In vitro interactions of malachite green and leucomalachite green with hepatic drug-metabolizing enzyme systems in the rainbow trout (Onchorhyncus mykiss)

- Malachite green depleted liver GSH and inhibited some drug metabolizing enzymes.
- EROD and GST were strongly inhibited with IC50 in the micromolar range.
- EROD inhibition was competitive.
- GST inhibition was competitive for GSH and mixed for the substrate CDNB.
- LMG did not affect GSH content or drug metabolizing enzymes.

Malachite green (MG) has been widely used in aquaculture to treat a number of microbial and parasitic diseases. It is currently banned in the EU because of the high cytotoxicity and carcinogenic activity, which is also shared by leucomalachite green (LMG), a reduced MG metabolite that can persist in fish tissues for months. There is scant information about the ability of either compound to interact with drug metabolizing enzymes in fish. Therefore we evaluated the in vitro effects of MG and LMG (25, 50 and 100 μM) on some DMEs and glutathione (GSH) content in rainbow trout liver subfractions. LMG did not affect any of the examined parameters. In contrast, MG proved to deplete GSH and to depress to a various extent the activities of NAD(P)H cytochrome c reductase, 7-ethoxycoumarin O-deethylase, 1-naphthol uridindiphosphoglucuronyl-transferase and maximally those of 7-ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST) accepting 1-chloro2,4-dinitrobenzene (CDNB) as substrate. The inhibition mechanisms of EROD and GST were investigated by means of non-linear Michaelis-Menten kinetics and Lineweaver-Burk plots using 0.175-8 μM MG. The calculated IC50 for EROD was 7.1 μM, and the inhibition appeared to be competitive (Ki 2.78 ± 0.24 μM). In the case of GST, the calculated IC50 was 0.53 μM. The inhibition was best described as competitive toward GSH (Ki 0.39 ± 0.02 μM) and of mixed-type toward CDNB (Ki 0.64 ± 0.06 μM). Our findings indicate that, contrary to LMG, MG behaves as a relatively strong inhibitor of certain liver DMEs and can reversibly bind GSH.

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