Distribution of aflatoxin B1 in tissues of mink (Mustela vison).

Seven female mink (Mustela vison) were injected intraperitoneally with a single dose of 100 mug aflatoxin B1 (14 C-label and unabeled). They were sacrificed 1, 2, 4, and 24 h after dosing. Liver, instestines, stomach, lung, kidney, brain, pancrease, spleen, urinary bladder, uterus, and bile were removed and examined for the retained radioactivity. 1 h after dosing, intestines and their contents retained the largest amount of 14C-radioacivity (18.9% of the amount that was administered) which was followed by liver (13.2%) and the bile (10.8%). At this time all other tissues retained less than 1% of the administered radioactivity. Generally, the amount of radioactivity retained in all tissues declined with time. Only 1.2 and 0.6% of the administered radioactivity was found in testines and bile, respectively, 24 h after dosing; however, the liver still contained 6.6% of the initial radioactivity. Examination of subcellular fractions of liver revealed that at all time intervals most of the radioactivity was associated with the micrososmal supernatant fluid.     

Phytoestrogens alter the reproductive organ development in the mink (Mustela vison)

The aim of the present study was to examine the reproductive effects of two perorally applied phytoestrogens, genistein (8 mg/kg/day) and beta-sitosterol (50 mg/kg/day), on the mink (Mustela vison) at human dietary exposure levels. Parental generations were exposed over 9 months to these phytoestrogens and their offspring were exposed via gestation and lactation. Parents and their offspring were sampled 21 days after the birth of the kits. Sex hormone levels, sperm quality, organ weights, and development of the kits were examined. The exposed females were heavier than the control females at the 1st postnatal day (PND). The control kits were heavier than the exposed kits from the 1st to the 21st PND. Phytoestrogens did not affect the organ weights of the adult minks, but the relative testicular weight of the exposed kits was higher than in the control kits. The relative prostate weight was higher and the relative uterine weight lower in the beta-sitosterol-exposed kits than in the control kits. Moreover, the plasma dihydrotestosterone levels were lower in the genistein-exposed male kits compared to the control male kits. This study could not explain the mechanisms behind these alterations. The results indicate that perinatal phytoestrogen exposures cause alterations in the weight of the reproductive organs of the mink kits.     

Chronic fuel oil toxicity in American mink (Mustela vison): systemic and hematological effects of ingestion of a low-concentration of bunker C fuel oil

Petroleum oil enters the coastal marine environment through various sources; marine mammals such as sea otters that inhabit this environment may be exposed to low concentrations of petroleum hydrocarbons through ingestion of contaminated prey. The inability to perform controlled studies in free-ranging animals hinders investigations of the effects of chronic petroleum oil exposure on sea otter morbidity and mortality, necessitating the development of a reliable laboratory model. We examined the effects of oral exposure to 500 ppm bunker C fuel oil over 113-118 days on American mink, a species phylogenetically related to the sea otter. Hematological parameters and organs were examined for fuel oil-associated changes. Hepatic cytochrome P4501A1 mRNA expression and fecal cortisol concentrations were also measured. Ingestion of fuel oil was associated with a decrease in erythrocyte count, hemoglobin concentration (Hgb), hematocrit (HCT), and an increase in mean corpuscular volume (MCV). Total leukocytes were elevated in the fuel oil group from increases in neutrophils, lymphocytes, and monocytes. Significant interactions between fuel oil and antigen challenge were found for erythrocyte parameters, monocyte and lymphocyte counts. Liver and adrenal weights were increased although mesenteric lymph node weights were decreased in the fuel oil group. Hepatic cytochrome P4501A1 mRNA was elevated in the fuel oil group. Fecal cortisol concentration did not vary between the two groups. Our findings show that fuel oil exposure alters circulating leukocyte numbers, erythrocyte homeostasis, hepatic metabolism and adrenal physiology and establish a framework to use mink as a model for sea otters in studying the systemic effects of marine contaminants.     

Methods for measuring mutagenicity in urine of rats dosed with [14C]di(2-ethylhexyl)phthalate

Di(2-ethylhexyl)phthalate (DEHP) is extensively used as a plasticizer for vinyl plastic articles. It has been found to be positive in an NCI rodent bioassay but has generally given negative results in in vitro genotoxicity tests. We therefore decided to test the urine of rats fed [14C]DEHP for mutagenic activity in the Ames Salmonella test. The recovery of radioactivity from the urine of rats dosed with [14C]DEHP was examined by solvent extraction and XAD-2 resin absorption procedures. Both of these procedures were inadequate for quantitative recovery of urinary metabolites required for subsequent mutagenicity testing using the Ames Salmonella/microsome procedure. Recoveries of less than 5% were observed using standard solvent extraction techniques whereas the XAD-2 adsorption technique gave about 67% at high resin/urine ratios. Treatment of the urine with beta-glucuronidase/aryl sulfatase did not affect these recoveries. The direct urine plating procedure represents a viable alternative to the above concentration procedures for this phthalate ester. The effects of L-histidine and the beta-glucuronidase/aryl sulfatase preparation on the background reversion frequencies of the Ames tester strains is discussed.     

Residues of aflatoxin B1 in broiler meat: Effect of age and dietary aflatoxin B1 levels

This study describes the effect of dietary levels of aflatoxin B1 (AFB1) and age of the birds upon the residue level in liver and muscles of broiler chicks. In three different experiments broiler chicks of 7, 14 and 28 days of age were kept for 7 days on contaminated rations having 1600, 3200 and 6400 μg/kg AFB1. AFB1 residues were detected earlier in younger birds and those fed high AFB1 dietary levels. The highest residue levels in liver and muscles of young chicks fed 6400 μg/kg AFB1 was 6.97 ± 0.08 and 3.27 ± 0.05 ng/g, respectively. Maximum residue concentration was high in birds of young age and those kept on high AFB1 ration. After withdrawal of AF contaminated rations, residues clearance was slow and AFB1 was detectable in liver and muscles of birds for longer duration in younger birds and those fed high AFB1 dietary levels. AFB1 residues in poultry tissues may buildup to high levels in areas with no regulatory limits on AFB1 levels of poultry feed and may pose a risk to consumers health.     

Partitioning and kinetics of methylmercury among organs in captive mink (Neovison vison): A stable isotope tracer study

Despite the importance of methylmercury (MeHg) as a neurotoxin, we have relatively few good data on partitioning and kinetics of MeHg among organs, particularly across the blood–brain barrier, for mammals that consume large quantities of fish. The objective of this study was to determine the partition coefficients between blood and brain, liver and kidney and fur for MeHg under steady-state conditions and to measure the half-lives for MeHg in these organs. Captive mink (Neovison vison) were fed a diet enriched with two stable isotopes of Hg, Me¹⁹⁹Hg and Me²⁰¹Hg for a period of 60 days. After a period of 10 days the diet was changed to contain only Me²⁰¹Hg so that, between days 10 and 60, we were able to measure both uptake and elimination rates from blood, brain, liver kidney and fur. Liver and kidney response was very rapid, closely following changes in blood concentrations but there was a small lag time between peak blood concentrations and peak brain concentrations. Half-lives for MeHg were 15.4, 10.2 and 13.4 days for brain, liver and kidney, respectively. There was no measurable conversion of the MeHg to inorganic Hg (IHg) in the brain over the 60 day period, unlike in liver and kidney.     

Metabolism and excretion of [14C]taranabant,a cannabinoid-1 inverse agonist,in humans

1. Taranabant (N-[(1S,2S)-3-(4-Chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-methyl-2-{[5-(trifluoromethyl)pyridin-2-yl]oxy}propanamide or MK-0364) is an orally active inverse agonist of the cannabinoid 1 (CB-1) receptor that was under development for the management of obesity. The metabolism and excretion of taranabant were investigated following a single oral dose of 5?mg/201 μCi [¹⁴C]taranabant to six healthy male subjects. The overall excretion recovery of the administered radioactivity was nearly quantitative (～?92%), with the majority of the dose (～?87%) excreted into faeces and a much smaller fraction (～?5%) into urine.

2. Taranabant was absorbed rapidly, with C_max of radioactivity attained at 1–2-h postdose. The parent compound and its monohydroxylated metabolite, M1, were the major radioactive components circulating in plasma and comprised ～?12–24% and 33–42%, respectively, of the plasma radioactivity for up to 48?h. A second monohydroxylated metabolite, designated as M1a, represented ～?10–12% of the radioactivity in the 2- and 8-h postdose plasma profiles.

3. Metabolite profiles of the faeces samples consisted mainly of the (unabsorbed) parent compound and multiple diastereomeric carboxylic acid derivatives derived from oxidation of the geminal methyl group of the parent compound and of the hydroxylated metabolite/s. These data suggest that, similar to rats and monkeys, taranabant is primarily eliminated in humans via oxidative metabolism and excretion of metabolites via the biliary/faecal route.

     

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.     

Effect of butylated hydroxytoluene on the disposition of [14C]aflatoxin B1 in the lactating rat

The distribution and metabolism of an ip dose of [14C]aflatoxin B1 (AFB1) were studied in lactating Sprague-Dawley rats fed for the previous 13 days on a diet containing 0.5% butylated hydroxytoluene (BHT). Compared with ingestion of a BHT-free diet, treatment with BHT increased the biotransmission of AFB1 metabolites, predominantly aflatoxin M1 (AFM1), into the mammary gland and its content of milk, decreased AFB1 binding to liver nuclear DNA and enhanced the excretion of water-soluble metabolites of AFB1, all measured 6 hr after an oral dose of [14C]AFB1. These changes are related to the induction by BHT of hepatic enzymes involved in the transformation and detoxification of AFB1. The results suggest that exposure to BHT may protect the lactating animal from the carcinogenic effect of AFB1 but may increase the risk of exposure of the newborn infant to the carcinogenic metabolite AFM1.     

Reduction in binding of [14C] aflatoxin B1 to rat liver macromolecules by phenobarbitone pretreatment.

Various dietary regimens have been shown to alter the acute and chronic toxicity of aflatoxin B₁ (AFB₁) in rats^1–4. Phenobarliitone (PB), an inducer of liver mixed function oxidases⁵ reduces the liver carcinogenioity of AFB₁ contaminated peanut meal⁶, decreases the LD₅₀ after an intraperitoneal injection of AFB₁ from 1 to 5 mg/kg (Gamer, Miller and Miller, unpublished) and reduces the inhibitory action of AFB₁ on rat liver ribonucleic acid synthesis⁷. The protective effect of PB contrasts with $\text{in vitro}$ findings that AFB₁ metabolism is increased by this enzyme-inducing agent, both to detoxification products such as aflatoxin B_2a⁸ and aflatoxin M₁⁹ as well as to AFB₁ 2, 3-oxide, the probable ultimate carcinogenic form of this compound¹⁰. To investigate this apparent discrepancy further the macro-molecular binding of [¹⁴C] AFB₁ after a single intraperitoneal injection has been studied in control and PB pretreated animals.     

Clinical features of aflatoxin B1-exposed patients with liver cancer and the molecular mechanism of aflatoxin B1 on liver cancer cells

Aflatoxin B1 (AFB1) induces hepatocellular carcinoma (HCC) through consumption of contaminated food in Southern China. Aldo-keto reductase-7A (AKR7A) functionally plays a potent role in the biodetoxification in the liver. In addition, hepatocellular lipid disorder has found to be closely linked to the development of HCC. This study was, therefore, designed to investigate the potent bioeffect of AKR7A on the lipid metabolism in AFB1-exposed hepatocellular carcinoma cells through assaying human cancerous samples and cell culture. In the baseline data, the HCC patients showed increased contents of AFB1 in sera and cancerous samples. In the clinical parameters, the HCC patients demonstrated changed lipid settings in sera. As revealed by immunostaining and immunoblotting, AFB1-elevated HCC sections showed marked down-regulation of AKR7A expression, accompanied with reduced ApoB expression and increased CD36, S6K1 expressions in the HCC. Studies in the human hepatocarcinoma line HepG2 also showed AFB1-exposure to increase ApoA1, LDL, TC, and TG contents; induce cell proliferation; and reduce hepatocellular AKR7A expression. Furthermore, AKR7A bioactivity was inactivated after treatment with perfluorooctane sulfonate (PFOS), an ApoB activator, in AFB1-dosed HepG2 cells. Collectively, our current findings suggest that hepatocellular AKR7A has a protective role against AFB1-induced cytotoxicity through the regulation of CD36, S6K1 and ApoB expression through the reduction of lipid utilization in malignant liver cells.     

Metabolism and distribution of [14C]glucose in rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

Male Sprague-Dawley rats were given a single, usually lethal, dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 125 micrograms/kg ip in corn oil), or vehicle alone. Twenty-four hours after ip administration of TCDD the animals received an ip injection of 14C-labeled glucose, and the time course and amount of exhalation of 14CO2 were monitored for 8 h continuously and once daily for 20 min for the subsequent 5 d. TCDD treatment reduced the amount of 14CO2 exhaled within 8 h after the injection of [14C]glucose by 33%, as compared to pair-fed controls. Blood levels of radioactivity were affected by TCDD accordingly. No particular organ appeared to act as a sink for the radioactivity not exhaled during these 8 h by the treated animals. TCDD (125 micrograms/kg) induced significant changes in the disposition of radioactivity in heart and brown adipose tissue between 25 and 125 min after the iv injection of [14C]glucose. The areas under the curve of [14C]glucose-derived radioactivity were the same after either iv or ip injection in the blood of TCDD-treated rats, allowing a direct comparison of experiments with iv or ip injection of [14C]glucose. The half-lives of radioactivity in the exhaled air and in feces of treated animals were greatly elevated during the 5 d following administration of [14C]glucose. These results indicate that TCDD induces in rats, within 24 h after dosing, alterations in the metabolism of glucose that preceded changes in insulin homeostasis, because hypoglycemia and hypoinsulinemia in rats do not occur until about a week after TCDD treatment. Since overt signs of acute toxicity (reduced feed intake and body weight loss) are also not noticeable until several days after a lethal dose of TCDD, it is probable that this earlier disturbance of glucose metabolism is part of the biological changes that result in wasting away and eventually in death.     

Distribution of [14C]-Labelled Aflatoxin B1 in Mice

Abstract The distribution of [¹⁴C]-labelled aflatoxin B₁ has been studied in mice with the aid of whole-body autoradiography. In addition to the localisation of labelled aflatoxin B₁ and/or its metabolites in the liver, bile, kidney, lung and urine an uptake of ¹⁴C in the pigment of the Harderian gland and the eye was observed. Uptake of radioactivity was also found in the eyes of the foetuses although their livers did not accumulate radioactivity.     

[14C]Dimethyltitanocene and [14C]Methyl(methyltrimethylsilyl) titanocene–Reagents for the [14C]olefination of carbonyl compounds: synthesis of [14C]Aprepitant

A neurokinin (NK‐1) receptor antagonist, [¹⁴C]Aprepitant, was synthesized using two labeled olefination reagents: [¹⁴C]dimethyltitanocene 1 and [¹⁴C]methyl (methyltrimethylsilyl)titanocene 7. Both reagents can be readily prepared from [¹⁴C]methyllithium and have been shown to convert a variety of carbonyls to [¹⁴C]methylenes in good radiochemical yields. Copyright © 2009 John Wiley & Sons, Ltd.     

Cause-effect linkages between chemicals and populations of mink (Mustela vison) and otter (Lutra canadensis) in the Great Lakes basin

Following outbreaks of reproductive failure in commercial ranching operations, laboratory experiments showed that mink are extremely sensitive to organochlorine chemicals, particularly PCBs and dioxins. The purpose of this paper is to test the hypothesis that, since wild mink are exposed to these compounds through consumption of Great Lakes fish, they might exhibit reproductive dysfunction and population declines. The otter, another piscivorous animal, should show the same effects. The available information is reviewed according to five epidemiological criteria. Harvest data are presented as a surrogate for the population status of mink and otters in certain locations around the Great Lakes. Data from Ohio show that the mink harvest between 1982 and 1987 from contaminated counties bordering Lake Erie was consistently lower (380 animals per year) than those from counties removed from Lake Erie (850 animals per year), suggesting an effect of chemicals on the status of mink populations. Preliminary studies from Ontario also suggest that mink harvest is lower in potentially "high PCB exposure areas" compared with lower exposure areas. Evidence is also presented on the harvest data for otters taken from four New York State counties adjacent to Lake Ontario and the St. Lawrence River. The harvest data from these four counties show that between 1960 and early 1970 otter harvest remained stable but has since increased. Increased harvest is consistent with improved water quality in Lake Ontario during the past 15 yr. Data relating to strength of association between chemicals and populations of mink and otter are weak and need to be further analyzed. The specificity of the effects of the chemicals on mink reproduction and mortality is well established from toxicological experiments, but there is poor resolution of the information on effects using field data. The strongest case for a causal relationship comes from consideration of the coherence criterion. In conclusion, before a causal link can be drawn between the status of mink and otter populations and exposure to organochlorine chemicals from the Great Lakes, a large amount of research and data analysis needs to be undertaken.     

Identification and quantitation of 7-(benzo[a]pyren-6-yl)guanine in the urine and feces of rats treated with benzo[a]pyrene

The major identified benzo[a]pyrene (BP)-DNA adduct formed by cytochrome P-450 contains BP bound at the C-6 position to the N-7 position of guanine (BP-N7Gua). This adduct is rapidly depurinated from DNA. When rats were treated with [14C]BP, about 0.02% of the administered dose of BP was excreted as BP-N7Gua in feces and urine within 5 days. Chloroform extracts of the urine and feces were analyzed by high-pressure liquid chromatography. The structure of the adduct was established by cochromatography with electrochemically prepared BP-N7Gua and by fluorescence line narrowing spectrometry. This study represents the first demonstration that BP-N7Gua is formed in vivo in animals treated with BP.     

Zinc inhibits aflatoxin B1-induced cytotoxicity and genotoxicity in human hepatocytes (HepG2 cells)

Aflatoxin B1 (AFB1) has strong carcinogenicity. Consumption of AFB1-contaminated agricultural products and the occurrence of hepatocellular carcinoma have received widespread attention. The aim of this paper was to investigate whether zinc supplementation could inhibit AFB1-induced cytotoxicity and genotoxicity in HepG2 cells and the mechanism of this inhibition. Our data suggest that zinc sources can relieve a certain degree of AFB1-induced cytotoxicity and genotoxicity by protecting against apoptotic body formation and DNA strand breaks, affecting S phase cell cycle arrest, reducing 8-OHdG formation, inhibiting global DNA hypomethylation and regulating gene expression in antioxidation, zinc-association and apoptosis processes. Consequently, zinc stabilizes the integrity of DNA and improves cell survival.These data provides new insights into the protective role of zinc in alleviating AFB1-induced cytotoxicity and mediating epigenetic changes in hepatocytes, demonstrating that zinc sources have detoxification properties in mycotoxin-induced toxicity.     

Exposure to aflatoxin B1 in late gestation alters protein kinase C and apoptotic protein expression in murine placenta

Mycotoxins are chemicals with diverse toxicities that are produced by fungi. Aflatoxin B1 is commonly found in plant food, and is generally regarded as one of the most toxic mycotoxins. In the present study, pregnant ICR mice were given p.o. daily doses of aflatoxin B1 at 0, 0.05, 0.5, 5 mg/kg for 4 days (from E13.5 to E16.5). Compared to the control group, time of delivery was shortened and low birth weight was induced in mice treated with 0.5 and 5 mg aflatoxin B1/kg, respectively. Placental tissue isolated from pregnant mice at E17.5 showed that the mRNA expression of crh was increased in aflatoxin-treated groups. This upregulation might signify premature delivery. Further analysis indicated that Pkc proteins were activated and Bcl-2 was reduced in the placental tissue of the aflatoxin-treated groups. Reduction of the anti-apoptotic proteins, on the other hand, might affect the morphorgenesis and maintenance of the placenta.     

Changes in hepatic cytosolic glutathione S-transferase activity and expression of its class-P during prenatal and postnatal period in rats treated with aflatoxin B1

The effect of aflatoxin B1 (AFB1) on the expression of glutathione S-transferase-P (GST-P) which is the major isoform of GST in developmental stages has been investigated in rat liver during prenatal and postnatal stages. Following administration of AFB1 (0, 0.5, 1.0, 2.0, 3.0 or 4.0 mg/kg bw) injected I.P on day 8.5 of gestation the number of dead or reabsorbed fetuses and malformed embryos were recorded. Then the fetal livers were processed for measurement of total GST and GST-P activities, using 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (ETA) as substrates respectively. RT-PCR using rat GST-P specific primers was performed on mRNA extracted from livers. Besides, the effects of AFB1 on hepatic GST and GST-P were assessed in groups of suckling rats directly injected with the toxin. The results show that a single dose of AFB1 (1.0 or 2.0 mg/kg bw) caused approximately 50–60% depletion in fetal liver GST towards CDNB. Postnatal experiments revealed that liver GST (using CDNB as substrate) was significantly induced (~40%) in suckling rats injected with a single dose of AFB1 (3.0 mg AFB1/kg) 24 h before killing. Liver GST-P expression was unaffected due to AFB1 exposures of rats before and after the birth. This finding was substantiated by western blotting and RT-PCR techniques. These data suggest that AFB1-related induction in rat liver total GST after birth may be implicated in protective mechanisms against AFB1. In contrast, inhibition of this enzyme in fetal liver following placental transfer of the carcinogen may explain high susceptibility of fetal cells to transplancental aflatoxins. Furthermore, lack of influence of AFB1 on GST-P expression in developmental stages can role out the involvement of this class of GST in AFB1 biotransformation.     

A clonal cell line (BME-UV1) as a possible model to study bovine mammary epithelial metabolism: metabolism and cytotoxicity of aflatoxin B1

Despite the toxicological risks to which humans and animals are exposed due to the transfer of toxic xenobiotic metabolites into milk of domestic animals, studies on the metabolizing mechanisms occurring in ruminant mammary gland are totally lacking. To investigate the possible biotransformation capabilities of a bovine mammary epithelial cell line (BME-UV1), monolayers were exposed to aflatoxin B1 (AFB1 - 1.0-8.0 μM). Starting from 4 h of exposure, the hydroxylate metabolite aflatoxin M1 (AFM1) was detected in media by high performance liquid chromatography. AFM1 concentration increased linearly with time for 36-48 h and the percent biotransformation of AFB1 (2-4 μM) at 48 h was about 12-14%. Parallel cytotoxicity assays (neutral red uptake-NRU and MTT assays) were performed to investigate the possible interference of AFB1 cytotoxicity with cellular metabolism. MTT assay (from 24 h of cell exposure) and NRU assay (from 16 h of cell exposure) showed time-dependent and time/concentration-dependent decrease of cell viability, respectively, and the former assay being more successful at revealing cytotoxic effects (NRU: CC₅₀ at 48 h = 12.00 ± 2.66 μM; MTT: CC₅₀ at 72 h = 20.42 ± 7.30 μM). The results suggest that BME-UV1 cells express metabolizing enzymes having catalytic activity, thus representing a potential in vitro model for studying biotransformation in bovine mammary gland.