Comparative In Vitro Cytotoxicity of 20 Potential Food Ingredients in Canine Liver, Kidney, Bone Marrow-Derived Mesenchymal Stem Cells, and Enterocyte-like Cells

Abstract

To begin development of a mechanistically relevant humane alternative platform for safety assessment of dog food ingredients, comparative in vitro cytotoxicity of 20 ingredients was assessed in four canine cell types relevant for toxicity assessments. Previously, we described the toxicity of 13 compounds (clove leaf oil, eugenol, guanosine monophosphate [GMP], GMP plus inosine monophosphate, sorbose, ginger root extract, cinnamon bark oil, cinnamaldehyde, thyme oil, thymol, lemon grass oil, xylitol, and citric acid) using in vitro primary canine cell culture models for liver, kidney, bone marrow-derived mesenchymal stem cells (BMSC), and enterocyte-like cells (ELC). In this report, dose-response cytotoxicity studies and LC 50 using alamar blue assays are reported for seven additional compounds: denatonium benzoate, eucalyptol, hexahydroisohumulone, tetrahydroisohumulone, green tea catechin extract, epigallocatechin gallate, and sodium copper chlorophyllin. Data across 20 compounds were compared between different cell types and responses were not parallel, precluding the use of a single cell line for in vitro ingredient hazard assessment. Hepatocytes were most resistant to all compounds, consistent with their xenobiotic detoxification functions. BMSC and ELC showed an increase in sensitivity to the essential oils eucalyptol, eugenol, and thymol compared to renal cells and hepatocytes. These studies provide a baseline of acute cytotoxicity of 4 canine cell types to 20 different food components that begin to illustrate how such an in vitro panel could be used for hazard assessment.

Mechanistic Toxicity Assessment of Hexahydroisohumulone in Canine Hepatocytes, Renal Proximal Tubules, Bone Marrow-Derived Mesenchymal Stem Cells, and Enterocyte-like Cells

“…Briefly, canine hepatocytes, CPTC, BMSC and ELC in 96-well plates were treated with 0-2.12 mg/mL of HEX for the canine hepatocytes and CPTC, and 0-0.05 mg/mL of HEX for BMSC and ELC. In this study, the various concentration ranges were selected based on the cytotoxic responses of HEX and tetrahydroisohumulone (TRA) in canine hepatocytes (LC 50 , 0.12 and 0.29 mg/mL); for CPTC 0.03 and 0.01 mg/mL; for BMSC 0.04 and 0.03 and mg/mL; for ELC 0.02 and 0.01 mg/mL, and a metabolic inhibitory potency of Isoxanthohumol on human CYP450 enzymes (IC 50 values, 10 μM) that have been previously reported [14,38]. The plasma concentrations of other hop ingredients, dihydroisohumulone (0.21 to 0.76 mg/mL) in white rabbits with a single IV dose of 25 mg/kg were also considered for a selection of HEX dose level; for TRA 0.2 to 0.65 mg/mL; and for isohumulone 0.04 to 0.65 mg/mL [39].…”

“…HEX at cytotoxic concentrations modulated the expression of different target genes involved in cell death and survival with various magnitudes of transcriptional levels, in hepatocytes and to a less extent CPTC (Table 2). These results suggest that the increase in ROS/RNS levels (Figure 1 and 3) may be the primary cause of HEX-mediated cytotoxicity in canine hepatocytes, but the secondary cause in CPTC because its concentration corresponding to the increase in ROS/RNS levels in CPTC is greater than its cytotoxic level (LC 50 , 0.03 mg/mL) [14]. Previous study reported that other hop components such as humulone and β-acids hop didn't induce cell survival-related molecules, BCL-2 and BCL-X but activated cell death-related CASP3 and CASP9 as well as cytochrome c release in a human leukemia HL-60 cell line [48].…”

“…However, the ELC exposed to HEX showed an increase in proapoptotic genes such as ABL1 and TP53. When these results were compared to the cytotoxicity of HEX after 24 hr derived from in vitro canine cell systems, the extent of gene alteration was inversely correlated with the degree of HEX toxicity in hepatocytes (113 genes at 0.12mg/mL of LC 50 value of HEX), in CPTC (80 genes at 0.037 mg/mL) and in ELC (18 genes at 0.023 mg/mL) [14]. These results suggest that the hepatocytes exert more extensive metabolic function and biochemical defense against HEX compared to the excretory proximal tubule epithelial cells and the absorptive enterocytes.…”

“…There are three different reduced isohumulones; humulone) inhibited COX-2-mediated prostaglandin E 2 (PGE 2 ) production in antibiotic A23187-stimulated human whole blood [13]. In contrast, we recently reported that HEX in dogs caused a cytotoxic response in canine hepatocytes, canine proximal tubule cells (CPTC), bone marrow-derived mesenchymal stem cells (BMSC) and enterocyte-like cells (ELC) [14]. HEX at 100 mg/kg causes an increase in serum alkaline phosphatase levels in beagle dogs, not purportedly due to liver toxicity, and was considered to be the no observed adverse effect level (NOAEL) [2].…”

The 3Rs in research: a contemporary approach to replacement, reduction and refinement

“…The ability to generate complex cell cultures, including stem cell technologies, three-dimensional (3D) approaches and tissue engineering all contribute to better modelling of the digestive system in vitro. Monteiro-Riviere et al (12) used four canine cell types to study potential toxicity of ingredients commonly used in dog food, illustrating how an in vitro panel can be used for hazard assessment. 3D intestinal organoids (mini-guts) appear to faithfully replicate healthy gut physiology as well as helping us to understand host-parasite interactions (13) and diseases such as cystic fibrosis (14) .…”