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


METHODS
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-h single exposures as well as continuous daily repeated doses of metals for 48-h and 96-h.


RESULTS
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-h acute and 48- and 96-h 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.


CONCLUSIONS
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.
Calabro
Anthony R
AR
St. John's University, College of Pharmacy and Allied Health Professions, Department of Pharmaceutical Sciences, Toxicology Division, Queens, New York 11439, USA.
Gazarian
Dmitry I
DI
Barile
Frank A
FA
D000199:Actins / Q000032:analysis / Q000096:biosynthesis*; D001692:Biological Transport; D018938:Caco-2 Cells; D018929:Cell Culture Techniques; D002463:Cell Membrane Permeability / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D017097:Electric Impedance; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D005998:Glycine / Q000378:metabolism; D006801:Humans; D007413:Intestinal Mucosa / Q000187:drug effects*; D008670:Metals / Q000494:pharmacology*; D014176:Protein Biosynthesis / Q000187:drug effects*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D019108:Tight Junctions / Q000187:drug effects / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D000199:Actins; D008670:Metals; D012333:RNA, Messenger; D014443:Tyrosine; D005998:Glycine
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