Screening a Strain of Aspergillus niger and Optimization of Fermentation Conditions for Degradation of Aflatoxin B1

Aflatoxin B₁, a type of highly toxic mycotoxin produced by some species belonging to the Aspergillus genus, such as Aspergillus flavus and Aspergillus parasiticus, is widely distributed in feed matrices. Here, coumarin was used as the sole carbon source to screen microorganism strains that were isolated from types of feed ingredients. Only one isolate (ND-1) was able to degrade aflatoxin B₁ after screening. ND-1 isolate, identified as a strain of Aspergillus niger using phylogenetic analysis on the basis of 18S rDNA, could remove 26.3% of aflatoxin B₁ after 48 h of fermentation in nutrient broth (NB). Optimization of fermentation conditions for aflatoxin B₁ degradation by selected Aspergillus niger was also performed. These results showed that 58.2% of aflatoxin B₁ was degraded after 24 h of culture under the optimal fermentation conditions. The aflatoxin B₁ degradation activity of Aspergillus niger supernatant was significantly stronger than cells and cell extracts. Furthermore, effects of temperature, heat treatment, pH, and metal ions on aflatoxin B₁ degradation by the supernatant were examined. Results indicated that aflatoxin B₁ degradation of Aspergillus niger is enzymatic and this process occurs in the extracellular environment.     

Trp266 determines the binding specificity of a porcine aflatoxin B1 aldehyde reductase for aflatoxin B1-dialdehyde

Aflatoxin B₁ (AFB₁) is a severe threat to human and animal health. The aflatoxin B₁ aldehyde reductase (AFAR) family specifically catalyzes AFB₁-dialdehyde, a toxic metabolic intermediate of AFB₁, producing a nontoxic dialcohol. Although several AFARs have been found and characterized, the binding specificity of the family for AFB₁-dialdehyde remains unclear. Herein, according to the published sequence, we cloned a porcine AFAR gene. Recombinant porcine AFAR was expressed and purified from Escherichia coli as hexa-histidine tagged fusion protein. Using the cloned porcine AFAR as a model, site-directed mutagenesis combined with high performance liquid chromatography studies revealed that the substitution of Trp266 with Ala resulted in almost complete loss of catalytic activity for AFB₁-dialdehyde. Interestingly, the substitution of Met86 with Ala exhibited an obviously increased activity to the dialdehyde. Based on these results and by using molecular docking simulations, this work provides a structural explanation for why the AFAR family exhibits high specificity for AFB₁-dialdehyde. The Trp266 residue in porcine AFAR plays a critical role in stabilizing the binding of AFB₁-dialdehyde in the active pocket through the hydrophobic interaction of the side-chain indole ring of Trp266 with the fused coumarin rings of the dialdehyde molecule. The enhanced activity of M86A may be attributed to the formed π–π stacking interaction between Trp266 and the dialdehyde. In addition, other hydrophobic residues (e.g. Phe and Trp) around the dialdehyde molecule also stabilize the substrate binding. The findings may contribute to understanding the substrate specificity of the AFAR family for AFB₁-dialdehyde.     

Aflatoxin M1 in Raw Milk,Pasteurized Milk and Cottage Cheese Collected along Value Chain Actors from Three Regions of Ethiopia

Milk is a highly nutritious and perfect natural food for humans. However, when lactating animals feed on Aflatoxin B₁ (AFB₁)-containing feed, the hydroxyl metabolite aflatoxin M₁ (AFM₁) contaminates the milk and dairy products. The objective of the current study was to assess the level of AFM₁ in raw milk, normally pasteurized milk and Ethiopian cottage cheese collected from value chain actors (producers, collectors, processors and retailers). Cross-sectional study and simple random techniques were used to collect primary samples. A total of 160 composite samples was collected; raw milk (n = 64), pasteurized milk (n = 64) and cheese (n = 32) was analyzed. Quantitative analysis of AFM₁ was conducted using enzyme-linked immunosorbent assay (ELISA). The results indicate that AFM₁ was detected in all milk products. Results along value chains show that the concentration of AFM₁ in raw milk from collectors was significantly higher than from producers, and in pasteurized milk from processors and retailers (p < 0.05). However, no significant (p > 0.05) difference was observed in cottage cheese value-chain actors in all regions. Comparison of AFM₁ mean values among all dairy products shows that raw milk had a significantly higher concentration of AFM₁ followed by pasteurized milk and cottage cheese. However, there was no significant difference between raw and pasteurized milk (p > 0.05). The mean AFM₁ contamination in milk products ranged from 0.137 to 0.319 µg/L (mean value 0.285 µg/L). The contamination percentages of AFM₁ in raw milk (62.50%), pasteurized milk (67.20%) and cottage cheese (25%) were above the regulatory limit set by the European Union (EU) (0.05 µg/L). According to USA/Ethiopian Standard (US/ES) (0.50 µg/L), 21.87%, 25% and 1% exceeded the regulatory limit for the above products, respectively. The overall prevalence (56.88%) was above the EU regulatory limit and 19.38% over US/ES regulations. Therefore, to provide accurate information about the health risk to consumers, there is a need to conduct risk assessment studies in consumers of milk and dairy products at different age groups.     

Risk Assessment on Dietary Exposure to Aflatoxin B1 in Post-Harvest Peanuts in the Yangtze River Ecological Region

Based on the 2983 peanut samples from 122 counties in six provinces of China’s Yangtze River ecological region collected between 2009–2014, along with the dietary consumption data in Chinese resident nutrition and health survey reports from 2002 and 2004, dietary aflatoxin exposure and percentiles in the corresponding statistics were calculated by non-parametric probability assessment, Monte Carlo simulation and bootstrap sampling methods. Average climatic conditions in the Yangtze River ecological region were calculated based on the data from 118 weather stations via the Thiessen polygon method. The survey results found that the aflatoxin contamination of peanuts was significantly high in 2013. The determination coefficient (R²) of multiple regression reflected by the aflatoxin B₁ content with average precipitation and mean temperature in different periods showed that climatic conditions one month before harvest had the strongest impact on aflatoxin B₁ contamination, and that Hunan and Jiangxi provinces were greatly influenced. The simulated mean aflatoxin B₁ intake from peanuts at the mean peanut consumption level was 0.777–0.790 and 0.343–0.349 ng/(kg·d) for children aged 2–6 and standard adults respectively. Moreover, the evaluated cancer risks were 0.024 and 0.011/(100,000 persons·year) respectively, generally less than China’s current liver cancer incidence of 24.6 cases/(100,000 persons·year). In general, the dietary risk caused by peanut production and harvest was low. Further studies would focus on the impacts of peanut circulation and storage on aflatoxin B₁ contamination risk assessment in order to protect peanut consumers’ safety and boost international trade.     

Occurrence of aflatoxins in mahua (Madhuca indica Gmel.) seeds: Synergistic effect of plant extracts on inhibition of Aspergillus flavus growth and aflatoxin production

Occurrence of aflatoxin in Madhuca indica Gmel. seeds was determined by competitive ELISA. Eighty percent of mahua seed samples were found to be contaminated with aflatoxin. Total aflatoxin content ranged from 115.35 to 400.54 ppb whereas the concentration of AFB₁ was in the range of 86.43 to 382.45 ppb. Mahua oil was extracted by cold press expeller and analysed for contamination of aflatoxin in both the oil and cake samples. Total aflatoxin and aflatoxin B₁ were 220.66 and 201.57 ppb in oil as compared to that in cake samples where it was 87.55 and 74.35 ppb, respectively. Various individual and combined plant extracts were evaluated for their efficacy against growth of Aspergillus flavus and aflatoxin production in vitro. Combination of botanicals were found to be more effective in controlling fungal growth and aflatoxin production than individual extracts. Results of the present study suggests that synergistic effect of plant extracts can be used for control of fungal growth and aflatoxin production. These natural plant products may successfully replace synthetic chemicals and provide an alternative method to protect mahua as well as other agricultural commodities of nutritional significance from toxigenic fungi such as A. flavus and aflatoxin production.     

Metabolism of aflatoxin B1 in turkey liver microsomes: the relative roles of cytochromes P450 1A5 and 3A37

The extreme sensitivity of turkeys to aflatoxin B₁ (AFB₁) is associated with efficient epoxidation by hepatic cytochromes P450 (P450) 1A5 and 3A37 to exo-aflatoxin B₁-8,9-epoxide (exo-AFBO). The combined presence of 1A5 and 3A37, which obey different kinetic models, both of which metabolize AFB₁ to the exo-AFBO and to detoxification products aflatoxin M₁ (AFM₁) and aflatoxin Q₁ (AFQ₁), respectively, complicates the kinetic analysis of AFB₁ in turkey liver microsomes (TLMs). Antisera directed against 1A5 and 3A37, thereby individually removing the catalytic contribution of these enzymes, were used to identify the P450 responsible for epoxidating AFB₁ in TLMs. In control TLMs, AFB₁ was converted to exo-AFBO in addition to AFM₁ and AFQ₁ confirming the presence of functional 1A5 and 3A37. Pretreatment with anti-1A5 inhibited exo-AFBO formation, especially at low, submicromolar (~ 0.1 μM), while anti-3A37, resulted in inhibition of exo-AFBO formation, but at higher (> 50 μM) AFB₁ concentrations. Metabolism in immunoinhibited TLMs resembled that of individual enzymes: 1A5 produced exo-AFBO and AFM₁, conforming to Michaelis-Menten, while 3A37 produced exo-AFBO and AFQ₁ following the kinetic Hill equation. At 0.1 μM AFB₁, close to concentrations in livers of exposed animals, 1A5 contributed to 98% of the total exo-AFBO formation. At this concentration, 1A5 accounted for a higher activation:detoxification (50:1, exo-AFBO: AFM₁) compared to 3A37 (0.15: 1, exo-AFBO: AFQ₁), suggesting that 1A5 is high, while 3A4 is the low affinity enzyme in turkey liver. The data support the conclusion that P450 1A5 is the dominant enzyme responsible for AFB₁ bioactivation and metabolism at environmentally-relevant AFB₁ concentrations in turkey liver.     

Chemical profile,antifungal, antiaflatoxigenic and antioxidant activity of Citrus maxima Burm. and Citrus sinensis (L.) Osbeck essential oils and their cyclic monoterpene, dl-limonene

The study deals with antifungal, antiaflatoxigenic and antioxidant activity of Citrus maxima and Citrus sinensis essential oils (EOs) and their phytochemical composition. The EOs were obtained by hydrodistillation and their chemical profile was determined through GC and GC–MS analysis. Both the EOs and their 1:1 combination showed broad fungitoxic spectrum against different food contaminating moulds. The EOs and their combination completely inhibited aflatoxin B₁ (AFB₁) production at 500 ppm, whereas, dl-limonene, the major component of EOs showed better antiaflatoxigenic efficacy even at 250 ppm. Both the oils exhibited antioxidant activity as DPPH free radical scavenger in dose dependent manner. The IC₅₀ for radical scavenging efficacy of C. maxima and C. sinensis oils were to be 8.84 and 9.45 μl ml⁻¹, respectively. The EOs were found non-mammalian toxic showing high LD₅₀ for mice (oral, acute). The oils may be recommended as safe plant based antimicrobials as well as antioxidants for enhancement of shelf life of food commodities by checking their fungal infestation, aflatoxin production as well as lipid peroxidation.     

Simplified Synthesis and Stability Assessment of Aflatoxin B1-Lysine and Aflatoxin G1-Lysine

Aflatoxins B₁ (AFB₁) and G₁ (AFG₁) are carcinogenic mycotoxins that contaminate crops such as maize and groundnuts worldwide. The broadly accepted method to assess chronic human aflatoxin exposure is by quantifying the amount of aflatoxin adducted to human serum albumin. This has been reported using ELISA, HPLC, or LC-MS/MS to measure the amount of AFB₁-lysine released after proteolysis of serum albumin. LC-MS/MS is the most accurate method but requires both isotopically labelled and unlabelled AFB₁-lysine standards, which are not commercially available. In this work, we report a simplified synthetic route to produce unlabelled, deuterated and ¹³C₆ ¹⁵N₂ labelled aflatoxin B₁-lysine and for the first-time aflatoxin G₁-lysine. Additionally, we report on the stability of these compounds during storage. This simplified synthetic approach will make the production of these important standards more feasible for laboratories performing aflatoxin exposure studies.     

DNA binding,adduct characterisation and metabolic activation of aflatoxin B1 catalysed by isolated rat liver parenchymal,Kupffer and endothelial cells

In vitro studies with rat liver parenchymal, Kupffer and endothelial cells isolated from male Sprague-Dawley rats were undertaken to investigate cell-specific bioactivation of aflatoxin B₁, DNA binding and adduct formation. In the mutagenicity studies, using homogenates of all three separated liver cell populations (co-incubated with NADP⁺ and glucose-6-phosphate as cofactors for the cytochrome P-450 monooxygenase system) parenchymal, Kupffer and endothelial cells were able to activate aflatoxin B₁ to a metabolite mutagenic to Salmonella typhimurium TA 98. In the case of nonparenchymal cells (i.e. Kupffer and endothelial cells) 10-fold higher concentrations of aflatoxin B₁ had to be used to obtain a similar number of revertants to that observed with parenchymal cells. Induction studies with Aroclor 1254 led to a striking decrease in the activation of aflatoxin B₁ in parenchymal cells, whereas nonparenchymal cells had a slightly enhanced metabolic activation capacity for aflatoxin B₁. Metabolism studies with microsomes from induced and noninduced cells using testosterone as substrate revealed comparable results: after induction with Aroclor 1254, parenchymal cells showed a 60% decrease in the formation rate of 2-hydroxytestosterone, whereas the formation rate of this metabolite remained unchanged in nonparenchymal cells; 2-hydroxytestosterone is specifically formed by cytochrome P-450 IIC11, which also catalyses the activation of aflatoxin B₁ to its epoxide. When freshly isolated, intact cells were incubated with tritiated aflatoxin B₁, a dose-dependent aflatoxin B₁ binding to DNA in parenchymal and nonparenchymal cells was observed. HPLC analysis of DNA acid hydrolysates of all three cell types showed the major adduct to be 8,9-dihydro-8-(N⁷-guanyl)-9-hydroxyaflatoxin B₁.This project was supported by the Deutsche Forschungsgemeinschaft (SFB 302). B. Schlemper was the recipient of a European Science Foundation Scholarship     

Stable expression of human kinin B1 receptor in 293 cells: pharmacological and functional characterization

1. We compared the binding properties of [³H]-desArg¹⁰-[Leu⁹]-kallidin, a radiolabelled kinin B₁ receptor antagonist, to membranes from IMR-90 human embryonic fibroblasts and from 293 cells transiently or stably transfected with the human B₁ receptor.
2. The dissociation constant (K_D) of [³H]-desArg¹⁰-[Leu⁹]-kallidin and the affinity of several kinin receptor agonists and antagonists were similar between the native and cloned receptor, either transiently or stably expressed in 293 cells. In IMR-90 cells, the rank order of potency was that expected for a kinin B₁ receptor.
3. The receptors transiently or stably expressed in 293 cells were fully functional with respect to their signalling properties. Phosphoinositide hydrolysis was increased in a concentration-dependent manner by the B₁ receptor agonist, desArg¹⁰-kallidin. Functional coupling to the calcium pathway was also demonstrated for the native and stably expressed human B₁ receptor.
4. In conclusion, the established stable and functional 293 cell clone may provide an important tool for further analysis of the molecular mechanisms involved in binding, activation, and coupling of the kinin B₁ receptor.

     

Removal of Aflatoxin B1 by Edible Mushroom-Forming Fungi and Its Mechanism

Aflatoxins (AFs) are biologically active toxic metabolites, which are produced by certain toxigenic Aspergillus sp. on agricultural crops. In this study, five edible mushroom-forming fungi were analyzed using high-performance liquid chromatography fluorescence detector (HPLC-FLD) for their ability to remove aflatoxin B₁ (AFB₁), one of the most potent naturally occurring carcinogens known. Bjerkandera adusta and Auricularia auricular-judae showed the most significant AFB₁ removal activities (96.3% and 100%, respectively) among five strains after 14-day incubation. The cell lysate from B. adusta exhibited higher AFB₁ removal activity (35%) than the cell-free supernatant (13%) after 1-day incubation and the highest removal activity (80%) after 5-day incubation at 40 °C. In addition, AFB₁ analyses using whole cells, cell lysates, and cell debris from B. adusta showed that cell debris had the highest AFB₁ removal activity at 5th day (95%). Moreover, exopolysaccharides from B. adusta showed an increasing trend (24–48%) similar to whole cells and cell lysates after 5- day incubation. Our results strongly suggest that AFB₁ removal activity by whole cells was mainly due to AFB₁ binding onto cell debris during early incubation and partly due to binding onto cell lysates along with exopolysaccharides after saturation of AFB₁ binding process onto cell wall components.     

Butylated hydroxytoluene chemoprevention of aflatoxicosis – Effects on aflatoxin B1 bioavailability, hepatic DNA adduct formation, and biliary excretion

The extreme sensitivity of turkeys to aflatoxin B₁ (AFB₁) is associated with efficient hepatic cytochrome P-450 (P450)-mediated bioactivation, and deficient glutathione S-transferase (GST) mediated detoxification. Butylated hydroxytoluene (BHT) protects against AFB₁ toxicity in turkeys through mechanisms that include competitive inhibition of P450-mediated AFB₁ bioactivation. To test whether dietary BHT alters hepatic AFB₁–DNA adduct formation, excretion, and bioavailability of AFB₁ in vivo, turkeys were given diets with BHT (4000 ppm) for 10 days, given a single oral dose of [³H]-AFB₁ (0.05 μg/g; 0.02 μCi/g), then sampled at intervals up to 24 h. Radiolabel in serum, red blood cells, liver, and breast meat was frequently lower in BHT-treated compared to control. Hepatic AFB₁–DNA adducts in BHT-treated turkeys were significantly lower at 12 and 24 h. BHT-fed birds had significant higher bile efflux, though biliary radiolabel excretion was not different from control. The amount of aflatoxin M₁ (AFM₁) excreted in the bile was lower than in control, but BHT had no effect on the biliary excretion of AFB₁, aflatoxin Q₁ or glucuronide and sulfate conjugates. Thus, the chemopreventive properties of BHT may also occur through a reduction in AFB₁ bioavailability in addition to inhibition of bioactivation.     

Toxigenic Potential of Aspergillus Species Occurring on Maize Kernels from Two Agro-Ecological Zones in Kenya

Two agro-ecological zones in Kenya were selected to compare the distribution in maize of Aspergillus spp. and their toxigenicity. These were Nandi County, which is the main maize growing region in the country but where no human aflatoxicoses have been reported, and Makueni County where most of the aflatoxicosis cases have occurred. Two hundred and fifty-five households were sampled in Nandi and 258 in Makueni, and Aspergillus was isolated from maize. Aspergillus flavus and A. parasiticus isolates were tested for the presence of aflD and aflQ genes. Positive strains were induced to produce aflatoxins on yeast extract sucrose and quantified using liquid chromatography-tandem mass spectrometry (LCMSMS). Aspergillus flavus was the most common contaminant, and the incidence of occurrence in Nandi and Makueni was not significantly different (82.33% and 73.26%, respectively). Toxigenic strains were more prevalent than non-toxigenic strains. All the toxigenic strains from Makueni were of the S-type while those from Nandi belonged to the L-type. Quantitative differences in aflatoxin production in vitro between isolates and between strains were detected with S strains producing relatively larger amounts of total aflatoxins, B toxins and lower values for G toxins. This was in accord with the frequent aflatoxicosis outbreaks in Makueni. However some L strains produced considerable amounts of B toxins. Given the widespread distribution of toxigenic strains in both regions, the risk of aflatoxin poisoning is high when favorable conditions for toxin production occur.     

Mitochondrial F(0) F(1) -ATP synthase is a molecular target of 3-iodothyronamine, an endogenous metabolite of thyroid hormone

BACKGROUND AND PURPOSE

3-iodothyronamine (T1AM) is a metabolite of thyroid hormone acting as a signalling molecule via non-genomic effectors and can reach intracellular targets. Because of the importance of mitochondrial F₀F₁-ATP synthase as a drug target, here we evaluated interactions of T1AM with this enzyme.

EXPERIMENTAL APPROACH

Kinetic analyses were performed on F₀F₁-ATP synthase in sub-mitochondrial particles and soluble F₁-ATPase. Activity assays and immunodetection of the inhibitor protein IF₁ were used and combined with molecular docking analyses. Effects of T1AM on H9c2 cardiomyocytes were measured by in situ respirometric analysis.

KEY RESULTS

T1AM was a non-competitive inhibitor of F₀F₁-ATP synthase whose binding was mutually exclusive with that of the inhibitors IF₁ and aurovertin B. Both kinetic and docking analyses were consistent with two different binding sites for T1AM. At low nanomolar concentrations, T1AM bound to a high-affinity region most likely located within the IF₁ binding site, causing IF₁ release. At higher concentrations, T1AM bound to a low affinity-region probably located within the aurovertin binding cavity and inhibited enzyme activity. Low nanomolar concentrations of T1AM increased ADP-stimulated mitochondrial respiration in cardiomyocytes, indicating activation of F₀F₁-ATP synthase consistent with displacement of endogenous IF_1,, reinforcing the in vitro results.

CONCLUSIONS AND IMPLICATIONS

Effects of T1AM on F₀F₁-ATP synthase were twofold: IF₁ displacement and enzyme inhibition. By targeting F₀F₁-ATP synthase within mitochondria, T1AM might affect cell bioenergetics with a positive effect on mitochondrial energy production at low, endogenous, concentrations. T1AM putative binding locations overlapping with IF₁ and aurovertin binding sites are described.     

Multi-Toxic Endpoints of the Foodborne Mycotoxins in Nematode Caenorhabditis elegans

Aflatoxins B₁ (AFB₁), deoxynivalenol (DON), fumonisin B₁ (FB₁), T-2 toxin (T-2), and zearalenone (ZEA) are the major foodborne mycotoxins of public health concerns. In the present study, the multiple toxic endpoints of these naturally-occurring mycotoxins were evaluated in Caenorhabditis elegans model for their lethality, toxic effects on growth and reproduction, as well as influence on lifespan. We found that the lethality endpoint was more sensitive for T-2 toxicity with the EC₅₀ at 1.38 mg/L, the growth endpoint was relatively sensitive for AFB₁ toxic effects, and the reproduction endpoint was more sensitive for toxicities of AFB₁, FB₁, and ZEA. Moreover, the lifespan endpoint was sensitive to toxic effects of all five tested mycotoxins. Data obtained from this study may serve as an important contribution to knowledge on assessment of mycotoxin toxic effects, especially for assessing developmental and reproductive toxic effects, using the C. elegans model.     

Determination of aflatoxin B1 levels in Iranian rice by ELISA method

This study was carried out to detect the presence of aflatoxin B₁ (AFB₁) in 40 samples of Tarom rice from Iran. Enzyme-linked immunosorbent assay (ELISA) was applied to analyze AFB₁ in the samples. All the analyses were conducted twice. Aflatoxin B₁ was found in all rice samples, the concentration of AFB₁ ranged from 0.29 to 2.92?µg/kg. The AFB₁ concentration mean in the rice samples produced in 2013 was higher (P < 0.05) than the findings in rice in 2012.. However, 25 of the 40 samples exceeded the maximum prescribed limit, i.e. 2?µg/kg of European Union Regulations and also none of the samples reached the maximum prescribed limit 5?µg/kg of the Institute of Standards and Industrial Research of Iran (ISIRI) for aflatoxin B₁. Although, rice is ranked the second among cereal staples consumed food in Iran and many countries, it can make a serious health problem for people even for a small amount of aflatoxin.     

Identification of alpha 1L-adrenoceptor in mice and its abolition by alpha 1A-adrenoceptor gene knockout

Background and purpose:

The α_1L-adrenoceptor has pharmacological properties that distinguish it from three classical α₁-adrenoceptors (α_1A, α_1B and α_1D). The purpose of this was to identify α_1L-adrenoceptors in mice and to examine their relationship to classical α₁-adrenoceptors.

Experimental approach:

Radioligand binding and functional bioassay experiments were performed on the cerebral cortex, vas deferens and prostate of wild-type (WT) and α_1A-, α_1B- and α_1D-adrenoceptor gene knockout (AKO, BKO and DKO) mice.

Key results:

The radioligand [³H]-silodosin bound to intact segments of the cerebral cortex, vas deferens and prostate of WT, BKO and DKO but not of AKO mice. The binding sites were composed of two components with high and low affinities for prazosin or RS-17053, indicating the pharmacological profiles of α_1A-adrenoceptors and α_1L-adrenoceptors. In membrane preparations of WT mouse cortex, however, [³H]-silodosin bound to a single population of prazosin high-affinity sites, suggesting the presence of α_1A-adrenoceptors alone. In contrast, [³H]-prazosin bound to two components having α_1A-adrenoceptor and α_1B-adrenoceptor profiles in intact segments of WT and DKO mouse cortices, but AKO mice lacked α_1A-adrenoceptor profiles and BKO mice lacked α_1B-adrenoceptor profiles. Noradrenaline produced contractions through α_1L-adrenoceptors with low affinity for prazosin in the vas deferens and prostate of WT, BKO and DKO mice. However, the contractions were abolished or markedly attenuated in AKO mice.

Conclusions and implications:

α_1L-Adrenoceptors were identified as binding and functional entities in WT, BKO and DKO mice but not in AKO mice, suggesting that the α_1L-adrenoceptor is one phenotype derived from the α_1A-adrenoceptor gene.     

BGC20-1531, a novel,potent and selective prostanoid EP4 receptor antagonist: a putative new treatment for migraine headache

Background and purpose:

Prostanoid EP₄ receptor antagonists may have therapeutic utility in the treatment of migraine since EP₄ receptors have been shown to be involved in prostaglandin (PG)E₂-induced cerebral vascular dilatation, which may be an important contributor to migraine pain. This study reports the pharmacological characterization of BGC20-1531, a novel EP₄ receptor antagonist.

Experimental approach:

BGC20-1531 was characterized in radioligand binding and in vitro functional assays employing recombinant and native EP₄ receptors. Changes in canine carotid haemodynamics were used to assess the pharmacodynamic profile of BGC20-1531 in vivo.

Key results:

BGC20-1531 exhibited high affinity at recombinant human EP₄ receptors expressed in cell lines (pK_B 7.6) and native EP₄ receptors in human cerebral and meningeal artery (pK_B 7.6–7.8) but showed no appreciable affinity at a wide range of other receptors (including other prostanoid receptors), channels, transporters and enzymes (pKi < 5). BGC20-1531 competitively antagonized PGE₂-induced vasodilatation of human middle cerebral (pK_B 7.8) and meningeal (pK_B 7.6) arteries in vitro, but had no effect on responses induced by PGE₂ on coronary, pulmonary or renal arteries in vitro. BGC20-1531 (1–10 mg·kg⁻¹ i.v.) caused a dose-dependent antagonism of the PGE₂-induced increase in canine carotid blood flow in vivo.

Conclusions and implications:

BGC20-1531 is a potent and selective antagonist at EP₄ receptors in vitro and in vivo, with the potential to alleviate the symptoms of migraine that result from cerebral vasodilatation. BGC20-1531 is currently in clinical development for the treatment of migraine headache.     

Aflatoxin B1 and fumonisin B1 affect the oxidative status of bovine peripheral blood mononuclear cells

Mycotoxins are secondary metabolites having a high cytotoxic potential. They are produced by molds and released in food and feed. To date, the mechanisms underlying the mycotoxin-induced cytotoxicity have not been fully clarified. The induction of oxidative stress, as a possible mechanism, has been postulated. This in vitro study was focused on the effect of two widely occurring mycotoxins, aflatoxin B₁ (AFB₁) and fumonisin B₁ (FB₁), on the oxidative status of bovine peripheral blood mononuclear cells (PBMC) incubated for 2 and 7 days at different levels of AFB₁ (0, 5 and 20 μg/ml) and FB₁ (0, 35 and 70 μg/ml). Reactive oxygen metabolites (ROM), intracellular thiols (SH), malondialdehyde (MDA) and gene expression of cytoplasmic superoxide dismutase (SOD) and glutathione peroxidase (GSHPX-1) were measured on PBMC after incubation. The highest concentration of AFB₁ and all concentrations of FB₁ caused an increase (p < 0.05) of intracellular ROM without any time dependent effect. Intracellular SH decreased with 20 μgAFB₁/ml (p < 0.05) and the effect was particularly marked after 7 days of exposure. Intracellular SH were not affected by FB₁ even though a lower (p < 0.05) SH level after 2 days exposure than after 7 days was observed. MDA increased (p < 0.05) in AFB₁ or FB₁ treated PBMC. The exposure to FB₁ for 7 days increased MDA (p < 0.05) only in cells treated with 70 μg/ml. Exposure of PBMC to AFB₁ reduced SOD mRNA while FB₁ decreased both SOD and GSHPX-1 mRNA abundance. These results demonstrate that, even though by different mechanisms, AFB₁ and FB₁ may induce cytotoxicity through an impairment of the oxidative status of PBMC.