Dioxin congener patterns in commercial catfish from the United States and the indication of mineral clays as the potential source.

Since 1991 the US Department of Agriculture (USDA) has conducted annual surveys of pesticide residues in foods under the Agricultural Marketing Service's Pesticide Data Program (PDP). To assess chemical residues in domestically marketed catfish products, 1479 catfish samples were collected during the 2008-2010 PDPs. A subset of 202 samples was analysed for 17 toxic polychlorinated dibenzo-p-dioxins and furans (PCDD/Fs). The average pattern of the individual PCDD/F congener concentrations in the catfish was rather unique in that it had almost no measurable amounts of polychlorinated dibenzofurans (PCDFs), but all PCDDs were present. This pattern was more dominant in the domestically produced catfish products than in the imported products (China/Taiwan). Comparison of the pattern to known sources of PCDD/Fs showed strong similarities to the pattern of PCDD/Fs found in kaolin clays which have often been used as anti-caking agents in animal feeds. To investigate whether catfish feeds may be the source of the PCDD/Fs found in the catfish, archived catfish feed data from a US Food and Drug Administration (USFDA) database were examined. In 61 out of 112 feed samples, the PCDD concentrations were 50 times higher than the PCDF concentrations and resembled the pattern found in the catfish products and in clays mined in the south-eastern United States. Although the source of PCDD/Fs in domestically marketed catfish products cannot be definitively established, mined clay products used in feeds should be considered a likely source and, given the wide concentration range of PCDD/Fs that has been found in clays, a critical control point for PCDD/Fs entrance to the food supply.

The impact of different housing systems on egg safety and quality.

A move from conventional cages to either an enriched cage or a noncage system may affect the safety or quality, or both, of the eggs laid by hens raised in this new environment. The safety of the eggs may be altered either microbiologically through contamination of internal contents with Salmonella enterica serovar Enteritidis (Salmonella Enteritidis) or other pathogens, or both, or chemically due to contamination of internal contents with dioxins, pesticides, or heavy metals. Quality may be affected through changes in the integrity of the shell, yolk, or albumen along with changes in function, composition, or nutrition. Season, hen breed, flock age, and flock disease-vaccination status also interact to affect egg safety and quality and must be taken into account. An understanding of these different effects is prudent before any large-scale move to an alternative housing system is undertaken.

Absorption, distribution, metabolism and excretion (ADME) study with 2,2',4,4',5,6'-hexabromodiphenyl ether (BDE-154) in male Sprague-Dawley rats.

A metabolism study of orally administered 2,2',4,4',5,6'-hexabromodiphenyl ether (BDE-154; 11.3 micromoles kg(-1)) was conducted in conventional and bile duct-cannulated male Sprague-Dawley rats. In conventional rats, approximately 31% of the radiolabelled dose was retained at 72 h, and lipophilic tissues were the preferred sites for disposition. Urinary excretion of BDE-154 was very low (1.0%), and parent compound was detected. Cumulative biliary excretion was 1.3%, and glutathione conjugates were suggested. Over 62% of the dose in conventional male rats was excreted in faeces, and was composed of parent compound (7.3%), free metabolites (13.1%), and covalently bound residues (41.4%). Faecal metabolites characterized by gas chromatography/mass spectrometry included multiple isomers of monohydroxylated hexa-/penta-/tetrabromodiphenyl ethers, and di-hydroxylated hexa/pentabromodiphenyl ethers. The adipose tissue 14C was extractable BDE-154, but 40% of liver 14C was bound to macromolecules. The study demonstrated the importance of performing individual polybrominated diphenyl ether (PBDE) metabolism studies to understand fully PBDE pharmacokinetics.

Effects of short-chain nitrocompounds against Campylobacter jejuni and Campylobacter coli in vitro.

Effects of 2-nitro-1-propanol, 2-nitroethanol, nitroethane, and 2-nitro-methyl-propionate (0, 10, and 20 mM) on growth of Campylobacter jejuni were tested during culture in Bolton broth adjusted to pH 5.6, 7.0, or 8.2. The nitrocompounds were similarly tested against C. coli but at pH 8.2 only. Viable cell counts measured during incubation revealed main effects (P < 0.05) of all nitrocompounds on the survivability of C. jejuni. An effect of pH (P < 0.05) on the survivability of C. Jejuni during incubation with nitrocompounds was observed, with greater inhibition observed at pH 8.2 than at pH 5.6 or 7.0 for nitroethane, 2-nitro-l-propanol, and 2-nitroethanol, but not for 2-nitro-methyl-propionate, which showed greatest inhibition at pH 5.6. Except for 2-nitro-methyl-propionate, which was ineffective, all nitrocompounds elicited similar effects on C. coli. The effect of nitroethane and 2-nitro-l-propanol (10 mM) on naturally occurring Campylobacter was investigated during incubation of porcine fecal suspensions, where Campylobacter concentrations decreased more rapidly (P < 0.05) in suspensions with added 2-nitro-l-propanol than in unsupplemented or nitroethane-supplemented suspensions, thus reiterating the superior inhibitory effect of 2-nitro-l-propanol.

Effect of sodium [36Cl]chlorate dose on total radioactive residues and residues of parent chlorate in growing swine.

An experimental chlorate-based product has been shown to be efficacious in eliminating economically important, Gram-negative human pathogens in the gastrointestinal tracts of food animals. Prior to the commercial marketing of such a product, the magnitude and chemical nature of residues remaining in edible tissues must be determined. Thus, the objective of this study was to determine the tissue distribution and elimination of sodium [36Cl]chlorate in orally dosed swine. Three sets of pigs, each consisting of a barrow and a gilt, were orally dosed with a total of 20, 40, or 60 mg of sodium [36Cl]chlorate per kg body weight via the drinking water. Urine and feces were collected throughout the 30 h study. Twenty-four hours after the last exposure to [36Cl]chlorate, each pig was harvested and both edible and inedible tissues were collected. Urine and tissue samples were analyzed for total radioactive residues and for chlorate metabolites. Elimination of radioactivity in urine averaged 81.6, 83.7, and 83.9% of the total dose for the low, medium, and high doses, respectively. Fecal elimination of radioactivity averaged 1.1% of the dosed radiochlorine across all doses. Parent chlorate always represented greater than 97.4% of the urinary radiochlorine with the remaining radiochlorine being excreted as chloride ion. Chlorate represented 39-77% of fecal radioactivity, depending upon dose. Chlorate concentrations in edible tissues ranged from 0.01 to 0.49 ppm, with residues in liver and skeletal muscle generally lower than those in kidney and adipose tissue. Chlorate residues were concentrated in thyroid tissues (7.7-25.4 ppm) relative to edible tissues. No evidence for the presence of chlorite was observed in excreta or in tissues. Results of this study suggest that further development of chlorate as a preharvest food safety tool in swine merits consideration.

Levels of polychlorinated dibenzo-p-dioxins and dibenzofurans in cattle raised at agricultural research facilities across the USA and the influence of pentachlorophenol-treated wood.

Adipose tissue samples from 158 cattle raised locally at experiment stations across the USA were analysed for polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/F). While 80% of the samples had PCDD/F concentrations that fell within the range of a previous US survey of beef animals (not detected -4.1 ppt toxic equivalency), several animals had exceptionally high concentrations (8-54 ppt toxic equivalency). The investigations of three facilities where highly contaminated animals were raised found pentachlorophenol-treated wood at each site. The congener pattern in the animals' tissues and the lack of elevated PCDD/F levels in other environmental samples, i.e. hay and soil, indicated that the treated wood was the source of contamination. A congener pattern similar to that of pentachlorophenol-exposed animals was seen for the means and medians of the entire data, i.e. OCDD, HpCDD and 1,2,3,6,7,8-HxCDD dominated, the PCDD concentrations equalled or exceeded the furan concentrations, and the concentration of 1,2,3,6,7,8-HxCDD was six times that of the other HxCDD isomers. This suggested that pentachlorophenol-treated wood contributed measurably to many of the animals in this survey. The largest contributors to the median toxic equivalencies were 1,2,3,7,8-PeCDD (40%) and 1,2,3,6,7,8-HxCDD (16%). No clear geographical trends emerged from the data.

On the isolation of polychlorinated dibenzo-p-dioxins and furans from serum samples using immunoaffinity chromatography prior to high-resolution gas chromatography-mass spectrometry.

Immunoaffinity chromatography (IAC) for the purification of polychlorinated dibenzo-p-dioxins and furans (PCDD/Fs) from biological samples was explored as a means to simplify the cleanup procedure and thereby decrease the time and cost of dioxin analysis. A monoclonal antibody (DD3) was used to produce IAC columns and to isolate the PCDD/Fs from serum. Native and 13C-labeled PCDD/Fs were spiked at the ppq to ppt range into serum. Quantitation of the PCDD/Fs was performed by a standard dioxin analytical method, i.e. high-resolution gas chromatography-mass spectrometry (GC-MS), which was easily compatible with IAC. Five of the most toxic PCDD/Fs consistently showed acceptable recoveries (>25%) and were reliably quantitated. The congeners specifically recovered by this method represent almost 80% of the toxic equivalency of dioxins and furans present in the serum samples. Dioxin-like polychlorinated biphenyls (PCBs) were not recognized by this antibody column. Compared to conventional dioxin cleanup methods, IAC decreased solvent usage by 1.5 l/sample and took only 2 h to process a sample for analysis.

An investigation of the in vivo formation of octachlorodibenzo-p-dioxin.

The in vivo formation of dioxins from chemical precursors was investigated in rats. Sprague-Dawley rats were fed pentachlorophenol or a predioxin in peanut oil for 14 days. Mass balance calculations indicated that pentachlorophenol was not converted to dioxins; however, the predioxin, nonachloro-2-phenoxyphenol, was converted to OCDD. Conversion of the predioxin ranged from 0.5% to 153% and depended on the amount of predioxin and OCDD present in the diet. The analytical procedures used for sample preparation did not appear to cause conversion of the predioxin to OCDD. The mechanism for biological conversion may be enzymatic or spontaneous.

Chlorinated dibenzo-p-dioxin and dibenzofuran concentrations in beef animals from a feeding study.

Four calves were fed polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans for 120 days at levels somewhat higher than what may be found in forage near some waste incinerators and manufacturing plants. Four calves were fed identical diets but without the chemicals. Using bioelectrical impedance measurements of total body fat, 30-50% of the dosed 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD, and 2,3,4,7,8-PeCDF was estimated to be retained by the animals. Although these same congeners were bioconcentrated in adipose tissue (BCF approximately 10), consumer products such as ribeye showed concentrations less than what were found in the animal feed (BCF approximately 0.1). Distribution of the dioxins and furans into various lipid compartments appeared to be rather uniform in back fat, perirenal fat, and ribeye for tetra to hexa congeners. Ribeye, serum, and liver lipids had higher concentrations of the higher chlorinated congeners, due in part to not reaching a steady state. An unexpected source of dioxin and furan contamination was discovered during the experiment, resulting in the control animals having concentrations of some congeners that were equal to or in some cases greater than those of the dosed animals. Pentachlorophenol-treated wood components in the pole barn where the feeding experiment was conducted were found to have contributed to the animals' exposure.

Intermediary metabolism of pentachloronitrobenzene in the control and germ-free rat and rat with cannulated bile ducts.

1. Nearly 70% of single oral doses of 14C-labelled pentachloronitrobenzene (PCNB) was excreted in bile within 24 h. 2. The characterized biliary metabolites of PCNB were either mercapturic acid pathway metabolites or catabolites thereof (thiols, methylthiols, S-glucuronides). 3. A major biliary metabolite was S-(aminotetrachlorophenyl)glutathione. 4. Conjugation with glutathione with subsequent catabolism to bis-methylthiotetrachlorobenzene was the major pathway in the control rat. 5. Germ-free experiments showed that only nitro- group displacement occurred, and no nitro- group reduction was detected.

Metabolism and disposition of 1,4,7,8-tetrachlorodibenzo-p-dioxin in rats.

Metabolism studies of 1,4,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a relatively nontoxic dioxin congener, were undertaken to gain a better understanding of mammalian metabolism of dioxins without the problems associated with the use of the most toxic congener, 2,3,7,8-TCDD. 14C-1,4,7,8-TCDD was dosed to conventional and bile-cannulated rats at a level of 8 mg/kg. The 14C was excreted almost entirely in 72 hours with the major routes of excretion feces and bile. Metabolites were identified from the feces, bile, and urine by GC-MS or negative ion FAB MS and 1H NMR. The two major fecal metabolites were hydroxylated tetra- and triCDDs. Glucuronide and sulfate conjugates of these hydroxyl metabolites were found in the urine and bile. Minor metabolites included dichlorocatechol, dihydroxylated tetra- and triCDDs, and conjugates of these compounds.

Use of an immunoaffinity column for tetrachlorodibenzo-p-dioxin serum sample cleanup.

Covalently linking 1-amino-3,7,8-trichlorodibenzo-p-dioxin with either keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA) provided antigens that generated antibodies in chickens. Competitive ELISA analysis demonstrated that the antibodies isolated from egg yolk (IgY) bound with 1,3,7,8-tetrachlorodibenzo-p-dioxin (1,3,7,8-TCDD). The antibodies were linked to CNBr-Sepharose to generate an immunoaffinity column. Radiolabeled 1,3,7,8-TCDD in a 0.05% Tween 20 solution was retained by the column and could be eluted by increasing the Tween 20 concentration. The binding efficiency for 10.7 ng per ml gel matrix ranged from 85 to 97%. Immunoaffinity columns generated by this method did not effectively bind 14C-1,3,7,8-TCDD from serum samples. Diluting the serum 1:20 with 0.05% Tween 20 increased the binding efficiency. Alternately, ethanol-hexane extraction followed by solid phase extraction on a carbon column using a fat removal protocol also provided an appropriate preaffinity column cleanup for serum samples. After this preaffinity column cleanup, spiked serum samples applied to the immunoaffinity column showed binding efficiencies of over 90%.

Identification of NIH-shifted metabolites of 1,3,7,8-tetrachlorodibenzo-p-dioxin in the rat by NMR comparison with synthesized isomers.

In the rat, 1,3,7,8-tetrachlorodibenzo-p-dioxin was oxidatively metabolized to the NIH-shifted products 2-hydroxy-1,4,7,8-tetrachlorodibenzo-p-dioxin and 3-hydroxy-1, 2,7,8-tetrachlorodibenzo-p-dioxin. The chlorine substitution patterns were determined by comparison with 1H NMR spectra of six synthesized isomers in CDCl3, CD3OD, and acetone-d6. Glucuronide and glucuronide-sulfate conjugates of the monohydroxy dioxins were identified in the bile by FAB-MS. The dominance of the NIH-shift products in the metabolism of tetrachlorodibenzo-p-dioxins indicates that the same isomers may be produced from differently substituted chlorinated dioxins.

An investigation of the binding site of alpha 2u-globulin using isotopically labeled ligands and inverse nuclear magnetic resonance techniques.

A series of isotopically labeled ligands were bound to the protein alpha 2u-globulin. These protein complexes were studied using 13C, 19F, and selective inverse detection NMR experiments to determine chemical shifts and nuclear Overhauser effect correlations for the labeled sites of the ligands. The NMR data indicate that the labeled portions of the ligands are located in a highly aromatic region of the alpha 2u-globulin binding pocket. Molecular modeling based on the NMR data and a medium resolution X-ray crystal structure of alpha 2u-globulin predicts a model for ligand binding which is consistent with experimental observations and calculated ring current effects. Conformational changes in the aromatic region of the binding site upon binding these ligands in solution may be supported by this model.

Glutathione-mediated methylthio-turnover and sex differences in the metabolism of pentachlorothioanisole by rat.

1. Sex differences observed in the metabolism of pentachlorothioanisole in rat were due to: (1) greater excretion in urine by females, and greater biliary excretion by males; (2) formation of pentachlorophenyl mercapturic acid pathway metabolites by females; and (3) redox-cycling between methylthio and methylsulphoxyl oxidation congeners in intermediary metabolites by females. 2. Three methylthio-turnover processes are proposed in the intermediary metabolism of pentachlorothioanisole.

Metabolism and disposition of ractopamine hydrochloride by turkey poults.

Ractopamine HCl, ((1R*,3R*),(1R*,3S*)-4-hydroxy-alpha-[[[3-(4- hydroxyphenyl)-1-methylpropyl]-amino]methyl]benzenemethanol hydrochloride), is a beta-adrenergic agonist that is under evaluation as a nutrient repartitioning agent in livestock. Because ractopamine metabolism has not been evaluated in turkeys, the objectives of this study were to synthesize and identify products of ractopamine metabolism and to determine the stereoselective metabolism and tissue distribution of [14C]ractopamine HCl in orally dosed turkey poults. Glucuronides of diastereoisomeric [14C]ractopamine, and the (1R,3R) and (1R,3S) stereoisomers of ractopamine were synthesized by use of microsomal proteins immobilized on Sepharose beads. Monoglucuronides conjugated to the phenols at C-10 or C-10' were isolated and identified by 1H-NMR and negative ion FAB/MS. Urine and feces were collected from colostomized turkey poults after oral dosing with 20 mg of [14C]ractopamine HCl (9.28 microCi). Radioactive residues in tissues were highest in the gallbladder and liver. Radioactivity was not detectable in blood 48 hr after dosing and was slightly above background (< 100 dpm/g tissue) in skeletal and cardiac muscle. Urine contained 47.5% of the administered radioactivity by 16 hr after dosing, and by 48 hr 52.0% of the radioactivity was excreted in the urine. Feces contained 36.6% and 41.5% of the dose 16 and 48 hr after dosing, respectively. Unmetabolized ractopamine represented only 8% of the urinary radioactivity; ractopamine glucuronides represented 72% of the urinary radioactivity. Glucuronides conjugated to the C-10 phenol of ractopamine represented 59.8% of the urinary metabolites and were composed of all four ractopamine stereoisomers. Glucuronides conjugated to the C-10' phenol of ractopamine represented 12.7% of the urinary metabolites and were composed of the (1R,3R) and (1R,3S) stereoisomers. Ractopamine was rapidly eliminated from turkeys after oral dosing, and glucuronidation was the major pathway of metabolism. Regioselective glucuronidation occurred favoring the C-10 phenol of ractopamine; glucuronidation of the C-10' phenol of ractopamine was specific for the (1R,3R) and (1R,3S) stereoisomers.

Artifact formation during gas chromatographic-mass spectrometric analysis of a methylsulfinyl-containing metabolite.

During gas chromatographic-mass spectrometric analysis using a heated injector, 1-methylthio-4-methylsulfinyltetrachlorobenzene degraded to form tetrachlorothioanisole. Similar reductive defunctionalizations have been reported during in vivo metabolisms. Caution should be used to distinguish metabolites from artifacts which may be formed during the analysis of methylsulfoxides.

Conjugation of polychlorinated agrochemical sulphoxides and sulphones by glutathione.

1. Pentachlorophenyl methyl sulphoxide and pentachlorophenyl methyl sulphone were found to be substrates for microsomal and cytosolic glutathione-S-transferase of rabbit, monkey, chicken and human liver, covalently immobilized on beaded sepharose. 2. Protein was immobilized with greater than 95% transferase activity, measured by dinitrochlorobenzene. Immobilized rabbit liver microsomal transferase activity was more stable than immobilized cytosolic activity. 3. The sulphoxide moiety was displayed by glutathione in the presence of chicken liver microsomal protein. The sulphone moiety was displayed by glutathione in the formation of a diglutathione under catalysis by rhesus monkey liver cytosolic and microsomal protein. 4. Chlorine was displaced by transferases from all species to form regioisomeric monoglutathiones. 5. Qualitative and quantitative differences were observed in product distributions between species and between microsomal and cytosolic protein.

Metabolism of 1,2,4-trichlorobenzene in rats: examination of thiol formation.

1. More than 60% of oral doses of 14C-1,2,4-trichlorobenzene (ca. 21 mg/kg) administered to rats were excreted in bile as S-trichlorophenyl-mercapturic acid pathway metabolites. 2. The biliary metabolites were ultimately excreted in urine mainly as the isomeric mercapturic acids. 3. An acetylated glutathione conjugate was isolated as a major metabolite in bile (8% dose). The acetyl group was shown by mass spectrometry to be on the glutamyl moiety. 4. A glutamylcysteine conjugate of trichlorobenzene was also isolated from bile as a major metabolite (8% dose). 5. Trichlorothiophenols were deduced not to be intermediates or end-products of enzymic metabolism of trichlorobenzene in rats because 14C-2,4,5-trichlorothiophenol dosed i.p. to rats was excreted as the S-glucuronide (17% dose) and as S-(methylsulphonyl-dichlorophenyl)-mercapturic acid (36% dose).

Studies on the displacement of methylthio groups by glutathione.

1. 14C-Methylthio-labelled 2-methylthio-4-ethylamino-6-tert-butylamino-sym-triazine (terbutryn), pentachlorothioanisole (PCTA), and 1,4-bis(methylthio)tetrachloro-benzene (bis-MTTCB) and their methylthio-oxidation congeners were reacted with glutathione (GSH) in the presence and absence of immobilized liver microsomal enzymes. 2. 13C-Methylthio-labelled terbutryn sulphoxide and terbutryn sulphone were used to study displacement of the methylthio moiety by GSH using 13C-n.m.r. 3. Methanesulphinic acid was identified as the group displaced by GSH from the methyl sulphones in vitro. 4. Methanesulphenic acid is proposed to be the group displaced by GSH from methyl sulphoxides forming methyl mercaptan, methyl glutathionyl disulphide and methane-sulphinic acid in vitro. 5. Rats given 14C-methylthio-labelled terbutryn, PCTA, bis-MTTCB, and their methylthio-oxidation congeners excreted 14CO2 and 14C-methanesulphinic acid in varying amounts. These results were compared to the in vitro data.