realgar ( 90% as as4s4 ) has been used in traditional chinese medicines [ 1 , 2 ] and in indian ayurvedic medicines   for thousands of years and claimed to have therapeutic effects in these remedies . however , arsenic ( as ) is a highly toxic substance and its risk in traditional remedies is of concern [ 46 ] . hundreds of traditional medicines are forbidden in the usa or european market because of the contents of as are higher than the allowable limits for food and drugs , and over 20% of online - sold ayurvedic medicines were demanded for rigorous regulation for heavy metal contents . the chinese pharmacopeia committee has reduced the allowable as contents in traditional medicine recipes by as much as 65% , but as contents are still thousands - fold over the health food standards . more studies are therefore recommended to evaluate the true risk of metal - containing traditional medicines . niu - huang - jie - du pian ( nhjd ,   ) is a popular realgar - containing traditional chinese medicine ( http://www.wickpedia.org/ ) , which can be easily obtained in chinese grocery stores worldwide . nhjd is composed of realgar ( 6.4% ) , niu - huang ( calculus bovis ) , huang - qin ( radix scutellariae , rhubarb ) , ju - geng ( platycodon grandiflorum ) , gan - cao ( radix glycyrrhizae uralensis , licorice root ) , gypsum ( calcium sulfate ) , and bing - pian ( borneol ) , and it is used for antipyretic , cold , gingivitis and other inflammatory diseases . there is a general perception that the use of toxic metals in medicines is an unacceptable risk , but an opposing opinion holds that realgar - containing traditional medicines are not necessarily toxic at clinical doses [ 2 , 7 ] . we have recently shown that chemical form of realgar ( as4s4 ) is a major determinant for its disposition and toxicity . for example , realgar - containing an - gong - niu - huang wan (  ) was much less toxic in cultured cells , in acute animal studies , and in subchronic toxicology studies [ 10 , 11 ] . the present study was undertaken to evaluate the true risk of a realgar - containing nhjd , a most popular patent chinese medicine , in cultured cells and in intact animals , as compared to common arsenicals . the results fortified our prior conclusions that chemical forms of metals are very important in determining the disposition and toxicity of metal - containing traditional medicines . realgar ( > 90% of as4s4 ) , and realgar ( 6.4%)-containing nhjd were obtained from beijing tong - ren - tang technologies co. , ltd . , beijing , china ; the common arsenicals as2s2 , as2o3 , naaso2 , and na2haso47h2o were purchased from sigma chemical company ( st . human pharyngeal carcinoma fadu cells were obtained from shanghai institute of biochemistry and cell biology ( shanghai , china ) and cultured in dmem media supplemented with 10% fetal bovine serum ( fbs ) with penicillin and streptomycin . all chemicals were dissolved in dimethyl sulfoxide ( dmso ) in 1030  mm and serially diluted in culture media before addition to the cultures at 5 - 6 different concentrations as indicated . for cytotoxic assay , chemicals were added to 96-wells when initiating the culture , and 48  h later , the cytotoxicity was measured by the mts assay as described previously . for as uptake and accumulation studies , cells were exposed to the same concentration ( 100  m ) for 30120  min , and cellular as was determined after thoroughly washing . adult male and female kunming mice ( 22  2  g ) were purchased from the animal center of the third military medical university ( chongqing , china ) . mice were kept in a regulated environment ( 22  1c , 50  2% humidity ) with a 12  h  :  12  h light : dark cycle . all animal procedures follow the who guidance of humane care and use of laboratory animals . mice were orally administered with nhjd ( 600  mg / kg ) , realgar ( 600  mg / kg ) , and the equal amount of as doses as sodium arsenite ( 36  mg / kg ) and sodium arsenate ( 88  mg / kg ) . the nhjd 600  mg / kg is approximately 2 times of clinical dose ( 2000  mg / day/60  kg person , taken mouse - human extrapolation factor of 10 ) . animals were monitored closely for clinical symptoms after gavage , and tissues were harvested for analysis 8  hrs later . the doses of arsenicals selection were based on our recent publications [ 9 , 12 ] . serum was separated from whole blood by standing for 1  hr , and blood biochemistry was determined with an autoanalyzer ( glamour1600 ) . the serum activities of alanine aminotransferase ( alt ) and concentrations of blood urea nitrogen ( bun ) were quantified to evaluate the hepatotoxicity and nephrotoxicity of animals treated with nhjd and various arsenicals . total arsenic contents in cells and tissues were analyzed by atomic fluorescence spectrometry ( afs ) as described previously . briefly , tissues were completely digested in nitric acid at 170c for 2.5  hrs and brought to 25  ml with distilled water , and 5  ml of the sample was mixed with 1  ml 5% thiourea - ascorbic acid solution . following 30  min incubation , aliquots were used for quantification of as contents with atomic fluorescence spectrometry ( kechuang haiguan instrument co. ltd , beijing , china ) . these assays were performed at the guizhou chemical analysis center of chinese academia of sciences . rna isolation and real - time rt - pcr analysis approximate 50100  mg tissue was homogenized in 1  ml trizol agent ( invitrogen , carlsbad , calif , usa ) , and total rna was extracted according to manufacturer 's instructions , followed by purification with rneasy columns ( qiagen , valencia , calif , usa ) . the quality of rna was determined by the 260/280 ratios , and by gel electrophoresis to visualize the integrity of 18s and 28s bands . the pcr primers were designed with primer express software ( applied biosystems , foster city , calif , usa ) as mt-1 ( bc027262 ) , forward : aatgtgcccagggctgtgt ; reverse : gctgggttggtccgatactatt . the power sybr green mater mix ( applied biosystems , foster city , ca , usa ) was used for real - time rt - pcr analysis . the cycle threshold ( ct ) values of the interested genes were first normalized with -actin of the same sample and expressed as percentage of controls . for cytotoxicity analysis , means and standard error were calculated from 5 separate cultures , and the lc50 values were estimated via graphics data were analyzed using a one - way analysis of variance ( anova ) , followed by duncan 's multiple range test . differential cytotoxicity between realgar - containing an - gong - niu - huang wan and arsenicals was evident in human nasopharyngeal carcinoma fadu cells . thus , the cytotoxicity potential of nhjd and arsenicals was examined in this cell line . figure 1 illustrated that the lc50 at 48  h for realgar - containing nhjd was 1200  m , for realgar was 2000  m . the most toxic arsenicals was sodium arsenite ( 35  m ) , sodium arsenate ( 400  m ) , arsenic disulfide ( as2s2 , 2000  m ) , and arsenic trioxide ( as2o3 , 280  m ) . confluent fadu cells were exposed to nhjd and arsenicals , all at the 100  m concentrations for 30  min , and cultures were thoroughly washed and cellular uptake of as were determined as described in methods . the results ( figure 2 ) show dramatic difference in cellular arsenic accumulation , nhjd , and realgar treatments resulted in 100200  ng  as / mg cellular protein , while sodium arsenite and sodium arsenate resulted in 1360 and 470  ng  as / mg protein , respectively . similar results were also obtained after 60  min and 120  min exposure ( data not shown ) . mice were orally administered with nhjd ( 600  mg / kg ) , realgar ( 600  mg / kg , equivalent to reaglar in nhjd ) , and the equal amount of as doses of sodium arsenite ( 36  mg / kg ) and sodium arsenate ( 88  mg / kg ) . the results ( table 1 ) show that the elevations of serum alt and bun after arsenite and arsenate , but these parameters were unaltered after nhjd , and realgar . histopathology was consistent with blood biochemistry , showing more severe liver and kidney damage after arsenite and arsenate , but mild or absent after nhjd and realgar . mice were orally administered with nhjd ( 600  mg / kg ) , realgar ( 600  mg / kg , equivalent to realgar in nhjd ) , and the equal amount of as doses of sodium arsenite ( 36  mg / kg ) and sodium arsenate ( 88  mg / kg ) . the results ( figure 3 ) show that the dramatic difference in tissue arsenic accumulation , nhjd and reaglar treatments resulted in approximately 200  ng  as / g liver , while arsenite ( 6200  ng / g liver ) and arsenate ( 3320  ng / g liver ) produced significant as accumulation . renal as contents were under detection limits for nhjd and realgar , but reached 3350  ng / g kidney after arsenite and 1500  ng / g kidney after arsenate . mt is a small , cysteine - rich , metal - binding protein playing an important role in metal detoxication . mt overexpression is a sensitive biomarker for arsenic - induced stress . as shown in figure 4 , hepatic mt-1 was increased 57-folds after arsenite and arsenate , but was not altered after nhjd and realgar . as shown in figure 4 , bottom , renal mt-1 transcript levels were also increased after arsenite ( 5-fold ) and arsenate ( 2-fold ) , respectively . in comparison , nhjd and realgar did not produce significant elevation in renal mt-1 transcripts . the present study clearly demonstrated that realgar and realgar - containing nhjd were much less toxic than sodium arsenite and arsenate in cultured cells and in mice , as evidenced by lc50 values , the elevated serum alt and bun concentrations in mice . toxicokinetically , much less as was accumulated in the cells or in tissues after realgar and nhjd . furthermore , the expression of stress - related genes , namely , mt-1 , was increased 27-folds after sodium arsenite and arsenate in the liver and kidneys , but was basically unaltered after realgar and nhjd , consistent with tissue damage . the present study fortified our recent observations in cultured cells   and in intact animals [ 913 ] , indicating that realgar and realgar - containing nhjd are different from sodium arsenite and arsenate and clearly demonstrate that the chemical forms of arsenicals underlies their disposition and toxicity potentials . in addition to the use of arsenic compounds in cancer chemotherapy [ 16 , 17 ] , realgar has been included in 22 oral patent traditional remedies according to pharmacopeia of china , and was claimed to have many beneficial effects for various diseases [ 13 , 16 , 17 ] . however , the major concern is their toxicity potential , which limits many realgar - containing remedies . nhjd is a most popular realgar - containing traditional chinese medicine ( http://www.wickpedia.org/ ) available in chinese grocery stores in the usa and in the europe and is used for many inflammatory diseases . to critically evaluate its toxicity potential is important for safely use of realgar and realgar - containing traditional medicines in the treatment of various inflammatory diseases . arsenic exists in the trivalent ( as , arsenite ) and pentavalent ( as , arsenate ) forms and is widely distributed in nature . in general , sodium arsenate ( ld50 112175  mg / kg ) is 4 - 5 times less acutely toxic than sodium arsenite ( ld50 1544  mg / kg ) , and the pentavalent organic arsenicals mma , dma , and tma are 40100 times less acutely toxic than arsenite . arsenicals in seafood mainly exist as organic forms , such as arsenobetaine , arsenosugar , and arsenocholine , with acute oral ld50 values 100500-fold above arsenite or arsenate . in traditional medicines , the oral ld50 for arsenic trioxide ( i.e. , arsenolite ) in mice is 3239  mg / kg , but the ld50 for realgar is 3.2  g / kg , a 100-fold difference in acute toxicity . thus , arsenical toxicity is highly dependent on the chemical form , and realgar ( as4s4 ) is much less acutely toxic than arsenic trioxide ( as2o3 ) and is also much less acutely toxic than sodium arsenite ( naaso2 ) and arsenate ( na2haso4 ) in the present study . it is generally assumed that the severity of poisoning is related to the total amount of poison ingested , and assessment of health risk associated with arsenic exposure from human ingestion of traditional medicines has typically taken this tactic [ 47 ] . however , the present study clearly showed that realgar was poorly accumulated into the cells and tissues and was unable to reach a critical concentration to cause tissue damage as compared to the same amount of arsenic given as arsenite and arsenate , with tremendous differences in their tissue contents . the disposition of these arsenicals in the body depends on various key factors including solubility , absorption , distribution , and excretion . i.e. , 70,000  ppm ) , corresponding to 28  mg arsenic per pill , of which only 1  mg arsenic finds its way into the blood stream , that is , only 4% of intake realgar is bioavailable . compared to realgar , 80% of orally given sodium arsenite and arsenate can be absorbed from the gastrointestinal tract , with much higher plasma as levels and tissue distribution [ 2 , 18 ] . thus , it is the amount of toxicants to the target organ , rather than the amount ingested or inhaled that makes a poison . various stresses have been proposed as an important mechanism involved in arsenic toxicity and carcinogenesis . mt is a small , cysteine - rich , metal - binding protein playing an important role in metal toxicity . induction of mt by arsenicals is dependent on arsenic forms , with sodium arsenite is the most potent and efficient inducer followed by arsenate and organic arsenicals , such as mma and dma , and mt - null mice were susceptible to chronic arsenic - induced hepatotoxicity and nephrotoxicity . mt induction is considered an adaptive mechanism to toxic metal - induced oxidative stress [ 14 , 15 ] and can be used as an indicator for arsenic - induced stress . in the present study , hepatic and renal mt was induced by sodium arsenite and arsenate , but was basically unaltered by realgar and nhjd , fortifying the observations from blood biochemistry that realgar and nhjd are much less acutely toxic than sodium arsenite and arsenate . traditional medicines are based on empirical experience and have their own theory and are generally safe at clinical doses . the regulation of metal - containing traditional medicines has been a topic of debate [ 27 , 13 , 2325 ] . we have been invited to write two reviews on this topic for arsenic ( j pharmcol exp ther ) and for hg ( exp biol med   and have challenged this bias by studying the relative safety of liu - shen - wan (  ,  as ) , an - gong - niu - huang - wan (  ,  as+hg ) [ 911 ] , and zhu - sha - an - shen - wan (  ,  hg ) . in the present study , nhjd is unique in that it is the most popular traditional medicine available not only in china , but also in chinese grocery stories worldwide . to study realgar is less toxic than arsenic oxide yet effective in cancer chemotherapy based on the recent literature . high dose of realgar for the long - term use did produce toxicity to the liver and kidneys , similarly , long - term use nhjd was reported to produce hepatotoxicity and nephrotoxicity . in the evaluation of realgar and realgar - containing nhjd in the treatment of various diseases , although realgar and nhjd are less toxic than sodium arsenite and arsenate , high - dose and long - term administration should be avoided to reduce undesired adverse effects and toxicity [ 2 , 18 ] .   in summary , the present study clearly demonstrated that realgar and realgar - containing nhjd are much less toxic to the cultured cells and to the intact animals , as compared to sodium arsenite and arsenate . the chemical forms of arsenicals determine their tissue accumulation and toxicity potentials , and thus the use of the total content of as to evaluate realgar - containing traditional medicines is inappropriate .
niu - huang - jie - du pian ( nhjd ) is a widely used traditional chinese medicine containing realgar ( as4s4 ) . 
 realgar has been included in many traditional medicines , but is often taken as arsenite for risk assessment . to evaluate true risk of realgar and realgar - containing nhjd , 
 their toxicity was compared with common arsenicals . in cultured cells , 
 the lc50 for nhjd ( 1200  m ) and realgar ( 2000  m ) was much higher than arsenite(35  m ) , arsenic trioxide ( 280  m ) , and arsenate ( 400  m ) . 
 acute toxicity in mice showed more severe liver and kidney injury after arsenite or arsenate , but was mild after realgar and nhjd , corresponding to cellular and tissue arsenic accumulation . 
 the expressions of arsenic - sensitive stress gene metallothionein-1 were increased 37-folds after arsenite or arsenate , but were unaltered after nhjd and realgar . 
 thus , realgar and nhjd are much less toxic than arsenite and arsenate . 
 the use of total arsenic to evaluate the safety of realgar and realgar - containing nhjd is inappropriate .