Characterization of hepatic metallothionein expression in channel catfish (Ictalurus punctatus) by reverse transcriptase polymerase chain reaction

Abstract

Measurements of metallothioneins (MTs) can be labour intensive and require expensive probes and or analytical instrumentation. In contrast, the reverse transcriptase polymerase chain reaction (RT-PCR0 has provided a relatively sensitive and simple mechanism to measure expression of MT in sentinel species in aquatic environments. The objectives of this study were to characterize expression of hepatic MT in a commonly occurring fish species of the southeastern United States, the channel catfish (Ictalurus punctatus). Expression of hepatic MT was assessed following exposure to several metals found naturally and in excess due to anthropogenic inputs. MT expression by RT PCR was confirmed by Southern blot with a cDNA probe (552 bp) encoding MT and the complete 3 untranslated region from the common carp (Cyprinus carpio) and a 388 bp cDNA probe encoding 49 bp of the 5′ untranslated region, the entire MT protein, and the complete 3′ untranslated region of winter flounder (Pleuronectes americanus). Each probe recognized a single band of approximately 550 bp which corresponded to the RT-PCR product from hepatic RNA of channel catfish 24 h after an intraperitoneal injection of cadmium chloride (10 mg kg^-1). Catfish were also injected with intraperitoneal administrations of zinc sulphate (10 mg kg^-1) and copper sulphate (2 mg kg^-1). After 24 h, zinc increased MT expression 16-fold over controls whereas cadmium or copper increased levels 13 and nearly 6-fold, respectively. A 1-week exposure to water borne 1 mg l^-1 sodium arsenite or monosodium methylarsonate (MSMA) induced hepatic MT expression nearly two fold, whereas water borne exposure to the same dose of sodium arsenate failed to increase MT levels. To examine the effect of dietary sub chronic methylmercury exposure on hepatic MT expression, Japanese medaka (Oryzias latipes), were lethally injected with solutions of methylmercuric chloride to provide a 0.1 mg kg^-1 daily dose to catfish. After 30 days, hepatic MT expression, condition factor and liver somatic indices were unchanged, although hepatic and muscle residues of total mercury were significantly increased following treatment. The results of this study show that MT expression can vary in aquatic organisms in response to different metal treatments and may be utilized as a biomarker of exposure and possibly effect in the channel catfish.

Keywords::

• Metallothionein, Rt Pcr, Cadmium, Zinc, Arsenic, Copper, Methylmercury, Channel Catfish, Ictalurus Punctatus, Liver