Oral administration of oxalate-enriched spinach extract as an improved methodology for the induction of dietary hyperoxaluric nephrocalcinosis in experimental rats.

Experimental induction of hyperoxaluria by ethylene glycol (EG) administration is disapproved as it causes metabolic acidosis while the oral administration of chemically synthesized potassium oxalate (KOx) diet does not mimic our natural system. Since existing models comprise limitations, this study is aimed to develop an improved model for the induction of dietary hyperoxaluria, and nephrocalcinosis in experimental rats by administration of naturally available oxalate rich diet. Male albino Wistar rats were divided into five groups. Group I, control; group II rats received 0.75% EG, group III rats fed with 5% KOx diet and group IV and V rats were administered with spinach extract of 250 and 500 mg soluble oxalate/day respectively, for 28 d. Urine and serum biochemistry were analyzed. After the experimental period, rats were sacrificed, liver and kidney tissue homogenates were used for antioxidant and lipid peroxidation assay. Relative change in expression of kidney injury molecule-1 (KIM-1) and crystal modulators genes in kidney tissues were evaluated. Tissue damage was assessed by histology studies of liver and kidney. Experimental group rats developed hyperoxaluria and crystalluria. Urine parameters, serum biochemistry, antioxidant profile, lipid peroxidation levels and gene expression analysis of experimental group II and III rats reflected acute kidney damage compared to group V rats. Histopathology results showed moderate hyperplasia in liver and severe interstitial inflammation in kidneys of group II and III than group V rats. Ingestion of naturally available oxalate enriched spinach extract successfully induced dietary hyperoxaluria and nephrocalcinosis in rats with minimal kidney damage.

Histamine, histamine intoxication and intolerance.

Excessive accumulation of histamine in the body leads to miscellaneous symptoms mediated by its bond to corresponding receptors (H1-H4). Increased concentration of histamine in blood can occur in healthy individuals after ingestion of foods with high contents of histamine, leading to histamine intoxication. In individuals with histamine intolerance (HIT) ingestion of food with normal contents of histamine causes histamine-mediated symptoms. HIT is a pathological process, in which the enzymatic activity of histamine-degrading enzymes is decreased or inhibited and they are insufficient to inactivate histamine from food and to prevent its passage to blood-stream. Diagnosis of HIT is difficult. Multi-faced, non-specific clinical symptoms provoked by certain kinds of foods, beverages and drugs are often attributed to different diseases, such as allergy and food intolerance, mastocytosis, psychosomatic diseases, anorexia nervosa or adverse drug reactions. Correct diagnosis of HIT followed by therapy based on histamine-free diet and supplementation of diamine oxidase can improve patient's quality of life.

Selenium and Iodine in Autoimmune Thyroiditis.

Selenium and iodine are essential for thyroid hormone synthesis and function. Selenium, in form of selenocysteine, is found either in the catalytic center of enzymes involved in the protection of the thyroid gland from free radicals originating during thyroid hormone synthesis, and in three different iodothyronine deiodinases catalyzing the activation and the inactivation of thyroid hormones. Iodine is an essential constituent of thyroid hormones and its deficiency causes different disorders that include goiter, hypothyroidism, reduced fertility and alteration in growth, physical and neurological development. These two micronutrients could be involved in the pathogenesis of autoimmune thyroid diseases, a spectrum of pathological conditions including Hashimoto's thryoiditis, post-partum thyroiditis, the so-called painless thyroiditis, Graves' disease and Graves' ophtalmopathy. Aim of this paper is to review the role played by selenium and iodine in autoimmune thyroiditis.

Heliotropium europaeum poisoning in cattle and analysis of its pyrrolizidine alkaloid profile.

Pyrrolizidine alkaloids (PAs) are carcinogenic and genotoxic phytochemicals found exclusively in angiosperms. The ingestion of PA-containing plants often results in acute and chronic toxicities in man and livestock, targeting mainly the liver. During February 2014, a herd of 15-18-month-old mixed-breed beef cattle (n = 73) from the Galilee region in Israel was accidently fed hay contaminated with 12% Heliotropium europaeum (average total PA intake was 33 mg PA/kg body weight/d). After 42 d of feed ingestion, sudden death occurred over a time period of 63 d with a mortality rate of 33%. Necropsy and histopathological examination revealed fibrotic livers and moderate ascites, as well as various degrees of hyperplasia and fibrosis of bile duct epithelial cells. Elevated γ-glutamyl-transferase and alkaline phosphatase levels were indicative of severe liver damage. Comprehensive PA profile determination of the contaminated hay and of native H. europaeum by LC-MS/MS revealed the presence of 30 PAs and PA-N-oxides, including several newly reported PAs and PA-N-oxides of the rinderine and heliosupine class. Heliotrine- and lasiocarpine-type PAs constituted 80% and 18% of the total PAs, respectively, with the N-oxides being the most abundant form (92%). The PA profile of the contaminated hay showed very strong resemblance to that of H. europaeum.

Effect of the combined probiotics with aflatoxin B1-degrading enzyme on aflatoxin detoxification, broiler production performance and hepatic enzyme gene expression.

In order to degrade aflatoxin B1 (AFB1), AFB1-degrading microbes (probiotics) such as Lactobacillus casei, Bacillus subtilis and Pichia anomala, and the AFB1-degrading enzyme from Aspergillus oryzae were selected and combined to make feed additive. Seventy-five 43-day-old male Arbor Acres broilers were randomly divided into 5 groups, 15 broilers for each group. The broilers were given with 5 kinds of diets such as the basal diet, 400 µg/kg AFB1 supplement without feed additive, and 200, 400, 800 µg/kg AFB1 supplement with 0.15% feed additive. The feeding experimental period was 30 d, which was used to determine production performance of broilers. In addition, serum, liver and chest muscle were selected for measuring AFB1 residues, gene expressions, microscopic and antioxidant analyses. The results showed that adding 0.15% feed additive in broiler diets could significantly relieve the negative effect of AFB1 on chicken's production performance and nutrient metabolic rates (P<0.05). It could also improve AFB1 metabolism, hepatic cell structure, antioxidant activity, and many hepatic enzyme gene expressions involved in oxidoreductase, apoptosis, cell growth, immune system and metabolic process (P<0.05). It could be concluded that the feed additive was able to degrade AFB1 and improve animal production.

Aquilegia vulgaris L. extract counteracts oxidative stress and cytotoxicity of fumonisin in rats.

Exposure to fumonisins (FB) is known to have toxic and carcinogenic effects in different animal species, and to express toxicity in cells via the induction of oxidative stress. The aim of the current study was to evaluate the protective effects of the ethanol extract of Aquilegia vulgaris L. against the oxidative stress and the genotoxicity using micronucleus assay and random amplified polymorphism DNA (RAPD-PCR) in FB-treated rats. Sixty mature female Sprague-Dawley were divided into six treatment groups and treated for 4 weeks as follow: the control group, the group fed FB-contaminated diet (200 mg/kg diet), the groups treated orally with the extract (5 and 10 mg/kg bw) and the groups fed FB-contaminated diet and treated with the extract at the two doses. The results showed that treatment with FB alone disturbed lipid profile in serum, increases Sa/So ratio, induces micronucleated polychromatic erythrocytes (Mn-PCEs) in bone marrow, increases DNA and RNA in liver accompanied with significant changes in histological picture The extract alone at the two tested doses did not induce any significant changes in the biochemical or histological picture. The combined treatment showed significant improvements in all biochemical, cytogenetic parameters tested and histological pictures in the liver tissues. Moreover, this improvement was more pronounced in the group received the high dose of the extract. It could be concluded that the ethanol extract of A. vulgaris induced its protective effect via the increase in the antioxidant capacity, inhibition of lipid peroxidation and scavenging of free radicals.

Generation of quinoneimine intermediates in the bioactivation of 3-(N-phenylamino)alanine (PAA) by human liver microsomes: a potential link between eosinophilia-myalgia syndrome and toxic oil syndrome.

Eosinophilia-myalgia syndrome (EMS) was an intoxication episode that occurred in the US in 1989 and affected 1,500 people. EMS was associated with the ingestion of manufactured L-tryptophan, and 3-(N-phenylamino)alanine (PAA) was identified as one of the contaminants present in the L-tryptophan batches responsible for intoxication. In previous studies (Martínez-Cabot et al., Chem Res. Toxicol., in press), we have shown that the incubation of 3-(N-phenylamino)propane-1,2-diol (PAP), a toxic biomarker of the oil batches that caused Toxic Oil Syndrome in Spain, with human liver microsomes generates a reactive quinoneimine intermediate. The structural similarity between PAA and PAP led Mayeno and co-workers (Mayeno et al. (1995) Chem. Res. Toxicol. 8, 911-916) to hypothesize that both xenobiotics could be linked to a common etiologic agent. We thus set about to study the bioactivation of PAA by human liver microsomes. Under these conditions, PAA is converted to its 4'-hydroxy derivative, an unstable intermediate that is rapidly transformed into the final metabolites 4-aminophenol and formylglycine, which were identified in the incubations by GC/MS using the H2(18)O-labeled medium. We also provide evidence that 4-aminophenol and formylglycine are formed from a quinoneimine intermediate via a pathway similar to that demonstrated for PAP bioactivation. This quinoneimine, in the absence of nucleophiles in the incubation medium, could isomerize to give the corresponding imine, which could undergo hydrolysis to yield the aforementioned final products. These findings establish that EMS and TOS are linked by a common toxic metabolite (4-aminophenol) and that they may be further linked by the concomitant release of potentially hazardous carbonyl species.

In vitro bioactivation of 3-(N-phenylamino)propane-1,2-diol by human and rat liver microsomes and recombinant P450 enzymes. Implications for toxic oil syndrome.

Toxic oil syndrome (TOS) was a massive food-borne intoxication that occurred in Spain in 1981. Epidemiological studies imputed 3-( N-phenylamino)propane-1,2-diol (PAP) derivatives as the toxic agents. The in vitro bioactivation of PAP by rat and human liver microsomes was studied. In both cases, 3-[ N-(4'-hydroxyphenyl)amino]propane-1,2-diol ( 1) was detected as the main metabolite. Inhibition studies with pooled human liver microsomes in the presence and absence of P450-specific inhibitors suggest that 2C8 and 2E1 are the main enzymes involved in PAP bioactivation, followed by 3A4/5, 1A1/2, and 2C9. Incubations of PAP with 10 different recombinant P450 enzymes showed that 2C8, 2C9, 2C18, 2D6, and 2E1 catalyzed PAP 4'-hydroxylation. Incubations of phenol 1 with rat and human liver microsomes in the presence of GSH resulted in the formation of a glutathione conjugate of a quinoneimine metabolite derived from 1. In rat liver microsomes, P450 enzymes play a key role in the bioactivation of 1, whereas in human liver microsomes, autoxidation appears to be the major mechanism. The implications of these results for toxic oil syndrome are discussed.

[Mercury and the risk of coronary heart disease]. / Rtéc a choroba niedokrwienna serca.

Results from recently published studies have shown an independent association between the mercury concentration in the human body and the risk of coronary heart disease. There are two major sources of exposure to mercury of the general population: fish consumption and dental amalgamats. Mercury may promote atheriosclerosis and hence it may increase the risk of acute coronary events in several ways. Mercury stimulates the production of free radicals, binds to sulfhydryl groups of enzymes and forms an insoluble complex with selenium. Thus mercury may induce lipid peroxydation and increase oxidized low-density lipoprotein concentration in blood.

Synthesis and stability studies of the glutathione and N-acetylcysteine adducts of an iminoquinone reactive intermediate generated in the biotransformation of 3-(N-phenylamino)propane-1,2-diol: implications for toxic oil syndrome.

Epidemiological studies have pointed to fatty acid mono- and diesters of 3-(N-phenylamino)propane-1,2-diol (PAP) as the biomarkers of the toxic oil batches that caused toxic oil syndrome (TOS), an intoxication episode that occurred in Spain in 1981, causing over 400 deaths and affecting more than 20000 people. The biotransformation of PAP administered intraperitoneally to two mouse strains produced potentially toxic metabolites. The identification of 3-(4'-hydroxyphenylamino)propane-1,2-diol among those metabolites was important because the compound can generate the quinoneimine intermediate 2. The potential toxicity of quinoneimines has been attributed primarily to their electrophilic character. Accordingly, the reactions of 2 with N-acetylcysteine, N-acetylcysteine methyl ester, and GSH were investigated. Quinoneimine 2 reacts with the N-acetylcysteine methyl ester to give the expected conjugate as a major product, accompanied by the corresponding bis and tris adducts. The monoadduct, when isolated in pure form, undergoes spontaneous oxidation to generate a new quinoneimine intermediate, which in turn rearranges and undergoes hydrolysis to afford the thiol adduct formally derived from the quinoneimine generated from p-aminophenol. The same overall pathway was observed for the reaction of 2 with N-acetylcysteine and GSH. Both thiol reagents reacted with the quinoneimine to give the corresponding adducts in which the addition took place at the ortho position with respect to the amino group. These conjugates were also unstable and ultimately afforded the corresponding adduct derived from p-aminophenol. The relevancy of these results to TOS, as well as their potential generalization for quinoneimines derived from other xenobiotics, is discussed herein.

Metabolism of R,S enantiomers of 3-phenylamino-1,2-propanediol, a compound associated with the toxic oil syndrome, in C57BL/6- and A/J-strain mice.

PAP, a very polar substance, is highly metabolized in mice and excreted principally in urine in the form of the 2-hydroxy-3-phenylaminopropanoic acid of each enantiomer. Thus, the major route of PAP elimination in these strains is alkyl chain oxidation. In particular, S-PAP is eliminated principally in the form of that metabolite, whereas R-PAP enantiomer showed further oxidized species at the aromatic ring and alkyl chain, yielding potential decarboxylated compounds and iminoquinones. All these metabolites may have toxicologic implications. On the other hand, OOPAP intestinal hydrolysis in favour of one PAP enantiomer might be expected since lipases show chiral hydrolysis (unpublished data, manuscript in preparation). In this respect, enantiomeric distribution and metabolic differences should be taken into account in the toxicokinetics of these compounds and their potential association with Toxic Oil Syndrome symptoms.

Fatty acid anilides: in vivo formation and relevance to toxic oil syndrome.

Toxic oil syndrome (TOS), a multisystemic epidemic outbreak in 1981 in Spain, was caused by the ingestion of a cooking oil mixture containing rapeseed oil denatured with aniline. The mechanisms and causative agents responsible for the TOS are still not known. Although primary lesions observed in TOS patients could not be reproduced experimentally, the levels of fatty acid anilides (FAAs) and aniline in TOS-related cooking oil were considered proximate markers of TOS. Aniline, available from free aniline and FAAs ingested with TOS-related cooking oil, and its reconjugation with endogenous fatty acids could be an early event leading to TOS. Therefore, the present study was undertaken to determine the formation of FAAs following an oral dose of 2 mmol/kg aniline hydrochloride (AH) via gavage in rats. Here, 16:0, 18:0, 18:1, 18:2, 18:3, and 20:4 FAAs were analyzed in the whole blood, brown fat, liver, and pancreas at 0 (control), 0.25, 0.5, 1, 3, 6, 12, 24, and 48 hours. Generally, 16:0 and 18:1 FAAs were detected in the whole blood, brown fat, and liver of AH-treated rats with highest mean levels at 0.25 or 0.5 hour, except 3 hours for the whole blood. Only 16:0 FAA was detectable in the pancreas of AH-treated animals. The 18:0 FAA was also detected frequently in the liver while other FAAs were either in trace amounts or not detectable in the tissues analyzed in the present study. Overall, highest formation of the 16:0 FAA was found in the liver followed by pancreas and of 18:1 FAA in the whole blood and brown fat. These results indicate a rapid formation and further metabolism and disposition of FAAs in rat model and support our previous findings that 18:1 and 16:0 fatty acids are better substrates for the conjugation with aniline. Surprisingly, a small or trace amount of a few FAAs also detected in the tissues of control rats indicates their endogenous biosynthesis and/or presence. Results of 18:1 fatty acid incubation and aniline in the presence of fatty acid ethyl ester synthase, purified to homogeneity from rat liver microsome, suggest that formation of FAAs is catalyzed by an enzyme involved in the conjugation of fatty acids with xenobiotic alcohols. Because the FAAs are known to exert a wide range of toxicity in experimental animals and primary cell cultures, in vivo formation of FAAs could be an early event leading to TOS.

Biotransformation of 3-(phenylamino)-1,2-propanediol to 3-(phenylamino)alanine: a chemical link between toxic oil syndrome and eosinophilia-myalgia syndrome.

During late 1989, the eosinophilia-myalgia syndrome (EMS) developed as an epidemic in the United States, with numerous additional cases reported in several other countries worldwide. Eight years earlier, a closely-related disease, the toxic oil syndrome (TOS), occurred in Spain as a massive food-borne epidemic. Although EMS was linked to the ingestion of tainted L-tryptophan, and TOS to aniline-denatured rapeseed oil, the etiologic agent(s) responsible for both diseases remains undetermined. Contaminants in these foodstuffs are believed to have triggered the diseases. Aniline contaminants, including 3-(phenylamino)-1,2-propanediol (PAP), have been reported in oil used by patients who developed TOS. A related aniline derivative, 3-(phenylamino)-L-alanine (PAA), was recently isolated from L-tryptophan associated with the onset of EMS. Here, we demonstrate the biotransformation of PAP into PAA by both rat hepatocytes and human liver tissue. The structural characterization of PAA was unequivocally determined using on-line HPLC coupled with atmospheric pressure chemical ionization tandem mass spectrometry (LC-APCI-MS/MS). This finding is the first reported chemical link between TOS and EMS and suggests that these two related diseases share a common etiology, namely, PAA.

[Protective effect of selenium in acute T-2 mycotoxicosis]. / Zashchitnoe deistvie selena pri ostrom T-2-mikotoksikoze.

Male Wistar rats were fed diets supplemented with selenium (0.5 and 2.5 mg/kg) for 6 weeks. When T-2 toxin was administered at 3.8 mg/kg body weight; sings of intoxication were less distinct in rats fed selenium-supplemented diets. Mortality caused by T-2 toxin was two times lower if selenium content amounted to 2.5 mg/kg. The level of cytochrome P-450 and the rate of deethylation of 7-ethoxycoumarin were considerably decreased in the liver of rats that received T-2 toxin and fed a diet with high selenium content. At the same time the activity of epoxide hydrolase and UDP-glucuronosyltransferase was significantly higher (1.6 and 1.7 times more) as compared with unsupplemented group. The results obtained allow to assume that there is a relationship between the decrease of T-2 toxin toxicity and the change in its metabolism in rats fed selenium-supplemented diets.