Cytotoxic effects of I۳C and TCDD on macrophages: Role of aryl hydrocarbon receptor target genes

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-۳-carbinol (I۳C), a dietary phytochemical derived from cruciferous vegetables, and ۲,۳,۷,۸-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 I۳C and TCDD on THP-۱-derived macrophages and to assess the consequent transcriptional regulation of three canonical AhR target genes: CYP۱A۱, CYP۱B۱, and AhRR. Methods: THP-۱ human monocytic cells were differentiated into macrophages using phorbol ۱۲-myristate ۱۳-acetate (PMA) and ۲-mercaptoethanol (۲-ME). Differentiated macrophages were treated with I۳C (۱۰ ng/µL) or TCDD (۱۰۰ nM), each dissolved separately in dimethyl sulfoxide (DMSO), for ۲۴ and ۴۸ hours. Lipopolysaccharide (LPS; ۱۰۰ ng/mL) was used as a positive control. Cell viability was assessed using the MTT colorimetric assay. Gene expression of CYP۱A۱, CYP۱B۱, 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-Value < ۰.۰۵. Results: TCDD demonstrated pronounced cytotoxicity, reducing cell viability by ۲۵% at ۲۴ hours (P-Value < ۰.۰۵) and by ۴۰% at ۴۸ hours (P-Value < ۰.۰۱) compared to untreated controls. I۳C induced significantly milder cytotoxicity, with viability reductions of ۱۰% and ۱۵% at ۲۴ and ۴۸ hours, respectively (P-Value < ۰.۰۵). DMSO vehicle control did not significantly affect cell viability. Real-time qRT-PCR analysis revealed that both ligands significantly upregulated CYP۱A۱, CYP۱B۱, and AhRR. TCDD induced greater upregulation of CYP۱A۱ (۵.۵-fold at ۴۸ h) and CYP۱B۱ (۴.۸-fold at ۴۸ h) compared to I۳C (۳.۲-fold and ۲.۷-fold, respectively; P-Value < ۰.۰۵ for all between-group comparisons). AhRR was upregulated to a similar extent by both ligands (~۲.۱-fold). Conclusion: TCDD exhibited markedly higher cytotoxicity than I۳C in THP-۱-derived macrophages, consistent with potent and sustained AhR activation. I۳C 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 I۳C as a candidate for future anti-inflammatory research.

کلیدواژه ها:

Aryl Hydrocarbon Receptors ، Aryl Hydrocarbon Receptor Repressor ، Cytochrome P-۴۵۰ CYP۱B۱ ، Cytochrome P-۴۵۰ CYP۱A۱ ، THP-۱ Cells ، Indole-۳-Carbinol ، Tetrachlorodibenzo-p-dioxin ، Protein ، Macrophage Cytotoxicity ، Immune Modulation