New aspects in deriving health-based guidance values for bromate in swimming pool water.

Bromate, classified as a EU CLP 1B carcinogen, is a typical by-product of the disinfection of drinking and swimming pool water. The aim of this study was (a) to provide data on the occurrence of bromate in pool water, (b) to re-evaluate the carcinogenic MOA of bromate in the light of existing data, (c) to assess the possible exposure to bromate via swimming pool water and (d) to inform the derivation of cancer risk-related bromate concentrations in swimming pool water. Measurements from monitoring analysis of 229 samples showed bromate concentrations in seawater pools up to 34 mg/L. A comprehensive non-systematic literature search was done and the quality of the studies on genotoxicity and carcinogenicity was assessed by Klimisch criteria (Klimisch et al., Regul Toxicol Pharmacol 25:1-5, 1997) and SciRAP tool (Beronius et al., J Appl Toxicol, 38:1460-1470, 2018) respectively. Benchmark dose (BMD) modeling was performed using the modeling average mode in BMDS 3.1 and PROAST 66.40, 67 and 69 (human cancer BMDL10; EFSA 2017). For exposure assessment, data from a wide range of sources were evaluated for their reliability. Different target groups (infants/toddlers, children and adults) and exposure scenarios (recreational, sport-active swimmers, top athletes) were considered for oral, inhalation and dermal exposure. Exposure was calculated according to the frequency of swimming events and duration in water. For illustration, cancer risk-related bromate concentrations in pool water were calculated for different target groups, taking into account their exposure using the hBMDL10 and a cancer risk of 1 in 100,000. Convincing evidence was obtained from a multitude of studies that bromate induces oxidative DNA damage and acts as a clastogen in vitro and in vivo. Since statistical modeling of the available genotoxicity data is compatible with both linear as well as non-linear dose-response relationships, bromate should be conservatively considered to be a non-threshold carcinogen. BMD modeling with model averaging for renal cancer studies (Kurokawa et al., J Natl. Cancer Inst, 1983 and 1986a; DeAngelo et al., Toxicol Pathol 26:587-594, 1998) resulted in a median hBMDL10 of 0.65 mg bromate/kg body weight (bw) per day. Evaluation of different age and activity groups revealed that top athletes had the highest exposure, followed by sport-active children, sport-active adults, infants and toddlers, children and adults. The predominant route of exposure was oral (73-98%) by swallowing water, followed by the dermal route (2-27%), while the inhalation route was insignificant (< 0.5%). Accepting the same risk level for all population groups resulted in different guidance values due to the large variation in exposure. For example, for an additional risk of 1 in 100,000, the bromate concentrations would range between 0.011 for top athletes, 0.015 for sport-active children and 2.1 mg/L for adults. In conclusion, the present study shows that health risks due to bromate exposure by swimming pool water cannot be excluded and that large differences in risk exist depending on the individual swimming habits and water concentrations.

Critical evaluation of human health risks due to hydraulic fracturing in natural gas and petroleum production.

The use of hydraulic fracturing (HF) to extract oil and natural gas has increased, along with intensive discussions on the associated risks to human health. Three technical processes should be differentiated when evaluating human health risks, namely (1) drilling of the borehole, (2) hydraulic stimulation, and (3) gas or oil production. During the drilling phase, emissions such as NOx, NMVOCs (non-methane volatile organic compounds) as precursors for tropospheric ozone formation, and SOx have been shown to be higher compared to the subsequent phases. In relation to hydraulic stimulation, the toxicity of frac fluids is of relevance. More than 1100 compounds have been identified as components. A trend is to use fewer, less hazardous and more biodegradable substances; however, the use of hydrocarbons, such as kerosene and diesel, is still allowed in the USA. Methane in drinking water is of low toxicological relevance but may indicate inadequate integrity of the gas well. There is a great concern regarding the contamination of ground- and surface water during the production phase. Water that flows to the surface from oil and gas wells, so-called 'produced water', represents a mixture of flow-back, the injected frac fluid returning to the surface, and the reservoir water present in natural oil and gas deposits. Among numerous hazardous compounds, produced water may contain bromide, arsenic, strontium, mercury, barium, radioactive isotopes and organic compounds, particularly benzene, toluene, ethylbenzene and xylenes (BTEX). The sewage outflow, even from specialized treatment plants, may still contain critical concentrations of barium, strontium and arsenic. Evidence suggests that the quality of groundwater and surface water may be compromised by disposal of produced water. Particularly critical is the use of produced water for watering of agricultural areas, where persistent compounds may accumulate. Air contamination can occur as a result of several HF-associated activities. In addition to BTEX, 20 HF-associated air contaminants are group 1A or 1B carcinogens according to the IARC. In the U.S., oil and gas production (including conventional production) represents the second largest source of anthropogenic methane emissions. High-quality epidemiological studies are required, especially in light of recent observations of an association between childhood leukemia and multiple myeloma in the neighborhood of oil and gas production sites. In conclusion, (1) strong evidence supports the conclusion that frac fluids can lead to local environmental contamination; (2) while changes in the chemical composition of soil, water and air are likely to occur, the increased levels are still often below threshold values for safety; (3) point source pollution due to poor maintenance of wells and pipelines can be monitored and remedied; (4) risk assessment should be based on both hazard and exposure evaluation; (5) while the concentrations of frac fluid chemicals are low, some are known carcinogens; therefore, thorough, well-designed studies are needed to assess the risk to human health with high certainty; (6) HF can represent a health risk via long-lasting contamination of soil and water, when strict safety measures are not rigorously applied.

Production of Highly Polarized Positrons Using Polarized Electrons at MeV Energies.

The Polarized Electrons for Polarized Positrons experiment at the injector of the Continuous Electron Beam Accelerator Facility has demonstrated for the first time the efficient transfer of polarization from electrons to positrons produced by the polarized bremsstrahlung radiation induced by a polarized electron beam in a high-Z target. Positron polarization up to 82% have been measured for an initial electron beam momentum of 8.19 MeV/c, limited only by the electron beam polarization. This technique extends polarized positron capabilities from GeV to MeV electron beams, and opens access to polarized positron beam physics to a wide community.

A novel method for measuring aromatase activity in tissue samples by determining estradiol concentrations.

Increasing scrutiny of endocrine disrupters has led to changes to European pesticide and biocide legislation and to the introduction of the Endocrine Disrupter Screening Program by the US EPA. One element of endocrine disrupter identification is to determine its effects on aromatase, but most available assays are limited as they depend on tritiated water production to indicate enzyme activity. Whilst acceptable for determining aromatase effects using a cell-free approach, this method is unreliable for cell or tissue-based investigations as other cytochrome P-450 isoenzyme activities can similarly produce tritiated water and consequently confound interpretation of the aromatase data. To address this lack of specificity an assay directly measuring the final estrogen product by incubating rat tissue protein with testosterone and measuring the resultant estradiol concentration was developed. Using this approach we demonstrated marked increases in enzyme activity in pregnant rat ovary samples and dose-related inhibitions when incubating non-pregnant rat ovary samples with known aromatase inhibitors. Hepatic aromatase activity was investigated using our method and by tritiated water production with microsomes from rats dosed with the antiandrogen 1,1-dichloro-2,2-bis(4 chlorophenyl)ethane. Additional cytochrome P-450s were also measured. Treatment-related increased tritiated water production and general hepatic enzyme activity were recorded but estradiol was not increased, indicating that the increased tritiated water was due to general enzyme activity and not aromatase activity. A simple and specific method has been developed that can detect aromatase inhibition and induction, which when applied to tissue samples, provides a means of generating relevant animal data concerning chemical effects on the aromatase enzyme.

The assessment of estrogenic or anti-estrogenic activity of chemicals by the human stably transfected estrogen sensitive MELN cell line: results of test performance and transferability.

The need for development and validation of in vitro hormone receptor transactivation assays as important alternative tools to study interactions with sex hormone receptors is outlined by international organisations, as such assays should be included in the OECD conceptual framework for the testing and assessment of endocrine active chemicals. Therefore as part of the European Union (EU)-sponsored 6th framework project ReProTect, the validation study with MELN cells, MCF-7 cells (ER+, estrogen receptor positive) which were stably transfected with the estrogen responsive gene ERE-betaGlob-Luc-SVNeo was set up. Standard operating procedures including a prescreen assay for unknown chemicals, an ER-agonist assay and an ER-antagonist assay were developed at the Flemish Institute for Technological Research, Belgium, and successfully transferred to Bayer Schering Pharma AG, Germany. Test results were obtained for 16 chemicals, and it was demonstrated that the MELN assay is transferable, robust and reproducible which allowed to rank chemical compounds according to their strong to weak affinity for the estrogen-alpha receptor, or identify negative chemicals within the test range up to 10(-5)M. Besides the screening for agonism, we demonstrated the suitability of MELN cells to test for antagonistic activity, which is of added value compared to current validated assays. As the MELN assay successfully passed the first modules of the ECVAM validation procedure, it now should be considered for further steps including the definition of a prediction model and application domain to get it accepted as an alternative screening assay, contributing to the 3R's with a reduction of animal experiments.

Screening for (anti)androgenic properties using a standard operation protocol based on the human stably transfected androgen sensitive PALM cell line. First steps towards validation.

Despite more than a decade of research in the field of endocrine active compounds targeting the androgen receptor (AR), and although suitable cell lines can be obtained, no validated human stably transfected androgen sensitive transactivation assay is available. Bayer Schering Pharma (BSP) and the Flemish Institute for Technological Research (VITO), partners within the EU-sponsored 6th framework project ReProTect, made first steps towards such a validation. A standard operation protocol (SOP) developed at BSP based on the androgen sensitive PALM cell line was transferred to VITO and its performance and transferability were thoroughly studied. The investigation followed a generic protocol prepared for all reporter gene assays evaluated within ReProTect, and in both laboratories at least three independent experiments were performed. The highest concentration to be tested was limited to 10 microM, if needed. A few compounds, 17alpha-methyltestosterone (17alpha-MT), vinclozolin and linuron, were studied using a real world scenario, i.e., assuming that their interaction with the AR was not known: A prescreening for agonism and true, competitive antagonism was used to select conditions such as the appropriate mode of action, and the working range excluding cytotoxicity for the final screening. All other compounds were tested according to the generic protocol: Compounds screened for agonism were the reference androgen 17alpha-methyldihydrotestosterone (MDHT), levonorgestrel, norethynodrel, progesterone, o,p'-DDT, and dibutylphthalate (DBP), while compounds screened for antagonism were the reference anti-androgen flutamide, prochloraz, o,p'-DDT, progesterone, norethynodrel, and DBP. Cytotoxicity was assessed in parallel as lactate dehydrogenase release. The prescreen classified 17alpha-MT as androgenic, vinclozolin and linuron as anti-androgenic and compounds were tested accordingly. In the absence of cytotoxicity, appropriate androgenic properties of reference and test compounds were detected by both laboratories, o,p'-DDT and DBP had no androgenic activity. Across the two laboratories EC(50)-values for MDHT, 17alpha-MT, and levonorgestrel varied by not more than a factor of 3.4, for norethynodrel by a factor of 9.7. Progesterone effects could not fully be evaluated, as frequently concentration response curves were incomplete. In the absence of cytotoxicity anti-androgenic properties of reference and test compounds were also detected in both laboratories. DBP, the putative negative reference compound, was inactive, norethynodrel rather showed agonistic properties. Progesterone was an antagonist at low concentrations, but agonistic properties were observed in one laboratory at high concentrations. Since the highest test concentration was limited to 10 microM, for some compounds no complete concentration response curves were obtained and estimation of EC(50)-values was less robust. Our data demonstrated that the SOP was transferable, and that the assay was able to rank compounds with strong, weak, and without affinity for the AR and to discriminate agonists and antagonists. The sensitivity of the assay could be improved further, if the limit of solubility or beginning cytotoxicity was chosen as the highest test concentration. The assay avoids the use of tissues from laboratory animals, and thus contributes to the 3R concept. Furthermore, it could be adjusted to an intermediate/high throughput format. On the whole, this PALM assay is a promising candidate for further validation.

Evaluation of the rodent Hershberger bioassay on intact juvenile males--testing of coded chemicals and supplementary biochemical investigations.

Under the auspices of the Organization for Economic Cooperation and Development (OECD) the Hershberger assay on juvenile intact male rats is being validated as a screen for compounds with anti-androgenic potential. We participated in the testing of coded chemicals. Compounds included the positive control flutamide (FLUT, 3 mg/kg), linuron (LIN, 10, 100 mg/kg), p,p'-DDE (16, 160 mg/kg), and two negative substances, 4-nonylphenol (NP, 160 mg/kg) and 2,4-dinitrophenol (DNP, 10 mg/kg). Compounds were administered for 10 consecutive days by gavage to testosterone propionate (TP, 1 mg/kgs.c.)-supplemented rats. Uncoding revealed these results: compared to vehicle controls, treatment with TP resulted in increased androgen-sensitive tissue (AST) weights of ventral prostate (VP), seminal vesicles (SV), levator ani and bulbocavernosus muscles (LABC), Cowper's glands, and epididymides, and in decreased testes weight. When assessing anti-androgenic potential in TP-supplemented rats, FLUT decreased all AST weights, and increased testes weight. p,p'-DDE at the high dose, decreased final body weight and all AST weights, whereas the low dose only affected SV weight. LIN slightly decreased final body weight and decreased absolute SV and LABC and relative SV weights only at the high dose. NP decreased final body weight and only absolute SV weights, DNP was ineffective. Investigations not requested by OECD included measurement of liver enzymes and revealed strong induction of testosterone-metabolizing and phase II conjugating enzymes by p,p'-DDE. Our findings suggest that in principle the juvenile intact male rat can be used in the Hershberger assay to screen for anti-androgenic potential thereby contributing to a refinement of the assay in terms of animal welfare. However, in our hands this animal model was somewhat less sensitive than the peripubertal castrated rat. Final conclusions, however, can only be drawn on the basis of all available validation data. Results obtained with the negative reference compound NP suggest that a treatment-related decrement in body weights may affect AST weights and represent a confounding factor when screening for anti-androgenic properties. Finally, p,p'-DDE may affect AST weights by several mechanisms including enhanced testosterone metabolism.

The use of metabolising systems for in vitro testing of endocrine disruptors.

Legislation and prospective legislative proposals in for instance the USA, Europe, and Japan require, or may require that chemicals are tested for their ability to disrupt the hormonal systems of mammals. Chemicals found to test positive are considered to be endocrine active substances (EAS) and may be putative endocrine disruptors (EDs). To date, there is still little or no experience with incorporating metabolic and toxicokinetic aspects into in vitro tests for EAS. This is a situation in sharp contrast to genotoxicity testing, where in vitro tests are routinely conducted with and without metabolic capacity. Originally prepared for the Organisation of Economic Cooperation and Development (OECD), this detailed review paper reviews why in vitro assays for EAS should incorporate mammalian systems of metabolism and metabolic enzyme systems, and indicates how this could be done. The background to ED testing, the available test methods, and the role of mammalian metabolism in the activation and the inactivation of both endogenous and exogenous steroids are described. The available types of systems are compared, and the potential problems in incorporating systems in in vitro tests for EAS, and how these might be overcome, are discussed. Lastly, some recommendations for future activities are made.

Evaluation of the rodent Hershberger bioassay: testing of coded chemicals and supplementary molecular-biological and biochemical investigations.

Under the auspices of the Organization for Economic Cooperation and Development (OECD) the Hershberger assay is being validated as an in vivo screen for compounds with (anti)androgenic potential. We participated in the final activity, the testing of coded chemicals. Test compounds included trenbolone (TREN; 1.5, 40 mg/kg), testosterone propionate (TP; 0.4 mg/kg), flutamide (FLUT; 3mg/kg), linuron (LIN; 10, 100mg/kg), 1,1-bis-(4-chlorophenyl)-2,2-dichloroethylene (p,p'-DDE; 16, 160 mg/kg), and two negative reference substances, i.e., compounds not considered to affect androgen-sensitive tissue weights (ASTWs) in the Hershberger assay, namely 4-nonylphenol (NP; 160 mg/kg) and 2,4-dinitrophenol (DNP; 10mg/kg); TREN, LIN, p,p'-DDE, NP, and DNP being used under code. Compounds were administered for 10 days by oral intubation or subcutaneous injection (TP). Additional investigations not mandatorily requested by OECD included organ gravimetry of the liver, gene expression analysis in prostate using quantitative RT PCR for prostate specific binding protein polypeptide C3 (PBPC3) and ornithine decarboxylase 1 (ODC1) and determination of testosterone metabolizing and phase II conjugating enzymes in the liver. After submission of all study reports to OECD by participants uncoding revealed the following results: (A) When assessing androgenic potential in castrated rats, administration of TREN increased the weights of ventral prostate (VP), seminal vesicles (SV), glans penis, levator ani and bulbocavernosus muscles, and Cowper's glands at the high dose. A similar or stronger (VP, SV) increase of ASTWs was observed for TP; NP and DNP were ineffective. TREN dose-dependently increased gene expression of ODC1 and PBPC3, TP induced expression of these genes even more strongly (almost) to the level of untreated intact animals, whereas NP and DNP were inactive. Liver enzyme activities depending on physiological androgen levels were lower in castrated than in intact rats and could not be restored by androgen treatment. (B) When assessing antiandrogenic potential in TP-supplemented castrated rats, administration of LIN and p,p'-DDE decreased ASTWs only at the high dose. FLUT even more effectively decreased ASTWs, NP and DNP were again without effect. Decreases in androgen-responsive gene expression in the prostate corresponding to the organ weight changes were only observed for p,p'-DDE (high dose) and flutamide (PBPC3 only). p,p'-DDE dose-dependently induced liver weights and most liver enzyme activities including androgen-dependent ones. Our study accurately reproduced ASTW changes obtained in previous studies also under code suggesting that the Hershberger assay is a robust tool to screen for an (anti)androgenic potential. Assessment of ODC1 and PBPC3 gene expression in prostate, however, may only represent a sensitive tool for the detection of an androgenic potential. Finally, p,p'-DDE may affect ASTWs by several mechanisms including enhanced testosterone metabolism.

Measurements of the proton elastic-form-factor ratio mu pG p E/G p M at low momentum transfer.

High-precision measurements of the proton elastic form-factor ratio, mu pG p E/G p M, have been made at four-momentum transfer, Q2, values between 0.2 and 0.5 GeV2. The new data, while consistent with previous results, clearly show a ratio less than unity and significant differences from the central values of several recent phenomenological fits. By combining the new form-factor ratio data with an existing cross-section measurement, one finds that in this Q2 range the deviation from unity is primarily due to G p E being smaller than expected.

O-phenylphenol and its sodium and potassium salts: a toxicological assessment.

Ortho-phenylphenol (OPP) and its sodium (SOPP) and potassium (POPP) salts are used as fungicides and disinfectants. Due to the widespread use of especially OPP and SOPP, the potential for consumer exposure and some "critical" findings the toxicological database is quite extensive and complex. In experimental animals toxicity after single oral and dermal administration of these compounds is low. For the skin and mucous membranes, OPP has to be considered as irritating, and SOPP and POPP as corrosive. A large number of chronic toxicity and reproduction studies did not show any indication of oestrogen-like or other endocrine effects of OPP in the mammalian organism. No teratogenic effects were observed after the administration of OPP or SOPP in rats, mice, and rabbits. In two-generation studies in rats, OPP did not affect reproduction. The available data do not suggest a relevant potential for immunotoxic properties. The administration of high dietary concentrations of OPP to mice up to 2 years induced hepatocellular changes indicative of adaptations to metabolic demands, zonal degeneration, focal hepatocellular necrosis, and/or pigmentation of the liver. Only in male mice of one study, using a strain prone to develop hepatocellular tumors at high spontaneous incidences, the incidence of hepatocellular adenomas was increased. The incidence of hepatocellular carcinomas was not affected by treatment. The urothel of the urinary bladder (at very high doses also of the renal pelvis and the papilla) is the main target tissue after the repeated oral exposure of rats. The changes initially consist of increased mitosis, followed by simple epithelial hyperplasia, developing to a papillary and/or nodular form, later on to papillomas and transitional carcinomas. Crystals or stones in the bladder do not play a decisive role in this cascade. SOPP is more effective than OPP in this respect. Male rats are much more sensitive than females. In mice, hamsters, guinea pigs, and dogs, urothelial lesions do not develop even at very high oral dose levels. The findings in rats explain why there is a large genotoxicity/mutagenicity data base not only for OPP and SOPP but also for their metabolites on nearly all kinds of endpoints/targets. The weight of evidence suggests that genotoxicity of OPP/SOPP or their metabolites does not play a decisive role for the carcinogenicity at the urothel. Among them are lack of DNA binding of OPP to the rat bladder epithelium, the differences between OPP and SOPP, between male and female rats, between rats and mice (despite roughly comparable toxicokinetics), as well as the fact that tumors develop only at dose levels inducing hyperplasias. In addition, the strong dependence of the incidence and severity of the nonneoplastic and neoplastic bladder changes on urinary pH values (modified by feeding of ammonium chloride or sodium hydrogen carbonate) is consistent with the hypothesis of a nongenotoxic mode of action. Finally, there is no correlation between the urinary concentration of OPP or its metabolites and the incidence of hyperplasias/tumors in the urinary bladder. Both tumorigenic effects in rats and male mice are considered to represent high-dose, sex- and/or species-specific phenomena, based on nongenotoxic mechanisms of action and therefore allow the conclusion that the conventional margin of safety approaches are appropriate when assessing the risk of applications of OPP and its salts.

Feasibility and potential gains of enhancing the subacute rat study protocol (OECD test guideline no. 407) by additional parameters selected to determine endocrine modulation. A pre-validation study to determine endocrine-mediated effects of the antiandrogenic drug flutamide.

Groups of five male and five female Wistar rats were treated by gavage with 0, 1, 10, and 100 mg flutamide/kg body weight for at least 28 days to investigate whether proposed enhancements to the current subacute rodent OECD test guideline no. 407 could be included into the testing routine, which of the current and/or additional parameters would detect endocrine-mediated effects of flutamide reliably and sensitively, and to provide information on intra-laboratory variability. Two identical studies were performed concurrently. The enhanced protocol requests the additional determination of the specific hormones triiodothyronine, thyroxine, thyroid stimulating hormone, follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol, prolactin, testosterone, corticosterone; of oestrus cyclicity and necropsy of all females in the dioestrus stage; of the number of homogenization-resistant testicular spermatids and the number, motility, viability, and morphology of cauda epididymal spermatozoa; of additional organ weights (pituitary, ovaries, uterus, thyroid, male accessory reproductive organs); and of the histopathology of additional organs (pituitary, epididymides, coagulation glands, pancreas, vagina). From a technical standpoint, it was possible to conduct a study according to the enhanced protocol, however, with substantial additional effort, an increase in costs by some 67%, and logistic problems. In line with the specific pharmacological effect of flutamide, treatment-related changes were mainly found in male rats, while females were hardly affected by 100 mg/kg. In male rats, 100 mg/kg was the maximal tolerated dose resulting in reduced body weight gain, but no or little other effects on clinical, haematological, clinico-chemical, or behavioral parameters, and 1 mg/kg was the no-observed-adverse-effect level. Antagonism of peripheral androgen receptors by flutamide resulted in decreased relative organ weights of male accessory reproductive organs, changes that were reliably detected in both studies at 100 mg/kg, but only in one of both studies at 10 mg/kg. Corresponding histopathological changes were also detected reliably at 100 mg/kg. Antagonism of central androgen receptors by flutamide increased LH and FSH levels. LH stimulation of testicular Leydig cells in turn increased testosterone and estradiol levels. Again, all these changes were detected reliably at 100 mg/kg, but only in one of both studies at 10 mg/kg. Corresponding histopathological alterations (increase of LH- and FSH-secreting cells, Leydig cell hypertrophy) were detected reliably and sensitively at 10 mg/kg. Studies on liver enzymes performed outside the scope of the enhanced protocol showed that flutamide at 100 mg/kg generally induced hepatic enzyme activities, but decreased the activity of the sex-specific testosterone-dependent liver enzyme CYP2C11 in male rats. The laboratory methods employed yielded reliable results, i.e., 93.6% of the quantitative measurements obtained in both studies were in agreement. Doubling the animal number from five to ten per sex and dose does not increase the sensitivity of detection of endocrine-mediated effects above the level already provided by histopathological examination of groups of five animals. Some of the proposed enhancements evaluated (additional organgravimetry and histopathology) were helpful in detecting the endocrine-mediated effects of flutamide reliably, while others did not contribute towards this aim (spermatology resulted in doubtful effects, female cyclicity was not affected, hormone determinations provided mechanistic information). Ongoing testing according to the revised version of the enhanced OECD test guideline no. 407 protocol and using ten compounds interfering with the endocrine system by different mechanisms will result in the identification of the most appropriate enhancements.

Differential response of immature rat uterine tissue to ethinylestradiol and the red wine constituent resveratrol.

The stilbene derivative resveratrol (RES) is a phytoalexin of grapes, peanuts and other fruits. It is structurally related to stilbene estrogens and an estrogenic potential of RES has recently been demonstrated in a number of in vitro studies. In this investigation, the uterotrophic responses of immature Wistar rats to subcutaneous administration of RES (18, 58, and 575 mg/ kg) and the reference estrogen ethinylestradiol (EE2; 0.3, 1, 3, 30 microg/kg) on three consecutive days were determined. Uterine weight, histopathological changes, immunohistochemical expression of nuclear estrogen receptor-alpha (ERalpha) and progesterone receptor (PR) protein, gene expression of ERalpha and PR at the messenger ribonucleic acid (mRNA) level and peroxidase induction were examined. EE2 dose dependently increased uterine weight, enlarged the uterine lumen and induced hypertrophy of epithelial, stromal and myometrial cells. Expression of ERalpha protein in epithelial, stromal and myometrial nuclei and of PR protein in epithelial nuclei was reduced in EE2-treated rats, while PR protein in stromal and myometrial nuclei was increased in a dose-dependent manner. EE2 increased messenger ribonucleic acid (mRNA) levels of uterine PR and induced peroxidase activity. In contrast, RES rather mildly decreased uterine weight, while histology did not reveal differences between controls and RES-treated rats. Expression of nuclear ERalpha protein was dose dependently decreased in epithelial, stromal and myometrial cells of RES-treated rats, while nuclear PR protein content was similar in controls and RES-treated rats. Following administration of RES, a trend toward reduced levels of ERalpha and PR mRNA was found, while no peroxidase induction occurred. Plasma levels of RES, 45 min after the administration of a single subcutaneous dose of 500 mg/kg, were in the range 1-2 microM. In summary, an estrogenic potential of RES could not be substantiated in this in vivo study, although the most effective route of administration and extremely high doses were used and plasma levels were in the range reported to be effective in vitro. Whether other pharmacological properties of RES could mediate the observed changes in RES-treated animals is discussed.

Inhibition of prostaglandin-H-synthase by o-phenylphenol and its metabolites.

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 microM ARA IC50-values were: 13 microM (OPP), 17 microM (PHQ), and 190 microM (PBQ). In cells cultured from OSV, which express high PHS activity, 40 microM 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 microM) 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.

Rapid, high performance liquid chromatography-based, periodate-coupled thiobarbituric acid method for the determination of sialic acid in bronchoalveolar lavage fluid.

A simple, rapid high performance liquid chromatographic adaptation of the periodate-coupled thiobarbituric acid (TBA) method was developed for the quantification of sialic acid (SIAC) in supernatants from bronchoalveolar lavage fluids (BALF). Malondialdehyde released from BALF samples in various amounts did not interfere with the accurate quantification of SIAC.