Products of Spontaneous Conjugation of Aflatoxins with Bovine Serum Albumin: Immunochemical Properties

Products of spontaneous conjugation of aflatoxins B₁, G₁, and G₂ with bovine serum albumin (BSA) were shown to interact with antibodies against aflatoxins. Solid-phase BSA conjugates inhibited the binding of aflatoxins by antiaflatoxin antibodies. Antisera against BSA–B₁, BSA–G₁, and BSA–G₂ were obtained and their specificity determined. The mechanisms of spontaneous binding of aflatoxins by proteins are discussed.     

Exposure measurement of aflatoxins and aflatoxin metabolites in human body fluids. A short review

Aflatoxins are highly toxic secondary fungal metabolites mainly produced by Aspergillus flavus and A. parasiticus. Human exposure to aflatoxins may result directly from ingestion of contaminated foods, or indirectly from consumption of foods from animals previously exposed to aflatoxins in feeds. This paper focuses on exposure measurement of aflatoxins and aflatoxin metabolites in various human body fluids. Research on different metabolites present in blood, urine, breast milk, and other human fluids or tissues including their detection techniques is reviewed. The association between dietary intake of aflatoxins and biomarker measurement is also highlighted. Finally, aspects related to the differences between aflatoxin determination in food versus the biomarker approach are discussed.     

Formation and biliary excretion of glutathione conjugates of bile acids in the rat as shown by liquid chromatography/electrospray ionization-linear ion trap mass spectrometry

Acyl-adenylates and acyl-CoA thioesters of bile acids (BAs) are reactive acyl-linked metabolites that have been shown to undergo transacylation-type reactions with the thiol group of glutathione (GSH), leading to the formation of thioester-linked GSH conjugates. In the current study, we examined the transformation of cholyl-adenylate (CA-AMP) and cholyl-coenzyme A thioester (CA-CoA) into a cholyl-S-acyl GSH (CA-GSH) conjugate by rat hepatic glutathione S-transferase (GST). The reaction product was analyzed by liquid chromatography (LC)/electrospray ionization (ESI)-linear ion trap mass spectrometry (MS). The GST-catalyzed formation of CA-GSH occurred with both CA-AMP and CA-CoA. Ursodeoxycholic acid, lithocholic acid, and 2,2,4,4-²H₄-labeled lithocholic acid were administered orally to biliary fistula rats, and their corresponding GSH conjugates were identified in bile by LC/ESI-MS². These in vitro and in vivo studies confirm a new mode of BA conjugation in which BAs are transformed into their GSH conjugates via their acyl-linked intermediary metabolites by the catalytic action of GST in the liver, and the GSH conjugates are then excreted into the bile.     

Aflatoxin and aflatoxicosis: V. The kinetic behaviour of dietary aflatoxins in colostrum drawn from cows postpartum

This work was conducted in order to study the kinetic behaviour of dietary aflatoxins in the colostrum of a pregnant cow exposed to contaminated feeds for a short period.In this study, two pregnant cows received a single dose of dietary aflatoxins in the form of rice powder contained 31.20 ppm aflatoxin B and 19.68 ppm aflatoxin G during the last stage of pregnancy, at about two weeks before parturition.Samples of colostrum were collected from dams and assayed for the presence of toxic metabolites as well as its conjugations by electrophoretic analysis.The results revealed that the intake of aflatoxins appeared in the colostrum pospartum as AFM₁ and also AFB_2a which is a non toxic metabolite. Moreover, it was found that the excreted metabolites including AFB_2a were conjugated to the immunoglobulin protein fraction of the colostrum.The significance of the obtained results to the newborn calf are discussed.     

Metabolic activation of aflatoxins related to their mutagenicity.

Rat hepatic microsome-mediated DNA-binding and mutagenesis to Salmonella typhimurium strain Ta-100 by various aflatoxins and some mixed function oxygenase-mediated metabolites of aflatoxin B₁ were studied. The data indicated a good correlation between the DNA-binding and mutagenesis; a requirement for the intact C₂–C₃ double bond in parent aflatoxins B₁ and G₁; and relative inactivity of aflatoxin B₁ metabolites with an otherwise intact C₂–C₃ double bond.     

Impact of linker and conjugation chemistry on antigen binding,Fc receptor binding and thermal stability of model antibody-drug conjugates

Antibody-drug conjugates (ADCs) with biotin as a model cargo tethered to IgG1 mAbs via different linkers and conjugation methods were prepared and tested for thermostability and ability to bind target antigen and Fc receptor. Most conjugates demonstrated decreased thermostability relative to unconjugated antibody, based on DSC, with carbohydrate and amine coupled ADCs showing the least effect compared with thiol coupled conjugates. A strong correlation between biotin-load and loss of stability is observed with thiol conjugation to one IgG scaffold, but the stability of a second IgG scaffold is relatively insensitive to biotin load. The same correlation for amine coupling was less significant. Binding of antibody to antigen and Fc receptor was investigated using surface plasmon resonance. None of the conjugates exhibited altered antigen affinity. Fc receptor FcγIIb (CD32b) interactions were investigated using captured antibody conjugate. Protein G and Protein A, known inhibitors of Fc receptor (FcR) binding to IgG, were also used to extend the analysis of the impact of conjugation on Fc receptor binding. H10_NPEG4 was the only conjugate to show significant negative impact to FcR binding, which is likely due to higher biotin-load compared with the other ADCs. The ADC aHIS_NLC and aHIS_TPEG8 demonstrated some loss in affinity for FcR, but to much lower extent. The general insensitivity of target binding and effector function of the IgG1 platform to conjugation highlight their utility. The observed changes in thermostability require consideration for the choice of conjugation chemistry, depending on the system being pursued and particular application of the conjugate.     

Destruction of aflatoxins B1 and G1 in bread making

The effect of processing steps as well preservatives used in French bread making namely propionic acid and/or potassium sorbate (0.2%) on the destruction of aflatoxins B₁ and G₁ was studied. Mixing and baking processes showed marked destruction of aflatoxins B₁ and G₁; being 71.2% and 52.5% for aflatoxin B₁ after mixing and baking steps, while reaching 73.9% and 54.5% for aflatoxin G₁. Fermentation step caused additional 15.3% and 15.0% destruction of aflatoxins B₁ and G₁. On the other hand, aflatoxin B₁ destruction was 79.2% and 50.7% when propionic acid was used and 75.3 and 56.7% in the presence of potassium sorbate and after mixing and baking steps respectively. Concerning aflatoxins G₁ it was found that mixing and baking steps showed destruction of 81.9% and 53.4% in the presence of propionic acid and 75.1 and 49.4% in the presence of potassium sorbate in this respective order. Generally, it can be concluded that using propionic acid as preservative appeared to be more effective on the destruction of aflatoxins B₁ and G₁ than potassium sorbate in French bread making.     

A review of the dose-response induction of DNA adducts by aflatoxin B1 and its implications to quantitative cancer-risk assessment

The induction of DNA adducts by aflatoxin B₁ in the liver has been extensively reviewed in a quantitative cancer-risk assessment of aflatoxins (CDHS, 1990). Rat is the most sensitive species for aflatoxin tumorigenesis and liver is the most sensitive site. In vitro DNA-adduct studies were mostly on adduct identification and specificity of binding. In vivo studies provided dose-response relationship of aflatoxin B₁, binding to DNA and DNA-adduct formation. Most in vivo studies were conducted in rats. The dose-response curves of DNA-adduct induction after ingestion or injection treatments in this species were reviewed. A linear dose-response relationship was observed in both injection and ingestion studies at low doses. For cancer-risk assessment, this observation is consistent with the assumption of the linear dose-response risk-assessment model for genotoxic agents, and justifies the use of this model for quantitative cancer-risk assessment for aflatoxins.     

Afaltoxin B1 transport in rat blood plasma. Binding to albumin in vivo and in vitro and spectrofluorimetric studies into the nature of the interaction

Binding of [³H]aflatoxin B₁ to rat plasma was investigated in vivo and vn vitro. Column chromatographic and polyacrylamide gel electrophoretic analysis clearly demonstrated that aflatoxin B₁ bound primarily plasma albumin. Very little binding activity was shown by other plasma proteins. Spectrofluorimetric studies were undertaken to gain some insight into the nature of the aflatoxin-albumin interaction. Quenching of the lone tryptophan fluorescence intensity upon aflatoxin binding was due, at least in part, to a ligand-induced conformational change in the albumin molecule. Aflatoxin B₁ binds an apolar site with an association constant of 30 mM⁻¹ at pH 7.4 and 20°C. Neither charcoal treatment of rat albumin nor the presence of 0.15 M NaCl had many significant effect on the interaction. The association constant was pH-dependent, increasing about 1.7-fold as the pH increased from 6.1 to 8.4. This pH dependence is ascribed to a pH-induced conformational change in the albumin molecule. Thermodynamic studies indicated that the aflatoxin-albumin interaction was exothermic (ΔH = −29.3 kJ·mol⁻), with a ΔS value of −13.8 J·mol⁻¹·K⁻¹.     

Impact of linker and conjugation chemistry on antigen binding,Fc receptor binding and thermal stability of model antibody-drug conjugates

Antibody-drug conjugates (ADCs) with biotin as a model cargo tethered to IgG1 mAbs via different linkers and conjugation methods were prepared and tested for thermostability and ability to bind target antigen and Fc receptor. Most conjugates demonstrated decreased thermostability relative to unconjugated antibody, based on DSC, with carbohydrate and amine coupled ADCs showing the least effect compared with thiol coupled conjugates. A strong correlation between biotin-load and loss of stability is observed with thiol conjugation to one IgG scaffold, but the stability of a second IgG scaffold is relatively insensitive to biotin load. The same correlation for amine coupling was less significant. Binding of antibody to antigen and Fc receptor was investigated using surface plasmon resonance. None of the conjugates exhibited altered antigen affinity. Fc receptor FcγIIb (CD32b) interactions were investigated using captured antibody conjugate. Protein G and Protein A, known inhibitors of Fc receptor (FcR) binding to IgG, were also used to extend the analysis of the impact of conjugation on Fc receptor binding. H10_NPEG4 was the only conjugate to show significant negative impact to FcR binding, which is likely due to higher biotin-load compared with the other ADCs. The ADC aHIS_NLC and aHIS_TPEG8 demonstrated some loss in affinity for FcR, but to much lower extent. The general insensitivity of target binding and effector function of the IgG1 platform to conjugation highlight their utility. The observed changes in thermostability require consideration for the choice of conjugation chemistry, depending on the system being pursued and particular application of the conjugate.     

Enhanced stability of β-galactosidase in parenchymal and nonparenchymal liver cells by conjugation with dextran

In order to enhance the stability of β-galactosidase, we conjugated the enzyme with dextran T-10 (M_r approx. 10 000). The conjugate contained 9–10 mol dextran/mol protein (β-galactosidase, M_r 68 000), and the specific activity retained after conjugation was 90 ± 4% (n = 3) of the initial activity. Uptake and degradation of native and conjugated β-galactosidase in isolated hepatocytes and nonparenchymal liver cells was studied. There was a marked increase in stability against degradation in both cell types when β-galactosidase was conjugated with Dextran. The degradation of dextran-conjugated enzyme was reduced by 35% in hepatocytes and by 43% in nonparenchymal cells, after 80 and 40 min, respectively, as compared with the free enzyme. However, there was insignificant difference between the uptake of native and conjugated enzyme into the liver cells. Upon intravenous infusion into rats, native and conjugated enzyme were cleared from plasma with only a slight difference in the clearance rate. The observed stability of dextran-conjugated β-galactosidase towards cellular degradation was in accordance with the in vitro experiments. The conjugate showed marked thermal stability at 50°C and enhanced resistance towards proteolysis by the broad specific protease subtilopeptidase A. This demonstrates that dextran conjugation may be used as a means of stabilizing lysosomal enzymes for therapeutic purposes.     

The role of pH and its control on effective conjugation of bovine hemoglobin and human serum albumin

Human serum albumin (HSA) and bovine hemoglobin (Hb) conjugate is a promising candidate as a blood substitute. However, preparation of the conjugate is problematic because both proteins tend to conjugate between themselves rather than crosslink each other. In this work, a facile process for conjugation of Hb and HSA was developed through control strategy of the reaction. The reaction was carried out in a buffer containing borax-borate and mannite. The borax-borate was used for pH buffering while mannite was used as a pH switch and a reaction promoter. As a result, self-conjugation of Hb and self-conjugation of HSA were minimized. After the one-step conjugation reaction in aqueous solution, followed by the one-step purification by ion-exchange chromatography, the conjugate of HSA and Hb was obtained with the total yield about 50%. The P₅₀ and the Hill coefficient for the product were 16.1 mmHg and 1.82, respectively.     

Invitro metabolism of aflatoxin B1 by rat liver nuclei

Rat liver nuclei were used to study the metabolism of aflatoxin B₁ (AFB₁). Nuclei were found to metabolize AFB₁ to aflatoxins M₁(AFM₁), Q₁(AFQ₁), and P₁(AFP₁); the formation of AFP₁ was relatively negligible. AFM₁ was preferentially formed when rats were pretreated with 3-methylcholanthrene (MC), whereas phenobarbital (PB) pretreatment enhanced the formation of both AFM₁ and AFQ₁. PB pretreatment also resulted in increased binding of AFB₁ metabolites to DNA and to other macromolecules in the nucleus. Following PB and MC pretreatment, induction specificities of the nuclear metabolism of AFB₁ were similar to those of the microsomal metabolism.     

Production and characterization of polyclonal and monoclonal antibodies against Aflatoxin B1 oxime-BSA in an enzyme-linked immunosorbent assay

From a single aflatoxin B₁ oxime — bovine serum albumin conjugate, polyclonal and monoclonal antibody preparations were produced. The four rabbit polyclonal antisera were specific for aflatoxin Bi in a microtitration plate enzyme — linked immunosorbent assay. The monoclonal antibodies showed a wide range of differing specificities, recognizing, for example, aflatoxins B₁, B₂, G₁ and G₂; B₁ and B₂; B₁ and G₁; and G₁ alone. No antibody preparations reacted with aflatoxin M₁. The significance of these results to the strategy of anti-aflatoxin antibody production for use in quantitative enzyme immunoassays is discussed.     

Aflatoxins B1 and G1 solubility in standard solutions and stability during cold storage

In the present work we study the use of different solvents to store aflatoxins B₁ and G₁ standard solutions. We have obtained significant differences between aflatoxin B₁ and G₁ In ethyl acetate, methanol and water, with aflatoxin G₁ being less stable. We recommend chloroform as the election solvent to store the aflatoxin solutions. The fact that aflatoxins are highly stable in water may have a potential use in experiments of biological activity.     

Hepatocyte metabolism of coenzyme Q1 (ubiquinone-5) to its sulfate conjugate decreases its antioxidant activity

Previous studies on the metabolism of coenyzme Q (CoQ) have focused on products found in the urine, bile or feces. However, the metabolites found in these samples were end products from a multitude of catabolic processes which did not necessarily reflect CoQ intracellular metabolism (e.g. in the liver, the major site of CoQ synthesis or metabolism). Using isolated rat hepatocytes, we have found that the sulfation of coenzyme Q1 (CoQ1) was the initial and dominant step following its reduction to the hydroquinone. This metabolic process is important as conjugation may occur on the hydroquinone metabolites of any coenzyme10 scission product retaining the quinone ring. By using rat liver cytosol, we were able to identify the monosulfated metabolite of CoQ1. The CoQ1 sulfate conjugate was identified by mass spectrometry followed by tandem mass spectrometry. The rate of formation of the CoQ1 sulfate conjugate was markedly increased by the addition of NADH and was prevented by dicumarol, a DT-diaphorase (NQO1) inhibitor. CoQ1 sulfate conjugate formation catalysed by cytosol was inhibited by the sulfotransferase 1A (SULT1A) inhibitor, pentachlorophenol (PCP) suggesting that sulfation was carried out by the SULT 1A isoform. CoQ1 sulfation in isolated hepatocytes and inversely CoQ1 hydroquinone formation were dependent on the concentration of inorganic sulfate in the media. Intracellular sulfation also decreased CoQ1 antioxidant and cytoprotective activity towards cumene hydroperoxide (CHP) induced cell death. Sulfotransferases may therefore play a significant role in endogenous CoQ metabolism following its degradation to short chain products.     

Zearalenone,deoxynivalenol and aflatoxin B<Subscript>1</Subscript> and their metabolites in pig urine as biomarkers for mycotoxin exposure

Methods to determine zearalenone (ZEA), deoxynivalenol (DON), aflatoxins (AF) and their metabolites in pig urine were developed as biomarkers for pig exposure to the mycotoxins in feed. Urine samples were incubated with β-glucuronidase to cleave conjugates, extracted and cleaned-up with solid phase and immunoaffinity columns, followed by HPLC with UV and fluorescence detection. Good recoveries (83–130%), low variation (2–10%), and low detection limits (0.3–9.9 ng/ml) were obtained. The results of controlled AFB₁ feeding trials found no difference in urine concentrations of AFB₁ or AFM₁ from pigs fed three different levels (127, 227, 327 μg/kg) of AFB₁ in diets. The excretion of AFB₁ and AFM₁ in urine was on average 30% of the oral dose and the ratio AFB₁ to AFM₁ was around 23%. The analysis of 15 Vietnamese pig urine samples indicate a relatively high exposure of ZEA, DON and AF, which were found as toxin or metabolites in 47, 73, and 80% of the urine samples, respectively.     

Multispecific Organic Cation Transporter 1 (OCT1) from Bos taurus Has High Affinity and Slow Binding Kinetics towards Prostaglandin E2

Organic cation transporter 1 (OCT1, SLC22A1), like many solute carrier 22 (SLC22) family members, is important for the disposition of clinically important drugs, metabolites and signaling molecules. Several studies suggest that SLC22 family (eg. organic anion transporters or OATs and OCTs) bind and possibly transport prostaglandins with relatively high affinity (submicromolar). The affinities of OCT1 and OATs toward PGE2 and PGF2a reported in these cell-based transport studies are considerably greater than for xenobiotics and natural metabolite substrates—in many cases over 100-fold higher. This raises the possibility that prostaglandins are key endogenous substrates and/or that they act on the transporter in a manner different from other substrates such as xenobiotics and lower affinity metabolites. To further investigate OCT1—prostaglandin interactions, we designed biophysical studies using purified bovine OCT1 (Bos taurus, btOCT1/SLC22A1) with PGE2 analogs, in fluorescently labeled and label-free formats. Using fluorescence polarization (FP), we detected a binding of btOCT1 to the PGE2-Rhodamine conjugate at submicromolar affinity, consistent with affinity data for PGE2 from cells over-expressing the related human OCT1. Using purified native btOCT1 as analyte and biotinylated PGE2 analog as ligand, our data from surface plasmon resonance (SPR) revealed that btOCT1 specifically interacts to PGE2 with K_D values in the hundred nanomolar range. BtOCT1 also demonstrated a slow association (k_a) in the range of 10³ M^-1s^-1 and an even slower dissociation rate (k_d) in the range of 10⁻⁴ s^-1 for PGE2, suggesting the possibility of a different mode of binding compared to other structurally unrelated transported substrates of low-affinity (eg. drugs, metabolites). Our results complement in vitro transport studies and provide direct evidence that OCT1—which is normally expressed in liver and other tissues—interacts with prostaglandin analogs. While it is not entirely clear from the published literature whether OCTs function as major prostaglandin transporters, the tight binding of the naturally occurring PGE2, as well as the slow dissociation rate, could conceivably affect the transport of lower affinity substrates such as drugs and metabolites by SLC22 transporters. More research is necessary to establish the extent to which individual SLC22 family members actually function as PG transporters in vitro and in vivo and to investigate whether PGs can, independent of being directly transported, alter the ability of SLC22 transporters to handle drugs and other substrates.     

Fungal flora and aflatoxin contamination of feedstuff samples in Beida Governorate,Libya

Fungi of 19 genera, 30 species, and one variety were isolated from 25 samples of sheep-, cattle- and camel feedstuffs collected from different farms in the Beida Governorate, Libya.Aspergillus, Penicillium andFusarium were the most common genera in the three substrates tested. TLC was used to establish the identity of aflatoxins in the chloroform extract of all samples and the ability to produce aflatoxins byAspergillus flavus in a synthetic liquid medium. Twenty samples out of 25 tested were naturally contaminated and 21 isolates ofA. flavus out of 30 produced at least one of the following aflatoxins: B₁; B₁, G₁; and B₁, B₂, G₁, G₂. This is the first report about the natural occurrence of aflatoxins and aflatoxin-producers of the genusAspergillus in Libya.     

Characterization of the conjugation system associated with the Staphylococcus aureus plasmid pJE1

The conjugation system of Staphylococcus aureus that is conferred, at least in part, by plasmid pJE1, requires cell-to-cell contact. Optimum transfer was found when the numbers of donor and recipient cells were equal. Certain antibiotics increased the conjugation frequency. Fragments of plasmid pJE1 were cloned into a staphylococcal plasmid vector; although separate clones were isolated that conferred ethidium bromide resistance and gentamicin resistance, none of the clones carried the ability to conjugate. Transposon mutagenesis with Tn551 was used to create 26 mutants of pJE1. These were analysed for the position of the insertion and for their ability to conjugate. The sites of insertion were non-random. Only six mutants were unaffected in their conjugability; one showed increased ability to conjugate whilst the rest were either unable to conjugate or showed a reduced frequency. It is concluded that there are at least two separate regions necessary for conjugation and that the system is not obviously similar to that reported in streptococci.