BIOANALITICAL
HIGH-THROUGHPUT MOLECULAR BIOASSAY FOR THE
DETERMINATION OF DIOXINs
AND PCBs IN MEAT AND ANIMAL FEEDS
Sang-hee Jeong. Joon-hyoung Cho, Michael S. Denison ', Ok-Kyung Kim
National Veterinary Research & Quarantine Service, 480, Anyang 6-dong, Anyang city, 430-016,
KOREA' Department of Environmental Toxicology, University of California, Davis, CA 95616-8588,
USA

Introduction
Polyhalogenated aromatic hydrocarbons (PHAHs) such as dioxins and PCBs induce a wide varietyof toxic effects including tumor promotion, immunotoxicity, birth defects and changes in hormone
metabolism'". The trials for limiting human exposure to dioxins is more important than ever
because 2,3,7,8-letrachlorodibenzo-p-dioxin (TCDD) has been recently re-classified as a Class 1
human carcinogen by International Agency for Research on Cancer (lARC)'. Animal products
including meal, milk and etc are major exposure sources of dioxin to human^ Such as, animal
feeds are major exposure sources lo livestock animals. Monitoring dioxin levels in food andanimal feeds is a useful basis for identification of contamination events in animal products andprovides a basis for assessing the level of current exposure lo human Ihrough animal diet''.
However, dioxin analysis through either conventional or modem analytical techniques involves
exhaustive sample preparation and long time consuming for analysis. This study is performed forlowering costs and saving time for the delermination of residual level of dioxins in meat andanimal feeds by bioassay system using recombinant cell line that is very sensitively andspecifically responding to dioxins and PCBs.
Methods and Materials
Chemicals
All chemicals used were of pesticide analysis or HPLC grade. 2,3,7,8-TCDD, 1,2,3,7,8-PnCDD,
1,2,3,4,7,8-HxCDD, 1,2,3,4,6,7,8-HpCDD, OCDD, 2,3,7,8-TCDF, OCDF, PCB126, PCB169,PCB 105, PCB 118, PCB 156 were purchased from ChemService(Weslchesler, USA). 17 different
2,3,7,8-congeners of PCDD/F (500ng TEQ/ml of nonane) was purchased from Cambridge Isotope
Laboratories Inc. (Massachusetts, USA)
Recombinant cell line and culture condition
Mouse hepatoma cell line (Hepalclc7) stably transfected wilh the pGudLucl.l plasmid containingluciferase reporter gene and four dioxin responsive elements was used'. The cells(HEPALUCl.l)
were grown in 24-well cell cullure plates in 0.5 ml minimal essential medium (a-MEM, Gibco)
wilh 10% heat-inactivated fetal bovine serum, 50 lU/ml penicillin and 50 ng/ml streptomycin. Thecells were grown in 24-well cell culture plales with 0.5ml growth medium. The cells were treatedwith test compounds or sample extracts solved in DMSO (1% of medium) for 4.5 hrs. Luciferase
activity was measured wilh luminometer.
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Preparation of spiked fat of meat or animal feed samples
Fal was prepared from beef containing large portion of fat or swine feed ihrough n-pentane
extraction. Clean fat passed through activated carbon was prepared. An aliquot of fal wasdissolved in 4 limes volume of A7-hexane/dielhylelher(97:3, v/v) and mixed wilh 17 differeni
2,3,7,8-congeners of PCDD/F lo final concentration of 100 pg TEQ/g fal. Standard samples forcalibration curve are made by dilution of slock fat wilh clean fat al six differeni dioxin levels of 0,0.2, 0.4, 2, 5 and 10 pgTEQ/0.5g fat.
Clean-up of meat fat or feed fat for bioassay
Meat fat or feed fal was extracted from ground meat (lOg) or feedsluffs (I50g), and then 0.5g fat
solved in 2ml distilled /7-hexane/dielhylether (97:3, v/v) was added to 8g of 33% acidified silicagel
colume. The final extract was solved in 5iil DMSO and treated to the recombinant cells for 4.5 hrs.
Luciferase synthesized in the exposed cells was measured. All procedures are described in Fig. 1.
Fat collection from samples (lOg meat or lOOg feed)
Grind thoroughly in mortar
Extract fal with 2 fold volume of/7-penlane
Repeat this exlraclion slep unlil the pentane remained colorless
Filler the exlract over glass mineral wool with anhydrous sodium sulfate
Wash the filter wilh /7-pentane
Evaporate the combined filtrate to dryness under vacuum
Clean-up of fat

Load aliquol of 0.5g fal in 2ml distilled rt-hexane/diethyl ether (97:3,v/v) lo
pre-rinsed 33% acid silicagel column
Elute dioxin wilh 18ml /7-hexane/dielhyl ether (97:3,v/v)
Dry the eluate by rotor-evaporation and then under a gentle flow of nitrogen
Add 5 id DMSO, mix with 0.5ml medium(l% vehicle/ml culture medium)
Treatment of extracts to cells for 4.5hrs
Determination ofthe Luciferase activity
Calculate the dioxin level against a dose-response curve generated from 17 mixeddioxins standards included in each test
Fig. 1. Flow scheme of dioxin bioassay
Statistics
For delerminalion of the TCDD equivalency factors (TEF) for PCDD, PCDF and PCBs in thebioassay, dose-response curves ofeach chemical in triplicate for every concentration were made.
EC50 ofeach chemical was obtained by fitting the dose-response curve using an sigmoidal fitting
method (Origin, version 6.0). It represenled the concentration of agonist giving a half-maximal
response of 2,3,7,8-TCDD. The TEF was calculated by dividing the EC50 value of 2,3,7,8-TCDD
by EC50 value ofthe compound of inlerest. TEQs from HR-GC/MS dala were calculated using

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17BIOANALITICAL
WHO TEF values (1998)^ Dioxin quantities obtained by bioassay were calculated by comparison
ofthe luciferase activity induced by a sample extract againsi a calibration curve generated from lheeach concentration of 17 mixed-dioxin standards spiked lo clean fat or elution solution followedsame procedure lo sample.
Results and Discussion

1. TCDD-inducible luciferase activity in recombinant cells
The dose dependence of luciferase induction in HEPALUCl.l cells was determined byincubation of the cells wilh increasing concentrations of TCDD for 4.5 hrs. Induction ofluciferase was dose dependent, with a maximal induction by 500pM TCDD (11 fold), EC50 of10.29 pM and a minimal detection limil of aboul 0.1 pM (Fig.2). 0.1 pM was equal lo 16fg per
0.5ml cullure medium (24 well). Wilh consideration of these resulls, the recombinant cell can beused for bioassay system of highly sensitive and throughput.

60000

.

50000

---••

•

•

•

/JC.:..,-...---...,:.
20000i•*"'"'.:•.•'• V;,%:;: '"•-•-^•^•:^f~~^1E-30.01.

—3 30000_i10000^

0.1

1101001000

TCDD Concentratlon(pM)

Fig.2. Dose-response curve for the induction of luciferase activity by 2,3,7,8-TCDD in
HEPALUCl.l cell.Dioxins- and FCBs-inducible luciferase activity in the recombinant cell line

The dose-responses for a number of olher selected dioxins and some coplanar PCBs were also
observed (Fig.2). The shape ofthe curves for these compounds was comparable lo that ofTCDD,
but shifted to a higher dose range. The maximum responses of these chemicals were also similar,
with the exception of PCB 126(22% higher response), and PCBs 105 and 118(67-69% responsesof that of TCDD). The EC50 values of dioxins and PCBs determined by sigmoidal fitting
melhod (Origin program 6.0) with fix value of maximal response of 500pM TCDD (Table I).
There was a good correlation between WHO-TEFs('98) and this bioassay TEFs. Also, luciferase
acfivity induced by TEQ dose ofthe 17 differeni 2,3,7,8-substiluled PCDDs and PCDFs wascomparable lo that by same dose ofTCDD (dala are not shown)

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BIOANALITICAL

70000 -—•- 2,3,7,8-TCDD• 1,2,3,7,8-PnCDD

1,2,3,4,7,8-HxCDD
1,2,3.4,6,7,8-HpCDD<> OCDD•< 2,3,7,8-TCDF
OCDF/•• PC8126* - PCB169• PCB105•.
-» PCB118
PCB 156,,•«•A

60000 -

60000 -

4000030000 -^'«•20000' / • ••S'i'A

•

/a ;
'•

0-

1

1E-4

1E-30.01••A-s«•I'.li.9/»—1E-5s,*//

10000-1E-6•. *• * • •0.1

*'"

410'

100 1000 10000

Concentration(nM)

Fig. 3. Dose-responsive curves obtained in the bioassay wilh a number of differeni dioxins andPCBs.
Dala points are means ofthe three independent measurements.

Table 1. Toxic Equivalency factors (bioassay-TEFs) for induction of luciferase in HEPALUCl.l cells of anumber of selected dioxins and coplanar PCBs
Compound
2,3,7,8-TCDD
1,2,3,7,8-PnCDD
1,2,3,4,7,8-HxCDD
1,2,3,4,6,7,8-HpCDD
OCDD
2,3,7,8-TCDF
OCDF
PCB 126
PCB 169
PCB 105
PCBl 18
PCB156ECSO (pM)*
10.29
14.80
139.2
1,185
98,601123.8113,921
120.8
1,267
110,972
64,245
18,423

WHO-TEF('98)

I

11
0.1

0.70
0.074
0.00870.00010.083
0.00009
0.085
0.00810.000093
0.000160.00056•* ECSO values were calculated by sigmoidal fitting melhod

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Bioassay-TEF

190.010.00010.1
0.0001

0.1
0.01
O.OOOl0.00010.0005

BIOANALITICAL
3. Limits of detection and quantitation in bioassay

Figure 4 shows the calibration curves for dioxins obtained from 8 independenl experimenls overa period of 2 months. 0, 0.2, 0.4, 2, 5, and 10 pgTEQ of 17 differeni 2,3,7,8-subslituled PCDDs
and PCDFs were added lo 2ml solution of hexane/diethyl ether (97:3) and then processed same
treatmeni lo sample. The luciferase aclivilies induced by spiked standards lo 0.5g of clean fal(from beef or animal feed producis) and then processed same trealmeni showed same luciferase
activities to those of spiked standards lo elution solution omitting clean fat (dala not shown).
With this reason, calibration curve wa's made from clean fat-omitted procedure. The withinlaboratory coefficients of variation (CVs) ranged from 1.8 to 6.3% and mean recoveries ofthe
spiked dioxins from the fal (or elution solution) ranged from 66.6 lo 143.2%(Table 2). These
values of CVs and recoveries are satisfied lo the recommendations for validation of analyticalmethods by CODEX, EU EMEA and USA FDA. The detecfion limit(LOD) and quanlilalionlimil(LOQ) were calculated from 8 different experiments(n=24) following lo the EU VICH
guideline on validation of analytical procedures(1999). LOD was 0.33 pgTEQ/0.5g fat and LOQ
was 1.00 pgTEQ/0.5g fat. The tolerances or guidelines for meats are from 3 to 5 pgTEQ/g fat
in European counlries. So, this bioassay can be used for the determination of dioxins and PCBs
in meat. In the case of animal feed, guidelines for dioxins in complete feeds is seltled as 0.75
35000 -300001-J

25000iDote
00.204
2510

RLU

94oa
9375
10443
14545
22898
32321

RLU(Er)591
492
341
5B244459G

Y - A • B • X
W t i g h t o<v«n by D i i a l _ S D 3 •tocoa20000-(015000 -Z'#23ir b a o .
EnofAB

46e9566 02885
2397 50962

243.86005
61.05607

RSON

P

0.99503

1.965096<0.0001

S

10791112

Dosetpg TEQ/O.Sg fat)ng/kg product in Belgium. The bioassay system can be also adopted lo determine dioxins inanimal feed producis.
Fig. 4. Calibration curve for 17 mixed-dioxins (n=24).

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BIOANALITICAL

Dioxin conieni(pg TEQ/0.5g fal)
00.20.425CV(%)

Recovery(%)

6.3
5.2
3.3
4.0
1.9

143.2 ±7.5122.3 ±6.1
72.1 ±2.461.2 ±1.967.6 ±1.4

1.866.4 ±1.2

10LOD"(pgTEQ/0.5gfat)

LOQ""(pgTEQ/0.5gfal)

0.33

1.00

n=24, LOD: detection limil = 3.3o/S(a,lhe standard devialion ofthe y-inlercept of regression line
. in calibration curve; S, the slope ofthe calibration curve), LOQ: quanlilalion limit = 3xL0DI

4. Correlation between Bioassay- and GC/MS-determined TEQ contents in meats and animal feeds
The meals used in bioassay and HR-GC/MS analysis were collected during 1999 from imported
or domeslic beefs. Contents ofthe 17 mixed-2,3,7,8-substiluted PCDD and PCDF congeners
were determined by HR-GC/MS analysis and then TEQ concentrations were calculated using
the WHO(1998)-TEFs. Correlation belween bioassay- and GC/MS-determined dioxin levels was0.85. The HR-GC/MS analysis revealed any samples that exceeded the tolerance of 5pgTEQ/g
fal. Bioassay-determined dioxin levels were approximately 1.5 limes higher than GC/MSdetermined level (Fig. 5).
The animal feeds used in bioassay and GC/MS analysis were collected during 1998 fromfarmland growing cattle, pig and poultry. The correlation between bioassay- and GC/MSdelermined dioxin levels was 0.75. Neither the GC/MS analysis, nor the bioassay revealed any
samples that exceeded the Belgian tolerance of 0.75 pgTEQ/g feed. Bioassay-determined dioxin
levels were approximately 2 times higher than GC/MS-determined level (Fig. 6). It can beconcluded that bioassay is a very valuable tool, allowing the screening of relatively large
number of meal or feed samples forthe conlaminalion of dioxins and dioxin-like PCBs.

///•

1 Sllll

/3J-.

.

r^.

JO.S

1.0.

1

1.5.

12.0

•

12.S

•

1

•

3.0

HR-GC/MS|pg

1

3.5.

0 Ills.

I p-3td

1.

4.0

TEQ/g

1

4.5r—,

5.0

1

1

5.5.

,6.0.

,0.5fat)Fig. 5. A comparison ofthe dioxin TEQ-contents determined by bioassay wilh those from HRGC/MS for 10 meat samples.

ORGANOHALOGEN COMPOUNDS
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21BIOANALITICALoUJ0.4w

0.2L n . i r R i B i a i i n n Ihrouoti e r q n to. O i r i l _ F :PirMnUlf

V.baEf«0)
B.9bl.tSll7

0.414140.1 0.0

•'•
0.0

•
0.1

RSDN

P

0.T4B19

0.1S314100.07124

0.2

0.3

HR-GC/MS (pg TEQ/g feed)

Fig. 6. A comparison ofthe dioxin TEQ-contents determined by bioassay wilh those from HRGC/MS for
10 animal feed samples.

Acknowledgements
This sludy is performed in Nalional Veterinary Research & Quarantine Service (NVRQS) andsupported wilh budgei. HR-GC/MS analysis in meat and animal feed producis was performed inDepartmeni of environmental engineering in University of Seoul and Environmeni Toxicology
Division of NVRQS, respectively.

Reference
1. Murk A. J., Legler J., Denison M.S., Giesy J.P. Van De Guchte Cand Brouwer A.(1996) Fundam
Appl Toxicol. 33, 149
2. Hayward D.(1999) Inside laboratory management of AOAC. 3, 14
3. Garrison RM., Tullis K., Aarts J.M.M.J.G, Brouwer J.P, Giesy J.P and Denison M.S.(1996)
Fundam Appl Toxicol. 30, 194
4. Bovee T. F.H., Hoogenboom L.A.P., Hamers A.R.M., Traag W.A., Zuidema T., Aarts
J.M.M.J.G.,Brouwer A., and Kuiper, H.A.(1998) Food Addi Contam. 15, 863
5. WHO ECEH, IPCS (1998) Executive summary. Assessment ofthe heallh risk of dioxins: reevaluation ofthe tolerable daily intake

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