A comparative evaluation of aflatoxin B1 genotoxicity in fish models using the Comet assay

Aflatoxin B₁ (AFB₁) is classified as a Group I hepatocarcinogen in humans by the International Agency for Research on Cancer (IARC). The alkaline Comet assay is a simple and rapid method by which DNA damage can be demonstrated as a function of tail moment. The present work is the first to evaluate the genotoxicity of AFB₁ in fish using the Comet assay. Two different species of fish were selected as models due to previously established sensitivity to AFB₁: rainbow trout (sensitive) and channel catfish (resistant). Fish were i.p. injected with 0.5 mg AFB₁/1 ml DMSO/1 kg body weight. The Comet assay was performed after 4 and 24 h on whole blood, liver, and kidney cells of both species. Trout blood and kidney tissue tested displayed significant (p<0.05) and extensive DNA damage (shown by increased tail moment) after 4 h which then decreased by 24 h. In liver cells, damage progressively increased over time. Conversely, similarly treated catfish showed no elevation in DNA damage over controls at the same doses. These results suggest that the Comet assay is a useful tool for monitoring the genotoxicity of mycotoxins such as AFB₁ and for evaluating organ specific effects of these agents in different species.     

Characterization of a bone-specific alkaline phosphatase in chick limb mesenchymal cell cultures

Previous morphometric and biochemical studies suggested that osteoblasts develop in cultures derived from phenotypically unexpressive stage 24 chick limb mesenchymal cells. Others have shown that osteoblast expression is marked by an increase in bone-specific alkaline phosphatase activity. Our results indicate that chick limb mesenchymal cells develop alkaline phosphatase activity that is identical to that of the chick embryonic bone-specific isoenzyme. The alkaline phosphatase isozymes were partially purified from samples of chick intestine, liver, stage 38 embryonic limbs, and cultures of stage 24 limb mesenchymal cells. These tissues were separately extracted with butanol, acetone precipitated, redissolved, and passed over a DEAE-Sephacel ion-exchange column and ion-filtration column (Sephadex A-25). From the data obtained during this purification scheme, we conclude that the alkaline phosphatase from stage 38 limbs (bones) and Day 4 cultures are identical, and this activity is different from the enzyme purified from intestine and liver. The cell culture isozyme has an apparent K_m, heat lability, response to specific inhibitors, electrophoretic mobility, and molecular weight similar to those of bone-specific alkaline phosphatase. These observations support the view that osteoblastic progenitor cells are present in the stage 24 limb mesenchyme and that under specific culture conditions, bone development can be uniquely observed in vitro.     

A signal-amplified electrochemical immunosensor for aflatoxin B1 determination in rice

A sensitive and simple electrochemical immunosensor based on enzymatic silver deposition amplification was constructed for the detection of aflatoxin B₁ (AFB₁) in rice. The immunosensor was based on an indirect competitive format between free AFB₁ and aflatoxin B₁-bovine serum albumin (AFB₁-BSA) conjugate immobilized on the electrode surface for binding to a fixed amount of anti-AFB₁ antibody. Then the alkaline phosphatase (ALP)-labeled anti-mouse immunoglobulin G (IgG) secondary antibody was bound to the electrode surface through reaction with primary antibody. Finally, ALP catalyzed the substrate, ascorbic acid 2-phosphate, into ascorbic acid that reduced silver ions in solution to metal silver deposited onto the electrode surface. Linear sweep voltammetry was carried out to quantify the metal silver, which indirectly reflected the amount of the analyte. The experimental parameters, such as the dilution ratio of antibody and the concentration of AFB₁-BSA conjugate, have been evaluated and optimized. At the optimal conditions, the working range of the electrochemical immunosensor was from 0.1 to 10 ng/ml with a detection limit of 0.06 ng/ml. Good recoveries were obtained for the detection of spiked rice samples. So, the proposed method in this article could find a good use for screening AFB₁ in real samples.     

Subcellular localization of enterokinase (Enteropeptidase EC 3.4.21.9) in rat small intestine

The subcellular localization of enterokinase is controversial. In this study, enterokinase was extracted from a soluble fraction and a brush border fraction of rat small intestine by differential centrifugation. The soluble fraction contained 41% of the initial enterokinase activity while the brush border fraction contained only 4.6% of the initial activity. In contrast, alkaline phosphatase monitored as a brush border marker, yielded 26.3 in the brush border fraction and only 6% in the soluble fraction. Further separation of the soluble fraction on a Sepharose 4B column revealed three peaks of enterokinase activity. One small peak (3%) of a bound enzyme (M_r, 2·10^?6) and two larger peaks of free enzyme (M_r, 3·10⁵ and 9·10⁵). In contrast, alkaline phosphatase major fraction was in a high molecular weight peak of bound enzyme. When the brush border fraction was chromatographed only a single peak of bound enterokinase and alkaline phosphatase were found. In the lower part of the small intestine, no brush border-bound enterokinase was found, while the peak of alkaline phosphatase was the same as in the upper intestine. These data suggest that enterokinase activity in the rat intestine is mainly in a free form localized in the mucin and soluble fraction and to a negligible extent in the brush border.     

Prevention of cardiotoxicity of aflatoxin B1 via dietary supplementation of papaya fruit extracts in rats

The aim of the current study was to evaluate the cardioprotective ability of water (WE) and ethanolic (EE) papaya fruits extracts against cardiotoxicity induced by aflatoxin B₁ (AFB₁) in rats. Forty two female Sprague–Dawley rats were divided into six treatment groups and treated orally for 2 weeks as follow: control group, the group treated with WE (250 mg/kg b.w), the group treated with EE (250 mg/kg b.w), the group treated orally with AFB₁ (17 μg/kg b.w) and the groups treated orally with AFB₁ plus WE or EE. The results indicated that treatment with AFB₁ resulted in oxidative stress in the heart manifested by the marked increase in cardiac malondialdehyde and calcium levels accompanied with a significant decrease in cardiac total antioxidant capacity. Serum nitric oxide and sodium levels, lactate dehydrogenase and creatine kinase isoenzyme activities were significantly increased, whereas, cardiac Na⁺/K⁺-ATPase activity and serum potassium were insignificantly affected. Supplementation with WE or EE effectively ameliorated most of the changes induced by AFB₁. It could be concluded that both extracts attenuated the oxidative stress induced in heart tissue by AFB₁ and WE was more pronounced due to the higher total phenolic contents than in the EE.     

Immunosuppressive effects of aflatoxin in growing rats

The immunosuppressive potential of aflatoxin B₁ (AFB₁), the carcinogenic metabolite ofAspergillus flavus, was evaluated in growing rats. The weanling rats were subchronically exposed to 60, 300 and 600 µg AFB₁/kg body weight for four weeks on alternate days by oral feeding. Various parameters of cell mediated immunity (CMI) and humoral immunity were assessed in control and treated animals. CMI was evaluated by measuring delayed type of hypersensitivity (DTH) response and humoral by plaque forming cell (PFC) assay. The lymphoproliferative response assay for T- and B-cells was also performed. It was observed that AFB₁ selectively suppressed cell mediated immunity in growing rats. AFB₁ suppressed CMI at the 300 and 600 µg dose levels only as measured by DTH response assay. It is concluded that continuous low level exposure of aflatoxin to growing host may enhance its susceptibility to infection and tumorigenesis.Abbreviations AF Aflatoxin - AFB₁ Aflatoxin B₁ - CMI Cell mediated immunity - CPM Counts per minute - DTH Delayed type of hypersensitivity - GST Glutathione-S-transferase - LPS Lipopolysaccharide - PFC Plaque forming cell - PHA Phytohemagglutinin - SRBC Sheep red blood cells     

Beta-1 integrins mediate substrate dependent effects of 1α,25(OH)2D3 on osteoblasts

Surface micron-scale and submicron scale features increase osteoblast differentiation and enhance responses of osteoblasts to 1,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃]. β₁ integrin expression is increased in osteoblasts grown on Ti substrates with rough microarchitecture, and it is regulated by 1α,25(OH)₂D₃ in a surface-dependent manner. To determine if β₁ has a role in mediating osteoblast response, we silenced β₁ expression in MG63 human osteoblast-like cells using small interfering RNA (siRNA). In addition, MG63 cells were treated with two different monoclonal antibodies to human β₁ to block ligand binding. β₁-silenced MG63 cells grown on a tissue culture plastic had reduced alkaline phosphatase activity and levels of osteocalcin, transforming growth factor β₁, prostaglandin E₂, and osteoprotegerin in comparison with control cells. Moreover, β₁-silencing inhibited the effects of surface roughness on these parameters and partially inhibited effects of 1α,25(OH)₂D₃. Anti β₁ antibodies decreased alkaline phosphatase but increase osteocalcin; effects of 1α,25(OH)₂D₃ on cell number and alkaline phosphatase were reduced and effects on osteocalcin were increased. These findings indicate that β₁ plays a major and complex role in osteoblastic differentiation modulated by either surface microarchitecture or 1α,25(OH)₂D₃. The results also show that β₁ mediates, in part, the synergistic effects of surface roughness and 1α,25(OH)₂D₃.     

An early effect of aflatoxin B1 administered in vivo on the growth of bone marrow CFU-GM and the production of some cytokines in rats

Our data demonstrate the granulopoietic toxicity of aflatoxin B₁ (AFB₁)in vivo and show an impact of this mycotoxin on the production of some humoral regulatory factors dealing with the granulopoietic developmental pathway (CSA, IL-1, IL-2). The dose of AFB₁ studied represented approximately 1/5 of LD₅₀ for young male rats. An early suppressive effect of AFB₁ towards CFU-GM was transient in treated animals. The peak in granulopoietic activity was preceded in time by an increased CSA and IL-1 formation. Elevated IL-2 synthesis and increased T cell activation paralleled the peak in granulopoietic activity.Abbreviations AFB₁ Aflatoxin B₁ - CFU-GM granulocyte-monocyte colony-forming unit - CSA colony-stimulating activity - CSF colony-stimulating factor - GM-CSF granulocyte-monocyte CSF - G-CSF granulocyte CSF - M-CSF monocyte CSF - IL-1–6 Interleukin 1–6 - TNF tumour necrosis factor - IFN Interferon     

In vitro regulation of 3-hydroxy-3-methylglutarylcoenzyme a reductase and acylcoenzyme a: Cholesterol acyltransferase activities by phoshorylation-dephosphorylation in rabbit intestine

The regulation of 3-hydroxy-3-methylglutarylcoenzyme A reductase and acylcoenzyme A: cholesterol acyltransferase activities by phosphorylation-dephosphorylation in rabbit intestine was studied in vitro. Preparing intestinal microsomes in the presence of 50 mM NaF caused a 64% decrease in the reductase activity. It had no effect on acyl-CoA: cholesterol acyltransferase activity. Microsomes that were prepared in NaF were incubated with intestinal cytosol, a partially purified phosphatase from cytosol, and Escherichia coli alkaline phosphatase. All three preparations increased 3-hydroxy-3-methylglutaryl-CoA reductase by two- or three-fold suggesting dephosphorylation and ‘reactivation’ of enzyme activity. Cytosol caused a 78% increase in acyl-CoA: cholesterol acyltransferase activity, but neither the partially purified phosphatase nor the E. coli alkaline phosphatase affected the acyltransferase activity. Microsomes incubated with increasing concentrations of MgCl₂ and ATP decreased both the activities of 3-hydroxy-3-methylglutaryl-CoA reductase and acylcoenzyme A: cholesterol acyltransferase in a step-wise fashion. Whereas this inhibitory effect was specific for reductase, the effect on acyl-CoA: cholesterol acyltransferase activity was secondary to the presence of ATP in the assay mixture. The 8500×g supernatant of intestinal whole homogenate from isolated intestinal cells or scraped mucosa was incubated with MgCl₂, ATP and NaF. In microsomes prepared from this supernatant, the activity of 3-hydroxy-3-methylglutaryl-CoA reductase was significantly decreased. Again, no change was observed in the acyltransferase activity. The rate of cholesterol esterification in isolated intestinal cells was not affected by 0.1 mM cAMP or 50 mM NaF. We conclude that under conditions which regulate 3-hydroxy-3-methylglutaryl-CoA reductase activity in rabbit intestine by phosphorylation-dephosphorylation, no regulation of acyl-CoA: cholesterol acyltransferase activity is observed.     

Stimulatory effect of menaquinone-7 (vitamim K2) on osteoblastic bone formation in vitro

Menaquinone-7, which is vitamin K₂ (menatetrenone) with seven isoprene units, is highly contained in the fermented soybean. The effect of menaquinone-7 (MK-7) on osteoblastic bone formation was investigated. Femoral-diaphyseal and metaphyseal tissues of young male rats (4 weeks old) were cultured for 48 h in a medium containing either vehicle or MK-7 (10^–7–10^–5 M). Calcium content, alkaline phosphatase activity, and deoxyribonuclic acid (DNA) content in the diaphyseal and metaphyseal tissues was significantly increased in the presence of MK-7 (10^–6 and 10^–5 M). The effect of MK-7 in increasing the diaphyseal and metaphyseal calcium content and alkaline phosphatase activity was completely prevented in the presence of cycloheximide (10^–6 M), an inhibitor of protein synthesis. Moreover, osteoblastic MC3T3-E1 cells after subculture were cultured for 24 h in a serum-free medium containing MK-7 (10^–7–10^–5 M). Protein content, alkaline phophatase activity, osteocalcin and DNA content in the cells was significantly increased in the presence of MK-7 (10^–6 and 10^–5 M). The effect of MK-7 in increasing protein content, alkaline phosphatase activity, and osteocalcin production in the cells was completely blocked by cycloheximide. This study demonstrates that MK-7 has an anabolic effect on bone tissue and osteoblastic MC3T3-E1 cells in vitro, suggesting that the compound can stimulate osteoblastic bone formation.     

The effect of large doses of Nematodirus battus on the histology and biochemistry of the small intestine of lambs

Coop R.L., Angus K.W. and Mapes C.J. 1973. The effect of large doses of Nematodirus battus on the histology and biochemistry of the small intestine of lambs. International Journal for Parasitology3: 349–361. Large single and multiple doses of Nematodirus battus alter the structure of the small intestine, producing a low flattened, convoluted type of mucosa with extensive cellular infiltration of the lamina propria. The brush borders of the epithelial cells on the surface of the villi are narrow and the microvilli sparse and distorted.There is a reduction in alkaline phosphatase and disaccharidase activity in the mucosa of infected lambs, whereas dipeptidase activity is unaffected. The changes in enzyme activity are thought to arise from damage to mature epithelial cells on the villi.Alkaline phosphatase and disaccharidase activity in the mueosa of lambs killed 18 days after infection is correlated with the dry matter content of the faeces.     

Acute effect of aflatoxin B1 on different inbred mouse strains II

The acute effects of Aflatoxin B₁(AFB₁) were evaluated on C57B1/6, CBA/J, B10A and Balb/c mice challenged with a single intraperitoneal dose of the mycotoxin (60 mg/Kg animal weight). 90 mice per strain were divided into three groups of 30 animals each: the intoxicated group and control groups I and II. Intoxicated mice were injected intraperitonealy with AFB₁ dissolved in corn oil, while control I mice received corn oil only (0.01 ml/g) by the same route. Lots of 10 animals from the intoxicated and control groups were sacrificed 24, 72 and 168 hours after challenge. Control mice II remained untreated and were used as standards of normality for biochemical (hepatic and renal function) and hematological evaluation. AFB₁ was detected in the liver of C57B1/6 and CBA/J mice 24 hours (1.46 and 0.75 ng/g, respectively), 72 hours (2.30 and 0.08 ng/g, respectively), and 168 hours (2.18 and 0.25 ng/g, respectively) after challenge. The mycotoxin was also observed in the liver of B10A mice (6.20 ng/g) 72 hours post-injection. The most evident histological lesions were observed 168 hours after treatment in C57B1/6 and B10A mice. Serum levels of alkaline phosphatase in intoxicated C57B1/6 and B10A mice were significantly higher than those of control I and II animals. The histopathologic lesions and biochemical changes were very discrete in Balb/c and CBA/J mice. It is included that strains C57B1/6 and B10A are more susceptible than strains CBA/J and Balb/c to the acute effects of AFB₁. Such difference probably reflects each strains's ability to biotransform and eliminate AFB₁ and its metabolites.     

Use of yeast (<Emphasis Type="Italic">Pichia kudriavzevii</Emphasis>) as a novel feed additive to ameliorate the effects of aflatoxin B<Subscript>1</Subscript> on broiler chicken performance

The aim of this study was to evaluate the efficacy of autochthonous Pichia kudriavzevii as a novel bioadsorbent for aflatoxin B₁ (AFB₁). The selection of this yeast was based on the AFB₁ adsorption capacity previously demonstrated in vitro (Magnoli et al. 2016). One-day-old Cobb broilers (n = 160) were randomly assigned to four dietary treatments (T1: basal diet (B); T2: B + 0.1% yeast; T3: B + AFB₁, 100 μg/kg; T4: B + 0.1% yeast + AFB₁, 100 μg/kg). Performance parameters (average daily weight gain body, average daily consumption, feed conversion ratio, carcass weight, and dead weight), biochemical parameters (albumin, globulin, and albumin/globulin), liver pathological changes, and AFB₁ residual levels in the liver and excreta were evaluated. Significant differences (P < 0.05) in performance parameters were observed among treatments and controls: T3 group showed the lowest average daily body weight gain value while in T4 group, the value of this parameter increased significantly (P < 0.05). T3 and T4 groups showed the lowest and highest values for average daily feed consumption, respectively. The feed conversion ratio (FC) showed no significant differences among treatments. T3 group showed the lowest dead weight and carcass weight compared with T1 group. The biochemical parameters showed no significant differences among treatments. T3 group showed macroscopic and microscopic liver changes compared to the control. Aflatoxin B₁ levels (μg/g) were detected in broiler livers and showed significant differences among treatments (P < 0.05). In conclusion, native P. kudriavzevii incorporation (0.1%) in broiler diets containing AFB₁ was shown to be effective in ameliorating the adverse effects of AFB₁ on production.     

Global DNA methylation in a population with aflatoxin B1 exposure

We previously reported that global DNA hypomethylation, measured as Sat2 methylation in white blood cells (WBC), and aflatoxin B₁ (AFB₁) exposure were associated with increased hepatocellular carcinoma risk. In this study, we assessed the association between AFB₁ exposure and global DNA methylation. We measured LINE-1 and Sat2 methylation in WBC DNA samples from 1140 cancer free participants of the Cancer Screening Program (CSP) cohort. Blood and urine samples were used to determine the level of AFB₁-albumin (AFB₁-Alb) adducts and urinary AFB₁ metabolites. In continuous models, we found reverse associations of urinary AFB₁ with LINE-1 and Sat2 methylation. The odds ratio (OR) per 1 unit decrease were 1.12 (95%CI = 1.03–1.22) for LINE-1 and 1.48 (95%CI = 1.10–2.00) for Sat2 methylation. When compared with subjects in the highest quartile of LINE-1, we found that individuals in the 2nd and 3rd quartiles were less likely to have detectable AFB₁-Alb adducts, with ORs (95%CI) of 0.61 (0.40–0.93), 0.61 (0.40-.94), and 1.09 (0.69–1.72), respectively. The OR for detectable AFB₁-Alb was 1.81 (95%CI = 1.15–2.85) for subjects in the lowest quartile of Sat2 methylation. The OR for detection of urinary AFB₁ for those with LINE-1 methylation in the lowest quartile compared with those in the highest quartile was 1.87 (95%CI = 1.15–3.04). The corresponding OR was 1.75 (95%CI = 1.08–2.82) for subjects in the lowest quartile of Sat2 methylation. The association between AFB₁ exposure and global DNA methylation may have implications for the epigenetic effect of AFB₁ on hepatocellular carcinoma development and also suggests that changes in DNA methylation may represent an epigenetic biomarker of dietary AFB₁ exposure.     

Effects of Sodium Selenite on Aflatoxin B1-Induced Decrease of Ileal IgA+ Cell Numbers and Immunoglobulin Contents in Broilers

This study was aimed to assess the protective effect of sodium selenite on the ileum mucosal immunologic injury induced by AFB₁. One hundred eighty-one-day-old healthy male Avian broilers were divided into four groups of three replicates and 15 birds per replicate and fed with basal diet (control group), 0.3 mg/kg AFB₁ (AFB₁ group), 0.4 mg/kg Se (+Se group), and 0.3 mg/kg AFB₁?+?0.4 mg/kg Se (AFB₁?+?Se group) respectively. The numbers of IgA⁺ cells of ileum were determined by immunohistochemistry as well as the contents of sIgA, IgA, IgG, and IgM in the mucosa of ileum by ELISA. Compared with those in the control group, the numbers of IgA⁺ cells as well as the sIgA, IgA, IgG, and IgM contents were decreased in the AFB₁ group. However, compared with those in the AFB₁ group, the numbers of IgA⁺ cells as well as the sIgA, IgA, IgG, and IgM contents were increased in the AFB₁?+?Se group, and these data had no difference between AFB₁?+?Se group and control group. It was concluded that 0.3 mg/kg AFB₁ could reduce the humoral immune function of the ileum mucosa, but 0.4 mg/kg supplemented dietary selenium could protect the mucosal humoral immune function from AFB₁-induced impairment.     

Effects of Sodium Selenite on Aflatoxin B1-Induced Decrease of Ileac T cell and the mRNA Contents of IL-2, IL-6, and TNF-α in Broilers

The aim of this work was to assess the protective effect of sodium selenite on the ileum mucosal immunologic toxicity induced by aflatoxin B₁ (AFB₁). One hundred and eighty one-day-old healthy male avian broilers were divided into four groups of three replicates and 15 birds per replicate and fed with basal diet (control group), 0.3 mg/kg AFB₁ (AFB₁ group), 0.4 mg/kg Se (+Se group), and 0.3 mg/kg AFB₁?+?0.4 mg/kg Se (AFB₁+Se group), respectively. The ileac T-cell subsets were determined by the methods of flow cytometry (FCM), and the mRNA contents of interleukin-2 (IL-2), interleukin-6(IL-6), and tumor necrosis factor-alpha (TNF-α) by quantitative real-time PCR. Compared with those in control group, the percentages of CD₃ ⁺, CD₃ ⁺CD₄ ⁺, CD₃ ⁺CD₈ ⁺ intraepithelial lymphocytes (IELs) and LPLs, the CD4⁺/CD8⁺ ratio of IELs, and the mRNA contents of IL-2, IL-6, and TNF-α were decreased in AFB₁ group. However, compared with those in AFB₁ group, these parameters of AFB₁+Se group were increased to be close to those in control group. It was concluded that 0.3 mg/kg AFB₁ could reduce the cellular immune function of the ileum mucosa, but 0.4 mg/kg supplemented dietary selenium showed protective effects on AFB₁-induced immunologic injury.     

Lactobacillus rhamnosus Strain GG Reduces Aflatoxin B1 Transport, Metabolism, and Toxicity in Caco-2 Cells

The probiotic Lactobacillus rhamnosus GG is able to bind the potent hepatocarcinogen aflatoxin B₁ (AFB₁) and thus potentially restrict its rapid absorption from the intestine. In this study we investigated the potential of GG to reduce AFB₁ availability in vitro in Caco-2 cells adapted to express cytochrome P-450 (CYP) 3A4, such that both transport and toxicity could be assessed. Caco-2 cells were grown as confluent monolayers on transmembrane filters for 21 days prior to all studies. AFB₁ levels in culture medium were measured by high-performance liquid chromatography. In CYP 3A4-induced monolayers, AFB₁ transport from the apical to the basolateral chamber was reduced from 11.1% ± 1.9% to 6.4% ± 2.5% (P = 0.019) and to 3.3% ± 1.8% (P = 0.002) within the first hour in monolayers coincubated with GG (1 × 10¹⁰ and 5 × 10¹⁰ CFU/ml, respectively). GG (1 × 10¹⁰ and 5 × 10¹⁰ CFU/ml) bound 40.1% ± 8.3% and 61.0% ± 6.0% of added AFB₁ after 1 h, respectively. AFB₁ caused significant reductions of 30.1% (P = 0.01), 49.4% (P = 0.004), and 64.4% (P < 0.001) in transepithelial resistance after 24, 48, and 72 h, respectively. Coincubation with 1 × 10¹⁰ CFU/ml GG after 24 h protected against AFB₁-induced reductions in transepithelial resistance at both 24 h (P = 0.002) and 48 h (P = 0.04). DNA fragmentation was apparent in cells treated only with AFB₁ cells but not in cells coincubated with either 1 × 10¹⁰ or 5 × 10¹⁰ CFU/ml GG. GG reduced AFB₁ uptake and protected against both membrane and DNA damage in the Caco-2 model. These data are suggestive of a beneficial role of GG against dietary exposure to aflatoxin.     

Cytotoxicity of aflatoxin B1 and its chemically synthesised epoxide derivative on the A549 human epithelioid lung cell line

Aflatoxin B₁ (AFB₁) is a carcinogenic mycotoxin found in feeds and in airborne grain dusts. Aflatoxin B₁ requires biotransformation to the AFB₁-8,9 epoxide (AFBO) by a bioactivation system and subsequent covalent binding to DNA or proteins, to exert its carcinogenic potential. The lung contains cytochrome P₄₅₀, prostaglandin-H-synthase, lipoxygenase, epoxide hydrolase and other bioactivation enzymes, and is thus a potential target for the effects of AFB₁ via the routes of inhalation and ingestion. The A549 human epithelioid lung cell line and the methylthiazol tetrazolium (MTT) bioassay were used to investigate the cytotoxicity of AFB₁ and its chemically synthesised epoxide (AFBO) in vitro. Statistical analysis of the MTT results indicated that there were overall significant differences between the control and both the AFB₁-treated (p < 0.0001) and AFBO-treated cells (p = 0.00 2). However, there was no significant difference between AFB₁ and AFBO-treated cells, when the entire range of concentrations were assessed against each other (p = 0.2877). Whenanalysed at each concentration, only at 0.01 mM was there a significant difference between the effects of AFB₁ and AFBO (p = 0.0358). The results of this investigation show that AFB₁ and AFBO are both cytotoxic in the A549 cell line.This revised version was published online in October 2005 with corrections to the Cover Date.     

Intestinal calcium transport: evidence for two distinct mechanisms of action of 1,25-dihydroxyvitamin D3

The response of the small intestine in the vitamin D-deficient rat to a single intrajugular injection of 1,25-dihydroxyvitamin D₃ has been studied. The time course of 1,25-dihydroxyvitamin D₃-induced transport suggests that two separate responses occur. The first or initial response reaches a maximum at 6 h after 1,25-dihydroxy vitamin D₃ administration, decays, and is effectively gone by 12 h postinjection. This response does not appear to be associated with alkaline phosphatase activity. The second or late response first appears roughly 12 h after dosing, reaches a maximum at 24 h, and remains elevated for up to 72 h. This response is accompanied by an elevation of alkaline phosphatase activity and appears to be mediated through the action of 1,25-dihydroxyvitamin D₃ on the absorptive cell during its normal differentiation and migration up the villus.     

In vitro evaluation of the capacity of zeolite and bentonite to adsorb aflatoxin B1 in simulated gastrointestinal fluids

Anin vitro study using single concentration and isotherm adsorption was carried out to evaluate the capacity of Vietnamese produced zeolite and bentonite to adsorb aflatoxin B₁ (AFB₁) in simulated gastrointestinal fluids (SGFs), and a commercial sorbent hydrated sodium calcium aluminosilicate (HSCAS) was used as reference. In this study, AFB₁ solution was mixed with sorbents (0.3, 0.4 and 0.5% w/v) in SGFs at pH 3 and pH 7 and shaken for 8 h, centrifuged and the supernatant measured by Vicam fluorometer. Adsorption of AFB₁ onto zeolite and bentonite varied according to the pH of SGFs and was lower than HSCAS. Linearity between the increased amount of AFB₁ adsorbed on sorbents and the decrease of sorbent concentration was observed for bentonite and HSCAS, except for zeolite in SGFs at pH 7. The observed maximum amounts of AFB₁ adsorbed on bentonite and HSCAS were 1.54 and 1.56 mg/g, respectively. The adsorption capacities of bentonite and HSCAS for AFB₁ were 12.7 and 13.1 mg/g, respectively, from fitting the data to the Freundlich isotherm equation. Improvement in processing and purification for bentonite is needed to enhance the surface area, which would probably result in better adsorptive capacity for this sorbent.