Global DNA Methylation in Cord Blood as a Biomarker for Prenatal Lead and Antimony Exposures.

DNA methylation is an epigenetic mechanism for gene expression modulation and can be used as a predictor of future disease risks. A prospective birth cohort study was performed to clarify the effects of neurotoxicants on child development, namely, the Tohoku Study of Child Development, in Japan. This study aimed to evaluate the association of prenatal exposure to five toxic metals-arsenic, cadmium, mercury, lead (Pb), antimony (Sb), and polychlorinated biphenyls (PCBs, N = 166)-with global DNA methylation in umbilical cord blood DNA. DNA methylation markers, 5-methyl-2'-deoxycytidine (mC) and 5-hydroxymethyl-2'-deoxycytidine (hmC), were determined using liquid chromatography-tandem mass spectrometry. The mC content in cord blood DNA was positively correlated with Pb and Sb levels (r = 0.435 and 0.288, respectively) but not with cord blood PCBs. We also observed significant positive correlations among Pb levels, maternal age, and hmC content (r = 0.155 and 0.243, respectively). The multiple regression analysis among the potential predictors demonstrated consistent positive associations between Pb and Sb levels and mC and hmC content. Our results suggest that global DNA methylation is a promising biomarker for prenatal exposure to Pb and Sb.

Selenium uptake through cystine transporter mediated by glutathione conjugation.

Selenium (Se) is an essential trace element and is regarded as a protective agent against cancer. In particular, antioxidant effects of selenoenzymes contribute to cancer prevention. Se can also produce reactive oxygen species and, thereby, exert cancer-selective cytotoxicity. Selenodiglutathione (SDG) is a primary Se metabolite conjugated to two glutathione (GSH) moieties. SDG increases intracellular Se accumulation and is more toxic than selenous acid (H2SeO3), but the mechanisms for importing Se compounds into cells are not fully understood. Here, we propose a novel mechanism for importing Se, in the form of SDG. Cellular intake of Se compounds was assessed based on Se accumulation, as detected by ICP-MS. SDG incorporation was decreased in the presence of thiols (GSH, cysteine or their oxidized forms, GSSG and cystine), whereas H2SeO3 uptake was increased by addition of GSH or cysteine. Cellular SDG uptake was decreased by pretreatment with specific inhibitors against gamma-glutamyl transpeptidase (GGT) or the cystine/glutamate antiporter (system xc-). Furthermore, siRNA against xCT, which is the light chain component of system xc-, significantly decreased SDG incorporation. These data suggest an involvement of SDG in Se incorporation, with SDG processed at the cell surface by GGT, leading to formation of selenodicysteine which, in turn, is likely to be imported via xCT. Because GGT and xCT are highly expressed in cancer cells, these mechanisms mediated by the cystine transporter might underlie the cancer-selective toxicity of Se. In addition, the system described in our study appears to represent a physiological transport mechanism for the essential element Se.

DNA methylation dynamics in mouse preimplantation embryos revealed by mass spectrometry.

Following fertilization in mammals, paternal genomic 5-methyl-2'-deoxycytidine (5 mC) content is thought to decrease via oxidation to 5-hydroxymethyl-2'-deoxycytidine (5 hmC). This reciprocal model of demethylation and hydroxymethylation is inferred from indirect, non-quantitative methods. We here report direct quantification of genomic 5 mC and 5 hmC in mouse embryos by small scale liquid chromatographic tandem mass spectrometry (SMM). Profiles of absolute 5 mC levels in embryos produced by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) were almost identical. By 10 h after fertilization, 5 mC levels had declined by ~40%, consistent with active genomic DNA demethylation. Levels of 5 mC in androgenotes (containing only a paternal genome) and parthenogenotes (containing only a maternal genome) underwent active 5 mC loss in the first 6 h, showing that both parental genomes can undergo demethylation independently. We found no evidence for net loss of 5 mC 10-48 h after fertilization, implying that any passive 'demethylation' following DNA replication was balanced by active 5 mC maintenance methylation. However, levels of 5 mC declined during development after 48 h, to 1% (measured as a fraction of G-residues) in blastocysts (~96 h). 5 hmC levels were consistently low (<0.2% of G-residues) throughout development in normal diploid embryos. This work directly quantifies the dynamics of global genomic DNA modification in mouse preimplantation embryos, suggesting that SMM will be applicable to other biomedical situations with limiting sample sizes.

Thiol-mediated multiple mechanisms centered on selenodiglutathione determine selenium cytotoxicity against MCF-7 cancer cells.

Selenium (Se) is an essential antioxidative micronutrient but can exert cancer-selective cytotoxicity if the nutritional levels are too high. Selenodiglutathione (GSSeSG) is a primary Se metabolite conjugated with two glutathione (GSH) moieties. GSSeSG has been suggested to be an important molecule for cytotoxicity. Here, we propose the underlying mechanisms for the potent cytotoxicity of GSSeSG: cellular intake; reductive metabolism; production of reactive oxygen species; oxidative damage to DNA; apoptosis induction. GSSeSG rather than selenite decreased cell viability and induced apoptosis accompanied by increases in intracellular Se contents. Therefore, GSSeSG-specific cytotoxicity may be ascribed to its preferable incorporation. Base oxidation and strand fragmentation in genomic DNA preceded cell death, suggesting that oxidative stress (including DNA damage) is crucial for GSSeSG cytotoxicity. Strand breaks of purified DNA were caused by the coexistence of GSSeSG and thiols (GSH, cysteine, homocysteine), but not the oxidized form or non-thiol reductants. This implies the important role of intracellular thiols in the mechanism of Se toxicity. GSH-assisted DNA strand breaks were inhibited by specific scavengers for hydrogen peroxide or hydroxyl radicals. The GSSeSG metabolite selenide induced some DNA strand breaks without GSH, whereas elemental Se did so only with GSH. These observations suggest involvement of Fenton-type reaction in the absence of transition metals and reactivation of inert elemental Se. Overall, our results suggest that chemical interactions between Se and the sulfur of thiols are crucial for the toxicity mechanisms of Se.

Oral administration of Brazilian propolis exerts estrogenic effect in ovariectomized rats.

Propolis, a natural product derived from plants by honeybees, is a mixture of several hundred chemicals, including flavonoids, coumaric acids, and caffeic acids, some of which show estrogen-like activity. In this study, the estrogenic activity of crude ethanolic extract of Brazilian propolis was determined using several in vitro and in vivo assays. Propolis was found to bind to human estrogen receptors (ERs). Furthermore, propolis induced the expression of estrogen-responsive genes in ER-positive MCF-7 and Ishikawa cells. These in vitro assays suggest that propolis exerts estrogenic activity; therefore, in vivo experiments were conducted using ovariectomized rats. Oral administration of propolis (55 or 550 mg/kg/day for 3 days) significantly increased uterine wet weight and luminal epithelium thickness in comparison with the corresponding values in the corn oil-treated control group. Moreover, propolis induced ductal cell proliferation in the mammary glands. These effects were completely inhibited by full ER antagonist ICI 182,780, confirming that the effects of propolis are mediated by the ER. Our data show that oral intake of propolis induces estrogenic activity in ER-expressing organs in vivo and suggest that Brazilian propolis is a useful dietary source of phytoestrogens and a promising treatment for postmenopausal symptoms.

[Neural differentiation of pluripotent stem cells and application for metal-induced neural toxicity study].

Metals are effectively used in biological systems under the strict regulation for exploiting their specific and broad reactivities. For example, manganese (Mn) can induce catecholamines-mediated oxidative biological damage in cooperation with iron (Fe) and/or copper (Cu). In children, the damage could induce developmental disorders such as attention deficit hyperactivity disorder (ADHD). We hypothesize that infant neurons are more labile to metals than adult ones due to the prematured protection systems and sensitive differentiating cells. An experimental system reconstituting neural differentiation is expected to assess the influences of endogenous/exogenous factors including metals. In this study, we investigated an impact of Mn together with Fe and dopamine (DA) on neural differentiation of mouse embryonic stem cells (mESCs). The differentiation of mESCs was initiated by embryoid bodies (EBs) formation in the presence of all-trans retinoic acid, and then EBs were treated with Mn, Fe and/or DA. Then, the mRNA levels of neural differentiation marker genes (Nestin, Emx2, Mtap2, Th, Olig2 and Gfap) were examined using realtime RT-PCR analysis. Mn or DA alone reduced Mtap2, Th and Olig2 expression levels and increased Nestin. Moreover, combined treatment of Mn and DA also increased Nestin expression level. On the other hand, Fe alone reduced Mtap2, Th and Olig2 expression levels, and increased Emx2. Combined treatments of Fe with Mn or DA also tended to increase Emx2 expression level. These effects emerged at about 100 times less concentration than that inducing cytotoxicity in human neuroblastoma. The present study showed that Mn inhibits neural development, and that our mESCs system can be a useful tool to elucidate the toxicity mechanism as well as to evaluate the effects of metals and chemicals on differentiating cells.

PRDM14 promotes active DNA demethylation through the ten-eleven translocation (TET)-mediated base excision repair pathway in embryonic stem cells.

Ten-eleven translocation (TET) proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). 5fC and 5caC can be excised and repaired by the base excision repair (BER) pathway, implicating 5mC oxidation in active DNA demethylation. Genome-wide DNA methylation is erased in the transition from metastable states to the ground state of embryonic stem cells (ESCs) and in migrating primordial germ cells (PGCs), although some resistant regions become demethylated only in gonadal PGCs. Understanding the mechanisms underlying global hypomethylation in naive ESCs and developing PGCs will be useful for realizing cellular pluripotency and totipotency. In this study, we found that PRDM14, the PR domain-containing transcriptional regulator, accelerates the TET-BER cycle, resulting in the promotion of active DNA demethylation in ESCs. Induction of Prdm14 expression transiently elevated 5hmC, followed by the reduction of 5mC at pluripotency-associated genes, germline-specific genes and imprinted loci, but not across the entire genome, which resembles the second wave of DNA demethylation observed in gonadal PGCs. PRDM14 physically interacts with TET1 and TET2 and enhances the recruitment of TET1 and TET2 at target loci. Knockdown of TET1 and TET2 impaired transcriptional regulation and DNA demethylation by PRDM14. The repression of the BER pathway by administration of pharmacological inhibitors of APE1 and PARP1 and the knockdown of thymine DNA glycosylase (TDG) also impaired DNA demethylation by PRDM14. Furthermore, DNA demethylation induced by PRDM14 takes place normally in the presence of aphidicolin, which is an inhibitor of G1/S progression. Together, our analysis provides mechanistic insight into DNA demethylation in naive pluripotent stem cells and developing PGCs.

Modulation of oxidative DNA damage and DNA-crosslink formation induced by cis-diammine-tetrachloro-platinum(IV) in the presence of endogenous reductants.

Platinum(IV) [Pt(IV)] complex, satraplatin, is currently in clinical trials for the treatment of various cancers. As a key step of the anti-cancer effect exertion, satraplatin is supposed to be reduced by endogenous reductants to platinum(II) [Pt(II)] complex. In this study, we investigated the interaction of DNA, Pt(IV), and the endogenous reductants such as ascorbic acid (AsA) and glutathione (GSH). As a model Pt(IV) compound, cis-diammine-tetrachloro-Pt(IV) [cis-Pt(IV)], which is a prodrug of cisplatin [cis-diammine-dichloro-Pt(II), cis-Pt(II)], was incubated with calf thymus DNA in the presence of AsA or GSH. In the presence of AsA, cis-Pt(IV) induced oxidative DNA damage. Hydroxyl radical scavengers suppressed the AsA-associated oxidative damage, thereby suggesting that hydroxyl radicals are involved in the DNA oxidation. cis-Pt(II)-like CD spectral change and crosslink formation in calf thymus DNA were also observed during this DNA oxidation, suggesting cis-Pt(IV) reduction by AsA and DNA conformational change induced by the newly formed cis-Pt(II) binding to DNA. GSH did not induce oxidative DNA damage likely due to its own hydroxyl radical scavenging ability. Further, GSH suppressed the Pt(II)-mediated DNA conformational change and crosslink formation, suggesting that GSH sequesters the cis-Pt(II) away from DNA by GSH-cis-Pt(II) complex formation.

Different mechanisms between copper and iron in catecholamines-mediated oxidative DNA damage and disruption of gene expression in vitro.

Catechols produce reactive oxygen species (ROS) and induce oxidative DNA damage through reduction-oxidation reactions with metals such as copper. Here, we examined oxidative DNA damage by neurotransmitter catecholamines in the presence of copper or iron and evaluated the effects of this damage on gene expression in vitro. Dopamine induced strand breaks and base oxidation in calf thymus DNA in the presence of Cu(II) or Fe(III)-NTA (nitrilotriacetic acid). The extent of this damage was greater for Cu(II) than for Fe(III)-NTA. For the DNA damage induced by dopamine, the responsible reactive species were hydrogen peroxide and Cu(I) for Cu(II) and hydroxyl radicals and Fe(II) for Fe(III)-NTA. Cu(II) induced DNA conformational changes, but Fe(III)-NTA did not in the presence of dopamine. These differences indicate different modes of action between Cu and Fe-NTA with regard to the induction of DNA damage. Expression of the lacZ gene coded on plasmid DNA was inhibited depending on the extent of the oxidative damage and strand breaks. Endogenous catecholamines (dopamine, adrenaline, and noradrenaline) were more potent than catechols (no aminoalkyl side chains) or 3,4-dihydroxybenzylamine (aminomethyl side chain). These results suggest that the metal-mediated DNA damage induced by dopamine disrupts gene expression, and leukoaminochromes (further oxidation products of O-quinones having aminoethyl side chain) are involved in the DNA damage. These findings indicate a possibility that metal (especially iron and copper)-mediated oxidation of catecholamines plays an important role in the pathogenesis of neurodegenerative disorders including Parkinson's disease.

Carbonyl side-chain of catechol compounds is a key structure for the suppression of copper-associated oxidative DNA damage in vitro.

Catechol is possibly carcinogenic to humans (International Agency for Research on Cancer, IARC). The key mechanism could include its oxidative DNA-damaging effect in combination with reductive-oxidative metals like Cu. We found that DNA damage was suppressed by introducing an α-carbonyl group to catechol at C4-position to produce carbonyl catechols. During the oxidative DNA-damaging process, catechols but not carbonyl catechols were oxidized to o-quinone; however, coexisting Cu(II) was reduced to Cu(I). Carbonyl catechols were possibly arrested at the oxidation step of semiquinones in the presence of Cu(II). Cu(I)-binding to DNA was stronger than Cu(II)-binding, on the basis of the circular dichroism spectral change. None of the carbonyl catechols induced such change, suggesting sequestration of Cu(I) from DNA. Solid-phase extraction experiments and spectrophotometric analyses showed the formation of semiquinone chelates with Cu(I). Thus, chelate formation could explain the suppression mechanism of the Cu-catechol-dependent DNA damage by terminating the reduction-oxidation cycle. Structural modifications such as introducing an α-carbonyl group to catechol at C4-position would contribute to reducing the risk and improving industrial and medical potentials of aromatic/phenolic compounds sustaining our daily lives.

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.

Combined activation of methyl paraben by light irradiation and esterase metabolism toward oxidative DNA damage.

Methyl paraben (MP) is often used as a preservative in foods, drugs, and cosmetics because of its high reliability in safety based on the rapid excretion and nonaccumulation following administration. Light irradiation sometimes produces unexpected activity from chemicals such as MP; furthermore, there is ample opportunity for MP to be exposed to sunlight. Here, we investigated whether MP shows DNA damage after sunlight irradiation. Two major photoproducts, p-hydroxybenzoic acid (PHBA) and 3-hydroxy methyl paraben (MP-3OH), were detected after sunlight irradiation to an aqueous MP solution. Both photoproducts were inactive in the in vitro DNA damage assay that measures oxidized guanine formed in calf thymus DNA in the presence of divalent copper ion, a known mediator of oxidative DNA damage. Simulated MP metabolism using dermal tissues after light irradiation produced these two photoproducts, which reacted with a microsomal fraction (S9) of the skin. A metabolite from MP-3OH, not PHBA, caused distinct DNA damage in the in vitro assay. This active metabolite was identified as protocatechuic acid, a hydrolyzed MP-3OH product. In addition, NADH, a cellular reductant, enhanced DNA damage by approximately five times. These results suggest that reactive oxygen species generated by the redox cycle via metal ion and catechol autoxidation are participating in oxidative DNA damage. This study reveals that MP might cause skin damage involving carcinogenesis through the combined activation of sunlight irradiation and skin esterases.

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.

Formation of estrogenic products from benzophenone after exposure to sunlight.

Benzophenone (BP) is a suspected endocrine disrupter that is found in our environment. BP undergoes metabolic and photochemical activation. In this study, photoproducts of BP were identified using high-performance liquid chromatography and mass spectrometry and their estrogenic activity was determined using both in vitro and in vivo assays. Although BP showed no estrogenic activity, two estrogenic photoproducts were detected after irradiating an aqueous solution of BP with UV or sunlight. These active products were identified as 3-hydroxy BP (BP-3OH) and 4-hydroxyBP (BP-4OH). The formation of hydrogen peroxide H2O2) was detected with increasing levels of UV, and the addition of H2O2 to the BP solution increased BP-3OH and BP-4OH production under UV irradiation. BP hydroxylation was also observed in the reaction with the Fenton reagent generating hydroxyl radical without UV irradiation. These results suggest the involvement of photochemically generated H2O2 and hydroxyl radical in the BP hydroxylation. BP-4OH was more potent than BP-3OH for promoting estrogen receptor (ER)-mediated transcription and uterotrophic activity, although both of them showed same affinity in ER binding. In conclusion, BP can be converted into ring-hydroxylated derivatives that have estrogenic activity after exposure to light.

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.

Isoflavonoids with antiestrogenic activity from Millettia pachycarpa.

Three new isoflavonoids, named millewanins G (1) and H (2) and furowanin B (3), were isolated from the leaves of Millettia pachycarpa. Their structures were elucidated on the basis of spectroscopic analyses. The antiestrogenic activity in the yeast two-hybrid assay of these isoflavonoids was examined and shown to be comparable with that of 4-hydroxytamoxifen.

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.