lasiocarpine and senecionine

Pyrrolizidine alkaloids (PA) are secondary plant metabolites that occur as food and feed contaminants. Acute and subacute PA poisoning can lead to severe liver damage in humans and animals, comprising liver pain, hepatomegaly and the development of ascites due to occlusion of the hepatic sinusoids (veno-occlusive disease). Chronic exposure to low levels of PA can induce liver cirrhosis and liver cancer. However, it is not well understood which transcriptional changes are induced by PA and whether all hepatotoxic PA, regardless of their structure, induce similar responses. Therefore, a 28-day subacute rat feeding study was performed with six structurally different PA heliotrine, echimidine, lasiocarpine, senecionine, senkirkine, and platyphylline, administered at not acutely toxic doses from 0.1 to 3.3 mg/kg body weight. This dose range is relevant for humans, since consumption of contaminated tea may result in doses of ~ 8 µg/kg in adults and cases of PA ingestion by contaminated food was reported for infants with doses up to 3 mg/kg body weight. ALT and AST were not increased in all treatment groups. Whole-genome microarray analyses revealed pronounced effects on gene expression in the high-dose treatment groups resulting in a set of 36 commonly regulated genes. However, platyphylline, the only 1,2-saturated and, therefore, presumably non-hepatotoxic PA, did not induce significant expression changes. Biological functions identified to be affected by high-dose treatments (3.3 mg/kg body weight) comprise cell-cycle regulation associated with DNA damage response. These functions were found to be affected by all analyzed 1,2-unsaturated PA.In conclusion, 1,2-unsaturated hepatotoxic PA induced cell cycle regulation processes associated with DNA damage response. Similar effects were observed for all hepatotoxic PA. Effects were observed in a dose range inducing no histopathological alterations and no increase in liver enzymes. Therefore, transcriptomics studies identified changes in expression of genes known to be involved in response to genotoxic compounds at PA doses relevant to humans under worst case exposure scenarios.

Topics: Animals; DNA Damage; Gene Expression; Humans; Liver; Liver Neoplasms; Plants; Pyrrolizidine Alkaloids; Rats; Structure-Activity Relationship

Pyrrolizidine alkaloids (PA) are a group of secondary plant metabolites belonging to the most widely distributed natural toxins. PA intoxication of humans leads to severe liver damage, such as hepatomegaly, hepatic necrosis, fibrosis and cirrhosis. An acute consequence observed after ingestion of high amounts of PA is veno-occlusive disease (VOD) where the hepatic sinusoidal endothelial cells are affected. However, the mechanisms leading to VOD after PA intoxication remain predominantly unknown. Thus, we investigated PA-induced molecular effects on human umbilical vein endothelial cells (HUVEC). We compared the effects of PA with the effects of PA metabolites obtained by in vitro metabolism using liver homogenate (S9 fraction). In vitro-metabolized lasiocarpine and senecionine resulted in significant cytotoxic effects in HUVEC starting at 300 μM. Initial molecular effect screening using a PCR array with genes associated with endothelial cell biology showed PA-induced upregulation of the Fas receptor, which is involved in extrinsic apoptosis, and regulation of a number of interleukins, as well as of different enzymes relevant for prostanoid synthesis. Modulation of prostanoid synthesis was subsequently studied at the mRNA and protein levels and verified by increased release of prostaglandin I

Topics: Animals; Cyclooxygenase 2; Cytochrome P-450 Enzyme System; Epoprostenol; Gene Expression Regulation; Hepatic Veno-Occlusive Disease; Human Umbilical Vein Endothelial Cells; Humans; Inactivation, Metabolic; Liver; Male; Prostaglandins; Pyrrolizidine Alkaloids; Rats, Wistar; Thromboxane A2

Plants producing dehydropyrrolizidine alkaloids (DHPAs) are found throughout the world and they are dangerous to human and animal health. Several DHPAs are carcinogenic but only riddelliine has been classified as a potential human carcinogen by the National Toxicology Program. As DHPA-related carcinogenicity is probably linked to cytotoxicity, a model of CRL-2118 chicken hepatocyte cytotoxicity was developed to compare equimolar DHPA exposures between 19 and 300 μM. Alkaloid-related cytotoxicity was estimated using cytomorphology, cell viability reflected by mitochondrial function and cellular degeneration reflected by media lactate dehydrogenase activity. Lasiocarpine induced cytotoxicity and decreased cell viability in a concentration dependent manner at 24 h. At similar concentrations and exposures of 48 and 72 h, seneciphylline, senecionine, monocrotaline and riddelliine were cytotoxic. None of the DHPA-N-oxides were significantly cytotoxic at these concentrations. Using graphic analyses the median cytotoxic concentration (DHPA concentration that produced ½ the maximum response) were estimated. The estimated descending order of cytotoxicity was lasiocarpine, seneciphylline, senecionine, heliotrine, riddelliine, monocrotaline, riddelliine-N-oxide, lycopsamine, intermedine, lasiocarpine-N-oxide and senecionine-N-oxide. This comparison identifies DHPAs that were more cytotoxic than carcinogenic riddelliine. Additional studies to better characterize the carcinogenic potential of these alkaloids are essential to better determine the risk they each may pose for human and animal health.

Topics: Animals; Cattle; Cell Line, Tumor; Cell Survival; Chickens; Cyclic N-Oxides; Cytotoxins; HEK293 Cells; Hep G2 Cells; Humans; In Vitro Techniques; Molecular Structure; Monocrotaline; Pilot Projects; Pyrrolizidine Alkaloids; Tetrazolium Salts; Thiazoles

The biological mechanisms by which pyrrolizidine alkaloids contribute to initiation and nodule selection (promotion) steps in hepatic carcinogenesis were studied in male Fischer 344 rats. Lasiocarpine at single or double dosages (up to 80 mumol/kg) delayed hepatic regeneration for at least 8 weeks after partial hepatectomy (PH). This regimen of lasiocarpine and PH had a strong selective influence on the growth of gamma-glutamyltranspeptidase (gamma-GT)-positive hepatocyte nodules in rats previously initiated with diethylnitrosamine. However, both lasiocarpine (up to 80 mumol/kg) and senecionine (up to 160 mumol/kg) were inactive as initiators of gamma-GT-positive nodules in rats exposed to a similar selection regimen consisting of 2-acetylaminofluorene and PH. When lasiocarpine or senecionine was given 12 h after PH, very few nodules were initiated. Lasiocarpine pretreatments reduced the initiating activity of diethylnitrosamine and N-nitrosomethylurea in rats subsequently selected with 2-acetylaminofluorene and PH. Resistant nodules selected with lasiocarpine had the typical resistant nodule phenotype (positive for gamma-GT and epoxide hydrolase) and also lacked pyrrolizidine alkaloid-induced megalocytosis. Lasiocarpine treatment also resulted in small regenerative nodular proliferations of hepatocytes that were distinct from resistant nodules because they were negative for gamma-GT and epoxide hydrolase and unrelated to diethylnitrosamine pretreatments. These studies suggest that the hepatocarcinogenicity of pyrrolizidine alkaloids can be better explained by their strong selection (promotion) influence on initiated hepatocytes, rather than by their very weak initiating activity.

Topics: Animals; Carcinogens; Diethylnitrosamine; DNA; Drug Resistance; gamma-Glutamyltransferase; Hepatectomy; Liver Neoplasms, Experimental; Liver Regeneration; Male; Methylnitrosourea; Pyrrolizidine Alkaloids; Rats; Rats, Inbred F344

The influences of in vivo pretreatment with phenobarbitone (PB), 3-methylcholanthrene (3-MC), 2,2',4,4',5,5'-hexachlorobiphenyl (HCBP), and 3,3',4,4'-tetrachlorobiphenyl (TCBP) on cytocidal hepatotoxicity of two pyrrolizidine alkaloids, lasiocarpine (LC) and senecionine (SC), were compared in short-term primary cultures of rat hepatocytes. Toxicity was measured by release of lactate dehydrogenase (LDH) into culture medium at 24 h. LC was slightly more toxic to control hepatocytes than SC in the graded response range of 10-160 microM. PB and HCBP (a PB-type polychlorobiphenyl inducer) similarly potentiated toxicity of SC, and each diminished the degree to which cell killing by LC and SC was inhibited by SKF-525-A. By comparison, 3-MC and TCBP (a 3-MC-type PCB inducer) each diminished toxicity of SC but had little effect on toxicity of LC. Alpha-naphthoflavone (ANF) potentiated toxicity of both LC and SC in hepatocytes induced by 3-MC or TCBP but had little effect on responses of hepatocytes induced by either PB or HDBP. These results indicate that xenobiotics that induce similar patterns of cytochrome P-450 isozymes have qualitatively similar modulating influences on cytocidal hepatotoxicity of pyrrolizidine alkaloids in primary cultures. However, the observed modulating effects could not be explained solely on the basis of altered activation rates by the cytochrome P-450 species known to be induced by the various xenobiotics.

Topics: Animals; Carcinogens; Cells, Cultured; Cytochrome P-450 Enzyme System; Enzyme Induction; Liver; Male; Methylcholanthrene; Phenobarbital; Polychlorinated Biphenyls; Proadifen; Pyrrolizidine Alkaloids; Rats; Rats, Inbred F344