Establishment of a Novel Alternative Method Using the Gobiocypris rarus AhR for Toxicity Testing of the Dioxin-like Potency of Halogenated Polycyclic Aromatic Hydrocarbons

Abstract Halogenated polycyclic aromatic hydrocarbons (HPAHs) often exhibit distinct toxicity and mechanisms relative to polycyclic aromatic hydrocarbons (PAHs), yet most remain unassessed, particularly for dioxin-like effects mediated by the aryl hydrocarbon receptor (AhR). This study characterized three rare minnow AhRs and established an efficient AhR-activation screening assay (using graAhR2, EC50 = 2.53 nM and Z′ factor = 0.58), and evaluated 40 HPAHs/PAHs with preliminary activity screening and ecotoxicity considered. HPAHs showed a median IC50 of 1.15 × 10–4 M for cellular viability (parent PAHs: 4.29 × 10–4 M), indicating greater cellular toxicity. Nine compounds were AhR-active. Notably, halogenated fluorene and pyrene exhibited AhR activation, whereas the parent did not. 4-bromobenz[a]anthracene exhibited 2028-fold higher activation potency than the parent. Acute toxicity (cell viability IC50) and AhR activation potency (EC50) exhibited a weak correlation, R2 = 0.21 and P = 0.22 in COS-7 cells, implying little AhR contribution to acute toxicity. All HPAHs/PAHs shared a similar binding direction within the AhR. AhR-active compounds possessed greater affinity than inactive ones (P = 0.0032). For instance, in fluorene-based HPAHs, a binding pose more similar to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) may confer stronger activation potency. The results support the need for a tiered risk evaluation of HPAHs, e.g., considering acute and long-term aspects, and integrating dioxin-like potency into relevant regulatory frameworks, e.g., the toxic equivalency factor (TEF) or similar ones.