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Cockle extracts: an in vitro cytotoxicity study of effect on lactate dehydrogenase leakage, ATP levels and apoptosis induction in Balb/c 3T3 and HepG2 cells

Project Code: B16006

12/11/2004

Centre for Environment, Fisheries & Agriculture Science (CEFAS) Lowestoft
Kumaravel, T

Cockle extracts (Positive and negative extracts) were tested for their ability to induce cytotoxicity as measured by their effects on lactate dehydrogenase (LDH) release, cellular adenosine triphosphate (ATP) levels and caspase 3/7 activation (induction of apoptosis) in Balb/c 3T3 and HepG2 cells, cultured on 96 well plates.

A series of eight concentrations (both extracts) separated by half log dilutions prepared from the maximum concentration (0.000316, 0.001, 0.00316, 0.01, 0.0316, 0.1, 0.316 and 1.0 % of neat extract as supplied by the sponsor) were tested in this study. The cells were exposed to various concentrations of Cockle extracts for 24 hours at 37ºC.

LDH Release

The percentage of LDH released in medium following treatment with Cockle extracts was determined using Roche Applied Sciences LDH kit.

Tween 20 (0.05 and 0.1% v/v; supplied by Sigma-Aldrich Chemical Company) was used as positive control compound. 1% Tween 60 (supplied by the Sponsor) was used as negative control. The treatment of cultures with the positive control compound (0.1%) resulted in a robust positive response (increase LHD release) and therefore the study was considered valid.

The LDH release in the Cockle extracts (negative and positive extracts) treated cultures (both cell types) were similar to and not significantly different from those observed in the concurrent negative control treated cultures.

ATP reduction

ATP levels were determined following exposure of cultures to Cockle extracts in both cell types using Bioluminescent Somatic Cell Assay kit obtained from Sigma Chemical Company.

Paracetamol (5.00 and 7.50 μg/mL) and Tamoxifen (10.0 and 12.5 μg/mL) were used as positive control compounds for Balb/c 3T3 and HepG2 cells respectively. 1% Tween 60 (supplied by the Sponsor) was used as negative control. The treatment of cultures with the positive control compound resulted in a robust positive response (decrease in ATP levels) and therefore the study was considered valid.

There was no significant reduction in ATP levels in Cockle extracts (negative and positive extracts) treated cultures (both cell types) and all values were similar to those observed in concurrent control cultures.

Caspase 3/7 activity

Caspase (3 and 7) activity in cells was determined following exposure of cultures to Cockle extracts in both cell types using Caspase-Glo™ 3/7 Kit from Promega, Southampton.

Stauroporine (0.1 μg/mL) was used as positive control compound. 1% Tween 60 (supplied by the Sponsor) was used as negative control. The treatment of cultures with the positive control compound resulted in a robust positive response (increase Caspase 3/7 activity) and therefore the study was considered valid.

There was no significant activation of Caspase 3/7 in Cockle extracts (negative and positive extracts) treated cultures (both cell types) and all values were similar to those observed in concurrent control cultures. However, it should be noted that a small increase in the caspase activity was observed at the highest concentration (1% of neat extract) tested in Balb/c 3T3 cells following treatment with both extracts. This increase was not associated with significant LDH release or decrease in ATP levels and is therefore not considered to be of biological relevance.

It is concluded that under the conditions described, Cockle extracts (both positive and negative extracts) did not induce cytotoxicity either in Balb/c 3T3 or HepG2 cells.

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