Non-additive hepatic gene expression elicited by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153) co-treatment in C57BL/6 mice

Abstract

Interactions between environmental contaminants can lead to non-additive effects that may affect the toxicity and risk assessment of a mixture. Comprehensive time course and dose-response studies with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), non-dioxin-like 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153) and their mixture were performed in immature, ovariectomized C57BL/6 mice. Mice were gavaged once with 30 μg/kg TCDD, 300 mg/kg PCB153, a mixture of 30 μg/kg TCDD with 300 mg/kg PCB153 (MIX) or sesame oil vehicle for 4,12, 24,72 or 168 h. In the 24h dose-response study, animals were gavaged with TCDD (0.3,1, 3, 6, 10, 15, 30, 45 μg/kg), PCB153 (3,10, 30, 60, 100, 150, 300, 450 mg/kg), MIX (0.3+3, 1+10, 3+30, 6+60, 10+100, 15+150, 30+300, 45 μg/kg TCDD+450 mg/kg PCB153, respectively) or vehicle. All three treatments significantly increased relative liver weights (RLW), with MIX eliciting significantly greater increases compared to TCDD and PCB153 alone. Histologically, MIX induced hepatocellular hypertrophy, vacuolization, inflammation, hyperplasia and necrosis, a combination of TCDD and PCB153 responses. Complementary lipid analyses identified significant increases in hepatic triglycerides in MIX and TCDD samples, while PCB153 had no effect on lipids. Hepatic PCB153 levels were also significantly increased with TCDD co-treatment. Microarray analysis identified 167 TCDD, 185 PCB153 and 388 MIX unique differentially expressed genes. Statistical modeling of quantitative real-time PCR analysis of Pla2g12a, Serpinb6a, Nqo1, Srxn1, and Dysf verified non-additive expression following MIX treatment compared to TCDD and PCB153 alone. In summary, TCDD and PCB153 co-treatment elicited specific non-additive gene expression effects that are consistent with RLW increases, histopathology, and hepatic lipid accumulation.

Figure 1

Temporal and dose-dependent changes in the relative liver weight following exposure to TCDD, PCB153, and MIX. (A) In the time course study, 30 μg/kg TCDD + 300 mg/kg PCB153 were co-administered (MIX, 1:10,000 TCDD to PCB153). (B) In the 24 h dose-response, MIX (μg/kg TCDD + mg/kg PCB153) was administered at 1:10,000 ratio of TCDD to PCB153, respectively. Results are displayed as mean ± standard error (SE) for 5 independent replicates. Data were analyzed by analysis of variance (ANOVA) followed by Tukey’s post hoc test: *p<0.05 vs. vehicle, ^ap<0.05 vs. PCB153 and ^bp<0.05 vs. TCDD. Time course increases in relative liver weight induced by individual TCDD and PCB153 treatments are from Kopec et al. (2010b).

Figure 2

Hepatic (A) TCDD and (B) PCB153 tissue levels per g liver wet weight measured by HRGC-HRMS (Kopec et al. 2010b). PCB153 levels were potentiated following co-administration with TCDD (in MIX) at 24 and 168 h. However no changes in TCDD hepatic levels were detected following co-treatment with PCB153 (in MIX). Results are displayed as mean ± SE for 3 independent replicates. Data were analyzed by ANOVA followed by Tukey’s post hoc test: *p<0.05 vs. time-matched PCB153 levels alone.

Figure 3

Standard hematoxylin and eosin staining of liver sections at 24 (top panel: A–D) and 168 h (bottom panel: E–H). (A&E) Sesame oil elicited minimal vacuolization. (B&F) 30 μg/kg TCDD elicited vacuolization and inflammation (solid arrow). (C&G) 300 mg/kg PCB153 resulted in minimal vacuolization and hypertrophy (asterisk). (D&H) MIX resulted in vacuolization, inflammation (solid arrow), hypertrophy (asterisk) and necrosis (dashed arrow). Bars = 50 μm.

Figure 4

Temporal microarray data sets for 30 μg/kg TCDD, 300 mg/kg PCB153 (Kopec et al. 2010b), and MIX (1:10,000 TCDD:PCB153) groups were compared using (A) stringent (|fold change|>1.5, P1(t)>0.999) and (B) relaxed (|fold change|>1.5, P1(t)>0.95) selection criteria. Numbers in the Venn diagram represent unique genes.

Figure 5

QRTPCR verification of AhR, CAR, and PXR-regulated genes. TCDD induced Cyp1a1 (AhR), while PCB153 induced Cyp2b10 and Cyp3a11 (CAR/PXR), and MIX induced all three genes. All fold changes were calculated relative to time-matched vehicle controls. The genes are represented by their official gene symbols. Results are displayed as mean ± SE for 5 independent replicates. Data were analyzed by ANOVA followed by Tukey’s post hoc test: *p<0.05 vs. time-matched vehicle, ^ap<0.05 vs. PCB153 and ^bp<0.05 vs. TCDD. TCDD and PCB153 temporal data are from Kopec et al. (2010b).

Figure 6

Temporal and dose-dependent QRTPCR verification of putative non-additive genes. Genes exhibiting putative non-additive interactions initially identified in the time course study were verified by QRTPCR in the time course study and 24 h dose-response study. The genes are represented by their official gene symbols. Results are displayed as mean ± SE for 5 independent replicates. Data were analyzed by ANOVA followed by Tukey’s post hoc test: *p<0.05 vs. time-matched vehicle, ^ap<0.05 vs. PCB153 and ^bp<0.05 vs. TCDD.

Figure 7

Non-linear logistic modeling of QRTPCR dose-response data. Graphs depict the additive model (dashed line) generated using TCDD and PCB153 data and the mixture model (solid line) fitted using MIX data (black dots). MIX data for Nqo1, Dysf, Pla2g12a, Serpinb6a, and Srxn1 fit the mixture model better than the additivity model, indicating a statistically significant (p<0.0001) synergistic interaction.