Occurrence of ochratoxin A in herbal drugs of Indian origin — a report

This paper contains a report of occurrence of ochratoxin A in some common herbal medicines collected from different store-houses and shop-keepers of Bihar, India. Of 129 samples of 9 plants, 55 were found to be contaminated with various levels of ochratoxin A. The level of ochratoxin A was found maximal in barks ofHolarrhena antidysenterica (1.14 – 2.34 μg/g) whereas it was minimal in rhizomes ofTacca aspera (0.3 – 0.74 μg/g).Aspergillus ochraceus, A sulphureus and Penicillium viridicatum isolates obtained from drug samples were also examined for their toxigenic potentials. 19 isolates ofA ochraceus, 13 ofA sulphureus and 37 isolates ofP viridicatum were found to be toxigenic out of 67, 33, and 107 isolates, respectively. The ochratoxin A produced by Aochraceus was in the range of 0.09 to 2.44 μg/mL, byA sulphureus 0.1 to 1.76 μg/mL, and byP viridicatum 0.14 to 2.78 μg/mL of the culture filtrate.     

Toxicity of ochratoxin A in aBrevibacillus brevis — Growth inhibition assay

Ochratoxin A (OTA) is a nephrotoxic, carcinogenic and immunosuppressive mycotoxin. It can be detoxified by various microorganisms, e.g. different yeast strains, via metabolisation into ochratoxin α (OTα). Within this study a growth inhibition assay was developed to compare the toxicity of OTA and its degradation product OTα. As an indicator organismBrevibacillus brevis was used. The assay was performed in microtiterplates. Growth inhibition was determined by comparing the optical density values ofBrevibacillus brevis cultures grown in medium supplemented with OTA/OTα and OTA/OTα-free medium, respectively. It could be shown thatB. brevis is sensitive to OTA (EC₁₀₀=0.5 mg/L±0.03 mg/L), which is not the case for its metabolite OTα. Therefore this bioassay is a useful tool to show the detoxification of OTA to OTα by microbial degradation. Presented at the 25^th Mykotoxin Workshop in Giessen, Germany, May 19–21, 2003     

Is there an influence of enzyme polymorphisms on ochratoxin A genotoxicity?

Primary cultured human urothelial cells derived from ureter specimens of urological patients were used to evaluate induction of DNA-damage by OTA in the alkaline single-cell gel electrophoresis (comet) assay. With the cultured cells from each donor a separate comet assay was performed and tail length of the damaged DNA was measured. A broad spectrum of effects was detected between the individual cell cultures with effects reaching from tail lengths on control level up to strongly enhanced tail lengths.All donors of urothelial tissue were additionally genotyped for several xenobiotic metabolising enzymes (cytochrome P450 1A2, glutathione S-transferases T1, M1, and P1, N-acetyltransferase 2) in lymphocyte DNA. The genotype was then correlated with the genotoxic effects obtained in the comet assay.No correlation was found with CYP1A2, GSTT1, and GSTM1 genotypes whereas for GSTP1 stronger genotoxic effects were found in cells from donors with hetero-and homozygously mutated (w/m, m/m) genotypes compared to homozygous wildtypes. The strongest hint for a correlation was found for NAT2, as cells from donors with homozygous mutated alleles (m/m), known as slow acetylators, displayed a higher susceptibility to OTA in the comet assay than cells from donors with the heterozygously mutated or wildtype alleles (rapid acetylators).     

Vorkommen von ochratoxin A und B in gewürzen

A total of 681 samples of spices, which comprised more than 50 different spice commodities were analysed for the natural occurrence of the mycotoxins ochratoxin A (OTA) and ochratoxin B (OTB). The analytical method involved chloroform extraction, clean-up by immunoaffinity column and HPLC determination of both mycotoxins. OTA and OTB were detected in 143 (21%) and 68 (10%) of the samples, respectively. The highest frequency of occurrence of both mycotoxins detected were in chili (100% for OTA and 55% for OTB), paprika (41% and 15%, respectively) and pepper (23% and 44%, respectively). The toxin concentrations ranged between the detection limit (0.01 ng/g) and 41.8 ng OTA (2.7 ng OTB)/g of chili, 18.9 ng OTA (1.4 ng OTB)/g of paprika and 3.8 ng OTA (4.6 ng OTB)/g of pepper. One sample of a extract of vanilla was found to be positive for OTB at 15 ng/g. However, median values of most samples showed to be below the detection limit. Comparison of the geographical origin of the samples showed that the predominant number of contaminated spices was from Southeast-Asia and India. Highly contaminated paprika samples were found to come from Israel.     

Occurrence of ochratoxin A in spices commercialized in Abidjan (Côte d’Ivoire)

Ochratoxin A (OTA) is a mycotoxin produced mostly by several species of Aspergillus and Penicillium. OTA is nephrotoxic in all animal species in which it has been tested and is cancerogenic in rodents. It is associated with Balkan endemic nephropathy. It is naturally present in many crop products such as cereals (barley, wheat, maize) and dried fruits, spices, coffee, wine, olives, and cocoa. The aim of this study was to assess the contamination of three Ivoirian spices with OTA (ginger, chili, and pepper) widely consumed by the population. A total of 90 spice samples (ginger: n?=?30; chili: n?=?30; pepper n?=?30) was taken from various sales outlets of Abidjan. OTA was quantified using an HPLC apparatus coupled with a fluorimetric detector. The chili and ginger samples were contaminated with OTA at a mean concentration of 57.48?±?174 and 0.12?±?0.15 μg/kg, respectively. No contamination of the pepper samples was detected. Eight (26.67 %) of the chili samples exceeded the maximum limit of 15 μg/kg established by European regulation. These results should serve as an alert on the risk to the consumer population of these products that are highly contaminated with OTA.     

Occurrence of the toxigenic fungi (producers of aflatoxins and ochratoxin A) in foodstuffs in the Czech Republic 1999–2000

The occurrence of toxigenic fungi producing aflatoxins and ochratoxin A in foodstuffs was studied in the Czech Republic. Twenty five commodities were collected at twelve collection places in the Czech Republic (300 food samples). The presence of potentially toxigenicAspergillus flavus was observed in 28% of the sampled foods (black pepper, caraway seeds, fruit tea, black tea, oat flakes, fine flour, rolled oat flakes and semolina) in the year 1999, and in 25% of the sampled foods (black pepper, black tea, fine flour) in the year 2000.A tamarii (aflatoxins producer) was found in 3 black pepper samples (25%) in both years. Aflatoxins were detected in black pepper and caraway seed samples in the year 1999 and in sweet red pepper in the year 2000.A parasiticus andA nomius were not isolated. Aspergillus section Nigri (potential producer of ochratoxin A) was detected in some foodstuffs. Ochratoxin A was detected in raisins.Penicillium verrucosum andA ochraceus were not isolated from foodstuffs.     

Determinatıon of ochratoxin A and total aflatoxin levels in corn samples from Turkey by enzyme-linked immunosorbent assay

Forty-seven samples of corn were collected from various street bazaars and market outlets in different regions of Turkey and total aflatoxin (AF) and ochratoxin A (OTA) levels were determined by enzyme-linked immunosorbent assay (ELISA) following sample preparation. Levels of AF and OTA in corn samples ranged between 1.75–120.3 μg/kg and 1.08–8.57 μg/kg, respectively. Although 53% of the samples analysed had no detectable levels of AF, 4% of similar samples were found to contain AFs above the acceptable limit of 10 μg/kg in Turkey. For OTA, 4% of the corn samples had levels above the acceptable limit (3 μg/kg) in Turkey, with over 43% samples not found to contain this mycotoxin. Although the levels of mycotoxins analysed in this study were not found to be high and the percentage of samples contaminated above permitted limits were low, the importance of overall daily dietary intake should not be underestimated and control of these fungal metabolites in corn must be explored to minimise the hazards they may cause in humans.     

In vitro induction of tumor necrosis factor-α by ochratoxin A (OTA) from rat liver: role of Kupffer cells

Tumor necrosis factor-α (TNF-α) is released from blood-free perfused rat liver by the fungal metabolite ochratoxin A. Here we have identified Kupffer cells as the sole source of OTA-mediated cytokine release. If single cell preparation of Kupffer cells, hepatocytes, or sinusoidal endothelial cells were prepared from rat livers, only Kupffer cells released TNF-α upon incubation with 2.5 μmol/l OTA. OTA failed to induce TNF-α release in the blood-free perfused isolated rat liver when Kupffer cells were blockedin vitro by 15 μmol/l gadolinium chloride. When rats were pretreatedin vivo with the Kupffer cell depleting clodronate liposomes, OTA-mediated TNF-α release was abrogated in the isolated perfused liver model.     

Golgi β-glucuronidase of androgen-stimulated mouse kidney

The equilibrium constant of the phosphoglyceromutase reaction was determined over a range of pH (5.4-7.9), in solutions of different ionic strength (0.06-0.3) and in the presence of Mg(2+), at 30 degrees C and at 20 degrees C. The values obtained (8.65-11.65) differ substantially from previously published values. The third acid dissociation constants were redetermined for 2- and 3-phosphoglycerate, and in contrast with previous reports the pK values (7.03 and 6.97 respectively at zero ionic strength) were closely similar. The Mg(2+)-binding constants were measured spectrophotometrically and the values, 286mm(-1) and 255mm(-1) for 2- and 3-phosphoglycerate at pH7 and ionic strength 0.02, were also very similar. From the relative lack of effect of temperature, pH and ionic strength it is concluded that the equilibrium constant differs from unity largely because of entropic factors. At low ionic strength, in the neutral region, the pH-dependence can be attributed to the small difference in the acid dissociation constants, but the difference in dissociation constants does not explain the pH-dependence in the acid region or at high ionic strength. Within physiological ranges of pH, Mg(2+) concentration and ionic strength there will be little variation in equilibrium constant.     

A "two-hit" model of cystogenesis in autosomal dominant polycystic kidney disease?

An intriguing feature of autosomal dominant polycystic kidney disease (ADPKD) is the focal and sporadic nature of individual cyst formation. Typically, only a few renal cysts are detectable in an affected individual during the first two decades of life. By the fifth decade, however, hundreds to thousands of renal cysts can be found in most patients. Additionally, significant intra-familial variability of ADPKD has been well documented. Taken together, these findings suggest that factor(s) in addition to the germline mutation of a polycystic kidney disease gene might be required for individual cyst formation. Indeed, recent studies have provided compelling evidence in support of a "two-hit" model of cystogenesis in ADPKD. In this model, inactivation of both copies of a polycystic kidney disease gene by germline and somatic mutations within an epithelial cell provides growth advantages for it to proliferate clonally into a cyst. This article highlights key findings of these recent studies and discusses the controversies and implications of the "two-hit" model in ADPKD.     

The kidney form of Trypanosoma musculi: A distinct stage in the life cycle?

Trypanosoma musculi is a parasite specific to mice, which resides in the blood and lacks intracellular stages. After immune clearance of the flagellates from the general circulation, mice are resistant to reinfection. Yet, long after parasites are no longer detected in the peripheral blood, they persist in the vasa recta of the kidneys and it has been proposed that this is an immunologically privileged site for T. musculi. This relationship provides a useful model for studies of latent or chronic infections in immune hosts. Here, Fernando Monroy and Donald Dusanic consider the immune responses of mice to T. musculi and compare characteristics of the parasites from the vasa recta (kidney forms, KFs) of mice with latent infections to trypanosomes from the peripheral blood (bloodstream forms, BSFs) of animals during active infections. They consider how KFs evade immune destruction and suggest that these sequestered parasites represent a distinct stage in the life cycle.     

The daily dietary intake of ochratoxin a — Results of a duplicate portion study with Bavarian schoolchildren

The daily dietary intakes of ochratoxin A (OTA) were estimated for 28 school-children at the age of seven and eight years using duplicate portions of the total diets collected on three successive days. For most children and collection days the dietary OTA intakes were below the PTDI-value of 5 ng/kg bw/day. The mean dietary intake was 1.52 ng/kg bw/day for all children. According to sex the mean intakes were 1.21 ng/kg bw/day for boys and 1.83 ng/kg bw/day for girls.     

Mannose 6-phosphate receptor and sortilin mediated endocytosis of α-galactosidase A in kidney endothelial cells

Prominent vasculopathy in Fabry disease patients is caused by excessive intracellular accumulation of globotriaosylceramide (GL-3) throughout the vascular endothelial cells causing progressive cerebrovascular, cardiac and renal impairments. The vascular lesions lead to myocardial ischemia, atherogenesis, stroke, aneurysm, thrombosis, and nephropathy. Hence, injury to the endothelial cells in the kidney is a key mechanism in human glomerular disease and endothelial cell repair is an important therapeutic target. We investigated the mechanism of uptake of α-galactosidase A (α-Gal A) in renal endothelial cells, in order to clarify if the recombinant enzyme is targeted to the lysosomes via the universal mannose 6-phosphate receptor (M6PR) and possibly other receptors. Immunohistochemical localization of infused recombinant α-Gal A in a renal biopsy from a classic Fabry disease patient showed that recombinant protein localize in the endothelial cells of the kidney. Affinity purification studies using α-Gal A resins identified M6PR and sortilin as α-Gal A receptors in cultured glomerular endothelial cells. Immunohistochemical analyses of normal human kidney with anti-sortilin and anti-M6PR showed that sortilin and M6PR were expressed in the endothelium of smaller and larger vessels. Uptake studies in cultured glomerular endothelial cells of α-Gal A labeled with fluorescence and (125)I showed by inhibition with RAP and M6P that sortilin and M6PR mediated uptake of α-Gal A. Biacore studies revealed that α-Gal A binds to human M6PR with very high affinity, but M6PR also binds to sortilin in a way that prevents α-Gal A binding to sortilin. Taken together, our data provide evidence that sortilin is a new α-Gal A receptor expressed in renal endothelial cells and that this receptor together with the M6PR is able to internalize circulating α-Gal A during enzyme replacement therapy in patients with Fabry disease.     

Presence of an α-galactolipid on the cell surfaces of endothelial cells of human kidney

Summary The presence of an -galactolipid was investigated with a peroxidase-labelled lectin fromGriffonia simplicifolia (GSA-I) with specific binding for terminal -d-galactose residues. Normal kidney tissue was obtained from patients undergoing nephrectomy for renal neoplasms. For light microscopy, tissue was snap-frozen; 4 µm-thick sections were briefly fixed in paraformaldehyde and incubated with GSA (0.025 mg ml^–1). The peroxidase activity was developed with 3-amino-9-ethylcarbazole. Adjacent sections were stained at the same time after lipid extraction with 3:1 (v/v) chloroform/methanol. For electron microscopy, 0.2–0.5 mm-thick paraformaldehyde-fixed blocks, with or without lipid extraction, were stained with peroxidase-labelled GSA. The label was developed with diaminobenzidine and osmium tetroxide. Some structures, such as tubular epithelia, stained both in lipid-extracted and non-extracted tissues, suggesting that glycoproteins were most likely involved. In addition, tissue stained immediately after fixation showed GSA reactivity on endothelial cell surfaces of intertubular capillaries and larger vessels. In lipid-extracted tissues, however, tubular epithelium was still positive for GSA but endothelial cells failed to stain. These findings suggest that a glycolipid, bearing a terminal -galactose residue, is present on the endothelial cells in human kidney and possibly on tubular epithelia. Our data may explain the preferential storage of -galactolipid in endothelial cells of patients with Fabry's disease and other biological phenomena such asEscherichia coli adhesion.     

Characteristics of antibody inhibition of rat kidney (Na+−K+)-ATPase

Summary Antibodies which were raised against highly purified membrane-bound (Na⁺–K⁺)-ATPase from the outer medulla of rat kidneys inhibit the (Na⁺–K⁺)-ATPase activity up to 95%. The antibody inhibition is reversible. The time course of enzyme inhibition and reactivation is biphasic in semilogarithmic plots.In the purified membrane-bound (Na⁺–K⁺)-ATPase negative cooperativity was observed (a) for the ATP dependence of the (Na⁺–K⁺)-ATPase activity (n=0.86), (b) for the ATP binding to the enzyme (n=0.58), and (c) for the ouabain inhibition of the (Na⁺–K⁺)-ATPase activity (n=0.77). By measuring the Na⁺ dependence of the (Na⁺–K⁺-ATPase reaction, a positive homotropic cooperativity (n=1.67) was found.As reactivation of the antibody-inhibited enzyme proceeds very slowly (t _0.5=5.2hr), it was possible to measure characteristics of the antibody-(Na⁺–K⁺)-ATPase complex: The antibodies exerted similar effects on the ATP dependence of the (Na⁺–K⁺)-ATPase reaction and on the ATP binding of the enzyme.V _max of the (Na⁺–K⁺)-ATPase reaction and the number of ATP binding sites were reduced whileK _{0.5 ATP} for the (Na⁺–K⁺)-ATPase activity and for the ATP binding were increased by the antibodies. The Hill coefficients for the ATP binding and for the ATP dependence of the enzyme activity were not significantly altered by the antibodies. The antibodies increased theK _0.5 value for the Na⁺ stimulation of the (Na⁺–K⁺)-ATPase activity, but they did not alter the homotropic interactions between the Na⁺-binding sites. The negative cooperativity which was observed for the ouabain inhibition of the (Na⁺–K⁺)-ATPase activity was abolished by the antibodies.The data are tentatively explained by the following model: The antibodies bind to the (Na⁺–K⁺)-ATPase from the inner membrane side, reduce the ATP binding symmetrically at the ATP binding sites and reduce thereby also the (Na⁺–K⁺)-ATPase activity of the enzyme. The antibodies may inhibit the ATP binding by a direct interaction or by means of a conformational change at the ATP binding sites. This may possibly also lead to the alteration of the Na⁺ dependence of the (Na⁺–K⁺)-ATPase activity and to the observed alteration of the dose response to the ouabain inhibition.     

Intracellular transport of mouse kidney β-glucuronidase induced by gonadotrophin

1. The response of renal beta-glucuronidase with time to the injection of gonadotrophin was investigated in each submicrosomal fraction of rough and smooth microsomal fractions of mouse kidney homogenate. 2. The increase in beta-glucuronidase activity appeared initially in membranes of the rough microsomal fraction, 24h after injection. 3. Afterwards the newly synthesized enzyme appeared in the contents of the rough microsomal fraction and was subsequently found in the smooth microsomal fraction, reaching a maximum concentration in this fraction at 72h. 4. At this juncture, a decrease in the enzyme activity was observed in rough microsomal contents whereas the lysosomal fraction had reached its maximum value. 5. The time-course of the appearance of beta-glucuronidase in the submicrosomal fractions after the gonadotrophin stimulation suggests that the newly synthesized enzyme at the site of membrane-bound ribosomes is transferred across the membrane into cisternae of the rough endoplasmic reticulum, and then is transported into lysosomes via the smooth endoplasmic reticulum. 6. The properties of microsomal and lysosomal beta-glucuronidases were compared.     

Progressive induction of β-glucuronidase in individual kidney epithelial cells

The magnitude and kinetics of β-glucuronidase induction in mouse kidney are determined by a cis-acting regulatory gene, Gus-r, that is closely linked to the enzyme structural gene. The accumulation of β-glucuronidase mRNA during induction is much slower than the turnover time of the mRNA, suggesting progressive acquisition of mRNA synthesizing capacity during induction. Counts of the numbers of induced cells present at various times of induction in strains carrying three different alleles of Gus-r show that all potentially responsive cells respond immediately. The level of induction is progressive in individual cells and does not involve continued recruitment of new cells into the induced population. It appears that during induction each chromosome becomes progressively more active in directing the synthesis of β-glucuronidase.