Arsenolipids in oil from blue whiting Micromesistius poutassou--evidence for arsenic-containing esters.

Arsenic-containing lipids in the oil from the blue whiting fish (Micromesistius poutassou) were separated into three broad polarity groups and investigated by HPLC and mass spectrometry. A total of 11 arsenolipids including 4 new compounds were identified. The polar lipid fraction constituting 24% of the total arsenolipid content (which totalled 2.16 µg As/g) contained four known dimethylarsinoyl fatty acids and three known dimethylarsinoyl hydrocarbons. The less polar fraction (ca 30% of the total arsenolipids) contained four new dimethylarsinoyl hydrocarbons with chain lengths 22-30 carbons, in addition to more complex arsenicals that hydrolysed to known dimethylarsinoyl fatty acids suggesting they were conjugated carboxylic acids, presumably esters. The rest of the lipid-soluble arsenic (ca 45% of the total) remained in the non-polar fraction together with the bulk of the fish oil lipids, a complex mixture of compounds that precluded identification of the small amounts of arsenolipids.

Arsenic-containing lipids are natural constituents of sashimi tuna.

Arsenic occurs naturally in many types of seafood as water- and fat-soluble organoarsenic compounds. Although water-soluble compounds have been well characterized, the fat-soluble compounds, so-called arsenolipids, have until recently remained unknown. We report that sashimi-grade tuna fish, with a total arsenic content of 5.9 microg of As/g dry mass, contains approximately equal quantities of water- and fat-soluble arsenic. The water-soluble arsenic comprised predominantly arsenobetaine (>95%) with a trace of dimethylarsinate. Two fat-soluble compounds, which together accounted for about 40% of the lipid-arsenic, were isolated and characterized. The first was identified as 1-dimethylarsinoylpentadecane [(CH(3))(2)As(O)(CH(2))(14)CH(3)] by comparison of HPLC/mass spectrometric data and accurate mass data with those of an authenticated synthesized standard. The second arsenolipid was postulated as 1-dimethylarsinoyl all-cis-4,7,10,13,16,19-docosahexane from mass spectrometric data and analogy with non-arsenic-containing lipids found in fish. The remaining fat-soluble arsenic consisted of less polar arsenolipids of currently unknown structure. This is the first identification of arsenolipids in commonly consumed seafood.

Identification of arsenolipids with GC/MS.

Arsenic-containing hydrocarbons have recently been reported as natural constituents of fish oil. We report a simple method for determining these compounds by GC/MS. Application of the methodology will delineate the distribution of these novel arsenic compounds in foods, and facilitate an assessment of the toxicological implications.

Arsenic-containing hydrocarbons: natural compounds in oil from the fish capelin, Mallotus villosus.

Arsenic-containing hydrocarbons have been identified for the first time as natural components of fish oil.

Degradation of arylarsenic compounds by microorganisms.

Microorganisms were not directly accumulated when soil contaminated to about 0.5 mM with diphenylarsinic acid (DPAA) was used as the sole source of carbon. However, using toluene as the carbon source yielded several isolates, which were then used in cultivation with DPAA as the sole source of carbon. By these methods, Kytococcus sedentarius strain NK0508, which can grow in up to 0.038 mM DPAA, was isolated. The toxicity of DPAA retarded the growth of K. sedentarius and the direct accumulation of DPAA-utilizing microorganisms from environmental samples. This strain can utilize about 80% of DPAA and phenylarsonic acid as the sole source of carbon for 3 days. Degradation products of DPAA were determined to be cis, cis, muconate and arsenic acid. When K. sedentarius was cultivated with methylphenylarsinic acid and diphenylmethylarsine, about 90% and 10% degradation of the two compounds, respectively, were observed. Diphenylmethylarsine oxide, possibly synthesized by methylation of DPAA, was detected as one of the transformation products. These results suggest that degradation is initiated by splitting of the phenyl groups from the arylarsenic compounds with subsequent hydroxylation of the phenyl groups and ring opening to yield cis, cis, muconate.

Very high concentrations of DDE and toxaphene residues in crocodiles from the Ord River, Western Australia: an investigation into possible endocrine disruption.

Organochlorine pesticide concentrations, particularly those of the DDT family and of toxaphene, were measured by gas chromatography in samples of liver and body fat taken from Australian freshwater crocodiles Crocodylus johnstoni at three locations along the Ord River in Western Australia. The three sampling sites were the irrigation area, downstream of the irrigation area, and well upstream of the irrigation area; the last site serving as the control. DDT and toxaphene were applied in large and known quantities to cotton grown in the Ord Irrigation Area from 1964 to 1974. Thus the residues in the crocodile tissues are representative of the situation almost thirty years after the use of DDT and toxaphene ceased in the area. Very high concentrations of p,p'-DDE and toxaphene were found in the lipid-rich tissues that were examined. Livers and body fat from estuarine crocodiles Crocodylus porosus from the downstream site were also analysed. As p,p'-DDE and toxaphene are both known to be disruptive of endocrine systems, a range of blood parameters, including estradiol and testesterone concentrations, were also measured for all the animals studied. The ovaries and testes of the freshwater crocodiles were also examined histologically. There were no obvious effects on blood chemistry or gonad histology of the large burden of pesticides and their metabolites carried by exposed animals, although the limited number of samples and the variability of the breeding state of the animals examined may have masked possible effects. The isolation of the area, the accurately known applications of DDT and toxaphene, and the simplicity of the drainage system make the lower Ord River a unique natural laboratory for studying the long term breakdown and effects of pesticides applied in a tropical environment.

Aerial oxidation of the glucocorticoid side-chain under pH control.

The outcome of aerial oxidation of the glucocortico-steroid side-chain (as exemplified by dexamethasone) has been shown to be subject to strict pH control. At pH 7.4 the glyoxal is the only product; at pH values of 8 and 9.2 the etioacid is formed, and at pH values of 13 or above the epimeric glycolic acids are produced. The glycolic acid epimer that predominates by a factor of 2 and is more stable has been shown by an X-ray crystal structural analysis to be the 20R compound. The presence of arsenite changes the course of the reaction and only the glycolic acids are yielded at pH values of 8 and above.

Diastereoisomerism of thiol complexes of arsenic acids and pseudoasymmetry of arsenic: a 1H and 13C NMR study.

Diastereoisomeric complexes of methylphenylarsinic acid and (L)-glutathione could be partially separated by HPLC, but the separated compounds rapidly racemized, presumably by pyramidal inversion at the arsenic atom. Hydrolysis of the diastereoisomeric complexes yielded methylphenylarsinous acid as a pair of enantiomers revealed by a 1H NMR study with an asymmetric lanthanide shift reagent. Methylphenylarsinous acid was also synthesized as an enantiomeric pair, shown by an asymmetric shift reagent experiment, by the hydrolysis of iodomethylphenylarsine. 1H and 13C NMR spectroscopy were used to demonstrate that complexing of phenylarsonic acid with (R,S)-3-mercapto-1,2-propanediol and with (R,S)-1-mercapto-2-propanol yielded, in each case, a pair of enantiomers, PhAs[(R)-ligand)]2, PhAs[(S)-ligand)]2, in which the homomorphic ligands were diastereotopic, and a pair of diastereoisomers, PhAs[(R)-ligand][(S)-ligand], which differed from each other in the configuration about the pseudoasymmetric arsenic atom.

Microbial treatment of bis (2-ethylhexyl) phthalate in polyvinyl chloride with isolated bacteria.

In this study, we investigated the use of microbes to degrade and remove bis (2-ethylhexyl) phthalate (DEHP), a common plasticizer and a suspected endocrine disruptor, exuding from polyvinyl chloride. Four species of bacteria that utilize DEHP as their sole carbon source were isolated from garden soil, one of which, strain NK0301, was markedly more efficient than the others in degrading DEHP and was chosen for further studies. Strain NK0301 was a coryneform bacterium (1.5x1.0 microm) identified as Mycobacterium sp. from its 16S rDNA sequencing homology. It readily degraded DEHP to two major products determined by gas chromatography/mass spectrometry to be 2-ethylhexanol and 1,2-benzenedicarboxylic acid. Other phthalate esters, suspected of being endocrine disruptors, were also tested and all except two could be utilized by strain NK0301 as their sole source of carbon. When strain NK0301 was cultivated on polyvinyl chloride sheets containing DEHP as the plasticizer, it removed up to 90% of DEHP in 3 d. Following this treatment, the polyvinyl chloride sheets did not exude DEHP to artificial saliva.

Estrogenic activity of impurities in industrial grade bisphenol A.

The estrogenicities of 10 compounds found as impurities in industrial grade bisphenol A (BPA) were measured by yeasttwo-hybrid assays incorporating the human estrogen receptor alpha(hERalpha) orthe medaka fish (Oryziaslatipes) estrogen receptor alpha (medERalpha). Five impurities showed greater activity than BPA itself in an agonist assay for hERalpha. p-Cumylphenol, the most active of the impurities in the hERalpha assay, was 12 times as active as BPA. The REC10 (10% relative effective concentration: 10% of the activity of 10(-8)M 17,B-estradiol) was 710 nM. Five impurities showed greater activity than BPA in an agonist assay for medERa: 4,4'-(1,3-dimethylbutylidene) bisphenol and 2-(4'-hydroxy-phenyl)-2,4,4-trimethylchroman were nearly equipotent and 9 times as active as BPA, and the REC10 values of these compounds in the medERalpha assay were 280 and 320 nM, respectively. Comparison of the experimentally determined estrogenicities of mixtures of BPA and 4,4'-(1,3-dimeth-ylbutylidene) bisphenol and those calculated by the concentrations addition (CA) method confirmed the suitability of the method for the prediction of the estrogenicities of the mixtures of BPA and its phenolic analogues. The measured estrogenicities of four samples of industrial grade BPA and laboratory grade (pure) BPA were not significantly different in either the hERalpha assay or the medERalpha assay (p > 0.05 in each case). We conclude that the impurities in industrial grade BPA, although some are of much higher estrogenic activity than BPA itself, do not significantly increase the estrogenicity of the industrial compound and therefore do not increase possible adverse health effects from such activity.

Degradation of dioxins by cyclic ether degrading fungus, Cordyceps sinensis.

Use of the cyclic ether degrading fungus, Cordyceps sinensis strain A to degrade dibenzo-p-dioxin (DD), 2,3,7-trichlorodibenzo-p-dioxin (2,3,7-triCDD) and octachlorodibenzo-p-dioxin (octaCDD) has revealed a new degradation pathway for dioxins. Catechols and other possible degradation products were synthesized to facilitate the identification, detection and quantification of these products, and phenylboronate was used for the derivatization and analysis of dihydroxylated degradation products. Degradation of DD yielded catechol, which was further metabolized to cis,cis-muconate. 2,3,7-triCDD yielded mono- and dichloro-catechol as well as catechol itself; and the cis,cis-muconates were also detected. octaCDD gave mono- to trichloro-catechol as well as catechol, and the cis,cis-muconates were also found. For all tested dioxin samples dechlorination resulted in replacement of chlorine with hydrogen, and no hydroxylation was observed. It appeared that dechlorination may occur in the degradation of octaCDD to catechols and possibly in the subsequent degradation of chlorinated catechols and/or chlorinated cis,cis-muconates to further breakdown products.

Complexes of diphenylarsinic acid and phenylarsonic acid with thiols: a 1H and 13C NMR study.

The high toxicity of diphenylarsinic acid, found in ground water and well water as a probable consequence of the inappropriate disposal of warfare agents, prompted us to study the reaction, monitored by 1H and 13C NMR spectroscopy, of the compound and its monophenyl analogue, phenylarsonic acid, with cellular thiols as represented, in particular, by glutathione. Glutathione reduced the phenylarsenic acids to trivalent forms and complexed them: diphenylarsinic acid to a monoglutathione adduct and phenylarsonic acid to a diglutathione adduct. The complexes were characterized by 1H and 13C NMR spectroscopy and mass spectrometry. The NMR spectra showed the diastereotopic nature of the two phenyl groups in the diphenylarsinic acid-glutathione compound, and of the two glutathione residues in the phenylarsonic acid-diglutathione complex. The stereochemistry of thiol compounds of phenylarsonic acid was further explored by 1H and 13C NMR spectroscopy of the L-cysteine complex. The diphenylarsinic acid-glutathione complex was stable below pH 12 but at higher pH the complex dissociated into diphenylarsinous acid and reduced glutathione. The released diphenylarsinous acid then oxidized to diphenylarsinic acid with a half-life of about 7 h at pH 13 and at room temperature.

Degradation of 1,4-dioxane and cyclic ethers by an isolated fungus.

By using 1,4-dioxane as the sole source of carbon, a 1,4-dioxane-degrading microorganism was isolated from soil. The fungus, termed strain A, was able to utilize 1,4-dioxane and many kinds of cyclic ethers as the sole source of carbon and was identified as Cordyceps sinensis from its 18S rRNA gene sequence. Ethylene glycol was identified as a degradation product of 1,4-dioxane by the use of deuterated 1,4-dioxane-d8 and gas chromatography-mass spectrometry analysis. A degradation pathway involving ethylene glycol, glycolic acid, and oxalic acid was proposed, followed by incorporation of the glycolic acid and/or oxalic acid via glyoxylic acid into the tricarboxylic acid cycle.

Deuterium exchange in arsenobetaine and dimethylarsinoylacetic acid.

Arsenobetaine occurs naturally in almost all marine animals and it is assumed to be the unreactive end-product of a detoxification pathway. To investigate the properties of arsenobetaine and its likely immediate biogenic precursor, dimethylarsinoylacetic acid, we studied the exchanges of the C-2 methylene protons of these compounds in D2O solution and showed them to be pH dependent first-order reactions. For arsenobetaine, the rate of exchange was highest at high pH values although exchange also occurred at low pH values. For dimethylarsinoylacetic acid, the rate was highest at low pH values although there was also exchange at high pH values. The half-life of the reaction was maximum for arsenobetaine at pH values of 5-6, and for dimethylarsinoylacetic acid at 6.5-8.5. Mechanisms are suggested for the exchange reactions involved.

Structural requirements for the interaction of 91 hydroxylated polychlorinated biphenyls with estrogen and thyroid hormone receptors.

Estrogenic and thyroid activities of 91 monohydroxylated PCBs were measured with two-hybrid assays using yeast cells containing the human estrogen receptor ERalpha or human thyroid receptor TRalpha. Estrogenic activity of 30 of the 91 compounds, including all compounds active in the yeast two-hybrid assay, were also measured by a reporter gene assay employing Chinese hamster ovary cells. The mammalian cell assay was more sensitive than the yeast assay but the rank order of estrogenicities of the compounds were in broad agreement for the two assays. Results for estrogenicity and thyroid activity were analyzed by inspection and those for estrogenicity by a theoretical treatment. Inspection indicated para-hydroxyl was more likely to be estrogenically active than meta-, which was more likely to be active than ortho-; one ortho-chlorine was important for activity but additional ortho-chlorines did not increase activity; and 2 lateral chlorines or 2,4,6-chloro- substitution of the non-phenol ring were favorable. In contrast, thyroid activity appeared not to depend strongly on the position of the hydroxyl group although ortho-hydroxyls occurred in the most active compounds. Activity was usually associated with at least one ortho-chlorine, with 2 chlorines in the phenolic ring and, importantly, two chlorines in the non-phenolic ring, and with 1 or 2 chlorines ortho to the hydroxyl group. Examination of the torsion angle between the rings, in the theoretical examination of estrogenicity, suggested that perpendicular orientation (i.e., rigidity) was not essential for activity. Intramolecular hydrogen bonding of the phenolic groups to adjacent chlorines or to the pi-electron cloud of the non-phenol ring possibly decreased activity--the hydroxyl should be free of intramolecular interactions for maximum activity. It was difficult to predict the estrogenic activity of a congener from its obtained potential energy curve (PEC). In general, estrogenically active congeners had large values for the sum of the atomic charges on the carbon atoms of the hydroxylated ring, and on the oxygen atom. Hydroxyl substitution at the para-position allowed the compounds to become more polarizable in the x-axis (molecular axis), whereas OH substitution at the ortho-position made the congeners less polarizable in the same direction. However, no general statement about polarizability and estrogenic activity was possible.

Microbial production and vaporization of mono-(2-ethylhexyl) phthalate from di-(2-ethylhexyl) phthalate by microorganisms inside houses.

Laboratory workers were bothered by an irritation that caused coughing during the cultivation of microorganisms that degraded di-(2-ethylhexyl) phthalate (DEHP). The authors found that mono-(2-ethylhexyl) phthalate (MEHP), a known cause of asthma, was released during the degradation of DEHP. At its highest production and vaporization rate, the amount was almost equal to that of the DEHP starting material. It appeared that transport into the atmosphere depended on its adsorption on dust particles. The authors attempted to cultivate several microorganisms from house materials, especially those composed of rotting polyvinyl chloride. And microorganisms produced MEHP in the culture medium. In addition, MEHP was produced from DEHP by several stock microorganisms. Thus, MEHP could easily be produced from DEHP by microorganisms in the environment. In Japan, there are many cases of asthma with unknown causes. If MEHP is one of causes, then preventive measures against some cases of asthma could be taken.

Glycosylation of bisphenol A by tobacco BY-2 cells.

Tobacco BY-2 cells in suspension culture absorbed and transformed bisphenol A dissolved in the culture medium. Major products were bisphenol A mono-O-beta D-gentiobioside and the trisaccharide bisphenol A mono-O-beta-D-glucopyranosyl-(1-->4)-[beta-D-glucopyranosyl-(1 --> 6)] beta-D-glucopyranoside. Also produced were the mono- and di- O-beta-D-glucopyranosides. As glycosides of bisphenol A lack the estrogenic activity of the parent compound, these findings enhance the possibilities of phytoremediation of natural waters contaminated by bisphenol A. .

The reaction of bisphenol A 3,4-quinone with DNA.

The 3,4-quinone of the estrogen-active compound bisphenol A (BPA), characterized by a single crystal X-ray structure determination, has been shown by (1)H NMR spectroscopy to react with herring testes DNA, and with deoxyguanosine (dG), in aqueous buffer at pH 7, to form a BPA 3,4-quinone-guanine-N7 adduct (BPAQ-N7-Gua). Presumably this adduct resulted from decomposition (by loss of deoxyribose) of an initially formed, but unstable, BPAQ-N7-dG adduct. Chemical synthesis if BPAQ-N7-Gua, in up to 60% yield, was achieved by the reaction of BPAQ and dG in aqueous acetic acid. Characterization of this product, by NMR spectroscopy and high resolution mass spectrometry, allowed the monitoring (by (1)H NMR spectroscopy) of the reaction of BPAQ with DNA and with dG. The relevance of this adduct formation to the potential mutagenicity and carcinogenicity of BPA will depend upon confirmation of the necessary metabolic oxidative transformation of BPA in vivo.

An origin for arsenobetaine involving bacterial formation of an arsenic-carbon bond.

Lysed-cell extract of a Pseudomonas sp. was shown to catalyse bioconversion of dimethylarsinoylacetate to arsenobetaine and dimethylarsinate. Provision of the universal methyl donor S-adenosylmethionine to bioconversion mixtures promoted both the rate and extent of arsenobetaine formation. These findings suggest that in the proposed biosynthesis of arsenobetaine from dimethylarsinoylethanol, oxidation (i.e. the formation of the carboxymethyl group of dimethylarsinoylacetate) would precede the reduction and methylation at the arsenic atom. The presence of enzyme(s) capable of methylating dimethylarsinoylacetate in a bacterial isolate from marine mussel (Mylitus edulis), highlights a possible direct involvement of prokaryotic organisms in the biosynthesis of organoarsenic compounds within marine animals.