Flow cytometric analysis of chronic and acute toxicity of copper(II) on the marine dinoflagellate Amphidinium carterae.
BACKGROUND
Copper(II) is a heavy metal whose levels have increased in some marine ecosystems to polluting levels. Dinoflagellates, an important phytoplankton group, are at the base of aquatic food chains and bioaccumulation of copper by these microorganisms can result in complex ecosystem alterations, so we investigated how copper disturbs those cells.


METHODS
Cytotoxic effects of sublethal and lethal copper concentrations ranging from 4.2 nM (control condition) to 3.13 microM estimated labile copper were studied in batch cultures of Amphidinium carterae. Cell morphology, motility, autofluorescence, and fluorescein diacetate (FDA)-dependent fluorescence generation were evaluated by flow cytometry (FCM) and microscopy.


RESULTS
Exposure of A. carterae to toxic levels of copper impaired cell mobility, delayed cell proliferation, led to increased green autofluorescence, and at 3.13 microM labile copper also induced encystment and death. Chlorophyll fluorescence, however, was not affected. Kinetic FCM assay of FDA-dependent fluorescence generation showed a dose-dependent enhancement of fluorescein fluorescence immediately after copper addition and in cultures with sustained exposure to this toxicant.


CONCLUSIONS
Our data suggest that copper toxicity occurs quickly at the membrane level in relation to oxidative stress generation. Based on fluorescence kinetic studies, the Na(+)/H(+) antiporter seemed to be affected by copper, thereby affecting intracellular pH.
Lage
O M
OM
Departamento de Botânica da Faculdade de Ciências and Centro Interdisciplinar de Investigação Marinha e Ambiental, Universidade do Porto, Porto, Portugal. olga.lage@clix.pt
Sansonetty
F
F
O'Connor
J E
JE
Parente
A M
AM
D000818:Animals; D002413:Cations, Divalent; D003300:Copper / Q000494:pharmacology*; D004141:Dinoflagellida / Q000737:chemistry / Q000187:drug effects* / Q000254:growth & development; D005434:Flow Cytometry / Q000379:methods; D005452:Fluoresceins / Q000032:analysis; D005453:Fluorescence; D007700:Kinetics; D013997:Time Factors
D002413:Cations, Divalent; D005452:Fluoresceins; D003300:Copper; C018506:diacetylfluorescein