چکیده: (2492 مشاهده)
Background: Macrophages are central effectors of innate immunity and exhibit remarkable functional plasticity in response to environmental stimuli. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that plays a critical role in regulating macrophage function, immune homeostasis, and xenobiotic metabolism. Two well-characterized AhR ligands, indole-3-carbinol (I3C), a dietary phytochemical derived from cruciferous vegetables, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a persistent environmental pollutant, exert divergent biological effects despite engaging the same receptor. However, their differential cytotoxic impact on macrophages and the extent to which they activate downstream AhR target genes remain poorly characterized. This study aimed to evaluate and compare the cytotoxic effects of I3C and TCDD on THP-1-derived macrophages and to assess the consequent transcriptional regulation of three canonical AhR target genes: CYP1A1, CYP1B1, and AhRR.
Methods: THP-1 human monocytic cells were differentiated into macrophages using phorbol 12-myristate 13-acetate (PMA) and 2-mercaptoethanol (2-ME). Differentiated macrophages were treated with I3C (10 ng/µL) or TCDD (100 nM), each dissolved separately in dimethyl sulfoxide (DMSO), for 24 and 48 hours. Lipopolysaccharide (LPS; 100 ng/mL) was used as a positive control. Cell viability was assessed using the MTT colorimetric assay. Gene expression of CYP1A1, CYP1B1, and AhRR was quantified by real-time quantitative RT-PCR (qRT-PCR) using SYBR Green chemistry, with GAPDH as the reference gene. Data were analyzed by one-way ANOVA followed by Tukey’s post-hoc test; significance was set at p < 0.05.
Results: TCDD demonstrated pronounced cytotoxicity, reducing cell viability by 25% at 24 hours (P-Value < 0.05) and by 40% at 48 hours (P-Value < 0.01) compared to untreated controls. I3C induced significantly milder cytotoxicity, with viability reductions of 10% and 15% at 24 and 48 hours, respectively (P-Value < 0.05). DMSO vehicle control did not significantly affect cell viability. Real-time qRT-PCR analysis revealed that both ligands significantly upregulated CYP1A1, CYP1B1, and AhRR. TCDD induced greater upregulation of CYP1A1 (5.5-fold at 48 h) and CYP1B1 (4.8-fold at 48 h) compared to I3C (3.2-fold and 2.7-fold, respectively; P-Value < 0.05 for all between-group comparisons). AhRR was upregulated to a similar extent by both ligands (~2.1-fold).
Conclusion: TCDD exhibited markedly higher cytotoxicity than I3C in THP-1-derived macrophages, consistent with potent and sustained AhR activation. I3C induced comparatively milder cytotoxic and transcriptional responses, suggesting more controlled AhR engagement. These results provide mechanistic insights into ligand-dependent AhR signaling in macrophages and underscore the immunotoxicological risks of TCDD exposure, while highlighting I3C as a candidate for future anti-inflammatory research.
Methods: THP-1 human monocytic cells were differentiated into macrophages using phorbol 12-myristate 13-acetate (PMA) and 2-mercaptoethanol (2-ME). Differentiated macrophages were treated with I3C (10 ng/µL) or TCDD (100 nM), each dissolved separately in dimethyl sulfoxide (DMSO), for 24 and 48 hours. Lipopolysaccharide (LPS; 100 ng/mL) was used as a positive control. Cell viability was assessed using the MTT colorimetric assay. Gene expression of CYP1A1, CYP1B1, and AhRR was quantified by real-time quantitative RT-PCR (qRT-PCR) using SYBR Green chemistry, with GAPDH as the reference gene. Data were analyzed by one-way ANOVA followed by Tukey’s post-hoc test; significance was set at p < 0.05.
Results: TCDD demonstrated pronounced cytotoxicity, reducing cell viability by 25% at 24 hours (P-Value < 0.05) and by 40% at 48 hours (P-Value < 0.01) compared to untreated controls. I3C induced significantly milder cytotoxicity, with viability reductions of 10% and 15% at 24 and 48 hours, respectively (P-Value < 0.05). DMSO vehicle control did not significantly affect cell viability. Real-time qRT-PCR analysis revealed that both ligands significantly upregulated CYP1A1, CYP1B1, and AhRR. TCDD induced greater upregulation of CYP1A1 (5.5-fold at 48 h) and CYP1B1 (4.8-fold at 48 h) compared to I3C (3.2-fold and 2.7-fold, respectively; P-Value < 0.05 for all between-group comparisons). AhRR was upregulated to a similar extent by both ligands (~2.1-fold).
Conclusion: TCDD exhibited markedly higher cytotoxicity than I3C in THP-1-derived macrophages, consistent with potent and sustained AhR activation. I3C induced comparatively milder cytotoxic and transcriptional responses, suggesting more controlled AhR engagement. These results provide mechanistic insights into ligand-dependent AhR signaling in macrophages and underscore the immunotoxicological risks of TCDD exposure, while highlighting I3C as a candidate for future anti-inflammatory research.
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