traditional kava is an aqueous extract 
 of the roots of piper methysticum and serves as a 
 ceremonious and daily 
 beverage or an herbal remedy for south pacific islanders .
kava had also been used clinically to treat mild 
 and moderate anxiety , based on results of numerous clinical trials .
anxiolytic kava was typically prepared as an organic extract of 
 kava root with ethanol or acetone , instead of the traditional aqueous 
 preparation .
anxiolytic kava had been banned in europe and a few other 
 countries since 2002 because of its risk to induce hepatotoxicity , 
 and it is listed on the usa fda advisory board , but 
 germany s federal administrative court negated the ban in june 
 2014 .
various causes have been proposed 
 for kava s hepatotoxic 
 risk , but none have been validated so far .
first of all , in response 
 to high demand , anxiolytic kava may have included nonroot toxic plant 
 parts .
it has also been postulated that 
 some kava roots were not properly dried , resulting in hepatotoxin 
 contamination .
usage of nontraditional 
 cultivars could be another cause ; different kava cultivars have diverse 
 chemical profiles while traditional kava is prepared from only a few 
 of them . due to preparation difference ,
traditional 
 and anxiolytic kavas have distinct composition profiles , which may impose different hepatotoxic risks as well .
furthermore , 
 90% of the purported hepatotoxic cases associated with kava 
 usage involved concomitant consumption of other drugs or dietary supplements , suggesting that kava s hepatotoxic risk may be mediated via 
 herb  herb or herb  drug interactions .
in addition 
 to kava s anxiolytic benefit , one epidemiological 
 survey suggested that traditional kava usage may be able to reduce 
 cancer risk , which was supported by results 
 from several laboratory animal tumorigenesis models .
moreover , despite its ban and being on usa fda s advisory 
 list , kava consumption has experienced a global resurgence based on 
 the amount of kava exported from the major kava producing nations 
 ( the republic of vanuatu , fiji , and tonga ) between 2008 and 2013 . with the recent overturn of the kava ban in 
 germany
our recent 
 metabolomics and cellular cytotoxicity analyses of an array of current 
 commercial kava products revealed that they were diverse in chemical 
 profile and cellular cytotoxicity , and 
 likely distinct in their health benefit and risk .
considering 
 the increasing human exposure and the diverse chemical 
 composition of current kava products , the hepatotoxic risk of kava 
 needs to be clarified and the responsible chemicals need to be identified , 
 which is the focus of this study .
our results showed that kava was 
 safe when given alone but significantly enhanced acetaminophen ( apap)-induced 
 hepatotoxicity in c57bl/6 mice .
chalcone - based flavokawains a ( fka ) 
 and b ( fkb ) recapitulated kava s potentiation of apap - induced 
 hepatotoxicity while dihydromethysticin ( dhm ) lacked such a risk .
an ethanolic extract of the 
 wild crafted kava root from vanuatu was purchased from gaia herbs , 
 inc .
( brevard , nc , standardized to 150 mg / ml total kavalactones ) . 

dhm was purified from this kava product using normal phase silica 
 gel chromatography as described earlier .
kava and all compounds were 
 completely dried under vacuum to remove any solvent residue .
the desired drug 
 formulations were prepared by mixing kava or pure compounds with peg-400 
 and stored at 4 c until use .
all animal studies were performed 
 in compliance with the institutional animal care and use committee 
 at the university of minnesota guidelines .
six - week - old female c57bl/6j 
 mice ( jackson laboratories , me ) were housed at specific pathogen - free 
 animal facilities of research animal resources , university of minnesota , 
 with free access to standard rodent food and water .
mice were 
 gavaged with dose formulations at the indicated doses and times , and 
 euthanized by co2 overdosing with necropsy performed by 
 experienced researchers .
the long - term study was designed to 
 evaluate the hepatotoxicity of kava alone .
mice in the control group were given peg-400 
 ( 200 l ) on a daily basis via gavage , 6 days a week , for 14 
 weeks .
mice in the kava treatment group were given kava at a dose 
 of 500 mg / kg bodyweight on a daily basis via gavage , 6 days a week , 
 for 14 weeks .
the chosen kava dose was based on the recent safety 
 studies of another kava product performed by the national toxicology 
 program .
final bodyweight was measured and serum 
 from each mouse was analyzed for alanine aminotransferase ( alt ) and 
 aspartate aminotransferase ( ast ) , two major biomarkers of liver function .
the short - term combination studies were designed to evaluate the 
 potential synergism of kava and its chemicals to apap - induced hepatotoxicity . 

c57bl/6 mice were randomized ( 815 mice per group ) and were 
 administered with peg-400 ( 200 l ) , kava ( 500 mg / kg bodyweight ) , 
 dhm or fka , and fkb in peg-400 ( 200 l ) at the indicated doses 
 daily via oral gavage for 2 days . on the third day
, mice in the respective 
 groups were coadministered with apap ( 800 mg / kg bodyweight ) in peg-400 
 ( 200 l ) .
appropriately fixed tissues 
 were processed into paraffin blocks using standard histological techniques , 
 and 5 m sections were cut and stained with hematoxylin and 
 eosin ( h&e ) .
histological slides were examined using light microscopy 
 by an experienced a.c.v.p board certified pathologist ( m.g.os . ) 
 under blinded conditions , with liver lesions graded on a 0 to 4 scale 
 based on the extent of necrosis ( 0 = absent , 1 = minimal , 2 = mild , 
 3 = moderate , 4 = severe ) .
the clinical 
 chemistry data were 
 reported as mean  sd ( n = 415 ) . for 
 the long - term kava alone
study , the two - tailed student t - test was used to compare the means between the control and treatment 
 groups .
one - way analysis of variance ( anova ) was used to compare 
 the means among different groups in the short term combination studies . 

dunnett s test was used for comparisons of apap and other treatment 
 groups when the one - way anova analysis was statistically significant .
at the tested dose ( 500 mg / kg bodyweight ) , 
 daily kava treatment did not affect mouse growth ( data not shown ) . 

there were also no statistically or biologically significant differences 
 between control and kava - treated mice with respect to alt and ast 
 ( figure 1a and 1b ) .
effect 
 of 14-week daily kava treatment ( 500 mg / kg bodyweight ) via 
 gavage on mouse serum alt ( a ) and ast ( b ) .
p values 
 were given with comparison between the control group ( n = 4 ) and the kava treatment group ( n = 4 ) using 
 the two - tailed student t - test . since 90% of the human kava hepatotoxic cases involved 
 concurrent consumption of other medications or dietary supplements , herb  drug interactions
based on this and on a recent report that kava enhanced the 
 toxicity of apap in vitro , this study was designed to evaluate the effect of kava on apap - induced 
 hepatotoxicity in vivo .
the treatment regimen was 
 designed to mimic potential scenarios in humans  kava was consumed 
 on a daily basis while apap was used occasionally .
as expected , kava 
 treatment alone had no effect on alt and ast while apap treatment 
 significantly increased serum alt and ast activities ( figure 2a ) .
kava and apap combination caused further increase 
 in serum alt and ast activities ( 3-fold increase relative 
 to apap alone , figure 2a ) , and these increases 
 were statistically significant in comparison to apap treatment alone . 

histopathological analyses of the liver tissues revealed no lesions 
 in control and kava treated mice ( figure 2b ) , 
 confirming the lack of hepatotoxicity by kava treatment alone . the 
 lesions from apap - treated mice evenly distributed among different 
 severity categories ( 0 being no lesion and 4 being the highest grade 
 lesion ) while kava and apap combination markedly increased the number 
 of mice with the highest liver lesion ( figure 2b ) , supporting the notion that the increases in alt and ast activities 
 were biologically significant .
these clinical chemistry data and histopathological 
 findings for the first time demonstrate that kava enhanced apap - induced 
 hepatotoxicity in vivo , and may reflect the purported 
 kava hepatotoxicity cases in humans .
the histopathological lesion 
 severity also nicely correlated positively with the clinical chemistry 
 results ( figure 2c ) .
effect of 3-day daily 
 kava treatment ( 500 mg / kg bodyweight ) via 
 gavage on mouse serum alt and ast and liver lesions with / without apap 
 treatment ( 800 mg / kg bodyweight ) via gavage .
( c ) 
 the relationships among serum alt , ast , and the grades of liver lesions . 
 for a
, comparisons were made with the apap treatment group by dunnett s 
 test when one - way anova was statistically significant ( n = 815 ) .
this experiment was designed to explore the potential 
 of dhm and fkb ( figure 3 ) to synergize the 
 hepatotoxicity of apap following the same kava and apap cotreatment 
 regimen .
thirteen chemicals have been isolated and quantified from 
 the kava product used in this study with no detection of pipermethystine .
dhm and fkb were selected for this initial evaluation 
 because they are representatives of kavalactones and chalcones , respectively , 
 two major classes of chemicals in kava .
in addition dhm has been recently 
 demonstrated to potently and effectively block nnk - induced lung tumorigenesis 
 in mice while fkb has been identified 
 as the most cytotoxic compound in kava to various cancerous cells .
the dosages for dhm ( 37.5 mg / kg ) and fkb ( 11.5 mg / kg ) were based 
 on their abundance ( 7.5% and 2.3% , respectively ) in this kava product 
 at a dose of 500 mg / kg .
dhm and fkb individually 
 caused no effect on serum alt and ast ( figure 4 ) .
dhm had no effect on serum alt and ast as well when combined with 
 apap ( figure 4 ) .
fkb on the other hand when 
 combined with apap moderately increased the serum levels of alt and 
 ast , and the increase in ast was statistically significant ( figure 4 ) , suggesting that fkb contributes to kava s 
 potentiation of apap - induced hepatotoxicity .
effect of 3-day daily dhm ( 37.5 mg / kg ) or fkb ( 11.5 mg / kg ) via 
 gavage on mouse serum alt and ast with / without apap treatment .
comparisons 
 were made with the apap treatment group by dunnett s test when 
 one - way anova was statistically significant ( n = 
 615 ) .
given that the kava 
 product used in this study contains flavokawain a ( fka ) of similar 
 abundance as fkb ( figure 3 ) , this experiment 
 was designed to evaluate the dose  response effect of fka and 
 fkb together on apap s hepatotoxicity following the same treatment 
 regimen .
the final dosages of fka and fkb were 1 , 2 , and 4 times their 
 abundance ( 1.6% and 2.3% , respectively ) of a kava dose at 500 mg / kg 
 bodyweight .
fka and fkb together did not induce any changes on serum 
 alt and ast at the three tested dosages ( figure 5a and b ) . when combined with apap , fka and fkb dose - dependently potentiated 
 the increase in alt and ast induced by apap ( figure 5a and b ) . of note ,
one mouse with the treatment of the highest 
 dose of fka and fkb in combination with apap died 0.52 
 h before necropsy ( i.e. , 22 to 23.5 h after the combined dose of apap 
 with fka and fkb ) .
its serum alt and ast levels were the highest 
 among all mice ( figure 5c ) , and 23 
 times higher than the next highest values .
histopathological examination 
 revealed multifocal and coalescing acute centrilobular necrosis in 
 the liver of this mouse ( figure 5d , panel b ) , 
 whereas livers from a control mouse ( figure 5d , panel a ) and a mouse treated with fka and fkb alone ( not shown ) 
 were histologically within normal limits .
response 
 effect of 3-day daily fka and fkb via gavage 
 on mouse serum alt and ast and livers with / without apap treatment . 

comparisons were made with the apap treatment 
 group by dunnett s test when one - way anova was statistically 
 significant ( n = 5 ) .
( c ) serum level of alt and ast 
 from the dead mouse in the high - dose fka and fkb groups with apap 
 cotreatment .
( d ) photomicrographs of h- and e - stained livers from 
 a control mouse ( panel a ) and a mouse treated with fka and fkb plus 
 apap ( panel b ) .
note extensive karyorrhexis ( arrow ) reflecting acute 
 necrosis of hepatocytes ( increased eosinophilia ) in mouse treated 
 with fka , fkb , and apap ( panel b ) .
kava has demonstrated anxiolytic activity 
 in the clinic and potentially 
 reduces cancer risk in humans . on the other hand , kava usage has been 
 speculated to be associated with rare but severe hepatotoxicity .
various 
 mechanisms have been proposed and different chemicals have been postulated 
 with no confirmation . given kava s global resurgence and the 
 diverse chemical composition among current kava products , it is urgent 
 and important to recapitulate kava s hepatotoxicity in an in vivo model , which can help identify the responsible chemicals 
 and guide the development of strategies to minimize and ideally eradicate 
 such an adverse potential .
the results from this study demonstrated 
 that kava when administered 
 alone via gavage in c57bl/6 mice induced no adverse effect even at 
 a fairly high dose ( 500 mg / kg bodyweight daily ) in a chronic manner , 
 as reflected in mouse growth and serum levels of alt and ast ( figure 1 ) .
on the other hand , kava significantly potentiated 
 the hepatotoxicity of apap in c57bl/6 mice , as indicated by the increase 
 in serum alt and ast , and the increased severity of liver lesions 
 ( figure 2 ) .
the treatment regimen was designed 
 to mimic potential circumstances among human kava users that kava 
 would be consumed on a daily basis while other medications , apap in 
 this case , were used occasionally when needed .
since the majority 
 of kava - associated hepatotoxic cases consumed other medications or 
 dietary supplements concomitantly , the results from this study may 
 have direct indication to the observed hepatotoxicity among kava users . 

it remains to be determined whether kava usage can potentiate the 
 hepatotoxic risk of other medications or hepatotoxins , such as alcohol 
 consumption .
it also remains to be determined whether other kava treatment 
 regimens , such as prolonged kava usage or in a fasted stage ( recommended 
 for traditional kava usage ) , may potentiate its hepatotoxic risk even 
 at lower kava dosages . with the c57bl/6 mouse model that captures 
 kava s hepatotoxic 
 risk in vivo
the results demonstrated that a chalcone - based compound 
 in kava , fkb , moderately potentiated apap s hepatotoxicity 
 while dhm , a representative of kavalactones in kava , lacked such a 
 risk when they were evaluated at a dose equivalent to kava at a dose 
 of 500 mg / kg bodyweight ( figure 4 ) .
as the 
 kava product contains fka , an analog of fkb , at similar abundance , 
 the combination of fka and fkb was evaluated , which dose - dependently 
 enhanced apap - induced hepatotoxicity ( figure 5 ) .
indeed , the one mouse that died early , and which had the highest 
 alt and ast levels ( figure 5c ) , reflecting 
 extensive acute hepatocellular necrosis ( figure 5d , panel b ) , was in the apap cotreatment group at the highest dose 
 of fka and fkb .
these data overall indicate that fka and fkb are the 
 responsible compounds in kava that potentiate apap - induced hepatotoxicity 
 while dhm is free of this risk . besides fka and fkb , flavokawain
c 
 ( fkc ) has been reported in other kava products but was not detectable in the kava product used in this 
 study .
our recent analysis of a set of kava products on the current market 
 demonstrates that the abundance of fka and fkb can vary 20-fold .
similarly , a recent study analyzed the abundance 
 of fka , fkb , and fkc in different kava cultivars .
cultivars not recommended for traditional use were found 
 to contain higher abundance of fka , fkb , and fkc than the traditionally 
 consumed cultivars .
further studies therefore 
 are warranted to evaluate whether cultivars or kava products with 
 higher content of fka , fkb , and fkc would impose a higher hepatotoxic 
 risk .
future studies are also needed to elucidate the molecular mechanisms 
 of the observed hepatotoxicity enhancement , such as the depletion 
 of glutathione .
such knowledge will help 
 guide the preparation of kava products for human use with higher health 
 benefit and minimal adverse effects .
 anxiolytic 
 kava products have been associated with rare but severe 
 hepatotoxicity in humans . 
 this adverse potential has never been captured 
 in animal models , and the responsible compound(s ) remains to be determined . 
 
 the lack of such knowledge greatly hinders the preparation of a safer 
 kava product and limits its beneficial applications . in this study 
 
 we evaluated the toxicity of kava as a single entity or in combination 
 with acetaminophen ( apap ) in c57bl/6 mice . 
 kava alone revealed no 
 adverse effects for long - term usage even at a dose of 500 mg / kg bodyweight . 
 on the contrary a three - day kava pretreatment potentiated apap - induced 
 hepatotoxicity , resulted in an increase in serum alt and ast , and 
 increased severity of liver lesions . 
 chalcone - based flavokawains a 
 ( fka ) and b ( fkb ) in kava recapitulated its hepatotoxic synergism 
 with apap while dihydromethysticin ( dhm , a representative kavalactone 
 and a potential lung cancer chemopreventive agent ) had no such effect . 
 these results , for the first time , 
 demonstrate the hepatotoxic risk 
 of kava and its chalcone - based fka and fkb in vivo and suggest that herb  
 drug interaction may account for the 
 rare hepatotoxicity associated with anxiolytic kava usage in humans . 
Introduction
Materials
and Methods
Results
Discussion