Cytotoxicity of anisidine in hepatic cell lines

Humans are exposed to azo dyes and its degradation products like aromatic amines through different consumer product. The ECHA classified the aromatic amine o-anisidine as carcinogen (Carc. 1B) and mutagen (Muta 2) due to increased rates of bladder and kidney tumors in mice and rats. In contrast, p-anisidine was classified with specific organ toxicity (STOT RE 2) only. Hepatotoxic effects of o- and p-anisidine were studied using HepG2 and Huh-7 cells.

Cells were grown in DMEM (Huh-7) or RPMI medium (HepG2), supplemented with 10% FCS, 5 mM L-glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin. EC50 values were determined using the MTT assay, reactive oxygen species (ROS) were detected using 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), and changes in the gene expression were quantified using PCR.

In HepG2 cells the EC50 values were 16 ± 1 mM and 18 ± 1 mM for o- and p-anisidine, respectively. However, in Huh 7 cells EC50 values were 11 ± 1 mM and 16 ± 1 mM for o- and p-anisidine, respectively. At 1 mM both anisidine isomers induced the generation of ROS in either cell line, which could be suppressed by adding N-acetylcysteine. Moreover, the carcinogenic o-anisidine upregulated the expression of CYP1A1 and NQO1 in both cell lines and TNFα in HepG2 cells.

While tumors mainly occur in kidney and bladder tissues, our study shows that human hepatic cells are as well significantly affected by o-anisidine with regard to ROS induction and altered gene expression and – to a lesser extent – also by p-anisidine.

Corresponding author: Zellmer, Sebastian

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