Polymethoxy-1-Alkenes Screening of Chlorella and Spirulina Food Supplements Coupled with In Vivo Toxicity Studies.

Selected species of cyanobacteria and green algae have been reported to produce lipophilic polymethoxy-1-alkenes (PMAs) which were shown to exhibit in vivo teratogenicity. Considering that information on PMAs in Arthospira sp. (known commercially as Spirulina) and Chlorella sp. cultivated for food supplement production was essentially lacking, the present study screened Chlorella (n = 10) and Spirulina (n = 13) food supplements registered in the European Union. Mass spectrometry analysis of column fractionated extracts was performed. None of the four variants previously reported in some cyanobacteria and green algae, nor any potentially related structures were detected in the studied samples. Since the isolated lipophilic fractions contained various compounds, they were further screened for in vivo teratogenicity in Danio rerio embryo, and for the potential to induce oxidative stress and genotoxicity in the liver and neurotoxicity in the brain of adult zebrafish. None of the tested food supplements had detectable levels of PMAs or any potentially related structures. No teratogenicity was revealed except for spinal curvature induced by fractions obtained from two Chlorella products. Selected fractions revealed cytotoxicity as indicated by an increased level of reactive oxygen species, catalase activity, lipid peroxidation and increased frequency of DNA strand breaks in hepatic tissue. The majority (60%) of Chlorella fractions induced an increase in cholinesterase activity in zebrafish brain homogenate while exposure to 61.5% of Spirulina fractions was associated with its decrease. The present study confirms that Chlorella and Spirulina food supplements are free of teratogenic PMAs, although the observed in vivo toxicities raise questions regarding the quality of selected products.

Effects of Sargaquinoic Acid in Sargassum Serratifolium on Inducing Brown Adipocyte-like Phenotype in Mouse Adipocytes In Vitro.

A previous study showed that the meroterpenoid-rich fraction of an ethanolic extract of Sargassum serratifolium (MES) stimulated adipose tissue browning and inhibited diet-induced obesity and metabolic syndrome. Sargaquinoic acid (SQA) is a major component in MES. We investigated the effects of SQA on the differentiation of preadipocytes to the beige adipocytes. SQA was treated in 3T3-L1 adipocytes differentiated under a special condition that has been reported to induce the browning of adipocytes. SQA at 10 µM reduced lipid accumulation by approximately 23%. SQA at 2.5 - 10 µM induced the differentiation of white adipocytes to beige adipocytes partially by increasing the mitochondrial density and the expression of beige/brown adipocyte markers. In addition, SQA activated lipid catabolic pathways, evidenced by the increased expression levels of perilipin, carnitine palmitoyltransferase 1, and acyl-CoA synthetase long-chain family member 1. As a partial mechanism, biochemical and in silico analyses indicate that SQA activated AMP-activated protein kinase signaling in adipocytes.

Dual-mixed/CMC model for screening target components from traditional Chinese medicines simultaneously acting on EGFR & FGFR4 receptors.

Radix Salviae Miltiorrhiae (also known as DanShen (DS) in China), a popular herbal drug in traditional Chinese medicine (TCM) for promoting blood circulation and treating blood stasis, has been reported to possess potential anti-tumor effects. The aim of the study was to develop an effective and practical method for screening and identifying bioactive compounds from Radix Salviae Miltiorrhiae. In this work, the epidermal growth factor receptor (EGFR) and fibroblast growth factor receptors 4 (FGFR4) dual-mixed/cell membrane chromatography (CMC) coupled with high performance liquid chromatography-electrospray ionization-ion trap-time of flight-multistage mass spectrum (HPLC-ESI-IT-TOF-MSn) was established and successfully used to identify the active components from Radix Salviae Miltiorrhiae. Salvianolic acid C (SAC), tanshinone I (Tan-I), tanshinone IIA (Tan-IIA), and cryptotanshinone (C-Tan) were identified as bioactive components with EGFR and FGFR4 activities. MTT and kinase assay were performed to investigate inhibitory effects of these compounds against EGFR and FGFR4 cells growth in vitro. Both cell viability and kinase activity showed that cryptotanshinone acting on EGFR receptor and tanshinone IIA acting on FGFR4 receptor. In conclusion, the EGFR & FGFR4 dual-mixed/CMC can simultaneously screen the bioactive components from TCMs that act on both EGFR and FGFR4 receptors, which significantly improve the efficiency of specific bioactive components identification from a complex system.

The termiticidal activity of Sextonia rubra (Mez) van der Werff (Lauraceae) extract and its active constituent rubrynolide.

BACKGROUND: Termites are degradation agents that inflict severe damage on wood. Some long-lasting Amazonian trees can resist these insects by producing toxic secondary metabolites. These metabolites could potentially replace synthetic termiticidal products which are becoming more restricted to use. RESULTS: Sextonia rubra is resistant to termite-induced degradation. It has been demonstrated that this species naturally produces an ethyl-acetate-soluble termiticidal metabolite, rubrynolide, to protect its wood. Assays in the presence of tropical and invasive termites established that both rubrynolide and crude ethyl acetate extract from S. rubra wood can be used as a treatment for the protection of sensitive woods against termites. CONCLUSION: Rubrynolide and S. rubra extract are promising candidates for the replacement of synthetic termiticides.

Safety evaluation of Trikatu, a generic Ayurvedic medicine in Charles Foster rats.

Chemical characterization and acute and sub-acute toxicity study of Trikatu, a generic herbal formulation of Indian system of medicine, was carried out in Charles Foster (CF) rats for safety profiling. In acute toxicity experiment, Trikatu at 2,000 mg/kg body weight once orally was well tolerated by the experimental animals (both male and female) and no changes were observed in mortality, morbidity, gross pathology, gain in weight, vital organ weight, hematological (total white blood cells (WBC) and red blood cells (RBC) count), biochemical parameters such as serum creatinine, serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), serum lipid profile and tissue biochemical parameters such as reduced glutathione and malonaldehyde content as oxidative stress markers. In sub-acute experiment, Trikatu was administered at 5, 50 and 300 mg/kg body weight once daily for 28 days in female CF rats, and non-significant changes were found in most of the parameters studied such as acute experiment except significant increase in low density lipoprotein (LDL) cholesterol level at 50 and 300 mg/kg body weight, decrease in high density lipoprotein (HDL) cholesterol level at 300 mg/kg body weight, increase in SGPT activity at 50 mg/kg body weight and decrease in WBC count at 300 mg/kg body weight on 28(th) day post treatment.

Molecular mechanisms of toxicity of important food-borne phytotoxins.

At present, there is an increasing interest for plant ingredients and their use in drugs, for teas, or in food supplements. The present review describes the nature and mechanism of action of the phytochemicals presently receiving increased attention in the field of food toxicology. This relates to compounds including aristolochic acids, pyrrolizidine alkaloids, beta-carotene, coumarin, the alkenylbenzenes safrole, methyleugenol and estragole, ephedrine alkaloids and synephrine, kavalactones, anisatin, St. John's wort ingredients, cyanogenic glycosides, solanine and chaconine, thujone, and glycyrrhizinic acid. It can be concluded that several of these phytotoxins cause concern, because of their bioactivation to reactive alkylating intermediates that are able to react with cellular macromolecules causing cellular toxicity, and, upon their reaction with DNA, genotoxicity resulting in tumors. Another group of the phytotoxins presented is active without the requirement for bioactivation and, in most cases, these compounds appear to act as neurotoxins interacting with one of the neurotransmitter systems. Altogether, the examples presented illustrate that natural does not equal safe and that in modern society adverse health effects, upon either acute or chronic exposure to phytochemicals, can occur as a result of use of plant- or herb-based foods, teas, or other extracts.

Beyond TPH: health-based evaluation of petroleum hydrocarbon exposures.

The term "total petroleum hydrocarbons" (TPH) is a widely used, but loosely defined, parameter quantified by a number of different methodologies for expressing the aggregate amount of petroleum hydrocarbon compounds (PHCs) in a sample. Because of the shortcomings associated with comparing data from different methods, and the difficulty of assessing potential toxicities of complex mixtures of hydrocarbons, a new approach at more fully and explicitly defining the PHC composition of samples and predicting human noncancer health risks from those exposures has been developed. This new approach is the subject of this paper. This method can be used to perform site-specific risk assessments or to develop health-based cleanup standards for petroleum hydrocarbons. The technique divides the broad chemical classes of PHC (i.e., saturated versus unsaturated) into subgroups of compounds based on numbers of carbon atoms in the compounds within each subgroup. The mass of compounds in each subgroup is then translated into discrete estimates of health risk for specified exposure scenarios. The subgroups were identified from qualitative and quantitative changes in the nature of noncancer toxicities recorded in the literature. For saturated compounds, toxicity changes as carbon chain length increases (measured by numbers of carbon atoms). A "reference compound" was chosen for each range of compounds, usually because its toxicity was relatively well characterized. A published oral reference dose (RfD) was identified for these compounds, or in the absence of a published value, an oral dose-response value was developed from available toxicity information. For saturated PHCs (alkanes, cycloalkanes, and isoalkanes) the subgroups' reference compounds and assigned toxicity value used are C5 to C8 (n-hexane, 0.06 mg/kg/day); C9 to C18 (n-nonane, 0.6 mg/kg/day); and C19 to C32 (eicosane, 6.0 mg/kg/day). For unsaturated compounds (aromatics), one reference RfD was identified for all compounds: C9 through C32 (pyrene, 0.03 mg/kg/day). Dependent upon the analytical technique used for separation of compounds, the unsaturated alkenes may be grouped and subsequently quantified with either the saturate or unsaturate groups. The implications of possible association with either group and contributions to risk estimates are probably not significant. Alkenes make up a small fraction of most fuel products, and they bear structural similarity to the alkanes and are not particularly toxicologically active. If grouped analytically with the aromatics the alkene contribution to toxicity estimates would likely be minor and not be an underestimate of its true toxicity. The mass of PHC in each segment of a chromatogram is quantified and converted to a medium-specific concentration which is then entered into standard medium intake equations to arrive at a daily dose of PHC. This dose is then used with the toxicity value identified for the particular segment of the chromatogram to derive a hazard quotient. The quotients can then be summed across fractions to yield a total hazard index. The noncancer health risks from the aromatics benzene, toluene, and xylenes are evaluated separately using standard risk assessment techniques.

Separation of the toxic and glutathione-enhancing effects of the naturally occurring nitrile, cyanohydroxybutene.

Cyanohydroxybutene (CHB) is reported to be hepatotoxic in male Fischer 344 rats at an oral dose of 300 mg/kg and, while no longer hepatotoxic, pancreatotoxic at 200 mg/kg. In addition, the 200 mg/kg dose causes a persistent elevation in hepatic and pancreatic glutathione (GSH). This study was conducted to determine if smaller doses of CHB could cause GSH elevation in the absence of toxicity. A single oral dose of 100 mg/kg or multiple lower doses (50 mg/kg daily for 3 days or 30 mg/kg for 6 days) caused a significant and persistent increase in pancreatic GSH, although hepatic levels were unchanged. Ten milligrams per kilogram, even daily for 24 days, was without effect on hepatic or pancreatic GSH. Neither a single oral dose of 100 mg/kg nor multiple lower doses were associated with toxicity. However, when either 100 or 50 mg/kg were administered intravenously, pancreatic apoptosis was observed. In animals dosed with 100 mg/kg iv, mixed histiocytic and suppurative inflammation and frank pancreatic necrosis also developed and were associated with elevated plasma lipase and amylase. The animals receiving CHB intravenously also exhibited elevated GSH levels in both pancreas and liver. This study shows that oral doses between 30 and 100 mg CHB/kg can be used to elevate GSH levels without any pancreatotoxicity. However, a single 50 mg CHB/kg dose given intravenously causes apoptosis, while 100 mg/kg causes severe pancreatotoxicity with necrosis.

The acute toxicity evaluation of a low-temperature hydraulic fluid.

A low-temperature version of MIL-H-83282 (LT 83282) is a candidate hydraulic fluid to be used as a replacement for the current low-temperature fluid used on Strategic Air Command aircraft. A single neat dose of 0.1 mL LT 83282 into New Zealand White (NZW) rabbit eyes resulted in slight conjunctival irritation for up to 24 hr after treatment in two of nine rabbits. Rinsing the eyes after treatment appeared beneficial. A single treatment of 0.5 mL neat LT 83282 to rabbit skin produced no irritation. A total of 40% of the guinea pigs receiving repeated dermal application of the fluid demonstrated a positive sensitization response. A single oral dose of 5 g LT 83282/kg body weight given to five male and five female Fischer 344 (F-344) rats and a single dermal application of 2 g LT 83282/kg body weight applied to five male and five female NZW rabbits resulted in no deaths. Inhalation exposures to aerosol concentrations of LT 83282 resulted in an LC50 of 2.13 and 1.50 mg/L for male and female F-344 rats, respectively. No clinical signs of acute delayed neurotoxicity were observed in hens twice dosed at limit levels (5 g/kg) and observed for 21 days.

The acute pancreatotoxic effects of the plant nitrile 1-cyano-2-hydroxy-3-butene.

The effects of synthetic 1-cyano-2-hydroxy-3-butene (CHB), a racemic mixture of the (R)- and (S)-enantiomers, were studied in adult male rats. The compound given by gavage in olive oil at doses of 25-200 mg/kg causes toxic effects on the pancreas that resemble those seen when naturally occurring CHB is given to rats. At 6 h after dosing, pancreatic edema is seen with doses of 100 mg/kg and greater. The edema fluid had a high protein content, indicating a marked increase in macromolecular permeability of the pancreatic microcirculation. A loss of zymogen granules from the acinar cells and a lacy supranuclear vacuolation of the acinar cell cytoplasm was observed. At 4 h after dosing, pancreatic nonprotein thiols were depleted and rebounded at 24 h to three times control values. At 120 h nonprotein thiol levels decreased but were still elevated compared with control values. Glutathione-S-transferase activity in the pancreas had a similar pattern of change with initial reduction, followed by elevation at 24 h. In rats with pancreatic and biliary fistulas, intraduodenal CHB caused a transient early stimulation of pancreatic juice secretion followed by a return to control values in the case of the lower doses of CHB and depression of flows at larger doses. All doses of CHB caused a dose-related depression of protein concentration in pancreatic juice. Pancreatic juice flow was almost abolished at doses of 200 mg/kg. CHB caused a dose-dependent choleresis accompanied by a marked reduction in bile acid concentrations in bile.(ABSTRACT TRUNCATED AT 250 WORDS)

Preclinical toxicological evaluation of fostriecin, a novel anticancer antibiotic, in rats.

Fostriecin, a novel anticancer antibiotic produced by Streptomyces pulveraecus, is believed to act via inhibition of topoisomerase II. Single-dose intravenous administration to rats at dose levels of 8.8 to 48 mg/kg resulted in lethality at dose levels of 35 mg/kg and higher. Major toxic effects were observed primarily at 17.5 mg/kg and higher, were reversible, and consisted of bone marrow hypocellularity, leukopenia, neutropenia, thrombocytopenia, and diffuse necrosis of various lymphoid tissues. The kidney was also identified as a target organ. Renal effects were observed primarily at 20 mg/kg, were reversible, and included increases in serum BUN, creatinine, and 24-hr glucose excretion. Twenty-four-hour excretion of Na+, K+ and urine osmolality were decreased postdosing at 10 and 20 mg/kg. Renal lesions, observed primarily at 20 mg/kg, consisted of vacuolization and necrosis of proximal and distal tubular epithelium at the corticomedullary junction extending into the medulla. Repeated daily intravenous administration of fostriecin for 5 days to rats at dose levels of 2.5 to 26.5 mg/kg resulted in death at 10 mg/kg and above and similar hematologic, bone marrow, lymphoid tissue, and renal changes as observed in the single-dose study. Hematological, bone marrow, lymphoid, and renal changes observed in rats were consistent with the cytotoxic mechanism of action of the compound.

Enhancement of pancreatic and hepatic glutathione levels in rats during cyanohydroxybutene intoxication.

1-Cyano-2-hydroxy-3-butene (CHB), a cruciferous plant product, is hepatotoxic, pancreatotoxic, and elevates glutathione (GSH) in liver and pancreas. Whether GSH elevation is preceded by a depletion related to toxic insult, or whether toxicity and GSH elevation are unrelated, is not known. To evaluate the temporal relationship between toxicity and GSH levels, male Fisher 344 rats (6/group) were given CHB (200 mg/kg po) and killed up to 96 hr after dosing. At death, histological and ultrastructural evaluations and GSH/GSSG determinations were performed on liver and pancreas. In pancreas, dilatation of the cisternae of the rough endoplasmic reticulum (RER) was evident from 2 hr, becoming progressively more severe 4 and 6 hr after CHB. Frank apoptosis and loss of zymogen granules was evident by 6 hr, becoming widespread by 12 hr. Recovery had commenced by 72 hr, and 50% of treated rats had normal pancreata by 96 hr. No hepatic lesions were observed at this dose. Pancreatic GSH was depressed below 20% at 2 and 4 hr, rose to a maximum of 540% by 12 hr, and remained elevated in treated rats throughout the study (275% at 96 hr). Hepatic GSH only fell to 50%, rose to 150-180%, and returned to normal by 96 hr. While this pattern of depletion and rebound following exposure to hepatotoxins is common, the exaggerated and persistent elevation of pancreatic GSH is unprecedented.

The relationship of vehicle to target organ toxicology induced by the naturally occurring nitrile 1-cyano-2-hydroxy-3-butene.

The effects of gavage vehicle on the acute toxicity of the naturally occurring nitrile 1-cyano-2-hydroxy-3-butene (CHB) were investigated by oral administration of 200 mg/kg body wt/day CHB to male CDF (F-344/Crl BR) rats for 2 days. The vehicles studied here were distilled water, 5% aqueous Tween 20, and corn oil. Liver, kidney, and pancreas were examined histologically and the differences in lesion incidence and severity were assessed. The effects of gavage vehicle on nitrile-induced elevations of daily urinary thiocyanate excretion and tissue glutathione concentrations were also assessed. The pancreatotoxicity of CHB was present regardless of vehicle and consisted of apoptosis of pancreatic acinar cells, infiltration of pancreatic lobules by macrophages, and acinar atrophy and disorganization. CHB in water alone was associated with the least pancreatotoxic effect, whereas the aqueous Tween vehicle was associated with more severe CHB-induced pancreatic lesions. CHB-induced elevations of tissue nonprotein thiol and glutathione concentrations occurred in all treatment groups, but the values were elevated significantly less in the pancreata of CHB/Tween-treated rats than in those of rats given CHB in water or corn oil. By contrast, the greatest elevation in daily urinary thiocyanate excretion occurred in rats given CHB in aqueous Tween, indicating increased biotransformation of CHB to cyanide when Tween 20 was used as a vehicle. These results illustrate the difficulty of identifying suitable vehicles for administration of lipophilic compounds in toxicology studies.

Selective pancreato-toxicity in the rat induced by the naturally occurring plant nitrile 1-cyano-2-hydroxy-3-butene.

The acute toxicity of 1-cyano-2-hydroxy-3-butene (CHB), a nitrile derived from many cruciferous plants, was investigated. Young male CDF (F-344/CrlBr) rats were treated by gavage once daily with 200 mg (2.1 mmol) CHB/kg body weight for 0-4 days and killed 24 hr after the final dose. Lesions were confined to the exocrine pancreas and characterized by individual acinar cell death, inflammation and acinar atrophy and disorganization. Ultrastructural alterations included dilation of cisternae of the acinar cell endoplasmic reticulum, acinar cell death resembling apoptosis, macrophage phagocytosis of acinar cell debris and regenerative changes in remaining acinar cells. Pancreatic, hepatic and renal non-protein thiol concentrations were elevated, suggesting an enhancement of tissue glutathione concentrations and an alteration in glutathione metabolism. Urinary thiocyanate (SCN-) excretion was modestly elevated, indicating some in vivo cyanide release from this nitrile. The results of this study indicate that CHB is a selective pancreatotoxin, inducing changes consistent with apoptosis. CHB is also a possible inducer of tissue glutathione in the liver and kidneys as well as in the pancreas, even at toxic doses.

Anticancer activity of the structurally novel antibiotic Cl-920 and its analogues.

Cl-920 is a structurally novel phosphate ester antibiotic that contains an unsaturated lactone and a conjugated triene system. It has potent antileukemic activity in mice. At doses of 25 mg/kg given i.p. once daily for 5 days to mice bearing approximately 10(7) L1210 leukemia cells, Cl-920 is curative in about 10% of the mice. Life span increases in noncured mice are typically in excess of 150%. The unsaturated lactone and phosphate ester moieties are required for activity against L1210 leukemia. Ring hydroxylation or removal of the terminal hydroxyl group have only modest effects on activity. Schedule studies suggest that prolonged exposure to low levels of Cl-920 is considerably more toxic than is daily or intermittent administration. Daily administration produces optimal activity against L1210 leukemia. Administration i.p. and i.v. of Cl-920 produce roughly equal toxicity and equal activity against an i.p. implant of L1210 leukemia. Cl-920 is inactive when given p.o. or s.c. Cl-920 failed to show confirmed activity against the following tumors in mice: M5076 sarcoma, B16 melanoma, and Ridgway osteogenic sarcoma. The lack of solid tumor activity in mice may be caused by a transport deficiency similar to that found with methotrexate.

Novel antitumor agents CI-920, PD 113,270 and PD 113,271. I. Taxonomy, fermentation and biological properties.

CI-920 (PD 110,161) and two analogues (PD 113,270 and PD 113,271) are novel antitumor compounds produced by a new actinomycete characterized as Streptomyces pulveraceus subsp. fostreus ATCC 31906. The antitumor compounds are predominantly produced during the stationary (idiophase) growth phase of the organism. CI-920 is active versus the murine P388 lymphocytic and L1210 lymphoid leukemia with T/C values of 246 and 207, respectively. This compound has no significant antimicrobial activity.

Toxic effects of Iomex on rat after repeated oral administration.

Administration of Iomex, petroleum fraction, at a dose level of 1 ml/kg for a period of 4 weeks did not produce any toxic symptoms in albino rats. At higher doses, however, the animals showed high incidence of mortality, reduction in food and water intake and loss in body weight. At necropsy, lungs were found to be congested and intestines hyperaemic. Histopathological examination revealed slight abnormalities in lungs, liver and kidney. The absolute organ weights decreased (liver, kidney, heart, spleen, lung, gonad, brain and adrenal), whereas relative weights increased. There was a significant fall in total erythrocyte (RBC) and total leucocyte (WBC) counts and haemoglobin content (Hb) was reduced. Increased lymphocytes with correspondingly decreased neutrophils were also observed. Serum and liver transaminases (GOT and GTP) showed enhanced activity.

Toxicological studies on hydrocarbons--Iomex.

Hematological changes and concurrent enzymatic activities in various tissues were determined in rats injected parentally with Iomex, a petroleum derivate and proposed weedicide. Among the hematological parameters studied only the differential leucocytic count showed significant alterations. This was indicated by a significant decrease in the number of lymphocytes and an increase in those of neutrophils. Significant differences were not observed in the levels of acid phosphatase, glutamic oxaloacetic, and glutamic pyruvate transaminase activities in brain and liver of the experimental group of animals.