Effect of metals on β-actin and total protein synthesis in cultured human intestinal epithelial cells

Abstract

As an important structural protein, β-actin is associated with anchoring of tight junctions (TJs) to the cell scaffold. Caco-2 cells, an immortal intestinal epithelial cell line, rely on β-actin to form intact monolayers with high transepithelial electrical resistance in cell culture inserts. We examined the effect of six metals on expression of β-actin mRNA and β-actin synthesis, on total and net production of newly synthesized proteins, on paracellular transport of TJ markers, and on cell viability in confluent monolayers. [ 3 H]-glycine and [ 3 H]-tyrosine were used as indicators of newly synthesized proteins in the absence or presence of increasing concentrations of arsenic, cadmium, copper, manganese, mercury and nickel. The monolayers were exposed to 24-hr single exposures as well as continuous daily repeated doses of metals for 48-hr and 96-hr. Results suggest that decreases in newly synthesized proteins, in which β-actin represents about 10%, correlated with 2-to 5-fold higher expression of β-actin mRNA for the higher concentrations of metals. Interestingly, IC 50 s calculated for each chemical for 24-hr acute and 48-and 96-hr repeated dosing experiments, using the MTT viability assay and paracellular permeability markers, decreased newly synthesized and total proteins to 10% and 40% of control, respectively. Overall, the results indicate that, at equivalent concentrations, the metals affect β-actin mRNA and newly synthesized proteins before cell viability and paracellular permeability are compromised. Consequently the results help in elucidating mechanisms of metal cytotoxicity that lead to understanding the relationship between tight junction integrity, paracellular transport, and cell viability.

The Blood-Testis Barrier and Its Implications for Male Contraception

“…These findings are also in agreement with studies in epithelia other than the testis, such as the small intestine, kidney, liver, and skin, which imply that cell junctions are the target of different classes of reproductive toxicants, including cadmium (Fiorini et al, 2004;Zefferino et al, 2008;Pinton et al, 2009;Choi et al, 2010;Li et al, 2010). In addition, there is mounting evidence that both AJ (and/or TJ) (Prozialeck and Lamar, 1999;Prozialeck, 2000;Prozialeck et al, 2003;Jacquillet et al, 2007;Thompson et al, 2008;Siu et al, 2009b,c;Calabro et al, 2011) and gap junction (Fukumoto et al, 2001;Jeon et al, 2001;Guan et al, 2007;Tang et al, 2009;Vinken et al, 2010) are targets of cadmium toxicity in multiple epithelia in different organs, such as the kidney, heart, liver, ovary, and testis.…”

Regulation of Sirt1/Nrf2/TNF-α signaling pathway by luteolin is critical to attenuate acute mercuric chloride exposure induced hepatotoxicity

“…Researches have shown that, in mammals, mercury can induce a wide range of adverse effects on systems and tissues12345. Aplastic anemia is another potential consequence of inorganic mercury exposure6.…”

Epigenetic Modifications and Stem Cell Toxicology: Searching for the Missing Link

“…The increasing awareness of the influence of growth factors (GFs) and basement membrane substrata has improved our understanding of the role of cell regeneration in wound repair and in preneoplastic tumor growth. For instance, in their ability to form tight junction (TJ) strands, differentiating epidermal and epithelial cells act to prevent free exchange of most solutes between luminal and interstitial fluids along the paracellular route (Calabro, Gazarian, and Barile, 2011). Breakdown in the barrier functions of these membranes may contribute to cellular neoplasia.…”