Anthraquinones in the aqueous extract of Cassiae semen cause liver injury in rats through lipid metabolism disorder.

BACKGROUND: Cassiae semen has been used as the tea or medicine component to treat hyperlipidemia or for hepatoprotection. However, Cassiae semen was reported to be a potentially hepatotoxic herb, and the underlying hepatotoxicity mechanisms or specific hepatotoxic components of Cassiae semen are unknown. PURPOSE: In this study, we aimed to explore the potential hepatotoxicity mechanisms and the hepatotoxic components of Cassiae semen. METHODS: Both young adult male and female SD rats were orally administrated with the aqueous extract of the seeds of Senna obtusifolia (L.) H.S.Irwin & Barneby at doses of 4.73, 15.75, 47.30 g/kg for 28 days, and the body weight, liver coefficient, bile acids, histopathology, serum levels of TC, TG, LDL, HDL, ALP, ALT, AST, and LDH were examined. Lipidomic analysis of rat serum was performed by LC-MS to investigate the specifically changed lipids caused by the aqueous extract treatment. The components absorbed in plasma were detected by UHPLC-Q-Exactive-MS. MTT assay was used to evaluate the cytotoxicity of these components absorbed in plasma. RESULTS: The serum levels of ALP, AST, ALT, LDH were increased on day 7 with some of which gradually dropped to normal level on day 28. In high dose of the aqueous extract treated group, the histopathological changes were observed based on the cytoplasmic vacuolation in the liver and the increase of bile acids, indicating the hepatotoxicity of the aqueous extract. The changes of TC, TG, LDL, HDL indicated the disorder of lipid metabolism. By comparing the difference in lipids between high dose group and control group, the results showed that the alterations were primarily focused on glycerophospholipid metabolism in both male and female rats. In addition, the glycerolipid metabolism in female rats also changed. Further analyses found that PC (18:2/20:4) and LysoPC 18:0 were significantly increased. Among these phytochemicals detected in plasma, nine components in the aqueous extract were considered to have the highest concentrations, particularly some types of anthraquinones (AQs) existing in Cassiae semen (AQs-in-CS), such as obtusifolin, aurantio-obtusin, and obtusin. The MTT assay showed that emodin, obtusifolin, rhein, aurantio-obtusin, and obtusin inhibited cell viability. Considering plasma concentrations and cytotoxicity of these components, our study indicates that the AQs-in-CS (obtusifolin, aurantio-obtusin and obtusin), emodin and rhein are the potential hepatotoxic phytochemicals in the aqueous extract.

Excretion and toxicity evaluation of 131I-Sennoside A as a necrosis-avid agent.

1. Sennoside A (SA) is a newly identified necrosis-avid agent that shows capability for imaging diagnosis and tumor necrosis targeted radiotherapy. As a water-soluble compound, 131I-Sennoside A (131I-SA) might be excreted predominately through the kidneys with the possibility of nephrotoxicity. 2. To further verify excretion pathway and examine nephrotoxicity of 131I-SA, excretion and nephrotoxicity were appraised. The pharmacokinetics, hepatotoxicity and hematotoxicity of 131I-SA were also evaluated to accelerate its possible clinical translation. All these studies were conducted in mice with ethanol-induced muscular necrosis following a single intravenous administration of 131I-SA at 18.5 MBq/kg or 370 MBq/kg. 3. Excretion data revealed that 131I-SA was predominately (73.5% of the injected dose (% ID)) excreted via the kidneys with 69.5% ID detected in urine within 72 h post injection. Biodistribution study indicated that 131I-SA exhibited initial high distribution in the kidneys but subsequently a fast renal clearance, which was further confirmed by the results of autoradiography and single-photon emission computed tomography-computed tomography (SPECT-CT) imaging. The maximum necrotic to normal muscle ratio reached to 7.9-fold at 48 h post injection, which further verified the necrosis avidity of 131I-SA. Pharmacokinetic parameters showed that 131I-SA had fast blood clearance with an elimination half-life of 6.7 h. Various functional indexes were no significant difference (p > 0.05) between before administration and 1 d, 8 d, 16 d after administration. Histopathology showed no signs of tissue damage. 4. These data suggest 131I-SA is a safe and promising necrosis-avid agent applicable in imaging diagnosis and tumor necrosis targeted radiotherapy.

The injury of serotonin on intestinal epithelium cell renewal of weaned diarrhoea mice.

Diarrhoea is a common cause of death in children and weaned animals. Recent research has found that serotonin (5-HT) in the gastrointestinal tract plays an important role in regulating growth and the maintenance of mucosa, which protect against diarrhoea. To determine the influence of 5-HT on intestinal epithelium cell renewal under weaned stress diarrhoea, a weaned-stress diarrhoea mouse model was established with senna infusion (15 mL/Kg) via intragastric administration and stress restraint (SR). Mice with an increase in 5-HT were induced by intraperitoneal injection with citalopram hydrobromide (CH, 10 mg/Kg). The results demonstrated that compared with the control animals, diarrhoea appeared in weaned stress mice and the 5-HT content in the small intestine was significantly increased (P<0.05). Further, the caspase-3 cells and cells undergoing apoptosis in the small intestine were significantly increased, but the VH (villus height), V/C (villus height /crypt depth), and PCNA-positive rate significantly decreased. Compared with the control animals, CH increased the intestinal 5-HT content, caspase-3 cells and cells undergoing apoptosis but decreased the VH and V/C. Compared with both control and weaned stress animals, weaned stress animals that were pre-treated with CH showed higher 5-HT concentrations, positive caspase-3 cells and cells undergoing apoptosis but lower VH, V/C and PCNA-positive rate. In vitro, a low concentration of 5-HT inhibit, IEC-6 cell line apoptosis but a higher concentration of 5-HT promoted it. Therefore, weaned stress diarrhoea mice were accompanied by a 5-HT increase in the small intestine and vice versa, and the increase in 5-HT induced by CH caused diarrhoea. In brief, 5-HT and diarrhoea slowed the intestinal epithelium cell renewal and injured the abortion function and mucosal barrier by decreasing VH, V/C and proliferation and increasing epithelium cell apoptosis.

Immunological changes in the intestines and skin after senna administration.

CONTEXT: It has been reported that chronic sennoside use is associated with the development of melanosis coli, colonic adenoma, and/or carcinomas. OBJECTIVES: In this study, we investigated the immunological changes in the colon and skin after the administration of senna. MATERIALS AND METHODS: In this study, we investigated the colon and epidermis of C57/BL6j mice after a single administration of 10 mg/kg of senna [Cassia angustifolia (Caesalpiniaceae); 3, 6, 12, and 24 h after administration] and after repeated once per week administrations (on days 3, 5, 7, 14, and 21 of administration). The LD50 and ED50 of senna used in this experiment were 165 mg/kg and 13 g/kg, respectively. RESULTS: We demonstrated that the DOPA-positive cells in the colon increased at 12 h after single administration and were further increased from at 5-28 d after repeated administration. We also studied the physiological changes of the small intestine using the charcoal meal test. We found that there was a tendency for peristalsis to be inhibited after repeated senna administration. In the epidermis, we investigated the number of Langerhans cells, because they are important immune cells of the skin. The number of these cells decreased, especially after repeated administration. DISCUSSION AND CONCLUSION: The present findings suggested that it is necessary to pay attention to not only the intestine but also the skin, during long-term senna treatment.

Toxicology and carcinogenesis study of senna in C3B6.129F1-Trp53 tm1Brd N12 haploinsufficient mice.

Senna is a pod or leaf of Senna alexandrina P. Mill and is used as a stimulant laxative. In the large intestine, bacterial enzymes reduce sennosides to rhein-9-anthrone, the active form for the laxative effect. To determine the potential toxic effects of senna, a 5-week dose range finding study in the C57BL/6N mouse and a 40-week toxicology and carcinogenesis study in the C3B6.129F1-Trp53 (tm1Brd) N12 haploinsufficient (p53(+/-)) mouse were conducted. In the 5-week study, C57BL/6N mice were exposed to up to 10,000 ppm senna in feed. Increased incidences of epithelial hyperplasia of the cecum and colon were observed in males and females exposed to 5,000 or 10,000 ppm senna. These intestinal lesions were not considered to be of sufficient severity to cause mortality and, thus, in the p53(+/-) mouse 40-week study, the high dose of 10,000 ppm was selected. Significant increases in the incidences of epithelial hyperplasia of the colon and cecum were observed at 10,000 ppm in p53(+/-) males and females, and the incidence of hyperplasia of the colon was significantly increased at 3,000 ppm in females. In conclusion, the large intestine was the major target of senna-induced toxicity in both wild-type and the p53(+/-) mouse model. There was no neoplastic change when senna was administered to p53(+/-) mouse.

Assessment of antimutagenic and genotoxic potential of senna (Cassia angustifolia Vahl.) aqueous extract using in vitro assays.

Senna (Cassia angustifolia Vahl.) is widely used as a laxative, although potential side effects, such as toxicity and genotoxicity, have been reported. This study evaluated genotoxic and mutagenic effects of senna aqueous extract (SAE) by means of four experimental assays: inactivation of Escherichia coli cultures; bacterial growth inhibition; reverse mutation test (Mutoxitest) and DNA strand break analysis in plasmid DNA. Our results demonstrated that SAE produces single and double strand breaks in plasmid DNA in a cell free system. On the other hand, SAE was not cytotoxic or mutagenic to Escherichia coli strains tested. In effect, SAE was able to avoid H(2)O(2)-induced mutagenesis and toxicity in Escherichia coli IC203 (uvrA oxyR) and IC205 (uvrA mutM) strains, pointing to a new antioxidant/antimutagenic action of SAE.

An oral carcinogenicity and toxicity study of senna (Tinnevelly senna fruits) in the rat.

Senna (Tinnevelly senna fruits), a known laxative derived from plants, was administered by gavage to Sprague-Dawley (Crl:CD (SD) BR) rats once daily at dose levels of 0, 25, 100 and 300 mg/kg/day for up to 104 consecutive weeks. Based upon clinical signs related to the laxation effect of senna, the highest dose (300 mg/kg/day) was considered to be a maximum tolerated dose. Sixty animals per sex were assigned to the control and dose groups. Assessments included clinical chemistry, hematology, full histology (control and high-dose groups; in addition, low and mid dose: intestinal tract, adrenals, liver, kidneys, brain and gross lesions) and toxicokinetics. The primary treatment-related clinical observation was mucoid feces seen at 300 mg/kg/day. When compared to controls, animals administered 300 mg/kg/day had slightly reduced body weights, increased water consumption and notable changes in electrolytes in serum (increases in potassium and chloride) and urine (decreases in sodium, potassium and chloride). The changes in electrolytes are most likely physiologic adaptations to the laxative effect of senna. At necropsy, dark discoloration of the kidneys was observed in animals in all treated groups. Histological changes were seen in the kidneys of animals from all treated groups and included slight to moderate tubular basophilia and tubular pigment deposits. In addition, for all treated groups, minimal to slight hyperplasia was evident in the colon and cecum. These histological changes, together with the changes seen in the evaluation of clinical chemistry and urine parameters, have been shown to be reversible in a previous 13-week rat study of senna. No treatment-related neoplastic changes were observed in any of the examined organs. Based upon these data, it is concluded that senna is not carcinogenic even after daily administration for 2 years at dosages of up to 300 mg/kg/day in Sprague-Dawley rats.

A 13-week oral toxicity study of senna in the rat with an 8-week recovery period.

Senna was administered by gavage to Sprague Dawley rats once daily at dose levels of 0, 100, 300, 750 or 1500 mg/kg for up to 13 consecutive weeks followed by an 8-week recovery period for selected animals. Dose- and treatment-related clinical signs included abnormal feces, which were seen to varying degrees from animals at 300 mg/kg per day and more. Animals receiving 750 or 1500 mg/kg per day had significantly reduced body weight gain (males only) and, related to the laxative properties of senna, increased water consumption and notable changes in electrolytes in both serum and urine. At both the terminal and recovery phase necropsy, an increase in absolute and relative kidney weights was seen for male and female animals receiving 750 and/or 1500 mg/kg per day. A dark discoloration of the kidneys was observed at necropsy along with histopathological changes in the kidneys (slight to moderate tubular basophilia and pigment deposits) at 300 mg/kg and above. However, there were no indications in laboratory parameters of any renal dysfunction. In addition, for all treated groups, minimal to slight hyperplasia was recorded in the forestomach and large intestine. Following 8 weeks of recovery, with the exception of the brown pigment in the kidneys, there were no histopathological abnormalities. Thus, the biochemical and morphological changes seen following 13 weeks of treatment of senna significantly reversed following 8 weeks of recovery.

Distribution of COX-negative mitochondria in myofibers of rats intoxicated with Senna occidentalis seeds.

We have described that administration of seeds or parts of the seed of Senna occidentalis (coffee senna) for long periods, induces histochemical changes in the skeletal muscles of hens and rats that are characteristic of a mitochondrial myopathy--as decrease of SDH and COX activity, with some COX negative fibers. In this experimental model of mitochondrial myopathy, as in many human mitochondrial diseases, there is a random distribution of COX negative fibers. Some fibers are completely COX negative while others are partially negative and others are completely positive. In the present work we have studied the distribution of COX negative mitochondria at transmission electron microscopy in skeletal muscle of rats in this experimental myopathy. In myofibers of intoxicated animals the expression of COX was heterogeneous. The histochemical reaction was observed in the internal membrane (more evident in mitochondrial cristae) of all mitochondria of some myofibers, while it was almost absent in other myofibers. In these myofibers the great part of the mitochondria were negative for COX reaction while other ones had a weak expression of this enzyme (dot or focal expression of COX). Our results indicated that the COX mitochondrial activity is heterogeneously impaired in myofibers of rats intoxicated with S. occidentalis. These abnormalities remember those observed in some types of human mitochondrial myopathies.

Mitochondrial metabolism impairment in muscle fibres of rats chronically intoxicated with Senna occidentalis seeds.

The chronic administration of S. occidentalis seeds was found to induce a mitochondrial myopathy in hens. This study was undertaken to determine if the chronic treatment with S. occidentalis seeds of rats (as a mammalian model) would induce a mitochondrial myopathy similar to those described in humans and to determine if the histological changes could be correlated with the amount of ingested seeds. Twenty-one days old rats were fed S. occidentalis seeds at different diet concentrations (1, 2, 3%). Rats fed 1% S. occidentalis seeds had only a few COX-negative muscle fibers in the pectoralis major muscle. Rats fed 3% Senna occidentalis seeds had a greater number of COX-negative fibers. Rats fed 2% had an intermediate number of COX-negative fibers. Activity of SDH and NADH-tr were decreased in rats of groups 2% and 3%. Our data indicate that a progressive mitochondrial metabolism impairment can be produced in rats fed S. occidentalis seeds and that this impairment can be correlated with the amount of ingested seeds.

No clastogenic activity of a senna extract in the mouse micronucleus assay.

In previous studies, an analytically well-defined senna extract, commonly used as a laxative, gave positive responses in vitro in the Ames test and in the CHO assay. Therefore, the objective of this study was to investigate the genotoxic activity of the same senna extract in an in vivo genotoxicity assay by means of the generally acknowledged MNT. After administration of an oral dose of 2000 mg senna extract/kg to NMRI mice of both genders, which is equivalent to 119 mg potential rhein/kg, 5.74 mg potential aloeemodin/kg and 0. 28 mg potential emodin/kg, there were no elevated levels of micronuclei in bone marrow cells. Kinetic studies were performed in parallel to demonstrate target organ availability. Highest concentrations in the plasma were reached after 1 h with 3.4 microg rhein/ml and 0.065 microg aloeemodin/ml. In all cases, emodin was below the limit of quantification. From the results, the in vitro clastogenic activity of the senna extract could not be confirmed in the mouse micronucleus assay. Together with further negative in vivo genotoxicity studies with anthranoids, the conclusion can be drawn that there is no indication so far demonstrating a genotoxic risk for patients taking senna laxatives.

Genotoxicity of sennosides on the bone marrow cells of mice.

Preparations of a number of plants which contain hydroxyanthraquinones as active constituents are used worldwide for their laxative effect. Anthraquinone glycosides of Cassia angustifolia and C. fistula were investigated for their ability to induce a clastogenic effect on the bone marrow cells of Swiss albino mice. The endpoints screened were chromosomal aberrations and frequency of aberrant cells. Oral exposure to doses of these anthraquinones and their equivalent amount in leaf and pod extracts did not induce significant numbers of chromosomal aberrations or aberrant cells. The results indicate that anthraquinone sennoside B and rhein are weakly genotoxic.

Muscle atrophy induced in broiler chicks by parts of Senna occidentalis seeds.

Senna occidentalis (formerly Cassia occidentalis) is a common contaminant of agricultural commodities. It is toxic to cattle and poultry, reportedly being responsible for skeletal myodegeneration in these animals. All parts of the plant present toxicity, but the seeds are the most toxic. The toxin(s) responsible for the myodegeneration have not been definitively identified, nor is it known which part of the seeds is most toxic. Intoxication by this plant leads to weight loss with considerable economic repercussions. The effects of the whole seed and of parts of S. occidentalis seeds (1% in commercial feed) were compared on the pectoralis major muscle of broiler chicks intoxicated from birth until 22 days of life. There were severe clinical signals and reduced body weight in birds that received the external tegment of the seed, whereas no adverse effects were observed in birds that received the whole seed or other parts of the seed. Histological and morphometric studies showed an intense muscle fibre atrophy (both type 1 and type 2 fibres were affected) in the group that received 1% external tegment. This study may be the first step to identifying the substance(s) involved in this pathological process.

Toxic peripheral neuropathy of chicks fed Senna occidentalis seeds.

Plants of the genus Senna (formerly Cassia) are poisonous to livestock and other laboratory animals, leading to a syndrome of a widespread muscle degeneration, incoordination, recumbence, and death. The main histologic lesion is necrosis of skeletal muscle fibers. Recently, a mitochondrial myopathy with ragged-red and cytochrome oxidase (COX)-negative muscle fibers was recognized in hens chronically intoxicated with parts of seeds of S. occidentalis. The purpose of the present work was to investigate if there was peripheral nerve involvement in the acute intoxication of chicks with S. occidentalis seeds. Teasing of individual fibers revealed signs of extensive axonal damage with myelin ovoids. Ultrathin sections confirmed the axonal damage. Axons were filled with membranes, some residual disorganized filaments, and enlarged mitochondria. In some instances the axon disappeared and there was secondary degeneration of the myelin sheath. The present work is the first description of the neurotoxic effect of S. occidentalis intoxication. Future work should attempt to determine the mechanisms involved in this neuropathy.

Mitochondrial myopathy in Senna occidentalis-seed-fed chicken.

Plants of the genus Senna (formerly Cassia) have been recognized as the cause of a natural and experimental syndrome of muscle degeneration frequently leading to death in animals. Histologically, it demonstrated skeletal and cardiac muscle necrosis, with floccular degeneration and proliferation of sarcolemmal nuclei. Recently, it was described as an experimental model of mitochondrial myopathy in hens chronically treated with Senna occidentalis. Currently, skeletal muscles of chicks intoxicated with seeds of the poisonous plant S. occidentalis were studied by histochemistry and electron microscopy. Since birth, the birds were fed ground dried seeds of this plant with a regular chicken ration at a dose of 4% for 11 days. Microscopic examination revealed, besides muscle-fiber atrophy, lipid storage in most fibers and a moderate amount of cytochrome oxidase-negative fibers. By electron microscopy, enlarged mitochondria with disrupted or excessively branched cristae were seen. This picture was characteristic of mitochondrial myopathy. These findings have hitherto remained unnoticed in skeletal muscle of young birds treated with S. occidentalis.

The role of constitutive and inducible nitric oxide synthase in senna- and cascara-induced diarrhoea in the rat.

The role of constitutive and inducible nitric oxide (NO) synthase in rats treated with senna and cascara was studied. Senna (60 mg/kg p.o.) and cascara (800 mg/kg p.o.) ex vivo significantly increased Ca(2+)-dependent constitutive NO synthase activity in the rat colon. Induction of NO synthase (12% of the total NO synthase) was associated with cascara, but not senna, administration. Dexamethasone (0.03-0.3 mg/kg i.p.), which inhibits the expression of inducible NO synthase, significantly and dose-dependently reduced cascara-(but not senna-) induced diarrhoea and colonic fluid secretion. These findings suggest that senna probably exerts its laxative effect through stimulation of the constitutive isoform of NO synthase, while the inducible isoform of NO synthase also seems to be involved in the laxative effect of cascara.

Evaluation of the potential carcinogenic activity of Senna and Cascara glycosides for the rat colon.

Anthraquinone glycosides of Senna and Cascara were investigated for their ability to induce aberrant crypt foci (ACF) in the rat colon mucosa, which are considered putative preneoplastic lesions. Dietary exposure to high doses of these glycosides for 56 successive days did not cause the appearance of ACF or increase in incidence of ACF induced by 1,2-dimethyl-hydrazine (DMH). However, in rats treated with both DMH and the highest dose of glycosides, the average number of aberrant crypts per focus, considered a consistent predictor of tumor outcome, was higher than in rats given DMH alone. These findings suggest that Senna and Cascara glycoside might behave as weak promoters in rat colon carcinogenesis.

The genotoxicity status of senna.

Genotoxicity tests were performed by several laboratories with the drug fructus sennae, senna extract, sennosides, rhein and aloe-emodin. The drug fructus sennae, the sennosides and rhein did not increase mutation frequencies in the following test systems: bacterial systems (Salmonella reverse mutation test and/or Escherichia coli forward mutation test); mammalian cell cultures [hypoxanthine guanine phosphoribosyl transferase (HGPRT) test; mouse lymphoma test; chromosome aberration test with Chinese hamster ovary cells]; bone marrow (micronucleus test; chromosome aberration test); melanoblast cells (mouse spot test) of rodents. With aloe-emodin mutagenic effects were observed only in vitro in the chromosome aberration test with CHO cells and in the Salmonella reverse mutation test (frameshift mutations in strains TA 1537, TA 1538 and TA 98). In the in vitro gene mutation test with V79 cells (HGPRT test) no mutagenic potential of aloe-emodin was observed. In in vivo studies [micronucleus test with bone marrow cells of NMRI mice, chromosome aberration test with bone marrow cells of Wistar rats, mouse spot test (crossing DBA/2J x NMRI) no indication for a mutagenic activity of aloe-emodin was found. The relevance of the absence of a mutagenic potential in in vivo test systems was strengthened by the fact that aloe-emodin could be found in the blood serum after oral administration. Additional information on the interaction of aloe-emodin with DNA was obtained from an ex vivo unscheduled DNA synthesis test performed with hepatocytes of male Wistar rats: aloe-emodin did not induce unscheduled DNA synthesis as expression of DNA damage.(ABSTRACT TRUNCATED AT 250 WORDS)