In vitro safety assessment of food ingredients in canine renal proximal tubule cells

• In vivo kidney injury markers (e.g. KIM-1) are not applicable to an in vitro model
• Volatile ingredients (CINA, CBO, LGO) studied are more toxic than the nephrotoxicant 4-AP
• Feasibility of extrapolating data from canine cells for human toxicity studies
• Lemongrass oil (LGO) induces high levels of oxidative stress represented by GSTA3
• Gene relationship and TEM analyses predict the LGO-induced cellular response

In vitro models are useful tools to initially assess the toxicological safety hazards of food ingredients. Toxicities of cinnamaldehyde (CINA), cinnamon bark oil, lemongrass oil (LGO), thymol, thyme oil (TO), clove leaf oil, eugenol, ginger root extract (GRE), citric acid, guanosine monophosphate, inosine monophosphate and sorbose (SORB) were assessed in canine renal proximal tubule cells (CPTC) using viability assay and renal injury markers. At LC50, CINA was the most toxic (0.012 mg/ml), while SORB the least toxic (>100 mg/ml). Toxicities (LC50) of positive controls were as follows: 4-aminophenol (0.15 mg/ml in CPTC and 0.083 mg/ml in human PTC), neomycin (28.6 mg/ml in CPTC and 27.1 mg/ml in human PTC). XYL displayed lowest cytotoxic potency (LC50 = 82.7 mg/ml in CPTC). In vivo renal injury markers in CPTC were not significantly different from controls. The LGO toxicity mechanism was analyzed using qPCR and electron microscopy. Out of 370 genes, 57 genes (15.4%) were significantly up (34, 9.1%) or down (23, 6.2%) regulated, with the most upregulated gene gsta3 (∼200-fold) and the most affected pathway being oxidative stress. LGO induced damage of mitochondria, phospholipid accumulation and lack of a brush border. Viability assays along with mechanistic studies in the CPTC model may serve as a valuable in vitro toxicity screening tool.