Inducing Effects of Dioxin-like Polychlorinated Biphenyls on CYP1A in the Human Hepatoblastoma Cell Line HepG2, the Rat Hepatoma Cell Line H4IIE, and Rat Primary Hepatocytes: Comparison of Relative Potencies

M. Zeiger*, R. Haag{dagger}, J. Höckel{dagger}, D. Schrenk* and H.-J. Schmitz*,1

* Food Chemistry and Environmental Toxicology, University of Kaiserslautern, Erwin-Schroedinger-Str. 52, D-67663 Kaiserslautern, Germany; and {dagger} TÜV Süddeutschland Bau und Betrieb GmbH, Umweltservice, Kompetenzzentrum Analytik, D-73072 Donzdorf, Germany

Received February 26, 2001; accepted June 7, 2001

ABSTRACT

Polychlorinated biphenyls (PCBs) are a group of widespread environmental pollutants. Some non-ortho-substituted congeners with a high likelihood of coplanarity of both aromatic rings have been shown to act like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as agonists of the aryl hydrocarbon receptor (AhR) subsequently leading to adverse effects, such as immunosuppression and tumor promotion. Although there is a broad base of experimental data concerning the toxicity of PCBs in laboratory animals and animal-derived primary cells and cell lines, only few experimental data are available for cells of human origin. As a parameter of AhR activation, induction of CYP1A-mediated 7-ethoxyresorufin O-deethylase (EROD) activity was determined in the human hepatoblastoma cell line HepG2 treated with the PCBs IUPAC Nos. 77, 81, 105, 114, 118, 123, 126, 156, 157, 167, 169, and 189, and with TCDD as a positive control. Compared with results in rat primary hepatocytes and the rat hepatoma cell line H4IIE, treated HepG2 cells showed lower specific EROD activities maximally inducible by TCDD and PCBs, and EC50 values were shifted to higher concentrations. Furthermore, relative potency factors (REPs) for some congeners such as PCBs 81, 126, and 169 greately differed from those observed in cells derived from rats. Northern blot analyses showed that EROD activities run parallel to changes in CYP1A-specific mRNA contents. The considerable differences in EROD-derived REPs between cells of human and rat origin indicate the need for further investigations in experimental models from different species including humans in order to extend the database of biochemical and toxic responses to PCBs.

Key Words: PCB; dioxin; TCDD; Ah receptor; CYP1A; EROD; human; rat.

Polychlorinated biphenyls (PCBs) are a family of 209 compounds (congeners) differing in extent and position of chlorination of their aromatic rings. They were extensively used in industrial applications, e.g., because of their insulating and flame retardant properties, leading to widespread distribution in soil and water. Because of their lipophilic character, chemical stability, and slow rate of degradation they tend to accumulate in adipose tissue of animals and humans, and although production and use have been terminated they are still present in the food chain and environmental matrices (Danse et al., 1997Go).

Some of the PCB congeners, especially those with non- and mono-ortho-chlorine substitution show patterns of toxicity in laboratory animals resembling those of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), including teratogenicity, endocrine disorders, tumor promotion and adverse effects on skin, immune system, and reproduction (Safe, 1994Go). There is sufficient evidence that these compounds share a common mechanism of action of toxicity, involving agonist binding to the cytosolic aryl hydrocarbon receptor (AhR; Poland and Knutson, 1982). The ligand-activated receptor forms a heterodimer with the nuclear protein ARNT ("AhR nuclear translocator"), and after binding to specific DNA elements (xenobiotic responsive elements [XRE]) increases transcription of several dioxin-activated genes (AhR gene battery) (Bock, 1993Go). Among these genes, increased expression of cytochrome P450 1A1 (CYP1A1) is a well-understood example frequently used as a parameter for the potency of AhR agonists (Whitlock, 1993Go). The 7-ethoxyresorufin O-deethylase (EROD) activity of CYP1A isozymes is widely accepted to be used for measuring induction by dioxin-like compounds (Leece et al., 1985Go).

To describe the relative potency of dioxin-like compounds, the concept of Toxic Equivalency Factors (TEFs) has been developed (Eadon et al., 1986Go; Birnbaum, 1999Go) with TCDD as one of the strongest AhR agonists attributed with a TEF value of 1. The TEFs published by the World Health Organization (WHO) for risk assessment in humans and wildlife are consensus values derived from available experimental in vivo and in vitro data by scientific consideration and evaluation (Van den Berg et al., 1998Go). Following the recommendation of the WHO expert group, the values of our experiments determining the range of CYP1A-inducing potency of dioxin-like PCBs are referred to as relative potencies (REPs).

Usually, REPs are ratios of EC50 values or other hallmarks of effects of individual congeners compared to TCDD (Van den Berg et al., 1998Go) in a single experimental model based on a series of experiments carried out under reproducible conditions. Dose-response curves running parallel under ideal conditions allow the calculation of EC50 values including confidence intervals (DeVito et al., 1997Go). In in vivo experiments, different pharmacokinetic properties including absorption, distribution, metabolism, and elimination of congeners can affect REPs (DeVito and Birnbaum, 1995Go), whereas in tissue culture, different binding affinities to the Ah receptor seem to play a predominant role (Safe, 1994Go).

In recent years, the TEF/REP concept has been extended to dioxin-like PCBs comprising non-ortho- and mono-ortho-chlorinated congeners (Ahlborg et al., 1994Go; Safe, 1994Go; Van den Berg et al., 1998Go). A number of studies have been published presenting REPs for dioxin-like PCBs using CYP1A induction as a surrogate parameter for AhR activation. These include studies in vivo in rodents (DeVito et al., 1993Go,2000Go; Safe, 1990Go) and in vitro in rat hepatocytes and hepatoma cells (Schmitz et al., 1995Go), in hepatocytes from other species (van der Burght et al., 1999Go,2000Go), and in human cell lines (Pang et al., 1999Go).

Previously, we found considerable species differences in the potency of polychlorinated dibenzo-p-dioxins (PCDDs) to induce CYP1A isozymes in tissue culture (Lipp et al., 1992Go; Schrenk et al., 1991Go,1995Go). Therefore, investigations of the CYP1A-inducing potencies of most dioxin-like PCBs were done in the human hepatoblastoma cell line HepG2 in comparison to the rat hepatoma cell line H4IIE, and in rat primary hepatocytes. For a number of dioxin-like PCBs pronounced differences were found between cells of human and rat origin.

MATERIALS AND METHODS

Chemicals.
PCB 81 (3,4,4',5-TetraCB) was kindly provided by L. Robertson (University of Kentucky, Lexington, KY). PCBs 77 (3,3',4,4'-TetraCB), 105 (2,3,3',4,4'-PentaCB), 114 (2,3,4,4',5-PentaCB), 118 (2,3',4,4',5-PentaCB), 123 (2',3,4,4',5-PentaCB), 126 (3,3',4,4',5-PentaCB), 156 (2,3,3',4,4',5-HexaCB), 157 (2,3,3',4,4',5'-HexaCB), 167 (2,3',4,4',5,5'-HexaCB), 169 (3,3',4,4',5,5'-HexaCB), 189 (2,3,3',4,4',5,5'-HeptaCB), and 2,3,7,8-TCDD were purchased from Promochem (Wesel, Germany). All other chemicals were purchased from Sigma (Deissenhofen, Germany). The purity of the commercially purchased PCBs exceeded 99% according to the supplier. In addition, HPLC analyses were carried out that confirmed the supplier's information. For the chemical analysis of PCB 81 the preparation was dissolved and the solution was diluted in toluene. This solution was analyzed by high-resolution mass spectrometry. Quantification was carried out after addition of 13C-labelled PCB standards. A contamination with 3.9% PCB 39 and 0.7% of another unidentified trichlorinated biphenyl was found. Other PCBs and PCDD/Fs could not be detected.

Preparation of TCDD and PCB solutions.
Solutions for treatment of cell cultures were obtained by dissolving PCBs in dry dimethyl sulfoxide (DMSO) and preparation of stepwise dilutions resulting in final DMSO concentrations of 0.5 % in the culture medium.

Cell culture and treatment.
Fetal bovine serum (FBS) was from Gibco (Karlsruhe, Germany), all other components of the culture medium were from Biochrom (Berlin, Germany). Type I collagen for coating was prepared from rat tail tendon (Elsdale and Bard, 1972Go).

The human hepatoblastoma cell line HepG2, and the rat hepatoma cell line H4IIE were a kind gift from F. Wiebel (GSF, Munich, Germany). Hepatocytes were prepared from male Wistar rats (Charles River, Kisslegg, Germany) as described earlier (Schmitz et al., 1995Go). Preparations showing viability > 90% were seeded on collagenated Petri dishes (6 cm diameter) at a density of 70,000 cells/cm2 in Dulbecco's modified Eagle's medium (DMEM) containing 20% FBS, 0.1 µM dexamethasone, 100 U/ml penicillin, and 100 µg/ml streptomycin. After 3 h, medium was replaced by fresh medium, PCBs were added, and the cells were incubated for additional 48 h. DMSO alone served as a negative control. Cell lines were seeded at a density of 10,000 cells/cm2 in the same medium. After reaching 50% confluency, medium was replaced by fresh medium and cells were treated as described above.

EROD assay.
After incubation, cells were washed with ice-cold saline and scraped off with ice-cold Tris-buffered sucrose solution (10 mM Tris-HCl, 250 mM sucrose; pH 7.4). After centrifugation, cells were homogenized by disruption with a sonifier (Braun, Reutlingen, Germany) at 50 watts on ice. EROD activity in the homogenates was measured according to the method of Burke and Mayer (1974) modified by Pohl and Fouts (1980) using a fluorescence sprectrometer (Perkin-Elmer LS 50). Protein concentrations were determined according to Lowry et al. (1951).

Northern analysis.
Total cellular RNA was isolated from rat primary hepatocytes and H4IIE cells as described previously (Schmitz et al., 1997Go; Schrenk et al., 1994Go), from HepG2 cells according to Chomczynski and Sacchi (1987).

After electrophoresis in a 1% agarose gel containing 2.2 M formaldehyde, RNA was blotted to nylon membranes (Magnagraph, MSI, Westborough, MA) using capillary transfer with 10x SSC. As a size standard, an RNA ladder of fragment size of 0.24–9.5 kb (Gibco) was used.

Analysis of CYP1A mRNA expression was achieved by hybridization to a 2 kb mouse CYP1A2 cDNA probe (Gonzalez et al., 1984Go) hybridizing with rat and human CYP1A1 and 1A2 mRNAs. Loading controls were performed using a 249 bp cDNA probe for the rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (Fort et al., 1985Go). The probes were labeled with [{alpha}-32P]dCTP using a random primer labeling kit (Roche, Mannheim, Germany). Membranes were prehybridized and hybridized in 50% deionized formamide, 6x SSC, 5x Denhardt's solution, and 1% sodium dodecyl sulfate (SDS). Hybridized mRNA was visualized by autoradiography at –80°C for 1 to 2 days and quantified using a scanning software (Stratagene Eagle Sight).

Statistical analysis.
Treatments were carried out in duplicate in 3 independent experiments. Dose-response curves, EC50 values, and SDs were calculated using a computerized log-probit procedure (Origin 5.0, Microcal, Northampton, MA). Calculated no-effect concentrations (CNEC) were determined analogous to the calculation of no-effect levels (CNEL) of TCDD in a subchronic dose-response study by Van Birgelen et al. (1995). Briefly, a margin of 2 standard deviations was added to the EROD activity of untreated controls (EROD of control + 2 SDs). The corresponding concentration value was calculated from the output parameters of the logistic regression equation for each concentration-response curve. The 95% confidence limits were calculated from the error values of these parameters. This procedure was carried out with each cell type investigated and for any single PCB leading to EROD induction.

RESULTS

The non-ortho PCBs 77, 81, 126, the mono-ortho PCB 114, and TCDD as the reference compound induced EROD activity in human HepG2 cells as shown in Figure 1Go. The EC50-based potencies were in the rank order TCDD > PCB 81 > PCB 126 > PCB 77 > PCB 114. The maximal efficacies of most inducing PCBs were lower than that of TCDD while the non-ortho PCB 77 exhibited an extraordinary low maximal efficacy. In contrast to cells of rat origin, the other mono-ortho PCB congeners (105, 118, 123, 156, 157, 167, and 189) and the non-ortho congener PCB 169 did not induce EROD activity in HepG2 cells. The EC50 value for TCDD in HepG2 cells was within the same order of magnitude as the earlier published value, i.e., 0.68 nM in this vs. 0.31 nM in a previous report (Lipp et al., 1992Go). A summary of EC50 values is presented in Table 1Go.



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FIG. 1. EROD-inducing effects of TCDD or the PCBs 77, 81, 114, and 126 in HepG2 cells after treatment over 48 h. PCBs 105, 118, 123, 156, 157, 167, 169, and 189 did not induce EROD activities in HepG2 cells. Data were obtained from 3 independent experiments, and concentration-effect curves fitted from data sets obtained at 6–8 different concentrations of each congener were calculated using a log-probit software.

 

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TABLE 1 EC50 Values ± S.D. (nM) for Induction of EROD Activity in HepG2 Cells, H4IIE Cells, and Rat Primary Hepatocytes
 
To better take into consideration different shapes of concentration-response curves (slope and maximum), a benchmark concept of CNEC similar to the CNEL described by Van Birgelen et al. (1995) for a subchronic dose-response study with TCDD was applied. In contrast to that study, our calculations are based upon a logistic regression model because, with our data, other models did not result in narrower 95% confidence intervals (data not shown).

The rank order of CNEC-based potencies was TCDD > PCB 81 {approx} PCB 126 > PCB 114 > PCB 77, revealing an even lower potency of the non-ortho PCB 77 compared with the mono-ortho PCB 114, and similar potencies for both the non-ortho congeners PCB 81 and PCB 126. A summary of CNEC values is presented in Table 2Go.


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TABLE 2 Calculated No Effect Concentrations (CNEC; nM) for Induction of EROD Activity in HepG2 Cells, H4IIE Cells, and Rat Primary Hepatocytes
 
To determine whether the effects on EROD activity in HepG2 cells were due to changes in CYP1A mRNA contents or to posttranslational events, Northern blot analyses were performed. Figure 2Go demonstrates that increasing EROD activities run parallel to specific mRNA contents. An increase in CYP1A mRNA could not be detected after treatment with PCB 77 and with those PCBs that failed completely to induce EROD activity (PCBs 105, 118, 123, 156, 157, 169, and 189) although GAPDH controls were positive (data not shown).



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FIG. 2. Representative Northern analyses of HepG2 cells after treatment with TCDD or the PCBs 81, 114, and 126 over 48 h. CYP1A mRNA could not be detected after treatment with PCBs 77, 105, 118, 123, 156, 157, 167, 169, and 189 (GAPDH control positive; data not shown). Blots were probed with a mouse CYP1A2 cDNA, and a rat GAPDH cDNA as loading control. Autoradiographs were scanned and analyzed densitometrically. Intensities (arbitrary units) of CYP1A mRNA (verticle bars) were calculated relative to corresponding GAPDH mRNA intensities.

 
For the analysis of EROD induction in rat primary hepatocytes and the rat hepatoma cell line H4IIE, part of the data were taken from a previous publication (Schmitz et al., 1995Go). The effects of the non-ortho PCB 81 and the mono-ortho PCBs 114, 123, 157, 167, and 189 were investigated in this study to complete the existing set of data.

Figures 3 and 4GoGo show the fitted concentration-effect curves for EROD induction by selected non-ortho and mono-ortho PCBs in H4IIE cells. The EC50-based potencies were in the rank order TCDD > PCB 126 > PCB 81 > PCB 169 > PCB 114 {approx} PCB 157 > PCB 77 {approx} PCB 156 > PCB 105 {approx} PCB 167 > PCB 118 {approx} PCB123. The maximal efficacies of TCDD and most inducing PCBs, in particular the non-ortho PCB 77, were considerably higher than in HepG2 cells. The mono-ortho PCB 189 failed to induce detectable EROD activity in H4IIE cells.



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FIG. 3. EROD-inducing effects of TCDD or the non-ortho-substituted PCBs 77, 81, 126, and 169 in H4IIE cells after treatment over 48 h. Data for PCB 81 were obtained from 3 independent experiments, data for the other non-ortho PCBs were from a previous publication (Schmitz et al., 1995Go). Concentration-effect curves fitted from data sets obtained at 6–8 different concentrations of each congener were calculated using a log-probit software.

 


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FIG. 4. EROD-inducing effects of TCDD or the mono-ortho-substituted PCBs 105, 114, 118, 123, 156, 157, and 167 in H4IIE cells after treatment over 48 h. PCB 189 did not induce EROD activity in H4IIE cells. Data for PCBs 114, 123, 157, 167, and 189 were obtained from 3 independent experiments, data for the other mono-ortho-substituted PCBs were from a previous publication (Schmitz et al., 1995Go). Concentration-effect curves fitted from data sets obtained at 6–8 different concentrations of each congener were calculated using a log-probit software.

 
The CNEC-based potencies were in the rank order TCDD > PCB 126 > PCB 81 > PCB 77 > PCB 169 > PCB 156 > PCB 105 > PCB 114 > PCB 157 > PCB 118 > PCB 167 > PCB 123, i.e., all non-ortho congeners showed higher potencies than the mono-ortho congeners, and among mono-ortho congeners, the potencies of PCB 77 and PCB 169 were in the same order of magnitude.

Figures 5 and 6GoGo show the fitted concentration-effect curves for EROD induction by selected non-ortho and mono-ortho PCBs in rat primary hepatocytes. All congeners tested including PCB 189 led to a significant induction. The maximal efficacy of most inducers was considerably higher than in H4IIE cells. Furthermore, no inducers with extraordinary low maximal efficacy as observed in the hepatoma cells lines were found. The EC50-based potencies were in the rank order TCDD > PCB 126 > PCB 81 > PCB 169 > PCB 114 > PCB 157 > PCB 156 > PCB 77 > PCB 105 > PCB 118 > PCB 123 > PCB 167 > PCB 189, thus very similar to the EC50-based rank order in H4IIE cells. The maximal efficacies of TCDD and most inducing PCBs, in particular PCB 77, were considerably higher than in HepG2 cells.



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FIG. 5. EROD-inducing effects of TCDD or the non-ortho-substituted PCBs 77, 81, 126, and 169 in rat primary hepatocytes after treatment over 48 h. Data for PCB 81 were obtained from 3 independent experiments, data for the other non-ortho-substituted PCBs were from a previous publication (Schmitz et al., 1995Go). Concentration-effect curves fitted from data sets obtained at 6–8 different concentrations of each congener were calculated using a log-probit software.

 


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FIG. 6. EROD-inducing effects of TCDD or the mono-ortho-substituted PCBs 105, 114, 118, 123, 156, 157, 167, and 189 in rat primary hepatocytes after treatment over 48 h. Data for PCBs 114, 123, 15, 167, and 189 were obtained from 3 independent experiments, data for the other mono-ortho-substituted PCBs were from a previous publication (Schmitz et al., 1995Go). Concentration-effect curves fitted from data sets obtained at 6–8 different concentrations of each congener were calculated using a log-probit software.

 
The CNEC-based potencies were in the rank order TCDD > PCB 126 > PCB 81 > PCB 169 > PCB 77 > PCB 156 > PCB 105 {approx} PCB 114 > PCB 157 > PCB 118 > PCB 123 > PCB 189 > PCB 167, also similar to CNEC-based rank order in H4IIE cells. Again all non-ortho congeners had higher potencies than the mono-ortho congeners.

The fitted concentration-response curves did not strictly meet the requirements for a direct comparison of EC50 values. In particular, the maximal efficacies (maximum response) were very different, in some cases, for individual congeners. In addition, some indications for different slopes of the fitted curves were obtained, which led us to use a second benchmark concept better considering curve shapes, i.e., CNEC as a means for comparison of the different cell types. The analysis of both the benchmarks (Tables 1 and 2GoGo) revealed marked differences in REP values (Tables 3 and 4GoGo) for the non-ortho PCBs 81 and 126 between hepatoma cells of rat and human origin, and a complete lack of inducing potency in human HepG2 cells for PCB 169 and the mono-ortho PCBs tested with the exception of PCB 114.


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TABLE 3 EROD-Specific EC50-Based REP (EC50-REP) Values of Dioxin-Like PCBs in HepG2 Cells, H4IIE Cells, and Rat Primary Hepatocytes, and Corresponding WHO-TEFs for Human Risk Assessment and Mammals
 

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TABLE 4 EROD-Specific CNEC (Calculated No-Effect Concentration)-Based REP (CNEC REP) Values of Dioxin-Like PCBs in HepG2 Cells, H4IIE Cells, and Rat Primary Hepatocytes, and Corresponding WHO-TEFs for Human Risk Assessment and Mammals
 
DISCUSSION

Our data show that the inducibility of CYP1A activity by TCDD in the human HepG2 line is at least 1 order of magnitude below that in the rat hepatoma cell line H4IIE. This finding is in accordance to observations by Wiebel et al. (1996) who found that HepG2 cells were 20 times less sensitive with respect to induction of CYP1A-related aryl hydrocarbon hydroxylase than H4IIE cells. The rank order of sensitivity of the cells towards TCDD in terms of EROD induction was rat hepatocytes > H4IIE > HepG2. Parallel to the differences in EC50 values run different maxima of CYP1A induction in the 3 cell types resulting in the same rank order. These findings were completed by according differences in CNEC.

The fitted concentration-response curves did not strictly meet, for all PCBs, the requirements for a direct comparison of EC50 values. In particular, the maximal efficacies (maximum response) were very different for individual congeners. In addition, some indications for different slopes of the fitted curves were obtained. A comprehensive review of studies with dose-response curves for different biological endpoints of PCBs revealed that different shapes of the curves usually do not compromise the TEF concept, i.e., measured TCDD equivalencies are according to a dose additive model (van den Berg et al., 1998Go). This statement corresponds to our own experimental findings with relative potencies of PCB mixtures (Schmitz et al., 1995Go). Nevertheless, a second benchmark concept better considering curve shapes was used in this study to compare cell types and congeners, i.e., the calculation of CNEC from untreated control EROD activities, acoording to a method described by Van Birgelen et al. (1995).

A striking result of this analysis were the different EC50:CNEC ratios for different congeners and cell types, e.g., the CNEC of TCDD was 1 order of magnitude below the EC50 in HepG2 cells but 2 in H4IIE cells and even 3 orders of magnitude in rat primary hepatocytes. The reason for these differences are the slopes of the left branch of the sigmoidal induction curves. A steeper slope of the left branch means a greater effect at low concentrations leading to a lower CNEC value and a higher CNEC-based potency compared to the corresponding EC50-based pontency. This effect is independent of the maximum response of the cell type to an inducing agent and the median slope of the corresponding induction curve and may be of great importance for the evaluation of inducers in a low concentration range. On the other hand, measurable effects of TCDD at extremely low concentrations (fmolar range in cells of rat origin) and thus an corresponding low CNEC of TCDD may lead to CNEC-based REP values significantly different from the corresponding EC50-based ones and even more different from TEFs. This was the case, in particular, for the mono-ortho PCBs with weak inducing potencies, no matter which cell type was used. The CNEC-based potency of PCB 123 e.g., was 2 orders of magnitude lower than the EC50-based potency and even 3 orders of magnitude lower than the proposed TEF value. Likewise, PCBs showing a slope of the induction curve at low concentrations similar to TCDD did not reveal a great discrepancy between EC50-based and CNEC-based REP values.

The PCBs investigated showed much higher EC50 values and CNEC values in human than in rat cells, i.e., they were less potent. Among the non-ortho congeners, PCBs 126 and 169 elicited the most extensive species differences. The EC50 value of PCB 126 was found to be 2, the CNEC values even 3 to 4 orders of magnitude higher in human than in rat cells, whereas PCB 169 failed completely to induce EROD activity. Furthermore, PCB 81 (non-ortho) was the most potent inducer in HepG2 cells among the PCB congeners tested resulting in an EC50-based REP of 0.02. It also showed a high potency at low concentrations leading to the same CNEC-based REP value. This finding is in accordance to a previous report by Pang et al. (1999) who found PCB 81 to be the most potent inducer among the non-ortho PCBs tested of CYP1A1-dependent estrogen metabolism in HepG2 cells. In other experimental systems REPs for EROD induction by PCB 81 of 0.0069 in wild-type H4IIE cells (Sanderson et al., 1996Go) and 0.2 in chicken hepatocytes (Kennedy et al., 1996Go) were reported suggesting a high potency of this congener in birds. On the other hand, it should be taken into consideration that PCB 81 can be metabolized relatively easily due to the presence of 2 pairs of adjacent nonsubstituted carbon atoms. One possible explanation for its high potency in HepG2 cells may therefore be a reduced specific metabolic activity in this cell line, provided there is sufficient time for metabolization during 48 h of incubation. PCB 81 is found in mother's milk (Hong et al., 1994Go), and in a variety of environmental samples including fish (Echols et al., 1997Go). Further work is required to analyze if the TEF of 0.0005 currently used for PCB 81 may understimate the potency of this congener in humans or if the elevated potency in HepG2 is a special feature of this cell line.

The third non-ortho PCB acting as EROD inducer in HepG2, PCB 77, showed an extremely low maximal efficacy and was inactive as an inducer of CYP1A mRNA expression. This corresponds to the even higher CNEC value compared to the mono-ortho PCB 114 and to results of other investigators who found no EROD induction in HepG2 cells at 50 µM PCB 77 (Dubois et al., 1996Go).

As a possible explanation for a reduced maximal efficacy (maximal induction) of EROD inducers reported in various cell culture systems (Kennedy et al., 1996Go; Lipp et al., 1992Go; Schrenk et al., 1991Go; van der Burght et al., 1999Go) reduced receptor activation/transactivation has been suggested (van der Burght et al., 1999Go). However, Abnet et al. (1999) have shown that the human AhR transfected into COS-7 cells showed a higher transactivating efficacy on a CYP1A reporter plasmid than those derived from rainbow trout or zebrafish. Furthermore, transactivation capacities with PCB 77 and TCDD via the human receptor were almost equivalent.

Our results with PCB 77 in HepG2 cells rule out that the extremely low maximal efficacy concerning EROD induction is due to CYP1A inhibition. Since no CYP1A mRNA was found, a suppression of the nuclear transactivation/transcription of AhR/CYP1A in HepG2 cells is a likely explanation. Possibly, these effects are not mimicked in an extranuclear reporter gene model for the human AhR.

In contrast, the marked decrease in EROD activity frequently found at higher concentrations of CYP1A inducers may be related to different mechanisms. It has been proposed that cytotoxicity, or inhibition of the catalytic activity of CYP1A may explain this effect (Hahn et al., 1993Go; Pang et al., 1999Go; van der Burght et al., 1999Go). Furthermore, a possible role of porphyrin accumulation has been suggested (Tysklind et al., 1995Go).

The fourth congener showing EROD-inducing potency in HepG2 cells was PCB 114 (mono-ortho). The EC50-based REP calculated as 0.0006 was in the same range as the REPs found for EROD induction in hepatocytes from pigs (van der Burght et al., 2000Go) and monkeys (van der Burght et al., 1999Go). The relatively low CNEC of PCB 114 (25 nM), i.e., the relatively high inducing potency at low concentrations compared to the non-ortho PCB 77, supports the observation in Northern RNA analysis. However, other mono-ortho PCBs exhibiting a comparable (PCBs 105, 118, 123, 156, 157, and 189) or even higher (PCB 167) potency in pig hepatocytes were compeletely inactive as inducers in HepG2. Furthermore, those PCBs being inactive in HepG2 cells (PCB 105, 118, and 167) showed REPs > 0.0001 in hepatocytes from Cynomolgus monkeys. These data suggest that PCB 114 may have distinct properties that make it an AhR agonist in the HepG2 system in contrast to a variety of other mono-ortho congeners.

The EC50 values for PCBs 81, 114, and 126 in HepG2 were confirmed by Northern blotting. In addition, these data revealed that treatment with congeners not inducing EROD activity did not lead to an increase in CYP1A mRNA.

The reason for the highly reduced potencies of most dioxin-like PCBs in HepG2 cells may either be related to differences in uptake, metabolism, etc. or be based on a reduced ability to activate the AhR complex. The uptake of PCBs through the membrane is mainly governed by their lipophilic character and molecular size. The fact that only congeners with a limited range of chlorine atoms (4 to 7) were investigated leads to the conclusion that differnces in uptake are unlikely to be of importance in this study. Additionally, the experimental time period of 48 h is probably too short to cause any significant metabolization of PCBs. Therefore differences in activation of the Ah receptor complex and/or transcription may be dominating factors explaining the variation in the in-vitro systems investigated. The structure of the XREs of human CYP1A genes alone cannot hold fully responsible for reduced induction since the difference in the case of TCDD is only 10-fold. Furthermore, it should be taken into account that HepG2 as a tumor cell line may have developed an aberrant AhR-XRE activation and/or transcription pathway. Future in vitro studies with human primary hepatocytes may elucidate this possibility. Lower levels of AhR that were found in human hepatocytes (Safe, 1986Go) or species differences in the hepatic expression patterns of the CYP isozymes 1A1 and 1A2 (Xu et al., 2000Go) may also contribute to the reduced sensitivity of human cells.

In summary, our results demonstrate striking differences between the rat and human hepatoma cell lines H4IIE and HepG2 with respect to both types of REPs of EROD induction for most dioxin-like PCBs. In particular, the non-ortho-substituted PCB 169 was inactive, and PCB 126 was less potent by 2 orders of magnitude in HepG2 cells. These findings and findings in hepatocytes of other primate species (van der Burght et al., 1999Go) show the necessity of widening the base of experimental data for human risk assessment of PCBs by screening their toxicity in cells of human origin.

The benchmark concept of CNEC used in this study is not completely independent of the shape of induction curves. Since a logistic regression model proved best to fit our data the slope of the left branch of the curve (i.e., at low concentrations) has a dominating influence on potencies based upon CNEC values. Whereas EC50-based REPs showed sufficient additivity in many studies despite of different curve shapes, the great differences between EC50-based and CNEC-based REPs, particularly for dioxin-like PCB congeners present at higher concentrations in technical and environmental samples, make the latter values less applicable for prescreening and assessing the TEQ of mixtures of inducers. On the other hand, the CNEC concept may provide an appropriate means to support the assessment of low-concentration effects of inducing agents.

ACKNOWLEDGMENTS

The authors wish to thank Dr. Larry Robertson for providing PCB 81, Dr. Frank Gonzalez for providing the CYP1A2 cDNA, and Ms. Silke Ripp and Ms. Tina Blauth for expert technical assistance. This work was supported by the Stiftung Innovation für Rheinland-Pfalz.

NOTES

1 To whom correspondence should be addressed. Fax: +49-631-205-4398. E-mail: hschmitz{at}rhrk.uni-kl.de. Back

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