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.     

Reproductive and neurobehavioural effects in three-generation toxicity study of piperonyl butoxide administered to mice

Piperonyl butoxide (PB) was administered continuously to mice from 5 weeks of age in the F₀ generation to weaning of the F₂ generation. PB was administered in the diet at levels of 0 (control), 0.1, 0.2, 0.4 and 0.8%. Selected reproductive, developmental and behavioural parameters were measured. Litter size and litter weight were reduced in higher-dosed groups, and the body weight of the pups in the lactation period was reduced in dosed pups in each generation. The survival index at postnatal day 21 of the group receiving 0.8% PB was reduced in each generation. The developmental and behavioural parameters in the lactation period were little different from those of the controls, apart from olfactory orientation in the F₁ generation. However, in the F₂ generation mice, surface righting, cliff avoidance and olfactory orientation were adversely affected in treatment groups. The results suggest that PB had adverse effects on reproductive, developmental and behavioural parameters of mice, with increasing effects in subsequent generations of offspring.     

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.     

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.     

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.     

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.     

Aflatoxin deposition and clearance in the eggs of laying hens

Hens fed a diet containing 3310 μg of AFB₁ and 1680 μg of AFB₂ per kg feed for 28 days showed a significant decrease in egg production and egg weights by wk 3 and 4 of feeding, respectively. Transfer of aflatoxins to the eggs occurred rapidly, reaching maximum levels after 4–5 days, and remained relatively constant throughout aflatoxin feeding. The mean values for combined residue levels in eggs were less than 0.5 μg/kg. Levels of AFB₂, AFM₁ and AFM₂ were similar in yolk and albumen while levels of B₁ and B_2a were higher in the yolk. Upon removal of the aflatoxin-containing diet, residues in eggs decreased rapidly. Clearance of aflatoxin residues from the albumen occurred faster than from the yolk. Thus, no residues were detected in the albumen and in the yolk after 5 and 7 days of withdrawal, respectively. No aflatoxin residues could be recovered from whole eggs after feeding the aflatoxin-free diet for 4 days.     

Effectiveness of ammonia treatment in detoxification of fumonisin-contaminated corn

Corn throughout the world is frequently contaminated by the fungus Fusarium moniliforme, which produces toxic fumonisins. Ammonia has been shown to detoxify effectively aflatoxins in corn and cottonseed. Since corn can be contaminated by both fumonisins and aflatoxins, we investigated the effects of ammoniation of corn either cultured with or naturally contaminated by F. moniliforme. Fumonisin B₁ levels in the culture material and in naturally contaminated corn were reduced by 30 and about 45%, respectively, by the ammonia treatment. Despite the apparent reduction in fumonisin content, the toxicity of the culture material in rats was not altered by ammoniation. Reduced weight gains, elevated serum enzyme levels and histopathological lesions, typical of F. moniliforme toxicity, occurred in rats fed either the ammoniated or non-ammoniated culture material. Atmospheric ammoniation of corn does not appear to be an effective method for the detoxification of F. moniliforme-contaminated corn.     

Effects of dietary methylmercury on zebrafish skeletal muscle fibres

Mercury and more specifically methylmercury have been reported as hazardous environmental pollutants able to accumulate along the aquatic food chain with severe risk for animal and human health. Adult male zebrafish (Danio rerio) were distributed in two groups: a control group (fed with uncontaminated food) and a MeHg-contaminated group (fed with food containing 13.5 μg Hg g⁻¹ (dry wt)). Five fish per condition were removed after 7, 21 and 63 days. Bioaccumulation of mercury was determined and muscle samples from control and exposed groups were fixed for histological and ultrastructural studies. In contaminated muscles were observed a decrease of the inter-bundle surface, mitochondria with variable shapes, sizes and cristae disorganization, also decreasing the surface area and inter-bundle surfaces. Indeed, damage in the endoplasmic reticulum cisternae was observed. For statistical evaluation the damages in mitochondria was quantified by image. According to the current results, methylmercury affects the structure of fibre cells of D. rerio after trophic and low dose exposure.     

Reproductive and neurobehavioural effects of phloxine administered to mice

The colour additive phloxine was given in the diet to provide dietary levels of 0 (control), 0.1, 0.3 and 0.9%, from 5 wk of age of the F₀ generation to 8 wk of age of the F₁ generation in mice, and selected reproductive and neurobehavioural parameters were measured. There was little effect of phloxine on either litter size or weight, or sex ratio, whereas the body weight of the pups in the lactation period was significantly increased in all treatment groups. Among the neurobehavioural parameters measured, surface righting at postnatal day 4 of male pups was significantly reduced in all treatment groups. Some parameters of the motor activity of pups at 3 wk of age differed from those of the controls; in particular, the average speed of movement male pups was significantly reduced in all treatment groups. The dose levels of phloxine in this study produced a few adverse effects in reproductive and neurobehavioural parameters in mice.     

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.     

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.     

Host resistance to Trypanosoma cruzi infection is enhanced in mice fed Fusarium verticillioides (=F. moniliforme) culture material containing fumonisins

Fumonisins, metabolites of Fusarium verticillioides (=F. moniliforme) and related fungi that occur naturally on corn, elicit various organ- and species-specific toxicities. However, immunologic effects of fumonisins are not well characterized. BALB/c mice were fed diets containing F. verticillioides culture material (CM) providing 50 (LD) or 150 (HD) ppm fumonisins (FB₁+FB₂) beginning 1 week before and continuing 5 weeks after challenge with the myotropic Brazil strain of T. cruzi. A control group (ZD) was fed a diet lacking CM. The LD and HD diets caused increases in tissue sphinganine/sphingosine ratios and minimum to mild hepatotoxicity, both of which are typically induced by fumonisins. Nitric oxide (NO) production by peritoneal macrophages from HD mice was significantly higher than by peritoneal macrophages from ZD mice on day 14 after challenge. NO production also was stimulated in macrophages from ZD mice, but the peak response did not occur until day 26 after challenge. Compared with ZD mice, LD and HD mice exhibited reduced parasitemia and decreased numbers of pseudocysts in cardiac muscle. Thus, the CM increased host resistance to T. cruzi by accelerating NO production by macrophages or otherwise enhancing the immune response. The findings provide additional evidence that fumonisins modulate immune function.     

右美托咪定复合丙泊酚在宫颈锥切手术麻醉中的应用

目的探讨右美托咪定(Dex)复合丙泊酚用于宫颈锥切手术麻醉的可行性。方法择期行宫颈锥切术患者75例,ASAⅠ级或Ⅱ级,随机分为3组,每组25例。对照组(C组)、小剂量Dex组(D_1组)、大剂量Dex组(D_2组)于手术开始前15 min分别以微量泵缓慢输注氯化钠注射液10 mL、Dex 0.4μg·kg~(-1)、Dex 0.8μg·kg~(-1),继之启动靶控输注丙泊酚,术中根据患者反应和脑电双频指数(BIS)的变化调整丙泊酚靶浓度,维持合适的麻醉深度,并根据需要注射小剂量芬太尼。记录入室、局麻时、手术开始、术中10 min、术中20 min、术毕以及清醒时的呼吸和血流动力学参数、不良反应发生情况和苏醒时间。结果 3组手术时间、苏醒时间无显著差异(P>0.05)。D_2组丙泊酚c_e(1.5±0.4)mg·L~(-1)、丙泊酚总量(184±54)mg、芬太尼用量(28±11)μg,均显著低于C组[(2.2±0.3)mg·L~(-1)、(259±59)mg、(75±21)μg,P<0.01]和D_1组[(2.0±0.4)mg·L~(-1)、(234±41)mg、(42±16)μg,P<0.01],D_1组芬太尼用量也低于C组(P<0.05)。D_2组脉搏血氧饱和度维持满意,C组和D_1组部分患者出现呼吸抑制,分别有14、4例需要放置通气道或辅助呼吸。C组发生低血压10例,高于D_1组(5例)和D2组(2例),差异非常显著(P<0.01)。而D_2组发生心动过缓12例,高于C组(2例)和D_1组(7例),有显著差异(P<0.05)。结论宫颈锥切手术麻醉中复合应用Dex不仅可减少丙泊酚和芬太尼用量,而且能维持更加稳定的血流动力学和呼吸功能。     

Effects of amaranth on F1 generation mice.

The color additive, amaranth, was given in the diet to provide dietary levels of 0 (control), 0.03, 0.09 and 0.27%, from 5 weeks of age in F₀ generation mice to 9 weeks of age in F₁ generation mice, and some reproductive, developmental and behavioral parameters were measured. There was no effect on the parameters of litters, litter size, pup weight and litter weight. The body weight of pups during the lactation period in the treatment groups increased less significantly, and the survival index at postnatal day (PND) 21 of the amaranth 0.27% group was reduced. Developmental parameters, direction of swimming on PND 4 in male pups and olfactory orientation in each sex were significantly reduced in the treatment groups. The dose levels of amaranth in this study influenced some reproductive, developmental and behavioral parameters in mice.     

Effects of sarin on the operant behavior of guinea pigs

The present study evaluated the dose–response effects of subacute exposure to sublethal doses of the organophosphorus (OP) chemical warfare nerve agent (CWNA) sarin (GB) on the operant behavior of guinea pigs. Dietary restricted guinea pigs, trained to respond for food under a progressive ratio (PR) schedule of reinforcement, were injected five times per week (Monday–Friday) for 2 weeks with fractions (0.1, 0.2, and 0.4) of the established LD₅₀ of GB (42 μg/kg). Changes in body weight, whole blood (WB) acetylcholinesterase (AChE) levels, and operant performances were monitored over the 2 weeks of GB exposure and for an additional 2 weeks following the termination of exposures. There were dose-related changes in body weight and WB AChE levels throughout the exposure and post-exposure periods. Several parameters of PR performance were disrupted during exposure to 0.4 LD₅₀ GB, however, concurrent weight loss indicated the presence of overt toxicity. PR performance recovered following the termination of exposures. Lower doses (0.1 and 0.2 LD₅₀) of GB failed to produce reliable effects on operant performance during the exposure period. Overall responding decreased during exposure to 0.4 LD₅₀ GB, resulting in reduced response rates and break points. The decrease in overall response rates was attributed to an increase in pausing since there was no decrease in running rate. Motor effects of 0.4 LD₅₀ GB were evident as an increase in the proportion of lever press durations ≥ 1.0 s. In the present study, doses of GB lower than 0.4 LD₅₀ produced no marked alteration of operant performance in guinea pigs, although WB AChE levels were maximally inhibited to 20% of control.     

Hepatotoxicity and renal toxicity in rats of corn samples associated with field cases of equine leukoencephalomalacia

Currently there is no convenient bioassay to determine the potential toxicity of corn naturally contaminated with Fusarium moniliforme. A short-term bioassay would be useful for future investigations aimed at isolating as yet unidentified toxins produced by this fungus. Two groups of five male Sprague-Dawley rats were each fed one of two F. moniliforme contaminated corn samples, designated CS-1 and CS-2, that were associated with separate field cases of equine leukoencephalomalacia (ELEM). A control group, also consisting of five male rats, was fed uncontaminated seed corn. All animals survived to the end of the study and there were no apparent differences in appearance or behaviour among groups. Weight loss and irregular food consumption occurred in all groups and probably resulted from nutritional deficiencies inherent in the corn diets. Hepatocellular degeneration, necrosis and hyperplasia as well as biliary hyperplasia were found in the test groups only and were attributed to F. moniliforme. Serum transaminase and alkaline phosphatase activities in animals fed CS-1 and CS-2 for 4 wk were significantly increased compared with the controls, while serum bilirubin concentration was increased only in the CS-1 group. Tubular nephrosis was also present in the renal cortex of all animals fed CS-1 and CS-2. These effects may have been related to fumonisins B1 and B2, recently discovered metabolites of F. moniliforme, that were found in both CS-1 and CS-2. Short-term studies of this type may be useful in screening naturally-contaminated grains and other materials for hepatotoxic metabolites produced by F. moniliforme.     

A comparison of the selectivities of SCH 23390 with BW737C89 for D1, D2 and 5-HT2 binding sites both in vitro and in vivo

The in vitro affinities (K_Is) for SCH 23390 in D₁, D₂ and 5-HT₂ binding assays were 0.4, 631 and 20 nM as compared with 0.3, 79 and 79 nM for BW737C89. The K_B values, derived from their abilities to right-shift dopamine-mediated dose-dependent increases in striatal adenylyl cyclase activity, were 0.8 and 0.5 nM for SCH 23390 and BW737C89, respectively. Thus, BW737C89 was a highly potent dopamine D₁ receptor antagonist and, although it was less D₁/D₂-selective than SCH 23390, it was more D₁/5-HT₂-selective. Both SCH 23390 and BW737C89 (0.1–100 μmol/kg s.c.) exhibited a selective dose-dependent protection of D₁, but not D₂, binding, from inactivation by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ, 8 mg/kg s.c.) as measure by [³H]SCH 23390 (D₁) and [³H]spiperone (D₂) binding. The ED₅₀ values for this selective protection of D₁ binding were similar and were between 1 and 3 μmol/kg s.c. BW737C89 showed no protective effect at all on the inactivation of [³H]ketanserin (5-HT₂) binding by EEDQ whereas SCH 23390 started to show protection at doses of 10 μmol/kg. s.c. and above. A direct comparison of the time course of the effects of pretreatment of a dose of 30 μmol/kg s.c. of both compounds to protect 5-HT₂ binding was carried out. This study confirmed the complete lack of protective effect of BW737C89 from 1 to 4 h of pretreatment whereas SCH 23390 exhibited 62, 29 and 28% protection at 1,2 and 4 h pretreatment respectively. Thus, these data clearly show that BW737C89 is a potent and selective D₁ antagonist which is more selective for D₁ receptors in vivo than is SCH 23390.     

Immunological evaluation of the mycotoxin patulin in female b6C3F1 mice

Patulin is a mycotoxin produced by many fungal species of the genera Penicillium, Aspergillus and Bryssochamys. Previous literature reports have suggested that patulin is toxic to the immune system. The studies presented were conducted to provide a comprehensive assessment of the effects of patulin on the immune system. Unlike previous reports, the doses of patulin used (0.08, 0.16, 0.32, 0.64, 1.28 and 2.56 mg/kg) were based on predicted human exposure levels. Female B6C3F₁ mice were exposed orally to patulin for 28 days. Effects were not observed on final body weight or body weight gain. Relative weight of the liver, spleen, thymus, kidneys with adrenals, and lungs was not affected. Peripheral blood leucocyte and lymphocyte counts were decreased by approximately 30% in the two highest dose groups. The leucocyte differential was not altered. Total spleen cell, total T-cell (CD3⁺), helper T-cell (CD4⁺CD8⁻), B-cell (surface immunoglobulin⁺) and monocyte (MAC-3⁺) counts were not changed. Cytotoxic T-cell (CD8⁺CD4⁻) counts were increased 50% only by the highest dose. Natural killer cell (NK1.1⁺CD3⁻) and monocyte (MAC-1⁺) counts were increased 30% and 24%, respectively, only in the 0.08 mg/kg group. Humoral immune function as assessed by antibody-forming cell response and serum IgM titre to sheep erythrocytes, and cell-mediated immune function evaluated utilizing natural killer cell activity and the mixed lymphocyte reaction were not altered. Oral exposure to patulin for 28 days did not alter the ability of female B6C3F₁ mice to mount either a cell-mediated or humoral immune response.