The
triphenylmethane dye,
malachite green (MG), is used to treat and prevent fungal and
parasitic infections in the aquaculture industry. It has been reported that the reduced metabolite of MG,
leucomalachite green (LMG), accumulates in the tissues of fish treated with MG. MG is structurally related to other
triphenylmethane dyes (e.g.,
gentian violet and
pararosaniline) that are carcinogenic in the liver, thyroid, and other organs of experimental animals. The ability of LMG to inhibit
thyroid peroxidase (TPO), the
enzyme that catalyzes the iodination and coupling reactions required for
thyroid hormone synthesis, was determined in this study. LMG inhibited TPO-catalyzed
tyrosine iodination (half-maximal inhibition at ca. 10 microM). LMG also inhibited the TPO-catalyzed formation of
thyroxine in low-
iodine human
goiter thyroglobulin (half-maximal inhibition at ca. 10 microM) using a model system that measures simultaneous iodination and coupling. Direct inhibition of the coupling reaction by LMG was shown using a coupling-only system containing chemically preiodinated
thyroglobulin as the substrate. Incubation of LMG with TPO,
iodide, and
tyrosine in the presence of a H2O2-generating system yielded oxidation products that were identified by using on-line LC/APCI-MS as desmethyl LMG, 2desmethyl LMG, 3desmethyl LMG, MG, and MG N-
oxide. Similar products from LMG were observed in incubations with TPO and H2O2 alone. These findings suggest that the anti-thyroid effects (increased serum
thyroid-stimulating hormone and decreased serum
thyroxine) observed in rats treated with LMG result from blockade of
hormone synthesis through alternate substrate inhibition and that chronic exposure could cause thyroid follicular cell
tumors through a hormonal mechanism. The observed TPO-catalyzed oxidative demethylation of LMG to a primary arylamine also suggests a genotoxic mechanism for
tumor formation is possible.