Copper Acetate(WSDTY) assay mainly reflects the damage to cellular mitochondria, and it is commonly used for in vitro cytotoxicity (including skin irritation) measurement. Our results indicated that neither GHK nor GHK-Cu showed cytotoxic activity after 72h exposure to HaCaT cells. CuCl2 and Cu(OAc)2 were only found to be cytotoxic at 5800μM and cell viability decreased significantly after 48h treatment at 580μM. At 58μM and 580μM, cytotoxicity was not observed following 24h treatment with all the samples. The results of the Copper Acetate assay were consistent with the results of LDH assay as CuCl2 and Cu(OAc)2 were able to induce significant cytotoxicity in the HaCaT keratinocytes within 24hours, but not for GHK and GHK-Cu. Lactate dehydrogenase (LDH) is a cytosolic enzyme and its presence in the media is often used as an indicator for cellular toxicity. Large amount of LDH in the media is linked to the leaky, damaged and compromised cellular membrane integrity.
The measurement of LDH in media has therefore been a commonly used and validated method for assessing cytotoxicity among many in vitro based cell model studies. The LDH assay was performed to validate the results obtained from the Copper Acetate assay. In this assay, the 30min incubation duration was chosen to be optimal because of the inherently cytotoxic nature of CuCl2 and Cu(OAc)2 towards HaCaT keratinocytes, as they were observed to cause substantial cell death at a duration longer than 30mins. This may result in a false negative result as the media containing the LDH was replaced with fresh serum-free media and incubated for another 24?h and most of the LDH released into the media would have been removed in the process. The replacement of the media is essential for removing the CuCl2 and Cu(OAc)2 interference before assaying the LDH levels as they are shown to reduce the fluorescence signal read-out within treated wells. Our results indicated that neither GHK nor GHK-Cu showed cytotoxic activity at the range of concentrations tested while CuCl2 and Cu(OAc)2 showed substantial cytotoxicity to the HaCaT cells at the highest concentration assayed, i.e., at 5800μM, after 30?mins treatment.
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