An in vitro comparison of the cytotoxic potential of selected dehydropyrrolizidine alkaloids and some N-oxides

Authors: FIELD, REUEL - US Department Of Agriculture (USDA); Stegelmeier, Bryan; Colegate, Steven; BROWN, AMMON - Us Army Research; Green, Benedict - Ben

Submitted to: Toxicon
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/5/2015
Publication Date: 2/7/2015
Publication URL: http://handle.nal.usda.gov/10113/62901
Citation: Field, R.A., Stegelmeier, B.L., Colegate, S.M., Brown, A.W., Green, B.T. 2015. An in vitro comparison of the cytotoxic potential of selected dehydropyrrolizidine alkaloids and some N-oxides. Toxicon. 97:36-45.

Interpretive Summary: Many plants contain toxic dehydro-pyrrolizidine alkaloids (PAs). These plants or their toxins can contaminate of feed and food. Several PAs cause cancer (carcinogen) and others toxic PA have that potential. As the mechanisms of toxicity and producing cancer are probably similar models to directly compare small quantities of purified PA are needed. The objectives of this study are to determining and compare the relative cytotoxicity of select purified PAs. Initial studies identified CRL-2118 chicken hepatocytes as the cell type most likely to be a sensitive good model. CRL-2118 cells were exposed to equilmolar doses of PAs for 24, 48, and 72 hrs. Alkaloid related cytotoxicity was estimated using microscopy and several cell viability assays. At 24 hours of lasiocarpine induced cell cytotoxicity and decreased cell viability in a concentration dependant manner. At slightly higher concentrations and longer exposure times seneciphylline, senecionine, monocrotaline, and riddelliine were also cytotoxic. Cytotoxicity and degeneration were confirmed microscopically. Riddelliine n-oxide, intermedine, intermedine, lasiocarpine n-oxide and senecionine n-oxide produced minimal cytotoxicity. Using graphically and mathematically derived effective doses the alkaloids were ranked in descending order of toxicity as lasiocarpine, senciphylline, senecionine, heliotine, riddelliine, monocrotaline, riddelliine n-oxide, intermedine, lycopsamine, lasiocapine n-oxide and senecionine n-oxide. In summary, this direct comparison of PA cytotoxicity has identified 4 PAs more cytotoxic than riddelliine (the only PA currently classified as a potential human carcinogen). As these other PAs are also likely to be carcinogenic, additional studies to better characterize the carcinogenic potential these alkaloids is essential to better determine the risk they each may pose for human and animal health.

Technical Abstract: Plants containing dehydro-pyrrolizidine alkaloids (PAs) can be found throughout the world and their invasive, weedy nature often results in PA contamination of feed and food. Other PA-containing plants may be purposefully or accidentally included in food or herbal preparations. Poisoning can be acute or chronic depending on the dose, duration and species. Though direct poisoning presents a serious risk to human and animal health, subclinical poisoning may be even more dangerous. Several PAs are carcinogenic and initial observations suggest the incidence of PA-induced neoplastic transformation is increased in both clinical and subclinical poisoning. As the mechanism of carcinogenicity is similar or linked to direct toxicity, a direct comparison of alkaloid specific cytotoxicity may be useful to predict carcinogenicity and develop models to identify alkaloids for further studies and better estimate potential risk from these structurally different PAs. The objectives of this study are to determining and compare the relative cytotoxicity of select purified free base and n-oxide retronecine and heliotridine base PAs. Initial studies identified CRL-2118 chicken hepatocytes as uniquely capable of PA activation and subsequent cytotoxicity. Cultured CRL-218 hepatocytes were exposed to equilmolar doses of PAs for 24, 48, and 72 hrs. Alkaloid related cytotoxicity was estimated using cytomorphology, cell viability reflected as a factor of mitochondrial function (tetrazolium dye reduction assay) and cellular degeneration or membrane damage reflected as changes in media lactate dehydrogenase activity. At 24 hours of PA exposure to heliotridine based lasiocarpine induced cell cytotoxicity and decreased cell viability in a concentration dependant manner. Later cytotoxicity and degeneration were also seen in similar concentrations of retronecine based seneciphylline, senecionine, monocrotaline, and riddelliine. Cytotoxicity and degeneration were confirmed morphologically as cellular swelling with cytoplasmic vacuolation. At these concentrations and exposures none of the PA N-oxides were significantly cytotoxic to chicken hepatocytes. Using graphically and mathematically derived effective doses the alkaloids were ranked in descending order of toxicity as lasiocarpine, senciphylline, senecionine, heliotine, riddelliine, monocrotaline, riddelliine n-oxide, intermedine, lycopsamine, lasiocapine n-oxide and senecionine n-oxide. In summary, this direct comparison of PA cytotoxicity has identified 4 PAs more cytotoxic than riddelliine. As these are likely to be carcinogenic, additional studies to better characterize the carcinogenic potential these alkaloids is essential to better determine the risk they each may pose for human and animal health.