Urinary biomarkers of aflatoxin exposure in young children from Egypt and Guinea.

Aflatoxins are a major risk factor for hepatocellular carcinoma (HCC), and thus understanding the pattern of aflatoxin exposure in different regions is important in order to develop targeted intervention strategies. Given the early onset of HCC in many countries early life exposures may be important. This study investigated aflatoxin exposure in Egyptian children (n=50, aged 1-2.5 years) by assessing urinary aflatoxin metabolite (AFM(1), AFB(1), AFB(2), AFG(1), AFG(2)) levels. Samples from Guinean children (n=50, aged 2-4 years) were analyzed in parallel providing a comparison to a region of established frequent aflatoxin exposure. Aflatoxins were isolated from urine using C18-cartridges followed by immunoaffinity clean-up, and quantified by HPLC with fluorescence detection. Overall aflatoxins were less frequently present in Egyptian (38%) than Guinean urine samples (86%) (p<0.001), which was particularly related to differences in detection rates of AFM(1) (8% compared to 64%, respectively, (p<0.001)). For AFM(1) the geometric mean level in Guinea (16.3 pg/ml; 95% CI: 10.1, 26.6 pg/ml) was 6-fold higher (p<0.001) than in Egypt (2.7 pg/ml; 95% CI: 2.5, 2.8 pg/ml). Urinary aflatoxins from healthy children in these two regions have not previously been reported, and exposure appears modest in Egypt compared to Guinea. These data suggest that measures to reduce aflatoxin exposure in both regions are important, though particularly in Guinea.     

Aflatoxin B1 Occurrence in Children under the Age of Five’s Food Products and Aflatoxin M1 Exposure Assessment and Risk Characterization of Arab Infants through Consumption of Infant Powdered Formula: A Lebanese Experience

Aflatoxin M1 (AFM1) is a salient metabolite that can be used to assess Aflatoxin B₁ (AFB1) exposure in humans and animals. The carcinogenic potency of AFB1 and AFM1 was severely reported. The aims of this study were (1) to survey the contamination level of AFM1 in the most traded infant powdered formula brands (IPF) (n = 42) along with the AFB1 level in under 5’s children food brands (biscuits, cornflakes, and cereals) (n = 42) and (2) to assess the estimated daily intake (EDI), the hazard quotient (HQ) and the margin of exposure (MOE) of AFM1 among infants (0–12 months) in Lebanon. All of the samples were analyzed using ELISA technique. AFB1 was below detection limit in all of the children’s food brands samples. Out of 42 IPF samples 9.5% were AFM1-positive in the range of 29.54–140.16 ng/L and exceeded the maximum tolerable limit (MTL) set by the European commission (25 ng/kg). The overall average contamination level was 5.72 ± 0.014 ng/L. The EDI of AMF1 for male was in the range of 0.37–0.78 ng/kg/b.w./day and 0.40–0.87 ng/kg/b.w./day for females. Similarly, the HQ calculation resulted in an average of 3.05 for males and 3.28 for females. MOE calculations were far lower from 10,000 in both genders which indicates a high risk of genotoxicity and carcinogenicity. Our findings show that AFM1’s EDI, HQ and MOE scored high among Lebanese infants. As infants consume more IPF relative to their body weight, the persistence of IPF with high AFM1 levels threatens their health. Thus, infant’s exposure risk to AFM1 in IPF should be a continuous focus of attention.     

Cytochrome P450 2A13 mediates aflatoxin B1-induced cytotoxicity and apoptosis in human bronchial epithelial cells

Cytochrome P450 (CYP) 2A13 is mainly expressed in the respiratory system and has the ability to metabolize aflatoxin B(1) (AFB(1)). However, the role of CYP2A13-mediated AFB(1) metabolism and its consequences in human lung epithelial cell is not clear. Therefore, the objectives of this study were to investigate the significance of CYP2A13 in AFB(1)-induced cytotoxicity, DNA adducts, and apoptosis. To achieve these objectives, CYP2A13 was stably over-expressed in immortalized human bronchial epithelial BEAS-2B cells (B-2A13) and its significance in AFB(1)-induced cytotoxicity, DNA adducts, and apoptosis was compared to cells with stably expression of CYP1A2 (B-1A2), the predominant AFB(1) metabolizing enzyme in liver, as well as CYP2A6 (B-2A6) as controls. AFB(1) induced B-2A13 cytotoxicity and apoptosis in a dose- and time-dependent manner. The cytotoxic and apoptotic effects of AFB(1) were significantly remarkable in B-2A13 cells than those of B-1A2 and B-2A6 cells. The increased expression of pro-apoptotic proteins, such as C-PARP, C-caspase-3, and Bax, and decreased expression of anti-apoptotic proteins, such as caspase-3, Bcl-2, and p-Bad further confirmed the data of AFB(1)-induced cytotoxicity and apoptosis. Furthermore, increased DNA adduct was observed in B-2A13 after AFB(1) treatment as compared to B-1A2 cells and B-2A6 cells. Finally, treatment with nicotine, a competitor of AFB(1), and 8-methoxypsoralen (8-MOP), an inhibitor of CYP enzyme, further confirm the critical role of CYP2A13 in AFB(1)-induced cytotoxicity and apoptosis. Collectively, these findings suggest adverse effects of AFB(1) in respiratory diseases mediated by CYP2A13.     

Effects of aflatoxin B(1) and fumonisin B(1) on blood biochemical parameters in broilers

The individual and combined effects of dietary aflatoxin B(1 )(AFB(1)) and fumonisin B(1) (FB(1)) on liver pathology, serum levels of aspartate amino-transferase (AST) and plasma total protein (TP) of broilers were evaluated from 8 to 41 days of age. Dietary treatments included a 3 × 3 factorial arrangement with three levels of AFB(1 )(0, 50 and 200 μg AFB(1)/kg), and three levels of FB(1 )(0, 50 and 200 mg FB(1)/kg). At 33 days post feeding, with the exception of birds fed 50 mg FB(1 )only, concentrations of AST were higher (p < 0.05) in all other treatment groups when compared with controls. Plasma TP was lower (p < 0.05) at six days post feeding in groups fed 200 μg AFB(1)/kg alone or in combination with FB(1). At day 33 days post feeding, with the exception of birds fed the highest combination of AFB(1 )and FB(1 )which had higher plasma TP than control birds(, )plasma TP of birds fed other dietary treatments were similar to controls. Broilers receiving the highest levels of AFB(1) and FB(1) had bile duct proliferation and trabecular disorder in liver samples. AFB(1) singly or in combination with FB at the levels studied, caused liver damage and an increase in serum levels of AST.     

Sodium tanshinone IIA sulfonate derived from Danshen (Salvia miltiorrhiza) attenuates hypertrophy induced by angiotensin II in cultured neonatal rat cardiac cells

Recent studies support the view that in addition to its effect on both phase I and phase II xenobiotic metabolizing enzymes, the synthetic chemopreventive agent oltipraz also increases the nucleotide excision repair (NER) which represents the major pathway of elimination of chemical carcinogen DNA adducts. Since most carcinogens are activated in the liver, we investigated the influence of oltipraz on NER activity of this target tissue by using two different approaches. First, we employed an assay based on the measurement of DNA repair in cisplatin-damaged plasmid DNA incubated in the presence of cell-free extracts prepared from either rat liver or human hepatoma HepG2 cells treated by oltipraz. Secondly, we analyzed the removal of aflatoxin B(1)-derived DNA adducts formed in primary human hepatocytes exposed to oltipraz after treatment with this mycotoxin. Whatever the strategy used, NER activity was not altered in liver cells. These data demonstrated that liver cells actively repair bulky DNA adducts by NER and that oltipraz does not influence their NER activity neither in vivo nor in vitro, consequently strongly suggesting that the chemopreventive agent oltipraz is acting before the initiation step of cancer development.     

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin administration and high-fat diet on the body weight and hepatic estrogen metabolism in female C3H/HeN mice

We studied the effect of administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by i.p. injection once every 2 weeks in combination with a high-fat (HF) diet for 8 or 16 weeks on the body and organ weight changes as well as on the hepatic enzyme activity for estrogen metabolism in C3H/HeN female mice. Administration of TCDD at 100 microg/kg b.w. once every 2 weeks for 8 weeks increased the body weight by 46% in the HF diet-fed animals, but not in the regular diet-fed animals. This is the first observation suggesting that TCDD at a high dose (100 microg/kg b.w.), but not at lower doses (1 or 10 microg/kg b.w.), may have a strong obesity-inducing effect in C3H/HeN mice fed an HF diet. While TCDD increased liver weight and decreased thymus weight in animals, these effects were enhanced by feeding animals an HF diet. Metabolism studies showed that TCDD administration for 8 or 16 weeks increased the liver microsomal activity for the 2- and 4-hydroxylation of 17 beta-estradiol in animals fed a control diet, but surprisingly not in animals fed an HF diet. Treatment with TCDD dose-dependently increased the hepatic activity for the O-methylation of catechol estrogens in both control and HF diet-fed animals, and it also decreased the levels of liver microsomal sulfatase activity for hydrolysis of estrone-3-sulfate. TCDD did not significantly affect the hepatic enzyme activity for the glucuronidation or esterification of endogenous estrogens. It is suggested that enhanced metabolic inactivation of endogenous estrogens by hepatic estrogen-metabolizing enzymes in TCDD-treated, control diet-fed animals contributes importantly to the reduced incidence of estrogen-associated tumors in animals treated with TCDD.     

Expression of human microsomal epoxide hydrolase in Saccharomyces cerevisiae reveals a functional role in aflatoxin B1 detoxification.

The metabolism and genotoxicity of the carcinogenic mycotoxin, aflatoxin B1 (AFB), was studied in the lower eukaryotic yeast Saccharomyces cerevisiae. Recombinant strains of yeast were engineered to express human cDNAs for CYP1A1, CYP1A2, and microsomal epoxide hydrolase (mEH). Coexpression of mEH with CYP1A1 or CYP1A2 resulted in significant decreases in measurements of AFB genotoxicity. In cells expressing CYP1A2 and mEH, the level of AFB-DNA adducts was decreased by 50% relative to cells expressing CYP1A2 alone. Mitotic recombination, as assayed by gene conversion at the trp5 locus, was diminished by 50% or greater in cells coexpressing mEH and CYP1A2 compared to CYP1A2 alone. The mutagenicity of AFB in the Ames assay was also decreased by approximately 50% when AFB was incubated with microsomes containing CYP1A1 or CYP1A2 and mEH versus CYP1A1 or CYP1A2 alone. The biotransformation of AFB by CYPs is known to involve the generation of a reactive epoxide intermediate, AFB-8,9-epoxide, but previous direct biochemical and kinetic studies have failed to demonstrate any functional role for mEH in AFB detoxification. By reconstructing a metabolic pathway in intact yeast, we have shown, for the first time, that mEH may play a role in mitigating the carcinogenic effects of AFB.     

Determination of aflatoxin B(1) levels in deep-red ground pepper (isot) using immunoaffinity column combined with ELISA.

Deep-red ground pepper, a variety of red ground pepper, is a special spices belonging to Sanliurfa and consumed both in Sanliurfa and other provinces of Turkey. The aim of this study was to determine the aflatoxin B(1) (AFB(1)) levels of deep-red ground pepper. For this purpose, 75 samples of deep-red ground pepper (isot) marketed in Sanliurfa (Turkey) were purchased from bazaars and herbal shops. The occurrence and concentration range of AFB(1) in the samples were investigated by microtitre plate Enzyme Linked Immunosorbent Assay (ELISA) method using immunoaffinity columns. Seventy-two of the 75 ground deep-red pepper samples (96%) contained AFB(1) in the range of 0.11-24.7 microg/kg. Eleven (14.7%) samples were above the regulatory limits used in the European Union and in Turkey. More precaution should be taken on hygiene controls in order to prevent microbiological and chemical hazards.     

Adsorbents Reduce Aflatoxin M1 Residue in Milk of Healthy Dairy Cow Exposed to Moderate Level Aflatoxin B1 in Diet and Its Exposure Risk for Humans

This study investigated the effect of moderate risk level (8 µg/kg) AFB₁ in diet supplemented with or without adsorbents on lactation performance, serum parameters, milk AFM₁ content of healthy lactating cows and the AFM₁ residue exposure risk in different human age groups. Forty late healthy lactating Holstein cows (270 ± 22 d in milk; daily milk yield 21 ± 3.1 kg/d) were randomly assigned to four treatments: control diet without AFB₁ and adsorbents (CON), CON with 8 μg/kg AFB₁ (dry matter basis, AF), AF + 15 g/d adsorbent 1 (AD1), AF + 15 g/d adsorbent 2 (AD2). The experiment lasted for 19 days, including an AFB₁-challenge phase (day 1 to 14) and an AFB₁-withdraw phase (day 15 to 19). Results showed that both AFB₁ and adsorbents treatments had no significant effects on the DMI, milk yield, 3.5% FCM yield, milk components and serum parameters. Compared with the AF, AD1 and AD2 had significantly lower milk AFM₁ concentrations (93 ng/L vs. 46 ng/L vs. 51 ng/L) and transfer rates of dietary AFB₁ into milk AFM₁ (1.16% vs. 0.57% vs. 0.63%) (p < 0.05). Children aged 2–4 years old had the highest exposure risk to AFM₁ in milk in AF, with an EDI of 1.02 ng/kg bw/day and a HI of 5.11 (HI > 1 indicates a potential risk for liver cancer). Both AD1 and AD2 had obviously reductions in EDI and HI for all population groups, whereas, the EDI (≥0.25 ng/kg bw/day) and HI (≥1.23) of children aged 2–11 years old were still higher than the suggested tolerable daily intake (TDI) of 0.20 ng/kg bw/day and 1.00 (HI). In conclusion, moderate risk level AFB₁ in the diet of healthy lactating cows could cause a public health hazard and adding adsorbents in the dairy diet is an effective measure to remit AFM₁ residue in milk and its exposure risk for humans.     

DNA-damaging effects of genotoxins in mixture: modulation of covalent binding to DNA.

Modulation of DNA adduct formation by pre-existing adducts was examined in synthetic oligonucleotides and genomic DNA (calf thymus); genotoxins studied were N-acetoxy-acetylaminofluorene (N-AcO-AAF), aminofluorene (AF), aflatoxin B1-8,9-epoxide (AFB1-8,9-epoxide), and dimethylsulfate (DMS). Oligodeoxynucleotides containing either guanine-C8-AAF (Gua-C8-AAF) or Gua-C8-AF adducts and a neighboring unadducted guanine (G) (target G), located 1, 2, or 4 nucleotides from the adduct, were reacted, as single- (ss) or double-stranded (ds) substrates, with dimethylsulfate (DMS) or AFB1-8,9-epoxide. A modified Maxam-Gilbert technique showed that the presence of the AAF adduct lowered the extent to which AFB1-8,9-epoxide, but not DMS, reacted with target G. Binding of AFB1-8,9-epoxide to the target G was attenuated (> or =5-fold) when the target was located immediately adjacent to an AAF, but not AF, adduct in ds-DNA. Reaction with AFB1-8,9-epoxide increased when the target G was located 2 or 4 nucleotides from the AAF adduct. Pretreatment of calf thymus DNA with AAF (0-1.8% nucleotides modified) reduced levels of Gua-N7-AFB1 adducts formed after subsequent treatment with AFB1-8,9-epoxide. Pretreatment of calf thymus DNA with AFB1 did not alter levels of adducts formed after subsequent treatment with N-AcO-AAF. The supposition that aflatoxin B1-binding to DNA may be altered by conformational changes in the helix, due to the presence of a pre-existing AAF adduct, is supported by the absence of an effect by AF and confirmation of local denaturation of the oligomer helix by use of chemical probes hydroxylamine and diethylpyrocarbonate. Nonetheless, the importance of changes in the nucleophilicity of neighboring nucleotides and local steric effects cannot be ruled out.     

Metabolomics of the Bio-Degradation Process of Aflatoxin B1 by Actinomycetes at an Initial pH of 6.0

Contamination of food and feed by Aflatoxin B1 (AFB1) is a cause of serious economic and health problems. Different processes have been used to degrade AFB1. In this study, biological degradation of AFB1 was carried out using three Actinomycete species, Rhodococcus erythropolis ATCC 4277, Streptomyces lividans TK 24, and S. aureofaciens ATCC 10762, in liquid cultures. Biodegradation of AFB1 was optimised under a range of temperatures from 25 to 40 °C and pH values of 4.0 to 8.0. An initial concentration of 20 µg/mL of AFB1 was used in this study. The amount of AFB1 remaining was measured against time by thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC), coupled with UV and mass spectrometry (LC-MS). All species were able to degrade the AFB1, and no significant difference was found between them. AFB1 remained in the liquid culture for R. erythropolis, S. lividans and S. aureofaciens were 0.81 µg/mL, 2.41 µg/mL and 2.78 µg/mL respectively, at the end of the first 24 h. Degradation occurred at all incubation temperatures and the pH with the optimal conditions for R. erythropolis was achieved at 30 °C and pH 6, whereas for S. lividans and S. aureofaciens the optimum conditions for degradation were 30 °C and pH 5. Analysis of the degradative route indicated that each microorganism has a different way of degrading AFB1. The metabolites produced by R. erythropolis were significantly different from the other two microorganisms. Products of degradation were identified through metabolomic studies by utilizing high-resolution mass spectral data. Mass spectrometric analysis indicated that the degradation of AFB1 was associated with the appearance of a range of lower molecular weight compounds. The pathway of degradation or chemical alteration of AFB1 was followed by means of high resolution Fourier transform mass spectrometry (HR-FTMS) analysis as well as through the MS² fragmentation to unravel the degradative pathway for AFB1. AFB1 bio-degradation was coupled with the accumulation of intermediates of fatty acid metabolism and glycolysis. A plausible mechanism of degradation of AFB1 by Rhodococcus was hypothesized.     

Effect of gender and cigarette smoking on urinary excretion of etheno DNA adducts in humans measured by isotope dilution gas chromatography/mass spectrometry

Endogenous formation of the promutagenic DNA adducts 1,N(6)-ethenoadenine (epsilon Ade) and 3,N(4)-ethenocytosine (epsilon Cyt) has been considered as biomarkers originated from lipid peroxidation. Elevated levels of epsilon Ade and epsilon Cyt were observed in cancer-prone tissues, suggesting the validity of these adducts in cancer risk assessment. The presence of DNA base adducts in biological fluids is considered to derive primarily from base excision repair (BER) systems. In this study, a modified gas chromatography/mass spectrometry (GC/MS) method is developed for simultaneous analysis of epsilon Ade and epsilon Cyt in human urine. After adjusting for creatinine concentration, urinary excretion of epsilon Ade, as well as epsilon Cyt, is much higher in 18 male smokers than in 10 male nonsmokers (p=0.003 for epsilon Ade and p=0.04 for epsilon Cyt). Furthermore, excretion of epsilon Ade and epsilon Cyt in 14 female nonsmokers is much higher than in 10 male nonsmokers (p=0.002 for epsilon Ade and p=0.005 for epsilon Cyt). These results suggest a statistically significant association between gender, as well as smoking, and excretion of epsilon Ade and epsilon Cyt. Moreover, urinary excretion of epsilon Ade in these 42 subjects correlates with that of epsilon Cyt (R(2)=0.6846, p<0.0001). Measurement of urinary epsilon Ade and epsilon Cyt excretion should provide valid noninvasive biomarkers for carcinogenesis and chemoprevention studies.     

Mechanisms of butylated hydroxytoluene chemoprevention of aflatoxicosis--inhibition of aflatoxin B1 metabolism

Chemoprevention of toxicoses and/or cancer through the use of nutrients or pharmacologic compounds is the subject of intense study. Among the many compounds examined, food additives such as antioxidants are being considered due to their ability to reduce disease formation by either induction or inhibition of key enzyme systems. One such compound, butylated hydroxytoluene (BHT), has been found to protect against cancer formation caused by exposure to aflatoxin B₁ (AFB₁) in rodents. We have shown that dietary BHT protects against clinical signs of aflatoxicosis in turkeys, a species that is very susceptible to this mycotoxin. In this study, the effect of BHT on AFB₁ metabolism and other cytochrome P450 (CYP)-related enzyme activities in turkey liver microsomes was examined to discern possible mechanisms of BHT-mediated protection against aflatoxicosis. Ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), prototype activities for CYP1A1 and 1A2, respectively, were decreased in the BHT fed (4000 ppm) animals, while oxidation of nifedipine, a prototype activity for CYP3A4, was increased. However, BHT added to microsomal incubations inhibited these CYP activities in a concentration-related manner. Importantly, BHT inhibited conversion of AFB₁ to the reactive intermediate AFB₁-8,-9-epoxide (AFBO), exhibiting Michaelis-Menton competitive inhibition kinetics (Ki = 0.81 μM). Likewise, microsomes prepared from turkeys fed BHT were significantly less active in AFBO formation compared to those from control birds. When turkeys were fed BHT for up to 40 days, residual BHT was present in liver, breast meat, thigh meat and abdominal fat in concentrations substantially below U.S. FDA guidelines for this antioxidant, but in concentrations greater than the Ki, likely sufficient to inhibit bioactivation of AFB₁in vivo. BHT-induced hydropic degeneration in the livers of BHT fed animals was significantly greater in birds that remained on BHT treatment for up to 30 days, but this lesion diminished in animals fed for 40 days or when returned to a control diet. The data indicate that the observed chemopreventive properties of BHT in turkeys may be due, at least in part, to its ability to inhibit hepatic AFB₁ epoxidation and also that the BHT-induced hydropic degeneration is reversible and does not appear to cause long-term effects.     

Bacterial lipopolysaccharide exposure alters aflatoxin B(1) hepatotoxicity: benchmark dose analysis for markers of liver injury.

Aflatoxin B(1) (AFB(1)) is a fungal toxin that causes both acute hepatotoxicity and hepatocellular carcinoma in humans and experimental animals. Previous studies demonstrated that a small, noninjurious dose of bacterial lipopolysaccharide (LPS) augments the hepatotoxicity of AFB(1) through activation of inflammatory cells and production of soluble inflammatory mediators (Barton et al., 2000b, 2001). This study was conducted to examine the effect of LPS on the dose-response relationship for AFB(1)-induced liver injury. Male Sprague-Dawley rats (250-350g) were treated with AFB(1) (0.1 mg/kg-6.3 mg/kg, ip) and 4 h later with a noninjurious dose of E. coli LPS (7.4 x 10(6) EU/kg, iv). Twenty-four h after AFB(1) administration, hepatic parenchymal cell injury was estimated from elevations in serum alanine aminotransferase and aspartate aminotransferase activities. Injury to intrahepatic bile ducts was evaluated from increased serum gamma-glutamyl transferase and alkaline phosphatase activities. Based on benchmark dose (BMD) analysis, the AFB(1) BMD for parenchymal cell injury was decreased 10-fold by LPS cotreatment, whereas AFB(1) BMDs for bile duct injury were decreased nearly 20-fold. The data suggest that concurrent inflammation renders the liver considerably more sensitive to the hepatotoxic effects of AFB(1).     

Glycine N-methyltransferase affects the metabolism of aflatoxin B1 and blocks its carcinogenic effect

Previously, we reported that glycine N-methyltransferase (GNMT) knockout mice develop chronic hepatitis and hepatocellular carcinoma (HCC) spontaneously. For this study we used a phosphoenolpyruvate carboxykinase promoter to establish a GNMT transgenic (TG) mouse model. Animals were intraperitoneally inoculated with aflatoxin B₁ (AFB₁) and monitored for 11 months, during which neither male nor female GNMT-TG mice developed HCC. In contrast, 4 of 6 (67%) male wild-type mice developed HCC. Immunofluorescent antibody test showed that GNMT was translocated into nuclei after AFB₁ treatment. Competitive enzyme immunoassays indicated that after AFB₁ treatment, the AFB₁-DNA adducts formed in stable clones expressing GNMT reduced 51.4% compared to the vector control clones. Experiments using recombinant adenoviruses carrying GNMT cDNA (Ad-GNMT) further demonstrated that the GNMT-related inhibition of AFB₁-DNA adducts formation is dose-dependent. HPLC analysis of the metabolites of AFB₁ in the cultural supernatants of cells exposed to AFB₁ showed that the AFM₁ level in the GNMT group was significantly higher than the control group, indicating the presence of GNMT can enhance the detoxification pathway of AFB₁. Cytotoxicity assay showed that the GNMT group had higher survival rate than the control group after they were treated with AFB₁. Automated docking experiments showed that AFB₁ binds to the S-adenosylmethionine binding domain of GNMT. Affinity sensor assay demonstrated that the dissociation constant for GNMT-AFB₁ interaction is 44.9 μM. Therefore, GNMT is a tumor suppressor for HCC and it exerts protective effects in hepatocytes via direct interaction with AFB₁, resulting in reduced AFB₁-DNA adducts formation and cell death.     

Molecular Mechanisms of Lipoic Acid Protection against Aflatoxin B1-Induced Liver Oxidative Damage and Inflammatory Responses in Broilers

Alpha-lipoic acid (α-LA) was evaluated in this study for its molecular mechanisms against liver oxidative damage and inflammatory responses induced by aflatoxin B₁ (AFB₁). Birds were randomly allocated into four groups with different diets for three weeks: a basal diet, a 300 mg/kg α-LA supplementation in a basal diet, a diet containing 74 μg/kg AFB₁, and 300 mg/kg α-LA supplementation in a diet containing 74 μg/kg AFB₁. In the AFB₁ group, the expression of GSH-P_X mRNA was down-regulated (p < 0.05), and the levels of lipid peroxide and nitric oxide were increased (p < 0.05) in the chicken livers compared to those of the control group. Additionally, the mRNA level of the pro-inflammatory factor interleukin-6 was up-regulated significantly (p < 0.05), the protein expressions of both the nuclear factor kappa B (NF-κB) p65 and the inducible nitric oxide synthase were enhanced significantly (p < 0.05) in the AFB₁ group. All of these negative effects were inhibited by α-LA. These results indicate that α-LA may be effective in preventing hepatic oxidative stress, down-regulating the expression of hepatic pro-inflammatory cytokines, as well as inhibiting NF-κB expression.     

Fumonisin B(1): oxidative status and DNA damage in rats

Fumonisin B(1) (FB(1)) is a carcinogenic mycotoxin involved in several animal diseases and assumed to be involved in the etiology of some human tumors. FB(1) disturbs the metabolism of sphinganine (Sa) and sphingosine (So), increasing the ratio of their concentrations (Sa/So). FB(1) is mutagenic in cell cultures, but the mechanism of its genotoxicity is not understood. The aim of this study was to see whether DNA lesions in kidney and liver cells of rats treated with FB(1) were related to the changes in the oxidative status or to the disturbance of the sphingolipid metabolism. Male Wistar rats were receiving either FB(1) (0.5 mg/kg b.w./day, i.p. for 2 or 7 days) or solvent only and were sacrificed 24 h after the last treatment. The ratio of Sa and So concentrations and parameters of oxidative status (catalytic activity of catalase and the concentrations of protein carbonyls and malondialdehyde, MDA) were measured in plasma and liver and kidney homogenates, while DNA damage was measured in liver and kidney using the comet assay. In plasma and liver and kidney homogenates catalase activity and the concentrations of protein carbonyls and MDA were not affected by the 2-day treatment with FB(1), but the ratio of Sa and So in plasma and liver and kidney homogenates was significantly higher than in controls (0.99+/-0.27 versus 0.38+/-0.08, 1.05+/-0.12 versus 0.59+/-0.09 and 4.51+/-0.51 versus 0.54+/-0.17, respectively) (p<0.05). After the 2-day treatment, the tail length and tail intensity measured with the comet assay in the liver homogenate did not change, while in the kidney homogenate, the difference between the treated and control animals was significant in both the tail length (26.4+/-0.7 microm versus 14.6+/-0.1 microm) and tail intensity (8.0+/-0.4% versus 1.7+/-0.02% DNA) (p<0.05). After the 7-day treatment all measured parameters significantly differed from controls (p<0.05). This study showed that FB(1) causes DNA lesions in the kidney of experimental animals before affecting the catalytic activity of catalase and the concentration of protein carbonyls and MDA. The ratio of Sa and So significantly increases in all tissues already after 2-day treatment thus indicating that the metabolism of sphingolipids may have an important role in the DNA damage caused by FB(1).     

Elevation of 8-hydroxydeoxyguanosine in DNA from isolated mouse lung cells following in vivo treatment with aflatoxin B(1).

Aflatoxin B(1) (AFB(1)) is a mycotoxin produced by some strains of Aspergillus and is a recognized pulmonary and hepatic carcinogen. The most widely accepted mechanism of AFB(1) carcinogenicity involves bioactivation to AFB(1)-8,9-exo-epoxide and binding to DNA to form AFB(1)-N(7)-guanine. Another potential cause of DNA damage is AFB(1)-mediated stimulation of reactive oxygen species formation, leading to oxidation of DNA bases. The objective of this study was to determine the ability of AFB(1) to cause oxidative DNA damage in lung cell types of the A/J mouse. The formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in freshly isolated mouse lung alveolar macrophages, alveolar type II cells, and nonciliated bronchial epithelial (Clara) cells was assessed by high-performance liquid chromatography with electrochemical detection. An approximately 3-fold increase in 8-OHdG formation occurred in both alveolar macrophage and Clara cell preparations isolated from A/J mice 2 h following treatment with a single tumorigenic dose of 50 mg/kg AFB(1) ip (n = 3, p < 0.05). Prior treatment with 300 kU/kg polyethylene glycol-conjugated catalase prevented the AFB(1)-induced increase in 8-OHdG levels in all mouse lung cell preparations (n = 3, p < 0.05). These results support the possibility that oxidative DNA damage in mouse lung cells contributes to AFB(1) carcinogenicity.     

The effects of mycotoxins,fumonisin B1 and aflatoxin B1, on primary swine alveolar macrophages

Mycotoxins were fungal metabolites that were widely present in feed and food; some of them were known to associate with human and animal disease. In the present study, the effects of fumonisin B1 (FmB1) and aflatoxin B1 (AFB1) on swine alveolar macrophages (AM) were examined by exposing primary cultures of swine AM to various concentrations of mycotoxins. Incubation of AM with 5 microg/ml of FmB1 for 72 h led to a reduction in the number of viable cells to 65% of the control levels. In the presence of 1.5 microg/ml of AFB1, the viability of AM falls to less than 41% of controls after 24 h exposure. FmB1, but not AFB1, induced the apoptosis of swine AM with evidence of DNA laddering and nuclear fragmentation. However, both FmB1 and AFB1 exposure induced the expression of apoptosis-related heat shock protein 72 (HSP 72) in AM. Swine AM treated with 50 ng/ml of FmB1 and 100 ng/ml of AFB1 for 24 h led to a reduction in phagocytic ability to approximately 55 and 36% of the control levels, respectively. Incubation of AM with FmB1 (2 and 10 microg/ml) for 24 h dramatically decreased the mRNA levels of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha). However, AFB1 treatment did not affect the expression of IL-1beta and TNF-alpha mRNA. The results suggest that both FmB1 and AFB1 are immunotoxic to swine AM but that they exert their toxic effects via different biochemical mechanisms.