Date Awarded

1989

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Virginia Institute of Marine Science

Advisor

Beverly A. Weeks

Abstract

The effects of tributyltin (TBT) on phorbol myristate acetate (PMA)- and calcium ionophore (A23187)-stimulated peritoneal macrophages from oyster toadfish (Opsanus tau) were evaluated in a luminol-dependent chemiluminescence (CL) assay. PMA-stimulated CL responses were increased by in vitro exposure to 50 ug/L TBT, but depressed to baseline values by 500 ug/L TBT. A23187-stimulated CL increased with 5 ug/L and 50 ug/L TBT, and depressed by 500 ug/L TBT. Responses stimulated by synergistic doses of PMA and A23187 were increased by 50 ug/L TBT and depressed by 500 ug/L TBT. Enhanced PMA- and A23187-stimulated CL following TBT exposure returned to baseline values after extracellular calcium was removed. In the absence of PMA or A23187, 50 ug/L TBT stimulated a significant CL response that was inhibited by chelation of extracellular calcium. Uptake of calcium-45 was greatly increased with 50 ug/L TBT but depressed by 500 ug/L TBT. Results suggest that low TBT concentrations stimulate calcium influx and enhancement of reactive oxygen intermediate formation, while high TBT concentrations cause membrane dysfunctions which inhibit calcium flux and depress activation sequences. The physiological basis of TBT-stimulated macrophage activation was investigated in vitro. Inhibition of TBT-stimulated CL by sodium benzoate (.OH scavenger) and superoxide dismutase (.O&\sb2& scavenger) was most similar to PMA-stimulated CL, indicating a role of NADPH oxidase activity in TBT-stimulated CL. Inhibition by nordihydroquaiaretic acid (lipoxygenase antagonist) was similar to PMA-, PMA + A23187-, and A23187-stimulated CL, indicating a common role of leukotrienes in activation responses. Inhibition by indomethacin (cyclooxygenase antagonist) was most similar to PMA + A23187-, and A23187-stimulated CL, indicating a common role of prostaglandins in activation responses. Inhibition by isobutylmethylxanthine (phosphodiesterase antagonist) was most similar to PMA- and A23187-stimulated CL, indicating a role of cyclic neucleotides in activation regulation. Inhibition of TBT-stimulated CL by verapamil (calcium channel antagonist) and trifluoperazine (calmodulin antagonist) were most similar to PMA + A23187- and A23187-stimulated CL, indicating the role of calcium in TBT-stimulated macrophage activation. Therefore, TBT-stimulated macrophage activation is initiated by an increased plasma-membrane calcium flux. The influence of in vivo TBT exposure was compared with the effects of in vitro exposure. Fish were treated (i.p. injections) weekly for six weeks with either sham, vehicle, 0.250, 0.750, or 2.5 mg/kg TBT. Macrophages were isolated and PMA + A23187- and TBT (50 ug/L)-stimulated CL was compared between treatment groups. Chemiluminescence was reduced in a dose-dependent manner in response to both PMA + A23187 and TBT (50 ug/L). It is concluded that TBT supresses macrophage function following both in vivo and in vitro exposure. The mode of action appears to be an interference with plasma membrane calcium transport, therefore inhibiting both intracellular and intercellular signal transduction.

DOI

https://dx.doi.org/doi:10.25773/v5-w8vz-7736

Rights

© The Author

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