Cytotoxicity Studies of Anchorage-independent LS-L-929 Mouse Fibroblasts Using Membrane Integrity, ATP Content and ATP/ADP Ratio as Determinants: Including the Xenobiotic Effects of Components in an Experimental Shampoo Formulation

Abstract

Cell death (CD50), as measured by loss of membrane integrity using fluorescein diacetate and ethidium bromide, and reduction in ATP content (ATP50), were used as endpoints in a comparative study of the cytotoxicity of 40 chemicals (supplied by FRAME) to anchorage-independent LS-L-929 mouse fibroblasts. The rank order correlation of the two tests was strong (r = 0.98), but the ATP50 values only showed a weak correlation with rat acute lethality (LD50) data (r = 0.43). More-mechanistic endpoints were sought in order to characterise cellular energy status, and the effects of acetaminophen, acetylsalicylic acid, diazepam, digoxin and ethanol on ATP/ADP ratio, phosphorylation potential and adenylate energy charge were examined in detail. The first two ratios were very sensitive to toxic insult to the cells and gave results which more closely mimicked differences in human acute oral lethality than did the ATP50 and CD50. In addition, there was evidence that digoxin and diazepam affected oxidative phosphorylation, which suggested an alteration to mitochondrial function. Energy charge appeared less sensitive to the xenobiotics, probably owing to the dampening effect of the denominator in the ratio. The effects of individual components of an experimental detergent-based formulation on cell death were examined. It was found that one detergent gave different results, depending on the presence or absence of a second detergent. A commercial cream containing varying amounts of glyceryl monostearate contaminated with varying levels of sodium dodecyl sulphate gave results which reinforced the point that cytotoxic assays are valuable in quality assurance.