Gerald L. Larsen

Tissue disposition, excretion and metabolism of 2,2',4,4',6-pentabromodiphenyl ether (BDE-100) in male Sprague-Dawley rats.

The absorption, disposition, metabolism and excretion study of orally administered 2,2′,4,4′,6-pentabromodiphenyl ether (BDE-100) was studied in conventional and bile-duct cannulated male rats. In conventional rats, >70% of the radiolabelled oral dose was retained at 72 h, and lipophilic tissues were the preferred sites for disposition, i.e. adipose tissue, gastrointestinal tract, skin, liver and lungs. Urinary excretion of BDE-100 was very low (0.1% of the dose). Biliary excretion of BDE-100 was slightly greater than that observed in urine, i.e. 1.7% at 72 h, and glucuronidation of phenolic metabolites was suggested. Thiol metabolites were not observed in the bile as had been reported in other PBDE metabolism studies. Almost 20% of the dose in conventional male rats and over 26% in bile-duct cannulated rats was excreted in the faeces, mainly as the unmetabolized parent, although large amounts of non-extractable radiolabel were also observed. Extractable metabolites in faeces were characterized by mass spectrometry. Monohydroxylated pentabromodiphenyl ether metabolites were detected; mono- and di-hydroxylated metabolites with accompanying oxidative debromination were also observed as faecal metabolites. Tissue residues of [14C]BDE-100 in liver, gastrointestinal tract and adipose tissue contained only parent material. The majority of the 0–72-h biliary radioactivity was associated with an unidentified 79-kDa protein or to albumin.

Development of an ultra-high-pressure liquid chromatography-tandem mass spectrometry multi-residue sulfonamide method and its application to water, manure slurry, and soils from swine rearing facilities.

An analytical method was developed using ultra-high-pressure liquid chromatography-triple quadrupole-tandem mass spectrometry (UHPLC-TQ-MS/MS) to simultaneously analyze 14 sulfonamides (SA) in 6 min. Despite the rapidity of the assay the system was properly re-equilibrated in this time. No carryover was observed even after high analyte concentrations. The instrumental detection limit based on signal-to-noise ratio (S/N)>3, was below 1 pg/microL (5 pg on column) for all SAs except sulfachloropyridazine. Surface water, ground water, soil, and slurry manure contained in storage ponds in and around swine [Sus scrofa domesticus] rearing facilities were analyzed. Sample cleanup for ground water and surface water included using solid phase extraction (SPE) using Oasis hydrophilic-lipophilic balance (HLB) cartridges. The soil and slurry manure required tandem strong anion exchange (SAX) and HLB solid phase extraction cartridges for sample cleanup. With few exceptions, the recoveries ranged from 60 to 100% for all matrices. The minimum detectable levels were below 2.0 ng/L for water, 30 ng/L for slurry manure, and 45 ng/kg for soil except for sulfachloropyridazine. The coefficient of variation (CV) was within 20% for most of the compounds analyzed. Using this method, sulfamethazine concentrations of 2250-5060 ng/L, sulfamethoxazole concentrations of 108-1.47 x 10(6)ng/L, and sulfathiazole concentrations of 785-1700 ng/L were found in the slurry manure. Sulfadimethoxine (2.0-32 ng/L), sulfamethazine (2.0-5.1 ng/L), and sulfamethoxazole (20.5-43.0 ng/L) were found in surface water and ground water. In top soil (0-15 cm), sulfamethazine ranged 34.5-663 ng/kg dry weight in those locations that received slurry manure as a nutrient; no SAs were found in the soil depths between 46 and 61 cm. The speed makes the method practical for medium to high throughput applications. The sensitivity and positive analyte identification make the method suitable for the demanding requirements for real world applications.

Metabolism of mercapturic acid-pathway metabolites of 2-chloro-N-isopropylacetanilide (propachlor) by gastrointestinal bacteria

1. Mercapturic acid pathway metabolites of propachlor, labelled with 14C, were incubated with pig caecal contents, small and large intestinal contents, and pure cultures of gastrointestinal bacteria. 2. The glutathione, cysteine, N-acetylcysteine, and the S-oxide of the N-acetylcysteine conjugates of propachlor (2-chloro-N-isopropylacetanilide) were each metabolized by mixed pig caecal micro-organisms to 2-mercapto-N-isopropylacetanilide. 3. The extent of formation of 2-mercapto-N-isopropylacetanilide by mixed pig caecal micro-organisms from mercapturic acid-pathway metabolites of 14C-propachlor was as follows: glutathione (43.4%), cysteine (32.6%), N-acetylcysteine (5.2%), and the S-oxide of the N-acetylcysteine conjugate of propachlor (6.1%). The S-oxide of the N-acetylcysteine conjugate of propachlor was converted by the mixed pig caecal micro-organisms to N-acetylcysteine and cysteine conjugates of propachlor. 4. Small intestinal contents metabolized the glutathione conjugate of propachlor to the cysteine conjugate; little C-S lyase activity was present in the small intestinal contents. 5. The cysteine conjugate of propachlor was metabolized to 2-mercapto-N-isopropylacetanilide by Fusobacterium necrophorum, Bacteroides vulgatus, Megasphaera elsdenii and Eubacterium aerofaciens.

Distribution of cysteine conjugate β-lyase in gastrointestinal bacteria and in the environment

A cysteine conjugate beta-lyase was detected in 24 of 43 gastrointestinal bacteria tested, and from mixed populations of bacteria obtained from lake, river and soil samples. These bacterial cysteine conjugate beta-lyases catalyse the cleavage of the thioether linkage of both an S-alkyl- and an S-aryl-linked cysteine conjugate (2-S-cysteinyl-N-isopropylacetanilide (cysteine conjugate of propachlor) and S-(2-benzothiazolylcysteine), respectively). A cysteine conjugate beta-lyase was isolated from Eubacterium limosum at levels at least 16-fold greater than those from any other gastrointestinal bacterial tested. This enzymic activity was present from the late lag phase through the stationary phase of growth of the bacteria. Maximum activity occurred in the mid log phase. A cysteine conjugate beta-lyase isolated from animal and plant tissues cleaved only the S-aryl-linked cysteine conjugate (S-(2-benzothiazolyl)cysteine) and generally had less enzymic activity than enzymes isolated from the bacteria. Glutathione-S-transferase activity was not detected in the 43 gastrointestinal bacteria tested, except for a low level (0.6 nmol/min per mg) of activity in Escherichia coli. No S-methyl transferase activity was detected in the gastrointestinal bacteria or mixed populations of bacteria tested.

Enterohepatic circulation in formation of propachlor (2-chloro-N-isopropylacetanilide) metabolites in the rat

1. Bile secreted from rats given single oral doses of 2-chloro-N-isopropylacetanilide (propachlor) contained 58% dose as metabolites from the mercapturic acid pathway (glutathione, mercapturate, cysteine conjugates, and a sulphoxide of the mercapturate). 2. Bile secreted from rats given single oral doses of the cysteine conjugate of propachlor contained glucuronide conjugates of hydroxylated 2-methylsulphonyl-N-isopropylacetanilides. 3. In contrast, when the intestinal microflora were bypassed by intravenous administration of the cysteine conjugate of propachlor, the bile contained only the mercapturate and the sulphoxide of the mercapturate. 4. Rats fed an antibiotic-containing diet and given single oral doses of either propachlor or the cysteine conjugate of propachlor excreted only mercapturic acid pathway metabolites in the urine, bile, and faeces, and no methylsulphonyl-containing metabolites. Faecal 14C from the antibiotic-fed rats given either propachlor or the cysteine conjugate of propachlor was extractable, in contrast to previously reported unextractable faecal 14C residues from untreated rats given propachlor orally. 5. From these results, we conclude that metabolism by the microflora was necessary for production of the methylsulphonyl-containing metabolites excreted by the rat. Enterohepatic circulation of the xenobiotic moiety of these mercapturic acid pathway metabolites is influenced by the presence of a microbial C-S lyase.

Occurrence and pathways of manure-borne 17β-estradiol in vadose zone water

The hormone 17beta-estradiol (E2) can cause endocrine disruption in sensitive species at part per trillion concentrations. The persistence and transport pathways of manure-borne E2 in agricultural soils were determined by comparing its occurrence with the transfer of water and the transport of non-sorbing fluorobenzoic acid (FBA) tracers. This comparison was done using capillary wick lysimeters installed 0.61m beneath three corn (Zea mays L.) plots that receive swine (Sus scrofa domesticus) manure from various sources. An additional control plot was included that received no manure. Soil water transfer was modeled to compare actual versus predicted percolation. On average, lysimeters collected 61% of the expected percolation and 8% of the FBA. There were frequent E2 detections, where there were an average of 8 detections for the 11 sample events. The average detection was 21ngL(-1) and its range was 1-245ngL(-1). 17beta-Estradiol was detected before manure was applied and also in the control plot lysimeters. Furthermore, the average mass recovery of E2 in all the lysimeters was >50%, which was greater than the FBA tracer recovery. Results indicated that tracer was transported with precipitated water infiltrating into the soil surface and percolating down through the soil profile. There was substantial evidence for antecedent E2, which was persistent and mobile. The persistence and mobility of the E2 may result from its associations with colloids, such as dissolved organic matter. Furthermore, this antecedent E2 appeared to overwhelm any observable effect of manure management on E2 fate and transport.

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 μmoles 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.

Metabolism of 2,6-Dichlorobenzonitrile, 2,6-Dichlorothiobenzamide in Rodents and Goats

1. Twelve 14C-labelled metabolites were isolated from either urine or bile from either rats (11 metabolites) or goats (7 metabolites) given single oral doses of 2,6-dichlorobenzo[14C]nitrile (DCBN). Five of these metabolites were also excreted in urine from rats dosed orally with 2,6-dichlorothiobenz[14C]-amide (DCTBA). 2. All metabolites from either DCBN or DCTBA were benzonitriles with the following ring substituents: Cl2, OH (three isomers); Cl2, (OH)2; Cl, (OH)2; Cl, OH, SH; Cl, OH, SCH3; SCH3, SOCH3, OH; Cl2, S-(N-acetyl)cysteine; Cl, S-(N-acetyl)cysteine; Cl, OH, S-(N-acetyl)cysteine. 3. The thiobenzamide moiety of DCTBA was converted to the nitrile in all the excreted urinary metabolites. No hydrolysis of the nitrile in DCBN to either an amide or an acid was detected. 4. Urine was the major route for excretion; however, enterohepatic circulation occurred. 5. Whole-body autoradiography of 14C-DCBN and 14C-DCTBA in mice showed the presence of bound residues in the mucosa of the nasal cavity, trachea, tongue, oesophagus, the kidney, liver and the intestinal contents.

Cysteine conjugate β-lyase in the gastrointestinal bacterium Fusobacterium necrophorum

A cysteine conjugate beta-lyase from the anaerobic gastrointestinal bacterium Fusobacterium necrophorum was purified 51-fold by heat treatment, ammonium sulphate fractionation, gel-filtration chromatography, and anion-exchange chromatography. This enzyme catalyses the cleavage of the thioether linkage in cysteine conjugates of the following S-alkyl- or S-aryl-linked compounds: cysteine conjugate of propachlor (2-S-cysteinyl-N-isopropylacetanilide); 1,2-dihydro-1-hydroxy-2-S-cysteinylnaphthalene and S-(2-benzothiazolyl)cysteine. 2-Mercapto-N-isopropylacetanilide, pyruvic acid and ammonia were produced from the beta-lyase cleavage of the cysteine conjugate of propachlor in equimolar ratios. The apparent Km values for the cysteine conjugate of propachlor and S-(benzothiazolyl)cysteine were 1.1 and 1.0 mM, respectively. Pyridoxal phosphate was required for enzymic activity. Ammonium ion activated enzymic activity, while hydroxylamine completely inhibited the enzyme. Dithiothreitol and bovine serum albumin had no effect on enzymic activity.

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%.