Analysis of the CYP1A1 mRNA dose-response in human keratinocytes indicates that relative potencies of dioxins, furans, and PCBs are species and congener specific

Abstract

Reports indicate that toxic equivalency factors (TEFs) based primarily on rodent data do not accurately predict in vitro human responsiveness to certain dioxin-like chemicals (DLCs). To investigate this in cells responsive to dioxins and relevant to chloracne, normal human epidermal keratinocytes were treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and several DLCs, each with a TEF value of 0.1, representing three classes of congeners. We estimated half maximal effective concentration (EC50)-based donor-specific relative potency (REP) values for cytochrome P450 1A1 (CYP1A1) messenger RNA (mRNA) induction for TCDD, 1,2,3,6,7,8-hexachlorodibenzo-p-dioxin (HxCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 1,2,3,6,7,8-hexachlorodibenzofuran (HxCDF), and 3,3',4,4',5-pentachlorobiphenyl (PCB 126). We also determined EC50-based population-level REP values (n = 4) for CYP1A1 mRNA induction for TCDD, HxCDF, and PCB 126. Furthermore, an alternative factor, the relative threshold factor (RTF) based on the low end (threshold) of the dose-response curve, was calculated. Our results demonstrated that HxCDF had a population-based REP value of 0.98, 9.8-fold higher than its assigned TEF value of 0.1. Conversely, PCB 126 had an REP value of 0.0027 and an RTF of 0.0022, 37-fold and 45-fold less than its assigned TEF of 0.1, respectively. The REP values for HxCDD and TCDF were 0.24 and 0.10, respectively, similar to their assigned value of 0.1. Therefore, although the DLCs tested in the current study all possessed the same assigned TEF value of 0.1, congener-specific differences in REPs and RTFs were observed for human keratinocytes. These congener-specific discrepancies are likely because of differences in interspecies factors that have yet to be defined.

FIG. 1.

(A) NHEKs were treated with control vehicle (DMSO, 0.7%), TCDD (100nM), TCDF (300nM), HxCDD (300nM), HxCDF (300nM), or PCB 126 (10,000nM) for 48 h. Cell viability was determined using the MTT assay as described in Materials and Methods section, which measures absorbance at 570nM. The results are presented as mean ± SD (n = 3). (B) NHEKs were treated with TCDD (10nM) or PCB 126 (10,000nM) for 0, 3, 6, 12, 24, and 48 h. Real-time PCR was used to determine the level of CYP1A1 mRNA. Levels of mRNA are expressed in units relative to the maximum, given a value of 1.

FIG. 2.

CYP1A1 mRNA increases in NHEKs from four donors treated with DLCs modeled as individual donors. NHEKs from donor 1 were treated with DMSO (0.7%) or increasing concentrations of TCDD, TCDF, HxCDD, HxCDF, or PCB 126 for 48 h (see Materials and Methods section for concentrations). NHEKs from donors 2–4 were treated with DMSO (0.7%) or increasing concentrations of TCDD, HxCDF, or PCB 126 for 48 h. The individual culture data points were graphed relative to the average maximum TCDD response reached by each individual donor. Curves represent predicted responses generated by the models described in the Materials and Methods section.

FIG. 3.

CYP1A1 mRNA increases in NHEKs from four donors treated with TCDD, HxCDF, and PCB 126 modeled together as a population and compared with the rat liver cell line H4IIE. Relative CYP1A1 levels from NHEKs from each donor and from H4IIE cells after 48 h treatment with TCDD (upper panel), HxCDF (middle panel), and PCB 126 (lower panel) are plotted as indicated. Each point in the graphs represents an individual sample. Data were modeled as described in the Materials and Methods section, resulting in a population-level prediction curve for NHEKs (solid line) and the predicted response of rat H4IIE cells (dashed line).

FIG. 4.

Models of apparent population thresholds for NHEKs treated with TCDD, HxCDF, and PCB 126. Relative CYP1A1 mRNA increases from each of the four donors treated with TCDD (upper panel), HxCDF (middle panel), and PCB 126 (lower panel) were plotted and analyzed as described in Materials and Methods section. Each point in the graphs represents an individual sample, and curves are the population-level response predicted by the threshold model. Note that both axes are log₁₀ transformed.