The comparative cytotoxicity of riddelliine in primary mouse, rat, and chick hepatocytes

Authors: Stegelmeier, Bryan; RESEAGER, W - University Of Arizona; COLEGATE, S - Retired ARS Employee

Submitted to: Poisonous Plant Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/4/2020
Publication Date: 5/12/2020
Citation: Stegelmeier, B.L., Reseager, W.S., Colegate, S.W. 2020. The comparative cytotoxicity of riddelliine in primary mouse, rat, and chick hepatocytes. Poisonous Plant Research. 3:43-57. https://doi.org/10.26077/p9te-bv07.
DOI: https://doi.org/10.26077/p9te-bv07

Interpretive Summary: The dehydro-pyrrolizidine alkaloids (DHPAs) are plant poisons that affect poison livestock, humans and wildlife. There are nearly a thousand different DHPAs with unique toxicokinetics, metabolisms and toxicities. Many are difficult to purify or synthesize. Consequently direct toxicologic comparison has been difficult. Due to its availability, DHPA riddelliine has been used to characterize toxicity and carcinogenicity and often serves as a comparison bench mark. This study’s objectives were to develop in vitro primary hepatocyte cultures from mice, rats, and chickens and to compare their response to riddelliine toxicity. After establishing viable cultures, the hepatocytes were exposed for 24 hours to riddelliine (from 0.1 µM to 1.2 mM) and cytotoxicity was estimated using a mitochondrial function assay (MTT). Cytotoxic riddelliine concentrations (CD50s) were calculated. Riddelliine cytotoxicity in mouse and rats hepatocytes were similar to previous reports. Chick hepatocytes were highly sensitive to riddelliine cytotoxicity (IC50 = 0.4 µM), but the response was biphasic and some resistant cells appeared unaffected. A marker enzymes thought to activate DHPA in many species (hepatocyte cytochrome P450 3A4 activities did not correlate with riddelliine induced cytotoxicity. These findings suggest chick hepatocyte cultures are uniquely sensitive to DHPA cytotoxicity and cytotoxicity does not appear to be related to CYP3A4 activity. As some chick hepatocytes appeared to be resistant to riddelliine toxicity, more work is needed to better characterize these differences. With further development or better isolation to test only the highly sensitive primary chick hepatocytes, this model may be useful to evaluate the toxicity of small amounts of rare DHPAs.

Technical Abstract: Dehydropyrrolizidine alkaloid (DHPA) producing plants commonly poison livestock, wildlife and humans. Poisoning occurs when DHPAs are ingested as feed or food, or when they contaminate medicinal or herbal products. Direct toxicologic comparison of individual DHPAs is essential to estimate their actual health risks. This has been problematic due to varying models and difficulties in DHPA isolation or synthesis. In contrast, the macrocyclic DHPA riddelliine is readily isolated and it has been used as a benchmark to characterize different models of toxicity and carcinogenicity. Following earlier work with immortalized cell lines, the objective of this study was to characterize the effect of riddelliine on primary mouse, rat and chick hepatocyte cultures with the aim of developing a suitable, sensitive model for assessing DHPA-related cytotoxicity. After establishing viable cultures, the hepatocytes were exposed for 24 hours to riddelliine (from 0.1µM to 1.2mM) and cytotoxicity (CT­­50) was estimated using a mitochondrial function assay (MTT). Despite a biphasic response, possibly attributable to a sub-population of resistant chick hepatocytes, chick hepatocyte cultures were highly sensitive (CT50 0.9 µM) to riddelliine cytotoxicity relative to rat (CT50 289 µM) and mouse (CT50 627 µM) hepatocytes. Chick, mouse and rat hepatocyte cytochrome P450 3A4 activities did not correlate with riddelliine-induced cytotoxicity. With further development to utilize the highly sensitive primary chick hepatocytes, this model may be useful to directly compare panels of DHPAs, including rare or difficult to isolate alkaloids.