Tissue disposition and excretion of 14C-labelled aflatoxin B1 after oral administration in channel catfish.

The pharmacokinetics, tissue distribution and excretion of ¹⁴C-labelled aflatoxin B₁ (AFB₁) were examined after oral administration (250 μg/kg body weight) in channel catfish (Ictalurus punctatus). Plasma concentrations of parent AFB₁ were best described by a one-compartment pharmacokinetic model, in which peak plasma concentration (503 ppb) occurred at 4.1 hr after dosing. The absorption and elimination half-lives were 1.5 and 3.7 hr, respectively. AFB₁ was highly bound (95%) to plasma proteins. Concentrations of ¹⁴C (in AFB₁ equivalents) measured in the tissues were highest at 4 hr, ranging from 596 ppb in the plasma to 40 ppb in the muscle. AFB₁ residues were rapidly depleted; at 24 hr the concentrations in the plasma and muscle were 32 and <5 ppb, respectively. Concentrations in the bile exceeded 2000 ppb (at 24 hr), whereas the highest concentration in the urine was 51 ppb (4–6-hr collection interval). Renal and biliary excretion accounted for <5% of the administered dose, indicating incomplete absorption. Pharmacokinetic modelling and tissue data demonstrate a very low potential for the accumulation of AFB₁ and its metabolites in the edible flesh of channel catfish through the consumption of AFB₁-contaminated feed.     

Fate of aflatoxin M1 in Iranian white cheese processing

Aflatoxin M₁ (AFM₁) is an important mycotoxin frequently found in milk and dairy products. AFM₁ is a major metabolic product of Aflatoxin B₁ and is usually excreted in the milk and urine of dairy cattle that have consumed aflatoxin-contaminated feed.

The aim of this study was to determine the AFM₁ concentration in curd and whey of Iranian white cheese. The cheese milk samples were artificially contaminated with AFM₁ in six levels (0.25, 0.5, 0.75, 1, 1.25, and 1.75 μg L⁻¹). Cheese was produced according to Iranian traditional recipe. AFM₁ distribution between curd, whey and cheese was determined by high performance liquid chromatography (HPLC) using immunoaffinity column clean up and florescence detection. AFM₁ was recovered in whey, curd and cheese in the concentrations of 0.43, 1.47 and 1.57 μg L⁻¹,respectively. The level of Aflatoxin M₁ in curd and cheese obtained 3.12- and 3.65-fold more than that in whey that shows the affinity of Aflatoxin M₁ to the protein fraction of milk.     

Activity of some respiratory enzymes and cytochrome contents in rat hepatic mitochondria following aflatoxin B1 administration

Effects of aflatoxin B₁ (AFB₁) administration (7 mg/kg body weight i.p.) on rat hepatic mitochondrial respiratory components have been examined. Succinoxidase and cytochrome oxidase activities were decreased in liver mitochondria isolated from rats 12–24 h after AFB₁ treatment. Both enzyme activities returned to normal levels after 48 h. Glutamate dehydrogenase and β-hydroxybutyrate dehydrogenase activities did not show any alterations up to 24 h and thereafter increased at 48–72 h. Succinate dehydrogenase activity was impaired by 41% at 12 h and thereafter was found to be normal. The intramitochondrial cytochrome b content declined at 24–72 h, whereas cytochrome aa₃ content was decreased maximally at 72 h after AFB₁ administration. These observations on mitochondrial enzyme activities and cytochrome contents correlate well with our earlier observations made on hepatic mitochondrial respiratory rates after AFB₁ treatment. The impairment of respiratory functions possibly results from membrane damage and selective modification of gene expression in mitochondria imparted by AFB₁.     

Effects of dietary fat level and aflatoxin B1 treatment on rat hepatic lipid composition

Sprague-Dawley rats were given either ten daily doses of aflatoxin B₁ (AFB₁) or the solvent tricaprylin intragastrically over a 2-wk period and were fed diets containing either 1.6 or 20% corn oil throughout the study. Hepatic lipid composition was analysed in groups of five rats both 3 and 13 wk after the start of treatment, in order to determine short-term and longer-term alterations. Total lipid and cholesterols (total, free and esterified) increased on the high-fat diet at wk 3. At wk 13 only total and esterified cholesterol were increased by 20% corn oil. AFB₁ treatment resulted in large intra-group variations in total lipid and cholesterol at wk 3, but these were no longer apparent by wk 13. AFB₁ produced various alterations in the fatty acid composition of hepatic phosphatidylcholine (PC) and phosphatidylethanolamine (PE), apparent at wk 3 but not at wk 13. The unsaturation index decreased but no changes were seen in the saturated fatty acids. Only in animals fed 20% corn oil did AFB₁ result in significant changes in 18:2, 20:3 and 22:6 fatty acids, while 20:4 and 22:5 tended to decrease and 18:1 to increase in response to AFB₁ treatment with both diets in both phospholipids. The high-corn oil diet was found to increase 18:2, 22:6, and total unsaturation in PC and PE, while the ratio of 20:4 to 18:2 tended to decrease in these phospholipids, γ-Glutamyltranspeptidase, an indicator of liver damage, was significantly increased in AFB₁-treated animals, with the greatest increase over controls in those fed the high-fat diet.     

Immunohistochemical study of proliferating cell nuclear antigen (PCNA) in duckling liver fed with aflatoxin B1 and esterified glucomannan

The effect of esterified glucomannan on aflatoxin B₁ toxicity in ducklings was studied by immunohistochemical staining of proliferating cell nuclear antigen (PCNA) in hepatic cells on formalin-fixed paraffin-embedded liver samples. Cherry Valley ducklings were divided into five groups, 20 birds in each. One of the groups was fed with conventional feed, and the other groups were fed with diet containing 100 ppb aflatoxin B₁, that containing 0.05% esterified glucomannan, or that containing 100 ppb aflatoxin B₁ supplemented with 0.05 or 0.1% esterified glucomannan, from five days of age for one month, and subsequently all the groups were fed with conventional feed for 20 days. Four birds of each group were sacrificed on the 30th, 35th, 40th, 45th and 50th day of feeding, and PCNA on the liver tissue sections was quantitatively analyzed by immunohistochemical staining. The percentage of PCNA-positive hepatocytes was significantly higher in the group given diet containing aflatoxin B₁ than in the other groups, which were not significantly different from each other. The results demonstrate that supplementation of feed with esterified glucomannan is effective in reduction of aflatoxin B₁-induced hepatic injury in ducklings.     

Studies on the in vitro metabolism of aflatoxin B1 and G1 in the liver of rats fed at various levels of vitamin C

Male and female weanling rats, divided into three groups, were maintained for 30, 60 and 90 days on an experimental diet without vitamin C. The drinking water containing or devoid of vitamin C was offered each day. The first group had no vitamin C; the second group or control had normal vitamin C and the third group had above-normal vitamin C in the water. Twenty-four hours after the last feeding, the animals were decapitated and the liver microsomes plus soluble fraction (9000 g) were prepared. This preparation was used for O-demethylase and the hydroxylase assays of aflatoxins B₁ and G₁ (AFB₁ and AFG₁). Results from O-demethylation in both control and experimental groups of both sexes, showed that O-demethylase activity is directly related to ascorbic acid content of the water. But the hydroxylase activity in male and female animals maintained on the same diet showed only slight differences. In the male, there was an increase with time of hydroxylase activity with AFB₁ up to 60 days and a decrease at 90 days. Hydroxylation assay revealed that in the female, metabolism of AFB₁ increased at 30 days but decreased at 60 and 90 days.     

Evaluation of a method to determine the natural occurrence of aflatoxins in commercial traditional herbal medicines from Malaysia and Indonesia

Traditional herbal medicines, popularly known as ‘jamu’ and ‘makjun’ in Malaysia and Indonesia, are consumed regularly to promote health. In consideration of their frequent and prolonged consumption, the natural occurrence of aflatoxins (AF) in these products was determined using immunoaffinity column clean-up and high-performance liquid chromatography with pre-column derivatization. The evaluated method, which entails dilution of sample extracts with Tween 20–phosphate buffered saline (1:9, v/v) and a chromatographic system using isocratic mobile phase composed of water–methanol–acetonitrile (70:20:10, v/v/v), was effective in separating AFB₁, AFG₁ and AFG₂ from interference at their retention times. Results were confirmed using post-column derivatization with photochemical reactor. For 23 commercial samples analyzed, mean levels (incidence) of AFB₁, AFB₂ and AFG₁ in positive samples were 0.26 (70%), 0.07 (61%) and 0.10 (30%) μg/kg, respectively; one sample was positive for AFG₂ at a level of 0.03 (4%) μg/kg. In contrast to the high levels of AF in crude herbal drugs and medicinal plants reported previously by other researchers, the low contamination levels reported in this study may be attributed to the higher selectivity to AF of the method applied. Based on the AFB₁ levels and the daily consumption of positive samples, a mean probable daily intake of 0.022 ng/kg body weight was calculated.     

HPLC residues of enrofloxacin and ciprofloxacin in eggs of laying hens

Eggs of 12 laying hens with 5 mg/kg/day oral administration of 5% enrofloxacin (EFX) or ciprofloxacin (CFX) solution during 5 days contained residues from 0.02 to 1.98 μg/g (EFX) or 0.14 to 0.28 μg/g (CFX). At identical dosage regime High Performance Liquid Chromatograhy (HPLC) residues of EFX were 6-fold greater than CFX ones. Maximun concentrations were detected at the second day after the administration withdrawal. The limits of detection were 0.019 μg/g for EFX and 0.156 μg/g for CFX. The recovery was 36–50% for CFX and 49–85% for EFX. The withdrawal treatment periods in hens are six days for EFX and five days for CFX in order to avoid violative levels of egg residues.     

Changes in egg composition of American kestrels exposed to dietary polychlorinated biphenyls

Changes in the quality of eggs of birds exposed to polychlorinated biphenyls (PCBs) have been described, but have never been directly attributed to PCBs. Polychlorinated biphenyl residues in eggs have been associated with reduced reproductive success and embryonic deformities in wild birds. Egg size and composition, specifically the amount of albumen, yolk, and water in an egg, also influence the growth and viability of embryos and hatchlings, and consequently the reproductive success of birds. To deter mine whether PCB exposure of adult birds affected the size and composition of their eggs, 25 pairs of captive American kestrels (Falco sparverius) were fed a mixture of PCB-spiked (1248:1254:1260) food to give an approximate exposure of 7 mg/kg body weight/d, beginning 1 mo prior to pairing, and continuing throughout the courtship, egg-laying, and incubation periods. This dietary level in the adult female kestrels resulted in mean total PCB residues in the eggs of 34.1 microg/g wet weight (geometric mean), which is environmentally relevant. PCB residues in eggs increased with the time of female exposure to the contaminated diet and laying date. Variation in egg size within PCB clutches was significantly greater than within control clutches, although absolute egg mass and volume did not differ markedly by treatment. Only infertile eggs and only one egg per clutch were used for egg composition analysis. Yolks in the PCB-contaminated eggs were heavier, with less wet and dry albumen relative to control eggs. Water content and eggshell thickness were not significantly affected by PCB exposure. These results suggest that eggs from the PCB treatment have relatively more lipid and less protein available for embryonic development. Changes in egg composition were not associated with egg size, lay date, ambient temperature, humidity, or precipitation, which are factors known to affect these variables in bird eggs. The PCB-induced changes in egg composition described here provide insight into possible mechanisms contributing to reduced reproductive performance in wild birds exposed to PCBs.     

A mixture of antioxidants and fatty acids improves the viability of cultured rat hepatocytes untreated or treated with doxorubicin

The objective of this study was to determine whether medium supplementation by antioxidants and fatty acids would improve the viability of cultured rat hepatocytes and protect them against doxorubicin toxicity. We examined the effects of three agents: vitamin E, sodium pyruvate and egg yolk (the combination of vitamin E, sodium pyruvate and fatty acids is a proprietary, patented technology of Warner Lambert called CRT) 0.3% (v/v) as a source of fatty acids, on cell viability measured by the dehydrogenase-dependent bioreduction of a tetrazolium salt (MTS). Untreated hepatocytes and hepatocytes treated with carbon tetrachloride (CCl₄, EC₅₀ 5.7 m ) or doxorubicin (1 and 30 μ ) were exposed to different amounts of a mixture of antioxidants and fatty acids. The mixture, identified as 1X, provided a final concentration of 5 units of vitamin E, 0.1% egg yolk and 10 m sodium pyruvate while the 3X and 5X mixtures contained proportionately higher concentrations of these components. The mixtures were added 18 hr prior to, simultaneously with or following treatment with doxorubicin and just simultaneously with CCl₄. Neither vitamin E, sodium pyruvate nor egg yolk alone improved viability. However, the viability of untreated hepatocytes improved significantly when the 3X mixture was added after 18 hr as indicated by determination of MTS reduction activity 24 hr later. The viability of doxorubicin treated cultures (1 and 30 μ ) increased significantly when exposed either to the 3X or 5X mixtures simultaneously. A significant increase in viability was also seen when cells were exposed to the 3X mixture following doxorubicin (1 μ ). The mixtures did not protect against toxicity caused by CCl₄, perhaps due to the overwhelming level of damage at its EC₅₀ concentration. It is proposed that the antioxidant properties of vitamin E and sodium pyruvate protect the cells from low levels of reactive oxygen species generated spontaneously in culture and by doxorubicin metabolism while the fatty acids help to maintain the integrity of hepatocyte membranes, resulting in greater viability of the hepatocytes.     

Cyclopiazonic acid inhibits mutagenic action of aflatoxin B(1)

Cyclopiazonic acid and aflatoxin B₁ are mycotoxins which can both be produced by the same moulds. Men can be exposed to these mycotoxins directly via ingestion of plant-derived food, as well as, indirectly via consumption of animal products. Although it is well known that aflatoxin B₁ is mutagenic, contradictory results exist on the mutagenicity of cyclopiazonic acid. Using the Ames test cyclopiazonic acid was not found to be mutagenic either with or without metabolic activation by S9-mix of Arochlor treated rats. However, the mutagenicity of aflatoxin B₁ was inhibited in the presence of cyclopiazonic acid. Since cyclopiazonic acid inhibited the formation of certain metabolites of caffeine and testosterone, it was concluded that the reduction of the mutagenicity of aflatoxin B₁ in the presence of cyclopiazonic acid results from the inhibition of the bioactivation of aflatoxin B₁ by certain cytochrome P450 enzymes.     

Levels of fumonisin B1 in corn naturally contaminated with aflatoxins

Aflatoxins and fumonisin B₁ are hepatotoxic and carcinogenic metabolites produced by Aspergillus flavus and Fusarium moniliforme, respectively. These fungi are common natural contaminants of corn, and both aflatoxins and fumonisin B₁ have been implicated as aetiological agents in animal and human diseases. To determine whether these mycotoxins co-exist on corn under natural conditions, 28 samples from the 1991 Georgia (USA) corn crop were assayed for (total) aflatoxin and fumonisin B₁. 27 samples were positive for aflatoxin, 24 samples were positive for fumonisin B₁, and 23 samples had detectable levels of both. In the positive samples, the mean aflatoxin concentration was 73 ppb (SD = 86), and the average fumonisin B₁ concentration was 0.87 ppm (SD = 0.65). A correlation between aflatoxin and fumonisin B₁ concentrations was not evident. None the less, these results demonstrate that exposure to both mycotoxins can occur simultaneously by consumption of co-contaminated corn.     

Retention of aluminium in the tissues of rats after the discontinuation of oral exposure to aluminium

Two studies were conducted to determine whether the aluminium deposited in the bones, muscles and kidneys of young growing rats fed diets supplemented with A1(OH)₃ (989 or 1070 μg A1/g diet) for 16 days was retained after the A1(OH)₃ was removed from the diets. Al levels in the tissues of test rats decreased significantly (P 0.01) 3 days after withdrawal of the Al(OH)₃ from the diet, and 7 days after withdrawal the tissue concentrations of A1 were similar in the test and control animals. Ingestion of A1 had no effect on tissue levels of phosphorus, calcium, magnesium, zinc or iron.     

安神益脑丸中黄曲霉毒素残留量测定及LC-QQQ/MS确证

目的：建立高效液相色谱-荧光检测器测定安神益脑丸中黄曲霉毒素G₂、G₁、B₂、B₁的方法;分析安神益脑丸中黄曲霉毒素的污染情况。方法：待测样品采用80%乙腈作为提取溶剂,经免疫亲和柱净化、高效液相色谱分离、光化学柱后衍生后,通过荧光检测器测定其中黄曲霉毒素的含量;采用超高效液相色谱串联三重四极杆质谱法对以上实验测定结果进行进一步的确证。结果：在优化条件下,黄曲霉毒素G₂、G₁、B₂、B₁分别在0.19~0.95,0.53~2.65,0.22~1.08,0.51~2.55 ng·mL^-1的范围内有良好的线性关系,r>0.999 5,回收率在86.2%~104.9%之间。结论：该方法操作简便,灵敏度高、重复性好、结果准确,可运用于安神益脑丸中黄曲霉毒素的检测。5批样品中有2批检出黄曲霉毒素,检出率为40%,建议增订该品种黄曲霉毒素的检查项。     

CHANGES IN EGG COMPOSITION OF AMERICAN KESTRELS EXPOSED TO DIETARY POLYCHLORINATED BIPHENYLS

Changes in the quality of eggs of birds exposed to polychlorinated biphenyls (PCBs) have been described, but have never been directly attributed to PCBs. Polychlorinated biphenyl residues in eggs have been associated with reduced reproductive success and embryonic deformities in wild birds. Egg size and composition, specifically the amount of albumen, yolk, and water in an egg, also influence the growth and viability of embryos and hatchlings, and consequently the reproductive success of birds. To deter mine whether PCB exposure of adult birds affected the size and composition of their eggs, 25 pairs of captive American kestrels (Falco sparverius) were fed a mixture of PCB-spiked (1248:1254:1260) food to give an approximate exposure of 7 mg/kg body weight/d, beginning 1 mo prior to pairing, and continuing throughout the courtship, egg-laying, and incubation periods. This dietary level in the adult female kestrels resulted in mean total PCB residues in the eggs of 34.1 µg/g wet weight (geometric mean), which is environmentally relevant. PCB residues in eggs increased with the time of female exposure to the contaminated diet and laying date. Variation in egg size within PCB clutches was significantly greater than within control clutches, although absolute egg mass and volume did not differ markedly by treatment. Only infertile eggs and only one egg per clutch were used for egg composition analysis. Yolks in the PCB-contaminated eggs were heavier, with less wet and dry albumen relative to control eggs. Water content and eggshell thickness were not significantly affected by PCB exposure. These results suggest that eggs from the PCB treatment have relatively more lipid and less protein available for embryonic development. Changes in egg composition were not associated with egg size, lay date, ambient temperature, humidity, or precipitation, which are factors known to affect these variables in bird eggs. The PCB-induced changes in egg composition described here provide insight into possible mechanisms contributing to reduced reproductive performance in wild birds exposed to PCBs.     

A pharmacological study of dopaminergic receptors in planaria

The dopaminergic receptors of planaria have been studied with pharmacological and biochemical criteria. Dopamine D₁ selective agonists (CY 208243 (10 μg/ml) and SKF 38393 (10 μg/ml)) induced in planaria typical screw-like hyperkinesias, that were inhibited by a D₁ antagonist (SCH 23390 (10 μg/ml)), but not by a D₂ antagonist (sulpiride (1000 μg/ml)). Dopamine D₂ selective agonists (PHNO (5 μg/ml), lisuride (5 μg/ml)) on the contrary induced a typical “C” like curling, that was inhibited by pretreatment with D₂ selective blocking agents, but not by D₁ selective blocking agents. With agonists with a D₁ /D₂ mixed action (apomorphine 60 μg/ml) or with amphetamine (100 μg/ml), the D₁ type movements appeared to be more evident.

Dopamine D₁-selective agonists, mixed action agonists or D₂-selective agonists, all induced a significant increase in levels of cAMP, that was prevented by pretreatment with the specific DA blocking agent.     

Supraspinal mu 2-opioid receptors mediate spinal/supraspinal morphine synergy.

TRIMU-5 (Tyr-D-Ala-Gly-NHC₂H₄CH(CH₃)₂) is a potent μ₂-opioid agonist/μ₁-opioid antagonist. A supraspinal dose (0.5 μg i.c.v.) of TRIMU-5 which is not analgesic when given alone antagonizes the analgesia produced by intracerebroventricular (i.c.v.) morphine, a μ₁ action. In contrast, in a synergy model consisting of the simultaneous administration of intrathecal morphine (0.1 μg) with multiple doses of i.c.v. morphine, the same supraspinal TRIMU-5 dose (0.5 μg i.c.v.) enhances analgesia. Supraspinal TRIMU-5 also potentiates spinal morphine directly, shifting its dose-response to the left. These results imply that within the brainstem μ₁ receptors mediate supraspinal analgesia while μ₂ receptors mediate the synergy with spinal μ systems.     

Voltammetric determination of vitamins in a pharmaceutical formulation

Direct current polarography and differential pulse polarographic methods have been developed for the qualitative as well as quantitative analysis of vitamin B₁, B₂ and B₆. Thiamin (Vitamin B₁) produced a well-defined wave in 0.1 M KCl at pH 5.2 with E_1/2=−1.2 V and E_p=−1.22 V versus saturated calomal electrode (SCE). Riboflavin (Vitamin B₂) gave two distinct waves in Britton Robinson buffer at pH 1.8 with E_1/2 VALUES=−0.13 and −0.34 V versus SCE and at pH 6.5 with E_1/2=−1.10 V and E_p=−1.2 V versus SCE. Pyridoxin (Vitamin B₆) produced a well-defined wave in Britton Robinson buffer at pH 6.5 with E_1/2=−1.7 V and E_p=−1.68 V versus SCE. All the three Vitamins under study are reversibly reduced at the electrode surface. The number of electrons involved in the electrode process for vitamin B₁ and B₆ is one in each case where as for the two waves of B₂ it is one and two, respectively. This has been confirmed by the measurement of E_3/4–E_1/4 values and also from the log plot slopes for the reduction waves. The wave height of polarogram was found to be proportional to the vitamin concentration. The developed methods have been standardised for the determination of these compounds in pharmaceutical formulation. The concentration of Vitamin B₁, B₂ and B₆ are found to be 9.96, 9.92 and 3.01 mg, respectively in 240 mg of capsule powder of a standard company (name has not been disclosed due to secrecy purpose). The results have been found to be in excellent agreement to that claimed by the manufacturer. The observed data has been subjected to statistical analysis, which revealed high reliability and precision.     

Effect of cooking on the concentration of Vitamins B in fortified meat products

B vitamins fortification of meat products is useful to compensate the loss of these compounds occurring during the heat treatment. The objective of this study was to evaluate the influence of heat treatments on the B vitamins concentration in fortified meat products. A rapid and reliable method for the simultaneous determination of Vitamins B₁, B₆ and B₁₂ in homogenized boiled ham and in various fortified burgers was set up. Extraction procedure and HPLC method ensure low detection limits, good sensitivity and resolution. Results showed that cooking processes caused a decrease in the B vitamins content both in mild (70–90 °C) and severe (120 °C) conditions. Performing a fortification of 25 μg g⁻¹ the residual concentration of B vitamins after cooking allow to reach the recommended daily allowance, thus suggesting that B vitamins fortification of meat product is an useful practice.