EDC IMPACT: Reduced sperm counts in rats exposed to human relevant mixtures of endocrine disrupters.

Human semen quality is declining in many parts of the world, but the causes are ill defined. In rodents, impaired sperm production can be seen with early life exposure to certain endocrine-disrupting chemicals, but the effects of combined exposures are not properly investigated. In this study, we examined the effects of early exposure to the painkiller paracetamol and mixtures of human relevant endocrine-disrupting chemicals in rats. One mixture contained four estrogenic compounds; another contained eight anti-androgenic environmental chemicals and a third mixture contained estrogens, anti-androgens and paracetamol. All exposures were administered by oral gavage to time-mated Wistar dams rats (n = 16-20) throughout gestation and lactation. In the postnatal period, testicular histology was affected by the total mixture, and at the end of weaning, male testis weights were significantly increased by paracetamol and the high doses of the total and the anti-androgenic mixture, compared to controls. In all dose groups, epididymal sperm counts were reduced several months after end of exposure, i.e. at 10 months of age. Interestingly, the same pattern of effects was seen for paracetamol as for mixtures with diverse modes of action. Reduced sperm count was seen at a dose level reflecting human therapeutic exposure to paracetamol. Environmental chemical mixtures affected sperm count at the lowest mixture dose indicating an insufficient margin of safety for the most exposed humans. This causes concern for exposure of pregnant women to paracetamol as well as environmental endocrine disrupters.

Low-dose developmental exposure to bisphenol A alters the femoral bone geometry in wistar rats.

BACKGROUND: Bisphenol A (BPA) is a chemical produced in large volumes for use in manufacturing of consumer products and industrial applications, and an endocrine disruptor known to affect several hormonal systems. Bone produces hormones and is additionally a sensitive hormone target tissue, and is thus potentially sensitive to low doses of endocrine disruptors such as BPA, especially during development. METHODS: 110 pregnant Wistar rats were gavaged with 0; 25 µg; 250 µg; 5000 µg or 50,000 µg BPA/kg bodyweight (bw)/day from gestational day 7 until weaning at postnatal day 22. The three-month-old offspring were sacrificed and right femurs collected for length measurements, geometrical measurements by peripheral quantitative computed tomography (pQCT), as well as for analyses of biomechanical properties using the three-point-bending method. RESULTS: The femur was elongated in female offspring of dams exposed to 25 or 5000 µg BPA/kg bw/day (1.8% and 2.1%, respectively), and increased cortical thickness (4.7%) was observed in male offspring of dams exposed to 25 µg BPA/kg bw/day, compared to controls (p < 0.005). The biomechanical properties of the bone were not significantly altered. CONCLUSIONS: In utero and lactational exposure to the lowest BPA dose used in this study altered femoral geometry in both male and female offspring. This was observed at 25 µg BPA/kg bw/day, a dose lower than the Human Equivalent Dose (HED) applied by EFSA to set a temporary TDI (609 µg BPA/kg bw/day), and far lower than the No-Observed-Adverse-Effect-Level (NOAEL) (5000 µg BPA/kg bw/day) on which the US FDA TDI is based.

Multiple Endocrine Disrupting Effects in Rats Perinatally Exposed to Butylparaben.

Parabens comprise a group of preservatives commonly added to cosmetics, lotions, and other consumer products. Butylparaben has estrogenic and antiandrogenic properties and is known to reduce sperm counts in rats following perinatal exposure. Whether butylparaben exposure can affect other endocrine sensitive endpoints, however, remains largely unknown. In this study, time-mated Wistar rats (n = 18) were orally exposed to 0, 10, 100, or 500 mg/kg bw/d of butylparaben from gestation day 7 to pup day 22. Several endocrine-sensitive endpoints were adversely affected. In the 2 highest dose groups, the anogenital distance of newborn male and female offspring was significantly reduced, and in prepubertal females, ovary weights were reduced and mammary gland outgrowth was increased. In male offspring, sperm count was significantly reduced at all doses from 10 mg/kg bw/d. Testicular CYP19a1 (aromatase) expression was reduced in prepubertal, but not adult animals exposed to butylparaben. In adult testes, Nr5a1 expression was reduced at all doses, indicating persistent disruption of steroidogenesis. Prostate histology was altered at prepuberty and adult prostate weights were reduced in the high dose group. Thus, butylparaben exerted endocrine disrupting effects on both male and female offspring. The observed adverse developmental effect on sperm count at the lowest dose is highly relevant to risk assessment, as this is the lowest observed adverse effect level in a study on perinatal exposure to butylparaben.

Low-dose effects of bisphenol A on mammary gland development in rats.

Bisphenol A (BPA) is widely used in food contact materials, toys, and other products. Several studies have indicated that effects observed at doses near human exposure levels may not be observed at higher doses. Many studies have shown effects on mammary glands at low doses of BPA, however, because of small number of animals or few doses investigated these data have not been used by EFSA as point of departure for the newly assessed tolerable daily intake (TDI). We performed a study with perinatal exposure to BPA (0, 0.025, 0.25, 5, and 50 mg/kg bw/day) in rats (n = 22 mated/group). One of the aims was to perform a study robust enough to contribute to the risk assessment of BPA and to elucidate possible biphasic dose-response relationships. We investigated mammary gland effects in the offspring at 22, 100, and 400 days of age. Male offspring showed increased mammary outgrowth on pup day (PD) 22 at 0.025 mg/kg BPA, indicating an increased mammary development at this low dose only. Increased prevalence of intraductal hyperplasia was observed in BPA females exposed to 0.25 mg/kg at PD 400, but not at PD 100, and not at higher or lower doses. The present findings support data from the published literature showing that perinatal exposure to BPA can induce increased mammary growth and proliferative lesions in rodents. Our results indicate that low-dose exposure to BPA can affect mammary gland development in male and female rats, although higher doses show a different pattern of effects. The observed intraductal hyperplasia in female rats could be associated with an increased risk for developing hyperplastic lesions, which are parallels to early signs of breast neoplasia in women. Collectively, current knowledge on effects of BPA on mammary gland at low doses indicates that highly exposed humans may not be sufficiently protected.

Low-dose effect of developmental bisphenol A exposure on sperm count and behaviour in rats.

Bisphenol A is widely used in food contact materials and other products and is detected in human urine and blood. Bisphenol A may affect reproductive and neurological development; however, opinion of the European Food Safety Authority (EFSA) on bisphenol A (EFSA J, 13, 2015 and 3978) concluded that none of the available studies were robust enough to provide a point of departure for setting a tolerable daily intake for bisphenol A. In the present study, pregnant Wistar rats (n = 17-21) were gavaged from gestation day 7 to pup day 22 with bisphenol A doses of 0, 25 µg, 250 µg, 5 mg or 50 mg/kg bw/day. In the offspring, growth, sexual maturation, weights and histopathology of reproductive organs, oestrus cyclicity and sperm counts were assessed. Neurobehavioural development was investigated using a behavioural testing battery including tests for motor activity, sweet preference, anxiety and spatial learning. Decreased sperm count was found at the lowest bisphenol A dose, that is 25 µg/kg/day, but not at the higher doses. Reproductive organ weight and histology were not affected and no behavioural effects were seen in male offspring. In the female offspring, exposure to 25 µg/kg bw/day bisphenol A dose resulted in increased body weight late in life and altered spatial learning in a Morris water maze, indicating masculinization of the brain. Decreased intake of sweetened water was seen in females from the highest bisphenol A dose group, also a possible sign of masculinization. The other investigated endpoints were not significantly affected. In conclusion, the present study using a robust experimental study design, has shown that developmental exposure to 25 µg/kg bw/day bisphenol A can cause adverse effects on fertility (decreased sperm count), neurodevelopment (masculinization of spatial learning in females) and lead to increased female body weight late in life. These results suggest that the new EFSA temporary tolerable daily intake of 4 µg/kg bw/day is not sufficiently protective with regard to endocrine disrupting effects of bisphenol A in humans.

Mixtures of endocrine disrupting contaminants modelled on human high end exposures: an exploratory study in rats.

By diminishing the action of androgens during gestation, certain chemicals can induce irreversible demasculinization and malformations of sex organs in the male rat after gestational exposure. Studies with mixtures of such anti-androgens have shown that substantial combined effects occur even though each individual chemical is present at low, ineffective doses, but the effects of mixtures modelled based on human intakes have not previously been investigated. To address this issue for the first time, we selected 13 chemicals for a developmental mixture toxicity study in rats where data about in vivo endocrine disrupting effects and information about human exposures was available, including phthalates, pesticides, UV-filters, bisphenol A, parabens and the drug paracetamol. The mixture ratio was chosen to reflect high end human intakes. To make decisions about the dose levels for studies in the rat, we employed the point of departure index (PODI) approach, which sums up ratios between estimated exposure levels and no-observed-adverse-effect-level (NOAEL) values of individual substances. For high end human exposures to the 13 selected chemicals, we calculated a PODI of 0.016. As only a PODI exceeding 1 is expected to lead to effects in the rat, a total dose more than 62 times higher than human exposures should lead to responses. Considering the high uncertainty of this estimate, experience on lowest-observed-adverse-effect-level (LOAEL)/NOAEL ratios and statistical power of rat studies, we expected that combined doses 150 times higher than high end human intake estimates should give no, or only borderline effects, whereas doses 450 times higher should produce significant responses. Experiments indeed showed clear developmental toxicity of the 450-fold dose in terms of increased nipple retention (NR) and reduced ventral prostate weight. The 150-fold dose group exhibited significantly increased NR. These observations suggest that highly exposed population groups, especially women of reproductive age, may not be protected sufficiently against the combined effects of chemicals that affect the hormonal milieu required for normal male sexual differentiation.

Perinatal ethinyl oestradiol alters mammary gland development in male and female Wistar rats.

Increased attention is being paid to human mammary gland development because of concerns for environmental influences on puberty onset and breast cancer development. Studies in rodents have showed a variety of changes in the mammary glands after perinatal exposure to endocrine disrupting chemicals, indicating progressed development of mammary glands when exposed to oestrogens early in life. However, laboratories use different parameters to evaluate the development of mammary glands, making studies difficult to compare. Moreover, studies of whole mounts in Wistar rats are lacking. In the present study, Wistar rats were exposed to 0, 5, 15 or 50 µg/kg of ethinyl oestradiol per day during gestation and lactation. A wide range of morphological parameters were evaluated in whole mounts of mammary glands from male and female offspring PD21-22. This study showed that in both male and female pre-pubertal Wistar rats, mammary gland development was accelerated after perinatal oestrogen exposure with increase in size, density and number of terminal end buds (TEBs). In female rats, the most sensitive parameters were the distance to the fifth gland, the relative growth towards the lymph node and the overall density. The sensitive endpoints in male rats were TEB numbers, both in the whole gland and in the zone C, the overall- and the highest density. The overall density was sensitive in both male and female rats and was considered a good representative of both branching and budding of the gland. The number of TEBs in zone C was representative of the number of TEBs in the whole gland. Further studies in older Wistar rats and with weak oestrogenic compounds could be performed to validate mammary gland examination as an endpoint in reproductive toxicity studies and to examine how early life environmental exposures may alter mammary gland development, disrupt lactation and alter susceptibility to breast cancer.

Combined exposure to endocrine disrupting pesticides impairs parturition, causes pup mortality and affects sexual differentiation in rats.

Risk assessment is currently based on the no observed adverse effect levels (NOAELs) for single compounds. Humans are exposed to a mixture of chemicals and recent studies in our laboratory have shown that combined exposure to endocrine disrupters can cause adverse effects on male sexual development, even though the doses of the single compounds are below their individual NOAELs for anti-androgenic effects. Consequently, we have initiated a large project where the purpose is to study mixture effects of endocrine disrupting pesticides at low doses. In the initial range-finding mixture studies, rats were gavaged during gestation and lactation with five doses of a mixture of the fungicides procymidone, mancozeb, epoxyconazole, tebuconazole and prochloraz. The mixture ratio was chosen according to the doses of each individual pesticide that produced no observable effects on pregnancy length and pup survival in our laboratory and the dose levels used ranged from 25 to 100% of this mixture. All dose levels caused increased gestation length and dose levels above 25% caused impaired parturition leading to markedly decreased number of live born offspring and high pup perinatal mortality. The sexual differentiation of the pups was affected at 25% and higher as anogenital distance was affected in both male and female offspring at birth and the male offspring exhibited malformations of the genital tubercle, increased nipple retention, and decreased prostate and epididymis weights at pup day 13. The results show that doses of endocrine disrupting pesticides, which appear to induce no effects on gestation length, parturition and pup mortality when judged on their own, induced marked adverse effects on these endpoints in concert with other pesticides. In addition, the sexual differentiation of the offspring was affected. This as well as the predictability of the combination effects based on dose-additivity modelling will be studied further in a large dose-response study.

Combined exposure to anti-androgens causes markedly increased frequencies of hypospadias in the rat.

The incidence of hypospadias is increasing in young boys, but it remains unclear whether human exposure to endocrine disrupting chemicals plays a role. Risk assessment is based on estimation of no-observed-adverse-effect levels for single compounds, although humans are exposed to combinations of several anti-androgenic chemicals. In a mixture (MIX) study with three androgen receptor antagonists, vinclozolin, flutamide and procymidone, rats were gavaged during gestation and lactation with several doses of a MIX of the three chemicals or the chemicals alone. External malformations of the male reproductive organs were assessed on PND 47 using a score from 0 to 3 (normal to marked) for hypospadias. Markedly increased frequencies were observed after exposure to a MIX of the three chemicals compared to administration of the three chemicals alone. Anogenital distance at PND 1, nipple retention at PND 13, and dysgenesis score at PND 16 were highly correlated with the occurrence of hypospadias, and MIX effects were seen at doses where each of the individual chemicals caused no observable effects. Therefore, the results indicate that doses of anti-androgens, which appear to induce no hypospadias when judged on their own, may induce a very high frequency of hypospadias when they interact in concert with other anti-androgens.

BioWAP, mobile Internet service for bioinformatics.

UNLABELLED: We have developed a new Internet service, which provides mobile access to bioinformatics databases and software tools. The BioWAP service facilitates access to basic bioinformatics databases and analysis tools from everywhere without a PC or a laptop computer. Both open source bioinformatics program suites and Internet services, which are not designed for mobile Internet access, were utilized in the BioWAP service. AVAILABILITY: The BioWAP service starting page can be browsed with any WAP terminal from http://bioinf.uta.fi/wml/welcome.wml.

Effects of the multiple stressors copper and reduced salinity on the metabolism of the hermatypic coral Porites lutea.

This study investigates the physiological responses in the hermatypic coral Porites lutea when exposed to a combination of reduced salinity (from ambient 30 psu to 20 psu) and two concentrations of copper (CuS04), 10 microg 1(-1) and 30 microg 1(-1). Corals were exposed for 14 h and changes in metabolism in terms of primary production rate per chlorophyll a and respiration per surface area (cm2) were used as measures of stress. The results showed no significant changes in respiration rate in any of the treatments compared with controls, or between treatments. The primary production rate, however, displayed a more complex pattern. Corals exposed to reduced salinity, 30 microg 1(-1) copper, and the combination of the two stressors significantly reduced the production rate, whereas corals exposed to 10 microg 1(-1) only, remained unaffected. However, adding 10 microg 1(-1) copper to reduced salinity did not affect the production rate thus indicating an antagonistic effect.

Spouses as adjuncts in fluency therapy.

Couples with one partner who stutters completed questionnaires about the fluent partners' awareness of their spouses' stuttering; their general knowledge about stuttering; how fluent spouses had helped their partners; and the nature and benefits of spousal participation in therapy. Findings strongly suggested that spouses may be helpful in therapy. Considerations and suggestions for involving spouses are made on the basis of these findings and those of surveys completed by speech-language pathologists (SLPs) who include spouses in fluency therapy.

Accurate prediction of protein secondary structural class with fuzzy structural vectors.

The prerequisites for accurate prediction of protein secondary structural class (all-alpha, all-beta, alpha+beta, alpha/beta or multidomain) were studied, and a new similarity-based method is presented for the prediction of the secondary structural class of a protein from its sequence. The new method uses representatives of nuclear families as a learning set. For the sequence to be predicted, the method produces a vector of certainty factors called a fuzzy structural vector. Validation with independent test sets shows that the prediction accuracy of the proposed method has clear dependency on the representativity of the learning set. The representatives obtained from the nuclear families of the Brookhaven Protein Data Bank (PDB) were shown to give accurate predictions for PDB proteins, whilst the amino acid composition-based methods used previously achieve their maximum predictability with relatively limited learning sets, and they remain inaccurate even with highly representative learning sets. The usability of the new method is increased further by the fuzzy structural vectors, which substantially reduce the risk of misclassification and realistically describe vague secondary structural tendencies.

Representative selection of proteins based on nuclear families.

The selection of unbiased representatives from a large database is complicated by the requirement for the chosen entries to be not only genuinely different from each other but also typical for the family of related entries. A method satisfying this 2-fold objective was developed by equipping complete linkage clustering with a novel noise elimination procedure to deal with overlapping cluster structure. A total of 200 nuclear families of truly related Brookhaven Protein Data Bank structures were generated, from which any entry can be chosen to represent its family.

Skeletally metastatic malignant melanoma of the foot.

Malignant melanoma involving the foot has typically been documented in cases whereby the primary lesion was subungually located. Characteristic clinical features usually assist the physician in diagnosing the suspicious lesion. The case study that follows illustrates an atypical presentation of skeletally metastatic malignant melanoma of the foot where the primary lesion was not identified.

Hallux set angle. A new radiographic measurement for hallux valgus.

The authors introduce a unique and clinically oriented technique for evaluation of hallux abducto valgus deformity and its operative correction. A new concept in the application of the Austin osteotomy is also presented.

Selection of a representative set of structures from Brookhaven Protein Data Bank.

Reliable structural and statistical analyses of three dimensional protein structures should be based on unbiased data. The Protein Data Bank is highly redundant, containing several entries for identical or very similar sequences. A technique was developed for clustering the known structures based on their sequences and contents of alpha- and beta-structures. First, sequences were aligned pairwise. A representative sample of sequences was then obtained by grouping similar sequences together, and selecting a typical representative from each group. The similarity significance threshold needed in the clustering method was found by analyzing similarities of random sequences. Because three dimensional structures for proteins of same structural class are generally more conserved than their sequences, the proteins were clustered also according to their contents of secondary structural elements. The results of these clusterings indicate conservation of alpha- and beta-structures even when sequence similarity is relatively low. An unbiased sample of 103 high resolution structures, representing a wide variety of proteins, was chosen based on the suggestions made by the clustering algorithm. The proteins were divided into structural classes according to their contents and ratios of secondary structural elements. Previous classifications have suffered from subjective view of secondary structures, whereas here the classification was based on backbone geometry. The concise view lead to reclassification of some structures. The representative set of structures facilitates unbiased analyses of relationships between protein sequence, function, and structure as well as of structural characteristics.

The effects of beta blockade with (epanolol) and without (atenolol) intrinsic sympathomimetic activity in stable angina pectoris. The Visacor Study Group.

Beta blockade constitutes efficient therapy for stable angina pectoris. The effects of lowering blood pressure and heart rate with such treatment are not always desired. Epanolol is a beta 1-selective partial agonist with minor effects on blood pressure and heart rate at rest. Atenolol is a pure beta 1-selective antagonist with more pronounced effects on blood pressure and heart rate at rest. The effects of epanolol, 200 mg o.d., and atenolol, 100 mg o.d., were compared in 173 middle-aged patients with stable angina pectoris in a randomized, double-blind, parallel group-controlled study for one year. No significant differences were shown in angina attack rate, nitrate consumption, or exercise performance. Resting heart rate and blood pressure were significantly lower on atenolol. Epanolol tended to be better tolerated than atenolol, as shown by visual analogue scales of well-being, activity, energy, and warm extremities, further supported by fewer reports on possible adverse reactions. In conclusion, epanolol appears to be as effective as atenolol and better tolerated in patients with stable angina pectoris.

The distal akin osteotomy: a new approach.

The authors describe a modification of the Akin procedure using a distal oblique osteotomy with rigid internal fixation. The anatomic, biomechanic, and physiologic advantages of this modification are discussed. Its proper use and an analysis of results is included.