Absence of overt toxicity from feeding the flavonol, quercetin, to rainbow trout (Salmo gairdneri)

Absence of overt toxicity from feeding the flavonol, quercetin, to rainbow trout (Salmo gairdneri)

Fd(hem low~ Vol 23 No 12 pp 1077 1080 1985 Printed m Great Britain All rights reserved 0278-6915 85 $3{)0+000 Copvnght i 1985 Pergamon Pre~,, Lid AB...
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Fd(hem low~ Vol 23 No 12 pp 1077 1080 1985 Printed m Great Britain All rights reserved

0278-6915 85 $3{)0+000 Copvnght i 1985 Pergamon Pre~,, Lid

ABSENCE OF OVERT TOXICITY FROM FEEDING THE FLAVONOL, QUERCETIN, TO RAINBOW TROUT

(SALMO GAIRDNERI) S M PLAKAS*t, T - C LEL~: and R E WOLKE* *Department of Fisheries, Aquaculture and Pathology, and .~Department of Food Science and Technology, Nutrmon and Dletehcs, Umverslty of Rhode Island. Kingston, RI 02881 USA (Renewed 2 Januarl 1985 reztgtons recewed 30 Aprd 1985)

A~tract--The toxicJty of the plant flavonol quercetm, to rainbow trout (Sahno gazrdnert) was investigated Quercetm, which had been confirmed to be mutagenlc in the Ames Salmonellamammahan microsome test, was fed to trout at levels of 1 or 5% in the diet for 8 months Surwval, growth and feed conversion efficiency, selected haematologlcal parameters and the relative weights of heart, liver and spleen were unaffected by the ingestion of quercetm, and there were no histopathologlcal changes m any of the tissues examined

INTRODUCTION

Quercetln (3,Y,4',5,7-pentahydroxyflavone) is a ubNultous plant flavonold existing in both free (aglycone) and glycoside (e g rutm, quercetrln) forms in nature Quercetm and ~ts glycosides are c o m m o n flavonold compounds of food plants (Herrmann, 1976), and may enter the diet of humans and animals Quercetm and other flavonols have received considerable attenuon since the appearance of reports of their mutagenlc activity Brown & Dietrich (1979) estimated that 50 mg of promutagemc flavonol glycosides, in quercetm equivalents, may be consumed by man per day Some food substances from which flavonolds that are mutagenlc or potentially mutagenlc (with glycoside hydrolysis) have been extracted include certain pickled vegetables, onions, spices, tea and citrus fruit Juices (Fernandez & Vega. 1981, Mazaki. Ishll & Uyeta. 1982, Seino. Nagao, Yahagl et al 1978, Takahashi, Nagao. Fujino et al 1979, Uyeta, Taue & Mazakl, 1981) Quercetln has been found to induce frameshlft-type mutations in Sahnonella o'phtmurlum in the Ames test (Bjeldanes & Chang, 1977, Hardlgree & Epler, 1978, M a c G r e g o r & Jurd, 1978, Suglmura, Nagao, Matsushima et al 1977) Quercetln glycones have shown mutagenlc activity in the Ames test when exposed to glycosldases such as have been extracted from human faeces (Tamura, Gold, Ferro-Luzzl & Ames. 1980) and from rat caecal contents (Brown & Dietrich. 1979) Quercetln has also been shown to be mutagenlc and cytotoxlc by other test systems (Meltz & MacGregor. 1981, Sahu, Basu & Sharma, 1981. Umezawa, Matsushlma, Suglmura et al 1977. Watson, 1982) Investigations of the carcinogenlelty of quercetln have yielded conflicting results Hlrono, Ueno, Hosaka et al (1981) found no evidence that quercetln tPresent address Fishery Research Branch, Food and Drug Admmlstratlon, P O Box 158, Dauphin Island, AL 36528. USA

was carcinogenic to rats at relatively high dietary levels (up to 10%) in long-term feeding studies whereas Pamukcu, Yalclner. Hatcher & Bryan I1980) reported that quercetin was carcinogenic to rats at a much lower dietary level (0 1%) Quercetln was not carclnogemc when fed to hamsters (Morlno Matsukura. Kawachl et al 1982) or mice (Hosaka & Hlrono. 1981) Quercetln could enter the diet of fish or other aquatic animals through plant materials, either m the natural environment or under aquaculture conditions Depending on the results of comprehensive toxicity tests, quercetm might also be intentJonally introduced into the diet to provide the advantage of some useful property, such as ~ts ant~oxidant capacity (Herrmann. 1976. Kuhnau. 1976. Younes & Slegers 1982) In the present study we ,nvestlgated the toxicity of high dietary levels of quercetln to rainbow trout in an intermediate-term feeding study EXPERIMENTAl

Animals and mamtenance Rainbow trout (Sahno galrdnerl) of disease-free stock were obtained from

the North Attleboro National Fish Hatchery. North Attleboro. M A The fish were randomly distributed into three groups of fourteen fish Each group was kept in a 250-hire fibreglass tank and the tanks were supphed with chlorine-free city water m a flow-through system The average water temperature was 12 C and the dissolved oxygen level was maretamed at > 5 mg/htre Diets The c o m p o s m o n of the basal d~et is given m Table 1 The two test diets were identical to the basal diet except that an equal weight of the cellulose component was replaced by 1 or 5% quercetln (3,3',4',5,7-pentahydroxyflavone, Aldrich Chemical C o , Milwaukee, Wl) We confirmed this compound to be mutagenlc by the Ames Salmonella/mammahan mlcrosome mutagenlc~ty assay (Maron & Ames, 1983), using strains TA98 and TA100 The diets were processed into a pelleted form containing approximately 30% moisture using a Hobart Kitchen Aid

1077

1078

% M PI_AKAS~'l ,#[ Table I ( o m p o , q i t o n ut the ba'~al dtct Component*

L~.~.I (",,) m diet

Casein Gelatin Dexiro',e C o d - h ' ~ e r oil C o r n oil V i t a n l m nl i -,~) Mineral mi\{ :<-Cellulo~e~ '&aler

:;2 II S II l 0 II 6 (I 2 0 2 q 4t) ~ ~, l(J (1

*All c.omponents ~,ere obtained Irom ICN Nutritional Blochemit.aN (Cleveland OHI except the corn Ol1 ( Mazola C PC lni~.rnatlonal Englev, ood Cliff"> N J) "l'A,> ,>peLihed b,, the National Research Council 1197t1 7he mix ,>upphed the following per 100g el diet thiamine H ( I 6 4 m g riboflavin 1 4 4 i n g mac.mamlde iIol 0 ]q g l,>Ltirbic acid 0 12g "vitamin ~l 250(111' "~ltamm [2 S(IIU c.hohnc t.hlorldt. llSg ++Modlhed BernharI Tomarelh ,>alt mittur~. ~An equal amount el ~-~.ellulo~e ,aas replaced with I or S",, qucict+tm in the te,>t diets

food mixer ( H o b a r t C o r p , Tro> O H ) The diets were p r e p a r e d m o n t h l y and stored at - 2 0 C until they were used Quercetln has previously been found to be stable in a dr$, diet for 2 m o n t h s at 4 C ( H i r o n o et al 1981)

~ a s killed after I 2 el 3 m o n t h s of leedlng and was examined histologically At the end el the stud) a b o u t 20 hr after the last teed six lish Irom each group were randolnl.,, selected tot detailed examination The remaining llsh in ~.ach tank ~ete given a gross e x a m i n a t i o n &t the time of killing the fish selected for detailed ~tud3 were anaesthetized \~lth a 200-rag htte solution el MS 222 [ttic,unc nlethaneMtlphonate Sigma Chemical ( o St [ o u i s , M e ) and I 5 m l of whole blood was extracted at the ~.audal pedtincle using hepartmzed syringes HaernatocJlt and total haemoglobin d e t e r m i n a t i o n s l Sigma ('heroical ( o Technical Bulletin No 525) wcrc p e t l o i m c d untnedlatelx The temalndei el cac.h blood sample was then c.entnluged at 1300g tel 15tmn and the plasma was w i t h d r a w n and refrigerated Plasma glutaml~. p\ruxtc t r a n s a m m a s e dCtl%lt~ V~,IS measured on the same d, D b> the m e t h o d glxen in Sigma Technical Bulletin No 55-['V and based tin tile method el Wroblewski & LaDuc (1956l The plasma samples V ele then r i o i 0 n Ioi a m a \ l m u m o1+2 \~k tliltil l u t t h e r anJl}qlS Alter blood s a m p h n g ~hole-body x~,elghts and h xer spleen and he,u t \~elghts ~ c l e l ecorded All Inalor tissues (heart lixm spleen k~dne) inusc.lc grin and g a s t t o - l n t e q l n a l tract} v~ete then fixed in a 10"o buffered l\wlnahn solution iOuilnely sectioned at 6 j i m and Mairied ~ l t h h a e m a t o \ ' , h n cosin lol o \ a m i n a t i o n under a hght mlCiOscopo PlaMna I t e m the ~.ontrol group and the g l o u p gl\OI1 tile 5"o quercetln-supplemented diet ~,~.,is also analysed for the following total plotein using blulel reagent (Sigma Chemical ( e l by the method el ( m r n a l l Barda,alll & D a \ l d t1949) albumui using b l o m cresol green reagent (Sigma Chemical C o l b,, the m e t h o d of D o u m a s & Blggs 11c)72) total dmle',ieiol and trlglycerides b b the enzymatic a~s
E~pet zmental destgn and ~ondu~ t The diets were fed to the three groups of fish once a day, 5 days/wk at a rate of 1% of body weight, da5 The fish were mdlvtduall) ~ e l g h e d once e~er~ 2 wk a n d the feeding a m o u n t s were adjusted according b for each group Based on this feeding rate the daily rate of c o n s u m p t i o n of quercetm w as approximately 100 or 500 m g l k g b e d ) weight for fish fed the I and 5 % q u e r c e t t n - s u p p l e m e n t e d diets, respecmel> G r o w t h a n d feed efliclencj, data were t a b u l a t e d after 6 m o n t h s of feeding the experimental diets However, the fish c o n t i n u e d to be led at the same rate for the r e m a i n d e r of the feeding trial which was t e r m i n a t e d at 8 m o n t h s One fish from each group

b~

Sigma

KIt

Nos

I", 5°0

ie-

The present stud} re\ealed no exert toxic effects from feeding quercetin to rainbow trout No hsh lrom any of the test groups died during the feeding trial, and there was no exldence el an,; Io~s of appetite associated w~th the presence of quercetm m the diet G r o w t h and feed c o m e r s l o n were slmthtr with all three diets (Table 2) Ambrose R o b b l n s &

Mean bod~, weight (g)* Diet

U~5-I, IV

RF%t,IT% %NI) 1)1%(I%%1()N

Table 2 Grov, th and teed um,,erslon efhc.lcm.', el I nnbov, flout lt.d the h isal diet or diet} ,>upph.mented with I or 5% quert.etm |or 24 wk

Basal Ouer~.etm

"45(I a n d

spectixel) and plasma gmt
Initial

Final

~,elght ganl C ,, )

St ~ 2+'t s0 ~ IR 79 I- 16

222 + 21 211 + 43 2(16 + 28

161 164 161

Fl_t.d c.on\ trslon cfllclcn,-\ 4I Iq I IS I 21

*Value,, are mean,, ± SD (n - 14 for initial hod', weight n I I lot lm d hod'+ weight) There ,sere no `>lgmhcant dlfferenu.',

gl,,en the quer~.etm-,,upplemt.nted dlLt,> Lind tilt. ~.onttol group m LIther initial or hnal mean bod~, 'aelght +Total weight ot teed ~on,>umed total v, mght gain

Absence ol touclt) o f quercetm to trout D e E d s (1952) found no mh~bltmn o f g r o w t h m a l b m o rats fed diets c o n t a , m n g up to I°o quercetln for > 5 8 w k However, Pamukcu et al (1980) noted a stgmficant dechne m the growth rate o f albino rats after 20 ~ k o f feeding a diet c o n t a m , n g 0 1% quercetln N o significant d,fferences ( P ~> 0 05) were found between the quercetm-treated and the control groups m the relative weights o f the hver, spleen and heart after 32 wk o f feedmg (Table 3) Similarly, A m b r o s e et al (1952) found no significant d~ffcrences between the weights o f the major organs o f rats fed a control dmt and o f those fed dints containing quercetln F u r t h e r m o r e , we found no significant differences ( P ~> 0 05 by Student s t test) between fish fed the 5"/,, quercetln diet and those led the basal dmt w~th regard to h a e m a t o c n t and haemoglobin determinations or plasma ~alues for total protein, albumin, glutamtc oxalaceuc transamlnase, glutam~c py~uxm transammase, trtglyccrtdes or cholesterol (~alues not reportedl No gross or h~stologmal les~ons were noted m any o f the fish examined d u r m g or at the end o f the study A l t h o u g h the mutagentc~ty o f quercetm ~s well d o c u m e n t e d and the findings are in general agreement. conflicting results have been o b t a m e d m t o y tctty studms m rats Pamukcu et al (1980) showcd quercetm to be an obvious mtestmal and bladder eDthehal carcmogen m albino rats led the flavonold at a dietary level o f 0 I % for 406 days However A m b r o s e et al (1952) d e m o n s t r a t e d no toxic effects m association ~tth feeding albmo rats a diet contalnmg 1% quercetln f o r 4 1 0 days H~rono et al (1981) & d not find quercetm to be carcmogen,c to ACI strata rats when ~t was fed at dietary lexels o f 1 or 5?0 for 540 days or at 10% for 850 days Smgle and multtplc oral doses o f quercetln (as high as 2 g,'kg bod3 weight) administered to pregnant rats had no teratogemc effects (Wlllh~te, 1982) N o evidence o f toxicity was found when quercetm was administered m smgle ~ doses (100 mg,'kg body w,mght) to rabbits ( A m b r o s e el al 1952) The efficmnt metabolism and excretion o f quercetm, as d e m o n s t r a t e d m rats (Brov~n & Grtffiths 1983, Ueno, N a k a n o & H~rono, 1983), may explain the a p p a r e n t lack o f toxicity that has been observed m most an,mal sludms Several useful properties o f quercetm have been ~dennfied Basarkar & Nath (19gl & 1983) found ~t to have hypocholesterolaemtc and h y p o h p t d a e m t c activity when administered orally to rats fed cholesterol-supplemented diets Quercetm has been d e m o n s t r a t e d to ha~e a n n - t u m o u r dCtl'~,lty (Molnar Beladt, D o m o n k o s et al 1981), antwtral activity Table "~ Relative organ weights ol rambo'a trout led the basal diet or diets supplemented with I or 5% quercenn lor q2wk Relatp, e wmght (% of total body ',,,mght) ot Dmt Basal

Quercenn

1% 5",,

Lp~ er

Spleen

I 21 +_024 0 9 7 ± 0 12 0 9 6 + 0 19

009 +0t)~ 008 "-002 006 k002

Heart I) 1 8 + 0 0 ~ 0 18+005 020±003

Values are means ± S D tor subgroups o f sl~, hsh r a n d o m b selected from each treatment group There 'aere no slatls.call,, slgmflcant differences ( P ~> 0 05 h~, Student s t test) bctv, een the quercetm-treated and the control group', in the relatr, e veelghts o f the h',cr spleen or heart

Ft

[

21 12

D

1079

(Veckenstedt & Pusztal 1981), annoxJdant capacity (Younes & Stagers, 1982) and the abdlty to inhibit both the release o f histamine and the biosynthesis o f a slow-reactmg substance o f anaphylaxm (Hope Welton, Fmdler-Nagy el a/ 1983) Further study may indicate other beneficial actwtttes and a p p h c a n o n s o f quercetm and related c o m p o u n d s ,l¢l~nolah'~&ement~--The authors ackno~xledge the cooperation and assistance of Mr P Mullane (North Attleboro National Fish Hatchery) in supplying the fish of T Miller and S Polol\kx (Marine Patholog? Laboratory Um,~erslt) of Rhode Island) m microscopical preparauons and of A Senecal (Department of Food Science and Nutrition, Unp~erslty of Rhode Island) m microbiological assays This work ~s published ascontrlbunon no 2110 of the Rhode Island &grlcultmal Experiment Station and sponsored b? NOA& Olhcc ol Sea Grant U S Depamnenl o f ( o m m e r c e under (want No N Ag5-4&-D-SG-094 REI"E R F N ( F S

Ambrose A M Robbms D J & DeEds F (1952) Comparative tO'ClCllles of quercetln and querc~tnn J 4m phwrn 4s~ 41, 119 Basarkar P W & Nath N (198l) Cholesterol lowering a c t i o n o f ' ~ l l d m l n P-hke compounds m rats ]tldtotl J e \ p Bud 19, 787 Basarkar P W & Nath N (1983I H.'~pocholesterolemlc and h~pohpldemlc actwlt~ ol querceun--,t ~ltalnm P-hke compound in rats hi&an J reed Res 77, 122 Bjeldanes L F & Chang G W (1977) Mutag~.mc ,tctlwty of quercetm and related compounds Stteme, N } 197, 577 Brown J P & Dlemch P S (1979) Mutagemclt,¢ of plant fiavonols In the Salmonella'mature,than mmrosome test Acmahon of flaxonol gl,~cosldes b) mixed gljcosldases from rat cecal bacteria and other sources IlUla[lOll Rt's 6@ 223 Brown S &Gnffiths L ¢ (198"~) New metabohtes ol the naturally-occurring mutagen quercctln the pro-mutagen, rutln and of taxlfohn ExperwnHu 39, 198 Doumas B T & Blgg~ H G (1972) 5tandmd lh,thods o/ Elmual Chemt~ttt Vol 7 Edited b) G & Cooper, p 175 Academic Press New "York Fernandez M & Vega F A (1981) Innocuous alteranons in preserxed onions 4/hum ~epa L J Fd S~z 46, 1966 Gornall A G Bardawlll ( J & David M M (1949) Determination of serum proteins b 3 means of the bluret reacnon J htol Chem 177, 751 Hardlgree A A & Epler I L (1978) ( o m p a r a m e mutageneszs of plant flavono~ds m m~croNa] systems I/utatton Res 58, 231 Herrmann K (1976) Flaxonols and lla~ones in tood plants a review J Fd fe~hnol It, 433 Hlrono I Ueno l , Hosaka S Takanashl H Matsushlma T , Suglmura T & Natorl S (198[) Carcmogenlclty examination of quercetm and rutm in ACt rats (ameJ Lett 13, 15 Hope W ( , Welton A F Ficdler-Nag'~ ( BatulaBernardo C & (ofte,, J W 11983) In ~ltro mhlbmon ol the biosynthesis ol slow ruactmg substance ofanaph,,laxls (SRS-A) and hpox>genase acnvlt~ b3 quercetm &oehem Phatma¢ 32, 367 Hosaka S & Hirono l (1981) Carcmogenn.lty test ol quercetln b~ pulmonary-adenoma btoassav m strata A mice Oann 72, 327 KarmenA I1955) Anoteoqthespectrophotometncassav of glutam~c oxalacetlc transammase m human blood serum J (Im bTtesl 34, 131 Kuhnau J (1976) The flaxonmds A class of seml-essennal food componcnts their role in human nutrmon ~l M Ret ,~eutr Diet 24, 117 MacGregor J T & Jurd L (1978) Mutagemc~t~, of plant

lO80

S M PLAK~.S el al

fiavonmds structural reqmrements for mutagemc activity m Salmonelkl t~phtmurmm Mutatwn Re~ 54, 297 Maron D M & Ames B N (1983) Re,,lsed methods for the Salmonella mutagemclt> test Mutatum Re~ il3, 173 Mazakl M Ishn T & Uyeta M (1982) Mutagemmty of hxdrol~sates of citrus fruit juices Mutatum Res 101,283 M d t z M L & M a c G r e g o r J T (19{?I) Actp~lty of the plant flavanol quercetln m the mouse lymphoma L517g'~ FK' mutat,on. DNA single-strand break, and Balb,'~. tT3 ~.hcmlcal translk~rmatmn assays Mutatton Re~ 88, 117 Molnar J Beladl l . Domonkos K , Foldeak S Boda K & Veckenstedt & (1981) Antltumor activity of flavonolds on NK.L 3 ascltes tumor cells Neopla~ma 28, I 1 Mo~mo K Matsukura N , Kawach) T Ohgakl H , Suglmura T & Hirono 1 (1982) Carcmogenlclty test of qucr~.etm and rutm m golden hamsters by oral administration C ar~ mogenest~ 3, 93 Natmnal Research Council (1973) Nutrient Reqmrements of Trout Salmon and Catfish p 49 National Academ~ ol Scmnces. Washington, DC PamukcuA M YalcmerS, HatcherJ F & BryanG T (198(I) Quercetm a rat intestinal and bladder carcinogen present m bracken fern (Ptertdmrn aqudmum) ( a m e r Re~ 40, ~,468 Sahu R K Basu R & Sharma A (1981) Genet,c tox,colog~cal testing of some plant flavonmds b~, the toreronucleus test ,~tutatwn Res 89, 69 Semo Y Nagao M Yahag~ T , Sug~mura T . Yasuda T & Nlshlmura S (1978) Identification of a mutagemc substant.e m a spice sumac, as quercetln mutatton Res 58, 22s Sugm~ura T , Nagao M , Matsushtma T , Yahagl T Semo

Y Shlral A . Sa,aamura M N a t o n S, "roshlhlra K Fukuoka M & Kuroyanagl M (1977) Mutagemclt~ of fla~one dertvate, cs Pro~ Jap A~ad 53(B), 194 Takahashl Y , Nagao M , Fujmo T Yamalzuml Z & Suglmura T (1979l Mutagens m Japanese plt.kle Identified as flavonolds Mutatum Re~ 68, 117 Tamura G Gold C Ferro-Luzzl A & Ames B N (1980) Fecalase a model for activation ol dmtar,, gl~cosldes q~ mutagens by intestinal flora Pro( It~llt'l l(~,'d S~ t ~' g) 4 77, 4961 Ueno I . N a k a n o N & H l r o n o 1 (198t) Metaboh~.lateol ()4C) quercetm m the A(_l rat Jap J t'xp ~h'd 5~, 41 Umeza,aa K . Matsushlma T Suglmura T , Ihrakawa T Tanaka M , Katoh Y & Takayama S (1977l In vitro transformation o[ hamster embryo ~.clls bv qucrct.tm Toxt~olog~ Left i, 175 Uyeta M , Taue S & Mazakl 1%1 (1981) Mutag~.mclt) ol hydrol)sates of tea infusions ,~lutatum Res 88, 293 VeckenstedtA & P u s z t a i R (1981) Me~.hamsmofantlxlral action of quercetln against ~.ardlovlrus mfe~.tlon m nnee 4ntwtral Res I, 249 Watson W A F (1982) The mutagemc a~.tv~lt> ol quercetm and kaempferol m Drosophtla m(la~7ok,a~tct ,~lutatwn Re~ 103, 145 Willhlte C C (1982) Teratogemc potential c~f quercctm m the rat Fd C/win Toxu 20, 75 Wroblewskl F & LaDue J S (1956) Serum glutamlc pyruvlc transammase m cardiac and hepatic d~sease Pro< So~ exp Btol ~led 91, 569 Younes M & Slegers C - P (1982) Inhibition b,, some fla,,onolds of enhanced spontaneous hpld peroxldatJon in wtro duc to glutathlone depletmn in '¢1",o 4t~hs T o x . o l Suppl 5, p 335