Arch Toxicol (1992) 66:220-223

Archives of

To:dcology 9 Springer-Verlag 1992

Short communication

Assessment of biological activities of mixtures of polychlorinated dibenzo-p-dioxins (PCDDs) and their constituents in human HepG2 cells Hans-Peter Lipp], Dieter Schrenk], Thomas Wiesmiiller2, Hanspaul Hagenmaier e, and Karl Walter Bock 1 1 Institute of Toxicology, University of Ttthingen, Wilhelmstrasse 56, W-7400 Tfibingen, Federal Republic of Germany 2 Institute of Organic Chemistry, University of TiJbingen, Auf der Morgenstelle 18, W-7400 Ttibingen, Federal Republic of Germany Received 24 July 1991/Received after revision 28 October 1991/Accepted 29 October 1991

Abstract. Dose-response curves of the induction of P4501Al-dependent 7-ethoxyresorufin O-deethylase (EROD) were analyzed in human hepatoma HepG2 cells treated with defined mixtures of polychlorinated dibenzop-dioxins (PCDDs) and their 2,3,7,8-substituted constituents, similar to previous studies with rat hepatocytes and H4IIE cells (Schrenk et al. 1991). PCDDs appear to act less potent in human HepG2 cells in comparison with rat cells. For example, EC50 values of 2,3,7,8-C14DD were 8-fold and 19-fold higher than in rat H4IIE cells and hepatocytes, respectively. EC50 values of PCDDs were compared with that of 2,3,7,8-C14DD and expressed as 2,3,7,8-C14DD equivalents (TEs). Although the rank order of PCDD potencies was similar, TEs for some PCDDs (1,2,3,7,8C15DD; TE = 0.75 and 1,2,3,4,7,8-C16DD; TE = 0.61) were found to be higher than in the rat system. In contrast to rat cells no significant induction of EROD could be detected with C18DD in HepG2 cells up to its limit of solubility. Experimentally determined TEs of PCDD mixtures containing 49 constituents were found to be largely due to additive effects of their 2,3,7,8-substituted constituents. Key words: PCDD mixtures - Induction of P450IA1 2,3,7,8-C14DD equivalents - Human hepatoma HepG2 cells

Introduction Assessment of biological activities of mixtures of polychlorinated dibenzo-p-dioxins (PCDDs), which are widespread environmental pollutants, represents a great chal-

Dedicated to Professor Dr. rer. nat. Ernst Bayer on the occasion of his 65th birthday Offprint requests to: K.W. Bock

lenge for toxicology. Many of the biological and toxic responses of PCDDs have been shown to be mediated through the Ah or dioxin receptor (Poland and Knuts0n 1982; Safe 1986; Landers and Bunce 1991). The di0xin receptor is thought to be a member of the erb-A superfamily of proteins (Evans 1988). Considerable progress has been made in elucidating the initial sequence of events, including ligand binding to the receptor, binding of the receptor-ligand complex to DNA enhancer elements, and expression of specific genes and their translation (Poland and Knutson 1982; Safe 1986; Whitlock 1990; Lande~ and Bunce 1991). However, a large gap in our knowledge still exists between these initial events and the signs 0[ overt toxicity. Studies in rat hepatoma H4IIE cells revealed that the potency of a number of PCDDs to induce P4501A1 was in good agreement with their toxicity in rats (Safe 1986). Based on these findings ECs0 values of P4501A1 induction are widely used to assess biological activities of PCDDs and PCDD mixtures (Barnes et al. 1966). Usually biological activities of PCDDs are compared to those of the most potent congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-C14DD), and expressed as 2,3,7,8-ChDD equiv. alents (TEs; BGA 1985; Kutz et al. 1990). An intriguing aspect of PCDD-induced toxicity is the large species-dependence in the susceptibility to various toxic reactions. The susceptibility of humans is unknown. However, based on a comparison of clinical findings in exposed people and their tissue levels of PCDDs with toxicity data in rodents, it seems reasonable to conclude that humans are probably less susceptible to the effect of PCDDs than most laboratory rodents (Kimbrough 1990; Landers and Bunce 1991). Previously, biological activities of PCDDs, such as P4501A1 induction, have been compared in rat hepat0cytes and rat hepatoma H4IIE cells using several defined PCDD mixtures and their constituents (Schrenk et al. 1991). It was found that biological activities of the mixtures were largely due to additive effects of their 2,3,7,8substituted constituents. The previous study in rat models has been extended to the human hepatoma cell line,

221 Table 1. Composition of the defined PCDD mixtures used in induction experimentsa Mixture 1

Congener

H e p G 2 . H e p G 2 cells h a v e p r o v e n to r e p r e s e n t a suitable h u m a n cell m o d e l . T h e y contain the h u m a n r e c e p t o r controlling the i n d u c t i o n o f P 4 5 0 1 A I (Cresteil et al. 1987; G r a n t et al. 1988; L a b r u z z o et al. 1989; Roberts et al. 1990). Synthesis o f s e r u m proteins and cell surface recept o r - m e d i a t e d functions such as l i p o p r o t e i n u p t a k e ( w h i c h p r o b a b l y plays a role in the u p t a k e o f P C D D s ; S h i r e m a n and W e i 1987) are c l o s e l y s i m i l a r to those in n o r m a l hepat o c y t e s ( K n o w l e s et al. 1980; H a v e k e s et al. 1983). H o w ever, species differences in d i o x i n r e c e p t o r p r o p e r t i e s h a v e also b e e n noted (Roberts et al. 1990; P o l a n d et al. 1990). T h e results with 2 , 3 , 7 , 8 - P C D D s and c o m p l e x P C D D m i x tures s u g g e s t that P C D D s a p p e a r to be less p o t e n t in h u m a n cells in c o m p a r i s o n with rat cells.

Mixture 2 (%)

1368 1379 1378 1369;1247/8 1268 1478 2378 1246/9 1237/8;1234 1236;1279 1278;1469 1239 1269 1267 1289 sumof C14DDs

0.07 0.09 0.15 0.14 0.07 0.05 0.05 0.04 0.08 0.11 0.07 0.04 0.05 0.02 0.02 1.06

0.21 0.23 0.46 0.88 0.30 0.19 0.11 0.41 0.57 0.63 0.48 0.29 0.31 0.11 0.21 5.38

12479;12468 12368 12478 12379 12469;12347 /2378 12369 12467 12489 12346 12367 12389 sumof ClsDDs

1.32 0.54 0.58 0.48 0.74 0.45 0.41 0.38 0.46 0.10 0.19 0.40 6.05

3.25 1.21 1.54 1.39 1.96 1.20 1.23 1.17 1.45 0.41 0.95 1.36 17.12

124679;124689;123468 123679;123 689 123478 12367R 123469 123789 123467 sumof C16DDs

4.12 4.21 0,51 0,67. 0.65 0.95 0.89 12.02

10.23 10.76 _l.,.8_2 1.66 2.32 2.54 30.61

Statistical analysis. Dose-response curves were analysed with a computerized log probit procedure (SAS Institute, Car)', NC, USA; technical report P- 179).

1234679 1234678 sumof C17DDs

15.51 10.61 26.12

20.25 13.75 34.00

Results

12346789

54,75

'

Materials a n d m e t h o d s Chemicals. Eagle's minimal essential medium was from Seromed (Berlin, FRG), dexamethasone from Sigma (St Louis, USA), fetal calf serum and calf serum from Gibco BRL (Eggenstein, FRG), and 7-ethoxyresorufin from Boehringer (Mannheim, FRG). Preparation of PCDD mixtures 1 and 2 and their composition, listed in Table 1, was identical to those investigated previously (Schrenk et al. 1991). EROD activity was assayed as described (Burke and Mayer 1974). Culture of HepG2 cells. The cell line was grown on 90 • 20 mm petri dishes in Eagle's minimal essential medium (with L-glutamine) supplemented with 10% fetal calf serum, 10% calf serum, penicillin (100 units/ml) and streptomycin (100/.tg/ml) in a humidified atmosphere of 5% CO2 in air at 37~ (Beck and Bock-Hennig 1987). Cells were seeded at 5 • 105 cells/plate in 7 ml medium. After 24 h, medium was replaced by fresh medium, PCDDs were added in DMSO (0.5%, final concentration) and cells were harvested after additional 48 h.

In H e p G 2 cells P 4 5 0 1 A 1 (reflected by E R O D activity) w a s m a x i m a l l y i n d u c e d b y P C D D s within 48 h (not shown), s i m i l a r to findings in rat h e p a t o c y t e s a n d H 4 I I E cells ( S c h r e n k et al. 1991). In contrast to the rat s y s t e m , a d d i t i o n o f d e x a m e t h a s o n e (10-7 M ) d i d not affect P 4 5 0 1 A 1 induction. M a x i m a l E R O D activities o b t a i n e d with each

2378-substituted congeners are underlined

too J > 0

/ ji(-123478-CI~DD

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P C D D

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Fig. 1. Dose-response curves of EROD induction by 2,3,7,8-substituted PCDDs in HepG2 ceils. Ceils (5 • 105/plate) were incubated with 2,3,7,8-substituted PCDDs for 48 h at the concentrations indicated. EROD activity was determined in cell homogenates. Data represent means of four experiments

222 Table 2. Comparisonof EC50values and TEs for EROD induction and of TEs by PCDDs in human and rat cellsa

PCDD congener

HepG2 ECs0

H4IIEb (TE)

ECs0

(TE)

Rat hepatocytesb ECs0 (TE)

100 w

,.,

50

o

(pg/plate) 2 378-C14DD 12378-ClsDD 123478-C16DD 123789-C16DD 123678-C16DD 1234678-C17DD C18DD

706 (1.00) 946 (0.75) 1150 (0.61) 10150(0.07) 16080(0.04) 16700(0.04) >106

89 (1.00) 37 (1.00) 492 (0.18) 224 (0.17) 483 (0.18) 505 (0.07) 1447 (0.06) 901 (0.04) 2072 (0.04) 1207 (0.03) 1904 (0.05) 2088 (0.02) 635 000 (0.0001) 11 480 (0.003)

Mixture 1

109000 (0.007)

11588(0.008)

7031 (0.005)

Mixture 2

20800 (0.034)

5636 (0.016)

4064 (0.009)

values were calculated from the dose-response curves given in Figs. 1 and 2. TE values were calculated from the ECs0 values b Takenfrom Schrenk et al. (1991) a EC50

Table 3. Comparison of experimentally determined and calculated TE-

values of PCDD mixtures and their constituents with human HepG2cells PCDD congener

Mixture 1

Mixture 2

(TE • % content)a 2 378-ChDD 12378-C15DD 123478-C16DD 123 789-C16DD 123 678-C16DD 1 234 678-C17DD ClsDD

0.050 0.338 0.305 0.047 0.042 0.446 -

0.110 0.900 0.960 0.127 0.102 0.578 -

Sum of TE • % content

1.225

2.777

TE, calculatedb

0.012

0.028

TE, experimental

0.007

0.034

a The TE values were taken from Table 2 and % content from Table 1 b Sum ofTE x % content/100

congener did not differ significantly (2170__+235 pmol/min/mg homogenate protein, four experiments each for the six 2,3,7,8-substituted congeners). Dose-response curves of EROD induction for different PCDDs (Fig. 1) were used to calculate the corresponding EC50 values (Table 2). The EC50 value for 2,3,7,8-C14DD was markedly higher (706 pg/plate) than that determined in rat H4IIE cells (89 pg/plate) or rat hepatocytes (37 pg/plate). The rank order of TEs for different PCDDs was similar to findings in rat cells. However, in HepG2 cells 1,2,3,7,8C15DD (TE = 0.75) and 1,2,3,4,7,8-C16DD (TE = 0.61) were almost as potent as 2,3,7,8-C14DD. With C18DD, the most abundant congener in human tissues and breast milk, no or only slight EROD induction (up to 30% of maximal EROD activity) was detectable using C18DD concentrations up to 1 ktg/plate (Fig. 1). This concentration represents the limit of solubility of C18DD. Slight induction may be attributed to 0.5% contamination of C18DD by 1,2,3,4,6,7,8-C17DD. Therefore a TE could not be calculated for CI8DD in HepG2 cells.

0

i

i

i

i i iii

I

t~

i

i

i

iiiii

I

t

t

i

Itlll

5

j

I

6

log PCDO concentration (pglplate)

Fig. 2. Dose-response curves of EROD induction by PCDD mixtures

Contents of 2,3,7,8-substituted congeners of mixtures 1 and 2 are listed in Table 1. Culture conditions were identical to those given in Fig. 1 Data represent means of four experiments

The composition of the PCDD mixtures 1 and 2 is listed in Table 1. In brief, mixtures 1 and 2 contained 49 PCDD constituents and 55 % and 13% ClsDD, respectively. Doseresponse curves used for calculation of ECs0 values of the PCDD mixtures are given in Fig. 2. Experimental TE values for the mixtures were found to be in fair agreement with those calculated from the sum of 2,3,7,8-substituted congeners (Table 3). Suspected antagonistic effects of C18DD were not observed in mixing experiments of C18DD with either 2,3,7,8-C14DD, 1,2,3,4,7,8-C16DD or 1,2,3,4,6,7,8-C17DD (not shown). Discussion

Previous studies with rat cells were extended to human HepG2 cells which have proven to represent a suitable human cell model to investigate Ah receptor-controlled P4501A1 induction by PCDDs. Suitability of a hepatoma cell line is further supported by previous findings of a close correspondence of data obtained in rat hepatocytes and rat hepatoma H4IIE cells (Schrenk et al. 1991). Two points have been addressed, (a) the potency of PCDDs in human compared to rat cells and (b) assessment of biological activites of PCDD mixtures. For most PCDDs, such as 2,3,7,8-C14DD and the PCDD mixtures a lower sensitivity was obtained in HepG2 cells than in rodent cells (Roberts et al. 1990). This finding is consistent with dioxin receptor studies in other human tissues and cells, such as lung (Harper et al. 1986; Roberts et al. 1986), placenta (Manchester et al. 1987), the A431 human squamous cell carcinoma line (Harper et al. 1988) and tonsils (Lorenzen and Okey 1991). In all these cases the human Ah receptor appeared to exhibit a lower affinity for 2,3,7,8-C14DD as compared to the rodent receptors. While the rank order of potencies of 2,3,7,8-substituted PCDDs in HepG2 cells was similar to previous findings in rat cells (Schrenk et al. 1991) some notable exceptions were found: 1,2,3,7,8-C15DD and 1,2,3,4,7,8-C16DD elicited markedly higher relative potencies (TEs) in HepG2 cells. Furthermore, with C18DD no induction of EROD was detectable in HepG2 cells. The reason for these differences is unclear. They may be due to species differences in the structure of the Ah receptor (Golas et al. 1990; Poland et al. 1991).

223

In agreement with previous findings in the rat model, biological effects of the complex PCDD mixtures 1 and 2 containing 49 constituents appear to be largely due to additive effects of their 2,3,7,8-substituted constituents in human HepG2 cells. In general, potencies of PCDDs were found to be less than in rat cells. It has to be emphasized, however, that studies on dioxin receptor-mediated P4501A1 induction do not obviate the need for bridging the gap between enzyme induction in vitro and PCDD-induced chronic toxicity in vivo. Acknowledgements. The authors wish to thank Dr. Friedrich Wiebel (GSF MiJnchen, FRG) for providing HepG2 cells and Ms. S. Pahl for expert technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft and the Landesforschungsschwerpunkt 27, 13aden-Wtirttemberg.

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