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Background: It has been suggested that phytoestrogens and dietary fiber can affect puberty timing. Objective: We examined whether intake of isoflavone and fiber in healthy white children before their pubertal growth spurt [age at take-off (ATO)] was associated with puberty timing. Design: Multivariate regression analyses were performed in 227 DONALD (DOrtmund Nutritional and Anthropometric Longitudinally Designed) Study participants with 3-d weighed dietary records and information on potential confounders at baseline (1 and 2 y before ATO). In a subsample (n = 111), urinary isoflavones were determined in 24-h urine samples by gas chromatography-mass spectrometry analysis. Puberty timing was examined by using ATO and chronologic ages at pubertal stage 2 for breast development (B2) or gonadal development, peak height velocity (PHV), and menarche or voice break. Results: Girls whose diet was in the highest dietary isoflavone tertile experienced Tanner stage 2 for breast development [approximately equal to]0.7 y later and reached PHV [approximately equal to]0.6 y later than did girls whose diet was in the lowest isoflavone tertile [age (95% CI) at B2:10.7 y (10.4, 10.9 y) compared with 10.0 y (9.7, 10.3 y), respectively; P for trend = 0.04; age at PHV: I 1.9 y (11.6, 12.2 y) compared with 11.3 y (11.0, 11.6 y), respectively; P for trend = 0.04; adjusted for body mass index z score and fiber intake]. In boys, dietary isoflavones were not associated with pubertal markers. Urinary isoflavone and dietary fiber intakes were not associated with pubertal markers. Conclusions: Girls, but not boys, with higher prepubertal isoflavone intakes appear to enter puberty at a later age. Fiber intake in this sample of healthy white girls and boys was not relevant for puberty timing. doi: 10.3945/ajcn.2010.29394

Infants are particularly susceptible toward the toxic effects of food contaminants, including mycotoxins. However, multimycotoxin exposure assessment in breast milk has received very limited attention so far, resulting in a poor understanding of coexposures during early life. Here, we present the development and application of a highly sensitive, specific, and quantitative assay assessing up to 28 mycotoxins, including regulated (aflatoxins, ochratoxin A, deoxynivalenol, zearalenone) and emerging mycotoxins as well as key metabolites by LC-MS/MS. After careful optimization of the sample preparation procedure, a QuEChERS protocol combined with a freeze-out step was validated in-house. The limits of quantification varied between 0.009 and 2.9 ng/mL, and for most analytes extraction recovery (74–116%) and intermediate precision (2–20%) were satisfactory. The method was applied to examine multiple breast milk samples obtained from 22 women (n= 75 in total) from Ogun State, Nigeria. Most samples were either entirely free of mycotoxins or contaminated to a minimal extent with beauvericin (56%), enniatin B (9%), ochratoxin A (15%), and aflatoxin M1(1%). The most abundant mycotoxin was beauvericin, which was not reported in this biological fluid before, with concentrations up to 0.019 ng/mL. In conclusion, the method demonstrated to be fit for purpose to determine and quantify low background contaminations in human breast milk. On the basis of the high sensitivity of the novel analytical method, it was possible to deduce that tolerable daily intake values were not exceeded by breastfeeding in the examined infants.

Lead compounds are known genotoxicants, principally affecting the integrity of chromosomes. Lead chloride and lead acetate induced concentration‐dependent increases in micronucleus frequency in V79 cells, starting at 1.1 μM lead chloride and 0.05 μM lead acetate. The difference between the lead salts, which was expected based on their relative abilities to form complex acetato‐cations, was confirmed in an independent experiment. CREST analyses of the micronuclei verified that lead chloride and acetate were predominantly aneugenic (CREST‐positive response), which was consistent with the morphology of the micronuclei (larger micronuclei, compared with micronuclei induced by a clastogenic mechanism). The effects of high concentrations of lead salts on the microtubule network of V79 cells were also examined using immunofluorescence staining. The dose effects of these responses were consistent with the cytotoxicity of lead(II), as visualized in the neutral‐red uptake assay. In a cell‐free system, 20–60 μM lead salts inhibited tubulin assembly dose‐dependently. The no‐observed‐effect concentration of lead(II) in this assay was 10 μM. This inhibitory effect was interpreted as a shift of the assembly/disassembly steady‐state toward disassembly, e.g., by reducing the concentration of assembly‐competent tubulin dimers. The effects of lead salts on microtubule‐associated motor‐protein functions were studied using a kinesin‐gliding assay that mimics intracellular transport processes in vitro by quantifying the movement of paclitaxel‐stabilized microtubules across a kinesin‐coated glass surface. There was a dose‐dependent effect of lead nitrate on microtubule motility. Lead nitrate affected the gliding velocities of microtubules starting at concentrations above 10 μM and reached half‐maximal inhibition of motility at about 50 μM. The processes reported here point to relevant interactions of lead with tubulin and kinesin at low dose levels. Environ. Mol. Mutagen., 2005. © 2005 Wiley‐Liss, Inc.

The tumor promoter 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is known to increase the expression of prostaglandin endoperoxide H synthase (PGHS)-2. This study focused on the regulatory mechanism of TCDD-mediated transcriptional activation of PGHS-2. Treatment of rat hepatocytes with TCDD led to a dose-dependent induction of PGHS-2 mRNA levels associated with an increased synthesis of prostaglandin E2, whereas expression of PGHS-1 was not affected. In vitro experiments with c-Src inhibitors, such as herbimycin A and geldanamycin, and in vivo studies with c-Src-deficient mice indicated that up-regulation of PGHS-2 but not the cytochrome P450 gene CYP1A1 by TCDD is mediated via a c-Src-dependent pathway. Transient transfection studies with different reporter constructs of the murine PGHS-2 promoter mutated in the xenobiotic-responsive element (XRE) or CCAAT/enhancer binding protein (C/EBP) element revealed that a C/EBP-binding site is an important regulatory cis-acting factor for trans-activation of the PGHS-2 gene by TCDD. Consistent with transfection studies, gel mobility shift assays showed that TCDD led to an enhanced DNA-binding activity of C/EBPβ transcription factor. The experimental data presented in this article reveal a XRE-independent and c-Src-mediated activation of the PGHS-2 gene by TCDD through the C/EBP response element located in its promoter region.

Von einer Reihe natürlicher und anthropogener Substanzen ist bekannt, dass sie östrogenartige Wirkungen aufweisen können. Es stellt sich daher die Frage, ob mit der Wirkung dieser Stoffe auf die Hormonsysteme von Mensch und Tier Risiken verbunden sind. Für eine Risikoabschätzung werden Daten über die tatsächliche Bioverfügbarkeit dieser Substanzen benötigt, zudem muss berücksichtigt werden, dass neben östrogenmimetischen auch antiöstrogene sowie antiandrogene Effekte auftreten können.

Abstract: Female DA/Han rats were administered p-tert-octylphenol [OP; p-(1,1,3,3-tetramethylbutyl)-phenol], either intravenously (5 mg/kg body wt.) or orally by gavage (50 or 200 mg/kg body wt.). After i.v. administration the blood concentration-time curve of OP was fitted to a tri-exponential model, resulting in a final half-life (γ-phase) of 36.1 h. This contrasts to much more rapid eliminations previously reported in male Wistar rats. The oral bioavailability of 50 mg/kg OP was 12.3% and of 200 mg/kg 8.4%. The higher dose (200 mg/kg) was absorbed slower than the smaller dose, probably due to low solubility of OP in aqueous media. Maximal OP blood levels in female DA/Han rats receiving 50 and 200 mg OP/kg body wt. were 4.5 and 3 times higher than previously reported in male Wistar rats. The blood concentration-time curves after oral administration of OP to female DA/Han rats revealed pronounced interindividual differences, indicating extensive enterohepatic circulation of OP in this rat strain. In contrast to male Wistar rats, after application of high doses of OP to female DA/Han rats the compound was not completely eliminated within 48 h; under these conditions some bioaccumulation might therefore occur. The experimental toxicokinetics of OP appears as a relevant subject to be integrated into extrapolation of toxicological data, from in vitro to in vivo, and into systems of risk assessment of endocrine modulating activity which are currently being developed.

Abstract: Chronic administration of o-phenylphenol (OPP) is known to induce urinary bladder tumours in the Fischer rat. The underlying toxic mechanism is poorly understood. Recently, arachidonic acid (ARA)-dependent, prostaglandin-H-synthase (PHS)-catalysed metabolic activation of the OPP metabolite phenylhydroquinone (PHQ) to a genotoxic species was suggested to be involved in OPP toxicity. To investigate this hypothesis in more detail, we have studied the effects of OPP and its metabolites on PHS. When microsomal PHS from ovine seminal vesicles (OSV) was used as enzyme source, both OPP, PHQ, and 2-phenyl-1,4-benzoquinone (PBQ) inhibited PHS-cyclooxygenase. The inhibitory potency was inversely related to the ARA concentration in the assay; at 7 μM ARA IC50-values were: 13 μM (OPP), 17 μM (PHQ), and 190 μM (PBQ). In cells cultured from OSV, which express high PHS activity, 40 μM OPP almost completely suppressed prostaglandin formation. Studies with microsomal PHS demonstrated that PHQ was an excellent substrate for PHS-peroxidase; both ARA and hydrogen peroxide supported oxidation to PBQ. OPP was only a poor substrate for PHS, but inhibited the ARA-mediated and to a lesser extent also the hydrogen peroxide-mediated in␣vitro oxidation of PHQ. Moreover, PHQ at up to moderately cytotoxic concentrations (50 μM) did not induce micronuclei in OSV cell cultures. Taken together, our findings do not provide evidence for an ARA-dependent, PHS-catalysed formation of genotoxic species from PHQ. Moreover, it seems to be questionable whether such activation can effectively occur in vivo, since OPP and PHQ turned out to be efficient cyclooxygenase inhibitors, and high levels of OPP and PHQ were found at least in the urine of OPP-treated rats. On the other hand, inhibition of the formation of cytoprotective prostaglandins in the urogenital tract may play a crucial role in OPP-induced bladder carcinogenesis.

Prostaglandin endoperoxide H synthases (PGHS-1 and PGHS-2) catalyze an intermediate step in the biosynthesis of prostaglandins and thromboxanes. Recently, it was observed that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) modulates the expression of PGHS-2 mRNA in different cell lines. The main aim of this study was to examine whether PGHS-2 mRNA expression can be changed by acute TCDDin vivoand, second, we were also interested in whether modulation of PGHS-2 is mediated by the aryl hydrocarbon receptor (AhR) which is known to be involved in the transcriptional control of TCDD-induced phase 1 and phase 2 enzymes. Initially C57BL/6J mice were treated with a single dose of 10,000 ng TCDD/kg and the PGHS-1 and PGHS-2 mRNAs were analyzed in liver, lung, thymus, kidney, and spleen. In all tissues examined the expression of PGHS-1 mRNA was not affected by TCDD. However, TCDD treatment enhanced the PGHS-2 mRNA levels in lung and spleen. No effect of TCDD on PGHS-2 expression was found in liver and kidney. For dose–response studies C57BL/6J and DBA/2J mice were treated for 24 h with various doses of TCDD (1–50,000 ng/kg) and the PGHS-2 mRNA increases were analyzed in lungs and spleens. A significant increase of PGHS-2 mRNA in lungs of C57BL/6J mice was found at a dose of 100 ng TCDD/kg, whereas a nearly 100-fold higher TCDD dose was needed to increase PGHS-2 in DBA/2J mice. A similar dose-dependent induction of PGHS-2 was found in spleens of C57BL/6J mice; however, no significant increase of PGHS-2 was found in spleens of DBA/2 mice. These results indicate an involvement of AhR in TCDD-mediated changes of PGHS-2 expression. This suggestion is supported by studies in AhR-deficient animals which showed that TCDD had no effect on PGHS-2 mRNA. When changes of PGHS-2 mRNA expression are compared with those of CYP1A1 between 4 and 72 h after TCDD, it is noteworthy that TCDD led to a delayed and more transient increase of PGHS-2. These data suggest that the mechanism of modulation of both genes by TCDD may be different.

Abstract:  Drug metabolizing enzyme activities have been determined in cultured porcine urinary bladder epithelial cells (PUBEC) in order to evaluate this system as an in vitro model for studies of urinary bladder carcinogens. Activities of several phase I and II enzymes were measured in cells cultured for various periods and compared with the activities determined in freshly isolated PUBEC. Prostaglandin H synthase mediated production of prostaglandin E2 was found both in freshly isolated and in cultured PUBEC, whereas cytochrome P450 1A1-associated EROD activity was only detectable in freshly isolated bladder cells. The latter activity was not inducible by benz(a)anthracene or 3-methylcholanthrene in PUBEC cultures. N-acetyltransferase (NAT) activity measured with p-aminobenzoic acid, a diagnostic substrate for human NAT-1, was stable and even higher during the culture period compared to freshly isolated cells. In contrast, isoniazid (a substrate for NAT-2) was not acetylated either in fresh or cultured PUBEC. Glutathione S-transferases activity determined with 1-chloro-2,4-dinitrobenzene decreased gradually to 50% after 1 week and to 20% after 4 weeks in culture compared to fresh cells. A similar decline was also observed for UDP-glucuronyltransferase activities measured with 1-naphthol. In accordance with the reported lack of sulfotransferases in pigs, no sulfation of 1-naphthol or 2-naphthylamine was detected in PUBEC. Our results show that cultured porcine urinary bladder epithelial cells maintain several enzyme activities required for the biotransformation of xenobiotics. In future investigations on the mechanism of action of bladder carcinogens PUBEC cultures may thus provide a useful in vitro model for this target tissue.