Production of polyselenodipenicillamines, unique selenium compounds.

Selenite (H(2)SeO(3)) reacts with thiol compounds (RSH) under acidic conditions to form selenotrisulfides (RSSeSR, i.e. monoselenodithiols). The stoichiometry of the reaction is proposed as 4RSH+H(2)SeO(3)-->RSSeSR+RSSR+3H(2)O. Surprisingly, we found novel polynuclear selenium-containing compounds, i.e. polyselenodipenicillamines (PenSSe(2-4)SPen), in the reaction of D-penicillamine (PenSH) with H(2)SeO(3). The selenium-centered features of PenSSe(2-4)SPen were determined by (1)H-NMR and LC-MS/MS analyses, showing that the selenium isotope abundance patterns of the compounds were in good agreement with the theoretically-calculated ones. In order to better understand the mechanisms for PenSSe(2-4)SPen production, various molar ratio of H(2)SeO(3) (1/8 to 4 times of PenSH) was reacted with PenSH, and the concentration of the products was calculated from integral values of dimethyl proton signals for PenSSe(1-2)SPen as compared with methyl proton signals for acetic acid (an internal standard). Total PenSSe(1-2)SPen concentration was increased with increasing of H(2)SeO(3), in which concomitant decrease of PenSSPen (disulfide form of PenSH) was observed. Based on these results, we proposed the PenSSe(2-4)SPen production mechanisms being involved in penicillamine selenopersulfides (PenSSe(1-2)H).

Effects of phthalate ester derivatives including oxidized metabolites on coactivator recruiting by PPARalpha and PPARgamma.

Phthalate esters (PEs), a group of environmental chemicals, affect biological systems via endocrine and lipid metabolism modulations. These effects are believed to be mediated in part by peroxisome proliferator-activated receptors (PPARs). Evaluations of PE activities as ligands toward PPARs have been investigated in many studies on their primary metabolites, monoesters. However, the activities of various other metabolites, including oxidized derivatives, remain to be determined. Here, we have evaluated the PPAR ligand activities of these PE derivatives by in vitro coactivator recruiting assay. Mono(2-ethyl-5-hydroxyhexyl)phthalate, the most abundant metabolite of di-(2-ethylhexyl)phthalate (DEHP), was less active than mono(2-ethylhexyl)phthalate (MEHP) as a PPAR ligand. Other derivatives oxidized at the alkyl group and benzene ring of DEHP, MEHP, dibutyl phthalate and its monoester were also investigated and some affected PPAR activities. Unexpectedly, MEHP as well as its further oxidized metabolite did not show clear activity for PPARalpha, although MEHP is believed to interact with PPARalpha. This might imply indirect PPAR-mediated mechanisms that lead to observed biological effects such as peroxisome proliferation.

Structural properties of estrogen receptor ligand obtained by study of hydroxylated phthalate ester derivatives.

Phthalate esters (PEs), a group of plasticizers, are suspected to be endocrine-disrupting chemicals. Here, PE derivatives were used as probes for elucidating the structural properties of estrogen receptor (ER) ligands. A comprehensive study was performed using more than 40 PE derivatives including ring-/alkyl-hydroxylated and nonsymmetrical diesters possessing independently altered alkyls of C1-C8. Estrogenic activity of these derivatives is determined with three assays for ER-binding, coactivator-recruiting and transactivation. Phenolic hydroxylation increased activity, while hydroxylation of the ester alkyl group had no distinct effect on ER binding or transcription coactivator recruitment. Ring-hydroxylated PE derivatives harboring different ester alkyls revealed that the length of both alkyls independently affects transactivation of ER. These comprehensive data would be useful for the better understanding of structure-activity relationship of endocrine-disrupting chemicals.

Toxicity of di(2-ethylhexyl)phthalate (DEHP) and di(2-ethylhexyl)adipate (DEHA) under conditions of renal dysfunction induced with folic acid in rats: enhancement of male reproductive toxicity of DEHP is associated with an increase of the mono-derivative.

F344 male rats were given five consecutive weekly subcutaneous injections of folic acid for induction of chronic renal dysfunction and then di(2-ethylhexyl)phthalate (DEHP) or di(2-ethylhexyl)adipate (DEHA) in the diet at a concentration of 0, 6000 or 25,000 ppm for 4 weeks in order to investigate whether male reproductive toxicity of the two chemicals might be enhanced under conditions of renal disease. Control animals also received DEHP or DEHA in the same manner but without folic acid pretreatment. Decreased testicular weights, seminiferous atrophy with vacuolization of sertoli cells and diminished sperm counts were more prominent in rats given folic acid and then 25,000 ppm DEHP as compared to those exposed to DEHP alone. No such reproductive toxicity was evident in rats given 6000 ppm DEHP or either dose of DEHA. An increased concentration of the mono-derivative of DEHP (mono(2-ethylhexyl)phthalate, MEHP) in the blood, testis and urine was considered relevant to the enhanced reproductive toxicity observed with DEHP.

Formation of estrogenic products from environmental phthalate esters under light exposure.

Phthalate esters (PEs) have been suspected to be environmental endocrine disruptors and the detailed mechanism remains unclear. The activities of these chemicals can be enhanced through chemical modification under the environmental conditions. We demonstrate that PEs acquire unequivocal estrogenic activity by light exposure. Through UV exposure of an aqueous PE solution, one active photoproduct, identified as 4-hydroxyPE (PE-4OH) based on its characteristic UV and mass spectra, was detected in an estrogen receptor alpha-dependent transactivation assay. PE-4OH was effectively generated by UV 290 nm. The PE-4OH production accompanied H2O2 generation in a UV dose-dependent manner. Both PE and UV irradiation were indispensable in the generation of H2O2. Addition of H2O2 to the PE solution increased PE-4OH production under UV irradiation. The PE-4OH production was also observed in the PE reaction with the Fenton reagent generating hydroxyl radical without UV irradiation. The proposed mechanism for PE-4OH production based on these results is such that by PE-mediated photosensitization H2O2 is generated from O2 and H+ and decomposed to hydroxyl radical, thus oxidizing the PE benzene ring. The PEs-4OH are remarkably active estrogenic products of PEs and would be involved in ER-mediated endocrine disruption.

Testicular toxicity of DEHP, but not DEHA, is elevated under conditions of thioacetamide-induced liver damage.

As part of an investigation of possible enhancement by liver disease of testicular toxicity caused by phthalates, we tested the effects of di(2-ethylhexyl)phthalate (DEHP) and di(2-ethylhexyl)adipate (DEHA) in a thioacetamide (TAA)-induced rat liver damage model. Male, 6-week-old, F344 rats (n=60) were divided into ten groups. Animals of groups 1-5 received TAA (200 mg/kg, intraperitoneal, three times per week) for 4 weeks, and groups 6-10 served as controls without TAA. After a 1 week interval, at week 5, powder diet containing DEHP or DEHA was provided to the animals of groups 1 and 6 (DEHP 25000 ppm), groups 2 and 7 (DEHP 6000 ppm), groups 3 and 8 (DEHA 25000 ppm) and groups 4 and 9 (DEHA 6000 ppm), while groups 5 and 10 received basal diet. All animals were sacrificed at week 9. Significant decrease in sperm numbers and motility and increase in morphology abnormalities were evident in group 1 as compared to groups 5 and 6 (p<0.01). However, DEHA treatment was not associated with any apparent testicular toxicity in either TAA- or vehicle-treated animals. Histopathological examination of the testes revealed severe atrophy and degeneration of testicular tubules in all animals given TAA and DEHP at high dose, only mild to moderate lesions being found with DEHP alone. We conclude that liver toxicity induced by TAA is associated with the enhancement of testicular toxicity of DEHP, but not DEHA, in rats.

Metabolic activation of carcinogenic ethylbenzene leads to oxidative DNA damage.

Ethylbenzene is carcinogenic to rats and mice, while it has no mutagenic activity. We have investigated whether ethylbenzene undergoes metabolic activation, leading to DNA damage. Ethylbenzene was metabolized to 1-phenylethanol, acetophenone, 2-ethylphenol and 4-ethylphenol by rat liver microsomes. Furthermore, 2-ethylphenol and 4-ethylphenol were metabolically transformed to ring-dihydroxylated metabolites such as ethylhydroquinone and 4-ethylcatechol, respectively. Experiment with 32P-labeled DNA fragment revealed that both ethylhydroquinone and 4-ethylcatechol caused DNA damage in the presence of Cu(II). These dihydroxylated compounds also induced the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine in calf thymus DNA in the presence of Cu(II). Catalase, methional and Cu(I)-specific chelator, bathocuproine, significantly (P<0.05) inhibited oxidative DNA damage, whereas free hydroxyl radical scavenger and superoxide dismutase did not. These results suggest that Cu(I) and H2O2 produced via oxidation of ethylhydroquinone and 4-ethylcatechol are involved in oxidative DNA damage. Addition of an endogenous reductant NADH dramatically enhanced 4-ethylcatechol-induced oxidative DNA damage, whereas ethylhydroquinone-induced DNA damage was slightly enhanced. Enhancing effect of NADH on oxidative DNA damage by 4-ethylcatechol may be explained by assuming that reactive species are generated from the redox cycle. In conclusion, these active dihydroxylated metabolites would be involved in the mechanism of carcinogenesis by ethylbenzene.

Unequivocal estrogen receptor-binding affinity of phthalate esters featured with ring hydroxylation and proper alkyl chain size.

The effect of plasticizers phthalate esters (PEs) on health is a controversial subject. PEs are likely to be estrogenic, but the results on the potency obtained by many investigators are still inconsistent and the endocrine disrupting mechanism remains to be clarified. Here, we show that PEs acquire unequivocal binding affinities for human estrogen receptors (ERs) through ring hydroxylation that is possible in the environment and through metabolism. Unexpectedly, the acquired affinities of hydroxylated PEs (PEs-OH) were enhanced by elongation and branching of the ester alkyl chains. PEs-OH with alkyl chains more than six carbons may grope for a new binding site, which is inaccessible to PEs-OH with short chains. The strongest ER-binding affinity among the tested PEs-OH was close to that of diethylstilbestrol, the most potent synthetic ER-binder. Ring hydroxylation would be a new clue to the clarification of the endocrine disruption mechanism of PEs.

DNA damage by ethylbenzenehydroperoxide formed from carcinogenic ethylbenzene by sunlight irradiation.

Ethylbenzene, widely used in human life, is a non-mutagenic carcinogen. Sunlight-irradiated ethylbenzene caused DNA damage in the presence of Cu2+, but unirradiated ethylbenzene did not. A Cu+ -specific chelator bathocuproine inhibited DNA damage and catalase showed a little inhibitory effect. The scopoletin assay revealed that peroxides and H(2)O(2) were formed in ethylbenzene exposed to sunlight. These results suggest that Cu+ and alkoxyl radical mainly participate in DNA damage, and H(2)O(2) partially does. When catalase was added, DNA damage at thymine and cytosine was inhibited. Ethylbenzenehydroperoxide, identified by GC/MS analysis, induced the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine and caused DNA damage at consecutive guanines, as observed with cumenehydroperoxide. Equimolar concentrations of H(2)O(2) and acetophenone were produced by the sunlight-irradiation of 1-phenylethanol, a further degraded product of ethylbenzene. These results indicate a novel pathway that oxidative DNA damage induced by the peroxide and H(2)O(2) derived from sunlight-irradiated ethylbenzene may lead to expression of the carcinogenicity.

Phthalate esters detected in various water samples and biodegradation of the phthalates by microbes isolated from river water.

Phthalate esters (PEs), especially di-n-butyl phthalate (DBP) and di-(2-ethylhexyl) phthalate (DEHP) were detected in various water samples such as river water, well water and tap water. On degradation tests of PEs, Tempaku River water degraded almost 100% of diethyl phthalate (DEP), di-isobutyl phthalate and DBP, and approximately 70% of DEHP. All eight isolates from Tempaku River water (R1-R7, D1) did not degrade dimethyl phthalate (DMP), but showed biodegrading ability for the other PEs. The DBP-degrading ability was particularly high for the isolates R1-R3 and D1 of Acinetobacter iwoffii. Crude enzyme solutions prepared from bacterial cells of these isolates showed a higher degrading activity for DEHP compared with that for microbially-degradable DBP. Particularly high DEHP-degrading activity was found for crude enzyme solutions of the isolate D1. As metabolites from the river water and bacterial isolates, DMP and an unknown diester were produced from DEP. DMP, DEP, monomethyl phthalate, monobutyl phthalate (MBP) and an unknown diester were produced from DBP. DBP, DEP, DMP and an unknown diester were produced from DEHP. As metabolites by the crude enzyme solutions, DMP, MBP and an unknown diester derivative were produced from DBP. DBP, mono-(2-ethylhexyl) phthalate and an unknown diester derivative were produced from DEHP. Diesters with shortened alkyl carbon chains were also found as metabolites by the isolates and their crude enzyme solutions. The results suggest that the alkyl chains in the diesters are also decomposed in addition to monoester formation from DBP or DEHP at the first step reported for animals and some types of bacteria.