Determination of 2,3,7,8-TCDD toxic equivalent factors (TEFs): support for the use of the in vitro AHH induction assay

Chemosphere, Vol.16, No.4, pp 791-802, Printed in Great Britain

1987

0 0 4 5 - 6 5 3 5 / 8 7 $2.0© ~ .©C P e r g a m o n JourneIs Ltd.

DETERMINATION OF 2,3,7,8-TCDD TOXIC EQUIVALENT FACTORS (TEFs): SUPPORT FOR THE USE OF THE IN VITRO AHH INDUCTION ASSAY S. Safe Department of Physiology and Pharmacology College of Veterinary Medicine Texas A&M University College Station, TX 77843

ABSTRACT The in vitro induction of the cytochrome P1-450-dependent monooxygenases, aryl hydrocarbon hydroxylase (AHH) or ethoxyresorufin O-deethylase (EROD) by 2,3,7,8-TCDD and related toxic halogenated aryl hydrocarbons in rat hepatoma H-4-II E cells has been developed as a short term quantitative bioassay for these toxic chemicals. There was a linear correlation between the -log ECgo (in vitro) AHH induction vs the -log ED50 (in vivo) for body weight loss, thymic atrSphy, hepatic AHH and EROD induction in the rat for several polychlorinated biphenyl, dibenzo-p-dioxin and dibenzofuran congeners and mixtures. These data clearly support the utility of the in vitro AHH induction assay as a short term test system for quantitating the "toxic or 2,3,7,8-TCDD equivalents" in an extract containing toxic halogenated aromatics. The bioassay method is rapid, relatively accurate and much more cost effective than conventional analytical methods such as gas-chromatography-mass spectrometry from which it is difficult to determine the levels of 2,3,7,8-TCDD equivalents in specific analytes. BACKGROUND - ENVIRONMENTAL SIGNIFICANCE Polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs) and biphenyls (PCBs) are a class of highly stable lipophilic aromatic chemicals which have been either widely used in industry (PCBs) or are found as by-products in industrial chemical preparations (PCDFs and PCDFs) and in combustion processes (PCDDs, PCDFs and PCBs) (I-13). Jensen and coworkers first reported PCBs in environmental extracts in the late 1960's (14) and subsequent analytical studies have demonstrated the presence of PCBs in almost every component of the global ecosystem including the air, water, sediments, fish, wildlife and humans (14-18).

Since PCDFs and PCDDs are present only as by-products in industrial

chemicals (e.g., PCBs, chlorinated phenols and benzenes) and as by-products of organic waste incineration, relatively low levels have entered the environment.

During the past

decade there has been increasing concern about the high toxicity of some PCDFs and PCDDs and their potential adverse human health effects.

This fact, coupled with significant

advances in analytical methodologies for the identification and quantitation of PCDDs and PCDFs, has resulted in several studies which have confirmed that these chemicals are also

ubiquitous environmental contaminants (19-29).

PCDDs and PCDFs have been identified in

aquatic sediments (19-21), marine and freshwater biota including fish and wildlife (21-25), and in human adipose tissue, blood and milk (25-29). Human exposure to PCBs, PCDFs and PCDFs occurs not only

via environmental

uptake of these toxins but also through

ccupational and accidental routes of exposure. The effects of PCBs, PCDFs and PCDDs on 791

792

occupationally exposed workers have been described and numerous reports on Seveso, Yusho poisoning, Yu Cheng poisoning and PCB fires have documented the effects of accidental poisoning by these halogenated aryl hydrocarbons (29-38). COMMON ANALYTICAL AND TOXICOLOGICAL PROBLEMS The chlorination of biphenyl, dibenzofuran and dibenzo-p-dioxins can lead to a complex series of (a) congeners within each class which differ in their degree of chlorination and (b) isomers which differ in the orientation of their CI substituents. the number of isomers and congeners for all the PCBs, PCDDs and PCDFs. the PCB composition

of the commercial

industrial

environmental extracts is highly complex (40-42).

Table I summarizes Not surprisingly,

products (e.g., Aroclors) and

Moreover, due to the preferential

degradation, uptake and retention of specific PCB isomers and congeners, the pattern of FCBs present in human adipose tissue, blood and milk and in Yusho or Yu Cheng patients is different than the PCB c~nposition of the commercial products (31,31,41). The PCDDs and/or PCDFs which have been identified in Yusho oil, commercial PCBs, most c~nmercial chlorinated phenols and as combustion products are also highly complex mixtures (19-31). Therefore with one exception, individuals are exposed to multiple isomers and congeners. The major exception is associated with individuals exposed to 2,4,5-trichlorophenol,

2,4,5-T (Agent

Orange) and derived products which contain a single major PCDD by-product, namely the highly toxic 2,3,7,8-tetrachlorodibenzo-p-dioxin Table I. No. of C1 atoms

(TCDD) (34,38).

PCB, PCDF and PCDD Isomers and Congeners. I

2

3

4

5

6

7

8

9

10

Total

PCBs

3

12

24

42

46

42

24

12

3

I

209

PCDFs

4

16

28

38

28

16

4

I

135

PCDDs

2

10

14

22

14

10

2

I

75

Toxicologic studies have shown that 2,3,7,8-TCDD is the most toxic halogenated aryl hydrocarbon (43-49) and it is apparent that the toxicity of halogenated aryl hydrocarbons is remarkaoly dependent on their structure (43-55). For example, the body weight loss EDso values for two TCDD isomers, 2,3,7,8- and 1,3,7,8-TCDD in the rat are 5 x 10 -8 and 1.3 x 10-4 mol/kg respectively (53) and clearly demonstrate that the shift of a single C1 group from C-2 to C-I in the dibenzo-p-dioxin ring can result in a > 2,500-fold

decrease in

toxicity. Thus it is apparent that risk assessment of any PCB, PCDD or PCDF mixtures must require prior information on the identities of the specific toxic congeners and their

quantitation in any analyte. A strict analytical approach to this problem requires the availability of all the appropriate PCB, PCDF and PCDD congeners (209, 135 and 75 respectively) and the development of methods which can separate and quantitate each individual compound in a mixture. Several laboratories have made significant progress in this field using sophisticated cleanup procedures followed by high resolution capillary gas

793

chromatography-mass

spectrometry (GC-MS) analysis which can separate,

identify and

quantitate individual PCDD, PCB and PCDF congeners (41,56-59). These analytical techniques require highly sophisticated equipment and training and are also costly. The development of monoclonal antibodies to PCBs and PCDFs is clearly another rational approach to this problem (60,61). However, since there are a multitude of toxic PCBs, PCDFs and PCDDs, antibodies will have to be prepared either for each toxic compound or for the families of toxic PCB, PCDF and PCDD congeners.

The feasibility of this approach has

not yet been fully determined. PCBs, PCDDs and PCDF elicit a number of common toxic and biologic responses which include, chloracne and dermal toxicity, thymic atrophy, immunotoxicity, reproductive toxicity, porphyria, organ/tissue-specific tion,

body weight

monooxygenases

hypo- and hyperplastic responses, tumor promo-

loss and the induction of cytochrome

PI-450

(P-450c)-dependent

(43-49). Poland and coworkers first reported the identification of a high

affinity cytosolic receptor protein (46) and subsequent mechanistic studies support the role of this receptor in mediating the toxic and biologic effects evoked by 2,3,7,8-TCDD and related toxic halogenated aryl hydrocarbons.

Not surprisingly,

interactions with the

2,3,7,8-TCDD (or Ah) receptor are highly stereoselective and this accounts for the marked effects of structure on the biologic and toxic potencies of PCBs, PCDDs and PCDFs (4446,49,51-55). Several groups have developed the use of bioassays for toxic halogenated aryl hydrocarbons (52,53,55,61-69). These assay systems utilize mammalian cells in culture or a cytosolic receptor preparation which measures a specific response (e.g., keratinization, receptor binding or enzyme induction) which is associated only with and PCDD congeners.

the toxic PCB, PCDF

This review will briefly discuss the use and validation of the

induction bioassay for the detection and quantitation of 2,3,7,8-TCDD and related toxic halogenated aromatics. VALIDATION OF THE AHH/EROD INDUCTION ASSAY IN RAT HEPATOMA H-4-II E CELLS FOR DETERMINING 2,3,7,8-TCDD EQUIVALENTS Qualitative structure-activity relationships (SARs) for PCBs, PCDDs and PCDFs have been determined for receptor binding avidities and for several receptor-mediated responses inciuding keratinization (in vitro), body weight loss, immunotoxicity, LD50 , tumor promotion, reproductive toxicity and/or teratogenicity, thymic atrophy and the induction of the cytochrome P-450-dependent monooxygenases,

aryl hydrocarbon hydroxylase (AHH) and ethoxy-

resorufin O-deethylase (EROD) (43-55). These responses have been measured in diverse species and mammalian cells in culture and the SARs for these halogenated aryl hydrocarbons were comparable for all the effects noted above. Bradlaw

and coworkers (55,65) first reported that PCBs, PCDDs and PCDFs readily

induced AHH in rat hepatoma H-4-II E cells in culture and they demonstrated the utility of this assay system for detecting toxic halogenated ar~natics in diverse matrices including fish extracts, PCB/PCDF contaminated rice oil, diverse food extracts including gelatin samples containing pentachlorophenol and trace levels of higher chlorinated PCDDs.

794

Recent studies in our laboratory (51-53,61-64,68) have focused on validating the utility of the in vitro AHH/EROD induction bioassay as a quantitative method for estimating the relative in vivo toxic potencies of individual halogenated aromatic hydrocarbons, reconstituted mixtures and environmental extracts containing these compounds.

The male

Wistar rat was chosen as the in vivo animal model; the test compounds were administered in a dose-response fashion and the ED50 values for the following receptor-mediated responses were determined; induction. congeners

thymic atrophy, body weight loss, hepatic microsomal AHH and EROD

Table 2 summarizes the in vivo ED50 values for several PCB, PCDD and PCDF and

a reconstituted

mixture

of PCDFs

(2,3,7,8-tetra-,

1,2,4,7,8-penta-,

1,2,3,7,8-penta-, 2,3,4,7,8-penta- and 1,2,3,4,7,8-hexachlorodibenzofuran) which resembled the composition of these compounds identified in Yusho victims (70). Table 3 summarizes the in vitro EC50 values for receptor binding (rat hepatic cytosol), AHH and EROD induction in rat hepatoma H-4-II E cells for the same group of chemicals.

A plot of the -log EC50

values for AHH induction (in vitro) vs. body weight loss (in vivo), thymic atrophy (in vivo) and AHH induction (in vivo) is illustrated

in Figures I-3.

The correlation

coefficients (r) between the in vitro -log EC50 (AHH induction) and the in vivo -log ED50 for body weight loss and thymic atrophy were 0.93 and 0.92 respectively and clearly demonstrate the predictive utility of the in vitro bioassays.

The coefficient (r) was

lower for the correlation with in vivo AHH induction (r=0.83); since the in vivo enzyme induction activities were determined 14 days after administration of the toxic halogenated aryl hydrocarbons, the deviations from linearity are related to the differential rates of metabolism of these compounds.

These results clearly validate the use of the rat hepatoma

H-4-II E bioassay for determining the toxic or 2,3,7,8-TCDD equivalents (e.g., Table 3) for PCB, PCDF and PCDF congeners and reconstituted mixtures.

8

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-=3 ,,1[

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5 6 7 8 9 10 II BODY WEIGHT LOSS i - l o g ED 501

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795

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THYI'IIC ATROPHY l - l o g EO 501 Figure 2. A plot of the -log ECso values for A ~ induction (in vitro) vs -log ED~o thymic atrophy in the rat for several PCBs, PCDD~ and PCDFs.

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•

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AHH INDUCTION (-log El) 50) IN VlVO Figure 3. A plot of the -log EC50 values for AHH induction (in vitro) vs -log EDso AHH induction (in vivo) or several PCBs, PCDDs and PCDFs.

79~

Table 2.

A Summary of the In Vivo Biologic and Toxic Effects of Several PCDD, PCDF and PCB Congeners in the Immature Male Wistar Rat (50-52). IN VIVO ED50 Inhibition of Body Weight Gain

(umol/kg) Thymic Atrophy

B[a]P Hydroxylase

4-CB Hydroxylase

2,3,4,7,8-PeCDF

1.04

0.21

.037

.018

1,2,3,4,7,8-HCDF

1.3

0.5

.293

.240

1,2,3,7,8-HCDF

2.64

1.76

2,3,4,6,7,8-HDCF

2.8

0.93

.265

.213

1,2,3,6,7,8-HCDF

3.2

0.93

.347

.213

2,3,7,8-TCDF

3.2

3.60

1,3,4,7,8-PeCDF

26.1

0.70

2,3,4,7,9-PeCDF

22.0

5.5

2,3,4,7-TCDF

34.0

7.84

1,2,3,7,9-PeCDF

49.3

23.0

1,2,4,7,8-PeCDF

49.3

46.4

1,2,3,7-TCDF

86.9

110

2,3,4,8-TCDF

1.47

.652 3.49 6.96

1.47

.462 2.47 6.20

46.1

27.5

14.7

13.9

7.80

4.14

110

110

137

> 150

1,2,4,6,7-PeCDF

> 150

> 150

> 150

ND

> 150

ND

1,2,3,6-TCDF

> 250

> 250

> 160

168

2,3,7,8-TCDD

0.05

0.09

0.004

.003

1,2,3,7,8-PeCDD

0.62

0.17

0.031

---

1.07

0.03

1,3,7,8-TCDD

1,2,3,4,7,8-HCDD

132

1.63

100

31.2

1,2,4,7,8-PeCDD

34.0

11.0

2,3,4,4',5-PeCB

180

200

30

--

2,3,3',4,4',5'-HCB

220

225

6

--

2.82

---

2,3,3',4,4',5-HCB

180

180

25

--

2,3,3',4,4'-PeCB

750

1030

65

--

2,3'4,4',5-PeCB

1120

1550

165

--

2',3,4,4',5-PeCB

370

2790

3,3',4,4',5,5'-HCB

15

8.9

130

--

0.50

--

3,3',4,4',5-PeCB

3.3

0.95

1.10

--

PCDF Mixture

0.52

0.65

0.16

--

797

Table 3.

A Summary of the In Vitro Receptor Binding Affinities and AHH and EROD Induction Potencies of Several PCB, PCDF and PCDD Congeners (50-52). IN VITRO AHH Induction

2,3,4,7,8-PeCDF

2.56 x 10 -10

EC50

(M) EROD Induction 1.34 x 10 -10

Receptor Binding 1.5 x 10 -8

1,2,3,4,7,8-HCDF

3.56 x 10 -10

3.79 x 10 -10

2.3 x 10 -7

1,2,3,7,8-HCDF

2.54 x 10 -9

3.06 x 10 -9

7.45 x 10 -8

2,3,4,6,7,8-HDCF

6.87 x 10 -10

5.75 x 10-10

4.70 x 10 -9

1,2,3,6,7,8-HCDF

1.47 x 10 -9

1.24 x 10 -9

2.7

x 10 -7

2,3,7,8-TCDF

3.91 x 10 -9

2.02 x 10 -9

4.1

x 10 -8

1,3,4,7,8-PeCDF

1.60 x 10-9

1.40 x 10 -9

2.00 x 10 -7

2,3,4,7,9-PeCDF

7.90 x 10 -9

5.80 x 10 -9

2.00 x 10 -7

2,3,4,7-TCDF

1.79 x 10 -8

1.48 x 10 -8

2.51 x 10 -8

1,2,3,7,9-PeCDF

8.60 x 10-8

8.60 x 10 -8

3.98 x 10 -7

1,2,4,7,8-PeCDF

1.O6 x 10 -7

1.48 x 10 -7

1.3

x 10 -6

1,2,3,7-TCDF

2.70 x 10 -5

6.30 x 10 -5

1.12 x 10 -7

2,3,4,8-TCDF

4.14 x 10 -8

3.76 x 10 -8

2.00 × 10 -7

1,2,4,6,7-PeCDF

1.0

1.2

3.09 x 10 -6

x 10 -5

x 10 -5

1,2,3,6-TCDF

> 10-4

> 10 -4

2,3,7,8-TCDD

7.23 x 10-11

1.85 x 10-10

1.0

3.54 x 10 -7 x 10 -8

1,2,3,7,8-PeCDD

1.10 x 10 -8

1.70 x 10 -8

7.90 x 10 -8

1,2,3,4,7,8-HCDD

2.10 x 10 -9

4.10 x 10 -9

2.80 x 10 -7

1,3,7,8-TCDD

5.9

3.2

7.90 x 10 -7

1,2,4,7,8-PeCDD

2.10 x 10 -8

1.10 x 10 -8

1.10 x 10 -6

2,3,4,4',5-PeCB

9.73 x 10 -7

5.65 x 10 -7

4.1

× 10 -6

x 10 -7

x 10 -7

2,3',3,4,4',5'-HCB

1.33 x 10 -5

9.00 x 10 -6

5.0

× 10 -6

2,3,3',4,4',5-HCB

2.07 x 10 -6

8.96 x 10 -7

7.1

x 10 -6

2,3,3'4,4'-PeCB

8.75 x 10 -8

1.20 x 10 -7

4.3

x 10 -6

2,3',4,4',5-PeCB

1.15 x 10 -5

8.86 x 10 -6

9.1

x 10 -6

2',3,4,4',5-PeCB

3.91 x 10 -6

1.11 x 10 -6

1.4

× 10 -5

3,3',4,4',5,5'-HCB

6.01 x 10 -8

2.41 x 10 -8

3,3',4,4',5-PeCB

2.40 x 10-10

2.48 x 10-10

PCDF Mixture

3.23 x 10 -10

1.02 x 10 -10

insol. 1.2

x 10 -7 --

79~ For both the PCBs and PCDFs the only compounds which did not exhibit the linear correlation were those which contained two adjacent unsubstituted carbon atoms and were therefore readily metabolized (e.g., 3,3',4,4'-tetrachlorobiphenyl,

2,3,3',4,4'-penta-

chlorobiphenyl, 2,3,4,7-, 1,2,3,7-, 2,3,4,8- and 1,2,3,6-tetrachlorodibenzofuran).

Thus

the estimated 2,3,7,8-TCDD equivalents for these c~npounds derived from the in vitro AHH induction assay would overestimate their potential in vivo toxicity. However, it should be noted that the toxic halogenated aryl hydrocarbons which persist in animal tissues (including humans) are usually those compounds which are resistant to metabolic breakdown. This group of persistent PCDFs, PCDDs and PCBs are precisely those chemicals which exhibit a linear correlation between their in vitro and in vivo biologic and toxic potencies as illustrated in the Figures

Current research in our laboratory and others is focused on

further validation of the utility of the short term in vitro induction assay system for

quantitatively determining the "2,3,7,8-TCDD equivalents" in a sample extract of PCBs, PCDDs and PCDFs. Preliminary studies with a limited number of PCDFs and PCDDs show that there is also a linear correlation between their in vitro -log EC50 values for enzyme induction and the -log EDs0 values body weight loss (guinea pigs, M. Holcomb, S. Safe and coworkers, unpublished results and teratogenicity (C57BL/6 mice, L. Birnbaum and coworkers, unpublished results). In collaboration with B. Chittim and J. Madge (71), we have recently determined the "2,3,7,8-TCDD equivalents"present in a cleaned up fly ash extract containing a complex mixture of PCDD/PCDF isomers and congeners.

The isomer homolog distribution pattern is

highly complex and contains tetra-octachlorodibenzofurans (5.52 ug/g, total) and tetraoctachlorodibenzo-p-dioxins

(3.83 ug/g, total). The dose-response activity of this mixture

to induce AHH and EROD in rat hepatoma H-4-II E cells was determined and comparison of this data with the results obtained for 2,3,7,8-TCDD showed that this mixture contained 105 ng/g (fly ash) of "2,3,7,8-TCDD equivalents."

The in vivo dose-response induction of AHH by

this mixture and 2,3,7,8-TCDD were also determined in the immature male Wistar rat and the results showed that the extract contained 75 ng/g (fly ash) of "2,3,7,8-TCDD equivalents." (Note:

due to the lack of sufficient material only the in vivo AHH/EROD induction

activities were determined for this extract; however, Figures I-3 illustrate that this receptor-mediated response can also be used to predict in vivo toxicities.) These results clearly demonstrate that the in vitro bioassay can also be used to estimate the in vivo activity of a highly c~nplex PCDD/PCDF mixture and validates the use of this short term in vitro test system.

SUMMARY I.

The results summarized in this review demonstrate that for several individual PCDD and PCDF congeners there was an excellent linear correlation between their

PCB, in

vitro -log EC50 values for AHH (and EROD) induction and their in vivo -log ED50 values for several receptor mediated responses (e.g., body weight loss, thymic atrophy, hepatic AHH and EROD induction) in the rat.

Preliminary studies also suggest that

this correlation also holds for other toxic responses and other species (e.g., mouse-

799

teratogenicity; guinea pig-body weight loss).

2.

The only halogenated aromatics which do not fit this correlation are congeners which are readily metabolized in vivo.

None of these compounds are 2,3,7,8-substituted

PCDDs and PCDFs and they would be expected to be much less active in the in vitro induction assays.

The presence of the more readily metabolized compounds in crude

extracts containing complex mixtures of PCDDs and PCDFs may result in s~ne overestimation of the potential in vivo toxicity by the in vitro induction assay system. However, since some PCDD and PCDF congeners act as receptor antagonists and inhibit AHH/EROD induction (72), the potential for overestimation of toxic potencies by this assay may be somewhat ameliorated (see #3 below).

.

The utility of the in vitro induction assay has also been validated using (a) a reconstituted mixture of PCDFs resembling the composition of those compounds which persist in Yusho patients and (b) a highly complex PCDD/PCDF fraction from a fly ash extract.

For both mixtures there was an excellent quantitative correlation between

the 2,3,7,8-TCDD equivalents determined using the in vitro induction assay and their in vivo effects in the rat.

4.

Comparable SARs have been observed for toxic halogenated aryl hydrocarbons in several species and for many receptor-mediated effects.

Therefore, the data summarized in

this paper suggest that the TEFs estimated from their relative in vitro AHH induction potencies may be useful as an interim method for estimating potential toxic potencies of halogenated aryl hydrocarbons.

Further in vivo studies are required to show that

the in vitro data can be used to estimate carcinogenic potencies since the precise role of the Ah receptor in this process has not been delineated.

5.

The results summarized in this report validate the utility of the in vitro AHH induction assay for quantitatively estimating the potential toxicity of PCB, PCDF and PCDD mixtures in environmental extracts. This assay can be carried out rapidly, is relatively inexpensive and utilizes the established (and stable) rat hepatoma H-4-II E cells in culture which can detect 20-50 pg of 2,3,7,8-TCDD equivalents per plate.

ACKNOWLEDGEMENTS Several colleagues have collaborated on this project, and I would like to thank T. Sawyer, O. Hutzinger, B. Keys, G. Mason, M. Kelley, J. Piskorska-Pliszczynska, L. Safe, B. Zmudzka, K. Farrell, R. Bannister, J.A. Madge, B. Chittim, M.A. Denomme, K. Homonko, M.A. Li, B. Leece and R. Towner for their contributions.

The financial assistance of the Texas

Agricultural Experiment Station, the United States Environmental Protection Agency, the National

Institutes of Health

this work possible.

and the Center for Energy and Mineral Resources has made

800

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(Received

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in Germany

17 O c t o b e r

A.B. Okey, S. Bandiera and S. Safe,

J.F. Gierthy,

1986;

12, 529

accepted

J.B. Silkworth

29 O c t o b e r

and C. Tumasonis,

J.A. Madge and B.

1986)