Identification of hydroxylated metabolites in 2,2',4,4'-tetrabromodiphenyl ether exposed rats

Faeces from day 1-5 of orally administered 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) in rat have been analysed for hydroxylated metabolites. Six hydroxylated tetrabrominated diphenyl ethers, as well as three hydroxylated tribrominated diphenyl ethers found, were structurally identified. They were 2'-hydroxy-2,4,4'-tribromodiphenyl ether, 3'-hydroxy-2,4,4'-tribromodiphenyl ether, 4'-hydroxy-2,2',4-tribromodiphenyl ether, 6-hydroxy-2,2',4,4'-tetrabromodiphenyl ether, 2'-hydroxy-2,3',4,4'-tetrabromodiphenyl ether, 3-hydroxy-2,2',4,4'-tetrabromodiphenyl ether, 5-hydroxy-2,2',4,4'-tetrabromodiphenyl ether, 4'-hydroxy-2,2',4,5'-tetrabromodiphenyl ether and 4-hydroxy-2,2',3,4'-tetrabromodiphenyl ether. The analysis was performed using gas chromatography-mass spectrometry (GC-MS). The identification of the hydroxylated polybrominated diphenyl ether (OH-PBDE) metabolites in the rat faeces was supported by similar relative retention times (RRTs) versus 2,2',3,4,4',5-hexabromodiphenyl ether (BDE-138) on two columns of different polarities compared to the authentic references. The identification of the OH-PBDE metabolites was also supported by full scan electron ionisation mass spectra. Two of the identified OH-PBDE metabolites have identical structures as natural products, which previously have been isolated from marine sponges and an ascidian.     

Structure of the major metabolite of 1-propyl-3,5-diethyl-6-chlorouracil in rabbits

The major metabolite of 1-propyl-3,5-diethyl-6-chlorouracil has been identified as 6,8-diethyl-2-methyl-tetrahydrooxazolo[3,2-c]pyrimidine-5,7(4H,6H)-dione. The biotransformation to this bicyclic barbituric acid derivative takes place via substitution of the chlorine by ß- and not by α-hydroxy group of the intermediate hydroxypropane.     

Urinary and serum metabolites of di-n-pentyl phthalate in rats

Di-n-pentyl phthalate (DPP) is used mainly as a plasticizer in nitrocellulose. At high doses, DPP acts as a potent testicular toxicant in rats. We administered a single oral dose of 500 mg kg⁻¹ bw of DPP to adult female Sprague-Dawley rats (N = 9) and collected 24-h urine samples 1 d before and 24- and 48-h after DPP was administered to tentatively identify DPP metabolites that could be used as exposure biomarkers. At necropsy, 48 h after dosing, we also collected serum. The metabolites were extracted from urine or serum, resolved with high performance liquid chromatography, and detected by mass spectrometry. Two DPP metabolites, phthalic acid (PA) and mono(3-carboxypropyl) phthalate (MCPP), were identified by using authentic standards, whereas mono-n-pentyl phthalate (MPP), mono(4-oxopentyl) phthalate (MOPP), mono(4-hydroxypentyl) phthalate (MHPP), mono(4-carboxybutyl) phthalate (MCBP), mono(2-carboxyethyl) phthalate (MCEP), and mono-n-pentenyl phthalate (MPeP) were identified based on their full scan mass spectrometric fragmentation pattern. The ω − 1 oxidation product, MHPP, was the predominant urinary metabolite of DPP. The median urinary concentrations (μg mL⁻¹) of the metabolites in the first 24 h urine collection after DPP administration were 993 (MHPP), 168 (MCBP), 0.2 (MCEP), 222 (MPP), 47 (MOPP), 26 (PA), 16 (MPeP), and 9 (MCPP); the concentrations of metabolites in the second 24 h urine collection after DPP administration were significantly lower than in the first collection. We identified some urinary metabolic products in the serum, but at much lower levels than in urine. Because of the similarities in metabolism of phthalates between rats and humans, based on our results and the fact that MHPP can only be formed from the metabolism of DPP, MHPP would be the most adequate DPP exposure biomarker for human exposure assessment. Nonetheless, based on the urinary levels of MHPP, our preliminary data suggest that human exposure to DPP in the United States is rather limited.     

Effect of the trichothecene deoxynivalenol on brain biogenic monoamines concentrations in rats and chickens

Male Sprague-Dawley rats (180 g) and 28-day-old Single Comb White Leghorn Cockerels (300 g) were orally dosed with deoxynivalenol (DON) at 2.5 mg kg-1 body weight. In the first experiment, whole brains were collected at 2, 6, 12, 24 and 48 hours after the toxin treatment and analyzed for brain biogenic monoamines by high-performance liquid chromatography with electrochemical detection. Although several interesting trends were observed, DON did not influence whole brain concentrations of monoamine neurotransmitters or their metabolites in either species, at any time. In a second experiment, brains were collected 24 hours postdosing, dissected into 5 brain regions (pons and medulla oblongata, cerebellum, hypothalamus, hippocampus and cerebral cortex), and analyzed. DON treatment resulted in significantly elevated concentrations of serotonin (HT) and 5-hydroxyindole-3-acetic acid (HIAA) in all brain regions of the rat. However, this was not seen in poultry, where DON treatment resulted in a decrease in norepinephrine (NE) in the hypothalamus and hippocampus, and a decrease in dopamine (DA) in the pons and medulla oblongata region. These results suggest that DON influences brain biogenic amine metabolism, and that there may be intraspecies differences in the central effects of this mycotoxin.     

Identification and dose dependency of ibuprofen biliary metabolites in rainbow trout

The biotransformation of the anti-inflammatory drug ibuprofen (IBF) was studied by exposing rainbow trout (Oncorhynchus mykiss) to IBF via intraperitoneal (i.p.) injection, and via water at four (0.17, 1.9, 13 and 145 μg L⁻¹) exposure levels for 4 d. Following exposure, the bile was collected and analyzed by LC–MS/MS methods. The identification of the formed metabolites in i.p. injected fish bile was based on the exact mass determinations by a time-of-flight mass analyzer (Q–TOF–MS) and on the studies of fragments and fragmentation patterns of precursor ions by ion trap mass analyzer (IT-MS). In addition to unmetabolized IBF, several phase I and phase II metabolites were found in the bile. The main metabolites were acyl glucuronides and taurine conjugates of IBF and of hydroxylated IBFs. The bioconcentration factors (BCF_bile), defined as the ratio of the sum of IBF and its metabolites in fish bile to the concentration of IBF in water, was determined following enzymatic deconjugation and was found to range from 14 000 to 49 000. The highest BCF_bile was found at the lowest exposure concentration (0.17 μg L⁻¹). The results show that rainbow trout has a high capacity for biotransformation of IBF, and the exposure of fish to sub μg L⁻¹ concentrations of IBF can be determined by the analyses of the biliary metabolites of the compound.     

Effect of phenobarbital, naphthoflavone and diethlmaleate on the sex dependent urinary pattern of hexachlorobenzene metabolites in the rat

The sex related difference in the urinary excretion of pentachlorothiophenol (PCThP) was investigated in rats after a single oral dose of hexachlorobenzene (HCB) and compared to that obtained after a single oral dose of pentachloronitrobenzene (PCNB). A ten fold difference was obtained in both cases in favour of females. The effect of phenobarbital (PB) and β-naphtoflavone (β–NF) and the effect of diethylmaleate (DM) on the urinary pattern of HCB metabolites was investigated in male and female rats. PB enhanced the urinary excretion of pentachlorophenol (PCP) and tetrachlorohydroquinone (TCHQ) but had no effect on the excretion of PCThP. β-NF did not have any effect on any of the three main metabolites while an hepatic glutathione depletion induced by DM significantly reduced the excretion of PCThP in females. The results suggest that, after HCB or PCNB intake, a common sex dependent biotransformation step leads to the formation of PCThP, after conjugation with glutathione, independently of the pathway leading to the formation of PCP which is under the control of a PB inducible form of cytochrome P-450.     

Identification and significance of phenazone drugs and their metabolites in ground- and drinking water

Residues of three phenazone-type pharmaceuticals have been identified in routine analyses of groundwater samples from selected areas in the north-western districts of Berlin, Germany. Phenazone, propiphenazone, and dimethylaminophenazone have been detected in some wells at concentrations up to the low microg/l-level. Additionally, three phenazone-type metabolites namely 1-acetyl-1-methyl-2-dimethyl-oxamoyl-2-phenylhydrazide (AMDOPH), 1-acetyl-1-methyl-2-phenylhydrazide, and dimethyloxalamide acid-(N'-methyl-N-phenyl)-hydrazide have also been identified in these groundwater samples. The residues are suspected to originate from former production spills of a pharmaceutical plant located in a city north of Berlin. It was observed that with the exception of AMDOPH all other residues were efficiently removed during conventional drinking water treatment. The drug metabolite AMDOPH deriving from dimethylaminophenazone residues was found at concentrations of 0.9 microg/l in finished drinking water. However, a following study on the toxicological relevance of the AMDOPH residues has shown that there is no toxicological harm for humans at the low concentrations of AMDOPH observed in Berlin drinking water.     

Relation of dietary inorganic arsenic exposure and urinary inorganic arsenic metabolites excretion in Japanese subjects

Inorganic arsenic (InAs) is a ubiquitous metalloid that has been shown to exert multiple adverse health outcomes. Urinary InAs and its metabolite concentration has been used as a biomarker of arsenic (As) exposure in some epidemiological studies, however, quantitative relationship between daily InAs exposure and urinary InAs metabolites concentration has not been well characterized. We collected a set of 24-h duplicated diet and spot urine sample of the next morning of diet sampling from 20 male and 19 female subjects in Japan from August 2011 to October 2012. Concentrations of As species in duplicated diet and urine samples were determined by using liquid chromatography-ICP mass spectrometry with a hydride generation system. Sum of the concentrations of urinary InAs and methylarsonic acid (MMA) was used as a measure of InAs exposure. Daily dietary InAs exposure was estimated to be 0.087 µg kg^?1 day^?1 (Geometric mean, GM), and GM of urinary InAs+MMA concentrations was 3.5 ng mL^?1. Analysis of covariance did not find gender-difference in regression coefficients as significant (P > 0.05). Regression equation Log ₁₀ [urinary InAs+MMA concentration] = 0.570× Log ₁₀ [dietary InAs exposure level per body weight] + 1.15 was obtained for whole data set. This equation would be valuable in converting urinary InAs concentration to daily InAs exposure, which will be important information in risk assessment.     

Synthesis of anti-12-hydroxyendrin and 12-ketoendrin,the two major mammalian metabolites of endrin

The first synthesis of $\text{syn}$ and $\text{anti}$ 12-hydroxylated derivatives of endrin (and thence 12-ketoendrin) has been accomplished in three steps starting from hexachlorocyclopentadiene and 7-benzoyloxy [2.2.1]hepta-2,5-diene.     

Acetochlor mineralization and fate of its two major metabolites in two soils under laboratory conditions

The degradation of the herbicide acetochlor, in a neoluvisol and in a calcosol were studied as a function of depth (0-25cm and 25-50cm) and temperature (25 degrees C and 15 degrees C) under controlled laboratory conditions during 58 and 90 days, respectively. The surface and sub-surface soil samples were respectively spiked with 1 and 0.01mgkg(-1) of 14C-acetochlor, the concentrations observed in previous field monitoring. The half-lives (DT50) varied from 1.4 to 14.9 days depending on the soil, temperature and applied concentration. The maximal mineralization (24%) was observed for the surface calcosol at 25 degrees C. The comparison of results obtained for sterilized and non-sterilized soils, the decrease of DT50 with the increase of temperature, the shape of CO2 emissions and the increase of number of aerobic endogenous microflora through the experiment suggested that biological process are dominant in degradation. A particular attention was paid to the formation and dissipation of metabolites ESA (ethanesulphonic acid) and OA (oxanilic acid) during the whole experiment. At 25 degrees C, ESA and OA were observed after three days, but as ESA concentration decreased over time in surface calcosol, it remained constant in surface neoluvisol. A difference in ESA/OA ratio depends on the soil with a predominance of OA in surface neoluvisol and a disappearance of OA in surface calcosol.     

Relationship of urinary arsenic metabolites to intake estimates in residents of the Red River Delta, Vietnam

This study investigated the status of arsenic (As) exposure from groundwater and rice, and its methylation capacity in residents from the Red River Delta, Vietnam. Arsenic levels in groundwater ranged from <1.8 to 486 μg/L. Remarkably, 86% of groundwater samples exceeded WHO drinking water guideline of 10 μg/L. Also, estimated inorganic As intake from groundwater and rice were over Provisional Tolerable Weekly Intake (15 μg/week/kg body wt.) by FAO/WHO for 92% of the residents examined. Inorganic As and its metabolite (monomethylarsonic acid and dimethylarsinic acid) concentrations in human urine were positively correlated with estimated inorganic As intake. These results suggest that residents in these areas are exposed to As through consumption of groundwater and rice, and potential health risk of As is of great concern for these people. Urinary concentration ratios of dimethylarsinic acid to monomethylarsonic acid in children were higher than those in adults, especially among men, indicating greater As methylation capacity in children.     

Stimulating effect of activated charcoal beads on fecal excretion of 2,3,4,7,8-pentachlorodibenzofuran in rats

We investigated by using rats whether activated charcoal beads can stimulate the fecal excretion of and reduce the toxicity of 2,3,4,7,8-pentachloro-dibenzofuran (PenCDF), one of the most important causal agents of Yusho. The hypertrophy of liver and atrophy of thymus caused by an oral administration of PenCDF at a dose of 1 mg/kg were suppressed significantly by activated charcoal beads treatment for 3 weeks after a week interval from the day of PenCDF administration. Fecal excretion of PenCDF was stimulated approximately 3-fold, and the PenCDF level in the liver and blood tended to decline by this treatment.     

Identification of oxidized TNT metabolites in soil samples of a former ammunition plant

Water extracts of soil samples of the former ammunition plant “Tanne” near Clausthal-Zellerfeld, Lower Saxony, Germany, were investigated for highly polar oxidized 2,4,6-trinitrotoluene (TNT) metabolites. 0.4 to 9.0 mg/kg dry soil 2,4,6-trinitrobenzoic acid (TNBA) and 5.8 to 544 mg/kg dry soil 2-amino-4,6-dinitrobenzoic acid (2-ADNBA) were found. In addition to the oxidized metabolites, TNT, 4- and 2-aminodinitrotoluene (4- and 2-ADNT), and 2,4-dinitrotoluene (2,4-DNT) were extractable with water. Most interestingly, in one sample, 2-ADNBA represented the main contaminant. The origin of the oxidized nitroaromatics is unknown at this time. They might be generated chemically or photochemically. Furthermore, a biological synthesis seems possible.     

Structure of the major metabolite of 5-ethyl-2′-deoxyuridine in rats

The major metabolite of 5-ethyl-2′-deoxyuridine in rats has been isolated and purified by preparative thick layer and column chromatography. By mass and ¹H NMR spectroscopy, it was identified as 5-(1-hydroxyethyl) uracil; the structure was confirmed by comparison with an authentic sample.     

Neurotoxic effects of leptophos (PhosvelR) in chickens and rats following chronic low-level feeding.

Leptophos (O-[4-bromo-2,5 dichlorophenyl] O-methyl phenylphosphonothioate) (PhosvelR) was administered orally to chickens and rats in doses of 0.5 and 5.0 mg/kg/day for 26 weeks. Hens fed 5.0 mg/kg, except one, showed ataxia and became paralysed in the legs at varying times from 8 to 19 weeks. A fifth hen showed ataxia early in the experiment but recovered fully for the remainder of the experiment. Rats fed both doses and chickens fed 0.5 mg/kg showed no signs of delayed neurotoxicity. All hens fed 5.0 mg/kg stopped laying by about the third week. Animals of both species fed 5.0 mg/kg either lost weight (chickens) or gained less weight (rats) than the others. Erythrocyte acetylcholinesterase (AChE) of the chickens given both doses was significantly depressed at first, then increased, and later dropped to control levels. AChE of rats fed 0.5 mg/kg was significantly inhibited but soon recovered to within control levels. On the other hand, the AChE of rats fed 5.0 mg/kg was inhibited throughout the experiment. Plasma cholinesterase (ChE) of both species was first inhibited and then recovered erratically for both insecticide concentrations. Histological alterations in the spinal cord of paralysed hens included axon and myelin degeneration in the ventral, lateral and posterior columns. In the paralysed hens, 79% of the neurotoxic esterase in the brain were inhibited, whereas in the non-paralysed hens (including the one non-paralysed hen receiving 5.0 mg/kg/day) and all rats only about half as much was inhibited.     

Identification of phase II in vivo metabolites of alkyl-anthracenes in rainbow trout (Oncorhynchus mykiss)

Metabolism of xenobiotics is a two-step process that increases the polarity of compounds to facilitate their excretion. In previous work, the major in vitro phase I metabolites of alkyl-anthracenes by rainbow trout (Oncorhynchus mykiss) CYP enzymes were shown to be predominantly ring hydroxylated metabolites. Here, we present the first report on the identification of in vivo phase II metabolites of alkyl-anthracenes in juvenile rainbow trout. Bile was collected from trout injected with individual alkyl-anthracenes with, in some cases, a co-injection of β-naphthoflavone (BNF). Some samples were digested with the β-glucuronidase enzyme to confirm the presence of glucuronide conjugates. The metabolites were separated using a water-acetonitrile gradient on a HPLC system equipped with a C₁₈ column and a UV-diode array detector. Trout with endogenous and BNF-induced enzymes produced the same metabolites, but higher concentrations of metabolites were detected after enzyme induction. Alkyl-anthracenes were metabolized predominantly on the rings as evidenced by the UV spectral analysis. Likewise, mass spectrometry and UV spectral analysis confirmed a predominance of glucuronide conjugates for all systems investigated.     

Identification of a novel hydroxylated metabolite of 2,2′,3,5′,6-pentachlorobiphenyl formed in whole poplar plants

Polychlorinated biphenyls (PCBs) are a group of persistent organic pollutants consisting of 209 congeners. Oxidation of several PCB congeners to hydroxylated PCBs (OH-PCBs) in whole poplar plants has been reported before. Moreover, 2,2′,3,5′,6-pentachlorobiphenyl (PCB95), as a chiral congener, has been previously shown to be atropselectively taken up and transformed in whole poplar plants. The objective of this study was to determine if PCB95 is atropselectively metabolized to OH-PCBs in whole poplar plants. Two hydroxylated PCB95s were detected by high-performance liquid chromatography-mass spectrometry in the roots of whole poplar plants exposed to racemic PCB95 for 30 days. The major metabolite was confirmed to be 4′-hydroxy-2,2′,3,5′,6-pentachlorobiphenyl (4′-OH-PCB95) by gas chromatography-mass spectrometry (GC-MS) using an authentic reference standard. Enantioselective analysis showed that 4′-OH-PCB95 was formed atropselectively, with the atropisomer eluting second on the Nucleodex β-PM column (E2-4′-OH-PCB95) being slightly more abundant in the roots of whole poplar plants. Therefore, PCB95 can at least be metabolized into 4′-OH-PCB95 and another unknown hydroxylated PCB95 (as a minor metabolite) in whole poplar plants. Both atropisomers of 4′-OH-PCB95 are formed, but E2-4′-OH-PCB95 has greater atropisomeric enrichment in the roots of whole poplar plants. A comparison with mammalian biotransformation studies indicates a distinctively different metabolite profile of OH-PCB95 metabolites in whole poplar plants. Our observations suggest that biotransformation of chiral PCBs to OH-PCBs by plants may represent an important source of enantiomerically enriched OH-PCBs in the environment.     

Identification of 2,6-diisopropylnaphthalene metabolites in carp part 1. In vivo experiment

The metabolism of diisopropylnaphthalene (DIPN) in fish was studied from an ecotoxicological viewpoint to clarify the environmental fate of DIPN, a solvent of carbonless paper which is widely used as a substitute for PCB. This study identified the following metabolites in carp by in vivo experiment; 2-(1-hydroxy-1-methyl) ethyl-6-isopropylnaphthalene, 2-(1-methyl-2-hydroxyethyl)-6-isopropyl-naphthalene, 2,6-(1-hydroxy-1-methyl) ethyl-naphthalene, 2-(1-hydroxy-1-hydroxymethyl) ethyl-6-isopropylnaphthalene and α-[2-(6-isopropylnaphthyl)] propionic acid. Identification was based on UV, IR, MS, and NMR analyses. The metabolism of 2,6-DIPN in carp was concluded to mainly through oxidation of the isopropyl chain.     

Monoterpenes and microbial metabolites in the soil

Volatile microbial metabolites were analysed in the soil beneath 15 H year-old Picea abies which had been fumigated for 5 consecutive years with charcoal-filtered air enriched with ozone and sulphur dioxide. Major compounds which could be attributed to the metabolism of basidiomycete fungi were oct-1-en-3-ol, oct-1-en-3-one and 1,3-octadiene. They were accompanied by other C(8)-compounds. The lower amounts of C(8)-compounds observed in the soil of the sulphur dioxide and ozone treatment chambers indicate a reduced development of basidiomycetes. The higher amounts of geosmin and 2-methylisoborneol found in the soil of the ozone treatment chamber indicate an increase in actinomycete populations. The differences of the monoterpene patterns in the particular soils were not large enough to recognize trends unequivocally.     

Determination of fecal contamination origin in reclaimed water open-air ponds using biochemical fingerprinting of enterococci and fecal coliforms

Low levels of fecal indicator bacteria (FIB) were recently detected in two reclaimed water open-air ponds used to irrigate a golf course located in Northeastern Spain. The aim of this study was to evaluate the feasibility of a biochemical fingerprinting method to track the origin of fecal contamination in water with low FIB levels, as in the aforementioned ponds. We also aimed to determine whether FIB presence was due to regrowth of the reclaimed water populations or to a contribution of fecal matter whose source was in the golf facility. Three hundred and fifty enterococcal strains and 308 fecal coliform strains were isolated from the ponds and reclamation plant, and they were biochemically phenotyped. In addition, the inactivation of several microbial fecal pollution indicators (fecal coliforms, total bifidobacteria, sorbitol-fermenting bifidobacteria, somatic bacteriophages, and bacteriophages infecting Bacteroides thetaiotaomicron) was studied using a mesocosm in situ in order to obtain information about their decay rate. Although FIB concentration was low, the biochemical fingerprinting provided evidence that the origin of the fecal contamination in the ponds was not related to the reclaimed water. Biochemical fingerprinting thus proved to be a successful approach, since other microbial source-tracking methods perform poorly when dealing with low fecal load matrices. Furthermore, the mesocosm assays indicated that none of the microbial fecal indicators was able to regrow in the ponds. Finally, the study highlights the fact that reclaimed water may be recontaminated in open-air reservoirs, and therefore, its microbial quality should be monitored throughout its use.