Microplastics in grey seal (Halichoerus grypus) intestines: Are they associated with parasite aggregations?

Between 2012 and 2015, 13 grey seals were recovered from trammel nets targeting monkfish and rays off the south coast of Ireland. Incidence and distribution of microplastics were investigated along the intestines of bycaught seals. No macrodebris items were found, whereas microplastics were detected in all seals. A total of 363 microplastics items were identified (85% fibers, 14% fragments, 1% films). Estimation of microplastic ingestion based on prey ingestion (245 particles) was lower than the observed data. Acantocephala parasites (n = 1543) were found in 12 seals, with an average of 74.5 ±â€¯67.7 parasites per seal. Distribution of microplastics varied between seals, although microplastics tended to accumulate in areas where more parasites were aggregated; however, there was no significant relationship between the number of parasites and microplastics was found. Seals recovered from nets appear to be a good source to monitor the incidence of microplastic pollution within the coastal food webs.

Current and Prospective Use of Musculoskeletal Diagnostic Ultrasound Imaging at Chiropractic Teaching Institutions: A Worldwide Survey of Diagnostic Imaging Staff.

OBJECTIVE: The purpose of this study was to survey the use of musculoskeletal diagnostic ultrasound imaging (MSK-DUSI) at chiropractic educational programs worldwide and to elicit opinions of academic diagnostic imaging staff of its prospective use at their teaching institutions. METHODS: An electronic questionnaire was delivered in 2014 using SurveyMonkey and notifications were disseminated by e-mail to 127 diagnostic imaging staff at chiropractic programs worldwide. The questionnaire consisted of 27 items using multiple-choice, Likert-type, and open-ended questions. Descriptive statistics were used for basic demographic data and the results of the numerical scales used in each item. RESULTS: Fifty-nine respondents (46.5%) from 24 (24/41) chiropractic programs returned questionnaires. The reported use of MSK-DUSI at chiropractic programs is low (n = 5/24); however, respondents from 9 institutions stated that it is planned to be implemented. Few respondents stated they had formal MSK-DUSI qualifications (4/59); however, 7 respondents stated they were in the process of becoming certified. Most respondents expressed an interest in the prospect of incorporating MSK-DUSI at their chiropractic program. Sixty-five percent stated that chiropractic programs should provide MSK-DUSI training to chiropractic students, and 75% of respondents stated that chiropractic programs should be providing accredited postgraduate MSK-DUSI courses. CONCLUSIONS: The current use of MSK-DUSI among chiropractic programs that responded to this survey is low. The opinions of diagnostic imaging staff who responded suggest a positive attitude to its use and possible growth in its use if foundational work, including gaining funding, accreditation, and acceptance of within the scope of chiropractic practice, is undertaken.

A genomewide catalogue of single nucleotide polymorphisms in white-beaked and Atlantic white-sided dolphins.

The field of population genetics is rapidly moving into population genomics as the quantity of data generated by high-throughput sequencing platforms increases. In this study, we used restriction-site-associated DNA sequencing (RADSeq) to recover genomewide genotypes from 70 white-beaked (Lagenorhynchus albirostris) and 43 Atlantic white-sided dolphins (L. acutus) gathered throughout their north-east Atlantic distribution range. Both species are at a high risk of being negatively affected by climate change. Here, we provide a resource of 38,240 RAD-tags and 52,981 nuclear SNPs shared between both species. We have estimated overall higher levels of nucleotide diversity in white-sided (π = 0.0492 ± 0.0006%) than in white-beaked dolphins (π = 0.0300 ± 0.0004%). White-sided dolphins sampled in the Faroe Islands, belonging to two pods (N = 7 and N = 11), showed similar levels of diversity (π = 0.0317 ± 0.0007% and 0.0267 ± 0.0006%, respectively) compared to unrelated individuals of the same species sampled elsewhere (e.g. π = 0.0285 ± 0.0007% for 11 Scottish individuals). No evidence of higher levels of kinship within pods can be derived from our analyses. When identifying the most likely number of genetic clusters among our sample set, we obtained an estimate of two to four clusters, corresponding to both species and possibly, two further clusters within each species. A higher diversity and lower population structuring was encountered in white-sided dolphins from the north-east Atlantic, in line with their preference for pelagic waters, as opposed to white-beaked dolphins that have a more patchy distribution, mainly across continental shelves.

The accuracy of diagnostic ultrasound imaging for musculoskeletal soft tissue pathology of the extremities: a comprehensive review of the literature.

Musculoskeletal diagnostic ultrasound imaging (MSK-DUSI) has been growing outside the traditional radiology speciality. Increased use of this technology has been reported in several healthcare settings, however an apparent gap in the knowledge of the accuracy of this diagnostic technology indicated a review was warranted. We undertook a structured review of the literature to assess the accuracy of MSK-DUSI for the diagnosis of musculoskeletal soft tissue pathology of the extremities. An electronic search of the National Library of Medicine's PubMed database (1972 to mid-2014) was conducted. All relevant systematic reviews of diagnostic studies, all diagnostic studies published after the date of the latest systematic reviews and relevant diagnostic studies outside the scope the systematic reviews that directly compared the accuracy of MSK-DUSI (the index test) to an appropriate reference standard for the target condition were included. A fundamental appraisal of the methodological quality of studies was completed. The individual sensitivity, specificity and likelihood ratio data were extracted and entered into diagnostic accuracy tables. A total of 207 individual studies were included. The results show that MSK-DUSI has acceptable diagnostic accuracy for a wide spectrum of musculoskeletal conditions of the extremities. However, there is a lack of high quality prospective experimental studies in this area and as such clinicians should interpret the results with some caution due to the potential for overestimation of diagnostic accuracy.

CT of the neonatal head.

Computed tomography (CT) is used less often than other techniques on neonatal units. However, in the acute setting, CT can be invaluable in diagnosing or excluding potentially life-threatening conditions and guiding initial management in neonates. Common indications for scanning include trauma, suspected non-accidental injury, infection, or an acute hypoxic or metabolic event. The aim of this review is to provide an overview of the normal neonatal head at CT and compare this to the common pathological abnormalities. Several key features of each condition will be highlighted. It is important to note that some pathological conditions can have overlapping features at CT and, therefore, the clinical history and additional investigations are also of key importance in determining the diagnosis.

Atypical panmixia in a European dolphin species (Delphinus delphis): implications for the evolution of diversity across oceanic boundaries.

Despite the scarcity of geographical barriers in the ocean environment, delphinid cetaceans often exhibit marked patterns of population structure on a regional scale. The European coastline is a prime example, with species exhibiting population structure across well-defined environmental boundaries. Here we undertake a comprehensive population genetic study on the European common dolphin (Delphinus delphis, based on 492 samples and 15 loci) and establish that this species shows exceptional panmixia across most of the study range. We found differentiation only between the eastern and western Mediterranean, consistent with earlier studies, and here use approximate Bayesian computations to explore different scenarios to explain the observed pattern. Our results suggest that a recent population bottleneck likely contributed significantly to the differentiation of the Eastern Mediterranean population (in Greek waters). This interpretation is consistent with independent census data that suggest a sharp population decline in the recent past. The implication is that an unperturbed population may currently show panmixia across the full study range. This exception to the more typical pattern of population structure seen for other regional dolphin species (and for common dolphin populations elsewhere in the world) suggests particular ecological or life-history traits distinct to this species in European waters.

hPMC2 is required for recruiting an ERbeta coactivator complex to mediate transcriptional upregulation of NQO1 and protection against oxidative DNA damage by tamoxifen.

In the presence of ERbeta, trans-hydroxytamoxifen (TOT) protects cells against 17beta-estradiol (E(2))-induced oxidative DNA damage (ODD) and this correlates with increased expression of the antioxidative enzyme quinone reductase (QR). Here, we investigate the molecular mechanism responsible for ERbeta-mediated protection against ODD. We observe constitutive interaction between ERbeta and the novel protein hPMC2. Using a combination of breast epithelial cell lines that are either positive or negative for ERalpha, we demonstrate TOT-dependent recruitment of both ERbeta and hPMC2 to the EpRE (electrophile response element)-regulated antioxidative enzyme QR. We further demonstrate TOT-dependent corecruitment of the coactivators Nrf2, PARP-1 (poly (ADP-ribose) polymerase 1) and topoisomerase IIbeta, both in the presence and absence of ERalpha. However, absence of either ERbeta or hPMC2 results in nonrecruitment of PARP-1 and topoisomerase IIbeta, loss of antioxidative enzyme induction and attenuated protection against ODD by TOT even in the presence of Nrf2 and ERalpha. These findings indicate minor role for Nrf2 and ERalpha in TOT-dependent antioxidative gene regulation. However, downregulation of PARP-1 attenuates TOT-dependent antioxidative gene induction. We conclude that ERbeta and hPMC2 are required for TOT-dependent recruitment of coactivators such as PARP-1 to the EpRE resulting in the induction of antioxidative enzymes and subsequent protection against ODD.

Bioaccumulation of persistent organic pollutants in female common dolphins (Delphinus delphis) and harbour porpoises (Phocoena phocoena) from western European seas: geographical trends, causal factors and effects on reproduction and mortality.

Concentrations of polychlorinated biphenyls (PCBs) in blubber of female common dolphins and harbour porpoises from the Atlantic coast of Europe were frequently above the threshold at which effects on reproduction could be expected, in 40% and 47% of cases respectively. This rose to 74% for porpoises from the southern North Sea. PCB concentrations were also high in southern North Sea fish. The average pregnancy rate recorded in porpoises (42%) in the study area was lower than in the western Atlantic but that in common dolphins (25%) was similar to that of the western Atlantic population. Porpoises that died from disease or parasitic infection had higher concentrations of persistent organic pollutants (POPs) than animals dying from other causes. Few of the common dolphins sampled had died from disease or parasitic infection. POP profiles in common dolphin blubber were related to individual feeding history while those in porpoises were more strongly related to condition.

Biological and ecological factors related to trace element levels in harbour porpoises (Phocoena phocoena) from European waters.

Selected trace elements (Cd, Cu, Hg, Se, Zn) were measured in the kidneys and the liver of 104 harbour porpoises (Phocoena phocoena) stranded along the coasts of France, Galicia (Spain), Ireland, Scotland (UK), and the Netherlands. Generally, relatively low concentrations of toxic elements were encountered in the tissues of European porpoises, except for two individuals, which displayed high hepatic Hg concentrations. Also, elevated Cd levels obtained in Scottish porpoises could be related to their feeding preferences and this result suggests an increase of the proportion of cephalopods in their diet with latitude. Moreover, significant geographical differences were seen in hepatic Zn concentrations; the elevated Zn concentrations displayed by porpoises from the Netherlands may relate their poor health status. Variation in metal concentrations within porpoises from the North Sea is likely to reflect a long-term segregation between animals from northern (Scotland) and southern areas (the Netherlands), making trace elements powerful ecological tracers.

Protective roles of quinone reductase and tamoxifen against estrogen-induced mammary tumorigenesis.

We previously reported that antiestrogen-liganded estrogen receptor beta (ERbeta) transcriptionally activates the major detoxifying enzyme quinone reductase (QR) (NAD(P)H:quinone oxidoreductase). Further studies on the functional role of ERbeta-mediated upregulation of antioxidative enzymes indicated protective effects against estrogen-induced oxidative DNA damage (ODD). We now report on in vivo and in vitro studies that show that ERbeta-mediated upregulation of QR are involved in the protection against estrogen-induced mammary tumorigenesis. Using the August Copenhagen Irish (ACI) model of estrogen-induced carcinogenesis, we observed that increased ODD and decreased QR expression occur early in the process of estrogen-induced mammary tumorigenesis. Prevention of ACI mammary gland tumorigenesis by tamoxifen was accompanied by decreased ODD and increased QR levels. These correlative findings were supported by our findings that downregulation of QR levels led to increased levels of estrogen quinone metabolites and enhanced transformation potential of 17beta-estradiol treated MCF10A non-tumorigenic breast epithelial cells. Concurrent expression of ERbeta and treatment with 4-hydroxytamoxifen decreased tumorigenic potential of these MCF10A cells. We conclude that upregulation of QR, through induction by tamoxifen, can inhibit estrogen-induced ODD and mammary cell tumorigenesis, representing a possible novel mechanism of tamoxifen prevention against breast cancer.

Potential biomarker for early risk assessment of prostate cancer.

BACKGROUND: Catechol estrogen quinones (CEQ) derived from 4-hydroxyestrone (4-OHE1) and 4-hydroxyestradiol (4-OHE2) react with DNA to form depurinating--N7Gua and--N3Ade adducts. This damage leads to mutations that can initiate breast and prostate cancer. To determine whether this damage occurs in humans, urine samples from men with prostate cancer and benign urological conditions, and healthy controls were analyzed. The objective was determining whether any of the cancer patients had formed the depurinating 4-OHE1(E2)-1-N3Ade adducts. METHODS: The adducts were extracted from samples by using affinity columns equipped with a monoclonal antibody developed for detecting 4-OHE1(E2)-1-N3Ade adducts. Eluted extracts were separated by capillary electrophoresis with field-amplified sample stacking and/or ultraperformance liquid chromatography. Absorption/luminescence spectroscopies and mass spectrometry were used to identify the adducts. RESULTS: 4-OHE1-1-N3Ade was detected at higher levels in samples from subjects with prostate cancer (n = 7) and benign urological conditions (n = 4) compared to healthy males (n = 5). CONCLUSION: This is the first demonstration that CEQ-derived DNA adducts are present in urine samples from subjects with prostate cancer.

Lead contamination of small cetaceans in European waters--the use of stable isotopes for identifying the sources of lead exposure.

Lead concentrations and isotopic composition have been measured in bone and teeth of small cetaceans belonging to three species (Delphinus delphis, Phocoena phocoena and Stenella coeruleoalba), to evaluate the toxicological risk and to determine sources of lead in the European waters. Lead concentrations, far lower than threshold value inducing toxic effects in human, were higher in teeth than in bones, but highly correlated between the two tissues (r=0.92, p<0.001). Large variations of 206Pb/207Pb values in bone tissue showed that cetaceans must be submitted to various atmospheric influences. No geographical differences appeared which is consistent with studies on their distribution indicating seasonal movements between Brittany waters and the Bay of Biscay. The negative correlation between 206Pb/207Pb ratios and age of the individuals reflected the decrease in the production of alkyl lead in Europe, i.e., the increasing use of unleaded gasoline.

Development of monoclonal antibodies to 4-hydroxyestrogen-2-N-acetylcysteine conjugates: immunoaffinity and spectroscopic studies.

Catechol estrogen quinones (CEQ) derived from oxidation of the catechol estrogens 4-hydroxyestrone (4-OHE1) and 4-hydroxyestradiol (4-OHE2) can conjugate with glutathione (GSH), a reaction that prevents damage to DNA and can provide biomarkers of exposure to CEQs. Monoclonal antibodies (MAb) to 4-OHE1(E2)-2-N-acetylcysteine [4-OHE1(E2)-2-NAcCys] were developed and characterized by immunological and spectroscopic studies. The NAcCys conjugate is the hydrolytic product of the corresponding conjugate with GSH, followed by N-acetylation of cysteine. MAbs were produced by immunizing mice with 4-OHE1(E2)-2-NAcCys attached to an appropriate linker that was conjugated to keyhole limpet hemocyanin (KLH). Hybridoma cell lines were screened using 4-OHE1(E2)-2-NAcCys conjugated to ovalbumin (OA). There is no immunological cross-reactivity between KLH and OA. Hence, positive hybridoma cell lines secreting antibody against 4-OHE1(E2)-2-NAcCys could be rapidly identified using OA-4-OHE1(E2)-2-NAcCys. An affinity column was developed and used to purify MAb against 4-OHE1(E2)-2-NAcCys. The purified MAb was immobilized on an agarose bead column. This column was used to capture and preconcentrate the hapten of interest out of urine samples. A number of structurally related standards were used to estimate the selectivity and specificity of the chosen MAb. Capillary electrophoresis (CE) with field-amplified sample stacking in absorbance detection mode and laser-induced low temperature luminescence measurements were used to identify and quantitate the 4-OHE1(E2)-2-NAcCys conjugates and related compounds released from the affinity column. Femtomole detection limits have been demonstrated. Future prospects in clinical diagnostics for testing human exposure to CEQ by urine analysis are briefly addressed.

Genotoxic metabolites of estradiol in breast: potential mechanism of estradiol induced carcinogenesis.

Long term exposure to estradiol increases the risk of breast cancer in a variety of animal species, as well as in women. The mechanisms responsible for this effect have not been firmly established. The prevailing theory proposes that estrogens increase the rate of cell proliferation by stimulating estrogen receptor-mediated transcription and thereby the number of errors occurring during DNA replication. An alternative hypothesis proposes that estradiol can be metabolized to quinone derivatives which can react with DNA and then remove bases from DNA through a process called depurination. Error prone DNA repair then results in point mutations. We postulate that these two processes, increased cell proliferation and genotoxic metabolite formation, act in an additive or synergistic fashion to induce cancer. If correct, aromatase inhibitors would block both processes whereas anti-estrogens would only inhibit receptor-mediated effects. Accordingly, aromatase inhibitors would be more effective in preventing breast cancer than use of anti-estrogens. Our studies initially demonstrated that catechol estrogen (CE) quinone metabolites are formed in MCF-7 human breast cancer cells in culture. Measurement of estrogen metabolites and conjugates involved utilization of an HPLC separation coupled with an electrochemical detector. We then utilized an animal model that allows dissociation of estrogen receptor-mediated function from that of the effects of estradiol metabolites. Wnt-1 transgenic mice harboring a knock-out of ERalpha provides a means of examining the effect of estrogen deprivation in the absence of the ER in animals with a high incidence of breast tumors. ERbeta was shown to be absent in the breast tissue of these animals by RNase protection assay. In the breast tissue of these estrogen receptor alpha knock-out (ERKO)/Wnt-1 transgenic mice, we demonstrated formation of genotoxic estradiol metabolites. The ERKO/Wnt-1 breast extracts contained picomole amounts of the 4-catechol estrogens, but not their methoxy conjugates nor the 2-CE or their methoxy conjugates. The 4-CE conjugates with glutathione or its hydrolytic products (cysteine and N-acetylcysteine) were detected in picomole amounts in both tumors and hyperplastic mammary tissue, demonstrating the formation of CE-3,4-quinones. These results are consistent with the hypothesis that mammary tumor development is primarily initiated by metabolism of estrogens to 4-CE and, then, to CE-3,4-quinones, which may react with DNA to induce oncogenic mutations. The next set of experiments examined the incidence of tumors formed in Wnt-1 transgenic mice bearing wild type ERalpha (ER+/+), the heterozygous combination of genes (ER+/ER-) or ERalpha knock-out (ER-/-). To assess the effect of estrogens in the absence of ER, half of the animals were oophorectomized on day 15 and the other half were sham operated. Castration reduced the incidence of breast tumors in all animal groups and demonstrated the dependence of tumor formation upon estrogens. A trend toward reduction in tumor number (not statistically significant at this interim analysis) occurred in the absence of functional ER since the number of tumors was markedly reduced in ERKO animals which were castrated early in life. In aggregate, our results support the concept that metabolites of estradiol may act in concert with ER mediated mechanisms to induce breast cancer.

Spectral characterization of catechol estrogen quinone (CEQ)-derived DNA adducts and their identification in human breast tissue extract.

Estrogens, including the natural hormones estrone (E(1)) and estradiol (E(2)), are thought to be involved in tumor induction. Catechol estrogen quinones (CEQ) derived from 4-hydroxyestrone (4-OHE(1)) and 4-hydroxyestradiol (4-OHE(2)) react with DNA and form depurinating N7Gua and N3Ade adducts that might be responsible for tumor initiation (Cavalieri, E. L., et al. (2000) J. Natl. Cancer Inst. Monogr. 27, 75). Current detection limits for the CEQ-derived DNA adducts by high-performance liquid chromatography with multichannel electrochemical detection are in the picomole range. To improve the limit of detection (LOD) for CEQ-derived DNA adducts, spectrophotometric monitoring was investigated. Spectroscopic studies of 4-OHE(1)-1-N3Ade, 4-OHE(1)-1-N7Gua, 4-OHE(2)-1-N3Ade, and 4-OHE(2)-1-N7Gua adduct standards were performed at 77 and 300 K. Upon laser excitation at 257 nm, the 4-OHE(1)- and 4-OHE(2)-derived N7Gua and N3Ade adducts are strongly phosphorescent at T = 77 K. No phosphorescence was observed at 300 K. Both N3Ade and N7Gua adduct types have weak phosphorescence origin bands near 383 and 385 nm, respectively. The corresponding phosphorescence lifetimes are 1.11 +/- 0.05 and 0.37 +/- 0.05 s. The LOD, based on phosphorescence measurements, is in the low femtomole range. The concentration LOD is approximately 10(-9) M, i.e., similar to that recently obtained for CEQ-derived N-acetylcysteine conjugates (Jankowiak, R., et al. (2003) Chem. Res. Toxicol. 16, 304). The LOD in capillary electrophoresis (CE) with field-amplified sample stacking and absorbance detection is about 3 x 10(-8) M. To verify whether CEQ-derived DNA adducts are formed in humans or not, tissue extracts from two breast cancer patients were analyzed by CE interfaced with room temperature absorption and low temperature (laser-excited) phosphorescence spectroscopies. For the first time, formation of CEQ-derived DNA adducts is shown in humans. For example, the level of 4-OHE(1)-1-N3Ade in the breast tissue extract from a patient with breast carcinoma (8.40 +/- 0.05 pmol/g of tissue) is larger by a factor of about 30 than that in the breast tissue sample from a woman without breast cancer (0.25 +/- 0.05 pmol/g of tissue). In contrast, similar amounts of 4-OHE(2)-1-N3Ade were observed in both types of tissue. Although more breast tissue samples from women with and without breast cancer need to be studied, these results suggest that the N3Ade adducts could serve as biomarkers to predict the risk of breast cancer.

Marine mammals from northeast Atlantic: relationship between their trophic status as determined by delta13C and delta15N measurements and their trace metal concentrations.

The relationship between trophic position through delta13C and delta15N and trace metal concentrations (Zn, Cd, Cu and Hg) was investigated in the tissues of six marine mammal species from the Northeast Atlantic: striped dolphin Stenella coeruleoalba, common dolphin, Delphinus delphis, Atlantic white-sided dolphin Lagenorhynchus acutus, harbour porpoise Phocoena phocoena, white beaked-dolphin Lagenorhynchus albirostris, grey seal Halichoerus grypus stranded on French Channel and Irish coasts. White-beaked dolphins, harbour porpoises, white-sided dolphins, common and striped dolphins display the same relative and decreasing trophic position, as measured by delta15N values, along both the Irish and French channel coasts, reflecting conservative trophic habits between these two places. Hepatic and renal Cd concentrations were significantly correlated to muscle delta13C and delta15N values while Hg, Zn and Cu did not. These results suggest that Cd accumulation is partly linked to the diet while other factors such as age or body condition might explain Hg, Zn or Cu variability in marine mammals. Combined stable isotope and trace metal analyses appear to be useful tools for the study of marine mammal ecology.

Initiation of cancer and other diseases by catechol ortho-quinones: a unifying mechanism.

Exposure to estrogens is a risk factor for breast and other human cancers. Initiation of breast, prostate and other cancers has been hypothesized to result from reaction of specific estrogen metabolites, catechol estrogen-3,4-quinones, with DNA to form depurinating adducts at the N-7 of guanine and N-3 of adenine by 1,4-Michael addition. The catechol of the carcinogenic synthetic estrogen hexestrol, a hydrogenated derivative of diethylstilbestrol, is metabolized to its quinone, which reacts with DNA to form depurinating adducts at the N-7 of guanine and N-3 of adenine. The catecholamine dopamine and the metabolite catechol (1,2-dihydroxybenzene) of the leukemogen benzene can also be oxidized to their quinones, which react with DNA to form predominantly analogous depurinating adducts. Apurinic sites formed by depurinating adducts are converted into tumor-initiating mutations by error-prone repair. These mutations could initiate cancer by estrogens and benzene, and Parkinson's disease by the neurotransmitter dopamine. These data suggest a unifying molecular mechanism of initiation for many cancers and neurodegenerative diseases and lay the groundwork for designing strategies to assess risk and prevent these diseases.

Polybrominated diphenyl ethers in two species of marine top predators from England and Wales.

During an earlier study, polybrominated diphenyl ethers were detected at high concentrations in fish and sediments downstream of a manufacturing site in NE England. We have now undertaken analysis of 14 tri- to heptabromodiphenyl ether congeners in tissues of two species of marine top predators exposed to these compounds through their consumption of fish. In this paper we report data for 47 cormorants and 60 harbour porpoises from England and Wales, sampled during the period 1996-2000. Concentrations of the summed congeners ranged from 1.8 to 140 microg kg(-1) wet weight in cormorant livers, and from not detected to 6900 microg kg(-1) wet weight in porpoise blubber. The major congeners present were generally BDE47, BDE99 and BDE100. There was little correlation between concentrations of chlorobiphenyls (as the sum of 25 individual congeners) and the sum of the 14 BDE congeners determined, particularly for the porpoises.

Evidence that a burst of DNA depurination in SENCAR mouse skin induces error-prone repair and forms mutations in the H-ras gene.

Treatment of SENCAR mouse skin with dibenzo[a,l]pyrene results in abundant formation of abasic sites that undergo error-prone excision repair, forming oncogenic H-ras mutations in the early preneoplastic period. To examine whether the abundance of abasic sites causes repair infidelity, we treated SENCAR mouse skin with estradiol-3,4-quinone (E(2)-3,4-Q) and determined adduct levels 1 h after treatment, as well as mutation spectra in the H-ras gene between 6 h and 3 days after treatment. E(2)-3,4-Q formed predominantly (> or =99%) the rapidly-depurinating 4-hydroxy estradiol (4-OHE(2))-1-N3Ade adduct and the slower-depurinating 4-OHE(2)-1-N7Gua adduct. Between 6 h and 3 days, E(2)-3,4-Q induced abundant A to G mutations in H-ras DNA, frequently in the context of a 3'-G residue. Using a T.G-DNA glycosylase (TDG)-PCR assay, we determined that the early A to G mutations (6 and 12 h) were in the form of G.T heteroduplexes, suggesting misrepair at A-specific depurination sites. Since G-specific mutations were infrequent in the spectra, it appears that the slow rate of depurination of the N7Gua adducts during active repair may not generate a threshold level of G-specific abasic sites to affect repair fidelity. These results also suggest that E(2)-3,4-Q, a suspected endogenous carcinogen, is a genotoxic compound and could cause mutations.

Catechol estrogen metabolites and conjugates in mammary tumors and hyperplastic tissue from estrogen receptor-alpha knock-out (ERKO)/Wnt-1 mice: implications for initiation of mammary tumors.

A novel model of breast cancer was established by crossing mice carrying the Wnt-1 transgene (100% of adult females develop spontaneous mammary tumors) with the ERKO mouse line, in which mammary tumors develop despite a lack of functional estrogen receptor-alpha. To begin investigating whether metabolite-mediated genotoxicity of estrogens may play an important role in the initiation of mammary tumors, the pattern of estrogen metabolites and conjugates was examined in ERKO/Wnt-1 mice. Extracts of hyperplastic mammary tissue and mammary tumors were analyzed by HPLC with identification and quantification of compounds by multichannel electrochemical detection. Picomole amounts of the 4-catechol estrogens (CE) were detected, but their methoxy conjugates, as well as the 2-CE and their methoxy conjugates, were not. 4-CE conjugates with glutathione or its hydrolytic products (cysteine and N-acetylcysteine) were detected in picomole amounts in both tumors and hyperplastic mammary tissue, demonstrating the formation of CE-3,4-quinones. These preliminary findings show that the estrogen metabolite profile in the mammary tissue is unbalanced, in that the normally minor 4-CE metabolites were detected in the mammary tissue and not the normally predominant 2-CE. These results are consistent with the hypothesis that the mammary tumor development is primarily initiated by metabolism of estrogens to 4-CE and, then, to CE-3,4-quinones, which may react with DNA to induce oncogenic mutations.