Cytotoxic and genotoxic effects of (R)- and (S)-ricinoleic acid derivatives.

(R)-ricinoleic acid is the main component of castor oil from Ricinus communis L. Due to the presence of the hydroxyl group in homoallylic position and asymmetrically substituted carbon atom, it may undergo a number of chemical and biochemical transformations resulting in the products with some specific bioactivities. Conversion of (R)-ricinoleic acid into its (S)-enantiomer enables synthesis of both (R)- and (S)-ricinoleic acid derivatives and comparison of their biological activities. In the present research, (R)- and (S)-ricinoleic acid amides synthesized from methyl ricinoleates and ethanolamine or pyrrolidine as well as acetate derivatives of ethanolamine amides were studied to demonstrate their biological activities using HT29 cancer cells. Double staining of cells with fluorochromes (Hoechst 33258/propidium iodide) as well as 2,'7'-dichlorodihydrofluorescein (DCF) and comet assays were performed. Both the tested amides and acetates caused DNA damage and induced apoptotic and necrotic cell death. In the case of (R)- and (S)-enantiomers of one of the tested acetates, significant difference in the ability to induce DNA damage was observed, which showed the impact of the stereogenic center on the activities of these compounds.

Oleic acid and derivatives affect human endothelial cell mitochondrial function and vasoactive mediator production.

BACKGROUND: Inhalation of common air pollutants such as diesel and biodiesel combustion products can induce vascular changes in humans which may contribute to increased mortality and morbidity associated with fine particulate matter exposures. Diesel, biodiesel, and other combustion byproducts contain fatty acid components capable of entering the body through particulate matter inhalation. Fatty acids can also be endogenously released into circulation following a systemic stress response to some inhaled pollutants such as ozone. When in the circulation, bioactive fatty acids may interact with cells lining the blood vessels, potentially inducing endothelial dysfunction. To examine whether fatty acids could potentially be involved in human vascular responses to air pollutants, we determined the effects of fatty acids and derivatives on important vascular cell functions. METHODS: Human umbilical vein endothelial cells (HUVEC) were exposed in vitro to oleic acid (OA) or OA metabolites for 4-48 h. Cytotoxicity, vasodilator production (by ELISA measurement), mitochondrial function (using Sea Horse assays), and iron metabolism (inferred by ICP-OES measurements) were examined, with standard statistical testing (ANOVA, t-tests) employed. RESULTS: Dose-dependent cytotoxicity was noted at 24 h, with 12-hydroxy OA more potent than OA. Mitochondrial stress testing showed that 12-hydroxy OA and OA induce mitochondrial dysfunction. Analysis of soluble mediator release from HUVEC showed a dose-dependent increase in prostaglandin F2α, a lipid involved in control of vascular tone, at 24 h (85% above controls) after OA-BSA exposure. RT-PCR analysis revealed OA did not induce changes in gene expression at noncytotoxic concentrations in exposed HUVEC, but 12-OH OA did alter ICAM and COX2 gene expression. CONCLUSIONS: Together, these data demonstrate that FA may be capable of inducing cytotoxic effects and altering expression of mediators of vascular function following inhalation exposure, and may be implicated in air pollutant-induced deaths and hospitalizations. (267 of max 350 words).

Suppression of ricinoleic acid toxicity by ptl2 overexpression in fission yeast Schizosaccharomyces pombe.

We previously succeeded to obtain a high content of ricinoleic acid (RA), a hydroxylated fatty acid with great values as a petrochemical replacement, in fission yeast Schizosaccharomyces pombe by introducing Claviceps purpurea oleate Δ12-hydroxylase gene (CpFAH12). Although the production was toxic to S. pombe cells, we identified plg7, encoding phospholipase A2, as a multicopy suppressor that restored the growth defect by removing RA from phospholipids and induced secretion of a part of the released free RA into culture media. In this study, we extended our analysis and examined the effect of triglyceride (TG) lipase overexpression on the tolerance to RA toxicity and RA productivity. S. pombe has three TG lipase genes, ptl1, ptl2, and ptl3, which have high protein sequence similarities to each other and to Saccharomyces cerevisiae counterparts TGL3, TGL4, and TGL5, but only ptl2 overexpression suppressed the growth defect induced by RA production, and the culture grown at 20 °C secreted free RA into media like plg7 overexpression. Suppression by ptl2 was independent of plg7, and a large amount of free RA was accumulated in the cells concomitant with the decrease in RA moieties in phospholipids. Furthermore, the suppression by ptl2 was attenuated by bromoenol lactone (BEL), a phospholipase A2 specific inhibitor, suggesting that Ptl2p may have phospholipase activity. Simultaneous overexpression of ptl2 and plg7 in the FAH12 integrant increased secretion and intracellular accumulation of RA 1.2- and 1.3-fold, respectively, compared to those with single overexpression of plg7 on day 10 at 20 °C.

Toxicity of ricinoleic acid production in fission yeast Schizosaccharomyces pombe is suppressed by the overexpression of plg7, a phospholipase A2 of a platelet-activating factor (PAF) family homolog.

In an effort to produce ricinoleic acid (RA), an important natural raw material with great values as a petrochemical replacement, in Schizosaccharomyces pombe, we introduced Claviceps purpurea oleate Δ12-hydroxylase gene (CpFAH12) to S. pombe, putting it under the control of an inducible nmt1 promoter. However, RA was toxic to S. pombe and the cells expressing CpFAH12 grew poorly at the normal growth temperature 30 °C. To address its toxic mechanism in S. pombe, we screened for a S. pombe cDNA library and identified plg7, which encodes a phospholipase A2, as a suppressor that restored the growth defect without affecting the RA production. A lacZ fusion experiment showed that the expression of plg7 was inducible by RA. Thin layer chromatographic analysis confirmed a reduction in RA moiety in phospholipids and a concomitant increase in free RA in the plg7 overexpressed strain. Since RA is synthesized at the sn-2 position of phosphatidylcholine by Fah12p, and phospholipase A2 hydrolyzes the sn-2 acyl bond of phospholipids, we speculate that plg7 is a stress-responsive gene, and removal of RA moieties from phospholipids, major components of lipid bilayer membrane, by Plg7p would be its suppression mechanism.

Rhipicephalus sanguineus (Acari: Ixodidae) female ticks exposed to castor oil (Ricinus communis): an ultrastructural overview.

Tick control has been accomplished through the use of synthetic acaricides, which has created resistant individuals, as well as contaminating the environment and nontarget organisms. Substances of plant origin, such as oils and extracts of eucalyptus and neem leaves, have been researched as an alternative to replace the synthetic acaricides. Ricinoleic acid esters from castor oil have recently been shown as a promising alternative in eliminating bacterial contamination during ethanol fermentation, by acting as an effective biocide. The same positive results have been observed when these esters are added to the food given to tick-infested rabbits. This study tested the effect of these substance on the reproductive system of Rhipicephalus sanguineus females, added to rabbit food, more specifically on oogenesis. For this, four groups were established: four control groups (CG1, CG2, CG3, and CG4) and four treatment groups (TG1, TG2, TG3, and TG4) with one rabbit in each (New Zealand White), used as hosts. After full 4 days feeding (semi-engorgement), the females were collected and had their ovaries extracted. In this study, it was observed that R. sanguineus females exposed to esters had their ovaries modified, which was demonstrated through transmission electron microscopy techniques. The addition of ricinoleic esters to the diet of tick-infested rabbits revealed how toxic such substances are for the cytoplasmic organelles of oocytes and pedicel cells. These compounds can change the morphophysiology of germ and somatic cells, consequently influencing their viability and, therefore, confirming that the ricinoleic acid esters from castor oil are a promising substance in the control of R. sanguineus.

Toxicology and pharmacology of sodium ricinoleate.

Ricinoleic acid constitutes approximately 90% of the fatty acid content of castor oil. Castor oil is known for its purgative effects and can be used to induce labor. Both castor oil and ricinoleic acid are approved for use in food. The mechanistic basis for purgative actions likely includes the membrane-disruptive effects of detergent-like molecules, such as sodium ricinoleate (a 'soap'). These effects have been shown to be dose-related and to exhibit a threshold below which no laxative response was evident, in both animals and in humans. Castor oil was not toxic in subchronic feeding studies in rodents at doses ranging up to 10-20% of the diet. Sodium ricinoleate, as a surfactant, demonstrates predictable skin and mucus membrane irritant effects, and may induce a Type IV dermal sensitization response in those previously sensitized to it. However, food-grade castor oil and sodium ricinoleate are prepared in such a manner as to be free of the castor bean constituents that have been proven to be the source of reported Type I immediate hypersensitivity responses. Feeding studies with castor oil in rodents provide a basis for a no observable adverse effect level (NOAEL) estimate of 7,500 mg/kg/day and 5,000 mg/kg/day in mice and rats, respectively (). Applying an uncertainty factor of 100 to the lesser of these NOAELs, one can thus estimate an acceptable daily intake (ADI) in man to be 50 mg/kg, or 3,000 mg of castor oil per day in an average 60 kg person. As ricinoleic acid constitutes approximately 90% of castor oil, applying this calculation to the 3,000 mg/day estimated ADI in humans for castor oil (given the rapid hydrolysis of castor oil glyceride in the gastrointestinal tract), the acceptable daily intake of ricinoleic acid may be as high as 2,400 mg/person.

Poly(sebacic acid-co-ricinoleic acid) biodegradable carrier for paclitaxel: in vitro release and in vivo toxicity.

Polyesteranhydrides synthesized by the transesterification of ricinoleic acid and sebacic acid followed by anhydride polymerization were examined as potential controlled delivery carrier for paclitaxel. Solid and liquid polymers were used. Polymers containing 30% ricinoleic acid are solid whereas polymers containing 70% ricinoleic acid are liquid at body temperature and semisolid at room temperature. It was found that upon addition of the liquid polymer to water it solidifies to form a stable semisolid. Paclitaxel, a potent antitumor agent, was incorporated in the polymers (5-20% w/w) and its release in buffer solution was monitored. Paclitaxel was released for over 100 days while the polymer carrier was being degraded. The release rate was affected by the paclitaxel content; the higher the content, the slower was the release. The toxicity of the polymers and formulations with paclitaxel was examined by subcutaneous injection of liquid polymer samples or implantation of solid polymer specimens to mice for different time periods. Histopathological examination of the tissue surrounding the implant showed minor inflammation 1 week after the injection and no inflammation 3 weeks after implantation. Injection of the polymer without paclitaxel showed no adverse effects.

Overview of the preparation, use and biological studies on polyglycerol polyricinoleate (PGPR).

The esterification of condensed castor oil fatty acids with polyglycerol gives a powerful water-in-oil emulsifier which is used by the food industry in tin-greasing emulsions and as an emulsifier with lecithin in chocolate couverture and block chocolate. A safety evaluation programme was undertaken in the late 1950s and early 1960s to determine whether this food emulsifier polyglycerol polyricinoleate (PGPR). (Quest International trade name ADMUL WOL) presented any health implications for consumers. This programme included acute toxicity tests, subacute rat and chicken toxicity studies, a rat chronic toxicity/multigeneration reproduction study, rodent metabolism, carcinogenicity testing in rat and mouse and a human clinical evaluation. PGPR was found to be 98% digested by rats and utilized as a source of energy superior to starch and nearly equivalent to groundnut oil. There was no interference with normal fat metabolism in rats or in the utilization of fat-soluble vitamins. Despite the intimate relationship with fat metabolism, no evidence was found of any adverse effects on such vital processes as growth, reproduction and maintenance of tissue homeostasis. PGPR was not carcinogenic in either 2-year rat or 80-week mouse feeding studies. The human studies showed no adverse effects on tolerance, liver and kidney function, and fat balance at levels up to 10 g/day PGPR. The acceptable daily intake for PGPR which was set by JECFA in 1974 and the EC/SCF in 1979 is 7.5 mg/kg body weight/day. The UK FAC in 1992 estimated that the maximum per capita mean daily intake of PGPR is 2.64 mg/kg body weight/day. It can be concluded that the use of ADMUL WOL brand of PGPR in tin-greasing emulsions or in chocolate couverture does not constitute a human health hazard.

A three-generation reproduction study on polyglycerol polyricinoleate (PGPR) in Wistar rats.

A series of toxicology studies were conducted in the 1950s and 1960s to investigate the toxicity of ADMUL WOL, a brand of polyglycerol polyricinoleate (PGPR). Included as part of these investigations was a three-generation reproduction study in rats. The control rats received a commercial pelletted stock diet and the treated rats were given the same diet ground with 1.5% (w/w) PGPR. A continuous breeding protocol was adopted, in which the breeding pairs were maintained until the female had produced five litters or when it became evident that breeding had ceased. The main focus of the study design was to observe any effect on breeding. The parameters measured in each of the three generations included number of litters per dam, average litter size, average weaning weights of males and females, litters per group showing 100% survival and total survival (%) at day 21. Growth was normal throughout the three generations and there were no deaths or clinical signs associated with the consumption of PGPR. The only significant change in breeding performance was a reduction in the percentage of animals weaned in the second generation, but as this occurred in the control group to a similar extent it was concluded that this was due to an unknown environmental factor and was not treatment related. A histological examination of selected tissues from those rats continued for 1 year failed to show any lesions which could be ascribed to the consumption of PGPR. In conclusion, rats fed 1.5% (w/w) PGPR showed no evidence of a cumulative effect on breeding performance over three generations.

Assessment of the carcinogenic potential of polyglycerol polyricinoleate (PGPR) in rats and mice.

The carcinogenic potential of the food emulsifier ADMUL WOL brand of polyglycerol polyricinoleate (PGPR) was evaluated in rats and mice. Groups of 60 male and 60 female rats were given purified diets containing 5% of either PGPR or groundnut oil for 2 years. Groups of 25 male and 25 female mice were given purified diets containing 5% of either PGPR or groundnut oil for 80 weeks. No carcinogenic effect of PGPR was observed. In addition, dietary PGPR had no adverse effect on growth, food consumption, longevity and haematology. Organ weight analysis revealed an increase in liver and kidney weight in both male and female rats and female mice. Histological analysis of tissues revealed no treatment related adverse effects.

Ultrastructural changes in the ovary cells of engorged Rhipicephalus sanguineus female ticks treated with esters of ricinoleic acid from castor oil (Ricinus communis).

Rhipicephalus sanguineus is a widely distributed tick species that has adapted to the urban environment, and the dog is its main host. This species is also known as a vector and reservoir of diseases caused by bacteria, protozoa, and viruses. Currently, acaricides of synthetic chemical origin have been widely and indiscriminately used, leading to the development of resistance to these products by ticks and causing damage to the environment. Thus, these issues have made it necessary to seek other forms of controlling these ectoparasites. R. sanguineus was artificially infested in host New Zealand White rabbits, which were divided into four treatment groups: control (CG1 and CG2) and treatment (TG1 and TG2) groups. TG1 and TG2 hosts were provided with feed supplemented with esters of ricinoleic acid from castor oil at a concentration of 5 g/kg of feed for 7 and 15 days. Afterward, the ovaries of the female ticks were removed for analysis by transmission electron microscopy. The results showed ultrastructural changes in the somatic and germ cells of ovaries from TG1 and TG2 females, particularly with respect to chorion deposition, a protective membrane of the oocyte, as well as in the transport process of vitellogenic materials via the hemolymph and pedicel cells. Moreover, the mitochondria were less electron-dense and had cristae that were more disorganized than the mitochondria from CG1 and CG2 individuals. Thus, this study demonstrated the action of esters on the ovaries of R. sanguineus, signaling the prospect of a way to control this ectoparasite without affecting nontarget organisms or the environment.

Biocompatibility and safety evaluation of a ricinoleic acid-based poly(ester-anhydride) copolymer after implantation in rats.

The aim of this study was to evaluate the safety and tissue compatibility of an injectable biodegradable poly(ester-anhydride) copolymer of ricinoleic acid (RA) and sebacic acid (SA) in rats. The absorbable biomaterial containing 70% w/w of RA and 30% w/w of SA [P(SA-RA) 3:7] was implanted in rats in two separate studies: (1) at high doses subcutaneously (SC) and intramuscularly (IM) simultaneously into the same animal and (2) intracranially (IC). The safety was established in the high-dose administration experiment. No systemic tissue damage, polymer-related lesions, or abnormalities could be detected in the animals. The histopathological evaluation of the SC and IM P(SA-RA) 3:7 implanted sites suggested a typical foreign body reaction (FBR) to biomaterials, and was characterized by excellent tissue repair and good tissue tolerance. In the second experiment, no neurological deficits or behavior changes suggestive of systemic or localized toxicity were observed in the animals implanted IC with the polymer. Only minimal, well-demarcated inflammatory response was observed on days 14 and 21 and consisted of glia cells. No abnormalities were noted in the brain tissue parenchyma located further from the edges of the implant. These results demonstrated that the P(SA-RA) 3:7 copolymer was tolerated well by the animals and compatible with rat subcutaneous, muscle and brain tissues. The biodegradable polymeric system described here could be used as a scaffold for varied applications in localized and sustained delivery of therapeutic agents.

Translocation of indigenous bacteria from the gastrointestinal tract of mice after oral ricinoleic acid treatment.

A single dose of ricinoleic acid, the active component of castor oil, administered intragastrically to specific pathogen-free mice produced significant alterations in the proximal small intestinal mucosa. Two hours after drug administration, the duodenal villi were markedly shortened with massive exfoliation of columnar and goblet cells. This disruption of the mucosal barrier resulted in continuity between the intestinal lumen and the lamina propria of the villi. Because of the loss of the mucosal barrier, bacteria of the indigenous gastrointestinal flora translocated from the gastrointestinal lumen to the mesenteric lymph nodes, spleen, and liver. The peak incidence of bacterial translocation occurred 4 days after the ricinoleic acid treatment. Strictly anaerobic bacteria, which normally colonize the gastrointestinal tract at greater levels than aerobic or facultatively anaerobic bacteria, were translocated at a greater incidence to the mesenteric lymph nodes than were the other indigenous bacteria. The mucosa began regenerating within 4 h after the ricinoleic acid treatment and viable translocated bacteria were no longer cultured from the mesenteric lymph nodes by 7 days after treatment.

Ricinoleic acid-based biopolymers.

Polyanhydrides synthesized from pure ricinoleic acid half-esters with maleic and succinic anhydrides possess desired physicochemical and mechanical properties for use as drug carriers. Ricinoleic acid maleate or succinate diacid half-esters were prepared from the reaction of crude ricinoleic acid (85% content) with succinic or maleic anhydride. The pure diacid monomers were obtained by chromatography purification through silica gel using petroleum ether/ethyl acetate/acetic acid (80/30/1 v/v/v) mixture as eluent. The pure diacid monomers (>99%) were polymerized by melt condensation to yield film-forming polymers with molecular weights exceeding 40,000 with a polydispersity of 2. Extensive biocompatibility study demonstrated their toxicological inertness and biodegradability. Their rate of elimination from rats in the course of about 4-6 weeks was faster than that found for similar fatty acid-based polyanhydrides previously tested. In vitro studies showed that these polymers underwent rapid hydrolytic degradation in 10 days. Methotrexate release from the polymers was not affected by the initial polymer molecular weight in the range of 10,000-35,000. The in vitro drug release correlated with the degradation of the polymers. The fatty acid ester monomers were further degraded to its counterparts, ricinoleic acid and succinic or maleic acid.

Perfusion of rabbit colon with ricinoleic acid: dose-related mucosal injury, fluid secretion, and increased permeability.

Morphology of the rabbit colon was examined by light and scanning electron microscopy after perfusion of the organ with 2.5, 5.0, 7.5, and 10 mM sodium ricinoleate. Colons perfused with control buffer showed the expected normal appearances, whereas ricinoleate produced desquamation of surface epithelial cells. Surface changes in the colon were comparable with those reported after similar treatment of the rabbit ileum. Concomitant with these histological changes was loss of DNA into the lumen of the colon. Dose-related changes in net fluid transport and mucosal permeability (as assessed by lumen to plasma flux of low molecular weight polyethylene glycols and plasma to lumen flux of urea and creatinine) were also associated with ricinoleate perfusion. These structural and functional alterations may contribute to intraluminal accumulation of fluid and catharsis that can result from administration of ricinoleic acid (castor oil). The findings might also pertain to the pathophysiology of steatorrheal diseases, because dietary fatty acids of similar chemical structure are known to have comparable effects on the intestinal mucosa.

Surfactants selectively ablate enteric neurons of the rat jejunum.

Surfactants, a group of nonspecific membrane perturbating substances, can cause nerve damage. Various concentrations of the cationic surfactants benzalkonium chloride (BAC) and benzethonium chloride, the anionic surfactants sodium ricinoleate, dioctyl sodium sulfosuccinate and sodium lauryl sulfate and the nonionic surfactant Triton X-100 were applied to the serosal surface of the rat jejunum every 5 min for 0.5 hr and then rinsed off with saline. Thirty days after surfactant application, the treated and an untreated segment of jejunum were removed and examined histologically. All surfactants which were tested significantly reduced the number of ganglion cells in the myenteric plexus. In addition, sodium ricinoleate significantly reduced the number of ganglion cells in the submucosal plexus. Higher concentrations of the cationic agents BAC and benzethonium chloride caused a generalized tissue damage including disruption of the smooth muscle, lymphocytic infiltration, intestinal perforation and death. Using BAC as a prototype surfactant, peptidergic neuron distribution and gut electrical activity were examined. BAC treatment markedly reduced the immunoreactivity of somatostatin, substance P, met-enkephalin and vasoactive intestinal peptide in the myenteric plexus. In addition, the electric properties of the smooth muscle were altered. BAC treatment resulted in an erratic, markedly distorted basic electric rhythm and an alteration in spike potential generation. These studies demonstrate that surfactants in appropriate concentrations selectively ablate the myenteric neurons and alter peptidergic neuron distribution and gut electrical parameters in the rat jejunum.