/or KRE (100 mg/kg) for 11 days. Bar graphs represent the densitometry scans of 278-bp TNF-a amplicons and are expressed as a ratio to the housekeeping gene, l3-actin. Values are expressed as the mean + S.D. for 5 rats in each treatment group, *p<0.05.
50
120
" ~ -! '
Control PM KRE PM+KRE
Figure 4.5. Hepatic mitochondrial uncoupling protien 2 (UCP-2) protein expression in F-344 rats treated with doses of PM (10 mg/kg) and/or KRE (100 mg/kg) for 11 days. Protein expression of UCP-2 was determined by western analysis. The bar graph represents arbitrary units of the densitometry scan and are expressed as a % of control. Values are expressed as the mean ± S.D. for 5 rats in each group, *p<0.05.
51
(A)
:a- 300
c ~ 250 ]is e'5 200 Co
~ ~ 150 ~c ~.Q 100
U I 50 1il o
(C)
o
• •
Control PM KRE PM+KRE
Control PM KRE PM+KRE
(8)
Control PM KRE PM+KRE
(D)
:a- 150
c E 120 ]is e'5 90 Co
;f~ 60 ~.Q ul 30 Co
1il 0
Control PM KRE PM+KRE
Figure 4.6. Hepatic microsomal cytochrome P450 (CYP) protein expression in F-344 rats treated with doses of PM (10 mg/kg) and/or KRE (100 mg/kg) for 11 days. Protein expression of CYP1 A2 (A), 2E1 (8), 2D6 (C), and 3A4 (D) were determined by western analysis. The bar graph represents arbitrary units of the densitometry scan and are expressed as % of control. Values are expressed as the mean + S.D. for 5 rats in each group, *p<0.05.
52
Discussion
In contrast to earlier in vitro studies on human hepatoma cell line HepG2,
PM failed to induce liver injury as determined by body weight, feed intake, liver
weight, AST and ALT in F-344 rats. Although PM induced a significant cytotoxic
effect in HepG2, differences between cell and animal models as well as higher
metabolic rates in rats as compared to humans may be responsible for the lack of
toxicity, in vivo.
Kava rhizome extract also failed to cause any changes in the markers of liver
damage tested; however, these results are in agreement with a recent in vivo study
by Singh and Devkota (2003), who reported that rats receiving up to 500mg
KL/kglday displayed a lack of changes in liver injury markers serum AST, ALT,
ALP and LDR. The National Toxicology Program (NTP) also performed studies
in rodents with oral doses of kava extracts between 0.125 and 2.0 glkglday
(National Toxicology Program, 2005). Although some mortality was observed in
the 1.0 and 2.0 glkglday groups during the early stages of the experiments, overall
the effects were considered to be minimal to mild, causing some hepatocyte and
centrilobular hypertrophy but no hepatocellular necrosis (National Toxicology
Program, 2005). This is in contrast to a clinical study in New Caledonia where
significantly elevated AST and ALT levels were observed with consumption of
high concentrations of kava beverage (Russmann et al., 2003). However, in larger
53
clinical studies liver function tests were only moderately and reversibly elevated in
kava consuming populations (Clough et aI., 2003a; Russmann et aI., 2003).
The mRNA expression of pro- and anti-apoptotic proteins Bcl-2 and Bax
were unchanged in both PM and KRE treated rats, showing a lack of an apoptosis.
A similar lack of apoptosis was observed in HepG2 as measured by caspase
activity and DNA fragmentation (data not shown).
A wide variety of environmental stimuli including chemical stress are
known to increase the production of reactive oxygen species leading to oxidative
damage to cellular proteins, lipid membranes and DNA (Kroemer and Reed, 2000).
Increases in antioxidant enzymes are part of an adaptive response which protects
against ROS-induced oxidative stress (Koch et aI., 2004). Superoxide dismutase
(SOD) and reduced glutathione (GSH) are key antioxidants in the pathways
responsible for conversion of superoxide radical to water and oxygen (Koch et aI.,
2004; Femandez-Checa and Kaplowitz, 2005). The observed increases in hepatic
activity of cytosolic and mitochondrial SOD, in addition to the increases in total
liver GSH, may· be an adaptive response to PM and KRE induced production of
ROS. In addition, GSH up regulation is thought to be an adaptive response to
CYP2El-dependent oxidative stress and ethanol toxicity(Caro and Cederbaum,
2004). Our results indicate that KRE induces CYP2El expression but does not
cause oxidative stress, which may be due to the protective effects of GSH.
54
Oxidative stress occurs when the cell's equilibrium between ROS generation and
its antioxidant defenses shift in favor to the former, resulting in an excess of ROS,
which are capable of causing damage to proteins, DNA and lipids. Common
markers of oxidative damage to phospholipids in cellular membranes are MDA and
HNE. Aconitase activity is also a sensitive marker of oxidative stress. Our data
indicates that PM and KRE failed to cause oxidative stress as measured by MDA,
HNE and aconitase activity, which may have been due to protection from
antioxidant defenses.
Mitochondria are responsible for crucial cellular processes including energy
production and cell death (Van Houten et al., 2006). They are also a large source
of ROS that can lead to oxidative damage to mitochondrial DNA (mtDNA) (Van
Houten et al., 2006). Damage to mitochondrial DNA can lead to a vicious cycle
due to loss of essential proteins of the electron transport chain and enhanced ROS
production (Van Houten et al., 2006). Previous studies showed that both PM and
KRE induced mitochondrial dysfunction in HepG2 cells by depleting cellular ATP.
However, the same response was not observed with PM and KRE treatment in vivo
in F-344 rats (Figure 4.1). An opposite non-significant increase> in ATP was seen
in all treatments. Furthermore, UCP-2 protein levels were decreased in KRE
containing treatment groups. This may be an adaptive response to increased
cellular stress requiring greater energy expenditure to detoxification pathways.
55
Environmental toxins have been shown to cause an increase in protective
chaperone proteins such as heat shock proteins HSP-70 (Carnevali and Maradonna,
2003). This study showed no changes in hepatic mitochondrial or cytosolic HSP-
70 with PM and KRE treatment, further supporting a lack of severe kava
associated hepatic stress.
Many recent studies using in vitro models have shown that KL are strong
inhibitors of CYP450 enzymes (Mathews et al., 2002; Unger et al., 2002; Zou et
aI., 2002). Therefore in a recent review, it was hypothesized that kava-drug
interactions are highly likely to occur due to inhibition of CYP450 enzymes, which
are responsible for metabolizing a majority of the pharmaceutical drugs on the
market (Anke and Ramzan, 2004). Our data showed that PM and KRE treatment
results in an increase in hepatic CYP450 enzyme levels. Increases in CYP450
enzymes could result in faster metabolism of co-administered drugs, which could
lead to decreased effectiveness of medications or increased production of toxic
metabolites. Although different from previous studies, the increases observed in
our studies may be due to use of an in vivo rat model rather than cell culture.
56
CHAPTERS
GENERAL DISCUSSION
Pipermethystine (PM) caused a significant increase in reactive oxygen
species production, decrease in mitochondrial membrane potential and ATP and
induced cell death in human hepatoma cell line, HepG2. Subsequent studies
testing the effects of kava compounds in rat cell line H4IIEC3 showed similar
results when treated with equivalent concentrations. This suggests that differences
in metabolism between rats and humans does not seem to affect the toxicity of PM
and kavalactones.
Therefore, we tested the effects of PM and kavalactones using rodent models
as an indication of potential toxicity in humans. Long term treatment of C57BL6
mice with PM and KSPE failed to induce liver toxicity. However, low dosage and
amelioration of toxicity from the coconut milk vehicle likely contributed to the
lack of toxicity. Short term administration of PM and acetonic kava extracts also
failed to induce liver toxicity in F-344 rats, even at doses much higher than would
be consumed by humans. However, an initial antioxidant stress response was
induced as well as an increase in CYP450 enzyme expression. In vivo, there may
be a self-induction of CYP450 enzymes such as CYP2El and CYPIA2 which may
be metabolizing PM and kava extracts to non toxic metabolites. HepG2 and
57
H4IIEC3 are known to be deficient in cytochrome P450 enzymes, possibly
increasing their susceptibility to PM and kava extracts.
Our data corroborates with accumulating evidence supporting the lack of
toxicity associated with kavalactones. However, PM potentially contributed to the
liver toxicity seen in human cases as shown by its negative effects on human and
rat liver cell function and health. Although treatment of PM and kava rhizome
extracts failed to cause liver injury in the mouse and rat models studied, there was
an induction of an early stress response and expression of CYP450 proteins. From
the data obtained, it is unlikely that PM and kavalactones are the main cause of the
toxicity observed in humans. There are probably a number of factors that
contributed to the toxicity associated with kava rather than just a single cause. The
future research with kava should focus on the possible interactions of kava with
commonly used medications and herbs.
58
CHAPTER 6
MATERIALS AND METHODS
Isolation of PM, DHM and DMY. PM was isolated from stem peelings of
Piper methysticum cv. Isa (Dragull et aI., 2003), and the kavalactones DHM and
DMY were purified from commercial root powder. Preparative liquid
chromatography was performed on silica gel (Mallinckrodt Baker Inc.,
Phillipsburg, NJ); all solvents used were HPLC grade (Fisher Scientific, Fair
Lawn, NJ). Ethyl acetate (EtOAc) extracts of plant material were chromatographed
with a gradient of EtOAc/n-hexane mixtures (Shao, 1998) running from a ratio of
1:9 to 1:0 (Dragull et al., 2003). Crude target compounds were further purified by
repeated isocratic flash chromatography using the same packing material and
EtOAc/n-hexane (1:1). PM was obtained as an oil (98% pure by gas
chromatography-flame ionization detector (GC-FID» and kavalactones were
obtained as crystals (98% pure by GC-FID). The identities of the compounds were
confIrmed by GC-MS and high resolution mass spectroscopy.
Preparation of Kava Stem PeeUng Extract (KSPE) and Kava Rhizome
Extract (KRE). Mahakea stem peelings or rhizome were cut into chips, dried at
60°C at normal pressure and stored at -18°C until further processing. Before
extraction, the chips were re-dried at 50°C and -50 kPa for 1 h and milled. The
59
powder was fIrst extracted with ethyl acetate and supernatant was stored. The
particulate matter was further extracted by shaking at 150 rpm for 8 h with 75%
(v/v) acetone/water and acetone was removed from the supernatant under reduced
pressure. The aqueous phase was further partitioned with ethyl acetate 1:1 (v/v)
three times. All of the ethyl acetate extracts were combined, dried over sodium
sulfate and filtered through silica gel. KL concentration (sum of six major KL:
kavain (K), dihydrokavain (DHK), methysticin (M), dihydromethysticin (DHM),
yangonin (y), and desmethoxyyangonin (DMY» in the solvent-free kava rhizome
extract (KRE) was 63.6% ± 7.2% (n=5), as determined by GC-FID.
HepG2 cell cultures. Human hepatocellular carcinoma cell line HepG2
(American Type Culture Collection, ATCC, Manassas, VA) was cultured using T-
75 flasks (Coming Incorporated, Coming, NY) in minimum essential medium
eagle (MEME) containing 10% fetal bovine serum (PBS) and antibiotics. The cells
were grown in a 37°C/5% C02 incubator, split before confluence and used before
passage 12. HepG2 cells were trypsinized and seeded in clear ii-well and 96-well
culture plates (Coming Incorporated, Coming, NY) unless otherwise specifIed.
Cells were allowed 24 h to re-adhere and recover before.
C57BL6 mouse treatment procedure. All animal procedures were
performed in accordance with the University of Hawaii's Institutional Animal Care
60
& Use Committee (IACUC) guidlines. 10-week old male C57BL6 mice (22-28g,
Jackson Laboratory, Wilmington, MA) were housed individually and maintained in
an environment of 12h darkl 12h light cycle at 20°C to 22°C. Food and water was
provided ad libitum. The mice were randomly split into 5 treatment groups
containing 5 animals each: 1) untreated control, 2) coconut milk vehicle, 3) PM
(2mg/kg BW) 4) kava stem peeling extract (KSPE) (8.75mg/kg BW), 5) PM +
KSPE (respectively 2mg/kg+8.75mg/kg BW). Both PM and KSPE were mixed in
coconut milk and administered orally for 10 weeks daily. Body weight and feed
intake was monitored as marker of liver toxicity. Animals were fasted overnight
before being sacrificed in a carbon dioxide chamber. Blood was collected by
cardiac puncture, allowed to clot at room temperature for 30 minutes and serum
was collected by centrifugation at 1500 x g for 15 min at 4°C. Serum ALT, AST
and LDH were analyzed using commercial kits. Livers were excised, weighed and
examined for any gross changes. The liver was immediately snap frozen in liquid
nitrogen and stored in aliquots at -80°C until further analysis.
F-344 rat treatment procedure. All animal procedures were performed in
accordance with the University of Hawaii's Institutional Animal Care & Use
Committee (IACUC) guidlines. Male Fischer-344 rats (200-220g) were obtained
from Charles River Laboratory (Wilmington, MA), housed individually and
maintained in an environment of 12h darkl 12h light cycles at 68°F to nOF. Food
61
and water was provided ad libitum. The rats were randomly split into 4 treatment
groups containing 5 animals each: 1) com oil vehicle control (3.33mL/kglday), 2)
PM (10mg/kglday), 3) KRE (100 mg/kglday equivalent to 63 mg total KL/kglday)
4) PM+KRE. Both PM and KRE were mixed in com oil and administered by
intragastric gavage for 11 days. Animals were fasted overnight before being
sacrificed in a carbon dioxide chamber. Blood was collected by cardiac puncture,
allowed to clot at room temperature for 30 minutes and serum was collected by
centrifugation at 1500 x g for 15 min. at 4°C. Serum alanine aminotrasferase
(ALl) and aspartate aminotransferase (ASl) were analyzed by Diagnostic
Laboratory Services (DLS, Honolulu, HI). Livers were excised, weighed and
examined for any gross changes. Fresh liver was used to measure hepatic reduced
glutathione (GSH) content. The remaining liver was snap frozen in liquid nitrogen
and stored in aliquots at -BO°C until further analysis.
HepG2 cell treatments. HepG2 cells were treated with PM, DHM, DMY
and KRE at concentrations of 1, 10, 25, 50, 100, and 200,uM for various time
points up to 24 h. PM, DHM, DMY and KRE were dissolved in dimethylsulfoxide
(DMSO) and added to the culture media at the appropriate concentrations. The
final concentration of DMSO in the media was below 0.2% which was shown to
have no effects on the cells. Controls were treated with an equal amount of DMSO
as the highest treatment group. Culture media was collected off of the cells to
62
measure the release of LDH and living cells were used to detennine ROS,
mitochondrial membrane potential and cellular ATP content. Cells were harvested
for mRNA isolation and protein extraction, which was used for PCR and western
blotting analysis. All the assays were read using the Victor2 multilabel reader
(Perkin Elmer Life Sciences, Boston, MA).
Cytotoxicity assay (WH release). Cell viability was assayed
fluorimetrically by measuring LDH leakage from the cell into the culture media.
The LDH activity was measured with an enzymatic assay that results in the
conversion of resazurin into resorufm, using the commercial CytoTox-ONE assay
kit (Promega, Madison, WI). The product is measured with an excitation
wavelength of 535 nm and an emission wavelength of 590 nm.
ROS production. The production of ROS was estimated flourometrically
using an oxidant-sensitive probe, 2',7' -dihydrodichlorofluorescin diacetate
(H2DCFDA, Molecular Probes, Eugene, OR). HepG2 cells were treated with PM
or a combination of DHM and DMY for 1, 2, 3, 4 and 6 h. The cells were then
washed with phosphate-buffered saline (PBS) and further incubated in 20 roM
H2DCFDA in culture medium for 30 min at 37°C (Osseni et aI., 2000). The dye
was removed and the cells were washed with warm PBS. Fluorescence was
measured at 485ex/535em nm after adding 200 m1 of fresh PBS to the wells.
63
H2DCFDA is a cell permeable dye that is cleaved by endogenous esterases and the
product 2',7' -dichlorofluorescein (DCF) is oxidized by reactive oxygen species
which produces a fluorescent product. The fluorescence is proportional to the
amount of ROS production within the cell.
Mitochondrial membrane potential (!:i",). ~'I' was measured using a
membrane permeable, lipophilic cationic probe 5,5',6,6'- tetrachloro-
1,1' ,3,3 'tetraethylbenzinidazolylcarbocyanine iodide (J C-l; Molecular Probes,
Eugene, OR), according to published protocols (Mukherjee et al., 2002) with slight
modifications. Cells were washed twice in PBS and stained with lO,uM JC-l for
30 min in the dark at 37°C. Cells were washed once with PBS and then 200uL of
PBS was added to all wells. Fluorescence was measured at 485ex/535em nm and
then at 535ex/590em nm. The ratio of the emission values 590/535 nm is
equivalent to the relative !:i'l', with a decrease in 590/535 nm corresponding to
mitochondrial depolarization. Valinomycin (Molecular Probes, Eugene, OR) at a
final concentration of 100 ,uM was used as a positive control.
Cellular ATP levels. Total cellular ATP levels were measured using the
ATPLite kit (Perkin Elmer Life Sciences, Boston, MA). Briefly, cells were plated
in white 96-well clear-view culture plates (Packard BioScience, Meriden, CI).
Cells were exposed to treatments for 3 and 6 h, washed twice with PBS and
64
assayed for total cellular ATP according to the manufacturer's directions. The
principle of the assay is based on production of light when luciferase is oxidized to
oxyleuciferin. Luminescence is proportional to ATP levels, which are calculated
from a standard curve generated with each experiment and expressed as pM
ATP/mg protein.
Serum liver injury markers. Common markers of liver injury were assayed
in the serum within 7 days of collection. AST and ALT were measure using
commercial enzymatic assay kits (Biotron, Hemet, CA) and LDH was assayed
using the Cytotox-One Homogeneous Membrane Integrity Assay (Promega,
Madison, WI) as manufacturer's instructions. All spectrophotometric assays were
performed in a 96-well plate (Coming International, Coming, NY) using the
Victor2 multilabel reader (Perkin Elmer, Boston, MA)
Total Hepatic ATP levels. Frozen livers were homogenized in 10 volumes
of O.25M sucrose buffer with 15 strokes using a dounce glass homogenizer.
Homogenates were heated to 90°C for 5 minutes and centrifuged at 10,000 x g for
10 minutes. The supernatant was collected and assayed for ATP levels using the
Perkin Elmer ATPlite kit according to the manufacturer's directions with slight
modifications. Luminescence was proportional to ATP levels which was
calculated from a standard curve generated with each set of experiments and was
expressed as pM ATP/mg protein.
65
Hepatic Glutathione (GSH). Hepatic GSH was measured according to
published protocol with slight modifications (Hissin and Hilf, 1976). In brief,
fresh liver was homogenized in phosphate buffer (100mM phosphate, pH 8.3,
5mM EDTA, 20% w/v) and centrifuged at 10,000 x g for 15 minutes. Supernatants
were deproteinated with an equal volume of 10% trichloroacetic acid (TeA) and
frozen until analysis. The fmal reaction was performed in a 96 well plate by adding
deproteinated liver samples to phosphate buffer and O-phthalaldehyde solution.
Fluorescence was read at 350/420 in the Victor2 multilabel plate reader (Perkin
Elmer, Boston, MA). Each fluorescence value was compared to reduced
glutathione standards prepared freshly with each batch of samples and adjusted to
,uM GSH/g liver.
Hepatic Superoxide Dismutase Activity (SOD) and Lipid Peroxidation
(LPO). Liver homogenates were prepared in 10 volumes of ice-cold HEPES
buffer (20mM HEPES, 1mM EGTA, 210mM mannitol, 70mM sucrose, pH 7.2)
and centrifuged at 1500 x g for 5 minutes. The supernatant was further centrifuged
at 10,000 x g for 15 minutes. The resulting supernatant contained the cytosolic
SOD and the pellet contained the mitochondrial SOD. The mitochondrial pellet
was resuspended in 600uL of HEPES buffer. Both fractions were frozen at -80g C
for up to 1 month and cytosolic and mitochondrial SOD activities were analyzed
66
using commercial Superoxide Dismutase Assay kit (Cayman Chemicals, Ann
Arbor, MI).
Lipid peroxidation was determined by measuring the amounts of
malondialdehyde (MDA) and 4-Hydroxyalkenals (HAE) using the Bioxytech
LPO-S86 kit (Oxis Research, Portland, OR). In brief, liver homogenates were
prepared with 2.5 volumes of ice-cold PBS (20mM, pH 7.4) containing fresh
butylated hydroxytoluene (BHT) (SmM final concentration), centrifuged at SOOO x
g for 10 minutes and supernatants were used to analyze MDA and HNE.
Hepatic Aconitase activity. Liver tissue was homogenized at 1% (w/v) in
ice-cold sodium citrate buffer (0.2mM sodium citrate, SOmM Tris-HC!, pH 7.4)
and centrifuged at 800xg for 10 minutes to pellet tissue debris. The supernatant
was assayed fresh for aconitase activity using a commercial assay kit (Oxis
Research, Portland, OR).
Cellular Uncoupling Protein-2 mRNA Expression. UCP-2 mRNA gene
expression was determined by semi-quantitative RT-PCR. HepG2 cells were
plated and allowed to grow until confluence in clear 6-well culture plates. Cells
were treated with SO,uM PM for 24 h. After two washes with PBS, cells were
collected using a cell scraper. RNA was extracted following the manufacturer's
protocol using the RNA-bee isolation reagent (Iso-Tex Diagnostics, Inc,
67
Friendswood, TX). RNA (2 Ilg) was reverse transcribed into complementary DNA
(cDNA). UCP-2 mRNA expression was determined using published primers.
Semiquantitation of Bax, Bcl-2 and TNF-alpha Gene Expression. Bax,
Bcl-2 and TNF-alpha mRNA gene expression was determined by semi-quantitative
RT-PCR. Total RNA was extracted using RNA-Bee (Tel-Test, Friendswood, TX).
RNA (2 Ilg) was reverse transcribed into complementary DNA (cDNA). Bcl-2
expressions levels were quantified using commercial primers (Sigma, Saint Louis,
MO, cataIog# APO-PCR), while Bax and TNF-alpha expression was quantified
using published primers and cycling conditions (Wang et aI., 2004; Kono et al.,
2005). PCR reactions were performed in a GeneAmp PCR System 9700 (Applied
Biosciences), amplicons size-fractionated on a 2% agarose gel and visualized with
ethidium bromide staining. Bax, Bcl-2 and TNF-alpha gene expression was
semiquantitated with Kodak ID image analysis software and the intensity of the
amplicons were expressed as a ratio of the gene of interest against a housekeeping
gene, GAPDH and ~-actin.
Hepatic Lipids extraction. Frozen liver was homogenized in 20 volumes of
homogenizing buffer A (0.3M Sucrose, 25nM 2-mercaptoethanol, lOmM EDTA at
pH 7.0) with 20 strokes in a dounce homogenizer. The homogenate was combined
with 2.5 volumes of Chloroform, vortexed and incubated for 30 minutes at room
68
temperature. An additional 2.5 volumes of Chloroform:0.15M NaCI (1:1) was
added and the resulting solution was vortexed and incubated at room temperature
for 1h. Homogenates were centrifuged at 3000 x g for 10min to allow for phase
separation. The bottom chloroform layer was dried under a vacuum and the lipid
extract was re-suspended in 95% Ethanol. Lipids were stored for no more than
seven days at 4QC until triglyceride assay was performed.
Triglyceride (TG) assay. TG levels were determined in hepatic lipid
extracts using the Infinity Triglyceride Reagent kit (Thermo Electron Corporation,
Waltham, MA) following manufacturer's instructions. Lipid extracts were
combined with the reagent which produces an absorbance that can be detected at
570 nm. Absorbance values were proportional to TG levels, which were calculated
from a standard curve generated with each experiment.
Mitochondrial and cytosolic extract preparation for HepG2. HepG2 cells
were plated to confluence in T-25 flasks. Cells were treated for 48 h with 50,uM
PM and 100,uM KRE with fresh treatment media being added every 24 h. After
48 h of treatment, cells were collected and washed twice with PBS. The cell pellet
was homogenized in 10 volumes of mitochondrial isolation buffer (20mM HEPES,
210mM mannitol, 1mM EGTA, 70mM sucrose, 2mM Tris-HCl, pH 7.2)
containing complete protease inhibitor cocktail (Roche) with 6 strokes in a dounce
69
glass homogenizer. The homogenate was centrifuged at 750 x g for 10 minutes to
pellet cell debris. The supernatant was subjected to a centrifugation of 10,000 x g
for 15 minutes to pellet mitochondrial fraction. The supernatant was collected
(cytosolic fraction) while the pellet (mitochondrial fraction) was resuspended in
mitochondrial isolation buffer. Mitochondrial and cytosolic fractions were stored
at -80aC until use.
Liver Microsome preparation. Microsomal extracts were prepared as
previously described with slight modifications (Nelson et al., 2001). Frozen livers
were homogenized in 2.5 volumes of ice-cold homogenization buffer (0.1 M
potassium phosphate, 0.125 M KCI, 0.25 M Sucrose, 1 mM EDTA, pH 7.4).
Homogenates were diluted to 4 volumes of the liver sample weight and centrifuged
at 12,000 x g for 20 minutes to pellet mitochondria and cell debris. Resulting
supernatant was further centrifuged at 138,000 x g for 60 minutes to pellet the
microsomal fraction. The pellet was resuspended in microsomal buffer (0.1 M
Tris-Base, 0.125 M KCI, pH 7.4) and centrifuged again at 138,000 x g for 60
minutes. The supernatant was discarded and the pellet was suspended in
microsomal buffer and stored at -80 DC until use.
Mitochondrill and Cytosol Preparation. Frozen liver was homogenized in
10 volumes of mitochondrial isolation buffer (20mM HEPES, 210mM mannitol,
lmM EGTA, 70mM sucrose, 2mM Tris-HCl, pH 7.2, containing complete
70
protease inhibitor cocktail (Roche Applied Sciences, Indianapolis, IN)) with 6
strokes in a Dounce homogenizer. The homogenate was centrifuged at 750 x g for
10 minutes to pellet cell debris. The supernatant was subjected to a centrifugation
of 10,000 x g for 15 minutes to pellet mitochondrial fraction. The supernatant was
collected as the cytosolic fraction while the pellet was resuspend as the
mitochondrial fraction in mitochondrial isolation buffer. Mitochondrial and
cytosolic fractions were stored at -80 gc until use.
Protein determination. Protein concentrations were determined by the
Lowry method using a commercial protein assay kit (Bio-Rad Laboratories,
Hercules, CA) according to manufacturer's instructions with bovine serum
albumin as a standatd.
Western blotting for mitochondrial and cytosolic Apoptosis.inducing
Factor (AlF) and cytochrome c. Western analysis was applied to both
mitochondrial and cytosolic extracts by the method of Laemmli (1970) for
determining protein expression of AIF and cytochrome c. Mitochondrial proteins
(25 and 30 ILgIlane for AIF and cytochrome c, respectively) and cytosolic proteins
(75 and 20 ILgIlane for AIF and cytochrome c, respectively) were sepatated by
SDS-PAGE gels and transferred to a nitrocellulose membrane, blocked in 10%
non-fat dry milk, and incubated overnight with specific primary antibodies against
71
AIF and cytochrome c (Santa Cruz Biotechnology, Santa Cruz, CA). After
washing, blots were probed with appropriate secondary antibodies for 2 h at room
temperature (Santa Cruz Biotechnology, Santa Cruz, CA). Proteins were detected
using AP color development reagents (Biorad Laboratories, Hercules,CA).
Western blotting for Hydroxy-nonenol (HNE) adduct. HepG2 cells were
homogenized in ice-cold homogenizing buffer (50mM Tris-HCI, pH 8.0, 0.5%
Triton X-100, supplemented with fresh protease inhibitors) with 25 strokes in a
dounce glass homogenizer. The whole homogenate was centrifuged at 10,000 x g
for 15 minutes and the resulting supernatant was subjected to Western analysis.
Whole cell protein extracts (5.ug/lane) were separated on a 10% sodium dodecyl
sulfate-polyacrilaminde gel (SDS-PAGE) and transferred to a nitrocellulose
membrane. Blots were blocked in 10% non-fat dry milk and incubated with anti
HNE antiserum (Alpha Diagnostics International, San Antonio, TX) overnight.
After washing, blots were probed with appropriate secondary antibodies for 2 h at
room temperature (Santa Cruz Biotechnology, Santa Cruz, CA). Proteins were
detected using AP color development reagents (Biorad Laboratories, Hercules,
CA).
Western blotting of microsomal CYP2El. Microsomal proteins (1.25!lg)
were separated on 10% SDS-PAGE by the method of Laemmli (1970). The gels
72
were subjected to electrophoretic transfer onto nitrocellulose membrane, blocked
with 1 % bovine serum albumin (BSA) in TBS, and incubated overnight with
specific primary antibodies against CYP2E1 (Research Diagnostics, Flanders, NJ).
After washing, blots were probed with horseradish peroxidase conjugated anti
rabbit IgG for 2h at room temperature (Santa Cruz Biotechnology). Proteins were
detected by Enhanced Chemiluminescence (ECL) Western Blotting Detection
Reagents (Amersham Biosciences, Piscataway, NJ).
Western Blot Analysis of CYP450 Proteins, UCP-2 and HSP-70. Western
analysis was used to determine expression of CYP450 enzymes in liver
microsomes, HSP-70 in mitochondrial and cytosolic extracts and UCP-2 in
mitochondrial extracts. Protein concentrations were determined using commercial
protein assay reagent according to the manufacturer's instructions (Bio-Rad
Laboratories, Hercules, CA). Proteins (0.1, 1.25, 2.5, 11, 25 and 100 ""g!1ane for
CYP-2D6, -2E1, -3A4, -1A2, HSP-70 and UCP-2, respectively) were separated on
10% to 12% SDS-PAGE and transferred to a nitrocellulose membrane, blocked in
1 % bovine serum albumin (BSA) or 5% non-fat dry milk. Blots were incubated
overnight with primary antibodies against CYP1A2 and CYP2E1 (Research
Diagnostics, Flanders, NJ), CYP2D6 (BD Biosciences, Bedford, MA), CYP3A4
(Affinity Bioreagents, Golden CO), UCP-2 and HSP-70 (Santa Cruz
Biotechnology, Santa Cruz, CA). Blots were washed and probed with HRP-
73
conjugated secondary antibodies for 2 h at room temperature (Santa Cruz
Biotechnology, Santa Cruz, CA). Proteins were detected using ECL Western
Blotting Detection Reagents (Amersham Biosciences, Piscataway, NJ).
Statistical Antllysis. All data are presented as mean + SD. All the
biochemical and molecular analysis were performed in duplicates or triplicates.
Statistical significance was analyzed using Student's t-test and two-way Anova
after data normalization and logistic regression. P-values :s; 0.05 were considered
significant.
74
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