riddelliine and Liver-Neoplasms

Dehydropyrrolizidine alkaloids (DHPA) are a large, structurally diverse group of plant-derived protoxins that are potentially carcinogenic. With worldwide significance, these alkaloids can contaminate or be naturally present in the human food supply. To develop a small animal model that may be used to compare the carcinogenic potential of the various DHPAs, male heterozygous p53 knockout mice were administered a short-term treatment of riddelliine 5, 15 or 45 mg kg(-1) bodyweight day(-1) by oral gavage for 14 days, or dosed a long-term treatment of riddelliine 1 mg kg(-1) bodyweight day(-1) in pelleted feed for 12 months. Exposure to riddelliine increased the odds of tumor development in a dose-responsive manner (odds ratio 2.05 and Wald 95% confidence limits between 1.2 and 3.4). The most common neoplastic process was hepatic hemangiosarcoma, which is consistent with published lifetime rodent riddelliine carcinogenesis studies. Angiectasis (peliosis hepatis) and other previously unreported lesions were also identified. The results of this research demonstrate the utility of the heterozygous p53 knockout mouse model for further investigation of comparative carcinogenesis of structurally and toxicologically different DHPAs and their N-oxides.

Topics: Administration, Oral; Animals; Carcinogenicity Tests; Dose-Response Relationship, Drug; Hemangiosarcoma; Heterozygote; Liver Neoplasms; Male; Mice, Knockout; Pyrrolizidine Alkaloids; Senecio; Tumor Suppressor Protein p53

Pyrrolizidine alkaloids (PAs) are the most common plant constituents that poison livestock, wildlife and humans. Riddelliine is a prototype genotoxic PA and has been nominated to be classified as a reasonably anticipated human carcinogen by the US National Toxicology Program (NTP) in the 12th Report on Carcinogens. Riddelliine's nomination is due to the high incidence of liver tumours that were observed in both mice and rats in the NTP tumourigenicity bioassay study. In this current study, we explored whether riddelliine treatment could alter microRNA (miRNA) expression in rat liver and whether the possible deregulation of miRNA was related to mutagenicity and carcinogenicity of riddelliine. Groups of six rats were administered riddelliine at a mutagenic dose of 1 mg/kg body weight or with control vehicle 5 days a week for 12 weeks. A group of six rats treated with aristolochic acid, a renal carcinogen, was used as a tissue-specific negative control. The animals were sacrificed 1 day after the last treatment and the livers were isolated for miRNA expression analysis using miRNA microarrays. miRNA expression was significantly altered by riddelliine treatment. Principal component analysis and hierarchical clustering analysis showed that the miRNA expression profiles were clearly classified into two groups, riddelliine treatment versus other samples. Forty-seven miRNAs were significantly dysregulated by riddelliine treatment, among which 38 were up-regulated and 9 were down-regulated. Functional analysis of these differentially expressed miRNAs by riddelliine revealed that these miRNAs were involved in liver carcinogenicity and toxicity, such as liver proliferation, liver necrosis/cell death, hepatocellular carcinoma, liver hepatomegaly, liver inflammation and liver fibrosis. These results suggest that miRNAs actively respond to a mutagenic dose of riddelliine and the pattern of miRNA expression has the potential to be used as a biomarker of genotoxicity and carcinogenicity for riddelliine and possibly other PAs.

Topics: Animals; Carcinogens; Cluster Analysis; Dose-Response Relationship, Drug; Gene Expression Profiling; Gene Expression Regulation; Liver; Liver Neoplasms; Microarray Analysis; MicroRNAs; Pyrrolizidine Alkaloids; Rats; Rats, Inbred F344; Rats, Transgenic; RNA

Topics: Adenoma, Liver Cell; Animals; Carcinogens; Cattle; Chickens; Cricetinae; DNA Adducts; Female; Guinea Pigs; Hemangiosarcoma; Humans; Liver; Liver Neoplasms; Lung; Lung Diseases; Male; Mice; Organ Specificity; Pyrrolizidine Alkaloids; Rabbits; Rats; Rats, Wistar; Species Specificity

Riddelliine is a naturally occurring pyrrolizidine alkaloid that induces liver hemangiosarcomas in male and female F344 rats and male B6C3F1 mice. We previously reported that eight dehydroretronecine (DHR)-derived DNA adducts were formed in liver DNA of rats treated with riddelliine. In order to examine the relationship between DNA adduct levels and the incidence of hemangiosarcomas, we have measured DHR-derived DNA adduct levels in purified rat and mouse liver endothelial cells, the cells of origin for the hemangiosarcomas. F344 rats and B6C3F1 mice were treated by gavage 5 days per week for 2 weeks with riddelliine at 1.0 mg/kg for rats and 3.0 mg/kg for mice. One, 3, 7, and 28 days after the last dose, liver parenchymal and endothelial cell fractions were isolated, and the quantities of DHR-derived DNA adducts were determined by 32P-postlabeling/HPLC. The DHR-derived DNA adduct levels in the endothelial cells were significantly greater than in the parenchymal cells. The DNA adduct levels in rat endothelial cells were greater than in the mouse endothelial cells. These results indicate that the levels of riddelliine-induced DNA adducts in specific populations of liver cells correlate with the preferential induction of liver hemangiosarcomas by riddelliine.

Topics: Animals; Area Under Curve; Carcinogens; Cells, Cultured; Chromatography, High Pressure Liquid; DNA; DNA Adducts; Endothelial Cells; Female; Hemangiosarcoma; Liver; Liver Neoplasms; Male; Mice; Models, Chemical; Pyrrolizidine Alkaloids; Rats; Rats, Inbred F344; Sex Factors; Time Factors

In recent studies, riddelliine, a pyrrolizidine alkaloid, was found to increase rates of replication and apoptosis and induce hemangiosarcoma in the liver of rats and mice. To analyze DNA replication and apoptosis data taken from the same animals, we have developed a predictive mathematical model for describing BrdU labeling and apoptotic processes. The model allows the incorporation of simple diurnal patterns in cellular kinetics and is applied to data on hepatocytes and endothelial cells taken from riddelliine exposed rats. Predictions from the model were used with multivariable nonlinear regression techniques to estimate replication and apoptotic rate constants for both cell types and all treatment groups. Hypothesis tests were used with the predicted rates to separate the competing effects of riddelliine on replication and apoptosis of hepatocytes and endothelial cells as well as compare replication rates between cell types. That estimated replication rates were found to be significantly higher for endothelial cells supports the supposition of induction of hemangiosarcoma by riddelliine in the liver.

Topics: Algorithms; Animals; Apoptosis; Carcinogens; Cell Count; Cell Proliferation; Cells; DNA Replication; Endothelium; Hemangiosarcoma; Hepatocytes; Least-Squares Analysis; Liver Neoplasms; Male; Models, Statistical; Pyrrolizidine Alkaloids; Rats; Rats, Inbred F344

Riddelliine alters hepatocellular and endothelial cell kinetics and function including stimulating an increase in hepatocytic vascular endothelial growth factor (VEGF) in the absence of increased serological levels of VEGF (NYSKA et al. 2002). The objective of this study was to further assess hepatic VEGF and KDR/flk-1 synthesis and expression by hepatic cells under riddelliine treatment conditions. Forty-two male F344/N rats were dosed by gavage with riddelliine (0, 1.0, and 2.5 mg/kg/day) for 6 weeks. Seven animals/group were sacrificed after 8 consecutive daily doses; remaining rats were terminated after 30 daily doses, excluding weekends. Hepatic tissues were evaluated by immunohistochemistry and in situ hybridization. The results showed that VEGF mRNA expression was observed in control and treated animals; however, qualitative differences were noted. Treated animals exhibited VEGF mRNA in clustered, focal hepatocytes and bile duct epithelium, whereas VEGF mRNA in hepatocytes from vehicle control rats was distributed evenly across all hepatocytes. Results evaluating the distribution of the VEGF cognate receptor, KDR/flk-1 showed that randomly distributed, rare sinusoidal endothelium, including those demonstrating karyomegaly and cytomegaly expressed KDR/flk-1. Phosphorylation of KDR/flk-1 at pTyr996 and pTyr1054/1059, but not pTyr951, was also detected, evidence that endothelial cell KDR/flk-1 was activated. These results suggest that both hepatocytes and endothelial cells are targets of riddelliine-induced injury. We speculate that damage to both populations of cells may lead to dysregulated VEGF synthesis by hepatocytes and activation of KDR/flk-1 by endothelium leading to the induction of sustained endothelial cell proliferation, culminating in the development of hepatic hemangiosarcoma.

Topics: Administration, Oral; Animals; Bile Ducts, Intrahepatic; Dose-Response Relationship, Drug; Endothelium, Vascular; Epithelial Cells; Hemangiosarcoma; Hepatocytes; Immunoenzyme Techniques; In Situ Hybridization; Liver; Liver Neoplasms; Male; Pyrrolizidine Alkaloids; Rats; Rats, Inbred F344; RNA Probes; RNA, Messenger; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

Riddelliine belongs to a class of toxic pyrrolizidine alkaloids and is isolated from plants of the genera Crotalaria, Amsinckia, and Senecio that grow in the western United States. Cattle, horses, and sheep that ingest these plants succumb to their toxic effects. Riddelliine residues have been found in meat, milk, and honey, and the plants may contaminate human food sources. Riddelliine was nominated for study by the Food and Drug Administration because of its potential for human exposure and its economic impact on the livestock industry and because the toxicity of other pyrrolizidine alkaloids suggests riddelliine may be carcinogenic. Male and female F344/N rats and B6C3F1 mice received riddelliine (approximately 92% pure) by gavage. Female rats and male and female mice were dosed for 2 years; due to high mortality, the study in male rats was terminated at week 72. In vitro genetic toxicology studies were conducted in Salmonella typhimurium and in cultured Chinese hamster ovary (CHO) cells. In addition, riddelliine was evaluated in vivo for induction of micronuclei in mouse bone marrow and peripheral blood erythrocytes and for induction of S-phase DNA synthesis and unscheduled DNA synthesis in the liver of rats and mice. Riddelliine-induced DNA adduct levels were determined in liver tissue obtained from female rats admininstered riddelliine for 3 or 6 months. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female rats were administered 0 or 1 mg riddelliine/kg body weight in sodium phosphate buffer by gavage 5 days per week; additional groups of 50 female rats received 0.01, 0.033, 0.1, or 0.33 mg/kg. A wide dose range was used in female rats to better characterize the dose-response curve. Females were dosed for 105 weeks; due to high mortality, male rats were terminated at week 72. All but three 1 mg/kg males died before week 70, and all 1 mg/kg females died before week 97. Mean body weights of 1 mg/kg males and females were less than those of the vehicle controls throughout most of the study. The only clinical finding related to riddelliine administration was a general debilitation of the animals prior to death. Hemangiosarcomas were present in the liver of 86% of males and 76% of females in the 1 mg/kg groups, and this neoplasm was considered the cause of the large number of early deaths in these groups. The incidences of hepatocellular adenoma and mononuclear cell leukemia in 1 mg/kg males and females were significantly increased. Nonneoplastic lesion. Riddelliine was mutagenic in S. typhimurium strain TA100 with, but not without, S9 activation; no significant mutagenic activity was detected in strain TA98 or TA1535,ed in strain TA98 or TA1535, with or without S9. A small, dose-related increase in mutant colonies seen in strain TA97 with S9 was judged to be equivocal. Riddelliine induced sister chromatid exchanges in cultured CHO cells with and without S9. Chromosomal aberrations were induced in CHO cells only in the presence of S9. Following 4 or 13 weeks of daily gavage treatment with riddelliine, no increases in the frequency of micronucleated erythrocytes were noted in the peripheral blood of male or female B6C3F1 mice. Use of a single intraperitoneal injection protocol, however, produced a small but significant increase in the frequency of micronucleated eryth-rocytes in peripheral blood of male Swiss mice 48 hours after injection; bone marrow analysis 24 hours after injection demonstrated a small but insignificant increase in the frequency of micronuclei. Unscheduled DNA synthesis was detected in cultured hepatocytes from male and female rats and mice following 5 or 30 days of riddelliine treatment by gavage. In addition, an S-phase DNA synthesis was observed in cultured hepatocytes of male and female rats treated for either time period.. Under the conditions of these studies, there was clear evidence of carcinogenic activity of riddelliine in male and female F344/N rats based primarily on increased incidences of hemangiosarcoma in the liver. The increased incidences of hepatocellular adenoma and mononuclear cell leukemia in male and female rats were also considered to be treatment related. There was clear evidence of carcinogenic activity of riddelliine in male B6C3F1 mice based on increased incidences of hemangiosarcoma in the liver. There was clear evidence of carcinogenic activity in female B6C3F1 mice based on increased incidences of alveolar/bronchiolar neoplasms. Administration of riddelliine by gavage resulted in nonneoplastic lesions in the liver and kidney of male and female rats; the liver and kidney of male and female mice; and the lung and arteries (multiple tissues) of female mice. Decreased incidences of hepatocellular neoplasms in male and female mice were related to riddelliine administration.

Topics: Adenoma, Liver Cell; Administration, Oral; Animals; Body Weight; Carcinogenicity Tests; Carcinogens; Female; Kidney; Liver; Liver Neoplasms; Male; Mice; Mice, Inbred Strains; Mutagenicity Tests; Pyrrolizidine Alkaloids; Rats; Rats, Inbred F344; Sex Factors; Survival Analysis; Time Factors

These investigations of riddelliine analyzed potential carcinogenesis and the utility of the female-rat/male-mouse design in bioassays and dose-response. Groups of 50 Fischer rats and B6C3F1 mice were gavage-administered riddelliine 5 days per week for 105 weeks. The dose levels for male rats were 0 or 1.0 mg/kg body weight; female rats 0, 0.01, 0.033, 0.1, 0.33, or 1.0 mg/kg; male mice 0, 0.1, 0.3, or 1.0, 3.0 mg/kg; and female mice 0 or 3.0 mg/kg. The dose groups were purposely designed to evaluate the dose-response relationship only in female rats and male mice. In rats, liver hemangiosarcoma, hepatocellular adenoma, and mononuclear cell leukemia were significantly increased in the 1.0 mg/kg male and female dose groups. Non-neoplastic lesions occurred in the liver and kidney of male and female rats. In mice, hemangiosarcomas increased significantly in the liver of males in the 3.0 mg/kg dose group. Alveolar/bronchiolar neoplasms in the 3.0 mg/kg dose group of female mice were significantly increased. Hepatocellular neoplasms were significantly decreased in the 1.0 mg/kg dose group of male and 3.0 mg/kg dose groups of male and female mice. Non-neoplastic lesions occurred in the liver and kidney of male and female, and lung and arteries of female mice. These studies demonstrate toxicity and carcinogenicity of riddelliine in rats and mice, and a dose-response relationship in female rats and male mice under the experimental conditions employed.

Topics: Animals; Carcinogens; Carcinoma, Hepatocellular; Dose-Response Relationship, Drug; Female; Hemangiosarcoma; Kidney; Leukemia; Liver; Liver Neoplasms; Male; Mice; Pyrrolizidine Alkaloids; Rats; Rats, Inbred F344; Sex Factors

Toxicity studies of riddelliine, a member of a class of pyrrolizidine alkaloids, were conducted because riddelliine has been found to contaminate human food sources. Groups of male and female Fischer rats were administered riddelliine by gavage in phosphate buffer at doses up to 10 mg/kg, and B6C3F1 mice at doses up to 25 mg/kg, five times a week. The animals were necropsied after 13 weeks of treatment or after a 7 or 14 week recovery period. Body weight gains were inversely related to dose in both rats and mice. Body weight of the 1.0 and 3.3 mg/kg female rats and 10.0 and 25.0 mg/kg mice remained depressed during the 14 week recovery period. At 13 weeks, significant findings included dose-related hepatopathy and intravascular macrophage accumulation in rats and hepatocytomegaly in mice. During the 14 week recovery period these lesions persisted and hepatic foci of cellular alteration in male rats and bile duct proliferation in female rats and male and female mice increased in severity. In the 10 mg/kg group of female rats adenomas of the liver occurred in two of ten at 13 weeks and in one of five at the 14 week recovery period. In separate studies, the frequency of micronucleated erythrocytes in peripheral blood was increased in male mice administered a single dose (150 mg/kg) of riddelliine. Increases in unscheduled DNA and S-phase syntheses were detected in primary hepatocytes from rats and mice treated with riddelliine at doses up to 25.0 mg/kg for 5 or 30 days. In mating trials in rats and mice, pup weights from treated dams at birth and during suckling were lower than controls. Thus, riddelliine is genotoxic and carcinogenic and may cross the placenta and/or be found in milk, causing developmental toxicity in rodents.

Topics: Adenoma, Liver Cell; Administration, Oral; Animals; Body Weight; Carcinogens; Cells, Cultured; DNA; Dose-Response Relationship, Drug; Female; Image Processing, Computer-Assisted; Liver; Liver Neoplasms; Macrophages; Male; Mice; Micronucleus Tests; Microscopy, Fluorescence; Mutation; Organ Size; Pyrrolizidine Alkaloids; Rats; Rats, Inbred F344; Reproduction