Differential Expression of Metallothionein Isoforms in Terrestrial Snail Embryos Reflects Early Life Stage Adaptation to Metal Stress

Abstract

The aim of this study was to analyze the expression of three metallothionein (MT) isoform genes (CdMT, CuMT and Cd/CuMT), already known from adults, in the Early Life Stage (ELS) of Cantareus aspersus. This was accomplished by detection of the MT isoform-specific transcription adopting Polymerase Chain Reaction (PCR) amplification and quantitative Real Time (qRT)-PCR of the three MT genes. Freshly laid eggs were kept for 24 hours under control conditions or exposed to three cadmium (Cd) solutions of increasing concentration (5, 10, and 15 mg Cd/L). The transcription of the three MT isoform genes was detected via PCR in 1, 6 and 12-day-old control or Cd-exposed embryos. Moreover, the transcription of this isoform genes during development was followed by qRT-PCR in 6 and 12-day-old embryos. Our results showed that the CdMT and Cd/CuMT genes, but not the CuMT gene, are expressed in embryos at the first day of development. The transcription of the 3 MT genes in control embryos increased with development time, suggesting that the capacities of metal regulation and detoxification may have gradually increased throughout embryogenesis. However in control embryos, the most highly expressed MT gene was that of the Cd/CuMT isoform, whose transcription levels greatly exceeded those of the other two MT genes. This contrasts with the minor significance of this gene in adult snails and suggests that in embryos, this isoform may play a comparatively more important role in metal physiology compared to adult individuals. This function in adult snails appears not to be related to Cd detoxification. Instead, snail embryos responded to Cd exposure by over-expression of the CdMT gene in a concentration-dependent manner, whereas the expression of the Cd/CuMT gene remained unaffected. Moreover, our study demonstrates the ability of snail embryos to respond very early to Cd exposure by up-regulation of the CdMT gene.

High-level dietary cadmium exposure is associated with global DNA hypermethylation in the gastropod hepatopancreas

“…It will also be of interest to study the effects of cadmium on specific genes in gastropods. In this context, the Cd-selective metallothionein ( Cd-MT ) gene deserves particular consideration since it can be activated by dietary Cd levels as low as 2 mg/L [ 54 , 55 ] and is generally inducible, unlike constitutively expressed genes with housekeeping functions [ 56 ]. The Cd-MT gene contains CG sites [ 57 ], but there is no information available about their methylation status.…”

Cadmium Accumulation and Pathological Alterations in the Midgut Gland of Terrestrial Snail Helix pomatia L. from a Zinc Smelter Area: Role of Soil pH

“…The studies demonstrated that pathological changes typical for Cd toxicity, specifically in the midgut gland, include an increased number of excretory cells with residual bodies (lipofuscin granules), programmed cell death, the disruption of mitochondrial membranes, and inflammation (Chabicovsky et al 2004 ; Hödl et al 2010 ; Amachree et al 2013 ; Sheir et al 2013 ). These alterations are thought to occur at Cd concentrations exceeding the capacity of metallothionein (MT), a low-molecular weight Cd-binding protein which is responsible for detoxification of the metal in the midgut gland of H. pomatia and other species (Dallinger et al 1997 ; Chabicovsky et al 2004 ; Hödl et al 2010 ; Baurand et al 2015 ). The non-MT-bound Cd ions are believed to generate reactive oxygen species and peroxidation of membrane lipids, thereby producing toxicity (Roesijadi et al 1997 ; Liu et al 2009 ; Amachree et al 2013 ).…”

Genotoxicity assessment of pesticides on terrestrial snail embryos by analysis of random amplified polymorphic DNA profiles

“…aspersus embryos [47](unpublished data). The CuMT gene is known to be up-regulated during Cu stress in adults [48].…”